Fragile X-associated Tremor/ataxia Syndrome Patients Research Articles

Mitochondrial dysfunction in brain tissues and Extracellular Vesicles Fragile X-associated tremor/ataxia syndrome.

Mitochondrial impairments have been implicated in the pathogenesis of Fragile X-associated tremor/ataxia syndrome (FXTAS) based on analysis of mitochondria in peripheral tissues and cultured cells. We sought to assess whether mitochondrial abnormalities present in postmortem brain tissues of patients with FXTAS are also present in plasma neuron-derived extracellular vesicles (NDEVs) from living carriers of fragile X messenger ribonucleoprotein1 (FMR1) gene premutations at an early asymptomatic stage of the disease continuum. We utilized postmortem frozen cerebellar and frontal cortex samples from a cohort of eight patients with FXTAS and nine controls and measured the quantity and activity of the mitochondrial proteins complex IV and complex V. In addition, we evaluated the same measures in isolated plasma NDEVs by selective immunoaffinity capture targeting L1CAM from a separate cohort of eight FMR1 premutation carriers and four age-matched controls. Lower complex IV and V quantity and activity were observed in the cerebellum of FXTAS patients compared to controls, without any differences in total mitochondrial content. No patient-control differences were observed in the frontal cortex. In NDEVs, FMR1 premutation carriers compared to controls had lower activity of Complex IV and Complex V, but higher Complex V quantity. Quantitative and functional abnormalities in mitochondrial electron transport chain complexes IV and V seen in the cerebellum of patients with FXTAS are also manifest in plasma NDEVs of FMR1 premutation carriers. Plasma NDEVs may provide further insights into mitochondrial pathologies in this syndrome and could potentially lead to the development of biomarkers for predicting symptomatic FXTAS among premutation carriers and disease monitoring.

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.

Digital gait markers to potentially distinguish fragile X-associated tremor/ataxia syndrome, Parkinson's disease, and essential tremor.

Fragile X-associated tremor/ataxia syndrome (FXTAS), a neurodegenerative disease that affects carriers of a 55-200 CGG repeat expansion in the fragile X messenger ribonucleoprotein 1 (FMR1) gene, may be given an incorrect initial diagnosis of Parkinson's disease (PD) or essential tremor (ET) due to overlapping motor symptoms. It is critical to characterize distinct phenotypes in FXTAS compared to PD and ET to improve diagnostic accuracy. Fast as possible (FP) speed and dual-task (DT) paradigms have the potential to distinguish differences in gait performance between the three movement disorders. Therefore, we sought to compare FXTAS, PD, and ET patients using quantitative measures of functional mobility and gait under self-selected (SS) speed, FP, and DT conditions. Participants with FXTAS (n = 22), PD (n = 23), ET (n = 20), and controls (n = 20) underwent gait testing with an inertial sensor system (APDM™). An instrumented Timed Up and Go test (i-TUG) was used to measure movement transitions, and a 2-min walk test (2MWT) was used to measure gait and turn variables under SS, FP, and DT conditions, and dual-task costs (DTC) were calculated. ANOVA and multinomial logistic regression analyses were performed. PD participants had reduced stride lengths compared to FXTAS and ET participants under SS and DT conditions, longer turn duration than ET participants during the FP task, and less arm symmetry than ET participants in SS gait. They also had greater DTC for stride length and velocity compared to FXTAS participants. On the i-TUG, PD participants had reduced sit-to-stand peak velocity compared to FXTAS and ET participants. Stride length and arm symmetry index during the DT 2MWT was able to distinguish FXTAS and ET from PD, such that participants with shorter stride lengths were more likely to have a diagnosis of PD and those with greater arm asymmetry were more likely to be diagnosed with PD. No gait or i-TUG parameters distinguished FXTAS from ET participants in the regression model. This is the first quantitative study demonstrating distinct gait and functional mobility profiles in FXTAS, PD, and ET which may assist in more accurate and timely diagnosis.

Exploration of SUMO2/3 Expression Levels and Autophagy Process in Fragile X-Associated Tremor/Ataxia Syndrome: Addressing Study Limitations and Insights for Future Research.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in adult FMR1 premutation carriers. The neuropathological hallmark of FXTAS is an intranuclear inclusion in neurons and astrocytes. Nearly 200 different proteins have been identified in FXTAS inclusions, being the small ubiquitin-related modifier 2 (SUMO2), ubiquitin and p62 the most highly abundant. These proteins are components of the protein degradation machinery. This study aimed to characterize SUMO2/3 expression levels and autophagy process in human postmortem brain samples and skin fibroblast cultures from FXTAS patients. Results revealed that FXTAS postmortem brain samples are positive for SUMO2/3 conjugates and supported the idea that SUMO2/3 accumulation is involved in inclusion formation. Insights from RNA-sequencing data indicated that SUMOylation processes are significantly upregulated in FXTAS samples. In addition, the analysis of the autophagy flux showed the accumulation of p62 protein levels and autophagosomes in skin fibroblasts from FXTAS patients. Similarly, gene set analysis evidenced a significant downregulation in gene ontology terms related to autophagy in FXTAS samples. Overall, this study provides new evidence supporting the role of SUMOylation and autophagic processes in the pathogenic mechanisms underlying FXTAS.

Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

Screening for the FMR1 premutation in Greek patients with late-onset movement disorders

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset, X-linked, neurodegenerative disorder that affects premutation carriers of the FMR1 gene. FXTAS is often misdiagnosed as spinocerebellar ataxia (SCA) or Parkinson's disease (PD). Herein, we sought to investigate the frequency, genotypic and phenotypic profile of FXTAS in two cohorts of Greek patients with late-onset movement disorders, one with cerebellar ataxia and the other with PD. In total, 90 index patients with late-onset cerebellar ataxia and 171 with PD were selected. None of the cases had male-to-male transmission. Genetic screening for the FMR1 premutation was performed using standard methodology. The FMR1 premutation was detected in two ataxia patients (2.2%) and two PD patients (1.2%). Additional clinical features in FXTAS patients from the ataxia cohort included neuropathy, mild parkinsonism, cognitive impairment and pyramidal signs. The FXTAS patients from the PD cohort had typical PD. We conclude that, in the Greek population, the FMR1 premutation is an important, albeit rare, cause of late-onset movement disorders. Routine premutation screening should be considered in SCA panel-negative late-onset ataxia cases. Directed premutation screening should be considered in all ataxia and PD cases with additional features suggestive of FXTAS. Our study highlights the importance of FMR1 genetic testing in the diagnosis of late-onset movement disorders.

Mechanistic convergence across initiation sites for RAN translation in fragile X associated tremor ataxia syndrome.

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.

Allopregnanolone Improves Locomotor Activity and Arousal in the Aged CGG Knock-in Mouse Model of Fragile X-Associated Tremor/Ataxia Syndrome.

Carriers of the fragile X premutation (PM) can develop a variety of early neurological symptoms, including depression, anxiety and cognitive impairment as well as being at risk for developing the late-onset fragile X-associated tremor/ataxia syndrome (FXTAS). The absence of effective treatments for FXTAS underscores the importance of developing efficacious therapies to reduce the neurological symptoms in elderly PM carriers and FXTAS patients. A recent preliminary study reported that weekly infusions of Allopregnanolone (Allop) may improve deficits in executive function, learning and memory in FXTAS patients. Based on this study we examined whether Allop would improve neurological function in the aged CGG knock-in (CGG KI) dutch mouse, B6.129P2(Cg)-Fmr1tm2Cgr/Cgr, that models much of the symptomatology in PM carriers and FXTAS patients. Wild type and CGG KI mice received 10 weekly injections of Allop (10 mg/kg, s.c.), followed by a battery of behavioral tests of motor function, anxiety, and repetitive behavior, and 5-bromo-2′-deoxyuridine (BrdU) labeling to examine adult neurogenesis. The results provided evidence that Allop in CGG KI mice normalized motor performance and reduced thigmotaxis in the open field, normalized repetitive digging behavior in the marble burying test, but did not appear to increase adult neurogenesis in the hippocampus. Considered together, these results support further examination of Allop as a therapeutic strategy in patients with FXTAS.

Cellular Bioenergetics and AMPK and TORC1 Signalling in Blood Lymphoblasts Are Biomarkers of Clinical Status in FMR1 Premutation Carriers.

Fragile X Associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder affecting carriers of premutation alleles (PM) of the X-linked FMR1 gene, which contain CGG repeat expansions of 55–200 range in a non-coding region. This late-onset disorder is characterised by the presence of tremor/ataxia and cognitive decline, associated with the white matter lesions throughout the brain, especially involving the middle cerebellar peduncles. Nearly half of older male and ~ 20% of female PM carriers develop FXTAS. While there is evidence for mitochondrial dysfunction in neural and some peripheral tissues from FXTAS patients (though less obvious in the non-FXTAS PM carriers), the results from peripheral blood mononuclear cells (PBMC) are still controversial. Motor, cognitive, and neuropsychiatric impairments were correlated with measures of mitochondrial and non-mitochondrial respiratory activity, AMPK, and TORC1 cellular stress-sensing protein kinases, and CGG repeat size, in a sample of adult FXTAS male and female carriers. Moreover, the levels of these cellular measures, all derived from Epstein- Barr virus (EBV)- transformed and easily accessible blood lymphoblasts, were compared between the FXTAS (N = 23) and non-FXTAS (n = 30) subgroups, and with baseline data from 33 healthy non-carriers. A significant hyperactivity of cellular bioenergetics components as compared with the baseline data, more marked in the non-FXTAS PMs, was negatively correlated with repeat numbers at the lower end of the CGG-PM distribution. Significant associations of these components with motor impairment measures, including tremor-ataxia and parkinsonism, and neuropsychiatric changes, were prevalent in the FXTAS subgroup. Moreover, a striking elevation of AMPK activity, and a decrease in TORC1 levels, especially in the non-FXTAS carriers, were related to the size of CGG expansion. The bioenergetics changes in blood lymphoblasts are biomarkers of the clinical status of FMR1 carriers. The relationship between these changes and neurological involvement in the affected carriers suggests that brain bioenergetic alterations are reflected in this peripheral tissue. A possible neuroprotective role of stress sensing kinase, AMPK, in PM carriers, should be addressed in future longitudinal studies. A decreased level of TORC1—the mechanistic target of the rapamycin complex, suggests a possible future approach to therapy in FXTAS.

Characterization of the Cerebrospinal Fluid Proteome in Patients with Fragile X-Associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS), first described in 2001, is a neurodegenerative and movement disorder, caused by a premutation in the fragile X mental retardation 1 (FMR1) gene. To date, the biological mechanisms causing this condition are still not well understood, as not all premutation carriers develop FXTAS. To further understand this syndrome, we quantitatively compared the cerebrospinal fluid (CSF) proteome of FXTAS patients with age-matched controls using mass spectrometry. We identified 415 proteins of which 97 were altered in FXTAS patients. These proteins suggest changes in acute phase response signaling, liver X receptor/ retinoid X receptor (LXR/RXR) activation, and farnesoid X receptor (FXR)/RXR activation, which are the main pathways found to be affected. Additionally, we detected changes in many other proteins including amyloid-like protein 2, contactin-1, afamin, cell adhesion molecule 4, NPC intracellular cholesterol transporter 2, and cathepsin B, that had been previously noted to hold important roles in other movement disorders. Specific to RXR pathways, several apolipoproteins (APOA1, APOA2, APOA4, APOC2, and APOD) showed significant changes in the CSF of FXTAS patients. Lastly, CSF parameters were analyzed to investigate abnormalities in blood brain barrier function. Correlations were observed between patient albumin quotient values, a measure of permeability, and CGG repeat length as well as FXTAS rating scale scores.

Mechanistic Convergence Across Initiation Sites for RAN Translation in Fragile X Associated Tremor Ataxia Syndrome

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

Eye Movements in Fragile X-Associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder characterized by ataxia, tremor, and parkinsonism. Eye motility abnormalities on the clinical examination of FXTAS patients have not been formally studied. A case-control study with fragile X gene mutation carriers with and without FXTAS and normal controls was conducted and included a videotaping of ocular items of the International Cooperative Ataxia Rating Scale (ICARS). A neuro-ophthalmologist blinded to gene status rated nystagmus, ocular pursuit, and saccades. Forty-four cases and controls were recruited, with an average age of 55.2 years (±7.4) and 57% women. Gaze-evoked nystagmus was increased in fragile X gene carriers (odds ratio 1.44, 95% confidence interval: 0.33-7.36) but was not statistically significant. There was no difference in ocular pursuit nor saccade dysmetria between cases and controls. The results show that clinical examination findings of ocular abnormalities, using the ICARS oculomotor disorders movement subscale, are not more common in FXTAS or FMR1 premutation carriers than normal controls on examination in the clinic. Examining a larger cohort of patients with FXTAS would be an ideal next step.

