Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3.

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6ß and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus.

[GDF5: a therapeutic candidate for combating sarcopenia]. / GDF5 - Un candidat thérapeutique dans la lutte contre la sarcopénie.

Sarcopenia is a complex age-related muscular disease affecting 10 to 16 % of people over 65 years old. It is characterized by excessive loss of muscle mass and strength. Despite a plethora of studies aimed at understanding the physiological mechanisms underlying this pathology, the pathophysiology of sarcopenia remains poorly understood. To date, there is no pharmacological treatment for this disease. In this context, our team develop therapeutic approaches based on the GDF5 protein to counteract the loss of muscle mass and function in various pathological conditions, including sarcopenia. After deciphering one of the molecular mechanisms governing GDF5 expression, we have demonstrated the therapeutic potential of this protein in the preservation of muscle mass and strength in aged mice.

Title: GDF5 - Un candidat thérapeutique dans la lutte contre la sarcopénie. Abstract: La sarcopénie est une maladie musculaire complexe liée à l'âge qui affecte entre 10 à 16 % des personnes âgées de plus 65 ans. Elle se caractérise par une perte excessive de la masse musculaire et de la force. Malgré la multitude d'études visant à comprendre les mécanismes physiologiques qui sous-tendent cette pathologie, la physiopathologie de la sarcopénie reste encore mal comprise. A ce jour, il n'existe pas de traitement pharmacologique pour lutter contre cette pathologie. Dans ce contexte, notre équipe développe des approches thérapeutiques basées sur l'utilisation de la protéine GDF5 pour contrecarrer la perte de la masse et de la fonction musculaire dans diverses conditions pathologiques dont la sarcopénie. Après avoir décrypté un des mécanismes moléculaires régulant l'expression du GDF5, nous avons démontré le potentiel thérapeutique de cette protéine dans la préservation de la masse et la force musculaire chez les souris âgées.

RhoA Is a Crucial Regulator of Myoblast Fusion.

Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here we show that RhoA in SCs is indispensable to have correct muscle regeneration and hypertrophy. In particular, the absence of RhoA in SCs prevents a correct SC fusion both to other RhoA-deleted SCs (regeneration context) and to growing control myofibers (hypertrophy context). We demonstrated that RhoA is dispensable for SCs proliferation and differentiation; however, RhoA-deleted SCs have an inefficient movement even if their cytoskeleton assembly is not altered. Proliferative myoblast and differentiated myotubes without RhoA display a decreased expression of Chordin, suggesting a crosstalk between these genes for myoblast fusion regulation. These findings demonstrate the importance of RhoA in SC fusion regulation and its requirement to achieve an efficient skeletal muscle homeostasis restoration.

New Insights in CaVß Subunits: Role in the Regulation of Gene Expression and Cellular Homeostasis.

The voltage-gated calcium channels (CaVs or VGCCs) are fundamental regulators of intracellular calcium homeostasis. When electrical activity induces their activation, the influx of calcium that they mediate or their interaction with intracellular players leads to changes in intracellular Ca2+ levels which regulate many processes such as contraction, secretion and gene expression, depending on the cell type. The essential component of the pore channel is the CaVα1 subunit. However, the fine-tuning of Ca2+-dependent signals is guaranteed by the modulatory role of the auxiliary subunits ß, α2δ, and γ of the CaVs. In particular, four different CaVß proteins (CaVß1, CaVß2, CaVß3, and CaVß4) are encoded by four different genes in mammalians, each of them displaying several splice variants. Some of these isoforms have been described in regulating CaVα1 docking and stability at the membrane and controlling the channel complex's conformational changes. In addition, emerging evidences have highlighted other properties of the CaVß subunits, independently of α1 and non-correlated to its channel or voltage sensing functions. This review summarizes the recent findings reporting novel roles of the auxiliary CaVß subunits and in particular their direct or indirect implication in regulating gene expression in different cellular contexts.

Report and Abstracts of the 18th Meeting of the Interuniversity Institute of Myology: Virtual meeting, October 21-24, 2021.

