Oxidative Stress in the Brain Novel Cellular Targets That Govern 2005

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Oxidative stress in the brain: Novel cellular targets that governsurvival during neurodegenerative disease

Zhao Zhong Chong a, Faqi Li a, Kenneth Maiese a,b,c,*

aDivision of Cellular and Molecular Cerebral Ischemia, Wayne State University School of Medicine, Detroit, MI 48201, USAbDepartment of Neurology and Anatomy & Cell Biology, Center for Molecular Medicine and Genetics, Institute of Environmental Health Sciences,

Wayne State University School of Medicine, 8C-1 UHC, 4201 St. Antoine, Detroit, MI 48201, USAc

Center for Molecular Medicine and Genetics, Institute of Environmental Health Sciences,

Wayne State University School of Medicine, Detroit, MI 48201, USA

Received 29 July 2004; accepted 16 February 2005

Abstract

Despite our present knowledge of some of the cellular pathways that modulate central nervous system injury, complete therapeutic

prevention or reversal of acute or chronic neuronal injury has not been achieved. The cellular mechanisms that precipitate these diseases are

more involved than initially believed. As a result, identification of novel therapeutic targets for the treatment of cellular injury would be

extremely beneficial to reduce or eliminate disability from nervous system disorders. Current studies have begun to focus on pathways of

oxidative stress that involve a variety of cellular pathways. Here we discuss novel pathways that involve the generation of reactive oxygen

species and oxidative stress, apoptotic injury that leads to nuclear degradation in both neuronal and vascular populations, and the early loss of

cellular membrane asymmetry that mitigates inflammation and vascular occlusion. Current work has identified exciting pathways, such as the

Wnt pathway and the serinethreonine kinase Akt, as central modulators that oversee cellular apoptosis and their downstream substrates that

include Forkhead transcription factors, glycogen synthase kinase-3b, mitochondrial dysfunction, Bad, and Bcl-xL. Other closely integrated

pathways control microglial activation, release of inflammatory cytokines, and caspase and calpain activation. New therapeutic avenues that

are just open to exploration, such as with brain temperature regulation, nicotinamide adenine dinucleotide modulation, metabotropicglutamate system modulation, and erythropoietin targeted expression, may provide both attractive and viable alternatives to treat a variety of

disorders that include stroke, Alzheimers disease, and traumatic brain injury.

# 2005 Elsevier Ltd. All rights reserved.

www.elsevier.com/locate/pneurobioProgress in Neurobiology 75 (2005) 207246

Abbreviations: Ab, b-amyloid; AD, Alzheimers disease; AIF, apoptosis-inducing factor; ALS, amyotrophic lateral sclerosis; Apaf-1, apoptotic protease-

activating factor; APC, adenomatous polyposis coli; APP, amyloid precursor protein; BrdU, bromodeoxyuridine; CARD, caspase recruitment domain; CDK,

cyclin-dependent kinase; CNS, central nervous system; CPCR, G protein-coupled receptor; CREB, cAMP-response element-binding protein; CTMP, carboxy-

terminal modulator protein; EC, endothelial cell; eIF2B, the translation initiation factor 2B; EPO, erythropoietin; FADD, Fas-associated protein with death

domain; FLIP, Fas-associated death domain-like interleukins 1b converting enzyme-like inhibitory protein; FRAT1, frequently rearranged in advanced T-cell

lymphoma type 1; GSK-3b, glycogen synthase kinase-b; HD, Huntingtons disease; 4-HNE, 4-hydroxynonenal; IAP, inhibitor of apoptosis protein; IKK, IkB

kinase; JNK, c-Jun-amino terminal kinases; Lef, lymphocyte enhancer factor; LPR,lipoprotein related protein; MPP

+

, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NF-kB, nuclear factor-kB; NO, nitric oxide; 6-OHDA, 6-hydroxydopamine; OHdG, 8-hydroxy-2-deoxyguano-

sine; OGD, oxygen-glucose deprivation; PARP, poly(ADP-ribose) polymerase; PCD, programmed cell death; PCNA, proliferating cell nuclear antigen; PD,

Parkinsons disease; PDK1, phosphoinositide-dependent kinase-1; PI 3-K, phosphoinositide 3 kinase; PIP 2, phosphatidylinositol 3,4-bisphosphate; PIP3,

phosphatidylinositol 3,4,5-trisphosphate; PKB, protein kinase B; PKC, protein kinase C; PP2A, protein phosphatase 2A; PS, phosphatidylserine; PS1,

presenilin 1; PTEN, the phosphatase and tensin homolog deleted from chromosome 10; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SN,

substantia nigra; SOD, superoxide dismutase; Tcf, T cell factor; TCL1, the T cell leukemia/lymphoma 1; TNF, tumor necrosis factor; WISP-1, Wnt-1 induced

secreted preotein-1

* Corresponding author at: Department of Neurology, Department of Anatomy & Cell Biology, Center for Molecular Medicine and Genetics, Institute of

Environmental Health Sciences, Wayne State University School of Medicine, 8C-1 UHC, 4201 St. Antoine, Detroit, MI 48201, USA. Tel.: +1 313 966 0833;

fax: +1 313 966 0486.

E-mail address: [email protected] (K. Maiese).

0301-0082/$ see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2005.02.004


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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

1.1. The population at risk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

1.2. Elucidating novel targets within the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

1.3. The biology of oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

2. Oxidative stress and neurodegenerative disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

2.1. Acute . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2102.2. Chronic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

3. Early and late apoptotic programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

4. Microglial activation and inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

5. Attempted cell cycle induction in post-mitotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

6. Induction of the Wnt pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

7. Akt as an essential regulatory element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

7.1. Activation and expression of Akt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

7.2. Akt as a modulator apoptotic injury and inflammation during ROS exposure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

7.3. Akt can provide the stimulus for altering the course of neurodegenerative disease . . . . . . . . . . . . . . . . . . . . . . . . . 219

8. Downstream cellular targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

8.1. The Forkhead transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

8.2. GSK-3b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

8.3. Bad, Bcl-xL, and NF-kB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2238.4. Mitochondrial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

8.5. Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

8.6. Calpains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

9. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

1. Introduction

1.1. The population at risk

At present, over 23 million people in the United Statessuffer from central nervous system (CNS) disorders.

Globally, this number reaches a level of 368 million people.

These disorders predominantly consist of neurodegenerative

diseases that include presenile dementia, Alzheimers

disease (AD), and Parkinsons disease (PD). Intimately

linked to the development of CNS degeneration are also a

variety of injuries associated with traumatic brain injury

(TBI). For example, both penetrating head injuries and blast

injuries without direct head trauma have been shown to

result in subsequent neurotrauma as a result of potential

elevations in nervous system oxidative stress and free radical

levels (Cernak et al., 2000). In addition to direct head

trauma, diffuse neuronal degeneration can ensue as a result

of an increased load of kinetic energy from the original

insult (Carey et al., 1984). Furthermore, tangential cranial

injuries are susceptible to acute ischemic neuronal injury

with intracerebral hemorrhage (Elron et al., 1998). Finally,

environmental toxin exposure also may foster oxidative

neuronal and vascular damage (Miller et al., 2002) (Table 1).

In the general population, the cost of physician services,

hospital and nursing home care, and medications continues

to rise dramatically. In addition, these medical costs for

neurodegenerative disease parallel a progressive loss of

economic productivity with rising morbidity and mortality,

ultimately resulting in an annual deficit to the economy that

is greater than $ 380 billion. Interestingly, the most

significant portion of this economic loss is composed of

only a few neurodegenerative disease entities, such as

ischemic disease and AD. The annual cost per patient withAD is estimated at $ 174,000 with an annual population

aggregate cost of $ 100 billion (McCormick et al., 2001;

Mendiondo et al., 2001).

1.2. Elucidating novel targets within the cell

Despite our present knowledge of some of the cellular

pathways that modulate CNS injury, complete therapeutic

prevention or reversal of acute or chronic neuronal injury has

not been achieved. As a result, identification of novel

therapeutic targets for the treatment of neuronal injury

would be extremely beneficial to reduce or eliminate

disability from CNS disorders. Current studies have begun to

focus on pathways of oxidative stress that involve a variety

of cellular pathways. Here we describe the unique capacity

of intrinsic cellular mechanisms that may offer novel therapy

for a variety of acute and chronic disorders in both neuronal

and vascular systems. Oxidative stress leads to apoptotic

injury that involves early loss of cellular membrane

asymmetry as well as the eventual destruction of genomic

DNA. These dynamic stages of apoptosis can be associated

with an ill-fated attempt to enter the cell cycle, particularly

in post-mitotic neurons. Subsequent cellular pathways can

originate from the proto-oncogene Wnt and the serine

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threonine kinase Akt and involve mechanisms linked to

inflammatory activation of microglia, Forkhead transcrip-

tion factors, glycogen synthase kinase-3b activation, loss of

mitochondrial membrane permeability, and the eventual

induction of caspases and calpains. Understanding these

processes may ultimately serve to elucidate robust

therapeutic strategies linked to brain temperature, cellular

metabolism, genomic DNA repair, metabotropic glutamate

modulation, and cytokine regulation that allow future

clinical strategies to mature from bench side prediction

to daily practice.

