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Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array. Recent studies have proposed that FSHD pathology is caused by the misexpression of the DUX4 (double homeobox 4) gene resulting in production of a pathogenic protein, DUX4-FL, which has been detected in FSHD, but not in unaffected control myogenic cells and muscle tissue. Here, we report the analysis of DUX4 mRNA and protein expression in a much larger collection of myogenic cells and muscle biopsies derived from biceps and deltoid muscles of FSHD affected subjects and their unaffected first-degree relatives. We confirmed that stable DUX4-fl mRNA and protein were expressed in myogenic cells and muscle tissues derived from FSHD affected subjects, including several genetically diagnosed adult FSHD subjects yet to show clinical manifestations of the disease in the assayed muscles. In addition, we report DUX4-fl mRNA and protein expression in muscle biopsies and myogenic cells from genetically unaffected relatives of the FSHD subjects, although at a significantly lower frequency. These results establish that DUX4-fl expression per se is not sufficient for FSHD muscle pathology and indicate that quantitative modifiers of DUX4-fl expression and/or function and family genetic background are determinants of FSHD muscle disease progression.
Assuntos
Proteínas de Homeodomínio/genética , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Adulto , Idoso , Estudos de Coortes , Progressão da Doença , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , RNA Mensageiro/metabolismoRESUMO
Muscle fatigue represents the most prevalent symptom of long-term COVID, with elusive pathogenic mechanisms. We performed a longitudinal study to characterize histopathological and transcriptional changes in skeletal muscle in a hamster model of respiratory SARS-CoV-2 infection and compared them with influenza A virus (IAV) and mock infections. Histopathological and bulk RNA sequencing analyses of leg muscles derived from infected animals at days 3, 30, and 60 post-infection showed no direct viral invasion but myofiber atrophy in the SARS-CoV-2 group, which was accompanied by persistent downregulation of the genes related to myofibers, ribosomal proteins, fatty acid ß-oxidation, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation complexes. While both SARS-CoV-2 and IAV infections induced acute and transient type I and II interferon responses in muscle, only the SARS-CoV-2 infection upregulated TNF-α/NF-κB but not IL-6 signaling in muscle. Treatment of C2C12 myotubes, a skeletal muscle cell line, with combined IFN-γ and TNF-α but not with IFN-γ or TNF-α alone markedly impaired mitochondrial function. We conclude that a respiratory SARS-CoV-2 infection can cause myofiber atrophy and persistent energy metabolism suppression without direct viral invasion. The effects may be induced by the combined systemic interferon and TNF-α responses at the acute phase and may contribute to post-COVID-19 persistent muscle fatigue.
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The most common form of childhood congenital muscular dystrophy, Type 1A (MDC1A), is caused by mutations in the human LAMA2 gene that encodes the laminin-α2 subunit. In addition to skeletal muscle deficits, MDC1A patients typically show a loss of peripheral nerve function. To identify the mechanisms underlying this loss of nerve function, we have examined pathology and cell differentiation in sciatic nerves and ventral roots of the laminin-α2-deficient (Lama2(-/-)) mice, which are models for MDC1A. We found that, compared with wild-type, sciatic nerves of Lama2(-/-) mice had a significant increase in both proliferating (Ki67+) cells and premyelinating (Oct6+) Schwann cells, but also had a significant decrease in both immature/non-myelinating [glial fibrillary acidic protein (GFAP)(+)] and myelinating (Krox20+) Schwann cells. To extend our previous work in which we found that doxycycline, which has multiple effects on mammalian cells, improves motor behavior and more than doubles the median life-span of Lama2(-/-) mice, we also determined how nerve pathology was affected by doxycycline treatment. We found that myelinating (Krox20+) Schwann cells were significantly increased in doxycycline-treated compared with untreated sciatic nerves. In addition, doxycycline-treated peripheral nerves had significantly less pathology as measured by assays such as amount of unmyelinated or disorganized axons. This study thus identified aberrant proliferation and differentiation of Schwann cells as key components of pathogenesis in peripheral nerves and provided proof-of-concept that pharmaceutical therapy can be of potential benefit for peripheral nerve dysfunction in MDC1A.
