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

Abstract 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.


| INTRODUCTION
The primary targets of autoimmune injury in acute inflammatory demyelinating polyneuropathy (AIDP), the demyelinating form of Guillain-Barré syndrome (GBS), are Schwann cell membranes. These comprise the internodal compacted myelin membranes, abaxonal membranes and the specialised non-myelinated membranes of the nodal complex. Whilst the range of Schwann cell antigens in AIDP has yet to be fully defined, it is widely believed that complement fixing autoantibodies to glycolipids, including gangliosides, sulphated glycolipids and possibly other unidentified molecules, mediate the injury. [1][2][3] Ultimately, Schwann cell injury results in conduction block due to nodal disorganisation, usually accompanied by segmental demyelination. [4][5][6] The recovery rates in the acute motor axonal neuropathy (AMAN) variant of GBS and in AIDP are dependent upon the extent and site of axon degeneration. 7,8 In AMAN, where the axonal injury is the primary pathological event, recovery varies from very poor to complete, depending on the site and extent of axonal injury. In pure AIDP without axonal injury, segmental remyelination is an efficient process in peripheral nerve with variable and complete restoration of function. 9,10 When AIDP is complicated by secondary axon degeneration, this may result in permanent denervation, especially when the axon loss occurs proximally, and consequential long-term functional loss. 3,7 The mechanisms by which axons undergo secondary axon degeneration in AIDP are unknown. Autopsy studies of AIDP patients have shown deposits of complement including the terminal membrane attack complex (MAC) over glial membranes, 11 accompanied by infiltration of macrophages which phagocytose myelin debris. 12 In AIDP patients with secondary axonal degeneration, studies have suggested a variety of causative mechanisms including compression of the axon due to infiltration of macrophage processes 13 and rises in endoneurial fluid pressure at critical anatomical sites that might induce ischaemic and other injuries to axons. 14 In rat models of experimental allergic neuritis (EAN) in which immune attack is targeted to myelin, degradation of the axon-glial unit at nodes of Ranvier (NoR) with disruption of adhesion molecules and ion channels is a prominent feature, 4 but the causal mechanisms linking these events to the concomitant axon degeneration have not been elucidated. In non-inflammatory demyelinating neuropathies, notably Charcot-Marie-Tooth diseases, secondary axonal degeneration is widespread over a long timescale and attributed to energy failure, metabolic and neurotrophic factor deprivation. 15 Many lines of evidence thus support the symbiotic interdependency between Schwann cells and axons that are essential for each other's survival and function, and that will vary considerably in different developmental and pathological contexts.
We recently reported the mouse model of Schwann cell membrane-directed injury, in which anti-GM1 antibody (Ab) plus complement targets the glial membrane, resulting in deposition of MAC pores at the distal paranodal glial membranes, and the disruption of nodal architecture. 5 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][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. Herein we describe one putative mechanism by which this secondary axonal degeneration occurs.
The following commercial antibodies were used: rat anti-myelin

| Mice
Studies were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Wild-type mice used in these studies were C57 Bl/6 mice. All genetically altered mice used for acute and extended ex vivo glial injury paradigms are described in Table 1 and all are on a C57BL/6 background. Mice used were both male and female sexes and aged between 4 and 10 weeks.

| Experimental design and statistics
Sample size for ex vivo models was estimated based on a priori power calculations from pilot studies with G*power analysis software (v.3.1.9.4). For studies using anti-GM1 antibody, an effect size of 1.78, an alpha error probability of 0.05 and a power of 0.8 was used to calculate group sizes. For studies using GAME-G3 antibody, an effect size of 3.51, an alpha error probability of 0.05 and a power of 0.8 was used to calculate group sizes.
Immunofluorescent images were analysed blinded by a single observer for each complete experimental dataset. All data are displayed as mean ± SE of the mean (SEM) and relevant statistical tests and mouse numbers used for each analysis are described in the figure legends. Statistics were performed using GraphPad Prism (version 6.07), as was the generation of all graphs

