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Apoptosis, or programmed cell death [30], is characterized histologically by cells positive for terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) that exhibit DNA laddering. Necrotic cells, in contrast, show mitochondrial and nuclear swelling, dissolution of organelles, nuclear chromatin condensation, followed by rupture of nuclear and cytoplasmic membranes, and the degradation of DNA by random enzymatic cuts. Cell type, cell age, and brain location render cells more or less resistant to apoptosis or necrosis. Mild ischemic injury preferentially induces cell death via an apoptotic-like process rather than necrosis, although "aponecrosis" more accurately describes the pathology.

Apoptosis occurs via caspase-dependent as well as caspase-independent mechanisms (Fig. 1.3). Caspas-es are protein-cleaving enzymes (zymogens) that belong to a family of cysteine aspartases constitutively expressed in both adult and especially newborn brain cells, particularly neurons. Since caspase-dependent cell death requires energy in the form of ATP, apopto-sis predominantly occurs in the ischemic penumbra (which sustains milder injury) rather than in the ischemic core, where ATP levels are rapidly depleted [31]. The mechanisms of cleavage and activation of caspases in human brain are believed to be similar to those documented in experimental models of stroke, trauma, and neurodegeneration [32].Apopto-genic triggers [33] include oxygen free radicals [34], Bcl2, death receptor ligation [35], DNA damage, and possibly lysosomal protease activation [36]. Several mediators facilitate cross communication between

Penumbra Anatomy
Figure 1.3

Cell death pathways relevant to an apoptotic-like mechanism in cerebral ischemia. Release of cytochrome c (Cyt.c) from the mitochondria is modulated by pro- as well as anti-apoptotic Bcl2 family members. Cytochrome c release activates downstream caspases through apoptosome formation (not shown) and caspase activation can be modulated by secondary mitochondria-derived activator of caspase (Smac/Diablo) indirectly through suppressing protein inhibitors of apoptosis (IAP). Effector caspases (caspases 3 and 7) target several substrates,which dismantle the cell by cleaving home-ostatic,cytoskeletal, repair, metabolic,and cell signaling proteins.Caspases also activate caspase-activated deoxyribonu-clease (CAD) by cleavage of an inhibitor protein (ICAD). Caspase-independent cell death may also be important. One mechanism proposes that poly-ADP(ribose)polymerase activation (PARP) promotes the release of apoptosis-inducing factor (AIF), which translocates to the nucleus, binds to DNA, and promotes cell death through a mechanism that awaits clarification. (From Lo et al., Nat Rev Neurosci 2003,4:399-415)

cell death pathways [37, 38], including the calpains, cathepsin B [39], nitric oxide [40, 41], and PARP [42]. Ionic imbalances, and mechanisms such as NMDA receptor-mediated K+ efflux, can also trigger apoptotic-like cell death under certain conditions [43, 44]. This inter-relationship between glutamate excitotoxicity and apoptosis presents an opportunity for combination stroke therapy targeting multiple pathways.

The normal human brain expresses caspases-1, -3, -8, and -9, apoptosis protease-activating factor 1 (APAF-1), death receptors, P53, and a number of Bcl2 family members, all of which are implicated in apop-tosis. In addition, the tumor necrosis factor (TNF) superfamily of death receptors powerfully regulates upstream caspase processes. For example, ligation of Fas induces apoptosis involving a series of caspases, particularly procaspase-8 and caspase-3 [45]. Cas-

pase-3 has a pivotal role in ischemic cell death. Cas-pase-3 cleavage occurs acutely in neurons and it appears in the ischemic core as well as penumbra early during reperfusion [46]. A second wave of caspase cleavage usually follows hours to days later, and probably participates in delayed ischemic cell death. Emerging data suggest that the nucleus - traditionally believed to be simply the target of apoptosis - is involved in releasing signals for apoptosis. However, the mitochondrion plays a central role in mediating apoptosis [47,48]. Mitochondria possess membrane recognition elements for upstream proapoptotic signaling molecules such as Bid, Bax, and Bad. Four mi-tochondrial molecules mediate downstream cell-death pathways: cytochrome c, secondary mitochondria-derived activator of caspase (Smac/Diablo), apoptosis-inducing factor, and endonuclease G [49]. Apoptosis-inducing factor and endonuclease G mediate caspase-independent apoptosis,which is discussed below. Cytochrome c and Smac/Diablo mediate caspase-dependent apoptosis. Cytochrome c binds to Apaf-1, which, together with procaspase-9, forms the "apoptosome," which activates caspase-9. In turn, caspase-9 activates caspase-3. Smac/Diablo binds to inhibitors of activated caspases and causes further caspase activation. Upon activation, executioner caspases (caspase-3 and -7) target and degrade numerous substrate proteins including gelsolin, actin, PARP-1, caspase-activated deoxyribonuclease inhibitor protein (ICAD), and other caspases, ultimately leading to DNA fragmentation and cell death (Fig. 1.3).

Caspase-independent apoptosis was recently recognized to play an important role in cell death and probably deserves careful scrutiny as a novel therapeutic target for stroke. NMDA receptor perturbations activate PARP-1, which promotes apoptosis-in-ducing factor (AIF) release from the mitochondria [42]. AIF then relocates to the nucleus, binds DNA, promotes chromatin condensation, and kills cells by a complex series of events. Cell death by AIF appears resistant to treatment with pan-caspase inhibitors but can be suppressed by neutralizing AIF before its nuclear translocation.

A number of experimental studies have shown that caspase inhibition reduces ischemic injury [50].

Caspase-3 inhibitors [51], gene deletions of Bid or caspase-3 [52],and the use of peptide inhibitors,viral vector-mediated gene transfer, and antisense oligonucleotides that suppress the expression and activity of apoptosis genes have all been found to be neuro-protective [50]. However, caspase inhibitors do not reduce infarct size in all brain ischemia models, perhaps related to the greater severity of ischemia, limited potency or inability of the agent to cross the blood-brain barrier, relatively minor impact of apoptosis on stroke outcome, and upregulation of caspase-independent or redundant cell death pathways. Ultimately, it may be necessary to combine caspase inhibitors and other inhibitors of apoptosis with therapies directed towards other pathways, for successful neuroprotection.

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