Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Interdepartmental Neuroscience Program

First Advisor

De Camilli, Pietro

Abstract

Biallelic loss-of-function mutations in VPS13C cause early-onset Parkinson’s Disease (PD), and the VPS13C locus is a GWAS hit for sporadic PD risk. VPS13C is a member of the VPS13 family, which in humans contains three other proteins, VPS13A, VPS13B, and VPS13D, all with ties to neurological diseases. Mutations in VPS13A cause chorea-acanthocytosis a Huntington’s like syndrome with dysmorphic erythrocytes, mutations in VPS13B cause the neurodevelopmental disorder Cohen syndrome, and mutations in VPS13D cause spastic ataxia with varying presentation. The molecular function of these proteins, why their loss cause neurological diseases, and why they are each associated with distinct diseases despite their homology have all been open questions. In yeast, the single Vps13 protein localizes to contact sites between the mitochondria and vacuole, the yeast lysosome, and at the nuclear-vacuolar junction (NVJ), where multiple lines of indirect evidence has hinted that it may play a role in lipid transfer between these organelles. To understand whether human VPS13 proteins have diverged in their subcellular localization, we employed a combination of light and electron microscopy to demonstrate that VPS13A localizes to contact sites between the endoplasmic reticulum (ER) and mitochondria, while VPS13C localizes to contact sites between the ER and late endosomes/lysosomes. Both proteins also share a localization at ER-lipid droplet contact sites. We further show that the N-terminal portion of VPS13 forms a novel, tubular, hydrophobic cavity that can solubilize and transport glycerolipids between membranes. These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggested lipid transport roles for these proteins as well, which has since been demonstrated. We next investigated the cellular phenotypes of VPS13C loss-of-function in an attempt to shed light on the pathophysiology of VPS13C-associated PD. We used CRISPR-Cas9 to generate VPS13C-knockout (VPS13CKO) HeLa cells. These cells have more lysosomes compared to WT, with accumulation of both membrane and luminal lysosomal proteins. These lysosomes have an altered lipid profile, including a substantial decrease in ether-linked phospholipids and an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS/STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation and place VPS13C in pathways relevant to PD pathogenesis. Further exploration of these pathways has the potential to yield new mechanistic understanding and novel therapeutic strategies for this debilitating illness.

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