In 2021, as the situation due to COVID-19 pandemic was still uncertain, the 18 th annual meeting of the Interuniversity Institute of Myology (IIM), took place on a virtual platform, following the same organization already tested for the previous edition. Participants from Italy, European countries, Canada and USA included clinicians, scientists, pharmaceutical companies and representatives of patient organizations. Four keynote speakers presented new insights into the modulation of muscle stem cell self-renewal in the treatment of neuromuscular disease, the role of nuclear positioning in muscle function, regeneration and tumorigenesis in the heart and advances on therapies of muscular dystrophies. Young PhD students and trainees presented oral communications distributed in five scientific sessions and posters in two poster sessions. On October 21, 2021, selected young scientists participated in the "High Training Course on Advanced Myology", organized with the University of Perugia, Italy. This course consisted of lectures on muscle regeneration and therapeutic perspectives by internationally recognized speakers, followed by roundtable discussions on "Omics technologies in myology" and "New therapeutic approaches", plus the meeting itself. Young trainees, winners of past IIM conferences, forming the Young IIM Committee, selected one of Keynote speakers and were involved in the organization of scientific sessions and roundtable discussions. The friendly welcoming of the meeting, which has strongly characterized this event and is of great help in facilitating scientific exchanges and stimulating novel collaborations, was the hallmark of the conference this year again, even on virtual platform. Breakthrough studies showing interdisciplinary works are fostering new avenues in the field of myology. This year again, scientists and students attended the meeting at the huger number, challenging the difficulties due to the COVID-19 pandemic. All participants shared the wish to continue and implement IIM meeting with new insights on muscle biology, perspectives in the understanding of the muscle-related diseases and in novel therapeutic approaches. We report here abstracts of the meeting describing basic, translational, and clinical research contributing to the large field of myology.

The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin.

Ataxia with oculomotor apraxia (AOA) type 2 (AOA2 MIM 606002) is a recessive subtype of AOA characterized by cerebellar atrophy, oculomotor apraxia, early loss of reflexes, and peripheral neuropathy. Various mutations either in homozygous or compound heterozygous condition were so far identified in the associated gene SETX (MIM 608465). SETX encodes a large protein called senataxin with a DNA-RNA helicase domain and a putative N-terminus protein interaction domain. Here, we report the identification of two novel homozygous mutations in SETX gene, c.340_342delCTT (p.L114Del) and c.1669C > T (p.R557X), in two AOA2 families. The characterization of the mutant lymphoblastoid cell lines for sensitivity to oxidative DNA-damaging agents indicates that the p.L114Del deletion confers an increased sensitivity to H2O2, camptothecin, and mitomycin C, previously found to induce death in lymphoblasts harbouring other SETX mutations; the cells carrying the nonsense mutation display instead values within the normal range. Further analysis of a neuronal cell model SKNBE, transfected with the mutant senataxin proteins, reveals increased sensitivity also to staurosporine and excitotoxicity associated with the p.L114Del mutant only. We also demonstrate that the sensitizing effect of p.L114Del on apoptosis can be reversed by senataxin silencing. The ability of a single amino acid deletion to sensitize cells to death by different agents, compared to the lack of effect of a whole protein deletion, seems to exclude a protective role played by the native protein while suggesting that a specific mutation confers to the protein the ability to enhance the toxic effect of various cell damaging agents.

A novel CLN8 mutation in late-infantile-onset neuronal ceroid lipofuscinosis (LINCL) reveals aspects of CLN8 neurobiological function.

