Fragile X Syndrome: Lessons Learned and What New Treatment Avenues Are on the Horizon.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading single-gene form of autism spectrum disorder, encompassing cognitive, behavioral, and physical forms of clinical involvement. FXS is caused by large expansions of a noncoding CGG repeat (>200 repeats) in the FMR1 gene, at which point the gene is generally silenced. Absence of FMR1 protein (FMRP), important for synaptic development and maintenance, gives rise to the neurodevelopmental disorder. There is, at present, no therapeutic approach that directly reverses the loss of FMRP; however, there is an increasing number of potential treatments that target the pathways dysregulated in FXS, including those that address the enhanced activity of the mGluR5 pathway and deficits in GABA pathways. Based on studies of targeted therapeutics to date, the prospects are good for one or more effective therapies for FXS in the near future.

Hypermobile Ehlers-Danlos syndrome (hEDS) phenotype in fragile X premutation carriers: case series.

BACKGROUND: While an association between full mutation CGG-repeat expansions of the Fragile X Mental Retardation 1 (FMR1) gene and connective tissue problems are clearly described, problems in fragile X premutation carriers (fXPCs) CGG-repeat range (55-200 repeats) of the FMR1 gene may be overlooked. OBJECTIVE: To report five FMR1 fXPCs cases with the hypermobile Ehlers-Danlos syndrome (hEDS) phenotype. METHODS: We collected medical histories and FMR1 molecular measures from five cases who presented with joint hypermobility and loose connective tissue and met inclusion criteria for hEDS. RESULTS: Five cases were female and ranged between 16 and 49 years. The range of CGG-repeat allele sizes ranged from 66 to 150 repeats. All had symptoms of hEDS since early childhood. Commonalities in molecular pathogenesis and coexisting conditions between the fXPCs and hEDS are also presented. The premutation can lead to a reduction of fragile X mental retardation protein, which is crucial in maintaining functions of the extracellular matrix-related proteins, particularly matrix metallopeptidase 9 and elastin. Moreover, elevated FMR1 messenger RNA causes sequestration of proteins, which results in RNA toxicity. CONCLUSION: Both hEDS phenotype and premutation involvement may co-occur because of related commonalities in pathogenesis.

Fragile X-associated tremor/ataxia syndrome: pathophysiology and management.

PURPOSE OF REVIEW: The purpose of this paper is to review the prevalence, pathophysiology, and management of fragile X-associated tremor/ataxia syndrome (FXTAS). RECENT FINDINGS: The pathophysiology of FXTAS involves ribonucleic acid (RNA) toxicity due to elevated levels of the premutation-expanded CGG (eoxycytidylate-deoxyguanylate-deoxyguanylate)-repeat FMR1 mRNA, which can sequester a variety of proteins important for neuronal function. A recent analysis of the inclusions in FXTAS demonstrates elevated levels of several proteins, including small ubiquitin-related modifiers 1/2 (SUMO1/2), that target molecules for the proteasome, suggesting that some aspect(s) of proteasomal function may be altered in FXTAS. Recent neuropathological studies show that Parkinson disease and Alzheimer disease can sometimes co-occur with FXTAS. Lewy bodies can be found in 10% of the brains of patients with FXTAS. Microbleeds and iron deposition are also common in the neuropathology, in addition to white matter disease (WMD) and atrophy. SUMMARY: The premutation occurs in 1:200 females and 1:400 males. Penetrance for FXTAS increases with age, though lower in females (16%) compared to over 60% of males by age 70. To diagnose FXTAS, an MRI is essential to document the presence of WMD, a primary component of the diagnostic criteria. Pain can be a significant feature of FXTAS and is seen in approximately 50% of patients.

