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Studies of intracellular cascades mediating neuronal damage in two animal models of neurodegeneration

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Studies of intracellular cascades mediating neuronal damage in two animal models of neurodegeneration

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Content 1

STUDEIS OF INTRACELLULAR CASCADES MEDIATING
NEURONAL DAMAGE IN TWO ANIMAL MODELS OF
NEURODEGENERATION

By
Xiaobo Xu

University of Southern California

2

Contents
Chapter I: General Introduction .......................................................................................... 6
1. Alzheimer’s disease .................................................................................................... 7
1.1 General features .................................................................................................... 7
1.2. 3xTg-AD Mice Model ....................................................................................... 10
1.3. Oxidative Stress and AD .................................................................................... 13
1.4. Antioxidants in AD treatment ............................................................................ 15
1.5. SOD/Catalase Mimetics ..................................................................................... 16
2. A model of seizure activity: systemic kainic acid injection in rats ........................... 17
2.1 KA targets. .......................................................................................................... 17
2.2. Role of Calpain in cell death and neurodegeneration ........................................ 19
2.3. mTOR pathway .................................................................................................. 21
2.4. Arc...................................................................................................................... 22
2.5. mTOR pathway and epilepsy ............................................................................. 23
2.6. Different effects of seizure activity in adult and immature animals .................. 25
3. Questions addressed in my dissertation work ........................................................... 27
3

Chapter II: Effects of the Superoxide Dismutase/Catalase Mimetic EUK-207 in a Mouse
Model of Alzheimer’s Disease: Interruption of Progression of Amyloid and Tau
Pathology and Cognitive Decline ..................................................................................... 29
Abstract ......................................................................................................................... 30
Introduction ................................................................................................................... 31
Materials and Methods ................................................................................................. 35
Chemicals .................................................................................................................. 35
Mice and treatment ................................................................................................... 35
Behavioral analysis ................................................................................................... 36
Analysis of Alzheimer’s disease pathology and oxidative stress ............................. 37
Statistical Analysis .................................................................................................... 37
Results ........................................................................................................................... 38
1. Effects of SOD/catalase mimetic EUK-207 on fear-conditioning learning in 3xTg-
AD mice .................................................................................................................... 38
2. Effects of SOD/catalase mimetic EUK-207 on brain oxidative stress in 3xTg-AD
mice ........................................................................................................................... 41
3. Effects of SOD/catalase mimetic EUK-207 on beta-amyloid pathology in 3xTg-
AD mice .................................................................................................................... 46
4. Effects of SOD/catalase mimetic EUK-207 on tau pathology in 3xTg-AD mice 49
5. Correlation between behavioral performance and oxidative stress ...................... 53
Discussion ..................................................................................................................... 55
4

Chapter III: Differential effects of kainate-induced seizure activity on PTEN, mTOR and
Arc in hippocampus of adult and neonatal rats ................................................................. 60
Abstract ......................................................................................................................... 61
Introduction ................................................................................................................... 62
Materials and Methods .................................................................................................. 64
Animals and treatment .............................................................................................. 64
Immunohistochemistry ............................................................................................. 64
Western Blots ............................................................................................................ 65
Statistics .................................................................................................................... 66
Results ........................................................................................................................... 66
1. Changes in drebrin, PTEN, mTOR and Arc in hippocampus 4 h after KA-induced
seizure in 2 month-old rats ........................................................................................ 66
2. Effects of calpeptin injection on changes in drebrin, PTEN, p-mTOR and Arc in
hippocampus 4 h after KA-induced seizures. ........................................................... 74
3. Changes in drebrin, PTEN, mTOR, and Arc in hippocampus after KA-induced
seizure in juvenile rats .............................................................................................. 84
Discussion ..................................................................................................................... 89
Chapter IV: Discussion and Conclusions ......................................................................... 94
1. Role of oxidative stress in AD pathogenesis ............................................................ 94
2. Role of calpain activation and its regulation of the mTOR pathway in KA-induced
seizures .......................................................................................................................... 99
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3. Role of oxidative stress in KA-induced seizures .................................................... 105
4. Role of calpain-mediated mTOR pathway activation in AD pathogenesis ............ 106
5. Cross-talks between oxidative stress and calpain-mediated mTOR activation ...... 108
References ....................................................................................................................... 111
6

Chapter I: General Introduction

Many different cascades have been proposed to contribute to neurodegeneration by first
triggering the progressive loss of structure or function of neurons and ultimately a
sequence of events leading to cell death. Two major cascades have been particularly
linked to neurodegeneration: (1) mitochondrial damage, due to increased
formation/accumulation of reactive oxygen species (ROS) and increased Ca
2+
influx,
leading to cytochrome C release into the cytosol, and the resulting activation of apoptotic
cascades, which could in turn exacerbate oxidative stress, thereby producing a positive
feed-back loop, and (2) increased intracellular Ca
2+
concentration, as well as activation of
mitogen-activated protein kinases and extracellular signal-regulated kinases
(MAPK/ERK), which would activate calcium-dependent proteases, triggering further
downstream pathways, including the mammalian target of rapamycin (mTOR) pathway.
In my dissertation, I focused on these two cascades in two models of
pathology/neurodegeneration: a mouse model of Alzheimer’s disease (AD), and the
kainate model of seizure-induced neuronal damage in rats. The triple-transgenic mouse
model of AD (3xTg-AD) was developed by inserting the mutated presenilin gene,
PS1M146V, the amyloid protein precursor gene, APPSwe, and the mutated tau gene,
tauP301L in mice, thus generating mice expressing mutant forms of the amyloid-β
precursor protein, presenilin 1, and microtubule-associated protein tau (Oddo et al., 2003).
These mice exhibit both Aβ and tau pathology, as well as cognitive deficits and neuronal
loss, and are widely used as a model of human AD.
7

Kainic acid (KA) is an analog of glutamate, derived from a seaweed, with agonist
property at several glutamate receptors, which has been shown to have strong agonist
efficacy due to lack of receptor desensitization (Johnston, et al.1974), and is therefore a
potent neurotoxin (Bleakman D et al.,1998). A systemic injection of KA in both adult and
neonatal rats rapidly elicits prolonged seizure activity that lasts several hours (Leite et al.,
2002).
I will first describe in greater details these 2 models of pathology/neurodegeneration, and
discuss the general questions that my dissertation work attempted to answer. In Chapter 2,
I will report the results obtained by using a synthetic SOD/Catalase mimetic, EUK-207,
in the 3xTg-AD mouse model of AD, and the conclusions from these studies. Chapter 3
will be devoted to the results obtained with the KA model of seizure activity focusing in
the early events taking place during the seizures that could lead to the later
neurodegeneration observed in this model. Finally, the general conclusion will discuss
the significance of the results and the proposal that the two pathways that are involved in
neurodegeneration are closely interlinked in a degenerative loop.
1. Alzheimer’s disease
1.1 General features
Alzheimer’s disease (AD) is an age-related neurodegenerative disease, with symptoms of
memory loss in the early stage, and severe dementia later on. The diagnosis is usually
confirmed with behavioral assessments and cognitive tests, but final diagnosis cannot be
made until post-mortem analysis of the brain. While in its mild form, age-related decline
in memory function is not life-threatening, it becomes much more dramatic in the
8

pathological form exhibited by patients with AD. The number of Americans inflicted
with AD is expected to reach nearly 15 million over the next several decades (Salmon et
al., 2002).
AD is characterized by two pathological markers, neuritic plaques, composed of the
amyloid peptide Aβ and neurofibrillary tangles (NFTs), composed of filamentous
aggregates of hyperphosphorylated tau protein (Selkoe et al., 2001). The 37–43 amino
acid Aβ peptides are produced from the proteolysis of a transmembrane protein, the
amyloid precursor protein (APP) (Haass et al., 1992), which requires the sequential
action of the aspartic protease, BACE1 (β-secretase), and γ-secretase, a multiprotein
complex, encoded by presenilin genes (Haass et al., 2004). The length of Aβ peptides is
determined by different BACE1 cleavage sites. The most common forms of Aβ found in
AD brains are Aβ1-40 and Aβ1-42. Aβ1-42 is most prone to aggregation and thus most
susceptible to conformational changes to form amyloid plaques. However, the insoluble
Aβ peptides found in amyloid plaques are less pathogenic than soluble and nonfibrillar
forms of Aβ (Rangachari et al., 2007). Aβ peptides may not play critical roles under
normal physiological condition, since the absence of Aβ does not result in any loss of
physiological function (Luo et al., 2003). The toxicity of Aβ accumulation has been
proposed to be due to several mechanisms, including interruption of ion homeostasis by
elevated intracellular calcium (Mattson et al., 1992) and formation of ion channels
(Arispe et al., 1993), induction of apoptosis (Yu et al., 2006), and ROS formation
(Schilling and Eder, 2011). The neurotoxic properties of Aβ require assembly of the
peptide into oligomers, which indicates that toxicity involves an abnormal regulation of
the processes clearing the peptide and its aggregated variants. The induction of apoptosis
9

via the p53-Bax cell death pathway is probably involved in the process and it also
interferes with ability of Brain-derived neurotrophic factor (BDNF) to activate
downstream pathway to protect cells for damage. One hypothesis for amyloid toxicity is
the “channel hypothesis”, which postulates that the aggregated peptide increases
membrane ion permeability by forming large ion channels, which are voltage-
independent and poorly selective, admitting Ca++, Na+ and K+, disrupt ion gradients,
and trigger apoptosis (Kadowaki H. et al. 2005) A recent study found a non-apoptotic
baseline caspase-3 activity in hippocampal dendritic spines and an enhancement of this
activity at the onset of memory decline in the Tg2576-APPswe mouse model of
Alzheimer's disease, which overexpresses Aβ, indicating that a caspase-3-dependent
mechanism might be involved in the APPSwe mutation-induced neuronal pathology
(D'Amelio M, 2011). Upon activation of caspase-3, calcineurin is activated and triggers
dephosphorylation and removal of the GluR1 subunit of AMPA receptors from
postsynaptic sites, leading to alterations in glutamatergic synaptic transmission and
plasticity, resulting in a deficit in learning and memory (D'Amelio M, 2011). Tau
pathology has also been proposed to be downstream of Aβ peptide pathogenesis.
Mutations in APP or presenilins that result in the accumulation of Aβ peptides could lead
to tau hyperphosphorylation and NFTs formation (Busciglio et al., 1994). Under normal
conditions, the microtubule-associated protein tau participates in the regulation of
microtubule assembly and transport. The neurofibrillary tangles, which are made up of
hyperphosphorylated tau, not only disrupt its normal function, but also exhibit a gain of
toxic function by destabilizing microtubules and impairing axonal transport (Morris et al.,
2011). Despite these findings, the cause of the disease is not yet understood.
10

While the majority of the cases are sporadic, only less than 5% are due to an inherited
familial genetic mutation (Blennow et., 2006). Several gene mutations contribute to AD
pathogenesis. In early 1993, the genetic data showed that the APP gene mutation is one
of the risk factors that lead to early onset Alzheimer's disease (Lannfelt et al, 1993).
APP670/671 (APPSwe) switches metabolism on the cleavage point, and accelerates the
rate of Aβ production (Lannfelt et al, 1993). Presenilins (PS), consisting of two
homologous proteins, PS1 and PS2, play an important role in γ-secretase cleavage of
multiple type I membrane proteins, including APPs and Notch, and in regulating
intracellular protein trafficking and turnover (Annaert, 2002). Mutations in PS1 can lead
to autosomal dominant inheritance of familial Alzheimer's disease (FAD) and result in
impaired hippocampus-dependent associative learning (Wang et al., 2004). Studies
showed that PS1 mutations lead to increased APP expression and greater production of
Aβ1-42 due to alterations in γ-secretase activity (Siman et al., 2000; Moehlmann et al.,
2002). Additionally, studies have also suggested that the apolipoprotein E4 (ApoE 4)
genotype is also a risk factor in in late-onset familial and sporadic AD (Miyata and Smith,
1996; Mazur-Kolecka et al., 2002). The ApoE4 isoform slows down Aβ and NFTs
clearance by binding with senile plaques and NFTs and promotes oxidative stress, thus
accelerating AD progression (Mazur-Kolecka et al., 2002). Genetic screening could
potentially reveal individuals who might be at risk for developing hereditary forms of AD.

1.2. 3xTg-AD Mice Model
The laboratory of Frank LaFerla at the University of California, Irvine, developed a
triple-transgenic mouse model of AD (3xTg-AD). Because AD pathology is associated
11

with both amyloid plaques and neurofibrillary tangles, the triple-transgenic model was
derived by harboring the presenilin mutation, PS1M146V, the amyloid precursor protein
mutation, APPSwe, and the tau mutation, P301L, which resulted in the expression of
human mutant forms of presenilin 1, APP and tau. Therefore, this model is unique as
compared to the previous models, as mice exhibit both plaque and tangle pathology. Two
transgenes were microinjected into single-cell embryos from homozygous PS1M146V
knock-in mice, generating mice with the same genetic background, which progressively
developed plaques and tangles. Since this transgenic model is homozygous, it is easy to
keep the consistency in the colony. This model has been widely used as a model for
human AD. These mice also exhibit cognitive deficits, although they do not exhibit
neuronal loss. Aβ accumulation occurs prior to tangles formation (Mastrangelo and
Bowers, 2008), in a pattern that is very similar to that seen in human AD patients
(Mesulam et al., 2000). These mice begin to show intraneuronal Aβ accumulation in
neocortex at 2 months of age (Mastrangelo and Bowers, 2008), and in cortex, amygdala
and the CA1 region of hippocampus by 6 months of age. Extracellular Aβ plaques also
appear in frontal cortex at 6 months of age, and in hippocampus and other regions of the
cerebral cortex by 12 months of age. Tau pathology in 3xTg-AD mice is also present, but
starts at 12 months of age in neurons of the CA1 region of hippocampus. In our previous
studies, hyperphosphorylated tau proteins were mostly present in the ventral part of
hippocampus. In addition, 3xTg-AD mice also show synaptic dysfunction in the CA1
hippocampal region.
In AD patients the most common symptoms of the disease are cognitive deficits, memory
loss in the early stage, or severe dementia later on as well as psychological changes. The
12

