| Abstract |
Myelinated fibers are divided in discrete subdomains around the Nav-enriched nodes of Ranvier: the paranodes, where axoglial interactions occur, the juxtaparanodes, where voltage-gated potassium channels (VGKCs) are aggregated, and the internode. The integrity of these domains is crucial for the function of the axon and the propagation of axon potentials along its length. Perinodal changes have been reported in Multiple Sclerosis (MS) with functional consequences for the axon. TAG-1 (Transient Axonal Glycoprotein-1 or Contactin2 – Cntn2), both an axonal and glial protein, is also found enriched at juxtaparanodes and is responsible for the recruitment and maintenance of VGKC and Caspr2 at these sites, while it was also identified as an autoantigen in a subset of MS patients. Furthermore, the protein has been assigned with crucial roles in mediating processes such as migration, fasciculation and maturation in a variety of neuronal subtypes in the developing central nervous system (CNS).
In the first part of the study, in an effort to further study its role in myelination, we focused on the role of TAG-1 in demyelination and remyelination in two different murine models. In the first one, the EAE (Experimental Autoimmune Encephalomyelitis) model, we induced demyelination after activation of the immune system directed against a myelin component, driving autoimmunity which results in spinal cord white matter (WM) pathology and associated clinical symptoms. TAG-1 absence resulted in a delay in the development of neurological symptoms, linked to a reduced recruitment of Tregs in the spinal cord. The second model utilizes a toxin (lysophospatidylοcholine - LPC) to induce focal demyelination in the corpus callosum (cc). Following demyelination, TAG-1 affects microglial and astroglial recruitment to the lesioned area, driving also OPC differentiation. Last but not least, in the absence of TAG-1 another mechanism is driven to cluster VGKCs at JXPs during remyelination.
In the second part of our study, we report on alterations of the juxtaparanodal proteins TAG-1, Caspr2 and VGKCs in normal appearing white matter (NAWM), perilesion and chronic lesion areas in post-mortem white matter tissue from MS patients compared to control white matter. We show that the molecular organization and maintenance of JXPs is affected in lesions, perilesions and NAWM in chronic MS through protein and mRNA expression as well as immunohistochemistry. The three molecules analyzed were differentially altered.
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TAG-1 clustering at JXPs was reduced in NAWM; TAG-1 and Caspr2 are diffused in perilesions and absent in lesion areas. VGKCs were no longer enriched at juxtaparanodes either at the NAWM or the perilesion and demyelinated plaques. While the protein levels of the three molecules showed only a tendency of reduction in the plaques, there was a significant upregulation of Caspr2 mRNA in the lesions accompanied by a transcriptional increase of paranodal Caspr, indicating an axonal homeostatic mechanism.
Overall, our study points to novel roles of TAG-1 in demyelination and remyelination in mice, such as a pivotal role in driving autoimmunity, microglial and astroglial recruitment, as well as differentiation of oligodendrocyte precursor cells (OPCs) to mature, myelin-producing, oligodendrocytes (OLs). In chronic MS tissue, the comparative analysis of the juxtaparanodal complex revealed for the first time differential alterations of the three components and a juxtaparanodal vulnerability in NAWM, an area of diffuse pathology lacking demyelination.
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Investigation of the role of the glial isoform of TAG-1 protein in the organization of mammalian myelinated fibers
