Mechanisms of Pharmacological and Cellular Regulators of Mitophagy

Citation

Rosencrans, William Max (2025) Mechanisms of Pharmacological and Cellular Regulators of Mitophagy. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/zvm6-0682. https://resolver.caltech.edu/CaltechTHESIS:06022025-150650052

Abstract

Autophagy is a highly conserved cellular process that isolates and degrades damaged or unnecessary intracellular structures. Mitochondria, well known as metabolic centers, provide ATP and critical metabolites essential for life. Unchecked mitochondrial damage impairs metabolism, releases immunogenic mitochondrial DNA, and triggers apoptosis. Mitophagy, the selective removal of mitochondria via autophagy, is vital to preventing these harmful outcomes. The PINK1/Parkin pathway detects damaged mitochondria and targets them for mitophagy. Dysfunction in this pathway underlies certain subset Parkinson’s Disease (PD) cases. Efforts to understand the mechanistic basis of this pathway and its regulators provides a pathway to development of potentially disease modifying therapeutics for PD. In this thesis, I characterize the mechanism of action of a series of clinical stage mitophagy activating drugs. We find that these compounds reduce the threshold for which mitochondrial stress initiates mitophagy. However, contrary to reported literature, I demonstrate that these compounds do not directly activate PINK1 or Parkin. Rather, they act as weak mitochondrial toxins sensitizing cells to mitochondrial insult. I reveal that this phenomenon is characteristic of any weak mitochondrial toxin, revealing a potent pitfall for current drug discovery campaigns. Next, I detail a novel endogenous regulator of PINK1/Parkin mitophagy, the immune-related protein TNIP1. We show through a series of cell, biochemistry, and biophysical assays, that TNIP1 competes for autophagy machinery to slow down mitophagy. These data center TNIP1 as a important regulator of autophagic processes, and a unique negative regulator of mitophagy. Finally, I describe a biophysical analysis of the mitochondrial ion channel, VDAC2, critical in apoptosis and PINK1/Parkin pathways. Using a series of single-molecule approaches, I reveal how its structural plasticity regulates its interactions with protein partners. These finding provide a mechanistic basis for understanding its role in disparate cellular processes. I also explore the biological impact of KO of each VDAC isoform on mitochondrial and cell function. Unexpectedly, we find that the lowest expressed isoform, VDAC3, has an outsized impact on mitochondrial function.

Thesis Files