Amyloid precursor protein gene mutated at Swedish 670/671 sites in vitro induces changed expression of nicotinic acetylcholine receptors and neurotoxicity

In order to investigate the influence of amyloid precursor protein (APP) over-expression on the levels of nicotinic acetylcholine receptors (nAChRs), the pCDNA 3.0 carrying the Swedish 670/671 APP double mutation (APPSWE) gene was transfected into human neuroblastoma (SH-SY5Y) cells and primary culture of rat hippocampal neurons. The mRNA level of APP, and nAChR α3, α4 and α7 subunits were detected by real-time PCR, and their corresponding proteins as well as α-secreted APP (αAPPs) by Western blotting. [3H]Epibatidine binding sites were measured by the receptor binding assay. The results showed that significantly concomitant increases in mRNA and protein levels of SH-SY5Y cells and primary cultured neurons transfected with APPSWE were observed. Interestingly, a decreased αAPPs level was detected in both cells treated with APPSWE transfection. In addition, decreases in mRNA and protein levels of α3 nAChR subunit in SH-SY5Y cells or α4 subunit in primary cultured neurons with APPSWE transfection were observed. For α7 nAChR, the increased protein and mRNA levels were found in SH-SY5Y cells and primary cultured neurons with APPSWE transfection. The number of cholinergic receptor binding site of [3H]epibatidine was decreased in the SH-SY5Y cells transfected with APPSWE. Elevations in the activities of AChE and BuChE and in the level of lipid peroxidation were detected in both types of cultured cells transfected with APPSWE. These results indicated that the over-expression of APPSWE gene can influence the expression of nAChRs and resulted in neurotoxicity, in which this process might play an important role in the pathogenesis of Alzheimer's disease.

Research highlights▶ Transfection with APPSWE in cultured neuronal cells significantly induces concomitantly increased expressions at mRNA and protein levels of APP. ▶ Over-expression of APP decreases the expressions of α3 and α4 nAChR subunits at protein and mRNA levels, and whereas increases the expression of α7; and increases the activity of AChE and BuChE. ▶ Over-expression of APP decreases αAPPs and MTT reduction, and increases lipid peroxidation.