3xTg-AD mouse model also provides a good model to investigate cognitive function
impairment in AD, because learning and memory decline, cognitive deficits and
behavioral changes are also present in the triple-transgenic model. These mice show
deficits in synaptic plasticity, including long-term potentiation, before extracellular Aβ
accumulation and the occurrence of tau pathology. Studies showed that the earliest
cognitive impairment was manifested at 4 months of age as a deficit in long-term
retention, when there was intraneuronal Aβ accumulation in hippocampus and amygdala
(Billings et al., 2005). As intraneuronal Aβ continued to accumulate in hippocampus and
amygdala at 6 month age, these mice showed cognitive deficits in the Morris Water Maze
and passive fear avoidance task and also impaired contextual learning, which requires
both amygdala and hippocampus (Billings, 2005). In our previous studies, the 3xTg-AD
mice showed lower level of freezing rate, as compared to the control group both at 9
months and 13 months and both in contextual and cue test, indicating a deficit in learning
and memory. On the other hand, human AD patients also show emotional and
psychological changes, which are the early indicators of developing AD (Piccininni et al.,
2005). Sterniczuk also observed a higher level of anxiety in the open field, elevated plus
maze, horizontal ladder and passive avoidance tasks in the 3xTg-AD mouse model at
ages ranging from 7.5 to 11 months (Sterniczuk et al., 2010). The emotional changes are
probably due to altered functioning of amygdala resulting from both Aβ and tau
pathology. All these cognitive and emotional changes in the 3xTg-AD mice model
parallels well with the symptoms in human AD patients.
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1.3. Oxidative Stress and AD
Reactive oxygen species (ROS) consist of molecules and free radicals with unpaired
electrons derived from molecular oxygen. There are three main ways to generate ROS.
ROS can be produced through oxidative phosphorylation, which is the last step of cellular
respiration to produce ATP. Normally, an electrochemical gradient is generated and
electrons generated in the electron transport chain, are ultimately added to molecular
oxygen to produce a water molecule by Complex IV of the electron transport chain.
However, when electrons are released from the protein complexes to react with oxygen
with incomplete oxygen reduction, superoxide is generated mainly in Complex I (Turrens
et al., 1980), Complex II (Zhang et al., 1998), and Complex III (Cadenas et al., 1977).
Superoxide can also be generated by nicotinamide adenine dinucleotide phosphate
(NADH) oxidases. NADH oxidases are located in the plasma membrane and transfer an
electron from inside the cell across the membrane to a molecule of oxygen to form a
superoxide anion. Finally, 5-lipoxygenase can also produce superoxide anion. The
superoxide can rapidly form hydrogen peroxide, and collectively, superoxide and
hydrogen peroxide are called reactive oxygen species. As byproducts of cellular
respiration, ROS normally are present at a very low level due to a variety of defense
mechanisms within the mitochondria. Superoxide dismutase (SOD) catalyzes the
conversion of superoxide anions into hydrogen peroxide and oxygen; catalase then
converts hydrogen peroxide into water and oxygen (McCord and Fridovich, 1968).
However, during oxidative stress, the large amount of ROS produced surpasses the
capacity of ROS defense system. For instance, when a large amount of glutamate is
released from the presynaptic terminal and binds to postsynaptic glutamate receptors, it
14

induces a large influx of calcium in postsynaptic cells, which ultimately gets absorbed by
the mitochondria. This large influx of calcium into the mitochondria could result in
mitochondria dysfunction, leading to enhanced ROS production, reduced ATP production
and mitochondria dysfunction (Bubber et al., 2005); this would exacerbate oxidative
stress, resulting in a vicious circle, eventually causing increase mitochondrial membrane
permeability, release of cytochrome c and thereby activation of the apoptotic machinery.
In addition, mitochondrial oxidative stress could trigger numerous redox reactions,
reversibly affecting a variety of biochemical reactions.
A number of studies have shown that AD pathogenesis is associated with increase of
ROS formation in brain. Brains from AD patients exhibit significantly increased levels of
protein oxidation, lipid peroxidation, and DNA and RNA oxidation (Good et al., 1996;
Smith et al., 1997; Butterfield, et al 2001). However, the mechanisms underlying age-
related increase in oxidative stress in AD pathogenesis are still poorly understood, and
this effect could be due to either an increase in free radical production, or an impairment
in free radical defense mechanisms, or a combination of both. This observed age-
dependent increase in free radical production is most likely due to an increase in
mitochondrial produced ROS because mitochondria preparations from the brains of
3xTg-AD mice exhibit a significant age-dependent increase in superoxide and hydrogen
peroxide production. The reason for this age-associated accelerated reactive oxygen
species (ROS) production seen in mitochondria is most likely due to mitochondrial
dysfunction brought on by age-dependent damage to the mitochondria. On the other hand,
several other studies have also reported decrease in the activity and expression of certain
antioxidant molecules in AD. Superoxide dismutase activity and catalase activity both
15

decrease with age in the brain of rats, as do the mRNA levels that encode these two
enzymes (Rao et al., 1990).
While ROS clearly play some role in AD pathogenesis, it is still unknown whether ROS
is a causal factor or a consequence of pathology. A number of AD studies have suggested
that oxidative stress is a downstream mediator of Aβ toxicity, as Aβ could stimulate ROS
accumulation in cultured neurons (Yatin et al., 1999) and astrocytes (Harris et al., 1996).
Other lines of evidence showed that oxidative stress could serve as an initiator of the
pathology, since mitochondrial dysfunction precedes the development of AD pathology
(Monte et al., 2000), and continuous mitochondrial dysfunction and increased ROS
production participate in the progression of AD pathology (Mancuso et al., 2010).
1.4. Antioxidants in AD treatment
Reducing oxidative stress and mitochondrial dysfunction has been extensively attempted
to treat several neurodegenerative diseases, including AD. In both preclinical and clinical
studies, numerous dietary antioxidants and anti-inflammatory compounds have been
investigated regarding their efficacy on both cognitive functions and AD pathogenesis.
Thus, several dietary supplements, including Vitamin E, Coenzyme Q10 (CoQ10),
resveratrol (trans-3,5,4-trihydroxystilbene), curcumin, etc., which are directed at reducing
oxidative stress, have been tested as potential therapy. These anti-oxidants contribute to
the electron transfer in the mitochondrial oxidative respiratory chain, or counters the
activity of free radicals, by giving away an hydrogen atom from an intact hydroxyl group,
thus preventing oxidative damage by free radicals in the mitochondrial membrane
(Matthews et al., 1998). In addition some of the antioxidants also play an anti-
inflammatory role in several neurodegenerative disease pathogenesis (Martin et al., 1999).
16

In animal studies, mice that were fed with a clinically tolerated dosage of resveratrol for
45 days showed diminished plaque formation in medial cortex, stratum and hypothalamus,
indicating that dietary resveratrol may protect against Aβ-induced neuronal damage
(Karuppagounder et al., 2009). In in vitro studies, treatment of hippocampal neurons with
resveratrol significantly reduced ROS production, mitochondrial membrane-potential
disruption and neurotoxicity induced by Aβ by inhibiting AMPK phosphorylation (Kwon
et al., 2010). In clinical trials, the efficacy of the Ginkgo biloba extract, EGb 761, in
dementia of the Alzheimer type and multi-infarct dementia was confirmed in a
randomized, double-blind, placebo-controlled study with 216 AD patients with a daily
oral dose of 240 mg EGb 761 or placebo for a 24-week treatment period (Kanowski et al.,
1996). However, given that most of the antioxidant compounds react on a one-to-one
basis with free radical molecules, to apply such treatments to humans would require
enormous amounts of antioxidant compounds, which might by far exceed the safety
dosage. Therefore, it is urgent to search for a potent antioxidant in AD treatment.
1.5. SOD/Catalase Mimetics
The SOD/catalase mimetic compound EUK-207, is a member of a class of molecules
known as salen-Mn complexes, which 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, it can protect
against damage caused by superoxide, hydrogen peroxide and reactive nitrogen species.
A previous study from our laboratory showed that EUK-207 showed a beneficial effect
for treating pathologies associated with age-related oxidative stress. A 3-month chronic
treatment with EUK-207 starting at 8 months of age significantly improved cognitive
17

function and reduced lipid peroxidation and protein oxidation in mouse brain (Liu et al.,
2003). In addition, in the prevention study with the 3xTg-AD mice, a 5-month chronic
treatment with EUK-207 starting at 4 months of age significantly prevented cognitive
impairment and AD pathological alterations in AD mice by the age of 9 months.
2. A model of seizure activity: systemic kainic acid injection in rats
2.1 KA targets.
Kainic acid (KA), a cyclic compound with a rigid structure analogous to glutamate, is
derived from seaweed and has been shown to have strong potency at glutamate receptors,
due the lack of receptor desensitization and is therefore a potent neuroexitant (Johnston,
et al., 1974) and neurotoxin (Bleakman et al., 1998). A systemic injection of KA in both
adult and adolescent rats elicits prolonged seizures, with characteristic of “wet dog
shakes” within half an hour to an hour, mild to severe intermittent seizures for the next
four to five hours or even death due to respiratory muscle spasm, and this is followed by
long-term spontaneous recurrent seizures (SRS) later, depending on the injection protocol
( Leite et al.,2002). KA has widely been used as a seizure model both in vitro and in vivo
(Carreño et al., 2009; Raedt et al., 2009; Lee, et al., 2011; Bausch, et al., 2006).
The mechanism of KA-induced seizure has been postulated to be due to the binding of
KA to several types of glutamate receptors, including the AMPA and Kainate receptors.
Back in the 1970s, John Olney postulated that KA acted directly on excitatory receptors
(Olney et al., 1979). KA has been reported to be less effective as a striatal neurotoxin
following ablation of glutamergic corticostriatal tract, indicating that KA may act on
presynaptic glutamate receptors (McGeer et al. 1978). Similarly, John Olney suggested
18

that most neurons receive some glutamergic inputs and, therefore, are all sensitive to KA
(Olney, 1979). Later it was shown that KA binds to the AMPA/KA receptors, subtypes of
the ionotropic glutamate receptors (Bleakman et al., 1998). Similar to the glutamate
neurotoxicity pathway, KA toxicity involves first binding to the AMPA/KA receptors,
resulting in membrane depolarization and a large influx of Ca
2+
, which would trigger a
series of cascade reactions, including oxidative stress, mitochondrial dysfunction and
eventually cell death. Pyramidal neurons in CA1 and CA3 of hippocampus exhibit
delayed cell death after KA-induced seizures and the cell death process is preceded by
down-regulation of GluR2, a subunit of AMPA receptors that controls calcium influx by
reducing Ca
2+
permeability (Bleakman et al., 1998). In contrast, GluR2 expression
remains unchanged in the dentate gyrus (DG) granule cells, which are resistant to KA
(Pellegrini-Giampietro et al., 1997; Friedman et al., 1998). This process is also
accompanied by oxidative stress. An increase in ROS production and lactate
dehydrogenase after KA injection indicate a loss of cell membrane integrity and
mitochondrial dysfunction (Cheng et al., 1994; Gluck, 2000; Milatovic et al., 2002). As a
result, cytochrome C is released into the cytoplasm, triggering apoptotic pathways that
lead to neuronal death (Weiss, 2000). Condensed nuclei were observed in adult mouse
brain after intraventricular infusion of KA, suggesting apoptosis in the pyramidal cell
layer of the hippocampus (Nishiyama et al., 1996). Studies also showed that caspase-3
activation occurred at 30 h after KA administration (Faherty et al., 1999). On the other
hand, KA injection also interferes with inhibitory neural transmission. In particular,
sprouting of GABAergic terminals has been observed following intrahippocampal KA
injection, indicating that anomalous inhibitory synapses may also contribute to the
19

chronic KA hippocampal hyperexcitability (Davenport et al., 1990). Finally, KA-induced
seizures also cause both apoptotic and necrotic death of neurons, suggesting the existence
of multiple death pathways (Van Lookeren, 1995; Humphrey, 2002; Montgomery, 1999).
2.2. Role of Calpain in cell death and neurodegeneration
The calpain family comprises calcium-dependent, non-lysosomal cysteine proteases,
expressed ubiquitously in mammal tissues and other organisms. The two main isoforms,
μ-calpain and m-calpain are defined by their calcium requirements, which are micro- and
nearly millimolar concentrations of Ca
2+
, respectively. In the brain, while μ-calpain is
found mainly in the cell body and dendrites of neurons and activated by synaptic NMDA
receptors (Lenzlinger, et al., 2000), m-calpain is mainly localized in glia and a small
amount in axons and can be activated by extrasynaptic NMDA receptors (Belcastro et al.,
1996). Both isoforms cleave numerous cellular proteins and thus contribute to many
physiological and pathological processes, including cell motility, cell cycle progression,
aging and apoptotic cell death. We recently reported that, while µ-calpain is calcium
dependent, m-calpain can be activated via mitogen-activated protein kinase and
extracellular signal-regulated kinase (MAPK/ERK)-mediated phosphorylation,
independently of calcium (Zadran et al., 2010). Moroever, we also recently found that m-
calpain selectively mediates the degradation of the phosphatase and tensin homolog
deleted on chromosome 10 (PTEN), thus resulting in the stimulation of local protein
synthesis through the mammalian target of rapamycin (mTOR) pathway (Briz et al.,
2013).
Increasing evidence suggests that calpain plays critical roles in neuronal injuries and
neurodegneration, including AD, Parkinson’s diseases, Huntington’s disease, cerebral
20

ischemia and epilepsy (Bernath et al., 2006; Wang et al., 2013). Although the exact
mechanisms by which calpain is involved in these pathologies are not very well
understood, several pathways have been proposed. It is well known that calcium overload
could lead to significant neuronal injury (Barsukova et al., 2011) and the ERK/MAPK
pathway has also been reported to be activated in neurodegeneration (Colucci-D'Amato et
al., 2003). Calpain activation as been postulated to trigger intrinsic apoptotic cascades
through: 1) cleavage of the sodium and calcium exchanger, resulting in mitochondrial
calcium accumulation; 2) truncation of various cytoskeletal proteins, leading to increase
in plasma membrane permeability; 3) cleaving the apoptotic proteins Bax and Bid,
producing cytochrome C release (Oh et al., 2004); 4) cleavage of p53, another protein
implicated in neuronal cell death (Saulle et al., 2004). On the other hand, recent studies
also suggest that calpain contributes to neurodegeneration via regulation of the cyclin-
dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase. Cdk5 is activated
by a neuron specific activator, p35. Calpain cleaves p35 to p25, which binds Cdk5 to
form a hyperactive and more stable complex, resulting in aberrant phosphorylation of
various substrates, and enhancing misfolded protein accumulation (Shukla et al., 2011).
While calpain exhibits a wild range of pathologic properties, it remains unclear which
isoform of calpain participate in neuropathology. High intracellular levels of calcium and
ERK activation could probably occur under pathological conditions, and several authors
have suggested that m-calpain might be implicated in pathology. Alternatively, µ-calpain
has been proposed as a pathologic isoform, as knockdown studies have shown that down-
regulation of μ-calpain attenuated spectrin proteolysis following transient forebrain
ischemia, prevented the loss of hippocampal CA1 neurons, and preserved
21

electrophysiological function, while m-calpain knockdown was less effective in reducing
calpain activity and improving the survival of hippocampal CA1 pyramidal neurons
(Bevers et al., 2010). However, the roles of different calpain isoforms in
neurodegeneration remain to be determined. Because calpain could play an important role
in controlling cell death and neurodegeneration, several laboratories have developed
calpain inhibitors as a therapeutic strategy for neurodegenerative diseases (Buki et al.,
2003; Carragher, 2006). However, so far, this approach has failed and there are no
calpain inhibitors in clinical trials, or in early stages clinical development.
2.3. mTOR pathway
The mammalian target of rapamycin (mTOR) pathway regulates multiple cellular
functions, which may influence neuronal excitability and epileptogenesis, including
protein synthesis, cell growth and proliferation, synaptic plasticity and cell death
(Sarbassov et al., 2005; Sandsmark et al., 2007; Tsang et al., 2007). mTOR has two
complexes, mTORC1, which can be inhibited by rapamycin, containing raptor
(regulatory associated protein of mTOR), mLST8 (also known as GβL) and PRAS40
(proline-rich Akt substrate 40 kDa), and mTORC2, which is not sensitive to rapamycin,
containing the mLST8 protein, rictor (rapamycin-insensitive companion of mTOR) and
sin1 (stress-activated protein kinase-interacting protein) proteins (Huang et al., 2009).
The mTOR pathway is regulated through several cellular signaling pathways, including
growth factors, nutrients, hormones, cellular metabolic rates and stress conditions (Russo
et al., 2012). The major pathway is through growth factor receptor activation, including
insulin-like growth factor receptor (IGFR), platelet-derived growth factor receptor
(PDGFR), and epidermal growth factor receptor (EGFR) (Fingar and Blenis, 2004). The
22

activation of growth factor receptors directly stimulate the PI3K/AKT signal transduction
pathway. PI3K catalyzes the phosphorylation of membrane-bound phosphatidylinositol
(4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 then
works on AKT. In addition, AKT can also be phosphorylated by phosphoinositide-
dependent kinase-1 (PDK-1). PTEN inhibits the PI3K/AKT pathway, serving as an
upstream inhibitor of the mTOR pathway. PTEN has a phosphatase domain and a C2
domain. The C2 domain brings the enzyme and binds the phospholipid membrane and the
phosphatase domain de-phosphorylates PIP3 to PIP2 (Lee et al., 1999). AKT indirectly
activates mTOR via dissociation of the TSC1/TSC2 complex (tuberous sclerosis
complex), thus releasing the TSC2-mediated inactivation of the Ras family of small
GTPases, known as the Ras homolog enriched in brain (RHEB). On the other hand,
mTOR can also be negatively regulated through Adenosine 5′-monophosphate-activated
protein kinase (AMPK), which senses the ratio of cellular adenosine 5′-monophosphate
(AMP)/adenosine triphosphate (ATP). Therefore, in a catabolic state, activation of
AMPK phosphorylates TSC2, thus inhibiting mTOR activity (Russo et al., 2012). In
response to hypoxia, REDD1 also acts on TSC2 to inhibit mTOR. Downstream of the
mTOR pathway, mTORC1 initiates mRNA translation via activation of the p70
ribosomal S6 kinase 1 (S6K1) and the eukaryotic initiation factor 4E binding protein-1
(4E-BP1) (Ryther and Wong. 2012).
2.4. Arc
Arc, activity-regulated cytoskeletal protein, an unusual plasticity-associated molecule, is
also downstream of the mTOR pathway and has been postulated to play a critical role in
activity-induced synaptic plasticity associated with LTP consolidation. Following
23

