Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions

Neurological comorbidities

Two years ago, only one form of presynaptic CMS had been identified, linked to loss-of-function mutations in the gene encoding the presynaptic choline acetyltransferase enzyme ChAT (Fig. 1). The number of presynaptic forms of CMS is now 7, with an extremely low frequency of occurrence in all of them which, with the exception of CHAT and SLC5A7 related forms (Table 1), have only been reported in isolated individuals. SLC5A7 encodes the hemicholinium sensitive-high-affinity choline transporter 1 (CHT1), expressed by all cholinergic neurons, which imports choline, generated by hydrolysis of ACh by acetylcholinesterase (AChE), from the synaptic cleft into the nerve terminal (Fig. 1). One dominant-negative frameshift mutation in this gene has been reported to cause hereditary motor neuropathy [16]. On the other hand, loss-of-function mutations (missense and nonsense) in SLC5A7 cause CMS with episodic apnea (EA) during early infancy (CMS-EA), as do loss-of-function mutations in CHAT. A third gene recently shown to cause CMS-EA is SLC18A3, which encodes the vesicular ACh transporter (VAChT) required for uptake of newly synthesized ACh into presynaptic vesicles [18, 19].

These three presynaptic proteins, critical for cholinergic metabolism, are expressed in all cholinergic neurons. It is well known that nicotinic cholinergic neurotransmission plays an important role in “higher-level” functions such as behavior and cognition [20]. Reflecting this role, five of the eight infants with CMS-EA due to SLC5A7 or SLC18A3 mutations suffer from severe cognitive impairment (Table 2). Whether this comorbidity results from episodes of brain hypoxia during EA, or is a direct consequence of defective central cholinergic neurons, has to be further investigated. Sudden apneic episodes are one of the most severe clinical features of CMS. They have sometimes been reported in CMS as a result of mutations in genes encoding subunits of AChR (fast channel syndrome), rapsyn and the skeletal muscle voltage-gated sodium channel Na_V1.4. However, the systematic association of EA with loss-of-function in genes encoding neuronal proteins critical for presynaptic cholinergic homeostasis (uptake, local synthesis and vesicular upload of the neurotransmitter, Table 2) suggests that these episodes result from the critical role of cholinergic transmission in respiratory functions during development [21, 22].

A further presynaptic group of CMS, due to mutations in genes encoding members of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex, have also emerged from high-throughput sequencing of human samples. This complex drives the docking or fusion of the synaptic vesicles (SV) with the presynaptic motoneuronal membrane (Fig. 1). Dominant mutations in SYT2 and SNAP25B genes, encoding the synaptotagmin 2 and the SNAP25B proteins of the vesicle-associated (v-SNARE) and presynaptic membrane-associated (t-SNAREs) complex respectively, were shown to cause CMS without (SYT2) or with (SNAP25B) neurological comorbidities in individuals with isolated forms of CMS (cortical hyperexcitability, cerebellar ataxia, and intellectual disability, Table 2) [14, 23].

The absence of signs suggesting defective high-level functions in individuals with SYT2 dominant mutations may be due to the functional redundancy of synaptotagmin paralogs and to the restricted expression of the B isoform of synaptotagmin 2 encoded by this gene. However, they presented with a Charcot-Marie Tooth-like clinical pattern in two unrelated families [13, 23]. In one family, affected members developed foot deformities in childhood including pes cavus and hammer toes. In the second family, congenital hip dislocation was reported in addition to foot deformities. Neurophysiological study showed a Lambert-Eaton Myasthenic Syndrome-like pattern with low-amplitude of CMAP and marked increment after brief exercise (see below). In addition, a slight reinnervation pattern on needle electromyography of distal muscles, consistent with motor neuropathy was recorded in one family [13]. This CMS-like form benefited both at the clinical and neurophysiological level from treatment with 3,4-diaminopyridine (DAP), a chemical inhibitor of presynaptic K⁺ channels.

