Slow-channel Myasthenic Syndrome Research Articles

Caspase-9: A Multimodal Therapeutic Target With Diverse Cellular Expression in Human Disease.

Caspase-9, a cysteine-aspartic protease known for its role as an initiator of intrinsic apoptosis, regulates physiological cell death and pathological tissue degeneration. Its nonapoptotic functions, including regulation of cellular differentiation/maturation, innate immunity, mitochondrial homeostasis, and autophagy, reveal a multimodal landscape of caspase-9 functions in health and disease. Recent work has demonstrated that caspase-9 can drive neurovascular injury through nonapoptotic endothelial cell dysfunction. CASP9 polymorphisms have been linked with various cancers, neurological disorders, autoimmune pathologies and lumbar disc disease. Clinical reports suggest alterations in caspase-9 expression, activity or function may be associated with acute and chronic neurodegeneration, retinal neuropathy, slow-channel myasthenic syndrome, lumbar disc disease, cardiomyopathies, atherosclerosis and autoimmune disease. Healthy tissues maintain caspase-9 activity at low basal levels, rendering supraphysiological caspase-9 activation a tractable target for therapeutic interventions. Strategies for selective inhibition of caspase-9 include dominant negative caspase-9 mutants and pharmacological inhibitors derived from the XIAP protein, whose Bir3 domain is an endogenous highly selective caspase-9 inhibitor. However, the mechanistic implications of caspase-9 expression and activation remain indeterminate in many pathologies. By assembling clinical reports of caspase-9 genetics, signaling and cellular localization in human tissues, this review identifies gaps between experimental and clinical studies on caspase-9, and presents opportunities for further investigations to examine the consequences of caspase activity in human disease.

Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome

Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

A mouse model of the slow channel myasthenic syndrome: Neuromuscular physiology and effects of ephedrine treatment

In the slow channel congenital myasthenic syndrome mutations in genes encoding the muscle acetylcholine receptor give rise to prolonged ion channel activations. The resulting cation overload in the postsynaptic region leads to damage of synaptic structures, impaired neuromuscular transmission and fatigable muscle weakness. Previously we identified and characterised in detail the properties of the slow channel syndrome mutation εL221F. Here, using this mutation, we generate a transgenic mouse model for the slow channel syndrome that expresses mutant human ε-subunits harbouring an EGFP tag within the M3–M4 cytoplasmic region, driven by a ~1500bp region of the CHRNB promoter. Fluorescent mutant acetylcholine receptors are assembled, cluster at the motor endplates and give rise to a disease model that mirrors the human condition. Mice demonstrate mild fatigable muscle weakness, prolonged endplate and miniature endplate potentials, and variable degeneration of the postsynaptic membrane. We use our model to investigate ephedrine as a potential treatment. Mice were assessed before and after six weeks on oral ephedrine (serum ephedrine concentration 89±3ng/ml) using an inverted screen test and in vivo electromyography. Treated mice demonstrated modest benefit for screen hang time, and in measures of compound muscle action potentials and mean jitter that did not reach statistical significance. Ephedrine and salbutamol show clear benefit when used in the treatment of DOK7 or COLQ congenital myasthenic syndromes. Our results highlight only a modest potential benefit of these β2-adrenergic receptor agonists for the treatment of the slow channel syndrome.

Skeletal Muscle IP3R1Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.

Effect of fluoxetine on neuromuscular function in acetylcholinesterase (AChE) knockout mice

Congenital myasthenic syndromes (CMS) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. The causal mutations have been described in number of cases. The slow channel myasthenic syndrome (slow-channel-CMS) results in a marked prolongation of channel opening in stimulated receptors (nAChR) and the end plate acetylcholinesterase (AChE) deficiency congenital myasthenic syndrome (ColQ-CMS) results in an increased action of acetylcholine (ACh) at the synapse. Anticholinesterase medication is detrimental in these cases. The successful treatment of slow-channel-CMS patients with the antidepressant serotonin re-uptake inhibitor fluoxetine has been reported. At high concentration it has a non-depolarizing effect on nicotinic receptors. This led us to the idea that fluoxetine could protect AChR from a relative excess of ACh. We investigated the possible use of fluoxetine as treatment in the AChE KO mouse. Treatment at 6 mg/kg from 3 weeks to 2 months increased slightly the daily weight gain but not the final weight at 2 months in AChE−/− mice. Isometric force production of Tibialis anterior in response to electric nerve stimulation was measured in situ in AChE−/− and wild type mice treated or not by fluoxetine. The results show that the maximum twitch force in response to a single nerve stimulation, the maximal tetanic force (P0) in response to repetitive nerve stimulation and the tetanic fade are not changed in AChE−/− mice treated with fluoxetine versus control AChE−/− mice.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Slow-channel mutation in acetylcholine receptor alphaM4 domain and its efficient knockdown.

