Small molecule 1a reduces FMRpolyG-mediated toxicity in in vitro and in vivo models for FMR1 premutation.

Fragile X-associated tremor and ataxia syndrome (FXTAS) is a late-onset, progressive neurodegenerative disorder characterized by tremors, ataxia and neuropsychological problems. This disease is quite common in the general population with approximately 20 million carriers worldwide. The risk of developing FXTAS increases dramatically with age, with about 45% of male carriers over the age of 50 being affected. FXTAS is caused by a CGG-repeat expansion (CGGexp) in the fragile X mental retardation 1 (FMR1) gene. CGGexp RNA is translated into the FMRpolyG protein by a mechanism called RAN translation. Although both gene and pathogenic trigger are known, no therapeutic interventions are available at this moment. Here, we present, for the first time, primary hippocampal neurons derived from the ubiquitous inducible mouse model which is used as a screening tool for targeted interventions. A promising candidate is the repeat binding, RAN translation blocking, small molecule 1a. Small molecule 1a shields the disease-causing CGGexp from being translated into the toxic FMRpolyG protein. Primary hippocampal neurons formed FMRpolyG-positive inclusions, and upon treatment with 1a, the numbers of FMRpolyG-positive inclusions are reduced. We also describe for the first time the formation of FMRpolyG-positive inclusions in the liver of this mouse model. Treatment with 1a reduced the insoluble FMRpolyG protein fraction in the liver but not the number of inclusions. Moreover, 1a treatment had a reducing effect on the number of Rad23b-positive inclusions and insoluble Rad23b protein levels. These data suggest that targeted small molecule therapy is effective in an FXTAS mouse model and has the potential to treat CGGexp-mediated diseases, including FXTAS.

Selective rescue of heightened anxiety but not gait ataxia in a premutation 90CGG mouse model of Fragile X-associated tremor/ataxia syndrome.

A CGG-repeat expansion in the premutation range in the Fragile X mental retardation 1 gene (FMR1) has been identified as the genetic cause of Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder that manifests with action tremor, gait ataxia and cognitive impairments. In this study, we used a bigenic mouse model, in which expression of a 90CGG premutation tract is activated in neural cells upon doxycycline administration-P90CGG mouse model. We, here, demonstrate the behavioural manifestation of clinically relevant features of FXTAS patients and premutation carrier individuals in this inducible mouse model. P90CGG mice display heightened anxiety, deficits in motor coordination and impaired gait and represent the first FXTAS model that exhibits an ataxia phenotype as observed in patients. The behavioural phenotype is accompanied by the formation of ubiquitin/FMRpolyglycine-positive intranuclear inclusions, as another hallmark of FXTAS, in the cerebellum, hippocampus and amygdala. Strikingly, upon cessation of transgene induction the anxiety phenotype of mice recovers along with a reduction of intranuclear inclusions in dentate gyrus and amygdala. In contrast, motor function deteriorates further and no reduction in intranuclear inclusions can be observed in the cerebellum. Our data thus demonstrate that expression of a 90CGG premutation expansion outside of the FMR1 context is sufficient to evoke an FXTAS-like behavioural phenotype. Brain region-specific neuropathology and (partial) behavioural reversibility make the inducible P90CGG a valuable mouse model for testing pathogenic mechanisms and therapeutic intervention methods.

Reversibility of neuropathology and motor deficits in an inducible mouse model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.

PRKAR1B mutation associated with a new neurodegenerative disorder with unique pathology.

