High Molecular Weight Neurofilament Proteins Are Physiological Substrates of Adduction by the Lipid Peroxidation Product Hydroxynonenal

Protein adducts of the lipid peroxidation product<i>trans</i>-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimer's disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. <i>In vitro</i>, HNE treatment of permeabilized sciatic nerve showed the same specificity, <i>i.e.</i> NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1–33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.