BIOPTERIN, CATECHOLAMINES, & NO IN REGULATING APOPTOSIS

描述：(逐字摘自申请人的摘要)大脑中的多巴胺神经元 帕金森氏病患者黑质过早死亡和细胞凋亡 已经在死后被检测到。细胞凋亡的原因尚不清楚。两个突出的， 然而，未经证实的假设是，细胞凋亡是由氧化介导的。 应激或不适当地重新进入细胞周期；这两种情况都可能是 由纹状体靶细胞的神经营养支持不足引发。我们 将通过研究细胞凋亡的潜在机制来解决这一知识差距 培养儿茶酚胺细胞，那里的条件可以更严格地控制 而不是在动物身上。待研究的细胞包括嗜铬细胞瘤(PC12)、 交感神经元和胎儿黑质神经元。这些模型在体内有 相关性，因为黑质多巴胺神经元在发育过程中的凋亡死亡 可能是由于纹状体靶细胞的营养支持不足；这 机制已被认为是导致帕金森病的细胞死亡因素 疾病。四氢生物蝶呤(BH4)是一种重要的调节辅因子 酪氨酸羟基酶和一氧化氮合酶在合成中的作用 儿茶酚胺和NO，如果缺乏BH4就不会发生这种情况。儿茶酚胺 细胞中含有最高浓度的BH4。BH4的代谢， 儿茶酚胺和一氧化氮会产生破坏性的活性氧(ROS)。我们的 初步数据显示，分化的神经元样PC12的凋亡性死亡 停用神经生长因子期间的细胞数与 BH4的胞内水平。因此，我们假设内生水平是 BH4支持正常情况下的儿茶酚胺细胞功能，并促进 当营养支持取消时，细胞凋亡性死亡。我们的工作假设和 具体目的是：细胞内BH4介导细胞凋亡和死亡 儿茶酚胺细胞失去营养支持。虽然这个复杂的过程 凋亡发生在许多细胞类型，我们将集中在儿茶酚胺细胞和 BH4与细胞凋亡关键介质的直接和间接相互作用 在营养支持不足的情况下这些细胞的数量。 这些介质包括氧化应激，即可引发致命死亡的癌基因 重新进入细胞周期，以及关键的半胱氨酸蛋白酶(Caspase) 介导细胞凋亡。我们的研究将确定BH4参与的机制 通过测试：1)ROS升高和来源(BH4， 儿茶酚胺，或NO)；2)调节细胞凋亡的表达改变 癌基因、PS3、c-myc和bc1-2家族(bax、bak、bc1-2、bclxl)和3) Caspase 2、3和9的激活。将通过以下方式监测细胞凋亡：a)数量 活细胞和死亡细胞；以及b)不同时间细胞的荧光定量 细胞凋亡的不同阶段。 这些研究可能会揭示新的细胞凋亡调控机制，并提供 促进神经元存活和预防的治疗方法新途径 使人衰弱的神经系统疾病，如帕金森氏症。

DESCRIPTION: (Verbatim from the Applicant's Abstract) Dopamine neurons in the substantia nigra die prematurely in Parkinson's disease, and apoptotic death has been detected postmortem. The cause of apoptosis is unknown. Two prominent, yet unsubstantiated hypotheses are that apoptosis is mediated by oxidative stress or inappropriate reentry into the cell cycle; both conditions may be initiated by insufficient neurotrophic support from striatal target cells. We will address this knowledge gap by studying mechanisms underlying apoptosis in cultured catecholamine cells, where conditions can me more tightly controlled than in animals. Cells to be studied include pheochromocytoma (PC12), sympathetic neurons, and fetal nigral neurons. These models have in vivo relevance, since apoptotic death of nigral dopamine neurons during development may be due to insufficient trophic support from striatal target cells; this mechanism has been suggested as a causal cell death factor in Parkinson's disease. Tetrahydrobiopterin (BH4) is an essential regulatory cofactor for tyrosine hydroxylase and nitric oxide (NO) synthase in the synthesis of catecholamines and NO, which will not occur if BH4 is lacking. Catecholamine cells contain among the highest concentrations of BH4. The metabolism of BH4, catecholamines, and NO can generate damaging reactive oxygen species (ROS). Our preliminary data shows that apoptotic death of differentiated neuron-like PC12 cells during nerve growth factor withdrawal is directly proportional to the intracellular level of BH4. Thus, we hypothesize that the endogenous level of BH4 supports catecholamine cell functions under normal conditions and promotes apoptotic death when trophic support is withdrawn. Our working hypothesis and specific aim is: Intracellular BH4 mediates apoptosis and death of catecholamine cells deprived of trophic support. While the complex processes of apoptosis occur in many cell types, we will focus on catecholamine cells and the direct and indirect interactions of BH4 with pivotal mediators of apoptosis of these cells during insufficient trophic support. These mediators include oxidative stress, oncogenes that can initiate fatal reentry into the cell cycle, and critical cysteine proteases (caspases) mediating apoptosis. Our studies will identify the mechanism of BH4 involvement in apoptosis by testing for: 1) elevation of ROS and the source (BH4, catecholamines, or NO); 2) altered expression of the apoptotic-mediating oncogenes, pS3, c-myc, and the bc1-2 family (bax, bak, bc1-2, bcl-xl), and 3) activation of caspases 2,3, and 9. Apoptosis will be monitored by: a) number of living and dead cells; and b) fluorescent quantitation of cells at different stages of apoptosis. These studies may will reveal novel apoptotic regulatory mechanisms and provide new avenues for therapeutic approaches to promote neuronal survival and prevent debilitating neurological diseases, such as Parkinson's disease.