Ricerca di nuovi target terapeutici nella distrofia muscolare da deficit di distrofina e alfa-sarcoglicano

Con il termine di distrofie muscolari si indica un gruppo di gravi malattie neuromuscolari, ad esordio prevalentemente nell\u2019et\ue0 pediatrica, geneticamente determinate a carattere degenerativo che causano progressiva atrofia della muscolatura scheletrica. Il numero dei soggetti affetti da questi disordini costituisce circa il 10% di tutti i malati neurologici. Tali patologie, tra le quali la Distrofia Muscolare di Duchenne (DMD) rappresenta la forma pi\uf9 frequente, sono dovute a mutazioni in geni che codificano proteine associate alla membrana plasmatica della cellula muscolare e la cui perdita comporta una membrana muscolare pi\uf9 fragile e suscettibile al danno indotto dalla contrazione. Nonostante il grande sforzo per identificare una terapia per queste patologie, una cura specifica non \ue8 al momento disponibile. Per tale motivo, nuovi approcci terapeutici connessi con la complessa patologia dei difetti di membrana, sono oggetto di ricerca. Razionale: Nelle distrofie muscolari da difetto delle proteine di membrana, l\u2019instabilit\ue0 del sarcolemma si traduce in una serie di processi secondari degenerativi quali l\u2019infiltrazione di cellule infiammatorie e la ridotta rigenerazione. In questo scenario, l\u2019ATP extracellulare (eATP) rilasciato dalle miofibre in necrosi induce un\u2019attivazione protratta dei recettori P2X(1-7) determinando un effetto diretto legato ad alterazione dell\u2019omeostasi del calcio intracellulare e un effetto indiretto tramite il \u2018triggering\u2019 della fase iniziale della risposta immunitaria e l\u2019inibizione delle cellule T regolatorie (Treg) ad azione immunosoppressoria. Lo scopo del lavoro \ue8 quindi quello di valutare gli effetti dell\u2019inibizione dei recettori purinergici in modelli murini di distrofie da difetto di proteine di membrana

Clinical and molecular consequences of exon 78 deletion in DMD gene

We present a 13-year-old patient with persistent increase of serum Creatine Kinase (CK) and myalgia after exertion. Skeletal muscle biopsy showed marked reduction of dystrophin expression leading to genetic analysis of DMD gene by MLPA, which detected a single deletion of exon 78. To the best of our knowledge, DMD exon 78 deletion has never been described in literature and, according to prediction, it should lead to loss of reading frame in the dystrophin gene. To further assess the actual effect of exon 78 deletion, we analysed cDNA from muscle mRNA. This analysis confirmed the absence of 32 bp of exon 78. Exclusion of exon 78 changes the open reading frame of exon 79 and generate a downstream stop codon, producing a dystrophin protein of 3703 amino acids instead of 3685 amino acids. Albeit loss of reading frame usually leads to protein degradation and severe phenotype, in this case, we demonstrated that deletion of DMD exon 78 can be associated with a functional protein able to bind DGC complex and a very mild phenotype. This study adds a novel deletion in DMD gene in human and helps to define the compliance between maintaining/disrupting the reading frame and clinical form of the disease

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, beta-dystroglycan, and alpha-sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663-1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in human

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy

Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Pias\u3b3) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients