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Age-related androgen depletion and the development of Alzheimer's disease
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Age-related androgen depletion and the development of Alzheimer's disease
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Content
AGE-RELATED ANDROGEN DEPLETION AND THE DEVELOPMENT OF
ALZHEIMER’S DISEASE
by
Emily R. Rosario
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)
May 2007
Copyright 2007 Emily Rosario
ii
DEDICATION
To my loving and supportive husband, family, and friends
especially my parents who have always been there for me and believed in me
iii
ACKNOWLEDGEMENTS
I would like to thank my advisor, Christian Pike, for his support and guidance
throughout my graduate studies and for sharing his knowledge and expertise. His
enthusiasm for science is infectious, making the lab a motivating and stimulating
work environment. Through Dr. Pikes’ mentorship I have learned many valuable
lessons that will assist me in achieving my future goals.
A big thank you needs to go to the past and present members of the Pike lab, Martin
Ramsden, Ming-Zhong Yao, Jenna Carroll, Anusha Jarayaman, Thuy-Vi Nguyen,
Radha Aras, and Myriam Cordey. Not only are these people my colleagues, but they
have also been my friends and support system the past 4 years, to go to for scientific
discussion, as well as to celebrate and commiserate with. I would like to say a
special thank you to Martin Ramsden and Jenna Carroll who I worked most closely
with as the other in vivo people in the lab. I could not have asked for better scientists
and friends to work with.
I would like to thank my committee members, Roberta Brinton, Larry Swanson,
Richard Thompson, and Margaret Gatz for their knowledge and expertise, for helpful
discussion about my research, and most importantly for their valuable time which
they have given to be a part of my committee.
iv
I would like to thank Professor Vern Bengston and Professor Eileen Crimmins for
allowing me the wonderful opportunity of participating on their NIA funded
Multidisciplinary Training grant on Aging. In addition to providing my funding, I
was able to interact and get to know other scientists from several multidisciplinary
fields, which has enriched me as a scientist and provided training and experiences I
will carry throughout my career.
I would like to acknowledge the National Institute of Neurological Disease and
Stroke (NINDS) for funding my NRSA predoctoral grant and providing my funding
the last year. Not only was writing the grant a valuable experience but it also
afforded me the opportunity to travel to international meetings which were an
invaluable experience.
I need to thank my colleagues from the Finch lab and Davies lab for the use of lab
equipment, supplies and reagents when needed. In addition, I would like to say a
special thanks to Dr. Todd Morgan and Dr. Irina Rozovsky for their knowledge and
always having time to answer my questions. I would also like to thank for my
collaborators Dr. Frank Stanczyk, Dr. Lilly Chang, Dr. M. Paul Murphy, and Dr.
Elizabeth Head for their help and assistance with my projects.
I would like to acknowledge the tissue repositories at the Alzheimer’s Disease
Research Centers at the University of Southern California (AG05142), University of
v
California Irvine (AG16573), University of California San Diego (AG05131), and
Duke University Kathleen Price Bryan Brain Bank (AG05128) for the human
postmortem tissue.
I would like to thank the Journal of the American Medical Association, (JAMA),
Brain Research, and the Journal of Neuroscience for copyright permission.
vi
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES ix
LIST OF FIGURES x
ABSTRACT xii
CHAPTER ONE: Introduction 1
1. Female Sex Steroid Hormones and Alzheimer’s disease (AD) 2
Women and the risk for Alzheimer’s disease 3
Role of hormone therapy in the treatment and prevention of AD 4
Estrogen and progesterone neuroprotection 8
Estrogen regulation of β-amyloid 11
2. Androgen depletion during normal aging 16
Synthesis, metabolism, and regulation of sex steroid hormones 16
Age-related androgen depletion in men 18
The HPG axis with age 19
Androgen deficiency in aging men 19
3. Androgen actions in the brain 20
Androgen receptors in the brain 21
Beneficial neural actions of androgens 21
Androgen actions through androgen and estrogen mediated 25
pathways
4. Androgens and cognition 26
5. Androgens and Alzheimer’s disease in men 28
Age-related androgen depletion and the risk for Alzheimer’s 28
disease
Gonadotropins and other risk factors for AD 30
6. Androgens and neuroprotection 32
Androgens promote neuron viability 32
Androgen regulation of tau hyperphosphorylations 37
7. Androgen regulation of Aβ levels 38
Androgen regulation of β-amyloid 38
Mechanism of androgen regulation of Aβ 39
LH and other factors that may affect Aβ 41
8. Hypothesis and experimental paradigm 43
vii
CHAPTER TWO: Age-Related Testosterone Depletion and the Development 45
of Alzheimer Disease
Introduction 46
Methods 46
Results 47
Discussion 49
CHAPTER THREE: Age-related changes in brain levels of sex steroid 51
hormones in women and men and their relationship to Alzheimer’s disease
Chapter Three Abstract 52
Introduction 53
Methods 55
Results 59
Discussion 70
CHAPTER FOUR: Age-related testosterone decline and increased Aβ in 77
brown Norway rat brain
Chapter Four Abstract 78
Introduction 79
Methods 82
Results 84
Discussion 89
CHAPTER FIVE: Androgens regulate development of neuropathology in a 95
triple transgenic mouse model of Alzheimer’s disease
Chapter Five Abstract 96
Introduction 97
Methods 99
Results 102
Discussion 108
CHAPTER SIX: Androgen regulation of Alzheimer-like neuropathology 114
in male 3XTg-AD mice is through androgen and estrogen mediated pathways
Chapter Six Abstract 115
Introduction 116
Methods 118
Results 121
Discussion 127
CHAPTER SEVEN: Progestins inhibit the neuroprotective effects of 134
estrogen in rat hippocampus
Chapter Seven Abstract 135
Introduction 135
viii
Methods 136
Results 139
Discussion 143
CHAPTER EIGHT: Conclusions and future directions 146
REFERENCES 162
ix
LIST OF TABLES
1. Estrogen and androgen levels in female cases across 59
neuropathological status
2. Estrogen and androgen levels in male cases across neuropathological status 62
3. Estrogen and androgen levels across neuropathological status in male cases 67
over 80 years of age
4. Correlations and partial correlations between sex steroid hormones 68
and soluble levels of Aβ
5. Changes in androgen levels following GDX in 3mo old rats 85
6. Efficacy of hormone treatment for 30 days in wild-type mice 122
7. Assessment of hormone treatments in ovariectomized rats 139
x
LIST OF FIGURES
1. Brain Levels of Testosterone and Estradiol in Elderly Men 48
2. Brain Levels of Testosterone and Estradiol in Men, by 49
Neuropathological Diagnosis
3. Brain levels of estrogens and androgens remain the same during normal 60
aging in postmenopausal women
4. Brain levels of estrogen are decreased in AD 61
5. Brain levels of androgens decrease with age 63
6. Low androgens are a risk factor for AD in male cases 60-80 years of age 66
7. Low brain levels of androgens correlate with increased soluble Aβ1-42 69
8. Changes in circulating levels of androgen with increasing age 87
9. Androgen depletion increases brain levels of soluble Aβ 88
10. Androgen treatment in aged rats has partial effect on Aβ levels 89
11. Aβ accumulation increases with age in male 3xTg-AD mice 103
12. Aβ accumulation is regulated by androgens 105
13. Accumulation of APP C-terminal fragments (CTF) does not change 106
with age
14. Brain levels of soluble Aβ increase with age 108
15. Intraneuronal Aβ increases following 4 months of androgen depletion 124
16. Quantification of Aβ load and extracellular deposition following 125
androgen depletion.
17. Age and hormone-related changes in AT8 positive neurons 126
xi
18. Progestins block estrogen neuroprotection against k!ainate lesion 141
in female rats
19. Hormone manipulations do not reduce seizure activity induced 142
by !kainite
xii
ABSTRACT
Advancing age is the most significant risk factor for the development of
Alzheimer's disease (AD), however, which age-related changes underlie this effect
remains unclear. In men, one normal consequence of aging is a robust decline in the
circulating levels of the sex steroid hormone testosterone. Testosterone depletion
leads to functional impairments in androgen-responsive tissues that are often
manifested as the clinical syndrome ‘androgen deficiency in aging males’. Although
the brain is an androgen-responsive tissue unknown is (1) whether brain levels of T
decline during aging, and if so, (2) whether low brain T levels place the aging brain
at increased risk for AD, and if so (3) what do androgens regulate that may modulate
the increased risk for AD. My thesis work investigated these questions and others
examining the relationships between sex steroid hormones, advancing age, and
development of AD. In Chapters Two and Three we observed that brain levels of
androgens but not estrogens are significantly lower in men with moderate to severe
AD in comparison to normal men. To examine how low testosterone levels may
contribute to AD development we examined androgen regulation of Aβ, a causal
factor in the development of AD. In Chapters, Three through Six we investigated the
effects of androgens on regulation and development of Aβ pathology. We found that
low levels of testosterone, both in humans and a rodent model of male reproductive
aging, correlated with increased levels of soluble Aβ. Using a transgenic mouse
model of AD we found that depletion of endogenous androgens resulted in increased
xiii
accumulation of Aβ pathology and behavioral impairments. Replacement of
androgens in these mice was able to prevent this increased accumulation. These
findings suggest the use of androgen replacement therapy in men with low levels of
androgens.
1
CHAPTER ONE
Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative illness that
affects over four and a half million people in the United States, a number that is
projected to grow (Brookmeyer et al., 1998; Hebert et al., 2003). Advancing age is
the most significant risk factor for the development of AD (Rocca et al., 1986; Jorm
et al., 1987; Evans et al., 1989) however, what age-related changes underlie this
effect remain unclear. There is a large body of work that has investigated estrogen’s
beneficial effects in the brain and the relationship between estrogen loss and the
development of AD (Henderson, 1997; Cholerton et al., 2002). Recently, androgens
have also been found to have beneficial actions in the brain and the relationship
between androgen loss in men and AD has begun to be investigated (for review see
(Pike et al., 2006)).
Men experience a slow but significant decline in androgen levels during
normal aging (Purifoy et al., 1981; Gray et al., 1991; Morley et al., 1997; Feldman et
al., 2002). This decrease in androgens has been associated with disease and
dysfunction in androgen responsive tissue such as reproductive tissues, muscle, bone,
and brain (Morley, 2001b). Androgens have been found to have several beneficial
actions in the brain, and the loss of androgens has been associated with an increased
2
risk for AD in men (Hogervorst et al., 2001; Hogervorst et al., 2002; Moffat et al.,
2004). My thesis work investigated the relationship between sex steroid hormones
and AD using several different experimental paradigms such as human tissue, wild-
type rats (Sprague Dawley, Brown Norway), and transgenic mice. In this
introduction (Chapter One), I will first discuss the relationship between estrogen and
AD. Next, I will review androgen actions in the brain, the relationship between
androgen depletion and AD, and the potential androgen actions responsible for
modulating the increased risk for the development of AD. In Chapters Two through
Seven, I will discuss the experiments and studies performed to test my hypotheses
regarding the relationships between sex steroid hormones and AD, and in Chapter
Eight, I will discuss the results of these studies and conclusions that come from this
work.
1. Female sex steroid hormones and Alzheimer ’s disease (AD)
One normal age-related change all women experience is the almost complete
loss of their primary sex steroid hormones, estrogen and progesterone, during
menopause. The loss of estrogen leads to several clinical disorders in hormone
responsive tissues such as osteoporosis (Kleerekoper and Sullivan, 1995; Burger et
al., 1998). The brain is also a hormone responsive tissue, and estrogen has been
found to have several beneficial actions in the brain (for review see (Cholerton et al.,
2002). Epidemiological evidence has linked the loss of estrogen with an increased
risk for the development of AD (for review see (Henderson, 1997; Cholerton et al.,
3
2002). Despite the epidemiological data and basic research suggesting beneficial
actions of estrogen, clinical studies examining the role of sex steroid hormones in the
development of AD have provided conflicting results on the effectiveness of
hormone therapy.
Women and the risk for Alzheimer’s disease
Women are 1.5 times more likely than men to develop AD (Gao et al., 1998).
The reason for this difference between men and women may be due in part to the
loss of estrogen associated with menopause. This idea is supported by a case-control
study using the Leisure World Cohort of aging, (Paganini-Hill and Henderson,
1994). Since the loss of estrogen was found to increase the risk of AD, future studies
investigated the role of estrogen replacement for the treatment and prevention of AD.
Results from several studies found that estrogen therapy (ET) significantly reduced
the risk for AD, a risk that is further decreased with an increased duration of ET use
(Paganini-Hill and Henderson, 1996; Tang et al., 1996). A prospective study using
the Baltimore Longitudinal Study of Aging (BLSA) indicated that women taking ET
were at reduced risk for AD, however, this study differed from previous studies in
that it did not find an effect between duration of estrogen use and risk of AD (Kawas
et al., 1997). Interestingly, not all studies examining the effects of estrogen and AD
found ET to be protective. A case-control study using subjects from the Group
Health Cooperative in Seattle, Washington found that unopposed estrogen use had
no impact on the risk of AD (Brenner et al., 1994). A more recent prospective study
4
using the Cache County cohort found an increased risk of AD in women as
compared to men in the same age range (80 years and older) and a reduced risk of
AD in subjects using hormone therapy (HT; estrogen + progestin) (Zandi et al.,
2002). Interestingly, this study provided evidence that HT use had a variable effect
on the risk of AD depending on the duration of use. Specifically, HT was found to
have no effect except when duration of treatment exceeded 10 years (Zandi et al.,
2002). This result is supported by a previous study, which found that an increase in
the duration of HT corresponds to a decreased risk in AD (Waring et al., 1999).
Taken together, these studies suggest there may be a critical period for the initiation
and/or duration of hormone replacement, which may help explain why some studies
have not found a beneficial relationship between HT use and AD.
Role of hormone therapy in the treatment and prevention of AD
There are a great number of studies examining the role of HT in the
development of AD. However, variability in treatment type and duration leads to
conflicting findings between studies. Similar to these findings is the effect of HT on
cognition; some studies have found HT to be beneficial against cognitive decline
while others have shown no effect (for review see (Cholerton et al., 2002; Maki and
Hogervorst, 2003)). Therefore, the best way to investigate the effects of HT is
through randomized, double-blind, placebo-controlled clinical trials which evaluate
both cognitive decline and AD pathology.
5
There have been several smaller clinical trials that vary in duration (2 weeks
to 1 year), treatment type (estrogen alone or with a progestin), and age of
participants, and different cognitive tests, which provide conflicting results
(Cholerton et al., 2002; Maki and Hogervorst, 2003). Due to the large amount of
variability among studies, it is difficult to draw overall conclusions about the
effectiveness of HT on cognitive status. In 1995, an extensive clinical study
(Women’s Health Initiative, WHI) was started to determine the effects of HT and ET
on coronary heart disease, stroke, pulmonary embolism, breast cancer, colon cancer,
and osteoporatic fractures (Shumaker et al., 1998). In 1996, the Women’s Health
Initiative Memory Study (WHIMS) was started to determine the result of HT on the
cognitive function, impairment, and dementia (Shumaker et al., 1998). The
treatment paradigm for this study was a combination of 0.625 mg conjugated equine
estrogens (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) (Rapp et al., 2003;
Shumaker et al., 2003). All subjects were 65 years of age or older. The large
number of participants makes WHIMS the largest clinical study to date examining
the effects of HT on dementia and cognition (Rapp et al., 2003; Shumaker et al.,
2003). While HT was shown to have positive effects on colon cancer and
osteoporatic fractures, this study was stopped prematurely due to an increased risk of
pulmonary embolism, heart disease, stroke, and breast cancer in participants taking
HT as compared to those taking placebo (Rapp et al., 2003; Shumaker et al., 2003).
At the time the study was ended, the effects of HT on cognitive function were not
positive. In fact, more women in the HT group showed a decline in their MMSE
6
(modified mini-mental state examination) score as compared to the placebo treated
controls (Rapp et al., 2003). In addition, subjects in the HT group also saw an
increased rate of dementia, and HT did not prevent mild cognitive impairments
(Shumaker et al., 2003).
A progesterone component is important in HT treatment to reduce the risk of
breast and uterine cancer caused by unopposed estrogen (Gambrell, 1986; Hirvonen,
1996). It has been hypothesized that the negative effects observed by HT may be
due to the use of medroxyprogesterone acetate (MPA) as the progestin component.
In vitro studies have found that MPA does not follow the same neuoprotective
properties of progesterone and may even block the beneficial effects estrogen in
brain (Nilsen and Brinton, 2002). In addition to the HT clinical trial, WHIMS also
started a trial using unopposed estrogen (ET) in women who had a prior
hysterectomy. The purpose of this trial, in light of the HT results, was to determine
if MPA was blocking estrogens beneficial effects in the brain. This trial was also
stopped early due to an increased risk of stroke observed in the ET group as
compared to the placebo group (Espeland et al., 2004; Shumaker et al., 2004) but
seemed to suggest that estrogen alone did not reduce incidence of either mild
cognitive impairments or dementia (Shumaker et al., 2004). An undesirable effect
was also observed on cognition; subjects in the ET group who presented with low
cognitive scores at the time of entry experienced the greatest decline (Espeland et al.,
2004). The results from this large study are consistent with four other studies that
7
found the use of unopposed estrogen had no benefit on cognition (Henderson et al.,
2000; Mulnard et al., 2000; Wang et al., 2000; Levine, 2004).
Results from the WHI clinical trial suggest that neither HT nor ET should be
used as the risks associated with taking theses hormones are greater than the benefits
(Rapp et al., 2003; Shumaker et al., 2003; Espeland et al., 2004; Shumaker et al.,
2004). The hope of the large WHIMS study was to answer questions about the role
of HT in AD raised by conflicting results in other studies. However, several
criticisms have been made about this study such as the hormone preparation (CEE +
MPA) and the age of initiation of all patients (mean age 65y). Results from the
WHIMS study tell us that HT and ET are not effective in women over the age of
65y. We can still hypothesize however, that if hormone replacement was started at
the time of menopause, it may still be effective. The Cache county study found HT
to be most effective after 10 years and when replacement began at or near
menopause (Zandi et al., 2002). This is consistent with another report that found a
reduction in cognitive decline when HRT was started at the time of menopause
(Matthews et al., 1999). Perhaps if the timing of treatment initiation corresponds
with the initial drop in estrogen during menopause, ET will be able to reduce the
other negative factors observed with the WHI trial.
In addition to the issue of timing, apolipoprotein E (apoE) genotype may be
complicating the relationship between estrogen and AD. Adding to the previously
discussed risk factors (gender and age), apoE genotype is a significant risk factor in
the older population (Corder et al., 1998). There are three apoE alleles, ε2, ε3, and
8
ε4. The ε4 allele increases the risk of developing AD three fold more than those
without an ε4 allele (Seshadri et al., 1995; Strittmatter and Roses, 1995). ApoE has
been shown to be important in maintenance and regeneration of neurons (Poirier,
1994). Experimental studies in mice have found that apoE expression in different
brain regions varied according to estrous cycle (Struble et al., 2003) suggesting a role
for estrogen in apoE expression. Consistent with this idea, a recent report found that
neurite outgrowth is dependent on apoE and interestingly; the type of apoE allele
plays a role in this effect (Nathan et al., 2004). In some human studies, the presence
of an apoE ε4 allele has negated the beneficial effects observed with ET (Yaffe et al.,
2000). This effect is not universal and other studies have shown inconclusive results
due to low sample size (Zandi et al., 2002). A recent study from a cohort of 181
postmenopausal women found no differences in certain aspects of memory between
apoE ε4 positive cases that did and did not use ET (Burkhardt et al., 2004). Women
who did not have the apoE ε4 allele and were taking ET scored highest, suggesting
ET may only benefit those without an apoE ε4 allele (Burkhardt et al., 2004). ApoE
genotype was not reported in the WHIMS study therefore apoE along timing could
all have compounded to produce the results reported.
Estrogen and progesterone neuroprotection
Estrogen has several beneficial actions in the brain that may modulate the
risk for the development of AD such as increased neuron viability, synaptic
plasticity, and attenuation of oxidative stress (for review see (Cholerton et al., 2002;
9
Wise, 2003)). A variety of cell culture and animal models have found estrogen to
have neuroprotective effects throughout the brain. Cell culture studies have
established that estrogen is neuroprotective against a variety of toxic insults,
including β-amyloid peptides (Fitzpatrick et al., 2002; Cordey et al., 2003), oxidative
stressors (Behl et al., 1995), and excitotoxins (Singer et al., 1996; Regan and Guo,
1997; Sawada et al., 1998; Zaulyanov et al., 1999). Supporting these findings in cell
culture, studies in vivo have found that estrogen modulates neuron viability in brain.
In these animal models, endogenous estrogen levels are depleted by ovariectomy
(OVX) causing increased neuronal vulnerability to excitotoxic and ischemic lesions
(Azcoitia et al., 1998; Dubal et al., 1999; Veliskova et al., 2000; Dubal et al., 2001).
Further, estrogen replacement protects hippocampal neurons against kainate
excitotoxicity (Azcoitia et al., 1998; Azcoitia et al., 1999a; Reibel et al., 2000;
Veliskova et al., 2000). In a recent study no effect of estrogen was observed on
seizure severity (Hoffman et al., 2003) suggesting that the mechanism of estrogen
neuroprotection involves neuroprotective cell signaling pathways (for review see
(Wise, 2002)) and is independent of an effect on seizure severity.
Previous studies have found that progesterone is also neuroprotective against
kainate lesion (Azcoitia et al., 1999b; Ciriza et al., 2004). Results from a recent
study found progesterone-induced neuron viability was through attenuation of
seizure activity (Hoffman et al., 2003). With increasing concentrations of constant
progesterone treatment, the progesterone-induced anti-seizure effect decreased.
Therefore, when low concentrations or short-term progesterone treatment is used,
10
seizure severity is decreased resulting in decreased cell death (Hoffman et al., 2003).
Allopregnanolone, a metabolite of progesterone, is a potent modulator of the
GABA
A
receptor (Baulieu et al., 1996) and desensitization of the GABA
A
receptor
can occur with prolonged treatment of either progesterone or allopregnanolone
(Gulinello et al., 2001; Wohlfarth et al., 2002). If progesterone attenuation of seizure
activity is acting through the GABA
A
receptor, this helps to explain why only low
doses or shorter time periods of progesterone treatment are effective. In addition to
kainic acid-induced excitotixicity, other models of injury have observed similar
neuroprotective effects of progesterone. Progesterone has been found to be
neuroprotective after glutamate toxicity (Nilsen and Brinton, 2002), protect against
spinal cord injury (Labombarda et al., 2003), and prevent neuron loss after ischemic
insult (Roof and Hall, 2000). Conversely, progesterone has also been found to have
no effect against glutamate toxicity (Singer et al., 1996). Following MCAO,
differing doses of progesterone can either result in protection (Chen et al., 1999), no
protection, or exacerbation of infarct volume (Murphy et al., 2000).
Unlike estrogen’s neuroprotective properties, there has been significantly less
research conducted examining the effects of various types of progestins in brain.
Medroxyprogesterone acetate (MPA), the progestin used in the WHIM study, was
originally used to antagonize estrogenic effects on breast and uterus in HT.
Experimentally, MPA has not been shown to have neuroprotective properties (Nilsen
and Brinton, 2002). However, differences in P4 and MPA signaling have been shown
which may indicate P4 and MPA do not act the same in the induction of cellular
11
responses (Nilsen and Brinton, 2002). Because progesterone levels also decrease
following menopause, and progestins are important in blocking the negative effects
of estrogen on breast and uterine cancer, it is important to investigate whether
progesterone has beneficial actions in the brain and, what happens when estrogen
and progesterone are replaced together. As part of my thesis work I investigated the
effects of estrogen, progesterone, and MPA, both alone and in combination, on
neuron loss in rats. In this study, presented in Chapter Seven, I examined the effects
of sex steroid hormones on kainiate-induced neuronal loss in the hippocampus of
female rats.
Estrogen regulation of β-amyloid
Another important action of estrogen in the brain that may modulate the risk
for AD is the regulation of β-amyoid (Aβ) levels. Endogenous levels of Aβ have
been found to decrease in response to 17β-estradiol (E2) in several in vitro models
such as an estrogen receptor rich cell line (Jaffe et al., 1994), neuroblastoma cells
from primary rat cultures (Xu et al., 1998), and an αAPP transfected kidney cell line
(Chang et al., 1997). Estrogen regulation of Aβ levels has also been observed in
animal and human studies. Schonknecht et al. measured estrogen levels in cerebral
spinal fluid of AD patients and found they were significantly lower as compared to
non-AD control subjects. Within this AD group, estrogen levels were inversely
correlated with levels of Aβ
1-42
(Schonknecht et al., 2001). In a more recent clinical
study, 20 women were given either a placebo or transdermal estrogen for 8 weeks.
12
While estrogen was not responsible for lowering Aβ during this short replacement
period, subjects who had never previously used HT had a higher baseline level of Aβ
as compared to those who had used HT. Aβ levels in those who received HRT for
the first time during this study experienced a significant reduction in Aβ levels
(Baker et al., 2003).
The effect of estrogen on regulation of Aβ levels in animal models has
produced conflicting results. Petanceska et al. used guinea pigs to determine if the
depletion of endogenous estrogen levels, by ovariectomy (OVX), increases Aβ
levels, and if so, will estrogen replacement reduce Aβ levels to that of sham-OVX
placebo treated animals. Eight weeks after OVX, animals were given either a
placebo or differing doses of E2 for 10 days. A significant increase in Aβ levels was
observed in the OVX groups as compared to the intact controls (Petanceska et al.,
2000). In addition, estrogen replacement for 10 days significantly reduced the OVX-
induced increase in Aβ levels (Petanceska et al., 2000). Transgenic mouse models
that overproduce Aβ, have also been used to investigate the role of estrogen in
regulation of Aβ. Using APPswe transgenic mice, Zheng et al. found that estrogen
depletion through OVX significantly increased levels of Aβ
1-40
and Aβ
1-42
. After
animals were replaced with estrogen, this effect was reversed and Aβ levels returned
to control values or lower depending on the amount of E2 used (Zheng et al., 2002).
Similar results were found using the APPswe + PS1 double transgenic mice. E2
replaced mice experienced a significant decrease in Aβ
1-40
and an even more
13
significant decrease in Aβ
1-42
(Zheng et al., 2002). Using a different APPswe line,
comparable results demonstrated a 50% increase in Aβ levels 5 months after OVX
and a 27% decrease in Aβ levels after E2 replacement (Levin-Allerhand et al., 2002).
Brain levels of estrogen also correlate with Aβ pathology. Specifically, aromatase
deficient mice have significantly increased accumulation of Aβ deposition compared
to APP transgenic mice beginning at 6 mo of age. Interestingly, in this study OVX
did not result in increased Aβ pathology (Yue et al., 2005). This finding is supported
by a study by Heikkinen et al. that found that OVX and subsequent estrogen
replacement had no effect on Aβ levels. In this study which used APP + PS1
transgenic mice, an OVX-induced increase in hippocampal Aβ levels was not
observed, and E2 replacement had no effect on Aβ levels at several different time
points (Heikkinen et al., 2004). Supporting this study is recent work by Green et al.
which found that long-term estrogen treatment in PDAPP transgenic mice did not
decrease levels of soluble Aβ (Green et al., 2005). There are several reasons for
differences in these studies, including the strain of animals, experimental parameters
such as age, dose of estrogen, Aβ sample, method of measuring Aβ, and duration of
hormonal manipulation.
The mechanism of estrogen regulation of Aβ may be through either
decreased deposition or increased clearance of Aβ. Initial studies documenting the
effect of estrogen on Aβ levels also measured levels of sAPPα and found that while
estrogen decreased levels of Aβ, it increased levels of sAPPα (Jaffe et al., 1994).
14
sAPPα is formed through the cleavage of APP by α-secretase. Therefore, up-
regulation or stimulation of α-secretase would result in increased levels of sAPPα.
Increased sAPPα in response to estrogen was observed using primary cortical
neurons, which over-express human APP (Vincent and Smith, 2000). This E2-
induced increase in sAPPα supports the idea that estrogen regulation of Aβ is
through metabolism of APP, which prevents the formation of Aβ and promotes the
release of sAPPα (Jaffe et al., 1994; Vincent and Smith, 2000).
The mechanism of this estrogen action is unclear but may be due to estrogen
activation of protein kinase C (PKC). Studies have found that estrogen regulates
PKC activity (Drouva SV, 1990; Maizels ET, 1992; Maeda T, 1993) and PKC has
been shown to increase sAPPα through mediating APP processing (Xu H, 1995).
There are several different isoforms of PKC which may be activated by estrogen and
responsible for regulation of Aβ production. PKCβ is a conventional, calcium-
dependent PKC (Nishizuka, 1992), which has been found to play a role in APP
processing through studies which either over-expressed or inhibited PKCα
(Kinouchi T, 1995; Benussi L, 1998; Jolly-Tornetta C, 2000). PKCε is a novel,
calcium-independent PKC (Nishizuka, 1992; Dekker LV, 1993) which has also been
found to regulate APP metabolism (Yeon SW, 2001; Zhu G, 2001). In addition to
the estrogen mediated role of PKC on APP processing, estrogen mediated regulation
of sAPPα has been found to occur through estrogen phosphorylation of extracellular-
regulated kinase 1 and 2 (ERK1/2) which is a part of the mitogen-activated protein
kinase-signaling (MAPK) pathway (Manthey et al., 2001). This study indicates that
15
increased sAPPα in response to estrogen activation of the MAPK pathway is a rapid,
estrogen receptor independent action (Manthey et al., 2001).
Not all studies have observed an increase in sAPPα in response to estrogen.
In fact, no changes in sAPPα were seen after guinea pigs were given estrogen
following OVX (Petanceska et al., 2000). These results are supported by another in
vivo study where an estrogen-induced effect on sAPPα was not observed (Savage et
al., 1998). In this study, mice were treated with phorbol 12-myristate 13-acetate
(PMA - activates PKC), which resulted in a significant decrease in Aβ levels.
Decreased levels of APPα were also observed signifying a reduction in APP
cleavage by α-secretase (Savage et al., 1998). In this case estrogen may be working
to decrease Aβ levels by stimulating accelerated trafficking of βAPP. Estrogen has
been shown to increase the formation and trafficking of vesicles, which contain
βAPP from the trans-golgi network therefore preventing the majority of Aβ
formation and causing a decrease in Aβ levels (Greenfield et al., 2002).
Results have shown that estrogen acts to block production and deposition of
Aβ and recently it has been also shown that estrogen may increase clearance of Aβ.
Neprilysin (NEP) is a major Aβ-catabolizing enzyme in the brain (Iwata et al., 2000;
Marr et al., 2003a; Newell et al., 2003). NEP has been found to be decreased in
OVX rats and when E2 was replaced, NEP levels returned to sham levels (Huang et
al., 2004). This study suggests estrogen may also play a role in clearance of Aβ
16
however, more research investigating the relationship between NEP, estrogen, and
Aβ levels needs to be done to further investigate this possibility.
2. Androgen depletion during normal aging
While the relationship between estrogen loss and AD has been well
established, the relationship between androgen loss in men and AD remains unclear.
The main focus of my thesis work has been to investigate the relationships between
androgen loss during normal aging, and the development of AD. For the remainder
of this introduction I will focus on androgens, the loss of androgens during normal
aging, the beneficial actions of androgens in the brain, and the relationship between
androgen loss and AD.
Synthesis, metabolism, and regulation of sex steroid hormones
Synthesis and metabolism of sex steroid hormones from cholesterol is
regulated by the hypothalamic-pituitary-gonadal axis (HPG). The HPG regulation of
sex steroid hormones involves a negative feedback loop beginning with the
hypothalamus. Hormones produced in the hypothalamus, gonadotropin releasing
hormone (GnRH), act on the anterior pituitary resulting in production and release of
the gonadotropins, luteinizing hormone (LH), and follicular stimulating hormone
(FSH) (Finkelstein et al., 1991a; Finkelstein et al., 1991b). LH and FSH act on the
ovaries and testes to produce estrogens and androgens (for review see (Kaufman and
Vermeulen, 2005)). Estrogens and androgens can then act back on the hypothalamus
17
and pituitary decreasing LH and FSH, and subsequently estrogen and androgen
levels. Circulating androgens include dehydroepiandrosterone (DHEA), DHEA-S,
androstenedione, and the primary male sex steroid hormones, testosterone and its
active metabolite, dihydrotestosterone (DHT) (Kaufman and Vermeulen, 2005).
Metabolism of androgens occurs via two major pathways aromatization and 5α-
reduction. Androstenedione and testosterone are aromatized to form the estrogens,
estrone and estradiol and testosterone can be reduced to form DHT. DHT can be
further metabolized to 3β-diol and 3α-diol, with the 3β-diol metabolites exhibiting
estrogen activity (Lund et al., 2006).
The testes secrete the majority of testosterone, with only a small percent
being produced in the adrenals. In contrast to testosterone, the testes secrete only
20% of DHT with the rest resulting from metabolism of testosterone in tissue
(Toorians et al., 2003). Circulating testosterone is bound to either sex hormone
binding globulin (SHBG) or albumin resulting in only 1-2% free testosterone
(Vermeulen and Verdonck, 1968; Dunn et al., 1981). Bioavailable testosterone,
which is readily available for transport into the tissue, represents the loosely,
albumin-bound testosterone and free testosterone (Pardridge, 1985, 1986).
Aromatization of testosterone in the tissue is the main source of estrogen in males
with about 20% of estrogen being secreted from the testes (Gooren and Toorians,
2003).
18
Age-related androgen depletion in men
With advancing age, men experience a significant decrease in circulating
levels of testosterone (Swerdloff and Wang, 1993; Morley et al., 1997). The annual
decline in total testosterone levels is between 0.2%-1% and begins in the thirties
(Gray et al., 1991; Feldman et al., 2002). Due to an age-related increase in sex
hormone binding globulin (SHBG) at a rate of 1.1%-1.6% (Purifoy et al., 1981; Gray
et al., 1991; Vermeulen et al., 1996; Harman et al., 2001; Feldman et al., 2002),
levels of free, non-bound, testosterone decrease at a higher rate (2%-3% per year)
than total testosterone (Gray et al., 1991; Feldman et al., 2002; Muller et al., 2003).
While it remains unclear whether DHT levels change with age in men (Feldman et
al., 2002; Gray et al., 1991), decreases in DHEA, and androstendione are observed
with increasing age (Gray et al., 1991; Vermeulen et al., 1996; Feldman et al., 2002;
Muller et al., 2003). The age-associated decrease in testosterone is not reflected in
estradiol levels, due to increasing aromatase activity with age and an age-associated
increase in fat mass. Free estradiol decreases very slightly in males with advancing
age (Davidson et al., 1983; Vermeulen et al., 1996; Muller et al., 2003).
Although circulating levels of hormones generally parallel tissue levels,
factors such as sex hormone binding globulin, transport across the blood brain
barrier, and conversion enzymes present in the tissue can affect tissue levels of
hormones (Manni et al., 1985; Pardridge, 1985, 1986). There are few studies that
have examined brain levels of sex steroid hormones, and none have examined age-
related changes of these hormones in the brain. In Chapters Two and Three, I will
19
examine whether similar to circulating levels, brain levels of androgens also decrease
with increasing age.
The HPG axis with age
The depletion of androgens with age is due to both primary and secondary
hypogonadism (for review see (Korenman et al., 1990; Morley, 2001b; Swerdloff
and Wang, 2002; Kaufman and Vermeulen, 2005). Primary hypogonadism is
characterized by a decrease in the number of Leydig cells in the testes (Neaves et al.,
1984) and reduced levels of steroidogenic acute regulatory protein (STAR) in Leydig
cells (Leers-Sucheta et al., 1999), resulting in a decreased ability of the testes to
produce testosterone. Secondary hypogonadism results from dysfunction of the
hypothalamic pituitary-gonadal axis. Specifically, asynchronous release of GnRH
from the hypothalamus leads to decreased responsiveness of the pituitary to GNRH.
The pituitary then secretes LH more frequently and with lower amplitude, and FSH
is secreted at higher basal levels (reviewed in Morley 2001 and 2003). Therefore in
aged men levels of LH and FSH are significantly elevated (Morley et al., 1997) (for
review see (Morley, 2001b; Swerdloff and Wang, 2002)).
Androgen deficiency in men
Normal age-related testosterone depletion has been associated with functional
impairments in androgen-responsive tissues such as bone, muscle, and heart (Meier
et al., 1987; Burger et al., 1998; Baumgartner et al., 1999; Ferrando et al., 2002;
20
Jones et al., 2003; Sheffield-Moore and Urban, 2004). The resultant dysfunction and
disease due to testosterone loss has been collectively recognized as a clinical
syndrome termed ‘androgen deficiency in aging males’ (Morley, 2001b). Since the
brain is also an androgen responsive tissue, it may be susceptible to age-related
androgen loss. Although studies have reported alterations in mood, libido, and
cognition resulting from androgen depletion (Swerdloff and Wang, 1993; Morley,
2001b; Swerdloff and Wang, 2002; Gooren, 2003; Kaufman and Vermeulen, 2005),
all the consequences of ADAM on the aging brain still remains unknown. One
hypothesis of my thesis work is that brain levels of androgens decrease with age,
similar to circulating levels of androgens, placing the brain at increased risk for the
development of AD.
3. Androgen actions in the brain
Androgens, testosterone and its active metabolite dihydrotestosterone (DHT),
have several important actions in androgen responsive tissue throughout the body.
Androgen responsive tissue has significant expression of androgen receptors (AR),
which when activated by T and DHT are involved in regulating androgen actions
(Mooradian et al., 1987). One of these androgen responsive regions in the body with
significant localization of AR is the brain (Simerly et al., 1990; Kerr et al., 1995;
Tohgi et al., 1995).
21
Androgen receptors in the brain
Androgen receptors (AR) are distributed throughout the brain. The densest
expression of AR is in the amygdala with the posterodorsal part of the medial
nucleus and amygdalohippocampal area showing the greatest degree of AR
expression (Simerly et al., 1990). Other areas with high AR expression are the bed
nucleus of the stria terminalis and the medial and central parts of the preoptic
nucleus in hypothalamus. Sensory and motors areas of the brainstem express low to
medium levels of AR (Simerly et al., 1990). The hippocampus also expresses low to
medium levels of AR except for the dentate gyrus, which has not been shown to
express AR (Simerly et al., 1990). Consistent with the findings by Simerly et al.,
another study has found similar trends of AR expression in the hippocampus and
hypothalamus (Kerr et al., 1995). The thalamus has fairly low levels of AR and in
some areas it appears that there is no AR present (Simerly et al., 1990). The
cerebellar cortex has high expression of AR and the basal ganglia has medium but
consistent levels of AR expression (Simerly et al., 1990). Since very few areas in the
brain do not express AR, one can assume androgens, like estrogens, are very
important throughout the brain.
Beneficial neural actions of androgens
Androgens are responsible for several important actions in the brain
including classic roles in development and sexual differentiation (Cooke et al., 1998;
22
Forger, 2006). Masculinization of the brain during development occurs through
either androgen pathways involving androgen receptors, or estrogen pathways via
estrogen receptors following aromatization of testosterone (Shaw et al., 1988;
Renfree et al., 2001). Therefore, both testosterone and estrogen are responsible for
the development of neural systems that lead to male specific reproductive behaviors
(Arnold and Gorski, 1984; Balthazart and Foidart, 1993). Sexual differentiation
during development results from androgen regulation of apoptosis in several sexually
dimorphic nuclei, including the sexually dimorphic nuclei of the preoptic area in
hypothalamus, and the spinal nucleus of the bulbocavernosus (SNB) and dorsolateral
nucleus of the spinal cord (Nordeen et al., 1985; Lund et al., 2000; Nuñez et al.,
2000).
In addition to development and sexual differentiation, androgens also have
important beneficial neural actions in the brain. Testosterone, but not estrogen, has
been found to stimulate neuron differentiation in hypothalamic neurons (Beyer and
Hutchison, 1997). Specifically, testosterone exposure resulted in longer neurites,
increased neuritic branching, and enlarged somas through an androgen receptor-
dependent mechanism (Beyer and Hutchison, 1997). Testosterone also effects length
of dendrites, soma size and synapse number in motor neurons of the SNB
(Matsumoto, 1997). A recent study suggests that a possible mechanism of
testosterone action on neuron plasticity and morphology may involve AR-dependent
up-regulation of neuritin, a protein in neurons that contributes to the development
and regeneration (Naeve et al., 1997; Marron et al., 2005).
23
Androgens have also been shown to increase spine density (Kovacs et al.,
2003; Leranth et al., 2003; Leranth et al., 2004a; Leranth et al., 2004b). Studies have
observed a significant decrease in the density of spine synapses following
gonadectomy (GDX) in male rats. (Kovacs et al., 2003). This effect of androgen
depletion was reversed with acute testosterone replacement (Kovacs et al., 2003).
Leranth et al. also found that GDX significantly decreased spine density (Leranth et
al., 2003). In this study, male rats were replaced with testosterone and DHT
following GDX, and both androgens were able to return spine density to control
levels (Leranth et al., 2003). Androgens have also been found to regulate spine
density in other brain regions besides hippocampus. For example, spine density in
the ventromedial nucleus of the hypothalamus is significantly decreased when male
rats are castrated the day they are born (Matsumoto and Arai, 1986).
Since androgen actions include neuronal plasticity and synaptic changes, one
might predict that androgens may play a role in long-term potentiation (LTP).
Interestingly, the initial studies to examine the effects of androgens on LTP found
that peripubertal androgen depletion resulted in an increase in degree and duration of
potentiation and chronic exposure to testosterone and DHT resulted in a decrease of
magnitude and duration of LTP (Harley et al., 2000). Results from this study suggest
androgens act to decrease synaptic plasticity. However, it is interesting to note that
rats castrated during the beginning of puberty had enhanced LTP while rats castrated
at a later time point experienced weaker LTP (Harley et al., 2000). This finding
suggests the time of castration may affect the mechanism of androgen action. A
24
study by Hebbard et al. attempted to address this issue. This study found that rats
given testosterone during puberty had reduced LTP, in fact, a shift towards LTD was
observed. Rats that were not exposed to testosterone during puberty had normal
induction of LTP (Hebbard et al., 2003). Adult rats given testosterone exhibited a
normal LTP pattern with increased magnitude and duration of LTP as compared to
GDX adult rats (Hebbard et al., 2003). Results from this study suggest that androgen
exposure in purbertal rats has different effects on LTP and synaptic plasticity than in
adult rats. If androgens are replaced after the critical period, T has beneficial effects
on LTP (Hebbard et al., 2003). Similar results were observed by Sakata et al., who
found that in GDX mice LTP is attenuated. However, when mice are replaced with
testosterone LTP is restored to normal. (Sakata K, 2000). Results from studies
examining the role of androgens in LTP have found that it is likely they do enhance
LTP although timing of hormone withdrawal and replacement is important. In
support of these studies are findings that testosterone increases excitatory post-
synaptic potentials (EPSP) (Pouliot et al., 1996; Smith et al., 2002a). Results from
these studies found that EPSP duration was decreased following androgen depletion,
and testosterone and DHT exposure significantly increased EPSP amplitude (Pouliot
et al., 1996; Smith et al., 2002a).
In addition to androgen actions in neurons, testosterone has been found to
down-regulates reactive astrogliosis (Day et al., 1998). Glial fibrillary acidic protein
(GFAP), is a marker of astrogliosis which has been shown to be increased during
normal brain aging and following neural injury (Little and O'Callaghan, 2001).
25
Testosterone depletion correlates with increased GFAP in aged rats (Nichols et al.,
1993), an effect prevented with androgen treatment (Day et al., 1998).
Androgens have also been found to have actions which may play a direct role
in development of neurological disease such as regulation of Aβ and increasing
neuronal viability (Pike, 2001; Ramsden et al., 2003a) which will be discussed in
more detail later in the introduction.
Androgen actions through androgen and estrogen pathways
As discussed previously, androgen actions may be mediated by both
androgen and estrogen pathways. Androgens can act directly through androgen
receptors (AR) by testosterone and DHT. DHT is a more potent ligand and has a
higher affinity for androgen receptors than testosterone (Zhou et al., 1994; Singh et
al., 2000). Testosterone can also be aromatized to 17β-estradiol and act through
estrogen receptors (Gooren and Toorians, 2003). Androgen control of development
and sexual differentiation is through both direct androgen effects involving androgen
receptors, and aromatization of testosterone to estrogen and subsequent estrogen
pathways. In some cases when testosterone is used, it is unclear whether androgen
or estrogen pathways are responsible for testosterones’ actions. For example,
androgen depletion in male rats has been found to result in decreased spine synapse
number (Leranth et al., 2003) but whether androgen regulation of synapse number is
androgen-or estrogen-mediated is unclear. Interestingly in this study DHT was able
to prevent GDX induced decreases in spine synapse number while estrogen
26
replacement was not, suggesting in this particular case that androgen pathways are
responsible and estrogen does not appear to be playing a role (Leranth et al., 2004a).
Further, the effect of DHT and testosterone on spine density has also been studied in
female rats. Results from this study indicated that acute androgen replacement has a
positive effect on spine density (Leranth et al., 2004a). Together, these studies find
there is gender specific regulation of spine density by androgens and estrogens in
male and female brains. In the CA1 region of male hippocampus only androgens are
responsible for regulating spine synapse density suggesting actions of testosterone in
male brain are through an androgen pathway, not through aromatized estrogen.
Since androgen receptors are present in the hippocampus (Simerly et al., 1990; Kerr
et al., 1995) one can predict this androgen pathway is through testosterone or DHT
activation of androgen receptors
4. Androgens and cognition
Since androgens decrease with age, and androgens are involved with several
beneficial actions in the brain we predict that low levels of androgen will place the
brain at increased risk for the development of disease and dysfunction. Androgens
have been shown to have an effect on cognition including spatial abilities (Gouchie
and Kimura, 1991; Janowsky et al., 1994) and verbal fluency (Alexander et al.,
1998). Low levels of androgens have been associated with impaired cognitive
performance in some but not all studies (Moffat et al., 2002; Haren et al., 2005).
Men with a higher free testosterone index have been found to perform better on
27
visual and verbal memory and had better long-term memory (Barrett-Connor et al.,
1999) while those with low FTI’s had decreased visual memory, visuomotor
scanning, verbal memory, and visuospatial processing (Moffat et al., 2002).
Androgen therapies have been used for the treatment of hypogonadism in men (for
review see (Tan and Culberson, 2003)). A clinical study using hypogonadal and
eugonadal men found an androgen treatment of testosterone enanthate or sublingual
testosterone cyclodextrin may increase verbal fluency (Alexander et al., 1998). A
small clinical study using men who were recently diagnosed with AD found
testosterone contributed to improvement in cognition (Tan and Pu, 2003). In
particular, the MMSE and clock drawing test (CDT) were used to assess visual
spatial skills. Those subjects receiving testosterone treatment improved on both the
MMSE and CDT (Tan and Pu, 2003). Androgen replacement in cases with AD, or
mild cognitive impairments have had beneficial results in some but not all studies
(Tan and Pu, 2003; Cherrier et al., 2005; Lu et al., 2006).
Another use of androgen therapies has been in the treatment of prostate
cancer. Leuprorelin, goserlin, and cyproterone acetate are used for the treatment of
prostate cancer because they block androgen actions either through negative
feedback on the hypothalamic-pituitary axis or through androgen receptors. The use
of these drugs has been associated with impaired memory (Green, 2002; Salminen et
al., 2004). In a separate study, the discontinuation of these drugs resulted in
improved cognitive performance, specifically, verbal memory (Almeida and
Papadopoulos, 2003). Prostate cancers can be treated with a course of 6-9 months
28
anti-androgen treatment, followed by a 3 month period where testosterone levels are
allowed to return to normal. In a study using this paradigm, androgen blockade had
a negative effect on spatial memory but not on other aspects of cognition leading to
the conclusion that timing of androgen suppression and replacement is important on
cognitive skills (Cherrier et al., 2003).
5. Androgens and Alzheimer’s disease in Men
Age-related androgen depletion and the risk for Alzheimer’s disease
As discussed above advancing age is the most significant risk factor for the
development of AD and like women, men experience a robust decline in the
circulating levels of their sex steroid hormones testosterone and DHT as a
consequence of normal aging (Morley et al., 1997). This androgen depletion is due
to a combination of primary (gonads) and secondary (hypothalamic-pituitary-
gonadal axis) hypogonadism (Swerdloff and Wang, 1993). Decreases in testosterone
lead to functional impairments in androgen-responsive tissues such as bone, muscle,
and heart that are often manifested as the clinical syndrome ‘androgen deficiency in
aging males’ (Morley, 2001a). Since the brain has also been established as an
androgen responsive tissue we would hypothesize the loss of these important
hormones could place the brain at risk for damage and disease. Unlike in women,
where there is a wealth of studies investigating the relationship between estrogen and
AD, little is known about the relationship between androgens and the risk for AD.
29
Several studies have found low circulating levels of total testosterone in men
under the age of 80 with AD compared with age-matched non-demented men
(Hogervorst et al., 2001; Hogervorst et al., 2002; Rasmuson et al., 2002; Hogervorst
et al., 2003; Watanabe, 2004). Low levels of free testosterone has also been
observed in AD cases compared with controls (Hogervorst et al., 2003; Paoletti et al.,
2004). In contrast to these studies, there has been some work that has not found
differences in testosterone levels between AD cases and controls, however sample
size is small in these cases perhaps contributing to the conflicting results (Twist et
al., 2000; Pennanen et al., 2004). The risk of AD associated with low testosterone
levels appears to be stronger when other factors are involved. Similar to women, the
apoE ε4 allele is associated with an increased risk in AD in men. Interestingly, the
same study which found that low circulating levels of testosterone increased the risk
of AD, also found that cases which had at least one ε4 allele had lower levels of
testosterone than those cases without the ε4 allele (Hogervorst et al., 2002). This
study is supported by an animal study where male mice had deficits in learning and
memory after the androgen receptor had been blocked (Raber et al., 2002). The
androgen receptor has also been found to be associated with a risk for AD. Using the
Oxford cohort men were determined to be at increased risk for AD if they had an
androgen receptor polymorphism (less than 20 CAG repeats) (Lehmann et al., 2003).
Lower testosterone levels are also seen in those with this AR polymorphism
(Lehmann et al., 2004). However, like with apoE, it is hard to determine if these
factors are working together or independently to increase the risk for AD.
30
Although theses studies have identified a relationship between low
testosterone and AD, they are unable to clearly determine whether low testosterone is
a result of the disease process or rather, is contributing to it. In a prospective
longitudinal study, subjects from the Baltimore Longitudinal Study on Aging were
studied for anywhere from 4 to 37 years with a mean of 19 years. While all subjects
were free of AD at the time of first testosterone assessment, those that went on to
develop AD had lower free testosterone levels. Interestingly, low testosterone levels
were observed up to 5 years prior to AD diagnosis (Moffat et al., 2004). These
results suggest low testosterone may be responsible for the increased risk of AD. In
Chapters Two and Three, I will continue to investigate whether low testosterone is a
risk factor for AD, or resulting from the disease process. In addition, all the previous
studies examining the relationship between androgen and AD have been based on a
clinical diagnosis of AD. In the following chapters, I will also determine if brain
levels of androgens decrease with age using postmortem tissue, and if so, does this
place the brain at increased risk for a neuropathological diagnosis of AD.
Gonadotropins and other risk factors for AD
Recently gonadotropin levels, specifically luteinizing hormone (LH), have
been linked to an increased risk of AD (Bowen et al., 2000; Short et al., 2001).
Studies in men have found circulating levels of gonadotropins are increased in AD
cases as compared to control (non-AD) cases (Bowen et al., 2000; Short et al., 2001).
Another study however, found no difference in LH or FSH levels between AD and
31
control male cases but a significant decrease in testosterone levels in AD cases
(Hogervorst et al., 2003). One major difference between these studies is the age
difference. The latter study, which found no change in LH levels across disease
state, had a mean age of 75 years old while the study by Bowen et al. had a mean age
of 85 years. LH does not significantly increase in men till after 80 years of age
(Morley et al., 1997), suggesting a combination of age and disease state may be
responsible for the different effects observed between these studies. Although there
is not a great deal of evidence to support this idea there is a possibility that
gonadotropins may be at least partially responsible. A study separating the decrease
in sex steroid hormones from the increase in gonadotropins needs to be done to
determine if one hormone is solely responsible or if a combination of the two events
causes the increased risk of AD.
If sex steroid hormones are a primary risk factor for AD that sex hormone
binding globulin (SHBG) may be playing a role in preventing efficacy of HT.
SHBG is responsible for binding estrogens and testosterone; unbound hormone is
known as bioavailable and able to bind to hormone receptors (Pardridge, 1985).