| Ex vivo injury models
Triangularis sterni nerve-muscle explants from wild-type, GalNAc-T À/À -Tg(glial) (referred to hereafter as Glial), SARM1 KO or hCAST Â Glial mice were dissected and maintained in oxygenated Ringer's solution. Acute and extended glial injury was performed as described previously. 5 Briefly, explants were incubated with either anti-sulfatide antibody or anti-GM1 antibody at 100 μg/mL alone (control) or with 40% normal human serum (NHS) as a source of complement (injury) in Ringer's. The antibody used was dependent on the ganglioside expression in the mice; Glial and hCAST Â Glial only express complex gangliosides on glial cells and therefore anti-GM1 antibody DG2 could be used to induce glial injury. In all other genotypes, gangliosides are expressed ubiquitously, therefore the anti-sulfatide antibody GAME-G3 was used to target injury to the glial membrane (Table 1). Antibodies to both GM1 and sulfatide are clinically relevant antibodies in AIDP patients, with varying proportions of patients being seropositive for these antibodies. 2,3,23,24 For acute injury, explants were incubated in control or injury solution For extended injury, explants were incubated for 20 h at room temperature (RT) then washed and fixed as above. To decipher T A B L E 1 Genetically altered mice used in this study and anti-glycolipid antibodies used for each genotype.

Mouse Genetic alteration Purpose
Original reference Anti-glycolipid antibody used GalNAc-T À/À -Tg(glial) (Referred to as Glial) GalNAc-T expression driven by Plp promoter on GalNAc-T À/À background For dye application studies, extended glial injury was performed as described above, then tissue was washed, and, prior to fixation, dextran dyes were added at 1 mg/mL for 2 h at 37 C, followed by 4Â 30 min washed with Ca 2+ free Ringer's solution to reduce membrane activity. Explants were then fixed as described above.
Following fixation and washes, explants were incubated for 10 min in freezing ethanol at À20 C then primary antibodies were added overnight in PBS containing 3% NGS and 0.5% Triton X-100.
The following day, subtype-specific secondary antibodies were added for 2 h at RT then explants were washed and mounted on APEScoated slides in Citifluor antifadent mounting medium (Citifluor, USA).

| Immunofluorescent image capture
Fluorescent images were captured using a Zeiss AxioImager Z1. For illustrative images, an Apotome attachment was used.

| Live calcium imaging studies
Nerve-muscle explants were maintained in fresh Ringer's solution. Baseline images were taken (described below) then explants were incubated with monoclonal anti-sulfatide antibody GAME-G3 (100 μg/mL) along with 40% NHS in Ringer's for 4 h at RT. Explants were washed 3Â with Ringer's solution before every subsequent image was taken and returned to Ab/NHS solution in between images.
Images were taken up to 10 h post-addition of antibody and NHS. Explants were then fixed as described above and TS muscles were removed and frozen down at À80 C. Images were captured as previously described. 18 Briefly, Z-stacks of explant regions replete with neuromuscular junctions (ranges included in figure legends) were acquired using a Zeiss LSM7 MP system, with a 20Â/1.0NA water-immersion objective lens and a tuneable tita-

| Image analysis and quantification
Average intensity of GFP was measured along a 10 μm section spanning the final distal NoR determined by a gap in MBP staining. This was achieved using the line feature in the Fiji distribution of ImageJ software (version 1.52b). 26 For presence and absence of other immunofluorescent markers or dyes, images were analysed blinded by a single investigator. For pan-Neurofascin staining, two neurofascins have distinct staining patterns allowing their discrimination. Here we quantified two outputs: (1) Immunostaining that appeared normal and consisted of both NF155 and NF186 (if only nodal NF186 was present, these were counted as absent); (2) Length of immunostaining present. If staining was completely absent no measurements were made.
Axonal calcium presence was measured by calculating ratios of citrine/CFP in Thy1-TNXXL mice as previously described. 18 The morphology of axons was classed as either "normal", "swollen" or "fragmented" at each timepoint measured. Fragmented axons were those displaying separation between segments of the axon even after the image stack was contrast adjusted. Calcium FRET images are displayed as ratiometric images which were created as previously described. 18 3 | RESULTS

