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FMR1premutation cytosine-guanine-guanine repeat expansion alleles are relatively common mutations in the general population that are associated with a neurodegenerative disease (fragile X-associated tremor/ataxia syndrome), reproductive health problems and potentially a wide range of additional mental and general health conditions that are not yet well-characterised. The International Fragile X Premutation Registry (IFXPR) was developed to facilitate and encourage research to better understand theFMR1premutation and its impact on human health, to facilitate clinical trial readiness by identifying and characterising diverse cohorts of individuals interested in study participation, and to build community and collaboration among carriers, family members, researchers and clinicians around the world. Here, we describe the development and content of the IFXPR, characterise its first 747 registrants from 32 countries and invite investigators to apply for recruitment support for their project(s). With larger numbers, increased diversity and potentially the future clinical characterisation of registrants, the IFXPR will contribute to a more comprehensive and accurate understanding of the fragile X premutation in human health and support treatment studies.

Repeat-associated non-AUG (RAN) translation of expanded repeat-mutation mRNA produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based and Drosophila models, we examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, DENR and MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters and its reduction improved the survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest these may serve as novel therapeutic targets in neurodegenerative disease.

In response to the COVID-19 global pandemic, the largest mass vaccination campaign on record was initiated. In the U.S., the Pfizer-BioNTech BNT162b2 and Moderna mRNA-1273 messenger RNA (mRNA) vaccines received Emergency Use Authorization (EUA) in December 2020. Since immunosuppressed patients including liver transplant recipients (LTR) were excluded from these vaccine trials, safety and efficacy of COVID-19 vaccination in LTR are largely unknown. Herein, we report a case of acute inflammatory demyelinating polyneuropathy (AIDP), the most common subtype of Guillain-Barre syndrome (GBS), following initial dose of mRNA COVID-19 vaccine in an adult LTR.

BackgroundThe original Fragile X-associated Tremor Ataxia Syndrome Rating Scale (FXTAS-RS) contained 61 items, some requiring modifications to better meet recommendations for patient-focused rating scale development.PurposeProvide initial validation of a revised version of the FXTAS-RS for motor signs.MethodWe conducted a two-phase mixed-method approach. In Phase 1, revision, we implemented a Delphi technique identifying pertinent domains/subdomains and developing items through expert consensus. In Phase 2, content validation, we conducted cognitive pretesting assessing comprehensibility, comprehensiveness, and relevance of items to FXTAS motor signs.ResultsAfter five rounds of Delphi panel and two rounds of cognitive pretesting, the revised version of the FXTAS-RS was established with 18 items covering five domains and 13 subdomains of motor signs. Cognitive pretesting revealed adequate content validity for the assessment of FXTAS motor signs.ConclusionThe revised FXTAS-RS has been successfully validated for content and it is now ready for large-scale field validation.

Background Premutation-sized (55-200) CGG repeat expansions in the FMR1 gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Most studies of premutation carriers utilized reverse ascertainment to identify patients, leading to a selection bias for larger repeats. As shorter CGG premutation repeats are common in the population, understanding their impact on health outcomes has a potentially large public health footprint. Objective The study's objective was to compare an unselected group of premutation carriers (n = 35, 55-101 CGG repeats) with matched controls (n = 61, 29-39 CGG repeats) with respect to FXTAS-type signs using structured neurological assessments. Methods Three neurologists independently rated signs, using an adapted version of the FXTAS Rating Scale (Leehey MA, Berry-Kravis E, Goetz CG, et al. FMR1 CGG repeat length predicts motor dysfunction in premutation carriers. Neurology. 2008). This was a double-blind study, as genetic status (premutation vs. control) was known neither by the participants nor by any of the neurologists. Analyses controlled potentially confounding comorbid conditions in the electronic health record (eg, osteoarthritis and stroke) and probed the association of age with signs. Results Although there was no overall difference between carriers and controls, among individuals without any potentially confounding comorbid diagnoses, there was a statistically significant age-associated elevation in FXTAS-type signs in premutation carriers compared to controls. Conclusions Among those who do not have other comorbid diagnoses, women who have CGG repeats at the lower end of the premutation range may be at greater risk for ataxia and parkinsonism than their age peers, although their overall risk of developing such clinical features is low. This study should provide reassurance to those who share characteristics with the present cohort. © 2021 International Parkinson and Movement Disorder Society.

CGG repeat expansions in the FMR1 5’UTR cause the neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). These repeats form stable RNA secondary structures that support aberrant translation in the absence of an AUG start codon (RAN translation), producing aggregate-prone peptides that accumulate within intranuclear neuronal inclusions and contribute to neurotoxicity. Here, we show that the most abundant RAN translation product, FMRpolyG, is markedly less toxic when generated from a construct with a non-repetitive alternating codon sequence in place of the CGG repeat. While exploring the mechanism of this differential toxicity, we observed a +1 translational frameshift within the CGG repeat from the arginine to glycine reading frame. Frameshifts occurred within the first few translated repeats and were triggered predominantly by RNA sequence and structural features. Short chimeric R/G peptides form aggregates distinct from those formed by either pure arginine or glycine, and these chimeras induce toxicity in cultured rodent neurons. Together, this work suggests that CGG repeats support translational frameshifting and that chimeric RAN translated peptides may contribute to CGG repeat-associated toxicity in FXTAS and related disorders.

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)subn/subrepeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive.We investigated whether (AGAGGG)subn/subrepeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases.XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR.We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)subn/subsequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells.In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)subn/subrepeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Fragile X–associated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 58–70 (2015); S. Jacquemont et al., JAMA 291, 460–469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55–200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.

Significance Expansion of 55-200 CGG repeats in the 5′ untranslated region of FMR1 predisposes carriers to fragile X–associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder. FXTAS demonstrates incomplete penetrance, which strongly suggests the presence of genetic modifiers. We performed whole-genome sequencing (WGS) on male premutation carriers (CGG 55–200 ) followed by a functional screen in Drosophila and identified PSMB5 as a strong suppressor of CGG-associated neurodegeneration, thereby presenting a therapeutic strategy for FXTAS.

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5'-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders.

Transcribed CGG repeat expansions cause neurodegeneration in Fragile X‐associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear foci and undergo repeat‐associated non‐AUG (RAN) translation into toxic peptides. To identify proteins involved in these processes, we employed a CGG repeat RNA‐tagging system to capture repeat‐associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine‐rich) proteins that interact selectively with CGG repeats basally and under cellular stress. These proteins modify toxicity in a Drosophila model of FXTAS. Pharmacologic inhibition of serine/arginine protein kinases (SRPKs), which alter SRSF protein phosphorylation, localization, and activity, directly inhibits RAN translation of CGG and GGGGCC repeats (associated with C9orf72 ALS/FTD) and triggers repeat RNA retention in the nucleus. Lowering SRPK expression suppressed toxicity in both FXTAS and C9orf72 ALS/FTD model flies, and SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. Together, these findings demonstrate roles for CGG repeat RNA binding proteins in RAN translation and repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders., Fragile X‐associated tremor/ataxia syndrome (FXTAS) is an adult‐onset neurodegenerative disease caused by a CGG trinucleotide repeat expansion in FMR1. This study identified CGG repeat RNA binding proteins and their kinases that regulate repeat associated non‐AUG (RAN) translation and neurotoxicity.

Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.

Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.

Summary: eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36–G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length–dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.

GGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG (RAN) translation of this expansion generates toxic proteins that accumulate in patient brains and contribute to disease pathogenesis. The DEAH-Box Helicase 36 (DHX36/G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. We found that DHX36 depletion suppresses RAN translation from reporter constructs in a repeat length dependent manner while overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Taken together, these results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeats expansion disorders.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a transcribed CGG repeat expansion in the 5’ UTR ofFMR1. Expanded CGG repeat RNAs both sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic homopolymeric peptides. RBPs that interact with CGG repeats may play a pivotal role in foci formation and/or RAN translation. Here we employed a CGG repeat RNA-tagging system to capture and identify CGG repeat binding RBPsin vivounder different cellular conditions. We found that several SR (serine/arginine-rich domain) proteins interact with CGG repeat RNAs basally and under cellular stress. These same proteins strongly modify toxicity in a Drosophila model of FXTAS, improving eye degeneration and survival. Furthermore, genetic or pharmacological targeting of the serine/arginine protein kinases (SRPKs) suppresses RAN translation in cellular reporters and toxicity in fly models of FXTAS and C9orf72 ALS/FTD. Finally, pharmacological targeting of SRPK1 supressed CGG repeat toxicity and enhanced survival in rodent neurons. Taken together, these findings demonstrate roles for CGG repeat RNA binding proteins in both RAN translation and repeat toxicity and suggest SRPK inhibition may serve as a possible therapeutic strategy in repeat expansion disorders.

