Effects of HNE-modification induced by Aβ on neprilysin expression and activity in SH-SY5Y cells

The cerebral accumulation of beta-amyloid (A beta) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in A beta accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves A beta in vivo, is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected A beta, SH-SY5Y cells were treated with HNE or A beta, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted A beta levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in A beta 1-40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with A beta 1-42 also induced HNE adduction of NEP. In an apparent compensatory response, A beta-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced A beta cleavage and enhanced A beta accumulation.