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Extensive loss of connexins in Baló’s disease: evidence for an auto-antibody-independent astrocytopathy via impaired astrocyte–oligodendrocyte/myelin interaction

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Abstract

Extensive aquaporin-4 (AQP4) loss without perivascular deposition of either activated complement or immunoglobulins is a characteristic of Baló’s disease. Our aim in this study was to investigate the relationship between astrocytopathy and demyelination in Baló’s disease, focusing on connexins (Cx), which form gap junctions among glial cells and myelin. Autopsied specimens from four cases that provided seven actively demyelinating concentric lesions infiltrated with numerous CD68+ macrophages were immunohistochemically examined for the astrocyte markers glial fibrillary acidic protein (GFAP), AQP4, Cx43, Cx30 and megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1). Specimens were also stained for oligodendrocyte/myelin markers, namely Cx32, Cx47, myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-specific protein (OSP) and Nogo-A. Serum samples from six patients that had undergone magnetic resonance imaging, confirming a diagnosis of Baló’s disease, were assayed for the presence of anti-Cx43, -Cx32 and -AQP4 antibodies. Despite the presence of numerous GFAP- and MLC1-positive astrocytes, there was a marked decrease in the levels of Cx43, Cx32 and Cx47. At the leading edges, Cx43 and AQP4 were mostly absent despite positive GFAP, MLC1, Cx32, Cx47, MOG, MAG, and OSP immunoreactivity. Of the six Baló’s disease patients, none were positive for anti-Cxs or -AQP4 antibodies. Baló’s disease is characterized by extensive loss of Cxs and AQP4, and a lack of auto-antibodies to Cxs and AQP4. Loss of Cx43 and AQP4 in the presence of other oligodendrocyte/myelin proteins at the leading edges suggests the possibility that auto-antibody-independent astrocytopathy may contribute to disease pathology via the disruption of astrocyte–oligodendrocyte/myelin interactions.

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Acknowledgments

This work was supported in part by a Health and Labour Sciences Research Grant on Intractable Diseases (H22-Nanchi-Ippan-130 and H23- Nanchi-Ippan-017) from the Ministry of Health, Labour, and Welfare, Japan, by a Scientific Research B Grant (No. 22390178) and a Challenging Exploratory Research Grant (No. 23659459) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan), by the Kaibara Morikazu Medical Science Promotion Foundation (Japan), and by the Japanese Multiple Sclerosis Society. We thank Professor Artemio T. Ordinario, Department of Neurology and Psychiatry, University of Santo Tomas (Philippines), for providing the Baló’s disease samples, and Ms. Sachiko Koyama and Mr. Takaaki Kanemaru, Department of Neuropathology and Morphology Core Unit, Kyushu University (Japan), for their excellent technical assistance.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Katsuhisa Masaki, Takuya Matsushita, Tomomi Yonekawa, Takeshi Matsuoka, Noriko Isobe, Kyoko Motomura & Jun-ichi Kira

  2. Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Satoshi O. Suzuki, Takeshi Matsuoka & Toru Iwaki

  3. Department of Neurology, Jiangxi Provincial People’s Hospital, Nanchang, China

    Xiao-Mu Wu

  4. Department of Diagnosis, Prevention and Treatment of Dementia, Graduate School of Medicine, Juntendo University, Tokyo, Japan

    Takeshi Tabira

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  1. Katsuhisa Masaki
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Supplementary material 1 (DOC 49 kb)

401_2012_972_MOESM2_ESM.pdf

Suppl. Fig. 1 Double immunofluorescence staining for GFAP and astrocytic Cxs in a case of myasthenia gravis (MG) (n = 1 case). Punctate staining for Cx43 is distributed in astrocytic cell bodies and processes in the cerebral white matter (a, arrowheads), glial limiting membrane (b, arrow) and perivascular foot processes in the cerebral white matter (c, arrowheads). Cx30 is diffusely expressed in the gray matter of the pons (d). Merged image shows punctate staining for Cx30 frequently abutting from GFAP-immunopositive astrocytic processes. Arrowheads indicate neurons in the gray matter (d). Scale bar: 10 μm (a), 50 μm (b, d), 20 μm (c) (PDF 391 kb)

