Pathogenesis Of Fragile X-associated Tremor/ataxia Syndrome Research Articles

Neurodegeneration of White and Gray Matter in the Hippocampus with FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects older premutation carriers (55-200 CGG repeats) of the fragile X gene. Despite the high prevalence of the FXTAS disorder, neuropathology studies of individuals affected by FXTAS are limited. We performed hematoxylin and eosin (H&E) staining in the hippocampus of 26 FXTAS cases and analyzed the tissue microscopically. The major neuropathological characteristics were white matter disease, intranuclear inclusions in neurons and astrocytes, and neuron loss. Astrocytes contained more and larger inclusions than neurons. There was a negative correlation between age of death and CGG repeat length in cases over the age of 60. The number of astroglial inclusions (CA3 and dentate gyrus) and the number of CA3 neuronal inclusions increased with elevated CGG repeat length. In the two cases with a CGG repeat size less than 65, FXTAS intranuclear inclusions were not present in the hippocampus, while in the two cases with less than 70 (65-70) CGG repeat expansion, neurons and astrocytes with inclusions were occasionally identified in the CA1 sub-region. These findings add hippocampus neuropathology to the previously reported changes in other areas of the brain in FXTAS patients, with implications for understanding FXTAS pathogenesis.

Structure and Alternative Splicing of the Antisense FMR1 (ASFMR1) Gene.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by an expansion of 55-200 CGG repeats (premutation) in the 5'-UTR of the FMR1 gene. Bidirectional transcription at FMR1 locus has been demonstrated and specific alternative splicing of the Antisense FMR1 (ASFMR1) gene has been proposed to have a contributing role in the pathogenesis of FXTAS. The structure of ASFMR1 gene is still uncharacterized and it is currently unknown how many isoforms of the gene are expressed and at what level in premutation carriers (PM) and if they may contribute to the premutation pathology. In this study, we characterized the ASFMR1 gene structure and the transcriptional landscape by using PacBio SMRT sequencing with target enrichment (IDT customized probe panel). We identified 45 ASFMR1 isoforms ranging in sizes from 523bp to 6Kb, spanning approximately 59kb of genomic DNA. Multiplexing and sequencing of six human brain samples from PM samples and normal control (HC) were carried out on the PacBio Sequel platform. We validated the presence of these isoforms by qRT-PCR and Sanger sequencing and characterized the acceptor and donor splicing site consensus sequences. Consistent with previous studies conducted in other tissue types, we found a high expression of ASFMR1 isoform Iso131bp in brain samples of PM as compared to HC, while no differences in expression levels were observed for the newly identified isoforms IsoAS1 and IsoAS2. We investigated the role of the splicing regulatory protein Sam68 which we did not observe in the alternative splicing of the ASFMR1 gene. Our study provides a useful insight into the structure of ASFMR1 gene and transcriptional landscape along with the expression pattern of various newly identified novel isoforms and on their potential role in premutation pathology.

Metabolomic Biomarkers Are Associated With Area of the Pons in Fragile X Premutation Carriers at Risk for Developing FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele (55–200 CGG repeats; PM) in the fragile X mental retardation (FMR1) gene. It is currently unknown how the observed brain changes are associated with metabolic signatures in individuals who develop the disorder over time. The primary objective of this study was to investigate the correlation between longitudinal changes in the brain (area of the pons, midbrain, and MCP width) and the changes in the expression level of metabolic biomarkers of early diagnosis and progression of FXTAS in PM who, as part of an ongoing longitudinal study, emerged into two distinct categories. These included those who developed symptoms of FXTAS (converters, CON) at subsequent visits and those who did not meet the criteria of diagnosis (non-converters, NCON) and were compared to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern Blot and PCR analysis. Magnetic Resonance Imaging (MRIs) acquisition was obtained on a 3T Siemens Trio scanner and metabolomic profile was obtained by ultra-performance liquid chromatography, accurate mass spectrometer, and an Orbitrap mass analyzer. Our findings indicate that differential metabolite levels are linked with the area of the pons between healthy control and premutation groups. More specifically, we observed a significant association of ceramides and mannonate metabolites with a decreased area of the pons, both at visit 1 (V1) and visit 2 (V2) only in the CON as compared to the NCON group suggesting their potential role in the development of the disorder. In addition, we found a significant correlation of these metabolic signatures with the FXTAS stage at V2 indicating their contribution to the progression and pathogenesis of FXTAS. Interestingly, these metabolites, as part of lipid and sphingolipid lipids pathways, provide evidence of the role that their dysregulation plays in the development of FXTAS and inform us as potential targets for personalized therapeutic development.

