Alterations in RNA homeostasis and RNA binding protein functions underlie critical aspects of pathogenesis in neurodegeneration. For RNA dominant disorders caused by nucleotide repeats, the effects of RNA are direct, with the repeats themselves eliciting toxicity via the sequestration of certain proteins. For other neurodegenerative conditions, including amyotrophic lateral sclerosis (ALS), RNA binding proteins such as TDP-43 form neuronal aggregates that serve as pathologic hallmarks of disease. Recently, a novel intronic GGGGCC nucleotide repeat expansion (C9ALS) was identified as the most common known cause of ALS. C9ALS is associated with TDP-43 pathology but also exhibits accumulation of repeat RNA into nuclear foci. We propose a set of studies that take advantage of our expertise in RNA dominant disorders to interrogate how the GGGGCC repeat elicits disease in C9ALS. We hypothesize that GGGGCC repeats are inherently toxic as RNA, and that they elicit toxicity by sequestering specific RNA binding proteins. The immediate goals of this study are to generate and characterize a drosophila model of C9ALS and to identify proteins which interact with the GGGGCC repeat in vitro and in vivo. Research Plan/Methods: To better understand C9ALS pathogenesis, we will first generate an GGGGCC repeat model in Drosophila that allows us to track both the splicing and localization of the intron within expressing tissues. We will determine if GGGGCC RNA repeats confer neurodegeneration and affect lifespan and motor neuron function. We will then take a candidate approach to identify genetic suppressors of GGGGCC repeat toxicity. In a parallel set of studies, we will use in vitro and cell culture based techniqes to empirically identify proteins that bind to the GGGGCC repeat. We will then validate that these candidates colocalize with RNA inclusions in Drosophila and patient cells. Impact/Clinical Relevance: All forms of ALS and FXTAS are currently untreatable and impart significant morbidity and mortality upon Veterans. The C9ALS repeat discovery provides a unique opportunity to rapidly translate recent advances in RNA dominant diseases to this new disorder. The long term goal of this project is to identify small molecules that block interactions between GGGGCC repeat RNA with its cognate RNA binding proteins. Such compounds will then be evaluated for their ability to prevent GGGGCC RNA toxicity in animal models and eventually utilized to treat patients with C9ALS. Taken together, our approach is novel, feasible, and of high potential impact to patients with ALS and related GGGGCC associated neurological disorders.
ALS and other neurodegenerative disorders lead to significant death and disability in US Veterans. Recently, a novel RNA repeat mutation was identified as the most common cause of ALS and Frontotemporal Dementia (C9ALS). The RNA repeat is thought to cause toxicity because it binds to specific proteins and prevents them from performing their normal functions. This proposal will first identify which proteins are bound by the toxic RNA. It will then generate valuable new disease model of C9ALS in the fruit fly, drosophila and take a candidate genetic approach to determine how this RNA repeat elicits toxicity. Together, these two approaches will lay the groundwork for future studies aimed at developing small molecule therapeutics for this currently untreatable disease.