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Tau impacts overall axonal transport particularly when overexpressed by interfering with translocation of kinesin along microtubules (MTs) and/or as a cargo of kinesin by outcompeting other kinesin cargo. To discern between which of these mechanisms was more robust during axonal outgrowth, we overexpressed phosphomimetic (E18; which is incapable of MT binding), phospho-null (A18) or wild-type (WT) full-length human tau conjugated to EGFP, the latter two of which bind MTs. Expression of WT and A18 displayed increased acetylated MTs and resistance to colchicine, while expression of E18 did not, indicating that E18 did not contribute to MT stabilization. Expression of all tau constructs reduced overall levels of neurofilaments (NFs) within axonal neurites, and distribution of NFs along neurite lengths. Since NFs are another prominent cargo of kinesin during axonal neurite outgrowth, this finding is consistent with WT, A18 and E18 inhibiting NF transport to the same extent by competing as cargo of kinesin. These findings indicate that tau can impair axonal transport independently of association with MTs in growing axonal neurites.

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.

Neurofilaments (NFs) are thought to provide structural support to mature axons via crosslinking of cytoskeletal elements mediated by the C-terminal region of the high molecular weight NF subunit (NF-H). Herein, we inhibited NF-H expression in differentiating mouse NB2a/d1 cells with shRNA directed against murine NF-H without affecting other NF subunits, microtubules or actin. shRNA-mediated NF-H knockdown not only in compromised of late-stage axonal neurite stabilization but also compromised early stages of axonal neurite elongation. Expression of exogenous rat NF-H was able to compensate for knockdown of endogenous NF-H and restored the development and stabilization of axonal neurites. This rescue was prevented by simultaneous treatment with shRNA that inhibited both rat and murine NF-H, or by expression of exogenous rat NF-H lacking the C-terminal sidearm during knockdown of endogenous NF-H. Demonstration of a role for NF-H in the early stages of axonal elaboration suggests that axonal stabilization is not delayed until synaptogenesis, but rather that the developing axon undergoes sequential NF-H-mediated stabilization along its length in a proximal–distal manner, which supports continued pathfinding in distal, unstabilized regions.