Human Cerebral Cortex Proteome of Fragile X-Associated Tremor/Ataxia Syndrome.

Background: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder associated with premutation CGG-repeat expansions (55–200 repeats) in the 5′ non-coding portion of the fragile X mental retardation 1 (FMR1) gene. Core features of FXTAS include progressive tremor/ataxia, cognitive decline, variable brain volume loss, and white matter disease. The principal histopathological feature of FXTAS is the presence of central nervous system (CNS) and non-CNS intranuclear inclusions.Objective: To further elucidate the molecular underpinnings of FXTAS through the proteomic characterization of human FXTAS cortexes.Results: Proteomic analysis of FXTAS brain cortical tissue (n = 8) identified minor differences in protein abundance compared to control brains (n = 6). Significant differences in FXTAS relative to control brain predominantly involved decreased abundance of proteins, with the greatest decreases observed for tenascin-C (TNC), cluster of differentiation 38 (CD38), and phosphoserine aminotransferase 1 (PSAT1); proteins typically increased in other neurodegenerative diseases. Proteins with the greatest increased abundance include potentially novel neurodegeneration-related proteins and small ubiquitin-like modifier 1/2 (SUMO1/2). The FMRpolyG peptide, proposed in models of FXTAS pathogenesis but only identified in trace amounts in the earlier study of FXTAS inclusions, was not identified in any of the FXTAS or control brains in the current study.Discussion: The observed proteomic shifts, while generally relatively modest, do show a bias toward decreased protein abundance with FXTAS. Such shifts in protein abundance also suggest altered RNA binding as well as loss of cell–cell adhesion/structural integrity. Unlike other neurodegenerative diseases, the proteome of end-stage FXTAS does not suggest a strong inflammation-mediated degenerative response.

Expression of expanded FMR1-CGG repeats alters mitochondrial miRNAs and modulates mitochondrial functions and cell death in cellular model of FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats located within 5′UTR of FMR1.These CGG repeats are transcribed into RNAs, which sequester several RNA binding proteins and alter the processing of miRNAs. CGG repeats are also translated into a toxic polyglycine-containing protein, FMRpolyG, that affects mitochondrial and nuclear functions reported in cell and animal models and patient studies. Nuclear-encoded small non-coding RNAs, including miRNAs, are transported to mitochondria; however, the role of mitochondrial miRNAs in FXTAS pathogenesis is not understood. Here, we analyzed mitochondrial miRNAs from HEK293 cells expressing expanded CGG repeats and their implication in the regulation of mitochondrial functions. The analysis of next generation sequencing (NGS) data of small RNAs from HEK293 cells expressing CGG premutation showed decreased level of cellular miRNAs and an altered pattern of association of miRNAs with mitochondria (mito-miRs). Among such mito-miRs, miR-320a was highly enriched in mitoplast and RNA immunoprecipitation of Ago2 (Argonaute-2) followed by Droplet digital PCR (ddPCR)suggested that miR-320a may form a complex with Ago2 and mitotranscripts. Finally, transfection of miR-320a mimic in cells expressing CGG permutation recovers mitochondrial functions and rescues cell death. Overall, this work reveals an altered translocation of miRNAs to mitochondria and the role of miR-320a in FXTAS pathology.

