Advances in Stroke 2007
Advances in Neuronal Cell Death 2007
Maged M. Harraz, MD, PhD; Ted M. Dawson, MD, PhD; Valina L. Dawson, PhD
I
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neuronal injury has been controversial, which may be, in part,
due to nonspecific reagents and the variability of the experimental models. In this study, calpain I cleaves an N-terminal
fragment from AIF, leading to its liberation from HSP10 in
the mitochondria, which may then render AIF available for
release from the mitochondria. Calpastatin, an inhibitor of
calpain activity, was shown to provide neuroprotection, as
was knockdown of AIF during cerebral transient ischemia,
further supporting a role for calpain in the release of AIF and
subsequent neuronal injury after stroke.5 The question that
remains is whether calpain-mediated release of AIF is a
parallel pathway independent of PAR or whether PAR
participates with calpain to release AIF. There is suggestive
data, in mouse embryonic fibroblasts, treated with DNA
damaging alkylating agents, that PARP1 and AIF-dependent
cell death may involve p53, Bax and calpains, but not Bak,
caspases or PARP2.6 The study used a series of genetically
modified cells and time course analysis to define a molecular
signaling sequence of PARP1, calpain activation, Bax and
AIF translocation in cell death due to DNA damage. These
data are provocative, but the study was conducted in engineered mitotic cells. Further work in experimental neuronal
systems is required.
Another player in the field of AIF release in stroke is
cyclophilin A, a protein that is highly expressed in the
cytoplasm and nucleus of neurons. Cyclophilin A has been
implicated in various models of cell death and, in the
Rice-Vannucci model of experimental ischemic stroke performed in 9 day old mice, cyclophilin A deficiency is
neuroprotective and AIF does not translocate to the nucleus.
These data suggest that cyclophilin A may physically interact
with AIF after ischemic injury, but not under normal physiological conditions.7
n a stroke lesion, there is a core of necrotic cell death
surrounded by a zone of tissue at risk, termed the penumbra. In the penumbra, there is usually less severe tissue
damage. Programmed cell death (PCD) pathways have been
documented to be present in the penumbra. While many
studies have used the term apoptosis as equivalent to PCD,
the absence of complete biochemical and morphological
characteristics of apoptosis, coupled with the ineffectiveness
of caspase inhibitors, indicates that there are other important
cell death pathways activated during stroke. A few caspaseindependent cell death pathways have been identified and
there are likely more to be described.
Parthanatos
Stroke injury leads to the activation of a cell death program,
dependent on poly(ADP-ribose) polymerase-1 (PARP1) activation1 and culminating in apoptosis inducing factor (AIF)
mediated cell death.2 Poly(ADP-ribose) (PAR) polymer is the
death signal in this pathway and Thanatos is the Greek
personification of death and mortality, hence, the name
parthanatos. In this form of cell death that occurs in many
different organ systems during ischemia-reperfusion injury,
PAR polymer translocates from the nucleus to the cytoplasm
and mitochondria. This leads to release of AIF from the
mitochondria, an effect that is inhibited by poly(ADP-ribose)
glycohydrolase (PARG), an enzyme that degrades PAR
polymer.3 After cerebral focal ischemia reperfusion injury,
mice with reduced PARG levels had higher infarct volumes
than controls. On the other hand, mice overexpressing PARG
had significantly lower infarct volumes. Combined, the current data indicate that PAR is a powerful death signal. What
is not yet understood are the mechanisms of PAR translocation out of the nucleus and PAR-mediated AIF release from
the mitochondria. Parthanatos is caspase-independent and is
biochemically and morphologically distinct from apoptosis.4
Regulation of PAR signaling may be a useful therapeutic
intervention to reduce stroke-mediated damage.
Other Caspase-Independent Cell Death
BNIP3 belongs to a growing family of mitochondrial death
signals. It is not detectable in neurons under physiological
conditions, but BNIP3 protein expression is induced by
hypoxia. After 90 minutes of middle cerebral artery occlusion
in rats, BNIP3 is upregulated 48 hours after focal brain
ischemia reperfusion in ischemic tissue, but not in nonischemic tissue.8 Knockdown of BNIP3 expression in primary
cortical cultures delays neuronal cell death after hypoxia;
Apoptosis-Inducing Factor
Additional investigations this year have explored the role of
AIF during stroke. In an elegant piece of work, investigators
studied the actions of the calcium-dependent protease, calpain
I, on the release of AIF. The role of calpains in mediating
Received December 5, 2007; accepted December 6, 2007.
From the Institute for Cell Engineering (M.M.H., T.M.D., V.L.D.), Departments of Neurology (M.M.H., T.M.D., V.L.D.), Neuroscience (T.M.D.,
V.L.D.), and Physiology (V.L.D.), The Johns Hopkins University School of Medicine, Baltimore, Md, USA.
Correspondence to Valina L. Dawson, PhD, Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 North Broadway, Suite
731, Baltimore, MD 21205, USA. E-mail [email protected]
(Stroke. 2008;39:286-288.)
© 2008 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.107.511857
286
Harraz et al
however, a role for BNIP3 in oxygen-glucose deprivation,
more commonly used in vitro as a culture model of middle
cerebral artery occlusion, was not explored. It is possible that
BNIP3 might mediate EndoG translocation from the mitochondria. However, there is concern regarding the specificity
of the EndoG antibodies, which are not well characterized.
It is difficult to understand how a matrix protein, such as
EndoG, could have a specific release mechanism involving
BNIP3 or whether translocation occurs after rupture of
mitochondrial membranes.8
Energy
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PARP1-dependent cell death is believed to result, at least
partially, from NAD⫹ depletion. Both genotoxic stress and
nutrient deprivation activate PARP1 and result in NAD⫹
depletion. However, mitochondrial NAD⫹ levels are not
reduced following those stressors.9 Nampt, an enzyme involved in NAD⫹ biogenesis, protects cells from death by
genotoxic stress, an effect that is mediated by regulating
mitochondrial NAD⫹ levels. In addition, Nampt inhibited
AIF translocation to the nucleus following genotoxic stress.9
These studies emphasize the importance of investigating the
subcellular levels of NAD⫹ versus total levels in experimental models of stroke. It will be interesting to determine if
Nampt plays a role in parthanatos after ischemia reperfusion
injury in the brain and whether it has a neuroprotective action
in stroke.
Endoplasmic Reticulum Stress and Autophagy
Endoplasmic reticulum (ER) stress induced neuronal cell
death plays an important role in stroke pathophysiology.10
BBF2H7 is a novel ER stress transducer with interesting
potential as a candidate for neuroprotection. BBF2H7 appears
to be expressed in the peri-infarction zone after permanent
focal brain ischemia, but not in the nonischemic neurons.11 In
neuroblastoma cells, overexpression of BBF2H7 suppresses
and conversely knockdown of BBF2H7 promotes ER stressinduced cell death. Taken together, these data suggest a role
for BBF2H7 and ER stress in ischemic injury; however,
changes in protein expression do not necessarily define a
functional role in ischemic injury and may instead be bystander events triggered by severe stress. Additionally, the
cell death assays were performed in tumor cell lines and have
not yet been replicated in neurons or the brain. Thus, while
intriguing, any interpretation must be cautiously made.
The role of autophagy in neuronal cell death is an emerging
area of cell death research. Autophagy can be triggered by ER
stress and oxidative stress, poising it to be a key player in
ischemic cell death. Autophagy is a lysosomal pathway
important for the turnover of organelles and proteins that can
be induced in neurons.12 Autophagy has only recently been
recognized as an important process that may be a key
regulator of neurodegenerative processes. Recent studies
implicate a role for autophagy in the prenumbra after experimental stroke. Beclin 1 and microtubule-associated protein 1
light chain 3, which are known to promote autophagy are
induced in the penumbra of rats after 90 minutes middle
cerebral artery occlusion.13 Both genetic disruption and
chemical inhibition of autophagy inhibit necrosis induced by
Advances in Neuronal Cell Death 2007
287
40 minutes of hypoxia in C elegans.14 Taken together with the
observation of autophagic morphology following the modified Levine/Vannucci procedure in adult mice,15 these early
studies suggest that autophagic cell death may be a critically
important form of cell death in the nervous system that has,
until recently, been overlooked.
Summary
“Death Hath so Many Doors to Let Out Life.”
—John Fletcher & Francis Beaumont
While life and death are binary events, there are many
different pathways for cells to die. We are just coming to the
realization that there is more to cell death than apoptosis and
necrosis, which implies that our understanding of cell death in
the brain is severely restricted. Until we understand the
primary pathways that result in cell death after ischemia, we
will not be able to define new and effective therapies. Several
advances this year are predictive of future studies. When AIF
was first identified, it was thought that AIF mediated a very
specific and restricted form of caspase-independent cell
death. We are now finding that AIF belongs to the growing
family of mitochondrial death effectors and that many different signaling events can lead to AIF release. AIF can be
released as a commitment point to cell death, but it can also
be released later in the cell death cycle. Nuclear AIF is a
promiscuous marker for cell death; therefore, it is important
to determine whether AIF translocation is a primary causal
event in the cell death process by blocking AIF translocation.
Additional work is necessary to fully understand the role of
AIF in neuronal cell death. There is a growing number of
caspase-independent cell death effectors being identified in
the brain, but a lot of work is required in order to understand
which are primary causal events, toward which therapeutics
might be targeted, and which are secondary and perhaps less
important events. Along these lines, PAR signaling appears to
be an important primary event that would be amenable for
therapeutic development. Lastly, autophagy is likely to be a
very fruitful frontier for understanding the response of the
brain to stress and injury. Cell death due to autophagy may
turn out to be one of the more important cell death signaling
events in the brain and is a relatively untapped area of
research.
Acknowledgment
We thank K. Quinn Tyler for editorial assistance.
Sources of Funding
This work was supported by USPHS NS039148, DA000266. T.M.D.
is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases.
Disclosures
None.
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KEY WORDS: apoptosis
䡲
cell death
Advances in Neuronal Cell Death 2007
Maged M. Harraz, Ted M. Dawson and Valina L. Dawson
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Stroke. 2008;39:286-288; originally published online January 10, 2008;
doi: 10.1161/STROKEAHA.107.511857
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Copyright © 2008 American Heart Association, Inc. All rights reserved.
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