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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.

Facioscapulohumeral dystrophy (FSHD) is the most frequent muscular disease in adults. FSHD is characterized by a weakness and atrophy of a specific set of muscles located in the face, the shoulder, and the upper arms. FSHD patients may present different genetic defects, but they all present epigenetic alterations of the D4Z4 array located on the subtelomeric part of chromosome 4, leading to chromatin relaxation and, ultimately, to the aberrant expression of one gene called DUX4. Once expressed, DUX4 triggers a cascade of deleterious events, eventually leading to muscle dysfunction and cell death. Here, we review studies on DUX4 expression in skeletal muscle to determine the genetic/epigenetic factors and regulatory proteins governing DUX4 expression, with particular attention to the different transcripts and their very low expression in muscle.

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS—using mitochondria-targeted Paraquat (MitoPQ)—recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features., Graphical abstract fx1, Highlights • Exogenous and endogenous synaptic inactivity models increase mitochondrial ROS. • Manganese porphyrins rescue synaptic inactivity induced motor deficits. • Mitochondrial ROS may select inactive synapses for pruning during development.

The neuromuscular junction (NMJ) defines the functional interface between nerve and muscle. Reactive oxygen species (ROS) are linked to pathological denervation in multiple neurodegenerative diseases, but whether and how ROS cause denervation is unclear. Our hypothesis is that ROS reiteratively repeat the signaling pathways of developmental denervation to cause pathological denervation in multiple diseases. The stage 37-38 Xenopus Laevis model allows us to “hi-jack” the systems of functional denervation and track cellular changes in vivo. To address our hypothesis, we used SPARC to induce developmental denervation. SPARC induced both denervation and increased ROS levels. MnTE-2-PyP5+ and MnTnBuOE -2-PyP5+, two antioxidants, block ROS to abolish SPARC induced denervation. Next, we used α-Latrotoxin, α-Bungarotoxin and Mito-Paraquat to mimic pathological nerve, muscle and environmental toxin induced denervation, respectively. Analogous to SPARC, each pathological denervation model increases ROS. Strikingly, MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+ block ROS to abolish pathological denervation in each of the three models. We conclude that ROS are a shared biochemical feature of pathological denervation, recapitulating an endogenous redox regulated developmental denervation pathway.