MECHANISMS OF APOPTOTIC DEATH IN DOPAMINERGIC NEURONS

帕金森病（PD）是一种神经退行性疾病，影响北美100万人，可能是由于暴露于环境中未知的化学物质引起的。 PD的标志是黑质延髓部多巴胺能神经元的丢失。 已知在体内产生PD样病理的一种化学物质是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换在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（PC 12）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的渗透性转换孔的打开而引起线粒体渗透性转换。 线粒体渗透性转变导致离子渗透性增加、线粒体膜电位崩溃和氧化磷酸化解偶联。 这些变化进一步引起大幅度的线粒体基质肿胀，导致线粒体外膜破裂，促凋亡线粒体蛋白从膜间隙释放，如细胞色素c，和凋亡性细胞死亡。 根据这一假设，阻断线粒体通透性转换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释放的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在暴露于MPP+在单个活细胞原位凋亡的发生。 我还将确定是否抗凋亡蛋白，如Bc 1 -2，防止线粒体渗透性转换和随后的线粒体外膜断裂，细胞色素c释放和凋亡的发生。 2)兴奋性毒性通过增加线粒体Ca 2+和氧自由基的形成而有助于MPP+诱导的线粒体通透性转变的开始。 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PC 12细胞。 我预计，这些受体的过度刺激将增加线粒体游离Ca 2+，导致Ca 2+依赖性的活性氧的形成和线粒体渗透性转变，去极化，细胞色素c释放和细胞凋亡的开始加速。 用PC 12细胞获得的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，以改善帕金森病的治疗和预防疾病的进展。

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.