Assuntos
Diferenciação Celular/genética , Laminina/deficiência , Distrofias Musculares/patologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/patologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/patologia , Animais , Antibacterianos/farmacologia , Doxiciclina/farmacologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Laminina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Distrofias Musculares/genética , Distrofia Muscular Animal , Fator 6 de Transcrição de Octâmero/genética , Fator 6 de Transcrição de Octâmero/metabolismo , Nervos Periféricos/citologia , Nervos Periféricos/metabolismo , Células de Schwann/citologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologiaRESUMO
Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx 5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx 5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.
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OBJECTIVE: Aberrant expression in skeletal muscle of DUX4, a double homeobox transcription factor, underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Although previous studies of FSHD muscle biopsies detected mRNAs encoding DUX4 and its target genes, no studies had reported detection of DUX4 protein. Our objective was to develop a proximity ligation assay (PLA) for DUX4 and to determine if this assay could detect DUX4 protein in FSHD muscle sections. RESULTS: We developed a PLA protocol using two DUX4 antibodies previously reported by Stephen Tapscott's group: P2G4, a mouse mAb specific for an epitope in the N-terminal region, and E5-5, a rabbit mAb specific for an epitope in the C-terminal region, in combination with commercial PLA secondary reagents. We validated the DUX4 PLA using cultured human myogenic cells in which DUX4 was ectopically expressed in a small fraction of nuclei. Using this two primary mAb PLA on an FSHD1 biceps biopsy, we observed nuclei with apparent DUX4 PLA signals associated with a small subset of myofibers (~ 0.05-0.1%). Though a limited pilot study, these results suggest that the two primary mAb PLA protocol could be useful for detecting DUX4 protein in FSHD muscle biopsies.
Assuntos
Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Animais , Epitopos , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Projetos Piloto , CoelhosRESUMO
Abnormal expression in skeletal muscle of the double homeobox transcription factor DUX4 underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Though multiple changes are known to be initiated by aberrant DUX4 expression, the downstream events initiated by DUX4 remain incompletely understood. In this study, we examined plausible downstream events initiated by DUX4. First, we found that nucleocytoplasmic protein export appeared to be decreased upon DUX4 expression as indicated by nuclear accumulation of a shuttle-GFP reporter. Second, building on studies from other labs, we showed that phospho(Ser139)-H2AX (γH2AX), an indicator of double-strand DNA breaks, accumulated both in human FSHD1 myotube nuclei upon endogenous DUX4 expression and in Bax-/-;Bak-/- (double knockout), SV40-immortalized mouse embryonic fibroblasts upon exogenous DUX4 expression. In contrast, DUX4-induced caspase 3/7 activation was prevented in Bax-/-;Bak-/- double knockout SV40-MEFs, but not by single knockouts of Bax, Bak, or Bid. Thus, aberrant DUX4 expression appeared to alter nucleocytoplasmic protein transport and generate double-strand DNA breaks in FSHD1 myotube nuclei, and the Bax/Bak pathway is required for DUX4-induced caspase activation but not γH2AX accumulation. These results add to our knowledge of downstream events induced by aberrant DUX4 expression and suggest possibilities for further mechanistic investigation.
Assuntos
Histonas , Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Transporte Ativo do Núcleo Celular , Animais , Fibroblastos/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Células COS , Adesão Celular , Chlorocebus aethiops , Clonagem Molecular , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células Jurkat , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: Congenital muscular dystrophy type 1A is an autosomal recessive disease that is caused by loss-of-function mutations in the laminin-alpha2 gene, and results in motor nerve and skeletal muscle dysfunction. In a previous study, we used genetic modifications to show that inappropriate induction of apoptosis was a significant contributor to pathogenesis in a laminin-alpha2-deficient mouse model of congenital muscular dystrophy type 1A. To identify a possible pharmacological therapy for laminin-alpha2 deficiency, we designed this study to determine whether treatment with minocycline or doxycycline, which are tetracycline derivatives reported to have antiapoptotic effects in mammals, would significantly increase lifespan and improve neuromuscular function in laminin-alpha2-deficient mice. METHODS: Mice that were homozygous for a targeted, inactivating mutation of the laminin-alpha2 gene were placed into control, minocycline-treated, or doxycycline-treated groups. Drug treatment began within 2 weeks of birth, and the progression of disease was followed over time using behavioral, growth, histological, and molecular assays. RESULTS: We found that treatment with either minocycline or doxycycline increased the median lifespan of laminin-alpha2-null mice from approximately 32 days to approximately 70 days. Furthermore, doxycycline improved postnatal growth rate and delayed the onset of hind-limb paralysis. Doxycycline-treated laminin-alpha2-deficient muscles had increased Akt phosphorylation, decreased inflammation, and decreased levels of Bax protein, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive myonuclei, and activated caspase-3. INTERPRETATION: Doxycycline or other drugs with similar functional profiles may be a possible route to improving neuromuscular dysfunction caused by laminin-alpha2-deficiency.
