Structure Of Neuromuscular Junction Research Articles

Adipose mesenchymal stem cells-derived extracellular vesicles exert their preferential action in damaged central sites of SOD1 mice rather than peripherally.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.

Alterations in neuromuscular junction morphology with ageing and endurance training modulate neuromuscular transmission and myofibre composition.

Both ageing and exercise training affect the neuromuscular junction (NMJ) structure. Morphological alterations in the NMJ have been considered to influence neuromuscular transmission and myofibre properties, but the direct link between the morphology and function has yet to be established. We measured the neuromuscular transmission, myofibre composition and NMJ structure of 5-month-old (young) and 24-month-old untrained (aged control) and trained (aged trained) mice. Aged trained mice were subjected to 2 months of endurance training before the measurement. Neuromuscular transmission was evaluated in vivo as the ratio of ankle plantar flexion torque evoked by the sciatic nerve stimulation to that by direct muscle stimulation. The torque ratio was significantly lower in aged mice than in young and aged trained mice at high-frequency stimulations, showing a significant positive correlation with voluntary grip strength. The degree of pre- to post-synaptic overlap of the NMJ was also significantly lower in aged mice and positively correlated with the torque ratio. We also found that the proportion of fast-twitch fibres in the soleus muscle decreased with age, and that age-related denervation occurred preferentially in fast-twitch fibres. Age-related denervation and a shift in myofibre composition were partially prevented by endurance training. These results suggest that age-related deterioration of the NMJ structure impairs neuromuscular transmission and alters myofibre composition, but these alterations can be prevented by structural amelioration of NMJ with endurance training. Our findings highlight the importance of the NMJ as a major determinant of age-related deterioration of skeletal muscles and the clinical significance of endurance training as a countermeasure. KEY POINTS: The neuromuscular junction (NMJ) plays an essential role in neuromuscular transmission and the maintenance of myofibre properties. We show that neuromuscular transmission is impaired with ageing but recovered by endurance training, which contributes to alterations in voluntary strength. Neuromuscular transmission is associated with the degree of pre- to post-synaptic overlap of the NMJ. Age-related denervation of fast-twitch fibres and a shift in myofibre composition toward a slower phenotype are partially prevented by endurance training. Our study provides substantial evidence that age-related and exercise-induced alterations in neuromuscular transmission and myofibre properties are associated with morphological changes in the NMJ.

Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.

Neuronal synapse dysfunction is a key characteristic of several neurodegenerative disorders, such as Alzheimer's disease, spinocerebellar ataxias, and Huntington's disease. Modeling these disorders to study synaptic dysfunction requires a robust and reproducible method for assaying the subtle changes associated with synaptopathies in terms of structure and function of the synapses. Drosophila melanogaster neuromuscular junctions (NMJs) serve as good models to study such alterations. Further, modifications in the microenvironment of synapses can sometimes reflect in the behavior of the animal, which can also be assayed in a high-throughput manner. The methods outlined in this chapter highlight assays to study the behavioral changes associated with synaptic dysfunction in a spinocerebellar ataxia type 3 (SCA3) model. Further, molecular assessment of alterations in NMJ structure and function is also summarized, followed by effects of autophagy pathway upregulation in providing neuroprotection. These methods can be further extended and modified to study the therapeutic effects of drugs or small molecules in providing neuroprotection for any synaptopathy models.

Transcriptome profile of subsynaptic myonuclei at the neuromuscular junction in embryogenesis.

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.

Novel DPAGT1 Gene Mutation in Two Twins with Congenital Myasthenic Syndrome and a Review of the Literature.

Congenital myasthenic syndromes (CMS) are rare diseases caused by mutation in genes coding for proteins involved in neuromuscular junction structure and function. DPAGT1 gene mutations are a rare cause of CMS whose clinical evolution and pathophysiological mechanisms have not been clarified completely. We present the case of two twins displaying an infancy-onset predominant limb-girdle phenotype and carrying a novel DPAGT1 mutation associated with unusual histological and clinical findings. CMS can mimic paediatric and adult limb-girdle phenotype, hence neurophysiology plays a fundamental role in the differential diagnosis.

