One goal is to understand the mechanism of the selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which causes parkinsonism by killing certain dopaminergic neurons. Our studies will be performed on the clonal cell line PC12 and on a PC12 variant that is resistant to MPTP. MPTP kills wild type PC12 cells, which are dopaminergic, and it does so by inhibiting mitochondrial respiration. The PC12 variant is resistant to MPTP because of an alteration in energy metabolism. We will characterize energy metabolism in the variant to understand its mechanism of MPTP resistance. These studies are important brain dopaminergic neurons unaffected by MPTP may have a similar mechanism or resistance. We will also use the variant to identify, characterize, and determine the function of heretofore unrecognized proteins involved in neuronal storage, release, and reuptake of neurotransmitter. Presumably unrelated to its MPTP resistance, the variant has difficiencies in several neurotransmitter-related activities, e.g., neurotransmitter release and reuptake. Furthermore, the variant exhibits markedly decreased expression of multiple genes, and it is likely that most of the genes that are expressed in wild type PC12 but poorly or not at all in the variant code for proteins involved in neurotransmitter metabolism, storage, release, and transport. We have identified several of these genes by surveying some rat brain-specific cDNA clones and by screening a PC12 cDNA library. After sequencing these cDNA clones, we propose to synthesize the corresponding peptides, raise antibody aganist the peptides, and identify and characterize the natural protein products of these genes. Using the antisense RNA technique (by which it is possible to reduce the expression in a cell of one gene at a time) we will isolate several PC12 strains each of which is normal except for having a reduced level of the protein product of one of these genes. We will then determine which cellular activity pertaining to neurotransmitter storage, transport, or release is lost as a consequence of the low level of this protein. Three of the PC12 proteins not found in the MPTP- resistant variant are calmodulin-binding proteins. Because there is evidence that calmodulin mediates the Ca2+-triggered release of neurotransmitters, we will purify and characterize these three calmodulin-binding proteins.
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