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Brain, Vol. 126, No. 11, 2497-2509, November 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg254

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

Graham M. O’Hanlon1, Peter D. Humphreys1, Rebecca S. Goldman1, Susan K. Halstead1, Roland W. M. Bullens3,4, Jaap J. Plomp3,4, Yuri Ushkaryov2 and Hugh J. Willison1

1 University Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow, 2 Department of Biological Sciences, Imperial College, London, UK and 3 Departments of Neurophysiology and 4 Neurology, Leiden University Medical Centre, The Netherlands

Correspondence to: Hugh J. Willison, Division of Clinical Neurosciences, University of Glasgow Department of Neurology, Southern General Hospital, Glasgow G51 4TF, UK E-mail: h.j.willison{at}udcf.gla.ac.uk

Received April 29, 2003. Revised June 4, 2003. Accepted June 5, 2003.


   Summary
Top
 Summary
Introduction
 Material and methods
 Results
 Discussion
 References
 
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.

Keywords: Miller Fisher syndrome; neuromuscular junction; anti-ganglioside antibody; {alpha}-latrotoxin; calpain inhibitor

Abbreviations: ACh = acetylcholine; BTx = {alpha}-bungarotoxin; CI-V = calpain inhibitor V; EM = electron microscopy; GBS = Guillain–Barré syndrome; LTx = {alpha}-latrotoxin; MAC = membrane attack complex; MEPP = miniature end-plate potential; NF = neurofilament; NHS = normal human serum; NMJ = neuromuscular junction; pSC = perisynaptic Schwann cell


   Introduction
Top
 Summary
 Introduction
Material and methods
 Results
 Discussion
 References
 
The functional transection of peripheral motor axons is a major cause of chronic disability in a wide variety of peripheral nervous system disorders. Such transection may occur through antibody-dependent complement-mediated injury, as seen in axonal forms of Guillain–Barré syndrome (GBS), where the targeting of neuronal gangliosides by autoantibodies precipitates a pro-inflammatory cascade (O’Hanlon et al., 2002Go; Willison and Yuki, 2002Go). In the in vitro mouse hemidiaphragm, anti-GQ1b ganglioside antibodies associated with the GBS variant termed Miller Fisher syndrome produce a complement-dependent effect at the neuromuscular junction (NMJ) that is electrophysiologically similar to that of the spider neurotoxin, {alpha}-latrotoxin (LTx). Both cause massive asynchronous release of acetylcholine (ACh), manifested as elevated frequencies of miniature end-plate potentials (MEPPs), the small postsynaptic depolarizations that result from the spontaneous presynaptic release of single quanta of ACh packaged in a single vesicle. The increased MEPP frequency leads to asynchronous muscle fibre twitching, which is rapidly followed by paralysis (Goodyear et al., 1999Go; Plomp et al., 1999Go; Willison and O’Hanlon, 1999Go). At physiological Ca2+ concentrations, there is also a severe loss of presynaptic cytoskeletal proteins and degenerative change at the NMJ (Okamoto et al., 1971Go; Duchen et al., 1981Go; O’Hanlon et al., 2001Go). Both LTx and the activated complement product, membrane attack complex (MAC, comprising components C5b-9), form non-selective pores within the target membrane that allow the uncontrolled passage of ions and small molecules (Acosta et al., 1996Go; Davletov et al., 1998Go; Newsholme et al., 1999Go; Ashton et al., 2000Go). In both situations, it is likely that the presynaptic NMJ cytoskeletal injury is mediated, at least in part, by uncontrolled Ca2+ influx with subsequent activation of calpains. This is supported by the observation that neurofilament (NF) damage can be produced by the treatment of nerves with a Ca2+ ionophore (Schlaepfer, 1977Go; Chan et al., 1998Go). Calpains, a family of Ca2+-activated cysteine proteases, cleave a wide variety of cytoskeletal proteins including NF (Chan and Mattson, 1999Go) and have been implicated in many aspects of neural development and in neurodegenerative change (Ono et al., 1998Go; Wells and Bihovsky, 1998Go; Chan and Mattson, 1999Go; Squier et al., 1999Go; Glass et al., 2002Go). Calpain inhibitor administration was shown to elicit an accumulation of NF at the nerve terminal, suggesting that such proteases are important in the normal regulation of synaptic structure (Roots, 1983Go). Since calpain-mediated proteolysis is a feature of many models of neurodegenerative disease, the therapeutic value of calpain inhibitors has been investigated. Small cell-permeant inhibitors have been found to exert a neuroprotective effect in a variety of in vitro and ex vivo models of ischaemia, hypoxia and toxicity (Rami and Krieglstein, 1993Go; Kampfl et al., 1996Go; Wang KKW et al., 1996Go; Chen et al., 1997Go; Jiang and Stys, 2000Go; Wang MS et al., 2000Go; Caba et al., 2002Go). Encouragingly, the administration of inhibitors in vivo resulted in decreased neuronal damage and preserved function after brain injury (Bartus et al., 1994Go; Saatman et al., 1996Go; Posmantur et al., 1997Go) and proved both to be safe and to have a beneficial effect on repair after nerve transection (Badalamente et al., 1989Go, 1995Go).