RNAs derived from expanded nucleotide repeats form detectable foci in patient cells and these foci are thought to contribute to disease pathogenesis. The most widely used method for detecting RNA foci is fluorescence in situ hybridization (FISH). However, FISH is prone to low sensitivity and photo-bleaching that can complicate data interpretation. Here we applied hybridization chain reaction (HCR) as an alternative approach to repeat RNA foci detection of GC-rich repeats in two neurodegenerative disorders: GGGGCC (G4C2) hexanucleotide repeat expansions in C9orf72 that cause amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD) and CGG repeat expansions in FMR1 that cause Fragile X-associated tremor/ataxia syndrome. We found that HCR of both G4C2 and CGG repeats has comparable specificity to traditional FISH, but is >40x more sensitive and shows repeat-length dependence in its intensity. HCR is better than FISH at detecting both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. We used HCR to determine the impact of integrated stress response (ISR) activation on RNA foci number and distribution. G4C2 repeat RNA did not readily co-localize with the stress granule marker G3BP1, but ISR induction increased both the number of detectible nuclear RNA foci and the nuclear/cytoplasmic foci ratio in patient fibroblasts and patient derived neurons. Taken together, these data suggest that HCR can be a useful tool for detecting repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

CGG repeat expansions within the premutation range (55–200) of the FMR1 gene can lead to Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders. These CGG repeats are translated into a toxic polyglycine-containing protein, FMRpolyG. Pathology of Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders comprises FMRpolyG- and p62-positive intranuclear inclusions. Diagnosing a FMR1-premutation carrier remains challenging, as the clinical features overlap with other neurodegenerative diseases. Here, we describe two male cases with Fragile X-associated neuropsychiatric disorders-related symptoms and mild movement disturbances and novel pathological features that can attribute to the variable phenotype. Macroscopically, both donors did not show characteristic white matter lesions on MRI; however, vascular infarcts in cortical- and sub-cortical regions were identified. Immunohistochemistry analyses revealed a high number of FMRpolyG intranuclear inclusions throughout the brain, which were also positive for p62. Importantly, we identified a novel pathological vascular phenotype with inclusions present in pericytes and endothelial cells. Although these results need to be confirmed in more cases, we propose that these vascular lesions in the brain could contribute to the complex symptomology of FMR1-premutation carriers. Overall, our report suggests that Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders may present diverse clinical involvements resembling other types of dementia, and in the absence of genetic testing, FMRpolyG can be used post-mortem to identify premutation carriers.

Repeat associated non-AUG (RAN) initiated translation of FMR1 5’ UTR CGG repeats produces toxic homo-polymeric proteins that accumulate within ubiquitinated inclusions in Fragile X-associated tremor/ataxia syndrome (FXTAS) patient brains and model systems. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG codon located 5’ to the repeat. Methods to accurately measure FMRpolyG in FXTAS patients are lacking. Here we used data dependent acquisition (DDA) and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides (SIS) to identify potential signature FMRpolyG fragments in patient cells and tissues. Following immunoprecipitation enrichment, we detected FMRpolyG signature peptides by PRM in transfected cells, FXTAS human samples and patient derived stem cells, but not in controls. Surprisingly, we identified two amino-terminal peptides: Ac-MEAPLPGGVR, initiating at an ACG, and Ac-TEAPLPGGVR, initiating at a GUG, along with evidence for RAN translation initiation within the CGG repeat itself. Initiation at all three sites was upregulated in response to cellular stress, downregulated following eIF1 overexpression and dependent on the initiation factor helicase, eIF4A. Use of the two initiation sites upstream of the repeat decreased following loss of the eIF2 alternative factor eIF2A. These data demonstrate that FMRpolyG is quantifiable in human samples and that RAN translation on FMR1 initiates via similar mechanisms at both near-cognate codons and within the repeat itself through processes dependent on available initiation factors and the cellular environment.

Repeat associated non-AUG (RAN) translation of FMR1 5’ UTR CGG repeats produces toxic homo-polymeric proteins that accumulate within ubiquitinated inclusions in Fragile X-associated tremor/ataxia syndrome (FXTAS) patient brains and model systems. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG codon located just 5’ to the repeat. Methods to accurately measure FMRpolyG in FXTAS patients are lacking. Here we used data dependent acquisition (DDA) and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides (SIS) to identify potential signature FMRpolyG fragments in patient cells and tissues. Following immunoprecipitation (IP) enrichment, we detected FMRpolyG signature peptides by PRM in transfected cells, FXTAS human samples and patient derived stem cells, but not in controls. Surprisingly, we identified two amino-terminal peptides: one beginning with methionine (Ac-MEAPLPGGVR) initiating at an ACG, and a second beginning with threonine (Ac-TEAPLPGGVR), initiating at a GUG. Abundance of the threonine peptide was enhanced relative to the methionine peptide upon activation of the integrated stress response. In addition, loss of the eIF2 alternative factor, eIF2A, or enhanced expression of initiation factor eIF1, preferentially suppressed GUG initiated FMRpolyG synthesis. These data demonstrate that FMRpolyG is quantifiable in human samples and that RAN translation on FMR1 initiates at specific near cognate codons dependent on available initiation factors and cellular environment.

