Delayed expression of the NFH subunit in differentiating P19 cells.

Pluripotent embryonal carcinoma (P19) cells differentiate into a neural phenotype in response to retinoic acid (RA). Expression of the low and medium molecular weight neurofilament subunits, but not the high molecular weight subunit (NFH), has been reported following RA treatment. In this study NFH expression was detected by Western blotting and immunofluorescence microscopy, but lagged behind the expression of the other subunits in a manner similar to that reported during in vivo neuronal development.

Pathogenetic studies of hexane and carbon disulfide neurotoxicity.

Two commonly employed solvents, n-hexane and carbon disulfide (CS2), although chemically dissimilar, result in identical neurofilament-filled swellings of the distal axon in both the central and peripheral nervous systems. Whereas CS2 is itself a neurotoxicant, hexane requires metabolism to the gamma-diketone, 2,5-hexanedione (HD). Both HD and CS2 react with protein amino functions to yield initial adducts (pyrrolyl or dithiocarbamate derivatives, respectively), which then undergo oxidation or decomposition to an electrophile (oxidized pyrrole ring or isothiocyanate), that then reacts with protein nucleophiles to result in protein cross-linking. It is postulated that progressive cross-linking of the stable neurofilament during its anterograde transport in the longest axons ultimately results in the accumulation of neurofilaments within axonal swellings. Reaction with additional targets appears to be responsible for the degeneration of the axon distal to the swellings.

Dimethylhexanedione impairs the movement of neurofilament protein subunits, NFM and NFL, in the optic system.

Exposure to the neurotoxic gamma-diketone 3,4-dimethyl-2,5-hexanedione (DMHD) leads to the accumulation of neurofilaments within the proximal axon and to an inhibition in the rate of anterograde transport of recently synthesized neurofilaments. These effects of DMHD are similar to those of the neurotoxic nitrile 3,3'-iminodipropionitrile (IDPN), which is also characterized by formation of neurofilamentous swellings within the proximal axon and an inhibition of transport, both of newly synthesized neurofilaments and those already in transit in more distal regions of the axon. Due to the similarities between these compounds, DMHD also might be expected to inhibit neurofilament transport in the distal axon. The objective of this study was to examine the effects of DMHD on the movement of labeled neurofilament proteins which were in transit at the time of intoxication. Proteins in the optic system, pulse labeled with 35S-methionine, underwent transport for two weeks prior to the start of intoxication. Neurofilament transport was assessed by SDS-PAGE fluorography and computerized densitometry. At two and five weeks in control animals, the peaks of NFL and NFM neurofilament subunits had broadened and flattened from their original proximal location and assumed a more uniform, proximodistal distribution (peak dispersion). In contrast, in DMHD-treated animals, the radiolabeled NFL and NFM remained near their position at the start of intoxication, retaining a peak of radiolabeled protein. A proportion of each of the subunits, however, had entered the distal axon during intoxication suggesting that a population of filaments may remain transport competent.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure.

The neurotoxicant 2,5-hexanedione (HD) causes the accumulation of neurofilaments in the distal axon and an acceleration of neurofilament transport proximal to the site of their accumulation. It has been proposed that the acceleration of transport is due to the direct reaction of HD with neurofilament proteins and, conversely, that this acceleration is a secondary response to the axon to injury. The objective of this study was to determine whether the response of axons to HD intoxication includes acceleration of neurofilament transport. Pulse labelling was used to analyze neurofilament transport in age-matched rats exposed to HD or PBS. The animals receiving HD were exposed either throughout the period of radiolabel transport, or prior to the pulse labeling of neurofilament proteins. If acceleration of the rate of neurofilament transport was due to the direct reaction of HD with proteins, then neurofilaments synthesized after the exposure period should travel at control rates, since these proteins would not have been exposed to the toxicant. After 28 days of transport, optic nerve proteins were examined using SDS-PAGE, fluorography, and computerized densitometry. In both HD-treated groups, neurofilament transport was accelerated relative to age-matched control animals. In addition, the amount of NFH was decreased relative to other neurofilament subunits. The combination of accelerated transport and a diminished proportion of NFH is similar to the observations of neurofilament axonal transport during growth and development. These observations suggest that this persistent, secondary effect is a reparative response to injury that recapitulates axonal growth and development.

The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione.

Exposure to the gamma-diketone, 2,5-hexanedione (HD), results in the accumulation of neurofilaments within the distal axon and is associated with acceleration of neurofilament transport within the proximal axon. The epsilon-amino groups of lysyl residues react with HD forming pyrrole adducts, followed by pyrrole-mediated protein crosslinking. Both reaction steps have been proposed as mechanisms causing neurofilament accumulation and acceleration of transport. In order to assess the importance of these steps on neurofilament transport, we compared transport in the optic system of rats exposed to HD and 3-acetyl-2,5-hexanedione (AcHD), a non-toxic analog of HD which forms pyrroles but does not crosslink proteins. Control, HD-treated, and AcHD-treated rats received intraoptic injections of [35S]-methionine and were exposed to saline, HD, or AcHD by intraperitoneal injections before and during the period of neurofilament transport. Neurofilament triplet proteins in the optic nerve and tract were identified by polyacrylamide gel electrophoresis followed by fluorography. The rate of neurofilament transport was accelerated in HD-treated animals over that of controls. However, despite higher levels of protein-bound pyrroles in AcHD-treated animals, the rate of transport was indistinguishable from that of controls. These findings indicate that pyrrole formation alone is not sufficient to cause acceleration of neurofilament transport.