The therapeutic applicability of calpain inhibitors in acute inflammatory nerve or nerve terminal disorders has not been explored previously. Therefore, we have assessed the ability of calpain inhibitors to prevent axonal cytoskeletal loss at the NMJ due to both complement- and LTx-mediated injury. We show that calpain inhibitors block such degeneration and conclude that analysis of NMJ integrity in the mouse hemidiaphragm preparation presents an easily quantifiable system in which to test neuroprotective strategies.


   Material and methods
Top
 Summary
 Introduction
 Material and methods
Results
 Discussion
 References
 
Calpain inhibitors
Five milligrams of calpeptin or 1 mg of calpain inhibitor V (CI-V) (both from Calbiochem, La Jolla, CA, USA) were dissolved in 250 µl of DMSO and diluted in Ringer solution (116 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 1 mM NaH2PO4, 23 mM NaHCO3, 11 mM glucose, pH 7.4) to provide 6.2, 12.5, 25, 50 and 100 µg/ml stock solutions (7.5–276 and 15–242 µM, respectively). Corresponding ‘No Inhibitor’ solutions, consisting of DMSO in Ringer solution, were also prepared. Coded pairs of solutions, comprising ‘Inhibitor/No Inhibitor’, ‘Inhibitor/Inhibitor’ and ‘No Inhibitor/No Inhibitor’ pairs, were stored at –20°C prior to testing in the two hemidiaphragm preparations from one mouse.

Antibodies and serum
The mouse monoclonal anti-GQ1b IgM, CGM3 (100 µg/ml) (Goodyear et al., 1999Go), and normal human serum (NHS) were dialysed against Ringer solution for 24 h and then diluted to final concentrations (CGM3, 50 µg/ml; NHS, 1: 2) with the coded ‘Inhibitor’ and ‘No Inhibitor’ solutions described above. NHS was taken from a single donor stock that had been freshly frozen and stored in aliquots at –80°C to preserve complement activity (Plomp et al., 1999Go).

LTx
LTx was purified from the freeze-dried venom of Latrodectus lugubris using the three-step procedure described previously (Ashton et al., 2000Go). The toxin was diluted to a working concentration of 4 nM in Ringer solution, with the addition of calpeptin at 50 µg/ml where appropriate. Controls consisting of Ringer solution without LTx were also included. Samples were coded prior to administration and analysis. In some instances, samples were prepared using Ca2+-free Ringer solution containing 2 mM EGTA. For the electrophysiological study (conducted at the Leiden University Medical Centre), LTx was purchased from Alomone Labs (Jerusalem, Israel).

In vitro NMJ electrophysiology
To investigate whether calpain inhibition affected the massive increase of asynchronous quantal ACh release induced by LTx or CGM3 and NHS, electrophysiological recordings were made at hemidiaphragm NMJs in vitro. Male Swiss mice (3–4 weeks old, 15–20 g) were killed by CO2 inhalation according to local guidelines (Leiden DEC 01055). Left and right hemidiaphragms were dissected and pinned out in a 2 ml dish in Ringer solution at room temperature (20–22°C). Randomly within the preparation, muscle fibres were impaled near the NMJ with a 10–20 M{Omega} glass microelectrode filled with 3 M KCl. Intracellular recordings of MEPPs were made at NMJs using standard recording equipment at room temeprature. Signals were digitized and stored for later off-line analysis. In LTx-treated tissue, the MEPP frequency was monitored before and after the addition of 4 nM toxin. For the complement-induced lesion, MEPP frequency was monitored before and after the addition of NHS (1: 2) at room temperature to tissue preincubated with CGM3 at 50 µg/ml (3 h at 32°C and then 30 min at 4°C) according to the previously published protocol (Plomp et al., 1999Go). Both experimental strategies were conducted in the presence and the absence of calpeptin at 50 µg/ml. Spontaneous asynchronous fibre twitches resulting from high MEPP frequencies were blocked by the addition of 0.5–1 µM tetrodotoxin (Alomone Labs).

Morphological studies
Hemidiaphragm preparations were prepared from male BALB/c mice (5–14 weeks old, mean age 9.1 weeks) killed by CO2 inhalation subject to local and UK Home Office guidelines. Left and right hemidiaphragms and attached phrenic nerves were pinned out in a dish containing Ringer solution at room temperature, pre-gassed with O2/CO2 (95%/5%). Prior to the addition of test reagents, the ventral-most quarter of each hemidiaphragm was removed and immediately processed for immunohistology. This tissue constituted the untreated standard control to which other results were subsequently ratiometrically compared.

