Axolemmal nanoruptures arising from paranodal membrane injury induce secondary axon degeneration in murine Guillain-Barré syndrome.

The major determinant of poor outcome in Guillain-Barré syndrome (GBS) is axonal degeneration. Pathways leading to primary axonal injury in the motor axonal variant are well established, whereas mechanisms of secondary axonal injury in acute inflammatory demyelinating polyneuropathy (AIDP) are unknown. We recently developed an autoantibody-and complement-mediated model of murine AIDP, in which prominent injury to glial membranes at the node of Ranvier results in severe disruption to paranodal components. Acutely, axonal integrity was maintained, but over time secondary axonal degeneration occurred. Herein, we describe the differential mechanisms underlying acute glial membrane injury and secondary axonal injury in this model. Ex vivo nerve-muscle explants were injured for either acute or extended periods with an autoantibody-and complement-mediated injury to glial paranodal membranes. This model was used to test several possible mechanisms of axon degeneration including calpain activation, and to monitor live axonal calcium signalling. Glial calpains induced acute disruption of paranodal membrane proteins in the absence of discernible axonal injury. Over time, we observed progressive axonal degeneration which was markedly attenuated by axon-specific calpain inhibition. Injury was unaffected by all other tested methods of protection. Trans-axolemmal diffusion of fluorescent proteins  and live calcium imaging studies indirectly demonstrated the presence of nanoruptures in the axon membrane. This study outlines one mechanism by which secondary axonal degeneration arises in the AIDP variant of GBS where acute paranodal loop injury is prominent. The data also support the development of calpain inhibitors to attenuate both primary and secondary axonal degeneration in GBS.

The endogenous calpain inhibitor calpastatin attenuates axon degeneration in murine Guillain-Barré syndrome.

Axon degeneration accounts for the poor clinical outcome in Guillain-Barré syndrome (GBS), yet no treatments target this key pathogenic stage. Animal models demonstrate anti-ganglioside antibodies (AGAb) induce axolemmal complement pore formation through which calcium flux activates the intra-axonal calcium-dependent proteases, calpains. We previously showed protection of axonal components using soluble calpain inhibitors in ex vivo GBS mouse models, and herein, we assess the potential of axonally-restricted calpain inhibition as a neuroprotective therapy operating in vivo. Using transgenic mice that over-express the endogenous human calpain inhibitor calpastatin (hCAST) neuronally, we assessed distal motor nerve integrity in our established GBS models. We induced immune-mediated injury with monoclonal AGAb plus a source of human complement. The calpain substrates neurofilament and AnkyrinG, nerve structural proteins, were assessed by immunolabelling and in the case of neurofilament, by single-molecule arrays (Simoa). As the distal intramuscular portion of the phrenic nerve is prominently targeted in our in vivo model, respiratory function was assessed by whole-body plethysmography as the functional output in the acute and extended models. hCAST expression protects distal nerve structural integrity both ex and in vivo, as shown by attenuation of neurofilament breakdown by immunolabelling and Simoa. In an extended in vivo model, while mice still initially undergo respiratory distress owing to acute conduction failure, the recovery phase was accelerated by hCAST expression. Axonal calpain inhibition can protect the axonal integrity of the nerve in an in vivo GBS paradigm and hasten recovery. These studies reinforce the strong justification for developing further animal and human clinical studies using exogenous calpain inhibitors.

Complement inhibition prevents glial nodal membrane injury in a GM1 antibody-mediated mouse model.

