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In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic defects that limit regenerative potential or are disrupted by their degenerative/regenerative environment is unclear. We investigated cell behavior and gene expression in human using MuSCs derived from DMD or healthy muscles. We found that proliferation, differentiation and fusion were not altered in DMD-MuSCs, but with time, they lost their myogenic identity twice as fast as healthy MuSCs. The rapid drift towards a fibroblast-like cell identity was observed at the clonal level, and resulted from the altered expression of epigenetic enzymes required to maintain the myogenic cell fate. Indeed, the re-expression ofCBX3,SMC3,H2AFVandH3F3Bprevented the MuSC identity drift. Amongst the epigenetic changes, a closing of chromatin at the gene encoding the transcription factorMEF2Bcaused a down-regulation of its expression and a loss of the myogenic fate. Thus, MEF2B is a key mediator of the myogenic identity in human MuSCs, that is altered in DMD pathology.

Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis (RM) episodes precipitated by fasting. Since starvation promotes autophagy induction, we wondered whether TANGO2-related muscle symptoms result from autophagy insufficiency to meet cellular demands in stress conditions. Autophagy functioning was analyzed in vitro, in primary skeletal muscle cells from TANGO2 patients in basal and fasting conditions. In addition, wce developed a tango2 morphant zebrafish model to assess the effect of tango2 knockdown (KD) on locomotor function and autophagy efficiency in vivo. We report that TANGO2 mutations are associated with decreased LC3-II levels upon starvation in primary muscle cells, but not in fibroblasts. In zebrafish larvae, tango2 knockdown induces locomotor defects characterized by reduced evoked movements which are exacerbated by exposure to atorvastatin, a compound known to cause RM. Importantly, RM features of tango2 KD are also associated with autophagy defects in zebrafish. Calpeptin treatment, a known activator of autophagy, is sufficient to rescue the locomotor function and improves autophagy in zebrafish. LC3-II levels of primary muscle cells of TANGO2 patients are also ameliorated by calpeptin treatment. Overall, we demonstrate that TANGO2 plays an important role in autophagy, and that autophagy efficiency is critical to prevent RM, thus giving rise to new therapeutic perspectives in the prevention of these life-threatening episodes in the context of TANGO2 pathology.

TANGO2 disease is a severe inherited disorder associating multiple symptoms such as metabolic crises, encephalopathy, cardiac arrhythmias, and hypothyroidism. The mechanism of action of TANGO2 is currently unknown. Here, we describe a cohort of 20 French patients bearing mutations in the TANGO2 gene. We found that the main clinical presentation was the association of neurodevelopmental delay (n = 17), acute metabolic crises (n = 17) and hypothyroidism (n = 12), with a large intrafamilial clinical variability. Metabolic crises included rhabdomyolysis (15/17), neurological symptoms (14/17), and cardiac features (12/17; long QT (n = 10), Brugada pattern (n = 2), cardiac arrhythmia (n = 6)) that required intensive care. We show previously uncharacterized triggers of metabolic crises in TANGO2 patients, such as some anesthetics and possibly l-carnitine. Unexpectedly, plasma acylcarnitines, plasma FGF-21, muscle histology, and mitochondrial spectrometry were mostly normal. Moreover, in patients' primary myoblasts, palmitate and glutamine oxidation rates, and the mitochondrial network were also normal. Finally, we found variable mitochondrial respiration and defective clearance of oxidized DNA upon cycles of starvation and refeeding. We conclude that TANGO2 disease is a life-threatening disease that needs specific cardiac management and anesthesia protocol. Mechanistically, TANGO2 disease is unlikely to originate from a primary mitochondrial defect. Rather, we suggest that mitochondrial defects are secondary to strong extrinsic triggers in TANGO2 deficient patients.

Background and ObjectivesTo determine common clinical and biological traits in 2 individuals with variants in ISCU and FDX2, displaying severe and recurrent rhabdomyolyses and lactic acidosis.MethodsWe performed a clinical characterization of 2 distinct individuals with biallelic ISCU or FDX2 variants from 2 separate families and a biological characterization with muscle and cells from those patients.ResultsThe individual with FDX2 variants was clinically more affected than the individual with ISCU variants. Affected FDX2 individual fibroblasts and myoblasts showed reduced oxygen consumption rates and mitochondrial complex I and PDHc activities, associated with high levels of blood FGF21. ISCU individual fibroblasts showed no oxidative phosphorylation deficiency and moderate increase of blood FGF21 levels relative to controls. The severity of the FDX2 individual was not due to dysfunctional autophagy. Iron was excessively accumulated in ISCU-deficient skeletal muscle, which was accompanied by a downregulation of IRP1 and mitoferrin2 genes and an upregulation of frataxin (FXN) gene expression. This excessive iron accumulation was absent from FDX2 affected muscle and could not be correlated with variable gene expression in muscle cells.DiscussionWe conclude that FDX2 and ISCU variants result in a similar muscle phenotype, that differ in severity and skeletal muscle iron accumulation. ISCU and FDX2 are not involved in mitochondrial iron influx contrary to frataxin.

In Duchenne muscular dystrophy (DMD) patients, absence of dystrophin causes muscle wasting by impacting both the myofiber integrity and the properties of muscle stem cells (MuSCs). Investigation of DMD encompasses the use of MuSCs issued from human skeletal muscle. However, DMD-derived MuSC usage is restricted by the limited number of divisions that human MuSCs can undertake in vitro before losing their myogenic characteristics and by the scarcity of human material available from DMD muscle. To overcome these limitations, immortalization of MuSCs appears as a strategy. Here, we used CDK4/hTERT expression in primary MuSCs and we derived MuSC clones from a series of clinically and genetically characterized patients, including eight DMD patients with various mutations, four congenital muscular dystrophies and three age-matched control muscles. Immortalized cultures were sorted into single cells and expanded as clones into homogeneous populations. Myogenic characteristics and differentiation potential were tested for each clone. Finally, we screened various promoters to identify the preferred gene regulatory unit that should be used to ensure stable expression in the human MuSC clones. The 38 clonal immortalized myogenic cell clones provide a large collection of controls and DMD clones with various genetic defects and are available to the academic community.

International audience; Letter to the Editor

International audience; Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines (90-8, 88-14 and 96-2), STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive "active-site" mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies.