Construction of SH-EP1-a4b2-hAPP695 Cell Line and Effects of Nicotinic Agonists on b-amyloid in the Cells

1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. a4b2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor a4 subunit and b2 subunit gene, to observe effects of a4b2 receptors activation on b-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-a4b2 cells. Function of the transfected a4b2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective a4b2 nicotinic receptor agonist) on b-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using realtime PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1a4b2 cells; Nicotine (1 lM) and epibatidine (0.1 lM) decreased intracellular and secreted b-amyloid in the cells; and activation of a4b2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor a4 subunit and b2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Ab level induced by activation of a4b2 nAChR in our study might occur at a post-translational level.