Because of the association of oxidative stress with widespread debilitating disorders of the CNS, it is imperative to identify how signaling systems are affected by oxidative stress. This project is focused on testing specific hypotheses concerning the effects of oxidative stress on signaling systems linked to cholinergic muscarinic receptors. These receptors are coupled to the phosphoinositide (PI) signal transduction system, increases in protein tyrosine phosphorylation, and downstream transcription factor modulation. Deficient PI signaling has been reported in Alzheimer's disease, which is centered on dysfunction of the G- proteins that mediate signal transduction. Human neuroblastoma SH-SY5Y cells provide an optimal model system to study because they express m3 muscarinic receptors which mediate robust stimulation of the PI signal transduction system, intracellular increases in protein tyrosine phosphorylation, and activation of transcription factors such as AP-1 and NFkappaB, each of which appears to be important in cellular responses to oxidative stress. The overall goal is to test the hypothesis that oxidative stress modulates specific sites in these three signaling components, PI hydrolysis, protein tyrosine phosphorylation, and transcription factor activation. Three complementary approaches will be used to model oxidative stress, exposure of SH-SY5Y cells to (a) H2O2, or (b) peroxynitrite, or (c) the use of """"""""cybrid"""""""" SH-SY5Y cells in which endogenous mitochondria have been replaced with mitochondria from Alzheimer's disease or matched control subjects. The Alzheimer's disease-derived cybrid cells thus possess defective cytochrome c oxidase and produce excessive reactive oxygen species.
Specific Aim 1 will test the hypotheses that oxidative stress impairs muscarinic receptor-induced PI hydrolysis and that inhibition of the G-protein Gq/11 is a critical site of action.
Specific Aim 2 will test the hypotheses that oxidative agents impair the palmitoylation of cysteines on the G-protein Gq/11 and that inhibition of Gq/11 palmitoylation impairs PI hydrolysis.
Specific Aim 3 will test the hypotheses that oxidative stress alters intracellular protein tyrosine phosphorylation, including substrates responding to muscarinic stimulation.
Specific Aim 4 will test the hypothesis that oxidative agents inhibit the tyrosine phosphorylation of Gq/11 and will test if this is associated with inhibition of Gq/11 palmitoylation.
Specific Aim 5 will terst the hypothesis that oxidative agents modulate transcription factor activation, including those activated by muscarinic receptor stimulation.
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