RESUMO
Mutations in the Matrin 3 (MATR3) gene have been identified as a cause of amyotrophic lateral sclerosis (ALS) or vocal cord and pharyngeal weakness with distal myopathy (VCPDM). This study investigated the mechanism by which mutant MATR3 causes multisystem proteinopathy (MSP) including ALS and VCPDM. We first analyzed the muscle pathology of C57BL/6 mice injected with adeno-associated viruses expressing human WT or mutant (S85C) MATR3. We next generated transgenic mice that overexpress mutant (S85C) MATR3, driven by the CMV early enhancer/chicken ß-actin promoter, and evaluated their clinicopathological features. Intramuscular injection of viruses expressing WT and mutant MATR3 induced similar myogenic changes, including smaller myofibers with internal nuclei, and upregulated p62 and LC3-II. Mutant MATR3 transgenic mice showed decreased body weight and lower motor activity. Muscle histology demonstrated myopathic changes including fiber-size variation, internal nuclei and rimmed vacuoles. Spinal cord histology showed a reduced number of motor neurons, and activation of microglia and astrocytes. Comprehensive proteomic analyses of muscle demonstrated upregulation of proteins related to chaperones, stress response, protein degradation, and nuclear function. Overexpression of WT and mutant MATR3 similarly caused myotoxicity, recapitulating the clinicopathological features of MSP. These models will be helpful for analyzing MSP pathogenesis and for understanding the function of MATR3. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Esclerose Lateral Amiotrófica/genética , Miopatias Distais/genética , Doenças da Laringe/genética , Músculo Esquelético/metabolismo , Mutação , Proteínas Associadas à Matriz Nuclear/genética , Doenças Faríngeas/genética , Proteínas de Ligação a RNA/genética , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Miopatias Distais/metabolismo , Miopatias Distais/patologia , Miopatias Distais/fisiopatologia , Análise da Marcha , Técnicas de Transferência de Genes , Predisposição Genética para Doença , Humanos , Doenças da Laringe/metabolismo , Doenças da Laringe/patologia , Doenças da Laringe/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Doenças Faríngeas/metabolismo , Doenças Faríngeas/patologia , Doenças Faríngeas/fisiopatologia , Proteínas de Ligação a RNA/metabolismo , Teste de Desempenho do Rota-Rod , Proteína Sequestossoma-1/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Redução de PesoRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by ptosis, dysphagia, and weakness of proximal limbs. OPMD is caused by the expansion of polyalanine in poly(A)-binding protein, nuclear 1 (PABPN1). Although mitochondrial abnormality has been proposed as the possible etiology, the molecular pathogenesis is still poorly understood. The aim of the study was to specify the mechanism by which expanded PABPN1 causes mitochondrial dysfunction in OPMD. We evaluated whether transgenic mouse model of OPMD, by expressing expanded PABPN1, indeed causes mitochondrial abnormality associated with muscle degeneration. We also investigated the mechanism by which expanded PABPN1 would cause mitochondrial dysfunction in the mouse and cell models of OPMD. Mitochondrial localization of PABPN1 was observed in the muscle fibers of patients with OPMD. Moreover, abnormal accumulation of PABPN1 on the inner membrane of mitochondria and reduced expression of OXPHOS complexes were detected in the muscle fibers of the transgenic mice expressing expanded human PABPN1 with a 13-alanine stretch. In cells expressing PABPN1 with a 10-alanine or 18-alanine stretch, both types of PABPN1 accumulated in the mitochondria and interacted with TIM23 mitochondrial protein import complex, but PABPN1 with 18-alanine stretch decreased the cell viability and aggresome formation. We proposed that the abnormal accumulation of expanded PABPN1 in mitochondria may be associated with mitochondrial abnormality in OPMD.
Assuntos
Mitocôndrias Musculares/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Expansão das Repetições de Trinucleotídeos , Animais , Estudos de Casos e Controles , Sobrevivência Celular , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias Musculares/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Oculofaríngea/patologia , Proteínas Mutantes/química , Fosforilação Oxidativa , Proteína I de Ligação a Poli(A)/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
OBJECTIVE: Sporadic inclusion body myositis (sIBM), an intractable progressive muscle disease, frequently occurs in older persons. sIBM pathogenesis may involve protein degradation dysfunction and immune abnormalities. Autoantibodies recognizing cytosolic 5'-nucleotidase 1A (cN1A) were found in plasma and serum from sIBM patients. However, whether anti-cN1A autoantibodies play a pathogenic role in sIBM is controversial. This study investigated the pathogenic properties of anti-cN1A autoantibodies in sIBM pathogenesis. METHODS: We developed a cell-based assay to detect anti-cN1A autoantibodies, which we found in serum from patients with neuromuscular diseases including sIBM. We also investigated the clinicopathological differences between sIBM patients with and without the autoantibodies. We used passive in vitro and in vivo immunization models to evaluate the pathogenic role of the autoantibodies. RESULTS: Of 67 patients with sIBM, 24 (35.8%) possessed anti-cN1A autoantibodies as determined via our cell-based assay. In the anti-cN1A-positive group, the percentage of patients with hepatitis C virus antibodies was significantly lower and the mean area of type 2 myofibers was significantly smaller compared with the autoantibody-negative group. In the in vitro passive immunization model, p62/SQSTM1 significantly increased in anti-cN1A-positive sIBM immunoglobulin G (IgG)-supplemented cells. In the in vivo passive immunization model, anti-cN1A-positive sIBM IgG-injected mice demonstrated p62/SQSTM1-positive sarcoplasmic aggregates in myofibers, associated with macrophage infiltration. INTERPRETATION: Our cell-based assay is useful for anti-cN1A autoantibodies detection. Patients with anti-cN1A autoantibodies demonstrated unique clinicopathological features. In vitro and in vivo passive immunization model results suggest that anti-cN1A autoantibodies may affect protein degradation in myofibers. Ann Neurol 2017;81:512-525.
