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Estrogen regulation of bioenergetics and mitochondrial function: implications for Alzheimer's disease risk and therapeutics

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Estrogen regulation of bioenergetics and mitochondrial function: implications for Alzheimer's disease risk and therapeutics

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ESTROGEN REGULATION OF BIOENERGETICS AND
MITOCHONDRIAL FUNCTION:
IMPLICATIONS FOR ALZHEIMER’S DISEASE RISK AND THERAPEUTICS

by
Jia Yao

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2010

Copyright 2010 Jia Yao
ii

Dedication
To my beloved parents, words cannot express how fortunate I am to have the
world’s best parents. I would not have achieved so far without your support.
To my grandma for her coming 82nd birthday, grandma is the spirit for the whole
family. Her wisdom of life is a great treasure to us all.

iii

Acknowledgements
First, I would like to thank my doctoral dissertation advisor, Professor Roberta
Diaz Brinton. In the past a few years, Robbie has set up an outstanding example of
scientist for me to follow. Her inspiring thoughts, her passion for science, her
perseverance in seeking the truth have taught me an invaluable lesson that will benefit me
life long. In addition to the extraordinary laboratory environment that she provided me,
she also set the highest standards of science and ethics for me to reach for as a graduate
student. My thanks also go to my Ph.D. thesis committee members, Dr. Enrique Cadenas,
Dr. Ronald Alkana, and Dr. Michel Baudry. I sincerely thank Dr. Cadenas for allowing
me conduct part of my research in his lab. Dr. Cadenas is a world-renown expert of
mitochondrial bioenergetics and oxidative stress and his expertise and sharp insights have
made my training more rewarding than a doctoral degree. Dr. Alkana and Dr. Baudry are
always nice and ready to offer me help at any time. I would like to thank my colleague
Dr. Ronald Irwin for his helpful collaborations in conducting mitochondrial isolation and
transgenic mouse work. Special thanks to Dr. Shuhua Chen for all her kind help on tissue
culture and immunohistochemistry. Indeed, I would like to thank all of the past and
present Brinton Research team members and coworkers, including Dr. Jon Nilsen, Dr.
Liqin Zhao, Dr. Junming Wang, Dr. Ryan Hamilton, Dr. Cristal Gama, Dr. Lifei Liu,
Jennifer Mao, Fan Ding, Jamaica Rettberg, Martha Hernandez, Eric Hernandez, Claudia
Lopez, Jimmy To, Michelle Wong, Yureli Lopez, and all those not listed who have
iv

helped me to achieve my research goals. It is a great honor and fortune for me to choose
to study at USC, a wonderful community of scholars where creativity and knowledge
spark.
I would like to specially thank Dr. Jon Nilsen. Jon devoted much of his time and
energy to my training experience at USC. He and Dr. Brinton have guided me into the
realm of mitochondrial biology and gonadal hormone actions in the brain. I felt really
lucky to start my research with his mentorship and the discussions with him have
contributed enormously to my success over the past several years.

v

Table of Contents
Dedication........................................................................................................................... ii
Acknowledgements............................................................................................................iii
List of Tables .................................................................................................................... vii
List of Figures..................................................................................................................viii
Abbreviations...................................................................................................................viii
Abstract............................................................................................................................xiii
Chapter I. Overview of Estrogen, Mitochondria, Brain Metabolism, and Alzheimer’s
Disease ................................................................................................................................ 1
Introduction ..................................................................................................................... 1
Significance ..................................................................................................................... 8
Specific Aims .................................................................................................................. 9
Chapter I References: .................................................................................................... 11

Chapter II. Mitochondrial Bioenergetic Deficit Precedes Alzheimer’s Pathology in
Female Mouse Model of Alzheimer’s Disease................................................................. 18
Abstract.......................................................................................................................... 18
Introduction ................................................................................................................... 19
Results ........................................................................................................................... 22
Discussion......................................................................................................................33
Material and Methods.................................................................................................... 39
Chapter II References .................................................................................................... 46

Chapter III. Estrogen Regulation of Mitochondrial Bioenergetics, Oxidative Stress
and Mitochondrial Localization of Alzheimer’s Pathology.............................................. 59
Abstract.......................................................................................................................... 59
Introduction ................................................................................................................... 60
Results ........................................................................................................................... 63
Discussion......................................................................................................................77
Materials and Methods .................................................................................................. 84
Chapter III References................................................................................................... 92

Chapter IV. Mitochondrial Scheme of Estrogen Induced Neuroprotection. ................... 97
Abstract.......................................................................................................................... 97
Introduction ................................................................................................................... 99
Results ......................................................................................................................... 101
Discussion.................................................................................................................... 115
Material and Methods.................................................................................................. 120
Chapter IV References................................................................................................. 126
vi

Chapter V. Therapeutic Implications of Regulating Mitochondrial Bioenergetics
in AD............................................................................................................................... 130
Abstract........................................................................................................................ 130
Introduction ................................................................................................................. 132
Results ......................................................................................................................... 133
Discussion.................................................................................................................... 142
Material and Methods.................................................................................................. 145
Chapter V References.................................................................................................. 151

Chapter VI. Conclusions................................................................................................. 153
Chapter VI References................................................................................................. 156

References....................................................................................................................... 157

vii

List of Tables
Table II-1. Mitochondrial enzyme function in the aging female nonTg and
3xTgAD mouse brain 25

Table V-1. Body weight change in ctrl and 2-DG group 138
viii

List of Figures
Figure I-1: Estrogen-induced signaling pathways mediating
neuroprotection and plasticity 5

Figure I-2: Overview of specific aims I-IV 9

Figure II-1: Age-related increase in Aβ immunoreactivity in
female 3xTgAD mice 22

Figure II-2: 3xTgAD female mice have decreased PDH E1α and COX
IV protein levels relative to age-matched nonTg female mice 24

Figure II-3: 3xTgAD female mice exhibit higher oxidative stress
than age-matched nonTg female mice 26

Figure II-4: 3xTgAD female mice exhibit higher lipid peroxidation
than age-matched nonTg female mice 27

Figure II-5: 3xTgAD female mice exhibit decreased mitochondrial
respiration relative to age-matched nonTg female mice 29

Figure II-6: Hippocampal neurons derived from 3xTgAD mouse brain
exhibit decreased mitochondrial respiration and increased glycolysis 31

Figure II-7: Female 3xTgAD mice have increased ABAD and mitochondrial
Aβ protein levels relative to age-matched nonTg female mice 32

Figure III-1:Uterine weight change with estrogen manipulation 63

Figure III-2: OVX induced decrease in mitochondrial respiration and E2
treatment prevented OVX-induced decrease in mitochondrial respiration 66

Figure III-3: OVX induced decrease in enzyme activity and E2
treatment prevented OVX-induced decrease in enzyme activity 67

Figure III-4: OVX induced increase in oxidative stress in brain and
E2 treatment prevented OVX-induced increase in oxidative damage 69

Figure III-5: OVX induced increase in brain and mitochondrial amyloid
load and E2 treatment prevented OVX-induced increase in amyloid load 71
ix

Figure III-6: OVX significantly increased amyloid pathology in 3xTgAD
mouse brains whereas E2 treatment prevented the OVX-induced increase 72

Figure III-7: Mitochondrial biogenesis was unaffected by estrogen
manipulation 73

Figure III-8: Estradiol differentially regulates mitochondrial
bioenergetics in neurons and mixed glia 76

Figure IV-1: Dose response of mitochondrial inhibitors 102

Figure IV-2: Therapeutic efficacy of E2 induced neuroprotection
against mitochondrial inhibitors 104

Figure IV-3: Estrogen protects against 3-NPA by inhibiting apoptosis
and reducing oxidative stress 105

Figure IV-4: E2 induced neuroprotection against Aβ toxicity 108

Figure IV-5: Aβ  induced mitochondrial DNA damage 109

Figure IV-6: E2 does not directly activate DNA repair pathway 110

Figure IV-7: Both ER α and ER β regulates mitochondrial function 112

Figure IV-8: E2 and P4 regulation of mitochondrial respiration 114

Figure IV-9: ERE and CRE in genes regulated by E2 117

Figure V-1: Different SCOT profile in nonTg and 3xTgAD mice 134

Figure V-2: Metabolic profile of neurons and mixed glia on
different substrates 136

Figure V-3: Study design 137

Figure V-4: Induction of ketogenesis by 2-DG diet 138

Figure V-5: Decreased amyloid pathology in 2-DG treated 3xTgAD mice 140
x

Figure V-6: 2-DG induction of enzymes in ketone metabolism and
α Secretase 141

Figure V-7: 2-DG induction of enzymes in ketone metabolism and
reduction in mouse APP 141

Figure V-8: Nutrient components of AIN93G and 2-DG (0.04%) diet 150

xi

Abbreviations
17-beta Estradiol E2
Adenosine 5’-diphosphate ADP
Adenosine 5’-triphosphate ATP
Alzheimer’s disease AD
Amyloid Binding Alcohol Dehydrogenase ABAD
amyloid precursor protein APP
Atractyloside ATR
central nervous system CNS
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
Familial AD FAD
Hydroxyacyl-Coenzyme A Dehydrogenase/
3-ketoacyl-Coenzyme A Thiolase/
Enoyl-Coenzyme A Hydratase (trifunctional protein), alpha subunit HADHA
Kilo/Milli/Micro gram kg/mg/μg
Milli/Micro molar mM/μM
medroxyprogesterone acetate MPA
mitochondria/mitochondrial mito
mitochondrial DNA mtDNA
mitochondrial permeability transition MPT
Non-transgenic nonTg
ovariectomy OVX
Peroxiredoxin V Prdx V
Potential of Hydrogen PH
Presenilin 1 PENS1/PS1
Presenilin 2 PENS2/PS2
Progesterone P4
Propyl Pyrazole Trisphenol PPT
Pyruvate Dehydrogenase PDH
Reactive Oxygen Species ROS
Respiratory Control Ratio RCR
Sodium Dodecyl Polyacrylamide Gel Electrophoresis SDS-PAGE
Sporadic AD SAD
Triple Transgenic Alzheimer’s disease mouse model 3xTgAD
Voltage Dependent Anion Channel VDAC
xii

Women’s Health Initiative WHI
WHI Memory Study WHIMS
xiii

Abstract

Presented here are a collection of studies that build on our existing knowledge of
estrogen actions on brain mitochondria. Previous studies from our lab as well as other
colleagues have demonstrated that estrogen (E2, 17β-estradiol) is neuroprotective against
neurotoxic insults including exposure to glutamate and amyloid beta (Aβ) (Nilsen et al,
2006; Simpkins and Dykens, 2007; Vina et al, 2007b). Further, the E2 induced
neuroprotective mechanisms converge on mitochondria (Brinton, 2008b; Nilsen and Diaz
Brinton, 2003; Nilsen et al, 2007). In the current research project, we have hypothesized
that mitochondria, particularly mitochondrial bioenergetics play a pivotal role in the
pathogenesis of age-related Alzheimer’s disease (AD) whereas estrogen proactively
sustains and enhances mitochondrial function that was compromised in aging and AD. To
test our hypotheses, we have conducted a combination of in vitro and in vivo analyses
which address four specific aims. Specific Aim 1 (Chapter 2) serves to investigate the
role of mitochondrial bioenergetic deficits in AD pathogenesis using the triple transgenic
Alzheimer’s mouse model. Specific Aim 2 (Chapter 3) serves to determine the impact of
E2 deficiency on mitochondrial function and AD progression. Specific Aim 3 (Chapter 4)
seeks to investigate the underlying mechanism of estrogen induced neuroprotection as
well as to examine the role of different estrogen receptors in estrogen action in the brain.
Specific Aim 4 (Chapter 5) focuses on the therapeutic implications of the findings from
the previous three aims and introduces a novel therapeutic strategy targeting
mitochondria for preventing AD and/or delaying the progression of AD.
xiv

Data from these four specific aims have demonstrated that: 1) Mitochondrial bioenergetic
deficit precedes Alzheimer’s pathology in the 3xTgAD mouse model. 2) Loss of ovarian
hormones either due to age-related reproductive senescence or surgical ovariectomy
(OVX) significantly exacerbates mitochondrial dysfunction that occurs in aging or AD.
3) E2 treatment initiated at the time of OVX prevented the OVX-induced mitochondrial
deficits by sustaining mitochondrial bioenergetic capacity, decreasing oxidative stress,
and preventing mitochondrial amyloid and ABAD induction. 4) Therapeutic strategies
targeting mitochondria could alter AD progression by manipulating brain mitochondrial
bioenergetic profile.
We believe that findings from these studies will expand our understanding of the
mechanism of AD pathogenesis as well as the pharmacological pathway of estrogen
induced neuroprotection against AD. Our research will also enable the development of
novel therapeutics that target mitochondrial bioenergetics for the prevention and
treatment of AD and other neurodegenerative diseases that could be partially attributed to
mitochondrial dysfunction.
1

Chapter I Overview of Estrogen, Mitochondria, Brain Metabolism, and
Alzheimer’s Disease
Introduction

Estrogen has long been demonstrated to protect neurons against a variety of
neurotoxic insults. Estrogen, particularly E2 (17β-estradiol, the most potent form of
endogenous estrogen steroid hormones) is neuroprotective against various neurotoxic
insults including exposure to glutamate and amyloid beta (Aβ) (Nilsen et al, 2006;
Simpkins et al, 2007; Vina et al, 2007b). Further, estrogen induced neuroprotective
mechanisms converge on mitochondria and depend on intact mitochondrial function
(Brinton, 2008b; Nilsen et al, 2003; Nilsen et al, 2007). The current study builds on and
expands our knowledge of estrogen action in the brain by investigating estrogen
regulation of brain mitochondrial bioenergetics in the aging and AD process. Briefly, in
this study we sought to 1) address the role of mitochondria in AD pathogenesis; 2)
determine the impact of estrogen loss on brain mitochondria in AD and aging; 3)
investigate the underlying mechanism of estrogen induced neuroprotection; and 4)
explore the therapeutic potential of sustaining brain mitochondrial bioenergetics to
prevent AD or delay disease progression. Findings from this study contribute to a large
scheme of estrogen action in the brain where a combination of different estrogenic
actions leads to the convergence of neuronal survival with the maintenance of
mitochondrial function.
2

Mitochondria and Brain Metabolism
Mitochondria evolved from a symbiotic relationship between aerobic bacteria and
primordial eukaryotic cells(McBride et al, 2006). Mitochondria produce the majority of
ATP molecules in oxidative phosphorylation (OXPHOS). Other important roles of
mitochondria in cellular activities include maintaining cellular Ca
2+
homeostasis and
determining cell death through the convergence of many apoptotic pathways onto the
mitochondrial permeability transition (MPT)(Abramov et al, 2004). In addition,
mitochondria are the major sources of Reactive Oxygen Species (ROS), a byproduct of
OXPHOS (Trushina and McMurray, 2007).
Brain, as the center for thought, emotion, and memory, is among the organs with
the highest energy demand. While only accounting for 2% of total body weight, brain
consumes up to 20% of the energy. Under normal conditions, brain exclusive relies on
glucose for energy production via aerobic oxidation. Upon uptake, glucose is converted
into pyruvate through a series of glycolytic pathways. The oxidation of pyruvate yields
the energy rich compound acetyl CoA, which either condenses with oxaloacetate into
Krebs cycle coupled with OXPHOS for ATP generation or participates in the formation
of the neurotransmitter acetylcholine or the biosynthesis of cholesterol (Blass, 2001;
Hoyer, 2000). In addition to serving the regular cellular and molecular activities such as
biomolecule synthesis, transport, and degradation, a huge amount of ATP molecules are
utilized to maintain homeostasis or restore electrochemical gradient for synaptic function.

3

Alzheimer’s Disease Etiology
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease among
the aged population. The disease is symptomatically characterized by progressive
memory deficits, cognitive impairments and personality change, which are caused by
progressive synaptic dysfunction and the subsequent loss of neurons in certain area of the
brain, including the neocortex, the limbic system and the subcortical regions (Fassbender
et al, 2001). From a histo-pathological view, AD is characterized by senile plaques and
neurofibrillary tangles in the medial temporal lobe and cortical areas of the brain together
with neurodegeneration and loss of synaptic functions (Hansson et al, 2006). According
to the genetics of the disease, AD has been categorized into two major forms: familial
AD (FAD) and sporadic AD (SAD) or age-related AD, with the latter being the leading
cause of dementia, accounting for about 50~60% of all cases (Brookmeyer et al, 1998).
FAD is an autosomal dominant disorder with the onset age before 65. The majority of
FAD cases have been attributed to mutations in three genes, the amyloid precursor
protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2)(Chen and Yan, 2007). In
terms of SAD, age has been identified as the greatest risk factor (von Strauss et al, 1999).
The prevalence of AD increases exponentially with age in people aged 65 or older
(Hansson et al, 2006). Other candidate risk factors include ApoE4 alleles, Neprilysin
(NEP) and gender. Noticeably, post-menopause women have a much higher risk of
developing SAD than age-matched men. Currently, the cause of SAD has not been fully
identified. The suggested causes of SAD include amyloid beta overproduction, TAU
4

hyperphosphorylation, oxidative stress, cholesterol deregulation, and metabolic
dysfunction (Dolev and Michaelson, 2004; Hoyer, 2000; Oddo et al, 2003b; Sorrentino
and Bonavita, 2007).
Mitochondrial dysfunction in Alzheimer’s disease
Previous studies have demonstrated that compared to age-matched control
individuals, many tissues from AD patients have significantly lower mitochondrial
respiratory function. The decline of mitochondrial function is especially amplified in
tissues with high energy demand, such as brain, heart and liver. In the central nervous
system (CNS), neurons rely heavily on mitochondria for energy production and the
maintenance of ionic homeostasis. In AD patients, the following activities have been
suggested to cause the loss of synaptic activity and cognitive decline, which are all
closely related with mitochondrial dysfunction(Reddy, 2008; Reddy and Beal, 2008a;
Reddy et al, 2008).
i) The decrease of metabolic functions has been observed in frontal areas, temporal
and parietal cortices.
ii) The deregulation of calcium in synapses has been suggested to be the primary
cause of AD.
iii) Activation of apoptotic pathways in vulnerable neuron populations has been
observed in AD patients.
iv) Overproduction of reactive oxygen species and higher oxidative stress have been
observed in AD brains (Atamna and Frey, 2007).
5

Estrogen-induced Neuroprotection: Current Models and Remaining Challenges

Figure I-1. Estrogen-induced signaling pathways Mediating Neuroprotection and
Plasticity. Estrogen mechanisms of action converge upon the mitochondria. Estrogen
(17β-estradiol; E2) binding to a membrane-associated estrogen receptor (ER) undergoes a
protein–protein interaction with the regulatory subunit of PI3K, p85, to activate the diver
gent but coordinated activation of the Akt and MAPk signaling cascades. These E2-
induced signaling pathways in hippocampal and cortical neurons converge upon the
mitochondria to enhance glucose uptake and metabolism, aerobic glycolysis and pyruvate
dehydrogenase to couple aerobic glycolysis to acetyl-CoA production and tricarboxylic
acid cycle (TCA) -coupled oxidative phosphorylation and ATP generation. In parallel, E2
increases antioxidant defense and antiapoptotic mechanisms. Estrogen receptors at the
membrane, in mitochondria and within the nucleus are well positioned to regulate
coordinated mitochondrial and nuclear gene expression required for optimal bioenergetics.
Enhancing and sustaining glycolysis, aerobic metabolism and mitochondrial function
would be predicted to prevent the shift to alternative fuel sources and the hypometabolism
characteristic of Alzheimer’s disease (Brinton, 2008a).
6

Both in vitro and in vivo animal studies have provided robust evidence of estrogen
induced neuroprotection against a broad range of neurotoxic insults, including free
radicals generators, excitotoxicity, amyloid beta induced neurotoxicity and hypoxia
conditions in ischemia and stroke (McClean and Nunez, 2008; Tripanichkul et al, 2007;
Valles et al, 2008; Wang et al, 2006). The mechanisms of estrogen induced
neuroprotection include, but not limited to, activation of both the classical nuclear
receptors and novel membrane receptor, which subsequently initiate signaling cascades
and increase the expression of certain neurotrophic genes, including brain derived
neurotrophic factor (BDNF) and the anti-apoptotic proteins Bcl-2 and Bcl-xL (Fig.I-1). In
addition, epidemiological analyses indicate that estrogen promotes neurological health
and reduces the risk of Alzheimer’s disease (Kawas et al, 1997; Lerner et al, 1997;
Lerner, 1997; Paganini-Hill, 1996; Paganini-Hill and Henderson, 1994, 1996a, b; Yaffe et
al, 1998a; Yaffe et al, 1998b).
Despite all the positive evidence from animal studies and epidemiological
analyses, the outcome from the Women’s Health Initiative Memory Study (WHIMS) was
surprisingly unanticipated (Shumaker et al, 2004; Shumaker et al, 2003). Results from
the study revealed that women receiving the hormone therapy of a combination of
conjugated equine estrogens and medroxyprogesterone acetate (MPA) had a two-fold
higher risk of developing AD than women in the placebo arm. In addition, women
receiving the estrogen-only therapy were not statistically different from women receiving
the placebo, but there was a trend of greater risk for AD and mild cognitive impairment.
7

The apparent conflicts between basic science and clinical outcome were rather disturbing
and could partially be attributed to the incapability of animal models to completely
recapitulate the conditions in human beings. Nevertheless, as our understanding of the
mechanisms of estrogen-induced neuroprotection accumulates, new insights have
developed arguing that the interpretation of the WHIMS is limited due to the hormone
preparation used, the regimen of hormone administration and the advanced age of
subjects under study (Bieber and Cohen, 2001; Dunne and Seaton, 2001; Grady et al,
2002; Hogervorst et al, 2002a, b; Huang et al, 2006; Kurt et al, 2006; Lavi et al, 2007;
Lethaby et al, 2008). Re-evaluation of the WHIMS and other similar studies has
suggested that the beneficial outcome of hormone therapy depends stringently on the time
of therapy initiation (Honjo et al, 2005). Moreover, our in vitro analyses indicate that E2

treatment of hippocampal neurons prior to amyloid insults protects neurons against
amyloid induced neuron death whereas post-insult exposure to E2

actually shows no
benefits or even exacerbates neuronal death (Chen and Chan, 2006a; Chen and Yan,
2006c).
In summary, current knowledge strongly supports a neuroprotective action of E2

against AD insults. As the understanding of E2

induced neuroprotection expands, basic
science discoveries will be better translated and applied to clinical practice.
Estrogen Regulation of Mitochondrial Function
Previous studies have identified mitochondria as the major target for estrogen
regulation (Duckles et al, 2006; Nilsen and Brinton, 2004; Simpkins et al, 2005).
8

Estrogen has been demonstrated to increase the capacity of mitochondria to
accommodate calcium overload (Nilsen et al, 2003). In addition, estrogen protects cells
from ROS by either acting directly as a free radical scavenger or by increasing the
expression of anti-oxidant proteins, such as MnSOD and peroxiredoxin (Irwin et al,
2008a; Nilsen et al, 2007; Simpkins et al, 2005). Further, estrogen regulates
mitochondrial respiration by increasing the expression of key mitochondrial proteins,
including Pyruvate Dehydrogenase (PDH), Cytochrome c Oxidase, and ATP synthase
(Nilsen et al, 2007). Estrogen also increases anti-apoptotic proteins, such as Bcl-2 and
Bcl-xL. The coordinated actions of estrogen on the cell proteome converge upon
mitochondria and regulate mitochondrial functions.
Significance

From a basic science perspective, the presented studies contribute to a better
understanding of (1) the mechanism of AD pathogenesis and (2) the pharmacological
pathway of estrogen induced neuroprotection against AD.
From a translational perspective, the presented studies expand the knowledge of the
neuroprotective mechanisms of estrogen, which, in turn, will facilitate the development
of new therapeutics against AD and other age-related neurodegenerative diseases.
From a clinical perspective, the health benefits and risks of estrogen replacement
therapy (ERT) remain a topic of controversy. Elucidation of the sites and the targets of
estrogen action will have a definite impact on the use of hormone replacement therapy.
9

Specifically, results from our studies will provide new targets for the development of
novel molecules that are efficacious in the prevention and treatment of neurodegenerative
diseases and aging.