In the series of experiments using LTx, hemidiaphragms were incubated for 1 h in normal or Ca2+-free Ringer solution with or without calpeptin at 50 µg/ml, followed by incubation with LTx in normal or Ca2+-free Ringer solution with or without calpeptin for a further 1 h.

In the series of experiments using anti-GQ1b antibody, hemidiaphragms were incubated with antibody in the presence or absence of inhibitor solutions for 3 h at 32°C, followed by 30 min at 4°C, as described above. The preparations were rinsed briefly in Ringer solution at room temperature and then incubated with diluted NHS for 1 h at room temperature. At the end of the incubation protocols, half of the remaining hemidiaphragm was processed for immunohistology. The tissue samples were mounted in Lipshaw’s M-1 mounting medium (Lipshaw, Pittsburgh, PA, USA), and longitudinal cryostat sections (8 µm) were cut onto 3-aminopropyltriethoxysilane-coated slides, allowed to air-dry and stored at –20°C. Samples consisting of the preincubation standard and corresponding experimentally treated tissue were mounted together on the same slide.

The remaining portion of the hemidiaphragm was fixed in situ for electron microscopy (EM) analysis with 4% paraformaldehyde for 2 min, after which the tissue was removed for overnight immersion in 2% paraformaldehyde, 2.5% glutaraldehyde, and then stored in PBS at 4°C prior to resin embedding.

Immunostaining
Complement deposition at the NMJ was quantified in antibody-treated tissue by analysis of C3c immunostaining (O’Hanlon et al., 2001Go). C3c is common to both the classical and alternative complement pathways, and under physiological conditions it correlates with the presence of MAC (G. M. O’Hanlon and S. K. Halstead, unpublished observations). Unfixed cryostat tissue sections were incubated with FITC-labelled anti-complement C3c (diluted 1: 300; Dako, Ely, UK) in PBS containing 10% goat serum. To localize NMJs, Texas Red-labelled {alpha}-bungarotoxin (BTx; diluted 1: 2000; Molecular Probes, Leiden, The Netherlands), which binds to postsynaptic ACh receptors, was included in the incubation medium. The sections were incubated for 1 h at 4°C, rinsed and then mounted in Citifluor antifade (Citifluor, Canterbury, UK).

Our earlier studies into the complement-mediated lesion showed a profound loss of NF (O’Hanlon et al., 2001Go), a well-documented calpain substrate. To quantify cytoskeletal structural changes at the NMJ, two anti-200 kDa NF antibodies were used (Affiniti Research Products, Exeter, UK): the mouse monoclonal antibody (mAb) 1217 (clone SMI 32), reactive with non-phosphorylated NF (diluted 1: 750); and the rabbit polyclonal serum 1211, reactive with highly phosphorylated NF (diluted 1: 750).

Sections of unfixed tissue were preincubated for 1 h at room temperature with BTx (1: 2000), rinsed and then incubated for 1 h at room temperature and then overnight at 4°C with one of the two anti-NF antibodies in a staining solution of PBS containing 10% goat serum and 0.1% Triton X-100. The sections were then rinsed, incubated for 1 h at 4°C with goat anti-mouse or anti-rabbit IgG (1: 300; Southern Biotechnology Associates, Birmingham, AL, USA) and mounted in Citifluor antifade.

A minimum of two staining runs of each marker was performed on tissue from individual hemidiaphragm preparations.

Image acquisition
Digital images were captured by a Zeiss Pascal confocal microscope. Morphometric measurements were made using Scion Image (Scion, Frederick, MD) or Aequitas IA (Dynamic Data Links, Cambridge UK) image analysis software. Bitmap processing and annotation were conducted on PhotoMagic (Micrographx, Richardson, TX, USA) and Powerpoint (Microsoft).

Image analysis of the NMJ
NMJs were identified by BTx staining, and images of the BTx and associated C3c stain were recorded under standardized camera conditions. In the case of NF staining, signals were compared in each individual experiment with untreated standard control tissue collected as described above (the ventral-most quarter of each hemidiaphragm), and thus the camera settings were adjusted for each individual staining run. Scion image processing and archiving software, running a macro programme written in-house, was used to determine the C3c or NF signal of individual end-plates as a percentage of the area of the underlying NMJ as delineated by BTx staining (O’Hanlon et al., 2001Go). The threshold for BTx staining was varied by the operator to select a position just before the background became visible, after which preset threshold values were used to determine the C3c or NF signal overlaying the BTx. These values were expressed as a percentage of the mean value for the combined untreated standard tissues. When stained for complement, the amount of C3c deposited at the NMJ in untreated standard samples was close or equal to zero, so the levels at mAb-treated end-plates were expressed in absolute terms rather than a percentage of these values. Throughout this study, and consistent with earlier observations (O’Hanlon et al., 2001Go), a strong and significant correlation between the signals for phosphorylated and non-phosphorylated NF was observed (Spearman’s rank correlation coefficient {rho}s = 0.75, P < 0.001; data not shown), indicating that NF loss occurs with both phosphorylated and non-phosphorylated filaments. Therefore, with the exception of the study correlating complement deposition with NF loss, in which data from both antibodies were used, the data presented herein represent non-phosphorylated NF only.

