RESUMO
BACKGROUND: Myotonic dystrophy is a multisystem disorder characterized by widespread organic involvement including central nervous system symptoms. Although myotonic dystrophy disease types 1 (DM1) and 2 (DM2) cover a similar spectrum of symptoms, more pronounced clinical and brain alterations have been described in DM1. Here, we investigated brain volumetric and white matter alterations in both disease types and compared to healthy controls (HC). METHODS: MRI scans were obtained from 29 DM1, 27 DM2, and 56 HC. We assessed macro- and microstructural brain changes by surface-based analysis of cortical thickness of anatomical images and tract-based spatial statistics of fractional anisotropy (FA) obtained by diffusion-weighted imaging, respectively. Global MRI measures were related to clinical and neuropsychological scores to evaluate their clinical relevance. RESULTS: Cortical thickness was reduced in both patient groups compared to HC, showing similar patterns of regional distribution in DM1 and DM2 (occipital, temporal, frontal) but more pronounced cortical thinning for DM1. Similarly, FA values showed a widespread decrease in DM1 and DM2 compared to HC. Interestingly, FA was significantly lower in DM1 compared to DM2 within most parts of the brain. CONCLUSION: Comparisons between DM1 and DM2 indicate a more pronounced cortical thinning of grey matter and a widespread reduction in microstructural integrity of white matter in DM1. Future studies are required to unravel the underlying and separating mechanisms for the disease courses of the two types and their neuropsychological symptoms.
Assuntos
Distrofia Miotônica , Humanos , Distrofia Miotônica/patologia , Distrofia Miotônica/diagnóstico por imagem , Distrofia Miotônica/complicações , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Substância Branca/diagnóstico por imagem , Substância Branca/patologia , Afinamento Cortical Cerebral/diagnóstico por imagem , Afinamento Cortical Cerebral/patologia , Imageamento por Ressonância Magnética , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Adulto Jovem , Testes Neuropsicológicos , AnisotropiaRESUMO
We report on genetic and environmental modulation of social cognition abilities and brain volume correlates in two monozygotic twins (Twin1 and Twin2) with genetically confirmed myotonic dystrophy-type1 who grew up in different environmental settings. They both underwent neuropsychological assessment (i.e., Intelligent Quotient [IQ], theory of mind, emotion recognition tests), and MRI scanning to evaluate regional brain volumetrics compared to 10 gender and sex-matched healthy controls. Against a normal IQ level in both patients, Twin1 was more impaired in emotional processing and Twin2 in cognitive aspects of social cognition. Both patients showed grey matter (GM) atrophy in Brodmann Areas 23/31 (BA23/31) and BA7 bilaterally, while Twin2 showed additional GM loss in right BA46. Both patients showed a similar pattern of white matter atrophy involving the thalamus, basal ganglia, and uncinate fasciculus. White matter atrophy appeared to be mostly driven by genetics, while grey matter volumes appeared associated with different impairments in social cognition and possibly modulated by environment.
Assuntos
Encéfalo , Imageamento por Ressonância Magnética , Distrofia Miotônica , Testes Neuropsicológicos , Fenótipo , Gêmeos Monozigóticos , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Distrofia Miotônica/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Masculino , Feminino , Adulto , Atrofia/patologia , Substância Cinzenta/patologia , Substância Cinzenta/diagnóstico por imagem , Pessoa de Meia-Idade , Substância Branca/diagnóstico por imagem , Substância Branca/patologia , Cognição SocialRESUMO
Congenital myotonic dystrophy (CDM) is a genetic disease caused by an abnormally long CTG repeat expansion in the DMPK gene, which generally increases in size following intergenerational transmission. CDM is the rarest and most severe form of myotonic dystrophy type 1, yet an important number of patient-derived cells are needed to study this heterogeneous disease. Therefore, we have reprogrammed lymphoblastoid cells derived from a 3-year-old male with CDM into induced pluripotent stem cells (iPSCs; CBRCULi015-A) featuring 1800 CTG repeats and characterized their pluripotent state. This cell line constitutes an important resource to study CDM and potential treatments in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Miotônica , Humanos , Distrofia Miotônica/patologia , Distrofia Miotônica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pré-Escolar , Linhagem Celular , Diferenciação Celular , Miotonina Proteína Quinase/genéticaRESUMO
Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.
