Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States.
Laboratory for Fluorescence Dynamics, Biomedical Engineering, University of California, Irvine, California 92697, United States.
ACS Chem Neurosci. 2022 Aug 17;13(16):2473-2482. doi: 10.1021/acschemneuro.2c00313. Epub 2022 Jul 27.
Aβ oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17-36 of Aβ as model systems for studying Aβ oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, indicating the presence of oligomers within cells. Collectively, these multiple complementary techniques help better understand the complex behavior of the 2AT-L model system.
β淀粉样蛋白寡聚体在阿尔茨海默病观察到的神经退行性变中起核心作用。我们的实验室已经开发出了源自 Aβ 残基 17-36 的共价稳定三聚体，作为研究 Aβ 寡聚体的模型系统。在当前的研究中，我们应用新兴的荧光寿命成像显微镜（FLIM）和天然质谱（native MS）技术，以更好地了解寡聚体模型系统 2AT-L 在水溶液中和与细胞中的组装和相互作用。2AT-L 和荧光标记的 2AT-L 类似物在 SDS-PAGE 的膜样环境中组装，显示出处于平衡状态的寡聚物的弥散带。2AT-L 的天然离子淌度-质谱（native IM-MS）允许在溶液中鉴定离散的寡聚物，并显示 2AT-L 和荧光标记类似物之间形成寡聚物的相似模式。用 SH-SY5Y 细胞进行荧光显微镜研究表明，荧光标记的 2AT-L 类似物在溶酶体中共定位。用相分析进行的 FLIM 研究进一步阐明了 2AT-L 在细胞内的组装，并确定了 FRET 的发生，表明细胞内存在寡聚物。总之，这些多种互补技术有助于更好地理解 2AT-L 模型系统的复杂行为。