A Chinese case of fragile X-associated tremor/ataxia syndrome (FXTAS) with orthostatic tremor:case report and literature review on tremor in FXTAS

BackgroundFragile X-associated tremor/ataxia syndrome (FXTAS) is a late onset, X-linked genetic, neurodegenerative disorder caused by a “premutation (PM)” in the fragile X mental retardation 1 (FMR1) gene. Here we report a case of FXTAS from mainland of China who presented with rare orthostatic tremor. A review of tremor of FXTAS in the literature is also included.Case presentationA 67-year-old right-handed farmer started with tremor of both legs 8 years ago which was present while standing but absent when sitting or lying and progressed with unsteady gait one and a half years ago. The brain MRI showed high intensity signal in the bilateral middle cerebellar peduncles (MCP) in T2-weighted and fluid-attenuated inversion recovery (FLAIR) images and gene test for premutation for FMR1 was positive with 101 CGG repeats. The patient met the the diagnosis of definite FXTAS. Clonazepam and topiramate were administered to control tremor. We reviewed the literature and identified 64 cases with detailed clinical and genetic information. Orthostatic tremor associated with FXTAS is very rare. We found 85.2% patients reported tremor,42.6% with intention tremor,36.1% with kinetic tremor,32.8% with rest tremor and 29.5% with posture tremor. 37.7% of patients who have tremor showed at least two types of tremor. There were 6 patients with isolated rest tremor. There was 2 patient with voice tremor and 6 with head tremor. We also found that 74.6% FXTAS patients had family history of FMR1 gene associated diseases including Fragile X syndrome (FXS), FXTAS or fragile X-associated primary ovarian insufficiency (FXPOI).ConclusionsAdding our data to the available literature suggests that orthostatic tremor could be a rare initial manifestation of FXTAS and the review will increasing our understanding the phenotype of tremor in FXTAS. Family history of FMR1 gene associated diseases might be an important clue to the diagnosis.

Parkinsonism Versus Concomitant Parkinson's Disease in Fragile X-Associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder associated with premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is characterized by the presence of ubiquitin-positive inclusions in neurons and astrocytes and by cerebellar tremor and ataxia. Parkinsonism has been reported in FXTAS, but most patients lack the characteristic rest tremor and severe rigidity seen in idiopathic Parkinson's disease (PD). To describe the frequency of concomitant PD in FXTAS. We reviewed the medical record of 40 deceased patients diagnosed with FXTAS and performed a pathology analysis to confirm both FXTAS and PD. Clinical histories indicated that 5 FXTAS patients were diagnosed with idiopathic PD and 2 with atypical parkinsonian syndrome. After pathological examination, we found that 7 patients in the PD clinical diagnosis group had dopaminergic neuronal loss; however, only 2 of 7 presented Lewy bodies (LBs) in the substantia nigra. Therefore, a total of 5% of the 40 cohort patients met the pathologic criteria for the concomitant diagnosis of FXTAS and PD. In addition, 2 patients not clinically diagnosed with PD also had nigral neuronal loss with LBs in substantia nigra. In total 10% of these 40 patients had LBs. This report expands our understanding of clinical symptoms and unusual presentations in patients with FXTAS and the concept that the parkinsonism found in FXTAS is sometimes indistinguishable from PD. We propose that FMR1 should be recognized as one of the exceptional genetic causes of parkinsonism with presynaptic dopaminergic loss and LBs.

