BIOPTERIN, CATECHOLAMINES, & NO IN REGULATING APOPTOSIS

生物蝶呤、儿茶酚胺、

• 批准号: 6407212
• 负责人: ROBERT A LEVINE
• 金额: $ 3.64万
• 依托单位: HENRY FORD HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-28 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6407212
• 关键词: PC12 cells; Parkinson's disease; apoptosis; catecholamines; cysteine endopeptidases; free radical oxygen; gene expression; laboratory rat; neural degeneration; neurons; nitric oxide; oncogenes; substantia nigra; tetrahydrobiopterin; tissue /cell culture

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BH4 supports catecholamine cell functions under normal conditions and promotes apoptotic death when trophic support is withdrawn.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.

描述：（逐字来自申请人的摘要） 帕金森病患者黑质过早死亡， 是在死后发现的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， 压力或不适当的重新进入细胞周期;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缺口， 培养的儿茶酚胺细胞，在那里条件可以更严格地控制， 比在动物身上。待研究的细胞包括嗜铬细胞瘤（PC 12）， 交感神经元和胎儿黑质神经元。这些模型在体内 相关性，因为在发育过程中黑质多巴胺神经元的凋亡性死亡 可能是由于纹状体靶细胞的营养支持不足;这 在帕金森氏症中，这种机制被认为是导致细胞死亡的因素。 疾病四氢生物蝶呤（BH 4）是一种重要的调节辅因子， 酪氨酸羟化酶和一氧化氮（NO）合酶在合成 儿茶酚胺和NO，如果缺乏BH 4，则不会发生。儿茶酚胺 细胞中含有最高浓度的BH 4。BH 4的代谢， 儿茶酚胺和NO可以产生破坏性的活性氧（ROS）。我们 初步数据显示分化的神经元样PC 12的凋亡性死亡 在神经生长因子撤退期间的细胞是成正比的， 细胞内BH 4水平。因此，我们假设， BH 4在正常条件下支持儿茶酚胺细胞功能， 当营养支持被撤销时细胞凋亡。我们的工作假设和 具体目的是：细胞内BH 4介导细胞凋亡和死亡 缺乏营养支持的儿茶酚胺细胞。虽然复杂的过程， 凋亡发生在许多细胞类型中，我们将重点关注儿茶酚胺细胞， BH 4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，这些细胞的生长。 这些介质包括氧化应激，致癌基因，可以启动致命的 重新进入细胞周期，和关键的半胱氨酸蛋白酶（半胱天冬酶） 介导细胞凋亡。我们的研究将确定BH 4参与的机制 通过测试：1）ROS的升高和源（BH 4， 儿茶酚胺，或NO）; 2）改变表达的促肾上腺皮质激素介导的 癌基因，pS3，c-myc，和bc 1 -2家族（bax，巴克，bc 1 -2，bcl-xl），和3） 半胱天冬酶2、3和9的活化。将通过以下方式监测细胞凋亡： 活细胞和死细胞;和B）在不同温度下对细胞进行荧光定量 凋亡的阶段。 这些研究可能会揭示新的凋亡调控机制， 治疗方法的新途径，以促进神经元存活和预防 使人衰弱的神经系统疾病，如帕金森病。