Assuntos
Doenças Mitocondriais , Distrofia Miotônica , Humanos , Masculino , Feminino , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Estudos Transversais , Proteômica , RNA , DNA Mitocondrial/genética , Doenças Mitocondriais/genéticaRESUMO
Dysregulated mRNA splicing is involved in the pathogenesis of many diseases including cancer, neurodegenerative diseases, and muscular dystrophies such as myotonic dystrophy type 1 (DM1). Comprehensive assessment of dysregulated splicing on the transcriptome and proteome level has been methodologically challenging, and thus investigations have often been targeting only few genes. Here, we performed a large-scale coordinated transcriptomic and proteomic analysis to characterize a DM1 mouse model (HSALR) in comparison to wild type. Our integrative proteogenomics approach comprised gene- and splicing-level assessments for mRNAs and proteins. It recapitulated many known instances of aberrant mRNA splicing in DM1 and identified new ones. It enabled the design and targeting of splicing-specific peptides and confirmed the translation of known instances of aberrantly spliced disease-related genes (e.g., Atp2a1, Bin1, Ryr1), complemented by novel findings (Flnc and Ywhae). Comparative analysis of large-scale mRNA and protein expression data showed quantitative agreement of differentially expressed genes and splicing patterns between disease and wild type. We hence propose this work as a suitable blueprint for a robust and scalable integrative proteogenomic strategy geared toward advancing our understanding of splicing-based disorders. With such a strategy, splicing-based biomarker candidates emerge as an attractive and accessible option, as they can be efficiently asserted on the mRNA and protein level in coordinated fashion.
Assuntos
Distrofia Miotônica , Proteogenômica , Camundongos , Animais , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Processamento Alternativo/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Myotonic dystrophy type 1 (DM1) patients are at risk for metabolic abnormalities and commonly experience overweight and obesity. Possibly, weight issues result from lowered resting energy expenditure (EE) and impaired muscle oxidative metabolism. OBJECTIVES: This study aims to assess EE, body composition, and muscle oxidative capacity in patients with DM1 compared to age-, sex- and BMI-matched controls. METHODS: A prospective case control study was conducted including 15 DM1 patients and 15 matched controls. Participants underwent state-of-the-art methodologies including 24âh whole room calorimetry, doubly labeled water and accelerometer analysis under 15-days of free-living conditions, muscle biopsy, full body magnetic resonance imaging (MRI), dual-energy x-ray absorptiometry (DEXA), computed tomography (CT) upper leg, and cardiopulmonary exercise testing. RESULTS: Fat ratio determined by full body MRI was significantly higher in DM1 patients (56 [49-62] %) compared to healthy controls (44 [37-52] % ; pâ=â0.027). Resting EE did not differ between groups (1948 [1742-2146] vs (2001 [1853-2425>] kcal/24âh, respectively; pâ=â0.466). In contrast, total EE was 23% lower in DM1 patients (2162 [1794-2494] vs 2814 [2424-3310] kcal/24âh; pâ=â0.027). Also, DM1 patients had 63% less steps (3090 [2263-5063] vs 8283 [6855-11485] steps/24âh; pâ=â0.003) and a significantly lower VO2 peak (22 [17-24] vs 33 [26-39] mL/min/kg; pâ=â0.003) compared to the healthy controls. Muscle biopsy citrate synthase activity did not differ between groups (15.4 [13.3-20.0] vs 20.1 [16.6-25.8] µM/g/min, respectively; pâ=â0.449). CONCLUSIONS: Resting EE does not differ between DM1 patients and healthy, matched controls when assessed under standardized circumstances. However, under free living conditions, total EE is substantially reduced in DM1 patients due to a lower physical activity level. The sedentary lifestyle of DM1 patients seems responsible for the undesirable changes in body composition and aerobic capacity.
Assuntos
Composição Corporal , Metabolismo Energético , Músculo Esquelético , Distrofia Miotônica , Estresse Oxidativo , Humanos , Músculo Esquelético/patologia , Distrofia Miotônica/patologia , Estudos de Casos e Controles , Estudos Prospectivos , Estudos Transversais , Masculino , Feminino , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou maisRESUMO
Reduced brain volume including atrophy in grey and white matter is commonly seen in myotonic dystrophy type 1 (DM1). DM1 is caused by an expansion of CTG trinucleotide repeats in the 3' untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Mutant DMPK mRNA containing expanded CUG RNA (DMPK-CUGexp) sequesters cytoplasmic MBNL1, resulting in morphological impairment. How DMPK-CUGexp and loss of MBNL1 cause histopathological phenotypes in the DM1 brain remains elusive. Here, we show that BDNF-TrkB retrograde transport is impaired in neurons expressing DMPK-CUGexp due to loss of cytoplasmic MBNL1 function. We reveal that mature BDNF protein levels are reduced in the brain of the DM1 mouse model EpA960/CaMKII-Cre. Exogenous BDNF treatment did not rescue impaired neurite outgrowth in neurons expressing DMPK-CUGexp, whereas overexpression of the cytoplasmic MBNL1 isoform in DMPK-CUGexp-expressing neurons improved their responsiveness to exogenous BDNF. We identify dynein light chain LC8-type 2, DYNLL2, as an MBNL1-interacting protein and demonstrate that their interaction is RNA-independent. Using time-lapse imaging, we show that overexpressed MBNL1 and DYNLL2 move along axonal processes together and that MBNL1-knockdown impairs the motility of mCherry-tagged DYNLL2, resulting in a reduced percentage of retrograde DYNLL2 movement. Examination of the distribution of DYNLL2 and activated phospho-TrkB (pTrkB) receptor in EpA960/CaMKII-Cre brains revealed an increase in the postsynaptic membrane fraction (LP1), indicating impaired retrograde transport. Finally, our neuropathological analysis of postmortem DM1 tissue reveals that reduced cytoplasmic MBNL1 expression is associated with an increase in DYNLL2 and activated pTrkB receptor levels in the synaptosomal fraction. Together, our results support that impaired MBNL1-mediated retrograde BDNF-TrkB signaling may contribute to the histopathological phenotypes of DM1.
Assuntos
Distrofia Miotônica , Animais , Camundongos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Expansão das Repetições de Trinucleotídeos , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , RNA/genética , Encéfalo/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
AIM: Myotonic dystrophy type 1 (DM1) is the second most common muscular dystrophy after Duchenne and is the most prevalent muscular dystrophy in adults. DM1 patients that participate in aerobic exercise training experience several physiological benefits concomitant with improved muscle mitochondrial function without alterations in typical DM1-specific disease mechanisms, which suggests that correcting organelle health is key to ameliorate the DM1 pathology. However, our understanding of the molecular mechanisms of mitochondrial turnover and dynamics in DM1 skeletal muscle is lacking. METHODS: Skeletal muscle tissue was sampled from healthy and DM1 mice under sedentary conditions and at several recovery time points following an exhaustive treadmill run. RESULTS: We demonstrate that DM1 patients exhibit an imbalance in the transcriptional apparatus for mitochondrial turnover and dynamics in skeletal muscle. Additionally, DM1 mice displayed elevated expression of autophagy and mitophagy regulators. A single dose of exercise successfully enhanced canonical exercise molecular pathways and skeletal muscle mitochondrial biogenesis despite failing to alter the cellular pathology in DM1 mice. However, treadmill running stimulated coordinated organelle fusion and fission signaling, as well as improved alternative splicing of Optic atrophy 1. Exercise also evoked autophagy and mitophagy pathways in DM1 skeletal muscle resulting in the normalized expression of autophagy- and lysosome-related machinery responsible for the clearance of dysfunctional organelles. CONCLUSION: Collectively, our data indicate that mitochondrial dynamics and turnover processes in DM1 skeletal muscle are initiated with a single dose of exercise, which may underlie the adaptive benefits previously documented in DM1 mice and patients.