FMR1 locus isoforms: potential biomarker candidates in fragile X-associated tremor/ataxia syndrome (FXTAS)

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder that affects movement and cognition in male and female carriers of a premutation allele of 55–200 CGG repeats in the Fragile X mental retardation (FMR1) gene. It is currently unknown if and when an individual carrier of a premutation allele will develop FXTAS, as clinical assessment fails to identify carriers at risk before significant neurological symptoms are evident. The primary objective of this study was to investigate the alternative splicing landscape at the FMR1 locus in conjunction with brain measures in male individuals with a premutation allele enrolled in a very first longitudinal study, compared to age-matched healthy male controls, with the purpose of identifying biomarkers for early diagnosis, disease prediction and, a progression of FXTAS. Our findings indicate that increased expression of FMR1 mRNA isoforms, including Iso4/4b, Iso10/10b, as well as of the ASFMR1 mRNAs Iso131bp, are present in premutation carriers as compared to non-carrier healthy controls. More specifically, we observed a higher expression of Iso4/4b and Iso10/10b, which encode for truncated proteins, only in those premutation carriers who developed symptoms of FXTAS over time as compared to non-carrier healthy controls, suggesting a potential role in the development of the disorder. In addition, we found a significant association of these molecular changes with various measurements of brain morphology, including the middle cerebellar peduncle (MCP), superior cerebellar peduncle (SCP), pons, and midbrain, indicating their potential contribution to the pathogenesis of FXTAS. Interestingly, the high expression levels of Iso4/4b observed both at visit 1 and visit 2 and found to be associated with a decrease in mean MCP width only in those individuals who developed FXTAS over time, suggests their role as potential biomarkers for early diagnosis of FXTAS.

Composition of the Intranuclear Inclusions of Fragile X-associated Tremor/Ataxia Syndrome

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder associated with a premutation repeat expansion (55–200 CGG repeats) in the 5′ noncoding region of the FMR1 gene. Solitary intranuclear inclusions within FXTAS neurons and astrocytes constitute a hallmark of the disorder, yet our understanding of how and why these bodies form is limited. Here, we have discovered that FXTAS inclusions emit a distinct autofluorescence spectrum, which forms the basis of a novel, unbiased method for isolating FXTAS inclusions by preparative fluorescence-activated cell sorting (FACS). Using a combination of autofluorescence-based FACS and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based proteomics, we have identified more than two hundred proteins that are enriched within the inclusions relative to FXTAS whole nuclei. Whereas no single protein species dominates inclusion composition, highly enriched levels of conjugated small ubiquitin-related modifier 2 (SUMO 2) protein and p62/sequestosome-1 (p62/SQSTM1) protein were found within the inclusions. Many additional proteins involved with RNA binding, protein turnover, and DNA damage repair were enriched within inclusions relative to total nuclear protein. The current analysis has also allowed the first direct detection, through peptide sequencing, of endogenous FMRpolyG peptide, the product of repeat-associated non-ATG (RAN) translation of the FMR1 mRNA. However, this peptide was found only at extremely low levels and not within whole FXTAS nuclear preparations, raising the question whether endogenous RAN products exist at quantities sufficient to contribute to FXTAS pathogenesis. The abundance of the inclusion-associated ubiquitin- and SUMO-based modifiers supports a model for inclusion formation as the result of increased protein loads and elevated oxidative stress leading to maladaptive autophagy. These results highlight the need to further investigate FXTAS pathogenesis in the context of endogenous systems.

Fragile X-associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in at least one-third of adult carriers of a premutation (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.

The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG-repeat expansion in the 5′ UTR of the FMR1 gene on the X-chromosome. Both elevated levels of the expanded FMR1 mRNA and aberrant expression of a polyglycine protein (FMRpolyG) from the CGG-repeat region are hypothesized to trigger the pathogenesis of FXTAS. While increased expression of FMRpolyG leads to higher toxicity in FXTAS models, the pathogenic effect of this protein has only been studied in the presence of CGG-containing mRNA. Here we present a model that allows measurement of the effect of FMRpolyG-expression without co-expression of the corresponding CGG mRNA hairpin. This allows direct comparison of the effect of the FMRpolyG protein per se, vs. that of the FMRpolyG protein together with the CGG mRNA hairpin. Our results show that expression of the FMRpolyG, in the absence of any CGG mRNA, is sufficient to cause reduced cell viability, lamin ring disruption and aggregate formation. Furthermore, we found FMRpolyG to be a long-lived protein degraded primarily by the ubiquitin-proteasome-system. Together, our data indicate that accumulation of FMRpolyG protein per se may play a major role in the development of FXTAS.

