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Characteristic ultrastructural lesions of the paranodal regions in CIDP associated with

anti-Neurofascin 155 antibodies.

Running head: Paranodal alterations in Nfasc155 CIDP

Jean-Michel Vallat,1 Nobuhiro Yuki,2 Kenji Sekiguchi,3 Norito Kokubun,4 Nobuyuki Oka,5

Diane L. Sherman,6 Peter J. Brophy,6 and Jérôme J. Devaux.7

From 1 Department of Neurology and ‘Centre de Référence des neuropathies rares’, CHU

Limoges; 2 Department of Neurology, Mishima Hospital, Niigata, Japan; 3 Division of

neurology, Kobe University Graduate School of Medicine, Kobe, Japan; 4 Department of

Neurology, Dokkyo Medical University, Tochigi, Japan; 5 Department of Neurology, National

Hospital Organization Minami-Kyoto Hospital, Joyo, Kyoto, Japan; 6 Centre for

Neuroregeneration, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom; 7 Aix-

Marseille Université, CNRS, CRN2M-UMR 7286, Marseille, France;

Corresponding author: Pr. Vallat, Department of Neurology, Centre de référence

“neuropathies périphériques rares”, CHU Limoges, 2 Avenue Martin Luther-King, 87042

Limoges, France.

E-mail: jean-michel.vallat@unilim.fr

Author email addresses: JMV, jean-michel.vallat@unilim.fr;

NY, gbs.yuki.cidp@gmail.com; KS, sekiguch@med.kobe-u.ac.jp;

NK, kokubun@dokkyomed.ac.jp; NO, dkajo904@kyoto.zaq.ne.jp; DLS,

Diane.Sherman@ed.ac.uk; PJB, Peter.Brophy@ed.ac.uk;

JJD, JD jerome.devaux@univ-amu.fr

Title, 120 characters with spaces; running head, 38 characters with spaces; abstract, 197

words; manuscript, 1524 words; figures, 2; tables, 1; references, 18.

Study funding: Supported by the Agence Nationale pour la Recherche (ACAMIN; JJD)

under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases, and the

Association Française contre les Myopathies (MNM1 2012-14580; JJD) and by the Medical

Research Council and the Wellcome Trust (DLS and PJB).

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Keywords: autoantibody, chronic inflammatory demyelinating polyneuropathy, NF155,

nodes of Ranvier, myelin

Abbreviations: Caspr1 = Contactin-associated protein-1, CIDP = chronic inflammatory

demyelinating polyneuropathy, EM = electron microscopy, Nfasc155 = Neurofascin 155

Financial disclosure statement: JMV, KS, NK, NO, DLS, PJB, and JJD report no relevant

disclosures. NY serves as an editorial board member of Expert Review of Neurotherapeutics,

The Journal of the Neurological Sciences, The Journal of Peripheral Nervous System,

Journal of Neurology, Neurosurgery & Psychiatry, and Journal of Alzheimer’s Disease.

Author contributions: Drafting/revising the manuscript for content, including medical

writing for content, JMV, NY, KS, NK, NO, DLS, PJB, and JJD. Study concept and design,

JMV, NY and JD. Acquisition of data, JMV, NY, KS, NK, NO, and JJD. Analysis and

interpretation of data, JMV, NY, KS, NK, NO, DLS, PJB, and JJD.

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ABSTRACT

Antibodies to Contactin-1 and Neurofascin 155 (Nfasc155) have recently been associated

with subsets of patients with chronic inflammatory demyelinating polyneuropathy (CIDP).

Contactin-1 and Nfasc155 are cell adhesion molecules that constitute the septate-like

junctions observed by electron microscopy in the paranodes of myelinated axons. Antibodies

to Contactin-1 have been shown to affect the localization of paranodal proteins both in patient

nerve biopsies and in animal models after passive transfer. However, it is unclear whether

these antibodies alter the paranodal ultrastructure. We examined by electron microscopy sural

nerve biopsies from two patients presenting with anti-Nfasc155 antibodies, and also four

patients lacking antibodies, three normal controls, and five patients with other neuropathies.

We found that patients with anti-Nfasc155 antibodies presented a selective loss of the septate-

like junctions at all paranodes examined. Further, cellular processes penetrated into the

expanded spaces between the paranodal myelin loops and the axolemma in these patients.