The clinical association between CMS and neurological disorders due to defective docking or exocytosis of SV has been further emphasized by the recent identification of loss-of-function mutations in the genes encoding VAMP1 and Munc13-1 (Fig. 1, Table 2). VAMP1 is a member of the synaptobrevin family (v-SNARE). Four individuals with CMS from two unrelated families, were shown to possess loss-of-function mutations in VAMP1 [4]. All presented severe impairment of developmental milestones. Munc13-1 is a partner of syntaxin 1B – a t-SNARE component – and is critical for the assembly of the SNARE complex, which promotes priming of the SV in preparation for release in response to Ca²⁺ entry [1]. Loss-of-function of UNC13A has been reported in one patient with CMS, microcephaly and cortical hyperexcitability that could be related to the expression of Munc13-1 in glutamatergic neurons [24]. Interestingly, one amino-acid substitution exerting a gain-of-function effect in Munc13-1 has subsequently been reported in one isolated individual with dyskinesia, developmental delay and autism [25]. The impact of this gain of function mutation on neuromuscular transmission was not investigated.

The gene most recently identified as harboring mutations causing CMS with possible primary presynaptic dysfunction is LAMA5, which encodes the α-5 isoform of laminin, a basal lamina protein [26]. In vitro analyses showed that the pathogenic amino-acid substitution decreased binding of laminin α5 to the vesicular protein SV2, and impaired neural cell-adhesion and extension of cell projections. Whether this isoform of laminin is expressed in the central nervous system is unknown, and the individual with the LAMA5 loss-of-function mutation suffered from myopia and facial tics but no intellectual disability. As emphasized above, most of these presynaptic forms are observed at a very low frequency. This could be due to undiagnosed individuals suffering from forms of CMS masked by most prominent neurological signs.

Muscular comorbidities

The clinical presentations of CMS are highly variable, and one well-known differential diagnosis of CMS is congenital disorders of skeletal muscles, including the congenital myopathies (CM) and the congenital muscular dystrophies and their later onset limb-girdle muscular dystrophy (LGMD) forms. What was already a complex picture has recently become even more challenging with new clinical presentations that confound standard anatomical classifications. Atypical phenotypes associated with classical CMS genes resembling other types of neuromuscular diseases (LGMD, distal myopathies) have been observed. Conversely, significant fatigue and a positive response to AChE inhibitors have been reported in individuals with CM/CMD diagnosed at the molecular level. This striking overlap may be explained by the fact that numerous molecules involved in NMJ formation, maintenance and functioning are expressed in other regions of the muscle fiber, playing critical roles in key cell functions such as membrane trafficking, nucleus migration, or sarcolemma stability.

Electrophysiological investigation of the NMJ

Clinical evidence of muscle fatigability resulting from impaired neuromuscular transmission is mainly obtained from EMG investigation. Decrement of the fifth CMAP, in response to 2–5 Hz RNS, by more than 10% of the initial value, is usually taken to confirm the diagnosis of CMS, in the knowledge that the amplitude of the CMAP in normal individuals changes little at these frequencies [13]. Because the CMAP reflects the summed effect of the AP of many muscle fibers, it may not detect ‘subthreshold’ impairment of transmission at individual NMJs during RNS. By contrast Single Fiber EMG (SFEMG), which measures the temporal instability, or ‘jitter’, of the activation of individual muscle fibers by the nerve, is a highly sensitive, but unspecific, method that can detect subthreshold impaired neurotransmission at even 1-2 NMJs [41].

While these approaches can provide diagnostically useful evidence of impaired neuromuscular transmission, they are generally unable to identify which steps in the complex sequence of events leading to transmission are primarily affected. Detail at this level can only be obtained by making intracellular recordings of synaptic events from NMJs in isolated motor point biopsy samples e.g. [42–44]. Unfortunately, very few centers world-wide are equipped to do this on human muscle biopsies, with the result that we remain ignorant of the exact nature and site of neurotransmission impairment in many forms of CMS.