Specific Aims

In the current study, we set up four specific aims, each of which serves to
accomplish a specific goal as shown in Fig.I-2.
Figure I-2. Overview of specific aims I-IV. Focused on mitochondria, each specific aim
addresses a specific question. Specific aim 1 investigate the relationship between
mitochondrial bioenergetic deficits and AD pathogenesis and provides the basis for specific
aim II; Specific aim II focuses on estrogen regulation of mitochondrial function and
pathology in AD; Specific aim III aims to mechanistically investigate the underlying
mechanism of estrogen regulation of mitochondrial function; Specific aim IV is based on
the previous specific aims and investigates the therapeutic potential of manipulating brain
mitochondrial function.
10

Specific aim 1 investigates the relationship between mitochondrial bioenergetic
deficits and AD pathogenesis and provides the basis for specific aim II; Specific aim II
focuses on estrogen regulation of mitochondrial function and pathology in AD; Specific
aim III aims to mechanistically investigate the underlying mechanism of estrogen
regulation of mitochondrial function; Specific aim IV is based on the previous specific
aims and investigates the therapeutic potential of manipulating brain mitochondrial
function.
The ultimate goal of this study is to expand our knowledge of estrogen
neuroprotective action in the brain, to better understand the pathogenesis of AD, and to
facilitate the development of novel therapeutics that target mitochondria for AD
prevention and treatment.
11

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18

Chapter II Mitochondrial Bioenergetic Deficit Precedes Alzheimer’s
Pathology in Female Mouse Model of Alzheimer’s Disease

Jia Yao, Ronald W. Irwin, Liqin Zhao, Jon Nilsen, Ryan T. Hamilton and Roberta Diaz
Brinton
Published in Proceedings of the National Academy of Sciences, 2009 Aug
25;106(34):14670-5. Epub 2009 Aug 10
Chatper II Abstract

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative
diseases, including Alzheimer’s disease (AD). To address whether mitochondrial
dysfunction precedes the development of AD pathology, we conducted mitochondrial
functional analyses in female triple transgenic Alzheimer’s mice (3xTgAD) and age-
matched non-transgenic (nonTg). Mitochondrial dysfunction in the 3xTgAD brain was
evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase
(PDH) protein level and activity as early as 3 months of age. 3xTgAD mice also
exhibited increased oxidative stress as manifested by increased hydrogen peroxide
production and lipid peroxidation. Mitochondrial amyloid beta (Aβ) level in the 3xTgAD
mice was significantly increased at 9 months and temporally correlated with increased
level of amyloid beta binding to alcohol dehydrogenase (ABAD). Embryonic neurons
derived from 3xTgAD mouse hippocampus exhibited significantly decreased
19

mitochondrial respiration and increased glycolysis. Results of these analyses indicate that
compromised mitochondrial function is evident in embryonic hippocampal neurons,
continues unabated in females throughout the reproductive period and is exacerbated
during reproductive senescence. In non-transgenic control mice, oxidative stress was
coincident with reproductive senescence and accompanied by a significant decline in
mitochondrial function. Reproductive senescence in the 3xTgAD mouse brain markedly
exacerbated mitochondrial dysfunction. Collectively, the data indicate significant
mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse
model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the
hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for
prevention of AD.
Introduction

The essential role of mitochondria in cellular bioenergetics and survival has been
well established (Beal, 2007; Duchen, 2004; Wallace, 2008). Previous studies have
suggested that mitochondrial dysfunction plays a central role in the pathogenesis of
neurodegenerative disorders, including Alzheimer’s disease (AD) (Beal, 2007; Brinton,
2008b). Alzheimer’s pathology is accompanied by a decrease in expression and activity
of enzymes involved in mitochondrial bioenergetics, which would be expected to lead to
compromised electron transport chain complex activity and reduced ATP synthesis (Blass
et al, 2000). Further, in AD there is a generalized shift from glycolytic energy production
20

towards use of an alternative fuel, ketone bodies. This is evidenced by a 45% reduction in
cerebral glucose utilization in AD patients (Ishii et al, 1997), which is paralleled by
decrease in the expression of glycolytic enzymes coupled to a decrease in the activity of
the pyruvate dehydrogenase complex (Blass et al, 2000). Patients with incipient AD
exhibit a utilization ratio of 2:1 glucose to alternative fuel whereas comparably aged
controls exhibit a ratio of 29:1 while young controls exclusively use glucose as with a
ratio of 100:0 ratio (Hoyer, 1991). In addition to the lowered mitochondrial bioenergetic
capacity, impairment of oxidative phosphorylation is associated with increased free
radical production and the resultant oxidative damage. Overproduction of reactive
oxygen species and higher oxidative stress is characteristic of brains from AD (Atamna et
al, 2007). Increased oxidative stress, coupled with dysregulation of calcium homeostasis
and resulting apoptosis of vulnerable neuronal populations are proposed to underlie the
loss of synaptic activity and initiate cognitive decline (Reddy and Beal, 2008b).
In addition to the mitochondrial dysfunction in the clinically confirmed AD cases,
multiple analyses have demonstrated that mitochondrial dysfunction is a plausible
contributing factor in the pathogenesis of sporadic AD. The “cybrid model” of AD has
provided evidence for mitochondrial dysfunction in AD pathogenesis (King and Attardi,
1989). AD cybrid cells exhibit decreased COX activity, decreased mitochondrial
membrane potential, decreased mitochondrial mobility and motility, increased oxidative
stress, over activation of caspase-3, and increased Aβ production (Cardoso et al, 2004;
Khan et al, 2000; Swerdlow et al, 1997). Further, increased risk of AD occurs in
21

offspring of women with AD suggesting human maternal mitochondrial inheritance
(Apostolova et al, 2008; Mosconi et al, 2009). Collectively, these observations suggest a
role for mitochondrial dysfunction in the pathogenesis of sporadic AD.
To determine whether mitochondrial bioenergetic mechanisms are associated with
AD pathogenesis, we assessed mitochondrial function in the triple transgenic AD mouse
model (3xTgAD) developed by Oddo, LaFerla and colleagues (Oddo et al, 2003a; Oddo
et al, 2003b). This transgenic mouse strain bears mutations in three genes(human
APP
SWE
, Tau
P301L
, and PS1
M146V
genes) linked to AD and fronto-temporal dementia
(FTD) and exhibits an age-related neuropathological phenotype including both amyloid
beta deposition and tau hyperphosphorylation (Carroll et al, 2007; Oddo et al, 2003a). To
characterize the change in mitochondrial functions in this model, we conducted
biochemical and functional assays on whole brain mitochondria isolated from both
3xTgAD and nonTg female mice at different ages. The results presented herein indicate
that mitochondrial dysfunction, especially that leading to compromised energy
production, precedes plaque formation, the hallmark histopathology of AD. Collectively
current clinical findings and our data are suggestive of a potential causal role of
mitochondrial dysfunction in AD pathogenesis. From a therapeutic aspect, these data
indicate a new strategy that targets mitochondrial bioenergetics to prevent or delay the
development of AD.

22

Results

Age-dependent development of AD-like pathology in female 3xTgAD mice. The triple-
transgenic AD mouse model has been demonstrated to exhibit an age-related
neuropathological progression pattern(Carroll et al, 2007; Oddo et al, 2003a). To
determine the temporal correlation between mitochondrial dysfunction and AD
histopathology, we characterized the age-dependent development of amyloid pathology
in the triple-transgenic mouse model. Aβ accumulation in the hippocampal CA1 region
was assessed in both gonadally intact 3xTgAD and nonTg female mice at 3, 6, 9 and 12
months of age. We observed minimal Aβ-Immunoreactivity (IR) in the hippocampal CA1
region in the 3xTgAD mice at 3 months. Aβ-IR increases in an age-dependent way in
3xTgAD mice and at 12 months, overt amyloid plaque formation is observed, whereas no
Aβ-IR is observed in nonTg mice at all age groups (Fig.II-1).

Figure II-1. Age-related increase in Aβ immunoreactivity in female 3xTgAD mice.
Representative images show Aβ-IR in hippocampus CA1 region from female 3xTgAD
mice at 3, 6, 9 and 12 months and female nonTg mice at 12 month. (scale: 100 μm)

23

Decreased expression and activity of key regulatory enzymes of oxidative
phosphorylation in female 3xTgAD mice. Decreased expression and activity of
cytochrome c oxidase and pyruvate dehydrogenase have been observed in postmortem
brain tissue derived from Alzheimer’s patients (Blass et al, 2000). To determine if
3xTgAD mice recapitulated these mitochondrial defects and to identify the temporal
correlation with AD histopathology, hippocampal proteins were isolated from another set
of intact female 3xTgAD and nonTg mice at 3, 6, 9 and 12 months of age. Expression of
pyruvate dehydrogenase (PDH; PDH E1α) and cytochrome c oxidase (COX; COX
subunit IV) was assessed by Western blot analysis. PDH E1α expression in 3xTgAD
mice was decreased relative to age-matched nonTg mice (Fig. II-2A; P<0.05, n=6).
Significantly decreased PDH E1α expression was evident as early as 3 months of age and
was greatest at 12 months of age (Fig.II-2A). Likewise, COX IV expression was
decreased in 3xTgAD mice compared to age-matched nonTg mice and was significantly
decreased at 9 months of age (Fig.II-2B).
To confirm that the changes in protein expression were indicative of changes in
enzyme activity, PDH and COX activities were assessed in mitochondria isolated from
whole forebrain of the same set of 3xTgAD and nonTg mice utilized for the hippocampal
PDH E1α and COX IV expression. Both PDH and COX activities were decreased in the
aging 3xTgAD mice as compared to the age-matched nonTg mice (Table II-1). While
PDH E1α expression was significantly decreased as early as 3 months of age in the
3xTgAD mice, significant decline in PDH activity was first evident at 9 months of age.
24

The preserved PDH enzyme activity relative to the PDH E1α subunit expression in the 3
and 6 month old 3xTgAD mice may be indicative of compensatory upregulation of PDH
activity by posttranslational modification. As with PDH activity the decline in COX
activity was also significantly decreased at 9 months of age (Table II-1).

Figure II-2. 3xTgAD female mice have decreased PDH E1α and COX IV protein
levels relative to age-matched nonTg female mice. Equal amount of hippocampal (for
PDH E1 α) and mitochondrial (for COXIV) samples from both 3xTgAD and nonTg mice
of different age groups were loaded onto the gel. Expression of (A) PDH E1α subunit and
(B) COX IV subunit were determined by Western blot analysis. Bars represent mean
relative expression ± S.E.M. (*=p<0.05 compared to age-matched nonTg group; n=6).
25

Table II-1. Decreased PDH and COX activity in female 3xTgAD Mice.
AGE
(MONTHS)
PDH ACTIVITY
(NMOL/MIN/MG PROTEIN)
RELATIVE COX ACTIVITY
(NORMALIZED TO 3 MONTH
NONTG)
NONTG 3XTGAD NONTG 3XTGAD
3 116.45±9.89 126.2±5.46 100±7.25 105.8±6.07
6 83.73±5.37 76.91±5.12 94.81±7.48 92.22±7.33
9 107.07±6.14 86.94±4.54*100.2±11.62 74.65±9.48*
12 79.43±5.66 56.59±1.37* 69.1±2.60 52.7±0.63*

Increased oxidative stress in brain mitochondria of 3xTgAD mice. Mitochondrial
dysfunction is associated with oxidative stress and development of AD neuropathology.
To determine the oxidative load of brain mitochondria in the 3xTgAD mice we assessed
the rate of hydrogen peroxide production and magnitude of lipid peroxidation in
mitochondria isolated from whole forebrain of female 3xTgAD and nonTg mice at 3, 6, 9
and 12 months of age. The Amplex Red hydrogen peroxide assay was used to determine
the rates of hydrogen peroxide production of mitochondria in both state 4 (in the absence
of ADP) and state 3 (in the presence of ADP) respiration. There was an age-associated
Table II-1. Mitochondrial enzyme function in the aging female nonTg and
3xTgAD mouse brain. Brain mitochondria isolated from both nonTg and
3xTgAD mice were assessed for PDH and COX activity. Relative COX Activity
was presented as the relative value normalized to that of 3 month nonTg female
mice. Mean ± SEM (*=p<0.05 compared to age-matched nonTg group, n=6).
26

increase in the rate of state 4 hydrogen peroxide production in both the 3xTgAD and
nonTg mice (Fig.II-3; p<0.05; n=6). More importantly, there was a significant increase in
the rate of state 4 hydrogen peroxide production in the 3xTgAD mice as compared to
age-matched nonTg mice that was evident as early as 3 month of age and was most
pronounced at 12 months of age (Fig.II-3; p<0.05; n=6).

Production of hydrogen peroxide in the nonTg female mice increased at 9 months
of age to a level comparable to, and thus not significantly different than, that generated
by comparable aged 3xTgAD mice.
Figure II-3. 3xTgAD female mice exhibit higher oxidative stress than age-matched
nonTg female mice. Whole brain mitochondria were isolated from intact female mice and
rates of hydrogen peroxide production were determined in State 4 respiration by Amplex-
Red hydrogen peroxide assay. Bars represent mean hydrogen peroxide production rates ±
S.E.M. (*=p<0.05 compared with age-matched nonTg group; #=p<0.05 compared between
different age group within the same genotype, n=6).
27

Increased hydrogen peroxide production would be expected to lead to a rise in
oxidative damage to cellular components. Therefore, we measured lipid peroxidation as
an indicator of overall oxidative stress. Both whole forebrain mitochondria and
hippocampal lysates were used to determine lipid peroxidation which yielded comparable
results. Correlated with the increased rate of hydrogen peroxide production, there was
significant age-related increase in lipid peroxidation in both the 3xTgAD and nonTg mice
in both isolated mitochondria (Fig. II-4; p<0.05; n=6) and hippocampal lysates. Further,
there was a significant increase in lipid peroxidation in the 3xTgAD mice as compared to
age-matched nonTg mice in both isolated mitochondria (Fig.II-4; p<0.05; n=6) and
hippocampal lysates.

Decreased brain mitochondrial respiratory efficiency in 3xTgAD mice. Decreased
expression and activity of the key mitochondrial regulatory enzymes, PDH and COX,
would be expected to result in decreased oxidative phosphorylation and impaired
mitochondrial respiratory efficiency. To determine if oxidative phosphorylation was
Figure II-4. 3xTgAD female
mice exhibit higher lipid
peroxidation than age-
matched nonTg female mice.
Whole brain mitochondria were
isolated from intact female
mice and lipid peroxide levels
were determined by the
leucomethylene blue assay.
Bars represent mean ± S.E.M.
(*=p<0.05 compared with age-
matched nonTg group; n=6).

28

altered in the 3xTgAD mice, mitochondrial respiration was determined in freshly isolated
whole forebrain mitochondria from female 3xTgAD and nonTg mice at 3, 6, 9 and 12
months of age. Respiratory rate of isolated whole brain mitochondria was first
determined 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. In
nonTg group, an age-related decline in the respiratory control ratio (RCR; state 3: state
4
o
) was apparent from 3 to 9 month and reached statistical significance at 12 month when
compared to 3 month. Similarly in 3xTgAD mice, there was also an age-related decline in
RCR, which also reached statistical significance at 12 month when compared to either 3,
6, or 9 month (Fig.II-5A, p<0.05; n=6). More importantly, compared with the age-
matched nonTg group, 3xTgAD mice showed decreased RCR at each age, and this
genotype-related impairment of mitochondrial respiration deteriorated with age and was
most pronounced at 12 months of age (Fig.II-5A, p<0.05; n=6).
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 by COX enzyme. Similar to the change in RCR, in both 3xTgAD
and nonTg mice, the age-related increase in the free radical leak with malate/glutamate
plus ADP (state 3) was apparent and reached statistical significance at 12 months when
compared to either 3, 6 or 9 month (Fig.II-5B, p<0.05; n=6). More importantly, the free
29

radical leak was significantly increased in the 3xTgAD mice as compared to age-matched
nonTg mice at 3, 6 and 12 months of age (Fig. II-5B; p<0.05; n=6). As with hydrogen
peroxide generation, the lack of statistically significant effect between nonTg and
3xTgAD females at 9 months of age was due to the rise in the free radical leak of the
nonTg to a level comparable to that of the 3xTgAD mice (Fig.II-5B).
To determine the cellular contribution to the mitochondrial deficits of 3xTgAD
mouse brain, basal cellular respiration and glycolysis in primary neuronal cultures from
3xTgAD and nonTg mice harvested on embryonic day 14 and cultured for 1 week in
vitro. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR)
were determined using the Seahorse XF-24 metabolic flux analyzer. In primary neuronal
cultures >80% of the OCR measured was due to oxidative phosphorylation (Fig. II-6A)
and >89% of the ECAR measured was due to lactic acid production via glycolysis.
Figure II-5. 3xTgAD female mice exhibit decreased mitochondrial respiration relative
to age-matched nonTg female mice. Whole brain mitochondria were isolated from intact
female mice and state 3/state 4 respiration was determined. Oxygen electrode
measurements of respiration using isolated brain mitochondria from 3xTgAD and nonTg
mice were conducted 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. Bars
represent the mean ± S.E.M. of the respiratory control ratio (RCR, state3/state 4
respiration) (*=p<0.05 as compared to age-matched nonTg group; #=p<0.05 compared
between different age group within the same genotype, n=6).
30

Neurons derived from the 3xTgAD mice exhibited significantly lower OCR relative to
the OCR of nonTg neurons (Fig.II-6A; p<0.05; n=3). Correlated with the decreased
oxygen consumption was increased glycolysis, as evidenced by a significant increase in
the ECAR (Fig.II-6C). The addition of the ATP synthase inhibitor oligomycin (1 µM)
resulted in ~60% decrease in OCR in neurons from both 3xTgAD and nonTg mice (Fig.
II-6), indicating that the measured oxygen consumption was largely driven by oxidative
phosphorylation-coupled ATP generation. That the decrease in OCR in response to
oligomycin was similar in both nonTg and 3xTgAD groups indicates that the decline in
oxygen consumption was not due to a direct impairment of ATP synthesis. The decrease
in oxygen consumption in response to oligomycin was correlated with an increase in
ECAR (Fig.II-6C), indicating a shift to ATP production through glycolysis via the
Pasteur effect (Guppy et al, 1995). The addition of a mitochondrial uncoupler (FCCP; 1
µM) resulted in a dramatic increase in OCR, as expected, giving an estimation of the
maximal respiratory capacity of the mitochondria. Both the direct measurement of OCR
as well as the percent increase over baseline in response to FCCP were significantly
lower in hippocampal neurons derived from the 3xTgAD mice as compared to the nonTg
mice (Fig.II-6A&B; p<0.05; n=3). These data suggest an impairment of the reserve
respiratory capacity in the 3xTgAD neurons that would potentiate mitochondrial
dysfunction in the face of increasing metabolic demand. Further, the decreased maximal
respiratory capacity is consistent with the impaired COX activity in the 3xTgAD mice.
The addition of the complex I inhibitor rotenone resulted in a further reduction in OCR
31

values to about 15% of baseline in neurons from both 3xTgAD and nonTg mice (Fig.II-
5). The ~25% difference in OCR values between oligomycin and rotenone exposure in
both 3xTgAD and nonTg neurons indicates that both groups had equivalent oxygen
consumption due to proton leakage. The residual OCR capacity in the presence of
rotenone most likely represents cellular oxygen consumption by non-mitochondrial
pathways.