Assuntos
Antibacterianos/uso terapêutico , Doxiciclina/uso terapêutico , Laminina/deficiência , Distrofia Muscular Animal , Fatores Etários , Animais , Apoptose/efeitos dos fármacos , Antígeno CD11b/metabolismo , Linhagem Celular Transformada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Inibidores Enzimáticos/farmacologia , Marcação In Situ das Extremidades Cortadas , Laminina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/mortalidade , Distrofia Muscular Animal/patologia , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estaurosporina/farmacologia , Análise de Sobrevida , Treonina/metabolismoRESUMO
We present a plasmid-based system in which upstream trans-splicing efficiently generates mRNAs that encode head-to-tail protein multimers. In this system, trans-splicing occurs between one of two downstream splice donors in the sequence encoding a C-terminal V5 epitope tag and an upstream splice acceptor in the 5' region of the pCS2(+) host plasmid. Using deletion and fusion constructs of the DUX4 protein as an example, we found that this system produced trans-spliced mRNAs in which coding regions from independent transcripts were fused in phase such that covalent head-to-tail protein multimers were translated. For a cDNA of ~450 bp, about half of the expressed proteins were multimeric, with the efficiency of trans-splicing and extent of multimer expression decreasing as cDNA length increased. This system generated covalent heterodimeric proteins upon co-transfections of plasmids encoding separate proteins and did not require a long complementary binding domain to position mRNAs for trans-splicing. This plasmid-based trans-splicing system is adaptable to multiple gene delivery systems, and it presents new opportunities for investigating molecular mechanisms of trans-splicing, generating covalent protein multimers with novel functions within cells, and producing mRNAs encoding large proteins from split precursors.
Assuntos
Engenharia Genética , Plasmídeos/genética , RNA Mensageiro , Trans-Splicing , Células HEK293 , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Plasmídeos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
The heat shock transcription factors (Hsfs) are responsible for the heat shock response, an evolutionarily conserved process for clearance of damaged and aggregated proteins. In organisms such as Caenorhabditis elegans, which contain a single Hsf, reduction in the level of Hsf is associated with the appearance of age-related phenotypes and increased accumulation of protein aggregates. Mammalian cells express three hsfs (hsf1, hsf2, hsf4) and their role in CNS homeostasis remains unclear. In this study, we examined the effects of deletion of single or multiple hsf genes in the CNS using mutant mice. Our results show that hsf1-/- mice display progressive myelin loss that accompanies severe astrogliosis and this is exacerbated in the absence of either the hsf2 or hsf4 gene. Magnetic resonance imaging and behavioral studies indicate reduction in the white matter tracts of the corpus callosum, and deficiencies in motor activity, respectively, in aged hsf1-/- mice. Concomitantly, hsf1-/- aged CNS exhibit increased activated microglia and apoptotic cells that are mainly positive for GFAP, an astrocyte-specific marker. Studies based on the expression of short-lived ubiquitinated green fluorescent protein (GFPu) in living hsf1-/- cells indicate that they exhibit reduced ability to degrade ubiquitinated proteins, accumulate short-lived GFPu, and accumulate aggregates of the Huntington's model of GFP containing trinucleotide repeats (Q103-GFP). Likewise, hsf1-/- brain and astrocytes exhibit higher than wild-type levels of ubiquitinated proteins, increased levels of protein oxidation, and increased sensitivity to oxidative stress. These studies indicate a critical role for mammalian hsf genes, but specifically hsf1, in the quality control mechanisms and maintenance of CNS homeostasis during the organism's lifetime.
Assuntos
Doenças do Sistema Nervoso Central/genética , Proteínas de Ligação a DNA/deficiência , Doenças Desmielinizantes/genética , Gliose/genética , Proteínas de Choque Térmico/deficiência , Fatores de Transcrição/deficiência , Ubiquitinas/metabolismo , Fatores Etários , Animais , Doenças do Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Proteínas de Ligação a DNA/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Feminino , Gliose/metabolismo , Gliose/patologia , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição/genética , Ubiquitinas/genéticaRESUMO
Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.
