University of Southern California Dissertations and Theses

Estrogen and progesterone-based hormone therapy and the development of Alzheimer's disease

(USC Thesis Other)

Estrogen and progesterone-based hormone therapy and the development of Alzheimer's disease

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content ESTROGEN AND PROGESTERONE-BASED HORMONE
THERAPY AND THE DEVELOPMENT OF ALZHEIMER’S DISEASE
by
Jenna C. Carroll
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
August 2009
Copyright 2009 Jenna C. Carroll
ii
DEDICATION
To my mom, who always believes in me, even when I don’t believe in myself
iii
ACKNOWLEDGEMENTS
Most importantly, I would like to thank my advisor, Dr. Christian Pike. Without
him, none of this work would have been possible. He provided me with invaluable
guidance, support, and encouragement. He always pushed me to strive for the top and
taught me all the important skills I will need to have a successful scientific career. He
has been an excellent role model and is the kind of PI I hope to be in the future. I greatly
respect him as a scientist and I truly admire the way the gears in his mind are always
turning out new project ideas. Most importantly, I want to thank him for always having
my back and looking out for my best interest, through good times and bad. And of
course, I thank him for putting up with my “New Jersey attitude.”
I would like to thank the past and present members of the Pike lab; Dr. Martin
Ramsden, Dr. Emily Rosario, Dr. Mingzhong Yao, Mrs. Anusha Jayaraman and Dr.
Anna Barron. Thank you for your support and friendship. Also, I would like to thank our
past and present undergraduate students, Miss Tina Huang, Kelly McMullen, Sara
Krieimer, Holly Villamagna and Meg Brown for their hard work on so many projects.
In particular, I would like to thank Dr. Emily Rosario for being the best friend and
colleague I could ever have asked for. I can’t begin to express how thankful I am that
our lives crossed paths. You taught me everything from how to use a pipette, to all the
surgical techniques, to how to trick Christian into reading our emails. You have cried
when I cried and celebrated when I celebrated and I would not have made it through
grad school without you. Thank you from the bottom of my heart.
In addition, I would like to thank my committee members, Dr. Caleb Finch, Dr.
Richard Thompson, Dr. Carol Miller, and Dr. Wendy Mack. I am honored to have been
iv
mentored by such an esteemed group of faculty. I thank you for all of your helpful
criticisms, support, and wisdom, both in the lab and in terms of my career.
I would like to thank the National Institute of Aging (NIA) and Dr. Caleb Finch for
the opportunity to be part of the Training Grant entitled “The Endocrinology and
Neurobiology of Aging.” In addition to providing me with 2 years of funding, this group
provided me with important scientific critiques and opportunities.
I would also like to thank the National Institute of Neurological Disorders and
Stroke (NINDS) for funding my NRSA Predoctoral grant entitled “Estrogen, Progesterone
and Alzheimer’s Disease.” Learning the grant-writing and submission process was an
invaluable training opportunity that will help me throughout my scientific career.
Furthermore, I would like to thank my colleagues and my collaborators. Thank
you to the Finch and Davies lab members for the use of their equipment and supplies.
Thank you to Milton and Darryl for taking care of my mice. Thank you to Dr. Frank
Stanczyk and Dr. Lilly Chang for help with serum samples. Thank you to Dr. Roberta
Brinton and the members of the Progesterone Program Project Grant for allowing me to
be involved with such a wonderful collaboration. And special thanks to Todd Morgan for
countless discussions, wonderful collaborations and help learning exciting techniques.
Lastly, I would like to thank my family and friends. Thanks mom, dad and
Jonathan for all your love and support my entire life. Thank you mom for talking me out
of quitting when my western blots weren’t working and for encouraging me to try my
hardest and stay true to myself. I couldn’t have done any of this without you. Thanks to
my friends who were there for me whether it was for a celebratory beer at the Parlor or
Diddy Riese to ease my sorrows. And especially, thank you to my friend and roommate
Andrea, who was there through it all and always supported me.
v
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
ABSTRACT xi
CHAPTER ONE Introduction 1
1. Alzheimer’s disease 1
Classic AD neuropathology 1
2. Gender difference in AD 3
Prevalence and incidence 3
Gender differences in transgenic mice 4
3. Menopause, hormone therapy and AD 4
Loss of hormones at menopause 4
Hormone therapy and AD 5
4. Estrogen neuroprotection 7
Synthesis and bioavailability of sex steroid 7
hormones in brain
Estrogen regulates neuron viability 8
Estrogen receptor-mediated actions 13
5. Estrogen regulation of AD insults 16
Estrogen regulation of β–amyloid accumulation 16
Estrogen regulation of tau hyperphosphorylation 19
6. Aging effects on estrogen responsiveness 20
7. Effects of progestins in the brain 25
Progesterone regulation of neuron viability 26
Progesterone regulation of β-amyloid 27
8. Interactive effects of estrogens and progestins in the brain 28
Clinical observations 28
Progesterone inhibits estrogen neuroprotection 29
Cyclic versus continuous progesterone treatment 31
9. Male sex steroid hormones and AD 34
Age-related androgen depletion and AD 34
Testosterone neuroprotection and AD insults 37
Testosterone regulation of β-amyloid accumulation 39
vi
10. Hypothesis and Experimental Paradigms 40
The 3xTg-AD mouse 41
Kainate lesion model 44
CHAPTER TWO Progesterone blocks estrogen neuroprotection 45
from kainate in middle-aged female rats
Chapter two abstract 45
Introduction 45
Methods 47
Results 49
Discussion 52
CHAPTER THREE Progesterone and estrogen regulate 57
Alzheimer-like neuropathology in female 3xTg-AD mice
Chapter three abstract 57
Introduction 58
Methods 58
Results 64
Discussion 77
CHAPTER FOUR Continuous and cyclic progesterone 83
differentially effect estrogen regulation of Alzheimer-like pathology
in female 3xTg-AD mice
Chapter four abstract 83
Introduction 84
Methods 85
Results 90
Discussion 96
CHAPTER FIVE Selective estrogen receptor modulators differentially 102
regulate Alzheimer-like changes in female 3xTg-AD mice
Chapter five abstract 102
Introduction 103
Methods 104
Results 106
Discussion 111
CHAPTER SIX Perinatal sex steroid hormones alter adult development 114
of Alzheimer’s disease-like neuropathology in 3xTg-AD mice
Chapter six abstract 114
Introduction 114
Methods 117
vii
Results 122
Discussion 128
CHAPTER SEVEN Conclusions and future directions 135
REFERENCES 154
viii
LIST OF TABLES
1. Neuroprotective effects of E
2
through ERα and ERβ 15
2. Progesterone antagonizes many neuroprotective effects of estradiol 29
3. Confirmation of TP treatment in female mice 125
4. Confirmation of flutamide treatment in male mice 126
ix
LIST OF FIGURES
1. Estrogens activate neuroprotective pathways that may attenuate AD 8
2. Treatment conditions did not significantly affect the severity and 50
latency of kainate-induced seizures
3. Progesterone blocks the neuroprotective effect of estrogen 52
4. Age-related increase in Aβ immunoreactivity 65
5. Quantification of age-related increase in Aβ accumulation 66
6. CTF levels do not change across conditions in 3xTg-AD mice 67
7. Age-related increase in tau hyperphosphorylation 68
8. Age-related decline in spontaneous alternation behavior 69
9. E
2
regulates Aβ accumulation 71
10. E
2
regulation of tau hyperphosphorylation 72
11. E
2
regulates spontaneous alternation behavior 73
12. P4 attenuates the effects of E
2
on Aβ accumulation 75
13. P4 regulates tau hyperphosphorylation 76
14. P4 regulation of spontaneous alternation behavior 77
15. Bioassay confirmation of hormone treatments 90
16. Behavioral confirmation of hormone treatments 92
17. Continuous, but not cyclic P4 attenuates E
2
reduction 93
of Aβ accumulation
18. Continuous, but not cyclic P4 attenuates E
2
increase in 95
working memory
x
19. E
2
, Continuous P4 and cyclic P4 reduce tau hyperphosphorylation 96
20. Accumulation of Aβ is differentially regulated by E
2
and the 107
SERMs PPT and DPN, across brain regions
21. Quantitative comparisons of E
2
, PPT, and DPN treatments on Aβ 108
accumulation
22. Assessment of working memory performance using the SAB in 109
female 3xTg-AD mice
23. PPT and DPN mimic the effect of E
2
in preventing the OVX-induced 110
increase in immobility in the forced swim test (FST)
24. Female 3xTg-AD mice demonstrate higher levels of Aβ 123
immunohistochemistry than males, particularly at 12-14 mo of age
25. Females perform more poorly on hippocampal-dependent working 124
memory task (SAB) than male 3xTg-AD mice, particularly at
12-14 mo of age
26. Perinatal flutamide treatment feminizes male 3xTg-AD adult brains 127
and TP treatment masculinizes female brains in terms of AD-like
neuropathology development in a region-specific manner
27. Perinatal flutamide treatment, but not TP treatment alters 128
hippocampal-dependent working memory on the Y-maze
xi
ABSTRACT
This dissertation seeks to investigate the broad issue of the effects of sex steroid
hormones on neuroprotective measures related to aging and the prevention of the
progression of Alzheimer’s disease (AD). In particular, this dissertation will focus on the
effects of female sex steroid hormones, estrogen and progesterone both alone and in
combination on AD in women. Abundant evidence implicates sex steroid depletion in
postmenopausal women as a risk factor for the development of AD. However, there
appears to be a disconnect between experimental data clearly establishing multiple
estrogen protective functions, and clinical findings showing that estrogen-based
hormone therapy fails to prevent and slow progression of cognitive decline and AD.
Furthermore, while estrogen’s many beneficial effects in the brain have been well
established, the effects of progesterone, both alone and in combination with estrogen,
are currently unclear and under-investigated. The Women’s Health Initiative Memory
Study (WHIMS) raised several questions regarding the actions of progestins in the brain
and the efficacy of hormone therapy in lowering the risk of AD in post-menopausal
women. These questions are the basis behind the experiments described in this
dissertation; a) the effects of estrogen and progesterone, both alone and in combination
on AD-like neuropathology, b) the difference between various, clinically-relevant
progestin exposures in combination with estrogen, c) the “critical window hypothesis” of
aging brain responsiveness, d) the effects of clinically-relevant selective estrogen
receptor modulators (SERMs) and e) the effects of activational vs. organizational sex
steroid hormone exposure. The data demonstrate that aged rodents have altered
neuroprotective responsiveness to the hormone treatment, that continuous progesterone
xii
treatment attenuates estrogen neuroprotection against kainate lesion and Aβ
accumulation, cyclic progesterone has more beneficial effects than continuous
progesterone treatment, synthetic estrogen-like compounds can mimic estrogen action,
and that gender differences in AD pathogenesis can be regulated by perinatal hormone
action. Overall, these results demonstrate that estrogen and progesterone therapies
remain important and potentially powerful preventive and intervention strategies against
AD and other age-related neurodegenerative disorders. Understanding the effects of
both estrogen and progesterone in the brain is essential for future optimization of
estrogen-based hormone therapy for the potential prevention and treatment of AD in
post-menopausal women.
1
CHAPTER ONE
Introduction
1. Alzheimer’s Disease
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder and the
leading cause of dementia in the elderly population. Currently, the Alzheimer’s Disease
Association estimates that approximately 4.5 million Americans have AD, a number that
could quadruple in the next 45 years (http://www.alz.org/). The prevalence of AD is
estimated to double every 5 years after the age of 65, making age the largest risk factor
for the development of AD (Brookmeyer et al., 1998).
Classic AD neuropathology
Neuropathologically, AD is characterized by brain region-specific deposition of β-
amyloid protein (Aβ) which creates senile plaques, hyperphosphorylation of the
cytoskeletal protein tau that forms lesions called neurofibrillary tangles and neuropil
threads, glial activation which is associated with inflammatory responses, and both
synaptic and neuronal loss (Braak and Braak, 1990; Hardy, 1997; Akiyama et al., 2000;
Hardy and Selkoe, 2002; LaFerla and Oddo, 2005). Although the mechanisms of AD
pathogenesis remain to be fully resolved, the leading hypothesis posits that the disease
is initiated and driven by prolonged elevation of Aβ levels (Hardy and Higgins, 1992). Aβ
is a proteolytic byproduct of the metabolism of amyloid precursor protein, a widely
expressed protein with numerous functions ranging from axonal transport to gene
transcription (Turner et al., 2003). As a consequence of amyloid precursor protein
expression, Aβ is normally found as a soluble protein at low levels in fluids and tissues
throughout the body. In theory, alterations in either the production or clearance of Abeta
2
that sway Aβ homeostasis towards increased neural levels will promote the development
of AD (Hardy and Selkoe, 2002). The accumulation of Aβ encourages its abnormal
assembly into oligomeric species that exhibit an altered structural conformation and can
induce a range of neurodegenerative effects (Haass and Selkoe, 2007). Consequently,
enormous effort has been expended on identifying factors that regulate Aβ accumulation
and or affect its neurodegenerative properties.
Outwardly, AD patients suffer from progressive cognitive decline, declarative
memory deficits, and a decline in language function. Upon autopsy, AD patients display
significant neuronal atrophy, specifically the cholinergic neurons in regions crucial for
memory and cognitive function including the hippocampus and neocortex. This
devastating cell loss can be as severe as 70% of pyramidal hippocampal neurons in
end-stage disease cases (West et al., 1994). Presumably before cell death, cells in the
AD brain also suffer from morphological changes and damage at the synapse level such
as loss of dendritic spines (Spires and Hyman, 2004). Both diffuse and aggregated
plaques, primarily composed of various species of β-amyloid, are first observed in the
neocortex and progressively spread to the hippocampus and associated cortical areas
(Urbanc et al., 1999). NFTs are caused by intraneuronal, paired helical filaments of
hyperphosphorylated tau protein (Brion et al., 1985) and are present throughout the
brain, progressing from the entorhinal cortex through subiculum, CA1 of hippocampus,
and cortical areas (Braak and Braak, 1997).
The primary pathological feature of AD that causes the disease and/or correlates
most closely with cognitive decline and memory loss is a topic of debate. Synapse loss,
a pathological feature presumed to occur before the appearance of dense, aggregated
amyloid plaques, was found to be correlate well with cognitive decline. The number of
3
NFTs and extent of cell loss, but not β-amyloid deposition, have been shown to correlate
with age-related cognitive decline (Arriagada et al. 1992). Insoluble aggregated species
of β-amyloid were found to correlate poorly with cognitive decline compared to soluble
species of β-amyloid (Wang et al., 1999). Despite the weak correlation between amyloid
plaques and cognitive decline, the amyloid cascade hypothesis (Hardy and Selkoe,
2002) is the leading theory regarding AD pathogenesis and points to Aβ as the key
causative factor of AD pathology.
2. Gender differences in AD
Prevalence and incidence
Converging lines of evidence indicate a potentially important role of estrogens in
regulating AD pathogenesis. Preliminary clues suggesting this possibility stemmed from
reports of sex differences in AD risk, with women showing higher prevalence and
incidence. Although sex differences in AD are difficult to interpret due to gender
differences in life expectancy, many studies of various cohorts indicate that women are
at greater risk of AD (Molsa et al., 1982; Rocca et al., 1986; Jorm et al., 1987; Bachman
et al., 1992; Andersen et al., 1999; Ruitenberg et al., 2001; Corder et al., 2004). Further,
there is some evidence that AD pathogenesis may be more severe in women as
indicated by sex differences in cognitive deficits and neuropathology (Buckwalter et al.,
1993; Henderson and Buckwalter, 1994), although other studies indicate men have
higher levels of tau pathology (Schultz et al., 1996; Schultz et al., 1999). Further, there is
a stronger association between the apolipoprotein E ε4 allele and sporadic AD in women
compared to men (Duara et al., 1996; Hyman et al., 1996; Corder et al., 2004) and the
ε4 allele has been shown to be associated with greater hippocampal atrophy memory
4
impairments in women compared to men (Fleisher et al., 2005). When considered
together, these epidemiological and neuropathological studies indicate sex differences in
AD, suggesting that women may be more vulnerable to AD than men.
Gender differences in transgenic mice
Several transgenic mouse models exhibit sex differences in AD-like
neuropathology that appears to parallel that observed in human AD cases. For example,
female Tg2576 mice display a higher plaque load burden and higher levels of both
soluble and insoluble Aβ40 and Aβ42 than age-matched males (Callahan et al., 2001).
Similarly, female APP
swe
xPS1 transgenic mice have higher Aβ load burden and plaque
number than age-matched males (Wang et al., 2003). The same pattern of greater Aβ
deposition in female versus male mice is also observed in the 3xTg-AD triple transgenic
mouse (Hirata-Fukae et al., 2008). These studies in transgenic mouse models of AD
suggest that the female brain may be more vulnerable to AD pathogenesis and will be
discussed at length in Chapter Six.
3. Menopause, hormone therapy and AD
Loss of hormones at menopause
The increased risk of AD in women is presumed to be associated with the
precipitous loss of estrogens and progesterone at menopause. Consistent with this
position, plasma levels of the estrogen 17β-estradiol (E
2
) (Manly et al., 2000) are
reported to be lower in women with AD in comparison to age-matched controls. If the
depletion of ovarian hormones at menopause contributes to women’s increased risk of
AD, then one would predict that estrogen-based HT would be effective in the prevention
and or treatment of AD. This critical issue remains unresolved with solid and persuasive
5
arguments both for and against the use of HT for AD. An early study of this issue found
that AD risk was lower in women who used HT relative to nonusers and this risk
decreased significantly as both dose and duration of HT use increased (Paganini-Hill
and Henderson, 1994). Similarly, findings from several other case control and
prospective studies suggest that postmenopausal women with a history of HT use were
at reduced risk of AD (Henderson et al., 1994; Paganini-Hill and Henderson, 1996; Tang
et al., 1996; Kawas et al., 1997; Waring et al., 1999; Carlson et al., 2001; Zandi et al.,
2002). Further, a meta-analysis of studies found that HT was associated with decreased
risk of cognitive dysfunction (Sherwin, 1988; Ohkura et al., 1994; Hogervorst et al., 1999;
LeBlanc et al., 2001). Collectively, these studies suggest a potential protective role of
estrogen against the development of AD.
Hormone therapy and AD
Despite indications of benefits, the potential protective of HT against AD remains
controversial. Arguing against a protective role, several studies found that HT use was
not associated with reduced risk of AD (reviewed in Haskell et al., 1997) or failed to yield
significant cognitive benefits (Barret-Connor and Kritz-Silverstein, 1993; Brenner et al.,
1994; Goebel et al., 1995; Polo-Kantola et al., 1998; Henderson et al., 2000; Mulnard et
al., 2000; Wang et al., 2000; Binder et al., 2001; Yaffe et al., 2001; Almeida et al., 2006).
One possible explanation for this discrepancy is suggested by findings from the Cache
County Study, which demonstrated that the association between HT use and reduced
risk of AD was strengthened in long-term HT users (Zandi et al., 2002). Interpretations of
these findings include the concept that HT may have a largely preventive role against
AD and or the hypothesis that early initiation of HT is essential as women who took HT
for longer periods likely began treatment nearer the time of menopause.
6
The notion that estrogen-based HT can effectively reduce the risk of AD and
improve age-related deficits in cognition has been challenged by findings from the
Women’s Health Initiative Memory Study (WHIMS). WHIMS was a randomized, multi-
center, double-blind, placebo-controlled study of ~4500 women between 65-79 yrs of
age that evaluated effects of HT consisting of conjugated equine estrogen (CEE) alone
or CEE with the progestin medroxyprogesterone acetate (MPA). This study reported that
neither CEE nor CEE+MPA significantly improved cognition versus placebo in women
showing cognitive decline associated with normal aging (Rapp et al., 2003; Espeland et
al., 2004) or dementia (Shumaker et al., 2003; Shumaker et al., 2004). In the CEE alone
arm, there was no significant difference in dementia incidence between HT or placebo
groups although there was a non-significant trend towards increased risk in the HT
group (Espeland et al., 2004; Shumaker et al., 2004). In the CEE+MPA arm, they found
that women receiving HT had a higher risk of probable dementia (Shumaker et al.,
2003). HT use was also associated with increased incidence of stroke and breast
cancer, suggesting that the risks of HT may outweigh its benefits. Further, the important
differences between the CEE alone arm and the CEE+MPA arm raise several issues
about the inclusion of a progestin component.
Although the WHIMS findings raise serious concerns over HT use, many
challenge the interpretation that these data dismiss the potential efficacy of HT in
reducing the risk of AD (Resnick and Henderson, 2002; Maki and Hogervorst, 2003;
Craig et al., 2005; Gleason et al., 2005; Pinkerton and Henderson, 2005; Henderson,
2006). A variety of issues have been identified that may have affected HT outcomes in
the WHIMS compared to the many observational studies such as differences in
methodological techniques, outcome measures, hormone exposures, menopausal
7
symptoms, and the timing of hormone use (Henderson, 2006). In particular, neural
sensitivity to sex steroid hormones may diminish during the menopause transition,
resulting in a critical window in which to initiate HT in order to realize benefits (reviewed
in Craig et al., 2005). Because HT was initiated many years after menopause in the
WHIMS study, the study’s design may have inadvertently focused on an age group in
which estrogen is minimally active in brain and thus was unlikely to detect potential
cognitive benefits. In addition, as suggested by prior epidemiological and clinical
findings, estrogen may be most effective in preventing rather than treating AD. In this
case, the relatively advanced age of WHIMS subjects would also be biased against
positive outcomes. Additional issues include HT formulation, route of administration, and
treatment regime (e.g., cyclic versus continuous hormone delivery). In order to unravel
this conundrum, a greater understanding of the neuroprotective actions of estrogens and
progestogens are needed, as well as their limitations in the context of aging.
4. Estrogen neuroprotection
Synthesis and bioavailability of sex steroid hormones in brain
Both male and female sex steroid hormones are locally produced in the brain,
and act of their metabolites through receptors in these brain regions. Both estrogen and
progesterone are synthesized from cholesterol through a complex metabolic pathway.
Estrogen is converted from testosterone by aromatase. Progesterone is converted by
3α,5α-THP(allopregnanolone; ALLO). While these hormones are primarily produced in
the ovary, is has recently been demonstrated that they are produced de novo from glia
and neurons in several regions of the CNS (Stoffel-Wagner, 2003; Garcia-Ovejero et al.,
2005), especially the hippocampus (Baulieu, 1997; Ibanez et al., 2003; Kawato, 2003).
8
Estrogen regulates neuron viability
An established neural action of estrogens that may contribute to a protective role
against AD is promotion of neuron viability. Estrogen is neuroprotective against a variety
of insults in several cell culture and rodent paradigms of injury and neurodegenerative
disease. Of particular interest is the ability of estrogen to protect against neuronal loss
induced by Aβ. Reports from several groups demonstrate that estrogen can protect
cultured neurons and neural cell lines from Aβ mediated toxicity (Behl et al., 1995;
Goodman et al., 1996; Green et al., 1996; Gridley et al., 1997; Mook-Jung et al., 1997;
Pike, 1999). These pathways are illustrated in Figure 1.

