Reduction of caspase-8 and -9 cleavage is associated with increased c-FLIP and increased binding of Apaf-1 and Hsp70 after neonatal hypoxic/ischemic injury in mice overexpressing Hsp70

Background and purpose: Caspase-8 and caspase-9 are essential proteases of the extrinsic and intrinsic apoptotic pathways, respectively. We investigated whether neuroprotection associated with overexpression of heat-shock protein 70 (HSP70), a natural cellular antiapoptotic protein, is mediated by Caspase-8 and caspase-9 signaling in the neonatal mouse brain after hypoxia/ischemia (H/I) injury.

Methods: Postnatal day 7 transgenic mice overexpressing rat HSP70 (HSP70 Tg) and their wild-type (Wt) littermates underwent unilateral common carotid artery ligation followed by 30 minutes of exposure to 8% O2. The expression of apoptotic proteins was quantified by Western blot analysis, and the specific interaction between HSP70 and apoptotic protease activating factor 1 (Apaf-1) was determined by coimmunoprecipitation.

Results: HSP70 overexpression reduced cytosolic translocation of cytochrome c without affecting the levels of Apaf-1 and pro-caspase-9 24 hours after H/I. The expression of these apoptotic proteins in the naïve neonatal brains was also not affected by HSP70 overexpression. Reduced caspase-9 cleavage occurred in HSP70 Tg mice compared with Wt littermates 24 hours after H/I and correlated with increased binding of HSP70 and Apaf-1. Increased cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) expression and decreased Caspase-8 cleavage were also observed in HSP70 Tg compared with Wt mice 24 hours after H/I.

Conclusions: Our results suggest that the extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of HSP70 overexpression in neonatal H/I, specifically by upregulating FLIP and sequestering Apaf-1, leading to reduced cleavage of Caspase-8 and caspase-9.