Project Summary: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects the carriers of premutation alleles (55–200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Common features of FXTAS include progressive intention tremor, gait ataxia, Parkinsonism, and cognitive decline. The neuropathological hallmarks of FXTAS include ubiquitin-positive intranuclear inclusions throughout brain and marked dropout of Purkinje neurons in cerebellum. The long-term goal of this project is to understand the molecular pathogenesis of FXTAS and develop effective therapeutic interventions for FXTAS. At the molecular level, FMR1 CGG premutation carriers exhibit a 2 to 8-fold increase in FMR1 mRNA compared to control individuals. Expression of mutant mRNAs containing long (~100) CGG triplets has been shown to be toxic in cell and animal models. Currently, data support two non-mutually exclusive molecular pathogenesis mechanisms for FXTAS: 1) RNA gain-of-function, in which rCGG repeat-binding proteins (RBPs) become functionally limited through sequestration by lengthy rCGG repeats, and 2) Repeat- associated non-AUG (RAN) translation, whereby translation through the CGG (or antisense CCG) repeats leads to the production of toxic homo-polypeptides, such as FMRpolyG, which in turn interfere with a variety of cellular functions. Our previous work has identified two known RNA-binding proteins, Pur and hnRNP A2/B1, as RBPs affected by expression of rCGG. We showed that increased expression of either protein could modulate rCGG-mediated toxicity, supporting the RNA-mediated sequestration model of FXTAS. To determine the contributions of both mechanisms to FXTAS pathogenesis, we have generated transgenic lines of mice that express hnRNP A2/B1 and suppress rCGG repeat-mediated toxicity. In parallel, we have taken both whole genome sequencing and global metabolic profiling approaches combined with fly genetic screens to identify potential additional genetic modifiers of FXTAS. We have found that PSMB5 and the sphingolipid metabolic pathway could modulate rCGG repeat toxicity. In this proposal, we plan to further test the hypothesis that FXTAS results from abnormal RNA metabolism stemming from inappropriate association of RBPs with the RNA produced by FMR1 premutation alleles, as well as determine whether these additional candidate modifiers can modulate FXTAS pathogenesis. Successful completion of these studies should significantly advance our understanding the molecular pathogenesis of FXTAS. Identifications of genes and pathways involved in FXTAS will provide valuable targets for future pharmacological research aimed at developing drugs for therapy.