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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37MH038633-12
Application #
2244651
Study Section
Special Emphasis Panel (NSS)
Project Start
1983-12-01
Project End
1996-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
12
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Chou, A H; Zheng, S; Itsukaichi, T et al. (2000) Wnt-1 inhibits nerve growth factor-induced differentiation of PC12 cells by preventing the induction of some but not all late-response genes. Brain Res Mol Brain Res 77:232-45
Palos, T P; Zheng, S; Howard, B D (1999) Wnt signaling induces GLT-1 expression in rat C6 glioma cells. J Neurochem 73:1012-23
Zheng, S; Ramachandran, B; Haigh, J R et al. (1996) The induction of ret by Wnt-1 in PC12 cells is atypically dependent on continual Wnt-1 expression. Oncogene 12:555-62
Wu, C F; Howard, B D (1995) K252a-potentiation of EGF-induced neurite outgrowth from PC12 cells is not mimicked or blocked by other protein kinase activators or inhibitors. Brain Res Dev Brain Res 86:217-26
Houben, K; Dardashti, K; Howard, B D (1994) PC12 variants deficient in norepinephrine transporter mRNA have wild type activities of several other related transporters. Neurochem Res 19:743-51
Rozenberg, Y Y; Howard, B D (1994) Contrasting morphological changes in PC12 flat cells expressing two different forms of exogenous oncogenic ras. Exp Cell Res 211:59-67
Wu, C F; Zhang, M; Howard, B D (1993) K252a potentiates epidermal growth factor-induced differentiation of PC12 cells. J Neurosci Res 36:539-50
Ramachandran, B; Houben, K; Rozenberg, Y Y et al. (1993) Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells. J Biol Chem 268:23891-7
Dudley, M W; Howard, B D; Cho, A K (1990) The interaction of the beta-haloethyl benzylamines, xylamine, and DSP-4 with catecholaminergic neurons. Annu Rev Pharmacol Toxicol 30:387-403
Howard, B D; Cho, A K; Zhang, M B et al. (1990) Covalent labeling of the cocaine-sensitive catecholamine transporter. J Neurosci Res 26:149-58

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