PROJECT SUMMARY Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3) are inherited aging-associated diseases that have a devastating impact on patients and family caretakers relative to their prevalence in the general population. These conditions belong to a family of polyglutamine (polyQ) expansion diseases caused by mutations resulting in the pathological expansion of trinucleotide (CAGn) repeats in distinct genes. Increases in CAG repeat length give rise to proteins containing aberrantly expanded polyQ tracts, which interfere with normal protein function and promote misfolding. Toxicity in these diseases is thought to arise in part from the formation of pathological inclusion bodies comprised of aberrantly conformed mutant proteins, a hallmark observed in numerous aging-associated neurodegenerative diseases. Despite extensive efforts to decipher the mechanisms underlying toxicity in polyQ diseases, however, little progress has been made towards identifying targets for therapeutic intervention. Recently, the post-translational modification (PTM), AMPylation, has emerged as a novel regulator of HSP70 family chaperones, crucial components of the cell’s protein quality control machinery that buffer against protein misfolding stress. Protein AMPylation is carried out by the fic-type AMPylase, FICD in humans, and its ortholog FIC-1, in C. elegans, respectively. Work in our lab has established that FIC-1-mediated AMPylation directly alters polyQ aggregation dynamics and toxicity. Further, my preliminary data as presented in this proposal identifies fic-1 deficiency as sufficient to rescue survival of C. elegans expressing aggregation-prone polyQs during development in a polyQ length-dependent manner. Taken together, these findings suggest that the loss of FIC- 1/FICD-mediated AMPylation bolsters proteostasis network capacity to alleviate toxicity induced by polyQ protein aggregation. This project will utilize cross-disciplinary approaches to generate a holistic characterization of FICD/FIC-1- mediated AMPylation in polyQ diseases. To this end, I will harness the powerful genetics of C. elegans to uncover novel pathway(s) that promote survival in the face of pathogenic polyQ aggregation (Aim 1). In tandem, I will employ functional assays in neurons derived from HD and SCA3 patient stem cells to profile how FICD activity affects polyQ aggregation and toxicity in these disease models (Aim 2). The results of these studies will advance our knowledge of how AMPylation regulates proteostasis in polyQ diseases. The ultimate goal of my research is to capitalize on these findings to develop translatable therapeutic approaches for aging-associated diseases.

PROJECT NARRATIVE Polyglutamine (polyQ) expansion diseases are a family of inherited aging-associated diseases characterized by a failure of the cell’s protein quality control machinery and a subsequent accumulation of misfolded protein aggregates. Recent work has highlighted a potential beneficial role for the novel post-translational modification AMPylation, in regulating the aggregation and toxicity of polyQ disease proteins, but the mechanisms underlying this process remains unknown. This proposal will utilize complementary approaches to (1) uncover how AMPylation deficiency facilitates suppression of polyQ toxicity in C. elegans, and (2) determine how AMPylation levels impact polyQ dynamics in physiologically relevant in vitro models of Huntington’s disease and SCA3.