Structure and function of the NMJ The NMJ transmits impulses from a motor neuron onto a skeletal muscle fibre (Ruff, 2003; Slater, 2008). Myelinated axons originating from motor neuron somata in the spinal cord anterior horn travel through a peripheral nerve into a muscle, branch off and innervate multiple fibres. Generally, each fibre has only one, single-innervated, NMJ (although during embryonic and perinatal development, multiple innervation occurs on a large scale; it remains present only at some special muscles, e.g. extraocular muscle). The distal axon ending (< 100 μm) loses myelin but, instead, is covered by 3–5 perisynaptic Schwann cells (Figs 2A and 3), involved in stabilization, regeneration and possibly also transmitter release modulation (Auld & Robitaille, 2003). Presynaptic terminals synthesize and release the neurotransmitter ACh in a tightly controlled way. A mouse motor nerve terminal contains 250 000–350 000 synaptic vesicles, each containing ∼10 000 ACh molecules (a ‘quantum’). The smaller human nerve terminals probably contain fewer vesicles (Slater, 2008). ACh is synthesized from cytosolic choline and acetylcoenzyme A and loaded into vesicles by a specific transferase. It is exocytosed at active zones by a release machinery composed of several structural and functional proteins (Sudhof, 2004) (Figs 2C and 3). A crucial functional protein is the voltage-gated Ca2+ channel (Cav2.1, at mammalian NMJs), which allows for Ca2+ influx that stimulates the neuroexocytotic machinery. Spent vesicles are recycled, probably in two different pools, depending on transmitter release rate (Perissinotti et al. 2008). A proportion of the released ACh is degraded by acetylcholinesterase in the synaptic cleft. The remainder binds and opens AChRs, clustered at high density (∼10 000–20 000 μm−2) at the tops of the typical postsynaptic membrane foldings (Figs 2 and 3). This allows the influx of positive charge, gating voltage-gated Nav1.4 channels in the folds. This causes an action potential along the muscle fibre which stimulates the contraction mechanism (Beam et al. 1989). AChR-mediated depolarizations can be measured as miniature endplate potentials (MEPPs, ∼1 mV), resulting from spontaneous release of single ACh quanta, and endplate potentials (EPPs, ∼20–40 mV), upon release of multiple quanta (‘quantal content’) by a nerve impulse (Fig. 2B and C). These events can be measured electrophysiologically with relative ease in muscle–nerve preparations from experimental animals or in biopsied human tissue (Fig. 2A and B). EPPs, not MEPPs, will normally trigger an action potential. A 3–5 × safety factor exists, i.e. the EPP is much larger than required to trigger an action potential (Wood & Slater, 2001), ensuring robustness of transmission, even at high rate when EPP rundown occurs.
Figure 3. Overview of the ultrastructural and electrophysiological deleterious effects of anti-ganglioside antibodies when targeting either the motor nerve terminal alone, the perisynaptic Schwann cells alone, or both of these structures Effects of anti-ganglioside antibodies and complement in ex vivo incubation studies on NMJs of mouse muscle–nerve preparations. For details see Halstead et al. (2005b). Electron microscopy shows that immunotargeted motor axon terminals become disorganized and swollen with a reduced synaptic vesicle density and damaged mitochondria. Immunotargeted Schwann cells are ultrastructurally characterized by a swollen, electron-lucent appearance and damaged organelles. Electrophysiologically, nerve terminal damage was hallmarked by a temporary dramatic increase in spontaneous uniquantal ACh release (measured as miniature endplate potential frequency), leading to block of evoked release (measured as endplate potentials) upon nerve stimulation. The moment of stimulation in the signals of the right column is indicated by a dot and a stimulus artefact can be seen. Interestingly, acute damage of perisynaptic Schwann cells did not change the presynaptic release or the postsynaptic effect of ACh, indicating that any modulatory effect of these cells on synaptic transmission is only to occur in the longer term. Scale bars in electron micrographs, 500 nm.
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Experimental evidence for synaptopathic actions of anti-ganglioside antibodies at the NMJOverview of our experimental mouse NMJ studies using patient sera and mouse monoclonal antibodies against gangliosides. First experimental indication that anti-ganglioside antibodies can harm NMJs came from Roberts who exposed ex vivo mouse diaphragms to three anti-GQ1b-positive MFS sera and observed transmission block (Roberts et al. 1994). Extensive studies by us on many MFS sera as well as purified IgG and human anti-GQ1b monoclonal antibody (mAb) elucidated pathophysiology in detail (Plomp et al. 1999). Firstly, immunohistochemistry showed that anti-GQ1b IgG and IgM bind to the NMJ. Secondly, electrophysiological analysis revealed a temporary (tens of minutes) but dramatic rise in MEPP frequency, peaking at a several 100-fold higher level than normal (Fig. 3), and that nerve stimulation-evoked ACh release became permanently blocked, paralysing the muscle. This effect resembled that of α–latrotoxin, from the black widow spider, which binds NMJs presynaptically and stimulates neuroexocytosis via second messengers as well as by forming pores that conduct ions, including Ca2+, causing massive ACh release and structural damage (Sudhof, 2001; Ushkaryov, 2002). Therefore, we coined the effect of anti-GQ1b antibodies as the ‘α–latrotoxin-like effect’. These effects were completely dependent on the activation of the complement cascade that is triggered by bound antibody (Walport, 2001). This was shown by loss of pathophysiological potency of sera by heating (30 min, 56°C), known to inactivate complement. Furthermore, anti-GQ1b IgG or IgM mAb only exerted pathophysiological effects when normal human serum was added as a complement source. In addition, complement deposition at NMJs was shown immunohistologically. Asynchronous twitching of muscle fibres paralleled the dramatically increased MEPP frequency. This disappeared upon AChR block by d–tubocurarine, excluding muscle fibre membrane malfunction as cause. Electrophysiological recordings showed that MEPPs frequently became superimposed due to high frequency and that summed potentials occasionally reached threshold and triggered an action potential. This twitching was used as easy readout in a bioassay where we tested synaptopathic potency of large numbers of MFS and GBS sera (Jacobs et al. 2002). Muscle strips were pre-incubated with heated serum and twitching was scored during incubation with normal human serum as complement source. In this way we found ∼80% of anti-GQ1b-positive MFS sera to induce NMJ synaptopathy, as well as 10% of the (non-MFS) GBS sera, 80% of them being anti-GQ1b-positive.
We immunized mice with lipo-oligosaccharides from GBS-associated C. jejuni and cloned mAbs that bound both lipo-oligosaccharides and gangliosides GQ1b, GT1a and GD3 (Goodyear et al. 1999). Besides showing that molecular mimicry is a likely mechanism for the generation of autoantibodies in MFS/GBS, this yielded a valuable set of anti-GQ1b/GD3/GT1a mAbs. At mouse diaphragm NMJs, these mAbs potently induced identical synaptopathic effects as produced earlier with anti-GQ1b-positive MFS and GBS sera and the human anti-GQ1b mAb. Again, antibody and complement deposits at NMJs were clearly shown (Fig. 4A and B). Subsequently, we investigated whether block of evoked ACh release (measured as EPPs) either occurred directly, or secondarily after the dramatic increase in MEPP frequency (Bullens et al. 2000). Before and after incubation of NMJs with purified anti-GQ1b-positive MFS IgG or the mouse anti-GQ1b/GD3 mAb CGM3, we measured the evoked ACh release. However, no change was found, showing that antibodies alone do not block evoked ACh release but that this occurs either as a complement-dependent effect, subsequent or parallel to the dramatic increase in MEPP frequency. It may occur either by transmitter vesicle depletion or presynaptic damage. While the first seems excluded because high frequency MEPPs stay for some time after block of evoked release (Plomp et al. 1999), the latter was clearly shown in immunofluorescence and electron microscopy (O’Hanlon et al. 2001). The electrophysiological effects coincide with loss of the cytoskeletal proteins neurofilament (heavy, 200 kDa) and type III β–tubulin (Fig. 4C and D). Ultrastructurally, disorganized terminals with a reduced synaptic vesicle density and swollen and damaged mitochondria were observed (Fig. 3), with synaptic clefts often infiltrated by Schwann cells. Immunogold labelling demonstrated a specific presynaptic binding of anti-GQ1b antibody (Halstead et al. 2004), confirming the electrophysiological absence of postsynaptic effects, e.g. MEPP amplitude reduction due to AChR block. Together, these observations suggested that anti-GQ1b antibody and complement destroy the presynaptic membrane and that ensuing Ca2+ influx activates proteases, degrading intraterminal cytoskeletal proteins.
