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Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury

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Abstract

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35–55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139–151-induced relapsing–remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury.

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Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft, CRC1080 Project B6 to MKES and FZ. The anti-L1 antibody clones 555 and 324 were developed in the laboratory of Melitta Schachner and a generous gift from Peter Altevogt (DKFZ, Heidelberg, Germany). We gratefully acknowledge the technical assistance of Tobias Hirnet, Christine Oswald and Heike Ehrengard and proofreading of the manuscript by Dr. Cheryl Ernest (all UMC Mainz, Germany).

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    Authors and Affiliations

    1. Department of Anesthesiology, University Medical Center of the Johannes-Gutenberg-University Mainz, Mainz, Germany

      Lutz Menzel, Wiesia Bobkiewicz & Michael K. E. Schäfer

    2. Department of Neurology, University Medical Center of the Johannes-Gutenberg-University Mainz, Mainz, Germany

      Magdalena Paterka, Stefan Bittner, Esther Witsch & Frauke Zipp

    3. Institute of Physiology, University Medical Center of the Johannes-Gutenberg-University Mainz, Mainz, Germany

      Robin White

    4. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

      Jack van Horssen

    5. Center for Neuroscience, Shantou University Medical College, Shantou, China

      Melitta Schachner

    6. Institute of Neuropathology, University Hospital Münster, Münster, Germany

      Tanja Kuhlmann

    7. Focus Program Translational Neuroscience (FTN) and Rhine Main Neuroscience Network (rmn²), Mainz, Germany

      Stefan Bittner, Frauke Zipp & Michael K. E. Schäfer

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    Frauke Zipp and Michael K. E. Schäfer are equally contributing co-senior authors.

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    401_2016_1607_MOESM1_ESM.tif

    Supplemental Fig. S1. CD4+ T cell subsets express L1 and cause reduction of neuronal L1 mRNA expression. (a) Flow cytometry showing similar L1 expression on CD4+ T cell subsets isolated from the CNS of C57BL/6J mice at the peak of disease upon MOG35-55-induced EAE (n=6). (b) Neuronal L1 mRNA expression is reduced upon 2 h of co-culturing with CD4+ T cell subsets, differentiated from spleen-derived CD4+ T cells (n=6/subset, normalized to βIII-tubulin and control, set as 1, which represents neurons cultured alone). Data are expressed as mean ± SEM. βIII-tubulin immunofluorescence is normalized to control conditions which represent neurons cultured alone (set as 100 %). Generation of CD4+ T cell subpopulations: Antigen-presenting cells (APC) from spleen of 8-10-week-old C57BL6/J mice were enriched by T cell depletion using the CD90+ MicroBeads kit (Miltenyi), irradiated (30 Gy) and cultured in full culture medium (10 % FCS, 0,01 % β-mercaptoethanol, 1 % glutamate, 1 % penicillin/streptomycin, 1 % HEPES). Naïve T cells were isolated using the CD4+CD62L+ MicroBeads kit (Miltenyi) and cultured with APCs and anti-CD3 antibodies (2 µg/mL). Differentiation of CD4 subpopulations was stimulated using subset-specific cytokine combinations (all cytokines purchased from R&D Systems) and respective antibodies (all Bio X cell) as followed: Th1 cells (IL-12, 50 ng/ml; IL-18, 25 ng/ml; anti-IL-4 antibody, 10 µg/ml), Th2 cells (IL-4, 10 ng/ml; anti-IL-12 antibody, 10 µg/mL; anti-IFNγ antibody, 10 µg/mL), Th17 cells (IL-23, 20 ng/mL; IL-6, 20 ng/mL; TGFβ, 3 ng/mL; anti-IL-4 antibody, 10 µg/mL; anti-IFNγ antibody, 10 µg/mL), regulatory T cells (TGFβ, 3 ng/mL; anti-IL-12 antibody, 10 µg/mL; anti-IFNγ antibody, 10 µg/mL). Th1, Th2 and regulatory T cells were used after 5 days of differentiation, Th17 cells were re-stimulated for additional 5 days before use. All subpopulations were routinely tested for subset-specific cytokine expression using intracellular staining and flow cytometry analysis (data not shown). (TIFF 1741 kb)

    401_2016_1607_MOESM2_ESM.tif

    Supplemental Fig. S2. Axonal loss in spinal cord tissue. (a) Representative image showing a spinal cord hemisphere of C57BL/6J mice at the peak of disease, immunolabeled with antibodies specific to the axonal marker neurofilament (anti-NF) and counterstained with DAPI to visualize cell nuclei. Boxes indicate regions with high and low cell densities. (b, c) High-power magnifications showing NF/DAPI-double labeling. The number of axons is strongly reduced in tissue regions with high cell densities. Arrows depict NF-labeled axons. Scale: 200 µm (a), 20 µm (b). (TIFF 3800 kb)

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    Menzel, L., Paterka, M., Bittner, S. et al. Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury. Acta Neuropathol 132, 703–720 (2016). https://doi.org/10.1007/s00401-016-1607-4

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