Calcium dysregulation and Cdk5-ATM pathway involved in a mouse model of fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurological disorder that affects premutation carriers with 55-200 CGG-expansion repeats (preCGG) in FMR1, presenting with early alterations in neuronal network formation and function that precede neurodegeneration. Whether intranuclear inclusions containing DNA damage response (DDR) proteins are causally linked to abnormal synaptic function, neuronal growth and survival are unknown. In a mouse that harbors a premutation CGG expansion (preCGG), cortical and hippocampal FMRP expression is moderately reduced from birth through adulthood, with greater FMRP reductions in the soma than in the neurite, despite several-fold elevation of Fmr1 mRNA levels. Resting cytoplasmic calcium concentration ([Ca2+]i) in cultured preCGG hippocampal neurons is chronically elevated, 3-fold compared to Wt; elevated ROS and abnormal glutamatergic responses are detected at 14 DIV. Elevated µ-calpain activity and a higher p25/p35 ratio in the cortex of preCGG young adult mice indicate abnormal Cdk5 regulation. In support, the Cdk5 substrate, ATM, is upregulated by 1.5- to 2-fold at P0 and 6 months in preCGG brain, as is p-Ser1981-ATM. Bax:Bcl-2 is 30% higher in preCGG brain, indicating a greater vulnerability to apoptotic activation. Elevated [Ca2+]i, ROS, and DDR signals are normalized with dantrolene. Chronic [Ca2+]i dysregulation amplifies Cdk5-ATM signaling, possibly linking impaired glutamatergic signaling and DDR to neurodegeneration in preCGG brain.

Fragile X syndrome and connective tissue dysregulation.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disabilities and autism spectrum disorders, and it is an X-linked disorder in which there is a deficiency of the fragile X mental retardation 1 protein. This protein is crucial in regulating translation of mRNAs related to dendritic maturation and cognitive development. The phenotype of FXS is characterized by neurobehavioral alterations, social deficits, communication difficulties, and findings which suggest an alteration of connective tissue, especially in the ligaments and muscles, cardiovascular system and genitourinary system. Connective tissue connects and supports all other tissues of the body and is composed of cells and extracellular matrix (ECM). Several proteins have been involved in the connective tissue abnormalities associated with the FXS, such as matrix metalloproteinase 9, which plays an important role in the homeostasis of the ECM, being a potential therapeutic target for certain tetracycline antibiotics that have shown beneficial effects in FXS. Here, we review connective tissue problems described in FXS.

Microglial cell activation and senescence are characteristic of the pathology FXTAS.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with premutation alleles of the FMR1 gene. Expansions of more than 200 CGG repeats give rise to fragile X syndrome, the most common inherited form of cognitive impairment. Fragile X-associated tremor/ataxia syndrome is characterized by cerebellar tremor and ataxia, and the presence of ubiquitin-positive inclusions in neurons and astrocytes. It has been previously suggested that fragile X-associated tremor/ataxia syndrome is associated with an inflammatory state based on signs of oxidative stress-mediated damage and iron deposition. OBJECTIVE: Determine whether the pathology of fragile X-associated tremor/ataxia syndrome involves microglial activation and an inflammatory state. METHODS: Using ionized calcium binding adaptor molecule 1 and cluster differentiation 68 antibodies to label microglia, we examined the number and state of activation of microglial cells in the putamen of 13 fragile X-associated tremor/ataxia syndrome and 9 control postmortem cases. RESULTS: Nearly half of fragile X-associated tremor/ataxia syndrome cases (6 of 13) presented with dystrophic senescent microglial cells. In the remaining fragile X-associated tremor/ataxia syndrome cases (7 of 13), the number of microglial cells and their activation state were increased compared to controls. CONCLUSIONS: The presence of senescent microglial cells in half of fragile X-associated tremor/ataxia syndrome cases suggests that this indicator could be used, together with the presence of intranuclear inclusions and the presence of iron deposits, as a biomarker to aid in the postmortem diagnosis of fragile X-associated tremor/ataxia syndrome. An increased number and activation indicate that microglial cells play a role in the inflammatory state present in the fragile X-associated tremor/ataxia syndrome brain. Anti-inflammatory treatment of patients with fragile X-associated tremor/ataxia syndrome may be indicated to slow neurodegeneration. © 2018 International Parkinson and Movement Disorder Society.

