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

Abstract

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.