Anti-GD1a antibodies activate complement and calpain to injure distal motor nodes of Ranvier in mice.

The motor axonal variant of Guillain-Barré syndrome is associated with anti-GD1a immunoglobulin antibodies, which are believed to be the pathogenic factor. In previous studies we have demonstrated the motor terminal to be a vulnerable site. Here we show both in vivo and ex vivo, that nodes of Ranvier in intramuscular motor nerve bundles are also targeted by anti-GD1a antibody in a gradient-dependent manner, with greatest vulnerability at distal nodes. Complement deposition is associated with prominent nodal injury as monitored with electrophysiological recordings and fluorescence microscopy. Complete loss of nodal protein staining, including voltage-gated sodium channels and ankyrin G, occurs and is completely protected by both complement and calpain inhibition, although the latter provides no protection against electrophysiological dysfunction. In ex vivo motor and sensory nerve trunk preparations, antibody deposits are only observed in experimentally desheathed nerves, which are thereby rendered susceptible to complement-dependent morphological disruption, nodal protein loss and reduced electrical activity of the axon. These studies provide a detailed mechanism by which loss of axonal conduction can occur in a distal dominant pattern as observed in a proportion of patients with motor axonal Guillain-Barré syndrome, and also provide an explanation for the occurrence of rapid recovery from complete paralysis and electrophysiological in-excitability. The study also identifies therapeutic approaches in which nodal architecture can be preserved.

C5 inhibitor rEV576 protects against neural injury in an in vitro mouse model of Miller Fisher syndrome.

Guillain-Barré syndrome and its clinical variants, including the anti-GQ1b ganglioside-mediated Miller Fisher syndrome (MFS), comprise the world's leading cause of acute neuromuscular paralysis. Presently, no specific drug therapies exist. The complement cascade, which is activated in these patients, forms an attractive drug target. In this study, we tested whether the complement C5-inhibiting recombinant protein, rEV576, was able to prevent neural injury in a previously developed in vitro mouse model for MFS. Mouse hemidiaphragm preparations were treated with anti-GQ1b antibody and normal human serum as a source of complement with added rEV576 or control protein. Immunohistology in control tissue showed deposition of C3c and membrane attack complex at neuromuscular junctions (NMJs), along with terminal motor axonal neurofilament degradation as well as ethidium homodimer-2 staining showing perisynaptic Schwann cell (pSC) injury. Electrophysiological and functional analyses showed block of synaptic transmission at the NMJ after an initial period of a dramatically high level of asynchronous acetylcholine release. In tissue treated with rEV576, all these indicators of motor neuronal damage were absent, except for the presence of C3c, indicating effective inhibition of C5. These results demonstrate that rEV576 effectively prevents development of neuronal and pSC damage in experimental murine neuropathy.

Eculizumab prevents anti-ganglioside antibody-mediated neuropathy in a murine model.

Anti-GQ1b ganglioside antibodies are the serological hallmark of the Miller Fisher syndrome (MFS) variant of the paralytic neuropathy, Guillain-Barré syndrome, and are believed to be the principal pathogenic mediators of the disease. In support of this, we previously showed in an in vitro mouse model of MFS that anti-GQ1b antibodies were able to bind and disrupt presynaptic motor nerve terminals at the neuromuscular junction (NMJ) as one of their target sites, thereby causing muscle paralysis. This injury only occurred through activation of complement, culminating in the formation and deposition of membrane attack complex (MAC, C5b-9) in nerve membranes. Since this step is crucial to the neuropathic process and an important convergence point for antibody and complement mediated membrane injury in general, it forms an attractive pharmacotherapeutic target. Here, we assessed the efficacy of the humanized monoclonal antibody eculizumab, which blocks the formation of human C5a and C5b-9, in preventing the immune-mediated motor neuropathy exemplified in this model. Eculizumab completely prevented electrophysiological and structural lesions at anti-GQ1b antibody pre-incubated NMJs in vitro when using normal human serum (NHS) as a complement source. In a novel in vivo mouse model of MFS generated through intraperitoneal injection of anti-GQ1b antibody and NHS, mice developed respiratory paralysis due to transmission block at diaphragm NMJs, resulting from anti-GQ1b antibody binding and complement activation. Intravenous injection of eculizumab effectively prevented respiratory paralysis and associated functional and morphological hallmarks of terminal motor neuropathy. We show that eculizumab protects against complement-mediated damage in murine MFS, providing the rationale for undertaking clinical trials in this disease and other antibody-mediated neuropathies in which complement activation is believed to be involved.

