NEURONAL SIGNALING--OXIDANTS AND ALZHEIMERS DISEASE

神经信号传导——氧化剂和阿尔茨海默病

基本信息

批准号：
6393968
负责人：
RICHARD S. JOPE
金额：
$ 17.77万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2003-06-30
项目状态：
已结题

项目摘要

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.

由于氧化应激与中枢神经系统的普遍使人衰弱的疾病的关联，因此必须确定信号系统如何受到氧化应激的影响。该项目的重点是测试有关氧化应激对与胆碱能毒蕈碱受体相关的信号系统影响的特定假设。这些受体与磷酸肌醇（PI）信号转导系统耦合，蛋白酪氨酸磷酸化的增加和下游转录因子调节。已经报道了阿尔茨海默氏病的PI信号传导不足，该疾病以介导信号转导的G蛋白功能障碍为中心。人类神经母细胞瘤SH-SY5Y细胞提供了一个最佳的研究模型系统，因为它们表达了M3毒素受体，可介导对PI信号转导系统的鲁棒刺激，蛋白质酪氨酸磷酸化的细胞内增加，并激活转换因子（如AP-1和NFKAPPAB），每个蛋白质的转换因子（如细胞中都具有重要的氧气反应）。总体目标是检验以下假设：氧化应激调节了这三个信号成分，PI水解，蛋白酪氨酸磷酸化和转录因子激活中的特定位点。将使用三种互补方法来模拟氧化应激，SH-SY5Y细胞暴露于（a）H2O2或（b）过氧亚硝酸盐，或（c）使用“ Cybrid” SH-Sy5Y细胞的使用，其中内源性线粒体已被Alzheimer病的Mitochondria替换为来自Alzheimer病的Mitochondria。因此，阿尔茨海默氏病的衍生的cybrid细胞具有有缺陷的细胞色素C氧化酶，并产生过多的活性氧。具体目标1将检验氧化应激会损害毒蕈碱受体诱导的PI水解的假设，并且抑制G蛋白GQ/11是关键的作用部位。特定的目标2将检验氧化剂损害GQ/11棕榈酰化抑制GQ/11棕榈酰化会损害PI水解的假设。具体目标3将测试氧化应激会改变细胞内蛋白酪氨酸磷酸化的假设，包括对毒蕈碱刺激反应的底物。具体目标4将检验以下假设：氧化剂抑制GQ/11的酪氨酸磷酸化，并将测试这是否与GQ/11棕榈酰化的抑制有关。具体目标5将涉及氧化剂调节转录因子激活的假设，包括毒蕈碱受体刺激激活的转录因子。