EM analysis
Tissue samples were prepared for EM analysis using standard procedures and were observer blinded. Briefly, fixed tissue was treated with 1% osmium tetroxide for 45 min, rinsed and then taken through a series of graded alcohols (25, 50, 75 and 100%; 2 x 10 min each) prior to treatment with 100% propylene oxide (2 x 10 min). After infiltration with propylene oxide/araldite (1: 1 and 1: 3) for 4 h, the tissue was placed in mould with fresh araldite and cured overnight at 60°C.

Longitudinal ultrathin sections (80–100 nm) of the NMJ-containing region of the muscle were mounted onto copper grids and stained with 5% uranyl acetate and Reynolds lead citrate.

Ultrastructural quantification
NMJs were identified by the presence of postsynaptic junctional folds, and images recorded at x10 000 and x30 000 magnification were used for morphological assessment. The analysis essentially follows that detailed previously (O’Hanlon et al., 2001Go). Individual NMJs were composed of one or more separate nerve terminal profiles, presented in a range of orientations, and for each profile a series of measurements and counts were made.

Profile perimeter, muscle contact (contact length as a percentage of perimeter) and roundness (a measure of the profile’s approximation to a circle, scored 1 for a true circle to infinity for a straight line) were measured using image analysis software. Measurements of these parameters were made using Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA).

The percentage of mitochondria within a nerve profile showing breakdown of internal structures was recorded for each terminal, as was the incidence of mitochondria touching the presynaptic membrane (scored 1 or 0). The presence of bundles of cytoskeletal fibres (scored 1 or 0) was assessed for each profile.

The number of vesicles present in a 200 x 200 nm box placed over the presynaptic nerve terminal opposite the opening of a junctional fold (a position considered to coincide with the active zone) gave an indication of the level of vesicle depletion. The numerical value for each profile was the mean of up to eight counts, and no figure was recorded where no junctional fold was observed or where the intervening cleft was blocked by perisynaptic Schwann cell (pSC) processes.

The presence of pSC processes that separate a junctional fold from the nerve terminal (scored 1 or 0) and the incidence of pSC processes forming a ‘full wrap’ that completely separates a nerve profile from the muscle (scored 1 or 0) was assessed for each profile.

Statistical analysis
The data shown for each of the NF immunostaining assays was pooled from multiple staining runs obtained from each repeat of the experimental condition. EM analysis was performed on one or, in a few cases, two hemidiaphragms for each experimental condition. Unless otherwise stated, statistical comparisons were made using a two-tailed Student’s t-test that employed a 1% level of significance.


   Results
Top
 Summary
 Introduction
 Material and methods
 Results
Discussion
 References
 
Calpain inhibition does not prevent the induction of neuroexocytosis by LTx or activated complement components
In vitro electrophysiological experiments revealed that calpain inhibition by calpeptin did not block the massive increase of asynchronous quantal ACh release (assessed by measuring MEPP frequency) induced by either LTx or the anti-GQ1b mAb CGM3 with subsequent NHS (Fig. 1). Unexpectedly, compared with inhibitor-free samples, calpeptin-treated hemidiaphragms showed much less or none of the asynchronous muscle fibre twitching usually observed in conjunction with increased MEPP frequency after LTx or CGM3/NHS incubation. Although that is an interesting observation, pointing to an unknown postsynaptic action of calpeptin possibly affecting the firing threshold, further analysis of this phenomenon falls beyond the scope of the present study.



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  		Fig. 1 In vitro electrophysiological analysis of the effect of calpain inhibition on the neuroexocytosis induced by {alpha}-latrotoxin (LTx) or anti-GQ1b monoclonal antibody CGM3 and complement. Miniture end-plate potentials (MEPPs) were measured at neuromuscular junctions (NMJs) before and after incubation of mouse hemidiaphragm preparations either with 4 nM LTx or with CGM3 at 50 µg/ml followed by normal human serum (diluted 1: 2) as the source of complement. (A) Calpeptin at 50 µg/ml did not affect the increase in MEPP frequency induced by these treatments (2–3 muscles, 10–15 NMJs per muscle). (B) Examples of MEPPs recorded after the addition of LTx or CGM3 plus complement, in either the presence or absence of calpeptin.

