doi:10.1093/brain/awq119
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BRAIN
A JOURNAL OF NEUROLOGY
Anti-GD1a antibodies activate complement and
calpain to injure distal motor nodes of Ranvier
in mice
Rhona McGonigal,1 Edward G. Rowan,2 Kay N. Greenshields,1 Susan K. Halstead,1
Peter D. Humphreys,1 Russell P. Rother,3 Koichi Furukawa4 and Hugh J. Willison1
1
2
3
4
Division of Clinical Neurosciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, UK
Strathclyde Institute of Pharmacy and Biomedical Sciences, Sir John Arbuthnott Building, University of Strathclyde, Glasgow G4 0NR, UK
Alexion Pharmaceuticals, Cheshire, CT 06410, USA
Department of Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-0065, Japan
Correspondence to: Prof. Hugh J. Willison,
University of Glasgow Division of Clinical Neurosciences,
Glasgow Biomedical Research Centre,
Room B330, 120 University Place,
Glasgow G12 8TA,
Scotland
E-mail: h.j.willison@clinmed.gla.ac.uk
The motor axonal variant of Guillain–Barré syndrome is associated with anti-GD1a immunoglobulin antibodies, which are
believed to be the pathogenic factor. In previous studies we have demonstrated the motor terminal to be a vulnerable site.
Here we show both in vivo and ex vivo, that nodes of Ranvier in intramuscular motor nerve bundles are also targeted by
anti-GD1a antibody in a gradient-dependent manner, with greatest vulnerability at distal nodes. Complement deposition is
associated with prominent nodal injury as monitored with electrophysiological recordings and fluorescence microscopy.
Complete loss of nodal protein staining, including voltage-gated sodium channels and ankyrin G, occurs and is completely
protected by both complement and calpain inhibition, although the latter provides no protection against electrophysiological
dysfunction. In ex vivo motor and sensory nerve trunk preparations, antibody deposits are only observed in experimentally
desheathed nerves, which are thereby rendered susceptible to complement-dependent morphological disruption, nodal protein
loss and reduced electrical activity of the axon. These studies provide a detailed mechanism by which loss of axonal conduction
can occur in a distal dominant pattern as observed in a proportion of patients with motor axonal Guillain–Barré syndrome, and
also provide an explanation for the occurrence of rapid recovery from complete paralysis and electrophysiological in-excitability.
The study also identifies therapeutic approaches in which nodal architecture can be preserved.
Keywords: anti-GD1a antibody; GD1a ganglioside; node of Ranvier; acute motor axonal neuropathy; complement; calpain
Abbreviations: CAP = compound action potential; CFP = cyan fluorescent protein; Nav1.6 = voltage-gated sodium channel;
NrCAM = neuronal cell adhesion molecule
Received January 5, 2010. Revised March 24, 2010. Accepted April 14, 2010. Advance Access publication May 30, 2010
ß The Author (2010). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Anti-GD1a antibody-mediated nodal injury
Introduction
The motor axonal variant of Guillain–Barré syndrome, acute motor
axonal neuropathy (Feasby et al., 1986; McKhann et al., 1993;
Hughes and Cornblath, 2005) characteristically follows
Campylobacter jejuni infection and is associated with serum
anti-GM1, -GD1a and -GalNAc-GD1a ganglioside antibodies
(Lugaresi et al., 1997; Ho et al., 1999; Ogawara et al., 2000).
Gangliosides have diverse functions related to neural development,
maintenance and regeneration, including stabilizing the axoglial
junction at the node of Ranvier (Sheikh et al., 1999b; Susuki
et al., 2007a; Silajdzic et al., 2009). GD1a has been identified in
the motor nerve terminal and nodal axolemma (Sheikh et al.,
1999a; De Angelis et al., 2001; Gong et al., 2002; Goodfellow
et al., 2005), sites that correspond to those predicted from clinical,
electrophysiological and pathological data to be affected in motor
axonal forms of Guillain–Barré syndrome (Griffin et al., 1996; Ho
et al., 1997; Kuwabara et al., 2004).
The distal motor nerve, including nerve terminals and the spinal
roots, are relatively accessible to circulating factors that may
account for vulnerability of these regions to injury compared
with nerve trunks, owing to blood nerve barrier permeability variations (Burkel, 1967; Saito and Zacks, 1969; Olsson, 1990). As
such, the rapid clinical recovery seen in some patients with
acute motor axonal neuropathy could be due to very distal
axonal conduction injury and block, a site with the capacity to
readily regenerate (Ho et al.,1997; Goodfellow et al., 2005).
Conversely, severe proximal axonal injury resulting in widespread
axonal degeneration would inevitably lead to permanent motor
axonal deficits, as is seen in some acute motor axonal neuropathy
cases (Hiraga et al., 2005a, b).
Anti-ganglioside antibody-mediated mouse and rabbit models of
acute motor axonal neuropathy have been generated that focus
on sciatic nerve and ventral root axons, or on axonal components
of neuromuscular junctions (Susuki et al., 2003; Sheikh et al.,
2004; Goodfellow et al., 2005). In a rabbit ventral root model,
destabilization of nodal and paranodal structures including loss of
voltage-gated sodium channels (Nav1.6) was interpreted as the
consequence of antibody and complement-mediated axoglial disruption (Susuki et al., 2007b), which could be protected with a
complement inhibitor (Phongsisay et al., 2008).
Complement mediated effects at the nodes of Ranvier in the
ventral root mirror those demonstrated in patient autopsy tissue
(Hafer-Macko et al., 1996). As the node of Ranvier is vital for
impulse propagation, understanding acute motor axonal neuropathy immunopathology at this site is important. The node of
Ranvier is organized into three subdomains—the nodal gap, the
paranode and the juxtaparanode (Scherer, 1996; Poliak and Peles,
2003). Nav1.6 is expressed at the node (Caldwell et al., 2000)
along with the cytoskeletal protein ankyrin G and the cell adhesion
molecules neurofascin 186 and neuronal cell adhesion molecule
(NrCAM). At the paranode, the axoglial junction is formed by
the axolemmal proteins contactin and Caspr, while neurofascin
155 is the glial receptor to this complex. At the juxtaparanode,
voltage-gated potassium channels localized on the axon in complex with Caspr 2 and Tag1, play a role in repolarization of the
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resting membrane potential following an action potential (Wang,
1993). Glycosyltransferase knockout mouse studies indicate that
GD1a or related gangliosides modulate the structural and functional integrity of this site, although the precise mechanisms are
poorly understood (Sheikh et al., 1999b; Susuki et al., 2007a;
Silajdzic et al., 2009).
In our ex vivo mouse model of acute motor axonal neuropathy,
motor nerve terminals develop severe functional and pathological
injury when exposed to anti-GD1a antibody with complement activation (Goodfellow et al., 2005). In this model GD1a is enriched
in axons through using the GD3 synthase knockout mouse and
thus becomes more vulnerable to anti-GD1a antibody mediated
injury than normal mice. How this relates more precisely to motor
axonal GD1a level in man is unknown. This model also requires a
heterologous complement source (human serum) in order to
achieve the pathological effects, as previously reported (Willison
et al., 2008). The pore forming action of complement is central to
the development of this injury and that mediated by other
anti-ganglioside antibodies, in part through allowing uncontrolled
calcium influx into the nerve terminals, with subsequent Ca2+-dependent protease, calpain, activation and cleavage of structural
proteins in the axon terminal (O’Hanlon et al., 2003).
This study set out to assess whether anti-GD1a-antibody
mediated injury could be observed to occur at the nodes of
Ranvier in the distal portions of the axon, upstream from the
motor nerve terminal. If present, we also intended to determine
the mechanism of action and functional effects of any observed
injury that might lead to therapeutic intervention, analogous to
our previous approach at the neuromuscular junction.
Materials and methods
Mice
Male GD3 synthase knockout mice (GD3s / ) (Okada et al., 2002)
were crossed with B6/Cg-TgN(Thy1-CFP) Dilute Brown non-Agouti
(DBA/1) mice that endogenously express cyan fluorescent protein
(CFP) in their axons (Feng et al., 2000), herein termed GD3s / /
CFP. GD3s / mice relatively overexpress GD1a compared with
wild-type counterparts, bind anti-GD1a antibody strongly and deposit
complement resulting in injury, as reported (Goodfellow et al., 2005).
To confirm this in the crossed strain used in this study, we assessed
triangularis sterni muscles from both GD3s / /CFP and wild-type/CFP
mice for anti-GD1a antibody immunoreactivity. GD1a levels were significantly greater in GD3s / /CFP compared with wild-type/CFP mice
(P50.05). Consequently, wild-type mice were found to be relatively
insensitive to anti-GD1a antibody mediated injury compared with
GD3s / mice and the latter were used for all subsequent experiments. Mice aged 6–12 weeks were killed by CO2 inhalation.
Experiments complied with UK Home Office guidelines.
Antibodies and reagents
Anti-GD1a IgG2b monoclonal antibody (previously termed MOG35,
herein termed ‘anti-GD1a antibody’) was generated as described
(Bowes et al., 2002; Boffey et al., 2005). By way of control for the
specificity of the anti-GD1a antibody, GalNAc transferase / mice that
lack GD1a showed no binding or pathological effects from antibody
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exposure, as previously reported (Goodfellow et al., 2005). Antibodies
to channels, other proteins and membrane attack complex, C5b-9 are
detailed in the online Supplementary Table 1. Eculizumab, a humanized anti-human C5 monoclonal antibody that binds plasma C5 to
prevent membrane attack complex formation, and ALXN3300 (the
isotype-matched control monoclonal antibody) were supplied by
Alexion Pharmaceuticals (Cheshire, USA). The synthetic peptide
AK295 binds calpain I, II and cathepsin B to prevent their activation
and proteolytic action (Li et al., 1996). Toxins were used as follows:
-bungarotoxin (Molecular Probes, UK); -latrotoxin (Alomone Labs,
Israel) at 12 nM; tetrodotoxin (Biotium Inc., USA) at 5 mM; and vecuronium (Organon Laboratories Ltd, Cambridge, UK) at 5 mM.
Secondary antibodies conjugated to Alexa Fluor 488 and 647 were
applied at 1 : 200 dilution.
Ex vivo and in vivo muscle and nerve
permeability studies
Triangularis sterni muscle, phrenic and sural nerve were maintained
alive in Ringer’s medium (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), pre-gassed with 95% O2/5% CO2 at room temperature
(20 C). Muscle and nerve (desheathed by slitting and opening the
epineurium with a fine needle, or left intact) were incubated with
100 mg/ml anti-GD1a antibody for 2 h at 32 C, 30 min at 4 C and a
final 10 min at room temperature, plus -bungarotoxin to label the
neuromuscular junctions. Antibody control preparations were incubated with Ringer’s alone. Preparations were rinsed in Ringer’s prior
to fixation in 4% paraformaldehyde (20 min, room temperature).
Tissue was then rinsed in phosphate buffered saline, 0.1 M glycine
and phosphate buffered saline. Tissue was incubated with
anti-IgG2b-488 (1 : 200) and the pan anti-neurofascin antibody
NFC2 (1 : 1000) with 0.5% Triton X-100 in blocking solution
(1% goat serum and 1% L-lysine) overnight at 4 C. Intramuscular
nerve bundles were categorized as follows: single fibres, small bundles
(515 mm), medium bundles (15–35 mm) and large bundles (435 mm).
Nodes of Ranvier were identified by neurofascin staining and the
anti-GD1a antibody immunofluorescence quantitated and compared
to control tissue. For in vivo studies, triangularis sterni muscle was
removed from mice injected i.p. 16 h previously with 3 mg
anti-GD1a antibody. Phosphate buffered saline was used for control
groups.
For phrenic and sural nerves, isolated nerves were incubated ex vivo
with anti-GD1a antibody under intact and desheathed conditions. It
was thereby established that desheathing was essential for achieving
anti-GD1a antibody binding at nodes of Ranvier in nerve trunks and
that under these conditions antibody binding levels were equivalent to
intramuscular nerve nodes of Ranvier (data not shown). All studies on
nerve trunks were thus conducted on desheathed nerves.
Ex vivo preparations for complement
activation and nodal protein disruption
Muscle and nerve preparations were processed as above, with the
additional step of incubation with 40% normal human serum
(source of complement) for 3 h at room temperature prior to fixation.
Ten micrometre cryosections were stained for membrane attack complex, nodal channels and other proteins overnight at 4 C. In order to
identify nodes of Ranvier, fluoromyelin green (1: 400), which labels
lipids, or dystrophin (1: 200), which labels the myelin sheath, were
applied. Secondary antibodies were applied for 3 h at room
R. McGonigal et al.
temperature as follows: anti-rabbit IgG-647 (1 : 300) for Nav1.6,
Caspr, NFC2, Kv1.1, neurofilament; anti-mouse IgG1-647 (1 : 300)
for ankyrin G, moesin, NrCAM and dystrophin; anti-mouse
IgG2a-555 (1 : 200) for membrane attack complex. Nodes of Ranvier
with a normal immunostaining pattern for nodal proteins were scored
as present or absent/abnormal. To control for the possible confounding effect of synaptic injury that occurs with anti-GD1a antibody application to nerve muscle preparations (Plomp and Willison, 2009),
Nav1.6 immunostaining at nodes of Ranvier was compared between
antibody treated and -latrotoxin (2 nM) treated tissue.
To assess the contribution of membrane attack complex to injury,
eculizumab (100 mg/ml) was added to normal human serum 10 min
prior to incubation with the muscle (Halstead et al., 2008a). To investigate the contribution of calpain, AK295 (100 mM), was added concurrently with normal human serum. AK295 was first assessed in dose
ranging studies (25–200 mM) and the lowest fully protective concentration was used. In eculizumab-treated and -unprotected intramuscular axons, the presence of axonal CFP was used to monitor axonal
integrity. After AK295 treatment, the intensity of neurofilament immunoreactivity was quantified at the nerve terminal as delineated by
-bungarotoxin staining and compared to AK295-unprotected tissue
levels. The efficacies of eculizumab and AK295, as monitored by
immunostaining profiles, were expressed as the percentage of protected versus unprotected signals at the relevant node of Ranvier sites.
