Jeanine Koenig

Spontaneous contractile activity and the presence of the 16 S form of acetylcholinesterase in rat muscle cells in culture: Reversible suppressive action of tetrodotoxin

Abstract Multiple molecular forms of acetylcholinesterase are found in rat primary muscle cell cultures (mainly 4 S, 6.5 S, 10 S, 12 S, and 16 S). During differenciation (myoblasts → immature myotubes → spontaneously contracting, mature myotubes) a precise developmental pattern in total AChE activity in the relative proportion of the different molecular forms have been found. Total AChE increases when myoblasts fusion occurs: later the 16 S form is induced (about 1 day after spontaneous contractions are first noticed in culture). Tetrodotoxin (TTX), which reversibly blocks the contractile activity, acts on the presence of the 16 S form. The 16 S form disappears upon addition of 1 μ M TTX, with a half-life of about 1 day. After removal of TTX, it reappears into the myotubes, is fully restored, after about 2 days, to control levels, and does not seem to be focally distributed on the surface of the myotubes. This culture system seems to be very well suited for studies on the cellular regulation of the high molecular weight forms of AChE.

Single acetylcholine-activated channel currents in developing muscle cells☆

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for greater than 95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and -80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2-3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1-2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 micron ACh.

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.

Possible founder effect of rapsyn N88K mutation and identification of novel rapsyn mutations in congenital myasthenic syndromes

Congenital myasthenic syndromes (CMS) constitute a group of rare diseases heterogeneous both in terms of their mode of hereditary transmission (recessive and dominant forms) and their pathophysiology (with presynaptic, synaptic, and postsynaptic defects). They are responsible for dysfunction of neuromuscular transmission giving rise to a condition of muscle weakness which is accentuated by exertion. In most cases, CMS begin in early childhood but later onset in adulthood is possible. Severity also varies from severe with respiratory failure to mild expression.1,2 The majority of CMS primarily affect postsynaptic function and are the result of mutations located in the muscle acetylcholine receptor (AChR) subunit genes that lead to kinetic abnormalities or to AChR deficiency. For example, an increased response occurs in the slow channel syndromes associated with dominant mutations in AChR subunits delaying channel closure or increasing the affinity of the receptor for acetylcholine. However, the most commonly encountered CMS is a deficiency in AChR which occurs with recessive mutations. Most of these mutations are located in the AChR e subunit.3,4 Recent advances have shown that mutations in rapsyn are also involved in recessive forms of postsynaptic CMS and cause AChR deficiency.5 Rapsyn is a 43 kDa postsynaptic protein involved in development and maintenance of the molecular architecture of the postsynaptic membrane by participating in the clustering of AChR after binding of neural agrin to its muscle specific receptor tyrosine kinase, MuSK.6,7 The rapsyn gene ( RAPSN ) has been mapped to chromosome 11p11.2 p11.1 and comprises eight exons.8 The primary structure of rapsyn is predicted to contain several functional domains such as an N-terminus myristoylation signal, seven tetratricopeptide repeats (residues 6-279), a coiled-coiled domain (298-331), a cysteine rich RING-H2 domain (363-402), and a serine phosphorylation site at codon 406.9,10 The aim of this …

Biochemical Stability of the AChE Molecular Forms after Cytochemical Staining: Postnatal Focalization of the 16S AChE in Rat Muscle

A biochemical analysis of rat muscle acetylcholinesterase (AChE) is possible after a cytochemical staining at pH7 (Koelle and Friedenwald method). The solubilization properties, the physicochemical characteristics (sedimentation coefficients) of the multiple molecular forms of AChE are similar before and after the cytochemical procedure without fixation and without ammonium sulfide treatment. This is in contrast to the partial inactivation obtained after conventional pH 5 cytochemical staining, which selectively inactivates 16S AChE. We found that pH 7 cytochemical staining in these same conditions afford a satisfying morphological visualization of the motor end-plate AChE. The combined cytochemical-biochemical techniques allow a very precise dissection of motor end-plate containing (neural) and free (aneural) regions. In rat muscle (sternomastoid), high levels of 16S AChE are present in the aneural region in the first stages of postnatal development. At the end of the 1st month after birth, the 16S AChE becomes restricted to the neural (motor end-plate) region.

