Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Estradiol regulation of cerebral metabolism: implications for neuroprotection and mitochondrial bioenergetics
(USC Thesis Other)
Estradiol regulation of cerebral metabolism: implications for neuroprotection and mitochondrial bioenergetics
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
ESTRADIOL REGULATION OF CEREBRAL METABOLISM: IMPLICATIONS
FOR NEUROPROTECTION AND MITOCHONDRIAL BIOENERGETICS
by
Ronald W. Irwin
____________________________________________________________________
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
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2008
Copyright 2008 Ronald W. Irwin
Dedications
To my lovely wife Kristina, words cannot express how much you mean to me.
Thank you for taking me into your life and making me part of your family. The sky’s
the limit! Mom, Dad, Natalie, Jeff and the whole family – I would never have made
it this far without your infinite support. You all deserve a Ph.D. for putting up with
me! Cheers!
Completion of my doctoral work ended on a bittersweet note for our family. We
were struck with the passing of my wife’s grandmother “Baba” Leposava Tomi ć and
my grandfather George R. Swenson within a week of my graduation ceremony. We
deeply miss both of them and have many fond memories. Our spirits have been
revived by the birth of Evan Willis Irwin. He is our pride and joy.
ii
Acknowledgments
First, I would like to sincerely thank my doctoral dissertation advisor, Professor
Roberta Diaz Brinton. Robbie provided me with the scientific input, laboratory
environment and encouragement that made training with her far more rewarding than
a doctoral degree. She set the highest of scientific and ethical standards for me to
reach for as a graduate student. Robbie explained to me early on that the Doctor of
Philosophy is the ultimate academic degree. The primary purpose as a Ph.D. is to
seek the truth. As scientists we must stand on the shoulders of those that have made
solid discoveries in the past. I would like to acknowledge Professor Enrique Cadenas
for providing his mentorship and allowing me to conduct part of my research in his
laboratory including mitochondrial isolations, 2-dimensional gels, and respiration
measurements. Cadenas was one of the first researchers in the world to demonstrate
that mitochondria are the primary source of oxidative stress. I would like to thank my
colleague and friend Dr. Ryan Hamilton for his helpful collaborations, measuring
lipid peroxides, and for enduring graduate school with me every step of the way.
Special thanks to soon-to-be doctor Jia Yao for his help and enthusiasm to continue
on with the mitochondrial project and transgenic mouse work. Indeed, I would like to
thank all of the past and present Brinton Laboratory members and coworkers
including Dr. Jon Nilsen, Dr. Jun Ming Wang, Dr. Shuhua Chen, Dr. Liqin Zhao, Dr.
Paolo Mannella, Dr. Tzu-wei Wu, Dr. Junegoo Lee, Dr. Lixia Zhao, Dr. Xiaobing
Jiang, Jia Yao, Lifei Liu, Anna Tran, Rana Masri, Tino Sanchez, Sean Iwamoto,
Jimmy To, Jennifer Mao, and all those not listed whom have helped me to obtain my
iii
research goals. I really lucked out in choosing to study at USC because of the
wonderful community of scholars that have shared with me both challenges and
success stories. I would also like to acknowledge Professor Caleb Finch for enriching
my training experience through guidance of the NIH-NIA sponsored predoctoral
training grant in the Neuroendocrinology of Aging. Of his many fascinating
dimensions, his teaching style was a perfect example of how to think outside the box
and to integrate knowledge across many levels and disciplines. Much gratitude to
Professor Frances Richmond for introducing me to the field of Regulatory Science
and the nitty gritty details of biomedical product development. Thanks to the USC
Proteomics Core guided by Dr. Timothy Gallaher for mass-spectrometry
identification of individual protein spots from 2-D gels. I would also like to
acknowledge my previous mentors Dr. Stanley Parsons at UCSB and Drs. Diane
Harris, Sergio Huerta, David Heber, and Bill Go at UCLA.
I would like to specially thank Dr. Jon Nilsen. Jon devoted much of his time and
energy to my training experience at USC and his mentorship was instrumental to my
success over the past several years. Jon taught me a tremendous amount about
mitochondrial biology and gonadal hormone actions in the brain which have become
my primary interests. Much of my doctoral work was carried out under Jon and
Robbie’s guidance and collaboration. I am also grateful to Dr. Jun Ming Wang who
was always there to encourage me and sharpen my understanding of molecular and
stem cell biology.
iv
I would like to thank my dissertation committee of Dr. Roberta Brinton, Dr.
Michel Baudry, and Dr. Enrique Cadenas. My gratitude also goes to my qualifying
exam committee which consisted of the aforementioned final dissertation committee
in addition to Dr. Nouri Neamati and Dr. Alex Sevanian. I sincerely wish Dr.
Sevanian could have witnessed the completion of my doctoral degree. Dr. Sevanian
played a direct role in persuading me to join the Department at USC. I first
introduced myself to him one morning while he was an invited speaker at crosstown
UCLA, where I was working as a Research Associate in early 2002. I vividly
remember that day and the superb lecture he delivered on antioxidants and the free
radical chemistry of lipids.
v
Table of Contents
Dedications ii
Acknowledgments iii
List of Tables x
List of Figures xi
Abbreviations xiii
Abstract xv
Chapter I: Overview of Estrogen, Mitochondria,
and the Brain 1
Introduction 1
Cerebral Oxidative Metabolism 2
Basis for Estrogen Therapeutics to Prevent Alzheimer’s Disease 3
Estrogen Receptors in Mitochondria 6
Selective Estrogen Receptor Modulators 7
The Estrogen Treatment Paradigm 9
Significance 9
Specific Aims 11
Chapter I References 13
Chapter II: Estradiol Regulation of Brain
Mitochondrial Proteome In Vivo 15
Abstract 15
Introduction 16
Materials and Methods 18
Chemicals 18
Animals 18
Mitochondrial Isolation 19
Mitochondrial Purity Assessment 20
Two-Dimensional Gel Electrophoresis 21
Protein Identification by Mass Spectrometry 22
Western Blot Analysis 24
Respiratory Measurements 24
Complex IV/Cytochrome c Oxidase Activity 25
RNA Isolation and RT-PCR 26
vi
Statistics 26
Results 26
17β-Estradiol-Induced Regulation of Mitochondrial Proteins 32
17β-Estradiol-Induced Mitochondrial Functional Responses 38
Discussion 42
Chapter II References 47
Chapter III: Progesterone and Estrogen Regulate
Oxidative Metabolism in Brain Mitochondria 52
Abstract 52
Introduction 53
Materials and Methods 55
Chemicals 55
Animals 56
Mitochondrial Isolation 57
Respiratory Measurements 58
Complex IV/Cytochrome C Oxidase Activity 59
Western Blot Analysis 59
RNA Isolation and RT-PCR 60
Mitochondrial Biogenesis 60
Mitochondrial Peroxide Production 60
Free Radical Leak 60
Lipid Peroxidation 61
Statistics 61
Results 62
Progesterone and 17 β-estradiol enhanced brain mitochondrial
respiratory activity 62
Enhanced cytochrome c oxidase activity and expression in
hormone replaced rats 64
Enhanced Complex V expression in hormone replaced rats 66
Mitochondrial biogenesis was unaffected by hormone replacement 67
Effects of progesterone and 17 β-estradiol on reactive oxygen
species production by brain mitochondria 68
Progesterone and 17 β-estradiol reduced lipid peroxidation of
brain mitochondria 71
Progesterone and 17 β-estradiol alter the antioxidant profile
of brain mitochondria 74
Discussion 74
Acknowledgements 83
Chapter III References 84
vii
Chapter IV: Selective estrogen receptor modulators
potentiate brain mitochondrial function 88
Abstract 88
Introduction 89
Materials and Methods 91
Chemicals 91
Animals 92
Mitochondrial Isolation 93
Respiration Measurements 94
Lipid Peroxides 94
Cytochrome c Oxidase activity and expression 95
Mitochondrial Membrane Potential 95
Mitobiogenesis measurement 95
Estrogen Receptor protein expression 96
2D Gel/Western 96
Statistics 96
Results 96
Respiration 96
Lipid Peroxidation 98
Cytochrome Oxidase 99
Mitochondria membrane potential 101
Mitobiogenesis 102
Estrogen Receptors in Mitochondria 103
Discussion 105
Chapter IV References 108
Chapter V: Triple transgenic Alzheimer’s disease
mouse model and age-related effects on the brain
mitochondrial proteome 109
Abstract 109
Introduction 110
Materials and Methods 111
Chemicals 111
Animals 111
Mitochondrial Isolation 111
Respiration Measurements 113
Lipid Peroxides 113
Cytochrome c Oxidase activity and expression 113
Estrogen Receptor protein expression 114
2D Gel Electrophoresis 114
Results and Discussion 117
Chapter V References 122
viii
Chapter VI: Integration of Specific Aims 1-4 and
Concluding Remarks 123
Discussion 123
Specific Aim 1: Estradiol Regulation of the Mitoproteome 123
Specific Aim 2: Estradiol and Progesterone Regulation of
Cerebral Oxidative Metabolism 125
Specific Aim 3: Selective Estrogen Receptor Modulators
(SERMs) Potentiate Mitochondrial Function using both
ER-alpha and ER-beta Selective Estrogen Receptor Modulators 126
Specific Aim 4: Age and transgene studies in the 3xTg-AD
mouse model 128
Chapter VI References 129
Concluding Remarks 130
Complete List of References 134
ix
List of Tables
Table II-1. Mitochondrial Proteome Identification 31
Table III-1. Mitochondrial Respiration. 64
x
List of Figures
Figure I-1. Estradiol neuroprotective actions converge upon
the mitochondria. 4
Figure I-2. Overview of Specific Aims 1-4 11
Figure II-1. Mitochondrial purity assessment 27
Figure II-2. Schematic overview of experimental design 28
Figure II-3. 2-D analysis of brain mitochondrial proteome 30
Figure II-4. Comparison of protein expression in 2-D analysis 36
Figure II-5. Quant. analysis of brain mitoproteome expression
patterns 37
Figure II-6. 17 β -estradiol enhances mitochondrial respiratory
activity 39
Figure II-7. 17 β-estradiol enhances cytochrome c oxidase activity
and expression 41
Figure III-1. Progesterone and 17 β-estradiol enhance
mitochondrial respiratory activity. 63
Figure III-2. Progesterone and 17 β-estradiol enhance
cytochrome c oxidase activity and expression. 66
Figure III-3. Progesterone and 17 β-estradiol increase expression
of Complex V subunit α protein. 67
Figure III-4. Progesterone and 17 β-estradiol do not alter
mitochondrial biogenesis. 68
Figure III-5. Ovarian hormones reduce rate of peroxide
production and free radical leak in brain mitochondria. 70
Figure III-6. Progesterone and 17 β-estradiol reduce lipid
peroxidation of brain mitochondria. 72
xi
Figure III-7. Progesterone and 17 β-estradiol alter the
antioxidant profile of brain mitochondria. 73
Figure IV-1. Chemical structures of Estradiol (E2),
ER α selective agonist (PPT), and ER β selective agonist (DPN). 91
Figure IV-2. E2, PPT, and DPN increase mito. respiration 97
Figure IV-3. Uterine weight to demonstrate proliferative
effect of estradiol 24 hrs post-treatment. 98
Figure IV-4. Lipid Peroxides measured by Leucomethylene
blue method 99
Figure IV-5. E2, PPT, and DPN increase
cytochrome c oxidase (COX) activity in mitochondria. 100
Figure IV-6. E2, PPT, and DPN mediated upregulation of
COX IV and II protein. 101
Figure IV-7. Mitochondrial Membrane Potential
Increased by Estrogen Receptor Agonists. 102
Figure IV-8. Lack of increased mitochondrial biogenesis
in response to in vivo estradiol treatment. 103
Figure IV-9. Estrogen receptors in purified mitochondria
from OVX rat forebrain. 104
Figure IV-10. 2-D/Western blot of rat brain mitochondria. 105
Figure V-1. ER-alpha and ER-beta expressed in
brain mitochondria associated with age and transgene status. 115
Figure V-2. Oxygen consumption of brain mitochondria measured
as respiratory control ratio (RCR). 116
Figure V-3. Lipid peroxides - male vs female and 3xTg vs nonTg 117
Figure V-4A and V-4B. 2-D gel mitoproteomic profile. 120
Figure V-5. Proposed schematic model of estradiol
prevention of AD 121
xii
Abbreviations
17-beta Estradiol E
2
Adenosine 5’-diphosphate ADP
Adenosine 5’-triphosphate ATP
Alzheimer’s disease AD
Atractyloside ATR
Collapsin-response mediator protein isoform 1 Crmp-1
Complementary strand deoxyribonucleic acid cDNA
Control (vehicle) C or Ct
Cytochrome c oxidase COX
Diarylpropionitrile DPN
Electron transport chain ETC
Estrogen receptor alpha ER α
Estrogen receptor beta Er β
Estrogen receptor splice variants ERsv
Kilogram kg
Liquid Chromatography/Mass Spectrometry LC-MS/MS
Messenger ribonucleic acid mRNA
Malate Dehydrogenase MDH
Manganese superoxide dismutase MnSOD
Medroxyprogesterone acetate MPA
Microgram μg
xiii
Milligram mg
Micromolar μM
Millimolar mM
Mitochondria mito
Non-transgenic mouse nonTg
Peroxiredoxin isoform V Prdx V
Potential of Hydrogen pH
Progesterone P4
Propyl pyrazole trisphenol PPT
Pyruvate Dehydrogenase PDH
Respiratory Control Ratio RCR
Reverse transcription-polymerase chain reaction RT-PCR
Sodium dodecyl polyacrylamide gel electrophoresis SDS-PAGE
Triple transgenic Alzheimer’s disease mouse model 3xTg-AD
Two-dimensional gel electrophoresis 2-DE
Voltage Dependent Anion Channel VDAC
Women’s Health Initiative WHI
WHI Memory Study WHIMS
xiv
Abstract
Herein are a collection of studies that build on our existing knowledge of
estrogen actions in the brain. We extend the efforts of current neuroprotective
strategies by demonstrating that estrogenic molecules promote cell survival
mechanisms governed by neuronal mitochondria. Estrogen (E
2
, 17β-estradiol)
protects neurons from a series of age-related risk factors for developing Alzheimer’s
disease (AD) supported by basic science, clinical, and epidemiological data.
However, there exists a window of opportunity for E
2
as a preventive therapy and
our findings are not intended for hopeful treatments of pre-existing pathologies but
rather to support the proposed healthy-cell bias therapeutic approach (Brinton 2005).
Our preclinical pharmacology research work covers biochemistry, molecular biology
and cell imaging of rodent brain. Controlled in vitro and in vivo studies are
organized under four specific aims that test our hypotheses in brain tissues with a
focus on the hippocampus and cortex regions involved in learning and memory and
adversely affected in age-related dementias. Specific Aim 1 serves to determine the
presence of estrogen receptors in mitochondria and the effects of E
2
on the brain
mitochondrial proteome. Specific Aim 2 focuses on E
2
and P4 actions on the
mitochondria and the mechanistic role of these hormones on mitochondrial function.
Specific Aim 3 extends these basic science discoveries to test selective estrogen
receptor modulators. Specific Aim 4 introduces the triple-transgenic mouse model of
xv
Alzheimer’s disease as a tool to test hormone therapies and intervene in cases of
mitochondrial dysfunction. We believe that our experimental approach and
subsequent findings will forge the way for a new class of E
2
-based mitochondrial
therapeutics to reduce the risk of Alzheimer’s disease and other diseases attributed to
mitochondrial malfunction.
xvi
Chapter I: Overview of Estrogen, Mitochondria, and the
Brain
Introduction
The mission of our laboratory is to elucidate fundamental cellular
mechanisms of cognitive function and neuron survival and to translate our
discoveries into therapeutics that sustain cognitive function during aging and to
prevent neurodegenerative disease. The practical application phase of this mission is
aimed at translating these basic science discoveries into therapeutics to prevent
neurodegenerative diseases including Alzheimer’s (AD) and to restore neurological
function. Our research has focused on the most potent endogenous estrogen steroid
hormone, 17 β-estradiol (E
2
). Results of these studies, along with those from our
colleagues, have demonstrated at the most basic cellular levels that E
2
is
neuroprotective against neurotoxic insults including exposure to glutamate and β-
amyloid (McEwen and Alves 1999; Brinton 2001; Kim, Bang et al. 2001; Behl 2002;
Mattson and Kroemer 2003; Nilsen and Brinton 2003). E
2
actions are attributed
primarily to binding and activation of the estrogen receptor (ER). ER protein has two
main isoforms, ER α and ER β that arise from separate and distinct genes but have
converged to bind E
2
with essentially equal affinity. Both ER α and ER β exhibit
similar binding affinity for E
2
with Ki values of 0.30 nM and 0.90 nM, respectively
(Sun, Meyers et al. 1999). However, the two receptors behave differently when
bound to E
2
(Weihua, Andersson et al. 2003). Several alternatively spliced variants
1
of ER have evolved in each brain region and possess distinct localization patterns
and functions, providing a plausible hypothesis for rapid estrogen induced
neuroprotection (Price, Lorenzon et al. 2000). Specifically, membrane associated ER
is involved with rapid signal transduction pathways. The “classical” genomic actions
of ER activate transcription in the nucleus via full length ERs that bind DNA
directly, or bind with other transcription factors such as AP-1 to indirectly activate
transcription (Paech, Webb et al. 1997). Recently, it has been proposed that a
mitochondrial estrogen receptor (mtER) exists. Some evidence suggests that the
brain may harbor these mtERs but the precise location and function has not been
determined (Yang, Liu et al. 2004; Milner, Ayoola et al. 2005; Stirone, Duckles et al.
2005). It remains to be determined whether mtERs are involved in estrogenic actions
afforded by E
2
. Our hypothesis is that mtERs coordinate with both nuclear and
nongenomic pathways initiated by E
2
that provide neuroprotection convergent upon
mitochondrial function.
Cerebral Oxidative Metabolism
The brain almost exclusively utilizes glucose and oxygen via aerobic
oxidation. Following glucose uptake, the enzymatic process of glycolysis converts
glucose into pyruvate which enters the mitochondrial matrix and pyruvate
dehydrogenase (PDH) converts pyruvate to acetyl CoA and further oxidation occurs
via the citric acid cycle. Oxidative phosphorylation within mitochondria utilizes
electron flow from reduced substrates to molecular oxygen. The end result of glucose
utilization is energy production as ATP is generated from precursors ADP and P
i
by
2
the F1-F0 ATPase anchored to the inner mitochondrial membrane. In the absence of
oxygen, the glycolytic cycle is completed by conversion of pyruvate to lactic acid in
the cytoplasm. No other fuel source is sufficient to maintain normal brain function
and structure. In fact the brain consumes oxygen at a rate of 156 μmol/100 g
tissue/min. Likewise, carbon dioxide production matches this rate 1:1 (Sokoloff
1960; Sokoloff and Kety 1960). Only under extreme conditions of ketosis can the
brain use ketone bodies produced by the liver. It is well established that AD patients
have reduced glucose utilization and shrinkage of brain tissues (Meltzer, Zubieta et
al. 1996; Hoffman, Welsh-Bohmer et al. 2000). These insights have led us to study
the relationship between estradiol neuroprotection and cerebral oxidative
metabolism.
Basis for Estrogen Therapeutics to prevent Alzheimer’s Disease
Clinical studies of estrogen therapy/hormone therapy (ET/HT) have been
divided in recent years. It remains unclear as to why such strong evidence for
therapeutic benefit observed with in vitro and in vivo basic science studies does not
correlate well with recent reports of women that opt to use ET/HT (Turgeon,
McDonnell et al. 2004; Brinton 2005). There are convincing reports that demonstrate
E
2
protects against the development but not progression of AD (Sherwin 1988;
Resnick, Metter et al. 1997; Yaffe, Sawaya et al. 1998). In addition to cognitive
studies, there have been several small- and large-scale studies conducted to evaluate
ET/HT efficacy and safety in coronary heart disease, osteoporosis, stroke, cancer,
diabetes, etc. including the largest multi-center study: the Women’s Health Initiative
3
Trial (WHI) (www.nhlbi.nih.gov/whi/) which was scheduled to last 15 years and
included more than 161,000 postmenopausal women. An ancillary study to the WHI,
called the Memory Study (WHIMS) was aimed to track the development and
progression of dementia symptoms. However, since the NIH halted the WHI studies
early, women have turned to alternative medicines. The focus for ET/HT should not
only be the dosing cycle of E
2
but also ER molecular biology: tissue specific
expression, alternatively spliced variants, and subcellular localization.
Figure I-1. Estradiol neuroprotective actions converge upon the mitochondria.
These neurprotective actions provide increased calcium load tolerance, respiratory
efficiency, antioxidant defense, and ATP generation. Estrogen receptors are located
at or near the plasma membrane, in the cytosol, nucleus, and mitochondria.
One neuroprotective E
2
hypothesis proposed by our lab (Figure 1), involves
calcium buffering by the mitochondria and increased antiapoptotic bcl-2 expression,
4
(Nilsen and Brinton 2002; Nilsen and Brinton 2003; Nilsen and Brinton 2004).
Excitotoxic glutamate raises the intracellular calcium ion concentration [Ca
2+
]
i
to
levels that would trigger apoptosis in neurons that not pre-conditioned with E
2
. In a
subset of neurons, E
2
bound mER can activatee PI3K, which then activates an
isoform of PKC that is calcium independent, PKC δ. This kinase then phosphorylates
the L-type calcium channel to trigger a calcium influx to the cytoplasm. High
concentrations of intracellular calcium activate calcium dependent PKCs. These
PKCs can then phosphorylate Src to activate the Src/MEK/ERK cascade. ERK can
then enter the nucleus where it phosphorylates CREB. CREB is then able to
stimulate transcription of spinophilin, Bcl-2, and Bcl-xL. E
2
has been shown to
stimulate Bcl-2 expression (Nilsen and Brinton 2003; Zhao, Wu et al. 2004). Bcl-2
expression potentiates the intramitochondrial calcium capacity (Murphy, Bredesen et
al. 1996; Nilsen and Brinton 2003). Acting as a buffer system, mitochondria are able
to protect cells from triggering death pathways. Mitochondrial dysfunction is
associated with but not exclusive to neurodegenerative diseases, ischemia-
reperfusion injury in stroke and heart attack, diabetes, and aging. Healthy
mitochondria are able to lower the bulk free calcium by sequestration. Both ERs
were found to be neuroprotective in our laboratory (Zhao, Wu et al. 2004). In
addition, we recently demonstrated that E
2
treatment promotes mitochondrial
viability in primary hippocampal neurons as well as in ovariectomized rat brain
mitochondrial preparations and can protect from toxic levels of calcium (Nilsen,
Chen et al. 2006).
5
Estrogen Receptors in Mitochondria
In 2001 and 2002, the first known reports of ER β identification in subcellular
compartments and most notably the mitochondria of rabbit uterus and ovary were
published by Monje and Boland (Monje and Boland 2001; Monje and Boland 2002).
In 2004, the Simpkins Laboratory published results in PNAS that showed ER β
localization in differentiated primary rat myocytes, primary rat hippocampal neurons,
and clonal mouse hippocampal HT22 cells by immunocytochemical imaging. They
further demonstrated that purified human heart mitochondrial fractions contain ER β
via Western blot and mass spectrometry analysis (Yang, Liu et al. 2004). However,
more recently the mass spectrometry results of Yang et al. have been challenged and
suggested to be false positives by Gustafsson’s group (Schwend and Gustafsson
2006). Around the same time that Yang et al. published their findings in 2004,
JinQiang Chen from James Yager’s group at Johns Hopkins published data showing
both ER α and ER β localized to mitochondria in the human breast cancer cell line,
MCF-7. This work included an estimate of mitochondrial ER α and ER β expression
at 10 and 18% respectively of total expression following E
2
treatment (Chen,
Delannoy et al. 2004). Yager and colleagues also found that E
2
-treated cultured
female rat hepatocytes have increased transcript levels of the mitochondrial genes
cytochrome oxidase subunits I, II, and III. Mitochondrial respiratory chain activity
was reflected by increased mitochondrial superoxide generation. Increased levels of
Bcl-2 protein and mitochondrial glutathione were observed (Chen, Delannoy et al.
2003). Their work in MCF-7 cells has been extended by reports that recombinant
6
ERs bind to mtDNA response elements and suggest that mtERs can directly
transcribe mtDNA (Chen and Yager 2004). Milner and colleagues have also
observed ERs located in subcellular compartments including mitochondria in
dendritic spines and axon terminals of rat hippocampus (Milner, Ayoola et al. 2005;
Milner, Lubbers et al. 2008).
Despite these early attempts, it is still undetermined how ER is associated
with the mitochondrion and what function mtER provides to the cell from this
location. As the existence of mtER is still debated, we initiated our own studies on
the subject. The existence of mtER in brain is crucial to our understanding of the
physiological role and relevance of mtER in an attempt to pinpoint pharmacological
intervention possibilities for diseases of the central nervous system.
Selective Estrogen Receptor Modulators
The 7 α-analog ICI 182,780 (7α -[9-(4,4,5,5,5
pentafluoropentylsulfinyl)nonyl] estra-1,3,5(10 )- triene-3,17 beta-diol) was
developed by Imperial Chemical Industries and marketed by AstraZeneca as
Fulvestrant or “Faslodex” for hormone receptor-positive breast cancer that does not
respond to tamoxifen therapy (Wakeling, Dukes et al. 1991). ICI 182,780 has high
affinity for ER α (Kd ~ 1.0 nM) and ER β (Kd ~ 3.6 nM) (Sun, Meyers et al. 1999).
The literature suggests that ICI 182,780 is a full ER antagonist or antiestrogen
molecule. However, in our hands, data suggests that ICI 182,780 is exceptionally
neurotrophic and neuroprotective at low concentrations (1-30nM). It has been
demonstrated by our laboratory that ICI 182, 780 is a full mER agonist, activating
7
ERK and increasing the expression of spinophilin as well as protecting against
glutamate or amyloid toxicity (Zhao, O'Neill et al. 2006). Keeping with this finding,
ICI-like molecules that share the chemical features of alkyl chain addition at the 7- α
position, were designed to selectively activate (or deactivate) non-genomic ER
targets of E
2
action that reside in cytosol (Zhao, Jin et al. 2007). The unique aspect of
the ICI 182,780 chemical structure is the side chain “tail” addition at the 7 α- position
of the E
2
backbone. The long side chain of ICI 182,780 does not sterically hinder the
ligand binding pocket of ER. Moreover, the tail serves to prevent ER-ER
dimerization (Borras, Laios et al. 1996) and nuclear translocation (Dauvois, White et
al. 1993). Hypothetically ICI-like compounds will preferentially activate mER while
avoiding nucER transcriptional events.
If mtER is expressed in both isoforms, α and β, or splice variants thereof,
rational based drug design may lead to highly specific agonists to control ER protein
function. For the nucERs, selective agonists have been designed by the
Katzenellenbogen group (Sun, Meyers et al. 1999; Katzenellenbogen, Sun et al.
2001; Sun, Baudry et al. 2003). PPT was found to act on ER α as a full agonist.
Likewise, DPN, acts as an ER β agonist. Their recent work involves the discovery of
selective antagonists for either ER α (MPP) or ER β (R,R-THC). Both selective
agonists and antagonists, are potentially useful SERMs for developing effective
ET/HT and eliminating potentially negative side effects. To summarize, a rationally
designed SERM ideally has the following properties: 1.) uptake in the cell to provide
therapeutically relevant concentrations; 2.) specific ER binding affinity; 3.) crosses
8
the BBB; 4.) neuroprotective. In Specific Aim 3, SERMs (PPT and DPN) were
evaluated for mitochondrial functional efficacy.