| Terminal complement pores disrupt glial membrane integrity
Following acute immune-mediated injury to the glial membranes, we show deposits of MAC at the distal NoR, particularly at paranodal regions ( Figure 1A). We have previously shown that CFP expressed in axonal cytoplasm is lost through MAC pores when axons are directly targeted with anti-ganglioside antibody and a source of complement. 27,28 Using mice that express cytosolic GFP driven by the S100B promoter, and therefore present in Schwann cell cytoplasm, we here demonstrate that following acute (4 h) glial injury, deposits of MAC on the paranodal glial membrane are accompanied by a significant decrease in GFP fluorescent intensity, indicating a loss of GFP likely through the complement pores ( Figure 1A, two-tailed paired t test, P < 0.05). As previously observed, MBP disruption was often noted in this model 5 as was S100 staining of Schwann cells (data not shown).

| Glial calpains disrupt glial cytoskeletal and cell adhesion molecules
To determine the role of calpain in the disruption of the paranode following acute immune-mediated injury to the glial membrane, we used genetic and exogenous calpain inhibition strategies and studied the structural and cell adhesion paranodal proteins AnkyrinB and NF155, respectively. As shown previously, AnkyrinB immunostaining is disrupted when glial membranes are targeted compared to control. 5 There is a significant reduction in AnkyrinB in all injured tissue compared to control ( Figure 1B,  There was a significant reduction in total pan-neurofascin length in all injured tissue compared to control. Again, there was a comparable but incomplete improvement compared to injury in the presence of calpain inhibition by both hCAST expression and application of AK295.

| Axonal calpain inhibition attenuates secondary loss following glial injury
At the acute timepoints described above, axonal protein NF-H immunostaining was previously shown to be unaffected. 5 Here, we show again that NF-H is intact following acute glial injury, but that NF-H is progressively lost over extended periods of injury, first undergoing fragmentation and then disappearing ( Figure 2A). We next assessed the various mechanisms that might underlie this secondary NF-H immunostaining loss following extended glial injury ex vivo, using a range of biochemical mediators and transgenic mice ( Figure 2B,C, pairings were compared by individual one-tailed, paired student's ttests  Results are represented as the mean ± SEM. n = 6/ treatment; on average 29 NoR/mouse were analysed (range 19-51). Data were compared by a paired two-tailed t-test, * signifies P < 0.05. (B) Nerve-muscle preparations from Glial or hCAST Â Glial mice were treated acutely ex vivo with anti-GM1 Ab alone (Control) or with a source of complement (Injury) in the presence or absence of exogenous calpain inhibitor AK295. Loss of nodal protein (magenta) immunostaining at the NoR due to injury was assessed; the site of expected staining is indicated by arrowheads. Representative images demonstrate nodal protein localisation coinciding with complement deposition (green). AnkyrinB (AnkB) immunostaining was significantly reduced compared to control for each treatment. A significant protection was conferred by AK295. (C) A pan-neurofascin (pNfasc) antibody was used to assess paranodal NF155 and nodal NF186. NoR with normal paranodal pNFasc immunostaining significantly decreased following Glial injury and was partially restored by both hCAST expression and AK295. Scale bars = 5 μm. Results are represented as the mean ± SEM. n = 3/treatment; on average 24 NoR/mouse were analysed (ranges 12-44 AnkB, 16-34 pNFasc). One-way ANOVA, followed by Tukey post-hoc tests were used. *** signifies P < 0.001, ** signifies P < 0.01 and * signifies P < 0.05. As the loss of intra-axonal cytoplasmic CFP is not prevented, even in the presence of axonal calpain inhibition, we considered that nanoruptures in the axolemmal membrane might exist, through which CFP was leaking into the extracellular milieu. This would also be consistent with calcium influx through axolemmal membrane pores, as opposed to the release of calcium from intracellular stores. We sought to confirm this and determine the relative size of the axonal membrane pores by using different molecular weight dextran dyes following extended glial injury. We analysed the presence of dyes at distal axons ( Figure 3F . Student's t-test and one-way ANOVA, followed by Tukey's post-hoc tests were used to assess significant differences. *** signifies P < 0.001, ** signifies P < 0.01 and * signifies P < 0.05.
calpain-mediated paranodal loop distortion leading to an indirect effect on axonal binding partners and axonal integrity. In this study, we investigate the mechanisms leading to axonal injury following subacute glial injury. Here, we show that, axonal calpain inhibition has no glial protective effect acutely, whereas both glial and axonal protection is seen with AK295. The partial protection seen with AK295 is likely due to incomplete calpain inhibition resulting from insufficient tissue penetration or be concentration dependent. Immunohistological readouts may also be affected by tissue swelling and molecular distortion.

