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1.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
2.
Nat Commun ; 11(1): 2755, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488011

RESUMO

Fragile X Syndrome results from a loss of Fragile X Mental Retardation Protein (FMRP). We now show that FMRP is a member of a Cav3-Kv4 ion channel complex that is known to regulate A-type potassium current in cerebellar granule cells to produce mossy fiber LTP. Mossy fiber LTP is absent in Fmr1 knockout (KO) mice but is restored by FMRP(1-297)-tat peptide. This peptide further rapidly permeates the blood-brain barrier to enter cells across the cerebellar-cortical axis that restores the balance of protein translation for at least 24 h and transiently reduces elevated levels of activity of adult Fmr1 KO mice in the Open Field Test. These data reveal that FMRP(1-297)-tat can improve function from the levels of protein translation to synaptic efficacy and behaviour in a model of Fragile X syndrome, identifying a potential therapeutic strategy for this genetic disorder.


Assuntos
Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Canais Iônicos/metabolismo , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/patologia , Masculino , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Biossíntese de Proteínas
3.
Clin Interv Aging ; 15: 285-292, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32161452

RESUMO

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that usually begins in the early 60s and affects carriers of premutation expansion (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Additional disorders can co-occur with FXTAS including Alzheimer's disease (AD). Here we discuss a case report of a male with 67 CGG repeats in FMR1 who had mild late-onset FXTAS symptoms followed by neurocognitive disorder symptoms consistent with AD. The patient has developed tremor and ataxia that are the two characteristic symptoms of FXTAS. In addition, he shows rapid cognitive decline, brain atrophy most substantial in the medial temporal lobe, and decreased metabolism in the brain regions that are the characteristic findings of AD. The purpose of this study is to describe a patient profile with both diseases and review the details of an overlap between these two diseases.


Assuntos
Doença de Alzheimer/complicações , Doença de Alzheimer/metabolismo , Ataxia/diagnóstico , Ataxia/metabolismo , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/metabolismo , Tremor/diagnóstico , Tremor/metabolismo , Idoso , Ataxia/complicações , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Tremor/complicações
4.
J Ovarian Res ; 13(1): 22, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32101156

RESUMO

BACKGROUND: Fragile 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. RESULTS: FMRpolyG 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. CONCLUSIONS: Corresponding 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.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Células da Granulosa/metabolismo , Adulto , Animais , Ataxia/genética , Ataxia/metabolismo , Modelos Animais de Doenças , Feminino , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismo , Transfecção , Tremor/genética , Tremor/metabolismo
5.
Gene ; 731: 144359, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31935509

RESUMO

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.


Assuntos
Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Processamento de RNA/fisiologia , Adulto , Processamento Alternativo/fisiologia , Estudos de Casos e Controles , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Éxons/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Células HEK293 , Células HeLa , Humanos , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribuição Tecidual
6.
Eur J Paediatr Neurol ; 24: 100-104, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31926845

RESUMO

Many pathways have been involved in pathophysiology of the fragile X syndrome, one of the more frequent genetic causes of intellectual disability and autism. This review highlights the recent insights in the role the abnormalities in the GABAergic system play in the disorder. Since the initial observations showed that the expression of specific subunits of the GABA(A) receptor were underexpressed in the fragile X knockout mouse model more than a decade ago, evidence has accumulated that the expression of approximately half of the GABAergic system is compromised in multiple species, including in fragile X patients. Functional consequences of the GABAergic deficiencies could be measured using whole-cell voltage clamp recordings. Pharmalogical treatment with agonist of the receptor was been able to restore several behavioral deficits in the fragile X mouse model, including seizures, marble burying and, in part, prepulse inhibition. Trials in patients with the same agonist have demonstrated encouraging post-hoc results in the most severely affected patients, although no effect could be demonstrated in the patient group as a whole. In conclusion, there can be little doubt that the GABAergic system is compromised in the fragile X syndrome and that these abnormalities contribute to the clinical abnormalities observed. However, at the moment the difference in treatment effectiveness of agonist of the receptor in animal models as opposed to in patients remains unexplained.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Receptores de GABA/genética , Animais , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Camundongos
7.
Hum Genet ; 139(2): 227-245, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31919630

RESUMO

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.


Assuntos
Ataxia/patologia , Metilação de DNA , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Mutação , Proteoma/análise , Tremor/patologia , Ataxia/genética , Ataxia/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , RNA Interferente Pequeno/genética , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tremor/genética , Tremor/metabolismo
8.
Glia ; 68(3): 495-508, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31626382

RESUMO

During development, oligodendrocytes in the central nervous system extend a multitude of processes that wrap axons with myelin. The highly polarized oligodendrocytes generate myelin sheaths on many different axons, which are far removed from the cell body. Neurons use RNA binding proteins to transport, stabilize, and locally translate mRNA in distal domains of neurons. Local synthesis of synaptic proteins during neurodevelopment facilitates the rapid structural and functional changes underlying neural plasticity and avoids extensive protein transport. We hypothesize that RNA binding proteins also regulate local mRNA regulation in oligodendrocytes to promote myelin sheath growth. Fragile X mental retardation protein (FMRP), an RNA binding protein that plays essential roles in the growth and maturation of neurons, is also expressed in oligodendrocytes. To determine whether oligodendrocytes require FMRP for myelin sheath development, we examined fmr1-/- mutant zebrafish and drove FMR1 expression specifically in oligodendrocytes. We found oligodendrocytes in fmr1-/- mutants developed myelin sheaths of diminished length, a phenotype that can be autonomously rescued in oligodendrocytes with FMR1 expression. Myelin basic protein (Mbp), an essential myelin protein, was reduced in myelin tracts of fmr1-/- mutants, but loss of FMRP function did not impact the localization of mbpa transcript in myelin. Finally, expression of FMR1-I304N, a missense allele that abrogates FMRP association with ribosomes, failed to rescue fmr1-/- mutant sheath growth and induced short myelin sheaths in oligodendrocytes of wild-type larvae. Taken together, these data suggest that FMRP promotes sheath growth through local regulation of translation.


Assuntos
Proteína do X Frágil de Retardo Mental/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Bainha de Mielina/genética , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Peixe-Zebra
9.
J Pharmacol Toxicol Methods ; 101: 106656, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31734279

RESUMO

INTRODUCTION: FXS is the leading cause of intellectual disabilities in males and a major monogenic cause of ASD (Autism spectrum disorders). It occurs due to the loss of FMRP, whose role in early development is not well understood. In this study, we have used a novel DNAzyme based approach to create a larval model of FXS in zebrafish with specific focus on the early developmental window. METHODS: Fmr1specific DNAzymes were electroporated into embryos to create the knockdown. Changes in RNA and protein levels of FMRP and relevant biomarkers were measured in the 0-7dpf window. Behavioral tests to measure anxiety, cognitive impairments and irritability in the larvae were conducted at the 7dpf stage. Drug treatment was carried out at various time points in the 0-7dpf window to identify the critical window for pharmacological intervention. RESULTS: The DNAzyme based knockdown approach led to a significant knockdown of FMRP in the zebrafish embryos, accompanied by increased anxiety, irritability and cognitive impairments at 7dpf, thus creating a robust larval model of FXS. Treatment with the Mavoglurant was able to rescue the behavioral phenotypes in the FXS larvae, and found to be more efficacious in the 0-3dpf window. DISCUSSION: The results from this study have revealed that a) a DNAzyme based knockdown approach can be used to create robust larval zebrafish model of disease, in a high-throughput manner and b) optimal window for therapeutic intervention for FXS as well as other pediatric diseases with a monogenic cause can be identified using such a model.


Assuntos
DNA Catalítico/genética , Síndrome do Cromossomo X Frágil/metabolismo , Técnicas de Silenciamento de Genes , Animais , Escala de Avaliação Comportamental , Modelos Animais de Doenças , Larva , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
J Neurosci ; 40(6): 1355-1365, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31882402

RESUMO

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability, resulted from the silencing of the Fmr1 gene and the subsequent loss of fragile X mental retardation protein (FMRP). Spine dysgenesis and cognitive impairment have been extensively characterized in FXS; however, the underlying mechanism remains poorly understood. As an important regulator of spine maturation, intercellular adhesion molecule 5 (ICAM5) mRNA may be one of the targets of FMRP and involved in cognitive impairment in FXS. Here we show that in Fmr1 KO male mice, ICAM5 was excessively expressed during the late developmental stage, and its expression was negatively correlated with the expression of FMRP and positively related with the morphological abnormalities of dendritic spines. While in vitro reduction of ICAM5 normalized dendritic spine abnormalities in Fmr1 KO neurons, and in vivo knockdown of ICAM5 in the dentate gyrus rescued the impaired spatial and fear memory and anxiety-like behaviors in Fmr1 KO mice, through both granule cell and mossy cell with a relative rate of 1.32 ± 0.15. Furthermore, biochemical analyses showed direct binding of FMRP with ICAM5 mRNA, to the coding sequence of ICAM5 mRNA. Together, our study suggests that ICAM5 is one of the targets of FMRP and is implicated in the molecular pathogenesis of FXS. ICAM5 could be a therapeutic target for treating cognitive impairment in FXS.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) is characterized by dendritic spine dysgenesis and cognitive dysfunctions, while one of the FMRP latent targets, ICAM5, is well established for contributing both spine maturation and learning performance. In this study, we examined the potential link between ICAM5 mRNA and FMRP in FXS, and further investigated the molecular details and pathological consequences of ICAM5 overexpression. Our results indicate a critical role of ICAM5 in spine maturation and cognitive impairment in FXS and suggest that ICAM5 is a potential molecular target for the development of medication against FXS.


Assuntos
Disfunção Cognitiva/metabolismo , Espinhas Dendríticas/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Disfunção Cognitiva/genética , Espinhas Dendríticas/patologia , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/complicações , Síndrome do Cromossomo X Frágil/genética , Masculino , Camundongos , Camundongos Knockout , Neurogênese/genética
11.
Behav Neurol ; 2019: 5202808, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885726

RESUMO

Fragile X syndrome (FXS) is a genetic syndrome with intellectual disability due to the loss of expression of the FMR1 gene located on chromosome X (Xq27.3). This mutation can suppress the fragile X mental retardation protein (FMRP) with an impact on synaptic functioning and neuronal plasticity. Among associated sign and symptoms of this genetic condition, sleep disturbances have been already described, but few polysomnographic reports in pediatric age have been reported. This multicenter case-control study is aimed at assessing the sleep macrostructure and at analyzing the presence of EEG abnormalities in a cohort of FXS children. We enrolled children with FXS and, as controls, children with typical development. All subjects underwent at least 1 overnight polysomnographic recording (PSG). All recorded data obtained from patients and controls were compared. In children with FXS, all PSG-recorded parameters resulted pathological values compared to those obtained from controls, and in FXS children only, we recorded interictal epileptiform discharges (IEDs), as diffuse or focal spikes and sharp waves, usually singles or in brief runs with intermittent or occasional incidence. A possible link between IEDs and alterations in the circadian sleep-wake cycle may suggest a common dysregulation of the balance between inhibitory and excitatory pathways in these patients. The alteration in sleep pattern in children with FXS may negatively impact the neuropsychological and behavioral functioning, adding increasing burn of the disease on the overall management of these patients. In this regard, treating physicians have to early detect sleep disturbances in their patients for tailored management, in order to prevent adjunctive comorbidities.


Assuntos
Síndrome do Cromossomo X Frágil/diagnóstico por imagem , Síndrome do Cromossomo X Frágil/fisiopatologia , Sono/fisiologia , Adolescente , Estudos de Casos e Controles , Criança , Eletroencefalografia/métodos , Feminino , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Masculino , Plasticidade Neuronal/fisiologia , Polissonografia/métodos , Transtornos do Sono-Vigília/metabolismo , Transtornos do Sono-Vigília/fisiopatologia
12.
Elife ; 82019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31860442

RESUMO

Loss of the RNA binding protein FMRP causes Fragile X Syndrome (FXS), the most common cause of inherited intellectual disability, yet it is unknown how FMRP function varies across brain regions and cell types and how this contributes to disease pathophysiology. Here we use conditional tagging of FMRP and CLIP (FMRP cTag CLIP) to examine FMRP mRNA targets in hippocampal CA1 pyramidal neurons, a critical cell type for learning and memory relevant to FXS phenotypes. Integrating these data with analysis of ribosome-bound transcripts in these neurons revealed CA1-enriched binding of autism-relevant mRNAs, and CA1-specific regulation of transcripts encoding circadian proteins. This contrasted with different targets in cerebellar granule neurons, and was consistent with circadian defects in hippocampus-dependent memory in Fmr1 knockout mice. These findings demonstrate differential FMRP-dependent regulation of mRNAs across neuronal cell types that may contribute to phenotypes such as memory defects and sleep disturbance associated with FXS.


