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

摘要 α-突触核蛋白(a-syn)聚集成不溶性纤维在糖尿病的发病机制中起关键作用。 路易体痴呆症(DLB)、帕金森氏病(PD)和其他突触核病。尽管 A-突触核蛋白作为路易小体包裹体的一种成分的记录超过25年， 在蛋白质聚集体与神经退行性变之间的因果联系机制方面存在着重大的知识空白。近期 遗传学研究表明，溶酶体降解系统与DLB和PD的发病机制有关。其中 最强的遗传风险因素是功能丧失突变溶酶体β-葡萄糖脑苷酶(GCase) 由GBA1编码，表明溶酶体功能受损可能在神经退行性变中起直接作用。 在我们之前的资助期间，我们发现a-syn聚集体是通过相互作用在溶酶体内启动的。 在失去GCase时积累的神经鞘糖脂底物。一旦形成，这些聚集体就会扰乱 蛋白平衡途径的多个基本分支，包括内质网(ER)中的折叠 和后急诊室贩卖在顺位高尔基。这进一步增强了a-syn聚合，创建了自传播的 致病循环。上一个资助期发现了新的机制和生物目标，以增强 水解酶的运输和溶酶体的功能。在这里，我们将在以前工作的基础上，研究如何- SYN聚集物扰乱蛋白质在内质网折叠、N-连接的糖基化在内质网中的作用及其下游效应 关于溶酶体的功能。我们将开发新的分子来恢复这些关键的蛋白平衡途径。我们的研究 将采用患者来源的PD iPSC-神经元培养、突触核素症小鼠模型和 人脑。我们先前发现，a-syn在帕金森病患者神经元中的积聚诱导了内质网的碎裂和 隐藏了细胞识别内质网中错误折叠的蛋白质的能力，导致未成熟蛋白质聚集 GCase.由于内质网中错误折叠的蛋白质通常通过未折叠蛋白反应(UPR)识别，在 目的1，我们将研究UPR、GCase溶解性和转运到溶酶体之间的联系。我们会 确定PD患者触发UPR是否能恢复溶酶体功能，减少a-syn。在目标2中，我们将测试 假设葡萄糖流量减少以及GCase和其他水解酶的蛋白质N-糖基化有助于 帕金森病患者溶酶体耗竭和功能障碍。在目标3中，我们将在先前研究的基础上再接再厉，这些研究表明 增强SNARE蛋白ykt6可以挽救溶酶体的功能。我们将测试新的小分子 体外和体内ykt6-溶酶体生物发生途径的激活物。通过研究基本的生物学机制 在蛋白质运输方面，我们有一个独特的机会将基本的细胞蛋白平衡途径与疾病联系起来 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 生物学靶点，并进一步开发治疗方法以增强溶酶体的生物发生，以恢复帕金森病的蛋白稳定 和DLB。

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.