|
Pan-Neurofascin staining was moderately protected by both axonal calpain inhibition in hCAST mice, and by exogenous AK295. The localisation of pan-Neurofascin staining correlates with the paranodal region and, therefore, assumed to be NF155, was absent in injury.
Consequently, it seems that NF155 is disrupted by glial membrane injury and that this is in part mediated by calpains. NF155 is partially tethered by AnkyrinB at the glial paranodal membrane 33 ; however, during development there may be redundancy in the requirements for this interaction. 34 Nevertheless, in our severe acute injury model, loss of AnkyrinB by calpain cleavage and membrane distortion does also appear to result in the loss of detectable NF155 immunostaining at the paranodes, although the precise mechanism for this remains unknown. The partial rescue by exogenous calpain inhibition may therefore result from the partial protection of AnkyrinB. However, axonal calpains must also play a role in NF155 loss, perhaps due to the disruption of axonal anchoring proteins such as Caspr, which we previously showed was lost following acute glial injury. 5 This would explain the partial protection of pan-neurofascin staining by axonal hCAST expression. The mechanism of axonal calpain involvement at this early timepoint is unknown as no detectable axonal calcium flux is present at acute time points, and NF-H staining is also intact, suggesting a more subtle and complicated contribution to NF155 loss. It is possible that low intracellular calcium concentrations are sufficient to activate calpain 1 and affect axonal proteins at this early stage and that this is undetectable by TNXXL calcium imaging. The interpretation of this result is confounded by the use of pan-neurofascin antibody as the partial protection of neurofascin staining could also be a spread of NF186 from the axonal NoR.
F I G U R E 3 Following extended glial injury, pores form in axonal membranes allowing influx of calcium with pathological consequences. Triangularis sterni nerve-muscle explants from Thy1-TNXXL mice were imaged at baseline (Pre) then treated ex vivo for 4 h with anti-sulfatide antibody (Ab) alone (Control) or with a source of complement (Injury). Subsequent images were captured hourly up to 10 h. (A) Ratiometric calcium images showing axonal calcium signals. Colour key demonstrates relative calcium levels. Scale bar = 50 μm. (B) Calcium rose significantly compared to controls at motor nerve terminals (MNTs; arrows) after 6 h and at distal axons at 8 h. (C) Average peak calcium levels for each mouse showed that injured mice had significantly higher axonal calcium than control counterparts at both MNTs and distal axons. (D) normal morphology was reduced at both measured sites in injured explants. n = 4 control, n = 5 injury; on average 15 axons were measured per mouse (range [11][12][13][14][15][16][17][18][19][20][21]. Two-way ANOVA followed by Sidak's post-hoc tests were used to assess significant differences. ****signifies P < 0.0001, ***signifies P < 0.001, **signifies P < 0.01 and *signifies P < 0.05. (E) Peak levels of calcium were significantly higher in "fragmented" axons by the conclusion of the experiment than those whose morphology remained normal. n = 5 for normal and swollen, n = 4 fragmented. One-way ANOVA with Tukey's post-hoc test was used to assess significant differences. *signifies P < 0.05. (F) Explants from B6.Cg-Tg(Thy1-CFP/S100B-GFP) with single fluorescence for axonal cyan fluorescent protein (CFP) were incubated with either anti-sulfatide Ab alone (Control) or with a source of complement (Injury) for 20 h. Explants were then incubated with different-sized dextrans for 2 h. No differences were observed between dextrans in control tissues. In Injured tissues, significantly less penetration was seen with 70 kDa dextran than with 3 kDa dextran. Illustrative images are shown of injured tissue with differently sized dextrans. White arrowheads show 3 kDa Dextran colocalising with remaining axonal CFP. 3 kDa Dextran also does not closely associate with areas of MAC or MBP positive staining. n = 3 3 kDa, n = 4 70 kDa; on average 22 single axons/mouse (range 16-33) and 40 total axons (range 20-54) were analysed per mouse. Two-way ANOVA followed by Sidak's post-hoc tests were used to assess significant differences. **signifies P < 0.01. All results are represented as the mean ± SEM. Scale bar = 10 μm.

