Deletion of the Thrombin Proteolytic Site in Neurofascin 155 Causes Disruption of Nodal and Paranodal Organization.

Dipankar J Dutta,R Douglas Fields

doi:10.3389/fncel.2021.576609

Abstract

In the central nervous system, myelin is attached to the axon in the paranodal region by a trimolecular complex of Neurofascin155 (NF155) in the myelin membrane, interacting with Caspr1 and Contactin1 on the axolemma. Alternative splicing of a single Neurofascin transcript generates several different Neurofascins expressed by several cell types, but NF155, which is expressed by oligodendrocytes, contains a domain in the third fibronectinIII-like region of the molecule that is unique. The immunoglobulin 5–6 domain of NF155 is essential for binding to Contactin1, but less is known about the functions of the NF155-unique third fibronectinIII-like domain. Mutations and autoantibodies to this region are associated with several neurodevelopmental and demyelinating nervous system disorders. Here we used Crispr-Cas9 gene editing to delete a 9 bp sequence of NF155 in this unique domain, which has recently been identified as a thrombin binding site and implicated in plasticity of the myelin sheath. This small deletion results in dysmyelination, eversion of paranodal loops of myelin, substantial enlargement of the nodal gap, a complete loss of paranodal septate junctions, and mislocalization of Caspr1 and nodal sodium channels. The animals exhibit tremor and ataxia, and biochemical and mass spectrometric analysis indicates that while NF155 is transcribed and spliced normally, the NF155 protein is subsequently degraded, resulting in loss of the full length 155 kDa native protein. These findings reveal that this 9 bp region of NF155 in its unique third fibronectinIII-like domain is essential for stability of the protein.

Highlights

Neurofascins are a family of cell-surface proteins of the immunoglobulin superfamily generated by alternative splicing of a single Neurofascin (NF) transcript (Hassel et al, 1997)
Neurofascin 155 (NF155) is a member of the L1-CAM family of cell adhesion molecules, with 6 immunoglobulin-like extracellular domains and four fibronectin type III-like (FNIII) extracellular regions anchored to the membrane by a short transmembrane segment (Hortsch, 1996)
Having determined the exact coordinates of the NF gene corresponding to the thrombin cleavage site in the NF155 protein, we proceeded to determine in silico, the putative effects of various mutations and deletions in this site to NF155 structure

Summary

Introduction

Neurofascins are a family of cell-surface proteins of the immunoglobulin superfamily generated by alternative splicing of a single Neurofascin (NF) transcript (Hassel et al, 1997). In the CNS, oligodendroglial Neurofascin 155 (NF155) interacts with the axonal Caspr1-Contactin complex to form septate-like cell-adhesion junctions that attach uncompacted loops of myelin to the paranodal axon. These junctions are essential for neural impulse conduction by securing the uncompacted. NF155 is a member of the L1-CAM family of cell adhesion molecules, with 6 immunoglobulin-like extracellular domains and four fibronectin type III-like (FNIII) extracellular regions anchored to the membrane by a short transmembrane segment (Hortsch, 1996). NF155 is produced by alternative splicing of Neurofascin into a NF186 form, expressed on neurons, and NF155, expressed by oligodendrocytes and localized to the paranodal region of myelinated axons (Davis et al, 1996). Binding of QKi to an RNA element (QRE2) in Nfasc intron 21 is required to promote inclusion of exons 21/22, which encodes the third FNIII-like domain that is unique to NF155 (Darbelli et al, 2016)

Methods

Results

Conclusion