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Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease., (© 2024 Lee et al.)

Purpose of Review: Congenital myasthenia syndromes (CMS) are treatable, inherited disorders affecting neuromuscular transmission. We highlight that the involvement of an increasing number of proteins is making the understanding of the disease mechanisms and potential treatments progressively more complex., Recent Findings: Although early studies identified mutations of proteins directly involved in synaptic transmission at the neuromuscular junction, recently, next-generation sequencing has facilitated the identification of many novel mutations in genes that encode proteins that have a far wider expression profile, some even ubiquitously expressed, but whose defective function leads to impaired neuromuscular transmission. Unsurprisingly, mutations in these genes often causes a wider phenotypic disease spectrum where defective neuromuscular transmission forms only one component. This has implications for the management of CMS patients., Summary: Given the widening nonneuromuscular junction phenotypes in the newly identified forms of CMS, new therapies need to include disease-modifying approaches that address not only neuromuscular weakness but also the multisystem involvement. Whilst the current treatments for CMS are highly effective for many subtypes there remains, in a proportion of CMS patients, an unmet need for more efficacious therapies., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Erectile dysfunction (ED) incidence is higher in patients with obstructive sleep apnea (OSA). Studies have suggested that ED and OSA may activate similar pathways; however, few have investigated the links between their underlying genotypic profiles. Therefore, we conducted an in-silico analysis to test whether ED and OSA share genetic variants of risk and to identify any molecular, cellular and biological interactions between them. Two gene lists were manually curated through a literature review based on a PUBMED search, which resulted in one gene list associated with ED (total of 205 genes) and the other with OSA (total of 2622 genes). Between those gene sets, 35 were common for both lists (Fisher exact test, p-value = 0.027). The Protein-protein interaction (PPI) analysis using the intersect list as input showed that 3 of them had direct interactions (LPL, DGKB and PLCB1). In addition, the biological function of the genes contained in the intersect list suggested that pathways related to lipid metabolism and the neuromuscular junction were commonly found in the genetic basis of ED and OSA. From the shared genes between both conditions, the biological pathways highlighted in this study may serve as preliminary findings for future functional investigations on OSA and ED association., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10 P80L variant (zebrafish: Chchd10 P83L ). Larval chchd10 P83L/P83L fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ). These deficits were not accompanied by changes in transcripts involved in the integrated stress response (ISR), phenocopying previous findings in our knockout (chchd10 -/- ). Adult, 11-month old chchd10 P83L/P83L zebrafish, displayed smaller slow- and fast-twitch muscle cell cross-sectional areas compared to wild type zebrafish muscle cells. Motoneurons in the spinal cord of chchd10 P83L/P83L zebrafish displayed similar cross-sectional areas to that of wild type motor neurons and significantly fewer motor neurons were observed when compared to chchd2 -/- adult spinal cords. Bulk RNA sequencing using whole spinal cords of 7-month old fish revealed transcriptional changes associated with neuroinflammation, apoptosis, amino acid metabolism and mt-DNA inflammatory response in our chchd10 P83L/P83L model. The findings presented here, suggest that the CHCHD10 P80L variant confers an ALS-like phenotype when expressed in zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future., (© 2024. The Author(s).)

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins. In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth, and cell survival. However, the upstream regulatory mechanisms of Dprs and DIPs are not clear. On the other hand, while transcription factors have been implicated in target recognition, their downstream cell surface proteins remain mostly unknown. We conduct an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We identify huckebein (hkb), a transcription factor previously implicated in target recognition of the dorsal Is motor neuron. We show that hkb genetically interacts with DIP-α and loss of hkb leads to complete removal of DIP-α expression specifically in dorsal Is motor neurons. We then confirm that this specificity is through the dorsal Is motor neuron specific transcription factor, even-skipped (eve), which acts downstream of hkb. Analysis of the genetic interaction between hkb and eve reveals that they act in the same pathway to regulate dorsal Is motor neuron connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons., (© 2024. The Author(s).)

Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative β-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where β-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where β-catenin serves as a transcriptional coactivator in Wnt/β-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/β-catenin signaling that regulate the formation of the embryonic NMJ., (© 2023 International Society for Neurochemistry.)

Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and β2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

Muscle-specific kinase (MuSK) is the key regulator of neuromuscular junction development. MuSK acts via several distinct pathways and is responsible for pre- and postsynaptic differentiation. MuSK is unique among receptor tyrosine kinases as activation and signaling are particularly tightly regulated. Initiation of kinase activity requires Agrin, a heparan sulphate proteoglycan derived from motor neurons, the low-density lipoprotein receptor-related protein-4 (Lrp4) and the intracellular adaptor protein Dok-7. There is a great knowledge gap between MuSK activation and downstream signaling. Recent studies using omics techniques have addressed this knowledge gap, thereby greatly contributing to a better understanding of MuSK signaling. Impaired MuSK signaling causes severe muscle weakness as described in congenital myasthenic syndromes or myasthenia gravis but the underlying pathophysiology is often unclear. This review focuses on recent advances in deciphering MuSK activation and downstream signaling. We further highlight latest break-throughs in understanding and treatment of MuSK-related disorders and discuss the role of MuSK in non-muscle tissue., Competing Interests: Declaration of Competing Interest The authors declare no competing financial or non-financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sarcopenia is common in patients with Parkinson's disease (PD), showing mitochondrial oxidative stress in skeletal muscle. The aggregation of α-synuclein (α-Syn) to induce oxidative stress is a key pathogenic process of PD; nevertheless, we know little about its potential role in regulating peripheral nerves and the function of the muscles they innervate., Methods: To investigate the role of α-Syn aggregation on neuromuscular system, we used the Thy1 promoter to overexpress human α-Syn transgenic mice (mThy1-hSNCA). hα-Syn expression was evaluated by western blot, and its localization was determined by confocal microscopy. The impact of α-Syn aggregation on the structure and function of skeletal muscle mitochondria and neuromuscular junctions (NMJs), as well as muscle mass and function were characterized by flow cytometry, transmission electron microscopy, Seahorse XF24 metabolic assay, and AAV9 in vivo injection. We assessed the regenerative effect of mitochondrial-targeted superoxide dismutase (Mito-TEMPO) after skeletal muscle injury in mThy1-hSNCA mice., Results: Overexpressed hα-Syn protein localized in motor neuron axons and NMJs in muscle and formed aggregates. α-Syn aggregation increased the number of abnormal mitochondrial in the intramuscular axons and NMJs by over 60% (P < 0.01), which inhibited the release of acetylcholine (ACh) from presynaptic vesicles in NMJs (P < 0.05). The expression of genes associated with NMJ activity, neurotransmission and regulation of reactive oxygen species (ROS) metabolic process were significantly decreased in mThy1-hSNCA mice, resulting in ROS production elevated by ~220% (P < 0.05), thereby exacerbating oxidative stress. Such process altered mitochondrial spatial relationships to sarcomeric structures, decreased Z-line spacing by 36% (P < 0.05) and increased myofibre apoptosis by ~10% (P < 0.05). Overexpression of α-Syn altered the metabolic profile of muscle satellite cells (MuSCs), including basal respiratory capacity (~170% reduction) and glycolytic capacity (~150% reduction) (P < 0.05) and decreased cell migration and fusion during muscle regeneration (~60% and ~40%, respectively) (P < 0.05). We demonstrated that Mito-TEMPO treatment could restore the oxidative stress status (the complex I/V protein and enzyme activities increased ~200% and ~150%, respectively), which caused by α-Syn aggregation, and improve the ability of muscle regeneration after injury. In addition, the NMJ receptor fragmentation and ACh secretion were also improved., Conclusions: These results reveal that the α-synuclein aggregation plays an important role in regulating acetylcholine release from neuromuscular junctions and induces intramuscular mitochondrial oxidative stress, which can provide new insights into the aetiology of muscle atrophy in patients with Parkinson's disease., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Canonical Wnt signaling is involved in skeletal muscle cell biology. The exact way in which this pathway exerts its contribution to myogenesis or neuromuscular junctions (NMJ) is a matter of debate. Next to the common co-receptors of canonical Wnt signaling, Lrp5 and Lrp6, the receptor tyrosine kinase MuSK was reported to bind at NMJs WNT glycoproteins by its extracellular cysteine-rich domain. Previously, we reported canonical Wnt signaling being active in fast muscle fiber types. Here, we used conditional Lrp5 or Lrp6 knockout mice to investigate the role of these receptors in muscle cells. Conditional double knockout mice died around E13 likely due to ectopic expression of the Cre recombinase. Phenotypes of single conditional knockout mice point to a very divergent role for the two receptors. First, muscle fiber type distribution and size were changed. Second, canonical Wnt signaling reporter mice suggested less signaling activity in the absence of Lrps. Third, expression of several myogenic marker genes was changed. Fourth, NMJs were of fragmented phenotype. Fifth, recordings revealed impaired neuromuscular transmission. In sum, our data show fundamental differences in absence of each of the Lrp co-receptors and suggest a differentiated view of canonical Wnt signaling pathway involvement in adult skeletal muscle cells.