Protein metabolism plays central roles in age-related decline and neurodegeneration. While a large body of research has explored age-related changes in protein degradation, alterations in the efficiency and fidelity of protein synthesis with aging are less well understood. Age-associated changes occur in both the protein synthetic machinery (ribosomal proteins and rRNA) and within regulatory factors controlling translation. At the same time, many of the interventions that prolong lifespan do so in part by pre-emptively decreasing protein synthesis rates to allow better harmonization to age-related declines in protein catabolism. Here we review the roles of translation regulation in aging, with a specific focus on factors implicated in age-related neurodegeneration. We discuss how emerging technologies such as ribosome profiling and superior mass spectrometric approaches are illuminating age-dependent mRNA-specific changes in translation rates across tissues to reveal a critical interplay between catabolic and anabolic pathways that likely contribute to functional decline. These new findings point to nodes in posttranscriptional gene regulation that both contribute to aging and offer targets for therapy. This article is categorized under: Translation > Translation Regulation Translation > Ribosome Biogenesis Translation > Translation Mechanisms.

CGG repeat expansions within the premutation range (55–200) of the FMR1 gene can lead to Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders. These CGG repeats are translated into a toxic polyglycine-containing protein, FMRpolyG. Pathology of Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders comprises FMRpolyG- and p62-positive intranuclear inclusions. Diagnosing a FMR1-premutation carrier remains challenging, as the clinical features overlap with other neurodegenerative diseases. Here, we describe two male cases with Fragile X-associated neuropsychiatric disorders-related symptoms and mild movement disturbances and novel pathological features that can attribute to the variable phenotype. Macroscopically, both donors did not show characteristic white matter lesions on MRI; however, vascular infarcts in cortical- and sub-cortical regions were identified. Immunohistochemistry analyses revealed a high number of FMRpolyG intranuclear inclusions throughout the brain, which were also positive for p62. Importantly, we identified a novel pathological vascular phenotype with inclusions present in pericytes and endothelial cells. Although these results need to be confirmed in more cases, we propose that these vascular lesions in the brain could contribute to the complex symptomology of FMR1-premutation carriers. Overall, our report suggests that Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders may present diverse clinical involvements resembling other types of dementia, and in the absence of genetic testing, FMRpolyG can be used post-mortem to identify premutation carriers., FMR1-premutation carriers can present clinically with a range of symptoms, including neuropsychiatric symptoms. Pathologically, the nuclear inclusions are positive for FMRpolyG and in the donors discussed here, also present in the vasculature. This has not been described before and this finding possibly contributes to the complex phenotype., Graphical Abstract Graphical Abstract

An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5’ UTR of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5’ UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR carboxyl terminus was observed when repeats were expressed in rodent neurons. The presence of the native carboxyl-termini across all three reading frames was partly protective. Taken together, these findings suggest that carboxyl terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.

ObjectiveTo determine the utilization of genetic testing in patients seen by a neurologist within a large private insurance population.MethodsUsing the Optum health care claims database, we identified a cross-sectional cohort of patients who had been evaluated by a neurologist no more than 30 days before initial genetic testing. Within this group, we then categorized genetic testing between 2014 and 2016 on the basis of the Current Procedural Terminology (CPT) codes related to molecular and genetic testing. We also evaluated the International Classification of Disease Version 9 Clinical Code Classifications (ICD-9 CCS) associated with testing.ResultsFrom 2014 to 2016, a total of 45,014 claims were placed for 29,951 patients who had been evaluated by a neurologist within the preceding 30 days. Of these, 29,926 (66.5%) were associated with codes that were too nonspecific to infer what test was actually performed. Among those claims where the test was clearly identifiable, 7,307 (16.2%) were likely obtained for purposes of neurologic diagnosis, whereas the remainder (17.2%) was obtained for non-neurological purposes. An additional 3,793 claims (8.4%) wherein the test ordered could not be clearly identified were associated with a neurology-related ICD-9 CCS.ConclusionsAccurate assessment of genetic testing utilization using claims data is not possible given the high prevalence of nonspecific codes. Reducing the ambiguity surrounding the CPT codes and the actual testing performed will become even more important as more genetic tests become available.

