Activation and Cleavage of Caspase-3 in Apoptosis Induced by Experimental Cerebral Ischemia

Abstract

We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.

Mice overexpressing human caspase 3 appear phenotypically normal but exhibit increased apoptosis and larger lesion volumes in response to transient focal cerebral ischaemia

“…Caspase 3 activation has been detected following focal (and global) ischaemic insults to the brain [14][15][16][17][18][19] where it coincides with areas of enhanced apoptotic cell death 14 and treatment with caspase 3 inhibitors has been reported to reduce infarct size following MCA occlusion. 14,[56][57][58] Moreover, studies in caspase 3-deficient mice indicate that these animals are relatively resistant to ischaemic insults with smaller lesions than their WT littermates.…”

“…10 The decreased apoptosis observed in the brain suggested a critical role for caspase 3 in the development of the central nervous system. Caspase 3 has also been implicated in cell death following a number of neurodegenerative insults including global ischaemia, 12,13 focal ischaemia, [14][15][16][17][18][19] transient ischaemia of the spinal cord, 20 neonatal cerebral hypoxia-ischaemia, [21][22][23] traumatic brain injury, [24][25][26] Alzheimer's disease, [27][28][29] Huntington's disease, [30][31][32] and Parkinson's disease. [33][34][35][36][37] It was therefore hypothesized that overexpression of caspase 3 may sensitize the animal to normal (physiological) apoptotic processes that occur during development and natural ageing and confer particular susceptibility to neurodegenerative insults.…”

Caspase activation in fetal rat brain following experimental intrauterine inflammation

“…In mature animals, Namura and colleagues showed that activated caspase-3 protein was detected within 12 h after stroke while peak caspase-3 activity was observed within 1 h [30]. In a similar model, Benchoua and colleagues demonstrated that caspase-8 activation occurs in the acute time periods after stroke while delayed cell death (after 24 h) occurs concurrently with activation of caspase-9, suggesting differential activation of the two caspase cascades [5].…”

Caspases on the brain

“…The timing of death varies among regions of the brain, from the early embryonic to the early postnatal periods. Mice lacking many of the other caspases, such as caspase 1, 2, 6, 11, or 12, do not have an obvious neuronal phenotype (Kuida et al, 1995;Bergeron et al, 1998;Namura et al, 1998;Wang et al, 1998;Zheng et al, 2000), but these caspases may play a role in regional pruning of neurons or in the plasticity of the nervous system.…”