Altered lysosomal degradation in PD

PD 中溶酶体降解的改变

• 批准号: 6812924
• 负责人: David Sulzer
• 金额: $ 32.94万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6812924
• 关键词: Parkinson' s disease; alpha synuclein; autophagy; cell death; dopamine; electrochemistry; gene mutation; genetically modified animals; intracellular transport; intravital microscopy; laboratory mouse; lysosomes; neural degeneration; pathologic process; protein degradation; protein transport; substantia nigra; Parkinson' s disease; alpha synuclein; autophagy; cell death; dopamine; electrochemistry; gene mutation; genetically modified animals; intracellular transport; intravital microscopy; laboratory mouse; lysosomes; neural degeneration; pathologic process; protein degradation; protein transport; substantia nigra

We propose that some proteins involved in Parkinson's Disease (PD) are poorly degraded by autophagic/lysosomal pathways, resulting in disruption of normal cytosolic dopamine homeostasis, intracellular oxyradical stress, and selective degeneration of substantia nigra (SN) neurons. The central proposals are 1) reactions requiring oxidized dopamine underlie the selectivity of neuronal death in the SN, 2) aberrant autophagic degradation of alpha-synuclein dysregulates cytosolic dopamine. To test these hypotheses, we will use a range of biochemical, genetic, and physiological approaches. First, using SN cultures derived from tyrosine hydroxylase (TH) knockouts, alpha-synuclein knockouts, and their sibling wild-type animals, we will confirm if the presence of dopamine is required for selective dopaminergic neuronal death. Second, as most long-lived cytosolic proteins are degraded through lysosomal (a.k.a. autophagic) pathways, we will determine the half-lives and proteolytic pathways followed by proteins implicated in familiar PD and determine whether proteins implicated in familial PD, including alpha-synuclein are normally degraded in lysosomes. Third, as amphiphilic proteins at sufficient levels can damage intracellular membranes, we will assay effects of these proteins on membrane integrity and adapt methods to reconstruct fusion between AGs and between AGs and lysosomes. We will also use real time vital microscopy to document changes in fusion events related to changes in intracellular levels of PD mutant proteins in cultured neurons. Finally, as we propose an interaction between alpha-synuclein mutations, aberrant protein degradation, and ultimately, that a pathogenic increase in cytosolic dopamine may underlie PD we will use a new approach, intracellular patch electrochemistry (IPE), to directly measure cytosolic catecholamines under a range of conditions. In summary, this series of experiments will test the possibility that altered lysosomal degradation of alpha-synuclein and consequent alterations in cytosolic dopamine levels provides an initial, upstream cause of at least some forms of PD.

我们建议一些参与帕金森氏病（PD）的蛋白质因 自噬/溶酶体途径，导致正常的胞质多巴胺稳态，细胞内用氧应激和质原质（SN）神经元的选择性退化。中心提案是1）需要氧化多巴胺的反应是SN中神经元死亡的选择性，2）α-突触核蛋白的异常自噬降解，可鼻核蛋白的异常中调胞质多巴胺。为了检验这些假设，我们将使用一系列生化，遗传和生理方法。首先，使用源自酪氨酸羟化酶（TH）敲除，α-突触核蛋白敲除及其兄弟式野生型动物的SN培养物，我们将确认是否需要多巴胺来进行选择性多巴胺能神经元死亡。其次，由于大多数长寿命的胞质蛋白通过溶酶体（又称自噬）途径降解，因此我们将确定与熟悉的PD有关的蛋白质的半衰期和蛋白水解途径，并确定蛋白质与家族性PD有关，包括α-突触蛋白（包括α-突触蛋白）是否含有α-突触蛋白是lysossomess in lysossomess中的。第三，由于具有足够水平的两亲性蛋白会损害细胞内膜，因此我们将测定这些蛋白质对膜完整性的影响，并适应方法在AGS和AGS和溶酶体之间重建融合。我们还将使用实时重要显微镜来记录与培养神经元中PD突变蛋白的变化有关的融合事件的变化。最后，由于我们提出了α-突触核蛋白突变，异常蛋白质降解和最终之间的相互作用，最终，胞质多巴胺的致病性增加可能是基础 PD我们将使用一种新方法，细胞内斑块电化学（IPE），直接在一系列条件下直接测量胞质儿茶酚胺。 总而言之，这一系列实验将测试改变α-突触核蛋白溶酶体降解的可能性 随之而来的胞质多巴胺水平的改变为AT提供了初始的上游原因 至少有一些形式的PD。