Understanding the functional status of signal transduction systems in Alzheimer's disease (AD) and the endogenous regulatory processes for these signal transduction systems is vital for the treatment of this disorder. In AD, cholinergic neurons degenerate but postsynaptic muscarinic receptors remain intact, which led to the therapeutic use of cholinergic agonists, including cholinesterase inhibitors. however, the functional state of the receptors, which are coupled to phosphoinositide (PI) hydrolysis, is unknown. We recently developed a method to measure agonist-induced, GTP- dependent PI hydrolysis in postmortem human brain membranes. Therefore we can now study the functional status of the therapeutically targeted PI- linked receptors in AD and initial studies revealed impaired cholinergic- stimulated PI hydrolysis in AD. Therefore this is the focus of the first specific aim. Understanding how the activity of the PI second messenger system is regulated and is impaired in AD is of critical importance. Neither aging nor lesions cause decreased PI activity, as is seen in AD, but glucocorticoid hormones (e.g., cortisol), which are elevated in AD and which exacerbate age- and toxin-related neurodegeneration, impair the Pi response. Therefore identifying modulatory interactions of glucocorticoid hormones on signal transduction systems associated with PI hydrolysis is the focus of the second specific aim. The first specific aim is to test the hypothesis that agonist-induced PI metabolism can be measured in postmortem human brain, that it is impaired in AD, and that G-protein dysfunction contributes to the impairment. PI hydrolysis will be measured in response to activation of phospholipase C, G-proteins, and several receptor subtypes by agonists in SAD and age- matched control brain regions, using a variety of experimental methods. Western blots will b used to measure protein levels of components of this system, including phospholipase C, G-protein subtypes, and protein kinase C, and G-protein function will be measured. The second specific aim is to test the hypothesis that elevated glucocorticoid hormones impair rat brain signal transduction systems associated with the PI second messenger system, especially when coupled with excitotoxic lesions. Rats will be adrenalectomized, administered glucocorticoids and/or kainate. Cortical and hippocampal IP metabolism, G- protein levels and function, protein kinase C, and gene expression will be measured. The results of this project will greatly increase our knowledge of the function of the PI signal transduction system in AD, will identify specific sites impaired in AD, will identify how glucocorticoids modulate its activity in rat rain, and will identify consequences of glucocorticoid effects at the level of gene expression.
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