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

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

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ROLE OF OXIDATIVE STRESS IN AGE-ASSOCIATED MILD
COGNITIVE IMPAIRMENT AND ALZHEIMER’S DISEASE

by

Aaron Clausen

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
(NEUROSCIENCE)

May 2011

Copyright 2011 Aaron Clausen
ii
Acknowledgements

During the pursuit of my PhD I received invaluable support, guidance, and
instruction from a number of different USC faculty, fellow graduate students, USC staff,
family members, and senior researchers. The road leading to the completion of this
dissertation was often challenging and required a lot of hard work at times and I was
lucky enough to be surrounded by a good group of people that were willing to help me
along the way when I needed it. My time at USC for the most part was a pleasurable
experience and I will never forget the people I have met and the knowledge that I have
gained while pursuing my PhD.
I first would like to thank my thesis advisor, Dr. Michel Baudry. Dr. Baudry was
gracious enough to allow me to pursue my PhD in his lab after I decided that the first lab
I joined was not a fit for me. Dr. Baudry made the environment in lab one in which I
could grow as an independent scientist. He allowed me to overcome problems and
develop ideas and experiments on my own, but he was always readily available if I
needed his assistance. Dr. Baudry was always eager to go over my work with me and
always made time to help me with whatever I needed. Dr. Baudry is also a nice person
with a good sense of humor who understands that life is not just centered on research.
This made the working environment a pleasurable one, in which get-togethers outside of
lab were regular, and the word vacation was not blasphemy. Dr. Baudry was also very
patient with regards to getting techniques and experiments to work and obtaining results.
I would also like to thank Dr. Baudry for his generosity in providing me with financial
support for the last four years. This definitely made it a lot easier to focus on research and
iii
pursue my PhD. I feel that Dr. Baudry’s guidance has given me the tools that are
necessary for being a successful scientist.
Secondly, I would like to thank the members of my thesis and qualifying exam
committees, Dr. Richard F. Thompson, Dr. John P. Walsh, Dr. Roger F. Duncan, and Dr.
Enrique Cadenas. I really appreciated the time they spent preparing and grading my
qualifying exam, evaluating and guiding me with my thesis proposal, and reviewing my
dissertation. In addition, I would like to thank the Programs in Biomedical and Biological
Sciences Graduate program at the USC health science campus for giving me an
opportunity to pursue my PhD at USC and generously providing me with funding for the
first year while I selected the lab I was going to do my thesis in. They also did a great job
exposing me to all the research opportunities available at USC. I would also like to
especially thank the National Institute on Aging and the Alzheimer's Association because
they provided the funding for the research that is presented in my thesis.
Neuroscience research requires a lot of techniques, equipment, and knowledge
from a number of different scientific disciplines, thus a lot of my research required
materials, reagents, and expertise that my laboratory did not possess, but luckily I was
surrounded by labs and people that were willing to lend me a helping hand. I would like
to thank Dr. Peter Z. Qin’s lab for letting me use their fluorimeter and Dr. Alan G. Watt’s
lab for allowing me to use their well maintained microtome and microscope and also
being generous with their reagents and supplies. I would also like to thank the lab of Dr.
Richard F. Thompson for allowing me to perform my fear conditioning experiments in
their lab and for lending me any anything I needed for my research. I especially would
like to thank Dr. Arshad M. Khan at USC for helping me set up my
iv
immunohistochemistry protocols, teaching me how to use the microscope and microtome,
and always being available to answer any technical questions I might have. Xiaoning
Bi’s lab at the Western University of Health Sciences also generously provided me with
antibodies and assistance with immunohistochemistry and western blot techniques.
Several people were also very instrumental in helping me complete my
dissertation projects. I would like to thank our research specialist at the time, Anna
Knize, for helping out with the survival surgeries that were performed in Chapter 2 of my
thesis and Dr. Xiaoning Bi from the Western University of Health Sciences for carrying
out the immunohistochemistry that was performed in Chapter 3. I want to also thank
Xiaobo Xu, a fellow graduate student in our lab, for helping out with the brain sectioning
and immunostaining carried out in Chapter 4. In addition, I would like to thank Dr. Susan
Doctrow from Proteome Systems Incorporated for assisting with the development of the
experimental design used in Chapter 2.
I would also like to say thank you to my fellow lab members who created a great
working environment and were eager to help me when I needed it. Dr. Karoline
Rostamiani and Dr. Miou Zhou always helped me with technical issues and were an
invaluable resource when it came to performing western blots. Dr. Hussam Jourdi has a
great deal of expertise in the biological sciences and therefore he always provided a lot of
helpful suggestions when I discussed experiments and results with him. Dr. Michael
Grammer taught me how to perform fear conditioning and Anna Knize showed me how
to run western blots. Alexis Seegan, Eric Hu, and Genoveva Aguilar played a large part
in maintaining the lab, ordering reagents and supplies, and making buffers and other
stock solutions that I routinely used. Yu-tien Hsu and Maggie Chou were great to work
v
with in the lab and were always willing to listen to me and provide good suggestions.
Xiaobo Xu, Dr. Ludwig Hamo, Dr. Reymundo Dominguez, Dr. Claudia Aguirre, Dr.
Tetsushi Oka, and Dr. Wei Xu also played a vital role in maintaining the lab and made
the lab a nice place to be. I had a great time in Dr. Baudry’s lab and I leave the lab with
many new friends and good memories.
Lastly, I would like to thank my family. My family has changed quite a bit since
I started graduate school. My grandma and grandpa, who I hold very dear to my heart,
have both past away. I got married to the woman of my dreams and am now also a father.
I would like to thank my Mom and Dad for providing me with everything I needed to
reach this goal, always allowing me to follow my dreams, and supporting me in whatever
decisions I make. I would especially like to thank my wife for bringing so much joy and
happiness to my life, standing by me and supporting me while I pursued my PhD, and
tolerating those late nights when I was away in lab. I also want to thank my son Dylan,
who always lifts my spirits and brings so much excitement to my life.

vi
Table of Contents

Acknowledgements………………………………………………………………………..ii
List of Tables……………………………………………………………………………..ix
List of Figures…………………………………………………………………………......x
Abstract…………………………………………………………………………………..xii
Chapter 1. Introduction………………………………………………………………........1
1.1. Oxidative stress……………………………………………………………………...2
1.2. Age-associated increase in oxidative stress………………………………………....4
1.3. Age-related cognitive impairment is associated with oxidative stress……………...7
1.4. Anti-oxidant compounds as potential therapeutic agents for age associated
cognitive decline…………………………………………………………………….8
1.5. SOD/catalase mimetics……………………………………………………………...9
1.6. Cognitive targets of oxidative stress……………………………………………….13
1.7. Free radicals and cell death………………………………………………………...14
1.8 Age-associated oxidative stress might affect cellular mechanisms responsible for
maintaining neuron homeostasis……………………………………………………15
1.9. Alzheimer’s disease………………………………………………………………..16
1.10. Oxidative stress and Alzheimer’s disease………………………………………...17
1.11 Dissertation outline………………………………………………………………..19

Chapter 2. Prevention of cognitive deficits and brain oxidative stress with superoxide
dismutase/catalase mimetics in aged mice……………………………………………….21
2.1. Introduction………………………………………………………………………...21
2.2. Materials and methods……………………………………………………………..23
2.2.1. Materials………………………………………………………………………..23
2.2.2. Mice and treatments………………………………………………………….....23
2.2.3. Behavioral analysis……………………………………………………………..25
2.2.3.1. Fear conditioning……………………………………………………………25
2.2.3.2. Behavioral analysis………………………………………………………….26
2.2.4. Assays for lipid peroxidation, nucleic acid oxidation, and ROS content………27
2.2.5. Statistical analysis……………………………………………………………....30
2.3. Results……………………………………………………………………………...30
2.3.1. Effects of SOD/catalse mimetics on fear conditioning learning………………..30
2.3.2. Effects of SOD/catalse mimetics on brain oxidative stress…………………….33
2.3.3. Correlation between cognitive function and brain levels of markers for
oxidative stress………………………………………………………………….36
2.4. Discussion………………………………………………………………………….39

vii
Chapter 3. Age-related changes in autophagy-lysosome system and microglial
function are reversed by chronic treatment with SOD/catalase mimetics……………….44
3.1. Introduction………………………………………………………………………...44
3.2. Materials and methods……………………………………………………………..49
3.2.1. Materials………………………………………………………………………..49
3.2.2. Mice and treatments…………………………………………………………….49
3.2.3. Analysis of autophagy-lysosome system and microglia function by Western
blot……………………………………………………………………………...51
3.2.4. Analysis of autophagy-lysosome system and microglia function by
immunohistochemistry………………………………………………………….52
3.2.5. Statistical analysis………………………………………………………………53
3.3. Results……………………………………………………………………………...54
3.3.1. Effects of SOD/catalase mimetics on brain autophagy…………………………54
3.3.2. Effects of SOD/catalase mimetics on brain lysosome function………………...59
3.3.3. Effects of SOD/catalase mimetics on microglia status…………………………64
3.4. Discussion………………………………………………………………………….67

Chapter 4. The superoxide dismutase/catalase mimetic EUK-207 protects against
beta-amyloid and tau pathology and cognitive decline in a mouse model of
Alzheimer’s disease………………………………………………………………….......75
4.1. Introduction………………………………………………………………………...75
4.2. Materials and methods……………………………………………………………..80
4.2.1. Materials………………………………………………………………………..80
4.2.2. Mice and treatments…………………………………………………………….80
4.2.3. Behavioral analysis……………………………………………………………..82
4.2.3.1. Fear conditioning……………………………………………………………82
4.2.3.2. Behavioral analysis………………………………………………………….83
4.2.4. Analysis of Alzheimer’s disease pathology and oxidative stress………………84
4.2.4.1. A β
1-42
ELISA………………………………………………………………..84
4.2.4.2 Lipid peroxidation assay……………………………………………………..86
4.2.4.3. Immunohistochemistry……………………………………………………...86
4.2.5. Statistical Analysis……………………………………………………………...88
4.3. Results……………………………………………………………………………...88
4.3.1. Effects of SOD/catalase mimetic EUK-207 on fear-conditioning
learning in 3xTg-AD mice……………………………………………………...88
4.3.2. Effects of SOD/catalase mimetic EUK-207 on brain oxidative stress in
3xTg-AD mice………………………………………………………………….94
4.3.3. Effects of SOD/catalase mimetic EUK-207 on beta-amyloid pathology in
3xTg-AD mice………………………………………………………………...101
4.3.4. Effects of SOD/catalase mimetic EUK-207 on tau pathology in 3xTg-AD
mice……………………………………………………………………………109
4.4. Discussion………………………………………………………………………...118

viii
Chapter 5. Conclusion…………………………………………………………………..126
5.1. Brain oxidative stress plays a significant role in age-associated mild cognitive
impairment………………………………………………………………………..127
5.2. Age-associated changes in the autophagy-lysosome pathway and microglia
function are dependent upon brain oxidative stress………………………………128
5.3. Brain oxidative stress is critically involved in the pathogenesis of Alzheimer’s
disease…………………………………………………………………………….132
5.4. Superoxide dismutase/catalase mimetics can potentially be used to protect
individuals against age-associated mild cognitive imapairment and
Alzheimer’s disease………………………………………………………………134
5.5. Age-associated mild cognitive impairment and Alzheimer’s disease might
result from age-dependent brain oxidative stress disrupting the autophagy-
lysosome system and microglia function…………………………………………136
5.6. Closing remarks…..……………………………………………………………....140

References………………………………………………………………………………141

ix
List of Tables
Table 1. Summary of studies assessing the effects of exogenous antioxidants on age-
associated cognitive decline and oxidative stress in rodents…………………………….11

Table 2. Body weight, nociception, and vision test results for SOD-catalase treated
mice……………………………………………………………………………………....33

Table 3. Body weight, nociception, and vision test results for EUK-207 treated mice….94

x
List of Figures
Figure 1. Effects of 3-month chronic treatment with EUK-189 or EUK-207 on context
fear conditioning........................…....................…............................................................32

Figure 2. Effects of 6-month chronic treatment with EUK-189 or EUK-207 on context
fear conditioning...........................……….........................................................................32

Figure 3. Effects of chronic treatment with EUK-189 or EUK-207 on lipid
peroxidation in brain homogenates..........………………………………………………..34

Figure 4. Effects of chronic treatment with EUK-189 or EUK-207 on oxidized nucleic
acids in brain homogenates……………………........................……................................35

Figure 5. Effects of chronic treatment with EUK-189 or EUK-207 on reactive oxygen
species (ROS) levels in brain homogenates.......................…….....…...............................36

Figure 6. Correlation between performance in the contextual fear conditioning task
and brain levels of free radicals and lipid peroxidation………………………….............38

Figure 7. Effects of chronic treatment with EUK-189 or EUK-207 on ATG7 levels in
brain homogenates and Beclin-1 expression in field CA1 of Hippocampus…………….56

Figure 8. Effects of chronic treatment with EUK-189 or EUK-207 on LC3 – II to
LC3 – I ratio in brain homogenates and total LC3 expression in CA1 of the
hippocampus………………………………………………………………………..........58

Figure 9. Effects of chronic treatment with EUK-189 or EUK-207 on LAMP1
expression in brain homogenates and in CA1 of the hippocampus.…..........……………61

Figure 10. Effects of chronic treatment with EUK-189 or EUK-207 on LAMP2
expression in brain homogenates…..........…………….....................................................63

Figure 11. Effects of chronic treatment with EUK-189 or EUK-207 on CD11b
expression in brain homogenates and in CA1 of the Hippocampus……………..............65

Figure 12. Effects of chronic treatment with EUK-189 or EUK-207 on CD11b and
Cathepsin D immunoreactivity in various brain regions……….………………..............66

Figure 13. Absence of deficit in contextual or cued fear conditioning in 3xTg-AD
mice at 7 months of age...............…………......................................................................90

Figure 14. Chronic treatment with EUK-207 starting at 4 months of age protects
against deficits in contextual and cued fear conditioning in 9 month old 3xTg-AD
mice…………........................………………………..………………..............................93
xi

Figure 15. Chronic treatment with EUK-207 starting at 4 months of age significantly
reduces lipid peroxidation in brain homogenates from 9 month-old 3xTg-AD mice……95

Figure 16. Chronic treatment with EUK-207 starting at 4 months of age reduces
oxidized guanine levels in hippocampus of 9 month-old 3xTg-AD mice……………….98

Figure 17. Chronic treatment with EUK-207 starting at 4 months of age reduces
oxidized guanine levels in amygdala of 9 month-old 3xTg-AD mice………………….100

Figure 18. Chronic treatment with EUK-207 starting at 4 months of age reduces
6E10 staining in hippocampus of 9 month-old 3xTg-AD mice. ……………………….104

Figure 19. Chronic treatment with EUK-207 starting at 4 months of age reduces
6E10 staining in amygdala of 9 month-old 3xTg-AD mice……………………………106

Figure 20. Chronic treatment with EUK-207 starting at 4 months of age significantly
reduces detergent soluble A β
1-42
in brain homogenates from 9 month-old 3xTg-AD
mice……………………..…………………………..…………………………..............108

Figure 21. Chronic treatment with EUK-207 starting at 4 months of age reduces tau
accumulation in hippocampus of 9 month-old 3xTg-AD mice………………………...111

Figure 22. Chronic treatment with EUK-207 starting at 4 months of age reduces tau
accumulation in amygdala of 9 month-old 3xTg-AD mice……………………….........113

Figure 23. Chronic treatment with EUK-207 starting at 4 months of age reduces
hyperphosphorylated tau in ventral CA1 pyramidal cells of 9 month-old 3xTg-AD
mice…………..................................................................................................................115

Figure 24. Chronic treatment with EUK-207 starting at 4 months of age reduces
hyperphosphorylated tau in amygdala of 9 month-old 3xTg-AD mice………………...117

Figure 25. Schematic diagram illustrating the potential relationship between the
disruption of the autophagy-lysosome system and microglia function by age-
associated brain oxidative stress and the pathogenesis of age-associated mild
cognitive impairment and Alzheimer’s disease...............................................................139

xii
Abstract

Continuous decline in cognitive performance accompanies the natural aging
process in humans, and multiple studies in both humans and animal models have
indicated that this decrease in cognitive function is associated with an age-related
increase in oxidative stress. Treating aging mammals with exogenous free radical
scavengers has generally been shown to attenuate age-related cognitive decline and
oxidative stress. I assessed the effectiveness of the superoxide dismutase/catalase
mimetics EUK-189 and EUK-207 on age-related decline in cognitive function and
increase in oxidative stress. C57/BL6 mice received continuous treatment via osmotic
minipumps with either EUK-189 or EUK-207 for 6 months starting at 17 months of age.
At the end of treatment, markers for oxidative stress were evaluated by analyzing levels
of free radicals, lipid peroxidation and oxidized nucleic acids in brain tissue. In addition,
cognitive performance was assessed after 3 and 6 months of treatment with fear
conditioning. Both EUK-189 and EUK-207 treatments resulted in significantly decreased
lipid peroxidation, nucleic acid oxidation, and reactive oxygen species (ROS) levels. In
addition, the treatments also significantly improved age-related decline in performance in
the fear-conditioning task. My results thus confirm a critical role for oxidative stress in
age-related decline in learning and memory and strongly suggest a potential usefulness
for salen–manganese complexes in reversing age-related declines in cognitive function
and oxidative load.
A large body of evidence indicates that oxidative stress plays a critical role in
normal aging, and in particular in age-related impairment of brain function.
Accumulation of oxidatively damaged organelles, proteins, and other macromolecules
xiii
within various cell types are likely to be involved, as treatment with different types of
antioxidants has been shown to reverse some of these effects. Whether cause or
consequence, aging is also associated with impairment of the cellular mechanisms
involved in removing damaged cellular components, such as the ubiquitin-proteosome
and autophagy-lysosome pathways. Furthermore, age-related changes in astrocytes and
microglia have also been repeatedly reported. The causal relationships between these
multiple age-related changes are far from being clear. I therefore carried out a study
directed at testing the role of oxidative stress in age-related changes in autophagy-
lysosome function and microglia status. Starting at 17 months of age, C57/BL6 mice
received continuous treatment via subcutaneous osmotic pumps with either EUK-189 or
EUK-207, two superoxide-dismutase/catalse mimetics that I have previously found to
significantly reduce age-dependent oxidative stress and cognitive decline. After 6
months of treatment, markers for autophagy-lysosomal function, and microglia were
determined in forebrain. The ratio of LC3-II to LC3-I, which is widely used as a marker
of autophagy, was markedly enhanced in brains of 23 month-old mice as compared to
that in 16 month-old mice. Expression of the autophagy chaperone protein APG7
decreased with age and lysosomal proteins, such as LAMP-1 and LAMP-2, were
significantly reduced in aged brains. These age-related changes in autophagy-lysosme
system were reversed by treatment with EUK-189 and EUK-207. In the CA1 region of
the hippocampus, EUK-189 and EUK-207 treatment increased expression of the
lysosomal protease cathepsin D, but decreased expression of the autophagy mediator
Beclin-1 and lysosomal associated membrane protein LAMP-1. Brain levels of CD11b
and numbers of CD11b-imunopositive microglia were decreased in aged brains,
xiv
suggesting a decline in microglia function. This effect was also reversed by treatment
with the SOD/catalase mimetics. Both EUK-189 and EUK-207 have been previously
shown to reverse age-related increase in brain oxidative stress, thus these results
demonstrate that oxidative stress is causally related to age-related alterations in
autophagy-lysosome function and microglia function.
Alzheimer’s disease is an age-associated neurodegenerative disorder that
currently affects the lives of about 5.3 million Americans. Alzheimer’s disease (AD) is
characterized by progressive memory loss and cognitive deficits that appear to be
associated with the accumulation of beta-amyloid plaques and intracellular
neurofibrillary tangles within the brain, and neuronal death. A small percentage of AD
cases are hereditary and result from mutations in the genes that encode for beta-amyloid
precursor protein or proteins responsible for beta-amyloid processing. However, the vast
majority of AD cases are sporadic and the underlying molecular mechanisms of AD
pathogenesis in these non-hereditary forms remain unclear. A number of studies have
shown that, in addition to beta-amyloid and tau pathology, mitochondrial dysfunction and
free radical damage are also hallmarks of AD brain, suggesting that oxidative stress
might be important in AD pathology. I set out to define the role oxidative stress plays in
AD pathogenesis by chronically treating mice that model human AD with the superoxide
dismutase (SOD)/catalase mimetic, EUK-207, which I have previously shown to protect
against age-associated cognitive impairment and oxidative stress in mice. 3xTg-AD
mice, which exhibit cognitive decline as well as A β and tau pathology in an age-
dependent manner, and wild-type mice were chronically treated with EUK-207 via
subcutaneous micro-osmotic pumps for 5 months. Treatment began at 4 months of age
xv
because AD pathology and cognitive deficits are still negligible in 3xTg-AD mice at this
time point and continued until 9 months of age, when the AD phenotype has been shown
to be significant. After 5 months of treatment, cognitive performance was assessed using
a fear conditioning paradigm that tests both contextual and cued fear memories. In
addition, brain beta-amyloid and tau pathology as well as oxidative stress were analyzed.
At 9 months of age, 3xTg-AD mice exhibited a sharp decline in performance in both
contextual and cued fear memory tasks as compared to wild-type mice; however, 3xTg-
AD mice administered EUK-207 did not display deficits in fear conditioning
performance. Furthermore, chronic treatment with EUK-207 reduced the expression of
beta-amyloid, tau and hyperphosphorylated tau accumulation in amygdala and
hippocampus of 3xTg-AD mice, and protected against increased levels of A β42, oxidized
nucleic acids and lipid peroxidation in brain. My results thus confirm a critical role for
oxidative stress in AD pathogenesis and strongly suggest a potential usefulness for salen-
manganese complexes in protecting against AD development.

1
Chapter 1
Introduction
The average life span of humans has been steadily on the rise due to advances in
modern medicine and to the standard of living industrial societies provide. With
increased life span comes a variety of pathological conditions associated with aging that
reduce the quality of life in the elderly and place a heavy burden to the health care system
of the society; it is therefore important to understand the biological processes that
underline age-related pathologies in order to some day minimize the effects aging has on
the body and enhance the quality of life in the aging population. Among the age-
associated problems that we are facing today, decline in cognitive function and increased
prevalence of Alzheimer’s disease (AD) represent major scientific and financial
problems.
A decline in cognitive function associated with aging has been well documented
in a variety of different mammals. Administering a verbal word recall test to humans
from different age groups revealed an inverse and linear relationship between age and
memory performance (Davis et al., 2003). Aged mice and rats also perform poorly
compared to young mice in a variety of behavioral tests that assess locomotion, motor
coordination, and learning and memory (Barnes et al., 1990, Forster et al., 1996). Age-
associated decline in learning and memory is often referred to as mild cognitive
impairment (MCI). Although MCI is not life threatening it becomes much more dramatic
in the pathological form exhibited by patients with Alzheimer's disease. In addition,
humans that exhibit MCI have a greater chance of developing Alzheimer’s disease than
2
the general population (Petersen et al., 2001b). The biological mechanisms that underlie
MCI and Alzheimer’s disease pathogenesis are still not clearly understood, but a great
deal of evidence points towards age-associated oxidative stress.
1.1. Oxidative stress
Reactive oxygen species (ROS) include: superoxide, hydrogen peroxide, and
hydroxyl radicals and are mostly generated during oxidative phosphorylation. Oxidative
phosphorylation is the last step of cellular metabolism that is responsible for producing
the majority of cellular ATP under aerobic conditions. Oxidative phosphorylation
requires the generation of an electrochemical proton gradient across the mitochondrial
inner membrane, in order to drive ATP synthase. During oxidative phosphorylation,
electrons that were produced by glycolysis, the Krebs cycle, and fatty acid and amino
acid metabolism are transferred to the electron transport chain, which consists in several
enzyme complexes embedded in the inner mitochondrial membrane. These complexes
are made up of a number of different proteins, including electron carriers and proton
pumps. NADH transfers electrons to Complex I, which then passes them along to
coenzyme Q and the electrons ultimately get transferred down the electron transport
chain. This transfer of electrons through the electron transport chain is what drives the
transport of protons into the mitochondrial inner membrane space, resulting into the
generation of the electrochemical gradient that drives ATP synthase (Alberts, 2002).
Ultimately, the electrons combined with protons and oxygen to produce water in
Complex IV of the electron transport chain (Adam-Vizi and Chinopoulos, 2006).
However, a significant fraction of electrons continuously escapes from the complexes
that reside in the electron transport chain before they are combined into water (Turrens,
3
2003). These electrons that leaked out of the electron transport chain can then directly
react with oxygen to produce the superoxide ion (Turrens, 2003). In vitro studies have
shown that superoxide can be generated by Complex I (Turrens and Boveris, 1980),
Complex II (Zhang et al., 1998), and Complex III (Cadenas et al., 1977) of the electron
transport chain under the right conditions. However, since these experiments were done
in mitochondria removed from the cell it is hard to know which one of these complexes
contributes to ROS production in vivo. In addition to the enzyme complexes that
constitute the electron transport chain, alpha-ketoglutarate dehydrogenase, which is part
of the KREBS cycle, has also been shown to produce both superoxide and hydrogen
peroxide, when the levels of the electron carrier NAD+ are low (Starkov et al., 2004).
The formation of free radicals during normal cellular respiration usually results in
a low steady state level of ROS (Boveris and Chance, 1973, Finkel and Holbrook, 2000),
which can be handled by the various antioxidant defense mechanisms present within cells
and mitochondria. The cell contains a number of mechanisms to neutralize free radicals.
These include enzymatic scavengers, such as superoxide dismutase (SOD), catalase, and
glutathione peroxidase (Halliwell, 1991, Brigelius-Flohe, 1999). Three different forms of
SOD are found in mammals; SOD1 is located in the cytoplasm, SOD2 is specific to the
mitochondria, and SOD3 is extracellular. SOD converts superoxide and hydrogen to
hydrogen peroxide, which can then be transformed into water by either catalase or
glutathione peroxidase. In addition, a number of non-enzymatic free radical scavengers,
such as ascorbate (vitamin C), urate, glutathione, tocopherol (vitamin E), carotenoids, and
flavonoids, interact stoichiometrically with ROS to eliminate them.
4
During oxidative stress the levels of ROS production increase and overcome the
capacity of endogenous free radical scavengers, such as superoxide dismutase and
catalase, and several macromolecules, including lipids, proteins, and nucleic acids
become vulnerable to oxidation. The brain is especially sensitive to oxidative stress
because it utilizes high levels of oxygen, contains large amounts of lipids that free
radicals can readily react with, and exhibits lower levels of antioxidants as compared to
other tissues (Halliwell, 1992). Severe oxidative stress in neurons can be brought on by
an excitotoxic event such as stroke. During excitotoxicity, large amounts of glutamate
are released from presynaptic cells, which bind and activate postsynaptic glutamate
receptors (Choi, 1987). Rapid activation of glutamate receptors results in a large calcium
influx in postsynaptic cells (Sattler and Tymianski, 2001), which ultimately gets absorbed
by mitochondria (Tymianski et al., 1993). This large influx of calcium into the
mitochondria finally can result in mitochondria dysfunction (Nicholls et al., 2003), which
leads to enhanced ROS production (Luetjens et al., 2000) due to impairment of the
electron transport chain. In addition, significant oxidative stress in the brain is also
associated with the natural aging process.
1.2. Age-associated increase in oxidative stress

