Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration.

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.

How Inflammation Pathways Contribute to Cell Death in Neuro-Muscular Disorders.

Neuro-muscular disorders include a variety of diseases induced by genetic mutations resulting in muscle weakness and waste, swallowing and breathing difficulties. However, muscle alterations and nerve depletions involve specific molecular and cellular mechanisms which lead to the loss of motor-nerve or skeletal-muscle function, often due to an excessive cell death. Morphological and molecular studies demonstrated that a high number of these disorders seem characterized by an upregulated apoptosis which significantly contributes to the pathology. Cell death involvement is the consequence of some cellular processes that occur during diseases, including mitochondrial dysfunction, protein aggregation, free radical generation, excitotoxicity and inflammation. The latter represents an important mediator of disease progression, which, in the central nervous system, is known as neuroinflammation, characterized by reactive microglia and astroglia, as well the infiltration of peripheral monocytes and lymphocytes. Some of the mechanisms underlying inflammation have been linked to reactive oxygen species accumulation, which trigger mitochondrial genomic and respiratory chain instability, autophagy impairment and finally neuron or muscle cell death. This review discusses the main inflammatory pathways contributing to cell death in neuro-muscular disorders by highlighting the main mechanisms, the knowledge of which appears essential in developing therapeutic strategies to prevent the consequent neuron loss and muscle wasting.

Alleviating Sepsis-Induced Neuromuscular Dysfunction Linked With Acetylcholine Receptors by Agrin.

BACKGROUND: Abnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by sepsis can lead to neuromuscular dysfunction. Here, we asked whether neural agrin regulates nAChRs to ameliorate muscle function, which could be associated with the agrin/muscle-specific kinase pathway. METHODS: Rats were subjected to cecal ligation and puncture (CLP) group, sham group, or control group to observe the alteration caused by sepsis. To verify the effect of improving function, rats were injected with agrin or normal saline intramuscularly after CLP. Electromyogram was used to measure neuromuscular function. Cytokines levels of serum and the expression of related proteins and mRNA were tested after treatment. RESULTS: Compared with the rats in control or sham group, CLP-treated rats showed an acute inflammatory status and a reduction of neuromuscular dysfunction in tibialis anterior muscle, which was associated with abnormal expression in agrin/muscle-specific kinase pathway and increased expression of γ- and α7-nAChR. Exogenous agrin alleviated neuromuscular dysfunction and decreased the expression of γ- and α7-nAChR through agrin-related signaling pathway. CONCLUSIONS: The decreased expression of agrin may lead to skeletal muscle dysfunction. Early enhancement of intramuscular agrin levels after sepsis may be a potential strategy for the treatment of sepsis-induced muscle dysfunction.

Synaptic Homeostasis and Its Immunological Disturbance in Neuromuscular Junction Disorders.

In the neuromuscular junction, postsynaptic nicotinic acetylcholine receptor (nAChR) clustering, trans-synaptic communication and synaptic stabilization are modulated by the molecular mechanisms underlying synaptic plasticity. The synaptic functions are based presynaptically on the active zone architecture, synaptic vesicle proteins, Ca2+ channels and synaptic vesicle recycling. Postsynaptically, they are based on rapsyn-anchored nAChR clusters, localized sensitivity to ACh, and synaptic stabilization via linkage to the extracellular matrix so as to be precisely opposed to the nerve terminal. Focusing on neural agrin, Wnts, muscle-specific tyrosine kinase (a mediator of agrin and Wnts signalings and regulator of trans-synaptic communication), low-density lipoprotein receptor-related protein 4 (the receptor of agrin and Wnts and participant in retrograde signaling), laminin-network (including muscle-derived agrin), extracellular matrix proteins (participating in the synaptic stabilization) and presynaptic receptors (including muscarinic and adenosine receptors), we review the functional structures of the synapse by making reference to immunological pathogenecities in postsynaptic disease, myasthenia gravis. The synapse-related proteins including cortactin, coronin-6, caveolin-3, doublecortin, R-spondin 2, amyloid precursor family proteins, glia cell-derived neurotrophic factor and neurexins are also discussed in terms of their possible contribution to efficient synaptic transmission at the neuromuscular junction.

