It is generally believed that a relative hyperactivity of dopaminergic transmission is characteristic of schizophrenia. We, like many other research groups, have used the dopaminoceptive medium spiny neurons of the dorsal neostriatum as a model system for the study of dopaminergic transmission. This grant application is to support a multi-disciplinary study of signal transduction pathways in these medium spiny neurons. (Biochemical Analysis of Basal Ganglia Phosphoproteins) will include: characterization of phosphorylation and dephosphorylation of DARPP-32 and inhibitor-1, which are inhibitors of protein phosphatase-1 (PP-1); characterization of the structure of DARPP-32 and PP1 and analysis of their interaction; regulation of PP1 by cyclin-dependent protein kinases; regulation of PP1 localization in striatal neurons; characterization of the phosphorylation and dephosphorylation of Na+K+-ATPase; characterization of novel substrates for PP1. (Pharmacological Regulation of Basal Ganglia-Enriched Phosphoproteins) will include: identification of first messenger and signal transduction pathways which regulate the phosphorylation of DARPP-32 and inhibitor-1 in intact cell preparations; use of intact animals to evaluate the physiological regulation of DARPP-32 and inhibitor-1 phosphorylation; evaluation of the regulation of PP-1 by first messengers in intact cells; evaluation of the roles of protein phosphatase 2B and casein kinase I and casein kinase II phosphorylation sites, in regulating phosphorylation and activity of DARPP-32 and inhibitor 1 using gene-targeting and transgenic techniques; examination of possible differences in the levels of DARPP- 32, inhibitor-1, PP-1 and other signal transduction components in post mortem brain tissue from schizophrenic patients and normal controls. (Physiological and Anatomical Studies of Phosphoproteins in the Basal Ganglia) will include: characterization of the regional and subcellular distribution of protein phosphatase-1 (PP1) and of effector proteins in the neostriatum; study of the regulation of phosphorylation and dephosphorylation of effector proteins in the neostriatum; study of the physiological significance of the regulation of phosphorylation and dephosphorylation of effector proteins in the neostriatum by means of electrophysiological and cell biological methods. (Molecular Biology of Basal Ganglia Specific Phosphoproteins) will include: use of gene-targeting as a means to inactivate the genes for protein phosphatase-2B (PP-2B), PP-1alpha and PP-1gamma; characterization of the phenotypes of DARPP-32, inhibitor-1 and DARPP-32/inhibitor-1 double knock-out mice; generation of mice containing phospho-site mutations and deletions in DARPP-32; identification and characterization of proteins interacting with the catalytic subunits of PP-1 and PP-2B; generation of immortalized cell lines that express basal ganglia enriched phosphoproteins and manipulation of the levels of these -molecules via antisense technology. (Scientific Core) will include the production and supply of key reagents for the other sections of this Program Project.
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