Complement inhibition abrogates nerve terminal injury in Miller Fisher syndrome.

A large body of clinical and experimental data indicate that complement activation is an important mechanism for neuronal and glial injury in Guillain-Barré syndromes. Inhibition of complement activation therefore might be expected to limit the progression of the disease. Using in vitro and in vivo models of the Guillain-Barré syndrome variant, Miller Fisher syndrome, we have shown previously that anti-GQ1b ganglioside antibodies target the presynaptic motor nerve terminal axon and surrounding perisynaptic Schwann cells, thereby mediating destructive injury through deposition of membrane attack complex. Here, we have used this model to investigate the effects of a novel therapeutic inhibitor of complement activation, APT070 (Mirococept), both in vitro and in vivo. In these models, APT070 completely prevents membrane attack complex formation, and thereby has a major neuroprotective effect at the nerve terminal, as assessed by immunohistology of perisynaptic Schwann cell and axonal integrity. These data provide a rationale for considering clinical trials of APT070 in Guillain-Barré syndrome, its variant forms, and other complement dependent neuromuscular disorders.

Anti-disialosyl antibodies mediate selective neuronal or Schwann cell injury at mouse neuromuscular junctions.

The human paralytic neuropathy, Miller Fisher syndrome (MFS) is associated with autoantibodies specific for disialosyl epitopes on gangliosides GQ1b, GT1a, and GD3. Since these gangliosides are enriched in synaptic membranes, anti-ganglioside antibodies may target neuromuscular junctions (NMJs), thereby contributing to disease symptoms. We have shown previously that at murine NMJs, anti-disialosyl antibodies induce an alpha-latrotoxin-like effect, electrophysiologically characterized by transient massive increase of spontaneous neurotransmitter release followed by block of evoked release, resulting in paralysis of the muscle preparation. Morphologically, motor nerve terminal damage, as well as perisynaptic Schwann cell (pSC) death is observed. The relative contributions of neuronal and pSC injury to the paralytic effect and subsequent repair are unknown. In this study, we have examined the ability of subsets of anti-disialosyl antibodies to discriminate between the neuronal and glial elements of the NMJ and thereby induce either neuronal injury or pSC death. Most antibodies reactive with GD3 induced pSC death, whereas antibody reactivity with GT1a correlated with the extent of nerve terminal injury. Motor nerve terminal injury resulted in massive uncontrolled exocytosis with paralysis. However, pSC ablation induced no acute (within 1 h) electrophysiological or morphological changes to the underlying nerve terminal. These data suggest that at mammalian NMJs, acute pSC injury or ablation has no major deleterious influence on synapse function. Our studies provide evidence for highly selective targeting of mammalian NMJ membranes, based on ganglioside composition, that can be exploited for examining axonal-glial interactions both in disease states and in normal NMJ homeostasis.

Overexpression of GD1a ganglioside sensitizes motor nerve terminals to anti-GD1a antibody-mediated injury in a model of acute motor axonal neuropathy.

Anti-GD1a ganglioside antibodies (Abs) are the serological hallmark of the acute motor axonal form of the post-infectious paralysis, Guillain-Barre syndrome. Development of a disease model in mice has been impeded by the weak immunogenicity of gangliosides and the apparent resistance of GD1a-containing neural membranes to anti-GD1a antibody-mediated injury. Here we used mice with altered ganglioside biosynthesis to generate such a model at motor nerve terminals. First, we bypassed immunological tolerance by immunizing GD1a-deficient, beta-1,4-N-acetylgalactosaminyl transferase knock-out mice with GD1a ganglioside-mimicking antigens from Campylobacter jejuni and generated high-titer anti-GD1a antisera and complement fixing monoclonal Abs (mAbs). Next, we exposed ex vivo nerve-muscle preparations from GD1a-overexpressing, GD3 synthase knock-out mice to the anti-GD1a mAbs in the presence of a source of complement and investigated morphological and electrophysiological damage. Dense antibody and complement deposits were observed only over presynaptic motor axons, accompanied by severe ultrastructural damage and electrophysiological blockade of motor nerve terminal function. Perisynaptic Schwann cells and postsynaptic membranes were unaffected. In contrast, normal mice were not only unresponsive to immunization with GD1a but also resistant to neural injury during anti-GD1a Ab exposure, demonstrating the central role of membrane antigen density in modulating both immune tolerance to GD1a and axonal susceptibility to anti-GD1a Abmediated injury. Identical paralyzing effects were observed when testing mouse and human anti-GD1a-positive sera. These data indicate that anti-GD1a Abs arise via molecular mimicry and are likely to be clinically relevant in injuring peripheral nerve axonal membranes containing sufficiently high levels of GD1a.

