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.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

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.

Androgen-dependent impairment of myogenesis in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disease caused by expansion of a polyglutamine (polyQ) tract in the androgen receptor (AR). SBMA is triggered by the interaction between polyQ-AR and its natural ligands, testosterone and dihydrotestosterone (DHT). SBMA is characterized by the loss of lower motor neurons and skeletal muscle fasciculations, weakness, and atrophy. To test the hypothesis that the interaction between polyQ-AR and androgens exerts cell-autonomous toxicity in skeletal muscle, we characterized the process of myogenesis and polyQ-AR expression in DHT-treated satellite cells obtained from SBMA patients and age-matched healthy control subjects. Treatment with androgens increased the size and number of myonuclei in myotubes from control subjects, but not from SBMA patients. Myotubes from SBMA patients had a reduced number of nuclei, suggesting impaired myotube fusion and altered contractile structures. The lack of anabolic effects of androgens on myotubes from SBMA patients was not due to defects in myoblast proliferation, differentiation or apoptosis. DHT treatment of myotubes from SBMA patients increased nuclear accumulation of polyQ-AR and decreased the expression of interleukin-4 (IL-4) when compared to myotubes from control subjects. Following DHT treatment, exposure of myotubes from SBMA patients with IL-4 treatment rescued myonuclear number and size to control levels. This supports the hypothesis that androgens alter the fusion process in SBMA myogenesis. In conclusion, these results provide evidence of an androgen-dependent impairment of myogenesis in SBMA that could contribute to disease pathogenesis.

Inhibition of mitochondrial fission favours mutant over wild-type mitochondrial DNA.

Biased segregation of mitochondrial DNA variants has been widely documented, but little was known about its molecular basis. We set out to test the hypothesis that altering the balance between mitochondrial fusion and fission could influence the segregation of mutant and wild-type mtDNA variants, because it would modify the number of organelles per cell. Therefore human cells heteroplasmic for the pathological A3243G mitochondrial DNA mutation were transfected with constructs designed to silence Drp1 or hFis1, whose gene products are required for mitochondrial fission. Drp1 and hFis1 gene silencing were both associated with increased levels of mutant mitochondrial DNA. Thus, the extent of the mitochondrial reticular network appears to be an important factor in determining mutant load. The fact that the level of mutant and wild-type mitochondrial DNA can be manipulated by altering the expression of nuclear encoded factors involved in mitochondrial fission suggests new interventions for mitochondrial DNA disorders.

A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function.

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

Development and characterization of polyspecific anti-mitochondrion antibodies for proteomics studies on in toto tissue homogenates.

We describe the characterization of polyclonal antibodies directed against the whole mitochondrial subproteome, as obtained by hyperimmunization of rabbits with an organelle fraction purified from human skeletal muscle and lysed by sonication. After 2-DE separations with either blue native electrophoresis or IPG as first dimension and blotting, the polyspecific antibodies detect 113 proteins in human muscle mitochondria, representative of all major biochemical pathways and oxidative phosphorylation (OXPHOS) complexes, and cross-react with 28 proteins in rat heart mitochondria. Using as sample cryosections of human muscle biopsies lysed in urea/thiourea/CHAPS, the mitochondrial subproteome can be detected against the background of contractile proteins. When comparing with controls samples from mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes patients, immunoblotting shows in the latter a drastic reduction for the subunits of OXPHOS complex I as well as an increase of several enzymes, including ATP synthase. This finding is the first evidence at the proteomic level of massive up-regulation in a number of metabolic pathways by which the affected tissues try to compensate for the deficit in the OXPHOS machinery.

Cultured muscle cells display defects of mitochondrial myopathy ameliorated by anti-oxidants.

The mitochondrial DNA A3243G mutation causes neuromuscular disease. To investigate the muscle-specific pathophysiology of mitochondrial disease, rhabdomyosarcoma transmitochondrial hybrid cells (cybrids) were generated that retain the capacity to differentiate to myotubes. In some cases, striated muscle-like fibres were formed after innervation with rat embryonic spinal cord. Myotubes carrying A3243G mtDNA produced more reactive oxygen species than controls, and had altered glutathione homeostasis. Moreover, A3243G mutant myotubes showed evidence of abnormal mitochondrial distribution, which was associated with down-regulation of three genes involved in mitochondrial morphology, Mfn1, Mfn2 and DRP1. Electron microscopy revealed mitochondria with ultrastructural abnormalities and paracrystalline inclusions. All these features were ameliorated by anti-oxidant treatment, with the exception of the paracrystalline inclusions. These data suggest that rhabdomyosarcoma cybrids are a valid cellular model for studying muscle-specific features of mitochondrial disease and that excess reactive oxygen species production is a significant contributor to mitochondrial dysfunction, which is amenable to anti-oxidant therapy.

Human papillomavirus type 77 E7 protein is a weak deregulator of cell cycle.

Human Papillomavirus type 77 is a skin type found in non-melanoma skin cancers of immuno-compromised individuals. Although, the HPV77 E6 oncoprotein has been well studied, nothing is known about E7. Studies on mucosal HPV types (e.g. HPV16) showed that E7 deregulates the cell cycle by binding to and promoting degradation of retinoblastoma protein (pRb). Here, we characterized the impact of HPV77 E7 on the cell cycle. We observed that HPV77 E7 associated with pRb with a lower affinity than HPV16 E7, promoting weakly its degradation. Although, HPV16 E7 led to cellular proliferation and accumulation of the cell cycle inhibitor p16(INK4a), both events were not clearly observed in HPV77 E7 cells. Together, these data indicate that HPV77 E7 does not efficiently deregulate the cell cycle, in contrast to several E7s of mucosal HPV types.

In vitro development of engineered muscle using a scaffold based on the pressure-activated microsyringe (PAM) technique.

The development of new human skeletal muscle tissue is an alternative approach to the replacement of tissue after severe damage, for example in the case of traumatic injury, where surgical reconstruction is often needed following major loss of natural tissue. Treatment to date has involved the transfer of muscle tissue from other sites, resulting in a functional loss and volume deficiency of donor sites. Approaches that seek to eliminate these problems include the relatively new solution of skeletal muscle engineering. Here there are two main components to consider: (a) the cells with their regenerative potential; and (b) the polymeric structure onto which cells are seeded and where they must perform their activities. In this paper we describe well-defined two- and three-dimensional polymeric structures able to drive the myoblast process of adhesion, proliferation and differentiation. We examine a series of polymers and protein adhesions with which to functionalize the structures, and cell-seeding methods, with a view to defining the optimal protocol for engineering skeletal muscle tissue. All polymer samples were tested for their mechanical and biological properties, to support the validity of our results in the real context of muscle tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

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.

Altered Ca2+ homeostasis and endoplasmic reticulum stress in myotonic dystrophy type 1 muscle cells.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.