We showed that gamma irradiation of the developing mouse brain with 2 Gy induced a massive apoptosis of neural precursors but not of neurons within 24 h. Successive phosphorylation and dephosphorylation of histone H2AX have been linked to DNA breaks and repair. Similar numbers of nuclear foci of phosphorylated H2AX (gamma-H2AX) were found 1 h postirradiation in neural precursors and in neurons, suggesting that differences in radiosensitivity were not related to variations in the numbers of DNA double-strand breaks induced by radiation. Surviving neural precursors like neurons totally lost gamma-H2AX within 24 h after irradiation, but they had a slower kinetics of loss of gamma-H2AX foci. This suggests that the DNA repair machinery processed damage more slowly in these neural precursors in relation to their greater radiosensitivity. We also found a bright and diffuse gamma-H2AX staining of nuclei of cells at an early stage of apoptosis, whereas cells at later stages of apoptosis were unstained. This was probably related to phosphorylation and subsequent degradation of H2AX in the course of DNA fragmentation during apoptosis. Detection of gamma-H2AX-bright nuclei may thus be a useful marker of neural cells at an early stage of apoptosis.
We investigated the role of tumor suppressor p53 and Fas (CD95/APO-1), a member of the tumor necrosis factor receptor family, in neural progenitors response to cirradiation exposure. Telencephalic cells were obtained from wild-type C57Bl/6, or p53À/À or fasÀ/À, 15-dayold mouse embryos. They were cultured in conditions allowing neural progenitors to form proliferating clusters (neurospheres). A 2 Gy c-irradiation induced a G1 cell cycle arrest and triggered apoptosis in wild-type neural progenitor cultures in correlation with an enhanced expression of p53 and of its downstream target p21 WAF1 , both of them acquiring a nuclear localization. These effects did not occur in p53À/À neural progenitors demonstrating the central role played by p53 in their response to ionizing radiation. Furthermore, the monoclonal antibody Jo2 directed against Fas induced apoptosis of wild type but not of fasÀ/À neural progenitors, indicating the existence of a functional Fas signaling pathway in neural progenitors. Ionizing radiation induced an increase of Fas membrane expression related to a p53-dependent increase of fas mRNA expression in wild-type neural progenitors. Moreover, fasÀ/À neural progenitors exhibited delayed radiation-induced apoptosis compared to wild-type cells. Therefore, these findings establish a role for Fas/CD95 related to p53 in the response of neural progenitors to c-radiation exposure. Similar mechanisms could be triggered in neural progenitors in case of different stresses during brain development or in the course of various diseases affecting the adult brain.
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