Perineural and extracellular recordings
Triangularis sterni nerve-muscle preparations were set up for
electrophysiological recordings in Ringer’s at room temperature
(20–22 C) using 2 M NaCl-filled micro-electrodes with a resistance
of 25–45 MV. Recordings were made from nerve terminals and
small and large intramuscular nerve bundles after anti-GD1a antibody
incubation followed by normal human serum for 3 h. Perineural waveforms associated with nerve terminal action potentials were made as
previously described (Braga et al., 1991). Muscles were paralysed with
5 mM vecuronium to prevent twitching. In some experiments the same
microelectrode was used to measure muscle resting membrane potentials. Signals were amplified, recorded and analysed as per the nerve
extracellular recordings below.
For extracellular recordings, nerves were mounted in a Perspex recording block across three chambers and sealed in with vacuum
grease. Nerves were stimulated at 1 Hz and supramaximal voltage
(Grass S88 stimulator). Signals were amplified (CED1902), digitized
(NIDAQ-MX A/D converter, National Instruments, Austin, TX, USA)
and analysed using WinWCP version 4.1.0. Phrenic nerves and sural
nerves remained in the recording chamber throughout the experiments
and recordings were made for 2 h on the application of normal human
serum. At termination, 5 mM tetrodotoxin was applied to confirm that
the recorded waveform originated from the opening of sodium channels. A representative graph of the positive peak value of compound
action potential (CAP) over time was plotted to convey conduction. A
minimum of 200 control waveforms were averaged prior to the addition of normal human serum. Absolute CAP values vary between experiments and thus the percentage of the starting CAP peak value was
calculated for each of 3–5 preparations and averaged for each treatment group. A 2-sample t-test was used for comparison of phrenic
and sural nerve conduction.
Image acquisition and analysis
Fluorescent images were captured on both a Zeiss Axio Imager Z1
with ApoTome attachment and a Zeiss Pascal confocal microscope
Anti-GD1a antibody-mediated nodal injury
and analysed using ImageJ software. For quantitation of antibody and
membrane attack complex deposition, the fluorescence signal at the
region of the node of Ranvier was measured with background fluorescence subtracted. Where relevant, measurements were categorized
by bundle size as described above. Quantitation of the neurofilament
signal over the motor endplate was performed as reported (Halstead
et al., 2008a). Measurements were pooled from three experiments.
Non-parametric data are presented as box and whisker plots. Mann–
Whitney mean rank test was used to compare possible statistical differences between groups (1% level of significance). For comparison of
nodal protein immunostaining, nodes of Ranvier positive for individual
markers were counted for each bundle category and the chi-squared
test used at a 1% level of significance.
Results
Anti-GD1a antibodies are preferentially
deposited at distal motor nerve nodes of
Ranvier
Anti-GD1a antibody was applied to ex vivo triangularis sterni
muscle preparations from GD3s / /CFP mice and its deposition
immunolocalized and quantified at the nodes of Ranvier.
Anti-GD1a antibody also binds intensely at the motor nerve terminal of GD3s / mice (Fig. 1A; upper area of image) as shown in
previous studies (Goodfellow et al., 2005). Anti-GD1a antibody
deposits are prominent at the nodes of Ranvier of distal intramuscular axons, identified by the nodal and paranodal marker,
pan-neurofascin antibody (Fig. 1A). Juxta-terminal nodes of
Ranvier bear the most prominent antibody deposits, the fluorescence intensity of anti-GD1a antibody deposits at nodes of
Ranvier decreasing with increasing distance from the nerve terminal. In order to quantitatively assess this, intramuscular nerve bundles were categorized into three groups (Fig. 1B). The single arrow
indicates a single fibre, double arrow a small bundle (515 mm) and
triple arrow a medium bundle (15–35 mm). A further category of
large nerve bundles (not evident in Fig. 1B) was assigned for
bundle diameters exceeding 35 mm. Anti-GD1a antibody applied
to triangularis sterni muscle preparations ex vivo were deposited at
significantly higher levels at single fibre nodes of Ranvier compared to all other bundle categories and control tissue (Fig. 1C
and D; P50.001). This was also evident for small (P50.001) and
medium (P = 0.0023) bundles. Large bundles had an insignificant
anti-GD1a antibody deposition level, comparable to control tissue,
suggesting anti-GD1a antibody was unable to gain access to bundles of this size following topical application. In order to assess
anti-GD1a antibody penetration to these intramuscular nerve compartments when delivered through the vascular bed (as opposed
to organ bath incubation), anti-GD1a antibody was injected intraperitoneally and 16 h later the triangularis sterni muscle was
removed for antibody quantification, as for ex vivo preparations
above. Equivalent results to the ex vivo findings were observed,
with antibody deposits being greatest in the distal part of the
nerve in a gradient-dependent manner when categorized by
bundle size (Fig. 1E). In order to establish that these differences
were not due to a proximal to distal gradient of GD1a expression
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at nodes of Ranvier in nerve, frozen sections of permeabilized
intramuscular nerve bundles in which antibody access is expected
to be uniform were stained with anti-GD1a antibody, and the
signal intensity was found to be the same, irrespective of the
nerve bundle size (data not shown). These ex vivo and in vivo
findings demonstrate that anti-GD1a antibody is able to bind to
intramuscular nerve nodes of Ranvier in a distal to proximal downward gradient, presumed due to the relatively increasing impermeability of the blood nerve barrier to antibody as bundle size
increases.
Nodal proteins are disrupted and distal
motor nerves are rendered inexcitable
by anti-GD1a antibody directed
complement activation
Membrane attack complex deposition was demonstrated at nodes
of Ranvier of the distal intramuscular nerves in response to the
addition of an exogenous source of human complement in the
form of normal human serum (Fig. 2A). As with anti-GD1a antibody, membrane attack complex deposition as assessed by fluorescence intensity for anti-membrane attack complex antibody was
gradient-dependent, with significantly higher levels at single fibre
nodes of Ranvier compared to all other categories (Fig. 2B;
P50.001) and significantly higher levels at small bundle nodes
of Ranvier compared to larger categories (Fig. 2B; P50.001).
Axonal injury was further characterized by the complete loss of
the endogenous axonal CFP signal, both at the nerve terminal and
along the distal axon as illustrated in Fig. 2C. Even in large bundles, the CFP signal was relatively attenuated in antibody plus
normal human serum treated preparations.
