Heat-Shock Protein 70 Attenuates Caspase-3 Activation and DNA Fragmentation Following Focal Cerebral Ischemia

J Korean Neurol Assoc > Volume 22(6); 2004 > Article

Journal of the Korean Neurological Association 2004;22(6): 623-630.

국소뇌허혈 모델에서 열충격단백질70의 카스파아제-3 활성화 및 디옥시리보핵산 분절화 억제에 의한 신경보호효과

이승훈, 권형민 김영주 이경미 김만호 윤병우

서울대학교 의과대학 신경과학교실, 서울대학교병원 임상의학연구소

Heat-Shock Protein 70 Attenuates Caspase-3 Activation and DNA Fragmentation Following Focal Cerebral Ischemia

Seung-Hoon Lee

Department of Neurology, Seoul National University College of Medicine, Seoul; Neuroscience Research Institute, SNUMRC and Clinical Research Institute, Seoul National University Hospital, Seoul, Korea

Abstract

Background: Recently, HSP70 has been shown to act as an inhibitor of apoptotic pathways in the cell culture following heat shock; however, little information is available on the mechanism of neuroprotection after cerebral ischemia. In this study, our purpose is to investigate whether the HSP70 protein can protect apoptotic cell death after focal cerebral ischemia.
Methods: hsp70.1 knockout (KO) and wild-type (WT) mice were subjected to transient middle cerebral artery occlusion for 2 hours. At 22 hours, we measured infarction volumes, and detected DNA fragmentation with TUNEL staining. HSP70 and hsp70.1 mRNA expression were analyzed by Western blots and Northern blots, respectively. Caspase-3 activation was examined with Western blots and caspase-3 activity assay.
Results: hsp70.1 mRNA was not detected in hsp70.1 KO mice after ischemia, and HSP70 expression was markedly suppressed in KO mice versus WT mice. The infarction volume was significantly larger in the KO (82.1±9.5 mm3) than in the WT (58.4±10.3 mm3; p<0.05) 24hours later. Caspase-3 activation was also significantly enhanced in KO mice versus WT mice, as evidenced by higher levels of activated caspase-3 and cleaved gelsolin, as determined by Western blotting and caspase-3 activity assay. TUNEL-positive apoptotic cells were much higher in the KO (1.94±0.61×103/mm2) than in the WT (1.05±0.35 ×103/mm2; p<0.05) in the cortex, but not in the striatum.
Conclusions: We conclude that that HSP70 acts as a strong inhibitor of apoptosis via blocking caspase-3 activation following focal cerebral ischemia.Key Words: Heat-shock protein 70, Apoptosis, Brain ischemia, Cytoprotection