A number of studies have demonstrated that aging is accompanied by an increase
in oxidative stress in mammalian brain. Levels of oxidized proteins are much higher in
the frontal and occipital poles of aged human brains as compared to young individuals
(Smith et al., 1991). Age-related increases in protein oxidation have also been reported
in the brain of rats (Cini and Moretti, 1995) and the hippocampus of mice (Sohal et al.,
1994). Lipid peroxidation is also significantly higher in hippocampus and cortical tissue
5
of aged rats (O'Donnell and Lynch, 1998, Rodrigues Siqueira et al., 2005). In addition,
aged rats also exhibit increased levels of lipid peroxidation in cerebellum, striatum, and
substantia nigra (Calabrese et al., 2004), and nuclear DNA from the brains of rats and
mice also shows signs of increased oxidation with age (Hamilton et al., 2001).
The reason for age-related increases in oxidative stress in the brain of aged
mammals is still unclear, but it might be due to an age-associated increase in steady-state
levels of ROS in the brain. In comparison to young rodents, aged rodents exhibit
considerably higher levels of free radicals in brain. Thus, 11 month-old rats have
significantly greater levels of superoxide in hippocampus, cortex, and striatum as
compared to 3 month-old rats (Antier et al., 2004). Furthermore, old- and middle-aged
gerbils also exhibit a significant age-dependent increase in hydroxyl radicals in
hippocampus, cortex, and striatum (Zhang et al., 1993), and 24 month-old rats also
exhibit an age-dependent increase in reactive oxygen species in hippocampus (Rodrigues
Siqueira et al., 2005).
The underlying cause for this significant rise in steady-state ROS levels in brain
of aged rodents has not been fully elucidated, but it could be due to either an increase in
free radical production, or a decrease in free radical defense mechanisms, or a
combination of both processes. Hippocampal slices from aged rats generate substantially
higher levels of hydrogen peroxide than slices from young rats (Auerbach and Segal,
1997), and basal levels of ROS production are significantly greater in frontal cortex,
striatum, hippocampus, and cerebellum of 24 month-old rats compared to 3 to 6 month
old rats (Driver et al., 2000). This age-dependent increase in free radical production is
most likely due to an increase in mitochondrial ROS production because mitochondria
6
preparations from brain of aged rats exhibit a significant age-dependent increase in
superoxide and hydrogen peroxide production (Sawada and Carlson, 1987, Sohal et al.,
1994). Age-associated mitochondrial dysfunction resulting from genetic mutations or
damage is probably the underlying cause of accelerated ROS production observed in
mitochondria from aged rodent brain (Sastre et al., 2003).
Several studies have also reported an age-dependent decrease in activity and
expression of certain antioxidant molecules. Superoxide dismutase and catalase activity
both decreased with age in rat brain, as well as the mRNA levels for these two enzymes
(Rao et al., 1990). O’Donnel et al. also found a significant age-dependent decrease in
catalase activity and in levels of the free radical scavenger glutathione in hippocampus
(O'Donnell et al., 2000).
Age-associated oxidative stress in brain also appears to be linked to cellular
metabolism, as age-dependent brain oxidative stress is significantly reduced by caloric
restriction. Rats fed a caloric restricted diet starting at 6 months of age did not exhibit
age-related increase in brain protein oxidation and lipid peroxidation at 18 or 24 months
of age (Hyun et al., 2006). Furthermore, 18 and 24 month-old caloric restricted rats have
significantly lower levels of brain lipid peroxidation and protein oxidation as compared to
age-matched controls (Hyun et al., 2006). Similar findings were obtained in studies with
mice, as mice fed a caloric restricted diet starting at 4 months of age exhibited
significantly reduced protein carbonyl levels in cortex, striatum, midbrain, cerebellum,
and hindbrain at 15 months of age (Dubey et al., 1996).
7
1.3. Age-related cognitive impairment is associated with oxidative
stress
In addition to the strong correlation between aging and oxidative stress, a number
of studies have demonstrated a link between age-related oxidative stress and cognitive
impairment. The decline in learning and memory exhibited by aged mice tested in the
spatial swim maze has been correlated to increased protein carbonyl content in cerebral
cortex, and the poor motor coordination these aged mice also displayed was correlated to
increased protein oxidation in cerebellum (Forster et al., 1996). In humans, lipid
peroxidation is also significantly higher in hippocampus and inferior parietal lobule of
elderly individuals who exhibit mild cognitive impairment (Butterfield et al., 2006).
Furthermore, assessing the learning ability of young rats compared to old rats using the
Morris water maze revealed an age-related decline in cognitive performance, which was
accompanied by increased lipid peroxidation levels in synaptic membranes from
hippocampus (Fukui et al., 2001). Impaired spatial learning in old rats is also
accompanied by a significant increase in levels of oxidized nucleic acids and oxidized
proteins in hippocampus (Nicolle et al., 2001). In addition, aged rats that perform just as
well as young rats in spatial learning tasks do not show the increased levels of nucleic
acid and protein oxidation in hippocampus that aged cognitive impaired rats do (Nicolle
et al., 2001).

8
1.4. Anti-oxidant compounds as potential therapeutic agents for
age-associated cognitive decline
The direct correlation between brain oxidative stress and age-associated cognitive
impairment has stimulated a number of studies evaluating the effects of exogenous anti-
oxidant compounds on age-associated cognitive decline and oxidative stress in rodents
(Table 1). A six month long chronic treatment in mice beginning at 14 months of age
with a green tea catechin mixture rich in the antioxidant (–)-epigallo-catechin-3-gallate
appreciably reduced both age-associated lipid peroxidation and protein oxidation in the
hippocampus (Qiong et al., 2010). Treatments for shorter periods of time with DL- α-
lipoic acid and L-carnitine produced similar effects on age-dependent brain oxidative
stress in aged rats (Muthuswamy et al., 2006.) Administration of the free radical spin
trapping compound N-tert-butyl-alpha-phenylnitrone to aged gerbils for 14 days not only
significantly reduced the age-related increase in brain protein carbonyl levels, but also
drastically decreased the age-related impairment in temporal and spatial memory (Carney
et al., 1991). Treating 24 month-old rats with the antioxidant deprenyl for 21 days or
grape seed extract, which contains high levels of poly-phenol antioxidants, for 30 days
also improved learning and memory and reduced brain oxidative stress (Kiray et al.,
2006, Balu et al., 2005). Melantonin has also been shown to improve the performance of
middle-aged mice in a passive-avoidance task and the elevated plus maze, but its ability
to reduce brain oxidative stress were not extensively evaluated (Raghavendra and
Kulkarni, 2001). Supplementing the diets of rats with Vitamin E for 8 months beginning
at 6 months of age significantly reduced an age-dependent increase in brain free radical
9
levels and improved spatial memory (Joseph et al., 1998), however, chronic long-term
treatment with vitamin E beginning at 20 months of age in mice had no effect on age-
associated brain oxidative stress or age-dependent performance in the Morris water maze
(Sumien et al., 2004). Moreover, several human population studies have demonstrated a
significant association between the dietary and plasma levels of the antioxidant vitamins
C, E, A, and D, and cognitive performance in the elderly (Berr, 2000).
1.5. SOD/catalase mimetics

The antioxidant studies mentioned above demonstrate a strong link between age-
associated cognitive decline and oxidative stress, and indicate that antioxidants could be
potential therapeutic agents in combating age-dependent cognitive decline. However, the
effective doses of most of these compounds appear to be very high (Table 1) and to apply
such treatments to humans would require enormous amounts of free radical scavengers.
This is most likely due to the fact that these types of antioxidants react on a one-to-one
basis with free radical molecules. Thus, the search for small synthetic molecules that can
function like the enzymes superoxide dismutase and catalase and prevent the
accumulation of oxidative damage over prolonged periods of time has been pursued
intensely by several laboratories. A family of molecules developed by a small biotech
company called Eukarion has shown promising results in this quest. These compounds
belong to a class of molecules know as salen-Mn complexes and exhibit superoxide
dismutase activity (Baudry et al., 1993), catalase activity (Doctrow et al., 2002), and
reactive nitrogen species scavenging activities (Doctrow et al., 2002, Sharpe et al., 2002).
Thus, they not only protect against damage caused by superoxide, but also hydrogen
peroxide and reactive nitrogen species. In addition, these compounds appear to penetrate
10
the blood brain barrier and the mitochondria, as evidenced by the fact that severe
neurodegeneration in mice lacking the mitochondrial form of superoxide dismutase
(SOD2) was significantly reduced by chronic i.p. injection with these molecules (Melov
et al., 2001, Hinerfeld et al., 2004). These characteristics make this group of compounds
an excellent tool to use for elucidating the relationship between age-dependent brain
oxidative stress and neurological diseases associated with aging. Our laboratory has
already shown that 2 members of this family showed great promise in treating
pathologies associated with age-related oxidative stress. Chronically treating mice with
either EUK-189 or EUK-207 starting at 8 months of age for 3 months reversed the
cognitive impairment observed in 11 month-old mice during contextual and cued fear
conditioning, and significantly reduced levels of lipid peroxidation and protein oxidation
in brain (Liu et al., 2003). In addition, both EUK-189 and EUK-207 were effective at
doses that could be reasonably applied to humans (Liu et al., 2003). A carboxyfullerene
SOD mimetic has also been shown to protect against age-associated brain oxidative stress
and learning and memory, but its effective dose was still about 67 times greater then
EUK-189 or EUK-207 (Quick et al., 2008, Liu et al., 2003) (Table 1).

11
Table 1. Summary of studies assessing the effects of exogenous antioxidants on age-
associated cognitive decline and oxidative stress in rodents.

Antioxidant

Treatment

Effect on age-
associated Brain
Oxidative Stress
Effect on age-
associated
impaired
learning and
memory

Reference
Green tea catechins:
71 % (–)-epigallo-
catechin-3-gallate

≈ 80 mg/kg body
weight/day for 6
months in female
C57BL/6J mice
beginning @ 14
months of age
↓ Lipid peroxidation in
hippocampus
↓ Protein carbonyls in
hippocampus

Not assessed

Qiong et al., 2010

Carboxyfullerene
SOD mimetic

10 mg/kg body
weight/day for ≈ 12
months in C57BL6
mice beginning @ 12
months of age

↓ Free radical levels
↓ Mitochondrial free
radical production

Improved spatial
learning and
memory in Morris
Water Maze

Quick et al., 2008

Deprenyl

1 mg/kg body
weight/day for 21
days in 24 month-old
male Wistar Rats

↓ Lipid peroxidation in
hippocampus,
striatum, & prefrontal
cortex

Improved spatial
learning in Morris
Water Maze

Kiray et al., 2006

DL- α-lipoic acid

100 mg/kg body
weight/day for 30
days in aged male
albino Wistar rats

↓ Lipid peroxidation in
hippocampus,
striatum, & cortex
↓ Protein carbonyls in
hippocampus,
striatum, & cortex
↓ DNA-protein cross-
links in hippocampus,
striatum, & cortex

Not assessed

Muthuswamy et al.,
2006

L-carnitine

300 mg/kg body
weight/day for 30
days in aged male
albino Wistar rats

↓ Lipid peroxidation in
hippocampus,
striatum, & cortex
↓ Protein carbonyls in
hippocampus,
striatum, & cortex
↓ DNA-protein cross-
links in hippocampus,
striatum, & cortex

Not assessed

Muthuswamy et al.,
2006

Grape Seed Extract

100 mg/kg body
weight/day for 30
days in 24–26
month-old male
albino Wistar Rats

↓ Free radical levels in
hippocampus,
striatum, cerebral
cortex & spinal cord
↓ Protein carbonyls in
hippocampus,
striatum, cerebral
cortex & spinal cord

Improved memory
performance in T-
maze test

Balu et al., 2005

12
Table 1 (continued). Summary of studies assessing the effects of exogenous
antioxidants on age-associated cognitive decline and oxidative stress in rodents.

Antioxidant

Treatment

Effect on age-
associated Brain
Oxidative Stress
Effect on age-
associated
impaired
learning and
memory

Reference
Vitamin E 200 mg/kg body
weight/day for 13
weeks in C57BL/6
mice beginning @ 20
months of age
No effect on lipid
peroxidation or protein
carbonyls in cerebral
cortex
No improvement
in spatial learning
or memory in
Morris Water
Maze after 4
weeks of
treatment
Sumien et al., 2004

SOD/catalase
mimetic EUK-189

≈ 0.15 mg & 1.5
mg/kg body
weight/day for 3
months in female
C57BL/6N mice
beginning @ 8
months of age

↓ Lipid peroxidation
↓ Protein carbonyls

Improved
performance in
contextual and
cued fear
conditioning

Liu et al., 2003

SOD/catalase
mimetic EUK-207

≈ 0.15 mg & 1.5
mg/kg body
weight/day for 3
months in female
C57BL/6N mice
beginning @ 8
months of age

↓ Lipid peroxidation
↓ Protein carbonyls

Improved
performance in
contextual and
cued fear
conditioning

Liu et al., 2003

Melatonin

0.1, 1.0, & 10 mg/kg
body weight/day for
30 days in 14 month
–old male Balb/C
mice

No effect on lipid
peroxidation in
forebrain (However,
lipid peroxidation was
the only marker
assessed and no
difference in forebrain
lipid peroxidation was
observed between
young and old mice)

All three doses
improved
performance in
passive-avoidance
task and elevated
plus maze

Raghavendra et al.,
2001

Vitamin E, 500 IU

1 g/kg feed for 8
months in male
Fischer 344 rats
beginning @ 6
months of age

↓ Free radical levels in
striatum and
cerebellum

Improved spatial
memory in Morris
Water Maze

Joseph et al., 1998

Spin-trapping
compound N-
tert-butyl-
α-pheynlnitrone

64 mg/kg body
weight/day for 14
days in 15–18
month-old male
Mongolian gerbils

↓ Protein carbonyls

Improved
temporal and
spatial memory in
eight-arm radial
arm maze

Carney et al., 1991

13
1.6. Cognitive targets of oxidative stress

While a role for oxidative stress in age-related cognitive impairment has been
repeatedly suggested, the actual mechanisms by which cognitive decline results from
oxidative damage are still not fully understood. Nevertheless, a number of studies have
indicated that oxidative stress might target molecular and cellular mechanisms involved
in synaptic plasticity. Long Term Potentiation, or LTP, is widely regarded as a
molecular/cellular mechanism that underlies certain forms of learning and memory, as
disrupting several of the molecular components of LTP produces impairment in learning
and memory (Young et al., 1994). A number of reports have demonstrated that age-
related cognitive impairment is accompanied by impaired LTP (Landfield et al., 1978,
Barnes, 1979, Tombaugh et al., 2002). In addition, dietary supplementation with
exogenous antioxidants not only reverses age-related cognitive deficits and oxidative
stress, but also reduces age-related impairment in LTP (Murray and Lynch, 1998,
McGahon et al., 1999a, McGahon et al., 1999b), thus indicating the existence of a direct
relationship between age-associated oxidative stress and impaired synaptic plasticity.
Several studies have revealed that glutamate release (Halliwell, 1992, Lynch and Voss,
1994, Mullany et al., 1996, Murray et al., 1997, McGahon et al., 1999a),RNA synthesis
(Poon et al., 2006a), actin polymerization (Huang et al., 2006, Poon et al., 2006a, Poon et
al., 2006b) and cell membrane composition (Kornberg et al., 1955, Lynch and Voss,
1994, McGahon et al., 1997) might be targets of oxidative stress that impinge on synaptic
plasticity.

14
1.7. Free radicals and cell death
Although cognitive decline caused by oxidative stress could be the result of
damage to key molecular and cellular components that underlie cognitive function, age-
associated cognitive decline could also be the result of ROS-induced neuronal cell death.
Reactive oxygen species (ROS) play a role in a number of cellular processes. They have
been implicated in the regulation of transcription factors (Flohe et al., 1997, Brigelius-
Flohe, 1999), and of several protein kinases and phosphatases (Klann and Thiels, 1999).
In addition, reactive oxygen species also play a positive role in memory and synaptic
plasticity. Mice lacking NADPH oxidase, an enzyme that produces superoxide, exhibit
memory impairment (Kishida et al., 2006), and so do young mice overexpressing
superoxide dismutase (Gahtan et al., 1998, Levin et al., 1998, Thiels et al., 2000).
Furthermore, young mice overexpressing superoxide dismutase also exhibit impaired
hippocampal long-term potentiation (Kamsler and Segal, 2003), and superoxide has been
shown to play a role in synaptic plasticity in vitro (Klann, 1998, Klann et al., 1998).
However, ROS also play an important role in apoptotic cell death. Hydrogen peroxide
has been shown to induce apoptosis in vitro via the activation of the mitochondrial
permeability transition pore and cytochrome c release (Stridh et al., 1998, Takeyama et
al., 2002). In addition, ROS-induced apoptosis might also include the death receptor Fas
and its ligand Fas-L (Facchinetti et al., 2002) as well as transcription factors that
stimulate proapoptotic gene expression, such as p53 (Uberti et al., 1999), NF-kB (Gloire
et al., 2006), and AP-1 (Sen and Packer, 1996). Although hydrogen peroxide has been
shown to induce apoptosis in a number of different cell types (Simon et al., 2000), high
15
doses of hydrogen peroxide have also been shown to induce necrosis (Buttke and
Sandstrom, 1994).
1.8. Age-associated oxidative stress might affect cellular
mechanisms responsible for maintaining neuron homeostasis.
Autophagy is a lysosomal degradation pathway that appears to be critically
involved in cell survival, cell differentiation, development, and cellular homeostasis
(Rajawat et al., 2009). Macroautophagy is primarily responsible for degrading damaged
or misfolded proteins, defective or unwanted organelles, protein aggregates, intracellular
pathogens, and other cellular debris (Levine and Kroemer, 2008). Most of the time
autophagy is occurring at low basal levels within cells and serves as a degradation
pathway for maintaining normal cellular homeostasis (Levine and Kroemer, 2008). Like
oxidative stress, the autophagy pathway also undergoes significant age-associated
changes in the mammalian brain. These changes include increased autophagosome
formation (Gamerdinger et al., 2009) and elevated expression and activity of lysosomal
cysteine proteases (Nakanishi et al., 1997, Gamerdinger et al., 2009, Nakanishi and Wu,
2009). In addition, autophagy plays a prominent role in maintaining normal neuron
function. This is evident by the fact that the disruption of autophagy in mice results in
significant neurodegeneration (Komatsu et al., 2005, Hara et al., 2006, Komatsu et al.,
2006), and altered brain autophagy is prevalent in age-associated neurodegenerative
diseases in humans such as Alzheimer’s disease and Parkinson’s disease (Anglade et al.,
1997, Nixon et al., 2005). Thus age-associated changes in brain autophagy might be
linked to age-dependent alterations in oxidative stress and cognitive impairment.
16
Microglia function as the immune response cells of the central nervous system
(Miller and Streit, 2007). However, they are also responsible for repairing tissue and
healing wounds following injury in the central nervous system (Streit et al., 2005). In
addition, they maintain neuron function by providing trophic support, secreting
extracellular matrix molecules, and phagocytizing macromolecules and other cellular
debris (Chamak et al., 1994, Rabchevsky and Streit, 1997, Nakajima K., 2002, Streit et
al., 2005). The role microglia play in neuron homeostasis is made even clearer by the fact
that neurodegenerative diseases are also accompanied by significant changes in microglia
morphology and activation (McGeer et al., 1987, Lassmann et al., 1995, v Eitzen et al.,
1998, Yang et al., 1998, Streit, 2004, Streit et al., 2004, Wierzba-Bobrowicz et al., 2004).
In addition to oxidative stress and autophagy, microglia also undergo age-dependent
changes. These include morphological modifications (Streit et al., 2004), reduced mitotic
activity (Prowse and Greider, 1995, Coviello-McLaughlin and Prowse, 1997), and
alterations in microglia density and the number of activated microglia (Rogers et al.,
1988, Perry et al., 1993, Ogura et al., 1994, Streit and Sparks, 1997, Sheffield and
Berman, 1998, Ma et al., 2003). Thus, like autophagy, age-associated oxidative stress
might be critically linked to age-associated alterations in microglia function.
1.9. Alzheimer’s disease
While in its mild form, age-related decline in memory function is not life-
threatening, it becomes much more dramatic in the pathological form exhibited by
patients with Alzheimer's disease. Alzheimer’s disease is the most prominent form of
dementia found in the elderly and the number of Americans inflicted with Alzheimer’s
disease is expected to reach nearly 15 million over the next several decades (Katzman
17
and Saitoh, 1991, Salmon et al., 2002). This neurodegenerative disorder is characterized
by progressive memory loss and cognitive deficits that ultimately lead to severe dementia
(Selkoe, 2001). The neuropathology associated with Alzheimer’s disease consists of the
presence in the brain of senile plaques consisting of aggregated extracellular β-amyloid
peptide and of intracellular neurofibrillary tangles mainly composed of
hyperphosphorylated tau protein (Salmon et al., 2002). Alzheimer’s disease is also
characterized by synapse loss (DeKosky and Scheff, 1990) and neuronal death (LeBlanc,
2005) in selective brain regions. The pathogenesis of Alzheimer’s disease is still not
clear but genetic studies using families with familial forms of the disease point to
mutations in β-amyloid metabolism as one of the initiating factors. Inherited forms of
Alzheimer’s disease consist of mutations in the genes for the amyloid precursor protein,
APP (Goate et al., 1991), presenilin-1 and presenilin-2 (Cruts et al., 1998), which result
in increased levels of fibrous β-amyloid peptide in brain (Hardy and Selkoe, 2002).
However, the number of cases of familial forms of Alzheimer’s disease is relatively small
when compared to the number of sporadic Alzheimer’s disease patients (Oddo et al.,
2003b). Other studies have shown that significant oxidative stress is also associated with
beta-amyloid plaques and neurofibrillary tangles in AD brain; thus, oxidation might be a
critical factor in AD pathology.
1.10. Oxidative stress and Alzheimer’s disease
Oxidative stress not only plays a role in age-associated cognitive impairment, but
also in Alzheimer’s disease. Brains from AD patients exhibit significantly increased
levels of protein oxidation (Good et al., 1996, Butterfield, 1997, Smith et al., 1997), lipid
peroxidation (Sayre et al., 1997, Butterfield et al., 2001, Butterfield and Lauderback,
18
2002), and DNA and RNA oxidation (Mecocci et al., 1994, Gabbita et al., 1998,
Nunomura et al., 1999, Lovell and Markesbery, 2001). The exact cause of brain
oxidative stress in AD is still unclear, but several pieces of evidence point toward the A β
peptide itself. A β has been shown to induce ROS production in both neuronal (Harris et
al., 1995, Yatin et al., 1999) and astrocyte cell cultures. In addition, cultured
hippocampal neurons and synaptosomes exhibit increased protein oxidation when
incubated with various A β peptides (Varadarajan et al., 2000). Furthermore, treating rat
synaptic plasma membranes with either A β(1-40) or A β(25-35) also increased lipid
peroxidation (Avdulov et al., 1997). However, A β might not be the only culprit for
oxidative stress in Alzheimer’s disease. Brains from AD patients exhibit damaged
mitochondria and reduced ATP production (Gibson et al., 1998, Castellani et al., 2002),
and electron transport chain deficiencies (Parker et al., 1994), thus supporting a role for
mitochondria in AD-related oxidative stress. As discussed above, AD-related oxidative
stress could be responsible for the cognitive deficits observed in Alzheimer’s disease
because it might damage key molecular and cellular components underlying cognitive
function. In addition, oxidative stress could also be a mediator in neuron cell death in
Alzheimer’s disease, since ROS production accompanies A β-induced neuronal apoptosis
and the inhibition of A β-induced neuronal apoptosis by the antioxidants α-tocopherol and
N-acetylcysteine (Tamagno et al., 2003). While there is ample evidence demonstrating a
relationship between Alzheimer’s disease and oxidative stress, whether or not oxidative
stress is an initiator or a consequence of its pathogenesis remains an unanswered
question. Evidence from in vitro studies with A β suggests that oxidative stress is a
downstream mediator of A β toxicity, while other results suggest that A β might have a
19
role as an antioxidant and therefore its accumulation in brains of AD patients could be the
result of oxidative stress (Zhu et al., 2007).
1.11. Dissertation outline
As discussed above, a number of questions remain unanswered. While there are
clear relationships between brain oxidative stress and cognitive impairment during
normal aging and in AD patients, the causal relationship between these parameters
remains to be unequivocally addressed. In my studies I set out to address this question.
In Chapter 1, I evaluated the relationship between oxidative stress and cognitive
impairment in aged mice by testing the effects of two SOD/catalase mimetics, EUK-189
and EUK-207, previously used in somewhat younger mice, on age-associated brain
oxidative stress and cognitive function. My results indicated that about 60 % of the age-
dependent decline in cognitive performance could be attributed to an increase in brain
lipid peroxidation and free radical levels. In Chapter 2, I further evaluated the role of
oxidative stress in age-related cognitive impairment by evaluating the relationship
between oxidative stress and the autophagic-lysosomal pathway and microglia, both of
which are critical in maintaining normal neuronal homeostasis. My results indicate that
both the autophagic-lysosomal pathway and microglia function appear to be targets of
age-associated brain oxidative stress. In Chapter 3, I attempted to define the role of
oxidative stress in the pathogenesis of Alzheimer’s disease. This was accomplished by
using a mouse model of Alzheimer’s disease, the triple transgenic mouse, and testing the
effects of the SOD/catalase mimetic, EUK-207, on learning and memory, oxidative stress
and Alzheimer’s disease pathology. The results from these studies support the idea that
oxidative stress plays a critical role in the development of Alzheimer’s disease.
20
Furthermore, they suggest that SOD/catalase mimetics could be potential therapeutic
agents for treating and preventing age-associated mild-cognitive impairment and
Alzheimer’s disease.

