Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons that is rapidly progressive and uniformly fatal. In 90-95% of cases, ALS is sporadic with no clearly associated risk factors. The other 5-10% of ALS is inherited with a genetic cause. The most common inherited cause of ALS, a GGGGCC hexanucleotide repeat expansion (HRE) found in the gene chromosome 9 open reading frame 72 (C9orf72), accounts for about half of all familial cases and about 8% of sporadic cases of ALS (called ?C9- ALS?). This HRE is hypothesized to be caused by a gain-of-function either by expression of repeat RNA and sequestration of RNA-binding proteins, or through the production of dipeptide repeat proteins (DPRs) through repeat-associated non-AUG translation (RANT). Using a genetic screen for modifiers of GGGGCC-mediated toxicity in Drosophila, we have identified p62/SQSTM1 as a strong genetic modifier of c9orf72 ALS. In addition to being a genetic modifier, p62-positive aggregates are a key pathology in C9-ALS, and p62/SQSTM1 mutations have rarely been shown to cause ALS in patients. Furthermore, p62 plays a key role in protein homeostasis (proteostasis) and protein degradation, which have been strongly implicated in the pathogenesis of ALS. Although p62 appears to be a key genetic target, its role in ALS pathogenesis is poorly understood. This project will determine 1) the mechanism by which protein homeostasis is disrupted in C9-ALS and 2) the role that p62 plays in C9-ALS pathogenesis. First, I will use novel transgenic lines to determine whether DPRs or GGGGCC RNA are responsible for neurodegeneration and protein homeostasis defects by selectively degrading each DPR. Next, I will determine the mechanism by which p62 regulates GGGGCC-mediated neurodegeneration using molecular and genetic tools in Drosophila to determine which protein homeostatic and signaling pathways contribute to p62-mediated toxicity in GGGGCC expressing Drosophila. Lastly, I will use CRISPR to generate novel alleles of p62 in order to assess the role of p62 in motor neuron degeneration. These alleles will be used to study the interactions between p62 and GGGGCC expression as well as other ALS-causing genes in order to provide insight into the genetic basis of ALS.
Disruption of protein homeostasis is key to the pathogenesis of ALS. p62/SQSTM1, a regulator of protein degradation, strongly modifies neurodegeneration in a Drosophila model of c9-ALS. Studying the mechanisms by which p62/SQSTM1 modifies protein homeostasis disruption in ALS will give us insight into the mechanism of disease and reveal new targets for therapeutic intervention.
