Altered lysosomal degradation in PD

我们认为，参与帕金森氏病(PD)的一些蛋白质在 自噬/溶酶体途径，导致正常的胞质多巴胺稳态被破坏，细胞内氧自由基应激，以及黑质(SN)神经元选择性变性。中心建议是：1)需要氧化的多巴胺的反应是黑质神经元死亡的选择性基础，2)α-突触核蛋白的异常自噬降解使胞浆中的多巴胺失去调节。为了检验这些假设，我们将使用一系列生化、遗传学和生理学方法。首先，使用来自酪氨酸羟化酶(TH)基因敲除、α-突触核蛋白基因敲除的SN培养物，以及它们的兄弟野生型动物，我们将证实多巴胺的存在是否是选择性多巴胺能神经元死亡所必需的。其次，由于大多数长寿命的胞浆蛋白都是通过溶酶体(又名：自噬)途径，我们将确定蛋白的半衰期和蛋白分解途径，以及与家族性帕金森病有关的蛋白，包括α-突触核蛋白，是否在溶酶体中正常降解。第三，由于足够水平的两亲性蛋白可以损伤细胞内膜，我们将检测这些蛋白对膜完整性的影响，并采用方法重建AGs之间以及AGs与溶酶体之间的融合。我们还将使用实时生命显微镜来记录与培养神经元中PD突变蛋白细胞内水平变化相关的融合事件的变化。最后，当我们提出α-突触核蛋白突变、异常蛋白降解和最终细胞质多巴胺致病增加之间的相互作用时，可能是其基础。 PD我们将使用一种新的方法，细胞内斑片电化学(IPE)，在一系列条件下直接测定胞浆儿茶酚胺。 总而言之，这一系列实验将测试改变的溶酶体降解α-突触核蛋白的可能性 随之而来的细胞质多巴胺水平的变化提供了AT的最初上游原因 至少是某种形式的帕金森病。

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.