The long-term objective of this proposal is to define mechanisms of abnormal cell cycle regulation leading to selective neuronal degeneration. This will be investigated by in vivo studies of expression, post-translational modifications and biological function of cell cycle regulatory genes in animal models of neuronal apoptosis.
Specific Aim 1 will analyze the effects of kainic acid-induced seizures on the post-translational phosphorylation and DNA binding of the p53 transcription factor, a major regulator of the cell cycle that has been implicated in neuronal apoptosis. A solid-phase kinase assay will be used to isolate and measure p53 kinase activity in hippocampal lysates. DNA binding activity will be analyzed by gel mobility shift assay.
Specific Aim 2 will analyze the activation of p53-regulated cell cycle genes during adrenalectomy- induced apoptosis of hippocampal granule cells. The cell cycle genes to be studied include cyclin D1, p21 WAF1 and the retinoblastoma susceptibility gene, RB. Wild type mice and mice that are either homozygous (p53-/-) or heterozygous (p53+/-) for the p53 null allele, will be used to elucidate the roles of these genes as effectors of p53-mediated neuronal death. To identify neuronal populations that constitutively express p53, Specific Aim 3 will define regional and cellular patterns of p53 gene expression in the normal postnatal, adult and aged central nervous system. Gene expression will be analyzed by in situ hybridization and immunohistochemistry. The degree of cellular injury will be assessed by histological staining techniques. The results of these studies will add significantly to our knowledge of mechanisms of selective neuronal degeneration related to aberrant cell cycle control, and will provide insight into the pathophysiology of neurodegenerative diseases.