SHBG increases with aging men (Morley et al., 1997) and decreases in
postmenopausal women (Skalba et al., 2001). A recent report found women with
AD had increased levels of SHBG as compared to subjects without AD (Hoskin et
al., 2004). This increase in SHBG in aged male and female cases with AD may play
a role in the effectiveness of hormone therapies by binding to the hormones and
making them less available for transport into tissues such as the brain.
32
6. Androgens and neuroprotection
Androgens promote neuron viability
One beneficial action of androgens, that may modulate the increased risk for
AD, is their role in neuroprotection. We previously discussed that during
development androgens regulate apoptosis in several sexually dimorphic nuclei
(Nordeen et al., 1985; Lund et al., 2000; Nuñez et al., 2000). Similar to androgen
regulation of apoptosis during development, androgens have also been found to
promote neuron viability in adult brain following mechanical injury and disease-
related toxicity. Several studies have examined the effects of androgens on
motorneurons following axotomy (for review see (Jones et al., 2001)). Testosterone
and DHT have been found to accelerate the rate of cranial nerve regeneration (Yu
and Srinivasan, 1981; Yu, 1982) and attenuate motor neuron loss following axotomy
(Yu, 1989). Similarly, following facial nerve crush in male hamsters, testosterone
increased the rate of axonal growth and functional recovery (Kujawa et al., 1989;
Kujawa et al., 1991). Interestingly in this study testosterone treatment was long-term
continuous exposure instead of injections. Long-term continuous release testosterone
exposure was the most effective treatment (Kujawa et al., 1989), suggesting the
importance of long-term androgen treatment for activation of androgen pathways.
Testosterone treatment accelerates the rate of nerve regeneration, and attenuates
neuron loss (Yu and Srinivasan, 1981; Yu, 1982; Kujawa et al., 1989; Kujawa et al.,
33
1991; Kujawa et al., 1993; Kujawa et al., 1995; Tanzer and Jones, 1997; Jones et al.,
2001; Huppenbauer et al., 2005). The mechanism behind this action appears to be
through testosterone regulation of trophic factors (Yu and Srinivasan, 1981; Yu,
1989). In addition, the anti-androgen flutamide was able to block testosterone’s
neuroprotective effects on motor neurons (Kujawa et al., 1995). Recently, studies
have found that in addition to long-term treatment, short-term testosterone, DHT,
and estrogen treatment is protective; these findings suggest a more direct mechanism
of hormone action (Huppenbauer et al., 2005).
In addition to androgen effects on motor neurons, androgens have also 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 vulnerable to AD and rich in androgen receptors as
discussed above (Simerly et al., 1990). In a study by Azocitia et al., acute
testosterone treatment attenuates neuron loss in the hilus of the dentate gyrus
following excitotoxic lesion in GDX male mice (Azcoitia et al., 2001). Interestingly,
acute treatment of estradiol is also protective while DHT treatment does not protect
again neuron loss in androgen depleted mice. Furthermore, the protective effect of
testosterone was blocked by an aromatase inhibitor, suggesting that in this model of
acute hormone treatment, estrogen is responsible for the neuroprotection (Azcoitia et
al., 2001). A study by Ramsden et al. investigated the effect of long-term hormone
replacement on excitotoxic lesion-induced cell death. In this study, removal of
endogenous androgens following GDX resulted in increased cell death compared
34
with sham kainite-lesioned rats. Following two weeks of testosterone and DHT
treatment, significant cell loss was attenuated and androgens were found to be
neuroprotective (Ramsden et al., 2003a). In contrast to the results of Azcoitia et al.,
rats treated with estrogen did not experience a decrease in cell loss following KA
lesion. This suggests that androgen regulation of neuroprotection in the
hippocampus is a result of androgen pathways not estrogen. In addition behavioral
responses to seizures were measured and no differences in hormone treated groups
were observed suggesting that neuroprotection was not a result of increased seizure
severity (Ramsden et al., 2003a). Although Ramsden et al. did not find a significant
effect of hormone treatment on seizure severity, thus providing a mechanism of
neuroprotection, other studies have found a significant effect of androgen and
androgen metabolites on seizure severity. In studies by Frye and colleagues
testosterone decreased neuron loss by decreasing seizure activity (Frye and Bayon,
1998; Frye et al., 2001). Subsequent studies found that the testosterone metabolite
DHT is further metabolism to 5α-androstane-3α, 17α-diol (3α-diol) which acts on
GABAa receptors resulting in GABA activation and subsequent decreased excitatory
signaling, antagonizing seizure activity (Edinger and Frye, 2004; Rhodes and Frye,
2004; Edinger and Frye, 2005).
Studies have found that androgens are neuroprotective, and there have been three
potential mechanisms proposed by in vivo studies: estrogen pathways, androgen
pathways, and anti-seizure effects via the DHT metabolite 3α-diol. Cell culture
models of toxicity have also examined the effects of testosterone on neuron viability
35
and are able to investigate the mechanism more thoroughly. Cell culture models of
disease-related cell death study the effects of hormones against serum deprivation
(Brooks et al., 1998; Hammond et al., 2001) Aβ toxicity (Pike, 2001; Zhang et al.,
2004) and oxidative damage (Ahlbom et al., 2001). Testosterone has been found to
be neuroprotective against serum deprivation induced neuronal apoptosis through an
androgen receptor dependent mechanism (Hammond et al., 2001). Specifically, the
anti-androgen flutamide attenuated protection while an aromatase inhibitor 4-
androsten-4-OL-3,17-dione, had no effect on neuron viability (Hammond et al.,
2001). Consistent with this androgen-mediated mechanism of androgen
neuroprotection is a study by Pike (2001) which found that testosterone
neuroprotection against toxicity induced by extracellular Aβ results from DHT not
estrogen (Pike, 2001). When DHT is used in this paradigm it is equally as protective
as testosterone, but use of an anti-estrogen droloxifene failed to block protection
suggesting androgen pathways are responsible for neuroprotection (Pike, 2001).
While it appears that the mechanism of androgen neuroprotection involves
androgen receptors, the mechanism of androgen action downstream of AR has also
been investigated and several different mechanisms have arisen. There is evidence
that androgen neuroprotection may be mediated through attenuation of oxidative
stress (Ahlbom et al., 2001). However, the concentration of testosterone used to
examine the effects of androgens on oxidative stress was at supra-physiological
levels, a concentration that when used with estrogen combats oxidative stress (Behl
et al., 1997). Another potential mechanism involves heat shock proteins. Androgens
36
have been found to attenuate Aβ induced toxicity through increasing levels of heat
shock protein 70, which is known to participate in neuroprotective responses against
cellular stress and neurodegeneration (Zhang et al., 2004). Recent studies from
Nguyen et al. have identified another mechanism involving an AR dependent
mechanism involving activation of a mitogen-activated protein kinase (MAPK) /
extracellular signal regulated kinase (ERK) pathway (Nguyen et al., 2005).
Testosterone and DHT activate MAPK, which in turn activates ERK followed by
activation of p90kDa ribosomal S6 kinase (Rsk), which phosphorylates the pro-
apoptotic protein Bad. Phosphorylation of Bad results in inactivation of Bad and
increased cell viability. Blocking any step of this pathway prevents phosphorylation
of Bad and androgen neuroprotection (Nguyen et al., 2005). Interestingly, a recent
study from Gatson et al. using C6 glial cells found DHT activation of ERK and
AKT, effectors in the MAPK and PI3K pathways, an effect blocked by flutamide
supporting the findings Nguyen et al of an AR dependent mechanism involving
MAPK signaling (Nguyen et al., 2005; Gatson et al., 2006). However, DHT
conjugated to BSA, and supraphysiological levels of T had no effect or resulted in
decreased expression of ERK and AKT, suggesting different AR signaling pathways
(Gatson et al., 2006).
Findings by Gatson et al., are supported by several studies that have found
androgens, instead of improving survival, may actually have no effect or even
exacerbate neuron loss. A study by Dluzen et al. (1996), found that estrogen but not
testosterone reduces methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
37
nigrostriatal dopaminergic toxicity (Dluzen, 1996). In addition, following 3-
nitropropionate acid (3-NP)-induced damage to the striatum and CA1 of
hippocampus, androgens had no effect on cell death and even exacerbated neuron
loss (Nishino et al., 1998).
Androgen regulation of tau hyperphosphorylation
While not a direct mechanism of cell viability, regulation of tau
hyperphosphorylation is very important for the development of AD. Along with
neuron loss and extracellular amyloid plaques, nerurofibrillary tangles are a
pathological hallmark of AD (Henderson and Finch, 1989). There has been little
research done to investigate the relationship between androgen loss and tau
hyperphosphorylation. Papsozomenos and colleagues have examined the effects of
testosterone and estrogen on tau hyperphosphorylation using a model of heat shock-
induced phosphorylation. In this model, testosterone, but not estrogen, prevented
hyperphosphorylation induced by heat shock (Papasozomenos, 1997;
Papasozomenos and Papasozomenos, 1999; Papasozomenos and Shanavas, 2002).
Androgen prevention of tau hyperphosphorylation may be through inhibition of GSK
(Papasozomenos and Shanavas, 2002). The relationship between androgens and AD-
like tau pathology had not been previously investigated in transgenic mice. I will
investigate the relationship between androgens and hyperphosphoylation of tau in
Chapter Six.
38
7. Androgen regulation of Aβ levels
Another beneficial action of androgens in the brain, which may modulate the
risk for AD in men is androgen regulation of Aβ. Senile plaques, which contain
mainly aggregated beta-amyloid (Aβ) peptides, are one of the major hallmarks of
Alzheimer’s disease (AD) pathology (Masters et al., 1985; Braak and Braak, 1991).
Aβ peptides result from the cleavage of amyloid precursor protein (APP) by β-
secretase (BACE) at the N terminus and γ-secretase at the C terminus (Hardy, 1997).
Cleavage of APP by α-secretase prevents the formation of Aβ by cleaving between
Lys16 and Lys17 residues on the Aβ domain (Sisodia, 1992). This action causes the
release of soluble APPα (sAPPα), which studies have found to be neuroprotective
(Mattson et al., 1993; Mattson, 1997). I predict that androgen loss during normal
aging increases the risk for AD through regulation of Aβ, and androgen depletion
will accelerate the progression of AD pathology (Chapter Four).
Androgen regulation of β-amyloid
Similar to estrogen, androgens have been shown to have beneficial actions in
the brain including regulation of Aβ levels in animal and human studies. Three
studies using prostate cancer patients measured Aβ levels while patients were
receiving a treatment of flutamide to block androgen receptors and the GNRH
agonist, leuprolimide, which causes decreased levels of testosterone, DHT, E2 and
gonadotropins. All these studies found that Aβ levels increased when androgens
39
were blocked (Gandy et al., 2001; Almeida and Papadopoulos, 2003; Gillett et al.,
2003). Unfortunately, these studies do not tell us if the effect is androgen regulated
or if it is due to the aromatization of testosterone to E2. In cell culture studies, a
reduction in Aβ levels with testosterone replacement was observed along with an
increase in α-secretase cleavage of APP (Gouras et al., 2000). However, since
replacement of testosterone was at pharmacological levels, the possibility of
testosterone being aromatized to E2 was likely. In fact, when aromatase inhibitors
were used Aβ levels did not decrease and cleavage of APP by α-secretase did not
change (Goodenough et al., 2000). The use of non-aromatizable DHT will determine
if regulation of Aβ in males is controlled by estrogens or androgens. A recent study
by Ramsden et al. was able to answer this question using rats that were GDX and
given either placebo or DHT for four weeks. A significant increase in soluble Aβ
1-40
levels was observed in brain lysates from GDX animals (Ramsden et al., 2003c).
Replacement of DHT in GDX rats for 4 weeks caused a significant reduction is brain
Aβ levels while rats replaced with E2 had no effect (Ramsden et al., 2003c). Results
from this study suggest androgens are responsible for regulation of Aβ in males
while estrogen is responsible in females.
Mechanism of androgen regulation of Aβ levels
Cellular functions of androgens such as neuron viability and modulation of
Aβ levels support the hypothesis that age-related androgen depletion may increase
the risk of developing AD. Androgens regulate Aβ either by decreasing production
40
or increasing clearance of Aβ. Estrogen appears to decrease deposition of Aβ by
precluding its formation (Jaffe et al., 1994; Vincent and Smith, 2000), but a recent
study has suggested estrogen may also play a role in clearance of Aβ (Huang et al.,
2004). Initial studies examining the effect of testosterone on APP processing
discovered their effect was due to estrogen, and not androgens (Goodenough et al.,
2000). One study has investigated the effect of androgens on APP processing by
using non-aromatizable DHT. In this study no differences were seen in either
sAPPα or full length APP, suggesting a mechanism by which androgens modulate
Aβ levels not through deposition, but increased clearance (Ramsden et al., 2003c).
If androgen regulation of Aβ is due to increased clearance, androgens must
be regulating an Aβ catabolizing enzyme. Neprilysin (NEP), is a major Aβ
catabolizing enzyme in the brain, (Iwata et al., 2000; Marr et al., 2003a; Newell et
al., 2003) and has been shown to be regulated by estrogen (Huang et al., 2004). NEP
is also of particular interest for androgens because the promoter region of the NEP
gene contains two functional steroid hormone response elements: an androgen
response element and an androgen response region (Shen et al., 2000b). This
suggests that NEP is a target for androgen transcriptional regulation. In fact, recent
data from a prostate cancer cell line suggests that DHT increases both expression and
activity of NEP (Shen et al., 2000a).
Another candidate mechanism may involve insulin-degrading enzyme (IDE),
another Aβ catabolizing enzyme that binds to AR and increases transcriptional
activity (Hamel et al., 1998). Other studies have found that an AR peptide fragment
41
can bind to IDE (Kupfer et al., 1994). Elucidation of the mechanism of androgen-
mediated modulation of Aβ levels will help determine the role of androgens in AD
pathogenesis and open the door to important therapeutic preventions and treatments.
LH and other factors that may affect Aβ
As discussed above, estrogen and androgens may have different mechanisms
in the regulation of Aβ with estrogen responsible for Aβ regulation in females and
androgens responsible in males. A recent study using double transgenic APPswe +
PS1 mice supports the idea of gender differences in regulation of Aβ. In this study,
Wang et. al. found female mice had an increased amyloid burden and plaque number
as compared to age-matched males. The female mice also accumulate amyloid at an
earlier age than male mice (Wang et al., 2003). These apparent gender differences
are most likely due to their different primary sex steroid hormones and the
mechanism through which they regulate Aβ.
There are also gender differences apparent in the apoE genotype. ApoE has
been shown to play a role in the accumulation of Aβ (Bales et al., 1997) and those
with at least one apoE ε4 allele are at increased risk to accumulate significantly more
Aβ than those without any apoE ε4 alleles (Johnson et al., 1998). Hogervorst and
colleagues found a significant relationship between low testosterone levels, apoE ε4,
and risk for AD in men (Hogervorst et al., 2002). Also women without any apoE ε2
alleles were found to have the highest Aβ load while men with at least 1 apoE ε2
allele had the lowest amount of Aβ load (Johnson et al., 1998). It can be predicted
42
that once Aβ deposition has begun accumulation of Aβ may be dependent on apoE
genotype and gender (Johnson et al., 1998).
Recently, levels of gonadotropins, specifically LH has been linked to an
increased risk of AD (Bowen et al., 2000; Short et al., 2001). Some believe HRT has
not been effective because the risk of AD associated with decreased levels of sex
steroid hormones is not due to the decrease in E2, but to the increase in LH (Webber
et al., 2004). A recent study sought to link LH levels with Aβ. A GNRH agonist
(Leuprolide acetate) was used to suppress LH levels in female mice. This treatment
reduced levels of Aβ1-42 and Aβ1-40 in brain after 4 weeks of treatment and an
even greater decrease in Aβ1-42 was observed at 8 weeks. To determine how LH
may be acting to regulate Aβ levels, a neuroblastoma cell line was used to observe
the effects of gondaotropins on APP. LH did not affect APP levels but levels of
secreted Aβ were increased. Next it was determined that increased LH levels
decreased levels of sAPPα, suggesting LH plays a role in APP processing of Aβ by
decreasing α-secretase cleavage and promoting α-secretase and γ-secreatase
cleavage of APP (Bowen et al., 2004). This study supports the idea of a novel
therapeutic target against the development of AD. Then again, when GNRH
agonists were used in the treatment of prostate cancer patients, different results were
observed. In these studies, the use of a GNRH agonist (decreased LH, E2, T, and
DHT) resulted in increased Aβ levels which were returned to normal when treatment
was stopped and sex steroid hormone levels had returned to normal (Gandy et al.,
43
2001; Almeida and Papadopoulos, 2003). More research in this area is needed to
fully determine the effects of gonadotropins on regulation of Aβ.
8. Hypothesis and experimental paradigm
Androgens have several beneficial and important actions in the body and
brain. Androgens decrease as a consequence of normal aging, and the loss of these
hormones results in disease and dysfunction in hormone responsive tissue such as
bone, muscle, and heart. Since the brain is also an androgen responsive region and
several beneficial actions have been identified, one would predict that the loss of
androgens with age might place the brain at increased risk for neurodengenerative
disease. In my thesis, I investigated the relationship between androgens, advancing
age, and the development of Alzheimer’s disease pathology. Specifically, I focused
on androgen regulation of Aβ using several different paradigms to determine
whether androgen loss increases the risk for AD through a mechanism involving
regulation of Aβ. The following chapters will outline the studies performed to
investigate my hypothesis. In Chapters Two and Three I examined whether brain
levels of androgens, similar to circulating levels, also decrease with increasing age,
and if so, does this place the brain at increased risk for the development of
Alzheimer’s disease? In Chapter Four, I used a rat model of male reproductive aging
to examine the relationship between circulating and brain levels of androgens and
their decline with age. I also started to investigate the mechanism of androgen
regulation of AD by examining the effect of age-related androgen depletion on Aβ.
44
In Chapter Five, I used a transgenic mouse model of AD to experimentally
investigate the effects of androgen depletion and replacement on the development of
AD-like pathology. In Chapter Six, I continued to investigate androgen-regulation of
AD pathology using the triple transgenic mice. In this study I investigated whether
androgen regulation of AD pathology was mediated through androgen or estrogen
pathways. In Chapter Seven, I examined the effect of estrogen and progesterone on
neuron viability following an excitotoxic lesion in female rats. Taken together,
results from these studies will provide useful insight into the relationships between
sex steroid hormones and the development of AD.
45
CHAPTER TWO
Age-related testosterone depletion and the development of Alzheimer
disease
Advancing age is the most significant risk factor for the development of
Alzheimer’s disease, however, which age-related changes underlie this effect
remains unclear. Men experience a significant decline in testosterone with
increasing age, which has been found to result in disease and dysfunction in several
androgen responsive regions. In this study we wanted to investigate two hypotheses,
1) that similar to circulating levels, brain levels of testosterone decrease with
increasing age in men and 2) if brain levels of testosterone do decrease with
advancing age, does this loss place the brain at increased risk for the development of
Alzheimer’s disease. We found that brain levels of testosterone decrease with age
while there is no change in brain levels of estradiol. In addition, significantly low
levels of testosterone were observed in cases with advanced Alzheimer’s disease,
and those with mild neuropathological changes as compared with
neuropathologically normal cases, suggesting that low levels of testosterone is a risk
factor for the development of AD. These results were published in 2004 in the
Journal of the American Medical Association (JAMA).
46
INTRODUCTION
Normal male aging is associated with declines in serum levels of the sex
steroid hormone testosterone, which contributes to a range of disorders including
osteoporosis and sarcopenia (Morley, 2001b). Unknown is how this relationship
applies to age-related disorders in the brain, an androgen-responsive tissue. We
hypothesize that testosterone levels in the brain are depleted as a normal
consequence of male aging and that low brain levels of testosterone increase the risk
of developing Alzheimer disease (AD). Recent data suggest a correspondence
between reduced serum levels of testosterone and the clinical diagnosis of AD
(Hogervorst et al., 2001; Hogervorst et al., 2002). However, it is unclear whether
testosterone depletion contributes to or results from the disease process. To
investigate this issue, testosterone and estradiol levels were analyzed in postmortem
brain tissue of elderly men and compared with their neuropathological diagnoses.
METHODS
Brain tissue from men who had provided informed consent was collected at
autopsy by repositories associated with Alzheimer’s Disease Research Centers at
University of Southern California; University of California, Irvine; University of
California, San Diego; and Duke University. Tissue was collected between 1997 and
2003, with postmortem delay less than 8 hours (mean delay=4.6 hours). Subjects
47
with conditions associated with altered testosterone levels (eg, endstage renal
disease, liver disease, alcoholism, and diabetes) were excluded from the study.
Included subjects satisfied 1 of the following neuropathological diagnoses: (1)
neuropathologically normal (controls) (Braak stage 0-1 without evidence of other
degenerative changes, and lacking a clinical history of cognitive impairment; n=17),
(2) AD (Braak stage 5-6 with neuropathological diagnosis of AD in the absence of
other neuropathology; n=19), and (3) mild neuropathological changes (Braak stage
2-3 in the absence of discrete neuropathology; n=9). No subjects were
neuropathologically diagnosed with Braak stage 4. Testosterone and estradiol levels
were measured by radioimmunoassay of homogenates of brain samples from the
midfrontal gyrus following organic extraction and celite column partition
chromatography (Goebelsmann, 1979). Hormone levels expressed as hormone
weight per wet tissue weight were statistically compared using analysis of covariance
with age as the covariate. This study was performed with institutional review board
approval from the University of Southern California.
RESULTS
We observed that brain levels of testosterone but not estradiol (Figure 1)
were inversely correlated with age in men aged 50 to 97 years who were diagnosed
as neuropathologically normal.
48
Figure 1. Brain Levels of Testosterone and Estradiol in Elderly Men.
The estradiol comparison shows only 16 data markers as there are 2 subjects aged 70
years with the same estradiol value (0.06 ng).
To investigate whether this depletion of brain testosterone may be a risk factor for
the development of AD, we compared hormone levels among elderly men who
exhibited no neuropathology, mild neuropathological changes, or moderate to severe
AD. Men in the 3 groups were of similar age, ranging from 60 to 80 years with mean
(SD) ages of 70.9 (5.3), 72.9 (5.7), and 72.0 (6.5) years, respectively, using analysis
of variance (F=0.002, P=.97). We found that brain levels of testosterone but not
estradiol (Figure 2) are significantly lower in AD subjects compared with the control
subjects. We found that brain levels of testosterone are also significantly reduced in
men with mild neuropathology consistent with early stage AD (Figure 2).
49
Figure 2. Brain Levels of Testosterone and Estradiol in Men, by Neuropathological
Diagnosis
Left, Testosterone levels in age-matched control subjects (controls), subjects with
mild neurological changes (MNC), and subjects with Alzheimer disease (AD). Right,
Estradiol levels in age-matched controls, subjects with MNC, and subjects with AD.
For testosterone levels, P<.05 is the comparison of age-matched controls vs subjects
with MNC and of age-matched controls vs subjects with AD by t test following
analysis of covariance with age as a covariate. Error bars indicate SEM.
DISCUSSION
Brain levels of testosterone significantly decrease with age in men who lack
any evidence of neuropathology, suggesting that neural androgen depletion is a
normal consequence of aging. In comparison with the control subjects, men with AD
exhibit significantly lower testosterone levels in the brain. In contrast, the data
suggest that estrogen levels in the male brain are affected by neither advancing age
nor AD diagnosis. Notably, testosterone depletion likely precedes and thus may
50
contribute to rather than result from the development of AD, since low brain
testosterone is observed in men with early indications of AD neuropathology.
Although it remains possible that low testosterone may reflect an unmeasured
correlate of AD rather than be a contributing factor, we controlled for established
causes of low testosterone by our exclusion criteria and statistical adjustment. How
testosterone depletion may contribute to AD development is unknown. However, we
have recently reported that androgen depletion in male rodents increases brain levels
of β-amyloid (Ramsden et al., 2003c), the protein implicated as a causal factor in AD
pathogenesis, and decreases neuronal survival upon exposure to toxic insult
(Ramsden et al., 2003a). Collectively, these findings suggest that normal, age-
related testosterone depletion in the male brain may impair beneficial neural actions
of androgens and thereby act as a risk factor for the development of AD.
51
CHAPTER THREE
Age-related changes in brain levels of sex steroid hormones in women
and men and their relationship to Alzheimer’s disease
In this study we wanted to continue to examine the relationship between low
levels of androgens and the development of Alzheimer’s disease (AD). We
expanded on our previous findings that brain levels of testosterone decrease with
increasing age, and low levels of testosterone are a risk factor for the development of
AD by including examination of other androgens and estrogens, dihydrotestosterone,
the active testosterone metabolite, and estrone, a precursor of estradiol. We found
that brain levels of androgens decrease with age while estrogens do not change. We
also observed low levels of testosterone and DHT in cases with advanced
Alzheimer’s disease, and those with mild neuropathological changes as compared
with neuropathologically normal cases, suggesting that low levels of androgens are a
risk factor for the development of AD. We also examined brain levels of Aβ, a
protein implicated as a causal factor in the development of AD. We found a strong
correlation between low levels of androgens and increased levels of Aβ. These
results have been recently submitted for publication.
52
CHAPTER THREE ABSTRACT
Advancing age is the most significant risk factor for the development of
Alzheimer's disease (AD). One consequence of normal aging in men and women
is a significant decline in circulating levels of their primary sex steroid hormones.
Several studies have identified a relationship between AD and estrogen loss
following menopause in women. Recent reports have also linked low levels of
androgens with an increased risk of AD in men. To further investigate these
relationships, we examined brain levels of sex steroid hormones in postmortem
tissue neuropathologically characterized as normal (Braak 0-I), advanced AD
pathology (Braak V-VI), and mild neuropathological changes (Braak II-III).
Hormones were isolated, purified by Celite column chromatography, and
analyzed using radioimmunoassay. In postmenopausal women, we observed
decreased levels of estradiol (E2) and estrone (E1) in cases with advanced AD
pathology. In men, we observed a robust decrease in androgen levels during
normal aging and significantly lower levels of androgens in men with moderate
to severe AD, supporting our previous findings. To investigate one possible
mechanism of this androgen-mediated risk for AD, we measured brain levels of
soluble Aβ, a protein implicated as a causal factor for AD. We found that low
brain levels of androgens in cases with mild neuropathological changes correlate
53
with increased levels of soluble Aβ protein. Collectively, these findings suggest
that normal, age-related androgen depletion in men acts as a risk factor for the
development of AD.
INTRODUCTION
Increasing age is the most significant risk factor for the development of
Alzheimer’s disease (AD) (Rocca et al., 1986; Jorm et al., 1987; Evans et al., 1989).