In order to assess the functional effect of membrane attack
complex deposition to the distal axonal region, electrophysiological
assessment of local ion currents was performed by recording perineural currents at the nerve terminal, small and large nerve bundles. In control tissue (anti-GD1a antibody without normal human
serum), biphasic waveforms were observed that correspond to
currents flowing through Na+ and K+ channels, respectively
(Fig. 2D; top panels). After treatment of tissue with anti-GD1a
antibody plus normal human serum as a complement source,
there was a complete loss of both K+ (broken arrow) and Na+
(solid arrow) current flow at the nerve terminal and nerve bundles,
with the exception of preserved Na+ current in large bundles
(Fig. 2D; lower panel, arrow).
To investigate for structural alterations, the node of Ranvier was
analysed for appearance under phase microscopy and by immunostaining for node of Ranvier proteins located at various nodal
sub-domains. The percentage of nodes of Ranvier with intact
staining for Nav1.6, the sodium channel isoform expressed at the
peripheral nerve nodes of Ranvier (Caldwell et al., 2000) was
reduced (590%) in single fibres and small bundles after treatment,
compared to control (Fig. 3A; P50.001). The cytoskeletal protein
ankyrin G was similarly affected (Fig. 3B; P50.001), as was the
paranodal axolemmal protein Caspr (Fig. 3C; P50.001). Staining
for neurofascin was partially lost at single fibre and small bundle
nodes of Ranvier (Fig. 3D; P50.001 and P = 0.001, respectively),
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R. McGonigal et al.
CFP axon
Merge
A
B
anti-GD1a Ab
pNFasc
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CFP axons
BTx
pNFasc
anti-GD1a Ab
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Control
Anti-GD1a Ab exposed
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IgG fluorescence intensity
(arbitrary units)
D
IgG fluorescence intensity
(arbitrary units)
anti-GD1a Ab
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Anti-GD1a Ab exposed
Figure 1 Anti-GD1a antibody is deposited at nodes of Ranvier in a gradient-dependent manner in distal intramuscular nerves. Triangularis
sterni muscle was treated ex vivo with anti-GD1a antibody (100 mg/ml for 160 min) and antibody deposits localized and quantified.
(A) Anti-GD1a antibody (magenta) binds at the nodes of Ranvier of distal motor axons as determined by co-localization with neurofascin
(green) and a narrowing of the endogenously expressed axonal CFP (blue). (B) Nerve fibres and bundles were categorized by size for
quantification. Single arrow = single fibre; double arrow = small bundle; triple arrow = medium bundle. (C) Intensity of anti-GD1a antibody
binding was assessed according to bundle size; image shows antibody at a single fibre node of Ranvier compared to that seen at small
bundle node of Ranvier. (D) Single fibres showed significantly higher fluorescence intensity at nodes of Ranvier compared to small bundles,
small bundles compared to medium bundles and medium bundles compared to large bundles. Single fibre, small bundle and medium
bundle nodes of Ranvier all had significantly increased levels compared to control (no antibody) tissue. (E) Sixteen hours after injection of
anti-GD1a antibody (i.p. total dose 3 mg), fluorescence intensity at nodes of Ranvier showed the same gradient-dependent binding
pattern as that seen in ex vivo antibody treated tissue compared to control mice injected with phosphate buffered saline. #P50.05,
compared to small, medium, large bundles and control; *P50.05 compared to medium, large bundles and control; **P50.05, compared
to large bundles and control. Scale bar = 20 mm.
Anti-GD1a antibody-mediated nodal injury
Brain 2010: 133; 1944–1960
A
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Merge
DIC
MAC
MAC fluorescence intensity
(arbitrary units)
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Injured
Nerve terminal
Small bundle
Large bundle
1 mV
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Injured
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Figure 2 Complement activation at distal nerve nodes of Ranvier is associated with marked attenuation of endogenous CFP and loss of
perineural currents. Ex vivo triangularis sterni preparations exposed to anti-GD1a antibody or Ringer’s control, followed by 40% normal
human serum as a source of complement, were examined for membrane attack complex (MAC) deposition at nodes of Ranvier, the
distribution of axonal CFP and perineural current recordings. (A) Illustrative image of a node of Ranvier in a small nerve bundle coated with
membrane attack complex deposits. (B) Quantification of membrane attack complex deposits demonstrated significantly higher levels at
single fibre nodes of Ranvier, and small bundle nodes of Ranvier, compared to all other categories. (C) Illustrative low power images of
intramuscular CFP axon bundles (blue) terminating at -bungarotoxin delineated neuromuscular junction (magenta) in control tissue
exposed to Ringer’s followed by normal human serum (left panel), and anti-GD1a antibody followed by normal human serum, the latter
showing marked attenuation (right panel). (D) Perineural recordings from control (Ringer’s followed by normal human serum) and treated
(anti-GD1a antibody followed by normal human serum) tissue demonstrate intact Na+ (solid arrow) and K+ (broken arrow) currents at
nerve terminals, small bundles and large bundles in control nerves. These currents are completely attenuated in treated nerves, with the
exception of the Na+ currents in large bundles. *P50.05, compared to small, medium, large bundles and control; #P50.05 compared to
medium, large bundles and control. Scale bar = 10 mm (A) and 20 mm (C). DIC = differential interference contrast.
1950
| Brain 2010: 133; 1944–1960
A
% Immunostained NoR
Nav1.6
B
% Immunostained NoR
Nav1.6
40
20
*
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Small Medium Large
Bundle size
10um
ankyrin G
CFP
axons
ankyrin G
Myelin
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Caspr
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axons
Caspr
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pNFasc
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Large
Caspr
% Immunostained NoR
% Immunostained NoR
Nav1.6
Dystrophin
Control
Injured
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Ankyrin G
100
0
80
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Injured
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*
*
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% Immunostained NoR
Nav1.6
CFP axon
BTx
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Injured
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100
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E
R. McGonigal et al.
Single
Small Medium Large
Bundle size
Pan-neurofascin
pNFasc
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axons
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Kv1.1
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80
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Small Medium
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Large
Figure 3 Immunohistological appearance of nodal markers at the nodes of Ranvier (NoR) of distal intramuscular nerves following
exposure to anti-GD1a antibody and normal human serum (treated), compared with Ringer’s and normal human serum (control). Ex vivo
triangularis sterni muscles were incubated with 100 mg/ml anti-GD1a antibody and 40% normal human serum as a source of complement.
The percentages of nodes of Ranvier positive for immunostaining in each bundle category for five nodal proteins were determined.
(A–D) Nav1.6, ankyrin G, Caspr and neurofascin immunostaining was significantly reduced at single fibre and small bundle nodes of
Ranvier after treatment, compared to controls. (E) Kv1.1 immunostaining remained unchanged after treatment in all bundle categories.
Merged illustrations are shown for control tissue; and both single and merged illustrations for treated tissue. *P50.05, compared to
control counterpart. Scale bar = 10 mm. Arrows indicate location of the nodal region.
Anti-GD1a antibody-mediated nodal injury
and at nodes of Ranvier where it was preserved, the pattern was
disrupted. Immunostaining to the potassium channel Kv1.1 localized to the juxtaparanode was unaffected in all bundle categories (Fig. 3E).
