ORCID

http://orcid.org/0000-0002-6860-8739

Date of Award

Spring 5-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Protein inclusions such as β-amyloid, tau, α-synuclein, and TDP-43 are considered the pathologic hallmarks of many neurodegenerative diseases. These proteins are prone to misfold, aggregate, and template new aggregates. Accumulating evidence suggests that those proteins in their high-molecular-weight forms can serve as a "seed", spread through an interconnected brain network, and induce new inclusions. Therefore, it is essential to understand the mechanism of proteopathic seeding. In this dissertation, we performed a whole genomic CRISPR-Cas9 KO screening to identify gene modifiers of αS seeding. Within the screening, we found several hits of endolysosomal function and trafficking, including VCP. VCP is a versatile protein required for protein homeostasis. Mutations in VCP are associated with type 1 Multisystem proteopathy (MSP1), whose patients can develop multiple neurodegenerative diseases with proteopathic protein inclusions, including Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Parkinson and Alzheimer's. We validated that either VCP dysfunction (inhibition or RNAi knockdown) or VCP disease mutations can exacerbate αS seeding in both αS biosensor cells and hippocampal neurons, in which both VCP knockdown and VCP mutation knock-in neurons show increasing αS aggregation following seeding. Additionally, mice carrying a VCP disease mutation exhibit more αS aggregates than C57 control 90 days after intrastriatal injection of αS pre-formed fibrils in vivo. We showed that VCP aggravates endolysosomal membrane damage with the proteopathic seeds. Similarly, seeding activities increased when we co-treated the cells with seeds and LLOMe, a drug specifically permeabilizes the endolysosome. Previously, in our lab, we demonstrated VCP involved in the autophagy-mediated clearance of damaged endolysosome, called lysophagy. Screening different VCP cofactors, we identified that only lysophagy-related cofactor UBXD1 modifies the α-synuclein seeding the same as VCP. We proposed that VCP might regulate αS seeding via UBXD1-dependent lysophagy. This dissertation also developed two novel TDP-43 seeding assays, which phenocopies hyper-phosphorylated TDP-43 aggregations in patients. We observed the same protective effect of VCP on intracellular TDP-43 seeding and tau. Overall, our results support VCP as a gatekeeper for different intracellular proteopathic seeding in a merging pathway.

Language

English (en)

Chair and Committee

Conrad C. Weihl

Committee Members

Yuna M. Ayala

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