One age-related change all women experience is the almost complete loss of their
primary sex steroid hormone estrogen. Men also experience a robust decrease in
circulating levels of their sex steroid hormones: testosterone (T) and its active
metabolite dihydrotestosterone (DHT) during normal aging (Vermeulen et al., 1996;
Morley et al., 1997). The loss of sex steroid hormones during normal aging may
result in disease and dysfunction in hormone-responsive tissues (Stampfer et al.,
1990; Kleerekoper and Sullivan, 1995; Burger et al., 1998; Baumgartner et al., 1999;
Morley, 2001b). Since the brain is also a hormone-responsive tissue, it is logical to
predict that this loss may also affect hormone-mediated actions in the brain. In fact,
epidemiological evidence has linked estrogen loss during menopause with an
increased risk for the development of AD in women (for review see (Henderson,
1997; Cholerton et al., 2002)). However, studies examining estrogen levels in
postmenopausal women have produced conflicting results (Manly et al., 2000; Twist
et al., 2000; Cunningham et al., 2001) as have clinical studies trying to determine the
54
effectiveness of hormone replacement therapy in the prevention and treatment of AD
(Rapp et al., 2003; Shumaker et al., 2003; Espeland et al., 2004; Shumaker et al.,
2004).
In men, low circulating levels of total and free testosterone have been associated
with an increased risk for the development of AD (Hogervorst et al., 2001;
Hogervorst et al., 2002; Hogervorst et al., 2003; Hogervorst et al., 2004; Paoletti et
al., 2004; Watanabe, 2004). While these studies established a relationship between
androgens and AD, they were unable to distinguish whether low T levels are
contributing to, or a result of the disease process. Two recent reports suggest that
low androgen levels are a risk factor for the development of AD. One study revealed
that men with low circulating T levels are at an increased risk to develop AD as
determined by a cognitive diagnosis. (Moffat et al., 2004) Previously published
results from our laboratory found low brain levels of T in male cases with AD
pathology. We also found low T levels in cases with mild neuropathological
changes, supporting the idea that low androgen levels are a risk factor for the
development of AD (Rosario et al., 2004).
In this study, we expanded on our previous findings to examine the relationship
between brain levels of sex steroid hormones, advancing age, and AD in women and
men including DHT, the active metabolite of T, and estrone (E1), a precursor of 17β-
estradiol (E2). We also wanted to begin to investigate a possible mechanism through
which low levels of androgens increase the risk for AD in men. Sex steroid
hormones in both men and women have been shown to have numerous beneficial
55
actions in the brain. Several studies have demonstrated that estrogen promotes
neuron viability, synaptic plasticity, differentiation, and regulation of Aβ (Azcoitia et
al., 1999b; Woolley, 1999; Roof and Hall, 2000; Cholerton et al., 2002; Wise, 2002;
Hoffman et al., 2003). Studies have also revealed that androgens have beneficial
actions in the brain including increasing neuron viability, (Pike, 2001; Ramsden et
al., 2003a) plasticity, (Matsumoto, 1997) excitability, (Smith et al., 2002b) neuronal
differentiation, (Beyer and Hutchison, 1997) and regulation of β-amyloid (Aβ)
(Ramsden et al., 2003c). In this study we measured brain levels of Aβ, a protein
associated with the development of AD, in male cases to establish if low androgen
levels correlate with increased levels of soluble Aβ.
METHODS
Human cases
Male and female brain tissue from midfrontal gyrus with a postmortem
interval of less than 10 hours was acquired from tissue repositories associated with
Alzheimer's disease Research Centers at the University of Southern California,
University of California Irvine, University of California San Diego, and Duke
University. Subjects with potentially altered androgen or estrogen levels resulting
from conditions such as end stage renal disease, liver disease, breast cancer, and
prostate cancer were excluded from the study (approximately 7% of cases).
56
Female cases were divided into two groups according to neuropathological
diagnosis: i) neuropathologically normal (Braak stage 0-I without evidence of
degenerative changes, and lacking a clinical history of cognitive impairment; n=12;
mean age = 81.3 + 2.5), ii) AD (Braak stage V-VI with neuropathological diagnosis
of AD in the absence of other neuropathologies; n=32; mean age = 74.3 + 1.6).
Male cases were divided into three groups according to age and
neuropathological diagnosis: i) neuropathologically normal (Braak stage 0-I, without
evidence of degenerative changes, and lacking a clinical history of cognitive
impairment; n=8), ii) AD (Braak stage V-VI with neuropathological diagnosis of AD
in the absence of other neuropathologies; n=22), and iii) mild neuropathological
changes, MNC, (Braak stage II-III in the absence of discrete neuropathologies; n=7).
Because the greatest amount of heterogeneity in androgen levels was observed
between 60-80 years of age, when the development of AD is likely to begin, we
analyzed cases between the ages of 60 and 80 separately from those over 80 years of
age. The mean ages (+SEM) for the neuropatholoigically normal, AD, and mild
neuropathological changes, MNC, groups in the conserved age range of 60-80 years
were 71.1 + 2.2, 73.0 + 1.23, and 71.9 + 2.2 years, respectively ( F=0.002, p=0.966
by ANOVA). The mean ages for the male cases over the age of 80 were 88.9 + 1.4,
85.6 + 1.2, and 83.8 + 1.2 for the neuropathologically normal (n=11), AD (n=8) and
mild neuropathological changes (n=11) groups respectively.
57
Hormone assays
Steroid hormones were purified from frozen postmortem brain tissue (mid-
frontal gyrus) by radioimmunoassay (RIA) following organic extraction and Celite
column partition chromatography as previously described (Goebelsmann, 1979).
Frozen (-80°C) brain tissue was thawed and weighed. 0.3-0.4 grams of tissue was
homogenized in 1 ml ice-cold PBS. An aliquot of homogenate was taken for protein
quantification and the remainder used for hormone quantification. 3H-labeled
hormones (~500 cpm/tube) were included as internal standards to correct for
procedural losses. The analytes were extracted with hexane:ethyl acetate (3:2) and
separated from interfering steroids by use of Celite column partition chromatography
with ethylene glycol as the stationary phase. DHT and T were eluted with 10% and
35% toluene in isooctane, respectively, whereas elution of E1 and E2 was carried out
with 15% and 40% ethyl acetate in isooctane, respectively. Quantification of
hormones was determined by RIA’s. These assays have been shown to be sensitive,
accurate, precise, and specific, based on our analysis of inter and intra-assay
variability (>10%). Raw data were presented from triplicate readings and as a
fraction of starting tissue weight (protein correction yielded comparable data).
Aβ ELISA
Soluble Aβ levels from postmortem tissue of normal and MNC cases, were
determined by ELISA. Aβ was sequentially extracted in DEA buffer (50mM sodium
chloride, 0.2% DEA, and 1x protease inhibitor cocktail) at 1 ml buffer/100mg wet
58
weight tissue and centrifuged at 4° C at 20,000 rpm for 30 min. After centrifugation,
the supernatant was collected and stored at –80°C until assayed. Brain samples were
run in triplicate on ELISA plates coated with a monoclonal anti-Aβ1-16 antibody
(kindly provided by Dr. William Van Nostrand, Stony Brook University, Stony
Brook, NY) and detection was by monoclonal HRP conjugated anti-Aβ
1-40
(MM32-
13.1.1) and anti-Aβ
1-42
(MM40-21.3.1) antibodies (kindly provided by Dr.
Christopher Eckman, Mayo Clinic Jacksonville, Jacksonville, CA) (Das, 2003;
Kukar, 2005; McGowan, 2005).
Statistical analysis
Linear regression was used to analyze the interaction between raw hormone
levels (expressed as hormone weight per wet tissue weight) and increasing age. An
analysis of covariance (ANCOVA) with age as the covariate followed by between
group comparisons using the Fisher LSD test was used to determine if there were
changes in hormone levels across neuropathological status. Due to the results from
the analysis of hormone levels and age, male cases were split into two groups, 60-80
and over 80, for separate analysis. Correlations followed by Spearman rank analyses
were used to determine the relationship between hormone levels and soluble Aβ.
Since increasing age has an independent effect on soluble Aβ, partial correlations
were performed to adjust for age.
59
RESULTS
Brain levels of sex steroid hormones during aging and across neuropathological
diagnoses in women
To examine if brain levels of hormones in postmenopausal women correlate
with increasing age, female cases were characterized according to neuropathological
status with the absence of other neurodegenerative diseases and a post-mortem
interval (PMI) less than 10 hours (Table 1).
Table 1. Estrogen and androgen levels in female cases across neuropathological
status
Control Alzheimer’s
disease
p=
Sample size 12 32
Mean age, years 81.3 + 2.5 72.4 + 1.6 0.004
Mean PMI, hours 5.9 + .6 5.1 + .4 .23
In neuropathologically normal postmenopausal female cases, we did not observe any
changes in brain levels of androgens, T and DHT, or estrogens, E2 and E1, with
increasing age (T, r= -.07, p=0.82; DHT, r= -0.003, p= 0.99; E2, r= -0.27, p= 0.39;
E1, r= -0.24, p= 0.46; Figure 3).
60
Figure 3. Brain levels of estrogens and androgens remain the same during normal
aging in postmenopausal women. Steroid hormones were purified from frozen
postmortem female brain tissue (mid-frontal gyrus) using Celite chromatography and
then quantified by RIA. Values show results from triplicate readings, corrected for
procedural losses by radio-labeled hormone tracers, and presented as a fraction of
starting tissue weight (protein correction yields comparable data). Subjects (age 63-
95years) were rated neuropathologically normal with Braak staging value 0 or I.
Correlation coefficients for aging versus estradiol, estrone, testosterone and DHT
levels are r = -0.07, r = -0.003, r = -0.27, and -0.24 respectively.
To investigate whether hormonal status in postmenopausal women is
associated with neuropathological diagnosis, we compared hormone levels in aged
women that exhibited no neuropathology, and severe AD pathology. We did not
61
observe any changes in brain levels of androgens between the neuropathologically
normal and AD cases (ANCOVA, age as a covariate; T, p=0.30; DHT, p=0.28,
Figure 4 A,B). We did observe decreased levels of E2 and E1 in cases with
moderate to advanced AD pathology (E2, p=0.08; E1, p=0.04, Figure 4 C,D).
Figure 4. Brain levels of estrogen are decreased in AD.
Brain levels of estradiol (C) and estrone (D) in women are lower in AD cases in
comparison to age-matched controls with no neuropathological changes. No changes
in T (A) or DHT (B) levels were observed in women. Steroid hormones were
purified from frozen postmortem male brain tissue (mid-frontal gyrus) using Celite
chromatography then quantified by RIA. Values show results from triplicate
readings, corrected for procedural losses by radio-labeled hormone tracers, and
presented as a fraction of starting tissue weight.
62
Brain levels of sex steroid hormones normal during aging and across
neuropathological diagnoses in men
To determine if brain levels of sex steroid hormones change in men during
normal aging, male cases were characterized according to neuropathological status
with the absence of other neurodegenerative diseases, and a PMI less than 10 hours
(Table 2).
Table 2. Estrogen and androgen levels in male cases across neuropathological status
Control Alzheimer’s
disease
Mild
neuropathological
changes
p=
Sample size 7 22 7
Mean age, years 71.1 + 2.2 73.0 + 1.23 71.9 + 2.2 0.35
Mean PMI, hours 5.8 + 1.1 3.4 + 0.9 5.5 + 1.3 0.19
Brain levels of T (r= -0.71, p< 0.01, Figure 5A) and DHT (r= -0.57, p=0.013, Figure
5B) are inversely correlated with age in neuropathologically normal men (N=18, age
range 50-97 years). However, no relationship between increasing age and either
estradiol (r=-0.035, p=0.96, Figure 5C) or estrone was observed (r= -0.09, p=0.73,
Figure 5D).
63
Figure 5. Brain levels of androgens decrease with age.
Brain levels of androgens are depleted during normal aging but brain levels of
estrogens remain the same. Steroid hormones were purified from frozen postmortem
male brain tissue (mid-frontal gyrus) using Celite chromatography and then
quantified by RIA. Values show results from triplicate readings, corrected for
procedural losses by radio-labeled hormone tracers, and presented as a fraction of
starting tissue weight (protein correction yields comparable data). Subjects (age 50-
97 years) were rated neuropathologically normal with Braak staging value 0 or I.
Correlation coefficients for aging versus estradiol, estrone, testosterone and DHT
level are r = -0.035, r = -0.09, r = -0.71 and r = -0.57 respectively.
64
To investigate whether the depletion of brain androgen levels we observed in
men may be a risk factor for the development of AD, we compared hormone levels
in aged men that exhibited no neuropathology, moderate to severe AD pathology,
and mild neuropathological changes. Male cases were age-matched within a
conserved range from 60-80 years with mean ages of 71.1 + 2.2, 73.0 + 1.23, and
71.9 + 2.2 years, respectively (ANOVA; F=0.002, p=0.97). We found that brain
levels of testosterone (ANCOVA, age as a covariate; F=4.7, p=0.02, Figure 6A) are
significantly lower in AD cases in comparison to neuropathologically normal men.
Importantly, we found that brain levels of testosterone were also significantly
reduced in men with mild neuropathology consistent with early stage AD. DHT
(ANCOVA, age as a covariate; F=2.4, p=0.25, Figure 6C) followed a similar trend to
that of T but does not reach significance. In contrast to T, no changes in brain levels
of E2 were observed between groups (ANCOVA, age as a covariate; F=0.22, p=0.7,
Figure 6E). There was a trend towards increased brain levels of E1, however, levels
did not reach significance (ANCOVA, age as a covariate; F=4.25, p=0.06, Figure
6G).
65
Figure 6. Low androgens are a risk factor for AD in male cases 60-80 years of age.
Brain levels of T in men are decreased in MNC and AD cases 60-80 years of age
(A), a similar trend is observed with DHT (C). Brain levels of estradiol remain the
same across neuropathological status (E) while brain levels of estrone are inceased in
AD (G). In men over 80 years of age brain levels of androgens and estrogens do not
change across neuropathological status (B,D,F,H). Steroid hormones were purified
from frozen postmortem male brain tissue (mid-frontal gyrus) using Celite
chromatography then quantified by RIA. Values show results from triplicate
readings, corrected for procedural losses by radio-labeled hormone tracers, and
presented as a fraction of starting tissue weight.
66
67
Hormone levels in male cases over 80 years of age were also analyzed to
determine whether a relationship between androgens and AD still exists in older age
when androgens are at their lowest levels in all men. Cases were again divided into
three groups that exhibited either no neuropathology, mild neuropathological
changes, or moderate to severe AD pathology with mean ages of 88.9 + 1.4, 85.6 +
1.2, and 83.8 + 1.2 respectively (Table 3).
Table 3. Estrogen and androgen levels across neuropathological status in male cases
over 80 years of age
Control Alzheimer’s
disease
Mild
neuropathological
changes
p=
Sample size 8 11 10
Mean age, years 87.2 + 1.6 84.6 + 1.2 85.0 + 1.2 0.42
Mean PMI, hours 3.9 + 1.0 4.4 + 0.8 4.1 + 0.7 0.9
No significant changes between neuropathological diagnosis and hormone levels
were observed (ANCOVA, age as a covariate; T, F=2.5, p=0.10; DHT, F=2.6,
p=0.09; E2, F=0.4, p=0.64; E1, F=0.4, p=0.66, Figure 6B, D, F, H).
Correlations between Aβ, age, and sex steroid hormones
To begin to elucidate the mechanism of androgen action in the development
of AD, brain levels of Aβ were measured in neuropathologically normal cases and
cases with mild neuropathological changes. Brain levels of Aβ were undetectable in
68
the neuropathologically normal cases; therefore, all correlations between hormones
and Aβ were performed using cases with mild neuropathology. Consistent with
previous studies that have reported increased levels of Aβ in plasma CSF with
increasing age (Fukuyama et al., 2000; Fukumoto et al., 2003; Mayeux et al., 2003),
we observed a mild trend towards increased levels of Aβ in the brain with increasing
age (r = 0.3, p=0.3). Since there is a small relationship between Aβ levels in the
brain and age, we ran both correlations and partial correlations to examine the
relationship between levels of hormones and Aβ in the brain. Interestingly, age had
little or no effect on the relationship between Aβ and hormone levels (Table 4).
Table 4. Correlations and partial correlations between sex steroid hormones and
soluble levels of Aβ
Hormone AGE Aβ42 Aβ42 /AGE
Testosterone (ng/g
WT)
-0.42 -0.49* -0.42
Dihydrotestosterone
(ng/g WT)
-0.2 -0.3 -0.26
Estradiol
(ng/g WT)
0.11 -0.3 -0.3
Estrone
(ng/g WT)
0.13 -0.14 -0.17
AGE
0.3
* P < 0.05
We observed a strong correlation between Aβ1-42 and T (-0.49), which was only
slightly decreased when age was partialed out (-0.42, Figure 7A). A similar
69
however, non-significant correlation was observed between DHT and Aβ, and E2
and Aβ (Table 7B,C). No relationship between estrone and Aβ1-42 levels was
observed in these cases (Figure 7D).
Figure 7. Low brain levels of androgens correlate with increased soluble Aβ1-42.
Low androgen levels correlate with increased levels of Aβ (A,B). Estradiol also
weakly correlates with Aβ however, there is no relationship between estrone levels
and Aβ. Steroid hormones from MNC cases were purified from postmortem tissue
using celite chromatography then quantified by RIA. Brain levels of soluble Aβ
were measured from DEA lysates using human specific Aβ ELISA.
70
Aβ1-40 was also measured in cases with mild neuropathological changes and
produced a similar trend as observed with Aβ1-42 for each hormone but did not
reach significance (data not shown).
DISCUSSION
In this study we investigated the relationship between brain levels of sex
steroid hormones in men and women during normal aging and across
neuropathological status. As predicted, we did not observe an age-related decline in
brain levels of sex steroid hormones in neuropathologically normal postmenopausal
women, we did however, observe a significant reduction in brain levels of E2 and E1
in female cases with AD. In men, we found that brain levels of androgens decrease
as a consequence of normal aging. Additionally, men with moderate to advanced
AD neuropathology had significantly lower levels of androgens compared with
neuropathologically normal men. Low androgen levels appear to be a risk factor for
the development of AD rather than a result of the disease process since we also
observed significantly reduced levels of androgens in cases with mild pathology
consistent with early AD. Examination of Aβ levels revealed that low levels of
testosterone correlate with increased levels of Aβ, suggesting a possible mechanism
for the role of androgen depletion in the development of AD.
Although epidemiological evidence suggests that estrogen loss at menopause is
a risk factor for the development of AD, (for review see (Henderson, 1997;
71
Cholerton et al., 2002)) few studies have examined the relationship between sex
steroid hormone levels and AD in postmenopausal women. In this study we found a
strong trend towards decreased brain levels of estrogen in cases with advanced AD
pathology compared with neuropathologically normal control cases. Our findings
are consistent with a report by Manly and colleagues, which reported significantly
lower circulating levels of estradiol in AD cases (Manly et al., 2000) and a recent
study in women with down syndrome which found that subjects with low levels of
estrogen were 4 times as likely to develop AD (Schupf et al., 2006). However, not all
studies have found similar results. Cunningham et at., found no difference in
circulating levels of estrogen between control and AD subjects (Cunningham et al.,
2001). Differences between these studies make direct comparisons difficult. There
are also important differences between these studies and our current findings, which
include the use of a pathological diagnosis of AD versus a clinical diagnosis, and the
measurement of brain levels of hormones, which represent an accurate measurement
of bioavailable hormone levels in the brain. Although tissue levels of sex steroid
hormones generally parallel circulating levels this is not always the case. Therefore,
to obtain the most accurate measure, hormone levels must be measured in the tissue
of interest, which in our case is the brain (Pardridge, 1985, 1986). There is one study
that measured brain levels of estradiol in AD cases, but they did not observe
differences between control and AD cases (Twist et al., 2000). While it is important
that they measured brain levels of estradiol, the small sample size and long post-
mortem delay makes the results difficult to compare with our study.
72
The steady but significant loss in circulating levels of androgens men experience
during normal aging differs from the abrupt loss of estrogen women experience
during menopause (Nankin and Calkins, 1986; Gray et al., 1991; Vermeulen et al.,
1996; Morley et al., 1997; Harman et al., 2001; Feldman et al., 2002; Rosario, 2004).
While it has been established that this abrupt and nearly complete loss in circulating
levels of estrogen places women at an increased risk for the development of AD,
(Henderson, 1997; Cholerton et al., 2002) the relationship between androgen
depletion and AD in men is not as clearly defined. Initial studies found a
relationship between low circulating levels of androgens and a cognitive diagnosis of
AD (Hogervorst et al., 2001; Hogervorst et al., 2002; Hogervorst et al., 2003;
Hogervorst et al., 2004). Interestingly, low T levels were also observed in men with
at least one ApoE ε4 allele, (Hogervorst et al., 2002) another risk factor for the
development of AD, which we were unable to examine due to the small number of
apoE4 cases in our control group. In addition to analysis of circulating levels of
androgens and AD, brain levels of androgens from frontal and temporal cortex were
measured in normal and AD cases (Twist et al., 2000). Similar to our findings but
not reaching significance, Twist et al found a trend towards decreased levels of T in
AD cases but the small sample size and long post-mortem delay prevent definitive
conclusions. While these studies support a relationship between low levels of
androgens and AD, they do not discriminate whether low androgen levels are a risk
factor for AD or a result of the disease process. Recently, a prospective, longitudinal
study using subjects from the Baltimore Longitudinal Study on Aging (BLSA)
73
addressed this issue. In this study, subjects were followed for anywhere from 4 to 37
years (with a mean of 19 years) and were diagnosed as cognitively normal at the time
of the first T assessment. Subjects that proceeded to develop a cognitive diagnosis of
AD had lower levels of free T. Interestingly, low T levels were observed up to 5
years prior to the AD diagnosis, suggesting that low T may be a risk factor for the
development of AD. (Moffat et al., 2004) The significant reduction in androgens we
observed in cases with mild neuropathological changes, consistent with the earliest
indications of AD pathology, as well as cases with advanced AD pathology, suggest
that androgen depletion occurs prior to the development of AD and may therefore
contribute to AD neuropathology rather than result from it. Since our study
examined hormone levels in the brain, specifically in mid-frontal gyrus a region
affected by AD, our results yield a more accurate measurement of what is occurring
in the brain in comparison to serum levels. While sex steroid hormone levels in brain
generally parallel circulating levels, differences can be created by age-related
alterations in hormone metabolic enzyme activities (Serafini et al., 1985; Roselli et
al., 1986) and the ability of hormones to cross the blood brain barrier (Pardridge,
1985).
Since low androgen levels appear to be a risk factor for the development of AD,
the next step is to begin to elucidate the mechanism of this androgen regulated effect.
Androgens have been found to have several beneficial actions in brain. Two of these
androgen-mediated actions relevant to the progression of AD pathology are neuronal
viability (Pike, 2001; Ramsden et al., 2003b) and the regulation of Aβ (Ramsden et
74
al., 2003c). In cell culture studies, T replacement reduced Aβ levels and increased
α-secretase cleavage of APP (Gouras et al., 2000). However, since replacement of T
was at pharmacological levels, it was likely that T was being aromatized to E2, and
thus this effect could have occurred through the estrogen pathway, E2. In fact, when
aromatase inhibitors were used, Aβ levels did not decrease and cleavage of APP by
α-secretase did not change (Goodenough et al., 2000). A study from our laboratory
using Spague-dawley rats determined that castration for 4 weeks significantly
increased soluble levels of Aβ in comparison to sham control rats (Ramsden et al.,
2003c). Replacement of DHT in GDX rats for 4 weeks caused a significant
reduction in brain Aβ levels while rats replaced with E2 experienced no such effect.
Results from this study suggest androgens are responsible for regulation of Aβ in
male rats (Ramsden et al., 2003c).
To examine this effect in the human brain, we measured levels of soluble Aβ to
determine if there is a correlation between androgen and Aβ levels in the human
brain. In cases with mild neuropathological changes, we found a strong correlation
between low levels of testosterone and increased levels of Aβ suggesting that
testosterone may regulate Aβ levels contributing to the increased risk of AD in men
with low levels of androgens. Our findings are supported by several studies in
humans, which used prostate cancer patients on altered hormone treatments. In these
studies, Aβ levels were measured while patients were receiving a treatment of
flutamide to block androgen receptors, and leuprolimide, a GNRH agonist, which
resulted in decreased levels of T, DHT, E2 and gonadotropins. All these studies
75
found that when androgens are blocked or decreased, Aβ levels increase (Gandy et
al., 2001; Almeida and Papadopoulos, 2003; Gillett et al., 2003). In conjunction with
our results and previous findings, it appears that androgen-regulation of Aβ levels
may contribute to the increased risk of AD in men with low levels of androgens.
Estrogen does not appear to be responsible for the progression of AD pathology
in men. Consistent with the literature, we did not observe any changes in brain
levels of estrogen during normal male aging or across neuropathological status in
men. We did however, see a trend towards increased brain levels of estrone in men
with advanced AD neuropathology. Since we did not observe changes in brain
levels of estrone during normal aging and levels were only elevated in AD cases, it
appears that increased estrone levels are a result of the disease process. Interestingly,
17β-HSD type 10, the enzyme responsible for the conversion of estradiol to estrone,
was increased in a mouse model for AD, (He et al., 2002) suggesting that AD may
lead to changes in enzyme activity that result in increased conversion of estradiol to
estrone. While we did not observe a similar effect in the female cases, one study has
reported increased circulating levels of estrone in female cases with AD
(Cunningham et al., 2001).
Similar to estrogens and androgens the gonadotropin, lutenizing hormone (LH),
has also been implicated as a risk factor for AD (Short et al., 2001; Bowen et al.,
2000). Due to changes in the hypothalamic-pituitary gonadal axis with age (Morley,
2001) gonadotropins (LH and FSH) increase as sex steroid hormones decrease.
Studies in men have found circulating levels of gonadotropins to be increased in AD
76
cases as compared to control (non-AD) cases (Bowen et al., 2000; Short et al., 2001).
LH has also been shown to regulate levels of Aβ. Leuprolide acetate, a GNRH
agonist, was used to suppress LH levels in female mice resulting in reduced levels of
Aβ1-42 and Aβ1-40 (Bowen et al., 2004). Although these studies support the idea of
a novel therapeutic target for the development of AD, other studies have found no
difference in LH or FSH levels between AD and control male cases but a significant
decrease in T levels in AD cases (Hogervorst et al., 2003). In addition, in men being
treated for prostate cancer with GnRH agonists responsible for reducing levels of LH
have resulted in increased levels of Aβ and cognitive impairment (Gandy et al.,
2001; Almeida et al., 2003). Since LH does not significantly increase in men till
after 80 (Morley et al., 1997), it may not become a significant risk factor till old age.
