Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by SMN gene defects. It leads to motor neuron death and muscle weakness. Without treatment, most affected children don't survive past age two. Recently, new gene therapies help SMA children survive, but treated patients now face ongoing muscle atrophy and functional deficits, creating a novel clinical presentation. Over the last years, treatments of various animal models of neuromuscular disorders have shown the ability of inhibitors of the non-conventional histone deacetylase 6 (HDAC6) to reduce muscle atrophy. This study examines HDAC6 inhibition's impact on muscle cell differentiation and tests in vivo if combining it with new standard SMA treatments improves muscle and overall condition in SMA mice. Here, we report that HDAC6 controls myotube formation and maturation in vitro. In particular, HDAC6 inhibition increases the size of SMA patients-derived muscle primary myotubes. In vivo, when combined with ASOs inducing exon-7 inclusion in SMN2 RNA, HDAC6 systemic inhibition strongly improved muscle strength, mass, function, and longevity of SMA-like mice model. These findings provide evidence that selective inhibition of HDAC6 improves myogenic progression. Hence, HDAC6 inhibitors are good candidates to ameliorate persisting symptoms of SMA patients treated with the new standard of care.

The remarkable success of mRNA-lipid nanoparticles (LNP) vaccines during the SARS-CoV-2 pandemic have highlighted the critical role of this cutting-edge technology as a cornerstone for contemporary vaccine innovation. Efforts to enhance mRNA-LNP vaccine efficacy have driven specific interest in optimizing nanoparticle design to improve immune cell transfection. Nevertheless, the precise mechanisms driving immune responses, including cell identity and their activation states within immune and local tissues, remain unclear and system-dependent. Muscle tissue is an ideal site for mRNA vaccine administration due to its ribosome abundance, ensuring efficient translation of the delivered mRNA into the encoded protein. Additionally, the localized nature of muscle injections ensures controlled biodistribution and minimizes systemic side effects, making it a safe and effective route for generating robust immune responses. In line with these observations, we developed a lipid-polymer hybrid nanosystem that effectively complexes mRNA, demonstrating high efficiency in transfecting muscle cells and tissue. Importantly, this delivery resulted in a robust adaptive immune response, characterized by both potent humoral and cellular immunity, highlighting the effectiveness of this nanosystem for mRNA-based vaccination. Overall, these findings highlight the need to consider the role of muscle cells as potential antigen-producing reservoirs in immune modulation when designing mRNA vaccines.

Lipid metabolism is a key process required for muscle stem cell (MuSCs) function during regenerative myogenesis. However, the molecular pathway responsible of such regulation is still unknown. Studies of lysine demethylase PHF2 have reported its critical role in lipid metabolism in pathological processes, although there are no data regarding its role during MuSC fate transition. Here we show that PHF2 controls lipid droplet homeostasis in MuSCs during regenerative myogenesis, by promoting the contact between lipid droplets and mitochondria. Consistently, in absence of PHF2, myocytes accumulate lipid droplets, leading to mitochondrial dysfunction and impaired regeneration. Interestingly, such phenotype is rescued by AMPKa2-PHF2 phospho-mimetic mutant expression. Our findings provide evidence that PHF2 is part of AMPKa2 signaling and underscore the critical role of AMPKa2/PHF2 axis in regulating lipid droplets homeostasis during MuSC fate. ### Competing Interest Statement The authors have declared no competing interest.

Organoids have emerged as innovative three-dimensional (3D) in vitro models capable of reproducing the essential structural and functional characteristics of human organs. They offer an alternative between traditional two dimensions (2D) cell cultures and animal models. Derived from stem cells, organoids have an intrinsic capacity for self-organisation and morphogenesis, recapitulating the processes of embryonic development. Three elements are crucial for their generation: the origin of the stem cells, the extracellular matrix, and controlled exposure to morphogens. Among the most promising applications, neuromuscular organoids (NMOs) enable the co-differentiation of motor neurons, skeletal muscle cells, and Schwann cells from neuro-mesodermal progenitors. 3D NMO models and simplified 2D versions have been developed, complemented by assembloid-type approaches or microfluidic devices, facilitating the study of inter-cellular interactions and pharmacological testing. Produced from patients' iPS cells (induced pluripotent stem cells), NMOs offer a relevant platform for disease modelling, study of functional phenotypes and pharmacological screening in personalized medicine. Despite these advances, limitations remain, hindering the routine use of organoids. The standardization of protocols and the automation of analyses will enable the full translational potential of organoids to be exploited in the future.

