The role of a-synuclein accumulation in lysosomal hydrolase trafficking and function

α-突触核蛋白积累在溶酶体水解酶运输和功能中的作用

• 批准号: 10539942
• 负责人: Joseph R Mazzulli
• 金额: $ 61.85万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10539942
• 关键词: Biogenesis; Biological; Biology; Brain; Cell Death; Cell physiology; Cells; Clinic; Data; Dementia with Lewy Bodies; Disease; Documentation; Endoplasmic Reticulum; Enhancers; Failure; Foundations; Functional disorder; Funding; Genetic study; Glucose; Glycolipids; Glycosphingolipids; Golgi Apparatus; Hexosamines; Human; Hydrolase; In Vitro; Inclusion Bodies; Induced pluripotent stem cell derived neurons; Knowledge; Lead; Lewy Bodies; Link; Lysosomes; Membrane Fusion; Methods; Modeling; Mutation; Nerve Degeneration; Nervous system structure; Neurons; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Phenotype; Play; Process; Proteins; Quality Control; Role; SNAP receptor; Solubility; Structure; System; Testing; Toxic effect; Transducers; Work; age related; alpha synuclein; base; clinical development; efficacy testing; genetic risk factor; genetic variant; glucosylceramidase; glycosylation; improved; in vivo; induced pluripotent stem cell; link protein; loss of function; loss of function mutation; misfolded protein; mouse model; neurotoxicity; new therapeutic target; novel; novel therapeutic intervention; prevent; protein aggregation; protein folding; protein transport; proteostasis; response; sensor; small molecule; sound; synucleinopathy; therapy development; trafficking

Abstract The aggregation of a-Synuclein (a-syn) into insoluble fibrils plays a key role in the pathogenesis of Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and other synucleinopathies. Despite the documentation of a-synuclein as a component of Lewy body inclusions for over 25 years, there remains a significant knowledge gap in the mechanisms that causally link protein aggregates to neurodegeneration. Recent genetic studies have implicated the lysosomal degradation system into the pathogenesis of DLB and PD. Among the strongest genetic risk factors are loss-of-function mutations lysosomal β-glucocerebrosidase (GCase) encoded by GBA1, indicating that compromised lysosomal function may play a direct role in neurodegeneration. During our previous funding period, we found that a-syn aggregates are initiated inside lysosomes by interacting with glycosphingolipid substrates that accumulate upon loss of GCase. Once formed, these aggregates perturb multiple, essential branches of the proteostasis pathway, including the folding in the endoplasmic reticulum (ER) and post-ER trafficking at the cis-Golgi. This further augments a-syn aggregation, creating a self-propagating pathogenic cycle. The previous funding period uncovered novel mechanisms and biological targets that enhance the trafficking of hydrolases and lysosomal function. Here, we will build on our previous work to examine how a- syn aggregates perturb protein folding in the ER, N-linked glycosylation in the ER, and the downstream effect on lysosomal function. We will develop novel molecules to restore these key proteostasis pathways. Our studies will employ a combination of patient-derived PD iPSC-neuron cultures, synucleinopathy mouse models, and human brain. We previously found that a-syn accumulation in PD patient neurons induced ER fragmentation and concealed the cell’s ability to recognize misfolded proteins in the ER, resulting in aggregation of immature GCase. Since misfolded proteins in the ER are usually recognized by the unfolded protein response (UPR), in aim 1, we will examine the link between the UPR, GCase solubility, and trafficking to the lysosome. We will determine if triggering the UPR in PD can restore lysosomal function and reduce a-syn. In aim 2, we will test the hypothesis that reduced glucose flux and protein N-glycosylation of GCase and other hydrolases contributes to lysosomal depletion and dysfunction in PD. In aim 3, we will build upon our prior studies, which showed that lysosomal function can be rescued by enhancing the SNARE protein ykt6. We will test novel small molecule activators of the ykt6-lysosomal biogenesis pathway in vitro and in vivo. By studying basic biology mechanisms of protein trafficking, we have a unique opportunity to link essential cellular proteostasis pathways to disease pathogenesis. Our studies may impact the field by discovering novel pathogenic mechanisms, identifying new biological targets, and further develop therapies to enhance lysosomal biogenesis to restore proteostasis in PD and DLB.

摘要 α-突触核蛋白（a-syn）聚集成不溶性纤维在脑梗死的发病机制中起关键作用。 路易体痴呆（DLB）、帕金森病（PD）和其他突触核蛋白病。尽管 虽然α-突触核蛋白作为路易体内含物的组分的文献超过25年，但仍然存在α-突触核蛋白， 在将蛋白质聚集体与神经变性因果联系起来的机制方面存在重大知识缺口。最近 遗传学研究表明溶酶体降解系统参与DLB和PD的发病机制。之间 最强的遗传风险因素是溶酶体β-葡萄糖脑苷脂酶（GCase）功能丧失突变 由GBA 1编码，表明受损的溶酶体功能可能在神经变性中起直接作用。 在我们之前的资助期间，我们发现a-syn聚集体通过相互作用在溶酶体内启动， 与GCase丧失后积累的鞘糖脂底物。一旦形成，这些聚集体就会扰乱 蛋白质稳态途径的多个重要分支，包括内质网（ER）中的折叠 以及ER后顺式高尔基体的运输。这进一步增强了a-syn聚合，创建了一个自传播 致病循环上一个资助期发现了新的机制和生物靶点， 水解酶的运输和溶酶体功能。在这里，我们将建立在我们以前的工作，以研究如何- syn聚集体干扰ER中的蛋白质折叠、ER中的N-连接糖基化以及下游效应 对溶酶体功能的影响我们将开发新的分子来恢复这些关键的蛋白酶抑制途径。我们的研究 将采用患者来源的PD iPSC神经元培养物、突触核蛋白病小鼠模型和 人脑我们先前发现PD患者神经元中的α-syn积累诱导ER片段化， 隐藏了细胞识别内质网中错误折叠蛋白的能力，导致未成熟的 GCase。由于ER中的错误折叠蛋白通常由未折叠蛋白反应（UPR）识别， 目的1，我们将检查UPR，GCase溶解度和运输到溶酶体之间的联系。我们将 确定触发PD中的UPR是否可以恢复溶酶体功能并减少α-syn。在目标2中，我们将测试 假设GCase和其它水解酶的葡萄糖通量和蛋白质N-糖基化的减少有助于 溶酶体耗竭和功能障碍。在目标3中，我们将建立在我们先前的研究基础上，这些研究表明， 可以通过增强SNARE蛋白ykt 6来挽救溶酶体功能。我们将测试新的小分子 体外和体内YKT 6-溶酶体生物合成途径的激活剂。通过研究基本的生物学机制 蛋白质运输，我们有一个独特的机会，连接必要的细胞蛋白质代谢途径的疾病 发病机制我们的研究可能会通过发现新的致病机制，识别新的 生物靶点，并进一步开发增强溶酶体生物合成的疗法，以恢复PD中的蛋白质稳态 的DLB。