 
The requirement for Ca2+ in LTx-mediated nerve terminal injury
In the presence of normal Ringer solution containing 2 mM Ca2+, LTx produced a 70% loss of NF in comparison with Ringer-treated control tissue (one-tailed U-test, P < 0.001); however, in Ca2+-free Ringer solution, LTx produced no significant loss in NF. A small but statistically significant increase in the NF signal was observed (one-tailed U-test, P < 0.001; Fig. 2A), possibly related to an increased availability of anti-NF antibody binding sites on NF molecules, thereby increasing the fluorescence signal. The ultrastructural correlates of the LTx-induced NF loss and the protection afforded by Ca2+ withdrawal were assessed by EM analysis (Table 1; Fig. 2B). In normal Ringer solution, the addition of LTx for 1 h caused significant morphological disruption. Consistent with the reduction in NF immunoreactivity, there was a reduction in the number of cytoskeletal bundles (P < 0.0001) and an increase in mitochondrial damage (P < 0.0001). As expected, after toxin-evoked exocytosis, the vesicle density was reduced (P < 0.0001) and individual nerve profiles became more rounded, with less of their surface in contact with the postsynaptic surface. The latter feature was due in part to an increased frequency of Schwann cell processes within the synaptic cleft. In hemidiaphragms exposed to LTx in Ca2+-free Ringer solution, vesicle counts were also reduced (P < 0.0001), indicating that the exocytic effect had occurred, and the profile perimeter was enlarged (P < 0.0001). However, the number of cytoskeletal bundles was not different from control levels (P > 0.1), nor was the profile roundness (P > 0.1) or muscle contact (P > 0.01). Abnormal mitochondria were present but were qualitatively different from those found in samples treated with LTx in normal Ringer solution. In the latter, mitochondria were swollen, with degeneration of christae. In Ca2+-free Ringer solution, the mitochondria were not swollen, but the christae had a compressed appearance (Fig. 3C).



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  		Fig. 2 (A) Analysis of the neurofilament (NF) signal (means ± SEM) observed at neuromuscular junctions (NMJs) in Ringer solution or {alpha}-latrotoxin (LTx)-treated tissue, with or without the inclusion of Ca2+ or calpeptin at 50 µg/ml. NF loss after LTx treatment was blocked by Ca2+ removal or the presence of calpeptin. *Significantly different from Ringer controls at 1% level (one-tailed Mann–Whitney U-test). BTx = {alpha}-bungarotoxin. Boxed numbers = n-values. (B) Ultrastructural parameters (means ± SEM) measured from the LTx-mediated injury in the presence or absence Ca2+ or calpeptin at 50 µg/ml. *Significantly different from control tissue at 1% level (two-tailed t-test). More extensive ultrastructural data for these samples are presented in Table 1.

 

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  		Table 1 Ultrastructural parameters (means ± SEM) measured from longitudinally sectioned mouse hemidiaphragms under various experimental paradigms
 


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  		Fig. 3 (A and B) Electron micrographs of the normal mouse diaphragm neuromuscular junction. In regions opposing junctional folds (jf) on the muscle, the nerve terminal displays regions of densely packed synaptic vesicles (sv). Mitochondria (m) are positioned away from the presynaptic membrane and appear electron dense with well-defined christae. Bundles of cytoskeletal structures (cyt) are set back from the presynaptic membrane. Schwann cell processes (sc) cover non-junctional surfaces of the terminal and are rarely found within the synaptic cleft. (C) In tissue treated with {alpha}-latrotoxin in Ca2+-free Ringer solution, vesicle density was reduced; in many mitochondria, the christae had a compressed appearance. (D and E) In CGM3- and complement-treated tissue, vesicle density was greatly reduced and cytoskeletal bundles were absent. Schwann cell processes were frequently inserted into the synaptic cleft and, in D, have formed a ‘full wrap’, in which the nerve terminal is completely separated from the postsynaptic membrane. Mitochondria were swollen with degenerating internal structures and frequently found in contact with the presynaptic membrane (E). (F) In tissue exposed to antibody and complement in the presence of the calpain inhibitor calpeptin, the structural damage described above was similar. However, cytoskeletal bundles were present at control levels, although they take on a wispy appearance. All scale bars = 250 nm.

 
Calpain inhibitors attenuate LTx-mediated cytoskeletal injury
When hemidiaphragms were exposed to LTx in normal Ringer solution in the presence of calpeptin at 50 µg/ml, NF immunoreactivity was preserved at levels seen with Ringer-treated tissue (one-tailed U-test, P > 0.01; Fig. 2A). Ultrastructural analysis confirmed a normal frequency of cytoskeletal bundles, although these had a ‘wispy’ appearance (Fig. 3F). Vesicle depletion was less pronounced in the presence of calpeptin (Table 1; Fig. 2B; P < 0.01), but vesicle counts were still significantly lower than control levels (P > 0.0001). In other respects, the nerve terminal did not differ significantly from hemidiaphragms treated with LTx in normal Ringer solution in the absence of calpeptin (Table 1; Fig. 2B).

The effect of calpain inhibitors on anti-GQ1b antibody-dependent complement-mediated injury
To test whether calpain inhibitors protected the nerve terminal against complement-mediated injury, hemidiaphragm preparations were exposed to calpeptin and CI-V in conjunction with the anti-GQ1b mAb CGM3 and the source of complement provided by NHS. NMJs were then quantitatively assessed for deposits of complement cleavage product, C3c and immunoreactive NF.

