Covalent crosslinking of neurofilaments in the pathogenesis of n-hexane neuropathy

These studies test the hypothesis that in n-hexane neuropathy the gamma-diketone metabolite 2,5-hexanedione (2,5-HD) results in covalent crosslinking of neurofilaments via nucleophilic attack on oxidized pyrrole rings formed from the reaction of 2,5-HD with epsilon-amino groups of lysyl residues. The 2,5-HD analogue and gamma-diketone,3,4-dimethyl-2,5-hexanedione (DMHD), was found to result in more rapid pyrrole formation, pyrrole autoxidation, and protein crosslinking when compared with 2,5-HD. DMHD was 20-30 times more potent than 2,5-HD in producing hindlimb paralysis. Following 2,5-HD intoxication the neurofilament filled axonal swellings were found in the distal, subterminal axon. After treatment with DMHD, swellings were present in the proximal axon, similar to those seen after intoxication with beta,beta'-iminodipropionitrile (IDPN). DMHD was proposed as a connecting link between the proximal neurofilamentous axonopathy caused by IDPN and the distal neurofilamentous axonopathies from n-hexane, acrylamide, and carbon disulfide intoxication. [14C]DMHD was found to alkylate nerve protein and to result in polymers of radiolabeled protein too large to pass through nitrocellulose filters with pore sizes as large as 12 nm. An even greater proportion of radiolabeled protein was retained by nitrocellulose filters when DMHD was reacted with nerve in which SCa (slow component a of axonal transport) had been pulse-labeled with [35S] methionine. Radiolabeled nerve proteins acylated with [125I]Bolton-Hunter reagent were minimally retained by nitrocellulose filters, suggesting that filter retention reflects polymerization rather than non-specific adsorption.