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Neural conditioning to scenarios of muscle disuse is undoubtedly a cause of functional decrements that typically exceed losses of muscle size. Yet establishing the relative contribution of neural adaptation and the specific location in the motor pathway remains technically challenging. Several studies of healthy humans have targeted this system and have established that motor unit firing rate is suppressed following disuse, with a number of critical caveats. It is suppressed in the immobilized limb only, at relative and absolute force levels, and preferentially targets lower‐threshold motor units. Concomitantly, electrophysiological investigation of neuromuscular junction transmission (NMJ) stability of lower‐threshold motor units reveals minimal change following disuse. These findings contrast with numerous other methods, which show clear involvement of the NMJ but are unable to characterize the motor unit to which they belong. It is physiologically plausible that decrements observed following disuse are a result of suppressed firing rate of lower‐threshold motor units and impairment of transmission of the NMJ of higher‐threshold motor units. As such, motor units within the pool should be viewed in light of their varying susceptibility to disuse. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Botulinum neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action would significantly ameliorate its pharmacological effects with beneficial outcomes for clinical use.Here, we combined BoNT/A with two fast‐acting inhibitors of excitation–contraction coupling inhibitors (ECCI), either the μ‐conotoxin CnIIIC or dantrolene, and tested the effect of their co‐injection on a model of hind‐limb paralysis in rodents using behavioural, biochemical, imaging and electrophysiological assays.The BoNT/A‐ECCI combinations accelerated the onset of muscle relaxation. Surprisingly, they also potentiated the peak effect and extended the duration of the three BoNT/A commercial preparations OnabotulinumtoxinA, AbobotulinumtoxinA and IncobotulinumtoxinA. ECCI co‐injection increased the number of BoNT/A molecules entering motoneuron terminals, which induced a faster and greater cleavage of SNAP‐25 during the onset and peak phases, and prolonged the attenuation of nerve‐muscle neurotransmission during the recovery phase. We estimate that ECCI co‐injection yields a threefold potentiation in BoNT/A pharmacological activity.Overall, our results show that the pharmacological activity of BoNT/A can be combined and synergized with other bioactive molecules and uncover a novel strategy to enhance the neuromuscular effects of BoNT/A without altering the neurotoxin moiety or intrinsic activity, thus maintaining its exceptional safety profile. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Females with generalized myasthenia gravis (gMG) report lower quality of life (QoL) and have more severe disease than males. Sex differences in disease characteristics exist, however whether there are sex differences in the treatment of gMG that may contribute to QoL disparities is unknown. Our objective is to determine whether there are sex differences in the treatment of gMG. Methods: We performed a single‐center retrospective study of people diagnosed with gMG at the University of Calgary between 1997 and 2021. Primary outcome was proportion starting treatment and secondary outcome was time from diagnosis to treatment initiation. Treatments included pyridostigmine, prednisone, steroid sparing therapies (azathioprine, mycophenolate mofetil [MMF], methotrexate [MTX], or tacrolimus), intravenous immunoglobulin (IVIg), plasmapheresis, rituximab, eculizumab, cyclosporine, stem cell transplantation, and thymectomy. Multivariable logistic and Cox proportional hazards regression models were used to examine treatment associations with sex, adjusted for time from onset to diagnosis, age at diagnosis, presence of thymoma, and antibody status. Results: A total of 179 people with gMG were included (41.9% female). Odds of starting treatment were not statistically associated with sex after adjustment for confounders and correction for multiple testing. Results of the secondary analysis using time to treatment initiation as the outcome were similar. Discussion: We found no sex differences in odds of starting treatment or time to treatment initiation that might explain previously observed sex‐based differences in QoL. Future work should capture physician and patient treatment preferences that may influence disease management. [ABSTRACT FROM AUTHOR]

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin‐B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3–6 months) were bred for EC‐specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non‐injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post‐injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s–1, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. Key points: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult.To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle.Capillary regeneration was reduced at 5 days post‐injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups.Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function. [ABSTRACT FROM AUTHOR]

Aims Methods Results Conclusion Age‐associated muscle loss, termed sarcopenia is the major cause of physical disability in patients with congestive heart failure (CHF). Angiotensin‐converting enzyme inhibitors (ACEi) are commonly used to treat CHF patients; however, their impacts on the neuromuscular junction (NMJ) and sarcopenia in CHF patients remain poorly understood. We aim to investigate the potential impact of ACEi on NMJ and CHF‐induced sarcopenia.The cardiac function, short physical performance battery, handgrip strength (HGS), appendicular skeletal mass index, gait speed (GS) and plasma c‐terminal agrin fragment‐22 (CAF22), a marker of NMJ degradation, were assessed in controls (n = 81) and CHF patients treated with (n = 134) or without (n = 145) ACEi.Irrespective of treatment, HGS and GS, indicators of sarcopenia, were profoundly declined in the patients with CHF vs. controls. However, patients on ACEi demonstrated significantly better HGS and GS compared to non‐ACEi patients (P < .001). The level of CAF22 was significantly lower (P < .0001) in the ACEi‐treated compared to non‐ACEi CHF patients. Further, the level of CAF22 was inversely correlated (R2 = .33, P < .0001) with HGS in both ACEi and non‐ACEi CHF patients, while CAF22 was inversely correlated with GS and short physical performance battery only in ACEi‐treated but not in patients on other therapies without ACEi. The receiver operating characteristic curve analysis revealed CAF22 as a potential diagnostic marker (95% confidence interval: 0.785–0.883; P < .0001) for CHF.Collectively, these findings strongly suggest that ACEi limits CHF‐induced neuromuscular disjunction and physical disability in CHF. CAF22 has shown diagnostic implications for CHF. [ABSTRACT FROM AUTHOR]

Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut‐released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long‐term effects of UnAG in age‐associated muscle atrophy and contractile dysfunction in mice. Four‐month‐old and 18‐month‐old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%–30% with age, which was partially protected against by UnAG. Specific force, force per cross‐sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA‐seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia. [ABSTRACT FROM AUTHOR]

Tetanus neurotoxin (TeNT) causes spastic paralysis by inhibiting neurotransmission in spinal inhibitory interneurons. TeNT binds to the neuromuscular junction, leading to its internalisation into motor neurons and subsequent transcytosis into interneurons. While the extracellular matrix proteins nidogens are essential for TeNT binding, the molecular composition of its receptor complex remains unclear. Here, we show that the receptor-type protein tyrosine phosphatases LAR and PTPRδ interact with the nidogen-TeNT complex, enabling its neuronal uptake. Binding of LAR and PTPRδ to the toxin complex is mediated by their immunoglobulin and fibronectin III domains, which we harnessed to inhibit TeNT entry into motor neurons and protect mice from TeNT-induced paralysis. This function of LAR is independent of its role in regulating TrkB receptor activity, which augments axonal transport of TeNT. These findings reveal a multi-subunit receptor complex for TeNT and demonstrate a novel trafficking route for extracellular matrix proteins. Our study offers potential new avenues for developing therapeutics to prevent tetanus and dissecting the mechanisms controlling the targeting of physiological ligands to long-distance axonal transport in the nervous system. Synopsis: Tetanus neurotoxin (TeNT) causes long-lasting spastic paralysis by blocking neurotransmission in the spinal cord and is a major health burden in developing countries. This work shows that the receptor-type protein tyrosine phosphatases LAR and PTPRδ act as receptors of TeNT, thereby mediating its uptake and delivery into the nervous system. LAR and PTPRδ bind to the extracellular matrix proteins nidogens, which function as TeNT co-receptors. Binding of LAR and PTPRδ to the nidogen-TeNT complex is mediated by their immunoglobulin and fibronectin III domains. These domains can be leveraged to inhibit TeNT entry into motor neurons in culture and protect mice from TeNT-induced paralysis. The function of LAR in neuronal uptake of the nidogen-TeNT complex is independent of its role in regulating TrkB receptor activity. LAR and PTPRδ mediate tetanus toxin uptake and delivery into the nervous system. [ABSTRACT FROM AUTHOR]

