Early activation of caspase-6 and cleavage of caspase-6 substrates in an in vitro model of stroke

In Canada, the US and Europe, stroke is the leading cause of long term adult disability and the 3rd leading cause of death. Activation of caspase-6 (casp6) in response to ischemia-induced injury has been observed in several rodent models of ischemia. In addition, activated casp6 has been observed in post-mortem tissue from brains of patients who died following ischemic stroke. Importantly, mice with a targeted deletion of casp6 demonstrate reduced stroke injury and improved sensorimotor abilities. These data provide important validation for the activation of casp6 as critical step in the pathogenesis of stroke however important details of the hierarchy of caspase activation events has been lacking. There is strong evidence to support a critical role for NMDA receptor-mediated excitotoxicity as a mechanism underlying neuronal damage post stroke. Inappropriate caspase activation in stroke could result from increases in intracellular calcium levels due to excitotoxic stress and lead to generation of active proteolytic fragments as a result of caspase substrate proteolysis. Using the well-characterized in vitro model of stroke (rat primary cortical neurons ± NMDA) we demonstrate that at the early time point post NMDA (1h) no increase in levels of LDH (cell death) is observed. However at 24h a significant increase in cell death is detected (ANOVA p < 0.0001). Assessment of caspase mRNA levels demonstrates that increased casp6 mRNA is observed at 1h and 24hs post NMDA (ANOVA p = 0.016), casp3 mRNA is significantly increased at 24h post NMDA (ANOVA p = 0.03) whereas no differences in casp8 or casp9 mRNA levels were observed. Increased casp6 activity post NMDA was also detected (ANOVA p = 0.055, post hoc 0h vs. 1h, p < 0.05). Preliminary data also shows that cleavage of the novel casp6 substrate, STK3, a proapoptotic kinase, and the casp6 substrate huntingtin [neuroprotective protein], occurs early and prior to cell death. These data provide a clear link between excitotoxicity and proteolysis and suggest that activation of casp6 is an early event in the pathogenesis of stroke.