Department of Clinical Neurosciences, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 1N4.
Department of Chemistry, Faculty of Science, University of Calgary, Calgary, Alberta, Canada, T2N 1N4.
ACS Chem Neurosci. 2021 Apr 7;12(7):1273-1280. doi: 10.1021/acschemneuro.1c00101. Epub 2021 Mar 11.
Protein aggregation is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Small organic fluorophores such as Congo Red preferentially bind to cross-β-sheet-rich deposits and have been used to label amyloid plaques and tau tangles in histological samples. However, distinguishing between different conformations of protein aggregates is not trivial. Using silkworm and spider silks (prototypical amyloids) and transgenic AD mouse (5XFAD) and human AD brain samples, we report how spectral confocal microscopy allowed for improved detection and differentiation of protein aggregates based on the unexpected photophysical behavior of the amyloid-specific dye K114. The pH and excitation power had pronounced effects on the emission spectrum and intensity of amyloid-bound K114 fluorescence. When bound to β-sheet-rich assemblies, the emission spectrum of K114 was governed by the local pH of the binding pockets much more than by the pH of the mounting medium, likely due to ionization of titratable phenols. Unexpectedly, exposure to high excitation power caused a permanent increase in fluorescence intensity and a spectral blue-shift. These light-induced fluorescence changes were dependent in a complex manner on laser power, exposure time, pH, and amyloid type examined. The above-mentioned phenomena were observed in silk fibers and Alzheimer brain sections from mouse and human, indicating that this may be a general characteristic of K114 when bound to tightly aggregated macromolecules. Potential mechanisms are discussed, likely involving photoinduced electron transfer. Our findings illustrate how the complex photophysical behavior of amyloid-bound K114 can be exploited for improved detection and differentiation of protein aggregates.
蛋白质聚集是阿尔茨海默病（AD）和许多其他神经退行性疾病的标志。小分子荧光染料如刚果红优先结合富含交叉-β 片层的沉积物，并已被用于标记组织学样本中的淀粉样斑块和 tau 缠结。然而，区分不同构象的蛋白质聚集体并非易事。我们使用家蚕和蜘蛛丝（典型的淀粉样蛋白）以及转基因 AD 小鼠（5XFAD）和人 AD 脑组织样本，报告了光谱共聚焦显微镜如何通过淀粉样蛋白特异性染料 K114 的意外光物理行为来改善对蛋白质聚集体的检测和区分。pH 值和激发功率对结合在淀粉样蛋白上的 K114 荧光的发射光谱和强度有显著影响。当结合到富含β 片层的组装体上时，K114 的发射光谱受结合口袋的局部 pH 值影响比受 mounting 介质的 pH 值影响更大，这可能是由于可滴定酚的离解。出乎意料的是，暴露于高激发功率会导致荧光强度的永久性增加和光谱蓝移。这些光诱导的荧光变化以复杂的方式依赖于激光功率、暴露时间、pH 值和所检查的淀粉样蛋白类型。上述现象在家蚕丝纤维和来自小鼠和人类的阿尔茨海默病脑组织切片中均有观察到，表明这可能是 K114 与紧密聚集的大分子结合时的普遍特征。讨论了潜在的机制，可能涉及光诱导电子转移。我们的研究结果说明了如何利用结合在淀粉样蛋白上的 K114 的复杂光物理行为来改善对蛋白质聚集体的检测和区分。
