MECHANISMS OF APOPTOTIC DEATH IN DOPAMINERGIC NEURONS

多巴胺能神经元细胞凋亡的机制

基本信息

批准号：
6032705
负责人：
ANNA-LIISA NIEMINEN
金额：
$ 20.32万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
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：
1999-12-25 至 2003-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6032705
关键词：
NMDA receptors PC12 cells Parkinson's disease apoptosis calcium flux dopamine free radical oxygen laboratory rat membrane permeability membrane potentials methylphenyltetrahydropyridine mitochondrial membrane substantia nigra tissue /cell culture transfection

项目摘要

Parkinson s disease (PD) is a neurodegenerative disorder that affects one million people in North America, which is likely caused by exposure to as yet unknown chemicals in the environment. The hallmark of PD is loss of dopaminergic neurons in the substantia nigra pars compacta. One chemical known to produce PD-like pathology in vivo is 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). My overall goal is to understand the role of the mitochondrial permeability transition in MPP+-induced dopaminergic cell death. In cultured rat pheochromocytoma-12 (PC12) dopaminergic cells, I will evaluate the following hypotheses: 1) MPP+ causes a mitochondrial permeability transition by promoting opening of permeability transition pores in the inner mitochondrial membranes. The mitochondrial permeability transition leads to increased ion permeability, collapse of the mitochondrial membrane potential and uncoupling of oxidative phosphorylation. These changes further cause large amplitude mitochondrial matrix swelling leading to rupture of the mitochondrial outer membrane, release of pro-apoptotic mitochondrial proteins from the intermembrane space, such as cytochrome c, and apoptotic cell death. According to this hypothesis, interventions that block the mitochondrial permeability transition prevent all these MPP+-induced events. To address this hypothesis directly, I will monitor mitochondrial membrane permeability, mitochondrial membrane potential, mitochondrial swelling and cytochrome c release by confocal microscopy of parameter-indicating fluorophores and transfected green fluoroscent protein fusion proteins in relation to onset of apoptotic death during exposure to MPP+ in single living cells in situ. I will also determine whether anti-apoptotic proteins, such as Bc1-2, prevent onset of the mitochondrial permeability transition and subsequent mitochondrial outer membrane breakage, cytochrome c release and apoptosis. 2) Excitotoxicity contributes to the MPP+-induced onset of the mitochondrial permeability transition by increasing mitochondrial Ca2+ and oxygen radical formation. To test this hypothesis, I will transfect PC12 cells with fully functional N-methyl-D-aspartate receptors. I expect that overstimulation of these receptors will increase mitochondrial free Ca2+, leading to Ca2+-dependent formation of reactive oxygen species and acceleration of the onset of the mitochondrial permeability transition, depolarization, cytochrome c release and apoptosis. The findings obtained with PC12 cells will be further translated to dopaminergic neurons isolated from the substantia nigra. These experiments will yield new information to improve therapy of Parkinson s disease and to prevent the disease s progression.

帕金森病 (PD) 是一种神经退行性疾病，影响着北美一百万人，这可能是由于接触环境中未知的化学物质引起的。 PD 的标志是黑质致密部多巴胺能神经元的丧失。一种已知在体内产生 PD 样病理的化学物质是 1-甲基-4-苯基-1,2,3,6-四氢吡啶 (MPTP)。我的总体目标是了解线粒体通透性转变在 MPP+ 诱导的多巴胺能细胞死亡中的作用。在培养的大鼠嗜铬细胞瘤-12 (PC12) 多巴胺能细胞中，我将评估以下假设：1) MPP+ 通过促进线粒体内膜中通透性转变孔的打开而引起线粒体通透性转变。线粒体通透性转变导致离子通透性增加、线粒体膜电位崩溃和氧化磷酸化解偶联。这些变化进一步导致线粒体基质大幅肿胀，导致线粒体外膜破裂，从膜间隙释放促凋亡线粒体蛋白（例如细胞色素c）和凋亡细胞死亡。根据这一假设，阻断线粒体通透性转变的干预措施可以防止所有这些 MPP+ 诱导的事件。为了直接解决这一假设，我将通过参数指示荧光团和转染的绿色荧光蛋白融合蛋白的共聚焦显微镜监测线粒体膜通透性、线粒体膜电位、线粒体肿胀和细胞色素 c 释放，这些参数与暴露于 MPP+ 期间细胞凋亡死亡的发生有关。原位单个活细胞。我还将确定抗凋亡蛋白（例如 Bc1-2）是否可以防止线粒体通透性转变以及随后的线粒体外膜破裂、细胞色素 c 释放和细胞凋亡的发生。 2) 兴奋性毒性通过增加线粒体 Ca2+ 和氧自由基的形成，促进 MPP+ 诱导的线粒体通透性转变。为了验证这一假设，我将用功能齐全的 N-甲基-D-天冬氨酸受体转染 PC12 细胞。我预计这些受体的过度刺激将增加线粒体游离 Ca2+，导致 Ca2+ 依赖性活性氧的形成，并加速线粒体通透性转变、去极化、细胞色素 c 释放和细胞凋亡的发生。用 PC12 细胞获得的研究结果将进一步转化为从黑质分离的多巴胺能神经元。这些实验将产生新的信息来改善帕金森病的治疗并防止疾病的进展。