Figure 4. Immunohistochemical characterization of the deleterious effects of anti-ganglioside antibodies Mouse neuromuscular junctions in ex vivo muscle nerve preparations were incubated with anti-GQ1b antibody CGM3 and subsequently exposed to normal human serum as complement source. Immunohistochemical analyses showed antibody (A, green) and complement (B, green) deposition at the neuromuscular junctions, delineated by acetylcholine receptor staining using fluorescently labelled α–bungarotoxin (red). Nerve terminal damage led to loss of neurofilament staining (green) at the terminal portion of intramuscular axon branches (C, control condition; D, after treatment; acetylcholine receptor staining in red in both panels). Schwann cell death occurred, indicated by ethidium-homodimer–1 staining (E, red; acetylcholine receptors stained green). For details see O’Hanlon et al. (2001); Bullens et al. (2002); Halstead et al. (2005b). Scale bars, 20 μm.
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We characterized complement in anti-GQ1b antibody-induced mouse NMJ synaptopathy and showed C1q, C3c, C4 and membrane attack complex (MAC) deposition (Halstead et al. 2004). MAC, the final complement product, is a membrane pore-forming conformation of factors C5b-9 (Walport, 2001), allowing uncontrolled ion fluxes. Three complement pathways exist which may each contribute: the classical, alternative and lectin pathway (Walport, 2001). When anti-GQ1b and complement were applied in Ca2+-free medium, no C4 or MAC deposition occurred, ruling out the alternative pathway, which is Ca2+ independent. Thus, one or both of the remaining pathways cause the effects. Proof for MAC as ultimate effector came from experiments with C6-deficient serum as complement source, which did not induce synaptopathy in C6-deficient tissue unless purified C6 was added.
Anti-GQ1b antibodies, as defined in ELISA, may in principle also exert NMJ synaptopathic effects by cross-reacting to other (sialylated) antigens, such as related glycolipids or membrane proteins. To investigate this possibility, we performed experiments at NMJs from GM2s-KO mice (see above) which lack GQ1b (and GT1a, to which most anti-GQ1b antibodies cross-react in ELISA) (Bullens et al. 2002). Monospecific anti-GQ1b IgM mAb, EM6, as well as a MFS serum induced NMJ synaptopathy in wild-type and heterozygous controls, and antibody and complement deposits were shown. However, this was not observed at NMJs of homozygous GM2s-KO mice, showing that GQ1b is the real target of ELISA-defined anti-GQ1b antibodies. We also studied monospecific mAbs against GD3, a remaining ganglioside in GM2s-KO mice. They induced little or no effect at controls, while at GM2s-KO NMJs they elicited clear synaptopathic effects. This indirectly showed that GD3 is upregulated at GM2s-KO NMJs, as in GM2s-KO brain (Takamiya et al. 1996), or becomes available for antibody binding due to the absence of steric hindering by the missing gangliosides. More importantly, they show that GD3 can substitute for GQ1b as an antigenic target in mediating synaptopathic effects at NMJs. We subsequently hypothesized that any specific anti-ganglioside mAb could induce synaptopathy, given the presence of a sufficiently high presynaptic density of the specific ganglioside. This hypothesis was tested by exposing NMJs from GD3s-KO mice (lacking b- and c-series gangliosides, and having upregulated GD1a) and GM2s-KO mice (lacking GD1a) to anti-GD1a mAb MOG35 (Goodfellow et al. 2005). This mAb readily induced the expected complement-mediated effects at GD3s-KO NMJs but failed to do so at GM2s-KO and wild-type NMJs. Anti-GD1a antibodies are often present in AMAN-GBS. GD3s-KO NMJs exposed to anti-GD1a-positive AMAN sera clearly developed synaptopathy whilst wild-type NMJs only showed moderately increased MEPP frequency without transmission block. This suggested that NMJ dysfunction may be a factor in the motor symptoms of AMAN. Recently, we also studied anti-GM1 and -GD1b mAbs and showed that they too can induce complement-dependent synaptopathy, at GD3s-KO and wild-type mouse NMJs, respectively. We made the interesting observation that for some anti-GM1 mAbs the antigenic GM1 is shielded by neighbouring gangliosides in the living neuronal membrane and only becomes available for binding under certain conditions such as created by freezing or fixation (Greenshields et al. 2009). Together, all these experimental studies show that many anti-ganglioside antibodies are capable of inducing complement-dependent neuropathogenic effects at the mouse motor nerve terminal, as long as the antigenic ganglioside is expressed at high enough density and is accessible for antibody.
Recently we generated the first paralytic in vivo mouse model for MFS (Halstead et al. 2008b). Intraperitoneal injection of anti-GQ1b/GD3 mAb CGM3 caused respiratory paralysis within hours and excised diaphragms appeared almost completely paralysed. This was due to antibody and complement causing transmission block at many (∼70%) NMJs, shown by morphological and electrophysiological analyses. This in vivo model required co-injection of human serum as complement source. Apparently, mouse complement was insufficiently activated to induce synaptopathy. The molecular explanation for this is unknown.
