Biochimica et Biophysica Acta 1802 (2010) 92–99

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a d i s

Review

Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia

Kuniyasu Niizuma, Hideyuki Yoshioka, Hai Chen, Gab Seok Kim, Joo Eun Jung, Masataka Katsu,
Nobuya Okami, Pak H. Chan ⁎
Department of Neurosurgery, Department of Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Stanford, CA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria
Received 24 April 2009 overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide
Received in revised form 26 August 2009 dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell.
Accepted 8 September 2009
Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to
Available online 12 September 2009
mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as
Keywords:
cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia
Mitochondria remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production
Cerebral ischemia and by regulating mitochondria-dependent apoptosis pathways.
SOD1 © 2009 Elsevier B.V. All rights reserved.
Reactive oxygen species
Neuronal death
PIDD

1. Introduction as intracellular messengers to transduce signals of various pathways,

including cell death pathways [4,5], similar to the way in which
Mitochondria are the powerhouse of the cell. Their primary reactive nitrogen species transduce signals in endothelial cells or
physiological function is to generate adenosine triphosphate through neurons [6,7].
oxidative phosphorylation via the electron transport chain, which Besides triggering molecular signals by overproduction of ROS,
contains five multi-subunit enzyme complexes, I–V. Reactive oxygen mitochondria regulate apoptotic pathways by sequestering Ca2+,
species (ROS) are generated in complex I and complex III during storing and releasing pro-apoptotic proteins such as cytochrome c
mitochondrial respiration [1]. Therefore, oxygen metabolism can be a and apoptosis-inducing factor (AIF), and probably by opening the
potential threat to tissues and cells. permeability transition pore [8,9]. In this review, we discuss the roles
Numerous studies have shown the roles ROS play in the of ROS generated in mitochondria and mitochondria-dependent
pathophysiology of neurological disorders, including ischemia, trau- apoptotic pathways in several in vivo models of cerebral ischemia.
ma, and degenerative diseases. ROS cause macromolecular damage
such as lipid peroxidation, protein oxidation, and DNA oxidation, all of
which can lead to cell injury and death [2,3]. In addition, ROS can act 2. The roles of ROS generated by mitochondria

2.1. Generation and clearance of ROS under normal physiological

conditions
Abbreviations: pFCI, permanent focal cerebral ischemia; tFCI, transient focal
cerebral ischemia; tGCI, transient global cerebral ischemia; APE, apurinic/apyrimidinic
endonuclease; ATF-4, activating transcription factor-4; CHOP, C/EBP homologous
Because mitochondria generate superoxide anions (O− 2 ) and

protein; DNP, 2,4-dinitrophenylhydrazone; GRP78, glucose-regulated protein 78; ILK, hydrogen peroxide (H2O2) during mitochondrial respiration under
integrin-linked kinase; MCP-1, monocyte chemoattractant protein 1; MIP-1α, macro- normal physiological conditions [1], oxygen metabolism poses a
phage inflammatory protein-1α; MMP, matrix metalloproteinase; NF-κB, nuclear potential threat to cells. It is, nevertheless, essential for cell survival.
factor-κB; pAkt, phosphorylated Akt; PERK, phosphorylation of RNA-dependent protein
Pro-oxidant enzymes, such as nitric oxide synthases (NOS), cycloox-
kinase-like endoplasmic reticulum eukaryotic initiation factor 2α kinase; PARP, poly
(ADP-ribose) polymerase; pGSK-3β, phosphorylated glycogen synthase kinase-3β; ygenases, xanthine dehydrogenase, xanthine oxidase, NADPH oxi-
pPRAS, phosphorylated proline-rich Akt substrate; PUMA, p53-upregulated modulator dase, myeloperoxidase, and monoamine oxidase, generate the ROS
of apoptosis; Ref-1, redox factor-1; Smac, second mitochondria-derived activator of O−
2 , H2O2, nitric oxide, and lipid peroxides.
caspases; XIAP, X chromosome-linked inhibitor of apoptosis protein To detoxify such ROS, cells develop ROS clearance systems.
⁎ Corresponding author. Neurosurgical Laboratories, Stanford University, 1201
Welch Road, MSLS #P314, Stanford, CA 94305-5487, USA. Tel.: +1 650 498 4457; fax:
Superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and
+1 650 498 4550. catalase contribute to scavenging these ROS. SOD has three isoforms:
E-mail address: [email protected] (P.H. Chan). copper/zinc SOD (SOD1), manganese SOD (SOD2), and extracellular