Figure II-6. Hippocampal neurons derived from
3xTgAD mouse brain exhibit decreased
mitochondrial respiration and increased glycolysis.
Primary embryonic neurons derived from both 3xTgAD
and nonTg mice were cultured in Neurobasal medium +
B27 supplement for 7 days. Oxygen consumption rate
(OCR) and extracellular acidification rate (ECAR) were
determined using Seahorse XF-24 Metabolic Flux
analyzer. (A) Oxygen consumption rates (OCR) in
primary neurons from 3xTgAD mice (black) have
lower basal rates of mitochondrial respiration than
primary neurons derived from nonTg mice (gray).
Vertical lines indicate time of addition of mitochondrial
inhibitors A: oligomycin (1 µM), B: FCCP (1 µM) or
C: rotenone (1 µM). The maximal respiratory capacity
(FCCP) is significantly lower in neurons from 3xTgAD
mice than those from nonTg mice. (B) Percent change
in mitochondrial respiration in response to
mitochondrial inhibitors. Bars represent the mean
change in OCR from baseline ± S.E.M. (*=p<0.05 as
compared to age-matched nonTg group; n=3). C).
Temporal bioenergetic profiling (ECAR vs. OCR) of
primary hippocampal neurons following exposure to
mitochondrial inhibitors A: oligomycin, B: FCCP and
C: Rotenone. (*=p<0.05 as compared to nonTg
cultures; n=3).
32

Increased mitochondrial Aβ levels in 3xTgAD mice. Previous studies indicated that
amyloid beta (Aβ) interacts with the mitochondrial protein, amyloid beta binding alcohol
dehydrogenase (ABAD) and that binding of Aβ contributes to mitochondrial dysfunction
(Lustbader et al, 2004; Yan and Stern, 2005). To determine the relationship between
mitochondrial Aβ and the above observed mitochondrial dysfunction, mitochondrial and
hippocampal lysates from female 3xTgAD and nonTg female mice at 3, 6, 9 and 12
months of age were analyzed by Western blot for ABAD and mitochondrial Aβ levels.
ABAD protein levels decreased with age in nonTg mice whereas ABAD increased with
age in 3xTgAD mice, which was clearly apparent and significant by 9 and 12 months of
age the 3xTgAD (Fig.II-7A; p<0.05; n=6). Likewise, at 9 month there was a significantly
Figure II-7. Female 3xTgAD mice have increased ABAD and mitochondrial Aβ protein
levels relative to age-matched nonTg female mice. Equal amount of hippocampal (for
ABAD) and mitochondrial (for Aβ) samples from both 3xTgAD and nonTg mice of different
age groups were loaded onto the gel. Expression of ABAD and 16 kD mitochondrial Aβ
oligomer were determined by Western blot analysis. A, 3xTgAD mice have increased ABAD
level than age-matched nonTg mice; B, Increased mitochondrial Aβ oligomer in the 3xTgAD
mice at 9 month. Bars represent mean relative expression ± S.E.M. (*=p<0.05 compared to
age-matched nonTg group; n=6).
33

higher level of Aβ oligomer level in the mitochondria of 3xTgAD mice as compared to
age-matched nonTg mice (Fig.II-7B; p<0.05; n=6).
Discussion

Increasing evidence implicates mitochondrial dysfunction in multiple
neurodegenerative disorders (Blalock et al, 2004). In this report, we demonstrate that the
female 3xTgAD mouse brain recapitulates multiple indicators of mitochondrial
dysfunction found in the human AD patients, including decreased mitochondrial
bioenergetics, increased oxidative stress, and increased mitochondrial amyloid load in the
3xTgAD mouse model (Brinton, 2008b; Reiman et al, 2004). Moreover, mitochondrial
dysfunction evident in embryonic neurons was sustained throughout postnatal and
reproductive ages and was most apparent following reproductive senescence at 12
months of age. Of particular importance is that the onset of mitochondrial dysfunction in
the 3xTgAD female mouse precedes the onset of plaque formation, the classical
histopathological marker of AD pathology. Although, dysfunction of each of the
individual components was not observed at the same time, mitochondrial dysfunction is
more than the sum of its components. Sustained disturbances in the pathway balances or
functional efficiency can lead to systems-level defects not observable in additional
individual components until later. Such early imbalances can lead to accumulated
changes in the mitochondria that later emerge as observable changes in other aspects of
the system. Collectively, our findings indicate a critical link between the transgenes
34

APP
swe
, PS1
M146V
and Tau
P301L
and mitochondrial dysfunction during early
neuropathogenesis.
Both the expression and activity of pyruvate dehydrogenase and cytochrome c
oxidase in 3xTgAD mitochondria were significantly decreased. Reduced mitochondrial
efficiency occurred in neurons as evidenced by the shift from oxidative phosphorylation
to lactic acid producing glycolysis in primary neurons from 3xTgAD mice. As oxidative
phosphorylation is driven by the activity of terminal enzyme COX and PDH serves as the
regulatory switch coupling glucose utilization to oxidative phosphorylation, we propose
that in 3xTgAD mice, deficits in the activity of these two enzymes reduces the substrate
input and driving force for oxidative phosphorylation, resulting in increased ATP demand
via other pathways. This unbalanced metabolic state coupled with the oxidative stress due
to reduced ETC efficiency leads to impaired bioenergetics that impairs neuronal function
and exacerbates neurodegeneration. The occurrence of these metabolic impairments prior
to plaque formation recapitulates findings derived from Alzheimer brain tissue and
indicates that mitochondrial dysfunction is an important early factor in the development
of AD-like pathology.
The concurrent increase in glycolysis in 3xTgAD neurons could be a compensatory
response in which neurons upregulate glycolytic ATP production to compensate for
declining mitochondrial respiration and OXPHOS energy production. In addition,
decreased PDH function could also contribute to the correlated decrease in mitochondrial
respiration and increase in glycolysis. PDH is the key rate-limiting enzyme in
35

mitochondria to convert pyruvate, the end product of glycolysis, into acetyl-CoA, which
subsequently condenses with oxaloacetate to initiate the TCA cycle for energy
production. Compromised PDH function will lead to the accumulation of pyruvate and
thus should stimulate anaerobic metabolism to lactic acid and cause an increase in
extracellular acidification, as indicated by the increase in ECAR in neurons derived from
3xTgAD neurons. Meanwhile, compromised PDH function in 3xTgAD neurons leads to
a deficit in Acetyl-CoA and consequently decreased OXPHOS activity as indicated by
the decrease in OCR in 3xTgAD neurons. These findings suggest a potential antecedent
role of mitochondrial bioenergetic deficits in AD pathogenesis and are consistent with
previous PET (positron emission tomography) metabolic analyses in persons with
increased risk of AD, mild cognitive impairment (MCI) or incipient to late AD, in which
decreased glucose uptake and utilization was demonstrated as among the earliest
symptoms of AD occurring far before the onset of AD (Cutler, 1986; Drzezga et al, 2003;
Jagust et al, 1991; Liang et al, 2008). These findings are also consistent with microarray
analyses of aging, incipient AD and AD human samples and rodent models
demonstrating that genes involved in mitochondrial bioenergetics are among those altered
early in AD or MCI patients (Blalock et al, 2004; Rowe et al, 2007). Consistent with
mitochondrial dysfunction, decreased mitochondrial bioenergetics has been demonstrated
to cause amyloid production and nerve cell atrophy (Meier-Ruge and Bertoni-Freddari,
1997; Velliquette et al, 2005).
36

In addition to compromised mitochondrial bioenergetics in 3xTgAD mice, elevated
oxidative stress was among the earliest indicators of dysfunction in the female 3xTgAD
mouse model. This finding is in agreement with previous studies from both the AD
animal models and the human subjects that showed elevated oxidative stress plays an
important role in AD pathogenesis (Sanz et al, 2006; Shi and Gibson, 2007). The data
also suggest that multiple aspects of mitochondrial dysfunction are closely related to the
pathogenesis of AD. Oxidative damage to mitochondrial membranes and proteins is well
documented to impair mitochondrial OXPHOS efficiency and result in increased electron
leak as observed by increased hydrogen peroxide levels and higher oxidative stress
(Reddy et al, 2008b, Beal, 2005 #6)
A direct link between Aβ-induced toxicity and mitochondrial dysfunction in AD has
been suggested by the interaction between mitochondrial Aβ and a mitochondrial protein,
ABAD (Amyloid-binding alcohol dehydrogenase, HSD17B10, 17β−hydroxysteroid
dehydrogenase) (Lustbader et al, 2004; Yan et al, 2005). As in previous reports, our
analyses show that the increase in mitochondrial Aβ correlated with the increase in
ABAD level in the 3xTgAD mouse brain. These observations confirmed the neurotoxic
role of mitochondrial deposits of Aβ. Nevertheless, the rise in ABAD and mitochondrial
accumulation of Aβ was subsequent to the first symptom of mitochondrial dysfunction
which was the decline in mitochondrial bioenergetic activity. A decline in mitochondrial
respiration and enzymes required for bioenergetics in vivo occurred in 3xTgAD mice as
37

early as 3 months of age. Supporting the hypothesis of mitochondrial dysfunction as an
antecedent event to AD pathology, are the in vitro findings derived from embryonic
neurons of 3xTgAD mouse hippocampus. Embryonic hippocampal neurons exhibited
significant deficits in mitochondrial respiratory function. Although we cannot rule out the
possibility that the transgenes themselves could lead to mitochondrial dysfunction in the
3xTgAD neurons, it is likely that decreased mitochondrial bioenergetics coupled with the
increase in oxidative stress contributes to the over-production of Aβ, which when binding
to ABAD forms an autocatalytic propagation of ROS further inducing mitochondrial
dysfunction (Frederikse et al, 1996).
In summary, mitochondrial dysfunction and deficits in bioenergetics occur early in
pathogenesis and precede the development of observable plaque formation in female
mouse model of AD. Further, age of reproductive senescence markedly exacerbated
mitochondrial and bioenergetic dysfunction which is coincident with marked increases in
AD pathology. If mitochondrial dysfunction is a causal link to Alzheimer’s, the
susceptibility of mitochondria to environmental and genetic risks factors should be a
critical factor in the development of late onset sporadic AD. This postulate is supported
by the relationship between brain hypometabolism and increased risk of AD in offspring
with maternal family history of AD (Liang et al, 2008; Mosconi et al, 2009). As the
mitochondrial genome is maternally inherited, this provides strong evidence of the
potential causal role of mitochondrial dysfunction in AD pathogenesis. From a
therapeutic perspective, the findings of this and other studies indicate a novel therapeutic
38

strategy to prevent AD by sustaining mitochondrial metabolic function. Therapeutics,
such as estrogen (Brinton, 2008b; Simpkins and Singh, 2008; Singh et al, 2008), that
sustain and enhance mitochondrial functions by upregulating key regulatory enzymes
involved in brain metabolism, efficiency of mitochondrial bioenergetics while
suppressing oxidative stress could prevent late onset AD.
39

Materials and Methods
Transgenic Mice. Colonies of 3xTgAD and nonTg mouse strain (C57BL6/129S; The
Jackson Laboratory, Bar Harbor, ME) (Oddo et al, 2003a; Oddo et al, 2003b) were bred
and maintained at the University of Southern California (Los Angeles, CA) following
National Institutes of Health guidelines on use of laboratory animals and an approved
protocol by the University of Southern California Institutional Animal Care and Use
Committee. Mice were housed on 12 h light/dark cycles and provided ad libitum access
to food and water. Only intact female mice at the age of 3, 6, 9, and 12 months were used
for the experiments. All animals were synchronized on estrus cycle prior to sacrifice.
Brain Tissue Preparation and Mitochondrial Isolation. Intact female mice of both
3xTgAD and nonTg groups at different age groups (3, 6, 9, and 12 months) were
sacrificed by decapitation and the brains were quickly dissected on ice. Cerebellum and
brain stem was removed from each brain and the hippocampus within the left hemisphere
was harvested. Brain mitochondria were isolated from the remainder (whole brain minus
cerebellum and brain stem) following previously established protocol (Irwin et al,
2008b). The brain was rapidly minced and homogenized at 4
o
C in mitochondrial isolation
buffer (MIB) (PH 7.4), containing sucrose (320 mM), EDTA (1 mM), Tris-HCl (10mM),
and Calbiochem’s Protease Inhibitor Cocktail Set I (AEBSF-HCl 500μM, 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, rehomogenized, and centrifuged again at 1500 X g for 5 min. The postnuclear
40

supernatants from both centrifugations were combined, and crude mitochondria 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 9000 X g for 8 min. The
resulting mitochondrial pellet was resuspended in MIB to an approximate concentration
of 1mg/ml. The resulting mitochondrial samples were used immediately for respiratory
measurements and hydrogen peroxide production or stored at -80
o
C for later protein and
enzymatic assays. During mitochondrial purification, aliquots were collected for
confirmation of mitochondrial purity and integrity, Western blot analysis was performed
for mitochondrial anti-VDAC (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).
Immunohistochemistry. For immunohistochemistry study, animals were sacrificed.
Brains were perfused with pre-chilled PBS buffer (pH 7.2) and immersion fixed in 4%
paraformaldehyde for 48 h and then stored in 4°C in PBS/1% sodium azide until use.
Fixed brains were sent to Neuroscience Associates (NSA, Knoxville, TN) for coronal
41

sectioning at 35μm, and then processed for immunohistochemistry using a standard
protocol. Briefly, every 12th section was blocked (1h at RT, PBS with 5% goat serum
and 0.3 % trinton x-100), immunostained using antibody directed against Aβ (6E10,
Signet 1:1000 dilution 4
o
C overnight) followed by washing and secondary antibody
Fluorescein goat anti-mouse (1:500, Chemicon, Ramona, CA, 1h at RT). Sections were
mounted with anit-fade mounting medium with DAPI (Vector Laboratories, Burlingame,
CA). Antigen unmasking treatment, consisting of 5 min rinse in 99% formic acid was
performed to enhance Aβ immunoreactivity (IR). Fluorescent images were taken using a
fluorescent microscope, normalized and analyzed with the slide book software
(Intelligent Imaging Innovations Inc, Santa Monica, CA).
Western Blot Analysis. Protein concentrations were determined by using the BCA
protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (20 μg/well) were
loaded in each well of a 15% SDS-PAGE gel, electrophoresed with a Tris/glycine
running buffer, and transferred to a 0.45μm pore size polyvinylidene difluoride (PVDF)
membrane and immunobloted with PDH E1 alpha antibody (1:1000, Mitosciences,
Eugene, OR), COX IV antibody (1:2000, Mitosciences, Eugene, OR), ABAD antibody
(1:1000, Abcam, Cambridge, MA), β-actin antibody (1:4000, Chemicon, Ramona, CA)
and porin/VDAC antibody (1:500, Mitosciences, Eugene, OR). Mitochondrial Aβ
oligomer (16KD) level was determined in isolated mitochondrial samples (20μg/well)
and blotted by specific Anti-Aβ monoclonal antibody (6E10, Signet). HRP-conjugated
42

anti-rabbit antibody and HRP-anti-mouse antibody (Vector Laboratories, Burlingame,
CA) were used as secondary antibodies. Immunoreactive bands were visualized by Pierce
SuperSignal Chemiluminescent Substrates (Thermo Scientific) and captured by
Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA). All band intensities
were quantified using Un-Scan-it software.
Enzyme Activity Assay. PDH activity was measured by monitoring the conversion of
NAD+ to NADH by following the change in absorption at 340 nm as previously
described (Gohil and Jones, 1983). Isolated brain mitochondria were dissolved in 2%
CHAPS buffer to yield a final concentration of 15 μg/μl and incubated at 37
o
C in PDH
Assay Buffer (35mM KH
2
PO
4
, 2 mM KCN, 0.5 mM EDTA, 5 mM MgCl
2
, (pH 7.25 with
KOH), 200mM Sodium Pyruvate, 2.5 mM Rotenone, 4mM Sodium CoA, 40 mM TPP).
The reaction was initiated by the addition of 15mM NAD
+
. COX activity was measured
on isolated mitochondria (20 μg) using Rapid Microplate Assay kit for Mouse Complex
IV Activity (Mitosciences, Eugene, OR) following the manufacturer’s instructions.
Hydrogen peroxide Production. The rate of hydrogen peroxide production by fresh
isolated mitochondria was determined by the Amplex Red Hydrogen
Peroxide/Peroxidase Assay kit (Invitrogen, Carlsbad, CA) following the manufacturer’s
instructions.
Lipid Peroxidation. Lipid peroxides in brain mitochondria and hippocampal lysates were
measured using the leucomethylene blue assay (Blass et al, 2000), using tert-butyl
hydroperoxide as a standard, by monitoring the 650nm absorbance after 1 h incubation at
43

RT. The aldehyde product or termination production of lipid peroxidation in brain
mitochondria was determined by measuring thiobarbituric acid reactive substances
(TBARS). Samples were mixed with 0.15 M phosphoric acid. After the addition of
thiobarbituric acid, the reaction mixture was heated to 100
o
C for 1 h. After cooling and
centrifugation, the formation of TBARS was determined by the absorbance of the
chromophore (pink dye) at 531 nm using 600nm as the reference wavelength.
Respiratory Measurement. Mitochondrial oxygen consumption was measured
polarographically using a Clarke-type electrode. 100 µg of isolated mitochondria were
placed in the respiration chamber at 37
o
C in respiratory buffer (130 mM KCl, 2 mM
KH
2
PO
4
, 3mM HEPES, 2mM MgCl
2
, 1 mM EGTA) to yield a final concentration of 200
μg/mL After a 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
0
respiration was induced by the addition of three
pulses 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 defined by dividing the rate of oxygen
consumption/min for state 3 (presence of ADP) by the rate of oxygen consumption/min
for state 4
0
respiration (Absence of ADP by addition of atractyloside).
Free Radical Leak. Free radical leak was determined as previously described (Irwin et
al, 2008b; Sanz et al, 2005). Hydrogen peroxide production and O
2
consumption were
44

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 the respiratory chain (the percent free radical leak) instead of
reaching cytochrome oxidase to reduce O
2
to water. Since two electrons are needed to
reduce 1 mole of O
2
to hydrogen peroxide 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 hydrogen
peroxide production divided by two times the rate of O
2
consumption, and the result was
multiplied by 100.
Seahorse XF-24 Metabolic Flux Analysis. Primary hippocampal neurons from day 14
(E14) embryos of both 3xTgAD and nonTg mice were cultured on Seahorse XF-24 plates
at a density of 75,000 cells/well. Neurons were grown in Neurobasal Medium +B27
supplement for 7 days prior to experiment. On the day of metabolic flux analysis, cells
were changed to unbuffered DMEM (DMEM Base medium supplemented with 25 mM
glucose, 1 mM sodium pyruvate, 31 mM NaCl, 2 mM GlutaMax; pH 7.4) and incubated
at 37
o
C in a non-CO
2
incubator for 1 h. All medium and injection reagents were adjusted
to pH 7.4 on the day of assay. Four baseline measurements of OCR and ECAR were
taken before sequential injection of mitochondrial inhibitors. Three readings were taken
following each addition of mitochondrial inhibitor prior to injection of the subsequent
inhibitors. The mitochondrial inhibitors used were oligomycin (1 µM), FCCP (1 µM),
and Rotenone (1 µM). OCR and ECAR were automatically calculated and recorded by
the Seahorse XF-24 software. After the assays, plates were saved and protein readings
45

were measured for each well in order to confirm equal cell numbers per well. The
percentage of change compared to the basal rates was calculated as the value of change
divided by the average value of baseline readings.
Statistics. Statistically significant differences between groups were determined by an
ANOVA followed by a Newman-Keuls post-hoc analysis.
46

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Chapter III. Estrogen Regulation of Mitochondrial Bioenergetics,
Oxidative Stress and Mitochondrial Localization of Alzheimer’s
Pathology
Chapter III Abstract

Previously, we demonstrated that mitochondrial bioenergetic deficits precede
Alzheimer’s pathology in the female triple transgenic Alzheimer’s mouse model
(3xTgAD). Further, reproductive senescence paralleled a dramatic decline in
mitochondrial function in normal aging mice and exacerbated mitochondrial dysfunction
in 3xTgAD mice. In this study, we investigated the long term impact of 17β-estradiol
(E2) deficiency on mitochondrial function in both nonTg and 3xTgAD female mouse
brain. Compared to gonadally intact mice, E2 depletion by ovariectomy (OVX)
significantly decreased brain mitochondrial respiration and enzyme activities in both
nonTg and 3xTgAD mice. Loss of E2 also induced increased oxidative stress manifested
by greater lipid peroxidation and RNA oxidation. In 3xTgAD mice, OVX significantly
increased mitochondrial amyloid beta (Aβ) level which was correlated with a significant
increase in Aβ  binding alcohol dehydrogenase (ABAD) expression. Estradiol treatment
at the time of OVX, prevented OVX-induced mitochondrial deficit by sustaining
mitochondrial bioenergetic capacity, decreasing oxidative stress, and preventing
mitochondrial amyloid and ABAD induction. Collectively, these data demonstrate that
60

ovarian hormone loss induced a significant decline in mitochondrial bioenergetics
paralleled by increased oxidative stress and mitochondrial localization of AD pathology,
which all were prevented by E2 treatment at the time of OVX. Results are relevant to
potential mechanisms of AD development in postmenopausal women and potential
therapeutic prevention of mitochondrial deficits associated with neurodegenerative
disease.
Introduction