401_2012_972_MOESM3_ESM.pdf

Suppl. Fig. 2 Double immunofluorescence staining for Cxs and other myelin markers in cases of Pick’s disease (a, b) and myasthenia gravis (MG) (c, d, e) (n = 2 cases). In the spinal cord, Cx32 is localized to the outer layer of myelin sheaths adjacent to GFAP-immunopositive astrocytic processes (a). MAG is localized along the surface of neurofilament-immunopositive axons (b). Immunostaining for Cx32 shows patchy expression along MBP-immunopositive transverse pontine fibers (c). Cx47 is typically expressed around intrafascicular oligodendrocytes in the MBP-immunopositive myelin fibers of the cerebral white matter (d, arrowheads). Double staining for Cx43 and Cx47 shows partial juxtaposition or colocalization suggestive of GJ plaque formation around intrafascicular oligodendrocytes (lower insets) (e). Scale bar: 10 μm (a, d, e), 20 μm (b, c) (PDF 331 kb)

401_2012_972_MOESM4_ESM.pdf

Suppl. Fig. 3 Astrocytic features in cases with other neurological disorders. (a–f) Subacute cerebral infarction. KB staining shows diffuse myelin pallor across the whole area of the lesion (a), but well preserved myelin in the surrounding area (a, upper). CD68 immunostaining shows massive infiltration of macrophages (b). GFAP-positive astrocytes are almost absent across the lesion (c), whereas many reactive astrocytes appear in the surrounding area (c, upper). (d–f) Higher magnification of the lesion (corresponding to Suppl. Fig. 3c, arrowhead). Immunoreactivities for AQP4, MLC1 and Cx43 in the perivascular foot processes are mostly preserved. (g–r) Cx43, Cx30, Cx32 and Cx47 expression in astrogliosis. (g–l) A case of limbic encephalitis. GFAP- (g) and MLC1-positive (h) gemistocytes exhibit membrane staining for Cx43 (i). MLC1 is intensely expressed in the perivascular foot processes and weakly expressed in the cytoplasm of gemistocytes. Immunoreactivity for Cx30 is not evident in gemistocytes (j). Loss of Cx47 and Cx32 are not observed in this astrogliotic lesion (k, l). Arrows indicate the gemistocytes (k, l). (m–r) A case of spastic paraplegia type 2 (SPG2). GFAP- (m) and MLC1-positive (n) reactive astrocytes in a case with chronic fibrillary gliosis show upregulation of Cx43 (o). MLC1 is intensely expressed in perivascular foot processes, and weakly expressed in glial fibers. Immunoreactivity for Cx30 is not evident in this lesion (p), whereas Cx47 and Cx32 expression is preserved (q, r). Scale bar: 200 μm (a–c), 50 μm (d–i), 20 μm (j, m–r), 10 μm (k, l) (PDF 1173 kb)

401_2012_972_MOESM5_ESM.pdf

Suppl. Fig. 4 Measurement of anti-Cx43 and anti-Cx32 antibodies in the sera of patients with Baló’s disease. (a–c) Immunostaining of HEK-293 cells transfected with a GFP-Cx43 fusion protein expression vector. The expressed Cx43-GFP is mainly localized to the plasma membrane (green) and forms gap junction channels at cell–cell interfaces (arrows) with some punctate intracellular distribution. (b) Positive control using an anti-Cx43 antibody. Immunostaining with the anti-Cx43 antibody shows the most intense fluorescence in the region of the gap junction (red). (c) Serum from a patient with Baló’s disease is negative for auto-antibodies against Cx43. (d, e) Immunostaining of HEK-293 cells transfected with a Cx32-GFP fusion protein expression vector. (d) Positive control using an anti-Cx32 antibody. Immunostaining with the anti-Cx32 antibody shows intense fluorescence at cell–cell interfaces (red). (e) Serum from a patient with Baló’s disease is negative for auto-antibodies against Cx32. Scale bar: 10 μm (a–e). DIC: differential interference contrast. (PDF 260 kb)

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Masaki, K., Suzuki, S.O., Matsushita, T. et al. Extensive loss of connexins in Baló’s disease: evidence for an auto-antibody-independent astrocytopathy via impaired astrocyte–oligodendrocyte/myelin interaction. Acta Neuropathol 123, 887–900 (2012). https://doi.org/10.1007/s00401-012-0972-x

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