| Secondary axon degeneration is prevented by axonal calpain inhibition
Following acute injury to the paranodal glial membranes we previously showed early changes to the axonal paranodal (Caspr) and nodal proteins (NF186, Nav1.6, AnkyrinG), but preservation of the major axonal structural protein NF-H, indicating a maintenance of axonal integrity at this early timepoint. 5 [37][38][39][40][41] Addition of exogenous L-Lactate can therefore compensate for deficits in Schwann cell metabolic support to axons. 36 We tested this in our model, but lactate supplementation did not attenuate axonal changes.
ROS may cause axon degeneration in neuropathies. 42 Lipid-rich myelin has a high rate of oxidative metabolism and therefore is susceptible to the oxidative stress that produces free radicals like ROS. In GBS patients, levels of some antioxidants have been shown to be reduced in the blood 43,44 though how these changes correlate to oxidative myelin or axonal damage has not been fully explored. Injured axons in an in vitro model of axonal GBS were shown to release H 2 O 2 from mitochondria due to calcium overload. 45 We tested whether Secondary axon degeneration could occur as a result of a programmed process of axon degeneration. SARM1 is an injury-induced NADase. Deletion of the SARM1 protein has been shown to protect against axon degeneration in many peripheral neuropathy models, including chemotherapy-induced peripheral neuropathies, diabetic neuropathies as well as central nervous system models of demyelinating disease. 47,48 However, it has had mixed effectiveness in preventing secondary axon degeneration in models of different variants of genetic demyelinating neuropathies. 49,50 We have previously shown that in our model of primary axonal injury, use of WLD s mice, which also show delayed Wallerian degeneration, did not protect from axonal injury. 51 This has also been demonstrated in SARM1 KO mice in our primary axonal injury model (unpublished observations). Here we show that secondary axonal degeneration in our model is not prevented in SARM1 KO mice and therefore is likely also not a programmed event.
Calpain inhibition prevents both programmed and nonprogrammed axon degeneration and is therefore a final executioner for many types of axon degeneration. [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. We, therefore, demonstrate a converging pathway in both primary and secondary axon degeneration. As calpain is a calcium-activated protease we next sought to demonstrate the events preceding calpain activation.

