| Current
Neurovascular Research
ISSN: 1567-2026
Current Neurovascular Research
Volume 2, Number 1, January 2005
Contents
From the Editor's Perspective: Rational Approaches for Radical
Entities Pp. 1-2
K. Maiese
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Original Articles
Amyloid Beta Peptide 1-40 Stimulates the Na+/Ca2+
Exchange Activity of SNCX Pp. 3-12
Menjor Tino Unlap, Corey Williams, Darryl Morin, Brian
Siroky,Attila Fintha, Amanda Fuson, Layla Dodgen, Gergely
Kovacs,Peter Komlosi, William Ferguson and Phillip Darwin
Bell
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Differential Susceptibility of Naive and Differentiated
PC-12 Cells to Methylglyoxal-Induced Apoptosis: Influence
of Cellular Redox Pp. 13-22
Masahiro Okouchi, Naotsuka Okayama and Tak Yee Aw
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Brief Communication
Stroke Outcomes in Mice Lacking the Genes for Neuronal Heme
Oxygenase-2 and Nitric Oxide Synthase Pp. 23-27
Khodadad Namiranian, Raymond C. Koehler, Adam Sapirstein,
Sylvain Dore
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Review Articles
Pathogenesis of Stroke-Like Episodes in MELAS: Analysis
of Neurovascular Cellular Mechanisms Pp. 29-45
Takahiro Iizuka and Fumihiko Sakai
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Article]
Blood-Brain Barrier Alterations in MDX Mouse,
An Animal Model of the Duchenne Muscular Dystrophy Pp.
47-54
Beatrice Nico, Luisa Roncali, Domenica Mangieri and Domenico
Ribatti
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Employing New Cellular Therapeutic Targets for
Alzheimer's Disease: A Change for the Better? Pp.
55-72
Zhao Zhong Chong, Faqi Li and Kenneth Maiese
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Article]
Cell Culture Models of Oxidative Stress and Injury
in the Central Nervous System Pp. 73-89
Marina V. Aksenova, Michael Y. Aksenov, Charles F. Mactutus
and Rosemarie M. Booze
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Abstracts
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From the Editor's Perspective: Rational Approaches for Radical
Entities
K. Maiese
It is estimated that almost 400 million individuals suffer
from nervous system disorders in the world. These disorders
consist of both acute and chronic neurodegenerative diseases
that include stroke, traumatic brain injury, presenile dementia,
Alzheimer's disease, and Parkinson's disease. In addition,
environmental toxin exposure also has become increasingly
prominent as a precipitant of degenerative disease in the
nervous system.
Intimately coupled to each of these disorders is the generation
of reactive oxygen species (ROS) in the brain. ROS consist
of oxygen free radicals and other chemical entities that include
superoxide free radicals, hydrogen peroxide, nitric oxide,
and peroxynitrite. Under normal physiological conditions,
these species are produced at low levels and are scavenged
by endogenous antioxidant systems of the body that include
superoxide dismutase, glutathione peroxidase, and catalase.
Yet, oxidative stress in the brain occurs when the generation
of ROS overrides the ability of the endogenous antioxidant
system to remove excess oxygen free radicals. Oxidative stress
is a formidable factor for injury in the nervous system, since
it leads to the dysfunction of both neuronal and vascular
cells as well as inciting inflammatory cellular demise. Oxidative
stress leads to the peroxidation of cellular membrane lipids,
cleavage of genomic DNA, and the oxidation of a variety of
cellular proteins. The generation of ROS also can block complex
enzymes in the electron transport chain of the mitochondria
to halt mitochondrial respiration. During the release of free
radicals in the brain, microglia also become activated to
begin the process of phagocytosis for the removal of injured
cells. As a result, microglia are believed to lead to cellular
damage of neighboring neurons and vascular cells, partly through
the generation of ROS products as well as through the production
of cytokines.