To identify the genetic basis of a slow-channel myasthenic syndrome, characterize functional properties of the mutant receptor, and selectively silence the mutant allele. We performed nutation analysis, cloning, and patch-clamp analysis of the functional properties of the mutant receptor; screening for a small interfering RNA with check plasmid; and assessed of the efficacy of small interfering RNA at the messenger RNA, protein, and functional levels. We traced the cause of a slow-channel myasthenic syndrome to a C418W mutation in the M4 domain of the acetylcholine receptor alpha subunit. The mutation is the first one to occur spontaneously in an M4 domain of the receptor, and it is positioned within a stripe of hydrophobic residues facing the lipid bilayer. Kinetic analysis shows that alphaC418W enhances the channel opening equilibrium constant 26-fold without altering agonist affinity. Using a check plasmid as a screening tool, we identified a small interfering RNA that markedly suppresses the mutant but not the wild-type allele at the messenger RNA, protein, and functional levels. alphaC418W occurring in humans causes a slow-channel syndrome by enhancing the relative stability of the channel open state. Efficient and selective knockdown of the mutant allele holds promise of therapeutic gene silencing.

A human congenital myasthenia‐causing mutation (ɛL78P) of the muscle nicotinic acetylcholine receptor with unusual single channel properties

A mutation in the epsilon subunit of the human nicotinic acetylcholine receptor (epsilonL78P) is known to cause a congenital slow channel myasthenic syndrome. We have investigated the changes in receptor function that result in the mutant receptor producing prolonged endplate currents, and consequent muscle damage. The rate constants for channel gating and for the binding and dissociation of acetylcholine were investigated by analysis of single ion channel recordings. A conventional mechanism with two non-equivalent binding sites, and variations upon this mechanism, were fitted to data using a maximum likelihood method that uses the Hawkes-Jalali-Colquhoun (HJC) treatment of missed brief events. The mutant receptor produced prolonged activations, bursts of openings that cause a slow decay of simulated synaptic currents. The main reason for the longer bursts of openings seen with mutant receptor was a decrease in the rate of ACh dissociation from diliganded receptors, though the lifetime of individual openings was somewhat increased too. As well as producing long bursts, the mutant receptor also produced many very short openings, though these carry little current. The burst structure for the mutant receptor at low ACh concentration is unusual in that most long bursts appear to start in a very brief monoliganded open state that then usually binds another ACh molecule to produce a long diliganded activation. The first opening is so short that it will usually be missed (together with the shut time that follows it), so the true burst length is likely to be underestimated.

Mechanistic diversity underlying fast channel congenital myasthenic syndromes.

A host of missense mutations in muscle nicotinic receptor subunits have been identified as the cause of congenital myasthenic syndromes (CMS). Two classes of CMS phenotypes have been identified: slow channel myasthenic syndromes (SCCMSs) and fast channel myasthenic syndromes (FCCMSs). Although both have similar phenotypic consequences, they are physiologic opposites. Expression of the FCCMS phenotype requires the missense mutation to be accompanied by a second mutation, either a null or a missense mutation, in the second allele encoding the same receptor subunit. This seemingly rare scenario has arisen with surprisingly high incidence over the past few years, and analyses of the syndromes have revealed a diverse array of mechanisms underlying the pathology. This review focuses on new mechanisms underlying the FCCMS.

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant epsilonL221F, inferred from maximum likelihood fits

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, eL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The eL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the eL221F mutation the lower affinity is increased so the sites become more similar.

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant epsilonL221F, inferred from maximum likelihood fits.

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.

Introduction

Introduction

Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation, Desensitization, and Agonist Binding Affinity Attributable to Mutation in the M2 Domain of the Acetylcholine Receptor α Subunit

We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation alphaV249F in the propositus and mosaicism for alphaV249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, alphaV249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the alphaV249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.

Block of the endplate acetylcholine receptor channel by the sympathomimetic agents ephedrine, pseudoephedrine, and albuterol

Recent observations suggest that some patients with congenital myasthenic syndromes respond favorably to ephedrine, pseudoephedrine, or albuterol. Conventional microelectrode studies, however, provide no clear explanation for a beneficial effect of ephedrine in endplate diseases. To gain further insight into how these drugs affect neuromuscular transmission, we investigated their effects on the kinetic properties of the acetylcholine (ACh) receptor. Single channel currents were recorded from rat lumbrical muscles endplates using low concentrations of ACh and 2.5–100 μM of drugs. Between 10–100 μM, each drug progressively increased the rate of channel closure in a concentration dependent manner, consistent with an open-channel block. Albuterol acted as a sequential fast-acting channel blocker, increasing the mean burst duration in a concentration dependent manner without altering the total open time per burst or the duration of intraburst blockages. Increasing concentrations of ephedrine and pseudoephedrine also increased the number of intraburst closures but decreased the total open time per burst. None of the drugs altered single channel conductance. The channel blocking effects of ephedrine and pseudoephedrine might reduce the synaptic overactivity that occurs in the slow-channel myasthenic syndromes or in endplate ACh esterase deficiency, but these effects occur at concentrations not attainable in clinical practice.

Mutation of the acetylcholine receptor α subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the α subunit, converting codon 153 from glycine to serine (αG153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered αG153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and αG153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.