These patients presented with important nerve conduction slowing and demyelination. Also,

the reactivity of anti-Nfasc155 antibodies from these patients was abolished in neurofascin-

deficient mice, confirming that the antibodies specifically target paranodal proteins. Our data

indicate that anti-Nfasc155 destabilizes the paranodal axo-glial junctions and may participate

in conduction deterioration.

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INTRODUCTION

Chronic inflammatory demyelinating polyneuropathy (CIDP) is the commonest acquired

immune-mediated neuropathy worldwide and is clinically heterogeneous (Vallat et al., 2010).

Anti-Neurofascin 155 (Nfasc155) and anti-Contactin-1 IgG4 antibodies have recently been

associated with subgroups of CIDP patients showing ataxia, tremor, and a poor response to

intravenous immunoglobulin (Ng et al., 2012; Querol et al., 2012; Querol et al., 2014; Miura

et al., 2015; Devaux et al., 2016). These autoantibodies may serve as biomarkers to improve

patient diagnosis and to guide treatments (Querol et al., 2015). Antibodies to Contactin-1

appear to induce paranodal alterations in skin biopsies from CIDP patients (Doppler et al.,

2015) and in animals models (Manso et al., 2016). However, the pathogenic effects of anti-

Nfasc155 IgG4 are yet unclear.

We present here characteristic ultrastructural modifications of the paranodal junctions in sural

nerve biopsies from two patients with anti-Nfasc155 IgG4 antibodies. These lesions have not

been described before in nerves of CIDP patients and are likely to contribute significantly to

the conduction deficits observed.

METHODS

Nerve biopsies. A portion of each sural nerve biopsy from two CIDP patients with anti-

Nfasc155 antibodies was fixed in buffered glutaraldehyde, embedded in Epon, and prepared

for light (semi-thin sections) and ultrastructural examination. To study specifically the node of

Ranvier, several longitudinal sections of each nerve biopsy were systematically examined by

electron microscopy (EM) and compared with longitudinal sections of sural nerve biopsies

from three normal controls, five patients with non-CIDP acquired neuropathies (two cases of

vasculitic neuropathy, one case of nutritional neuropathy, and two cases of lymphomatous

neuropathy), and from four CIDP patients showing no reactivity to paranodal proteins.

Nerve staining. Teased fibers from sciatic nerves of 7-day-old neurofascin-deficient

(Nfasc-/-) and Nfasc+/+ mice were prepared as previously described (Sherman et al., 2005).

Teased fibers were immersed in -20°C acetone for 10 min, blocked for 1 hr in blocking

solution containing 5% fish skin gelatin, 0.1% Triton X-100 in phosphate buffer saline and

incubated overnight at 4°C with anti-Nfasc155 CIDP sera diluted at 1:200, rabbit antisera

against Contactin-associated protein-1 (Caspr1; 1:2,000) and chicken antibody against Myelin

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Protein Zero (1:2,000; Abcam). The slides were washed and incubated with the appropriate

Alexa-conjugated secondary antibodies (1:500; Jackson Immunoresearch, West Grove, PA).

Slides were mounted and examined using an ApoTome fluorescence microscope (Carl Zeiss

MicroImaging GmbH).

Biochemistry. Brains from 7-day-old Nfasc+/+ and Nfasc-/- mice were homogenized as

previously described (Devaux, 2010). 100 µg of proteins were loaded on 7.5% SDS-PAGE

gels, transferred, and immunoblotted with a rabbit recognizing all Neurofascin isoforms

(PanNfasc; 1:2,000; Abcam), a rabbit antiserum against Actin (1:2,000; Sigma-Aldrich) or

anti-Nfasc155 CIDP sera (1:500). Immunoreactivity was revealed using peroxidase-coupled

secondary antibodies (1:5,000; Jackson ImmunoResearch) and BM chemiluminescence kit

(Roche).

RESULTS

In a previous study, we detailed the clinical data of two CIDP patients showing IgG4

antibodies to Nfasc155 (patients 10 and 24; Devaux et al., 2016). The serum IgG from these

patients stained the paranodal regions of mouse sciatic nerve fibers (Fig. 1A). To further

confirm the specificity of this reactivity in these patients, we tested these samples on nerves

from the Nfasc-/- mice (Sherman et al., 2005). As expected, CIDP sera did not stain

paranodes from Nfasc-/- mice (Fig. 1A), and immunoreactivity to Nfasc155 was abolished in

immunoblots of brain samples from Nfasc-/- mice (Fig. 1B).