In order to understand abnormal gait, this article will first review normal gait, discuss how neuromuscular diseases disturb gait patterns and review orthotic interventions. In normal gait, concentric contractions accelerate and eccentric contractions decelerate the limb. Neuromuscular gait disorders can be grouped into (1) proximal weakness, (2) distal weakness, (3) nonlength‐dependent or generalized weakness, (4) asymmetric weakness, and (5) sensory disorders. Identification of gait disturbance type in neuromuscular diseases leads to the appropriate orthotic prescription since orthotic strategies are grouped into (1) proximal weakness, (2) distal weakness, and (3) sensory disturbances. Orthotics is not indicated in all types of gait disturbance. Weakness in proximal hip musculature can be managed with gait aids such as walkers. In contrast, distal muscle weakness can be managed with orthotics. Preservation of gait assists in maintenance of daily function and integration in society. [ABSTRACT FROM AUTHOR]

Background and Purpose Methods Results Conclusions Acetylcholine receptor antibody (AChR‐Ab) detection is crucial in myasthenia gravis (MG) diagnosis and, currently, the radioimmunoassay (RIA) is the gold standard. However, RIA may detect AChR‐Ab against nonpathogenic intracellular epitopes. In this study, we performed fixed cell‐based assay (F‐CBA) in RIA‐AChR‐Ab positive subjects without MG symptoms, to assess whether F‐CBA could show a higher specificity compared to RIA in detecting pathogenic Abs.We reviewed medical records of patients referred to our MG outpatient clinic because of RIA‐AChR‐Ab detection. MG diagnosis was based on clinical examination, electrophysiology and Ab detection. AChR‐Abs were tested by RIA in the whole cohort. Serum samples from RIA‐positive asymptomatic subjects were retested by F‐CBA.Of 605 subjects who tested RIA‐AChR‐Ab positive, MG diagnosis was confirmed in 599. Six subjects were RIA‐AChR‐Ab positive although they had never had MG symptoms; in four of these subjects AChR‐Abs were not detected by F‐CBA, whereas the remaining two (both non‐MG thymoma cases) were positive also by F‐CBA.RIA false positivity for AChR‐Ab is very rare. Previous literature has demonstrated that F‐CBA has higher sensitivity than RIA for MG, especially in ocular cases. Our preliminary results show that, in rare instances, F‐CBA may be more specific than RIA for MG diagnosis. [ABSTRACT FROM AUTHOR]

Myofibres serve as the functional unit for locomotion, with the sarcomere as fundamental subunit. Running the entire length of this structure are hundreds of myonuclei, located at the periphery of the myofibre, juxtaposed to the plasma membrane. Myonuclear specialisation and clustering at the centre and ends of the fibre are known to be essential for muscle contraction, yet the molecular basis of this regionalisation has remained unclear. While the 'myonuclear domain hypothesis' helped explain how myonuclei can independently govern large cytoplasmic territories, novel technologies have provided granularity on the diverse transcriptional programs running simultaneously within the syncytia and added a new perspective on how myonuclei communicate. Building upon this, we explore the critical cellular and molecular sources of transcriptional and functional heterogeneity within myofibres, discussing the impact of intrinsic and extrinsic factors on myonuclear programs. This knowledge provides new insights for understanding muscle development, repair, and disease, but also opens avenues for the development of novel and precise therapeutic approaches. [ABSTRACT FROM AUTHOR]

Neuromuscular junction (NMJ) degeneration is one of pathological factors of sarcopenia. Low‐magnitude high‐frequency vibration (LMHFV) was reported effective in alleviating the sarcopenia progress. However, no previous study has investigated treatment effects of LMHFV targeting NMJ degeneration in sarcopenia. We first compared morphological differences of NMJ between sarcopenic and non‐sarcopenic subjects, as well as young and old C57BL/6 mice. We then systematically characterized the age‐related degeneration of NMJ in SAMP8 against its control strain, SAMR1 mice, from 3 to 12 months old. We also investigated effects of LMHFV in SAMP8 on the maintenance of NMJ during the onset of sarcopenia with respect to the Agrin‐LRP4‐MuSK‐Dok7 pathway and investigated the mechanism related to ERK1/2 signaling. We observed sarcopenic/old NMJ presented increased acetylcholine receptors (AChRs) cluster fragmentation and discontinuity than non‐sarcopenic/young NMJ. In SAMP8, NMJ degeneration (morphologically at 6 months and functionally at 8 months) was observed associated with the sarcopenia onset (10 months). SAMR1 showed improved NMJ morphology and function compared with SAMP8 at 10 months. Skeletal muscle performance was improved at Month 4 post‐LMHFV treatment. Vibration group presented improved NMJ function at Months 2 and 6 posttreatment, accompanied with alleviated morphological degeneration at Month 4 posttreatment. LMHFV increased Dok7 expression at Month 4 posttreatment. In vitro, LMHFV could promote AChRs clustering in myotubes by increasing Dok7 expression through suppressing ERK1/2 phosphorylation. In conclusion, NMJ degeneration was observed associated with the sarcopenia onset in SAMP8. LMHFV may attenuate NMJ degeneration and sarcopenia progression by increasing Dok7 expression through suppressing ERK1/2 phosphorylation. [ABSTRACT FROM AUTHOR]

The goal of this report is to explore how K2P channels modulate axonal excitability by using the crayfish ventral superficial flexor preparation. This preparation allows for simultaneous recording of motor nerve extracellular action potentials (eAP) and intracellular excitatory junctional potential (EJP) from a muscle fiber. Previous pharmacological studies have demonstrated the presence of K2P‐like channels in crayfish. Fluoxetine (50 µM) was used to block K2P channels in this study. The blocker caused a gradual decline, and eventually complete block, of motor axon action potentials. At an intermediate stage of the block, when the peak‐to‐peak amplitude of eAP decreased to ∼60%–80% of the control value, the amplitude of the initial positive component of eAP declined at a faster rate than that of the negative peak representing sodium influx. Furthermore, the second positive peak following this sodium influx, which corresponds to the after‐hyperpolarizing phase of intracellularly recorded action potentials (iAP), became larger during the intermediate stage of eAP block. Finally, EJP recorded simultaneously with eAP showed no change in amplitude, but did show a significant increase in synaptic delay. These changes in eAP shape and EJP delay are interpreted as the consequence of depolarized resting membrane potential after K2P channel block. In addition to providing insights to possible functions of K2P channels in unmyelinated axons, results presented here also serve as an example of how changes in eAP shape contain information that can be used to infer alterations in intracellular events. This type of eAP‐iAP cross‐inference is valuable for gaining mechanistic insights here and may also be applicable to other model systems. [ABSTRACT FROM AUTHOR]

Muscle‐specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter‐patient and inter‐muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by μ‐Conotoxin‐GIIIB, a blocker of skeletal muscle NaV1.4 voltage‐gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co‐expression of (μ‐Conotoxin‐GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno‐compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, μ‐Conotoxin‐GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High‐ and intermediate‐dosed mice showed clear, progressive myasthenia, not seen in low‐dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low‐dosed mouse diaphragms showed a much higher degree of μ‐Conotoxin‐GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed μ‐Conotoxin‐GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect. [ABSTRACT FROM AUTHOR]

