The goal of this project is to investigate the effects of cell membrane cholesterol on caveolin-G protein interactions in a PC12 cell model system. The rationale is to create a model neuronal cell system that will permit hypothesis testing regarding mechanisms underlying age-related impairments in signal transduction mediated through muscarinic receptor regarding mechanisms underlying age-related impairments in signal transduction mediated through muscarinic receptor activation of G proteins of the Gq/11 subtype in brain. Gq/11 mediates its effects through downstream activation of phospholipase C and production of the second messengers, inositol trisphosphate and diacylglycerol. In preliminary studies we have shown that basal and muscarinic receptor activation of Gq/11 is decreased with aging and that this correlates to an apparent increase in Gq/11alpha caveolar localization as well as Gq/11alpha- caveolin-1 binding in the F344 rat model. The primary working hypothesis is that age-related changes in cell membrane cholesterol increase caveolin-1 binding to Gq/11alpha in caveolae-enriched membrane domains resulting in down-regulation of signaling. Experiments to test these hypotheses will utilize methyl-beta- cyclodextrin-cholesterol complexes to modulate cholesterol in the PC 12 cells.
The specific aims are to: (1) determine the relationship between cell membrane cholesterol content and Gq/11 targeting to caveolae-enriched membranes, binding of caveolin-1 to Gq/11, and expression of caveolin-1 mRNA in POC12 cells; (2) investigate the relationship between cell membrane cholesterol and the magnitude of basal and muscarinic cholinergic agonist-stimulated GTP binding, low Km GTPase activity and inositol phospholipid production in PC12 cells; and (3) determine the effects of hydrogen peroxide-induced oxidative stress on Gq/11 targeting to caveolae-enriched membranes, binding of caveolin-1 to Gq/11, expression of caveolin-1 mRNA and on basal- and agonist-stimulated GTP binding in cholesterol-enriched and cholesterol depleted PC12 cells. The resulting data should provide important insights into the role that cell membrane cholesterol alterations play in the down-regulation of Gq/11 mediated signaling found in normal aging brain as well as age-related pathological states including Alzheimer's Disease.
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| Cutler, Roy G; Kelly, Jeremiah; Storie, Kristin et al. (2004) Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease. Proc Natl Acad Sci U S A 101:2070-5 |
