Assembly and turnover of neurofilaments in growing axonal neurites

Abstract

Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered.

Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics

“…They are distinguished from small bundles of NFs that normally occur in neurons. They also differ from the NF bundles described by others that are enriched in phosphorylated NFH and immunoreactive with monoclonal antibody RT97 (43,44) in being hypophosphorylated (7).…”

Intermediate filaments in developing neurons: Beyond structure

“…The majority of hippocampal neurons express some NFs (NF‐L, ‐M, and ‐I/INA) by DIV3, but expression patterns are heterogeneous. Interestingly, IFs are enriched in the distal regions of very early (DIV 1) growing neurites in cultured neurons, suggesting possible roles of IFs in axon growth and growth cone responses (Figure ) (Bott et al, ; Boumil, Vohnoutka, Lee, Pant, & Shea, ; Okabe, Miyasaka, & Hirokawa, ; Uchida & Brown, ; Yan, Yang, Bian, & Jing, ). What then is the evidence for IF roles in neurodevelopment, including axon growth?…”

“…It was initially shown that early axons are mainly composed of microtubules with only low levels of NF-M and NF-L before axons reach their synaptic targets (Perrot et al, 2008). Recent work showed that cortical neurons express VIM and nestin at 24 hr in culture, but expression of these IF subtypes is transient, but some adult neurons continue to express VIM and nestin (Bott et al, 2019a;Guo et al, 2014;Hendrickson et al, 2011;Su et al, 2013;Zhu et al, 2011) (Figure 3) (Bott et al, 2019a;Boumil, Vohnoutka, Lee, Pant, & Shea, 2018;Okabe, Miyasaka, & Hirokawa, 1993;Uchida & Brown, 2004;Yan, Yang, Bian, & Jing, 2001). What then is the evidence for IF roles in neurodevelopment, including axon growth?…”

Neurofilaments form flexible bundles during neuritogenesis in culture and in mature axons in situ

“…The early stages of neuritogenesis rely on transient expression of vimentin followed by alpha‐internexin (Boyne, Fischer, & Shea, ; Shea & Beermann, ; Shea, Paskevich, & Beermann, ; Yabe et al, ). Continued axonal maturation is accompanied by the appearance of neurofilaments (NFs), which are the major intermediate filament of mature neurons (Boumil, Vohnoutka, Lee, & Shea, ; de Waegh, Lee, & Brady, ; Lee & Shea, ; Shea & Beermann, ; Shea & Lee, ; Yuan, Rao, Veeranna, & Nixon, ). Unlike vimentin, which is downregulated during progressive neuritic outgrowth and maturation (Yabe et al, ), alpha‐internexin remains functionally associated with NFs (Yuan et al, ).…”

“…Extensive phosphorylation induces cation‐dependent NF‐NF associations that compete with transport and induce formation of NF bundles (Boumil, Vohnoutka, Lee, Pant, & Shea, ; Kushkuley et al, ; Lee, Sunil, & Shea, ; Shea & Lee, ; Sunil, Lee, & Shea, ; Yabe, Chan, Chylinski, Pimenta, & Shea, ). Bundles are likely to provide support to developing and mature axonal neurites (Boumil et al, ; Lee et al, ; Yuan et al, ). NFs are present within the first few days of neuritogenesis and are thought to provide progressive proximal‐distal support during axonal pathfinding (Lee & Shea, ).…”