Response of terminal Schwann cells following volumetric muscle loss injury

An often-overlooked component of traumatic skeletal muscle injuries is the impact on the nervous system and resultant innervation of the affected muscles. Recent work in a rodent model of volumetric muscle loss (VML) injury demonstrated a progressive, secondary loss of neuromuscular junction (NMJ) innervation, supporting a role of NMJ dysregulation in chronic functional deficits. Terminal Schwann cells (tSCs) are known to be vital for the maintenance of NMJ structure and function, in addition to guiding repair and regeneration after injury. However, the tSC response to a traumatic muscle injury such as VML is not known. Thus, a study was conducted to investigate the effect of VML on tSC morphological characteristics and neurotrophic signaling proteins in adult male Lewis rats that underwent VML injury to the tibialis anterior muscle using a temporal design with outcome assessments at 3, 7, 14, 21, and 48 days post-injury. The following salient observations were made; first, although there is a loss of innervation over time, the number of tSCs per NMJ increases, significantly so at 48 days post-injury compared to control. The degree of NMJ fragmentation was positively correlated with tSC number after injury. Moreover, neurotrophic factors such as NRG1 and BDNF are elevated after injury through at least 48 days. These results were unanticipated and in contrast to neurodegenerative disease models, in which there is a reduction in tSC number that precedes denervation. However, we found that while there are more tSCs per NMJ after injury, they cover a significantly smaller percent of the post-synaptic endplate area compared to control. These findings support a sustained increase in neurotrophic activity and tSC number after VML, which is a maladaptive response occurring in parallel to other aspects of the VML injury, such as over-accumulation of collagen and aberrant inflammatory signaling.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inducible deletion of endothelial cell EfnB2 attenuates neuromuscular regeneration in mouse skeletal muscle

Background: Acute injury of skeletal muscle disrupts myofibers, microvessels and neuromuscular junctions (NMJs). While myofiber regeneration is well-characterized, little is known of concurrent events in microvessels and motor nerves following injury. Endothelial cell [EC] ephrin B2 [EfnB2] is required for sprouting angiogenesis during embryonic development, for promoting vascularization of tumors in the adult, and for microvascular-dependent guidance of axon regeneration following nerve resection. Hypothesis: We hypothesized that following acute injury to skeletal muscle 1) EC EfnB2 is required for restoring microvascular volume; and 2) loss of EC EfnB2 would impair recovery of neuromuscular structure and function. Methods: Mice (male and female, age 3-5 months) were bred for EC-specific conditional knockout [CKO] of EfnB2 following tamoxifen injection; noninjected CKO mice served as “wildtype” controls [WT]. Following injury by local injection of the myotoxin BaCl2, the gluteus maximus [GM] muscle was studied for microvascular analysis or the tibialis anterior [TA] muscle was evaluated for recovery of neuromuscular function. Mice were studied (n=4-6/group) at 3 timepoints for vascular studies (0, 5, and 10 days post injury [dpi]) and 2 timepoints for neuromuscular structure and function (0 and 10 dpi). Results: Intravascular staining with fluorescent wheat germ agglutinin revealed a diminished microvascular area in the GM of CKO mice at 5 dpi vs. 10 dpi (means ± SE; CKO 5 dpi 35.5% ± 2.5 vs. CKO 10 dpi 49.8% ± 3.9, P<0.01). No difference in vascular area was recorded in WT groups at 5 vs. 10 dpi (WT 5 dpi 45.5% ± 2.6 vs. WT 10 dpi 46.7% ± 2.2, P=0.99). Direct electrical stimulation of the TA resolved a reduction in maximal force at 10 dpi when compared to 0 dpi for both WT and CKO (WT 0 dpi 128.6 g ± 4.1 vs. WT 10 dpi 64.7 g ± 3.0, P<0.0001) (CKO 0 dpi 112.7 g ± 5.0 vs. CKO 10 dpi 65.9 g ± 5.0, P<0.0001). Indirect stimulation (sciatic nerve) resolved initial neuromuscular transmission failure (INMTF) reduced submaximal force in CKO but not WT (CKO 0 dpi 2.0 ± 4.3% vs. CKO 10 dpi -30.0 ± 6.5 %, P<0.001), which correlated with perturbed recovery of NMJ innervation; (R2=0.328, P=0.003), endplate area; (R2=0.479, P=0.001), and nicotinic receptor clusters; (R2=0.207, P=0.025). Conclusion: Following acute injury of skeletal muscle in the adult mouse, loss of EfnB2 in ECs delays microvessel regeneration in the GM and disrupts the recovery of NMJ structure and function in the TA. Support: NIH grant F32 HL-152558 (NLJ), APS Postdoctoral Fellowship (ABM), NIH LRP (ABM), Margaret Proctor Mulligan Fellowship, MU (NLJ, SSS), NIH R01 AR078045 (DDC), School of Medicine Development Award, MU (SSS), NIH MERIT Award R37 HL-041026 (SSS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