The Estrogen Treatment Paradigm
Several studies suggest that E
2
is most effective in prevention models rather
than as a treatment. Our laboratory has revealed that hippocampal neurons exposed
to E
2
prior to exposure to β-Amyloid are protected much more effectively than
simultaneous exposure. In addition, neurons treated with E
2
after β-Amyloid insult,
were found to have decreased survival in comparison to β-Amyloid alone (Brinton
2005). This research mirrors what is observed in the clinic by several studies.
Prevention studies in women that are near or at their menopause indicate a lower risk
for AD in later years. The studies that include postmenopausal women that receive
ET/HT later in life, find that E
2
is not neuroprotective and may negatively influence
health including the brain and heart as found in the WHI studies
(www.nhlbi.nih.gov/whi/). This treatment paradigm suggests that E
2
must be given
within a window of opportunity to fully benefit the patient.
Significance
The recent discoveries of estrogen receptors localized to mitochondria
provide a target for yet unexplored mechanisms of estrogen action. Results of the
basic science studies herein provide a look at 24 hour treatment effects on ER action
indirectly or directly influencing neuroprotective mitochondrial endpoints. These
studies are directed towards explaining the mitochondrial contributions to
9
neuroprotective mechanisms of E
2
as well as progesterone, P4. The role of ER in
terms of its usefulness as a therapeutic target for prevention of neurodegenerative
diseases should be considered. Results from these studies are not only relevant to
neuroscientists but to all people that seek to understand the complex role of
neuroendocrine hormones and those affected by disease of the central nervous
system. The results will also benefit basic and clinical investigators focused on heart
diseases, epithelial cancers, and bone diseases among others. To practicing
clinicians, it can be gleaned from the results of this work that the way they assess
health benefits and risks of ET/HT should be further studied to tailor the way they
prescribe ET/HT to postmenopausal women.
10
Specific Aims
Figure I-2. Overview of Specific Aims 1-4. Aims 1-3 of this work were directed at
determining the role of estradiol (E2), progesterone (P4), and SERMs (PPT and
DPN) on mitochondrial function. Specific Aim 4 examines the triple transgenic
(3xTg-AD) mouse model of AD-like pathology and its potential for further studies
on hormone therapies to delay onset of AD-like pathology by preventing
mitochondrial dysfunction.
We sought to address four specific aims to elucidate the biology of
neurodegenerative diseases influenced by E
2
(Figure 2). We focus on developing
basic mechanisms for mitochondria-directed preventive strategies for men and
women. Specific Aim 1 served to determine the presence of estrogen receptors in
mitochondria and the effects of E
2
on the brain mitochondrial proteome. Specific
Aim 2 focused on E
2
and P4 actions on the mitochondria and the mechanistic role of
these hormones on mitochondrial function. Specific Aim 3 extended these basic
science discoveries to test selective estrogen receptor modulators. Specific Aim 4
introduced the triple-transgenic mouse model of Alzheimer’s disease as a tool to test
hormone therapies and intervene in cases of mitochondrial dysfunction. We believe
that our experimental approach and subsequent findings will forge the way for a new
11
class of E
2
-based mitochondrial therapeutics to reduce the risk of Alzheimer’s
disease. The ultimate aim of this work is to add to the knowledge of E
2
actions that
converge upon the mitochondria and the proteomic profile that gives rise to
neuroprotection.
12
Chapter I References
Behl, C. (2002). "Oestrogen as a neuroprotective hormone." Nat Rev Neurosci 3(6):
433-42.
Brewer, G. J., J. D. Reichensperger, et al. (2005). "Prevention of age-related
dysregulation of calcium dynamics by estrogen in neurons." Neurobiol
Aging.
Brinton, R. D. (2001). "Cellular and molecular mechanisms of estrogen regulation of
memory function and neuroprotection against Alzheimer's disease: recent
insights and remaining challenges." Learn Mem 8(3): 121-33.
Brinton, R. D. (2005). "Investigative Models for Determining Hormone Therapy-
Induced Outcomes in Brain: Evidence in Support of a Healthy Cell Bias of
Estrogen Action." Ann NY Acad Sci 1052(1): 57-74.
Kim, H., O. Y. Bang, et al. (2001). "Neuroprotective effects of estrogen against beta-
amyloid toxicity are mediated by estrogen receptors in cultured neuronal
cells." Neurosci Lett 302(1): 58-62.
Mattson, M. P. and G. Kroemer (2003). "Mitochondria in cell death: novel targets for
neuroprotection and cardioprotection." Trends Mol Med 9(5): 196-205.
McEwen, B. S. and S. E. Alves (1999). "Estrogen actions in the central nervous
system." Endocr Rev 20(3): 279-307.
Milner, T. A., K. Ayoola, et al. (2005). "Ultrastructural localization of estrogen
receptor beta immunoreactivity in the rat hippocampal formation." J Comp
Neurol 491(2): 81-95.
Nilsen, J. and R. D. Brinton (2002). "Impact of progestins on estradiol potentiation
of the glutamate calcium response." Neuroreport 13(6): 825-30.
Nilsen, J. and R. D. Brinton (2003). "Mechanism of estrogen-mediated
neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression."
Proc Natl Acad Sci U S A 100(5): 2842-7.
Nilsen, J. and R. D. Brinton (2004). "Mitochondria as therapeutic targets of estrogen
action in the central nervous system." Curr Drug Targets CNS Neurol Disord
3(4): 297-313.
Nilsen, J., S. Chen, et al. (2006). "Estrogen protects neuronal cells from amyloid
beta-induced apoptosis via regulation of mitochondrial proteins and
function." BMC Neurosci 7(1): 74.
13
Paech, K., P. Webb, et al. (1997). "Differential ligand activation of estrogen
receptors ERalpha and ERbeta at AP1 sites." Science 277(5331): 1508-10.
Price, R. H., Jr., N. Lorenzon, et al. (2000). "Differential expression of estrogen
receptor beta splice variants in rat brain: identification and characterization of
a novel variant missing exon 4." Brain Res Mol Brain Res 80(2): 260-8.
Resnick, S. M., E. J. Metter, et al. (1997). "Estrogen replacement therapy and
longitudinal decline in visual memory. A possible protective effect?"
Neurology 49(6): 1491-7.
Sherwin, B. B. (1988). "Estrogen and/or androgen replacement therapy and cognitive
functioning in surgically menopausal women." Psychoneuroendocrinology
13(4): 345-57.
Stirone, C., S. P. Duckles, et al. (2005). "Estrogen increases mitochondrial efficiency
and reduces oxidative stress in cerebral blood vessels." Mol Pharmacol.
Sun, J., M. J. Meyers, et al. (1999). "Novel ligands that function as selective
estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-
beta." Endocrinology 140(2): 800-4.
Turgeon, J. L., D. P. McDonnell, et al. (2004). "Hormone therapy: physiological
complexity belies therapeutic simplicity." Science 304(5675): 1269-73.
Weihua, Z., S. Andersson, et al. (2003). "Update on estrogen signaling." FEBS Lett
546(1): 17-24.
Yaffe, K., G. Sawaya, et al. (1998). "Estrogen Therapy in Postmenopausal Women:
Effects on Cognitive Function and Dementia." Jama 279(9): 688-695.
Yang, S. H., R. Liu, et al. (2004). "Mitochondrial localization of estrogen receptor
beta." Proc Natl Acad Sci U S A 101(12): 4130-5.
Zhao, L., C. Jin, et al. (2007). "Design, synthesis, and estrogenic activity of a novel
estrogen receptor modulator--a hybrid structure of 17beta-estradiol and
vitamin E in hippocampal neurons." J Med Chem 50(18): 4471-81.
Zhao, L., K. O'Neill, et al. (2006). "Estrogenic agonist activity of ICI 182,780
(Faslodex) in hippocampal neurons: implications for basic science
understanding of estrogen signaling and development of estrogen modulators
with a dual therapeutic profile." J Pharmacol Exp Ther 319(3): 1124-32.
14
Chapter II: Estradiol Regulation of Brain Mitochondrial
Proteome In Vivo
Jon Nilsen*, Ronald W. Irwin*, Timothy K. Gallaher, Roberta Diaz Brinton
*These authors contributed equally to this work.
Published: The Journal of Neuroscience. Dec 19, 2007 27(51).
Abstract
We utilized a combined proteomic and functional biochemical approach to
determine the overall impact of 17 β-estradiol (E
2
) on mitochondrial protein
expression and function. To elucidate mitochondrial pathways activated by E
2
in
brain, 2-D gel electrophoresis was conducted to screen the mitoproteome.
Ovariectomized adult female rats were treated with a single injection of E
2
.
Following 24 hrs of E
2
exposure, mitochondria were purified from brain and 2-D
analysis and LC-MS/MS protein identification were conducted. The results of
proteomic analyses indicated that of the 499 protein spots detected by image
analysis, a total of 66 protein spots had a two-fold or greater change in expression.
Of these, 28 proteins were increased in expression following E
2
treatment whereas
38 proteins were identified as decreased in expression relative to control. E
2
regulated key metabolic enzymes including pyruvate dehydrogenase, aconitase, and
ATP-synthase. To confirm that E
2
-inducible changes in protein expression translated
into functional consequences, we determined the impact of E
2
on the enzymatic
activity of the mitochondrial electron transport chain. In vivo, E
2
-treatment enhanced
15
brain mitochondrial efficiency as evidenced by increased respiratory control ratio,
elevated cytochrome-c oxidase activity and expression while simultaneously
reducing free radical generation in brain. Results of these analyses provide insights
into E
2
mechanisms of regulating brain mitochondria, which have potential for
sustaining neurological health and prevention of neurodegenerative diseases
associated with mitochondrial dysfunction such as Alzheimer’s disease.
Introduction
Basic science analyses indicate that estrogens induce protection of neurons
against neurodegenerative insults both in vitro and in vivo (Brinton 2005). Moreover,
estrogen in the same model systems activated biochemical, genomic, cellular and
behavioral mechanisms of memory (Singh, Meyer et al. 1994; Simpkins, Rajakumar
et al. 1997; Woolley 1999; Toran-Allerand 2000; Brinton 2001; McEwen 2002;
Brinton 2004). Estrogen’s neuroprotective effects are multifaceted, encompassing
chemical, biochemical, and genomic mechanisms and falling into three mechanistic
categories: antioxidant, defense, and viability (Nilsen and Brinton 2004; Morrison,
Brinton et al. 2006). Our findings demonstrate that a protein/protein interaction
between estrogen receptor and the regulatory subunit p85 activates the PI3K
signaling cascade, simultaneously activating both the Akt and ERK pathways which
ultimately converge upon mitochondria (Mannella and Brinton 2006). Activation of
this complex signaling cascade results in a proactive defense state conferring
significant protection against Ca2+ dysregulation induced by neurodegenerative
16
insults, leading to greater survival of E
2
responsive neurons (Nilsen and Brinton
2003; Brewer, Reichensperger et al. 2006; Chen, Nilsen et al. 2006; Mannella and
Brinton 2006). E
2
regulation of mitochondrial function is a pivotal convergence point
upon which estrogen neuroprotection depends.
Results of basic science, clinical, and epidemiological analyses demonstrate
that E
2
protects against age-related risk factors for developing Alzheimer’s disease
(AD) (Brinton 2005). More recently, E
2
has been found to regulate metabolic
functions sustaining the energetic demands of neuronal activation (Bishop and
Simpkins 1995; Yang, Liu et al. 2004; Nilsen, Chen et al. 2006; Singh, Dykens et al.
2006; Simpkins and Dykens 2007). Mitochondria are the primary energy source for
cells, converting nutrients into energy through cellular respiration via the electron
transport chain (Murphy, Bredesen et al. 1996; Cadenas and Davies 2000; Nicholls
and Budd 2000). In parallel, mitochondria are also the key regulators of the intrinsic
apoptotic cascade (Lin and Beal 2006). Many components of the mitochondrial
bioenergetic network are vulnerable to oxidative stress, which can impair
mitochondrial and cellular function as well as increasing apoptotic vulnerability
(Nicholls and Budd 2000; Lin and Beal 2006).
Based on these findings and because mitochondria appear to be a
convergence point for mechanisms underlying estrogen-induced neuroprotection
(Nilsen and Brinton 2003; Nilsen and Brinton 2004; Nilsen, Chen et al. 2006), we
sought to determine mechanisms whereby E
2
promotes mitochondrial function. To
address this issue, we conducted a proteomic analysis of brain-derived mitochondria
17
from female rats treated with E
2
. Mitochondria, by some estimates, contain up to
1500 proteins (Lopez, Kristal et al. 2000), a number that is amenable to examination
by two-dimensional gel electrophoresis coupled with LC-MS/MS protein
identification. In this study, 2D-gel electrophoresis was used to investigate the
influence of E
2
on the mitoproteome profile in brain tissue.
The results of the current investigation reveal that E
2
significantly regulates
the mitoproteome to promote enhanced function of metabolic pathways: pyruvate
oxidation, the tricarboxylic acid cycle, and mitochondrial respiration. To our
knowledge, this is the first documentation of proteomic profiling of brain
mitochondria following E
2
treatment in vivo.
Materials and Methods
Chemicals
Chemicals were from Sigma (St. Louis, MO) unless otherwise noted. 17 β-
estradiol (Steraloids; Newport, RI) was suspended in sesame oil vehicle. Deep Purple
gel stain was from Amersham/GE Healthcare. All other 2-DE reagents were from
Bio-Rad (Hercules, CA).
Animals
Use of animals was approved by the Institutional Animal Care and Use
Committee of University of Southern California (protocol number 10256). 4-6 month
ovariectomized female Sprague-Dawley rats were purchased from Harlan
18
(Indianapolis, IN) and housed under controlled conditions of temperature (22°C),
humidity, and light (14/10 h light/dark cycle); water and food were available ad
libitum until treatment. Pathogen-free rats were used two weeks following
ovariectomy to allow for equilibration to hormone deprivation. Ovariectomized rats
were treated subcutaneously with E
2
(30 µg/kg) or sesame-oil vehicle control, and
fasted 24 hours prior to sacrifice and dissection. The cerebellum, pineal gland, and
brainstem were removed and forebrain was utilized for mitochondrial isolations. At
time of sacrifice, uterei were removed and weighed to confirm biological efficacy of
E
2
-treatment. Prior studies demonstrated that E
2
plasma and brain levels after a 30-
µg/kg dose produced levels in OVX rats of 42 pg/g brain tissue (wet weight) E
2
in
brain tissue and 44 pg/ml E
2
in serum (Wang, Irwin et al. 2006).
Mitochondrial Isolation
Non-synaptosomal mitochondria were isolated in ice-cold Mitochondria
Isolation Buffer (MIB: pH 7.4, containing Sucrose (320 mM), EDTA (1 mM), Tris-
HCl (10 mM), and Calbiochem’s Protease Inhibitor Cocktail Set I (AEBSF-HCl 500
μM, Aprotonin 150 nM, E-64 1 μM, EDTA disodium 500 μM, Leupeptin
hemisulfate 1 μM), Phosphatase Inhibitor Cocktail Set I ((-)-p-
Bromotetramisoleoxalate 2.5 mM, Cantharidin 500 μM, Microcystin LR 500nM)) by
discontinuous density gradient centrifugation consisting of 15, 23, and 40% Percoll
colloidal silica particles of 15-30 nm coated with polyvinylpyrrolidone (Sigma) as
previously described (Schroeter, Boyd et al. 2003). Briefly, one rat forebrain was
homogenized into 5 mL MIB using a Dounce homogenizer. The homogenized brain
19
was centrifuged at 1,500 g for 5 min. BSA was not added to the MIB to avoid spot
interference with 2-D gel and protein identification. The supernatant was collected
and the pellet resuspended, re-homogenized in 2.5 mL MIB, and centrifuged at 1500
g for 5 min. The supernatants were collected and combined and the pellet discarded.
The resulting cytosolic homogenate was centrifuged at 21,000 g for 10 min to yield
the crude mitochondrial pellet. The crude pellet was resuspended in 15% Percoll and
centrifuged at 21,000 g for 10 min to remove excess myelin. The remaining crude
mitochondria, in 15% Percoll, were layered onto a pre-formed 23%/40%
discontinuous Percoll gradient and centrifuged at 31,000 g for 10 min. The purified
mitochondria were extracted from the gradient and washed with 10 mL MIB at
16,700 g for 13 min. The remaining loose pellet was collected and washed once
more with 1 mL MIB in an Eppendorf tube at 10,000 g for 8 min. The purified
mitochondrial pellet was then stored at -80 degrees until use.
Mitochondrial Purity Assessment
During the mitochondrial purification process, aliquots were collected of crude
homogenate, crude mitochondria, and highly purified mitochondria after density
gradient centrifugation. Protein concentration was determined by bicinchoninic acid
(BCA) protein assay (Pierce Biotechnology; Rockford, IL). Western blot analysis for
several subcellular markers was carried out to verify mitochondrial integrity and
purity. Proteins were separated by 10% SDS-PAGE, electrotransferred to
polyvinylidene difluoride membrane (Millipore; Billerica, MA), and probed with
primary antibody followed by horseradish peroxidase-conjugated with either horse
20
anti-mouse (1:20,000; Vector Laboratories; Burlingame, CA) or goat anti-rabbit IgG
(1:20,000; Vector Laboratories) depending on the primary antibody. Bands were
visualized by 3,3',5,5'-Tetramethylbenzidine Peroxidase Substrate kit (Vector
Laboratories). Twenty micrograms of protein were loaded into each lane. Equal
loading was confirmed by Coomassie blue staining (data not shown). Antibodies
raised against subcellular markers were used to detect mitochondrial anti-
Vdac1/porin (1:500; MitoSciences, Eugene, OR), nuclear anti-histone H1 (1:250;
AE-4; Santa Cruz Biotechnology, Santa Cruz, CA), endoplasmic reticulum anti-
calnexin (1:2000; SPA 865; Assay Designs, Ann Arbor, MI), and myelin basic
protein (1:500; clone 2, RDI, Concord, MA). Each membrane was stripped with
stripping buffer containing beta-mercaptoethanol and reprobed with each antibody
sequentially.
Two-Dimensional Gel Electrophoresis
Four-hundred micrograms of protein from purified mitochondria were
solubilized for 30 minutes with Bio-Rad ReadyPrep 2-D rehydration/sample buffer
(7 M urea, 2 M thiourea, 1% ASB-14, 40 mM Tris) and 2% Bio-Rad IPG buffer pH
3-10. Bio-Rad 17 cm ReadyStrip pH 3-10NL IPG strips were used to separate
proteins according to charge. Solubilized mitochondrial proteins were adsorbed into
the gel strip overnight and were then focused according to their isoelectric point with
the Bio-Rad Protean IEF System. The program utilized was the following: 250 V
rapid voltage ramping for 30 min, 10,000 V slow voltage ramping for 60 min, and
10,000 V rapid voltage ramping for 50KVhrs. The strips were incubated first in
21
Equilibration buffer I with 6 M urea, 20% glycerol, 2% SDS, 2% DTT, and 0.375 M
Tris (pH 8.8) for 10 min at room temperature, then in Equilibration buffer II with 6
M urea, 20% glycerol, 2% SDS, 2% iodoacetamide, and 0.375 M Tris (pH 8.8). The
strips were then loaded onto 12% SDS-PAGE gels and run at 50 V overnight to
complete the second dimension of protein separation. ReadyPrep Overlay Agarose
was added on top of the strip to secure it and included Bromophenol blue tracking
dye. A molecular standard was used to estimate relative mass (M
r
). Gels were fixed
with 7.5% acetic acid, 10% methanol solution, washed with 200 mM sodium
carbonate, then stained overnight with Deep Purple 1:200 diluted from stock as
stated in manufacturers protocol. Gels were destained in 7.5 % acetic acid, rinsed
with water, and fluorescence scanned with the Typhoon 8600 system (Molecular
Dynamics). Gel image analyses were carried out with PD Quest software (Bio-Rad).
Protein Identification by Mass Spectrometry
Protein spots were excised and tryptic digests were analyzed in the USC
Proteomics Core Facility by liquid chromatography-tandem mass spectrometry (LC-
MS/MS) as described previously (Gallaher, Wu et al. 2006). Briefly, protein spots
from 2-D gel were visualized with UV light, excised from the gels, and destained
with 50% acetonitrile in 50 mM ammonium carbonate. In-gel tryptic digest was
carried out using reductively methylated trypsin (Promega, Madison, WI). Prior to
digestion, samples were reduced with DTT (10 mM in 50 mM ammonium carbonate
for 60 minutes at 56°C) and subsequently alkylated with iodoacetamide (55 mM in
50 mM ammonium carbonate for 45 minutes in the dark at room temperature). The
22
digestion reaction was carried out overnight at 37°C. Digestion products were
extracted from the gel with a 5% formic acid/50% acetonitrile solution (2X) and one
acetonitrile extraction followed by evaporation using an APD SpeedVac
(ThermoSavant). The dried tryptic digest samples were cleaned with ZipTip
(Millipore CB18B) prior to analysis by tandem mass spectrometry for protein
identification.
The digested sample was resuspended in 10 µL of 60% acetic acid, injected via
autosample (Surveyor, ThermoFinnigan) and subjected to reverse phase liquid
chromatography using ThermoFinnigan Surveyor MS-Pump in conjunction with a
BioBasic-18 100 × 0.18 mm reverse-phase capillary column (ThermoFinnigan, San
Jose, CA). Mass analysis was done using a ThermoFinnigan LCQ Deca XP Plus ion
trap mass spectrometer equipped with a nanospray ion source (ThermoFinnigan)
employing a 4.5-cm long metal needle (Hamilton, 950-00954) in a data-dependent
acquisition mode. Electrical contact and voltage application to the probe tip took
place via the nanoprobe assembly. Spray voltage of the mass spectrometer was set to
2.9 kV and heated capillary temperature at 190°C. The column was equilibrated for 5
min at 1.5 μL/min with 95% solution A and 5% solution B (A, 0.1% formic acid in
water; B, 0.1% formic acid in acetonitrile) prior to sample injection. A linear
gradient was initiated 5 min after sample injection ramping to 35% A and 65% B
after 50 min and 20% A and 80% B after 60 min. Mass spectra were acquired in the
m/z 400–1800 range.
23
Protein identification was carried out with the MS/MS search software
Mascot 1.9 (Matrix Science) with confirmatory or complementary analyses with
TurboSequest as implemented in the Bioworks Browser 3.2, build 41
(ThermoFinnigan) (Gallaher, Wu et al. 2006).
Western Blot Analysis
Equal amounts of mitochondrial protein (20 µg/well) were loaded in each
well of a 10% SDS-PAGE gel, electrophoresed with a Tris/glycine running buffer,
and transferred to a polyvinylidine difluoride membrane (PVDF). The blots were
probed with anti-COXIV (1:1000; MitoSciences; Eugene, OR), anti-Peroxiredoxin-V
(1:500; BD Biosciences, San Jose, CA), anti-Vdac1 (1:500; MitoSciences; Eugene
OR), anti-Crmp1 (1:1000; Exalpha Biologicals, Maynard, MA) and a HRP-
conjugated horse anti-mouse secondary antibody (Vector; Burlingame, CA) as
appropriate. Antigen-antibody complexes were visualized with the SuperSignal West
Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL). Band intensities were
determined using the ChemiDoc XRS Gel Documentation System and Quantity One
Software (Bio-Rad).
Respiratory Measurements
Mitochondrial oxygen consumption was measured polygraphically using a
Clarke-type electrode. 100 µg of isolated mitochondria was placed in the respiration
chamber at 37ºC in respiratory buffer (25 mM sucrose, 75 mM mannitol, 5 mM
KH2PO4, 100 mM KCl, 0.05 mM EDTA, 20 mM HEPES, 5 mM MgCl2, freshly
24
added 0.5% BSA, protease inhibitor (Calbiochem), pH 7.4 with KOH) to yield a
final concentration of 200 µg/mL. Following 1 min baseline recording, mitochondria
were energized by the addition of glutamate (5 mM) and malate (5 mM) as
substrates. State 3 respiration was stimulated by the addition of ADP (410 µM).
State 4o respiration was induced by the addition of the adenine nucleotide
translocator (ANT) inhibitor atractyloside (50 µM) to deplete ADP. The rate of
oxygen consumption was calculated based on the slope of the response of isolated
mitochondria to the successive administration of substrates. The Respiratory Control
Ratio (RCR) was determined by dividing the rate of oxygen consumption/min for
state 3 (presence of ADP) by the rate of oxygen consumption/min for state 4o
respiration (absence of ADP by addition of atractyloside).
Complex IV/Cytochrome c Oxidase Activity
Cytochrome c oxidase activity was measured spectrophometrically by
monitoring change in absorbance (550nm) of reduced cytochrome C by
permeabilized mitochondria. Mitochondria were permeabilized in 0.2 mL of 75 mM
potassium phosphate buffer (pH 7.5) at 25ºC. The reaction was started by the
addition of 0.05 mL of 5% cytochrome c previously reduced with sodium
hydrosulfite. Cytochrome c oxidase (COX) activity was calculated in nanomoles of
oxidized cytochrome c per minute per mg protein and reported as rate relative to the
mean rate from vehicle control-treated animals.
25
RNA Isolation and RT-PCR
Total RNA was isolated from rat brain tissue with TRIzol reagent
(Invitrogen, Carlsbad, CA). Expression of mRNA for cytochrome c oxidase subunits
I, II, III and IV (COXI, COXII, COXIII and COXIV) were assessed by SYBRGreen
based real time RT-PCR. cDNA was synthesized from 10 µg total RNA by reverse
transcription using SuperScriptII Reverse Transcription kit (Invitrogen, Carlsbad,
CA). cDNA was amplified by PCR on a iCycler (Bio-Rad, Hercules, CA) using Bio-
Rad iScript SYBR Green reaction buffer.
Statistics
Statistically significant differences were determined by Student’s t-test.
Results
Prior to 2D gel electrophoresis (2DE) the purity of the mitochondrial prep
was assessed by Western blot analysis for expression of subcellular markers in crude
homogenate, crude mitochondria, and highly purified mitochondria after density
gradient centrifugation. The results of this analysis demonstrated that the Percoll
purified mitochondria was not immunoreactive for the nuclear (histone H1),
cytosolic (myelin basic protein), or endoplasmic reticulum (calnexin) markers (Fig.
1).
26
Figure II-1. Mitochondrial purity assessment
Figure II-1. Mitochondrial purity assessment
The purity of the mitochondrial sample was assessed by Western blot analysis for
expression of subcellular markers in crude homogenate, crude mitochondria, and
highly purified mitochondria after density gradient centrifugation. Antibodies raised
against subcellular markers were used to detect mitochondrial anti-porin, nuclear
anti-histone H1, endoplasmic reticulum anti-calnexin, and myelin basic protein. The
same membrane was stripped with stripping buffer containing beta-mercaptoethanol
and reprobed with subsequent antibodies.
To facilitate quantitative detection and to maximize identification of changes
in the proteome induced by 17 β-estradiol (E
2
) treatment, isolated whole brain
mitochondria were subjected to a 2D-gel electrophoresis LC-MS/MS proteomic
analysis (Fig. 2). The mitoproteome is highly complex, with proteins encoded from
both mitochondrial and genomic DNA that undergo post-transcriptional and post-
translational modifications, creating a challenge for proteomic analyses. Further,
many of the proteins are membrane bound or transiently translocated to
mitochondria. One limitation of 2-D gels is that only proteins absorbed into the first
dimension gel are detectable, which precludes profiling of all mitochondrial proteins
27
and thus is limited to those soluble in the first dimension gel. Since it was not
feasible to sequence all resolved proteins, the selection criteria for sequence was
based on gel image quantitative analysis of the 2D analysis with the goal of
obtaining a broad scope of the proteins regulated and unregulated by E
2
-treatment.
