Decoding pathogenesis of slow-channel congenital myasthenic syndromes using recombinant expression and mice models

Abstract

Despite the fact that they are orphan diseases, congenital myasthenic syndromes (CMS) challenge those who suffer from it by causing fatigable muscle weakness, in the most benign cases, to a progressive wasting of muscles that may sentence patients to a wheelchair or even death. Compared to other more common neurological diseases, CMS are rare. Nevertheless, extensive research in CMS is performed in laboratories such as ours. Among the diverse neuromuscular disorders of CMS, we are focusing in the slow-channel congenital myasthenic syndrome (SCS), which is caused by mutations in genes encoding acetylcholine receptor subunits. The study of SCS has evolved from clinical electrophysiological studies to in vitro expression systems and transgenic mice models. The present review evaluates the methodological approaches that are most commonly employed to assess synaptic impairment in SCS and also provides perspectives for new approaches. Electrophysiological methodologies typically employed by physicians to diagnose patients include electromyography, whereas patient muscle samples are used for intracellular recordings, single-channel recordings and toxin binding experiments. In vitro expression systems allow the study of a particular mutation without the need of patient intervention. Indeed, in vitro expression systems have usually been implicated in the development of therapeutic strategies such as quinidine- and fluoxetine-based treatments and, more recently, RNA interference. A breakthrough in the study of SCS has been the development of transgenic mice bearing the mutations that cause SCS. These transgenic mice models have actually been key in the elucidation of the pathogenesis of the SCS mutations by linking IP-3 receptors to calcium overloading, as well as caspases and calpains to the hallmark of SCS, namely endplate myopathy. Finally, we summarize our experiences with suspected SCS patients from a local perspective and comment on one aspect of the contribution of our group in the study of SCS.

Figure 1

Slow-channel congenital myasthenic syndrome mutations and acetylcholine receptor structure. A) Top view of the AChR. The stereo-stoichiometry relationship of the five subunits, 2α₁β₁δγ, of the AChR are highlighted by colors. B) Side view of the AChR. The δ subunit was omitted for clarity. Some SCS mutations are shown and identified in the insert (the insert zooms in the transmembrane region). C) Top view of the transmembrane domains of the AChR The majority of the SCS mutations are clustered into the ion channel pore, M1 and M2 domains. The SCS mutations are colored according to legend.

Figure 2

Classical electrophysiological features of slow-channel congenital myasthenic syndrome. A) Compound muscle action potentials (CMAPs) recorded over the hypothenar muscles evoked by single nerve stimulus. As is typical for SCS, there is a repetitive response to the single stimulus. B) CMAPs evoked by repetitive stimulation. The amplitude of each of four successive responses decreases, indicating failure of neuromuscular transmission. C) Superimposed miniature endplate currents recorded from anconeus muscle of control and the proband. The prolonged waveform of the patient miniature endplate current (MEPC) has a decay time constant of 31 msec, compared with control of 3.2 msec. The MEPC amplitude of the patient is about 50% of the control. Scale bars = A: 5 mV, 50 msec; B: 1 mV, 50 msec; C: 1 nA, 10 msec. (Figure 2 is reproduced by permission from Gómez et al. (2002) Annals of Neurology 51: 102–112).

