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

随着人口的迅速老龄化，预计神经退行性疾病如帕金森病（PD） 到2030年将上升到25%，对社会提出了巨大的经济和情感挑战。虽然大多数PD病例 是散发性的，大约10-15%是家族性的。导致早发性PD的少数几个基因之一是最近 VPS 13 C（蛋白质分选13同源物C）。Vps 13 C中的常染色体隐性突变结果 在蛋白质截短和功能丧失中，患者表现出α-突触核蛋白的路易体病理学 在多巴胺能和皮质神经元中的聚集。虽然Vps 13 C最近被卷入了 非神经元细胞中的内溶酶体途径，其神经元功能以及这种功能的丧失如何导致PD， 患者神经元仍有待阐明。通过基于无偏质谱的筛选，我们 最近发现Vps 13 C是Ykt 6的一种新的相互作用因子，Ykt 6是一种可溶性N-乙基马来酰亚胺敏感因子附着物 蛋白质受体（SNARE）蛋白质，主要参与内溶酶体途径并与病理生物学相关 α-synuclein。有趣的是，我们进一步发现Ykt 6和Vps 13 C的相互作用是由 钙调神经磷酸酶的活性，钙离子信号的主要调节和毒性的几个关键球员， PD模型。重要的是，我们证明了Ykt 6中钙调神经磷酸酶依赖的磷酸化位点是一个关键的磷酸化位点。 自噬过程中介导自噬体与溶酶体融合的调节步骤，Vps 13 C可以进一步 调节自噬。根据强有力的初步数据，本提案的目标是调查 Vps 13 C通过与Ykt 6（Aim 1）的相互作用调节神经元自噬及其功能依赖 钙调磷酸酶活性介导的钙动力学（目的2）在PD。我们的中心假设是Vps 13 C的缺失 神经元自噬和Ca 2+动力学失调，导致患者神经元PD发病。的 拟进行的研究将使用诱导多能干细胞（iPSC）衍生的人类中脑来验证我们的假设 来自携带VPS 13 C截短突变的患者的多巴胺（DA）神经元，以及iPSC-人DA神经元 CRISPR/Cas9产生的VPS 13 C敲除系。为了解决这个问题，我们将实现：1）微图案化 基质，我们已经产生，允许单独分离的神经元培养超过延长 2）Vps 13 C相互作用物组的亲和纯化偶联质谱（AP-MS）分析 在基础和应激条件下，3）使用神经元状态的Vps 13 C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）先进的细胞器特异性Ca 2+和脂质传感器成像 技术在PD患者衍生的DA神经元中进一步阐明Vps 13 C功能。这些研究将提供一个 Vps 13 C在患者神经元中的自噬和Ca 2+信号传导中的作用的机制理解。而且 拟议的研究是重要的，因为它提供了新的见解，内溶酶体的作用，Vps 13 C的关系， PD致病表型，目的是潜在地突出PD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.