Metabolic pathways modulate the neuronal toxicity associated with fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects premutation carriers (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Much remains unknown regarding the metabolic alterations associated with FXTAS, especially in the brain, and the most affected region, the cerebellum. Investigating the metabolic changes in FXTAS will aid in the identification of biomarkers as well as in understanding the pathogenesis of disease. To identify the metabolic alterations associated with FXTAS, we took advantage of our FXTAS mouse model that expresses 90 CGG repeats in cerebellar Purkinje neurons and exhibits the key phenotypic features of FXTAS. We performed untargeted global metabolic profiling of age-matched control and FXTAS mice cerebella at 16-20 weeks and 55 weeks. Out of 506 metabolites measured in cerebellum, we identified 186 metabolites that demonstrate significant perturbations due to the (CGG)90 repeat (P<0.05) and found that these differences increase dramatically with age. To identify key metabolic changes in FXTAS pathogenesis, we performed a genetic screen using a Drosophila model of FXTAS. Out of 28 genes that we tested in the fly, 8 genes showed significant enhanced neuronal toxicity associated with CGG repeats, such as Schlank (ceramide synthase), Sk2 (sphingosine kinase) and Ras (IMP dehydrogenase). By combining metabolic profiling with a Drosophila genetic screen to identify genetic modifiers of FXTAS, we demonstrate an effective method for functional validation of high-throughput metabolic data and show that sphingolipid and purine metabolism are significantly perturbed in FXTAS pathogenesis.

Impaired Mitochondrial Function and Dynamics in the Pathogenesis of FXTAS

Mitochondrial involvement plays an important role in neurodegenerative diseases. At least one-third of adult carriers of a FMR1 premutation (55-200 CGG repeats) are at risk of presenting an adult-onset neurodegenerative disorder known as fragile X-associated tremor/ataxia syndrome (FXTAS). In an attempt to provide new insights into the mechanisms involved in the pathogenesis of FXTAS, we characterized mitochondrial function and dynamics by the assessment of oxidative respiratory chain function, mitochondrial content, oxidative stress levels, and mitochondrial network complexity. Regarding mitochondrial function, we found that mitochondrial respiratory capacity is compromised in skin fibroblasts whereas in blood, no differences were observed between the FXTAS and control groups. Furthermore, fibroblasts from FXTAS patients presented altered mitochondrial architecture, with more circular and less interconnected mitochondria being observed. Mitochondrial function and dynamics deregulation and characteristic of neurological disorders are present in FXTAS patients. These features might be limiting temporal and spatial bioenergetics cells supply and thus contributing to disease pathogenesis.

Molecular Pathophysiology of Fragile X-Associated Tremor/Ataxia Syndrome and Perspectives for Drug Development.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder manifesting in carriers of 55 to 200 CGG repeats in the 5' untranslated region (UTR) of the fragile X mental retardation gene (FMR1). FXTAS is characterized by enhanced FMR1 transcription and the accumulation of CGG repeat-containing FMR1 messenger RNA in nuclear foci, while the FMRP protein expression levels remain normal or moderately low. The neuropathological hallmark in FXTAS is the presence of intranuclear, ubiquitin-positive inclusions that also contain FMR1 transcript. Yet, the complete protein complement of FXTAS inclusions and the molecular events that trigger neuronal death in FXTAS remain unclear. In this review, we present the two most accepted toxicity mechanisms described so far, namely RNA gain-of-function and protein gain-of-function by means of repeat-associated non-AUG translation, and discuss current experimental and computational strategies to better understand FXTAS pathogenesis. Finally, we review the current perspectives for drug development with disease-modifying potential for FXTAS.

Cerebellar Mild Iron Accumulation in a Subset of FMR1 Premutation Carriers with FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with premutation alleles of the FMR1 gene. Iron is essential for many facets of cell metabolism in the brain but when altered is likely to contribute to the development of neurodegenerative diseases. We previously reported that iron accumulates in the choroid plexus and the putamen in FXTAS and that the level and distribution of key iron-binding proteins are also altered, suggesting a potential alteration of iron metabolism in the brain. Here, we hypothesize that iron metabolism is also altered in the FXTAS cerebellum. To test this hypothesis, we used cerebellum samples collected from FXTAS and control subjects and measured the amount of iron contained within the cerebellar cortex and dentate nucleus. We found that the number of iron deposits increased in the cerebellum onlyin a subset of cases of FXTAS. This accumulation is likely to be mediated by factors other than or in addition to CGG-repeat coupled pathology. Thus, iron deposition in the cerebellum cannot be used as a hallmark of FXTAS pathogenesis.

Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) Motor Dysfunction Modeled in Mice.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects some carriers of the fragile X premutation (PM). In PM carriers, there is a moderate expansion of a CGG trinucleotide sequence (55-200 repeats) in the fragile X gene (FMR1) leading to increased FMR1 mRNA and small to moderate decreases in the fragile X mental retardation protein (FMRP) expression. The key symptoms of FXTAS include cerebellar gait ataxia, kinetic tremor, sensorimotor deficits, neuropsychiatric changes, and dementia. While the specific trigger(s) that causes PM carriers to progress to FXTAS pathogenesis remains elusive, the use of animal models has shed light on the underlying neurobiology of the altered pathways involved in disease development. In this review, we examine the current use of mouse models to study PM and FXTAS, focusing on recent advances in the field. Specifically, we will discuss the construct, face, and predictive validities of these PM mouse models, the insights into the underlying disease mechanisms, and potential treatments.

Fragile X-Associated Tremor-Ataxia Syndrome: Linking Ca2+ Dysregulation and DNA Damage Responses

Fragile X-associated tremor-ataxia syndrome (FXTAS) is a late-onset, neurodegenerative disorder that affects carriers of the premutation CGG-repeat allele (preCGG; 55-200 repeats) of the fragile X mental retardation 1 (FMR1) gene. Early abnormal growth and Ca2+ dynamics in FMR1 premutation preCGG knock-in mice and human iPSCs-derived preCGG neurons precede neurodegeneration and FXTAS. Whether intra-nuclear inclusions containing DNA damage response (DDR) proteins, pathologic hallmark of FXTAS, causally link Ca2+ dysregulation with abnormal neuronal growth and survival is unknown. We hypothesize a role for CdK5 and ATM in FXTAS pathogenesis, two key signaling kinases involved in both DDR and synaptic signaling. FMRP expression in postnatal day 0 (P0), 6-week- and 6-month-old preCGG mouse brain is reduced 60-, 50- and 30% compared to wt, respectively. In primary hippocampal neurons, FMRP reduction is more pronounced in preCGG soma (80%, p<0.0001) compared to neurites (94%, p=0.0177). Resting cytoplasmic calcium concentration ([Ca2+]i) in preCGG hippocampal neurons is chronically elevated 3-fold compared to wt at 7 DIV and 14 DIV, and can be reversed by acute exposure to 10μM dantrolene. Elevated [Ca2+]i-dependent calpain activity (120%-140%) and p25/p35 (120%) at 6-weeks and 6-months in preCGG cortex indicate abnormal CdK5 regulation. CdK5 substrate ATM is upregulated 150 to 200% and P-Ser1981-ATM is 150% (p= p<0.01-0.007) of wt at P0 and 6 months in preCGG brain. We propose that chronic Ca2+ dysregulation amplifies Cdk5/ATM signaling pathway activity possibly linking impaired DDR and neurodegeneration leading to FXTAS with early abnormal [Ca2+]i and synaptic activity.

Fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects some but not all carriers of small, noncoding CGG-repeat expansions (55-200 repeats; premutation) within the fragile X gene (FMR1). Principal features of FXTAS include intention tremor, cerebellar ataxia, Parkinsonism, memory and executive function deficits, autonomic dysfunction, brain atrophy with white matter disease, and cognitive decline. Although FXTAS was originally considered to be confined to the premutation range, rare individuals with a gray zone (45-54 repeats) or an unmethylated full mutation (>200 repeats) allele have now been described, the constant feature of the disorder remaining the requirement for FMR1 expression, in contradistinction to the gene silencing mechanism of fragile X syndrome. Although transcriptional activity is required for FXTAS pathogenesis, the specific trigger(s) for FXTAS pathogenesis remains elusive, highlighting the need for more research in this area. This need is underscored by recent neuroimaging findings of changes in the central nervous system that consistently appear well before the onset of clinical symptoms, thus creating an opportunity to delay or prevent the appearance of FXTAS.