Co-occurrence of multiple neuropathologic changes is a common phenomenon, most prominently seen in Alzheimer’s disease (AD) and Parkinson’s disease (PD), complicating clinical diagnosis and patient management. Reports of co-occurring pathological processes are emerging in the group of genetically defined repeat-associated non-AUG (RAN)-translation related diseases. Here we report a case of Fragile X-associated tremor-ataxia syndrome (FXTAS) with widespread and abundant nuclear inclusions of the RAN-translation related FMRpolyG-peptide. In addition, we describe prominent neuronal and glial tau pathology representing changes seen in progressive supranuclear palsy (PSP). The highest abundance of the respective pathological changes was seen in distinct brain regions indicating an incidental, rather than causal correlation.

A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context—namely, those with non-AUG start codons—to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Background Upstream open reading frames (uORFs) initiate translation within mRNA 5′ leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5′ leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in a model of neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Results Using a spectral coherence algorithm (SPECtre), we identify 4954 consistently translated uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. On a population basis, the global impact of both AUG and non-AUG initiated uORFs on basal CDS translation were small, even when analysis is limited to conserved and consistently translated uORFs. However, uORFs did alter the translation of a subset of genes, including the Diamond-Blackfan Anemia associated ribosomal gene RPS24. With retinoic acid induced differentiation, we observed an overall positive correlation in translational shifts between uORF/CDS pairs. However, CDSs downstream of uORFs show smaller shifts in TE with differentiation relative to CDSs without a predicted uORF, suggesting that uORF translation buffers cell state dependent fluctuations in CDS translation. Conclusion This work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation. Electronic supplementary material The online version of this article (10.1186/s12864-019-5775-1) contains supplementary material, which is available to authorized users.

Objective: Repeat associated non-AUG (RAN) translation drives production of toxic proteins from pathogenic repeat sequences in multiple untreatable neurodegenerative disorders. Fragile X-associated tremor/ataxia syndrome (FXTAS) is one such condition, resulting from a CGG trinucleotide repeat expansion in the 5' leader sequence of the FMR1 gene. RAN proteins from the CGG repeat accumulate in ubiquitinated inclusions in FXTAS patient brains and elicit toxicity. In addition to the CGG repeat, an antisense mRNA containing a CCG repeat is also transcribed from the FMR1 locus. We evaluated whether this antisense CCG repeat supports RAN translation and contributes to pathology in FXTAS patients. Methods: We generated a series of CCG RAN translation specific reporters and utilized them to measure RAN translation from CCG repeats in multiple reading frames in transfected cells. We also developed antibodies against predicted CCG RAN proteins and used immunohistochemistry and immunofluorescence on FXTAS patient tissues to measure their accumulation and distribution. Results: RAN translation from CCG repeats is supported in all three potential reading frames, generating polyproline, polyarginine, and polyalanine proteins, respectively. Their production occurs whether or not the natural AUG start upstream of the repeat in the proline reading frame is present. All three frames show greater translation at larger repeat sizes. Antibodies targeted to the antisense FMR polyproline and polyalanine proteins selectively stain nuclear and cytoplasmic aggregates in FXTAS patients and colocalize with ubiquitinated neuronal inclusions. Interpretation: RAN translation from antisense CCG repeats generates novel proteins that accumulate in ubiquitinated inclusions in FXTAS patients. This article is protected by copyright. All rights reserved.

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.

Highlights • Fragile X Tremor Ataxia Syndrome (FXTAS) is caused by expanded CGG repeats. • Mechanisms of CGG repeat toxicity include RNA protein titration and RAN translation. • A key pathogenic event could be the near-cognate codon translation of CGG repeats., Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset inherited neurodegenerative disorder characterized by progressive intention tremor, gait ataxia and dementia associated with mild brain atrophy. The cause of FXTAS is a premutation expansion, of 55 to 200 CGG repeats localized within the 5′UTR of FMR1. These repeats are transcribed in the sense and antisense directions into mutants RNAs, which have increased expression in FXTAS. Furthermore, CGG sense and CCG antisense expanded repeats are translated into novel proteins despite their localization in putatively non-coding regions of the transcript. Here we focus on two proposed disease mechanisms for FXTAS: 1) RNA gain-of-function, whereby the mutant RNAs bind specific proteins and preclude their normal functions, and 2) repeat-associated non-AUG (RAN) translation, whereby translation through the CGG or CCG repeats leads to the production of toxic homopolypeptides, which in turn interfere with a variety of cellular functions. Here, we analyze the data generated to date on both of these potential molecular mechanisms and lay out a path forward for determining which factors drive FXTAS pathogenicity.

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.