Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of voluntary muscle control [1]. The Gitler lab?where I will be conducting this research?discovered that a mutation in the ataxin-2 gene (ATXN2) is a relatively common genetic risk factor for ALS[2]. The mutation is an intermediate-length expansion of a CAG repeat in the ATXN2 coding region, leading to longer polyglutamine tracts in the Ataxin-2 protein. Reduction of the wild-type Atxn2 transcript extended survival and reduced pathology in a mouse model of ALS, as did crossing this mouse with the Ataxin-2 knockout mouse [3]. Despite these promising results, little is known about how wild-type Ataxin-2 contributes to ALS. Defects in RNA metabolism has emerged as a central mechanism in ALS[4-6]. Ataxin-2 is a regulator of mRNA translation, however transcripts under its control have only been identified on a case-by-case basis [7-12]. First, I am interested in exploring how knockout of Ataxin-2 elicits deficits in translation, and if this affords motor neurons protection in the transgenic TDP-43 (TDP-43tg/tg) ALS mouse model. I will use the expertise I gained during graduate school to perform genome-wide and biochemical translation assays but combine this with a new set of techniques for investigating mRNA dynamics in complex tissue. I will perform TRAP-seq, a technique for gauging the level of translation on individual transcripts by purifying mRNA bound to translating ribosomes [13]. This will allow me to determine transcripts with differential translation in TDP-43tg/tg mouse motor neurons, and how that is affected by the Ataxin-2 knockout. Ataxin-2 is an integral component of specialized messenger ribonucleoprotein (mRNP) granules and interacts with TDP-43 through RNA association [2, 8, 14]. mRNP granules are involved in the transport of mRNA to various parts of the cell for proper posttranscriptional processing [15, 16]. Deficits in axonal mRNA localization have been detected in both cultured peripheral neurons and mouse embryonic stem cell-derived motor neurons from multiple transgenic ALS mouse models, but never directly from tissue as the technology was not previously available [17, 18]. I will employ a novel technique called APEX-seq to determine the composition of mRNA transcripts spatially constricted to peripheral motor axons in WT and TDP-43tg/tg mice, and how this changes when crossed to the Ataxin-2 knockout[19, 20]. As described in my second aim, I will perform a genome-wide siRNA screen in human cells to discover regulators of Ataxin-2 that will illuminate pathways that work upstream to control its expression. The Gitler lab is proficient in large-scale approaches to identifying disease modifiers [21-23]. The goal of this aim is to harness our results to devise novel therapeutic strategies and to expand my training to include genome-wide screening. This project allows me the opportunity to expand my expertise in the topic of RNA metabolism in neurological disease, the topic I plan to make my career in researching, and to decipher the most promising targets for therapeutic development and future study.
Reduction of Ataxin-2 ameliorates neurodegeneration and prolongs lifespan in a mouse model of amyotrophic lateral sclerosis (ALS), a devastating human disease that primarily affects motor neurons in the cerebral cortex and spinal cord. The goal of this project is to define the function of Ataxin-2 in motor neurons by investigating its defined role as an RNA binding protein and posttranscriptional regulator, and to find negative regulators of Ataxin-2 in order to expand our list of therapeutic targets. I will accomplish this by taking novel transcriptomics approaches to studying Ataxin-2 in mouse motor neurons, and by performing a whole-genome siRNA screen to define regulators of Ataxin-2 levels in human cells.
