Current
Volume 4, Number 2, 2004
Contents
Molecular
and Cellular Mechanisms of Ischemic Cell Death in the Brain
Excitotoxic Versus Apoptotic Mechanisms of
Neuronal Cell Death in Perinatal Hypoxia/Ischemia Pp.77-85
Chainllie
Young, Tatyana Tenkova, Krikor Dikranian and John W. Olney
Rethinking the Excitotoxic Ionic Milieu: The
Emerging Role of Zn2+ in Ischemic Neuronal Injury Pp.87-111
S.L.
Sensi and J.-M. Jeng
White Matter Injury Mechanisms Pp.113-130
Peter
K. Stys
The Rise and Fall of NMDA Antagonists for
Ischemic Stroke Pp.131-136
L.
Hoyte, P.A. Barber, A.M. Buchan and M.D. Hill
Molecular Mechanisms Underlying Specificity
of Excitotoxic Signaling in Neurons Pp.137-147
Michelle
M. Aarts and Michael Tymianski
Mitochondrial Dysfunction and Glutamate
Excitotoxicity Studied in Primary Neuronal Cultures Pp.149-177
D.G. Nicholls
Nitric Oxide and its Role in Ischaemic Brain
Injury Pp.179-191
Robert G. Keynes and John Garthwaite
Astrocyte Influences on Ischemic Neuronal
Death Pp.193-205
Raymond A. Swanson, Weihai Ying and Tiina M. Kauppinen
What have Genetically Engineered Mice Taught
Us About Ischemic Injury?
Pp.207-225
Dong Liang, Ted M. Dawson and Valina L. Dawson
Abstracts
[Back to top] Excitotoxic Versus Apoptotic Mechanisms of
Neuronal Cell Death in Perinatal Hypoxia/Ischemia
Chainllie
Young, Tatyana Tenkova, Krikor Dikranian and John W. Olney
Hypoxic/ischemic
(H/I) neuronal degeneration in the developing central nervous system (CNS) is
mediated by an excitotoxic mechanism, and it has also been reported that an
apoptosis mechanism is involved. However, there is much disagreement regarding
how excitotoxic and apoptotic cell death processes relate to one another. Some
authors believe that an excitotoxic stimulus directly triggers apoptotic cell
death, but this interpretation is largely speculative at the present time. Our
findings support the interpretation that excitotoxic and apoptotic
neurodegeneration are two separate and distinct cell death processes that can
be distinguished from one another by ultrastructural evaluation. Here we review
evidence supporting this interpretation, including evidence that H/I in the
developing CNS triggers two separate waves of neurodegeneration, the first
being excitotoxic and the second being apoptotic. The first (excitotoxic) wave
destroys neurons that would normally provide synaptic inputs or synaptic
targets for the neurons that die in the second (apoptotic) wave. Since neurons,
during the developmental period of synaptogenesis, are programmed to commit
suicide if they fail to achieve normal connectivity, this explains why
neuroapoptosis occurs following H/I in the developing CNS. However, it does not
support the interpretation that H/I directly triggers apoptotic
neurodegeneration. Rather, it documents that H/I directly triggers excitotoxic
neurodegeneration, and apoptotic neurodegeneration ensues subsequently as the
natural response of developing neurons to a specific kind of deprivation - loss
of the ability to form normal synaptic connections.
[Back to top] Rethinking the Excitotoxic Ionic Milieu: The
Emerging Role of Zn2+ in Ischemic Neuronal Injury
S.L.
Sensi and J.-M. Jeng
Zn2+ plays an
important role in diverse physiological processes, but when released in excess
amounts it is potently neurotoxic. In vivo trans-synaptic movement and
subsequent post-synaptic accumulation of intracellular Zn2+ contributes to the
neuronal injury observed in some forms of cerebral ischemia. Zn2+ may enter
neurons through NMDA channels, voltage-sensitive calcium channels, Ca2+-permeable AMPA/kainate (Ca-A/K) channels, or
Zn2+-sensitive membrane
transporters. Furthermore, Zn2+ is also released from intracellular sites such
as metallothioneins and mitochondria. The mechanisms by which Zn2+ exerts its
potent neurotoxic effects involve many signaling pathways, including
mitochondrial and extra-mitochondrial generation of reactive oxygen species
(ROS) and disruption of metabolic enzyme activity, ultimately leading to
activation of apoptotic and/or necrotic processes.
As is the case
with Ca2+, neuronal mitochondria take up Zn2+ as a way of modulating cellular
Zn2+ homeostasis. However, excessive mitochondrial Zn2+ sequestration leads to
a marked dysfunction of these organelles, characterized by prolonged ROS
generation. Intriguingly, in direct comparison to Ca2+, Zn2+ appears to induce
these changes with a considerably greater degree of potency. These effects are
particularly evident upon large (i.e., micromolar) rises in intracellular Zn2+
concentration ([Zn2+]i), and likely hasten necrotic neuronal death. In
contrast, sub-micromolar [Zn2+]i increases promote release of pro-apoptotic
factors, suggesting that different intensities of [Zn2+]i load may activate
distinct pathways of injury. Finally, Zn2+ homeostasis seems particularly
sensitive to the environmental changes observed in ischemia, such as acidosis
and oxidative stress, indicating that alterations in [Zn2+]i may play a very
significant role in the development of ischemic neuronal damage.