Linkage of Stress with Neuromuscular Disorders.

Aging is associated with a progressive loss of muscle strength and mass, and a decline in neurophysiologic functions, which are characteristic features of neuromuscular disorders (NMDs). Understanding aging induced neuromuscular junction (NMJ) dysfunction is very crucial to understand the mechanism underlying NMDs. Morphological and physiological changes result in remodelling of the motor unit and a decline in the number of motor neuron muscle fibres. These alterations lead to excitation-contraction uncoupling and a loss of communication between the neuromuscular system, causing a decline in skeletal muscle strength and muscle mass. Understanding the molecular basis of NMJ dysfunction is essential in search for new treatment options. Besides structural and molecular studies, search for animal models to establish connection between brain and muscle is needed. Among various factors it has been observed that stress is one of the leading causes of NMDs. In the present review, we aim to explore various factors linking stress and NMDs neuromuscular disorders which gets aggravated by aging, with a special emphasis on mitochondrial connection. This in turn will help us gain new insights in the treatment of NMDs by aiding in improved symptoms, increased mobility and prolonged life.

Structural and Functional Abnormalities of the Neuromuscular Junction in the Trembler-J Homozygote Mouse Model of Congenital Hypomyelinating Neuropathy.

Mutations in peripheral myelin protein 22 (PMP22) result in the most common form of Charcot-Marie-Tooth (CMT) disease, CMT1A. This hereditary peripheral neuropathy is characterized by dysmyelination of peripheral nerves, reduced nerve conduction velocity, and muscle weakness. APMP22 point mutation in L16P (leucine 16 to proline) underlies a form of human CMT1A as well as the Trembler-J mouse model of CMT1A. Homozygote Trembler-J mice (Tr(J)) die early postnatally, fail to make peripheral myelin, and, therefore, are more similar to patients with congenital hypomyelinating neuropathy than those with CMT1A. Because recent studies of inherited neuropathies in humans and mice have demonstrated that dysfunction and degeneration of neuromuscular synapses or junctions (NMJs) often precede impairments in axonal conduction, we examined the structure and function of NMJs in Tr(J)mice. Although synapses appeared to be normally innervated even in end-stage Tr(J)mice, the growth and maturation of the NMJs were altered. In addition, the amplitudes of nerve-evoked muscle endplate potentials were reduced and there was transmission failure during sustained nerve stimulation. These results suggest that the severe congenital hypomyelinating neuropathy that characterizes Tr(J)mice results in structural and functional deficits of the developing NMJ.

Congenital myopathies with secondary neuromuscular transmission defects; a case report and review of the literature.

Congenital myopathies are a clinically and genetically heterogeneous group of disorders characterized by early onset hypotonia, weakness and characteristic, but not pathognomonic, structural abnormalities in muscle fibres. The clinical features overlap with muscular dystrophies, myofibrillar myopathies, neurogenic conditions and congenital myasthenic syndromes. We describe a case of cap myopathy with myasthenic features due to a mutation in the TPM2 gene that responded to anticholinesterase therapy. We also review other published cases of congenital myopathies with neuromuscular transmission abnormalities. This report expands the spectrum of congenital myopathies with secondary neuromuscular transmission defects. The recognition of these cases is important since these conditions can benefit from treatment with drugs enhancing neuromuscular transmission.

Chronic proximal axonopathy in rats is associated with long-standing neurofilament depletion in neuromuscular junctions and behavioral deficits.

In rodents exposed to 3,3'-iminodipropionitrile (IDPN), neurofilaments (NFs) accumulate in swollen proximal axon segments; this also occurs in motor neurons of patients with amyotrophic lateral sclerosis. We hypothesized that early loss of NFs in neuromuscular junctions (NMJs) in IDPN proximal neuropathy would result in neuromuscular dysfunction and lead to neuromuscular detachment. Adult male rats were given 0 or 15 mmol/L IDPN in drinking water for up to 1 year. The IDPN-exposed rats dragged their tails and had impaired endurance in a grip test. Neuromuscular junctions and distal axons were examined in the levator auris longus muscle after 3, 6, 9, and 12 months. Neuromuscular junctions showed a progressive reduction in NF immunolabeling, which became undetectable in up to 70% of the NMJs after 12 months. Neurofilament labeling was also reduced in preterminal axons and in a more proximal axon level within the muscle. Triple-label analysis with antisyntaxin demonstrated that the terminals remained in place and usually contained a few minute NF bundles. Electron microscopy revealed the disappearance of terminal NFs, reduced content in synaptic vesicles, and accumulation of multilamellar bodies, but scant degeneration. Thus, IDPN proximal neurofilamentous axonopathy is associated with NF depletion in motor terminals; motor weakness and structural changes in the NMJs suggest impaired synaptic function despite long-term preservation of the NMJs.

Morphological analysis of neuromuscular junction development and degeneration in rodent lumbrical muscles.

BACKGROUND: The neuromuscular junction (NMJ) is a specialised synapse formed between a lower motor neuron and a skeletal muscle fibre, and is an early pathological target in numerous nervous system disorders, including amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA). Being able to accurately visualise and quantitatively characterise the NMJ in rodent models of neurological conditions, particularly during the early stages of disease, is thus of clear importance. NEW METHOD: We present a method for dissection of rodent deep lumbrical muscles located in the hind-paw, and describe how to perform immunofluorescent morphological analysis of their NMJs. RESULTS: These techniques allow the temporal assessment of a number of developmental and pathological NMJ phenotypes in lumbrical muscles. COMPARISON WITH EXISTING METHODS: Small muscles, such as the distal hind-limb lumbrical muscles, possess a major advantage over larger muscles, such as gastrocnemius, in that they can be whole-mounted and the entire innervation pattern visualised. This reduces preparation time and ambiguity when evaluating important neuromuscular phenotypes. CONCLUSIONS: Together, these methods will allow the reader to perform a detailed and accurate analysis of the neuromuscular system in rodent models of disease in order to identify pertinent features of neuropathology.

Motor endplate disease affects neuromuscular junction maturation.

Postnatal formation of the neuromuscular synapse requires complex interactions among nerve terminal, muscle fibres and terminal Schwann cells. In motor endplate disease (med) mice, neuromuscular transmission is severely impaired without alteration of axonal conduction and a lethal paralytic phenotype occurs during the postnatal period. The med phenotype appears at a crucial stage of the neuromuscular junction development, corresponding to the increase in terminal Schwann cell number, the elimination of the multiple innervations and the pre- and postsynaptic maturation. Here we investigated the early cellular and molecular consequences of the med mutation on neuromuscular junction development. We observed that cellular defects preceded overt clinical phenotype. The first detectable cellular effect of the mutation at the onset of the clinical phenotype was a drastic reduction in the number of terminal Schwann cells, in part due to an increase in glial apoptosis, and a delayed maturation of motor endplates. We also showed that, in terminally ill animals, mono-innervation was not achieved, synaptic vesicles had accumulated in the presynaptic compartment and, finally, the size of motor endplates was reduced. All together, our findings suggested that the clinical weakness in these mutant mice was likely to be related to postnatal structural abnormalities of the neuromuscular junction maturation.

Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice.

Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.

Motor neuron, nerve, and neuromuscular junction disease.

