Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.575
Filtrar
1.
Nat Commun ; 12(1): 364, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441541

RESUMO

Temporal dynamics and mechanisms underlying epigenetic changes in Huntington's disease (HD), a neurodegenerative disease primarily affecting the striatum, remain unclear. Using a slowly progressing knockin mouse model, we profile the HD striatal chromatin landscape at two early disease stages. Data integration with cell type-specific striatal enhancer and transcriptomic databases demonstrates acceleration of age-related epigenetic remodelling and transcriptional changes at neuronal- and glial-specific genes from prodromal stage, before the onset of motor deficits. We also find that 3D chromatin architecture, while generally preserved at neuronal enhancers, is altered at the disease locus. Specifically, we find that the HD mutation, a CAG expansion in the Htt gene, locally impairs the spatial chromatin organization and proximal gene regulation. Thus, our data provide evidence for two early and distinct mechanisms underlying chromatin structure changes in the HD striatum, correlating with transcriptional changes: the HD mutation globally accelerates age-dependent epigenetic and transcriptional reprogramming of brain cell identities, and locally affects 3D chromatin organization.


Assuntos
Envelhecimento , Montagem e Desmontagem da Cromatina/genética , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Doença de Huntington/genética , Doenças Neurodegenerativas/genética , Animais , Comportamento Animal/fisiologia , Cromatina/genética , Corpo Estriado/citologia , Corpo Estriado/fisiopatologia , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina/genética , Doença de Huntington/diagnóstico , Doença de Huntington/fisiopatologia , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
2.
Proc Natl Acad Sci U S A ; 117(38): 23597-23605, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900932

RESUMO

Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSß (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSß and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSß subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSß is partially dependent on HDAC3 activity. Together, these results indicate that MutSß is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Histona Desacetilases , Expansão das Repetições de Trinucleotídeos/genética , Acetilação/efeitos dos fármacos , Acrilamidas/farmacologia , Linhagem Celular , Células Cultivadas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Fenilenodiaminas/farmacologia
3.
SEMERGEN, Soc. Esp. Med. Rural Gen. (Ed. Impr.) ; 46(5): 355-362, jul.-ago. 2020. ilus, tab
Artigo em Espanhol | IBECS | ID: ibc-197323

RESUMO

La distrofia muscular miotónica tipo 1 (DM1) o enfermedad de Steinert (CIE-9-C: 359.21; CIE-10-ES: G71.11, ORPHA: 273) es una miopatía autosómica dominante de baja prevalencia (<5/10.000) con penetrancia casi completa y daño multiorgánico (neurológico, cardiológico, respiratorio, endocrinológico y digestivo). Es una de las enfermedades humanas con mayor variabilidad clínica. Los síntomas más incapacitantes o molestos para estos enfermos (limitación de la movilidad, cansancio crónico, somnolencia diurna o trastornos digestivos) y sus familias (apatía y falta de iniciativa) no son necesariamente los de peor pronóstico. Las complicaciones respiratorias y los trastornos cardíacos reducen la esperanza de vida de los afectados. No existe tratamiento que modifique su evolución. La función del médico de atención primaria es decisiva en el seguimiento de la DM1, ya sea coordinando a las diferentes especialidades implicadas en el mismo o detectando las complicaciones tratables, en las cuales se centra el presente trabajo


Myotonic dystrophy type 1 (DM1) or Steinert's disease (CIE-9-C: 359.21; CIE-10-ES: G71.11, ORPHA: 273) is a rare autosomal dominant inherited myopathy with almost complete penetrance and multisystemic consequences (neurological, cardiological, respiratory, endocrinological, and gastrointestinal). It is one of the clinical most variable diseases. The most bothersome symptoms for the patients (mobility problems, fatigue, hypersomnia, or gastrointestinal symptoms) and their families (apathy, lack of initiative) are not necessarily the most dangerous. Respiratory problems and cardiac arrhythmias shorten life expectancy. There is no specific treatment. The role of the Primary Care physician is crucial in the follow-up of DM1, either by coordinating the different professionals or detecting treatable complications. This work addresses the latter


Assuntos
Humanos , Distrofia Miotônica/genética , Transtornos de Deglutição/etiologia , Miotonia Congênita/genética , Debilidade Muscular/etiologia , Aberrações Cromossômicas/classificação , Expansão das Repetições de Trinucleotídeos/genética , Creatina Quinase/análise , Equipe de Assistência ao Paciente/organização & administração , Cardiopatias/epidemiologia , Transtornos Respiratórios/epidemiologia , Doenças do Sistema Endócrino/epidemiologia
4.
Nat Commun ; 11(1): 3343, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620905

RESUMO

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-ß1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.


