Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Antibodies to MOG have a demyelination phenotype and affect oligodendrocyte cytoskeleton

doi: 10.1212/NXI.0000000000000012

Figure Lengend Snippet: F-actin (upper images) and β-tubulin (lower images) immunolabelings in human fixed (A) or live (B) oligodendroglial MO3.13 MOG+ cells incubated with purified healthy control (HC) IgG or MOG antibody–positive DEM IgG. Representative data are shown (volume projection of entire Z-stack acquired using deconvolution microscopy). Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Bar: 10 μm. Dotted lines on right images represent contour of cells as determined by differential interference contrast images (not shown). (C) Quantification of change in distribution of thin filament (F-actin, upper scatter plot) and microtubule network (β-tubulin, lower scatter plot) organization using 3D deconvolution microscopy. F-actin and β-tubulin were majorly affected in live cells incubated with DEM IgG. Forty different cells (1 cell = 1 diamond) from 2 HC and 2 DEM patients out of 3 independent experiments are shown. Results are expressed as ratio of the relative enrichment (RE) between cytoplasm and nucleus and values shown are percentages relative to HC IgG FIX (100%). Red bars represent mean. Note that there also was a loss of organization of the thin filaments after HC IgG incubation on live cells compared to fixed conditions, but to a lesser extent than after DEM IgG. (D) Cell viability assay by flow cytometry on live human oligodendroglial MO3.13 MOG+ cells incubated 45 minutes with 6 μg of purified HC IgG or DEM IgG. No difference in cell death was observed. Dead cell percentages are noted in the legend. 7AAD = 7-aminoactinomycin D; DEM = demyelinating diseases; Ig = immunoglobulin; MOG = myelin oligodendrocyte glycoprotein.

Article Snippet: In order to visualize effects of protein G–purified human IgGs on single cells, MO3.13 cells were seeded at low density and incubated with 6 μg of protein G–purified human IgGs from patient or control sera and HEK293 MOG+ -immunoabsorbed MOG antibody–positive serum for 45 minutes at room temperature followed by goat anti-human IgG secondary antibody for 15 minutes at room temperature., After washing, cells were fixed and permeabilized, and cytoskeleton filamentous actin (F-actin, marker of cytoskeleton thin filaments) and β-tubulin (marker of cytoskeleton microtubule) were quantified using 3D deconvolution microscopy (detailed methodology in appendix e-1).

Techniques: Incubation, Purification, Control, Microscopy, Staining, Viability Assay, Flow Cytometry