To determine whether these autoantibodies are associated with specific morphological

abnormalities, we examined sural nerve biopsies from these two patients. On semi-thin

sections, there was a decreased number of both large and small diameter myelinated fibers

compared to controls (Fig. 2A-B). This decrease was heterogeneous between fascicles. Some

myelinated axons presented thin myelin sheaths suggestive of demyelinating/remyelinating

process. Myelin ovoids attesting to nerve fiber destruction were also seen. No inflammatory

cells were noted. EM examination confirmed these aspects. Myelin debris in the cytoplasm of

some Schwann cells and in a few macrophages in the endoneurial space were present. No

proliferation of Schwann cell profiles like onion bulbs was seen. On longitudinal sections, we

observed frequent and significant widenings of the nodes of Ranvier (Fig. 2C). High

magnification study of all the paranodal regions showed an absence of transverse bands at

paranodes in Nfasc155-positive patients (Fig. 2D-E). In these areas there was a loss of the

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attachment of the myelin loops to the axon, and an irregular widening of the periaxonal space

by comparison to controls. Transverse bands could be readily observed at paranodes in nerve

biopsies from normal controls or CIDP patients lacking antibodies to paranodal proteins (Fig.

2F). In case 2 (patient 24), cellular processes often penetrated the paranodal region perhaps

contributing to the dissociated attachment between the axon and the paranodal loops (Fig.

2D). In case 1 (patient 10), only a few small axonal processes penetrated the first paranodal

loops located near the nodal area. No such alterations were ever seen in normal controls, in

non-CIDP acquired neuropathies or CIDP patients lacking antibodies to paranodal proteins.

The nerve conduction studies showed diffuse, but heterogeneous conduction slowing in motor

nerves as all nerves were not similarly affected. F-waves were detectable with prolonged

latency in both patients (Table 1). Sensory nerve action potentials could not be detected in all

tested nerves in both patients. In case 2, prolonged distal latencies and reduced conduction

velocities with probable conduction block were found in the forearm segment of the median

nerve (Table 1). These results indicated that the demyelinating process was diffuse along the

nerve.

DISCUSSION

Nfasc155 is a cell adhesion molecule playing an essential function in paranodal formation and

in nerve conduction (Sherman et al., 2005; Zonta et al., 2008). Nfasc155 is localized at the

terminal loops of myelin and associates with the axonal cell adhesion molecules Contactin-1

and Caspr1 (Charles et al., 2002). This ternary complex of glycoproteins is required for the

formation and stability of the paranodal transverse bands also known as septate-like junctions

(Bhat et al., 2001; Boyle et al., 2001; Sherman et al., 2005; Zonta et al., 2008). Recently, we

screened a large cohort of CIDP patients and identified anti-Nfasc155 IgG4 antibodies in a

subgroup of CIDP patients (Devaux et al., 2016). Here, we further show using Nfasc-/- mice

that the reactivity to Nfasc155 is specific and that autoantibodies target Nfasc155 at

paranodes. In addition, we describe paranodal alterations in nerve biopsies from two of these

patients.

At the ultrastructural level on longitudinal sections from control human nerve biopsies, we

observed the presence of transverse bands, which correspond to small linear structures

spiraling around the axon between the terminal myelin loops and the axolemma. In the two

nerve biopsies from CIDP patients with anti-Nfasc155 antibodies, these transverse bands were

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absent in all the paranodes that we have examined. In addition, the periaxonal space between

the axon and the paranodal loops was irregularly widened. In case 2, cytoplasm processes

were detected in this widened space penetrating between the axons and the loops. In this case,

the nerve biopsy was performed several years after the first clinical signs appeared. In case 1,

this penetration of cell cytoplasm between the paranodal loops and the axon seemed to be just

beginning. This could be due to the shorter duration of the polyneuropathy (about 5 months)

at the time of the nerve biopsy. These alterations were highly reminiscent of the alterations

found in mice deficient for Contactin-1, Caspr1, or Neurofascin (Bhat et al., 2001; Boyle et

al., 2001; Sherman et al., 2005). Altogether, these data indicate that in human nerves, the

transverse bands are also responsible for the narrow and regular bindings of myelin paranodal

loops to the axon. The loss of the paranodal axo-glial junctions may allow the penetration of

cellular processes between the paranodal loops and the axon. These results suggest that anti-

Nfasc155 antibodies destabilize transverse bands and thereby disrupt the paranodal barrier.

