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

Abstract Study question Does repeat-associated non-AUG (RAN) translation lead to accumulation of polyglycine- containing protein (FMRpolyG) in human lymphocytes and mural granulosa cells of FMR1 premutation carriers? Summary answer Lymphocytes and granulosa cells from FMR1 premutation carriers contain intracellular inclusions that stain positive for both FMRpolyG and ubiquitin. What is known already: Fragile-X-associated-Primary-Ovarian-Insufficiency (FXPOI) is characterized by oligo/amenorrhea and hypergonadotropic hypogonadism associated with the expansion of CGG-repeats in the 5’UTR of FMR1, called premutation (PM) (n: 55–200). Approximately 20% of women carrying a FMR1-premutation (PM) allele develop FXPOI. RAN-translation dependent on variable CGG-repeat length is hypothesized to cause FXPOI due to the production of a polyglycine-containing FMR1-protein, FMRpolyG. Recently, FMRpolyG inclusions were found in neuronal brain cells of FXTAS patients and stromal cells of the ovary of an FXPOI patient. Study design, size, duration: Lymphocytes and granulosa cells (GCs) from women with PM (6) and women without PM (10) (controls) were analyzed by immunofluorescence (IF) staining for the presence of inclusions positive for ubiquitin and FMRpolyG. Cell lysis and protein extraction samples were subjected to Fluorescent Western Blot (WB) analysis to detect FMRP and FMRpolyG Participants/materials, setting, methods Human GCs were obtained from follicular fluid after oocyte retrieval and lymphocytes were isolated from peripheral blood using Ficoll-Paque. Cells suspended in PBS were adhered to a glass-coverslip placed at the bottom of the 6-well culture plate, via gravity sedimentation. Adhered cells were fixed, IF staining for FMRpolyG and ubiquitin was performed and analyzed by fluorescence microscopy. Fluorescent WB was used to demonstrate the expression of FMRP, FMRpolyG in extracted protein from lymphocytes and GCs. Main results and the role of chance FMRP was successfully detected by fluorescence WB in both lymphocytes and GCs. FMRP is mainly present in cytoplasm and was expressed in greater amount in GCs than in leukocytes. Moreover, FMRP expression was significantly decreased in GCs from FMR1-PM compared with controls. Lymphocytes from PM-carriers and controls were immunostained for FMRpolyG and ubiquitin. In PM-carriers, FMRpolyG was present as aggregates, whereas in controls only a weak signal without inclusions was detectable. The expression pattern of FMRpolyG in GCs was similar to that in lymphocytes with a significant increase in PM-carriers. There, the FMRpolyG-aggregates additionally demonstrated as ubiquitin-positive inclusions. These may resemble the toxic potential of these protein fractions involved the ovarian damage in developing FXPOI. Limitations, reasons for caution More patients are needed to support the present findings. Further investigation into the possible consequences of these FMRpolyG-positive inclusions in PM-carriers is also advisable. Wider implications of the findings: We found for the first time FMRpolyG-accumulation in lymphocytes and GCs from FMR1-PM-carriers in ubiquitin-positive inclusions. Future experiments evaluating consistency in more patients and elucidating the impact on fertility and prospective value for individual ovarian reserve are therefore in preparation. Trial registration number Not applicable

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CGG repeat RNA G-quadruplexes interact with FMRpolyG to cause neuronal dysfunction in fragile X-related tremor/ataxia syndrome

Science Advances ◽

10.1126/sciadv.abd9440 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabd9440

Author(s):

Sefan Asamitsu ◽

Yasushi Yabuki ◽

Susumu Ikenoshita ◽

Kosuke Kawakubo ◽

Moe Kawasaki ◽

...

Keyword(s):

Therapeutic Strategy ◽

Aminolevulinic Acid ◽

Fragile X ◽

Protoporphyrin Ix ◽

Neuronal Dysfunction ◽

Cgg Repeat ◽

Repeat Expansions ◽

Ataxia Syndrome ◽

And Behavior ◽

Ran Translation

Fragile X-related tremor/ataxia syndrome (FXTAS) is a neurodegenerative disease caused by CGG triplet repeat expansions in FMR1, which elicit repeat-associated non-AUG (RAN) translation and produce the toxic protein FMRpolyG. We show that FMRpolyG interacts with pathogenic CGG repeat-derived RNA G-quadruplexes (CGG-G4RNA), propagates cell to cell, and induces neuronal dysfunction. The FMRpolyG polyglycine domain has a prion-like property, preferentially binding to CGG-G4RNA. Treatment with 5-aminolevulinic acid, which is metabolized to protoporphyrin IX, inhibited RAN translation of FMRpolyG and CGG-G4RNA–induced FMRpolyG aggregation, ameliorating aberrant synaptic plasticity and behavior in FXTAS model mice. Thus, we present a novel therapeutic strategy to target G4RNA prionoids.