Complement product C3c deposits
None of the 24 untreated standard samples contained C3c deposits, whereas there were complement deposits in each of the 24 CGM3/NHS-treated samples (Fig. 4A). In experimental pairs exposed to anti-GQ1b antibodies and complement in the presence or absence of calpain inhibitors, there was no significant difference in C3c staining in the left versus right hemidiaphragm (one-tailed paired t-test, P > 0.1; data not shown) or in the presence or absence of calpain inhibitor (one-tailed paired t-test, P > 0.1). This indicates that there was no anatomical bias between the two sides of the diaphragm and that calpain inhibitors did not affect complement activation. As reported previously (O’Hanlon et al., 2002Go), complement deposits at the NMJ were seen to encompass the cell bodies of pSCs (Fig. 4A and B), but the significance of this remains unclear.



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  		Fig. 4 Fluorescent light micrographs of the mouse diaphragm neuromuscular junction (NMJ) after immunostaining for complement or neurofilament (NF). (A) NMJ stained with Texas Red-labelled {alpha}-bungarotoxin (BTx) to delineate the postsynaptic acetylcholine receptors and FITC-labelled anti-complement C3c, with a phase-contrast image of the same NMJ (B). The complement deposits are clearly presynaptic, concentrated in an area close to the postsynaptic apparatus, but they also encompass perisynaptic Schwann cells. The cell body of one such cell is delineated by C3c staining (arrow). (C and D) The mouse NMJ stained for BTx (red) and NF (green). In control tissue (C), the pre-terminal axon is smooth and continuous, with a terminal arborization extending over the region stained by BTx. After treatment with anti-ganglioside GQ1b antibodies and complement, the terminal NF signal is largely lost, with little NF stain evident over that of BTx. Associated nerve bundles appear fragmented, suggesting that the lesion also extends into the pre-terminal axon. All scale bars = 10 µm.

 
Immunoreactive NF
NF levels were quantitated in hemidiaphragm preparations exposed to anti-GQ1b antibodies and complement, with and without calpain inhibitors, over a range of inhibitor concentrations from 0 to 50 µg/ml (Fig. 4). No anatomical bias in NF signal between the two sides of the diaphragm was observed (combined phosphorylated and non-phosphorylated NF data, one-tailed paired t-test, P > 0.1; data not shown). However, in paired samples treated with antibody and complement with and without calpain inhibitor, there was highly significant preservation of NF in the presence of inhibitor (one-tailed paired t-test, P < 0.001; data not shown).

In the absence of calpain inhibitors, there was an inverse correlation between C3c deposition and NF staining (Spear mans rank correlation coefficient {rho}s = –0.80, P < 0.001; Fig. 5), consistent with the NF loss resulting from a complement-dependent process. In comparison, the NF signals from samples exposed to calpeptin and CI-V at 50 µg/ml showed no significant correlation with C3c (combined data; {rho}s = 0.25, P > 0.1), indicating the loss of this relationship.



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  		Fig. 5 The relationship between complement C3c deposition and neurofilament (NF) loss at neuromuscular junctions treated with CGM3 plus normal human serum (NHS). There is an inverse correlation between complement signal and NF signal (phosphorylated and non-phosphorylated) in tissue treated with antibody and NHS in the absence of calpain inhibitors. Where inhibitors are included (calpeptin or calpain inhibitor V; 50 µg/ml), this relationship was lost. Linear trend-lines have been included for illustration. BTx = {alpha}-bungarotoxin.

 
A significantly higher NF signal was observed at all concentrations of calpeptin (12.5–50 µg/ml) in comparison with the corresponding inhibitor-free preparations (one-tailed U-test, P < 0.01). With calpeptin at 50 µg/ml, there was no significant reduction in the NF signal in comparison with untreated standard tissues (one-tailed U-test, P > 0.01; Fig. 6). Thus, calpeptin is able to reduce and, at 50 µg/ml, eliminate immunohistological changes in nerve terminal NF caused by antibody-dependent complement-mediated injury. At 50 µg/ml, CI-V also produced a similar significant neuroprotective effect (one-tailed U-test, P < 0.01; Fig. 6) but at lower concentrations, and this was not statistically significant.



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  		Fig. 6 (A) Analysis of the neurofilament (NF) signal (means ± SEM) observed at neuromuscular junctions (NMJs) in tissue treated either with Ringer solution or with CGM3 plus normal human serum (NHS) with or without the inclusion of the calpain inhibitors calpeptin or calpain inhibitor V (CI-V) at 50 µg/ml. In the presence of antibody and complement, the inclusion of inhibitor had a preservative effect on the NF signal. *Significantly different from the ‘No Inhibitor’ sample at 1% level; {ddagger}significantly different from untreated standard at 1% level (one-tailed Mann–Whitney U-test). BTx = {alpha}-bungarotoxin. Boxed numbers = n-values. (B) Ultrastructural NMJ parameters (means ± SEM) measured after CGM3/NHS treatment in the presence or absence of the calpain inhibitors calpeptin or CI-V at 50 µg/ml. *Significantly different from control tissue at 1% level; (two-tailed t-test). More detailed ultrastructural data are presented in Table 1.

