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(Stroke. 2006;37:507.)
© 2006 American Heart Association, Inc.

Original Contributions

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

Yasuhiko Matsumori, MD; Frances J. Northington, MD; Shwuhuey M. Hong, BS; Takamasa Kayama, MD; R. Ann Sheldon, MS; Zinaida S. Vexler, PhD; Donna M. Ferriero, MD; Philip R. Weinstein, MD Jialing Liu, PhD

From the Department of Neurological Surgery (Y.M., S.M.H., P.R.W., J.L.), University of California, San Francisco, and San Francisco Veterans Affairs Medical Center (Y.M., S.M.H., P.R.W., J.L.), San Francisco, Calif; Department of Neurological Surgery (Y.M., T.K.), Yamagata University, School of Medicine, Japan; Eudowood Neonatal Pulmonary Division, Department of Pediatrics (F.J.N.), The Johns Hopkins University, School of Medicine, Baltimore, Md; and Departments of Neurology (R.A.S., Z.S.V., D.M.F.) and Pediatrics (R.A.S., Z.S.V., D.M.F.), University of California at San Francisco.

Correspondence to Dr Jialing Liu, Department of Neurological Surgery (112C), University of California, San Francisco, and Department of Veterans Affairs Medical Center, 4150 Clement St, San Francisco, CA 94121. E-mail miro{at}itsa.ucsf.edu

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-1ß–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.

Key Words: apoptosis • mitochondria • stress proteins

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