[Congenital myasthenic syndromes: difficulties in the diagnosis, course and prognosis, and therapy--The French National Congenital Myasthenic Syndrome Network experience]. / Syndromes myasthéniques congénitaux: difficultés diagnostiques, évolution et pronostic, thérapeutique--l'expérience du réseau national "Syndromes Myasthéniques Congénitaux".

Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders caused by genetic defects affecting neuromuscular transmission and leading to muscle weakness accentuated by exertion. Three different aspects have been investigated by members of the national French CMS Network: the difficulties in making a proper diagnosis; the course and long-term prognosis; and the response to therapy, especially for CMS that do not respond to cholinesterase inhibitors. CMS diagnosis is late in most cases because of confusion with other entities such as: congenital myopathies, due to the frequent presentation in patients of myopathies such as permanent muscle weakness, atrophy and scoliosis, and the abnormalities of internal structure, diameter and distribution of fibers (type I predominance, type II atrophy) seen on biopsy; seronegative autoimmune myasthenia gravis, when CMS is of late onset; and metabolic myopathy, with the presence of lipidosis in muscle. The long-term prognosis of CMS was studied in a series of 79 patients recruited with the following gene mutations: CHRNA; CHRNE; DOK7; COLQ; RAPSN; AGRN; and MUSK. Disease-course patterns (progressive worsening, exacerbation, stability, improvement) could be variable throughout life in a given patient. DOK7 patients had the most severe disease course with progressive worsening: of the eight wheelchair-bound and ventilated patients, six had mutations of this gene. Pregnancy was a frequent cause of exacerbation. Anticholinesterase agents are the first-line therapy for CMS patients, except for cases of slow-channel CMS, COLQ and DOK7. In our experience, 3,4-DAP was a useful complement for several patients harboring CMS with AChR loss or RAPSN gene mutations. Ephedrine was given to 18 patients (eight DOK7, five COLQ, four AGRN and one RAPSN). Tolerability was good. Therapeutic responses were encouraging even in the most severely affected patients, particularly with DOK7 and COLQ. Salbutamol was a good alternative in one patient who was allergic to ephedrine.

Long-term follow-up of patients with congenital myasthenic syndrome caused by COLQ mutations.

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagen-like tail subunit gene (COlQ) cause recessive forms of synaptic CMS with end plate AChE deficiency. We present data on 15 COLQ -mutant CMS carrying 16 different mutations (9 novel ones identified) followed-up for an average period of 10 ears. The mean age at the first examination was 19 ears old (range from 3 to 48). We report relapses during short or long-term periods characterized by worsening of muscle weakness sometimes associated with respiratory crises. All the relapses ended spontaneously or with 3-4 DAP or ephedrine with no residual impairment. The triggering factors identified were esterase inhibitors, effort, puberty or pregnancy highlighting the importance of hormonal factors. There was no genotype-phenotype correlation. At the end of the follow-up, 80% of patients were ambulant and 87% of patients had no respiratory trouble in spite of severe relapses.

Phenotype genotype analysis in 15 patients presenting a congenital myasthenic syndrome due to mutations in DOK7.

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. Mutations of DOK7 have recently been described in recessive forms of CMS. Dok-7 is a cytoplasmic post-synaptic protein co-activator of the muscle-specific receptor-tyrosine kinase (MuSK) involved in neuromuscular synaptogenesis and maintenance. We report clinical, morphological and molecular data on 15 patients with mutations in DOK7. Eleven different mutations (5 novel) were identified and all patients but one were found to carry at least the common c.1124_1127dupTGCC mutation. Patients with DOK7 mutations have a particular limb-girdle pattern, without tubular aggregates but a frequent lipidosis on the muscle biopsy. Changes in pre- and post-synaptic compartments of the neuromuscular junction were also observed in muscle biopsies: terminal axons showed defective branching which resulted in a unique terminal axon contacting en passant postsynaptic cups. Clinical features, muscle biopsy findings or response to therapy were confusing in several patients. Characterization of this distinct phenotype is essential to provide clues for targeted genetic screening and to predict the therapeutic response to anticholinesterase treatments or ephedrine as has been suggested.

[Experimental and pathological changes of the neuromuscular junction]. / Remaniements expérimentaux et pathologiques de la jonction neuromusculaire.

The objective of our study was to investigate the cellular communication between the axon and its postsynaptic targets in the synapse. We used the neuromuscular junction (NMJ) model, which is a highly specialized structure between the nerve, the muscle, and the Schwann cell terminal where the motor neuron orders the muscle to contract. We used experimental models of motor nerve reimplantation in a denervated muscle to determine whether 1) the formation of new NMJ could participate in reinnervation of the muscle necessary to contraction or 2) the blockage of neurotransmitter release using botulinum toxin could be compensated by the formation of new NMJ. We also studied human genetic diseases that affect neuromuscular transmission--congenital myasthenic syndromes--to identify the mutations in the genes coding for synaptic molecules and to analyze the compensatory processes involved in NMJ dysfunction so that muscle contraction can occur in these conditions.

[Tools and techniques dedicated to neuromuscular junction observation]. / Modalités et outils d'observation de la jonction neuromusculaire.

A few decades ago, the neuromuscular junction (NMJ) concept was reduced to two elements: the nerve ending and the facing muscular zone. This description has since changed substantially based on recent studies conducted on the molecular aspects of neurotransmission. The aim of this paper is to provide a synthetic view of the major morphological, molecular and electrophysiological tools used in the analysis of NMJ architecture and its functional characterization.

[Structural and molecular organization, development and maturation of the neuromuscular junction]. / Organisation structurale, moléculaire, formation et maturation de la jonction neuromusculaire.

The neuromuscular junction is made up of the apposition of highly differentiated domains of three types of cell: the motor neuronal ending, the terminal Schwann cell and the muscle postsynaptic membrane. These three components are surrounded by a basal lamina, dedicated to molecular signal exchanges controlling neuromuscular formation, maturation and maintenance. This functional and structural differentiated complex conducts synaptic neurotransmission to the skeletal muscle fiber. Nerve and muscle have distinct roles in synaptic compartment differentiation. The initial steps of this differentiation and the motor endplate formation require several postsynaptic molecular agents including agrin, the tyrosine kinase receptor MuSK. Neuregulin is essentially involved in Schwann cell survival and guidance for axonal growth.

[Morphological study of CNS lesions and the consequences on rat neuromuscular junction and peripheral nerve using confocal laser scanning microscopy and Koelle's technique]. / Existe-t-il un retentissement sur la jonction neuromusculaire de rat lors de lésions du système nerveux central ? Etude morphologique en microscopie confocale et technique de Koelle.

STATE OF THE ART: In humans, it is currently believed that peripheral nerves remain intact after central nervous system (CNS) injuries. This should lead us to observe a lack of amyotrophy in the peripheral projection areas of CNS damage. Nevertheless, the appearance of amyotrophy, described as underuse amyotrophy, is common in victims of CNS injury. Its pathophysiology remains poorly understood and is currently being debated. Amyotrophy could result directly from the structural deterioration of a nervous fiber in the muscular area corresponding to the CNS injury caused by neuromuscular junction (NMJ) changes. AIMS OF THIS STUDY: The aims of this study were to assess the repercussions of a CNS injury on the NMJ and peripheral nerve complex and to evaluate the involvement of peripheral nerves and NMJs in plasticity. METHODOLOGY: Peripheral nerve and muscle biopsies were collected from a group of 35 female Wistar rats that had previously undergone a thoracic spinal cord hemisection (15 rats at the T2 level (group 1), 15 rats at the T6 level (group 2), and 5 matched rats used as controls). We studied the localization and expression of the NMJ molecular components in muscle specimens by immunohistochemistry using confocal microscopy. We also searched for signs of nerve and muscle degeneration using light and electron microscopy. RESULTS: We observed nonpathologic NMJs coexisting with completely denervated and partially reinnervated NMJs. We also found characteristics of embryonic behavior in rat axons secondary to axonal caliber distortions. Some authors associate this decrease in axonal activity with physiological denervation. CONCLUSION: This project was designed to improve the understanding of the mechanisms involved in the interactions between the first and second motoneurons after different types of CNS injuries, with variable functional repercussions. Our results strongly suggest that CNS injuries lead to both morphological and functional repercussions at the NMJ and the peripheral nerve.

The CHRNE 1293insG founder mutation is a frequent cause of congenital myasthenia in North Africa.

