The endogenous calpain inhibitor calpastatin attenuates axon degeneration in murineGuillain‐Barrésyndrome

Abstract

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.

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

“…In the acute phase of this model, both ex vivo and in vivo, we observed the loss of many glial nodal complex markers, indicating paranodal disruption. Ultrastructural analysis also showed greatly swollen and distorted paranodal regions likely due to the influx of extracellular fluid and ions via MAC pores that activate calpain cleavage pathways, mechanistically similar to that occurring in our AMAN models 16‐19 . At the acute timepoint in this glial model, axonal integrity remained intact; however, over time following more extended glial injury, both ex vivo and in vivo, secondary axonal degeneration developed.…”

“…We have demonstrated the central role of calpain and calpain inhibition in preventing the neurofilament breakdown occurring after extended glial membrane injury. We also observe this calpain pathway in a model of primary axonal injury, where axons directly targeted by anti‐GM1 antibody are protected by expression of axonal calpastatin and recover more rapidly 19 . Calpain inhibition is thus a promising candidate for therapeutic intervention in primary axonal forms of GBS and may also attenuate secondary axon degeneration in demyelinating forms of GBS.…”

“…Calpain inhibition prevents both programmed and non‐programmed axon degeneration and is therefore a final executioner for many types of axon degeneration 52‐56 . Calpain‐mediated injury is responsible for the primary axon degeneration seen in our AMAN models 16‐19 . Therefore, we anticipated a role for calpain in secondary axon degeneration.…”

“…Ultrastructural analysis also showed greatly swollen and distorted paranodal regions likely due to the influx of extracellular fluid and ions via MAC pores that activate calpain cleavage pathways, mechanistically similar to that occurring in our AMAN models. [16][17][18][19] At the acute timepoint in this glial model, axonal integrity remained intact; however, over time following more extended glial injury, both ex vivo and in vivo, secondary axonal degeneration developed. This experimental paradigm allows us to investigate potential mechanisms by which secondary axonal degeneration occurs following extended glial injury.…”

“…[52][53][54][55][56] Calpain-mediated injury is responsible for the primary axon degeneration seen in our AMAN models. [16][17][18][19] Therefore, we anticipated a role for calpain in secondary axon degeneration. Using axonal calpain inhibition, we observed a major attenuating effect on secondary axonal degeneration as assessed by NF-H loss following extended glial injury.…”