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Neuropathology of RAN translation proteins in Fragile X-associated Tremor/Ataxia Syndrome

10.1101/672444 ◽

2019 ◽

Cited By ~ 1

Author(s):

Amy Krans ◽

Geena Skariah ◽

Yuan Zhang ◽

Bryana Bayly ◽

Peter K. Todd

Keyword(s):

Hippocampal Neurons ◽

Neurodegenerative Disorder ◽

Fragile X ◽

Antisense Strand ◽

Common Component ◽

Cgg Repeat ◽

Neuronal Nuclei ◽

Sense Strand ◽

Ataxia Syndrome ◽

Ran Translation

AbstractCGG 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 were infrequent components of FMRpolyG+ inclusions. In conclusion, RAN translated FMRpolyG is a common component of ubiquitin and p62 positive inclusions in FXTAS patient brains.

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

Acta Neuropathologica Communications ◽

10.1186/s40478-019-0782-7 ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 7

Author(s):

Amy Krans ◽

Geena Skariah ◽

Yuan Zhang ◽

Bryana Bayly ◽

Peter K. Todd

Keyword(s):

Hippocampal Neurons ◽

Neurodegenerative Disorder ◽

Fragile X ◽

Antisense Strand ◽

Common Component ◽

Cgg Repeat ◽

Neuronal Nuclei ◽

Sense Strand ◽

Ataxia Syndrome ◽

Ran Translation

Abstract 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.

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Utilizing CGG repeat length to determine Anti-Müllerian Hormone (AMH) levels in women without the Fragile X premutation

10.26226/morressier.573c1514d462b80296c98b39 ◽

2016 ◽

Author(s):

Christine Mullin

Keyword(s):

Fragile X ◽

Repeat Length ◽

Cgg Repeat ◽

Fragile X Premutation

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Introducing direct CGG repeat analysis in preimplantation genetic diagnosis (PGD) for fragile X syndrome: an overview of clinical outcomes for 116 patients

10.26226/morressier.5af300b2738ab10027aa98fe ◽

2018 ◽

Author(s):

Rachael Cabey

Keyword(s):

Fragile X Syndrome ◽

Clinical Outcomes ◽

Preimplantation Genetic Diagnosis ◽

Fragile X ◽

Genetic Diagnosis ◽

Cgg Repeat ◽

Repeat Analysis

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Faculty Opinions recommendation of CGG repeat-associated translation mediates neurodegeneration in fragile X tremor ataxia syndrome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718009175.793482222 ◽

2013 ◽

Author(s):

Richard J Maraia ◽

Nathan Blewett

Keyword(s):

Fragile X ◽

Cgg Repeat ◽

Ataxia Syndrome

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Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

Scientific Reports ◽

10.1038/s41598-021-87097-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yogeeshwar Ajjugal ◽

Narendar Kolimi ◽

Thenmalarchelvi Rathinavelan

Keyword(s):

Rna Binding ◽

Trinucleotide Repeat ◽

Rna Binding Proteins ◽

Fragile X ◽

Repeat Expansion ◽

Mobility Shift ◽

Cgg Repeat ◽

Dna And Rna ◽

Structural Choice ◽

Sense Strand

AbstractCGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

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Autism Profiles of Males With Fragile X Syndrome

American Journal on Mental Retardation ◽

10.1352/2008.113:427-438 ◽

2008 ◽

Vol 113 (6) ◽

pp. 427-438 ◽

Cited By ~ 251

Author(s):

Susan W. Harris ◽

David Hessl ◽

Beth Goodlin-Jones ◽

Jessica Ferranti ◽

Susan Bacalman ◽

...

Keyword(s):

Fragile X Syndrome ◽

Fragile X ◽

Autism Diagnosis ◽

Cgg Repeat ◽

Molecular Features ◽

Fmr1 Mrna ◽

Two Measures ◽

Dsm Iv ◽

Relationship Of ◽

The Relationship

Abstract Autism, which is common in individuals with fragile X syndrome, is often difficult to diagnose. We compared the diagnostic classifications of two measures for autism diagnosis, the ADOS and the ADI-R, in addition to the DSM-IV-TR in 63 males with this syndrome. Overall, 30% of the subjects met criteria for autistic disorder and 30% met criteria for PDD-NOS. The classifications on the ADOS and DSM-IV-TR were most similar, whereas the ADI-R classified subjects as autistic much more frequently. We further investigated the relationship of both FMRP and FMR1 mRNA to symptoms of autism in this cohort and found no significant relationship between the measures of autism and molecular features, including FMRP, FMR1 mRNA, and CGG repeat number.

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