Figure 1. Estrogens activate neuroprotective pathways that may attenuate AD. Estrogens including
17β- estradiol (E 2) reduce neuronal apoptosis by (i) non-genomic signaling cascades, including activation of
PI3K, protein kinase C (PKC) and Src/ERK pathways, and (ii) genomic pathways utilizing CREB response
elements (CRE) and estrogen response elements (ERE) on members of the Bcl-2 family of genes including
Bcl-2, Bcl-x, Bcl-w, and Bim. Similarly, estrogens decrease levels of the AD-related protein Aβ by (i)
nongenomic signaling that promotes non-amyloidgenic processing of the Aβ precurser protein (APP), and
perhaps (ii) classic genomic mechanisms that involve ERE, CRE and/or other steroid response elements
(SRE) on the Aβ-catabolizing enzymes neprilysin (NEP) and insulin degrading enzyme (IDE).
9
Estrogen may potentially protect against Aβ-induced neurotoxicity at several
steps in the degenerative process. The leading theory of Aβ toxicity posits a pathologic
assembly of Aβ involving adoption of a β-sheet conformation, resulting in a change in
protein structure that is associated with a toxic gain of function (Pike et al., 1992),
(reviewed Walsh and Selkoe, 2007). Consistent with this working hypothesis of Aβ
neurotoxicity, our prior work has shown that Aβ is toxic only in an assembled state (Pike
et al., 1991; Pike et al., 1993). Assembled Aβ in the form of soluble oligomers and
insoluble fibrils can induce neuronal death (Cotman et al., 1992; Walsh et al., 2002;
Yankner and Lu, 2009) degeneration of neurites (Pike et al., 1992; Ivins et al., 1998) and
synaptic dysruption (Hartley et al., 1999; Walsh et al., 2002; Kamenetz et al., 2003;
Rowan et al., 2007) leading to impaired learning and memory (Lesne et al., 2006; Cheng
et al., 2007). Interaction of Aβ assemblies with neurons initiates a cascade of upstream
signaling mechanisms associated with cell death, including calcium dysregulation (Weiss
et al., 1994; Mattson et al., 1998), oxidative stress (Goodman and Mattson, 1994; Behl
et al., 1995; Mark et al., 1995), and activation of pro-inflammatory pathways promoting
chronic gliosis (Pike et al., 1994; El Khoury et al., 1996). Most evidence suggests that
the plethora of upstream signaling cascades elicited by Aβ ultimately mediate
neurotoxicity by downstream activation of neuronal apoptosis pathways (Cotman and
Anderson, 1995; Cotman, 1998). In particular, our lab has found that Aβ-induced
neuronal apoptosis involves activation of JNK signaling and consequent dysregulation of
the Bcl-2 family of apoptosis-related proteins (Yao et al., 2005).
Since neuronal apoptosis is an important downstream mediator of Aβ
neurotoxicity, regulation of cell apoptosis is predicted to be a key mechanism of estrogen
protection from Aβ. Consistent with this hypothesis, estrogen has been implicated in the
10
regulation of Bcl-2 family members in neurons (Garcia-Segura et al., 1998; Singer et al.,
1998; Dubal et al., 1999; Patrone et al., 1999; Pike, 1999; Stoltzner et al., 2001; Nilsen
and Diaz-Brinton, 2003; Koski et al., 2004). The Bcl-2 family includes both proteins that
promote cell survival (e.g., Bcl-2, Bcl-x
L
, and Bcl-w) and others that antagonize it (e.g.,
Bax, Bad, Bak, Bik, Bid, BNIP3, and Bim) (reviewed in Antonsson and Martinou, 2000;
Cory and Adams, 2002). Physiological levels of E
2
inhibit neuronal apoptosis at least in
part by increasing expression of anti-apoptotic Bcl-x
L
(Pike, 1999; Stoltzner et al., 2001)
and Bcl-w (Yao et al., 2007) while down-regulating expression of the pro-apoptotic Bim
(Yao et al., 2007). The observed effects of E
2
on Aβ mediated apoptosis are mediated by
ER dependent mechanisms, since the anti-apoptotic effects of E
2
were blocked by pre-
treatment with an ER antagonist (Pike, 1999; Yao et al., 2007). Supporting this, it has
been previously demonstrated that both ERα and ERβ are crucial in regulating Bcl-2
expression and neuronal survival (Zhao et al., 2004) and recently it has been shown that
E
2
can also increase Bcl-2 through Akt-dependent CREB activation (Yune et al., 2008).
Interestingly, estrogen dependent regulation of the Bcl-2 family of proteins has
also been implicated in neuroprotection against excitotoxicity, a form of neuronal injury
implicated in AD neurodegeneration (reviewed in Riederer and Hoyer, 2006;
Bezprozvanny and Mattson, 2008). Evidence suggests that in AD, Aβ toxicity and
glutamate excitotoxicity may cooperatively activate pathways leading neuronal death.
Glutamate-induced excitotoxic injury is potentiated by Aβ. In cell culture paradigms, the
combination of sub-lethal concentrations of glutamate combined with sub-lethal levels
Aβ yields robust neuronal loss (Koh et al., 1990; Mark et al., 1995). Such degenerative
interactions are predicted to occur in AD because the brain can experience both Abeta
accumulation and evidence of glutamate injury. Whether upstream or downstream
11
pathways of Aβ and glutamate action are responsible for their synergistic toxic effects is
unclear. Glutamate excitotoxicity leads to calcium dysregulation and oxidative stress,
and excitotoxic neuron death is mediated in part by apoptosis (reviewed in Choi, 1987).
Several studies have demonstrated that estrogen reduces excitotoxic neuronal
death induced by glutamate agonists in cell culture (Singer et al., 1996; Regan and Guo,
1997; Singer et al., 1999; Singh et al., 1999; Brinton et al., 2000; Nilsen et al., 2002). For
example, Dorsa and colleagues found that estrogen inhibited neuronal death in murine
cortical cultures following excitotoxic insult, an effect that could be pharmacologically
blocked with the ER antagonist, tamoxifen (Singer et al., 1996). Further, Brinton and
collegues found estrogen to promote intracellular Ca
2+
accumulation in neuronal cultures
treated glutamate at physiological doses, while inhibiting intracellular Ca
2+
accumulation
following treatment with excitotoxic glutamate doses (Nilsen et al., 2002). Similar
observations of estrogen neuroprotection following excitotoxic challenge have been
reported (Goodman et al., 1996; Regan and Guo, 1997; Weaver et al., 1997).
Estrogen has also been shown to regulate the extent of excitotoxic injury in
rodent models (Azcoitia et al., 1998; Azcoitia et al., 1999; Rosario et al., 2006a). For
example, administration of exogenous E
2
to OVX rats has been reported to protect
against kainate-induced neuronal loss (Azcoitia et al., 1998). Additionally, rats during
proestrus increase susceptibility to excitotoxicity (Azcoitia et al., 1999). Similarly, our lab
has observed neuroprotection against kainate lesion following administration of estrogen
to OVX rats (Rosario et al., 2006a).
Interestingly, estrogen neuroprotection against excitotoxic injury shares
mechanistic similarities with a protection against Aβ-induced apoptosis. That is, estrogen
regulation of the Bcl-2 family is implicated in protective actions against glutamate-related
12
injury (Singer et al., 1998; Nilsen et al., 2002; Nilsen and Diaz-Brinton, 2003; Zhao et al.,
2004). Brinton and colleagues found that estrogen mediated neuroprotection against
glutamate excitotoxicity by promoting mitochondrial Ca
2+
sequestration, and this was
associated with increased expression of Bcl-2 (Nilsen and Diaz-Brinton, 2003). Estrogen
dependent modulation Bcl-2 following excitotoxicity may be mediated by rapid, non-
genomic ER-dependent signaling mechanisms (Zhao et al., 2004; Zhao and Brinton,
2007). Estrogen is thought to act of surface ER (ERα and ERβ) activating the
Src/ERK/CREB signaling pathway, leading to upreguation of Bcl-2 (Wu et al., 2005b). In
contrast, others suggest that estrogen may regulate Bcl-2 family expression through a
direct genomic mechanism. Supporting this we have described an estrogen responsive
element (ERE) on the Bcl-x gene (Pike, 1999), while others describe EREs on Bcl-2
(Dong et al., 1999; Perillo et al., 2000).
In addition to regulation of the Bcl-2 family of proteins, estrogen has also been
implicated in neuroprotection against many other prominent features of the AD-
neurodegenerative cascade including inflammation and oxidative stress. Many studies
indicate that Aβ may be contribute to AD-related oxidative stress (Goodman and
Mattson, 1994; Behl et al., 1995; Mark et al., 1995) and deposition of Aβ may activate
microglia and promote inflammation (Sastre et al., 2008; Vasto et al., 2008). Abundant
evidence indicates that estrogen is a potent inhibitor of oxidative damage (Vedder et al.,
1999) and hydrogen peroxide mediated neuronal death (Behl et al., 1995; Goodman et
al., 1996; Behl et al., 1997). Estrogen mediates these antioxidant effects through ER-
independent mechanisms, acting as a free radical scavenger because of its own
phenolic structure (Sugioka et al., 1987; Subbiah et al., 1993; Rice-Evans et al., 1996;
Rice-Evans et al., 1997). However, these neuroprotective effects are only elicited at
13
supraphysiological estrogen dosages (Liehr and Roy, 1990; Behl et al., 1997), thereby
limiting the clinical relevance of these neuroprotective actions of estrogens. In contrast,
the anti-inflammatory actions of estrogen are mediated by ER-dependent mechanisms
and may provide preventative or therapeutic benefit to a range of neurodegenerative
diseases where neuroinflammation is a major degenerative process. Pluripotent effects
of estrogen have been described on glial function and activation, Estrogen may both
suppress reactive gliotic responses associated with traumatic injury and
neurodegeneration (Garcia-Estrada et al., 1993; Garcia-Segura et al., 1998), while also
promoting the neuroprotective properties of astrocytes (Del Cerro et al., 1995; Struble et
al., 2007). Interestingly, E
2
has been found to attenuate microglial activation only when
given to the cultures prior to inflammatory insult (Vegeto et al., 2001). This suggests the
anti-inflammatory benefits may be limited to prevention of AD rather than treatment.
Estrogen receptor-mediated actions
As discussed above, the described neuroprotective actions of estrogen against
AD-related insults are largely mediated by activation of estrogen receptors (ER). It has
been well established that both ER subtypes are widely distributed in the brain, including
in brain regions affected in AD such as the hippocampus, frontal cortex, and amygdala
(Shughrue et al., 1997; Hart et al., 2001). Recently, changes in the subcellular
distribution of ERs in hippocampal neurons have been implicated in AD pathogenesis
(Lu et al., 2004). Specifically, the shift of ERα from the nucleus to the cytoplasm may
decrease the development of AD pathology in humans (Hestiantoro and Swaab, 2004)
and transgenic mice (Kalesnykas et al., 2005). In addition, recent studies have begun to
demonstrate that ERα levels in the frontal cortex correlated with mini-mental state
examination scores in women with end-stage AD (Kelly et al., 2008) and that specific
14
allele differences in ERα are correlated with an increased risk for AD in women with
Down syndrome (Schupf et al., 2008). Also, ERβ immunoreactivity is reportedly
increased in the hippocampus compared to age-matched controls (Savaskan et al.,
2001). Taken together, these studies suggest that the expression of ERα/β in the AD
brain may play an integral role in the neuroprotective actions of E
2
. This is discussed in
recent reviews (Brann et al., 2007; Ogiue-Ikeda et al., 2008) and Chapter Five.
Both ER subtypes ERα and ERβ are implicated in mediating estrogen
neuroprotection. Although their relative contributions have been incompletely defined,
what is known to date is summarized in Table 1. Selective expression of ERα versus
ERβ in neural cell lines has suggested a more important role of ERα in mediating
neuroprotection in some studies (Kim et al., 2001) but significant contributions from both
ERα and ERβ in others (Linford et al., 2000; Fitzpatrick et al., 2002; Mize et al., 2003).
Cell culture studies utilizing selective ERα (propylpyrazole triol, PPT), and ERβ agonists
(diarylpropionitrile; DPN) to study estrogen neuroprotection typically report similar levels
of protection from both agonists, although some evidence suggests greater activity of the
ERα agonist PPT (Benvenuti et al., 2005). Studies from our lab in primary neuron culture
indicate comparable levels of neuroprotection against Aβ from E
2
, PPT, and DPN,
(Cordey and Pike, 2005). However, our data also suggested potential differences
between ER subtypes in terms of protective mechanisms, with PPT but not DPN
inducing PKC-dependent neuroprotection (Cordey and Pike, 2005). Similarly, Brinton
and colleagues find that PPT and DPN closely mimic E
2
protection from glutamate
excitotoxicity, including activation of ERK signaling and upregulation of Bcl-2 expression
(Zhao et al., 2004; Zhao and Brinton, 2007). Collectively, available evidence suggests
15
that both ERα and ERβ likely contribute to neuroprotection against AD-related insults but
that each may mediate protection by preferentially activate different signaling pathways.
Table 1: Neuroprotective effects of E
2
through ERα and ERβ
Author/Reference Experimental Model Result
(Walf et al., 2004) Young OVX rats treated
with E
2
, PPT, DPN
E
2
, through ERβ, decreases
depression-related behaviors
(Lund et al., 2005) Young OVX rats treated
with E
2
, PPT, DPN
E
2
, through ERβ, decreases
anxiety behavior on EPM
(Rhodes and Frye,
2006)
Young OVX rats treated
with E
2
, PPT, DPN
E
2
, through ERβ, enhances
memory on water maze tasks
and inhibitory avoidance
(Cordey and Pike,
2005)
Primary hippocampal
neurons challenged by Aβ
treated with E
2
, PPT, DPN
E
2
is neuroprotective by PKC
activation through ERα
(Zhao et al., 2004) Primary hippocampal
neurons challenged by
excitotoxic glutamate
treated with E
2
, PPT, DPN
E
2
, through both ERα and ERβ
are neuroprotective and
increase Bcl-2 expression
(Benvenuti et al.,
2005)
Neuroblast cultures
challenged by Aβ treated
with E
2
, PPT, DPN
E
2
, through ERα is
neuroprotective
(Dubal et al.,
2001)
Young, OVX, ERα and ERβ
–knockout mice challenged
by ischemia treated with E
2
E
2
, through ERα is
neuroprotective
(Carswell et al.,
2004)
Young OVX mice
challenged by ischemia
treated with E
2
, PPT, DPN
E
2
, through ERβ is
neuroprotective
(Tsurugizawa et
al., 2005)
Primary hippocampal
neurons treated with E
2
,
PPT, DPN
E
2
, through ERα decreased
thorn density on pyramidal
neurons
(Jelks et al., 2007) Primary hp neurons treated
with E
2
, PPT, DPN
E
2
, through ERα increased
synaptic density
(Suzuki et al.,
2007)
Young, OVX, ERα and ERβ
–knockout mice challenged
by ischemia treated with E
2
E
2
, through both ERα and ERβ
are neuroprotective
(Harrington et al.,
2003)
Adult male rat hp. slices
treated with E
2
, PPT, DPN
E
2
, through ERα enhanced
LTD
(Morissette et al.,
2008)
MPTP mice treated with E
2
,
PPT, DPN
E
2
, through ERα, prevents the
reduction in pGSK3-β
(Morissette et al.,
2007)
MPTP mice treated with E
2
,
PPT, DPN
E
2
, through ERβ, prevents cell
death of dopaminergic neurons
16
5. Estrogen regulation of AD insults
Estrogen regulation of β-amyloid accumulation
In addition to increasing neuronal resistance to AD-related insults, estrogen may
also protect against AD by preventing the key initiator of AD pathogenesis, accumulation
of Aβ. Steady state levels of Aβ are influenced by opposing pathways of Aβ production
and Aβ clearance, both of which appear to be regulated by estrogen. Estrogen
regulation of Aβ was first suggested by cell culture experiments focused on Aβ
production. Early studies demonstrated that estrogen modulates processing of amyloid
precursor protein (APP), the transmembrane parent protein of Aβ (Gandy and
Petanceska, 2001).
The majority of APP is metabolized by two competing pathways, the
amyloidogenic and non-amyloidogenic pathways. In the amyloidogenic pathway, thought
to occur following endocytosis of cell-surface APP, APP is first cleaved by β-secretase
(BACE) to liberate β-APPs. The C-terminal fragment (C99/β-CTF) is left embedded in
the membrane and is cleaved by the γ-secretase enzyme liberating the Aβ40/Aβ42
peptides. It is thought that another fragment is also released termed the APP
intracellular domain (AICD), which can translocate to the nucleus and activate gene
transcription. In the non-amyloidogenic pathway, which is the predominant pathway,
APP is cleaved within the Aβ domain by α-secretase to liberate a neuroprotective,
secreted form of APP (α-APPs). A C-terminal fragment (C83/αCTF) is left embedded in
the membrane for further cleavage into non-amyloidogenic fragments (Selkoe et al.,
1996; Verdile et al., 2004).
Estrogen appears to regulate Aβ levels at least in part by promoting the non-
amyloidogenic cleavage of APP, precluding production of the Aβ peptide. In the human
17
kidney 293 cell line, E
2
has been shown to reduce the level of Aβ peptide in a
concentration-dependent manner (Chang et al., 1997). Further, some preliminary results
suggest that in a clinical setting, short-term E
2
treatment is able to reduce plasma levels
of Aβ in postmenopausal women naïve to HT (Baker et al., 2003). Although this was a
small, short-term study, it provides important results suggesting that E
2
may directly
lower Aβ in women.
Importantly, subsequent studies demonstrated that estrogen also functions as a
regulator of Aβ in animal models. For example, the depletion of endogenous estrogen by
ovariectomy (OVX) in guinea pigs increased the levels of soluble Aβ in brain, an effect
partially reversed by E
2
treatment (Petanceska et al., 2000). This estrogen action may be
sex-dependent since androgen but not E
2
treatment reduced elevated Aβ levels resulting
from orchiectomy (ORX) of adult male rats (Ramsden et al., 2003). A similar pattern of
E
2
regulation of Aβ has been observed in several transgenic mouse models of AD. That
is, OVX is associated with increased Aβ and E2 treatment with reduced Aβ levels in
Tg2576 (Zheng et al., 2002; Yue et al., 2005), APP
swe
(Levin-Allerhand et al., 2002),
Tg2576xPS1 (Zheng et al., 2002; Xu et al., 2006). The mechanism by which estrogen
regulates Aβ in vivo has yet to be elucidated. In the APP
swe
transgenic mouse model of
AD, E
2
treatment was associated with increased sAPPa, indicating increased a-
secretase APP processing (Levin-Allerhand et al., 2002). However, in WT guinea pigs,
experimental manipulation of estrogen status was not associated with corresponding
changes in sAPPa levels (Petanceska et al., 2000). Increased levels and activity of
BACE observed in aromatase knock out mice also suggests a potential role for estrogen
in the regulation of secretase expression and/or activity (Yue et al., 2005). Further work
will be needed to elucidate whether estrogen regulation of Aβ in animals involves APP
18
processing and, if so, to define the relevant upstream signaling components (e.g.,
MAPK, PKC).
Curiously, estrogen levels are associated with Aβ accumulation only in some but
not all transgenic mouse models of AD (Heikkinen et al., 2004; Green et al., 2005; Yue
et al., 2005; Golub et al., 2007). In the Tg2576, PDAPP, and APP
swe
xPS1 mouse
models, OVX was not associated with increased Aβ levels and E
2
treatment did not
reduce Aβ levels. Discrepancies in the effects of estrogen on Aβ levels across
transgenic mouse models may reflect several differences, ranging from molecular design
of the transgenic lines to variability in methodological parameters such as the timing and
dosing of hormone manipulations. One potentially important methodological difference
across the studies is their varying techniques for Aβ quantification, each of which
preferentially measures different pools of Aβ ranging from soluble monomeric Aβ to
oligomeric and deposited forms. Whether estrogen differentially regulates these various
Aβ pools is currently unknown. Future studies will be needed to determine exactly which
Aβ pool(s) estrogen is capable of regulating and whether this contributes to observed
differences in estrogen actions across models. However, an important first step is to
continue investigating the efficacy of E
2
in lowering Aβ in transgenic mice, and results
regarding this question will be discussed at length in Chapter Three.
Discrepancies between studies on the role of estrogen as a regulator of Aβ
accumulation also may indicate differences in brain levels of estrogen. Recent work
suggests that OVX has limitations as a strategy to fully deplete brain estrogens. For
example, in the estrogen-responsive element-luciferase mouse model, which was
engineered to express the non-mammalian luciferase protein in response to classical ER
activation, OVX resulted in relatively high brain estrogen activity in comparison to other
19
body regions (Ciana et al., 2001). In evaluating the role of brain estrogens in Aβ
regulation, Yue et al. found that in the APP
23
transgenic mouse model OVX alone was
not sufficient to remove all brain estrogen and did not result in elevated Aβ levels (Yue et
al., 2005). However, they reported elevated Aβ levels after preventing E
2
formation in
brain by crossing the APP
23
mice with aromatase knock-out mice (Yue et al., 2005).
Thus, brain levels of sex steroid hormones, which are affected not only by gonadal
hormone production but also by de novo steroid hormone synthesis in brain (i.e.,
neurosteroidogenesis), may be the critical factor in regulation of brain Aβ accumulation.
This hypothesized importance of brain hormone levels is supported by the finding that
brain levels of E
2
are lower in female AD patients in comparison to age-matched control
cases (Yue et al., 2005), a finding our lab has recently replicated [Rosario and Pike,
unpublished observations]. In addition, it has recently been shown that long-term OVX in
female mice significantly lowers E
2
levels in the hippocampus while increasing serum
levels of Aβ (Fukuzaki et al., 2008). Thus, estrogen regulation of Aβ may depend
primarily upon brain estrogen levels, which may be depend on both ovarian and brain
steroid production.
Estrogen regulation of tau hyperphosphorylation
Another potentially significant neuroprotective action of estrogen that is highly
relevant to AD and related neurodegenerative disorders is inhibition of pathological tau
hyperphosphorylation. Both estrogen and progesterone can modulate activities of
kinases and phosphatases involved in regulating levels of tau phosphorylation.
Specifically, E
2
and P4 regulate tau phosphorylation through the glycogen synthase
kinase-3β (GSK-3β) pathway (Alvarez-De-La-Rosa et al., 2005; Goodenough et al.,
2005). Estrogen can reduce phosphorylized GSK-3β activity (Goodenough et al., 2005),
20
and progesterone can decrease expression of both tau and GSK-3β. Estrogen can also
lower tau hyperphosphorylation through the wnt signaling pathway and a gene called
dickkopf-1 (Zhang et al., 2008) as well as through the protein kinase A pathway (Liu et
al., 2008). These results demonstrate some of the first insights into the mechanism
behind estrogen neuroprotection in tau-related disorders.
6. Aging effects on estrogen responsiveness
Whether the described neuroprotective effects of estrogen prove to have
therapeutic relevance to age-related neural diseases including AD will depend in part on
the brain’s responsiveness to estrogen with advancing age. One of the primary criticisms
of WHIMS and other clinical studies of estrogen-based HT is that the intervention may
have been initiated in beyond a critical window of opportunity (discussed in Resnick and
Henderson, 2002; Gleason et al., 2005; Pinkerton and Henderson, 2005). This notion
refers to the possibility that the aging brain age may lose responsiveness to sex steroid
hormones after an extensive period of low hormone levels, such as occurs following
menopause. According to this argument, HT may exert estrogenic effects only if begun
near the time of menopause. Since most participants in the WHIMS study were many
years beyond menopause, the critical window hypothesis could explain in part the
absence of beneficial neural actions of HT. Consistent with this position, several clinical
studies have noted that HT is associated with positive neural effects in women showing
menopause symptoms (e.g., flushing), suggesting retained estrogen responsiveness
(Sherwin, 1988; Hlatky et al., 2002; Henderson et al., 2003; Sherwin, 2003; Henderson
et al., 2005).
21
The critical window notion of an age-related loss in brain responsiveness to
estrogen is supported by studies in animal models. One experimental approach to
address this issue is the “gap paradigm” in which OVX animals are treated with E
2
replacement after short versus long periods of time, a design that assesses the effects of
prolonged hormone deprivation on subsequent hormone exposure. In general, results
from gap studies indicate diminished estrogen responses following many months of
hormone absence. For example, E
2
and E
2
plus progesteorne replacement was
associated with improved spatial memory on the delayed match task when administered
within 3 months post-OVX, but not when hormone treatment was delayed 10 months
post-OVX (Gibbs, 2000a). Further, E
2
given immediately after OVX in rats improved
spatial memory performance on radial arm maze while E
2
given after 5 months of OVX
did not (Daniel et al., 2006). Recently, it was reported that E
2
given immediately after
OVX but not after a 5 months delay was able to increase hippocampal ChAT protein
levels in middle-aged OVX WT rats (Bohacek et al., 2008). Although this topic requires
further work to elucidate the key factors and underlying mechanism(s), the data
generated thus far suggest that in laboratory animals, the brain can show reduced
hormone responsiveness after an extended period of hormone depletion.
Another approach to evaluate the role of aging in neural estrogen
responsiveness has been to simply compare the effects of estrogen in young adult
versus aging female rodents. Our laboratory has contributed towards this question in
Chapter Two. This type of study is critical in determining the efficacy of female sex
steroid hormones before and after the onset of reproductive senescence, the result of
normal reproductive aging in female rodents (reviewed in Chakraborty and Gore, 2004).
While reproductive aging in rodents does not mimic menopause, rodents do experience
22
a pattern of estrus cycle irregularity followed by reproductive senescence that is similar
in some respects with the perimenopause period in women (Felicio et al., 1984). Age-
related reproductive changes in female rodents typically become apparent between 9-11
months of age, depending upon species and strain. For example, the age-related estrus
cycle changes have been well characterized in the C57Bl6 mouse strain (Felicio et al.,
1984). Like women, these female mice demonstrate a large range of variability in cycle
irregularity and hormone levels during middle and old age. These mice experience cycle
cessation between 11-16 mo of age during which a substantial proportion enter a period
of persistent vaginal cornification lasting 2-4 months. After this variable period, all mice
enter an irreversible final stage of permanent diestrus characterized by low E
2
and
progesterone levels and elevated luteinizing hormone levels (Gee et al., 1983), ovarian
follicle depletion, and loss of reproductive capability (Gosden et al., 1983).
In estrogen neuroprotection studies, middle-aged female rodents undergoing
reproductive senescence show diminished effects in some studies but retained
protection in others. First, several reports suggest that the neural effects of OVX and E
2
treatment are diminished in aging female rodents. Studies by Sohrabji and colleagues
suggest that reproductive senescence in middle-aged female rats reduces protective
estrogen actions. For example, in assessing estrogen regulation of neurotrophin
expression, they found that E
2
treatment in OVX young adult female rats (age 3 mo)
increased levels of BDNF, trkA, and trkB in olfactory bulb and diagonal band of Broca,
whereas E
2
treatment of middle-aged (17 months) OVX reproductively senescent,
female rats showed either no increase or decreased expression of neurotrophins
(Jezierski and Sohrabji, 2001). Similar studies by this research group found that, in
comparison to young adult female rats, reproductively senescent female rats show
23
several alterations in estrogen-mediated effects including cytokine and growth factor
responses following injury (Nordell et al., 2003; Johnson et al., 2006) and blood-brain-
barrier permeability (Bake and Sohrabji, 2004). In another paradigm, Finch and
colleagues reported differences between young adult (3 mo) and middle-aged (18 mo)
female rats on estrogen regulation of compensatory neuronal sprouting following
entorhinal cortex lesion. In comparison to young rats, older rats no longer exhibited an
OVX-induced decrease in sprouting and showed differences in regulation of GFAP
mRNA (Stone et al., 2000). However, some neural actions of estrogen appear to remain
relatively robust during aging. For example, E
2
treatment in middle-aged OVX rats
enhanced performance on hippocampal-dependent spatial memory tasks (Daniel et al.,
2006) as well as altered the hippocampal expression of several genes (Aenlle et al.,
2007).
In neural injury paradigms, there is evidence of both retained and altered
estrogen neuroprotection. In reproductively senescent female rats, both E
2
and
progesterone reduced infarct size in a model of stroke injury (Alkayed et al., 2000). Wise
and colleagues reported similar neuroprotective effects of E
2
treatment in OVX young (3-
4 mo) and middle-aged (9-12 mo) rats in the middle cerebral artery occlusion (MCAO)
model of stroke (Dubal and Wise, 2001; Wise, 2006). In a model of spinal cord injury, the
effects of E
2
treatment in young (2 mo) versus middle-aged (12 mo) sham-OVX and
OVX female rats were investigated. Treatment with E
2
protected against several indices
of injury in both young and aging OVX rats, however in ovary-intact rats E
2
neuroprotection was lost in middle-aged rats (Chaovipoch et al., 2006).
Interestingly, emerging data suggest that patterns of reproductive aging in female
rats may contribute to altered estrogen responsiveness with age. A recent study
24
compared estrogen protection using the MCAO stroke model in middle-aged female rats
stratified by their stage of reproductive aging: reproductively senescent rats that had
entered a persistent acyclic state, and rats with normal but lengthened cycles. In
comparison to the middle-aged cycling rats, the reproductively senescent rats exhibited
larger lesions and no longer showed reduced lesion size following E
2
treatment
(Selvamani and Sohrabji, 2008). Thus, although additional studies are necessary to
define the relationships, it is reasonable to hypothesize that patterns of reproductive
aging affect estrogen neuroprotection. Such findings are consistent with the “critical
window” hypothesis of HT and have important implications for the future of clinical use of
estrogen-based therapies.
Why the brain shows is less responsive to estrogen with age is unclear, but age-
related decreases in ER expression as well as E
2
binding to ERs in aged rat brain have
been reported (Wise and Parsons, 1984; Rubin et al., 1986; Chakraborty and Gore,
2004). Estrogen actions also show age-related changes in other estrogen responsive
tissues, including uterus, bone and heart. For example, the uterus becomes less
responsive to estrogen with increasing age, showing smaller OVX-induced decreases in
uterine weight (Xu et al., 2004) and uterotrophic effects of estrogen only when treated
soon after OVX (Daniel et al., 2006). Further, while most studies demonstrate that the
trophic effects of E
2
on bone extend through middle-age, some studies suggest that as
aging progresses, this effect is altered from an ERα/β-mediated effect to only an ERα
mediated one (Islander et al., 2003). Interestingly, increased ERα predominance has
also been suggested to underlie age changes in neural estrogen responsiveness (Bake
et al., 2008).
25
Taken together, available experimental research suggests that although estrogen
neuroprotection is often observed in aging rats, it can also be significantly diminished.
Still uncertain is how the many AD-related facets of estrogen neuroprotection may be
impacted by aging. Additional studies are necessary to determine the extent to which
observed age-related changes in estrogen responsiveness may be delayed, prevented
and or reversed. This phenomenon has important implications for the future of HT in
postmenopausal women. Ongoing clinical and animal studies promise to shed new
insight into this issue in the next few years.
7. Effects of progestins in the brain
Since levels of both estrogens and progestins naturally fluctuate in women
across the ovarian cycle, understanding estrogen actions must include consideration of
progestins. Further, estrogen-based HT typically includes a progestin component to
antagonize tumorigenic effects of estrogen in uterus. Thus, although HT effects are
typically attributed to estrogen, progesterone also contributes to HT responses.
Unfortunately, in contrast to the well-established effects of estrogen in the brain, the
neural actions of progesterone are rather poorly understood. Investigators have
prioritized the understanding of progesterone’s effects on the CNS since the majority of
women in the US currently taking HT are being exposed to progestins without a full
understanding of its effects. In the past few years, an increasing focus on progesterone
neurobiology has begun to define its effects on cognition and neural plasticity and
elucidate its neural cell signaling pathways (Schumacher et al., 2007; Brinton et al.,
2008). Below is a brief review of the emerging literature linking progesterone with
regulation of two key parameters of AD neuropathology, neuron viability and
26
accumulation of Aβ and results from our own laboratory regarding this conundrum will be
revealed in Chapters Two, Three and Four.
Progesterone regulation of neuron viability
Progesterone’s actions in the brain have been perhaps most studied in various in
vitro and in vivo models of neuroprotection. Like estrogen, progesterone has been
reported to directly induce neuroprotection in some but not all cell culture models of
neurodegeneration. For example, in cultured hippocampal neurons, progesterone but
not medroxyprogesterone acetate (MPA) – the progestin most commonly used in HT –
reduced excitotoxicity induced by glutamate exposure (Nilsen and Brinton, 2002). Also
similar to estrogen, progesterone induces several neuroprotective cell signaling
pathways, including activation of Akt (Singh, 2001) and ERK (Nilsen and Brinton, 2002)
and upregulation of anti-apoptotic protein Bcl-2 (Nilsen and Brinton, 2002).
In animal models, progesterone induces neuroprotection against several but not
all challenges. Some studies have shown that P4 improves spatial memory performance
in rats (Roof et al., 1994) while other studies show that P4 has no effect (El-Bakri et al.,
2004; Tanabe et al., 2004). P4 has been shown to be neuroprotective in some rodent
models of neuronal injury in the cortex and hippocampus (Roof et al., 1994; Alkayed et
al., 2000; Hoffman et al., 2003; De Nicola et al., 2006), but not other models (Azcoitia et
al., 1999; Toung et al., 2004). MPA has been shown to increase synaptic density in CA1
neurons (Silva et al., 2000). However, MPA has not mimicked progesterone’s
neuroprotective effect in cell culture models of glutamate toxicity (Nilsen et al., 2002;
Nilsen and Diaz-Brinton, 2003). Progesterone treatment has been shown to reduce
neural injury and or neuron death associated with traumatic brain injury (Roof et al.,
1994; Stein et al., 2008), spinal cord injury (Labombarda et al., 2003), ischemia (Murphy
27
et al., 2002; Gibson et al., 2005), and seizure models (Rhodes and Frye, 2004).
However, conflicting studies suggest an absence of progesterone neuroprotection in
some related injury models, which hints that progesterone likely exerts less general and
or less robust neuroprotection than does estrogen. Furthermore, similar to findings in cell
culture studies, protective effects of progesterone in vivo are mimicked by only some
progestagens, findings that may provide mechanistic insight (Singh, 2007). For example,
the protective effects of progesterone, certain progesterone metabolites, and some
progestins against neural injury induced by pilocarpine, kainate, and other seizure-
inducing toxins likely reflects anxiolytic effects resulting from modulation of the GABA
A
receptor. The extent to which these pathways of progesterone neuroprotection may
synergize with protective estrogen-mediated cell signaling remains to be fully elucidated.
Progesterone regulation of β-amyloid
In contrast to the established Aβ-lowering effects of estrogen, the effect of
progesterone on Aβ accumulation is largely unknown. Because OVX-induced depletion
of estrogen and progesterone in wild type rodents and some mouse models of AD
results in elevated Aβ levels, it is reasonable to speculate that the loss of endogenous
progesterone may contribute to disruption in steady state Aβ levels. Further, estrogen
treatment of OVX rodents often only partially reverses the increase in Aβ, perhaps
suggesting that progesterone may be needed to complement estrogen action and fully
reduce Aβ levels. However, no studies had been done investigating the effects of
progesterone on AD-like neuropathology to date. Certainly, additional studies are
necessary to more thoroughly investigate the potential role of progesterone in regulating
Aβ and AD-related pathologies.
28
8. Interactive effects of estrogens and progestins in the brain
Clinical observations
Estrogen actions must also be considered with respect to the second major class
of ovarian hormones, progestogens. Progesterone has long been recognized as a
regulator of estrogen, particularly in the female reproductive system (reviewed in
Graham and Clarke, 1997), where it often antagonizes estrogen action. Clinically,
progestogens are typically a key element of HT that are thought to minimize deleterious
effects of estrogen. Perhaps most importantly, in experimental paradigms progesterone
can inhibit human endometrial cancer cell growth (Dai et al., 2002; Davies et al., 2004)
and in clinical studies progestogens are associated with reduced risk of endometrial
cancer (Persson et al., 1989; Grady et al., 1995).
In clinical studies of AD and dementia, most results indicate similar outcomes
with both estrogens alone (ie., CEE) and estrogens in combination with progestogens
(ie., CEE+MPA). In the WHIMS trial, a comparison between the CEE alone and
CEE+MPA arms of the study raised the question that the clinical efficacy of HT may be
dependent upon the hormone constituents within. Both arms failed to demonstrate a
protective effect and both actually increased the risk of dementia compared to women
receiving placebo (Shumaker et al., 2003). Notably, the CEE alone arm had a negative
impact on global cognitive function, however this negative impact was worsened when
pooled with the data from the CEE+MPA arm (Espeland et al., 2004). Interestingly,
short-term HT treatment in women with existing AD was associated with some benefits
on psychiatric symptoms in the CEE group but not CEE+MPA (Honjo et al., 2005).
These issues suggest that the inclusion of a progestogen may influence the effects of
CEE alone on cognitive outcomes and risk of dementia (Craig et al., 2005).
29
Despite the common use of the progestogen MPA in current HT paradigms,
researchers have only relatively recently begun to investigate the effects of progesterone
on the CNS in regards to aging and neurodegenerative diseases. Although compelling
experimental evidence indicates numerous protective actions when hormones are
delivered independently (Brinton et al., 2008; Schumacher et al., 2008), comparatively
less is known about interactions between estrogen and progesterone when they are
administered together. Interestingly, accumulating observations indicate that
progesterone often antagonizes rather than synergizes with estrogen.
Progesterone inhibits estrogen neuroprotection
Findings from an increasing body of research have begun to provide insight into
how beneficial neural actions of estrogen are affected by interactions with progesterone.
Findings from both in vitro and in vivo paradigms suggest that progesterone treatment
can antagonize estrogen neuroprotection and these findings are summarized in Table 2.
Table 2: Progesterone antagonizes many neuroprotective effects of E
2
Author/reference Experimental
model
Hormone
paradigm/doses
Result
Woolley, 1993
#359}
CA1
pyramidal
cells of adult
female rat
E
2
and P4 injections
in OVX rats
P4 initially enhances
E
2
-induced spine
density for first 6 hrs
then decreases by 18
(Garcia-Segura et
al., 1998)
2 mo Wistar
albino female
rats
1,10,100ug E
2
and/or 500ug P4
injections 24 hrs
before sacrifice
Blocks E
2
increase in
Bcl-2 immunoreactivity
in hypothalamus
(Murphy and
Segal, 2000)
Hippocampal
neuron
culture
Cells fixed 48 hrs
after a 30nM E
2
dose and a 10 ug/ml
P4 dose
P4 blocks E
2
-induced
increase in dendritic
spine density and
pCREB
(Bimonte-Nelson
et al., 2004)
24 mo old
Fisher-344
female rats
1.5mg/60day E
2
and
2x(200mg/60days)
P4 pellets
P4 blocks E
2
-induced
increase in
neurotrophins in
entorhinal cortex
30
Table 2: Progesterone antagonizes many neuroprotective effects of E
2,
Continued
(Rosario et al.,
2006a)
3 mo Sprague
Dawley female rats
Silastic capsules
filled with hormones
for 2 wks:
E
2
(~140pg/ml)
P4 (~25 pg/ml)
MPA (~2ng/ml)
Both P4 and
MPA block E
2
neuroprotection
from kainic acid
in hippocampus
(Bimonte-Nelson
et al., 2006)
Fisher 344 female
rats 12 mo old
E
2
: Low (0.25mg),
high (0.5mg) pellets.
constant (12 weeks)
or tonic (10ug E
2
injection 2 weeks).
P4: (400mg pellet)
P4 blocks E
2
-
induced
improvement in
spatial memory
on the Morris
Water Maze
(Harburger et al.,
2007)
22 mo old female
C57BL/6 mice
Single injections of
HBC-conjugated
0.2mg/kg E
2
and
20mg/kg P4
P4 blocks E
2
-
induced
improvement in
spatial memory
For example, long-term progesterone treatment in aged (23-24 mo) OVX female
rats blocked estrogen upregulation of brain derived neurotrophic factor, nerve growth
factor, and neurotrophin 3 (Bimonte-Nelson et al., 2004). In a similar paradigm of
hormone treatment in middle-aged OVX rats, estrogen-induced improvement in spatial
memory performance was blocked by co-administration of progesterone (Bimonte-
Nelson et al., 2006). More specific to neuron viability though are recent studies from our
laboratory demonstrating that progesterone blocks estrogen neuroprotection from
excitotoxic injury in female rats. In both young adult (3 mo) (Rosario et al., 2006a),
continuous E
2
treatment reduced kainate-induced neuron loss in hippocampus CA2/3
whereas continuous progesterone did not significantly affect neuron viability. Importantly,
when progesterone was included with E
2
, estrogen neuroprotection was no longer
observed (Rosario et al., 2006a). Similarly, Brinton and colleagues recently reported that
E
2
and progesterone administered independently to WT rats enhanced several markers
31
of brain mitochondrial function but, when replaced in combination, the hormones showed
attenuated rather than enhanced responses (Irwin et al., 2008).
According to these studies, we can conclude that independently estrogen and
progesterone can exert protective actions, but in combination they can inhibit each other
and thus fail to protect. Perhaps not unexpectedly, the interactive neuroprotective effects
of the two female sex steroid hormones are not quite so straightforward. Besides
antagonistic effects, progesterone can also synergistically interact with estrogen to
promote beneficial neural effects including increased spine density (Frankfurt et al.,
1990; Silva et al., 2000). In OVX female rats, acute progesterone treatment (2-6 hours)
was observed to augment the estrogen-induced increase in spine density, whereas
prolonged progesterone treatment (18 hours) blocked the estrogen effect (Woolley and
Bruce S. McEwen, 1993). Thus, a key factor in understanding estrogen neuroprotection
and perhaps its relevance to HT in postmenopausal women is elucidation of the
interactions between estrogen and progesterone.
Despite evidence of antagonistic neural effects of progesterone on estrogen
neuroprotective actions, progestogens are still deemed a necessary component of HT in
women with a uterus. Therefore, HT may need to be optimized to maximize the benefits
and minimize the unwanted consequences associated with estrogen-progestogen
interactions. One possible strategy is the use of cyclic hormone delivery rather than the
continuous, combined treatment that is currently common to HT.
Cyclic versus continuous progesterone treatment
The second most important question raised by the WHIMS study was the issue
of combined estrogen + progestin treatment and the paradigm of hormone exposure.
Unfortunately, the participants receiving CEE+MPA performed poorly on the cognitive
32
measures, suggesting that perhaps the progestin component antagonized the possible
beneficial effects of estrogen. Therefore, investigators acknowledge that more research
is needed to elucidate the antagonistic effects of progesterone on estrogen action in the
brain. This question has become a major area of investigation in the field of
neuroendocrinology and will be addressed in detail in Chapter Three and Four.
Alternatively, clinicians discussed the possibility of eliminating the progestin
component of hormone therapy altogether or altering the paradigm of progestin
exposure. However, natural or synthetic progestins are viewed as a necessary
component of HT owing to their ability to reduce the risk of endometrial cancer
associated with estrogen use. Given the apparent necessity of progestins in HT,
researchers have turned to the strategy of optimizing HT administration that maximizes
benefits and minimizes deleterious consequences associated with hormone interactions.
For example, this issue stemmed from cardiovascular and cancer research on HT
(reviewed in Whitehead et al., 1990; Hammond et al., 2001) as researchers argue for
using the lowest effective dose of P4 to minimize its negative effects on lipid metabolism
and retain its anti-estrogen effects on the endometrium and breast.
Clues provided by the research described above support a strategy of cyclic
hormone delivery rather than the continuous, combined treatment that is currently
common to HT. The WHIMS investigators chose to use a continuous progestin dose (the
same dose every day for the duration of the study). However, a constant, continuous
dose of progestin does not mimic the naturally cyclic pattern of progesterone secretion in
women. The normal human menstrual cycle is 28 days in length and follows an identical
pattern every month; after surges in follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), ovulation occurs and estradiol (E
2
) and progesterone (P4) levels peak
33
and then hormone levels become relatively stable until the next cycle. Therefore, many
researchers speculate that a cyclic progestin treatment schedule may compliment the
estrogen dose in a way that more closely mimics this 28-day cycle.
Although clinical investigation is certainly needed, also lacking is sufficient
understanding of estrogen and progesterone interactions at a basic neurobiological level
to rationally design cyclic hormone regimens with desired protection against AD-relevant
endpoints. Towards this end, our laboratory and others have formed a multi-group
collaborative research team that is investigating timing and dosing parameters in cyclic
and continuous estrogen, progesterone, and clinically relevant progestin treatment
regimens in transgenic mice. This research will be discussed in depth in Chapter Four.
We believe that the continued evaluation of hormone treatments in animal models of AD
will yield important insights into the design of future HT regimens that will safely and
effectively reduce the development of AD in postmenopausal women and answer
several of the questions raised by the WHIMS study.
Initial clinical evaluation of cyclic progestogen exposure has been completed and
more is underway. Several clinical HT trials have incorporated a comparison between
continuous versus cyclic progestogen in postmenopausal women on osteoporosis and
cognitive function. For example, two completed studies have both demonstrated that
long term HT with a cyclic progestogen dose was able to increase bone mineral density
in post-menopausal women (Castelo-Branco et al., 1999). Similarly, a continuous
estrogen plus cyclic progestogen paradigm is currently employed in the KEEPS (Kronos
Early Estrogen Prevention Study) Cognitive and Affective Study, a randomized, placebo-
controlled, double-blind study investigating the effects of HT in postmenopausal women
who are within 36 months of their final menstrual period (Wharton W, 2008). This and
34
similar new trials promise to provide important insight on the efficacy of cyclic hormone
delivery and the hypothesized importance of a critical window of hormone intervention.
Experimental studies in animal models lend support for the use of cyclic rather
than continuous progesterone to optimize estrogen neuroprotection. For example, Gibbs
and colleagues have demonstrated that short-term treatment with E
2
and progesterone
can improve cholinergic function (Gibbs, 2000b), with maximal benefit resulting from
cyclic administration of estradiol and progesterone and the least benefit from continuous
combined treatment (Gibbs, 2000a). These exciting new findings support the hypothesis
that estrogen-progesterone interactions can yield additive neuroprotection and that cyclic
hormone delivery may be a critical parameter.
9. Male sex steroid hormones and Alzheimer’s Disease
Age-related androgen depletion and AD
In parallel to the relationships between age-related estrogen loss in women and
increased AD risk, testosterone is depleted as a normal consequence of aging in men
and is linked with elevated risk of AD. Although men do not experience menopause per
se (i.e., a cessation of reproductive ability, nearly complete loss of sex steroid
hormones), men do experience a somewhat similar process termed androgen deficiency
in aging males (ADAM). ADAM refers to normal, age-related depletion of testosterone
and the corresponding constellation of symptoms that reflect dysfunction and
vulnerability to disease in androgen-responsive tissues including brain (Meier et al.,
1987; Burger et al., 1998; Baumgartner et al., 1999; Morley, 2001; Ferrando et al., 2002;
Gooren, 2003; Jones et al., 2003; Sheffield-Moore and Urban, 2004; Kaufman and
Vermeulen, 2005). The decline in testosterone levels begins in the 3
rd
decade and
35
continues at an annual rate of 0.2 – 1% for total testosterone and 2 - 3% for bioavailable
testosterone (Gray et al., 1991; Feldman et al., 2002; Muller et al., 2003). Unlike
menopause, aging men do not experience comparable levels of andropause. That is,
although all men exhibit significant age-related testosterone loss typically beginning in
the third decade of life, men vary in the extent of testosterone loss and the
corresponding severity of clinical manifestations (Swerdloff and Wang, 1993; Morley et
al., 1997). It is estimated that 30% - 70% of men aged 70 and older are hypogonadal,
resulting in at least 5 million aging men in the U.S. suffering the consequences of
andropause and only a small minority of those receive hormone treatment (Morley and
Perry, 2003; Haren et al., 2006). ADAM is associated with increased risk of sarcopenia,
osteoporosis, falls, frailty, and all cause mortality.
The brain is a highly androgen responsive tissue where androgens induce
several beneficial actions. For example, androgens have been shown to improve mood
and promote select aspects of cognition, including spatial abilities (Gouchie and Kimura,
1991; Janowsky et al., 1994) and verbal fluency (Alexander et al., 1998). For example,
men with a higher free testosterone index have been found to perform better on visual
and verbal memory and exhibited better long-term memory (Barrett-Connor et al., 1999),
while those with a low free testosterone index can show decreased visual memory,
visuomotor scanning, verbal memory, and visuospatial processing (Moffat et al., 2002).
ADAM has been associated with impaired cognitive performance in some but not all
studies (Moffat et al., 2002; Haren et al., 2005).
One recently established consequence of ADAM is an increased risk for the
development of AD. Several (Hogervorst et al., 2001; Hogervorst et al., 2002; Rasmuson
et al., 2002; Hogervorst et al., 2003; Watanabe et al., 2004) but not all (Pennanen et al.,
36
2004) studies have identified a relationship between low circulating levels of
testosterone and a clinical diagnosis of AD (Hogervorst et al., 2001; Hogervorst et al.,
2002; Hogervorst et al., 2003; Paoletti et al., 2004). Animal studies support a link
between testosterone and AD (Raber, 2004, 2008).
Although the majority of studies have identified a relationship between low
testosterone and increased AD risk in men, most were unable to determine whether low
testosterone contributes to the disease process or is merely a result of it. However, two
complementary studies suggest low testosterone occurs prior to or in the early stages of
AD pathogenesis, and thus likely acts a risk factor. The first study compared clinical
diagnosis of dementia with blood levels of testosterone in the prospective Baltimore
Longitudinal Study on Aging (Moffat et al., 2004). Interestingly, in those men with AD,
testosterone levels were reduced at check-ups 5 to 10 years prior to diagnosis (Moffat et
al., 2004), suggesting androgen loss occurred well before clinical manifestations of the
disease. Consistent with this study are findings from a study by our laboratory linking low
brain levels of testosterone with increased risk of AD in men because men with both AD
and mild neuropathological changes exhibited low testosterone levels (Rosario, 2004),
again indicating that testosterone loss occurs prior to robust pathology and thus may
contribute to the development of AD. Taken together, these results suggest age-related
testosterone depletion in men is a risk factor for AD.
Unlike the numerous clinical studies that have evaluated the efficacy of HT use in
treating and or preventing AD in postmenopausal women, comparatively few studies
have examined testosterone therapy in men for protective roles against age-related
cognitive decline and development of dementia and they have had mixed results. In a
small clinical study of men recently diagnosed with AD, testosterone treatment for up to
37
one year contributed to improvement in both overall cognitive ability and visual spatial
skills (Tan and Culberson, 2003). In contrast, other studies have not reported significant
benefits of testosterone therapy in men with mild cognitive impairment and AD (Cherrier
et al., 2005; Lu et al., 2006). As with the observed inconsistencies in the literature of HT
use in women, there are likely several factors that contribute to the observed differences
between testosterone studies, including cognitive domains, treatment type and duration,
and the age and other characteristics of the subjects.
Testosterone neuroprotection and AD insults
One beneficial action of androgens that is hypothesized to contribute to a role in
reducing risk of AD is neuroprotection. Androgens have been found to promote neuron
survival in brain regions vulnerable to neurodegenerative diseases such as Alzheimer’s
disease. These areas include the hippocampus and cortical regions, which are both
affected in AD and rich in androgen receptors (Simerly et al., 1990). For example, in
studies from both other labs (Azcoitia et al., 2001) as well as our own, androgens offer
neuroprotective effects after excitotoxic lesions (Ramsden et al., 2003).
While in vivo models provide valuable insight into androgen neuroprotection,
neuron culture models of toxicity have proven valuable in defining the underlying
molecular mechanisms. Cell culture models of neural injury have demonstrated
testosterone protection against serum deprivation (Brooks et al., 1998; Hammond et al.,
2001), Aβ toxicity (Pike, 2001; Zhang et al., 2004; Nguyen et al., 2005), and oxidative
damage (Ahlbom et al., 2001). Testosterone neuroprotection against serum deprivation-
induced apoptosis requires activation of an androgen receptor (AR) dependent
mechanism (Hammond et al., 2001). Specifically, the anti-androgen flutamide attenuated
protection while an aromatase inhibitor had no effect on neuron viability (Hammond et
38
al., 2001). Consistent with this androgen-mediated mechanism of androgen
neuroprotection is an early study from our laboratory, which found that testosterone
neuroprotection against toxicity induced by extracellular Aβ results from DHT not E
2
(Pike, 2001). Further, we observe androgen neuroprotection in PC12 cells transfected
with AR but not in either untransfected PC12 cells or those transfected with empty vector
(Nguyen et al., 2005).
In addition to direct neuroprotection, androgens also protect against another form
of neuropathology directly relevant to AD, hyperphosphorylation of tau. Abnormal,
excessive phosphorylation of the cytoskeletal protein tau in the form of neuropil threads
and neurofibrillary tangles is a defining neuropathological characteristic of AD and
several other neurodegenerative disorders (Wilhelmsen et al., 1999; Weaver et al.,
2000; Iqbal et al., 2005). Although relatively little research has examined the relationship
between androgens and tau hyperphosphorylation, available evidence does indicate that
androgens are protective against this pathology. Papasozomenos and colleagues have
examined the effects of testosterone and E
2
on tau hyperphosphorylation using a model
of heat shock-induced phosphorylation. In this paradigm, testosterone but not E
2
prevents tau hyperphosphorylation (Papasozomenos, 1997; Papasozomenos and
Papasozomenos, 1999; Papasozomenos and Shanavas, 2002). This may be through
inhibition of GSK signaling (Papasozomenos and Shanavas, 2002). Consistent with this
possibility, recent work in our laboratory defines an AR-dependent pathway involving
sequential activation of Akt and GSK3β that results in decreased tau phosphorylation
[unpublished observations]. Recent evidence suggests that androgens also regulate tau
cleavage (Park et al., 2007). Specifically, testosterone prevented calpain-mediated tau
cleavage preventing the generation of the 17-kDa tau fragment (Park et al., 2007).
39
Testosterone regulation of β-amyloid accumulation
In addition to classic neuroprotective actions, androgens may also protect the
brain from AD by regulating accumulation of Aβ. Initial work suggesting a relationship
between androgens and Aβ came from a small study evaluating men treated with anti-
androgen therapies for prostate cancer. Gandy and colleagues found that within several
weeks following initiation of anti-androgen therapy, circulating levels of testosterone and
E
2
were largely depleted whereas plasma levels of Aβ were significantly elevated
(Gandy et al., 2001). Martins and colleagues similarly reported an association between
androgen depletion and elevated Aβ levels in males receiving anti-androgen therapy for
the treatment of prostate cancer (Almeida and Flicker, 2003) and in older males suffering
from memory loss or dementia (Gillett et al., 2003). To what extent these observations
reflect effects of androgen versus estrogen pathways or perhaps reflect associated
changes in gonadotropins remains incompletely resolved (Atwood et al., 2005; Rosario
and Pike, 2008).
Consistent with the observations in aging men, experimental work in rodents also
indicates that androgens function as endogenous negative regulators of Aβ. Early
studies in our laboratory demonstrated that androgens but not estrogens reduce brain
levels of soluble Aβ in male rats (Ramsden et al., 2003). We observed similar findings on
the relationship between androgen levels and Aβ accumulation in the 3xTg-AD mouse
model of AD (parallel to the studies presented here using E
2
and P4). Male 3xTg-AD
mice were depleted of androgens by ORX at age 3 mo and exposed immediately to
either DHT or vehicle, treatments that were maintained continuously for the next 4
months. The elevated Aβ pathology and exacerbated behavioral impairments in the ORX
40
group were blocked in ORX mice treated with DHT, suggesting a preventive effect of
androgens in regulation of AD-related pathology.
10. Hypothesis and Experimental Paradigms
Based on the current knowledge on the effects of sex steroid hormones in the
brain, we have chosen to investigate several hypotheses regarding the effects of these
hormones on age-related and AD pathology-associated changes in the brain. Primarily,
we have chosen to investigate many of the questions raised by the afore-mentioned
WHIMS clinical trial such as a) the effects of estrogen and progesterone, both alone and
in combination on AD-like neuropathology, b) the difference between various, clinically-
relevant progestin exposures in combination with estrogen, c) the “critical window
hypothesis” of aging brain responsiveness, and d) the effects of clinically-relevant
selective estrogen receptor modulators (SERMs). I hypothesize that progesterone will
have antagonistic effects on estrogen’s beneficial actions in some, but not all exposure
paradigms. Further, I predict that the aging brain will loose hormone responsiveness
after the “critical window”. In addition, I predict that some, but not all SERMs will mimic
the beneficial effects of estradiol.
Beyond female sex steroid hormones, I will also investigate the effects of
androgens on AD-like neuropathology. I hypothesize that androgens positively regulate
AD-like neuropathology and that males will be more resistant to the development of AD-
like neuropathology than females. In addition, I hypothesize that this gender difference
may be due, in part, to sex steroid hormones. In this light, I hypothesize that perinatal
hormone exposure will alter levels of androgens and estrogens in young animals that will
alter the progression of AD-like neuropathology in adulthood.
41
The 3xTg-AD mouse
In order the study the effects of sex steroid hormones on AD-like neuropathology,
we utilized the 3xTg-AD transgenic mouse. The 3xTg-AD mouse is the most
comprehensive model of AD to date and has been applauded as an excellent model with
which to study AD-like neuropathology (Cole, 2006). This model is the first to
incorporate mutations from the three genes most commonly utilized to express AD-like
pathology; APP, PS-1 and FTDP-17 (Oddo et al., 2003b). Specifically, this mouse over-
expresses the APP
swe
, PS-1 (M146V) and tau (P301L) mutations, those most commonly
used and successfully expressed in previous models. As opposed to crossing two
independent genetic background strains, these mice were backcrossed to the same
strain (129/C57BL6), thereby reducing strain differences that can affect APP phenotype
(Carlson et al., 1997) and behavior (Crawley et al., 1997).
Based on the initial characterizations from several lab including our own, the
3xTg-AD mice display an age-dependent, region-specific progression of AD-like
pathology and memory deficits that is very consistent with the human AD condition
(Oddo et al., 2003a; Oddo et al., 2003b; Billings et al., 2005; Caccamo et al., 2005).
Intraneuronal Aβ deposits, dense extracellular Aβ deposits, tau hyperphosphorylation,
tau aggregates (although no true “tangles”), dystrophic neurites, and signs of astrogliosis
accumulate with age in only the following regions; cortex, CA1-3, subiculum, dentate
gyrus, entorhinal cortex, and amygdala. The first sign of AD-like pathology is a
progressive accumulation of intraneuronal Aβ deposits found in the neocortex at 3 mo of
age that spreads to the CA1 pyramidal neurons, subiculum, entorhinal cortex, and
amygdala; areas observed to be affected in human AD. Between 3-6 mo of age, the
mice exhibit long-term potentiation (LTP) deficits suggesting synaptic disruption and
42
learning and memory impairments. Moreover, 6 mo old 3xTg-AD mice begin to show
deficits in MWM (Billings 2005) and fear conditioning (Caccamo et al., 2006). By 6 mo of
age, the first extracellular Aβ deposits become apparent as diffuse plaques in layers 4
and 5 of the frontal cortex. The observation that memory deficits are apparent before
dense Aβ plaque formation is also consistent with observations of human AD patients.
Early markers of P-tau were apparent in the CA1 pyramidal neurons of the hippocampus
and co-localized to Aβ immunopositive neurons. With age, intraneuronal Aβ deposition
increases, Aβ40 and 42 levels measured by ELISA increase, and dense extracellular
deposits are observed by 12 mo of age in the hippocampus, subiculum, entorhinal cortex
and amygdala. Also by 12-15 mo, tau hyperphosphorylation is observed in the
hippocampus and by 18 mo of age, tau aggregates and dystrophic neurites have spread
to the cortex. That Aβ accumulation precedes tau aggregation is consistent with the
amyloid cascade hypothesis. Moreover, 18 mo old 3xTg-AD mice display signs of
astrogliosis as assessed by GFAP levels, another aspect of human AD pathogenesis
(Oddo et al., 2003a). Taken together, the 3xTg-AD mouse could be considered the most
comprehensive model of AD that most closely mimics the human AD condition in an
age-dependent, region-specific manner.
Despite its attributes, there are several concerns regarding the 3xTg-AD mouse
as an appropriate model for AD. Most importantly, the 3xTg-AD mouse model may lack
one crucial aspect of AD, cell loss. While no reports to date have done an exhaustive
stereological analysis to definitively determine neuron number, no obvious cell loss can
be observed upon gross observation. This has been a limitation of most of the
previously generated transgenic models since the mechanism of cell death in AD has yet
to be elucidated. It is puzzling that several of the previously described APPxPS-1
43
models that demonstrated neuron loss in CA1 over-express the same genetic mutations
as the 3xTg-AD mouse (Casas et al., 2004; Schmitz et al., 2004). However, the 3xTg-
AD mutations were expressed on different backgrounds, perhaps contributing to
neuronal survival. That the most convincing models of cell death remain to be those
over-expressing tau mutations independent of APP or PS-1 mutations suggests that tau
pathology correlates with cell death more closely than Aβ accumulation. While more
studies are needed to understand the mechanism of cell death in AD, it remains clear
that the 3xTg-AD mouse is an excellent model with which to study AD.
Our laboratory has employed a well-known paradigm of sex steroid hormone
manipulation in the 3xTg-AD mouse in order to study the effects of both male and female
sex steroid hormones. By surgically removing hormone-producing organs
(ovariectomy;OVX and gonadectomy; GDX), we can deplete endogenous circulating
hormone levels and then we can replace these levels by slow-release, subcutaneous,
pellets containing hormone at a certain dose and for a certain period of time. Using this
paradigm in female 3xTg-AD mice, we can investigate the effects of estrogen and
progesterone, both alone and in combination, and in various exposures on endpoints of
AD-like neuropathology. In male 3xTg-AD mice, we can use this paradigm to investigate
the effects of testosterone and dihydrotestosterone on AD-like neuropathology. We
have employed this model to test the theory that both estrogens and androgens protect
against the progression of AD-like neuropathology. Further, by investigating both male
and female mice, we can compare the gender differences in AD-like pathology and the
effects of postnatal hormone exposure on this gender difference.
44
Kainate lesion model
That the 3xTg-AD mice do not suffer from cell death highlights the need for
another animal model in which to assess the effects of sex steroid hormones in terms of
neuroprotection. We have chosen to utilize the kainate acid lesion model in female
Sprague-Dawley rats. Kainic acid injections cause excitotxicity in CA2/3 neurons and cell
death by of both direct actions of the toxin on glutamate receptors and indirect over-
activation of glutamate receptors caused by seizure-induced events (Coyle, 1983). This
model has been widely used by our lab and by other to assess the effects of both
estrogens and androgens on neuroprotection in an area of the hippocampus also
affected by AD (Ramsden et al., 2003; Rosario et al., 2006a).
We have also employed the paradigm of surgical hormone depletion to
investigate the effects of sex steroid hormones in WT rats against neurotoxicity from a
kainate lesion. By removing endogenous hormone levels by OVX and replacing those
levels with subcutaneous, slow-release, hormone-filled silastic capsules, we can
investigate the effects of estrogen and progesterone, both alone and in combination
against hippocampal cell death subsequently induced by a kainate injection. Taken
together, the 3xTg-AD mouse model and the kainate lesion model in WT rodents will
allow us to investigate the effects of sex steroid hormones estrogen and progesterone
on b o t h A D - r e l a t e d n e u r o p a t h o l o g y and n e u r o p r o t e c t i o n .
45
CHAPTER TWO
Progesterone blocks estrogen neuroprotection from kainate in middle-aged
female rats
Chapter Two Abstract
The neuroprotective effects of estrogen in young adult rodents are well
established. Less well understood is how estrogen neuroprotection is affected by aging
and interactions with progesterone. In this study, we investigated the effects of estrogen
and continuous progesterone, both alone and in combination, on hippocampal neuron
survival following kainate lesion in 14-month-old female rats entering reproductive
senescence. Our results show that ovariectomy-induced hormone depletion did not
significantly affect the extent of kainate-induced neuron loss. Treatment of
ovariectomized rats with estrogen significantly reduced neuron loss, however this effect
was blocked by co-administration of continuous progesterone. Treatment of
ovariectomized rats with progesterone alone did not significantly affect kainate toxicity.
These results provide new insight into factors that regulate estrogen neuroprotection,
which has important implications for hormone therapy in postmenopausal women. This
manuscript was published in Neuroscience Letters in 2008.

Introduction
The precipitous loss of estrogen and progesterone that occurs after menopause
is a significant risk factor for the development of Alzheimer’s disease (AD) in women
(Maki and Hogervorst, 2003). However, support for the use of estrogen-based hormone
therapy (HT) to reduce the risk of AD in postmenopausal women has been controversial.
46
Although prospective studies have demonstrated that HT can reduce the risk of AD, the
Women’s Health Initiative trial indicated that the incidence of dementia was not
significantly affected by HT consisting of only conjugated equine estrogen (CEE) initiated
several years after the onset of menopause but increased by HT combining CEE plus
medroxyprogesterone acetate (MPA). These conflicting results suggested that neural
estrogen actions and thus HT efficacy may be attenuated by a variety of factors, such as
the paradigm of continuous versus cyclic progesterone treatment (Maki and Hogervorst,
2003) and advancing age.
Accumulating evidence suggests that there may be a limited window of
opportunity for initiation of HT in women after which benefits are not realized
(Henderson, 2006). In experimental models, the brain shows age-related alterations in
estrogen responsiveness that appear to begin with the onset of reproductive
senescence. For example, many neural effects of estrogen are diminished in aged
female rats, including induction of brain-derived neurotrophic factor (Jezierski and
Sohrabji, 2001), compensatory sprouting (Stone et al., 2000), spatial memory (Bimonte-
Nelson et al., 2006) and neuroprotection from stroke (Wise, 2006). However, middle-
aged female rodents retain some estrogen responsiveness on cognition in women
(Sherwin and Henry, 2008) and rodents (Aenlle et al., 2007). Taken together, these
studies highlight the importance of investigating the neuroprotective effects of female
sex steroid hormones in the aging brain.
In addition to aging, neural estrogen actions are also regulated by progesterone,
interactions that may affect HT efficacy. For example, we recently reported that estrogen
attenuated ovariectomy-induced accumulation of β-amyloid in a mouse model of AD, an
effect that was blocked by co-administration of continuous progesterone (Carroll et al.,
47
2007b, Chapter Three). Similarly, continuous progesterone can block estrogen-induced
increases in the expression of neurotrophins (Bimonte-Nelson et al., 2004) and indices
of cholinergic function (Gibbs, 2000b). When delivered independently, both estrogen
(Wise, 2002) and progesterone (Schumacher et al., 2007) can exert protective effects in
models of neuronal injury such as excitotoxic lesions and ischemia. Co-administration of
estrogen and progesterone rescues neuronal loss in some rodent models of injury but
not others (Schumacher et al., 2007). For example, continuous exposure to
progesterone or the progestin medroxyprogesterone acetate attenuates estrogen
neuroprotection against kainate lesion in young adult female rats (Rosario et al., 2006a).
How aging affects interactions between estrogen and progesterone in regulation of
neuroprotective effects is not well understood. In this study, we begin to investigate in an
animal model how both aging and progesterone affect estrogen neuroprotection. We
examine the independent and combined effects of estrogen and progesterone on neuron
survival in hippocampus following kainate lesion in reproductively senescent female rats.
Methods
Retired female Sprague-Dawley rat breeders (Harlan Laboratories; Indianapolis,
IN) were obtained at 9 months of age. Vaginal smears were taken 7 consecutive days
every 21 days from 12-14 mo of age to identify irregular estrus cycles, a hallmark of
reproductive senescence (LeFevre and McClintock, 1988). At 14 months of age, rats
underwent ovariectomy (OVX) or sham OVX. Two weeks following surgery, animals
were subcutaneously implanted with silastic capsules (1.57 mm I.D., 3.18 mm O.D.; Dow
Corning) that were either empty (controls) or contained hormones (Sigma, St. Louis,
MO) in a crystalline form. Each group (n=10 per group) received either 1 mm x 5 mm
48
blank capsule, 1 mm x 5 mm 17β-estradiol (E
2
) capsule, 4 mm x 40 mm progesterone
(P4) capsule, or a combination of 1 mm x 5 mm E
2
and 4 mm x 40 mm P4 capsules
designed to maintain plasma E
2
and P4 at physiological levels (Febo et al., 2002;
Hoffman et al., 2003; Rosario et al., 2006a). On the day of sacrifice, uteri were
dissected, blotted, and weighed as a bioassay of E
2
action to confirm efficacy of
estrogen treatment (Korach and McLachlan, 1995). Further, to confirm efficacy of
progesterone treatment, blood was collected and serum P4 levels were measured by
ELISA (Demeditec Diagnostics, Germany); our data were consistent with the
manufacturer’s reported values for assay sensitivity (0.045ng/ml) and intra-assay (CV=
5.4%) and inter-assay (CV= 9.96%) variability.
Three weeks after initiation of hormone treatment, animals were injected (ip) with
either saline or kainate (Sigma; 10 mg/kg in sterile 0.85% NaCl). To assess latency and
severity of kainate-induced seizure, animals were monitored continuously for 3 h
following injection according to a previously described progressive rating scale ranging
from 0 (no seizure) to 5 (continuous seizure) (Rosario et al., 2006a). Animals that
displayed a seizure rating of 5 were immediately sacrificed and excluded from further
study. Forty-eight hours post-injection, animals were anesthetized with isoflurane and
killed by rapid decapitation. Brains were immediately removed, bisected midsagitally,
and immersion-fixed for 48 h in cold, freshly prepared 4% paraformaldehyde.
Hemibrains were exhaustively sectioned (40 µm) in the horizontal plane using a
vibratome. Every tenth section containing hippocampus (approximately 12 sections per
brain) was immunostained with the neuron-specific antibody NeuN (1:250, Chemicon;
Temecula, CA) using the ABC Elite immunohistochemistry kit (Vector; Burlingame, CA),
as previously described (Ramsden et al., 2003). The number of NeuN-immunoreactive
49
cells in the CA2/3 of the hippocampus was counted using a protocol previously
described (Ramsden et al., 2003). Briefly, an Olympus BX50 microscope equipped with
a motorized stage was computer-controlled by the CAST-Grid software (Olympus,
Ballerup, Denmark) so that unbiased, randomly oriented counting frames of 32 µm x 32
µm with X-Y steps of 210 µm x 210 µm were generated throughout the CA2/3 region.
The number of positively stained nuclei within each counting frame was recorded by an
experimenter blinded to the experimental conditions.
To evaluate treatment effects, raw data were statistically examined using ANOVA
and, when appropriate, between group comparisons were made using Fisher LSD tests.
Results
To confirm the efficacy of the hormone manipulations, we measured uterine
weight, a bioassay of E
2
action, and P4 serum values. We observed a significant overall
effect on uterine weight across treatment groups [F(4,36) = 8.61, p <0.001]. In
comparison to sham OVX animals, the OVX group had significantly lower uterine weight
(530±32 mg versus 230±80 mg; p <0.001). Relative to the OVX group, both of the E
2
treated groups, OVX+E
2
(376±30 mg) and OVX+E
2
+P4 (402±23 mg), exhibited
significantly higher uterine weights (p = 0.03 and p = 0.02, respectively), indicating
effective E
2
treatment. Progesterone replacement had no statistically significant effect on
uterine weight (284±43 mg; p = 0.42 compared to OVX). We also observed a significant
overall effect of treatment on P4 serum values [F(4,23) = 6.28, p = 0.001]. Specifically,
compared to sham OVX (36.1±11.2 ng/ml), OVX animals had significantly decreased
serum P4 level (9.0±3.5 ng/ml; p = 0.04). Serum P4 levels after P4 replacement
50
(54.7±5.1 ng/ml) and E2+P4 replacement (60.8±12.9 ng/ml) significantly increased
levels compared to OVX (p = 0.003 and p = 0.0004, respectively).
Because kainate-induced seizure severity is related to the extent of neuronal
injury, we determined whether hormone manipulations affected seizure parameters.
Lesioned animals were monitored for latency to seizure onset and seizure severity. All
kainate-treated animals exhibited robust seizures with seizure severity scores of at least
3 out of 5 and an overall mean seizure severity of 3.8. In animals used for neuron
survival analysis (<5 seizure score), there was no effect of hormone treatment on either
seizure severity [F(4,40) = 0.34, p = 0.85] (Figure 2A) or latency to seizure onset
[F(4,40) = 0.42, p = 0.79] (Figure 2B). The proportion of animals achieving maximal
seizure rating (score 5; immediate sacrifice and exclusion from study) was higher
(50.0%) in the OVX+P4 group compared to all other groups (26.8%).