The essential role of mitochondria in cellular bioenergetics and survival has been
well established (Wallace, 2005; Magistretti, 2006; Brinton, 2008). Further,
mitochondrial dysfunction has been suggested to play a pivotal role in neurodegenerative
disorders, including Alzheimer’s disease (AD) (Beal, 2005; Mattson and Magnus, 2006;
Brinton, 2008). Recently we demonstrated that mitochondrial bioenergetic deficits
precede Alzheimer’s pathology in the female triple transgenic mouse model of
Alzheimer’s disease (3xTgAD), indicating a potential causal role of mitochondrial
bioenergetic deficiency in AD pathogenesis. Mitochondrial dysfunction in the female
3xTgAD mouse brain was evidenced by decreased mitochondrial respiration, decreased
metabolic enzyme expression and activity, increased oxidative stress, and increased
mitochondrial Aβ load and Aβ binding alcohol dehydrogenase (ABAD) expression (Yao
et al., 2009). Further, we observed that reproductive senescence paralleled a dramatic
decline in mitochondrial function in normal aging mice and exacerbated mitochondrial
61

dysfunction in 3xTgAD mice. Consistent with findings in the animal model, clinically
menopause is associated with a decline in brain metabolism (Resnick et al., 2006;
Henderson, 2009a) whereas women on hormone therapy did not show a decline in brain
metabolism (Maki and Resnick, 2001; Maki et al., 2001; Resnick et al., 2009), suggesting
a critical role of estrogen in regulating mitochondrial bioenergetics.
Basic science analyses have demonstrated that the neuroprotective mechanisms
converge upon mitochondria. Estrogen enhances mitochondrial bioenergetics, protects
against free radical damage, and up-regulates of Aβ clearance (Carroll et al., 2007; Nilsen
et al., 2007; Brinton, 2009; Zhao et al., 2010). Estrogen increases expression and activity
of proteins involved in oxidative phosphorylation, including pyruvate dehydrogenase,
aconitase, and ATP synthase, etc (Nilsen et al., 2007). Further, results of epidemiological
analyses indicate that hormone therapy at the menopause transition have a reduced risk of
Alzheimer’s disease (Henderson et al., 2005; Henderson, 2009b) whereas women not
receiving hormone therapy following surgically-induced menopause are at increased risk
for Alzheimer’s and Parkinson’s disease (Yaffe, 2003; Morrison et al., 2006; Rocca et al.,
2007; Brinton, 2008; Hogervorst et al., 2009).
In the current study, we sought to determine the long term impact of ovarian
hormone loss on mitochondrial function in both nonTg and 3xTgAD female mouse
brains. We also investigated the efficacy of 17β-estradiol (E2) to prevent ovariectomy
(OVX) induced deficits in mitochondrial function. We further differentiated the cellular
contribution to estrogen regulation of metabolic activities between neurons and glia in
62

vitro. Results of these analyzes demonstrate that ovarian hormone loss induced a
significant decline in mitochondrial bioenergetics paralleled by a significant increase in
oxidative stress and induction of mitochondrial localization of Aβ which were all
prevented by E2 treatment at time of OVX. Because hypometabolism in brain is an early
indicator of increased risk for AD (Mosconi et al., 2006; Nakano and Matsuda, 2009),
findings from this study provide potential mechanisms for the higher life-time risk of AD
in postmenopausal women (2010). These data indicate a therapeutic strategy that targets
mitochondria bioenergetics to prevent or delay menopause associated mitochondrial
deficits associated with increased risk of AD and potentially Parkinson’s disease (Rocca
et al., 2008; Rocca et al., 2010).
63

Results
Brain independent confirmation of ovarian hormone status. Uterine weight was used a
bioassay to indirectly confirm depletion of ovarian hormones and E2 treatment in female
nonTg and 3xTgAD mice. Ovariectomy (OVX) was performed at age 3 months of age
and mice were treated subsequent to surgery with continuous E2 or steroid free pellet for
3 months. The efficacy of E2 delivery in the same animal model has been investigated
previously by direct measuring serum E2 levels (Carroll et al., 2007). In both nonTg and
3xTgAD mice, OVX-induced hormone depletion induced a significant decrease in
uterine weight compared to the Sham-OVX group whereas E2 treatment induced a
significant increase compared to both the OVX group (Fig.III-1, P<0.05). The changes in
uterine weight confirmed experimental manipulations of OVX and E2 in both nonTg
3xTgAD mice (Fig.III-1, P<0.05).
Figure III-1. Change in uterine weight with estrogen manipulation. Both female nonTg
and 3xTgAD were either Sham-OVXed or OVXed at the age of 3 months. OVXed mice
were treated with either vehicle (OVX group) or E2 (OVX+E2 group) for 3 month. At the
end of the treatment, mice were sacrificed and uteri were collected and weighted. BARs
represent mean uterine weight + SEM (*/#, P<0.05 compared to OVX group, N=10)
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Ovariectomy-induced decrease in mitochondrial respiration and prevention by 17β-
estradiol. Mitochondrial respiration is a direct indicator of overall metabolic activity. To
investigate the impact of loss of ovarian hormones and E2 (0.25mg/90 days pellet)
exposure on mitochondrial respiration, whole forebrain (whole brain minus cerebellum)
mitochondria were isolated from both nonTg and 3xTgAD female mice following 3
months of OVX or E2 exposure. State 4 respiratory rate of isolated whole brain
mitochondria was first determined using glutamate (5mM) and malate (5mM) as
respiratory substrates. State 3 respiration was initiated by the addition of ADP (410
μM) to the mitochondrial suspension initiated state 3 respiration. Respiratory control
ratio (RCR) was significantly decreased by OVX in both nonTg (~30%) and 3xTgAD
(~40%) group (Fig.III-2, P<0.05, compared to Sham-OVX group, n=7). E2 treatment
initiated immediately at time of OVX successfully prevented the OVX-induced decline in
mitochondrial respiration (Fig.III-2, P<0.05, compared to OVX, n=7). The changes in
RCR were due to deficit in state 3 respiration in both nonTg and 3xTgAD mice as OVX
significantly decreased the rate of state 3 respiration whereas there was no significant
difference in the rate of state 4 respiration between groups (Fig.III-2A and 2C).. E2
treatment prevented the OVX-induced decrease in state 3 respiration (Fig.III-2A and 2C).
These data indicate that E2 is the primary ovarian hormone regulating mitochondrial
function in brain as the OVX-induced deficit in State 3 respiration was completely
prevented by E2. Further, because OVX specifically decreased ADP dependent state 3
respiration which was prevented by E2 treatment, E2 functions to promote efficiency of
65

mitochondrial respiration rather than directly affecting coupling of electron transport
chain.

Ovariectomy-induced decrease in metabolic enzyme activity and prevention by 17β-
estradiol. To investigate the impact of ovariectomy and E2 on mitochondrial enzymes
involved in metabolism and respiration, we assessed activities of PDH, complex I
(NADH dehydrogenase), and complex IV (cytochrome c oxidase). In both nonTg and
3xTgAD female mice, OVX induced a significant decrease in both PDH and COX
activity (Fig.III-3A and 3D) which was prevented by E2. Notably, there was a similar but
not significant pattern of change in complex I activity with OVX or E2 treatment in both
nonTg and 3xTgAD mice (Fig.III-3B).

66

Figure III-2. OVX induced decrease in mitochondrial respiration and E2 treatment
prevented OVX-induced decrease in mitochondrial respiration. Whole brain
mitochondria were isolated from Sham OVX, OVX, and OVX+E2 group for both nonTg
and 3xTgAD mice at the end of the treatment. State 3/state 4 respiration was determined
using the Luxcel MitoXpress Oxygen-sensitive Fluorescent Dye. State 4 mitochondrial
respiration was measured in the presence of L-malate (5mM), L-glutamate (5mM); state 3
mitochondrial respiration was measured in the presence of L-malate (5mM), L-glutamate
(5mM), plus ADP (410µM). The Respiratory Control Ratio (RCR) is defined as state3/state
4 respiration. A&C, representative figure for State 3 and 4 brain mitochondrial respiration
curve for nonTg and 3xTgAD mice, respectively. B&D, OVX significantly decreased RCR
in both nonTg (B) and 3xTgAD (D) mice, whereas E2 treatment initiated at time of OVX
prevented the OVX induced decrease. (Bars represent mean value ± S.E.M */#, P<0.05
compared to the OVX group, N=7)

67

Figure III-3. OVX induced decrease in enzyme activity and E2 treatment prevented
OVX-induced decrease in enzyme activity. Brain mitochondria isolated from Sham
OVX, OVX, OVX+E2 groups of both nonTg and 3xTgAD mice were assessed for PDH,
Complex I and Complex IV (COX) activity. A, relative COX Activity was presented as the
relative value normalized to that of nonTg Sham OVX group; B, relative Complex I
activity was presented as the relative value normalized to that of nonTg OVX group; C,
representative COX activity curve of 3xTgAD mice; D, relative COX activity was
presented as the relative value normalized to that of nonTg Sham OVX group. BARs
represent mean enzyme activity value ± SEM (*/#, p<0.05 compared to the OVX group,
N=7).
68

Ovariectomy-induced increase in oxidative stress in brain and and prevention by 17β-
estradiol. Efficiency of coupled electron transport is directly related to generation of
oxidative damage in mitochondria. To determine the impact of loss of ovarian hormones
and E2 treatment on oxidative stress, we assessed the magnitude of lipid peroxidation of
mitochondria isolated from whole forebrain and hippocampal lysates as an indicator of
oxidative damage to cellular components. In addition, we assessed the serum level of
oxidized RNA as indicated by 8-oxoGuanine (8-OHG). Loss of ovarian hormones
induced a significant rise in lipid peroxidation of mitochondria in both the nonTg and
3xTgAD female mice relative to corresponding Sham-OVX group (Fig.III-4A, P<0.05,
N=7). Under all conditions, mitochondrial derived from 3xTgAD mice exhibited a higher
level of lipid peroxidation (Fig.III-4A P<0.05, N=7) relative to nonTg brain
mitochondria. E2 prevented OVX-induced lipid peroxidation in both nonTg and 3xTgAD
mice. Lipid peroxidation within hippocampal lysates yielded comparable results (data not
shown). Likewise, OVX induced a significant increase in serum 8-OHG level in both
nonTg and 3xTgAD mice, which was also prevented by E2 treatment (Fig.III-4B,
P<0.05, N=10).

69

Figure III-4. OVX induced increase in oxidative stress in brain and E2 treatment
prevented OVX-induced increase in oxidative damage. Brain mitochondria were
isolated and serum samples were collected from Sham OVX, OVX, OVX+E2 groups of
both nonTg and 3xTgAD mice. lipid peroxide levels were determined by the
leucomethylene blue assay. Serum 8-oxoGuanine levels were determined with the 8-
oxoGuanine assay kit. A, OVX significantly increased lipid peroxidation whereas E2
treatment prevented this increase in both nonTg and 3xTgAD mice; B, OVX induced
significant increase in serum 8-oxoGuanine level and E2 treatment prevented the OVX-
induced increase in both nonTg and 3xTgAD mice. Bars represent mean value ± S.E.M.
(*/#, p<0.05 compared with the OVX group; n=7)
70

Ovariectomy-induced increase in brain and mitochondrial amyloid load and prevention
by 17β-estradiol. Previous studies indicated that amyloid beta (Aβ) interacts with the
mitochondrial protein, amyloid beta binding alcohol dehydrogenase (ABAD) and that
binding of Aβ contributes to mitochondrial dysfunction (Lustbader et al., 2004; Takuma
et al., 2005; Yan and Stern, 2005; Chen et al., 2007). In this 3xTgAD mouse model, we
have previously shown that female 3xAD mice at 9 month exhibited overt mitochondrial
Aβ deposition and elevated ABAD expression (Yao et al., 2009). To investigate the
impact of loss of ovarian hormones and E2 treatment on mitochondrial Aβ load and
ABAD expression, mitochondrial samples from Sham OVX, OVX, and OVX+E2 groups
of 3xTgAD mice were analyzed by western blot for mitochondrial Aβ and ABAD
expression. Ovariectomy of 3xTgAD mice induced a significant increase in
mitochondrially localized Aβ and ABAD expression (Fig.III-5A and 5B, P<0.05, N=7).
Treatment with E2 prevented the OVX-induced increase in mitochondrial Aβ and ABAD
expression (Fig.III-5A and 5B, P<0.05, N=7). As loss of ovarian hormones is known to
increase Aβ load in brain (Petanceska et al., 2000; Carroll et al., 2007), we sought to
determine whether mitochondrial Aβ correlated with overall Aβ load in hippocampus.
Immunocytochemical labeling of Aβ was conducted in hippocampal brain sections
derived from Sham, OVX and OVX+E2 treated 3xTgAD mice. Similar to findings of
mitochondrial Aβ load, a significant increase in 6E10 labeling of Aβ was apparent in the
hippocampal CA1 region of the OVX group compared with Sham-OVX group, whereas
71

OVX+E2 group exhibited lower 6E10 immunoreactivity that was comparable to Sham-
OVX (Fig.III-6).

Figure III-5. OVX induced increase in brain and mitochondrial amyloid
load and E2 treatment prevented OVX-induced increase in amyloid
load. Equal amount of mitochondrial samples isolated from Sham OVX,
OVX, and OVX+E2 groups of both nonTg and 3xTgAD mice were load
onto the gel. Expression of mitochondrial 16 kD Aβ oligomer and ABAD
were determined by Western blot analysis. A, OVX induced significant
increase in mitochondrial 16 kD Aβ level and E2 treatment prevented the
OVX-induced increase; B, OVX induced significant increase in
mitochondrial ABAD level and E2 treatment prevented the OVX-induced
increase. Bars represent mean relative expression ± S.E.M. (*=p<0.05
compared to the OVX group; n=6).
72

Figure III-6. OVX significantly increased amyloid pathology in 3xTgAD mouse brains
whereas E2 treatment prevented the OVX-induced increase. Brains from Sham OVX,
OVX, and OVX+E2 groups of 3xTgAD mice were sectioned at 30μm. Sections were
stained with 6E10 antibody (green) and DAPI for nuclei (DAPI). Representative images
show Aβ-IR in hippocampus (top row) and CA1 (bottom 2 rows) region from Sham OVX,
OVX, and OVX+E2 groups of 3xTgAD mice (scale: 100 μm).

73

Mitochondrial Biogenesis was unaffected by estrogen manipulation
Changes in mitochondrial respiratory efficiency, enzyme expression and activity can
result from a global increase in mitochondrial biogenesis (Lopez-Lluch et al., 2006;
Civitarese et al., 2007). To investigate the impact of loss of ovarian hormones and E2
treatment on mitochondrial biogenesis, we assessed a marker of mitochondrial
biogenesis, the ratio of mitochondrial DNA (mtDNA) to nuclear DNA. Real-time PCR
for COXII (mitochondrial DNA encoded) and β-actin (nuclear DNA encoded) was
performed on total genomic DNA isolated from cortical region. Results of these analyses
indicated no significant difference on the ratio of COXII to β-actin between Sham-OVX,
OVX or OVX+E2 group in both nonTg and 3xTgAD mice (Fig.III-7, n=10), indicating
that neither ovariectomy nor E2 altered the rate of mitochondrial biogenesis.

Figure III-7. Mitochondrial biogenesis was unaffected by estrogen manipulation. Total
DNA was isolated from cortical tissues of Sham OVX, OVX, and OVX+E2 groups of both
nonTg and 3xTgAD mice. Samples were analyzed by realtime PCR. Mitochondrial biogenesis
was estimated as the relative levels of COXII DNA (mtDNA) to β-actin (nDNA).
74

Estrogen differentially regulates mitochondrial bioenergetics in neurons and glia.
To determine the cellular contribution to estrogen regulation of mitochondrial
bioenergetics, we assessed E2 regulation of mitochondrial respiration in primary neuronal
and glial cultures using the Seahorse XF-24 metabolic analyzer. In both primary neurons
and mixed glia >80% of the oxygen consumption rate (OCR) measured was due to
oxidative phosphorylation as indicated by the decrease (>80% decrease relative to basal
level) in OCR with the addition of rotenone. The addition of the ATP synthase inhibitor
oligomycin (1 µM) resulted in ~70% decrease in OCR in neurons and ~60% decrease in
OCR in mixed glia (Fig.III-8A), indicating that the measured oxygen consumption was
largely driven by oxidative phosphorylation-coupled ATP generation. Direct comparison
of primary neurons and mixed glia revealed a much higher basal OCR in neurons
compared to mixed glia indicating that neurons are highly oxidative whereas glial cells
exhibit a glycolytic phenotype. The addition of a mitochondrial uncoupler (FCCP, 1µM)
resulted in a dramatic increase in OCR in both neurons and glia (Fig.III-8A), as expected,
giving an estimation of the maximal respiratory capacity of the mitochondria. The
maximal respiratory capacity of neurons was significantly higher than mixed glia,
suggesting a larger capacity reserved for potential neuronal activities with high energy
demand (Fig.III-8A, P<0.05). To investigate E2 regulation on mitochondrial
bioenergetics, an increasing series of E2 doses (0.1ng/ml, 1ng/ml, 10ng/ml, 100ng/ml,
and 1000ng/ml for 24 hours) was tested in neurons and mixed glia separately. The dose
of 10ng/ml E2 treatment was selected as this E2 dose yielded the strongest and most
75

consistent response in both neurons and mixed glia. The comparison between neurons
and mixed glia demonstrated differential regulation of mitochondrial respiration by E2 in
neurons and mixed glia. In neurons, E2 significantly increased the maximal respiratory
capacity without effecting basal respiration (Fig.III-8C, P<0.05) whereas in mixed glia,
E2 significantly increased both the basal respiration and the maximal respiratory capacity
(Fig.III-8B, P<0.05)
76

Figure III-8. 17βEstradiol
differentially regulates
mitochondrial bioenergetics in
neurons and mixed glia. Primary
hippocampal neurons from day 18
(E18) embryos of female Sprague-
Dawley rats were cultured in
Neurobasal medium + B27
supplement for 10 days prior to
experiment. Mixed glia from day 18
(E18) embryos of female Sprague-
Dawley rats were cultured in growth
media (DMEM:F12 (1:1)+10%
FBS). Primary Oxygen
consumption rate (OCR) and
extracellular acidification rate
(ECAR) were determined using
Seahorse XF-24 Metabolic Flux
analyzer. Vertical lines indicate
time of addition of mitochondrial
inhibitors A: oligomycin (1 µM), B:
FCCP (1 µM) or C: rotenone (1
µM). A, Oxygen consumption rates
(OCR) in primary neurons (gray)
have higher basal rates of
mitochondrial respiration and
maximal mitochondrial respiratory
capacity than mixed glia (black)
(*,P<0.05 compared ; B, E2
treatment (black) significantly
increased both the basal respiration
and the maximal mitochondrial
respiratory capacity in mixed glia.
(*, P<0.05 compared to ctrl (gray));
C, E2 treatment (black) only
increased both the the maximal
mitochondrial respiratory capacity
but no the basal respiration in
neurons (*, P<0.05 compared to ctrl
(gray)).
77

Discussion

In this study, we investigated the long term impact of estradiol deficiency on brain
mitochondrial function, particularly mitochondrial bioenergetics, in both nonTg and
3xTgAD female mice. In agreement with our previous findings that reproductive
senescence paralleled a dramatic decline in mitochondrial function (Yao et al., 2009), we
observed that estradiol deficiency due to OVX induced a significant decrease in
mitochondrial function in both nonTg and 3xTgAD brains. Specifically, OVX decreased
brain mitochondrial respiration, decreased expression and activity of metabolic enzymes,
and induced greater oxidative stress in both nonTg and 3xTgAD brains. In 3xTgAD
mice, OVX also induced a significant increase in mitochondrial amyloid load, which was
paralleled by a significant induction of ABAD protein expression. Importantly, E2
treatment initiated at time of OVX successfully prevented the OVX-induced detrimental
impacts. Results herein indicate that E2 is critical in regulating and maintaining key
domains of brain metabolism, cellular energetics, oxidative stress and free radical
maintenance.

Mitochondria play a center role in cellular bioenergetics (McBride et al., 2006;
Atamna and Frey, 2007). Perturbations of brain mitochondrial bioenergetic activity,
especially mitochondrial respiration, will inevitably impact brain metabolism. The
observation from this study that estradiol deficiency led to a decrease in mitochondrial
78

respiration whereas E2 treatment at time of OVX prevented the OVX-induced decrease in
mitochondrial respiration is consistent with the findings from human clinical trials that
compared to estrogen therapy (ET) users, post-menopausal women who did not receive
estrogen showed significant differences in PET regional cerebral blood flow (Resnick et
al., 1998; Maki and Resnick, 2000, 2001) and displayed a significant decrease in
metabolism of the posterior cingulated cortex (Rasgon et al., 2005). In our study, we
further examined the impact of estradiol deficiency on metabolic enzymes involved in
ATP production, including PDH, Complex I, and Complex IV (COX). PDH is the key
enzyme linking glycolysis to oxidative phosphorylation (OXPHOS); Complex I controls
the entry of electron flow to the mitochondrial electron transport chain (mETC), hence
controlling the entry point of OXPHOS in brain; Complex IV is the terminal enzyme of
electron flow and reduces O
2
to H
2
O. The activity of COX and PDH has been shown to
decrease significantly in postmortem brain tissue derived from Alzheimer’s patients
(Blass et al., 2000; Martins and Hallmayer, 2004) whereas Complex I activity was mainly
found to be compromised in Parkinson’s disease (Beal, 2005). In this study, we observed
that OVX induced significant decrease in PDH and COX activity in both nonTg and
3xTgAD group from this study while E2 treatment at time of OVX significantly
prevented the changes in PDH and COX activities. Complex I activity, unlike PDH and
COX, exhibited a similar but insignificant trend with estradiol manipulation in both
nonTg and 3xTgAD mice, suggesting an indirect, macro-scale change in response to
estradiol status instead of a direct regulation of Complex I activity.
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Oxidative stress is closely associated with mitochondrial bioenergetic dieficts and
development of AD neuropathology (Moreira et al., 2007; Trushina and McMurray,
2007). Estrogen has long been demonstrated as important regulator of the antioxidative
defense system. The mechanisms of estrogen protection against oxidative damage include
both scavenging free radicals directly by estrogen (Simpkins and Dykens, 2007) and the
upregulation of antioxidant enzymes, such as MnSOD and Peroxiredoxin, through
genomic and non-genomic estrogen signaling pathway (Vina et al., 2005; Nilsen et al.,
2007; Razmara et al., 2007; Irwin et al., 2008). In this study, we demonstrated that
estradiol depletion by OVX significantly increased lipid peroxidation and RNA
oxidation, the two commonly observed markers in AD human patients (Potashkin and
Meredith, 2006; Khong et al., 2008; Nunomura et al., 2009), whereas E2 treatment
initiated at time of OVX prevented the OVX-induced increase in oxidative damage.
These findings further strengthened a critical role of estradiol in protecting cells from
oxidative damage.

