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Gap junction pathology in multiple sclerosis lesions and normal-appearing white matter

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Abstract

Oligodendrocyte gap junctions (GJs) are vital for central nervous system myelination, but their involvement in multiple sclerosis (MS) pathology remains unknown. The aim of this study was to examine alterations of oligodendrocyte and related astrocyte GJs in MS lesions and normal-appearing white matter (NAWM). Post-mortem brain samples from 9 MS and 11 age-matched non-MS control patients were studied. Tissue sections that included both chronic active and inactive lesions were characterized neuropathologically with Luxol Fast Blue staining and immunostaining for myelin oligodendrocyte glycoprotein (MOG) and the microglial marker Iba1. We analyzed the expression of Cx32 and Cx47 in oligodendrocytes and of Cx43, the major astrocytic partner in oligodendrocyte–astrocyte (O/A) GJs by quantitative immunoblot and real-time PCR. Formation of GJ plaques was quantified by immunohistochemistry. Compared to control brains, both Cx32 and Cx47 GJ plaques and protein levels were reduced in and around MS lesions, while Cx43 was increased as part of astrogliosis. In the NAWM, Cx32 was significantly reduced along myelinated fibers whereas Cx47 showed increased expression mainly in oligodendrocyte precursor cells (OPCs). However, OPCs showed only limited connectivity to astrocytes. Cx43 showed modestly increased levels in MS NAWM compared to controls, while GJ plaque counts were unchanged. Our findings indicate that oligodendrocyte GJs are affected not only in chronic MS lesions but also in NAWM, where disruption of Cx32 GJs in myelinated fibers may impair myelin structure and function. Moreover, limited O/A GJ connectivity of recruited OPCs in the setting of persistent inflammation and astrogliosis may prevent differentiation and remyelination.

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Acknowledgments

This project was funded by the Cyprus Research Promotion Foundation (Grants ACCESS/0308/11 and HEALTH/BIOS/0308/01 to KAK) and by Cyprus Telethon (2010-11 grant to KAK). Post-mortem human brain tissue samples were kindly provided by the UK Multiple Sclerosis Society Tissue Bank, Imperial College London, funded by the UK Multiple Sclerosis Society.

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Authors and Affiliations

  1. Neuroscience Laboratory, The Cyprus Institute of Neurology and Genetics, P.O. Box 23462, 1683, Nicosia, Cyprus

    Kyriaki Markoullis, Irene Sargiannidou, Natasa Schiza & Kleopas A. Kleopa

  2. Neurology Clinics, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

    Kleopas A. Kleopa

  3. Wolfson Neuroscience Laboratories, Imperial College Faculty of Medicine, London, UK

    Federico Roncaroli & Richard Reynolds

  4. Department of Molecular Pathology and Electron Microscopy, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

    Andreas Hadjisavvas

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  1. Kyriaki Markoullis
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  2. Irene Sargiannidou
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  3. Natasa Schiza
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  7. Kleopas A. Kleopa
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Correspondence to Kleopas A. Kleopa.

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K. Markoullis and I. Sargiannidou contributed equally to this paper.

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Suppl. Table 1 (PDF 13 kb)

401_2012_978_MOESM2_ESM.tif

Suppl. Figure 1: Visualization of glial GJ plaques with confocal microscopy. These are images captured with confocal microscopy of non-MS control fixed brain WM sections double immunostained for GJ proteins (green) as indicated and cell or axonal markers (red) including GFAP (b), Iba1 (e), PLP (h), and Kv1.2 (k), as indicated. Merged images are shown in the right column. Diffusely distributed Cx43 GJ plaques are shown in a, while Cx47 GJ plaques are typically concentrated around oligodendrocytes and their proximal processes (green arrowheads in d). Cx32 GJ plaques are diffusely expressed along myelinated fibers (g) but frequently also appear in a paranodal distribution (green arrowheads in j) surrounding juxtaparanodal Kv1.2 channels (red arrowheads in k). Scale bar: 20 μm (TIFF 16053 kb)

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Suppl. Figure 2: Example of glial GJ plaque counts in control and MS brain sections. Quantification was performed in defined areas of pathologically characterized sections (example schematic diagram in a) immunostained for each of the GJ proteins Cx32, Cx47, and Cx43. GJ plaques were counted in NAWM areas as well as in lesions (L) and periplaque (P1-P3) (example in b; for definitions see Methods) in order to examine the gradient of GJ pathology. GJ plaque counts were performed using the ImagePro Plus software. Representative images with counted GJ plaques (highlighted red) in different areas as indicated are shown for Cx32 (c), for Cx47 (d) and for Cx43 (e). Cell nuclei are stained with DAPI (blue). Scale bars: in b: 100 μm; in c-e: 50 μm (TIFF 16341 kb)

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Suppl. Figure 3: Gradient of inflammation from MS NAWM toward the lesions. Immunostaining for Iba1 (red) in the same fields in which GJ counts were obtained (control WM, NAWM, perilesion P1-3, and lesions) reveals a gradient of inflammation with increasing immunoreactivity of Iba1-positive (red) activated microglia toward the lesions, reaching a maximum in the perilesion area (a-f). Cell nuclei are stained with DAPI (blue). Scale bar: 50 μm. Quantitative analysis (g) of Iba1 immunoreactivity (average % of total area from multiple fields) from groups of non-MS control and MS samples (n = 6 WM areas from 4 control cases, 5 NAWM areas from 4 MS cases, and 3 lesions and perilesions from 3 MS cases) confirms the presence of significant inflammation in MS NAWM compared to control WM, which increases toward chronic active lesions, reaching a maximum at the immediate border of the lesions (P1) (*indicates significant p-values following Bonferroni-Dunn correction for multiple comparisons) (TIFF 14216 kb)

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Suppl. Figure 4: Identification and quantification of oligodendrocyte precursor cells. These are images of Images of control WM (a, c) and MS NAWM (b, d) immunostained with NG2 (a, b, green) or Olig2 (c, d, red) antibodies to identify oligodendrocyte precursor cells (OPCs). Cell nuclei are stained with DAPI (blue). Staining with both NG2 and Olig2 shows higher OPC numbers (white arrowheads) in MS compared to control WM. Scale bar: 20 μm. OPC counts (e) obtained in different areas of control WM (n = 9 areas from 3 cases) and MS NAWM (n = 9 areas from 3 cases) demonstrate that OPCs are significantly increased in MS brain (MS) compared to non-MS controls (C). Scale bar: 30 μm (TIFF 10344 kb)

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Suppl. Figure 5: Lack of Cx32 expression in oligodendrocyte precursor cells. Images of double immunostaining for Cx32 (a, d, green) and Olig2 (b, e, red) in control WM (a-c) and in MS NAWM (d-f). Cell nuclei are stained with DAPI and merged images are shown in the right collumn (c, f). Although many more OPCs are present in MS tissue (red arrows) compared to control WM, they do not colocalize with Cx32 immunoreactivity (green arrowheads), which is found mainly along myelinated fibers both in control WM and in MS NAWM. Scale bars: 10 μm (TIFF 6157 kb)

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Suppl. Figure 6: Increased Cx43 levels in MS lesions: Immunoblot analysis for Cx43 (a) in control WM (n = 2 cases), MS NAWM (n = 2 cases) and in 5 different lesions (L) (n = 5 cases) shows overall increased levels in MS, although with some variability. GAPDH blot was used for loading control. Quantitative analysis (b) shows significantly elevated Cx43 levels in MS lesions compared to control WM (*indicates significant p-values following Bonferroni-Dunn correction for multiple comparisons) (TIFF 3476 kb)

401_2012_978_MOESM8_ESM.tif

Suppl. Figure 7: Expression of Cx30 in the cortex of control and MS brain. These are merged (a-f) or separate and merged channels (g-l) images from immunostaining for Cx30 (red) and Cx43 (green) in MS and non-MS brain, as indicated. Cell nuclei are stained with DAPI (blue). At low magnification (a, d) it is apparent that Cx30 is almost exclusively expressed in the cortex (a), where it forms dense GJ plaques shown at higher magnification (b). Cx30 is not expressed in the WM, and not in or around MS lesions (d). This is in contrast to Cx43, which is most prominent in the WM (f) and NAWM (a), and in MS lesions (d). Higher magnification images of MS NAWM (c) and MS lesion (e) show prominent Cx43 expression but no Cx30 expression. Lack of Cx30 expression is also found in control WM (f), and is therefore not a feature of MS. Higher magnification separate channels of MS cortex (g-i) and NAWM (j-l) show the characteristic diffuse GJ plaque formation by Cx30 in the cortex, typically decorating the surface of neurons (red arrowheads in g), and lack of Cx30 GJs in the NAWM (j). Cx43 (green arrowheads) is more distinctly expressed forming GJ plaques around astrocytes and blood vessels both in the cortex (h) and NAWM (k). Scale bars in a and d: 100 μm; in b, c, e: 20 μm; in f and g-l as well as in insets of b and e: 10 μm (TIFF 14473 kb)

401_2012_978_MOESM9_ESM.tif

Suppl. Figure 8: Decreasing colocalization of Cx47 and Cx43 around lesions and in the NAWM. These are images (merged channels in a-d and both separate and merged channels at higher magnification in e & f) of double labelling for Cx47 (red) and Cx43 (green) in control WM (a, e) and in MS NAWM (b), perilesion (c, f), or lesion (d). Cell nuclei are stained with DAPI (blue). GJ plaques formed by Cx47 and Cx43 colocalize around oligodendrocytes in control WM (white arrowheads in a and e). This colocalization is decreased in MS NAWM (b) and perilesion (c, f), where smaller Cx47 GJ plaques (red arrowheads) can be seen that do not overlap with Cx43 immunoreactivity. This is despite increasing density of Cx43 GJ plaques (green arrowheads) that are frequently concentrated around activated astrocytes, most prominently in the lesion (white arrows in d), where Cx47 is almost absent. Scale bars: in a-d: 50 μm; in e–f: 10 μm (TIFF 5661 kb)

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Markoullis, K., Sargiannidou, I., Schiza, N. et al. Gap junction pathology in multiple sclerosis lesions and normal-appearing white matter. Acta Neuropathol 123, 873–886 (2012). https://doi.org/10.1007/s00401-012-0978-4

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  • DOI: https://doi.org/10.1007/s00401-012-0978-4

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