Pathological accumulation of intermediate filaments can be observed in neurodegenerative disorders, such as Alzheimer's disease, frontotemporal dementia and Parkinson's disease, and is also characteristic of neuronal intermediate filament inclusion disease. Intermediate filaments type IV include three neurofilament proteins (light, medium and heavy molecular weight neurofilament subunits) and α-internexin. The phosphorylation of intermediate filament proteins contributes to axonal growth, and is regulated by protein kinase A. Here we describe a family with a novel late-onset neurodegenerative disorder presenting with dementia and/or parkinsonism in 12 affected individuals. The disorder is characterized by a unique neuropathological phenotype displaying abundant neuronal inclusions by haematoxylin and eosin staining throughout the brain with immunoreactivity for intermediate filaments. Combining linkage analysis, exome sequencing and proteomics analysis, we identified a heterozygous c.149T>G (p.Leu50Arg) missense mutation in the gene encoding the protein kinase A type I-beta regulatory subunit (PRKAR1B). The pathogenicity of the mutation is supported by segregation in the family, absence in variant databases, and the specific accumulation of PRKAR1B in the inclusions in our cases associated with a specific biochemical pattern of PRKAR1B. Screening of PRKAR1B in 138 patients with Parkinson's disease and 56 patients with frontotemporal dementia did not identify additional novel pathogenic mutations. Our findings link a pathogenic PRKAR1B mutation to a novel hereditary neurodegenerative disorder and suggest an altered protein kinase A function through a reduced binding of the regulatory subunit to the A-kinase anchoring protein and the catalytic subunit of protein kinase A, which might result in subcellular dislocalization of the catalytic subunit and hyperphosphorylation of intermediate filaments.

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.

The clinical and pathological phenotype of C9ORF72 hexanucleotide repeat expansions.

There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39-76), and disease duration 7.6 ± 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation.

Widespread non-central nervous system organ pathology in fragile X premutation carriers with fragile X-associated tremor/ataxia syndrome and CGG knock-in mice.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder generally presenting with intention tremor and gait ataxia, but with a growing list of co-morbid medical conditions including hypothyroidism, hypertension, peripheral neuropathy, and cognitive decline. The pathological hallmark of FXTAS is the presence of intranuclear inclusions in both neurons and astroglia. However, it is unknown to what extent such inclusions are present outside the central nervous system (CNS). To address this issue, we surveyed non-CNS organs in ten human cases with FXTAS and in a CGG repeat knock-in (CGG KI) mouse model known to possess neuronal and astroglial inclusions. We find inclusions in multiple tissues from FXTAS cases and CGG KI mice, including pancreas, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart, and mitral valve, as well as throughout the associated autonomic ganglia. Inclusions were observed in the testes, epididymis, and kidney of FXTAS cases, but were not observed in mice. These observations demonstrate extensive involvement of the peripheral nervous system and systemic organs. The finding of intranuclear inclusions in non-CNS somatic organ systems, throughout the PNS, and in the enteric nervous system of both FXTAS cases as well as CGG KI mice suggests that these tissues may serve as potential sites to evaluate early intervention strategies or be used as diagnostic factors.

Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5'UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

Neuropathology of FMR1-premutation carriers presenting with dementia and neuropsychiatric symptoms.

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.

In silico, in vitro, and in vivo Approaches to Identify Molecular Players in Fragile X Tremor and Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative monogenetic disorder affecting carriers of premutation (PM) forms of the FMR1 gene, resulting in a progressive development of tremors, ataxia, and neuropsychological problems. This highly disabling disease is quite common in the general population with an estimation of about 20 million PM carriers worldwide. The chances of developing FXTAS increase dramatically with age, with about 45% of male carriers over the age of 50 being affected. Both the gene and pathogenic trigger, a mutant expansion of CGG RNA, causing FXTAS are known. This makes it an interesting disease to develop targeted therapeutic interventions for. Yet, no such interventions are available at this moment. Here we discuss in silico, in vitro, and in vivo approaches and how they have been used to identify the molecular determinants of FXTAS pathology. These approaches have yielded substantial information about FXTAS pathology and, consequently, many markers have emerged to play a key role in understanding the disease mechanism. Integration of the different approaches is expected to provide crucial information about the value of these markers as either therapeutic target or biomarker, essential to monitor therapeutic interventions in the future.

