Determining the contribution of acute viral infection on the onset and progression of ALS
Project Overview
Almost all amyotrophic lateral sclerosis (ALS) is accompanied by neuronal cytoplasmic aggregates of TAR DNA-binding protein-43 (TDP-43). TDP-43 is an RNA-binding protein that regulates RNA expression and function, including mRNA translation. The mechanisms by which TDP-43 pathology contributes to ALS remain incompletely understood, hampering the development of a rational therapy for this fatal disorder. Previous studies have shown that cytoplasmic TDP-43 decreases global protein translation, which may be a universal ALS disease mechanism. Work in Cashman lab using a cellular model of ALS has shown that knocking down a ribosomal protein, Receptor of Activated C-Kinase 1 (RACK1), disaggregates cytoplasmic TDP-43 inclusions, partially re-localizes TDP-43 back to the nucleus and alleviates suppressed protein translation. These results suggest a novel pathogenic mechanism underlying TDP-43-associated neurodegeneration through RACK1-mediated translational suppression. We hypothesize that increased ribosomal pausing is due to RACK1-dependent TDP-43 aggregates on ribosomes, leading to suppression of translation in ALS. In this study, we aim to elucidate the molecular mechanism by which RACK1 knockdown alleviates TDP-43 toxicity, with a focus on protein translation, using Drosophila melanogaster. Our first aim is to assess whether modulation of ribosomal pausing is the mechanism by which RACK1 knockdown alleviates TDP-43-induced translational suppression. This will be accomplished using ribosomal profiling on Drosophila mature oocytes. Next, we will test whether RACK1 on the ribosome is crucial for TDP-43 to decrease translation. This will be done by knocking down fly RACK1 and expressing human RACK1 DE (an engineered mutant that does not bind to ribosomes) with and without human TDP-43. Finally, we will validate that RACK1 knockdown in fly retina rescues neurodegeneration elicited by TDP-43 proteinopathy. To test this idea, we will create RACK1 knockout eyes using Eyeless-GAL4-UAS-FLP (EUGF). We will test whether RACK1 knockout rescues the phenotype induced by the expression of human TDP-43 in retinal neurons. The results from this project may lay a novel foundation for RACK1-based therapy for ALS. It will further open new research avenues to understand the pathological mechanism behind TDP-43-linked ALS, translation defects in neurodegenerative diseases, and the role of RACK1 in ALS.
Principal Investigator
Art Marzok , McMaster University
Partners and Donors
ALS Society of Canada