C9orf72 poly-GA proteins impair neuromuscular transmission.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease, in which lower motoneurons lose control of skeletal muscles. Degeneration of neuromuscular junctions (NMJs) occurs at the initial stage of ALS. Dipeptide repeat proteins (DPRs) from G4C2 repeat-associated non-ATG (RAN) translation are known to cause C9orf72-associated ALS (C9-ALS). However, DPR inclusion burdens are weakly correlated with neurodegenerative areas in C9-ALS patients, indicating that DPRs may exert cell non-autonomous effects, in addition to the known intracellular pathological mechanisms. Here, we report that poly-GA, the most abundant form of DPR in C9-ALS, is released from cells. Local administration of poly-GA proteins in peripheral synaptic regions causes muscle weakness and impaired neuromuscular transmission in vivo. The NMJ structure cannot be maintained, as evidenced by the fragmentation of postsynaptic acetylcholine receptor (AChR) clusters and distortion of presynaptic nerve terminals. Mechanistic study demonstrated that extracellular poly-GA sequesters soluble Agrin ligands and inhibits Agrin-MuSK signaling. Our findings provide a novel cell non-autonomous mechanism by which poly-GA impairs NMJs in C9-ALS. Thus, targeting NMJs could be an early therapeutic intervention for C9-ALS.

In Adult Skeletal Muscles, the Co-Receptors of Canonical Wnt Signaling, Lrp5 and Lrp6, Determine the Distribution and Size of Fiber Types, and Structure and Function of Neuromuscular Junctions.

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.

α-Synuclein aggregation causes muscle atrophy through neuromuscular junction degeneration.

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. 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. 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. 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.

E-cigarette aerosol impairs male mouse skeletal muscle force development and prevents recovery from injury.

To date, there has been a lag between the rise in E-cigarette use and an understanding of the long-term health effects. Inhalation of E-cigarette aerosol delivers high doses of nicotine, raises systemic cytokine levels, and compromises cardiopulmonary function. The consequences for muscle function have not been thoroughly investigated. The present study tests the hypothesis that exposure to nicotine-containing aerosol impairs locomotor muscle function, limits exercise tolerance, and interferes with muscle repair in male mice. Nicotine-containing aerosol reduced the maximal force produced by the extensor digitorum longus (EDL) by 30%-40% and, the speed achieved in treadmill running by 8%. Nicotine aerosol exposure also decreased adrenal and increased plasma epinephrine and norepinephrine levels, and these changes in catecholamines manifested as increased muscle and liver glycogen stores. In nicotine aerosol exposed mice, muscle regenerating from overuse injury only recovered force to 80% of noninjured levels. However, the structure of neuromuscular junctions (NMJs) was not affected by e-cigarette aerosols. Interestingly, the vehicle used to dissolve nicotine in these vaping devices, polyethylene glycol (PG) and vegetable glycerin (VG), decreased running speed by 11% and prevented full recovery from a lengthening contraction protocol (LCP) injury. In both types of aerosol exposures, cardiac left ventricular systolic function was preserved, but left ventricular myocardial relaxation was altered. These data suggest that E-cigarette use may have a negative impact on muscle force and regeneration due to compromised glucose metabolism and contractile function in male mice.NEW & NOTEWORTHY In male mice, nicotine-containing E-cigarette aerosol compromises muscle contractile function, regeneration from injury, and whole body running speeds. The vehicle used to deliver nicotine, propylene glycol, and vegetable glycerin, also reduces running speed and impairs the restoration of muscle function in injured muscle. However, the predominant effects of nicotine in this inhaled aerosol are evident in altered catecholamine levels, increased glycogen content, decreased running capacity, and impaired recovery of force following an overuse injury.

