The overall goal of the proposed research is to further our understanding of the ways by which neuronal cell type is determined, and by which postmitotic neurons survive and respond to damage. The applicant has selected the cerebellar Purkinje cell as an experimental system because the anatomy and development of the cerebellar cortex is well-known, because the loss of Purkinje cells leads to ataxia, an easily detectable behavioral deficit, and because the Pcp-2 promoter, a Purkinje cell specific regulatory element, can be used to direct expression of foreign genes in transgenic mice. The applicant has recently shown that expression of the oncogenic SV40 largeT-antigen in the Purkinje cells of transgenic mice causes the death of the Purkinje cells, apparently via apoptosis. The focus of the proposed research is to determine whether the death of the Purkinje cells is indeed an apoptotic response to oncogenic stimulation. There are 3 specific aims. First, control and oncogene-expressing Purkinje cells will be analyzed for DNA fragmentation, a hallmark of apoptotic cell death, using an in situ enzymatic technique. Second, an in vivo approach will utilize Pcp-2 to target expression of two proteins, Bcl-2 and E1B, which have been shown in other systems to protect against or inhibit apoptosis. For this aim, transgenic Bcl-2 or E1B mice will be bred to the SV40 large T-antigen transgenic mice previously made by the applicant. A lengthening of the onset of ataxia and a reduction in the Purkinje cell death will be interpreted as support for the hypothesis that the Purkinje cell death is apoptotic. Third, dissociated cerebellar cell cultures from transgenic mice expressing various oncogenes (SV40 large T antigen, Bcl-2, E1B) within the Purkinje cells will be established. The goal of this aim will be to generate a proliferating cell line by which to study Purkinje cell transcription. The rationale is that transgene expression will be required for the cells to grow in vitro and, since the transgene expression is driven by the Pcp-2 promoter, which is Purkinje cell specific, at least some Purkinje cell specific transcription must be activ
