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Fig. 6.1 Delayed neuronal cell death in the ischemic penumbra and correlation with nuclear AIF following transient focal cerebral ischemia in mice, (a), Following 60 min of middle cerebral artery occlusion (MCAo) the majority of neurons (~70%) in the ischemic penumbra, i.e. the cerebral cortex, stay alive for at least 4 h. Despite sufficient blood flow 24 h after MCAo, over 90% of neurons that were viable 2 h after ischemia display altered membrane and nuclear morphology indicating cell death. (b), Correlation of neurons displaying pathological morphology with cells showing nuclear AIF (Culmsee et al. 2005)
b peptide inhibitors. Post-ischemic neuronal cell death was prevented and neuronal function was improved when caspase activation was inhibited up to 6 h following reperfusion from 30 min MCAo (Endres et al. 1998). The ultimate mechanistic link between caspase-3 activation and post-ischemic DNA fragmentation was established by Cao and co-workers by showing that caspase-activated Dnase (CAD), a molecule known to be cleaved and thereby activated by caspase-3, was responsible for post-ischemic DNA-fragmentation (Cao et al. 2001).
In consequence, many research groups concentrated on the upstream mechanisms of caspase-3 activation. Due to very low expression and activation levels of potentially involved molecules it turned out to be technically very challenging to identify respective mechanisms. Caspase-8, a molecule able to cleave caspase-3 in nonneuronal cells, was found to be activated following experimental stroke. However, caspase-8 was described to be activated in a population of neurons (lamina V) distinct from that where active caspase-3 was observed (lamina II/III) (Velier et al. 1999) and a direct link between caspase-8 and caspase-3 activation could never be demonstrated in models of cerebral ischemia. Further, upstream factors in the cascade of caspase activation such as Fas/CD95 receptors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were found to be upregulated following MCAo, and lpr mice, which express dysfunctional Fas receptors, were protected from focal ischemic brain damage (Martin-Villalba et al. 1999). Despite these interesting findings, it still remained unclear how caspase-3 was activated following cerebral ischemia until 2001, when it was demonstrated that the BH3-only Bcl-2 family member Bid, which has a caspase-8 specific cleavage site, was truncated after experimental stroke (Plesnila et al. 2001). Cleaved/truncated Bid (tBid) translocates from the cytoplasm to the outer mitochondrial membrane where together with Bax it induces the formation of an oligomeric membrane pore (Zha et al. 2000) thereby releasing cytochrome c from mitochondria (Wei et al. 2000). After focal cerebral ischemia, mitochondria of Bid-deficient mice released far less cytochrome c and cortical infarction was significantly reduced compared to wildtype littermates, thereby demonstrating the prominent role of mitochondria in post-ischemic cell death. These data further imply that after focal cerebral ischemia caspase-3 may be activated through the mitochondrial pathway, i.e. by the mitochondrial release of cytochrome c (Fujimura et al. 2000) and apoptosome formation (Plesnila et al. 2001; Yin et al. 2002; Plesnila 2004). Not much later, however, this view was challenged by the fact that caspase-3 knock out mice, which became available at that time, showed much less neuroprotection than expected based on the anticipated prominent role of caspase-3 activation for ischemic neuronal cell death (Le et al. 2002). Together with the pronounced neuroprotective effect achieved by interactions with mitochondrial cell death signaling, (Martinou et al. 1994; Wiessner et al. 1999; Plesnila et al. 2001; Cao et al. 2002; Kilic et al. 2002), i.e. mechanisms upstream of caspase-3 activation, it became clear that alternative cell death pathways distinct from caspase-3 may be present downstream of mitochondria.
The hypothesis that caspase-independent neuronal cell death signaling exists downstream of mitochondria was also suggested by in vitro experiments showing that caspase inhibition provided only transient neuroprotection, which was followed by a more delayed type of DNA-fragmentation-related cell death [see (Rideout and Stefanis 2001) for review]. It was Ruth Slack and her colleagues who identified a mitochondrial protein, apoptosis-inducing factor (AIF), to be one of the most potent molecular candidates for caspase-independent death in neurons (Cregan et al.
2002). AIF translocation from mitochondria to the nucleus was detected in damaged neurons in vitro in models of neuronal cell death relevant to the pathology of ischemic brain damage, such as glutamate toxicity, DNA damage or oxygen-glucose deprivation, whereas neutralizing AIF antibodies, pharmacological inhibition of AIF release or AIF siRNA prevented neuronal cell death in these in vitro approaches (Cao et al. 2002; Cregan et al. 2002; Culmsee et al. 2005; Becattini et al. 2006).
AIF is a 67 kDa flavoprotein with significant homology to bacterial and plant oxidoreductases located in the mitochondrial intermembranous space (Susin et al. 1999). Upon release from mitochondria, AIF migrates to the nucleus where it induces large-scale (~50 kbp) DNA fragmentation and cell death by a yet not completely understood, but certainly caspase-independent mechanism (Daugas et al. 2000).
In the brain, AIF was shown to be expressed in all so far investigated cell types, i.e. neurons and glial cells (Cao et al. 2003; Zhu et al. 2003). The expression in normal neuronal cells was confined to the mitochondria as shown by co-immunostaining with the mitochondrial marker cytochrome oxidase (Plesnila et al. 2004). Interestingly, in contrast to the expression pattern of many other apoptotic proteins, the expression of AIF protein increases gradually with brain maturation and peaks in adulthood, indicating that in contrast to, e.g. caspase-3, AIF may exert its main function in adult neurons (Cao et al. 2003).
The first pathological condition where AIF was shown to play an important role for neuronal damage was cerebral hypoxia-ischemia, a model for stroke in newborn children. Hypoxia-ischemia in 7-day-old rats induced by ligation of the left carotid artery for 55 min, together with the reduction of ambient oxygen to 7.7% in a hypoxia chamber, resulted in AIF release from mitochondria and translocation to the nucleus in neurons displaying DNA fragmentation and pyknosis (Zhu et al.
2003). Since AIF translocation was not influenced by inhibition of caspases by the pan-caspase inhibitor BAF, these experiments stressed the caspase-independent manner of AIF-induced cell death. Similar findings were also observed following cardiac arrest induced brain damage in rats, i.e. following transient global ischemia. Following 15 min of four-vessel occlusion (4-VO), AIF was found to translocate from mitochondria to the nucleus in hippocampal CA1 neurons. The temporal profile of AIF translocation coincided with the induction of large-scale DNA fragmentation (50 kbp; 24-72 h after 4-VO), a well-characterized hallmark of delayed neuronal cell death (Cao et al. 2003). In line with findings in the rodent models of transient hypoxia-ischemia in immature animals, treatment with an caspase-3 inhibitor had no effect on nuclear AIF accumulation and did not provide any long-lasting neuroprotective effects after global ischemia in adult rats (Cao et al. 2003).
At about the same time, we demonstrated the translocation of AIF from mitochondria to the nucleus following transient focal cerebral ischemia, an experimental model of ischemic stroke followed by reperfusion (Plesnila et al. 2004). Nuclear AIF was detected in single neuronal cells very early, i.e. within one hour after 45 min of middle cerebral artery occlusion (MCAo) and peaked 24 h thereafter. The time course of AIF translocation paralleled mitochondrial cytochrome c release and apoptosis-like DNA damage as identified by hair-pin probe (HPP) staining, indicating ischemia-induced mitochondrial permeabilization and AIF-induced DNA fragmentation (Plesnila et al. 2004). Further, we showed that in the same experimental paradigm of ischemic stroke that AIF nuclear translocation was mainly found in neurons (Culmsee et al. 2005) and that the number of cells displaying pathological morphology following cerebral ischemia correlated very well (r2 = 0.99) with the number of neurons showing nuclear AIF (Fig. 6.1b).
That nuclear translocation of AIF was indeed responsible for post-ischemic cell death and not only a byproduct of the morphological changes associated with neuronal cell death was first shown in 2005. Small interfering RNA (siRNA)-mediated downregulation of AIF expression (~80%) in HT22 hippocampal neurons and in primary cultured neurons resulted in a significant reduction of glutamate and oxygen-glucose deprivation-induced neuronal cell death, respectively (Fig. 6.2). Reduction of cell death was associated with a lack of nuclear AIF translocation, thereby demonstrating that AIF plays a causal role for excitotoxic and hypoxic-hypoglycemic cell death in vitro (Culmsee et al. 2005). In the same study, we demonstrated that AIF is also relevant for post-ischemic cell death in vivo. Harlequin mutant mice carry a pro-viral insertion in the AIF-gene, thereby expressing only 10-20% of normal AIF protein levels (Klein et al. 2002). These mutant mice show significantly reduced post-ischemic brain damage as compared to their wild-type littermates, which express AIF at normal levels (Culmsee et al. 2005) (Fig. 6.3).
In vivo nuclear AIF translocation was dependent on poly(ADP-ribose) polymerase-1 (PARP-1) activation, as shown by using the specific PARP-1 inhibitor PJ-34 (Culmsee et al. 2005). Accordingly, these results suggest that PARP-1 activation is located upstream of AIF release from mitochondria and that AIF is the major factor mediating PARPl-induced cell death, findings also supported by other laboratories using different strategies to inhibit PARP, i.e. by cilostazol or gallotannin (Wei et al. 2007; Lee et al. 2007). Further, activation of neuronal nitric oxide synthase (nNOS) and formation of free radicals were linked to PARP activation and AIF-mediated neuronal cell death following experimental stroke. Gene deletion of nNOS or application of a metalloporphyrin-based superoxide dismutase mimic reduced post-ischemic cell death together with a reduction of the number of neurons displaying nuclear AIF, thereby suggesting that free radical and peroxynitrite formation may cause direct or indirect mitochondrial damage and subsequent AIF release, nuclear translocation, and large-scale DNA fragmentation (Lee et al. 2005; Li et al. 2007). Results from our and other laboratories on the direct upstream mechanisms responsible for the release of AIF from mitochondria suggest that proteins of the Bcl-2 family of cell death proteins play an important role for this process. Small molecule inhibitors of Bid, a pro-apoptotic BH3-only member of the bcl-2 family, prevented cell death together with translocation of AIF from mitochondria to the nucleus in primary cultured neurons following oxygen-glucose deprivation and completely preserved cell and nuclear morphology following glutamate toxicity in HT22 hippocampal cells (Culmsee et al. 2005; Landshamer et al. 2008).
In conclusion, the current literature suggests that AIF-mediated caspase-independent signaling pathways are of major importance for delayed neuronal cell
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Fig. 6.3 Less ischemic brain damage in mice with reduced AIF expression. (a), Infarct volume of Harlequin mutant mice (HQ) which have a reduced expression of AIF protein due to a proviral insertion in the aif gene. Infarct areas were evaluated histomorphometrically on 11 consecutive Nissl-stained brain sections (500 |im apart) throughout the infarct of HQ animals and their wild type littermates (Control). The infarcted brain area in HQ mice was reduced on each investigated section as compared to controls. (b), In HQ mice, the infarct volume, calculated on the basis of the histomorphometric data from the individual sections, showed a 43% reduction as compared to wild type littermates (n = 5, *p < 0.03) (Culmsee et al. 2005)
death following experimental stroke. Caspase activation occurs during this process, however, inhibition of caspases seems to only delay and not to prevent neuronal death following focal cerebral ischemia. These findings suggest that AIF may be a novel target for drug development aimed to mitigate cell death following stroke.
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