Cholinesterases in Tripartite Neuromuscular Synapse

The neuromuscular junction (NMJ) is a tripartite synapse in which not only presynaptic and post-synaptic cells participate in synaptic transmission, but also terminal Schwann cells (TSC). Acetylcholine (ACh) is the neurotransmitter that mediates the signal between the motor neuron and the muscle but also between the motor neuron and TSC. ACh action is terminated by acetylcholinesterase (AChE), anchored by collagen Q (ColQ) in the basal lamina of NMJs. AChE is also anchored by a proline-rich membrane anchor (PRiMA) to the surface of the nerve terminal. Butyrylcholinesterase (BChE), a second cholinesterase, is abundant on TSC and anchored by PRiMA to its plasma membrane. Genetic studies in mice have revealed different regulations of synaptic transmission that depend on ACh spillover. One of the strongest is a depression of ACh release that depends on the activation of α7 nicotinic acetylcholine receptors (nAChR). Partial AChE deficiency has been described in many pathologies or during treatment with cholinesterase inhibitors. In addition to changing the activation of muscle nAChR, AChE deficiency results in an ACh spillover that changes TSC signaling. In this mini-review, we will first briefly outline the organization of the NMJ. This will be followed by a look at the role of TSC in synaptic transmission. Finally, we will review the pathological conditions where there is evidence of decreased AChE activity.

Transmission-selective muscle pathology induced by active propagation of mutant huntingtin across the human neuromuscular synapse

A potential explanation for the spatiotemporal accumulation of pathological lesions in the brain of patients with neurodegenerative protein misfolding diseases (PMDs) is cell-to-cell transmission of aggregation-prone, misfolded proteins. Little is known about central to peripheral transmission and its contribution to pathology. We show that transmission of Huntington’s disease- (HD-) associated mutant HTT exon 1 (mHTTEx1) occurs across the neuromuscular junctions in human iPSC cultures and in vivo in wild-type mice. We found that transmission is an active and dynamic process, that happens prior to aggregate formation and is regulated by synaptic activity. Furthermore, we find that transmitted mHTTEx1 causes HD-relevant pathology at a molecular and functional level in human muscle cells, even in the presence of ubiquitous expression mHTTEx1. With this work we uncover a casual-link between mHTTEx1 synaptic transmission and pathology, highlighting the therapeutic potential in blocking toxic protein transmission in PMDs.

Effect of hydrogen sulfide on the characteristics of the neuromuscular synapse terminals of the rat diaphragm

Hydrogen sulfide is one of the gas-transmitters that also performs other biological functions. The antioxidant property of this substance is one of the important ones. The research was conducted on rats of both sexes between 6 and 18 days of age. We have shown that the offspring of females injected subcutaneously with hydrogen sulfide increased the area and luminescence of nerve terminals during postnatal ontogenesis, which can be further used to level the effects of hyperhomocysteinemia on synaptic transmission. Key words: neuromuscular synapse, fluorescent microscopy, hydrogen sulfide.

Comparison of neuromuscular junction dynamics following ischemic and aged skeletal muscle

Both aging and neuromuscular diseases lead to significant changes in the morphology and functionality of the neuromuscular synapse. Skeletal muscles display a remarkable regenerative capacity, however, are still susceptible to diseases of aging and peripheral nerve perturbations. In this study, we assessed how neuromuscular synapses differ in aged and injured skeletal muscle using an improved neuromuscular junction (NMJ) staining and imaging method. We found that both aged and ischemic skeletal muscle display Wallerian degeneration of the presynaptic motor axons and fragmentation of postsynaptic acetylcholine receptors (AChRs). Quantifiable measurements of various metrics of the NMJs provide a more concrete idea of the dynamics that are occurring in the muscle microenvironment. We questioned whether neuronal degradation precedes myofiber atrophy or vice versa. Previously, it was shown that a cellular crosstalk exists among the motor neurons, myofibers, vasculature, and mitochondria within the muscle microdomain. It is apparent that lack of blood flow to motor neurons in ischemic skeletal muscle disrupts the structure of NMJs, however it is unclear if the aging condition experiences similar dynamics. We demonstrated that both aged and ischemic skeletal muscle demonstrate similar patterns of degeneration, characterized by a smaller percentage overlap of presynaptic and postsynaptic sides, greater fragmentation of AChRs, and a smaller area of AChR clusters. Together, these results reveal high resolution, precise parallels between the aged and ischemic NMJs.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle

ABSTRACT The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Lithium causes differential effects on postsynaptic stability in normal and denervated neuromuscular synapses

