We previously reported that the thiol proteinase inhibitor, E-64-d, ameliorated amyloid beta (A beta)-induced reduction of soluble amyloid precursor protein alpha (sAPP alpha) secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells. In the present study, we showed that A beta (1-42) peptide enhanced diacylglycerol (DAG) production by phospholipase D (PLD) activation in these cells. We subsequently examined whether PLD was involved in A beta-induced reduction of sAPP alpha secretion and showed that 2 mu M CAY10593, which selectively inhibits PLD2, ameliorated reduction of sAPP alpha secretion, whereas 50 nM CAY10593, which selectively inhibits PLD1, did not. Moreover, 50 mu M propranolol, a phosphatidic acid phosphohydrolase inhibitor, also ameliorated A beta-induced reduction of sAPPa secretion, suggesting that DAG may be responsible for A beta-induced reduction of sAPP alpha. We subsequently examined whether DAG affects sAPPa secretion and showed that a DAG analog reduced sAPP alpha secretion in SH-SY5Y cells. In addition, DAG enhanced ceramide production by stimulating neutral sphingomyelinase (N-SMase) activity. We previously demonstrated that A beta stimulates N-SMase activity in SH-SY5Y cells. Here, we showed that inhibition of PLD2 by 2 mu M CAY10593 suppressed A beta-induced N-SMase activation. Taken together, the results suggest that DAG produced through the PLD pathway is involved in A beta-induced reduction of sAPP alpha secretion in SH-SY5Y cells. (C) 2014 Elsevier Inc. All rights reserved.