 
In order to more precisely describe the nature of the neuroprotective effect of calpeptin, the frequency distribution of the NF signal data was calculated for the combined untreated standards (i.e. normal NMJ) and for lesioned tissue at each dose of calpeptin (Fig. 7). In tissue injures in the absence of inhibitor, the distribution was skewed towards a low NF signal, consistent with NF loss. Comparisons between the different distributions demonstrated a progressing shift towards the standard (healthy) distribution with increasing dose. At the highest dose of calpeptin (50 µg/ml), there was a significant difference from the frequency distribution of tissue exposed to antibody and complement in the absence of calpeptin ({chi}2 test of independence, P < 0.01). Additionally, there was no statistical difference from antibody-naive, untreated standard tissue (P > 0.01). At a low dose (12.5 µg/ml), the reverse was true. At 25 µg/ml, the effect was intermediate, with the distribution differing from both untreated standard and inhibitor-free tissue (P < 0.01). Thus, with increasing calpeptin dose, the distribution of NF signals within the observed samples shifts from that of the highly disrupted inhibitor-free lesion to that of the untreated control.



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  		Fig. 7 Frequency distribution of the neurofilament (NF) signal at individual neuromuscular junctions expressed as a percentage of the mean untreated standard value. In the complement-mediated lesion without calpain inhibition (0 µg/ml), the distribution is skewed towards a smaller signal, differing significantly from the normal population (Std; included in all panels for comparison). As the concentration of calpeptin is increased, the distribution becomes more like that of the normal population, until, at 50 µg/ml, there is no significant difference between the two ({chi}2 test of independence, P > 0.01). *Significantly different from untreated standard; significantly different from the inhibitor-free lesion (0 µg/ml). BTx = {alpha}-bungarotoxin.

 
Ultrastructural analysis of the neuroprotective effect of calpain inhibitors
The nerve terminal lesion induced by CGM3 antibody-dependent complement activation in the absence of calpain inhibition was compared with control tissue and with the lesion generated in the presence of calpeptin or CI-V at 50 µg/ml (Table 1; Figs 3 and 6). In complement-injured tissue, irrespective of the presence or absence of inhibitor, the synaptic vesicle counts were markedly reduced in comparison with the values in tissues exposed to Ringer solution only (P < 0.001). This is consistent with the in vitro electrophysiological observation described above, in that the uncontrolled exocytosis occurring in the lesion is unaffected by calpain inhibitors. Similarly, the proportion of severely damaged mitochondria and the presence of mitochondria touching the presynaptic membrane were independent of the presence of calpeptin (P > 0.01 compared with the inhibitor-free lesion) and in both cases significantly higher than in controls (P < 0.001).

Several structural parameters were significantly different in the presence of calpeptin or CI-V in comparison with the lesion in their absence (P < 0.01; Table 1; Figs 3 and 6). Individual nerve terminal profiles were larger and less rounded, with normal numbers of cytoskeletal bundles, although subjectively these appeared disorganized in comparison with controls. The calpeptin-treated nerve terminals retained relatively more contact with the postsynaptic muscle membrane than in the inhibitor-free lesion, but with both inhibitors this value remained significantly reduced in comparison with Ringer control levels (P < 0.01). In the presence of calpain inhibitors, the incidence of pSC processes forming ‘full wraps’ was still significantly different from Ringer control levels (P < 0.01), indicating that Schwann cell activation with process extension had taken place.


   Discussion
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 Summary
 Introduction
 Material and methods
 Results
 Discussion
References
 
LTx and anti-GQ1b antibody-dependent complement activation represent two distinct but complementary models of acute motor nerve terminal injury. By using both models in the hemidiaphragm preparation, we have shown that calpain inhibitors can gain access to and markedly limit structural damage at the NMJ. Although other facets of the injury, such as uncontrolled exocytosis, continue unabated, the preservation of cytoskeletal elements may have a significantly beneficial influence on overall axonal integrity and repair. This experimental system provides a good and reproducible environment for testing the efficacy of therapeutic agents for limiting acute autoimmune attack and developing neuroprotective strategies.

