Paucity and disorganization of presynaptic membrane active zones in the lambert-eaton myasthenic syndrome

Presynaptic membrane active zones are related to synaptic vesicle exocytosis, and the large intramembrane particles in these zones may represent voltage-sensitive calcium channels. We tested the hypothesis that an abnormality of the zones is associated with the low probability of quantal release in the Lambert-Eaton myasthenic syndrome (LEMS). Freeze-fractured presynaptic membranes were studied in nine patients with LEMS (227 end-plates) and in 14 controls (148 end-plates). Satisfactory replicas of 94 LEMS and 83 control presynaptic membrane P-faces were obtained. Presynaptic membrane areas were estimated by stereometric analysis. The LEMS samples showed a marked decrease in active zones and active zone particles per unit area. The average number of particles per active zone was also reduced. Clusters of large particles were observed with increased frequency in the LEMS samples. These may have arisen by aggregation of active zone particles. There was no decrease in the overall density of intramembrane particles not associated with active zones or clusters. The distribution of these particles according to size resembled the control distribution except for a small decrease in the frequency of 5.3–5.8 nm particles. The findings can explain the reduced quantal release in LEMS, and strongly suggest that active zone particles are targets of the pathogenic autoantibodies recently demonstrated in this disease.     

Presynaptic effects of immunoglobulin G from patients with Lambert-Eaton myasthenic syndrome: their neutralization by intravenous immunoglobulins

Intravenous immunoglobulin (IVIg) treatment improves muscle strength in Lambert-Eaton myasthenic syndrome (LEMS), but its specific mode of action is unknown. We have delineated its mode of action on neuromuscular blocking properties of LEMS IgG. The effect of sera and purified IgG from six patients with LEMS on evoked quantal release was investigated after direct application to the motor nerve terminal by the perfused macro-patch-clamp electrode in mouse hemidiaphragms. The effect of LEMS IgG was analyzed alone and after coincubation with different concentrations of IVIg or its Fab fragments. All LEMS sera and purified LEMS IgG fractions taken before IVIg treatment inhibited evoked quantal release in a dose-dependent manner. When LEMS IgG was coincubated with a therapeutic IVIg preparation, presynaptic inhibitory activity of LEMS IgG was diminished in a dose-dependent fashion. Monovalent Fab fragments were as effective in neutralizing the activity of LEMS IgG as whole IVIg. These direct neutralizing effects of IVIg may explain its therapeutic efficacy.     

Effector mechanisms in myasthenia gravis: end-plate function after passive transfer of IgG, Fab, and F(ab')2 hybrid molecules

Using the quantitative ionophoresis technique and the mouse passive transfer model of myasthenia gravis, end-plate function was measured in mice transferred with myasthenic IgG, Fab, or F(ab')2 hybrid molecules prepared by recombination of one acetylcholine receptor (AChR) specific Fab and one Fab directed against irrelevant antigen. The Hill coefficient (a measure for the cooperativity between AChR subunits) and the apparent dissociation constant K for the ACh-AChR interaction were essentially unaltered after passive transfer of IgG or IgG fragments. In contrast, myasthenic IgG and Fab, but not control IgG or Fab, markedly reduced the mean number of end-plate channels. A similar effect was observed after passive transfer of F(ab')2 hybrid molecules. These results show that passive transfer of myasthenic IgG or IgG fragments leads to a quantitative reduction of ACh-controlled end-plate channels, whereas the function of the remaining receptors/channels remains unchanged. The results suggest further that cross-linking of AChR by divalent antibody may not be an absolute requirement for the reduction of AChR at the functional neuromuscular synapse.     