The involvement of the complement pathway in Guillain-Barré syndrome pathogenesis has been demonstrated in both patient biosamples and animal models. One proposed mechanism is that anti-ganglioside antibodies mediate neural membrane injury through the activation of complement and the formation of membrane attack complex pores, thereby allowing the uncontrolled influx of ions, including calcium, intracellularly. Calcium influx activates the calcium-dependent protease calpain, leading to the cleavage of neural cytoskeletal and transmembrane proteins and contributing to subsequent functional failure. Complement inhibition has been demonstrated to provide effective protection from injury in anti-ganglioside antibody-mediated mouse models of axonal variants of Guillain-Barré syndrome; however, the role of complement in the pathogenesis of demyelinating variants has yet to be established. Thus, it is currently unknown whether complement inhibition would be an effective therapeutic for Guillain-Barré syndrome patients with injuries to the Schwann cell membrane. To address this, we recently developed a mouse model whereby the Schwann cell membrane was selectively targeted with an anti-GM1 antibody resulting in significant disruption to the axo-glial junction and cytoplasmic paranodal loops, presenting as conduction block. Herein, we utilize this Schwann cell nodal membrane injury model to determine the relevance of inhibiting complement activation. We addressed the early complement component C2 as the therapeutic target within the complement cascade by using the anti-C2 humanized monoclonal antibody, ARGX-117. This anti-C2 antibody blocks the formation of C3 convertase, specifically inhibiting the classical and lectin complement pathways and preventing the production of downstream harmful anaphylatoxins (C3a and C5a) and membrane attack complexes. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex vivo and in vivo Schwann cell nodal membrane injury models. In parallel studies, C2 inhibition also protects axonal integrity in our well-established model of acute motor axonal neuropathy mediated by both mouse and human anti-GM1 antibodies. These data demonstrate that complement inhibition prevents injury in a Schwann cell nodal membrane injury model, which is representative of neuropathies associated with anti-GM1 antibodies, including Guillain-Barré syndrome and multifocal motor neuropathy. This outcome suggests that both the motor axonal and demyelinating variants of Guillain-Barré syndrome should be included in future complement inhibition clinical trials.

Schwann cell nodal membrane disruption triggers bystander axonal degeneration in a Guillain-Barré syndrome mouse model.

In Guillain-Barré syndrome (GBS), both axonal and demyelinating variants can be mediated by complement-fixing anti-GM1 ganglioside autoantibodies that target peripheral nerve axonal and Schwann cell (SC) membranes, respectively. Critically, the extent of axonal degeneration in both variants dictates long-term outcome. The differing pathomechanisms underlying direct axonal injury and the secondary bystander axonal degeneration following SC injury are unresolved. To investigate this, we generated glycosyltransferase-disrupted transgenic mice that express GM1 ganglioside either exclusively in neurons [GalNAcT-/--Tg(neuronal)] or glia [GalNAcT-/--Tg(glial)], thereby allowing anti-GM1 antibodies to solely target GM1 in either axonal or SC membranes, respectively. Myelinated-axon integrity in distal motor nerves was studied in transgenic mice exposed to anti-GM1 antibody and complement in ex vivo and in vivo injury paradigms. Axonal targeting induced catastrophic acute axonal disruption, as expected. When mice with GM1 in SC membranes were targeted, acute disruption of perisynaptic glia and SC membranes at nodes of Ranvier (NoRs) occurred. Following glial injury, axonal disruption at NoRs also developed subacutely, progressing to secondary axonal degeneration. These models differentiate the distinctly different axonopathic pathways under axonal and glial membrane targeting conditions, and provide insights into primary and secondary axonal injury, currently a major unsolved area in GBS research.

Real time imaging of intra-axonal calcium flux in an explant mouse model of axonal Guillain-Barré syndrome.

The acute motor axonal variant of Guillain-Barré syndrome is associated with the attack of motor axons by anti-ganglioside antibodies which activate complement on the axonal plasma membrane. Animal models have indirectly implicated complement pore-mediated calcium influx as a trigger of axonal damage, through the activation of the protease calpain. However, this calcium influx has never been imaged directly. Herein we describe a method to detect changes in intra-axonal calcium in an ex vivo mouse model of axonal Guillain-Barré syndrome and describe the influence of calcium on axonal injury and the effects of calpain inhibition on axonal outcome. Using ex vivo nerve-muscle explants from Thy1-TNXXL mice which axonally express a genetically encoded calcium indicator, we studied the effect of the binding and activation of complement by an anti-GD1b ganglioside antibody which targets the motor axon. Using live multiphoton imaging, we found that a wave of calcium influx extends retrogradely from the motor nerve terminal as far back as the large bundles within the muscle explant. Despite terminal complement pores being detectable only at the motor nerve terminal and, to a lesser degree, the most distal node of Ranvier, disruption of axonal proteins occurred at more proximal sites implicating the intra-axonal calcium wave. Morphological analysis indicated two different types of calcium-induced changes: acutely, distal axons showed swelling and breakdown at sites where complement pores were present. Distally, in areas of raised calcium which lacked detectable complement pores, axons developed a spindly, vacuolated appearance suggestive of early signs of degeneration. All morphological changes were prevented with treatment with a calpain inhibitor. This is the first investigation of axonal calcium dynamics in a mouse model of Guillain-Barré syndrome and demonstrates the proximal reach of calcium influx following an injury which is confined to the most distal parts of the motor axon. We also demonstrate that calpain inhibition remains a promising candidate for both acute and sub-acute consequences of calcium-induced calpain activation.

Perisynaptic Schwann cells phagocytose nerve terminal debris in a mouse model of Guillain-Barré syndrome.

In mouse models of acute motor axonal neuropathy, anti-ganglioside antibodies (AGAbs) bind to motor axons, notably the distal nerve, and activate the complement cascade. While complement activation is well studied in this model, the role of inflammatory cells is unknown. Herein we aimed to investigate the contribution of phagocytic cells including macrophages, neutrophils and perisynaptic Schwann cells (pSCs) to distal nerve pathology. To observe this, we first created a subacute injury model of sufficient duration to allow inflammatory cell recruitment. Mice were injected intraperitoneally with an anti-GD1b monoclonal antibody that binds strongly to mouse motor nerve axons. Subsequently, mice received normal human serum as a source of complement. Dosing was titrated to allow humane survival of mice over a period of 3 days, yet still induce the characteristic neurological impairment. Behaviour and pathology were assessed in vivo using whole-body plethysmography and post-sacrifice by immunofluorescence and flow cytometry. ex vivo nerve-muscle preparations were used to investigate the acute phagocytic role of pSCs following distal nerve injury. Following complement activation at distal intramuscular nerve sites in the diaphragm macrophage localisation or numbers are not altered, nor do they shift to a pro- or anti-inflammatory phenotype. Similarly, neutrophils are not significantly recruited. Instead, ex vivo nerve-muscle preparations exposed to AGAb plus complement reveal that pSCs rapidly become phagocytic and engulf axonal debris. These data suggest that pSCs, rather than inflammatory cells, are the major cellular vehicle for axonal debris clearance following distal nerve injury, in contrast to larger nerve bundles where macrophage-mediated clearance predominates.

Glial Sulfatides and Neuronal Complex Gangliosides Are Functionally Interdependent in Maintaining Myelinating Axon Integrity.

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knock-out, CST-/-) or complex gangliosides (ß-1,4-N-acetylegalactosaminyltransferase1 knock-out, GalNAc-T-/-) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T-/- phenotype, as shown by neuron- or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST-/-, GalNAc-T-/-, and axo-glial protein-deficient mice suggests that these glycolipids stabilize membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST-/- and GalNAc-T-/- genotypes were interbred. CST-/-× GalNAc-T-/- mice develop normally to postnatal day 10 (P10), but all die between P20 and P25, coinciding with peak myelination. Ultrastructural, immunohistological, and biochemical analysis of either sex revealed widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST-/- × GalNAc-T-/- mice exhibited a major reduction in MAG protein levels in CNS myelin compared with WT and single-lipid-deficient mice. The CST-/- × GalNAc-T-/- phenotype was fully restored to that of CST-/- mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes, respectively, act in concert to promote NF155 and MAG in maintaining the stable axo-glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENT Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalization of multiple membrane proteins. The axo-glial adhesion molecules neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) require membrane microdomains containing either sulfatides or complex gangliosides to localize and function effectively. The cooperative roles of these microdomains and associated proteins are unknown. Here, we show vital interdependent roles for sulfatides and complex gangliosides because double (but not single) deficiency causes a rapidly lethal phenotype at an early age. These findings suggest that sulfatides and complex gangliosides on opposing axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins NF155 and MAG, which interact to maintain the nodal complex.

Differential binding patterns of anti-sulfatide antibodies to glial membranes.

Sulfatide is a major glycosphingolipid in myelin and a target for autoantibodies in autoimmune neuropathies. However neuropathy disease models have not been widely established, in part because currently available monoclonal antibodies to sulfatide may not represent the diversity of anti-sulfatide antibody binding patterns found in neuropathy patients. We sought to address this issue by generating and characterising a panel of new anti-sulfatide monoclonal antibodies. These antibodies have sulfatide reactivity distinct from existing antibodies in assays and in binding to peripheral nerve tissues and can be used to provide insights into the pathophysiological roles of anti-sulfatide antibodies in demyelinating neuropathies.

Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected.

