Figure 5.

Hydrogen peroxide leads to tAIF-induced neuronal cell death via Cdk5-mediated phosphorylation of CHIP at Ser20. (a) Following treatment of primary cultures of cortical neurons with 200 μM hydrogen peroxide (H₂O₂) for the indicated time periods, cell lysates were harvested and subjected to IB analysis using the indicated antibodies. For in vitro kinase assay, immunoprecipitates with anti-Cdk5 antibody were reacted with 1 μg Histone H1 in the presence of [γ-³²P]ATP at 30 °C for 30 min. The reaction products were resolved by SDS-PAGE and subjected to autoradiography. Coomassie brilliant blue (CBB)-stained gel represents the level of Histone H1 in each lane. (b and c) After normalization against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), relative levels of (b) pS20 CHIP and (c) tAIF were expressed as a fold intensity over the levels found in cells treated with H₂O₂ for 120 min (value=1). Bar represents the means±S.D. of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; NS, not significant. (d) Cell lysates harvested from hydrogen peroxide-treated cortical neurons (200 μM for 60 min) in the presence or the absence of 10 μM roscovitine were subjected to IB analysis using the indicated antibodies. In vitro kinase assay was performed as described in (a). (e) After normalization against GAPDH, relative levels of pS20 CHIP and tAIF were expressed as a fold intensity over the levels found in cells treated with H₂O₂ alone (value=1). Bar represents the means±S.D. of three independent experiments. **P<0.01; ***P<0.001. (f) Cortical neurons cultivated for 60 min in the indicated conditions (200 μM H₂O₂, 10 μM roscovitine) were subjected to the large-scale DNA fragmentation assay by using a pulsed-field gel electrophoresis. (g) Cortical neurons were exposed to 200 μM H₂O₂ for the indicated time periods in the presence or the absence of 10 μM roscovitine. The rate of cell viability was determined by MTT reduction assay. Values are expressed as a percent survival over the untreated control cultures (100%). Data represent the means±S.E.M. from three independent experiments done in triplicate. **P<0.01; ***P<0.001