Duchenne muscular dystrophy (DMD) is a severe degenerative disorder caused by mutations in the dystrophin gene. Dystrophin-deficient muscles are characterised by progressive myofibre necrosis in ...which inflammation plays a deleterious role. However, the molecular mechanisms underlying inflammation-induced necrosis in muscle cells are unknown. Here we show that necroptosis is a mechanism underlying myofibre death in dystrophin-deficient muscle. RIPK1, RIPK3 and MLKL are upregulated in dystrophic mouse myofibres. In human DMD samples, there is strong immunoreactivity to RIPK3 and phospho-MLKL in myofibres. In vitro, TNFα can elicit necroptosis in C2C12 myoblasts, and RIPK3 overexpression sensitises myoblasts to undergo TNF-induced death. Furthermore, genetic ablation of Ripk3 in mdx mice reduces myofibre degeneration, inflammatory infiltrate, and muscle fibrosis, and eventually improves muscle function. These findings provide the first evidence of necroptotic cell death in a disease affecting skeletal muscle and identify RIPK3 as a key player in the degenerative process in dystrophin-deficient muscles.
Muscular dystrophies are characterized by weakness and wasting of skeletal muscle tissues. Several drugs targeting the myostatin pathway have been used in clinical trials to increase muscle mass and ...function but most showed limited efficacy. Here we show that the expression of components of the myostatin signaling pathway is downregulated in muscle wasting or atrophying diseases, with a decrease of myostatin and activin receptor, and an increase of the myostatin antagonist, follistatin. We also provide in vivo evidence in the congenital myotubular myopathy mouse model (knock-out for the myotubularin coding gene Mtm1) that a down-regulated myostatin pathway can be reactivated by correcting the underlying gene defect. Our data may explain the poor clinical efficacy of anti-myostatin approaches in several of the clinical studies and the apparent contradictory results in mice regarding the efficacy of anti-myostatin approaches and may inform patient selection and stratification for future trials.
Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise ...large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.
Facioscapulohumeral dystrophy (FSHD) is a skeletal muscle disease caused by the aberrant expression of the DUX4 gene in the muscle tissue. To date, different therapeutic approaches have been ...proposed, targeting DUX4 at the DNA, RNA or protein levels. The recent development of the clustered regularly interspaced short-palindromic repeat (CRISPR) based technology opened new avenues of research, and FSHD is no exception. For the first time, a cure for genetic muscular diseases can be considered. Here, we describe CRISPR-based strategies that are currently being investigated for FSHD. The different approaches include the epigenome editing targeting the DUX4 gene and its promoter, gene editing targeting the polyadenylation of DUX4 using TALEN, CRISPR/cas9 or adenine base editing and the CRISPR-Cas9 genome editing for SMCHD1. We also discuss challenges facing the development of these gene editing based therapeutics.
Background
Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong ...survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA.
Methods
The experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated.
Results
We show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase).
Conclusions
These data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs.
The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, ...using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.
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•The capacity of human muscle stem cells to enter quiescence diminishes with age•This reduced capacity to re-quiesce is associated with increased DNA methylation•DNA methylation suppresses SPRY1, a known regulator of quiescence•Senescence, a feature of late cell division counts, is not increased with age
Loss of muscle strength in old age is linked to diminution of the muscle stem cell pool. Bigot et al. show that the age-associated increase in global DNA methylation acts through the SPRY1 pathway to suppress human muscle stem cell entry into quiescence, thus impairing self-renewal of the stem cell pool.
Facioscapulohumeral dystrophy (FSHD) is characterized by the contraction of the D4Z4 array located in the sub-telomeric region of the chromosome 4, leading to the aberrant expression of the DUX4 ...transcription factor and the mis-regulation of hundreds of genes. Several therapeutic strategies have been proposed among which the possibility to target the polyadenylation signal to silence the causative gene of the disease. Indeed, defects in mRNA polyadenylation leads to an alteration of the transcription termination, a disruption of mRNA transport from the nucleus to the cytoplasm decreasing the mRNA stability and translation efficiency. This review discusses the polyadenylation mechanisms, why alternative polyadenylation impacts gene expression, and how targeting polyadenylation signal may be a potential therapeutic approach for FSHD.
Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at ...the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.
Background
Facioscapulohumeral dystrophy (FSHD) is caused by mutations leading to the aberrant expression of the DUX4 transcription factor in muscles. DUX4 was proposed to induce cell death, but the ...involvement of different death pathways is still discussed. A possible pro‐apoptotic role of DUX4 was proposed, but as FSHD muscles are characterized by necrosis and inflammatory infiltrates, non‐apoptotic pathways may be also involved.
Methods
We explored DUX4‐mediated cell death by focusing on the role of one regulated necrosis pathway called necroptosis, which is regulated by RIPK3. We investigated the effect of necroptosis on cell death in vitro and in vivo experiments using RIPK3 inhibitors and a RIPK3‐deficient transgenic mouse model.
Results
We showed in vitro that DUX4 expression causes a caspase‐independent and RIPK3‐mediated cell death in both myoblasts and myotubes. In vivo, RIPK3‐deficient animals present improved body and muscle weights, a reduction of the aberrant activation of the DUX4 network genes, and an improvement of muscle histology.
Conclusions
These results provide evidence for a role of RIPK3 in DUX4‐mediated cell death and open new avenues of research.
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle dystrophy typically affecting patients within their second decade. Patients initially exhibit asymmetric facial and humeral muscle ...damage, followed by lower body muscle involvement. FSHD is associated with a derepression of DUX4 gene encoded by the D4Z4 macrosatellite located on the subtelomeric part of chromosome 4. DUX4 is a highly regulated transcription factor and its expression in skeletal muscle contributes to multiple cellular toxicities and pathologies ultimately leading to muscle weakness and atrophy. Since the discovery of the FSHD candidate gene DUX4, many cell and animal models have been designed for therapeutic approaches and clinical trials. Today there is no treatment available for FSHD patients and therapeutic strategies targeting DUX4 toxicity in skeletal muscle are being actively investigated. In this review, we will discuss different research areas that are currently being considered to alter DUX4 expression and toxicity in muscle tissue and the cell and animal models designed to date.
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