Analysis of PINK1-Parkin-dependent signalling in neurons

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Over the last 30 years, mitochondrial dysfunction has emerged as a common feature of age-related neurodegenerative disease like Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) or amyotrophic lateral sclerosis (ALS). Parkinson’s disease is the second most common neurodegenerative disorder resulting from loss of dopaminergic neurons of the substantia nigra, in part due to mitochondrial dysfunction. In recent decades, the identification of numerous genes linked to PD have highlighted the importance of mitochondrial quality control in the etiology of the disease. The mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin, found mutated in familial early onset recessive forms of PD, play central roles in mitochondrial homeostasis, via a signaling cascade in which depolarization-induced PINK1 stabilization and activation on the mitochondrial surface promotes recruitment of Parkin, which in turn, ubiquitylates proteins on the mitochondrial outer membrane (MOM) and then initiates a downstream pathway that eventually leads to mitophagy, a mitochondria-specific type of autophagy. How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. We have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation–dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.
Date of Award2022
Original languageEnglish
SupervisorMiratul Muqit (Supervisor) & Dario Alessi (Supervisor)

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