Figure II-2. Schematic overview of experimental design
Figure II-2. Schematic overview of experimental design
Young adult female rats were injected with E
2
or vehicle control 2 weeks following
ovariectomy. 24 hr later whole brain mitochondria were isolated and subjected to 2-
D electrophoresis. PD Quest software was used to match spots and identify
expression patterns. Select protein spots were excised and sequenced by LC-MS/MS.
28
Of the 499 protein spots detected by gel image analyses, a total of 66 protein
spots were met the criterion for a two-fold or greater change in expression relative to
control. Of these, 28 proteins were increased in expression following E
2
treatment as
measured via fluorescent dye protein staining and imaging analyses. Conversely, 38
spots in the E
2
group were identified as having decreased expression relative to
control. For identification by LC-MS/MS peptide sequencing, 37 spots were selected
from three categories: 1) E
2
-induced increased (18 spots), 2) E
2
-induced decreased (9
spots) and 3) no change in response to E
2
(10 spots) (Fig. 3; Table 1). Of these
proteins, several were identified as redundant and several spots did not result in
successful protein identification. LC-MS/MS and Mascot database matching resulted
in successful identification of 29 different proteins listed in Table 1. Of the detected
proteins, nearly every protein identified localizes to mitochondria, thus providing
further validation to the purity of isolated mitochondria used in these experiments.
As a negative control, we identified a sampling of proteins unaffected by E
2
-
treatment and performed MS analysis on 10 of the conserved spots, yielding the
identity of 5 mitochondrial proteins unaffected by E
2
treatment (Table 1). Overall,
the mitoproteome profile of E
2
treated brain encompassed multiple functional
categories.
29
Figure II-3. 2-D analysis of brain mitochondrial proteome
Figure II-3. 2-D analysis of brain mitochondrial proteome
Isolated mitochondria from control (top left) and E
2
-treated (top right) rats were
analyzed by 2-D gel electrophoresis and spots were identified and matched (lower
left) with PD Quest software. Following protein identification, selected protein spots
were annotated (lower right) for gel comparisons.
30
Table II-1. Mitochondrial Proteome Identification
31
17 β-Estradiol-Induced Regulation of Mitochondrial Proteins
The majority of the identified changes occurred in mitochondrial proteins that
regulate cellular energetics represented by the tricarboxylic acid (TCA) cycle and the
electron transport chain (Fig. 4). Identified proteins involved in cellular energetics
that were increased included 3 of the 4 subunits that comprise the multimeric
pyruvate dehydrogenase complex, 2 enzymes of the TCA cycle, 2 oxidative
phosphorylation Complex I subunits and 2 ATP synthase F1 subunits. All three
major components of PDH (lipoamide beta (E1 component), dihyrolipoamide S-
acetyltransferase (E2 component), and dihydrolipoamide dehydrogenase (E3
component)) were increased by E
2
-treatment (Fig. 4; Table 1). As an indicator of the
selectivity of E
2
regulation of the mitochondrial proteome, several identified proteins
were unchanged by E
2
including the voltage dependent anion channel protein 1
(Vdac1/porin) (Fig. 5B; Table 1). Further implicating a regulatory role of E
2
in
mitochondrial energetics, the expression of the TCA cycle enzymes aconitase and
malate dehydrogenase (MDH) were observed to be up- and down-regulated,
respectively, by E
2
treatment (Fig. 4; Table 1). The increase in expression of a
characteristic cluster of aconitase spots was found to be significant by quantitating
the spot densities over 3 gels per condition (Fig. 5A; p<0.05).
In addition to the PDH complex and the TCA cycle enzymes, electron
transport chain (ETC) enzymes were regulated by E
2
(Fig. 4; Table 1). NADH
dehydrogenase (ubiquinone) Fe-S protein-2 (Ndufs2) and NADH dehydrogenase 1-
alpha subcomplex 10-like protein (Ndufs10) decreased in abundance, whereas
32
NADH dehydrogenase Fe-S protein-1 (Ndufs1) and protein-8 (Ndufs8) were both
unaltered (Table 1). Further, of the subunits that comprise the F1 component of
Complex V (ATP synthase), both ATP synthase H+ transporting F1-alpha and ATP
synthase F1-beta were increased with more than two-fold change in expression by E
2
treatment (Table 1). The E
2
inducible increase in F1-alpha and beta was specific for
these subunits as the F1-gamma subunit remained unchanged (Table 1). Several
proteins on the gel were identified as redundant ATP synthase subunits although
many were found at uncharacteristic migration distances and isoelectric points. Other
researchers have encountered the same phenomenon, particularly for the highly
abundant ATP synthase F1-alpha, by 2-D gel electrophoresis (Yang, Juranville et al.
2005).
Brain mitoproteome changes in response to E
2
were not limited to proteins
involved in bioenergetics. Changes in glutamate metabolism, ROS defense, and
chaperone and structural proteins were also detected (Fig. 4; Table 1). In the
glutamate metabolic pathway, glutamate oxaloacetate transaminase-2 and glutamate
dehydrogenase expression were both elevated in E
2
rat brain mitochondria (Fig. 4;
Table 1). Other proteins of this metabolic pathway were not identified in the current
analysis, thus it remains unknown whether they were coordinately regulated along
with these two proteins.
Of the numerous proteins regulating the oxidative balance of the
mitochondria, peroxiredoxin-V and manganese superoxide dismutase (MnSOD)
were identified in the 2D gel (Fig 4). E
2
induced a significant increase in
33
peroxiredoxin-V to 2.5 fold relative expression, as determined by quantitating the
spot densities over 3 gels per condition (Fig. 5A; p<0.05). In contrast, expression of
MnSOD, the other antioxidant enzyme identified in this analysis, was unaffected by
E
2
-treatment (Fig 4; Table 1). The changes in peroxiredoxin-V expression were
subsequently verified by Western blot analyses of isolated mitochondria (Fig. 5C).
E
2
treatment altered expression of three chaperone and structural proteins
(Fig. 4). Two spots revealed an increase in Hsp60 with more than two-fold change in
expression, whereas one spot indicated decreased Hsp70 levels following E
2
exposure (Fig. 4; Table 1). The current analysis demonstrated that E
2
-treatment
induced a decreased mitochondrial expression of collapsin response mediator
protein-1 (Crmp1), a member of a family of cytosolic phosphoproteins expressed
exclusively in the nervous system (NCBI Entrez Gene; Gene ID: 1400).
The remainder of the identified changes occurred in four proteins with
differing functions. E
2
increased isovaleryl CoA dehydrogenase and decreased 3-
hydroxyisobutyryl-Coenzyme A hydrolase (Fig. 4; Table 1). The implications of
changes in these two proteins associated with valine catabolism and two other
proteins – stomatin (decreased), and coiled-coil-helix-coiled-coil-helix domain
containing protein (decreased), represent novel E
2
mechanistic avenues to pursue.
Very few proteins identified indicated extramitochondrial contamination. Only two
proteins - brain abundant membrane attached signal protein-1 and myelin basic
protein, were found to be likely contaminants and both were found in equal
quantities between treatment groups (Table 1).
34
To validate the expression changes identified by the 2-DE LC-MS/MS
analysis, we utilized Western blot analysis to assess the expression of three identified
proteins. The proteins for confirmation included one upregulated, one
downregulated, and one unchanged to represent the three expression patterns
identified. Consistent with the 2-DE analysis (Fig. 5B), we demonstrated an
increased expression of peroxiredoxin-V, decreased expression of Crmp1, and no
change in expression of porin/Vdac1 (Fig. 5C).
35
Figure II-4. Comparison of protein expression in 2D analysis
Figure II-4. Comparison of protein expression in 2-D analysis
Individual protein spots are displayed in an expanded view for comparison of
treatment groups. Subsets of identified spots are grouped by function category to
represent protein expression patterns in the brain mitoproteome in response to E
2
treatment.
36
Figure II-5. Quantitative analysis of brain mitoproteome expression patterns
Figure II-5. Quantitative analysis of brain mitoproteome expression
A. Highly consistent protein spots were selected to quantitate expression changes.
The characteristic spots representing aconitase (top) and peroxiredoxin-V (bottom)
were identified in 2-D gels from 3 separate animals for densitometric measurements.
Bar graphs represent mean +/- S.E.M. (*=p<0.05 as compared to control; n=3). B.
Peroxiredoxin-V, Vdac1 and Crmp1 were selected to represent upregulated,
unchanged and downregulated proteins, respectively, for confirmation of 2-DE
expression analysis. C. Whole brain mitochondria from control and E
2
-treated rats
37
were assessed for protein expression by Western blot analysis (n=4; *=p<0.05 as
compared to control).
17 β-Estradiol-Induced Mitochondrial Functional Responses
To confirm that the above-identified changes in protein expression were
indicative of changes in functional activity, we conducted corresponding functional
analyses. As many of the proteins altered by E
2
-treatment are involved in regulation
of cellular energetics, we assessed the respiratory activity representative of the
electron transport chain (ETC).
We first determined the respiratory rate of isolated whole brain mitochondria
using glutamate (5 mM) and malate (5 mM) as respiratory substrates. ADP addition
to the mitochondrial suspension initiated state 3 respiration. Addition of the adenine
nucleotide transporter inhibitor, atractyloside, reduced the rate of O
2
consumption to
that of state 4o respiration, limited by proton permeability of the inner membrane. In
vivo treatment with E
2
resulted in a significant increase (35.6%) in the RCR of
isolated brain mitochondria (Fig. 6; Table 2; p<0.05 as compared to control; n=6).
There was a significant increase in rate of state 3 respiration and no significant
difference in the rate of state 4o respiration in the E
2
group (Fig. 6; Table 2). These
data indicate an increased efficiency of mitochondrial respiration rather than an
alteration in the coupling of the electron transport chain.
38
Figure II-6. E2 enhances mitochondrial respiratory activity.
Figure II-6. 17 β -estradiol enhances mitochondrial respiratory activity
Oxygen electrode measurements of respiration using isolated rat brain mitochondria.
A. Representative traces of mitochondrial oxygen consumption ± in vivo 17 β -
estradiol (E
2
, 30 µg/kg) or sesame oil vehicle control treatment in the presence of L-
malate (5 mM), L-glutamate (5 mM), ADP (410 µM) to initiate state 3 respiration,
and atractyloside to induce state 4o respiration. The traces are representative of six
separate experiments. B. State 3 and State 4o respiration of mitochondria from
control and E
2
-treated rats. The data represents mean ± S.E.M. of six separate
experiments (*=p<0.05 as compared to control; n=6). C. Percent changes in
mitochondrial respiratory control ratio (state 3/state 4o). The data represents mean ±
S.E.M. of six separate experiments (*=p<0.05 as compared to control; n=6).
39
The accelerated movement of electrons down the electron transport chain
would be expected to alter the activity of the terminal complex, cytochrome c
oxidase (COX; Complex IV). To test this hypothesis, we examined the enzymatic
activity of COX. Brain mitochondria isolated from E
2
-treated rats displayed a
significant 1.5-fold activity of COX (Fig. 7B; p<0.01 as compared to control; n=6).
The increased COX activity could be due to modulation of enzymatic activity or to
an alteration in expression levels of COX holoenzyme proteins. To determine if E
2
-
induced a change in COX subunit expression, we assessed the expression of COX
mRNA and protein by real time RT-PCR and Western blot analysis, respectively. As
the proteomic analysis indicated that E
2
regulated both nuclear and mitochondrial
encoded gene products, we assessed the expression of both mitochondrial and
nuclear encoded COX subunit mRNAs. The mitochondrially encoded subunits
COXI, COXII and COXIII were all significantly upregulated to ~3-fold relative
expression in the E
2
-treatment group (Fig. 7A; *=p<0.05 as compared to control;
n=6). Likewise the expression of the nuclear encoded subunit COXIV was
significantly increased to ~2.5-fold relative expression in the E
2
-treatment group
(Fig. 7A; *=p<0.05 as compared to control; n=6). Consistent with the increased
COXIV mRNA expression, E
2
-induced a significant increase in COXIV protein
expression (Fig. 7B; *=p<0.05 compared to control; n=6).
40
Figure II-7. 17 β-estradiol enhances
cytochrome c oxidase activity and
expression.
Figure II-7. 17 β-estradiol enhances cytochrome c oxidase activity and
expression
A. Relative expression of COXI-IV mRNA. Total RNA was isolated from brain
following 24 hr exposure to 17 β -estradiol (E
2
, 30 µg/kg) or sesame oil vehicle
control. Expression of COXI, COXII, COXIII and COXIV mRNA was assessed by
real-time RT-PCR. Bars represent mean relative expression ± S.E.M. from six
animals per group (*=p<0.05 as compared to control; n=6). B. Relative expression of
COXIV protein. Tissue homogenates were isolated from brain following 24 hr
exposure to 17 β-estradiol (E
2
, 30 µg/kg) or sesame oil vehicle control. Expression of
COXIV protein was assessed by Western blot analysis. Bars represent mean relative
expression ± S.E.M. from six animals per group (*=p<0.05 as compared to control;
n=6). C. Relative rate of cytochrome c oxidase activity of isolated whole brain
mitochondria ± in vivo 17 β-estradiol (E
2
, 30 µg/kg) or sesame oil vehicle control
treatment. The bars represent mean ± S.E.M. from 3 separate experiments with 2
animals per group for each experiment (*=p<0.05 as compared to control;
**=p<0.01 as compared to control; n=6).
41
Discussion
We investigated the impact of E
2
on the mitoproteome in brain using 2-D
electrophoresis with subsequent protein identification by mass spectrometry.
Identified changes in mitochondrial protein expression were then correlated with
biochemical analyses of mitochondrial enzymatic function. Based on our criteria of a
≥ two-fold change in expression, the results indicated that E
2
significantly regulated
key domains of cellular energetics, metabolism, free radical maintenance, and stress
responses.
E
2
regulation of cellular energetic pathways was evidenced by increased
expression of multiple subunits of the PDH enzyme complex. PDH is a key
regulatory enzyme linking the glycolytic metabolism to the tricarboxylic acid (TCA)
cycle by transforming pyruvate into acetyl CoA, which can, in turn, be utilized as a
substrate for the TCA cycle. In brain, PDH is further responsible for directing acetyl
CoA to either the TCA cycle or to acetylcholine synthesis (Holmquist, Stuchbury et
al. 2006). The mitoproteome profile induced by E
2
is reflective of enhanced
glycolytic activity (increased aconitase and decreased MDH) coupled with increased
glutamatergic turnover (increased glutamate dehydrogenase and glutamate
oxaloacetate transaminase-2). The latter can impact generation of neurotoxic free
ammonium, as well as direct reduction of excitotoxic free glutamate (Parihar and
Brewer 2007). Consistent with this finding, E
2
has been reported to increase activity
of the key glycolytic enzymes hexokinase, phosphofructokinase, and
phosphoglycerate kinase in rodent brain (Kostanyan and Nazaryan 1992). Together,
42
these findings indicate that E
2
promotes enhanced utilization of glucose, the main
energy source for the brain.
Oxidative phosphorylation and proteins within the electron transport chain
were increased in expression and activity, a result that was consistent with increases
in the glycolytic metabolic pathway. E
2
induced significant increases in both protein
expression and activity of Complex IV subunits I-IV, a finding consistent with
previous reports (Bettini and Maggi 1992; Stirone, Duckles et al. 2005). This E
2
-
induced increase is particularly relevant given that reduction in Complex IV is an
early marker of Alzheimer’s (Lin and Beal 2006). E
2
also increased expression of
ATP synthase F1 subunits alpha and beta, which is consistent with our previous
report of estrogen-induced increases in ATP levels in primary neuronal cultures
(Brinton, Chen et al. 2000). F0 subunits of ATP synthase were not identified, a
finding expected due to limitations of 2D gel resolution of integral membrane
proteins. In contrast to the increase in Complex IV and V subunit expression, E
2
induced downregulation of two subunits of Complex I, Ndufs2 and Ndufs10.
Complex I is composed of ~ 60 proteins and down regulation of two subunits with
no change in the two other Complex I subunits identified in our analysis represents a
small proportion of the large enzyme complex and the functional significance
remains undetermined. The decrease in Complex I subunits notwithstanding, E
2
induced regulation of proteins composing the ETC corresponds to shifts in
mitochondrial function. Increased mitochondrial respiration coupled with increased
43
glycolytic balance is reflective of an enhanced energetic efficiency of brain
mitochondria derived from E
2
treated rats.
E
2
-induced enhancement of energetic efficiency is further strengthened by the
changes in the free radical defense systems identified in the current proteomic
analysis. Increased expression of peroxiredoxin-V is consistent with the well-
documented antioxidant effects of estrogens (Behl, Widmann et al. 1995; Ejima,
Nanri et al. 1999; Urata, Ihara et al. 2006), including increased glutaredoxin
expression (Ejima, Nanri et al. 1999; Diwakar, Kenchappa et al. 2006; Urata, Ihara et
al. 2006). In contrast to the reported E
2
-induced increase in expression of MnSOD
(Pejic, Kasapovic et al. 2003; Strehlow, Rotter et al. 2003), we did not observe
significant changes in the MnSOD protein spot identified in the current 2D
proteomic analysis. This may be due to multiple distributions within the gel due to
posttranslational modifications or to selective regulation by E
2
during brain
development (Pejic, Kasapovic et al. 2003) or selective E
2
regulation within the
vasculature (Strehlow, Rotter et al. 2003). Selective regulation of different
antioxidant proteins could indicate protection against different oxidative stresses.
Free radical balance is maintained by reduction of the superoxide anion to hydrogen
peroxidase by superoxide dimutases. The resulting hydrogen peroxide can then be
neutralized by various peroxidases, including peroxiredoxin-V (Banmeyer,
Marchand et al. 2005). Reduction in reactive oxygen species contributes to
neuroprotection and can reduce the overall stress response. In this context we
identified significant alterations in the expression of two mitochondrial heat shock
44
proteins, Hsp70 and Hsp60, which are important in the proper import of nascent
proteins to the mitochondrial matrix.
E
2
regulated both mitochondrial and nuclear encoded gene products,
requiring coordinated control of mitochondrial and nuclear encoded gene
transcription. Estrogen receptors have been detected in mitochondria (Chen,
Delannoy et al. 2004; Yang, Liu et al. 2004; Stirone, Duckles et al. 2005; Yager and
Chen 2007) as well as in the nucleus of neurons (McEwen, Akama et al. 2001). In
addition to classical ERs, membrane sites of estrogen action (mER), which activate
the PI3K/PKC/Src/MEK/ERK signaling pathway, activating CREB, have been
identified as required for E
2
-inducible neuroprotection (Zhao, Wu et al. 2004; Wu,
Lenchik et al. 2005; Mannella and Brinton 2006). While the mechanisms whereby
ERs coordinate the complex signaling pathway between three signaling
compartments, membrane, mitochondria, and nucleus, remains to be determined, it is
striking that ERs are perfectly positioned to coordinate events at the membrane with
events in the mitochondria and nucleus. Coordinated action of E
2
and activation of
ERs leads to regulation of mitochondrial function and ultimately neural defense and
survival.
Overall, the identified mitoproteome profile induced by E
2
in the rodent brain
reflects a mitochondrial state that could act as a buffer against mitochondrial
functional decline. E
2
increased expression of key components of the PDH complex
and aconitase while simultaneously decreasing MDH. This would be expected to
result in enhanced coupling between glycolysis and the TCA cycle combined with
45
decreased MDH diverting excess malate outside the mitochondria thereby preventing
oxaloacetate buildup. In the cytosol, malate is converted to pyruvate by cytosolic
malic enzyme, which re-enters the TCA cycle by PDH conversion to Acetyl CoA.
We propose that this enzymatic buffer includes a shift towards enhanced glycolytic
energy production and a reduction in β-oxidation.
In conclusion, within 24 hours of exposure in vivo, E
2
significantly shifted
the mitochondrial proteome to a profile consistent with a glycolytic driven TCA
cycle: increased oxidative phosphorylation, increased ATP synthase, and decreased
beta-oxidation. The mitoproteome profile induced by E
2
was validated by enhanced
functional efficiency of the brain mitochondria. Collectively, these data provide a
plausible mechanistic rationale for estrogen therapy reduction in risk of Alzheimer’s
disease and suggest that the E
2
-inducible changes in the mitoproteome or
mitochondrial function could serve as potential biomarkers of therapeutic efficacy.
Further, E
2
-induced regulation of mitochondrial protein expression and function
provides insights into targets and mechanistic strategies to prevent neurodegenerative
diseases.
46
Chapter II References
Banmeyer I, Marchand C, Clippe A, Knoops B (2005) Human mitochondrial
peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen
peroxide. FEBS Lett 579:2327-2333.
Behl C, Widmann M, Trapp T, Holsboer F (1995) 17-beta estradiol protects neurons
from oxidative stress-induced cell death in vitro. Biochem Biophys Res Commun
216:473-482.
Bettini E, Maggi A (1992) Estrogen induction of cytochrome c oxidase subunit III in
rat hippocampus. J Neurochem 58:1923-1929.
Bishop J, Simpkins JW (1995) Estradiol enhances brain glucose uptake in
ovariectomized rats. Brain Res Bull 36:315-320.
Brewer GJ, Reichensperger JD, Brinton RD (2006) Prevention of age-related
dysregulation of calcium dynamics by estrogen in neurons. Neurobiol Aging 27:306-
317.
Brinton RD (2001) Cellular and molecular mechanisms of estrogen regulation of
memory function and neuroprotection against Alzheimer's disease: recent insights
and remaining challenges. Learn Mem 8:121-133.
Brinton RD (2004) Impact of estrogen therapy on Alzheimer's disease: a fork in the
road? CNS Drugs 18:405-422.
Brinton RD (2005) Investigative Models for Determining Hormone Therapy-Induced
Outcomes in Brain: Evidence in Support of a Healthy Cell Bias of Estrogen Action.
Ann NY Acad Sci 1052:57-74.
Brinton RD, Chen S, Montoya M, Hsieh D, Minaya J, Kim J, Chu HP (2000) The
women's health initiative estrogen replacement therapy is neurotrophic and
neuroprotective. Neurobiol Aging 21:475-496.
Cadenas E, Davies KJ (2000) Mitochondrial free radical generation, oxidative stress,
and aging. Free Radic Biol Med 29:222-230.
Chen JQ, Delannoy M, Cooke C, Yager JD (2004) Mitochondrial localization of
ERalpha and ERbeta in human MCF7 cells. Am J Physiol Endocrinol Metab
286:E1011-1022.
47
Chen S, Nilsen J, Brinton RD (2006) Dose and temporal pattern of estrogen exposure
determines neuroprotective outcome in hippocampal neurons: therapeutic
implications. Endocrinology 147:5303-5313.
Diwakar L, Kenchappa RS, Annepu J, Saeed U, Sujanitha R, Ravindranath V (2006)
Down-regulation of glutaredoxin by estrogen receptor antagonist renders female
mice susceptible to excitatory amino acid mediated complex I inhibition in CNS.
Brain Res 1125:176-184.
Ejima K, Nanri H, Araki M, Uchida K, Kashimura M, Ikeda M (1999) 17beta-
estradiol induces protein thiol/disulfide oxidoreductases and protects cultured bovine
aortic endothelial cells from oxidative stress. Eur J Endocrinol 140:608-613.
Gallaher TK, Wu S, Webster P, Aguilera R (2006) Identification of biofilm proteins
in non-typeable Haemophilus Influenzae. BMC Microbiol 6:65.
Holmquist L, Stuchbury G, Berbaum K, Muscat S, Young S, Hager K, Engel J,
Munch G (2007) Lipoic acid as a novel treatment for Alzheimer's disease and related
dementias. Pharmacol Ther 113:154-164.
Kostanyan A, Nazaryan K (1992) Rat brain glycolysis regulation by estradiol-17
beta. Biochim Biophys Acta 1133:301-306.
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases. Nature 443:787-795.
Lopez MF, Kristal BS, Chernokalskaya E, Lazarev A, Shestopalov AI, Bogdanova
A, Robinson M (2000) High-throughput profiling of the mitochondrial proteome
using affinity fractionation and automation. Electrophoresis 21:3427-3440.
Mannella P, Brinton RD (2006) Estrogen receptor protein interaction with
phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and
extracellular signal-regulated kinase 1/2 in the same population of cortical neurons: a
unified mechanism of estrogen action. J Neurosci 26:9439-9447.
McEwen B (2002) Estrogen actions throughout the brain. Recent Prog Horm Res
57:357-384.
McEwen B, Akama K, Alves S, Brake WG, Bulloch K, Lee S, Li C, Yuen G, Milner
TA (2001) Tracking the estrogen receptor in neurons: implications for estrogen-
induced synapse formation. Proc Natl Acad Sci U S A 98:7093-7100.
48
Morrison JH, Brinton RD, Schmidt PJ, Gore AC (2006) Estrogen, menopause, and
the aging brain: how basic neuroscience can inform hormone therapy in women. J
Neurosci 26:10332-10348.
Murphy AN, Bredesen DE, Cortopassi G, Wang E, Fiskum G (1996) Bcl-2
potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc
Natl Acad Sci U S A 93:9893-9898.
Nicholls DG, Budd SL (2000) Mitochondria and neuronal survival. Physiol Rev
80:315-360.
Nilsen J, Brinton RD (2003) Mechanism of estrogen-mediated neuroprotection:
regulation of mitochondrial calcium and Bcl-2 expression. Proc Natl Acad Sci U S A
100:2842-2847.
Nilsen J, Brinton RD (2004) Mitochondria as therapeutic targets of estrogen action in
the central nervous system. Curr Drug Targets CNS Neurol Disord 3:297-313.
Nilsen J, Chen S, Irwin RW, Iwamoto S, Brinton RD (2006) Estrogen protects
neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial
proteins and function. BMC Neurosci 7:74.
Parihar MS, Brewer GJ (2007) Mitoenergetic failure in Alzheimer disease. Am J
Physiol Cell Physiol 292:C8-23.
Pejic S, Kasapovic J, Cvetkovic D, Pajovic SB (2003) The modulatory effect of
estradiol benzoate on superoxide dismutase activity in the developing rat brain. Braz
J Med Biol Res 36:579-586.
Schroeter H, Boyd CS, Ahmed R, Spencer JP, Duncan RF, Rice-Evans C, Cadenas E
(2003) c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria
function: new target proteins for JNK signalling in mitochondrion-dependent
apoptosis. Biochem J 372:359-369.
Simpkins JW, Dykens JA (2007) Mitochondrial mechanisms of estrogen
neuroprotection. Brain Res Rev.
Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ, Bodor
N, Day AL (1997) Estrogens may reduce mortality and ischemic damage caused by
middle cerebral artery occlusion in the female rat. J Neurosurg 87:724-730.
Singh M, Dykens JA, Simpkins JW (2006) Novel mechanisms for estrogen-induced
neuroprotection. Exp Biol Med (Maywood) 231:514-521.
49
Singh M, Meyer EM, Millard WJ, Simpkins JW (1994) Ovarian steroid deprivation
results in a reversible learning impairment and compromised cholinergic function in
female Sprague-Dawley rats. Brain Res 644:305-312.
Stirone C, Duckles SP, Krause DN, Procaccio V (2005) Estrogen increases
mitochondrial efficiency and reduces oxidative stress in cerebral blood vessels. Mol
Pharmacol 68:959-965.
Strehlow K, Rotter S, Wassmann S, Adam O, Grohe C, Laufs K, Bohm M, Nickenig
G (2003) Modulation of antioxidant enzyme expression and function by estrogen.
Circ Res 93:170-177.