0925-4439/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbadis.2009.09.002
 K. Niizuma et al. / Biochimica et Biophysica Acta 1802 (2010) 92–99 93

SOD (SOD3) (Table 1). All three SOD isoforms dismutate O− 2 to H2O2 chromatography/fluorescence assay [15], in addition to a fluorescent
and molecular oxygen. Then, GSHPx scavenges H2O2 to water at the microscope study, may be required.
expense of glutathione. Catalase also dismutates H2O2 to water [2].
Other small molecular non-enzymatic antioxidants such as vitamin E 2.4. Transgenic and knockout studies of SOD
and vitamin C are also involved in the detoxification of free radicals
[10]. Although development of methodologies to detect and quantify
Oxidative stress is defined as the pathogenic outcome of ROS ROS have enabled researchers to investigate their roles after cerebral
overproduction beyond the capacity of ROS clearance in cells. After ischemia, their causative roles in ischemic brain injury remain
cerebral ischemia, the balance between ROS production and clearance unclear. Advances in transgene and gene knockout (KO) technology
shifts to the production side, resulting in induction of oxidative stress- have allowed us to investigate the contributions of ROS to molecular
induced signaling and cell injury. mechanisms of ischemic brain injury. Table 2 shows studies using
cerebral ischemia models with transgenic (Tg) animals that carry
2.2. Reperfusion injury and ROS human SOD genes or KO animals that are homozygously or
heterozygously deficient in SOD genes.
Reperfusion injury is brain damage caused by the return of blood SOD1 is neuroprotective. In heterozygous SOD1 Tg animals that
flow, resulting in progression of vasogenic edema, hemorrhagic carry the human SOD1 gene, SOD1 activity increased (a three-fold
transformation, and an increase in stroke volume. ROS involvement increase in SOD1 Tg mice [21] and an approximate four-fold increase
in reperfusion injury has been described since the early 1980s [11,12]. in SOD1 Tg rats [22]) compared with wild-type (Wt) animals. In SOD1
Numerous subsequent reports have presented the relationship Tg animals, a 35–50% decrease in infarct volume is usually observed
between reperfusion injury and ROS. In ischemic brain tissue, ROS after focal cerebral ischemia (FCI) [23,24]. After transient global
generation is accelerated by cytosolic pro-oxidant enzymes and by cerebral ischemia (tGCI), delayed neuronal cell death decreases to
mitochondria, inactivation of detoxification systems, consumption of about 50% in SOD1 Tg animals [25,26]. Regulation of various pathways
antioxidants, and failure to adequately replenish antioxidants [2]. contributes to neuroprotection, including activation of the phosphoi-
These overproduced ROS cause macromolecular damage and activa- nositide 3-kinase (PI3-K) pathway [27,28], and inhibition of the
tion of various pathways. mitogen-activated protein kinase (MAPK) related pathway [29,30],
and the p53 signaling [18,31], nuclear factor-κB [19,20,32], and
2.3. Detection and quantification of ROS mitochondria-dependent apoptotic [22,29,33] pathways. Moreover,
infarct volume and edema levels decrease after FCI in homozygous
To detect and quantify various ROS in the ischemic brain, an SOD1 KO mice [34–36], while cell death increases after tGCI [37].
indirect measurement method is required because of the short half- SOD2 also has important neuroprotective roles. Heterozygous
life of most ROS. One approach is to detect oxidative modification of SOD2 Tg mice carrying the human SOD2 gene showed decreased
biological targets of ROS such as lipid peroxidation, protein oxidation, injury [38] and reduced vascular endothelial cell death [39] after FCI.
or DNA oxidation. Another approach is to use reporter molecules, Moreover, infarct volume [40], brain edema [39], O− 2 production [17],
which are oxidized by ROS, resulting in the production of chromo- matrix metalloproteinase-9 activity [39], caspase-9 activation [41],
genic, fluorescent, or luminescent molecules. Hydroethidine (HEt), and cytochrome c release [42] increase after FCI in SOD2 KO mice
one such reporter molecule, has been used to detect O− 2 in cells and compared with Wt mice. Furthermore, hemorrhagic transformation
tissues [13,14]. “Ethidium fluorescence,” which is the red fluorescence after transient FCI (tFCI) significantly increases in SOD2 KO mice [39].
arising from oxidation of HEt, has been attributed to O− 2 trapping in Although only a few studies have used SOD3 Tg or KO mice in
cells [13,14]. However, a recent study revealed that ethidium could be cerebral ischemia models, they have shown that SOD3 has neuropro-
generated by other ROS [15]. To specifically detect O− 2 , 2-hydro- tective roles. Infarct volume after FCI decreases (−28%) in SOD3 Tg
xyethidium (2-HE), the two-electron oxidation product of HEt [16], is mice [43] that express a five-fold higher level of SOD3 in the brain
a more suitable diagnostic marker than HEt [15]. compared with Wt mice [44]. Neuronal death after tGCI also decreases
Although the fluorescence spectra from 2-HE and ethidium (−48%) in SOD3 Tg mice [45]. In contrast, infarct after FCI in
overlap and fluorescence from 2-HE cannot be separated under a homozygous SOD3 KO mice increases (+81%) [46]. In summary,
fluorescent microscope, red fluorescence caused by HEt oxidation is studies using various SOD Tg and KO animals imply that ROS have
still a powerful tool for detecting ROS, mainly O−2 . Upregulation of this important roles in activating various pathways and determining the
red fluorescence suggests that O− 2 affects signaling and injury after outcome after cerebral ischemia.
cerebral ischemia [17–20]. A disadvantage of HEt is that reliable
quantification cannot be provided with a fluorescent microscope. For 2.5. Mitochondrial NOS
specific and quantitative detection of O− 2 , a high-performance liquid
Three canonical isoforms of NOS are well known in mammals:
Table 1 neuronal NOS (nNOS), inducible NOS, and endothelial NOS. Recent
Mammalian superoxide dismutases. findings reveal that mitochondria contain their own isoform of NOS,
mitochondrial NOS (mtNOS), at their inner membrane [47,48]. Since
SOD1 (CuZnSOD) SOD2 (MnSOD) SOD3 (ECSOD)
NOS isoforms are encoded not by mitochondrial DNA, but by nuclear
Location Cytosol Mitochondria Extracellular space DNA, mtNOS is thought to be synthesized in the cytosol and
Molecular weight 32,000 88,000 120,000
translocated to mitochondria [49], although the mechanism of this
Structure Dimer Tetramer Tetramer
Metals, Cu 1, Zn 1 Mn 1 Cu 1, Zn 1 translocation remains unknown. mtNOS stays active because of
g-atoms/subunit mitochondrial Ca2+ content, in contrast to other nitric oxide sources.
Phenotype of transgenic Normal Normal Normal mtNOS continuously controls mitochondrial respiration [47,48] and is
mouse (+/+)
considered a key molecule of reperfusion injury [50].
Phenotype of knockout Normal Neonatal Normal
mutant (−/−) lethality The enzymatic activity of mtNOS was higher in hypoxic animals
Chromosome 21 (human) 6 (human) 4 (human) than in normoxic controls [51]. mtNOS is also considered a marker of
16 (mouse) 17 (mouse) 5 (mouse) brain aging. In aged mice, mtNOS activity was linearly correlated
11 (rat) 1 (rat) 14 (rat) with neurological performance and survival [52]. Since mtNOS
CuZn, copper, zinc; Mn, manganese; EC, extracellular. controls mitochondrial respiration and nitric oxide generation, it
94 K. Niizuma et al. / Biochimica et Biophysica Acta 1802 (2010) 92–99

Table 2
Transgenic and knockout studies of superoxide dismutases using in vivo cerebral ischemia models.