Lack of a Clear Behavioral Phenotype in an Inducible FXTAS Mouse Model Despite the Presence of Neuronal FMRpolyG-Positive Aggregates.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a rare neurodegenerative disorder caused by a 55-200 CGG repeat expansion in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. FXTAS is characterized by progressive cerebellar ataxia, Parkinsonism, intention tremors and cognitive decline. The main neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the brain. The molecular pathology of FXTAS involves the presence of 2 to 8-fold elevated levels of FMR1 mRNA, and of a repeat-associated non-AUG (RAN) translated polyglycine peptide (FMRpolyG). Increased levels of FMR1 mRNA containing an expanded CGG repeat can result in cellular toxicity by an RNA gain-of-function mechanism. The increased levels of CGG repeat-expanded FMR1 transcripts may create RNA foci that sequester important cellular proteins, including RNA-binding proteins and FMRpolyG, in intranuclear inclusions. To date, it is unclear whether the FMRpolyG-positive intranuclear inclusions are a cause or a consequence of FXTAS disease pathology. In this report we studied the relation between the presence of neuronal intranuclear inclusions and behavioral deficits using an inducible mouse model for FXTAS. Neuronal intranuclear inclusions were observed 4 weeks after dox-induction. After 12 weeks, high numbers of FMRpolyG-positive intranuclear inclusions could be detected in the hippocampus and striatum, but no clear signs of behavioral deficits related to these specific brain regions were found. In conclusion, the observations in our inducible mouse model for FXTAS suggest a lack of correlation between the presence of intranuclear FMRpolyG-positive aggregates in brain regions and specific behavioral phenotypes.

Gustatory plasticity in C. elegans involves integration of negative cues and NaCl taste mediated by serotonin, dopamine, and glutamate.

While naïve Caenorhabditis elegans individuals are attracted to 0.1-200 mM NaCl, they become strongly repelled by these NaCl concentrations after prolonged exposure to 100 mM NaCl. We call this behavior gustatory plasticity. Here, we show that C. elegans displays avoidance of low NaCl concentrations only when pre-exposure to NaCl is combined with a negative stimulus, e.g., a repellent, or in the absence of food. By testing serotonin and/or dopamine signaling mutants and rescue by exogenously supplying these neurotransmitters, we found that serotonin and dopamine play a role during the plasticity response, while serotonin is also required during development. In addition, we also show that glutamate plays an important role in the response to NaCl, both in chemoattraction to NaCl and in gustatory plasticity. Thus, C. elegans can associate NaCl with negative stimuli using dopaminergic, serotonergic, and glutamatergic neurotransmission. Finally, we show that prolonged starvation enhances gustatory plasticity and can induce avoidance of NaCl in most gustatory plasticity mutants tested. Only mutation of the glutamate-gated Cl(-) channel gene avr-15 affected starvation-enhanced gustatory plasticity. These results suggest that starvation induces avoidance of NaCl largely independent of the normal gustatory plasticity mechanism.

Reduction of Fmr1 mRNA Levels Rescues Pathological Features in Cortical Neurons in a Model of FXTAS.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55-200 CGG repeat expansion in the 5' UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches.

Refining the Spectrum of Neuronal Intranuclear Inclusion Disease: A Case Report.

Neuronal intranuclear inclusion disease (NIID) is a rare heterogeneous progressive neurodegenerative disease characterized by the presence of eosinophilic hyaline intranuclear inclusions in neuronal and glial cells of the CNS, peripheral cells of the autonomic nervous system, visceral organs and skin. The clinical presentation is broadly heterogeneous and includes limb weakness, dementia, seizures, ataxia, and parkinsonism. High-intensity signal in the corticomedullary junction on brain MRI is a characteristic finding in NIID. We describe a 65-year-old patient presenting with mild cognitive impairment, evolving in dementia with behavioral disturbances and parkinsonism. Brain MRI showed mild global cortical atrophy, more pronounced in the cingulate and temporal cortex and mild leukoaraiosis, but no high-intensity signal in corticomedullary junction on diffusion weighted imaging. Neuropathological examination showed p62- and optineurin-positive neuronal intranuclear inclusions in the hippocampus and in some subcortical structures. Glial cells did not present any intranuclear inclusions, and no spongiotic changes proximal to the U-fibers or diffuse myelin pallor were disclosed in the white matter. We report on a case with pathological features of NIID showing different neuroimaging and pathological findings. We noted an absence of typical MRI abnormalities, lack of intranuclear inclusions in glial cells, and prominent involvement of hippocampal neurons, refining the clinico-pathological spectrum of the disease.

Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology.

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5'-untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function.

CGG-repeat length and neuropathological and molecular correlates in a mouse model for fragile X-associated tremor/ataxia syndrome.