1.3. The biology of oxidative stress

Oxidative stress occurs when oxygen free radicals are

generated in excess through the reduction of oxygen.

Reactive oxygen species (ROS) consist of oxygen free

radicals and associated entities that include superoxide free

radicals, hydrogen peroxide, singlet oxygen, nitric oxide

(NO), and peroxynitrite. Several of these species are

produced at low levels during normal physiological

conditions and are scavenged by endogenous antioxidant

systems that include superoxide dismutase (SOD), glu-

tathione peroxidase, catalase, and small molecule sub-

stances such as Vitamins C and E. Superoxide radical is the

most commonly occurring oxygen free radical that produces

hydrogen peroxide by dismutation. Hydroxyl radical is the

most active oxygen free radical and is generated from

hydrogen peroxide through the HaberWeiss reaction in the

presence of ferrous iron. Hydroxyl radical alternatively may

be formed through an interaction between superoxide

radical and NO (Fubini and Hubbard, 2003). NO interacts

with superoxide radical to form peroxynitrite that can further

lead to the generation of peroxynitrous acid. Hydroxyl

radical is produced from the spontaneous decomposition of

peroxynitrous acid. NO itself and peroxynitrite are also

recognized as active oxygen free radicals. In addition to

directly altering cellular function, NO may work through

peroxynitrite that is potentially considered a more potent

radical than NO itself (Pfeiffer et al., 2001).

Oxidative stress in the brain occurs when the generation

of ROS overrides the ability of the endogenous antioxidant

system to remove excess ROS subsequently leading to

cellular damage. Several cellular features of the brain

suggest that it is highly sensitive to oxidative stress. For

example, the brain is known to possess the highest oxygenmetabolic rate of any organ in the body (Maiese, 2002). The

brain consumes approximately twenty percent of the total

amount of oxygen in the body. This enhanced metabolic rate

leads to an increased probability that excessive levels of

ROS will be produced. In addition, the brain tissues contain

increased amounts of unsaturated fatty acids that can be

metabolized by oxygen free radicals. Finally, the brain

contains high levels of iron which have been associated with

free radical injury (Herbert et al., 1994). Liposoluble iron

chelators have been reported to lead to a reduction in ROS

and protect neurons from permanent focal cerebral ischemia

(Demougeot et al., 2004). Yet, given the increased risk

factors for the generation of elevated levels of ROS in the

brain, it is interesting to note that the brain also may suffer

from an inadequate defense system against oxidative stress.

Catalase activity in the brain is significantly below other

body organs. If one compares the catalase activity of the

brain to the catalase activity in the liver, the brain has been

shown to contain only 10% of the catalase activity present in

the liver (Floyd and Carney, 1992).

Oxidative stress represents a significant pathway that

leads to the destruction of both neuronal and vascular cells in

the CNS. The production of ROS can lead to cell injury

through cell membrane lipid destruction and cleavage of

Z.Z. Chong et al. / Progress in Neurobiology 75 (2005) 207246 209

Table 1

Oxidative stress in central nervous system disorders

Diseases Demonstration Selected references

Acute

Cerebral ischemia/

reperfusion

Superoxide radical and peroxynitrite increased on microvessels;

impaired mitochondrial function; protection with

reactive oxygen species reduction

Bazan et al. (2002); Yamato et al. (2003);

Gursoy-Ozdemir et al. (2004) and

Demougeot et al. (2004)

Traumatic brain injury Reactive oxygen species increased; lipid peroxidation and protein

oxidation increased; antioxidant reserve decreased

Awasthi et al. (1997); Tyurin et al. (2000);

Marklund et al. (2001) and Bayir et al. (2002)

Chronic

Alzheimers disease Oxidation of lipids, DNA, and proteins increased; induction of reactive

oxygen species by amyloid-b; metal ion reduction in senile plaques;

formation of ion-permeable channels

Behl et al. (1994); Montine et al. (1999);

McGrath et al. (2001); Monji et al. (2001)

and Boland and Campbell (2003)

Parkinsons disease Oxidation of lipid, DNA, and proteins increased in substantia nigra Alam et al. (1997); Groc et al. (2001);

Zigmond et al. (2002) and Basso et al. (2004)

Huntingtons disease Oxidative DNA damage increased in the basal ganglia; reactive

oxygen species present

Browne et al. (1997); Bogdanov et al. (2001)

and Perez-Severiano et al. (2004)

Amyotrophic lateral sclerosis Reactive oxygen species increased; oxidation of lipids, DNA, and

proteins increased; mutant in copper zinc superoxide dismutase;

protection with reactive oxygen species reduction

Rosen et al. (1993); Liu et al. (1999)

and Jung et al. (2001)


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DNA (Vincent and Maiese, 1999b; Wang et al., 2003). ROS

result in the peroxidation of cellular membrane lipids (Siu

and To, 2002), peroxidation of docosahexaenoic acid, a

precursor of neuroprotective docosanoids (Mukherjee et al.,

2004), the cleavage of DNA during the hydroxylation of

guanine and methylation of cytosine (Lee et al., 2002), and

the oxidation of proteins that yield protein carbonylderivatives and nitrotyrosine (Adams et al., 2001). In

addition to the detrimental effects to cellular integrity, ROS

can inhibit complex enzymes in the electron transport chain

of the mitochondria resulting in the blockade of mitochon-

drial respiration (Yamamoto et al., 2002). In cerebral

vascular system, the cellular effects of ROS may lead to the

destruction of endothelial cell (EC) membranes and an

increase in endothelial cell permeability (Sakamaki, 2004).

2. Oxidative stress and neurodegenerative disease

2.1. Acute

Oxidative brain damage is considered to be a signi ficant

contributor to ischemic brain injury (Chong et al., 2004b).

During cerebral ischemia, ROS, such as superoxide radicals,

are released in significant quantities and have been

demonstrated at the interface of the cerebrovascular cell

membrane (Yamato et al., 2003). Sources such as

cyclooxygenase-2 (COX-2) and impaired mitochondrial

function can lead to the release of ROS in the brain during

cerebral ischemia and reperfusion (Bazan et al., 2002).

Oxygen free radicals subsequently lead to reperfusion-

induced injury following cerebral ischemia and areassociated with delayed ischemic neuronal damage (Kita-

gawa et al., 1990). Several mechanisms may account for the

cellular injury that results during exposure of ROS. Both

ischemia and the subsequent failure of energy metabolism in

the brain lead to the calcium-dependent activation of

phospholipase A2. Phospholipase A2 can then cleave

membrane phospholipids and release arachidonic acid

(Mrsic-Pelcic et al., 2002). Superoxide radical is then

produced with the metabolism of arachidonic acid by

cyclooxygenase and lipooxygenase that are activated during

reperfusion. Mitochondrial injury and the electron transport

impairment also contribute to the production of superoxide

radicals during focal cerebral ischemia and exacerbate brain

infarction. ROS can precipitate endoplasmic reticulum

damage during global brain ischemia that can be attenuated

by copper zinc SOD overexpression (Hayashi et al., 2003).

Cerebral ischemia also leads to NO production in the brain

(Zhu et al., 2002). Superoxide readily reacts with NO

leading to the formation of peroxynitrite that has been

considered as a main product of NO contributing to

reperfusion-induced brain damage following cerebral

ischemia (Eliasson et al., 1999). Alternatively, NO may

involve other signal transduction pathways such as protein

kinase A and protein kinas C (Maiese and Boccone, 1995;

Maiese et al., 1993). Following cerebral ischemia, reperfu-

sion leads to the significant formation of superoxide, NO,

and peroxynitrite on microvessels and surrounding end-feet.

These ROS are believed to disrupt microvascular integrity

resulting in cerebral hemorrhage and edema (Gursoy-

Ozdemir et al., 2004).

In addition to the evidence for the production of ROSduring the acute onset of cerebral ischemia and subsequent

reperfusion injury, the ability to protect both neuronal and

vascular tissue during cerebral ischemia with antioxidants or

scavengers of ROS offers further support for the involve-

ment of ROS during acute cerebral ischemia. For example,

biologically active SOD fusion proteins can prevent

hippocampal neuronal injury during transient forebrain

ischemia (Sik Eum et al., 2004). Furthermore, novel free

radical scavengers, such as 8-(4-fluorophenyl)-2-((2E)-3-

phenyl-2-propenoyl)-1,2,3,4-tetra-hydropyrazolo[5,1-

c][1,2,4]triazine (FR210575), can significantly reduce

cortical damage by almost 40% in a transient model of

cerebral ischemia and protect against apoptotic injury during

permanent cerebral injury (Iwashita et al., 2003).

Oxidative stress also has been suggested to play a crucial

role in the pathology of TBI. Following TBI, increased

ascorbyl free radical signals and reduced ascorbic acid has

been demonstrated in rats (Awasthi et al., 1997). Additional

investigations have shown an increase in lipid peroxidation,

production of peroxynitrite, and impairment of the

endogenous antioxidant system following TBI (Hall et al.,

2004; Tyurin et al., 2000). Similarly, a sustained decrease in

the total antioxidant reserve including ascorbate and

glutathione has been observed in the cerebrospinal fluid

in infants and children after severe TBI (Bayir et al., 2002).The level of free radical induced products of lipid

peroxidation and protein oxidation in the cerebrospinal

fluid also were increased following TBI (Bayir et al., 2002).

In contrast, scavenging of ROS after TBI can improve

neurological function and reduce cerebral injury (Marklund

et al., 2001).

2.2. Chronic

Oxidative stress is considered to play a significant role in

the onset and progression of AD (Maiese and Chong, 2004;

Mattson, 2004). AD leads to a progressive deterioration of

cognitive function with memory loss and is characterized by

two pathologic hallmarks that consist of extracellular

plaques of amyloid-b peptide aggregates and intracellular

neurofibrillary tangles composed of hyperphosphorylated

microtubular protein tau. The b-amyloid deposition that

constitutes the plaques is composed of a 3942 amino acid

peptide (Ab), which is the proteolytic product of the

amyloid precursor protein (APP) (Maiese and Chong, 2004).

The association of oxidative stress with AD is dependent

on several lines of evidence. The oxidative products of

lipids, protein, and DNA have been reported in patients with

AD. In the neocortex of the brain of individuals with AD, the



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end product of lipid peroxidation, malondialdehyde (MDA),

has been observed to be in significantly higher quantities

than in aged matched controls (Palmer and Burns, 1994).

Elevated levels of another product of lipid peroxidation,

4-hydroxynonenal (4-HNE), also has been shown to be

increased in the plasma of patients with AD (McGrath et al.,

2001). 4-HNE is an aldehyde product of lipid peroxidationthat can lead to caspase activation and apoptosis (Liu et al.,

2000). In addition, HNE can become conjugated to the

neuronal glucose transporters (Mark et al., 1997) and as a

result has been suggested to be linked to impaired cellular

glucose transport activity in AD (Masliah et al., 1996). Loss

of specific plasma proteins, such as apolipoprotein E (apoE),

also may play a pivotal role during oxidative stress induced

injury during AD. In studies that examined cortical

synaptosomes or neurons from transgenic mice lacking

apoE, samples from apoE knockout mice possessed

increased levels of oxidative stress and caspase activity

during Ab exposure (Keller et al., 2000) as well as enhanced

NO synthase activity (Law et al., 2003), suggesting a

protective role for apoE. Although some investigators argue

that observed lipid peroxidation in the brain of AD patients

does not appear to correlate with the extent of neuritic

plaques, neurofibrillary tangles, or apoE genotype, lipid

peroxidation does appear to directly coincide with pro-

gressive neuronal degeneration in AD patients (Montine

et al., 1999). Other observations further support a role for

ROS during AD. Selective oxidative modification of

intracellular proteins, such as increased protein carbonyl

levels in creatine kinase BB and b-actin, can be seen in AD

(Chong et al., 2005b).

Other evidence exists that suggests cellular injury duringAD may result from both ROS as well as from impaired

cellular repair mechanisms following oxidative injury. In

one study, 8-hydroxy-20-deoxyguanosine (8-OHdG), a

marker of oxidative damage in intact DNA and as a free

repair product during DNA repair mechanisms, was

examined in the cerebrospinal fluid of AD patients.

Significant elevations of 8-OHdG linked to intact DNA

were observed in the cerebrospinal fluid of AD patients,

suggesting that these patients suffer from impaired DNA

mechanisms. Yet, levels of free 8-OHdG, which are

generated during normal cellular repair mechanisms, were

found to be significantly depleted in the cerebrospinal fluid

of AD patients, further supporting the premise of deficient

DNA repair mechanisms in these patients (Lovell et al.,

1999).

The neurotoxicity of Ab, a major component of AD

pathogenesis, also is associated with cellular injury

following ROS exposure. In mice overexpressing APP,

the Ab deposits that are characteristically found in AD co-

localize with several oxidative stress markers (Smith et al.,

1998), suggesting that there exists a close correlation

between oxidative stress and Ab deposition. In addition,

agents that modulate ROS have been shown to reduce

cellular injury during Ab exposure. Application of the free

radical antioxidant Vitamin E has been demonstrated to

prevent neurotoxicity from Ab (Subramaniam et al., 1998).

Over the last decade, a body of work has been generated

to support the premise that Ab can directly lead to the

generation of ROS. Early studies have demonstrated that Ab

can lead to the generation of hydrogen peroxide and cell

death in primary neuronal cultures (Behl et al., 1994). Theability of Ab to generate ROS may be a result of its

methionine composition, since the substitution of methio-

nine by valine, or the removal of the methionine in Ab,

blocks ROS production, protein oxidation, and toxicity to

primary hippocampal neurons (Varadarajan et al., 1999).

Furthermore, free radical generation by Ab appears to be

strongly influenced by the aggregational state of the

peptides, such that inhibition of Ab aggregation can reduce

neuronal toxicity and free radical generation (Monji et al.,

2001; Tomiyama et al., 1996). The generation of hydrogen

peroxide by Ab may be mediated through mechanisms that

are related to metal ion reduction (Huang et al., 1999) and

the cellular ions of copper, zinc, and iron (Liu et al., 1999b)

that are significantly elevated in the senile plaques of

patients with AD and can accelerate aggregation of Ab

(Deibel et al., 1996).

Channel formation during oxidative stress also may be a

significant factor in the pathogenesis of AD and Ab toxicity.

Ab is able to spontaneously insert into planar lipid

membranes to form selective, voltage-dependent, ion-

permeable channels (Arispe et al., 1993; Mirzabekov

et al., 1994). The subsequent channels formed may be

calcium-permeable and lead to cellular toxicity through

impaired calcium homeostasis (Lin et al., 1999; Sanderson

et al., 1997) as well as through calpain activation (Bolandand Campbell, 2003). Aggregates of Ab can further interact

with the lipid bilayer and reduce membrane fluidity to

potentially impair cell function and promote cell injury

(Kremer et al., 2001). Association with membrane

phospholipids by Ab can be extensive in nature to disrupt

both endosomal and plasma membranes through a pH

dependent mechanism (McLaurin and Chakrabartty, 1996).

Cellular injury as a result of ROS appears to proceed

through apoptotic or programmed cell death (PCD)

mechanisms. Accumulating evidence has been obtained

from human and in vitro models of AD suggesting that

apoptosis contributes to the neuronal loss during the disease.

Data from in situ TUNEL (terminal deoxynucleotidyl

transferase nick-end labeling) assays of brain tissues from

individuals with AD demonstrate neuronal demise consis-

tent with PCD. A correlation between the incidence of

TUNEL-positive cells and plaque density also exists

(Colurso et al., 2003). Levels of the apoptotic marker

prostate apoptosis response-4 (Par-4) has been shown to be

significantly increased in the brains of patients with AD

(Guo et al., 1998). Other lines of evidence link apoptotic

cellular injury with APP and its proteolytic product Ab. I n i n

vitro studies, expression of familial AD mutants of APP

results in apoptotic neuronal injury (McPhie et al., 2003). It



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is the cytoplasmic domain of APP that can lead to sustained

apoptosis through c-Jun N-terminal kinase pathways

(Hashimoto et al., 2003b). Additional studies have

illustrated that direct application of Ab to neuronal cells

can lead to chromatin condensation characteristic of

apoptosis in cultured neurons.

PD is a movement disorder characterized by restingtremor, rigidity, and bradykinesia. The pathophysiological

basis of the symptoms rests upon the degeneration of

dopaminergic neurons in the substantia nigra (SN). In some

scenarios, it has been hypothesized that dopamine may even

be a culprit in precipitating disease progression (Maiese

et al., 2003). Dopamine may increase the rate of the

generation of ROS species and subsequent oxidative

products as well as decrease the reserve capacity of the

brain to inactivate ROS (Zigmond et al., 2002). Other

observations also support the premise that PD is a result of

ROS generation. Cerebral iron, which can be a catalyst for

the formation of hydroxyl radicals has been demonstrated to

be increased in the basal ganglia of individuals with PD

(Griffiths et al.,1999). Elevations in oxidative products, such

as lipid peroxides (Groc et al., 2001), protein carbonyls

(Alam et al., 1997), and products of nucleic acid 8-

hydroxyguanosine (Zhang et al., 1999), have been observed

in the SN of PD patients. Furthermore, when protein

expression was compared in the SN from patients with PD

and from controls, a total of 44 proteins expressed in the SN

were identified by peptide mass fingerprinting with several

representing mitochondrial and ROS scavenging proteins

supporting oxidative stress involvement (Basso et al., 2004).

As a correlation to the increased levels of these products in

the brain, a systemic increase of the oxidized products ofDNA, RNA, 8-hydroxyguanosine, and 8-hydroxy-20-deox-

yguanosine has been found in the serum and cerebrospinal

fluid of individuals with PD (Kikuchi et al., 2002). Given

these studies, new approaches to treat patients with PD

advocate the use of neuroprotective monoamine oxidase

inhibitors combined with iron chelation therapy (Youdim

et al., 2004).