Figure 2: Treatment conditions did not significantly affect the severity and latency of kainate-induced
seizures. Female rats treated systemically with kainate (KA) were monitored for both seizure severity (A)
and latency to seizure onset (B). Seizure scores show mean values (+SEM) of each group (n = 10/group)
and represent the severity of seizure-related behavioral indices based on a 0-5 scale with 5 representing the
highest level of seizure behavior. Seizure latency shows show mean values (+SEM) for each group of the
time period (in minutes) from KA injection to the onset of seizure-related behaviors.
51
To determine the effect of hormone treatments on the extent of kainate lesion,
neuronal survival in the hippocampus CA2/3 was determined. Sham OVX animals
suffered ~60% neuron loss. If sex steroids are endogenous regulators of neuron viability,
then hormone depletion resulting from OVX should exacerbate kainate-induced cell loss
and hormone treatments should protect relative to OVX. Overall, there was a significant
effect of treatment on neuron survival [F(5,28) = 8.65, p <0.0001]. However, in contrast
to observations in young adult female rats (Rosario et al., 2006a), the OVX group did not
exhibit a significant decrease in neuron survival relative to the sham OVX group (Figure
2). Treatment of OVX rats with E
2
(OVX+E
2
, p = 0.008) but not P4 (OVX+P4; p = 0.32)
significantly increased neuronal survival compared to the OVX group. However, when
replaced in combination, the OVX+E
2
+P4 group suffered significantly more neuron loss
compared to the OVX+E
2
alone group (p = 0.013), demonstrating that the addition of P4
blocked the neuroprotective effect of E
2
. Within treatment groups, we did not observe
significant correlations between neuron survival and either progesterone levels or uterine
weight (data not shown).
52

Figure 3: Progesterone blocks the neuroprotective effect of estrogen. The number of NeuN-immunoreactive
cells in hippocampal region CA2/3 was quantified in sham OVX rats and in OVX rats treated with either
vehicle (veh), E 2 and/or P4. Data show mean numbers of counted cells (± SEM) for treatment groups
exposed to KA (filled bars) and not exposed to KA (open bars). In all groups exposed to KA except OVX+E 2,
the number of neurons was significantly decreased relative to Sham-veh. * denotes p<0.05 between
indicated groups.
Discussion
The goal of this study was to begin investigating, in an animal model, two
variables that may affect efficacy of estrogen-based HT in postmenopausal women: age-
related changes in hormone responsiveness and regulatory interactions between
estrogen and progesterone. In comparison to our previous study in young adult female
rats (Rosario et al., 2006a), our current results suggest that the middle-aged female rat
shows altered responsiveness to estrogen but can still benefit from estrogen
neuroprotection. Further, our data extend to an aging model the growing evidence that
progesterone can antagonize at least some neural effects of estrogen.
53
We applied to reproductively senescent female rats the same paradigm that we
previously utilized to demonstrate progesterone antagonism of estrogen neuroprotection
in young adult female rats (Rosario et al., 2006a). In the middle-aged rats studied here,
we observed a more extensive lesion and more robust seizures than we had in 3-month-
old female rats treated with the same dose of kainate (Rosario et al., 2006a). This
observed age-related increase in kainate vulnerability is consistent with some (Wozniak
et al., 1991) but not all (Kesslak et al., 1995) prior studies. It is possible that the severity
of the lesion created a floor effect, potentially masking subtle hormone effects on neuron
survival.
Our data are consistent with the hypothesis that middle-aged rodents show
diminished estrogen responsiveness. First, we observed that uterine weight decreased
by <60% after OVX in 14-mo old rats, a modest effect in comparison to observations in
younger female rats. Previous studies also demonstrated that the uterus becomes less
responsive to estrogen with increasing age, showing smaller OVX-induced decreases in
uterine weight (Xu et al., 2004) and uterotrophic effects of estrogen only when treated
soon after OVX (Daniel et al., 2006). Second, we did not observe a significant increase
in neuron death in OVX rats compared to sham OVX animals, a finding inconsistent with
the established role of endogenous estrogen as an important regulator of neuron
survival (Wise, 2002). Similarly, Stone et al. found that OVX impaired compensatory
sprouting in young adult but to a lesser degree in middle-aged female rats (Stone et al.,
2000), suggesting that aging may affect the neural response to estrogen depletion.
Although OVX-induced hormone deprivation did not exacerbate kainate lesion,
estrogen treatment in middle-aged OVX rats was significantly neuroprotective. This
observation adds to a growing literature indicating that although estrogen often exerts
54
diminished effects in the middle-aged female brain, it can retain at least some of its
protective effects. In prior studies, estrogen treatment in middle-aged OVX rats was
shown to enhance working memory performance on hippocampal-dependent spatial
memory tasks (Daniel et al., 2006; Aenlle et al., 2007), regulate cholinergic function
(Kalesnykas et al., 2004), and protect against spinal cord (Chaovipoch et al., 2006) and
ischemic brain (Simpkins et al., 1997; Dubal et al., 1998; Wise, 2006) injuries. However,
as discussed above, other studies have demonstrated reduced or lost protective actions
of estrogen in brain, including regulation of interleukin-1 expression following excitotoxic
injury (Nordell et al., 2003), regulation of brain-derived neurotrophic growth factor
expression (Jezierski and Sohrabji, 2001), compensatory sprouting (Stone et al., 2000),
neuroprotection from stroke (Alkayed et al., 2000), and working memory (Daniel et al.,
2006).
The mechanism(s) underlying age-related changes in estrogen responsiveness
are not clear, but likely are affected by age changes in ovarian function. We observed
that responsiveness to estrogen protection appeared to depend upon the estrus cycle
history of middle-aged female rats. Specifically, we noted that estrogen was protective in
rats showing persistent vaginal cornification, but not in rats with irregular cycles. The
period of persistent vaginal cornification in mice has been characterized by a nearly
twofold E
2
:P4 ratio due to a significant reduction in circulating P4 levels (Nelson et al.,
1981). This increased E
2
:P4 ratio would expose the brain to a relatively unopposed
estrogen-rich environment, perhaps promoting a higher level of estrogen
responsiveness.
We also found that the neuroprotective effect of E
2
in middle-aged female rats
was blocked by co-treatment with P4. This result is consistent with two previous studies
55
from our laboratory. First, using a parallel experimental design, we found that both P4
and medroxyprogesterone acetate antagonize estrogen neuroprotection against kainate
in young adult female rats (Rosario et al., 2006a). Second, in young adult female 3xTg-
AD transgenic mice we found that continuous P4 blocked the β-amyloid lowering action
of E
2
(Carroll et al., 2007b, Chapter Three). Our current finding is also consistent with a
recent report that progesterone blocks estrogen upregulation of neurotrophins (Bimonte-
Nelson et al., 2004). The mechanism by which progesterone blocks the neuroprotective
effect of estrogen remains to be elucidated. Previous studies report that short-term P4
treatment provides neuroprotective effects by attenuating seizure severity however long-
term exposure to P4, as used in this study, is not associated with anxiolytic, seizure-
reducing effects (Rosario et al., 2006a). Interestingly, there is evidence that CEE+MPA
hormone treatment increases seizure frequency in epileptic women, but not in laboratory
rodents (Harden, 2008). One potential strategy with relevance to clinical use of HT is
cyclic rather than continuous progesterone exposure, an approach that can increase
rather than inhibit estrogen actions in rodent brain (Sherwin and Henry, 2008).
Although abundant experimental and clinical data strongly suggest that estrogen-
based HT should yield beneficial neural outcomes in postmenopausal women, the
unexpected negative findings from the recent Women’s Health Initiative clinical study
have raised serious concerns about the safety and efficacy of HT. Several factors,
including advanced age and the use of a continuous progestogen component, have
been suggested as potential liabilities that may underlie the recent clinical shortcomings
of HT. Our results support this hypothesis. In middle-aged, reproductively senescent
female rats our data indicate an altered responsiveness to E
2
but also demonstrate a
retained ability to exhibit neuroprotection in at least a subset of animals. Further, our
56
findings show that continuous P4 exposure attenuates E
2
actions. Additional studies in
animal models are needed to elucidate the mechanisms underlying hormone interactions
and how they are affected by aging, which should generate the novel insight necessary
to develop rational new strategies for effective HT in women.
57
CHAPTER THREE
Progesterone and estrogen regulate Alzheimer-like neuropathology in female
3xTg-AD mice
Chapter Three Abstract
Estrogen depletion in postmenopausal women is a significant risk factor for the
development of Alzheimer’s disease (AD) and estrogen-based hormone therapy may
reduce this risk. However, the effects of progesterone both alone and in combination
with estrogen on AD neuropathology remain unknown. In this study, we utilized the
3xTg-AD mouse model of AD to investigate the individual and combined effects of
estrogen and progesterone on Aβ accumulation, tau hyperphosphorylation, and
hippocampal-dependent behavioral impairments. In gonadally intact female 3xTg-AD
mice, AD-like neuropathology was apparent by 3 months of age and progressively
increased through age 12 months, a time course that was paralleled by behavioral
impairment. Ovariectomy-induced depletion of sex steroid hormones in adult female
3xTg-AD mice significantly increased Aβ accumulation and worsened memory
performance. Treatment of ovariectomized 3xTg-AD mice with estrogen, but not
progesterone prevented these effects. When estrogen and progesterone were
administered in combination, progesterone blocked estrogen’s beneficial effect on Aβ
accumulation but not on behavioral performance. Interestingly, progesterone significantly
reduced tau hyperphosphorylation when administered both alone and in combination
with estrogen. These results demonstrate that estrogen and progesterone independently
and interactively regulate AD-like neuropathology and suggest that an optimized
58
hormone therapy may be useful in reducing the risk of AD in postmenopausal women.
This manuscript was published in the Journal of Neuroscience in 2007.
Introduction
Alzheimer’s disease (AD) is an age-related neurodegenerative disorder
characterized by accumulation of β–amyloid (Aβ) and neurofibrillary tangles, progressive
neuron loss, and cognitive deficits (Hardy and Selkoe, 2002). Abundant evidence
suggests that the depletion of the sex steroid hormones estrogen and progesterone at
menopause is a significant risk factor for the development of AD in women (Paganini-Hill
and Henderson, 1994; Paganini-Hill and Henderson, 1996; Tang et al., 1996; Kawas et
al., 1997). Further, prospective and case-control studies have demonstrated that
hormone therapy (HT) can reduce the risk of AD in women (Paganini-Hill and
Henderson, 1996; Tang et al., 1996; Kawas et al., 1997). Given this background,
findings from the Women’s Health Initiative Memory Study (WHIMS) demonstrating a
higher incidence of dementia in subjects receiving estrogen-based HT in the absence
(Espeland et al., 2004) and presence (Rapp et al., 2003; Shumaker et al., 2003) of
progestin were unexpected. The WHIMS findings raised many important issues,
highlighting the needs to better understand the role of estrogen and progesterone in AD
pathogenesis and to optimize HT.
One of the important issues raised by the WHIMS trial is the role of progestogens
in HT (Brinton, 2004; Craig et al., 2005). Although the many beneficial actions of
estrogen in the brain are well established, the roles of progesterone, both alone and in
combination with estrogen, remain incompletely defined. Some evidence suggests that
progesterone improves hippocampal-dependent spatial memory (Roof et al., 1994) and
59
protects neurons in some (Roof et al., 1994; Alkayed et al., 2000; Hoffman et al., 2003;
De Nicola et al., 2006), but not all (Azcoitia et al., 1999; Toung et al., 2004) rodent
models of neuronal injury. Similarly, co-administration of estrogen and progesterone
rescues neuronal loss in some rodent paradigms of neural injury (Azcoitia et al., 1999;
Toung et al., 2004) but not others (Rosario et al., 2006a). The effects of progesterone,
both alone and in combination with estrogen, on neuropathology in animal models of AD
are not known.
In order to begin investigating the individual and combined effects of estrogen
and progesterone on AD-like neuropathology, we utilized the 3xTg-AD mouse model,
which exhibits many features of AD neuropathology (Oddo et al., 2003b). In this study,
we evaluated the effects of experimental manipulation of estrogen and progesterone
levels in adult female 3xTg-AD mice on the development of Aβ accumulation, tau
hyperphosphorylation and hippocampal-dependent memory performance.
Methods
Animals
Colonies of 3xTg-AD (Oddo et al., 2003b) and background strain, wild type
(C57Bl6/129S; Jackson Laboratory, Bar Harbor, ME) mice were bred and maintained at
the University of Southern California in accordance with NIH guidelines on use of
laboratory animals and an approved protocol by the University’s Institutional Animal
Care and Use Committee. Female mice used in these studies were housed individually
on 12h light on/off cycles and provided ad libitum access to food and water.
60
Experimental design
This study consists of three experiments that were conducted using separate
groups of animals, except where indicated.
Treatment groups for Experiment 1: To investigate the age-related development
of AD-like neuropathology in female 3xTg-AD mice, gonadally intact females were
randomly assigned to one of the following four groups (n = 7 per group) representing
age at sacrifice: 3, 6, 9, and 12 mo. Wild-type (WT) mice age 6 mo were used as a
comparison group.
Treatment groups for Experiment 2: To investigate the development of AD-like
neuropathology after estrogen manipulation, 3 mo old female 3xTg-AD and WT mice
were assigned to one of the following three treatment groups (n = 7 per group): sham
ovariectomized (OVX), OVX, and OVX+17β-estradiol (E
2
). Mice were bilaterally OVX
and immediately implanted with a subcutaneous, continuous-release 90 d pellet
(Innovative Research of America, Sarasota FL) containing either 0.25 mg E
2
(OVX+E
2
group) or placebo (sham OVX and OVX groups). Animals in the sham OVX group were
the same mice that comprised the 6 mo group in Experiment 1. All animals in
Experiment 2 were sacrificed at age 6 mo, 3 mo after the initiation of hormone
manipulations.
Treatment groups for Experiment 3: To investigate the development of AD-like
neuropathology after progesterone manipulation, 3 mo old female 3xTg-AD and WT
mice were divided into the following treatment groups (n = 7 per group): OVX,
OVX+progesterone (P4) and OVX+E
2
+P4. Mice were bilaterally OVX and immediately
implanted with a subcutaneous, continuous-release 90 d pellet containing 25.0 mg P4,
61
both 0.25 mg E
2
and 25 mg P4, or vehicle (Innovative Research of America, Sarasota
FL) and sacrificed at 6 mo of age.
The hormone pellet doses used in Experiments 2 and 3 have been shown to
produce serum levels of estrogen and progesterone in the physiological range (Pomp et
al., 1995; Kadish and Van Groen, 2002; Shultz et al., 2004). All hormone-treated mice
were sacrificed at 6 mo of age, 3 mo after initiation of hormone treatment. For all
experiments, animals were behaviorally tested on the morning of sacrifice. Afterwards,
they were deeply anesthetized (100 mg/kg sodium pentobarbital), transcardially
perfused with cold PBS, and sacrificed by decapitation. To confirm the efficacy of
hormone treatments, a) uteri were dissected, blotted, and weighed, and b) blood was
collected for analysis of serum hormone levels. Hemi-brains were immersion fixed in 4%
paraformaldehyde for 48 hrs then stored in 4°C in PBS/1% sodium azide until use.
Serum hormone levels
E
2
and P4 serum levels were measured using radioimmunoassay (RIA) as
previously described (Slater et al., 2001).
Immunohistochemistry
Fixed hemi-brains were blocked, sectioned (40 µm) exhaustively in the horizontal
plane using a vibratome, and then processed for immunohistochemistry using a
standard protocol (Pike, 1999; Rosario et al., 2006b). Briefly, every 8th section (~12 per
brain) was immunostained using antibodies directed against a) Aβ (#71-5800 Aβ 1:300
dilution, Zymed, San Francisco CA), b) Aβ precursor protein C-terminal fragments
(CTF’s) (CT20 1:16,000 dilution, Calbiochem, San Diego, CA), or c)
hyperphosphorylated tau (AT8 1:1000 dilution, Pierce, Rockford, IL) using ABC Vector
Elite and diaminobenzidine (DAB) kits (Vector, Burlingame, CA). Antigen unmasking
62
treatment, consisting of 5 min rinse in 99% formic acid was performed to enhance Aβ
immunoreactivity (Cummings et al., 2002).
Quantification of immunohistochemistry
Immunohistochemistry was quantified using two different methods by a
researcher blinded to experimental condition. First, levels of positive immunoreactivity
for Aβ and CTF antibodies were determined by immunohistochemistry load technique
(Cummings et al., 2002; Rosario et al., 2006b). Load values were determined from
selected 420 µm x 330 µm fields of immunolabeled sections that were captured and
digitized using a video capture system (B/W CCD camera coupled to an Olympus BX40
upright microscope). Using NIH Image software 1.61, digital grayscale images were
converted into binary positive/negative data using a constant threshold limit. The
percentage of positive pixels (i.e., immunoreactive area) was quantified for each image
then averaged across images for each brain region in each animal to generate
immunoreactive load values. Quantified fields were selected systematically using a
predetermined pattern to maximize analysis of immunoreactivity in each brain region.
For hippocampus CA1 and subiculum, every 11
th
horizontal section was analyzed across
the entire hippocampus (a total of approximately 12 sections per brain), beginning with a
randomly selected section from the first 11 sections of dorsal hippocampus. In
hippocampus CA1, the capture frame was centered over the pyramidal layer with the
first captured field corresponding to the narrow zone that marks the division between the
end of the regio inferior (CA2/3) and the start of regio superior (CA1) as defined by West
et al. (West et al., 1991). Progressing across CA1 towards the subiculum, the first 3
adjacent, but non-overlapping fields were captured for load analysis. In the subiculum,
we similarly captured 2 adjacent non-overlapping fields per section beginning at the
63
sharp transition from the large pyramidal cells of the CA1 to the smaller cells of the
presubiculum (West et al., 1991). In the frontal cortex, fields were captured from every
sixth coronal section beginning rostrally with the appearance of somatosensory cortex
(Franklin and Paxinos, 1997) for a total of six sections per brain. For each section, the
frame was centered over layers 4-5 and 5 adjacent non-overlapping fields were captured
beginning in cingulate cortex area 1 and progressing laterally to somatosensory cortex
(Franklin and Paxinos, 1997).
Second, the numbers of Aβ deposits and AT8-immunoreactive (IR) neurons were
counted, as previously described (Rosario et al., 2006b). Because of the relatively low
numbers of both Aβ deposits and AT8-IR cells, we counted all immunoreactive objects
meeting criteria within the combined CA1 hippocampus and subiculum regions, as
defined above, for every 11
th
section in each animal. AT8-IR neurons were defined as
cells showing strong AT8 immunolabeling over most of the cell surface. Aβ deposits
were defined as diffuse or dense extracellular accumulations of Aβ immunoreactivity with
approximate diameter at least twice the size of a neuron (to prevent misidentification of
intraneuronal Aβ-IR).
Spontaneous alternation behavior
Mice were tested for working memory using spontaneous alternation behavior
(SAB) in a Y-maze, as previously described (Rosario et al., 2006b). The maze was
constructed from black-colored plexiglass (short arms A and B: 6 in x 5 in x 3 in, long
arm C: 8 in x 5 in x 3 in). Each animal was placed in arm C of the maze and allowed to
explore freely for 8 minutes. The total numbers of arm choices and alternations were
recorded. An arm choice was defined as both front and hind paws fully entering the arm.
The maze was cleaned with 70% ethanol between animals to minimize odor cues. SAB
64
score was calculated as the proportion of alternations (an arm choice differing from the
previous 2 choices) to the total number of alternation opportunities (total arm entries-2)
(King and Arendash, 2002).
Statistical analyses
To statistically evaluate treatment effects, raw data were first analyzed by
ANOVA and then subjected to between group comparisons using the Fisher LSD test.
Effects achieving 95% probability (i.e., p < 0.05) were interpreted as statistically
significant.
Results
Experiment 1: Development of neuropathology in female 3xTg-AD mice
Age-related increase in Aβ accumulation
As a first step in determining the effects of E
2
and P4 on AD-like neuropathology
in female 3xTg-AD mice, we assessed the age-related development of Aβ accumulation
in gonadally intact female 3xTg-AD mice at 3, 6, 9 and 12 mo of age. Aβ accumulation
was evaluated in three hormone-responsive brain regions affected in AD: CA1 of
hippocampus, subiculum, and frontal cortex. We observed intraneuronal Aβ-
immunoreactivity (IR) in all three brain regions that was apparent at low levels by age 3
mo and increased through age 12 mo (Figure 4). In frontal cortex, Aβ-IR was largely
restricted to layers IV and V and in hippocampus was most prominent in CA1 pyramidal
layer. Relatively lower levels of Aβ-IR were observed in basolateral amygdala and
entorhinal cortex (data not shown). Only light, non-specific immunolabeling was
observed in wild type (WT) female mice in all brain regions at age 6 mo (Figure 4A, F, K)
and later ages (data not shown). Load quantification of Aβ-IR showed that Aβ levels
65
were greatest in subiculum, followed by frontal cortex, then hippocampus CA1 (Figure
5). Aβ accumulated most quickly in frontal cortex and exhibited the strongest trend for
continued accumulation beyond age 12 mo in subiculum (Figure 5). In addition to
accumulating intraneuronally, Aβ-IR was also observed in the form of extracellular,
plaque-like deposits beginning at age 9 mo and strongly increasing at age 12 mo (Figure
5D, E). Extracellular Aβ deposits were observed almost exclusively in subiculum through
age 12 mo, although they were also occasionally observed in hippocampus CA1 and
entorhinal cortex.

Figure 4. Age-related increase in Aβ immunoreactivity. Representative images show Aβ-IR in hippocampus
CA1 (A-E), subiculum (F-J), and layers IV-V of frontal cortex (K-O) from female 3xTg-AD mice at ages 3 mo
(B, G, L), 6 mo (C, H, M), 9 mo (D, I, N), and 12 mo (E, J, O), and from wild type (WT) female mice at age 6
mo (A, F, K). Specific Aβ-IR is not observed in WT mice but in 3xTg-AD is apparent intraneuronally by age 3
mo and extracellularly (arrows) beginning at age 9 mo. Scale bar = 100 µm.
66

Figure 5. Quantification of age-related increase in Aβ accumulation Aβ load values were quantified in
hippocampus CA1 (A), subiculum (B), and layers IV and V of frontal cortex (C) from WT mice at 6 mo of age
and from female 3xTg-AD mice at 3, 6, 9, and 12 mo of age. Data represent mean Aβ load values (+SEM)
from 2-3 non-overlapping fields per section, 6-12 sections per brain (n = 7 per condition). ANOVA showed
significant between group differences in Aβ load in hippocampus CA1 (F = 5.98, p = 0.008), subiculum (F =
31.85, p < 0.001), and frontal cortex (F = 7.6, p = 0.008). (D) Aβ-IR also included extracellular Aβ deposits
that were observed primarily in subiculum. Scale bar = 50 µm. (E) Data show mean counts (+SEM) of
extracellular Aβ deposits in animals. A significant age-related increase in Aβ deposits was observed in 3xTg-
AD mice (F = 9.70, p < 0.001). * p < 0.05 compared to 3 mo, # p < 0.05 compared to 6 mo.
67
To confirm that our evaluation of Aβ pathology reflects amyloidogenic species of
Aβ rather than non-amyloidogenic carboxyl-terminal fragments (CTFs) of amyloid
precursor protein, we examined CTF-IR and quantified CTF load in the subiculum. We
observed that CTF-IR exhibited markedly different staining pattern in comparison to Aβ-
IR; CTF-IR was localized to the periphery of the cell body (Figure 6H) while Aβ-IR was
localized throughout the cell body, in punctate distribution (Figure 6I). High levels of CTF
load were observed in female 3xTg-AD mice across all ages examined (Figure 6B-F).
Comparably low levels of CTF load were seen in 6 mo female WT mice (Figure 6A, G).

Figure 6. CTF levels do not change across conditions in 3xTg-AD mice. Representative images show CTF-
IR in the subiculum of 6 mo WT female mice (A) and female 3xTg-AD mice at ages 3 mo (B), 6 mo (C), 9 mo
(D), and 12 mo (E). (F) Quantification of CTF load across conditions shows high CTF levels in all 3xTg-AD
groups that dos not significantly vary by age (F = 0.14, p = 0.94). A similar, extranuclear distribution of CTF-
IR is observed at high magnification (400X) in WT mice (G) and 3xTg-AD mice (H) that qualitatively differs
from the more uniform and often punctate appearance of Aβ-IR in 3xTg-AD mice (I). Scale bar = 50 µm.
68
Age-related increase in tau hyperphosphorylation
To further investigate the development of AD-like neuropathology in female
3xTg-AD mice, we assessed the age-related progression of tau hyperphosphorylation.
We quantified the number of cells strongly immunoreactive with the AT8 antibody, which
recognizes pathological phosphorylation of tau at Ser 202 and 305 that is associated
with AD (Goedert et al., 1995). Intensely stained AT8-IR neurons were reliably detected,
albeit in low numbers, at 9 mo of age and increased in abundance through 12 mo of age
(Figure 7). AT8-IR neurons were most common in the subiculum, observed occasionally
in hippocampus CA1, but were not found in frontal cortex by age 12 mo.

Figure 7. Age-related increase in tau hyperphosphorylation. Tau hyperphosphorylation is absent in 6 mo
WT mice (A) but exhibits an age-related increase in female 3xTg-AD mice at ages 3 mo (B), 6 mo (C), 9 mo
(D), and 12 mo (E). Representative images show sections immunostained with AT8 antibody for
phosphorylated tau, and counterstained with cresyl violet. Scale bar = 50 µm. (F) Quantification of numbers
of intensely AT8-IR neurons in hippocampus CA1 and subiculum demonstrates the magnitude of this effect
(F = 5.59, p = 0.007). * p <0.05 relative to 3 mo, # p <0.05 relative to 6 mo 3xTg-AD groups.
Age-related decline in spontaneous alternation behavior
Finally, we evaluated working memory in female 3xTg-AD mice by assessing
performance of hippocampal-dependent spontaneous alternation behavior (SAB) in the
69
Y-maze. SAB performance of female 3xTg-AD mice at ages 3 and 6 mo was not
significantly impaired compared to WT female mice. However, SAB performance was
significantly and increasingly impaired at 9 and 12 mo of age (Figure 8A). In contrast,
female WT mice did not show a significant age-related decline in SAB performance
through age 12 mo (data not shown). To ensure that observed changes in SAB in 3xTg-
AD mice were not the result of changes in activity level, we measured the number of arm
entries in the Y-maze. SAB performance was not associated with the number of arm
entries (Figure 8B).

Figure 8. Age-related decline in spontaneous alternation behavior. Hippocampal-dependent working
memory performance in female 3xTg-AD mice was investigated by assessing spontaneous alternation
behavior (SAB) in a Y-maze. (A) In comparison to WT mice (open bars), SAB performance in female 3xTg-
AD mice (filled bars) showed impairment that began at age 6 mo and became statistically significant at ages
9 and 12 mo age (F = 16.2, p < 0.001). * p < 0.05 compared to WT. (B) The number of arm entries,
measured as a negative control for activity level, did not significantly vary across conditions (F = 1.14, p =
0.37).
70
Experiment 2: Effect of estrogen on neuropathology in female 3xTg-AD mice
Confirmation of estrogen treatment
To investigate the effects of hormone status on the development of AD-like
pathology, female 3xTg-AD mice were depleted of sex steroid hormones by OVX at age
3 mo and then treated immediately with continuous E
2
or placebo for a period of 3 mo.
Efficacy of these hormone manipulations was confirmed directly by measuring serum E
2
levels using radioimmunoassay and indirectly using a bioassay of uterine weight. Serum
E
2
levels were found to be within physiological range (sham OVX = 98 ± 7 pg/ml, OVX =
28 ± 1 pg/ml, OVX+E
2
= 104 ± 28 pg/ml; F = 4.28, p = 0.04). The OVX group had
significantly lower E
2
levels than the sham OVX group (p = 0.016), whereas the OVX+E
2
group had serum E
2
levels significantly higher than the OVX group (p = 0.012) but not
significantly greater than the sham OVX group (p = 0.81). Similar results were observed
with uterine weights (sham OVX = 86.6 ± 9.6 mg, OVX = 15.5 ± 1.9 mg, OVX+E
2
=
162.2 ± 31.1 mg; F = 12.86, p < 0.001). OVX-induced hormone depletion caused a
significant decrease in uterine weight compared to the sham OVX condition (p = 0.027)
and E
2
treatment caused a significant increase compared to both the OVX (p < 0.001)
and sham OVX (p = 0.01) groups.
Estrogen regulates Aβ accumulation
If E
2
beneficially regulates Aβ accumulation, then OVX-induced hormone
depletion should increase Aβ accumulation whereas E
2
treatment of OVX animals
should prevent this increase. To investigate this hypothesis, female 3xTg-AD mice were
assessed for Aβ accumulation at age 6 mo, following the 3 mo period of hormone
manipulation. Consistent with our prediction, we observed that, in comparison to the
sham OVX group, the OVX group exhibited a robust increase in Aβ load in hippocampus
71
CA1, subiculum, and frontal cortex (Figure 9B, F, J). E
2
treatment in OVX animals
partially attenuated the increased Aβ load in subiculum and entirely prevented it in
hippocampus CA1 and frontal cortex (Figure 9C, G, K). The second measure of Aβ
pathology, Aβ plaque number, was not counted in E
2
-manipulated mice due to the small
number of plaques present in 6 mo old animals. To confirm that estrogen regulation of
Aβ-IR reflects amyloidogenic species of Aβ rather than CTFs, we quantified and
compared CTF-IR in E2-manipulated mice. As expected, we observed that CTF load
was not significantly different across the sham OVX, OVX, and OVX+E
2
groups (F =
0.36, p = 0.70).

Figure 9. E2 regulates Aβ accumulation. Aβ-IR was visualized in female 3xTg-AD mice at age 6 mo in the
sham OVX (A, E, I), OVX (B, F, J), and OVX+E2 (C, G, K) groups in hippocampus CA1 (A-C), subiculum (E-
G), and frontal cortex (I-K), as shown in representative images. Scale bar = 100 µm. Data demonstrate that
mean Aβ load (+SEM) differed significantly across treatment groups in hippocampus CA1 (F = 4.40, p =
0.028) (D), subiculum (F = 6.77, p = 0.007) (H), and frontal cortex (F = 4.30, p = 0.031) (L). **p < 0.05
relative to sham OVX group, *p < 0.05 relative to OVX group.
72
Estrogen regulation of tau hyperphosphorylation
To further investigate estrogen regulation of AD-like neuropathology, we also
determined the effect of estrogen status on levels of tau hyperphosphorylation by
counting the combined number of AT8-IR neurons in subiculum and hippocampus CA1
of E2-manipulated female 3xTg-AD mice. We found that the number of AT8-IR neurons
was not significantly altered in the OVX group or the OVX+E
2
group (F = 2.29, p = 0.13)
(Figure 10).

Figure 10. E2 regulation of tau hyperphosphorylation. Representative images of AT8-IR (counterstained
with cresyl violet) show neurons with abnormal tau phosphorylation in female 3xTg-AD mice at age 6 mo in
sham OVX (A), OVX (B), and OVX+E2 (C) conditions. Scale bar = 50 µm. (D) Quantification of numbers of
intensely AT8-IR cells in hippocampus CA1 and subiculum show that neither OVX nor E2 treatment
significantly altered tau phosphorylation (F = 2.29, p = 0.13).
73
Estrogen effects on spontaneous alternation behavior
Next, we investigated the possibility that E
2
-regulation of AD-like neuropathology
in female 3xTg-AD mice may also affect memory function as measured by SAB
performance in the Y-maze test. We found that OVX resulted in a significant decline in
SAB performance and that E
2
treatment of OVX animals reversed this effect, restoring
SAB performance to the level of sham OVX animals (Figure 11A). These effects were
unrelated to general activity levels as the numbers of arm entries were similar across
treatment groups (Figure 11B). To confirm that the effects of hormone manipulations on
SAB reflected underlying neuropathology rather than direct hormone effects on SAB, we
also evaluated the effects of OVX with and without E
2
treatment on SAB in WT female
mice at age 6 mo. There were no significant differences across the sham OVX, OVX,
and OVX+E
2
groups of WT female mice in SAB performance (Figure 11C).

Figure 11. E 2 regulates spontaneous alternation behavior. Estrogen status significantly affected SAB
performance in 6 mo old female 3xTg-AD mice but not in age-matched WT female mice. (A) In comparison
to the sham OVX group (Sham) of 3xTg-AD mice, the OVX group exhibited significantly impaired SAB that
was prevented in the OVX+E 2 group (F = 14.48, p < 0.001). **p < 0.05 relative to Sham, *p < 0.05 from
relative to OVX. (B) The number of arm entries in the task did not significantly differ across 3xTg-AD
treatment groups (F = 1.87, p = 0.19). (C) In contrast to results in 3xTg-AD mice, there was no significant
difference in SAB performance across Sham, OVX, and OVX+E 2 groups in WT mice (F = 0.94, p = 0.41).
74
Experiment 3: Effect of progesterone on neuropathology in female 3xTg-AD mice
Confirmation of progesterone treatment
To investigate the effects of progesterone on the development of AD-like
neuropathology, female 3xTg-AD mice were depleted of endogenous sex steroid
hormones by OVX at age 3 mo and then treated immediately with either placebo (OVX
group) or continuous P4 in the absence (OVX+P4 group) or presence of continuous E
2
(OVX+E2+P4 group) for a period of 3 mo. Efficacy of P4 treatment was confirmed
directly by measuring serum P4 levels using RIA. Effects on uterine weight were also
determined. Serum levels of P4 resulting from hormone treatment were within the
expected physiological range and significantly greater than observed in gonadally intact
female mice (sham OVX = 1.6 ± ng/ml, OVX+P4 = 7.3 ± ng/ml, OVX+E
2
+P4 = 3.9 ±
ng/ml; F = 4.59, p = 0.003). Evaluation of uterine weights (OVX = 17.1 ± 1.4 mg,
OVX+P4 = 40.3 ± 5.1 mg, OVX+E
2
+P4 = 108.1 ± 20.4 mg; F = 12.84, p < 0.001) showed
that P4 treatment had a mild uterotrophic effect that did not reach statistical significance
(p = 0.19) although the E
2
+P4 treatment had a significant proliferative effect (p < 0.001)
in comparison to OVX mice.
Progesterone attenuates the effect of estrogen on Aβ accumulation
If P4 shares with E
2
the ability to reduce Aβ accumulation, then P4 treatment
would be predicted to prevent the increase in Aβ accumulation resulting from OVX-
induced hormone depletion. However, we observed no significant difference in Aβ load
between the OVX and OVX+P4 conditions in hippocampus CA1, subiculum, and frontal
cortex (Figure 12). We also investigated the clinically relevant issue of whether P4
affects the actions of E
2
to reduce the OVX-induced increase in Aβ accumulation. We
observed that unlike the Aβ-lowering effect of continuous E
2
alone, combined continuous
75
treatment with E
2
and P4 treatment for 3 mo did not significantly affect Aβ accumulation
in hippocampus CA1, subiculum, or frontal cortex in comparison to the OVX condition
(Figure 12). There were also no significant differences in CTF load across treatment
groups (F = 0.11, p = 0.90).

Figure 12. P4 attenuates the effects of E 2 on Aβ accumulation. Aβ-IR was visualized in female 3xTg-AD
mice at age 6 mo in the OVX (A, E, I), OVX+P4 (B, F, J), and OVX+E 2+P4 (C, G, K) groups in hippocampus
CA1 (A-C), subiculum (E-G), and frontal cortex (I-K), as shown in representative images. Scale bar = 100
µm. Data demonstrate that mean Aβ load (+SEM) did not differ significantly across treatment groups in
hippocampus CA1 (F = 0.85, p = 0.45) (D), subiculum (F = 2.10, p = 0.15) (H), and frontal cortex (F = 0.58, p
= 0.57) (L).
Progesterone regulates tau hyperphosphorylation
We also investigated the effect of P4 treatments on tau hyperphosphorylation by
counting the number of AT8-IR neurons in hippocampus CA1 and subiculum of female
3xTg-AD mice. Interestingly, P4 treatment robustly decreased the number of AT8-IR
76
neurons in comparison to the OVX group, an effect that persisted when P4 was
delivered in combination with E
2
(Figure 13).

Figure 13. P4 regulates tau hyperphosphorylation. Representative images of AT8-IR (counterstained with
cresyl violet) show neurons with abnormal tau phosphorylation in female 3xTg-AD mice at age 6 mo in the
OVX (A), OVX+P4 (B), and OVX+E 2+P4 (C) conditions. Scale bar = 50 µm. (D) Quantification of numbers of
intensely AT8-IR cells in hippocampus CA1 and subiculum show that P4 treatments significantly decreased
tau hyperphosphorylation in comparison to the OVX group (F = 9.71, p = 0.002). * p <0.05 relative to OVX
group.
Progesterone effects on spontaneous alternation behavior
Finally, we investigated whether the P4 treatments affected SAB performance. In
comparison to the poor SAB performance of OVX 3xTg-AD mice, we observed that the
OVX+E
2
+P4 group but not the OVX+P4 group showed improved performance (Figure
77
14A). As expected, SAB did not reflect changes in activity level, as the number of arm
entries was not significantly different across the 3xTg-AD treatment groups (Figure 14B).
To ensure that SAB performance was not significantly affected by direct effects of
female sex steroid hormones, we evaluated SAB in WT mice in the OVX, OVX+P4, and
OVX+E
2
+P4 conditions and found no significant between group differences (Figure
14C).