| Axonal calcium flux through nanoruptures precedes secondary axon degeneration
Following extended glial injury, we demonstrated the presence of an axonal calcium flux. This became most apparent 6 to 9 h post glial injury and demonstrates that a rise in axonal calcium precedes secondary axon degeneration in this model. Measured MNTs appeared to show rises in calcium at earlier timepoints than single axons, likely due to the terminal heminode being more vulnerable to our injury than more proximal NoR due to a weaker blood nerve barrier. 16 The source of axonal calcium could be from intracellular calcium stores, or be extracellular, entering via calcium channels. However, we have previously noted that endogenously expressed axonal cytoplasmic CFP is lost in our extended glial injury model. 5 Additionally, here we demonstrate that despite axonal structural integrity being protected (as judged by NF-H staining), axonal CFP loss still occurs, not being prevented by calpain inhibition. This implies that axon membrane integrity must remain impaired in extended glial injury, allowing bidirectional flow of molecules, with calcium entering and CFP exiting.
We, therefore, found it more likely that calcium entry was via a previously proposed and demonstrated pathway: nanoruptures in the axolemma. 54,57 To investigate this in the absence of any available direct imaging methods, we used labelled dextrans to functionally demonstrate the putative nanopores in the axon membrane and determine their relative sizes, concluding they are in the range of 2 to 14 nm. 58,59 This would be compatible with the leakage of small molecules such as CFP (27 kDa) and ions, whilst preserving major structural integrity. Dextran localisation appears to be axonal but low molecular weight dextrans accumulating through MAC pores on the paranode cannot be excluded.
The nanoruptures we observed are similar in size to those formed in mouse models of experimental allergic encephalomyelitis (EAE). 57 The exact cause of the formation and appearance of these nanoruptures is unknown, but the authors speculated several potential mechanisms of rupture formation in this model, including mechanical stress, production of cytotoxic ROS or release of toxic mediators by immune cells. In our model, we have ruled out axonal metabolic insufficiency and injury due to cytotoxic ROS release from injured Schwann cells and immune cell involvement is unlikely in this ex vivo paradigm. The axon cytoskeleton, comprising periodic repeating rings of spectrins and ankyrins is critical for axonal integrity by forming a scaffolding, which also aligns to paranodal cell adhesion molecules. 60 This cytoskeleton has a proposed role in buffering axons from mechanical stress. 61 Therefore, we propose that the combination of distortion of the paranode and axoglial interface and disruption of axonal nodal and paranodal protein interactions causes the underlying axonal cytoskeleton to be compromised (Figure 4). With a reduced ability to buffer mechanical stress, the paranodal swelling causes compression and shearing of the axon, a known mechanism of pore formation in lipid membranes. 62,63 We speculate that the axonal paranode is a particularly vulnerable region for nanoruptures because of the tight and complex interactions between axonal and glial proteins which are necessary for maintenance of a stable node of Ranvier. 60 The concept of nanoruptures that allows influx of extracellular calcium have been demonstrated before. 54,57 Compellingly, these studies demonstrated that calcium influx does not necessarily destine an axon for F I G U R E 4 Proposed mechanism of secondary axon degeneration in mouse model of extended glial injury. 1. Antibody binds to glial paranode. 2. Complement pore is deposited in glial membrane. 3. Influx of calcium. 4. Paranodal proteins disrupted by glial calpains and membrane distortion. 5. Axon cytoskeleton is disturbed. 6. Axon undergoes mechanical stress. 7. Nanoruptures form on axon membrane. 8. Extracellular calcium enters the axon. 9. Axonal calpains activate and begin to degrade NF-H. 10. NF-H loss by axonal calpain is complete. 11. Nanoruptures remain present on axon membrane. Created with BioRender.com transection with ensuing destruction of the distal stump. Spontaneous membrane resealing may return an axon to its normal state. This metastable state of axon pathology infers a tipping point or threshold for calpain activation in individual axons. 54,57,64 The resulting dichotomy in axonal fate may explain both why primary and secondary axon degeneration occurs to differing extents in AMAN and AIDP patients respectively.
The formation of axonal nanoruptures appears to be the driving cause of secondary axonal degeneration following selective glial membrane injury in this model system. As with primary axon degeneration, axonal calcium influx followed by the activation of calpain are the climactic events in secondary axon degeneration. Importantly, the secondary axon degeneration we have observed is not directly associated with complement cascade activation in the axolemma. The common pathway of both primary and secondary axon degeneration is, thus, at the level of calcium-activated calpain cleavage, not complement.
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. As MAC pores are shed and nanoruptures reseal, the expectation would be that this shifts the metastable state in favour of axonal survival rather than transection. 54 Calpain inhibition strategies are of potential benefit in many disorders including GBS, though the development of successful treatments has proven challenging. 65,66 In GBS, calpain inhibition as a neuroprotective strategy might be best used as a combinatorial approach along with other treatments including autoantibody depletion and complement inhibition, strategies which have been shown to be effective in both axonal and glial injury-targeted mouse models of GBS. 27,67,68