Post-exercise CMAP increment in presynaptic CMS

In the increasing number of presynaptic forms of CMS described above, as in virtually all forms of CMS, significant decrement of the CMAP is observed during RNS at 2–5 Hz. Two important exceptions to this rule are CMS caused by mutations in CHAT, in which there is no decrement at this low frequency in most of the individuals [2, 3], and in the individual with CMS related to VAMP1, in whom increment was observed in response to RNS [4]. Seemingly paradoxically, in a number of these presynaptic forms, voluntary exercise (e.g. for 10 s) or nerve stimulation at high frequency (e.g. 20 Hz for 20 seconds or 10 Hz for 5 min) results in a transient reduction of this decrement, and may even lead to the CMAP becoming substantially bigger [4, 5]. To interpret these somewhat confusing findings, it is helpful to review some basic features of neuromuscular transmission.

Neuromuscular transmission during repetitive activity in normal conditions

Nerve impulses (nerve action potentials, or ‘nAPs’) activate muscle fibers by the process of neuromuscular transmission [12]. Each nAP opens a number of Ca_V2.1 channels in the nerve terminal allowing an influx of Ca²⁺. The resulting transient increase in Ca²⁺ concentration within the nerve terminal causes the release of a number (20–200 in vertebrates) of multi-molecular ‘quanta’ of the transmitter, acetylcholine (ACh) into the synaptic cleft. These quanta correspond to the amount of ACh (about 10,000 molecules) contained in each SV. This release occurs by exocytosis at specialized structures in the presynaptic membrane, the ‘active zones’ (AZs). The action of transmitter on the muscle fiber causes a transient increase of its resting membrane potential by about 35–40 mV, the so-called endplate potential (EPP). The EPP is normally great enough to cause opening of the voltage-gated sodium channels (Na_V1.4) in the postsynaptic muscle membrane, thus triggering a mAP and activating contraction. In addition to this evoked release, transmitter quanta are also released spontaneously, at random, each one generating a small (about 1 mV) ‘miniature EPP’ (mEPP).

Most muscles are activated by short bursts, or longer trains, of nerve impulses at frequencies between 10–100 Hz [45]. During such activity, the amplitude of the EPP usually declines. However, because substantially more quanta are normally released than are required to trigger a mAP, the muscle is still effectively activated, i.e. transmission has a high ‘safety factor’ [46]. Most of the decline of EPP amplitude results from changes in the number of transmitter quanta released by each nAP, the so-called ‘quantal content’ (QC) of the evoked response. These changes occur over a wide range of time scales. A number of factors influence the value of the QC, and hence the amplitude of the EPP, at any moment. In Fig. 2 we show, very schematically, the likely relationships between some of the main factors, and their impact on the EPP and CMAP, in normal and pathological muscles. We emphasize that most of the changes in Ca²⁺ and RRP depicted represent our best estimate of what occurs rather than actual observations which, for the most part, have not been made. Collectively, these various forms of modulation of QC are often referred to as ‘short-term synaptic plasticity’ [47].

OPEN IN VIEWER

Fig.2

patterns of transmitter release from motor nerve terminals. This figure provides a qualitative view of how different patterns of NMJ response to repetitive activity (2–5 Hz and 10–20 Hz) may arise, and their impact on the CMAP. Top row, level of Ca²⁺ in the nerve terminal. Second row, size of ‘readily releasable pool’ (RRP) of transmitter quanta. Third row, mEPP followed by EPPs. Dotted horizontal line shows mAP threshold. Bottom row, CMAPs. At normal NMJs, at 2–5 Hz, each nAP causes a brief increase in Ca²⁺ which triggers release and generates large EPPs. These all trigger mAPs, so the CMAP shows no decrement. The RRP is almost fully replenished between the responses, although there may be a little decline. At 10–20 Hz, the individual Ca²⁺ transients summate, raising the level of Ca^2 + . However, now there is not time for the RRP to be fully replenished between responses, so it declines significantly. The net effect is a modest decline in quantal content (QC) and EPP amplitude. However, the EPPs remain suprathreshold, so there is no decrement of the CMAP. In LEMS, the individual Ca²⁺ transients are much smaller than normal, so the QC at low frequency is very low and few EPPs reach threshold, so the CMAP is greatly reduced. At higher frequency, the Ca²⁺ transients summate, so the Ca²⁺ level increases. At the same time, because the QC is low, there is little decline of the RRP. As a result, the increased Ca²⁺ level causes a significant increase in the QC and EPP amplitude, so more EPPs reach threshold and the CMAP increases. In forms of CMS in which presynaptic function is impaired (for reasons other than a decrease in Ca²⁺ entry), QC is reduced at low frequency, even though mEPP amplitude, Ca²⁺ entry and RRP size are all normal. As a result, there is little decline in the RRP. At high frequency, the build-up of Ca²⁺ causes enhanced release and some EPPs reach threshold, causing some increase in the CMAP. In forms of CMS that act on the postsynaptic membrane, the amplitude of the mEPPs (‘quantal size’) is generally much smaller than normal. During low frequency activity, although the QC is normal, the EPPs are small and shows some decline. As a result, many EPPs do not reach threshold so the CMAP is small and may show some decrement as the RRP gets smaller. At high frequency, the decline in RRP results in a decline in EPP amplitude, in spite of the increase in Ca²⁺ level, leading to significant decrement of the CMAP.

