Liu Qiang, Xie Xitao, Emadi Sharareh, Sierks Michael R, Wu Jie
Division of Neurology, Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA; Department of Neurology and Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
Department of Biochemistry & Molecular Biology, Mayo Clinic, Scottsdale, AZ 85259, USA.
Neuropharmacology. 2015 Oct;97:457-63. doi: 10.1016/j.neuropharm.2015.04.025. Epub 2015 May 7.
Loss of basal forebrain cholinergic neurons (BFCN) correlates with cognitive deficits in Alzheimer disease (AD). Our recent evidence suggests that chronic exposure to Aβ up-regulated neuronal α7-nAChRs and increased neuronal excitability in cultured hippocampal neurons. However, the impact of the up-regulated α7-nAChRs on Aβ-induced neurotoxicity remains unclear. In this study, we investigated the role of α7-nAChRs in the mediation of Aβ-induced neurotoxicity. The effects of Aβ exposure on α7-nAChRs and cytotoxicity were examined using whole-cell patch clamp recordings, atomic force microscope (AFM) imaging, immunoprecipitation, and lactate dehydrogenase (LDH) release assay in primary cultured hippocampal neurons as well as differentiated human neuroblastoma (SH-SY5Y) cells with cholinergic characteristics. We found that α7-nAChRs are necessary for Aβ-induced neurotoxicity in hippocampal neurons because chronic Aβ significantly increased LDH level in hippocampal cultures, which was prevented by either α7-nAChR antagonist methyllycaconitine (MLA) or by α7 subunit gene deletion (cultures prepared from nAChR α7 subunit KO mice), whereas β2-containing nAChR antagonist (dihydro-β-erythroidine, DhβE) or the genetic deletion of nAChR β2 subunit (cultures prepared from β2 KO mice) failed to prevent Aβ-induced toxicity. In SH-SY5Y cells, larger aggregates of Aβ preferentially up-regulated α7-nAChR expression and function accompanied by a significant decrease in cell viability. Co-treatment MLA, but not mecamylamine (MEC), prevented Aβ exposure-induced neurotoxicity. Our results suggest a detrimental role of upregulated α7-nAChRs in the mediation of Aβ-induced neurotoxicity.
基底前脑胆碱能神经元（BFCN）的丧失与阿尔茨海默病（AD）的认知缺陷相关。我们最近的证据表明，长期暴露于Aβ会上调神经元α7烟碱型乙酰胆碱受体（α7-nAChRs）并增加培养的海马神经元的神经元兴奋性。然而，上调的α7-nAChRs对Aβ诱导的神经毒性的影响仍不清楚。在本研究中，我们研究了α7-nAChRs在介导Aβ诱导的神经毒性中的作用。使用全细胞膜片钳记录、原子力显微镜（AFM）成像、免疫沉淀和乳酸脱氢酶（LDH）释放测定法，在原代培养的海马神经元以及具有胆碱能特征的分化人神经母细胞瘤（SH-SY5Y）细胞中，检测了Aβ暴露对α7-nAChRs和细胞毒性的影响。我们发现，α7-nAChRs是海马神经元中Aβ诱导的神经毒性所必需的，因为长期Aβ显著增加了海马培养物中的LDH水平，而α7-nAChR拮抗剂甲基lycaconitine（MLA）或α7亚基基因缺失（由nAChRα7亚基敲除小鼠制备的培养物）可阻止这种增加，而含β2的nAChR拮抗剂（二氢-β-刺桐碱，DhβE）或nAChRβ2亚基的基因缺失（由β2敲除小鼠制备的培养物）未能阻止Aβ诱导的毒性。在SH-SY5Y细胞中，较大的Aβ聚集体优先上调α7-nAChR的表达和功能，同时细胞活力显著降低。联合使用MLA而非美加明（MEC）可预防Aβ暴露诱导的神经毒性。我们的结果表明，上调的α7-nAChRs在介导Aβ诱导的神经毒性中起有害作用。