OBJECTIVE: Mutations in various genes of the neuromuscular junction cause congenital myasthenic syndrome (CMS). A single truncating mutation (epsilon1293insG) in the acetylcholine receptor epsilon subunit gene (CHRNE) was most often identified in CMS families originating from North Africa and was possibly a founder mutation. METHODS: Twenty-three families were studied with an early onset form of CMS and originating from Tunisia, Algeria, Morocco, and Libya. Screening for the mutation epsilon1293insG was performed by direct sequencing. Haplotype analysis was done with 9 (CA)n repeat microsatellite markers and 6 SNPs flanking epsilon1293insG on chromosome 17p13-p12. Dating was calculated using the ESTIAGE method for rare genetic diseases. RESULTS: The epsilon1293insG mutation was identified in 14 families (about 60% of the initial 23). The expression of the CMS in affected members of these families was relatively homogeneous, without fetal involvement or being life-threatening, with moderate hypotonia and oculobulbar involvement, mild and stable disease course, and good response to cholinesterase inhibitors. Haplotype analysis revealed a common conserved haplotype encompassing a distance of 63 kb. The estimated age of the founder event was at least 700 years. CONCLUSIONS: These results strongly support the hypothesis that epsilon1293insG derives from an ancient single founder event in the North African population. Identification of founder mutations in isolated or inbred populations may have important implications in the context of molecular diagnosis and genetic counseling of patients and families by detection of heterozygous carriers.

Towards the molecular elucidation of congenital myasthenic syndromes: identification of mutations in MuSK.

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and characterized by a dysfunction of the neurotransmission. They are heterogeneous at the pathophysiological level and can be classified in three categories according to their origin: presynaptic, synaptic or postsynaptic. The strategy for the diagnosis and characterization of CMS relies on the clinic, EMG, muscle biopsy, identification of mutations in genes known to be responsible for CMS and the demonstration that the gene mutations are the cause of the disease by using experimental approaches. As an example of such strategy, we report briefly here the characterization of the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed marked pre- and postsynaptic structural abnormalities of the neuromuscular junction as well as a severe decrease in acetylcholine receptor epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The results obtained strongly suggested that the missense mutation, in the presence of a null mutation on the other allele, was responsible for the severe synaptic changes observed in the patient and, hence, is causing the disease. However the molecular origin of a large number of CMS is still unknown. There are hundreds of molecules known to be present at the NMJ and mutations in the genes coding for these synaptic molecules are likely to be responsible for a neuromuscular block.

Congenital myasthenic syndrome due to rapsyn deficiency: three cases with arthrogryposis and bulbar symptoms.

We report the cases of 3 children with postsynaptic congenital myasthenic syndrome with acetylcholine receptor deficiency due to rapsyn deficiency. Symptoms began at the neonatal period with hypotonia, arthrogryposis, bulbar symptoms, and respiratory distress. Two of the 3 children needed tracheostomy and gastrostomy. Electromyograms showed a decremental response to repetitive stimulation. Muscle biopsies were normal or showed type I fiber preponderance. Genetic studies identified mutations in the rapsyn gene (RAPSN). The 3 patients were heterozygous for N88 K and a second mutation (either Y86X, 1083_1084 dupCT or IVS4-2 A > G). The patients responded favorably to anticholinesterase treatment, with a clear improvement of clinical symptoms, especially the bulbar symptoms of apneas and swallowing disturbances. This paper underlines the importance of anticholinesterase medication in patients with congenital myasthenic syndrome due to rapsyn deficiency.

[Congenital myasthenic syndromes due to mutations in the rapsyn gene]. / Syndromes myasthéniques congénitaux dus à des mutations du gène de la rapsyne.

Congenital myasthenic syndromes (CMS) are genetic diseases characterized by dysfunctional neuromuscular transmission and usually start during the neonatal period. Most are due to postsynaptic abnormalities, specifically to mutations in the acetylcholine receptor (AChR) genes. In 2002, the group of A Engel reported the first cases of CMS with mutations in the gene coding rapsyn, a postsynaptic molecule which stabilizes AChR aggregates at the neuromuscular junction. Since this first publication, more than 30 other cases, including six in France, have been reported. Study of these published cases allows us to distinguish three classes of phenotypes: 1) severe neonatal cases; 2) more benign cases, starting during infancy; 3) cases with facial malformations, involving Jewish patients originating from the Near-East. Comparison of the observations of other groups with our own has led us to the following conclusions: the N88K mutation is frequent (homozygous in 50% of cases); besides the N88K mutation, the second mutation varies considerably; heterozygous allelic cases (N88K + another mutation) are severe; there is probably a founder effect in the European population. There is phenotypic variability in the homozygous N88K cases, with benign cases and severe cases of early expression. A Engel and colleagues report that the seven cases of benign CMS with facial malformation, previously described in the Jewish population of Iraq and Iran, were caused by mutation in the promoter region of the rapsyn gene.

[Congenital myasthenic syndromes: phenotypic expression and pathophysiological characterisation]. / Syndromes myasthéniques congénitaux.