Lithium chloride has been widely used as a therapeutic mood stabilizer. Although cumulative evidence suggests that lithium plays modulatory effects on postsynaptic receptors, the underlying mechanism by which lithium regulates synaptic transmission has not been fully elucidated. In this work, by using the advantageous neuromuscular synapse, we evaluated the effect of lithium on the stability of postsynaptic nicotinic acetylcholine receptors (nAChRs) in vivo. We found that in normally innervated neuromuscular synapses, lithium chloride significantly decreased the turnover of nAChRs by reducing their internalization. A similar response was observed in CHO-K1/A5 cells expressing the adult muscle-type nAChRs. Strikingly, in denervated neuromuscular synapses, lithium led to enhanced nAChR turnover and density by increasing the incorporation of new nAChRs. Lithium also potentiated the formation of unstable nAChR clusters in non-synaptic regions of denervated muscle fibres. We found that denervation-dependent re-expression of the foetal nAChR γ-subunit was not altered by lithium. However, while denervation inhibits the distribution of β-catenin within endplates, lithium-treated fibres retain β-catenin staining in specific foci of the synaptic region. Collectively, our data reveal that lithium treatment differentially affects the stability of postsynaptic receptors in normal and denervated neuromuscular synapses in vivo, thus providing novel insights into the regulatory effects of lithium on synaptic organization and extending its potential therapeutic use in conditions affecting the peripheral nervous system.

KATP channels and NO dilate redundantly intramuscular arterioles during electrical stimulation of the skeletal muscle in mice

Functional hyperemia is fundamental to provide enhanced oxygen delivery during exercise in skeletal muscle. Different mechanisms are suggested to contribute, mediators from skeletal muscle, transmitter spillover from the neuromuscular synapse as well as endothelium-related dilators. We hypothesized that redundant mechanisms that invoke adenosine, endothelial autacoids, and KATP channels mediate the dilation of intramuscular arterioles in mice. Arterioles (maximal diameter: 20–42 µm, n = 65) were studied in the cremaster by intravital microscopy during electrical stimulation of the motor nerve to induce twitch or tetanic skeletal muscle contractions (10 or 100 Hz). Stimulation for 1–60 s dilated arterioles rapidly up to 65% of dilator capacity. Blockade of nicotinergic receptors blocked muscle contraction and arteriolar dilation. Exclusive blockade of adenosine receptors (1,3-dipropyl-8-(p-sulfophenyl)xanthine) or of NO and prostaglandins (nitro-L-arginine and indomethacin, LN + Indo) exerted only a minor attenuation. Combination of these blockers, however, reduced the dilation by roughly one-third during longer stimulation periods (> 1 s at 100 Hz). Blockade of KATP channels (glibenclamide) which strongly reduced adenosine-induced dilation reduced responses upon electrical stimulation only moderately. The attenuation was strongly enhanced if glibenclamide was combined with LN + Indo and even observed during brief stimulation. LN was more efficient than indomethacin to abrogate dilations if combined with glibenclamide. Arteriolar dilations induced by electrical stimulation of motor nerves require muscular contractions and are not elicited by acetylcholine spillover from neuromuscular synapses. The dilations are mediated by redundant mechanisms, mainly activation of KATP channels and release of NO. The contribution of K+ channels and hyperpolarization sets the stage for ascending dilations that are crucial for a coordinated response in the network.

Мyasthenia gravis complicated by comorbid pathology

Acquired myasthenia gravis is a rare pathology of the spectrum of neuromuscular disorders and is characterized by the production of autoantibodies to various components of the neuromuscular synapse. The clinical picture is based on typical symptoms: dynamic ptosis, oculomotor dysfunction, diplopia, masticatory muscle weakness, pathological muscle fatigue, in case of dominance of oropharyngeal muscle weakness, patients complain of dysphagia, dysarthria, dyspnea resulting from insolvency.. This variant of the disease is the most dangerous, as it is characterized by a high risk of myasthenic crisis. In addition, the course of myasthenia gravis may be complicated by the development of comorbid pathology, which can be classified into four main groups. These include causal conditions in the development of diseases with a single mechanism of development for myasthenia gravis, such as cross‑autoimmune damage to the heart muscle. The group of complications of myasthenia includes myasthenic, cholinergic and mixed crises. Unrelated diseases are competitive, so in the elderly, myasthenia gravis is accompanied by a wide range of chronic diseases, such as hypertension, diabetes and others. The last group that should be targeted is intercurrent diseases — acute pathologies that develop against the background of myasthenia. Infectious diseases involving the most vulnerable respiratory system are potentially dangerous in the latter group. Knowledge of these potential factors of decompensation of myasthenia gravis will predict the development and timely diagnose the pathology. Timely treatment in a life‑threatening condition can be critical, since myasthenia gravis differs significantly from other diseases of the neuromuscular system in the rate of critical decompensation and the difficulty of normalizing the patient’s severe condition.

Mechanism of disease and therapeutic rescue of Dok7 congenital myasthenia

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.