21
Chapter 2

Prevention of cognitive deficits and brain oxidative stress with
superoxide dismutase/catalase mimetics in aged mice.
2.1. Introduction
An age-associated decline in learning and memory has been well documented in
humans (Davis et al., 2003). While in its mild form, age-related decline in memory
function is not life-threatening, it becomes much more dramatic in the pathological form
exhibited by patients with Alzheimer's disease. In addition, individuals exhibiting mild
cognitive impairment have a greater chance of developing Alzheimer’s disease compared
to the general population (Petersen et al., 2001). Such age-related loss of memory is not
unique to humans, and is present in a variety of mammals, including rats and mice
(Barnes et al., 1990, Foster et al., 1996). While there is still no consensus regarding the
nature of the biological process(es) that underlies age-related decline in cognitive
function, it has been frequently proposed that the aging process itself is linked to the
accumulation of oxidative damage in neurons.
Reactive oxygen species (ROS) are formed as a by-product of cellular
metabolism. The formation of free radicals by normal cellular respiration usually results
in a low steady-state level of ROS (Boveris and Chance, 1973, Finkel and Holbrook,
2000), which can be handled by cellular antioxidant defense mechanisms. However,
during oxidative stress, ROS production increases and surpasses the capacity of
endogenous free radical scavengers such as superoxide dismutase and catalase. In
addition, the brain is especially sensitive to oxidative stress because it utilizes high levels
22
of oxygen, contains large amounts of lipids that free radicals can readily react with, and
exhibits a lower level of antioxidants compared to other tissues (Halliwell, 1992).
Indeed, aging is associated with increased free radical levels and damage
associated with oxidative stress in mammalian brain, including lipid peroxidation, protein
oxidation, and oxidized nucleic acids (Calabrese et al., 2004, Cini and Moretti, 1995,
Hamilton et al., 2001, O’Donnell and Lynch, 1998, Siqueira et al., 2005, Sohal et al.,
1994). The reason for this age-related increase in oxidative stress in the brains of aged
mammals is still unclear, although mitochondria preparations from the brains of aged
rodents exhibit a significant age-dependent increase in superoxide and hydrogen peroxide
production (Sawada and Carlson, 1987, Sohal et al., 1994), and several studies have
reported age-associated decreases in superoxide dismutase activity and catalase activity
in rat brain (Rao et al., 1990, O’Donnell et al., 2000). Whether age-related increase in
oxidative stress is directly responsible for age-dependent decline in memory and
cognitive function remains unclear. Nevertheless, age-associated cognitive deficits have
been correlated with increased oxidative stress in various regions of the mammalian brain
(Butterfield et al., 2006, Forster et al., 1996, Fukui et al., 2001, Nicolle et al., 2001), and
supplementing diets of aging mammals with antioxidants or free radical scavengers has
generally been shown to attenuate age-related cognitive decline and oxidative stress
(Carney et al., 1991, Raghavendra and Kulkarni, 2001, Stoll et al., 1994).
Such findings support the idea that free radical accumulation might indeed impair
memory function. It would therefore seem that antioxidant molecules should be
beneficial for treating age-associated declines in learning and memory in humans.
However, to apply such treatments to humans would require enormous amounts of free
23
radical scavengers as they react on a stoichiometric basis with reactive oxygen and
nitrogen species. An alternative approach would be to use low molecular weight
synthetic molecules that function like the enzymes superoxide dismutase and catalase,
that is, act catalytically against ROS. Two such molecules, EUK-189 and EUK-207, have
previously been shown to improve cognitive performance and decrease oxidative stress in
middle-aged wild type mice (Liu et al., 2003).
In the current study, the effects of these compounds on age-related learning and
memory impairment and on markers of oxidative stress in the brain were tested in older
mice, at a lower dose, and for longer periods of time. The results provide strong evidence
that ROS accumulation and oxidative stress are directly related to age-associated decline
in learning and memory. In addition, my results also support potential therapeutic
applications for synthetic superoxide dismutase and catalase mimetics for treating age-
related decline in cognitive function.
2.2. Materials and methods
2.2.1. Materials
EUK-189 and EUK-207 were synthesized as described previously (Doctrow et al.,
2002, (Malfroy-Camine; Bernard (Arlington, 2003)) The oxo8dG/oxo8G antibody was
purchased from QED Bioscience (San Diego, CA). All other chemicals were purchased
from Sigma, unless indicated otherwise.
2.2.2. Mice and treatments
Animals were treated in accordance with the principles and procedures of the
National Institutes of Health Guide for the Care and Use of Laboratory Animals; all
24
protocols were approved by the Institutional Animal Care and Use Committee of the
University of Southern California. I used 85 C57BL/6N Sim male mice obtained from the
National Institute on Aging. Before experiments, mice were housed 4–5 per cage and
placed in the same room with a 12-h light/12-h dark cycle and behavioral testing was
performed during the last 6 h of the light cycle. Mice were allowed free access to food
and water, and their weights ranged from 27 to 36 g. Before surgery, mice were randomly
assigned to five of the following groups (17 mice per group): vehicle control and EUK-
treated groups (1.5 or 0.15 mM EUK-189 or EUK-207). An additional group of 10 male
mice were used for the untreated 16-month control group.
Before implantation, Alzet 2004 miniosmotic pumps (Durect Corporation,
Cupertino, CA) were loaded with either EUK-189 or EUK-207 at 1.5 or 0.15 mM in 5 %
mannitol, or 5 % mannitol alone (as vehicle control group) and then primed for at least 40
hours in 5 % mannitol at 37 ºC. The minipumps were then implanted s.c. in the 17-
month-old mice according to the manufacturer’s recommendations. Briefly, mice were
anesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg) by i.p. injection. A small
1-cm incision was then made to the hip area of the mice and a small pocket was formed
by spreading the s.c. connective tissues apart. The pump was placed into the prepared
pocket, and the wound was then closed with sutures.
Pumps delivered the drugs at 0.25 µl/hour for a 28-day period, and the calculated
drug infusion rates were ≈9 nmol/day for the 1.5 mM doses of EUK-compounds and ≈0.9
nmol/day for the 0.15 mM doses of EUK compounds. The lower concentration is
equivalent to a dose of 15 and 16 µg/kg/day for EUK-189 and EUK-207, respectively
(assuming a 30 g mouse). This is a much lower dose than has ever been tested for
25
efficacy in previous studies. Control mice were implanted with minipumps filled with
vehicle alone (5% mannitol).
During the 6-month treatment, pumps were replaced 5 more times with new ones
at the original sites at the end of each 28-day period of implantation. In some of the mice,
the new pumps had to be placed on the opposite hip area during the 6-month treatment
due to skin damage at the original site of implantation. In addition, body weights were
recorded at the end of the 6-month treatment in order to assess the effects of EUK-189
and EUK-207 on overall health.
2.2.3. Behavioral analysis
2.2.3.1. Fear conditioning
Experiments were run in a conditioning chamber consisting of a Plexiglas cage
(29 cm × 29 cm × 29 cm) with a grid floor composed of 26 stainless steel rods (0.48 cm
in diameter; Coulbourn Instruments, Allentown, PA). The apparatus was located in a
sound-attenuating box located in a room that is separated from the main laboratory. A
personal computer controlled the experimental events and a video camera monitoring
system was used to continuously record behavior for off-line scoring of freezing. The
chamber was wiped with 70 % ethanol before and after each training and testing session
and each mouse was placed in the chamber individually. On day 1 of training, mice were
put in the chamber and after 3 min they received three separate tones that were
terminated with a foot-shock (tone: 20 s, 80 dB, 2 kHz; foot shock: 1 s, 0.8 mA; intertrial
interval, 1 min apart). Thirty seconds after the final foot shock, mice were returned to
their home cages. Twenty-four hours after training, mice were tested for conditioning to
26
the context by placing them into the conditioning chamber for 8 min, but neither foot
shock nor tone was given. When fear conditioning was performed at 23-months of age,
the visual properties of the conditioning chamber were changed and the chamber was
cleaned with 4 % acetic acid instead of 70 % ethanol in order to minimize any effects fear
conditioning at 20-months of age might have on fear-conditioning performance at 23-
months of age.
2.2.3.2. Behavioral analysis
A time-sampling procedure performed by a trained observer blind to the
experimental conditions was used. Briefly, every 10 s, each mouse is judged as either
freezing or active. Freezing is defined as the absence of all visible movement of the body
and vibrissae, aside from movement necessitated by respiration. The percentage of
freezing is then calculated for the 8 min trial by dividing the number of freezing episodes
by the total number of observations (48) and multiplying by 100. In addition, freezing
was also assessed on the training day prior to the delivery of the first shock in order to
quantify baseline freezing levels. Baseline freezing levels were subtracted from the
freezing responses measured during the context test in order to get percentage of
cumulative freezing time. For comparison, and to confirm age-associated decline in
cognitive function, fear conditioning was also measured in 16-month-old mice.
In order to control for potential sensory deficits, mice were also tested for
auditory and visual functions, and for nociception. Hearing was assessed by
administering an auditory startle threshold test. Briefly, mice were placed in a sound-
attenuating chamber individually, and after a 3-min acclimation period, they were
presented with a series of 1 s 2 or 4 kHz tones. Each series started out at 80 dB and
27
increased by 10 dB until 100 or 110 dB was reached. Each tone was separated by 15 s
and responses to every tone were recorded. As previously reported, we observed that
aged C57BL/6N Sim mice had hearing impairment, and this impairment precluded
testing of the animals in the cue test as is often performed in fear conditioning. Vision
was evaluated using a forepaw-reaching test. Mice were held by their tail and placed up
side down in mid air next to a platform, and their ability to correctly reach towards the
platform was assessed. Special care was taken in order to keep the whiskers away from
the platform, and each mouse was tested with two different types of platforms.
Nociception was evaluated with a tail-flick latency test. Every mouse was placed in a
beaker in order to restrain them, and after the mouse calmed down their tale was placed
on a 51 ºC hot plate. Tail-flick latency was defined as the length of time that elapsed
between placing the tail on the hot plate and tail flicking. Overall health of the animals
was assessed by daily inspection and by monitoring body weights.
2.2.4. Assays for lipid peroxidation, nucleic acid oxidation, and
ROS content
For biochemical studies, mice were anesthetized with isoflurane and killed by
decapitation. Brains were rapidly extracted, placed on a chilled platform, and the
cerebellum and pons were removed and discarded. The brains were then cut in half
sagitally, and each half was immediately frozen on dry ice, and stored at −70 ºC until
assayed. Lipid peroxidation, nucleic acid oxidation, and free radical content were
measured in brain homogenates from 23-month-old vehicle controls, EUK-189- or EUK-
207-treated C57 mice, and 16-month-old untreated control mice.
28
The levels of lipid peroxidation were quantified by the thiobarbituric acid-reactive
substances (TBARS) assay as previously described (Bruce and Baudry, 1995) with minor
modifications. One sagital half from each brain was homogenized in 2.5 % SDS
containing 6.25 µM deferoxamine and 12.5 µM probucol (to prevent further oxidation).
Four hundred microliters of homogenates were added to an aqueous solution consisting
of 375 µl of 20 % acetic acid solution (pH 3.5) and 225 µl of 1.33 % thiobarbituric acid,
and the mixture was heated at 95 ºC for 1 hour. One milliliter of a 15:1 1-
butanol/pyridine solution was added, and TBARS were extracted into the organic layer
by centrifugation at 4,000 × g for 10 min. The amounts of TBARS were determined by
spectrophotometry at 532 nm and were calculated as nanomolar malondialdehyde
equivalent per milligram of protein according to a standard curve prepared from
malonaldehyde bis(dimethyl acetal).
Oxidized nucleic acid content was determined using a DNA dot/blot procedure.
Briefly, genomic DNA was extracted from 205 µl of the homogenized brain tissue
mentioned above using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Ten
micrograms of extracted DNA was then digested with HindIII (New England Biolabs,
Ipswich, MA) overnight and then precipitated with 3 M sodium acetate and 100 %
ethanol. The resulting DNA pellet was then resuspended in 6 × SSC by heating to 95 ºC
for 10 min and then chilled on ice. One microgram of extracted digested DNA was then
blotted onto a NitroPure nitrocellulose membrane (GE Healthcare Life Sciences,
Piscataway, NJ) that had been previously soaked in 6 × SSC. After allowing the
membrane to dry at room temperature, the DNA was fixed onto the membrane using UV
irradiation. The blot was then developed using the avidin–biotin–horseradish peroxidase
29
complex (ABC) method with reagents and instructions of the VECTASTAIN Elite ABC
kit from Vector Laboratories (Burlingame, CA). The membrane was washed with PBS
and then incubated in 10 % normal horse serum diluted in PBS for 1 hour at room
temperature. The membrane was then incubated in the presence of a monoclonal antibody
to oxo8dG/oxo8G (1:1000) in 5 % horse serum diluted in PBS overnight at 4 ºC. The blot
was then washed in PBS, incubated in biotinylated anti-mouse IgG (1:200 in PBS with 5
% normal horse serum) for 2–3 h, washed in PBS, and incubated in the avidin–biotin
complex solution for 45 min. After the diaminobenzidine reaction, the membrane was
washed with water and then air dried. Blots were scanned and then quantified using
Image J Software (NIH, Bethesda, MD), and the results were expressed as percentage of
the corresponding values assayed in the 16-month-old control mice.
ROS levels were quantified via the 2’-7’-dichlorofluorescein-diacetate (DCFH-
DA) assay previously described (Siqueira et al., 2005) with some minor modifications.
Briefly, the remaining sagittal half of each brain was homogenized in 50mM phosphate
buffer at pH 7.4 (10%, w/v) and centrifuged at 11,000 × g for 15 min to sediment
insoluble materials. DCFH-DA was then added to a portion of the homogenate to a final
concentration of 100 µM and the reaction mixture was incubated for 30 min at 37 ºC. The
reaction was then stopped by placing it on ice and the formation of oxidized fluorescent
2’-7’-dichlorofluorescein (DCF) was measured with a fluorimeter (HORIBA Jobin Yvon
Fluoromax-3) using excitation and emission wavelengths at 488 and 525 nm,
respectively. The final results were corrected for protein concentration and then
expressed as percentage of the corresponding values in 16-month-old mice. All steps
were performed in the dark and DCF formation was also monitored immediately after
30
DCFH-DA was added to the homogenate (t = 0 min) in order to subtract background
autofluorescence.
2.2.5 Statistical analysis
All statistics were performed using GraphPad Prism 4.03 software (GraphPad
Software, La Jolla, CA). One-way ANOVA was used to test if the means of each
experimental group were significantly different and if the overall p value was <0.05, then
multiple comparisons between the experimental groups were tested using Newman–
Keuls post hoc analysis. Pearson’s correlation coefficients were determined for the linear
relationships between markers of oxidative stress and fear conditioning performance and
two-tailed p values were calculated for each Pearson correlation coefficient in order to
assess the significance of the relationship.
2.3. Results
2.3.1. Effects of SOD/catalase mimetics on fear-conditioning
learning
C57 BL/6N mice were treated continuously with EUK-189 or EUK-207
administered through subcutaneously implanted osmotic minipumps. After 3 and 6
months of continuous treatment, cognitive performance was assessed using a contextual
fear-conditioning paradigm. Vehicle control mice exhibited a significant decline in
freezing response during the context test at both 20 and 23 months of age when compared
to 16-month-old mice (Figs. 1 and 2), thus suggesting an age-associated decline in
learning and memory in this strain of mice. Chronic treatment with either EUK-189 or
EUK-207 at both concentrations significantly increased freezing responses exhibited by
31
20- and 23-month-old mice (Figs. 1 and 2). The 20-month-old treated groups performed
just as well as the 16-month-old mice; therefore it appears that EUK-189 and EUK-207
were able to block the cognitive deficits that arose between 16 and 20 months of age. The
effects of chronic treatment with either EUK-189 or EUK-207 on freezing response in
23-month-old mice were not as striking as those seen in 20-month-old mice, but the
treatments still significantly increased learning and memory performance in the fear-
conditioning task (Fig. 2).
There were no significant differences between the two compounds in their ability
to protect against age-associated cognitive decline, and both 1.5 and 0.15 mM
concentrations provided similar results (Figs. 1 and 2). In addition, no significant defects
in vision or nociception were present in all the groups tested (Table 2), and the prolonged
treatments with either compounds did not appear to produce any ill effects or weight
changes (Table 2), suggesting that age-related differences observed in contextual fear
conditioning were due to learning and memory deficits and not to differences in visual or
pain perception or overall health conditions.

Figure 1. Effects of 3-month chronic treatment with EUK-189 or EUK-207 on context fear
conditioning. 17 month-old mice were treated for 3 months with a low (0.15 mM) or a high (1.5 mM)
concentration of EUK-189 or EUK-207 and then trained in a contextual fear conditioning paradigm and
tested 24 h after training. Results were calculated as percent time the mouse expressed freezing behavior
during the 8-min observation period for context minus the percent time the mouse expressed freezing
behavior prior to training. Shown are means ± SEM of 9-17 mice. One-way ANOVA indicated that the
effect of age was significant (*p <0.05 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207
(**p <0.05 vs. vehicle control).

Figure 2. Effects of 6-month chronic treatment with EUK-189 or EUK-207 on context fear
conditioning. After 6 months of treatment, 23 month-old mice were retrained in a contextual fear
conditioning paradigm, but in a new context, and tested 24 h after training. Results were calculated as
percent time the mouse expressed freezing behavior during the 8-min observation period for context minus
the percent time the mouse expressed freezing behavior prior to training. Shown are means ± SEM of 9-14
mice. One-way ANOVA indicated that the effect of age was significant (*p <0.001 vs. 16 month-old mice),
as was the effect of EUK-189 and EUK-207 (**p <0.05 vs. vehicle control).
32
Table 2. Body weight, nociception, and vision test results for SOD-catalase treated
mice.

Forepaw reaching
test (% success)
Tail-flick
latency, mean ± S.D. (s)
Body weight,
mean ± S.D. (g) Treatment
100 1.32 ± 0.39 30.3 ± 2.9 0.15 mM EUK-207
100 1.30 ± 0.45 31.3 ± 2.7 1.5 mM EUK-207
100 1.55 ± 0.44 30.9 ± 2.0 0.15 mM EUK-189
100 1.45 ± 0.44 30.1 ± 2.3 1.5 mM EUK-189
100 1.50 ± 0.52 30.8 ± 2.7 Vehicle
Forepaw reaching
test (% success)
Tail-flick
latency, mean ± S.D. (s)
Body weight,
mean ± S.D. (g) Treatment
100 1.32 ± 0.39 30.3 ± 2.9 0.15 mM EUK-207
100 1.30 ± 0.45 31.3 ± 2.7 1.5 mM EUK-207
100 1.55 ± 0.44 30.9 ± 2.0 0.15 mM EUK-189
100 1.45 ± 0.44 30.1 ± 2.3 1.5 mM EUK-189
100 1.50 ± 0.52 30.8 ± 2.7 Vehicle
Summary of the data for body weights after 6 months of treatment, tail-flick latency
(nociception) and forepaw reaching test (vision). There were no statistically significant
differences for the three parameters between the different treatment groups.

2.3.2. Effects of SOD/catalase mimetics on brain oxidative stress.
In order to assess the effects of EUK-189 or EUK-207 on age-related oxidative
stress, mice treated as described above were sacrificed at the end of the 6 month long
treatment and their brains, minus cerebellum and pons, were harvested and homogenized.
Brain homogenates were then used to measure markers for oxidative stress, which
included lipid peroxidation (levels of equivalent malondialdehyde), ROS levels and
oxidized nucleic acids. For comparison, and to confirm age-associated increase in
oxidative stress, markers for oxidative stress were also measured in 16-month-old mice.
Lipid peroxidation significantly increased between 16 and 23 months of age (Fig.
3). A 6-month chronic treatment with either EUK-189 or EUK-207 at both concentrations
significantly decreased age-dependent increase in lipid peroxidation (Fig. 3). A similar
age-associated increase in oxidized guanine (Fig. 4), and free radical levels (Fig. 5) was
observed between 16 and 23 months of age. Treatment with either EUK-189 or EUK-207
almost completely reversed age-associated increases in both ROS levels and oxidized
33
guanine and at both concentrations. Like for the behavioral study, there were no
significant differences between the two compounds in their ability to protect against age-
associated oxidative stress. Treatment with both compounds at 1.5 and 0.15 mM
concentrations resulted in similar reduction in lipid peroxidation and ROS content in the
brains of these mice, although EUK-189 at a concentration of 0.15mM and EUK-207 at a
concentration of 1.5 mM produced a more significant reduction in oxidized guanine (Fig.
4).

Figure 3. Effects of chronic treatment with EUK-189 or EUK-207 on lipid peroxidation in brain
homogenates. At the end of the 6 month treatment, mice were decapitated and their brains (minus
cerebellum) were removed and homogenized . Lipid peroxidation was then quantified by the thiobarbituric
acid-reactive substances (TBARS) assay. Lipid peroxidation was also determined in brain homogenates
from 17 month-old control mice. Levels of lipid peroxidation were expressed as nmol malondialdehyde
equivalent per mg of protein. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the
effect of age was highly significant (*p <0.001 vs. 17 month-old mice), as was the effect of EUK-189 and
EUK-207 (**p <0.001 vs. vehicle control).

34

35

igure 4. Effects of chronic treatment with EUK-189 or EUK-207 on oxidized nucleic acids in brain F
homogenates. At the end of the 6-month treatment, oxidized guanine was quantified by extracting DNA
from brain homogenates and then blotting onto a membrane, which was then probed with an anti-
oxo8dG/oxoG antibody. Oxidized nucleic acid content was also determined in brain homogenates from 17
month-old control mice. (A) Representative 8oxoG dot blot. (B) Results were expressed as percentage of
17 month value. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the effect of age
was significant (*p <0.05 vs. 17 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.05
vs. vehicle control, ***p <0.01 vs. vehicle control).

igure 5. Effects of chronic treatment with EUK-189 or EUK-207 on reactive oxygen species (ROS)
.3.3. Correlation between cognitive function and brain levels of
markers for oxidative stress
In order to further define the relationship between learning and memory
performance and brain oxidative stress, I assessed the correlation between performance in
the contextual fear-conditioning paradigm and brain levels of ROS and lipid
peroxidation. Individual data for contextual fear conditioning and brain ROS content
(Fig. 6A) or lipid peroxidation (Fig. 6B) were plotted for 16-month-old control mice, 23-
month-old vehicle control mice, and 23-month-old EUK-189 and EUK-207 treated mice
(as there were no significant differences between the two doses of both compounds,
results for both doses of EUK-189 or EUK-207 were combined). The linear relationship

F
levels in brain homogenates. At the end of the 6-month treatment, brain ROS content was quantified by
incubating brain homogenates with 2'-7-dichlorofluorescein diacetate (DCFH-DA). ROS content was also
determined in brain homogenates from 17 month-old control mice. Results were expressed as percentage of
17 month value. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the effect of age
was highly significant (*p <0.001 vs. 17 month-old mice), as was the effect of EUK-189 and EUK-207
(**p <0.001 vs. vehicle control).

2
36
37
between contextual fear-conditioning performance and oxidative stress was then assessed
by determining the significance of the Pearson’s correlation coefficient.
I observed a significant negative correlation between contextual fear-conditioning
performance and brain ROS content (Fig. 6A, ρ = −0.68) and lipid peroxidation levels
(Fig. 6B, ρ = −0.76) in control mice. This indicates that oxidative load accounts for about
50 – 60 % of the variability in learning and memory in aged mice. However, this
correlation was lost in 23-month-old mice treated with either EUK-189 or EUK-207 (Fig.
6A and B).

Figure 6. Correlation between performance in the contextual fear conditioning task and brain levels
of free radicals and lipid peroxidation. Individual data for contextual fear conditioning and brain free
radical content (A) or lipid peroxidation (B) were plotted for 17-month-old control mice, 23-month-old
vehicle control mice, and 23-month-old EUK-189 and EUK-207 treated mice. Free radical content was
expressed as percentage of 17-month-old control value (A) and levels of lipid peroxidation were expressed
as nmol malondialdehyde equivalent per mg of protein (B). Contextual fear conditioning performance was
calculated as percent time the mouse expressed freezing behavior during the 8-min observation period for
context minus the percent time the mouse expressed freezing behavior prior to training (A & B).
Regression lines were plotted for the control animals and analysis indicated a significant negative
correlation between contextual fear conditioning performance and brain free radical content (Pearson’s
correlation coefficient ρ= -0.68, p <0.0054, n =15) (A) and lipid peroxidation levels (Pearson’s correlation
coefficient, ρ= -0.76, p <0.0011, n =15) (B). No significant correlation between performance in the
contextual fear conditioning task and brain levels of free radicals or lipid peroxidation was observed in 23-
month-old EUK-189 (n =17) and EUK-207 (n =19) treated mice (A & B).
38
39
2.4. Discussion

A large body of evidence supports a role for oxidative stress in age-dependent
decline in cognitive function. The decline in learning and memory aged mice exhibit
when tested with the spatial swim maze has been correlated with an increase in protein
carbonyl content in cerebral cortex (Forster et al., 1996). Lipid peroxidation is also
significantly much higher in hippocampus and inferior parietal lobule of elderly
individuals who exhibit mild cognitive impairment (Butterfield et al., 2006). Aged rats
that perform just as well as young rats in spatial learning tasks do not show the increased
levels of nucleic acid and protein oxidation in hippocampus that aged cognitively
impaired rats do (Nicolle et al., 2001). Furthermore, age-dependent cognitive decline in
rodents can be attenuated by treating them with antioxidants and free radical scavengers,
such as alpha-lipoic acid (Stoll et al., 1994), melatonin (Raghavendra and Kulkarni,
2001), and N-tertbutyl-alpha-phenylnitrone (Carney et al., 1991).
My results further strengthen the relationship between oxidative stress and age-
associated deficits in learning and memory by showing that the age-dependent increased
oxidative stress and cognitive decline that arise between 16 and 23 months of age in mice
are significantly reduced by chronic treatment with the SOD/catalase mimetics EUK-189
or EUK-207. Numerous studies have shown that aging is associated with decrease in fear
conditioning in rats and mice and my data are in good agreement with published results.
Although I trained and tested the 20- and 23-month-old groups 4 or 7 months after the
16-month-old control group, it is not likely that this time difference is responsible for the
observed effects, as there is no report in the literature regarding possible seasonal
influence on fear conditioning in mice. Furthermore, the critical finding in my study is
40
the reversal of cognitive deficit provided by the drug treatments. Both compounds
appeared to significantly decrease the deficits in learning and memory that take place
between 16 and 23 months of age, and reverse the increases in brain lipid peroxidation,
ROS content, and oxidized guanine occurring during this time period. Chronic treatment
with either EUK-189 or EUK-207 also significantly reduced the increased cognitive
decline that occurred between 20 and 23 months of age. Moreover, my results indicate
that oxidative load in aged mice accounts for about 50–60% of the variance in learning
ability. Interestingly, while treatment with either EUK-189 or EUK-207 produced almost
complete reversal of age-related increase in oxidative load, it decreased decline in
performance in fear conditioning by about 50 – 60 %. This result suggests that some
factors other than oxidative load account for the remaining of the decline in cognitive
function with aging.
Both concentrations of EUK-189 and EUK-207 were equally effective in
preventing age-associated cognitive decline and oxidative stress in aged mice. However,
previous work done in my laboratory suggested that the effectiveness of theses
compounds might decrease at high concentrations. Like in this present study, 1.5 mM of
both compounds inhibited age-dependent cognitive deficits and oxidative stress in
middle-aged mice (Liu et al., 2003). However, in the previous study, my lab also showed
that at higher concentrations (15 mM), EUK-207 was less effective than at 1.5 mM (Liu
et al., 2003). In the current study, in an attempt to eliminate potentially deleterious doses
as well as to better characterize dose-dependency of the compounds, I repeated the 1.5
mM dose and added a ten-fold lower dose group. In this study, while not yet identifying a
41
suboptimal dose, I report for the first time in any in vivo system that EUK-189 and EUK-
207 are active at a dose of 0.15 mM, that is approximately 15 µg/kg/day.
My results also indicate that the age-associated deficits in learning and memory I
observed might be brought on by oxidative damage to hippocampus, amygdala, or both.
Contextual fear conditioning is a learning and memory task that is dependent on both
amygdala and hippocampus (Phillips and LeDoux, 1992, Maren et al., 1997), and my
data show that increased levels of markers for oxidative stress coincides with deficits in
contextual fear conditioning. Furthermore, chronic treatment with either EUK-189 or
EUK-207 significantly alleviates these age-associated deficits in contextual fear
conditioning. These results are also in agreement with a similar study in middle-aged
mice (Liu et al., 2003). While it might have been interesting to determine changes in
oxidative load in hippocampus and amygdala, the small size of these structures and the
need for relatively large amounts of tissues for all the biochemical assays compelled me
to limit my tissue samples to the combined forebrain and midbrain.
This body of work and previous studies by others suggest that antioxidant
compounds could prove to be beneficial in treating age-dependent cognitive deficits in
humans. However, compounds that mimic SOD and catalase might prove to be even
more beneficial in humans because, unlike antioxidants, they do not react on a
stoichiometric basis. Earlier work with a carboxyfullerene SOD mimetic showed a
dramatic decrease in age-dependent learning and memory deficits and oxidative stress in
mice (Quick et al., 2008). The multiple catalytic activities of EUK-189 and EUK-207
might prove to be even more beneficial, and could account for the very low efficacious
doses, as compared to that of the carboxyfullerene tested. Thus, a dose of the EUK
42
compounds of about 15µg/kg/day was as potent as a dose of 10 mg/kg/day of
carboxyfullerene. In addition, it is important to note that the carboxyfullerene is only an
SOD mimetic whereas the EUK compounds tested can also protect against damage
caused by hydrogen peroxide, and through catalase-like mechanisms (Doctrow et al.,
2002, Sharpe et al., 2002), reactive nitrogen species.
Chronic treatment with EUK-189 or EUK-207 was initiated at a relatively late
stage in the lives of these mice, but my 6 month-long treatments were still able to provide
protection against cognitive declines that occurred between 16 and 23 months of age.
Thus, my results suggest that these compounds might prove to be beneficial in preventing
further cognitive impairment in relatively old individuals that already exhibit mild
cognitive impairment. In addition, while this study was not intended to address chronic
toxicity, it is well worth noting that long term, sustained treatment with these compounds
was beneficial to the mice without showing any indications of toxicity. Similar
observations have been made in other long-term treatment studies, for example, chronic
administration of EUK-189 in a mouse Alzheimer’s disease model (Melov et al., 2005).
Previous work done using SOD2 knock-out mice provided indirect evidence that
salen–manganese compounds such as EUK-8, EUK-134, or EUK-189 are able to cross
the blood brain barrier and to be mito-protective (Melov et al., 2001). EUK-207 is as
equally effective as EUK-189 in extending the lifespan of SOD2 knock-out mice
(unpublished data), but its ability to alleviate the neurological phenotype has not been
characterized.
In conclusion, my data demonstrate that chronic treatment with the superoxide
dismutase/catalase mimetics EUK-189 or EUK-207 significantly reduces age-related
43
cognitive impairment and age-associated oxidative stress in mice. These findings add
support to the hypothesis that oxidative stress plays a critical role in age-related learning
and memory dysfunction. Furthermore, they suggest that EUK-189 or EUK-207 have
potential value as a treatment for age-related cognitive dysfunction.