The complete and rapid disappearance of key nodal axolemmal
proteins as assessed by immunostaining following 3 h of normal
human serum exposure was striking. In order to assess this in
more detail for Nav1.6, intermediate stages of dissolution of
Nav1.6 immunostaining were qualitatively examined at 15 and
30 min after the addition of complement treatment. At 15 min
there was no alteration to staining; however by 30 min a proportion of the nodes of Ranvier developed punctuate and dispersed
Nav1.6 staining, indicating fragmentation and spread of Nav1.6
channel clusters bound by the anti-Nav1.6 antibody (Fig. 7D).
In this model, the severe and concomitant motor nerve terminal
injury might have more proximal motor axonal consequences. By
way of control, Nav1.6 staining at nodes of Ranvier was assessed
after -latrotoxin-induced injury and found to be unaffected (data
not shown).
Complement inhibition completely
protects nodes of Ranvier from
anti-GD1a antibody-mediated injury
In order to demonstrate the role for the membrane attack complex
component of complement activation, the C5 complement inhibitor eculizumab, that completely prevents membrane attack complex assembly, was studied. Eculizumab protected Nav1.6, ankyrin
G and Caspr immunostaining at nodes of Ranvier from injury
mediated by complement activation, compared with the isotype
control antibody ALXN3300. In quantitative analysis, the percentage of nodes of Ranvier with intact Nav1.6 staining is significantly
greater on the addition of eculizumab compared to the
isotype-matched control monoclonal antibody ALXN3300 at
single fibres and small bundles (Fig. 4A; P50.001). As demonstrated previously, there was no reduction in immunostaining at
medium and large bundles in response to complement and thus
complement inhibition could not further attenuate this. Single fibre
and small bundle nodes of Ranvier also had significantly preserved
ankyrin G and Caspr staining with eculizumab protection compared to ALXN3300 application (Fig. 4B and C; P50.001).
Additionally, endogenous CFP was maintained in axons and bundles with eculizumab treatment compared to ALXN3300, essentially maintaining the normal overall architecture with a normal
appearance (Fig. 4D). Functional protection of the distal nerve
was also assessed. The sodium current as determined by perineural
recordings in the distal nerve was preserved by application of
eculizumab to anti-GD1a antibody and complement treated
tissue and the results illustrated in Fig. 4D.
Calpain inhibition protects sodium
channel and axonal protein integrity
without preserving nerve currents
A consequence of membrane attack complex pore deposition in
plasma membranes is the formation of a bi-directional,
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non-specific ion and water pore. At the nodes of Ranvier, the
electrical function of the nodal axolemmal membrane is dependent
on tightly regulated ion homeostasis and the consequences of this
uncontrolled flux are likely to be considerable. To assess the consequence of the calcium component of ion influx, the protective
effect of the synthetic calpain inhibitor AK295 was investigated.
Neurofilament is a known calpain substrate (Chan and Mattson,
1999) and its protection by calpain inhibition in response to
anti-ganglioside antibody-mediated complement-dependent injury
at nerve terminals has been reported previously (O’Hanlon et al.,
2003). In the present study, neurofilament at the neuromuscular
junction was also significantly protected by 100 mm AK295 treatment compared to antibody and normal human serum treated,
AK295 unprotected tissue (Fig. 5).
At the nodes of Ranvier in ex vivo whole-mount triangularis
sterni muscle preparations exposed to antibody and normal
human serum with and without AK295, assessments of Nav1.6,
ankyrin G and Caspr immunostaining, and of perineural electrophysiological recordings were made. As expected, the extent of
membrane attack complex deposition at nodes of Ranvier was
completely unaffected by AK295 (data not shown). Nav1.6 immunostaining at single fibre and small bundle nodes of Ranvier was
almost completely protected by AK295 treatment compared to
unprotected treated muscle (Fig. 6A; P50.001). Ankyrin G and
Caspr immunostaining was equally protected by calpain inhibition
at single fibre and small bundle nodes of Ranvier (Fig. 6B and C;
P50.001). For all nodal markers, the staining at nodes of Ranvier
in medium and large bundles did not significantly differ as injury
does not occur at these more proximal nodes of Ranvier.
To assess the protective properties of AK295 functionally, perineural recordings were conducted as previously described
(Fig. 6D). Compared to normal control tissue currents, perineural
Na+ and K+ currents were adversely affected by anti-GD1a antibody plus normal human serum, despite the presence of AK295.
Thus, a similar loss of current flow to that seen in injured tissue as
shown in Fig. 2D (lower panel) and Fig. 4D was observed. AK295
applied acutely to otherwise normal preparations had no neurotoxic effects (peak amplitude at time 0 and 20 min of 33.7 and
29.7 mV, respectively). These data indicate that calpain inhibition
is able to prevent the destruction of major structural components
at nodes of Ranvier, including Nav1.6 channels, but that despite
this, loss of nodal conduction as assessed electrophysiologically still
occurs.
Nodes of Ranvier in the nerve trunks
are also vulnerable to anti-GD1a
antibody and membrane attack
complex-mediated calpain activation
Experiments on nerve trunks were conducted with anti-GD1a antibody and normal human serum as the complement source, in the
presence and absence of calpain inhibition with AK295. A predominantly motor nerve (phrenic; 70% of myelinated fibres
being motor, Langford and Schmidt, 1983) and purely sensory
nerve (sural) were investigated in recording chambers during experimental incubations with normal human serum as the
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R. McGonigal et al.
Figure 4 The complement inhibitor, eculizumab, neuroprotects the distal nerve nodes of Ranvier on treatment with anti-GD1a antibody and
normal human serum. Eculizumab (100 mg/ml) plus 40% normal human serum were admixed 10 min before addition to ex vivo triangularis sterni
muscle preparations and the protective effects on the immunostaining signal of Nav1.6 channel, ankyrin G, Caspr and endogenous axonal CFP
were compared to tissue treated with anti-GD1a antibody and normal human serum admixed with the isotype matched control monoclonal
antibody ALXN3300. (A–C) Nav1.6, ankyrin G and Caspr immunostaining was significantly preserved at single fibre and small bundle nodes of
Ranvier following eculizumab treatment; illustrative images below. *P50.05, compared to control counterpart. Scale bar = 10 mm. (D) Illustrative
low power images of intramuscular CFP axon bundles (white) in triangularis sterni muscle terminating at -bungarotoxin delineated neuromuscular junction (magenta) after treatment with eculizumab (top image) or ALXN3300 (lower image). The CFP signal is completely preserved by
eculizumab, but markedly attenuated with the isotype control antibody. (E) Perineural recordings demonstrate a protection of Na+ and K+ currents
at nerve terminals, small and main branches of nerve bundles, in triangularis sterni preparations protected by eculizumab (upper traces), compared
to no protection afforded by control antibody, ALXN3300, in which currents are abolished (lower traces). Scale bar = 100 mm. Arrows indicate
location of the nodal region.
