GENES THAT REGULATE NEURONAL DEATH

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DESCRIPTION: Programmed cell death (PCD) is a necessary developmental phenomenon that is widespread in the nervous systems. Recent evidence suggests that PCD also occur in several pathological conditions. We and other have hypothesized that neurotrophic factor deprivation induced PCD is mediated by key cell cycle regulators activated by the removal of survival promoting factors. Recently, we provided the first evidence for the increased expression of a specific gene, cyclin D1, in neurons undergoing PCD. The only previously described function for cyclin D1 is its role in progression through the G1-phase of the cell cycle. We shall investigate the specific hypothesis that cyclin D1 functions as a necessary part of the death program in neurons. Toward this objective, we shall assess directly the role of cyclin D1 in the death of nerve growth factor (NGF) deprived sympathetic neurons. Using intracellular microinjections, we shall express (1) inhibitors of cyclin D1 function, (2) antisense cyclin D1 sequences, or (3) neutralizing cyclin D1 antibodies to examine whether cyclin D1 expression is required for NGF deprivation-induced PCD. We shall also examine whether overexpression of cyclin D1 ectopically is sufficient to induce PCD in neurons maintained in the presence of NGF. Biochemical approaches, including protein kinase assays, immunoprecipitations, and immunoblotting, will be used to identify and characterize the molecules that interact with cyclin D1 during PCD. These experiments will address whether a cyclin-dependent protein kinase is activated in dying neurons or whether cyclin D1 interacts with the retinoblastoma protein as part of a mechanism for cell death. Lastly, we shall use reverse transcription-polymerase chain reaction technology to continue to catalog cell cycle gene expression in dying neurons. These studies will determine the significance of the increased expression of cyclin D1 during PCD and will test the general hypothesis that neuronal cell death involves the activation of cell cycle events. This work should further our long-term goals of elucidating the molecular mechanism of neuronal PCD and of developing the means to manipulate the process pharmacologically.