Hu Min, Schurdak Mark E, Puttfarcken Pamela S, El Kouhen Rachid, Gopalakrishnan Murali, Li Jinhe
Neuroscience Research, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6125, USA.
Brain Res. 2007 Jun 2;1151:227-35. doi: 10.1016/j.brainres.2007.03.051. Epub 2007 Mar 24.
beta-Amyloid peptide 1-42 (A beta(1-42)) is generated from amyloid precursor protein (APP) and associated with neurodegeneration in Alzheimer's disease (AD). A beta(1-42) has been shown to be cytotoxic when incubated with cultured neurons. However, APP transgenic mice over-expressing A beta(1-42) do not show substantial loss of neurons, despite deficits in learning and memory. It is thus emerging that A beta(1-42)-induced memory deficits may involve subtler neuronal alternations leading to synaptic deficits, prior to frank neurodegeneration in AD brains. In this study, high content screen (HCS) microscopy, an advanced high-throughput cellular image processing and analysis technique, was utilized in establishing an in vitro model of A beta(1-42)-induced neurotoxicity utilizing rat neonatal primary cortical cells. Neurite outgrowth was found to be significantly reduced by A beta(1-42) (300 nM to 30 microM), but not by the scrambled control peptide control, in a time- and concentration-dependent manner. In contrast, no reduction in the total number of neurons was observed. The A beta(1-42)-induced reduction of neurite outgrowth was attenuated by the NMDA receptor antagonist memantine and the alpha 7 nicotinic acetylcholine receptor (nAChR) selective agonist PNU-282987. Interestingly, the alpha 7 nAChR antagonist methyllycaconitine also significantly prevented reduction in A beta(1-42)-induced neurite outgrowth. The observed neuroprotective effects could arise either from interference of A beta(1-42) interactions with alpha 7 nAChRs or by modification of receptor-mediated signaling pathways. Our studies demonstrate that reduction of neurite outgrowth may serve as a model representing A beta(1-42)-mediated neuritic and synaptic toxicity, which, in combination of HCS, provides a high-throughput cell-based assay that can be used to evaluate compounds with neuroprotective properties in neurons.
β淀粉样肽1-42（Aβ(1-42)）由淀粉样前体蛋白（APP）生成，并与阿尔茨海默病（AD）中的神经退行性变相关。已证明Aβ(1-42)与培养的神经元一起孵育时具有细胞毒性。然而，过度表达Aβ(1-42)的APP转基因小鼠尽管存在学习和记忆缺陷，但并未显示出大量神经元丢失。因此，越来越明显的是，在AD大脑出现明显神经退行性变之前，Aβ(1-42)诱导的记忆缺陷可能涉及更细微的神经元改变，导致突触缺陷。在本研究中，利用高内涵筛选（HCS）显微镜这一先进的高通量细胞图像处理和分析技术，建立了一个利用大鼠新生原代皮层细胞的Aβ(1-42)诱导神经毒性的体外模型。发现Aβ(1-42)（300 nM至30 μM）以时间和浓度依赖性方式显著减少神经突生长，但乱序对照肽对照组则无此现象。相反，未观察到神经元总数减少。NMDA受体拮抗剂美金刚和α7烟碱型乙酰胆碱受体（nAChR）选择性激动剂PNU-282987可减弱Aβ(1-42)诱导的神经突生长减少。有趣的是，α7 nAChR拮抗剂甲基lycaconitine也显著阻止了Aβ(1-42)诱导的神经突生长减少。观察到的神经保护作用可能源于Aβ(1-42)与α7 nAChRs相互作用的干扰，或受体介导信号通路的改变。我们的研究表明，神经突生长减少可作为代表Aβ(1-42)介导的神经突和突触毒性的模型，结合HCS，可提供一种基于细胞的高通量检测方法，用于评估具有神经保护特性的化合物对神经元的作用。