Toran-Allerand CD (2000) Novel sites and mechanisms of oestrogen action in the
brain. Novartis Found Symp 230:56-69; discussion 69-73.
Urata Y, Ihara Y, Murata H, Goto S, Koji T, Yodoi J, Inoue S, Kondo T (2006)
17Beta-estradiol protects against oxidative stress-induced cell death through the
glutathione/glutaredoxin-dependent redox regulation of Akt in myocardiac H9c2
cells. J Biol Chem 281:13092-13102.
Wang JM, Irwin RW, Brinton RD (2006) Activation of estrogen receptor alpha
increases and estrogen receptor beta decreases apolipoprotein E expression in
hippocampus in vitro and in vivo. Proc Natl Acad Sci U S A 103:16983-16988.
Woolley CS (1999) Electrophysiological and cellular effects of estrogen on neuronal
function. Critical Reviews in Neurobiology 13:1-20.
Wu TW, Wang JM, Chen S, Brinton RD (2005) 17Beta-estradiol induced Ca2+
influx via L-type calcium channels activates the Src/ERK/cyclic-AMP response
element binding protein signal pathway and BCL-2 expression in rat hippocampal
neurons: a potential initiation mechanism for estrogen-induced neuroprotection.
Neuroscience 135:59-72.
Yager JD, Chen JQ (2007) Mitochondrial estrogen receptors--new insights into
specific functions. Trends Endocrinol Metab 18:89-91.
Yang JW, Juranville JF, Hoger H, Fountoulakis M, Lubec G (2005) Molecular
diversity of rat brain proteins as revealed by proteomic analysis. Mol Divers 9:385-
396.
Yang SH, Liu R, Perez EJ, Wen Y, Stevens SM, Jr., Valencia T, Brun-Zinkernagel
AM, Prokai L, Will Y, Dykens J, Koulen P, Simpkins JW (2004) Mitochondrial
localization of estrogen receptor beta. Proc Natl Acad Sci U S A 101:4130-4135.
50
Zhao L, Chen S, Wang J, Brinton RD (2004) 17Beta-estradiol induced nuclear and
dentritic Ca2+ rise and subsequent CREB activation in hippocampal neurons: A
potential initiation mechanism for estrogen neurotrophism. Neurosci.
51
Chapter III: Progesterone and Estrogen Regulate Oxidative
Metabolism in Brain Mitochondria
R.W. Irwin, J. Yao, R.T. Hamilton, E. Cadenas, R.D. Brinton, and J. Nilsen. 2008.
Published: Endocrinology. 2008 Feb 21; [Epub ahead of print]
Abstract
The ovarian hormones progesterone and estrogen have well-established
neurotrophic and neuroprotective effects supporting both reproductive function and
cognitive health. More recently, it has been recognized that these steroids also
regulate metabolic functions sustaining the energetic demands of this neuronal
activation. Underlying this metabolic control is an interpretation of signals from
diverse environmental sources integrated by receptor-mediated responses converging
upon mitochondrial function. In this study, to determine the effects of progesterone
(P4) and 17 β-estradiol (E
2
) on metabolic control via mitochondrial function,
ovariectomized rats were treated with P4, E
2
or E
2
+P4 and whole brain mitochondria
were isolated for functional assessment. Brain mitochondria from hormone-treated
rats displayed enhanced functional efficiency and increased metabolic rates. The
hormone-treated mitochondria exhibited increased respiratory function coupled to
increased expression and activity of the electron transport chain complex IV
(cytochrome c oxidase). This increase respiratory activity was coupled with a
decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a
systematic enhancement of brain mitochondrial efficiency. As such, ovarian
52
hormone replacement induces mitochondrial alterations in the central nervous system
supporting efficient and balanced bioenergetics reducing oxidative stress and
attenuating endogenous oxidative damage.
Introduction
The ovarian hormones estrogen and progesterone have well-established
neurotrophic effects supporting both reproductive function and cognitive health
(Nilsen and Brinton 2002; Nilsen and Brinton 2003; Morrison, Brinton et al. 2006).
More recently, it has been recognized that these steroids can regulate metabolic
functions sustaining the energetic demands of neuronal activation. Underlying this
metabolic control is an interpretation of signals from diverse environmental sources
integrated by receptor-mediated responses converging upon mitochondrial function.
Mitochondria are the primary energy sources of the cell that converts nutrients into
energy through cellular respiration via the electron transport chain.
Mitochondria consist of a tightly integrated functional network that regulates
the energy balance of the cell. The energy balance is required not only to regulate the
survival of the cell, but more importantly to regulate the many specialized
physiological functions of the cell. In the central nervous system, mitochondrial
energy balance is critical to establish membrane excitability and to execute the
complex processes of neurotransmission and plasticity. Neurons are highly
specialized cells requiring large amounts of ATP to maintain the plasma membrane
ionic gradients that allow for rapid propagation of neural signaling and
neurotransmission. Ionic transport processes, namely Na
+
/K
+
-ATPases and Ca
2+
-
53
ATPases, consume ~60-80% of ATP consumption in the CNS to maintain ionic
gradients allowing for neuronal excitability (Nicholls and Budd 2000). Thus, the
CNS has an immense energetic demand critically dependent upon mitochondrial
function and oxygen supply to support the generation of ATP by oxidative
phosphorylation.
Many of the components of this functional bioenergetic network are
vulnerable to oxidative stress that can impair cellular function or increase the chance
of cell death (Nicholls and Budd 2000; Cadenas 2004). Compromised mitochondrial
function has been linked to numerous diseases, including those of the metabolic,
cardiovascular and nervous systems. Many of these vulnerable components of
mitochondrial function are ovarian controlled as demonstrated by reported sex
differences or functional alterations in response to hormonal manipulation (Bettini
and Maggi 1992; Zhai, Eurell et al. 2000; Rodriguez-Cuenca, Pujol et al. 2002;
Borras, Sastre et al. 2003). It has been shown that the extended lifespan of female
mice is due in part to a reduced oxidative load that is ovarian controlled (Borras,
Sastre et al. 2003). Female rat brown adipose tissue mitochondria are larger and have
more cristae than males (Rodriguez-Cuenca, Pujol et al. 2002). In addition, there are
purported effects of estrogens on mitochondria function in various tissues, including
decreased mitochondrial respiratory activity as assessed by MTT reduction in
myocardium of ovariectomized rats (Zhai, Eurell et al. 2000). In the brain, the
cytochrome c oxidase subunit II (COXII) was identified as the single E
2
-induced
54
clone resulting from a differential screen of a cDNA library prepared rat
hippocampus (Bettini and Maggi 1992).
For the most part, estrogen regulation of mitochondrial function has been the
primary focus despite the fact that progesterone has important properties that can
enhance or degrade these modulatory pathways. In the current study, we investigated
the role of progesterone in regulating mitochondrial function in the central nervous
system, both alone and in conjunction with estradiol. We propose that in addition to
the well-documented protective effects, E
2
and P4 enhance energy production
through balanced respiration, maintaining better electron flow coupling and reduced
oxidative damage. To evaluate this hypothesis, we utilized the ovariectomized,
hormone replaced rat model. Our data reveal that E
2
and P4 potently enhance the
functional efficiency of whole brain mitochondria, as evidenced by increased
electron transport and reduced oxidative leak and damage.
Materials and Methods
Chemicals
All chemicals were from MP Biomed (Irvine, CA) unless otherwise noted. 17β-
estradiol and progesterone were obtained from Steraloids (Newport, RI). Steroids
were dissolved in ethanol and diluted in sesame oil with final ethanol concentration
<0.001%.
55
Animals
The use of animals for the study was approved by the Institutional Animal Care and
Use Committee (IACUC) at the University of Southern California (Protocol No.
10256). Young adult (3-6 month old) female ovariectomized Sprague-Dawley rats
purchased from Harlan (Indianapolis, Indiana) were housed under controlled
conditions of temperature (22
o
C), humidity and light (14h light, 10h dark) with water
and food available ad libitum. After at 2 weeks of habituation to the facilities and
surgery recovery (following ovariectomy), rats were injected subcutaneously with E
2
(30 µg/kg), P4 (30 µg/kg) or sesame oil vehicle control and fasted 24 hr prior to
sacrifice and forebrain dissection. The dose of E
2
(30 µg/kg body weight)
was chosen
as representative of a standard systemic E
2
therapy used clinically and in previous
studies, which ranges from 10 to 100
µg/kg. To confirm administration of steroid,
plasma and brain E
2
levels were analyzed by commercial ELISA (IBL-Hamburg;
Germany) following hexane:ethyl acetate extraction. The 30-µg/kg
dose produced E
2
levels in OVX rats of 42 pg/g in brain tissue
and 44 pg/ml in serum. Our previous in
vitro studies indicated that P4 was most neurally effective at the same concentration
as E
2
(Han, Williams et al. 2001). At time of sacrifice uteri were removed and
weighed to determine efficacy of estradiol treatment (Control=0.117+/-0.005 g;
E
2
=0.230+/-0.012 g; P4=0.133+/-0.024 g; E
2
P4=0.216+/-0.026 g wet weight). All
experiments were approved by the Institutional Animal Care and Use Committee.
56
Mitochondrial Isolation
Brain mitochondria were isolated from rats as previously described (Sanz, Caro et al.
2005). Rats were decapitated, and the whole brain minus the cerebellum was rapidly
removed, minced and homogenized at 4
o
C in mitochondrial isolation buffer (MIB:
pH 7.4, containing sucrose (320 mM), EDTA (1 mM), Tris-HCl (10 mM), and
Calbiochem’s Protease Inhibitor Cocktail Set I (AEBSF-HCl 500 μM, Aprotonin 150
nM, E-64 1 μM, EDTA disodium 500 μM, Leupeptin hemisulfate 1 μM). Single
brain homogenates were then centrifuged at 1500 x g for 5 min. The pellet was
resuspended in MIB, re-homogenized and centrifuged again at 1500 x g for 5 min.
The postnuclear supernatants from both centrifugations were combined and crude
mitochondrial were pelleted by centrifugation at 21,000 x g for 10 min. The resulting
mitochondrial pellet was resuspended in 15% Percoll made in MIB, layered over a
preformed 23%/40% Percoll discontinuous gradient and centrifuged at 31,000 x g for
10 min. The purified mitochondria were collected at the 23%/40% interface and
washed with 10 mL MIB by centrifugation at 16,700 x g for 13 min. The loose pellet
was collected and transferred to a microcentrifuge tube and washed in MIB by
centrifugation at 9,000 x g for 8 min. The resulting mitochondrial pellet was
resuspended in MIB to an approximate concentration of 1 mg/mL. The resulting
mitochondrial samples were used immediately for respiratory measurements or
stored at -70
o
C for later protein and enzymatic assays. During mitochondrial
purification, aliquots were collected and for confirmation of mitochondrial purity and
integrity by Western blot analysis for mitochondrial anti-porin (1:500; Mitosciences,
57
Eugene, OR), nuclear anti-histone H1 (1:250; Santa Cruz Biotechnology, Santa
Cruz, CA), endoplasmic reticulum anti-calnexin (1:2000; SPA 865, Stressgen, now a
subsidiary of Assay Designs, Ann Arbor, MI) and cytoplasmic anti-myelin basic
protein (1:500, clone 2, RDI, Concord, MA) (Data not shown).
Respiratory Measurements
Mitochondrial oxygen consumption was measured polarographically using a Clarke-
type electrode. 100 µg of isolated mitochondria was placed in the respiration
chamber at 37
o
C in respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES,
2 mM MgCl
2
, 1 mM EGTA) to yield a final concentration of 200 µg/mL. Following
1 min baseline recording, mitochondria were energized by the addition of glutamate
(5 mM) and malate (5 mM) as substrates. State 3 respiration was stimulated by the
addition of ADP (410 µM). State 4
o
respiration was induced by the addition of three
pulsesof the adenine nucleotide translocator (ANT) inhibitor atractyloside (50 µM)
to deplete ADP. The rate of oxygen consumption was calculated based on the slope
of the response of isolated mitochondria to the successive administration of
substrates. The Respiratory Control Ratio (RCR) was determined by dividing the rate
of oxygen consumption/min for state 3 (presence of ADP) by the rate of oxygen
consumption/min for state 4
o
respiration (absence of ADP by addition of
atractyloside).
58
Complex IV/Cytochrome C Oxidase Activity
Cytochrome oxidase activity was measured spectrophometrically by monitoring
change in absorbance (550nm) of reduced cytochrome C by permeabilized
mitochondria. Mitochondria were permeabilized in 0.2 mL of 75 mM potassium
phosphate buffer (pH 7.5) at 25
o
C. The reaction was started by the addition of 0.05
mL of 5% cytochrome c previously reduced with sodium hydrosulfite. Cytochrome c
oxidase activity was calculated in nanomoles of oxidized cytochrome c per minute
per mg protein and reported as rate relative to the mean rate from vehicle control-
treated animals.
Western Blot Analysis
Equal amounts of mitochondrial protein (10 μg/well) was loaded in each well of a
10% SDS-PAGE gel, electrophoresed with a Tris/glycine running buffer, and
transferred to a polyvinylidine difluoride membrane (PVDF). The blots will be
probed with anti-COXIV (1:1000; Mitosciences; Eugene, OR), anti-Complex V,
Subunit α (1:1000; Mitosciences), anti-MnSOD (1:500; Santa Cruz Biotechnologies,
Santa Cruz, CA) or anti-peroxiredoxin V (1:500; BD Biosciences, San Jose, CA) and
an HRP-conjugated horse anti-mouse secondary antibody (Vector; Burlingame, CA).
Antigen-antibody complexes were visualized with the TMB Substrate kit (Vector).
Band intensities were determined using the VersaDoc System (Biorad).
59
RNA Isolation and RT-PCR
Total RNA was isolated from brain tissue with TRIzol reagent. Expression of mRNA
for cytochrome c oxidase subunits I, II, III and IV (COXI, COXII, COXIII and
COXIV) were assessed by SYBRGreen based real time RT-PCR. cDNA was
synthesized from 10 µg total RNA by reverse transcription using SuperScriptII
Reverse Transcription kit (Invitrogen, Carlsbad, CA). cDNA was amplified by PCR
on a iCycler (BioRad, Hercules, CA) using BioRad iScript SYBR Green reaction
buffer.
Mitochondrial Biogenesis
Total DNA was isolated with TRIzol reagent and analyzed by real time PCR.
Mitochondrial biogenesis was estimated as the relative levels of beta actin to COXII
DNA.
Mitochondrial Peroxide Production
The rate of H
2
O
2
production by isolated mitochondria was determined by the
Amplex Red peroxidase assay.
Free Radical Leak
Free radical leak was determined as previously described (Lopez-Lluch, Hunt et al.
2006). H
2
O
2
production and O
2
consumption were measured in parallel in the same
brain mitochondria under similar experimental conditions. This allowed for the
calculation of the fraction of electrons out of sequence, which reduce O
2
to ROS at
60
the respiratory chain (the percent free radical leak) instead of reaching cytochrome
oxidase to reduce O2 to water. Since two electrons are needed to reduce 1 mole of O
2
to H
2
O
2
whereas four electrons are required to reduce 1 mole of O
2
to water, the
percent free radical leak was calculated as the rate of H
2
O
2
production divided by
two times the rate of O
2
consumption, and the result was multiplied by 100.
Lipid Peroxidation
Lipid peroxides in brain mitochondria were measured using the leucomethylene blue
assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm
absorbance after 1 hr incubation at RT. The aldehyde product or termination product
of lipid peroxidation in brain mitochondria was determined by measuring
thiobarbituric acid-reactive substances (TBARS). Brain mitochondria were mixed
with 0.15M phosphoric acid. After the addition of thiobarbituric acid, the reaction
mixture was heated to 100
o
C for 1 hr. After cooling and centrifugation, the formation
of TBARS was determined by the absorbance of the chromophore (pink dye) at 531
nm using 600 nm as the reference wavelength.
Statistics
Statistically significant differences were determined by one-way ANOVA followed
by Student-Neuman Keuls post hoc analysis.
61
Results
Progesterone and 17 β-estradiol enhanced brain mitochondrial
respiratory activity
To determine progesterone (P4) and 17β-estradiol (E
2
) regulation of brain
mitochondrial respiration, young adult female Sprague-Dawley rats were
ovariectomized to remove endogenous sources of ovarian hormones. The animals
were allowed to recover from surgery for 2 weeks to allow for clearance of residual
ovarian hormones and to equilibrate to the state of ovarian deprivation.
Ovariectomized rats were injected subcutaneously with 30 µg/kg of P4, 30 µg/kg of
E
2
, 30 µg/kg of each steroid or equivalent volume of sesame oil vehicle as a control.
Rats were sacrificed 24 hr later and whole brain mitochondria were isolated. We first
measured the respiratory rate of the isolated whole brain mitochondria using
glutamate (5 mM) and malate (5 mM) as respiratory substrates. ADP addition to the
mitochondrial suspension initiated state 3 respiration. Addition of the adenine
nucleotide transporter inhibitor, atractyloside, reduced the rate of O
2
consumption to
that of state 4
o
respiration, limited by proton permeability of the inner membrane.
There was a significant increase in the respiratory control ratio (RCR) in the P4- and
E
2
-treatment groups, but not in the E
2
/P4 group (Figure 1; Table 1). In vivo treatment
with P4 resulted in a 24.5% increase in the RCR of isolated brain mitochondria
(Table 1; Figure 1). In vivo treatment with E
2
resulted in a 13.4% increase in the
RCR of isolated brain mitochondria (Figure 1; Table 1). In contrast to the effects of
62
the two hormones alone, the co-administration of E
2
and P4 did not result in a
significant change in the RCR of isolated brain mitochondria (Figure 1; Table 1).
There was a non-significant increase in rate of state 3 respiration in the P4 and E
2
groups (Table 1). There was no difference in the rate of state 4
o
respiration in any
treatment group (Table 1). These data indicate an increased efficiency of
mitochondrial respiration rather than an alteration in the coupling of the electron
transport chain.
Figure III-1.
Progesterone and 17 β-
estradiol enhance
mitochondrial
respiratory activity.
Oxygen electrode
measurements of respiration
using isolated rat brain
mitochondria. A.
Representative traces of
mitochondrial oxygen
consumption ± in vivo 17β-
estradiol (E
2
, 30 µg/kg),
progesterone (P4, 30 µg/kg),
E
2
/P4 or sesame oil vehicle
control treatment in the
presence of L-malate (5
mM), L-glutamate (5 mM),
ADP (410 µM) to initiate
state 3 respiration, and
atractyloside to induce state
4
o
respiration. The traces are
representative of six to eight
separate experiments. B.
Percent changes in
mitochondrial respiratory control ratio (state 3/state 4
o
). The data represents mean ±
S.E.M. of eight separate experiments (*=p<0.05 as compared to control; n=8).
Figure III-1. Progesterone and estradiol enhance
RCR.
63
State 4
o
State 3 RCR
Control 4.56 +/- 0.53 33.42 +/- 2.91 7.85 +/- 0.69
Estradiol 5.21 +/- 0.57 43.94 +/- 3.50 8.90 +/- 0.71
Progesterone 4.54 +/- 0.70 40.16 +/- 4.18 9.77 +/- 0.92
E + P 4.30 +/- 0.51 32.63 +/- 2.77 8.16 +/- 0.76
Table III-1. Mitochondrial Respiration.
Oxygen electrode measurements of respiration using isolated rat brain mitochondria.
Enhanced cytochrome c oxidase activity and expression in hormone
replaced rats
The accelerated movement of electrons down the electron transport chain
would be expected to alter the activity of the terminal complex, cytochrome c
oxidase (COX; Complex IV). To test this hypothesis we examined the enzymatic
activity of COX. Brain mitochondria isolated from P4-treated rats displayed a
significant 1.55-fold increase in COX activity (p<0.01 as compared to control; n=4;
Fig. 2A). E
2
-treatment resulted in a significant 1.5-fold increase in brain
mitochondrial COX activity (p<0.01 as compared to control; n=8; Fig. 2A). In
contrast to the lack of effect of co-administration on rates of respiration, co-treatment
with E
2
and P4 significantly increased brain mitochondrial COX activity by 1.4-fold
(p<0.05 as compared to control; n=8).
64
The increased COX activity could be due to modulation of enzymatic activity
or to an alteration in expression levels of COX holoenzyme proteins. To determine if
there was an alteration in COX subunit expression, we assessed mRNA expression
by real time RT-PCR. As it is unknown whether P4 or E
2
regulation of mitochondrial
function occurs at a nuclear or mitochondrial site of origin, we assessed the
expression of both mitochondrial and nuclear encoded COX subunit mRNAs. The
mitochondria encoded subunits COXI, COXII and COXIII were all significantly
upregulated by ~3-fold in the P4- and E
2
-treatment groups (Fig. 2B; *=p<0.05 as
compared to control; n=8). Likewise the expression of the nuclear encoded subunit
COXIV was significantly increased by ~2.5-fold in the P4- and E
2
-treatment groups
(Fig. 2B; *=p<0.05 as compared to control; n=8). The co-administration of P4 and E
2
resulted in a significant increase in the expression of COXII (~2-fold), COXIII
(~1.5-fold) and COXIV (~1.5-fold) (Fig. 2B; p<0.05 as compared to control; n=8).
65
Figure III-2. P4 and E2 enhance COX activity and
expression.
Figure III-2. Progesterone and 17 β-estradiol enhance cytochrome c
oxidase activity and expression.
A. Relative rate of cytochrome c oxidase activity of isolated whole brain
mitochondria ± in vivo 17β-estradiol (E
2
, 30 µg/kg), progesterone (P4, 30 µg/kg),
E
2
/P4 or sesame oil vehicle control treatment. The bars represent mean ± S.E.M.
from 4 separate experiments with 2 animals per group for each experiment
(*=p<0.05 as compared to control; **=p<0.01 as compared to control; n=8). B. Total
RNA was isolated from brain following 24 hr exposure to 17β-estradiol (E
2
, 30
µg/kg), progesterone (P4, 30 µg/kg), E
2
/P4 or sesame oil vehicle control. Expression
of (i) COXI, (ii) COXII, (iii) COXIII and (iv) COXIV mRNA was assessed by real-
time RT-PCR. Bars represent mean relative expression ± S.E.M. from eight animals
per group (*=p<0.05 as compared to control; n=8).
66
Enhanced Complex V expression in hormone replaced rats
The accelerated movement of electrons down the electron transport chain
would be expected to be coupled to increased ATP production. Increased ATP in
response to E
2
in vitro has previously been reported (Morrison, Brinton et al. 2006).
To determine if there was an increase in ATP synthase/Complex V to mediate the
accelerated electron flow and ATP production, we assessed protein expression of
Complex V, Subunit α by Western blot analysis. Complex V, Subunit α was
significantly increased by ~1.5 to 2 fold in the P4-, E
2
-, and E
2
/P4-treatment groups
(Fig. 3; *=p<0.05 as compared to control; n=4).
Figure III-3.
Progesterone
and 17 β-
estradiol
increase
expression of
Complex V
subunit α
protein.
Total protein
was isolated
from brain
following 24 hr
exposure to
17β-estradiol
(E
2
, 30 µg/kg),
progesterone
(P4, 30 µg/kg), E
2
/P4 or sesame oil vehicle control. Expression of Complex V,
Subunit α was determined by Western blot analysis. Bars represent mean relative
expression ± S.E.M. from four animals per group (*=p<0.05 as compared to control;
n=4).
Figure III-3. ATPsynthase complex V,
subunit alpha expression.
67
Mitochondrial biogenesis was unaffected by hormone replacement
Mitochondrial respiratory efficiency can be induced by increasing the number
of mitochondria (Nilsen and Brinton 2004). Thus the increase in respiratory rate,
COX activity and expression of COX subunits may have resulted from a global
increase in mitochondrial biogenesis. To test this hypothesis, we assessed the effect
of E
2
and P4 treatment on a marker of mitochondrial biogenesis, the ratio of
mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). Real time PCR for COXII
(mitochondrial) and beta actin (nuclear) was performed on total brain DNA. There
was no effect of P4, E
2
or E
2
/P4 treatment on the ratio of COXII:beta actin DNA
(Fig. 4). These data indicate that neither E
2
nor P4 affect the rate of mitochondrial
biogenesis.
Figure III-4. Mitochondrial biogenesis.
Figure III-4. Progesterone and 17 β-estradiol do not alter mitochondrial
biogenesis.
Total DNA was isolated from brain following 24 hr exposure to 17β-estradiol (E
2
, 30
µg/kg), progesterone (P4, 30 µg/kg), E
2
/P4 or sesame oil vehicle control. The
relative copy number of COXII (mitochondrial) and βactin (nuclear) DNA was
assessed by real-time PCR and the ratio of COXII/ βactin was used as a marker of
relative mitochondrial number. Bars represent mean ± S.E.M. from eight animals per
group (*=p<0.05 as compared to control; n=8).
68
Effects of progesterone and 17 β-estradiol on reactive oxygen species
production by brain mitochondria
The increased number of electron flowing through the electron transport
chain represented by the observed increased respiratory activity could result in
increased free radical production due to the increased probability of electron leak.
However, the increased activity of the terminal complex, COX, would be expected to
deplete the electron-rich intermediaries such as ubisemiquinone, decreasing the
incidental one-electron reduction of oxygen. Thus, we hypothesized that P4 and E
2
would reduce mitochondrial ROS production. Fluorescent Amplex Red
measurements of H
2
O
2
were made with isolated brain mitochondria exposed to
additions malate/glutamate plus ADP (state 3). P4-treatment resulted in a significant
decrease in the rate of H
2
O
2
production by isolated brain mitochondria (Fig. 5A;
p<0.05 as compared to control; n=8). In contrast, there no significant effect on H
2
O
2
production by isolated brain mitochondria in the E
2
-treated group (Fig. 5A; n=8).
The co-administration of E
2
and P4 resulted in a significant decrease in the rate of
H
2
O
2
production by state 3 mitochondria (Fig. 5A; p<0.05 as compared to control;
n=8). The efficiency of mitochondria can also be assessed by determining the rate of
free radical leak, or the percent electron flow that reduces oxygen to ROS instead of
reducing O
2
to water at COX. The free radical leak with malate/glutamate plus ADP
(state 3) was significantly lower in the P4 (~33%), E
2
(~11%) and E
2
/P4 (~12%)
groups than in the control group (Fig. 5B; p<0.05 as compared to control; n=8).
69
Figure III-5. Ovarian hormones reduce rate of peroxide
production and free radical leak in brain mitochondria.
Figure III-5. Ovarian hormones reduce rate of peroxide production and
free radical leak in brain mitochondria.
A. Fluorescent Amplex Red measurements of H
2
O
2
production in isolated rat brain
mitochondria exposed in vivo to ovarian hormones. Spectrofluorometer
measurements of H
2
O
2
production by isolated brain mitochondria in the presence of
malate (5 mM) and glutamate (5 mM) plus ADP (410 µM) to initiate state 3
respiration. Values represent relative mean H
2
O
2
production rates ± S.E.M. of eight
separate experiments (*=p<0.05 as compared to control; n=8). B. The percent free
radical leak was calculated for isolated whole brain mitochondria following in vivo
17β-estradiol (E
2
, 30 µg/kg), progesterone (P4, 30 µg/kg), E
2
/P4 or sesame oil
vehicle control treatment. Free radical leak was calculated as the percentage of H
2
O
2
production to twice the rate of O
2
consumption. The bars represent mean ± S.E.M.
from 8 animals per group (*=p<0.05 as compared to control; **=p<0.01 as compared
to control; n=8).