A goal of modern neuroscience involves understanding how connections in the brain form and function. Such a knowledge is essential to inform how defects in the exquisite complexity of nervous system growth influence neurological disease. Studies of the nervous system in the fruit fly Drosophila melanogaster enabled the discovery of a wealth of molecular and genetic mechanisms underlying development of synapses-the specialized cell-to-cell connections that comprise the essential substrate for information flow and processing in the nervous system. For years, the major driver of knowledge was the neuromuscular junction due to its ease of examination. Analogous studies in the central nervous system lagged due to a lack of genetic accessibility of specific neuron classes, synaptic labels compatible with cell-type-specific access, and high resolution, quantitative imaging strategies. However, understanding how central synapses form remains a prerequisite to understanding brain development. In the last decade, a host of new tools and techniques extended genetic studies of synapse organization into central circuits to enhance our understanding of synapse formation, organization, and maturation. In this review, we consider the current state-of-the-field. We first discuss the tools, technologies, and strategies developed to visualize and quantify synapses in vivo in genetically identifiable neurons of the Drosophila central nervous system. Second, we explore how these tools enabled a clearer understanding of synaptic development and organization in the fly brain and the underlying molecular mechanisms of synapse formation. These studies establish the fly as a powerful in vivo genetic model that offers novel insights into neural development., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Congenital myasthenic syndromes caused by mutations in the COL13A1 gene are very rare and have a phenotype described as severe. We present the first case of congenital myasthenic syndrome described in Algeria and the Maghreb with a new mutation of this gene., Case Presentation: We present an 8-year-old Algerian female patient, who presented with a moderate phenotype with bilateral ptosis that fluctuates during the day and has occurred since birth. During the investigation, and despite the very probable congenital origin, we ruled out other diagnoses that could induce pathology of the neuromuscular junction. The genetic study confirmed our diagnosis suspicion by highlighting a new mutation in the COL13A1 gene., Conclusion: We report a case with a mutation of the Col13A1 gene, reported in the Maghreb (North Africa), and whose phenotype is moderate compared with the majority of cases found in the literature., (© 2022. The Author(s).)

Trk-fused gene (TFG) mutations have been identified in patients with several neurodegenerative diseases. In this study, we attempted to clarify the effects of TFG deletions in motor neurons and in muscle fibers, using tissue-specific TFG knockout (vMNTFG KO and MUSTFG KO) mice. vMNTFG KO, generated by crossing TFG floxed with VAChT-Cre, showed deterioration of motor function and muscle atrophy especially in slow-twitch soleus muscle, in line with the predominant Cre expression in slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) motor neurons. Consistently, denervation of the neuromuscular junction (NMJ) was apparent in the soleus, but not in the extensor digitorum longus, muscle. Muscle TFG expressions were significantly downregulated in vMNTFG KO, presumably due to decreased muscle IGF-1 concentrations. However, interestingly, MUSTFG KO mice showed no apparent impairment of muscle movements, though a denervation marker, AChRγ, was elevated and Agrin-induced AChR clustering in C2C12 myotubes was inhibited. Our results clarify that loss of motor neuron TFG is sufficient for the occurrence of NMJ degeneration and muscle atrophy, though lack of muscle TFG may exert an additional effect. Reduced muscle TFG, also observed in aged mice, might be involved in age-related NMJ degeneration, and this issue merits further study., (© 2022. The Author(s).)

Background: Presynaptic forms of congenital myasthenic syndromes (CMS) due to pathogenic variants in SLC18A3 impairing the synthesis and recycling of acetylcholine (ACh) have recently been described. SLC18A3 encodes the vesicular ACh transporter (VAChT), modulating the active transport of ACh at the neuromuscular junction, and homozygous loss of VAChT leads to lethality., Methods: Exome sequencing (ES) was carried out to identify the molecular genetic cause of the disease in a 5-year-old male patient and histological, immunofluorescence as well as electron- and CARS-microscopic studies were performed to delineate the muscle pathology, which has so far only been studied in VAChT-deficient animal models., Results: ES unraveled compound heterozygous missense and nonsense variants (c.315G>A, p.Trp105* and c.1192G>C, p.Asp398His) in SLC18A3 . Comparison with already-published cases suggests a more severe phenotype including impaired motor and cognitive development, possibly related to a more severe effect of the nonsense variant. Therapy with pyridostigmine was only partially effective while 3,4 diaminopyridine showed no effect. Microscopic investigation of the muscle biopsy revealed reduced fibre size and a significant accumulation of lipid droplets., Conclusions: We suggest that nonsense variants have a more detrimental impact on the clinical manifestation of SLC18A3 -associated CMS. The impact of pathogenic SLC18A3 variants on muscle fibre integrity beyond the effect of denervation is suggested by the build-up of lipid aggregates. This in turn implicates the importance of proper VAChT-mediated synthesis and recycling of ACh for lipid homeostasis in muscle cells. This hypothesis is further supported by the pathological observations obtained in previously published VAChT-animal models.