PURPOSE OF REVIEW: The aim is to review the most relevant findings published during the last year concerning clinical, genetic, pathogenic, and therapeutic advances in motor neuron disease, neuropathies, and neuromuscular junction disorders. RECENT FINDINGS: Studies on animal and cell models have improved the understanding of how mutated survival motor neuron protein in spinal muscular atrophy governs the pathogenetic processes. New phenotypes of SOD1 mutations have been described. Moreover, animal models enhanced the insight into the pathogenetic background of sporadic and familial amyotrophic lateral sclerosis. Novel treatment options for motor neuron disease have been described in humans and animal models. Considerable progress has been achieved also in elucidating the genetic background of many forms of inherited neuropathies and high clinical and genetic heterogeneity has been demonstrated. Mutations in MuSK and GFTP1 have been shown to cause new types of congenital myasthenic syndromes. A third type of autoantibodies (Lrp4) has been detected to cause myasthenia gravis. SUMMARY: Advances in the clinical and genetic characterization of motor neuron diseases, neuropathies, and neuromuscular transmission defects have important implications on the fundamental understanding, diagnosis, and management of these disorders. Identification of crucial steps of the pathogenetic process may provide the basis for the development of novel therapeutic strategies.

Defective neuromuscular junction organization and postnatal myogenesis in mice with severe spinal muscular atrophy.

A detailed pathologic analysis was performed on Smn(-/-);SMN2 mice as a mouse model for human type I spinal muscular atrophy (SMA). We provide new data concerning changes in the spinal cord, neuromuscular junctions and muscle cells, and in the organs of the immune system. The expression of 10 synaptic proteins was analyzed in 3-dimensionally reconstructed neuromuscular junctions by confocal microscopy. In addition to defects in postsynaptic occupancy, there was a marked reduction in calcitonin gene-related peptide and Rab3A in the presynaptic motor terminals of some, but not all, of the skeletal muscles analyzed. Defects in the organization of presynaptic nerve terminals were also detected by electron microscopy. Moreover, degenerative changes in muscle cells, defective postnatal muscle growth, and prominent muscle satellite cell apoptosis were also observed. All of these changes occurred in the absence of massive loss of spinal cord motoneurons. On the other hand, astroglia, but not microglia, increased in the ventral horn of newborn SMA mice. In skeletal muscles, the density of interstitial macrophages was significantly reduced, and monocyte chemotactic protein-1 was downregulated. These findings raise questions regarding the primary contribution of a muscle cell defect to the SMA phenotype.

Neuromuscular pathology in mice lacking alpha-synuclein.

This work was undertaken in order to study the possible role of alpha-synuclein in the function of the neuro-muscular junction in skeletal muscles. Repeated stimulation of skeletal muscle motor neurons revealed signs of neuromuscular pathology in alpha-synuclein null mutated (C57Bl/6JOlaHsd) and knockout (B6;129X1-Snca(tm1Rosl)/J) mice. This stimulation produced repetitive compound muscle action potentials in both lines of alpha-synuclein deficient mice. Muscle strength and muscle coordination during ambulation were unaffected, though motor learning was slower in alpha-synuclein deficient mice in the Rotarod test. We conclude that alpha-synuclein may play a role in acetylcholine compartmentalization at the neuromuscular junction, and in the fine control of activity of skeletal muscles.

A review of autoimmune diseases associated with cancer.

The focus of this review is on the relationships between autoimmune diseases and cancer from two closely related perspectives: 1.Those autoimmune diseases which are often associated with malignancies. 2.Those prevalent cancers which may increase the risks of developing autoimmune disorders. The review concludes with a brief discussion of some selected innovative approaches to cancer immunotherapy.