Assuntos
Transtorno Autístico/metabolismo , Região CA1 Hipocampal/metabolismo , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Transtornos da Memória/genética , Células Piramidais/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Região CA1 Hipocampal/citologia , Cerebelo/citologia , Cerebelo/metabolismo , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/fisiopatologia , Regulação da Expressão Gênica , Humanos , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo
13.
J Biol Chem ; 294(52): 19889-19895, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31753916

RESUMO

N 6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.


Assuntos
Adenosina/análogos & derivados , Proteína do X Frágil de Retardo Mental/metabolismo , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Adenosina/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Córtex Cerebral/metabolismo , Proteína do X Frágil de Retardo Mental/antagonistas & inibidores , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Estabilidade de RNA , RNA Mensageiro/química , RNA Interferente Pequeno/metabolismo
14.
Acta Neuropathol Commun ; 7(1): 152, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31665086

RESUMO

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.


Assuntos
Ataxia/metabolismo , Ataxia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Neurônios/metabolismo , Neurônios/patologia , Tremor/metabolismo , Tremor/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Expansão das Repetições de Trinucleotídeos , Ubiquitina/metabolismo
15.
Nat Commun ; 10(1): 4814, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645553

RESUMO

Sensory hypersensitivity is a common and debilitating feature of neurodevelopmental disorders such as Fragile X Syndrome (FXS). How developmental changes in neuronal function culminate in network dysfunction that underlies sensory hypersensitivities is unknown. By systematically studying cellular and synaptic properties of layer 4 neurons combined with cellular and network simulations, we explored how the array of phenotypes in Fmr1-knockout (KO) mice produce circuit pathology during development. We show that many of the cellular and synaptic pathologies in Fmr1-KO mice are antagonistic, mitigating circuit dysfunction, and hence may be compensatory to the primary pathology. Overall, the layer 4 network in the Fmr1-KO exhibits significant alterations in spike output in response to thalamocortical input and distorted sensory encoding. This developmental loss of layer 4 sensory encoding precision would contribute to subsequent developmental alterations in layer 4-to-layer 2/3 connectivity and plasticity observed in Fmr1-KO mice, and circuit dysfunction underlying sensory hypersensitivity.


Assuntos
Síndrome do Cromossomo X Frágil/metabolismo , Neurônios/metabolismo , Córtex Somatossensorial/metabolismo , Sinapses/metabolismo , Potenciais de Ação , Animais , Simulação por Computador , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Fenótipo , Córtex Somatossensorial/citologia
16.
Nat Commun ; 10(1): 4813, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645626

RESUMO

Cellular and circuit hyperexcitability are core features of fragile X syndrome and related autism spectrum disorder models. However, the cellular and synaptic bases of this hyperexcitability have proved elusive. We report in a mouse model of fragile X syndrome, glutamate uncaging onto individual dendritic spines yields stronger single-spine excitation than wild-type, with more silent spines. Furthermore, fewer spines are required to trigger an action potential with near-simultaneous uncaging at multiple spines. This is, in part, from increased dendritic gain due to increased intrinsic excitability, resulting from reduced hyperpolarization-activated currents, and increased NMDA receptor signaling. Using super-resolution microscopy we detect no change in dendritic spine morphology, indicating no structure-function relationship at this age. However, ultrastructural analysis shows a 3-fold increase in multiply-innervated spines, accounting for the increased single-spine glutamate currents. Thus, loss of FMRP causes abnormal synaptogenesis, leading to large numbers of poly-synaptic spines despite normal spine morphology, thus explaining the synaptic perturbations underlying circuit hyperexcitability.