Huntingtons disease (HD) is an autosomal dominant

neurodegenerative disease characterized by impairment of

involuntary movement and cognitive impairment. Selective

loss of neurons in the basal ganglia and cerebral cortex is one of

the anatomical hallmarks of this disease. In patients with HD,

the basal ganglia has increased levels of OHdG, suggestive of

oxidative DNA damage (Browne et al., 1997). Furthermore,

transgenic models of HD with R6/2 mice reveal increased

OHdG in urine, plasma, and striatal microdialysates (Bogda-

nov et al., 2001). In other studies with transgenic R6/1 mice,

dichlorofluorescein (DCF), an index of ROS formation, was

significantly increased in R6/1 mice at 11, 19, and 35 weeks of

age while the antioxidant catalase enzyme was significantly

depressed, suggesting an active role for ROS during the onset

and progression of HD (Perez-Severiano et al., 2004).

Amyotrophic lateral sclerosis (ALS), a disabling and

fatal neurodegenerative disease, is characterized by the

progressive loss of muscle power as a result of the selective

loss of motor neurons in the motor cortex, brainstem, and

spinal cord. Although approximately 10% of the reported

cases are associated with inheritance, approximately 23%

of observed ALS cases can be related to a mutation in the

antioxidant enzyme copper zinc SOD (Rosen et al., 1993).

ROS has been found to be increased in ALS in mice(Bogdanov et al., 1998). Additionally, the presence of

oxidative products of protein, DNA, and lipid in the brains of

ALS patients supports an involvement of ROS in the

pathology of ALS (Liu et al., 1999a). Reduction in ROS may

offer hope in providing some form of therapy for ALS. For

example, mice expressing human mutant SOD1 G93A with

EUK-8 and EUK-134, two synthetic SOD/catalase

mimetics, have been shown to reduce oxidative stress and

potentially prolong survival in animal models of ALS (Jung

et al., 2001).

3. Early and late apoptotic programs

Apoptosis, or PCD, is considered to be important for

tissue re-modeling during development. Yet, this active

process is recognized as a central pathway that can lead to a

cells demise in a variety of tissues and has recently been

identified in organisms as diverse as plants (Hatsugai et al.,

2004). PCD consists of two independent processes that

involve membrane phosphatidylserine (PS) exposure and

DNA fragmentation (Maiese et al., 2004). Apoptotic injury

is believed to contribute significantly to a variety of disease

states that especially involve the nervous system such as

ischemic stroke, AD, PD, and spinal cord injury (Chong andMaiese, 2004; Li et al., 2004b). Outside of the nervous

system, such as during cardiovascular injury, PCD also may

be a significant precipitant of cell death. Ischemic-

reperfusion injury can lead to apoptosis in cardiomyocytes

(Cai et al., 2003).

As an early event in the dynamics of cellular apoptosis,

the biological role of membrane PS externalization can vary

in different cell populations. In some cell systems, PS may

be required for embryogenesis (Bose et al., 2004). Yet, in

mature tissues, membrane PS externalization can become a

signal for the phagocytosis of cells (Hong et al., 2004).In the

nervous system, cells expressing externalized PS may be

removed by microglia (Chong et al., 2003c; Li et al., 2004b).

An additional role for membrane PS externalization in the

vascular cell system is the activation of coagulation

cascades. The externalization of membrane PS residues in

ECs can promote the formation of a procoagulant surface

(Chong et al., 2004a).

In contrast to the early externalization of membrane PS

residues, the cleavage of genomic DNA into fragments is

considered to be a delayed event that occurs late during

apoptosis (Dombroski et al., 2000; Jessel et al., 2002; Kang

et al., 2003b; Maiese and Vincent, 2000). Several enzymes

responsible for DNA degradation have been differentiated



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based on their ionic sensitivities to zinc (Torriglia et al.,

1997) and magnesium (Sun and Cohen, 1994). Calcium, a

critical independent component that can determine cell

survival (Weber, 2004), also may determine endonuclease

activity through calcium/magnesium-dependent endonu-

cleases such as DNase I (Madaio et al., 1996). Other

enzymes that may disassemble DNA include the acidic,cation independent endonuclease (DNase II) (Torriglia et al.,

1995), cyclophilins (Montague et al., 1997), and the 97 kDa

magnesium-dependent endonuclease (Pandey et al., 1997).

In the nervous system, three separate endonuclease activities

are present that include a constitutive acidic cation-

independent endonuclease, a constitutive calcium/magne-

sium-dependent endonuclease, and an inducible magne-

sium-dependent endonuclease (Vincent and Maiese, 1999b).

The physiologic characteristics of the magnesium-depen-

dent endonuclease, such as a pH range of 7.48.0, a

dependence on magnesium, and a molecular weight of 95

108 kDa, are consistent with a recently described consti-

tutive 97 kDa endonuclease in non-neuronal tissues.

Exposure to ROS can precipitate apoptosis in neurons and

ECs through multiple cellular pathways. Oxidative stress,

such as NO or hydrogen peroxide, results in nuclei

condensation and DNA fragmentation (Chong et al.,

2003b; Goldshmit et al., 2001; Pugazhenthi et al., 2003;

Vincent and Maiese, 1999b). In neurons, NO exposure

produces apoptotic death in hippocampal and dopaminergic

neurons (Chong et al., 2003a; Sharma and Ebadi, 2003;

Vincent and Maiese, 1999a; Witting et al., 2000). Injury

during NO exposure also can become synergistic with

hydrogen peroxide to render neurons more sensitive to

oxidative injury (de la Monte et al., 2003; Wang et al., 2003).Hydrogen peroxide also results in neuronal injury through

impaired mitochondrial function and increased levels of pro-

apoptotic gene products, such as CD95/Fas (de la Monte

et al., 2000; Pugazhenthi et al., 2003; Vaudry et al., 2002).

Externalization of membrane PS residues also occurs in

neurons during anoxia (Chong et al., 2002b), NO exposure

(Chong et al., 2003f), or during the administration of agents

that induce the production of ROS, such as 6-hydroxydo-

pamine (Salinas et al., 2003).

4. Microglial activation and inflammation

Modulation of extrinsic cell homeostasis through micro-

glial activation is as vital to cellular survival as the

maintenance of cellular DNA integrity. Microglia are

monocyte-derived immunocompetent cells that enter the

CNS during embryonic development and function similar to

peripheral macrophages for the phagocytic removal of

apoptotic cells. Some studies identify the generation of

annexin I and membrane PS exposure that appears to be

necessary to connect an apoptotic cell with a phagocyte

(Arur et al., 2003). Secreted factors by either apoptotic or

phagocytic cells, such as milk fat globule-EGF-factor 8

(Hanayama et al., 2004), fractalkine (Hatori et al., 2002),

and lipid lysophosphatidylcholine (Lauber et al., 2003), also

have been shown to assist with the phagocytic removal of

injured cells. Yet, the translocation of membrane PS residues

from the inner cellular membrane to the outer surface

appears to be essential for the removal of apoptotic cells

(Fadok et al., 2001; Kang et al., 2003b; Maiese and Vincent,2000). The phospholipids of the plasma membrane are

normally in an asymmetric pattern with the outer leaflet of

the plasma membrane consisting primarily of choline-

containing lipids, such as phosphatidylcholine and sphin-

gomyelin, and the inner leaflets consisting of aminopho-

spholipids that include phosphatidylethanolamine and PS.

The loss of membrane phospholipid asymmetry leads to the

externalization of membrane PS residues and serves to

identify cells for phagocytosis (Chong et al., 2003d;

Hoffmann et al., 2001; Kang et al., 2003b; Maiese and

Chong, 2003).

Expression of the phosphatidylserine receptor (PSR) on

microglia also functions with cellular membrane PS

externalization to activate microglia. Cells, such as neurons

or ECs, exposed to ROS can lead to the induction of both

microglial activation and microglial PSR expression.

Treatment with an anti-PSR neutralizing antibody in

microglia prevents this microglial activation (Chong

et al., 2003b; Kang et al., 2003a) and application of PS

directly results in microglial activation that can be blocked

by a PSR neutralizing antibody (Chong et al., 2003b; Kang

et al., 2003b), suggesting that both PS exposure in target

cells and PSR expression in microglia are necessary for

microglial recognition of apoptotic cells in the nervous

system. Recognition of cellular membrane PS by the PS-specific receptors on microglia may require cofactors, such

as Gas6 (Nakano et al., 1997) or other agents, such as

integrin and lectin (Witting et al., 2000).

Although microglia may assist with the removal of

injured cells and cellular debri, these cellular scavengers of

the brain may sometimes aggravate tissue inflammation.

Studies with microglia stimulated by phorbol myristate

acetate have demonstrated the release of superoxide

radicals. Application of scavenger agents for ROS, such

as SOD or deferoxamine mesylate, in the presence of

activated microglia can prevent cellular injury. These studies

suggest that oxidative stress generated by microglia can be

responsible for cellular injury (Tanaka et al., 1994).

Microglia may lead to cellular damage in disease entities,

not only through the generation of ROS products

(Sankarapandi et al., 1998) but also through the production

of cytokines and the demise of neighboring neurons and ECs

(Benzing et al., 1999; Mehlhorn et al., 2000). In HD and

ALS, significant microglial activation has been reported in

regions of the nervous system that are specific for these

disease entities (Obal et al., 2001; Singhrao et al., 1999).

During ischemic injury to cells, activation of microglia can

parallel the induction of cellular apoptosis and correlate well

with the severity of the ischemic insult (Chong et al., 2004a;



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Kang et al., 2003b). Microglia promote the production of

pro-inflammatory cytokines such as tumor necrosis factor-a

(TNF-a) and interleukin-1b, free radicals such as NO and

superoxide (Sankarapandi et al., 1998), and fatty acid

metabolites such as eicosanoids that can precipitate cell

death (Liu and Hong, 2003). TNF-a production by microglia

may be linked to neurodegeneration by increasing thesensitivity of neurons to free radical exposure (Combs et al.,

2001).

In several neurodegenerative diseases, microglial activa-

tion has been identified through glial cultures in autopsy

specimens (Lue et al., 1996). For example, expression of

markers that are indicative of microglial activation was

found to be significantly increased in patients with AD

(Rogers and Lue, 2001). Application of a position emission

tomography marker [11C](R)-PK11195 for microglial

activation in patients with mild and early AD also has

demonstrated microglial activation in regions of the

entorhinal, parietal, and cingulate cortex, suggesting that

microglial activation is an early event in the pathogenesis of

the disease (Cagnin et al., 2001).

One of the major pathogens of AD, namely Ab, has been

shown to lead to inflammatory cell injury through a variety

of routes. Ab can not only precipitate a significant

inflammatory response with microglial activation and the

secretion of TNF-a (Bornemann et al., 2001), but also Ab

can elicit the neuronal expression of inducible nitric oxide

synthase, peroxynitrite production, and neuronal apoptosis

during an acute inflammatory response (Combs et al., 2001).

Microglial cells also co-localize with the perivascular

deposits of Ab and microglial activation correlates with the

development of amyloid plaques (Sheng et al., 1997).Ultrastructural three-dimensional reconstruction of human

amyloid plaques in different stages of development

illustrates that the number of microglia parallels a

progressive increase in fibrillar deposition and the size of

fibrillar plaque (Wegiel et al., 2000). The generation of ROS

by microglia during events such as Ab deposition suggests

that microglia may play an important role during the

development of neurodegenerative diseases.

5. Attempted cell cycle induction in post-mitotic cells

Theattempted reentrance into thecell cyclein post-mitotic

neurons can trigger apoptosis (Becker and Bonni, 2004). In

the CNS, post-mitotic neurons are incapable of differentia-

tion, but they continue to possess the ability to enter into the

cell cycle. During a cellular insult, deregulation of cell cycle

proteins, such as cyclin, cyclin-dependent kinase (CDK), and

the retinoblastoma protein, can ensue (Padmanabhan et al.,

1999).The deficiency of several essential components for the

complete execution of the cell cycle in post-mitotic neurons is

believed to be deleterious to neurons. Several studies have

provided direct evidence that cell cycle induction in post-

mitotic neurons can activate cellular mechanisms that lead to

neuronal apoptosis (El-Khodor et al., 2003; Ino and Chiba,

2001; Konishiand Bonni,2003; Lin et al., 2001; Rideout et al.,

2003; Tetsu and McCormick, 1999). Investigations that

examine ROS as a stimulus for cell cycle induction reveal that

distinct components of apoptotic injury, membrane PS

exposure and genomic DNA fragmentation, occur in concert

with early and late phases of cell cycle induction (Lin et al.,2001).

Oxidative injury associated with ROS may lead to

attempted cell cycle induction in neurons. The induction of

oxidative stress in sympathetic neurons by either dopamine,

which produces free radicals during its metabolism, or by

hydrogen peroxide leads to the increased expression of cell

cycle related genes that include cyclin B and CDK5 prior to

the induction of neuronal apoptosis (Shirvan et al., 1998).

Furthermore, antioxidants that include N-acetyl-L-cysteine

(LNAC) and N-acetyl-D-cysteine (DNAC) can prevent DNA

fragmentation during trophic factor deprivation through

mechanisms that may involve the inhibition of cell cycle

progression in neuronal cell lines (Ferrari et al., 1995).

Acute injury paradigms have suggested a potential role for

ill-fated cell cycle induction in neurons. Cell cycle proteins

(cyclin A, cyclin D, CDK2, CDK4) have been co-localized

with apoptotic cells following middle cerebral artery

occlusion (Li et al., 1997b). Although evidence for cell cycle

induction during cerebral ischemia may be partially

associated with neurogenesis (Taguchi et al., 2004), ischemic

insults also can lead to aberrant cell cycle induction that

may have ramifications for both acute and long-term

consequences on cellular function (Wen et al., 2004).

Other neurodegenerative diseases, such as AD, also

appear to rely upon attempted cell cycle induction, at least inpart, to yield subsequent neuronal cell loss (Arendt et al.,

2000; Busser et al., 1998; Maiese, 2001; Raina et al., 2000 ).

In clinical specimens from AD patients, the cell cycle

regulators P16 and CDK4 have increased expression in

regions such as the hippocampus (McShea et al., 1997). In

addition, expression of other components of the cell cycle,

such as cyclin D, CDK4, proliferating cell nuclear antigen

(PCNA), and cyclin B1 have been shown to be present in

patients with AD in regions that include the hippocampus,

subiculum, locus coeruleus, and dorsal raphe nuclei. A close

association appears to exist between injured cells and cell

cycle protein expression, since staining for cell cycle

proteins have been shown to be absent in brain regions

without neuronal injury of AD patients and in age-matched

brains (Busser et al., 1998). Increased accumulation of cell

cycle kinases, such as CDK5, also has been found in neurons

that are developing neurofibrillary tangles (Pei et al., 1998).

Interestingly, in patients with mild cognitive impairment,

many of which can progress to develop AD (Bennett et al.,

2002), cell cycle proteins, such as cyclin D, cyclin B, and

PCNA, are significantly increased in the hippocampus and

basal nucleus (Yang et al., 2003).

Experimental models of AD have provided further

evidence that cell cycle induction in post-mitotic neurons



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can activate cellular mechanisms that lead to neuronal

apoptosis. For example, application of Ab (140), Ab (1

42), and its active fragment Ab (2535) in neurons can result

in the induction of cyclin D1, cyclins E and A, and the

phosphorylation of the retinoblastoma protein. The activa-

tion of the upstream cyclin-dependent kinases (CDK)4/5/6

appears to be required for the induction of apoptosis inneurons by Ab, since inhibition of CDKs can prevent Abinduced neuronal apoptosis (Alvarez et al., 2001; Giovanni

et al., 1999).

Cell cycle proteins can contribute to neurofibrillary

tangle development. Expression of familial AD mutants of

the APP in primary neurons can precipitate apoptotic injury

through cell cycle induction and p21 mediated pathways

(McPhie et al., 2003). CDK5 also has been identified as a

critical regulator of the tau protein which leads to

neurofibrillary tangles. CDK5 can phosphorylate tau

directly (Flaherty et al., 2000). Furthermore, phosphoryla-

tion of tau by Ab can be blocked by treatment with antisense

against p35, a protein that is cleaved to the truncated form

p25 which can activate CDK5. This work provides evidence

that Ab requires both the cleavage of p35 and the activation

of CDK5 to lead to tau phosphorylation (Town et al., 2002).

Correlative work has shown that p25 also accumulates in

neurons of patients with AD (Patrick et al., 1999). In

addition, overexpression of the p25/Cdk5 complex in

cultured primary neurons leads to cytoskeletal disruption,

the hyperphosphorylation of tau, and apoptosis (Patrick

et al., 1999), suggesting that induction of cell cycle proteins

can be a significant precipitant for neuronal degeneration.

6. Induction of the Wnt pathway

Wnt proteins, named after the Drosophilia protein

wingless and the mouse protein Int-1, represent a

large family of secreted cysteine-rich glycosylated proteins.

This novel family of proteins are intimately involved in

cellular signaling pathways that play a role in a variety of

processes that involve embryonic cell patterning, prolifera-

tion, differentiation, orientation, adhesion, survival, and

apoptosis (Chong and Maiese, 2004; Nelson and Nusse,

2004; Patapoutian and Reichardt, 2000).

Nineteen of the 24 Wnt genes that express Wnt proteins

have been identified in the human. In addition, greater than

80 target genes of Wnt signaling pathways have been

demonstrated in human, mouse, Drosophilia, Xenopus, and

Zebrafish. This representation encompasses several cellular

populations, such as neurons, cardiomyocytes, endothelial

cells, cancer cells, and pre-adipocytes (Nusse, 1999). Wnt

binds to Frizzled transmembrane receptors on the cell

surface to activate downstream signaling events (Fig. 1).

These involve at least two intracellular signaling pathways

that are considered of particular importance. One pathway

controls target gene transcription through b-catenin,

generally referred to as the canonical pathway that involves

Wnt1, Wnt3a, and Wnt8 and functions through b-catenin-

dependent pathways. Another pathway pertains to intracel-

lular calcium (Ca2+) release which is termed the non-

canonical or Wnt/Ca2+ pathway consisting primarily of

Wnt4, Wnt5a, and Wnt11 that functions through non-b-

catenin-dependent pathways, such as the planar cell polarity

(PCP) pathway and the Wnt-Ca2+-dependent pathways

(Kuhl et al., 2000; Nusse, 1999; Patapoutian and Reichardt,

2000).

As one of the best characterized members of the Wnt

family, Wnt1 was first identified as a proto-oncogene in

mammary carcinomas, but recently has been illustrated to

play a critical role in neuronal development (Tang et al.,

2002). Wnt functions by binding to the transmembrane

receptor Frizzled and the co-receptor lipoprotein related

proteins 5 and 6 (LRP-5/6) (Wehrli et al., 2000) followed by

recruitment of disheveled, the cytoplasmic bridging mole-

cule, to inhibit glycogen synthase kinase (GSK-3b) (Ikeda

et al., 1998; Papkoff and Aikawa, 1998). The inhibition of

GSK-3b prevents phosphorylation of b-catenin and its


Fig. 1. Modulation of apoptotic injury by Wnt and Akt pathways. The Wnt

canonical signaling pathway is initiated by activation of its transmembrane

receptor Frizzled (Friz) and the co-receptor lipoprotein related proteins 5

and 6 (LRP-5/6), resulting in the recruitment and activation of disheveled

which inhibits glycogen synthase kinase (GSK)-3b. When active, GSK-3bfunctions with adenomatous polyposis coli (APC) and the phosphorylation

of Axin to result in b-catenin phosphorylation and its subsequent degrada-

tion. In contrast, free b-catenin translocates to the nucleus and activates

lymphocyte enhancer factor (Lef) and T cell factor (Tcf) to stimulate Wnt-

responsive genes. The serinethreonine kinase Akt functions as a down-

stream target of phosphoinositide 3 kinase (PI 3-K). PI 3-K phosphorylates

glycerophospholipid phosphatidylinositol 4,5-bisphosphate, yielding phos-

phatidylinositol 3,4-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-

trisphosphate (PIP3). As a cytosolic protein, Akt translocates to the cell

membrane following its binding to PIP2 and PIP3 and becomes activated

through phosphorylation by phosphoinositide-dependent kinase 1 (PDK1).

Wnt also may activate Akt through the Wnt-1 induced secreted protein

(WISP-1). Akt targets GSK-3b through phosphorylation, resulting in the

inactivation of GSK-3b and blocking the degradation ofb-catenin. Further-

more, phosphorylation of the translation initiation factor 2B (eIF2B) isprevented to prevent the release of cytochrome c (Cyto c). In addition, Akt

inactivates FOXO3a and Bad to inhibit induction of Bim and restore Bcl-xLfunction.


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degradation. The free b-catenin translocates to the nucleus

where it activates lymphocyte enhancer factor (Lef) and T

cell factor (Tcf) (Ishitani et al., 2003) leading to stimulation

of Wnt-response genes (Fig. 1).

In some cell systems, Wnt1 signaling has been associated

with the control of apoptosis. Wnt-1 prevents apoptosis

through b-catenin/Tcf transcription mediated pathways(Chen et al., 2001; Rhee et al., 2002). Overexpression of

exogenous Wnt1 results in the protection of cells against

c-myc induced apoptosis through induction of b-catenin,

cyclooxygenase-2, and Wnt1 induced secreted protein

(WISP-1) (You et al., 2002). Wnt1 signaling also can

inhibit apoptosis through prevention of cytochrome c release

from mitochondria and the subsequent inhibition of caspase

9 activation (Chen et al., 2001). The adenomatous polyposis

coli (APC) gene, a member of the Wnt pathway, appears to

represent another mechanism that regulates PCD. The APC

gene functions to cleave b-catenin leading to the down-

regulation of transactivation of Tcf/Lef (Tetsu and McCor-

mick, 1999). Without Tcf/Lef activity, APC is then

permitted to increase the activities of caspase 3, caspase

7, and caspase 9 and lead to the cleavage of poly(ADP-

ribose) polymerase (PARP) to enhance the vulnerability of

cells to apoptosis (Chen et al., 2003).

In the nervous system, the non-canonical Wnt pathway

has been shown to be expressed in the hippocampus of mice

and can increase dendritic branching in cultured neurons

(Rosso et al., 2005). Wnt signaling through Wnt1 also is able

to guide early neural crest stem cells to develop into sensory

neural cells rather than maturing into other potential neural

crest cell derivatives (Lee et al., 2004). Yet, in regards to

cytoprotection in the brain that involves the Wnt pathway,limited studies are available. The work that is presently

available suggests that enhanced Wnt activity may function

through several cellular pathways to prevent apoptosis

during neuronal or vascular injury. Conditioned media with

Wnt3a activity or the application of a GSK-3b inhibitor can

block hydrogen peroxide induced mitochondrial dysfunc-

tion and apoptotic DNA fragmentation (Shin et al., 2004).

Other work illustrates that Wnt signaling may foster specific

protection against cellular destruction and inflammatory

injury by maintaining genomic DNA integrity and cellular

membrane PS asymmetry (Chong et al., 2004b; Maiese and

Vincent, 2000). Wnt1 overexpression in primary hippo-

campal neurons protects cells against oxidative stress or Ab

toxicity that increases cell survival and prevents PS exposure

and DNA degradation (Chong et al., 2004b). In addition,

agents that combine non-steroidal anti-inflammatory com-

pounds with a cholinesterase inhibitor are believed to

prevent neurotoxicity against Ab. The mechanism of

protection has been suggested to involve the enhancement

of non-amyloidogenic APP cleavage that leads to a

decreased production of endogenous Ab through the Wnt

pathway (Farias et al., 2005).

Loss of Wnt activity may lead to cellular injury or

dysfunction in the CNS during oxidative stress. Wnt1

expression has been demonstrated in the brains of

individuals affected by neuropsychiatric disorders (Miyaoka

et al., 1999). Furthermore, retinal degeneration during

retinitis pigmentosa with the progressive loss of photo-

receptors has been associated with increased secretion of

Frizzled-related protein-2, a Wnt inhibitory protein,

suggesting that loss of Wnt signaling may contribute toretinal neurodegeneration (Jones et al., 2000). Additional

work demonstrates that a mutation in the membrane-type

Frizzled-related protein gene may be involved in retinal

photoreceptor degeneration (Kameya et al., 2002).

During AD, neurotoxicity of Ab in hippocampal neurons

has been linked to increased levels of GSK-3b and loss ofb-

catenin. Decreased production of Ab can occur during the

enhancement of protein kinase C (PKC) activity (Savage

et al., 1998) which may be controlled by the Wnt pathway

(Garrido et al., 2002). The proteolytic processing of APP

during AD also has been closely linked to the Wnt pathway

through presenilin 1 (PS1) and disheveled. PS1 is required

for the processing of APP and has been shown to down-

regulate Wnt signaling and interact with b-catenin to

promote its turnover (Soriano et al., 2001). Disheveled, a

known downstream transducer of Wnt signaling pathway,

also can regulate the a-secretase cleavage of APP through

PKC/mitogen-activated protein kinase dependent pathways,

increasing soluble production of APP (sAPP) (Mudher et al.,

2001). Overexpression of mouse disheveled-1 and -2

inhibits GSK-3b mediated phosphorylation of tau protein

and may thus prevent formation of neurofibrillary tangles

during AD (Wagner et al., 1997). Thus, disheveled may

increase neuronal protection during neurodegenerative

disorders through sAPP production and reduction in tauphosphorylation.

7. Akt as an essential regulatory element

7.1. Activation and expression of Akt

Protein kinase B (PKB) is ubiquitously expressed in

mammals but is initially present at low levels in the adult

brain (Owada et al., 1997). Three family members of this

serine/threonine kinase are now known to exist that were

termed Akt after the molecular cloning of the oncogene

v-Akt and two human homologs (Staal, 1987; Staal et al.,

1988). Theyare PKBa or Akt1, PKBb or Akt2, and PKBg or

Akt3 (Chong et al., 2005a). Akt is part of the AGC (cAMP-

dependent kinase/protein kinase G/protein kinase C) super-

family of protein kinases and consists of three functional

domains. The N-terminal pleckstrin homology (PH) domain

provides binding sites for membrane phospholipids, which

are involved in the recruitment of Akt to the plasma

membrane (Frech et al., 1997). The catalytic domain of Akt

has specificity for serine or threonine residues of proteins

that are substrates for Akt. It is interesting to note that the

three isoforms of Akt share the same regulatory phosphor-



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ylation sites but that splice variants of Akt that lack the

C-terminal hydrophobic motif (HM) possess lower specific

activity than full-length isoforms, suggesting that the

C-terminal HM is vital to stimulate Akt activity (Brodbeck

et al., 2001; Yang et al., 2002) (Table 2).

Activation of Akt is dependent upon PI 3-K (Fig. 1). The

activation of the receptor tyrosine kinase (RTK) and the G

protein-coupled receptor (CPCR) are required to activate PI

3-K. Trophic factors or cytokines can stimulate the

recruitment of PI 3-K to the plasma membrane. Following

activation, PI 3-K phosphorylates membrane glyceropho-

spholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]

resulting in the production of phosphatidylinositol 3,4,5-

trisphosphate (PIP3) and phosphatidylinositol 3,4-bispho-

sphate (PIP2). Both PIP2 and PIP3 bind with equal affinity to

Akt and are required for Akt activation (Thomas et al.,

2002). The critical step for activation of Akt is its transitionfrom the cytosol to the plasma membrane, which is

accomplished by the binding of Akt to PIP2 and PIP3through its PH domain (Stephens et al., 1998). As a result of

this sequence of events, Akt becomes available for

phosphorylation by several upstream kinases.