Key points Mitochondrial dysfunctions are thought to contribute to muscle atrophy and weakness that develop during ageing and mechanical unloading caused by immobilization, bed rest and microgravity. Older adults are at greater risk of developing muscle and mitochondrial dysfunctions in response to unloading. Although exercise is well known to promote muscle and mitochondrial health, its protective effect during mechanical unloading in older adults remains largely unexplored. Here, we investigated the impact of 14 days of head‐down tilt bed rest (HDBR) with and without a multimodal exercise countermeasure in older men and women (55–65 years). Leg muscle volume was assessed using magnetic resonance imaging. Biopsies of the vastus lateralis were performed to assess markers of mitochondrial content, respiration, reactive oxygen species (ROS) production and calcium retention capacity (mCRC). Indices of mitochondrial quality control (MQC), including markers of fusion (MFN1 and 2), fission (Drp1), mitophagy (Parkin) and autophagy (p62 and LC3I and II) were measured using immunoblots. Muscle cross‐sections were stained for neural cell adhesion molecule (NCAM, a marker of denervation). HDBR triggered muscle atrophy, decreased mitochondrial content and respiration and increased mitochondrial ROS production. HDBR had no impact on mCRC or MQC markers but increased markers of autophagy and denervation. Exercise prevented the deleterious effects of HDBR on leg muscle volume, mitochondrial ROS production and markers of autophagy and denervation. Exercise also increased mitochondrial content and respiration without altering mCRC and MQC markers. Collectively, our results indicate that an exercise countermeasure that can be performed in bed is effective in protecting muscle and mitochondrial health during HDBR in older adults. Conditions associated with muscle unloading, such as immobilization, bed rest or microgravity, result in muscle atrophy and weakness, particularly in older adults. Mitochondrial dysfunctions are thought to contribute to muscle atrophy caused by unloading and ageing. However, whether exercise can counteract the deleterious effects of unloading in older adults remains largely unexplored. Here, we report that older adults exposed to 14 days of head‐down tilt bed rest (HDBR) displayed upper leg muscle atrophy, a decrease in mitochondrial content and respiration, an increase in H2O2 emission, and an increase in autophagy and denervation markers. No impact of HDBR on mitochondrial quality control was observed. A multimodal exercise countermeasure prevented the deleterious effects of HDBR on upper leg muscle volume, mitochondrial reactive oxygen species emission, and markers of autophagy and denervation and increased mitochondrial content and respiration. These findings highlight the effectiveness of exercise in promoting muscle and mitochondrial health in older adults undergoing bed rest. [ABSTRACT FROM AUTHOR]

With aging skeletal muscle fibers undergo repeating cycles of denervation and reinnervation. In approximately the 8th decade of life reinnervation no longer keeps pace, resulting in the accumulation of persistently denervated muscle fibers that in turn cause an acceleration of muscle dysfunction. The significance of denervation in important clinical outcomes with aging is poorly studied. The Study of Muscle, Mobility, and Aging (SOMMA) is a large cohort study with the primary objective to assess how aging muscle biology impacts clinically important traits. Using transcriptomics data from vastus lateralis muscle biopsies in 575 participants we have selected 49 denervation‐responsive genes to provide insights to the burden of denervation in SOMMA, to test the hypothesis that greater expression of denervation‐responsive genes negatively associates with SOMMA participant traits that included time to walk 400 meters, fitness (VO2peak), maximal mitochondrial respiration, muscle mass and volume, and leg muscle strength and power. Consistent with our hypothesis, increased transcript levels of: a calciumdependent intercellular adhesion glycoprotein (CDH15), acetylcholine receptor subunits (CHRNA1, CHRND, CHRNE), a glycoprotein promoting reinnervation (NCAM1), a transcription factor regulating aspects of muscle organization (RUNX1), and a sodium channel (SCN5A) were each negatively associated with at least 3 of these traits. VO2peak and maximal respiration had the strongest negative associations with 15 and 19 denervation‐responsive genes, respectively. In conclusion, the abundance of denervationresponsive gene transcripts is a significant determinant of muscle and mobility outcomes in aging humans, supporting the imperative to identify new treatment strategies to restore innervation in advanced age. [ABSTRACT FROM AUTHOR]

Ca2+‐dependent K+ (BK) channels at varicosities in Xenopus nerve–muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca2+]AZ resulting from different rates and durations of Ca2+ entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca2+ tail currents produce mean peak [Ca2+]AZ ~ 30 μM; with continued influx, [Ca2+]AZ reaches ~45–60 μM at different rates depending on Ca2+ driving force and duration of influx. Both IBK and release are dependent on Ca2+ microdomains composed of both N‐ and L‐type Ca channels. Domains collapse with a time constant of ~0.6 ms. We have constructed an active zone (AZ) model that approximately fits this data, and depends on incorporation of the high‐capacity, low‐affinity fixed buffer represented by phospholipid charges in the plasma membrane. Our observations suggest that in this preparation, (1) some BK channels, but few if any of the Ca2+ sensors that trigger release, are located within Ca2+ nanodomains while a large fraction of both are located far enough from Ca channels to be blockable by EGTA, (2) the IBK is more sensitive than the excitatory postsynaptic current (EPSC) to [Ca2+]AZ (K1/2–26 μM vs. ~36 μM [Ca2+]AZ); (3) with increasing [Ca2+]AZ, the IBK grows with a Hill coefficient of 2.5, the EPSC with a coefficient of 3.9; (4) release is dependent on the highest [Ca2+] achieved, independent of the time to reach it; (5) the varicosity synapses differ from mature frog nmjs in significant ways; and (6) BK channels are useful reporters of local [Ca2+]AZ. [ABSTRACT FROM AUTHOR]

This preface introduces the Journal of Neurochemistry special issue on Cholinergic Mechanisms that highlights the progress in the molecular, structural, neurochemical, pharmacological, toxicological, and clinical studies of the cholinergic system which underline its complexity and impact on health and disease. This issue comprises of (systematic) reviews and original articles, the majority of which have been presented at the 17th International Symposium on Cholinergic Mechanisms (ISCM2022) held in Dubrovnik, Croatia in May 2022. The symposium brought together leading "Cholinergikers" to shed new light on cholinergic transmission, ranging from the molecular to the clinical and cognitive mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (β4 spectrin) were reported to cause both neuropathy and myopathy. Although the role of β4 spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific β4 spectrin conditional knockout mouse, we show that β4 spectrin does not contribute to muscle function. In addition, we show β4 spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, β1 and β2 spectrins are found in skeletal muscle, with α2 and β1 spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and β2 spectrins had no effect on muscle health, function or the enrichment of β1 spectrin at the NMJ. Muscle specific deletion of β1 spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na+ channels. Together, our results suggest that muscle β1 spectrin functions independently of an associated α spectrin to maintain Na+ channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. Key points: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding β4 spectrin) is neurogenic in origin.β1 spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na+ channels.By contrast to the canonical view of spectrin organization and function, we show that β1 spectrin can function independently of an associated α spectrin.Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons. [ABSTRACT FROM AUTHOR]

Sarcopenia is a systemic skeletal muscle disease characterized by a decline in skeletal muscle mass and function. Originally defined as an age‐associated condition, sarcopenia presently also encompasses muscular atrophy due to various pathological factors, such as intensive care unit‐acquired weakness, inactivity, and malnutrition. The exact pathogenesis of sarcopenia is still unknown; herein, we review the pathological roles of the neuromuscular junction and mitochondria in this condition. Sarcopenia is caused by complex and interdependent pathophysiological mechanisms, including aging, neuromuscular junction impairment, mitochondrial dysfunction, insulin resistance, lipotoxicity, endocrine factors, oxidative stress, and inflammation. Among these, neuromuscular junction instability and mitochondrial dysfunction are particularly significant. Dysfunction in neuromuscular junction can lead to muscle weakness or paralysis. Mitochondria, which are plentiful in neurons and muscle fibers, play an important role in neuromuscular junction transmission. Therefore, impairments in both mitochondria and neuromuscular junction may be one of the key pathophysiological mechanisms leading to sarcopenia. Moreover, this article explores the structural and functional alterations in the neuromuscular junction and mitochondria in sarcopenia, suggesting that a deeper understanding of these changes could provide valuable insights for the prevention or treatment of sarcopenia. [ABSTRACT FROM AUTHOR]

BACKGROUND: Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V‐Type adenosine triphosphatase (V‐ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked. RESULTS: The bioassay revealed that Drosophila adults with different genetic backgrounds showed varying levels of susceptibility to PSP in the order: parats1 > parats1;DSC1−/− ≈ w1118 > DSC1−/−. Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration‐dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. CONCLUSION: Our findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V‐ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Despite prior efforts to understand and target dynapenia (age‐induced loss of muscle strength), this condition remains a major challenge that reduces the quality of life in the aged population. We have focused on the neuromuscular junction (NMJ) where changes in structure and function have rarely been systematically studied as a dynamic and progressive process. Our cross‐sectional study found neurotransmission at the male mouse NMJ to be biphasic, displaying an early increase followed by a later decrease, and this phenotype was associated with structural changes to the NMJ. A cross‐sectional characterization showed that age‐induced alterations fell into four age groups: young adult (3–6 months), adult (7–18 months), early aged (19–24 months), and later aged (25–30 months). We then utilized a small molecule therapeutic candidate, GV‐58, applied acutely during the later aged stage to combat age‐induced reductions in transmitter release by increasing calcium influx during an action potential, which resulted in a significant increase in transmitter release. This comprehensive study of neuromuscular ageing at the NMJ will enable future research to target critical time points for therapeutic intervention. Key points: Age‐induced frailty and falls are the leading causes of injury‐related death and are caused by an age‐induced loss of muscle strength due to a combination of neurological and muscular changes.A cross‐sectional approach was used to study age‐induced changes to the neuromuscular junction in a mouse model, and physiological changes that were biphasic over the ageing time course were found.Changes in physiology at the neuromuscular junction were correlated with alterations in neuromuscular junction morphology.An acutely applied positive allosteric gating modifier of presynaptic voltage‐gated calcium channels was tested as a candidate therapeutic strategy that could increase transmitter release at aged neuromuscular junctions.These results provide a detailed time course of age‐induced changes at the neuromuscular junction in a mouse model and test a candidate therapeutic strategy for weakness. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Human tonsils are a readily accessible source of stem cells for the potential treatment of skeletal muscle disorders. We reported previously that tonsil‐derived mesenchymal stem cells (TMSCs) can differentiate into skeletal muscle cells (SKMCs), which renders TMSCs promising candidates for cell therapy for skeletal muscle disorders. However, the functional properties of the myocytes differentiated from mesenchymal stem cells have not been clearly evaluated. In this study we investigated whether myocytes differentiated from TMSCs (skeletal muscle cells derived from tonsil mesenchymal stem cells [TMSC‐SKMCs]) exhibit the functional characteristics of SKMCs. Methods: To test the insulin reactivity of TMSC‐SKMCs, the expression of glucose transporter 4 (GLUT4) and phosphatidylinositol 3‐kinase/Akt was analyzed after the cells were treated for 30 minutes with 100 nmol/L insulin in normal or high‐glucose medium. We also examined whether these cells formed a neuromuscular junction (NMJ) when cocultured with motor neurons, and whether they were stimulated by electrical signals using whole‐cell patch clamping. Results: Skeletal muscle cells derived from tonsil mesenchymal stem cells expressed SKMC markers, such as MYOD, MYH3, MYH8, TNNI1, and TTN, at high levels, and exhibited a multinucleated cell morphology and a myotube‐like shape. The expression of the acetylcholine receptor and GLUT4 was confirmed in TMSC‐SKMCs. In addition, these cells exhibited insulin‐mediated glucose uptake, NMJ formation, and transient changes in cell membrane action potential, all of which are representative functions of human SKMCs. Discussion: Tonsil‐derived mesenchymal stem cells can be functionally differentiated into SKMCs and may have potential for clinical application for the treatment of skeletal muscle disorders. [ABSTRACT FROM AUTHOR]

Giant axonal neuropathy (GAN) is a fatal neurodegenerative disorder for which there is currently no treatment. Affecting the nervous system, GAN starts in infancy with motor deficits that rapidly evolve toward total loss of ambulation. Using the gan zebrafish model that reproduces the loss of motility as seen in patients, we conducted the first pharmacological screening for the GAN pathology. Here, we established a multilevel pipeline to identify small molecules restoring both the physiological and the cellular deficits in GAN. We combined behavioral, in silico, and high‐content imaging analyses to refine our Hits to five drugs restoring locomotion, axonal outgrowth, and stabilizing neuromuscular junctions in the gan zebrafish. The postsynaptic nature of the drug's cellular targets provides direct evidence for the pivotal role the neuromuscular junction holds in the restoration of motility. Our results identify the first drug candidates that can now be integrated in a repositioning approach to fasten therapy for the GAN disease. Moreover, we anticipate both our methodological development and the identified hits to be of benefit to other neuromuscular diseases. Synopsis: To date, there is no treatment for giant axonal neuropathy (GAN), a fatal neurodegenerative disease leading to loss of ambulation in young patients. Due to the lack of effective rodent models, the gan zebrafish was used to conduct the first pharmacological screening for therapeutic repurposing.The gan zebrafish is the first robust model of the GAN disease, exhibiting the loss of motility found in patients.A multi‐level methodology was designed to integrate behavioral (motility), computational (drug‐target functions) and cellular (NMJ compartments) readouts.A novel high‐content automated imaging analysis was developed to quantify the beneficial effect of the Hits at the NMJ.This tailored screening pipeline identified five Hits restoring the motility of the gan zebrafish through the stabilization of the NMJ. [ABSTRACT FROM AUTHOR]

Decline in neuromuscular function with aging is known to be a major determinant of disability and all‐cause mortality in late life. Despite the importance of the problem, the neurobiology of age‐associated muscle weakness is poorly understood. In a previous report, we performed untargeted metabolomics on frail older adults and discovered prominent alteration in the kynurenine pathway, the major route of dietary tryptophan degradation that produces neurotoxic intermediate metabolites. We also showed that neurotoxic kynurenine pathway metabolites are correlated with increased frailty score. For the present study, we sought to further examine the neurobiology of these neurotoxic intermediates by utilizing a mouse model that has a deletion of the quinolinate phosphoribosyltransferase (QPRT) gene, a rate‐limiting step of the kynurenine pathway. QPRT−/− mice have elevated neurotoxic quinolinic acid level in the nervous system throughout their lifespan. We found that QPRT−/− mice have accelerated declines in neuromuscular function in an age‐ and sex‐specific manner compared to control strains. In addition, the QPRT−/− mice show premature signs of frailty and body composition changes that are typical for metabolic syndrome. Our findings suggest that the kynurenine pathway may play an important role in frailty and age‐associated muscle weakness. [ABSTRACT FROM AUTHOR]

Background: The combination of botulinum toxin type A (BoNT/A) and energy equipment have been widely used in the clinic. Aims: To determine whether the energy of microneedle fractional radiofrequency (MFR) affects the efficacy of BoNT/A and to provide an optimal strategy for the energy device in combination with BoNT/A in the clinic. Methods: First, a total of 45 females with moderate‐to‐severe periorbital crow's feet wrinkles were enrolled and divided into three groups according to different treatment methods and intervals, including BoNT/A injection alone, BoNT/A injected immediately after MFR treatment and BoNT/A injected 7 days after MFR treatment. The photographs were compared before treatment and 4 weeks after treatment. Then, the mouse models were established by combining MFR with BoNT/A at different intervals, to evaluate muscle strength, muscle mass, muscle nutritional markers, and important cytokines levels. Results: All patients in each group had high satisfaction. The MFR + BoNT/A (immediately) group could improve dynamic wrinkles, but the others had more significant efficacy (p < 0.05). The results of mouse models showed that all BoNT/A groups induced different degrees of muscle paralysis in vivo, but the paralytic effect induced by the BoNT/A group, MFR + BoNT/A (interval of 3‐day) group, and MFR + BoNT/A (interval of 7‐day) group were higher than others and the expression levels of muscle nutritional markers in NMJ tissues were significantly upregulated. Conclusion: MFR has a certain reduction effect on the activity of BoNT/A, and this reduction effect would last for 3 days after MFR treatment. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a common neuromuscular junction disorder and autoimmune disease mediated by several antibodies. Several studies have shown that genetic factors play an important role in MG pathogenesis. To gain insight into the epigenetic factors affecting MG, we report here genome‐scale DNA methylation profiles of MG. DNA was extracted from eight MG patients and four healthy controls for genome‐wide DNA methylation analysis using the Illumina HumanMethylation 850K BeadChip. Verification of pyrosequencing was conducted based on differential methylation positions. Subsequently, C2C12 and HT22 cell lines (derived from mouse) were treated with demethylation drugs. Transcribed mRNA of the screened differential genes was detected using quantitative real‐time PCR. The control and MG group were compared, and two key probe positions were selected. The corresponding genes were CAMK1D and CREB5 (P < 0.05). Similarly, the myasthenic crisis (MC) and non‐MC group were compared and four key probe positions were selected. The corresponding genes were SAV1, STK3, YAP1, and WWTR1 (P < 0.05). Subsequently, pyrosequencing was performed for verification, revealing that hypomethylation of CAMK1D was significantly different between the MG and control group (P < 0.001). Moreover, transcription of CREB5, PKD, YAP1, and STK3 genes in the C2C12 cells was downregulated (P < 0.05) after drug treatment, but only YAP1 mRNA was downregulated in HT22 cells (P < 0.05). This is the first study to investigate genome‐scale DNA methylation profiles of MG using 850 K BeadChip. The identified molecular markers of methylation may aid in the prevention, diagnosis, treatment, and prognosis of MG. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Peripheral nerve injuries result in impaired neuromuscular interactions, leading to morphological and functional alterations. Adjuvant suture repair methods have been used to improve nerve regeneration and modulate the immune response. Heterologous fibrin biopolymer (HFB), a scaffold with adhesive properties, plays a critical role in tissue repair. The aim of this study is to evaluate neuroregeneration and immune response focusing on neuromuscular recovery, using suture‐associated HFB for sciatic nerve repair. Methods: Forty adult male Wistar rats were distributed into four groups (n = 10): C (control), only sciatic nerve location; D (denervated), neurotmesis and 6‐mm gap removal and fixation stumps in subcutaneous tissue; S (suture), neurotmesis followed by suture; and SB (suture + HFB), neurotmesis followed by suture and HFB. Analysis of M2 macrophages (CD206+), as well as the morphology and morphometry of nerves, soleus muscle, and neuromuscular junctions (NMJs), were performed at 7 and 30 days after surgery. Results: The SB group had the highest M2 macrophage area in both periods. After 7 days, SB was the only group similar to the C group regarding the number of axons; furthermore, after 30 days, the SB group was closer to the C group concerning blood vessel and central myonuclear numbers, NMJ angle, and connective tissue volume. After 7 days, increases in nerve area, as well as the number and area of blood vessels, were also observed in SB. Discussion: HFB potentiates the immune response, increases axonal regeneration, induces angiogenesis, prevents severe muscle degeneration, and assists in NMJ recovery. In conclusion, suture‐associated HFB has major implications for improved peripheral nerve repair. [ABSTRACT FROM AUTHOR]

Aim: Respiratory insufficiency can be a presenting symptom of congenital myasthenic syndromes (CMS) but is rarely recognised as such. In this study, we aim to raise awareness of CMS to paediatricians. Methods: We performed a retrospective case study of infants and preschool children treated in the past 5 years in Amalia Children's Hospital, Radboud University Medical Center in Nijmegen, the Netherlands for respiratory insufficiency as presenting symptom of CMS. Results: Five children aged 2 weeks to 5 years experienced severe to life‐threatening episodes of respiratory insufficiency, especially during viral infections, due to respiratory muscle weakness. During infections, they often also had progression of their otherwise mild ocular, facial, and limb muscle weakness. They were eventually diagnosed with genetically proven CMS. In these five children, treatment with pyridostigmine, an acetylcholinesterase inhibitor, resulted in clinical improvement. Conclusion: CMS should be considered in every patient with unexplained recurrent respiratory insufficiency, or with an unusually severe course of a normally mild respiratory infection, especially in combination with mild muscle weakness outside periods of illness. Early diagnosis of CMS is crucial for early treatment, which may help avoiding sudden infant death, severe respiratory insufficiency and further deterioration of the muscle strength. [ABSTRACT FROM AUTHOR]

Aims: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. Methods: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. Results: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. Conclusions: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays. [ABSTRACT FROM AUTHOR]

Background: Movement of the lower jaw, a common behavior observed among vertebrates, is required for eating and processing food. This movement is controlled by signals sent from the trigeminal motor nerve through neuromuscular junctions (NMJs) to the masticatory muscles. Dysfunctional jaw movements contribute to craniomandibular disorders, yet the pathophysiology of these disorders is not well understood, as limited studies have been conducted on the molecular mechanisms of jaw movement. Results: Using erc1b/kimm533 genetic loss of function mutant, we evaluated lower jaw muscle organization and innervation by the cranial motor nerves in developing zebrafish. Using time‐lapse confocal imaging of the erc1b mutant in a transgenic fluorescent reporter line, we found delayed trigeminal nerve growth and disrupted nerve branching architecture during muscle innervation. By automated 3D image analysis of NMJ distribution, we identified an increased number of small, disorganized NMJ clusters in erc1b mutant larvae compared to WT siblings. Using genetic replacement experiments, we determined the Rab GTPase binding domain of Erc1b is required for cranial motor nerve branching, but not NMJ organization or muscle attachment. Conclusions: We identified Erc1b/ERC1 as a novel component of a genetic pathway contributing to muscle organization, trigeminal nerve outgrowth, and NMJ spatial distribution during development that is required for jaw movement. Key Findings: A craniofacial genetic zebrafish mutant, kimble (kimm533), that carries a loss of function variant in the erc1b gene has jaw movement defects.Erc1b function is required for organization of the lower jaw muscles, including tendon attachments.Erc1b is required for trigemeinal nerve outgrowth, and nerve branching architecture during innervation of the jaw muscles.Erc1b/ERC1 is a novel component of a genetic pathway that is involved in the organization of craniofacial muscle attachments and cranial motor nerve innervation. [ABSTRACT FROM AUTHOR]

Background and Purpose: Donepezil, a piperidine inhibitor of acetylcholinesterase (AChE) prescribed for treatment of Alzheimer's disease, has adverse neuromuscular effects in humans, including requirement for higher concentrations of non‐depolarising neuromuscular blockers during surgery. Here, we examined the effects of donepezil on synaptic transmission at neuromuscular junctions (NMJs) in isolated nerve‐muscle preparations from mice. Experimental Approach: We measured effects of therapeutic concentrations of donepezil (10 nM to 1 μM) on AChE enzymic activity, muscle force responses to repetitive stimulation, and spontaneous and evoked endplate potentials (EPPs) recorded intracellularly from flexor digitorum brevis muscles from CD01 or C57BlWldS mice. Key Results: Donepezil inhibited muscle AChE with an approximate IC50 of 30 nM. Tetanic stimulation in sub‐micromolar concentrations of donepezil prolonged post‐tetanic muscle contractions. Preliminary Fluo4‐imaging indicated an association of these contractions with an increase and slow decay of intracellular Ca2+ transients at motor endplates. Donepezil prolonged spontaneous miniature EPP (MEPP) decay time constants by about 65% and extended evoked EPP duration almost threefold. The mean frequency of spontaneous MEPPs was unaffected but the incidence of 'giant' MEPPs (gMEPPs), some exceeding 10 mV in amplitude, was increased. Neither mean MEPP amplitude (excluding gMEPPs), mean EPP amplitude, quantal content or synaptic depression during repetitive stimulation were significantly altered by concentrations of donepezil up to 1 μM. Conclusion and Implications: Adverse neuromuscular signs associated with donepezil therapy, including relative insensitivity to neuromuscular blockers, are probably due to inhibition of AChE at NMJs, prolonging the action of ACh on postsynaptic nicotinic acetylcholine receptors but without substantively impairing evoked ACh release. [ABSTRACT FROM AUTHOR]