Synapses are particularly susceptible to the effects of advancing age, and mitochondria have long been implicated as organelles contributing to this compartmental vulnerability. Despite this, the mitochondrial molecular cascades promoting age-dependent synaptic demise remain to be elucidated. Here, we sought to examine how the synaptic mitochondrial proteome (including strongly mitochondrial associated proteins) was dynamically and temporally regulated throughout ageing to determine whether alterations in the expression of individual candidates can influence synaptic stability/morphology. Proteomic profiling of wild-type mouse cortical synaptic and non-synaptic mitochondria across the lifespan revealed significant age-dependent heterogeneity between mitochondrial subpopulations, with aged organelles exhibiting unique protein expression profiles. Recapitulation of aged synaptic mitochondrial protein expression at the Drosophila neuromuscular junction has the propensity to perturb the synaptic architecture, demonstrating that temporal regulation of the mitochondrial proteome may directly modulate the stability of the synapse in vivo.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by depletion of motor neurons (MNs), for which effective medical treatments are still required. Previous transcriptomic analysis revealed the up-regulation of C-X-C motif chemokine receptor 2 (CXCR2)-mRNA in a subset of sporadic ALS patients and SOD1G93A mice. Here, we confirmed the increase of CXCR2 in human ALS cortex, and showed that CXCR2 is mainly localized in cell bodies and axons of cortical neurons. We also investigated the effects of reparixin, an allosteric inhibitor of CXCR2, in degenerating human iPSC-derived MNs and SOD1G93A mice. In vitro, reparixin rescued MNs from apoptotic cell death, preserving neuronal morphology, mitochondrial membrane potential and cytoplasmic membrane integrity, whereas in vivo it improved neuromuscular function of SOD1G93A mice. Altogether, these data suggest a role for CXCR2 in ALS pathology and support its pharmacological inhibition as a candidate therapeutic strategy against ALS at least in a specific subgroup of patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Decreased acetylcholine receptor (AChR) clustering compromises signal transmission at the neuromuscular junction (NMJ) in myasthenia gravis, congenital myasthenic syndromes, and motor neuron diseases. Although the enhancement of AChR clustering at the NMJ is a promising therapeutic strategy for these maladies, no drug is currently available for this enhancement. We previously reported that zonisamide (ZNS), an anti-epileptic and anti-Parkinson's disease drug, enhances neurite elongation of the primary spinal motor neurons (SMNs). As nerve sprouting occurs to compensate for the loss of AChR clusters in human diseases, we examined the effects of ZNS on AChR clustering at the NMJ. To this end, we established a simple and quick co-culture system to reproducibly make in vitro NMJs using C2C12 myotubes and NSC34 motor neurons. ZNS at 1-20 μM enhanced the formation of AChR clusters dose-dependently in co-cultured C2C12 myotubes but not in agrin-treated single cultured C2C12 myotubes. We observed that molecules that conferred responsiveness to ZNS were not secreted into the co-culture medium. We found that 10 μM ZNS upregulated the expression of neuregulin-1 (Nrg1) in co-cultured cells but not in single cultured C2C12 myotubes or single cultured NSC34 motor neurons. In accordance with this observation, inhibition of the Nrg1/ErbB signaling pathways nullified the effect of 10 μM ZNS on the enhancement of AChR clustering in in vitro NMJs. Although agrin was not induced by 10 μM ZNS in co-cultured cells, anti-agrin antibody attenuated ZNS-mediated enhancement of AChR clustering. We conclude that ZNS enhances agrin-dependent AChR-clustering by upregulating the Nrg1/ErbB signaling pathways in the presence of NMJs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Motoneurons (MNs) control muscle activity by releasing the neurotransmitter acetylcholine (ACh) at the level of neuromuscular junctions. ACh is packaged into synaptic vesicles by the vesicular ACh transporter (VAChT), and disruptions in its release can impair muscle contraction, as seen in congenital myasthenic syndromes (CMS). Recently, VAChT gene mutations were identified in humans displaying varying degrees of myasthenia. Moreover, mice with a global deficiency in VAChT expression display several characteristics of CMS. Despite these findings, little is known about how a long-term decrease in VAChT expression in vivo affects MNs structure and function. Using Cre-loxP technology, we generated a mouse model where VAChT is deleted in select groups of MNs (mnVAChT-KD). Molecular analysis revealed that the VAChT deletion was specific to MNs and affected approximately 50% of its population in the brainstem and spinal cord, with alpha-MNs primarily targeted (70% in spinal cord). Within each animal, the cell body area of VAChT-deleted MNs was significantly smaller compared to MNs with VAChT preserved. Likewise, muscles innervated by VAChT-deleted MNs showed atrophy while muscles innervated by VAChT-containing neurons appeared normal. In addition, mnVAChT KD mice had decreased muscle strength, were hypoactive, leaner and exhibited kyphosis. This neuromuscular dysfunction was evident at 2 months of age and became progressively worse by 6 months. Treatment of mutants with a cholinesterase inhibitor was able to improve some of the motor deficits. As these observations mimic what is seen in CMS, this new line could be valuable for assessing the efficacy of potential CMS drugs., (© 2021 Federation of European Biochemical Societies.)

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca 2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis., Competing Interests: AG, EC, AG, NC, MA, VR, EG, CJ, CS, LL, CS, FJ, VJ, JT, ML, JC, JG, LS, VG No competing interests declared, (© 2021, Ghasemizadeh et al.)

Introduction/aims: We studied a patient with a congenital myasthenic syndrome (CMS) caused by a dominant mutation in the synaptotagmin 2 gene (SYT2) and compared the clinical features of this patient with those of a previously described patient with a recessive mutation in the same gene., Methods: We performed electrodiagnostic (EDX) studies, genetic studies, muscle biopsy, microelectrode recordings and electron microscopy (EM)., Results: Both patients presented with muscle weakness and bulbar deficits, which were worse in the recessive form. EDX studies showed presynaptic failure, which was more prominent in the recessive form. Microelectrode studies in the dominant form showed a marked reduction of the quantal content, which increased linearly with higher frequencies of nerve stimulation. The MEPP frequencies were normal at rest but increased markedly with higher frequencies of nerve stimulation. The EM demonstrated overdeveloped postsynaptic folding, and abundant endosomes, multivesicular bodies and degenerative lamellar bodies inside small nerve terminals., Discussion: The recessive form of CMS caused by a SYT2 mutation showed far more severe clinical manifestations than the dominant form. The pathogenesis of the dominant form likely involves a dominant-negative effect due to disruption of the dual function of synaptotagmin as a Ca 2+ -sensor and modulator of synaptic vesicle exocytosis., (© 2021 Wiley Periodicals LLC.)

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Introduction/aims: Terminal Schwann cells (tSCs) are nonmyelinating Schwann cells present at the neuromuscular junction (NMJ) with multiple integral roles throughout their lifespan. There is no known gene differentiating tSCs from myelinating Schwann cells, making their isolation and investigation challenging. In this work we investigated genes expressed within tSCs., Methods: A novel dissection technique was utilized to isolate the tSC-containing NMJ band from the sternomastoid muscles of S100-GFP mice. RNA was isolated from samples containing: (a) NMJ bands (tSCs with nerve and muscle), (b) nerve, and (c) muscle, and microarray genetic expression analysis was conducted. Data were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescent staining. To identify genes specific to tSCs compared with other NMJ components, analysis of variance and rank-order analysis were performed using the Partek Genomic Suite., Results: Microarray analysis of the tSC-enriched NMJ band revealed upregulation (by 4- to 12-fold) of several genes unique to the NMJ compared with muscle or nerve parts alone (P < .05). Among these genes, Tbx21 (or T-bet) was identified, which showed a 12-fold higher expression at the NMJ compared with sciatic nerve (P < .002). qRT-PCR analysis showed Tbx21 mRNA expression was over ninefold higher (P < .05) in the NMJ relative to muscle and nerve. Tbx21 protein colocalized with tSCs and was not noted in myelinating SCs from sciatic nerve., Discussion: We found TBX21 to be expressed in tSCs. Additional studies will be performed to determine the functional significance of TBX21 in tSCs. These studies may enhance the investigative tools available to modulate tSCs to improve motor recovery after nerve injury., (© 2021 Wiley Periodicals LLC.)