Assuntos
5'-Nucleotidase/imunologia , Autoanticorpos/sangue , Bioensaio/métodos , Imunização Passiva , Miosite de Corpos de Inclusão , Adulto , Idoso , Animais , Linhagem Celular , Citosol/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunização Passiva/estatística & dados numéricos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/imunologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Miosite de Corpos de Inclusão/fisiopatologiaRESUMO
Sporadic inclusion body myositis (sIBM) is the most commonly acquired myopathy in middle-aged and elderly people. The muscle histology is characterized by both inflammation and degeneration, including sarcoplasmic aggregation of TDP-43. Cylindromatosis (CYLD) is a deubiquitinating enzyme that targets Lys63-linked ubiquitin chains and negatively regulates signal transduction pathways, such as NF-κB signalling pathways. We examined localization of CYLD as well as phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in muscle tissues of sIBM patients and muscle-specific wild-type TDP-43 transgenic (TDP-43 TG) mice. We investigated whether overexpression of CYLD can affect muscle toxicity in the cell models treated by endoplasmic reticulum (ER) stress inducers tunicamycin and thapsigargin. CYLD expressed with phosphorylated TDP-43, phosphorylated p62, and Lys63-linked ubiquitin in the nuclear and perinuclear regions of muscle fibres of wild-type TDP-43 TG mice and the degenerative myofibres of sIBM patients with rimmed vacuoles and endomysial cellular infiltration. Although expression levels of CYLD decreased and cell viability was reduced in cells treated with ER stress inducers, wild-type CYLD, but not the catalytic mutant, substantially improved cell viability based on the deubiquitinase activity. Dysregulation of CYLD may reinforce myodegeneration in the pathophysiology of sIBM by attenuating autophagic clearance of protein aggregates. Regulating CYLD in muscle fibres might serve as a novel therapeutic strategy for sIBM treatment.
Assuntos
Enzima Desubiquitinante CYLD/fisiologia , Miosite de Corpos de Inclusão/fisiopatologia , Idoso , Animais , Enzima Desubiquitinante CYLD/genética , Estresse do Retículo Endoplasmático , Humanos , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/metabolismo , Fosforilação , Proteômica , Ubiquitina/metabolismo , Vacúolos/metabolismoRESUMO
Muscle histology of sporadic inclusion body myositis (sIBM) demonstrates inflammatory findings and degenerative features including accumulation of TAR DNA-binding protein of 43â¯kDa (TDP-43). However, whether sarcoplasmic accumulation of TDP-43 is a primary trigger of muscle degeneration or a secondary event resulting from muscle degeneration in the pathophysiology of sIBM remained unclear. Our study aimed to discover whether muscle-dominant expression of TDP-43 is a primary cause of muscle degeneration. We generated several lines of wild-type TDP-43 transgenic mice driven by a creatine kinase 8 promoter, and analyzed the phenotypes via biochemical, histological, and proteomic techniques. The mice showed increased serum levels of myogenic enzymes. Muscle histology demonstrated myopathic changes including fiber size variation, abundant tubular aggregates, and TDP-43 aggregation with upregulation of endoplasmic reticulum (ER) stress. Proteomic analysis with aggregated materials in degenerative myofibers identified increased sarcoplasmic reticulum (SR)/ER-resident proteins that regulated calcium homeostasis, as well as cytosolic 5'-nucleotidase 1A. Muscle-dominant wild-type TDP-43 expression indeed caused myotoxicity featuring tubular aggregates and TDP-43-positive inclusions. Our observation suggested that TDP-43 aggregates might not be sufficient to trigger the pathogenesis of sIBM although myofiber sarcoplasmic aggregation of TDP-43 led to myofiber degeneration via ER stress and possibly calcium dysregulation, independently of inflammatory process.