Neurofascin (NF) is a cell-adhesion molecule that is found at the nodes of Ranvier. The 186
kDa isoform of neurofascin (NF186) is expressed on the axon in the exposed node, and the
155 kDa isoform (NF155) is expressed on myelinating glia at the paranode. NF186 is essential
for clustering of sodium channels to the nodes while NF155 is needed for close paranodal interactions
between myelinating glia and axons. The neurofascins are found in both the peripheral
and central nervous system (PNS and CNS).
NF-specific autoantibodies were identified in serum of multiple sclerosis (MS) patients using
a proteomics approach with two-dimensional Western blotting of human myelin glycoproteins.
A monoclonal antibody (mAb) specific for NF was shown to induce axonal injury in an animal
model of MS, experimental autoimmune encephalomyelitis. This indicated that NF is a relevant
autoantibody target in patients with inflammatory diseases of the nervous system (central and
peripheral), but actual abundance of anti-NF autoantibodies is unknown.
The objectives of the thesis were the following: 1) Develop assays to detect autoantibodies
against human NF. 2) Determine the prevalence in patients with MS and with inflammatory
diseases of the PNS. 3) Characterize the reactivity by immunoglobulin isotyping, serial dilution,
epitope mapping, and staining of nodal structures in tissue sections. 4) Affinity purify anti-NF
antibodies from plasma exchange material. 5) Determine possible in vivo effects of anti-NF
antibodies in the PNS using a neuritis animal model.
First, we expressed the complete human NF155 and NF186 on the surface of stable human cell
lines, produced the complete extracellular portion of the NFs in HEK293 cells, and expressed
truncated variants of the NFs in E. coli. With these reagents, we set up three antibody detection
assays: cell-based assay by flow cytometry, ELISA, and Western blot. These assays were
validated using NF-specific monoclonal and polyclonal antibodies, and optimized with a test
cohort of serum samples.
We screened 687 serum and 48 plasma exchange samples from patients with MS (n = 233),
inflammatory diseases in the PNS (n = 294), and controls (n = 208). From serum analysis,
we observed low prevalence of anti-NF reactivity (3%) by flow cytometry and/or ELISA despite
broad reactivity in almost half of the serum samples analyzed by Western blot. Reactivity observed
by flow cytometry and by ELISA were congruent only in the patients with the highest
reactivities. The anti-NF antibodies were NF-isoform specific, mainly IgG subclasses, and at
high titres in some cases. Using truncated variants of NF fused to super green fluorescence
protein (sGFP), we showed that reactivity of anti-NF Abs was largely directed towards the membrane
proximal extracellular domains that are unique to each isoform, while the membrane
distal immunoglobulin-like domains and fibronectin domains were not recognized.
A small proportion (3%; 8/254) of patients with GBS and CIDP showed reactivity to human NF
by ELISA. A few showed a particularly high reactivity (up to 1:10 000 dilution) to NF. Two CIDP
patients showed a particularly high (up to 1:10 000 dilution) anti-NF155 reactivity by FACS and
ELISA, recognized paranodes in tissue sections, and exhibited dominant IgG4 subclass usage.
Another CIDP patient who benefited from plasma exchange had a persistent anti-NF155
reactivity by ELISA in serum, and after affinity purification, anti-NF186 and -NF155 reactivity by
FACS and ELISA were detected in addition. These antibodies were mainly IgG3, with minor
contribution of IgM and IgA. To investigate possible functions of anti-NF antibodies in inflammatory
PNS diseases, we injected two different monoclonal antibodies (mAbs) into a P2-peptide
induced experimental autoimmune neuritis (EAN) animal model at disease onset. We found
that while the anti-NF mAbs prolonged and exacerbated clinical disease in these animals, they
could not induce disease on their own.
We detected NF-reactivity in a small proportion of MS samples (3%; 7/225) by ELISA and flow
cytometry. We obtained follow-up material from two NF-reactive patients and saw a persistent
NF reactivity in one of them. To increase detection sensitivity, we affinity purified anti-NF antibodies
from plasma exchange material of patients with MS (n = 8). IgG, IgM, and IgA were
isolated from most of the samples; they were found to recognize NF155 and to a lower extent
NF186 by ELISA and in a few also by flow cytometry. This indicates that low levels of anti-NF
antibodies exist in a proportion of MS patients.
In conclusion, 3% of serum samples from patients with PNS inflammatory neuropathies (GBS
and CIDP) showed reactivity by ELISA and none of the controls. In an animal model of autoimmune
peripheral nerve inflammation, we showed, using two anti-NF mAbs, that antibody
targeting of NF can enhance and prolong disease course. This suggests that antibodies to NF
may be relevant for a small group of patients with peripheral inflammatory neuropathies. In MS
patients, 3% showed anti-NF reactivity by flow cytometry and ELISA. Furthermore, low levels of
anti-NF antibodies that could be detected by ELISA and flow cytometry after affinity purification
were additionally found in some MS patient samples that were unreactive by serum screening.
This raises the possibility that low levels of antibodies to NF are present in some MS patients
and may contribute to the pathogenesis of this chronic disease