Lambert-Eaton myasthenic syndrome: mouse passive-transfer model illuminates disease pathology and facilitates testing therapeutic leads

Abstract

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder caused by antibodies directed against the voltage-gated calcium channels that provide the calcium ion flux that triggers acetylcholine release at the neuromuscular junction. To study the pathophysiology of LEMS and test candidate therapeutic strategies, a passive-transfer animal model has been developed in mice, which can be created by daily intraperitoneal injections of LEMS patient serum or IgG into mice for 2-4 weeks. Results from studies of the mouse neuromuscular junction have revealed that each synapse has hundreds of transmitter release sites but that the probability for release at each one is likely to be low. LEMS further reduces this low probability such that transmission is no longer effective at triggering a muscle contraction. The LEMS-mediated attack reduces the number of presynaptic calcium channels, disorganizes transmitter release sites, and results in the homeostatic upregulation of other calcium channel types. Symptomatic treatment is focused on increasing the probability of release from dysfunctional release sites. Current treatment uses the potassium channel blocker 3,4-diaminopyridine (DAP) to broaden the presynaptic action potential, providing more time for calcium channels to open. Current research is focused on testing new calcium channel gating modifiers that work synergistically with DAP.

Keywords: GV-58; Lambert-Eaton myasthenic syndrome; active zone; voltage-gated calcium channels.

Figure 1

Structure and function of the mouse neuromuscular junction. (A) A single mouse neuromuscular junction stained for postsynaptic acetylcholine receptors using Alexa-594–labeled α-bungarotoxin (red) and presynaptic active zones using Alexa-488–labeled antibodies to bassoon (green puncta). One labeled active zone (AZ) is circled in white, and an artist rendering of the active zone is shown in (B). (B) Artist rendering of a single AZ, including two synaptic vesicles docked and ready to fuse (blue spheres), proteins involved in vesicle fusion (brown tube-like structures), presynaptic P/Q-type calcium channels (purple disks), and other active zone proteins (yellow disks). Note the ordered arrangement of active zone proteins into two double rows. (C) Average action potential–triggered endplate potential (EPP) recorded from a single neuromuscular junction (top) and two representative spontaneous miniature EPPs (mEPPs). (C) Diagram of predicted calcium influx into an active zone following a single action potential stimulus. Mammalian active zones contain 2–3 synaptic vesicles (spheres) and a small number of calcium channels (in this case, six are pictured). After action potential stimulations, only one of these channels may open, leading to a local cloud of calcium ion influx. Adapted from Refs. and .

Figure 2

A radioimmune assay for voltage-gated calcium channel (VGCC) antibodies in the serum of LEMS patients. In this plot of a representative sample of 10 patients diagnosed with LEMS (indicated by letter code), serum VGCC antibody titer varies between 0 and 14 fmol/L. Healthy control patients (Cont) have no detectable antibodies to VGCCs. Adapted from Ref. .

Figure 3

LEMS patient antibody effects on the mouse neuromuscular synapse. Top: The normally well-organized active zone is disrupted after LEMS passive transfer. Bottom: LEMS passive transfer to mice results in a strong reduction in the recorded endplate potential (EPP). Adapted from Ref. .

Figure 4

Freeze-fracture electron micrographs of active zone structure in control and LEMS passive-transfer mouse neuromuscular junctions. (A) Low-power view of control (left panel) and LEMS passive-transfer (right panel) presynaptic nerve terminal membranes that include several active zones (arrows and arrowheads). (B) High-power view of a control active zone that includes a rare slight disruption (X) in the ordered array of intramembraneous particles (tinted red). (C) Diagrams of the typical organization of two control active zones. Red dots represent hypothesized location of calcium channels based on analogy with published data at the frog neuromuscular junction. (D) High-power view of a LEMS passive-transfer active zone that includes a disrupted array of active zone particles (tinted red). (E) Diagrams that represent the range of organization of active zone particles after LEMS passive transfer (red dots represent hypothesized location of calcium channels). Adapted from Ref. .

Figure 5

The effects of DAP on calcium influx during an action potential can be modeled in cells that express calcium channels in vitro. A simulated action potential waveform is used as a voltage command in a voltage clamp recording of calcium current from a cultured neuron. The control action potential (cont; thin line) can be broadened to simulate the effects of DAP (broadened; thick line). The effects of this action potential broadening on calcium current are shown below. The current activated by the broadened action potential waveform is larger and lasts longer. Adapted from Ref. .

Figure 6

GV-58 is a novel calcium channel gating modifier that increases calcium flux during a depolarization and increases transmitter release in a manner that acts synergistically with DAP. (A) Structure of GV-58. (B) Calcium tail current (bottom) elicited by a voltage step (top) in a cultured cell expressing P/Q-type calcium channels. Control calcium currents decay quickly following voltage repolarization (black), but GV-58 significantly prolongs calcium current deactivation, leading to a much larger calcium influx (red). (C) Representative endplate potentials recorded from an ex vivo mouse neuromuscular junction. A mouse injected daily with serum from a healthy control patient has neuromuscular EPPs that are of normal large size (left; control serum). After passive transfer of LEMS to a mouse using daily injections of LEMS patient serum, the EPP size is greatly reduced (middle and right; LEMS). Acute exposure of LEMS passive-transfer synapses to 50 μM GV-58 increases EPP amplitude by about twofold (middle red trace). This is the same effect as can be observed after acute exposure to 1.5 μM DAP (right black traces). However, using a combination of GV-58 plus DAP leads to a synergistic effect that increases EPP amplitude by more than the sum of both drugs in isolation (right red trace). Adapted from Refs. , , and .