70
Progesterone and 17 β-estradiol reduced lipid peroxidation of brain
mitochondria
If the leakage of free radicals is reduced in mitochondria exposed to P4 and
E
2
, we reasoned that the oxidative damage to mitochondrial membranes should also
be reduced. Results of these analyses indicated that brain mitochondrial lipid
peroxidation was significantly lower in ovarian hormone replaced rats than in vehicle
control treated rats (Fig. 6). When assessed as lipid peroxides, there was a reduction
in mitochondrial lipid peroxidation of 13.8%, 43.7% and 22.5% in P4, E
2
and E
2
/P4
groups, respectively (Fig. 6; n=4; p<0.05 as compared to control). When assessed, as
the aldehyde product there was a reduction in TBARS of 25%, 41.7% and 37.5% in
P4, E
2
and E
2
/P4 groups, respectively (Fig. 6; n=4; p<0.05 as compared to control).
71
Figure III-6. Progesterone and 17 β-estradiol
decrease lipid peroxidation of brain mitochondria.
Figure III-6. Progesterone and 17 β-estradiol reduce lipid peroxidation of
brain mitochondria.
Whole brain mitochondria were isolated 24 hr following in vivo exposure to 17β-
estradiol (E
2
, 30 µg/kg), progesterone (P4, 30 µg/kg), E
2
/P4 or sesame oil vehicle
control treatment. Lipid peroxides in brain mitochondria were measured using the
leucomethylene blue assay or by measuring TBARS (data not shown). The bars
represent mean ± S.E.M. from 8 animals per group (*=p<0.05 as compared to
control; **=p<0.01 as compared to control; n=8).
72
Figure III-7. Progesterone and 17 β-estradiol
alter the antioxidant profile of brain mitochondria.
Figure III-7. Progesterone and 17 β-estradiol alter the antioxidant profile
of brain mitochondria.
Whole brain mitochondria were isolated 24 hr following in vivo exposure to 17β-
estradiol (E
2
, 30 µg/kg), progesterone (P4, 30 µg/kg), E
2
/P4 or sesame oil vehicle
control treatment. Expression of the mitochondrial antioxidant proteins (A) MnSOD
and (B) Peroxiredoxin V were measured using Western blot analysis. The bars
represent mean ± S.E.M. from 4 animals per group (*=p<0.05 as compared to
control; **=p<0.01 as compared to control; n=4).
73
Progesterone and 17 β-estradiol alter the antioxidant profile of brain
mitochondria
Even though we observed effects on H
2
O
2
production in only the P4-
treatment group, and not the E
2
-treatment group (Fig. 5), there was a pronounced
reduction in lipid peroxidation in the E
2
-treatment group (Fig. 6). This would
indicate activation of mechanisms beyond solely mitochondrial efficiency in
response to E
2
that may not be activated in response to P4. Thus we determined the
impact of P4 and E
2
on the protein expression of the mitochondrial antioxidant
proteins MnSOD and peroxiredoxin V (Fig. 7). There was a significant increase in
MnSOD expression of ~1.4-fold in the P4, E
2
and E
2
/P4 groups (Fig. 7; n=4; p<0.05
as compared to control). In contrast, the expression of peroxiredoxin V was only
increased in the E
2
-treatment group (Fig. 7; n=4; p<0.05 as compared to control), but
not in the P4- and E
2
/P4-treatment groups (Fig. 7; n=4). These results fit with the
above observations on lipid peroxidation, in that MnSOD is responsible for
generating H
2
O
2
from the superoxide anion and peroxiredoxin V is involved in
clearance of H
2
O
2
and prevention of peroxidative damage.
Discussion
The ovarian hormones, estradiol and progesterone, play significant
neuromodulatory and neuroprotective roles, influencing synaptogenesis, cerebral
blood flow and neuronal survival. These multiple actions during development and
74
aging regulate not only organismal reproductive physiology, but also regulate
cognitive health (Nilsen and Brinton 2002; Nilsen and Brinton 2002). More recently,
it has been recognized that E
2
also regulates the metabolic functions sustaining the
energetic demands of its neurotrophic effects. Underlying this metabolic control is an
interpretation of signals from diverse environmental sources integrated by receptor-
mediated responses converging upon mitochondrial function (Sato, Teramoto et al.
2003). In this study, we investigated the impact of P4 and E
2
on key mitochondrial
functions, oxidative respiration and free radical generation. Results of these analyses
indicate that both E
2
and P4 significantly increased mitochondrial respiration 24 hrs
following a single in vivo exposure. Consistent with an increase in oxidative
respiration, P4 and E
2
significantly increased COXIV enzyme activity and
expression of COXIV mRNA. Remarkably, both E
2
and P4 reduced free radical leak
indicating greater efficiency of electron transport. Consistent with reduced
generation of free radicals, P4 and E
2
induced a significant reduction in
mitochondrial lipid peroxidation. The data further indicate that P4 and E
2
directly
regulate mitochondrial function and that the effects are not due to an increase in the
number of mitochondria as neither P4 nor E
2
nor their combination induced evidence
for mitochondrial biogenesis. The mechanisms underlying this response are as yet
unknown. Although the serum steroid levels measured at 24 hr fall within the
physiological range, it is known that steroid levels following a single injection are
much higher at earlier time points. However, such ‘spiking’ is also observed with
other delivery methods including oral administration and the systemic dosing in this
75
study are comparable to the systemic doses observed for common oral administration
of 1-2 mg/day of micronized 17β-estradiol. Together, these data indicate that the
gonadal hormones, P4 and E
2
, are potent regulators of mitochondrial function to
increase both the magnitude and efficiency of mitochondrial respiration in brain.
Surprising to us was the efficacy of P4 and the lack of synergy when P4 and
E
2
were administered in combination. On all outcome measures, the combination of
P4 and E
2
resulted in a substantial decrement in response magnitude. Further, the
data indicate that the combination of P4 and E
2
is not synergistic and when
administered in combination leads to a lower level of response relative to either
gonadal hormone alone. Although the concentration of P4 was not sufficient to
inhibit E
2
-induced uterine proliferation, using the currently available data is not
possible to discern the mechanism for the antagonism on mitochondrial respiration
and future studies will be required to determine if it is an effect related to dosage or
competitive mechanisms of the two compounds. Regardless of the outcome on
mitochondrial respiration, there was a profound effect in the co-administration
groups on free radical leak and oxidative damage that coincides with the
neuroprotective ability of E
2
/P4 co-administration that we have previously reported
(Brinton, Chen et al. 2000). Another point of note is that the effects of the combined
treatment were not consistent across all outcome measures. In some outcomes, as in
mitochondrial respiration, there was no effect of the combined treatment relative to
control and in others, as in COX activity and expression, the combined treatment
significantly altered the response relative to control. Although this may be due to
76
methodological limitations, in the face of the different composite mitochondrial
outcome profile between E
2
and P4, it is more suggestive of alternative mechanisms
of steroidal regulation of mitochondrial function. Thus, the neuroprotective abilities
of E
2
and P4 do not involve all aspects of mitochondrial bioenergetics and the two
steroids probably act through two different sets of overlapping sets of mechanism.
These different mechanisms may act synergistically or antagonistically resulting in
the mixed profile of outcomes.
The primary function of mitochondria is to produce the ATP required for
cellular bioenergetics, which in the neuron includes the maintenance of membrane
potentials crucial for proper signaling and information processing. The effects of
ovariectomy in Sprague-Dawley rats have been characterized as progressive memory
deficits, central cholinergic nerve system degeneration and homeostatic imbalance
(Campian, Qian et al. 2004). These effects are a characteristic consequence of
neuronal ATP impairment, resulting from the loss of ovarian hormones. Further,
implicating the role of mitochondrial ATP production in the neuronal effects of E
2
,
we have previously demonstrated that estrogen-treatment of primary hippocampal
neuronal cultures results in increased ATP production (Campian, Qian et al. 2004). It
is unknown whether P4 exerts similar effects, but would be expected based upon the
increased mitochondrial functionality demonstrated in the current study.
Mitochondrial ATP production occurs through the coupling of oxidative
phosphorylation with respiration. The electron transport chain (ETC) is composed of
four enzyme complexes: NADH dehydrogenase (complex I), succinate
77
dehydrogenase (complex II), ubiquinol-cytochrome c reductase (complex III) and
cytochrome c oxidase (complex IV). The enzymatic complexes of the ETC act in
concert to couple the reduction of oxygen to water (respiration) with the generation
of a chemiosmotic gradient across the mitochondrial inner membrane, the latter of
which is utilized by ATP synthase to generate ATP. The coupled respiration occurs
by the sequential transfer of electrons through the enzymatic complex, terminating at
complex IV (COX) with the reduction of oxygen to water. Besides being the
terminal ETC complex, regulation of COX holds special prominence due to its
implication as a key regulatory point of oxidative phosphorylation (Bettini and
Maggi 1992; Puerta, Rocha et al. 1998; Stirone, Duckles et al. 2005). Although it
was earlier thought that COX activity was in excess of other components of the
electron transport chain, more recent experiments indicate that COX is in fact much
more tightly coupled to mitochondrial respiration (Jung, Agarwal et al. 2007). In the
current study, we demonstrated that complex IV activity is significantly increased in
response to ovarian hormone replacement. These findings are consistent with
reported results of other groups indicating increased COX activity with E
2
treatment
in various tissues (Abramson, Svensson-Ek et al. 2001), including brain in which E
2
-
treatment prevented ethanol withdrawal-induced declines in COX activity (Bettini
and Maggi 1992). Our study is the first that we know of to examine the COX activity
in the central nervous system in response to estradiol and progesterone.
The regulation of COX is of further interest from a transcriptional vantage
point due to its being encoded by a combination of mitochondrial and nuclear genes
78
that must be coordinately expressed. The catalytic core of the multimeric COX
holoenzyme comprises the two subunits COXI and COXII and is stabilized by a third
subunit, COXIII, all three of which are encoded by the mitochondrial DNA
(mtDNA). Ten additional subunits, including COXIV, are encoded by the nuclear
genome and are required for full assembly and function of the COX holoenzyme
(Zheng and Ramirez 2000). The increased COX activity can largely be explained by
the increased expression of COX subunits. Corresponding to the responses in COX
activity, we saw robust changes in COX expression in the E
2
and P4 alone groups,
with more modest increases in the E
2
/P4 co-administration group. These results are
consistent with the previously reported increases in COX III mRNA expression
(Keller, Germeyer et al. 1997; Zheng and Ramirez 1999; Massart, Paolini et al.
2002). It should also be noted that there was much more variability in the COX
transcript expression levels than was observed for COX holoenzyme activity. It is
unknown whether this was due to relative sensitivities of the assay methodologies or
if it represented unexamined regulatory control, including allosteric modulation of
COX activity or ancillary protein expression, which allowed for tighter modulation
of enzymatic activity relative to gene transcription.
In addition to oxidative enzymes, other mitochondrial proteins that regulate
mitochondrial respiration and ATP production may be under control of E
2
and P4.
Transport of substrates needed for oxidative phosphorylation, such as pyruvate, fatty
acids and phosphate may be regulated by P4 or E
2
, either through transcriptional
control or allosteric modulation. Some authors have suggested that E
2
can bind
79
directly to and modulate the activity of ATPase. It was demonstrated that E
2
-BSA
conjugates bind in vitro the oligomycin-sensitivity conferring protein (OSCP), which
forms the stalk region between F
0
and F
1
subunits of F
0
F
1
-ATPase in mitochondria
(Justo, Boada et al. 2005). Further in vitro studies demonstrated that estrogens can
enhance or inhibit ATPase activity in a tissue- and concentration dependent manner
(Van Itallie and Dannies 1988). It is unknown what effect in vivo exposure to E
2
or
P4 has on ATPase activity in brain or whether the alterations in respiratory activity,
ATP levels and oxidative stress are dependent upon a direct interaction of the
steroids with the OSCP.
Changes in the oxidative capacity are often due to differences in
mitochondrial number, size and density per cell as well as to changes in
mitochondrial components and/or activity. The mtDNA content, which can be
utilized as a suitable marker of mitochondrial number, was not different between any
of the treatment groups, the increased mitochondrial activity observed is not
explained by an increase in the number of mitochondria. Although, an effect of
mitochondrial size cannot be ruled out based on the current data, the greater
oxidative capacity induced by ovarian hormones could be linked to a greater
machinery per mitochondrion and therefore to higher efficiency of the individual
mitochondria. This lack of mitochondrial biogenesis is consistent with greater
oxidative capacity in female rat liver mitochondria without a sex difference in the
number of liver mitochondria (Calabrese, Scapagnini et al. 2001; Barja 2004; Reddy
2006). Further, the findings of Van Itallie and Dannies demonstrated an E
2
-induced
80
increase in COXII mRNA in pituitary tumor cells with no increase in COXII gene
copy number (Behl, Widmann et al. 1995; Shea and Ortiz 2003; Kii, Adachi et al.
2005). We observed an equivalent increase in the expression of both mitochondrial
and nuclear encoded COX subunits. Due to the tightly coordinated regulation of
mtDNA and nDNA encoded mitochondrial genes, it is not possible to identify
whether the hormones act at a nuclear site regulating COXIV that in turn signals for
increased COXI, II and III expression, or vice versa, or if the hormones act at both
sites. More detailed experiments are required to dissect the site of action.
Oxidative damage to mitochondria is posited to play a major role in aging
and in neuronal populations may underlie cognitive declines associated with aging
(Subramanian, Pusphendran et al. 1993). As electrons pass through the mitochondrial
ETC, some electrons leak out to molecular oxygen (O
2
) to form O
2
•-
,
which is
dismutated by manganese superoxide dismutase (MnSOD) to form H
2
O
2
. The
hydrogen peroxide can in turn be reduced to water by peroxidases such as
glutathione peroxidase or peroxiredoxins. A lack of sufficient peroxidase activity
results in the peroxidation of lipids and proteins.
Lipid peroxidation, the nonspecific
oxidation of polyunsaturated fatty acids in cellular membranes, is a radical-mediated
pathway and generates a number of harmful degradation products besides drastically
altering the structure and function of the membrane. Our results demonstrate that
ovarian hormone treatment reduced lipid peroxidation of whole brain mitochondria.
This result is consistent with a previous reports indicating that E
2
(Gridley, Green et
al. 1998; Nakamizo, Urushitani et al. 2000) and P4 (Nicotera, Leist et al. 2000) can
81
individually reduce oxidative stress in the brain. Here, we extend these findings by
demonstrating that the prevention of the lipid peroxidation is present at the level of
the mitochondrial membrane and is correlated with enhanced mitochondrial
functional efficiency represented by the lower values of free radical leak. The lower
levels of lipid peroxidation in the E
2
-treatment group in the absence of a change in
the rate of H
2
O
2
production are indicative of an enhanced cellular antioxidant
defenses. This is consistent with reports of E
2
-induced increases in expression of
MnSOD and interactions with the glutathione system (Nicholls and Budd 2000).
This E
2
- and P4-induced reduction in lipid peroxidation is due to a reduction the lipid
peroxidation-induced by the 2-week steroid deprivation following ovariectomy in
which we observed a ~3.3-fold induction in lipid peroxidation in the OVX compared
to sham OVX rats (data not shown).
Respiratory control is one facet of an interlocking network regulating
metabolic activity and energy production in which mitochondrial redox potential is
coupled with cytosolic signaling. This tightly coupled network integrates various
signals to determine cell fate based upon ATP levels, extracellular signals, and
energetic demands. The response of a cell to a death signal depends upon energetic
status and redox balance. Energetic status is crucial for full realization of the
apoptotic cascade as many of the steps involved are ATP dependent (Nilsen, Chen et
al. 2006). Redox balance can alter the sensitivity of the mitochondria to permeability
transition (Irwin, Yao et al. 2008). As such, efficient respiratory control can affect
not only energy production, but also response to toxic insults. In addition to the
82
defensive effects of E
2
and P4, including regulation of the Bcl-2 family proteins
(Han, Williams et al. 2001) the increased respiratory control efficiency and
decreased oxidative load demonstrated in this study could establish a buffer against
neuronal functional decline associated with aging and neurodegenerative diseases.
In summary, we have demonstrated that P4 and E
2
increase the oxidative
capacity of whole brain mitochondria. This increased respiratory activity is
correlated with decreased free radical leak and reduced lipid peroxidation. As such,
ovarian hormone replacement induces mitochondrial alterations in the central
nervous system supporting efficient and balanced bioenergetics reducing oxidative
stress and attenuating endogenous oxidative damage.
Acknowledgements
This study was supported by grants from the National Institute of Aging
(5PO1AG026572; Project 1 to J.N.), National Institutes of Mental Health (1RO1
MH67159-01 to R.D.B and J.N.), R.W.I. is supported by NIA training grant T32-
AG000093-24/25 (Finch, C.E., PI).
83
Chapter III References
Abramson, J., M. Svensson-Ek, et al. (2001). "Structure of cytochrome c oxidase: a
comparison of the bacterial and mitochondrial enzymes." Biochim Biophys
Acta 1544(1-2): 1-9.
Behl, C. (2002). "Oestrogen as a neuroprotective hormone." Nat Rev Neurosci 3(6):
433-42.
Bettini, E. and A. Maggi (1992). "Estrogen induction of cytochrome c oxidase
subunit III in rat hippocampus." J Neurochem 58(5): 1923-9.
Borras, C., J. Sastre, et al. (2003). "Mitochondria from females exhibit higher
antioxidant gene expression and lower oxidative damage than males." Free
Radic Biol Med 34(5): 546-52.
Brewer, G. J., J. D. Reichensperger, et al. (2005). "Prevention of age-related
dysregulation of calcium dynamics by estrogen in neurons." Neurobiol
Aging.
Brinton, R. D. (2001). "Cellular and molecular mechanisms of estrogen regulation of
memory function and neuroprotection against Alzheimer's disease: recent
insights and remaining challenges." Learn Mem 8(3): 121-33.
Brinton, R. D. (2005). "Investigative Models for Determining Hormone Therapy-
Induced Outcomes in Brain: Evidence in Support of a Healthy Cell Bias of
Estrogen Action." Ann NY Acad Sci 1052(1): 57-74.
Campian, J. L., M. Qian, et al. (2004). "Oxygen tolerance and coupling of
mitochondrial electron transport." J Biol Chem 279(45): 46580-7.
Han, D., E. Williams, et al. (2001). "Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space."
Biochem J 353(Pt 2): 411-6.
Irwin, R. W., J. Yao, et al. (2008). "Progesterone and Estrogen Regulate Oxidative
Metabolism in Brain Mitochondria." Endocrinology.
Jung, M. E., R. Agarwal, et al. (2007). Ethanol withdrawal posttranslationally
decreases the activity of cytochrome c oxidase in an estrogen reversible
manner. Neurosci. Lett. 416: 160-4.
84
Justo, R., J. Boada, et al. (2005). "Gender dimorphism in rat liver mitochondrial
oxidative metabolism and biogenesis." Am J Physiol Cell Physiol 289(2):
C372-8.
Kim, H., O. Y. Bang, et al. (2001). "Neuroprotective effects of estrogen against beta-
amyloid toxicity are mediated by estrogen receptors in cultured neuronal
cells." Neurosci Lett 302(1): 58-62.
Lopez-Lluch, G., N. Hunt, et al. (2006). "Calorie restriction induces mitochondrial
biogenesis and bioenergetic efficiency." Proc Natl Acad Sci U S A 103(6):
1768-73.
Mattson, M. P. and G. Kroemer (2003). "Mitochondria in cell death: novel targets for
neuroprotection and cardioprotection." Trends Mol Med 9(5): 196-205.
McEwen, B. S. and S. E. Alves (1999). "Estrogen actions in the central nervous
system." Endocr Rev 20(3): 279-307.
Milner, T. A., K. Ayoola, et al. (2005). "Ultrastructural localization of estrogen
receptor beta immunoreactivity in the rat hippocampal formation." J Comp
Neurol 491(2): 81-95.
Morrison, J. H., R. D. Brinton, et al. (2006). "Estrogen, menopause, and the aging
brain: how basic neuroscience can inform hormone therapy in women." J
Neurosci 26(41): 10332-48.
Nicholls, D. G. and S. L. Budd (2000). "Mitochondria and neuronal survival."
Physiol Rev 80(1): 315-60.
Nicotera, P., M. Leist, et al. (2000). "Energy requirement for caspase activation and
neuronal cell death." Brain Pathol 10(2): 276-82.
Nilsen, J. and R. D. Brinton (2002). "Impact of progestins on estradiol potentiation
of the glutamate calcium response." Neuroreport 13(6): 825-30.
Nilsen, J. and R. D. Brinton (2003). "Mechanism of estrogen-mediated
neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression."
Proc Natl Acad Sci U S A 100(5): 2842-7.
Nilsen, J. and R. D. Brinton (2004). "Mitochondria as therapeutic targets of estrogen
action in the central nervous system." Curr Drug Targets CNS Neurol Disord
3(4): 297-313.
85
Nilsen, J., S. Chen, et al. (2006). "Estrogen protects neuronal cells from amyloid
beta-induced apoptosis via regulation of mitochondrial proteins and
function." BMC Neurosci 7(1): 74.
Nilsen, J., R. W. Irwin, et al. (2007). "Estradiol in vivo regulation of brain
mitochondrial proteome." J Neurosci 27(51): 14069-77.
Oddo, S., A. Caccamo, et al. (2003). "Triple-transgenic model of Alzheimer's disease
with plaques and tangles: intracellular Abeta and synaptic dysfunction."
Neuron 39(3): 409-21.
Paech, K., P. Webb, et al. (1997). "Differential ligand activation of estrogen
receptors ERalpha and ERbeta at AP1 sites." Science 277(5331): 1508-10.
Price, R. H., Jr., N. Lorenzon, et al. (2000). "Differential expression of estrogen
receptor beta splice variants in rat brain: identification and characterization of
a novel variant missing exon 4." Brain Res Mol Brain Res 80(2): 260-8.
Resnick, S. M., E. J. Metter, et al. (1997). "Estrogen replacement therapy and
longitudinal decline in visual memory. A possible protective effect?"
Neurology 49(6): 1491-7.
Riddle, D. R. and M. E. Forbes (2005). "Regulation of cytochrome oxidase activity
in the rat forebrain throughout adulthood." Neurobiology of Aging 26(7):
1035-1050.
Sanz, A., P. Caro, et al. (2005). "Dietary restriction at old age lowers mitochondrial
oxygen radical production and leak at complex I and oxidative DNA damage
in rat brain." J Bioenerg Biomembr 37(2): 83-90.
Sato, T., T. Teramoto, et al. (2003). "Effects of ovariectomy and calcium deficiency
on learning and memory of eight-arm radial maze in middle-aged female
rats." Behav Brain Res 142(1-2): 207-16.
Sherwin, B. B. (1988). "Estrogen and/or androgen replacement therapy and cognitive
functioning in surgically menopausal women." Psychoneuroendocrinology
13(4): 345-57.
Stirone, C., S. P. Duckles, et al. (2005). "Estrogen increases mitochondrial efficiency
and reduces oxidative stress in cerebral blood vessels." Mol Pharmacol.
Subramanian, M., C. K. Pusphendran, et al. (1993). "Gestation confers temporary
resistance to peroxidation in the maternal rat brain." Neurosci Lett 155(2):
151-4.
86
Sun, J., M. J. Meyers, et al. (1999). "Novel ligands that function as selective
estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-
beta." Endocrinology 140(2): 800-4.
Turgeon, J. L., D. P. McDonnell, et al. (2004). "Hormone therapy: physiological
complexity belies therapeutic simplicity." Science 304(5675): 1269-73.
Van Itallie, C. M. and P. S. Dannies (1988). "Estrogen induces accumulation of the
mitochondrial ribonucleic acid for subunit II of cytochrome oxidase in
pituitary tumor cells." Mol Endocrinol 2(4): 332-7.
Weihua, Z., S. Andersson, et al. (2003). "Update on estrogen signaling." FEBS Lett
546(1): 17-24.
Yaffe, K., G. Sawaya, et al. (1998). "Estrogen Therapy in Postmenopausal Women:
Effects on Cognitive Function and Dementia." Jama 279(9): 688-695.
Yang, S. H., R. Liu, et al. (2004). "Mitochondrial localization of estrogen receptor
beta." Proc Natl Acad Sci U S A 101(12): 4130-5.
Zhai, P., T. E. Eurell, et al. (2000). "Effect of estrogen on global myocardial
ischemia-reperfusion injury in female rats." Am J Physiol Heart Circ Physiol
279(6): H2766-75.
Zhao, L., C. Jin, et al. (2007). "Design, synthesis, and estrogenic activity of a novel
estrogen receptor modulator--a hybrid structure of 17beta-estradiol and
vitamin E in hippocampal neurons." J Med Chem 50(18): 4471-81.
Zhao, L., K. O'Neill, et al. (2006). "Estrogenic agonist activity of ICI 182,780
(Faslodex) in hippocampal neurons: implications for basic science
understanding of estrogen signaling and development of estrogen modulators
with a dual therapeutic profile." J Pharmacol Exp Ther 319(3): 1124-32.
Zheng, J. and V. D. Ramirez (1999). "Purification and identification of an estrogen
binding protein from rat brain: oligomycin sensitivity-conferring protein
(OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase." J Steroid
Biochem Mol Biol 68(1-2): 65-75.
87
Chapter IV: Selective estrogen receptor modulators
potentiate brain mitochondrial function
Ronald W. Irwin, Jon Nilsen, Jia Yao, Ryan Hamilton, and Roberta Diaz
Brinton
Department of Pharmacology and Pharmaceutical Sciences, University of
Southern California, Los Angeles, CA, USA
Unpublished: In preparation to be submitted.
Abstract
We have previously shown that 17 β-estradiol (E
2
) regulates the mitoproteome
to potentiate the function of mitochondria isolated from whole brain (Nilsen, Irwin,
et al. J. Neurosci 2007). The purpose of this study was to determine the role of the
ER subtypes in regulation of mitochondrial function using the ER subtype selective
agonists PPT (ER α) and DPN (ER β). Adult ovariectomized rats were treated with E
2
(30 µg/kg), PPT (30 µg/kg), DPN (100 µg/kg) or vehicle control 24 hr prior to
isolation of whole brain mitochondria by discontinuous Percoll density
centrifugation. A Clarke-type electrode was used to record mitochondrial oxygen
consumption. After 24 hours all estrogenic treatments significantly increased
mitochondrial respiration relative to vehicle control as measured by the respiratory
control ratio (RCR). Likewise, cytochrome c oxidase activity increased following all
estrogenic treatments. RT-PCR and Western blots of whole brain mitochondria were
performed for COX II (mtDNA) and COX IV (nucDNA) expression. Lipid
88
peroxides were significantly reduced by E
2
, PPT, and DPN. These findings suggest
that activation of both ER α and ER β enhance mitochondrial function in brain. Future
synthetic estrogens and phytoestrogens used in hormone therapies may be tailored to
improve mitochondrial endpoints. We are currently following up these investigations
in cell cultures and transgenic mouse models relevant to neurodegenerative diseases.
Introduction
The purpose of these studies is to elucidate fundamental cellular mechanisms
of cognitive function and neuron survival and to translate these discoveries into
therapeutics that sustain cognitive function during aging and to prevent
neurodegenerative disease. Results of these studies, along with those from our
colleagues, have demonstrated at the most basic cellular levels that E
2
is
neuroprotective against neurotoxic insults including exposure to glutamate and β-
amyloid. Mitochondrial dysfunction is associated with but not exclusive to
neurodegenerative diseases, ischemia-reperfusion injury in stroke and heart attack,
diabetes, and aging. We plan to extend current neuroprotective strategies by
demonstrating that selective estrogenic molecules promote cell survival mechanisms
governed by neuronal mitochondria.