Lambert-Eaton myasthenic syndrome: II. Immunoelectron microscopy localization of IgG at the mouse motor end-plate

The autoimmune origin of the Lambert-Eaton myasthenic syndrome (LEMS) was documented by passive transfer of its electrophysiological features from humans to mice with IgG. Freeze-fracture electron microscopy has demonstrated a loss of active-zone particles in human LEMS and in its mouse passive transfer model. These data imply that the active zones are targets of the pathogenic LEMS autoantibodies. Immunolocalization of the antibodies has been hindered, however, by a paucity of active-zone particles (about 50/micron2 normally and still lower in LEMS) and by diffusion artifacts in the immunoperoxidase method. To obviate these problems, we employed sensitive avidin-biotin detection systems, both peroxidase and ferritin labels, and quantitative immunoelectron microscopy and end-plate morphometry. We compared mice treated with LEMS IgG, control IgG, and no IgG. In all mice, nonspecific background staining was found in the basal lamina covering the muscle fibers and Schwann cells. When a single 10-mg dose of IgG was injected intravenously, IgG samples from 12 patients produced significant immunostaining of the mouse active zones; from 7 patients they did not. Higher doses of intraperitoneally injected IgG (20 mg, three times a day for 2 days, or 10 mg/day for 15 days) from each of 4 patients (3 of whose IgG previously transferred LEMS to mice) caused significant immunostaining of mouse active zones: (1) the mean density (no./micron presynaptic membrane length) of positive active zones was 0.91 in the immunoferritin study and 0.72 in the immunoperoxidase study (control values, 0.12 and 0.02); and (2) 43% of the ferritin particles in the primary cleft were concentrated at the active zones and the rest were scattered randomly (control value, 5.3%). The findings indicate that LEMS IgG binds to the active zones of the presynaptic membrane.     

Lambert-Eaton myasthenic syndrome: I. Early morphological effects of IgG on the presynaptic membrane active zones

In the freeze-fractured presynaptic membrane of the motor end-plate, the active zones consist of two parallel arrays and each array contains 10- to 12-nm particles arranged in two rows. In the Lambert-Eaton myasthenic syndrome (LEMS) and in mice treated with 10 mg/day of LEMS IgG, administered intraperitoneally for several weeks, there was a paucity and disorganization of the active zones, and clusters of 10- to 12-nm particles appeared. To further define the changes in the active zones, mice were studied that had been treated over 2 days with 104 to 180 mg of IgG. Treatment transferred the physiological defect of LEMS. Control mice received normal human IgG or no IgG. The spacing and density (number/unit area) of active-zone particles were evaluated in presynaptic membrane P-faces using computer-assisted stereometry. In the normal active zone, the distance between particles in a given row and between adjacent rows of an array was less than, but the distance between the two arrays was greater than, the distance between the two antigen-binding sites on human IgG. In mice treated with LEMS IgG, the initial alteration in the active zone was a decrease in the distance between particles in a given row and between adjacent rows of an array; the distance between the two arrays remained unaltered. In more affected active zones, the parallel orientation of the rows was disturbed and the arrays became clusters. There was a significantly reduced density of active zones and of large-membrane particles associated with all active zones and clusters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoimmunity to the voltage-gated calcium channel underlies the Lambert-Eaton myasthenic syndrome, a paraneoplastic disorder

The Lambert-Eaton myasthenic syndrome (LEMS) is a disorder of neuromuscular transmission, often associated with small cell lung carcinoma (SCLC), and characterized by reduced quantal release of acetylcholine from the motor nerve terminals. Another neuromuscular transmission disorder, myasthenia gravis, has a well-understood autoimmunological cause. This review discusses the evidence for a similar autoimmunological effect in the development of LEMS. Injection of LEMS IgG into mice passively transfers the physiological and morphological abnormalities, which include paucity and disorganized arrangement of active zone particles believed to represent the voltage-gated calcium channels (VGCCs). Calcium influx via VGCCs into SCLC cells is reduced by LEMS IgG suggesting that in SCLC-associated LEMS, antibodies may be triggered by VGCCs expressed on these cells; this immunological cross-reactivity may lead to the neurological abnormality. Similar VGCCs on neuronally derived cells may trigger the disorder in those without a tumour. The disorder provides another example of the complicated relationships between the nervous and immune systems and tumorigenic processes.     

Specificity of Lambert-Eaton Myasthenic Syndrome immunoglobulin for nerve terminal calcium channels

Lambert-Eaton Myasthenic Syndrome (LEMS) is a presynaptic, neuromuscular disorder characterized by impaired nerve-evoked release of ACh. Repetitive nerve stimulation, which increases the probability of quantal release, improves the transmission defect. An autoantibody to Ca2+ channels of presynaptic motor nerve terminals is thought to mediate the pathogenesis of this disease. The goal of the present study was to examine the specificity of LEMS autoantibodies for nerve terminal Ca2+ channels as compared to other voltage-sensitive ion channels in nerve terminals, and to determine if non-specific membrane damage contributed to the pathogenesis of LEMS. The ion channel specificity of LEMS autoantibody was assessed by comparing the ability of acute application of IgG isolated from the plasma of a patient with LEMS to reduce depolarization-dependent uptake of 45Ca2+ and 22Na+ into or efflux of 86Rb+ from rat forebrain synaptosomes. The clinical diagnosis of LEMS was confirmed electrophysiologically by treatment of mice for 30 days with plasma (1.5 ml/day) taken from this patient. Characteristic reduction of quantal content elicited at 1 Hz and facilitation at 20 Hz was observed in mice treated with LEMS plasma compared to those treated with control plasma. One s, K(+)-stimulated 45Ca2+ uptake was inhibited 36.5 +/- 14.5% and 44.5 +/- 9.8% by acute application of 2 and 4 mg/ml LEMS IgG, respectively; IgG from patients with small cell carcinoma of the lung (SCC) had no effect on 45Ca2+ entry. The same concentrations of LEMS IgG affected neither voltage-dependent uptake of 22Na+ into veratridine-depolarized synaptosomes nor 86Rb+ efflux from K(+)-depolarized synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of action of voltage-gated K+ channel antibodies in acquired neuromyotonia

Acquired neuromyotonia (ANM) is associated with antibodies to voltage-gated K+ channels (VGKCs). ANM sera reduce the number of K+ currents in neuronal cell lines, but it is not clear how the antibodies act. Here, we show by using the NB-1 cell line that the reduction in K+ currents by IgG is independent of added complement. IgG Fc and Fab fragments from ANM sera had no effect, but three of four ANM F(ab')2 fragments significantly reduced K+ currents. Thus, cross-linking of the channels by divalent antibodies is likely to be an important mechanism in reducing K+ currents.     

Action of Lambert-Eaton myasthenic syndrome IgG at mouse motor nerve terminals

We have studied the electrophysiological effects of IgG obtained from four patients with Lambert-Eaton myasthenic syndrome (LEMS) (two with small cell carcinoma), using the mouse passive transfer model. Mice received LEMS or control IgG or plasma, 10 to 60 mg daily. Microelectrode intracellular recordings were made from diaphragm muscle. LEMS IgG and plasma decreased end-plate potential quantal content similarly, confirming IgG as the active factor. LEMS IgG was equally effective in C5-deficient mice, indicating that late complement components are not required. The time course of decline and recovery of quantal content closely followed that of the human IgG in the mouse serum, with time to half-maximal effect of about 1.5 days in each case. Binding/dissociation of IgG or down/up regulation of the antigenic determinants, possibly Ca2+ channels, has a half-life of between 2 and 36 hours. The results confirm our concept that IgG antibody to nerve terminal determinants underlies the disorder of transmitter release in LEMS.     

Passively transferred myasthenia gravis: protection of mouse endplates by Fab fragments from human myasthenic IgG.