Anti-GD1a antibody-mediated nodal injury
Brain 2010: 133; 1944–1960
AK295 unprotected
AK295 protected
Merge
Merge
Neurofilament
Neurofilament
100
Neurofilament signal
(% of control)
| 1953
*
*
80
60
40
20
0
AK295
Injured
Treatment
Figure 5 The calpain inhibitor AK295 protects neurofilament at the nerve terminal from degradation by anti-GD1a antibody and normal
human serum exposure. Ex vivo triangularis sterni muscle was treated with anti-GD1a antibody and normal human serum with or without
100 mM AK295. Neurofilament immunostaining (red) intensity over the motor endplate (delineated by -bungarotoxin, green) was
measured and expressed as a percentage of normal levels. In the images, extensive pruning of the distal neurofilament arborisation can be
seen in AK295 unprotected tissue (right), compared with protected tissue (left). *P50.05, compared to AK295 treatment. Scale
bar = 50 mm.
complement source, with continuous serial recordings followed by
end-point immunohistology. Anti-GD1a antibody and complement
(membrane attack complex) deposition were present at nodes of
Ranvier in both phrenic and sural nerve; however their appearance
was significantly different, being more elongated in distribution
across the nodes of Ranvier in phrenic nerve compared to sural
nerve (Fig. 7A and B; P50.001) . Furthermore, sural nerve nodes
of Ranvier with antibody and membrane attack complex deposits,
and yet intact Nav1.6 channel immunostaining, were often
observed (Fig. 7C), a finding not seen in the phrenic nerve or its
intramuscular branches. In terms of functional effects on CAP
amplitudes, sural nerve CAPs remained stable or only modestly
reduced over time (76.3 9.6%; Fig. 7B), which was not significantly different from the controls.
In phrenic nerves, immunostaining of Nav1.6, ankyrin G and
Caspr at nodes of Ranvier was quantified in response to
anti-GD1a and normal human serum exposure. Having demonstrated nodal protein loss upon normal human serum exposure,
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R. McGonigal et al.
Figure 6 Calpain inhibition preserves immunostaining profiles of node of Ranvier proteins, without protecting conduction of distal axons
after treatment with anti-GD1a antibody and normal human serum. Ex vivo triangularis sterni preparations were incubated with
anti-GD1a antibody and normal human serum with or without 100 mM AK295 and its protective effect on the immunostaining of proteins
quantified in different bundle categories. Perineural recordings were performed after 3 h of treatment. (A–C) Nav1.6 channel, ankyrin G
and Caspr immunostaining was significantly preserved by AK295 treatment in single fibres and small bundles compared to the same
categories in AK295 unprotected tissue. Illustrative images depict intact staining to the right of the corresponding graphs. (D) Perineural
current traces show Na+ (solid arrow) and K+ (broken arrow) ion currents in nerve terminals, small bundles and large bundles from
completely normal triangularis sterni tissue (control, upper traces) and in tissue treated with anti-GD1a antibody, normal human serum
and AK295. In AK295 treated preparations, no protection of perineural currents in single and small bundles is seen. *P50.05, compared to
AK295 treatment. Scale bar = 10 mm. BTx = bungarotoxin; NoR = nodes of Ranvier. Arrows indicate location of the nodal region.
Anti-GD1a antibody-mediated nodal injury
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A
IgG
CFP axon
Merge
IgG
CFP axon
Sural nerve
Phrenic nerve
Merge
Spread of IgG staining (µm)
B
C
12
Merge
IgG
10
8
*
6
MAC
Nav1.6
4
2
Phrenic nerve
Sural nerve
Nerve
D
Merge
DIC
Nav1.6
Merge
DIC
Nav1.6
Merge
DIC
Nav1.6
Figure 7 Differential anti-GD1a antibody binding at nodes of Ranvier in phrenic nerve (motor) and sural nerve (sensory). Phrenic and
sural nerves were desheathed and incubated with anti-GD1a antibody (100 mg/ml for 2 h) before the distribution of antibody across the
node of Ranvier was quantitated. (A) Illustrative images of staining profile. (B) There was a significantly greater spread of antibody in
phrenic nerve compared to sural nerve nodes of Ranvier. (C) In sural nerve treated with anti-GD1a antibody plus normal human serum,
deposits of IgG and membrane attack complex (MAC) were frequently seen at nodes of Ranvier without loss of Nav1.6 immunostaining,
which was very rarely seen in either phrenic nerve or distal motor nerve nodes of Ranvier in triangularis sterni preparations. (D) Three
examples of Nav1.6 channel immunostaining at phrenic nerve nodes of Ranvier after anti-GD1a antibody exposure and 30 min treatment
with normal human serum, conditions under which membrane attack complex deposits are extensive. Various stages of dissolution of
Nav1.6 immunostaining are evident (arrows), prior to its subsequent complete disappearance. *P50.05, compared to phrenic nerve.
DIC = differential interference contrast; Scale bars = 5 mm.
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| Brain 2010: 133; 1944–1960
experiments were also conducted in the presence and absence of
AK295 (calpain inhibition) as for the ex vivo triangularis sterni
preparation. Immunostaining of the extracellular domain of
NrCAM, and moesin (a Schwann cell microvillal component) was
also assessed, these being molecules within the nodal complex but
predicted to be unaffected by calpain cleavage directly. There was
a significant loss of immunostaining to Nav1.6, ankyrin G and
Caspr in nerve exposed to antibody and complement, compared
to control (Fig. 8A and C; P50.001). Unlike the reduction in
Nav1.6, ankyrin G and Caspr staining, the NrCAM and moesin
staining was retained but appeared mislocalized, being more diffusely spread throughout the node of Ranvier area in comparison
with the staining pattern in control tissue (Fig. 8A and C).
Nav1.6 channel and Caspr staining was significantly protected
by AK295 (Fig. 8B; P = 0.01 for both proteins), although this was
more modest when compared with the levels of protection
achieved at the distal nerve node of Ranvier. Protection of ankyrin
G staining followed the same trend but did not achieve significance (P = 0.14). The retained but disrupted pattern of NrCAM
and moesin immunostaining was not altered by AK295 treatment.
Under phase optics (differential interference contrast), a constant
feature observed in phrenic nerve subjected to membrane attack
complex deposition and injury was the swollen, granular appearance
of the node of Ranvier, in comparison with control tissue, as visible
in Fig. 8C. This subjective and unquantifiable appearance was unaffected by AK295 treatment (Fig. 8C, third and fourth column) but
was consistently present. Two examples of each antibody staining
pattern are shown for AK295 protected nodes of Ranvier (Fig. 8C,
right). Extracellular recordings of phrenic nerve CAPs showed a large
fall in amplitude over time (to 40.5 10.7%) after treatment with
anti-GD1a antibody and complement, in comparison with peak
amplitude of the CAP prior to complement exposure (Fig. 8D and
E). This fall in CAP amplitude was not significantly prevented by
AK295 treatment (15.0 8.7%, P = 0.3).
