This research to investigate the underlying molecular mechanisms that trigger neuronal apoptosis after DNA damage. While apoptosis is a cardinal feature of programmed cell death during neural development , it has been increasingly recognized that neurons also undergo apoptosis in response to injury. DNA damage produces apoptotic death in cultured mammalian central nervous system (CNS) neurons. Cell death is inhibited by two drugs which inhibit cyclin-dependent kinases. These drugs also protect neurons against apoptotic cell death after trophic factor withdrawal. This suggest that apoptosis in neurons due to both toxic injury and lack of trophic support required enzymes that normally control the cell cycle. Investigating this hypothesis is the major focus of this application. The first two specific aims will determine the role of immediate early genes and cycle-related proteins in apoptosis of cultured rat CNS neurons after DNA damage by irradiation or treatment with camptothecin. We will first determine the changes in immediate early genes (IEGs) and cell cycle-related proteins (cyclin- dependent kinases, Cdks) following DNA damage. We will then determine whether apoptosis is reduced by microinjection of antibodies directed against the IEGs that are increased following DNA damage or by injection of inhibitors or dominant negative mutants the Cdks and also by treatment with antisense oligonucleotides against the cyclins and Cdks that are increased following DNA damage. We will also evaluate the role of the p53 protein in neuronal apoptosis by utilizing neurons from p53 knockout mice. We will also utilize a series of Cdk-inhibitor drugs to determine a structure/activity relationship between Cdk inhibitory activity and reduction of neuronal apoptosis due to DNA damage and also for their ability to produce apoptosis in dividing PC12 cells. Finally, since campotothecin toxicity is reduced by inhibition of transcription, we will evaluate whether inhibition of transcription reduces toxicity of campothecin by reducing 1)DNA damage by camptothecin or 2) the apoptotic process itself or both. These studies should provide significant information about the underlying mechanisms that trigger apoptosis after DNA damage. Moreover, drugs that simultaneously kill dividing cells and protect neurons against toxicity from cancer chemotherapeutic agents or radiation may be exceedingly useful in therapy of brain cancers, in which survivors normally suffer a significant intellectual impairment.
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