Our relationship between androgens, Aβ, and AD is in men between the ages of 60
and 80 suggesting that LH is not playing a role in this study.
Results from this study suggest that low levels of androgens are a risk factor for
the development of AD and that this effect may be mediated through androgen-
regulation of Aβ levels. Further understanding the mechanism of androgen actions
in the brain and during the development of AD will lead to potential therapeutic
interventions.
77
CHAPTER FOUR
Age-related testosterone decline and increased Aβ in the brown Norway
rat brain
In humans, we have found that low levels of testosterone are a risk factor for
the development of AD. In this study we wanted to investigate age-related changes
in androgens and determine if circulating and brain levels of androgens correlate.
We hypothesized that age-related changes in androgens would correlate increased
levels of Aβ. To test these hypotheses we used brown Norway rats, which are a
good model of male reproductive aging. We measured brain levels of Aβ in young
and aged rats and treated aged rats with androgens to determine the brain maintains
hormone responsiveness with age. We found that circulating and brain levels of
testosterone decrease with increasing age. In addition, brain, but not circulating
levels of DHT also decrease with increasing age. We also observed a significant
increase in Aβ levels with age.
78
CHAPTER FOUR ABSTRACT
During normal aging, men experience a significant decline in their primary sex
steroid hormones, testosterone (T) and dihydrotestosterone (DHT). Low levels of
androgens have been found to result in disease and dysfunction in several androgen
responsive tissues such as the brain. Brain levels of androgens have also been found
to decrease with increased age. Low brain levels of androgens have been identified
as a risk factor for the development of Alzheimer’s disease (AD) in men. How low
levels of androgens increase the risk for AD has yet to be fully elucidated. However,
a potential mechanism is androgen regulation of β-amyloid (Aβ), a protein
implicated as a causal factor in the development of AD. Low levels of androgens
correlate with increased Aβ in both human and rodent models; however, the effects
of age-related T decline on Aβ have not been clearly examined. To investigate these
relationships, we first examined changes in circulating and brain levels of androgens
in Brown Norway rats. Brown Norway rats are an ideal model of male reproductive
aging because they experience both primary and secondary hypogonadism which
mirrors the human condition closely. Hormones were isolated from serum and brain
samples, purified by Celite column chromatography, and quantified using
radioimmunoassay. We observed reduced brain levels of T and DHT, and decreased
circulating levels of T with increasing age. To investigate a possible mechanism for
how androgen loss increases risk for developing AD, we measured brain levels of
soluble Aβ using ELISA. We found increased levels of soluble Aβ with increasing
79
age and following androgen depletion in young animals. Collectively, these findings
suggest that the age-related decline in brain levels of androgens, and subsequent
increase in levels of Aβ, increases the likelihood for the development of AD.
INTRODUCTION
Men experience a significant decline in circulating levels of their primary sex
steroid hormone, testosterone (T), with increasing age. Both longitudinal and cross-
sectional studies found a significant decrease in circulating levels of T with
advancing age (Baker et al., 1976; Morley et al., 1997). In addition, an even greater
decline has been observed in levels of free, non-sex hormone binding globulin
(SHBG) bound, testosterone (Feldman et al., 2002; Muller et al., 2003). This
significant loss in T has been found to result in disease and dysfunction in androgen-
responsive tissue such as bone and muscle (Morley, 2001b). The brain is also an
androgen-responsive tissue (Mooradian et al., 1987). Although tissue levels of sex
steroid hormones generally parallel circulating levels, differences in converting
enzymes present in tissue and the ability of a particular hormone to cross the blood
brain barrier may contribute to differences between serum and tissue levels
(Pardridge et al., 1980; Manni et al., 1985; Pardridge, 1986). Few studies have
examined tissue levels of androgens in comparison to circulating levels during
normal aging. T levels in pubic skin, and those of its active androgen metabolite,
dihydrotestosterone (DHT) have been shown to decrease with increasing age
80
(Deslypere and Vermeulen, 1981). Previous work from our laboratory found that
similar to circulating levels of T, brain levels of T also decrease with advancing age
(Rosario et al., 2004, Chapter Two and Three). The loss of androgens, and resultant
disease and dysfunction, lead to recent studies investigating the relationship between
low levels of T and the development of Alzheimer’s disease (AD). Several studies
by Hogervorst and colleagues found low circulating levels of T in cases with a
clinical diagnosis of AD (Hogervorst et al., 2001; Hogervorst et al., 2002;
Hogervorst et al., 2003). Two recent studies suggested that low levels of T are
contributing to the development of AD pathology and thus, are a risk factor for the
development of AD (Moffat et al., 2004; Rosario et al., 2004, Chapter Two).
Specifically, in human postmortem tissue, cases with advanced AD pathology had
significantly decreased brain levels of T; in addition, cases with mild
neuropathological changes also had significantly decreased levels of T, suggesting
that low brain levels of T are a risk factor for the development of AD (Rosario et al.,
2004, Chapter Two). Consistent with these findings, Moffat et al. observed low
levels of T in cases with a clinical diagnosis of AD, 5 and 10 years prior to the onset
of the disease (Moffat et al., 2004).
Previous work from our laboratory has begun to investigate how low levels
of androgens are increasing the risk for AD. Androgens have been found to have
several beneficial actions in the brain including increasing neuron viability
(Ramsden et al., 2003a), and synaptic plasticity (Leranth et al., 2003), promoting
neurite outgrowth and differentiation (Matsumoto, 1997; Marron et al., 2005) and
81
decreasing tau hyperphosphorylation (Papasozomenos, 1997). Another androgen-
mediated action relevant to the development of AD is regulation of β-amyloid (Aβ)
levels (Ramsden et al., 2003c). Aβ is a protein that has been implicated as a causal
factor for the development of AD (Hardy and Higgins, 1992). A previous study
from our lab using Sprague Dawley rats, found that androgen depletion by
gonadectomy (GDX) resulted in a significant increase in brain levels of soluble Aβ
(Ramsden et al., 2003c). Androgen treatment in these GDX rats returned Aβ levels
to those observed in sham animals (Ramsden et al., 2003c). Recently, using a
transgenic mouse model of AD, we observed a significant increase in accumulation
of intraneuronal and extracellular Aβ pathology following 4 months of androgen
depletion, an effect prevented with androgen treatment (Rosario, 2006, Chapter
Five). In men, plasma Aβ levels have been shown to increase following anti-
androgen treatment (Gandy et al., 2001; Almeida et al., 2004). In addition, in aged
men, low levels of androgens have been found to correlate with increased plasma
levels of Aβ (Gillett et al., 2003).
Although we have previously shown that complete androgen depletion
increases levels of Aβ (Ramsden et al., 2003c; Rosario, 2006, Chapter Five), it
remains unclear whether age-related androgen depletion will be sufficient in
magnitude to impair androgens' normal regulatory function on Aβ levels. In this
study, we examined age-related changes in circulating and brain levels of androgens,
and investigated whether normal, age-related androgen depletion affects regulation
of Aβ levels. To examine this question we used the Brown Norway (BN) rat model
82
of male reproductive aging. The role of primary and secondary hypogonadism in
mediating androgen depletion in BN rats matches the human condition very closely,
making BN rats a good model of normal reproductive aging in men (Wang et al.,
1993; Gruenewald et al., 1994; Wang et al., 2002). By 18 months of age, BN rats
exhibit a variety of deficits in both neural and reproductive tissues associated with a
decline in circulating levels of T, which are less than half of the levels observed in
adult 6 mo old males (Wang et al., 2002). We predict that the age-related changes in
circulating levels of androgens observed in male BN rats will also occur in the brain
and result in increased brain Aβ levels.
METHODS
Animals
In order to examine age-related changes in androgen and Aβ levels, we first
wanted to determine the effect of nearly complete androgen depletion on androgen
and Aβ levels. To investigate these relationships we used male BN rats supplied by
the National Institute of Aging (NIA). To examine the effect of gonadectomy
(GDX) on brain levels of androgens and Aβ levels, animals were sham GDX or
GDX at 3 mo of age under pentobarbital (50mg/kg) anesthesia. To assess age-
related changes in androgen and Aβ levels, we used BN rats at 3, 13, and 23 months
83
of age (N=7/group). After 1 month of hormone manipulations, animals (N=7 per
condition) under isoflurane anesthesia were sacrificed by decapitation. Brains were
rapidly dissected, halved midsagitally, and snap-frozen for Aβ ELISA and hormone
determination.
Hormone assays
Steroid hormones were purified from frozen brain tissue and serum using
Celite chromatography, and then quantified by RIA as previously described
(Goebelsmann, 1979; Slater et al., 2001). Briefly, frozen (-80°C) brain tissue was
thawed, weighed, and homogenized in 1 ml of ice cold PBS. 3H-labeled hormones
(~500 cpm/tube) were included as internal standards to correct for procedural losses.
Brain and serum samples were extracted with hexane:ethyl acetate (3:2) and
separated from interfering steroids by Celite chromatography with ethylene glycol as
the stationary phase. DHT and T were eluted with 10% and 35% toluene in
isooctane, respectively; whereas elution of estrone (E1) and 17β-estradiol (E2) was
carried out with 15% and 40% ethyl acetate in isooctane, respectively.
Quantification of steroid hormones was determined by sensitive, accurate, precise,
and specific RIA. Statistical analysis was performed using ANOVA followed by
between-group comparisons using the Fisher LSD test.
84
Aβ ELISA
One hemi-brain was processed for Aβ ELISA by extracting soluble protein
by homogenization (DEA buffer: 0.2% diethylamine, 50mM NaCl) using a polytron
for 1 min on ice. Homogenates were centrifuged at 100,000 g at 4°C for 1 h, the
supernatants were collected and neutralized (1/10th volume of 0.5 M Tris-HCl pH
6.8). Brain samples were analyzed by Aβ sandwich capture ELISA as previously
described (Suzuki et al., 1994; Murphy et al., 1999) with antibodies specific for
rodent. Briefly, Aβ was captured using antibody-coated plates, followed by
detection of Aβ1-40 peptides with the horseradish peroxidase-conjugated antibodies.
Plates were developed using TMB as substrate (reaction stopped by addition of 6%
phosphoric acid) and optical density read at 450 nm. Aβ values were determined on
the basis of a synthetic Aβ standard curve. Raw data (expressed as pmol Aβ/g
protein) was statistically analyzed by ANOVA followed by pairwise comparisons
using the Fisher LSD test.
RESULTS
Changes in circulating and brain levels of androgens following androgen depletion
in young rats
To determine the effect of androgen depletion in young rats, we measured
seminal vesicle weight, a common bioassay of androgen action, and circulating
levels of T and DHT. We found that following androgen depletion (GDX) for 1
85
month, seminal vesicle weight, calculated as percent of the body weight (seminal
vesicle wt/body weight), was significantly decreased, as were circulating levels of T
and DHT (Table 5). To examine the effects of androgen depletion on brain levels of
hormones, we also measured T and DHT in sham and GDX rats. We found that
similar to circulating levels, brain levels of T and DHT were also significantly
decreased (Table 5). In fact, brain levels of DHT in GDX rats were all below the
detectable range of the assay. Since T can be converted to DHT and 17β-estradiol
(E2), we also measured brain levels of E2 and found a small non-significant decrease
in brain levels of E2 (data not shown).
Table 5. Changes in androgen levels following GDX in 3mo old rats
3mo Sham 3mo GDX
Seminal vesicle wt
(seminal vesicle
wt/body wt) x10
1.09 + 0.03 0.37 + 0.02*
Serum testosterone
(ng/ml)
2.9 + 0.9 0.015 + 0.002*
Serum DHT (pg/ml) 52.8 + 11.8 12.0 + 1.5*
Brain testosterone
(ng/g wt)
2.18 + 0.39 0.07 + 0.3*
Brain DHT
(ng/g wt)
1.5 + 0.42 Below detectable range
* p<0.05 versus Sham group.
Circulating and brain levels of sex steroid hormones during aging
To determine if androgens decrease with increasing age in the BN rat, we
measured circulating and brain levels of androgens at 3, 13, and 23 months of age.
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We found that circulating levels of T decreased about 50% between 3 and 13 months
of age and continued to decline slightly between 13 and 23 months (Figure 8A).
Interestingly, circulating levels of DHT did not change with increasing age (Figure
8B). We observed a small but significant decrease in seminal vesicle weight with
age (F=5.7, p=0.01), calculated as a percent of the body weight due to increased
body weight with increasing age (seminal vesicle wt/body weight). Although tissue
levels of hormones generally parallel circulating levels, we nonetheless wanted to
measure brain levels of androgens, which are a more accurate measure of what is
occurring in the brain. We found a significant decrease in brain levels of both T and
DHT (Figure 8C,D). Since T can be converted to DHT and 17β-estradiol (E2), we
also measured brain levels of E2 and found a small non-significant decrease in brain
levels of E2 (3mo sham, 79.6 + 25 pg/g WT; 13mo sham 50.7 + 8.6 pg/g WT; 23mo
sham 35.6 + 9 pg/g WT; F=1.9, p=0.17).
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Figure 8. Changes in circulating levels of androgens with increasing age.
Circulating levels of T decrease with age, while no change was observed in DHT
levels. Brain levels of T and DHT decrease with age. T and DHT were purified from
serum and brain tissue using Celite chromatography, followed by quantification by
radioimmunoassay. * p<0.05 versus 3 mo group.
Age and androgen related changes in β-amyloid
To determine if age-related androgen depletion is sufficient in magnitude to
result in increased levels of Aβ
1-40
, we first measured the effects of nearly complete
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androgen depletion in young rats on Aβ levels. We found that GDX significantly
increased Aβ levels (Figure 9). To investigate the effect of age-related androgen
depletion on Aβ levels, we measured Aβ in 3, 13, and 23 mo old mice. We found a
significant increase in Aβ levels with increasing age (Figure 10).
Figure 9. Androgen depletion increases brain levels of soluble Aβ. Brain levels of
soluble Aβ were measured from DEA lysates using a rodent specific Aβ ELISA.
Androgen depletion in gondectomized (GDX) rats significantly increased soluble
levels of Aβ. Data show mean values (±SEM). * p<0.05
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Figure 10. Brain levels of soluble Aβ increased with age. Brain levels of soluble Aβ
were measured from DEA lysates using a rodent specific Aβ ELISA in 3, 13 and
23mo old rats. Soluble Aβ is significantly increased in 13 and 23mo old rats as
compared to 3mo old rats. Data show mean values (±SEM). * p<0.05 versus 3 mo
group.
DISCUSSION
In this study, we wanted to examine the relationships between circulating and
brain levels of androgens and Aβ following androgen depletion and with increasing
age. We found that T and DHT were depleted following gonadectomy in 3mo old
rats. A significant increase in Aβ levels was coincident with this loss of androgens.
Circulating levels of T, but not DHT, decrease with increasing age in Brown Norway
rats. Brain levels of T and DHT both significantly decreased with increasing age.
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Next, we examined the effects of age-related androgen depletion on Aβ levels and
found that Aβ levels decreased with increasing age.
The role of primary and secondary hypogonadism in mediating age-related
androgen depletion in BN rats matches the human condition very closely, making
BN rats a good model for normal reproductive aging in men (Gruenewald et al.,
1994; Wang et al., 2002). The loss of T in men is widely accepted as a combination
of both primary hypogonadism, a decreased number of Leydig cells in the testes
(Neaves et al., 1984) and reduced levels of steroidogenic acute regulatory protein
(STAR) in Leydig cells (Leers-Sucheta et al., 1999), and secondary hypogonadism,
dysfunction of the hypothalamic pituitary-gonadal axis resulting in the reduced
ability of the testes to produce testosterone (Morley, 2001b). Previous work has
found that as the BN rats age they exhibit a significant decline in circulating levels of
T between 6 and 28 mo of age (Wang et al., 2002). We observed a similar decrease
in circulating T levels between 3 and 13 mo of age, a decline we continued to
observe through 23 mo of age. However, we did not observe an age-related decline
in circulating levels of DHT. Interestingly, there is an inconsistent literature on age-
related changes in DHT in human studies. Some studies suggest there is a slight
decrease in free levels of DHT (Gray et al., 1991), while others have found there is
no change or even a slight increase in DHT levels with advancing age (Sparrow et
al., 1980; Couillard et al., 2000; Feldman et al., 2002). One reason for these
different findings could be the fact that circulating levels of DHT only comprise 20%
of DHT in the body. The majority of DHT is synthesized in peripheral tissue from
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5α-reduction from T (for review see (Kaufman and Vermeulen, 2005)), therefore
tissue levels of DHT would provide a more accurate measure of changes in DHT
levels with age.
Although tissue and circulating levels of sex steroid hormones are typically
similar, brain levels yield a more accurate measurement of what is occurring in brain
(Pardridge et al., 1980; Deslypere and Vermeulen, 1981; Manni et al., 1985;
Pardridge, 1985, 1986). Differences between brain and circulating levels of
androgens can be due to a variety of factors including age-related alterations in
hormone metabolic enzyme activities (Serafini et al., 1985; Roselli and Resko, 1986)
and the ability of hormones to cross the blood brain barrier (Pardridge, 1985). The
literature examining brain levels of sex steroid hormones is not extensive, and has
dealt primarily with human postmortem tissue, thus introducing other variables such
as postmortem delay. Studies have found some variability in sex steroid hormone
levels amongst brain regions. Although some of these early studies were limited by
a small sample size and confounding variables such as sex, and age, they observed
the greatest amount of T and E2 in the substantia nigra, hypothalamus and preoptic
area. Interestingly, serum T levels were similar to brain T levels, but serum E2 levels
differed from brain E2 levels, suggesting differences in peripheral conversion due to
aromatase activity in the brain (Bixo et al., 1986; Bixo et al., 1995). In previous
work from our lab, we examined brain levels of sex steroid hormones in frontal
cortex of aged men and found that brain levels of testosterone decreased with
increasing age; however, there was no change in brain levels of estradiol (Rosario et
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al., 2004, Chapter Two). Similar to our findings with the male BN rats, we also
found an age-related decrease in brain levels of DHT in men, suggesting tissue levels
of DHT better reflect age-related changes. A previous study examined tissue DHT
levels with age, but not in brain tissue. In this study, age-related decreases in T and
DHT levels were observed in pubic skin (Deslypere and Vermeulen, 1981). A study
by Hammond et al., did not examine age changes, but did find that brain levels of T
were similar to brain levels of DHT (Hammond et al., 1983). Our results using BN
rats were consistent with this previous work, as we observed similar levels of T and
DHT in the brain, and a similar age-related decline in brain levels of T and DHT
again supporting the idea that what occurs in brain levels of hormones is not always
the equivalent to circulating levels. Interestingly, a recent paper by Yue et al. found
that brain levels of estradiol did not correlate with circulating levels of estradiol (Yue
et al., 2005). In this study, circulating levels of estrogen were depleted following
ovariectomy, but no change in brain levels of estrogen was observed (Yue et al.,
2005). We found that circulating and brain levels of androgens were significantly
decreased following androgen depletion by gonadectomy.
Low levels of androgens have recently been identified as a risk factor for AD.
In a study by Twist et al., a non-significant trend toward decreased brain levels of T
was observed in AD cases (Twist et al., 2000). In previous work from our lab, we
found that AD cases had significantly decreased levels of T (Rosario et al., 2004,
Chapter Two). In addition, cases with mild neuropathological changes, consistent
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with early AD, also exhibited significantly decreased levels of T, suggesting that low
levels of T are a risk factor for the development of T (Rosario et al., 2004).
There are a few proposed mechanisms through which androgens may be
modulating this increased risk for the development of AD, including increased
neuron viability (Pike, 2001; Ramsden et al., 2003a) and regulation of Aβ (Ramsden
et al., 2003c; Rosario et al., 2006, Chapter Five). In this study, we predicted that
age-related androgen depletion observed in male BN rats would affect brain Aβ
levels. In human studies, Aβ levels correlate with age-related androgen depletion
(Gillett et al., 2003, Chapter Three). Similar to these studies, which found a
relationship between decreased levels of T and increased levels of Aβ, we found that
age-related testosterone loss resulted in increased Aβ levels in the BN rats.
Androgen regulation of Aβ levels has been observed in humans, cell culture studies,
and rodent models. A study using prostate cancer patients treated with anti-androgen
therapy found that circulating T and E2 levels were depleted and there was
approximately a two-fold increase in plasma levels of Aβ (Gandy et al., 2001).
However it is unclear whether T or E2 was having this effect on Aβ levels. Gouras
and colleagues reported that mixed neural cell cultures exposed to T exhibited
increased α-secretase cleavage of APP and reduced Aβ (Gouras et al., 2000).
However, the chronic treatment time with supra-physiological levels of T suggests
the likelihood of T aromatization to estradiol rather than a novel androgen action. In
fact, a subsequent study confirmed this possibility, showing that T mediates
increased α-secretase APP cleavage that is blocked by aromatase inhibitors
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(Goodenough et al., 2000). Previous work in our lab however, suggests that
androgens, not estrogen, are regulating Aβ levels (Ramsden et al., 2003c). Recently
we found that androgen depletion for 4 months in a transgenic mouse model of AD
resulted in increased accumulation of intraneuronal and extracellular Aβ pathology
(Rosario et al., 2006, Chapter Five).
This study found that brain levels of androgens correlate with circulating
levels of androgens, and that age-related androgen loss is enough to significantly
increase Aβ levels. These results suggest the potential use of testosterone therapy in
the treatment and prevention of AD. Clinical guidelines on the use of androgen
therapy have suggested that androgen therapy may be used, and is safe and effective
when used in men with very low levels circulating levels of androgens (Bhasin et al.,
2006). The fact that levels of testosterone in the brain are important in the
development of the AD and correlate strongly with circulating levels strengthens the
idea that men with low circulating levels of testosterone are at increased risk of
developing AD, and testosterone therapy may reduce this risk.
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CHAPTER FIVE
Androgens regulate development of neuropathology in a triple
transgenic mouse model of Alzheimer’s disease
Our previous work has found that low levels of androgens are a risk factor for
the development of AD, but we do not know how androgens are modulating this
increased risk. In this study we wanted to investigate the effect of androgen
depletion on the development of Alzheimer’s disease (AD) pathology in a transgenic
mouse model of AD. We measured intracellular and extracellular Aβ pathology and
behavior in 3xTg-AD transgenic mice under different conditions of androgen
exposure. We found that androgen depletion for 4 months significantly increased Aβ
pathology, an effect prevented by androgen treatment. The increased accumulation
of Aβ correlated with impaired behavioral performance, and androgen treatment was
able to prevent these behavioral deficits. These results were published in 2006 in the
Journal of Neuroscience.
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CHAPTER FIVE ABSTRACT
Normal, age-related testosterone depletion in men is a recently identified risk
factor for Alzheimer’s disease (AD), but how androgen loss affects development of
AD is unclear. To investigate the relationship between androgen depletion and AD,
we compared how androgen status affects progression of neuropathology in the
3xTg-AD triple-transgenic mouse model of AD. Adult male 3xTg-AD mice were
sham gonadectomized (GDX) or GDX to deplete endogenous androgens, and then
exposed for 4 months to either the androgen dihydrotestosterone (DHT) or placebo.
In comparison to gonadally intact 3xTg-AD mice, GDX mice exhibited robust
increases in accumulation of β-amyloid (Aβ), the protein implicated as the primary
causal factor in AD pathogenesis, in both hippocampus and amygdala. In parallel to
elevated levels of Aβ, GDX mice exhibited significantly impaired spontaneous
alternation behavior, indicating deficits in hippocampal function. Importantly, DHT
treatment of GDX 3xTg-AD mice attenuated both Aβ accumulation and behavioral
deficits. These data demonstrate that androgen depletion accelerates the development
of AD-like neuropathology, suggesting that a similar mechanism may underlie the
increased risk for AD in men with low testosterone. In addition, our finding that
DHT protects against acceleration of AD-like neuropathology predicts that
androgen-based hormone therapy may be a useful strategy for the prevention and
treatment of AD in aging men.
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INTRODUCTION
Advancing age is the most significant risk factor for the development of
Alzheimer’s disease (AD), however the full range of age-related factors underlying
this increased risk is not known. One recently identified age-related risk factor for
AD in men is low testosterone (Pike et al., 2006). Testosterone, the primary male sex
steroid hormone, is gradually depleted as a normal consequence of aging (Morley et
al., 1997). Age-related loss of testosterone can manifest as a clinical syndrome of
dysfunction and increased vulnerability to disease in androgen-responsive tissues
(Morley, 2001b). The brain is androgen-responsive, exhibits age-related testosterone
depletion (Rosario et al., 2004), and is vulnerable to senescent effects of androgen
loss (Janowsky, 2006). Recent studies show that men with AD have significantly
lower testosterone levels than aged men without AD (Hogervorst et al., 2001),
suggesting that one neural effect of age-related testosterone loss in men is increased
risk for AD. Importantly, testosterone depletion appears to occur well before clinical
(Moffat et al., 2004) and neuropathological (Rosario et al., 2004) diagnosis of AD,
suggesting that low testosterone contributes to AD pathogenesis rather than results
from it.
How testosterone depletion increases the risk of AD remains to be
established. Androgens exert several actions in brain potentially associated with
protection against AD, including neuroprotection (Pike, 2001) and attenuation of tau
hyperphosphorylation (Papasozomenos, 1997). In addition, recent experimental
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findings indicate that androgens may reduce levels of soluble β-amyloid (Aβ)
(Goodenough et al., 2000; Gouras et al., 2000; Ramsden et al., 2003c), the protein
widely implicated in the initiation of AD pathogenesis (Hardy, 2002). In aged men,
circulating levels of testosterone are inversely correlated with plasma levels of Aβ
(Gillett et al., 2003). Further, prostate cancer therapy that depletes endogenous
androgens and antagonizes androgen signaling results in elevated plasma levels of
Aβ (Gandy et al., 2001; Almeida et al., 2004). Together, these studies establish a
correlation between low testosterone and elevated Aβ levels, a finding consistent
with the possibility that testosterone depletion in aging men may act as a risk factor
for AD by increasing neural accumulation of Aβ.
To further investigate the relationship between testosterone and AD, we
assessed how the development of AD-like neuropathology in a triple-transgenic
mouse model of AD (3xTg-AD) (Oddo et al., 2003) is affected by androgen status.
We report that androgen depletion in male 3xTg-AD mice significantly accelerates
both Aβ deposition and behavioral impairment, effects that are prevented by
androgen treatment. These data represent the first experimental evidence directly
linking androgen depletion to development of AD-like neuropathology.