neuronal activity of the type seen with tetanic stimulation, the Arc mRNA is transported
out of the nucleus to the recently activated dendrites and synapses, translated into protein
and integrated into the N-methyl-D-aspartate (NMDA) receptor complex (Lyford et al.,
1995). Arc synthesis is also required for phosphorylation of cofilin, and the stable
expansion of the actin network (Bramham et al., 2009). In addition Arc can also
negatively regulate AMPA receptors through their removal from the plasma membrane
by interacting with dynamin and endophilin, and clathrin-mediated endocytosis
(Chowdhury et al., 2006). While increased Arc expression and synthesis have been well
documented following a large number of experimental conditions, there is no report of
changes in Arc levels in hippocampus following KA-induced seizures.
2.5. mTOR pathway and epilepsy
The mTOR pathway has been repeatedly involved in epileptogenesis. (Sarbassov et al.,
2005; Sandsmark et al., 2007; Tsang et al., 2007; Talos, 2012). Expression of several
upstream activators of the mTOR pathway, such as epidermal growth factor (EGF),
hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF), have
been reported to be enhanced in epilepsy in TSC patients, suggesting that enhanced
expression of these growth factors may contribute to proliferation and cellular growth
induced by epilepsy (Parker, 2011). Similarly, recent studies showed an increase in both
BDNF mRNA and protein levels in surgically resected hippocampi from epileptic
patients as compared to control (LaFrance, 2010). Increased growth factors or other
nutrients stimulate P13k/Akt and activate the mTOR pathway. In addition, PTEN, an
inhibitor of the P13k/Akt pathway, is also upstream from mTOR. PTEN gene mutations
have been associated with many abnormal phenotypes, including macrocephaly, mental
24

retardation, epilepsy, and cancers. We recently reported that PTEN was a selective
substrate for m-calpain, and that calpain-mediated PTEN degradation represents a
necessary step linking BDNF receptor activation with mTOR activation and stimulation
of local protein synthesis. Many studies have previously provided evidence that calpain is
activated after seizure onset. In particular, calpain activation, as assessed with increased
levels of a specific calpain-generated breakdown product of the cytoskeletal protein,
spectrin (sbdp), was demonstrated in different neuronal populations at various times after
seizure initiation (Bi et al., 1996). Thus, sbdp first appeared at 1 h after seizure initiation
in selected interneurons in stratum oriens and in the hilus of the dentate gyrus; 4 h after
seizure onset, sbdp was increased in the cell bodies of CA3 pyramidal neurons as well as
in neurons in thalamus and piriform cortex. Five days after seizures, sbdp was massively
increased in dendrites and cell bodies of pyramidal neurons in field CA1 and CA3 but not
in the dentate gyrus. Clinical studies have also shown increased μ-calpain expression in
the anterior temporal neocortex in patients with intractable epilepsy (Feng et al., 2011).
Treatment with the calpain inhibitor MDL28170 significantly prevented
neurodegeneration in CA1 region after status epilepticus (Araújo et al., 2008) and
neuronal death after KA-induced seizure mice was decreased in mice overexpressing the
endogenous calpain inhibitor, calpastatin, or increased in mice lacking calpastatin
(Higuchi et al., 2005). All these data clearly indicate that calpain is activated and plays an
important role in the hours and days following seizure activity. It was therefore logical to
assume that calpain-mediated PTEN degradation would lead to mTOR activation in
hippocampus following seizure onset.
25

Several downstream effectors of mTOR regulate protein synthesis and synaptic plasticity
after seizure onset. Increased levels of phospho-p70S6Kinase (p-p70S6K), a downstream
target of mTOR, were first observed about 1 h after seizure onset, with a peak at 3-6 h
and a return to baseline after 24 h in both hippocampus and neocortex, followed by a
second increase 3 days later, with a peak after 5-10 days, which lasted for several weeks
in both hippocampus and neocortex after KA-induced seizure, indicating that mTOR
signaling participates in both the acute phase of seizure activity and chronic
epileptogenesis (Zeng et al., 2009). In addition, p27, a cyclin-dependent kinase inhibitor,
which is also a calpain substrate and arrests cell cycle at the G1-S phase, has been shown
to regulate protein synthesis after seizures. P27 was down-regulated at 2 h after KA-
induced seizures in mice, and p27 knockout mice showed enhanced KA-induced seizures
and hippocampal degeneration (Ueyama et al., 2007). Accordingly, cell proliferation in
the dentate gyrus starts to increase 2 h after seizure onset and lasts for 40 days, probably
because of the shortened progenitor cell cycle due to decrease in p27 (Varodayan et al.,
2009). Local protein synthesis regulated by mTOR could participate in neural plasticity
and pathological changes through rapid changes in several proteins, including Arc. We
were therefore particularly interested in testing the role of calpain activation in the
regulation of pMTOR and Arc levels following KA-induced seizures.
2.6. Different effects of seizure activity in adult and immature animals
Systemic injection of kainate results in repetitive seizure activities in both adult and
immature rats but the mechanisms of epileptogenesis and the resulting pathological
alterations are quite different. It is well known that the immature brain is more prone to
seizures due to an unstable status of excitation and inhibition balance, and a decreased
26

seizure threshold (Holmes, 1997). A transient overexpression of glutamate receptors, and
a relative lack of GABAergic inhibitory transmission in immature brain have been shown
to contribute to increased susceptibility for seizures in immature brain (Jensen, 1999).
However, while kainate-induced seizure results in neurodegeneration in the limbic
system of adult rats, immature rats have been shown to be resistant to the neurotoxic
effects of kainate. It was noticed that there were more long-term deficits in learning and
memory, more cell loss and cell death in adult rats as compared to immature rats after
kainate-induced seizures. Numerous cell loss, degenerating dentate hilar neurons and
pyramidal cells in CA1 and CA3 regions in hippocampus have been observed in adult
rats two weeks following KA-induced seizure, but significantly less hippocampus
damage in 16 day old rats. (Haas et al., 2001). Long-term deficits in learning and memory
caused by status epilepticus in the immature P12 brain have been reported to be less
severe as compared to P20 brain, where cell loss and mossy fiber sprouting were
observed (Cillio et al., 2003). In addition, inflammation, angiogenesis and blood brain
barrier (BBB) leakage in P21 rats after seizures were observed and spontaneous seiures
were developed 1 week and 4 months after pilocarpine-induced status epilepticus but
none of the corresponding changes were found in P9 rats (Marcon et al., 2009). Likewise,
we previously reported that calpain activation in hippocampus was significantly different
following KA-induced seizure activity in postnatal and adult rats (Bi et al., 1997). It was
therefore interesting to investigate whether the KA-induced activation of the pathway
calpain-  PTEN----mTOR- local protein synthesis was similar or different in adult and
neonatal rat hippocampus.
27

3. Questions addressed in my dissertation work
During my dissertation studies, I attempted to address several general questions raised by
the studies discussed above. I was therefore particularly interested in answering the
following questions:
- Is ROS accumulation a consequence or a cause of pathology associated with
neurodegeneration? As discussed, many studies have shown that ROS accumulation is
present in many experimental models of neurodegeneration, and it has often been
assumed that ROS accumulation is a consequence of the neurodegeneration and results
from dysfunctional mitochondria, or increased activity of NADP oxidase. I will argue
that in fact, ROS accumulation is an early event in many models of neurodegeneration
and is in fact a causal factor in neurodegeneration.
- Does calpain activation precede ROS accumulation or is it due to ROS accumulation?
As briefly discussed above, calpain activation is also observed in many models of
neurodegeneration, and it is therefore important to understand whether calpain activation
precedes ROS accumulation and could be responsible for ROS accumulation or whether
calpain activation is a consequence of ROS accumulation.
- Are different isoforms of calpain associated with different neurodegeneration-associated
cascades? We now know that the 2 major calpain isoforms, µ- and m-calpain, often play
opposite effects in regulating cellular functions. It was therefore important to determine
whether these 2 isoforms play different roles in the neurodegeneration models I studied in
my dissertation work.
28

As will be discussed in Chapter II, in the case of AD, my studies show that oxidative
stress serves as a mediator in AD pathogenesis because ROS production accompanies Aβ
and tau pathology (Clausen et al., 2012); moreover, calpain activation has also been
shown to be present before the onset of pathology (Saito et al., 1993).
In contrast, Chapter III will show that following kainate-induced seizures, calpain
activation can be detected shortly after seizure onset, as were stimulation of several
cascades including the mTOR pathway; these events contrast with those observed in
neonatal rats, where there is no calpain activation and no obvious neurodegeneration.
However, whether ROS production/accumulation is upstream or downstream from
calpain activation is still unknown and the role of calpain as an initiator of the mTOR
pathway remains to be determined.

29

Chapter II: Effects of the Superoxide Dismutase/Catalase Mimetic EUK-207 in a
Mouse Model of Alzheimer’s Disease: Interruption of Progression of Amyloid and
Tau Pathology and Cognitive Decline

Xiaobo Xu, Aaron Clausen, Xiaoning Bi and Michel Baudry

Neuroscience Program
USC
Los Angeles, CA 90089-2520
and
Western University of Health Sciences
Pomona, CA 91766

30

Abstract
Alzheimer’s disease is characterized by progressive memory loss and cognitive deficits,
accumulation of ß-amyloid plaques and intracellular neurofibrillary tangles within the
brain, and neuronal death. In addition to ß-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. In the companion study we 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,
starting before the onset of pathology and cognitive deficits, and continuing until 9
months of age, when the AD phenotype is established. In the present study, we initiated
the treatment after the onset of pathology at 9 months of age. After 3 months of treatment,
cognitive performance, brain ß-amyloid and tau pathology as well as oxidative stress
were analyzed. At 12 months of age, 3xTg-AD mice exhibited a decline in performance
in both contextual and cued fear memory tasks as compared to wild-type mice; EUK-
207-treated 3xTg-AD mice did not display any deficit in fear conditioning performance,
as compared to wild-type controls. Chronic treatment with EUK-207 protected against
increased levels of oxidized nucleic acids and lipid peroxidation in brain and reduced ß-
amyloid, tau and hyperphosphorylated tau accumulation in amygdala and hippocampus
of 3xTg-AD mice. Our results thus confirm a critical role for oxidative stress in AD
progression and strongly suggest the potential usefulness for salen-manganese complexes
as a treatment for AD.

31

Introduction
Alzheimer’s disease (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 of senile plaques made up of aggregated extracellular
amyloid- ß (Aß) peptides and intracellular neurofibrillary tangles mainly composed of
phosphorylated tau protein (Selkoe, 2001). In addition, AD also results in synapse loss
and neuronal death in hippocampus, entorhinal cortex, basal forebrain, and neocortical
association cortices (Dekosky et al., 1996). Although the pathology associated with AD
has been well established, the mechanisms for initiation and progression of the disease
remain unclear. Genetic studies performed in families with hereditary forms of AD point
to impaired Aß metabolism, as mutations in the genes that encode for the amyloid- ß
protein precursor (AßPP) (Goate, et al., 1991), presenilin-1, and presenilin-2 (Cruts and
Van, 1998) result in increased Aß levels in brain (Hardy and Selkoe, 2002). However,
familial forms of AD account for less than 5% of all cases, and the vast majority of AD
patients are sporadic (Harman, 2006). Studies performed in transgenic mouse models of
AD have revealed that intraneuronal Aß might be the first pathological event in AD,
which is then followed by the deposition of extracellular Aß and finally neurofibrillary
alterations (Oddo et al,. 2003). These and other results have led to the development of
the “amyloid cascade hypothesis”, in which increased Aß formation triggers the
progression of the disease and is responsible for AD-associated cognitive dysfunction and
32

neurodegeneration. In vitro and in vivo experiments have demonstrated that the Aß
peptide is neurotoxic (Pike et al., 1993; Geula et al., 1998) and that intraneuronal Aß
accumulation is sufficient to disrupt synaptic function (Oddo et al,. 2003) and memory
formation (Oddo et al., 2003). However, multiple clinical trials directed at manipulating
this cascade have so far failed to produce convincing results in favor of this hypothesis
(see http://www.alz.org for a list of clinical trials discontinued or still ongoing).
Moreover, these studies do not explain what causes increased accumulation in
intraneuronal Aß nor do they identify possible downstream mediators of Aß -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. Protein oxidation (Good et al., 1996; Butterfield et al., 1997; Smith et
al., 1997) lipid peroxidation (Sayre et al., 1997; Butterfield et al., 2001; Butterfield et al.,
2001) and DNA and RNA oxidation (Gabbita et al., 1998; Nunomura et al., 1999; Lovell
et al., 2001; Nakanishi and Wu, 2009) are significantly elevated in AD brains. In addition,
the Aß peptide may be responsible for AD-associated oxidative stress, as it induces
production of reactive oxygen species (ROS) 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 peptides (see (Varadarajan et al., 2000) for review), and treating rat synaptic
plasma membranes with Aß 1-40 increased lipid peroxidation (Avdulov et al., 1997).
Oxidative stress could also potentially be responsible for the neurodegeneration observed
in AD because ROS production accompanies Aß-induced neuronal apoptosis and the
33

antioxidants alpha-tocopherol and N-acetylcysteine inhibit Aß-induced neuronal
apoptosis (Tamagno et al., 2003).
While there is strong evidence linking AD with oxidative stress, whether or not oxidative
stress is the initiator or a product and mediator of its pathogenesis remains unclear.
Mitochondrial abnormalities have been shown to precede the development of
neurofibrillary tangles in AD (Monte et al., 2000; Hirai et al., 2001) and in mouse models
of AD (Yao et al., 2009); moreover, recent reports indicate that superoxide might play a
causal role in several manifestations of AD (Ma et al., 2011; Massaad et al., 2009). Part
of the difficulty to test the role of oxidative stress in AD originates from the lack of
quantitative information regarding the levels of free radicals that are continuously
generated under physiological as well as pathological conditions, the low levels of
antioxidants that can be administered, and their limited brain penetration (Lee et al.,
2010). Because signs of oxidative stress appear before the development of neurofibrillary
tangles in AD and in mouse models of AD, it has been suggested that oxidative stress
contributes to the initiation of the disease. In addition, recent evidence indicate that the A
peptides directly interact with mitochondria (Borger et al., 2011; Tillement et al., 2011),
and it has also been proposed that continuous mitochondrial dysfunction and increased
ROS production participate in the progression of AD pathology (Mancuso et al., 2010;
Muller et al., 2010). In support of this mechanism, several reports have indicated that
antioxidants ameliorate mitochondrial function (Dragicevic et al., 2011; Manczak et al.,
2010; Picone et al., 2009; Rhein et al., 2010; Simpkins et al., 2010); however, very few
studies have convincingly demonstrated the usefulness of antioxidant treatment in mouse
34

models of AD or in human AD (Lee et al., 2010; Bonda et al., 2010; Darvesh et al., 2010;
Wang et al., 2008).
The present study utilized the 3xTg-AD mouse model to further assess the relationship
between AD pathogenesis and oxidative stress. These mice express mutant forms of the
amyloid- protein precursor 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. They exhibit deficits in various learning tasks and develop many
pathological features of AD before 9 months of age (Oddo et al, 2003). Thus we tested
the effects of chronic treatment of 3xTg-AD mice with the SOD/catalase mimetic EUK-
207, which we previously showed to significantly reduce age-associated cognitive
impairment and oxidative stress in middle aged and aged wild-type mice (Clausen et al.,
2010), starting after the occurrence of AD pathology (9 months of age), on learning and
memory and AD-like pathology. EUK-207 has a number of features that make it a perfect
tool to study the role of oxidative stress in the progression of the disease. It has
previously been shown to reduce age-related increase in oxidative stress and decrease in
cognitive impairment (Clausen et al., 2010), to act as a mito-protectant (Melov et al.,
2011; Morten et al., 2006) and to cross the blood-brain barrier (Melov et al., 2011) and
the use of minipumps to produce its continuous delivery provides for a constant level of
the molecule for long periods of time. The results demonstrate that oxidative stress is
crucial in AD development and progression and provide strong support for using
SOD/catalase mimetics to protect against AD progression.
35

Materials and Methods
Chemicals
EUK-207 was synthesized as described previously (Doctrow et al., 2002). All other
chemicals were purchased from Sigma, unless indicated otherwise.
Mice and treatment
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. Twenty-four 8-month-old C57BL/6J/129S male mice were
purchased from the Jackson Laboratory (Bar Harbor, ME) and 24 9-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 35 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 9-month-old mice according to
the manufacturer’s recommendations. Briefly, mice were anesthetized with ketamine (80
36

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).
During the 3-month treatment, pumps were replaced 2 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 3-month treatment in order to assess the effects of
repeated surgery and EUK-207 on overall health.
Behavioral analysis
The procedures for training, testing and analyzing fear conditioning learning were the
same as those described in the companion manuscript.
In order to control for potential sensory deficits, mice were also tested for visual function
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 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
37

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.
As shown in Table 1, there were no differences between all 4 groups of animals on all
these measures.
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 Aβ1-42 levels. The other half
was placed in 4 % paraformaldehyde in 1xPBS pH 7.4 and fixed for 24 hrs at 4 ºC for
immunohistochemical analysis of pathology and oxidative stress. All procedures for
evaluation of oxidative stress and amyloid and tau pathology were identical to those
described in the companion manuscript.
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.
38

Results
1. Effects of SOD/catalase mimetic EUK-207 on fear-conditioning learning in 3xTg-AD
mice
Table 1: Body weight, nociception, and vision test results for EUK-207 treated mice

Treatment Body weight (g),
mean ± S.D.
Tail-flick latency
(sec),
mean ± S.D.
Forepaw reaching
(% of success)
Vehicle Non-Tg 40.0 ± 7.0 2.12 ± 0.74 100
EUK-207 Non-Tg 40.2 ± 5.0 2.35 ± 0.67 100
Vehicle 3xTg-AD 41.1 ± 6.4 2.09 ± 0.42 100
EUK-207 3xTg-AD 38.1 ± 4.6 1.98 ± 0.37 100
Summary of the data for body weights, tail-flick latency on the hot plate (nociception)
and forepaw reaching (vision) after 5 months of treatment with vehicle or EUK-207.
There were no statistically significant differences for the three parameters between the
different animal groups.
39

Figure 1. Chronic treatment with EUK-207 starting at 9 months of age protects against
deficits in contextual and cued fear conditioning in 12 month-old 3xTg-AD mice.
Nine 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 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 and
cue test (*p <0.01 vs. 3xTg-AD vehicle), as was the effect of EUK-207 on context and
cue test († p <0.05 3xTg-AD EUK-207 vs 3xTg-AD vehicle).
40

3xTg-AD mice were treated continuously with EUK-207 administered through
subcutaneously implanted osmotic minipumps for 3 months starting at 9 months age.
Treatment began when AD pathology and cognitive impairments are already present
(Oddo et al, 2003). Cognitive performance was assessed at 12 months of age in Non-Tg
and 3xTg-AD using a contextual and cued fear-conditioning paradigm. Twelve 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. 1). Chronic treatment with EUK-207 significantly increased the freezing
response in 12 month-old 3xTg-AD mice in both the context and cue test. 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. 1), therefore suggesting that EUK-207 was able to
prevent the development of cognitive deficits. 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 12 months of age, 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. 1). No significant defects in vision or nociception were
present between Non-Tg and 3xTg-AD mice (Table 1), and the prolonged treatment with
EUK-207 did not appear to produce any ill effects or weight changes (Table 1),
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.
41

2. Effects of SOD/catalase mimetic EUK-207 on brain oxidative stress in 3xTg-AD mice

Figure 2. Chronic treatment with EUK-207 starting at 9 months of age significantly
reduces lipid peroxidation in brain homogenates from 12 month-old 3xTg-AD mice.
At the end of the 3-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 12 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. ** p < 0.01 vs Non-Tg Vehicle
(One-way ANOVA); † p < 0.01 vs 3xTg-AD Vehicle.
In addition to dense extracellular beta-amyloid plaques and intracellular neurofibrillary
tangles, brains from AD patients also exhibit significant oxidative stress. Similarly, 3xTg-
AD mice also exhibit a striking increase in brain oxidative stress beginning at about 3–5
months of age. Therefore, we assessed lipid peroxidation and oxidized nucleic acids in
42

the brains of 3xTg-AD mice that started receiving chronic EUK-207 treatment after 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 3 month-long treatment 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 lipid peroxidation (levels of
equivalent malondialdehyde). For comparison, lipid peroxidation was also assessed in
detergent-soluble fraction from 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 12 months of age (Fig. 2). 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. 2). Compared to Non-Tg vehicle control mice, Non-Tg mice
treated with EUK-207 also displayed a significant decrease in brain lipid peroxidation
levels (Fig. 2).
43

Figure 3. Chronic treatment with EUK-207 starting at 9 months of age reduces oxidized
guanine levels in hippocampus and amygdala of 12 month-old 3xTg-AD mice.
Chronic treatment with EUK-207 starting at 9 months of age reduces oxidized guanine
levels in hippocampus and amygdala of 12 month-old 3xTg-AD mice. At the end of the
3-month treatment, mice were sacrificed and brains were fixed using 4%
paraformaldehyde. The brains were sectioned at 30 um and labeled for oxidized guanine
using immunohistochemistry. Staining for oxidized guanine was assessed in the
hippocampus and amygdala of non-Tg and 3xTg-AD mice treated with vehicle or
EUK207 (A) and quantified in the soma of CA1 pyramidal cells (B) and in amygdala (C)
by counting pixels. Levels of oxidized guanine were expressed as % of non-Tg vehicle.
Insets are full size images of dorsal CA1 pyramidal cells acquired at 50 X (A). Levels of
44

oxidized guanine were expressed as % of non-Tg vehicle for the hippocampus (B) and
amygdala (C). Shown are means±SEM of 4 mice per group for hippocampus (B) and
amygdala (C). Bonferroni post-test analysis following two-way ANOVA indicated that
the difference in oxidized guanine levels between non-Tg vehicle and 3xTg-AD vehicle
mice was significant for the hippocampus (*p < 0.01 versus non-Tg vehicle) and
amygdala (*p < 0.01 versus non-Tg vehicle), as was the effect of EUK-207 on
hippocampus and amygdala (*p < 0.01 versus respective vehicle).
Oxidized DNA and RNA within the brains of 3xTg-AD mice were also assessed at 12
months of age. Mice were sacrificed after the 3 month-long treatment with EUK-207 and
their brains were fixed in 4 % paraformaldehyde and processed for
immunohistochemistry. Brains sections were then probed with an antibody specific for
oxidized guanine. For comparison, oxidized guanine was also assessed in brain sections
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. We 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 (Philips and LeDoux, 1992), whereas the hippocampus is critical for contextual
fear memories (Maren et al., 1997 ; Riedel et al, 1997 ; Kim and Fanselow, 1992, Phillips
and LeDoux, 1992). Oxidized guanine was present throughout the hippocampus of Non-
Tg and 3xTg-AD mice (Fig. 3). Oxidized guanine was present in dentate gyrus, and CA1,
CA2, and CA3 fields of hippocampus (Fig. 3). Staining was observed in both cytoplasm
45

and nucleus, thus indicating that nuclear DNA, mitochondrial DNA, and mRNA were
probably oxidized (Fig. 3). Although oxidized guanine was present in both Non-Tg and
3xTg-AD hippocampus, the intensity of staining was greater in hippocampus of 3xTg-
AD vehicle mice (Fig. 3). Chronic treatment with EUK-207 beginning at 9 months of age
reduced the level of oxidized guanine in hippocampus of 12 month-old 3xTg-AD mice.
Quantification of the staining intensity in the CA1 region of the hippocampus revealed
that these differences in staining were significant (Fig. 3). In addition, there was no
significant difference in oxidized guanine between 3xTg-AD mice treated with EUK-207
and Non-Tg vehicle mice (Fig. 3). Similar results for oxidized guanine were also
observed in the amygdala of Non-Tg and 3xTg-AD mice at 12 months of age (Fig. 3).
However, the increase in oxidized guanine labeling in the amygdala of 3xTg-AD mice
was greater and more significant than that observed in hippocampus (Fig. 3). These
results along with the lipid peroxidation data confirmed that like AD brains, brains of
3xTg-AD mice exhibit a considerable increase in oxidative stress. In addition, these
results clearly demonstrated that chronic treatment with EUK-207 starting after the onset
of AD pathology significantly reduced both lipid peroxidation and nucleic acid oxidation
in brains of 3xTg-AD mice.
46

3. Effects of SOD/catalase mimetic EUK-207 on beta-amyloid pathology in 3xTg-AD
mice

Figure 4. Chronic treatment with EUK-207 starting at 9 months of age reduces 6E10
staining in hippocampus and amygdala of 12 month-old 3xTg-AD mice.

At the end of the 3-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 and amygdala of vehicle and EUK-207-treated 3xTg-AD mice (A)
47

and quantified by counting pixels (B). Levels of oxidized guanine were expressed as % of
vehicle-treated 3xTg-AD (B). Shown are means ± SEM of 4 mice (B). *p <0.01
(Student’s t-test).
In 3xTg-AD mice, intraneuronal Aβ is present in hippocampus, cortex, and amygdala by
6 months of age, and extracellular Aβ deposits begin to form in the frontal cortex at 6
months of age and in hippocampus by 12 months of age (Oddo et al., 2003). Therefore,
Aβ pathology was assessed in brains from 3xTg-AD mice that started receiving chronic
EUK-207 treatment after the onset of AD pathology in order to better understand the link
between oxidative stress and the progression of AD pathology.
The localization and expression of ß-amyloid was assessed within the brains of 3xTg-AD
at 12 months of age. Mice were sacrificed after the 3-month long treatment with EUK-
207 and their brains were fixed in 4 % paraformaldehyde and processed for
immunohistochemistry. Brains sections were then probed with the antibody 6E10, which
recognizes APP and all mature forms of ß-amyloid peptides. Because we previously
found that there were very low levels of 6E10 staining in Non-Tg mice, we omitted these
groups for the analysis. Immunohistochemistry indicated that APP and ß-amyloid
peptides were accumulating in 12 month-old 3xTg-AD mice. (Fig. 4). Intraneuronal ß-
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 ß-
amyloid deposits were found throughout the brain. Like for oxidized guanine, we focused
on hippocampus and amygdala because these two brain regions are critical in associative
fear memories. 6E10-labeled neurons were found in CA1, CA2, and CA3 fields of
hippocampus, but not in the dentate gyrus of 3xTg-AD mice (Fig. 4). Staining was
48

localized mainly to the cell bodies of pyramidal cells within the hippocampus. Chronic
treatment with EUK-207 beginning at 9 months of age reduced 6E10 staining intensity as
well as the number of 6E10-positive neurons within the hippocampus of 12 month-old
3xTg-AD mice (Fig. 4). Quantification of the staining in the CA1 region of the
hippocampus revealed that these differences were significant (Fig. 4). Similar results for
6E10 staining were also observed in amygdala of 3xTg-AD mice at 12 months of age
(Fig. 4).

49

4. Effects of SOD/catalase mimetic EUK-207 on tau pathology in 3xTg-AD mice

Figure 5. Chronic treatment with EUK-207 starting at 9 months of age reduces Tau
accumulation in hippocampus and amygdala of 12 month-old 3xTg-AD mice.
At the end of the 3-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
50

by immunohistochemistry using the antibody HT7. Staining for HT7 was assessed in the
hippocampus and amygdala of vehicle and EUK-207-treated 3xTg-AD mice (A) and
quantified by counting pixels (B). Levels of oxidized guanine were expressed as % of
vehicle-treated 3xTg-AD (B). Shown are means ± SEM of 4 mice (B). *p <0.01
(Student’s t-test).
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., 2003). This extra
transgene produces AD-like tau pathology in 3xTg-AD mice (Oddo et al., 2003), 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., 2003). 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 12 month-old 3xTg-AD that
were chronically treated with EUK-207. Mice were sacrificed after the 3-month long
treatment and their brains were fixed in 4 % paraformaldehyde and processed for
immunohistochemistry. Brains sections were then probed with the antibody HT7, which
is specific for all forms of human tau. As for ß-amyloid pathology, tau pathology was not
assessed in sections from age-matched Non-Tg mice. HT7 staining was present in frontal
cortex, cortex, hippocampus and amygdala of 12 month-old 3xTg-AD mice. Tau protein
was found in both the cell bodies and processes of neurons. Like for oxidative stress and
51

beta-amyloid pathology, we also focused specifically on hippocampus and amygdala.
HT7 staining was only detected in the cell bodies and processes of pyramidal cells within
the CA1 region of the hippocampus (Fig. 5). Chronic treatment with EUK-207 starting at
9 months of age reduced the number of HT7-positive neurons within the hippocampus of
12 month-old 3xTg-AD mice (Fig. 5). Quantification of the staining in the CA1 region of
the hippocampus revealed that these differences were statistically significant (Fig. 6B).
Similar results for HT7 staining were also observed in the amygdala of Non-Tg and
3xTg-AD mice at 9 months of age (Fig. 5).

52

Figure 6. Chronic treatment with EUK-207 starting at 9 months of age reduces
hyperphosphorylated tau in ventral CA1 pyramidal cells and amygdala of 12 month-old
3xTg-AD mice.
At the end of the 3-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 and amygdala of vehicle and EUK-207-treated
3xTg-AD mice (A) and quantified by counting pixels (B). Levels of oxidized guanine
were expressed as % of vehicle-treated 3xTg-AD (B). Shown are means ± SEM of 4 mice
(B). *p <0.01 (Student’s t-test).
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 12 month-old 3xTg-AD mice, AT8 expression was only found in
ventral hippocampus, subiculum, and amygdala. AT8 staining was present in ventral
CA1 and CA3 pyramidal cells (Fig. 6), and like HT7, AT8 expression was located in both
cell bodies and processes (Fig. 6). Chronic treatment with EUK-207 starting at 9 months
of age reduced the number of AT8-positive neurons within ventral hippocampus of 12
month-old 3xTg-AD mice (Fig. 6). Quantification of AT8 labeling within the ventral
CA1 region of hippocampus revealed that these observations were statistically significant
(Fig. 6).
53

Similar observations for AT8 labeling were also observed in amygdala of 3xTg-AD mice
at 12 months of age (Fig. 6), and chronic EUK-207 treatment had also a significant effect
on AT8 expression in amygdala (Fig. 6). Both tau accumulation and expression of
hyperphosphorylated tau were reduced in hippocampus and amygdala of 12 month-old
3xTg-AD mice treated with EUK-207 starting at 9 months of age. This suggests that in
addition to beta-amyloid pathology, oxidative stress might also be linked to the
progression of tau pathology in AD.