VP.05 Innervation defect: new pathomechanism of centronuclear myopathy?

Mutations in <i>DNM2, BIN1</i> and <i>MTM1</i> cause centronuclear myopathy (CNM). T-tubule malformation and its resultant defects in excitation-contraction coupling has been suggested to be the pathomechanism of this disease. Recently, in biopsied skeletal muscles from some CNM patients, we noticed the circumferential localization of acetylcholinesterase (AChE) on the sarcolemma on histochemical stain for AChE, which may well be due to the failure of AChE clustering in neuromuscular junction (NMJ). In support of our notion, NMJ anomalies were recently described in <i>Dnm2</i>-mutant animals. To check whether circumferential AChE expression on the sarcolemma is associated with CNM, and if so, to know whether NMJ is structurally and electrophysiologically abnormal in patients with CNM. We re-reviewed muscle pathology slides of the 40 patients with genetically-confirmed CNM (34 <i>DNM2, 1BIN1</i>, and 5 <i>MTM1</i>) and evaluated the localization of AChE. We also analyzed the morphology of NMJ in CNM by electron microscopy (EM). We collected the data of repetitive nerve stimulation (RNS) study and the effects of administration of AChE inhibitors (AChE-I) in CNM patients from the physicians. Circumferential AChE activity on the sarcolemma was observed in all patients with CNM. The frequency of the fibers with circumferential AChE was 100% in all CNM_DNM2, CNM_BIN1 and CNM_MTM1. On EM, postsynaptic structure of NMJ was abnormal in CNM_DNM2 muscles. Decremental response was seen on RNS in all three patients with CNM_DNM2 who received RNS, indicating the presence of myasthenic feature in CNM. One of two patients who received AChE-I showed improvement in muscle manual testing. Abnormal NMJ formation and a failure in AChE clustering indicate defects in NMJ transmission in CNM skeletal muscles. Decremental muscle action potential amplitudes and effective AChE-I suggest that myasthenic features in CNM contribute to muscle weakness in this disease. NMJ transmission defect due to postsynaptic structural abnormality is a new pathomechanism of CNM.

KMT5B is required for early motor development.

Disruptive variants in lysine methyl transferase 5B (KMT5B/SUV4-20H1) have been identified as likely-pathogenic among humans with neurodevelopmental phenotypes including motor deficits (i.e., hypotonia and motor delay). However, the role that this enzyme plays in early motor development is largely unknown. Using a Kmt5b gene trap mouse model, we assessed neuromuscular strength, skeletal muscle weight (i.e., muscle mass), neuromuscular junction (NMJ) structure, and myofiber type, size, and distribution. Tests were performed over developmental time (postnatal days 17 and 44) to represent postnatal versus adult structures in slow- and fast-twitch muscle types. Prior to the onset of puberty, slow-twitch muscle weight was significantly reduced in heterozygous compared to wild-type males but not females. At the young adult stage, we identified decreased neuromuscular strength, decreased skeletal muscle weights (both slow- and fast-twitch), increased NMJ fragmentation (in slow-twitch muscle), and smaller myofibers in both sexes. We conclude that Kmt5b haploinsufficiency results in a skeletal muscle developmental deficit causing reduced muscle mass and body weight.

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle.