Using the mouse passive transfer model, the effects of purified human myasthenic immunoglobulin G and of the monovalent Fab fragment on neuromuscular junctions were investigated. Treatment with IgG markedly reduced amplitudes of miniature endplate potentials. When Fab fragments were transferred alone or with subsequent addition of IgG no neuromuscular transmission block was induced. Myasthentic IgG and Fab were specifically demonstrated at the neuromuscular junctions by immunohistochemistry. On electronmicroscopy endplate structure was normal in transfer experiments using IgG for up to 30 days. It is suggested that Fab fragments bind to acetylcholine receptors without affecting transmission and protect them from the attack of complete IgG antibodies.     

Neonatal Guillain-Barré syndrome: blocking antibodies transmitted from mother to child.

OBJECTIVE: To investigate the role of blocking antibodies in neonatal Guillain-Barré syndrome (GBS) occurring 12 days postpartum in a child born to a mother with ongoing GBS. METHODS: We studied plasma filtrate, purified IgG, and monovalent Fab fragments from the affected mother and serum from the neonate as well as serum samples after recovery from disease 3 months later. Experiments were performed on the hemidiaphragms of adult mice and neonatal and juvenile rats. Quantal endplate currents were recorded with the perfused macro-patch clamp electrode. RESULTS: A dual effect was seen. Serum from mother and infant depressed quantal content by approximately 90% and reduced the amplitude of postsynaptic currents by 30 to 40%. The antibody nature of the blockade could be confirmed by showing that monovalent Fab fragments were similarly effective as purified immunoglobulin (Ig) G. No IgG antibodies to gangliosides, fetal or adult nicotinic acetylcholine receptor, or voltage-gated calcium channels could be detected, but IgM antibodies to the ganglioside GM1 were present. After recovery from GBS no blocking activity was seen in the sera of mother and infant. To elucidate why neonatal disease onset was delayed we examined the possible influence of early developmental changes in functional properties of the neuromuscular junction and applied the mother's active serum to postnatal rats. Although blockade was present in 23-day-old rats, it was absent in 5-day-old rats. CONCLUSION: Transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction.     

Effect of Lambert-Eaton myasthenic syndrome antibodies on autonomic neurons in the mouse

Somatic muscle weakness and autonomic symptoms characterize the autoimmune Lambert-Eaton myasthenic syndrome (LEMS). The former results from IgG autoantibody–mediated down-regulation of P/Q-type voltage-gated calcium channels at motor nerve terminals and consequent reduction in acetylcholine release; the basis for the autonomic symptoms is unknown. Using ω-conotoxins GVIA and MVIIC and ω-agatoxin IVA that block N-, Q-, and P-type channels, we investigated ex vivo the calcium channels subserving transmitter release from postganglionic parasympathetic neurons in the bladder and from postganglionic sympathetic neurons in the vas deferens of mice injected with IgG from LEMS patients or from controls. Calcium influx through N-, P-, and Q-type channels subserved transmitter release from parasympathetic and sympathetic neurons in control mice. In test mice, the component of transmitter release subserved by P-type channels was abolished by four of four LEMS IgG preparations, that subserved by Q-type channels was significantly reduced by three, and that subserved by N-type channels by one. Thus, LEMS IgG impairs transmitter release from parasympathetic and sympathetic neurons through down-regulation of one or more subtypes of voltage-gated calcium channels. The results suggest that antibody-mediated interference with specific ion channel function may also underlie autonomic dysfunction occurring in other autoimmune diseases.     

Membrane associated particles of the presynaptic active zone in rat spinal cord. A morphometric analysis

The distribution of membrane-associated particles in the presynaptic membrane was investigated in the spinal cord of unanesthetized and anesthetized rats by freeze etching electron microscopy. Both ‘external’ face (EF) and ‘protoplasmic’ face (PF) were examined. Particles were classified according to size in two categories: small particles (diameter 5.0–8.7 nm) and large particles (diameter 8.7–13.7 nm). The presynaptic region was subdivided into an active and a surrounding zone, depending on specific ultrastructural criteria. The density of large particles in the PF was found to be significantly higher in the active as compared with the surrounding zone in both unanesthetized and anesthetized rats. Thus, the presence of large particles represents an important feature of the active zone. Considerably more large particles were found in the walking than in the barbiturized state. This difference is paralleled by a vast increase of vesicle attachtment sites in the presynaptic membrane of unanesthetized animals reported by Streit et al.²² and confirmed in the present study. It is suggested that the two phenomena could be interrelated and that the large particles may represent calcium channels and thus provide the morphological substrate for the mechanism of excitation-secretion coupling.     