FMRpolyG accumulates in FMR1 premutation granulosa cells

BackgroundFragile X premutation (Amplification of CGG number 55–200) is associated with increased risk for fragile X-Associated Premature Ovarian Insufficiency (FXPOI) in females and fragile X-associated tremor/ataxia syndrome (FXTAS) predominantly in males. Recently, it has been shown that CGG repeats trigger repeat associated non-AUG initiated translation (RAN) of a cryptic polyglycine-containing protein, FMRpolyG. This protein accumulates in ubiquitin-positive inclusions in neuronal brain cells of FXTAS patients and may lead to protein-mediated neurodegeneration. FMRpolyG inclusions were also found in ovary stromal cells of a FXPOI patient. The role of FMRpolyG expression has not been thoroughly examined in folliculogenesis related cells. The main goal of this study is to evaluate whether FMRpolyG accumulates in mural granulosa cells of FMR1 premutation carriers. Following FMRpolyG detection, we aim to examine premutation transfected COV434 as a suitable model used to identify RAN translation functions in FXPOI pathogenesis.ResultsFMRpolyG and ubiquitin immunostained mural granulosa cells from six FMR1 premutation carriers demonstrated FMRpolyG aggregates. However, co-localization of FMRpolyG and ubiquitin appeared to vary within the FMR1 premutation carriers’ group as three exhibited partial ubiquitin and FMRpolyG double staining and three premutation carriers demonstrated FMRpolyG single staining. None of the granulosa cells from the five control women expressed FMRpolyG. Additionally, human ovarian granulosa tumor, COV434, were transfected with two plasmids; both expressing 99CGG repeats but only one enables FMRpolyG expression. Like in granulosa cells from FMR1 premutation carriers, FMRpolyG aggregates were found only in COV434 transfected with expended CGG repeats and the ability to express FMRpolyG.ConclusionsCorresponding with previous studies in FXTAS, we demonstrated accumulation of FMRpolyG in mural granulosa cells of FMR1 premutation carriers. We also suggest that following further investigation, the premutation transfected COV434 might be an appropriate model for RAN translation studies. Detecting FMRpolyG accumulation in folliculogenesis related cells supports previous observations and imply a possible common protein-mediated toxic mechanism for both FXPOI and FXTAS.

Neuropathology of RAN translation proteins in fragile X-associated tremor/ataxia syndrome

CGG repeat expansions in FMR1 cause the neurodegenerative disorder Fragile X-associated Tremor/Ataxia Syndrome (FXTAS). Ubiquitinated neuronal intranuclear inclusions (NIIs) are the neuropathological hallmark of FXTAS. Both sense strand derived CGG repeats and antisense strand derived CCG repeats support non-AUG initiated (RAN) translation of homopolymeric proteins in potentially 6 different reading frames. However, the relative abundance of these proteins in FXTAS brains and their co-localization with each other and NIIs is lacking. Here we describe rater-blinded assessment of immunohistochemical and immunofluorescence staining with newly generated antibodies to different CGG RAN translation products in FXTAS and control brains as well as co-staining with ubiquitin, p62/SQSTM1, and ubiquilin 2. We find that both FMRpolyG and a second CGG repeat derived RAN translation product, FMRpolyA, accumulate in aggregates in FXTAS brains. FMRpolyG is a near-obligate component of both ubiquitin-positive and p62-positive NIIs in FXTAS, with occurrence of aggregates in 20% of all hippocampal neurons and > 90% of all inclusions. A subset of these inclusions also stain positive for the ALS/FTD associated protein ubiquilin 2. Ubiquitinated inclusions and FMRpolyG+ aggregates are rarer in cortex and cerebellum. Intriguingly, FMRpolyG staining is also visible in control neuronal nuclei. In contrast to FMRpolyG, staining for FMRpolyA and CCG antisense derived RAN translation products were less abundant and less frequent components of ubiquitinated inclusions. In conclusion, RAN translated FMRpolyG is a common component of ubiquitin and p62 positive inclusions in FXTAS patient brains.

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.

