Regulation of the transition from vimentin to neurofilaments during neuronal differentiation

Vimentin (Vm) is initially expressed by nearly all neuronal precursors in vivo, and is replaced by neurofilaments (NFs) shortly after the immature neurons become post-mitotic. Both Vm and NFs can be transiently detected within the same neurite, and Vm is essential for neuritogenesis at least in culture. How neurons effect the orderly transition from expression of Vm as their predominant intermediate filament to NFs remains unclear. We examined this phenomenon within growing axonal neurites of NB2a/d1 cells. Transfection of cells with a construct expressing Vm conjugated to green fluorescent protein confirmed that axonal transport machinery for Vm persisted following the developmental decrease in Vm, but that the amount undergoing transport decreased in parallel to the observed developmental increase in NF transport. Immunoprecipitation from pulse-chase radiolabeled cells demonstrated transient co-precipitation of newly synthesized NF-H with Vm, followed by increasing co-precipitation with NF-L. Immunofluorescent and immuno-electron microscopic analyses demonstrated that some NF and Vm subunits were incorporated into the same filamentous profiles, but that Vm was excluded from the longitudinally-oriented "bundle" of closely-apposed NFs that accumulates within developing axons and is known to undergo slower turnover than individual NFs. These data collectively suggest that developing neurons are able to replace their Vm-rich cytoskeleton with one rich in NFs simply by down-regulation of Vm expression and upregulation of NFs, coupled with turnover of existing Vm filaments and Vm-NF heteropolymers.