44
Chapter 3
Age-related changes in autophagy-lysosome system and microglial
function are reversed by chronic treatment with SOD/catalase
mimetics.
3.1. Introduction
The brain undergoes significant changes in morphology, biochemistry, and
physiology during aging. Thus, it comes as no surprise that changes in cognitive function
accompany the aging process. An age-associated decline in cognitive function has been
well documented in a variety of animals, including humans. Administering a verbal word
recall test to humans from different age groups revealed a linear relationship between an
increase in age and a decrease in memory performance (Davis et al., 2003). Aged mice
perform poorly compared to young mice in a variety of behavioral tests that assess
locomotion, motor coordination, and learning and memory (Forster et al., 1996). Testing
22 month old rats and 3-4 month old rats in an assortment of learning and memory tests
also revealed impairment in cognitive function associated with aging (Barnes et al.,
1990).
The underlying biological mechanisms responsible for this age-related decline in
cognitive performance are not yet fully understood, but one of the most striking age-
associated alterations in the brain is an increase in oxidized macromolecules, such as
lipids, proteins, and nucleic acids. The level of oxidized proteins is much higher in the
frontal and occipital poles of brains from Alzheimer’s disease patients and age-matched
45
controls compared to young individuals (Smith et al., 1991). Age-related increases in
protein oxidation and oxidized nuclear DNA have also been reported in the brain of rats
and mice (Sohal et al., 1994, Cini and Moretti, 1995, Hamilton et al., 2001). Lipid
peroxidation in cerebellum, striatum, hippocampus, and substantia nigra is also
significantly higher in aged rats. The reasons for this age-associated increase in brain
oxidative stress are still not clear, but age-dependent increase in free radical formation
has been reported (Auerbach and Segal, 1997, Driver et al., 2000), which could be the
result of age-associated mitochondrial dysfunction (Sawada and Carlson, 1987, Sohal et
al., 1994), and/or reduced expression and activity of endogenous free radical scavengers
(Rao et al., 1990, O'Donnell et al., 2000).
The age-associated decline in cognitive function is accompanied by a significant
age-associated increase in brain oxidative stress in mammals, suggesting that oxidative
stress might be the mechanism underlying age-dependent cognitive impairment. This
relationship between age-associated cognitive impairment and oxidative stress is further
strengthened by the fact that exogenous free radical scavengers such as
superoxide/dismutase catalase mimetics, melatonin, estrogen, and alpha-lipoic acid
significantly reduce age-dependent decline in cognitive function (Stoll et al., 1994,
Raghavendra and Kulkarni, 2001, Liu et al., 2003, Kiray et al., 2004). Increased
oxidative stress might disrupt cognitive function by interfering with the cellular
machinery responsible for maintaining neuronal homeostasis.
Autophagy is a lysosomal degradation pathway that appears to be critically
involved in cell survival, cell differentiation, development, and cellular homeostasis
(Rajawat et al., 2009). There are three types of autophagic pathways, which include:
46
macroautophagy, microautophagy, and chaperone-mediated autophagy (Levine and
Kroemer, 2008). Macroautophagy is primarily responsible for degrading damaged or
misfolded proteins, defective or unwanted organelles, protein aggregates, intracellular
pathogens, and other cellular debris (Levine and Kroemer, 2008). Macroautophagy
involves the formation of an isolation membrane by vesicle nucleation (Arstila and
Trump, 1968). The isolation membrane elongates and the ends of this membrane
ultimately fuse together around the cellular material to form the autophagosome (Arstila
and Trump, 1968). The autophagosome then fuses with a lysosome where the material it
engulfed is degraded by cysteine proteases and other hydrolases (Arstila and Trump,
1968). Most of the time autophagy is occurring at low basal levels within cells and
serves as a degradation pathway for maintaining normal cellular homeostasis (Levine and
Kroemer, 2008). However, during periods of cellular stress, such as starvation, autophagy
is significantly increased in order to degrade cellular material and generate free amino
and fatty acids that can be used to produce new proteins and ATP for the cell (Levine and
Kroemer, 2008).
Like oxidative stress, the autophagy pathway also undergoes significant age-
associated changes in mammalian brain. These changes include increased
autophagosome formation, and elevated expression and activity of lysosomal cysteine
proteases (Nakanishi et al., 1997, Gamerdinger et al., 2009, Nakanishi and Wu, 2009).
Thus, age-associated changes in brain autophagy might be linked to age-dependent
alterations in oxidative stress and cognitive impairment. This relationship is further
suggested by the fact that aging is accompanied by a significant increase in oxidation-
induced damage to proteins and other macromolecules and autophagy is the primary
47
degradation pathway for defective proteins and organelles. In addition, autophagy
appears to be critical in the selective removal of dysfunctional mitochondria (Kim et al.,
2007), and a significant increase in defective mitochondria producing high levels of free
radicals does accompany the aging process (Sawada and Carlson, 1987, Sohal et al.,
1994). Autophagy also plays a prominent role in maintaining normal neuron function.
This is evident by the fact that the disruption of autophagy in mice results in significant
neurodegeneration (Komatsu et al., 2005, Hara et al., 2006, Komatsu et al., 2006), and
altered brain autophagy is prevalent in age-associated neurodegenerative diseases, such
as Alzheimer’s and Parkinson’s diseases (Anglade et al., 1997, Nixon et al., 2005).
Microglia function as the immune response cells of the central nervous system
(Miller and Streit, 2007). They express antigens and receptors that are also present in
other macrophages, such as MHC class II and I antigens (Perry and Gordon, 1987,
Morioka et al., 1992), complement receptors (Graeber et al., 1988, Johnson et al., 1994),
chemokine receptors (Maciejewski-Lenoir et al., 1999), and cytokine receptors (Prinz and
Hanisch, 1999, Streit et al., 2000). Microglia can also secrete cytokines such as IL- β and
TNF α and function as antigen-presenting cells (Frei et al., 1987, Hetier et al., 1988,
Hickey and Kimura, 1988). In addition to serving as immunocompetent cells, they are
also responsible for repairing tissues and healing wounds following injury in the central
nervous system (Streit et al., 2005). Furthermore, microglia assist in maintaining
neuronal function by providing trophic support, secreting extracellular matrix molecules,
and phagocytizing macromolecules and other cellular debris (Chamak et al., 1994,
Rabchevsky and Streit, 1997, Nakajima K., 2002, Streit et al., 2005). The role microglia
play in neuronal homeostasis is made even clearer by the fact that neurodegenerative
48
diseases are also accompanied by significant changes in microglia morphology and
activation (McGeer et al., 1987, Lassmann et al., 1995, v Eitzen et al., 1998, Yang et al.,
1998, Streit, 2004, Streit et al., 2004, Wierzba-Bobrowicz et al., 2004).
In addition to oxidative stress and autophagy, microglia also undergo age-
dependent changes. Microglia in aged brain exhibit morphological modifications such as
the loss of fine processes, shortened twisted branches, cytoplasmic fragmentation, and
spheroid formation (Streit et al., 2004). Furthermore, microglia might have reduced
mitotic activity because their telomere length declines with age (Prowse and Greider,
1995, Coviello-McLaughlin and Prowse, 1997). Microglia density and number of
activated microglia have also been repeatedly reported to change with age (Rogers et al.,
1988, Perry et al., 1993, Ogura et al., 1994, Streit and Sparks, 1997, Sheffield and
Berman, 1998, Kullberg et al., 2001, Ma et al., 2003), as well as their expression of
certain antigens (Perry, 1993) and cytokines (Lukiw, 2004, Streit et al., 2004).
Since significant changes in oxidative stress, autophagy, and microglia all
accompany the aging process one might hypothesize that these age-dependent changes
might be related. The goal of the present study was to examine the relationship between
age-dependent modifications in oxidative stress, autophagy, and microglia. This was
carried out by chronically treating aged mice for six months with the SOD/catalase
mimetics EUK-189 and EUK-207. In chapter 2 these two compounds were shown to
significantly attenuate age-associated brain oxidative stress and cognitive impairment,
and it was therefore of interest to analyze autophagy and microglia function in the brains
of mice chronically treated with these compounds. The results indicate that age-
associated changes in both autophagy and microglia function were present at 23 months
49
of age and that these changes are related to brain oxidative stress because treatment with
either EUK-189 or EUK-207 markedly reduced these age-dependent modifications.
Thus, age-associated changes in autophagy might be dependent on alterations in
lysosomal function possibly resulting from oxidative stress, and microglia-mediated
phagocytosis might be hindered by oxidative stress in aged brains.
3.2. Materials and methods
3.2.1. Materials
EUK-189 and EUK-207 were synthesized as described previously (Doctrow et al.,
2002, Malfroy-Camine; Bernard (Arlington, 2003)All other chemicals were purchased
from Sigma, unless indicated otherwise.
3.2.2. Mice and treatments
Animals were treated in accordance with the principles and procedures of the
National Institutes of Health Guide for the Care and Use of Laboratory Animals; all
protocols were approved by the Institutional Animal Care and Use Committee of the
University of Southern California. I used 85 C57BL/6N Sim male mice obtained from the
National Institute on Aging. Before experiments, mice were housed 4–5 per cage and
placed in the same room with a 12-h light/12-h dark cycle and behavioral testing was
performed during the last 6 h of the light cycle. Mice were allowed free access to food
and water, and their weights ranged from 27 to 36 g. Before surgery, mice were randomly
assigned to five of the following groups (17 mice per group): vehicle control and EUK-
treated groups (1.5 or 0.15 mM EUK-189 or EUK-207). An additional group of 10 male
mice were used for the untreated 16-month control group.
50
Before implantation, Alzet 2004 miniosmotic pumps (Durect Corporation,
Cupertino, CA) were loaded with either EUK-189 or EUK-207 at 1.5 or 0.15 mM in 5 %
mannitol, or 5 % mannitol alone (as vehicle control group) and then primed for at least 40
hours in 5 % mannitol at 37 ºC. The minipumps were then implanted s.c. in the 17-
month-old mice according to the manufacturer’s recommendations. Briefly, mice were
anesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg) by i.p. injection. A small
1-cm incision was then made to the hip area of the mice and a small pocket was formed
by spreading the s.c. connective tissues apart. The pump was placed into the prepared
pocket, and the wound was then closed with sutures.
Pumps delivered the drugs at 0.25 µl/hour for a 28-day period, and the calculated
drug infusion rates were ≈9 nmol/day for the 1.5 mM doses of EUK-compounds and ≈0.9
nmol/day for the 0.15 mM doses of EUK compounds. The lower concentration is
equivalent to a dose of 15 and 16 µg/kg/day for EUK-189 and EUK-207, respectively
(assuming a 30 g mouse). This is a much lower dose than has ever been tested for
efficacy in previous studies. Control mice were implanted with minipumps filled with
vehicle alone (5% mannitol).
During the 6-month treatment, pumps were replaced 5 more times with new ones
at the original sites at the end of each 28-day period of implantation. In some of the mice,
the new pumps had to be placed on the opposite hip area during the 6-month treatment
due to skin damage at the original site of implantation.

51
3.2.3. Analysis of autophagy-lysosome system and microglia
function by Western blot
In order to assess autophagy, lysosome, and microglia status, mice were
anesthetized with isoflurane and killed by decapitation. Brains were rapidly extracted,
placed on a chilled platform, and the cerebellum and pons were removed and discarded.
The brains were then cut in half sagitally, and each half was immediately frozen on dry
ice, and stored at −70 ºC until homogenized. Protein markers for autophagy, lysosomes,
and microglia were measured in brain homogenates from 23-month-old vehicle controls,
EUK-189- or EUK-207-treated C57 mice, and 16-month-old untreated control mice via
Western blot.
One sagital half from each brain was homogenized in 2.5 % SDS supplemented
with 0.5 mM phenylmethylsulphonyl fluoride (PMSF) and 1:100 protease inhibitor
cocktail. Homogenates were briefly sonicated on ice in order to shear DNA, and protein
concentrations for each homogenate were determined using the bicinchoninic acid (BCA)
method (Pierce, Rockford, IL). 25 or 30 µg of protein (depending on protein of interest)
were loaded onto 10 % or 15 % Tris – glycine gels, separated by SDS-PAGE, and then
transferred onto 0.45 µm or 0.2 µm polyvinylidene difluoride (PVDF) membranes
(Millipore, Billerica, MA). The PVDF membranes were blocked for 1 h at room
temperature with 5 % non-fat milk in Tris – buffered saline, pH 7.5, containing 0.05 %
Tween – 20 and then probed with the following primary antibodies: LC3 (1:500 dilution,
Abgent, San Diego, CA), CD11b (1:1,000 dilution, AbD Serotec, Raleigh, NC), APG7
(1:1,000 dilution, Anaspec, Fremont, CA), LAMP1 (1:1,000 dilution, Anaspec, Fremont,
52
CA), LAMP2 (1:1,000 dilution, Anaspec, Fremont, CA), or Actin (1:10,000 dilution,
Millipore, Billerica, MA) overnight at 4 ºC. PVDF membranes were then incubated with
an HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West
Grove, PA) for 1 h at room temperature and then developed using Amersham ECL Plus
Western Blotting Detection Reagents (GE Healthcare Bio-Sciences Corp., Piscataway,
NJ).
Photographs of each immunoblot were scanned, imported, and converted to gray
scale images using AdobePhotoshop CS1 (Adobe Systems Incorporated, San Jose, CA).
Immunoblots were then quantified by densitometry using Image J Software (NIH,
Bethesda, MD), and the results were expressed as percentage of the corresponding values
assayed in the 16-month-old control mice.
3.2.4. Analysis of autophagy-lysosome system and microglia
function by immunohistochemistry.
3 – 4 mice from the 23-month-old vehicle control group and the EUK-189- or
EUK-207-treated groups were randomly selected for immunohistochemistry in order to
visualize protein markers for autophagy, lysosomes, and microglia. The selected animals
were anesthetized and fixed by transcardial perfusion with 4 % paraformaldehye in 0.1 M
Phosphate – buffered saline, pH 7.4. The brains were removed, cryoprotected with
sucrose, and then sectioned at 25 µm with a Leica SM 2400 Sliding Microtome (Leica
Microsystems Inc., Bannockburn, IL). Free-floating sections were blocked for 1 h at
room temperature in 3 % goat serum or 10 % horse serum diluted in Tris – buffered
saline, pH 7.4, and then probed with the following primary antibodies: Beclin-1 (1:500
53
dilution, Santa Cruz Biotechnology, Santa Cruz, CA), CD11b (1:500 dilution, AbD
Serotec, Raleigh, NC), LAMP1 (1:1,000 dilution, Abcam, San Francisco, CA), LC3
(1:2,000 dilution, Abgent, San Diego, CA), or Cathepsin D (1:200 dilution, Oncogene,
Cambridge, MA) overnight at 4 ºC. The sections were then incubated with secondary
antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 594 (1:1,500 dilution,
Invitrogen, Carlsbad, CA) for two h at room temperature. Sections were mounted on
gelatin-coated slides, air-dried, and coverslipped with VECTASHIELD mounting
medium (Vector Laboratories, Inc., Burlingame, CA). The perimeter of each slide was
sealed with nail polish prior to imaging. Stained sections were visualized at 50 X using a
Zeiss AxioImager.Z1 Upright Microscope (Carl Zeiss MicroImaging, Inc., Thornwood,
NY) with a mercury lamp. Images were captured digitally with a Hammatsu ORCA-ER
Digital Camera (Hammatsu Photonics k.k., Hammatsu City, Japan) and AxioVision
Version 4.8.1 software (Carl Zeiss MicroImaging, Inc., Thornwood, NY).
3.2.5. Statistical analysis
All statistics were performed using GraphPad Prism 4.03 software (GraphPad
Software, La Jolla, CA). One-way ANOVA was used to test if the means of each
experimental group were significantly different and if the overall p value was <0.05, then
multiple comparisons between the experimental groups were tested using Newman–
Keuls post hoc analysis.

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3.3. Results
3.3.1. Effects of SOD/catalase mimetics on brain autophagy
C57 BL/6N mice were treated continuously with EUK-189 or EUK-207
administered through subcutaneously implanted osmotic minipumps starting at 16
months of age. After 6 months of continuous treatment, mice were sacrificed and their
brains were harvested and homogenized for western blot analysis or fixed by transcardial
perfusion for immunohistochemistry. Brain homogenates and sections were then used to
assess the expression of proteins involved in autophagy induction and autophagosome
formation. For comparison, and to evaluate any age-associated changes in brain
autophagy activity, autophagy markers were also analyzed in brain homogenates from
16-month-old mice.
Autophagy is carried out by the coordination of a number of different proteins.
These proteins are encoded by genes that are highly conserved from yeast to mammals
(Mizushima and Klionsky, 2007). The majority of the proteins that participate in
autophagy interact with one another to form protein complexes that are responsible for
responding to upstream signals, vesicle nucleation, vesicle elongation, autophagosome
completion, and recycling of autophagic proteins (Ohsumi, 2001). ATG7 is similar to E1
Ubiquitin-ligase enzymes (McCray and Taylor, 2008), and appears to be critical for
autophagy induction. This is evident by the fact that mice with a neuron specific deletion
of the Atg7 gene posses neurons with ubiquitinated protein aggregates and cytoplasmic
inclusion bodies (Komatsu et al., 2006). Western blots of mouse brain homogenates
revealed a decline in ATG7 levels between 16 months and 23 months of age that was
55
reversed by chronic treatment with either EUK-189 or EUK-207 (Fig. 7A). However,
quantification of the blots determined that this age-associated decline in ATG7
expression was not statistically significant and only chronic treatment with 0.15 mM
EUK-207 produced a significant increase in brain ATG7 levels at 23 months of age (Fig.
7B). Beclin-1 (ATG6) associates with other proteins to form the PI(3) kinase class III
lipid kinase complex, which is required for the vesicle nucleation step of autophagy
(Levine et al., 2008). In addition, muscle tissues from mice with only one functional
copy of the Beclin-1 gene exhibit a significant reduction in autophagy during starvation
(Qu et al., 2003). Beclin-1 expression in CA1 region of hippocampus was higher in 23-
month-old control mice (Fig. 7C). Beclin-1 expression in 23-month-old control mice and
mice treated with EUK-189 or EUK-207 was primarily localized in the cell bodies of
neurons in CA1 (Fig. 7C).

Figure 7. Effects of chronic treatment with EUK-189 or EUK-207 on ATG7 levels in brain
homogenates and Beclin-1 expression in field CA1 of hippocampus. At the end of the 6 month
treatment, mice were decapitated and their brains (minus cerebellum and pons) were removed and
homogenized or fixed by transcardial perfusion using 4 % paraformaldehyde. ATG7 expression in the brain
homogenates was evaluated by western blot (A) and quantified by densitometry (B). APG7 was also
assessed in brain homogenates from 16 month-old control mice (A & B). ATG7 levels were divided by
actin and expressed as % of 16-month-old control (B). Shown are means ± SEM of 7-8 mice (B). One-way
ANOVA indicated that the effect of age was not significant, but the effect of 0.15 mM EUK-207 was
(*p<0.05 vs.vehicle control) (B). Beclin-1 expression was evaluated in 25 µm brain sections from 23-
month-old mice chronically treated with EUK-189 or EUK-207 for six months by immunohistochemistry
(C). Chronic treatment with 0.15 mM EUK-189 decreased the expression of beclin-1 in CA1 region of
hippocampus as compared to 23-month-old control mice (C). Similar results were observed in 3 mice from
each of the treated groups.
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57
When autophagy is triggered in mammals the protein LC3 – I is cleaved
generating LC3 – II (Kabeya et al., 2000). LC3 – II is then conjugated to lipid
phosphotidylethanolamine and inserted into the autophagophore (Kabeya et al., 2000). In
mammals, LC3 – II is the only known protein that remains associated with the mature
autophagosome, thus autophagic activity and autophagosome formation can be
determined by assessing the ratio of LC3 – II to LC3 – I (Klionsky et al., 2007). Western
blots of mouse brain homogenates indicated that the ratio of LC3 – II to LC3 – I
increased from 16 months to 23 months of age (Fig. 8A), thus suggesting a potential age-
associated increase in autophagic activity or autophagosome formation. This age-
dependent increase in LC3-II to LC3-I levels was reversed by chronic treatment with
EUK-189 or EUK-207 (Fig. 8A). Quantification of the blots determined that the age-
associated increase in the ratio of LC3 – II to LC3 – I was significant, as well as the
effects of both EUK-189 and EUK-207 on LC3 – II to LC3 – I levels (Fig. 8B). Both
lower doses of EUK-189 and EUK-207 had a more significant effect on LC3 – II to LC3
– I ratio than the higher doses (Fig. 8B). The expression and localization of total LC3
(LC3 – I and LC3 – II) protein within CA1 region of hippocampus was similar between
23-month-old mice administered vehicle and administered EUK-189 or EUK-207 (Fig.
8C).

58
Figure 8. Effects of chronic treatment with EUK-189 or EUK-207 on LC3 – II to LC3 – I ratio in
brain homogenates and total LC3 expression in CA1 of the hippocampus. At the end of the 6 month
treatment, mice were decapitated and their brains (minus cerebellum and pons) were removed and
homogenized or fixed by transcardial perfusion using 4 % paraformaldehyde. LC3 – I and LC3 – II
expression in brain homogenates was evaluated by western blot (A), quantified by densitometry, and the
ratio of LC3 – II to LC3 – I was determined (B). LC3 – II to LC3 – I ratio was also determined in brain
homogenates from 16 month-old control mice (A & B). Levels of LC3 – II to LC3 – I were expressed as %
of 16-month-old control (B). Shown are means ± SEM of 7-8 mice (B). One-way ANOVA indicated that
the effect of age was significant (*p <0.01 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-
207 (**p <0.05 vs. vehicle control, ***p <0.01 vs. vehicle control). (B). Total LC3 protein (LC3 – I and
LC3 – II) expression was evaluated in 25 µm brain sections from 23-month-old mice chronically treated
with EUK-189 or EUK-207 for six months by immunohistochemistry (C). Chronic treatment with 0.15 mM
EUK-189 had no effect on the expression of total LC3 protein in the CA1 region of the hippocampus
compared to 23 month control mice (C). Similar results were observed in 3 mice from each of the treated
groups.
59
3.3.2. Effects of SOD/catalase mimetics on brain lysosome
function

Auotphagy relies on lysosomes to carry out the degradation of cellular material
(Levine and Kroemer, 2008). During autophagy a vesicle expands and engulfs cytosolic
components targeted for degradation and then fuses together to form the autophagosome
(Arstila and Trump, 1968). The autophagosome must then fuse with a lysosome and
release the material into the lumen of the lysosome where the cytosolic components are
degraded by cysteine proteases (Levine and Kroemer, 2008).
In order to assess the effects of EUK-189 or EUK-207 on age-related changes in
lysosome function, mice treated as described above were sacrificed at the end of the 6
month long treatment and their brains were harvested and homogenized for western blot
analysis or fixed by transcardial perfusion for immunohistochemistry. Brain
homogenates and sections were then used to assess the expression of lysosome proteins.
For comparison, and to evaluate any age-associated changes in brain lysosome function
markers for lysosomes were also analyzed in brain homogenates from 16-month-old
mice.
LAMP1 and LAMP2 are lysosome-associated membrane proteins that account for
about half of all the proteins found in the lysosomal membrane (Hunziker et al., 1996).
LAMP1 does not appear to be critical for autophagy or lysosome function, as evidenced
by the fact that LAMP1-deficient mice are indistinguishable from wild – type mice and
exhibit lysosomes with normal characteristics (Andrejewski et al., 1999). Western blots
of mouse brain homogenates revealed a decline in LAMP1 expression between 16
months and 23 months of age that was reversed by chronic treatment with either EUK-
60
189 or EUK-207 (Fig. 9A). Quantification of the blots did determine that this age-
associated decline in LAMP1 expression was significant, but only chronic treatment with
0.15 mM EUK-207 produced a significant increase in brain LAMP1 levels at 23 months
of age (Fig. 9B). However, chronic treatment with EUK-189 or EUK-207 decreased the
expression of LAMP1 specifically in the cell bodies of neurons in CA1 region of
hippocampus at 23 months of age (Fig. 9C).