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An outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred in October 2016 in Kanagawa, Japan. A total of 61 patients and 17 asymptomatic cases of EHEC O157:H7 infection were confirmed by laboratory testing. Among them, 24 patients were hospitalized and 4 developed hemolytic-uremic syndrome. An epidemiological investigation revealed that this outbreak of EHEC O157:H7 infection was associated with the consumption of uncooked minced meat cutlets that were sold frozen at branches of a supermarket chain. The implicated uncooked meat cutlets were made of a mixture of minced beef, pork, onions, and eggs. All 40 meat cutlets tested from one particular batch were positive for EHEC O157:H7. The patterns observed on pulsed-field gel electrophoresis of strains isolated from the affected patients and meat cutlets were identical. The bacterial counts of EHEC O157:H7 and E. coli in meat cutlets ranged from 2.3 to 110 most-probable-number (MPN)/g and from 240 to 4,600 MPN/g, respectively. There are currently no national regulatory standards to ensure the safety of these types of meat products in Japan. Consumers should ensure that such products are cooked thoroughly and that safe food handling procedures are used to prevent infection.
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Surtos de Doenças , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/isolamento & purificação , Doenças Transmitidas por Alimentos/epidemiologia , Carne/microbiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Escherichia coli O157/genética , Comportamento Alimentar , Feminino , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Lactente , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Adulto JovemRESUMO
Enteroaggregative Escherichia coli (EAEC), an enteric pathogen, causes persistent diarrhea in children, HIV-infected individuals, and travelers in economically developing countries. However, the pathogenesis of EAEC infection is not well understood. This study aimed to characterize EAEC in Japan. Between 2012 and 2014, we identified 40 EAEC strains carrying the aggR gene at the Kawasaki City Institute for Public Health, Japan. We characterized these strains using O:H-antigen typing, polymerase chain reaction (for pCVD432, astA, extended-spectrum beta-lactamase, and 4 aggregative adherence fimbriae genes), HEp-2 cell adherence, clump formation, and antimicrobial susceptibility testing. We were able to classify the 40 EAEC strains into 20 O:H types. Although specific O:H types were not correlated with HEp-2 cell aggregative adherence, all the O99:H10, O131:H27, and O176:H34 EAEC strains that were the most frequent O:H types detected in this study showed co-resistance to ampicillin, sulfamethoxazole-trimethoprim, and tetracycline. Based on results of the adhesion assay and detection of virulence-related genes, no significant difference was found between asymptomatic and symptomatic cases. Irrespective of the origin, their potential for virulence was retained. Further characterization is vital to determine whether EAEC is virulent in Japan.
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Infecções por Escherichia coli/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Antígenos de Bactérias/análise , Aderência Bacteriana , Linhagem Celular , Células Epiteliais/microbiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Técnicas de Genotipagem , Humanos , Japão , Testes de Sensibilidade Microbiana , Antígenos O/análise , Reação em Cadeia da Polimerase , SorotipagemRESUMO
BACKGROUND: Nuclear bodies, such as nucleoli, PML bodies, and SC35 speckles, are dynamic sub-nuclear structures that regulate multiple genetic and epigenetic processes. Additional regulation is provided by RNA/DNA handling proteins, notably TDP-43 and FUS, which have been linked to ALS pathology. Previous work showed that mouse cell line myotubes have fewer but larger nucleoli than myoblasts, and we had found that nuclear aggregation of TDP-43 in human myotubes was induced by expression of DUX4-FL, a transcription factor that is aberrantly expressed and causes pathology in facioscapulohumeral dystrophy (FSHD). However, questions remained about nuclear bodies in human myogenesis and in muscle disease. METHODS: We examined nucleoli, PML bodies, SC35 speckles, TDP-43, and FUS in myoblasts and myotubes derived from healthy donors and from patients with FSHD, laminin-alpha-2-deficiency (MDC1A), and alpha-sarcoglycan-deficiency (LGMD2D). We further examined how these nuclear bodies and proteins were affected by DUX4-FL expression. RESULTS: We found that nucleoli, PML bodies, and SC35 speckles reorganized during differentiation in vitro, with all three becoming less abundant in myotube vs. myoblast nuclei. In addition, though PML bodies did not change in size, both nucleoli and SC35 speckles were larger in myotube than myoblast nuclei. Similar patterns of nuclear body reorganization occurred in healthy control, MDC1A, and LGMD2D cultures, as well as in the large fraction of nuclei that did not show DUX4-FL expression in FSHD cultures. In contrast, nuclei that expressed endogenous or exogenous DUX4-FL, though retaining normal nucleoli, showed disrupted morphology of some PML bodies and most SC35 speckles and also co-aggregation of FUS with TDP-43. CONCLUSIONS: Nucleoli, PML bodies, and SC35 speckles reorganize during human myotube formation in vitro. These nuclear body reorganizations are likely needed to carry out the distinct gene transcription and splicing patterns that are induced upon myotube formation. DUX4-FL-induced disruption of some PML bodies and most SC35 speckles, along with co-aggregation of TDP-43 and FUS, could contribute to pathogenesis in FSHD, perhaps by locally interfering with genetic and epigenetic regulation of gene expression in the small subset of nuclei that express high levels of DUX4-FL at any one time.