Glucose is the primary energy source of the brain. Following anaerobic
glycolysis in the cytosol, acetyl CoA is converted into energy through the citric acid
cycle and oxidative phosphorylation to produce ATP. Acting as a buffer system,
mitochondria are also able to protect cells from triggering death pathways (Nilsen
and Brinton, 2003; Nilsen and Brinton, 2004). E
2
has been shown to stimulate Bcl-2
89
expression (Nilsen and Brinton, 2003; Zhao et al., 2004). Bcl-2 expression
potentiates the intramitochondrial calcium capacity (Murphy et al., 1996; Nilsen and
Brinton, 2003).
Mitochondria have their own genome that contains the genetic information
for 13 essential enzyme subunits of the approximately 100 required for
mitochondrial respiration (about 10%). The rest of the estimated 1500 (low estimate
500) proteins that reside in the mitochondria are imported from the nucleus. E
2
has
been shown to regulate several critical mitochondrial components including
cytochrome c oxidase protein expression either directly or indirectly (Riddle and
Forbes 2005).
Activation of either or both estrogen receptors (ERs) was found to be
neuroprotective (Zhao et al., 2004). It has been proposed that a mitochondrial
estrogen receptor exists (Yang et al., 2004). Some evidence suggests that the brain
mitochondria may harbor these receptors but the precise function and protein isoform
composition has not been determined. Since E
2
has equal affinity for both estrogen
receptor subtypes, ER α and ER β, selective estrogen receptor modulators (SERMs)
were designed by the Katzenellenbogen group to distinguish ER α-guided
mechanisms from those of ER β. PPT acts as a full agonist of ER α whereas DPN acts
as an agonist of ER β. These SERMs may help to distinguish the functions of ER α
and ER β in promoting mitochondrial viability. The purpose of this study was to
distinguish estrogen receptor initiated pathways in the brain that converge upon
mitochondrial function. We propose that in addition to well-established
90
neuroprotective benefits, both estrogen receptor subtypes contribute to mitochondrial
efficiency and relative reduction of oxidative stress. This hypothesis was evaluated
in the ovariectomized rat model. Our data reveal that E
2
, PPT, and DPN enhance the
functional efficiency of brain mitochondria, as evidenced by increased electron
transport control and reduced oxidative damage.
Selective Estrogen Receptor Modulators
(SERMS)
N
N
OH
HO
HO
H
3
C
PPT
ER-alpha agonist
CN
OH
HO
DPN
ER-beta agonist
OH
CH
3
HO
17 β-estradiol (E2)
Equal affinity
for ER α and ER β
Figure IV-1. Chemical structures of Estradiol, PPT, and
DPN.
Figure IV-1. Chemical structures of Estradiol (E
2
), ER α selective agonist
(PPT), and ER β selective agonist (DPN).
Materials and Methods
Chemicals
All chemicals were from MP Biomed (Irvine, CA) unless otherwise noted. 17 β-
estradiol was obtained from Steraloids (Newport, RI). PPT and DPN were obtained
91
from Tocris Bioscience (Bristol, UK). E
2
, PPT or DPN were dissolved in ethanol and
diluted in sesame oil with final ethanol concentration <0.001%.
Animals
The use of animals for the study was approved by the Institutional Animal Care and
Use Committee (IACUC) at the University of Southern California (Protocol No.
10256). Young adult Sprague-Dawley female rats (4-6 months old) purchased from
Harlan (Indianapolis, Indiana) were ovariectomized. Rats were housed under
controlled conditions of temperature (22
o
C), humidity and light (14h light, 10h dark)
with water and food available ad libitum. After two weeks of recovery, animals were
injected subcutaneously with 17 β-estradiol (30 mg/kg), PPT (30 mg/kg), DPN (100
mg/kg each), or with a sesame oil control. The dose of E
2
(30 µg/kg body weight)
was chosen as representative of a standard systemic E
2
therapy used clinically and in
previous studies, which ranges from 10 to 100
µg/kg. To confirm administration of
steroid, plasma and brain E
2
levels were analyzed by commercial ELISA (IBL-
Hamburg; Germany) following hexane:ethyl acetate extraction. The 30-µg/kg
dose
produced E
2
levels in OVX rats of 42 pg/g in brain tissue
and 44 pg/ml in serum. Our
previous in vitro studies indicated that PPT and DPN were able to protect neurons
from various insults at the same concentration as E
2
(Nilsen and Brinton 2003). At
time of sacrifice uteri were removed and weighed to determine efficacy of estradiol
treatment (Control=0.117+/-0.005 g; E
2
= 0.192+/-0.009 g; PPT=0.1459+/-0.009 g;
DPN=0.1434+/-0.011 g wet weight). All experiments were approved by the
Institutional Animal Care and Use Committee.
92
Mitochondrial Isolation
24 hours after E
2
, PPT, DPN, or control injections, animals were sacrificed and
whole brain tissue dissected. Mitochondria were then isolated through differential
centrifugation and Percoll gradients. Brain mitochondria were isolated from rats as
previously described (Nilsen and Brinton 2004). Rats were decapitated, and the
whole brain minus the cerebellum was rapidly removed, minced and homogenized at
4
o
C in mitochondrial isolation buffer (MIB: pH 7.4, containing sucrose (320 mM),
EDTA (1 mM), Tris-HCl (10 mM), and Calbiochem’s Protease Inhibitor Cocktail
Set I (AEBSF-HCl 500 μM, Aprotonin 150 nM, E-64 1 μM, EDTA disodium 500
μM, Leupeptin hemisulfate 1 μM). Single brain homogenates were then centrifuged
at 1500 x g for 5 min. The pellet was resuspended in MIB, re-homogenized and
centrifuged again at 1500 x g for 5 min. The postnuclear supernatants from both
centrifugations were combined and crude mitochondrial were pelleted by
centrifugation at 21,000 x g for 10 min. The resulting mitochondrial pellet was
resuspended in 15% Percoll made in MIB, layered over a preformed 23%/40%
Percoll discontinuous gradient and centrifuged at 31,000 x g for 10 min. The purified
mitochondria were collected at the 23%/40% interface and washed with 10 mL MIB
by centrifugation at 16,700 x g for 13 min. The loose pellet was collected and
transferred to a microcentrifuge tube and washed in MIB by centrifugation at 9,000 x
g for 8 min. The resulting mitochondrial pellet was resuspended in MIB to an
approximate concentration of 1 mg/mL. The resulting mitochondrial samples were
used immediately for respiratory measurements or stored at -70
o
C for later protein
93
and enzymatic assays. During mitochondrial purification, aliquots were collected and
for confirmation of mitochondrial purity and integrity by Western blot analysis for
mitochondrial anti-porin (1:500; Mitosciences, Eugene, OR), nuclear anti-histone H1
(1:250; Santa Cruz Biotechnology, Santa Cruz, CA), endoplasmic reticulum anti-
calnexin (1:2000; SPA 865, Stressgen, now a subsidiary of Assay Designs, Ann
Arbor, MI) and cytoplasmic anti-myelin basic protein (1:500, clone 2, RDI, Concord,
MA) (Data not shown).
Respiration Measurements
Mitochondrial oxygen consumption was measured polygraphically using a Clarke-
type electrode. 100 µg of isolated mitochondria was placed in the respiration
chamber at 37
o
C in respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES,
2 mM MgCl
2
, 1 mM EGTA) to yield a final concentration of 200 µg/ml followed by
the addition of glutamate (5mM) and malate (5mM) as substrates, and ADP (410
µM) in order to stimulate state 3 respiration. State 4o was measured after the
depletion of ADP by 50 μM pulses of atractyloside (adenine nucleotide tranporter
inhibitor), and the Respiratory Control Ratio (RCR) was determined as a ratio of
state 3 to state 4o respiration.
Lipid Peroxides
Lipid peroxides in brain mitochondria were measured using the leucomethylene blue
assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm
absorbance after 1 hr incubation at RT.
94
Cytochrome c Oxidase activity and expression
Cytochrome c oxidase (COX) activity was measured spectrophometrically by
monitoring change in absorbance (550nm) of reduced cytochrome c by
permeabilized mitochondria (Nilsen and Brinton 2003). Mitochondria were
permeabilized in 0.2 mL of 75 mM potassium phosphate buffer (pH 7.5) at 25
o
C.
The reaction was started by the addition of 0.05 mL of 5% cytochrome c previously
reduced with sodium hydrosulfite. Cytochrome c oxidase activity was calculated in
nanomoles of oxidized cytochrome c per minute per mg protein and reported as rate
relative to the mean rate from vehicle control-treated animals. Cytochrome c
Oxidase subunit IV and II protein was determined by Western blot (COXIV:
Molecular Probes, A-21348, 1:2000, COXII: Molecular Probes, A6404, 1:1000)
Mitochondrial Membrane Potential
JC-1 dye (3 μM) was added to hippocampal neurons from rat embyonic primary
culture after 10DIV in Neural Basal Medium. Cells were pretreated with vehicle, E
2
,
PPT, or DPN (10nM each condition) for 48 hr.
Mitobiogenesis measurement
Total DNA was extracted from cortex and hippocampus of treated OVX rats.
Relative copy number of mtDNA (COXII) and nDNA ( β-actin) were determined by
real time PCR. The ratio of mtDNA/nDNA was used as a marker of the relative rate
of mitochondrial biogenesis.
95
Estrogen Receptor protein expression
Western blot analysis of ER was performed using anti-ER β, Santa Cruz H-150
(1:1000) and anti-ER α, Novocastra 6F11 (1:50). Rat ovary homogenate was used as
a positive control.
2D Gel/Western
Mitochondria were isolated from whole brain of treated OVX rats by percoll density
centrifugation. 400 μg of mitochondrial protein were subjected to isoelectric
focusing on 17 cm pH 3-10 NL strips followed by SDS-PAGE through 12%
acrylamide gels. Following the second dimension PAGE, the proteins were
transfered to PVDF membranes and then subsequently immunoblotted to determine
the presence of ER α and ER β in the mitochondria.
Statistics
Statistically significant differences were determined by Student's t-test or one-way
ANOVA with Student Newman Keuls post hoc analysis.
Results
Respiration
Selective estrogen receptor agonists PPT (ER α) and DPN (ER β) significantly
increased several outcome measures related to mitochondrial function. Mitochondrial
respiration measured as the ratio of ADP stimulated State 3 to basal oxygen
96
consumption in the absence of ADP State 4. The respiratory control ratio, a measure
of mitochondrial oxygen consumption efficiency, was increased by all three
treatments compared to control. Due to limitations of the technique, it was not
feasible to run dose responses with this measurement. However, previous studies
both in vivo and in vitro indicate that these doses are within the range of comparison
to high physiological level of estradiol during the peak of the estrous cycle. PPT and
DPN both displayed comparable increases in RCR. We demonstrated that there is an
increase in the efficiency of mitochondrial respiration with E
2
, PPT, or DPN rather
than an alteration in the coupling of the electron transport chain.
**
*
*
**
*
*
Figure IV-2. RCR normalized to Control.
Figure IV-2. E
2
, PPT, and DPN increase mitochondrial respiration.
Oxygen consumption of mitochondria isolated from whole brain of E
2
(30 μg/kg),
PPT (30 μg/kg), DPN (100 μ/kg) OVX rats treated for 24h. Respiration was
measured in the presence of Complex I substrates glutamate (5 mM)/malate (5 mM).
State 3 respiration was initiated by the addition of ADP (410 mM). Respiratory
control ratio (RCR) was measured as a ratio of state 3/state 4 respiration. Bar chart
depicts RCR data with state 4 measured after addition of the ANT inhibitor,
atractyloside, normalized to control. (*p<0.05, **p<0.01, n=4).
97
0
0.05
0.1
0.15
0.2
CE2 PPT DPN
Uterine weight (g)
***
**
0
0.05
0.1
0.15
0.2
CE2 PPT DPN
Uterine weight (g)
***
**
Figure IV-3. Uterine weight (g) following 24 hour treatment in 4-6
month OVX rats.
Figure IV-3. Uterine weight to demonstrate proliferative effect of
estradiol 24 hrs post-treatment.
Whole uterine tissue wet weight (grams) was measured to confirm efficacy of
treatment. The expected increase in uterine weight was found for E
2
versus control.
PPT and DPN had a minimal but significant effect on uterine weight.
Lipid Peroxidation
Interestingly, decreased lipid peroxidation was observed in mitochondrial fractions
for each treatment condition. Activation of either estrogen receptor subtype ER α or
ER β was able to elicit a reduction of mitochondrial lipid peroxides. PPT most
potently reduced lipid peroxide levels suggesting that activation of ER α is involved
in the antioxidant process. E
2
with equal affinity for each receptor and DPN, an ER β
agonist, were also able to significantly reduce lipid peroxides (Control 0.746+/-0.866
μM/ μg; E
2
0.516+/-0.0587 μM/ μg; PPT 0.310+/-0. 0487 μM/ μg; DPN 0.601+/-0.115
μM/ μg protein) in purified brain mitochondrial samples from OVX rats treated 24
hours prior to tissue isolation.
98
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Ct E2 PPT DPN
LP uM/ug mito protein
***
***
***
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Ct E2 PPT DPN
LP uM/ug mito protein
***
***
***
Figure IV-4. Lipid peroxides are decreased by E
2
, PPT, and DPN.
Figure IV-4. Lipid Peroxides measured by Leucomethylene blue method
The lipid peroxides content of isolated mitochondria was measured. E
2
, PPT, and
DPN each had significantly decreased levels of lipid peroxides. ***p<0.0001, n=7.
Cytochrome Oxidase
The mitochondrial respiratory chain protein cytochrome oxidase (complex IV) was
assayed for activity; COX subunits IV and II, nuclear and mitochondrial DNA
encoded genes respectively, were found to be regulated by estrogen and SERMs in
vivo. COX activity was significantly increased across all treatments nearly equally.
All three treatments displayed a significant 1.55-fold increase in COX activity
(p<0.01, n=4) as compared to control. COX subunits IV and II were found to be
99
increased. E
2
had the greatest effect on COX IV expression. PPT likewise had the
greatest influence on COX II expression.
Figure IV-5. E2, PPT, and DPN increase cytochrome c oxidase (COX) activity
in mitochondria.
Figure IV-5. E
2
, PPT, and DPN increase cytochrome c oxidase (COX)
activity in mitochondria.
Isolated mitochondria from steroid treated rats were added to a reduced cytochrome
c solution (0.07%). Activity was measured spectrophotometrically (550nm), and
defined as mM of reduced cytochrome c substrate oxidized/ml/mg of mitochondria
(22oC, pH 7). Significant increases in activity were measured in treated samples
compared to control (**p<0.01, n=4).
100
Figure IV-6. E2, PPT, and DPN mediated
upregulation of COX IV and II protein.
Figure IV-6. E
2
, PPT, and DPN mediated upregulation of COX IV and II
protein.
Mitochondria were isolated from OVX rat brain following a 24h treatment. COX IV
(nuclear DNA encoded) and COX II (mtDNA encoded) protein expression measured
by Western blot (n=2).
Mitochondria membrane potential
The fluorescent dye JC-1 was used to assess membrane potential in primary cultured
hippocampal cells from embryonic rat brain. The dye has a characteristic fluorescent
shift from green to red due to aggregation of the dye in the mitochondria. The
positively charged dye accumulates in the negatively charged matrix of the
mitochondria. We observed an increase in mitochondrial membrane potential as
101
indicated by imaging. The JC-1 dye accumulated in E
2
(10nM) treated cells and PPT
(10nM) treated cells much more than the control and DPN treated cells. This
indicates a likely ER α mediated pathway and a mechanistic explanation for E
2
’s
ability to increase the mitochondrial buffering capacity (Oddo, Caccamo et al. 2003).
Control E2 PPT (ER α)DPN (ER β)
CY3 CY3 + FITC
100 μm
Control E2 PPT (ER α)DPN (ER β)
CY3 CY3 + FITC
Control E2 PPT (ER α)DPN (ER β)
CY3 CY3 + FITC
100 μm
Figure IV-7. Mitochondrial membrane potential increased by ER agonists.
Figure IV-7. Mitochondrial Membrane Potential Increased by Estrogen
Receptor Agonists.
JC-1 dye (3uM) is converted to red aggregate in mitochondria within minutes.
Hippo 10 DIV, 48h pretreatment.
Mitobiogenesis
E
2
effects on protein and gene expression were not likely due to mitochondrial
biogenesis events as measured by a standard method of PCR (mtDNA encoded
COXII gene/ nucDNA encoded β-actin). The difference in mitochondrial biogenesis
between control, E
2
, PPT, and DPN treated rats was insignificant (n=3).
102
Figure IV-8. Mitochondrial biogenesis is unaltered by E2, PPT, and DPN. Relative
levels of mtDNA/nDNA were measured by PCR for COXII/actin genes
respectively.
Figure IV-8. Lack of increased mitochondrial biogenesis in response to in
vivo estradiol treatment.
Adult OVX rats were treated with E
2
, PPT, DPN or vehicle control for 24 h. Total
DNA was isolated from cortex and hippocampus. Relative mtDNA (COX II) and
nDNA ( β-actin) were determined by real time PCR and the ratio of mtDNA/nDNA
was calculated. (n=3)
Estrogen Receptors in Mitochondria
Estrogen receptor expression and/or translocation to mitochondria may be altered by
SERMs as indicated by Western blot analysis of purified mitochondria. Fractions
were collected during the Percoll density ultracentrifugation procedure to assess
estrogen receptor localization within brain cells. Crude cytosol (without nuclei and
unbroken cells), crude mitochondria, and pure mitochondria fractions were
compared. ER α or ER β immunoreactivity remained in the sequential purification
steps. It was observed that the level of nonspecific binding decreased considerably
between each purification step while the level of estrogen receptor content did not
103
decrease. While these studies are not definitive, they do indicate that estrogen
receptors may be located in or associated with mitochondria in rat brain.
Figure IV-9A.
Ovary
DPN
PPT
E2
Control
OVX Rat Brain Mitochondria
ER α
67kD
59kD ER β1
61kD ER β2
Ovary
DPN
PPT
E2
Control
OVX Rat Brain Mitochondria
ER α
67kD
Ovary
DPN
PPT
E2
Control
OVX Rat Brain Mitochondria
ER α
67kD
59kD ER β1
61kD ER β2
59kD ER β1
61kD ER β2
Fig. IV-9B.
Figure IV-9. Estrogen receptors in purified mitochondria from OVX rat
forebrain.
A. Crude brain homogenate, crude mitochondrial pellet, and Percoll density gradient
purified mitochondrial fractions were tested for immunoreactivity to estrogen
receptors. B. Purified brain mitochondria from E
2
, PPT, DPN treated OVX rats. Rat
ovary homogenate used as positive control for estrogen receptors.
104
Figure IV-10. 2-D/Western blot of rat brain mitochondria.
ER β antibody was used to detect individual spots that correspond to single protein
species. Immunoreactivity was observed at the correct size and pI for receptor
subtype with ER β being more abundant and compared with positive control at
~55kD for rat ovary (rOv) lysate. Two circled spots represent ER β1 (~55kD) and
ER β1 (~61kD) isoforms. ER α (~67kD) data not shown.
rOv pH 3.0 rat brain mito. 100 μg pH 10.0
59kD
ER β
rOv pH 3.0 rat brain mito. 100 μg pH 10.0
59kD
ER β
rOv pH 3.0 rat brain mito. 100 μg pH 10.0
59kD
ER β
rOv pH 3.0 rat brain mito. 100 μg pH 10.0
59kD
ER β
Figure IV-10.
Discussion
This study was aimed at determining the nature of estradiol neuroprotection
in relation to mitochondrial function. Specifically, our goal was to determine the role
of estrogen receptor subtypes, ER α and ER β, in mitochondrial gene and protein
regulation. These basic findings were conducted primarily in ovariectomized rats and
primary cultured hippocampal neurons. Regulation of expression patterns of proteins
that reside in the mitochondria suggest a plausible mechanism for estradiol enhanced
respiration and calcium ion buffering capacity. E
2
actions are integrated by receptor-
105
mediated responses converging upon mitochondrial function (Nilsen and Brinton
2003; Nilsen and Brinton 2004; Nilsen, Chen et al. 2006; Nilsen, Irwin et al. 2007;
Irwin, Yao et al. 2008). Our future work will focus on developing therapeutics that
selectively regulate mitochondrial respiration and in turn the control of a primary
source of oxidants in healthy and neurodegenerative diseased states.
In this study, we investigated the impact of E
2
, PPT, and DPN on key
mitochondrial functions, oxidative respiration and free radical generation. Results of
these analyses indicate that E
2
, PPT, and DPN significantly increased mitochondrial
respiration 24 hrs following a single in vivo exposure. Consistent with an increase in
oxidative respiration, E
2
, PPT, and DPN significantly increased COXIV enzyme
activity and expression of COXIV and COX II protein. Consistent with control of
oxygen consumption efficiency, E
2
, PPT, and DPN induced a significant reduction in
mitochondrial lipid peroxidation. The data further indicate that E
2
regulates
mitochondrial function through estrogen receptor mediated pathways. Effects are not
due to an increase in the number of mitochondria evidenced by a marker of
mitochondrial biogenesis. Although the E
2
, PPT, and DPN levels after 24 hours are
within therapeutic range, it is conceded that the initial levels following a single
injection were much higher. However, spiking is also observed with other delivery
methods including oral administration and the systemic dosing in this study is
comparable to the systemic doses observed for common oral administration of 1-2
mg/day of micronized 17 β-estradiol. Together, these data indicate that the gonadal
hormone E
2
and its synthetic selective analogs are potent regulators of mitochondrial
106
function to increase both the magnitude and efficiency of mitochondrial respiration
in brain.
Further, the data indicate that ER α and ER β activation are uniquely able to
elicit mitochondrial responses. While we only used a single dose of each estrogen
receptor agonist, the data indicate that further studies will be required to determine
the exact influence of each estrogen receptor subtype on the mechanism that induced
mitochondrial respiration benefits. Another point of note is that the effects of the
combined treatment were not consistent across all outcome measures. In some
outcomes, as in mitochondrial membrane potential, there was a substantial increase
relative to control with either E
2
or PPT but not DPN. Other measures were very
consistent among treatments such as the relative influences on COX activity and
expression compared with control.
In summary, we have demonstrated that E
2
and SERMs increase the
oxidative capacity of whole brain mitochondria and this is attributable to activation
of either or both ERs. This increased respiratory activity is correlated with reduced
lipid peroxidation and increased cytochrome oxidase activity. In conclusion, the
ovarian hormone E
2
and synthetic selective ER agonists PPT and DPN, induce
mitochondrial alterations in the central nervous system that support efficient and
balanced bioenergetics and reduced oxidative stress.
107
Chapter IV References
Han, D., E. Williams, et al. (2001). "Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space."
Biochem J 353(Pt 2): 411-6.
Irwin, R. W., J. Yao, et al. (2008). "Progesterone and Estrogen Regulate Oxidative
Metabolism in Brain Mitochondria." Endocrinology.
Nilsen, J. and R. D. Brinton (2003). "Mechanism of estrogen-mediated
neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression."
Proc Natl Acad Sci U S A 100(5): 2842-7.
Nilsen, J. and R. D. Brinton (2004). "Mitochondria as therapeutic targets of estrogen
action in the central nervous system." Curr Drug Targets CNS Neurol Disord
3(4): 297-313.
Riddle, D. R. and M. E. Forbes (2005). "Regulation of cytochrome oxidase activity
in the rat forebrain throughout adulthood." Neurobiology of Aging 26(7):
1035-1050.
108
Chapter V: Triple transgenic Alzheimer’s disease mouse
model and age-related effects on the brain mitochondrial
proteome
Abstract
We are currently investigating the cellular mechanisms of estradiol influences
on mitochondria in cell lines and rodent models relevant to neurodegenerative
diseases. The purpose of this study was to determine the mitochondrial protein
differences between young and aged triple-transgenic Alzheimer’s disease (3xTg-
AD) and non-transgenic (nonTg) female mice. 3xTg-AD and nonTg mice were
obtained from Dr. Frank LaFerla’s group at UC Irvine. It is known that in addition
to neurodegeneration, AD brains exhibit mitochondrial dysfunction. Pre- (3 mo.) and
post-onset AD (9 mo.) female mice were chosen for our study. We have previously
shown that estradiol significantly increases the respiratory control ratio in
mitochondria isolated from rat brain (Nilsen and Brinton, 2004). Thus ER activity
may influence mitochondrial function. For this study, mitochondria were isolated
from mouse forebrains by discontinuous Percoll density centrifugation. Protein
fractions taken from the isolation procedure were assessed using organelle specific
probes to verify mitochondrial purification. 1-Dimensional and 2-D gel
electrophoresis was followed by anti-estrogen receptor immunoblotting or 2D profile
analysis respectively. Mass spectrometric analysis will be used to identify the
109
proteins recognized by immunospecific antibodies. Western blots of forebrain
mitochondria, cortex and hippocampus homogenates were performed for ER α/ β
expression. Thus far our results indicate that in terms of ER β levels in brain
mitochondria: 9mo. 3xTg > 9mo. nonTg > 3mo. 3xTg >> 3mo. nonTg and were
verified by repetition. Estrogen receptor levels were found to be dependent on age
and disease status. 3 month old mice had less ER β than 9 month mice. Likewise,
nontransgenic background strain mice had less ER β than their transgenic
counterparts. These findings suggest that mitochondrial ER expression and/or
localization is influenced by age and AD-like pathological status. Our future work is
aimed at determining the function of ER in mitochondria.
Introduction
We are investigating the cellular mechanisms of estradiol influences on
mitochondria in cell lines and rodent models relevant to neurodegenerative diseases.
The purpose of this study was to characterize the mitochondrial deficits in young and
aged triple-transgenic Alzheimer’s disease (3xTg-AD) and non-transgenic (nonTg)
female mice. 3xTg-AD and nonTg mice were obtained from Dr. Frank LaFerla’s
group at UC Irvine (Han, Williams et al. 2001). It is known that in addition to
neurodegeneration, AD brains exhibit mitochondrial dysfunction. Pre- (3 mo.) and
post-onset AD (9 mo.) female mice were chosen for our study. We have previously
shown that estradiol significantly increases the respiratory control ratio in
mitochondria isolated from rat brain (Riddle and Forbes 2005). Thus ER activity
may influence mitochondrial function. We also aimed to determine the protein levels
110
of estrogen receptor (ER) subtypes α/ β using ER subtype selective antibodies. In this
study, mitochondria were isolated from mouse forebrains by discontinuous Percoll
density centrifugation. Protein fractions taken from the isolation procedure were
assessed using organelle specific probes to verify mitochondrial purification. 1-
Dimensional and 2-D gel electrophoresis was followed by anti-estrogen receptor
immunoblotting. Mass spectrometric analysis will be used to identify the proteins
recognized by immunospecific antibodies. Western blots of forebrain mitochondria,
cortex and hippocampus homogenates were performed for ER α / β expression. 2-D
gel of aged mouse brain mitochondria were analyzed and compared to know
proteome profiles from our previous studies (Bubber, Haroutunian et al. 2005).
Materials and Methods
Chemicals
All chemicals were from MP Biomed (Irvine, CA) unless otherwise noted.
Animals
The use of animals for the study was approved by the Institutional Animal Care and
Use Committee (IACUC) at the University of Southern California (Protocol No.
10256). Mice were housed under controlled conditions of temperature (22
o
C),
humidity and light (14h light, 10h dark) with water and food available ad libitum. At
time of sacrifice uteri were removed and weighed to determine hormone status. All
experiments were approved by the Institutional Animal Care and Use Committee.
