Rolle von Extrazellulären ATP in der Progression der Muskeldegeneration in Sarkoglykanopathien

Limb Girdle Muscular Dystrophy 2D (LGMD2D) is an inherited disorder characterized by progressive weakness and degeneration of skeletal muscle, loss of ambulation, respiratory insufficiency and, often, premature death. The disease results from mutations in the alpha-sarcoglycan (SG) gene, encoding a muscle membrane associated protein. A-SG loss-of-function causes a cell membrane fragility, which ultimately results in a tissue-specific increase of danger-associated molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular adenosine triphosphate (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the “triggering” of the immune response and inhibition of regulatory T cells (Tregs). Accordingly, pharmacological and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy (LGMD2D), eATP effects may be further amplified since αSG extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Here we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad spectrum P2XR–antagonist delayed the progression of the dystrophic phenotype in -SG null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of Forkhead-Box-Protein P3 (Foxp3)+ immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis and limited expression of pro-fibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in the muscle infiltrates, might provide a therapeutic approach to slow disease progression in LGMD2D. In this scenario, the consequences of purinoreceptor inhibition on the stability and function of Tregs cells are particularly intriguing given the potential clinical value of targeting this T cell subset in muscle diseases. Transition to cure of our approaches is feasible: Tregs immunosuppressive properties have prompted clinical trials in models of autoimmunity and human graft versus host disease; clinical trials with P2X7 antagonists have been completed for other diseases, showing acceptable safety and tolerability.Die Muskeldystrophie der Gliedmaßengürtel (LGMD2D) ist eine vererbte Erkrankung, die durch fortschreitende Muskelschwäche und Degeneration der Skelettmuskulatur, Verlust der Gehfähigkeit, Ateminsuffizienz und häufig vorzeitigen Tod gekennzeichnet ist. Die Krankheit resultiert aus Mutationen im alpha-Sarkoglycan (αSG) Gen, der für ein muskelmembranassoziiertes Protein kodieret. A-SG „Loss-of-Function“ verursacht die Schwäche der Muskelmembran, die zu einer gewebespezifischen Erhöhung der danger associated molecules (DAMPs) und zur Infiltration von Entzündungszellen führt. Das DAMP extrazelluläre Adenosintriphosphat (eATP), das durch das Absterben von Myofasern freigesetzt wird, aktiviert die purinergischen Rezeptoren der Muskulatur und des Immunsystems. eATP stellt einen doppelt negativen Effekt im Muskelgewebe: eine direkte Schädigung, mit einer veränderten intrazellulären Kalziumhomöostase in den Muskelzellen, und eine indirekte Toxizität durch das "Auslösen" der Immunantwort mitsamt der Hemmung der regulatorischen T-Zellen (Tregs). Dementsprechend verbessert die pharmakologische und genetische Hemmung der eATP-Signalgebung den klinischen Phänotyp in Modellen chronisch entzündlicher Erkrankungen. Bei der α-Sarkoglykanopathie, LGMD2D, können die eATP-Effekte weiter verstärkt werden, da die extrazelluläre Domäne der αSG eATP bindet und eine Ecto-ATPase-Aktivität zeigt, wodurch die eATP-Konzentration an der Zelloberfläche und die Stärke und / oder Dauer von eATP- induzierte Signale kontrolliert können werden. Hier zeigen wir, dass die In-vivo-Hemmung des eATP / P2X-Purinergiewegs durch einen breiten P2XR-Antagonisten das Fortschreiten des dystrophischen Phänotyps in αSG knock-out Mäuse verzögerte. Die eATP-Blockierung dämpfte die muskuläre Entzündungsreaktion und verstärkte die Rekrutierung von Forkhead-Box-Protein P3 (Foxp3) + immunsuppressiven regulatorischen CD4 + T-Zellen. Die Verbesserung der Entzündungsmerkmale ging einher mit erhöhter Stärke, verminderter Nekrose und eingeschränkter Expression pro-fibrotischer Faktoren, was darauf schließen lässt, dass ein pharmakologischer purinergischer Antagonismus, der die angeborene und anpassungsfähige Immunkomponente in den Muskelinfiltraten verändert, einen therapeutischen Ansatz zur Verlangsamung des Krankheitsverlaufs in LGMD2D bieten könnte. In diesem Szenario sind die Konsequenzen der Purinorezeptor-Hemmung auf die Stabilität und Funktion von Tregs-Zellen besonders interessant. Ein Übergang zur Heilung unserer Ansätze ist möglich: Tregs immunsuppressive Eigenschaften haben klinische Studien mit Modellen von Autoimmunerkrankungen veranlasst; klinische Studien mit P2X7-Antagonisten wurden für andere Krankheiten abgeschlossen und zeigen akzeptable Sicherheit und Verträglichkeit

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

β1-Syntrophin Modulation by miR-222 in mdx Mice

Background: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. Methodology/Principal Findings: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3′-UTR of β1-syntrophin and participates in the downregulation of β1-syntrophin. In addition, we documented an altered regulation of the 3′-UTR of β1-syntrophin in muscle tissue from dystrophic mice. Conclusion/Significance: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy. © 2010 De Arcangelis et al

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