Construction of SH-EP1-alpha4beta2-hAPP695 cell line and effects of nicotinic agonists on beta-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. alpha4beta2 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 alpha4 subunit and beta2 subunit gene, to observe effects of alpha4beta2 receptors activation on beta-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-alpha4beta2 cells. Function of the transfected alpha4beta2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective alpha4beta2 nicotinic receptor agonist) on beta-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-alpha4beta2 cells; Nicotine (1 muM) and epibatidine (0.1 muM) decreased intracellular and secreted beta-amyloid in the cells; and activation of alpha4beta2 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 alpha4 subunit and beta2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer's disease. Alteration of Abeta level induced by activation of alpha4beta2 nAChR in our study might occur at a post-translational level.
Nie
Huizhen
H
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Li
Zuoqing
Z
Lukas
Ronald J
RJ
Shen
Yinghua
Y
Song
Li
L
Wang
Xin
X
Yin
Ming
M
D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002999:Clone Cells; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D018722:Nicotinic Agonists / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D015203:Reproducibility of Results
D016564:Amyloid beta-Protein Precursor; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; C093598:amyloid beta-protein precursor 695 (681-695); C403531:nicotinic receptor alpha4beta2