In consideration of the significant risks free radical generation
can present to the nervous system, it is surprising to learn
that the brain is highly susceptible to oxidative stress injury
and has only limited capacity to avert cellular injury. A
variety of observations support this premise. For example,
the brain possesses the highest oxygen metabolic rate of any
organ in the body, consuming twenty percent of the total amount
of oxygen in the body and enhancing the possibility for the
aberrant generation of free radicals. In addition, the brain
is composed of significant amounts of unsaturated fats that
can readily serve as a source of oxygen free radicals. Interestingly,
given the increased risk factors for the generation of elevated
levels of ROS in the brain, the brain also may suffer from
an inadequate defense system against oxidative stress. Catalase
activity in the brain, an endogenous antioxidant, has been
reported to exist at levels markedly below other organs of
the body, sometimes approaching catalase levels of ten percent
in other organs such as the liver.
Given the vulnerability of the nervous system during ROS
generation, identifying the cellular pathways that determine
oxidative stress injury in the brain may significantly assist
in the development of therapeutic strategies to either prevent
or at least reduce disability from crippling neurodegenerative
disorders. With this objective, our original articles and
review manuscripts in this issue offer unique insights into
the cellular and molecular pathways that lead to oxidative
stress in the nervous system during a variety of neurodegenerative
disorders. Unlap et al. present in an original article of
this issue evidence that the activities of two sodium/calcium
exchangers, SNCX (salt sensitive sodium/calcium exchanger)
and RNCX (salt resistant sensitive sodium/calcium exchanger),
can influence cytosolic calcium homeostasis during ß-amyloid
exposure, suggesting that these channels, and in particular
the SNCX, may play a role during ß-amyloid cell toxicity
and oxidative stress in Alzheimer's disease.
Interestingly, insight into the vulnerability of the nervous
system during oxidative stress may be gained from the original
studies of Okouchi et al. The work implicates that in neuronal
cell lines oxidative perturbations in the glutathione-to-glutathione
disulfide redox ratio can be associated with increased cell
vulnerability and lead to decreased Bcl-2 expression, cytochrome
c release, and caspase activation. In a brief communication,
Namiranian et al. report that cerebral heme oxygenase-2 (HO-2),
considered to be cytoprotective, and neuronal nitric oxide
synthase (nNOS), viewed to be deleterious, may counter the
effects of one another during acute ischemic oxidative stress.
Although individual gene deletion of HO-2 can exacerbate cerebral
infarct size while knockout of only nNOS can reduce stroke
injury, animals that lack both HO-2 and nNOS do not demonstrate
a significant difference in infarct volume when compared to
normal animals without the double knockout, suggesting that
cytoprotective therapies may need to address multiple gene
and protein targets concurrently to achieve effective results.
Further insights into novel pathways that may precipitate
oxidative stress in the nervous system are outlined in our
review articles for this issue. Iizuka and Sakai discuss the
pathogenesis of stroke-like episodes in the syndrome of mitochondrial
encephalopathy, myopathy, lactic acidosis, and stroke-like
episodes (MELAS). They present evidence for the premise that
MELAS may be precipitated, at least in part, by non-ischemic
neurovascular events that involve increased capillary permeability
precipitated by heightened neuronal excitability in the presence
of mitochondrial capillary angiopathy leading to neuronal
injury and cellular death. The potential contribution of vascular
injury during neurodegenerative disease is further advanced
in the article of Nico et al. The authors outline endothelial
and astrocytic changes in the MDX mouse, an experimental model
of Duchenne muscular dystrophy, that may foster the onset
and progression of the disease as a result of perturbations
in brain osmotic equilibrium. The paper by Chong et al. expands
the list of potential cellular populations that may be targets
of oxidative stress as the authors offer insight into the
cellular complexities of Alzheimer's disease that can involve
the generation of ROS. They present for consideration novel
avenues for therapeutic development that focus upon the modulation
of inflammatory microglial activation, cellular metabolism,
cell-cycle regulation, G-protein regulated receptors, and
cytokine activation. In our final article for this issue,
Aksenova et al. highlight both the necessity and utility of
in vitro based experimental models for the investigation of
oxidative pathways that determine cellular damage as well
as cellular adaptation in a variety of clinical disorders
that include dementia, substance abuse, and human immunodeficiency
viral neurodegeneration.