Disruption of transverse bands have been previously documented in diabetic polyneuropathies

(Sima et al., 1986; Giannini and Dyck, 1996) or in experiment allergic neuritis (Allt, 1975).

These were mostly partial loss involving some paranodes or some paranodal loops. This was

not the case here, because all paranodes were affected. In addition, we carefully examined

serial sections at high magnification to make sure that transverse band alterations were not

preparation artifacts.

The patients presented with diffuse and heterogeneous nerve conduction slowing along the

nerves. Because all paranodes were affected, it is plausible that transverse band loss accounts

for the diffuse conduction defects. However, significant node widenings and demyelination

were also observed and likely participate in conduction slowing. Both cases also presented a

rarefaction of myelinated axons. The absence of sensory nerve action potentials may therefore

be due to both axonal loss and distal demyelination. Axonal loss is a common phenomenon in

CIDP and is likely related to the demyelination process. The patients showed a prominent

IgG4 response to Nfasc155, which did not activate the complement pathway (Devaux et al.,

2016) and are unlikely to induce axonal degeneration. In addition, no immune cell infiltration

was found in nerve biopsies.

In conclusion, our data indicate that patients with IgG4 antibodies to Nfasc155 present typical

electrophysiological and morphological aspects of CIDP, but in presence of specific

paranodal alterations. These results suggest that multiple mechanisms can be responsible for

the physiopathology of CIDP, including IgG4-mediated demyelination.

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REFERENCES

Allt G. The node of Ranvier in experimental allergic neuritis: an electron microscope study. J

Neurocytol 1975; 4: 63-76.

Bhat MA, Rios JC, Lu Y, Garcia-Fresco GP, Ching W, St Martin M, et al. Axon-glia

interactions and the domain organization of myelinated axons require Neurexin

IV/Caspr/Paranodin. Neuron 2001; 30(2): 369-83.

Boyle MET, Berglund EO, Murai KK, Weber L, Peles E, Ranscht B. Contactin orchestrates

assembly of the septate-like junctions at the paranode in myelinated peripheral nerve.

Neuron 2001; 30(2): 385-97.

Charles P, Tait S, Faivre-Sarrailh C, Barbin G, Gunn-Moore F, Denisenko-Nehrbass N, et al.

Neurofascin is a glial receptor for the paranodin/Caspr-contactin axonal complex at the

axoglial junction. Curr Biol 2002; 12(3): 217-20.

Devaux JJ. The C-terminal domain of betaIV-spectrin is crucial for KCNQ2 aggregation and

excitability at nodes of Ranvier. J Physiol 2010; 588(23): 4719-30.

Devaux JJ, Miura Y, Fukami Y, Inoue T, Manso C, Belghazi M, et al. Neurofascin-155 IgG4

in chronic inflammatory demyelinating polyneuropathy. Neurology 2016; 86(9): 800-7.

Doppler K, Appeltshauser L, Wilhelmi K, Villmann C, Dib-Hajj SD, Waxman SG, et al.

Destruction of paranodal architecture in inflammatory neuropathy with anti-contactin-1

autoantibodies. J Neurol Neurosurg Psychiatry 2015.

Giannini C, Dyck PJ. Axoglial dysjunction: a critical appraisal of definition, techniques, and

previous results. Microsc Res Technique 1996; 34(5): 436-44.

Manso C, Querol L, Mekaouche M, Illa I, Devaux JJ. Contactin-1 IgG4 antibodies cause

paranode dismantling and conduction defects. Brain 2016; 139(Pt 6): 1700-12.

Miura Y, Devaux JJ, Fukami Y, Manso C, Belghazi M, Wong AH, et al. Contactin 1 IgG4

associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia.

Brain 2015; 138(Pt 6): 1484-91.

Ng JK, Malotka J, Kawakami N, Derfuss T, Khademi M, Olsson T, et al. Neurofascin as a

target for autoantibodies in peripheral neuropathies. Neurology 2012; 79(23): 2241-8.

Querol L, Nogales-Gadea G, Rojas-Garcia R, Diaz-Manera J, Pardo J, Ortega-Moreno A , et

al. Neurofascin IgG4 antibodies in CIDP associate with disabling tremor and poor

response to IVIg. Neurology 2014; 82(10): 879-86.

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Querol L, Nogales-Gadea G, Rojas-Garcia R, Martinez-Hernandez E, Diaz-Manera J, Suarez-

Calvet X, et al. Antibodies to contactin-1 in chronic inflammatory demyelinating

polyneuropathy. Ann Neurol 2012; 73(3): 370-80.

