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

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

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

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.600840 ◽

2021 ◽

Vol 7 ◽

Author(s):

Katharine Nichole Holm ◽

Anthony W. Herren ◽

Sandra L. Taylor ◽

Jamie L. Randol ◽

Kyoungmi Kim ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Structural Integrity ◽

Rna Binding ◽

Neurodegenerative Disorder ◽

Fragile X ◽

Protein Abundance ◽

Cgg Repeat ◽

Phosphoserine Aminotransferase ◽

Brain Volume Loss ◽

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.

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Therapeutic Development for CGG Repeat Expansion-Associated Neurodegeneration

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.655568 ◽

2021 ◽

Vol 15 ◽

Author(s):

Keqin Xu ◽

Yujing Li ◽

Emily G. Allen ◽

Peng Jin

Keyword(s):

Neurological Disorders ◽

Rna Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Repeat Expansion ◽

Therapeutic Interventions ◽

Cgg Repeat ◽

Associated Diseases ◽

Novel Therapeutic Strategies

Non-coding repeat expansions, such as CGG, GGC, CUG, CCUG, and GGGGCC, have been shown to be involved in many human diseases, particularly neurological disorders. Of the diverse pathogenic mechanisms proposed in these neurodegenerative diseases, dysregulated RNA metabolism has emerged as an important contributor. Expanded repeat RNAs that form particular structures aggregate to form RNA foci, sequestering various RNA binding proteins and consequently altering RNA splicing, transport, and other downstream biological processes. One of these repeat expansion-associated diseases, fragile X-associated tremor/ataxia syndrome (FXTAS), is caused by a CGG repeat expansion in the 5’UTR region of the fragile X mental retardation 1 (FMR1) gene. Moreover, recent studies have revealed abnormal GGC repeat expansion within the 5’UTR region of the NOTCH2NLC gene in both essential tremor (ET) and neuronal intranuclear inclusion disease (NIID). These CGG repeat expansion-associated diseases share genetic, pathological, and clinical features. Identification of the similarities at the molecular level could lead to a better understanding of the disease mechanisms as well as developing novel therapeutic strategies. Here, we highlight our current understanding of the molecular pathogenesis of CGG repeat expansion-associated diseases and discuss potential therapeutic interventions for these neurological disorders.

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FMRP association with and regulation of Fragile X granules exhibit circuit-dependent requirements for the KH2 RNA binding domain

10.1101/462168 ◽

2018 ◽

Author(s):

Ellen C. Gingrich ◽

Katherine A. Shepard ◽

Molly E. Mitchell ◽

Kirsty Sawicka ◽

Jennifer C. Darnell ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Brain Regions ◽

Binding Domain ◽

Cell Type ◽

Related Disorder ◽

Rna Granules ◽

Rna Binding Domain

AbstractThe localization and translation of mRNAs is controlled by a diverse array of ribonucleoprotein particles (RNPs), multimolecular complexes containing mRNAs and RNA binding proteins. Fragile X granules (FXGs) are a family of RNPs that exemplify the diversity of RNA granules in the mammalian nervous system. FXGs are found in a conserved subset of neurons, where they localize exclusively to the axonal compartment. Notably, the specific RNA binding proteins and mRNAs found in FXGs depend on brain circuit and neuron type, with all forebrain FXGs containing Fragile X mental retardation protein (FMRP), the protein mutated in the human autism-related disorder Fragile X syndrome. FMRP negatively regulates FXG abundance but is not required for their association with ribosomes or mRNA. To better understand the circuit-dependent mechanisms whereby FMRP associates with and regulates FXGs, we asked how a disease-causing point mutation, I304N, in the KH2 RNA binding domain of FMRP affects these granules in two brain regions – cortex and hippocampus. We found that FMRPI304N had a reduced association with FXGs, as it was absent from approximately half of FXGs in cortex and nearly all FXGs in hippocampus. FXG abundance correlated with the number of FMRP-containing FXGs, suggesting that FMRP regulates FXG abundance by KH2-independent mechanisms that occur locally within the granules. Together, these findings illustrate that cell type-dependent mechanisms guide the assembly of similar RNA granules. Further, point mutations in RNA granule components may lead to cell type-dependent phenotypes that produce atypical forms of disorders that normally arise from more severe mutations.