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METHODS
Animals and hormone treatment
Colonies of male homozygous 3xTg-AD (APP
swe
, PS1
M146V
, tau
P301L
) (Oddo
et al., 2003) and wild-type (B6129SF2/J; Jackson Laboratory, Bar Harbor, ME) mice
were bred and maintained in our vivarium, where they were housed individually
under a 12 h light/12 h dark schedule with ad libitum access to food and water. At
age 3 mo, mice (N=6-8/group) under pentobarbital (50mg/kg) anesthesia were either
gonadectomized (GDX) to deplete endogenous testosterone or sham GDX and were
immediately implanted with a subcutaneous, 90-day continuous release hormone
pellet (IRA, Sarasota, FL) containing either 10 mg dihydrotestosterone (DHT) or
vehicle; animals received a second pellet 90 days later. Four months after initiation
of hormone treatment, or at 3, 7, and 13 mo of age for non-treatment groups, animals
were sacrificed and brains were collected, immersion fixed in fresh 4%
paraformaldehyde/0.1M PBS for 48 h, then stored at 4°C in 0.1M PBS/0.2% sodium
azide. Efficacy of androgen manipulations was assessed at time of death by weighing
dissected and blotted seminal vesicles.
Immunohistochemistry
Fixed hemi-brains were sectioned (40 µm) exhaustively in the horizontal
plane using a vibratome, then immunostained using a standard protocol. Briefly,
free-floating sections were immunolabeled with antibodies directed against Aβ (#71-
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5800, 1:300 dilution; Zymed, San Francisco, CA) and Aβ precursor protein C-
terminal fragments (CTFs) (anti-APP-CT20, 1:16,000 dilution; Calbiochem, San
Diego, CA) using ABC Vector Elite and DAB kits (Vector, Burlingame, CA). Prior
to Aβ immunostaining, sections were pretreated for 5 min with 95% formic acid to
enhance immunoreactivity.
Quantification of immunoreactivity
High magnification fields from immunolabeled sections were digitized using
a video capture system, then thresholded using NIH Image 1.61 software to separate
positive and negative immunolabeling and permit calculation of immunoreactive
load, the percent area occupied by immunoreactive label. Mean load values were
determined by sampling 2-3 non-overlapping, representative fields from each brain
region of interest (subiculum, hippocampus CA1, amygdala, and frontal cortex) in 5
separate sections per animal. Using this imaging technique, load values were also
quantified for individual, extracellular plaques (defined below) from 16-20 Aβ-
immunostained sections per animal.
Quantification of extracellular plaques
Plaques were defined as extracellular Aβ-immunoreactive deposits that
exhibited a spherical shape and morphology distinct from intraneuronal Aβ
immunoreactivity. For quantification, 16-20 Aβ-immunostained sections (beginning
from dorsal hippocampus, approximately 160-200µm apart) per brain were examined
101
under light microscopy and the total number of extracellular plaques counted. Also,
the area of each plaque was measured using NIH Image 1.61 software.
Spontaneous alternation behavior
Approximately one week prior to killing, all mice were tested for
spontaneous alternation behavior (SAB) in a Y-maze, a hippocampal-dependent task
of working memory. Arm choices (both front and hind paws entering arm) were
recorded while animals freely explored the maze for 8 minutes. Mice that made 10 or
fewer arm choices were excluded; only two animals across all groups were excluded.
SAB score was calculated as the proportion of alternations (an arm choice differing
from each of two previous choices) to the total number of alternation opportunities
(total arm entries - two), as described by (King and Arendash, 2002). For example,
the following arm choices A-A-B-C-B-B-A-C-C would be scored as 2 alternations
(ABC and BAC) out of 7 opportunities (9-2=7).
Statistical analyses
Raw data were analyzed by ANOVA followed by between group comparisons using
Fisher LSD test.
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RESULTS
Male 3xTg-AD mice show age-dependent increases in Aβ accumulation
In agreement with prior observations in the 3xTg-AD mouse (Oddo et al.,
2003), we observed an age-dependent increase in Aβ that appeared to begin with
neuronal accumulation and progress to extracellular deposition. Specifically, sham
gonadectomized (GDX) male mice showed very low levels of neuronal Aβ
immunoreactivity at age 3 mo that increased substantially by age 7 mo, with relative
abundance in the following order subiculum > amygdala > CA1 region of
hippocampus (Fig. 11). Aβ immunoreactivity was absent or present at comparatively
low levels in frontal cortex at these time points (data not shown). Quantification of
immunoreactive load confirmed a progressive, age-dependent increase in Aβ
accumulation across all three examined brain regions (Fig. 11D, H, L). Extracellular
plaque-like deposits appeared in some animals by age 7 mo and all animals by age
13 mo (F=12.6, p<0.01), being most abundant in subiculum (Fig. 11C).
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Figure 11. Aβ accumulation increases with age in male 3xTg-AD mice.
Representative photomicrographs of Aβ immunoreactivity in sham GDX 3xTgAD
mice at ages 3 (A, E, I), 7 (B, F, J), and 13 (C, G , K) mo in subiculum (A-C),
hippocampus CA1 (E-G), and amygdala (I-K). Arrows show extracellular Ab
deposits. Scale bars=100µm. Aβ-immunoreactivity in 3, 7, and 13 mo old 3xTgAD
mice was quantified by load values in subiculum (D), CA1 (H) and amygdala (L).
Data show mean load values (±SEM). * p<0.05 versus 3 mo group, # p<0.05 versus
7 mo group.
Androgen depletion accelerates Aβ accumulation
To investigate the effect of androgen status on development of
neuropathology, male 3xTg-AD mice were depleted of endogenous androgens by
GDX at age 3 mo and treated immediately with DHT or placebo. To assess the
efficacy of these experimental manipulations, we measured seminal vesicle weight, a
bioassay of androgen status. Seminal vesicle weight was significantly decreased in
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the GDX group and significantly increased above sham GDX levels in the
GDX+DHT group, suggesting supra-physiological DHT replacement (sham GDX =
83.2+9.9 mg, GDX = 15.8+4.7 mg, GDX+DHT = 149+24 mg; F=21.6, p<0.001).
If androgen loss is a risk factor for AD in men, then experimental androgen
depletion in male 3xTg-AD mice should accelerate development of AD-like
neuropathology. Consistent with this prediction, we observed that GDX 3xTg-AD
mice exhibited a robust increase in Aβ load in comparison to age-matched sham
GDX mice (Fig. 12). Importantly, DHT treatment of GDX animals completely
prevented the increase in Aβ load in subiculum, CA1, and amygdala (Fig. 12).
We also determined how androgens affect extracellular Aβ deposition by
comparing the number, load, and area of plaques across hormone conditions.
Although there are few extracellular Aβ deposits at age 7 mo, both the number
(F=4.3, p<0.05) and load (F= 4.6, p<0.05) of Aβ plaques were significantly
increased in GDX 3xTg-AD animals in comparison to sham GDX animals, effects
that were prevented by DHT treatment. In contrast, the mean plaque area did not
vary across the sham GDX, GDX and GDX+DHT groups (F=1.01, p=0.36),
suggesting androgen status does not significantly affect plaque size.
105
Figure 12. Aβ accumulation is regulated by androgens. Representative
photomicrographs show Aβ-immunoreactivity in male 3xTg-AD mice at age 7 mo in
the sham GDX (A, E, I), GDX (B, F, J), and GDX + DHT (C, G, K) conditions in
subiculum (A-C), CA1 (E-G), and amygdala (I-K). Scale bars = 100µm. Aβ-
immunoreactivity in 7 mo sham GDX (Sham), GDX, and GDX+DHT 3xTgAD mice
was quantified by load values (means±SEM) in subiculum (D), CA1 (H) and
amygdala (L). * p<0.05 versus 7 mo Sham group, # p<0.05 versus 7 mo GDX group.
Androgen depletion does not affect accumulation of APP C-terminal fragments
Prior work demonstrated that Aβ immunoreactivity in 3xTg-AD mice largely
represents oligomeric Aβ species rather than C-terminal fragments (CTFs) of the Aβ
precursor protein APP (Oddo et al., 2006a). To both verify this finding and evaluate
the potential effect of androgen status on CTF levels, we assessed CTF
immunoreactivity across all groups. We found that in contrast to the punctate
appearance of Aβ immunoreactivity in 3xTg-AD mice (Fig. 13C), CTF
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immunoreactivity is more even in appearance and largely restricted to the cell
perimeter (Fig. 13B). Although more abundant than levels in wild-type mice (Fig.
13A), CTF immunoreactivity in sham GDX 3xTg-AD mice did not show a
significant change with increasing age in either subiculum, CA1 (Fig. 13 D-G), or
amygdala (data not shown). Notably, androgen status did not significantly alter CTF
immunoreactive load (Fig. 13H).
Figure 13. Accumulation of APP C-terminal fragments (CTF) does not change with
age. Representative photomicrographs of CTF immunoreactivity in subiculum in (A)
wild-type male and (B) 3xTg-AD mice at age 7 mo. (C) Representative
photomicrograph of Ab immunoreactivity in subiculum in 7 mo 3xTg-AD mouse.
Scale bar=25 µm. Representative photomicrographs of CTF immunoreactivity in
subiculum of sham GDX 3xTg-AD mice at ages 3 (E), 7 (F), and 13 (G) mo. Scale
bar=50 µm. (D) Aβ-immunoreactivity in subiculum (solid bars) and hippocampus
CA1 (open bars) was quantified by load values (means + SEM) in (D) sham GDX
3xTgAD mice ages 3, 7 and 13 mo, and (H) age 7 mo 3xTgAD mice in the sham
GDX (Sham), GDX, and GDX+DHT conditions.
Androgen depletion worsens behavioral performance in 3xTg-AD mice
Next, we evaluated the effects of aging and androgen status on behavioral
performance in the 3xTg-AD mice using spontaneous alternation behavior (SAB), a
107
hippocampal-dependent behavior. We observed that in comparison to wild-type
mice, 3xTg-AD mice showed significantly impaired SAB that was apparent by age 7
mo and mildly worse by age 13 mo (Fig. 14A). Importantly, androgen status
significantly affected SAB: androgen depletion in GDX mice worsened performance
and this effect was prevented by DHT treatment (Fig. 14B). To confirm that
androgen regulation of SAB in 3xTg-AD mice reflected androgen-induced effects on
neuropathology rather than on behavior, we also assessed the effects of androgen
depletion on SAB in wild-type male mice. SAB scores of GDX wild-type mice
(69.0+8.2) were not significantly different from sham GDX wild-type mice
(73.1+7.7, p=0.09). Finally, there were no significant group differences in number of
arm entries (F=0.46, p=0.64), suggesting that activity levels were similar across the
conditions.
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Figure 14. Advancing age and androgen depletion increase behavioral deficits in
3xTg-AD mice. Spontaneous alternation behavior (SAB), expressed as % alternation
(+SEM), in wild-type (WT; open bars) and 3x-Tg-AD (solid bars) mice. (A) SAB
decreased with increasing age in sham GDX (Sham) 3xTg-AD mice. * p<0.05 versus
7 mo WT Sham group, # p<0.05 versus 7 mo Sham 3xTg-AD group. (B) SAB was
decreased by androgen depletion, an effect prevented by DHT treatment. * p<0.05
versus 7 mo Sham group, # p<0.05 versus 7 mo GDX group.
DISCUSSION
Recent studies in humans have implicated normal, age-related testosterone
depletion in men as a risk factor for the development of AD (Pike et al., 2006). In
this study, we report the first experimental investigation of this risk factor using an
animal model of AD. Specifically, we evaluated two hypotheses: (1) experimental
androgen depletion should increase development of neuropathology, and (2)
androgen treatment should prevent this acceleration of neuropathology. Consistent
with the proposed regulatory role of androgens in AD pathogenesis, we observed that
depletion of endogenous androgens in adult male 3xTg-AD mice significantly
109
accelerated the accumulation of Aβ and, in parallel, behavioral impairment.
Importantly, acceleration of both pathologies was prevented by continuous treatment
with the androgen DHT, although at apparently supraphysiological levels. Taken
together, our results demonstrate that androgens regulate the development of AD-like
neuropathology.
Androgens are endogenous regulators of Aβ
Although AD pathogenesis remains to be fully elucidated, the disease appears
to be initiated by genetic and environmental factors which ultimately result in
increased neural accumulation of Aβ (Hardy, 2002). Our data demonstrate that
depletion of endogenous androgens robustly increases Aβ accumulation in brain,
suggesting not only that a normal androgen function is regulation of neural Aβ
levels, but also that the loss of this function can promote AD pathogenesis. Our
findings are consistent with an emerging literature on sex steroid hormones and Aβ.
For example, estrogen alters metabolism (Jaffe et al., 1994) and trafficking
(Greenfield et al., 2002) of APP yielding reduced Aβ levels in cultured cells (Xu et
al., 1998) and wild-type rodents (Petanceska et al., 2000) by a mechanism that
involves MAPK (Manthey et al., 2001) and or PKC (Zhang et al., 2005) signaling
and may interact with other risk factors for Aβ accumulation including zinc (Lee et
al., 2004). Further, Aβ accumulation in females from some mouse models of AD is
increased following estrogen depletion induced by aromatase knockout (Yue et al.,
2005) or ovariectomy (Levin-Allerhand et al., 2002; Zheng et al., 2002).
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Testosterone can also alter APP processing and reduce Aβ levels in cultured cells
(Goodenough et al., 2000; Gouras et al., 2000). In adult male rats, GDX increases
brain levels of soluble Aβ, an effect prevented by the non-aromatizable DHT but not
by estrogen (Ramsden et al., 2003c). It remains unclear to what extent androgens
regulate Aβ in female animals.
There is evidence that androgens also regulate Aβ in men. For example, anti-
androgen therapy for the treatment of prostate cancer is associated with increased
levels of soluble Aβ in plasma (Gandy et al., 2001; Almeida et al., 2004). One
concern is that anti-androgen therapy decreases both testosterone and estrogen levels,
making it unclear which hormone(s) are predominantly responsible for regulating
Aβ. However, in aged men not undergoing anti-androgen therapy, plasma Aβ levels
inversely correlate with testosterone but not estrogen levels (Gillett et al., 2003).
The mechanism(s) by which androgens regulate Aβ is not known.
Testosterone can mediate cellular effects by three general pathways that are not
mutually exclusive: activation of androgen receptor-dependent pathways, indirect
activation of estrogen pathways via aromatization to estradiol, and modulation of
gonadotropin actions via regulation of the hypothalamic-pituitary-gonadal axis.
Previous studies provide evidence that the observed androgen regulation of Aβ may
involve individual or combined effects by all three pathways. Androgens are
reported to regulate Aβ levels by estrogen-independent androgen pathways in male
rodents (Ramsden et al., 2003c) as well as by indirect estrogen-mediated APP
metabolism in cultured cells (Goodenough et al., 2000). Also, recent work by
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Bowen et al. (2004) indicates Aβ regulation by the gonadotropin luteinizing
hormone. Although future studies will be necessary to distinguish which of these
pathways contribute to the present androgen effects, our finding that DHT treatment
prevented the acceleration of AD-like neuropathology in GDX 3xTg-AD mice is
consistent with an estrogen-independent mechanism since DHT is not aromatized to
estrogen. However, a recent report suggests that the DHT metabolite 5α-androstan-
3β, 17β-diol can have agonist actions on estrogen receptor β (Lund et al., 2006).
Regardless of the underlying mechanism(s), our results clearly demonstrate that a
loss of androgens leads to an increase in Aβ accumulation.
Androgens regulate progression of cognitive impairment
Because androgens regulate Aβ accumulation and Aβ impairs cognitive
function, androgens are predicted to affect development of cognitive dysfunction.
Prior work has established that, without causing neuron death, Aβ can disrupt the
synaptic plasticity necessary for normal cognitive functions including learning and
memory (Walsh and Selkoe, 2004). In 3xTg-AD mice, accumulation of Aβ does not
appear to cause significant neuron loss but does impair long-term potentiation (Oddo
et al., 2003) and various measures of behavioral performance (Billings et al., 2005).
In this study, Aβ was observed to accumulate at significant levels in limbic regions
by age 7 mo, the same time point at which hippocampal-dependent SAB became
impaired. Prevention of Aβ accumulation in 3xTg-AD mice attenuates behavioral
impairments (Billings et al., 2005), strengthening the link between Aβ accumulation
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and behavioral performance in this model. Thus, our observation that DHT treatment
blocked the GDX-induced decrease in SAB suggests that the androgen effect was
mediated by regulation of Aβ accumulation. Arguing against a significant, direct
androgen effect on SAB was our observation that androgen status did not affect SAB
in wild-type mice.
In both rodent and human studies, androgens can exert beneficial cognitive
effects (Cherrier et al., 2005; Janowsky, 2006). For example, in transgenic mice
overexpressing the AD genetic risk factor apolipoprotein E4, females exhibit
cognitive deficits that are prevented by testosterone, whereas males exhibit deficits
only if treated with an androgen receptor antagonist (Raber et al., 2002).
Interestingly, chronic androgen depletion in male apolipoprotein E4 mice impaired
performance on some behavioral tasks but improved performance on others
(Pfankuch et al., 2005), suggesting a complex relationship between androgens,
apolipoprotein E, and behavior. In men with clinically significant age-related
testosterone depletion, androgen supplementation can improve mood and some
cognitive abilities (Alexander et al., 1998). In contrast, reduction of androgen and
estrogen levels and or inhibition of androgen signaling by anti-androgen prostate
cancer therapies can impair some aspects of cognition (Green, 2002). Interestingly,
discontinuation of anti-androgen treatment resulted in restored plasma testosterone
levels that correlated with improved cognitive performance on some tasks and
reduced plasma levels of Aβ (Almeida et al., 2004). Thus, our findings extend a
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growing literature indicating that androgens can significantly benefit cognitive
abilities by mechanisms that may include reduction of Aβ levels.
Androgens and the prevention of Alzheimer’s disease
Androgen loss, which occurs as a consequence of normal aging in men, can
promote disease and dysfunction in androgen-responsive tissues including the brain
(Morley, 2001b). Recent studies have linked age-related testosterone depletion with
increased risk of AD (Hogervorst et al., 2001; Moffat et al., 2004; Rosario et al.,
2004). Our findings demonstrate that in an animal model of AD, experimental
depletion of endogenous androgens accelerates development of both AD-like
neuropathology and behavioral impairment. Importantly, our data also show that
androgen treatment prevents the increase in pathology progression. These findings
not only demonstrate a significant role of androgen depletion in AD pathogenesis,
but also predict that androgen-based therapeutics may function effectively in the
prevention of AD.
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CHAPTER SIX
Androgen regulation of Alzheimer-like neuropathology in male 3XTG-
AD mice is through androgen and estrogen mediated pathways
In the previous study, we found that androgen depletion increased Aβ
pathology, and DHT was able to prevent this effect. In this study we wanted to
investigate whether this androgen-mediated effect involves direct androgen
pathways, or indirect estrogen pathways. We also wanted to examine whether
androgens or estrogen regulate tau hyperphosphorylation. We measured Aβ and tau
pathology in male 3xTg-AD transgenic mice depleted of endogenous androgens, and
treatment with androgens or estrogen. We found that androgen depletion
significantly increased Aβ pathology, and testosterone and DHT were able to prevent
this increase. Estrogen was only able to prevent Aβ pathology in hippocampus.
Interestingly, we found that estrogen was able to decrease tau hyperphosphorylation
in male mice.
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CHAPTER SIX ABSTRACT
Normal, age-related, androgen depletion in men has been identified as a risk
factor for the development of Alzheimer’s disease (AD). Recently, using a triple-
transgenic mouse model of AD (3xTg-AD), we found that androgen depletion in
male mice results in a robust increase in the accumulation of β-amyloid (Aβ), the
protein implicated as the primary causal factor in AD pathogenesis while androgen
treatment, in the form of the non-aromatizable androgen, dihydrotestosterone (DHT),
prevented this increase. However, the mechanism of androgen regulation of Aβ
remains unclear. To examine whether androgen actions on Aβ accumulation involve
direct androgen and/or estrogen pathways, we examined the effects of testosterone
(T), and its metabolites, DHT, and 17β-estradiol (E2) in androgen depleted male
mice. Male 3xTg-AD mice were gonadectomized (GDX) at 3 months of age, and
exposed to subcutaneous, slow-release drug delivery pellets containing either
vehicle, 10 mg DHT, 10mg T, 0.025mg E2, or 0.01mg E2. After 4 months of
hormone treatment (at 7 months of age), mice were evaluated for severity of AD-like
neuropathology. Similar to our previous results, we observed a significant increase
in Aβ pathology in GDX mice in the subiculum and CA1 of hippocampus and
amygdala, an effect prevented by DHT treatment. Treatment with testosterone also
prevented increased accumulation of Aβ pathology in both hippocampus and
amygdala. Interestingly, estrogen prevented Aβ accumulation in CA1 of
hippocampus but had only partial effects in the subiculum and amygdala. Although
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androgen depletion did not significantly increase tau hyperphosphorylation, as
measured by AT8 positive neurons, T and the higher dose of E2 treatment reduced
the number of AT8 positive neurons below that observed in sham animals. These
findings suggest that androgen regulation of Aβ pathology is mediated mainly
through direct androgen pathways, and regionally by estrogen pathways. Regulation
of tau pathology in male mice appears to be through estrogen mediated pathways.
INTRODUCTION
Advancing age is the most significant risk factor for the development of
Alzheimer’s disease (AD) (Rocca et al., 1986; Jorm et al., 1987; Evans et al., 1989).
One age-related change that has been identified as a potential risk factor for the
development of AD in men is a significant loss in testosterone (T) levels (Morley et
al., 1997; Rosario et al., 2004). Initial studies investigating the relationship between
androgen levels and Alzheimer’s disease (AD) found that circulating levels of free
and total T were lower in men with AD in comparison to age-matched controls
(Hogervorst et al., 2003; Hogervorst et al., 2004; Hogervorst et al., 2001; Lehmann
et al., 2004; Paoletti et al., 2004), an effect strengthened in men who are ApoE ε4
positive (Hogervorst et al., 2002). In addition, low circulating and brain levels of
androgens have been found prior to a clinical and neuropathological diagnosis of
AD, suggesting that low levels of androgens are a risk factor for the development of
AD (Rosario et al., 2004; Moffat et al., 2004).
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Androgens have several beneficial actions in the brain relevant to the
development of AD including increased neuron viability (Pike, 2001; Ramsden et al.,
2003a) and synaptic plasticity (Leranth et al., 2003). One important action of
androgens in the brain is regulation of Aβ, a casual factor in the development of AD.
Specifically, studies investigating prostate cancer patients undergoing anti-androgen
treatment, found that the treatment decreased levels of androgens, estrogens, and
gonadotropins, and increased Aβ levels (Gandy et al., 2001; Almeida et al., 2003;
Gillett et al., 2003). Further, cell culture studies have reported a reduction in Aβ
levels following testosterone replacement (Gouras et al., 2000). Previous work from
our lab presented the first in vivo demonstration of androgen regulation of Aβ levels
(Ramsden et al., 2003c). A significant increase in soluble Aβ
1-40
levels was observed
in brain lysates from gonadectomized (GDX) rats as compared with sham-GDX
animals (Ramsden et al., 2003).
Since androgen actions can be mediated through either direct androgen
pathways, involving androgen receptors (AR), or indirect estrogen (through
aromatization of T to estrogen) pathways (Mooradian et al., 1987), the mechanism of
androgen regulation of Aβ remains unclear. However, our lab has previously shown
that replacement of dihydrotestosterone (DHT) in GDX rats for 4 weeks caused a
significant reduction is brain Aβ levels while estradiol treatment in male rats had no
effect (Ramsden et al., 2003). Results from this study suggest androgens are acting
through androgen-mediated pathways to regulate Aβ levels in males.
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We recently published findings, suggesting that experimental androgen
depletion in a transgenic mouse model of AD resulted in a significant increase in Aβ
pathology (Rosario et al., 2006, Chapter 5). This transgenic mouse model (3xTg-
AD) exhibit a region and age specific progression of both Aβ deposition and tau
hyperphosphorylation that is consistent with AD pathology (Oddo et al., 2003). In
our previous study, we determined that androgen depletion significantly increases
Aβ pathology and DHT treatment prevents this increased accumulation (Rosario et
al., 2006, Chapter 5). However, we were unable to clearly determine whether these
effects were through androgen or estrogen mediated pathways. The efficacy of DHT
suggests that a direct androgen pathway is involved because DHT is not aromatized
to estradiol. However, recently the DHT metabolite, 3β-diol, has been found to bind
to ERβ thereby activating estrogen pathways (Lund et al., 2006). Therefore, in this
study, we utilized both testosterone and 17β-estradiol (E2) to evaluate the
mechanism of androgen regulation of AD-like neuropathology.
METHODS
Animals and hormone treatment
The 3xTg-AD mice were created by Dr. Frank LaFerla’s laboratory as
previously described and harbor three mutations for AD (Oddo et al., 2003). All the
mice used in this study were bred in our laboratory, had ad libitum access to food
and water, and were housed individually under a 12hlight/12h dark schedule. 3, 7,
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ad 13mo old mice were used to examine age-related changes in tau pathology. To
examine the effect of hormonal manipulations, mice were divided into 6 groups (n =
6-7 per group) and all 3mo old mice were either sham gonadectomixed (GDX) or
GDX under pentobarbital (50mg/kg) anesthesia, and received either a placebo (Pl)
(90-day release pellet, IRA Sarasota, FL), DHT (10mg 90-day release pellet, IRA
Sarasota, FL), T (10mg 60-day release pellet IRA Sarasota, FL) or E2 (0.01mg,
0.025mg 60-day release pellet, IRA Sarasota, FL) pellet for 4 months. Mice were
anesthetized with 100mg/kg pentobarbital and perfused using room temperature
PBS. Seminal vesicles were dissected, blotted, and weighed to determine
effectiveness of hormone treatment. The brain was dissected and fixed for
immunohistochemical analysis.
Immunohistochemistry
Paraformaldehyde fixed hemi-brains were sectioned in the horizontal plane
(40µm) using a vibratome. Sections were immuno-labeled following a previously
described protocol (Pike, 1999). Briefly, sections were labeled using primary
antibody specific for either hyperphosphorylated tau (AT8 monoclonal antibody,
1:1000; Pierce, Rockford, IL) or Aβ (polyclonal antibody directed against human
Aβ1-43, 1:300 dilution; Zymed, San Fransisco, CA) and visualized with standard
ABC immunohistochemistry (Pike, 1999).