Aberrantly expressed fused in sarcoma (FUS) is a hallmark of FUS-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Wildtype FUS localises to synapses and interacts with mitochondrial proteins while mutations have been shown to cause to pathological changes affecting mitochondria, synapses and the neuromuscular junction (NMJ). This indicates a crucial physiological role for FUS in regulating synaptic and mitochondrial function that is currently poorly understood. In this paper we provide evidence that mislocalised cytoplasmic FUS causes mitochondrial and synaptic changes and that FUS plays a vital role in maintaining neuronal health in vitro and in vivo. Overexpressing mutant FUS altered synaptic numbers and neuronal complexity in both primary neurons and zebrafish models. The degree to which FUS was mislocalised led to differences in the synaptic changes which was mirrored by changes in mitochondrial numbers and transport. Furthermore, we showed that FUS co-localises with the mitochondrial tethering protein Syntaphilin (SNPH), and that mutations in FUS affect this relationship. Finally, we demonstrated mutant FUS led to changes in global protein translation. This localisation between FUS and SNPH could explain the synaptic and mitochondrial defects observed leading to global protein translation defects. Importantly, our results support the 'gain-of-function' hypothesis for disease pathogenesis in FUS-related ALS.

During the development of the nervous system, synaptogenesis occurs in excess though only the appropriate connections consolidate. At the neuromuscular junction, competition between several motor nerve terminals results in the maturation of a single axon and the elimination of the others. The activity-dependent release of transmitter, cotransmitters, and neurotrophic factors allows the direct mutual influence between motor axon terminals through receptors such as presynaptic muscarinic ACh autoreceptors and the tropomyosin-related kinase B neurotrophin receptor. In previous studies, we investigated the synergistic and antagonistic relations between these receptors and their downstream coupling to PKA and PKC pathways and observed a metabotropic receptor-driven balance between PKA (stabilizes multinnervation) and PKC (promotes developmental axonal loss). However, how much does each kinase contribute in the developmental synapse elimination process? A detailed statistical analysis of the differences between the PKA and PKC effects in the synapse elimination could help to explore this point. The present short communication provides this analysis and results show that a similar level of PKA inhibition and PKC potentiation would be required during development to promote synapse loss.

Mutations in the factor-induced-gene 4 (FIG 4) gene are associated with multiple disorders, including Charcot-Marie-Tooth disease (CMT), epilepsy with polymicrogyria, Yunis-Varón syndrome and amyotrophic lateral sclerosis. The wide spectrum of disorders associated with FIG 4 may be related to the dysregulated epigenetics. Using Gene Expression Omnibus, we found that HDAC1 binds to the FIG 4 gene locus in the genome of human CD4+ T cells. Rpd3 is a well-known Drosophila homolog of human HDAC1. We previously established Drosophila models targeting Drosophila FIG 4 (dFIG 4) that exhibited defective locomotive ability, abnormal synapse morphology at neuromuscular junctions, enlarged vacuoles in the fat body and aberrant compound eye morphology. Genetic crossing experiments followed by physiological and immunocytochemical analyses revealed that Rpd3 mutations suppressed these defects induced by dFIG 4 knockdown. This demonstrated Rpd3 to be an important epigenetic regulator of dFIG 4, suggesting that the inhibition of HDAC1 represses the pathogenesis of FIG 4-associated disorders, including CMT. Defects in epigenetic regulators, such as HDAC1, may also explain the diverse symptoms of FIG 4-associated disorders., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. Here we find that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, our results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, we find that Caper interacts with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, we show that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues-very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each other's structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; we surveyed multiple muscles for this reason. Here, we identified a cell-specific promoter that allows ablation of 1s MNs postinnervation and measured structural and functional responses of convergent 1b NMJs using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, we genetically blocked 1s innervation in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations. SIGNIFICANCE STATEMENT In complex neural circuits, multiple convergent inputs contribute to the activity of the target cell, but whether synaptic plasticity exists among these inputs has not been thoroughly explored. In this study, we examined synaptic plasticity in the structurally and functionally tractable Drosophila larval neuromuscular system. In this convergent circuit, each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase neuromuscular junction outgrowth and functional output. However, this is not a general feature as each motor neuron differentially compensates. Further, robust compensation requires initial coinnervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and disease states., (Copyright © 2021 the authors.)

