Early expression of the high molecular weight neurofilament subunit attenuates axonal neurite outgrowth

Phospho-dependent interactions of the C-terminal region of the high molecular weight NF subunit (NF-H) with each other and with other cytoskeletal elements stabilize the axonal cytoskeleton and contribute to an increase in axonal caliber. The same kinase cascades that mediate axonal pathfinding via growth cone dynamics are those that foster NF-mediated axonal stabilization, yet there is a developmental delay in the accumulation of NF C-terminal phosphorylation. Moreover, the phospho-mediated C-terminal NF-H interactions that stabilize the axonal cytoskeleton also inhibit axonal elongation. We hypothesized that a delay in expression and/or accumulation of NF-H within developing axons is essential to allow axonal elongation and pathfinding. We tested this hypothesis in differentiating NB2a/d1 cells. The first 3 days of differentiation of NB2a/d1 cells is normally accompanied by rapid elongation of axonal neurites. This period is followed by the accumulation of C-terminally phosphorylated NF-H, cessation of axonal elongation and an increase in axonal caliber. Herein, overexpression of GFP-tagged NF-H simultaneously with induction of differentiation fostered accumulation of C-terminally phosphorylated NF-H within developing axonal neurites within 48hr, which was accompanied by retardation of axonal elongation and a hastened increase in caliber. These effects were prevented by treatment with inhibitors of kinases that mediate the association of NFs with other cytoskeletal elements. Overexpression of GFP-NF-H lacking the C-terminal 187 amino acids (which mediate NF-NF interactions) did not retard elongation nor increase caliber. These findings support the hypothesis that a developmental delay in NF-H C-terminal phosphorylation is essential to allow appropriate axonal elongation prior to stabilization.