The 5'untranslated region (UTR) of the FMR1 gene contains a CGG-repeat, which may become unstable upon transmission to the next generation. When repeat length exceeds 200, the FMR1 gene generally undergoes methylation-mediated transcriptional silencing. The subsequent absence of the gene product Fragile X Mental Retardation Protein (FMRP)causes the mental retardation seen in fragile X patients. A CGG-repeat length between 55 and 200 trinucleotides has been termed the premutation (PM). Predominantly elderly male PM carriers are at risk of developing a progressive neurodegenerative disorder: fragile X-associated tremor/ataxia syndrome (FXTAS). All PM carriers have elevated FMR1 mRNA levels, in spite of slightly decreased FMRP levels. The presence of intranuclear ubiquitin-positive inclusions in many brain regions is a neuropathological hallmark of FXTAS. Studies in humans attempting to correlate neuropathological outcomes with molecular measures are difficult because of the limited availability of tissue. Therefore, we have used the expanded CGG-repeat knock-in mouse model of FXTAS to examine the relationship between the molecular and neuropathological parameters in brain. We present Fmr1 mRNA and Fmrp levels and the presence of intranuclear inclusions at different repeat lengths. Contrary to existing hypotheses, our results suggest that inclusion formation may not depend on the elevation per se of Fmr1 transcript levels in aged CGG mice.

Potential pathogenic mechanisms underlying Fragile X Tremor Ataxia Syndrome: RAN translation and/or RNA gain-of-function?

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disease caused by an expansion of 55-200 CGG repeats located in the FMR1 gene. The main clinical and neuropathological features of FXTAS are progressive intention tremor and gait ataxia associated with brain atrophy, neuronal cell loss and presence of ubiquitin-positive intranuclear inclusions in both neurons and astrocytes. At the molecular level, FXTAS is characterized by increased expression of FMR1 sense and antisense RNA containing expanded CGG or GGC repeats, respectively. Here, we discuss the putative molecular mechanisms underlying FXTAS and notably recent reports that expanded CGG and GGC repeats may be pathogenic through RAN translation into toxic proteins.

BC RNA Mislocalization in the Fragile X Premutation.

Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5' untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor-dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.

Paradoxical effect of baclofen on social behavior in the fragile X syndrome mouse model.

INTRODUCTION: Fragile X syndrome (FXS) is a common monogenetic cause of intellectual disability, autism spectrum features, and a broad range of other psychiatric and medical problems. FXS is caused by the lack of the fragile X mental retardation protein (FMRP), a translational regulator of specific mRNAs at the postsynaptic compartment. The absence of FMRP leads to aberrant synaptic plasticity, which is believed to be caused by an imbalance in excitatory and inhibitory network functioning of the synapse. Evidence from studies in mice demonstrates that GABA, the major inhibitory neurotransmitter in the brain, and its receptors, is involved in the pathogenesis of FXS. Moreover, several FXS phenotypes, including social behavior deficits, could be corrected in Fmr1 KO mice after acute treatment with GABAB agonists. METHODS: As FXS would probably require a lifelong treatment, we investigated the effect of chronic treatment with the GABAB agonist baclofen on social behavior in Fmr1 KO mice on two behavioral paradigms for social behavior: the automated tube test and the three-chamber sociability test. RESULTS: Unexpectedly, chronic baclofen treatment resulted in worsening of the FXS phenotypes in these behavior tests. Strikingly, baclofen treatment also affected wild-type animals in both behavioral tests, inducing a phenotype similar to that of untreated Fmr1 KO mice. CONCLUSION: Altogether, the disappointing results of recent clinical trials with the R-baclofen enantiomer arbaclofen and our current results indicate that baclofen should be reconsidered and further evaluated before its application in targeted treatment for FXS.

An Integrative Study of Protein-RNA Condensates Identifies Scaffolding RNAs and Reveals Players in Fragile X-Associated Tremor/Ataxia Syndrome.

Recent evidence indicates that specific RNAs promote the formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs). We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly contacted RNAs are structured, have long UTRs, and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3' UTR that harbors CGG expansions implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model, we validated the TRA2A-FMR1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post-mortem human samples. Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of therapeutic targets.