The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. However, the mechanisms balancing synaptic GluR subtypes remain unclear. Herein, we show that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other, as shown by super-resolution microscopy. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtype B decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, we performed an RNA interference screen of candidate genes and found that postsynaptic-specific knockdown of dunce , which encodes cAMP phosphodiesterase, increased levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. Our findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Caveolae are the cholesterol-rich small invaginations of the plasma membrane present in many cell types including adipocytes, endothelial cells, epithelial cells, fibroblasts, smooth muscles, skeletal muscles and cardiac muscles. They serve as specialized platforms for many signaling molecules and regulate important cellular processes like energy metabolism, lipid metabolism, mitochondria homeostasis, and mechano-transduction. Caveolae can be internalized together with associated cargo. The caveolae-dependent endocytic pathway plays a role in the withdrawal of many plasma membrane components that can be sent for degradation or recycled back to the cell surface. Caveolae are formed by oligomerization of caveolin proteins. Caveolin-3 is a muscle-specific isoform, whose malfunction is associated with several diseases including diabetes, cancer, atherosclerosis, and cardiovascular diseases. Mutations in Caveolin-3 are known to cause muscular dystrophies that are collectively called caveolinopathies. Altered expression of Caveolin-3 is also observed in Duchenne's muscular dystrophy, which is likely a part of the pathological process leading to muscle weakness. This review summarizes the major functions of Caveolin-3 in skeletal muscles and discusses its involvement in the pathology of muscular dystrophies.

Hereditary spastic paraplegias (HSPs) are genetic neurodegenerative diseases. HSPs are characterized by lower-extremity weakness and spasticity. However, there is no specific clinical treatment strategy to prevent or reverse nerve degeneration in HSPs. Mutations in receptor expression-enhancing protein 1 (REEP1) are well-recognized and relatively common causes of autosomal dominant HSPs. REEP1 modifies the endoplasmic reticulum (ER) shape, and is implicated in the ER stress response. Defects in the ER stress response seem to be crucial mechanisms underlying HSP neurodegeneration. Here, we report that REEP1 -/- mice exhibit progressive motor deficits, along with denervation of neuromuscular junctions and increased ER stress. Moreover, marked axonal degeneration and morphological abnormalities are observed. In this study, we treated both REEP1 -/- and wild-type (WT) mice with salubrinal, which is a specific inhibitor of ER stress, and we observed increased nerve-muscle connections and enhanced motor functions. Our data highlight the importance of ER homeostasis in HSPs, providing new opportunities for HSP treatment., Competing Interests: Competing interestsAll authors declare no actual or potential conflicts of interest, including any financial, personal or other relationships, with other people or organizations within three years of beginning the work submitted that could inappropriately influence (bias) their work., (© 2020. Published by The Company of Biologists Ltd.)

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival motor neuron (SMN1) gene. An important hallmark of disease progression is the pathology of neuromuscular junctions (NMJs). Affected NMJs in the SMA context exhibit delayed maturation, impaired synaptic transmission, and loss of contact between motor neurons and skeletal muscle. Protection and maintenance of NMJs remains a focal point of therapeutic strategies to treat SMA, and the recent implication of the NMJ-organizer Agrin in SMA pathology suggests additional NMJ organizing molecules may contribute. DOK7 is an NMJ organizer that functions downstream of Agrin. The potential of DOK7 as a putative therapeutic target was demonstrated by adeno-associated virus (AAV)-mediated gene therapy delivery of DOK7 in Amyotrophic Lateral Sclerosis (ALS) and Emery Dreyefuss Muscular Dystrophy (EDMD). To assess the potential of DOK7 as a disease modifier of SMA, we administered AAV-DOK7 to an intermediate mouse model of SMA. AAV9-DOK7 treatment conferred improvements in NMJ architecture and reduced muscle fiber atrophy. Additionally, these improvements resulted in a subtle reduction in phenotypic severity, evidenced by improved grip strength and an extension in survival. These findings reveal DOK7 is a novel modifier of SMA., Competing Interests: Declaration of competing interest C.L.L. is the co-founder and Chief Scientific Officer of Shift Pharmaceuticals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Exercise perturbs homeostasis, alters the levels of circulating mediators and hormones, and increases the demand by skeletal muscles and other vital organs for energy substrates. Exercise also affects bone and mineral metabolism, particularly calcium and phosphate, both of which are essential for muscle contraction, neuromuscular signaling, biosynthesis of adenosine triphosphate (ATP), and other energy substrates. Parathyroid hormone (PTH) is involved in the regulation of calcium and phosphate homeostasis. Understanding the effects of exercise on PTH secretion is fundamental for appreciating how the body adapts to exercise. Altered PTH metabolism underlies hyperparathyroidism and hypoparathyroidism, the complications of which affect the organs involved in calcium and phosphorous metabolism (bone and kidney) and other body systems as well. Exercise affects PTH expression and secretion by altering the circulating levels of calcium and phosphate. In turn, PTH responds directly to exercise and exercise-induced myokines. Here, we review the main concepts of the regulation of PTH expression and secretion under physiological conditions, in acute and chronic exercise, and in relation to PTH-related disorders.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: Doxorubicin (DOX) is a highly effective antitumor agent widely used in cancer treatment. However, it is well established that DOX induces muscular atrophy and impairs force production. Although no therapeutic interventions exist to combat DOX-induced muscle weakness, endurance exercise training has been shown to reduce skeletal muscle damage caused by DOX administration. Numerous studies have attempted to identify molecular mechanisms responsible for exercise-induced protection against DOX myotoxicity. Nevertheless, the mechanisms by which endurance exercise protects against DOX-induced muscle weakness remain elusive. In this regard, impairments to the neuromuscular junction (NMJ) are associated with muscle wasting, and studies indicate that physical exercise can rescue NMJ fragmentation. Therefore, we tested the hypothesis that exercise protects against DOX-induced myopathy by preventing detrimental changes to key proteins responsible for maintenance of the NMJ., Methods: Female Sprague-Dawley rats were assigned to sedentary or exercise-trained groups. Exercise training consisted of a 5-d treadmill habituation period followed by 10 d of running (60 min·d, 30 m·min, 0% grade). After the last training bout, exercise-trained and sedentary animals were paired with either placebo (saline) or DOX (20 mg·kg i.p.) treatment. Two days after drug treatment, the soleus muscle was excised for subsequent analyses., Results: Our results indicate that endurance exercise training prevents soleus muscle atrophy and contractile dysfunction in DOX-treated animals. These adaptations were associated with the increased expression of the following neurotrophic factors: brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, nerve growth factor, and neurotrophin-3. In addition, exercise enhanced the expression of receptor-associated protein of the synapse and the acetylcholine receptor (AChR) subunits AChRβ, AChRδ, and AChRγ in DOX-treated animals., Conclusion: Therefore, upregulating neurotrophic factor and NMJ protein expression may be an effective strategy to prevent DOX-induced skeletal muscle dysfunction.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1 G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dscam2 is a cell surface protein required for neuronal development in Drosophila; it can promote neural wiring through homophilic recognition that leads to either adhesion or repulsion between neurites. Here, we report that Dscam2 also plays a post-developmental role in suppressing synaptic strength. This function is dependent on one of two distinct extracellular isoforms of the protein and is autonomous to motor neurons. We link the PI3K enhancer, Centaurin gamma 1A, to the Dscam2-dependent regulation of synaptic strength and show that changes in phosphoinositide levels correlate with changes in endosomal compartments that have previously been associated with synaptic strength. Using transmission electron microscopy, we find an increase in synaptic vesicles at Dscam2 mutant active zones, providing a rationale for the increase in synaptic strength. Our study provides the first evidence that Dscam2 can regulate synaptic physiology and highlights how diverse roles of alternative protein isoforms can contribute to unique aspects of brain development and function., (© 2020 Odierna et al.)

Aim: The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered., Main Methods: We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups., Key Findings: Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1., Significance: These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression., Competing Interests: Declaration of competing interest The authors have no competing interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin-binding domain. Another, c.4744G>A - at the last nucleotide of exon 26 - caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias., (© 2020 Titlow et al.)

Regulation of post-synaptic receptors plays an important role in determining synaptic strength and plasticity. The Drosophila larval neuromuscular junction (nmj) has been used extensively as a model to understand some of these processes. In this context, we are interested in the role of Drosophila Monensin sensitivity protein 1 (DMon1) in regulating glutamate receptor (GluRIIA) levels at the nmj. DMon1 is an evolutionarily conserved protein which, in complex with calcium caffeine zinc sensitivity1 (CCZ1), regulates the conversion of early endosomes to late endosomes through recruitment of Rab7. C-terminal deletion mutants of Dmon1 (Dmon1 Δ181 ) exhibit lethality. The escapers have a short life span and exhibit severe motor defects. At the nmj, these mutants show defects in synaptic morphology and a strong increase in GluRIIA levels. The mechanism by which Dmon1 regulates GluRIIA is unclear. In this study, we have characterized an EMS mutant referred to as pog 1 and demonstrate it to be an allele of Dmon1. Further, we have examined the role of rab7 in regulating GluRIIA. We show that similar to Dmon1, knock-down of rab7 using RNAi in neurons, but not muscles, leads to an increase in GluRIIA. Loss of one copy each of Dmon1 and rab7 leads to a synergistic increase in receptor expression. Further, overexpression of an activated Rab7 can rescue the GluRIIA phenotype observed in Dmon1 Δ181 mutants. Together, these results highlight a neuronal role for Rab7 in GluRIIA regulation and underscore the importance of the endo-lysosomal pathway in this process.