Congenital Myasthenic Syndromes (CMS) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. The twenty five past Years saw major advances in identifying different types of CMS due to abnormal presynaptic, synaptic, and postsynaptic proteins. CMS diagnosis requires two steps: 1) positive diagnosis supported by myasthenic signs beginning in neonatal period, efficacy of anticholinesterase medications, positive family history, negative tests for anti-acetylcholine receptor (AChR) antibodies, electromyographic studies (decremental response at low frequency, repetitive CMAP after one single stimulation); 2) pathophysiological characterisation of CMS implying specific studies: light and electron microscopic analysis of endplate (EP) morphology, estimation of the number of AChR per EP, acetylcholinesterase (AChE) expression, molecular genetic analysis. Most CMS are postsynaptic due to mutations in the AChR subunits genes that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh resulting respectively in Slow Channel Syndrome (characterized by a autosomal dominant transmission, repetitive CMAP, refractoriness to anticholinesterase medication) and fast channel, recessively transmitted. AChR deficiency without kinetic abnormalities is caused by recessive mutations in AChR genes (mostly epsilon subunit) or by primary rapsyn deficiency, a post synaptic protein involved in AChR concentration. Recently, mutations in SCN4A sodium channel have been reported in one patient. AChE deficiency is identified on the following data: recessive transmission, presence of repetitive CMAP, refractoriness to cholinesterase inhibitors, slow pupillary response to light and absent expression of the enzyme at EP. This synaptic CMS is caused by mutations in the collagenic tail subunit (ColQ) that anchors the catalytic subunits in the synaptic basal lamina. The most frequent presynaptic CMS is caused by mutations of choline acetyltransferase. Several CMS are still not characterized. Many EP molecules are potential etiological candidates. In these unidentified cases, other methods of investigations are required: linkage analysis, when sufficient number of informative relatives are available, microelectrophysiological studies performed in intercostal or anconeus muscles. Prognosis of CMS, depending on severity and evolution of symptoms, is difficult to assess, and it cannot not be simply derived from mutation identification. Most patients respond favourably to anticholinesterase medications or to 3,4 DAP which is effective not only in presynaptic but also in postsynaptic CMS. Specific therapies for slow channel CMS are quinidine and fluoxetine that normalize the prolonged opening episodes. Clinical benefits derived from the full characterisation of each case include genetic counselling and specific therapy.

Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging.

Tubular aggregates are observed in various muscle disorders and appear as densely packed tubules believed to arise from sarcoplasmic reticulum of striated muscle. They are found both in human skeletal muscle, especially from patients suffering from 'tubular aggregate myopathy' and in fast twitch skeletal muscle of the male inbred mouse during aging. In this work, we studied tubular aggregates present in inbred male mouse skeletal muscle using electron microscopy as well as histochemistry and Western blotting with the main markers of the sarcoplasmic reticulum. We show that mouse tubular aggregates include the proteins SERCA 1, sarcalumenin (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum). We demonstrate also that 95 and 51 kDa triadin isoforms are present in mouse skeletal muscle and are both components of tubular aggregates. These results support the hypothesis that tubular aggregates form a tubular arrangement of a complete sarcoplasmic reticulum containing the junctional, cisternae and longitudinal components of sarcoplasmic reticulum implicated in calcium homeostasis. During mouse skeletal muscle aging, however, densitometry of Western blots reveals a persistent decrease in the expression of the calcium binding protein calreticulin as well as a continuous increase in calsequestrin-like protein expression which both appear unrelated to the tubular aggregate formation.

Expression of the thrombin receptor (PAR-1) during rat skeletal muscle differentiation.

The serine protease thrombin has been proposed to be involved in neuromuscular plasticity. Its specific receptor "protease activated receptor-1" (PAR-1), a G protein-coupled receptor, has been shown to be expressed in myoblasts but not after fusion (Suidan et al., 1996 J Biol Chem 271:29162-29169). In the present work we have investigated the expression of PAR-1 during rat skeletal muscle differentiation both in vitro and in vivo. Primary cultures of rat foetal skeletal muscle, characterized by their spontaneous contractile activity, were used for exploration of PAR-1 by RT-PCR, immunocytochemistry and Western blotting. Our results show that PAR-1 mRNA and protein are both present in myoblasts and myotubes. Incubation of myotubes loaded with fluo-3-AM in presence of thrombin (200 nM) or PAR-1 agonist peptide (SFLLRN, 500 microM), induced the intracellular release of calcium indicating the activation of PAR-1. Blockade of contractile activity by tetrodotoxin (TTX, 6 nM) did not modify either PAR-1 synthesis or its cellular localization. Investigation of PAR-1 on rat muscle cryostat sections at Day 18 of embryogenesis and postnatal Days 1, 5, and 10 indicated that this protein is first expressed in the cytoplasm and that it later localizes to the membrane. Moreover, its expression correlates with myosin heavy chain transitions occurring during post-natal period and is restricted to primary fibers. Taken together, these results suggest that PAR-1 expression is not related to contractile activity but to myogenic differentiation.