Induced expression of expanded CGG RNA causes mitochondrial dysfunction in vivo

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of premutation forms of the FMR1 gene, resulting in a progressive development of tremor, ataxia and neuropsychological problems. The disease is caused by an expanded CGG repeat in the FMR1 gene, leading to an RNA gain-of-function toxicity mechanism. In order to study the pathogenesis of FXTAS, new inducible transgenic mouse models have been developed that expresses either 11CGGs or 90CGGs at the RNA level under control of a Tet-On promoter. When bred to an hnRNP-rtTA driver line, doxycycline (dox) induced expression of the transgene could be found in almost all tissues. Dox exposure resulted in loss of weight and death within 5 d for the 90CGG RNA expressing mice. Immunohistochemical examination of tissues of these mice revealed steatosis and apoptosis in the liver. Decreased expression of GPX1 and increased expression of cytochrome C is found. These effects were not seen in mice expressing a normal sized 11CGG repeat. In conclusion, we were able to show in vivo that expression of an expanded CGG-repeat rather than overexpression of a normal CGG-repeat causes pathology. In addition, we have shown that expanded CGG RNA expression can cause mitochondrial dysfunction by regulating expression levels of several markers. Although FTXAS patients do not display liver abnormalities, our findings contribute to understanding of the molecular mechanisms underlying toxicity of CGG repeat RNA expression in an animal model. In addition, the dox inducible mouse lines offer new opportunities to study therapeutic interventions for FXTAS.

Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in Caenorhabditis elegans

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS): pathology and mechanisms

Since its discovery in 2001, our understanding of fragile X-associated tremor/ataxia syndrome (FXTAS) has undergone a remarkable transformation. Initially characterized rather narrowly as an adult-onset movement disorder, the definition of FXTAS is broadening; moreover, the disorder is now recognized as only one facet of a much broader clinical pleiotropy among children and adults who carry premutation alleles of the FMR1 gene. Furthermore, the intranuclear inclusions of FXTAS, once thought to be a CNS-specific marker of the disorder, are now known to be widely distributed in multiple non-CNS tissues; this observation fundamentally changes our concept of the disease, and may provide the basis for understanding the diverse medical problems associated with the premutation. Recent work on the pathogenic mechanisms underlying FXTAS indicates that the origins of the late-onset neurodegenerative disorder actually lie in early development, raising the likelihood that all forms of clinical involvement among premutation carriers have a common underlying mechanistic basis. There has also been great progress in our understanding of the triggering event(s) in FXTAS pathogenesis, which is now thought to involve sequestration of one or more nuclear proteins involved with microRNA biogenesis. Moreover, there is mounting evidence that mitochondrial dysregulation contributes to the decreased cell function and loss of viability, evident in mice even during the neonatal period. Taken together, these recent findings offer hope for early interventions for FXTAS, well before the onset of overt disease, and for the treatment of other forms of clinical involvement among premutation carriers.

Current Gaps in Understanding the Molecular Basis of FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder with defining clinical features that include kinetic tremor, gait ataxia, and parkinsonism, with associated features spanning medical, cognitive, and psychiatric clinical domains. The emerging model for the pathogenesis of FXTAS is that of RNA toxicity as a consequence of the sequestration of RNA binding proteins by the expanded CGG-repeat element within the FMR1 message, thus compromising the normal functions of those proteins. A principal challenge at this point is to determine precisely which proteins are involved in FXTAS pathogenesis and how to prevent or reverse this process. A second challenge is to determine why there is incomplete penetrance of FXTAS among premutation carriers with identical CGG-repeat lengths, and what the protective factors are in some carriers. Finally, the discovery in premutation mice of early neurodevelopmental abnormalities, some occurring even during late embryogenesis, raises the question of whether FXTAS is the end-stage of a life-long process of neuronal dysregulation. If an extended pre-clinical phase precedes the development of FXTAS, there is great potential for therapeutic intervention, years or even decades before its clinical features are manifest.