The Ca²⁺ that enters following each nAP is subsequently removed by a combination of processes: binding to soluble buffers, extrusion from the terminal by Ca²⁺-ATPases or Na⁺-Ca²⁺ transporters, and sequestration by mitochondria and components of the ER. Each of these processes has a distinctive time course, some requiring seconds [48]. As a result, during ‘physiological’ activity at frequencies of 10 Hz and more, there is a gradual build-up of the basal level of Ca²⁺ in the terminal (Fig. 2, top row, Normal). Since the relationship between Ca²⁺ concentration and QC is very non-linear [49], the effect of the ‘residual’ Ca²⁺ from previous activity enhances the effect of the Ca²⁺ entering during subsequent nAPs. This potentiating effect would be expected to cause an increase in the amplitude of the EPP and, hence, the efficacy of transmission.

In reality, the potentiating effect of Ca²⁺ build up is counteracted by a decline in the number of transmitter quanta capable of being released. These quanta normally represent only about 0.1% of the SVs present in the terminal and are considered to represent a distinct ‘pool’ of ‘readily releasable’ quanta, the RRP (Fig. 2, second row) [45]. Following depletion, the RRP is only relatively slowly replenished (1–10 seconds) [45, 50]. The RRP at mammalian NMJs is currently believed to consist of the 2-3 SVs closely associated with each AZ, making a total of several hundred SVs at a typical human NMJ [43, 50]. During low frequency activation (e.g. 0.1–1 Hz), about 5–10% of the initial RRP is released by each nerve impulse. During a train of 10 impulses, the RRP would be reduced to about 60% of its initial value. However, although the resting rate of RRP replenishment is relatively slow, it speeds up during activity, helping to maintain adequate release [50].

In clinical practice, these complex interacting effects on the efficacy of transmission are typically investigated by stimulating first at 2–5 Hz and then at 10–20 Hz or by recording CMAPs at 2–5 Hz after 10 s of voluntary activation of muscles.

Overview

The impairment of neuromuscular transmission that gives rise to the defining features of CMS is generally accompanied by structural changes in the cellular components of the NMJ. Because these are often more easily investigated than the functional defects themselves, they form an important component of many recent studies of CMS. Immunohistological as well as ultrastructural analyses help to confirm the diagnosis of CMS versus CM and, in some situations, may point clinicians to a specific form of CMS.

The mature NMJ is formed by the presynaptic nerve terminal of one α motor neuron closely apposed to the postsynaptic gutters of the skeletal muscle fiber membrane and ensheathed by several non-myelinating terminal Schwann cells. Most current knowledge of the NMJ in CMS comes from the investigation of mouse models in which key synaptic components have been deleted or inactivated. However, the human NMJ is quite distinct from the rodent NMJ in at least two aspects. The first concerns its development. The first steps of synaptic maturation, including the loss of polyaxonal innervation and the elaboration of the mature form of the NMJ, occur postnatally in mice but take place in utero in humans [61]. The second aspect concerns the architecture of the adult NMJ. The human NMJ is smaller in diameter (20μm vs 30μm in mice) and is composed of smaller and more distinct post-synaptic elements with an aspect called “cat’s paw” compared to the nice “pretzel-like form” observed in rodents [43]. In spite of these differences, morphological investigations of human samples show many similarities with observations made in mice, which is critical for developing precision therapeutics using this species as a preclinical model.