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.

Iron accumulation and dysregulation in the putamen in fragile X-associated tremor/ataxia syndrome.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome is an adult-onset disorder associated with premutation alleles of the FMR1 gene. This disorder is characterized by progressive action tremor, gait ataxia, and cognitive decline. Fragile X-associated tremor/ataxia syndrome pathology includes dystrophic white matter and intranuclear inclusions in neurons and astrocytes. We previously demonstrated that the transport of iron into the brain is altered in fragile X-associated tremor/ataxia syndrome; therefore, we also expect an alteration of iron metabolism in brain areas related to motor control. Iron is essential for cell metabolism, but uncomplexed iron leads to oxidative stress and contributes to the development of neurodegenerative diseases. We investigated a potential iron modification in the putamen - a structure that participates in motor learning and performance - in fragile X-associated tremor/ataxia syndrome. METHODS: We used samples of putamen obtained from 9 fragile X-associated tremor/ataxia syndrome and 9 control cases to study iron localization using Perl's method, and iron-binding proteins using immunostaining. RESULTS: We found increased iron deposition in neuronal and glial cells in the putamen in fragile X-associated tremor/ataxia syndrome. We also found a generalized decrease in the amount of the iron-binding proteins transferrin and ceruloplasmin, and decreased number of neurons and glial cells that contained ceruloplasmin. However, we found increased levels of iron, transferrin, and ceruloplasmin in microglial cells, indicating an attempt by the immune system to remove the excess iron. CONCLUSIONS: Overall, found a deficit in proteins that eliminate extra iron from the cells with a concomitant increase in the deposit of cellular iron in the putamen in Fragile X-associated tremor/ataxia syndrome. © 2017 International Parkinson and Movement Disorder Society.

Transcription-associated R-loop formation across the human FMR1 CGG-repeat region.

Expansion of a trinucleotide (CGG) repeat element within the 5' untranslated region (5'UTR) of the human FMR1 gene is responsible for a number of heritable disorders operating through distinct pathogenic mechanisms: gene silencing for fragile X syndrome (>200 CGG) and RNA toxic gain-of-function for FXTAS (∼ 55-200 CGG). Existing models have focused almost exclusively on post-transcriptional mechanisms, but co-transcriptional processes could also contribute to the molecular dysfunction of FMR1. We have observed that transcription through the GC-rich FMR1 5'UTR region favors R-loop formation, with the nascent (G-rich) RNA forming a stable RNA:DNA hybrid with the template DNA strand, thereby displacing the non-template DNA strand. Using DNA:RNA (hybrid) immunoprecipitation (DRIP) of genomic DNA from cultured human dermal fibroblasts with both normal (∼ 30 CGG repeats) and premutation (55

Concomitant occurrence of FXTAS and clinically defined sporadic inclusion body myositis: report of two cases.

This report describes unique presentations of inclusion body myositis (IBM) in two unrelated patients, one male and one female, with genetically and histologically confirmed fragile X-associated tremor/ataxia syndrome (FXTAS). We summarize overlapping symptoms between two disorders, clinical course, and histopathological analyses of the two patients with FXTAS and sporadic IBM, clinically defined per diagnostic criteria of the European Neuromuscular Centre. In case 1, a post-mortem analysis of available brain and muscle tissues is also described. Histopathological features (rimmed vacuoles) consistent with clinically defined IBM were detected in both presented cases. Postmortem testing in case 1 revealed the presence of an FMR1 premutation allele of 60 CGG repeats in both brain and skeletal muscle samples. Case 2 was a premutation carrier with 71 CGG repeats who had a son with FXS. Given that FXTAS is associated with immune-mediated disorders among premutation carriers, it is likely that the pathogeneses of IBM and FXTAS are linked. This is, to our knowledge, the first report of these two conditions presenting together, which expands our understanding of clinical symptoms and unusual presentations in patients with FXTAS. Following detection of a premutation allele of the FMR1 gene, FXTAS patients with severe muscle pain should be assessed for IBM.

CNS expression of murine fragile X protein (FMRP) as a function of CGG-repeat size.

Large expansions of a CGG-repeat element (>200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.

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.

Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene.

The human fragile X mental retardation 1 (FMR1) gene contains a (CGG)(n) trinucleotide repeat in its 5' untranslated region (5'UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, greater than 200 CGG repeats) and fragile X-associated tremor/ataxia syndrome (FXTAS; premutation range, 55-200 repeats). Study of these diseases has been limited by an inability to sequence expanded CGG repeats, particularly in the full mutation range, with existing DNA sequencing technologies. Single-molecule, real-time (SMRT) sequencing provides an approach to sequencing that is fundamentally different from other "next-generation" sequencing platforms, and is well suited for long, repetitive DNA sequences. We report the first sequence data for expanded CGG-repeat FMR1 alleles in the full mutation range that reveal the confounding effects of CGG-repeat tracts on both cloning and PCR. A unique feature of SMRT sequencing is its ability to yield real-time information on the rates of nucleoside addition by the tethered DNA polymerase; for the CGG-repeat alleles, we find a strand-specific effect of CGG-repeat DNA on the interpulse distance. This kinetic signature reveals a novel aspect of the repeat element; namely, that the particular G bias within the CGG/CCG-repeat element influences polymerase activity in a manner that extends beyond simple nearest-neighbor effects. These observations provide a baseline for future kinetic studies of repeat elements, as well as for studies of epigenetic and other chemical modifications thereof.

Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications.

A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA. In conjunction with the single-molecule real-time sequencing approach, this enrichment method enables direct readout of the methylation status and the CGG repeat number of the FMR1 allele(s) for a clonally derived cell line. The current method avoids potential biases introduced through chemical modification and/or amplification methods for indirect detection of CpG methylation events.

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 only in 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.

A Majority of FXTAS Cases Present with Intranuclear Inclusions Within Purkinje Cells.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder that affects carriers of a FMR1 premutation. Symptoms include cerebellar ataxia, tremor, and cognitive deficits. The most characteristic pathology of FXTAS is the presence of eosinophilic ubiquitin-positive intranuclear inclusions in neurons and astrocytes throughout the nervous system and non-nervous tissues. Inclusions are present in neurons throughout the brain but are widely believed not to be present in the Purkinje cells (PCs) of the cerebellum. However, we analyzed 26 postmortem cases of FXTAS and demonstrated that 65 % of cases presented with inclusions within PCs of the cerebellum. We determined that the presence or absence of inclusions in PCs is correlated with age and that those cases with PC inclusions were overall 11 years older than those with no PC inclusions. Half of the cases with PCs with inclusions presented with twin nuclear inclusions. This novel finding demonstrating the presence of inclusions within PCs provides an insight into the understanding of the FXTAS motor symptoms and provides a novel target for the development of therapeutic strategies.

Differential increases of specific FMR1 mRNA isoforms in premutation carriers.

BACKGROUND: Over 40% of male and ∼16% of female carriers of a premutation FMR1 allele (55-200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology. METHODS: To address this question, we have applied a long-read sequencing approach using single-molecule real-time (SMRT) sequencing and qRT-PCR. RESULTS: Our SMRT sequencing analysis performed on peripheral blood mononuclear cells, fibroblasts and brain tissue samples derived from premutation carriers and controls revealed the existence of 16 isoforms of 24 predicted variants. Although the relative abundance of all mRNA isoforms was significantly increased in the premutation group, as expected based on the bulk increase in mRNA levels, there was a disproportionate (fourfold to sixfold) increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14, specifically Iso10 and Iso10b, containing the complete exon 15 and differing only in splicing in exon 17. CONCLUSIONS: These findings suggest that RNA toxicity may arise from a relative increase of all FMR1 mRNA isoforms. Interestingly, the Iso10 and Iso10b mRNA isoforms, lacking the C-terminal functional sites for fragile X mental retardation protein function, are the most increased in premutation carriers relative to normal, suggesting a functional relevance in the pathology of FMR1-associated disorders.

