Neurofilament light as an immune target for pathogenic antibodies

Abstract

Antibodies to neuronal antigens are associated with many neurological diseases including paraneoplastic neurological disorders, epilepsy, amyotrophic lateral sclerosis and multiple sclerosis. Immunization with neuronal antigens such as neurofilament light (NF-L), a neuronal intermediate filament in axons, has been shown to induce neurological disease and spasticity in mice. Also, although antibodies to NF-L are widely used as surrogate biomarkers of axonal injury in amyotrophic lateral sclerosis and multiple sclerosis, it remains to be elucidated if antibodies to NF-L contribute to neurodegeneration and neurological disease. To address this, we examined the pathogenic role of antibodies directed to NF-L in vitro using spinal cord co-cultures and in vivo in experimental autoimmune encephalomyelitis (EAE) and optic neuritis animal models of multiple sclerosis. Here we show that peripheral injections of antibodies to NF-L augmented clinical signs of neurological disease in acute EAE, increased retinal ganglion cell loss in experimental optic neuritis and induced neurological signs following intracerebral injection into control mice. The pathogenicity of antibodies to NF-L was also observed in spinal cord co-cultures where axonal loss was induced. Taken together, our results reveal that as well as acting as reliable biomarkers of neuronal damage, antibodies to NF-L exacerbate neurological disease, suggesting that antibodies to NF-L generated during disease may also be pathogenic and play a role in the progression of neurodegeneration.

Keywords: antibodies; autoimmunity; axonal damage; neurodegeneration; neurofilament light.

© 2017 John Wiley & Sons Ltd.

Figures

Figure 1

Specificity of the monoclonal antibody NF‐L 10H9. (a) Western blot analysis illustrates the specificity of NF‐L 10H9 to neurofilament light (NF‐L) protein (68 000 MW). Lane 1 shows the reactivity to spinal cord and lane 2 the reactivity to mouse brain homogenate. (b) Reactivity to the recombinant NF‐L protein. MW, protein standard.

Figure 2

Retinal ganglion cell (RGC) density is significantly affected by NF‐L 10H9. (a) RGC density and (b) retinal nerve fibre layer (RNFL) thickness at 15 days (group A, grey bars, n = 3 mice per group, n = 2 for isotype control) or at 20 days (group B, black bars, n = 2 mice per group) after treatment with (un)labelled NF‐L 10H9 or isotype control IgG1. (a) Only mice in group B treated with unlabelled NF‐L 10H9 showed a statistically significant reduction in RGC density compared with mice treated with unlabelled isotype control IgG1 (Student's t‐test, P < 0·05). (b) No significant differences in percentage change in RNFL thickness were observed in NF‐L 10H9‐treated mice 15 or 20 days post‐induction. *P <0·05

Figure 3

Effect of an anti‐human neurofilament light (NF‐L) antibody on neuronal viability. Differentiated PC12 cells were treated for 24, 48 and 72 hr with 20 μg/ml rabbit anti‐human NF‐L antibody or rabbit IgG as a control of the assay. Cells were incubated with MTT (0·5 mg/ml) for the last 4 hr of incubation, then supernatants were discarded and 200 μl DMSO was added to solubilize the formazan crystals. Absorbance was determined at 590 nm using an ELISA reader. ***P < 0·001, **P < 0·01.

Figure 4

Monoclonal antibody (mAb) to neurofilament light (NF‐L) reduces axonal density in rat spinal cord co‐cultures. (a) Co‐cultures were treated with NF‐L 10H9 or isotype control for 0·5, 1, 2, 3, 16 or 48 hr with 10 μg/ml mAb. Subsequently, cultures were stained for SMI310 and axonal density was determined using imageJ and represented as percentage axonal loss relative to the isotype control IgG1. Axonal loss was significant after 1, 2, 3, 16 or 48 hr of treatment with NF‐L 10H9 (n = 4 for 0·5 and 48 hr, n = 3 for 1–16 hr, ***P < 0·001, **P < 0·01, *P < 0·05, Student's t‐test). Representative immunofluorescent images of cultures treated with (b) 10 μg/ml isotype control IgG1 or (c) NF‐L 10H9 for 16 hr and stained with SMI310 (scale bars = 35 μm). [Colour figure can be viewed at wileyonlinelibrary.com]