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RNA-Binding Proteins and Translation in Neurodegenerative Disease

The Oxford Handbook of Neuronal Protein Synthesis ◽

10.1093/oxfordhb/9780190686307.013.25 ◽

2020 ◽

Author(s):

Kent E. Duncan

Keyword(s):

Neurodegenerative Diseases ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X ◽

Potential Contribution ◽

Fused In Sarcoma ◽

Specific Mrnas ◽

Ataxia Syndrome ◽

Research Frontiers

Both RNA-binding proteins (RBPs) and translation are increasingly implicated in several neurodegenerative diseases, but their specific roles in promoting disease are not yet fully defined. This chapter critically evaluates the evidence that altered translation of specific mRNAs mediated by RNA-binding proteins plays an important role in driving specific neurodegenerative diseases. First, diseases are discussed where a causal role for RNA-binding proteins in disease appears solid, but whether this involves altered translation is less clear. The main foci here are TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Subsequently, diseases are presented where altered translation is believed to contribute, but involvement of RNA-binding proteins is less clear. These include Huntington’s and other repeat expansion disorders such as fragile X tremor/ataxia syndrome (FXTAS), where repeat-induced non-AUG-initiated (RAN) translation is a focus. The potential contribution of both canonical and non-canonical RBPs to altered translation in Parkinson’s disease is discussed. The chapter closes by proposing key research frontiers for the field to explore and outlining methodological advances that could help to address them.

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

Journal of Neurochemistry ◽

10.1111/j.1471-4159.2008.05747.x ◽

2008 ◽

Vol 107 (6) ◽

pp. 1671-1682 ◽

Cited By ~ 73

Author(s):

Judith R. Brouwer ◽

Karin Huizer ◽

Lies-Anne Severijnen ◽

Renate K. Hukema ◽

Robert F. Berman ◽

...

Keyword(s):

Mouse Model ◽

Fragile X ◽

Repeat Length ◽

Cgg Repeat ◽

Ataxia Syndrome

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Matrin3: Disorder and ALS Pathogenesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.794646 ◽

2022 ◽

Vol 8 ◽

Author(s):

Ahmed Salem ◽

Carter J. Wilson ◽

Benjamin S. Rutledge ◽

Allison Dilliott ◽

Sali Farhan ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Nuclear Dna ◽

Neurodegenerative Disorder ◽

Rna Binding Proteins ◽

Binding Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.

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Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function

10.1101/2020.09.04.263251 ◽

2020 ◽

Cited By ~ 2

Author(s):

Fiona Haward ◽

Magdalena M. Maslon ◽

Patricia L. Yeyati ◽

Nicolas Bellora ◽

Jan N. Hansen ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Rna Splicing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Small Body ◽

Splicing Factor ◽

Nuclear Retention ◽

Ciliary Ultrastructure ◽

Retention Signal

AbstractShuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

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Axonal localization of the fragile X family of RNA binding proteins is conserved across mammals

The Journal of Comparative Neurology ◽

10.1002/cne.24772 ◽

2019 ◽

Vol 528 (3) ◽

pp. 502-519 ◽

Cited By ~ 2

Author(s):

Katherine A. Shepard ◽

Lulu I T. Korsak ◽

Danielle DeBartolo ◽

Michael R. Akins

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Fragile X

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Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay

Scientific Reports ◽

10.1038/s41598-019-52411-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Aikaterini S. Papadopoulou ◽

Casandra Gomez-Paredes ◽

Michael A. Mason ◽

Bridget A. Taxy ◽

David Howland ◽

...

Keyword(s):

Mouse Model ◽

Neurodegenerative Disorder ◽

Simultaneous Detection ◽

Brain Regions ◽

Cag Repeat ◽

Highly Pathogenic ◽

Exon 2 ◽

Mouse Tissues ◽

Exon 1 ◽

The Brain

Abstract Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin (HTT) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon1 transcript, and the full-length HTT mRNA has three 3′UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA.

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