Expression levels of the α7 nicotinic acetylcholine receptor in the brains of patients with Alzheimer's disease and their effect on synaptic proteins in SH-SY5Y cells
Expression levels of the α7 nicotinic acetylcholine receptor in the brains of patients with Alzheimer's disease and their effect on synaptic proteins in SH-SY5Y cells
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阿尔茨海默病患者大脑α7烟碱乙酰胆碱受体的表达水平及其对SH-SY5Y细胞突触蛋白的影响
DOI：
10.3892/mmr.2020.11253
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发表时间：
2020-09-01
影响因子：
3.4
通讯作者：
Qi, Xiao-Lan
中科院分区：
文献类型：
--
作者：
Ren, Jia-Mou;Zhang, Shu-Li;Qi, Xiao-Lan
阿尔茨海默病(AD)是一种慢性神经退行性疾病，神经毒性β淀粉样蛋白(Aβ)多肽的异常聚集是AD的早期事件。本研究旨在确定AD患者脑内烟碱型乙酰胆碱受体α7亚单位(α7 nAChR)与Aβ的相关性，并探讨α7 nAChR表达水平的升高是否能改变Aβ的神经毒性。用免疫荧光法分析阿尔茨海默病患者和正常脑组织中α7、nAChR和Aβ的表达水平。此外，SH-SY5Y细胞被用来稳定地过表达或沉默α7nAChR的表达水平，在加或不加1mU摩尔/L Aβ(1-42)寡聚体(AβO)之前。采用逆转录-定量聚合酶链式反应和免疫印迹技术分析α7-nAChR、突触素(SYP)、突触后密度(PSD-95)和突触体相关蛋白(SNAP-25)的基因和蛋白表达水平。用分光光度法检测细胞内乙酰胆碱(ACh)浓度和乙酰胆碱酯酶(AChE)活性，用流式细胞仪检测细胞凋亡率。与健康对照组比较，AD患者脑内Aβ蛋白表达显著增加，而α7 nAChR蛋白表达显著降低。在体外，α7 nAChR的表达水平分别随着该基因的过度表达或沉默而显著增加或降低。与这些观察结果一致的是，SYP、PSD-95和SNAP-25的mRNA和蛋白表达水平也随着α7 nAChR的过表达而显著增加，而随着受体的遗传沉默而降低。在未转染或阴性对照细胞中，这些因子的表达水平和凋亡率在暴露于AβO后显著降低，α7 nAChR过表达可减弱这些因子的表达，而α7 nAChR RNA沉默可增强其凋亡率。然而，无论是ACh浓度还是AChE活性，转染组均未见明显差异。综上所述，这些发现提示，α7 nAChR可能对AD患者的脑起到保护作用，因为α7 nAChR的过度表达增加了SYP、SNAP-25和PSD-95的表达水平，并减弱了Aβ对这些突触蛋白表达和细胞凋亡的抑制作用。总体而言，这表明α7 nAChR可能在AD中发挥重要的神经保护作用。
Alzheimer's disease (AD) is a chronic neurodegenerative, and abnormal aggregation of the neurotoxic beta amyloid (A beta) peptide is an early event in AD. The present study aimed to determine the correlation between the nicotinic acetylcholine receptor alpha 7 subunit (alpha 7 nAChR) and A beta in the brains of patients with AD, and to investigate whether the increased expression levels of the alpha 7 nAChR could alter the neurotoxicity of A beta. The expression levels of alpha 7 nAChR and A beta in the brains of patients with AD and healthy brains were analyzed using immunofluorescence. Moreover, SH-SY5Y cells were used to stably overexpress or silence alpha 7 nAChR expression levels, prior to the treatment with or without 1 mu mol/l A beta(1-42) oligomer (A beta O). The mRNA and protein expression levels of alpha 7 nAChR, synaptophysin (SYP), postsynaptic density of 95 kDa (PSD-95) and synaptosomal-associated protein of 25 kDa (SNAP-25) were subsequently analyzed using reverse transcription-quantitative PCR and western blotting. In addition, the concentration of acetylcholine (ACh) and the activity of acetylcholinesterase (AChE) were analyzed using spectrophotometry, while the cell apoptotic rate was determined using flow cytometry. The expression of A beta in the brains of patients with AD was found to be significantly increased, whereas the expression of alpha 7 nAChR was significantly decreased compared with the healthy control group. In vitro, the expression levels of alpha 7 nAChR were significantly increased or decreased following the overexpression or silencing of the gene, respectively. Consistent with these observations, the mRNA and protein expression levels of SYP, PSD-95 and SNAP-25 were also significantly increased following the overexpression of alpha 7 nAChR and decreased following the genetic silencing of the receptor. In untransfected or negative control cells, the expression levels of these factors and the apoptotic rate were significantly reduced following the exposure to A beta O, which was found to be attenuated by alpha 7 nAChR overexpression, but potentiated by alpha 7 nAChR RNA silencing. However, no significant differences were observed in either the ACh concentration or AChE activity following transfection. Collectively, these findings suggested that alpha 7 nAChR may protect the brains of patients with AD against A beta, as alpha 7 nAChR overexpression increased the expression levels of SYP, SNAP-25 and PSD-95, and attenuated the inhibitory effect of A beta on the expression of these synaptic proteins and cell apoptosis. Overall, this indicated that alpha 7 nAChR may serve an important neuroprotective role in AD.