Thrombin receptor induction by injury-related factors in human skeletal muscle cells.

Thrombin is involved in tissue repair through its proteolytic activation of a specific thrombin receptor (PAR-1). Previous studies have shown that serine proteases and their inhibitors are involved in neuromuscular junction plasticity. We hypothesized that thrombin could also be involved during skeletal muscle inflammation. Thus we investigated the expression of PAR-1 in human myoblasts and myotubes in vitro and its regulation by injury-related factors. The functionality of this receptor was tested by measuring thrombin's ability to elicit Ca2+ signals. Western blot analysis and immunocytochemistry demonstrated the presence of PAR-1 in myoblasts but not in myotubes unless they were treated by tumor necrosis factor-alpha (10 ng/ml), interleukin-1beta (5 ng/ml), or transforming growth factor-beta(1) (10 ng/ml). The addition of 10 nM alpha-thrombin evoked a strong Ca2+ signal in myoblasts while a limited response in myotubes was observed. However, in the additional presence of injury-related factors, the amplitude of the Ca2+ response was significantly enhanced, representing 88, 65, 48% of their respective basal level, compared to 27% of that obtained in controls. Moreover, immunochemical studies on human skeletal muscle biopsies of patients suffering from inflammatory myopathies showed an overexpression of PAR-1. These results suggest that PAR-1 synthesis may be induced in response to muscle injury, thereby implicating thrombin signaling in certain muscle inflammatory diseases.

Developmental regulation of amyloid precursor protein at the neuromuscular junction in mouse skeletal muscle.

Amyloid precursor protein (APP), associated with Alzheimer's disease plaques, is known to be present in synapses of the brain and in the adult neuromuscular junction (NMJ). In the present study we examined protein and gene expression of APP during the development of mouse skeletal muscle. Using immunocytochemical approaches, we found that APP is first detected in myotube cytoplasm at embryonic day 16 and becomes progressively concentrated at the NMJ beginning at birth until adulthood. The colocalization between APP and acetylcholine receptors at the NMJ is only partial at birth, but becomes complete upon reaching adulthood. We observed that all APP isoforms, including the Kunitz-containing (protease inhibitor or KPI) forms, are up-regulated from birth to postnatal day 5 and then decreased to reach the low levels observed in the adult. This suggests the involvement of APP during the events which lead to a mature mono-innervated synapse. A 92-kDa band, characteristic of a cleaved APP695 isoform and not due to a new muscle-specific alternative spliced form, was observed from postnatal day 15 following completion of polyneuronal synapse elimination. Taken together, these data suggest that skeletal muscle APP may well play a role in the differentiation of skeletal muscle and in the formation and maturation of NMJs.

Thrombomodulin as a new marker of lesion-induced astrogliosis: involvement of thrombin through the G-protein-coupled protease-activated receptor-1.

Because injury of the CNS causes an astrogliosis, characterized by cell swelling and proliferation, similar to the effects of the serine protease thrombin on astrocytes, we hypothesized that a high level of thrombin at the site of injury might initially induce an astrocyte reaction and later increase the expression of its specific inhibitor, thrombomodulin. Thrombomodulin could then stabilize the astroglial scar through its adhesive properties. Here, we studied the in vivo injury response of astrocytes in the anterior medullary velum of adult rat by immunostaining and in situ hybridization of thrombomodulin. Thrombomodulin was poorly expressed on astrocytes in normal tissue, increased up to 2 d after injury, and was still highly expressed at 6 d. To check that thrombin had a direct effect on thrombomodulin expression by astrocytes, we used brain cortical astrocyte primary cultures treated with either thrombin or the agonist peptide thrombin receptor-activating peptide-6, known to activate directly the thrombin G-protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR-1). Modification of thrombomodulin expression was studied by Western blotting and quantitative reverse transcription-PCR. There was a dose-dependent increase in thrombomodulin after 48 hr of treatment, with gene expression peaking at 24 hr but falling to control levels by 48 hr. Together, these results show the following: (1) injury increases astrocyte thrombomodulin expression; (2) thrombin might mediate thrombomodulin expression via the specific receptor PAR-1; and (3) serine proteases, their inhibitors, and the new family of GPCR, PARs, are active on astrogliosis.