Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia.
Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia.
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DOI：
10.1007/s11481-012-9424-6
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发表时间：
2013-03
影响因子：
6.2
通讯作者：
Nichols, Michael R.
中科院分区：
文献类型：
--
作者：
Paranjape, Geeta S.;Terrill, Shana E.;Gouwens, Lisa K.;Ruck, Benjamin M.;Nichols, Michael R.
淀粉样蛋白-β (Aβ)的可溶性聚集形式最近因其在阿尔茨海默病（AD）中的作用而引起了极大的关注。原原纤维是这些可溶性物种的一个子集，被认为是成熟a β原纤维聚集途径的中间产物。生物学研究表明原纤维具有毒性和炎症活性。在这些体外研究中，重要的是使用适合细胞研究的溶液条件制备原原纤维，并有利于原原纤维的生物物理表征。本文描述了改性人工脑脊液（aCSF）中Aβ(1-42)原原纤维的制备和表征，并证明了它们与小胶质细胞的显著结合和活化。制备了一种简单的磷酸盐/碳酸氢盐缓冲体系，该缓冲体系保持了F-12培养基的离子强度和细胞相容性，但不含有大量干扰Aβ原原纤维光谱分析的添加剂。在aCSF中重构Aβ(1-42)，并通过粒径隔离色谱（SEC）进行分离，发现β-原原纤维呈曲线状，长度<100 nm，水动力半径为21 nm。BCA法测定原纤维浓度在aCSF中更为准确，这在F-12培养基中是不可能的。在aCSF中形成和分离的原纤维，而不是单体，显著刺激BV-2和初级小胶质细胞中tnf - α的产生，并大量结合到小胶质膜上。本报告证明了改良的aCSF系统用于制备sec分离的a β(1-42)原纤维的适用性，并强调了原纤维与小胶质细胞功能相互作用的独特能力。
Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia.
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影响因子：
5
作者：
Paranjape, Geeta S.;Gouwens, Lisa K.;Nichols, Michael R.
通讯作者：
Nichols, Michael R.
DOI：
10.1523/jneurosci.3537-10.2010
发表时间：
2010-10-27
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
--
作者：
O'Nuallain B;Freir DB;Nicoll AJ;Risse E;Ferguson N;Herron CE;Collinge J;Walsh DM
通讯作者：
Walsh DM
影响因子：
4.4
作者：
Liu, Shirong;Liu, Yang;Fassbender, Klaus
通讯作者：
Fassbender, Klaus
影响因子：
25
作者：
Cleary, JP;Walsh, DM;Ashe, KH
通讯作者：
Ashe, KH
影响因子：
2.9
作者：
Nichols, MR;Moss, MA;Rosenberry, TL
通讯作者：
Rosenberry, TL