Collagen VI is required for the structural and functional integrity of the neuromuscular junction.

The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.

SPP1 genotype and glucocorticoid treatment modify osteopontin expression in Duchenne muscular dystrophy cells.

Glucocorticoids are beneficial in Duchenne muscular dystrophy (DMD). Osteopontin (OPN), the protein product of SPP1, plays a role in DMD pathology modulating muscle inflammation and regeneration. A polymorphism in the SPP1 promoter (rs28357094) has been recognized as a genetic modifier of DMD, and there is evidence suggesting that it modifies response to glucocorticoid treatment. The effect of the glucocorticoid deflazacort on SPP1 mRNA and protein expression was investigated in DMD primary human myoblasts and differentiated myotubes with defined rs28357094 genotype (TT versus TG). Both healthy and DMD myoblasts/myotubes abundantly express OPN. In immunoblot, OPN was detected as a doublet of 55 and 50 kDa bands, with a shift towards the lighter isoform in the transition from myoblasts to myotubes and to mature muscle. A significant increase in OPN expression was observed in DMD myotubes carrying the TG compared to the TT genotype at rs28357094. Deflazacort treatment led to a significant increase of OPN only in myotubes carrying the TG genotype, leading to OPN overexpression. Our study shows a strong effect of the rs28357094 G allele in increasing OPN expression in the presence of deflazacort, and adds to the evidence that rs28357094 polymorphism may predict response to glucocorticoids in DMD.

Increased mitophagy in the skeletal muscle of spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by polyglutamine expansion in the androgen receptor (AR) and characterized by the loss of lower motor neurons. Here we investigated pathological processes occurring in muscle biopsy specimens derived from SBMA patients and, as controls, age-matched healthy subjects and patients suffering from amyotrophic lateral sclerosis (ALS) and neurogenic atrophy. We detected atrophic fibers in the muscle of SBMA, ALS and neurogenic atrophy patients. In addition, SBMA muscle was characterized by the presence of a large number of hypertrophic fibers, with oxidative fibers having a larger size compared with glycolytic fibers. Polyglutamine-expanded AR expression was decreased in whole muscle, yet enriched in the nucleus, and localized to mitochondria. Ultrastructural analysis revealed myofibrillar disorganization and streaming in zones lacking mitochondria and degenerating mitochondria. Using molecular (mtDNA copy number), biochemical (citrate synthase and respiratory chain enzymes) and morphological (dark blue area in nicotinamide adenine dinucleotide-stained muscle cross-sections) analyses, we found a depletion of the mitochondria associated with enhanced mitophagy. Mass spectrometry analysis revealed an increase of phosphatidylethanolamines and phosphatidylserines in mitochondria isolated from SBMA muscles, as well as a 50% depletion of cardiolipin associated with decreased expression of the cardiolipin synthase gene. These observations suggest a causative link between nuclear polyglutamine-expanded AR accumulation, depletion of mitochondrial mass, increased mitophagy and altered mitochondrial membrane composition in SBMA muscle patients. Given the central role of mitochondria in cell bioenergetics, therapeutic approaches toward improving the mitochondrial network are worth considering to support SBMA patients.

Beta-agonist stimulation ameliorates the phenotype of spinal and bulbar muscular atrophy mice and patient-derived myotubes.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of ß-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the ß-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that ß-agonist stimulation is a novel therapeutic strategy for SBMA.

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.

Glycolytic-to-oxidative fiber-type switch and mTOR signaling activation are early-onset features of SBMA muscle modified by high-fat diet.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The mechanism by which expansion of polyglutamine in AR causes muscle atrophy is unknown. Here, we investigated pathological pathways underlying muscle atrophy in SBMA knock-in mice and patients. We show that glycolytic muscles were more severely affected than oxidative muscles in SBMA knock-in mice. Muscle atrophy was associated with early-onset, progressive glycolytic-to-oxidative fiber-type switch. Whole genome microarray and untargeted lipidomic analyses revealed enhanced lipid metabolism and impaired glycolysis selectively in muscle. These metabolic changes occurred before denervation and were associated with a concurrent enhancement of mechanistic target of rapamycin (mTOR) signaling, which induced peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC1α) expression. At later stages of disease, we detected mitochondrial membrane depolarization, enhanced transcription factor EB (TFEB) expression and autophagy, and mTOR-induced protein synthesis. Several of these abnormalities were detected in the muscle of SBMA patients. Feeding knock-in mice a high-fat diet (HFD) restored mTOR activation, decreased the expression of PGC1α, TFEB, and genes involved in oxidative metabolism, reduced mitochondrial abnormalities, ameliorated muscle pathology, and extended survival. These findings show early-onset and intrinsic metabolic alterations in SBMA muscle and link lipid/glucose metabolism to pathogenesis. Moreover, our results highlight an HFD regime as a promising approach to support SBMA patients.

Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.