Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695

Abstract

Alpha-secretase (α-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-β (Aβ) sequence, resulting in the release of a secreted fragment of APP (αAPPs) and precluding Aβ generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Aβ generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0–10 μM) increased αAPPs release. Therefore, we evaluated two α-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-α converting enzyme (TACE)/ADAM17 inhibited the Hup A–induced rise in αAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic α-secretase pathway. Hup A had no effect on Aβ generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A–induced αAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer’s disease (AD).