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Keywords:

  • autoantibody;
  • ganglioside;
  • motor nerve terminal;
  • neuropathy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

The pre-synaptic motor nerve terminal is a highly complex and dynamic compartment within the lower motor neuron responsible for converting electrical signals into secreted chemicals. This self-renewing process of synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and the subsequent retrieval and recycling of vesicle components. Besides this conventional physiological role, the highly active process of vesicle fusion and re-uptake into endosomal sorting pathways acts as a conduit for entry of a range of substances into the intracellular compartment of the motor nerve terminal. Whilst this entry portal sub-serves many vital physiological processes, such as those mediated by neurotrophin trafficking, there is also the potential for substantial pathological consequences resulting from uptake of noxious agents, including autoantibodies, viruses and toxins. These may act locally to induce disease within the nerve terminal, or traffic beyond to the motor neuron cell body and central nervous system to exert their pathological effects. This review focuses on the recent evidence that the ganglioside-rich pre-synaptic membrane acts as a binding site for potentially neurotoxic serum autoantibodies that are present in human autoimmune motor neuropathies. Autoantibodies that bind surface antigens induce membrane lytic effects, whereas their uptake attenuates local injury and transfers any potential pathological consequences to the intracellular compartment. Herein the thesis is explored that a balance exists between local injury at the exofacial leaflet of the pre-synaptic membrane and antibody uptake, which dictates the overall level and site of motor nerve injury in this group of disorders.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

Lower motor neurons terminate their axons at the pre-synaptic membrane, localized in the synaptic cleft where neuromuscular transmission takes place, a site referred to as the neuromuscular junction (NMJ). At this distal site, the motor axon lies outside the protection of the blood nerve barrier (BNB), which is formed by tight junctions within vascular endothelial cells of endoneurial blood vessels that penetrate peripheral nerve fascicles, and by a relatively impermeable perineurium (Weerasuriya & Mizisin, 2011). The BNB maintains physiological homeostasis within the nerve compartment, and also protects against ingress of potentially harmful substances (Shlosberg et al. 2010; Mizisin & Weerasuriya, 2011). However, the motor nerve terminal (MNT) obtains its nutrient and oxygen supply via diffusion from adjacent vascular beds in muscle tissue rather than from the nerve fascicle blood supply, and lacks a perineurium. As a result, a very wide range of substances can freely diffuse into the synaptic cleft from the systemic circulation and extracellular fluid environment, and this BNB-deficient compartment is therefore a directly accessible gateway for both harmful and beneficial substances to enter the nervous system.

A second feature of the NMJ that enhances its vulnerability to uptake of circulating agents, including chemicals, macromolecules, viruses and toxins, is the very high rate of vesicle trafficking at the pre-synaptic membrane. This process is mediated by exocytotic fusion of synaptic vesicles (SVs), which are recovered and recycled by local endocytosis (Sudhof, 2004). Whereas the precise details underlying synaptic endocytosis in nerve terminals continue to be explored, there is substantial information available, especially in relation to molecular pathway uptake of toxins and viruses (Henaff & Salinas, 2010; Salinas et al. 2010). What has been less well studied is the uptake and fate of autoantibodies that bind the pre-synaptic membrane and are subsequently internalized, despite this being a widely recognized phenomenon. Long-standing studies have demonstrated that autoantibodies associated with human and experimental peripheral neuropathies are able to exert primary neuronal degeneration effects without demyelination (Engelhardt & Joo, 1986). Whether these antibody effects occur through local toxicity in the plasma membrane or through more remote effects following internalization remains the subject of considerable speculation. This review focuses on these studies in the light of recent data surrounding anti-ganglioside antibodies present in human peripheral nerve disorders, gangliosides being prominent glycolipid components of the pre-synaptic membrane.

Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

The cycle of neurotransmitter release and re-uptake at synapses is mediated by the exocytotic fusion of synaptic vesicles in active zones, followed by local endocytosis in adjacent but anatomically distinct membrane areas. Such a pathway facilitates rapid recovery and recycling of synaptic vesicle components for reuse, thereby maintaining synapse integrity and function. Pre-synaptic terminals synthesize and release the neurotransmitter acetylcholine (ACh) in a tightly controlled way. A mouse motor nerve terminal contains roughly 300 000 synaptic vesicles, each containing ~ 10 000 ACh molecules (a ‘quantum’). The smaller human nerve terminals likely 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, including the voltage-gated Ca2+ channel (Cav2.1, at mammalian NMJs), allowing for Ca2+ influx, which stimulates the neuroexocytotic machinery. Synaptic vesicles are recycled in nerve terminals via a variety of different endocytic pathways, each potentially using a different subset of the total endocytic machinery (Voglmaier & Edwards, 2007; Clayton & Cousin, 2009; Dittman & Ryan, 2009). These pathways include kiss-and-run (He & Wu, 2007; Rizzoli & Jahn, 2007), clathrin-dependent and clathrin-independent endocytosis (Jockusch et al. 2005; Granseth et al. 2007) and bulk endocytosis. Each of these, with the possible exception of the rapid and reversible kiss-and-run model, has the capacity to mediate uptake of plasma membrane-bound material. Neurons also undertake the less specific functions of pinocytosis and phagocytosis of extracellular material (Bowen et al. 2007; Ohka et al. 2009)

Clathrin-dependent endocytosis, sited within the periactive zone, is believed to be the dominant pathway of endocytosis at moderate rates of synaptic activity (Granseth et al. 2007). It is mediated by complex protein machinery that controls membrane lipid composition, cargo recognition, clathrin coat assembly, membrane invagination, membrane fission, and coat disassembly (Jung & Haucke, 2007). The endocytic process begins by recruitment of clathrin, adaptor proteins and different accessory proteins to the pre-synaptic membrane. The coated membrane grows into an invaginating pit, where dynamin finally assembles at the neck of the invaginated vesicle (Dittman & Ryan, 2009).

In general, the primary function of the endocytic system is to sort internalized ligands and receptors to different destinations. Whether SV endocytosis happens by clathrin-dependent endocytosis or ‘bulk endocytosis’ pathways, the immediate outcome of this process is the transport of SV vesicles directly to the SV reserve pool compartment or to the early endosome sorting station. One feature of synaptic vesicle recycling is the mechanism by which cargoes are targeted to the SV reserve pool or sorted to the retrograde axonal transport pathway. Shortly after the endocytosed vesicle pinches off, it sheds its clathrin coat and is transported to the early endosomes (EEs) where the components for a synaptic vesicle are sorted. This step is regulated by Rab5, a small GTPase which is known in eukaryotic cells to modulate early endocytic pathways including clathrin-dependent endocytosis, targeting of cargoes to early endosomes, and endosome fusion (Rink et al. 2005; Vonderheit & Helenius, 2005). Rab5 is present at neuromuscular synapses (Fischer von et al. 1994) and its genetic disruption in Drosophila leads to the impairment of endosomal trafficking at this synapse (Wucherpfennig et al. 2003). Once at the pre-synaptic EE compartment, endosomal cargoes are sorted either to the reserve recycling pool compartment or to the retrograde axonal transport route. The sorting of cargoes to the axonal transport route is regulated by Rab7, a small GTPase involved in the sorting and transport of cargoes between late endosome and lysosome in eukaryotic cells and responsible for long-range transport in motor neurons and dorsal root ganglion neuron (Rink et al. 2005). Both physiological (neurotrophins) and pathological (tetanus toxin) agents are able to adopt this pathway for trafficking to the cell body (Deinhardt et al. 2007). One specific feature is the neutral endosomal pH, which enables molecules involved in neuronal homeostasis, including nerve growth factor, brain-derived neurotrophic factor (BDNF) and their receptors p75NTR and TrkB, to be safely transported to the neuronal cell body.

Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

The endocytic machinery at the motor nerve terminal has the capacity to internalize harmful substances, the most widely studied and reviewed being bacterial toxins and viruses (Salinas et al. 2010). Botulinum toxins bind to ligands on the pre-synaptic membrane, become internalized in vesicular compartments, and translocate their catalytic subunit into the cytosol, where they exert their toxic activity by cleaving a protein substrate through their metalloproteinase activity, either at the terminal or following retrograde transport (Antonucci et al. 2008; Caleo et al. 2009; Salinas et al. 2010). Although toxins use the synaptic recycling machinery to enter neurons, evidence suggests that the endocytic cargoes used may be different. With respect to tetanus (TeNT) and botulinum (BoNT) neurotoxin, they both bind and are internalized at the NMJ. Whereas most serotypes of BoNT are locally retained at NMJ where they act by inhibiting the release of Ach, TeNT is sorted via the retrograde transport route to the neuronal cell body in the spinal cord, where it then is trafficked trans-synaptically to exert its toxic action on inhibitory spinal inter-neurons. Poliovirus and adenoviruses utilize a similar long-range pathway (Ohka et al. 2009).

A general description of retrograde axonal transport was originally provided by examining the transport of horseradish peroxidase (HRP; Tsukita & Ishikawa, 1980). It had been assumed that transport cargoes sorted from the early endosome at nerve terminal associate with the minus-end-directed motor proteins such as dynein and slide along the microtubule, using it as a rail. A second motor protein called dynactin, a multi-subunit protein, mediates the binding of dynein to selective cargoes and also associates with the membrane cytoskeleton (Schroer, 2004). The ability of dynactin to interact with both cytoplasmic dynein and the membrane cytoskeleton suggests a model in which dynactin links dynein to the membrane cytoskeleton, providing an anchor for dynein-mediated movement of axonal microtubules. In addition, experiments using antibodies against dynein have indicated that cytoplasmic dynein would be the primary motor for retrograde transport. Moreover, in other neurons such as sympathetic and sensory neurons, actin, microtubule and motor proteins, dynein is a key component of the retrograde transport architecture.

Although the axonal retrograde transport mechanism is the same for all exogenous materials destined to reach the CNS, there are differences in the type of retrograde motor used. The size, shape and target of cargoe vesicles may also trigger differences in axonal retrograde transport. One of the commonly used neuronal tracers to study retrograde transport of exogenous materials is TeNT. Morphological and kinetic analyses of retrograde carriers of TeNT have demonstrated their presence in round vesicles and fast tubular structures (Lalli et al. 2003). Moreover, TeNT carriers are not acidified during retrograde transport, indicating that the toxin escapes lysosomal targeting. Interestingly, TeNT and nerve growth factor (NGF) share the same retrograde machinery, an example of pathogens hijacking a retrograde transport route physiologically used by cell survival pathways.

Besides toxins, pathogenic viruses can exploit specific endocytic pathways at the nerve terminal to gain access to the axonal retrograde transport machinery. A striking example of exploiting the vesicular transport machinery provided by neurons in order to invade the CNS is provided by canine adenovirus type 2 (CAdV-2; also known as CAV-2) (Salinas et al. 2009). This virus binds at the pre-synaptic nerve terminal to specific receptors (coxsackievirus and adenovirus receptor, CAR) and is internalized along with the receptor in clathrin-coated vesicles. This process allows the virus to use the intracellular sorting machinery provided by endosomes which direct the virus-containing vesicle to the Rab7 sorting compartment, and finally recruit molecular motors for axonal retrograde transport machinery. Likewise, poliovirus together with its receptor, CD155 are internalized in endocytic vesicles which facilitate the sorting of the virus to axonal transport machinery (Ohka et al. 2009). Rabies virus binds the neuronal receptor p75NTR and enters the axonal retrograde transport similarly to poliovirus. In contrast to viruses that hijack neuronal vesicular endocytosis to access axonal retrograde transport pathways, some pathogens gain access to the retrograde transport system through other routes. For example, alpha herpes viruses fuse with the plasma membrane and enter the cytoplasm without endocytic vesicular protection (Diefenbach et al. 2008). Once in the cytoplasm, the virus recruits adaptor proteins and molecular motors to gain access to microtubular transport.

Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

Considering the extensive literature on growth factor, toxin and viral uptake at the NMJ, it is perhaps surprising that less attention has been paid to autoantibody uptake, especially when considering the range of autoimmune disorders in which autoantibodies target the pre- and post-synaptic membranes of the NMJ. The molecular architecture of the pre-synaptic membrane contains an abundance of potential antibody targets, including ion channels, receptors, glycan and glycolipid components of the membrane (Martin, 2003). In Lambert–Eaton myasthenic syndrome (LEMS) IgG antibodies are directed at P/Q type voltage-gated calcium channels (VGCCs) in the pre-synaptic membrane (Hirsch, 2007; Titulaer & Verschuuren, 2008). Antibody-mediated receptor cross-linking results in the disruption of the normal architecture of channels and reduces the active zone complex. This decreases calcium entry into the pre-synaptic terminal, which in turn attenuates activation of the exocytic machinery. Recently, P/Q type VGCCs have been implicated as a target for autoantibodies in sporadic motor neuron disease, possibly inducing injury through modulation of calcium currents (Gonzalez et al. 2011). In Isaac's syndrome, antibodies are directed towards accessible voltage-gated potassium channels in the distal portion of the motor nerve and its terminal arborizations. Here, suppression of outward potassium currents by antibodies induces a hyper-excitability syndrome, manifested clinically and electrophysiologically by neuromyotonia (Arimura & Watanabe, 2010). Recent evidence indicates proteins associated with the potassium channel, including LGI1 and Caspr2, are components of the autoantibody target, and are responsible for CNS manifestations of the disorder (Lancaster et al. 2011). What is less clear is the precise pathophysiological basis for these disorders, although channel cross-linking by autoantibody with or without internalization, rather than direct channel blockade, appears the most likely mechanism for the adverse functional effects, as recently shown for antibodies to nicotinic ACh receptor a3 subunits (Kobayashi et al. 2013). Furthermore, if autoantibody-channel complexes are internalized, their subsequent fate is unknown.

Interest in antibody internalization at motor nerve terminals followed on from the identification of lectin and bacterial toxin uptake at nerve terminals, cholera toxin being widely used for tract-tracing studies. Antisera generated in rabbits immunized with rat brain synaptosomes and then injected into rat muscle were found to be retrogradely transported to lower motor neuron cell bodies in the appropriate brain area, and also spread transynaptically into the surrounding neuropil. In these early experiments, the precise antibody specificity for individual antigens within the synaptosomal fraction was not known; however, pre-immune control sera were not transported, indicating some specificity was present (Ritchie et al. 1985). Subsequently, antibodies to the GPI-anchored protein Thy-1 that is enriched in synaptic membranes and is now known to be a raft-enriched component, were shown to be similarly retrogradely transported (Fabian, 1990). These studies predated the search for antibodies in motor neuron diseases (MND) that might bind motor nerve terminals, be transported and have neurotoxic effects (Appel et al. 1993; Smith et al. 1993; Gonzalez et al. 2011). Although these latter studies and subsequent work have show neurotoxic effects of human and experimental MND antisera, this interesting field still remains somewhat inconclusive, among other cell biological and genetic areas of mainstream MND research (Pagani et al. 2011).

Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

Autoantibody targeting of a specific antigen in the pre-synaptic membrane, with subsequent antibody uptake, has been most clearly demonstrated experimentally in motor neuropathies associated with anti-ganglioside antibodies (Plomp & Willison, 2009). Gangliosides are amphiphilic molecules residing in plasma membranes where the hydrophobic ceramide tail inserts in the membrane and the hydrophilic oligosaccharide moiety is displayed extracellularly, where it acts as an autoantibody target. The carbohydrate portion consists of a variable backbone chain of neutral sugars linking one or more negatively charged sialic acid residues which defines gangliosides as a distinct glycosphingolipid group. Ganglioside biosynthesis takes place in the Golgi complex in parallel enzymatic pathways by the addition of neutral sugar and sialic acid moieties to a glucosylceramide. The simple gangliosides GM3, GD3 and GT3 form the basis for complex gangliosides of the a-, b-, and c-series, respectively (Svennerholm, 1994; Maccioni, 2007). Although widely distributed in all cell types, gangliosides are particularly abundant in neurons where they compose 10–20% of the total lipid of the outer neuronal membrane layer (Ledeen, 1985). Membrane gangliosides are concentrated in dynamic membrane ‘rafts’ characterized by high concentrations of (glyco-)sphingolipids and cholesterol (Simons & Ikonen, 1997; Kasahara et al. 2000; van der Goot & Harder, 2001; Prinetti et al. 2001; Vyas et al. 2001; Pike, 2006; Fujita et al. 2007; Hanzal-Bayer & Hancock, 2007). Rafts also contain specific proteins, e.g. GPI-anchored proteins, G-proteins and kinases, suggesting raft-associated signaling functions (van der Goot & Harder, 2001). Relatively recently it was realized that gangliosides may play an active role in the formation of lipid membrane domains, instead of only being taken up passively (Sonnino et al. 2007; Silveira e Souza et al. 2008). Ganglioside functions include modulation of membrane proteins, neural development, cell–cell interaction/recognition, temperature adaptation, neuronal Ca2+ homeostasis, axonal growth, node of Ranvier stability and synaptic transmission (Ando, 1983; Ledeen, 1985; Thomas & Brewer, 1990; Rahmann et al. 1992; Wu & Ledeen, 1994; Lloyd & Furukawa, 1998; Ledeen & Wu, 2006; Susuki et al. 2007). Transgenic mice lacking ganglioside-synthesizing enzymes have allowed for investigations into the function of endogenous gangliosides (Sheikh et al. 1999; Kawai et al. 2001; Inoue et al. 2002; Okada et al. 2002; Jennemann et al. 2005; Sugiura et al. 2005; Zitman et al. 2008, 2011).

A role in synaptic function has been suggested from biochemical, morphological and in vitro functional studies (Thomas & Brewer, 1990; Ando et al. 1998, 2004). First, ganglioside density in synaptic membranes is high (Thomas & Brewer, 1990; Ando et al. 2004). Secondly, bath-applied GM1 and GQ1b increase K+-evoked neurotransmitter release from rat brain synaptosomes, presumably via Cav2.2 Ca2+ channels (Tanaka et al. 1997). Thirdly, synapse plasticity in brain slices is affected by bath-applied gangliosides (Wieraszko & Seifert, 1985; Ramirez et al. 1990; Egorushkina et al. 1993; Tanaka et al. 1997; Furuse et al. 1998; Fujii et al. 2002). Fourthly, gangliosides co-localize in lipid rafts with key transmitter release proteins (e.g. Ca2+ channels and SNAREs) (Chamberlain et al. 2001; Lang et al. 2001; Salaun et al. 2004; Taverna et al. 2004; Davies et al. 2006). Fifthly, polysialylated gangliosides bind Ca2+, a crucial ion in transmitter release (Rahmann et al. 1992). Lastly, it is predicted that gangliosides themselves undergo recycling through endosomal pathways during exocytosis and endocytosis in the pre-synaptic membrane and that some functional role is attributable to this. Despite the above evidence, detailed electrophysiological studies at the NMJ have concluded rather remarkably that although complex gangliosides may modulate temperature- and use-dependent fine-tuning of transmitter release, they are largely dispensable players in transmitter release. Since the glycosyltransferase-deficient mice used in these electrophysiology experiments still possess simple gangliosides including GM3, the degree to which transmitter release would be perturbed by the complete absence of all gangliosides remains an open question.

