Project Summary Intermediate expansion of a CGG repeat in the 5’ UTR of the FMR1 gene (between 55-200 repeats) underlies an age-related neurodegenerative disorder, Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). While full mutations (>200 repeat expansions) resulting in FXS typically lead to methylation and silencing of FMR1 expression, FXTAS patients express normal or elevated amounts of FMR1 mRNA but have reduced FMRP. Large transcribed CGG repeats are potentially toxic as RNA or by triggering Repeat associated non-AUG initiated (RAN) translation, allowing for production of toxic homopolymeric peptides that contribute to neurodegeneration. Therefore, effective therapies for FXTAS need to simultaneously block CGG RAN and enhance production of FMRP. Previous data provides evidence that RAN translation inhibits translation of downstream FMRP, but may also play a critical role in regulating mGluR-LTD, a form of synaptic plasticity. This finding was shown at normal repeat sizes in addition to the expanded condition. In this proposal, I will investigate the native role of RAN translation in regulating FMRP synthesis both basally and upon stimulation of the metabotropic glutamate receptor at the endogenous FMR1 locus in patient stem cell-derived neurons. This work will determine the potential of blocking RAN translation as a method to simultaneously reduce toxic RAN products and enhance FMRP. Additionally, I will directly test this therapeutic mechanism utilizing an antisense oligonucleotide that selectively targets RAN initiation sites (RAN ASOs) on the FMR1 transcript. In reporter systems and human cell lines, these ASOs suppress RAN translation at both normal and expanded repeat sizes. RAN ASOs also enhanced endogenous FMRP, reduced toxic RAN protein, FMRpolyG, and enhanced neuronal survival in neurons derived from a patient with an unmethylated full mutation (UFM), in which FMR1 is efficiently transcribed, but poorly translated. In this project, I will treat FXTAS patient stem cell-derived neurons with RAN ASOs and evaluate ASO-mediated amelioration of disease-specific deficits in neuronal survival, calcium signaling, and neurite outgrowth. Together, these experiments would demonstrate a native function for CGG RAN in regulating FMRP synthesis and demonstrate that targeting RAN translation has the potential to correct multiple disease relevant features in Fragile X-associated disorders. Furthermore, the training described in this proposal will prepare me for a future in translational research and modeling of neurological disease. The activities under this award will train me to thrive in a future research career and prepare me to train the next generation of scientists.

Project Narrative Expansion of repetitive elements in DNA cause human diseases, but the native function of these repeats is not clear. The CGG trinucleotide repeat in the Fragile X gene (FMR1) can expand to cause a neurodegenerative disease known as Fragile X-associated tremor/ataxia syndrome (FXTAS), but may also play a normal role in regulating the FMR1 gene, providing a potential reason for its existence and conservation in mammals. In this study, I will evaluate the normal regulatory function of CGG repeats and evaluate whether blocking translation of these repeats can improve the survival and function of FXTAS patient neurons.