Two novel mutations in the COLQ gene cause endplate acetylcholinesterase deficiency

Congenital myasthenic syndromes with endplate acetylcholinesterase deficiency are very rare autosomal recessive diseases, characterized by onset of the disease in childhood, general weakness increased by exertion, ophthalmoplegia and refractoriness to anticholinesterase drugs. To date, all reported cases are due to mutations within the gene encoding ColQ, a specific collagen that anchors acetylcholinesterase in the basal lamina at the neuromuscular junction. We identified two new cases of congenital myasthenic syndromes with endplate acetylcholinesterase deficiency. The two patients showed different phenotypes. The first patient had mild symptoms in childhood, which worsened at 46 years with severe respiratory insufficiency. The second patient had severe symptoms from birth but improved during adolescence. In both cases, the absence of acetylcholinesterase was demonstrated by morphological and biochemical analyses, and heteroallelic mutations in the COLQ gene were found. Both patients presented a novel splicing mutation (IVS1-1G-->A) affecting the exon encoding the proline-rich attachment domain (PRAD), which interacts with acetylcholinesterase. This splicing mutation was associated with two different mutations, both of which cause truncation of the collagen domain (a known 788insC mutation belonging to one patient and a novel R236X to the other) and may impair its trimeric organization. The close similarity of the mutations of these two patients with different phenotypes suggests that other factors may modify the severity of this disease.

A Mutation Causes MuSK Reduced Sensitivity to Agrin and Congenital Myasthenia

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient’s biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.

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 (ε1293insG) 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 ε1293insG was performed by direct sequencing. Haplotype analysis was done with 9 (CA)n repeat microsatellite markers and 6 SNPs flanking ε1293insG on chromosome 17p13-p12. Dating was calculated using the ESTIAGE method for rare genetic diseases. Results: The ε1293insG 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 ε1293insG 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.

Establishment of Neuromuscular Contacts in Cultures of Rat Embryonic Cells: Effect of Tetrodotoxin on Maturation of Muscle Fibers and on Formation and Maintenance of Acetylcholinesterase and Acetylcholine Receptor Clusters

The part played by muscle activity was mainly studied in relation to the formation and persistence of cytochemically detectable spots of acetylcholinesterase (AChE) during synaptogenesis. A correlation was found between AChE spot evolution and that of two other morphological synapse markers, silver salt-impregnated nerve endings and clusters of acetylcholine receptors (AChR) exposed to 125I-alpha-bungarotoxin and revealed by radioautography. The formation and persistence of AChE spots were activity-dependent but in the case of neuronally induced synaptic nerve endings and AChR clusters, both morphological markers were independent of such activity. The final evolution and maturation of neuromuscular contacts were linked to the differentiation stage reached by the muscle fibers at the time when they established contact with nerve endings. The fibers acquired this differentiated state independently of tetrodotoxin-blocked muscle activity.

Thrombin reduces MuSK and acetylcholine receptor expression along with neuromuscular contact size in vitro.

In the course of studies on thrombin and its inhibitor(s) in synaptic plasticity, we addressed the question of their roles in the formation of neuromuscular junctions (NMJ) and used a model of rat neuron–myotube cocultures. We report that the size of acetylcholinesterase (AChE) patches used as a marker of neuromuscular contacts was decreased in the presence of either thrombin or SFLLRN, the agonist peptide of the thrombin receptor PAR‐1, whereas it was increased with hirudin, a specific thrombin inhibitor. In an attempt to relate these neuromuscular contact size variations to molecular changes, we studied muscle‐specific tyrosine kinase receptor (MuSK), acetylcholine receptor (AChR) and rapsyn expression in the presence of thrombin. We showed that thrombin did not change rapsyn gene and protein expression. However, the expression of MuSK and surface AChR proteins was diminished in both myotube cultures and neuron–myotube cocultures. These reductions in protein expression were associated with a decrease in MuSK and AChR α‐subunit gene expression in myotube cultures but not in neuron–myotube cocultures. Moreover, the expression of the AChR ε‐subunit gene, specifically enhanced by neuron‐released factors, was not modified by thrombin in neuron–myotube cocultures. This suggests that thrombin did not affect the expression of synaptic AChRs enhanced by neuron‐released factors but rather reduced the level of extrasynaptic AChRs. Taken together, these results indicate that thrombin in balance with its inhibitor(s) could modulate the formation of neuromuscular contacts in vitro by affecting the expression of two essential molecules in NMJ postsynaptic differentiation, MuSK and AChR.