FMRpolyG alters mitochondrial transcripts level and respiratory chain complex assembly in Fragile X associated tremor/ataxia syndrome [FXTAS

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats (premutation) in FMR1. These CGG repeats are Repeat Associated non-ATG (RAN) translated into a small and pathogenic protein, FMRpolyG. The cellular and molecular mechanisms of FMRpolyG toxicity are unclear. Various mitochondrial dysfunctions have been observed in FXTAS patients and animal models. However, the causes of these mitochondrial alterations are not well understood. In the current study, we investigated interaction of FMRpolyG with mitochondria and its role in modulating mitochondrial functions. Beside nuclear inclusions, FMRpolyG also formed small cytosolic aggregates that interact with mitochondria both in cell and mouse model of FXTAS. Importantly, expression of FMRpolyG reduces ATP levels, mitochondrial transmembrane potential, mitochondrial supercomplexes assemblies and activities and expression of mitochondrial DNA encoded transcripts in cell and animal model of FXTAS, as well as in FXTAS patient brain tissues. Overall, these results suggest that FMRpolyG alters mitochondrial functions, bioenergetics and initiates cell death. The further study in this direction will help to establish the role of mitochondria in FXTAS conditions.

Cognitive Deficits and Associated ERP N400 Abnormalities in FXTAS With Parkinsonism

Objective: To examine cognitive deficits and associated brain activity in fragile X-associated tremor/ataxia syndrome (FXTAS) patients with parkinsonism (FXTp+), in relation to FXTAS patients without parkinsonism (FXTp-), and normal elderly controls (NC).Methods: Retrospective reviews were performed in 65 FXTAS patients who participated in the event-related brain potential (ERP) study and also had either a videotaped neurological examination or a neurological examination for extrapyramidal signs. Parkinsonism was defined as having bradykinesia with at least one of the following: rest tremor, postural instability, hypermyotonia, or rigidity. Eleven FXTp+ patients were identified and compared to 11 matched FXTp- and 11 NC. Main ERP measures included the N400 congruity effect, N400 repetition effect, and the late positive component (LPC) repetition effect.Results: When compared with FXTp- and NC, the FXTp+ group showed more severe deficits in executive function, cued-recall, recognition memory, along with a significantly reduced N400 repetition effect (thought to index semantic processing and verbal learning/memory) which was correlated with poorer verbal memory. Across all patients, FMR1 mRNA levels were inversely correlated with delayed recall on the California Verbal Learning Test (CVLT).Interpretation: The findings of more prominent executive dysfunction and verbal learning/memory deficits in FXTp+ than FXTp- are consistent with findings in Parkinson’s disease (PD), and may indicate that concomitant and/or synergistic pathogenetic mechanisms associated with PD play a role in FXTAS. These results have implications not only for understanding the cognitive impairments associated with the parkinsonism subtype of FXTAS, but also for the development of new interventions for these patients.

Quantitative Evaluation of Toxic Polyglycine Biosynthesis and Aggregation in Cell Models Expressing Expanded CGG Repeats.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGGexp) trinucleotides in the 5′UTR of the FMR1 gene encoding fragile X mental retardation protein (FMRP). The patients, with the number of the repeats ranging from 55 to 200, show specific manifestation of clinical symptoms that include intention tremor, gait ataxia, cognitive deficits, and brain atrophy. Accumulation of toxic polyglycine (FMRpolyG), a by-product of the CGGexp repeat-associated non-ATG (RAN) translation, is considered to be one of the main factors triggering neurodegenerative processes in FXTAS patients. Nevertheless, the nature of the FMRpolyG-induced cell damage, especially in the context of its soluble and inclusion-associated forms, is still elusive. Targeting either biosynthesis, cellular stability or aggregation capacity of toxic FMRpolyG could be considered as a potential therapeutic strategy for FXTAS. Therefore, we tested a variety of quantitative methods based on forced expression of genetic constructs carrying CGGexp repeats in the context of the FMR1 5′UTR fused to GFP, mCherry or Firefly luciferase gene in or out of frame to the polyglycine encoding sequence. We show that FMRpolyG translation either from native or an AUG-induced start codon as well as the translation yield of the FMRP open reading frame equivalent located downstream of the CGGexp element can be effectively estimated using fluorescence microscopy, flow cytometry or luciferase assay. We also quantitatively estimated soluble fraction and insoluble form of FMRpolyG aggregated in foci using an electrophoretic separation of cell lysates and fluorescence microscopy, respectively. Importantly, we show that dependent on a fusion tag, FMRpolyG has a different potential for aggregate formation. Our established protocols enable sensitive tracking of FMRP and FMRpolyG quantitative and qualitative changes after treatment with potential therapeutic agents for FXTAS. Furthermore, they can be modified for application to other RAN translation- and aggregation-related diseases.