Figure 9. Effects of chronic treatment with EUK-189 or EUK-207 on LAMP1 expression in brain
homogenates and in CA1 of the hippocampus. At the end of the 6 month treatment, mice were
decapitated and their brains (minus cerebellum and pons) were removed and homogenized or fixed by
transcardial perfusion using 4 % paraformaldehyde. LAMP1 expression in the brain homogenates was
evaluated by western blot (A) and quantified by densitometry (B). LAMP1 was also determined in brain
homogenates from 16 month-old control mice (A & B). LAMP1 levels were divided by actin and
expressed as % of 16-month-old control (B). Shown are means ± SEM of 8 mice (B). One-way ANOVA
indicated that the effect of age was significant (*p <0.01 vs. 16 month-old mice), as was the effect of 0.15
mM EUK-207 (**p <0.01 vs. vehicle control) (B). LAMP1 protein expression was evaluated in 25 µm
brain sections from 23-month-old mice chronically treated with EUK-189 or EUK-207 for six months by
immunohistochemistry (C). Chronic treatment with 0.15 mM EUK-189 decreased the expression of
LAMP1 in the CA1 region of the hippocampus compared to 23-month-old control mice (C). Similar results
were observed in 3 mice from each of the treated groups.
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62
Cells from mice lacking a functional copy of the gene that encodes for LAMP2
exhibit a significant build up of autophagosomes (Tanaka et al., 2000), thus LAMP2
might be critically involved in the fusion of autophagic vacuoles to lysosomes. Western
blots of mouse brain homogenates revealed a decline in LAMP2 expression between 16
months and 23 months of age that was reversed by chronic treatment with either EUK-
189 or EUK-207 (Fig. 10A). Quantification of the blots determined that this age-
associated decline in LAMP2 expression was significant, and that chronic treatment with
EUK-189 or EUK-207 produced a significant increase in brain LAMP2 levels at 23
months of age (Fig. 10B). However, EUK-189 at a concentration of 1.5 mM did not have
a significant effect on brain LAMP2 levels when compared to the 23 month control mice
(Fig. 10B) and the increase in LAMP2 levels at 23 months of age was more significant in
mice treated with EUK-207 at both 0.15 mM and 1.5 mM concentrations (Fig. 10B).

Figure 10. Effects of chronic treatment with EUK-189 or EUK-207 on LAMP2 expression in brain
homogenates. At the end of the 6 month treatment, mice were decapitated and their brains (minus
cerebellum and pons) were removed and homogenized. LAMP2 expression in the brain homogenates was
evaluated by western blot (A) and quantified by densitometry (B). LAMP2 was also determined in brain
homogenates from 16 month-old control mice (A & B). LAMP2 levels were divided by actin and
expressed as % of 16-month-old control (B). Shown are means ± SEM of 8 mice (B). One-way ANOVA
indicated that the effect of age was significant (*p <0.05 vs. 16 month-old mice), as was the effect of 0.15
mM EUK-189, 0.15 mM EUK-207 and 1.5 mM EUK-207 (**p <0.05 vs. vehicle control, ***p <0.01 vs.
vehicle control) (B).

Cathepsin D is a lysosomal cysteine protease that also appears to play an
important role in lysosome function and autophagosome clearance. Mice deficient in
cathepsin D exhibit disrupted lysosome storage, neurodegeneration, and accumulation of
autophagosomes (Koike et al., 2000, Shacka et al., 2007). Chronic treatment with EUK-
189 or EUK-207 increased the expression of cathepsin D at 23 months of age in the cell
bodies of neurons in CA1 region of hippocampus (Fig. 12), but EUK-189 or EUK-207
did not change cathepsin D levels or the number of cathepsin D-positive cells in the
dentate gyrus, lateral geniculate nucleus, and substantia nigra (Fig. 12).
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3.3.3. Effects of SOD/catalase mimetics on microglia status
In addition to serving as the brain’s immune system (Streit, 2002), microglia also
protect and support neurons by releasing neurotrophic factors and cytokines (Streit et al.,
1999) and phagocytosing macromolecules and other cellular debris (Streit et al., 2005).
CD11b or complement receptor 3 (CR3) appears to mediate phagocytosis in microglia
(Fallman et al., 1993, Reichert and Rotshenker, 2003, Choucair-Jaafar et al., 2010) and is
routinely used as a microglia marker. Western blots of mouse brain homogenates
indicated a decline in CD11b expression between 16 months and 23 months of age that
was reversed by chronic treatment with either EUK-189 or EUK-207 (Fig. 11A).
Quantification of the blots showed that this age-associated decline in CD11b expression
was significant, but only chronic treatment with 0.15 mM EUK-207 produced a
significant increase in brain CD11b levels at 23 months of age (Fig. 11B). However,
chronic treatment with EUK-189 or EUK-207 increased the expression of CD11b and the
number of CD11b-positive microglia in stratum oriens and stratum radiatum in CA1
region of the hippocampus at 23 months of age (Fig. 11C). In addition to hippocampus,
chronic treatment with EUK-189 or EUK-207 increased the expression of CD11b and the
number of CD11b-positive microglia at 23 months of age in the dentate gyrus, and lateral
geniculate nucleus (Fig. 12). However, CD11b immunoreactivity in the substantia nigra
at 23 months of age was not affected by EUK-189 or EUK-207 (Fig. 12).

65

Figure 11. Effects of chronic treatment with EUK-189 or EUK-207 on CD11b expression in brain
homogenates and in CA1 of the hippocampus. At the end of the 6 month treatment, mice were
decapitated and their brains (minus cerebellum and pons) were removed and homogenized or fixed by
transcardial perfusion using 4 % paraformaldehyde. CD11b expression in the brain homogenates was
evaluated by western blot (A) and quantified by densitometry (B). CD11b was also determined in brain
homogenates from 16 month-old control mice (A & B). CD11b levels were divided by actin and expressed
as % of 16-month-old control (B). Shown are means ± SEM of 8 mice (B). One-way ANOVA indicated
that the effect of age was significant (*p <0.05 vs. 16 month-old mice), as was the effect of 0.15 mM EUK-
207 (**p <0.001 vs. vehicle control) (B). CD11b expression was evaluated in 25 µm brain sections from
23-month-old mice chronically treated with EUK-189 or EUK-207 for six months by
immunohistochemistry (C). Chronic treatment with 0.15 mM EUK-189 increased the expression of CD11b
and CD11b positive microglia in the CA1 region of the hippocampus compared to 23-month-old control
mice (C). Similar results were observed in 3 mice from each of the treated groups.

Figure 12. Effects of chronic treatment with EUK-189 or EUK-207 on CD11b and Cathepsin D
immunoreactivity in various brain regions. At the end of the 6 month treatment, mice were fixed by
transcardial perfusion using 4 % paraformaldehyde and brains were sectioned at 25 µm. Sections were
labeled for CD11b (green) and Cathepsin D (red) by immunohistochemistry. Chronic treatment with 0.15
mM EUK-189 and 0.15 mM EUK-207 increased the expression of CD11b and CD11b positive microglia in
the CA1 region of the hippocampus, dentate gyrus, and lateral geniculate nucleus compared to 23-month-
old control mice. EUK-189 and EUK-207 treatment only increased cathepsin D expression in the CA1
region of the hippocampus compared to 23-month-old control mice. Abbreviations are as follows: Dentate
gyrus (DG), Lateral geniculate nucleus (LGN), and Substantia nigra (SN). Similar results were observed in
3 mice from each of the treated groups.
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3.4. Discussion
Increased oxidative stress is a prominent feature of the aged mammalian brain.
However, there are a number of other different changes that occur with brain aging. Two
significant features in the aged brain are modifications in autophagy and microglia. Both
autophagy and microglia play an important role in maintaining neuronal function and
thus age-associated alterations in either autophagy or microglia might impinge on
cognition. Since brain oxidative stress also accompanies the aging process, there might
be a significant link between oxidative stress and age-associated changes in autophagy
and microglia. This study was designed to find potential links between age-associated
oxidative stress, autophagy, and microglia. This was accomplished by chronically
treating aged mice with EUK-189 or EUK-207, which are two SOD/catalase mimetics
that have previously been shown to protect against age-associated oxidative stress and
cognitive impairment. After the treatment was over, autophagy and microglia status were
evaluated in the brains of these mice.
A significant increase in brain autophagy activity, as assessed by an increase in
the ratio of LC3 II to LC3 I, was observed between 16 months and 23 months of age.
This observation is in agreement with a previous study done in aged rats (Gamerdinger et
al., 2009). In addition, this age-associated increase in autophagic activity was blocked by
chronic treatment with either EUK-189 or EUK-207, thus suggesting a potential
relationship between age-associated oxidative stress and autophagy. Such a relationship
appears reasonable because autophagy is responsible for degrading damaged organelles,
proteins, lipids, and other macromolecules and oxidative stress can induce such damage.
Thus, as brain ages and becomes bombarded with more oxidized cellular material,
68
autophagic activity must increase in order to repair the oxidative damage. Autophagy
might be a potential mechanism in the brain that can be used to cope with increased
oxidative stress.
An age-dependent increase in the ratio of LC3 II to LC3 I could also be more
specifically interpreted as a significant increase in the number of autophagosomes within
the cell because LC3 II is the only known protein that remains associated with the mature
autophagosome (Klionsky et al., 2007). Autophagosmes are not commonly found in
neurons of healthy adult brain, but brains from patients with neurodegenerative diseases
such as Alzheimer’s, Parkinson’s, and Huntingtons’s disease contain neurons that exhibit
significant accumulation of autophagosomes (Anglade et al., 1997, Petersen et al., 2001a,
Nixon et al., 2005). Thus, increased levels of autophagosomes in brain of 23 month old
mice might suggest a degenerative state and impaired neuronal function similar to that
observed in neurodegenerative diseases. LC3 protein expression within the CA1 region
of hippocampus was similar between 23 month old mice treated with vehicle or EUK-207
and EUK-189, but this just suggests that total LC3 protein expression was similar in
treated and untreated mice because the antibody cannot distinguish between LC3 I and
LC3 II. The only way to truly tell if there is an actual increase in autophagosome number
would be to visualize autophagosomes under high magnification using electron
microscopy, but these brain sections were not processed for such a technique.
Beclin – 1 expression in CA1 region of hippocampus at 23 months of age was
much higher in mice not treated with EUK-189 or EUK-207. Beclin-1 is required for the
vesicle nucleation step of autophagy (Levine et al., 2008) and mice with only one
functional copy of the Beclin-1 gene display a significant reduction in autophagy during
69
starvation (Qu et al., 2003). Furthermore, cells obtained from patients with the lipid
storage disorder Niemann – Pick Type C disease contain significantly high levels of
beclin – 1 and exhibit increased autophagy (Pacheco et al., 2007). Thus, increased Beclin
– 1 immunoreactivity might also suggest an increase in autophagic activity at 23 months
of age. This observation also reflects the age-dependent increase in autophagic activity
determined by measuring LC3 II to LC3 I levels. Also, like with LC3 II to LC3 I ratio,
chronic treatment with either EUK-189 or EUK-207 markedly reduced the expression of
Beclin-1 thus strengthening the relationship between oxidative stress and autophagy.
However, ATG7 expression, which is also important for autophagic induction (Komatsu
et al., 2006), appeared to decline with age. This observation is somewhat contradictory to
the other results, but this age-associated decline in ATG7 was not significant and only a
0.15 mM concentration of EUK-207 produced a significant increase in ATG7 expression
at 23 months of age. In addition, the level of ATG7 expressed might not be directly
correlated to autophagic activity. ATG7 expression was also not assessed in specific
brain regions, and therefore significant age-associated changes in ATG7 level might still
exist in particular parts of the brain.
As mentioned previously, the autophagosome must fuse with a lysosome in order
for the contents of the autophagosome to be degraded. Therefore, age-associated changes
in lysosome function might also be related to oxidative stress. In brain homogenates, the
expression of LAMP1 and LAMP2 both significantly declined between 16 months and 23
months of age and this age-dependent decrease in LAMP1 and LAMP2 might be due to
oxidative stress because chronic treatment with EUK-189 or EUK-207 significantly
increased LAMP2 expression at 23 months of age and EUK-207 had the same effect on
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LAMP1 levels. Although both LAMP1 and LAMP2 expression significantly decreased
with age, they differ in their importance to autophagy and lysosomes. LAMP1 does not
appear to be critical for autophagy or lysosome function, as evidenced by the fact that
LAMP1 deficient mice are indistinguishable from wild – type mice and contain normal
functioning lysosomes (Andrejewski et al., 1999). However, cells from LAMP2 deficient
mice exhibit a significant build up of autophagosomes (Tanaka et al., 2000), thus unlike
LAMP1, LAMP2 appears to be critically involved in the fusion of autophagic vacuoles to
lysosomes. Therefore, oxidative stress might hinder the clearance of autophagosomes by
somehow disrupting LAMP2 levels since the SOD/catalase mimetics EUK-189 and
EUK-207 protected against this age-associated decline in LAMP2 expression. The
relationship between oxidative stress and possible age-associated lysosome dysfunction
was further enhanced by the fact that cathepsin D expression within the CA1 region of
hippocampus was also increased by chronic treatment with EUK-189 or EUK-207. Like
cells in LAMP2 deficient mice, cells from cathepsin D knock-out mice also exhibit a
massive accumulation of autophagosomes (Koike et al., 2000, Shacka et al., 2007), thus
cathepsin D might play a specific role in the turnover of autophagosomes.
Interestingly, LAMP1 levels significantly declined with age and EUK-207
provided significant protection against this age-associated decrease in expression;
however immunohistochemistry revealed that LAMP1 expression specifically increased
in the CA1 region of the hippocampus in mice treated with EUK-189 or EUK-207. This
difference could possibly be explained by the fact that the western blots assessed LAMP1
levels in the total forebrain, whereas immunohistochemistry was done in specific brain
regions. Increased LAMP1 expression in CA1 region of hippocampus might still agree
71
with the other results suggesting a relationship between oxidative stress and LAMP2 and
Cathepsin D expression. Increased LAMP1 expression in hippocampus could be
interpreted as an increase in the number of lysosomes resulting from a decline in the
ability of lysosomes to fuse with autophagosomes and degrade cellular material. Thus,
lysosomes would remain in the cytosol because they are not being utilized. This idea is
further supported by the following observations: cells from cathepsin D deficient mice
also accumulate lysosomes (Koike et al., 2000, Shacka et al., 2007), and cathepsin D
expression was also reduced in hippocampus of 23 month old mice not treated with EUK-
189 or EUK-207.
In light of these results, there appears to be an age-associated increase in
autophagic activity or autophagosomes within the brain that is possibly dependent on
oxidative stress. This age-associated increase in autophagic activity could be a
neuroprotective mechanism used to combat the increase in oxidative damage that
accompanies the aging process. However, accumulation of autophagosomes in aged
brain could also be due to impaired autophagy resulting from dysfunctional lysosomes.
In addition, age-associated modifications in lysosome function appear to be brought on
by oxidative stress. As disrupting autophagy in mouse CNS results in significant
neurodegeneration (Hara et al., 2006, Komatsu et al., 2006), it is possible that age-
associated impairment in autophagy could have severe effects on cognitive function.
In humans, long term use of anti-inflammatory drugs significantly reduces the risk
for Alzheimer’s and Parkinson’s disease (McGeer et al., 1996, Chen et al., 2005, Vlad et
al., 2008), and since microglia represent the brain inflammatory system, they have
recently received a lot of attention with regards to neurodegeneration. Many studies have
72
reported a significant increase in activated microglia in aged and Alzheimer’s diseased
brains (Vaughan and Peters, 1974, Peters et al., 1991, Perry et al., 1993, DiPatre and
Gelman, 1997). In addition, the expression of genes that encode for pro-inflammatory
proteins are up-regulated in aged brains (Colangelo et al., 2002). Activated microglia can
release a number of cytotoxic factors including reactive nitrogen and oxygen species
(Paris et al., 1999, Badie et al., 2000, Liu et al., 2001, He et al., 2002, Ryu et al., 2002).
Thus, many researchers believe that age-associated cognitive impairment is the result of
neurodegeneration produced by chronic brain inflammation. However, there is no
correlation between the localization of activated microglia and the localization of
neurodegeneration and it is very difficult to distinguish between activated and resting
microglia in the human brain (Conde and Streit, 2006). In addition, some researchers
believe that the morphology of microglia observed in aged Alzheimer’s diseased brains is
not that of an active state, but that of a degenerative state (Streit, 2004, Streit et al., 2004).
In the present study there was a significant increase in microglia number in
hippocampus and other brain regions of mice chronically treated with EUK-189 or EUK-
207; thus age-associated oxidative stress might play an important role in microglia status.
Besides playing a role in inflammation, microglia also assist in maintaining neuronal
health by providing trophic support, secreting extracellular matrix molecules, and
phagocytizing macromolecules and other cellular debris (Chamak et al., 1994,
Rabchevsky and Streit, 1997, Nakajima K., 2002). Therefore increased brain microglia
might stimulate neuronal function. It has been suggested that microglia might undergo
replicative senescence during aging because their telomere length declines with age in the
rat brain (Prowse and Greider, 1995, Coviello-McLaughlin and Prowse, 1997). Thus,
73
oxidative stress might directly target the mitotic capacity of microglia, because chronic
treatment with SOD/catalase mimetics increased microglia number in hippocampus.
Another study in mice also observed an age-associated decline in microglia number in
cortex, striatum, medial and lateral corpus cullosum, and thalamus; however Isolectin B4
instead of CD11b was used to label microglia. Other reports that did use CD11b found
no age-associated differences in microglia number in mouse hippocampus or rat brain
(Perry et al., 1993, Ogura et al., 1994, Long et al., 1998)
CD11b appears to mediate phagocytosis. Disrupting CD11b in microglia with
antibodies prevents phagocytosis of myelin, beta-amyloid, and opsonized yeast particles
in vitro (Fallman et al., 1993, Reichert and Rotshenker, 2003, Choucair-Jaafar et al.,
2010). In my study there was an age-associated decline in brain levels of CD11b,
suggesting that the ability of microglia to engulf foreign particles and other potentially
toxic cellular debris is impaired with age. Impaired phagocytosis in microglia might be
directly related to oxidative stress because chronic treatment with EUK-207 significantly
increased CD11b expression at 23 months of age and both EUK-189 and EUK-207
increased the number of CD11b-positive microglia in the brain. Reduced phagocytosis
might be very detrimental to neurons because it might increase their chances of being
exposed to cytotoxic material.
Chapter 2 demonstrated that both EUK-189 and EUK-207 at 0.15 and 1.5 mM
concentrations were equally effective in protecting against age-associated cognitive
impairment and brain oxidative stress. However, western blot analysis in this study
revealed that the lowest dose of EUK-207 was the most effective in increasing total brain
protein levels of ATG7, LAMP 1, and CD11b at 23 months of age. In addition, the lower
74
dosage of EUK-189 and EUK-207 had a significantly greater effect then the higher
dosage on reducing the LC3 II to LC3 I ratio in aged brains. The reasons for these
observations are not yet clearly understood, but one might postulate that EUK-189 and
EUK-207 at higher doses might promote autophagy induction or produce some minor
deleterious effect on microglia and autophagy function. Furthermore, EUK-207 appears
to be significantly more effective then EUK-189 in protecting acute hippocampal slices
against oxygen/glucose deprivation (Zhou et al., 2007), thus EUK-207 might actually be
a more potent molecule then EUK-189.
Oxidative stress accompanies the aging process and appears to be responsible for
age-associated cognitive impairment. However, the exact mechanisms by which age-
dependent oxidative stress impinge on brain function is still not clear. Autophagy and
microglia are both important in maintaining neuron homeostasis and both undergo
changes associated with aging and neurodegenerative disorders. Thus, autophagy activity
and microglia function might be linked to age-dependent brain oxidative stress. My
study indeed found that age-associated changes in microglia function and autophagy
taking place between 16 months and 23 months of age in mice could be significantly
reversed by chronic treatment with SOD/catalase mimetics. These results suggest that
age-associated oxidative stress plays a significant role in age-dependent changes in
autophagy and microglia.

