The single neurofilament subunit of the lamprey forms filaments and regulates axonal caliber and neuronal size in vivo

Abstract

Neurofilaments (NFs) are composed of a heteropolymer of three related subunits in mammalian neurons, where they are a major component of the cytoskeleton in large neurons and are thought to regulate axonal diameter. NFs in the lamprey, while ultrastructurally and functionally indistinguishable from mammalian NFs, are polymers of a single subunit protein, NF180. In this study, we use the simplicity of lamprey NFs and the accessibility of the lamprey central nervous system (CNS) to examine the effects of overproducing NFs in an identified giant neuron in vivo, and thus to elucidate the role of NFs in regulating neuronal size and axonal caliber in the vertebrate CNS. We show that overexpression of NF180 tagged with a variant of Green Fluorescent Protein (EYFP) in identified lamprey neurons (ABCs) and in human neuroblastoma (NB2a) cells results in the assembly of exogenous NF180 into ultrastructurally normal NFs that are tightly packed and unphosphorylated. These accumulate in the somata of NB2a cells and produce somatic swelling by 3 days post-transfection. NF180 overexpression in lamprey ABCs in vivo causes exogenous NFs to accumulate in ABC axons, somata, and dendrites, and induces a significant increase in axonal diameter without increasing axonal NF packing density. Overexpression of EYFP alone has none of these effects. We conclude that NF180 normally plays a critical role in determining axonal caliber in ABCs and may influence neuronal size in situations where NFs accumulate in the soma, such as after axonal injury.