Figure 14. P4 regulation of spontaneous alternation behavior. Progesterone treatment did not significantly
affect SAB performance in either 6 mo old female 3xTg-AD mice or in age-matched WT female mice. (A) In
comparison to the OVX group of 3xTg-AD mice, P4 significantly improved the impaired SAB performance
only when co-treated with E 2 (F = 4.36, p = 0.03). *p < 0.05 relative to OVX. (B) The number of arm entries
in the task did not significantly differ across 3xTg-AD treatment groups (F = 0.37, p = 0.70). (C) In WT mice,
there were no significant differences in SAB performance across OVX, OVX+P4, and OVX+E 2+P4 groups (F
= 1.68, p = 0.22).
Discussion
In this study, we investigated the relationship between the sex steroid hormones
estrogen and progesterone and the development of AD-like neuropathology in the 3xTg-
AD mouse model of AD. Consistent with previous findings (Oddo et al., 2003a), we
observed that AD-like pathologies in female 3xTg-AD mice, including Aβ accumulation,
tau hyperphosphorylation and deficits in working memory, appear within a few months of
age and progressively worsen. Specifically, intraneuronal Aβ accumulation occurred first,
78
followed by tau hyperphosphorylation, and then impaired SAB performance.
Significantly, we found that experimental depletion of endogenous estrogen and
progesterone by OVX exacerbated these pathologies, suggesting independent and or
combined actions of these hormones in regulating AD-like pathology. Consistent with a
protective role of estrogen, we found that E
2
treatment in OVX mice prevented the
worsening in Aβ accumulation and SAB impairment. However, estrogen status was not
associated with significant changes in tau phosphorylation. In contrast, P4 treatment in
OVX mice did not affect either Aβ accumulation or SAB, although it did strongly reduce
tau hyperphosphorylation. When P4 treatment was combined with E
2
, the protective
effect of E
2
on Aβ accumulation was attenuated, however both reduced tau
hyperphosphorylation and improved SAB were observed in comparison to OVX mice.
These data, which represent the first report of progesterone effects in an animal model
of AD, implicate both separate and combined actions of estrogen and progesterone in
regulation of AD-like pathologies.
Our results support the hypothesis that estrogen protects against development of
AD-like neuropathology. Similar to our findings, other rodent studies have reported
increased Aβ levels following OVX in WT guinea pigs (Petanceska et al., 2000) and in
transgenic mouse models of AD (Zheng et al., 2002). Further, E
2
treatment has been
associated with decreased brain levels of Aβ in guinea pigs (Petanceska et al., 2000) as
well as in the APP
SWE
(Levin-Allerhand et al., 2002), Tg2576 (Zheng et al., 2002), and
Tg2576xPS1 (Zheng et al., 2002) AD mouse models. In contrast, other studies have
reported that neither OVX nor E
2
treatment significantly altered Aβ levels in the PDAPP
(Green et al., 2005), APP
SWE
xPS1 (Heikkinen et al., 2004), and APP23 (Yue et al., 2005)
transgenic mouse models of AD. It is unclear why estrogen status is associated with Aβ
79
levels in some models but not others. There are several factors that vary between the
studies and may contribute to the inconsistent findings. One potentially important
difference across studies has been varying methods of Aβ quantification, each of which
preferentially detects different pools of Aβ ranging from soluble monomeric Aβ to other
oligomeric and deposited forms. The effect of estrogen on the various Aβ forms has yet
to be determined. In this study, we found that estrogen decreases Aβ as determined by
immunoreactive load method, which measures Aβ that accumulates both intra- and
extra-cellularly and presumably reflects relatively insoluble Aβ forms. Because
oligomeric Aβ is increasingly recognized as pathologically significant (Walsh et al.,
2005), we have begun to consider the potential hormone regulation of this Aβ pool. In
preliminary slot blot studies using an oligomeric-specific anti-Aβ antibody, we observed
elevated levels of oligomeric Aβ in aged female 3xTg-AD mice (unpublished
observations), consistent with a prior report (Oddo et al., 2006b). However, we did not
consistently observe oligomeric Aβ in female 3xTg-AD mice at age 6 months and thus
were unable to evaluate whether hormonal status affects levels of oligomeric Aβ. Future
studies will need to address not only how sex steroid hormones regulate Aβ, but also
which Aβ pool(s) they regulate.
Beyond investigating estrogen effects, this study is the first to directly examine
the effects of progesterone on AD-like neuropathology. The most significant findings
from this study are the observations that although continuous P4 treatment by itself had
no effect on Aβ accumulation, P4 attenuated the beneficial effect of E
2
treatment on
lowering Aβ accumulation in female 3xTg-AD mice. Our finding that P4 lacks
independent action but attenuates E
2
action is consistent with recent observations in
other paradigms. For example, we recently reported that while P4 treatment in female
80
OVX rats did not affect the extent of kainate-induced neuron loss, it completely blocked
estrogen neuroprotection (Rosario et al., 2006a). Similarly, continuous P4 treatment has
been reported to inhibit estrogen-mediated increases in neurotrophin expression
(Bimonte-Nelson et al., 2004) and spatial memory performance (Bimonte-Nelson et al.,
2006). One particularly interesting component of this relationship with a hypothesized
clinical relevance is the effect of continuous versus cyclic P4 treatment. Prior work by
Gibbs (Gibbs, 2000a) demonstrated that hippocampal cholinergic activity in OVX female
rats was increased by cyclic treatment with E
2
and P4 but decreased by continuous
treatment with E
2
and P4. Potential differences between continuous and cyclic P4
treatment in 3xTg-AD mice are currently under study.
In addition to the hormone-mediated effects on Aβ accumulation, we also
observed that progesterone significantly regulated tau hyperphosphorylation in female
3xTg-AD mice. While we observed a statistically nonsignificant trend of reduced tau
hyperphosphorylation in the presence of estrogen, we found that P4 treatment
significantly decreased tau hyperphosphorylation, reducing AT8-IR to levels even lower
than those observed in gonadally intact 3xTg-AD mice. These observations are
generally consistent with previous studies demonstrating that E
2
and P4 can modulate
activities of kinases and phosphatases involved in regulating levels of tau
phosphorylation, including glycogen synthase kinase-3β (GSK3β) (Alvarez-De-La-Rosa
et al., 2005; Goodenough et al., 2005). Specifically, E
2
has been reported to reduce
GSK-3β activity (Goodenough et al., 2005), a kinase that phosphorylates specific tau
sites that are associated with neuropathology (Ishizawa et al., 2003). Recent data
suggest that acute P4 treatment can decrease expression of both tau and GSK-3β in rat
cerebellum, however net increases in both GSK-3β activity and tau phosphorylation
81
were also observed (Guerra-Araiza et al., 2007). It is unclear how continuous P4 affects
GSK-3β, although our data predict that extended P4 exposure alters the balance of tau-
relevant kinase and phosphatase activities to yield a net decrease in pathological tau
phosphorylation. Interestingly, prior studies have demonstrated that GSK3β-dependent
regulation of tau hyperphosphorylation is related to behavioral performance in 3xTg-AD
mice (Billings et al., 2007), which suggests a potentially important connection between
the effects of progesterone on tau hyperphosphorylation and cognition in AD patients.
The results of this study also provide interesting insight into the relationships
between Aβ accumulation, tau hyperphosphorylation and behavioral impairment. In
gonadally intact 3xTg-AD females, we found a strong negative association between the
progression of Aβ accumulation in hippocampus CA1 and subiculum and performance in
the hippocampal-dependent SAB task. In addition, we observed a correlation between
E
2
-mediated changes in Aβ accumulation and behavioral impairment, further evidence of
a cause-and-effect relationship between these two pathologies. However, combined
E
2
+P4 treatment revealed that behavioral impairments in the 3xTg-AD mouse are not
simply a function of Aβ load. Combined E
2
+P4 treatment did not decrease Aβ load yet
reduced tau hyperphosphorylation and significantly improved SAB performance
suggesting that the relationship between tau hyperphosphorylation and behavioral
impairment is presumably dependent upon interactions with Aβ accumulation. For
example, despite strongly reducing AT8 immunoreactivity, independent P4 treatment did
not significantly affect either Aβ load or SAB performance. These results are consistent
with recent observations by Oddo and colleagues that improving behavioral performance
in the 3xTg-AD mouse model by Aβ immunotherapy strategies required not only
decreasing soluble Aβ levels, but also lowering soluble tau levels (Oddo et al., 2006a).
82
Together, these results suggest that behavioral impairment in 3xTg-AD mice involves
both Aβ accumulation and tau hyperphosphorylation, pathologies that are differentially
affected by E
2
and P4 treatments.
In summary, the results of this study provide novel insights into the roles of
estrogen and progesterone in regulating AD neuropathology. Although case-control and
prospective studies demonstrate that HT use in postmenopausal women can
significantly reduce the risk of developing AD (Paganini-Hill and Henderson, 1996; Tang
et al., 1996; Kawas et al., 1997; Hogervorst et al., 2000; Zandi et al., 2002; Maki, 2005),
the recent WHIMS study has raised several important issues that may significantly affect
the efficacy of HT, including the estrogen and progestogen composition of treatment, the
timing of treatment initiation, and cyclic versus continuous application of treatment
hormones (Brinton, 2004; Craig et al., 2005). Our findings support the hypothesis that
the loss of sex steroid hormones promotes development of AD neuropathology and that
hormone treatments can effectively prevent this effect. Our results are also consistent
with the complex, seemingly conflicting clinical data, which indicate both apparent
benefits and risks with HT use in women. In the 3xTg-AD mouse, our results indicate
that continuous E
2
treatment combats Aβ accumulation and behavioral impairments.
However, continuous P4 treatment had both a positive effect, reduction of tau
hyperphosphorylation, and a negative outcome, attenuation of Aβ-lowering by E
2
.
Further studies are needed to not only elucidate the relationships between estrogen and
progesterone interactions, but also to optimize the ability of E
2
and P4 treatments. We
believe that the continued evaluation of hormone treatments in animal models of AD will
yield important insights into the design of future HT regimens that will safely and
effectively reduce the development of AD in postmenopausal women.
83
CHAPTER FOUR
Continuous and cyclic progesterone differentially effect estrogen regulation of
Alzheimer-like pathology in female 3xTg-AD mice
Chapter Four Abstract
As the levels of female sex steroid hormones, estrogens and progestins decline
at menopause, women suffer an increased risk for the development of Alzheimer’s
disease (AD). Therefore, the development of a safe, effective estrogen + progestin
hormone therapy (HT) for post-menopausal women is of interest to slow the
development of Alzheimer-like neuropathology. We recently demonstrated that
continuous progesterone treatment attenuated the beneficial effects of continuous
estradiol treatment on β-amyloid accumulation in female AD transgenic mice. However,
the effects of variable progesterone treatment exposure in the brain, both alone and in
combination with estrogens are under-investigated. To that end, this study compared the
effects of continuous and cyclic progesterone treatment both alone and in combination
with estradiol in regulating β-amyloid accumulation in the brain of female 3xTg-AD mice.
We observed that cyclic progesterone treatment, which more closely mimics a women’s
natural, monthly hormone fluctuation, had a synergistic effect with estradiol to reduce β-
amyloid accumulation in hormone-depleted mice while continuous progesterone
attenuated the beneficial effects of estradiol. These results suggest that varying
paradigms of estrogens + progestin therapy may differentially alter the risk of AD
development in post-menopausal women and that future studies are necessary to
optimize the paradigm of hormone therapy.
84
Introduction
The Women’s Health Initiative Memory Study (WHIMS) was the first large-scale,
placebo-controlled, multi-center clinical trial investigating the effects of hormone therapy
(HT) on incidence of dementia, risk for Alzheimer’s disease (AD), cognitive function and
other age-related processes in post-menopausal women. Based on a wealth of evidence
from prospective and case-control studies demonstrating that hormone therapy can
reduce the risk of AD in women (Paganini-Hill and Henderson, 1996; Tang et al., 1996;
Kawas et al., 1997) the WHIMS investigators hypothesized that continuous conjugated
equine estrogen (CEE) + continuous treatment of the synthetic progestin,
medroxyprogesterone acetate (MPA) would reduce the incidence of dementia. However,
they demonstrated a higher incidence of dementia in subjects receiving CEE in the
absence (Espeland et al., 2004) and presence (Rapp et al., 2003; Shumaker et al.,
2004) of the progestin. Therefore, the WHIMS findings highlighted the need to better
understand the roles of estrogens and progestins in AD pathogenesis and to optimize
the delicate balance of estrogen and progestin exposure in HT.
The WHIMS trial underscored the lack of understanding regarding the effects of
progestins in the brain (Craig et al., 2005). Although the many beneficial neural actions
of estrogen are well established (Wise 2006), the roles of progesterone, both alone and
in combination with estrogen, remain incompletely defined. While much evidence
suggests a neuroprotective role for progestins in the female rodent brain (reviewed in
Brinton et al., 2008; Schumacher et al., 2008), many recent studies have demonstrated
that progestins attenuate the beneficial effects of estrogens in the brain. Specifically,
progestins block the estradiol-induced increase in important hippocampal spine density
(Woolley and Bruce S. McEwen, 1993), Bcl-2 expression (Garcia-Segura et al., 1998),
85
ChAT activity (Gibbs, 2000a), neurotrophin expression (Bimonte-Nelson et al., 2004),
neuron viability after kainate lesion (Rosario et al., 2006a; Carroll et al., 2008, Chapter
Two), and spatial memory (Bimonte-Nelson et al., 2006; Harburger et al., 2007).
Regarding Alzheimer’s disease directly, our lab recently investigated the effects of
progesterone on β-amyloid, the protein considered central to the neuropathological
development of AD (Hardy and Selkoe, 2002). We demonstrated that continuous
progesterone attenuates the beneficial effect of estradiol in lowering β-amyloid
accumulation in female AD transgenic mice (Carroll et al., 2007b, Chapter Three). Taken
together, these studies would suggest that progestins act antagonistically with estrogens
in the female brain.
However, the incorporation of a progestin component into HT is necessary due to
the ability of progestins to combat endometrial cancer associated with use of unopposed
estrogens. Therefore, it is of interest to develop a novel HT treatment paradigm using a
more efficacious exposure of progestins. In order to investigate the effects of varying
progesterone treatment paradigms on AD-like neuropathology, we utilized the 3xTg-AD
mouse model (Oddo et al., 2003b). Adult female 3xTg-AD mice were treated with either
continuous or cyclic progesterone, both alone and in combination with estradiol, and
were evaluated the development of Aβ accumulation, tau hyperphosphorylation and
hippocampal-dependent memory performance.
Methods
Experimental design
Colonies of 3xTg-AD (Oddo et al., 2003b) and background strain, wild type (WT)
(C57Bl6/129S; Jackson Laboratory, Bar Harbor, ME) mice were bred and maintained at
86
the University of Southern California in accordance with NIH guidelines on use of
laboratory animals and an approved protocol by the University’s Institutional Animal
Care and Use Committee. Female mice used in these studies were housed individually
on 12h light on/off cycles and provided ad libitum access to food and water
To assess the effects of both continuous and cyclic progesterone treatment, both
alone and in combination with estrogen, mice were given hormone treatments similar to
as described previously (Carroll et al., 2007b, Chapter Three). At 3 mo of age, both WT
and 3xTg-AD mice were divided into the following groups (n = 7/group); a) sham-
ovariectomized, b) OVX, c) OVX+E
2
(continuous estradiol, d) OVX+P4 (continuous
progesterone), e) OVX+E
2
+P4 (continuous estradiol and continuous progesterone), f)
OVX+p4 (cyclic progesterone), and g) OVX+E
2
+p4 (continuous estradiol and cyclic
progesterone). At this time, mice were OVX or sham-OVX and immediately replaced
with a subcutaneous, slow-release pellet containing hormone (Innovative Research of
America, Sarasota, FL). The sham-OVX group and the OVX group received a placebo
pellet. The OVX+E
2
group received a 90day, 0.025mg E
2
pellet. The OVX+P4 group
received a 90day, 25.0mg P4 pellet. The OVX+E
2
+P4 received both 90day 0.025mg E
2
and 25mg P4 pellets. The OVX+p4 group received a 10day, 2.8mg P4 pellet on the 20
th
day every 30 day segment for 90 days total. And finally, the OVX+E
2
+p4 group received
a 90day, 0.025mg E
2
pellet at the time of OVX and then received a 10day, 2.8mg P4
pellet on the 20
th
day every 30 day segment for 90 days total. The 10day P4 pellets were
inserted under Isoflow. The hormone pellet doses of E
2
and P4 were used previously
(Carroll et al., 2007b, Chapter Three; Carroll and Pike, 2008, Chapter Five) and have
been shown to produce serum levels of estrogen and progesterone in the physiological
range (Pomp et al., 1995; Kadish and Van Groen, 2002; Shultz et al., 2004). The 2.8mg,
87
10 day cyclic P4 dose was calculated based on the same daily dose of P4 delivered in
the 25.0mg, 90day pellet (0.28mg/day).
All mice were sacrificed 90 days following OVX at 6 mo of age. On the day of
sacrifice, 3xTg-AD mice were deeply anesthetized (100 mg/kg Nembutal), transcardially
perfused with PBS, and sacrificed by decapitation. One hemibrain was immersion fixed
in 4% paraformaldehyde for 48 hrs then stored in 4°C in PBS/1% sodium azide until use
before being sectioned exhaustively into 40µm sections on a Vibratome for
immunohistochemistry. WT mice were deeply anesthetized with Isoflow and decapitated.
To confirm efficacy of hormone treatment, in both WT and 3xTg-AD, uteri were
dissected, blotted and dry weight was measured. Further, a pilot experiment was
conducted to ensure that the 10-day cyclic P4 pellet in fact caused a physiological level
of circulating P4 for the desired length of time and no longer released P4 after the 10-
day period. In this pilot experiment, four groups (n = 5/group) WT female mice aged 4-6
mo were sham-OVX or OVX and immediately replaced with the same pellets mentioned
above containing either a) placebo, b) continuous P4 or c) cyclic P4. At each of the
following time points (day 5, 10, 20 and 30), four separate groups of each treatment
condition were evaluated for P4 efficacy using the Elevated Plus Maze and then
sacrificed to measure uterine weight.
Immunohistochemistry
As previously described (Carroll et al., 2007b, Chapter Three; Carroll and Pike,
2008, Chapter Five), every 8th section (12 per brain) was immunostained using
antibodies directed against Aβ (#71-5800 Aβ 1:300 dilution, Zymed, San Francisco CA),
or hyperphosphorylated tau (AT8 1:1000 dilution, Pierce, Rockford, IL) in the
hippocampus, subiculum, frontal cortex and amygdala, regions known to be affected in
88
AD. Sections were processed using ABC Vector Elite and diaminobenzidine (DAB) kits
(Vector, Burlingame, CA). Antigen unmasking treatment, consisting of 5 min rinse in
99% formic acid was performed to enhance immunoreactivity (Cummings et al., 2002).
Aβ immunoreactivity quantification
Aβ immunoreactivity load values were quantified based on a previously
described protocol (Carroll et al., 2007b, Chapter Three; Carroll and Pike, 2008, Chapter
Five). Briefly, in 12 sections per CA1 and subiculum and 6 sections in frontal cortex and
amygdala high magnification fields from immunolabeled sections were digitized using a
video capture system (B/W CCD camera coupled to an Olympus BX40 upright
microscope). Quantification was completed using NIH Image software 1.61 which
quantifies the percentage of image pixels detected over a threshold of 100 (percentage
of immunoreactive pixels) and termed “immunoreactive load”. Five images were
quantified per brain area so that the entire surface area of each brain region was
sampled. Experimenter was blind to treatment conditions.
AT8 (+) neuron quantification
Every 8
th
section (12 per brain) was counted for number of AT8-immunoreactive
(+) neurons in the CA1 and subiculum as previously described (Carroll et al., 2007b,
Chapter Three). AT8 (+) neurons were classified as neurons with >50% of the cell body
stained and stained dystrophic processes extending from the cell body.
Spontaneous Alternation Behavior
All 3xTg-AD mice were tested for working memory using spontaneous alternation
behavior (SAB) in a Y-maze, as previously described (Carroll et al., 2007b Chapter
Three; Carroll and Pike, 2008, Chapter Five) < 5 days prior to sacrifice. Briefly, each
animal was placed in the maze and allowed to explore freely for 8 minutes. SAB score
89
was calculated as the proportion of alternations to the total number of alternation
opportunities (total arm entries-2) (King and Arendash, 2002).
Elevated Plus Maze
All mice were tested on the Elevated Plus Maze (EPM) as a behavioral assay for
progesterone action as adapted from a previously described protocol (Boyce-Rustay and
Holmes, 2006; Carroll et al., 2007a) < 5 days before sacrifice, or 5 days after cyclic P4
pellet insertion in the case of mice from the OVX+p4 and OVX+E
2
+p4 groups. Briefly,
mice were tested for 5 minutes on the EPM maze (based on San Diego Instruments,
San Diego, CA) comprised 2 open arms (30 x 5 cm) and 2 closed arms (30 x 5 x 15 cm)
from a common central platform (5 x 5 cm). Behavior was scored for time spent in open
arms and the number of entries into the open and closed arms (an arm entry was
defined as all 4 paws in an arm).
Forced Swim Test
All mice were tested on the Forced Swim Test (FST) as a behavioral assay for
estrogen action as previously described (Boyce-Rustay and Holmes, 2006; Carroll and
Pike, 2008, Chapter Five) < 5 days prior to sacrifice. Briefly, the FST was conducted in a
20 cm-diameter cylinder filled to ~13 cm with 24 ±1.0°C water for 5 minutes. The mice
were acclimated to the cylinder for the first minute and then scored for freezing behavior
every 5 seconds for the remaining 4 minutes of the trial. FST scores were calculated as
the proportion of freezing incidents to the total incidents observed (Carroll and Pike,
2008, Chapter Five).
Statistical Analysis
Statistical analyses. To statistically evaluate treatment effects, raw data were first
analyzed by ANOVA and then subjected to between group comparisons using the Fisher
90
LSD test. Effects achieving 95% probability (i.e., p < 0.05) were interpreted as
statistically significant.
Results
Bioassay confirmation of hormone treatments
To investigate the effects of hormone status on the development of AD-like
neuropathology, female 3xTg-AD mice were OVX at age 3 mo and then treated
immediately or in a cyclic fashion with hormone or placebo for a period of 3 mo. Efficacy
was confirmed using a bioassay of uterine weight and were significantly different across
treatment groups (Figure 15). OVX-induced hormone depletion caused a significant
decrease in uterine weight and E
2
caused a significant increase. Similarly, E
2
+P4 and
E
2
+p4 were proliferative. Finally, P4 and p4 also had a uterotrophic effect, albeit small.

Figure 15. Bioassay confirmation of hormone treatments. Uterine weight was measured to confirm efficacy
of estrogen and progesterone treatments. Uterine weights were significantly different across treatment
groups (F = 15.19, p < 0.0001). OVX significant decreased uterine weight compared to the sham-OVX and
E 2 treatment reversed this effect. Similarly, OVX+E 2+P4 and OVX+E 2+p4 treatment had a significant
proliferative effect in comparison to OVX mice. Finally, OVX+P4 and OVX+p4 treatment also had a
uterotrophic effect compared to the OVX group albeit a much smaller effect. Data demonstrate uterine
weight (±SEM). Sham (Sham-OVX), O (OVX), E (OVX+E 2), P con (OVX+P4), E+P con (OVX+E 2+P4), P cyc
(OVX+p4), E+P cyc (OVX+E 2+p4). * p < 0.05 relative to sham-OVX, # p < 0.05 relative to OVX.
91
Behavioral confirmation of hormone treatments
As a second method of confirming hormone treatments, all mice were also
behaviorally analyzed on the Forced Swim Test (FST) and Elevated Plus Maze (EPM) to
measure efficacy of E
2
and P4 treatments respectively (Figure 16). Both behavioral tests
were conducted < 5 days before sacrifice with the exception of the OVX+p4 and
OVX+E
2
+p4 groups, which were tested on the EPM after 5 days of the second cyclic P4
pellet insertion. As expected, the FST (Figure 16A) demonstrated significant group
differences between the sham-OVX, OVX and OVX+E
2
groups. Hormone depletion
significantly increased depression and anxiety-related freezing behavior in the FST
compared to sham-OVX. Further, E
2
treatment significantly reversed this effect in all
three groups receiving E
2
treatment, OVX+E
2
, OVX+E
2
+P4 and OVX+E
2
+p4. In parallel,
the EPM (Figure 16B) demonstrated significant group differences in anxiety-related
behavior across treatment groups. Treatment with progesterone decreased anxiety-
related behavior compared to the OVX group by increasing time spent in open arms of
the EPM in the OVX+P4 group, OVX+p4, OVX+E
2
+P4 and OVX+E
2
+p4.
To further ensure that our cyclic P4 treatment had the desired effect of only 10
days of P4 exposure, a pilot experiment was also conducted on the EPM. WT female
mice aged 4-6 mo were sham-OVX or OVX and replaced with a) placebo, b) a 90 day
continuous P4, or c) a cyclic p4 10 day pellet and tested on the EPM 5, 10, 20 and 30
days after pellet insertion, respectively (Figure 16C). As expected, OVX induced a
significant decrease in the amount of time spent in open arms while progesterone
treatment significantly attenuated this effect when replaced in continuous form. Further,
when a cyclic, 10 day P4 pellet was given and tested at either 5 days or 10 days after
pellet insertion, P4 had the same positive effect as the continuous P4 pellet. However,
92
when tested 20 or 30 days post pellet insertion, P4 no longer had a positive effect on the
EPM, demonstrating that the 10 day, cyclic P4 pellets no longer distributed hormone
after 10 days as guaranteed by the manufacturer.

Figure 16. Behavioral confirmation of hormone treatments. The Forced Swim Test (FST) was conducted to
confirm efficacy of E 2 treatment while the Elevated Plus Maze (EPM) was conducted to confirm efficacy of
P4 treatment. The FST (A) demonstrated significant group differences (F = 4.15, p = 0.003). Data
demonstrate % time freezing (±SEM). Hormone depletion significantly increased depression and anxiety-
related freezing behavior in the FST and E 2 treatment significantly reversed this effect in all three E 2-treated
groups. In addition, the EPM (B) demonstrated significant group differences (F = 4.3, p = 0.0028). Data
demonstrate % time spent in open arms (±SEM). Treatment with progesterone decreased anxiety-related
behavior compared to the OVX group in the OVX+p4, OVX+E 2+P4 and OVX+E 2+p4 groups and nearing
significance in the OVX+P4 group (p=0.06). Sham (Sham-OVX), O (OVX), E (OVX+E 2), P con (OVX+P4),
E+P con (OVX+E 2+P4), P cyc (OVX+p4), E+P cyc (OVX+E 2+p4). Finally, the EPM pilot conducted in WT mice
demonstrated significant group differences (F = 13.5, p <0.0001). While treatment with either continuous P4
or cyclic P4 either 5 or 10 days after treatment initiation did attenuate the OVX-induced increase in anxiety,
when mice were tested either 20 (p = 0.78) or 30 (p = 0.88) days after cyclic P4 treatment initiation, P4 did
not remain effective. d5-30 (5-30 days after 10-day P4 pellet insertion). * p < 0.05 relative to sham-OVX, # p
< 0.05 relative to OVX.
Continuous, but not cyclic progesterone attenuates estrogen’s beneficial effect on Aβ
accumulation
If cyclic and continuous P4 differentially regulate the effect of E
2
on Aβ
accumulation, then E
2
+P4 treatment of OVX animals should not alter the OVX-induced
increase in Aβ accumulation while E
2
+p4 treatment should reduce it. To investigate this
93
hypothesis, female 3xTg-AD mice were assessed for Aβ accumulation at age 6 mo.
Consistent with our prediction, we observed that, in comparison to the sham-OVX group,
the OVX group exhibited a robust increase in Aβ load in all three brain regions and E
2
treatment attenuated this increase (Figure 17). Further, continuous P4 had no beneficial
effect when replaced alone and attenuated E
2
action when replaced in combination.
Interestingly, when cyclic P4 was replaced alone, it could reduce Aβ accumulation in the
frontal cortex. Most importantly, in contrast to E
2
+P4, when E
2
was replaced in
combination with cyclic P4, this treatment reduced the OVX-induced increase in Aβ
accumulation in both the hippocampus and frontal cortex but not amygdala (Figure 17).

Figure 17. Continuous, but not cyclic P4 attenuates E 2 reduction of Aβ accumulation. Aβ-IR was visualized
in female 3xTg-AD mice at age 6 mo across treatment groups in the subiculum (A-G), hippocampus (H-N)
and amygdala (O-U), as shown in representative images. Scale bar = 100 µm. Data demonstrate that mean
Aβ load (±SEM) differed significantly across treatment groups in the subiculum (F = 9.1, p < 0.0001) (V),
hippocampus (F = 4.39, p = 0.003) (W), and the amygdala (F = 4.17, p = 0.003) (X). In both the
hippocampus and frontal cortex, but not the amygdala, continuous P4 attenuated E 2 action while cyclic P4
did not. Sham (Sham-OVX), O (OVX), E (OVX+E 2), P con (OVX+P4), E+P con (OVX+E 2+P4), P cyc (OVX+p4),
E+P cyc (OVX+E 2+p4). * p < 0.05 relative to sham-OVX group, # p < 0.05 relative to OVX group.
94
Continuous, but not cyclic progesterone attenuates estrogen’s beneficial effect on
working memory
To further investigate progesterone regulation of AD-related neuropathology,
hippocampal-dependent working memory was assessed using the spontaneous
alternation behavior (SAB). We observed that SAB performance was significantly altered
across hormone status (Figure 18). OVX caused a significant decline in SAB
performance compared to sham-OVX while OVX+E
2
prevented this decline (Figure
18A). Neither continuous P4 nor cyclic P4 treatment alone had an effect on the OVX-
induced decline. However, when both hormones were replaced in combination,
continuous P4 attenuated the beneficial effect of E
2
(not significantly different than OVX)
while cyclic P4 augmented E
2
’s beneficial effect (Figure 18A). To ensure that the
observed changes in SAB were not the result of changes in activity level, we measured
the number of arm entries in the Y-maze. SAB performance was not associated with the
number of arm entries across treatment groups (F = 1.2, p = 0.32) (Figure 18B).
Furthermore, to ensure that the effects of hormone manipulations on SAB reflected
underlying neuropathology rather than direct hormone effects on SAB, we also
evaluated the effect of all hormone manipulations on SAB in WT female mice at age 6
mo. There were no significant differences across hormone-manipulate groups of WT
female mice in SAB performance (F = 1.2, p = 0.32) (Figure 18C) or in number of arm
entries (F = 0.29, p = 0.93) (Figure 18D).
95

Figure 18. Continuous, but not cyclic P4 attenuates E 2 increase in working memory. Estrogen and
progesterone status significantly affected SAB performance in 6 mo old female 3xTg-AD mice (A) but not in
age-matched WT female mice (C). SAB performance was significantly altered across hormone status in
3xTg-AD mice (F = 3.05, p = 0.016). OVX caused a significant decline in SAB performance while OVX+E 2
prevented this decline. When E 2 and P4 were replaced in combination, continuous P4 attenuated the
beneficial effect of E 2 (not significantly different than OVX, p = 0.30) while cyclic P4 augmented E 2’s
beneficial effect. (B) The number of arm entries in the task did not significantly differ across 3xTg-AD
treatment groups (F = 1.2, p = 0.32). (C) In contrast to results in 3xTg-AD mice, there was no significant
difference in SAB performance (F = 1.2, p = 0.32) nor in the number of arm entries (D) (F = 0.29, p = 0.93)
across treatment groups in WT mice. Data demonstrate % SAB or number of arm entries (±SEM). Sham
(Sham-OVX), O (OVX), E (OVX+E 2), P con (OVX+P4), E+P con (OVX+E 2+P4), P cyc (OVX+p4), E+P cyc
(OVX+E 2+p4). *p < 0.05 relative to sham-OVX, *p < 0.05 from relative to OVX.
Both continuous and cyclic progesterone lower tau hyperphosphorylation
To comprehensively investigate progesterone regulation of AD-like
neuropathology, we also determined the effect of both continuous and cyclic
96
progesterone status on levels of tau hyperphosphorylation by counting the combined
number of AT8-IR (+) neurons in the CA1 and subiculum of hormone-manipulated mice.
We found that the number of AT8-IR (+) neurons was significantly different across
hormone status (Figure 19). Hormone depletion by OVX significantly increased the
number of AT8-IR (+) neurons compared to sham-OVX. Further, all hormone treatments
containing continuous hormones significantly attenuated this effect. Only the cyclic P4
alone group offered a non-significant trend towards lowering the number of AT8-IR (+)
neurons (p = 0.09) (Figure 19).

Figure 19. E 2, Continuous P4 and cyclic P4 reduce tau hyperphosphorylation. Representative images of
AT8-IR (counterstained with cresyl violet) show neurons with abnormal tau phosphorylation in female 3xTg-
AD mice at age 6 mo in the sham-OVX OVX, OVX+E 2, OVX+P4, OVX+E 2+P4, OVX+p4, and OVX+E 2+p4
conditions. Scale bar = 50 µm. Quantification of numbers of intensely AT8-IR (+) cells in the CA1 and
subiculum show that all hormone treatments, with the exception of the OVX+p4, significantly decreased tau
hyperphosphorylation in comparison to the OVX group (F = 2.71, p = 0.028). Data demonstrate number of
AT8 (+) neurons (±SEM). Sham (Sham-OVX), O (OVX), E (OVX+E 2), P con (OVX+P4), E+P con (OVX+E 2+P4),
P cyc (OVX+p4), E+P cyc (OVX+E 2+p4). * p < 0.05 relative to sham-OVX group, # p < 0.05 relative to OVX
group.
Discussion
The goal of this study was to compare the effects of continuous versus cyclic
progesterone on estrogen regulation of indices of AD-like neuropathology in female
97
3xTg-AD mice. Our previous study demonstrated that continuous P4 attenuates
estrogen’s positive regulatory effect on Aβ accumulation (Carroll et al., 2007b, Chapter
Three). Therefore, to extend on those findings, we hypothesized that a cyclic regimen of
P4 that more closely mimics the natural monthly hormone fluctuations would regulate
estrogen’s effects differently. Our results largely support the hypothesis that cyclic P4
offers significant beneficial effects. Regarding Aβ accumulation, we have replicated our
previous findings by reporting that continuous P4 attenuates the effect of E
2
on lowering
Aβ load in the majority of brain regions investigated (Carroll et al., 2007b, Chapter
Three). Further, here we demonstrate that cyclic P4 does not attenuate E
2
’s beneficial
effect when both hormones are replaced in combination and cyclic P4 even offers some
Aβ-lowering effects when replaced alone. In parallel, we also report similar findings
regarding hippocampal-dependent working memory on the SAB test. We replicated our
previous demonstration that continuous P4 attenuated E
2
’s beneficial effects (Carroll et
al., 2007b, Chapter Three), and now we show that cyclic P4 augmented E
2
’s beneficial
effects. Lastly, regarding tau hyperphosphorylation, we again replicated our previous
findings by demonstrating that all treatments containing continuous P4 lowered the
number of AT8 (+) neurons (Carroll et al., 2007b, Chapter Three) and further show that
cyclic P4 both alone and in combination with E
2
also lower tau hyperphosphorlyation.
Taken together, these results demonstrate that in contrast to continuous P4, a cyclic P4
treatment schedule in combination with continuous E
2
replacement is very beneficial in
lowering indices of AD-like neuropathology such as Aβ accumulation, tau
hyperphosphorylation and working memory deficits. These results provide novel insights
into the simultaneous effect of cyclic P4 treatment and continuous estrogen in regulating
98
indices of AD-like neuropathology as this is the first study, to date, investigating such a
hormone regimen in a mouse model of AD.
Of particular interest is our finding regarding the replacement of cyclic P4 alone.
We demonstrate that cyclic P4 treatment of just three 10-day treatments over a three mo
time period was sufficient to prevent significant OVX-induced increases in tau
hyperphosphorylation and Aβ accumulation in some brain regions. These findings
support several recent investigations into the neuroprotective effects of progesterone in
brain regions related to AD after traumatic brain injury (Roof et al., 1994; Stein et al.,
2008), spinal cord injury (Labombarda et al., 2003), ischemia (Gibson et al., 2005), and
seizure (Rhodes and Frye, 2004) (reviewed in Brinton et al., 2008; Schumacher et al.,
2008). Taken together, results from our laboratory and others suggest that cyclic or short
term P4 alone may offer neuroprotective effects.
However, the most important result of this study describes the interactions
between estrogens and progestins, specifically that continuous progesterone
antagonizes estrogen action. These results are consistent with accumulating
observations such as clinical data suggesting that the progestin MPA may negate
beneficial effects of CEE on cognitive function in elderly women (Rice et al., 2000).
Further, in animal models, continuous progesterone antagonizes estrogen
neuroprotection against the excitotoxic agent kainate (Rosario et al., 2006a; Carroll et
al., 2008, Chapter Five), estrogen upregulation of the neurotrophic factors brain derived
neurotrophic factor, nerve growth factor, and neurotrophin 3 (Bimonte-Nelson et al.,
2004), estrogen increase in hippocampal spine density (Murphy and Segal, 2000),
estrogen up-regulation of Bcl-2 immunoreactivity (Garcia-Segura et al., 1998), and
99
estrogen enhancement of spatial memory performance (Bimonte-Nelson et al., 2006;
Harburger et al., 2007).
However, cyclic progesterone may offer a promising alternative to continuous
progesterone in augmenting estrogen’s beneficial neural effects. Our results are
consistent with previous studies also describing positive effects of cyclic, rather than
continuous progesterone. For example, acute treatment (2-6 hours) with progesterone
had the maximal effect by augmenting the estradiol increase in hippocampal spine
density, but more prolonged progesterone treatment (18 hours) blocks this effect
(Woolley and Bruce S. McEwen, 1993) and yielded the lowest cholinergic function. In
summary, the results presented here along with recent studies on the interaction
between estrogens and progestins in animal models suggest that neural benefits of
hormone treatment may be maximized by repeated, brief cycles of estrogens and
progestins and minimized by continuous combined treatment.
The mechanisms underlying progestin antagonism of estrogen neuroprotection
are not known, however receptor-mediated function may be involved. Since protein
levels of progesterone receptors are up-regulated by estrogen (Guerra-Araiza et al.,
2003), it is reasonable to hypothesize that P4 may be capable of regulating the
expression of both estrogen receptors, ERα and ERβ. In support of this theory, recent
cell culture data from our laboratory demonstrate that progesterone strongly reduces
neuronal expression of both ERα and ERβ, which is paralleled in a time-dependent
manner by decreases in estradiol-mediated genomic action and neuroprotection
(Jayaraman and Pike, 2009). By this action, it is possible that long-term, continuous
progesterone would be capable of attenuating the effects of E
2
. In parallel, it may
explain, in part the opposite effects of cyclic progesterone, as this natural, cyclic
100
hormone exposure may allow ER levels to be restored thus allowing E
2
to exert it’s
beneficial effects. The notion has been previously discussed (Toran-Allerand, 2006) and
may be an important insight into the mechanism behind estrogen and progesterone
action on AD-like neuropathology.
The most significant implication of the results presented here are the potential
clinical application of a continuous estrogen treatment paired with a cyclic progestin
exposure. The WHIMS study employed a continuous combined estrogen + progestin
treatment and demonstrated, as the results here demonstrate, that such hormone
replacement was not beneficial (Rapp et al., 2003; Shumaker et al., 2003). However, the
concept of transitioning from a continuous to a cyclic P4 exposure is already underway
in clinical research. Several clinical HT trials have incorporated a comparison between
continuous vs. cyclic (last 12d/mo) P4 in post-menopausal women on osteoporosis and
cognitive function (Castelo-Branco et al., 1999) as well as cardiovascular health in the
Postmenopausal Estrogen/Progestin Intervention trial (PEPI) (1995). Most importantly,
the Kronos Early Estrogen Prevention Study (KEEPS) is a large, multi-center,
randomized, double-blind, placebo-controlled clinical trial aimed at investigating the
effects of HT on vascular disease in post-menopausal women. This clinical trial is
utilizing a treatment paradigm of constant estrogen + cyclic P4 (the last 12d of each
month) which is almost identical to our hormone paradigm (continuous estrogen + cyclic
P4 during the last 10d of each month). While this trial is in its infancy, it will be extremely
informative to compare the future results to our report in female 3xTg-AD.
Taken together, the results presented here provide support for the continued use
of HT containing a progestin component, perhaps for reducing the risk of AD in post-
menopausal women. It was deemed necessary to add progesterone, a natural estrogen
101
antagonist, to the ERT formulation when it was demonstrated that oral progestins could
antagonize the estrogen-induced endometrial cancer that many post-menopausal
women were experiencing after long-term HT usage (Grady et al., 1995). Based on the
data presented here, a straightforward possibility for the modification of the progestin
component in HT would be to simply utilize a shorter, cyclic dose of P4 to use the lowest
effective dose of P4 to minimize its negative effects on lipid metabolism and retain its
anti-estrogen effects on the endometrium and breast (reviewed in Whitehead et al.,
1990; Hammond et al., 2001). Therefore, as in our model, this preparation would, in
theory, retain the beneficial P4 effects on tau hyperphosphorylation while avoiding the
antagonizing effects of P4 on E
2
neuroprotection in women. Such a preparation of HT is
modeled after the natural, cyclic hormone fluctuations of the female menstrual cycle. As
described for both women and rodents (Wu et al., 2005a), the natural cyclic fluctuation of
progesterone involves a high peak after ovulation for followed by a swift, steady decline
to baseline and low levels the remainder of the cycle. Our results argue for the use of
such a cyclic progestin component in future HT paradigms.
102
CHAPTER FIVE
Selective estrogen receptor modulators differentially regulate Alzheimer-like
changes in female 3xTg-AD mice
Chapter Five Abstract
Estrogen-based hormone therapy (HT) in postmenopausal women may reduce
the risk of Alzheimer’s disease (AD), although HT remains controversial. One key
concern with HT is the potential of adverse outcomes such as breast and uterine cancer.
A promising strategy to maximize HT benefits and minimize HT risks is the use of
selective estrogen receptor modulators (SERMs) that exert tissue-specific estrogenic
effects. To begin investigating the SERM approach in reducing the risk of AD, we
investigated whether AD-like neuropathology in the 3xTg-AD mouse model of AD is
regulated by the SERMs propylpyrazole triol (PPT) and diarylpropionitrile (DPN) that
exhibit relative specificity for estrogen receptors ERα and ERβ, respectively. Consistent
with our previous observations, we found that ovariectomy-induced hormone depletion in
adult female 3xTg-AD mice significantly increased accumulation of β-amyloid protein
(Aβ) and decreased hippocampal-dependent behavioral performance. Treatment with
17β-estradiol (E
2
) prevented the ovariectomized-induced worsening of both pathologies.
PPT treatment was similar to E
2
in terms of reducing Aβ accumulation in hippocampus,
subiculum, and amygdala but comparatively less effective in frontal cortex. In contrast,
DPN did not significantly reduce Aβ accumulation in hippocampus and subiculum, was
partially effective in frontal cortex, and nearly as effective as E
2
in amygdala.
Furthermore, PPT but not DPN mimicked the ability of E
2
to improve behavioral
103
performance. These findings provide initial evidence of beneficial actions of SERMs in a
mouse model of AD and support continued investigation of SERMs as an alternative to
estrogen-based HT in reducing the risk of AD in postmenopausal women. This
manuscript was published in Endocrinology in 2008.
Introduction
Hormone therapy (HT) has long been investigated as a therapeutic option for
postmenopausal women to reduce the risk of developing several age-related disorders,
including Alzheimer’s disease (AD). Prospective studies have demonstrated that HT use
in postmenopausal women can significantly lower the risk of AD (Kawas et al., 1997;
Zandi et al., 2002). Prolonged duration and or early initiation of HT appears to be
important for reducing AD risk (Kawas et al., 1997; Zandi et al., 2002), as HT initiated
several years after the onset of menopause can have adverse rather than beneficial
effects on cognition (Espeland et al., 2004). Despite its protective effect against AD, HT
is also associated with adverse effects in estrogen-responsive tissues including breast
and uterus (Warren, 2004). To minimize estrogen-related risks, progestins are typically
included in HT. However, progestins can blunt beneficial actions of estrogens, including
protective actions in brain that may be relevant to AD (Bimonte-Nelson et al., 2006;
Rosario et al., 2006a; Carroll et al., 2007b, Chapter Three). These findings underscore
the need to develop novel treatment strategies that realize the benefits but minimize the
risks associated with long-term HT use.
One alternative approach to estrogen-based HT is the use of selective estrogen
receptor agonists (SERMs), compounds that exert mixed agonist effects on estrogen
receptors (ERs) in a tissue-specific manner. Ideally, SERMs used to maximize neural
104
health would mimic beneficial estrogen actions in brain but exert negligible adverse
effects on non-neural estrogen-responsive tissues. Two recently developed SERMs that
show promise for mimicking neural benefits of estrogen are propylpyrazole triol (PPT)
and diarylpropionitrile (DPN), which exhibit relative selectivity for ERα and ERβ,
respectively (Stauffer et al., 2000; Meyers et al., 2001). Both PPT and DPN have been
shown to protect cultured neurons from toxic insults implicated in AD neurodegeneration
(Zhao et al., 2004; Cordey and Pike, 2005) and reduce neuron loss in rodent models of
stroke (Carswell et al., 2004; Dai et al., 2007). Further, PPT but not DPN increases
synapse number in hippocampal neurons (Jelks et al., 2007). Unknown are the effects of
PPT and DPN on the development of AD-like neuropathology.
To begin evaluating the efficacy of SERMs in attenuating the development of AD-
like changes, we utilized the 3xTg-AD mouse model of AD. Recently, we found that
17β-estradiol (E
2
) treatment in female 3xTg-AD mice effectively prevents the
acceleration of AD-like neuropathology and behavioral impairments caused by hormone
depletion (Carroll et al., 2007b, Chapter Three). In the present study, we compared the
effects of E
2
, PPT, and DPN on Aβ accumulation and hippocampal-dependent memory
performance in female 3xTg-AD mice.
Methods
Female 3xTg-AD mice were bred and maintained in accordance with NIH
guidelines and a protocol approved by our Institutional Animal Care and Use Committee.
At age 3 mo, female 3xTg-AD mice were randomly assigned to the following treatment
groups (N = 8 per group): sham ovariectomized (OVX), OVX, OVX+E
2
, OVX+PPT and
OVX+DPN. Mice were bilaterally OVX or sham OVX and immediately implanted with a
105
subcutaneous, 90 d continuous-release drug delivery pellet containing 0.025 mg E
2
, 0.25
mg PPT, 0.25 mg DPN, or vehicle (Innovative Research of America, Sarasota, FL). The
0.025 mg dose of E
2
was empirically determined based upon a comparison of the effects
of 0.25mg, 0.025mg and 0.01mg E
2
pellets on uterine weight, a bioassay of estrogen
action. PPT and DPN were delivered at a dose ten-fold higher than E
2
based upon their
lower transcriptional activity than E2 (Stauffer et al., 2000; Meyers et al., 2001) and
demonstrated effectiveness at such doses in previous in vivo studies (Harris et al., 2002;
Frasor et al., 2003; Le Saux and Di Paolo, 2005; Lund et al., 2005).
All mice were sacrificed at age 6 mo, which was 3 mo after initiation of hormone
treatment. Animals were behaviorally assessed on the morning of sacrifice. Mice were
evaluated for 8 minutes for spontaneous alternation behavior (SAB) in a Y-maze, a
hippocampal-dependent task of working memory, as previously described (Rosario et
al., 2006b; Carroll et al., 2007b, Chapter Three). SAB score was calculated as the
proportion of alternations to the total number of alternation opportunities. Total arm
entries were also counted as a measure of activity.
After behavioral assessment, mice were deeply anesthetized (100 mg/kg sodium
pentobarbital), transcardially perfused with cold PBS, and sacrificed by decapitation.
Brains were rapidly collected and immersion fixed in 4% paraformaldehyde/0.1 M PBS
for 48 h then stored in 4°C in PBS/1% NaZ until use. To assess the efficacy of hormone
treatments, uteri were dissected, blotted, and weighed.
Fixed hemibrains were blocked, sectioned (40 µm) exhaustively in the horizontal
plane using a vibratome, and then processed for immunohistochemistry using a
standard protocol (Rosario et al., 2006b; Carroll et al., 2007b, Chapter Three). Briefly,
every 8
th
section (~12 per brain) was immunostained using an antibody directed against
106
Aβ (#71-5800 Aβ 1:300 dilution, Zymed, San Francisco, CA) and ABC Vector Elite
immunohistochemistry and diaminobenzidine kits (Vector, Burlingame, CA). Levels of
Aβ immunoreactivity were quantified in frontal cortex (layers 4-5), basolateral amygdala,
subiculum, and hippocampus CA1 by immunohistochemistry load technique (Rosario et
al., 2006b; Carroll et al., 2007b, Chapter Three). In brief, high magnification fields (420 x
330 µm) from immunolabeled sections were captured and digitized. Using NIH Image
software 1.61, digital grayscale images were converted into binary positive/negative data
using a predetermined threshold limit that was held constant across all brain areas and
samples. The percentage of positive pixels was quantified for each image and is termed
immunoreactive load. Mean load values were calculated from analysis of 2-3 non-
overlapping, representative images per brain region from 6-12 sections (depending upon
brain region) per animal.
Raw data were analyzed by repeated measures ANOVA with the Wilks’ Lambda
test followed by ANOVA with the Fisher LSD test for between group comparisons.
Results
E
2
, PPT and DPN differentially regulate Aβ accumulation
To investigate the effects of SERMs on the development of AD-like
neuropathology, female 3xTg-AD mice were depleted of endogenous sex steroid
hormones by OVX at age 3 mo, treated immediately with E
2
, PPT, DPN, or vehicle then
assessed in 3 mo for complete Aβ accumulation in the following brain regions:
hippocampus CA1, subiculum, frontal cortex and amygdala. Repeated measures
ANOVA revealed a significant overall difference in Aβ load between brain regions F
(4,24) = 1.82, P < 0.0001 and a significant overall interaction of brain region by condition
107
F (3,22) = 17.42, P < 0.0001. As earlier (Carroll et al., 2007b, Chapter Three), we found
that OVX increased Aβ accumulation in all four brain regions and that E
2
treatment
largely prevented this (Figures 20, 21). If the SERMs also regulate Aβ, then treatment
with PPT and or DPN should also prevent the increased Aβ accumulation caused by
OVX. In hippocampus CA1 and subiculum, PPT treatment was as efficacious as E
2
in
reducing Aβ load, whereas DPN treatment had no significant effect on Aβ load (Figures
20, 21). Both PPT and DPN had intermediate effects in frontal cortex and were not
significantly different from either the sham OVX or OVX groups. In amygdala, PPT and
DPN were similar to E
2
with Aβ loads that were lower than the OVX group, but not
significantly different from the sham OVX and OVX+E
2
groups (Figures 20, 21).

Figure 20. Accumulation of β-amyloid protein (Aβ) is differentially regulated by E 2 and the SERMs PPT and
DPN across brain regions. Images show Aβ-immunoreactivity in hippocampus CA1 (A-E), subiculum (F-J),
frontal cortex (K-O), and amygdala (P-T) from female 3xTg-AD mice in sham OVX (A, F, K, P), OVX (B, G,
L, Q), OVX+E 2 (C, H, M, R), OVX+PPT (D, I, N, S), and OVX+DPN (E, J, O, T). Scale bar = 100 µm.
108

Figure 21. Quantitative comparison of E 2, PPT and DPN treatments on Aβ accumulation. Data show mean
(+SEM) Aβ loads for each treatment group in hippocampus CA1 (A), subiculum (B), frontal cortex (C) and
amygdala (D). Hormone and SERM treatments significantly altered Aβ load in the CA1 [F (4,29) = 7.26, P =
0.0004], subiculum [F (4,29) = 18.95, P < 0.0001], frontal cortex [F (4,30) = 3.80, P = 0.013] and amygdala
[F (4,26) = 6.06, P = 0.0014]. Compared to sham OVX, the OVX group had a significantly higher Aβ load in
all brain regions and E 2 treatment significantly reduced Aβ load (A-D). In CA1 and subiculum (A-B), PPT
treatment reduced Aβ load, whereas DPN treatment did not. Both PPT and DPN partially reduced Aβ loads
in the frontal cortex (C). In amygdala (D), both PPT and DPN significantly decreased Aβ load.* P < 0.05
compared to sham OVX group, # P < 0.05 compared to OVX group.
E
2
and PPT but not DPN rescue behavioral impairment
We have previously found that performance of female 3xTg-AD mice in
hippocampal-dependent spontaneous alternation behavior (SAB) is inversely associated
with Aβ levels in hippocampus CA1 and subiculum but not directly affected by levels of
sex steroid hormones in wild type mice (Carroll et al., 2007b, Chapter Three). To
determine whether the effects of SERMs on Aβ accumulation extrapolate to behavior,
we evaluated SAB across groups. Consistent with our prior findings in 3xTg-AD mice,
we found that the OVX group had significantly impaired SAB performance in comparison
to the sham OVX group, an effect that was prevented by E
2
treatment (Figure 22).
Importantly, we observed that PPT but not DPN significantly improved SAB performance
with the same efficacy as E
2
(Figure 22). The number of arm entries in the Y-maze did
not differ significantly between groups [F (4,27) = 1.90; P = 0.14], suggesting that group
differences in SAB performance were not associated with differences in activity levels.
109

Figure 22. Assessment of working memory performance using the spontaneous alternation behavior (SAB)
in female 3xTg-AD mice. Data show mean (+SEM) percentages of alternations in a Y-maze assessed during
an eight-minute trial on the day of sacrifice. Both E 2 and PPT, but not DPN treatment rescued the OVX-
induced impairment in SAB compared to sham OVX [F (4,27) = 9.22, P < 0.0001]. * P < 0.05 compared to
sham OVX group, # P < 0.05 compared to OVX group.
Assays of hormone and SERM treatments
Uterine weight was measured at time of sacrifice as a bioassay of hormone
treatments. In comparison to the sham OVX group, uterine weights were significantly
decreased in the OVX group and significantly elevated in the OVX+E
2
group (sham
OVX, 72.1 ± 4.1 mg, OVX, 16.1 ± 1.2 mg, and OVX+E2, 121 ± 13.6 mg; F (3,22) = 57.2,
P < 0.001). Uterine weight was not significantly increased by 3 mo treatment of OVX
mice with PPT or DPN (30.7 ± 3.9 mg and 23.8 ± 2.0 mg, respectively). We performed a
110
dose-response study using a small subset of wild type mice (n = 2 mice/condition). PPT
treatment in OVX mice had a transient uterotrophic effect, increasing uterine weight to
~50% that of sham OVX levels after 2 wks and peaking at ~75% of sham OVX levels
after 2 mo (data not shown). Conversely, DPN treatment had no uterotrophic effects as
uterine weight remained at ~25% that of sham OVX after 1 wk, 2 wks, and 2 mo (data
not shown). To confirm neural efficacy of PPT and DPN treatments, sham OVX and
OVX wild type mice treated with vehicle, E
2
, PPT, or DPN were evaluated on the forced
swim test, an assay of anxiety-related behavior. We observed that E
2
, PPT, and DPN
treatments significantly reduced the increase in anxiety behavior from OVX (Figure 23).