Enhanced asynchronous Ca(2+) oscillations associated with impaired glutamate transport in cortical astrocytes expressing Fmr1 gene premutation expansion.

BACKGROUND: FMR1 CGG expansion repeats in the premutation range have not been linked to astrocyte pathophysiology. RESULTS: Premutation cortical astrocytes display decreased Glu transporter expression/activity and enhanced asynchronous Ca(2+) oscillations. CONCLUSION: Glu transport and Ca(2+) signaling defects in premutation astrocytes could contribute to FXTAS neuropathology. SIGNIFICANCE: Premutation astrocytes may have an etiological role in FXTAS neuropathology. Premutation CGG repeat expansions (55-200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene can cause fragile X-associated tremor/ataxia syndrome. Defects in early neuronal migration and morphology, electrophysiological activity, and mitochondria trafficking have been described in a premutation mouse model, but whether preCGG mutations also affect astrocyte function remains unknown. PreCGG cortical astrocytes (∼170 CGG repeats) displayed 3-fold higher Fmr1 mRNA and 30% lower FMR1 protein (FMRP) when compared with WT. PreCGG astrocytes showed modest reductions in expression of glutamate (Glu) transporters GLT-1 and GLAST and attenuated Glu uptake (p < 0.01). Consistent with astrocyte cultures in vitro, aged preCGG mice cerebral cortex also displayed reduced GLAST and GLT-1 expression. Approximately 65% of the WT and preCGG cortical astrocytes displayed spontaneous asynchronous Ca(2+) oscillations. PreCGG astrocytes exhibited nearly 50% higher frequency of asynchronous Ca(2+) oscillations (p < 0.01) than WT, a difference mimicked by chronic exposure of WT astrocytes to l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC) or by partial suppression of GLAST using siRNA interference. Acute challenge with Glu augmented the frequency of Ca(2+) oscillations in both genotypes. Additionally, 10 µm Glu elicited a sustained intracellular Ca(2+) rise in a higher portion of preCGG astrocytes when compared with WT. Pharmacological studies showed that mGluR5, but not NMDA receptor, contributed to Glu hypersensitivity in preCGG astrocytes. These functional defects in preCGG astrocytes, especially in Glu signaling, may contribute to fragile X-associated tremor/ataxia syndrome neuropathology.

Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone.

Premutation CGG repeat expansions (55-200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca(2+) oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-ß-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABA(A) receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.

Signaling defects in iPSC-derived fragile X premutation neurons.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a leading monogenic neurodegenerative disorder affecting premutation carriers of the fragile X (FMR1) gene. To investigate the underlying cellular neuropathology, we produced induced pluripotent stem cell-derived neurons from isogenic subclones of primary fibroblasts of a female premutation carrier, with each subclone bearing exclusively either the normal or the expanded (premutation) form of the FMR1 gene as the active allele. We show that neurons harboring the stably-active, expanded allele (EX-Xa) have reduced postsynaptic density protein 95 protein expression, reduced synaptic puncta density and reduced neurite length. Importantly, such neurons are also functionally abnormal, with calcium transients of higher amplitude and increased frequency than for neurons harboring the normal-active allele. Moreover, a sustained calcium elevation was found in the EX-Xa neurons after glutamate application. By excluding the individual genetic background variation, we have demonstrated neuronal phenotypes directly linked to the FMR1 premutation. Our approach represents a unique isogenic, X-chromosomal epigenetic model to aid the development of targeted therapeutics for FXTAS, and more broadly as a model for the study of common neurodevelopmental (e.g. autism) and neurodegenerative (e.g. Parkinsonism, dementias) disorders.