Amyloid, the overt pathology marker of AD, has been demonstrated to
accumulate within mitochondria, interact with a mitochondrial protein, ABAD (Amyloid-
binding alcohol dehydrogenase, HSD17B10, 17β−hydroxysteroid dehydrogenase), and
lead to mitochondrial dysfunction in AD (Lustbader et al., 2004; Takuma et al., 2005;
Yan and Stern, 2005; Chen et al., 2007). We previously observed that brain mitochondria
derived from 3xTgAD mice at 9 month of age showed significantly higher amount 16kD
80

Aβ oligomer (Yao et al., 2009). In the current study, OVX induced loss of ovarian
hormones significantly increased mitochondrial Aβ load to a level comparable to 9 month
old 3xTgAD animals which was prevented by E2 treatment. Similarly, mitochondrial
ABAD expression was increased by OVX and prevented by E2 treatment at time of
OVX. Estrogen has been demonstrated to induces anti-amyloidogenic pathways
(Morinaga et al., 2007) and regulate key Aβ degrading enzymes, such as Neprilysin
(NEP) and Insulin Degrading Enzyme (IDE) (Xiao et al., 2009; Zhao et al., 2010). Loss
of estrogen leads to the decrease in NEP and IDE expression and consequently the
increase in Aβ level. More importantly, decreased mitochondrial bioenergetics coupled
with increased oxidative stress, as a result of estradiol deficiency, also contributes to the
overproduction of amyloid (Grieb et al., 2004; Velliquette et al., 2005). Results from this
study not only are consistent with the well-documented findings on estrogen regulation of
Aβ level (Petanceska et al., 2000), but also are indicative of a close relationship between
the perturbation of mitochondrial bioenergetics and the over-production of amyloid.

Changes in mitochondrial bioenergetic capacity could be due to differences in
mitochondrial number, size, and density per cell as well as to changes in mitochondrial
components, enzyme activity, and consequently altered mitochondrial efficiency. In this
study, using mtDNA content as a marker of mitochondrial number, we did not observe
any difference between different treatment groups for the period of this study, suggesting
that mitochondrial biogenesis is not impacted by relatively long term (3 month) change in
81

estrogen status. The increased mitochondrial bioenergetic activity observed is likely due
to increased mitochondrial efficiency rather than a direct increase in the number of
mitochondria. This finding is consistent with our previous findings that short-term
estrogen treatment did not alter mitochondrial number. Although we cannot completely
rule out the possible effect on mitochondrial size, the decrease in mitochondrial
bioenergetic capacity in response to estradiol deficiency could be linked to a decrease in
metabolic machinery per mitochondrion and therefore compromised mitochondrial
efficiency, which was prevented by E2 treatment at time of OVX.

Neurons and glia are different in many aspects, including morphology, function,
and energy consumption. In this study, we demonstrated in vitro that neurons, compared
to mixed glia, exhibited a much higher rate of both basal respiration and the maximal
respiratory capacity. This observation is consistent with the difference in function
between neurons and glia. Neurophysiological activities of neurons require high energy
input whereas glial cells are less involved in firing action potentials but more in
supporting neurons, performing glycolysis and providing substrates for neurons (Chih et
al., 2001; Pellerin, 2003; Magistretti, 2006). Moreover, we observed that estradiol
treatment differentially regulated mitochondrial bioenergetics in neurons and in mixed
glia. The fact that E2 treatment only increased the maximal mitochondrial respiratory
capacity but not the basal respiration indicates that estrogen does not elevate
mitochondrial bioenergetics of neurons at basal or resting level, rather, it enhances the
82

reserved mitochondrial capacity for high energy demanding or energy challenging
situations. Unlike neurons, both the basal respiration and the maximal respiratory
capacity of mixed glia were significantly increased with E2 treatment. Considering the
fact that glial cells are heavily involved in glycolysis, the increase in basal respiration is
likely due to E2-induced up-regulation in glycolytic pathways. Estrogen has been
reported to increase the activity of glycolytic enzymes including hexokinase,
phosphofructokinase, and phosphoglycerate kinase in rodent brains (Kostanyan and
Nazaryan, 1992). The increase in glycolytic activity of glia enlarges the substrate
reservoir for high energy demanding situations. Together, the data suggest a coordinated
estrogen action on mitochondrial bioenergetics between neurons and glia to ensure
sufficient substrates for mitochondrial respiration in energy challenging situations.
In conclusion, estradiol deficiency by OVX induced a significant decrease in
mitochondrial bioenergetics coupled with a significant increase in oxidative stress in both
nonTg and 3xTgAD mice, whereas E2 treatment initiated at time of OVX successfully
prevented these impacts. In the 3xTgAD mice, decreased mitochondrial bioenergetics is
also associated with a significant increase in mitochondrial amyloid load and ABAD
expression. Additional in vitro studies demonstrated a coordinated estrogen action on
neurons and glia to ensure sufficient mitochondrial bioenergetics for ATP production in
high energy demanding situations. From a clinical respective, results from this study
suggest a potential causal role of estrogen deficiency in brain hypometabolism and
decreased enzyme functions, and eventually increased risk for Alzheimer’s disease
83

observed in menopausal women whereas therapeutics that sustain and maintain
mitochondrial metabolic function while suppressing oxidative stress could prevent age-
related AD.
84

Materials and Methods
Transgenic Mice. Colonies of 3xTgAD and nonTg mouse strain (C57BL6/129S; The
Jackson Laboratory, Bar Harbor, ME) (Oddo et al, 2003a; Oddo et al, 2003b) were bred
and maintained at the University of Southern California (Los Angeles, CA) following
National Institutes of Health guidelines on use of laboratory animals and an approved
protocol by the University of Southern California Institutional Animal Care and Use
Committee. Mice were housed on 12 h light/dark cycles and provided ad libitum access
to food and water.
Experimental Design. To investigate the changes in mitochondrial function after
estrogen manipulation, 3 month old female 3xTgAD and nonTg mice were randomly
assigned to one of the following three treatment groups (n=10 per group): sham
ovariectomized (Sham-OVX), ovariectomized (OVX), and OVX plus 17β-estradiol
(OVX+E2). Mice were bilaterally OVXed and immediately implanted with a continuous
90 day release pellet (Innovative Research of America, Sarasota FL) containing either
0.25 mg of E2 (OVX+E2 group) or placebo (Sham-OVX and OVX groups). All the mice
were killed at 6 month, 90 days after the initiation of hormone treatment.
Brain Tissue Preparation and Mitochondrial Isolation. Upon completion of hormone
treatment, both 3xTgAD and nonTg groups (n=7 per group) were sacrificed by
decapitation and the brains were quickly dissected on ice. Cerebellum and brain stem was
removed from each brain and the hippocampus within the left hemisphere was harvested.
Brain mitochondria were isolated from the remainder (whole brain minus cerebellum and
85

brain stem) following previously established protocol (Irwin et al, 2008b). The brain was
rapidly minced and homogenized at 4
o
C in mitochondrial isolation buffer (MIB) (PH
7.4), containing sucrose (320 mM), EDTA (1 mM), Tris-HCl (10mM), and Calbiochem’s
Protease Inhibitor Cocktail Set I (AEBSF-HCl 500μM, 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,
rehomogenized, and centrifuged again at 1500 X g for 5 min. The postnuclear
supernatants from both centrifugations were combined, and crude mitochondria 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 9000 X g for 8 min. The
resulting mitochondrial pellet was resuspended in MIB to an approximate concentration
of 1mg/ml. The resulting mitochondrial samples were used immediately for respiratory
measurements and hydrogen peroxide production or stored at -80
o
C for later protein and
enzymatic assays. During mitochondrial purification, aliquots were collected for
confirmation of mitochondrial purity and integrity, Western blot analysis was performed
for mitochondrial anti-VDAC (1:500; Mitosciences, Eugene, OR), nuclear anti-histone
H1 (1:250; Santa Cruz Biotechnology, Santa Cruz, CA), endoplasmic reticulum anti-
86

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).
Immunohistochemistry. For immunohistochemistry studies, animals (n=3 per group)
were sacrificed, brains were perfused with pre-chilled PBS buffer (pH 7.2) and
immersion fixed in 4% paraformaldehyde for 48 h and then stored in 4°C in PBS/1%
sodium azide until use. Fixed brains were sent to Neuroscience Associates (NSA,
Knoxville, TN) for coronal sectioning at 35μm, and then processed for
immunohistochemistry using a standard protocol. Briefly, every 12th section was blocked
(1h at RT, PBS with 5% goat serum and 0.3 % trinton x-100), immunostained using
antibody directed against A (6E10, Signet 1:1000 dilution 4
o
C overnight) followed by
washing and secondary antibody Fluorescein goat anti-mouse (1:500, Chemicon,
Ramona, CA, 1h at RT). Sections were mounted with anti-fade mounting medium with
DAPI (Vector Laboratories, Burlingame, CA). Antigen unmasking treatment, consisting
of 5 min rinse in 99% formic acid was performed to enhance A immunoreactivity (IR).
Fluorescent images were taken using a fluorescent microscope, normalized and analyzed
with the slide book software (Intelligent Imaging Innovations Inc, Santa Monica, CA).
Western Blot Analysis. Protein concentrations were determined by using the BCA
protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (20 μg/well) were
loaded in each well of a 12% SDS-PAGE gel, electrophoresed with a Tris/glycine
running buffer, and transferred to a 0.45 m pore size polyvinylidene di fluoride (PVDF)
87

membrane and immunobloted with PDH E1 alpha antibody (1:1000, Mitosciences,
Eugene, OR), COX IV antibody (1:2000, Mitosciences, Eugene, OR), ABAD antibody
(1:1000, Abcam, Cambridge, MA), β-actin antibody (1:4000, Chemicon, Ramona, CA)
and porin/VDAC antibody (1:500, Cell Sigaling, Danvers, MA). Mitochondrial Aβ
oligomer (16KD) level was determined in isolated mitochondrial samples (20μg/well)
and blotted by specific Anti-Aβ monoclonal antibody (6E10, Signet). HRP-conjugated
anti-rabbit antibody and HRP-anti-mouse antibody (Vector Laboratories, Burlingame,
CA) were used as secondary antibodies. Immunoreactive bands were visualized by Pierce
SuperSignal Chemiluminescent Substrates (Thermo Scientific) and captured by
Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA). All band intensities
were quantified using Un-Scan-it software (Silk Scientific, Orem, UT).
Enzyme Activity Assay. PDH activity was measured by monitoring the conversion of
NAD+ to NADH by following the change in absorption at 340 nm as previously
described (Gohil et al, 1983). Isolated brain mitochondria were dissolved in 2% CHAPS
buffer to yield a final concentration of 15 μg/μl and incubated at 37
o
C in PDH Assay
Buffer (35mM KH
2
PO
4
, 2 mM KCN, 0.5 mM EDTA, 5 mM MgCl
2
, (pH 7.25 with
KOH), 200mM sodium pyruvate, 2.5 mM rotenone, 4mM sodium CoA, 40 mM TPP).
The reaction was initiated by the addition of 15mM NAD
+
. COX activity was measured
on isolated mitochondria (20 μg) using Rapid Microplate Assay kit for Mouse Complex
IV Activity (Mitosciences, Eugene, OR) following the manufacturer’s instructions.
88

Complex I Activity was measured on isolated mitochondrial samples (5μg) using
Complex I Enzyme Activity Dipstick Assay Kit (Mitosciences, MS130-60, Eugene, OR),
band density was captured and analyzed by the matching Mitosciences Dipstick reader
(Mitosciences, MS1000, Eugene, OR).
Lipid Peroxidation. Lipid peroxides in brain mitochondria and hippocampal lysates were
measured using the leucomethylene blue assay (Blass et al, 2000), using tert-butyl
hydroperoxide as a standard, by monitoring the 650nm absorbance after 1 h incubation at
RT. The aldehyde product or termination production of lipid peroxidation in brain
mitochondria was determined by measuring thiobarbituric acid reactive substances
(TBARS). Samples were mixed with 0.15 M phosphoric acid. After the addition of
thiobarbituric acid, the reaction mixture was heated to 100
o
C for 1 h. After cooling and
centrifugation, the formation of TBARS was determined by the absorbance of the
chromophore (pink dye) at 531 nm using 600nm as the reference wavelength.
8-oxoGuannine Quanitification. Serum 8-oxoGuanine level was measured using the
OxiSelect
TM
oxidative RNA Damage ELISA (8-oxoGuanine Quantitation) assay kit
(Cellbiolabs, San Diego, CA) following the product manual.
Respiratory Measurement. Mitochondrial respiration was measured using MitoXpress
TM

fluorescent dye from isolated mitochondria following a previously established
protocol(Will et al, 2008; Will et al, 2006). Briefly, 50 µg of isolated mitochondria were
diluted to 1 µg/µl with respiratory buffer (250mM sucrose, 15 mM KCl, 1mM EGTA,
5mM MgCl
2
, 30 mM KH
2
PO
4
, pH7.4) and added to test well. MitoXpress
TM
probe was
89

reconstituted into 1µM stock solution and further diluted 1:10 in respiration buffer. State
4 respiration was stimulated with the addition of glutamate (5 mM) and malate (5 mM)
as substrates. State 3 respiration was stimulated by the addition of glutamate (5 mM) and
malate (5 mM) plus ADP (410 μM). The rate of oxygen consumption was calculated
based on the slope of the response of isolated mitochondria to the successive
administration of substrates. 100µl mineral oil was added to each well promptly after the
addition of MitoXpress working solution into the well. MitoXprobe signal was measured
at 1min intervals for 60 mins using excitation and emission wavelength of 380nm and
650nm respectively. To determine to rate of respiration, fluorescence-time profiles were
linearized using the following coordinate scal: Abscissa, Y: I(t
0
)/(I(t)-I(t
0
)), where I(t
0
)
and I(t) represent fluorescence intensity signals at the start and at time t of monitoring,
respectively; Ordinate, X: 1/t, min
-1
; exclude zero time points and regions of signal
saturation, i.e., long monitoring times. Linear regression analysis was applied to the
transformed profiles and determine the slope and correlation coefficient for each of the
transformed profiles. The State 4 and state 3 respiration rate was calculated as the
reciprocal ratio of the above two calculated slopes, respectiviely. The respiratory control
ratio (RCR) was defined by dividing the rate of oxygen consumption/min for state 3
(presence of ADP) by the rate of oxygen consumption/min for state 4

respiration.
Mitochondrial Biogenesis. Total DNA was isolated with Wizard Genomic DNA
Purification Kit (Promega, Madison, WI) and analyzed by real-time PCR. Mitochondrial
biogenesis was estimated as the relative levels of COXII DNA to β-actin.
90

Seahorse XF-24 Metabolic Flux Analysis. Primary hippocampal neurons from day 18
(E18) embryos of female Sprague-Dawley rats were cultured on Seahorse XF-24 plates at
a density of 50,000 cells/well. Neurons were grown in Neurobasal Medium +B27
supplement for 10 days prior to experiment. Mixed glia from day 18 (E18) embryos of
female Sprague-Dawley rats were cultured in T75 flasks and grown in growth media
(DMEM:F12 (1:1) +10% FBS). 24 hour prior to the experiment, mixed Glia were
trypsinized and seeded onto Seahorse XF-24 plates at a density of 50,000 cells/well in
growth media. For basal respiration comparison between neurons and mixed glia, cells
were prepared for assay directly. To investigate the impact of E2 treatment on
mitochondrial respiration in cell cultures, cells were treated with vehicle or E2 and the
assays were conducted 24 hours post-treatment. Neurons were treated with vehicle or E2
in neurobasal+B27 media. For E2 treatment in mixed glial cells, full growth media were
substituted with DMEM: F12 (1:1) without FBS to eliminate the interference from serum.
On the day of metabolic flux analysis, cells were changed to unbuffered DMEM (DMEM
Base medium supplemented with 25 mM glucose, 1 mM sodium pyruvate, 31 mM NaCl,
2 mM GlutaMax; pH 7.4) and incubated at 37
o
C in a non-CO
2
incubator for 1 h. All
medium and injection reagents were adjusted to pH 7.4 on the day of assay. Four baseline
measurements of OCR and ECAR were taken before sequential injection of
mitochondrial inhibitors. Three readings were taken following each addition of
mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (1 µM), FCCP (1 µM), and rotenone (1 µM). OCR and
91

ECAR were automatically calculated and recorded by the Seahorse XF-24 software.
After the assays, plates were saved and protein readings were measured for each well in
order to confirm equal cell numbers per well. The percentage of change compared to the
basal rates was calculated as the value of change divided by the average value of baseline
readings.
Statistics. Statistically significant differences between groups were determined by an
ANOVA followed by a Newman-Keuls post-hoc analysis.
92

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97

Chapter IV Mitochondrial Scheme of Estrogen Induced
Neuroprotection
Chapter IV Abstract

Previously we have demonstrated that estrogen protects against Alzheimer’s disease by
proactively sustaining and enhancing mitochondrial function, particularly mitochondrial
bioenergetics. Studies have shown that estrogen potentiates mitochondrial function by
preventing the OVX-induced decrease in bioenergetics, maintaining cellular redox
homeostasis, and preventing mitochondrial amyloid deposition. In this section, we sought
to investigate the mitochondrial mechanisms of estrogen induced neuroprotection. We
first determined the neuroprotective efficacy of estrogen against a variety of
mitochondrial toxins targeting different sites throughout the electron transport chain to
map out the site of direct action of estrogen induced neuroprotection. Pretreatment with
E2 protects neurons against insults from 3-NPA and antimycin, but not from KCN or
Oligomycin. E2 pretreatment also provided a moderate protection against rotenone. E2
pretreatment successfully prevented the 3-NPA induced activation of apoptotic pathways
and prevented the 3-NPA induced depletion of cellular GSH. In addition, we also
investigated the neuroprotective efficacy of E2 against amyloid toxicity. E2 pretreatment
significantly protected neurons from amyloid toxicity and prevented the increase in DNA
oxidative damage induced by amyloid. Using selective estrogen receptor agonists, PPT
for ER alpha and DPN for ER beta, we investigated the role of different estrogen
98

receptors in estrogen regulation of mitochondrial function. Both PPT and DPN increased
maximal mitochondrial respiratory capacity, with DPN inducing a higher increase than
PPT or E2, suggesting that ER beta is a promising target for promoting mitochondrial
function in brain. Lastly, we investigated the interaction between E2 and P4 on regulation
of mitochondrial function. Both in vitro and in vivo assays showed that both E2 and P4
re-regulated brain mitochondrial function and increased mitochondrial respiration.
However, the co-administration of E2 and P4 showed minimal benefits on mitochondrial
function.
All together, data from this study contribute to the understanding of estrogen
action on mitochondrial function. From a translational perspective, data also contribute to
the development of potential selective estrogen receptor modulators specific for the brain
and optimal hormone therapy strategies to prevent Alzheimer’s disease.
99

Introduction
Basic science analyses using both in vitro and in vivo model systems indicate that
estrogen, typically 17β-estradiol but also conjugated equine estrogens, induce protection
of neurons against insults associated with Alzheimer’s disease (Brinton, 2005; Chen et al,
2006b). Pretreatment with E
2
can protect against a wide range of toxic insults including
free radical generators (Behl et al, 1995; Green et al, 1996; Green et al, 2001)
excitotoxicity, β amyloid-induced toxicity (Nilsen et al, 2006) and ischemia (Dubal et al,
1998; Green et al, 2001). Moreover, these same estrogens in the same model systems
activated biochemical, genomic, cellular and behavioral mechanisms of memory
(Brinton, 2001, 2004; Dubal et al, 1998; McEwen, 2002; Nilsen and Brinton, 2001;
Simpkins et al, 1997; Singh et al, 1994; Toran-Allerand, 2000; Wise, 2001; Woolley,
1999). We have previously shown that many of the neuroprotective mechanisms of
estrogen converge upon mitochondria. We have demonstrated that E
2
pretreatment can
prevent mitochondrial dysfunction by promoting the maintenance of mitochondrial Ca2+
homeostasis (Nilsen et al, 2002). Further, E
2
increases the oxidative capacity and
efficiency of brain mitochondria (Irwin et al, 2008b; Nilsen et al, 2007). This increased
oxidative efficiency is paralleled by increased expression of subunits within both
Complex IV and V and is correlated with increased MnSOD and Peroxiredoxin
expression and reduced lipid peroxidation. Consistent with these findings, E2 treatment
increased the activity of the key glycolytic enzymes hexokinase, phosphofructokinase
and phosphoglycerate kinase in rodent brain (Kostanyan and Nazaryan, 1992b).
100

In the current study, we sought to further investigate estrogen action within the
mitochondrial electron transport chain. Using mitochondrial inhibitors specific for each
of the complexes, we sought to map out estrogen regulation of the oxidative
phosphorylation machinery. We further sought to determine the neuroprotective efficacy
of estrogen against amyloid insult in vitro. In addition, we investigated the role of
different estrogen receptors, ER alpha and ER beta, in estrogen regulation of
mitochondrial function. Lastly, we examined the interaction between E2 and P4 in
regulating mitochondrial function.
Findings from this study contribute to further understanding the mechanisms of
estrogen induced neuroprotection; they also help to facilitate the development of
therapeutic molecules as well as optimal hormone therapy regimens to better protect
against AD.
101

Results
Dose dependent toxicity of different mitochondrial inhibitors. E18 Primary neurons
were cultured for 10 days prior to exposure to a series of increasing doses of various
mitochondrial inhibitors that target different sites throughout the electron transport chain
(ETC) (Fig.IV-1A). Rotenone binds and inhibits complex I specifically. 3-NPA is a
specific inhibitor for succinate dehydrogenase (SDH, complex II). Antimycin inhibits
complex III whereas KCN binds and inhibits complex IV, cytochrome c oxidase.
Oligomycin is an ATP synthase inhibitor and inhibits the synthesis of ATP from ADP.
Cell viability was measured 24 hours after treatment. All mitochondrial inhibitors
exhibited a dose dependent toxicity (Fig.IV-1B - 1F). The cell death induced by
mitochondrial inhibitors is likely due to energy inhibition coupled with increased
oxidative stress. The inhibition of the complexes in the ETC not only inhibits electron
flow through the ETC and hence inhibits the generation of proton gradient and ATP
production, but also induces increased free radical generation and oxidative stress. Based
on this dose response experiment, we chose the toxin concentration that induced about
30% cell death to perform subsequent E2 neuroprotection assays in the next step.
102

Figure IV-1. Dose response of mitochondrial inhibitors. Primary hippocampal neurons
were treated with various mitochondrial toxins at different doses for 24 hours. Cell viability
after toxin treatment was measured by Calcein Am fluorescent assay. A, inhibition sites of
selected mitochondrial toxins; B-F, cell viability assay with treatments of rotenone, 3-NPA,
antimycin, KCN, and Oligomycin respectively.
103

Estrogen induced neuroprotection against mitochondrial toxins. Upon determining the
toxicity profile of different mitochondrial inhibitors, we continued to investigate the
therapeutic efficacy of estrogen induced neuroprotection against these toxins. In this
experiment, we selected the optimal toxin dose that could induce about 30% cell death to
avoid the activation of the irreversible cell death induced by high toxin dose whereas too
low toxin dose would not be able to induce enough cell death. A 24 hour pretreatment of
E2 at different doses exerted neuroprotection against only some of the inhibitors,
including 3-NPA, antimycin, and rotenone, whereas E2 showed no protection or even
adverse effects against other inhibitors, such as KCN and Oligomycin (Fig.IV-2).

