Determining the mechanism of NF C-terminal mediated radial growth of myelinated axons
Authors:   , Downer Natalie L. 
Published: May 2024

Expansion of axonal diameter, referred to as radial growth, is important for rapid signal propagation along an axon. For myelinated axons, the diameter of the myelinated segments, referred to as the internodes, is increased relative to the unmyelinated segments. Neurofilaments (NFs) are the major cytoskeleton proteins in myelinated axons and consist of neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits. Upon myelination, NF-M and NF-H carboxy termini (C-termini) are phosphorylated on conserved lysine-serine-proline (KSP) repeats. It has been hypothesized that myelin-dependent phosphorylation of NF C-termini is required for radial axonal growth. Deletion of the entire NF-M C-terminus resulted in a significant decrease in axonal diameter as well as conduction velocity. However, a direct test of the phosphorylation hypothesis showed that phosphorylation of NF-M C-terminal KSP repeats was not essential for radial growth. These results suggested that the C-terminus of NF-M specifies radial growth and influences conduction velocity by a mechanism that was independent of KSP phosphorylation. Recent results suggested that the overall length of the C-terminus is required for radial growth as expansion of NF-M C-terminus resulted in an increase in the magnitude of radial growth. Gene targeted mice has given significant insight into the effects of NF-M Cterminus on radial growth. However, these analyses were performed primarily on motor axons. Due to the limited analysis of sensory axons, the contribution of NFs to radial growth of sensory axons is unclear. Therefore, I extended analysis of radial growth in sensory axons of existing mouse lines that express genetic modifications to NF-M Cterminus. Our results suggest that increased axonal diameter in sensory axons was selected for through a mechanism that was different from motor axons and was independent of NF C-termini. Molecular phylogenetic analysis suggested that the NF-M C-terminus was not a single domain, but instead, consisted of three sub-domains. Moreover, sequence analysis and mouse genetics have suggested that longer NF-M C-termini are associated with larger axon diameters. It was, therefore, hypothesized that lengthening the NF-M Cterminus was a mechanism utilized to increase axonal diameter and conduction velocity as mammals evolved larger body plans. To provide a direct test of this hypothesis in which the overall length of NF-M Cterminus was reduced, I generated a novel line of genetically modified mice in which gene replacement was applied to delete the first highly conserved NF-M C-terminal subdomain. This region of the NF-M C-terminus constitutes 85 amino acids that occur NH2- terminal to the KSP repeat sub-domain. Decreasing the length of NF-M C-terminus in mice increased axonal diameter in large motor axons and resulted in compensatory increases in other NF subunits. However, increased axonal diameter did not increase conduction velocity possibly due to lack of increased myelin thickness. As expected, mice expressing NF-M in which the first highly conserved C-terminal sub-domain was deleted did not show any behavioral deficits in response to noxious stimuli. These analyses suggested that the overall length of NF-M C-terminus is not required for radial growth.