Abstract
Protein adducts of the lipid peroxidation product trans-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. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. 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.
Highlights
One of the most striking and earliest changes noted with the development of Alzheimer’s disease (AD)1 is that cell bodies of vulnerable neurons uniformly show increased oxidative damage
These studies indicate that HNE adduction to neurofilament heavy subunit (NFH) is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and neurofilament medium subunit (NFM) modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage
Because preliminary immunoblotting studies of the brain and peripheral nerve of human, rat, rabbit, and mouse showed that the only proteins displaying readily detectable HNE adducts are neurofilament heavy subunit (NFH) and neurofilament medium subunit (NFM), we decided to focus the analysis on the axons of the sciatic nerve as a natural site rich in NFH
Summary
Specificity of the various antibodies was verified in all cases by immunoabsorption for 16 h at 4 °C with the competing antigens or to adducts prepared from the reaction of HNE with lysine, cysteine, or histidine at a 1:1 concentration of 0.8 mM Reaction of the latter two amino acids with HNE gives the corresponding Michael adduct nearly stoichiometrically, whereas the reaction of HNE with lysine leads to a time-dependent mixture of mainly the reversibly formed Michael adduct and several additional adducts derived from initial Schiff base formation at the C1 carbonyl group of HNE [9]. Equal protein loads were run on SDS-PAGE, electrotransferred to Immobilon, and immunoblotted with antisera to HNE-Michael, HNE-pyrrole, HNE-lysine-lysine, or monoclonal antibody SMI-34. Samples were separated on PAGE, electrotransferred to Immobilon (Millipore), and probed with antisera to HNE-Michael and monoclonal antibodies against phosphorylated and nonphosphorylated NFH. Quantitative analysis was performed by measuring the intensity of immunostained NFH bands by using digital scanning and KS300 software (Zeiss)
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