Anti-disialoside antibodies kill perisynaptic Schwann cells and damage motor nerve terminals via membrane attack complex in a murine model of neuropathy.

Anti-disialoside antibodies (Abs) that bind NeuAc(alpha2-8) NeuAc epitopes on GQ1b and related gangliosides are found in human autoimmune neuropathy sera and are considered to be pathogenic. In a model system in mice, one mechanism by which anti-disialoside Abs have been demonstrated to induce paralysis is through a complement dependent blocking effect on transmitter release at the neuromuscular junction, similar to the effects of alpha-latrotoxin. Although direct targeting of presynaptic neuronal membranes occurs in this model, concomitant injury to perisynaptic Schwann cells (pSC) could indirectly contribute to this paralytic effect by influencing nerve terminal function and survival. To examine this possibility and the specific complement components that might mediate these effects, we exposed neuromuscular junctions in vivo and in vitro to an anti-disialoside Ab in conjunction with intact and selectively deficient complement sources. Using immuno-electron microscopy, we observed Ab deposits equally distributed on both neuronal and pSC membranes, and ultrastructural evidence of injury at both sites. Presynaptic neuronal injury was demonstrated functionally with microelectrode recordings and histologically as neurofilament loss. As hypothesized, concomitant pSC injury occurred, as indicated by abnormal uptake of ethidium dimer into pSC nuclei. The pSC and nerve terminal damage indicators correlated well with deposition of the pore-forming terminal complement component, membrane attack complex (MAC) in pSC and nerve terminal membranes. Furthermore, both neuronal and pSC injury were exacerbated in tissues from mice lacking the inhibitory complement regulator, CD59, where MAC formation is increased. These data demonstrate that both presynaptic neuronal membranes and pSCs are targets for anti-disialoside Abs, and that the injury to both sites is mediated by MAC and further regulated by CD59. This is the first demonstration that complement mediated pSC injury occurs in a model of autoimmune neuropathy and provides a rationale for investigating the possibility of pSC injury in equivalent conditions in man.

Calpain inhibitors protect against axonal degeneration in a model of anti-ganglioside antibody-mediated motor nerve terminal injury.

Miller Fisher syndrome-associated anti-GQ1b ganglioside antibodies produce an acute complement-dependent neuroexocytic effect at the mouse neuromuscular junction (NMJ) that closely resembles the effect of alpha-latrotoxin (LTx). This pathophysiological effect is accompanied by morphological disruption of the nerve terminal involving the loss of major cytoskeletal components, including neurofilament. Both LTx and the membrane attack complex of complement form membrane pores that allow free ionic movement and we have previously hypothesized that Ca2+ ingress and the subsequent activation of Ca2+-dependent proteases, calpains, may lead to substrate degradation resulting in structural disorganization of the terminal. Here, we treated mouse NMJs in hemidiaphragm preparations with anti-GQ1b antibodies and complement, or with LTx in the presence and absence of extracellular Ca2+, and studied possible neuroprotective effects of the calpain inhibitors calpeptin and calpain inhibitor V. Both Ca2+ depletion and calpain inhibition protected the cytoskeleton from degradation, as assessed by immunohistological and ultrastructural analysis. Calpain inhibitors may therefore be useful therapeutically in limiting nerve terminal and axonal injury in autoimmune peripheral neuropathy and in human latrodectism.