The late-infantile-onset forms of neuronal ceroid lipofuscinosis (LINCL) are the most genetically heterogeneous group among the autosomal recessive neuronal ceroid lipofuscinoses (NCLs), with causative mutations found in CLN1, CLN2, CLN5, CLN6, CLN7 (MFSD8), and CLN8 genes. Homozygous mutations in CLN8 are associated with two distinct phenotypes: progressive epilepsy and mental retardation (EPMR), first identified in Finland; and a variant of late-infantile NCL (v-LINCL) described in a subset of Turkish and Italian patients. The function of the protein encoded by CLN8 is currently unknown. Here we report the identification of an Italian v-LINCL patient with a complete isodisomy of chromosome 8, leading to homozygosity of a maternally-inherited 3-bp deletion in CLN8 gene (c.180_182delGAA, p.Lys61del). Notably, uniparental disomy (UPD) has never been described associated with the NCLs. In addition, we provide evidence of the biological role of CLN8 characterized by expressing in different neuronal cell models the native protein, the protein carrying the mutation identified here, or three additional missense mutations previously described. Our results, validated through a gene silencing approach, indicate that CLN8 plays a role in cell proliferation during neuronal differentiation and in protection against cell death.

Nitric oxide boosts chemoimmunotherapy via inhibition of acid sphingomyelinase in a mouse model of melanoma.

Cisplatin is one of the most effective anticancer drugs, but its severe toxic effects, including depletion of immune-competent cells, limit its efficacy. We combined the systemic treatment with cisplatin with intratumor delivery of dendritic cells (DC) previously treated ex vivo with a pulse of nitric oxide (NO) released by the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate or isosorbide dinitrate. We found that this chemoimmunotherapy, tested in the B16 mouse model of melanoma, was significantly more efficacious than cisplatin alone, leading to tumor regression and animal survival at low doses of cisplatin that alone had no effect. Tumor cure was not observed when combining cisplatin with DCs not exposed to NO donors, indicating the key role of the pretreatment with NO. We investigated the mechanisms responsible for the synergic effect of NO-treated DCs and cisplatin and found that NO-treated DCs were protected both in vitro and in vivo from cisplatin-induced cytotoxicity. Cisplatin triggered DC apoptosis through increased expression and activation of acid sphingomyelinase; pretreatment of DCs with NO donors prevented such activation and inhibited activation of the downstream proapoptotic events, including generation of ceramide, activation of caspases 3 and 9, and mitochondrial depolarization. The effects of NO were mediated through generation of its physiologic messenger, cyclic GMP. We conclude that NO and NO generating drugs represent promising tools to increase the efficacy of chemoimmunotherapies in vivo, promoting the survival and increasing the function of injected cells by targeting a key pathway in cisplatin-induced cytotoxicity.

Nuclear defects in skeletal muscle from a Dynamin 2-linked centronuclear myopathy mouse model.

Dynamin 2 (DNM2) is a key protein of the endocytosis and intracellular membrane trafficking machinery. Mutations in the DNM2 gene cause autosomal dominant centronuclear myopathy (CNM) and a knock-in mouse model expressing the most frequent human DNM2 mutation in CNM (Knock In-Dnm2R465W/+) develops a myopathy sharing similarities with human disease. Using isolated muscle fibres from Knock In-Dnm2R465W/+ mice, we investigated number, spatial distribution and morphology of myonuclei. We showed a reduction of nuclear number from 20 weeks of age in Tibialis anterior muscle from heterozygous mice. This reduction is associated with a decrease in the satellite cell content in heterozygous muscles. The concomitant reduction of myonuclei number and cross-section area in the heterozygous fibres contributes to largely maintain myonuclear density and volume of myonuclear domain. Moreover, we identified signs of impaired spatial nuclear distribution including alteration of distance from myonuclei to their nearest neighbours and change in orientation of the nuclei. This study highlights reduction of number of myonuclei, a key regulator of the myofiber size, as a new pathomechanism underlying muscle atrophy in the dominant centronuclear myopathy. In addition, this study opens a new line of investigation which could prove particularly important on satellite cells in dominant centronuclear myopathy.

An embryonic CaVß1 isoform promotes muscle mass maintenance via GDF5 signaling in adult mouse.