Effects of botulinum neurotoxin and Lambert-Eaton myasthenic syndrome IgG at mouse nerve terminals

Summary The interaction between two presynaptically acting agents, Lambert-Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) and purified botulinum neurotoxin (BoNT) type A, was studied. Intracellular microelectrode recordings were carried out on mouse muscles after injection with LEMS IgG. BoNT was either injected before recordings were made or applied in vitro. The time course of the in vitro actions of BoNT on miniature end-plate potential and end-plate potential parameters were not affected by pretreatment with LEMS IgG. After in vivo injection of BoNT, end-plate potential quantal content was reduced to less than 2% of control values, whether or not LEMS IgG had also been previously given. Quantitative electron-microscope autoradiographical analysis showed that neither the binding of¹²⁵I-BoNT to acceptors on the nerve terminal membrane nor the pattern of its internalisation were affected by pretreatment with LEMS IgG. We conclude that the effects of BoNT are not affected by LEMS IgG, suggesting different presynaptic binding sites for the two agents.     

The action of Lambert-Eaton myasthenic syndrome immunoglobulin G on cloned human voltage-gated calcium channels

In the Lambert-Eaton myasthenic syndrome (LEMS), immunoglobulin G (IgG) autoantibodies to presynaptic voltage-gated calcium channels (VGCCs) at the neuromuscular junction lead to a reduction in nerve-evoked release of neurotransmitter and muscle weakness. We have examined the action of LEMS IgGs on cloned human VGCCs stably expressed in transfected human embryonic kidney (HEK293) cell lines: 10-13 (alpha(1A-2), alpha(2b)delta, beta(4a)) and C2D7 (alpha(1B-1), alpha(2b)delta, beta(1b)). All LEMS IgGs studied showed surface binding to [(125)I]-omega-CTx-MVIIC-labeled VGCCs in the alpha(1A) cell line and two of six IgGs showed surface binding to [(125)I]-omega-CTx-GVIA-labeled VGCCs in the alpha(1B) cell line. We next studied the effect of LEMS IgGs (2 mg/ml) on whole-cell calcium currents in the alpha(1A) and alpha(1B) cell lines. Overnight treatment of alpha(1A) (10-13) cells with LEMS IgGs led to a significant reduction in peak current density without alteration of the current-voltage relationship or the voltage dependence of steady-state inactivation. In contrast, LEMS IgGs did not reduce peak current density in the alpha(1B) cell line. Overall these data demonstrate the specificity of LEMS IgGs for the alpha(1A) cell line and suggest that LEMS IgGs bind to and downregulate VGCCs in this cell line. Although several LEMS IgGs can be shown to bind to the alpha(1B) (C2D7) cell line, no functional effects were seen on this channel.     

Inhibition or acceleration of fibrin polymerization by monoclonal immunoglobulins and immunoglobulin fragments

A prolongation of the thrombin clotting time by at least 25% was found in the presence of 8 of 26 IgG myeloma proteins, 2 of 5 IgA proteins and 1 of 10 macroglobulins, in a concentration of 10 mg/ml. Normal pooled immunoglobulin in concentrations up to 83 mg/ml were inactive. 2 IgG and 2 IgM monoclonal immunoglobulins caused an acceleration of the thrombin time by at least 20%; this phenomenon has not been reported before. Corresponding results were obtained with these proteins when the polymerization of des-AA and des-AABB fibrin monomers were investigated in a light scattering system. Studies with enzymatically produced Fab, F(ab')2 and Fc fragments from normal and monoclonal immunoglobulin suggested that the polymerization inhibitory activity resides in the Fab portion of the immunoglobulin molecule. While the ability to inhibit fibrin polymerization may be an immunoglobulin specific effect, the acceleration caused by certain other monoclonal immunoglobulins may be an unspecific effect similar to that seen with dextran or albumin.     

Lambert-Eaton myasthenic syndrome

Lambert-Eaton myasthenic syndrome (LEMS) is an idiopathic or paraneoplastic syndrome producing antibodies against presynaptic voltage-gated P/Q calcium channels. This decreases calcium entry into the presynaptic terminal, which prevents binding of vesicles to the presynaptic membrane and acetylcholine release. LEMS is most often associated with small cell lung cancer, although idiopathic presentations comprise approximately 40% of the cases. The most common initial complaint is proximal muscle weakness involving the lower extremities more than the upper extremities. Depressed deep tendon reflexes and autonomic dysfunction are frequently present. Involvement of the bulbar or respiratory muscles is rare. Diagnosis is confirmed by electrophysiological testing, which demonstrates small compound muscle action potentials and facilitation with exercise or 20-Hz repetitive stimulation. A serum test for voltage-gated calcium channel antibodies is commercially available. Treatment involves removing the cancer associated with the disease. If cancer is not found, immunosuppressive medications and acetylcholinesterase inhibitors are used with moderate success. Patients with idiopathic LEMS should be screened every 6 months with chest imaging for cancer.     

Lambert–Eaton myasthenic syndrome – Diagnosis,pathogenesis and therapy

Lambert–Eaton myasthenic syndrome (LEMS) describes a rare human autoimmune disorder of the neuromuscular junction (NMJ). Clinically, LEMS patients suffer from characteristic muscle weakness that is caused by the presence of antibodies directed against their voltage-gated calcium channels (VGCC). These channels are localized in the presynaptic membrane of their motor nerve terminals. Binding of autoimmune antibodies to the VGCCs leads to reduced neuromuscular transmission. In approximately 50% of the patients, LEMS is reflected by a paraneoplastic manifestation and most commonly associated with a small cell lung carcinoma (SCLC) whose cells also express VGCCs in their plasma membrane. Better understanding of the pathophysiological mechanisms of LEMS has helped with the development of new diagnostic approaches and has led to targeted symptomatic and immunosuppressive therapy. For LEMS patients with an underlying malignancy, tumor therapy is the first choice to date.     

Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers

The freeze-fracture technique was used to study differences in membrane structure which could explain differences in the number of quanta released from axon terminals on twitch and tonic muscle fibers in Anolis intercostal muscles. The protoplasmic leaflets of axon terminals facing lizard twitch muscle fibers have intramembrane particle specializations characterized by two parallel linear particle arrays each composed of two particle rows which lie perpendicular to the axis of shallow ridges in the axolemma. During K+ depolarization, vesicles open between the arrays, confirming that these structures are the active zones for synaptic vesicle opening. Active zones at axon terminals on tonic fibers are defined by one linear particle array composed of two parallel particle rows oriented along the axis of a shallow presynaptic ridge; vesicles open beside these arrays. Thus, there are more particles near active zone vesicles in terminals on twitch fibers. Even though terminals on twitch and tonic muscle fibers seem to have similar numbers of synaptic vesicles associated with their active zones, a presynaptic action potential is reported to release at least 10 times more quanta from terminals on twitch fibers. We postulate that the differences in quantal output are related to the observed differences in the number of active zone particles flanking synaptic vesicles at the active zone. Indeed, the correlation between the distribution of these particles and the level of transmitter release provides additional support for the idea that they are the calcium channels which couple transmitter release to the action potential.     

Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake,immunoglobulin-mediated calcium increase,and neurotransmitter release in motor neurons

Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals.