MICA: Multi-parametric and super-resolution imaging of amyloidogenic proteins

阿尔茨海默氏症和其他神经退行性疾病一样，是由蛋白质的异常形式引起的，这些蛋白质往往会积累和聚集成形状，据信对受影响患者的脑细胞是有毒的。被认为导致阿尔茨海默氏症的两种蛋白质被称为淀粉样β蛋白和tau蛋白。这些蛋白质聚集成有毒形式的确切机制尚不清楚，但越来越多的证据表明，形成聚集体的形状和大小与疾病的发生和发展有着错综复杂的联系。然而，目前还没有研究活细胞中有毒聚集体生长的工具，它们太小了，无法用传统技术观察到。对于开发治疗阿尔茨海默氏症的潜在疗法来说，对导致聚集的事件及其对受影响细胞的影响的微观了解至关重要。能够观察到脑细胞中的蛋白质聚集将使我们能够将蛋白质“团块”的出现与它们的毒性作用联系起来。受影响的细胞有故障吗？以什么方式？哪些聚合体物种会导致细胞发生故障？我们能确定导致细胞“行为不端”的有毒物种吗？我们能否通过添加潜在的治疗剂来抑制活细胞中“有毒形状”的形成？我们能阻止有毒物种从细胞到细胞的传播吗？这些问题的答案需要全新的方法，以前所未有的分辨率研究细胞。在我们团队中，我们最近开发了一种新的显微镜，即所谓的光学超分辨率显微镜，它的功能如此强大，能够直接在培养的脑细胞中可视化蛋白质聚集体的形状，我们将其用作疾病的模型系统。我们是第一批证明有可能获得神经元中有毒蛋白质形状的“图像”的研究人员，在这里，我们建议使用这一新开发的工具来阐明导致疾病发生和传播的机制。我们拟议的研究项目的目的是：1)识别神经元和大脑中存在的其他细胞中的特定种类的淀粉样β蛋白和tau，并将这些种类的形状和大小与疾病症状的出现联系起来。2)研究淀粉样β蛋白和tau蛋白在细胞内和细胞外的运输，并推断这对疾病进展的可能机制的影响。3)看看潜在的蛋白质聚集和繁殖抑制物是否会对我们在所研究的细胞内观察到的东西产生影响。我们将测试我们的合作者提供的几种有希望的抗聚集抗传播化合物。通过我们小组开发的先进显微成像技术的开发和应用，我们的目标是深入了解阿尔茨海默病和其他形式痴呆的核心分子水平的过程。我们的技术使我们能够以非常高的分辨率研究导致蛋白质错误折叠和扩散以及它们聚集到活细胞模型系统中有毒结构的机制，从而深入了解疾病及其传播的机制。这一认识可能有助于未来寻找潜在的治疗剂。这里产生的知识和工具将为生物标志物/诊断学的开发提供合理的基础，使医生能够：1)在只有生物化学变化的情况下，在疾病的最早阶段发现疾病；2)识别疾病的阶段，以便为个别患者量身定制治疗方法；以及3)监测对治疗的反应。对这些途径的了解也将为修复这些途径的治疗提供靶点，从而预防甚至逆转这种疾病。

Alzheimer's, like other neurodegenerative diseases, is caused by aberrant forms of proteins which tend to accumulate and aggregate into shapes, which are believed to be toxic to the brain cells of affected patients. The two proteins thought to cause Alzheimer's are called amyloid-beta and tau. The precise mechanisms by which these proteins aggregate into toxic forms are unknown, but there is accumulating evidence that the shape and size of forming aggregates is intricately linked to the onset and progression of disease. Yet there are no tools to study the growth of toxic aggregates in living cells, they are simply too small to be observed with traditional techniques. For the development of potential therapies against Alzheimer's, a microscopic understanding of the events leading to aggregation, and their consequence on affected cells, is crucial. Being able to observe protein aggregation in brain cells would permit us to relate the appearance of protein "clumps" with their toxic effect. Do affected cells malfunction? In what way? What aggregate species cause the cells to malfunction? Can we identify the toxic species leading cells to 'misbehave'? Can we inhibit the formation of "toxic shapes" in living cells through the addition of potential therapeutic agents? Can we stop the spreading of toxic species from cell-to-cell? Answers to these questions require radically new approaches to look into cells, at unprecedented resolution. In our group we have recently developed a new kind of microscope, a so called optical super-resolution microscope, that is so powerful that it is capable of visualising the shape of protein aggregates directly in cultured brain cells, which we use as model systems of disease. We were the first researchers to demonstrate that it is possible to obtain 'images' of toxic protein shapes in neurones, and here we propose to use this newly developed tool to shed new light on the mechanisms that lead to the onset and propagation of disease.The aims of our proposed research project are: 1) to identify specific species of amyloid-beta and tau in neurones and other cells present in the brain and to relate the shape, and size of these species to the appearance of disease symptoms. 2) to study amyloid-beta and tau trafficking in and out of cells and to infer on the implications of this on the possible mechanisms of disease progression. 3) to see whether potential inhibitors of protein aggregation and propagation have an effect on what we observe within the cells we study. We will test several promising anti-aggregation anti-propagation compounds that our collaborators provide.Through the development and application of advanced microscopic imaging techniques developed in our group we aim to gain insight into the molecular level processes that lie at the heart of Alzheimer's disease and other forms of dementia. Our techniques enable us to investigate the mechanisms that lead to the misfolding and spreading of proteins and their aggregation into toxic structures in live cell model systems at very high resolution, giving insight into the mechanisms of disease and its propagation. This understanding may help in the future search for potential therapeutic agents. The knowledge and tools generated here will provide a rational basis for the development of biomarkers/diagnostics that will enable physicians to: 1) detect the disease in its earliest stages when only biochemical changes are apparent; 2) identify the stage of the disease in order to tailor therapies for individual patients; and 3) monitor response to that treatment. Knowledge of these pathways will also provide targets for therapies to repair these pathways, thereby preventing or even reversing the disease.