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot–Marie–Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross‐species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole‐mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features. [ABSTRACT FROM AUTHOR]

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR‐containing plaques to elaborate and branched pretzel‐like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome‐like structures to define discrete low nAChR‐containing regions to consolidate a plaque‐to‐pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome‐like structures that drive the postsynaptic maturation of vertebrate NMJs. [ABSTRACT FROM AUTHOR]

Analysis of rodent muscles affords an opportunity to glean key insights into neuromuscular development and the detrimental impact of disease‐causing genetic mutations. Muscles of the distal leg, for instance the gastrocnemius and tibialis anterior, are commonly used in such studies with mice and rats. However, thin and flat muscles, which can be dissected, processed and imaged without major disruption to muscle fibres and nerve‐muscle contacts, are more suitable for accurate and detailed analyses of the peripheral motor nervous system. One such wholemount muscle is the predominantly fast twitch epitrochleoanconeus (ETA), which is located in the upper forelimb, innervated by the radial nerve, and contains relatively large and uniformly flat neuromuscular junctions (NMJs). To facilitate incorporation of the ETA into the experimental toolkit of the neuromuscular disease field, here, we describe a simple method for its rapid isolation (<5 min), supported by high‐resolution videos and step‐by‐step images. Furthermore, we outline how the ETA can be imaged in live, anaesthetised mice, to enable examination of dynamic cellular processes occurring at the NMJ and within intramuscular axons, including transport of organelles, such as mitochondria and signalling endosomes. Finally, we present reference data on wild‐type ETA fibre‐type composition in young adult, male C57BL6/J mice. Comparative neuroanatomical studies of different muscles in rodent models of disease can generate critical insights into pathogenesis and pathology; dissection of the wholemount ETA provides the possibility to diversify the repertoire of muscles analysed for this endeavour. [ABSTRACT FROM AUTHOR]

Aim: Despite males typically exhibiting greater muscle strength and fatigability than females, it remains unclear if there are sex‐based differences in neuromuscular recruitment strategies e.g. recruitment and modulation of motor unit firing rate (MU FR) at normalized forces and during progressive increases in force. Methods: The study includes 29 healthy male and 31 healthy female participants (18‐35 years). Intramuscular electromyography (iEMG) was used to record individual motor unit potentials (MUPs) and near‐fibre MUPs from the vastus lateralis (VL) during 10% and 25% maximum isometric voluntary contractions (MVC), and spike‐triggered averaging was used to obtain motor unit number estimates (MUNE) of the VL. Results: Males exhibited greater muscle strength (P <.001) and size (P <.001) than females, with no difference in force steadiness at 10% or 25% MVC. Females had 8.4% and 6.5% higher FR at 10% and 25% MVC, respectively (both P <.03), while the MUP area was 33% smaller in females at 10% MVC (P <.02) and 26% smaller at 25% MVC (P =.062). However, both sexes showed similar increases in MU size and FR when moving from low‐ to mid‐level contractions. There were no sex differences in any near‐fibre MUP parameters or in MUNE. Conclusion: In the vastus lateralis, females produce muscle force via different neuromuscular recruitment strategies to males which is characterized by smaller MUs discharging at higher rates. However, similar strategies are employed to increase force production from low‐ to mid‐level contractions. These findings of similar proportional increases between sexes support the use of mixed sex cohorts in studies of this nature. [ABSTRACT FROM AUTHOR]

4‐aminopyridine (4‐AP) is a potassium channel blocker that has been used to treat patients with multiple sclerosis and Lambert–Eaton disease. The concentration of this drug in the blood of patients was estimated to be in low or submicromolar range. Animal studies have shown that 4‐AP at such low concentration selectively blocks a subset of channels in Kv1 or Kv3 families. The crayfish opener neuromuscular junction and ventral superficial flexor (VSF) preparations were used to examine functions of K+ channels blocked by low concentrations of 4‐AP. At opener motor axons, intracellular recordings show that 4‐AP could increase action potential (AP) amplitude, duration, and after‐depolarization (ADP) at 10 μM. As 4‐AP concentration was increased, in twofold steps, AP amplitude did not increase further up to 5 mM. AP duration and ADP increased significantly mainly in two concentration ranges, 10–50 μM and 1–5 mM. The effects of 50 μM 4‐AP on the VSF were less consistent than that observed at the opener motor axons. 4‐AP did not change AP amplitude of motor axons recorded with an extracellular electrode and change in AP repolarizing potential was observed in ∼25% of the axons. EPSP recorded simultaneously with AP showed an increase in amplitude with 4‐AP treatment only in 30% of the axon‐EPSP pairs. 4‐AP also increased firing frequencies of ∼50% of axons. In four animals, 4‐AP "awakened" the firing of APs from an axon that was silent before the drug. The mixture of positive and negative 4‐AP effects summarized above was observed in the same VSF preparations in all cases (n = 8). We propose that there is a significant diversity in the density 4‐AP‐sensitive potassium channels among motor axons of the VSF. Functional significance in the differences of 4‐AP sensitivity of the two motor systems is discussed. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Motor function recovery is frequently poor after peripheral nerve injury. The effect of different numbers of nerve crushes and exercise on motor function recovery is unknown. We aimed to examine how different numbers of crushes of the rat sciatic nerve affects muscle reinnervation and plasticity of spinal circuits and the effect of exercise intervention. Methods: Single and multiple sciatic nerve crush models with different numbers of crushes were created in rats. Treadmill exercise was performed at 10 m/min for 60 min, five times a week. Muscle reinnervation and synaptic changes in L4‐5 motor neurons were examined by immunofluorescence staining. Behavioral tests were the sciatic functional index (SFI) and the pinprick tests. Results: The percentage of soleus muscle reinnervation was not significantly increased by the presence of exercise in single or multiple crushes. Exercise after a single crush increased the contact of motor neurons with VGLUT1‐containing structures (Exercised vs. Unexercised, 12.9% vs. 8.7%; p <.01), but after multiple crushes, it decreased with or without exercise (8.1% vs. 8.6%). Exercise after a single crush significantly improved SFI values from 14 to 24 days, and exercise after multiple crushes from 21 to 35 days (all p <.05). The pinprick test showed no difference in recovery depending on the number of crushes or whether or not exercised. Discussion: Different numbers of sciatic nerve crushes affect muscle reinnervation and motor neuron synaptic changes differently, but motor function recovery may improve with exercise regardless of the number of crushes. [ABSTRACT FROM AUTHOR]