111
Mitochondrial Isolation
3xTg and nonTg mice aged 3, 6, 9, 12, 18 months were sacrificed and whole brain
tissue dissected. Mitochondria were then isolated through differential centrifugation
and Percoll gradients. Brain mitochondria were isolated from mice as previously
described (Diaz Brinton, Chen et al. 2000). Mice were decapitated, and the whole
brain minus the cerebellum was rapidly removed, minced and homogenized at 4
o
C in
mitochondrial isolation buffer (MIB: pH 7.4, containing sucrose (320 mM), EDTA
(1 mM), Tris-HCl (10 mM), and Calbiochem’s Protease Inhibitor Cocktail Set I
(AEBSF-HCl 500 μM, Aprotonin 150 nM, E-64 1 μM, EDTA disodium 500 μM,
Leupeptin hemisulfate 1 μM). Single brain homogenates were then centrifuged at
1500 x g for 5 min. The pellet was resuspended in MIB, re-homogenized and
centrifuged again at 1500 x g for 5 min. The postnuclear supernatants from both
centrifugations were combined and crude mitochondrial were pelleted by
centrifugation at 21,000 x g for 10 min. The resulting mitochondrial pellet was
resuspended in 15% Percoll made in MIB, layered over a preformed 23%/40%
Percoll discontinuous gradient and centrifuged at 31,000 x g for 10 min. The purified
mitochondria were collected at the 23%/40% interface and washed with 10 mL MIB
by centrifugation at 16,700 x g for 13 min. The loose pellet was collected and
transferred to a microcentrifuge tube and washed in MIB by centrifugation at 9,000 x
g for 8 min. The resulting mitochondrial pellet was resuspended in MIB to an
approximate concentration of 1 mg/mL. The resulting mitochondrial samples were
used immediately for respiratory measurements or stored at -70
o
C for later protein
112
and enzymatic assays. During mitochondrial purification, aliquots were collected and
for confirmation of mitochondrial purity and integrity by Western blot analysis for
mitochondrial anti-porin (1:500; Mitosciences, Eugene, OR), nuclear anti-histone H1
(1:250; Santa Cruz Biotechnology, Santa Cruz, CA), endoplasmic reticulum anti-
calnexin (1:2000; SPA 865, Stressgen, now a subsidiary of Assay Designs, Ann
Arbor, MI) and cytoplasmic anti-myelin basic protein (1:500, clone 2, RDI, Concord,
MA) (Data not shown).
Respiration Measurements
Mitochondrial oxygen consumption was measured polygraphically using a Clarke-
type electrode. 100 µg of isolated mitochondria was placed in the respiration
chamber at 37
o
C in respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES,
2 mM MgCl
2
, 1 mM EGTA) to yield a final concentration of 200 µg/ml followed by
the addition of glutamate (5mM) and malate (5mM) as substrates, and ADP (410
µM) in order to stimulate state 3 respiration. State 4o was measured after the
depletion of ADP by 50 μM pulses of atractyloside (adenine nucleotide tranporter
inhibitor), and the Respiratory Control Ratio (RCR) was determined as a ratio of
state 3 to state 4o respiration.
Lipid Peroxides
Lipid peroxides in brain mitochondria were measured using the leucomethylene blue
assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm
absorbance after 1 hr incubation at RT.
113
Cytochrome c Oxidase activity and expression
Cytochrome c oxidase (COX) activity was measured spectrophometrically by
monitoring change in absorbance (550nm) of reduced cytochrome c by
permeabilized mitochondria (Nilsen, Irwin et al. 2007). Mitochondria were
permeabilized in 0.2 mL of 75 mM potassium phosphate buffer (pH 7.5) at 25
o
C.
The reaction was started by the addition of 0.05 mL of 5% cytochrome c previously
reduced with sodium hydrosulfite. Cytochrome c oxidase activity was calculated in
nanomoles of oxidized cytochrome c per minute per mg protein and reported as rate
relative to the mean rate from vehicle control-treated animals. Cytochrome c
Oxidase subunit IV and II protein was determined by Western blot (COXIV:
Molecular Probes, A-21348, 1:2000, COXII: Molecular Probes, A6404, 1:1000).
Estrogen Receptor protein expression
Western blot analysis of ER was performed using anti-ER β, Santa Cruz H-150
(1:1000) and anti-ER α, Novocastra 6F11 (1:50). Rat ovary homogenate was used as
a positive control.
2D Gel Electrophoresis
Mitochondria were isolated from whole brain of treated OVX rats by percoll density
centrifugation. 400 μg of mitochondrial protein were subjected to isoelectric
focusing on 17 cm pH 3-10 NL strips followed by SDS-PAGE through 12%
acrylamide gels. Following the second dimension PAGE, the proteins stained with
114
Deep Purple and then subsequently analyzed to determine the protein spot
differences between 18 month old 3xTg and nonTg mouse brain mitochondria.
ER- α
VDAC-1
VDAC-2
ER- β
Ovary
Purified Brain Mitochondria
nonTg 3xTg nonTg 3xTg
3mo. ♀ 3mo. ♀ 9mo. ♀ 9mo. ♀
ER- α
VDAC-1
VDAC-2
ER- β
Ovary
Purified Brain Mitochondria
ER- α
VDAC-1
VDAC-2
ER- β
Ovary
Purified Brain Mitochondria
nonTg 3xTg nonTg 3xTg
3mo. ♀ 3mo. ♀ 9mo. ♀ 9mo. ♀
Figure V-1.
Figure V-1. ER-alpha and ER-beta expressed in brain mitochondria
associated with age and transgene status.
ER-alpha (red box) is expressed in 3month nontg female brain mitochondria, not
apparent in 3month 3xTg, 9month nonTg or 3xTg. Highest levels of ER-beta are
observed with transgene and age increase. N=2
ER6F11 antibody 1:75. Mouse ovary positive control.
ERH150 antibody 1:500, VDAC antibody 1:500 loading control.
115
4.5
5
5.5
6
6.5
7
7.5
8
FnonTg
3mo.
F3xTg
3mo.
FnonTg
9mo.
F3xTg
9mo.
FnonTg
12mo.
F3xTg
12mo.
RCRATR
**
4.5
5
5.5
6
6.5
7
7.5
8
FnonTg
3mo.
F3xTg
3mo.
FnonTg
9mo.
F3xTg
9mo.
FnonTg
12mo.
F3xTg
12mo.
RCRATR
**
Figure V-2. Oxygen consumption of brain mitochondria
Figure V-2. Oxygen consumption of brain mitochondria measured as
respiratory control ratio (RCR).
A significant decline in respiration was observed at 12months in female 3xTg brain
mitochondria compared to nonTg control. (N=2 for 3 and 9 month; N=4 for 12
month).
116
16
12
8
4
0
F M
3 mo 3 mo 9 mo 9 mo
LP uM/ug mito protein
nonTg
3xTg-AD
16
12
8
4
0
16
12
8
4
0
F M
3 mo 3 mo 9 mo 9 mo
LP uM/ug mito protein
nonTg
3xTg-AD
F M
3 mo 3 mo 9 mo 9 mo
F M
3 mo 3 mo 9 mo 9 mo
LP uM/ug mito protein LP uM/ug mito protein
nonTg
3xTg-AD
nonTg
3xTg-AD
Figure V-3. Lipid Peroxides in Female and Male
nonTg (light bars) and 3xTg (dark bars).
Figure V-3. Lipid peroxides - male vs female and 3xTg vs nonTg
Lipid Peroxides in Female and Male nonTg (light bars) and 3xTg (dark bars). All
results were highly significant P<0.001. N=4
Results and Discussion
Thus far our results indicate that in terms of ER β levels in brain
mitochondria: 9mo. 3xTg > 9mo. nonTg > 3mo. 3xTg >> 3mo. nonTg and were
verified by repetition (Figure 1). Estrogen receptor levels were found to be
dependent on age and disease status. 3 month old mice had less ER β than 9 month
mice. Likewise, nontransgenic background strain mice had less ER β than their
transgenic counterparts. These findings suggest that mitochondrial ER expression
and/or localization is influenced by age and AD-like pathological status. From the 2-
117
D gel analysis, a global reduction in mitochondrial proteins was observed in 18
month 3xTg-AD mice compared to age
matched nonTg control (figure 4A and 4B). Several proteins of interest were
identified from the spot pattern compared with the rat brain mitoproteome in aim 1.
MnSOD, aconitase-2, and VDAC-1 were all identified by their unique spot locations.
Each of these three proteins was observed to be highly decreased in 3xTg brain
mitochondria. These findings are indicative of a serious mitochondrial dysfunction
that is expected to occur in these mice. We measured mitochondrial respiration and
found an age and transgene dependent decline in respiratory function. This decline
was significant by 12 months of age (Figure 2). Cytochrome oxidase activity was
surprisingly increased in 3 month old 3xTg mice possibly indicating a compensatory
effect to a mitochondrial malfunction due to transgene status. This increase is
overcome by 6 months of age as the pathology progresses (data not shown). Lipid
peroxides were measured in both male and female mice (Fig.3). We found that lipid
peroxides increase with age and transgene status as well. Male 3xTg mice at 9
months of age had a drastic increase in lipid peroxides. This observation may be
pronounced in male mice not protected by ovarian hormones may be explained by
the pathological progression of AD-like symptoms including mitochondrial
dysfunction and oxidative stress.
The data reveal that 3xTg-AD mice have compromised mitochondrial
function which may exacerbate the genetic status to produce more pathology such as
amyloid deposits. Therefore mitochondrial dysfunction precedes AD-like pathology
118
in this mouse model. In this model, therapeutic intervention may be evaluated to find
lead candidates for early drug development pipelines. In our future studies, we plan
to extend previous work with E2, P4, and SERMs (see Specific Aims 1-3 of this
document) to test the hypothesis that E2 therapy can improve mitochondrial function
such as oxidative phosphorylation efficiency and thereby slow the progression of
pathology in these genetically engineered mouse.
We have proposed a relationship between development of AD pathology and
hypometabolism in brain tissues (Figure 5). E
2
may protect against mitochondrial
dysfunction and Alzheimer’s disease pathology by regulating key enzymes in the
glycolytic and TCA cycle pathways. E
2
increases pyruvate dehydrogenase (PDH)
and aconitase protein expression coupled with a decrease in malate dehydrogenase
(MDH) protein level (Nilsen, Irwin, et al 2007). In contrast, enzyme activity of each
of these proteins is opposite in AD brain compared to age-matched controls (Bubber,
et al. 2005). The decrease in PDH activity concomitant with an increase in MDH
increases oxaloacetate, then shunted towards gluconeogenesis rather than glycolysis
thereby depriving neurons of a major source of energy. Mechanistically, E
2
promotes
the glycolytic pathway, which is coupled to an increase in glucose uptake to prevent
hypometabolism characteristic of Alzheimer’s disease.
119
NonTg
3xTg
Aconitase
MnSOD
VDAC
NonTg
3xTg
Aconitase
MnSOD
VDAC
Figure V-4A. 2-D gel mitoproteomic profile.
NonTg (left) and 3xTg (right) 18 month female mice.
nonTg 18mo 3xTg 18mo
Aconitase
MnSOD
VDAC
nonTg 18mo 3xTg 18mo
Aconitase
MnSOD
VDAC
Figure V-4B. Spots from 2-D gel show
decreased levels of key mitochondrial proteins
Aconitase, MnSOD, and VDAC.
Figure V-4A and V-4B. 2-D gel mitoproteomic profile.
NonTg (left) and 3xTg (right) 18 month female mouse brain mitochondria. Key
proteins are identified from the following categories: TCA (aconitase), reduction of
oxidative stress (mnSOD), and mitochondrial permeability (VDAC). The protein
spots analyzed are shown in Figure V-4B.
120
Figure V-5. Proposed schematic model of estradiol prevention of AD.
Figure V-5. Proposed schematic model of estradiol prevention of AD
Estrogen (E
2
; red) may protect against mitochondrial dysfunction and Alzheimer’s
disease (AD; brown) pathology by regulating key enzymes in the glycolytic and
tricarbolic acid cycle (TCA) cycle pathways. Specifically, AD results in enzyme
activity decreases for pyruvate dehydrogenase (PDH), aconitase, and increased
malate dehydrogenase (MDH) (Bubber et al. 2005). All three of these enzymes are
oppositely regulated by E2, measured by protein expression (Nilsen, Irwin, et al
2007). Decreased MDH activity may result in malate shunting to cytosol where
malic enzyme produces pyruvate to feed back into TCA cycle.
121
Chapter V References
Han, D., E. Williams, et al. (2001). "Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space."
Biochem J 353(Pt 2): 411-6.
Irwin, R. W., J. Yao, et al. (2008). "Progesterone and Estrogen Regulate Oxidative
Metabolism in Brain Mitochondria." Endocrinology.
Nilsen, J. and R. D. Brinton (2003). "Mechanism of estrogen-mediated
neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression."
Proc Natl Acad Sci U S A 100(5): 2842-7.
Nilsen, J. and R. D. Brinton (2004). "Mitochondria as therapeutic targets of estrogen
action in the central nervous system." Curr Drug Targets CNS Neurol Disord
3(4): 297-313.
Nilsen, J., S. Chen, et al. (2006). "Estrogen protects neuronal cells from amyloid
beta-induced apoptosis via regulation of mitochondrial proteins and
function." BMC Neurosci 7(1): 74.
Nilsen, J., R. W. Irwin, et al. (2007). "Estradiol in vivo regulation of brain
mitochondrial proteome." J Neurosci 27(51): 14069-77.
Oddo, S., A. Caccamo, et al. (2003). "Triple-transgenic model of Alzheimer's disease
with plaques and tangles: intracellular Abeta and synaptic dysfunction."
Neuron 39(3): 409-21.
Riddle, D. R. and M. E. Forbes (2005). "Regulation of cytochrome oxidase activity
in the rat forebrain throughout adulthood." Neurobiology of Aging 26(7):
1035-1050.
122
Chapter VI: Integration of Specific Aims 1-4 and
Concluding Remarks
Discussion
We investigated the impact of E
2
, P4, SERMs in a well-established rat model of
ovarian hormone replacement, as well as investigations on age and genetic
background in a mouse model of Alzheimer’s disease. The mitoproteome in brain
was studied using various biochemical techniques including 2-D gel electrophoresis,
Western blots, RT-PCR, enzyme activity assays, and hydrogen peroxide and lipid
peroxide oxidative stress measurements.
Specific Aim 1: Estradiol Regulation of the Mitoproteome
In specific aim 1, E
2
was shown to influence the protein profile of the brain
mitochondria in a pattern that indicates improved defense mechanisms and oxidative
metabolism. 2-D gel electrophoresis was employed to resolve proteins into uniquely
identifiable protein species. Protein identification by mass spectrometry revealed
several key components of key players in glucose metabolism, such as pyruvate
dehydrogenase complex subunits which were all upregulated following 24 hour
exposure to E
2
. Other findings included proteins that play a role in cellular
energetics, amino acid and neurotransmitter metabolism, free radical maintenance,
and stress responses were determined. Several proteins of interest were pursued
further to identify changes in protein expression by Western blot. These data were
123
then correlated with analyses of enzymatic function such as COX and PDH activity.
In brain, PDH is further responsible for directing acetyl CoA to either the TCA cycle
or to acetylcholine synthesis (Holmquist et al., 2007). We observed an interesting
inverse correlation between our data and a well received study on enzyme activity in
AD brain (Irwin, Yao et al. 2008). Specifically, our data showed that PDH and
aconitase were increased while malate dehydrogenase was decreased following E
2
exposure. In the case of AD, PDH and aconitase enzyme activities are lowered and
malate dehydrogenase is increased. E
2
increased expression of key components of
the PDH complex and aconitase-2 while simultaneously decreasing MDH. Increased
PDH and aco-2 combined with decreased MDH is expected to play a compensatory
role to divert excess malate to the cytosol, outside the mitochondria, to prevent
oxaloacetate buildup. Together, our data indicate that E
2
may have a role in
regulating metabolic enzyme levels that control glucose utilization and respiratory
efficiency and thereby prevent neurodegenerative processes. Two proteins of
glutamate metabolism were also altered by E
2
(increased glutamate dehydrogenase
and glutamate oxaloacetate transaminase-2). Glutamate metabolism is linked to the
TCA cycle by the enzyme alpha-ketoglutarate dehydrogenase which receives a
metabolite of glutamate to enter the TCA cycle. Glutamate is also important in
neurotransmission excess glutamate can cause neurotoxicity and excess can also
arise from breakdown of glutamine to glutamate with toxic ammonium as a
byproduct.
124
Oxidative phosphorylation and proteins within the electron transport chain
were increased in expression and activity. E
2
induced a significant increase activity
of Complex IV. Reduction in Complex IV is an early marker of Alzheimer’s and
therefore potentiating this enzyme is advantageous as a neuroprotectant (Lin and
Beal, 2006). E
2
also increased expression of ATP synthase F1 subunits alpha and
beta, which is consistent with our previous report of estrogen-induced increases in
ATP levels in primary neuronal cultures (Nilsen, Irwin et al. 2007). E
2
induced
regulation of proteins composing the ETC corresponds to improvement of
mitochondrial function and oxidative capacity and defense. These basic science
studies demonstrate that in hormone replaced rats, aerobic respiration is enhanced by
E
2
(Behl, Widmann et al. 1995). Overall, the mitoproteome changes induced by E
2
in
the rodent brain reflect a mitochondrial profile that is proposed to act as a buffer
against diseases with underlying mitochondrial dysfunction such as AD.
Specific Aim 2: Estradiol and Progesterone Regulation of the
Cerebral Oxidative Metabolism
Estradiol (E
2
) and progesterone (P4) were assessed for both there individual
and combined contributions to mitochondrial health (Pejic, Kasapovic et al. 2003;
Stirone, Duckles et al. 2005). Interestingly, one indicator of mitochondrial health, the
mitochondrial respiratory control ratio, was benefited by either hormone alone but
not in combination. This provided an unexpected outcome to our hypothesis which
was that the combination would prove to be synergistic, which by this measure was
proven incorrect. This could have profound implications on our understanding of
125
hormone therapies which usually contain at least one form of estrogen and a
progestin. We also extended these studies to look at proteins revealed by our
previous proteomic analysis described by addressed by Aim 2 (Nilsen 2008).
Increased expression of peroxiredoxin-V is consistent with the well-documented
antioxidant effects of estrogens (Stirone, Duckles et al. 2003; Chen, Delannoy et al.
2004; Chen, Eshete et al. 2004; Chen and Yager 2004; Yang, Liu et al. 2004; Chen,
Yager et al. 2005; Milner, Ayoola et al. 2005). In contrast to the 2-D gel data which
did not find significant increases in E
2
-induced expression of MnSOD, we did find
an increase by Western blot which is consistent with previous findings. Enhancement
of PrdxV and MnSOD by both E
2
and P4 could protect against oxidative stresses or
may be compensatory to increased oxidative phosphorylation. The observed
reduction in reactive oxygen species by these ovarian hormones is essential for
neuroprotection.
Specific Aim 3: Selective Estrogen Receptor Modulators (SERMs)
Potentiate Mitochondrial Function using both ER-alpha and ER-
beta Selective Estrogen Receptor Modulators
E
2
regulated mitochondrial-DNA and nuclear-DNA encoded subunits of
cytochrome oxidase require coordinated control of mitochondrial and nuclear
encoded gene transcription. Immunoreactivity to estrogen receptors in mitochondria
(mtER) has been reported in the literature. In addition to classical nucERs,
membrane sites of estrogen action (mER) activate the PI3K/PKC/Src/MEK/ERK
126
signaling pathway which then activate the nuclear transcription factor CREB,
identified by our lab as required for E
2
-inducible neuroprotection (Zhao, Wu et al.
2004; Mannella and Brinton 2006; Morrison, Brinton et al. 2006). The ER signaling
pathways between plasma membrane, mitochondria, and nucleus, remain unknown
and will require further pharmacological studies. We propose that coordinated E
2
activation events at ERs leads to regulation of mitochondrial function and ultimately
neuroprotection. It is open to discovery to find these coordinates and target ERs in
specific locations and for a precise duration to translate these findings into therapies
for hormone-related diseases including AD.
The selective ER agonists, PPT and DPN, were studied in comparison to E
2
treated ovariectomized rats. The results revealed that both ERs are involved in E
2
afforded mitochondrial potentiation. Measurements for oxygen consumption, COX
activity, lipid peroxides were used to determine the contribution of each subtype of
estrogen receptor. Mitobiogenesis was measured to determine if the observed effects
were due to difference in mitochondrial number. However, we did not see
significance is mitobiogenesis which may be due to the short duration, 24h, of the
experiments. Future experiments with long term treatment will be necessary to fully
assess this parameter.
The mitochondrial fractions were also examined for their ER content. The
still debated mtERs, were investigated in this model and under hormone replacement
conditions. ER α and ER β were observed in these highly purified brain mitochondrial
samples. ER β was examined following E
2
, PPT, and DPN treatment and was found
127
to be increased with DPN treatment. E
2
and PPT appear to slightly decrease mtER β.
These findings are the first attempts to look at mtER following E
2
and SERM
treatment in ovariectomized rat brain. The purified mitochondria were subjected to
2D/Western to approach the identification process in a more specific way. The
results revealed several spots that could correspond to ER β immunoreactivity. One
spot was observed for ER α immunoreactivity. These spots matched the size
migration of a positive control ovary lysate for both ER α and ER β. The results
suggest that there may be both estrogen receptors in the rodent brain. The exact brain
regions and cell types that harbor each receptor are still unknown.
Specific Aim 4: Age and transgene studies in the 3xTg-AD mouse
model
We have been interested in the 3xTg-AD mouse model that exhibits AD-like
pathologies including hyperphosphorylation of Tau and amyloid plaque buildup
created by Salvatore Oddo and Frank LaFerla’s laboratory at UC Irvine (Oddo,
Caccamo et al. 2003). This model develops AD-like neuropathology and is touted as
a useful model of therapeutic intervention. In our studies, we are interested in
ovarian hormone effects on neuroprotection and prevention of AD. This model is
expected to exhibit age-related loss of mitochondrial function but the onset and
extent were unknown. This aim was designed to address our goals of finding a
genetic model of AD that exhibits mitochondrial dysfunction with age. The results
show that as early as three months of age, 3xTgAD mice exhibit a slight reduction in
mitochondrial respiration compared to nonTg control. This effect is significantly
128
pronounced by 12 months of age in female mouse mitochondria isolated from brain.
Studies using purified brain mitochondria revealed in females that only young nonTg
mice possessed ER α in this organelle. 3xTgAD mice at 3, 6, and 9 months had no
ER α in brain indicating an early difference associated with transgene, age, and
estrogen receptor biology. We also observed an increase in ER β in purified brain
mitochondria with age and transgene status in these mice. The studies also included
the mitochondrial proteome assessment by 2-D gel electrophoresis. The initial results
revealed that in 18 month old female mice, the mitochondria of nonTg control mice
had higher levels of key proteins including aconitase, MnSOD, and VDAC. The
results suggest that energy production by the TCA cycle (aconitase), oxidative stress
(MnSOD), and permeability (VDAC) were all compromised in the 3xTgAD mouse
brain.
Chapter VI References
Oddo, S., A. Caccamo, et al. (2003). "Triple-transgenic model of Alzheimer's disease
with plaques and tangles: intracellular Abeta and synaptic dysfunction."
Neuron 39(3): 409-21.
Wagner, B. K., T. Kitami, et al. (2008). "Large-scale chemical dissection of
mitochondrial function." Nat Biotechnol 26(3): 343-51.
129
Concluding Remarks
These projects were a culmination of several years work and much
collaboration with excellent researchers in neuroscience, free radical biology, and
mitochondrial biochemistry. It was rewarding to work towards a common goal: to
understand the pharmacology of gonadal hormone actions in the brain and their
relation to mitochondrial function. These findings have advanced our understanding
of the role of estradiol and progesterone neuroprotective actions that converge upon
the mitochondria. It is my hope that these findings will be used to extend our
knowledge of hormone therapies, aging, and mitochondrial biology. I too hope to
continue adding to this work in the coming years. It is my intention to incorporate
the work from this dissertation into my upcoming postdoctoral studies and beyond. I
envision the development of these projects into therapeutic regimens that will
prevent or delay mitochondrial malfunctions with applications to several diseases
associated with aging and metabolic imbalances.
My dream is to see the development of therapeutics targeted to mitochondria
that will optimize metabolism and glucose utilization while minimizing oxidative
stress. I propose that a new class of therapeutics be developed that I have termed
“MitoSERMs.” These small molecules will possess the characteristics of a
mitochondrially targeted drug - including a lipophilic cation moiety attached to the
steroid ring structure at a position that does not inhibit the desired binding effect of
ligand and receptor – and promote beneficial estrogenic actions while reducing the
risk of unwanted side effects. The molecule may accumulate in the mitochondria at a
130
concentration 100-1000 times higher than in the cytosol or nucleus. The ideal
MitoSERM would be administered at a dose that is subnanomolar and accumulate in
mitochondria in the nanomolar or low micromolar range to promote cellular vitality.
The effects on direct ER-guided nuclear transcription associated with cell
proliferation would thus be minimal while the mitochondrial effects associated with
cellular vitality would be greatest. The tissues with the highest permeability, highest
mitochondrial membrane potential, and highest amount of mitochondria would
accumulate the drug first such as the brain, heart, and liver. The ideal MitoSERM
would activate mitochondrial responses that are beneficial to brain and heart while
avoiding negative side effects in breast and uterus. The findings from PPT and DPN
in Specific Aim 3 (Chapter IV) indicate that both ER α and ER β are possible targets
that may elicit beneficial effects. Another exciting avenue we identified is the
independent action of progesterone on mitochondrial function, demonstrated in
Specific Aim 2 (Chapter III). New hormone therapy formulations may take into
consideration the beneficial mitochondrial effects of E
2
and P4 alone while limiting
their negative convergence points on respiration. Work on the mitochondrial
proteome described in Specific Aim 1 (Chapter II) has directed our studies towards
research areas that would have otherwise been left unexplored. Some of the proteins
identified from the 2-D gels may turn out to be biomarkers for detection of early
events in estrogen deficiency-related diseases such as AD as well as biomarkers for
E
2
therapeutic efficacy. These initial proteomic studies on E2 actions in brain
131
mitochondria within 24 hours also suggest that the metabolomic profile will be
altered and in some cases predictable.
I hope to look back on this work as the start of a new research focus on E
2
-
based therapeutics targeted to the brain and more specifically the mitochondrial
compartment. In the future, as technologies develop, I await the opportunity to
develop these MitoSERMs with both a rationale-based and combinatorial chemistry
approach to allow synthesis of multiple chemical analogs. Not only may these
molecules be useful for therapeutic applications but some may turn out to be
excellent chemical probes for various mitochondrial functions. As high-throughput
technologies become accessible, it may become a reality to test a library of
MitoSERMs in cultured cells or purified receptors. It is now possible to perform
large-scale chemical dissection of mitochondrial functions such as cell viability,
mitochondrial membrane potential, dehydrogenase activities, ATP production, ROS
production, as well as expression of nuclear and mitochondrial encoded genes
(Wagner, Kitami et al. 2008). Other advancements that I foresee jumpstarting new
avenues in this research area are those related to new genetic models of diseases and
the study of mitochondrial function in early development such as in stem cells.
The overall thrust of this work is to extend the brain span to match the
lifespan of humans susceptible to neurodegenerative diseases. One of the most
obvious signs of aging and diseases related to aging is a reduction in gonadal
hormone production and responsiveness. Further, the research accomplishments
herein are the result of successful collaborations I haved shared with my mentors and
132
colleagues and we have demonstrated that estradiol and progesterone are critical
chemical activators of brain mitochondrial function. It is therefore important to
consider the effects of these hormones with respect to brain function and
neurological health. If the hypothesis is correct, that being the antecedent event of
AD is glucose hypometabolism, then promoting mitochondrial function is at the core
of the solution to this age-related disease. In addition to AD, prevention of
hypometabolism may hold promise for other diseases such as diabetes or cancer.