Estrogen prevents 3-NPA induced cell death by preventing cell GSH depletion and
activation of caspase 9. To further investigate the underlying mechanisms of E2 induced
neuroprotection, we continued to examine estrogen regulation of the oxidative
components as well as apoptotic pathways. We have previously demonstrated that E2
increases antioxidant enzymes, including peroxiredoxin and MnSOD and reduced lipid
peroxidation (Irwin et al, 2008b; Nilsen et al, 2007). In this experiment, we investigated
the generation of mitochondrial reactive oxygen species (ROS) as well as the cellular
Glutathione (GSH) level as an indicator of oxidative status. 3-NPA induced both cell
death and mitochondrial ROS generation in a dose dependent manner (Fig.IV-3A&3B).
The increase in oxidative stress together with the inhibition in energy production induced
the cleavage of pro-caspase 9 into the active form and hence activated the apoptotic
104

pathway. E2 pretreatment significantly inhibited the 3-NPA induced activation of
apoptotic caspase 9 (Fig.IV-3C). Further, E2 pretreatment significantly prevented the 3-
NPA induced depletion of cellular GSH level (Fig.IV-3D).
Figure IV-2. Therapeutic efficacy of E2 induced neuroprotection against mitochondrial
inhibitors. Primary hippocampal neurons were pretreated with increasing doses of E2 24
hours prior to exposure to different mitochondrial toxins at desired doses for another 24
hours. Cell viability after toxin treatment was measured by Calcein Am fluorescent assay. A-
E, Therapeutic efficacy of E2 induced neuroprotection against rotenone, 3-NPA, antimycin,
KCN, and Oligomycin, respectively.
105

Figure IV-3. Estrogen protects against 3-NPA by inhibiting apoptosis and reducing
oxidative stress. A, Dose response of 3-NPA toxicity, cell viability after toxin treatment was
measured by Calcein Am fluorescent assay; B, 3-NPA induces mitochondrial ROS
generation, mitochondrial ROS was measured by mtSOX fluorescent assay; C, E2
pretreatment prevented 3-NPA induced activation of caspase 9; D, E2 pretreatment restored
3-NPA induced depletion of cellular GSH, GSH level was measured by fluorescent
monochlorobimane assay.
106

Estrogen prevents neurons against amyloid toxicity. An ultimate goal of our research is
to develop therapeutics to prevent or treat AD. Therefore, we investigated estrogen
induced neuroprotection against amyloid toxicity in addition to mitochondrial inhibitors.
Amyloid is known to induce neurotoxicity by activating Ca
2+
channels and hence leading
to excitotoxicity (Arispe et al, 1994). Amyloid has also been shown to cause
mitochondrial dysfunction and induce oxidative stress (Abramov et al, 2004). Other
proposed mechanisms of amyloid induced toxicity include activation of apoptotic
pathways (Yao et al, 2005), hyper activation of inflammatory responses(Morales et al)
and, of note, inhibition of energy production (Canevari et al, 1999; McGowan et al, 2006;
Reddy, 2009; Reddy et al, 2008a). In this section of the study, we focused on the
oxidative components of amyloid. Amyloid exhibited a dose dependent toxicity (Fig.4A)
and induction of mitochondrial ROS generation (Fig.4B). E2 pretreatment successfully
protected neurons from amyloid toxicity at many doses (Fig.4C). The increase in
mitochondrial ROS generation could lead to increased oxidative damage on cellular
components. We have previously shown that OVX induces an increase in lipid
peroxidation (Irwin et al, 2008b; Nilsen et al, 2006). In the current study, we investigated
the impact of amyloid on DNA oxidative damage. Aβ treatment at both 0.5μM and
1.5μM induced increase in DNA damage with the higher dose of amyloid beta inducing
overtly more DNA damage (Fig.5A). Moreover, the punctate pattern of 8-oxodG staining
suggests the mitochondrial localization of oxidative DNA damage. To further confirm
that mitochondrial DNA (mtDNA) rather than nuclear DNA (nDNA) was the primary
107

target of amyloid induced DNA damage, we double stained 8-oxodG and a mitochondrial
marker, porin (VDAC). There was significant overlap between 8-oxoDG and porin
signals (Fig.IV-5B), suggesting that it was mtDNA that was mostly oxidatively modified
by Aβ treatment. Compared to the nucleus, mitochondria lack potent DNA repair systems
and thus render mtDNa more susceptible to oxidative damage. As expected, E2
pretreatment significantly reduced DNA oxidative damage induced by amyloid (Fig.IV-
5C).
Estrogen does not directly activate DNA damage/repair pathway. Reduction in DNA
damage could be attributed to two potential pathways, either by decreasing free radical
generation, thus promoting the antioxidant system and preventing oxidative damage or by
up-regulating DNA repair pathway. To investigate the mechanisms of the E2 induced
decrease in DNA damage, we performed the DNA repair/damage pathway oligoarray
assay. The oligoarray contains major genes involved in DNA repair. As shown in Fig.IV-
6, there was no significant change in the DNA repair pathway with E2 treatment,
suggesting a preventative role for E2 treatment in protecting amyloid induced DNA
damage rather than a reparative role for E2. Findings from this study are consistent with
previous findings that E2 treatment reduces ROS generation by increasing mitochondrial
efficiency and promotes antioxidant system by increasing antioxidant enzymes, such as
peroxiredoxin V (Prdx V) and Manganese Superoxide Dismutase (MnSOD) (Borras et al,
2003; Irwin et al, 2008b; Mann et al, 2007; Nilsen et al, 2004; Numakawa et al, 2007;
Simpkins et al, 2007).
108

Figure IV-4. E2 induced
neuroprotection against Aβ
toxicity. A, dose dependent
Aβ toxicity. Primary neurons
were treated with increasing
doses of Aβ for 3 days. Cell
viability after toxin treatment
was measured by MultiTox-
Glo Multiplex cytotoxicity
assay. B,Aβ induced increase
in mitochondrial ROS
generation. Primary neurons
were treated with increasing
doses of Aβ for 24 hours.
Mitochondrial ROS was
measured by mtSOX red
fluorescent assay. C,
Therapeutic efficacy of E2
induced neuroprotection
against Aβ toxicity. Primary
neurons were pretreated with
increasing doses of E2
(0.1ng/ml to 1000ng/ml) 24
hours prior to the exposure to
1.5μM Aβ for 3 days. . Cell
viability after toxin treatment
was measured by MultiTox-
Glo Multiplex cytotoxicity
assay.
109

Figure IV-5. Aβ  induced mitochondrial DNA damage. Primary neurons were treated with
different doses of Aβ for 3 days. 8-oxodG was stained. A, Dose dependent increase in DNA
oxidative damage induced by Aβ . (red, 8-oxodG staining, blue, DAPI staining); B,
mitochondrial localization of DNA damage. Primary neurons were treated with 1.5μM for 3
days. Cells were fixed with 4% PFA and staining for both 8-oxodG and mitochondrial marker,
porin. 8-oxodG staining (red) overlap with porin signal (green); C, E2 pretreatment prevented
Aβ induced DNA damage.
110

Figure IV-6. E2 Does Not Directly Activate DNA Repair Pathway. Primary
hippocampal neurons were treated with four different treatments, vehicle, E2
only, amyloid only and E2 pretreatment plus amyloid. RNA was isolated and the
expression profile of genes involved in DNA repair pathway was investigated
using DNA Repair Oligoarray.
111

Both ER α and ER β are involved in the regulation of mitochondrial function. To
investigate the role of different ER subtypes in estrogen induced regulation of
mitochondrial function, we used selective estrogen receptor agonist PPT (for ER α) and
DPN (for ER β) and measured the impact of these agonists on cellular metabolic flux.
Both PPT and DPN induced a significant increase in the maximal mitochondrial
respiratory capacity whereas the basal mitochondrial respiration did not change,
suggesting that both ER α and ER β agonists can increase mitochondrial respiration.
Further, DPN induced a higher increase in maximal respiratory capacity than E2 or PPT,
suggesting a potent but different role for ER β (Fig.IV-7A). We also investigated the
involvement of ER α and ER β in E2 regulation of anaerobic glycolysis. E2 and PPT
induced a similar increase in extracellular acidification rate (ECAR) both at basal level
and in the presence of mitochondrial inhibitors. The increase in ECAR in response to the
addition of mitochondrial inhibitors reflected a compensatory response where glycolysis
was up-regulated to compensate for the reduction in ATP generation due to inhibition of
mitochondrial oxidative phosphorylation. Compared to E2 and PPT, DPN did not induce
any increase of ECAR either at basal level or in the presence of mitochondrial inhibitors
(Fig.IV-7B), suggesting a different role of ER β in regulating mitochondrial function and
glucose metabolism aside from ER α. In summary, both ER α and ER β are capable of
mediating E2 induced increase in mitochondrial function; nevertheless ER α and
112

ERβ may elicit distinct pathways and therefore regulate mitochondrial function
differentially.
Figure IV-7. Both ERα and
ERβ regulates mitochondrial
function. Primary neurons
were cultured for 10 days and
treated with vehicle, PPT,
DPN, or E2 for 24 hours.
Cellular metabolic flux
activity was measured using
the Seahorse metabolic
analyzer. A, PPT, DPN, and
E2 all increased maximal
respiratory capacity , with
DPN induced the highest
response than E2 and PPT; B,
E2 and PPT increased
glycolysis relative to vehicle
treatment both at basal level
and in response to
mitochondrial inhibitors,
whereas DPN did not induce
any change in anaerobic
glycolysis.
113

Progesterone antagonizes estrogen induced increase in mitochondrial function. In
addition to estrogen, ovarian hormones such as progesterone also have well-established
neurotrophic effects (Brinton et al, 2000; Nilsen et al, 2002; Norman et al, 2004).
Mitochondria have been proposed to be the center for steroid action in the brain. It has
been demonstrated that estrogen and progesterone can regulate metabolic function and
sustains the energy demands of neuronal activities. Nevertheless, the interaction between
estrogen and progesterone in regulating mitochondrial function is yet to be fully
investigated. This part of the study summarizes both the in vitro and in vivo studies we
conducted to determine the interaction between estrogen and progesterone in regulating
mitochondrial function. In vitro metabolic assays showed that both estrogen and
progesterone increased maximal mitochondrial respiratory capacity; however the co-
administration of estrogen and progesterone had no effect on mitochondrial function
(Fig.IV-8A). These findings are consistent with our previous in vivo study where short
term E2 or P4 treatment both enhanced brain mitochondrial function as evidenced by
increased respiratory control ratios whereas the co-administration of E2 and P4 failed to
increase brain mitochondrial respiratory rate (Fig.IV-8B).
Taken together, data suggest that the combined E2+P4 therapy strategy may not
be optimal paradigm to sustain post-menopausal brain metabolic function.
114

Figure IV-8. E2 and P4 regulation of mitochondrial respiration. A, E2 and P4
regulation of mitochondrial function in vitro. Primary neurons were cultured for 10 days
and treated with vehicle, E2, P4 or E2+P4 for 24 hours. Cellular metabolic flux activity
was measured using the Seahorse metabolic analyzer. Both E2 and P4 increased
maximal respiratory capacity whereas E2+P4 co-administration had no effect on
mitochondrial; B, E2 and P4 regulation of brain mitochondrial function in vivo. The
traces are representative of six to eight separate experiments. B, Percent changes in
mitochondrial RCR (state 3/state 4o). The data represent mean + SEM of eight separate
experiments. *, P< 0.05, compared with control; n = 5.
115

Discussion

In this study, we sought to 1) investigate the detailed sites of estrogen action
throughout the mitochondrial electron transport chain; 2) investigate the neuroprotective
efficacy of E2 against amyloid induced neurotoxicity and DNA oxidative damage; 3)
investigate the role of estrogen receptors, ER α and ER β, in estrogen regulation of
mitochondrial function; 4) investigate the interaction between E2 and P4 on
mitochondrial function. The outcome from this study not only contributes to the basic
understanding of the mechanism of E2 induced neuroprotection but also helps facilitate
the translational development of therapeutics targeting ER β for AD prevention in the
brain with fewer or no off-target effects in the peripheral system. Elucidation of the E2
and P4 interaction will enable better design of hormone therapy paradigm.
In this study, we showed that E2 pretreatment only protected inhibitors that are
specific for certain complexes, namingly rotenone for Complex I, 3-NPA for complex II,
antimycin for complex III, but not KCN for complex IV or Oligomycin for complex V.
Estrogen has long been demonstrated to up-regulate mitochondrial proteins, including
ETC complex subunits (Baulieu, 1991; Bieber et al, 2001; Brinton, 2008a). The
mechanisms by which estrogen regulates mitochondrial proteome are multifaceted,
including activation of classic estrogen receptor ER α/ ER β pathway, and activation of
the novel membrane estrogen receptor coupled with CREB/ERK signaling pathway. The
classical estrogen receptor pathway requires estrogen response elements (ERE) in the
promoter region of the target genes whereas the CREB/ERK pathway requires CREB
116

response elements (CRE). In this study, we did a bioinformatical search on genes that
were previously shown to be regulated by estrogen. While some of them only contains
either ERE or CRE, the majority of them contain both elements, adding to the complicity
of estrogen regulation (Fig.IV-9). In this study, E2 pretreatment failed to protect neurons
from KCN toxicity, despite the fact that E2 has been reported to up-regulate complex IV
subunits. This inconsistency could be attributed to the characteristics of KCN. E2 was
reported to moderately up-regulate only part of the complex IV subunits. The increase in
complex IV subunits is unlikely to counteract KCN induced toxicity in both energy
inhibition and increased oxidative stress. In this study, E2 also failed to rescue neurons
from Oligomycin toxicity. Our previous study has shown that E2 increases complex V
(ATP synthase) subunit α expression (F1 α). However, Oligomycin inhibits ATP
synthesis by blocking the proton channel (F0 complex). The increase in CV α expression
by E2 cannot alleviate the inhibition of the proton channel and therefore cannot protect
neurons against Oligomycin toxicity.
Mitochondrial bioenergetics and oxidative stress are two closely related aspects of
mitochondrial function. The neuroprotective efficacy of E2 could come from both
sustaining energy production and preventing oxidative stress. In this study, we showed
that E2 prevents the 3-NPA induced depletion of cellular GSH, a common antioxidant.
The pro-energetic and anti-oxidant properties of E2 together mediate E2 prevention of the
3-NPA induced activation of apoptotic pathways.

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Amyloid has long been demonstrated to cause mitochondrial dysfunction, induce
oxidative stress and inhibit ATP production. In this study, E2 pretreatment significantly
protected neurons from Aβ insults. Moreover, data suggest that mitochondrial DNA is the
Figure IV-9. ERE and CRE in genes regulated by E2. Proteins that have been
demonstrated to be regulated by E2 are categorized into 4 functional groups, with ERE and
CRE in their promoter region identified and labeled. Blue: CRE in the promoter; Red, ERE in
the promoter; Mixed, both CRE and ERE; Blank, no CRE or ERE; Green mtDNA encoded,
ERE/CRE in the D-loop of mtDNA.
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primary target of Aβ induced DNA damage. Although mitochondria have their own DNA
and DNA replication machinery, there is limited DNA repair mechanism inside
mitochondria compared to nuclear DNA, rendering mtDNA more susceptible to oxidative
damage. In the study, E2 pretreatment prevented the Aβ induced increase in DNA
damage. Data from oligoarray assay ruled out the possibility of direct activation of the
DNA repair pathway by E2, suggesting that the reduction in DNA damage is likely due to
E2 induced prevention of oxidative stress, either by decreasing the production of reactive
oxygen species or by increasing the expression of anti-oxidant enzymes. Our data is
consistent with previous findings that estrogen increases the expression and/or activity of
anti-oxidant enzymes, such as MnSOD and Prdx5 (Irwin et al, 2008b; Razmara et al,
2007) and decreases free radical generation (Borras et al, 2003; Valles et al, 2008; Vina
et al, 2005; Vina et al, 2007a).
In this study, we also provided evidence of the differential roles for ER α and ER
β in regulating mitochondrial function. Compared to PPT, the ER β selective agonist
DPN induced a higher increase in mitochondrial respiration. Considering the relatively
high expression level of ER β in the brain, our findings provide rationale for designing
and developing ER β selective molecules to sustain and enhance brain mitochondrial
function, while minimizing the ER α mediated proliferative side effect in the peripheral
system.
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In the last part of this study, we investigated the interaction between E2 and P4 in
regulating mitochondrial function. It has recently been reported that the neuroprotective
efficacy of estrogen is largely dependent on the time and regimen of hormone therapy in
the patients (Brinton, 2005, 2008b; Sherwin and Henry, 2007). Most women with intact
ovaries receive estrogen plus progesterone or progestins to block the proliferative side
effect of estrogen in the peripheral system. Data from this study suggest that the co-
administration of estrogen and progesterone at the same time might not be the optimal
paradigm to sustain and promote mitochondrial function, as such, our studies may help
clarify the conflict between basic science discoveries and the recent clinical findings from
the WHIMS (Brinton, 2005; Honjo et al, 2005; Shumaker et al, 2004; Shumaker et al,
2003) study.
In summary, we have demonstrated that E2 pretreatment protects against some,
but not all, mitochondrial inhibitors. Further, E2 pretreatment also protects against Aβ
insults. E2 induced neuroprotection is likely due to the coordinated actions of E2 that
sustain mitochondrial bioenergetics and prevent oxidative stress, rather than a direct
activation of the DNA repair pathway. Our findings that ER β is at least equally, if not
more, potent as ER α to enhance mitochondrial function in vitro could contribute to the
design and development of potential brain selective therapeutics. Finally, the interaction
between E2 and P4 in regulating mitochondrial function suggests that the co-
administration of estrogen and progesterone might not be the optimal paradigm for
hormone therapy to prevent AD and other neurodegenerative diseases.
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Methods and Material
Tissue Culture. Primary hippocampal neurons from embryonic day 18 (E18) embryos of
female Sprague-Dawley rats were cultured as previously described and generated cultures
98% neuronal in phenotype (Nilsen et al, 2006). Briefly, embryonic rat hippocampi were
dissociated by passage through fire-polish constricted Pasteur pipettes. Neurons plated on
polyethylenimine precoated 96 well plates or 60 mm petri-dishes were grown in
Neurobasal Medium +B27 supplement at 37oC in humidified 5% CO2 atmosphere for
10-12 days prior to experimentation.
Estrogen Induced Neuroprotection Against Mitochondrial Toxins. To measure the
toxicity of mitochondrial inhibitors, neurons were cultured 12 days prior to the exposure
to either vehicle or individual mitochondrial toxins at different doses. Briefly, rotenone
doses include 5μM, 7.5μM, 10μM, 12.5μM, and 15μM of rotenone; 3-NPA doses
include 2.5mM, 5mM, 7.5mM, 10mM, and 15mM of 3-NPA; Antimycin doses include
62.5μM, 75μM, 100μM, 125μM, and 150μM of antimycin; KCN doses include 10mM,
15mM, 20mM, 25mM, and 30mM of KCN; Oligomycin doses include 1μM, 2μM, 3μM,
4μM, and 5μM of Oligomycin. 24 hours later, cell viability was measured and the toxin
dose that induced about 25% to 30% of cell death was chosen to measure estrogen
induced neuroprotection against these toxins. Briefly, neurons were cultured for 11 days
and treated with an ascending series of doses of E2 (0.1ng/ml, 1ng/ml, 10ng/ml,
100ng/ml, and 1000ng/ml) 24 hours prior to the exposure of toxins. After another 24 hour
121

