Maturation of the peripheral nervous system requires establishment of axonal diameter which has a significant influence on the rate of signal propagation along the axon. Expansion of axonal diameter is referred to as radial growth and is dependent upon both myelination and neurofilaments. Neurofilaments (NFs) are the most abundantly expressed cytoskeletal proteins in myelinated axons and consist of neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits. NF-M and NF-H were hypothesized to regulate axonal diameter through myelin-dependent phosphorylation of lysine-serine-proline (KSP) repeats. Gene deletion of NF-L suggested that NF-L is required for assembly of NF subunits into mature NFs. NF-H gene deletion resulted in small reductions in axonal diameter suggesting that NF-H was dispensable for radial axonal growth. Gene deletion of NF-M and its carboxy terminus (C-terminus) resulted in substantial reductions in axonal diameter and reduced nerve conduction velocity. However, prevention of NF-M KSP phosphorylation did not recapitulate the reduction in axonal diameter that was observed in NF-M C-terminal deleted mice. While NF-M was identified as the critical subunit and its C-terminus as the critical domain for radial axonal growth, KSP phosphorylation was not required. Therefore, the mechanism by which the NF-M C-terminus mediated radial axonal growth remained unresolved. It also remained unclear the role of NF-H expression in radial axonal growth.
Studies on radial axonal growth in NF-H gene deleted (NF-H-/- ) were limited to only the most proximal axonal regions of peripheral nerves. To address the role of NF-H expression in radial growth of peripheral axons, I analyzed radial growth of axonal segments along the length of the phrenic nerve in NF-H-/- mice. The analysis indicated that axonal diameter was reduced along the entire length of the phrenic nerve with only the most distal segments being unaffected. NF number was reduced in the proximal axonal segments and was unaffected in the most distal segment of the phrenic nerve. Taking into consideration previous observations, the current analysis of NF-H-/- mice suggest that distal to proximal development of peripheral axons requires NF-H expression. Distal axonal segments underwent radial growth prior to proximal segments. As NF-H expression increases, NFs accumulate and radial growth occurs in more proximal axonal segments which proceeds radial growth in distal segments.
To better understand the function of the NF-M C-terminus in mediating radial axonal growth, I performed phylogenetic sequencing analysis of the NF-M C-terminus across several clades of mammals. Sequencing analysis suggested that the C-terminus is divided into three sub-domains, two highly conserved regions flanking a highly variable region. Independent expansion events of the NF-M C-terminus occurred within multiple clades of mammals. All observed expansion events occurred by the addition of amino acids, including KSP repeats, in the variable sub-domain. NF-M C-terminal length correlated with axonal diameter in a subset of mammals. These data suggested that expansion of the NF-M C-terminus through addition of KSP repeats might have resulted in larger axonal diameters during mammalian evolution.
To further test whether expansion of the NF-M C-terminus increases axonal diameter, I performed gene replacement in mice to increase the length of the NF-M C-terminus. The endogenous NF-M C-terminus was replaced with the longer bovine NF-M C-terminus. Expression of the expanded NF-M C-terminus did not affect expression of NF-L or NF-H subunits and resulted in reduced NF-H phosphorylation. Increasing the length of the NF-M C-terminus increased radial axonal growth of large motor axons in mice. Despite increased axonal diameter, nerve conduction velocity was unaffected possibly due to a lack of increased myelin thickness. Failure of myelin to compensate suggest a lack of plasticity during the processes of myelination and radial axonal growth.
My dissertation has provided greater understanding of the mechanisms by which NF-H and NF-M regulate radial growth of axons. NF-H expression was developmentally delayed to facilitate distal to proximal radial growth of peripheral axons during postnatal development. Expansion of the NF-M C-terminus during mammalian evolution may have been a mechanism to increase radial axonal growth as larger mammals evolved. Expansion of the NF-M C-terminus in mice suggested that the length of the NF-M C-terminus determines the magnitude of radial growth in large motor axons.
|Advisor:||Garcia, Michael L.|
|School:||University of Missouri - Columbia|
|School Location:||United States -- Missouri|
|Source:||DAI-B 74/03(E), Dissertation Abstracts International|
|Keywords:||Axonal growth, Myelin, Neurofilaments, Radial growth|
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