Increasingly, cell cycle proteins are being recognized as important contributors to cellular processes beyond cell division. Developmental, target-related neural cell death is associated with the re-appearance of cell cycle proteins and DNA replication. In aging, the neurons at risk for cell death in Alzheimer's disease also re-express a number of cell cycle proteins. Not only are these proteins required for cell survival, the retinoblastoma tumor suppresser gene is also a key enabler of differentiation phenotypes, and the cyclin dependent kinase, Cdk5, while playing no overt role in the cell cycle is crucial for neuronal migration and axon outgrowth. The current application will pursue these findings in many ways.
The first aim i s to study the role of the retinoblastoma gene in neural development. Mouse chimeras will be constructed with retinoblastoma and wild-type embryos. Our hypothesis is that a variety of cell intrinsic differentiation events will be blocked in all Rb -/- cells even when genetically normal neighbors surround them. In the adult chimera, we predict that in several neuronal cell populations all mutant cells will die. A temporal analysis of both CNS and PNS cell types in such chimeras will define the developmental stage at which Rb is required. Cell cultures of Rb -/- dorsal root ganglion neurons will be transfected with TrkA constructs to determine whether the death of these cells is due to the absence of one crucial growth factor receptor or to a broader interference with the developmental program. We will explore this issue further by transfecting several Rb-1 mutants that fail to block cycling, but still permit differentiation to occur. The second specific aim will be to explore the roles of Cdk5 in CNS development. Chimeras of Cdk5-/- will be used to extend our finding in cerebellum that the failures of cell migration in the mutants are cell autonomous events. Detailed 'birthday' studies in these chimeras will allow us to tease apart the Cdk5-dependent and -independent forms of cell migration. Cell cultures of Cdk5-/- cell will be used to determine more precisely which cell biological processes related to migration and axon elongation are blocked in the different cell types. Taken together these studies are aimed at proof of the hypothesis that cell cycle components have been appropriated during evolution to function in a variety of cellular processes and are now inextricably linked to both cell survival and cellular differentiation in the nervous system.
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