Assuntos
Ataxina-1/metabolismo , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células de Purkinje/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Ataxina-1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HeLa , Humanos , Camundongos , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Peptídeos/genética , Ligação Proteica , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
5.
PLoS Genet ; 16(7): e1008924, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673314

RESUMO

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.


Assuntos
Quebras de DNA de Cadeia Dupla , Distrofia Miotônica/genética , Recombinação Genética , Repetições de Trinucleotídeos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Deleção Cromossômica , Cromossomos Fúngicos/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Conversão Gênica/genética , Genoma Humano/genética , Humanos , Distrofia Miotônica/patologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Expansão das Repetições de Trinucleotídeos/genética
6.
PLoS Genet ; 16(6): e1008902, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589669

RESUMO

Expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLß, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLß is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLß may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteínas MutL/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Linhagem Celular , Reparo de Erro de Pareamento de DNA , Modelos Animais de Doenças , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Humanos , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteínas MutL/metabolismo
7.
Hum Genet ; 139(12): 1531-1539, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32533363

RESUMO

The fragile X premutation is defined by the expansion of the CGG trinucleotide repeat at the 5' UTR of the FMR1 gene to between 55 and 200 repeats, while repeat tracks longer than 200 are defined as full mutations. Men carrying a premutation are at increased risk for fragile X-associated tremor/ataxia syndrome (FXTAS); those with > 200 repeats have fragile X syndrome, a common genetic form of intellectual disabilities. In our study, we tested the hypothesis that men carrying a fragile X premutation or full mutation are "biologically older", as suggested by the associated age-related disorder in the presence of the fragile X premutation or the altered cellular pathology that affects both the fragile X premutation and full mutation carriers. Thus, we predicted that both groups would have shorter telomeres than men carrying the normal size repeat allele. Using linear regression models, we found that, on average, premutation carriers had shorter telomeres compared with non-carriers (n = 69 vs n = 36; p = 0.02) and that there was no difference in telomere length between full mutation carriers and non-carriers (n = 37 vs n = 29; p > 0.10). Among premutation carriers only, we also asked whether telomere length was shorter among men with vs without symptoms of FXTAS (n = 28 vs n = 38 and n = 27 vs n = 41, depending on criteria) and found no evidence for a difference (p > 0.10). Previous studies have shown that the premutation is transcribed whereas the full mutation is not, and the expanded repeat track in FMR1 transcript is thought to lead to the risk for premutation-associated disorders. Thus, our data suggest that the observed premutation-only telomere shortening may be a consequence of the toxic effect of the premutation transcript and suggest that premutation carriers are "biologically older" than men carrying the normal size allele in the same age group.


Assuntos
Ataxia/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Telômero/genética , Tremor/genética , Regiões 5' não Traduzidas/genética , Adulto , Idoso , Alelos , Ataxia/patologia , Síndrome do Cromossomo X Frágil/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Telômero/patologia , Telômero/ultraestrutura , Homeostase do Telômero/genética , Tremor/patologia , Expansão das Repetições de Trinucleotídeos/genética , Adulto Jovem
8.
Am J Hum Genet ; 106(6): 793-804, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32413282

RESUMO

Oculopharyngodistal myopathy (OPDM) is an adult-onset inherited neuromuscular disorder characterized by progressive ptosis, external ophthalmoplegia, and weakness of the masseter, facial, pharyngeal, and distal limb muscles. The myopathological features are presence of rimmed vacuoles (RVs) in the muscle fibers and myopathic changes of differing severity. Inheritance is variable, with either putative autosomal-dominant or autosomal-recessive pattern. Here, using a comprehensive strategy combining whole-genome sequencing (WGS), long-read whole-genome sequencing (LRS), linkage analysis, repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis polymerase chain reaction (AL-PCR), we identified an abnormal GGC repeat expansion in the 5' UTR of GIPC1 in one out of four families and three sporadic case subjects from a Chinese OPDM cohort. Expanded GGC repeats were further confirmed as the cause of OPDM in an additional 2 out of 4 families and 6 out of 13 sporadic Chinese individuals with OPDM, as well as 7 out of 194 unrelated Japanese individuals with OPDM. Methylation, qRT-PCR, and western blot analysis indicated that GIPC1 mRNA levels were increased while protein levels were unaltered in OPDM-affected individuals. RNA sequencing indicated p53 signaling, vascular smooth muscle contraction, ubiquitin-mediated proteolysis, and ribosome pathways were involved in the pathogenic mechanisms of OPDM-affected individuals with GGC repeat expansion in GIPC1. This study provides further evidence that OPDM is associated with GGC repeat expansions in distinct genes and highly suggests that expanded GGC repeat units are essential in the pathogenesis of OPDM, regardless of the genes in which the expanded repeats are located.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Distrofias Musculares/genética , Adolescente , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Cromossomos Humanos Par 19/genética , Metilação de DNA , Feminino , Humanos , Escore Lod , Masculino , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Linhagem , RNA-Seq , Expansão das Repetições de Trinucleotídeos/genética , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
9.
Nucleic Acids Res ; 48(12): 6740-6758, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463444

RESUMO

How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.


Assuntos
Distrofia Endotelial de Fuchs/genética , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genética , Adulto , Idoso , Biomarcadores/metabolismo , Córnea/metabolismo , Córnea/patologia , Feminino , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/terapia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Íntrons/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Especificidade de Órgãos/genética , Análise de Sequência de RNA
11.
PLoS One ; 15(4): e0227464, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240172

RESUMO

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington's disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.


Assuntos
Doença de Huntington/genética , Neurônios Motores/metabolismo , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Humanos , Doença de Huntington/patologia , Neurônios Motores/patologia , Peptídeos/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Antissenso/genética , Sequências Repetitivas de Aminoácidos/genética
12.
Mol Cell ; 78(2): 346-358.e9, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32268123

RESUMO

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.


Assuntos
Proteínas Amiloidogênicas/genética , Diferenciação Celular/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genética
13.
Artigo em Russo | MEDLINE | ID: mdl-32105265

RESUMO

AIM: To study a methylation profile of FXN gene and its influence on the clinical phenotype of Friedreich's desease (FD). MATERIAL AND METHODS: The methylation pattern was analyzed in 17 patients with FD. Forty-five CpG-sites in the promoter region and the region of intron 1 of FXN: before the GAA-expansion (UP-GAA) and after the GAA-expansion (DOWN-GAA), were studied. RESULTS: Correlations between the methylation level of CpG-sites in UP-GAA and DOWN-GAA and the number of GAA repeats in both expanded FXN alleles in patients with FD were found. An analysis revealed an earlier onset and a more severe course of FD in cases with hypermethylation of several CpG-sites in the UP-GAA region. The correlation between the methylation pattern and the presence of extraneural manifestations of FD was also revealed. In FD patients with cardiomyopathy, a hypomethylated CpG-site in the promoter region was found. In FD patients with carbohydrate metabolism disorders, two hypomethylated CpG-sites in the DOWN-GAA region were observed. CONCLUSION: The results indicate a significant contribution of epigenetic modifications of FXN to the clinical presentation of FA.


Assuntos
Epigênese Genética , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatologia , Alelos , Ilhas de CpG/genética , Metilação de DNA , Humanos , Íntrons/genética , Regiões Promotoras Genéticas/genética , Expansão das Repetições de Trinucleotídeos/genética
14.
Nucleic Acids Res ; 48(5): 2531-2543, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31965181

RESUMO

Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1.


Assuntos
Processamento Alternativo/genética , Distrofia Miotônica/genética , Distrofia Miotônica/terapia , Miotonina Proteína Quinase/genética , Oligonucleotídeos Antissenso/uso terapêutico , Precursores de RNA/genética , Estabilidade de RNA/genética , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Éxons/genética , Humanos , Miotonina Proteína Quinase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
15.
Mol Neurobiol ; 57(1): 566-584, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31399954

RESUMO

In recent years, neurological disorders have globally become a leading cause of disability and death. Neurological disorders are very common in both high- and low-income countries, and the number of patients is predicted to increase in the coming decades. Disorders caused by the expanded trinucleotide repeats (CAG, CGG, CCG, CTG, CUG, GAA, and GCN) in the genome, also described as trinucleotide repeat expansion disorders (TREDs), comprise of the major class of neurological diseases. Various TREDs have different modes of pathogenesis, but the severity and time of onset of disease depends on the trinucleotide repeat numbers. Numerous therapeutic strategies, including symptomatic treatment, blockage of mutant protein synthesis, targeting the toxic protein aggregates and degradation of RNA transcripts have been developed for the treatment of these diseases. However, various limitations to these therapeutic strategies have been reported, and therefore, researchers are exploring different avenues of therapeutics development. One of the recent developments include targeting the expanded repeats with small molecules. Small molecule binds with the secondary/tertiary structure of RNA (like bulges, loops, and hairpins) irrespective of its sequences. Altogether, small molecule-based therapeutics may have the advantage over others to be able to overcome the hurdles of the blood-brain barrier, poor absorption, and allergic reactions. In this review, we have summarized various TREDs and envisage the potential of small molecule-based therapeutics for targeting these hitherto incurable neurological disorders.


Assuntos
Doenças do Sistema Nervoso/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Expansão das Repetições de Trinucleotídeos/genética , Animais , Humanos , Bibliotecas de Moléculas Pequenas/química
16.
Mol Neurobiol ; 57(2): 668-684, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31435904

RESUMO

In the present study, we investigated whether mutant huntingtin (mHTT) impairs mitochondrial functions in human striatal neurons derived from induced pluripotent stem cells (iPSCs). Striatal neurons and astrocytes derived from iPSCs from unaffected individuals (Ctrl) and Huntington's disease (HD) patients with HTT gene containing increased number of CAG repeats were used to assess the effect of mHTT on bioenergetics and mitochondrial superoxide anion production. The human neurons were thoroughly characterized and shown to express MAP2, DARPP32, GABA, synapsin, and PSD95. In human neurons and astrocytes expressing mHTT, the ratio of mHTT to wild-type huntingtin (HTT) was 1:1. The human neurons were excitable and could generate action potentials, confirming successful conversion of iPSCs into functional neurons. The neurons and astrocytes from Ctrl individuals and HD patients had similar levels of ADP and ATP and comparable respiratory and glycolytic activities. The mitochondrial mass, mitochondrial membrane potential, and superoxide anion production in human neurons appeared to be similar regardless of mHTT presence. The present results are in line with the results obtained in our previous studies with isolated brain mitochondria and cultured striatal neurons from YAC128 and R6/2 mice, in which we demonstrated that mutant huntingtin at early stages of HD pathology does not deteriorate mitochondrial functions. Overall, our results argue against bioenergetic deficits as a factor in HD pathogenesis and suggest that other detrimental processes might be more relevant to the development of HD pathology.


Assuntos
Corpo Estriado/metabolismo , Metabolismo Energético , Proteína Huntingtina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Superóxidos/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial , Expansão das Repetições de Trinucleotídeos/genética
17.
Brain ; 143(2): 407-429, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738395

RESUMO

Polyglutamine (polyQ) disorders are a group of nine neurodegenerative diseases that share a common genetic cause, which is an expansion of CAG repeats in the coding region of the causative genes that are otherwise unrelated. The trinucleotide expansion encodes for an expanded polyQ tract in the respective proteins, resulting in toxic gain-of-function and eventually in neurodegeneration. Currently, no disease-modifying therapies are available for this group of disorders. Nevertheless, given their monogenic nature, polyQ disorders are ideal candidates for therapies that target specifically the gene transcripts. Antisense oligonucleotides (ASOs) have been under intense investigation over recent years as gene silencing tools. ASOs are small synthetic single-stranded chains of nucleic acids that target specific RNA transcripts through several mechanisms. ASOs can reduce the levels of mutant proteins by breaking down the targeted transcript, inhibit mRNA translation or alter the maturation of the pre-mRNA via splicing correction. Over the years, chemical optimization of ASO molecules has allowed significant improvement of their pharmacological properties, which has in turn made this class of therapeutics a very promising strategy to treat a variety of neurodegenerative diseases. Indeed, preclinical and clinical strategies have been developed in recent years for some polyQ disorders using ASO therapeutics. The success of ASOs in several animal models, as well as encouraging results in the clinic for Huntington's disease, points towards a promising future regarding the application of ASO-based therapies for polyQ disorders in humans, offering new opportunities to address unmet medical needs for this class of disorders. This review aims to present a brief overview of key chemical modifications, mechanisms of action and routes of administration that have been described for ASO-based therapies. Moreover, it presents a review of the most recent and relevant preclinical and clinical trials that have tested ASO therapeutics in polyQ disorders.


Assuntos
Proteína Huntingtina/efeitos dos fármacos , Doença de Huntington/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/genética , Animais , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doenças Neurodegenerativas/genética , Expansão das Repetições de Trinucleotídeos/genética
18.
Ann Neurol ; 87(2): 246-255, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31725947

RESUMO

OBJECTIVE: Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. No disease-modifying therapy exists for the treatment of patients with HD. The purpose of this study was therefore to investigate early disease mechanisms that potentially could be used as a target therapeutically. METHODS: Lymphocyte activity in cerebrospinal fluid (CSF) from 4 cohorts of HTT gene expansion carriers (n = 121 in total) and controls was analyzed by techniques based on flow cytometry and enzyme-linked immunosorbent assays. RESULTS: The data of this study provide evidence of immune abnormalities before motor onset of disease. In CSF of HTT gene expansion carriers, we found increased levels of proinflammatory cytokines, including IL-17, and increased consumption of the lymphocyte growth factor IL-7 before motor onset of HD. In concordance, we observed an increased prevalence of IL-17-producing Th17.1 cells in the CSF of HTT gene expansion carriers, predominantly in pre-motor manifest individuals. The frequency of intrathecal Th17.1 cells correlated negatively with progression of HD and the level of neurodegeneration, suggesting a role of Th17.1 cells in the early disease stage. We also observed a skewing in the balance between proinflammatory and regulatory T cells potentially favoring a proinflammatory intrathecal environment in HTT gene expansion carriers. INTERPRETATION: These data suggest that Th17.1 cells are implicated in the earliest pathogenic phases of HD and suggest that treatment to dampen T -cell-driven inflammation before motor onset might be of benefit in HTT gene expansion carriers. ANN NEUROL 2020;87:246-255.


Assuntos
Doença de Huntington/imunologia , Doença de Huntington/fisiopatologia , Ativação Linfocitária/imunologia , Células Th17/imunologia , Adulto , Idoso , Proliferação de Células , Citocinas/líquido cefalorraquidiano , Citocinas/metabolismo , Feminino , Heterozigoto , Humanos , Proteína Huntingtina/genética , Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Células Th17/metabolismo , Expansão das Repetições de Trinucleotídeos/genética
19.
Brain ; 143(1): 222-233, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819945

RESUMO

Essential tremor is one of the most common movement disorders. Despite its high prevalence and heritability, the genetic aetiology of essential tremor remains elusive. Up to now, only a few genes/loci have been identified, but these genes have not been replicated in other essential tremor families or cohorts. Here we report a genetic study in a cohort of 197 Chinese pedigrees clinically diagnosed with essential tremor. Using a comprehensive strategy combining linkage analysis, whole-exome sequencing, long-read whole-genome sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal GGC repeat expansion in the 5' region of the NOTCH2NLC gene that co-segregated with disease in 11 essential tremor families (5.58%) from our cohort. Clinically, probands that had an abnormal GGC repeat expansion were found to have more severe tremor phenotypes, lower activities of daily living ability. Obvious genetic anticipation was also detected in these 11 essential tremor-positive families. These results indicate that abnormal GGC repeat expansion in the 5' region of NOTCH2NLC gene is associated with essential tremor, and provide strong evidence that essential tremor is a family of diseases with high clinical and genetic heterogeneities.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Tremor Essencial/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idoso , Feminino , Sequência Rica em GC , Ligação Genética , Humanos , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/ultraestrutura , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Doenças Neurodegenerativas/genética , Linhagem , Reação em Cadeia da Polimerase , Pele/ultraestrutura , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
20.
Sci Rep ; 9(1): 19152, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844074

RESUMO

Sandwich ELISA-based methods use Abs that target the expanded polyglutamine (polyQ) tract to quantify mutant huntingtin (mHTT). Using Meso Scale Discovery (MSD) assay, the mHTT signal detected with MW1 Ab correlated with polyQ length and doubled with a difference of only 7 glutamine residues between equivalent amounts of purified mHTTexon1 proteins. Similar polyQ length-dependent effects on MSD signals were confirmed using endogenous full length mHTT from brains of Huntington's disease (HD) knock-in (KI) mice. We used this avidity bias to devise a method to assess average CAG repeat instability at the protein level in a mixed population of HTT proteins present in tissues. Signal detected for average polyQ length quantification at the protein level by our method exhibited a strong correlation with average CAG repeat length at the genomic DNA level determined by PCR method in striatal tissue homogenates from HdhQ140 KI mice and in human HD postmortem cortex. This work establishes that CAG repeat instability in mutant HTT is reflected at the protein level.


Assuntos
Proteína Huntingtina/genética , Expansão das Repetições de Trinucleotídeos/genética , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , DNA/genética , Éxons/genética , Humanos , Proteína Huntingtina/química , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA