Mitochondrial myopathies and muscle stem cells

PhD ThesisMitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA) for which there is no effective treatment. Culturing of myoblasts from patients with sporadically occurring mitochondrial diseases has suggested that mtDNA mutations may be lower or absent in muscle stem cells (satellite cells). The activation of muscle satellite cells and subsequent repair of muscle fibres may favourably shift the balance of delete to wild-type (WT) mtDNA, thereby decreasing mtDNA mutation load in affected muscle. This research has investigated muscle precursor cells from patients with mitochondrial myopathy due to sporadically occurring mtDNA deletions. This was to determine if they will benefit from attempts to “gene shift” the balance of WT and mutated mtDNA in their muscles using high intensity resistance training. Fluorescently Activated Cell Sorting (FACS) on the basis of CD56 (NCam) was used to isolate satellite cells and real time PCR to analyse them. In all eight patients investigated mtDNA deletions were detected in satellite cells at levels similar to mature muscle. In most of these patients the mtDNA deletions were lost during the culturing of their myoblasts. In some patients, however, the mutation was maintained, although there was a gradual decline in mutation load as the myoblasts headed towards differentiation. It was hypothesised that this difference between patients in the maintenance or loss of mutations in their myoblasts was attributable to an mtDNA bottleneck effect at the point of satellite cell activation. A second selection point occurred during the process of myoblast proliferation, possibly mediated by segregation of WT and delete mtDNA after cell division. Daughter cells that inherit large amounts of delete mtDNA will be unable to continue to proliferate. If efforts to “gene shift” in these patients will involve the activation of satellite cells to repair damaged muscle, it is paramount that this process does not exhaust the muscle stem cell pool. Satellite cell numbers have been determined in patients harbouring sporadically occurring mtDNA deletions, who will be considered potential beneficiaries of exercise based interventions. No significant difference was observed in satellite cell numbers when patients were compared to controls. In addition, a single patient was examined for satellite cell numbers over eleven years and no reduction in numbers was found. Given that the large majority of single deletion patients will lose their mtDNA mutation during the process of muscle regeneration and that they will not suffer from an exhaustion of the satellite cell pool, “gene shifting” remains a viable therapy in these patients. However, the mechanisms behind the process are somewhat different to what was originally hypothesised

Mitochondrial myopathies and muscle stem cells

PhD ThesisMitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA) for which there is no effective treatment. Culturing of myoblasts from patients with sporadically occurring mitochondrial diseases has suggested that mtDNA mutations may be lower or absent in muscle stem cells (satellite cells). The activation of muscle satellite cells and subsequent repair of muscle fibres may favourably shift the balance of delete to wild-type (WT) mtDNA, thereby decreasing mtDNA mutation load in affected muscle. This research has investigated muscle precursor cells from patients with mitochondrial myopathy due to sporadically occurring mtDNA deletions. This was to determine if they will benefit from attempts to “gene shift” the balance of WT and mutated mtDNA in their muscles using high intensity resistance training. Fluorescently Activated Cell Sorting (FACS) on the basis of CD56 (NCam) was used to isolate satellite cells and real time PCR to analyse them. In all eight patients investigated mtDNA deletions were detected in satellite cells at levels similar to mature muscle. In most of these patients the mtDNA deletions were lost during the culturing of their myoblasts. In some patients, however, the mutation was maintained, although there was a gradual decline in mutation load as the myoblasts headed towards differentiation. It was hypothesised that this difference between patients in the maintenance or loss of mutations in their myoblasts was attributable to an mtDNA bottleneck effect at the point of satellite cell activation. A second selection point occurred during the process of myoblast proliferation, possibly mediated by segregation of WT and delete mtDNA after cell division. Daughter cells that inherit large amounts of delete mtDNA will be unable to continue to proliferate. If efforts to “gene shift” in these patients will involve the activation of satellite cells to repair damaged muscle, it is paramount that this process does not exhaust the muscle stem cell pool. Satellite cell numbers have been determined in patients harbouring sporadically occurring mtDNA deletions, who will be considered potential beneficiaries of exercise based interventions. No significant difference was observed in satellite cell numbers when patients were compared to controls. In addition, a single patient was examined for satellite cell numbers over eleven years and no reduction in numbers was found. Given that the large majority of single deletion patients will lose their mtDNA mutation during the process of muscle regeneration and that they will not suffer from an exhaustion of the satellite cell pool, “gene shifting” remains a viable therapy in these patients. However, the mechanisms behind the process are somewhat different to what was originally hypothesised

Mitochondrial myopathies and muscle stem cells

Mitochondrial myopathies are a group of progressive muscle disorders caused by mutations in the mitochondrial genome (mtDNA) for which there is no effective treatment. Culturing of myoblasts from patients with sporadically occurring mitochondrial diseases has suggested that mtDNA mutations may be lower or absent in muscle stem cells (satellite cells). The activation of muscle satellite cells and subsequent repair of muscle fibres may favourably shift the balance of delete to wild-type (WT) mtDNA, thereby decreasing mtDNA mutation load in affected muscle. This research has investigated muscle precursor cells from patients with mitochondrial myopathy due to sporadically occurring mtDNA deletions. This was to determine if they will benefit from attempts to “gene shift” the balance of WT and mutated mtDNA in their muscles using high intensity resistance training. Fluorescently Activated Cell Sorting (FACS) on the basis of CD56 (NCam) was used to isolate satellite cells and real time PCR to analyse them. In all eight patients investigated mtDNA deletions were detected in satellite cells at levels similar to mature muscle. In most of these patients the mtDNA deletions were lost during the culturing of their myoblasts. In some patients, however, the mutation was maintained, although there was a gradual decline in mutation load as the myoblasts headed towards differentiation. It was hypothesised that this difference between patients in the maintenance or loss of mutations in their myoblasts was attributable to an mtDNA bottleneck effect at the point of satellite cell activation. A second selection point occurred during the process of myoblast proliferation, possibly mediated by segregation of WT and delete mtDNA after cell division. Daughter cells that inherit large amounts of delete mtDNA will be unable to continue to proliferate. If efforts to “gene shift” in these patients will involve the activation of satellite cells to repair damaged muscle, it is paramount that this process does not exhaust the muscle stem cell pool. Satellite cell numbers have been determined in patients harbouring sporadically occurring mtDNA deletions, who will be considered potential beneficiaries of exercise based interventions. No significant difference was observed in satellite cell numbers when patients were compared to controls. In addition, a single patient was examined for satellite cell numbers over eleven years and no reduction in numbers was found. Given that the large majority of single deletion patients will lose their mtDNA mutation during the process of muscle regeneration and that they will not suffer from an exhaustion of the satellite cell pool, “gene shifting” remains a viable therapy in these patients. However, the mechanisms behind the process are somewhat different to what was originally hypothesised.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology

Salbutamol modifies the neuromuscular junction in a mouse model of ColQ myasthenic syndrome.

The β-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ-/- mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ-/- mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that β-adrenergic agonists lead to functional benefit in the ColQ-/- mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission

Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen alpha 3 (VI) (COL6A3) C-terminal domain (CTD). These Col6a3(CTT) mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3(CTT) mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3(CTT) mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control

Mitochondrial DNA deletions in muscle satellite cells: implications for therapies.

Progressive myopathy is a major clinical feature of patients with mitochondrial DNA (mtDNA) disease. There is limited treatment available for these patients although exercise and other approaches to activate muscle stem cells (satellite cells) have been proposed. The majority of mtDNA defects are heteroplasmic (a mixture of mutated and wild-type mtDNA present within the muscle) with high levels of mutated mtDNA and low levels of wild-type mtDNA associated with more severe disease. The culture of satellite cell-derived myoblasts often reveals no evidence of the original mtDNA mutation although it is not known if this is lost by selection or simply not present in these cells. We have explored if the mtDNA mutation is present in the satellite cells in one of the commonest genotypes associated with mitochondrial myopathies (patients with single, large-scale mtDNA deletions). Analysis of satellite cells from eight patients showed that the level of mtDNA mutation in the satellite cells is the same as in the mature muscle but is most often subsequently lost during culture. We show that there are two periods of selection against the mutated form, one early on possibly during satellite cell activation and the other during the rapid replication phase of myoblast culture. Our data suggest that the mutations are also lost during rapid replication in vivo, implying that strategies to activate satellite cells remain a viable treatment for mitochondrial myopathies in specific patient groups

Targeted therapies for metabolic myopathies related to glycogen storage and lipid metabolism: a systematic review and steps towards a 'Treatabolome'

Background: Metabolic myopathies are a heterogenous group of muscle diseases typically characterized by exercise intolerance, myalgia and progressive muscle weakness. Effective treatments for some of these diseases are available, but while our understanding of the pathogenesis of metabolic myopathies related to glycogen storage, lipid metabolism and β-oxidation is well established, evidence linking treatments with the precise causative genetic defect is lacking. Objective: The objective of this study was to collate all published evidence on pharmacological therapies for the aforementioned metabolic myopathies and link this to the genetic mutation in a format amenable to databasing for further computational use in line with the principles of the "treatabolome" project. Methods: A systematic literature review was conducted to retrieve all levels of evidence examining the therapeutic efficacy of pharmacological treatments on metabolic myopathies related to glycogen storage and lipid metabolism. A key inclusion criterion was the availability of the genetic variant of the treated patients in order to link treatment outcome with the genetic defect. Results: Of the 1,085 articles initially identified, 268 full-text articles were assessed for eligibility, of which 87 were carried over into the final data extraction. The most studied metabolic myopathies were Pompe disease (45 articles), multiple acyl-CoA dehydrogenase deficiency related to mutations in the ETFDH gene (15 articles) and systemic primary carnitine deficiency (8 articles). The most studied therapeutic management strategies for these diseases were enzyme replacement therapy, riboflavin, and carnitine supplementation, respectively. Conclusions: This systematic review provides evidence for treatments of metabolic myopathies linked with the genetic defect in a computationally accessible format suitable for databasing in the treatabolome system, which will enable clinicians to acquire evidence on appropriate therapeutic options for their patient at the time of diagnosis.AM received a Summer Studentship stipend from the Faculty of Medicine at the University of Ottawa. HL receives support from the Canadian Institutes of Health Research (Foundation Grant FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant NG2-170044 for NMD4C), the Canada Foundation for Innovation (CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279). RT receives support from the Canadian Institutes of Health Research (postdoctoral fellowship award MFE-171275). This review is part of the Solve-RD treatabolome initiative. The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 77925

Mitochondrial Mutations Can Alter Neuromuscular Transmission in Congenital Myasthenic Syndrome and Mitochondrial Disease.

Congenital myasthenic syndromes (CMS) are a group of rare, neuromuscular disorders that usually present in childhood or infancy. While the phenotypic presentation of these disorders is diverse, the unifying feature is a pathomechanism that disrupts neuromuscular transmission. Recently, two mitochondrial genes-SLC25A1 and TEFM-have been reported in patients with suspected CMS, prompting a discussion about the role of mitochondria at the neuromuscular junction (NMJ). Mitochondrial disease and CMS can present with similar symptoms, and potentially one in four patients with mitochondrial myopathy exhibit NMJ defects. This review highlights research indicating the prominent roles of mitochondria at both the pre- and postsynapse, demonstrating the potential for mitochondrial involvement in neuromuscular transmission defects. We propose the establishment of a novel subcategorization for CMS-mitochondrial CMS, due to unifying clinical features and the potential for mitochondrial defects to impede transmission at the pre- and postsynapse. Finally, we highlight the potential of targeting the neuromuscular transmission in mitochondrial disease to improve patient outcomes