METAL-CATALYZED OXIDATION OF BOVINE NEUROFILAMENTS IN-VITRO

Neurofilaments (NF) are important determinants of the shape and size o f nerve cells. The oxidation of NF, relevant to aging, neurodegenerati ve disorders, and axonal (Wallerian) degeneration, has not been studie d. In this investigation, we have combined biochemical and ultrastruct ural methods to study the metal-catalyzed oxidation (MCO) of bovine NF using an ascorbate/Fe+3/O-2 system. The oxidation of NF proteins was documented by increases in carbonyl content, which were time- and conc entration-dependent. Polyacrylamide gel electrophoresis (PAGE) acid im munoblot analyses revealed the fragmentation of oxidized NF proteins, predominantly NF-H and NF-M. Electron microscopy (EM) showed that oxid ized NF formed dense aggregates and bundles of laterally aggregated fi laments. Finally, we also demonstrated that oxidized NF proteins were more susceptible to calpain proteolysis. In view of the growing eviden ce supporting increased oxidative stress on the nervous system in agin g and the report of Cu/Zn superoxide dismutase mutation in familial mo tor neuron disease, oxidative injury of NF may be relevant to cell atr ophy and degeneration of nerve cells and to the formation of abnormal cytoskeletal structures.