Neuronal roles of Parkinsons Disease Vps13C in regulating autophagy and calcium dynamics

随着人口的迅速老龄化，预计会出现帕金森氏病(PD)等神经退行性疾病 到2030年将上升到25%，这将给社会带来巨大的经济和情感挑战。而大多数帕金森氏症病例 是零星的，大约10%-15%是家族性的。导致早发性帕金森病的少数基因之一是最近的 发现了VPS13C(液泡蛋白分类13同源C)。Vps13C结果的常染色体隐性突变 在蛋白质截断和功能丧失方面，患者表现为α-突触核蛋白路易体病理 多巴胺能神经元和皮质神经元的聚集。虽然Vps13C最近被牵连到 非神经细胞内溶酶体途径，其神经元功能以及该功能的丧失如何导致帕金森病 患者的神经元仍有待阐明。通过基于质谱学的无偏筛选，我们 最近发现Vps13C是可溶性N-乙基马来酰亚胺敏感因子附件Ykt6的一种新的相互作用因子 蛋白受体(SNARE)蛋白在内溶酶体途径中起关键作用，并与病理生物学相关 α-突触核蛋白。有趣的是，我们进一步发现Ykt6和Vps13C的相互作用受 钙调神经磷酸酶的活性，钙调神经磷酸酶是钙信号的主要调节者，也是几种生物毒性的关键因素 PD模型。重要的是，我们证明了Ykt6中钙调神经磷酸酶依赖的磷酸化位点是一个关键的 调节步骤在自噬过程中介导自噬小体到溶酶体的融合，而Vps13C可以进一步 调节自噬。基于强有力的初步数据，这项提案的目标是调查 Vps13C通过与Ykt6相互作用调节神经元自噬(Aim 1)及其功能依赖 钙调神经磷酸酶活性(AIM 2)介导的帕金森病患者的钙动力学我们的中心假设是Vps13C的丢失 错误调节神经元自噬和钙动力学，导致帕金森病患者神经元的发病。这个 拟议中的研究将使用诱导多能干细胞(IPSC)来源的人类中脑来检验我们的假设 携带VPS13C截断突变患者的多巴胺(DA)神经元以及IPSC-人DA神经元 CRISPR/Cas9生成的VPS13C淘汰线。为了解决这个问题，我们将实施：1)微图案化 我们已经产生的底物，允许在延长的时间内培养单独分离的神经元 时间段，2)亲和纯化与质谱联用(AP-MS)分析Vps13C的相互作用组 在基础状态和应激状态下，3)Vps13C动力学和功能的神经元成像 西北大学尼康成像中心显微核心提供的最先进的成像技术 包括活细胞超分辨率显微镜，以及4)先进的细胞器特定钙离子和脂质传感器成像 帕金森病患者来源的DA神经元的技术，以进一步阐明Vps13C功能。这些研究将提供一个 从机制上理解Vps13C在患者神经元自噬和钙信号中的作用。此外， 拟议的研究具有重要意义，因为它为Vps13C在内溶酶体中的作用提供了新的见解 帕金森病的致病表型，目的是潜在地突出帕金森病的新治疗角度。

With a rapidly aging population, neurodegenerative diseases such as Parkinson’s Disease (PD), are expected to rise to 25% by 2030, presenting a huge economical and emotional challenge to society. While most PD cases are sporadic, approximately 10-15% are familial. One of the few genes leading to early-onset PD is the recently discovered VPS13C (Vacuolar Protein Sorting 13 Homolog C). Autosomal-recessive mutations in Vps13C result in protein-truncation and loss of function, with patients demonstrating Lewy body pathology with α-synuclein aggregation in both dopaminergic and cortical neurons. While Vps13C was recently implicated in the endolysosomal pathway in non-neuronal cells, its neuronal function and how loss of this function leads to PD in patient neurons still remains to be elucidated. Through an unbiased mass-spectrometry based screen, we recently identified Vps13C as a novel interactor of Ykt6, a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein critically involved in the endolysosomal pathway and linked to the pathobiology of α-synuclein. Interestingly, we further found that the Ykt6 and Vps13C interaction was regulated by the phosphatase activity of Calcineurin, a master regulator of Ca2+ signaling and a key player of toxicity in several PD models. Importantly, we demonstrated that the Calcineurin-dependent phosphorylation site in Ykt6 is a critical regulatory step mediating autophagosome to lysosome fusion during autophagy, and Vps13C can further regulate autophagy. Based on strong preliminary data, the goals of this proposal are to investigate the role for Vps13C in regulating neuronal autophagy via its interaction with Ykt6 (Aim 1) and its functional dependence on Ca2+ dynamics mediated by Calcineurin activity (Aim 2) in PD. Our central hypothesis is that loss of Vps13C misregulates neuronal autophagy and Ca2+ dynamics, contributing to PD pathogenesis in patient neurons. The proposed studies will test our hypotheses using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from patients carrying VPS13C truncation mutations, as well as iPSC-human DA neuron CRISPR/Cas9-generated VPS13C knockout lines. To address this, we will implement: 1) micropatterned substrates which we have generated which allow for the culture of individually separated neurons over extended periods of time, 2) affinity purification coupled to mass spectrometry (AP-MS) analysis of Vps13C’s interactome under both basal and stressed conditions, 3) neuronal imaging of Vps13C dynamics and function using state-of- the-art imaging techniques available at Northwestern University’s Nikon Imaging Center Microscopy Core including live cell super-resolution microscopy, and 4) advanced organelle-specific Ca2+ and lipid sensor imaging techniques in PD patient-derived DA neurons to further elucidate Vps13C function. These studies will provide a mechanistic understanding of Vps13C’s role in autophagy and Ca2+ signaling in patient neurons. Moreover, the proposed research is significant as it offers novel insights into the endolysosomal role of Vps13C in relation to PD pathogenic phenotypes with the goal of potentially highlighting new therapeutic angles for PD.