The recent global outbreak of Zika virus (ZIKV) infection has been linked to severe neurological disorders affecting the peripheral and central nervous systems (PNS and CNS, respectively). The pathobiology underlying these diverse clinical phenotypes are the subject of intense research; however, even the principal neural cell types vulnerable to productive Zika infection remain poorly characterised. Here we used CNS and PNS myelinating cultures from wild type and Ifnar1 knockout mice to examine neuronal and glial tropism and short-term consequences of direct infection with a Brazilian variant of ZIKV. Cell cultures were infected pre- or post-myelination for various intervals, then stained with cell-type and ZIKV-specific antibodies. In bypassing systemic immunity using ex vivo culture, and the type I interferon response in Ifnar1 deficient cells, we were able to evaluate the intrinsic infectivity of neural cells. Through systematic quantification of ZIKV infected cells in myelinating cultures, we found that ZIKV infection is enhanced in the absence of the type I interferon responses and that CNS cells are considerably more susceptible to infection than PNS cells. In particular, we demonstrate that CNS axons and myelinating oligodendrocytes are especially vulnerable to injury. These results have implications for understanding the pathobiology of neurological symptoms associated with ZIKV infection. Furthermore, we provide a quantifiable ex vivo infection model that can be used for fundamental and therapeutic studies on viral neuroinvasion and its consequences.

Anti-ganglioside antibodies are removed from circulation in mice by neuronal endocytosis.

SEE VAN DOORN AND JACOBS DOI101093/BRAIN/AWW078 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : In axonal forms of Guillain-Barré syndrome, anti-ganglioside antibodies bind gangliosides on nerve surfaces, thereby causing injury through complement activation and immune cell recruitment. Why some nerve regions are more vulnerable than others is unknown. One reason may be that neuronal membranes with high endocytic activity, including nerve terminals involved in neurotransmitter recycling, are able to endocytose anti-ganglioside antibodies from the cell surface so rapidly that antibody-mediated injury is attenuated. Herein we investigated whether endocytic clearance of anti-ganglioside antibodies by nerve terminals might also be of sufficient magnitude to deplete circulating antibody levels. Remarkably, systemically delivered anti-ganglioside antibody in mice was so avidly cleared from the circulation by endocytosis at ganglioside-expressing plasma membranes that it was rapidly rendered undetectable in serum. A major component of the clearance occurred at motor nerve terminals of neuromuscular junctions, from where anti-ganglioside antibody was retrogradely transported to the motor neuron cell body in the spinal cord, recycled to the plasma membrane, and secreted into the surrounding spinal cord. Uptake at the neuromuscular junction represents a major unexpected pathway by which pathogenic anti-ganglioside antibodies, and potentially other ganglioside binding proteins, are cleared from the systemic circulation and also covertly delivered to the central nervous system.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

INTRODUCTION: Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. RESULTS AND CONCLUSIONS: In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Neuronal expression of GalNAc transferase is sufficient to prevent the age-related neurodegenerative phenotype of complex ganglioside-deficient mice.

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes ß-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.

The effects of age and ganglioside composition on the rate of motor nerve terminal regeneration following antibody-mediated injury in mice.

Gangliosides are glycosphingolipids highly enriched in neural plasma membranes, where they mediate a diverse range of functions and can act as targets for auto-antibodies present in human immune-mediated neuropathy sera. The ensuing autoimmune injury results in axonal and motor nerve terminal (mNT) degeneration. Both aging and ganglioside-deficiency have been linked to impaired axonal regeneration. To assess the effects of age and ganglioside expression on mNT regeneration in an autoimmune injury paradigm, anti-ganglioside antibodies and complement were applied to young adult and aged mice wildtype (WT) mice, mice deficient in either b- and c-series (GD3sKO) or mice deficient in all complex gangliosides (GM2sKO). The extent of mNT injury and regeneration was assessed immediately or after 5 days, respectively. Depending on ganglioside expression and antibody-specificity, either a selective mNT injury or a combined injury of mNTs and neuromuscular glial cells was elicited. Immediately after induction of the injury, between 1.5% and 11.8% of neuromuscular junctions (NMJs) in the young adult groups exhibited healthy mNTs. Five days later, most NMJs, regardless of age and strain, had recovered their mNTs. No significant differences could be observed between young and aged WT and GM2sKO mice; aged GD3sKO showed a mildly impaired rate of mNT regeneration when compared with their younger counterparts. Comparable rates were observed between all strains in the young and the aged mice. In summary, the rate of mNT regeneration following anti-ganglioside antibody and complement-mediated injury does not differ majorly between young adult and aged mice irrespective of the expression of particular gangliosides.