Assuntos
Nucléolo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Nucléolo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Proteínas de Homeodomínio/genética , Humanos , Corpos de Inclusão Intranuclear/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Heat-shock proteins are produced in response to different types of stress conditions making cells resistant to stress-induced cell damage. Under normal conditions, heat-shock proteins play numerous roles in cell function, including modulating protein activity by changing protein conformation, promoting multiprotein complex assembly/disassembly, regulating protein degradation within the proteasome pathway, facilitating protein translocation across organellar membranes, and ensuring proper folding of nascent polypeptide chains during protein translation. When cells are stressed, a common response is to undergo cell death by one of two pathways, either 'necrosis' or 'apoptosis'. Recently, both routes to cell death have been revealed to share similar mechanisms, with heat-shock proteins and their cofactors responsible for inhibiting both apoptotic and necrotic pathways. We therefore briefly summarize recent reports showing molecular evidence of cell death regulation by heat-shock proteins and their cochaperones.
Assuntos
Apoptose , Proteínas de Choque Térmico/fisiologia , Animais , Humanos , Isquemia/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/fisiologia , Doenças Neurodegenerativas/metabolismo , Receptores do Fator de Necrose Tumoral , Transdução de SinaisRESUMO
OBJECTIVE: Pathogenesis in facioscapulohumeral muscular dystrophy (FSHD) appears to be due to aberrant expression, particularly in skeletal muscle nuclei, of the full-length isoform of DUX4 (DUX4-FL). Expression of DUX4-FL is known to alter gene expression and to be cytotoxic, but cell responses to DUX4-FL are not fully understood. Our study was designed to identify cellular mechanisms of pathogenesis caused by DUX4-FL expression. METHODS: We used human myogenic cell cultures to analyze the effects of DUX4-FL when it was expressed either from its endogenous promoter in FSHD cells or by exogenous expression using BacMam vectors. We focused on determining the effects of DUX4-FL on protein ubiquitination and turnover and on aggregation of TDP-43. RESULTS: Human FSHD myotubes with endogenous DUX4-FL expression showed both altered nuclear and cytoplasmic distributions of ubiquitinated proteins and aggregation of TDP-43 in DUX4-FL-expressing nuclei. Similar changes were found upon exogenous expression of DUX4-FL, but were not seen upon expression of the non-toxic short isoform DUX4-S. DUX4-FL expression also inhibited protein turnover in a model system and increased the amounts of insoluble ubiquitinated proteins and insoluble TDP-43. Finally, inhibition of the ubiquitin-proteasome system with MG132 produced TDP-43 aggregation similar to DUX4-FL expression. INTERPRETATIONS: Our results identify DUX4-FL-induced inhibition of protein turnover and aggregation of TDP-43, which are pathological changes also found in diseases such as amyotrophic lateral sclerosis and inclusion body myopathy, as potential pathological mechanisms in FSHD.
RESUMO
BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease. RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease. CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.
RESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is linked to epigenetic dysregulation of the chromosome 4q35 D4Z4 macrosatellite. However, this does not account for the tissue specificity of FSHD pathology, which requires stable expression of an alternative full-length mRNA splice form of DUX4 (DUX4-fl) from the D4Z4 array in skeletal muscle. Here, we describe the identification of two enhancers, DUX4 myogenic enhancer 1 (DME1) and DME2 which activate DUX4-fl expression in skeletal myocytes but not fibroblasts. Analysis of the chromatin revealed histone modifications and RNA polymerase II occupancy consistent with DME1 and DME2 being functional enhancers. Chromosome conformation capture analysis confirmed association of DME1 and DME2 with the DUX4 promoter in vivo. The strong interaction between DME2 and the DUX4 promoter in both FSHD and unaffected primary myocytes was greatly reduced in fibroblasts, suggesting a muscle-specific interaction. Nucleosome occupancy and methylome sequencing analysis indicated that in most FSHD myocytes, both enhancers are associated with nucleosomes but have hypomethylated DNA, consistent with a permissive transcriptional state, sporadic occupancy, and the observed DUX4 expression in rare myonuclei. Our data support a model in which these myogenic enhancers associate with the DUX4 promoter in skeletal myocytes and activate transcription when epigenetically derepressed in FSHD, resulting in the pathological misexpression of DUX4-fl.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Desenvolvimento Muscular/genética , Distrofia Muscular Facioescapuloumeral/genética , Processamento Alternativo/genética , Células Cultivadas , Metilação de DNA , Fibroblastos/metabolismo , Histonas/genética , Humanos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
To explore possible mechanisms of pathology in facioscapulohumeral muscular dystrophy (FSHD), we generated a novel library of myogenic cells composed of paired cultures derived from FSHD subjects and unaffected first-degree relatives. We prepared cells from biopsies of both biceps and deltoid muscles obtained from each of 10 FSHD and 9 unaffected donors. We used this new collection to determine how family background and disease affected patterns of growth and differentiation, expression of a panel of candidate, and muscle-specific genes, and responses to exogenous stressors. We found that FSHD and unaffected cells had, on average, indistinguishable patterns of differentiation, gene expression, and dose-response curves to staurosporine, paraquat, hydrogen peroxide, and glutathione depletion. Differentiated FSHD and unaffected cultures were both more sensitive to glutathione depletion than proliferating cultures, but showed similar responses to paraquat, staurosporine, and peroxide. For stress responses, the sample size was sufficient to detect a 10% change in effect at the observed variability with a power of >99%. In contrast, for each of these properties, we found significant differences among cells from different cohorts, and these differences were independent of disease status, gender, or muscle biopsied. Thus, though none of the properties we examined could be used to reliably distinguish between FSHD and unaffected cells, family of origin was an important contributor to gene-expression patterns and stressor responses in cultures of both FSHD and unaffected myogenic cells.
Assuntos
Distrofia Muscular Facioescapuloumeral/genética , Mioblastos/citologia , Adulto , Idoso , Diferenciação Celular , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Mioblastos/metabolismoRESUMO
BAG3 protein binds to and regulates Hsp70 chaperone activity. The BAG3 protein contains a WW domain and a proline-rich region with SH3-binding motifs, suggesting that it may interact with proteins relevant to signal transduction, recruiting Hsp70 to signaling complexes and altering cell responses. BAG3 overexpression has been observed in human cancers. We show here that homozygous BAG3-deficient mouse embryonic fibroblasts (MEF) exhibit delayed formation of filopodia and focal adhesion complexes when freshly plated. BAG3-deficient MEFs show reduced cell motility in culture. We observed that endogenous BAG3 protein is highly expressed in many human epithelial cancer cell lines, especially adenocarcinomas. Gene transfer-mediated overexpression of BAG3 increased motility of Cos7 cell and several human cancer cell lines, including breast cancer MCF7 and prostate cancer DU145 and ALVA31 cell lines. Conversely, reduction of BAG3 protein by RNA interference (RNAi) decreased cell motility in four of four epithelial tumor lines tested. We observed an influence of BAG3 on cell adhesion in culture. In Cos7 kidney epithelial cells, BAG3 protein partially colocalizes with actin at the leading edge of migrating cells, wherein active actin polymerization and nucleation occur. RNAi-mediated reductions in BAG3 expression were associated with decreased Rac1 activity, suggesting a role for BAG3 in regulating this small GTPase involved in actin-cytoskeleton dynamics. In mice, RNAi-mediated reductions in BAG3 in a human tumor xenograft suppressed invasion and metastasis in vivo. Thus, the high levels of BAG3 protein seen in some epithelial cancer cell lines may be relevant to mechanisms of tumor invasion and metastasis.