Deciphering the mechanisms that govern skeletal muscle plasticity is essential to understand its pathophysiological processes, including age-related sarcopenia. The voltage-gated calcium channel CaV1.1 has a central role in excitation-contraction coupling (ECC), raising the possibility that it may also initiate the adaptive response to changes during muscle activity. Here, we revealed the existence of a gene transcription switch of the CaV1.1 ß subunit (CaVß1) that is dependent on the innervation state of the muscle in mice. In a mouse model of sciatic denervation, we showed increased expression of an embryonic isoform of the subunit that we called CaVß1E. CaVß1E boosts downstream growth differentiation factor 5 (GDF5) signaling to counteract muscle loss after denervation in mice. We further reported that aged mouse muscle expressed lower quantity of CaVß1E compared with young muscle, displaying an altered GDF5-dependent response to denervation. Conversely, CaVß1E overexpression improved mass wasting in aging muscle in mice by increasing GDF5 expression. We also identified the human CaVß1E analogous and show a correlation between CaVß1E expression in human muscles and age-related muscle mass decline. These results suggest that strategies targeting CaVß1E or GDF5 might be effective in reducing muscle mass loss in aging.

Endothelial nitric oxide synthase overexpression by neuronal cells in neurodegeneration: a link between inflammation and neuroprotection.

The roles of neuronal and inducible nitric oxide synthases in neurones have been extensively investigated; by contrast, the biological significance of endothelial nitric oxide synthase (eNOS) overexpression that occurs in several pathological conditions has not yet been studied. We have started addressing this issue in a cell model of neurodegeneration, i.e. human SKNBE neuroblastoma cells transfected with a mutant form of alsin, a protein causing an early-onset type of amyotrophic lateral sclerosis, ALS2. We found that eNOS, which is endogenously expressed by these cells, was activated by tumour necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that plays important roles in ALS2 and several neurodegenerative diseases. The TNF-alpha-dependent eNOS activation occurred through generation, by sphingosine-kinase-1, of sphingosine-1-phosphate, stimulation of its membrane receptors and activation of Akt, as determined using small interference RNA and dominant negative constructs specific for the enzymes and receptors. eNOS activation by TNF-alpha conferred cytoprotection from excitotoxicity and neurotoxic cues such as reactive oxygen species, endoplasmic reticulum stress, DNA damage, and mutated alsin itself. Our results suggest that overexpression of eNOS by neurones is a broad-range protective mechanism activated during damage and establish a link of pathophysiological relevance between this enzyme and inflammation accompanying neurodegenerative diseases. These findings also question the concept that high NO output in the presence of oxidative stress leads always to peroxynitrite formation contributing to neurodegeneration.

miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context.

BACKGROUND: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. METHODS: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3'UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. RESULTS: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3'UTR-luciferase assay, confirming their interaction with the NOS1-3'UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. CONCLUSION: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

In Vitro Differentiation of Mature Myofibers for Live Imaging.

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation. Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

Nitric oxide confers therapeutic activity to dendritic cells in a mouse model of melanoma.

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSµ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSµ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

Nitric oxide, ceramide and sphingomyelinase-coupled receptors: a tale of enzymes and messengers coordinating cell death, survival and differentiation.

The gaseous messenger nitric oxide plays a role in a variety of biological functions. Evidence accumulated over the last 7 years indicates that functions of nitric oxide in apoptosis growth and differentiation may originate in part from an interplay with signalling members of the sphingolipid family. Interactions between nitric oxide and sphingolipids have been shown to be multiple, to involve regulation of activity and expression of the enzymes responsible for the synthesis of nitric oxide and of those involved in the sphingolipid metabolic pathways. Recent evidence indicates that one of these interactions, namely the cross-talk of sphingomyelinases and their product ceramide with nitric oxide and its generating enzyme endothelial nitric oxide synthase, plays prominent roles during key patho-physiological processes such as inflammation, proliferation, death and differentiation.

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway.

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte-derived DCs to the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2 diolate (DETA-NO) during their maturation process induced by treatment with tumor necrosis factor alpha or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA-NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo-antigens, or cross-linking of the CD3-T cell receptor complex. This effect persists after removal of DETA-NO, depends on the generation of cyclic guanosine 5'-monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)-12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL-12 release was observed after DC exposure to DETA-NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.

N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.

Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.