Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy.

The present studies were carried out to investigate the comparative roles of protein cross-linking and alteration in protein phosphorylation in the accumulation of neurofilaments due to aliphatic hexacarbons. In these studies, rats were given 2,5-hexanedione (0, 0.1, 0.25 and 1.0%) for 70 days in their drinking water. In a separate study of in vitro protein phosphorylation rats were given 1% 2,5-hexanedione for 14 days in their drinking water. Spinal cord neurofilaments were isolated and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using anti-neurofilament antibodies, radioimmunoassays (RIAs) of phosphorylated epitopes on neurofilament proteins and protein phosphorylation. Protein cross-linking of neurofilaments was found in all animals treated with 2,5-hexanedione including the lowest dose (0.1%) which did not produce clinical signs of intoxication. Protein phosphorylation of neurofilament proteins, as well as MAP-2 was significantly decreased upon treatment. Protein staining revealed a decreased amount of neurofilament protein and immunoblotting demonstrated neurofilament protein cross-linking in these animals. Protein staining of glial fibrillary acidic protein (GFAP) was unaltered by this treatment. RIAs of phosphorylated and non-phosphorylated epitopes of neurofilament proteins indicated that in vivo phosphorylation of these proteins was also decreased. Two-dimensional gel electrophoresis indicated a shift of the neurofilament proteins to a basic pI, indicating a dephosphorylation of neurofilament proteins. Cross-linked neurofilament proteins also exhibited a pI which was more basic than any of the individual neurofilament proteins. This report demonstrates differential effects of 2,5-hexanedione on neurofilament proteins and indicates that several mechanisms may be responsible for their accumulation.