75
Chapter 4

The superoxide dismutase/catalase mimetic EUK-207 protects
against beta-amyloid and tau pathology and cognitive decline in a
mouse model of Alzheimer’s disease.
4.1. Introduction
Age-associated cognitive impairment can be just a mild daily inconvenience;
however, cognitive dysfunction associated with Alzheimer’s disease (AD) is very
dramatic and significantly affects patient and caretaker’s lives. AD is a progressive
neurodegenerative disorder characterized by memory loss and cognitive deficits that
ultimately lead to severe dementia (Selkoe, 2001). AD is the most prominent form of
dementia found in the elderly and the number of Americans inflicted with the disease is
expected to reach nearly 15 million over the next several decades (Katzman and Saitoh,
1991, Salmon et al., 2002). AD pathology is characterized by the presence in the brain of
senile plaques made up of aggregated extracellular β-amyloid peptide and intracellular
neurofibrillary tangles mainly composed of hyperphosphorylated tau protein (Selkoe,
2001). In addition, AD also results in synapse loss and neuron death in hippocampus,
entorhinal cortex, basal forebrain, and neocortical association cortices (DeKosky et al.,
1996). Since AD is such a severe neurodegenerative disorder affecting the lives of so
many people, it is imperative that we understand the biological processes that underlie
this disease in order to develop a cure for it.
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Although the brain pathology associated with AD has been well established, the
driving underlying mechanism remains unclear. Genetic studies performed in families
with hereditary forms of AD point to impaired β-amyloid metabolism. Familial forms of
AD consist of mutations in the genes that encode for the amyloid precursor protein
(Goate et al., 1991), presenilin-1 and presenilin-2 (Cruts et al., 1998), which all result in
increased levels of amyloidogenic β-amyloid peptides in brain (Hardy and Selkoe, 2002).
However, familial forms of AD account for less than 5% of all cases (Onyango and
Khan, 2006); thus the vast majority of AD patients appears to develop it sporadically.
Studies performed in transgenic mouse models of AD have revealed that intraneuronal
beta-amyloid might be the first pathological event in AD, which is then followed by the
deposition of extracellular beta-amyloid and finally neurofibrillary alterations (Oddo et
al., 2003a, Oddo et al., 2003b). In this regard, many researchers believe in a “beta-
amyloid cascade hypothesis”, in which beta-amyloid formation triggers the progression
of the disease and is responsible for AD-associated cognitive dysfunction and
neurodegeneration. Experiments done both in vitro and in vivo have demonstrated that
the A β peptide is toxic to neurons (Pike et al., 1993, Geula et al., 1998) and that
intraneuronal A β accumulation is sufficient to disrupt synaptic function (Oddo et al.,
2003b)and memory formation (Billings et al., 2005), thus also supporting the notion that
beta-amyloid is the primary mediator in AD progression. However, these studies do not
explain what causes the increased accumulation in intraneuronal beta-amyloid to begin
with nor do they shed light on possible downstream mediators of beta-amyloid induced
neurodegeneration and cognitive dysfunction. Over the past decade a number of studies
have revealed that oxidative stress might play a critical part in AD.
77
Protein oxidation (Good et al., 1996, Butterfield, 1997, Smith et al., 1997), lipid
peroxidation (Sayre et al., 1997, Butterfield et al., 2001, Butterfield and Lauderback,
2002), and DNA and RNA oxidation are significantly elevated in Alzheimer’s diseased
brains (Gabbita et al., 1998, Nunomura et al., 1999, Lovell and Markesbery, 2001,
Nakanishi and Wu, 2009). In addition, the Aβ peptide might be responsible for AD-
associated oxidative stress. A β has been shown to induce ROS production in both
neuronal (Harris et al., 1995, Yatin et al., 1999) and astrocyte cell cultures (Harris et al.,
1996). Protein oxidation is also significantly elevated in cultured hippocampal neurons
incubated with a number of different A β peptides (Varadarajan et al., 2000) and treating
rat synaptic plasma membranes with A β
1-40
increased lipid peroxidation (Avdulov et al.,
1997). Mitochondrial abnormalities are also present in Alzheimer’s diseased brains. In
AD, the activity of the electron transport chain enzyme cytochrome c oxidase is
significantly reduced (Kish et al., 1992, Mutisya et al., 1994), ATP production is
diminished (Gibson et al., 1998, Castellani et al., 2002), and there is a decrease in both
mitochondrial mass and mitochondrial DNA content (Hirai et al., 2001,
Anandatheerthavarada et al., 2003). All of these AD-associated changes in mitochondria
could possibly result in electron transport chain deficiencies and therefore increase ROS
production (Smith et al., 1996, Davis et al., 1997, Butterfield et al., 2002). In addition,
age-associated mild cognitive impairment appears to be directly correlated to oxidative
stress (Carney et al., 1991, Bickford et al., 1997, Oge et al., 2003, Kiray et al., 2004,
Kiray et al., 2006) and individuals exhibiting mild cognitive impairment have a greater
chance of developing AD compared to the general population (Petersen et al., 2001b). In
addition, the activity and expression of the superoxide producing enzyme NADPH
78
oxidase is significantly higher in the temporal gyri of individuals inflicted with mild
cognitive impairment (Bruce-Keller et al., 2010) and experiments performed in vitro have
demonstrated that NADPH oxidase is activated in astrocytes by A β and appears to play a
critical role in A β induced neuron death (Abramov et al., 2004). Oxidative stress could
also potentially be the primary mediator of neurodegeneration observed in AD because
ROS production accompanies A β-induced neuronal apoptosis and the antioxidants α-
tocopherol and N-acetylcysteine inhibit A β-induced neuronal apoptosis (Tamagno et al.,
2003). Apolipoprotein E (apoE) genotype might also be a risk factor for AD and the
particular allele for apoE that appears to be correlated with AD encodes for a form of
apoE that exhibits reduced antioxidant activity (Miyata and Smith, 1996, Mazur-Kolecka
et al., 2002).
While there is strong evidence demonstrating a relationship between AD and
oxidative stress, whether or not oxidative stress is the initiator or a product and mediator
of its pathogenesis remains in question. Evidence from A β studies performed in vitro
suggests that oxidative stress is downstream from A β; however, mitochondrial
dysfunction is also present in AD brains, therefore ß-amyloid and tau pathology could be
a response to oxidative stress resulting from disruptions in the electron transport chain.
There is some evidence that also suggest that A β and tau might have a role as
antioxidants and this could make sense with regards to the latter possibility (Zhu et al.,
2007). Furthermore, mitochondrial abnormalities have also been shown to precede the
development of neurofibrillary tangles in AD (de la Monte et al., 2000, Hirai et al.,
2001); therefore oxidative stress might be a result of A β-induced mitochondrial
dysfunction and neurofibrillary tangles could be a response to oxidative stress.
79
The laboratory of Frank LaFerla at the University of California, Irvine has
developed a triple-transgenic mouse model of AD (3xTg-AD), which is now widely used
as a model for human AD (Oddo et al., 2003a, Oddo et al., 2003b). 3xTg-AD mice
express the mutant forms of the amyloid- β precursor protein and presenilin 1 that are
found in hereditary types of AD, and a mutated form of the microtubule-associated
protein tau, associated with frontal temporal dementia (Oddo et al., 2003b). These mice
exhibit intraneuronal A β accumulation in the neocortex between 3 and 4 months of age
and in the cortex, amygdala and CA1 region of the hippocampus by 6 months of age
(Oddo et al., 2003b). Extracellular A β plaques are also present in the frontal cortex at 6
months of age and in the hippocampus and other regions of the cerebral cortex by 12
months of age (Oddo et al., 2003b). Tau pathology in 3xTg-AD mice is also present, but
arises later at 12 months of age in neurons of the CA1 region of the hippocampus (Oddo
et al., 2003b). In addition, 3xTg-AD mice exhibit synaptic dysfunction in CA1 and
cognitive deficits in the Morris Water Maze and in passive fear avoidance task by 6
months of age (Oddo et al., 2003b, Billings et al., 2005). Since 3xTg-AD mice
progressively develop pathology and cognitive impairments that closely resemble those
that are found in human AD subjects they provide a useful model for studying AD.
Furthermore, 3xTg-AD mice also exhibit significant brain oxidative stress compared to
Non-Tg Mice (Resende et al., 2008, Yao et al., 2009); thus they are also ideal for
assessing the relationship between oxidative stress and AD.
The present study utilized the 3xTg-AD mouse model to further assess the
relationship between AD pathogenesis and oxidative stress. This was accomplished by
chronically treating 3xTg-AD mice with the SOD/catalase mimetic EUK-207 starting
80
before the occurrence of AD pathology and cognitive dysfunction and ending the
treatment when the untreated 3xTg-AD mice exhibited significant learning and memory
impairment. The results from chapter 2 demonstrated that the SOD/catalase mimetic
EUK-207 can significantly reduce age-associated cognitive impairment and oxidative
stress in aged mice and similar results were also observed for middle aged mice (Liu et
al., 2003), thus making this compound well-suited for elucidating the relationship
between oxidative stress and AD. The experimental design allowed me to specifically
evaluate the role oxidative stress plays in AD development. Chronic EUK-207 treatment
in 3xTg-AD mice beginning before AD-like pathology was present significantly reduced
beta-amyloid and tau pathology and protected against cognitive impairment in 9 month-
old 3xTg-AD mice. These results demonstrate that oxidative stress is crucial in AD
development and provide strong support for using SOD/catalase mimetics to protect
against AD progression.
4.2. Materials and methods
4.2.1. Materials
EUK-207 was synthesized as described previously (Doctrow et al., 2002,
Malfroy-Camine; Bernard (Arlington, 2003). All other chemicals were purchased from
Sigma, unless indicated otherwise.
4.2.2. Mice and treatments
Animals were treated in accordance with the principles and procedures of the
National Institutes of Health Guide for the Care and Use of Laboratory Animals; all
protocols were approved by the Institutional Animal Care and Use Committee of the
81
University of Southern California. Twenty-four 4-month-old C57BL/6J/129S male mice
were purchased from the Jackson Laboratory (Bar Harbor, ME) and 24 4-month-old
3xTg-AD (C57BL/6J/129S background) male mice were obtained from an established
breeding colony at the University of Southern California. Before experiments, mice were
housed 4 – 5 per cage and placed in the same room with a 12-h light/12-h dark cycle.
Mice were allowed free access to food and water, and their weights ranged from 26 to 41
g. Before surgery, mice were randomly assigned to four of the following groups (12
mice per group): Non-Tg vehicle control, Non-Tg EUK-207 treated, 3xTg-AD vehicle
control, and 3xTg-AD EUK-207 treated.
Before implantation, Alzet 1004 micro-osmotic pumps (Durect Corporation,
Cupertino, CA) were loaded with either EUK-207 at 3.41 mM in 5 % mannitol, or 5 %
mannitol alone (as vehicle control group) and then primed for at least 40 hours in 5 %
mannitol at 37 ºC. The micro-pumps were then implanted s.c. in the 4-month-old mice
according to the manufacturer’s recommendations. Briefly, mice were anesthetized with
ketamine (80 mg/kg) and xylazine (12 mg/kg) by i.p. injection. A small 1-cm incision
was then made to the hip area of the mice and a small pocket was formed by spreading
the s.c. connective tissues apart. The pump was placed into the prepared pocket, and the
wound was then closed with sutures.
Pumps delivered the drug at 0.11 µl/hour for a 28-day period, and the calculated
drug infusion rate was ≈9 nmol/day for the 3.41 mM dose of EUK-207. This
concentration of EUK-207 is equivalent to a dose of 170 µg/kg/day (assuming a 30 g
mouse). Control mice were implanted with micro-pumps filled with vehicle alone (5%
mannitol).
82
During the 5-month treatment, pumps were replaced 5 more times with new ones
at the original sites at the end of each 28-day period of implantation. In addition, body
weights were recorded at the end of the 5-month treatment in order to assess the effects of
repeated surgery and EUK-207 on overall health.
4.2.3. Behavioral analysis
4.2.3.1. Fear conditioning
All behavioral experiments were performed during the last 6 h of the light cycle.
Experiments were run in a conditioning chamber consisting of a Plexiglas cage (29 cm ×
29 cm × 29 cm) with a grid floor composed of 26 stainless steel rods 0.48 cm in diameter
(Coulbourn Instruments, Allentown, PA). The apparatus was located in a sound-
attenuating box located in a room that is separated from the main laboratory. A personal
computer controlled the experimental events and a video camera monitoring system was
used to continuously record behavior for off-line scoring of freezing. The chamber was
wiped with 70 % ethanol before and after each training and testing session and each
mouse was placed in the chamber individually. On day 1 of training, mice were put in the
chamber and after 3 min they received three separate tones that were terminated with a
foot-shock (tone: 20 s, 80 dB, 2 kHz; foot shock: 1 s, 0.8 mA; intertrial interval, 1 min
apart). Thirty seconds after the final foot shock, mice were returned to their home cages.
Twenty-four hours after training, mice were tested for conditioning to the context
(context test) by placing them into the conditioning chamber for 8 min, but neither foot
shock nor tone was given. Forty-eight hours after training, mice were tested for
conditioning to the tone (cue test) by placing them into a new chamber that differed from
83
the training chamber in its visual, tactile, and olfactory properties (30 cm x 20 cm x 13
cm). Following a 3 min acclimation period in the new chamber, the conditioning tone
was presented for an 8 min test.
Several modifications were made when fear conditioning and testing were
performed at 9-months of age in order to minimize any effects fear conditioning at 7-
months of age might have on fear-conditioning performance at 9-months of age. These
included: changing the visual properties of the conditioning chamber, cleaning the
chamber with 4 % acetic acid instead of 70 % ethanol before and after training, using a
20 sec 95 dB 4 kHz 1 s pulse tone instead of a 20 sec 80 dB 2 kHz continuous tone, and
using an observation chamber for the cue test that was completely different from the one
used for the cue test at 7-months of age in its visual, tactile, and olfactory properties.
4.2.3.2. Behavioral analysis
A time-sampling procedure performed by a trained observer blind to the
experimental conditions was used. Briefly, every 10 s, each mouse is judged as either
freezing or active. Freezing is defined as the absence of all visible movement of the body
and vibrissae, aside from movement necessitated by respiration. The percentage of
freezing is then calculated for the 8 min trial by dividing the number of freezing episodes
by the total number of observations (48) and multiplying by 100.
In order to control for potential sensory deficits, mice were also tested for visual
functions and for nociception. Vision was evaluated using a forepaw-reaching test. Mice
were held by their tail and placed up side down in mid air next to a platform, and their
ability to correctly reach towards the platform was assessed. Special care was taken in
order to keep the whiskers away from the platform, and each mouse was tested with two
84
different types of platforms. Nociception was evaluated with a tail-flick latency test.
Every mouse was placed in a beaker in order to restrain them, and after the mouse calmed
down their tale was placed on a 51 ºC hot plate. Tail-flick latency was defined as the
length of time that elapsed between placing the tail on the hot plate and tail flicking.
Hearing impairments have not been reported in C57BL/6J/129S or 3xTg-AD mice and
mice appeared to associate the tone with the shock during training as evident by freezing
or jumping when the tone was presented, thus no auditory test was administered. Overall
health of the animals was assessed by daily inspection and by monitoring body weights.
4.2.4. Analysis of Alzheimer’s disease pathology and oxidative
stress
In order to analyze AD pathology and oxidative stress, mice were anesthetized
with isoflurane and killed by decapitation. Brains were rapidly extracted, placed on a
chilled platform, and then cut in half sagitally. One half was immediately frozen on dry
ice, and stored at −70 ºC for later analysis of lipid peroxidation and A β42 levels. The
other half was placed in 4 % paraformaldehyde in 1 X PBS pH 7.4 and fixed for 24 hrs at
4 ºC for immunohistochemical analysis of pathology and oxidative stress.
4.2.4.1. A β
1-42
ELISA
The pons, cerebellum, and olfactory blub were removed and discarded from the
sagital halves and the brains were homogenized in 2 % SDS in water containing 0.7
µg/ml Pepstatin A supplemented with Complete Mini protease inhibitor cocktail tablet
(Roche Diagnostics, Indianapolis, IN) and PhosSTOP phosphotase inhibitor cocktail
tablet (Roche Diagnostics, Indianapolis, IN) using a Kontes polypropylene pellet pestle
85
(Kimble Chase, Vineland, NJ). The brain homogenates were then sonicated briefly in
order to sheer DNA and then centrifuged at 4 ºC for 1 hour at 100,000 x g. The resulting
supernatant was collected and stored as the detergent soluble fraction. The left over
pellet was resuspended in 70 % formic acid, briefly sonicated, and then centrifuged again
at 4 ºC for 1 hour at 100,000 x g. Following the centrifugation, the supernatant was
collected and stored as the detergent insoluble fraction. Protein concentrations were
determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL) for the
detergent soluble fractions and the Bradford method (Bio-Rad, Hercules, CA) for the
detergent insoluble fractions.
The concentration of A β1-42 within the detergent soluble fractions was
determined using reagents and instructions from an A β1-42 colorimetric sandwich
ELISA kit from Invitrogen (Carlsbad, CA). Briefly, a portion of the soluble fraction was
diluted 1:4 with 1 X PBS pH 7.4 and then mixed with standard diluent buffer and
supplemented with AEBSF at a final concentration of 1 mM. Fifty microliters of each
sample and A β1-42 standard were loaded onto a 96-well plate pre-coated with a
monoclonal antibody against the NH
2
terminus region of beta-amyloid. A rabbit primary
antibody against A β1-42 was added to each well and then the plate incubated at 4 ºC
overnight. Wells were emptied, washed, and then incubated with an anti-rabbit IgG
secondary antibody conjugated to horse radish peroxidase for 30 minutes at room
temperature. The secondary antibody solution was removed, the wells were washed, and
color was developed at room temperature for 30 minutes using 3.3’,5,5’-
Tretramethylbenzidine as the chromagen. The reaction was stopped using the provided
stop solution and the absorbances were read at 450 nm using a plate reader (Molecular
86
Devices, Sunnyvale, CA). A β42 was calculated as picogram of A β1-42 per mg of protein
according to a four parameter algorithm standard curve prepared from a series of A β1-42
standards that were run in parallel.
4.2.4.2. Lipid Peroxidation Assay
The levels of lipid peroxidation were quantified in the detergent soluble brain
fraction obtained above by the thiobarbituric acid-reactive substances (TBARS) assay as
previously described (Bruce and Baudry, 1995) with minor modifications. A portion of
the detergent soluble fraction was diluted 1:2 with 2.5 % SDS containing 12.5 µM
deferoxamine and 5 µM probucol (to prevent further oxidation). Four hundred microliters
of the resulting mixture were added to an aqueous solution consisting of 375 µl of 20 %
acetic acid solution (pH 3.5) and 225 µl of 1.33 % thiobarbituric acid, and the mixture
was heated at 95 ºC for 1 hour. One milliliter of a 15:1 1-butanol/pyridine solution was
added, and TBARS were extracted into the organic layer by centrifugation at 4,000 × g
for 10 min. The amounts of TBARS were determined by spectrophotometry at 532 nm
and were calculated as nanomolar malondialdehyde equivalent per milligram of protein
according to a standard curve prepared from malonaldehyde bis(dimethyl acetal).
4.2.4.3. Immunohistochemistry
Sagital brain halves were fixed with 4 % paraformaldehyde and processed for
immunohistochemistry in order to assess the expression and localization of oxidized
guanine, beta-amyloid protein and microtubule-associated protein tau. Following
fixation, brain halves were cryoprotected with sucrose, and then sectioned at 30 µm with
a Leica SM 2400 Sliding Microtome (Leica Microsystems Inc., Bannockburn, IL).
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Immunhistochemistry was carried out on free-floating brain sections using the avidin-
biotin-horseradish peroxidase complex (ABC) method with reagents and instructions of
the VECTASTAIN Elite ABC kit from Vector Laboratories (Burlingame, CA). Free-
floating sections were blocked for 1 hour at room temperature in 10 % normal horse
serum diluted in 1 X TBS, pH 7.4 or 1 X PBS, pH 7.4 and then probed with the following
primary antibodies: oh
8
dG/oh
8
G (1:5,000 dilution, QED Bioscience, San Diego, CA),
6E10 (1:1,000 dilution, Covance, Emeryville, CA), HT7 (1:4,000 dilution, Thermo Fisher
Scientific, Rockford, IL), or AT8 (1:1,000, Thermo Fisher Scientific, Rockford, IL)
overnight at 4 ºC. The sections were washed with either TBS or PBS and then incubated
with biotinylated anti-mouse IgG (1:400 in TBS or PBS in 5 % horse serum) for 2 – 3
hrs. After being washed with TBS or PBS, the sections were then incubated in the
avidin-biotin horse radish peroxidase complex solution for 45 min. The sections were
washed in order to remove any unbound avidin-biotin horse radish peroxidase and then
the stain was developed using 3,3’-diaminobenzidine (DAB) substrate (Vector
Laboratories, Burlingame, CA). Sections were mounted on gelatin-coated slides, air-
dried, dehydrated in a series of graded ethanol, and coverslipped with DPX mounting
medium (Electron Microscopy Sciences, Hatfield, PA).
Stained sections were visualized at 50 X under brightfield using a Zeiss
AxioImager.Z1 Upright Microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY).
Gray scale images were captured digitally with a Hammatsu ORCA-ER Digital Camera
(Hammatsu Photonics k.k., Hammatsu City, Japan) and AxioVision Version 4.8.1
software (Carl Zeiss MicroImaging, Inc., Thornwood, NY). In order to quantify changes
in immunoreactivity for oxidized guanine, gray scale photomicrographs of beta-amyloid,
88
and tau in the CA1 region of the hippocampus and the amygdala were imported into
Image J Software (NIH, Bethesda, MD) and pixel count was measured after the images
were converted into binary data using a constant threshold limit. Pixel counts for each
animal were averaged across three similar sections and results were expressed as
percentage of the corresponding values measured in the Non-Tg vehicle control group.
4.2.5. Statistical Analysis
All statistics were performed using GraphPad Prism 4.03 software (GraphPad
Software, La Jolla, CA). One-way ANOVA was used to test if the means of each
experimental group were significantly different and if the overall p value was <0.05, then
multiple comparisons between the experimental groups were tested using Tukey post hoc
analysis with 95 % confidence intervals.
4.3. Results
4.3.1. Effects of SOD/catalase mimetic EUK-207 on fear-
conditioning learning in 3xTg-AD mice
3xTg-AD mice were treated continuously with EUK-207 administered through
subcutaneously implanted osmotic minipumps for 5 months starting at 4 months age.
Treatment began when AD pathology was negligible and no cognitive impairments were
present (Oddo et al., 2003a, Oddo et al., 2003b, Billings et al., 2005) and continued until
the 3xTg-AD mice exhibited deficits in learning and memory. This experimental design
allowed us to specifically analyze the relationship between oxidative stress and AD
pathogenesis in this mouse model of Alzheimer’s disease. It had been previously
reported that 3xTg-AD mice show significant memory impairments in both the Morris
89
water maze and passive fear avoidance task (Billings et al., 2005); cognitive performance
was assessed at 7 months of age in Non-Tg and 3xTg-AD using a contextual and cued
fear conditioning paradigm. However, there was no significant difference in fear
conditioning performance between Non-Tg and 3xTg-AD mice administered vehicle in
both the context and cue tests (Fig. 13A and 13B). Therefore, EUK-207 treatment was
extended for two more months in order for AD pathology to become more pronounced in
the 3xTg-AD mice.

Figure 13. Absence of deficit in contextual or cued fear conditioning in 3xTg-AD mice at 7 months of
age. 4 month-old Non-Tg and 3xTg-AD mice were treated for 3 months with EUK-207 and then trained in
a contextual and cued fear conditioning paradigm. Mice were tested 24 h after training for the context test
(A) and 48 h after training for the cue test (B). Results were calculated as percent time the mouse exhibited
freezing behavior during the 8-min observation period for the context test (A) and cue test (B). Shown are
means ± SEM of 11-12 mice. One-way ANOVA indicated that there was no significant difference in
performance between Non-Tg and 3xTg-AD mice in both the context (A) and cue test (B).

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Cognitive performance was assessed again in both Non-Tg and 3xTg-AD mice at
9 months of age using a contextual and cued fear conditioning paradigm, but a new
context and cue were used for conditioning in order to minimize any effects the previous
training at 7 months of age might have had on fear-conditioning performance at 9 months
of age. Nine month-old 3xTg-AD mice administered vehicle exhibited a significant
decline in freezing response during the context test and cue test when compared to Non-
Tg vehicle control mice (Fig. 14A & 14B), thus suggesting a decline in learning and
memory. Chronic treatment with EUK-207 significantly increased the freezing response
in 9 month-old 3xTg-AD mice in both the context and cue test (Fig. 14A and 14B). In
fact, the EUK-207 treated 3xTg-AD group performed just as well as the Non-Tg vehicle
group in both the context and cue test (Fig. 14A and 14B); therefore suggesting that
EUK-207 was able to prevent the development of cognitive deficits and implying that
oxidative stress plays a critical role in this development. No significant difference in
freezing performance was observed between EUK-207 treated Non-Tg mice and vehicle
Non-Tg control mice in both the context test and cue test at 9 months of age (Fig. 14A
and 14B), but the difference in performance in both the context and cue test between
EUK-207 treated Non-Tg and 3xTg-AD vehicle mice was more significant than the
difference between Non-Tg vehicle and 3xTg-AD vehicle mice (Fig. 14A and 14B).
Nine month-old EUK-207 treated Non-Tg mice performed slightly better then their
3xTg-AD counterparts during the context test, but it was not a significant difference (Fig.
14A).
Chronic treatment with EUK-207 increased the average freezing response in 9
month-old 3xTg-AD mice by about 25 % in both the context and cue test (Fig. 14A and
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14B), but EUK-207’s effect in 3xTg-AD mice was more significant in the cue test (Fig.
14A and 14B). In addition, no significant defects in vision or nociception were present
between Non-Tg and 3xTg-AD mice (Table 3), and the prolonged treatment with EUK-
207 did not appear to produce any ill effects or weight changes (Table 3), suggesting that
the differences observed in contextual and cued fear conditioning between Non-Tg and
3xTg-AD mice were due to learning and memory deficits and not to differences in visual
or pain perception or overall health conditions.

Figure 14. Chronic treatment with EUK-207 starting at 4 months of age protects against deficits in
contextual and cued fear conditioning in 9 month old 3xTg-AD mice. 4 month-old Non-Tg and 3xTg-
AD mice were treated for 5 months with EUK-207 and then trained in a contextual and cued fear
conditioning paradigm. Mice were tested 24 h after training for the context test (A) and 48 h after training
for the cue test (B). Results were calculated as percent time the mouse expressed freezing behavior during
the 8-min observation period for the context test (A) and cue test (B). Shown are means ± SEM of 11-12
mice. One-way ANOVA indicated that the difference in performance between Non-Tg vehicle and 3xTg-
AD vehicle mice was significant for the context test (*p <0.01 vs. 3xTg-AD vehicle) and cue test (*p <0.01
vs. 3xTg-AD vehicle), as was the effect of EUK-207 on context test (**p <0.05 vs. 3xTg-AD vehicle, ***p
<0.001 vs. 3xTg-AD vehicle) and cue test (**p <0.01 vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD
vehicle) performance.
93

94
able 3. Body weight, nociception, and vision test results for EUK-207 treated mice.
.3.2. Effects of SOD/catalase mimetic EUK-207 on brain
In addition to dense extracellular beta-amyloid plaques and intracellular
neurofibrillary tangles, brains from AD patients also exhibit significant oxidative stress.
This includes protein oxidation (Good et al., 1996, Butterfield, 1997, Smith et al., 1997),,
lipid peroxidation (Sayre et al., 1997, Butterfield et al., 2001, Butterfield and Lauderback,
2002 , and DNA and RNA oxidation (Gabbita et al., 1998, Nunomura et al., 1999, Lovell
and Markesbery, 2001, Nakanishi and Wu, 2009) Similarly, 3xTg-AD mice also exhibit
a striking increase in brain oxidative stress beginning at about 3 – 5 months of age
(Resende et al., 2008). Therefore, we assessed lipid peroxidation and oxidized nucleic
acids in the brains of 3xTg-AD mice that started receiving chronic EUK-207 treatment
before the onset of AD pathology in order to better understand the link between oxidative
stress and AD.
In order to assess the effects of EUK-207 on brain lipid peroxidation, 3xTg-AD
mice treated as described above were sacrificed at the end of the 5 month-long treatment
and their brains, minus cerebellum and pons, were harvested and homogenized. Brain
100 % 2.09 ± 0.42 41.1 ± 6.4 Vehicle 3xTg-AD
100 % 1.98 ± 0.37 38.1 ± 4.6 EUK-207 3xTg-AD
100 % 2.12 ± 0.74 40.0 ± 7.0 Vehicle Non-Tg
100 % 2.35 ± 0.67 40.2 ± 5.0 EUK-207 Non-Tg
test (% success) latency, mean ± S.D. (s) mean ± S.D. (g) Treatment
T

Forepaw reaching Tail-flick Body weight,

Summary of the data for body weights after 5 months of treatment, tail-flick latency
100 % 2.09 ± 0.42 41.1 ± 6.4 Vehicle 3xTg-AD
100 % 1.98 ± 0.37 38.1 ± 4.6 EUK-207 3xTg-AD
100 % 2.12 ± 0.74 40.0 ± 7.0 Vehicle Non-Tg
100 % 2.35 ± 0.67 40.2 ± 5.0 EUK-207 Non-Tg
test (% success) latency, mean ± S.D. (s) mean ± S.D. (g) Treatment
Forepaw reaching Tail-flick Body weight,
(nociception) and forepaw reaching test (vision). There were no statistically significant
differences for the three parameters between the different treatment groups.

4
oxidative stress in 3xTg-AD mice

homogenates were separated into detergent soluble and insoluble fractions and a portion
of the detergent soluble fraction was used to measure lipid peroxidation (levels of
equivalent malondialdehyde). For comparison, lipid peroxidation was also assessed in
brain homogenates from age-matched Non-Tg mice. There was a significant increase in
brain lipid peroxidation in 3xTg-AD mice compared to Non-Tg vehicle control mice at 9
months of age (Fig. 15). EUK-207 treatment significantly reduced lipid peroxidation in
the brains of 3xTg-AD mice and brought lipid peroxidation to levels that were not
significantly different from those found in Non-Tg vehicle control mice (Fig. 15).
Compared to Non-Tg vehicle control mice, Non-Tg mice treated with EUK-207
displayed a decrease in brain lipid peroxidation levels, but this difference was not
significant (Fig. 15).

Figure 15. Chronic treatment with EUK-207 starting at 4 months of age significantly reduces lipid
peroxidation in brain homogenates from 9 month-old 3xTg-AD mice. At the end of the 5 month
treatment, mice were decapitated and their brains (minus cerebellum) were removed, homogenized, and
divided into detergent soluble and insoluble fractions. Lipid peroxidation in the detergent soluble fraction
was then quantified by the thiobarbituric acid-reactive substances (TBARS) assay. Lipid peroxidation was
also determined in brain homogenates from 9 month-old Non-Tg mice. Levels of lipid peroxidation were
expressed as nmol malondialdehyde equivalent per mg of protein. Shown are means ± SEM of 11-12 mice.
One-way ANOVA indicated that the difference in brain lipid peroxidation between Non-Tg vehicle and
3xTg-AD vehicle mice was significant (*p <0.001 vs. 3xTg-AD vehicle), as was the effect of EUK-207
(**p <0.01 vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle).
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96
Oxidized DNA and RNA within the brains of 3xTg-AD mice were also assessed
at 9 months of age. Mice were sacrificed after the 5 month-long treatment with EUK-207
and their brains were fixed in 4 % paraformaldehyde and processed for
immunohistochemisitry. Brains sections were then probed with an antibody specific for
oxidized guanine. For comparison, oxidized guanine was also assessed in brain
homogenates from age-matched Non-Tg mice.
Oxidized guanine was present in the brains of Non-Tg and 3xTg-AD mice.
Staining for oxidized guanine was observed in a number of different brain regions,
including the cortex, hippocampus, substantia nigra, amygdala, and striatum. I decided to
focus specifically on the hippocampus and amygdala because these two brain regions are
known to be critical in associative fear memories. Lesion studies have revealed that the
amygdala plays a role in both context and cue related fear memories (Phillips and
LeDoux, 1992), whereas the hippocampus is critical for contextual fear memories (Kim
and Fanselow, 1992, Phillips and LeDoux, 1992, Maren et al., 1997, Riedel et al., 1997)
Oxidized guanine was present throughout the hippocampus of Non-Tg and 3xTg-AD
mice (Fig. 16A). Oxidized guanine was present in the dentate gyrus, and the CA1, CA2,
and CA3 fields (Fig. 16A). Staining was observed in both cytoplasm and nucleus, thus
indicating that nuclear DNA, mitochondrial DNA, and mRNA were probably oxidized
(Fig. 16A). Although oxidized guanine was present in both Non-Tg and 3xTg-AD
hippocampus, the intensity of staining was greater in the hippocampus of 3xTg-AD
vehicle mice (Fig. 16A). Chronic treatment with EUK-207 beginning at 4 months of age
reduced the level of oxidized guanine in the hippocampus of 9 month-old 3xTg-AD mice.
The intensity of oxidized guanine labeling in the hippocampus was similar between
97
EUK-207 treated Non-Tg and Non-Tg vehicle mice (Fig. 16A). Quantification of the
staining intensity in the CA1 region of the hippocampus revealed that these differences in
staining were significant (Fig. 16B). In addition, there was no significant difference in
oxidized guanine between 3xTg-AD mice treated with EUK-207 and Non-Tg vehicle
mice (Fig. 16B).

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Figure 16. Chronic treatment with EUK-207 starting at 4 months of age reduces oxidized guanine
levels in hippocampus of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were
sacrificed and brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and
labeled for oxidized guanine using immunohistochemistry. Staining for oxidized guanine was assessed in
the hippocampus of Non-Tg and 3xTg-AD mice (A) and quantified in the soma of CA1 pyramidal cells by
counting pixels (B). Levels of oxidized guanine were expressed as % of Non-Tg vehicle (B). Shown are
means ± SEM of 4 mice (B). One-way ANOVA indicated that the difference in oxidized guanine between
Non-Tg vehicle and 3xTg-AD vehicle mice was significant (*p <0.01 vs. Non-Tg vehicle), as was the
effect of EUK-207 (**p <0.01 vs. 3xTg-AD vehicle) (B).
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Similar results for oxidized guanine were also observed in the amygdala of Non-
Tg and 3xTg-AD mice at 9 months of age (Fig. 17A and 17B). However, the increase in
oxidized guanine labeling in the amygdala of 3xTg-AD mice was greater and more
significant than that observed in the hippocampus (Fig. 17B). These results along with
the lipid peroxidation data confirm that like AD brains, the brains of 3xTg-AD mice
exhibit a considerable increase in oxidative stress. In addition, these results clearly
demonstrate that chronic treatment with EUK-207 starting before the onset of AD
pathology significantly prevent both lipid peroxidation and nucleic acid oxidation in the
brains of 3xTg-AD mice.