Assuntos
Distrofias Musculares , Distrofia Miotônica , Camundongos , Animais , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disease caused by the expansion of a CTG repeat tract within the 3' untranslated region (3' UTR) of the dystrophia myotonica protein kinase gene (DMPK). Although DM1 is considered to be the most frequent myopathy of genetic origin in adults, DM1 patients exhibit a vast diversity of symptoms, affecting many different organs. Up until now, different in vitro models from patients' derived cells have largely contributed to the current understanding of DM1. Most of those studies have focused on muscle physiopathology. However, regarding the multisystemic aspect of DM1, there is still a crucial need for relevant cellular models to cover the whole complexity of the disease and open up options for new therapeutic approaches. This review discusses how human pluripotent stem cell-based models significantly contributed to DM1 mechanism decoding, and how they provided new therapeutic strategies that led to actual phase III clinical trials.
Assuntos
Distrofia Miotônica , Células-Tronco Pluripotentes , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Células-Tronco Pluripotentes/metabolismo , Descoberta de DrogasRESUMO
Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. Here, we use Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. We show that miR-1 down-regulation in the heart leads to dilated cardiomyopathy (DCM), a DM1-associated phenotype. We combined in silico screening for miR-1 targets with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. We identify Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.
Assuntos
Cardiomiopatia Dilatada , MicroRNAs , Distrofia Miotônica , Adulto , Animais , Humanos , Idoso , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Cardiomiopatia Dilatada/genética , Coração , MicroRNAs/genética , MicroRNAs/metabolismo , Drosophila/genética , Drosophila/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is an autosomal dominant hereditary disease caused by abnormal expansion of unstable CTG repeats in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. This disease mainly affects skeletal muscle, resulting in myotonia, progressive distal muscle weakness, and atrophy, but also affects other tissues and systems, such as the heart and central nervous system. Despite some studies reporting therapeutic strategies for DM1, many issues remain unsolved, such as the contribution of metabolic and mitochondrial dysfunctions to DM1 pathogenesis. Therefore, it is crucial to identify molecular target candidates associated with metabolic processes for DM1. In this study, resorting to a bibliometric analysis, articles combining DM1, and metabolic/metabolism terms were identified and further analyzed using an unbiased strategy of automatic text mining with VOSviewer software. A list of candidate molecular targets for DM1 associated with metabolic/metabolism was generated and compared with genes previously associated with DM1 in the DisGeNET database. Furthermore, g:Profiler was used to perform a functional enrichment analysis using the Gene Ontology (GO) and REAC databases. Enriched signaling pathways were identified using integrated bioinformatics enrichment analyses. The results revealed that only 15 of the genes identified in the bibliometric analysis were previously associated with DM1 in the DisGeNET database. Of note, we identified 71 genes not previously associated with DM1, which are of particular interest and should be further explored. The functional enrichment analysis of these genes revealed that regulation of cellular metabolic and metabolic processes were the most associated biological processes. Additionally, a number of signaling pathways were found to be enriched, e.g., signaling by receptor tyrosine kinases, signaling by NRTK1 (TRKA), TRKA activation by NGF, PI3K-AKT activation, prolonged ERK activation events, and axon guidance. Overall, several valuable target candidates related to metabolic processes for DM1 were identified, such as NGF, NTRK1, RhoA, ROCK1, ROCK2, DAG, ACTA, ID1, ID2 MYOD, and MYOG. Therefore, our study strengthens the hypothesis that metabolic dysfunctions contribute to DM1 pathogenesis, and the exploitation of metabolic dysfunction targets is crucial for the development of future therapeutic interventions for DM1.
Assuntos
Distrofia Miotônica , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Miotonina Proteína Quinase/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is a severe autosomal dominant neuromuscular disease in which the musculoskeletal system contributes substantially to overall mortality and morbidity. DM1 stems from a noncoding CTG trinucleotide repeat expansion in the DMPK gene. The human skeletal actin long repeat (HSALR) mouse model reproduces several aspects of the disease, but the muscle-wasting phenotype of this model has never been characterized in vivo. Herein, we used quantitative MRI to measure the fat and muscle volumes in the leg compartment (LC) of mice. These acquired data were processed to extract relevant parameters such as fat fraction and fat infiltration (fat LC/LC) in HSALR and control (FBV) muscles. These results showed increased fat volume (fat LC) and fat infiltration within the muscle tissue of the leg compartment (muscle LC), in agreement with necropsies, in which fatty clumps were observed, and consistent with previous findings in DM1 patients. Model mice did not reproduce the characteristic impaired fat fraction, widespread fat replacement through the muscles, or reduced muscle volume reported in patients. Taken together, the observed abnormal replacement of skeletal muscle by fat in the HSALR mice indicates that these mice partially reproduced the muscle phenotype observed in humans.
Assuntos
Distrofia Miotônica , Humanos , Camundongos , Animais , Distrofia Miotônica/diagnóstico por imagem , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Expansão das Repetições de Trinucleotídeos , Fenótipo , Imageamento por Ressonância MagnéticaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Miotônica , Humanos , Processamento Alternativo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotônica/patologia , Splicing de RNA , Modelos BiológicosRESUMO
AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Miotônica , Adulto , Humanos , Distrofia Miotônica/patologia , Proteínas de Ligação a RNA/metabolismo , Junção Neuromuscular/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/patologiaRESUMO
Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by expansion of CTG microsatellite repeats within DMPK. The most severe form, congenital myotonic dystrophy (CDM), has symptom onset at birth due to large intergenerational repeat expansions. Despite a common mutation, CDM individuals present with a distinct clinical phenotype and absence of common DM1 symptoms. Given the clinical divergence, it is unknown if the hallmark of DM1 pathology, dysregulation of alternative splicing (AS) due to sequestration of MBNL proteins within toxic CUG repeat RNAs, contributes to disease throughout pediatric development. To evaluate global transcriptomic dysregulation, RNA-seq was performed on 36 CDM skeletal muscle biopsies ages 2 weeks to 16 years, including two longitudinal samples. Fifty DM1 and adult/pediatric controls were also sequenced as comparative groups. Despite a large CTG expansion and shared age of onset, CDM individuals presented with a heterogenous, MBNL-dependent mis-splicing signature. Estimation of intracellular MBNL concentrations from splicing responses of select events correlated with total spliceopathy and revealed a distinct, triphasic pattern of AS dysregulation across pediatric development. CDM infants (< 2 years) possess severe mis-splicing that significantly improves in early childhood (2-8 years) independent of sex or CTG repeat load. Adolescent individuals (8-16 years) stratified into two populations with a full range of global splicing dysregulation. DMPK expression changes correlated with alterations in splicing severity during development. This study reveals the complex dynamics of the CDM muscle transcriptome and provides insights into new therapeutic strategies, timing of therapeutic intervention, and biomarker development.
Assuntos
Distrofia Miotônica , Pré-Escolar , Humanos , Distrofia Miotônica/patologia , Transcriptoma/genética , Miotonina Proteína Quinase/genética , Miotonina Proteína Quinase/metabolismo , Músculo Esquelético/metabolismo , Splicing de RNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is an inherited autosomal-dominant condition that induces altered splicing of transcripts, including MAPT, leading to a distinctive abnormal deposition of tau protein in the CNS. We characterized the tau isoforms of abnormal depositions in the brains of 4 patients with classic DM1 by immunohistochemistry using isoform-specific antibodies. All patients, including those of presenile age, showed numerous neurofibrillary tangles (NFTs) of both 3-repeat and 4-repeat tau in the limbic area and mild involvement in the cerebral cortex. Amyloid-ß deposition was only seen in 1 senile case while cortical tauopathy in all other cases was consistent with primary age-related tauopathy (PART). In the putamen and globus pallidus, only a few tau deposits were observed. Tau deposits in the brainstem frequently showed a DM1-specific pattern with 3-repeat tau dominant NFTs. Additionally, tau-positive astrocytes morphologically similar to tufted astrocytes and astrocytic plaques were occasionally observed in the brainstem; however, they were predominantly composed of 3-repeat tau. Thus, the classic DM1 showed both early onset of PART-like pathology in the limbic areas as a progeroid syndrome of DM1 and an abnormal splicing event in the brainstem leading to 3-repeat tau dominant accumulation with both neuronal and astrocytic involvement.