Balanced mTOR activity and iron levels are crucial for muscle integrity, with evidence suggesting mTOR regulates cellular iron homeostasis. In this study, we investigated iron metabolism in muscle-specific mTOR knockout mice (mTORmKO) and its relation to their myopathy. The mTORmKO mice exhibited distinct iron content patterns across muscle types and ages. Slow-twitch soleus muscles initially showed reduced iron levels in young mice, which increased with the dystrophy progression but remained within control ranges. In contrast, the less affected fast-twitch muscles maintained near-normal iron levels from a young age. Interestingly, both mTORmKO muscle types exhibited iron metabolism markers indicative of iron excess, including decreased transferrin receptor 1 (TFR1) and increased levels of ferritin (FTL) and ferroportin (FPN) proteins. Paradoxically, these changes were accompanied by downregulated Ftl and Fpn mRNA levels, indicating post-transcriptional regulation. This discordant regulation resulted from disruption of key iron metabolism pathways, including NRF2/NFE2L2, HIFs, and AKT/PKB signaling. Mechanistically, mTOR deficiency impaired transcriptional regulation of iron-related genes mediated by NRF2 and HIFs. Furthermore, it triggered ferritin accumulation through two NRF2 mechanisms: (1) derepression of ferritin translation via suppression of the FBXL5-IRP axis, and (2) autophagosomal sequestration driven by NCOA4-dependent ferritin targeting to autophagosomes, coupled with age-related impairments of autophagy linked to chronic AKT/PKB activation. Three-week spermidine supplementation in older mTORmKO mice was associated with normalized AKT/PKB-FOXO signaling, increased endolysosomal FTL and reduced total FTL levels in the dystrophic soleus muscle. These findings underscore mTOR's crucial role in skeletal muscle iron metabolism and suggest spermidine as a potential strategy to address impaired ferritinophagy due to autophagy blockade in dystrophic muscle.

Proteinopathies, such as amyotrophic lateral sclerosis (ALS), are marked by the accumulation of misfolded proteins that disrupt cellular processes. Eukaryotic cells have developed protein quality control systems to eliminate these aberrant proteins, but these systems often fail to differentiate between normal and misfolded proteins. In ALS, pathological inclusions primarily composed of misfolded TDP-43 are a hallmark of the disease. Recently, a novel unconventional secretion process called misfolding-associated protein secretion (MAPS) has been discovered to selectively export misfolded proteins. USP19, an Endoplasmic Reticulum-associated ubiquitin peptidase, plays a crucial role in this process. In this study, we investigated the impact of ER-anchored USP19 on the secretion of misfolded TDP-43. Here we found that USP19 overexpression significantly promotes the secretion of soluble and aggregated misfolded TDP-43, requiring both ER anchoring and ubiquitin peptidase activity. Characterization of the cellular and molecular mechanisms involved in this process highlighted the importance of early autophagosomal and late endosomal/amphisomal compartments, while lysosomes did not play a key role. By using dominant-negative mutants and small interfering RNAs, we identified that USP19-mediated secretion of misfolded TDP-43 is modulated by key factors involved in cellular trafficking and secretion pathways, such as ATG7, the ESCRT-O HGS/HRS, the Rab GTPases RAB11A, RAB8A, and RAB27A, and the v-SNARE VAMP7. We also confirmed the crucial role of the DNAJC5/CSPα cochaperone. Overall, this study provides new insights into how cells manage the secretion of misfolded TDP-43 proteins and potentially opens new avenues for therapeutic interventions in ALS and related disorders.

Prime Editing can rewrite genes in living cells by allowing point mutations, deletions, or insertion of small DNA sequences with high precision. However, its safe and efficient delivery into human stem cells remains a technical challenge. In this report, we engineer Nanoscribes, virus-like particles that encapsidate ribonucleoprotein complexes of the Prime Editing system and allow their delivery into recipient cells. We identify key features that unlock the potential of Nanoscribes, including the use of multiple fusogens, the improvement of pegRNAs structures, their encoding by a Pol II system and the optimization of Prime-Editors. Nanoscribes edit HEK293T with an efficiency of 68% at the HEK3 locus with increased fidelity over DNA-transfection and support pegRNA-multiplexing. Importantly, Nanoscribes permit editing of myoblasts, hiPSCs and hiPSCs-derived hematopoietic stem cells with an editing efficiency up to 25%. Nanoscribes is an asset for development of next generation genome editing approaches using VLPs.

