ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Wollongong, NSW 2522, Australia.
Department of Physics, Nagoya University, Higashiyama Campus, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
J Mol Biol. 2019 Jul 12;431(15):2687-2699. doi: 10.1016/j.jmb.2019.04.044. Epub 2019 May 7.
Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ species. The findings revealed that smaller Aβ species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to "permissive" binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.
在阿尔茨海默病的淀粉样蛋白假说中，目前的重点已经转移到淀粉样β (Aβ) 肽组装的早期阶段，涉及可溶性寡聚物和较小的聚集体，与它们形态明显不同的纤维形式相比，这些寡聚物和聚集体对细胞更具毒性。关键是要揭示寡聚化的分子水平动力学途径，从而确定导致疾病病因的 Aβ 寡聚物群体的罪魁祸首亚类或特定物种。在这里，我们应用高速原子力显微镜来直接观察单个 Aβ 寡聚物和聚集体之间的动态相互作用，在液体中具有纳米级结构和毫秒级时间分辨率的结合。尺寸分析揭示了除了单体之外，还有三种主要的 Aβ 物种分布，单体的出现表明其表面扩散速度比较大的 Aβ 物种快。重要的是，我们设计了一种新的基于高速原子力显微镜电影图像对比度的单分子分析方法，以量化不同 Aβ 物种之间相互作用的速率决定动力学常数。研究结果表明，较小的 Aβ 物种显示出寿命分布的指数衰减，这表明所有分子都经历相同的过程，具有单个明确定义的能量势垒。相比之下，较大的聚集体显示出随机的寿命，这表明必须克服相互作用能量/势垒的分布才能解离。我们将后者解释为“许可”结合，这是由于聚集体的不同构象状态以及各种可及的相互作用基团引起的。不可避免的是，这可能导致形成不同的复合物或同种型，这已知会导致识别 Aβ 寡聚物毒性的困难，并对阿尔茨海默病伴随的神经元死亡的机制产生影响。