Figure 17. Chronic treatment with EUK-207 starting at 4 months of age reduces oxidized guanine
levels in amygdala of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were
sacrificed and brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and
labeled for oxidized guanine using immunohistochemistry. Staining for oxidized guanine was assessed in
the amygdala of Non-Tg and 3xTg-AD mice (A) and quantified by counting pixels (B). Levels of oxidized
guanine were expressed as % of Non-Tg vehicle (B). Shown are means ± SEM of 4 mice (B). One-way
ANOVA indicated that the difference in oxidized guanine between Non-Tg vehicle and 3xTg-AD vehicle
mice was significant (*p <0.001 vs. Non-Tg vehicle), as was the effect of EUK-207 (**p <0.01 vs. 3xTg-
AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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4.3.3. Effects of SOD/catalase mimetic EUK-207 on beta-amyloid
pathology in 3xTg-AD mice
Senile plaques in AD brain are largely composed of aggregated beta-amyloid
protein. Beta-amyloid peptides range in length from 39-43 amino acids and are usually
present in the human brain at low levels (Canevari et al., 2004). Many researchers assume
that the A β peptide is responsible for the neurodegeneration and cognitive deficits
observed in Alzheimer’s disease because in vitro and in vivo studies have indicated that
some forms of the beta-amyloid are toxic to neurons (Pike et al., 1993, Geula et al.,
1998). The mechanisms for the significant increase in beta-amyloid levels in sporadic
AD brain are unclear; on the other hand, heritable forms of AD have been shown to be
due to mutations in beta-amyloid precursor protein (APP) or in enzymes processing it to
produce beta-amyloid peptides. The beta-amyloid peptide is produced by the sequential
cleavage of the amyloid precursor protein (APP) by β-secretase and γ-secretase (Canevari
et al., 2004). γ-secretase is composed of the proteins Presenilin 1 or Presenilin 2,
nicastrin, aph-1, and pen-2 (Kimberly et al., 2003). Mutations in the genes that encode
for APP, Presenilin 1 or Presenilin 2 are present in heritable forms of AD (Ritchie and
Lovestone, 2002) and appear to result in increased production of the more toxic and
aggregate prone form of beta-amyloid, A β
1-42
(Snyder et al., 1994, Naslund et al., 2000).

3xTg-AD mice express both a mutant form of the human Presenilin 1 gene and APP gene
that are found in familial forms of AD (Oddo et al., 2003b). In 3xTg-AD mice,
intraneuronal A β is present in the hippocampus, cortex, and amygdala by 6 months of
age, and extracellular A β deposits begin to form in the frontal cortex at 6 months of age
102
and in the hippocampus by 12 months of age (Oddo et al., 2003b). Therefore, A β
pathology was assessed in brains from 3xTg-AD mice that started receiving chronic
EUK-207 treatment before the onset of AD pathology in order to better understand the
link between oxidative stress and the development of beta-amyloid pathology in
Alzheimer’s disease.
The localization and expression of beta-amyloid was assessed within the brains of
3xTg-AD at 9 months of age. Mice were sacrificed after the 5 month long treatment with
EUK-207 and their brains were fixed in 4 % paraformaldehyde and processed for
immunohistochemisitry. Brains sections were then probed with the antibody 6E10,
which recognizes APP and all mature forms of beta-amyloid peptides. For comparison,
and to confirm that there is an increase in beta-amyloid levels in 3xTg-AD mice, beta-
amyloid pathology was also assessed in sections from age-matched Non-Tg mice.
Immunohistochemistry indicated that APP and beta-amyloid peptides were
increased in 9 month-old 3xTg-AD mice, as compared to non-Tg mice. Intraneuronal
beta amyloid was observed in a number of different brain regions in 3xTg-AD mice,
including the frontal cortex, cortex, hippocampus, and amygdala. However, no
extracellular beta-amyloid deposits were found throughout the brain. Like for oxidized
guanine, we decided to focus specifically on hippocampus and amygdala because these
two brain regions appear to be critical in associative fear memories. 6E10-labeled
neurons were found in the CA1, CA2, and CA3 fields of the hippocampus, but not in the
dentate gyrus of 3xTg-AD mice (Fig. 18A). Staining was localized mainly to the cell
bodies of pyramidal cells within the hippocampus (Fig. 18A). Very light staining for
6E10 was present in the hippocampus of 9 month-old Non-Tg mice, but it was
103
considerably lower than that observed in 3xTg-AD mice (Fig. 18A). Chronic treatment
with EUK-207 beginning at 4 months of age reduced 6E10 staining intensity as well as
the number of 6E10-positive neurons within the hippocampus of 9 month-old 3xTg-AD
mice (Fig. 18A). Quantification of the staining in the CA1 region of the hippocampus
revealed that these differences were significant (Fig. 18B). In addition, the difference in
6E10 staining intensity between 3xTg-AD mice treated with EUK-207 and Non-Tg
vehicle mice was not significant (Fig. 18B).

Figure 18. Chronic treatment with EUK-207 starting at 4 months of age reduces 6E10 staining in
hippocampus of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were sacrificed
and brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and labeled for
beta-amyloid by immunohistochemistry using the antibody 6E10. Staining for beta-amyloid was assessed
in the hippocampus of Non-Tg and 3xTg-AD mice (A) and quantified in the soma of CA1 pyramidal cells
by counting pixels (B). Levels of oxidized guanine were expressed as % of Non-Tg vehicle (B). Shown are
means ± SEM of 4 mice (B). One-way ANOVA indicated that the difference in 6E10 staining between
Non-Tg vehicle and 3xTg-AD vehicle mice was significant (*p <0.001 vs. Non-Tg vehicle), as was the
effect of EUK-207 (**p <0.05 vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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Similar results for 6E10 staining were also observed in the amygdala of Non-Tg
and 3xTg-AD mice at 9 months of age (Fig. 19A and 19B). However, one-way ANOVA
indicated that unlike in the hippocampus, the difference in 6E10 expression between
3xTg-AD mice treated with EUK-207 and Non-Tg vehicle mice was significantly
different (p<0.01) (Fig. 19B).

Figure 19. Chronic treatment with EUK-207 starting at 4 months of age reduces 6E10 staining in
amygdala of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were sacrificed and
brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and labeled for beta-
amyloid by immunohistochemistry using the antibody 6E10. Staining for beta-amyloid was assessed in the
hippocampus of Non-Tg and 3xTg-AD mice (A) and quantified in the amygdala by counting pixels (B).
Levels of beta-amyloid were expressed as % of Non-Tg vehicle (B). Shown are means ± SEM of 4 mice
(B). One-way ANOVA indicated that the difference in 6E10 staining between Non-Tg vehicle and 3xTg-
AD vehicle mice was significant (*p <0.001 vs. Non-Tg vehicle), as was the effect of EUK-207 (**p <0.05
vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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In order to further assess the relationship between oxidative stress and A β
pathology in Alzheimer’s disease A β
1-42
levels were measured in brains from 3xTg-AD
mice that started receiving chronic EUK-207 treatment before the onset of AD pathology.
A β
1-40
is the most prominent form of beta-amyloid in the brain; however, the levels of
A β
1-42
significantly increase in Alzheimer’s disease (Canevari et al., 2004). A β
1-42

aggregates faster and is more harmful then A β
1-40
(Snyder et al., 1994), thus implying that
A β
1-42
might be critical in the development of senile plaques and neurodegeneration in
Alzheimer’s disease. Detergent soluble A β
1-42
in the brains of 3xTg-AD mice has been
previously reported to begin to increase compared to Non-Tg mice at 5 – 7 months of age
and becomes very significant by 13 months (Oddo et al., 2003b).
In my study, 3xTg-AD mice were sacrificed after the 5 month long treatment with
EUK-207 and their brains, minus cerebellum and pons, were harvested and homogenized.
Brain homogenates were separated into detergent soluble and insoluble fractions and a
portion of the detergent soluble fraction was used to measure A β
1-42
levels using a
colorimetric sandwich ELISA specific for A β
1-42
. For comparison, A β
1-42
was also
assessed in brain homogenates from age-matched Non-Tg mice. Although the amount of
brain A β
1-42
was fairly low among Non-Tg and 3xTg-AD mice there was still a
significant increase in brain A β
1-42
levels in 3xTg-AD mice compared to Non-Tg vehicle
control mice at 9 months of age (Fig. 20). In addition, EUK-207 treatment significantly
reduced A β
1-42
expression in the brains of 3xTg-AD mice and brought A β
1-42
to levels
that were not significantly different from those found in Non-Tg vehicle control mice
(Fig. 20). A β
1-42
levels were slightly lower in Non-Tg vehicle control mice compared to
Non-Tg mice treated with EUK-207, but this effect was not significant (Fig. 20).

Figure 20. Chronic treatment with EUK-207 starting at 4 months of age significantly reduces
detergent soluble A β
1-42
in brain homogenates from 9 month-old 3xTg-AD mice. At the end of the 5
month treatment, mice were decapitated and their brains (minus cerebellum) were removed, homogenized,
and divided into detergent soluble and insoluble fractions. A β
1-42
levels in the detergent soluble fraction
were quantified using a sandwich ELISA specific for A β
42
. A β
1-42
was also determined in brain
homogenates from 9 month-old Non-Tg mice. Levels of A β
42
were expressed as pg of A β
1-42
per mg of
protein. Shown are means ± SEM of 11-12 mice. One-way ANOVA indicated that the difference in A β
1-42

between Non-Tg vehicle and 3xTg-AD vehicle mice was significant (*p <0.001 vs. 3xTg-AD vehicle), as
was the effect of EUK-207 (**p <0.001 vs. 3xTg-AD vehicle).

Brains from 9 month-old 3xTg-AD mice exhibited a significant increase in
intraneuronal beta-amyloid expression in both hippocampus and amygdala and contained
significantly higher levels of A β
1-42.
These results confirm an AD-like pathology in 3xTg-
AD mice at 9 months of age and underscore the important relationship between oxidative
stress and AD-associated beta-amyloid pathology, as chronic treatment with EUK-207
starting before the onset of AD pathology significantly protected against increases in
beta-amyloid and A β
1-42
expression in 3xTg-AD mice.

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4.3.4. Effects of SOD/catalase mimetic EUK-207 on tau pathology
in 3xTg-AD mice
In addition to senile plaques, the AD brain also contains neurofibrillary tangles,
which are mainly composed of hyperphosphorylated tau protein (11-2 & 11-3). Mouse
models of Alzheimer’s disease that express mutant forms of Presenilin 1, Presenilin 2 or
amyloid precursor protein exhibit a number of Alzheimer’s disease-like pathologies
(Price et al., 1998, Avila, 2006) including a variety of cognitive deficits, and A β plaques
(Eriksen and Janus, 2007). However, unlike familial cases of Alzheimer’s disease, these
mice do not exhibit any tau pathology (Eriksen and Janus, 2007). In addition to mutant
APP and presenilin 1, 3xTg-AD mice also contain a mutant tau gene that is found in
humans with frontal temporal dementia, which like AD, is also associated with
neurofibrillary tangles in the brain (Oddo et al., 2003a). This extra transgene produces
AD-like tau pathology in 3xTg-AD mice (Oddo et al., 2003a), thus making these mice a
good model for studying most pathologies associated with Alzheimer’s disease. Human
tau protein begins to accumulate in the hippocampus of 3xTg-AD mice by 6 months of
age, and neurofibrillary alterations associated with conformational changes and
hyperphosphorylation occur between 9 and 12 months of age (Oddo et al., 2003a).
In order to assess the relationship between tau pathology and oxidative stress the
localization and expression of total human tau and hyperphosphorylated tau was assessed
within the brains of 9 month-old 3xTg-AD that were chronically treated with EUK-207.
Mice were sacrificed after the 5 month long treatment and their brains were fixed in 4 %
paraformaldehyde and processed for immunohistochemisitry. Brains sections were then
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probed with the antibody HT7, which is specific for all forms of human tau. For
comparison, and to confirm that there is an increase in HT7 staining in 3xTg-AD mice,
tau pathology was also assessed in sections from age-matched Non-Tg mice. HT7
staining was present in the frontal cortex, cortex, hippocampus and amygdala of 9 month-
old 3xTg-AD mice. Tau protein was found in both the cell bodies and processes of
neurons. Like for oxidative stress and beta-amyloid pathology, we also decided to focus
specifically on the hippocampus and amygdala because these two brain regions appear to
be critical in associative fear memories. HT7 staining was only detected in the cell bodies
and processes of pyramidal cells within the CA1 region of the hippocampus (Fig. 21A).
No HT7 labeled cells were found in hippocampus of 9 month-old Non-Tg mice (Fig.
21A), which was expected because they do not express human tau protein (Fig. 21A).
Chronic treatment with EUK-207 starting at 4 months of age reduced the number of HT7-
positive neurons within the hippocampus of 9 month-old 3xTg-AD mice (Fig. 21A). HT7
staining in the hippocampus was similar between EUK-207 treated Non-Tg and Non-Tg
vehicle mice (Fig. 21A). Quantification of the staining in the CA1 region of the
hippocampus revealed that these observations were significant (Fig. 21B).

Figure 21. Chronic treatment with EUK-207 starting at 4 months of age reduces Tau accumulation in
hippocampus of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were sacrificed
and brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and labeled for
total human tau by immunohistochemistry using the antibody HT7. Staining for HT7 was assessed in the
hippocampus of Non-Tg and 3xTg-AD mice (A) and quantified in the soma and somatodendritic region of
CA1 pyramidal cells by counting pixels (B). Levels of tau were expressed as % of Non-Tg vehicle (B).
Shown are means ± SEM of 3-4 mice (B). One-way ANOVA indicated that the difference in HT7 staining
between Non-Tg vehicle and 3xTg-AD vehicle mice was significant (*p <0.01 vs. Non-Tg vehicle), as was
the effect of EUK-207 (**p <0.05 vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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Similar results for HT7 staining were also observed in the amygdala of Non-Tg
and 3xTg-AD mice at 9 months of age (Fig. 22A and 22B). However, one-way ANOVA
indicated that unlike in the hippocampus, the difference in HT7 expression between
3xTg-AD mice treated with EUK-207 and Non-Tg vehicle mice was significantly
different (p<0.001) (Fig. 22B).

Figure 22. Chronic treatment with EUK-207 starting at 4 months of age reduces tau accumulation in
amygdala of 9 month-old 3xTg-AD mice. At the end of the 5 month treatment, mice were sacrificed and
brains were fixed using 4 % paraformaldehyde. The brains were sectioned at 30 µm and labeled for total
human tau by immunohistochemistry using the antibody HT7. Staining for HT7 was assessed in the
amygdala of Non-Tg and 3xTg-AD mice (A) and quantified in the amygdala by counting pixels (B). Levels
of tau were expressed as % of Non-Tg vehicle (B). Shown are means ± SEM of 4 mice (B). One-way
ANOVA indicated that the difference in HT7 staining between Non-Tg vehicle and 3xTg-AD vehicle mice
was significant (*p <0.001 vs. Non-Tg vehicle), as was the effect of EUK-207 (**p <0.01 vs. 3xTg-AD
vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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Tau proteins phosphorylated at serine 202 and threonine 205 are a common
feature of neurofibrillary tangles in AD. The antibody AT8 is specific for this form of
hyperphosphorylated tau and is commonly used as a marker for neurofibrillary
abnormalities. In 9 month-old 3xTg-AD mice, AT8 expression was only found in the
ventral hippocampus, subiculum, and amygdala. AT8 staining was present in ventral
CA1 and CA3 pyramidal cells (Fig. 23A), and like HT7, AT8 expression was located in
both the cell bodies and processes (Fig. 23A). Very few AT8-labeled cells were found in
the ventral hippocampus of 9 month-old Non-Tg mice (Fig. 23A), thus suggesting that
the accumulation of this form of hyperphosphorylated tau is uniquely associated with the
tau mutation that the 3xTg-AD mice posses and AD pathology. Chronic treatment with
EUK-207 starting at 4 months of age reduced the number of AT8-positive neurons within
the ventral hippocampus of 9 month-old 3xTg-AD mice (Fig. 23A). AT8 staining in the
hippocampus was similar between EUK-207 treated Non-Tg and Non-Tg vehicle mice
(Fig. 23A). Quantification of AT8 labeling within the ventral CA1 region of the
hippocampus revealed that these observations were significant (Fig. 23B).

Figure 23. Chronic treatment with EUK-207 starting at 4 months of age reduces
hyperphosphorylated tau in ventral CA1 pyramidal cells of 9 month-old 3xTg-AD mice. At the end of
the 5 month treatment, mice were sacrificed and brains were fixed using 4 % paraformaldehyde. The brains
were sectioned at 30 µm and labeled for hyperphosphorylated tau by immunohistochemistry using the
antibody AT8. Staining for AT8 was assessed in the hippocampus of Non-Tg and 3xTg-AD mice (A) and
quantified in the soma and somatodendritic region of ventral CA1 pyramidal cells by counting pixels (B).
Levels of hyperphosphorylated tau were expressed as % of Non-Tg vehicle (B). Shown are means ± SEM
of 4 mice (B). One-way ANOVA indicated that the difference in AT8 staining between Non-Tg vehicle and
3xTg-AD vehicle mice was significant (*p <0.001 vs. Non-Tg vehicle), as was the effect of EUK-207 (**p
<0.05 vs. 3xTg-AD vehicle, ***p <0.001 vs. 3xTg-AD vehicle) (B).
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Similar observations for AT8 labeling were also observed in the amygdala of
Non-Tg and 3xTg-AD mice at 9 months of age (Fig. 24A and 24B). Both tau
accumulation and the expression of hyperphosphorylated tau were reduced in the
hippocampus and amygdala of 9 month-old 3xTg-AD mice treated with EUK-207
starting at 4 months of age. This suggests that in addition to beta-amyloid pathology,
oxidative stress might also be linked to the development of tau pathology in AD.

Figure 24. Chronic treatment with EUK-207 starting at 4 months of age reduces
hyperphosphorylated tau in amygdala of 9 month-old 3xTg-AD mice. At the end of the 5 month
treatment, mice were sacrificed and brains were fixed using 4 % paraformaldehyde. The brains were
sectioned at 30 µm and labeled for hyperphosphorylated tau by immunohistochemistry using the antibody
AT8. Staining for AT8 was assessed in the amygdala of Non-Tg and 3xTg-AD mice (A) and quantified in
the in the amygdala by counting pixels (B). Levels of hyperphosphorylated tau were expressed as % of
Non-Tg vehicle (B). Shown are means ± SEM of 4 mice (B). One-way ANOVA indicated that the
difference in AT8 staining between Non-Tg vehicle and 3xTg-AD vehicle mice was significant (*p <0.001
vs. Non-Tg vehicle), as was the effect of EUK-207 (**p <0.01 vs. 3xTg-AD vehicle, ***p <0.001 vs.
3xTg-AD vehicle) (B).
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4.4. Discussion
The Alzheimer’s diseased brain is riddled with oxidative stress. Lipid
peroxidation is significantly elevated in cortex and hippocampus (Butterfield, 1997,
Smith et al., 1997, Hensley et al., 1998) protein oxidation is drastically increased in both
hippocampus and neocortex (Shan et al., 2003), and considerable levels of mRNA
oxidation are present in AD brains (Shan et al., 2003). In vitro studies have shown that
the beta-amyloid peptide induces ROS production in neurons (Harris et al., 1995, Yatin et
al., 1999) and astrocytes (Harris et al., 1996) and increases protein oxidation and lipid
peroxidation in neurons and synaptosomes (Avdulov et al., 1997, Varadarajan et al.,
2000). In addition, A β-induced neuron death is inhibited with the antioxidants α-
tocopherol and N-acetylcysteine in vitro (Tamagno et al., 2003). These studies suggest
that increased oxidative stress is a result of A β accumulation in AD brains. In addition,
damaged mitochondria, reduced ATP production, and electron transport chain
deficiencies (Parker et al., 1994, Gibson et al., 1998, Castellani et al., 2002) are also
characteristics of AD brains; thus, the A β peptide might trigger oxidative stress by
specifically targeting mitochondrial function. However, mitochondrial dysfunction and
brain oxidative stress arise before AD pathology in a mouse model of AD (Yao et al.,
2009). Therefore, oxidative stress resulting from mitochondrial dysfunction might
actually be responsible for the development of ß-amyloid and tau pathology.
While a relationship between AD and brain oxidative stress is clear, whether or
not oxidative stress initiates AD pathogenesis or is a product/mediator of AD remains an
open question. The present study was designed to define the role oxidative stress plays in
AD pathogenesis. As EUK-207 has previously been shown two significantly reduce age-
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associated cognitive impairment and oxidative stress in middle-aged and aged wild-type
mice (Liu et al., 2003, Clausen et al., 2010), we used this compound as a tool to elucidate
the relationship between oxidative stress and AD. Chronic treatment with EUK-207
began at 4 months of age because cognitive deficits and AD pathology are not present in
3xTg-AD mice at this age, and was maintained until cognitive deficits became apparent
in vehicle-treated 3xTg-AD mice. Chronic EUK-207 treatment significantly reduced
intraneuronal accumulation of ß-amyloid peptide, tau and hyperphosphorlated tau in
hippocampus and amygdala of 9 month-old 3xTg-AD mice. In addition, EUK-207
treatment reversed the increased levels of brain lipid peroxidation and oxidized guanine
in hippocampus and amygdala of 3xTg-AD mice to those found in WT animals. EUK-
207 also reversed deficits in performance of 9 month-old 3xTg-AD mice in both the
context and cue fear conditioning tests; finally the treatment significantly decreased brain
A β
42
levels. These results clearly demonstrate that oxidative stress is a critical mediator in
the development of AD-like pathology and cognitive impairment.
Studies performed in the Tg2576 mouse model of AD also support my results.
Like the 3xTg-AD mice, the Tg2576 mice develop cognitive impairments and ß-amyloid
associated pathology in an age-dependent manner. Overexpressing the mitochondrial
form of superoxide dismutase in Tg2756 mice protects against learning and memory
impairments observed in the Morris water maze and in contextual and cued fear
conditioning and significantly reduces the number of ß-amyloid plaques in brain
((Massaad et al., 2009)). In addition, supplementing the diets of Tg2576 mice with the
antioxidant Vitamin E beginning at 4 months of age significantly reduced lipid
peroxidation, levels of A β
1-40
and

A β
1-42
, and the number of amyloid plaques in the brain
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of 13 month-old Tg2576 mice (Sung et al., 2004). However, the relationship between
oxidative stress and the development of AD-associated tau pathology was not evaluated
in these two reports because Tg2576 mice do not express mutant tau protein and therefore
do not exhibit any neurofibrillary alterations. Thus, this current study appears to be the
first of its kind to assess the relationship between brain oxidative stress and AD-
associated tau pathology in vivo using an Alzheimer’s mouse model. Interestingly, data
from this study demonstrate that in addition to beta-amyloid pathology, AD-associated
changes in tau also appear to be critically linked to brain oxidative stress. However this
relationship might be indirect because it has been previously reported that intraneuronal
A β arises before AD associated neurofibrillary alterations in 3xTg-AD mice (Oddo et al.,
2003a). Therefore, if hyperphosphorylated tau is downstream from the accumulation of
beta-amyloid within neurons then the decrease seen in hyperphosphorylated tau in the
hippocampus and amygdala of EUK-207 treated 3xTg-AD mice might be due to a EUK-
207 mediated decrease in intraneuronal A β and not a decrease in brain oxidative stress.
Chronic EUK-207 treatment also significantly reduced the expression of total human
mutant tau levels in the hippocampus and amygdala of 3xTg-AD mice, thus the decrease
in hyperphosphorylated tau observed in EUK-207 treated 3xTg-AD mice might also be
the result of decreased total human tau levels.
Only intraneuronal ß-amyloid peptide and hyperphosphorylated tau accumulation
was present in hippocampus and amygdala of the 3xTg-AD mice at the time when
impairments in both contextual and cued fear conditioning were evident. This
observation suggests that extracellular A β plaques are not necessary for cognitive
impairment in these mice. Another study in 3xTg-AD mice found that intraneuronal ß-
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amyloid accumulation was sufficient to impair memory function in both the Morris water
maze and fear conditioning tasks (Billings et al., 2005). In addition, assessing LTP in the
CA1 region of hippocampus at different ages in 3xTg-AD mice revealed that synaptic
dysfunction also only required the presence of intraneuronal A β (Oddo et al., 2003b).
Unlike the expression of ß-amyloid and total human tau, hyperphosphorylated tau was
only located in the ventral hippocampus and amygdala of 9 month-old 3xTg-AD mice.
This observation is in line with several studies indicating that the ventral hippocampus is
more susceptible than the dorsal hippocampus to neurodegeneration, such as following
kainic acid injection (Altar and Baudry, 1990) or exposure to acetylcholinesterase
inhibitors (Apland et al., 2010).
EUK-207 treatment in 3xTg-AD mice reversed brain oxidative stress to levels
that were similar to those found in Non-Tg mice; on the other hand, levels of ß-amyloid
peptide, tau, and hyperphosphorylated tau, while significantly reduced compared to those
in 9 month-old vehicle-treated 3xTg-AD mice, were still elevated. This suggests that
accumulation of these proteins is only partially due to oxidative stress, and that other
factors contribute to their accumulation. In particular, it is tempting to propose that EUK-
207 stimulates the autophagic/lysosomal pathway as shown in Chapter 3. Nevertheless,
although intraneuronal beta-amyloid and hyperphosphorylated tau were still present in
hippocampus and amygdala of EUK-207-treated mice, they still performed as well as the
Non-Tg vehicle mice in both the context and cue test. These results suggest that ß-
amyloid and hyperphosphorylated tau must reach critical levels within neurons before
impairing synaptic function and plasticity.
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My results indicate that administering exogenous antioxidants, such as EUK-207,
to at-risk individuals before AD development might be potentially useful for protecting
them against AD. Genetic screening could potentially reveal individuals who might be at
risk for developing hereditary forms of AD by evaluating mutations in amyloid precursor
protein and presenilin genes. Recent studies have also suggested that apolipoprotein E4
genotype is a risk factor for AD (Miyata and Smith, 1996, Mazur-Kolecka et al., 2002);
therefore this gene could also possibly be used as a screening test. Furthermore, humans
exhibiting mild cognitive impairment have a greater chance of developing AD than the
general population (Petersen et al., 2001b); thus, antioxidant treatment could be
potentially useful for protecting mild cognitively impaired individuals from developing
AD. However, treating individuals exhibiting mild cognitive impairment with vitamin E
for 3 years did not reduce their chances of developing AD (Petersen et al., 2005). While
these results appear to negate the usefulness of antioxidants for protecting against AD,
this study has numerous problems. First, Vitamin E administration might have started
when AD pathology was already significant, and therefore the treatment did not prevent
further AD progression and severe cognitive impairment. Vitamin E supplementation
studies in Tg2756 mice suggest that there might be a critical time frame when oxidative
stress can be reduced and disrupt AD progression. In particular, while Vitamin E
administration to young Tg2756 mice significantly reduced both ß-amyloid levels and
plaque formation at 13 months of age, vitamin E supplementation beginning at 14 months
did not reduce ß-amyloid pathology at 20 months of age (Sung et al., 2004). Second, the
effective dose of Vitamin E in the Alzheimer’s mouse model study was around 8 – 10
I.U. per day (Sung et al., 2004) and only a dose of 2 I.U. per day was used in the human
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clinical study (Petersen et al., 2005), so maybe the dose of Vitamin E was too low to have
any effect on mild cognitive impairment progressing into Alzheimer’s disease. Third,
vitamin E reacts on a one-to-one basis with free radical molecules, and it has been
repeatedly stressed that the necessary doses of typical antioxidants would have to be huge
in order to be effective. On the other hand, EUK-207 functions like the enzymes
superoxide dismutase and catalase, and also exhibits reactive nitrogen species scavenging
activities (Doctrow et al., 2002, Sharpe et al., 2002) and penetrates the blood brain barrier
and mitochondria (Hinerfeld et al., 2004). In addition, chronic vitamin E treatment is
effective in protecting against age-associated cognitive impairment and brain oxidative
stress in wild type rodents when treatment is initiated at a fairly young age (Joseph et al.,
1998), but not when treatment starts at a relatively old age (Sumien et al., 2004), where as
Chapter 2 demonstrated that EUK-207 is able to protect against age-associated cognitive
impairment and brain oxidative stress when chronically administered to aged wild type
mice. Therefore, EUK-207 might prove to be more beneficial than vitamin E in
protecting humans against AD.
Although this study appears to be the first of its kind to utilize the 3xTg-AD
Alzheimer’s disease mouse model in order to assess the effectiveness of anti-oxidants in
preventing Alzheimer’s disease associated cognitive dysfunction and both beta-amyloid
and tau pathology a number of other studies using the 3xTg-AD mouse line has revealed
many potential therapeutic approaches for treating or preventing Alzheimer’s disease.
This includes immunization against the beta-amyloid peptide ((Oddo et al., 2006),
promoting neurogenesis in the hippocampus and subgranular zone of the dentate gyrus
via neural stem cell transplantation (Blurton-Jones et al., 2009) or treatment with
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allopregnanolone (Wang et al., 2010), estrogen or androgen hormone therapy (Rosario et
al., 2006) (Carroll et al., 2007), diets rich in omega n-3 polyunsaturated fatty acids
(Green et al., 2007), treatment with methylene blue (Medina et al., 2010) or the N-
methyl-D-aspartate receptor antagonist memantine (Martinez-Coria et al., 2010),
supplementing diets with the non-steroidal anti-inflammatory drug ibuprofen (McKee et
al., 2008) or Chinese celery extract rich in L-3-n-Butylphthalide (Peng et al., 2010), and
using gene therapy to peripherally express the beta-amyloid degrading protease neprilysin
(Guan et al., 2009). However, human clinical trials using A β based immunotherapy were
suspended because of patients developing meningoencephalitis (Check, 2002), and
woman undergoing estrogen replacement therapy appear to be more prone to dementia
(Espeland et al., 2004). In addition, stem cell transplantation and gene therapy are very
technically demanding procedures that have not been completely optimized yet in
humans. SOD/catalase mimetics target reactive oxygen species and reactive nitrogen
species, and thus unlike hormones, N-methyl-D-aspartate receptor antagonist, and non-
steroidal anti-inflammatory drugs, there should be minimal physiological and
psychological side effects during treatment.
My results suggest that oxidative stress might participate in AD pathogenesis
since chronic treatment with EUK-207 initiated before the apparition of the major
pathological features significantly their further development and prevented cognitive
impairments. Results from another study also support this idea; 3xTg-AD mice exhibit
significant brain lipid peroxidation, decreased expression of the oxidative
phosphorylation regulatory enzyme pyruvate dehydrogenase, and increased free radical
leakage from mitochondria before AD pathology is present (Yao et al., 2009). Therefore,
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AD pathology might result from increased oxidative stress due to mitochondria
dysfunction. Several studies have also indicated that ß-amyloid and tau might function as
antioxidants (Smith et al., 2002), and therefore, their accumulation could represent a
mechanism to protect neurons against oxidative stress resulting from mitochondria
dysfunction. However, the A β peptide can also interact with mitochondria by binding to
the mitochondrial protein A β-alcohol dehydrogenase (ABAD), and the binding of A β to
ABAD results in mitochondrial dysfunction (Lustbader et al., 2004, Yan and Stern,
2005). In addition, ß-amyloid induces ROS production in neurons (Harris et al., 1995,
Yatin et al., 1999). Thus, there are still many questions to be answered, and the role of
oxidative stress as the initiator of AD is still not conclusive. AD could possibly be the
result of ß-amyloid and oxidative stress acting together in an additive fashion.
A number of previous studies have demonstrated a potential link between brain
oxidative stress and AD. Although many questions still remain regarding the exact
cellular and molecular mechanisms underlying the connection between brain oxidative
stress and AD, the results from this current study clearly demonstrate a critical
relationship between brain oxidative stress and AD pathogenesis. Furthermore, these
results suggest that brain oxidative stress might be significantly involved in initiating the
progression of AD and that the SOD/catalase mimetic EUK-207 could be useful as a
therapeutic agent for protecting individuals against AD.