Spinal muscular atrophy (SMA) is a rare, progressive and severe neuromuscular disease. It is mostly caused by mutations in the SMN gene, which lead to the death of spinal cord motor neurons. In the absence of treatment, more than half of affected children die before the age of two. Recently, groundbreaking gene therapies were developed, allowing children to survive. However, a new clinical presentation of the disease has emerged in treated patients, characterized by ongoing functional deficits and a disability mainly due to persistent muscle atrophy. Over the last years, treatments of various animal models of neuromuscular disorders have shown the ability of inhibitors of the non-conventional histone deacetylase 6 (HDAC6) to reduce inflammation, fibrosis and muscle atrophy, and to ameliorate acetylcholine receptor distribution at the neuromuscular junction, microtubule network and mitochondrial transport in axons, indicating potential interest for the treatment of neuromuscular disorders. The present study was designed to properly characterize the effect of HDAC6 inhibition on muscle cells proliferation and differentiation and to evaluate in vivo if HDAC6 inhibition combined with the new standard of care treatments of SMA could ameliorate skeletal muscle and general status of a SMA mouse model. Here, we report that HDAC6 and tubulin acetylation controls myotube formation and maturation in vitro. In particular, HDAC6 inhibition increases the size of SMA patients-derived muscle primary myotubes. In vivo, when combined with ASOs inducing exon 7 inclusion in SMN2 RNA in motoneurons, HDAC6 systemic inhibition strongly improved muscle strength, mass, function, and longevity of the Smn[Delta]7/[Delta]7; hSMN2+/- mouse model of SMA. These findings provide evidence that in muscle cells, HDAC6 is the only tubulin deacetylase and that selective inhibition of HDAC6 improves myogenic progression and that HDAC6 inhibitors are good candidates to ameliorate persisting symptoms of SMA patients treated with the new standard of care. ### Competing Interest Statement The authors have declared no competing interest.

Les organoïdes ont émergé comme des modèles innovants tridimensionnels (3D) in vitro , capables de reproduire des caractéristiques structurelles et fonctionnelles essentielles des organes humains. Ils constituent une alternative entre les cultures cellulaires en deux dimensions (2D) classiques et les modèles animaux. Dérivés de cellules souches, les organoïdes possèdent une capacité intrinsèque d’auto-organisation et de morphogenèse, récapitulant les processus du développement embryonnaire. Trois éléments sont cruciaux pour leur génération : l’origine des cellules souches, la matrice extracellulaire et l’exposition contrôlée à des morphogènes. Parmi les applications les plus prometteuses, les organoïdes neuromusculaires (NMO pour neuromuscular organoids ) permettent la co-différenciation de motoneurones, de cellules musculaires squelettiques et de cellules de Schwann à partir de progéniteurs neuro-mésodermiques. Des modèles NMO 3D et des versions simplifiées en 2D ont été développés, complétés par des approches de type « assembloïdes » ou des dispositifs microfluidiques, facilitant l’étude des interactions inter-cellulaires et les tests pharmacologiques. Produits à partir de cellules iPS ( induced pluripotent stem ) de patients, les NMO offrent une plateforme pertinente pour la modélisation des maladies, l’étude des phénotypes fonctionnels et le criblage pharmacologique en médecine personnalisée. Malgré ces avancées, des limitations persistent, freinant l’utilisation des organoïdes en routine. La standardisation des protocoles et l’automatisation des analyses permettront à l’avenir d’exploiter pleinement le potentiel translationnel des organoïdes.

We aimed to evaluate whether inherited mitochondrial dysfunction is associated with neuromuscular junction remodeling in patients with mitochondrial disorders. Muscle biopsies from 15 patients with mitochondrial disorders and 10 control patients were analyzed through immunostaining for various neuromuscular junction components. The patient group, with a mean age of 49.9 years, exhibited various mitochondrial disorders including chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome, and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Patients with mitochondrial disorders had a high percentage of remodeled (p= 0.0001), neoformed (p= 0.0049) and dilated (p= 0.016) endplates. There was a trend toward an increased proportion of neuromuscular junctions with terminal Schwann cell extension in these patients (p= 0.052). No significant difference was found in myofiber diameter between the groups. The observed neuromuscular junction defects varied widely across different mitochondrial disorder phenotypes and were present even without accompanying muscle weakness or neuropathy. This suggest that mitochondrial disorders are associated with a primary NMJ remodeling independent of muscle structural damage. Pathomechanisms underpinning this remodeling of the neuromuscular junction, as well as clinical factors predictive of this remodeling, remain to be fully characterized.

Abstract Aged female mice lacking the Terf2 gene, encoding the telomeric protective factor TRF2, in their skeletal myofibers exhibit a younger muscle phenotype with lack of ongoing or past regeneration and myofiber atrophy, preserved muscle strength, and moderate prolonged lifespan. Since this is accompanied by a slight pro-oxidant effect without telomere deprotection, we propose that the age-related TRF2 downregulation triggers a mito-hormetic response with beneficial effect in the skeletal muscle.

TAR DNA binding protein of 43 kDa (TDP-43)-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS.Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their fifth to seventh decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies.The G376V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue.The identification of individuals with TDP-43-related myopathy, but not ALS, implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype. Zibold et al. identify a new TDP-43 missense variant (G376V) in two French families affected by late-onset distal myopathy but not ALS. The findings support a primary role for TDP-43 in skeletal muscle pathophysiology and suggest that TARDBP screening should be included in the genetic work-up of patients with distal myopathy.

The Wnt/β-Catenin pathway plays a key role in cell fate determination during development and in adult tissue regeneration by stem cells. These processes involve profound gene expression and epigenome remodeling and linking Wnt/β-Catenin signaling to chromatin modifications has been a challenge over the past decades. Functional studies of the lysine demethylase LSD1/KDM1A converge to indicate that this epigenetic regulator is a key regulator of cell fate, although the extracellular cues controlling LSD1 action remain largely unknown. Here we show that β-Catenin is a substrate of LSD1. Demethylation by LSD1 prevents β-Catenin degradation thereby maintaining its nuclear levels. Consistently, in absence of LSD1, β-Catenin transcriptional activity is reduced in both MuSCs and ESCs. Moreover, inactivation of LSD1 in mouse muscle stem cells and embryonic stem cells shows that LSD1 promotes mitotic spindle orientation via β-Catenin protein stabilization. Altogether, by inscribing LSD1 and β-Catenin in the same molecular cascade linking extracellular factors to gene expression, our results provide a mechanistic explanation to the similarity of action of canonical Wnt/β-Catenin signaling and LSD1 on stem cell fate.

Exercise preserves neuromuscular function in aging through unknown mechanisms. Skeletal muscle fibroblasts (FIB) and stem cells (MuSC) are abundant in skeletal muscle and reside close to neuromuscular junctions, but their relative roles in motor neuron maintenance remain undescribed. Using direct cocultures of embryonic rat motor neurons with either human MuSC or FIB, RNA sequencing revealed profound differential regulation of the motor neuron transcriptome, with FIB generally favoring neuron growth and cell migration and MuSC favoring production of ribosomes and translational machinery. Conditioned medium from FIB was superior to MuSC in preserving motor neurons and increasing their maturity. Lastly, we established the importance of donor age and exercise status and found an age-related distortion of motor neuron and muscle cell interaction that was fully mitigated by lifelong physical activity. In conclusion, we show that human muscle FIB and MuSC synergistically stimulate the growth and viability of motor neurons, which is further amplified by regular exercise.

<p>Description of additional methods and procedures used in the study.</p>

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

Proximal spinal muscular atrophy (SMA) is defined by a degeneration of the anterior horn cells resulting in muscle weakness predominantly in the proximal lower limbs. While most patients carry a biallelic deletion in the SMN1 gene (localized in chromosome 5q), little is known regarding patients without SMN1 -mutation, and a genetic diagnosis is not always possible. Here, we report a cohort of 24 French patients with non-5q proximal SMA from five neuromuscular centers who all, except two, had next-generation sequencing (NGS) gene panel, followed by whole exome sequencing (WES) if gene panel showed a negative result. The two remaining patients benefited directly from WES or whole genome sequencing (WGS). A total of ten patients with causative variants were identified, nine of whom were index cases (9/23 families = 39%). Eight variants were identified by gene panel: five variants in DYNC1H1 , and three in BICD2 . Compound heterozygous causative variants in ASAH1 were identified directly by WES, and one variant in DYNC1H1 was identified directly by WGS. No causative variant was found using WES in patients with a previous panel with negative results (14 cases). We thus recommend using primarily NGS panels in patients with non-5q-SMA and using WES, especially when several members of the same family are affected and/or when trio analyses are possible, or WGS as second-line testing if available.

Spinal muscular atrophy is an autosomal recessive neuromuscular disease caused by mutations in the multifunctional protein Survival of Motor Neuron, or SMN. Within the nucleus, SMN localizes to Cajal bodies, which are associated with nucleoli, nuclear organelles dedicated to the first steps of ribosome biogenesis. The highly organized structure of the nucleolus can be dynamically altered by genotoxic agents. RNAP1, Fibrillarin, and nucleolar DNA are exported to the periphery of the nucleolus after genotoxic stress and, once DNA repair is fully completed, the organization of the nucleolus is restored. We find that SMN is required for the restoration of the nucleolar structure after genotoxic stress. During DNA repair, SMN shuttles from the Cajal bodies to the nucleolus. This shuttling is important for nucleolar homeostasis and relies on the presence of Coilin and the activity of PRMT1. DNA damage causes a major reorganization of the nucleolus. Here, the authors find that this structural restoration depends on the shuttling of the protein SMN from the Cajal bodies to the nucleolus, which requires coilin and PRMT1.

Wild-type and Mstn-/- tumor-bearing mice have similar caloric intakes, indicating that anorexia is not involved in the development of cancer cachexia.