This once again points to the mitochondria as the Achilles heal of our most vital
organs including the CNS, heart, and muscle. In the future, we hope to better
understand mitochondrial functions such as glucose utilization, ROS production and
elimination, apoptosis, energy production and thus to control vital steps in biological
processes.
133
Complete List of References
Abramson, J., M. Svensson-Ek, et al. (2001). "Structure of cytochrome c oxidase: a
comparison of the bacterial and mitochondrial enzymes." Biochim Biophys
Acta 1544(1-2): 1-9.
Banmeyer, I., C. Marchand, et al. (2005). "Human mitochondrial peroxiredoxin 5
protects from mitochondrial DNA damages induced by hydrogen peroxide."
FEBS Lett 579(11): 2327-33.
Barja, G. (2004). "Free radicals and aging." Trends Neurosci 27(10): 595-600.
Behl, C. (2002). "Oestrogen as a neuroprotective hormone." Nat Rev Neurosci 3(6):
433-42.
Behl, C., M. Widmann, et al. (1995). "17-beta estradiol protects neurons from
oxidative stress-induced cell death in vitro." Biochem Biophys Res Commun
216(2): 473-82.
Bettini, E. and A. Maggi (1992). "Estrogen induction of cytochrome c oxidase
subunit III in rat hippocampus." J Neurochem 58(5): 1923-9.
Bishop, J. and J. W. Simpkins (1995). "Estradiol enhances brain glucose uptake in
ovariectomized rats." Brain Research Bulletin 36(3): 315-320.
Borras, C., J. Sastre, et al. (2003). "Mitochondria from females exhibit higher
antioxidant gene expression and lower oxidative damage than males." Free
Radic Biol Med 34(5): 546-52.
Borras, M., I. Laios, et al. (1996). "Estrogenic and antiestrogenic regulation of the
half-life of covalently labeled estrogen receptor in MCF-7 breast cancer
cells." J Steroid Biochem Mol Biol 57(3-4): 203-13.
Brewer, G. J., J. D. Reichensperger, et al. (2006). "Prevention of age-related
dysregulation of calcium dynamics by estrogen in neurons." Neurobiol Aging
27(2): 306-17.
134
Brinton, R. D. (2001). "Cellular and molecular mechanisms of estrogen regulation of
memory function and neuroprotection against Alzheimer's disease: recent
insights and remaining challenges." Learn Mem 8(3): 121-33.
Brinton, R. D. (2004). "Impact of estrogen therapy on Alzheimer's disease: a fork in
the road?" CNS Drugs 18(7): 405-22.
Brinton, R. D. (2005). "Investigative Models for Determining Hormone Therapy-
Induced Outcomes in Brain: Evidence in Support of a Healthy Cell Bias of
Estrogen Action." Ann NY Acad Sci 1052(1): 57-74.
Brinton, R. D., S. Chen, et al. (2000). "The women's health initiative estrogen
replacement therapy is neurotrophic and neuroprotective." Neurobiol Aging
21(3): 475-96.
Bubber, P., V. Haroutunian, et al. (2005). "Mitochondrial abnormalities in Alzheimer
brain: mechanistic implications." Ann Neurol 57(5): 695-703.
Cadenas, E. (2004). "Mitochondrial free radical production and cell signaling." Mol
Aspects Med 25(1-2): 17-26.
Cadenas, E. and K. J. Davies (2000). "Mitochondrial free radical generation,
oxidative stress, and aging." Free Radic Biol Med 29(3-4): 222-30.
Calabrese, V., G. Scapagnini, et al. (2001). "Mitochondrial involvement in brain
function and dysfunction: relevance to aging, neurodegenerative disorders
and longevity." Neurochem Res 26(6): 739-64.
Campian, J. L., M. Qian, et al. (2004). "Oxygen tolerance and coupling of
mitochondrial electron transport." J Biol Chem 279(45): 46580-7.
Chen, J., M. Delannoy, et al. (2003). "Enhanced Mitochondrial Gene Transcript,
ATP, Bcl-2 Protein Levels, and Altered Glutathione Distribution in Ethinyl
Estradiol-Treated Cultured Female Rat Hepatocytes." Toxicol. Sci. 75(2):
271-278.
135
Chen, J. Q., M. Delannoy, et al. (2004). "Mitochondrial Localization of ER{alpha}
and ER{beta} in Human MCF-7 cells." Am J Physiol Endocrinol Metab:
00508.2003.
Chen, J. Q., M. Eshete, et al. (2004). "Binding of MCF-7 cell mitochondrial proteins
and recombinant human estrogen receptors alpha and beta to human
mitochondrial DNA estrogen response elements." J Cell Biochem 93(2): 358-
73.
Chen, J. Q. and J. D. Yager (2004). "Estrogen's effects on mitochondrial gene
expression: mechanisms and potential contributions to estrogen
carcinogenesis." Ann N Y Acad Sci 1028: 258-72.
Chen, J. Q., J. D. Yager, et al. (2005). "Regulation of mitochondrial respiratory chain
structure and function by estrogens/estrogen receptors and potential
physiological/pathophysiological implications." Biochim Biophys Acta
1746(1): 1-17.
Chen, S., J. Nilsen, et al. (2006). "Dose and temporal pattern of estrogen exposure
determines neuroprotective outcome in hippocampal neurons: therapeutic
implications." Endocrinology 147(11): 5303-13.
Dauvois, S., R. White, et al. (1993). "The antiestrogen ICI 182780 disrupts estrogen
receptor nucleocytoplasmic shuttling." J Cell Sci 106 ( Pt 4): 1377-88.
Diaz Brinton, R., S. Chen, et al. (2000). "The women's health initiative estrogen
replacement therapy is neurotrophic and neuroprotective." Neurobiol Aging
21(3): 475-96.
Diwakar, L., R. S. Kenchappa, et al. (2006). "Down-regulation of glutaredoxin by
estrogen receptor antagonist renders female mice susceptible to excitatory
amino acid mediated complex I inhibition in CNS." Brain Res 1125(1): 176-
84.
136
Ejima, K., H. Nanri, et al. (1999). "17beta-estradiol induces protein thiol/disulfide
oxidoreductases and protects cultured bovine aortic endothelial cells from
oxidative stress." Eur J Endocrinol 140(6): 608-13.
Gallaher, T. K., S. Wu, et al. (2006). "Identification of biofilm proteins in non-
typeable Haemophilus Influenzae." BMC Microbiol 6: 65.
Gridley, K. E., P. S. Green, et al. (1998). "A novel, synergistic interaction between
17 beta-estradiol and glutathione in the protection of neurons against beta-
amyloid 25-35-induced toxicity in vitro." Mol Pharmacol 54(5): 874-80.
Han, D., E. Williams, et al. (2001). "Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space."
Biochem J 353(Pt 2): 411-6.
Hoffman, J. M., K. A. Welsh-Bohmer, et al. (2000). "FDG PET imaging in patients
with pathologically verified dementia." J Nucl Med 41(11): 1920-8.
Holmquist, L., G. Stuchbury, et al. (2006). "Lipoic acid as a novel treatment for
Alzheimer's disease and related dementias." Pharmacol Ther.
Irwin, R. W., J. Yao, et al. (2008). "Progesterone and Estrogen Regulate Oxidative
Metabolism in Brain Mitochondria." Endocrinology.
Jung, M. E., R. Agarwal, et al. (2007). Ethanol withdrawal posttranslationally
decreases the activity of cytochrome c oxidase in an estrogen reversible
manner. Neurosci. Lett. 416: 160-4.
Justo, R., J. Boada, et al. (2005). "Gender dimorphism in rat liver mitochondrial
oxidative metabolism and biogenesis." Am J Physiol Cell Physiol 289(2):
C372-8.
Katzenellenbogen, B. S., J. Sun, et al. (2001). "Structure-function relationships in
estrogen receptors and the characterization of novel selective estrogen
receptor modulators with unique pharmacological profiles." Ann N Y Acad
Sci 949: 6-15.
137
Keller, J. N., A. Germeyer, et al. (1997). "17Beta-estradiol attenuates oxidative
impairment of synaptic Na+/K+-ATPase activity, glucose transport, and
glutamate transport induced by amyloid beta-peptide and iron." J Neurosci
Res 50(4): 522-30.
Kii, N., N. Adachi, et al. (2005). "Acute effects of 17beta-estradiol on oxidative
stress in ischemic rat striatum." J Neurosurg Anesthesiol 17(1): 27-32.
Kim, H., O. Y. Bang, et al. (2001). "Neuroprotective effects of estrogen against beta-
amyloid toxicity are mediated by estrogen receptors in cultured neuronal
cells." Neurosci Lett 302(1): 58-62.
Kostanyan, A. and K. Nazaryan (1992). "Rat brain glycolysis regulation by estradiol-
17 beta." Biochim Biophys Acta 1133(3): 301-6.
Lin, M. T. and M. F. Beal (2006). "Alzheimer's APP mangles mitochondria." Nat
Med 12(11): 1241-3.
Lin, M. T. and M. F. Beal (2006). "Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases." Nature 443(7113): 787-95.
Lopez-Lluch, G., N. Hunt, et al. (2006). "Calorie restriction induces mitochondrial
biogenesis and bioenergetic efficiency." Proc Natl Acad Sci U S A 103(6):
1768-73.
Lopez, M. F., B. S. Kristal, et al. (2000). "High-throughput profiling of the
mitochondrial proteome using affinity fractionation and automation."
Electrophoresis 21(16): 3427-40.
Mannella, P. and R. D. Brinton (2006). "Estrogen receptor protein interaction with
phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and
extracellular signal-regulated kinase 1/2 in the same population of cortical
neurons: a unified mechanism of estrogen action." J Neurosci 26(37): 9439-
47.
Massart, F., S. Paolini, et al. (2002). "Dose-dependent inhibition of mitochondrial
ATP synthase by 17 beta-estradiol." Gynecol Endocrinol 16(5): 373-7.
138
Mattson, M. P. and G. Kroemer (2003). "Mitochondria in cell death: novel targets for
neuroprotection and cardioprotection." Trends Mol Med 9(5): 196-205.
McEwen, B. (2002). "Estrogen actions throughout the brain." Recent Prog Horm Res
57: 357-84.
McEwen, B., K. Akama, et al. (2001). "Tracking the estrogen receptor in neurons:
implications for estrogen-induced synapse formation." Proc Natl Acad Sci U
S A 98(13): 7093-100.
McEwen, B. S. and S. E. Alves (1999). "Estrogen actions in the central nervous
system." Endocr Rev 20(3): 279-307.
Meltzer, C. C., J. K. Zubieta, et al. (1996). "Regional hypometabolism in
Alzheimer's disease as measured by positron emission tomography after
correction for effects of partial volume averaging." Neurology 47(2): 454-61.
Milner, T. A., K. Ayoola, et al. (2005). "Ultrastructural localization of estrogen
receptor beta immunoreactivity in the rat hippocampal formation." J Comp
Neurol 491(2): 81-95.
Milner, T. A., L. S. Lubbers, et al. (2008). "Nuclear and extranuclear estrogen
binding sites in the rat forebrain and autonomic medullary areas."
Endocrinology.
Monje, P. and R. Boland (2001). "Subcellular distribution of native estrogen receptor
alpha and beta isoforms in rabbit uterus and ovary." J Cell Biochem 82(3):
467-79.
Monje, P. and R. Boland (2002). "Expression and cellular localization of naturally
occurring beta estrogen receptors in uterine and mammary cell lines." J Cell
Biochem 86(1): 136-44.
Morrison, J. H., R. D. Brinton, et al. (2006). "Estrogen, menopause, and the aging
brain: how basic neuroscience can inform hormone therapy in women." J
Neurosci 26(41): 10332-48.
139
Murphy, A. N., D. E. Bredesen, et al. (1996). "Bcl-2 potentiates the maximal calcium
uptake capacity of neural cell mitochondria." Proc Natl Acad Sci U S A
93(18): 9893-8.
Nakamizo, T., M. Urushitani, et al. (2000). "Protection of cultured spinal motor
neurons by estradiol." Neuroreport 11(16): 3493-7.
Nicholls, D. G. and S. L. Budd (2000). "Mitochondria and neuronal survival."
Physiol Rev 80(1): 315-60.
Nicotera, P., M. Leist, et al. (2000). "Energy requirement for caspase activation and
neuronal cell death." Brain Pathol 10(2): 276-82.
Nilsen, J. (2008). "Estradiol and neurodegenerative oxidative stress." Front
Neuroendocrinol.
Nilsen, J. and R. D. Brinton (2002). "Impact of progestins on estradiol potentiation
of the glutamate calcium response." Neuroreport 13(6): 825-30.
Nilsen, J. and R. D. Brinton (2002). "Impact of progestins on estrogen-induced
neuroprotection: synergy by progesterone and 19-norprogesterone and
antagonism by medroxyprogesterone acetate." Endocrinology 143(1): 205-
12.
Nilsen, J. and R. D. Brinton (2003). "Mechanism of estrogen-mediated
neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression."
Proc Natl Acad Sci U S A 100(5): 2842-7.
Nilsen, J. and R. D. Brinton (2004). "Mitochondria as therapeutic targets of estrogen
action in the central nervous system." Curr Drug Targets CNS Neurol Disord
3(4): 297-313.
Nilsen, J., S. Chen, et al. (2006). "Estrogen protects neuronal cells from amyloid
beta-induced apoptosis via regulation of mitochondrial proteins and
function." BMC Neurosci 7(1): 74.
140
Nilsen, J., R. W. Irwin, et al. (2007). "Estradiol in vivo regulation of brain
mitochondrial proteome." J Neurosci 27(51): 14069-77.
Oddo, S., A. Caccamo, et al. (2003). "Triple-transgenic model of Alzheimer's disease
with plaques and tangles: intracellular Abeta and synaptic dysfunction."
Neuron 39(3): 409-21.
Paech, K., P. Webb, et al. (1997). "Differential ligand activation of estrogen
receptors ERalpha and ERbeta at AP1 sites." Science 277(5331): 1508-10.
Parihar, M. S. and G. J. Brewer (2007). "Mitoenergetic failure in Alzheimer disease."
Am J Physiol Cell Physiol 292(1): C8-23.
Pejic, S., J. Kasapovic, et al. (2003). "The modulatory effect of estradiol benzoate on
superoxide dismutase activity in the developing rat brain." Braz J Med Biol
Res 36(5): 579-86.
Price, R. H., Jr., N. Lorenzon, et al. (2000). "Differential expression of estrogen
receptor beta splice variants in rat brain: identification and characterization of
a novel variant missing exon 4." Brain Res Mol Brain Res 80(2): 260-8.
Puerta, M., M. Rocha, et al. (1998). "Changes in cytochrome oxidase activity in
brown adipose tissue during oestrous cycle in the rat." Eur J Endocrinol
139(4): 433-7.
Reddy, P. H. (2006). "Mitochondrial oxidative damage in aging and Alzheimer's
disease: implications for mitochondrially targeted antioxidant therapeutics." J
Biomed Biotechnol 2006(3): 31372.
Resnick, S. M., E. J. Metter, et al. (1997). "Estrogen replacement therapy and
longitudinal decline in visual memory. A possible protective effect?"
Neurology 49(6): 1491-7.
Riddle, D. R. and M. E. Forbes (2005). "Regulation of cytochrome oxidase activity
in the rat forebrain throughout adulthood." Neurobiology of Aging 26(7):
1035-1050.
141
Rodriguez-Cuenca, S., E. Pujol, et al. (2002). "Sex-dependent thermogenesis,
differences in mitochondrial morphology and function, and adrenergic
response in brown adipose tissue." J Biol Chem 277(45): 42958-63.
Sanz, A., P. Caro, et al. (2005). "Dietary restriction at old age lowers mitochondrial
oxygen radical production and leak at complex I and oxidative DNA damage
in rat brain." J Bioenerg Biomembr 37(2): 83-90.
Sato, T., T. Teramoto, et al. (2003). "Effects of ovariectomy and calcium deficiency
on learning and memory of eight-arm radial maze in middle-aged female
rats." Behav Brain Res 142(1-2): 207-16.
Schroeter, H., C. S. Boyd, et al. (2003). "c-Jun N-terminal kinase (JNK)-mediated
modulation of brain mitochondria function: new target proteins for JNK
signalling in mitochondrion-dependent apoptosis." Biochem J 372(Pt 2): 359-
69.
Schwend, T. and J.-A. Gustafsson (2006). "False positives in MALDI-TOF detection
of ER[beta] in mitochondria." Biochemical and Biophysical Research
Communications In Press, Corrected Proof.
Shea, T. B. and D. Ortiz (2003). "17 beta-estradiol alleviates synergistic oxidative
stress resulting from folate deprivation and amyloid-beta treatment." J
Alzheimers Dis 5(4): 323-7.
Sherwin, B. B. (1988). "Estrogen and/or androgen replacement therapy and cognitive
functioning in surgically menopausal women." Psychoneuroendocrinology
13(4): 345-57.
Simpkins, J. W. and J. A. Dykens (2007). "Mitochondrial mechanisms of estrogen
neuroprotection." Brain Res Rev.
Simpkins, J. W., G. Rajakumar, et al. (1997). "Estrogens may reduce mortality and
ischemic damage caused by middle cerebral artery occlusion in the female
rat." J Neurosurg 87(5): 724-30.
Singh, M., J. A. Dykens, et al. (2006). "Novel mechanisms for estrogen-induced
neuroprotection." Exp Biol Med (Maywood) 231(5): 514-21.
142
Singh, M., E. M. Meyer, et al. (1994). "Ovarian steroid deprivation results in a
reversible learning impairment and compromised cholinergic function in
female Sprague-Dawley rats." Brain Res 644(2): 305-12.
Sokoloff, L. (1960). "Quantitative measurements of cerebral blood flow in man."
Methods Med Res 8: 253-61.
Sokoloff, L. and S. S. Kety (1960). "Regulation of cerebral circulation." Physiol Rev
Suppl 4: 38-44.
Stirone, C., S. P. Duckles, et al. (2003). "Multiple forms of estrogen receptor-alpha
in cerebral blood vessels: regulation by estrogen." Am J Physiol Endocrinol
Metab 284(1): E184-192.
Stirone, C., S. P. Duckles, et al. (2005). "Estrogen increases mitochondrial efficiency
and reduces oxidative stress in cerebral blood vessels." Mol Pharmacol.
Strehlow, K., S. Rotter, et al. (2003). "Modulation of antioxidant enzyme expression
and function by estrogen." Circ Res 93(2): 170-7.
Subramanian, M., C. K. Pusphendran, et al. (1993). "Gestation confers temporary
resistance to peroxidation in the maternal rat brain." Neurosci Lett 155(2):
151-4.
Sun, J., J. Baudry, et al. (2003). "Molecular basis for the subtype discrimination of
the estrogen receptor-beta-selective ligand, diarylpropionitrile." Mol
Endocrinol 17(2): 247-58.
Sun, J., M. J. Meyers, et al. (1999). "Novel ligands that function as selective
estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-
beta." Endocrinology 140(2): 800-4.
Toran-Allerand, C. D. (2000). "Novel sites and mechanisms of oestrogen action in
the brain." Novartis Found Symp 230: 56-69; discussion 69-73.
Turgeon, J. L., D. P. McDonnell, et al. (2004). "Hormone therapy: physiological
complexity belies therapeutic simplicity." Science 304(5675): 1269-73.
143
Urata, Y., Y. Ihara, et al. (2006). "17Beta-estradiol protects against oxidative stress-
induced cell death through the glutathione/glutaredoxin-dependent redox
regulation of Akt in myocardiac H9c2 cells." J Biol Chem 281(19): 13092-
102.
Van Itallie, C. M. and P. S. Dannies (1988). "Estrogen induces accumulation of the
mitochondrial ribonucleic acid for subunit II of cytochrome oxidase in
pituitary tumor cells." Mol Endocrinol 2(4): 332-7.
Wagner, B. K., T. Kitami, et al. (2008). "Large-scale chemical dissection of
mitochondrial function." Nat Biotechnol 26(3): 343-51.
Wakeling, A. E., M. Dukes, et al. (1991). "A potent specific pure antiestrogen with
clinical potential." Cancer Res 51(15): 3867-73.
Wang, J. M., R. W. Irwin, et al. (2006). "Activation of estrogen receptor {alpha}
increases and estrogen receptor {beta} decreases apolipoprotein E expression
in hippocampus in vitro and in vivo." Proc Natl Acad Sci U S A.
Weihua, Z., S. Andersson, et al. (2003). "Update on estrogen signaling." FEBS Lett
546(1): 17-24.
Woolley, C. S. (1999). "Electrophysiological and cellular effects of estrogen on
neuronal function." Critical Reviews in Neurobiology 13(1): 1-20.
Wu, J., N. J. Lenchik, et al. (2005). "Functional characterization of two-dimensional
gel-separated proteins using sequential staining." Electrophoresis 26(1): 225-
37.
Yaffe, K., G. Sawaya, et al. (1998). "Estrogen Therapy in Postmenopausal Women:
Effects on Cognitive Function and Dementia." Jama 279(9): 688-695.
Yager, J. D. and J. Q. Chen (2007). "Mitochondrial estrogen receptors--new insights
into specific functions." Trends Endocrinol Metab 18(3): 89-91.
Yang, J. W., J. F. Juranville, et al. (2005). "Molecular diversity of rat brain proteins
as revealed by proteomic analysis." Mol Divers 9(4): 385-96.
144
Yang, S. H., R. Liu, et al. (2004). "Mitochondrial localization of estrogen receptor
beta." Proc Natl Acad Sci U S A 101(12): 4130-5.
Zhai, P., T. E. Eurell, et al. (2000). "Effect of estrogen on global myocardial
ischemia-reperfusion injury in female rats." Am J Physiol Heart Circ Physiol
279(6): H2766-75.
Zhao, L., C. Jin, et al. (2007). "Design, synthesis, and estrogenic activity of a novel
estrogen receptor modulator--a hybrid structure of 17beta-estradiol and
vitamin E in hippocampal neurons." J Med Chem 50(18): 4471-81.
Zhao, L., K. O'Neill, et al. (2006). "Estrogenic agonist activity of ICI 182,780
(Faslodex) in hippocampal neurons: implications for basic science
understanding of estrogen signaling and development of estrogen modulators
with a dual therapeutic profile." J Pharmacol Exp Ther 319(3): 1124-32.
Zhao, L., T. W. Wu, et al. (2004). "Estrogen receptor subtypes alpha and beta
contribute to neuroprotection and increased Bcl-2 expression in primary
hippocampal neurons." Brain Res 1010(1-2): 22-34.
Zheng, J. and V. D. Ramirez (1999). "Rapid inhibition of rat brain mitochondrial
proton F0F1-ATPase activity by estrogens: comparison with Na+, K+ -
ATPase of porcine cortex." Eur J Pharmacol 368(1): 95-102.
Zheng, J. and V. D. Ramirez (2000). "Inhibition of mitochondrial proton F0F1-
ATPase/ATP synthase by polyphenolic phytochemicals." Br J Pharmacol
130(5): 1115-23.
145
Abstract (if available)
Abstract
Herein are a collection of studies that build on our existing knowledge of estrogen actions in the brain. We extend the efforts of current neuroprotective strategies by demonstrating that estrogenic molecules promote cell survival mechanisms governed by neuronal mitochondria. Estrogen (E2, 17beta-estradiol) protects neurons from a series of age-related risk factors for developing Alzheimer 's disease (AD) supported by basic science, clinical, and epidemiological data. However, there exists a window of opportunity for E2 as a preventive therapy and our findings are not intended for hopeful treatments of pre-existing pathologies but rather to support the proposed healthy-cell bias therapeutic approach (Brinton 2005). Our preclinical pharmacology research work covers biochemistry, molecular biology and cell imaging of rodent brain. Controlled in vitro and in vivo studies are organized under four specific aims that test our hypotheses in brain tissues with a focus on the hippocampus and cortex regions involved in learning and memory and adversely affected in age-related dementias. Specific Aim 1 serves to determine the presence of estrogen receptors in mitochondria and the effects of E2 on the brain mitochondrial proteome. Specific Aim 2 focuses on E2 and P4 actions on the mitochondria and the mechanistic role of these hormones on mitochondrial function. Specific Aim 3 extends these basic science discoveries to test selective estrogen receptor modulators. Specific Aim 4 introduces the triple-transgenic mouse model of Alzheimer 's disease as a tool to test hormone therapies and intervene in cases of mitochondrial dysfunction. We believe that our experimental approach and subsequent findings will forge the way for a new class of E2-based mitochondrial therapeutics to reduce the risk of Alzheimer 's disease and other diseases attributed to mitochondrial malfunction.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Estrogen regulation of bioenergetics and mitochondrial function: implications for Alzheimer's disease risk and therapeutics
PDF
Bioenergetic fuel shift of reproductive aging: implication for late-onset Alzheimer's disease
PDF
From risk mitochondrial and metabolic phenotype towards a precision medicine approach for Alzheimer's disease
PDF
C-jun N-terminal Kinase (JNK) mediated inhibition of Pyruvate Dehydrogenase (PDH) activity and its effect on mitochondrial metabolism during brain aging
PDF
Estrogen and progesterone interactions in neurons: implications for Alzheimer's disease-related pathways
PDF
LDL protein nitration: implication for protein unfolding and mitochondrial function by p-JNK-2
PDF
Insulin sensitivity in cognition, Alzheimer's disease and brain aging
PDF
PI3K/AKT signaling and the regulation of the mitochondrial energy-redox axis
PDF
Energy metabolism and inflammation in brain aging: significance of age-dependent astrocyte metabolic-redox profile
PDF
Regulation of mitochondrial bioenergetics via PTEN (phosphatase and tensin homolog deleted on chromosome 10)/estrogen-related receptor alpha (ERRα) signaling
PDF
NO mediated neurotoxicity: redox changes and energy failure in a neuroinflammatory model
PDF
Perimenopausal transition increases blood brain permeability: implications for neurodegenerative diseases
PDF
Neuroinflammation and ApoE4 genotype in at-risk female aging: implications for Alzheimer's disease
PDF
The regulation of fatty acid oxidation by estrogen related receptor alpha
PDF
Mitochondrial dynamics regulate Leydig cell health and integrity
PDF
An iPSC-based biomarker strategy to identify neuroregenerative responders to allopregnanolone
PDF
The mitochondrial energy – redox axis in aging and caloric restriction: role of nicotinamide nucleotide transhydrogenase
PDF
Role of neuronal nitric oxide synthase in aging and neurodegeneration
PDF
Metabolic shift in lung alveolar cell mitochondria after exposure to environmental toxicants
PDF
The structure of loop 2 is important for agonist and ethanol sensitivity in glycine and GABA Alpha receptors
Asset Metadata
Creator
Irwin, Ronald W.
(author)
Core Title
Estradiol regulation of cerebral metabolism: implications for neuroprotection and mitochondrial bioenergetics
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology
Publication Date
06/02/2008
Defense Date
03/24/2008
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Alzheimer's disease,estradiol,mitochondria,OAI-PMH Harvest,proteomics
Language
English
Advisor
Brinton, Roberta Diaz (
committee chair
), Baudry, Michel (
committee member
), Cadenas, Enrique (
committee member
)
Creator Email
ronaldir@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m1252
Unique identifier
UC1143061
Identifier
etd-Irwin-20080602 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-72061 (legacy record id),usctheses-m1252 (legacy record id)
Legacy Identifier
etd-Irwin-20080602.pdf
Dmrecord
72061
Document Type
Dissertation
Rights
Irwin, Ronald W.
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Repository Name
Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
Alzheimer's disease
estradiol
mitochondria
proteomics