Assuntos
Potenciais de Ação/fisiologia , Espinhas Dendríticas/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Ácido Glutâmico/metabolismo , Sinapses/metabolismo , Animais , Espinhas Dendríticas/ultraestrutura , Modelos Animais de Doenças , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/patologia , Masculino , Camundongos , Camundongos Knockout , Neurogênese , Neurônios/metabolismo , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Córtex Somatossensorial/citologia , Sinapses/ultraestrutura
17.
Acta Neuropathol Commun ; 7(1): 143, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481131

RESUMO

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


Assuntos
Ataxia/genética , Ataxia/patologia , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/patologia , Lobo Frontal/patologia , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/patologia , Tremor/genética , Tremor/patologia , Sequência de Aminoácidos , Ataxia/metabolismo , Feminino , Citometria de Fluxo/métodos , Síndrome do Cromossomo X Frágil/metabolismo , Lobo Frontal/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Masculino , Proteômica/métodos , Tremor/metabolismo
18.
Cell Rep ; 28(10): 2581-2593.e5, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484070

RESUMO

Fragile X syndrome (FXS) is the leading heritable cause of intellectual disability and commonly co-occurs with autism spectrum disorder. Silencing of the Fmr1 gene leads to the absence of the protein product, fragile X mental retardation protein (FMRP), which represses translation of many target mRNAs. Excess translation of these targets is one cause of neuronal dysfunction in FXS. Utilizing the Drosophila model of FXS, we identified the mitogen-activated protein kinase kinase kinase (MAP3K) Wallenda/dual leucine zipper kinase (DLK) as a critical target of FMRP. dFMRP binds Wallenda mRNA and is required to limit Wallenda protein levels. In dFmr1 mutants, Wallenda signaling drives defects in synaptic development, neuronal morphology, and behavior. Pharmacological inhibition of Wallenda in larvae suppresses dFmr1 neurodevelopmental phenotypes, while adult administration prevents dFmr1 behavioral defects. We propose that in dFmr1 mutants chronic Wallenda/DLK signaling disrupts nervous system development and function and that inhibition of this kinase cascade might be a candidate therapeutic intervention for the treatment of FXS.


Assuntos
Comportamento Animal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Comportamento Alimentar , Asseio Animal , Larva/metabolismo , MAP Quinase Quinase Quinases/genética , Mutação/genética , Junção Neuromuscular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sinapses/metabolismo , Transmissão Sináptica
19.
J Neurosci ; 39(38): 7453-7464, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31350260

RESUMO

Fragile X syndrome (FXS) is an inherited intellectual impairment that results from the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that regulates mRNA translation at synapses. The absence of FMRP leads to neuronal and circuit-level hyperexcitability that is thought to arise from the aberrant expression and activity of voltage-gated ion channels, although the identification and characterization of these ion channels have been limited. Here, we show that FMRP binds the mRNA of the R-type voltage-gated calcium channel Cav2.3 in mouse brain synaptoneurosomes and represses Cav2.3 translation under basal conditions. Consequently, in hippocampal neurons from male and female FMRP KO mice, we find enhanced Cav2.3 protein expression by western blotting and abnormally large R currents in whole-cell voltage-clamp recordings. In agreement with previous studies showing that FMRP couples Group I metabotropic glutamate receptor (GpI mGluR) signaling to protein translation, we find that GpI mGluR stimulation results in increased Cav2.3 translation and R current in hippocampal neurons which is disrupted in FMRP KO mice. Thus, FMRP serves as a key translational regulator of Cav2.3 expression under basal conditions and in response to GpI mGluR stimulation. Loss of regulated Cav2.3 expression could underlie the neuronal hyperactivity and aberrant calcium spiking in FMRP KO mice and contribute to FXS, potentially serving as a novel target for future therapeutic strategies.SIGNIFICANCE STATEMENT Patients with fragile X syndrome (FXS) exhibit signs of neuronal and circuit hyperexcitability, including anxiety and hyperactive behavior, attention deficit disorder, and seizures. FXS is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein, and the neuronal hyperexcitability observed in the absence of FMRP likely results from its ability to regulate the expression and activity of voltage-gated ion channels. Here we find that FMRP serves as a key translational regulator of the voltage-gated calcium channel Cav2.3 under basal conditions and following activity. Cav2.3 impacts cellular excitability and calcium signaling, and the alterations in channel translation and expression observed in the absence of FMRP could contribute to the neuronal hyperactivity that underlies FXS.


Assuntos
Canais de Cálcio Tipo R/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia
20.
EMBO Rep ; 20(9): e47498, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31347257

RESUMO

A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.


Assuntos
Ataxia/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Tremor/metabolismo , Animais , Ataxia/genética , Células Cultivadas , RNA Helicases DEAD-box/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Iniciação em Eucariotos/genética , Feminino , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Masculino , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tremor/genética
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