H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TDP-43-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their 5th-7th decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376 V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376 V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy but not ALS implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Immune-Mediated Rippling Muscle Disease Associated With Thymoma and Anti-MURC/Cavin-4 Autoantibodies.

OBJECTIVES: Rippling muscle disease (RMD) is characterized by muscle stiffness, muscle hypertrophy, and rippling muscle induced by stretching or percussion. Hereditary RMD is due to sequence variants in the CAV3 and PTRF/CAVIN1 genes encoding Caveolin-3 or Cavin-1, respectively; a few series of patients with acquired autoimmune forms of RMD (iRMD) associated with AChR antibody-positive myasthenia gravis and/or thymoma have also been described. Recently, MURC/caveolae-associated protein 4 (Cavin-4) autoantibody was identified in 8 of 10 patients without thymoma, highlighting its potential both as a biomarker and as a triggering agent of this pathology. Here, we report the case of a patient with iRMD-AchR antibody negative associated with thymoma. METHODS: We suspected a paraneoplastic origin and investigated the presence of specific autoantibodies targeting muscle antigens through a combination of Western blotting and affinity purification coupled with mass spectrometry-based proteomic approaches. RESULTS: We identified circulating MURC/Cavin-4 autoantibodies and found strong similarities between histologic features of the patient's muscle and those commonly reported in caveolinopathies. Strikingly, MURC/Cavin-4 autoantibody titer strongly decreased after tumor resection and immunotherapy correlating with complete disappearance of the rippling phenotype and full patient remission. DISCUSSION: MURC/Cavin-4 autoantibodies may play a pathogenic role in paraneoplastic iRMD associated with thymoma.

Pharmacological inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulating TGF-ß via Smad3 acetylation.

The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy.

Simple Methods for Permanent or Transient Denervation in Mouse Sciatic Nerve Injury Models.

Our ability to move and breathe requires an efficient communication between nerve and muscle that mainly takes place at the neuromuscular junctions (NMJs), a highly specialized synapse that links the axon of a motor neuron to a muscle fiber. When NMJs or axons are disrupted, the control of muscle fiber contraction is lost and muscle are paralyzed. Understanding the adaptation of the neuromuscular system to permanent or transient denervation is a challenge to understand the pathophysiology of many neuromuscular diseases. There is still a lack of in vitro models that fully recapitulate the in vivo situation, and in vivo denervation, carried out by transiently or permanently severing the nerve afferent to a muscle, remains a method of choice to evaluate reinnervation and/or the consequences of the loss of innervation. We describe here a simple surgical intervention performed at the hip zone to expose the sciatic nerve in order to obtain either permanent denervation (nerve-cut) or transient and reversible denervation (nerve-crush). These two methods provide a convenient in vivo model to study adaptation to denervation. Graphical abstract.

[HDAC6, a very specific deacetylase with a potential therapeutic role]. / HDAC6, une désacétylase très spécifique porteuse d'espoir thérapeutique.

The cytoplasmic histone deacetylase 6 (HDAC6) is defined today as a new key player in the treatment of many diseases. Overexpression of HDAC6 was observed in a variety of diseases. Over the past ten years, plenty of new selective inhibitors of HDAC6 activity have been synthesized and characterized. Many studies have shown the high efficiency and beneficial effects of HDAC6 inhibitors in many diseases such as cancers, neurodegenerative, inflammatory, or neuromuscular diseases. The mechanisms of HDAC6 action that explain the benefit of its inhibition in various pathologies are still unknown. We have recently shown that HDAC6, via the regulation of the microtubule network, plays a role at the level of neuromuscular junctions by controlling acetylcholine receptor delivery.

Title: HDAC6, une désacétylase très spécifique porteuse d'espoir thérapeutique. Abstract: L'histone désacétylase 6 (HDAC6) est envisagée aujourd'hui comme une cible thérapeutique de choix dans le traitement de nombreuses maladies. L'expression de HDAC6 est fortement augmentée dans un ensemble varié de maladies. Depuis une dizaine d'années, une pléiade de nouveaux inhibiteurs sélectifs de l'activité de HDAC6 ont été synthétisés et caractérisés. De nombreuses études ont démontré l'efficacité et les effets bénéfiques des inhibiteurs de HDAC6 dans différents cancers, maladies neurodégénératives ou inflammatoires, ainsi que dans diverses maladies neuromusculaires. Tous les mécanismes d'actions de HDAC6 expliquant l'effet de son inhibition dans les pathologies ne sont pas encore connus. Nous avons récemment montré que HDAC6, via la régulation du réseau de microtubules, joue un rôle au niveau des jonctions neuromusculaires en contrôlant l'acheminement des récepteurs de l'acétylcholine.

HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions.

Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.

Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT in the Mouse.

Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.

Triadin and CLIMP-63 form a link between triads and microtubules in muscle cells.

In skeletal muscle, the triad is a structure comprising a transverse (T)-tubule and sarcoplasmic reticulum (SR) cisternae. Triads constitute the basis of excitation-contraction coupling as the cradle of the Ca2+ release complex. We have shown previously that triadin, a member of this complex, has shaping properties on reticulum membrane and is indirectly involved in a link between triads and microtubules. We have identified here that CLIMP-63 (also known as CKAP4), as the partner of triadin, is responsible for this association of triads and microtubules. Triadin and CLIMP-63 interact through their respective luminal domains and the shaping properties of triadin depend on the capacity of CLIMP-63 to bind microtubules with its cytosolic portion. In skeletal muscle, CLIMP-63 is localized in the SR, including triads, and is associated with the Ca2+ release complex through its interaction with triadin. Knockout of triadin in muscles results in the delocalization of CLIMP-63 from triads, its dissociation from the Ca2+ release complex and a disorganization of the microtubule network. Our results suggest that the association of triadin and CLIMP-63 could be involved in the shaping of SR terminal cisternae and in the guidance of microtubules close to the triads.

Role of triadin in the organization of reticulum membrane at the muscle triad.

The terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad.