RESEARCH SUPPORT, U.S. GOV'T, P.H.S.
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Neurofilament protein crosslinking in gamma-diketone neuropathy: in vitro and in vivo studies using the seaworm myxicola infundibulum.

Neurofilament (NF) protein crosslinking has been proposed as the ultimate pathogenetic mechanism underlying the neuropathies caused by the gamma-diketones 2,5-hexanedione (HD) and 3,4-dimethyl-2,5-hexanedione (DMHD). Mammalian models have been used to investigate this hypothesis, but alternative experimental models are needed. Myxicola infundibulum is a marine worm which is gaining popularity in neuroscience research because of its large syncytial axon. A model system using Myxicola has been developed to investigate NF crosslinking in worms exposed to neurotoxic agents whose putative mechanisms involve covalent crosslinking of NF proteins. In vitro studies using purified NF demonstrate that progressive alkylation of Myxicola NF with [2,5-14C]DMHD is accompanied by NF protein crosslinking. Rabbit anti-Myxicola NF antisera showed highly restricted activity for Myxicola axoplasm and NF and were employed for immunoblotting axoplasm from Myxicola treated in vivo with DMHD. A dramatic increase in high molecular weight material was demonstrated in the axoplasm of treated worms, as demonstrated by polyacrylamide gel electrophoresis, and the new high molecular weight bands stained with the anti-NF antisera, indicating the presence of anti-NF reactive material in the crosslinked protein. Further, there was progression of crosslinking after cessation of exposure in vivo, an observation which suggests oxidation of remaining pyrrolyl derivatives. These studies support previous observations which suggest that NF crosslinking is the molecular event which initiates NF aggregation in gamma-diketone neurotoxicity, and establish Myxicola infundibulum as a useful species in which to study certain neurotoxic compounds.

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