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 alpha-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 alpha-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.
|Effective start/end date||9/30/20 → 6/30/25|
- National Institute of Neurological Disorders and Stroke (1R01NS117750-01)