Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro

Abstract

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.

The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

“…We found that these neurons express NFL, NFM, peripherin and α-internexin but no detectable NFH. The absence of NFH in these cultures is consistent with previous observations that NFH expression remains at low levels in most neurons until after birth [Shaw and Weber, 1982;Carden et al, 1987] and that this delayed expression in vivo is recapitulated in cell culture [Lee, 1985;Shaw et al, 1985;Foster et al, 1987;Breen and Anderton, 1991;Benson et al, 1996].…”

Dynamics of neuronal intermediate filaments: A developmental perspective

“…Thus, NF-H subunits mediate the establishment of a stable supporting structure that prevents the rapid retraction of the neurite [Shea et al, 1991bl. Increased neurofilament gene expression is followed by a gradual and complex series of phosphate additions to the C-terminal tails of NF-H and NF-M, which begin during the period when axons expand radially to their full size and myelination is proceeding [Foster et al, 1987;Dahl et al, 1986;Bignami and Dahl, 1987;Carden et al, 19871. These phosphorylation events appear to be intimately involved in the formation of the stationary network of neurofilaments and other cytoskeletal proteins that predominates in central axons [Nixon, 1987;Nixon and Logvinenko, 19861 and likely contributes to stabilization of axon structure [Dahl, 1988;Bignami and Dahl, 19871 and, together with increased neurofilament synthesis and export [Hoffman et al, 1984, 19871, to the radial growth of the axon.…”

Partial sequence of the rat heavy neurofilament polypeptide (NF‐H) Identification of putative phosphorylation sites

“…Neurofilament phosphorylation has been shown to be related to the neuronal calibre [19] and is very specifically developmentally regulated [2,20]. In the perikarya and developing axons, NF-H is in a relatively unphosphorylated form.…”

“…70 kDa (NF-L), 160 kDa (NF-M) and 200 kDa (NF-H) and are involved in the maintenance of neuronal caliber [1]. Phosphorylation probably plays a major role in the functioning of the two larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in neurofilament function [2,3]. Both NF-M and NF-H contain a heavily phosphorylated carboxy region which may influence the formation of filament-filament axonal cross bridges [4,5].…”