Figure 23: PPT and DPN mimic the effect of E 2 in preventing the OVX-induced increase in immobility in the
forced swim test (FST). Hormone treatments performed in 3xTg-AD were replicated in wild-type female
C57Bl6/129S mice. In brief, wild-type mice at age 3 mo (N=6 per group) were bilaterally ovariectomized
(OVX) or sham OVX, then immediately treated with either placebo (Sham OVX, OVX groups), E 2 (OVX+E 2),
PPT (OVX+PPT), or DPN (OVX+DPN). Two weeks following hormone manipulations, animals were
evaluated for 5 minutes on the FST. Data show mean percentage of time spent immobile. The OVX group
exhibited significantly higher immobility. The OVX+E 2, OVX+PPT and OVX+DPN groups showed immobility
values that were significantly less than the OVX group but not significantly different from the Sham group [F
(4,22) = 2.96, P = 0.04]. * Denotes P < 0.05 compared to Sham-OVX, # P < 0.05 compared to OVX.
111
Discussion
In this study, we sought to investigate the efficacy of the SERMs PPT and DPN
in comparison to E
2
in regulating neuropathology in the 3xTg-AD mouse model of AD.
Consistent with our recent findings (Carroll et al., 2007b, Chapter Three), we observed
that E
2
treatment in OVX female 3xTg-AD mice significantly reduced both Aβ
accumulation and working memory deficits. We observed that PPT was effective in
lowering Aβ accumulation in most brain regions and attenuating impairments in working
memory, whereas DPN had only modest protective effects. Notably, at the utilized
doses, PPT exhibited only mild uterotrophic effects while DPN exhibited none.
These results are consistent with several studies demonstrating a protective role
of E
2
in regulating pathological processes relevant to AD. For example, most previous
studies in wild-type and transgenic rodents have demonstrated that OVX-induced
hormone depletion increases, and E
2
treatment reduces Aβ accumulation in brain
(Petanceska et al., 2000; Levin-Allerhand et al., 2002; Zheng et al., 2002; Carroll et al.,
2007b, Chapter Three). That E
2
does not significantly regulate Aβ in some studies
(Heikkinen et al., 2004; Green et al., 2005) may be explained by the recent observation
that brain levels of E
2
are critical to Aβ regulation and may not be fully depleted by OVX
(Yue et al., 2005). Our results demonstrate for the first time that Aβ accumulation in
brain can be effectively regulated not only by E
2
but also by SERMs, reinforcing the
therapeutic potential of estrogenic drugs in preventing AD-related neuropathology.
In comparing the relative efficacies of the SERMs PPT and DPN to E
2
, we found
brain region specific relationships in which PPT was generally effective and DPN
generally ineffective in reducing AD-like neuropathology. In hippocampus CA1 and
subiculum, PPT was equivalent with E
2
in terms of reducing OVX-induced Aβ
112
accumulation whereas DPN demonstrated no apparent benefit. In parallel, E
2
and PPT
but not DPN improved SAB performance, consistent with the hippocampal-dependent
nature of the SAB task. Interestingly, all three compounds were similarly effective in
reducing Aβ loads in the amygdala, but PPT and DPN were only partially effective
compared to E
2
in the frontal cortex. That the two SERMs showed discrete, region-
specific patterns of reducing Aβ loads suggests that E
2
may differentially regulate Aβ
accumulation through as yet undefined mechanisms that are dependent upon ERα and
ERβ. For example, since transcriptional studies have found that PPT is a preferential
ERα agonist (Stauffer et al., 2000), it is tempting to speculate that the relatively stronger
effects with PPT indicate a prominent ERα-dependent, genomic mechanism underlying
E
2
regulation of Aβ levels. However, this conclusion appears to be inconsistent with what
is known about ER distribution in the brain. Both human and rodent studies have
demonstrated a differential distribution of ERα and ERβ expression in brain regions
affected in AD, including hippocampus, frontal cortex, and amygdala. In situ
hybridization studies have demonstrated a higher density of ERβ-expressing cells than
ERα- expressing cells in hippocampus (Mehra et al., 2005) and layers 4-6 of cortex
(Shughrue et al., 1997; Mitra et al., 2003) and similarly high levels of ERβ- and ERα-
expressing cells in amygdala (Shughrue et al., 1997). Further complicating the issue is
that neither the mechanism(s) by which E
2
reduces Aβ levels nor the relative
contributions of ERα and ERβ have been clearly elucidated, although modulation of APP
processing may be involved. One possibility is that E
2
regulation of Aβ largely involves
classic non-genomic cell signaling rather than classic genomic pathways on which the
ER subtype specificity of PPT and DPN is largely based. However the ER-dependent
involvement in these signaling pathways remain unclear.
113
In addition to E
2
, the potential role of progesterone as a regulator of AD
neuropathology must also be considered. Both progesterone and E
2
are reduced as a
consequence of menopause in women and OVX in experimental paradigms. In our
paradigm, OVX-induced depletion of progesterone and E
2
resulted in increased Aβ
accumulation and diminished behavioral performance. However, E
2
appears to be the
most relevant hormone since E
2
treatment alone was effective in fully restoring Aβ load
and working memory performance seen in sham OVX mice. Also, we recently found that
progesterone treatment of OVX female 3xTg-AD mice neither reduced Aβ nor improved
working memory (Carroll et al., 2007b, Chapter Three). To the extent that such findings
extrapolate to human treatment where a progestin component is typically included in HT,
they support the continued development of neuroactive SERMs such as PPT.
Clinically, a primary concern regarding HT in postmenopausal women is the
increased risk of breast and uterine cancer associated with prolonged treatment with
estrogenic compounds. Therefore, development of SERMs that exert beneficial effects
on the brain, bone, and/or cardiovascular system but minimal effects on estrogen-
responsive, tumor susceptible tissue is of critical importance. Towards this end, recent
clinical trials have demonstrated that various SERMs offer protection against
cardiovascular disease and osteoporosis (reviewed in Cano et al., 2007; Gennari et al.,
2007) and perhaps AD (Yaffe et al., 2005).
By demonstrating effective attenuation of Aβ accumulation and behavioral
impairment by PPT in a mouse model of AD, our data confirm the potential of SERMs in
protecting against AD neuropathology and support the continued development and
investigation of neuroactive SERMs as an alternative strategy to HT in preventing and
perhaps treating age-related neurodegenerative disorders.
114
CHAPTER SIX
Perinatal sex steroid hormones alter adult development of Alzheimer’s disease-
like neuropathology in 3xTg-AD mice
Chapter Six Abstract
The risk of Alzheimer’s disease (AD) is higher in women than in men, a sex
difference that likely results from the effects of sex steroid hormones. To investigate this
relationship, we first compared progression of AD-like neuropathology in male and
female 3xTg-AD mice. We found that female 3xTg-AD mice exhibit greater β-amyloid
burden and larger behavioral deficits than males. Next, we evaluated how the
organizational effects of sex steroid hormones during postnatal development may affect
adult vulnerability to AD-like pathology. We observed that male 3xTg-AD mice feminized
during early development exhibit significantly increased β-amyloid accumulation in
adulthood. In contrast, female mice masculinized during early development exhibit a
more male-like pattern of β-amyloid pathology in adulthood. Taken together, these
results demonstrate significant sex differences in pathology in 3xTg-AD mice and
suggest that these differences may be mediated by organizational actions of sex steroid
hormone during development.
Introduction
Women may have a higher risk for the development of Alzheimer’s disease (AD)
than men. Epidemiological and observational studies have demonstrated a higher
prevalence (Rocca et al., 1986; Jorm et al., 1987; Bachman et al., 1992) and higher AD
incidence in women than men (Rocca et al., 1986; Andersen et al., 1999; Fratiglioni et
115
al., 2001; Ruitenberg et al., 2001). Further, women may experience a higher clinical
diagnosis for probable AD when compared to men with the same level of pathology
(Barnes et al., 2005). In addition, a comparison of >5000 AD autopsy cases
demonstrated that women suffered more senile plaque deposition than men (Corder et
al., 2004) and a higher proportion of neurons with pretangles (Swaab et al., 2001)
compared to men, although not all studies report this gender effect (Ganguli et al., 2000;
Ruitenberg et al., 2001). Thus, AD appears to affect men and women differently, with
women showing greater vulnerability to the disease.
The sex difference in AD risk may reflect differences between women and men in
the activational effects of sex steroid hormones estrogen and testosterone during
adulthood and the loss of these hormone effects during aging. In aging women, the
depletion of estrogen at menopause may promote AD pathogenesis as evidenced by
findings that estrogen-based hormone therapy in postmenopausal women is associated
with reduced risk of AD in some (Paganini-Hill and Henderson, 1996; Tang et al., 1996;
Kawas et al., 1997; Zandi et al., 2002) but not all studies (Rapp et al., 2003; Espeland et
al., 2004). Similarly, ovariectomy-induced hormone depletion in wild-type rodents
(Petanceska et al., 2000) and some transgenic mouse models of AD (Levin-Allerhand et
al., 2002; Zheng et al., 2002; Carroll et al., 2007b, Chapter Three) can increase levels of
the AD-related β-amyloid protein (Aβ), an effect that is attenuated by estrogen treatment.
In men, low testosterone, a consequence of normal male aging (Morley et al., 1997), is
linked with elevated levels of Aβ (Gillett et al., 2003) and increased AD risk (Hogervorst
et al., 2001; Moffat et al., 2004; Rosario, 2004). In male AD transgenic mice, castration-
induced testosterone depletion significantly accelerates the development of AD
pathology, which is prevented by androgen treatment (Rosario et al., 2006b). Taken
116
together, these studies suggest that the adult exposure of estrogens and androgens may
regulate the development of AD in women and men, respectively.
Another factor that may contribute to sex differences in AD risk is the
organizational effects of sex steroid hormones during early development. Differential
patterns estrogen and testosterone exposure during development induce numerous
structural differences between male and female brains (reviewed in Cosgrove et al.,
2007) that yield significant sex differences in many areas of brain function (reviewed
Cahill, 2006; Vagnerova et al., 2008). Importantly, men and women also exhibit different
vulnerabilities to several neurological disorders that occur prior to age-related hormone
depletion, including post-traumatic stress disorder, schizophrenia, multiple sclerosis,
autism, attention deficit disorder, Tourette’s syndrome, and anxiety-related disorders
(Cahill, 2006; Vagnerova et al., 2008). Thus, differences between male and female
brains established during early development may contribute to sex differences in
disease vulnerability in adulthood, specifically, women’s relatively greater risk of AD.
The higher risk of AD in women may involve sex differences in the activational
effects and or organizational effects of estrogen and testosterone. To investigate these
issues, we compared development of AD-like neuropathology in male and female 3xTg-
AD mice. Further, to evaluate the potential role of organizational effects of sex steroid
hormones, we investigated how AD-like pathology in adult 3xTg-AD mice was affected
by perinatal masculinizaiton of female pups and feminization of male.
117
Methods
Experimental design
Male and female 3xTg-AD mice (Oddo et al., 2003b) were maintained at the USC
Gerontology vivarium on 12h light on/off cycle and given ad libitum access to food and
water. Mice were handled in accordance with the NIH Health and Wellness of Animal
Subjects and IACUC procedures.
Experiment 1: To assess normal, age-related sex differences in AD-like
neuropathology in 3xTg-AD mice, male and female 3xTg-AD mice were divided into 6
groups according to sex and age (n=7 per group). Groups of both male and females
were sacrificed at 2-4 mo, 6-8 mo, and 12-14 mo of age.
Experiment 2: To assess the effects of perinatal hormone exposure on the
development of AD-like neuropathology, both male and female 3xTg-AD pups were
identified by sex at birth by measuring anogenital distance (pups with a distance from
the anterior edge of the anus to the base of the genital tubercle >1mm were classified as
male) (Hurd et al., 2008) and marked by toe clips. Males were divided into two groups
(n=7 per group) feminized males and control males. Males were feminized or
demasculinized by treatment with an androgen-receptor antagonist, flutamide (Flut), a
treatment that induces irreversible demasculization that persists in adulthood in both
reproductive behavior and function of the hypothalamo-pituitary-adrenal (HPA) axis
(Husmann and McPhaul, 1991; Isgor and Sengelaub, 1998; Dominguez-Salazar et al.,
2002; Anahara et al., 2004; Seale et al., 2005b). Flutamide (Sigma, St. Louis, MO) was
injected daily from postnatal day 1-20 at a dose of 50mg/kg/day (ip) while control males
were injected daily with vehicle (sesame oil, ip) (Sigma, St. Louis, MO). Such
feminization using flutamide treatment was based on previous protocols demonstrating
118
irreversible abnormalities in adulthood in both reproductive behavior and in the brain For
comparison, a group of 3 mo old C57Bl6/129S mice were depleted of endogenous
testosterone (T) by orchidectomized (ORX) at age 3 mo.
In parallel, on the day of birth, females were divided into masculinized and
control groups (n = 7 per group). Females were masculinized by treatment with
testosterone propionate (TP) (Steraloids, Newport, RI) at a dose of 100 µg /day (ip) from
postnatal day 1-7. This masculization protocol was based on pervious studies
demonstrating irreversible abnormalities in both behavior and in the brain (Iguchi and
Takasugi, 1981; Hutter and Gibson, 1988; Isgor and Sengelaub, 2003; Akhmadeev and
Kalimullina, 2005; Lansing and Lonstein, 2006; Meek et al., 2006). Control females were
injected (ip) daily with sesame oil vehicle. For comparison,we also included a group of
female C57Bl6/129S mice that were depleted of endogenous estrogens by removal of
ovariectomy (OVX) at age 3 mo. After postnatal hormone or vehicle treatment, both male
and female 3xTg-AD mice were then weaned at 3 weeks of age and housed with
littermates of the same-sex and treatment condition until sacrifice at age 7 mo.
For both Experiment 1 and 2, mice were deeply anesthetized (100 mg/kg
Nembutal) on day of sacrifice, transcardially perfused with PBS, and sacrificed by
decapitation. To confirm the efficacy of hormone treatments, blood was collected for
serum hormone analysis in females and seminal vesicles were dissected, blotted and
weighed in males. Estradiol (E
2
) serum levels were measured using radioimmunoassay
(RIA) as previously described (Slater et al., 2001). Brains were sagitally bisected then
immersion fixed for 48 h in 4% paraformaldehyde/0.1 M PBS and stored at 4°C in 0.1 M
PBS/0.2% sodium azide. Fixed brains were sectioned exhaustively into 40µm sections
using a vibratome.
119
Immunohistochemistry
Aβ accumulation was visualized by immunohistochemistry as previously
described (Carroll et al., 2007b, Chapter Three; Carroll and Pike, 2008, Chapter Five).
Briefly, every 8th section (~12 per brain) was stained for Aβ immunoreactivity (Aβ 1:300,
Invitrogen, Carlsbad, CA). Sections were stained using standard immunohistochemistry
techniques (Pike, 1999) according to the ABC method (Vector Laboratories, Burlingame,
CA) and developed using diaminobenzidine. Antigen unmasking consisting of a wash in
99% formic acid prior to application of primary antibodies was used to enhance Aβ
immunoreactivity.
Aβ immunoreactivity quantification
Levels of Aβ immunoreactivity were quantified using two approaches. First, all
stained sections were quantified for immunoreactive load or burden as previously
described (Carroll et al., 2007b, Chapter Three; Carroll and Pike, 2008, Chapter Five).
Briefly, high magnification fields from immunolabeled sections were collected and
digitized using a video capture system (B/W CCD camera coupled to an Olympus BX40
upright microscope) by an experimenter blinded to treatment groups. Immunoreactivity
was converted to positive and negative signal with a constant, predetermined threshold
using NIH Image software 1.61. The percentage of positive pixels detected is termed
immunoreactive load. Quantification was completed on 5 images per section on 12
sections for hippocampus and subiculum and 6 sections for frontal cortex. Second, for
some animals the numbers of extracellular plaque-like, Aβ-immunorreactive deposits
were quantified as previously described (Carroll et al., 2007b, Chapter Three). Every 8
th
section (~12 per brain) was counted for the number of Aβ deposits in the hippocampus,
120
subiculum, and entorhinal cortex. Aβ plaques were classified as diffuse or dense
congregations of immunoreactivity >2x the size of a neuron.
Spontaneous Alternation Behavior (SAB)
To measure working memory deficits, 3xTg-AD mice were tested for SAB in a Y-
maze < one week prior to sacrifice using a standard protocol (Carroll et al., 2007b
Chapter Three; Carroll and Pike, 2008, Chapter Five). SAB during an 8 minute test
period score was calculated as the proportion of alternations (an arm choice differing
from the previous two choices) to the total number of alternation opportunities (total arm
entries-2) (King and Arendash, 2002). For comparison, male and female WT
C57Bl6/129S mice were also tested at ages 2-4 mo, 6-8 mo and 12-14 mo.
Male reproductive behaviors
Male mice were tested on their sexual reproductive behavior using two
approaches, mounting and stimulus bedding preference. First, mounting behavior of
males when paired with a sexually receptive female was conducted as adapted from a
previously described procedure (Houtsmuller et al., 1994; Meek et al., 2006). At 7 mo of
age, mice were habituated to the testing room at 18:00 h at the beginning of “lights off.”
At 19:00 h, mice were introduced into a female cage containing a receptive female,
brought into estrus by treatment with injections of 35 µg (ip) of estradiol benzoate
(Steraloids) 48 h prior and 100 µg (ip) progesterone (Steraloids) 6 h prior to behavioral
testing (Nwagwu et al., 2005). Mice were evaluated on latency to mount the female and
number of attempted mounts during a 60 min testing period. Second, male mice were
also evaluated on their preference for female stimulus bedding as adapted from
preceding work (Kudwa et al., 2005). Mice were habituated to the testing room at 18:00
h at the beginning of “lights off.” At 19:00 h, mice were introduced into a small, glass
121
cage (8”x20”x10”) with three Petri dishes (10 cm) filled with approximately equal
amounts of either male-soiled bedding, female-soiled bedding, or neutral bedding. Male-
and female-soiled bedding was retrieved from cages housing sexually active mice not
involved in this study. Neutral bedding consisted of unused bedding squares (Ancare
Corp., Bellmore, NY). Male mice were placed in the testing chamber for 6 min and the
amount of time spent with male, female and neutral bedding was recorded. Preference
for female stimulus bedding was calculated as the proportion of the time spent with the
female bedding to the total time spent with any bedding.
Female reproductive behavior
Females were tested on their sexual reproductive behavior by evaluating
lordosis quotient as adapted from a previously described protocol (Meek et al., 2006).
Mice were habituated to the testing room at 18:00 h at the beginning of “lights off.” At
19:00 h, a sexually active male mouse was introduced into each of the females’ cage.
During each bout of mounting attempted by the male, the female lordosis position was
recorded, with lordosis being characterized as classic arching of the back (Meek et al.,
2006). Mice were observed for a maximum of 60 minutes or until 10 mounts were
attempted; the percentage of full lordosis behavior per mounting attempts (lordosis
quotient) was calculated.
Statistical Analysis
Raw data were statistically evaluated by ANOVA followed by between group
comparisons using the Fisher LSD test.
122
Results
Experiment 1: Comparison of pathology development in male and female 3xTg-AD mice
To assess normal, age-related sex differences in AD-like neuropathology in
3xTg-AD mice, both male and females were sacrificed at the following ages: 2-4 mo, 6-8
mo, and 12-14 mo. The average age of males and females at each time point were not
statistically different within age groups [average age (wks ± standard error)]: female 2-4
mo = 13.0 ± 0.0; male 2-4 mo = 13.4 ± 0.2 (p = 0.78); female 6-8 mo = 28.1 ± 1.0; male
6-8 mo = 29.5 ± 1.6 (p = 0.37); female 12-14 mo = 54.7 ± 1.4; male 12-14 mo = 57.7 ±
1.0) (p = 0.06).
Female 3xTg-AD mice display higher levels of Aβ immunoreactivity than males
To assess the development of Aβ pathology in male and female 3xTg-AD mice,
we measured the level of Aβ accumulation by quantifying Aβ immunoreactive load in
three brain regions; CA1 of hippocampus, subiculum, and frontal cortex. Both male and
female 3xTg-AD mice exhibit an age-related increase in Aβ load in all three regions.
However, we observed that female 3xTg-AD mice exhibited higher Aβ load compared to
males (Figure 24), a sex difference that was significant at the 12-14 mo age group in
both subiculum (Figure 24A-C) and hippocampus CA1 (Figure 24D-F). The sex
difference in Aβ load was most striking in the frontal cortex where it was significantly
higher in females in both the 6-8 mo and 12-14 mo ages (Figure 24G-I).
As another measure of Aβ pathology, we also compared the development of
extracellular Aβ deposition into plaque-like structures in males and females. The
number of extracellular Aβ plaques was totaled from the subiculum, hippocampus CA1,
and entorhinal cortex in both females and males. At 12-14 months of age when
123
extracellular Aβ plaques became obviously apparent, female 3xTg-AD mice exhibited
significantly higher plaques compared to age-matched males (Figure 24J-L).

Figure 24. Female 3xTg-AD mice demonstrate higher levels of Aβ immunohistochemistry than males,
particulary at 12-14 mo of age. Representative pictomicrographs from the 12-14 mo group of female and
male 3xTg-AD mice display Aβ immunoreactivity visualized in the subiculum (A,B), CA1 of hippocampus
(D,E), and frontal cortex (G,H). Scale bar = 100µm. Significant sex differences were observed in the
subiculum (F = 23.72, p < 0.0001) and the CA1 (F = 5.88, p = 0.0005), the most pronounced in the frontal
cortex (F = 9.76, p <0.0001). In particular, at 12-14 mo of age, female 3xTg-AD mice displayed a higher Aβ
immunohistochemistry load than male mice. These effects were quantified which revealed that female mice
suffered significantly higher Aβ immunohistochemistry load than male mice in the 12-14 mo age group in the
subiculum (C), CA1 of hippocampus (F), and frontal cortex (I). Further, Aβ plaques were counted and a
significant sex difference was again observed (F = 9.25, p < 0.0001). Female 3xTg-AD mice suffer higher
number of Aβ plaques at 12-14 mo of age (L), as visualized by representative pictomicrographs from female
(J) and male (K) 12-14 mo age group. Scale bar = 250µm. Data are represented as mean values ± SEM. * p
< 0.01 from opposite sex of same age group.
124
Females display worse working memory performance than males
To measure behavioral changes in male and female 3xTg-AD mice, we assessed
performance on spontaneous alternation behavior (SAB), a hippocampal-dependent task
of working memory. We observed an age-related decline in SAB performance in both
sexes. Notably, we observed poorer performance on SAB in females compared to
males, which was statistically significant in the 12-14 mo age group (Figure 25B). To
control for possible group differences in activity level, we measured the number of arm
entries and found they were not significantly different across groups (F = 0.79, p = 0.56)
(Figure 25C). Further, to confirm that these SAB changes correlate with changes in AD-
like pathology rather than simply increasing age, WT C57Bl6/129S mice in matching age
groups were also tested and no change was observed with increasing age (F = 1.6, p =
0.19) (Figure 25A).

Figure 25. Females perform more poorly on hippocampal-dependent working memory task (SAB) than male
3xTg-AD mice, particularly at 12-14 mo of age. To evaluate age-related working memory in both male and
female 3xTg-AD mice, the SAB test was used. We observed an age-related decline in performance in both
3xTg-AD sexes, however, a significant sex difference was observed (F = 6.58, p = 0.0001). 3xTg-AD
females suffered a faster acceleration in working memory decline than males, performing significantly worse
at age 12-14 mo (B). As a control measure of activity level, the number of arm entries was measured and
was not significantly different across groups (F = 0.79, p = 0.56) (C). Further, as a control measure for
normal, age-related decline in memory performance, WT C5Bl6 mice displayed no change across all groups
(F = 1.6, p = 0.19) (A). Data are represented as mean values ± SEM. * p < 0.05 from opposite sex of same
age group.
125
Experiment 2: Effects of perinatal hormone manipulations on adult pathology
To assess the effects of perinatal hormone exposure on the development of AD-
like neuropathology in adult male and female 3xTg-AD mice, male pups were feminized
by treatment with the androgen receptor antagonist flutamide (Flut) during postnatal
days 1-21 and female pups were masculinized by treatment with testosterone propionate
(TP) during postnatal days 1-7. Hormone-treated animals as well as vehicle-treated and
gonadectomized male and female control groups were sacrificed at 7 mo of age.
To confirm the masculinizing effect of TP treatment in females, female mice were
assessed on their reproductive behavior and serum E
2
levels. TP-treated females
exhibited significantly lower lordosis quotients than vehicle-treated females (Table 3),
demonstrating that they were effectively masculinized. Control OVX mice showed
similarly low lordosis quotients. However, OVX mice are largely depleted of endogenous
E
2
whereas the TP-treated females show intermediate levels of serum E
2
that are not
significantly lower than vehicle-treated females (p = 0.11) (Table 3).
Table 3: Confirmation of TP treatment in female mice
Condition Lordosis
quotient
E
2
(pg/ml)
Female veh 59.6 ± 17.4 55.5 ± 14.2
OVX 10.0 ± 4.1* 9.9 ± 2.2*
Female + TP 8.6 ± 5.7* 27.4 ± 5.2
Table 3: Confirmation of TP treatment in female mice. To confirm efficacy of TP treatment, female mice
were assessed on their reproductive capacity by measuring lordosis quotient and E 2 levels. Significant
differences in lordosis quotient were observed (F = 8.49, p = 0.005) as both OVX and TP-treated females
displayed significantly lower lordosis quotients than vehicle-treated females. Significant differences were
also observed in serum E 2 levels (F = 5.07, p = 0.034) however only OVX females had lower levels
compared to vehicle-treated females. Data are represented as mean values ± SEM. * p < 0.05 relative to
vehicle-treated group.
126
To confirm efficacy of flutamide treatment in feminizing males, seminal vesicle
weights were measured and mice were assessed on reproductive capacity by measuring
both mounting behavior and bedding preference. Similar to control androgen depleted
ORX mice, flutamide-treated males attempted to mount a female significantly fewer
times and spent less time with female bedding compared to vehicle-treated males.
However, unlike ORX males, flutamide-treated males did not have significantly lower
seminal vesicle weight (p = 0.16) (Table 4).
Table 4: Confirmation of flutamide treatment in male mice
Condition Number of
mounts
% Time with
female bedding
Seminal vesicle
weight (mg)
Male veh 10.8 ± 1.5 63.4 ± 4.8 102.5 ± 10.7
ORX 0.0 ± 0.0* 30.5 ± 1.7* 12.8 ± 1.7*
Male + Flut 3.1 ± 1.0* 42.4 ± 4.5* 85.4 ± 7.9
*<0.05 relative to male veh
Table 4: Confirmation of flutamide treatment in male mice. To confirm efficacy of flutamide treatment in
males, seminal vesicle weights were measured and mice were also assessed on reproductive capacity by
measuring both a) number of attempted mounts on a female and b) the % of time spent in proximity to
female bedding. Significant differences in mounting (F = 19.54, p = 0.0001) and time with female bedding (F
= 11.39, p = 0.001) were observed. Both ORX males and flutamide-treated males displayed significantly
lower number of mounts and time spent with female bedding compared to vehicle-treated males. Further,
significant differences were observed in seminal vesicle weight (F = 27.7, p < 0.0001). Unlike ORX mice,
flutamide-treated mice did not have significantly lower seminal vesicles compared to vehicle-treated mice.
Data are represented as mean values ± SEM. * p < 0.05 relative to vehicle-treated group.
Perinatal hormone manipulations alter Aβ accumulation in adulthood
Several months following the perinatal hormone treatments, the TP-
treated female mice and Flut-treated male mice were compared with each other and
vehicle-treated control mice for levels of Aβ accumulation. At age 7 mo, Aβ
immunoreactivity was quantified in the subiculum, CA1, and frontal cortex (Figure 26).
127
Flut treatment of male 3xTg-AD mice was associated with significant increases in Aβ
load in hippocampus CA1 and subiculum but did not alter Aβ levels in frontal cortex. The
effects of TP treatment in female 3xTg-AD mice on Aβ levels varied by brain region,
showing a significant reduction in frontal cortex, no effect in subiculum, and a significant
increase in hippocampus CA1.
Figure 26: Perinatal flutamide treatment feminizes male 3xTg-AD adult brains and TP treatment
masculinizes female brains in terms of AD-like neuropathology development in a region-specific manner.
Perinatal male and female 3xTg-AD mice were treated with flutamide (Flut) and TP respectively in order to
determine the effects of developmental hormone exposure on patterns of AD-like pathology development in
adult 3xTg-AD mice. Representative pictomicrographs display Aβ immunoreactivity visualized in the
subiculum (A-D), CA1 of hippocampus (F-I), and frontal cortex (K-N). Scale bar = 100µm. Significant
treatment differences were observed in quantification of Aβ immunohistochemistry load in the subiculum (E)
(F = 2.82, p = 0.05), the CA1 (J) (F = 17.11, p < 0.0001) and the frontal cortex (O) (F = 13.01, p < 0.0001).
TP treatment to females significantly altered Aβ load in the CA1 and frontal cortex while flutamide treatment
to males significantly altered Aβ load in the CA1 and subiculum. Data are represented as mean values ±
SEM. * p < 0.05 from opposite sex of same age group.
Perinatal hormone manipulations affect behavioral performance in adulthood
We also evaluated the effects of perinatal hormone manipualtions on SAB
performance. Compared to vehicle-treated male 3xTg-AD mice, Flut-treated male 3xTg-
AD performed significantly poorer on the SAB Y task (Figure 27A). In contrast, TP-
128
treated female 3xTg-AD did not perform significantly different from vehicle control
females. To confirm that SAB performance differences did not reflect changes in activity
level, the numbers of arm entries across groups was measured and was not found to be
statistical insignificant (F = 1.23, p = 0.30) (Figure 27B).

Figure 27: Perinatal flutamide treatment, but not TP treatment alters hippocampal-dependent working
memory on the Y-maze. Perinatally-hormone treated mice were also assessed for working memory
performance on the Y-maze and exhibited a significant overall treatment effect (F = 6.37, p = 0.003).
Flutamide treatment in males significantly worsened working memory while TP treatment in females had no
effect (A). The number of arm entries was not significantly different across groups (B). Data are represented
as mean values ± SEM. * p < 0.05 from opposite sex of same age group.
Discussion
In this study, we investigated sex differences in the progression of AD-like
neuropathology in male and female 3xTg-AD mice and how this relationship is affected
by alterations in sex steroid hormones during a critical perinatal period of neural
development and sexual differentiation. Consistent with our previous findings (Rosario et
al., 2006b; Carroll et al., 2007b, Chapter Three), we observed that Aβ accumulation and
deficits in working memory increase with age in adult male and female 3xTg-AD mice.
These observations are consistent with the original characterization of the 3xTg-AD
129
mouse model (Oddo et al., 2003a), which reported progressive accumulation of
intraneuronal Aβ in CA1 hippocampus, subiculum, entorhinal cortex, and amygdala
followed by deposition of extracellular Aβ by age 12 mo. This progressive accumulation
of Aβ has been associated with corresponding cognitive impairments (Oddo et al.,
2003a), which is also consistent with our present observations.
Importantly, we also observed sex differences with females demonstrating higher
levels of Aβ accumulation and a more robust deficit in working memory in comparison to
age-matched males. Interestingly, the most pronounced sex difference was observed in
the frontal cortex, where females displayed significantly higher levels of Aβ accumulation
by age 6-8 mo. These observed sex differences in 3xTg-AD mice are consistent with
findings from other studies. In the Tg2576 (Callahan et al., 2001) and APP/PS1 (Wang
et al., 2003) transgenic mouse models of AD, females are also reported to show higher
levels of Aβ deposition than males. Further, our results are consistent with a recent
report in the 3xTg-AD mouse that demonstrated higher plaque load burden in females
(Hirata-Fukae et al., 2008). When considered together, these data demonstrate a
common pattern of greater AD-like pathology in female mice exposed to the same
genetic factors, suggesting an inherent sex difference in vulnerability to AD
pathogenesis.
The increased pathology in AD transgenic mice is consistent with reports in
humans of greater neuropathological AD changes in women compared to age-matched
men (Swaab et al., 2001; Corder et al., 2004). Such sex differences are generally
thought to reflect activational, neuroprotective effects of sex steroid hormones in the
adult, which are diminished more abruptly and completely in women as a consequence
of menopause. In fact, our prior observations in the 3xTg-AD mice demonstrate a
130
significant role of activational effects of sex steroid hormones in adult male (Rosario et
al., 2006b) and female (Carroll et al., 2007b, Chapter Three; Carroll and Pike, 2008,
Chapter Five) mice as gonadectomy-induced loss of hormones worsens pathology in a
manner that is prevented by sex steroid hormone replacement. However, our current
data suggest that adult activational effects of hormones alone are unlikely to explain the
observed sex differences. Unlike women, female rodents do not experience menopause
and the associated depletion of estrogens and progesterone. Aged mice undergo
reproductive senescence that is characterized in part by persistent vaginal cornification
and serum E
2
levels that may be altered at the preovulatory surge between ages 10-12
mo, but are indistinguishable from basal levels of young, cyclic mice when measured at
approximately age 14 mo (Nelson et al., 1981). Middle-aged mice can show alterations
in E
2
-mediated activational effects that are linked to reproductive senescence (Alkayed
et al., 2000; Jezierski and Sohrabji, 2001; Nordell et al., 2003), however these changes
do not typically manifest until 14-16 mo of age, which is later than the female mice used
in this study. Furthermore, our data demonstrate significant sex differences in the frontal
cortex as early as 6-8 mo of age. Therefore, it is reasonable to hypothesize that although
adult activational effects of sex steroid hormones are significant regulators of AD-like
pathology in 3xTg-AD mice, other hormone effects also have significant roles.
One contributing factor for the observed sex differences in AD-like pathology in
3xTg-AD mice may be the organizational effects of sex steroid hormones in the
developing brain. The brain is particularly sensitive to hormones during critical
developmental periods (Slob et al., 1980; Weisz and Ward, 1980; Krohmer and Baum,
1989)when even small hormone alterations can permanently alter brain structure and
function (reviewed in Gore, 2008). Critical periods include late embryonic through the
131
first 10 days of life, a stage targeted in this study with flutamide and TP treatments.
Elevated testosterone and estrogen levels during this critical period affect structural
organization of several brain regions, particularly hypothalamic and structures (reviewed
in Morris et al., 2004; Gore, 2008; McCarthy et al., 2008). Experimental manipulations
that affect the normal developmental hormone levels results in neural changes that
persist in the adult brain (Houtsmuller et al., 1994; Becu-Villalobos et al., 1997;
Breedlove, 1997; Isgor and Sengelaub, 1998; McCormick et al., 1998; McCormick and
Mahoney, 1999; Dominguez-Salazar et al., 2002; Miyata et al., 2003; Yang et al., 2004;
Anderson et al., 2005; Seale et al., 2005a; Bakker and Baum, 2008). Our data show
that disruption of androgen signaling in perinatal male 3xTg-AD mice by administration
of the androgen receptor antagonist flutamide resulted in elevated Aβ levels in CA1
hippocampus and subiculum. This increased Aβ burden is a more female-like pattern of
Aβ accumulation and suggests that neonatal feminization of male 3xTg-AD mice altered
their adult vulnerability to AD-like pathology. In parallel, feminized male 3xTg-AD mice
exhibited significantly poorer working memory that was similar to age-matched female
3xTg-AD mice. Female 3xTg-AD mice pups that were masculinized by perinatal
testosterone exposure showed significantly lower Aβ levels in frontal cortex, which is a
more male-like pattern. Thus, our findings provide novel evidence that developmental
sexual differentiation of the brain significantly affects subsequent progression of AD-like
neuropathology.
Interestingly, organizational actions of sex steroid hormones did not uniformly
affect development of pathology. Feminization of male 3xTg-AD mice did not alter Aβ
levels in frontal cortex. Similarly, masculinization of female 3xTg-AD mice did not affect
Aβ levels in subiculum and actually elevated them in hippocampus CA1. These
132
seemingly inconsistent observations are likely related in part to the various, and as yet
incompletely defined, mechanisms by which neonatal hormones induce their effects. Sex
steroid hormones regulate apoptosis, cell proliferation, and developmental cell migration
in the neonatal brain in a manner that is at least partially mediated by aromatase action
(Morris et al., 2004; Gore, 2008; McCarthy et al., 2008), which converts testosterone to
E
2
. However, aromatase expression can vary across brain regions (Negri-Cesi et al.,
1996), with cortical neurons showing lower expression than hypothalamic neurons, for
example. Thus, manipulations of sex steroid hormones during the critical neonatal period
would be expected to differentially affect brain regions. Also, it is possible if not likely that
the observed sex differences in pathology of 3xTg-AD mice reflect influences beyond the
organizational and activational effects of sex steroid hormones. For example, recent
evidence shows that sexual differentiation of neonatal rodent brain is significantly
affected by Mullerian inhibitory substance independently of sex steroid hormones (Wang
et al., 2009). We suggest that the organizational effects of sex steroids and related
developmental factors including Mullerian inhibitory substance combine to significantly
affect neural development in a manner that makes the female brain more vulnerable to
AD pathogenesis than the male brain.
The concept that the male and female brains exhibit an inherent difference in
vulnerability to AD is consistent with the literature on sexual dimorphisms in brain
regions related to AD. For example, imaging studies have shown higher hippocampal
volumes in females compared to males (Goldstein et al., 2001) and sexually dimorphic
differences in hippocampal cholinergic function (Madeira and Lieberman, 1995). In the
amygdala, volumes have been shown to be greater in males than in females (Goldstein
et al., 2001) and a sexually dimorphic function has been demonstrated in the
133
consolidation of emotional memories (Cahill et al., 2001; Canli et al., 2002; Cahill, 2003;
Cooke and Woolley, 2005; Hamann, 2005). In the prefrontal cortex, there are many
reports of sex differences in memory performance (Speck et al., 2000; Duff and
Hampson, 2001; Shansky et al., 2004). In addition to sex differences in neural structure
and function, abundant evidence indicates that differential vulnerability of men and
women to neurological diseases that develop prior to age-related depletion of sex steroid
hormones (Cahill, 2006; Vagnerova et al., 2008). One example is schizophrenia (Arato
et al., 2004; Mendrek, 2007), which is characterized by an increased risk with
developmental feminization (Lutchmaya et al., 2004) and protective effects in adulthood
by estrogen (Bergemann et al., 2005) and possibly testosterone (Akhondzadeh et al.,
2006). Therefore, it is reasonable to hypothesize that the organizational and activational
effects of sex steroid hormones contribute not only to sexual dimorphisms in the brain’s
structure and function but also its vulnerability to diseases including AD.These results
suggest important implications for the powerful influences of both organizational and
activational hormone effects contributing to the differential vulnerability to adult disease.
That Alzheimer’s disease might be an age-related neurological disorder
influenced by developmental hormone exposure fits with another recently emerging
hypothesis (reviewed in Barker, 2003). His studies focus on the critical period of human
development (from conception to 2 years of age) during which birth weight, nutrition and
weight gain predict adult development of coronary heart disease and related disorders
(Eriksson et al., 2006; Barker, 2008; Barker et al., 2008). Therefore, using the example
of maternal under-nutrition predisposing the fetus to metabolic disorders later in life, he
first described this phenomenon of the developmental origin of adult disease. And it has
proven to be an important concept for the emerging link between developmental
134
neuroendocrinology and adult disease. While the mechanisms by which developmental
hormone exposure influence adult disease pathogenesis are currently elusive, perhaps
there is a link between the contributing mechanisms behind cardiovascular disease and
Alzheimer’s disease. Future studies are needed to investigate the potential relationship
between perinatal sex steroid hormone exposure and brain morphology and
neuroendocrine function in adult neurodegenerative diseases pathogenesis.
135
CHAPTER SEVEN
Conclusions and future directions
This thesis focused on the neuroprotective effects of estrogen and progesterone
and the efficacy of these hormones in decreasing Alzheimer’s-like neuropathology in the
female brain. These studies were done in the context of the most commonly prescribed
regimen of hormone therapy on the market today for post-menopausal women which
consists of a preparation of conjugated equine estrogen (CEE) plus a synthetic progestin
(MPA) given at a constant, oral dose. Further, these studies were done in the context of
the Women’s Health Initiative Memory Study (WHIMS) which actually demonstrated that
this formula of CEE+MPA was unable to reduce the risk of AD in and incidence of
cognitive decline in post-menopausal women (Rapp et al., 2003; Shumaker et al., 2003).
Within this context, several questions have been raised about HT such as the use of a
progestin component, the constant vs. cyclic nature of hormone exposure, and the
timing of HT initiation. Therefore, the goal of this thesis work was to investigate these
issues regarding HT and its efficacy in reducing AD-like neuropathology in the female
brain.
Summary of experimental observations
Several important observations regarding progesterone’s attenuating effects
have resulted from the studies described in this thesis work. The results from both
Chapters Two and Three demonstrate that while estrogen offers neuroprotective effects,
that progesterone counteracts some of these effects when administered in a constant
exposure similar to the way in which a progestin is administered in the current forms of
HT. In Chapter Two showed constant progesterone treatment blocks estrogen
136
neuroprotection in middle-aged female rat brain after kainate lesion. And Chapter Three
revealed that constant progesterone blocked estrogen-induced reduction in AD-like
neuropathology in female 3xTg-AD mice. Taken together, these two chapters suggest
that progesterone may be counteracting estrogen’s beneficial effects in the brain,
shedding a discouraging light on the future of using HT to protect post-menopausal
women from neurodegenerative diseases.
However, the results from both Chapter Four and Five offer alternate strategies
to the current available forms of HT used today. Specifically, Chapter Four revealed that
compared to a constant progesterone dose, a cyclic dose of progestin offers a more
beneficial effect on estrogen neuroprotection from AD-like pathology in female 3xTg-AD
mice. Further, Chapter Five revealed that a synthetic type of estrogen such as the
SERM named PPT, which also reduced AD-like neuropathology in female 3xTg-AD
brains and therefore was an alternate strategy that would eliminate the use of a
progestin component. It is interesting to hypothesize that these strategies may be among
those that may replace the current regimen of HT.
In addition, the results from these studies also demonstrated that timing of
hormone initiation might be a key factor in the efficacy of HT’s neuroprotective effects.
Chapter Two demonstrated that the addition of HT to middle-aged rodents at the
beginning of reproductive senescence remains neuroprotective. This result lends
credence to the hypothesis that a window of opportunity exists during the early stages of
menopause at which HT is still efficacious. Perhaps only in later ages, when the brain
has altered hormone responsivity will HT loose its neuroprotective effect. In addition to
the studies presented here describing the effects of hormones in the middle-aged brain,
this thesis also describes how hormone actions on the early, developing brain can also
137
alter the adult brain. The results from Chapter Six describe how early life actions of
estrogen and androgens in the developing brain alter vulnerability to neurodegenerative
diseases in the adult brain. Taken together, the results from all Chapters described
above lend various insights into the current and future preparations of HT given to post-
menopausal women. However, at a more basic level, these results reveal important
information regarding the action of estrogen and progesterone on neural function.
Hormone actions in the developing brain
At the basic level, we have begun to investigate the organizational effects of sex
steroid hormones in the developing brain beyond the activational effects of E
2
and P4 on
the adult brain. As discussed in Chapter Six, sex steroid hormones have profound and
permanent effects on the developing brain and an appealing hypothesis is that these
effects may alter the vulnerability to disease in adulthood (reviewed in Gore, 2008). It
has long been understood that during gestation as well as this period, estrogen is
critically involved in the reproductive development of the brain (reviewed in Gore, 2008;
McCarthy et al., 2008) by altering the hypothalamic-limbic neural networks through
estrogen-dependent pathways (discussed in Morris et al., 2004). In particular, the
anterovental periventricular nucleus (AVPV) and the sexually-dimorphic nucleus of the
preoptic area (SDN-POA), and are well known to become strongly and permanently
sexually dimorphic as a direct result of sex hormone exposure during the critical period.
In parallel, many previous studies demonstrate the effects of perinatal hormone
manipulations on the adult brain (Houtsmuller et al., 1994; Becu-Villalobos et al., 1997;
Breedlove, 1997; Isgor and Sengelaub, 1998; McCormick et al., 1998; McCormick and
Mahoney, 1999; Dominguez-Salazar et al., 2002; Miyata et al., 2003; Yang et al., 2004;
Anderson et al., 2005; Seale et al., 2005b; Bakker and Baum, 2008). Based on this
138
previous work, it is reasonable to hypothesize that male and female sex steroid
hormones could also have organizational influences over brain regions, perhaps related
to Alzheimer’s disease such as the hippocampus.
In Chapter Six, we demonstrated that perinatal TP and flutamide exposure
permanently altered Aβ accumulation, working memory performance and tau
hyperphosporylation in female 3xTg-AD mice. Most crucially, these hormone treatments
did not alter activational changes in hormone levels. Therefore, it is reasonable to
assume that the observed effects of perinatal hormone exposure were due to permanent
organizational changes in the adult brain rather than transient, activational changes of
adult circulating hormones. Based on this data and the example of schizophrenia,
(discussed in Chapter Six and reviewed in Mendrek, 2007) it is reasonable to
hypothesize that estrogen and testosterone may be able to have organizational or
activational effects on other diseases such as Alzheimer’s. These results suggest
important implications for the powerful effects of perinatal hormone exposure on both the
developing and adult brain.
However, as the results described in Chapter Six are novel, they are just a first
glance into understanding the effects of perinatal hormone exposure on the developing
brain. Therefore, the data are difficult to interpret. That TP and flutamide had differential
effects in different brain regions is curious but not suprising given the differential
expression of estrogen and testosterone receptors in these regions, as discussed in
Chapter One. In addition, the expression of aromatase may play a major role in the
function of both E
2
and T in these brain regions and it is unknown how perinatal hormone
manipulations effect aromatase expression both in the short or long term. Finally, as
discussed above (Gore, 2008), in the perinatal brain there is a very precise critical
139
window during which hormone exposure has long-lasting effects. Our hormone exposure
was aimed at just one of those particular times and it remains possible that either TP or
flutamide exposure during a slightly different time point during perinatal development
would have considerably different effects. Considered together, it is important to
evaluate our results regarding perinatal hormone exposure as only one clue towards
understanding the organizational effects of sex steroid hormones on the brain.
Another recently emerging hypothesis regarding the developmental origins of
adult disease has been put forth by Barker et al. (reviewed in Barker, 2003). Barker uses
the example of maternal under-nutrition predisposing the fetus to metabolic disorders
such as cardiovascular disease later in life. This could be an important link between
developmental neuroendocrinology and adult disease. For example, perhaps there is a
link between the contributing mechanisms behind cardiovascular disease and
Alzheimer’s disease. Future studies are needed to investigate the potential relationship
between perinatal sex steroid hormone exposure and brain morphology and
neuroendocrine function in adult neurodegenerative diseases pathogenesis.
Mechanisms of both estrogen and progesterone action
While the results presented here describe many actions of both E
2
and P4, the
studies described lack investigation into the mechanisms by which these hormones exert
their effects. However, the mechanisms by which E
2
and P4 induce neuroprotective
effects are currently unknown. Yet many different mechanisms have been proposed for
a wide variety of both E
2
and P4 effects in the brain as argued in the Introduction
Chapter and reviewed in (McEwen, 1991; Moss and Gu, 1999; Keefe, 2002;
Chakraborty and Gore, 2004; Melcangi and Panzica, 2006; Brinton et al., 2008;
Schumacher et al., 2008).
140
In terms of cognition, estrogenic effects are mediated by several mechanisms,
with the most significant actions involving synaptic plasticity (reviewed in Foy et al.,
2004). For example, estradiol increases the number of dendritic spines and spine
synapses (Woolley and McEwen, 1994), synaptic excitability (Wong and Moss, 1992),
enhances long-term potentiation (Bi et al., 2000), cholinergic function (Gibbs, 2000b),
and neurogenesis (Galea et al., 2006).
Neuroprotection mechanisms
In terms of neuroprotection, the mechanisms by which female sex steroid
hormones promote their effects is very broad, as discussed in Chapter One. In some
paradigms estradiol neuroprotection appears to be mediated by non-classical steroid
hormone mechanisms involving rapid induction of cell signaling pathways including
protein kinase C, extracellular signal regulated kinase, and phosphoinositol-3-kinase.
Through both rapid signaling pathways and classic genomic signaling, estradiol can also
increase neuron viability by altering expression of proteins involved in regulation of cell
death including pro- and anti-apoptotic members of the Bcl-2 family (Green and
Simpkins, 2000). Also similar to estradiol, progesterone induces several neuroprotective
cell signaling pathways, including activation of Akt (Singh, 2001) and ERK (Nilsen and
Brinton, 2002, 2003) and upregulation of anti-apoptotic protein Bcl-2 (Nilsen and Brinton,
2002).
Furthermore, similar to findings in cell culture studies, protective effects of
progesterone in vivo are mimicked by only some progestins, findings that may provide
mechanistic insight (Singh, 2007). For example, the protective effects of progesterone,
certain progesterone metabolites, and some progestins against neural injury induced by
141
pilocarpine, kainate, and other seizure-inducing toxins likely reflects anxiolytic effects
resulting from modulation of the GABA
A
receptor.
Aβ and tau pathology
How estrogen activates regulates APP processing is not clear, although multiple
pathways have been implicated. First, most data suggest that estrogen increases the α-
secretase pathway of APP processing (Jaffe et al., 1994; Xu et al., 1998; Manthey et al.,
2001; Zhang et al., 2005). There is evidence that estrogen can promote the α-secretase
pathway via activation of extracellular-regulated kinase 1 & 2 (ERK1 & ERK2) signaling
(Manthey et al., 2001), a well-established estrogen signaling pathway (Watters et al.,
1997; Singh et al., 1999; Toran-Allerand et al., 1999). The action of estrogen on the ERK
components of mitogen-activated protein kinase (MAPK) signaling pathway and APP
generation are rapid and may be ER independent (Manthey et al., 2001). Estrogen may
also regulate APP processing through protein kinase C (PKC)-dependent pathways.
PKC signaling is a strong activator of non-amyloidogenic APP processing (Koo, 1997;
Coughlan and Breen, 2000; Olariu et al., 2005). Further, estrogen is a significant
activator of PKC in both neuron culture (Cordey et al., 2003; Cordey and Pike, 2006) and
in brain (Ansonoff and Etgen, 1998; Qiu et al., 2003; Sétáló et al., 2005). Consistent with
this possibility, a recent cell culture study has shown that estrogen activation of α-
secretase APP processing is blocked by PKC inhibitors (Zhang et al., 2005). However,
not all studies demonstrate such straightforward results; such as a previous study in vitro
demonstrating that E
2
is capable of increasing the production of sAPPα but not reducing
the release of Aβ in cortical neurons over-expressing APP
swe
(Vincent and Smith, 2000).
Some evidence also suggests that estrogen may promote non-amyloidogenic
APP processing by altering APP trafficking. Specifically, (Greenfield et al., 2002)
142
reported that estrogen promoted the secretion of APP containing vesicles from the
primary site of amyloidogenic APP processing, the trans golgi nucleus, thereby
decreasing available APP substrate for Aβ formation (Greenfield et al., 2002). In addition
to promoting the non-amyloidogenic APP processing pathway, estrogen has also been
implicated in the modulation of APP levels through the regulation of alternative splicing
(Thakur and Mani, 2005) and APP over-expression post-injury (Shi et al., 1998), thereby
altering substrate for APP processing and subsequent Aβ production.
In addition to modulating the production of Aβ, estrogen may also promote Aβ
clearance. One mechanism of Aβ clearance is through microglial degradation (Rogers et
al., 2002). Estrogen has been shown to promote microglial phagocytosis (Pow et al.,
1989; Bruce-Keller et al., 2000) and E
2
treatment increases microglial internalization of
Aβ in microglia of both murine (Harris-White et al., 2001) and human origin (Li et al.,
2000). Estrogen treatment has also been found to reduce Aβ accumulation in rats
following intracerebroventricular Aβ injection (Harris-White et al., 2001).
Correspondingly, increased Aβ burden and impaired microglial Aβ clearance has been
reported in an estrogen deficient transgenic mouse model of AD (Yue et al., 2005).
Estrogen has also been implicated in the regulation of levels of two major Aβ degrading
enzymes, insulin degrading enzyme and neprilysin. Both of these enzymes are
significant regulators of Aβ levels and their regulation and activities are implicated in AD
pathogenesis (Tanzi et al., 2004; Vardy et al., 2005). A few recent studies dosuggest
that estrogen depletion by OVX can decrease neprilysin activity in female rat brain, an
effect reversed by E
2
replacement (Huang et al., 2004). Thus, estrogens may influence
Aβ clearance through regulation of neprilysin, although this pathway may involve an
androgen responsive element on the neprilysin gene (Xiao et al., 2009). Given the
143
significance of Aβ accumulation to AD pathogenesis, future studies must clearly define
the role of estrogen in regulating both Aβ production and clearance pathways and how
they are affected by differences in E
2
brain levels.
Furthermore, in terms of tau phosphorylation, as discussed in Chapters One and
Three, E
2
and P4 can modulate activities of kinases and phosphatases involved in
regulating levels of tau phosphorylation, including glycogen synthase kinase-3β (GSK3β)
(Alvarez-De-La-Rosa et al., 2005; Goodenough et al., 2005). Specifically, E
2
can reduce
GSK-3β activity (Goodenough et al., 2005), and P4 can decrease expression of both tau
and GSK-3β (Guerra-Araiza et al., 2007). That estrogen and progesterone have
activational effects on AD-like neuropathology in the middle-aged brain fits with the
conventional, recent data regarding the loss of hormones at menopause contributing to
the increased risk of AD in women.
Receptor-mediated hormone neuroprotective mechanisms
The many mechanisms by which estrogen and progesterone induce their effects
in the brain are probably inter-related and most likely through both receptor and non-
receptor mediated pathways. As discussed in Chapter One, E
2
acts through both ERα
and ERβ and has a wide variety of neuroprotective effects. Currently, the mechanism by
which progesterone regulates E
2
action through ER is unknown. However, it has
previously been shown that E
2
can up-regulate protein levels of both PR isoforms
(Guerra-Araiza et al., 2003), therefore it is reasonable to hypothesize that P4 may be
able to regulate ER expression. To this end, our laboratory began to investigate the
possibility that progesterone modulates estrogen function in part by regulating ER
expression. We have observed in primary neuron culture that low physiological
concentrations of progesterone rapidly down-regulate mRNA levels of both ERα and
144
ERβ (Jayaraman and Pike, 2009). Functionally, this progesterone-mediated decrease in
ER expression was associated with inhibition of both ERE-mediated transcriptional
activity and estrogen neuroprotection against Aβ (Jayaraman and Pike, 2009). These
results give the first suggestion that one of the potential mechanisms by which P4
regulates E
2
action may be by regulating ER. It would be interesting to conduct follow-up
studies from the results from Chapter Four investigating the differential effects of
constant and cyclic P4 on ER expression. According to the finding that cyclic P4 did not
block E
2
action to the same extent as continuous P4, one would speculate that cyclic P4
would not down-regulate ER expression to the same extent as when administered
continually.
The current use of a progestin in HT
Progesterone’s effect on the female reproductive system especially tissues such
as the uterus have been well known for many years (reviewed in Graham and Clarke,
1997), it has long been considered an antagonist to estrogen action, and it has been
used clinically for over 50 years in oral contraceptives and hormone replacement
therapy. Using female hormone extracts to prevent contraception was discovered in the
early 1900’s and in 1938, oral contraceptives were synthesized when researchers
determined how to increase the oral bioavilability and half-life of the various hormones
(reviewed in Hammond et al., 2001). However, it has been relatively recently that
researchers have begun to investigate the effects of progesterone on the central
nervous system in regards to aging and neurodegenerative diseases. This interest
began when it became clear that an estrogenic antagonist was needed to supplement
estrogen replacement therapy (ERT) due to an increased risk in endometrial carcinoma.
In the 1980’s, the risk of endometrial cancer in women taking ERT was over 2 times the
145
risk of “never-users” and this risk increased as a function of years taking ERT (Cramer
and Knapp, 1979).
To block this estrogenic effect, a natural estrogen antagonist, progesterone was
deemed necessary to add to the ERT formulation (Persson et al., 1989). It was
demonstrated that progesterone could inhibit human endometrial cancer cell growth (Dai
et al., 2002) and that oral progestins could antagonize the estrogen-induced endometrial
cancer that many post-menopausal women were experiencing after long-term HT usage
(Grady et al., 1995). When the synthetic progestin medroxyprogesterone acetate (MPA)
was shown to be safe and effective, it was added to conjugated equine estrogen (CEE)
to became the most widely prescribed HT regimen in the United States for post-
menopausal women (Thompson, 1995). Following MPA was norethindrone acetate and
norgestimate, both derivatives of 19-nortestosterone (reviewed in Hammond et al., 2001)
and all available in various formulations. These progestins are combined with CEE to
make up the three main formulations which are used today; a) estrogen alone in women
without a uterus, b) estrogen plus a progestin administered continuously (every day of
the month), or c) estrogen plus a progestin administered “sequentially” during 10-14
days of each month. Although an assortment of HT forms are available in the country, all
continuous and intermittent HT formulations block endometrial cancer. However, the
benefits of these HT preparations on other systems such as cardiovascular, nervous and
skeletal system needed to be further investigated.
Effects on osteoporosis
It has been well characterized that the loss of female sex steroid hormones
at menopause leads to significant bone deterioration in women. Further, for many years,
clinicians have been reporting that estrogen-based hormone therapy can prevent bone
146
deterioration (reviewed in Committee AsfRM, 2008). Four large-scale, randomized
clinical trials have evaluated the effectiveness of HT for prevention of clinical fractures
(Hulley et al., 1998; Komulainen et al., 1998; Rossouw et al., 2002). And in the above-
mentioned review, the authors summarized the findings of these clinical trials and
reported that the overall relative rate of fractures for women on HT was 0.64 compared
to women given placebo, suggesting that HT is in fact protective in preventing clinical
fractures. Further, one comprehensive trial investigated the effects of HT on bone
mineral density in the spine, proximal femur, and radius and reported that HT uniformly
maintained or improved bone mineral density in these regions (Speroff et al., 1996). In
addition, two now completed trials have investigated the use of long term HT with a
cyclic progestogen component and demonstrated that this form of HT was able to
increase bone mineral density in post-menopausal women (1995; Castelo-Branco et al.,
1999). In light of the vast evidence suggesting that HT is protective against bone density
loss and clinical fractures, today HT is still recommended for the prevention of
osteoporosis in aging women, despite debates over the cost-benefit ratio.
Cardiovascular effects
Despite the evidence for beneficial effects of HT on the skeletal system, the
effects of HT on the cardiovascular system are not well understood. The Heart and
Estrogen/progestin Replacement Study (HERS) and Women’s Health Initiative (WHI)
trials demonstrated that estrogen+progestin was in fact, detrimental to the risk of
coronary events, and stroke (Rossouw et al., 2002) and offered no benefits to overall
quality of life measures (Hays et al., 2003).
Unfortunately, the current status of HT seems discouraging. The HERS and WHI
trials demonstrated that both estrogen+progestin treatment increased breast cancer risk
147
by ~25% compared to the decreased risk of estrogen alone treatment by ~23%
(reviewed in Hulley and Grady, 2004). Now it is understood that progesterone can
“prime” endometrial cells for stimulation by estrogen-induced growth signals (Lange et
al., 1998). However, the relationship between progestins and breast cancer is still a
topic of debate as other studies suggest that progestins have positive, negative, or
neutral effects on cancel incidence in both clinical trials (Pasqualini, 2007) and both
human and mice (reviewed in Haslam et al., 2002). However, since the well-publicized
results of these HERS and WHI studies, the US Food and Drug Administration (FDA)
prohibited the prescription of Premarin (CEE+MPA) as a primary treatment for disease
prevention and relief of post-menopausal symptoms. Furthermore, many subsequent
studies have investigated the combined effects of estrogen+/-progestin treatment
regimens on various health-related outcomes such as cancer, cognition, and
cardiovascular disease. Several comprehensive meta-analysis have reported that the
increased risks associated with the HERS and WHI trials have been replicated in many
other clinical trials using the same or similar hormone regimens (reviewed in Warren,
2004). After these analyses, researchers began to consider alternative strategies to the
use of progestins in HT.
The future of hormone therapy
In the future, HT will need to undergo many changes in order to continue it’s safe
and effective use for post-menopausal women. Both clinicians and researchers agree
that the optimal formula of HT should be designed with several goals in mind; a) it
should be effective in reducing the risk of osteoporosis in post-menopausal women, b)
effective in reducing menopause-associated symptoms such as hot flashes, c) effective
in contributing neuroprotective effects to the aging female brain, d) avoid the increase
148
risk of endometrial carcinoma, and e) contain the smallest yet effective dose of
hormones to achieve these goals. In addition, several concerns need to be addressed
such as the bioavailability of both natural and synthetic hormones, the mode of
administration, the age of initial administration and the paradigm of hormone exposure.
In order to create a novel formulation of HT that satisfies all these criteria, more research
still needs to be done to understand basic function of estrogen and progesterone in the
brain as well as the body. However, the results described here contribute some
understanding and perhaps some novel possibilities in creating the optimal HT
paradigm.
Replicable preparations of CEE
One of the primary concerns with HT is a relatively easy problem to solve; the
issue of consistent and replicable preparations of CEE for equal and reliable distribution
to the public. The first step towards testing this hypothesis would be to determine the
most effective compounds of the many molecular components of CEE and stabilize the
relative amounts of these compounds in the clinically available form of CEE. While this
drug has been prescribed in the US for many years to attenuate the symptoms of
menopause, conjugated equine estrogen is actually comprised of 10 different kinds of
estrogens beyond estradiol that each have different, only partially understood actions
and are not necessarily found in the same ratio in each batch of manufactured CEE
(Rozovsky et al., 2002). Specifically, CEE is comprised of 50% estrone, which is only a
weak estrogen. On the other hand, 17β-estradiol (E
2
) only comprises 10% of the total
steroid concentration even though E
2
is the primary estrogen found in women. In
addition, CEE also contains small amounts of progestins and androgens. Cell culture
and rodent models could be an ideal system to specifically identify mechanistic effects of
149
each different component of CEE, identify the most efficacious form, which can interact
positively with estradiol, and possibly determine any negative cellular side effects.
Time of HT initiation
Another relatively simple problem to solve in order to optimize hormone therapy
in the future is to accelerate the age of HT administration. As discussed in the
Introduction Chapter, women enrolled in the WHIMS study were up to 79 years of age at
the time of HT initiation. As the average age of menopause for women in the USA is
approximately 51 yrs of age, this suggests that some women enrolled in the study were
~30 yrs post-menopausal and have passed the “critical window” during which HT would
be efficacious. However, the results demonstrated in Chapter Two suggest that E
2
is still
neuroprotective in middle-age at the beginning of reproductive senescence. In light of
these ideas, the most straightforward suggestion for future HT is to initiate treatment at
the beginning of menopause. This paradigm would allow the body to maintain hormone
responsiveness by regulating hormone values before they begin irreversible, age-related
hormone fluctuation alterations.
Clinically, this would be a relatively straightforward paradigm to implement.
Women most frequently consult their doctor in the beginning signs of menopausal
symptoms, suggesting that they are beginning the transition into peri-menopause
characterized by an elongation and irregularity of menstrual cycles. Levels of sex steroid
hormones remain high during this period, therefore women who initiate HT at this time
would effectively avoid the “critical window” and would not suffer from intolerable
menstrual symptoms such as hot flashes, insomnia and mood swings. However, if these
symptoms do occur, women will be more likely to consult their doctor for relief therefore
compliance with taking this future HT formulation should be relatively high.
150
Inclusion of a progestin
Perhaps the most significant concern regarding the future of HT is the debate
over the inclusion of a progestin component and in what form such a progestin should be
included. This debate has been argued significantly by researchers and clinicians the
same. As discussed in the Introduction Chapter, much evidence exists for the
neuroprotective effects of both natural (P4) and synthetic (MPA) progestins (reviewed in
Brinton et al., 2008; Schumacher et al., 2008). However, as also discussed above, there
is much evidence demonstrating antagonizing effects of these hormones. The results
presented in Chapter Three support the abandonment of the current, continuous dose of
progestin component in HT because these results suggest that such a constant P4 dose
blocks the beneficial effects of estrogen in reducing Aβ accumulation in the brains of
female AD transgenic mice. However, the same Chapter also demonstrates that P4 is
capable of reducing tau hyperphopsphorylation, the secondary hallmark of AD
neuropathology. Therefore, based on the summary of data from Chapter Three, these
results do not suggest total abandonment of a P4 component in HT. Instead they simply
suggest a modification of the current progestin formulation as an alternate strategy.
A straightforward possibility for the modification of the progestin component
would be to simply utilize a shorter, cyclic dose of P4. As discussed above, this idea
stemmed from cardiovascular and cancer research on HT (reviewed in Whitehead et al.,
1990; Hammond et al., 2001) as researchers argue for using the lowest effective dose of
P4 to minimize its negative effects on lipid metabolism and retain its anti-estrogen
effects on the endometrium and breast. Therefore, in our model, this preparation would,
in theory, retain the beneficial P4 effects on tau hyperphosphorylation while avoiding the
antagonizing effects of P4 on E
2
neuroprotection. Such a preparation of HT would be
151
modeled after the natural, cyclic hormone fluctuations of the female menstrual cycle. As
described for both women and rodents (Wu et al., 2005a), the natural cyclic fluctuation of
progesterone involves a high peak after ovulation for followed by a swift, steady decline
to baseline and low levels the remainder of the cycle. Such a preparation became the
model for the HT paradigm used in Chapter Four and demonstrated much more
beneficial effects in that is no longer antagonized E
2
’s beneficial effects on Abeta
accumulation. Therefore, as Chapter Four suggests, a cyclic paradigm of P4 exposure
would be a promising alternative to the constant P4 exposure currently available.
Interestingly, the concept of switching from a constant to a cyclic P4 exposure is
already underway in clinical research. Several clinical HT trials have incorporated a
comparison between constant vs. cyclic (last 12d/mo) P4 in post-menopausal women on
osteoporosis and cognitive function (Castelo-Branco et al., 1999) and the
Postmenopausal Estrogen/Progestin Intervention trial (PEPI) on cardiovascular health
(1995). Most importantly, this constant estrogen + cyclic (12d/mo) P4 paradigm is
currently employed in the Kronos Early Estrogen Prevention Study (KEEPS), a large,
multi-center, randomized, double-blind, placebo-controlled clinical trial investigating the
effects of HT on vascular disease in post-menopausal women. While this trial is in its
infancy, it will be extremely informative to compare the future results to that which we
have discovered in Chapter Four.
Neuroactive SERMs
Perhaps the most innovative alternative to a progestin-containing HT paradigm is
to utilize novel, synthetic, estrogenic compounds. As discussed in the Introduction
Chapter and summarized in Table 1, estrogen induces the majority of its neuroprotective
effects through various ERs. Therefore, the development of a SERM would be very
152
advantageous if it had neuroprotective actions without receptor-dependent effects on
other hormone-responsive tissue such as breast and uterus. Currently, effort is being
focused on next generation SERMs that exhibit more robust and specific neuroprotective
actions (Brinton, 2004b; Sheldahl et al., 2007). For example, Brinton and colleagues
recently developed a synthetic SERM with both estrogenic and antioxidant potential that
protects cultured neurons from cell death (Zhao and Brinton, 2007). Based on the results
in Chapter Five, PPT may be one such compound.
However, much more research is needed before PPT can be considered for
possible clinical use. Namely, its long-term estrogenic properties must be evaluated. In
the results presented in Chapter Five, our study only measured uterine weight as a
bioassay for estrogen action and this measure is not sophisticated enough to evaluate
changes in the uterus or endometrium. A full study must be undertaken to measure the
long-term effects of PPT on proliferation of breast and uterine tissue as well as long-term
effects in the brain against Aβ accumulation, tau hyperphospholylation, learning and
memory, and cell death. These studies should be conducted in both cell culture systems
as well as WT female rodents and finally in various AD transgenic models.
Currently the most studied and clinically relevant SERMs are tamoxifen and
raloxifene, synthetic compounds that exhibit tissue-dependent ER agonist and
antagonist actions. As a potent antagonist of estrogen action in breast tissue, tamoxifen
is best recognized as an antiestrogen used to treat breast cancer although it can exert
agonist ER effects on bone and lipids (Bryant and Dere, 1998; Shang and Brown, 2002).
Interestingly, low concentrations of tamoxifen can protect cultured neurons from toxicity
due to Aβ and glutamate (O'Neill et al., 2004a), suggesting the potential for a protective
role against AD. However, tamoxifen has also been observed to block E
2
–mediated
153
protection in cultured neurons (Chae et al., 2001; Zhang et al., 2001). Potential benefits
of tamoxifen use in postmenopausal women for prevention or treatment of AD has not
been well studied. Some studies indicate increased risk of cognitive deficits in tamoxifen
users (Paganini-Hill and Clark, 2000; Shilling et al., 2003), whereas another study
suggested tamoxifen may reduce AD risk (Breuer and Anderson, 2000). In brain,
raloxifene mimics some but not all protective estrogen actions. Raloxifene increases
choline acetyltransferase in hippocampus of OVX rats (Wu et al., 1999) and in cultured
neurons it increases neurite outgrowth (Nilsen et al., 1998) and can reduce Aβ toxicity
(O'Neill et al., 2004b). Conversely, E
2
but not raloxifene was effective in attenuating Aβ-
induced inflammatory reaction in OVX rats (Thomas et al., 2001). In postmenopausal
women, raloxifene use has been linked with reduced risk of cognitive impairment and
development of AD (Yaffe et al., 2005).
Conclusion: Future directions in hormone therapies for men and women
As reviewed in this manuscript, there are numerous neuroprotective actions of
estrogens and androgens that have direct relevance to AD pathogenesis and compelling
potential to prevent and possibly treat the disease. However, the promise of estrogen-
based and androgen based HTs in reducing AD risk have yet to be realized. As findings
and directions from clinical and basic science research become increasingly integrated,
it is anticipated that critical parameters affecting HT efficacy will be optimized, including
age of HT initiation as well as the formulation, regimen, and delivery of HT. As ongoing
research continues to address these crucial and immediate concerns, an emerging area
of investigation is the development of natural and synthetic hormone mimetics that will
preferentially activate estrogen and androgen neuroprotective mechanisms while
minimizing deleterious consequences in other tissues.
154
REFERENCES
Aenlle KK, Kumar A, Cui L, Jackson TC, Foster TC (2007) Estrogen effects on cognition
and hippocampal transcription in middle-aged mice. Neurobiol Aging.
Ahlbom E, Prins GS, Ceccatelli S (2001) Testosterone protects cerebellar granule cells
from oxidative stress-induced cell death through a receptor mediated mechanism.
Brain Research 892:255-262.
Akhmadeev AV, Kalimullina LB (2005) [Neonatal androgenization of female rats during
sex differentiation of the brain modifies neuronal organization of the amygdala
complex]. Ontogenez 36:64-67.
Akhondzadeh S, Rezaei F, Larijani B, Nejatisafa AA, Kashani L, Abbasi SH (2006)
Correlation between testosterone, gonadotropins and prolactin and severity of
negative symptoms in male patients with chronic schizophrenia. Schizophr Res
84:405-410.
Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom
P, Emmerling M, Fiebich BL (2000) Inflammation and Alzheimer's disease.
Neurobiology of Aging 21:383-421.
Alexander GM, Swerdloff RS, Wang C, Davidson T, McDonald V, Steiner B, Hines M
(1998) Androgen-behavior correlations in hypogonadal men and eugonadal men.
II. Cognitive abilities. Horm Behav 33:85-94.
Alkayed NJ, Murphy SJ, Traystman RJ, Hurn PD, Miller VM (2000) Neuroprotective
effects of female gonadal steroids in reproductively senescent female rats. Stroke
31:161-168.
Almeida OP, Flicker L (2003) Testosterone and dementia: too much ado about too little
data. J Br Menopause Soc 9:107-110.
Almeida OP, Lautenschlager NT, Vasikaran S, Leedman P, Gelavis A, Flicker L (2006) A
20-week randomized controlled trial of estradiol replacement therapy for women
aged 70 years and older: effect on mood, cognition and quality of life. Neurobiol
Aging 27:141-149.
Alvarez-De-La-Rosa M, Silva I, Nilsen J, Perez MM, Garcia-Segura LM, Avila J, Naftolin
F (2005) Estradiol prevents neural Tau hyperphosphorylation characteristic of
Alzheimer's disease. Annals of the New York Academy of Sciences 1052:210-
224.
155
Alves SE, McEwen BS, Hayashi S, Korach KS, Pfaff DW, Ogawa S (2000) Estrogen-
regulated progestin receptors are found in the midbrain raphe but not
hippocampus of estrogen receptor alpha (ER alpha) gene-disrupted mice. J Comp
Neurol 427:185-195.
Anahara R, Toyama Y, Mori C (2004) Flutamide induces ultrastructural changes in
spermatids and the ectoplasmic specialization between the Sertoli cell and
spermatids in mouse testes. Reprod Toxicol 18:589-596.
Andersen K, Launer LJ, Dewey ME, Letenneur L, Ott A, Copeland JRM, Dartigues J-F,
Kragh-Sorensen P, Baldereschi M, Brayne C, Lobo A, Martinez-Lage JM, Stijnen
T, Hofman A (1999) Gender differences in the incidence of AD and vascular
dementia: The EURODEM Studies. Neurology 53:1992-1997.
Anderson LI, Leipheimer RE, Dluzen DE (2005) Effects of neonatal and prepubertal
hormonal manipulations upon estrogen neuroprotection of the nigrostriatal
dopaminergic system within female and male mice. Neuroscience 130:369-382.
Ansonoff MA, Etgen AM (1998) Estradiol Elevates Protein Kinase C Catalytic Activity in
the Preoptic Area of Female Rats10.1210/en.139.7.3050. Endocrinology
139:3050-3056.
Antonsson B, Martinou JC (2000) The Bcl-2 protein family. Exp Cell Res 256:50-57.
Arato M, Frecska E, Beck C, An M, Kiss H (2004) Digit length pattern in schizophrenia
suggests disturbed prenatal hemispheric lateralization. Prog
Neuropsychopharmacol Biol Psychiatry 28:191-194.
Arendash GW, King DL (2002) Intra- and intertask relationships in a behavioral test
battery given to Tg2576 transgenic mice and controls. Physiol Behav 75:643-652.
Arriagada PV, Growdon JH, Hedley-Whyte ET, Hyman BT (1992) Neurofibrillary tangles
but not senile plaques parallel duration and severity of Alzheimer's disease.
Neurology 42:631-639.
Atwood CS, Meethal SV, Liu T, Wilson AC, Gallego M, Smith MA, Bowen RL (2005)
Dysregulation of the Hypothalamic-Pituitary-Gonadal Axis with Menopause and
Andropause Promotes Neurodegenerative Senescence. Journal of
Neuropathology and Experimental Neurology 64:93-103.
Azcoitia I, Sierra A, Garcia-Segura LM (1998) Estradiol prevents kainic acid-induced
neuronal loss in the rat dentate gyrus. Neuroreport 9:3075-3079.
Azcoitia I, Fernandez-Galaz C, Sierra A, Garcia-Segura LM (1999) Gonadal hormones
affect neuronal vulnerability to excitotoxin-induced degeneration. J Neurocytol
28:699-710.
156
Azcoitia I, Sierra A, Veiga S, Honda S, Harada N, Garcia-Segura LM (2001) Brain
aromatase is neuroprotective. Journal Of Neurobiology 47:318-329.
Bachman DL, Wolf PA, Linn R, Knoefel JE, Cobb J, Belanger A, D'Agostino RB, White
LR (1992) Prevalence of dementia and probable senile dementia of the Alzheimer
type in the Framingham Study. Neurology 42:115-119.
Bake S, Sohrabji F (2004) 17beta-estradiol differentially regulates blood-brain barrier
permeability in young and aging female rats. Endocrinology 145:5471-5475.
Bake S, Ma L, Sohrabji F (2008) Estrogen receptor-alpha overexpression suppresses
17beta-estradiol-mediated vascular endothelial growth factor expression and
activation of survival kinases. Endocrinology 149:3881-3889.
Baker LD, Sambamurti K, Craft S, Cherrier M, Raskind MA, Stanczyk FZ, Plymate SR,
Asthana S (2003) 17beta-estradiol reduces plasma Abeta40 for HRT-naive
postmenopausal women with Alzheimer disease: a preliminary study. Am J
Geriatr Psychiatry 11:239-244.
Bakker J, Baum MJ (2008) Role for estradiol in female-typical brain and behavioral
sexual differentiation. Front Neuroendocrinol 29:1-16.
Barker DJ (2003) The developmental origins of adult disease. Eur J Epidemiol 18:733-
736.
Barker DJ (2008) Human growth and cardiovascular disease. Nestle Nutr Workshop Ser
Pediatr Program 61:21-38.
Barker DJ, Bergmann RL, Ogra PL (2008) Concluding remarks. The Window of
Opportunity: Pre-Pregnancy to 24 Months of Age. Nestle Nutr Workshop Ser
Pediatr Program 61:255-260.
Barnes LL, Wilson RS, Bienias JL, Schneider JA, Evans DA, Bennett DA (2005) Sex
differences in the clinical manifestations of Alzheimer disease pathology. Arch
Gen Psychiatry 62:685-691.
Barret-Connor E, Kritz-Silverstein D (1993) Estrogen Replacement Therapy and
Cognitive Function in Older Women. The Journal of the American Medical
Association 269:2637-2641.
Barrett-Connor E, Goodman-Gruen D, Patay B (1999) Endogenous sex hormones and
cognitive function in older men. The Journal of Clinical Endocrinology and
Metabolism 84:3681-3685.
Baulieu EE (1997) Neurosteroids: of the nervous system, by the nervous system, for the
nervous system. Recent Prog Horm Res 52:1-32.
157
Baulieu EE, Robel P, Schumacher M (2001) Neurosteroids: beginning of the story. Int
Rev Neurobiol 46:1-32.
Baumgartner RN, Waters DL, Gallagher D, Morley JE, Garry PJ (1999) Predictors of
skeletal muscle mass in elderly men and women. Mech Ageing Dev 107:123-136.
Becu-Villalobos D, Gonzalez Iglesias A, Diaz-Torga G, Hockl P, Libertun C (1997) Brain
sexual differentiation and gonadotropins secretion in the rat. Cell Mol Neurobiol
17:699-715.
Behl C, Widmann M, Trapp T, Holsboer F (1995) 17-beta estradiol protects neurons
from oxidative stress-induced cell death in vitro. Biochem Biophys Res Commun
216:473-482.
Behl C, Skutella T, Lezoualc'h F, Post A, Widmann M, Newton CJ, Holsboer F (1997)
Neuroprotection against Oxidative Stress by Estrogens: Structure-Activity
Relationship. Molecular Pharmacology 51:535-541.
Benvenuti S, Luciani P, Vannelli GB, Gelmini S, Franceschi E, Serio M, Peri A (2005)
Estrogen and selective estrogen receptor modulators exert neuroprotective effects
and stimulate the expression of selective Alzheimer's disease indicator-1, a
recently discovered antiapoptotic gene, in human neuroblast long-term cell
cultures. J Clin Endocrinol Metab 90:1775-1782.
Bergemann N, Mundt C, Parzer P, Jannakos I, Nagl I, Salbach B, Klinga K, Runnebaum
B, Resch F (2005) Plasma concentrations of estradiol in women suffering from
schizophrenia treated with conventional versus atypical antipsychotics. Schizophr
Res 73:357-366.
Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the
pathogenesis of Alzheimer's disease. Trends Neurosci 31:454-463.
Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS,
Montori VM (2006) Testosterone therapy in adult men with androgen deficiency
syndromes: an endocrine society clinical practice guideline. J Clin Endocrinol
Metab 91:1995-2010.
Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X,
Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim
I, Shen R, Storer TW (2001) Testosterone dose-response relationships in healthy
young men. Am J Physiol Endocrinol Metab 281:E1172-1181.
Bi R, Broutman G, Foy MR, Thompson RF, Baudry M (2000) The tyrosine kinase and
mitogen-activated protein kinase pathways mediate multiple effects of estrogen in
hippocampus. Proc Natl Acad Sci U S A 97:3602-3607.
158
Billings LM, Green KN, McGaugh JL, LaFerla FM (2007) Learning decreases A beta*56
and tau pathology and ameliorates behavioral decline in 3xTg-AD mice. J
Neurosci 27:751-761.
Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla FM (2005) Intraneuronal Abeta
causes the onset of early Alzheimer's disease-related cognitive deficits in
transgenic mice. Neuron 45:675-688.
Bimonte-Nelson HA, Nelson ME, Granholm AC (2004) Progesterone counteracts
estrogen-induced increases in neurotrophins in the aged female rat brain.
Neuroreport 15:2659-2663.
Bimonte-Nelson HA, Francis KR, Umphlet CD, Granholm AC (2006) Progesterone
reverses the spatial memory enhancements initiated by tonic and cyclic oestrogen
therapy in middle-aged ovariectomized female rats. Eur J Neurosci 24:229-242.
Binder EF, Schechtman KB, Birge SJ, Williams DB, Kohrt WM (2001) Effects of
hormone replacement therapy on cognitive performance in elderly women.
Maturitas 38:137-146.
Bohacek J, Bearl AM, Daniel JM (2008) Long-term ovarian hormone deprivation alters
the ability of subsequent oestradiol replacement to regulate choline
acetyltransferase protein levels in the hippocampus and prefrontal cortex of
middle-aged rats. J Neuroendocrinol 20:1023-1027.
Boyce-Rustay JM, Holmes A (2006) Genetic inactivation of the NMDA receptor NR2A
subunit has anxiolytic- and antidepressant-like effects in mice.
Neuropsychopharmacology 31:2405-2414.
Braak H, Braak E (1990) Alzheimer's disease: striatal amyloid deposits and
neurofibrillary changes. J Neuropathol Exp Neurol 49:215-224.
Braak H, Braak E (1997) Staging of Alzheimer-related cortical destruction. International
Psychogeriatrics / Ipa 9 Suppl 1:257-261; discussion 269-272.
Brann DW, Dhandapani K, Wakade C, Mahesh VB, Khan MM (2007) Neurotrophic and
neuroprotective actions of estrogen: Basic mechanisms and clinical implications.
Steroids 72:381-405.
Breedlove SM (1997) Neonatal androgen and estrogen treatments masculinize the size
of motoneurons in the rat spinal nucleus of the bulbocavernosus. Cell Mol
Neurobiol 17:687-697.
Brenner DE, Kukull WA, Stergachis A, van Belle G, Bowen JD, McCormick WC, Teri L,
Larson EB (1994) Postmenopausal estrogen replacement therapy and the risk of
Alzheimer's disease: a population-based case-control study. Am J Epidemiol
140:262-267.
159
Breuer B, Anderson R (2000) The relationship of tamoxifen with dementia, depression,
and dependence in activities of daily living in elderly nursing home residents.
Women Health 31:71-85.
Brinton RD (2004a) Requirements of a brain selective estrogen: advances and
remaining challenges for developing a NeuroSERM. J Alzheimers Dis 6:S27-35.
Brinton RD (2004b) Impact of estrogen therapy on Alzheimer's disease: a fork in the
road? CNS Drugs 18:405-422.
Brinton RD, Chen S, Montoya M, Hsieh D, Minaya J (2000) The estrogen replacement
therapy of the Women's Health Initiative promotes the cellular mechanisms of
memory and neuronal survival in neurons vulnerable to Alzheimer's disease.
Maturitas 34 Suppl 2:S35-52.
Brinton RD, Thompson RF, Foy MR, Baudry M, Wang J, Finch CE, Morgan TE, Pike CJ,
Mack WJ, Stanczyk FZ, Nilsen J (2008) Progesterone receptors: form and
function in brain. Front Neuroendocrinol 29:313-339.
Brion JP, Couck AM, Passareiro E, Flament-Durand J (1985) Neurofibrillary tangles of
Alzheimer's disease: an immunohistochemical study. J Submicrosc Cytol 17:89-
96.
Brookmeyer R, Gray S, Kawas C (1998) Projections of Alzheimer's disease in the United
States and the public health impact of delaying disease onset. Am J Public Health
88:1337-1342.
Brooks BP, Merry DE, Paulson HL, Lieberman AP, Kolson DL, Fischbeck KH (1998) A
cell culture model for androgen effects in motor neurons. J Neurochem 70:1054-
1060.
Bruce-Keller AJ, Keeling JL, Keller JN, Huang FF, Camondola S, Mattson MP (2000)
Antiinflammatory effects of estrogen on microglial activation. Endocrinology
141:3646-3656.
Bryant HU, Dere WH (1998) Selective estrogen receptor modulators: an alternative to
hormone replacement therapy. Proc Soc Exp Biol Med 217:45-52.
Buckwalter JG, Sobel E, Dunn ME, Diz MM, Henderson VW (1993) Gender differences
on a brief measure of cognitive functioning in Alzheimer's disease. Archives of
Neurology 50:757-760.
Burger H, de Laet CE, van Daele PL, Weel AE, Witteman JC, Hofman A, Pols HA (1998)
Risk factors for increased bone loss in an elderly population: the Rotterdam Study.
Am J Epidemiol 147:871-879.
160
Caccamo A, Oddo S, Sugarman MC, Akbari Y, LaFerla FM (2005) Age- and region-
dependent alterations in Abeta-degrading enzymes: implications for Abeta-
induced disorders. Neurobiol Aging 26:645-654.
Caccamo A, Oddo S, Billings LM, Green KN, Martinez-Coria H, Fisher A, LaFerla FM
(2006) M1 receptors play a central role in modulating AD-like pathology in
transgenic mice. Neuron 49:671-682.
Cahill L (2003) Sex- and hemisphere-related influences on the neurobiology of
emotionally influenced memory. Prog Neuropsychopharmacol Biol Psychiatry
27:1235-1241.
Cahill L (2006) Why sex matters for neuroscience. Nat Rev Neurosci 7:477-484.
Cahill L, Haier RJ, White NS, Fallon J, Kilpatrick L, Lawrence C, Potkin SG, Alkire MT
(2001) Sex-related difference in amygdala activity during emotionally influenced
memory storage. Neurobiol Learn Mem 75:1-9.
Callahan MJ, Lipinski WJ, Bian F, Durham RA, Pack A, Walker LC (2001) Augmented
senile plaque load in aged female beta-amyloid precursor protein-transgenic mice.
Am J Pathol 158:1173-1177.
Camacho-Arroyo I, Guerra-Araiza C, Cerbon MA (1998) Progesterone receptor isoforms
are differentially regulated by sex steroids in the rat forebrain. Neuroreport 9:3993-
3996.
Canli T, Desmond JE, Zhao Z, Gabrieli JD (2002) Sex differences in the neural basis of
emotional memories. Proc Natl Acad Sci U S A 99:10789-10794.
Cano A, Hermenegildo C, Oviedo P, Tarin JJ (2007) Selective estrogen receptor
modulators and risk for coronary heart disease. Climacteric 10:97-111.
Carlson GA, Borchelt DR, Dake A, Turner S, Danielson V, Coffin JD, Eckman C,
Meiners J, Nilsen SP, Younkin SG, Hsiao KK (1997) Genetic modification of the
phenotypes produced by amyloid precursor protein overexpression in transgenic
mice. Hum Mol Genet 6:1951-1959.
Carlson MC, Zandi PP, Plassman BL, Tschanz JT, Welsh-Bohmer KA, Steffens DC,
Bastian LA, Mehta KM, Breitner JC (2001) Hormone replacement therapy and
reduced cognitive decline in older women: the Cache County Study. Neurology
57:2210-2216.
Carroll JC, Pike CJ (2008) Selective estrogen receptor modulators differentially regulate
Alzheimer-like changes in female 3xTg-AD mice. Endocrinology 149:2607-2611.
Carroll JC, Rosario ER, Pike CJ (2008) Progesterone blocks estrogen neuroprotection
from kainate in middle-aged female rats. Neurosci Lett 445:229-232.
161
Carroll JC, Boyce-Rustay JM, Millstein R, Yang R, Wiedholz LM, Murphy DL, Holmes A
(2007a) Effects of mild early life stress on abnormal emotion-related behaviors in
5-HTT knockout mice. Behav Genet 37:214-222.
Carroll JC, Rosario ER, Chang L, Stanczyk FZ, Oddo S, LaFerla FM, Pike CJ (2007b)
Progesterone and estrogen regulate Alzheimer-like neuropathology in female
3xTg-AD mice. J Neurosci 27:13357-13365.
Carswell HV, Macrae IM, Gallagher L, Harrop E, Horsburgh KJ (2004) Neuroprotection
by a selective estrogen receptor beta agonist in a mouse model of global
ischemia. Am J Physiol Heart Circ Physiol 287:H1501-1504.
Casas C, Sergeant N, Itier JM, Blanchard V, Wirths O, van der Kolk N, Vingtdeux V, van
de Steeg E, Ret G, Canton T, Drobecq H, Clark A, Bonici B, Delacourte A,
Benavides J, Schmitz C, Tremp G, Bayer TA, Benoit P, Pradier L (2004) Massive
CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42
accumulation in a novel Alzheimer transgenic model. Am J Pathol 165:1289-1300.
Castelo-Branco C, Figueras F, Sanjuan A, Pons F, Vicente JJ, Vanrell JA (1999) Long-
term postmenopausal hormone replacement therapy effects on bone mass:
differences between surgical and spontaneous patients. Eur J Obstet Gynecol
Reprod Biol 83:207-211.
Chae HS, Bach JH, Lee MW, Kim HS, Kim YS, Kim KY, Choo KY, Choi SH, Park CH,
Lee SH, Suh YH, Kim SS, Lee WB (2001) Estrogen attenuates cell death induced
by carboxy-terminal fragment of amyloid precursor protein in PC12 through a
receptor-dependent pathway. J Neurosci Res 65:403-407.
Chakraborty TR, Gore AC (2004) Aging-related changes in ovarian hormones, their
receptors, and neuroendocrine function. Exp Biol Med (Maywood) 229:977-987.
Chang D, Kwan J, Timiras PS (1997) Estrogens influence growth, maturation, and
amyloid beta-peptide production in neuroblastoma cells and in a beta-APP
transfected kidney 293 cell line. Adv Exp Med and Biol 429:261-271.
Chaovipoch P, Jelks KA, Gerhold LM, West EJ, Chongthammakun S, Floyd CL (2006)
17beta-estradiol is protective in spinal cord injury in post- and pre-menopausal
rats. J Neurotrauma 23:830-852.
Cheng IH, Scearce-Levie K, Legleiter J, Palop JJ, Gerstein H, Bien-Ly N, Puolivali J,
Lesne S, Ashe KH, Muchowski PJ, Mucke L (2007) Accelerating amyloid-beta
fibrillization reduces oligomer levels and functional deficits in Alzheimer disease
mouse models. J Biol Chem 282:23818-23828.
Cherrier MM, Matsumoto AM, Amory JK, Asthana S, Bremner W, Peskind ER, Raskind
MA, Craft S (2005) Testosterone improves spatial memory in men with Alzheimer
disease and mild cognitive impairment. Neurology 64:2063-2068.
162
Cherrier MM, Asthana S, Plymate S, Baker L, Matsumoto AM, Peskind E, Raskind MA,
Brodkin K, Bremner W, Petrova A, LaTendresse S, Craft S (2001) Testosterone
supplementation improves spatial and verbal memory in healthy older men.
Neurology 57:80-88.
Choi DW (1987) Ionic dependence of glutamate neurotoxicity. JNeurosci 7:369-379.
Ciana P, Di Luccio G, Belcredito S, Pollio G, Vegeto E, Tatangelo L, Tiveron C, Maggi A
(2001) Engineering of a Mouse for the in Vivo Profiling of Estrogen Receptor
Activity. Molecular Endocrinology 15:1104-1113.
Cole G (2006) A transgenic triple scores a home run. Nat Med 12:762-763; discussion
763.
Committee ASfRM (2008) Estrogen and progestogen therapy in postmenopausal
women. Fertil Steril 90:S88-102.
Cooke BM, Woolley CS (2005) Sexually dimorphic synaptic organization of the medial
amygdala. J Neurosci 25:10759-10767.
Corder EH, Ghebremedhin E, Taylor MG, Thal DR, Ohm TG, Braak H (2004) The
biphasic relationship between regional brain senile plaque and neurofibrillary
tangle distributions: modification by age, sex, and APOE polymorphism. Ann N Y
Acad Sci 1019:24-28.
Cordey M, Pike CJ (2005) Neuroprotective properties of selective estrogen receptor
agonists in cultured neurons. Brain Res 1045:217-223.
Cordey M, Pike CJ (2006) Conventional protein kinase C isoforms mediate
neuroprotection induced by phorbol ester and estrogen. J Neurochem 96:204-
217.
Cordey M, Gundimeda U, Gopalakrishna R, Pike CJ (2003) Estrogen activates protein
kinase C in neurons: role in neuroprotection. Journal of Neurochemistry 84:1340-
1348.
Cordey M, Gundimeda U, Gopalakrishna R, Pike CJ (2005) The synthetic estrogen 4-
estren-3 alpha,17 beta-diol (estren) induces estrogen-like neuroprotection.
Neurobiol Dis 19:331-339.
Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch.
Nat Rev Cancer 2:647-656.
Cosgrove KP, Mazure CM, Staley JK (2007) Evolving knowledge of sex differences in
brain structure, function, and chemistry. Biol Psychiatry 62:847-855.
Cotman CW, Anderson AJ (1995) A potential role for apoptosis in neurodegeneration
and Alzheimer's disease. Mol Neurobiol 10:19-45.
163
Cotman CW, Pike CJ, Copani A (1992) beta-Amyloid neurotoxicity: a discussion of in
vitro findings. Neurobiol Aging 13:587-590.
Cotman CW, Cribbs, D.H., Pike, C.J., Ivins, K.J (1998) Cell death in Alzheimer's death.
In: When Cells Die: A Comprehensive Evaluation of Apoptosis and Programmed
Cell Death.385-409.
Coughlan CM, Breen KC (2000) Factors influencing the processing and function of the
amyloid beta precursor protein — a potential therapeutic target in Alzheimer's
disease? Pharmacol Ther 86:111-144.
Coyle JT (1983) Neurotoxic action of kainic acid. J Neurochem 41:1-11.
Craig MC, Maki PM, Murphy DG (2005) The Women's Health Initiative Memory Study:
findings and implications for treatment. Lancet Neurol 4:190-194.
Cramer DW, Knapp RC (1979) Review of epidemiologic studies of endometrial cancer
and exogenous estrogen. Obstet Gynecol 54:521-526.
Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ,
Maxson SC, Miner LL, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R (1997)
Behavioral phenotypes of inbred mouse strains: implications and
recommendations for molecular studies. Psychopharmacology (Berl) 132:107-
124.
Cummings BJ, Mason AJ, Kim RC, Sheu PC, Anderson AJ (2002) Optimization of
techniques for the maximal detection and quantification of Alzheimer's-related
neuropathology with digital imaging. Neurobiol Aging 23:161-170.
Dai D, Wolf DM, Litman ES, White MJ, Leslie KK (2002) Progesterone inhibits human
endometrial cancer cell growth and invasiveness: down-regulation of cellular
adhesion molecules through progesterone B receptors. Cancer Res 62:881-886.
Dai X, Chen L, Sokabe M (2007) Neurosteroid estradiol rescues ischemia-induced deficit
in the long-term potentiation of rat hippocampal CA1 neurons.
Neuropharmacology 52:1124-1138.
Daniel JM, Hulst JL, Berbling JL (2006) Estradiol replacement enhances working
memory in middle-aged rats when initiated immediately after ovariectomy but not
after a long-term period of ovarian hormone deprivation. Endocrinology 147:607-
614.
Davies S, Dai D, Wolf DM, Leslie KK (2004) Immunomodulatory and transcriptional
effects of progesterone through progesterone A and B receptors in Hec50co
poorly differentiated endometrial cancer cells. J Soc Gynecol Investig 11:494-499.
164
De Nicola AF, Gonzalez SL, Labombarda F, Deniselle MC, Garay L, Guennoun R,
Schumacher M (2006) Progesterone treatment of spinal cord injury: Effects on
receptors, neurotrophins, and myelination. J Mol Neurosci 28:3-15.
Del Cerro S, Garcia-Estrada J, Garcia-Segura LM (1995) Neuroactive steroids regulate
astroglia morphology in hippocampal cultures from adult rats. Glia 14:65-71.
Dluzen DE (1996) Effects of testosterone upon MPTP-induced neurotoxicity of the
nigrostriatal dopaminergic system of C57/B1 mice. Brain Research 715:113-118.
Dominguez-Salazar E, Portillo W, Baum MJ, Bakker J, Paredes RG (2002) Effect of
prenatal androgen receptor antagonist or aromatase inhibitor on sexual behavior,
partner preference and neuronal Fos responses to estrous female odors in the rat
accessory olfactory system. Physiol Behav 75:337-346.
Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M, Samudio I, Kladde MP,
Vyhlidal C, Safe S (1999) Mechanisms of transcriptional activation of bcl-2 gene
expression by 17beta-estradiol in breast cancer cells. J Biol Chem 274:32099-
32107.
Duara R, Barker WW, Lopez-Alberola R, Loewenstein DA, Grau LB, Gilchrist D, Sevush
S, St George-Hyslop S (1996) Alzheimer's disease: interaction of apolipoprotein E
genotype, family history of dementia, gender, education, ethnicity, and age of
onset. Neurology 46:1575-1579.
Dubal DB, Wise PM (2001) Neuroprotective effects of estradiol in middle-aged female
rats. Endocrinology 142:43-48.
Dubal DB, Shughrue PJ, Wilson ME, Merchenthaler I, Wise PM (1999) Estradiol
modulates bcl-2 in cerebral ischemia: a potential role for estrogen receptors. J
Neurosci 19:6385-6393.
Dubal DB, Kashon ML, Pettigrew LC, Ren JM, Finklestein SP, Rau SW, Wise PM (1998)
Estradiol protects against ischemic injury. J Cereb Blood Flow Metab 18:1253-
1258.
Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, Kindy MS, Wise PM
(2001) Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated
protection against brain injury. Proc Natl Acad Sci U S A 98:1952-1957.
Duff K, Knight H, Refolo LM, Sanders S, Yu X, Picciano M, Malester B, Hutton M,
Adamson J, Goedert M, Burki K, Davies P (2000) Characterization of pathology
in transgenic mice over-expressing human genomic and cDNA tau transgenes.
Neurobiol Dis 7:87-98.
Duff SJ, Hampson E (2001) A sex difference on a novel spatial working memory task in
humans. Brain Cogn 47:470-493.
165
Edinger KL, Frye CA (2004) Testosterone's analgesic, anxiolytic, and cognitive-
enhancing effects may be due in part to actions of its 5alpha-reduced metabolites
in the hippocampus. Behav Neurosci 118:1352-1364.
Edinger KL, Frye CA (2005) Testosterone's anti-anxiety and analgesic effects may be
due in part to actions of its 5alpha-reduced metabolites in the hippocampus.
Psychoneuroendocrinology 30:418-430.
El Khoury J, Hickman SE, Thomas CA, Cao L, Silverstein SC, Loike JD (1996)
Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature
382:716-719.
El-Bakri NK, Islam A, Zhu S, Elhassan A, Mohammed A, Winblad B, Adem A (2004)
Effects of estrogen and progesterone treatment on rat hippocampal NMDA
receptors: relationship to Morris water maze performance. J Cell Mol Med 8:537-
544.
Emmelot-Vonk MH, Verhaar HJ, Nakhai Pour HR, Aleman A, Lock TM, Bosch JL,
Grobbee DE, van der Schouw YT (2008) Effect of testosterone supplementation
on functional mobility, cognition, and other parameters in older men: a
randomized controlled trial. Jama 299:39-52.
Eriksson JG, Osmond C, Kajantie E, Forsen TJ, Barker DJ (2006) Patterns of growth
among children who later develop type 2 diabetes or its risk factors. Diabetologia
49:2853-2858.
Espeland MA, Rapp SR, Shumaker SA, Brunner R, Manson JE, Sherwin BB, Hsia J,
Margolis KL, Hogan PE, Wallace R, Dailey M, Freeman R, Hays J (2004)
Conjugated equine estrogens and global cognitive function in postmenopausal
women: Women's Health Initiative Memory Study. Jama 291:2959-2968.
Estrada M, Uhlen P, Ehrlich BE (2006) Ca2+ oscillations induced by testosterone
enhance neurite outgrowth. J Cell Sci 119:733-743.
Febo M, Jimenez-Rivera CA, Segarra AC (2002) Estrogen and opioids interact to
modulate the locomotor response to cocaine in the female rat. Brain Res 943:151-
161.
Feldman HA, Longcope C, Derby CA, Johannes CB, Araujo AB, Coviello AD, Bremner
WJ, McKinlay JB (2002) Age Trends in the Level of Serum Testosterone and
Other Hormones in Middle-Aged Men: Longitudinal Results from the
Massachusetts Male Aging Study. Journal of clinical endocrinology and
metabolism 87:589-598.
Felicio LS, Nelson JF, Finch CE (1984) Longitudinal studies of estrous cyclicity in aging
C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal
cornification. Biol Reprod 31:446-453.
166
Ferrando AA, Sheffield-Moore M, Yeckel CW, Gilkison C, Jiang J, Achacosa A,
Lieberman SA, Tipton K, Wolfe RR, Urban RJ (2002) Testosterone administration
to older men improves muscle function: molecular and physiological mechanisms.
Am J Physiol Endocrinol Metab 282:E601-607.
Fillit H, Weinreb H, Cholst I, Luine V, McEwen B, Amador R, Zabriskie J (1986)
Observations in a preliminary open trial of estradiol therapy for senile dementia-
Alzheimer's type. Psychoneuroendocrinology 11:337-345.
Fisher DL, Morin N, Doree M (1999) A novel role for glycogen synthase kinase-3 in
Xenopus development: maintenance of oocyte cell cycle arrest by a beta-catenin-
independent mechanism. Development 126:567-576.
Fitzpatrick JL, Mize AL, Wade CB, Harris JA, Shapiro RA, Dorsa DM (2002) Estrogen-
mediated neuroprotection against beta-amyloid toxicity requires expression of
estrogen receptor alpha or beta and activation of the MAPK pathway. Journal of
Neurochemistry 82:674-682.
Fleisher A, Grundman M, Jack CR, Jr., Petersen RC, Taylor C, Kim HT, Schiller DH,
Bagwell V, Sencakova D, Weiner MF, DeCarli C, DeKosky ST, van Dyck CH, Thal
LJ (2005) Sex, apolipoprotein E epsilon 4 status, and hippocampal volume in mild
cognitive impairment. Arch Neurol 62:953-957.
Foy M, Baudry M, Thompson R (2004) Estrogen and hippocampal synaptic plasticity.
Neuron Glia Biol 1:327-338.
Frankfurt M, Gould E, Woolley CS, McEwen BS (1990) Gonadal steroids modify
dendritic spine density in ventromedial hypothalamic neurons: a Golgi study in the
adult rat. Neuroendocrinology 51:530-535.
Franklin KBJ, Paxinos G (1997) The Mouse Brain in Stereotaxic Coordinates. San
Diego: Academic Press.
Frasor J, Barnett DH, Danes JM, Hess R, Parlow AF, Katzenellenbogen BS (2003)
Response-specific and ligand dose-dependent modulation of estrogen receptor
(ER) alpha activity by ERbeta in the uterus. Endocrinology 144:3159-3166.
Fratiglioni L, Small BJ, Winblad B, Bäckman L (2001) The transition from normal
functioning to dementia in the ageing population. In: Alzheimer’s Disease:
Advances in Etiology, Pathogenesis and Therapeutics (Iqbal K, Sisodia S,
Winblad B, eds), pp 3–10. Chichester: John Wiley & Sons, Ltd.
Frye CA, Reed TA (1998) Androgenic neurosteroids: anti-seizure effects in an animal
model of epilepsy. Psychoneuroendocrinology 23:385-399.
Frye CA, Seliga AM (2001) Testosterone increases analgesia, anxiolysis, and cognitive
performance of male rats. Cogn Affect Behav Neurosci 1:371-381.
167
Fukuzaki E, Takuma K, Funatsu Y, Himeno Y, Kitahara Y, Gu B, Mizoguchi H, Ibi D,
Koike K, Inoue M, Yan SD, Yamada K (2008) Ovariectomy increases neuronal
amyloid-beta binding alcohol dehydrogenase level in the mouse hippocampus.
Neurochem Int 52:1358-1364.
Galea LA, Spritzer MD, Barker JM, Pawluski JL (2006) Gonadal hormone modulation of
hippocampal neurogenesis in the adult. Hippocampus 16:225-232.
Gandy S, Petanceska S (2001) Regulation of alzheimer beta-amyloid precursor
trafficking and metabolism. Adv Exp Med Biol 487:85-100.
Gandy S, Almeida OP, Fonte J, Lim D, Waterrus A, Spry N, Flicker L, Martins RN (2001)
Chemical andropause and amyloid-beta peptide. Journal of the American Medical
Association 285:2195-2196.
Ganguli M, Dodge HH, Chen P, Belle S, DeKosky ST (2000) Ten-year incidence of
dementia in a rural elderly US community population: the MoVIES Project.
Neurology 54:1109-1116.
Garcia-Estrada J, Del Rio JA, Luquin S, Soriano E, Garcia-Segura LM (1993) Gonadal
hormones down-regulate reactive gliosis and astrocyte proliferation after a
penetrating brain injury. Brain Research 628:271-278.
Garcia-Ovejero D, Azcoitia I, Doncarlos LL, Melcangi RC, Garcia-Segura LM (2005)
Glia-neuron crosstalk in the neuroprotective mechanisms of sex steroid
hormones. Brain Res Brain Res Rev 48:273-286.
Garcia-Segura LM, Cardona-Gomez P, Naftolin F, Chowen JA (1998) Estradiol
upregulates Bcl-2 expression in adult brain neurons. Neuroreport 9:593-597.
Gatson JW, Singh M (2007) Activation of a membrane-associated androgen receptor
promotes cell death in primary cortical astrocytes. Endocrinology 148:2458-2464.
Gatson JW, Kaur P, Singh M (2006) Dihydrotestosterone differentially modulates the
mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways
through the nuclear and novel membrane androgen receptor in C6 cells.
Endocrinology 147:2028-2034.
Gee DM, Flurkey K, Finch CE (1983) Aging and the regulation of luteinizing hormone in
C57BL/6J mice: impaired elevations after ovariectomy and spontaneous
elevations at advanced ages. Biol Reprod 28:598-607.
Gennari L, Merlotti D, Valleggi F, Martini G, Nuti R (2007) Selective estrogen receptor
modulators for postmenopausal osteoporosis: current state of development. Drugs
Aging 24:361-379.
168
Gibbs RB (2000a) Oestrogen and the cholinergic hypothesis: implications for oestrogen
replacement therapy in postmenopausal women. Novartis Found Symp 230:94-
107; discussion 107-111.
Gibbs RB (2000b) Effects of gonadal hormone replacement on measures of basal
forebrain cholinergic function. Neuroscience 101:931-938.
Gibson CL, Constantin D, Prior MJ, Bath PM, Murphy SP (2005) Progesterone
suppresses the inflammatory response and nitric oxide synthase-2 expression
following cerebral ischemia. Exp Neurol 193:522-530.
Gillett MJ, Martins RN, Clarnette RM, Chubb SA, Bruce DG, Yeap BB (2003)
Relationship between testosterone, sex hormone binding globulin and plasma
amyloid beta peptide 40 in older men with subjective memory loss or dementia. J
Alzheimers Dis 5:267-269.
Gleason CE, Carlsson CM, Johnson S, Atwood C, Asthana S (2005) Clinical
pharmacology and differential cognitive efficacy of estrogen preparations. Ann N
Y Acad Sci 1052:93-115.
Goebel JA, Birge SJ, Price SC, Hanson JM, Fishel DG (1995) Estrogen replacement
therapy and postural stability in the elderly. Am J Otol 16:470-474.
Goedert M, Jakes R, Vanmechelen E (1995) Monoclonal antibody AT8 recognises tau
protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett
189:167-169.
Goldstein JM, Seidman LJ, Horton NJ, Makris N, Kennedy DN, Caviness VS, Jr.,
Faraone SV, Tsuang MT (2001) Normal sexual dimorphism of the adult human
brain assessed by in vivo magnetic resonance imaging. Cereb Cortex 11:490-497.
Golub MS, Germann SL, Mercer M, Gordon MN, Morgan DG, Mayer LP, Hoyer PB
(2007) Behavioral consequences of ovarian atrophy and estrogen replacement in
the APPswe mouse. Neurobiology of Aging 29:1512-1523.
Goodenough S, Schleusner D, Pietrzik C, Skutella T, Behl C (2005) Glycogen synthase
kinase 3beta links neuroprotection by 17beta-estradiol to key Alzheimer
processes. Neuroscience 132:581-589.
Goodman Y, Mattson MP (1994) Secreted Forms of b-Amyloid Precursor Protein Protect
Hippocampal Neurons against Amyloid b-Peptide-Induced Oxidative Injury.
Experimental Neurology 128:1-12.
Goodman Y, Bruce AJ, Cheng B, Mattson MP (1996) Estrogens attenuate and
corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta-
peptide toxicity in hippocampal neurons. J Neurochem 66:1836-1844.
169
Gooren L (2003) Androgen deficiency in the aging male: benefits and risks of androgen
supplementation. J Steroid Biochem Mol Biol 85:349-355.
Gore AC (2008) Developmental programming and endocrine disruptor effects on
reproductive neuroendocrine systems. Front Neuroendocrinol 29:358-374.
Gosden RG, Laing SC, Felicio LS, Nelson JF, Finch CE (1983) Imminent oocyte
exhaustion and reduced follicular recruitment mark the transition to acyclicity in
aging C57BL/6J mice. Biol Reprod 28:255-260.
Gouchie C, Kimura D (1991) The relationship between testosterone levels and cognitive
ability patterns. Psychoneuroendocrinology 16:323-334.
Grady D, Gebretsadik T, Kerlikowske K, Ernster V, Petitti D (1995) Hormone
replacement therapy and endometrial cancer risk: a meta-analysis. Obstet
Gynecol 85:304-313.
Graham JD, Clarke CL (1997) Physiological action of progesterone in target tissues.
Endocr Rev 18:502-519.
Gray A, Feldman H, McKinlay J, Longcope C (1991) Age, disease, and changing sex
hormone levels in middle-aged men: results of the Massachusetts Male Aging
Study. Journal of clinical endocrinology and metabolism 73:1016-1025.
Green PS, Simpkins JW (2000) Neuroprotective effects of estrogens: potential
mechanisms of action. Int J Dev Neurosci 18:347-358.
Green PS, Gridley KE, Simpkins JW (1996) Estradiol protects against beta-amyloid (25-
35)-induced toxicity in SK-N-SH human neuroblastoma cells. Neuroscience
Letters 218:165-168.
Green PS, Bishop J, Simpkins JW (1997) 17 alpha-estradiol exerts neuroprotective
effects on SK-N-SH cells. J Neurosci 17:511-515.
Green PS, Bales K, Paul S, Bu G (2005) Estrogen therapy fails to alter amyloid
deposition in the PDAPP model of Alzheimer's disease. Endocrinology 146:2774-
2781.
Greenfield JP, Leung LW, Cai D, Kaasik K, Gross RS, Rodriguez_Boulan E, Greengard
P, Xu H (2002) Estrogen lowers Alzheimer beta-amyloid generation by stimulating
trans-Golgi network vesicle biogenesis. J Biol Chem 277:12128-12136.
Gridley KE, Green PS, Simpkins JW (1997) Low concentrations of estradiol reduce b-
amyloid (25-35)-induced toxicity, lipid peroxidation and glucose utilization in
human SK-N-SH neuroblastoma cells. Brain Research 778:158-165.
170
Guerra-Araiza C, Amorim MA, Camacho-Arroyo I, Garcia-Segura LM (2007) Effects of
progesterone and its reduced metabolites, dihydroprogesterone and
tetrahydroprogesterone, on the expression and phosphorylation of glycogen
synthase kinase-3 and the microtubule-associated protein tau in the rat
cerebellum. Dev Neurobiol 67:510-520.
Guerra-Araiza C, Villamar-Cruz O, Gonzalez-Arenas A, Chavira R, Camacho-Arroyo I
(2003) Changes in progesterone receptor isoforms content in the rat brain during
the oestrous cycle and after oestradiol and progesterone treatments. J
Neuroendocrinol 15:984-990.
Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons
from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol 8:101-112.
Hallonquist JD, Seeman MV, Lang M, Rector NA (1993) Variation in symptom severity
over the menstrual cycle of schizophrenics. Biol Psychiatry 33:207-209.
Hamann S (2005) Sex differences in the responses of the human amygdala.
Neuroscientist 11:288-293.
Hammond GL, Rabe T, Wagner JD (2001) Preclinical profiles of progestins used in
formulations of oral contraceptives and hormone replacement therapy. Am J
Obstet Gynecol 185:S24-31.
Harburger LL, Bennett JC, Frick KM (2007) Effects of estrogen and progesterone on
spatial memory consolidation in aged females. Neurobiol Aging 28:602-610.
Harden CL (2008) Hormone replacement therapy: will it affect seizure control and AED
levels? Seizure 17:176-180.
Hardy J (1997) Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci
20:154-159.
Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and
problems on the road to therapeutics. Science 297:353-356.
Hardy JA, Higgins GA (1992) Alzheimer's disease: the amyloid cascade hypothesis.
Science 256:184-185.
Haren MT, Wittert GA, Chapman IM, Coates P, Morley JE (2005) Effect of oral
testosterone undecanoate on visuospatial cognition, mood and quality of life in
elderly men with low-normal gonadal status. Maturitas 50:124-133.
Haren MT, Kim MJ, Tariq SH, Wittert GA, Morley JE (2006) Andropause: a quality-of-life
issue in older males. Med Clin North Am 90:1005-1023.
171
Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA, Katzenellenbogen
BS (2003) Activities of estrogen receptor alpha- and beta-selective ligands at
diverse estrogen responsive gene sites mediating transactivation or
transrepression. Mol Cell Endocrinol 206:13-22.
Harris HA, Katzenellenbogen JA, Katzenellenbogen BS (2002) Characterization of the
biological roles of the estrogen receptors, ERalpha and ERbeta, in estrogen target
tissues in vivo through the use of an ERalpha-selective ligand. Endocrinology
143:4172-4177.
Harris-White ME, Chu T, Miller SA, Simmons M, Teter B, Nash D, Cole GM, Frautschy
SA (2001) Estrogen (E2) and glucocorticoid (Gc) effects on microglia and Ab
clearance in vitro and in vivo. Neurochemistry International 39:435-448.
Hart SA, Patton JD, Woolley CS (2001) Quantitative analysis of ERalpha and GAD
colocalization in the hippocampus of the adult female rat. The Journal of
Comparative Neurology 440:144-155.
Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe
DJ (1999) Protofibrillar intermediates of amyloid beta-protein induce acute
electrophysiological changes and progressive neurotoxicity in cortical neurons. J
Neurosci 19:8876-8884.
Haskell SG, Richardson ED, Horwitz RI (1997) The effect of estrogen replacement
therapy on cognitive function in women: A critical review of the literature. Journal
of Clinical Epidemiology 50:1249-1264.
Haslam SZ, Osuch JR, Raafat AM, Hofseth LJ (2002) Postmenopausal hormone
replacement therapy: effects on normal mammary gland in humans and in a
mouse postmenopausal model. J Mammary Gland Biol Neoplasia 7:93-105.
Hays J, Ockene JK, Brunner RL, Kotchen JM, Manson JE, Patterson RE, Aragaki AK,
Shumaker SA, Brzyski RG, LaCroix AZ, Granek IA, Valanis BG (2003) Effects of
estrogen plus progestin on health-related quality of life. N Engl J Med 348:1839-
1854.
Heikkinen T, Kalesnykas G, Rissanen A, Tapiola T, Iivonen S, Wang J, Chaudhuri J,
Tanila H, Miettinen R, Puolivali J (2004) Estrogen treatment improves spatial
learning in APP + PS1 mice but does not affect beta amyloid accumulation and
plaque formation. Exp Neurol 187:105-117.
Henderson V, Buckwalter J (1994) Cognitive deficits of men and women with
Alzheimer's disease. Neurology 44:90-96.
Henderson VW (2006) Estrogen-containing hormone therapy and Alzheimer's disease
risk: understanding discrepant inferences from observational and experimental
research. Neuroscience 138:1031-1039.
172
Henderson VW, Paganini-Hill A, Emanuel CK, Dunn ME, Buckwalter JG (1994) Estrogen
replacement therapy in older women. Comparisons between Alzheimer's disease
cases and nondemented control subjects. Arch Neurol 51:896-900.
Henderson VW, Guthrie JR, Dudley EC, Burger HG, Dennerstein L (2003) Estrogen
exposures and memory at midlife: a population-based study of women. Neurology
60:1369-1371.
Henderson VW, Benke KS, Green RC, Cupples LA, Farrer LA (2005) Postmenopausal
hormone therapy and Alzheimer's disease risk: interaction with age. J Neurol
Neurosurg Psychiatry 76:103-105.
Henderson VW, Paganini-Hill A, Miller BL, Elble RJ, Reyes PF, Shoupe D, McCleary
CA, Klein RA, Hake AM, Farlow MR (2000) Estrogen for Alzheimer's disease in
women: randomized, double-blind, placebo-controlled trial. Neurology 54:295-301.
Hennessy RJ, Lane A, Kinsella A, Larkin C, O'Callaghan E, Waddington JL (2004) 3D
morphometrics of craniofacial dysmorphology reveals sex-specific asymmetries in
schizophrenia. Schizophr Res 67:261-268.
Hestiantoro A, Swaab DF (2004) Changes in estrogen receptor-alpha and -beta in the
infundibular nucleus of the human hypothalamus are related to the occurrence of
Alzheimer's disease neuropathology. J Clin Endocrinol Metab 89:1912-1925.
Hirata-Fukae C, Li HF, Hoe HS, Gray AJ, Minami SS, Hamada K, Niikura T, Hua F,
Tsukagoshi-Nagai H, Horikoshi-Sakuraba Y, Mughal M, Rebeck GW, LaFerla FM,
Mattson MP, Iwata N, Saido TC, Klein WL, Duff KE, Aisen PS, Matsuoka Y (2008)
Females exhibit more extensive amyloid, but not tau, pathology in an Alzheimer
transgenic model. Brain Res 1216:92-103.
Hlatky MA, Boothroyd D, Vittinghoff E, Sharp P, Whooley MA (2002) Quality-of-life and
depressive symptoms in postmenopausal women after receiving hormone
therapy: results from the Heart and Estrogen/Progestin Replacement Study
(HERS) trial. Jama 287:591-597.
Hoffman GE, Moore N, Fiskum G, Murphy AZ (2003) Ovarian steroid modulation of
seizure severity and hippocampal cell death after kainic acid treatment. Exp
Neurol 182:124-134.
Hogervorst E, Combrinck M, Smith AD (2003) Testosterone and gonadotropin levels in
men with dementia. Neuroendocrinology Letters 24:203-208.
Hogervorst E, Boshuisen M, Riedel W, Willeken C, Jolles J (1999) 1998 Curt P. Richter
Award. The effect of hormone replacement therapy on cognitive function in elderly
women. Psychoneuroendocrinology 24:43-68.
173
Hogervorst E, Williams J, Budge M, Riedel W, Jolles J (2000) The nature of the effect of
female gonadal hormone replacement therapy on cognitive function in post-
menopausal women: a meta-analysis. Neuroscience 101:485-512.
Hogervorst E, Lehmann DJ, Warden DR, McBroom J, Smith AD (2002) Apolipoprotein E
epsilon4 and testosterone interact in the risk of Alzheimer's disease in men. Int J
Ger Psych 17:938-940.
Hogervorst E, Williams J, Biudge M, Barnetson L, Combrinck M, Smith AD (2001) Serum
total testosterone is lower in men with Alzheimer's disease. Neuro Endocrinol Lett
22:163-168.
Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, Wright K, Saad I,
Mueller R, Morgan D, Sanders S, Zehr C, O'Campo K, Hardy J, Prada CM,
Eckman C, Younkin S, Hsiao K, Duff K (1998) Accelerated Alzheimer-type
phenotype in transgenic mice carrying both mutant amyloid precursor protein and
presenilin 1 transgenes. Nat Med 4:97-100.
Honjo H, Iwasa K, Kawata M, Fushiki S, Hosoda T, Tatsumi H, Oida N, Mihara M,
Hirasugi Y, Yamamoto H, Kikuchi N, Kitawaki J (2005) Progestins and estrogens
and Alzheimer's disease. J Steroid Biochem Mol Biol 93:305-308.
Houtsmuller EJ, Brand T, de Jonge FH, Joosten RN, van de Poll NE, Slob AK (1994)
SDN-POA volume, sexual behavior, and partner preference of male rats affected
by perinatal treatment with ATD. Physiol Behav 56:535-541.
Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G
(1996) Correlative memory deficits, Abeta elevation, and amyloid plaques in
transgenic mice. Science 274:99-102.
Huang J, Guan H, Booze RM, Eckman CB, Hersh LB (2004) Estrogen regulates
neprilysin activity in rat brain. Neurosci Lett 367:85-87.
Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E (1998)
Randomized trial of estrogen plus progestin for secondary prevention of coronary
heart disease in postmenopausal women. Heart and Estrogen/progestin
Replacement Study (HERS) Research Group. Jama 280:605-613.
Hulley SB, Grady D (2004) The WHI estrogen-alone trial--do things look any better?
Jama 291:1769-1771.
Huppenbauer CB, Tanzer L, DonCarlos LL, Jones KJ (2005) Gonadal steroid
attenuation of developing hamster facial motoneuron loss by axotomy: equal
efficacy of testosterone, dihydrotestosterone, and 17-beta estradiol. J Neurosci
25:4004-4013.
174
Hurd PL, Bailey AA, Gongal PA, Yan RH, Greer JJ, Pagliardini S (2008) Intrauterine
position effects on anogenital distance and digit ratio in male and female mice.
Arch Sex Behav 37:9-18.
Husmann DA, McPhaul MJ (1991) Time-specific androgen blockade with flutamide
inhibits testicular descent in the rat. Endocrinology 129:1409-1416.
Hutter HS, Gibson MJ (1988) Effect of neonatal androgenization on positive feedback in
female mice. Biol Reprod 38:636-638.
Hyman BT, Gomez-Isla T, Briggs M, Chung H, Nichols S, Kohout F, Wallace R (1996)
Apolipoprotein E and cognitive change in an elderly population. Ann Neurol 40:55-
66.
Ibanez C, Guennoun R, Liere P, Eychenne B, Pianos A, El-Etr M, Baulieu EE,
Schumacher M (2003) Developmental expression of genes involved in
neurosteroidogenesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4
isomerase in the rat brain. Endocrinology 144:2902-2911.
Iguchi T, Takasugi N (1981) Occurrence of permanent anovulation in mouse ovaries and
permanent changes in the vaginal and uterine epithelia following neonatal
treatment with large doses of 5 alpha-dihydrotestosterone. Endocrinol Jpn 28:207-
213.
Iqbal K, Alonso Adel C, Chen S, Chohan MO, El-Akkad E, Gong CX, Khatoon S, Li B,
Liu F, Rahman A, Tanimukai H, Grundke-Iqbal I (2005) Tau pathology in
Alzheimer disease and other tauopathies. Biochim Biophys Acta 1739:198-210.
Irwin RW, Yao J, Hamilton RT, Cadenas E, Brinton RD, Nilsen J (2008) Progesterone
and estrogen regulate oxidative metabolism in brain mitochondria. Endocrinology
149:3167-3175.
Isgor C, Sengelaub DR (1998) Prenatal gonadal steroids affect adult spatial behavior,
CA1 and CA3 pyramidal cell morphology in rats. Horm Behav 34:183-198.
Isgor C, Sengelaub DR (2003) Effects of neonatal gonadal steroids on adult CA3
pyramidal neuron dendritic morphology and spatial memory in rats. J Neurobiol
55:179-190.
Ishizawa T, Sahara N, Ishiguro K, Kersh J, McGowan E, Lewis J, Hutton M, Dickson
DW, Yen SH (2003) Co-localization of glycogen synthase kinase-3 with
neurofibrillary tangles and granulovacuolar degeneration in transgenic mice. Am J
Pathol 163:1057-1067.
Islander U, Erlandsson MC, Hasseus B, Jonsson CA, Ohlsson C, Gustafsson JA,
Dahlgren U, Carlsten H (2003) Influence of oestrogen receptor alpha and beta on
the immune system in aged female mice. Immunology 110:149-157.
175
Ivins KJ, Bui ET, Cotman CW (1998) Beta-amyloid induces local neurite degeneration in
cultured hippocampal neurons: evidence for neuritic apoptosis. Neurobiol Dis
5:365-378.
Jaffe AB, Toran_Allerand CD, Greengard P, Gandy SE (1994) Estrogen regulates
metabolism of Alzheimer amyloid beta precursor protein. J Biol Chem 269:13065-
13068.
Janowsky JS, Oviatt SK, Orwoll ES (1994) Testosterone Influences Spatial Cognition in
Older Men. Behavioral Neuroscience 108:325-332.
Janowsky JS, Chavez B, Orwoll E (2000) Sex steroids modify working memory. Journal
of Cognitive Neuroscience 12:407-414.
Jayaraman A, Pike CJ (2009) Progesterone attenuates oestrogen neuroprotection via
downregulation of oestrogen receptor expression in cultured neurones. J
Neuroendocrinol 21:77-81.
Jelks KB, Wylie R, Floyd CL, McAllister AK, Wise P (2007) Estradiol targets synaptic
proteins to induce glutamatergic synapse formation in cultured hippocampal
neurons: critical role of estrogen receptor-alpha. J Neurosci 27:6903-6913.
Jezierski MK, Sohrabji F (2001) Neurotrophin expression in the reproductively senescent
forebrain is refractory to estrogen stimulation. Neurobiol Aging 22:309-319.
Johnson AB, Bake S, Lewis DK, Sohrabji F (2006) Temporal expression of IL-1beta
protein and mRNA in the brain after systemic LPS injection is affected by age and
estrogen. J Neuroimmunol 174:82-91.
Jones KJ, Brown TJ, Damaser M (2001) Neuroprotective effects of gonadal steroids on
regenerating peripheral motoneurons. Brain Res Brain Res Rev 37:372-382.
Jones KJ, Coers S, Storer PD, Tanzer L, Kinderman NB (1999) Androgenic regulation of
the central glia response following nerve damage. Journal of Neurobiology
40:560-573.
Jones RD, Pugh PJ, Hall J, Channer KS, Jones TH (2003) Altered circulating hormone
levels, endothelial function and vascular reactivity in the testicular feminised
mouse. European Journal of Endocrinology / European Federation of Endocrine
Societies 148:111-120.
Jorm AF, Korten AE, Henderson AS (1987) The prevalence of dementia: a quantitative
integration of the literature. Acta Psychiatr Scand 76:465-479.
Kadish I, Van Groen T (2002) Low levels of estrogen significantly diminish axonal
sprouting after entorhinal cortex lesions in the mouse. J Neurosci 22:4095-4102.
176
Kalesnykas G, Puolivali J, Sirvio J, Miettinen R (2004) Cholinergic neurons in the basal
forebrain of aged female mice. Brain Res 1022:148-156.
Kalesnykas G, Roschier U, Puolivali J, Wang J, Miettinen R (2005) The effect of aging
on the subcellular distribution of estrogen receptor-alpha in the cholinergic
neurons of transgenic and wild-type mice. Eur J Neurosci 21:1437-1442.
Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S,
Malinow R (2003) APP processing and synaptic function. Neuron 37:925-937.
Kato J, Hirata S, Nozawa A, Yamada-Mouri N (1994) Gene expression of progesterone
receptor isoforms in the rat brain. Horm Behav 28:454-463.
Kaufman JM, Vermeulen A (2005) The decline of androgen levels in elderly men and its
clinical and therapeutic implications. Endocr Rev.
Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A, Bacal C,
Lingle DD, Metter E (1997) A prospective study of estrogen replacement therapy
and the risk of developing Alzheimer's disease: the Baltimore Longitudinal Study
of Aging. Neurology 48:1517-1521.
Kawato S (2003) [Neurosteroids are 4th generation neuromessengers which are
synthesized and enhance/suppress learning and memory in the brain]. Seikagaku
75:1530-1535.
Keefe DL (2002) Sex hormones and neural mechanisms. Arch Sex Behav 31:401-403.
Kelly JF, Bienias JL, Shah A, Meeke KA, Schneider JA, Soriano E, Bennett DA (2008)
Levels of estrogen receptors alpha and beta in frontal cortex of patients with
Alzheimer's disease: relationship to Mini-Mental State Examination scores. Curr
Alzheimer Res 5:45-51.
Kesslak JP, Yuan D, Neeper S, Cotman CW (1995) Vulnerability of the hippocampus to
kainate excitotoxicity in the aged, mature and young adult rat. Neurosci Lett
188:117-120.
Kim H, Bang OY, Jung MW, Ha SD, Hong HS, Huh K, Kim SU, Mook-Jung I (2001)
Neuroprotective effects of estrogen against beta-amyloid toxicity are mediated by
estrogen receptors in cultured neuronal cells. Neurosci Lett 302:58-62.
King DL, Arendash GW (2002) Behavioral characterization of the Tg2576 transgenic
model of Alzheimer's disease through 19 months. Physiol Behav 75:627-642.
Koh JY, Yang LL, Cotman CW (1990) Beta-amyloid protein increases the vulnerability of
cultured cortical neurons to excitotoxic damage. Brain Research 533:315-320.
177
Komulainen MH, Kroger H, Tuppurainen MT, Heikkinen AM, Alhava E, Honkanen R,
Saarikoski S (1998) HRT and Vit D in prevention of non-vertebral fractures in
postmenopausal women; a 5 year randomized trial. Maturitas 31:45-54.
Koo E (1997) Phorbol esters affect multiple steps in beta-amyloid precursor protein
trafficking and amyloid beta-protein production. Mol Med 3:204-211.
Korach KS, McLachlan JA (1995) Techniques for detection of estrogenicity. Environ
Health Perspect 103 Suppl 7:5-8.
Koski CL, Hila S, Hoffman GE (2004) Regulation of cytokine-induced neuron death by
ovarian hormones: involvement of antiapoptotic protein expression and c-JUN N-
terminal kinase-mediated proapoptotic signaling. Endocrinology 145:95-103.
Krohmer RW, Baum MJ (1989) Effect of sex, intrauterine position and androgen
manipulation on the development of brain aromatase activity in fetal ferrets. J
Neuroendocrinol 1:265-271.
Kudwa AE, Bodo C, Gustafsson JA, Rissman EF (2005) A previously uncharacterized
role for estrogen receptor beta: defeminization of male brain and behavior. Proc
Natl Acad Sci U S A 102:4608-4612.
Kujawa KA, Kinderman NB, Jones KJ (1989) Testosterone-induced acceleration of
recovery from facial paralysis following crush axotomy of the facial nerve in male
hamsters. Exp Neurol 105:80-85.
Kujawa KA, Emeric E, Jones KJ (1991) Testosterone differentially regulates the
regenerative properties of injured hamster facial motoneurons. J Neurosci
11:3898-3906.
Kujawa KA, Jacob JM, Jones KJ (1993) Testosterone regulation of the regenerative
properties of injured rat sciatic motor neurons. J Neurosci Res 35:268-273.
Kujawa KA, Tanzer L, Jones KJ (1995) Inhibition of the accelerative effects of
testosterone on hamster facial nerve regeneration by the antiandrogen flutamide.
Exp Neurol 133:138-143.
Labombarda F, Gonzalez SL, Deniselle MC, Vinson GP, Schumacher M, De Nicola AF,
Guennoun R (2003) Effects of injury and progesterone treatment on progesterone
receptor and progesterone binding protein 25-Dx expression in the rat spinal cord.
J Neurochem 87:902-913.
LaFerla FM, Oddo S (2005) Alzheimer's disease: Abeta, tau and synaptic dysfunction.
Trends Mol Med 11:170-176.
Lange CA, Richer JK, Shen T, Horwitz KB (1998) Convergence of progesterone and
epidermal growth factor signaling in breast cancer. Potentiation of mitogen-
activated protein kinase pathways. J Biol Chem 273:31308-31316.
178
Lansing SW, Lonstein JS (2006) Tyrosine hydroxylase-synthesizing cells in the
hypothalamus of prairie voles (Microtus ochrogaster): sex differences in the
anteroventral periventricular preoptic area and effects of adult gonadectomy or
neonatal gonadal hormones. J Neurobiol 66:197-204.
Le Saux M, Di Paolo T (2005) Chronic estrogenic drug treatment increases
preproenkephalin mRNA levels in the rat striatum and nucleus accumbens.
Psychoneuroendocrinology 30:251-260.
Le Saux M, Estrada-Camarena E, Di Paolo T (2006) Selective estrogen receptor-alpha
but not -beta agonist treatment modulates brain alpha-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid receptors. J Neurosci Res 84:1076-1084.
LeBlanc ES, Janowsky J, Chan BKS, Nelson HD (2001) Hormone replacement therapy
and cognition: Systematic review and meta-analysis. Journal of the American
Medical Association 285:1489.
LeFevre J, McClintock MK (1988) Reproductive senescence in female rats: a
longitudinal study of individual differences in estrous cycles and behavior. Biol
Reprod 38:780-789.
Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH
(2006) A specific amyloid-beta protein assembly in the brain impairs memory.
Nature 440:352-357.
Levin-Allerhand JA, Lominska CE, Wang J, Smith JD (2002) 17Alpha-estradiol and
17beta-estradiol treatments are effective in lowering cerebral amyloid-beta levels
in AbetaPPSWE transgenic mice. J Alzheimers Dis 4:449-457.
Li R, Shen Y, Yang LB, Lue LF, Finch C, Rogers J (2000) Estrogen enhances uptake of
amyloid beta-protein by microglia derived from the human cortex. Journal of
Neurochemistry 75:1447-1454.
Liehr JG, Roy D (1990) Free radical generation by redox cycling of estrogens. Free
Radical Biology and Medicine 8:415-423.
Linford N, Wade C, Dorsa D (2000) The rapid effects of estrogen are implicated in
estrogen-mediated neuroprotection. J Neurocytol 29:367-374.
Liu XA, Zhu LQ, Zhang Q, Shi HR, Wang SH, Wang Q, Wang JZ (2008) Estradiol
attenuates tau hyperphosphorylation induced by upregulation of protein kinase-A.
Neurochem Res 33:1811-1820.
Lu PH, Masterman DA, Mulnard R, Cotman C, Miller B, Yaffe K, Reback E, Porter V,
Swerdloff R, Cummings JL (2006) Effects of testosterone on cognition and mood
in male patients with mild Alzheimer disease and healthy elderly men. Arch Neurol
63:177-185.
179
Lu YP, Zeng M, Swaab DF, Ravid R, Zhou JN (2004) Colocalization and alteration of
estrogen receptor-alpha and -beta in the hippocampus in Alzheimer's disease.
Hum Pathol 35:275-280.
Lund TD, Rovis T, Chung WC, Handa RJ (2005) Novel actions of estrogen receptor-beta
on anxiety-related behaviors. Endocrinology 146:797-807.
Lutchmaya S, Baron-Cohen S, Raggatt P, Knickmeyer R, Manning JT (2004) 2nd to 4th
digit ratios, fetal testosterone and estradiol. Early Hum Dev 77:23-28.
Madeira MD, Lieberman AR (1995) Sexual dimorphism in the mammalian limbic system.
Prog Neurobiol 45:275-333.
Maki P, Hogervorst E (2003) The menopause and HRT. HRT and cognitive decline. Best
Pract Res Clin Endocrinol Metab 17:105-122.
Maki PM (2005) A systematic review of clinical trials of hormone therapy on cognitive
function: effects of age at initiation and progestin use. Ann N Y Acad Sci
1052:182-197.
Maki PM (2006) Hormone therapy and cognitive function: is there a critical period for
benefit? Neuroscience 138:1027-1030.
Maki PM, Ernst M, London ED, Mordecai KL, Perschler P, Durso SC, Brandt J, Dobs A,
Resnick SM (2007) Intramuscular testosterone treatment in elderly men: evidence
of memory decline and altered brain function. J Clin Endocrinol Metab 92:4107-
4114.
Manly JJ, Merchant CA, Jacobs DM, Small S, Bell K, Ferin M, Mayeux R (2000)
Endogenous estrogen levels and Alzheimer's disease among postmenopausal
women. Neurology 54:833-837.
Manthey D, Heck S, Engert S, Behl C (2001) Estrogen induces a rapid secretion of
amyloid b precursor protein via the mitogen-activated protein kinase pathway.
European Journal of Biochemistry 268:4285-4291.
Mark R, Hensley K, Butterfield D, Mattson M (1995) Amyloid beta-peptide impairs ion-
motive ATPase activities: evidence for a role in loss of neuronal Ca2+
homeostasis and cell death. Journal of Neuroscience 15:6239-6249.
Mattson MP, Partin J, Begley JG (1998) Amyloid beta-peptide induces apoptosis-related
events in synapses and dendrites. Brain Res 807:167-176.
McCarthy MM, Schwarz JM, Wright CL, Dean SL (2008) Mechanisms mediating
oestradiol modulation of the developing brain. J Neuroendocrinol 20:777-783.
180
McCormick CM, Mahoney E (1999) Persistent effects of prenatal, neonatal, or adult
treatment with flutamide on the hypothalamic-pituitary-adrenal stress response of
adult male rats. Horm Behav 35:90-101.
McCormick CM, Furey BF, Child M, Sawyer MJ, Donohue SM (1998) Neonatal sex
hormones have 'organizational' effects on the hypothalamic-pituitary-adrenal axis
of male rats. Brain Res Dev Brain Res 105:295-307.
McEwen BS (1991) Non-genomic and genomic effects of steroids on neural activity.
Trends Pharmacol Sci 12:141-147.
Meek LR, Schulz KM, Keith CA (2006) Effects of prenatal stress on sexual partner
preference in mice. Physiol Behav 89:133-138.
Mehra RD, Sharma K, Nyakas C, Vij U (2005) Estrogen receptor alpha and beta
immunoreactive neurons in normal adult and aged female rat hippocampus: a
qualitative and quantitative study. Brain Res 1056:22-35.
Meier DE, Orwoll ES, Keenan EJ, Fagerstrom RM (1987) Marked decline in trabecular
bone mineral content in healthy men with age: lack of association with sex steroid
levels. J Am Geriatr Soc 35:189-197.
Melcangi RC, Panzica GC (2006) Neuroactive steroids: old players in a new game.
Neuroscience 138:733-739.
Mendrek A (2007) Reversal of normal cerebral sexual dimorphism in schizophrenia:
evidence and speculations. Med Hypotheses 69:896-902.
Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS, Katzenellenbogen
JA (2001) Estrogen receptor-beta potency-selective ligands: structure-activity
relationship studies of diarylpropionitriles and their acetylene and polar analogues.
J Med Chem 44:4230-4251.
Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, Pfaff DW, Ogawa S,
Rohrer SP, Schaeffer JM, McEwen BS, Alves SE (2003) Immunolocalization of
estrogen receptor beta in the mouse brain: comparison with estrogen receptor
alpha. Endocrinology 144:2055-2067.
Miyata K, Yabushita S, Sano M, Miyashita K, Okuno Y, Matsuo M (2003) Effects of
perinatal exposure to flutamide on sex hormone responsiveness in F1 male rats. J
Toxicol Sci 28:149-163.
Mize AL, Young LJ, Alper RH (2003) Uncoupling of 5-HT1A receptors in the brain by
estrogens: regional variations in antagonism by ICI 182,780. Neuropharmacology
44:584-591.
181
Moffat SD, Zonderman AB, Metter EJ, Blackman MR, Harman SM, Resnick SM (2002)
Longitudinal assessment of serum free testosterone concentration predicts
memory performance and cognitive status in elderly men. J Clin Endo Metab
87:5001-5007.
Moffat SD, Zonderman AB, Metter EJ, Kawas C, Blackman MR, Harman SM, Resnick
SM (2004) Free testosterone and risk for Alzheimer disease in older men.
Neurology 62:188-193.
Molsa PK, Marttila RJ, Rinne UK (1982) Epidemiology of dementia in a Finnish
population. Acta Neurol Scand 65:541-552.
Mook-Jung I, Joo I, Sohn S, Kwon HJ, Huh K, Jung MW (1997) Estrogen blocks
neurotoxic effects of beta-amyloid (1-42) and induces neurite extension on B103
cells. Neurosci Lett 235:101-104.
Morissette M, Jourdain S, Al Sweidi S, Menniti FS, Ramirez AD, Di Paolo T (2007) Role
of estrogen receptors in neuroprotection by estradiol against MPTP toxicity.
Neuropharmacology 52:1509-1520.
Morissette M, Le Saux M, D'Astous M, Jourdain S, Al Sweidi S, Morin N, Estrada-
Camarena E, Mendez P, Garcia-Segura LM, Di Paolo T (2008) Contribution of
estrogen receptors alpha and beta to the effects of estradiol in the brain. J Steroid
Biochem Mol Biol 108:327-338.
Morley JE (2001) Androgens and aging. Maturitas 38:61-71; discussion 71-63.
Morley JE, Perry HM (2003) Androgen treatment of male hypogonadism in older males.
The Journal of Steroid Biochemistry and Molecular Biology 85:367-373.
Morley JE, Kaiser FE, Perry HM, Patrick P, Morley PM, Stauber PM, Vellas B,
Baumgartner RN, Garry PJ (1997) Longitudinal changes in testosterone,
luteinizing hormone, and follicle-stimulating hormone in healthy older men.
Metabolism: Clinical and Experimental 46:410-413.
Morris JA, Jordan CL, Breedlove SM (2004) Sexual differentiation of the vertebrate
nervous system. Nat Neurosci 7:1034-1039.
Moss RL, Gu Q (1999) Estrogen: mechanisms for a rapid action in CA1 hippocampal
neurons. Steroids 64:14-21.
Mukai H, Tsurugizawa T, Ogiue-Ikeda M, Murakami G, Hojo Y, Ishii H, Kimoto T, Kawato
S (2006) Local neurosteroid production in the hippocampus: influence on synaptic
plasticity of memory. Neuroendocrinology 84:255-263.
182
Mukai H, Tsurugizawa T, Murakami G, Kominami S, Ishii H, Ogiue-Ikeda M, Takata N,
Tanabe N, Furukawa A, Hojo Y, Ooishi Y, Morrison JH, Janssen WG, Rose JA,
Chambon P, Kato S, Izumi S, Yamazaki T, Kimoto T, Kawato S (2007) Rapid
modulation of long-term depression and spinogenesis via synaptic estrogen
receptors in hippocampal principal neurons. J Neurochem 100:950-967.
Muller M, den Tonkelaar I, Thijssen JH, Grobbee DE, van der Schouw YT (2003)
Endogenous sex hormones in men aged 40-80 years. Eur J Endocrinol 149:583-
589.
Mulnard RA, Cotman CW, Kawas C, van Dyck CH, Sano M, Doody R, Koss E, Pfeiffer
E, Jin S, Gamst A, Grundman M, Thomas R, Thal LJ (2000) Estrogen
replacement therapy for treatment of mild to moderate Alzheimer disease: a
randomized controlled trial. Alzheimer's Disease Cooperative Study. Jama
283:1007-1015.
Murphy DD, Segal M (2000) Progesterone prevents estradiol-induced dendritic spine
formation in cultured hippocampal neurons. Neuroendocrinology 72:133-143.
Murphy SJ, Littleton-Kearney MT, Hurn PD (2002) Progesterone administration during
reperfusion, but not preischemia alone, reduces injury in ovariectomized rats. J
Cereb Blood Flow Metab 22:1181-1188.
Negri-Cesi P, Poletti A, Celotti F (1996) Metabolism of steroids in the brain: a new
insight into the role of 5alpha-reductase and aromatase in brain differentiation and
functions. J Steroid Biochem Mol Biol 58:455-466.
Nelson JF, Felicio LS, Osterburg HH, Finch CE (1981) Altered profiles of estradiol and
progesterone associated with prolonged estrous cycles and persistent vaginal
cornification in aging C57BL/6J mice. Biol Reprod 24:784-794.
Nguyen T-VV, Yao M, Pike CJ (2005) Androgens activate mitogen-activated protein
kinase signaling: Role in neuroprotection. Journal of Neurochemistry 94:1639-
1651.
Nilsen J, Brinton RD (2002) Impact of progestins on estradiol potentiation of the
glutamate calcium response. Neuroreport 13:825-830.
Nilsen J, Diaz-Brinton R (2003) Mechanism of estrogen-mediated neuroprotection:
regulation of mitochondrial calcium and Bcl-2 expression. Proceedings of the
National Academy of Sciences of the United States of America 100:2842-2847.
Nilsen J, Brinton RD (2003) Divergent impact of progesterone and medroxyprogesterone
acetate (Provera) on nuclear mitogen-activated protein kinase signaling. Proc Natl
Acad Sci U S A 100:10506-10511.
Nilsen J, Mor G, Naftolin F (1998) Raloxifene induces neurite outgrowth in estrogen
receptor positive PC12 cells. Menopause 5:211-216.
183
Nilsen J, Chen S, Brinton RD (2002) Dual action of estrogen on glutamate-induced
calcium signaling: mechanisms requiring interaction between estrogen receptors
and src/mitogen activated protein kinase pathway. Brain Res 930:216-234.
Nordell VL, Scarborough MM, Buchanan AK, Sohrabji F (2003) Differential effects of
estrogen in the injured forebrain of young adult and reproductive senescent
animals. Neurobiol Aging 24:733-743.
Nwagwu MO, Baines H, Kerr JB, Ebling FJ (2005) Neonatal androgenization of
hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production
and spermatogenesis. Reprod Biol Endocrinol 3:48.
O'Neill K, Chen S, Brinton RD (2004a) Impact of the selective estrogen receptor
modulator, raloxifene, on neuronal survival and outgrowth following toxic insults
associated with aging and Alzheimer's disease. Exp Neurol 185:63-80.
O'Neill K, Chen S, Diaz Brinton R (2004b) Impact of the selective estrogen receptor
modulator, tamoxifen, on neuronal outgrowth and survival following toxic insults
associated with aging and Alzheimer's disease. Exp Neurol 188:268-278.
Oddo S, Caccamo A, Kitazawa M, Tseng BP, LaFerla FM (2003a) Amyloid deposition
precedes tangle formation in a triple transgenic model of Alzheimer's disease.
Neurobiol Aging 24:1063-1070.
Oddo S, Vasilevko V, Caccamo A, Kitazawa M, Cribbs DH, Laferla FM (2006a)
Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates
cognitive decline in transgenic mice with plaques and tangles. J Biol Chem.
Oddo S, Caccamo A, Tran L, Lambert MP, Glabe CG, Klein WL, LaFerla FM (2006b)
Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of
Alzheimer disease. A link between Abeta and tau pathology. J Biol Chem
281:1599-1604.
Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R,
Mattson MP, Akbari Y, LaFerla FM (2003b) Triple-transgenic model of Alzheimer's
disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.
Neuron 39:409-421.
Ogiue-Ikeda M, Tanabe N, Mukai H, Hojo Y, Murakami G, Tsurugizawa T, Takata N,
Kimoto T, Kawato S (2008) Rapid modulation of synaptic plasticity by estrogens
as well as endocrine disrupters in hippocampal neurons. Brain Res Rev 57:363-
375.
Ohkura T, Isse K, Akazawa K, Hamamoto M, Yaoi Y, Hagino N (1994) Evaluation of
estrogen treatment in female patients with dementia of the Alzheimer type. Endocr
J 41:361-371.
184
Olariu A, Yamada K, Nabeshima T (2005) Amyloid pathology and protein kinase C
(PKC): possible therapeutics effects of PKC activators. J Pharmacol Sci 97:1.
Orlando R, Caruso A, Molinaro G, Motolese M, Matrisciano F, Togna G, Melchiorri D,
Nicoletti F, Bruno V (2007) Nanomolar concentrations of anabolic-androgenic
steroids amplify excitotoxic neuronal death in mixed mouse cortical cultures. Brain
Res 1165:21-29.
Paganini-Hill A, Henderson VW (1994) Estrogen deficiency and risk of Alzheimer's
disease in women. American Journal of Epidemiology 140:256-261.
Paganini-Hill A, Henderson VW (1996) Estrogen replacement therapy and risk of
Alzheimer disease. Arch Intern Med 156:2213-2217.
Paganini-Hill A, Clark LJ (2000) Preliminary assessment of cognitive function in breast
cancer patients treated with tamoxifen. Breast Cancer Res Treat 64:165-176.
Paoletti AM, Congia S, Lello S, Tedde D, Orru M, Pistis M, Pilloni M, Zedda P, Loddo A,
Melis GB (2004) Low androgenization index in elderly women and elderly men
with Alzheimer's disease. Neurology 62:301-303.
Papasozomenos S, Shanavas A (2002) Testosterone prevents the heat shock-induced
overactivation of glycogen synthase kinase-3 beta but not of cyclin-dependent
kinase 5 and c-Jun NH2-terminal kinase and concomitantly abolishes
hyperphosphorylation of tau: implications for Alzheimer's disease. Proc Natl Acad
Sci U S A 99:1140-1145.
Papasozomenos SC (1997) The heat shock-induced hyperphosphorylation of tau is
estrogen-independent and prevented by androgens: implications for Alzheimer
disease. Proc Natl Acad Sci U S A 94:6612-6617.
Papasozomenos SC, Papasozomenos T (1999) Androgens Prevent the Heat Shock-
Induced Hyperphosphorylation but not Dephosphorylation of t in Female Rats.
Implications for Alzheimer's Disease. J Alzheimers Dis 1:147-153.
Park SY, Tournell C, Sinjoanu RC, Ferreira A (2007) Caspase-3- and calpain-mediated
tau cleavage are differentially prevented by estrogen and testosterone in beta-
amyloid-treated hippocampal neurons. Neuroscience 144:119-127.
Pasqualini JR (2007) Progestins and breast cancer. Gynecol Endocrinol 23 Suppl 1:32-
41.
Patrone C, Andersson S, Korhonen L, Lindholm D (1999) Estrogen receptor-dependent
regulation of sensory neuron survival in developing dorsal root ganglion. Proc Natl
Acad Sci U S A 96:10905-10910.
185
Pennanen C, Kivipelto M, Tuomainen S, Hartikainen P, Hanninen T, Laakso MP,
Hallikainen M, Vanhanen M, Nissinen A, Helkala EL, Vainio P, Vanninen R,
Partanen K, Soininen H (2004) Hippocampus and entorhinal cortex in mild
cognitive impairment and early AD. Neurobiol Aging 25:303-310.
PEPI WG (1995) Effects of estrogen or estrogen/progestin regimens on heart disease
risk factors in postmenopausal women. The Postmenopausal Estrogen/Progestin
Interventions (PEPI) Trial. . Jama 273:199-208.
Perillo B, Sasso A, Abbondanza C, Palumbo G (2000) 17beta-estradiol inhibits
apoptosis in MCF-7 cells, inducing bcl-2 expression via two estrogen-responsive
elements present in the coding sequence. Mol Cell Biol 20:2890-2901.
Persson I, Adami HO, Bergkvist L, Lindgren A, Pettersson B, Hoover R, Schairer C
(1989) Risk of endometrial cancer after treatment with oestrogens alone or in
conjunction with progestogens: results of a prospective study. Bmj 298:147-151.
Petanceska SS, Nagy V, Frail D, Gandy S (2000) Ovariectomy and 17beta-estradiol
modulate the levels of Alzheimer's amyloid beta peptides in brain. Exp Gerontol
35:1317-1325.
Pike CJ (1999) Estrogen modulates neuronal Bcl-xL expression and beta-amyloid-
induced apoptosis: relevance to Alzheimer's disease. J Neurochem 72:1552-
1563.
Pike CJ (2001) Testosterone attenuates beta-amyloid toxicity in cultured hippocampal
neurons. Brain Research 919:160-165.
Pike CJ, Cummings BJ, Cotman CW (1992) beta-Amyloid induces neuritic dystrophy in
vitro: similarities with Alzheimer pathology. Neuroreport 3:769-772.
Pike CJ, Walencewicz AJ, Glabe CG, Cotman CW (1991) Aggregation-related toxicity of
synthetic beta-amyloid protein in hippocampal cultures. Eur J Pharmacol 207:367-
368.
Pike CJ, Cummings BJ, Monzavi R, Cotman CW (1994) Beta-amyloid-induced changes
in cultured astrocytes parallel reactive astrocytosis associated with senile plaques
in Alzheimer's disease. Neurosci 63:517-531.
Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegeneration
induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J
Neurosci 13:1676-1687.
Pinkerton JV, Henderson VW (2005) Estrogen and cognition, with a focus on
Alzheimer's disease. Semin Reprod Med 23:172-179.
186
Polo-Kantola P, Portin R, Polo O, Helenius H, Irjala K, Erkkola R (1998) The effect of
short-term estrogen replacement therapy on cognition: a randomized, double-
blind, cross-over trial in postmenopausal women. Obstet Gynecol 91:459-466.
Pomp D, Geisert RD, Durham CM, Murray JD (1995) Rescue of pregnancy and
maintenance of corpora lutea in infertile transgenic mice expressing an ovine
metallothionein 1a-ovine growth hormone fusion gene. Biol Reprod 52:170-178.
Pow DV, Perry VH, Morris JF, Gordon S (1989) Microglia in the neurohypophysis
associate with and endocytose terminal portions of neurosecretory neurons.
Neuroscience 33:567-578.
Qiu J, Bosch MA, Tobias SC, Grandy DK, Scanlan TS, Ronnekleiv OK, Kelly MJ (2003)
Rapid Signaling of Estrogen in Hypothalamic Neurons Involves a Novel G-Protein-
Coupled Estrogen Receptor that Activates Protein Kinase C. J Neurosci 23:9529-
9540.
Raber J (2004) Androgens, apoE, and Alzheimer's disease. 2004:re2.
Raber J (2008) AR, apoE, and cognitive function. Horm Behav 53:706-715.
Ramsden M, Shin TM, Pike CJ (2003) Androgens modulate neuronal vulnerability to
kainate lesion. Neuroscience 122:573-578.
Rapp SR, Espeland MA, Shumaker SA, Henderson VW, Brunner RL, Manson JE, Gass
ML, Stefanick ML, Lane DS, Hays J, Johnson KC, Coker LH, Dailey M, Bowen D
(2003) Effect of estrogen plus progestin on global cognitive function in
postmenopausal women: the Women's Health Initiative Memory Study: a
randomized controlled trial. Jama 289:2663-2672.
Rasmuson S, Nasman B, Carlstrom K, Olsson T (2002) Increased levels of
adrenocortical and gonadal hormones in mild to moderate Alzheimer's disease.
Dementia and Geriatric Cognitive Disorders 13:74-79.
Regan RF, Guo Y (1997) Estrogens attenuate neuronal injury due to hemoglobin,
chemical hypoxia, and excitatory amino acids in murine cortical cultures. Brain
Research 764:133-140.
Resnick SM, Henderson VW (2002) Hormone therapy and risk of Alzheimer disease: a
critical time. Jama 288:2170-2172.
Rhodes ME, Frye CA (2004a) Progestins in the hippocampus of female rats have
antiseizure effects in a pentylenetetrazole seizure model. Epilepsia 45:1531-1538.
Rhodes ME, Frye CA (2004b) Androgens in the hippocampus can alter, and be altered
by, ictal activity. Pharmacol Biochem Behav 78:483-493.
187
Rhodes ME, Frye CA (2006) ERbeta-selective SERMs produce mnemonic-enhancing
effects in the inhibitory avoidance and water maze tasks. Neurobiol Learn Mem
85:183-191.
Rice MM, Graves AB, McCurry SM, Gibbons LE, Bowen JD, McCormick WC, Larson EB
(2000) Postmenopausal estrogen and estrogen-progestin use and 2-year rate of
cognitive change in a cohort of older Japanese American women: The Kame
Project. Arch Intern Med 160:1641-1649.
Rice-Evans C, Miller N, Paganga G (1997) Antioxidant properties of phenolic
compounds. Trends in Plant Science 2:152-159.
Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity relationships
of flavonoids and phenolic acids. Free Radical Biology and Medicine 20:933-956.
Riederer P, Hoyer S (2006) From benefit to damage. Glutamate and advanced glycation
end products in Alzheimer brain. J Neural Transm 113:1671-1677.
Rocca WA, Amaducci LA, Schoenberg BS (1986) Epidemiology of clinically diagnosed
Alzheimer's disease. Ann Neurol 19:415-424.
Rogers J, Strohmeyer R, Kovelowski C, Li RG (2002) Microglia and inflammatory
mechanisms in the clearance of amyloid beta peptide. Glia 40:260–269.
Roof RL, Duvdevani R, Braswell L, Stein DG (1994) Progesterone facilitates cognitive
recovery and reduces secondary neuronal loss caused by cortical contusion injury
in male rats. Exp Neurol 129:64-69.
Rosario ER, Pike CJ (2008) Androgen regulation of beta-amyloid protein and the risk of
Alzheimer's disease. Brain Res Rev 57:444-453.
Rosario ER, Ramsden M, Pike CJ (2006a) Progestins inhibit the neuroprotective effects
of estrogen in rat hippocampus. Brain Res 1099:206-210.
Rosario ER, Carroll JC, Oddo S, LaFerla FM, Pike CJ (2006b) Androgens regulate the
development of neuropathology in a triple transgenic mouse model of Alzheimer's
disease. J Neurosci 26:13384-13389.
Rosario ER, Chang, L., Stanczyk, F. Z., Pike, C. J. (2004) Age-related testosterone
depletion and the development of Alzheimer disease. Jama 292:1431-1432.
Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML,
Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J
(2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal
women: principal results From the Women's Health Initiative randomized
controlled trial. Jama 288:321-333.
188
Rowan MJ, Klyubin I, Wang Q, Hu NW, Anwyl R (2007) Synaptic memory mechanisms:
Alzheimer's disease amyloid beta-peptide-induced dysfunction. Biochem Soc
Trans 35:1219-1223.
Rozovsky I, Hoving S, Anderson CP, O'Callaghan J, Finch CE (2002) Equine estrogens
induce apolipoprotein E and glial fibrillary acidic protein in mixed glial cultures.
Neurosci Lett 323:191-194.
Rubin BS, Fox TO, Bridges RS (1986) Estrogen binding in nuclear and cytosolic extracts
from brain and pituitary of middle-aged female rats. Brain Res 383:60-67.
Ruitenberg A, Ott A, van Swieten JC, Hofman A, Breteler MMB (2001) Incidence of
dementia: does gender make a difference? Neurobiology of Aging 22:575-580.
Sastre M, Walter J, Gentleman SM (2008) Interactions between APP secretases and
inflammatory mediators. J Neuroinflammation 5:25.
Savaskan E, Olivieri G, Meier F, Ravid R, Muller-Spahn F (2001) Hippocampal estrogen
beta-receptor immunoreactivity is increased in Alzheimer's disease. Brain Res
908:113-119.
Schmitz C, Rutten BP, Pielen A, Schafer S, Wirths O, Tremp G, Czech C, Blanchard V,
Multhaup G, Rezaie P, Korr H, Steinbusch HW, Pradier L, Bayer TA (2004)
Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse
model of Alzheimer's disease. Am J Pathol 164:1495-1502.
Schultz C, Braak H, Braak E (1996) A sex difference in neurodegeneration of the human
hypothalamus. Neurosci Lett 212:103-106.
Schultz C, Ghebremedhin E, Braak E, Braak H (1999) Sex-dependent cytoskeletal
changes of the human hypothalamus develop independently of Alzheimer's
disease. Exp Neurol 160:186-193.
Schumacher M, Sitruk-Ware R, De Nicola AF (2008) Progesterone and progestins:
neuroprotection and myelin repair. Curr Opin Pharmacol 8:740-746.
Schumacher M, Guennoun R, Stein DG, De Nicola AF (2007) Progesterone:
Therapeutic opportunities for neuroprotection and myelin repair. Pharmacol Ther
116:77-106.
Schupf N, Winsten S, Patel B, Pang D, Ferin M, Zigman WB, Silverman W, Mayeux R
(2006) Bioavailable estradiol and age at onset of Alzheimer's disease in
postmenopausal women with Down syndrome. Neurosci Lett 406:298-302.
Schupf N, Lee JH, Wei M, Pang D, Chace C, Cheng R, Zigman WB, Tycko B, Silverman
W (2008) Estrogen receptor-alpha variants increase risk of Alzheimer's disease in
women with Down syndrome. Dement Geriatr Cogn Disord 25:476-482.
189
Schwendimann RN, Alekseeva N (2007) Gender issues in multiple sclerosis. Int Rev
Neurobiol 79:377-392.
Seale JV, Wood SA, Atkinson HC, Lightman SL, Harbuz MS (2005a) Organizational role
for testosterone and estrogen on adult hypothalamic-pituitary-adrenal axis activity
in the male rat. Endocrinology 146:1973-1982.
Seale JV, Wood SA, Atkinson HC, Harbuz MS, Lightman SL (2005b) Postnatal
masculinization alters the HPA axis phenotype in the adult female rat. J Physiol
563:265-274.
Selkoe DJ, Yamazaki T, Citron M, Podlisny MB, Koo EH, Teplow DB, C. Haass (1996)
The Role of APP Processing and Trafficking Pathways in the Formation of
Amyloid β-Proteina. Annals of the New York Academy of Sciences
777:57-64.
Selvamani A, Sohrabji F (2008) Reproductive age modulates the impact of focal
ischemia on the forebrain as well as the effects of estrogen treatment in female
rats. Neurobiol Aging.
Sétáló G, Singh M, Nethrapalli IS, Toran-Allerand CD (2005) Protein Kinase C Activity is
Necessary for Estrogen-Induced Erk Phosphorylation in Neocortical Explants.
Journal Neurochemical Research 30:779-790.
Shanavas A, Papasozomenos SC (2000) tau kinases in the rat heat shock model:
possible implications for Alzheimer disease. Proc Natl Acad Sci U S A 97:14139-
14144.
Shang Y, Brown M (2002) Molecular determinants for the tissue specificity of SERMs.
Science 295:2465-2468.
Shansky RM, Glavis-Bloom C, Lerman D, McRae P, Benson C, Miller K, Cosand L,
Horvath TL, Arnsten AF (2004) Estrogen mediates sex differences in stress-
induced prefrontal cortex dysfunction. Mol Psychiatry 9:531-538.
Sheffield-Moore M, Urban RJ (2004) An overview of the endocrinology of skeletal
muscle. Trends Endocrinol Metab 15:110-115.
Sheldahl LC, Marriott LK, Bryant DM, Shapiro RA, Dorsa DM (2007) Neuroprotective
effects of estrogen and selective estrogen receptor modulators begin at the
plasma membrane. Minerva Endocrinol 32:87-94.
Sherwin BB (1988) Estrogen and/or androgen replacement therapy and cognitive
functioning in surgically menopausal women. Psychoneuroendocrinology 13:345.
Sherwin BB (2003) Estrogen and Cognitive Functioning in Women. Endocrine Reviews
24:133-151.
190
Sherwin BB, Henry JF (2008) Brain aging modulates the neuroprotective effects of
estrogen on selective aspects of cognition in women: a critical review. Front
Neuroendocrinol 29:88-113.
Shi J, Panickar KS, Yang S-H, Rabbani O, Day AL, Simpkins JW (1998) Estrogen
attenuates over-expression of b-amyloid precursor protein messager RNA in an
animal model of focal ischemia. Brain Research 810:87-92.
Shilling V, Jenkins V, Fallowfield L, Howell T (2003) The effects of hormone therapy on
cognition in breast cancer. J Steroid Biochem Mol Biol 86:405-412.
Shughrue PJ, Merchenthaler I (2001) Distribution of estrogen receptor beta
immunoreactivity in the rat central nervous system. J Comp Neurol 436:64-81.
Shughrue PJ, Lane MV, Merchenthaler I (1997) Comparative distribution of estrogen
receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol
388:507-525.
Shultz JM, Zhu XD, Knopp RH, Leboeuf RC, Rosenfeld ME (2004) Norgestimate and
medroxyprogesterone acetate do not attenuate the atheroprotective effects of
17beta-estradiol in ovariectomized, apolipoprotein E-deficient mice. Fertil Steril 82
Suppl 3:1133-1139.
Shumaker SA, Legault C, Kuller L, Rapp SR, Thal L, Lane DS, Fillit H, Stefanick ML,
Hendrix SL, Lewis CE, Masaki K, Coker LH (2004) Conjugated equine estrogens
and incidence of probable dementia and mild cognitive impairment in
postmenopausal women: Women's Health Initiative Memory Study. Jama
291:2947-2958.
Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones
BN, 3rd, Assaf AR, Jackson RD, Kotchen JM, Wassertheil-Smoller S, Wactawski-
Wende J (2003) Estrogen plus progestin and the incidence of dementia and mild
cognitive impairment in postmenopausal women: the Women's Health Initiative
Memory Study: a randomized controlled trial. Jama 289:2651-2662.
Silva I, Mello LE, Freymuller E, Haidar MA, Baracat EC (2000) Estrogen, progestogen
and tamoxifen increase synaptic density of the hippocampus of ovariectomized
rats. Neurosci Lett 291:183-186.
Simerly RB, Chang C, Muramatsu M, Swanson LW (1990) Distribution of androgen and
estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization
study. The Journal of Comparative Neurology 294:76-95.
Simpkins JW, Green PS, Gridley KE, Singh M, de Fiebre NC, Rajakumar G (1997a)
Role of estrogen replacement therapy in memory enhancement and the
prevention of neuronal loss associated with Alzheimer's disease. Am J Med
103:19S-25S.
191
Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ, Bodor N,
Day AL (1997b) Estrogens may reduce mortality and ischemic damage caused by
middle cerebral artery occlusion in the female rat. J Neurosurg 87:724-730.
Singer CA, Rogers KL, Dorsa DM (1998) Modulation of Bcl-2 expression: a potential
component of estrogen protection in NT2 neurons. Neuroreport 9:2565-2568.
Singer CA, Rogers KL, Strickland TM, Dorsa DM (1996) Estrogen protects primary
cortical neurons from glutamate toxicity. Neuroscience Letters 212:13-16.
Singer CA, Figueroa_Masot XA, Batchelor RH, Dorsa DM (1999) The mitogen-activated
protein kinase pathway mediates estrogen neuroprotection after glutamate toxicity
in primary cortical neurons. The Journal of Neuroscience : the Official Journal of
the Society For Neuroscience 19:2455-2463.
Singh M (2001) Ovarian hormones elicit phosphorylation of Akt and extracellular-signal
regulated kinase in explants of the cerebral cortex. Endocrine 14:407-415.
Singh M (2007) Progestins and neuroprotection: are all progestins created equal?
Minerva Endocrinol 32:95-102.
Singh M, Setalo G, Jr, Guan X, Warren M, Toran-Allerand CD (1999) Estrogen-Induced
Activation of Mitogen-Activated Protein Kinase in Cerebral Cortical Explants:
Convergence of Estrogen and Neurotrophin Signaling Pathways. J Neurosci
19:1179-1188.
Slater CC, Zhang C, Hodis HN, Mack WJ, Boostanfar R, Shoupe D, Paulson RJ,
Stanczyk FZ (2001) Comparison of estrogen and androgen levels after oral
estrogen replacement therapy. J Reprod Med 46:1052-1056.
Slob AK, Ooms MP, Vreeburg JT (1980) Prenatal and early postnatal sex differences in
plasma and gonadal testosterone and plasma luteinizing hormone in female and
male rats. J Endocrinol 87:81-87.
Speck O, Ernst T, Braun J, Koch C, Miller E, Chang L (2000) Gender differences in the
functional organization of the brain for working memory. Neuroreport 11:2581-
2585.
Speroff L, Rowan J, Symons J, Genant H, Wilborn W (1996) The comparative effect on
bone density, endometrium, and lipids of continuous hormones as replacement
therapy (CHART study). A randomized controlled trial. Jama 276:1397-1403.
Spires TL, Hyman BT (2004) Neuronal structure is altered by amyloid plaques. Rev
Neurosci 15:267-278.
Spires TL, Hyman BT (2005) Transgenic models of Alzheimer's disease: learning from
animals. NeuroRx 2:423-437.
192
Stauffer SR, Coletta CJ, Tedesco R, Nishiguchi G, Carlson K, Sun J, Katzenellenbogen
BS, Katzenellenbogen JA (2000) Pyrazole ligands: structure-affinity/activity
relationships and estrogen receptor-alpha-selective agonists. J Med Chem
43:4934-4947.
Stein DG, Wright DW, Kellermann AL (2008) Does progesterone have neuroprotective
properties? Ann Emerg Med 51:164-172.
Stoffel-Wagner B (2003) Neurosteroid biosynthesis in the human brain and its clinical
implications. Annals of the New York Academy of Sciences 1007:64-78.
Stoltzner SE, Berchtold NC, Cotman CW, Pike CJ (2001) Estrogen regulates bcl-x
expression in rat hippocampus. Neuroreport 12:2797-2800.
Stone DJ, Rozovsky I, Morgan TE, Anderson CP, Lopez LM, Shick J, Finch CE (2000)
Effects of age on gene expression during estrogen-induced synaptic sprouting in
the female rat. Exp Neurol 165:46-57.
Strittmatter WJ, Roses AD (1995) Apolipoprotein E and Alzheimer disease. Proc Natl
Acad Sci U S A 92:4725-4727.
Struble RG, Nathan BP, Cady C, Cheng X, McAsey M (2007) Estradiol regulation of
astroglia and apolipoprotein E: An important role in neuronal regeneration.
Experimental Gerontology 42:54-63.
Subbiah MT, Kessel B, Agrawal M, Rajan R, Abplanalp W, Rymaszewski Z (1993)
Antioxidant potential of specific estrogens on lipid peroxidation. J Clin Endocrinol
Metab 77:1095-1097.
Sugioka K, Shimosegawa Y, Nakano M (1987) Estrogens as natural antioxidants of
membrane phospholipid peroxidation. FEBS Letters 210:37-39.
Suzuki S, Gerhold LM, Bottner M, Rau SW, Dela Cruz C, Yang E, Zhu H, Yu J, Cashion
AB, Kindy MS, Merchenthaler I, Gage FH, Wise PM (2007) Estradiol enhances
neurogenesis following ischemic stroke through estrogen receptors alpha and
beta. J Comp Neurol 500:1064-1075.
Swaab DF, Chung WC, Kruijver FP, Hofman MA, Ishunina TA (2001) Structural and
functional sex differences in the human hypothalamus. Horm Behav 40:93-98.
Swerdloff RS, Wang C (1993) Androgen deficiency and aging in men. The Western
Journal of Medicine 159:579-585.
Tan RS, Culberson JW (2003) An integrative review on current evidence of testosterone
replacement therapy for the andropause. Maturitas 45:15-27.
193
Tanabe F, Miyasaka N, Kubota T, Aso T (2004) Estrogen and progesterone improve
scopolamine-induced impairment of spatial memory. J Med Dent Sci 51:89-98.
Tang MX, Jacobs D, Stern Y, Marder K, Schofield P, Gurland B, Andrews H, Mayeux R
(1996) Effect of oestrogen during menopause on risk and age at onset of
Alzheimer's disease. Lancet 348:429-432.
Tanzer L, Jones KJ (1997) Gonadal steroid regulation of hamster facial nerve
regeneration: effects of dihydrotestosterone and estradiol. Exp Neurol 146:258-
264.
Tanzi RE, Moir RD, Wagner SL (2004) Clearance of Alzheimer's A[beta] Peptide: The
Many Roads to Perdition. Neuron 43:605-608.
Tariq SH, Haren MT, Kim MJ, Morley JE (2005) Andropause: is the emperor wearing
any clothes? Rev Endocr Metab Disord 6:77-84.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R
(1991) Physical basis of cognitive alterations in alzheimer's disease: Synapse loss
is the major correlate of cognitive impairment. Annals of Neurology 30:572-580.
Thakur MK, Mani ST (2005) Estradiol regulates APP mRNA alternative splicing in the
mice brain cortex. Neuroscience Letters 381:154-157.
Thomas T, Bryant M, Clark L, Garces A, Rhodin J (2001) Estrogen and Raloxifene
Activities on Amyloid--Induced Inflammatory Reaction. Microvascular Research
61:28-39.
Thompson W (1995) Estrogen replacement therapy in practice: trends and issues. Am J
Obstet Gynecol 173:990-993.
Toran-Allerand CD (2006) Reply to 'Hormone in the hot seat'. Nat Med 12:379-380.
Toran-Allerand CD, Singh M, Sétáló G (1999) Novel Mechanisms of Estrogen Action in
the Brain: New Players in an Old Story. Frontiers in Neuroendocrinology 20:97-
121.
Toung TJ, Chen TY, Littleton-Kearney MT, Hurn PD, Murphy SJ (2004) Effects of
combined estrogen and progesterone on brain infarction in reproductively
senescent female rats. J Cereb Blood Flow Metab 24:1160-1166.
Tsurugizawa T, Mukai H, Tanabe N, Murakami G, Hojo Y, Kominami S, Mitsuhashi K,
Komatsuzaki Y, Morrison JH, Janssen WG, Kimoto T, Kawato S (2005) Estrogen
induces rapid decrease in dendritic thorns of CA3 pyramidal neurons in adult male
rat hippocampus. Biochem Biophys Res Commun 337:1345-1352.
194
Turner PR, O'Connor K, Tate WP, Abraham WC (2003) Roles of amyloid precursor
protein and its fragments in regulating neural activity, plasticity and memory. Prog
Neurobiol 70:1-32.
Urbanc B, Cruz L, Buldyrev SV, Havlin S, Irizarry MC, Stanley HE, Hyman BT (1999)
Dynamics of plaque formation in Alzheimer's disease. Biophys J 76:1330-1334.
Vagnerova K, Koerner IP, Hurn PD (2008) Gender and the injured brain. Anesth Analg
107:201-214.
Vardy ERLC, Catto AJ, Hooper NM (2005) Proteolytic mechanisms in amyloid-[beta]
metabolism: therapeutic implications for Alzheimer's disease. Trends in Molecular
Medicine 11:464-472.
Vasto S, Candore G, Listi F, Balistreri CR, Colonna-Romano G, Malavolta M, Lio D,
Nuzzo D, Mocchegiani E, Di Bona D, Caruso C (2008) Inflammation, genes and
zinc in Alzheimer's disease. Brain Res Rev 58:96-105.
Vaughan C, Goldstein FC, Tenover JL (2007) Exogenous testosterone alone or with
finasteride does not improve measurements of cognition in healthy older men with
low serum testosterone. J Androl 28:875-882.
Vedder H, Anthes N, Stumm G, Wurz C, Behl C, Krieg J-C (1999) Estrogen Hormones
Reduce Lipid Peroxidation in Cells and Tissues of the Central Nervous System.
Journal of Neurochemistry 72:2531-2538.
Vegeto E, Bonincontro C, Pollio G, Sala A, Viappiani S, Nardi F, Brusadelli A, Viviani B,
Ciana P, Maggi A (2001) Estrogen Prevents the Lipopolysaccharide-Induced
Inflammatory Response in Microglia. Journal of Neuroscience 21:1809-1818.
Verdile G, Fuller S, Atwood CS, Laws SM, Gandy SE, Martins RN (2004) The role of
beta amyloid in Alzheimer’s disease: still a cause of everything or the only one
who got caught? Pharmacological Research 50:397-409.
Vincent B, Smith JD (2000) Effect of estradiol on neuronal Swedish-mutated beta-
amyloid precursor protein metabolism: reversal by astrocytic cells. Biochemical
and Biophysical Research Communications 271:82-85.
Wahjoepramono EJ, Wijaya LK, Taddei K, Martins G, Howard M, Ruyck Kd, Bates K,
Dhaliwald SS, Verdile G, Carruthers M, Martins RN (2008) Distinct Effects of
Testosterone on Plasma and Cerebrospinal Fluid Amyloid-b Levels. Journal of
Alzheimer's Disease 129:129-137.
Walf AA, Rhodes ME, Frye CA (2004) Antidepressant effects of ERbeta-selective
estrogen receptor modulators in the forced swim test. Pharmacol Biochem Behav
78:523-529.
195
Walsh D, Selkoe D (2007) A beta oligomers - a decade of discovery. Journal of
Neurochemistry 101:1172-1184.
Walsh DM, Klyubin I, Fadeeva JV, Rowan MJ, Selkoe DJ (2002) Amyloid-beta
oligomers: their production, toxicity and therapeutic inhibition. Biochem Soc Trans
30:552-557.
Walsh DM, Klyubin I, Shankar GM, Townsend M, Fadeeva JV, Betts V, Podlisny MB,
Cleary JP, Ashe KH, Rowan MJ, Selkoe DJ (2005) The role of cell-derived
oligomers of Abeta in Alzheimer's disease and avenues for therapeutic
intervention. Biochem Soc Trans 33:1087-1090.
Wang J, Dickson DW, Trojanowski JQ, Lee VM (1999) The levels of soluble versus
insoluble brain Abeta distinguish Alzheimer's disease from normal and pathologic
aging. Exp Neurol 158:328-337.
Wang J, Tanila H, Puolivali J, Kadish I, van Groen T (2003) Gender differences in the
amount and deposition of amyloidbeta in APPswe and PS1 double transgenic
mice. Neurobiol Dis 14:318-327.
Wang PN, Liao SQ, Liu RS, Liu CY, Chao HT, Lu SR, Yu HY, Wang SJ, Liu HC (2000)
Effects of estrogen on cognition, mood, and cerebral blood flow in AD: A
controlled study. Neurology 54:2061-2066.
Wang PY, Protheroe A, Clarkson AN, Imhoff F, Koishi K, McLennan IS (2009) Mullerian
inhibiting substance contributes to sex-linked biases in the brain and behavior.
Proc Natl Acad Sci U S A.
Waring SC, Rocca WA, Petersen RC, O_Brien PC, Tangalos EG, Kokmen E (1999)
Postmenopausal estrogen replacement therapy and risk of AD: a population-
based study. Neurology 52:965-970.
Warren MP (2004) A comparative review of the risks and benefits of hormone
replacement therapy regimens. Am J Obstet Gynecol 190:1141-1167.
Watanabe T, Koba S, Kawamura M, Itokawa M, Idei T, Nakagawa Y, Iguchi T, Katagiri T
(2004) Small dense low-density lipoprotein and carotid atherosclerosis in relation
to vascular dementia. Metabolism 53:476-482.
Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM (1997) Rapid
Membrane Effects of Steroids in Neuroblastoma Cells: Effects of Estrogen on
Mitogen Activated Protein Kinase Signalling Cascade and c-fos Immediate Early
Gene Transcription. Endocrinology 138:4030-4033.
Weaver CE, Jr., Park-Chung M, Gibbs TT, Farb DH (1997) 17beta-Estradiol protects
against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors. Brain
Res 761:338-341.
196
Weaver CL, Espinoza M, Kress Y, Davies P (2000) Conformational change as one of the
earliest alterations of tau in Alzheimer's disease. Neurobiol Aging 21:719-727.
Weiss JH, Pike CJ, Cotman CW (1994) Ca2+ channel blockers attenuate beta-amyloid
peptide toxicity to cortical neurons in culture. J Neurochem 62:372-375.
Weisz J, Ward IL (1980) Plasma testosterone and progesterone titers of pregnant rats,
their male and female fetuses, and neonatal offspring. Endocrinology 106:306-
316.
West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the
total number of neurons in thesubdivisions of the rat hippocampus using the
optical fractionator. Anat Rec 231:482-497.
West MJ, Coleman PD, Flood DG, Troncoso JC (1994) Differences in the pattern of
hippocampal neuronal loss in normal ageing and Alzheimer's disease. Lancet
344:769-772.
Westerman MA, Cooper-Blacketer D, Mariash A, Kotilinek L, Kawarabayashi T, Younkin
LH, Carlson GA, Younkin SG, Ashe KH (2002) The relationship between Abeta
and memory in the Tg2576 mouse model of Alzheimer's disease. J Neurosci
22:1858-1867.
Wharton W GC, Brinton E, Cedars M, Lobo RA, Manson J, Merriam GR, Miller VM,
Santoro N, Taylor HS, Harman SM, Asthana S (2008) Baseline cognitive and
demographic characteristics of women enrolled in the Kronos Early Estrogen
Prevention Study (KEEPS) Alzheimer's & Dementia: The Journal of the
Alzheimer's Association 4:T792.
Whitehead MI, Hillard TC, Crook D (1990) The role and use of progestogens. Obstet
Gynecol 75:59S-76S; discussion 81S-83S.
Wilhelmsen KC, Clark LN, Miller BL, Geschwind DH (1999) Tau mutations in
frontotemporal dementia. Dement Geriatr Cogn Disord 10 Suppl 1:88-92.
Wise PM (2002) Estrogens and neuroprotection. Trends Endocrinol Metab 13:229-230.
Wise PM (2006) Estrogen therapy: does it help or hurt the adult and aging brain?
Insights derived from animal models. Neuroscience 138:831-835.
Wise PM, Parsons B (1984) Nuclear estradiol and cytosol progestin receptor
concentrations in the brain and the pituitary gland and sexual behavior in
ovariectomized estradiol-treated middle-aged rats. Endocrinology 115:810-816.
Wong M, Moss RL (1992) Long-term and short-term electrophysiological effects of
estrogen on the synaptic properties of hippocampal CA1 neurons. J Neurosci
12:3217-3225.
197
Woolley CS, Bruce S. McEwen (1993) Roles of estradiol and progesterone in regulation
of hippocampal dendritic spine density during the estrous cycle in the rat. The
Journal of Comparative Neurology 336:293-306.
Woolley CS, McEwen BS (1994) Estradiol regulates hippocampal dendritic spine density
via an N-methyl-D-aspartate receptor-dependent mechanism. J Neurosci 14:7680-
7687.
Wozniak DF, Stewart GR, Miller JP, Olney JW (1991) Age-related sensitivity to kainate
neurotoxicity. Exp Neurol 114:250-253.
Wu JM, Zelinski MB, Ingram DK, Ottinger MA (2005a) Ovarian aging and menopause:
current theories, hypotheses, and research models. Exp Biol Med (Maywood)
230:818-828.
Wu TW, Wang JM, Chen S, Brinton RD (2005b) 17Beta-estradiol induced Ca2+ influx
via L-type calcium channels activates the Src/ERK/cyclic-AMP response element
binding protein signal pathway and BCL-2 expression in rat hippocampal neurons:
a potential initiation mechanism for estrogen-induced neuroprotection.
Neuroscience 135:59-72.
Wu X, Glinn MA, Ostrowski NL, Su Y, Ni B, Cole HW, Bryant HU, Paul SM (1999)
Raloxifene and estradiol benzoate both fully restore hippocampal choline
acetyltransferase activity in ovariectomized rats. Brain Res 847:98-104.
Xiao Z-M, Sun L, Liu Y-M, Zhang J-J, Huang J (2009) Estrogen Regulation of the
Neprilysin Gene Through A Hormone-Responsive Element. Journal Journal of
Molecular Neuroscience In press.
Xu H, Wang R, Zhang Y-W, Zhang X (2006) Estrogen, -Amyloid Metabolism/Trafficking,
and Alzheimer's Disease. Annals of the New York Academy of Sciences
1089:324-342.
Xu H, Gouras GK, Greenfield JP, Vincent B, Naslund J, Mazzarelli L, Fried G, Jovanovic
JN, Seeger M, Relkin NR, Liao F, Checler F, Buxbaum JD, Chait BT, Thinakaran
G, Sisodia SS, Wang R, Greengard P, Gandy S (1998) Estrogen reduces
neuronal generation of Alzheimer beta-amyloid peptides. Nat Med 4:447-451.
Xu Y, Armstrong SJ, Arenas IA, Pehowich DJ, Davidge ST (2004) Cardioprotection by
chronic estrogen or superoxide dismutase mimetic treatment in the aged female
rat. Am J Physiol Heart Circ Physiol 287:H165-171.
Yaffe K, Krueger K, Sarkar S, Grady D, Barrett-Connor E, Cox DA, Nickelsen T, The
Multiple Outcomes of Raloxifene Evaluation Investigators (2001) Cognitive
Function in Postmenopausal Women Treated with Raloxifene. New England
Journal of Medicine 344:1207-1213.
198
Yaffe K, Krueger K, Cummings SR, Blackwell T, Henderson VW, Sarkar S, Ensrud K,
Grady D (2005) Effect of raloxifene on prevention of dementia and cognitive
impairment in older women: the Multiple Outcomes of Raloxifene Evaluation
(MORE) randomized trial. Am J Psychiatry 162:683-690.
Yang SH, Perez E, Cutright J, Liu R, He Z, Day AL, Simpkins JW (2002) Testosterone
increases neurotoxicity of glutamate in vitro and ischemia-reperfusion injury in an
animal model. J Appl Physiol 92:195-201.
Yang SH, Liu R, Wen Y, Perez E, Cutright J, Brun-Zinkernagel AM, Singh M, Day AL,
Simpkins JW (2005) Neuroendocrine mechanism for tolerance to cerebral
ischemia-reperfusion injury in male rats. J Neurobiol 62:341-351.
Yang SL, Chen YY, Hsieh YL, Jin SH, Hsu HK, Hsu C (2004) Perinatal androgenization
prevents age-related neuron loss in the sexually dimorphic nucleus of the preoptic
area in female rats. Dev Neurosci 26:54-60.
Yankner BA, Lu T (2009) Amyloid {beta}-Protein Toxicity and the Pathogenesis of
Alzheimer Disease. J Biol Chem 284:4755-4759.
Yao M, Nguyen TV, Pike CJ (2005) Beta-amyloid-induced neuronal apoptosis involves
c-Jun N-terminal kinase-dependent downregulation of Bcl-w. J Neurosci 25:1149-
1158.
Yao M, Nguyen TV, Pike CJ (2007) Estrogen regulates Bcl-w and Bim expression: role
in protection against beta-amyloid peptide-induced neuronal death. J Neurosci
27:1422-1433.
Yu WH (1982) Effect of testosterone on the regeneration of the hypoglossal nerve in
rats. Exp Neurol 77:129-141.
Yu WH, Srinivasan R (1981) Effect of testosterone and 5 alpha-dihydrotestosterone on
regeneration of the hypoglossal nerve in rats. Exp Neurol 71:431-435.
Yue X, Lu M, Lancaster T, Cao P, Honda S, Staufenbiel M, Harada N, Zhong Z, Shen Y,
Li R (2005) Brain estrogen deficiency accelerates Abeta plaque formation in an
Alzheimer's disease animal model. Proc Natl Acad Sci U S A 102:19198-19203.
Yune TY, Park HG, Lee JY, Oh TH (2008) Estrogen-induced Bcl-2 expression after
spinal cord injury is mediated through phosphoinositide-3-kinase/Akt-dependent
CREB activation. J Neurotrauma 25:1121-1131.
Zandi PP, Carlson MC, Plassman BL, Welsh-Bohmer KA, Mayer LS, Steffens DC,
Breitner JC (2002) Hormone replacement therapy and incidence of Alzheimer
disease in older women: the Cache County Study. Jama 288:2123-2129.
199
Zhang L, Rubinow DRCA, Xaing G-q, Li B-S, Chang YH, Maric D, Barker JL, Ma W
(2001) Estrogen protects against b-amyloid-induced neurotoxicity in rat
hippocampal neurons by activation of Akt. Neuroreport 12:1919-1923.
Zhang QG, Wang R, Khan M, Mahesh V, Brann DW (2008) Role of Dickkopf-1, an
antagonist of the Wnt/beta-catenin signaling pathway, in estrogen-induced
neuroprotection and attenuation of tau phosphorylation. J Neurosci 28:8430-8441.
Zhang S, Huang Y, Zhu YC, Yao T (2005) Estrogen stimulates release of secreted
amyloid precursor protein from primary rat cortical neurons via protein kinase C
pathway. Acta Pharmacol Sin 26:171-176.
Zhang Y, Champagne N, Beitel LK, Goodyer CG, Trifiro M, LeBlanc A (2004) Estrogen
and androgen protection of human neurons against intracellular amyloid beta(1-
42) toxicity through heat shock protein 70. Journal of Neuroscience 24:5315-5321.
Zhao L, Brinton RD (2007) Estrogen receptor alpha and beta differentially regulate
intracellular Ca(2+) dynamics leading to ERK phosphorylation and estrogen
neuroprotection in hippocampal neurons. Brain Res 1172:48-59.
Zhao L, Wu TW, Brinton RD (2004) Estrogen receptor subtypes alpha and beta
contribute to neuroprotection and increased Bcl-2 expression in primary
hippocampal neurons. Brain Res 1010:22-34.
Zheng H, Xu H, Uljon SN, Gross R, Hardy K, Gaynor J, Lafrancois J, Simpkins J, Refolo
LM, Petanceska S, Wang R, Duff K (2002) Modulation of A(beta) peptides by
estrogen in mouse models. J Neurochem 80:191-196.