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Quantification of Aβ
Quantification of Aβ load was conducted as previously described
(Cummings, 2002). Briefly, high magnification fields from Aβ-immunolabeled
sections were digitized and stored on computer by a video capture system (B/W
CCD camera coupled to an Olympus BX40 upright microscope). Using NIH Image
1.61 software, gray scale images were thresholded creating a binary separation
between positive and negative immunoreactivity, and permitting calculation of the
percentage of section area occupied by immunoreactive label (ie. 'load'). Load
values in specific regions (subiculum, CA1, and amygdala) include a representative
sampling of 2 or 3 fields from each of 5 separate sections. Raw data (%
immunoreactive area) across groups was statistically analyzed by ANOVA, followed
by pairwise comparisons using Fisher LSD.
Quantification of AT8 immunoreactivity and extracellular plaques
Hyperphosphorylated tau was quantified by counting the number of AT8
positive neurons in hippocampus and subiculum from 11-12 sections separated by
280µm. Raw data (total number of AT8 positive neuron from all 12 sections) was
statistically analyzed by ANOVA, followed by pairwise comparisons using Fisher
LSD. Extracellular plaques (labeled with anti-Aβ antibody) were defined as
extracellular Aβ deposits with a spherical shape and morphology distinct from
intraneuronal Aβ immunoreactivity (Rosario et al., 2006). Plaque number was
counted and analyzed using the same method as for AT8 positive neurons.
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RESULTS
Efficacy of hormone manipulations
In this study we used a triple transgenic mouse model of AD (3xTg-AD) to
investigate the mechanism of androgen regulation of AD pathology. In order to
evaluate the efficacy of hormone manipulations we measured seminal vesicle weight.
Similar to our previous findings in male 3xTg-AD mice (Rosario et al., 2006,
Chapter 5), we observed a significant decrease in seminal vesicle weight in GDX
mice as compared to sham GDX mice. GDX + DHT treatment significantly
increased seminal vesicle weight as compared with GDX and sham GDX mice,
suggesting that DHT treatment resulted in high physiological or supraphysiological
DHT levels. Interestingly, T treatment in GDX transgenic mice had no effect on
seminal vesicle weight. However, upon further examination it appears that the T
pellets were depleted of hormone prior to sacrifice. When the 60-day release T
pellets were tested in wild-type mice 30 days after GDX, T significantly increased
seminal vesicle weight compared with GDX wild-type mice but not sham GDX
mice, suggesting physiological replacement of T (Table 6). E2 had no effect on
seminal vesicle weight in either the transgenic mice following 4 months of treatment,
or in the wild-type mice following 1 month of treatment (Table 6).
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Table 6. Efficacy of hormone treatment for 30 days in wild-type mice
Treatment
Seminal vesicle wt (mg) (+SEM)
Sham 63.5 + 9.2
GDX 13.3 1.43*
GDX + T 49.5 + 7.58
GDX + 0.01 E2 16.5 + 3.7*
GDX + 0.025 E2 24.2 + 3.4*
* p<0.05 versus Sham group.
Aβ deposition
To determine whether androgen regulation of Aβ pathology is mediated by
androgen or estrogen pathways, we examined the effect of hormone manipulation on
Aβ deposition in mice depleted of endogenous hormones and following various
hormone treatments. We observed a significant increase in Aβ immunoreactivity in
androgen depleted (GDX) mice in comparison to sham animals in the subiculum, and
CA1 of hippocampus and amygdala (Figure 15, 16). Interestingly, increased Aβ
accumulation was observed both in the intensity of staining and in the size of the
immunoreactive area. DHT replacement in GDX mice prevented increased Aβ
accumulation in subiculum, CA1 of hippocampus, and amygdala (Figure 15, 16).
We observed a similar prevention of Aβ accumulation following treatment with T in
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subiculum and CA1 of hippocampus and amygdala. While we observed that both
doses of E2 protected against increased Aβ pathology in CA1 of the hippocampus,
E2 was only partially effective in the subiculum, and failed to prevent increased Aβ
pathology in the amygdala (Figure 15 and 16).
In addition to Aβ load, we also counted extracellular plaques as a measure of
Aβ deposition. Similar to Aβ immunoreactivity, we observed a significant hormone-
related effect on extracellular plaques (Figure 16). Androgen depletion significantly
increased the number of extracellular plaques, an effect prevented by T and DHT
treatment. E2 had no effect on the number of extracellular plaques in GDX mice.
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Figure 15. Intraneuronal Aβ increases following 4 months of androgen depletion.
High magnification representative photomicrographs display Aβ immunoreactivity
in subiculum (A,D,G,J,M,P), CA1 of hippocampus (B,E,H,K,N,Q), and amygdala
(C,F,I,L,O,R) in Sham (A-C), GDX (D-F), GDX + DHT (G-I), GDX + T (J-L),
GDX + 0.01mg E2(M-O), and GDX + 0.025mg E2 (P-R) mice. Scale bars = 100
µm.
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Subiculum CA1 Amygdala Extracellular plaques
Figure 16. Quantification of Aβ load and extracellular deposition following
androgen depletion. Quantification of Aβ immunoreactivity in the subiculum (A),
CA1 of hippocampus (B), and amygdala (C) using load values. Briefly, load is
defined as the percentage of section area occupied by immunoreactive label. Load
values in for each region include a representative sampling of 2 or 3 fields from each
of 5 separate sections. (D) Androgen depletion increases the number of extracellular
Aβ deposits, an effect prevented by DHT and T treatment. Data show mean counts
(± SEM). * denotes p < 0.05 versus 7mo Sham group, # denotes p < 0.05 versus
7mo GDX + PL group.
Hyper-phosphorylated tau immunoreactivity
Neurons exhibiting tau pathology, specifically hyperphosphorylation of tau,
were measured using AT8, an antibody specific for pathology-related
hyperphosphorylation at Ser 202 and Thr 205. We first wanted to examine the
localization and age-related progression of tau hyperphosphorylation in these mice.
We observed a significant age-related increase in AT8 positive neurons, which were
predominately localized in the subiculum and to lesser extent in CA1 (Figure 17 A-
C). While androgen depletion had only a modest effect on tau
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hyperphosphorylation, T and E2 (0.025mg) significantly decreased the number of
AT8 positive neurons below levels observed in male sham mice (Figure 17F). DHT
and the low dose of E2 (0.01mg) had no effect on the number of AT8 positive
neurons.
Figure 17. Age and hormone-related changes in AT8 positive neurons
AT8 positive neurons were counted as a measure of tau hyperphosphorylation. (A-
C) Representative photomicrographs of AT8 immunoreactivity in hippocampus in 3,
7, and 13mo old mice. Scale bar = 100 µm. (D) High magnification
photomicrograph of an AT8 positive neuron. Scale bar = 25µm. (E) Quantification
of AT8 positive neurons with increasing age. Data show mean values (+SEM). *
denotes p < 0.05 versus 3mo group, # denotes p < 0.05 versus 7mo group. (F)
Quantification of AT8 positive neurons in sham, GDX +PL, GDX + DHT, GDX + T,
GDX + 0.01mg E2, and GDX + 0.025mg E2 mice. Data show mean values (+SEM).
* denotes p < 0.05 versus 7mo Sham group, # denotes p < 0.05 versus 7mo GDX +
PL group.
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DISCUSSION
In this study, we sought to examine the role of androgens and estrogens in
regulating AD pathology in male mice. We observed a significant increase in
intraneuronal and extracellular Aβ pathology following androgen depletion. Both
androgens (T and DHT) were able to prevent this increased accumulation. We found
that E2 decreased intraneuronal Aβ accumulation in CA1, but had no effect in
subiculum and amygdala. Tau hyperphosphorylationwas not effected by androgen
depletion but was significantly decreased below levels observed in sham animals
following E2 (0.025mg) and T treatment.
Sex steroid hormone regulation of Aβ
Consistent with our previous work (Rosario et al., 2006, Chapter 5), we
found that androgen depletion accelerates the development of Aβ pathology in a
transgenic mouse model of AD, and that DHT prevents this increased accumulation.
We also found that testosterone prevented the GDX-induced increases in Aβ
pathology. These findings are consistent with several studies, which have
demonstrated that androgens regulate Aβ pathology (Gouras et al., 2000; Gandy et
al., 2001; Ramsden et al., 2003c). In cell culture studies, testosterone has been
shown to reduce Aβ, an effect that was subsequently found to be through an estrogen
mediated pathway (Goodenough et al., 2000; Gouras et al., 2000). Since DHT is not
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aromatized to estrogen, its efficacy in regulating Aβ supports an estrogen-
independent mechanism. However, a recent report suggests that the DHT metabolite
5α-androstan-3β, 17 β-diol may have agonist actions on estrogen receptor β (Lund et
al., 2006).
To determine if androgen regulation of Aβ pathology involved androgen or
estrogen pathways, we examined the effects of estrogen in male 3xTg-AD mice.
Similar to testosterone, estrogen has been found to decrease Aβ levels in cultured
cells (Xu et al., 1998), and female wild-type rodents (Petanceska et al., 2000).
Estrogen has also been found to regulate Aβ in some, but not all transgenic models
of AD (Levin-Allerhand et al., 2002; Zheng et al., 2002; Heikkinen et al., 2004;
Green et al., 2005; Yue et al., 2005). In the 3xTg-AD mouse model, we found that
estrogen regionally regulated Aβ. Specifically, following androgen depletion,
estrogen reduces Aβ in CA1 of the hippocampus, but not in the subiculum or
amygdala. In addition, estrogen had no effect in reducing the number of
extracellular plaques throughout the brain, suggesting the possibility that there are
different mechanisms for the regulation of intraneuronal and extracellular Aβ.
Mechanism of androgen regulation of Aβ
In this study we found that both T and DHT reduce intracellular and
extracellular Aβ deposition in male mice. Previous work in male rodents measuring
soluble Aβ, found that DHT, but not estrogen, was able to reduce Aβ levels
(Ramsden et al., 2003c). In addition, low levels of androgens but not estrogen have
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been found to correlate with increased plasma levels of Aβ in aged men (Gillett et
al., 2003). Although testosterone has been found to regulate Aβ through increased
Aβ production in cell culture (Gouras et al., 2000), the efficacy of an aromatase
inhibitor suggested that this effect is through an estrogen-dependent mechanism
(Goodenough et al., 2000). While our results support the idea that androgens are
primarily responsible for androgen regulation of Aβ, they also suggest that estrogen
is still having an effect. Estrogen has been shown to have several important
functions in the male brain including control of development and sexual
differentiation (Cooke et al., 1998; Forger, 2006). Estrogen actions in the brain
involve two subtypes of estrogen receptors (ER), ER α and ERβ, which are
differentially localized in various regions of the brain (Shughrue PJ, 1997). Our
results support the role of ERβ in the regulation of Aβ since there is it has been
shown that less ERβ immunoreactivity is present in the amygdala as compared with
ERα. Conversely, expression of ERβ is higher in CA1 where we observe estrogen
regulation of Aβ (Shughrue PJ, 1997).
Downstream of androgen and estrogen receptors, mechanistic events in the
regulation of Aβ pathology may involve either increased clearance or decreased
production of Aβ. It has been proposed that estrogen regulates Aβ by regulating
metabolism of the amyloid precursor protein (APP). Specifically, studies have found
that estrogen enhances α-secretase cleavage of APP (Jaffe et al., 1994, Vincent,
2000) thus increasing sAPPα production and preventing the formation of Aβ (Jaffe
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et al., 1994, Vincent, 2000). Interestingly, effects of estrogen on sAPPα have not
been observed in all studies. In an in vivo study by Petancheska et al., Aβ was
reduced, but no changes in sAPPα were observed following OVX and estrogen
treatment (Petanceska et al., 2000). Another proposed mechanism of estrogen
regulation of APP processing is through stimulating accelerated trafficking of βAPP.
Estrogen has been shown to increase the formation and trafficking of vesicles which
contain βAPP from the trans-golgi network therefore preventing the majority of Aβ
formation and causing a decrease in Aβ levels (Greenfield et al., 2002). One study
has investigated the effects of estrogen on the clearance of Aβ, by examining the
effects of estrogen on neprilysin, an Aβ-catabolizing enzyme, in the brain (Iwata et
al., 2000; Marr et al., 2003a; Newell et al., 2003). In this study, NEP was decreased
in estrogen-depleted rats, and estrogen replacement prevented this decrease in NEP
(Huang J et al., 2004).
The mechanism of androgen regulation of Aβ, when acting through androgen
pathways, has not been determined. DHT treatment in male rats reduces soluble
levels of Aβ but has not been found to regulate sAPPα (Ramsden et al., 2003b).
Recent cell culture data from our lab has identified an androgen receptor-dependent
mechanism of androgen regulation of Aβ involving NEP, suggesting that androgens
act through increased clearance of Aβ (unpublished data). Androgen actions
through increased clearance of Aβ may help to explain why androgens are able to
regulate extracellular Aβ, while estrogen is not.
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Age and androgen regulation of tau hyperphosphorylation
Consistent with studies by Oddo et al., we observed a significant increase in
tau hyperphosphorylation (AT8 positive neurons) with increasing age (Oddo et al.,
2003). The relationship between androgens and AD-like tau pathology has not been
previously investigated in transgenic mice. However, work by Papzemenous found
that androgens prevent heat shock-induced hyperphosphorylation of tau
(Papasozomenos, 1997). In this same model, androgens have been shown to prevent
overactivation of glycogen synthase kinase 3β (GSK3β) (Papasozomenos, 2002).
We did not observe a significant relationship between tau pathology and androgen
depletion and replacement. However, we did find that T treatment in GDX mice
decreased the number of AT8 positive neurons significantly below what is observed
in sham GDX male mice. Estrogen treatment at the higher dose (0.025mg) was also
found to significantly decrease tau hyperphosphorylation. Estrogen has been found
to increase dephosphorylation of tau in cell culture, an effect blocked by anti-
estrogens (Alvarez-de-la-Rosa et al., 2005). Interestingly, Papzomenous found that
testosterone prevention of heat shock-induced hyperphosphorylation of tau was
estrogen independent (Papasozomenos, 1997).
In our study, it appears that while androgens are primarily responsible for the
effects on Aβ pathology, estrogen is regulating tau pathology. Estrogen regulation
of tau hyperphosphorylation involves either increasing phosphatase activity or
decreasing kinase activity. Determining a particular kinase or phosphatase that may
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be responsible is difficult since estrogen acts on both kinases and phosphatases for
various reasons throughout in the brain. Specifically, estrogen has been found to
increases protein phosphatases 2A and 2B (Belcher et al., 2005; Yi et al., 2005),
inhibit GSK3β (Goodenough et al., 2003; Goodenough et al., 2005), and increase in
protein kinase C (Cordey et al., 2003), suggesting several possibilities for the
mechanism of regulation of tau pathology.
It is interesting to note that we do not observe a correlation between tau and
Aβ pathology following androgen depletion and manipulations. Recent evidence
suggests that there is a strong relationship between tau and Aβ pathology in the
3xTg-Ad mouse model (Oddo et al., 2003; Oddo et al., 2004). These studies utilized
anti-Aβ immunotherapy to test if clearance of Aβ affects tau pathology (Oddo et al.,
2004). Anti-Aβ antibodies decrease intraneuronal and extracellular Aβ followed by
clearance of early hyperphosphorylated tau, as measured by HT7. Late
hyperphosphorylated tau as measured by AT8, was not reduced following anti-Aβ
treatment (Oddo et al., 2004). Perhaps if had we measured HT7 we would have
observed a correlation between Aβ and tau, however, we chose AT8 for its
specificity for tau pathology. Since tau pathology has been shown to be regulated
independently of Aβ pathology in this mouse model and other tau transgenic mouse
models (Ramsden et al., 2005) we can predict that different mechanism can regulate
tau and Aβ pathology.
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Androgen therapy and AD
With increasing age, men experience a significant decrease in androgen
levels, and this loss has been linked to an increased risk for the development of AD
(Hogervorst and Bandelow, 2004; Rosario et al., 2004; Moffat, 2005). Our findings
suggest that androgen regulation of Aβ levels is a potential mechanism for the
increased risk for the development of AD. Both T and DHT are able to reduce Aβ
levels in all brain regions which develop appreciable levels of AD pathology, while
estrogen treatment only decreased Aβ in CA1 of the hippocampus. However,
testosterone and estrogen were able to reduce hyperphosphorylation of tau.
Therefore, these results suggest that androgen therapy may be useful in the treatment
and prevention of AD. Recent clinical guidelines for the use of androgen therapy
suggest they are safe and may be used in men with very low levels of androgens,
which would be the population at increased risk for AD (Bhasin et al., 2006). It
appears from these results that to gain the greatest benefit of androgen treatment,
testosterone, or a selective androgen receptor modulator, which can be aromatized
and reduced, would be more effective than DHT
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CHAPTER SEVEN
Progestins inhibit the neuroprotective effects of estrogen in rat
hippocampus
Estrogen loss during menopause has been identified as a risk factor for the
development of Alzheimer’s disease; however, recent clinical trials found that
hormone therapy (HT) had detrimental effects on dementia and cognition. One
reason for this disconnect could be the use of a progestin component. The effects of
progestins in the brain both alone and with estrogen remain largely unclear. In this
study we wanted to investigate the effects of estrogen and progesterone, both alone
and in combination, on neuron viability following an excitotxic lesion. We used
3mo old rats that were depleted of estrogen and treated with a placebo, or estrogen
for 2 weeks prior to the lesion. We found that estrogen is neuroprotective while the
progestins were not. Further, the progestins attenuated estrogen neuroprotective
actions. The study was published in 2006 in Brain Research.
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CHAPTER SEVEN ABSTRACT
Although estrogen has beneficial actions in brain, recent clinical trials
demonstrated adverse neural effects of hormone therapy in postmenopausal women.
The cause(s) of this disconnect between experimental and clinical findings may
include unanticipated effects of progestins. We report that both natural progesterone
and the clinical progestin medroxyprogesterone acetate block estrogen
neuroprotection. These findings underscore the need to evaluate neural actions of
progestins in the rational design of hormone therapy.
INTRODUCTION
Depletion of the sex steroid hormones estrogen and progesterone in
postmenopausal women is an established risk factor for the development of
Alzheimer’s disease (AD) (Paganini-Hill and Henderson, 1994; Tang et al., 1996;
Henderson, 1997; Zandi et al., 2002). However, the recent Women’s Health
Initiative (WHI) clinical trial found that hormone therapy (HT) increased rather than
decreased the risk of dementia in postmenopausal women (Shumaker et al., 2003).
This outcome was unanticipated in part owing to a wealth of experimental data
demonstrating that estrogen has numerous beneficial actions in brain (Wise, 2002;
Green, 1997; Brinton RD, 2000; Gibbs, 1999; McEwen, 1994; Petanceska, 2000)
suggestive of a protective role against neurodegenerative disorders, including AD.
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One particularly relevant estrogen action is neuroprotection. A shortcoming of the
existing estrogen neuroprotection literature is that the potential modulatory role of
progesterone in regulating neural actions of estrogen has been inadequately
evaluated. Further, animal studies have largely employed the physiologically
relevant hormones 17b-estradiol (E2) and progesterone (P4). In contrast, HT in the
WHI consisted of conjugated equine estrogens typically in combination with the
synthetic progestin medroxyprogesterone acetate (MPA) (Rossouw et al., 2002).
The composition of HT, in particular the use of MPA, is one of several problematic
factors that may have contributed to the unexpectedly negative findings of the WHI
(Breitner and Zandi, 2003; Brinton and Nilsen, 2003; Birkhaeuser, 2005; Phillips and
Langer, 2005). Thus, the disconnect between experimental predictions of protective
E2 actions in brain and deleterious clinical outcomes may reflect in part i)
interactions between E2 and P4, and ii) potential functional differences between
natural P4 and synthetic progestins such as MPA. To begin to evaluate these
possibilities, we assessed E2 protection against neuron loss in the presence and
absence of P4 and MPA using a kainate lesion model in female rats.
METHODS
Adult female rats (Harlan; Indianapolis, IN) (N = 6-8 per group) were
ovariectomized (OVX) then two weeks later implanted subcutaneously with Silastic
capsules (Dow Corning, Midland, MI) (ID 1.57 mm x OD 3.18 mm) that were either
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empty or contained hormones in crystalline forms (Sigma; St. Louis, MO) at doses
established to yield near physiological circulating levels. Specifically, E2 was
delivered in 1 x 5 mm capsule (~140 pg/ml) (Febo et al., 2002), P4 in 4 x 40 mm
capsules (~25 ng/ml) (Hoffman et al., 2003), and MPA in 1 x 5 mm capsule (~2
ng/ml) (McNeill et al., 2002). MPA was delivered at a lower dose than P4 to reflect
both its greater potency (Lundeen, et al., 2001) and its approximate levels in
therapeutic use (Hiroi, et al., 1975). Efficacy of hormonal manipulations were
assessed by i) weight of blotted uterus and i) vaginal cytology (i.e., presence of
cornified cells) (Adler and Nelson, 1988).
Two weeks after initiation of hormone treatment, all animals received
systemic kainate (Ocean Produce International; Nova Scotia, Canada) (10 mg/kg in
sterile 0.85% NaCl, i.p.), a treatment that induces excitotoxic neuron death in select
brain regions, including the hilus of the dentate gyrus (Ben-Ari and Cossart, 2000).
Animals were continuously monitored for 3 h following kainate administration to
assess latency to seizure onset and severity of behavioral indices of seizure (common
0-5 rating scale of increasing severity) (Ramsden et al., 2003a). Only animals that
exhibited moderate seizure severity, grades 2-4, were utilized for cell counts.
Animals with seizure grades of 0-1 have absent or very mild seizures that typically
are not associated with significant neuron loss, and thus were excluded. Animals that
experience a grade 5 seizure are euthanized immediately, according to requirements
of our animal protocol, and are not suitable for analysis.
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Three days after kainate injection, animals were sacrificed and brains rapidly
removed and fixed with 4% paraformaldehyde. Fixed hemi-brains were sectioned
then processed for immunohistochemistry with the neuron-specific antibody NeuN
(Chemicon; Temecula, CA), as previously described (Ramsden et al., 2003).
Estimates of neuron number in the hilus of the dentate gyrus were determined using
an unbiased optical disector method, according to a previously described protocol
(Ramsden et al., 2003). Briefly, cells were counted in randomly orientated, 32 µm x
32 µm counting frames with X-Y steps of 95 µm x 95 µm using an Olympus BX50
microscope equipped with a microcator and motorized stage computer-guided by
CAST-Grid software (Olympus; Ballerup, Denmark). Only cells with intact nuclear
morphology that exhibited NeuN immunoreactivity and came into focus within the
disector height (z = -10 µm to -30 µm) were counted. From these counts, neuronal
estimates (N) were calculated using the equation N = ΣQ
-
x t/h x 1/asf x 1/ssf (West,
et al., 1991), where ΣQ
-
is total number of neurons counted, t is section thickness (40
µm), h is disector height (20 µm), asf is area sampling fraction (counting frame area
divided by X-Y step area) and ssf is section sampling fraction (1/10). Raw data from
uterine weights, onset latency and neuron quantification were statistically analyzed
with ANOVA followed by pair-wise comparisons using the Fisher LSD test.
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RESULTS
To confirm biological efficacy of the hormone treatments, we assayed both
uterine weight and vaginal cytology. As expected, E2 alone treatment induced a
robust increase in uterine weight. P4 modestly but significantly increased uterine
weight when delivered alone, and partially attenuated estrogen’s growth-promoting
effect on uterus when delivered with E2 (Table 7). The presence of MPA, either
alone or in combination with E2, did not significantly alter uterine weight (Table 7).
Analysis of vaginal cytology confirmed the anticipated action of E2 treatment in
increasing the proportion of cornified cells, an effect largely antagonized by
combining E2 with either P4 or MPA (Table 7).
Table 7. Assessment of hormone treatments in ovariectomized rats
Without estrogen With estrogen
Veh P4 MPA Veh P4 MPA
Uterine
weight (mg)
94 + 4 152 + 6
a
115 + 5 414 + 11
a
344 + 7
a, b
405 + 41
a
Cornified
cells
- - - +++ + +
Veh = vehicle
a
p < 0.05 relative to veh without estrogen condition
b
p < 0.05 relative to veh with estrogen condition
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Two weeks following the initiation of hormone treatments rats were
systemically exposed to the glutamate agonist kainate. Quantitative analysis of
neuron number in the hilus showed that E2 treatment provided a modest but
significant increase in neuron survival in comparison to the OVX group (Fig. 18A,
B). In contrast, neither P4 nor MPA significantly increased neuron survival when
delivered alone. Notably, E2 neuroprotection against kainate was completely blocked
by co-administration with either P4 or MPA (Fig. 18).
Because some steroid hormones, including P4, can induce anxiolytic effects
that attenuate seizures (Frye and Scalise, 2000), we evaluated how the hormone
treatments affected both the onset latency and behavioral severity of kainate-induced
seizures. No treatment group exhibited increased latency or decreased severity, either
of which would indicate seizure reduction (Fig. 19). The only significant effect was a
modest decrease is onset latency in the E2 plus P4 group (Fig. 19A).
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Figure 18. Progestins block estrogen neuroprotection against kainate lesion in
female rats. (A) Representative photomicrographs of NeuN immunoreactivity in
hilus of the dentate gyrus of kainate-lesioned, OVX rats treated with either no
hormones (Veh), E2 alone (E2), P4 alone (P4), MPA alone (MPA), E2 plus P4
(E2+P4), or E2 plus MPA (E2+MPA). Scale bar = 50 µm. (B) Quantification of
NeuN-immunoreactive in the hilus using optical disector counts. Data show mean
counts (± SEM) for treatment groups in the presence (filled bars) and absence (open
bars) of E2. Coefficient of error values across the treatment groups ranged from
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0.073 to 0.092. Coefficient of variation values ranged from 0.1 and 0.2 across the
groups. Asterisk denotes p < 0.05 versus OVX condition.
Figure 19. Hormone manipulations do not reduce seizure activity induced by
kainate. Data show mean values (+SEM) for onset to seizure (A) and severity of
seizure (B) for treatment groups in the presence (filled bars) and absence (open bars)
of E2. Asterisk denotes p < 0.05 versus OVX condition.
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DISCUSSION
Consistent with prior studies, we found that estrogen is neuroprotective,
specifically against hippocampal neuron loss induced by the excitotoxin kainate.
Previously, estrogen neuroprotection has been observed in several brain regions
challenged with a variety of toxic insults, often in response to only acute E2
treatment (for review see (Wise, 2002). For example, (Azcoitia et al., 1999a) found
that a single E2 injection attenuated subsequent loss of hilar neurons induced by
kainate. In our paradigm, we chose a prolonged, two-week regimen of continuous E2
exposure to more closely model the chronic estrogen exposure of HT in
postmenopausal women. The common finding of estrogen neuroprotection across
multiple experimental paradigms testifies to the robust nature of this hormone effect.
In contrast to the neuroprotective effect of estrogen, we observed that the
progestins P4 and MPA failed to significantly improve survival of kainate-
challenged neurons and actually inhibited estrogen neuroprotection. Cell culture
studies suggest that P4 but not MPA can directly induce neuroprotection following
glutamate toxicity (Nilsen and Brinton, 2002, 2003). P4 exerts neuroprotection in
some animal models but not others (for review see (Stein and Hoffman, 2003). Why
P4 lacked neuroprotective effects in our paradigm remains to be definitively
determined, but may reflect acute versus continuous hormone exposure. For
example, short-term P4 treatment can protect against neuron loss caused by seizure-
inducing toxins such as kainate by attenuating seizure activity (Frye and Bayon,
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1998; Frye and Scalise, 2000; Hoffman et al., 2003). This anxiolytic effect of P4 is
likely mediated by the P4 metabolite allopregnanolone (Frye and Bayon, 1998; Frye
and Scalise, 2000), which is a potent modulator of the GABA
A
receptor (Baulieu et
al., 1996). However, prolonged P4 exposure, as occurs with HT and is modeled in
our paradigm, likely reduces its anxiolytic effects (Gulinello et al., 2001). Consistent
with this position, we observed that continuous, two-week treatment with P4 did not
decrease either seizure latency or severity. Of particular interest is our observation
that continuous P4 and MPA blocked estrogen neuroprotection. Although the
mechanism of this effect is unclear, both P4 and MPA are known to antagonistically
modulate some estrogen actions. For example, progesterone has been found to
attenuate the effect of estrogen on neurotrophins such as, BDNF, NGF, and NT3, in
aged female rats (Bimonte-Nelson et al., 2004). In addition, detrimental cognitive
change has been observed in post-menopausal women taking estrogen and a
progestin while unopposed estrogen users experienced a modest benefit (Rice et al.,
2000).
Outcomes of the recent WHI clinical trials have seriously questioned the
therapeutic utility of HT in the treatment and prevention of age-related disorders in
women, including AD. This unexpected result contradicts abundant data indicating
neural benefits of estrogen-based HT. Efforts to reconcile these divergent
observations have lead to the argument that the efficacy of HT is adversely affected
by several parameters, including the hormonal formulation of HT and the continuous
versus cyclic delivery of hormone components. In this study, our results support the
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established finding that estrogen is neuroprotective. However, our data also show
that the protective actions of estrogen are blocked when it is co-administered
continuously with either P4 or MPA, the progestin component used in the WHI trial.
Together, our data support the beneficial neural potential of HT but highlight the
need to optimize not only the choice of progestin but also the temporal pattern of its
delivery.
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CHAPTER EIGHT
Conclusions and future directions
The studies presented in this thesis investigated the relationship between sex
steroid hormones, aging, and Alzheimer’s disease (AD). The most significant risk
factor for AD is advancing age (Rocca et al., 1986; Jorm et al., 1987; Evans et al.,
1989). The loss of estrogen women experience during normal aging has been
identified as a risk factor for the development of AD (Henderson, 1997; Cholerton et
al., 2002). Less work has been done to investigate the relationship between
androgen loss in men during normal aging and in the development of AD. In
addition, how androgen depletion increases the risk for AD has not been determined.
This dissertation work examined whether low levels of androgens are a risk factor
for the development of AD and how androgens regulate this risk.
The study in Chapter Two investigated whether brain levels of androgens
decrease with age. We found a significant age-related decline in brain levels of
testosterone. We also observed a relationship between low levels of testosterone and
an increased risk for the development of AD (Rosario et al., 2004, Chapter Two).
Investigation of this relationship continued in Chapter Three, where analysis was
expanded to include examination of sex steroid hormones in postmenopausal
women, the metabolites and precursors of testosterone and estrogen, as well as Aβ, a
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causal factor in the development of AD. In postmenopausal women, we found that
brain levels of estrogen were decreased in cases with AD. Low levels of androgens
were observed in male cases with advanced AD pathology, and cases with mild
neuropathological changes, suggesting low levels of androgens place the brain at
increased risk for the development of AD. To begin to examine how low levels of
androgens increase the risk for AD, we examined changes in Aβ, a protein widely
implicated as a causal factor for AD. We found that low levels of androgen in cases
with mild neuropathological changes correlate with increased levels of Aβ. In order
to gain experimental control to examine more closely changes in androgens with age,
and the relationship with Aβ, we utilized an animal model of male reproductive
aging, the Brown Norway rats in Chapter Four. In these rats, we observed a
significant decrease in brain and circulating levels of testosterone with increasing
age. Interestingly, brain levels of DHT decreased with age, while circulating levels
of DHT did not change underling the importance of tissue levels of hormones when
investigating hormone regulated actions in the tissue. With Increasing age, and
following complete androgen depletion in young rats, we observed a significant
increase in Aβ levels suggesting that similar to complete androgen depletion in
young rats, age-related androgen depletion also regulates Aβ.
To further examine how low levels of androgens are increasing the risk for
AD, we experimentally investigated the effects of androgen depletion on the
development of AD pathology in a transgenic mouse model of AD (Chapter Five,
Six). In these mice, androgen depletion significantly increased Aβ pathology which
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correlated with behavioral impairments. Androgen replacement, in the form of
DHT, prevented increased accumulation of Aβ pathology and behavioral deficits
(Rosario et al., 2006 ) (Chapter Five). Since the mechanism of androgen actions can
be through either direct androgen pathways or indirect estrogen pathways in Chapter
Six we examined the effect of estrogen on AD pathology in male mice. Testosterone
was found to be as effective as DHT in preventing Aβ pathology, while estrogen was
able to regionally decrease intraneuronal Aβ pathology, but had no effect on
extracellular deposition. Estrogen and testosterone, but not DHT, were able to
decrease hyperphosphorylation of tau, suggesting an estrogen-dependent mechanism.
While estrogen is regulating hyperphosphorylation of tau, androgens appear to
regulate intraneuronal and extracellular Aβ pathology through androgen-dependent
pathways.
Recent results from the Women’s Health Initiative Memory study (WHIMS)
found that hormone therapy was not effective in preventing cognitive impairment
and increased the rate of dementia (Rapp et al., 2003; Shumaker et al., 2003). One
possibility as to why this study did not produce the expected results is the use of a
progestin component. There has been little research done to investigate the effects of
progesterone, both alone and in combination with estrogen, in the brain. Chapter
Seven examined the effect of estrogen and progesterone on neuroprotection, a factor
that may modulate the risk for AD. We found that estrogen is neuroprotective
following excitotoxic insult, and that progesterone has no effect. Further,
progesterone blocks estrogens protective effect against cell death.
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Taken together, these studies present the foundation for novel mechanisms of
hormone action against the risk of AD, and underlie the hypothesis for the use of
hormone therapy in the treatment and prevention of AD.
From basic research to clinical trials: lessons learned and future directions
The protective and beneficial role of estrogens in the brain is well
documented (Cholerton et al., 2002; Wise, 2002). We found that postmenopausal
women with advanced AD pathology had low levels of estrogen compared with
neuropathologically normal controls (Chapter Three). Epidemiological studies have
found that estrogen loss at menopause is a risk factor for the development of AD
(Tang et al., 1996; Henderson, 1997). Despite these findings, there is a disconnect
between these basic science studies, epidemiological studies, and clinical studies
trying to determine the role of sex steroid hormones in AD. The large WHIMS
clinical trial found that hormone therapy, both estrogen alone and estrogen + a
progestin, had either no effect or detrimental effects on cognition and dementia
(Rapp et al., 2003; Shumaker et al., 2003; Espeland et al., 2004; Shumaker et al.,
2004). Future studies, both clinical trials and basic research, need to address the
issues which have come up in light of the WHIMS trial such as mode of
administration, age of treatment initiation, duration, dose, apoE genotype, and
composition of HT including preparation with a progestin component.
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Progestin component
While estrogen actions in the brain have been widely studied (for review see
(Cholerton et al., 2002; Wise, 2003)), progesterone actions in the brain, both alone
and in combination with estrogen are much less researched. Some studies have
found that progesterone has beneficial actions in the brain (Roof et al., 1994; Roof et
al., 1996; Azcoitia et al., 1999b; Nilsen and Brinton, 2002; Hoffman et al., 2003;
Ciriza et al., 2004), while other have not (Singer et al., 1996; Chen et al., 1999;
Murphy et al., 2000). We found that following an excitotoxic lesion, progesterone
was not protective and blocked estrogens neuroprotective effect (Rosario et al., 2006,
Chapter Seven). This study suggests that constant administration of both estrogen
and progesterone would not be a recommended treatment method against AD. More
research needs to be done to fully examine the effects of estrogen and progesterone
in combination. For example, progesterone has been found to be neuroprotective by
decreasing seizure severity (Hoffman et al., 2003). However, prolonged
progesterone treatment, or high doses of progesterone results in down-regulation of
GABA receptors, which are responsible for modulating the anti-seizure effect
(Gulinello et al., 2001; Wohlfarth et al., 2002). Therefore, a cyclic treatment
paradigm of estrogen and progesterone may be more effective for neuroprotection,
and consequently for HT in women.
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Age of initiation and duration
Recently, clinical trials by the Woman’s Health Initiative found HT and ET
were not effective in decreasing cognitive decline and dementia (Rapp et al., 2003;
Shumaker et al., 2003; Espeland et al., 2004; Shumaker et al., 2004). Hormone
treatment in these trials started 10 to 20 years after menopause. There is evidence to
suggest that the brain losses responsiveness to hormones following menopause.
Specifically, animal studies have found that aged female rat brain has decreased
responsiveness to estrogen (Gibbs, 1998; Miranda et al., 1999; Alkayed et al., 2000;
Stone et al., 2000; Wise, 2002). Estrogen is still beneficial in young OVX animals
compared with aged “reproductively senescent” animals where estrogen treatment
has no effect (reviewed in (Sohrabji, 2005)). Changes in estrogen receptors with age
and disease could account for the decreased responsiveness to estrogen. For example
both ERα and β decrease with age in CA1-3 of hippocampus (Tohgi et al., 1995;
Adams et al., 2002; Mehra et al., 2005). Results from the WHIMS study suggests
the importance for more experimental studies examining this issue, and clinical
studies which start at the time of menopause before, age-related changes occur that
will result in altered responsiveness of the brain to hormone treatment. This idea is
supported by results from the Cache county study suggesting that initiation of HT at
the time of menopause, is more efficacious (Zandi et al., 2002).
In addition to decreased responsiveness of the brain following menopause,
initiation of hormone therapy at the time of hormone loss may also be important for
the mechanism of estrogen action in preventing or regulating AD pathology. For
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example, there is evidence that during estrogen depletion Aβ pathology increases,
and estrogen treatment reduces these elevated levels by acting on APP processing to
reduce Aβ production (Jaffe et al., 1994; Savage et al., 1998; Xu et al., 1998;
Petanceska et al., 2000; Vincent and Smith, 2000; Greenfield et al., 2002; Levin-
Allerhand et al., 2002; Zheng et al., 2002). If estrogen is only acting to decrease
production of Aβ, a significant accumulation of Aβ will likely occur during the gap
between estrogen loss and replacement, and HT would not be able to reduce this
accumulation. However, if estrogen is acting to decrease Aβ levels through
increased clearance of Aβ, HT may be beneficial in reducing some accumulation, but
this would also depend on how the brain responds to the hormone treatment. Based
on the proposed mechanism of estrogen regulation of Aβ, and the maintained
responsiveness of the brain to estrogen, one could predict that HT would be most
effective if started at the time of hormonal loss.
In conclusion, the beneficial actions of estrogen observed in experimental
studies warrant the continued investigation into hormone therapies for the treatment
and prevention of AD in women. It can be anticipated that future studies, both
experimental and clinical, will build upon the existing knowledge and bring us closer
to understanding the relationship between sex steroid hormones and their role in the
development of AD.
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Androgens and Alzheimer’s disease: understanding the relationship and
moving forward
The focus of my thesis work has been investigating androgens in the brain
during normal aging and in the development of AD. Initially, a relationship between
low levels of androgens and AD was observed in cases with a clinical diagnosis of
AD (Hogervorst et al., 2001). We found that low levels of androgens are a risk
factor for the development of AD (Rosario et al., 2004) (Chapter Two, Three). Since
androgens have been identified as a risk factor for the development of AD, the effect
of low levels of androgens on this increased risk was the next area of investigation.
Androgens have several beneficial actions in the brain relevant to the development of
AD such as neuroprotection and regulation of Aβ levels (Pike, 2001; Ramsden et al.,
2003c; Ramsden et al., 2003a). Using human postmortem tissue, wild-type rats, and
transgenic mice, we observed a relationship between low testosterone levels and
increased levels Aβ pathology (Chapter Three, Four, Five, and Six). Understanding
the mechanism of androgen actions on AD pathology will allow for the development
of specific therapeutic interventions. In order to fully understand the benefit of
androgens, large, randomized, clinical trials are needed to determine the
effectiveness of androgen therapy in the treatment and prevention of AD.
Mechanism of androgen regulation of Aβ
Several studies have investigated the effects of sex steroid hormones on
regulation of Aβ. Studies have found that androgens are endogenous regulators of
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Aβ (Gillett et al., 2003; Ramsden et al., 2003c; Rosario et al., 2006). Androgen
actions in the brain, such as regulation of Aβ pathology, can be through direct
androgen pathways, or indirect estrogen or gonadotropin pathways (Mooradian et al.,
1987). Research has found that estrogen regulates levels of Aβ by decreasing
production and deposition of Aβ (Jaffe et al., 1994; Savage et al., 1998; Vincent and
Smith, 2000; Greenfield et al., 2002). This mechanism of action appears to be
through the increased α-secretase cleavage of APP resulting in the sAPPα (Jaffe et
al., 1994; Vincent and Smith, 2000; Manthey et al., 2001). Studies have found that
sAPPα regulates neuronal excitability, enhances synaptic plasticity and learning and
memory (Scheuner et al., 1996). Testosterone has also been shown to regulate levels
of Aβ (Gouras et al., 2000). However, it was determined that this effect was not
through androgen pathways but instead due to the aromatization of T to E2
(Goodenough et al., 2000). Previous work from our lab indicates that androgens are
responsible for the regulation of Aβ levels in males (Ramsden et al., 2003c). Aβ
levels were significantly increased following GDX, an effect reduced by DHT
treatment (Ramsden et al., 2003). However, E2 did not have an effect on Aβ levels
in this study (Ramsden et al., 2003). Interestingly, we found that estrogen does
regulate intraneuronal Aβ but only regionally (Chapter Six), suggesting that a
specific ER subtype may be regulating Aβ in the male brain. In addition, previous
studies suggest that androgens and estrogens are acting to regulate Aβ pathology
through different mechanisms. Regulation of intraneuronal and extracellular Aβ is
155
likely also regulated by different mechanisms as estrogen acts to reduce
intraneuronal Aβ in CA1 but not extracellular Aβ.
Unlike estrogen, androgens do not appear to be regulating Aβ levels through
APP processing (Ramsden et al., 2003c). If androgens are not acting to decrease Aβ
production, they are likely acting to increase clearance of Aβ levels. Recent work
from our lab found that neprilysin (NEP), an Aβ catabolizing enzyme, is regulated
by androgens. In this cell culture study, androgen regulation of Aβ was androgen
receptor (AR) dependent and involved NEP. When NEP was blocked, androgens
were not able to reduce Aβ levels. In vivo work from our lab found that androgen
depletion resulted in decreased levels of NEP, and DHT replacement restored NEP to
levels observed in sham GDX animals. Also, in these animals, androgen depletion
resulted in increased Aβ levels, suggesting that androgen regulation of soluble Aβ in
rats involves NEP. Overexpression of NEP through intrahippocampal injection has
been shown to significantly decrease Aβ levels (Marr et al., 2003b). Interestingly,
recent work by Hersch et al. found that NEP overexpression in the white blood cells
also significantly decreased Aβ in the brain, suggesting that Aβ may be cleared in
the blood and that Aβ from the brain is pulled into the blood in a “sink” effect. Aβ
immunotherapy studies in the 3xTg-AD support the idea of an “Aβ sink theory” as
both active and passive immunotherapy first clears intraneuronal Aβ followed by
extracellular deposition (Oddo et al., 2004; Oddo et al., 2006b). One month
following anti-Aβ treatment, intraneuronal Aβ is first to reappear followed by
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extracellular deposition. If androgen regulation of Aβ involves NEP, both
circulating and brain androgens could be acting to regulate NEP and reduce Aβ.
In addition to sex steroid hormones, luteinizing hormone (LH) has also been
shown to have a regulatory effect on Aβ levels (Bowen et al., 2004). Although our
work suggests that androgens are responsible for the regulation of AD-like
pathology, there is evidence to suggest that gonadotropins may also play a role in the
development of AD. As sex steroid hormones decrease with age, the hypothalamic-
pituitary axis is disrupted due to decreased negative feedback of estrogen and
androgens on the hypothalamus and anterior pituitary. This results in increased
levels of the gonadotropins LH and FSH (Morley, 2001b). LH has been implicated
as a risk factor for the development of AD (Bowen et al., 2000; Short et al., 2001).
Studies in men have found circulating levels of gonadotropins to be increased in AD
cases as compared to control (non-AD) cases (Bowen et al., 2000; Short et al., 2001).
LH has also been shown to regulate levels of Aβ. Leuprolide acetate, a GNRH
agonist, was used suppress LH levels in female mice and resulted in reduced levels
of Aβ1-42 and Aβ1-40 (Bowen et al., 2004). Although these studies support the idea
of a novel therapeutic target for the development of AD, other studies have found no
difference in LH or FSH levels between AD and control male cases but a significant
decrease in T levels in AD cases (Hogervorst et al., 2003). In addition, in men being
treated for prostate cancer, GnRH agonists responsible for reducing levels of LH
have resulted in increased levels of Aβ and cognitive impairment (Gandy et al.,
2001; Almeida and Papadopoulos, 2003). Since LH does not significantly increase
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in men until after 80 (Morley et al., 1997), it may not become a significant risk factor
until old age. We would like to further examine the effect between LH and AD in
our transgenic model of AD. Although there is conflicting data, it is important to
continue research in this area to determine the extent to which gonadotropins may be
involved in Aβ regulation and thus development of AD. Since hormones associated
with the hypothalamic-pituitary axis (E2, T, and LH) have been strongly linked to an
increased risk for developing AD, it is important to determine the role of these
hormones. The mechanism of Aβ regulation by each hormone needs to be
determined to ascertain the best and most effective way to prevent Aβ pathology for
the treatment and prevention of AD.
Androgens, estrogens, and tau hyperphosphorylation
In Chapter Six, androgen depletion did not significantly increase tau
hyperphosphorylation, as measured by AT8 positive neurons. This is a late marker
of phosphorylation so potentially an earlier marker would have produced different
results. However, most markers specific for early phosphorylation changes are not
pathology specific and we wanted to use a pathology specific marker of tau
phosphorylation to assure we were measuring pathology-related changes in tau.
Although we did not observe an effect of androgen depletion, we did find that
estrogen and testosterone were able to significantly reduce tau hyperphosphorylation
below what is found in sham animals. Since DHT was not effective at reducing tau
pathology, we predict that this is an estrogen-mediated effect. The mechanism of
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estrogen regulation of tau pathology would be expected to involve either increased
phosphatase activity or decreased kinase activity. Elucidating the potential
mechanism poses difficulties since estrogen has been shown to increase protein
phosphatases 2A and 2B (Belcher et al., 2005; Yi et al., 2005), inhibit GSK3β
(Goodenough et al., 2003; Goodenough et al., 2005), and increase protein kinase C
(Cordey et al., 2003), suggesting several possibilities for the mechanism of
regulation of tau pathology. Future research needs to be done to determine the
mechanism and potential areas for therapeutic interventions.
Increasing age and androgen loss
During normal aging, both men and women experience changes in their
brains that may place them at increased risk for the development of AD or other
neurodegenerative diseases and disorders. In addition, depletion of sex steroid
hormones during normal aging results in decreased brain levels of hormones, which
as we know, also places the brain at increased risk for the development of AD.
Androgens, through androgen and estrogen pathways, have been found to have a
beneficial effect on Aβ and tau pathology. Therefore, androgen loss may be directly
acting on tau and Aβ pathology, to make the brain more vulnerable to insult and
damage. Several factors may be working together to increase the risk for AD such
as, age-related changes in the brain and androgen-related changes in the brain such
as oxidative damage with advancing age. Glutathione (GSH), a natural anti-oxidant,
has been shown to decrease with increased age (Rebrin et al., 2003; Zhu et al., 2006;
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Rebrin et al., 2007). As GSH decreases, signs of oxidative damage increases (Shi et
al., 1993; Wullner et al., 1999; Schulz et al., 2000). Gender differences in GSH
metabolism have been observed, specifically decreased levels of GSH are observed
in male cases with AD (Liu et al., 2005). Increased oxidative stress has been found
to result in increased BACE (Tamagno et al., 2005). Aβ peptides result from the
cleavage of amyloid precursor protein (APP) by β-secretase (BACE) at the N
terminus and γ-secretase at the C terminus (Hardy, 1997). In addition to the
proposed decreases in NEP which androgens regulate, there could be other pathways
converging that modulate the risk for AD.
Androgen therapy for the treatment and prevention of AD
As discussed above, my thesis work investigated the relationships between
androgen loss, advancing age, and AD. Findings from this work underlie the
importance of androgens in the treatment and prevention of AD in men. Recent
clinical guidelines for the use of androgen therapy suggest they are safe and may be
used in men with very low levels of androgens, which would also be the population
at increased risk for AD (Bhasin et al., 2006). Since we found that androgens were
effective in preventing intraneuronal and extracellular Aβ, and estrogen and
testosterone were able to decrease hyperphosphorylation of tau, it appears that to
gain the greatest benefit of androgen treatment, testosterone, or an androgen that can
be aromatized or reduced would be most effective.
160
There are some risks associated with androgen therapy. These risks may
include, prostatic carcinoma, atherosclerosis, breast carcinoma, and hypertension (for
review see (Kaufman and Vermeulen, 2005)). The principal concern is the
development of prostate cancer since the majority of prostate cancers are androgen
sensitive, at least at the beginning (Goldenberg et al., 1995). Despite these potential
risks, to date there is no direct evidence between androgen therapy and increased
incidence of prostate cancer (Raynaud, 2006, Kaufman, 2005). In fact, a recent
clinical trial found that short-term androgen therapy for 6 months was sufficient to
regulate serum levels of androgens in hypogonadal men, but had no adverse effects
on prostate tissue (Marks et al., 2006). However, the effects of short-term androgen
therapy for the treatment of AD are still unclear. In women, results examining
estrogen therapy suggest a longer duration may be important (Zandi et al., 2002) but
it is unclear if this would be the same for men and androgen therapy.
Another way to circumvent potential risks of androgen therapy would be to
construct selective androgen receptor modulators (SARMS) that have beneficial
actions in brain but have no or minimal effect on prostate tissue. Since DHT is a
more potent androgen, and is linked more closely to potential negative effects in
prostate, and because we observed that testosterone and DHT were equally effective
in reducing Aβ pathology in the 3xTg-AD mice, a SARM that had direct androgen
activity through androgen receptors but is not reduced to a more potent androgen,
like DHT, would be beneficial against AD, but not as high a risk for prostate cancer.
In addition, since we observed beneficial actions of estrogen on tau pathology, a
161
SARM that could be aromatized would allow for potential beneficial actions on both
Aβ and tau pathology. Future clinical trials will be important to test the
effectiveness of androgen therapy in the treatment and prevention of AD, and the
risks and benefits of this treatment will have to be determined.
In conclusion, results from this work suggest that low levels of androgens are
a risk factor for the development of AD, and that androgen treatment is effective in
preventing AD pathology in rodent models. Therefore, I predict that androgen
therapy in hypogonadal, aged men would be a beneficial intervention against the
development of AD. Future research should focus on two major areas: 1)
understanding and elucidating the mechanism of these androgen regulated actions, to
identify other potential areas for therapeutic intervention, and 2) generating SARMS
to be used in androgen therapy that will increase the benefits of androgen treatment
and diminish the risks.
162
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Abstract (if available)
Abstract
Advancing age is the most significant risk factor for the development of Alzheimer's disease (AD), however, which age-related changes underlie this effect remains unclear. In men, one normal consequence of aging is a robust decline in the circulating levels of the sex steroid hormone testosterone. Testosterone depletion leads to functional impairments in androgen-responsive tissues that are often manifested as the clinical syndrome 'androgen deficiency in aging males'. Although the brain is an androgen-responsive tissue unknown is (1) whether brain levels of T decline during aging, and if so, (2) whether low brain T levels place the aging brain at increased risk for AD, and if so (3) what do androgens regulate that may modulate the increased risk for AD. My thesis work investigated these questions and others examining the relationships between sex steroid hormones, advancing age, and development of AD. In Chapters Two and Three we observed that brain levels of androgens but not estrogens are significantly lower in men with moderate to severe AD in comparison to normal men. To examine how low testosterone levels may contribute to AD development we examined androgen regulation of A[beta], a causal factor in the development of AD. In Chapters, Three through Six we investigated the effects of androgens on regulation and development of A[beta] pathology. We found that low levels of testosterone, both in humans and a rodent model of male reproductive aging, correlated with increased levels of soluble A[beta]. Using a transgenic mouse model of AD we found that depletion of endogenous androgens resulted in increased accumulation of A[beta] pathology and behavioral impairments. Replacement of androgens in these mice was able to prevent this increased accumulation. These findings suggest the use of androgen replacement therapy in men with low levels of androgens.
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Creator
Rosario, Emily R.
(author)
Core Title
Age-related androgen depletion and the development of Alzheimer's disease
School
College of Letters, Arts and Sciences
Degree
Doctor of Philosophy
Degree Program
Neuroscience
Publication Date
04/05/2007
Defense Date
02/01/2007
Publisher
University of Southern California
(original),
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Tag
Alzheimer's disease,androgen,OAI-PMH Harvest
Language
English
Advisor
Pike, Christian J. (
committee chair
), Brinton, Roberta Diaz (
committee member
), Gatz, Margaret (
committee member
), Swanson, Larry W. (
committee member
), Thompson, Richard (
committee member
)
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erosario@usc.edu
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Alzheimer's disease
androgen