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Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

ABSTRACT: Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-? and TNF-?, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.

SUBMITTER: Farini A

PROVIDER: S-EPMC5154478 | biostudies-other | 2016 Nov

REPOSITORIES: biostudies-other

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Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

Farini Andrea A Sitzia Clementina C Cassani Barbara B Cassinelli Letizia L Rigoni Rosita R Colleoni Federica F Fusco Nicola N Gatti Stefano S Bella Pamela P Villa Chiara C Napolitano Filomena F Maiavacca Rita R Bosari Silvano S Villa Anna A Torrente Yvan Y

Molecular therapy : the journal of the American Society of Gene Therapy 20160810 11

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a f ...[more]

PMID: 27506451

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Project description:BackgroundDuchenne muscular dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofibre actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca2+ homeostasis, activation of proteases, mitochondrial damage, and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation are yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD.MethodsWe sequenced plasma miRNA in a DMD cohort, comprising 54 DMD patients treated or not by glucocorticoid, compared with 27 healthy controls, in three groups of the ages of 4-8, 8-12, and 12-20 years. We developed an original approach for the biological interpretation of miRNA dysregulation and produced a novel hypothesis concerning metabolic perturbation in DMD. We used the mdx mouse model for DMD for the investigation of this hypothesis.ResultsWe identified 96 dysregulated miRNAs (adjusted P-value <0.1), of which 74 were up-regulated and 22 were down-regulated in DMD. We confirmed the dysregulation in DMD of Dystro-miRs, Cardio-miRs, and a large number of the DLK1-DIO3 miRNAs. We also identified numerous dysregulated miRNAs yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP-1 and SREBP-2), perturbation of the mevalonate pathway, and the accumulation of cholesterol in the dystrophic muscles. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol-reducing agent, normalized these perturbations and partially restored the dystrophic parameters.ConclusionsThis investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.

Project description:BackgroundAdiponectin (ApN) is a hormone known to exhibit insulin-sensitizing, fat-burning, and anti-inflammatory properties in several tissues, including the skeletal muscle. Duchenne muscular dystrophy (DMD) is a devastating disease characterized by dystrophin deficiency with subsequent chronic inflammation, myofiber necrosis, and impaired regeneration. Previously, we showed that transgenic up-regulation of ApN could significantly attenuate the dystrophic phenotype in mdx mice (model of DMD). Recently, an orally active ApN receptor agonist, AdipoRon, has been identified. This synthetic small molecule has the advantage of being more easily produced and administrable than ApN. The aim of this study was to investigate the potential effects of AdipoRon on the dystrophic muscle.MethodsFour-week-old mdx mice (n = 6-9 per group) were orally treated with AdipoRon (mdx-AR) for 8 weeks and compared with untreated (mdx) mice and to control (wild-type) mice. In vivo functional tests were carried out to measure the global force and endurance of mice. Ex vivo biochemical and molecular analyses were performed to evaluate the pathophysiology of the skeletal muscle. Finally, in vitro tests were conducted on primary cultures of healthy and DMD human myotubes.ResultsAdipoRon treatment mitigated oxidative stress (-30% to 45% for 4-hydroxy-2-nonenal and peroxiredoxin 3, P < 0.0001) as well as inflammation in muscles of mdx mice (-35% to 65% for interleukin 1 beta, tumour necrosis factor alpha, and cluster of differentiation 68, a macrophage maker, P < 0.0001) while increasing the anti-inflammatory cytokine, interleukin 10 (~5-fold, P < 0.0001). AdipoRon also improved the myogenic programme as assessed by a ~2-fold rise in markers of muscle proliferation and differentiation (P < 0.01 or less vs. untreated mdx). Plasma lactate dehydrogenase and creatine kinase were reduced by 30-40% in mdx-AR mice, reflecting less sarcolemmal damage (P < 0.0001). When compared with untreated mdx mice, mdx-AR mice exhibited enhanced physical performance with an increase in both muscle force and endurance and a striking restoration of the running capacity during eccentric exercise. AdipoRon mainly acted through ApN receptor 1 by increasing AMP-activated protein kinase signalling, which led to repression of nuclear factor-kappa B, up-regulation of utrophin (a dystrophin analogue), and a switch towards an oxidative and more resistant fibre phenotype. The effects of AdipoRon were then recapitulated in human DMD myotubes.ConclusionsThese results demonstrate that AdipoRon exerts several beneficial effects on the dystrophic muscle. This molecule could offer promising therapeutic prospect for managing DMD or other muscle and inflammatory disorders.

Project description:Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA's metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (∼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.

Dataset Information

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

Publications

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

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