The phosphorylation of two major residues, Thr308 and

Ser473, are considered necessary for the activation of Akt.

The site of Thr308 is located within the activation T-loop of

Akt1. For Akt2 and Akt3, the equivalent residues are Thr309

and Thr305, respectively (Walker et al., 1998). These

phosphorylation sites are believed to be critical for the

activation of Akt. Yet, the phosphorylation of Ser473 at the

C-terminal HM domain also is necessary for the complete

activation of Akt (Bellacosa et al., 1998). The phosphoryla-

tion of Thr308 is dependent upon its upstream kinase, 3-

phosphoinositide-dependent kinase-1 (PDK1) (Wick et al.,

2000). PDK1 cannot directly phosphorylate Ser473, but a

distinct phosphoinositide-dependent kinase PDK2 (Ser473

kinase) has been postulated to promote Akt phosphorylation

on Ser473. The existence of PDK2 is pending further

confirmation.

A number of pathways can control the biological activity

of Akt. Some lipid phosphatases have been shown to

negatively modulate the activity of Akt. The phosphatase

and tensin homolog deleted from chromosome 10 (PTEN)

appears to be a critical regulator of PI 3-K signaling. PTEN

can dephosphorylate tyrosine-, serine-, and threonine-

phosphorylated peptides (Lee et al., 1999). PTEN negatively

regulates PI 3-K pathways by specifically dephosphorylat-

ing PIP2 and PIP3 at the D3 position (Maehama and Dixon,

1998). As a result, a reduction in the membrane

phospholipid pool that is necessary for the recruitment of

Akt can ensue during PTEN activity.

Other lipid phosphatases, such as SHIP (SH2 domain-

containing inositol phosphatase), can regulate Akt activity.

SHIP is an inositol 50-phosphatase that dephosphorylates

inositides and phosphoinositides on the 50-position resulting

in the transformation of PIP3 into PIP2. The SHIP2 gene

appears to modulate insulin signaling, since targeted

disruption of this gene leads to increased insulin sensitivity

that occurs as a result of enhanced phosphorylation of Akt2at the plasma membrane (Sasaoka et al., 2004). In other cell

systems that involve hematopoietic proliferation, SHIP also

functions to block activation of Akt (Carver et al., 2000).

The Src homology domain 2 (SH2)-containing tyrosine

phosphatases (SHP) also have been implicated in the control

of the Akt pathway. In regards to SHP1 and SHP2, SHP1 is

predominantly expressed in hematopoietic cells, but SHP2 is

more ubiquitously expressed and occurs in the nervous

system (Chong et al., 2003f). Through the activation of Akt,

SHP1 can selectively bind and dephosphorylate PTEN to

reduce the stability of this protein (Lu et al., 2003). SHP2

also appears to modulate the activation of Akt (Ivins Zito

et al., 2004) to prevent cellular death from apoptosis through

inhibition of either caspase 1- or 3-like activities (Chong

et al., 2003f; Ivins Zito et al., 2004).

Alternate cellular systems are responsible for the

enhancement of Akt activity. Carboxyl-terminal modulator

protein (CTMP) also can negatively regulate the activity of

Akt. CTMP is a 27 kDa protein that binds specifically to the

carboxyl-terminal regulatory domain of Akt1 at the plasma

membrane (Maira et al., 2001). The binding of CTMP to

Akt1 decreases the activity of Akt1 by inhibiting the

phosphorylation of Akt1 on Ser473 and Thr308 (Maira et al.,

2001). The T cell leukemia/lymphoma 1 (TCL1) protein


Table 2

Substrates of Akt that determine apoptotic cell injury

Substrate Function Selected references

FOXO3a Activation leads to apoptotic injury, cell cycle progression;

contributes to oxidative stress; possesses caspase 3 cleavage sequence

Brunet et al. (1999); Medema et al. (2000);

Kops et al. (2002) and Chong et al. (2004c)

GSK-3b Phosphorylates b-catenin, eIF2B, CREB, and tau protein to result

in apoptosis and the formation of neurofibrillary tangles; promotescytochrome c release, caspase activation

Somervaille et al. (2001); Kirschenbaum et al. (2001);

Pap and Cooper (2002) and Koh et al. (2003)

Bad Oxidative stress activates Bad;, phosphorylation of Bad by Akt blocks

apoptotic injury, prevents cytochrome c release

Datta et al. (1997); Simakajornboon et al. (2001);

Chong et al. (2003b) and Uchiyama et al. (2004)

NF-kB Leads to the induction of multiple anti-apoptotic genes; blocks caspase

activity; protects through activation of Bcl-xL

Wang et al. (1998); Chen et al., (2000);

De Smaele et al. (2001) and Tang et al. (2001)

Note: CREB, cAMP-response element-binding protein; eIF2B, the translation initiation factor 2B; GSK-3b, glycogen synthase kinase-b; IKK, IkB kinase;

JNK, c-Jun-amino terminal kinase; NF-kB, nuclear factor-kB.


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functions as a co-activator of Akt. TCL1 can stabilize

mitochondrial membrane potential and promote cell

proliferation and survival (Laine et al., 2000). TCL1 binds

to Akt1 and increases Akt1 kinase activity to promote its

nuclear translocation (Pekarsky et al., 2000). Additional

work has shown that TCL1 binds to the PH domain of Akt

and the formation of TCL1 trimers facilitate the formation ofthe Akt/TCL1 complex. Within this complex, Akt is

phosphorylated and activated in vivo (Laine et al., 2000).

Akt activity also can be facilitated by a 90 kDa heat shock

protein (Hsp90). Hsps are characterized by their mass in

kilodaltons, are induced in response to heat in essentially all

organisms, and are highly conserved between different

species. Hsps, such as Hsp90, can be cytoprotective, such

as preventing cell injury against heat thermal stress (Beere

et al., 2000; Kalwy et al., 2003; Latchman,2004).Aktbindsto

Hsp90 through its 229309 residues resulting in stabilization

of thephosphorylatedAkt. Inhibition of Akt binding to Hsp90

leads to dephosphorylation of Akt by protein phosphatase 2A

(PP2A) and induction of apoptosis (Sato et al., 2000).

Intracellular Akt also can become complexed with Hsp90 and

Cdc37. As a result of this association,increased Akt activity is

present but is closely dependentuponthe presence of Hsp90in

the complex (Basso et al., 2002).

The cellular expression of Akt can vary in a variety of

tissues and cells. Akt1 is the most highly expressed isoform.

Although Akt2 is expressed at a lower level than Akt1,

significant expression of Akt2 occurs in insulin-responsive

tissues, such as skeletal muscle, liver, heart, kidney, and

adipose tissue (Altomare et al., 1995). In the CNS, the

expression of Akt1 and Akt2 can be observed at increased

levels during development but is gradually decreased duringpostnatal development (Owada et al., 1997). Yet, in the adult

brain, expression of Akt1 and Akt2 is initially weak with a

dramatic increase in the expression of Akt1 mRNA and Akt1

protein in cells that are subjected to injury (Chong et al.,

2004a; Kang et al., 2003b; Owada et al., 1997), suggesting

that Akt may play an important role during cell injury. In

contrast to Akt1 and Akt2, Akt3 is expressed only in a

limited number of tissues, such as in the brain and testes,

with lower expression evident in skeletal muscle, pancreas,

heart, and kidney (Nakatani et al., 1999).

7.2. Akt as a modulator apoptotic injury and

inflammation during ROS exposure

Akt is a critical survival factor that can modulate cellular

pathways in both the central and peripheral nervous systems.

Early studies have demonstrated that overexpression of Akt

in CNS neurons prevents apoptosis during growth factor

withdrawal (Datta et al., 1997). Similar investigations that

employed superior cervical ganglion neurons also illustrated

that Akt was necessary to prevent cell death during nerve

growth factor withdraw (Philpott et al., 1997). Additional

studies have shown that Akt can be both necessary and

sufficient for the survival of neurons, since expression of a

dominant-negative Akt or inhibition of PI 3-K yields

apoptotic cell death during trophic factor administration

(Crowder and Freeman, 1998) and precipitates cell death

during oxidative stress (Kang et al., 2003a,b). Akt also

impacts upon the function and survival of cerebral vascular

ECs. Recent investigations have shown that Akt modulates

cerebral blood flow and vasomotor tone (Luo et al., 2000)and prevents apoptotic injury during compromises in

mitochondrial function and caspase regulation (Chong

et al., 2002b, 2004a). Further work has illustrated an

important role for Akt for the survival of cells during a

number of injury paradigms. Enhanced Akt activity can

foster cell survival during free radical exposure (Chong

et al., 2003b; Matsuzaki et al., 1999), matrix detachment

(Rytomaa et al., 2000), neuronal axotomy (Namikawa et al.,

2000), DNA damage (Chong et al., 2002b, 2004a; Henry

et al., 2001; Kang et al., 2003a), anti-Fas antibody

administration (Suhara et al., 2001), oxidative stress (Chong

et al., 2003b; Kang et al., 2003a,b; Yamaguchi and Wang,

2001), hypoxic preconditioning (Wick et al., 2002), Ab

exposure (Martin et al., 2001), and transforming growth

factor-b (TGF-b) application (Conery et al., 2004).

