Objective
Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a frequent autoimmune neuropathy with a heterogeneous clinical spectrum. Clinical and experimental evidence suggests that ...autoantibodies may be involved in its pathogenesis, but the target antigens are unknown. Axoglial junction proteins have been proposed as candidate antigens. We examined the reactivity of CIDP patients' sera against neuronal antigens and used immunoprecipitation for antigen unraveling.
Methods
Primary cultures of hippocampal neurons were used to select patients' sera that showed robust reactivity with the cell surface of neurons. The identity of the antigens was established by immunoprecipitation and mass spectrometry, and subsequently confirmed with cell‐based assays, immunohistochemistry with teased rat sciatic nerve, and immunoabsorption experiments.
Results
Four of 46 sera from patients with CIDP reacted strongly against hippocampal neurons (8.6%) and paranodal structures on peripheral nerve. Two patients' sera precipitated contactin‐1 (CNTN1), and 1 precipitated both CNTN1 and contactin‐associated protein 1 (CASPR1). Reactivity against CNTN1 was confirmed in 2 cases, whereas the third reacted only when CNTN1 and CASPR1 were cotransfected. No other CIDP patient or any of the 104 controls with other neurological diseases tested positive. All 3 patients shared common clinical features, including advanced age, predominantly motor involvement, aggressive symptom onset, early axonal involvement, and poor response to intravenous immunoglobulin.
Interpretation
Antibodies against the CNTN1/CASPR1 complex occur in a subset of patients with CIDP who share common clinical features. The finding of this biomarker may help to explain the symptoms of these patients and the heterogeneous response to therapy in CIDP. ANN NEUROL 2013;73:370–380
Dermatomyositis (DM) is inflammatory myopathy or myositis characterized by muscle weakness and skin manifestations. In the differential diagnosis of DM the evaluation of the muscle biopsy is of ...importance among other parameters. Perifascicular atrophy in the muscle biopsy is considered a hallmark of DM. However, perifascicular atrophy is not observed in all patients with DM and, conversely, perifascicular atrophy can be observed in other myositis such as antisynthetase syndrome (ASS), complicating DM diagnosis. Retinoic acid inducible-gene I (RIG-I), a receptor of innate immunity that promotes type I interferon, was observed in perifascicular areas in DM. We compared the value of RIG-I expression with perifascicular atrophy as a biomarker of DM.
We studied by immunohistochemical analysis the expression of RIG-I and the presence of perifascicular atrophy in 115 coded muscle biopsies: 44 patients with DM, 18 with myositis with overlap, 8 with ASS, 27 with non-DM inflammatory myopathy (16 with polymyositis, 6 with inclusion body myositis, 5 with immune-mediated necrotizing myopathy), 8 with muscular dystrophy (4 with dysferlinopathy, 4 with fascioscapulohumeral muscle dystrophy) and 10 healthy controls.
We found RIG-I-positive fibers in 50% of DM samples vs 11% in non-DM samples (p < 0.001). Interestingly, RIG-I staining identified 32% of DM patients without perifascicular atrophy (p = 0.007). RIG-I sensitivity was higher than perifascicular atrophy (p < 0.001). No differences in specificity between perifascicular atrophy and RIG-I staining were found (92% vs 88%). RIG-I staining was more reproducible than perifascicular atrophy (κ coefficient 0.52 vs 0.37).
The perifascicular pattern of RIG-I expression supports the diagnosis of DM. Of importance for clinical and therapeutic studies, the inclusion of RIG-I in the routine pathological staining of samples in inflammatory myopathy will allow us to gather more homogeneous subgroups of patients in terms of immunopathogenesis.
The type I interferon (IFN-I) pathway is implicated in the pathophysiology of dermatomyositis, but whether its blockade would have therapeutic benefits is unknown. Ladislau et al. show that the Janus ...kinase (JAK) 1/2 inhibitor ruxolitinib inhibits IFN-I pathway activation and prevents IFN-I-mediated damage in skeletal muscle cells and dermatomyositis patients.
Abstract
Dermatomyositis is an acquired auto-immune disease characterized by skin lesions and muscle-specific pathological features such as perifascicular muscle fibre atrophy and vasculopathy. Dermatomyositis patients display an upregulation of type I interferon-inducible genes in muscle fibres, endothelial cells, skin and peripheral blood. However, the effect of type I interferon on muscle tissue has not yet been determined. Our aim was to study the pathogenicity of type I interferon in vitro and to evaluate the efficacy of the type I interferon pathway blockade for therapeutic purposes. The activation of type I interferon in differentiating myoblasts abolished myotube formation with reduced myogenin expression while in differentiated myotubes, we observed a reduction in surface area and an upregulation of atrophy-associated genes. In vitro endothelial cells exposure to type I interferon disrupted vascular network organization. All the pathogenic effects observed in vitro were abolished by ruxolitinib. Finally, four refractory dermatomyositis patients were treated with ruxolitinib and improvement ensued in skin lesions, muscle weakness and a reduced serum type I interferon levels and interferon-inducbile genes scores. We propose JAK inhibition as a mechanism-based treatment for dermatomyositis, a finding that is relevant for the design of future clinical trials targeting dermatomyositis.
Background
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late‐onset myopathy characterized by ptosis, dysphagia, and progressive proximal limb muscle weakness. The disease is ...produced by a short expansion of the (GCN)n triplet in the PABPN1 gene. The size of expansion has been correlated to the disease onset and severity. We report the clinical features of a large cohort of OPMD patients harboring the (GCN)15 allele from the Canary Islands.
Methods
A retrospective observational study was performed analyzing the clinical, demographic, and genetic data of 123 OPMD patients. Clinical data from this cohort were compared with clinical data collected in a large European study including 139 OPMD patients.
Results
A total of 113 patients (94.2%) carried the (GCN)15 expanded PABN1 allele. Age of symptoms’ onset was 45.1 years. The most frequent symptom at onset was ptosis (85.2%) followed by dysphagia (12%). The severity of the disease was milder in the Canary cohort compared to European patients as limb weakness (35.1% vs. 50.4%), the proportion of patients that require assistance for walking or use a wheelchair (9.3% vs. 27.4%), and needed of surgery because of severe dysphagia (4.6% vs. 22.8%) was higher in the European cohort.
Conclusions
Nearly 95% of patients with OPMD from the Canary Islands harbored the (GCN)15 expanded allele supporting a potential founder effect. Disease progression seemed to be milder in the (GCN)15 OPMD Canary cohort than in other cohorts with shorter expansions suggesting that other factors, apart from the expansion size, could be involved in the progression of the disease.
Nearly 95% of OPMD patients from the Canary Islands harbored the expanded allele (GCN)15 supporting a possible founder effect. Disease progression and severity appeared to be milder, suggesting that factors other than the size of the expansion, could be involved in the progression of the disease.
Duchenne muscular dystrophy (DMD) is characterized by a progressive loss of muscle fibers, and their substitution by fibrotic and adipose tissue. Many factors contribute to this process, but the ...molecular pathways related to regeneration and degeneration of muscle are not completely known. Platelet-derived growth factor (PDGF)-BB belongs to a family of growth factors that regulate proliferation, migration, and differentiation of mesenchymal cells. The role of PDGF-BB in muscle regeneration in humans has not been studied. We analyzed the expression of PDGF-BB in muscle biopsy samples from controls and patients with DMD. We performed in vitro experiments to understand the effects of PDGF-BB on myoblasts involved in the pathophysiology of muscular dystrophies and confirmed our results in vivo by treating the mdx murine model of DMD with repeated i.m. injections of PDGF-BB. We observed that regenerating and necrotic muscle fibers in muscle biopsy samples from DMD patients expressed PDGF-BB. In vitro , PDGF-BB attracted myoblasts and activated their proliferation. Analysis of muscles from the animals treated with PDGF-BB showed an increased population of satellite cells and an increase in the number of regenerative fibers, with a reduction in inflammatory infiltrates, compared with those in vehicle-treated mice. Based on our results, PDGF-BB may play a protective role in muscular dystrophies by enhancing muscle regeneration through activation of satellite cell proliferation and migration.
Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem ...cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss.
Background
The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro‐adipose tissue. Although the role of some growth factors in the process ...of fibrogenesis has been studied, pathways activated by PDGF‐AA have not been described so far. Our aim was to study the molecular role of PDGF‐AA in the fibrotic process of DMD.
Methods
Skeletal muscle fibro‐adipogenic progenitor cells (FAPs) from three DMD treated with PDGF‐AA at 50 ng/mL were analysed by quantitative mass spectrometry‐based proteomics. Western‐blot, immunofluorescence, and G‐LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF‐AA on the activation of RhoA pathway using two inhibitors, C3‐exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J‐mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue.
Results
Mass spectrometry revealed that RhoA pathway proteins were up‐regulated in treated compared with non‐treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7‐fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF‐AA (n = 3, 1.88‐fold increase, P < 0.01) and both C3‐exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF‐AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3‐exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non‐treated dba/2J‐mdx and n = 6 treated dba/2J‐mdx, 1.76‐fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen‐I expression area (n = 5 non‐treated dba/2J‐mdx and n = 6 treated dba/2J‐mdx, P < 0.01).
Conclusions
Our results suggest that PDGF‐AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.
Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb‐girdle ...muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O‐glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O‐glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle‐specific α‐dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch‐dependent loss of satellite cells.
Synopsis
A protein O‐glucosyltransferase 1 (POGLUT1) homozygous D233E mutation underlies a novel autosomal recessive muscular dystrophy, wherein altered Notch signaling affects muscle regeneration and, as a consequence, α‐dystroglycan glycosylation.
POGLUT1 D233E exhibits decreased enzymatic activity toward Notch EGF repeats.
POGLUT1 D233E leads to Notch activity downregulation, which affects muscle regeneration due to satellite cell (SC) loss of quiescence, depletion of PAX7+ cells, and premature and enhanced differentiation.
Reduced Notch signaling accelerates muscle differentiation and disrupts the progressive and coordinated process of α‐dystroglycan glycosylation during differentiation, and hence, mild α‐dystroglycan hypoglycosylation is observed in skeletal muscle from POGLUT1 D233E patients.
Defective regeneration, combined with α‐dystroglycan hypoglycosylation, likely results in skeletal muscle degeneration and finally gives rise to muscular dystrophy.
A protein O‐glucosyltransferase 1 (POGLUT1) homozygous D233E mutation underlies a novel autosomal recessive muscular dystrophy, wherein altered Notch signaling affects muscle regeneration and, as a consequence, α‐dystroglycan glycosylation.
Dermatomyositis is an inflammatory myopathy characterized by symmetrical proximal muscle weakness and skin changes. Muscle biopsy hallmarks include perifascicular atrophy, loss of intramuscular ...capillaries, perivascular and perimysial inflammation and the overexpression of IFN-inducible genes. Among them, the retinoic-acid inducible gene 1 (RIG-I) is specifically overexpressed in perifascicular areas of dermatomyositis muscle. The aim of this work was to study if RIG-I expression may be modulated by hypoxia using an in vitro approach. We identified putative hypoxia response elements (HRE) in RIG-I regulatory regions and luciferase assays confirmed that RIG-I is a new HIF-inducible gene. We observed an increase expression of RIG-I both by Real time PCR and Western blot in hypoxic conditions in human muscle cells. Cell transfection with a constitutive RIG-I expression vector increased levels of phospho-IRF-3, indicating that RIG-I promotes binding of transcription factors to the enhancer sequence of IFN. Moreover, release of IFN-β was observed in hypoxic conditions. Finally, HIF-1α overexpression was confirmed in the muscle biopsies and in some RIG-I positive perifascicular muscle fibres but not in controls. Our results indicate that hypoxia triggers the production of IFN-I in vitro, and may contribute to the pathogenesis of DM together with other inflammatory factors.
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