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (α-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and α-BTX, and in the second group, samples were stained with NF200, α-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

Neuromuscular Synapse Dynamics in Aged and Injured Skeletal Muscle

It is evident that both aged and diseased skeletal muscle exhibit interesting neuromuscular dynamics, characterized by changes in morphology and functionality of the synapse. Although they display a high regenerative capacity, skeletal muscles are still susceptible to nervous system perturbations and impacts of aging. Aged neuromuscular junctions (NMJs) demonstrate a diseased phenotype following some form of muscle atrophy, indicating the presence of paracrine signaling between myofibers and neuronal cells. In this study, we investigated differences in neuromuscular dynamics in injured and aged skeletal muscle using whole‐mount immunostaining and imaging. It has previously been shown that disruption of one component of the muscle microenvironment, such as vasculature, disrupts the structure of NMJs, however it is unclear if aged skeletal muscle experiences similar changes. We hypothesized that both aged and injured NMJs would display similar morphological dynamics, characterized by denervation and fragmentation of postsynaptic acetylcholine receptors (AChRs). We demonstrated that aged and injured NMJs exhibit fragmentation of AChRs, Wallerian degeneration of the presynaptic motor neurons, decreased overlap of pre‐ and post‐synaptic sides, and decreased area of AChR clusters. Due to the prevalence of neuromuscular disorders in the aging population, it is pertinent to understand nerve‐muscle interactions in the healthy aging state in order to hypothesize how these dynamics may be altered in the age‐induced injury states. Ultimately, this will allow us to better comprehend the molecular and cellular mechanisms that are responsible for neuromuscular degeneration in order to develop strategies to improve mobility and quality of life.

Expression and Roles of Lynx1, a Modulator of Cholinergic Transmission, in Skeletal Muscles and Neuromuscular Junctions in Mice.

Lynx1 is a glycosylphosphatidylinositol (GPI)-linked protein shown to affect synaptic plasticity through modulation of nicotinic acetylcholine receptor (nAChR) subtypes in the brain. Because of this function and structural similarity to α-bungarotoxin, which binds muscle-specific nAChRs with high affinity, Lynx1 is a promising candidate for modulating nAChRs in skeletal muscles. However, little is known about the expression and roles of Lynx1 in skeletal muscles and neuromuscular junctions (NMJs). Here, we show that Lynx1 is expressed in skeletal muscles, increases during development, and concentrates at NMJs. We also demonstrate that Lynx1 interacts with muscle-specific nAChR subunits. Additionally, we present data indicating that Lynx1 deletion alters the response of skeletal muscles to cholinergic transmission and their contractile properties. Based on these findings, we asked if Lynx1 deletion affects developing and adult NMJs. Loss of Lynx1 had no effect on NMJs at postnatal day 9 (P9) and moderately increased their size at P21. Thus, Lynx1 plays a minor role in the structural development of NMJs. In 7- and 12-month-old mice lacking Lynx1, there is a marked increase in the incidence of NMJs with age- and disease-associated morphological alterations. The loss of Lynx1 also reduced the size of adult muscle fibers. Despite these effects, Lynx1 deletion did not alter the rate of NMJ reinnervation and stability following motor axon injury. These findings suggest that Lynx1 is not required during fast remodeling of the NMJ, as is the case during reformation following crushing of motor axons and development. Instead, these data indicate that the primary role of Lynx1 may be to maintain the structure and function of adult and aging NMJs.

A neuromuscular perspective of sarcopenia pathogenesis: deciphering the signaling pathways involved.

The escalation of life expectancy is accompanied by an increase in the prevalence of age-related conditions, such as sarcopenia. Sarcopenia, a muscle condition defined by low muscle strength, muscle quality or quantity, and physical performance, has a high prevalence among the elderly and is associated to increased mortality. The neuromuscular system has been emerging as a key contributor to sarcopenia pathogenesis. Indeed, the age-related degeneration of the neuromuscular junction (NMJ) function and structure may contribute to the loss of muscle strength and ultimately to the loss of muscle mass that characterize sarcopenia. The present mini-review discusses important signaling pathways involved in the function and maintenance of the NMJ, giving emphasis to the ones that might contribute to sarcopenia pathogenesis. Some conceivable biomarkers, such as C-terminal agrin fragment (CAF) and brain-derived neurotrophic factor (BDNF), and therapeutic targets, namely acetylcholine and calcitonin gene-related peptide (CGRP), can be retrieved, making way to future studies to validate their clinical use.

Neuromuscular Development and Disease: Learning From in vitro and in vivo Models.

The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.