Figure 3

Single-channel analysis of wild-type mouse and δS268F acetylcholine receptor. A) Current-voltage relationships for single-channel currents recorded from wild-type (WT) (open squares) or δS268F-acetylcholine receptor (AChR) (open circles) expressed in X. laevis oocytes. B) WT mouse and δS268F AChR single-channel currents recorded at low acetylcholine (ACh) concentration. AChR single-channel currents recorded from cell-attached patches on X. laevis oocytes expressing WT AChR (left) and AChRs expressing the δS268F mutation (right). All channels were recorded at a holding potential of 100 mV, 4 μM ACh, filtered at 5 kHz, sampling rate at 20 μsec per point and temperature of 22°C. Openings are shown as downward deflections, with close state denoted by the c, and the open state denoted by o. Representative histograms for current amplitude and open time distributions are shown for each of the AChR tested. Amplitude histograms were constructed from events list files created to include all event amplitudes that could be detected. The resulting distribution data was fitted with a Gaussian function with the appropriate number of components. Both the WT and mutant AChR show a single amplitude component of 7.874 ± 0.883 pA and 7.371 ± 0.465 pA, respectively. Open time histograms were fitted with exponential functions of the appropriate number of components using the maximum likelihood algorithm. The WT AChR shows two open time constants: τo1 = 0.411 msec (f = 0.229) and τo2 = 1.435 (f = 0.711), while the δS268F AChR shows two open time components of τo1 = 0.826 msec (f = 0.164) and τo2 = 10.582 msec (f = 0.896). Data were filtered at 5 kHz for each current sample display. Scale values are 5 pA for the vertical bar and 10 msec for the horizontal bar. C) Kinetics of activation of WT and δS268F AChR at high ACh concentrations. (Left) Single-channel currents elicited by the indicated concentration of ACh recorded from cell-attached patches from X. laevis oocytes expressing adult mouse AChRs containing the WT δ (upper) or mutated δS268F (lower) subunit. Recordings were done at holding potential of 100 mV, filtered at 5 kHz, sampling rate at 20 μsec per point and temperature of 22°C. Channel openings are shown as downward deflections. (Center and right) Open and closed time duration histograms for adult mouse AChRs containing the WT δ (upper) or mutated δS268F (lower) subunits generated and fitted using pSTAT6. Fitting was done using the simplex least-squares algorithm. Single-channel currents displayed were filtered at 4 kHz. (Figure 3 is reproduced by permission from Gómez et al. (2002) Annals of Neurology 51: 102–112).

Figure 4

εL269F-mice have degeneration of the neuromuscular synapse. Neuromuscular junctions from forelimb flexor muscles of 4-month-old εL269F-transgenic mice. To facilitate orientation all nerve termini are indicated using white arrowheads. In all three views the postsynaptic folds are simplified, whereas the adjacent the nerve termini (white arrowheads) seem normal. A) Large vacuoles containing membranous or granular debris fill the junctional sarcoplasm. The composition of the vacuoles can be seen more clearly in the enlargement from the boxed section. B) The mitochondria in the muscle fiber in (A) and in (B) (inset) are greatly enlarged compared with those in the nerve termini. Black arrows indicate what seem to be two degenerating mitochondria. Three mitochondria are massively enlarged and have densely packed cristae (black arrowheads). C) A degenerating myonucleus (black arrow) filled with autophagic debris and cytoplasmic contents lies immediately beneath a neuromuscular junction and adjacent to a relatively normal-appearing nucleus (white arrow). Calibration bars: A, 20 nM; B, 10 nM; C, 15 nM. (Figure 4 is reproduced by permission from Gómez et al. (1997) Journal of Neuroscience 17: 4170–4179).

Figure 5

Electromyogram of a patient with suspected slow-channel congenital syndrome. A) Representative example of a repetitive compound action potential on a patient with a suspected slow channel syndrome. B) On repetitive nerve stimulation at low frequency (3 Hz) it is possible to observe an abnormal decrement (17%) in the amplitude between the first and fifth elicited compound motor action potential. (Amplitude 1st potential = 2.37 mV; amplitude 5th potential = 1.96 mV; decrement of 17%).

Figure 6

Imaging of a bungarotoxin-labeled mouse endplates. Three-dimensional confocal image and Imaris-generated surface from an FVB mouse endplate (green) on dissociated muscle fiber (red), highlight the capacity of an in-depth analysis of endplate structure using confocal microscopy. Images of bungarotoxin-labeled AChR were collected on a Zeiss LSM 510 Confocal Microscope as a series of optical slices (Z-stack). Three-dimensional reconstructions of Z-stacks were generated using Imaris x64 6.2.0 which enables accurate determination of mean fluorescence intensity of voxels (three-dimensional pixel) as well as endplate volume. Lines in grids are separated by 5 μm.