Discussion
This study presents three major findings in relation to anti-GD1a
ganglioside antibody-mediated acute neuropathy models. First, we
demonstrate the increased vulnerability of very distal intramuscular
nodes of Ranvier to antibody and complement mediated injury, in
comparison with more proximal nodes that are relatively protected
by the blood nerve barrier. Secondly, we show that axolemmal
membrane attack complex pores at nodes of Ranvier result in
calpain activation, which in turn causes (i) immunodetectable
loss of key protein components of the nodal complex including
Nav1.6 channels, most likely by protein cleavage leading to fragmentation (Iwata et al., 2003; von Reyn et al., 2009); and (ii) loss
of function as demonstrated by the inability to record nerve terminal action potentials in distal axons. Thirdly, we show that electrical inexcitability of the nodes of Ranvier induced by membrane
attack complex pores can occur in the presence of preserved gross
structural integrity including that of key protein components
(Nav1.6, ankyrin, Caspr), suggesting that failure of the axolemmal
membrane to maintain ionic homeostasis when punctured by
R. McGonigal et al.
membrane attack complex pores is the critical factor in mediating
axonal conduction block in this model.
The study has been facilitated by exploiting the intramuscular
motor nerve node of Ranvier as a simple site relatively devoid
of blood nerve barrier restrictions for analysing the pathological
effects of locally or systemically delivered autoantibodies.
Identifying the antibody access gradient allowed us to focus attention on the most vulnerable distal sites in intramuscular nerve
bundles. We also demonstrated that any distal node of Ranvier
effects did not result from concomitant latrotoxin-like,
pre-synaptic injury that occurs in this model (Plomp and
Willison, 2009). The preparation usefully permits dual experimentation on neuromuscular junctions and nodes of Ranvier, although
the former site was not assessed in this study as it has been previously addressed (Goodfellow et al., 2005). The ex vivo focus of
this study has allowed us to investigate very early pathological and
electrophysiological features that result from membrane attack
complex injury to nodes of Ranvier and their therapeutic responsiveness, events that would be untractable in man or animal studies in vivo.
The application of perineural recordings to monitor nodes of
Ranvier electrophysiologically in our studies was essential as the
motor end plate is concomitantly paralysed in this model, and
endplate or muscle action potential measurement is thus not possible. The perineural recording technique allows for the recording
of local electrical signals, resulting from the opening of ion channels from the pre-terminal, terminal and axonal regions of motor
neurons (Mallart, 1985). When an electrode is inserted through
the perineural sheath of a motor nerve close to nerve terminals, a
waveform composed of two negative spikes can be recorded upon
nerve stimulation. The first negative spike is attributed with inward
Na+ current (INa, sensitive to tetrodotoxin) at the nodes of Ranvier
in the axonal trunk, and the second negative spike represents the
net local circuit current generated by the large outward current of
K+ (IK) and a relatively small inward Ca2+ current at motor nerve
terminals. The loss of recordable perineural currents in a
distal-dominant pattern correlated with our immunohistological
findings. At the distal node of Ranvier, the absence of recordable
currents indicates a severe disruption of the ability of the node of
Ranvier and motor nerve terminal to generate Na+ and K+ currents, respectively. This may either be due to calpain cleavage of
the channels directly or due to the inability of the injured axon to
maintain a resting membrane potential in the presence of membrane attack complex pores. The perineural current data obtained
in the presence of calpain inhibition, in which channel integrity is
preserved, indicate the latter mechanism is more likely. In the large
intramuscular nerve bundles, which are relatively resistant to
injury, the Na+ current was relatively preserved whereas the K+
current was reduced or absent, and interpreted as an inability to
generate or propagate an action potential in the severely affected
distal motor nerve that would be required to activate the terminal’s voltage dependent K+ channels (Braga et al., 1992).
Our previous studies have shown that the nerve terminal in this
mouse model of anti-GD1a antibody-mediated acute motor
axonal neuropathy is dependent upon membrane attack complex
deposition (Goodfellow et al., 2005), and can be completely
attenuated by the C5 neutralizing antibody, eculizumab
Anti-GD1a antibody-mediated nodal injury
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Figure 8 Phrenic nerve nodes of Ranvier immunostaining profiles of nodal proteins after exposure to anti-GD1a antibody plus normal
human serum are partially protected by AK295. Phrenic nerve was desheathed and treated with anti-GD1a antibody or Ringer’s, plus
normal human serum, with and without AK295, and the effect on nodal protein immunostaining was quantified. (A) Anti-GD1a antibody
treated phrenic nerve has significantly less Nodes of Ranvier that were immunopositive for Nav1.6 channel, ankyrin G and Caspr, than
controls. (B) AK295 does not protect ankyrin G and only modestly protects Nav1.6 channel and Caspr immunostaining from
complement-mediated injury. (C) Moesin and NrCAM staining profiles are not significantly altered in intensity after anti-GD1a antibody
exposure; however they both show an abnormal distribution as highlighted in the images. Examples of normal control staining of all of the
proteins (magenta) versus treated and AK295 protected nerve. Note occurrence of swollen morphology of treated nerves in differential
interference contrast that is not ameliorated by AK295 treatment. (D) Extracellular recordings of anti-GD1a antibody-treated phrenic
Continued
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| Brain 2010: 133; 1944–1960
(Halstead et al., 2008a) and other blockers of membrane attack
complex formation (Halstead et al., 2008b). Here we also
demonstrate the pivotal role for complement in mediating the
disorganization of the nodes of Ranvier. This study does not
directly rule out a possible contribution from C5a, whose formation is also blocked by eculizumab; however our previous studies
on terminal axons have shown severe axonal injury is abrogated
under C6 deficiency conditions (in which C5a is still formed),
making a critical role for C5a unlikely. Furthermore, blockade earlier in the complement pathway, prior to C5, is also neuroprotective in our nerve terminal model (Halstead et al., 2005; Plomp and
Willison, 2009). Nevertheless, the importance of other components of the complement pathway in peripheral nerve injury and
repair should not be overlooked (Ramaglia et al., 2008). Our
findings are supported by data from a chronic immunization
model of acute motor axonal neuropathy in rabbits in which ventral root nodes of Ranvier are targeted by anti-GM1 antibody and
complement that can be inhibited by nafamostat mesilate, although the precise mechanism(s) underlying this protection are
likely to be different (Phongsisay et al., 2008). In the rabbit
model, conduction block was attributed to lengthening of nodes
of Ranvier accompanied by Nav1.6 channel loss or dispersion;
whereas in our acute model, we conclude the conduction block
arises principally as a result of node of Ranvier ionic imbalance due
to axolemmal membrane attack complex pores. Irrespective of the
precise mechanism, both studies raise the therapeutic prospect of
using eculizumab in patients with acute motor axonal neuropathy
and Guillain–Barré syndrome, as has been achieved in other membrane attack complex-mediated disorders (Hillmen et al., 2006).
One advantage of the reliance of this mouse model on a heterologous (human) source of complement is that eculizumab is specific for human C5 and ineffective at neutralizing mouse C5.
Therefore, therapeutic effects in a ‘humanized’ complement dependent injury using human therapeutic agents can be tested. It
remains unknown why mouse complement cannot be sufficiently
fixed in this model to cause pathological effects, in comparison
with human complement, as previously discussed in detail
(Willison et al., 2008).
The membrane attack complex pore comprises a transmembrane 5 nm pore allowing unselective, bidirectional flow of
water, ions and soluble intracellular constituents of molecular
weights up to 35 kDa (Podack et al., 1982; Simone and
Henkart, 1982), including CFP (28 kDa). Thus in this model, the
outward flow of CFP and its subsequent dilution in the extracellular environment is the most likely explanation for its disappearance, as it is not a calpain substrate and appears to be a
very sensitive marker of pore formation. Even in the large intramuscular nerve bundles in which membrane attack complex is at
undetectable levels, the CFP signal is attenuated, although this
may alternatively be due to diffusion down axon with subsequent
leakage in the more distally injured region.
R. McGonigal et al.
At the nodes of Ranvier under physiological conditions, extracellular Ca2+ will flow intracellularly through membrane attack
complex pores where one effect will be to activate calpain, as
occurs at nerve terminals (O’Hanlon et al., 2003).
Calpain-mediated proteolysis cleaves cytoskeletal and membrane
proteins (Vosler et al., 2008), including sodium channels, as
observed here. We used two Nav.1.6 antibodies that bind to different peptide domains on intracellular cytoplasmic loops between
transmembrane channel subunits. Thus, the apparent ‘disappearance’ of Nav1.6 labelled by two antibodies observed here, over
such a short timeframe, most probably equates to cleavage of the
cytoplasmic loop(s), rather than more global disintegration, internalization or shedding of Nav1.6. Moreover, proteolysis of Nav1.6
intracellular loops may not significantly affect channel function
since activation is preserved, the dominant effect being a failure
of inactivation (von Reyn et al., 2009). Since ankyrin G links
Nav1.6 to the cytoskeleton, it is also possible that the un-tethered
Nav1.6 becomes mislocalized through lateral diffusion. The mislocalized but preserved immunostaining of Kv1.1 at the juxtaparanode, moesin in the Schwann cell microvilli and the extracellular
domain of NrCAM at the node of Ranvier, alongside the phase
optics images of the node of Ranvier, suggest that highly selective
injury to the node of Ranvier axolemma was accompanied by local
swelling and disorganization, with grossly preserved structural integrity of the axoglial unit over this timeframe. The mechanistic
similarities between this model and the Nav1.6 loss at ventral root
nodes of Ranvier reported in a chronic rabbit model of acute
motor axonal neuropathy are unknown (Susuki et al., 2007b) .
Functional performance of the nodes of Ranvier under these
injurious conditions was assessed with particular attention to
Nav1.6, owing to its central role in nodal conduction. Injured
nodes of Ranvier rapidly became electrically inactive, even when
Nav1.6 and other calpain substrates were protected by AK295
treatment; indicating that failure to maintain ionic and water
homeostasis owing to the presence of membrane attack complex
pores, leading to membrane depolarization and inactivation of
Nav1.6 channels, was the most likely mechanism, rather than
Nav1.6 disruption. Ideally, ultrastructural examination of nodes
of Ranvier would inform this; however electron micrographs of
both control and affected nodes of Ranvier all showed
fixation-related artefacts owing to the extended periods of time
the nerve was maintained ex vivo, and were not suitable for
analysis.
Our studies on phrenic and sural nerve trunks excluded the
possibility that the vulnerability to anti-GD1a antibody-mediated
injury was due to a distal to proximal GD1a antigen gradient
in nerves (rather than a reflection of antibody access) and that
concomitant, latrotoxin-like nerve terminal injury was responsible
for any disruption to the juxtaterminal nodes of Ranvier. It also
identified differential sensitivity of motor (phrenic) and sensory
(sural) nerves to membrane attack complex-mediated injury that
Figure 8 Continued
nerve show a reduction in CAP amplitudes over time that are unaffected by AK295. Anti-GD1a antibody was added for 2 h; subsequently
normal human serum was added for 2 h, starting at 0 min. Arrows indicate the addition of 5 mM tetrodotoxin to terminate the experiment.
(E) CAP amplitudes are expressed as the percentage of the starting value, there being no significant difference between AK295 treated or
untreated nerves. *P50.05, compared to control or AK295 counterpart. Scale bar = 5 mm.
Anti-GD1a antibody-mediated nodal injury
remains unexplained and may in part account for difficulties in
interpreting this study in the context of previous work from us
and others (Paparounas et al., 1999). Whether this quantitative
or qualitative resistance of sural nerve to membrane attack
complex-mediated injury provides insights into human acute
motor axonal neuropathy, in which motor nerves are selectively
affected, is unknown. In the currently used GD3s / mouse
model, the sural nerve contains GD1a that is sufficiently available
for antibody binding with complement activation, whereas in man,
the levels of GD1a available for antibody targeting may be greater
in motor than sensory nerve (De Angelis et al., 2001; Gong et al.,
2002).
The above model describing very distal injury as a feature of
acute motor axonal neuropathy corresponds well with existing
clinical data, notwithstanding the co-occurrence in some cases of
severe proximal injury (McKhann et al., 1991; Ho et al., 1997). In
terms of underlying molecular mechanisms, the acute and severe
motor nodes of Ranvier injury in this model may correspond to the
initial phases of axonal conduction block seen in human acute
motor axonal neuropathy, in which rapid onset but potentially
reversible pathophysiology develops, prior to any cellular infiltration or axonal degeneration. The extent to which such events
occur in man cannot be readily determined at a pathophysiological
level as clinical and electrophysiological interrogation is very limited; however recovery in acute motor axonal neuropathy may be
either very rapid and complete, or very prolonged with poor outcome, owing to extensive proximal axonal degeneration
(McKhann et al., 1993; Hiraga et al., 2005b). The events at the
nodes of Ranvier described here would correspond well with the
early injury phase of a dichotomous outcome model (Gabriel,
2005). Critically, inhibition of the terminal complement product,
membrane attack complex, as an early intervention seems essential from these data to limiting both acute injury and the development of more destructive long term pathology, while the
inhibition of calpain activation downstream from membrane
attack complex may also offer some partially additive benefit
(Wang et al., 2004). Models addressing the role of eculizumab
in more advanced stages of the pathological progression would
also be useful. The expectation that clinical trials of complement
or calpain inhibition in Guillain–Barré syndrome and its variants will
inform this further is considerable.
Acknowledgements
B6/Cg-TgN(Thy1-CFP) mice were kindly provided by
Dr W. Thompson, Austin, TX, USA. AK295 was kindly provided
by Dr J. Powers and J. Glass, Atlanta, Georgia. Dr Jaap Plomp is
thanked for critical reading of the article.
Funding
The Wellcome Trust (#077041/Z/05/Z to H.J.W.). R.M. and
K.N.G. were supported by the Medical Research Council doctoral
training account awarded to the University of Glasgow.
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| 1959
Supplementary material
Supplementary material is available at Brain online.
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