It is clear that disorders of the nervous system continue
to burden the planet's population with not only increasing
morbidity and mortality, but also with a significant financial
drain through increasing medical care costs coupled to a progressive
loss in economic productivity. As we seek to hone potential
therapeutic approaches for nervous system disorders mediated
by excessive ROS generation, elucidating the mechanisms of
oxidative stress induced injury as exemplified by the thought
provoking original papers and review manuscripts presented
in this issue of Current Neurovascular Research will
hopefully foster the maturation of these investigations into
"rational approaches for radical entities".
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Amyloid Beta Peptide 1-40 Stimulates the Na+/Ca2+ Exchange
Activity of SNCX
Menjor Tino Unlap, Corey Williams, Darryl Morin,
Brian Siroky, Attila Fintha, Amanda Fuson, Layla Dodgen, Gergely
Kovacs, Peter Komlosi, William Ferguson and Phillip Darwin
Bell
The Na+/Ca2+ exchangers, RNCX and SNCX,
were cloned from mesangial cells of salt sensitive and salt
resistant Dahl/Rapp rats, respectively, and differ at amino
acid 218 (RNCXi/SNCXf) and in the exons
expressed at the alternative splice site (RNCXB, D/SNCXB,
D, F). These isoforms are also expressed in myocytes,
neurons, and astrocytes where they maintain cytosolic calcium
homeostasis. We demonstrated that cells expressing SNCX were
more susceptible to oxidative stress than cells expressing
RNCX. Others demonstrated that amyloid β peptide (Aβ)
augments the adverse effects of oxidative stress on calcium
homeostasis. Therefore, we sought to assess the effect of
Aβ 1-40 on the abilities of OK-PTH cells stably expressing
RNCX and SNCX and human glioma cells, SKMG1, to regulate cytosolic
calcium homeostasis. Our studies showed that Aβ 1-40
(1 µM) did not affect RNCX activity, as assessed by
changes in [Ca2+]i (Δ[Ca2+]i,
260 ± 10 nM to 267 ± 8 nM), while stimulating
exchange activity 2.4 and 3 fold in cells expressing SNCX
(100 ± 8 to 244 ± 12 nM) and in SKMG1 cells
(90 ± 11 nM to 270 ± 18 nM), respectively. Our
results also showed that Aβ 1- 40, while not affecting
the rate of Mn2+ influx in cells expressing RNCX,
stimulated the rate of Mn2+ influx 2.8 and 2.9
fold in cells expressing SNCX and in SKMG1 cells. Thus, our
studies demonstrate that Aβ-induced cytosolic calcium
increase is mediated through certain isoforms of the Na+/Ca2+
exchanger and reveals a possible mechanism by which Aβ
1-40 can alter cytosolic calcium homeostasis.
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Differential Susceptibility of Naive and Differentiated PC-12
Cells to Methylglyoxal-Induced Apoptosis: Influence of Cellular
Redox
Masahiro Okouchi, Naotsuka Okayama and Tak Yee
Aw
Neuropathologies have been associated with neuronal de-differentiation
and oxidative susceptibility. To address whether cellular
states determines their oxidative vulnerability, we have challenged
naive (undifferentiated) and nerve growth factor-induced differentiated
pheochromocytoma (PC12) with methylglyoxal (MG), a model of
carbonyl stress. MG dose-dependently induced greater apoptosis
(24h) in naive (nPC12) than differentiated (dPC12) cells.
