MECHANISMS OF APOPTOTIC DEATH IN DOPAMINERGIC NEURONS

帕金森S病(PD)是一种影响北美100万人的神经退行性疾病，很可能是由于接触到环境中未知的化学物质而引起的。帕金森病的特点是黑质致密部多巴胺能神经元的丢失。已知的一种化学物质是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通透性转换孔的开放而导致线粒体通透性转换。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，氧化磷酸化解偶联。这些变化进一步导致线粒体基质的大量膨胀，导致线粒体外膜破裂，细胞色素c等促凋亡线粒体蛋白从膜间隙释放，并导致细胞凋亡。根据这一假设，阻止线粒体通透性转变的干预措施可以阻止所有这些MPP+诱导的事件。为了直接解决这一假说，我将通过指示参数的荧光团和转染绿色荧光蛋白融合蛋白的共聚焦显微镜来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的释放与单个活细胞在MPP+暴露过程中发生凋亡死亡的关系。我还将确定抗凋亡蛋白，如Bc1-2，是否阻止线粒体通透性转变的开始以及随后的线粒体外膜断裂、细胞色素c释放和细胞凋亡。2)兴奋性毒性通过增加线粒体Ca~(2+)和氧自由基的形成而参与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会增加线粒体的游离钙离子，导致钙依赖的活性氧物种的形成，并加速线粒体通透性转变、去极化、细胞色素c释放和细胞凋亡的开始。在PC12细胞上获得的发现将进一步转化为从黑质分离的多巴胺能神经元。这些实验将为改进帕金森S病的治疗和防止疾病S的进展提供新的信息。

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.