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7/31/2019 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
Z.Z. Chong et al. / Progress in Neurobiology 75 (2005) 207246 215
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
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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.
Z.Z. Chong et al. / Progress in Neurobiology 75 (2005) 207246 219
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