The neuroprotective factor Wld(s) fails to mitigate distal axonal and neuromuscular junction (NMJ) defects in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disorder in humans. Amongst the earliest signs of neurodegeneration are severe and progressive defects of the neuromuscular synapse. These defects, characterized by poor terminal arborization and immature motor endplates, presumably result in a loss of functional synapses. The slow Wallerian degeneration (Wld(s)) mutation in rodents has been shown to have a protective effect on mouse models of motor neuron disease by retarding axonal die-back and preventing neuromuscular synapse loss. In this study we tested the effects of the Wld(s) mutation on the disease phenotype of SMA model mice. Consistent with previous reports, the mutation slows axon and neuromuscular synapse loss following nerve injury in wild-type as well as in SMA mice. However, the synaptic defects found in severely affected SMA patients and model mice persist in the double (Wld(s);SMA) mutants. No delay in disease onset was observed and survival was not significantly altered. Finally, Wld(s) had no effect on the striking phrenic nerve projection defects that we discovered in SMA model mice. Our results indicate that the reported protective effects of Wld(s) are insufficient to mitigate the neuromuscular phenotype due to reduced SMN protein, and that the mechanisms responsible for distal defects of the motor unit in SMA are unlikely to be similar to those causing neurodegeneration in genetic mutants such as the pmn mouse which is partially rescued by the Wld(s) protein.

The spectrum of intermediate syndrome following acute organophosphate poisoning: a prospective cohort study from Sri Lanka.

BACKGROUND: Intermediate syndrome (IMS) is a major cause of death from respiratory failure following acute organophosphate poisoning. The objective of this study was to determine repetitive nerve stimulation (RNS) predictors of IMS that would assist in patient management and clinical research. METHODS AND FINDINGS: Seventy-eight consenting symptomatic patients with organophosphate poisoning were assessed prospectively with daily physical examination and RNS. RNS was done on the right and left median and ulnar nerves at 1, 3, 10, 15, 20, and 30 Hz. The study was conducted as a prospective observational cohort study in the Central Province, Sri Lanka. IMS was diagnosed in ten out of 78 patients using a priori clinical diagnostic criteria, and five of them developed respiratory failure. All ten patients showed progressive RNS changes correlating with the severity of IMS. A decrement-increment was observed at intermediate and high frequencies preceding the onset of clinical signs of IMS. As the patient developed clinical signs of IMS, decrement-increment was progressively noted at low and intermediate frequencies and a combination of decrement-increment and repetitive fade or severe decrement was noted at high frequencies. Severe decrement preceded respiratory failure in four patients. Thirty patients developed forme fruste IMS with less severe weakness not progressing to respiratory failure whose RNS was characterized by decrement-increment or a combination of decrement-increment and repetitive fade but never severe decrements. CONCLUSIONS: Characteristic changes in RNS, preceding the development of IMS, help to identify a subgroup of patients at high risk of developing respiratory failure. The forme fruste IMS with the characteristic early changes on RNS indicates that IMS is a spectrum disorder. RNS changes are objective and precede the diagnosis and complications of IMS. Thus they may be useful in clinical management and research.

Atrophy and degeneration in sciatic nerve of presymptomatic mice carrying the Huntington's disease mutation.

Huntington's disease (HD) is a progressive neurological disorder characterised by motor impairments caused by degeneration in the striatum. The mechanism by which the HD mutation leads to the neurodegenerative pathology of HD is still unknown. Recently it was shown that, in HD patients, early pathological changes in white matter precede selective cell death in the striatum. We wondered whether axonal pathology is also an early pathological feature in a transgenic mouse model carrying the HD mutation (R/2 line). R6/2 mice show brain atrophy, a progressive neurological deterioration and skeletal muscle atrophy that resemble those seen in HD patients. However, there is very little neuronal cell loss seen in these animals, even when they show severe symptoms. Here we used sciatic nerve to look for evidence of early neurodegenerative changes in axons of the R6/2 mouse at an ultrastructural level. We observed ultrastructural changes that preferentially affected large myelinated fibres of the sciatic nerve in 10-week-old asymptomatic R6/2 mice. The changes included a significant decrease in the axoplasm diameter of myelinated neurons and an increase in the number of degenerating myelinated fibres compared to age-matched wild type littermates. Myelin thickness and unmyelinated fibre diameter were not affected. The abnormalities described here precede the appearance of overt motor symptoms in the R6/2 mouse and occur in parallel with pathophysiological changes at the neuromuscular junction. We suggest that degenerative changes in axons are likely to contribute to the early pathological phenotype in HD, even in the absence of frank neuronal cell loss.

Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition.

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.