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Visualization of the Breakdown of the Axonal Transport Machinery: a Comparative Ultrastructural and Immunohistochemical Approach

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Abstract

Axonal damage is a major factor contributing to disease progression in multiple sclerosis (MS) patients. On the histological level, acute axonal injury is most frequently analyzed by anti-amyloid precursor protein immunohistochemistry. To what extent this method truly detects axonal injury, and whether other proteins and organelles are as well subjected to axonal transport deficits in demyelinated tissues is not known. The aim of this study was to correlate ultrastructural morphology with the immunohistochemical appearance of acute axonal injury in a model of toxin-induced oligodendrocyte degeneration. C57BL/6J mice were intoxicated with 0.25% cuprizone to induce demyelination. The corpus callosum was investigated by serial block-face scanning electron microscopy (i.e., 3D EM), immunohistochemistry, and immunofluorescence microscopy. Brain tissues of progressive MS patients were included to test the relevance of our findings in mice for MS. Volumes of axonal swellings, determined by 3D EM, were comparable to volumes of axonal spheroids, determined by anti-APP immunofluorescence stains. Axonal swellings were present at myelinated and non-myelinated axonal internodes. Densities of amyloid precursor protein (APP)+ spheroids were highest during active demyelination. Besides APP, vesicular glutamate transporter 1 and mitochondrial proteins accumulated at sites of axonal spheroids. Such accumulations were found as well in lesions of progressive MS patients. In this correlative ultrastructural-immunohistochemical study, we provide strong evidence that breakdown of the axonal transport machinery results in focal accumulations of mitochondria and different synaptic proteins. We provide new marker proteins to visualize acute axonal injury, which helps to further understand the complex nature of axonal damage in progressive MS.

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Acknowledgments

We thank Christiane Nolte and Helmut Kettenmann for providing hGFAP/EGFP transgenic mice (Max-Delbrueck Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany). We thank Sarah Wübbel, Astrid Baltruschat, and Beate Aschauer for their excellent and valuable technical assistance.

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Authors and Affiliations

  1. Department of Neuroanatomy, Ludwig-Maximilians-University of Munich, Pettenkoferstr. 11, 80336, Munich, Germany

    Sebastian Rühling, Franziska Kramer, Selina Schmutz, Zhan Jiangshan, Christoph Schmitz, Markus Kipp & Tanja Hochstrasser

  2. Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

    Sandra Amor

  3. Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

    Sandra Amor

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  1. Sebastian Rühling
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Correspondence to Tanja Hochstrasser.

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Rühling, S., Kramer, F., Schmutz, S. et al. Visualization of the Breakdown of the Axonal Transport Machinery: a Comparative Ultrastructural and Immunohistochemical Approach. Mol Neurobiol 56, 3984–3998 (2019). https://doi.org/10.1007/s12035-018-1353-9

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