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Chapter 5

Conclusion
Age-associated cognitive impairment is a major dilemma facing today’s society,
and since a significant proportion of the population will live longer then previous
generations this problem will only worsen. Therefore, it is imperative to understand the
molecular and cellular mechanisms underlying age-associated cognitive dysfunction, so
that one day a therapy may be available to protect the human population against this
devastating disease. A number of studies have demonstrated a potential link between
brain oxidative stress and age-associated cognitive impairment; however, many questions
remain unanswered. The experiments that were presented in the previous chapters were
designed to specifically define the relationship between brain oxidative stress and mild
age-associated cognitive impairment and the role of brain oxidative stress in Alzheimer’s
disease pathogenesis. The conclusions from these studies clearly show that brain
oxidative stress not only underlies mild age-associated cognitive decline, but also the
more serious form of age-related cognitive dysfunction that is associated with
Alzheimer’s disease. In addition, my results demonstrate that age-dependent alterations
in the autophagy-lysosome pathway and microglia function are linked to brain oxidative
stress and that SOD/catalase mimetics could potentially be used as a viable therapy for
protecting individuals against age-associated mild cognitive impairment and Alzheimer’s
disease.

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5.1. Brain oxidative stress plays a significant role in age-associated
mild cognitive impairment
In addition to a significant decline in cognitive function, a dramatic increase in
oxidized macromolecules within the brain also accompanies the natural aging process in
humans (Smith et al., 1991). Thus, brain oxidative stress might potentially underlie age-
associated cognitive dysfunction. In Chapter 2, I addressed the relationship between
brain oxidative stress and age-dependent cognitive decline by chronically administering
the superoxide dismutase/catalase mimetics EUK-189 or EUK-207 to 17 month-old wild
type mice for 6 months and assessing brain oxidative stress and cognitive function
following treatment. Aged mice administered vehicle alone exhibited a dramatic decline
in cognitive function after 3 months and 6 months of treatment when compared to 16
month-old mice; however, aged-mice administered EUK-189 or EUK-207 did not display
this significant age-associated decline in memory performance after 3 months of
treatment and their cognitive function was considerably better then vehicle treated mice
following 6 months of treatment. In addition, vehicle administered mice exhibited a
dramatic age-dependent increase in lipid peroxidation, oxidized nucleic acids, and free
radical levels within their brains, where as the levels of markers for oxidative stress in the
brains of EUK-189 or EUK-207 treated mice were virtually identical to that found in 16
month-old wild type mice. Furthermore, when memory performance was plotted against
total brain lipid peroxidation or total brain free radical levels for mice that were not
treated with EUK-189 or EUK-207 a significant correlation between brain oxidative
stress and cognitive performance was discovered. The results from this study strongly
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suggest that age-dependent brain oxidative stress plays a critical role in age-associated
cognitive impairment. However, since EUK-189 and EUK-207 administration offered
complete protection against age-associated brain oxidative stress, but only partially
protected against age-dependent cognitive decline, brain oxidative stress might not be the
only significant factor responsible for age-associated cognitive impairment.
5.2. Age-associated changes in the autophagy-lysosome pathway
and microglia function are dependent upon brain oxidative stress
Increased oxidative stress is not the only significant alteration present in the aged
mammalian brain. Age-associated changes occurring within the autophagy-lysosome
pathway and micorglia have also been previously reported. Such age-related changes
include increased autophagosome formation, increased expression and activity of
lysosomal proteases, alterations in microglia morphology and activation, and microglia
density (Rogers et al., 1988, Perry et al., 1993, Nakanishi et al., 1997, Streit et al., 2004,
Gamerdinger et al., 2009). These age-associated changes could potentially be linked to
age-related cognitive impairment because the auotphagic pathway and microglia are
critically involved in maintaining normal neuronal homeostasis. Furthermore, since
aging is also accompanied by brain oxidative stress one might suggest that age-dependent
changes in the autophagy-lysosome system and micorglia function are possibly linked to
an age-associated increase in brain oxidative stress. This hypothesis makes further sense
by the fact that the autophagy pathway and microglia both play critical roles in removing
damaged and potentially toxic cellular debris and one of the consequences of an age-
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associated increase in brain oxidative stress is a dramatic increase in macromolecules
damaged by oxidation.
Chapter 2 demonstrated that the SOD/catalase mimetics EUK-189 and EUK-207
are potent free radical scavengers that can offer significant protection against age-
associated brain oxidative stress, thus these two compounds are excellent tools to use for
assessing the relationship between brain oxidative stress and age-associated changes in
the autophagy-lysosome system and microglia function. Therefore, in Chapter 3 I used
the same treatment paradigm as in Chapter 2 in aged wild type mice except this time
alterations in the autophagy pathway and microglia function were assessed after 6 months
of treatment with EUK-189 or EUK-207. Vehicle treated mice exhibited a significant
increase in autophagosome formation within the brain after 6 months of treatment
compared to 16 month old control mice as assessed by LC3 – I to LC3 – II conversion.
In addition, the expression of the autophagy induction protein beclin-1 was also higher in
the CA1 region of the hippocampus compared to EUK treated groups; however, ATG7
expression, which is also involved in autophagy induction, exhibited an age-dependent
decrease, although it was not significant. Since Chapter 2 revealed that increased brain
oxidative stress does accompany the ageing process one might expect to see such results
because increased autophagosome formation and autophagy induction in the aged brain
might be a response to increased oxidatively damaged proteins and other cellular
components that need to be removed from the cell. In addition to significantly protecting
against age-associated changes in brain oxidative stress, both EUK-189 and EUK-207
also offered significant protection against the age-associated increase in autophagosome
formation that was found in 23 month old vehicle treated mice and reduced the
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expression of Beclin-1 in the CA1 region of the hippocampus, thus demonstrating a
critical relationship between autophagosome formation and autophagy induction and age-
associated brain oxidative stress.
The lysosomal associated protein LAMP1 was also noticeably higher in the CA1
region of the hippocampus of vehicle treated mice compared to EUK treated mice, which
would strengthen the argument that increased brain oxidative stress induces increased
autophagy induction because the lysosome fuses with the autophagosome and is
responsible for degrading the material within the autophagosme. However, western blot
analysis revealed that total brain levels of LAMP1 and LAMP2 actually dramatically
decreased with age and previous reports have shown that unlike LAMP1, the lysosomal
associated protein LAMP2 appears to be necessary for the fusion of the autophagosome
to the lysosome (Andrejewski et al., 1999, Tanaka et al., 2000) Therefore, the age-
associated increase that was observed in autphagosome formation might not be due to an
age-dependent increase in autophagy induction, but more likely to an age-associated
disruption in the ability of lysosomes to fuse with the autophagosome and degrade its
contents. If the latter is the case, then autophagosomes would build up within neurons and
there would appear to be an increase in autophagosome formation, but in reality there is
an increase in autophagosomes because they are not being cleared away sufficiently by
the lysosomes. Furthermore, chronic treatment with EUK-189 or EUK-207 also
significantly protected against this age-associated decline in LAMP2 expression and
increased the expression of the lysosomal cysteine protease cathepsin D in the CA1
region of the hippocampus, which like LAMP2 also appears to be necessary for
autophagosome clearance (Koike et al., 2000 Shacka et al., 2007). Thus, it might be
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reasonable to speculate that there is most likely an accumulation of autophagosomes
within the neurons of the aged brain that is brought on by impaired lysosome function
due to aging, and age-associated brain oxidative stress might specifically disrupt
lysosome function because exogenous SOD/catalase mimetics can protect against age-
associated changes in lysosome function and autophagosome formation.
In addition to assessing age-associated changes to the autophagy-lysosome
pathway, microglia function was also analyzed at the end of the 6 month long chronic
treatment with EUK-189 or EUK-207. The expression of the microglia marker CD11b
(complement receptor 3) was evaluated in the brains of the treated mice because it
mediates phagocytosis in microglia (Fallman et al., 1993, Reichert and Rotshenker, 2003,
Choucair-Jaafar et al., 2010) and since phagocytosis is critically involved in removing
damaged and cytotoxic material it might potentially be involved in removing damage
brought on by brain oxidative stress. Interestingly, total brain expression of CD11b
significantly decreased between 16 months and 23 months of age. Furthermore, EUK-207
offered significant protection against this age-associated decrease in total CD11b levels
within the brain, and compared to vehicle control mice, both EUK-189 and EUK-207
treatment increased the expression of CD11b and the number of CD11b-positive
microglia in the hippocampus, dentate gyrus, and lateral geniculate nucleus at 23 months
of age. These findings suggest that that capacity of microglia to engulf and remove
deleterious material from the brain might become significantly reduced with age. In
addition, since treatment with EUK-207 or EUK-189 protected against this age-
associated change in CD11b expression, the phagocytosis function of microglia, like the
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autophagy-lysosme system, might be disrupted specifically by age-dependent oxidative
damage.
The results I obtained with regards to brain oxidative stress and age-related
changes in the autophagy pathway and microglia function are very interesting because
they suggest that age-associated brain oxidative stress might specifically disrupt the
cellular mechanisms responsible for removing damaged and toxic cellular material.
Thus, damage to neurons brought on by age-associated brain oxidative stress could
potentially be very deleterious because the cellular systems that are in place to remove
the damage are also impaired by brain oxidative stress. In addition, the observed age-
associated increase in free radicals within the mammalian brain might be the result of an
increase in the number of defective mitochondria within the brain and if the autophagy
system is impaired then the ability to remove sick mitochondria will be significantly
reduced and therefore the potential source of ROS will not be removed, thus amplifying
the problem.
5.3. Brain oxidative stress is critically involved in the pathogenesis
of Alzheimer’s disease
Although some cases of AD are hereditary and are due to genetic mutations
affecting the proteins responsible for beta-amyloid processing, the vast majority of AD
cases appear to be sporadic and the mechanisms underlying these sporadic forms of AD
remain unclear. A number of studies performed in vitro have demonstrated a potential
relationship between brain oxidative stress and A β (Harris et al., 1995, Yatin et al., 1999)
and AD brains are marked by damaged mitochondria (Gibson et al., 1998) and increased
133
oxidative stress (Good et al., 1996, Sayre et al., 1997, Gabbita et al., 1998). Thus, age-
associated brain oxidative stress appears to be linked to Alzheimer’s disease; however,
whether or not brain oxidative stress is the cause or consequence of AD remains in
question. In Chapter 2, I demonstrated that EUK-189 or EUK-207 could protect against
the rise in age-associated brain oxidative stress; therefore these SOD/catalase mimetics
could potentially be used to elucidate the relationship between brain oxiditave stress and
AD pathogenesis in vivo. In Chapter 4, I utilized the 3xTg-AD transgenic mouse model
for Alzheimer’s disease, which exhibits both beta-amyloid and tau pathology and
cognitive dysfunction in an age dependent manner. The same SOD/catalase mimetic
chronic treatment paradigm that was used in Chapters 2 and 3 was also employed,
however only EUK-207 was administered this time. The treatment began at 4 months of
age in 3xTg-AD mice, when AD pathology and cognitive impairment was insignificant,
and continued until 9 months of age because at this age there was a significant difference
in cognitive function between vehicle treated wild type and 3xTg-AD mice. In addition
to a significant deficit in cognitive function, 9 month old 3xTg-AD mice also exhibited
significantly higher total brain lipid peroxidation and oxidized nucleic acids in the
hippocampus and amygdala compared to 9 month old wild type mice. Furthermore, there
was no significant decline in cognitive performance in 3xTg-AD mice that received
chronic treatment with EUK-207. Chronic EUK-207 treatment also offered significant
protection against brain oxidative stress and significantly reduced intraneuronal levels of
beta-amyloid and hyperphosphorylated tau protein in the hippocampus and amygdala as
well as significantly reducing total brain levels of the more amyloidogenic protein A β
1-42
.
These results strongly suggest that brain oxidative stress is a critical mediator in the
134
pathogenesis of AD; therefore brain oxidative stress might actually initiate AD
progression. In addition, both AD associated beta-amyloid and tau pathology might be
linked to brain oxidative stress because the SOD/catalase mimetic EUK-207 was able to
significantly reduce both forms of AD brain pathology. Interestingly, it appears that only
intraneuronal beta-amyloid deposits are necessary for impaired cognitive function
because no extracellular beta-amyloid plaques were present at 9 months of age in the
3xTg-AD mouse.
5.4. Superoxide dismutase/catalase mimetics can potentially be
used to protect individuals against age-associated mild cognitive
impairment and Alzheimer’s disease
The results obtained from Chapters 2 and 4 strongly support a relationship
between brain oxidative stress, mild age-associated cognitive impairment, and
Alzheimer’s disease. Thus, one could possibly target age-associated brain oxidative
stress in order to defend against both of these age-related diseases. In light of these
conclusions, administering exogenous antioxidants might potentially be one therapeutic
approach used to overcome the increased oxidative burden of the ageing brain and
therefore protect against age-associated cognitive impairment and AD. In Chapter 2, the
SOD/catalase mimetics EUK-189 and EUK-207 significantly reduced both age-
associated brain oxidative stress and cognitive impairment, and in Chapter 4, EUK-207
dramatically reduced AD associated brain oxidative stress, cognitive dysfunction, and
both beta-amyloid and tau pathology within the brain. Therefore, EUK-189 and EUK-
207 would make ideal candidates for protecting individuals against mild age-associated
135
cognitive impairment and AD. Indeed, other types of antioxidants have been shown to
curb age-associated brain oxidative stress and cognitive impairment (Table 1); however,
the effective doses of EUK-189 or EUK-207 are significantly lower then other
antioxidants, and therefore their doses would be easily extrapolated for use in humans.
Furthermore, unlike typical antioxidants such as Vitamin E, EUK-189 and EUK-207
function like the enzymes superoxide dismutase and catalase, and also exhibit reactive
nitrogen species scavenging activities and penetrate the blood brain barrier and
mitochondria. Vitamin E has been used before in humans to stop mild cognitive
impairment from progressing into AD (Petersen et al., 2005), however it was
unsuccessful, but compared to other studies, EUK-189 and EUK-207 appear to be more
effective then vitamin E in protecting aged rodents against age-associated cognitive
impairment, thus they might be more beneficial then vitamin E in protecting against AD.
The conclusions from my studies demonstrate that treatment with SOD/catalase mimetics
could possibly protect humans from mild age-associated cognitive impairment and AD,
therefore they might potentially only be useful as preventative therapies and not capable
of reversing either of these disorders. Thus, it would be imperative to identify at risk
individuals and began treatment before the disease manifest itself. Individuals with mild
cognitive impairment have a higher chance of developing AD then do the rest of the
general population (Petersen et al., 2001b); therefore patients with mild cognitive
impairment could potentially benefit from treatment with SOD/catalase mimetics.
However, there are no real clear indicators that a person might develop mild cognitive
impairment; thus, it might prove to be more difficult to prevent mild cognitive
impairment with SOD/catalase mimetics.
136
5.5. Age-associated mild cognitive impairment and Alzheimer’s
disease might result from age-dependent brain oxidative stress
disrupting the autophagy-lysosome system and microglia function
The conclusions that were obtained in Chapters 2 through 4 provide evidence for
a potential biological pathway that underlies both age-associated mild cognitive
impairment and AD (Fig. 25). Chapter 2 revealed that there is an age-associated increase
in brain oxidative stress and evidence from Chapter 2 demonstrated that this is most
likely due to a significant age-associated rise in free radical levels within the brain. The
underlying mechanisms behind this age-associated increase in brain free radicals is still
unclear, however previous studies have reported that the aged mammalian brain is also
marked by a rise in the number of damaged and electrochemically deficient mitochondria
that produce significantly higher levels of free radicals (Sawada and Carlson, 1987, Sohal
et al., 1994). Damaged mitochondria are also a hallmark of the human AD brain (Gibson
et al., 1998, Castellani et al., 2002, Parker et al., 1994), therefore, like mild age-
associated cognitive impairment; deficient mitochondria might also potentially be the
source of reactive oxygen species in Alzheimer’s disease.
Chapter 3 showed that age-associated brain oxidative stress might specifically
hinder the clearance of autophagosomes and microglia mediated phagocytosis, and
therefore the mechanisms by which the brain uses to remove damaged and potentially
toxic cellular material from neurons and the surrounding environment might become
significantly disrupted with age. Thus, brain oxidative stress might effect neuron
function and therefore impede on cognitive performance by specifically disrupting the
137
turnover and clearance of damaged organelles, proteins, and other cellular debris from
neurons and the surrounding environment. The fact that exogenous antioxidants could
protect against age-associated brain oxidative stress, significantly reduce age-associated
cognitive impairment, and protect against age-associated alterations in the autophagy-
lysosome system and microglia function further strengthen this relationship between
increased brain oxidative stress, impaired autophagy and microglia function, and age-
associated cognitive dysfunction.
By utilizing an exogenous SOD/catalase mimetic in a mouse model of AD I was
also able to conclude that like mild age-associated cognitive impairment, brain oxidative
stress also underlies the pathogenesis of AD. Since AD also appears to be the result of
the accumulation of A β and neurofibrillary tangles within neurons, one might speculate
that AD is a result of reduced clearance of intraneuronal beta-amyloid and
hyperphosphorylated tau. Therefore, like age-associated mild cognitive impairment,
impaired autophagy and microglia function might also be critically linked to AD
pathogenesis. Furthermore, since chronic treatment with the SOD/catalase mimetic EUK-
207 reduced brain oxidative stress, intraneuronal A β accumulation and
hyperphosphorylated tau in a mouse model of AD, one might also speculate that age-
associated brain oxidative stress significantly impairs both autophagy and microglia
function during AD pathogenesis. Interestingly, previous reports have shown that
autophagy and microglia function are altered in the human AD brain (McGeer et al.,
1987, Lassmann et al., 1995, Yang et al., 1998, Nixon et al., 2005) and individuals with
mild age-associated cognitive impairment have a higher risk of developing AD compared
to the general population (Petersen et al., 2001b). In addition, beta-amyloid can be
138
cleared by phagocytosis mediated by microglia in vitro (Choucair-Jaafar et al., 2010).
Thus, it seems very likely that impaired autophagy and microglia function brought on by
age-associated brain oxidative stress are involved in both mild age-associated cognitive
impairment and AD pathogenesis. Mild age-associated cognitive impairment might just
be the beginning stages of AD and the more severe dementia associated with AD could
be the result of intraneuronal beta-amyloid and hyperphosphorylated tau reaching a
critical limit that is detrimental to the cell because they are not being appropriately
cleared away.
This proposed mechanism is quite striking because it would mean that age-
associated oxidative damage to the brain could amplify itself by impairing the cellular
mechanisms responsible for removing oxidative damage. Furthermore, autophagy is
responsible for removing damaged and unhealthy mitochondria (Kim et al., 2007), which
are potential sources of free radicals, and if brain oxdative stress impedes on autophagy
function then the ability of the cell to remove the source of reactive oxygen species will
be greatly reduced and thus brain oxidative stress will increase further and cause even
more damage to neurons.

Figure 25. Schematic diagram illustrating the potential relationship between the disruption of the
autophagy-lysosome system and microglia function by age-associated brain oxidative stress and the
pathogenesis of age-associated mild cognitive impairment and Alzheimer’s disease. There is an
apparent age-associated rise in brain oxidative stress resulting from an increase in steady state levels of free
radicals. Previous reports have suggested that this age-dependent rise in brain free radicals is possibly due
to an increase in dysfunctional mitochondria within the aged mammalian brain. Age-associated brain
oxidative stress appears to significantly impair the autophagy-lysosme system and microglia function and
therefore there might be a reduction in the ability of the brain to remove damaged and potentially toxic
cellular material from neurons and the surrounding environment, thus disrupting neuron function and
inducing mild cognitive impairment. Like age-associated mild cognitive impairment, Alzheimer’s disease
pathogenesis also appears to be linked to brain oxidative stress. Furthermore, individuals exhibiting mild
age-associated cognitive impairment have a greater risk of developing AD then do the general population.
Therefore, AD might also result from impaired autophagy and microglia function. Thus, clearance of
intraneuronal beta-amyloid and hyperphosphorylated tau might be significantly disrupted during the
progression of mild cognitive impairment into AD and result in the accumulation of beta-amyloid and
neurofibrillary tangles within neurons leading to the more severe form of dementia associated with AD.

139
140
5.6. Closing remarks
The research presented in this thesis strongly supports a role for brain oxidative
stress in both mild age-associated cognitive impairment and Alzheimer’s disease
pathogenesis. Although a lot of insightful knowledge has been gained from these studies,
there are still a lot of questions surrounding the relationship between age-associated
cognitive impairment and brain oxidative stress. Although I did provide some evidence
demonstrating that microglia function and the autophagy-lysosome system might be the
underlying mechanisms involved in cognitive dysfunction induced by brain oxidative
stress, there is still a lot more knowledge to be gained with regards to what other proteins
and cellular components involved in neuron function are also disrupted by brain oxidative
stress. Furthermore, the exact mechanisms by which brain oxidative stress increases with
age still need to be definitively pinned down. In addition, although I did show that
oxidative stress is critically involved in the pathogenesis of AD and I speculated that this
is a result of impaired clearance of A β and hyperphosphorylated tau, the specific
relationship between beta-amyloid accumulation, neurofibrillary tangles and brain
oxidative stress needs to be further assessed. As one can see, the journey to solving the
problems regarding brain oxidative stress and age-associated cognitive impairment is far
from over. However, the conclusions drawn from this dissertation provide a number of
excellent directions to pursue.

141
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Abstract (if available)

Abstract Continuous decline in cognitive performance accompanies the natural aging process in humans, and multiple studies in both humans and animal models have indicated that this decrease in cognitive function is associated with an age-related increase in oxidative stress. Treating aging mammals with exogenous free radical scavengers has generally been shown to attenuate age-related cognitive decline and oxidative stress. I assessed the effectiveness of the superoxide dismutase/catalase mimetics EUK-189 and EUK-207 on age-related decline in cognitive function and increase in oxidative stress. C57/BL6 mice received continuous treatment via osmotic minipumps with either EUK-189 or EUK-207 for 6 months starting at 17 months of age. At the end of treatment, markers for oxidative stress were evaluated by analyzing levels of free radicals, lipid peroxidation and oxidized nucleic acids in brain tissue. In addition, cognitive performance was assessed after 3 and 6 months of treatment with fear conditioning. Both EUK-189 and EUK-207 treatments resulted in significantly decreased lipid peroxidation, nucleic acid oxidation, and reactive oxygen species (ROS) levels. In addition, the treatments also significantly improved age-related decline in performance in the fear-conditioning task. My results thus confirm a critical role for oxidative stress in age-related decline in learning and memory and strongly suggest a potential usefulness for salen–manganese complexes in reversing age-related declines in cognitive function and oxidative load.

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Asset Metadata

Creator Clausen, Aaron David (author)

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

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 05/02/2011

Defense Date 08/31/2010

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

Tag aging,Alzheimer's disease,antioxidants,autophagy,beta-amyloid,cognitive impairment,fear-conditioning,free radicals,Learning and Instruction,lysosome,microglia,OAI-PMH Harvest,oxidative stress,superoxide dismutase/catalase mimetics,Tau

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Baudry, Michel (committee chair), Duncan, Roger F. (committee member), Thompson, Richard F. (committee member), Walsh, John P. (committee member)

Creator Email aarroonn45@hotmail.com,aclausen@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3840

Unique identifier UC1200737

Identifier etd-Clausen-4117 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-457660 (legacy record id),usctheses-m3840 (legacy record id)

Legacy Identifier etd-Clausen-4117.pdf

Dmrecord 457660

Document Type Dissertation

Rights Clausen, Aaron David

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

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