A motor unit (MU) comprises the neuron cell body, its corresponding axon and each of the muscle fibres it innervates. Many studies highlight age‐related reductions in the number of MUs, yet the ability of a MU to undergo remodelling and to expand to rescue denervated muscle fibres is also a defining feature of MU plasticity. Remodelling of MUs involves two coordinated processes: (i) axonal sprouting and new branching growth from adjacent surviving neurons, and (ii) the formation of key structures around the neuromuscular junction to resume muscle–nerve communication. These processes rely on neurotrophins and coordinated signalling in muscle–nerve interactions. To date, several neurotrophins have attracted focus in animal models, including brain‐derived neurotrophic factor and insulin‐like growth factors I and II. Exercise in older age has demonstrated benefits in multiple physiological systems including skeletal muscle, yet evidence suggests this may also extend to peripheral MU remodelling. There is, however, a lack of research in humans due to methodological limitations which are easily surmountable in animal models. To improve mechanistic insight of the effects of exercise on MU remodelling with advancing age, future research should focus on combining methodological approaches to explore the in vivo physiological function of the MU alongside alterations of the localised molecular environment. [ABSTRACT FROM AUTHOR]

Objectives: To present phenotype features of a large cohort of congenital myasthenic syndromes (CMS) and correlate them with their molecular diagnosis. Methods: Suspected CMS patients were divided into three groups: group A (limb, bulbar or axial weakness, with or without ocular impairment, and all the following: clinical fatigability, electrophysiology compatible with neuromuscular junction involvement and anticholinesterase agents response), group B (limb, bulbar or axial weakness, with or without ocular impairment, and at least one of additional characteristics noted in group A) and group C (pure ocular syndrome). Individual clinical findings and the clinical groups were compared between the group with a confirmed molecular diagnosis of CMS and the group without molecular diagnosis or with a non‐CMS molecular diagnosis. Results: Seventy‐nine patients (68 families) were included in the cohort: 48 in group A, 23 in group B and 8 in group C. Fifty‐one were considered confirmed CMS (30 CHRNE, 5 RAPSN, 4 COL13A1, 3 DOK7, 3 COLQ, 2 GFPT1, 1 CHAT, 1 SCN4A, 1 GMPPB, 1 CHRNA1), 7 probable CMS, 5 non‐CMS and 16 unsolved. The chance of a confirmed molecular diagnosis of CMS was significantly higher for group A and lower for group C. Some individual clinical features, alterations on biopsy and electrophysiology enhanced specificity for CMS. Muscle imaging showed at least mild alterations in the majority of confirmed cases, with preferential involvement of soleus, especially in CHRNE CMS. Conclusions: Stricter clinical criteria increase the chance of confirming a CMS diagnosis, but may lose sensitivity, especially for some specific genes. [ABSTRACT FROM AUTHOR]

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic alterations effect neuromuscular transduction and muscle contractility, which ultimately dictate behavioural output. Here we develop and use a force transducer system to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs), examining how specific neuronal and muscle manipulations disrupt muscle contractility. Muscle contraction force increased with motoneuron stimulation frequency and duration, showing considerable plasticity between 5 and 40 Hz and saturating above 50Hz. Endogenous recordings of fictive contractions revealed average motoneuron burst frequencies of 20-30 Hz, consistent with the system operating within this plastic range of contractility. Temperature was also a key factor in muscle contractility, as force was enhanced at lower temperatures and dramatically reduced with increasing temperatures. Pharmacological and genetic manipulations of critical components of Ca2+ regulation in both pre- and postsynaptic compartments affected the strength and time course of muscle contractions. A screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation and modulation to alterations in excitation-contraction coupling. [ABSTRACT FROM AUTHOR]

The exon junction complex controls the translation, degradation, and localization of spliced mRNAs, and three of its core subunits also play a role in splicing. Here, we show that a fourth subunit, Barentsz, has distinct functions within and separate from the exon junction complex in Drosophila neuromuscular development. The distribution of mitochondria in larval muscles requires Barentsz as well as other exon junction complex subunits and is not rescued by a Barentsz transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We find that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles, and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex. Synopsis: Barentsz is best known as a subunit of the exon junction complex, and its effect on mitochondrial distribution in larval muscles in Drosophila is mediated by this complex. However, it acts independently of the exon junction complex to promote the growth of neuromuscular synapses by increasing Activin signaling. EJC‐independent functions of Barentsz can be identified using a transgenic form of the protein that fails to bind to the core subunit eIF4AIIIBarentsz acts through the EJC to allow mitochondria to be interspersed between larval muscle fibersBarentsz acts independently of the EJC to regulate the activin Dawdle, which enables normal neuromuscular synapse growth [ABSTRACT FROM AUTHOR]

Sarcopenia is an important health problem associated with adverse outcomes. Although the etiology of sarcopenia remains poorly understood, factors apart from muscle fibers, including humoral factors, might be involved. Here, we used cytokine antibody arrays to identify humoral factors involved in sarcopenia and found a significant increase in levels of milk fat globule epidermal growth factor 8 (MFG‐E8) in skeletal muscle of aged mice, compared with young mice. We found that the increase in MFG‐E8 protein at arterial walls and neuromuscular junctions (NMJs) in muscles of aged mice. High levels of MFG‐E8 at NMJs and an age‐related increase in arterial MFG‐E8 have also been identified in human skeletal muscle. In NMJs, MFG‐E8 is localized on the surface of terminal Schwann cells, which are important accessory cells for the maintenance of NMJs. We found that increased MFG‐E8 at NMJs precedes age‐related denervation and is more prominent in sarcopenia‐susceptible fast‐twitch than in sarcopenia‐resistant slow‐twitch muscle. Comparison between fast and slow muscles further revealed that arterial MFG‐E8 can be uncoupled from sarcopenic phenotype. A genetic deficiency in MFG‐E8 attenuated age‐related denervation of NMJs and muscle weakness, providing evidence of a pathogenic role of increased MFG‐E8. Thus, our study revealed a mechanism by which increased MFG‐E8 at NMJs leads to age‐related NMJ degeneration and suggests that targeting MFG‐E8 could be a promising therapeutic approach to prevent sarcopenia. [ABSTRACT FROM AUTHOR]

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. Key points: Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite.Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content).Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+‐channel function.The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following intentional ingestion of agricultural organophosphorus insecticides. Our findings also extend understanding of the effects of alcohols on synaptic transmission and homeostatic synaptic function. [ABSTRACT FROM AUTHOR]