Querol L, Rojas-Garcia R, Diaz-Manera J, Barcena J, Pardo J, Ortega-Moreno A, et al.

Rituximab in treatment-resistant CIDP with antibodies against paranodal proteins. Neurol

Neuroimmunol Neuroinflamm 2015; 2(5): e149.

Sherman DL, Tait S, Melrose S, Johnson R, Zonta B, Court FA, et al. Neurofascins are

required to establish axonal domains for saltatory conduction. Neuron 2005; 48(5): 737-42.

Sima AF, Lattimer SA, Yagihashi S, Greene DA. Axo-glial dysjunction: novel structural

lesion that accounts for poorly reversible slowing of nerve conduction in the spontaneously

diabetic bio-breeding rat. J Clin Invest 1986; 77: 474-84.

Vallat JM, Sommer C, Magy L. Chronic inflammatory demyelinating

polyradiculoneuropathy: diagnostic and therapeutic challenges for a treatable condition.

Lancet Neurol 2010; 9(4): 402-12.

Zonta B, Tait S, Melrose S, Anderson H, Harroch S, Higginson J, et al. Glial and neuronal

isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the

central nervous system. J Cell Biol 2008; 181(7): 1169-77.

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FIGURE LEGENDS

Figure 1: Autoantibodies in CIDP patients specifically bind Nfasc155 at paranodes.

(A) These are teased sciatic nerve fibers from 7 day old Nfasc+/+ (upper panel) and Nfasc-/-

mice (lower panel) stained with CIDP sera (red), Caspr1 (green), and Myelin Protein Zero

(P0; blue). IgG from Nfasc155 CIDP patients stained the paranodal regions of wild-type

animals, but not in Nfasc-/- mice. Scale bars: 10 μm.

(B) Brain proteins (100 µg) from Nfasc+/+ and Nfasc-/- mice were immunoblotted with a

Nfasc155 CIDP serum, a rabbit antiserum recognizing all Neurofascin isoforms (PanNfasc),

or a rabbit antiserum against Actin. The CIDP serum recognized two protein bands in

Nfasc+/+ but not in Nfasc-/- mice. These two bands likely represent Nfasc155 high and low

forms. The PanNfasc antibody revealed several protein bands corresponding to Nfasc186 and

Nfasc155 in Nfasc+/+ but not in Nfasc-/- mice. Molecular weight markers are indicated in

kDa on the left.

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Figure 2: Paranodal alterations in sural nerve biopsy from Nfasc155 CIDP patients.

(A-B) These are transverse semithin sections of sural nerve biopsy from a CIDP patient with

anti-Nfasc155 antibodies (A) and normal nerve biopsy of a patient of the same age (B). Note

the significant rarefaction of large myelinated axons by comparison to the normal nerve.

(C, D, E) Longitudinal ultrathin sections showing ultrastructural alterations at paranodes in

CIDP patients with anti-Nfasc155 antibodies (case 2). Widening of nodes of Ranvier (C) were

observed suggesting demyelinating process. Paranodal regions were severely disrupted and

cellular processes (colored in blue) interposed between the paranodal myelin loops (ml) and

the axon (a). The periaxonal space between the axon and the paranodal loops were abnormally

widened (D-E) and lacked transverse bands (arrow).

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(F) Longitudinal sections of a paranode showing transverse bands in a normal control nerve.

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Table 1: Electrophysiological characteristics of CIDP patients with anti-Nfasc155 IgG4

Case 1 Case 2

Median nerve

DML (ms) 6.5 6.9

dCMAP amplitude (mV) 4.2 5.0

MCV (m.s-1) 24 21

F wave latency (ms) 62 71.8

SNAP amplitude (µv) ND ND

Ulnar nerve

DML (ms) 4.0 5.1

dCMAP amplitude (mV) 8.2 7.3

MCV (m.s-1) 25 22

F wave latency (ms) 55 -

SNAP amplitude (µv) 2.0 ND

Tibial nerve

DML (ms) ND 15.4

dCMAP amplitude (mV) ND 0.4

MCV (m.s-1) ND Unmeasurable

F wave latency (ms) ND ND

Sural nerve

SNAP amplitude (µv) 1.5 ND

dCMAP = distal compound muscle action potential; DML = distal motor latency; MCV =

motor conduction velocity; ND = not detected; SNAP = sensory nerve action potential.