Fragile X-associated Tremor Ataxia Syndrome (FXTAS) results from a CGG repeat expansion in the 5’UTR of FMR1. This repeat is thought to elicit toxicity as RNA yet disease brains contain ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that CGG repeats trigger repeat associated non-AUG initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders where RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration.

Sleep is critical for proper memory consolidation. The locus coeruleus (LC) releases norepinephrine throughout the brain except when the LC falls silent throughout rapid eye movement (REM) sleep and prior to each non-REM (NREM) sleep spindle. We hypothesize that these transient LC silences allow the synaptic plasticity that is necessary to incorporate new information into pre-existing memory circuits. We found that spontaneous LC activity within sleep spindles triggers a decrease in spindle power. By optogenetically stimulating norepinephrine-containing LC neurons at 2 Hz during sleep, we reduced sleep spindle occurrence, as well as NREM delta power and REM theta power, without causing arousals or changing sleep amounts. Stimulating the LC during sleep following a hippocampus-dependent food location learning task interfered with consolidation of newly learned locations and reconsolidation of previous locations, disrupting next-day place cell activity. The LC stimulation-induced reduction in NREM sleep spindles, delta, and REM theta and reduced ripple-spindle coupling all correlated with decreased hippocampus-dependent performance on the task. Thus, periods of LC silence during sleep following learning are essential for normal spindle generation, delta and theta power, and consolidation of spatial memories.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG trinucleotide repeat expansion in the 5' UTR of the Fragile X gene, FMR1. FXTAS is thought to arise primarily from an RNA gain-of-function toxicity mechanism. However, recent studies demonstrate that the repeat also elicits production of a toxic polyglycine protein, FMRpolyG, via repeat-associated non-AUG (RAN)-initiated translation. Pathologically, FXTAS is characterized by ubiquitin-positive intranuclear neuronal inclusions, raising the possibility that failure of protein quality control pathways could contribute to disease pathogenesis. To test this hypothesis, we used Drosophila- and cell-based models of CGG-repeat-associated toxicity. In Drosophila, ubiquitin proteasome system (UPS) impairment led to enhancement of CGG-repeat-induced degeneration, whereas overexpression of the chaperone protein HSP70 suppressed this toxicity. In transfected mammalian cells, CGG repeat expression triggered accumulation of a UPS reporter in a length-dependent fashion. To delineate the contributions from CGG repeats as RNA from RAN translation-associated toxicity, we enhanced or impaired the production of FMRpolyG in these models. Driving expression of FMRpolyG enhanced induction of UPS impairment in cell models, while prevention of RAN translation attenuated UPS impairment in cells and suppressed the genetic interaction with UPS manipulation in Drosophila. Taken together, these findings suggest that CGG repeats induce UPS impairment at least in part through activation of RAN translation.

RNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a noncoding r(CGG) repeat expansion (r(CGG)exp) that (i) sequesters proteins involved in RNA metabolism in nuclear foci, causing dysregulation of alternative pre-mRNA splicing, and (ii) undergoes repeat associated non-ATG translation (RANT), which produces toxic homopolymeric proteins without using a start codon. Here, we describe the design of two small molecules that inhibit both modes of toxicity and the implementation of various tools to study perturbation of these cellular events. Competitive Chemical Cross Linking and Isolation by Pull Down (C-Chem-CLIP) established that compounds bind r(CGG)exp and defined small molecule occupancy of r(CGG)exp in cells, the first approach to do so. Using an RNA GFP mimic, r(CGG)exp-Spinach2, we observe that our optimal designed compound binds r(CGG)exp and affects RNA localization by disrupting preformed RNA foci. These events correlate with an improvement of pre-mRNA splicing defects caused by RNA gain of function. In addition, the compounds reduced levels of toxic homopolymeric proteins formed via RANT. Polysome profiling studies showed that small molecules decreased loading of polysomes onto r(CGG)exp, explaining decreased translation.