5. Correlation between behavioral performance and oxidative stress

54

Figure 7. Correlation between behavioral performance and levels of lipid peroxidation in
Non-Tg and 3xTg-AD mice.
Performance in the context or cue fear conditioning paradigm were plotted against the
levels of lipid peroxidation in various groups of mice.
A. Data from all the animals (Non-Tg and 3xTg-AD, treated with vehicle or EUK-207) in
the study clearly indicate the highly significant and negative correlation between
behavioral performance in the context test (expressed as cumulative freezing time), and
the levels of brain lipid peroxidation (expressed as pmol malondialdehyde/mg protein). B.
Data from all the vehicle-treated animals (Non-Tg and 3xTg-AD) indicate that whereas
there is a trend for a negative correlation in the Non-Tg mice, there is no correlation in
the 3xTg-AD mice. C. Data from the 3xTg-AD animals alone. Note that there is no
significant correlation between the 2 parameters. D. Correlation between the performance
in the cue test and the levels of 8oxoguanine in the amygdala for all the animals. Note the
significant correlation between these parameters.
As the various assays we performed were individualized, we were able to determine
potential correlations between numerous measures related to learning performance,
oxidative stress, and pathological features. We first analyzed the correlation between
animal performance in the context fear conditioning paradigm and the brain levels of
lipid peroxidation (Fig. 7). Taking into account all the animals, there was a highly
significant and negative correlation between these 2 parameters (Fig. 7). These results
reproduced those we had already obtained in aged animals (Clausen et al., 2010), and in
the companion study. The correlation was not present when analyzing only the vehicle-
55

treated animals, although it appeared to still be present in the Non-Tg animals (Fig. 7).
The correlation was also not present when analyzing EUK-207-treated animals (Fig. 7).
On the other hand, a significant correlation was also observed when we considered the
performance of the animals in the cue fear conditioning paradigm, and changes in lipid
peroxidation in the amygdala (Fig. 7).
Discussion
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 and its progression. As EUK-207 has previously been shown to
significantly reduce age associated cognitive impairment and oxidative stress in middle-
aged and aged wild-type mice (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 initiated at 9 months of age and continued for 3 months significantly reduced brain
levels of lipid peroxidation assessed in 12 month-old mice, whether non-Tg or 3xTg-AD.
Interestingly, the levels of brain lipid peroxidation determined at 12 month of age in the
3xTg-AD mice were significantly higher than the values determined at 9 month, and
EUK-207 treatment reversed them to those found in the 9 month-old vehicle-treated non-
Tg mice. These results indicate that this regimen of EUK-207 treatment almost
completely prevent the increased in lipid peroxidation taking place in the 3xTg-AD
between 9 and 12 months. Similarly, EUK-207 treatment significantly reduced the levels
of DNA oxidation in various brain structures in both non-Tg and 3xTg-AD. We
particularly analyzed these changes in hippocampus and amygdala, as both structures are
56

implicated in fear conditioning. Although it is difficult to compare the levels of oxidized
DNA observed in this study and in the previous study, it is worth noticing that the percent
increase between the Non-Tg and the 3xTg-AD was higher in the present study,
suggesting that the increase in DNA oxidation follows a similar pattern as that of lipid
peroxidation. However, in the case of DNA oxidation, it appears that EUK-207 treatment
only partially reversed the increase in both hippocampus and amygdala. The reason for
this difference between lipid peroxidation and DNA oxidation is not clear at this point,
and could be due to regional differences in EUK-207 distribution or access to different
subcellular compartments where these different reactions (lipid peroxidation and DNA
oxidation) are taking place. In any event, these results clearly indicate that EUK-207
treatment is effective in reducing oxidative stress in both Non-Tg and in 3xTg-AD. As in
the companion manuscript, this reduction in oxidative load was associated with decreased
intraneuronal accumulation of ß-amyloid peptide, as well as tau and hyperphosphorylated
tau levels in hippocampus and amygdala of 12 month-old 3xTg-AD mice, as compared to
vehicle-treated 3xTg-AD mice. From the previous studies with these mice, accumulation
of ß-amyloid peptide and hyperphosphorylated tau is a continuous process once it is
initiated, and older animals exhibit larger increases than younger ones. While it is
difficult to assess the absolute degree of changes in ß-amyloid peptide and
hyperphosphorylated tau in this study as compared to the companion study, it is
interesting to note that the effect of EUK-207 appeared to be somewhat larger at 12
month than at 9 month, suggesting that the treatment does result in a slowing down of the
accumulation of both ß-amyloid peptide and hyperphosphorylated tau. It is thus tempting
to conclude that continuous production of reactive oxygen species is causally related to
57

accumulation of ß-amyloid peptide and hyperphosphorylated tau. These results also
consolidate the argument made in the companion manuscript that EUK-207 might
stimulate the lysosomal/autophagy pathway, which is in turn responsible for the reduction
of the accumulation of ß-amyloid peptide and hyperphosphorylated tau. Finally, EUK-
207 treatment almost completely reversed the deficits in performance of 12 month-old
3xTg-AD mice in both the context and cue fear conditioning tests to values close to those
found in vehicle-treated Non-Tg mice. Our results therefore clearly demonstrate that
oxidative stress is a critical mediator in the development of cognitive impairment. a
continuous process once it is initiated, and older animals.
Studies performed in the Tg2576 mouse model of AD also support our results. Like the
3xTg-AD mice, the Tg2576 mice develop cognitive impairments and Aß -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 Aß 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 Aß 1-40
and Aß 1-42, and the number of amyloid plaques in the brain 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.
58

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 Aßpeptides, tau, and
hyperphosphorylated tau, while significantly reduced compared to those in 12 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 might
contribute to their accumulation. Nevertheless, although intraneuronal Aß 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 Aß and hyperphosphorylated tau must reach
critical levels within neurons before impairing synaptic function and plasticity.
Our results indicate that administering exogenous antioxidants, such as EUK-207, might
be potentially useful for stopping the progression of AD pathology. We previously
discussed the limitations of the currently available antioxidants and the problems raised
by the previous attempts to test the oxidative stress hypothesis of AD. In particular, we
stressed the point that 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).
Overall, our results indicate that the initiation and progression of AD pathology might
result from increased oxidative stress due to mitochondria dysfunction. As discussed
previously, the Aβ peptide can interact with mitochondria by binding to the
mitochondrial protein Aβ-alcohol dehydrogenase (ABAD), and the binding of Aβ to
ABAD results in mitochondrial dysfunction (Lustbader et al., 2004, Yan and Stern, 2005).
59

In addition, ß-amyloid induces ROS production in neurons (Harris et al., 1995, Yatin et
al., 1999). Thus, these results are consistent with the idea that AD involves a vicious
circle in which an initial event or existing mutation might produce increased oxidative
stress, resulting in mitochondrial dysfunction and later on in lysosomal dysfunction.
These cellular alterations would then produce increased accumulation of ß-amyloid
peptide and hyperphosphorylated tau, further fueling the cycle of cell dysfunction. 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 our 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 both the initiation and the progression
of AD and that the SOD/catalase mimetic EUK-207 could be useful as a therapeutic
agent for protecting and treating individuals against AD.

These results have now been published:
Clausen A, Xu X, Bi X and Baudry M (2012) Effects of the superoxide
dismutase/catalase mimetic EUK-207 in a mouse model of Alzheimer’s disease:
Protection against and interruption of progression of amyloid and tau pathology and
cognitive decline. JAD 30: 183-208.
60

Chapter III: Differential effects of kainate-induced seizure activity on PTEN,
mTOR and Arc in hippocampus of adult and neonatal rats

Xiaobo Xu, Xiaoning Bi and Michel Baudry
Neuroscience Program
USC
Los Angeles, CA 90089-2520
and
Western University of Health Sciences
Pomona, CA 91766

61

Abstract
Systemic injection of kainate produces repetitive seizure activity in both adult and
juvenile rats. However, while kainate-induced seizure results in neurodegeneration in the
limbic system of adult rats, juvenile rats have been repeatedly shown to be immune to the
neurotoxic effects of kainate. The mechanisms underlying this differential effect of
seizure activity in adult and juvenile rat brain are not clear. We previously reported that
kainate-induced seizure activity differentially affected calpain activation in neonatal and
adult rat brain. We recently discovered that calpain could truncate the phosphatase PTEN,
resulting in mTOR activation and stimulation of protein synthesis, including Arc
synthesis. In the present study, we evaluated the effects of kainate-induced seizure
activity on levels of calpain, PTEN, mTOR, and Arc in hippocampus from adult and
postnatal rats. In adult rats, seizure activity rapidly stimulated calpain-2, as evidenced by
decreased in drebrin and PTEN levels throughout the hippocampus. It was also associated
with increased mTOR and Arc levels in fields CA1 and CA3 4 h after seizure initiation.
PTEN truncation was blocked by systemic injection of the calpain inhibitor calpepetin
before KA injection, but mTOR and Arc levels were not affected. Interestingly, in p12-14
rats, seizure activity was not associated with a rapid calpain activation and PTEN loss in
any field of hippocampus, possibly due to lack of ERK activation. However, levels of
mTOR were increased 1 day after seizure initiation, but levels of PTEN and Arc
remained unchanged. These results indicate that seizure activity produces very different
effects on the calpainPTEN  mTOR and Arc cascade in hippocampus from juvenile
and adult rats. Whether these different effects are related to the different
neuropathological consequences of seizure activity remains to be determined.
62

Introduction
Systemic injection of kainic acid (KA) has been widely used to elicit seizure activities in
both adult and postnatal rats. However, while both adult and neonatal rats exhibit seizure
activity, the neuropathological consequences of seizures are drastically different between
adult and neonatal rats. Extensive neuronal degeneration takes place in the limbic system
in adult rats, while neonatal rats do not exhibit any neuronal damage. The mechanisms
underlying this difference in KA response between adult and neonatal rats are not yet
understood. Numerous cellular cascades have been shown to be involved in KA-induced
neurodegeneration, including calpain activation (Bi et al., 1996; Feng et al., 2011; Aroujo
et al., 2008; Higuchi et al., 2005), increased reactive oxygen species (ROS) formation
(ref), microglial activation (ref), and the generation of neurotoxic lipid metabolites (ref).
More recently, the mammalian target of rapamycin (mTOR) pathway has been proposed
to be involved in seizure activity (Sarbassov et al., 2005; Sandsmark et al., 2007; Tsang
et al., 2007; Talos et al., 2012). Activation of the extracellular regulated kinase (ERK)
was also observed after kainic acid-induced seizure in adult rats (Otani et al., 2003). The
mTOR pathway regulates multiple cellular functions, which may influence neuronal
excitability and epileptogenesis, including protein synthesis, cell growth and proliferation,
synaptic plasticity and cell death (Sarbassov et al., 2005; Sandsmark et al., 2007; Tsang et
al., 2007). Upstream of the mTOR pathway, growth factors such as epidermal growth
factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)
and brain-derived neurotrophic factor (BDNF), stimulate PI3k/Akt resulting in mTOR
activation (Fingar and Blenis, 2004). Phosphatase and tensin homolog (PTEN),
63

dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) into
phosphatidylinositol (4,5)-biphosphate (PIP2), resulting in inhibition of AKT signaling
pathway (Jaworski et al., 2005). Deletion of PTEN gene causes seizures (Backman et al.,
2001). Many downstream factors of mTOR pathway such as phosopho-S6 (PS6) and
4EBPs are also activated after seizures (Panja et al., 2009; Pende et al., 2004). Finally,
the expression and synthesis of the activity-regulated cytoskeleton-associated protein
(Arc), a plasticity protein were also observed after KA-induced seizures.
The two main calpain isoforms, μ-calpain and m-calpain, aka calpain-1 and calpain-2,
are ubiquitously distributed in the brain, and until recently their respective functions in
neurodegneration were not well understood. We recently reported that calpain-2 could be
activated via mitogen-activated protein kinase and extracellular signal-regulated kinases
(MAPK/ERK)-mediated phosphorylation, independently of calcium (Zadran et al., 2010).
In addition, we also found that calpain-2 selectively degrades the phosphatase and tensin
homolog, PTEN, resulting in mTOR activation and stimulation of local protein synthesis
(Briz et al., 2013). This study was therefore directed at testing the hypothesis that calpain
activation following KA-induced seizures could stimulate local protein synthesis,
including Arc synthesis, through PTEN degradation and mTOR activation. The role of
calpain was further examined by testing the effect of the brain-penetrating calpain
inhibitor, calpeptin. Finally because calpain activation following involved KA-induced
seizures is different in adult and postnatal rats (Bi et al., 1997), we also determined the
effects of KA injection on the PTEN- mTOR--  ARC pathway in neonatal animals.

64

Materials and Methods
Animals and treatment
Sprague–Dawley rats, at postnatal day 12 or 2 month old, were kept under standard
laboratory conditions with a 12 h:12 h light/ dark cycle and were injected i.p. with either
vehicle (1.0% DMSO in saline) or calpeptin (50 or 250 µg/kg) twice, one day before and
half an hour before i.p. injection with either vehicle (1.0% DMSO in saline) or kainic
acid (2 or 12mg/kg). 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
Committees of the University of Southern California, and of . Western University of
Health Sciences. Following KA injection, animals were observed for behavioral
alterations, and only those exhibiting limbic seizures were further processed. Seizure
behavior was characterized by the presence of repeated head nodding, wet-dog shaking,
repeated unilateral and bilateral limb scratching movements, and in some animals of
tonic-clonic forelimb movements. Control rats and rats exhibiting seizure activity were
killed at different time points from 4 h to 1 day after seizure onset.
Immunohistochemistry
Animals were anaesthetized with ketamine (80 mg/kg) and xylazine (12 mg/kg) by i.p.
injection and perfused intracardially with phosphate-buffered saline (PBS) followed by
freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). After
perfusion, brains were removed and were immersed in 4% paraformaldehyde at 4 ºC for
post-fixation, then in 15% and 30% sucrose for 3-5 days respectively at 4 ºC for
cryoprotection. Forty µm coronal sections were prepared with a microtome and stored in
65

cryoprotectant at -20 ºC. Immunohistochemistry was performed on free-floating brain
sections using the avidin-biotin-horseradish peroxidase complex (ABC) method with
VECTASTAIN Elite ABC kit from Vector Laboratories (Burlingame, CA). Free-floating
sections were blocked for 1 h at room temperature in 10% normal goat serum diluted in
1x TBS, pH 7.4 or 1x PBS, pH 7.4, and then incubated with the following primary
antibodies: PTEN, p-mTOR, p-ERK, Arc and Drebrin, overnight at 4 ºC. After washing
with either TBS or PBS, sections were incubated in biotinylated secondary antibodies
(1:400 in TBS or PBS in 5 % goat serum) for 2 h at room temperature. Sections were
washed again with either TBS or PBS and incubated in the avidin-biotin horseradish
peroxidase complex solution for 45 min. After washing, staining was developed using
3,3’-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA). Sections
were mounted on gelatin-coated slides, dehydrated in a series of graded ethanol, and
coverslipped with DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA).
Western Blots
Hippocampi were rapidly dissected and homogenized in cold lysis buffer (50 mM Tris-
HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 mM NaF, and
1 mM Na3VO4), containing protease inhibitor cocktail (Thermo Scientific). Fifteen-
twenty μg of proteins were loaded on 8-15% SDS gels, separated by gel electrophoresis
and transferred onto polyvinylidene difluoride membranes. Membranes were blocked
with 5% Odyssey blocking buffer for 1 h at room temperature and incubated overnight in
PTEN and drebrin primary antibodies at 4 °C. After washing to remove free primary
antibodies, membranes were incubated in secondary fluorescent antibodies for 30 min at
66

20 °C. After a final wash, membranes were scanned by Odyssey image scanner (LI-COR
Biosciences).
Statistics
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 performed using Tukey post hoc
analysis with 95 % confidence intervals.
Results
1. Changes in drebrin, PTEN, mTOR and Arc in hippocampus 4 h after KA-induced
seizure in 2 month-old rats
We previously reported that KA-induced seizures in adult rats were associated with
calpain activation, as assessed by changes in the selective spectrin breakdown product
generated by calpain-mediated cleavage, in selected neuronal populations at early time
points after seizure onset, which spread throughout the pyramidal cells of CA3 and CA1
5 days later. In the present study, we used a different calpain substrate, drebrin, to assess
the distribution of calpain activation at 4 h after seizure onset in adult rats. Surprisingly,
KA-induced seizures were associated with a widespread decrease in drebrin levels
throughout pyramidal neurons of CA1 and CA3 (Fig. 1). No changes in drebrin levels
were observed in the granule cells of the dentate gyrus.

67

Drebrin CA1
control
4hrs
0.0
0.5
1.0
1.5
Drebrin /control

Drebrin CA3
control
4hrs
0.0
0.5
1.0
1.5
Drebrin /control

4hrs Control
**
**
68

Figure 1: Changes in drebrin levels in hippocampus of adult rats 4 h after KA-induced
seizures.
Adult rats were injected with KA; 4 hours after seizure onset, rats were sacrificed and
fixed brain sections were stained with antibodies against drebrin. Staining levels were
quantified with Image J and were expressed as ratios of values found in control animals.
They represent means ± S.E.M. of 4 animals. T-test revealed significant differences in
drebrin levels in CA1 and CA3 (**<0.01, as compared to control).
Adjacent sections were then stained with PTEN antibodies (Fig. 2). PTEN was detected
in both cell bodies and dendrites throughout the hippocampus in control rats. PTEN
levels were also significantly decreased in CA1 and CA3 4 h after KA-induced seizures.
Like drebrin, PTEN levels were not modified in the granule cells of the dentate gyrus.