Overview of the experimental mouse NMJ studies of other groups. Other groups have, sometimes in different experimental settings, also investigated the effects of anti-ganglioside antibodies at mouse NMJs. Kishi and associates found increases in MEPP frequency, muscle fibre twitches and sometimes concomitant transmission block at NMJs exposed to sera from eight GBS variant patients, of which seven were anti-GQ1b-positive and one anti-GD1b-positive (Kishi et al. 2003). These effects were similar to those described by us (see above). With an extracellular recording pipette through which IgG and field stimulation was applied, Buchwald and colleagues observed complement-independent, reversible and near-complete inhibition of evoked ACh release from presynaptic motor nerve terminals by IgG from anti-GQ1b-positive or -negative MFS plasmas (Buchwald et al. 1995, 1998b). Furthermore, amplitude reduction of uniquantal responses was seen, suggesting effects on postsynaptic AChRs (Buchwald et al. 1998b). They also showed similar ACh release-inhibiting effects of IgG from several GBS sera with positivity for GM1, GQ1b or neither ganglioside, and of mouse mAbs against GM1, GD1a or GD1b (Buchwald et al. 1998a, 2007). Part of the GBS IgGs (but none of the mAbs) were reported to have a similar postsynaptic effect to the MFS IgGs, and direct competitive block of AChRs by low-affinity antibodies was hypothesized. From an ultrastructural NMJ study it was concluded that MFS IgG binds pre- and postsynaptic membranes (Wessig et al. 2001). Subsequent study of cells expressing muscle-type AChRs showed that GBS sera (either with activity against GQ1b, GM1 or neither) reduce ACh-induced currents (but only in part of the experiments) presumably through direct, competitive block of AChRs (Krampfl et al. 2003).
Santafé and colleagues studied the effects at mouse NMJs of serum and purified IgM mAb from a chronic demyelinating neuropathy patient with specificity against GM2, GalNAc-GD1a and GalNAc-GM1b, which share a terminal sugar epitope (Ortiz et al. 2001; Santaféet al. 2005, 2008). They found reversible complement-independent reduction (∼40%) of evoked ACh release and a complement-dependent increase in spontaneous release. No postsynaptic effects were noted, as judged by unchanged MEPP amplitude. Chronic mAb injection into the levator auris longus muscle of live mice greatly reduced quantal content at NMJs, as determined ex vivo, without an accompanying rise in MEPP frequency (Santaféet al. 2005, 2008). No mouse complement deposition was shown. It was hypothesized that anti-GM2 mAb inhibits presynaptic Cav2.1 Ca2+ channel function, possibly by disrupting a physiological role of GM2. A similar inhibiting action of (rabbit) anti-GalNAc-GD1a IgG was shown on Ca2+ current elicited in nerve growth factor-differentiated phaeochromocytoma cells (Nakatani et al. 2007). Anti-GM1-positive AMAN sera and additional specificity against (combinations of) GM2, GD1a, GD1b and GalNAc-GD1a, reduced Cav2.1 Ca2+ current in cerebellar Purkinje cells by 30–40%, without altering activation or inactivation kinetics, thus suggesting direct pore block (Nakatani et al. 2009). In addition, selective inhibition of intensely used Cav2.1 channels by anti-GM1 and -GD1a antibodies was suggested from studies in olfactory bulb neurons (Buchwald et al. 2007). Similarly, reversible and complement-independent block of Ca2+ channels by anti-GalNAc-GD1a antibodies was hypothesized from the observation that these antibodies blocked NMJ activity-mediated muscle fibre spikes in rat nerve–muscle co-cultures (Taguchi et al. 2004).
Together, our and other's studies indicate that NMJs may form targets of anti-ganglioside antibodies in GBS. However, a unifying mechanism is not yet established. Particularly, complement involvement in the diverse effects of anti-ganglioside antibodies is unclear and possibly complicated by the rather low complement activity in many mouse strains (Rice, 1950; Ebanks & Isenman, 1996). Neuropathy-associated anti-ganglioside antibodies are IgG–1, IgG–3 and IgM and binding of such isotypes will inevitably activate complement. Complement-independent effects, therefore, might initially take place at patient NMJs, but will probably be overwhelmed soon thereafter by complement activation, culminating in the devastating effects of MAC pore insertion.