The classical Koelle and Friedenwald histochemical method was one of the first methods used to visualize the motor endplate by revealing the synaptic cholinesterase activity [62]. The use of immuno-histo-fluorescent techniques to label the innervated zone of myofibers teased from skeletal muscle biopsies can give a global structural view of several NMJs by simultaneously staining the terminal axons (anti-neurofilaments), the Schwann cells (anti-cytoplasmic S100 glial protein), and the post-synaptic AChRs (fluorescent α−bungarotoxin conjugates). Ultrastructural analyses complete this investigation by exploring the fine structure of the presynaptic, synaptic and postsynaptic elements in a smaller number of synapses.

Application to CMS

Morphological investigation of the NMJ sometimes suggest a particular form of CMS, and also give essential cues about the functional plasticity of this giant cholinergic synapse in pathological situations. For example, the form of CMS due to loss-of-function mutations in the gene encoding the ColQ collagenic tail of the synaptic form of AChE was specifically recognized by the lack of AChE staining, despite the presence of nerve terminals and well defined synaptic gutters at the NMJ, well before the identification of the causative gene [63]. Whatever the primary functional impact of the defective protein on the NMJ may be most, if not all, forms of CMS are eventually associated with a global dysfunctional organization due to the complex and dynamic molecular dialogs occurring between its three cell components [64].

Presynaptic CMS-EA

The global architecture of the NMJ has been investigated by immunostaining of teased muscle fibers for only a few patients with a presynaptic form of CMS. Consistent with the critical role of ACh for NMJ formation and maintenance, CMS-EA due to loss-of-function of CHT1 is associated with several axons innervating a single NMJ, suggesting persisting polyaxonal innervation, and a poorly-differentiated post-synaptic apparatus and generally defective maintenance of the NMJ, suggestive of repeated denervation-reinnervation events, as illustrated in Fig. 3 [17]. Interestingly, NMJs in this form of CMS display an increased level of synaptic butyryl cholinesterase (BChE), whose synaptic functions have not been investigated in detail. This form of cholinesterase is bound to perisynaptic Schwann cells and is believed to finely tune synaptic transmission by regulating the binding of ACh to AChRs located on these glial cells [65]. How a decrease of CHT1 level results in increased expression of BChE remains to be investigated, as does the specificity of this inverse correlation, since the synaptic level of BChE has never been investigated in muscle samples from individuals with ChAT or VAChT-related forms of CMS-EA.

OPEN IN VIEWER

Fig.3

morphological changes occurring at the NMJ in some pre- and post-synaptic forms of CMS. Representative whole-mount pictures of neuromuscular biopsies of individuals with CMS due to mutations in genes encoding presynaptic, synaptic, and postsynaptic proteins. Dilacerated muscle fibers were used for immunostaining of motor axons using an anti-neurofilament (NF 165 kDa, in green) and for fluorescent staining of postsynaptic AChRs using tetra-rhodamine-conjugated α-bungarotoxin (in red). In one adult control sample, the terminal axonal branches typically end as a fork innervating a well-defined nAChR structure. Morphological analysis of NMJs from one young adult suffering from presynaptic CMS related to CHT1 shows a disorganized post-synaptic element with fragmented synaptic gutters partially innervated by thin terminal axons. An unusual spike-like area pattern of nAChR staining (arrow) contacted by thin terminal axonal branch was observed in individual with synaptic CMS related to agrin. Strong decrease of axonal branching with innervation “en passant” contacting small isolated synaptic cups was observed in one adult suffering from post-synaptic CMS related to DOK7. One muscle sample from one adolescent suffering from congenital myopathy due to an homozygous missense mutation in the STAC3 gene, encoding a component of the excitation-contraction coupling machinery (6), is shown for comparison with congenital myopathy without evidence for secondary synaptopathy. In this myopathic condition, the NMJs look nearly normal, with terminal axonal branches contacting well-defined synaptic gutters. Scale bar = 10μm (applies to all pictures).

