The long-term goal of the studies proposed here is to identify genes that play roles in the degeneration and death of dopaminergic neurons. Such genes and their products have the potential to serve as targets for therapeutic intervention. To accomplish this objective, a powerful technique designated SAGE (Serial Analysis of Gene Expression), will be used for comprehensive analysis of gene regulation in a convenient cell culture model of monoaminergic neuronal death, the 6-OHDA-treated rat PC12 cell. This approach will be used to compare the genes expressed in neuronally differentiated PC12 cells before and at several times after exposure to 6-OHDA. Analysis of the SAGE data will indicate the degree to which specific genes are regulated and will provide sequence information that can be used for their identification and/or cloning. A following set of studies will determine whether such regulated genes are present in dopaminergic neurons in the normal rat brain and whether their expression is regulated during death. This will be achieved by using in situ localization and immunocytochemistry to detect the transcripts and/or their products in neurons within the substantia nigra of normal rats and in rats in which death of such neurons occur during normal development or is evoked by exposure to 6-OHDA or by target injury. Regulated genes detected in the SAGE PC12 cell screen and also in rat brain substantia nigra neurons will then be examined for presence and regulation in Parkinson's Disease. This will be carried out by immunocytochemical localization using material available through the Neuropathology Core of our Center for Research on Alzheimer's Disease and other Neurodegenerative Disorders. Material to be examined will include sections from PD brains, age-matched controls without neurodegenerative disease and brains from patients with other neurodegenerative disorders. A fourth set of studies will exploit cell cultures of PC12 cells and sympathetic neurons to examine the roles of the regulated genes in death evoked by 6-OHDA and other means. This will be accomplished by a variety of strategies including over-expression, down-regulation and interference with function. Because it is anticipated that a large number of regulated transcripts may be detected, a number of criteria will be used to prioritize those genes that will be studied at each step of the project.
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