PROJECT SUMMARY Mitochondria are central to cellular metabolism, signaling, protein homeostasis, immunity, and apoptosis. One of the ways by which mitochondria respond effectively to changing mitochondrial and cellular needs is with calcium signaling. Ca2+ ions enter the mitochondrial matrix through a protein channel called the mitochondrial calcium uniporter (MCU) that resides on the inner membrane of the mitochondria. Altered MCU-mediated mitochondrial calcium signaling is involved in a variety of human neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and hereditary spastic paraplegia. Furthermore, these same diseases have been shown to exhibit the hallmarks of mitochondrial protein stress, including elevated protein levels of mitochondrial chaperones. Mitochondrial protein stress can be caused by misfolded or unfolded proteins accumulating within the mitochondria. Preliminary studies in the Sancak Lab suggest that MCU-mediated calcium signaling plays a critical role in facilitating the mitochondrial protein stress response. Data showing increased MCU mRNA and protein during mitochondrial protein stress suggests that MCU is upregulated as part of a mitochondrial protein stress response. Even more intriguingly, relative to WT and MCU rescue cell lines, MCU knockout cells have lower baseline levels of mitochondrial chaperones and ATF4, a transcription factor central to cellular stress responses. Furthermore, when mitochondrial protein stress is induced, the mRNA levels of ATF4 and these chaperones increase much less than in WT or rescue cells. The central hypothesis of this proposal is that altered MCU regulation and calcium signaling comprise a heretofore uncharacterized pathway that regulates transcription in response to mitochondrial protein stress. Aim 1 will offer new insight about the molecular mechanisms regulating the transcription, translation and protein stability of MCU. The functional studies of Aim 2 will reveal how mitochondrial protein stress alters calcium signaling and transcription. Successful completion of this proposal will advance the field by identifying novel players in the poorly understood mitochondria-to-nuclear signaling pathway. These findings will also uncover a new function for mitochondrial calcium signaling in the regulation of a transcriptional response central to the mitochondrial protein stress response, and will reveal potential therapeutic targets for the treatment of neurodegenerative disease. Completion of Aim 1 will provide valuable training in pairing gene editing with pharmacological tools to dissect the function and regulation of MCU, a protein with great disease relevance. Completion of Aim 2 will provide training in using laser-scanning confocal microscopy and live imaging techniques to study mitochondrial stress. The University of Washington is very well funded and well equipped. Its top tier faculty, staff, and technicians, and variety of state-of-the-art facilities will ensure the success of this proposed work.

PROJECT NARRATIVE Mitochondrial protein homeostasis (proteostasis) is essential for health and its loss is implicated in neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and hereditary spastic paraplegia. Mitochondrial protein stress initiates signaling between the mitochondria and the nucleus, which triggers a response that works to restore proteostasis. By investigating the role of mitochondrial calcium uniporter-mediated calcium signaling in this signaling cascade, this proposal has the potential to identify new molecular players and therapeutic targets for treatment of diseases featuring mitochondrial protein stress.