[Back to top] White Matter Injury Mechanisms
Peter
K. Stys
White matter of
the brain and spinal cord is susceptible to anoxia, ischemia, trauma and
autoimmune attack. Irreversible injury to this tissue can have serious
consequences for the overall function of the CNS through disruption of signal
transmission. Like neurons, central myelinated axons are critically dependent
on a continuous supply of oxygen and glucose.
Injury causes failure of the Na-K-ATPase and accumulation of axoplasmic
Na through non-inactivating Na channels, which, together with membrane
depolarization, promotes reverse Na-Ca exchange and axonal Ca overload. An
equally important source of deleterious Ca originates from intracellular
stores, released in part by a mechanism similar to
“excitation-contraction coupling” in muscle, involving activation
of ryanodine receptors by L-type Ca channels. Excitotoxic mechanisms also play
an important role: glutamate released by reversal of Na-dependent glutamate
transporters activates AMPA/kainate receptors to cause injury to glia and
myelin. Excessive accumulation of cytosolic Ca in turn activates various
Ca-dependent enzymes such as calpains, phospholipases and others resulting in
irreversible injury. Reoxygenation paradoxically accelerates injury in many
axons, and promotes cytoskeletal degradation. Blockers of voltage-gated Na
channels represent an attractive therapeutic target because of their ability to
simultaneously interfere indirectly with several Ca sourcing pathways.
Alternatively, or additionally, AMPA/kainate receptor inhibition has also been
shown to be neuroprotective in several white matter injury paradigms. In the clinical
setting, optimal protection of the CNS as a whole in common disorders such as
stroke, traumatic brain and spinal cord injury, will likely require combination
therapy aimed at unique steps in gray and white matter regions, or intervention
at common points in the injury cascades.
[Back to top] The Rise and Fall of NMDA Antagonists for
Ischemic Stroke
L.
Hoyte, P.A. Barber, A.M. Buchan and M.D. Hill
It has long been
accepted that high concentrations of glutamate can destroy neurons, and this is
the basis of the theory of excitotoxicity during brain injury such as stroke.
Glutamate N-methyl-D-aspartate (NMDA) receptor antagonists such as Selfotel,
Aptiganel, Gavestinel and others failed to show neuroprotective efficacy in
human clinical trials or produced intolerable central nervous system adverse
effects. The failure of these agents has been attributed to poor studies in
animal models and to poorly designed clinical trials. We also speculate that
NMDA receptor anatagonism may have hindered endogenous mechanisms for neuronal
survival and neuroregeneration. It remains to be proven in human stroke whether
NMDA receptor antagonism can be neuroprotective.
[Back to top] Molecular Mechanisms Underlying Specificity
of Excitotoxic Signaling in Neurons
Michelle M. Aarts and Michael Tymianski
The central role
of glutamate receptors in mediating excitotoxic neuronal death in stroke,
epilepsy and trauma has been well established. Glutamate is the major excitatory
amino acid transmitter within the CNS and it’s signaling is mediated by a
number of postsynaptic ionotropic and metabotropic receptors. Although calcium
ions are considered key regulators of excitotoxicity, new evidence suggests
that specific second messenger pathways rather than total Ca2+ load, are
responsible for mediating neuronal degeneration. Glutamate receptors are found
localized at the synapse within electron dense structures known as the
postsynaptic density (PSD). Localization at the PSD is mediated by binding of
glutamate receptors to submembrane proteins such as actin and PDZ containing
proteins. PDZ domains are conserved motifs that mediate protein-protein
interactions and self-association. In addition to glutamate receptors PDZ-containing
proteins bind a multitude of intracellular signal molecules including nitric
oxide synthase. In this way PDZ proteins provide a mechanism for clustering
glutamate receptors at the synapse together with their corresponding signal
transduction proteins. PSD organization may thus facilitate the individual
neurotoxic signal mechanisms downstream of receptors during glutamate
overactivity. Evidence exists showing that inhibiting signals downstream of
glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic
insult. Furthermore we have shown that uncoupling the interaction between
specific glutamate receptors from their PDZ proteins protects neurons against
glutamate-mediated excitotoxicity. These findings have significant implications
for the treatment of neurodegenerative diseases using therapeutics that
specifically target intracellular protein-protein interactions.
[Back to top] Mitochondrial Dysfunction and Glutamate
Excitotoxicity Studied in Primary Neuronal Cultures
D.G.
