Preventing expression of the nicotinic receptor subunit α7 in SH-SY5Y cells with interference RNA indicates that this receptor may protect against the neurotoxicity of Aß.

The present aim was to characterize the influence of the α7 nicotinic acetylcholine receptor (nAChR) on BACE, the enzyme that cleaves the amyloid precursor protein (APP) at the ß-site, as well as on the oxidative stress induced by amyloid-ß peptide (Aß). To this end, human neuroblastoma SH-SY5Y cells were transfected with siRNAs targeting the α7 nAChR subunit and/or exposed to Aß1-42. For α7 nAChR, BACE1 (cleaving at the ß-site of APP) and BACE2 (cleaving within the Aß domain), α-secretase (ADAM10), and the two components of γ-secretase, PS and NCT, the mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. The level of Aß1-42 in the cell culture medium was determined by an ELISA procedure. The extent of lipid peroxidation and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were assayed spectrophotometrically. In the transfected SH-SY5Y cells, expression of α7 nAChR was reduced; the level of BACE1 increased and that of BACE2 decreased; the amount of ADAM10 lowered; and the level of PS raised. Moreover, the level of Aß1-42 in the culture medium was elevated. Treatment of non-transfected cells with Aß elevated the level of malondialdehyde (MDA) and lowered the activities of SOD and GSH-Px and these changes were potentiated by inhibiting expression of α7 nAChR. These results indicate that α7 nAChR plays a significant role in amyloidogenic metabolism of APP and the oxidative stress evoked by Aß, suggesting that this receptor might help protect against the neurotoxicity of Aß.

The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-ß peptide and ß-secretase.

To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of ß-secretase and the concomitant level of amyloid-ß (Aß), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the ß-site of APP) and BACE2 (cleaving in the Aß domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aß(1-42) in culture media were determined by an Elisa procedure. In SH-SY5Y cells transfected with siRNA, the levels of α3 nAChR mRNA and protein were reduced by 96% and 88%, respectively; the levels of BACE1 mRNA and protein were significantly enhanced, while those of BACE2 were reduced; and the level of Aß in the culture medium was elevated. In contrast, when untransfected SH-SY5Y cells were exposed to nicotine, the levels of both α3 nAChR mRNA and protein were enhanced; while the levels of BACE1 mRNA and protein were diminished and the corresponding levels of BACE2 enhanced; and the level of Aß in the culture medium was attenuated. These results indicate that the α3 subunit of nAChR inhibits the production of Aß by reducing the expression of BACE1 and elevating the expression of BACE2, suggesting that this subunit might play an important neuroprotective role in connection with the pathogenesis of AD.