Study Animal Model Main findings References

SOD1 +/− Mouse pFCI Decreased cortical infarct (−35%) [23]

SOD1 +/− Mouse pFCI No protection [84]
SOD1 +/− Mouse tFCI Decreased infarct [85]
SOD1 +/− Mouse tFCI Sustained hsp70 mRNA expression [86]
SOD1 +/− Mouse tFCI Sustained c-fos mRNA expression [87]
SOD1 +/− Mouse tGCI Induction of hsp 70 [88]
SOD1 +/− Mouse tFCI Decreased injury (−50%) [24]
SOD1 +/− Rat tGCI Decreased injury (−50%) [25]
SOD1 +/− Mouse tGCI Decreased injury (−50%) [26]
SOD1 +/− Mouse tFCI Decreased DNA fragmentation [89]
SOD1 +/− Mouse tFCI Decreased cytochrome c release [90]
SOD1 +/− Mouse tFCI Decreased NF-κB expression [32]
SOD1 +/− Mouse tFCI Decreased activation of activator protein-1 [91]
SOD1 +/− Mouse pFCI No difference in infarct volume [92]
SOD1 +/− Rat tGCI Decreased active caspase-3, -9 [22]
SOD1 +/− Mouse tFCI Decreased ERK activation [29]
SOD1 +/− Mouse tFCI Decreased Bad activation [33]
SOD1 +/− Mouse tFCI Increased pAkt expression; decreased DNA fragmentation [27]
SOD1 +/− Mouse tFCI Decreased PARP activation [93]
SOD1 +/− Mouse tFCI Decreased lesion size and edema; decreased MMP-2, -9 expression [36]
SOD1 +/− Rat tGCI Decreased injury, PERK phosphorylation and GRP78 release [94]
SOD1 +/− Mouse tFCI Decreased injury, PERK phosphorylation and GRP78 release [95]
SOD1 +/− Mouse tFCI Decreased binding of XIAP/DNP, Smac/DNP and caspase-9/DNP [96]
SOD1 +/− Mouse tFCI Decreased Omi/HtrA2 activation [96]
SOD1 +/− Mouse tFCI Increased ILK expression and ILK/Akt complex [97]
SOD1 +/− Rat tGCI Inhibited ATF-4 induction and CHOP expression; decreased endoplasmic reticulum damage [98]
SOD1 +/− Mouse tFCI Inhibited ATF-4 induction and CHOP expression [98]
SOD1 +/− Mouse tFCI Increased proteasome activity and MDM2 activation; decreased nuclear p53 [31]
SOD1 +/− Rat tGCI Inhibited APE/Ref-1 decrease; decreased injury [99]
SOD1 +/− Mouse tFCI Decreased MCP-1 and MIP-1α expression [100]
SOD1 +/− Mouse tFCI Decreased level of O−2 ; decreased NF-κB activation and phosphorylation [20]
SOD1 +/− Mouse tFCI Increased pPRAS, pPRAS/pAkt binding and pPRAS/14-3-3 protein binding [101]
SOD1 +/− Rat tGCI Increased pAkt and pGSK-3β expression [28]
SOD1 +/− Rat tGCI Decreased p53 translocation to mitochondria [58]
SOD1 +/− Mouse tFCI Decreased level of O−2 ; inhibited persistent upregulation of NF-κB [19]
SOD1 +/− Rat tFCI with hyperglycemia Decreased MMP activity and Evans blue leakage [102]
SOD1 +/− Rat tFCI Decreased activity of p38, phospho-p38, Evans blue leakage, edema and infarct [30]
SOD1 +/− Rat tGCI Decreased PUMA activation and injury; decreased level of O− 2 [18]
SOD1 −/− Mouse tFCI Increased infarct (+ 40%) [34]
SOD1 −/− Mouse tFCI Increased lesion size and edema [35]
SOD1 −/− Mouse tGCI Increased cell death [37]
SOD1 −/− Mouse pFCI No difference in infarct volume [92]
SOD1 −/− Mouse tFCI Increased edema [36]
SOD2 +/− Mouse tFCI Decreased injury (−50%) [38]
SOD2 +/− Mouse tFCI Decreased vascular endothelial cell death [39]
SOD2 −/+ Mouse pFCI Increased infarct (+ 66%) [40]
SOD2 −/+ Mouse pFCI Increased active caspase-9 [41]
SOD2 −/+ Mouse tFCI Increased cytochrome c release [42]
SOD2 −/+ Mouse pFCI Increased O−2 production [17]
SOD2 −/+ Mouse tFCI Increased MMP-9 expression [103]
SOD2 −/+ Mouse tFCI Increased MMP activity, edema, inflammation and hemorrhagic transformation [39]
SOD3 +/− Mouse tFCI Decreased infarct (−28%) [43]
SOD3 +/− Mouse tGCI Decreased injury (−48%) [45]
SOD3 −/− Mouse tFCI Increased infarct (+ 81%) [46]

+/−, heterozygous transgenic animals carrying human SOD genes; −/+, heterozygous knockout mutant of SOD genes; −/−, homozygous knockout mutant of SOD genes.

may correlate with apoptosis after stroke. Further studies may reveal stored in the mitochondrial intermembrane space, followed by
the roles of mtNOS after stroke and may provide novel therapeutic neuronal apoptosis. This pathway is called the ‘intrinsic pathway.’
strategies.
3.2. Bcl-2 family protein interactions

3. Ischemic neuronal apoptotic pathways (Fig. 1) The Bcl-2 protein family, which is a principal regulator of
mitochondrial membrane integrity and function, is classified into
3.1. The intrinsic pathway three subgroups according to structural homology: the anti-apoptotic
proteins such as Bcl-2, Bcl-XL, and Bcl-w; the pro-apoptotic proteins
After mitochondria trigger various signaling pathways by over- such as Bax and Bak; and the BH3-only proteins including Bad, Bid,
production of ROS, some, but not all, apoptotic signals return to Bim, Noxa, and p53-upregulated modulator of apoptosis (PUMA).
mitochondria with the help of BH3-only proteins. Then, Bcl-2 family Since neurons lack full-length Bak, Bax is the only pro-apoptotic
proteins (such as cytochrome c, AIF, endonuclease G [Endo G], and protein in neurons. In response to apoptotic stimuli, specific BH3-only
second mitochondria-derived activator of caspase [Smac]) interact proteins are activated and transduce apoptotic signals to mitochon-
with each other, resulting in the release of pro-apoptotic proteins dria. Studies have shown that after cerebral ischemia, BH3-only
 K. Niizuma et al. / Biochimica et Biophysica Acta 1802 (2010) 92–99 95

Fig. 1. Mitochondria-dependent pathways of apoptosis in cerebral ischemia and reperfusion. After cerebral ischemia, various pathways, such as the death receptor pathway, p53
pathway, c-Jun N-terminal kinase (JNK) pathway, PI3-K pathway, and the MAPK pathway, are activated. Most signaling pathways induce apoptosis with the help of pro-apoptotic
proteins, such as cytochrome c, Endo G, AIF, and Smac, which are stored in mitochondria.

proteins were upregulated, meaning cerebral ischemia activates support the hierarchy model. After interacting with other Bcl-2 family
various apoptotic pathways. proteins, Bax is oligomerized and activated, which triggers release of
Currently, two main ideas can explain Bcl-2 protein family apoptotic proteins stored in the mitochondrial intermembrane space,
interaction: the ‘direct model’ and the ‘hierarchy model’ (Fig. 2). In leading to neuronal apoptosis [8,56].
the direct model, anti-apoptotic proteins trap pro-apoptotic proteins.
BH3-only proteins disrupt this interaction, resulting in liberation of
pro-apoptotic proteins and subsequent apoptosis (Fig. 2A). 3.3. Bcl-2 family downstream interactions
Recently, Kim et al. [53] advocated the ‘hierarchy model.’ In this
model, BH3-only proteins are subdivided into two groups: ‘activator’ Proteins in the mitochondrial intermembrane space, including
and ‘inactivator.’ Bim, PUMA, and truncated Bid (tBid) belong to the cytochrome c [64,65], Smac [66], AIF [67], and Endo G [68], are
activator group and other BH3-only proteins belong to the inactivator released after cerebral ischemia, at which time they cause transduc-
group. Activator BH3-only proteins are trapped by anti-apoptotic tion of apoptotic signals. Release of these proteins leads to ‘the point
proteins, whereas pro-apoptotic proteins are not. Inactivator BH3- of no return.’ Cytochrome c interacts with apoptosis activating factor-
only proteins disrupt this interaction, resulting in liberation of 1, deoxyadenosine triphosphate, and procaspase-9, and forms the
activator BH3-only proteins. Liberated activator BH3-only proteins apoptosome, which activates procaspase-9 [69–71]. Caspase-9 acti-
interact with pro-apoptotic proteins, followed by apoptosis (Fig. 2B). vates procaspase-3, then caspase-3 cleaves inhibitor of caspase-
The Bcl-2 family plays various roles in cerebral ischemia. BH3-only activated DNase, which is an inhibitor and a chaperone of caspase-
proteins, including Bad [33,54,55], Bim [56,57], Noxa [57,58], PUMA activated DNase. Liberated caspase-activated DNase damages DNA
[18,59], and tBid [18,60], contribute to cell death after cerebral and induces apoptosis. Caspase-3 can also activate other effector
ischemia, mainly through interactions with other Bcl-2 family caspases, which activate crucial substrates, including poly(ADP-
members. Bax increases after tGCI [61] or FCI [62], and translocates ribose) polymerase (PARP), after cerebral ischemia [72,73]. Although
from the cytosol to mitochondria, mediated by c-Jun N-terminal PARP is involved in both apoptotic and non-apoptotic cell death, 89-
kinase with BimL [56]. Bim [56], tBid [63], and PUMA [18] have been and 21-kDa fragments are cleaved by caspases and are related to
reported to interact with Bax after cerebral ischemia, which may apoptosis after cerebral ischemia [73,74].
96 K. Niizuma et al. / Biochimica et Biophysica Acta 1802 (2010) 92–99

3.4.2. p53 signaling pathway

Since a number of Bcl-2 family proteins such as Bax, Bid, Noxa,
PUMA, and p53AIP1 are the products of p53, p53 plays important roles
in the intrinsic pathway. These Bcl-2 family proteins increase and
regulate cell death after cerebral ischemia, as described in section 3.2.
Recent findings suggest that p53 can activate the intrinsic pathway in
a transcription-independent manner, as well as in a transcription-
dependent manner [58]. p53 translocates to mitochondria and
interacts with anti-apoptotic Bcl-XL, which precedes cytochrome c
release after tGCI [58]. A p53 inhibitor, pifithrin-α, decreased the
translocation of p53, and resulted in neuroprotection in the
hippocampal CA1 subregion after tGCI [58]. In summary, p53 acts as
a BH3-only protein in this transcription-independent manner in
addition to transcription of apoptosis-related proteins such as Bcl-2
family proteins.

3.4.3. PIDD signaling pathway

p53 and caspase-2 are involved with stress-induced apoptosis.
However, the key molecules connecting them have not been
determined. Tinel and Tschopp [77] reported that p53-induced
protein with a death domain (PIDD), which is a target of p53, formed
a high-molecular weight protein complex with RAIDD and procas-
pase-2. This molecular complex is referred to the ‘PIDDosome,’ in
which caspase-2 is activated, similar to caspase-9 activation in the
apoptosome [77]. After tGCI, the PIDDosome increased in the
Fig. 2. Two models of Bcl-2 protein family interaction. (A) The direct model for Bax hippocampal CA1 subregion, followed by caspase-2 activation and
activation. After apoptotic stimuli, specific BH3-only proteins are activated and inhibit
Bid cleavage, which preceded neuronal death [78].
anti-apoptotic Bcl-2 family proteins. Liberated Bax oligomerizes and triggers the release
of pro-apoptotic proteins stored in the mitochondrial intermembrane space. (B) The Recently, in vitro studies have presented new findings regarding
hierarchy model for Bax activation. After apoptotic stimuli, specific inactivator BH3- this PIDD pathway. One finding is that caspase-2 can directly interact
only proteins are activated and inhibit anti-apoptotic Bcl-2 family proteins. Then, with mitochondria and activate the mitochondria-dependent apo-
liberated activator BH3-only proteins interact with Bax, resulting in the release of pro- ptotic pathway [79,80]. Interestingly, this interaction occurs inde-
apoptotic proteins stored in the mitochondrial intermembrane space.
pendently of its proteolytic activity. Another finding is that a cleaved
fragment of PIDD (PIDD-C) forms protein complexes that differ from
Smac also contributes to activation of caspases. Smac released the PIDDosome. The protein complex containing PIDD-C and nuclear
from mitochondria binds to and neutralizes the effect of the X factor-κB has an anti-apoptotic role in response to genotoxic stress
chromosome-linked inhibitor-of-apoptosis protein, which prevents [81]. These interactions after cerebral ischemia are unknown and
procaspase activation and inhibits activities of activated caspases require further study.
[66,74] after cerebral ischemia.
Recent reports show the importance of the caspase-independent 3.4.4. Crosstalk between the intrinsic pathway and the extrinsic pathway
pathways. AIF translocates from mitochondria to the nucleus and The extrinsic pathway is the death receptor-mediated pathway
induces apoptosis after tFCI [67]. In mutant mice that express low- that receives extracellular signals and transduces them to intracellular
level AIF, infarct volume decreased (−43%) after tFCI [67]. PARP signals. Recent studies have shown that the death receptor pathway
helped nuclear translocation of AIF [75]. Endo G is also known to has various physiological functions as well as apoptotic roles.
translocate to the nucleus, causing DNA fragmentation after tFCI [68]. The Fas pathway (Fas is a death receptor) is involved in apoptosis
after cerebral ischemia. mRNA and protein levels of both Fas and the
3.4. Upstream of the intrinsic pathway Fas ligand are upregulated after cerebral ischemia [82,83]. Mutant
mice that have a loss-of-function mutation for Fas show reduced
ROS activate a number of pathways, including PI3-K, MAPK, and infarct volume after FCI [82]. Fas, Fas-associated death domain, and
p53 pathways. These pathways modulate the intrinsic pathway. procaspase-8 form a protein complex that is referred to as the death-
inducing signaling complex (DISC). DISC activates procaspase-8,
3.4.1. Kinase pathway similar to procaspase-9 activation by the apoptosome. Caspase-
Akt is a key molecule for neuronal death and survival after cerebral 8 activation is followed by activation of caspase-3 and caspase-10
ischemia [27]. Akt is a serine/threonine kinase and a major after cerebral ischemia [83].
downstream target of PI3-K. Akt phosphorylates and inactivates Bad There is crosstalk between the intrinsic pathway and the extrinsic
after cerebral ischemia [55]. Since phosphorylated Bad is unable to pathway. The key molecule involved in this crosstalk is Bid, which is
inhibit the pro-survival Bcl-2 family proteins, Bad phosphorylation also a key molecule for the p53–caspase-2 pathway as described
results in inactivation of the apoptotic pathway. Akt also phosphor- above. Bid is truncated by caspase-8, translocates to mitochondria,
ylates procaspase-9 and caspase-9 on serine-196, Procaspase-9 and interacts with other Bcl-2 family proteins, which causes
phosphorylation inhibits activation of procaspase-9, and caspase-9 cytochrome c release followed by apoptotic cell death [60].
phosphorylation inhibits protease activity [76]. Akt modulates p53
degradation through MDM2 phosphorylation [31]. 4. Conclusions
Other kinases also have regulative roles in the intrinsic pathway.
Phosphorylated extracellular signal-regulated kinase, which also Numerous reports show the involvement of ROS in cell death after
phosphorylates and inactivates Bad, is upregulated after tFCI [29]. cerebral ischemia. ROS contribute not only to injury of macromole-
Protein kinase A phosphorylates and inactivates Bad after cerebral cules, but also to transduction of apoptotic signals. Although it is well
ischemia [33]. known that various factors, including necrosis, are involved in the
 K. Niizuma et al. / Biochimica et Biophysica Acta 1802 (2010) 92–99 97

mechanisms of cell death after cerebral ischemia, mitochondria [21] P.H. Chan, C.J. Epstein, H. Kinouchi, H. Kamii, S. Imaizumi, G. Yang, S.F. Chen,
J. Gafni, E. Carlson, SOD-1 transgenic mice as a model for studies of
contribute to cell death by activating signaling pathways through neuroprotection in stroke and brain trauma, Ann. N. Y. Acad. Sci. 738 (1994)
ROS production and by regulating intrinsic apoptosis pathways. 93–103.
Future studies of these cell death mechanisms after ischemia may [22] T. Sugawara, N. Noshita, A. Lewén, Y. Gasche, M. Ferrand-Drake, M. Fujimura, Y.
Morita-Fujimura, P.H. Chan, Overexpression of copper/zinc superoxide dis-
provide unique information regarding molecular targets for thera- mutase in transgenic rats protects vulnerable neurons against ischemic damage
peutic strategies in clinical stroke. by blocking the mitochondrial pathway of caspase activation, J. Neurosci. 22
(2002) 209–217.
[23] H. Kinouchi, C.J. Epstein, T. Mizui, E. Carlson, S.F. Chen, P.H. Chan, Attenuation of
Acknowledgments focal cerebral ischemic injury in transgenic mice overexpressing CuZn
superoxide dismutase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 11158–11162.
[24] H. Kamii, S. Mikawa, K. Murakami, H. Kinouchi, T. Yoshimoto, L. Reola, E. Carlson,
This work was supported by National Institutes of Health grants
C.J. Epstein, P.H. Chan, Effects of nitric oxide synthase inhibition on brain
P50 NS014543, RO1 NS025372, RO1 NS036147, and RO1 NS038653. infarction in SOD-1-transgenic mice following transient focal cerebral ischemia,
The content is solely the responsibility of the authors and does not J. Cereb. Blood Flow Metab. 16 (1996) 1153–1157, doi:10.1097/00004647-
199611000-00009.
necessarily represent the official views of the NIH. We thank Liza
[25] P.H. Chan, M. Kawase, K. Murakami, S.F. Chen, Y. Li, B. Calagui, L. Reola, E. Carlson,
Reola and Bernard Calagui for technical assistance and Cheryl C.J. Epstein, Overexpression of SOD1 in transgenic rats protects vulnerable
Christensen for editorial assistance. neurons against ischemic damage after global cerebral ischemia and reperfusion,
J. Neurosci. 18 (1998) 8292–8299.
[26] K. Murakami, T. Kondo, C.J. Epstein, P.H. Chan, Overexpression of CuZn-
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