Abstract (if available)

Abstract This dissertation seeks to investigate the broad issue of the effects of sex steroid hormones on neuroprotective measures related to aging and the prevention of the progression of Alzheimer’s disease (AD). In particular, this dissertation will focus on the effects of female sex steroid hormones, estrogen and progesterone both alone and in combination on AD in women. Abundant evidence implicates sex steroid depletion in postmenopausal women as a risk factor for the development of AD. However, there appears to be a disconnect between experimental data clearly establishing multiple estrogen protective functions, and clinical findings showing that estrogen-based hormone therapy fails to prevent and slow progression of cognitive decline and AD. Furthermore, while estrogen’s many beneficial effects in the brain have been well established, the effects of progesterone, both alone and in combination with estrogen, are currently unclear and under-investigated. The Women’s Health Initiative Memory Study (WHIMS) raised several questions regarding the actions of progestins in the brain and the efficacy of hormone therapy in lowering the risk of AD in post-menopausal women. These questions are the basis behind the experiments described in this dissertation

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Estrogen and progesterone interactions in neurons: implications for Alzheimer's disease-related pathways

PDF

Estrogen, progesterone and BDNF interactions: roles in neuroprotection

PDF

Age-related androgen depletion and the development of Alzheimer's disease

PDF

Estrogen and progesterone interaction on synaptic transmission and LTP in rodent hippocampus

PDF

From risk mitochondrial and metabolic phenotype towards a precision medicine approach for Alzheimer's disease

PDF

TREM2 and C1q signaling regulates immunoproteostasis in Alzheimer's disease

PDF

Sex hormones and atherosclerosis in postmenopausal women

PDF

The impact of hormone therapy on Alzheimer’s disease biomarkers in early and late postmenopausal women

PDF

Vascular contributions to brain aging along the Alzheimer's disease continuum

PDF

The role of inflammation in mediating effects of obesity on Alzheimer's disease

PDF

Gonadal steroid hormones promote neuroplasticity in models of health and disease

PDF

Exploring the relationship between menopausal hot flushes and Alzheimer's disease biomarkers: a cross-sectional analysis in postmenopausal women

PDF

Role of oxidative stress in age-associated mild cognitive impairment and Alzheimer's disease

PDF

Using neuroinformatics to identify genomic and proteomic markers of suboptimal aging and Alzheimer's disease

PDF

Hormone therapy timing hypothesis and atherosclerosis

PDF

An overview of Alzheimer’s disease and the potential for APP-beta secretase-interaction as a therapeutic target

PDF

Neuroinflammation and ApoE4 genotype in at-risk female aging: implications for Alzheimer's disease

PDF

Sex differences in the TgF344-AD rat model: Investigating the behavior, pathology, and neuroanatomical structures

PDF

The effects of hormonal exposures on ovarian and breast cancer risk

PDF

The effects of a fasting mimicking diet (FMD) on mouse models of Alzheimer's and Parkinson's disease

Asset Metadata

Creator Carroll, Jenna C. (author)

Core Title Estrogen and progesterone-based hormone therapy and the development of Alzheimer's disease

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 08/08/2009

Defense Date 05/07/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Alzheimer's disease,beta amyloid,Estrogen,OAI-PMH Harvest,progesterone,Tau,transgenic

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Pike, Christian J. (committee chair), Finch, Caleb E. (committee member), Mack, Wendy J. (committee member), Miller, Carol (committee member), Thompson, Richard (committee member)

Creator Email jccarrol@usc.edu,jennacarroll@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2547

Unique identifier UC1492459

Identifier etd-Carroll-2964 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-177259 (legacy record id),usctheses-m2547 (legacy record id)

Legacy Identifier etd-Carroll-2964.pdf

Dmrecord 177259

Document Type Dissertation

Rights Carroll, Jenna C.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu