Inducible HSP70 overexpression protects cultured cerebeilar granule neurons against apoptosis induced by low potassium

Aim To observe effects of adenovirus-mediated human inducible HSP70 overexpression on apoptosis of primary cultured rat cerebellar granule neurons (CGN) induced by low potassium. Methods CGN in vitro at 5 d were coinfected with adenoviruses containing human inducible HSP70 and green fluorescent protein (GFP) (AdTR5/HSP70-GFP) on separate cistrons, as well as a tetracycline-regulated promoter (AdCMV/tTA). Control cells were coinfected only with AdTR5/GFP and AdCMV/tTA. After 48 h, the expression of HSP70 was measured by immunofluorescence analysis and Western blot analysis. Apoptosis was induced by switching CGN to serum-free medium containing 5 mmol·L~ -1 KCl. After 24 h, the neuronal viability was measured by MTT methods, the morphology of neurons was observed by phase-contrast microscopy, Hoechst 33258 nucleus staining and DNA agarose gel electrophoresis fragmentation were used to analyzeneuronal apoptosis. Results HSP70 was overexpressed in CGN coinfected with AdCMV/tTA and AdTR5/HSP70-GFP. HSP70 overexpression protected CGN from apoptosis induced by low potassium. The neuronal viability was elevated from 45.5%±5.2% to 82.3%±5.2%(P0.01), nuclear condensation/aggregation was reduced and a typical apoptotic DNA ladder almost disappeared. Conclusion Adenovirus-mediated human inducible HSP70 overexpression inhibits apoptosis of CGN induced by low potassium.