Defective protein translation: a causative pathway in ALS?
Aperçu du projet
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.
Chef d'équipe
Amrita Verma , University of British Columbia
Partenaire et Donateurs
ALS Society of Canada