Akt possesses the ability to offer a broad level of

cytoprotection in cells through both intrinsic cell mechan-

isms that involve the maintenance of genomic DNA and the

exposure of membrane PS residues. Through the over-

expression of a myristoylated (active) form of Akt and a

kinase-deficient dominant-negative Akt, recent work has

shown that Akt is both necessary and sufficient to protect

cells, such as neurons and ECs from injury associated with

oxidative stress (Chong et al., 2003b; Kang et al., 2003a,b).

Overexpression of myr-Akt significantly protects cells fromfree radical injury and prevents degradation of genomic

DNA (Fig. 2). Yet, cells with a dominant-negative

overexpression that lack kinase activity suffer a significant

loss in cell survival during oxidative stress. Further studies

have suggested that through the inhibition of PI 3-K

phosphorylation of Akt or through the overexpression of a

kinase-deficient dominant-negative Akt, endogenous cellu-

lar reserves of Akt also can provide an additional level of

protection during cell injury that can function in concert

with the exogenous activation of Akt to achieve increased

cellular protection (Chong et al., 2004a; Kang et al.,

2003a,b). It is important to note that activation of Akt is not

always desirable. Under some conditions, enhanced cellular

survival during Akt activation in cells that are targeted for

destruction, such as in neoplastic cells, could undermine

treatment as well as foster the growth of a neoplasm. As a

result, recent work has identified Akt as a potential target to

block during the treatment of non-small cell lung cancers

that contain mutations in the epidermal growth factor

(Sordella et al., 2004).

Akt prevents inflammatory cell demise through extrinsic

cellular mechanisms that involve membrane PS exposure

and the subsequent activation of microglia. Enhanced Akt

activity can prevent cellular membrane PS externalization in



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both neurons and ECs during a variety of insults that involve

anoxia, free radical exposure, and oxygen-glucose depriva-

tion (Chong et al., 2002b, 2004a; Maiese et al., 2004). In

addition, Akt appears to employ the modulation of

membrane PS externalization to prevent microglial activa-

tion (Kang et al., 2003b). Activation of Akt can prevent

membrane PS exposure on injured cells and block the

activation of microglia that are exposed to media taken from

cells that overexpress active, phosphorylated Akt during

cellular injury (Kang et al., 2003a,b). Cytoprotective agents,

such as nicotinamide and erythropoietin (EPO), also employ

mechanisms that involve Akt to regulate microglial

activation and proliferation (Chong et al., 2003d; Liet al., 2004b; Maiese et al., 2004). These protective agents

block membrane PS exposure on cells and possibly prevent

the shedding of membrane PS residues that is known to

occur during apoptosis (Simak et al., 2002). In addition to

targeting the activity of membrane PS exposure and

microglial activation, Akt also may directly address cellular

inflammation by inhibiting several pro-inflammatory cyto-

kines, such as TNF-a (Fontaine et al., 2002).

In addition to its ability to protect cells against apoptotic

and inflammatory injury, Akt can function to either reduce or

prevent cellular destruction from ROS. For example, ROS

generated by hydrogen peroxide can lead to the endogenous

activation of Akt in several cell lines such as Hela, A549, and

MCF-7 cells (Wang et al., 2000). In human glioblastoma cell

lines, hydrogen peroxide also leads to a marked phosphor-

ylation of Akt (Sonoda et al., 1999). Generation of

peroxynitrite by sodium nitrite and acidic hydrogen

peroxide also results in a time and dose-dependent activation

of Akt followed by inactivation of GSK-3b in human skin

primary fibroblast cells (Klotz et al., 2000). Akt activation

during ROS in several neuronal and vascular cell systems

has been demonstrated in neuronal cell lines (Kang et al.,

2003a,b; Salinas et al., 2003), primary hippocampal and

cortical neurons (Chong et al., 2003b,e; Crossthwaite et al.,

2002; Matsuzaki et al., 1999) and cerebral vascular ECs

(Chong et al., 2002b, 2004a).

7.3. Akt can provide the stimulus for altering the

course of neurodegenerative disease

As a result of the broad protective nature of Akt, it may

come as no surprise to learn that many agents or growth

factors appear to prevent apoptotic cellular injury through

Akt activation. In the vascular system, angiopoietin-1 is an

endothelium-specific ligand essential for embryonic vas-

cular stabilization, branching, morphogenesis, and post-

natal angiogenesis. Angiopoietin-1 also supports endothelialcell survival and prevents apoptosis through the activation of

Akt that requires a PI 3-K dependent pathway (Papape-

tropoulos et al., 2000). Furthermore, in the cardiovascular

system, myocardial protection by insulin during myocardial

ischemia/reperfusion is abolished by PI 3-K inhibition,

suggesting that cardioprotection of insulin is mediated by

Akt activation (Jonassen et al., 2001; Li et al., 2004a; Maiese

et al., 2005). The involvement of the PI 3-K/Akt pathway

also has been demonstrated during the protection of retinal

ganglion cells from axotomy (Kermer et al., 2000).

A number of trophic factors and cytokines, such as EPO,

may depend upon Akt to offer cellular protection ( Maiese

et al., 2003). EPO can phosphorylate Akt and is dependent

upon the activation of PI 3-K and Janus kinase 2 (Jak2)

(Chong et al., 2002b; Witthuhn et al., 1993). Activation of

Jak2 promotes the phosphorylation of tyrosine residues in

the intracellular portion of the EPO receptor (Witthuhn et al.,

1993). Phosphorylation of the last tyrosine of the EPO

receptor initiates binding of the 85 kDa regulatory subunit of

PI 3-K, a heterodimer consisting of a 110 kDa catalytic

subunit and an 85 kDa regulatory subunit. As a result of the

binding of the 85 kDa regulatory subunit, the 110 kDa

catalytic subunit becomes active and leads to the phosphor-

ylation of Akt.


Fig. 2. Overexpression of Akt1 prevents cellular injury and DNAdegradation during oxidative stress. Representative images illustrate DNAfragmentationwith

TUNEL and cell survival with a trypan blue dye exclusion (TB) methods in both wild-type and myristolated (myr)-Akt1 (active Akt1) transfected cerebral

microvascular endothelial cells (ECs) 24 h followingexposure to a NO donor (NOC-9, 1000mM).NO induced DNA fragmentation andTB stainingwas evident

in wild-type cells (wild-type/NO), but is absent in cells overexpressing Akt1 (myr-Akt1/NO).


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Central to the ability of EPO to prevent cellular apoptosis

is the activation of Akt by EPO (Maiese et al., 2004). During

anoxia or free radical exposure, expression of the active

form of Akt (phospho-Akt) is increased (Kang et al.,

2003a,b). EPO can significantly enhance the activity of Akt

during oxidative stress and prevent inflammatory activation

of microglia (Chong et al., 2003a,b,e). This up-regulation ofAkt activity during injury paradigms appears to be vital for

EPO protection, since prevention of Akt phosphorylation

blocks cellular protection by EPO (Chong et al., 2003a,b,e).

Through the regulation of the PI 3-K/Akt dependent

pathway, EPO can prevent cellular apoptosis following N-

methyl-D-aspartate toxicity (Dzietko et al., 2004), hypoxia

(Chong et al., 2002b), and oxidative stress (Chong et al.,

2003a,b,e).

Given the intimate association between Akt and

cytoprotective agents, Akt may be viewed as an essential

target for therapeutic strategies against a number of diseases

that involve apoptotic cell death. The association of familial

AD with mutations in APP suggests that wild-type APP may

have a protective ability against toxic insults to cells, since

mutations in APP impair its ability to offer resistance against

oxidative stress. Recent work has suggested that protection

by wild-type APP against ROS may require the PI 3-K/Akt

pathway, since dominant-negative forms of Akt eliminated

the protective capacity of wild-type APP (Kashour et al.,

2003). Furthermore, overexpression of Akt1 can attenuate

apoptosis during Ab exposure (Martin et al., 2001). In

models of Parkinsons disease that employ the neurotoxin 1-

methyl-4-phenylpyridinium (MPP+), apoptosis was pre-

vented and a reduction in ROS was observed in cells

overexpressing an active form of Akt (Salinas et al., 2001).Activation of Akt during acute cellular insults also

appears to be necessary to foster cell survival. Phosphoryla-

tion of Akt has been observed in the brain following either

focal or global cerebral ischemia (Friguls et al., 2001; Yano

et al., 2001). Sublethal ischemic induction during pre-

conditioning experiments leads to the phosphorylation of

Akt in the hippocampal CA1 region. This activation of Akt

was not present in ischemic animals that did not receive

sublethal ischemic preconditioning and led to a greater

degree of cerebral infarction, suggesting that Akt activation

provides an important mechanism for ischemic precondi-

tioning (Yano et al., 2001). Cell culture experiments also

have supported that hypoxic preconditioning may be

mediated by the activation of Akt (Wick et al., 2002).

8. Downstream cellular targets

At this point of time, no definitive therapy for either acute

or chronic neurodegenerative diseases is available. Yet,

investigations into the cellular pathways that determine

oxidative stress and apoptotic injury have begun to elucidate

pathways that provide us with a clearer understandin

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Oxidative Stress in the Brain Novel Cellular Targets That Govern 2005