Background: The discovery of adrenoceptors, which mediate the effects of the sympathetic nervous system neurotransmitter norepinephrine on specific tissues, sparked the development of sympathomimetics that have profound influence on skeletal muscle mass. However, chronic administration has serious side effects that preclude their use for muscle‐wasting conditions such as sarcopenia, the age‐dependent decline in muscle mass, force, and power. Devising interventions that can adjust neurotransmitter release to changing physiological demands will require understanding how the sympathetic nervous system affects muscle motor innervation and muscle mass, which will prevent sarcopenia‐associated impaired mobility, falls, institutionalization, co‐morbidity, and premature death. Here, we tested the hypothesis that prolonged heart and neural crest derivative 2 (Hand2) expression in peripheral sympathetic neurons (SNs) ameliorates sympathetic muscle denervation, motor denervation, and sarcopenia in geriatric mice. Methods: We delivered either a viral vector encoding the transcription factor Hand2 or an empty vector (EV) driven to SNs by the PRSx8 promoter by injecting the saphenous vein in 16‐month‐old C57BL/6 mice that were sacrificed 10–11 months later. Studies relied on sympathetic and muscle immunohistochemistry analysed by confocal microscopy, nerve and muscle protein expression assessed by immunoblots, nerve‐evoked and muscle‐evoked maximal muscle contraction force, extensor digitorum longus (EDL) muscle RNA sequencing, SN real‐time PCR, and tests of physical performance using an inverted‐cling grip test and in an open‐arena setting. Results: Examining the mice 10–11 months later, we found that inducing Hand2 expression in peripheral SNs preserved (i) the number of neurons (EV: 0.32 ± 0.03/μm2, n = 6; Hand2: 0.92 ± 0.08/μm2, n = 7; P < 0.0001) and size (EV: 279 ± 18 μm2, n = 6; Hand2: 396 ± 18 μm2, n = 7; P < 0.0001); (ii) lumbricalis muscle sympathetic innervation (EV: 1.4 ± 1.5 μm/μm2, n = 5; Hand2: 12 ± 1.8 μm/μm2, n = 5; P < 0.001); (iii) tibialis anterior, gastrocnemius, EDL, and soleus muscles weight and whole‐body strength (EV: 48 ± 6.4 s, n = 6; Hand2: 102 ± 6.8 s, n = 6; P < 0.001); (iv) EDL type IIb, IIx, and II/IIx and soleus type I, IIa, IIx, IIa/IIx, and IIb/IIx myofibre cross‐sectional area; (v) nerve‐evoked (EV: 16 ± 2.7 mN; Hand2: 30 ± 4.4 mN; P < 0.001) and muscle‐evoked (EV: 24 ± 3.8 mN, n = 5; Hand2: 38 ± 3.0 mN, n = 8; P < 0.001) muscle force by 150 Hz–3 s pulses; and (vi) motor innervation assessed by measuring presynaptic/postsynaptic neuromuscular junction area overlay. Conclusions: Preserving Hand2 expression in SNs from middle‐aged to very old mice attenuates decreases in muscle mass and force by (i) maintaining skeletal muscle sympathetic and motor innervation, (ii) improving membrane and total acetylcholine receptor stability and nerve‐evoked and muscle‐evoked muscle contraction, (iii) preventing the elevation of inflammation and myofibrillar protein degradation markers, and (iv) increasing muscle autophagy. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress and damage are associated with a number of ageing phenotypes, including age‐related loss of muscle mass and reduced contractile function (sarcopenia). Our group and others have reported loss of neuromuscular junction (NMJ) integrity and increased denervation as initiating factors in sarcopenia, leading to mitochondrial dysfunction, generation of reactive oxygen species and peroxides, and loss of muscle mass and weakness. Previous studies from our laboratory show that denervation‐induced skeletal muscle mitochondrial peroxide generation is highly correlated to muscle atrophy. Here, we directly test the impact of scavenging muscle mitochondrial hydrogen peroxide on the structure and function of the NMJ and muscle mass and function in a mouse model of denervation‐induced muscle atrophy CuZnSOD (Sod1−/− mice, Sod1KO). Methods: Whole‐body Sod1KO mice were crossed to mice with increased expression of human catalase (MCAT) targeted specifically to mitochondria in skeletal muscle (mMCAT mice) to determine the impact of reduced hydrogen peroxide levels on key targets of sarcopenia, including mitochondrial function, NMJ structure and function, and indices of muscle mass and function. Results: Female adult (~12‐month‐old) Sod1KO mice show a number of sarcopenia‐related phenotypes in skeletal muscle including reduced mitochondrial oxygen consumption and elevated reactive oxygen species generation, fragmentation, and loss of innervated NMJs (P < 0.05), a 30% reduction in muscle mass (P < 0.05), a 36% loss of force generation (P < 0.05), and a loss of exercise capacity (305 vs. 709 m in wild‐type mice, P < 0.05). Muscle from Sod1KO mice also shows a 35% reduction in sarco(endo)plasmic reticulum ATPase activity (P < 0.05), changes in the amount of calcium‐regulating proteins, and altered fibre‐type composition. In contrast, increased catalase expression in the mMCAT × Sod1KO mice completely prevents the mitochondrial and NMJ‐related phenotypes and maintains muscle mass and force generation. The reduction in exercise capacity is also partially inhibited (~35%, P < 0.05), and the loss of fibre cross‐sectional area is inhibited by ~50% (P < 0.05). Conclusions: Together, these striking findings suggest that scavenging of mitochondrial peroxide generation by mMCAT expression efficiently prevents mitochondrial dysfunction and NMJ disruption associated with denervation‐induced atrophy and weakness, supporting mitochondrial H2O2 as an important effector of NMJ alterations that lead to phenotypes associated with sarcopenia. [ABSTRACT FROM AUTHOR]

The article discusses the use of sugammadex, a drug used to reverse neuromuscular blockade during anesthesia, in infants. The authors highlight the need for larger doses of sugammadex in children under 2 years old and explain that this can be attributed to developmental changes in neuromuscular physiology and the pharmacokinetics of the drug rocuronium. They emphasize the importance of conducting formal pharmacokinetic-pharmacodynamic studies to better understand the interaction between sugammadex and rocuronium in this population. The authors acknowledge that there is limited knowledge in this area and call for further research to improve the dosing of sugammadex in infants. [Extracted from the article]

Neuromuscular junction (NMJ) transmission stability (i.e. NMJ function), determined by the electrophysiological evaluation of the near fibre MUP jiggle, was not significantly affected by the immobilisation protocol, similarly to what was reported with a 10-day ULLS study (Sarto et al., 2022). Keywords: firing rate; immobilisation; intramuscular electromyography; neuromuscular junction; physical inactivity; unloading EN firing rate immobilisation intramuscular electromyography neuromuscular junction physical inactivity unloading 4811 4813 3 11/17/22 20221115 NES 221115 Skeletal muscle is a highly plastic tissue that quickly adapts to changes in mechanical load. [Extracted from the article]

Recent development of novel therapies has improved mobility and quality of life for people suffering from inheritable neuromuscular disorders. Despite this progress, the majority of neuromuscular disorders are still incurable, in part due to a lack of predictive models of neuromuscular junction (NMJ) breakdown. Improvement of predictive models of a human NMJ would be transformative in terms of expanding our understanding of the mechanisms that underpin development, maintenance, and disease, and as a testbed with which to evaluate novel therapeutics. Induced pluripotent stem cells (iPSCs) are emerging as a clinically relevant and non‐invasive cell source to create human NMJs to study synaptic development and maturation, as well as disease modeling and drug discovery. This review will highlight the recent advances and remaining challenges to generating an NMJ capable of eliciting contraction of stem cell‐derived skeletal muscle in vitro. We explore the advantages and shortcomings of traditional NMJ culturing platforms, as well as the pioneering technologies and novel, biomimetic culturing systems currently in use to guide development and maturation of the neuromuscular synapse and extracellular microenvironment. Then, we will explore how this NMJ‐in‐a‐dish can be used to study normal assembly and function of the efferent portion of the neuromuscular arc, and how neuromuscular disease‐causing mutations disrupt structure, signaling, and function. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is an acquired autoimmune disorder caused by autoantibodies binding acetylcholine receptors (AChR), muscle‐specific kinase (MuSK), agrin or low‐density lipoprotein receptor‐related protein 4 (Lrp4). These autoantibodies inhibit neuromuscular transmission by blocking the function of these proteins and thereby cause fluctuating skeletal muscle weakness. Several reports suggest that these autoantibodies might also affect the central nervous system (CNS) in MG patients. A comprehensive overview of the timing and localization of the expression of MG‐related antigens in other organs is currently lacking. To investigate the spatio‐temporal expression of MG‐related genes outside skeletal muscle, we used in silico tools to assess public expression databases. Acetylcholine esterase, nicotinic AChR α1 subunit, agrin, collagen Q, downstream of kinase‐7, Lrp4, MuSK and rapsyn were included as MG‐related genes because of their well‐known involvement in either congenital or autoimmune MG. We investigated expression of MG‐related genes in (1) all human tissues using GTEx data, (2) specific brain regions, (3) neurodevelopmental stages, and (4) cell types using datasets from the Allen Institute for Brain Sciences. MG‐related genes show heterogenous spatio‐temporal expression patterns in the human body as well as in the CNS. For each of these genes, several (new) tissues, brain areas and cortical cell types with (relatively) high expression were identified suggesting a potential role for these genes outside skeletal muscle. The possible presence of MG‐related antigens outside skeletal muscle suggests that autoimmune MG, congenital MG or treatments targeting the same proteins may affect MG‐related protein function in other organs. [ABSTRACT FROM AUTHOR]