Nicholls
Primary
dissociated neuronal cultures have been intensively exploited for the past 15
years as model systems to investigate excitotoxic neuronal degeneration. Even
this simplified system contains a complex web of interactions between calcium
homeostasis, ATP production and the generation and detoxification of reactive
oxygen species. There is increasing realization that the mitochondrion occupies
the center stage in these processes. This review covers the normal bioenergetics
of the cultured neuron, the ways in which mitochondrial dysfunction impacts
upon the ability of the neuron to withstand excitotoxic stress, the nature of
the stresses imposed by NMDA receptor activation and possible molecular
mechanisms of excitotoxic cell death.
[Back to top] Nitric Oxide and its Role in Ischaemic Brain
Injury
Robert
G. Keynes and John Garthwaite
The role of the
neural messenger nitric oxide (NO) in cerebral ischaemia has been investigated
extensively in the past decade. NO may play either a protective or destructive
role in ischaemia and the literature is plagued with contradictory findings.
Working with NO presents many unique difficulties and here we review the
potential artifacts that may have contributed to discrepancies and cause future
problems for the unwary investigator. Recent evidence challenges the idea that
NO from neurones builds up to levels (micromolar) sufficient to directly elicit
cell death during the post-ischaemic period. Concomitantly, the case is
strengthened for a role of NO in delayed death mediated post-ischaemia by the
inducible NO synthase. Mechanistically it seems unlikely that NO is released in
high enough quantities to inhibit respiration in vivo; the formation of
reactive nitrogen species, such as peroxynitrite, represents the more likely
pathway to cell death. The protective and restorative properties of NO have
become of increasing interest. NO from endothelial cells may, via stimulating
cGMP production, protect the ischaemic brain by acutely augmenting blood flow,
and by helping to form new blood vessels in the longer term (angiogenesis).
Elevated cGMP production may also stop cells dying by inhibiting apoptosis and
help repair damage by stimulating neurogenesis. In addition NO may act as a
direct antioxidant and participate in the triggering of protective gene
expression programmes that underlie cerebral ischaemic preconditioning. Better
understanding of the molecular mechanisms by which NO is protective may
ultimately identify new potential therapeutic targets.
[Back to top] Astrocyte Influences on Ischemic Neuronal Death
Raymond
A. Swanson, Weihai Ying and Tiina M. Kauppinen
Glutamate
excitotoxicity, oxidative stress, and acidosis are primary mediators of
neuronal death during ischemia and reperfusion. Astrocytes influence these
processes in several ways. Glutamate uptake by astrocytes normally prevents
excitotoxic glutamate elevations in brain extracellular space, and this process
appears to be a critical determinant of neuronal survival in the ischemic
penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate
uptake, volume sensitive organic ion channels, and other routes may contribute
to extracellular glutamate elevations. Glutamate activation of neuronal
N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine:
both of these neuromodulators are transported by astrocytes, and D-serine
production is localized exclusively to astrocytes. Astrocytes influence
neuronal antioxidant status through release of ascorbate and uptake of its
oxidized form, dehydroascorbate, and by indirectly supporting neuronal
glutathione metabolism. In addition, glutathione in astrocytes can serve as a
sink for nitric oxide and thereby reduce neuronal oxidant stress during
ischemia. Astrocytes probably also influence neuronal survival in the
post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFa,
matrix metalloproteinases, and other factors that can contribute to delayed
neuronal death, and facilitate brain edema via aquaporin-4 channels localized
to the astrocyte endfoot-endothelial interface. On the other hand
erythropoietin, a paracrine messenger in brain, is produced by astrocytes and
upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2
(JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to
prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes
also secrete several angiogenic and neurotrophic factors that are important for
vascular and neuronal regeneration after stroke.
[Back to top] What have Genetically Engineered Mice Taught Us About Ischemic Injury?
Dong
Liang, Ted M. Dawson and Valina L. Dawson
Stroke, is the
third leading cause of death and disability in the Western world. Stroke refers
to set of ischemic conditions resulting from the occlusion or hemorrhage of
blood vessels supplying the brain. Loss of blood flow to the brain results in
neuronal injury due to both oxygen and nutrient deprivation and the activation
of injurious signal cascades. Ultimately cerebral ischemia results in death and
dysfunction of brain cells, and neurological deficits that reflect the location
and size of the compromised brain area. Injury due to ischemic stroke occurs by
a highly choreographed series of complex spatial and temporal events that
evolve over hours to days. These events involve complex interactions between
fundamental cell injury mechanisms including excitotoxicity and ionic
imbalance, oxidative and nitrosative stress, apoptotic-like cell death and
inflammatory responses. Genetically engineered mice have been valuable tools to
probe putative mechanisms of neuronal death and uncover potential strategies
that might render neurons resistant to ischemic injury. Findings from
experimental stroke studies in genetically engineered animals are discussed.