Role of the Neurofascins in targeting voltage-gated sodium channels in myelinated nerves
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The nodes of Ranvier are short, periodical interruptions in the myelin sheath of myelinated axons, at which voltage-gated sodium channels are highly concentrated. The correct targeting of sodium channels to the nodes of Ranvier permits rapid propagation of action potentials in myelinated axons. The nodes of Ranvier contain a unique set of ion channels, cell-adhesion molecules, and cytoplasmic adaptor proteins. Neurofascins are cell adhesion molecules of the immunoglobulin superfamily and previous work has shown they are involved in the assembly of the node of Ranvier. The Neurofascin (Nfasc) gene is subject to extensive alternative splicing. RT-PCR studies have suggested that there were several different Neurofascin (Nfasc) transcripts. Thus far, research on the Neurofascins has concentrated on two isoforms, Nfasc186 and Nfasc155, which are expressed in neurons and glia respectively. A third Neurofascin isoform, Nfasc140, lacking the Mucin domain and two of the fibronectin repeats was originally identified in the laboratory of V. Bennett. However, neither the location nor function of this protein was known. By RT-PCR I successfully cloned the Nfasc140 cDNA and determined its domain composition, which was confirmed by a series of Western blots using domain-specific antibodies. The developmental expression of Nfasc140 revealed that it is the predominate isoform of Neurofascin during the embryonic stage. Using cell-type-specific conditional Neurofascin knock-out mice, I have also found that Nfasc140 is a neuronal isoform, like Nfasc186. I have used transgenic mouse lines to characterize the location and function of Nfasc140. Like Nfasc186, Nfasc140 is targeted to the nodes of Ranvier and axonal initial segment. Also Nfasc140 alone can reconstitute the nodal complex in Neurofascin knock-out mice in CNS and PNS in the absence of Nfasc186 and Nfasc155. It can also partially restore the electrophysiological function of PNS nerves. In order to address the role of the paranodes in sodium channel clustering, I generated a new neuronal-Cre-expressing transgenic line which, when bred with floxed Nfasc mice, generated early neuronal Neurofascin knock-out mice. Using those animals I have shown that after the ablation of all neuronal Neurofascins, when only glial Nfasc155 is presented, sodium channels can still target to the nodes of Ranvier in both PNS and CNS. These conditional knock-out mice have a longer life span than pan-Neurofascin knock-out mice. This indicates the importance of paranodal junctions, in addition to nodal neuronal Neurofascins, in clustering sodium channels at the node.