Calpain activation is critically involved in structural neurodegeneration of motor nerve terminals by LTx or pathogenic antibodies and complement
We and others have extensively studied anti-GQ1b antibody-mediated effects on NMJs in the in vitro mouse hemidiaphragm (Roberts et al., 1994Go; Buchwald et al., 1995Go, 2001Go; Goodyear et al., 1999Go; Plomp et al., 1999Go; Kishi et al., 2001Go; O’Hanlon et al., 2001Go). In our model, increasing evidence suggests that presynaptic membrane insertion of the ‘pore forming’ complement complex MAC is required for cytoskeletal damage to occur (G. M. O’Hanlon and S. K. Halstead, unpublished observations). It is thus likely that antibody-mediated complement activation at the motor nerve terminal leads to unregulated Ca2+ ingress through the non-specific pores formed by MAC. The subsequent rise in intracellular [Ca2+] would thus be an important factor in triggering the uncontrolled increase in MEPP frequency, calpain activation and much of the subsequent morphological damage observed at the NMJ. Clearly, this flux cannot be limited to Ca2+, since other constituents, including Na+, K+ and water, could also travel bi-directional through these pores, producing interrelated additional effects. In a parallel manner, and shown here directly by using Ca2+-free Ringer solution, it seems likely that the similar structural damage seen after LTx application results from Ca2+ influx through toxin pores (Orlova et al., 2000Go). LTx forms pores independently of Ca2+ (Ashton et al., 2000Go; Volynski et al., 2000Go), but its action is complex and involves both Ca2+-dependent and -independent exocytic effects (Henkel and Sankaranarayanan, 1999Go). Here, we have shown that the use of LTx in Ca2+-free Ringer solution prevents the loss of NF immunostaining and, ultrastructurally, the loss of cytoskeletal bundles, in contrast to the lesion produced by LTx in the presence of Ca2+. In Ca2+-free Ringer solution, toxin-induced neurosecretion still occurred, and vesicle depletion was still observed in the LTx samples. Thus, it is clear that structural changes cannot be directly and solely linked to the process of exocytosis. In addition, by showing that calpain inhibitors blocked the degradation of nerve terminal cytoskeletal components in both LTx- and complement-mediated injury, we have provided evidence that the activation of the Ca2+-dependent protease calpain is centrally involved in this aspect of the injury. Since Ca2+ ingress was not blocked in these latter experiments, the Ca2+-dependent part of uncontrolled exocytosis and mitochondrial injury (Gorio et al., 1978Go; Calupca et al., 1999Go) still occurred in the presence of calpain inhibitors. Although it would support our hypothesis to directly measure Ca2+ influx into the nerve terminal as distinct from the pSC (which may also be targeted by anti-GQ1b antibodies and sustain complement injury), this is not technically trivial.

In both the LTx and complement-injured NMJ, the overlying pSCs become involved in the fragmentation and removal of the damaged nerve terminal. The incidence of pSC processes separating presynaptic membrane from junctional folds, or forming ‘full wraps’ around nerve terminal profiles, did not change significantly with the addition of calpain inhibitors, indicating that pSC activation is not purely a secondary phenomenon arising from structural failure of the underlying terminal. This also suggests that calpain inhibitors do not have a profound effect on cell motility in this injury, as has been observed in other situations (Xu and Mellgren, 2002Go). It is noteworthy that pSC involvement appeared reduced when LTx was administered in Ca2+-free Ringer solution. Since there are intimate metabolic links between nerve terminals and their associated glial cells (Castonguay et al., 2001Go; Rochon et al., 2001Go), and pSCs are able to respond to nerve terminal activity and neurotransmitter release via ACh and adenosine A1 receptors (Rochon et al., 2001Go), it remains unclear whether this observation represents a direct effect of calcium withdrawal on the pSC or is secondary to changes in the underlying nerve terminal.

The relevance of the in vitro mouse model for preventing neurodegeneration in human neuropathy
In the in vitro hemidiaphragm preparation, our data show that the cytoskeletal abnormalities arise very rapidly, being evident in the motor nerve terminal and pre-terminal axon only 15 min into the incubation with LTx or complement (G. M. O’Hanlon, unpublished observations). This timescale is consistent with that reported in calpain-mediated cytoskeletal loss after spinal injury in rats (Schumacher et al., 1999Go). The uncontrolled exocytosis can be electrophysiologically demonstrated and visually observed as muscle fibre twitches after only 5 min incubation (Goodyear et al., 1999Go; O’Hanlon et al., 2001Go). Since the addition of calpain inhibitors does not influence the initial Ca2+ ingress, but affects downstream events, the uncontrolled exocytosis is unaffected and the preparation thus still becomes refractive to evoked stimulation. The long-term benefits of cytoskeletal preservation in such circumstances remain unclear and cannot be addressed in this short-term in vitro model, whose physiological survival in vitro is limited to <6 h. However, it seems reasonable to expect that neuronal repair mechanisms might advance more rapidly if cytoskeletal integrity is preserved. This issue can be further investigated in vivo in animal models for human neuropathies such as GBS, irrespective of whether or not the nerve terminal or more proximal portions of the axon are the principal targets. The long-term beneficial effects of leupeptin in nerve-transection studies in monkeys (Badalamente et al., 1989Go, 1995Go) lead us to believe that calpain inhibitors could play an important therapeutic role in the treatment of complement-mediated peripheral axonal injury, in either proximal or distal sites. When such agents become available for therapy in humans, early efforts should be made to test their benefit in acute inflammatory peripheral axonal injury, as occurs in forms of GBS, and also in human latrodectism.


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 Discussion
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