Molecular Geriatrics, Department of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden.
PLoS One. 2012;7(2):e32014. doi: 10.1371/journal.pone.0032014. Epub 2012 Feb 15.
Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.
可溶性淀粉样β（Aβ）聚集体大小不一，从二聚体到大型原纤维都有，与阿尔茨海默病（AD）的神经毒性和突触功能障碍有关。为了研究具有生物学相关性的 Aβ 物种的特性，用密度梯度超速离心法在天然条件下，从淀粉样蛋白β前体（AβPP）转基因小鼠和 AD 患者的脑提取物以及合成 Aβ 制剂中分离出大小不同的物质。然后用原子力显微镜（AFM）、ELISA 和 MTT 细胞活力测定法对分级的样品进行分析。根据 AFM 外观和对我们的原纤维选择性抗体 mAb158 的免疫反应性，合成 Aβ42 被分为 4 个部分，其中大聚集体位于第 1 部分，最小的物质位于第 4 部分。来自第 2 和第 3 部分的合成 Aβ 聚集体在 MTT 测定中证明是最具毒性的。在 AβPP 转基因小鼠脑中，第 2 部分中发现了最丰富的可溶性 Aβ 物质，主要由 Aβ40 组成。在 AD 脑中，Aβ 主要存在于第 2 部分，但主要是 Aβ42。第 2 部分的所有生物来源的 Aβ 都与 mAb158 免疫区分开来。因此，通过密度梯度超速离心法分离出的具有生物学活性的可溶性 Aβ 的主要物质，与神经毒性的 80-500 kDa 合成 Aβ 原纤维的大小相匹配，并且同样可以用 mAb158 检测到。
