Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.