This enhanced nPC12 susceptibility was correlated with a high
basal oxidized cellular glutathione-to-glutathione disulfide
(GSH/GSSG) redox and an MG-induced GSH-to-Disulfide (GSSG
plus protein-bound SSG) imbalance. The loss of redox balance
occurred at 30 min post-MG exposure, and was prevented by
N-acetylcysteine (NAC) that was unrelated to de novo
GSH synthesis. NAC was ineffective when added at 1h post-MG,
consistent with an early window of redox signaling. This redox
shift was kinetically linked to decreased BcL-2, increased
Bax, and release of mitochondrial cytochrome c which preceded
caspase-9 and -3 activation and poly ADP-ribose polymerase
(PARP) cleavage (1-2h), consistent with mitochondrial apoptotic
signaling. The blockade of apoptosis by cyclosporine A supported
an involvement of the mitochondrial permeability transition
pore. The enhanced vulnerability of nPC12 cells to MG and
its relationship to cellular redox shifts will have important
implications for understanding differential oxidative vulnerability
in various cell types and their transition states.
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Stroke Outcomes in Mice Lacking the Genes for Neuronal Heme
Oxygenase-2 and Nitric Oxide Synthase
Khodadad Namiranian, Raymond C. Koehler, Adam
Sapirstein, Sylvain Dore
Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective
enzyme in a variety of in vivo experimental models.
HO-2, the constitutive isozyme, is enriched in neurons and,
under normal conditions, accounts for nearly all of brain
HO activity. HO-2 deletion (HO-2-/-) leads to increased
neurotoxicity in cultured brain cells and increased damage
following transient cerebral ischemia in mice. Moreover, pharmacologic
inhibition of HO activity significantly augments focal ischemic
damage in wildtype (WT) mice, but does not further exacerbate
it in HO-2-/- mice. The HO system shares some similarities
with nitric oxide synthase (NOS), notably their syntheses
of carbon monoxide (CO) and nitric oxide (NO), respectively,
which are diffusible gases with numerous biological actions,
including neurotransmission and vasodilation. While deletion
of HO-2 results in greater stroke damage, the pharmacologic
inhibition of neuronal nitric oxide synthase (nNOS), or its
gene deletion, confers neuroprotection in animal models of
transient cerebral ischemia. To investigate the interactions,
the outcome of focal cerebral ischemia-reperfusion in double
knockout (HO-2-/- X nNOS-/-) mice lacking
both genes was compared to control WT mice. Wildtype and double
knockout male mice underwent intraluminal middle cerebral
occlusion for 2 hours, followed by reperfusion for 22 hours.
Outcomes in neurologic deficits and infarct size were determined.
No difference was observed between WT and double knockout
mice in the volume of infarction, neurologic signs, decrease
in relative cerebral blood flow during ischemia, or core body
temperature. The results suggest that the deleterious action
of nNOS would counteract the role of HO-2 in neuroprotection.
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Pathogenesis of Stroke-Like Episodes in MELAS: Analysis of
Neurovascular Cellular Mechanisms
Takahiro Iizuka and Fumihiko Sakai
The pathogenesis of stroke-like episodes in mitochondrial
encephalopathy, myopathy, lactic acidosis and stroke-like
episodes (MELAS) is not fully understood although two main
theories have been proposed; ischemic vascular hypothesis
caused by “mitochondrial angiopathy” and generalized
cytopathic hypothesis caused by “mitochondrial cytopathy”.
Crucial molecular mechanism includes the lack of taurine modification
at the wobble uridine of mutant transfer RNAsLeu(UUR) resulting
in defective translation of cognate codons due to a defect
in codon-anticodon interaction. Whereas recent clinical studies
have shed light on the neuronal hyperexcitability, which may
potentially initiate a cascade of stroke-like events. Stroke-like
episodes are characterized by neuronal hyperexcitability,
neuronal vulnerability, increased capillary permeability,
and focal hyperaemia. It is recognized that stroke-like lesions
not only evolve in the area incongruent to a vascular territory,
but also potentially spread into the surrounding cortex with
concomitant vasogenic edema presumably provoked by prolonged
epileptic activities.
Based on the clinical observations, we speculate that stroke-like
episodes appear to be non-ischemic neurovascular events; once
neuronal hyperexcitability developed in a localized brain
region as a result from either mitochondrial dysfunction in
the capillary endothelial cells, or in neurons or astrocytes,
epileptic activities may depolarize the adjacent neurons leading
to propagation of epileptic activities in the surrounding
cortex. Increased capillary permeability provoked by epileptic
activities in the presence of mitochondrial capillary angiopathy
may cause unique edematous brain lesions predominantly involving
the cortex. As a consequence, susceptible neuronal population
in the cortex may result in neuronal loss with a laminar or
pseudo-laminar distribution.
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Blood-Brain Barrier Alterations in MDX Mouse, An Animal Model
of the Duchenne Muscular Dystrophy
Beatrice Nico, Luisa Roncali, Domenica Mangieri
and Domenico Ribatti
This article reviews recent studies on the alterations occurring
in the brain vessel wall of the mdx mouse, an animal model
with genetic defects in a region homologous with the human
Duchenne muscular dystrophy (DMD) gene. These alterations
affect both endothelial and astroglial cells and are associated
with opened tight junctions, swollen perivascular astrocyte
processes and a reduction in the expression of tight junctions
associated proteins, ie. Zonula occludens and of a specific
water channel i.e. aquaporin-4, suggesting that some neurological
dyspfunctions of mdx mice and DMD patients could be associated
with changes in brain osmotic equilibrium.
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Employing New Cellular Therapeutic Targets for Alzheimer's
Disease: A Change for the Better?
Zhao Zhong Chong, Faqi Li and Kenneth Maiese
Alzheimer's disease is a progressive disorder that results
in the loss of cognitive function and memory. Although traditionally
defined by the presence of extracellular plaques of amyloid-b
peptide aggregates and intracellular neurofibrillary tangles
in the brain, more recent work has begun to focus on elucidating
the complexities of Alzheimer's disease that involve the generation
of reactive oxygen species and oxidative stress. Apoptotic
processes that are incurred as a function of oxidative stress
affect neuronal, vascular, and monocyte derived cell populations.
In particular, it is the early apoptotic induction of cellular
membrane asymmetry loss that drives inflammatory microglial
activation and subsequent neuronal and vascular injury. In
this article, we discuss the role of novel cellular pathways
that are invoked during oxidative stress and may potentially
mediate apoptotic injury in Alzheimer's disease. Ultimately,
targeting new avenues for the development of therapeutic strategies
linked to mechanisms that involve inflammatory microglial
activation, cellular metabolism, cell-cycle regulation, G-protein
regulated receptors, and cytokine modulation may provide fruitful
gains for both the prevention and treatment of Alzheimer's
disease.
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Cell Culture Models of Oxidative Stress and Injury in the
Central Nervous System
Marina V. Aksenova, Michael Y. Aksenov, Charles
F. Mactutus and Rosemarie M. Booze
Constantly growing body of evidence suggests that hallmarks
of oxidative stress are present in various central nervous
system (CNS) disorders. Technological advantages in cell culturing
made it possible to use neural cell/tissue cultures as experimental
models for investigation of molecular mechanisms which underlie
the development of oxidative stress condition, damage and
adaptive responses to oxidative insults. This review is focused
on the application of cell culture methodology for studies
of oxidative stress condition in the brain. The review describes
studies of biomarkers of oxidative stress-dependent cell damage
and adaptive responses in various kinds of brain cell culture
models. It discusses the use of cell/tissue culture models
for elucidation of the role and pathogenesis of oxidative
stress in neurodegenerative brain disorders, AIDS-associated
brain pathology, drug abuse, and aging. The review underscores
the importance of cell/tissue-based studies for testing of
new antioxidants and development of therapeutic strategies
for amelioration of oxidative damage in the CNS. The impact
of new advances in gene and protein expression analysis on
the cell/tissue culture-based research of oxidative stress
in the CNS is also discussed.
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