The absence of a vital role for at least complex gangliosides in the pre-synaptic membrane does not diminish their importance as targets for autoantibodies (and toxins), with subsequent pathological effects, as seen in models of Guillain-Barré syndrome (GBS) and related neuropathies. Besides damaging axons and myelin, it was hypothesized 15 years ago that GBS-associated anti-ganglioside antibodies may target the NMJ, particularly since GBS symptoms overlap with those of known NMJ disorders/intoxications such as botulism, where Clostridial botulinum neurotoxins bind to pre-synaptic gangliosides at NMJs (Bullens et al. 2002). GBS is an acute, immune-mediated neuropathy affecting the peripheral nervous system (PNS), usually triggered by preceding infectious events including Campylobacter jejuni enteritis (van Doorn et al. 2008). This syndrome is subdivided in distinct variants. Acute motor axonal neuropathy (AMAN) is most usually initiated by infection with C. jejuni and characterized by early axonal degeneration of motor nerves. In addition, Miller-Fisher syndrome (MFS), which may also be triggered by C. jejuni infections, is characterized by acute onset of unsteadiness of gait (ataxia), areflexia, and an inability to move the eyes, often associated with nonreactive pupils (ophthalmoplegia). In contrast, the acute inflammatory demyelinating polyradiculoneuropathy (AIDP) GBS variant is characterized by myelin-directed injury, resulting in demyelination as the characteristic hallmark. With respect to C. jejuni infection, it is well documented that the lipoligosaccharides on the bacteria surface initiate the production of antibodies against a range of neuronal ganglioside epitopes via a molecular mimicry mechanism (Yuki & Koga, 2006). Binding of anti-ganglioside antibodies to the pre-synaptic nerve terminal at NMJ and specific nerve membranes, such as the node of Ranvier, initiates activation of the complement cascade that destroys the nerve tissue (Plomp & Willison, 2009).

Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

The motor nerve terminal is considered an ideal target for binding by anti-ganglioside antibodies in GBS. Substantial experimental evidence supports a role for pre-synaptic disruption in GBS pathology (Buchwald et al. 1998; O'Hanlon et al. 2001, 2002). However, clinical electrophysiological evidence to support the view that the pre-synaptic nerve terminal at NMJ is either the earliest or the primary target is less strong (Ho et al. 1997; Kuwabara et al. 2007). In early studies, we tested the vulnerability of the motor nerve terminal by co-incubating mouse hemidiaphragm or triangularis sterni preparations with various different anti-ganglioside antibodies and normal human serum as a source of complement (Plomp et al. 1999; Plomp & Willison, 2009). We observed dramatic injury to the nerve terminal closely resembling the effects seen with the black widow spider venom, alpha-latrotoxin. Electrophysiological investigation revealed a brief but massive increase in miniature endplate potential (MEPP) frequency, followed by block of synaptic transmission, as measured by reduction in the evoked endplate potential (EPP). The same findings were observed when human AMAN and MFS sera and a source of complement were used. These animal data suggested that a defect at pre-synaptic motor nerve terminals might contribute to the paralytic clinical features of human GBS. Since evidence in humans for distal motor nerve terminal injury is inconclusive, we considered that the motor nerve terminal might be relatively protected by antibody internalization, as it has such active uptake mechanisms in place. To address this experimentally, we used a range of monoclonal antibodies known to be associated with GBS, recognizing GM1, GD1a and GD1b ganglioside epitopes present at the pre-synaptic membrane (Fewou et al. 2012). Over a half-hour period, we observed a substantial reduction of surface immune-reactivity of all three antibodies upon incubation at 37 °C of antibody-labeled PC12 cells and triangularis sterni preparations. The reduction in surface immunoreactivity was accompanied by a massive decrease of cellular cytotoxicity and the terminal pore-forming complement product, membrane attack complex (MAC) deposition in the cell and tissue system, respectively. This was also found in in vivo murine studies. These results suggest that at physiological temperature, surface anti-ganglioside antibody deposits are rapidly cleared by vesicular endocytosis and that this phenomenon provides relative protection of the pre-synaptic nerve terminal against antibody-mediated complement-dependent injury. The fate of endocytosed antibody still remains unknown, but it is possible that it either enters the recycling pathway and is subsequently extruded back into the extracellular space, as has been demonstrated for anti-GD3 antibodies (Iglesias-Bartolome et al. 2006), or that it is retrogradely transported to the cell body. Here it might either be destined for lysosomal degradation, spread trans-synaptically, or exert local toxicity within the motor neuron cell body. One key principle emerging from these studies is that antibody that remains on the exofacial leaflet of the plasma membrane still has the capacity to fix complement, and thereby be highly neurotoxic, in contrast to endocytosed antibody. Once any membrane attack complex pores form, the consequence will be an uncontrolled calcium influx which will activate calpain and thereby cause paralysis of endocytic machinery, leading to further build up of surface antibody (O'Hanlon et al. 2003). Thus an equilibrium state must exist between antibody synthesis, membrane binding, and clearance at which normal physiological function can continue, but which is also vulnerable to dysfunctional perturbations. The variables on which this depends have not been fully explored and these issues are thus the subject of ongoing studies (Fig. 1).

image

Figure 1. Antibody uptake mechanism that underlies the attenuation of pre-synaptic nerve terminal injury in anti-ganglioside antibody-mediated motor axonal neuropathy. (1) Following an infection (e.g. Camplyobacter enteritis), B lymphocytes are stimulated to produce anti-microbial antibodies which through molecular mimicry also bind to self gangliosides. (2) Circulating anti-ganglioside antibodies bind ganglioside-rich neural membranes in accessible sites lying outside the blood nerve barrier, including the pre-synaptic nerve terminal membrane and nodal axolemma in distal motor nerves. (3) At the pre-synaptic membrane, where vesicular uptake pathways are highly active, bound antibodies are internalized by clathrin-mediated endocytosis. The extent to which antibody uptake may also occur during synaptic vesicle recycling is unknown (?). In contrast to the pre-synaptic membrane, at the nodal axolemma antibodies remain on the surface. (4) At 4 °C, endocytosis is inhibited and antibody uptake does not take place. Antibody remaining on the plasma membrane is available for complement fixation and subsequent lytic injury, whereas endocytosed antibody becomes cryptic to complement fixation. (5) Following uptake, endocytic vesicles are depleted from the clathrin coating and sorted to the early endosome. (6) Endocytic vesicles at the early endosome are not targeted to the synaptic recycling pool (7) but are rather sorted to the retrograde transport pathway (8). The extent of local recycling of antibody to the plasma membrane is unknown (?) (9) Vesicles containing antibodies are transported to the neuronal cell body and targeted to the lysosome for degradation. For reference see Fewou et al. (2012).

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Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References

Antibody binding at motor nerve terminals that exerts local neurotoxic effects has been described in a range of autoimmune disorders affecting motor nerves. The data on antibody uptake and subsequent retrograde transport are less well studied from a functional perspective. In contrast to uptake of toxins or viruses and their retrograde transport to a particular intracellular compartment, the intracellular fate of most of these autoantibodies is not known. It is also unknown whether these intracellularly sequestered antibodies contribute to the disease pathology. One can only speculate on the intracellular fate of endocytosed immunoglobulins. If the antibody is exclusively directed against a membrane receptor which mediates its uptake, its final fate is most likely to be lysosomal degradation, unless it is recycled to the membrane and expelled. In contrast, if an antibody can bind to an intracellular antigen, one might predict that binding could cause neuronal dysfunction or interference with neuronal physiology. In the case of anti-ganglioside antibodies, we consider it likely that they recognize only pre-synaptic membrane gangliosides and that their uptake through endocytic mechanisms at the synapse leads to their sorting and retrograde transport to lysosomes as a final destination for degradation and clearance. However, the possibility that they exert intracellular cytotoxic affects cannot be excluded on the current evidence, and merits further exploration.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Synaptic vesicle recycling: exocytosis and endocytosis at the neuromuscular junction
  5. Exogenous toxins and viruses use the synaptic vesicle recycling mechanism and axonal retrograde transport as a gateway to the nervous system
  6. Autoimmune diseases and autoantibodies in relation to the pre-synaptic nerve terminal
  7. Ganglioside function and anti-ganglioside antibody binding at the pre-synaptic membrane
  8. Anti-ganglioside antibody internalization modulates the pathology of the synaptopathy
  9. Concluding remarks
  10. Acknowledgements
  11. References