69

PTEN CA1
Control
4hrs
0.0
0.5
1.0
1.5
PTEN /control
PTEN CA3
control
4hrs
0.0
0.5
1.0
1.5
PTEN /control

Figure 2: Changes in PTEN levels in hippocampus of adult rats 4 h after KA-induced
seizures.
Adult rats were injected with KA; 4 hours after seizure onset, rats were sacrificed and
fixed brain sections were stained with antibodies against PTEN. Staining levels were
quantified with Image J and were expressed as ratios of values found in control animals.
They represent means ± S.E.M. of 4 animals. T-test revealed significant differences in
PTEN levels in CA1 (** p <0.01) and CA3 (* p <0.05), as compared to control.
4hrs Control
* **
70

Adjacent brain sections were also stained with antibodies against p- mTOR.
Phosphorylated mTOR was detected in both cell bodies and dendrites throughout the
hippocampus in control rats (Fig. 3). Levels of p-mTOR were significantly increased in
pyramidal neurons of CA1 and CA3 4 h after KA-induced seizures. Under these
conditions, p-mTOR levels were also increased in the granule cells of the dentate gyrus..

4hrs
Control
71

p-mTOR CA1
control
4hrs
0
1
2
3
4
p-mTOR /Control

p-mTOR CA3
control
4hrs
0.0
0.5
1.0
1.5
2.0
2.5
p-mTOR /Control

Figure 3: Changes in p-mTOR levels in hippocampus of adult rats 4 h after KA-induced
seizures.
Adult rats were injected with KA; 4 hours after seizure onset, rats were sacrificed and
fixed brain sections were stained with antibodies against p-mTOR. Staining levels were
quantified with Image J and were expressed as ratios of values found in control animals.
They represent means ± S.E.M. of 4 animals. T-test revealed significant differences in in
levels of p-mTOR in CA1 (** p <0.01) and CA3 (* p <0.05), as compared to controls.
**
*
72

Finally, fixed brain sections were stained with antibodies against Arc. Arc was detected
in both cell bodies and dendrites throughout the hippocampus in control rats (Fig. 4). Arc
levels were significantly increased in CA1, CA3 and especially in the dentate gyrus 4 h
after KA-induced seizures.

4hrs
Control
73

Arc CA1
control
4hrs
0
1
2
3
4
Arc /control

Arc CA3
control
4hrs
0.0
0.5
1.0
1.5
2.0
2.5
Arc /control

Arc Dentate Gyrus
control
4hrs
0
1
2
3
4
Arc /control

**
**
**
74

Figure 4: Changes in Arc levels in hippocampus of adult rats 4 h after KA-induced
seizures.
Adult rats were injected with KA; 4 h after seizure onset, rats were sacrificed and fixed
brain sections were stained with antibodies against Arc. Staining levels were quantified
with Image J and were expressed as ratios of values found in control animals. They
represent means ± S.E.M. of 4 animals. T-test revealed significant differences in in levels
of Arc in in CA1, CA3, and dentate gyrus (** p <0.01) as compared to, control..

2. Effects of calpeptin injection on changes in drebrin, PTEN, p-mTOR and Arc in
hippocampus 4 h after KA-induced seizures.
In order to determine the role of calpain in changes in drebrin, PTEN, p-mTOR and Arc
after KA induced seizures, adult rats were injected with the calpain inhibitor, calpeptin,
before KA injection. Calpeptin is a useful cell-penetrative calpain inhibitor that binds to
the active site of calpain and reversibly inactivates both μ-calpain and m-calpain
(Tsujinaka et al., 1988). In addition, ip injection of calpeptin has been shown to inhibit
inflammation, cell death, and axonal damage in animal models of multiple sclerosis,
suggesting that calpeptin enters the CNS (Guyton et al., 2010). Rats were pretreated with
calpeptin before receiving ip KA injection, and were sacrificed 4 h after seizure onset.
Hippocampi were homogenized and aliquots of homogenates were processed for
immunobloting with antibodies against drebrin and PTEN. Western blots showed that
both PTEN and drebrin levels were significantly decreased following KA injection (Fig.
5). Pretreatment with calpeptin significantly reduced PTEN and drebrin loss after KA-
induced seizure. Immunohistochemistry was also performed in additional groups of rats,
75

and indicated that the loss of PTEN staining in both cell bodies and dendritic fields in
CA1 and CA3 following KA injection was significantly reduced by calpeptin
pretreatment (Fig. 6). No changes in PTEN or drebrin levels were found in calpeptin
alone group as compared to the control group.

Drebrin western
Control
calp
KA Calp
KA
0.0
0.5
1.0
1.5
Drebrin /control

Western PTEN
Control
calp
KA Calp
KA
0.0
0.5
1.0
1.5
PTEN /control

**
*
*
*
76

77

PTEN CA1
Control
Calpeptin
Calp+KA
KA
0.0
0.5
1.0
1.5
PTEN / control

PTEN CA3
Control
Calpeptin
Calp+KA
KA
0.0
0.5
1.0
1.5
PTEN /control

Figure 5: Effects of calpeptin pretreatment on drebrin and PTEN levels in hippocampus
of adult rats 4 h after KA injection.
Adult rats were pretreated with calpeptin before KA injection, and were sacrificed 4 h
after seizure onset. Western blots were performed using hippocampal homogenates with
*
**
**
**
78

PTEN and drebrin antibodies. Levels of drebrin and PTEN were expressed as ratios of
control values and represent means ± SEM of 3-4 animals. One way ANOVA revealed
significant differences in levels of drebrin (* p <0.05 control vs KA, * p <0.05 calpeptin
plus KA vs KA) and PTEN (** p <0.01 control vs KA, * p <0.05 calpeptin plus KA vs
KA).
Figure 6: Effects of calpeptin pretreatment on drebrin and PTEN staining in hippocampus
of adult rats 4 h after KA injection.
Adult rats were pretreated with calpeptin before KA injection, and were sacrificed 4 h
after seizure onset. Animals were perfused and fixed brain sections were stained with
antibodies against PTEN. Staining levels were quantified with Image J and were
expressed as ratios of values found in control animals. They represent means ± S.E.M. of
3 or 4 animals. One way ANOVA revealed significant differences in levels PTEN
staining in CA1 (** p <0.01 control vs KA, ** p <0.01 KA plus calpeptin vs KA), and
CA3 (** p <0.01 control vs KA, * p <0.05 calpeptin plus KA vs KA).
Adjacent sections were stained for p-mTOR, and as shown above, p-mTOR staining was
increased in both cell bodies and dendritic fields in CA1 and CA3 following KA injection
(Fig. 7). Calpeptin injection alone did not modify the levels of p-mTOR staining.
However, calpeptin injection did not modify the increase in p-mTOR elicited by KA-
induced seizures.
79

80

p-mTOR CA1
Control
Calpeptin
Calp+KA
KA
0.0
0.5
1.0
1.5
p-
mTOR /Control

p-mTOR CA3
Control
Calpeptin
Calp+KA
KA
0.0
0.5
1.0
1.5
p-mTOR /Control

Figure 7: Effects of calpeptin pretreatment on p-mTOR staining in hippocampus of adult
rats 4 h after KA injection.
Adult rats were treated with calpeptin before KA injection. 4 hours after seizure onset,
rats were sacrificed. Animals were perfused and fixed brain sections were stained with
antibodies against p-mTOR. Staining levels were quantified with Image J and were
expressed as ratios of values found in control animals. They represent means ± S.E.M. of
**
**
** **
81

4 animals. One way ANOVA revealed significant differences in levels of p-mTOR levels
in CA1 (** p <0.01 control vs KA, ** p <0.01 control vs calpeptin plus KA) and CA3 (*
p <0.01, control vs KA, ** p <0.01 control vs calpeptin plus KA) but no significant
difference between KA and calpeptin plus KA groups.

Finally, we determined the effect of calpeptin pretreatment on KA-induced changes in
Arc levels of hippocampus (Fig. 8). While KA injection resulted in increased staining of
Arc throughout the hippocampus, and especially the dentate gyrus, calpeptin pretreatment
did not modify the changes in Arc produced by KA-induced seizure activity. Calpeptin
injection alone did not modify Arc staining.

82

Arc CA1
control
calpeptin
calp+KA
KA
0.0
0.5
1.0
1.5
Arc /control

Arc CA3
control
calpeptin
calp+KA
KA
0.0
0.5
1.0
1.5
Arc /control

** **
** **
83

Arc Dentate Gyrus
control
calpeptin
calp+KA
KA
0.0
0.5
1.0
1.5
2.0
Arc /control

Figure 8: Effects of calpeptin pretreatment on Arc staining in hippocampus of adult rats 4
h after KA injection.
Adult rats were treated with calpeptin before KA injection. 4 hours after seizure onset,
rats were sacrificed. Animals were perfused and fixed brain sections were stained with
antibodies against Arc. Staining levels were quantified with Image J and were expressed
as ratios of values found in control animals. They represent means ± S.E.M. of 4 animals.
One-way ANOVA revealed significant differences in levels of Arc in CA1 (** p <0.01
control vs 4 h, ** p <0.01 control vs calpeptin plus KA), CA3 (** p <0.01 control vs 4 h,
** p <0.01 control vs calpeptin plus KA) and dentate gyrus (* p <0.01, control vs 4 h,,
** p <0.01 control vs calpeptin plus KA) but no significant difference between KA and
calpeptin plus KA group.
**
**
84

3. Changes in drebrin, PTEN, mTOR, and Arc in hippocampus after KA-induced seizure
in juvenile rats
We previously reported that KA-induced seizures in juvenile rats did not activate calpain,
as assessed with the truncation of spectrin, we repeated the experiments and assessed
calpain activity with the truncation of drebrin and PTEN. Postnatal day 12-14 rats were
injected with 2-4 mg/kg KA, and were sacrificed 4 h after seizure onset. Animals were
perfused and fixed brain sections were stained with antibodies against drebrin. Drebrin
staining was observed in both cell body layer and dendritic fields of hippocampus in both
control and KA-treated rats (Fig. 9). In contrast to the large drebrin loss after KA-induced
seizures in adult rats, no significant differences in drebrin levels were found after KA
injection in neonatal rats.

85

Drebrin CA1
Control
KA
0.0
0.5
1.0
1.5
Drebrin /control

drebrin CA3
Control
KA
0.0
0.5
1.0
1.5
Drebrin /control

Figure 9: Effects of KA-induced seizures on drebrin levels in hippocampus of P12-14 rats.
Postnatal day 12-14 rats were injected with KA; 4 hours after seizure onset, rats were
sacrificed and fixed brain sections were stained with antibodies against drebrin. Staining
levels were quantified with Image J and were expressed as ratios of values found in
control animals. They represent means ± S.E.M. of 4 animals. T-test revealed no
significant differences in levels of drebrin between groups. Adjacent sections were
stained with antibodies against PTEN, and like for drebrin, KA injection did not result in
PTEN decrease either at 4 h or 1 day after seizure onset (Fig. 10).

86

PTEN CA1
control
4h
1d
0.0
0.5
1.0
1.5
2.0
PTEN /control

PTEN CA3
control
4h
1d
0.0
0.5
1.0
1.5
PTEN /control

Figure 10: Effects of KA-induced seizures on PTEN levels in hippocampus of P12-14
rats.
P12-14 rats were injected with KA; 4 hours after seizure onset, rats were sacrificed and
fixed brain sections were stained with antibodies against PTEN. Staining levels were
quantified with Image J and were expressed as ratios of values found in control animals.
They represent means ± S.E.M. of 4 animals T-test revealed no significant differences in
levels of PTEN between groups.
We then determined changes in p-mTOR levels in neonatal rats 4 h after seizure onset.
Neonatal rats exhibited no significant differences in p-mTOR levels 4 h after seizure
onset, although a significant increase in p-mTOR was observed in CA1 and dentate gyrus
1 day after KA-induced seizures (Fig. 11).,
87

p-mTOR CA1
control
4h
1d
0
1
2
3
4
p-
mTOR /Control

p-mTOR CA3
control
4h
1d
0.0
0.5
1.0
1.5
2.0
p-
mTOR /Control

Figure 11: Effects of KA-induced seizures on p-mTOR levels in hippocampus of P12-14
rats.
P12-14 rats were injected with KA; 4 h and 1 day after seizure onset, rats were sacrificed
and fixed brain sections were stained with antibodies against p-mTOR. Staining levels
were quantified with Image J and were expressed as ratios of values found in control
animals. They represent means ± S.E.M. of 4 animals. One way ANOVA revealed no
significant differences in levels of p-mTOR between control and the 4 h group but
significant increase in CA1 (** p <0.01 control vs 1d) 1 day after KA-induced seizure.
We also examined changes in Arc levels in neonatal rats following KA-induced seizures.
In contrast to what was observed in adult rats, no significant differences were found in
Arc levels at either 4 h or 1 day following KA injection (Fig. 12).
**
88

Arc CA1
control
4h
1d
0.0
0.5
1.0
1.5
2.0
Arc /control

Arc CA3
control
4h
1d
0.0
0.5
1.0
1.5
Arc /control

Arc Dentate Gyrus
control
4h
1d
0.0
0.5
1.0
1.5
Arc /control

Figure 12: Effects of KA-induced seizures on p-mTOR levels in hippocampus of P12-14
rats.
89

P12-14 rats were injected with KA; 4 hours after seizure onset, rats were sacrificed and
fixed brain sections were stained with antibodies against Arc. Staining levels were
quantified with Image J and were expressed as ratios of values found in control animals.
They represent means ± S.E.M. of 4 animals One way ANOVA revealed no significant
differences in levels of Arc between groups.
Discussion
The goal of the present study was to determine whether calpain activation following
KA-induced seizures was responsible for mTOR activation, which has been shown to
occur shortly after seizure onset under these conditions (Zeng et al., 2009). As increased
Arc expression and synthesis have also been shown to occur rapidly after KA-induced
seizures (Li et al., 2005), we were also interested in evaluating the potential role of
calpain activation in this response to seizures. In our previous study, we used spectrin
degradation as an index of calpain activation and we found increased of calpain-mediated
spectrin degradation only in some interneurons 4 h after seizure onset, while massive
spectrin degradation was present throughout cell bodies and dendritic fields at 5 days
following KA treatment (Bi et al., 1996). In this study, we used analyzed changes in 2
different calpain substrates to evaluate the role of calpain at early stages after seizure
onset. We recently found that Drebrin A, a neuron-specific F-actin binding protein, is a
calpain substrate, a finding corroborating results indicating that drebrin levels were
decreased in a pilocarpine model of epilepsy (Ferhat, 2012). We also recently reported
that PTEN was a selective substrate of calpain-2, and that calpain-2-mediated PTEN
degradation was necessary for BDNF-induced dendritic protein synthesis through
activation of the mTOR pathway in rat hippocampal slices, cortical synaptoneurosomes,
90

and cultured neurons (Briz et al., 2013). Surprisingly, our results indicated a widespread
activation of calpain, as assessed by drebrin and PTEN degradation, 4 h after KA-induced
seizures. While spectrin degradation was restricted to a subset of interneurons, drebrin
and PTEN degradation was present throughout the cell bodies and dendrites in CA1 and
CA3. Note that no changes in drebrin and PTEN were observed in the granule cells of the
dentate gyrus. That this decrease was due to calpain activation was further demonstrated
by its blockade when rats were pretreated with the calpain inhibitor calpeptin. Since
PTEN is a selective substrate of calpain-2, our results indicate that calpain-2 is widely
activated 4 h after KA-induced seizures. The differential pattern of spectrin degradation,
as compared to PTEN and drebin would therefore suggest that spectrin might be
preferentially degraded by calpain-1, and that this event will be delayed as compared to
calpain-2 activation. While calpain-1 activation requires calcium, calpain-2 can be
activated via mitogen-activated protein kinase and extracellular signal-regulated kinases
(MAPK/ERK)-mediated phosphorylation, independently of calcium (Zadran et al., 2010).
MAPK/ERK pathway is activated as early as 15 min, and until 6 h after KA-induced
seizure in adult rats (Otani et al., 2003), while calcium accumulation begins at 4 h and is
not prominent until 7 days after KA-induced seizure (Sztriha et al., 1986). Thus, the
differential pattern of spectrin and PTEN and drebrin degradation could reflect the
differential temporal activation of calpain-1 and calpain-2. Alternatively, it is possible
that spectrin and PTEN and drebrin have differential subcellular localizations and their
differential degradation could result from the differential activation of calpain-1 and
calpain-2 in these subcellular compartments. In parallel with the decrease in PTEN, p-
mTOR was significantly increased in CA1 and CA3 4 h after seizure onset in adult rats.
91

This finding is consistent with the increase in phospho-S6 (P-S6), an mTOR downstream
protein, starting approximately 1 h after seizure onset, with a peak at 3-6 h (Zeng et al.,
2009). Arc mRNA, a member of the immediate-early gene (IEG) family can be rapidly
transported and transcribed in recently activated synaptic sites in an NMDA receptor-
dependent manner (Steward and Worley, 2001). In agreement with previous results, we
also found Arc levels to be increased throughout the hippocampus, and especially in the
dentate gyrus, 4 h after KA-induced seizures. However, although both western blot and
immunohistochemistry indicated that PTEN degradation was blocked by calpain
inhibition, calpeptin injection did not have any effect on mTOR activation or Arc
synthesis. Since inhibition of mTORC1 with rapamycin did not impair Arc synthesis
(Rao et al., 2006), it is likely that the mTOR pathway is not essential for Arc synthesis
induced by seizure. Likewise, the mTOR pathway can be activated by mechanisms
unrelated to PTEN and the PI3K pathway. In particular, AKT can also be activated
through the phospholipid-dependent kinase-1 (PDK-1), which activates mTORC1 via I-
kappaB kinase (IKK); Akt can phosphorylate the proline rich Akt substrate 40 kDa
(PRAS40) thereby releasing mTORC1 from its inhibition by PRAS40 (Russo et al.,
2012). Therefore, our data indicate that seizure activity-induced mTOR activation and
Arc synthesis are not related to calpain activation and PTEN degradation. Although
PTEN deletion has been shown to produce seizures, the significance of calpain-mediated
PTEN truncation following KA injection is not clear.
Kainic acid treatment produces seizure activities in both adult and postnatal rats.
However the mechanisms of epileptogenesis and pathological alterations are quite
different. It is well known that the immature brain is more prone to seizures due to an
92

unstable status of excitation and inhibition balance, which decreases seizure threshold
(Holmes, 1997; Kim, 2010; Jensen, 1999). However, while kainate-induced seizures
results in neurodegeneration in the limbic system of adult rats, immature rats have been
shown to be resistant to the neurotoxic effects of kainate (Haas et al., 2001; Wang et al.,
2004; Sarkisian et al., 1997; Jandová et al., 2006; Stafstrom et al., 1992). In our previous
study, it was found that during the first two postnatal weeks, spectrin degradation was
decreased in neuronal cell bodies and increased in dendritic fields of hippocampus 4 h
after KA-induced seizures (Bi et al., 1996). In contrast, neither PTEN nor drebin
degradation was observed 4 h after KA-induced seizures in neonatal hippocampus. These
results would suggest that in neonatal hippocampus, calpain-1 is activated in the
dendrites of pyramidal neurons shortly after seizure onset, but calpain-2 is not.
Interestingly, while ERK was rapidly activated following KA-induced seizures in adult
hippocampus, it was not activated in neonatal hippocampus (not shown). This result
could account for the lack of calpain-2 activation in neonatal hippocampus following KA
injection. These results suggested that calpain-2 is not activated after KA induced seizure
in postnatal rats, possibly due to lack of ERK/AMPK pathway activation. Since calpain
also contributes to neurodegeneration in various experimental models (Squier et al.,
1994), the lack of calpain activation in postnatal rats could account for the lack of
neurodegeneration following KA-induced seizures in neonatal rats. These results further
support the idea that calpain-2 activation is an important factor in neurodegeneration.
In conclusion, our results showed that different calpain substrates exhibited different
patterns of degradation following KA-induced seizures: drebrin and PTEN were degraded
rapidly, as early as 4 h after seizure onsets while spectrin exhibited a delayed degradation,
93

possibly because calpain-1 and calpain-2 are activated at different time points. By using
the KA model of seizures, our study confirmed that calpain activation results in PTEN
degradation in vivo. On the other hand, mTOR activation and Arc expression were not
related to calpain activation. Finally, as calpain-2 is not activated following KA-induced
seizures in neonatal rats, our results further support the notion that calpain-2 activation is
critical factor in neurodegeneration.

94

Chapter IV: Discussion and Conclusions
1. Role of oxidative stress in AD pathogenesis
A number of studies have demonstrated that AD pathogenesis is associated with brain
increases in ROS, as well as in protein oxidation, lipid peroxidation, and DNA and RNA
oxidation (Good et al., 1996; Smith et al., 1997; Butterfield, et al 2001). However, the
mechanisms underlying age-related increase in oxidative stress in AD pathogenesis are
still poorly understood, and could involve either an increase in free radical production, or
an impairment in free radical defense mechanisms, or a combination of both. In particular,
age-dependent increase in free radical production is most likely due to an increase in
mitochondrially-produced ROS because mitochondria preparations from the brains of
3xTg-AD mice exhibit a significant age-dependent increase in superoxide and hydrogen
peroxide production. In turn, age-associated accelerated reactive oxygen species (ROS)
production in mitochondria is most likely due to mitochondrial dysfunction brought on by
age-dependent damage to mitochondria. On the other hand, several studies have also
reported decreases in the activity and expression of a number of antioxidant molecules in
AD. Superoxide dismutase activity and catalase activity both decreased with age in the
rat brain, as do mRNA levels for these two enzymes (Rao et al., 1990).
While it is clear that ROS plays a role in AD pathogenesis, it remains unknown whether
ROS is a causal factor or a consequence of pathology. Several studies have suggested that
oxidative stress is a downstream mediator of Aβ toxicity, as Aβ can stimulate ROS
production in cultured neurons (Yatin et al., 1999) and astrocytes (Harris et al., 1996).
95

However, evidence also showed that oxidative stress could serve as an initiator of the
pathology, since mitochondrial dysfunction precedes the development of AD pathology
(Monte et al., 2000), and continuous mitochondrial dysfunction and increased ROS
production participate in the progression of AD pathology (Mancuso et al., 2010). In
support of this idea, several reports have indicated that antioxidants ameliorate
mitochondrial function (Dragicervic et al., et al; Manczak et al., 2010; Picone et al., 2009;
Simpkins et al., 2010). However, very few of these studies have convincingly
demonstrated the usefulness of antioxidant treatment in mouse models of AD or in
human AD.
Given the strong link between oxidative stress and AD pathology, my study was designed
to determine whether or not ROS is a causal factor in AD pathogenesis in the 3xTg-AD
mouse model of AD. The SOD/catalase mimetic compound, EUK-207, is a member of a
class of molecules known as salen-Mn complexes, which 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, it
can protect against damage caused by superoxide, hydrogen peroxide and reactive
nitrogen species. Previous studies in our laboratory showed that EUK-207 provided
beneficial effects for treating pathologies associated with age-related oxidative stress. In
particular, an 8-month chronic treatment with EUK-207 starting at 3 months of age
significantly improved cognitive function and reduced lipid peroxidation and protein
oxidation in the brain of mice by the age of 11 months (Liu et al., 2003). In my study, the
effects of a 3-month chronic treatment with EUK-207 of 3xTg-AD mice were evaluated,
starting at 9 months of age, i.e., after the occurrence of pathology.
96

Our results clearly showed that oxidative stress plays a causal role in AD-associated
pathology, as chronic treatment with EUK-207 reduced amyloid accumulation and tau
hyperphosphorylation and improved cognitive function the 3xTg-AD mice. The levels of
oxidative guanine were significantly higher in both non-Tg and 3xTg-AD mice at 13
month of age compared to 9 month old mice, indicating that oxidative stress was a
consequence of brain aging. A 3-month chronic treatment with EUK-207 significantly
reduced the levels of lipid peroxidation and oxidative guanine in brains of 3xTg- AD
mice. This result is consistent with several studies indicating that other anti-oxidant
compounds and dietary supplements, including Vitamin E, Coenzyme Q10 (CoQ10),
resveratrol (trans-3, 5, 4-trihydroxystilbene), and curcumin have reduced oxidative stress
in brain (Morris et al., 2005).
Chronic treatment with EUK-207 interrupted the progression of AD pathogenesis, as
evidenced by reduced levels of amyloid and hyperphosphorylated tau, clearly indicating
that oxidative stress is a causal factor in the pathological process. Interestingly, a recent
study showed that a 5-month chronic treatment of APPswePS1dE9 transgenic mice with
micronized zeolites, starting at 7 months of age also protected neurons against ROS-
induced cell death, reduced amyloid levels and plaques (Montinaro et al., 2013).
However, the mechanism through which oxidative stress leads to accumulation of Aß
peptides and hyperphosphorylated tau is still unknown. Several possible cascades have
been postulated to contribute to the development of amyloid and tau pathology induced
by oxidative stress:
1) Amyloid peptides are formed from the proteolysis of the amyloid precursor protein
(APP) via the combined actions of BACE1 (β-secretase) and γ-secretase. During
97

oxidative stress, the ability of mitochondria to produce ATP decreases due to ROS
damage, resulting in a low metabolic rate. Decreased metabolic activity could possibly
activate β-secretase, which, in turn, would increase Aβ deposition as a secondary
response (Robert et al., 2010).
2) Impaired autophagy due to oxidative stress could lead to accumulation of damaged
mitochondria and misfolding proteins, thus promoting the formation of amyloid plaques
and neurofibrillary tangles (Rubinsztein, 2006).
3) Widespread calpain activation is associated with neurofibrillary tangles, senile plaques,
and dystrophic neurites (Saito et al., 1993), possibly due to perturbed calcium
homeostasis as well as MAPK/ERK pathway activation (Zadran et al., 2012). Calpain
degrades numerous downstream targets to trigger further detrimental events. Cyclin-
dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is activated by
neuron specific p35. Calpain cleaves p35 into p25, which binds to Cdk5 and form a
hyperactive and more stable complex, resulting in aberrant phosphorylation of various
substrates, thereby enhancing amyloid and tau pathology (Shukla et al., 2011).
4) Amyloid and tau can form noncovalent aggregates with limited lifetime, which can be
easily degraded. However, oxidation can modify the structure of these proteins, resulting
in more stable oxidated covalent crosslinks, thus preventing their removal (Friguet et al.,
1994). In addition, amyloid peptides and hyperphosphorylated tau themselves are
neurotoxic. Aβ peptides produce neuroinflammation, activate microglia (McGeer and
McGeer, 2010), and interact with mitochondria by binding to the mitochondrial protein
A-alcohol dehydrogenase (ABAD) (Lustbader et al., 2004; Yan and Stern, 2005),
resulting in mitochondrial dysfunction which, in turn, elicits further oxidative stress. High
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Aβ levels can further increase mTOR activity through the PI3K/AKT pathway (Martín et
al., 2001), and the mTOR pathway has been shown to reduce autophagy (Simonsen et al.,
2008), which in turn promotes oxidative stress and Aβ deposition. Under normal
conditions, tau regulates microtubule assembly and axonal transport. Neurofibrillary
tangles not only disrupt tau normal function, but also exhibit a gain of toxic function by
destabilizing microtubules and impairing axonal transport (Morris et al., 2011).
Interestingly, it has been shown that Aβ and tau exhibit antioxidant properties in response
to oxidative stress and their accumulation could represent a mechanism to protect neurons
against oxidative damage (Smith et al., 2002). Overall, oxidative stress resulting from
impaired mitochondrial function could produce compensatory changes, including high
levels of Aβ peptides, and hyperphosphorylated tau in an attempt to restore normal cell
function. However, these compensatory changes may themselves further disrupt normal
cellular functions and have toxic effects, resulting in a vicious cycle that further
exacerbates oxidative stress.
While few of the tested antioxidants reversed the cognitive decline and AD pathogenesis
in AD mouse models, the 3-month chronic treatment with EUK-207 showed promising
therapeutic effects by reversing cognitive impairment and AD pathology, including
amyloid and tau pathology. It is well demonstrated that there is a linear relationship
between aging and decrease in learning and memory performance (Forster et al., 1996).
Our previous results showed a clear decrease in cognitive performance, as measured by
contextual fear conditioning, between 16 and 20 months of age, and between 16 and 23
months of age. Chronic treatment with either EUK-189 or EUK-207 reversed the
cognitive deficits exhibited by the 20 month-old vehicle control mice (Clausen et al.,
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2010). Based on our results, we could propose that EUK-207 might be a reasonable
therapeutic candidate to be tested in AD patients. However, several issues need to be
overcome for this idea to be further tested. First, while many drug candidates have shown
potentials in mouse models of AD, they have all failed in human clinical trials. In
addition, EUK-207 is not orally available, and we had to use ALzet mini-pumps to
perform chronic treatment. While this might not be an insurmountable obstacle, it
represents an additional difficulty for the clinical development of this molecule.
In conclusion, our study provided evidence that oxidative stress serves as an initiator in
AD pathogenesis. Exogenous superoxide dismutase/ catalase compound could
significantly reverse the pathology and rescue the cognitive decline in 3xTg-AD mice.
Therefore, despite potential difficulties, EUK-207 could be useful as a therapeutic agent
for protecting individuals against AD.

2. Role of calpain activation and its regulation of the mTOR pathway in KA-
induced seizures
A systemic injection of KA in rats induce a rapid onset of seizures, with characteristic
“wet dog shakes” within half an hour to an hour, followed by mild to severe intermittent
seizures for the next four to five hours or even death due to respiratory muscle spasms,
and ultimately with an epileptic state characterized with spontaneous recurrent seizures
(SRS), depending on the protocol used to administer KA (Leite et al.,2002). KA binds to
the AMPA/KA receptors, subtypes of the ionotropic glutamate receptors (Bleakman et al.,
1998). Upon binding to the receptors, KA initiates membrane depolarization, resulting in
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a large influx of Ca2+, and triggering a series of cascade reactions, including oxidative
stress, mitochondrial dysfunction and even cell death.
The calpain family, a group of calcium-dependent cysteine proteases, are thought to play
critical roles in long term potentiation (LTP), through the partial truncation of various
substrates, resulting in structural modifications of dendritic spines, modifications of
presynaptic terminals and regulation of local protein synthesis (Baudry et al., 2011). We
recently reported that calpain-2, which requires millimolar calcium concentration to be
activated in vitro, can be activated via phosphorylation mediated by the mitogen-
activated protein kinase and extracellular signal-regulated kinases (MAPK/ERK),
independently of calcium (Zadran et al., 2010); we also found that calpain-2 selectively
mediates the degradation of the phosphatase and tensin homolog (PTEN) phosphatase.
This effect is responsible for BDNF-induced stimulation of local protein synthesis
through the mammalian target of rapamycin (mTOR) pathway (Briz et al., 2013). We
previously reported that following KA-induced seizure activity, calpain activation was
limited to a small subset of interneurons shortly after seizure onset, before spreading
throughout fields CA3 and CA1 by 5 days after KA injection (Bi et al., 1996). These
results were obtained by analyzing the formation of a specific breakdown product of
spectrin, one of the best known calpain substrates. Surprisingly, the present study
revealed a widespread calpain activation as early as 4 h after KA injection when
analyzing the degradation of another calpain substrates, drebrin. .Previous studies have
shown that the MAPK/ERK pathway is activated rapidly as early as 15 min, and
remained activated until 6 hours after KA-induced seizure activities in adult rats (Otani et
al., 2003), while calcium accumulation begins at 4 hours and is not prominent until 7
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days after KA-induced seizure (Sztriha et al., 1986). One possibility to account for these
seemingly contradictory findings is that the different degradation patterns of different
calpain substrates are due to the differential activation of different calpain isoforms
following KA-induced seizures: as we also observed PTEN degradation shortly after KA
injection, we postulate that the early drebrin degradation is due to the early activation of
calpain-2 resulting from MAPK/ERK-mediated phosphorylation. In contrast, calpain-1
would get activated later, possibly due to increased intracellular calcium concentration,
and would lead to spectrin degradation. Alternatively, it is possible that calpain-1 and
calpain-2 exhibit different subcellular localizations and could be activated by different
signaling cascades. In particular, we noted that calpain-1 and calpain-2 have different
PDZ binding domains, which would cluster them with different sets of binding partners.
As calpain inhibitors have been proposed to be useful for treating neurodegenerative
disorders, these results indicate that more remains to be done to understand the respective
roles of calpain-1 and calpain-2 in neurodegeneration.
The mTOR pathway is also activated shortly after KA-induced seizure (Zeng et al., 2009).
As mTOR regulates multiple cellular mechanisms that influence neuronal excitability and
epileptogenesis, including protein synthesis, cell growth and proliferation, synaptic
plasticity and cell death (Sarbassov et al., 2005; Sandsmark et al., 2007; Tsang et al.,
2007), it is important to understand the upstream pathways leading to its activation.
Protein synthesis regulated via mTOR pathway was shown to be critical in learning and
memory and long term potentiation (LTP) consolidation (Casadio et al., 1999), by
contributing to morphological changes including the increases in postsynaptic surface
area, spine number and area. The mTOR signaling is mediated by two mTOR complexes:
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mTORC1 and mTORC2. mTORC2 regulates cytoskeleton and controls cell survival.
Upstream of mTORC1 pathway, enhanced expression of growth factors such as
epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial
growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) stimulate PI3k/Akt
and activate the mTOR pathway (Fingar and Blenis, 2004). Expressions of epidermal
growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth
factor (VEGF) are enhanced in epileptic TSC patients, indicating that enhanced
expression of these growth factors may contribute to cell proliferation and growth
induced by epilepsy (Parker, 2011). Similarly, recent studies showed an increase in both
BDNF mRNA and protein levels in surgically resected hippocampi from epileptic
patients, as compared to controls (LaFrance, 2010). PTEN dephosphorylates
phosphatidylinositol (3,4,5)-trisphosphate (PIP3) into phosphatidylinositol (4,5)-
biphosphate (PIP2), resulting in inhibition of the AKT signaling pathway (Jaworski et al.,
2005). Deletion of the PTEN gene causes seizures (Backman et al., 2001). We also
observed a very large decrease in PTEN 4 hours after KA-induced seizure in adult rats.
PTEN truncation was blocked by calpain inhibition, a result consistent with our previous
study showing that calpain-2 degrades PTEN following BDNF treatment in rat
hippocampal slices, cortical synaptoneurosomes, and cultured neurons (Briz et al., 2013).
It is important to stress that calpeptin injection did not modify the onset or the intensity of
seizures, indicating that calpain activation is not involved in the generation of seizure
activity. Phosphorylated mTOR was significantly increased in both CA1 and CA3
regions of hippocampus 4 h after seizure onsets in adult rats. In addition, AMP-activated
Protein Kinase (AMPK) detects AMP/ATP ratio from energy deprivation and stress,
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leading to mTOR inhibition. mTORC1 initiates cap-dependent protein translation,
activates ribosomal S6 kinase-1 (S6K1), promoting ribosomal biogenesis and protein
translation, and inhibits the elongation factor 4E binding protein 1 (4EBP1), triggering
protein synthesis (Fingar et al., 2002). An increase in phospho-S6 (PS6) expression was
first noted about one hour after seizure onset, with a peak at 3-6 hours and then a return
to baseline after 24 hours in both hippocampus and neocortex; this was followed by a
second increase 3 days later, with a peak at 5-10 days, which lasted for several weeks in
both hippocampus and neocortex, after KA-induced seizure, indicating that mTOR
signaling participates in both the acute phase of seizure activity and the chronic
epileptogenesis phase (Zeng et al., 2009). We also found that the activity-regulated
cytoskeleton-associated protein (Arc), a plasticity protein, was rapidly increased after
KA-induced seizures. Since Arc protein functions in clathrin-mediated endocytosis,
leading to the removal of AMPA receptors from the plasma membrane, increased Arc
levels could represent a homeostatic response to seizure activities (Chowdhury et al.,
2006). Calpeptin injection before KA treatment significantly blocked PTEN and drebrin
degradation in CA1 and CA3 regions of hippocampus 4 h after KA -induced seizure,
indicating that calpain truncates PTEN after seizures. However, calpeptin injection did
not have any effect on mTOR activation or Arc synthesis. Since inhibition of mTORC1
with rapamycin did not impair Arc synthesis (Rao et al., 2006), it is possible that mTOR
pathway is not involved in Arc synthesis induced by seizures. In addition, calpain-
mediated PTEN truncation is not sufficient to activate mTOR following seizures, as its
blockade did not prevent mTOR activation. This is not completely unexpected, as there
are many pathways other than the PIP3/AKT to activate mTOR.
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Although systemic injection of kainate results in repetitive seizure activities in both adult
and immature rats, the mechanisms of epileptogenesis and resulting pathological
alterations are quite different in adult and immature rats. It is well known that the
immature brain is more prone to seizures due to an unstable balance between excitation
and inhibition, decreasing seizure threshold (Holmes, 1997). A transient overexpression
of glutamate receptors, and a relative lack of GABAergic inhibitory transmission in
immature brain have been shown to contribute to the increased susceptibility to seizures
in immature brains (Jensen, 1999). However, while kainate-induced seizure results in
neurodegeneration in the limbic system of adult rats, immature rats have been shown to
be resistant to the neurotoxic effects of kainate (Holmes, 1997). There are more long-
term deficits in learning and memory, more cell loss and cell death in adult rats compared
to immature rats after kainate-induced seizures. Numerous cell loss, degenerating dentate
hilar neurons and pyramidal cells in CA1 and CA3 regions in hippocampus have been
observed in adult rats two weeks following KA-induced seizure, but significantly less
hippocampus damages in 16 day old rats (Haaz et al., 2001). Long-term deficits in
learning and memory caused by status epilepticus in the immature P12 brain have been
reported to be less severe, as compared to P20 brain where cell loss and mossy fiber
sprouting were observed (Cillio et al., 2003). In addition, inflammation, angiogenesis and
blood brain barrier (BBB) leakage in P21 rats were observed after seizures, and
spontaneous seizures developed 1 week and 4 months after pilocarpine-induced status.
While calpain was activated in adult rats after KA-induced seizures, it was not after KA-
induced seizures in p12-14 rats, although basal calpain activity was higher, as evidenced
by increased levels of spectrin breakdown product in hippocampus (Bi et al., 1997). As
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mentioned above, calpain-2 can be activated through MAPK/ERK-mediated
phosphorylation (Zadran et al., 2010); however, MAPK/ERK was not activated following
KA-induced seizures in neonatal rats, suggesting that the lack of calpain-2 activation is
due to the absence of MAPK/ERK activation. Independently of the exact mechanism
underlying the lack of calpain activation following seizure in neonatal rats, these results
strengthen the argument that calpain-2 activation plays a critical role in
neurodegeneration, as its presence is associated with neuronal death and its absence with
no neuronal death.
3. Role of oxidative stress in KA-induced seizures
Oxidative stress has also been shown to accompany KA-induced seizures (Yalcin et al.,
2004, Cheng et al., 1994; Rong et al., 1999; Gluck 2000; Milatovic et al., 2002). The
redox status of hippocampus was shown to be reduced following KA-induced seizures in
a time –dependent manner (Liang and Patel, 2006). As a potent agonist of glutamate, KA
by acting on AMPA/KA receptors produces a strong depolarization, which results in the
activation of the NMDA receptors, a large calcium influx and an increase in intracellular
calcium concentration. These events trigger ROS formation, causing oxidative stress and
mitochondrial damage (Chang and Yu, 2010; Liang and Patel, 2006). Aconitase, an
enzyme of the Krebs cycle, is inactivated after KA-induced seizures, thus impairing
mitochondrial ATP synthesis (Liang et al., 2000). It was also reported that levels of lipid
peroxidation and protein oxidation were significantly increased in hippocampus and
piriform cortex at 8 and 16 h after KA-induced seizure activity in adult rats (Bruce and
Baudry, 1995). Mitochondrial damage and ROS generation were prevented by the KA
receptor-selective antagonist DNQX (Dabbeni-Sala et al., 2001). In addition,
106

pretreatment of granule neurons with melatonin, a direct ROS scavenger, or with the
reduced glutathione (GSH) delivery agent GSH ethylester, prevented mitochondrial
complex II damage (Dabbeni-Sala et al., 2001). These studies provide strong support to
the existence of a causal link between oxidative stress and KA-induced neuronal damage.
Oxidative stress leads to further release of endogenous glutamate in brain, resulting in a
vicious cycle, as mentioned above. Numerous antioxidants have been studied against
KA-induced seizures. Treatment with vineatrol and curcumin as antioxidants could
significantly reduce neurotoxicity and the incidence of convulsion induced by KA (Gupta
and Briyal, 2006; Gupta et al., 2009). In addition, our laboratory also showed that
treatment with EUK-134, another member of the Mn-salen synthetic superoxide
dismutase/catalase mimetics, could also prevent neuronal damage resulting from KA-
induced seizures (Rong et al., 1999).

4. Role of calpain-mediated mTOR pathway activation in AD pathogenesis
As the mTOR pathway regulates protein synthesis and alterations in protein synthesis in
AD have been reported decades ago (Langstrom et al., 1989; Sajdel-Sulkowska et al.,
1984), alterations in the mTOR pathway have been implicated in AD pathogenesis
(Tischmeyer et al., 2003; Caccamo et al., 2013; Pei and Hugon, 2008). In support of this
idea, mTOR signaling is significantly increased in brains of human AD patients (Li et al.,
2005). In addition, mTOR enzymatic activity and levels of phosphorylated p7056K were
significantly increased in cortex and hippocampus of 6 month-old 3xTg-AD mice
(Caccamo et al., 2010). mTOR hyperactivity also plays a role in learning and memory
impairments and cognitive deficits observed in AD patients. Rapamycin treatment
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reduced mTOR signaling levels in the brains of 3xTg-AD mice to those found in wild-
type mice and rescued the early spatial learning and memory deficits (Caccamo et al.,
2010). Upstream of the mTOR pathway, increased MAPK activity was reported at a very
early stage in human AD brains (Munoz and Ammit, 2010). Widespread activation of
calpain was found associated with neurofibrillary tangles, senile plaques, and dystrophic
neurites (Saito et al., 1993), possibly due to perturbed calcium homeostasis, as well as to
increased MAPK/ERK activity (Zadran et al., 2010). Calpain has been proposed to play a
critical role in numerous neurodegenerative diseases (Bernath et al., 2006; Wang et al.,
2013), possibly as a trigger for the intrinsic apoptotic cascade . A recent study showed a
potential therapeutic effect of an orally active calpain inhibitor (A-705253) in AD.
Chronic treatment with this inhibitor ameliorated Aβ and p-tau pathology and improved
mice performance in the hippocampus-dependent Morris water maze test, in the cortex-
dependent novel object recognition task, and in the contextual fear conditioning task in
3xTg-AD mice (Getz, 2012; Medeiros et al., 2012). Interestingly, levels of PTEN, a
selective calpain-2 substrate (Briz et al., 2013), were reduced in AD brains, and
mutations in PTEN or deficiency in its phosphatase activity result in tauopathies (Zhang
et al., 2006). The role of the mTOR pathway in the formation of Aβ plaques and
neurofibrillary tangles, the two hallmarks of AD pathology, however seems more
complicated than simply protective or neurotoxic in the progression of the disease.
Decreased activity of the mTOR pathway correlated with Aβ neurotoxicity in APP/PS1
knock-in mice (An et al., 2003; Li et al., 2004), while increased mTOR pathway activity
was associated with increased levels of phosphorylated tau (Pei and Hugon, 2008).
Inhibition of mTOR signaling with rapamycin ameliorated tau pathology and the
108

associated behavioral deficits (Caccamo et al., 2013), but exacerbated Aβ-induced
neurotoxicity and cell death (Lafay-Chebassier et al., 2006). Since mTOR pathway also
functions in autophagy inhibition (Díaz-Troya et al., 2008), and dysfunction of the
autophagy-lysosome system, which facilitates Aβ clearance, could be a potential cause of
aggregation of misfolding protein pathology in AD (Rubinsztein, 2006). High Aβ levels
can further increase mTOR activity through the PI3K/AKT pathway (Martín et al., 2001),
resulting in another vicious cycle, eventually producing higher levels of Aβ. In Oddo’s
study, It was also shown that autophagy induction was necessary for rapamycin-mediated
decrease in Aβ levels (Caccamo et al., 2010). Pei et al., suggested that the dual role of
mTOR in regulating AD pathology was possibly due to differential cellular susceptibility
to various cellular stresses in AD, with one group of neurons exhibiting the protective
pathway, while mTOR activation in another group lead to cell death (Pei and Hugon
2008).
5. Cross-talks between oxidative stress and calpain-mediated mTOR activation
Since many studies have shown that both oxidative stress and calpain-mediated activation
of the mTOR pathway participate in neurodegeneration, it is logical to ask whether these
two cascades act independently and in parallel or in a closely interacting manner. Indeed,
many studies have provided evidence for the existence of crosslinks and interactions
between oxidative stress and calpain-induced mTOR activation. Thus, MAPK/ERK
signaling and intracellular increase in calcium concentration lead to oxidative stress and
calpain activation (Gaitanaki et al., 2003). ROS may also facilitate calpain activation
through upregulation of BDNF (Mattson, 2005). Up-regulation of BDNF by ROS also
triggers some downstream factors, such as the transcription factor cAMP response
109

element binding protein (CREB), leading to DNA binding of CREB and activation of
inducible transcription factors, such as NF-kB, c-Fos, and Jun (Radak et al., 2007). ROS
produced from gp91phox-NADPH oxidase resulted in calpain activation in
cardiomyocytes during norepinephrine stimulation (Li et al., 2009). Correspondingly,
blocking ROS generation by the superoxide dismutase/ catalase compound EUK-134 in
KA-induced seizures significantly decreased levels of spectrin breakdown product,
indicating that reducing oxidative stress prevents calpain activation (Rong et al., 1999).
In addition, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran), a
scavenger of ROS, could significantly reduce photoreceptor apoptosis induced by SNP by
preventing calpain activation (Sanvicens et al., 2004). All these lines of evidence
suggests that calpain activation is downstream of oxidative stress.
However, mTOR pathway signaling is inhibited by oxidative stress. Chen et al.(2010)
found that hydrogen peroxide-induced ROS inhibited mTOR upstream kinases, Akt and
phosphoinositide-dependent kinase 1 (PDK1), but not PTEN. In addition, the negative
mTOR regulator, AMPK, was also activated (Chen et al., 2010). On the other hand,
mTOR pathway down-regulates autophagy and autophagy protects against free radical
damage by reducing accumulation of oxidized proteins (Simonsen et al., 2008).
Insulin/insulin-like growth factor (IGF-1) enhances mTOR pathway activation and
Igf1r+/− mice are much more resistant to oxidative damage than wild-type mice,
indicating that IGF-1 may exacerbate ROS damage (Cohen et al., 2009). Therefore,
mTOR pathway could actually enhance oxidative stress. In support of this, inhibition of
mTOR pathway with rapamycin produced a protective effect against ROS formation and
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oxidative stress. This suggests that upregulation of autophagy protects against free radical
damage (Iglesias-Bartolome et al., 2012).
Overall, oxidative stress and calpain-mediated mTOR pathway have been shown to play
critical roles in many biological and pathological processes, including AD and seizures.
Understanding the role of these two cascades will therefore shed light on learning and
memory as well as neurodegenerative diseases, such as AD and epilepsy.

111

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Abstract (if available)

Abstract Alzheimer’s disease is characterized by progressive memory loss and cognitive deficits, accumulation of ß-amyloid plaques and intracellular neurofibrillary tangles within the brain, and neuronal death. In addition to ß-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. In the companion study we 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, starting before the onset of pathology and cognitive deficits, and continuing until 9 months of age, when the AD phenotype is established. In the present study, we initiated the treatment after the onset of pathology at 9 months of age. After 3 months of treatment, cognitive performance, brain ß-amyloid and tau pathology as well as oxidative stress were analyzed. At 12 months of age, 3xTg-AD mice exhibited a decline in performance in both contextual and cued fear memory tasks as compared to wild-type mice

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Creator Xu, Xiaobo (author)

Core Title Studies of intracellular cascades mediating neuronal damage in two animal models of neurodegeneration

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 10/07/2013

Defense Date 07/18/2013

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

Tag Alzheimer's disease,kainic acid,OAI-PMH Harvest,oxidative stress,seizures

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Language English

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Advisor Walsh, John P. (committee chair), Baudry, Michel (committee member), Dane, Joseph A. (committee member), Watts, Alan G. (committee member)

Creator Email teresabmc@gmail.com,xiaoboxu@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-335637

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