Particular challenges

Many of the most recently identified mutations causing CMS are ‘private’, known to occur in only 1 or 2 people. In these situations there are major challenges, both technical and experimental, arising from the high number of variants existing for each of us and from the mandatory demonstration of pathogenicity of the candidate variant when the causative gene to be searched for has no known physiological relationship with synapses [75]. Moreover, not only are most of the remaining individuals with undiagnosed fatigable muscle weakness sporadic cases, but this complex trait may have a polygenic origin with association with susceptibility variants, whose identification remains challenging in the field of rare diseases. This challenging situation is a feature of all forms of orphan diseases, and international programs of data sharing, such as matchmaker exchange and RD-connect platforms, have now emerged to favor emergence of pertinent bioinformatics tools, and to facilitate transfer of clinical genomic information and biological samples between research teams to resolve this critical issue [76, 77].

Therapeutics

Identifying the pathogenic mechanism of each individual suffering from CMS is also critical to guide therapy since it is well know that drugs that benefit one form of CMS can be harmful in another form [10, 11]. From the earliest recognition of CMS as a distinct, if heterogeneous, group of conditions, the main therapeutic tools have been the use of anticholinesterases [78]. The second main tool has been the drug 3,4-DAP, which prolongs the nAP by blocking the neuronal K⁺ channels whose opening terminates the nAP [79]. By using these drugs in tandem, it is often possible to use lower doses, resulting in fewer side effects. However, a number of conditions have been identified in which anticholinesterase drugs are not only ineffective, but may actually exacerbate the symptoms, e.g. inherited AChE deficiency and ‘slow-channel’ CMS, in which the prolonged AChR-gated channel openings lead to harmful entry of Ca²⁺ into the postsynaptic region and secondary degeneration of the post-synaptic element [10].

A further new therapeutic dimension, based on the initially empirical use of sympathomimetics, has emerged in recent years from efforts to treat patients whose symptoms are refractory to cholinesterase inhibitors. Treatment with a range of β₂-adrenergic agonists (ephedrine, albutamol, salbutamol, ...) has proven to be effective in autoimmune myasthenia as well as in several forms of CMS [10, 80–83]. These drugs, as well as adrenaline itself, are known to have a variety of short-term (minutes) effects on neuromuscular transmission in vitro, some apparently presynaptic and others postsynaptic [84–86]. However, these effects occur only at concentrations that are an order of magnitude or so greater than the concentrations attainable in humans [85, 86]. Furthermore, most of the therapeutic benefits that have been reported for sympathomimetics in patients with CMS require days, weeks or even months to be fully achieved, suggesting that these drugs enhance neuromuscular transmission by inducing long-term structural changes. The mechanism(s) underlying their effects, or even whether their main action is on the central or peripheral neurons, the skeletal muscle, the Schwann cells, or even the vasculature, is not known.

The success of sympathomimetics on impaired neuromuscular transmission has prompted reinvestigation of the role of sympathetic innervation of skeletal muscle. A recent study confirms the association of unmyelinated sympathetic neurons and NMJs in mouse leg muscles [87]. It further reports that stimulation of the appropriate sympathetic nerve activates postsynaptic β2-adrenoceptors and elevates postsynaptic cAMP production at NMJs. Interestingly, chemical sympathectomy was found to cause a reduction in NMJ size, AChR density and CMAP amplitude, and these deleterious effects were largely reversed by systemic treatment with clenbuterol for 10 days [87]. These findings seem to demonstrate significant effects of sympathetic innervation on the maintenance of NMJs. It will be important to learn more about how these effects are mediated and how they relate to the clinically beneficial effects of sympathomimetics on neuromuscular transmission.