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

Cunningham, Madeleine E.; McGonigal, Rhona; Barrie, Jennifer A.; Yao, Denggao; Willison, Hugh J.

doi:10.1016/j.expneurol.2022.114127

Cited by 5 publications

(15 citation statements)

References 48 publications

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“…Significantly, ankyrin G (AnkG), the axonal anchoring protein responsible for tethering and maintaining voltage-gated sodium (Nav) channels in conjunction with neurofascin 186 (NF186) to the actin-spectrin cytoskeleton at the NoR ( 17 ), was also disturbed in these models. Later, the direct axonopathic mechanism was shown to be mediated through membrane attack complex (MAC) pore formation, calcium influx ( 18 ), and activation of the calcium-dependent protease calpain ( 16 , 19 ) for which neurofilament, AnkG, actin, and spectrin are substrates.…”

Section: Introductionmentioning

confidence: 99%

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

McGonigal¹,

Barrie²,

Yao³

et al. 2022

Journal of Clinical Investigation

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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 axon 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 (NoR) occurred. Following glial injury, axon disruption at nodes also developed sub-acutely, progressing to secondary axon degeneration. These models differentiate the distinctly different axonopathic pathways under axonal and glial membrane targeting conditions, and provide insights into primary and secondary axon injury, currently a major unsolved area in GBS research.

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Section: Introductionmentioning

confidence: 99%

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

McGonigal¹,

Barrie²,

Yao³

et al. 2022

Journal of Clinical Investigation

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“…1 Human tissue, and rabbit and mouse models of AMAN have shown complement deposition over the motor nerve terminals (MNT) and both proximal and distal motor axonal NoR. Activation of the complement cascade culminates in the formation of the membrane attack complex (MAC) pore, leading to bi-directional movement of water and ions, including Ca 2+ ions, 12 which coincides with pre-synaptic and nodal dysfunction and axonal conduction block. 8,9,13,14 From these models, it has been shown that at the MNT and NoR, neurofilament immunostaining, an indicator of disruption to axonal integrity preceding axon degeneration, is lost following AGAb complement-mediated injury.…”

Section: Introductionmentioning

confidence: 99%

“…We previously showed in our ex vivo mouse model, through the exogenous application of soluble calpain inhibitors, that the observed structural disturbances are mediated by activation of calpain due to the influx of Ca 2+ ions through MAC pores. 8,12,27 To explore the benefit of calpain inhibition in vivo, we assessed axonal protection using a transgenic calpastatin over-expression paradigm. Calpastatin is a calcium-dependent endogenous inhibitor specific for calpains 28 which are widely expressed in the mammalian nervous system.…”

Section: Introductionmentioning

confidence: 99%

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

McGonigal

Cunningham

Smyth

et al. 2022

J Peripheral Nervous Sys

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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.

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“…Binding of anti-GM1 mAb to the Schwann cell membrane, particularly at the paranode, activates the classical complement pathway and the formation of MAC pores in the membrane. We know from previous studies that these MAC pores, in addition to directly affecting ionic homeostasis, are notably associated with an influx of calcium 10 which then activates the calcium-dependent protease calpain. 12 Many components of the underlying cytoskeleton are known calpain substrates, including actin and AnkB 13 which are present in the cytoplasmic paranodal loops.…”

Section: Discussionmentioning

confidence: 99%

“… 8 These pores allow the bi-directional flow of ions and water, disrupting ionic homeostasis and culminating in swelling and cell lysis. 9 A consequence of this is an intracellular calcium influx causing a retrograde calcium wave 10 and the subsequent activation of the calcium-dependent protease calpain, 11 , 12 known to cleave neural cytoskeletal structural proteins such as neurofilament, actin and ankyrin. 13 , 14 This distal motor nerve injury results in distal axonal and neuromuscular transmission blocks, presenting as paralysis.…”

Section: Introductionmentioning

confidence: 99%

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

McGonigal

Barrie

Delaere

et al. 2022

Brain Communications

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The involvement of the complement pathway in Guillain-Barré syndrome pathogenesis has been demonstrated in both patient bio-samples 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 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 injury 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 utilise 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 humanised 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 complex. Here, we demonstrate that C2 inhibition significantly attenuates injury to paranodal proteins at the node of Ranvier and improves respiratory function in ex 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, 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.

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

Cited by 5 publications

References 48 publications

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

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

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

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

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