exposure to the toxins (10μM rotenone, 7.5mM 3-NPA, 125μM antimycin, 20mM KCN,
and 3μM Oligomycin, respectively), cell viability was measured.
Cell Viability Assays. Neuronal viability was assessed by Calcein AM staining
monitoring fluorescence (Ex: 485 nm/Em: 530 nm) on a fluorescent plate reader. Each
experiment consisted of 8 wells per condition. Means were normalized to the control
values for comparison across experiments. Data is presented as means +/- S.E.M. from at
least 3 independent experiments. Neuroprotective efficacy was defined as (Via
E2
-
Via
tox
)/(Via
veh
-Via
Tox
)*100%.
Western Blot Analysis. Cells were collected and protein was extracted using the M-Per
(Mammalian protein extract Reagent, Pierce, Rockford, IL). Protein concentrations were
determined by using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of
proteins (20 μg/well) were loaded in each well of a 12% SDS-PAGE gel, electrophoresed
with a Tris/glycine running buffer, and transferred to a 0.45μm pore size polyvinylidene
difluoride (PVDF) membrane and immunobloted with caspase 9 antibody (1:200, Santa
Cruz, Santa Cruz, CA), β-tubulin antibody (1:5000, Abcam, Cambridge, MA). HRP-
conjugated anti-rabbit antibody and HRP-anti-mouse antibody (Vector Laboratories,
Burlingame, CA) were used as secondary antibodies. Immunoreactive bands were
visualized by Pierce SuperSignal Chemiluminescent Substrates (Thermo Scientific) and
captured by Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA). All
band intensities were quantified using Un-Scan-it software.
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Mitochondrial ROS Generation. Mitochondrial ROS production was estimated by
loading cells with mitoSOX Red (Invitrogen, CA) fluorescent assay (Ex: 510 nm/Em:
580 nm) on a fluorescent plate reader. Each experiment consisted of 8 wells per
condition. Means were normalized to the control values for comparison across
experiments. Data is presented as means +/- S.E.M. from at least 3 independent
experiments.
Amyloid Induced Injury and E2 Neuroprotection. To measure amyloid toxicity, neurons
were treated with vehicle or different doses of A β1-42 (0.5 μM, 1μM, 1.5μM, 2μM, and
2.5 μM) for 3 days. Due to the auto fluorescence from A β, cytotoxicity was measured by
MultiTox-Glo Multiplex cytotoxicity assay (Promega, Madison, WI) instead of Calcein
Am cell viability assays. To measure E2 induced neuroprotection against A β, we chose
1.5 μM A β dose. Neurons were cultured 11 days and then exposed to an ascending series
of E2 doses (0.1ng/ml to 1000ng/ml) 24 hours prior to A β exposure. Cells were treated
with 1.5μM A β for 3 days in the continued presence of E2 and cell viability was
measured. Neuroprotective efficacy was defined as (Via
E2
-Via
A β
)/(Via
veh
-Via
A β
)*100%.
Immunocytochemistry. For immunocytochemistry study, cells grown in chamber slides
were treated as above and fixed in 4% paraformaldehyde for 15 mins and then blocked
(1h at RT, PBS with 5% goat serum and 0.3 % trinton x-100), immunostained using
antibody directed against 8-oxodG (6E10, Signet 1:1000 dilution 4
o
C overnight) and
porin (Mitosciences, Eugene, OR) followed by washing and secondary antibody
Fluorescein goat anti-mouse (1:500, Chemicon, Ramona, CA, 1h at RT) and CY3 goat
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anti-Rabbit (1:1000, Chemicon, Ramona, CA) 1h at RT. Slides were mounted with anit-
fade mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent
images were taken using a fluorescent microscope, normalized and analyzed with the
slide book software (Intelligent Imaging Innovations Inc, Santa Monica, CA).
Cellular GSH Measurement. To measure cellular GSH level, we use monochlorobimane
fluorescent assay as previously described (Chatterjee et al, 1999; Kamencic et al, 2000).
Briefly, cells were loaded with 100μM of monochlorobimane and washed with PBS 3
times. The GSH-monochlorobimane adduct was measured by monitoring the fluorescent
with excitation at 380nm and emission at 470nm. Means were normalized to the control
values for comparison across experiments. Data is presented as means +/- S.E.M. from at
least 3 independent experiments.
DNA Repair Oligoarray. Total RNA was isolated with Qiagen RNeasy mini RNA
extract kit (Qiagen, Valencia, CA). Changes in the expression of genes involved in DNA
repair pathway was investigate with DNA repair Oligoarray (SAbiosciences, Frederick,
MD) by following manufacturer’s protocol and data were analyzed by the suite soft from
SAbiosciences.
Seahorse XF-24 Metabolic Flux Analysis. Primary hippocampal neurons from day 18
(E18) embryos of female Sprague-Dawley rats were cultured on Seahorse XF-24 plates at
a density of 50,000 cells/well. Neurons were grown in Neurobasal Medium +B27
supplement for 10 days prior to experiment. Neurons were treated with vehicle, E2, PPT,
and DPN, respectively in neurobasal+B27 media 24 hours prior to the assay. On the day
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of metabolic flux analysis, cells were changed to unbuffered DMEM (DMEM Base
medium supplemented with 25mM glucose, 1mM sodium pyruvate, 31mM NaCl, 2mM
GlutaMax; pH 7.4) and incubated at 37
o
C in a non-CO
2
incubator for 1 h. All medium
and injection reagents were adjusted to pH 7.4 on the day of assay. Four baseline
measurements of OCR and ECAR were taken before sequential injection of
mitochondrial inhibitors. Three readings were taken following each addition of
mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (1 µM), FCCP (1 µM), and rotenone (1 µM). OCR and
ECAR were automatically calculated and recorded by the Seahorse XF-24 software.
After the assays, plates were saved and protein readings were measured for each well in
order to confirm equal cell numbers per well. The percentage of change compared to the
basal rates was calculated as the value of change divided by the average value of baseline
readings. To investigate the impact of E2 and P4 co-administration of cellular metabolic
flux activity, primary hippocampal neurons from day 18 (E18) embryos of female
Sprague-Dawley rats were cultured on Seahorse XF-24 plates at a density of 50,000
cells/well. Neurons were grown in Neurobasal Medium +B27 supplement for 10 days
prior to exposure of E2 (10ng/ml), P4 (100ng/ml), and E2 (10ng/mL)+P4 (100ng/mL),
respectively. Metabolic flux activity was measured 24 hours later. On the day of
metabolic flux analysis, cells were changed to unbuffered DMEM (DMEM Base medium
supplemented with 25mM glucose, 1mM sodium pyruvate, 31mM NaCl, 2mM
GlutaMax; pH 7.4) and incubated at 37
o
C in a non-CO
2
incubator for 1 h. All medium
125

and injection reagents were adjusted to pH 7.4 on the day of assay. Four baseline
measurements of OCR and ECAR were taken before sequential injection of
mitochondrial inhibitors. Three readings were taken following each addition of
mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (1 µM), FCCP (1 µM), and rotenone (1 µM). OCR and
ECAR were automatically calculated and recorded by the Seahorse XF-24 software.
After the assays, plates were saved and protein readings were measured for each well in
order to confirm equal cell numbers per well. The percentage of change compared to the
basal rates was calculated as the value of change divided by the average value of baseline
readings.
Statistics. Statistically significant differences between groups were determined by an
ANOVA followed by a Newman-Keuls post-hoc analysis.

126

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130

Chapter V Therapeutic Implications of Regulating
Mitochondrial Bioenergetics in AD
Chapter V Abstract

It has been shown that brain metabolism is declined in AD patients at least a
decade before disease diagnosis (Brinton, 2008a, b; Costantini et al, 2008; Wang et al,
2007) . This antecedent decline in metabolism indicates a important role of mitochondrial
bioenergetics in AD pathogenesis and disease progression (Yao et al, 2009). In the
current study, we sought to investigate the therapeutic potential of manipulating brain
metabolic profile in the triple transgenic mouse model. Using in vitro metabolic assays,
we demonstrated that both neurons and mixed glia were able to utilize alternative
substrates such as ketones and fatty acid for ATP production in energy demanding
situations. Moreover, at 3 months of age, 3xTgAD mice showed increased SCOT
expression, indicating a compensatory mechanism to up-regulate ketone utilization and
offset the bioenergetic deficits in AD. We further investigated the therapeutic efficacy of
ketone induction to delay AD progression. Using 2-deoxyglucose, we induced
ketogenesis in both nonTg and 3xTgAD female mice. The induction in ketone generation
showed a moderate beneficial effect against AD progression. In nonTg and 3xTgAD
mice, 2-DG induced increase in mitochondrial enzymes involved in ketogenesis and
ketone utilization. In 3xTgAD mice, the induction of ketogenesis was paralleled by the
reduction of brain amyloid load as evidenced by decreased both APP, 56* Aβ level, and
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Aβ immunoreactivity in the hippocampus. The decrease in amyloid pathology was likely
due to the up-regulation of non-amyloidogenic α secretase pathway. In nonTg mice, 2-
DG also reduced mouse APP protein level.
Together, findings from this study suggest a potential therapeutic strategy to
compensate brain bioenergetic deficits by providing alternative substrates and therefore
delay AD progression.
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Introduction
Previously it has been shown that mitochondrial bioenergetic deficits play a
critical role in AD pathogenesis (Yao et al, 2009). Further, mitochondrial function
deteriorates with AD progression (Lustbader et al, 2004; Takuma et al, 2005). The
trajectory of the decline in mitochondrial bioenergetics provides a potential therapeutic
window to compensate the energy loss and hence delay disease progression. Ketone body
has been demonstrated to serve as an alternative substrate aside from glucose for the
brain. Large scale epidemiology studies have suggested a potential neuroprotective
benefit of ketone bodies (Gasior et al, 2006; Guzman and Blazquez, 2004).
2-deoxyglucose (2-DG) is a glucose analog with the 2-hydroxyl group replaced
by hydrogen. Due to the structural similarity between 2-DG and glucose, it is uptaken by
glucose transporters of the cell. However, with no 2-hydroxyl group, 2-DG cannot be
phosphorylated by hexokinase and therefore cannot undergo further glycolysis. 2-DG
has been broadly used as a ketogenic compound. Diet containing a wide range of various
2-DG doses has been demonstrated to induce ketogenesis.
In the current study, we sought to investigate the therapeutic efficacy of
ketogenesis in delaying AD progression. Briefly, we treated both nonTg and 3xTgAD
female mice at 6 months with either the ctrl diet or the 2-DG diet (ctrl+ 0.04% 2-DG
w/w) for up to 7 weeks. We examined the impact of ketogenesis on AD pathology as well
as mitochondrial function in both nonTg and 3xTgAD mice. 2-DG diet increased the
expression of enzymes involved in ketone metabolism and reduced AD like pathology in
133

3xTgAD mice. Findings from this study suggest a potential therapeutic strategy to supply
alternative substrates to compensate the decline in brain metabolism and delay the
progression of AD.
Results

Compensatory up-regulation of SCOT expression early in AD. We have previously
shown that the decrease in mitochondrial bioenergetics, particularly glucose metabolism,
is an antecedent event in AD pathogenesis. Protein expression of important enzymes such
as PDH was compromised as early as 3 months. Moreover, primary neurons derived from
3xTgAD mice showed declined mitochondrial respiration when fed with
glucose/pyruvate substrates (Yao et al, 2009). The decline in glucose driven OXPHOS
could activate compensatory pathways, such as up-regulation of anaerobic glycolysis or
induction of ketogenesis as alternative fuel substrates. SCOT (Succinyl-CoA:3-ketoacid
transferase) is a key mitochondrial enzyme involved in ketone body catabolism. Change
in SCOT expression reflects ketogenic activity. We measured SCOT expression in the
hippocampal lysates from 3, 6, 9, and 12 month old female nonTg and 3xTgAD mice
collected in previous studies presented in chapter II. At 3 months of age, 3xTgAD mice
showed significant higher SCOT expression relative to age-matched nonTg mice,
suggesting a compensatory mechanism to up-regulate ketogenic pathway and compensate
the decline in brain glucose utilization. SCOT expression declined with age from 3 month
to 9 month in the 3xTgAD mice, indicating the diminishing of the ketogenic
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compensation with AD progression, whereas in nonTg mice, SCOT level stays largely
the same. There was a moderate increase in SCOT level from 9 month to 12 month in
nonTg and 3xTgAD mice, which paralleled the occurrence of reproductive senescence,
which again indicates a critical role for estrogen in sustaining and enhancing glucose
driven brain metabolism. Loss of estrogen due to reproductive senescence induced the
compensatory activation of ketogenic pathway. Noticeably, at 12 month nonTg mice had
significantly higher level of SCOT relative to 3xTgAD mice, further manifesting the
difference in brain metabolic profiles between nonTg and 3xTg-AD mice with advanced
AD pathology (Fig.V-1).

Figure V-1. Different SCOT profile in nonTg and 3xTgAD mice. Both female nonTg and
3xTgAD mice at 3, 6, 9, and 12 months of age were sacrificed and hippocampal SCOT level
was determined by western blot. 3xTgAD mice showed increased SCOT protein level relative
to nonTg mice at 3 months; at 12 month, nonTg mice had significantly higher SCOT protein
level (n=3 per group).
135

Ability to use ketone bodies as alternative fuel by neurons and mixed glia. Under
normal conditions, brain exclusively relies on glucose/pyruvate for energy production
(Brinton, 2008a, b). However, under long term starvation or certain disease conditions,
brain can use an alternative substrate, ketone bodies, for ATP generation (Costantini et al,
2008; Guzman et al, 2004; Morris, 2005). Ketone bodies are mainly synthesized in the
liver through fatty acid oxidation (FAO) and reserved as substrates for energy production
in the heart, brain and other organs. Additionally, brain has recently been demonstrated to
possess its own ketogenic machinery (Auestad et al, 1991; Guzman et al, 2004). To
investigate the metabolic profile of neurons and mixed glia on alternative substrates, we
measured mitochondrial respiration using different substrates, including pyruvate, ketone
body (acetoacetate), and fatty acid (palmitate). Neurons and mixed glia showed a similar
metabolic profile on different substrates. The addition of substrates alone did not increase
basal oxygen consumption rate (OCR) in neurons or mixed glia. However, with the
addition of a mitochondrial uncoupler, FCCP, all substrates showed increased OCR
relative to BSA control, with pyruvate yielding the highest OCR value and acetoacetate
and palmitate yielding a moderate increase in OCR (Fig.V-2). Data suggest that despite
the preference for glucose/pyruvate substrates, both neurons and mixed glia are able to
use ketones for ATP generation under energy demanding situation (mimicked by FCCP
addition). The difference in OCR value between neurons and mixed glia reflects the
different characteristics of neurons and glia as discussed in chapter III.

136

Induction of ketogenesis by 2-DG. 2-Deoxyglucose is a molecule that is structurally
similar to glucose but competitively inhibits the glycolysis pathway by binding and
inhibiting hexokinase. It has been used as a ketogenic compound (Numakawa et al,
2007). To investigate the impact of 2-DG induced ketogenesis on brain mitochondrial
function and Alzheimer’s pathology, we designed the experiment as described in Fig.V-3.
Figure V-2. Metabolic Profile
of Neurons and Mixed Glia
on Different Substrates.
Primary hippocampal neurons
from day 18 (E18) embryos of
female Sprague-Dawley rats
were cultured in Neurobasal
medium + B27 supplement for
10 days prior to experiment.
Mixed glia from day 18 (E18)
embryos of female Sprague-
Dawley rats were cultured in
growth media (DMEM:F12
(1:1)+10% FBS). Primary
Oxygen consumption rate
(OCR) was determined using
Seahorse XF-24 Metabolic
Flux analyzer. Vertical lines
indicate time of addition of
substrates and mitochondrial
inhibitors. Substrates: pyruvate,
acetoacetate, BSA ctrl and
BSA-conjugated palmitate;
oligomycin (1 µM); C: FCCP
(1 µM). A, metabolic profile of
neurons; B, metabolic profile
of mixed glia.
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Briefly, both nonTg and 3xTgAD mice at the age of 6 months were treated with either
ctrl diet (AIN93G) or 2-DG (AIN93G +0.04% 2-DG) diet up to 7 weeks (N=12 per
group). We expected to observe induction of ketogenesis in mice on the 2-DG diet upon
completion of the study. Body weight was measured as the first indicator of ketogenesis.

To our surprise, nonTg mice showed higher sensitivity to 2-DG diet than 3xTgAD
mice. There were 7 nonTg mice dropped out due to severe body weight loss (>40%)
compared to only 2 3xTgAD mouse. The difference in sensitivity to 2-DG between
nonTg and 3xTgAD indicated an intrinsic shift in metabolic profile of 3xTgAD mice
towards ketogenic phenotype. At the end of the study, all groups on the 2-DG diet lost
significant amount (~20%) of body weight, whereas mice on the ctrl diet showed no
significant change in body weight (Table V-1). We further measured the serum level of
β-hydroxybutyrate as a direct indicator of ketogenesis. 2-DG induced significant increase
Figure V-3. Study Design. Both nonTg and 3xTgAD female mice were treated with ctrl
diet (AIN93G) or 2-DG diet (AIN93G+0.04% 2-DG w/w). Upon completion, mice were
sacrificed and both pathological and mitochondrial metabolic parameters were analyzed.
138

in serum β-hydroxybutyrate level in 3xTgAD mice relative to ctrl diet, with a similar but
not significant increase in serum β-hydroxybutyrate level in nonTg mice (Fig.V-4).
Table V-1. Body Weight Change in Ctrl and 2-DG group

Body
Weight (g)
3xTgAD
Ctrl
3xTgAD
2-DG
nonTg
Ctrl
nonTg
2-DG
Week 0 24.6 + 0.95 26.8 + 1.36 24.6 + 0.84 26.9 + 1.03
Week 7 23.1 + 0.75 21.8 + 1.62 * 23.5 + 0.69 20.4 + 1.27 *
Change
(%)
-6.40% -18.61% -4.21% -23.82%
Figure V-4. Induction of ketogenesis by 2-DG diet. Serum β-hydroxybutyrate
level was measured by LIQUICOLOR β-hydroxybutyrate quantification kit as an
indicator of ketogenesis. 2-DG diet induced ketogenesis in both nonTg and
3xTgAD mice.
Table V-1. Body weight change in ctrl and 2-DG group. Both nonTg and 3xTgAD female
mice at 6 months of age were randomly assigned to either Ctrl (AIN93G) or 2-DG
(AIN93G+0.04% 2-DG) group. Body weight was monitor once per week. Percent of Change
is defined as (BW
07
-BW
00
)/ BW
00
*100%. 2-DG induced body weight decrease in both nonTg
and 3xTgAD mice. *, P<0.05, at the end of the study, body weight is significantly lower than
the beginning of the study.
139

Reduction of AD pathology by 2-DG. We have previously shown that bioenergetic
deficits precedes AD pathology (Yao et al, 2009), suggesting a potential causal
relationship between mitochondrial energy deficits and AD pathogenesis (Lin and Beal,
2006; Loeb et al, 2005; Reddy, 2006; Yao et al, 2009). Thus ketogenesis induction and
the subsequent energy compensation provided by ketones should modify AD progression.
To test this hypothesis, we examined the pathological markers in 3xTgAD mice on both
ctrl and 2-DG diet. Western from hippocampal tissue lysate demonstrated that there was a
significant decrease in both APP expression and the 56* Aβ oligomer level in 3xTgAD
mice treated with 2-DG compared to the ctrl diet group (Fig.V-5A). The reduction in
amyloid load was further confirmed by the decreased Aβ immunoreactivity of 6E10
staining (recognizing all forms of Aβ) in the CA1 region (Fig.V-5B). The simultaneous
reduction in APP and 56* Aβ oligomer level suggested an increase in the non-
amyloidogenic (α secretase) pathway instead of the amyloidogenic (β secretase and γ
secretase) pathway. This was further confirmed with western blot data showing that
ADAM10, the catalytic subunit of α secretase, was up-regulated in the 2-DG group
(Fig.V-6). As expected, there was a paralleled increase in enzymes involved in
mitochondrial ketogenic pathway as well as OXPHOS, including ERAB, SCOT and CV
α (Fig.V-6), suggesting a close relationship between mitochondrial bioenergetics and AD
pathology.

140

Figure V-5. Decreased amyloid pathology in 2-DG treated 3xTgAD mice. A,
Hippocampal tissue lysate from ctrl and 2-DG treated 3xTgAD mice were blotted for APP
and 56*Aβ level. 2-DG reduced both APP and 56*Aβ level. B, reduction of Aβ
immunoreactivity by 2-DG. Brains from the ctrl and 2-DG group of both 3xTgAD and nonTg
mice were sectioned at 40μm. Sections were stained with 6E10 antibody (green), IBA1
antibody (Red) and DAPI for nuclei (DAPI). Representative images show Aβ-IR in CA1
(bottom 2 rows) region (scale: 100 mm).
141

In nonTg mice, 2-DG induced a comparable increase in enzymes involved in
ketogenesis including ERAB, HADHA, and SCOT (Fig.V-7B). In addition, 2-DG
reduced mouse APP levels (Fig.V-7A). Due to the nature of mouse Aβ, we were not able
to measure the oligomer form. In both nonTg and 3xTgAD group, APP was reduced by
2-DG treatment.
Figure V-6. 2-DG induction of enzymes in ketone metabolism and α Secretase.
Hippocampal tissue lysate from ctrl and 2-DG treated 3xTgAD mice were blotted for
SCOT, ERAB, CV alpha and ADAM-10 levels. Proteins were normalized to β-actin.
Figure V-7. 2-DG induction of enzymes in ketone metabolism and reduction in mouse
APP. Hippocampal tissue lysate from ctrl and 2-DG treated 3xTgAD mice were blotted for
SCOT, ERAB, CV alpha, HADHA, and mouse APP levels. Proteins were normalized to β-
actin. *,P<0.05 compared to the ctrl group.
142

Discussion
In previous chapters, we focused on estrogen regulation of mitochondrial
bioenergetics and its role in sustaining and enhancing mitochondrial deficits for potential
prevention of AD. Recent clinical and basic researches have suggested a critical
therapeutic window for estrogen induced “PREVENTION” rather than treatment against
AD (Sherwin and Henry, 2007). The healthy cell bias hypothesis proposed by Brinton
and her colleagues also emphasized on the different outcome of estrogen actions on
neurons prior and post insults (Brinton, 2008b). We herein proposed a preventative role
for estrogen to prevent AD rather than a reparative estrogen action in AD treatment.
In the current study, we extended our effort to investigate the therapeutic potential
of manipulating brain mitochondrial bioenergetics to modify the disease progression. At
6 months of age, 3xTgAD mice exhibit intraneuronal amyloid pathology, memory
deficits, and fairly advanced mitochondrial dysfunction, a stage similar to early-to-middle
stage of AD in human. We initiated 2-DG treatment as an intervention aiming to delay
the progression of AD rather than prevention. At this age, the intrinsic compensatory
ketogenic pathway in the 3xTgAD mice that was most obvious at 3 months starts to
deminish. We hypothesized that 2-DG diet will promote ketogenesis, alleviate the energy
deficits in 3xTgAD brain and delay the progression of AD pathology. Data so far
supported this hypothesis. 2-DG promoted ketone generation, increased expression of
mitochondrial OXPHOS enzymes, and reduced amyloid pathology relative to the ctrl
diet. Our findings are consistent with previous epidemiological analysis that ketogenic
143

diet has moderate neuroprotective benefits (Guzman et al, 2004; Stafstrom et al, 2008).
Nevertheless, we are aware that ketogenesis is by large a compensatory and temporary
mechanism that only aims at delaying the progression of AD instead of preventing AD or
reversing AD. Ketones offer short-term marginal benefits when glucose metabolism is
declined or compromised but cannot completely replace glucose for energy production in
the brain. The length of benefits of ketogenesis in AD progression requires further
investigation. Interestingly, in this study nonTg mice showed a much higher sensitivity
towards 2-DG diet than 3xTgAD mice. This again suggests a shift in the metabolic
profile of 3xTgAD brains towards ketogenic phenotype early in the disease progression
whereas adult nonTg mice are exclusively dependent on glucose for energy production.
Thus a drastic switch to 2-DG diet caused greater body weight loss and higher mortality
rate in nonTg mice than in 3xTgAD mice.
In the current study, we also observed that the reduction of amyloid pathology
paralleled with the increase in α secretase pathway, suggesting that the activation of non-
amyloidogenic pathway likely accounts for the reduction in amyloid generation. Many
factors have been demonstrated to regulate the competition between the amyloidogenic
(β and γ secretase) and non-amyloidogenic (α secretase) pathways, among which
mitochondrial bioenergetic failure is of special importance. It has been shown that energy
inhibition activates the β secretase pathway and increases amyloid pathology (Bertoni-
Freddari et al, 1997; Meier-Ruge and Bertoni-Freddari, 1997; Velliquette et al, 2005).
In addition to directly regulating APP processing, changes in mitochondrial bioenergetics
144

also impact cholesterol synthesis, which subsequently regulates the co-localization of
APP processing machinery (Benarroch, 2008; Colell et al, 2009; Etgen, 2008). Further,
mitochondrial bioenergetics is tightly coupled with oxidative stress. Increased oxidative
damage usually magnifies the deficits in bioenergetics and leads to hyper-activation of
amyloid production. Centered on mitochondrial bioenergetics, all these factors contribute
to amyloid production and AD pathogenesis.
Taken together, data from this study suggest a potential disease modifying
strategy by providing alternative substrates to compensate the energy deficits in AD brain
and therefore delay the disease progression.
145

Material and Methods.
Transgenic Mice. Colonies of 3xTgAD and nonTg mouse strain (C57BL6/129S; The
Jackson Laboratory, Bar Harbor, ME) (Oddo et al, 2003a; Oddo et al, 2003b) were bred
and maintained at the University of Southern California (Los Angeles, CA) following
National Institutes of Health guidelines on use of laboratory animals and an approved
protocol by the University of Southern California Institutional Animal Care and Use
Committee. Mice were housed on 12 h light/dark cycles and provided ad libitum access
to food and water.
Experimental Design. To investigate the impact of 2-DG on mitochondrial function and
Alzheimer’s pathology, 6 month old female 3xTgAD and nonTg mice were randomly
assigned to either the ctrl diet (AIN93G) group or the 2-DG diet group. Detailed
components of the ctrl and 2-DG diet are listed in Fig V-8. Mice were on the Ctrl or 2-
DG diet for up to 7 weeks. Upon completion of the treatment, mice were sacrificed.
Mouse body weight was monitor once per week till the completion of the study.
Brain Tissue Preparation and Tissue Collection. Upon completion of hormone
treatment, both 3xTgAD and nonTg groups (n=7 to 12 per group) were sacrificed. Serum
was collected and stored at -70
o
C till use. Brains were perfused with pre-chilled PBS
buffer (pH 7.2) and the right hemisphere was immersion fixed in 4% paraformaldehyde
for 48 h and then stored in 4°C in PBS/1% sodium azide until use. The left hemisphere
was quickly dissected on ice and hippocampal and cortical tissues were harvested
separately and stored at -70
o
C for later use.
146

Immunohistochemistry. For immunohistochemistry studies, fixed hemispheres were
coronally sectioned at 40μm, and then processed for immunohistochemistry using a
standard protocol. Briefly, every 12th section was blocked (1h at RT, PBS with 5% goat
serum and 0.3 % trinton x-100), immunostained using antibody directed against
Aβ (6E10, Signet 1:1000 dilution 4
o
C overnight) and anti-IBA (Chemicon, Ramona, CA,
1:1000 dilution 4
o
C overnight) followed by washing and secondary antibody Fluorescein
goat anti-mouse (1:500, Chemicon, Ramona, CA, 1h at RT) and Cy3 conjugated goat
anti-rabbit (1:1000, Chemicon, Ramona, CA, 1h at RT). Sections were mounted with
anti-fade mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Antigen
unmasking treatment, consisting of 5 min rinse in 99% formic acid was performed to
enhance Aβ immunoreactivity (IR). Fluorescent images were taken using a fluorescent
microscope, normalized and analyzed with the slide book software (Intelligent Imaging
Innovations Inc, Santa Monica, CA).
Western Blot Analysis. Protein was extracted from the hippocampal tissue collected
previously using the tissue protein extraction reagent (Perce, Rockford, IL) plus 1%
protease inhibitor and 1% phosphatase inhibitor cocktail. Protein concentrations were
determined by using the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of
proteins (20 μg/well) were loaded in each well of a 12% SDS-PAGE gel, electrophoreses
with a Tris/glycine running buffer, and transferred to a 0.45μm pore size polyvinylidene
difluoride (PVDF) membrane and immunobloted with PDH E1 alpha antibody (1:1000,
Mitosciences, Eugene, OR), COX IV antibody (1:2000, Mitosciences, Eugene, OR),
147

ABAD antibody (1:1000, Abcam, Cambridge, MA), β-actin antibody (1:4000,
Chemicon, Ramona, CA), SCOT antibody (1:200, Santa Cruz, Santa Cruz, CA), Anti-Aβ
monoclonal antibody (6E10, Signet), rodent specific Aβ antibody (1:1000, M3.2
Covance) and HADHA antibody (1:1000, Abcam, Cambridge, MA). HRP-conjugated
anti-rabbit antibody and HRP-anti-mouse antibody (Vector Laboratories, Burlingame,
CA) were used as secondary antibodies. Immunoreactive bands were visualized by Pierce
SuperSignal Chemiluminescent Substrates (Thermo Scientific) and captured by
Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA). All band intensities
were quantified using Un-Scan-it software (Silk Scientific, Orem, UT).
Seahorse XF-24 Metabolic Flux Analysis. Primary hippocampal neurons from day 18
(E18) embryos of female Sprague-Dawley rats were cultured on Seahorse XF-24 plates at
a density of 50,000 cells/well. Neurons were grown in Neurobasal Medium +B27
supplement for 10 days prior to experiment. Mixed glia from day 18 (E18) embryos of
female Sprague-Dawley rats were cultured in T75 flasks and grown in growth media
(DMEM:F12 (1:1) +10% FBS). 24 hour prior to the experiment, mixed Glia were
trypsinized and seeded onto Seahorse XF-24 plates at a density of 50,000 cells/well in
growth media. To investigate the potential of neurons and glia to use alternative fuel
source (ketones and fatty acid) other than glucose/pyruvate, On the day of metabolic flux
analysis, cells were changed to unbuffered KHB (Krebs Henseleit Buffer, 111mM NaCl,
4.7mM KCl, 2mM MgSO
4
,

1.2mM Na
2
HPO4, 2.5mM glucose, and 0.5mM Carnitien, pH
7.4) and incubated at 37
o
C in a non-CO
2
incubator for 1 h. All medium and injection
148

reagents were adjusted to pH 7.4 on the day of assay. Four baseline measurements of
OCR and ECAR were taken before sequential injection of substrates and mitochondrial
inhibitors. Three readings were taken following each addition of mitochondrial inhibitor
prior to injection of the subsequent inhibitors. The substrates used were ketones
(acetoacetate), BSA ctrl, BSA conjugated palmitate, and pyruvate, respectively. The
mitochondrial inhibitors used were oligomycin (1µM), FCCP (1µM), and rotenone
(1µM). OCR and ECAR were automatically calculated and recorded by the Seahorse XF-
24 software. After the assays, plates were saved and protein readings were measured for
each well in order to confirm equal cell numbers per well. The percentage of change
compared to the basal rates was calculated as the value of change divided by the average
value of baseline readings. To investigate the impact of E2 and P4 co-administration of
cellular metabolic flux activity, primary hippocampal neurons from day 18 (E18)
embryos of female Sprague-Dawley rats were cultured on Seahorse XF-24 plates at a
density of 50,000 cells/well. Neurons were grown in Neurobasal Medium +B27
supplement for 10 days prior to exposure of E2 (10ng/ml), P4 (100ng/ml), and E2
(10ng/mL)+P4 (100ng/mL), respectively. Metabolic flux activity was measured 24 hours
later. On the day of metabolic flux analysis, cells were changed to unbuffered DMEM
(DMEM Base medium supplemented with 25mM glucose, 1mM sodium pyruvate, 31mM
NaCl, 2mM GlutaMax; pH 7.4) and incubated at 37
o
C in a non-CO
2
incubator for 1 h. All
medium and injection reagents were adjusted to pH 7.4 on the day of assay. Four baseline
measurements of OCR and ECAR were taken before sequential injection of
149

mitochondrial inhibitors. Three readings were taken following each addition of
mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial
inhibitors used were oligomycin (1µM), FCCP (1µM), and rotenone (1µM). OCR and
ECAR were automatically calculated and recorded by the Seahorse XF-24 software.
After the assays, plates were saved and protein readings were measured for each well in
order to confirm equal cell numbers per well. The percentage of change compared to the
basal rates was calculated as the value of change divided by the average value of baseline
readings.
Serum Ketone Quatification. Serum levels of β-hydroxybutyrate were measured using
the LIQUICOLOR β-hydroxybutyrate quantification kit as an indicator of ketogenesis by
following the manufacturer’s direction.
Statistics. Statistically significant differences between groups were determined by an
ANOVA followed by a Newman-Keuls post-hoc analysis.

150

Figure V-8. Nutrient components of AIN93G and 2-DG (0.04%) diet.
151

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Benarroch EE (2008). Brain cholesterol metabolism and neurologic disease. Neurology 71(17):
1368-1373.

Bertoni-Freddari C, Fattoretti P, Paoloni R, Caselli U, Meier-Ruge W (1997). Impaired dynamic
morphology of cerebellar mitochondria in physiological aging and Alzheimer's disease. Ann N Y
Acad Sci 826: 479-482.

Brinton RD (2008a). Estrogen regulation of glucose metabolism and mitochondrial function:
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Brinton RD (2008b). The healthy cell bias of estrogen action: mitochondrial bioenergetics and
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Colell A, Fernandez A, Fernandez-Checa JC (2009). Mitochondria, cholesterol and amyloid beta
peptide: a dangerous trio in Alzheimer disease. J Bioenerg Biomembr.

Costantini LC, Barr LJ, Vogel JL, Henderson ST (2008). Hypometabolism as a therapeutic target
in Alzheimer's disease. BMC Neurosci 9 Suppl 2: S16.

Etgen AM (2008). Estrogens and Alzheimer's disease: is cholesterol a link? Endocrinology
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Gasior M, Rogawski MA, Hartman AL (2006). Neuroprotective and disease-modifying effects of
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Guzman M, Blazquez C (2004). Ketone body synthesis in the brain: possible neuroprotective
effects. Prostaglandins, leukotrienes, and essential fatty acids 70(3): 287-292.

Lin MT, Beal MF (2006). Mitochondrial dysfunction and oxidative stress in neurodegenerative
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Loeb LA, Wallace DC, Martin GM (2005). The mitochondrial theory of aging and its relationship
to reactive oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci U S A
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Morris AA (2005). Cerebral ketone body metabolism. J Inherit Metab Dis 28(2): 109-121.

Numakawa Y, Matsumoto T, Yokomaku D, Taguchi T, Niki E, Hatanaka H, et al (2007). 17beta-
estradiol protects cortical neurons against oxidative stress-induced cell death through reduction in
the activity of mitogen-activated protein kinase and in the accumulation of intracellular calcium.
Endocrinology 148(2): 627-637.

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tangle formation in a triple transgenic model of Alzheimer's disease. Neurobiology of aging
24(8): 1063-1070.

Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, et al (2003b). Triple-
transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and
synaptic dysfunction. Neuron 39(3): 409-421.

Reddy PH (2006). Mitochondrial oxidative damage in aging and Alzheimer's disease:
implications for mitochondrially targeted antioxidant therapeutics. J Biomed Biotechnol 2006(3):
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selective aspects of cognition in women: A critical review. Front Neuroendocrinol.

Stafstrom CE, Roopra A, Sutula TP (2008). Seizure suppression via glycolysis inhibition with 2-
deoxy-D-glucose (2DG). Epilepsia 49 Suppl 8: 97-100.

Takuma K, Yao J, Huang J, Xu H, Chen X, Luddy J, et al (2005). ABAD enhances Abeta-
induced cell stress via mitochondrial dysfunction. Faseb J 19(6): 597-598.

Velliquette RA, O'Connor T, Vassar R (2005). Energy inhibition elevates beta-secretase levels
and activity and is potentially amyloidogenic in APP transgenic mice: possible early events in
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Wang X, Su B, Perry G, Smith MA, Zhu X (2007). Insights into amyloid-beta-induced
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153

Chapter VI Conclusions

The above chapters presented here summarize and culminates several years of my
research on estrogen regulation of mitochondrial function. The project is designed on the
basis of our existing knowledge of estrogen action in the brain. In addition to confirming
and expanding previous findings on estrogen induced neuroprotection against AD, the
current research provides insights into potential causes of AD pathogenesis. Findings
from this project also help facilitate the development of novel therapeutic strategies
targeting mitochondrial bioenergetics to prevent AD and other age-related
neurodegenerative diseases. The classic amyloid hypothesis of AD proposes a cascade of
amyloid toxicity as the initial cause, in which mitochondrial dysfunction is downstream
and secondary to amyloid toxicity. Nevertheless, findings from specific aim I suggest an
early and potential causal role for mitochondrial bioenergetic deficits in the pathogenesis
of AD. These findings are in agreement with recent clinical reports on human patients
that brain hypometabolism occurs at least a decade prior to the onset of AD (Cutler,
1986; Drzezga et al, 2003; Liang et al, 2008).
In specific aim I, we also demonstrated that reproductive senescence paralleled a
dramatic decline in mitochondrial function in both nonTg and 3xTgAD mice, indicating a
critical role of estrogen in maintaining mitochondrial function. Therefore, we continued
to investigate estrogen regulation of mitochondrial function in specific aim II. Specific
aim II expanded the discoveries from specific aim I and focused on the impact of ovarian
hormone loss, particularly loss of estrogen, on mitochondrial function. By surgical
154

removal of the ovaries, we constructed another estrogen deficiency model in addition to
age-related reproductive senescence. We successfully demonstrated that estrogen is
critical in sustaining and maintaining mitochondrial function in both wild type and AD
transgenic mice. Further, estrogen depletion has led to mitochondrial deposition of Aβ
pathology in the 3xTgAD mice, which in turn exacerbates the deficits in mitochondrial
function and initiates a vicious cycle, in which mitochondrial dysfunction and increased
AD pathology propagate each other and activate the entire downstream detrimental
cascade. Considering the fact that women, especially menopausal women, have a much
higher chance of developing AD, the findings from specific aim II not only explain the
increased AD risk in aged women, but also justify the rationale of estrogen therapy in AD
prevention.
In specific aim III, we extended our search to investigate the mechanism of
estrogen induced neuroprotection. Findings from this specific aim contribute to the
development of therapeutics targeting ER β for AD intervention. The fact that co-
administration of E2 and P4 abolished the benefits of E2 or P4 alone has important
clinical relevance in that it suggests current treatment paradigms of hormone therapy
might not be optimal.
As a lab focused on translational research, one of our continuous endeavors is to
develop novel therapeutics or therapeutic strategies to prevent AD or modify AD
progression. In specific aim IV, we conducted an initial trial in which we induced
ketogenesis to provide AD brain with alternative fuel sources. Noticeably, we chose this
155

strategy as an intervention targeting at early-to-middle stage of AD, where brain
mitochondrial bioenergetics is already compromised in the 3xTgAD mice and
intraneuronal AD pathology is observable. The treatment generated moderate but
significant benefits and provided the scientific basis for future development of strategies
to delay AD progression.
Taken together, from a therapeutic perspective, targeting mitochondrial and brain
bioenergetics provides a novel way to prevent AD or delay AD progression. First of all,
therapeutics, such as estrogen or molecules with estrogenic effects, will help sustain and
enhance mitochondrial function and therefore prevent the initiation of a bioenergetic
crisis which subsequently leads to AD. Later on, supplying alternative substrates to brain,
such as ketone bodies, provides additional disease modifying strategy to compensate for
the energy loss and brain hypometabolism and hence delay the progression of AD.
Different from the AD medicines available on the market, which only offers moderate
symptom relief, these strategies either aim at prevention or modify disease progression
and therefore shall offer better efficacy.
156

Chapter VI References
Cutler NR (1986). Cerebral metabolism as measured with positron emission tomography (PET) and [18F]
2-deoxy-D-glucose: healthy aging, Alzheimer's disease and Down syndrome. Prog Neuropsychopharmacol
Biol Psychiatry 10(3-5): 309-321.

Drzezga A, Lautenschlager N, Siebner H, Riemenschneider M, Willoch F, Minoshima S, et al (2003).
Cerebral metabolic changes accompanying conversion of mild cognitive impairment into Alzheimer's
disease: a PET follow-up study. Eur J Nucl Med Mol Imaging 30(8): 1104-1113.

Liang WS, Reiman EM, Valla J, Dunckley T, Beach TG, Grover A, et al (2008). Alzheimer's disease is
associated with reduced expression of energy metabolism genes in posterior cingulate neurons. Proc Natl
Acad Sci U S A 105(11): 4441-4446.

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Abstract (if available)

Abstract Presented here are a collection of studies that build on our existing knowledge of estrogen actions on brain mitochondria. Previous studies from our lab as well as other colleagues have demonstrated that estrogen (E2, 17β-estradiol) is neuroprotective against neurotoxic insults including exposure to glutamate and amyloid beta (Aβ) (Nilsen et al, 2006

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Asset Metadata

Creator Yao, Jia (author)

Core Title Estrogen regulation of bioenergetics and mitochondrial function: implications for Alzheimer's disease risk and therapeutics

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 05/04/2010

Defense Date 03/24/2010

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

Tag Alzheimer's disease,bioenergetics,Estrogen,mitochondria,OAI-PMH Harvest,therapeutics

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Brinton, Roberta Diaz (committee chair), Alkana, Ronald (committee member), Baudry, Michel (committee member), Cadenas, Enrique (committee member)

Creator Email jiayao@usc.edu,uscjiayao@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2999

Unique identifier UC1443595

Identifier etd-Yao-3656 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-317029 (legacy record id),usctheses-m2999 (legacy record id)

Legacy Identifier etd-Yao-3656.pdf

Dmrecord 317029

Document Type Dissertation

Rights Yao, Jia

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