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.

SummaryFragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5′ UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS.

Summary A repeat expansion in C9ORF72 causes frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). RNA of the expanded repeat (r(GGGGCC)exp) forms nuclear foci or undergoes repeat-associated non-ATG (RAN) translation producing “c9RAN proteins”. Since neutralizing r(GGGGCC)exp could inhibit these potentially toxic events, we sought to identify small molecule binders of r(GGGGCC)exp. Chemical and enzymatic probing of r(GGGGCC)8 indicate it adopts a hairpin structure in equilibrium with a quadruplex structure. Using this model, bioactive small molecules targeting r(GGGGCC)exp were designed and found to significantly inhibit RAN translation and foci formation in cultured cells expressing r(GGGGCC)66 and neurons trans-differentiated from fibroblasts of repeat expansion carriers. Finally, we show that poly(GP) c9RAN proteins are specifically detected in c9ALS patient cerebrospinal fluid. Our findings highlight r(GGGGCC)exp-binding small molecules as a possible c9FTD/ALS therapeutic, and suggest c9RAN proteins could potentially serve as a pharmacodynamic biomarker to assess efficacy of therapies that target r(GGGGCC)exp.

Repeat-associated non-AUG (RAN) translation allows for unconventional initiation at disease-causing repeat expansions. As RAN translation contributes to pathogenesis in multiple neurodegenerative disorders, determining its mechanistic underpinnings may inform therapeutic development. Here we analyze RAN translation at G4C2 repeat expansions that cause C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9RAN) and at CGG repeats that cause fragile X-associated tremor/ataxia syndrome. We find that C9RAN translation initiates through a cap- and eIF4A-dependent mechanism that utilizes a CUG start codon. C9RAN and CGG RAN are both selectively enhanced by integrated stress response (ISR) activation. ISR-enhanced RAN translation requires an eIF2α phosphorylation-dependent alteration in start codon fidelity. In parallel, both CGG and G4C2 repeats trigger phosphorylated-eIF2α-dependent stress granule formation and global translational suppression. These findings support a model whereby repeat expansions elicit cellular stress conditions that favor RAN translation of toxic proteins, creating a potential feed-forward loop that contributes to neurodegeneration.

SUMMARY Repeat associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ALS and Fragile X Tremor/Ataxia Syndrome (FXTAS). How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5′ leader of Fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30–40% as efficient as AUG initiated translation, is m7G-cap and eIF4E-dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model where RAN translation at CGG repeats utilizes cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection.

Fragile X syndrome (FXS) is a common inherited cause of mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP is thought to regulate the translation of target mRNAs, including its own transcript. Here we show that the levels of FMRP are rapidly up-regulated in primary cortical neurons in response to the type-I metabotropic glutamate receptor (mGluR) agonist S-3,5-dihydrophenylglycine. These changes require new protein synthesis but not transcription and are specific to mGluR activation. We also demonstrate that the mRNA for PSD-95, a scaffolding protein involved in synaptic plasticity, contains a highly conserved canonical binding site for FMRP within its 3 UTR. Furthermore, PSD-95 is rapidly translated in response to S-3,5-dihydrophenylglycine. Finally, we show that these mGluR-dependent changes in PSD-95 expression are lost in neurons derived from FMRP knockout mice, a model of FXS. Taken together, these studies suggest that FMRP is required for mGluRdependent translation of PSD-95 and provide insights into the pathophysiology of FXS.F ragile X syndrome is the most common inherited cause of mild to moderate mental retardation (1, 2). The syndrome typically results from transcriptional silencing of the fragile X mental retardation gene, FMR1, and loss of the fragile X mental retardation protein (FMRP) (for a review, see ref.3). Through RGG and KH domains, FMRP binds up to 4% of total brain mRNA, including its own transcript (4-7). FMRP is highly expressed in the cytoplasm and dendrites of neurons (8), often in large messenger ribonucleoprotein particles, implicating FMRP in translational regulation (9-12).FMRP is translated in response to activation of metabotropic glutamate receptors (mGluRs) (13,14) and may be required for mGluR-dependent translation (15). Moreover, FMRP is required for normal type-I mGluR-dependent long-term depression, a process that requires protein synthesis (16). Thus, we hypothesized that FMRP-bound mRNAs would be translated in cultured neurons in response to application of a type-I mGluR agonist.Here we show that application of the type-I mGluR agonist S-3,5-dihydrophenylglycine (DHPG) onto cortical cultures leads to the rapid and robust translation of both FMRP and the synaptic scaffolding protein PSD-95, whose mRNA contains a canonical FMRP binding site. Furthermore, the mGluRdependent translation of PSD-95 is lost in cultures derived from FMR1 knockout (KO) mice. Taken together, these findings support a role for FMRP in activity-dependent translation and may provide insights into mGluR-dependent synaptic plasticity. Figs. 1-3) and used at 14-15 days in vitro. Western blots were performed and analyzed as described (14) with anti-FMRP (mAb 2160, Chemicon), anti-PSD-95 (7E3-1B8, Affinity BioReagents, Neshanic Station, NJ), anti-mGluR 1 and 5 (Upstate Biotechnology, Lake Placid, NY), and anti-␤-actin (Sigma). For FMRP, only the top band was quantitated and normalized to ␤-actin. MethodsImmunofluorescence and Immunoblotting. For immunoflu...

SUMMARY Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55–200 CGG repeats in the 5′ UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of micro-RNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.

Protein degradation is an essential cellular function that, when dysregulated or impaired, can lead to a wide variety of disease states. The two major intracellular protein degradation systems are the ubiquitin-proteasome system (UPS) and autophagy, a catabolic process that involves delivery of cellular components to the lysosome for degradation. While the UPS has garnered much attention as it relates to neurodegenerative disease, important links between autophagy and neurodegeneration have also become evident. Furthermore, recent studies have revealed interaction between the UPS and autophagy, suggesting a coordinated and complementary relationship between these degradation systems that becomes critical in times of cellular stress. Here we describe autophagy and review evidence implicating this system as an important player in the pathogenesis of neurodegenerative disease. We discuss the role of autophagy in neurodegeneration and review its neuroprotective functions as revealed by experimental manipulation in disease models. Finally, we explore potential parallels and connections between autophagy and the UPS, highlighting their collaborative roles in protecting against neurodegenerative disease.