Assuntos
Tronco Encefálico , Hipocampo , Distrofia Miotônica , Tauopatias , Proteínas tau , Humanos , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Emaranhados Neurofibrilares/patologia , Isoformas de Proteínas/metabolismo , Proteínas tau/metabolismo , Tauopatias/patologiaRESUMO
Muscular dystrophies are a group of genetic muscular diseases characterized by impaired muscle regeneration, which leads to pathological inflammation that drives muscle wasting and eventually results in weakness, functional dependency, and premature death. The most known causes of death include respiratory muscle failure due to diaphragm muscle decay. There is no definitive treatment for muscular dystrophies, and conventional therapies aim to ameliorate muscle wasting by promoting physiological muscle regeneration and growth. However, their effects on muscle function remain limited, illustrating the requirement for major advancements in novel approaches to treatments, such as nanomedicine. Nanomedicine is a rapidly evolving field that seeks to optimize drug delivery to target tissues by merging pharmaceutical and biomedical sciences. However, the therapeutic potential of nanomedicine in muscular dystrophies is poorly understood. This review highlights recent work in the application of nanomedicine in treating muscular dystrophies. First, we discuss the history and applications of nanomedicine from a broader perspective. Second, we address the use of nanoparticles for drug delivery, gene regulation, and editing to target Duchenne muscular dystrophy and myotonic dystrophy. Next, we highlight the potential hindrances and limitations of using nanomedicine in the context of cell culture and animal models. Finally, the future perspectives for using nanomedicine in clinics are summarized with relevance to muscular dystrophies.
Assuntos
Distrofia Muscular de Duchenne , Distrofia Miotônica , Animais , Músculo Esquelético/patologia , Músculos/patologia , Atrofia Muscular/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Miotônica/patologia , Nanomedicina , Preparações FarmacêuticasRESUMO
The myotonic dystrophies (DM1 and DM2) are dominantly inherited disorders that cause pathological changes throughout the body. Many individuals with DM experience cognitive, behavioral and other functional central nervous system effects that impact their quality of life. The extent of psychological impairment that will develop in each patient is variable and unpredictable. Hence, it is difficult to get strong supervision information like fully ground truth labels for all cognitive involvement patterns. This study is to assess cognitive involvement among healthy controls and patients with DM. The DM cognitive impairment pattern observation is modeled in a weakly supervised setting and supervision information is used to transform the input feature space to a more discriminative representation suitable for pattern observation. This study incorporated results from 59 adults with DM and 92 control subjects. The developed system categorized the neuropsychological testing data into five cognitive clusters. The quality of the obtained clustering solution was assessed using an internal validity metric. The experimental results show that the proposed algorithm can discover interesting patterns and useful information from neuropsychological data, which will be be crucial in planning clinical trials and monitoring clinical performance. The proposed system resulted in an average classification accuracy of 88%, which is very promising considering the unique challenges present in this population.
Assuntos
Disfunção Cognitiva , Distrofia Miotônica , Adulto , Análise por Conglomerados , Disfunção Cognitiva/diagnóstico , Humanos , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/patologia , Testes Neuropsicológicos , Qualidade de VidaRESUMO
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder that affects many organs. It is caused by the expansion of a cytosine-thymine-guanine triplet repeat in the 3' untranslated region of the human dystrophia myotonica protein kinase (hDMPK) gene, which results in a toxic gain of function of mutant hDMPK RNA transcripts. Antisense oligonucleotides (ASOs) have emerged in recent years as a potential gene therapy to treat DM1. However, the clinical efficacy of the systemic administration of ASOs is limited by a combination of insufficient potency and poor tissue distribution. In the present study, we assessed the potential of a new ligand-conjugated ASO (IONIS-877864; C16-HA-ASO) to target mutant hDMPK mRNA transcripts in the DMSXL mouse model of DM1 carrying over 1000 CTG pathogenic repeats. DMSXL mice were treated subcutaneously for 9 weeks with either IONIS-877864 (12.5 or 25 mg/kg) or IONIS-486178 (12.5 or 25 mg/kg), an unconjugated ASO with the same sequence. At 25 mg/kg, IONIS-877864 significantly enhanced ASO delivery into the striated muscles of DMSXL mice following systemic administration compared with the unconjugated control. IONIS-877864 was also more efficacious than IONIS-486178, reducing mutant hDMPK transcripts by up to 92% in the skeletal muscles and 78% in the hearts of DMSXL mice. The decrease in mutant hDMPK transcripts in skeletal muscles caused by IONIS-877864 was associated with a significant improvement in muscle strength. IONIS-877864 was nontoxic in the DMSXL mouse model. The present study showed that the C16-HA-conjugated ASO is a powerful tool for the development of gene therapy for DM1.
Assuntos
Distrofia Miotônica , Animais , Modelos Animais de Doenças , Humanos , Ligantes , Camundongos , Músculo Esquelético/metabolismo , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , RNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Increasing loss of structure and function of neurons and decline in cognitive function is commonly seen during the progression of neurologic diseases, although the causes and initial symptoms of individual diseases are distinct. This observation suggests a convergence of common degenerative features. In myotonic dystrophy type 1 (DM1), the expression of expanded CUG RNA induces neurotransmission dysfunction before axon and dendrite degeneration and reduced MBNL2 expression associated with aberrant alternative splicing. The role of loss of function of MBNL2 in the pathogenesis of neurodegeneration and the causal mechanism of neurodegeneration-reduced expression of MBNL2 remain elusive. Here, we show that increased MBNL2 expression is associated with neuronal maturation and required for neuronal morphogenesis and the fetal to adult developmental transition of RNA processing. Neurodegenerative conditions including NMDA receptor (NMDAR)-mediated excitotoxicity and dysregulated calcium homeostasis triggered nuclear translocation of calpain-2, thus resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to developmental patterns. Nuclear expression of calpain-2 resembled its developmental pattern and was associated with MBNL2 degradation. Knock-down of calpain-2 expression or inhibition of calpain-2 nuclear translocation prevented neurodegeneration-reduced MBNL2 expression and dysregulated RNA processing. Increased calpain-2 nuclear translocation associated with reduced MBNL2 expression and aberrant RNA processing occurred in models for DM1 and Alzheimer's disease (AD) including EpA960/CaMKII-Cre mice of either sex and female APP/PS1 and THY-Tau22 mice. Our results identify a regulatory mechanism for MBNL2 downregulation and suggest that calpain-2-mediated MBNL2 degradation accompanied by re-induction of a developmental RNA processing program may be a converging pathway to neurodegeneration.SIGNIFICANCE STATEMENT Neurologic diseases share many features during disease progression, such as cognitive decline and brain atrophy, which suggests a common pathway for developing degenerative features. Here, we show that the neurodegenerative conditions glutamate-induced excitotoxicity and dysregulated calcium homeostasis induced translocation of the cysteine protease calpain-2 into the nucleus, resulting in MBNL2 degradation and reversal of MBNL2-regulated RNA processing to an embryonic pattern. Knock-down or inhibition of nuclear translocation of calpain-2 prevented MBNL2 degradation and maintained MBNL2-regulated RNA processing in the adult pattern. Models of myotonic dystrophy and Alzheimer's disease (AD) also showed calpain-2-mediated MBNL2 degradation and a developmental RNA processing program. Our studies suggest MBNL2 function disrupted by calpain-2 as a common pathway, thus providing an alternative therapeutic strategy for neurodegeneration.