水木未来·视界iss.7丨基于冷冻电镜的神经退行研究

神经退行性疾病指那些进展缓慢且无法治愈、严重影响患者身体和精神的衰弱性疾病。痴呆症是神经退行性疾病最常见的症状，其特征是记忆丧失和认知及语言能力下降。根据全球脑健康研究所和世界卫生组织的数据，到2030年，全球痴呆症的预计治疗费用将增加到2万亿美元，到2050年，痴呆症患者的数量预计将增加两倍，达到1.5亿。由于疏于预防并缺乏有效的治疗，神经退行是一个迫在眉睫的全球健康问题。

Neurodegenerative diseases are slow-progressing, incurable, and debilitating disorders affecting patients’ physical movements and mental functions. Dementia is the most common symptom of neurodegeneration, with hallmarks of memory loss and reduced cognitive and language skills. According to the Global Brain Health Institute and the World Health Organization, the global cost of dementia is estimated to increase to $2 trillion by 2030, and the number of people with dementia is expected to triple to 150 million by 2050. Due to the lack of prevention, a cure, or effective treatment, neurodegeneration is an imminent global health problem.

本文转载自: The-Scientist

"revolutionizing Neurodegeneration Research with Cryo-EM"

神经退行性疾病中的蛋白质聚集体

Protein Aggregates in Neurodegeneration

阿茨海默症、帕金森症和亨廷顿症是常见的神经退行性疾病，他们具有神经元死亡加速和大脑功能连接丧失的特点。这些神经退行性疾病共同的组织病理学标志是由特定的毒性蛋白产生的长纤维和斑块，它们在患者大脑中形成大的蛋白聚集体。例如，淀粉样斑块和涛蛋白丝是阿茨海默病患者大脑中的独特结构，其中异常的淀粉样ß蛋白在细胞外空间聚集，涛蛋白在神经元上聚集。在亨廷顿病患者中，突变的亨廷顿蛋白形成复杂的聚集物，而在帕金森病患者中，ɑ-synuclein聚集体在神经元中聚集并形成路易体。

Alzheimer’s, Parkinson’s, and Huntington’s diseases are common neurodegenerative disorders with accelerated neuronal death and loss of functional brain connections. Common histopathological hallmarks of these neurodegenerative diseases are long filaments and plaques that arise from disease-specific toxic proteins and form large protein aggregates in patient brains. For example, amyloid plaques and neurofibrillary tubulin-associated unit (tau) filaments are distinct structures in the brains of patients with Alzheimer’s disease, where abnormal amyloid ß proteins accumulate in extracellular spaces and tau proteins cluster on neurons. In Huntington’s patients, mutant huntingtin protein forms complex aggregates, and ɑ-synuclein aggregates clump in neurons to form Lewy bodies in Parkinson’s patients.

蛋白质聚集和神经退行之间的联系仍然不清楚，且受到争议。一般来说，蛋白质的结构和功能是相关的。通过了解这些蛋白质聚集物的结构特征，科学家们可以了解它们如何形成，如何与细胞环境相互作用，以及如何改变大脑功能。利用结构生物学工具，研究人员开始以原子分辨率解开蛋白质聚集体的结构特征，以确定它们在脑组织中的病理学。

The link between protein aggregates and neurodegeneration remains unclear and controversial. In general, protein structure and function correlate. By understanding the structural features of these protein aggregates, scientists can address how they form, interact with the cellular environment, and alter brain function. Using structural biology tools, researchers begin to unravel the structural features of protein aggregates at atomic resolution to determine their pathology in brain tissue.

神经退行性疾病的结构生物学研究

Structural Biology for Neurodegenerative Diseases

目前有几种结构生物学技术可以解决三维蛋白质结构：如X射线晶体学、核磁共振和冷冻电镜（cryo-EM）。通过这些技术，结构生物学家能够以原子级分辨率对分子进行成像，突破传统方法的分辨率极限。

There are several structural biology techniques that resolve three-dimensional protein structures. Incorporating different methods such as x-ray crystallography, nuclear magnetic resonance (NMR), and cryogenic electron microscopy (cryo-EM), structural biologists map the atomic-level structures of molecules that are otherwise not visible with traditional high-resolution microscopy techniques.

自1953年首次使用以来，X射线晶体学一直是结构生物学研究中的一个主流方法。研究人员对目的蛋白进行纯化和结晶，并使用X射线生成原子级分辨率的蛋白质分子图像。这些图像提供了诸如界面结构、化学键、结合点和蛋白质的相互作用等信息。然而，生成高质量的晶体是具有挑战性的，而且目前无法对丝状或缠结形式的复杂蛋白质聚合体进行结晶。在这种情况下，核磁共振波谱法为不适合结晶的蛋白质结构解析提供了替代方法。

Since its first use in 1953, x-ray crystallography has been a dominant method in structural biology research. Researchers purify and crystallize proteins of interest and use x-rays to generate an image of the protein molecules at atomic resolution. These structural images provide information such as structural interfaces, chemical bonds, binding sites, and protein-protein interactions. However, generating high-quality crystals is challenging, and complex protein aggregates in the form of filaments or tangles are impossible to crystallize. NMR spectroscopy offers an alternative to x-ray crystallography for proteins not amenable to crystallization.

然而，核磁共振方法仅限于分子量低于50kDa的蛋白质，所以它并不适合解析大分子量的蛋白质聚集体，如在神经退行性疾病中发现的那些：例如，研究人员采用核磁共振波谱来确定涛蛋白的结构，但该方法并不适用于分子量大且结构复杂的涛蛋白缠结。最近，冷冻电镜的发展促进了对大分子蛋白质的结构解析。使用冷冻电镜的科学家们在成像前无需对蛋白质进行结晶，而是将蛋白质溶液快速冷冻成玻璃状的水合状态。在样品进入电镜后，一束加速的电子就会击中蛋白质样品。这时，蛋白质分子会散射电子，从而产生显示蛋白质的原子组成和排列的图像。

However,this method is limited to proteins with molecular weights below 50kDa, so it is not compatible with high molecular weight protein aggregates like those found in neurodegenerative diseases. For example, researchers employed NMR spectroscopy to determine tau protein structure, but the method is not applicable for high molecular weight, complex tau tangles. Recent developments in cryo-EM facilitate the profiling of large macromolecular proteins. Instead of crystallizing proteins, scientists performing cryo-EM flash-freeze (vitrify) protein solutions into a glass-like, hydrated state prior to imaging. Once in the electron microscope, a beam of accelerated electrons hits the protein sample. The protein molecules scatter the electrons, which produces images that show the atomic composition and arrangement of the protein.

更大的样品成像区域，更好的电子捕获探测器，以及更快的图像处理：种种优势表明冷冻电镜是在神经退行性疾病研究中观察并解析复杂蛋白质结构的理想选择。结构解析对于了解蛋白质如何工作，在疾病中蛋白质为何失常，以及后续的靶点成药性筛选是非常有用的。神经退行性疾病的研究人员受益于冷冻电镜革命，发现了许多在神经退行性疾病中错误折叠的蛋白原子结构，如涛蛋白丝、ɑ-synuclein纤维、淀粉样ß聚合体，以及与这些蛋白聚合体结合的小分子候选药物。

With advancements in stable and multibeam microscopes to image larger sample areas, improved direct capture electron detectors, and faster software for image processing, cryo-EM is ideal for investigating the complex protein structures observed in neurodegenerative diseases. Such structural analyses are useful for understanding how proteins work, how they malfunction in disease, and how to target them with therapeutics. Researchers studying neurodegenerative diseases benefitted from the cryo-EM revolution and uncovered the atomic structures of numerous proteins that misfold in neurodegenerative diseases such as tau filaments, ɑ-synuclein fibrils, and amyloid ß aggregates, as well as small molecule drug candidates that bind to these protein aggregates.

观察阿茨海默症的大脑

Peeking into the Alzheimer’s Brain

细胞外的淀粉样β蛋白在阿尔茨海默病患者的大脑中形成高度不溶性、密集的丝状物。这些蛋白质聚集成长纤维，形成斑块的组成部分。斑块积聚在神经元之间，破坏了大脑功能。这些信息有助于解释阿尔茨海默病的病理生理学。冷冻电镜使研究人员能够了解淀粉样斑块的结构特征以及它们是如何形成的。

Extracellular amyloid β proteins form highly insoluble, densely-packed filaments in the brains of people with Alzheimer’s disease. The proteins aggregate into long fibers forming a building block for plaques. The plaques accumulate between neurons and disrupt brain function, which contributes to the pathophysiology of Alzheimer’s disease. Cryo-EM enables researchers to understand the structural features of amyloid plaques and how they form.

长按并扫描上方二维码，下载来自赛默飞世尔科技和The Scientist的完整电子书，了解冷冻电镜的进步如何使研究人员能够确定蛋白质聚合体的原子结构并推进神经退行性疾病的研究。

Download this ebook from Thermo Fisher Scientific and The Scientist to learn how advancements in cryogenic electron microscopy (cryo-EM) allow researchers to determine protein aggregate atomic structures, transforming neuro-degeneration research.

《水木未来x赛默飞联合公开课第十一讲》

北京大学PI 郭强博士

公开课简介：冷冻电子断层扫描技术是目前唯一可以在细胞生理环境下对生物大分子及亚细胞结构进行分子分辨率(1-10纳米)结构分析的技术手段。世界卫生组织(WHO)预测在2040年神经退行性疾病将取代癌症成为人类第二大致死疾病。至今为止还没有任何针对这类疾病的有效治疗方案，因此了解这类疾病的致病机理的需求尤为迫切。包括阿兹海默症(Alzheimer’s disease)、帕金森(Parkinson’s disease)、亨廷顿(Huntington’s disease)、以及肌萎缩侧索硬化症(ALS)在内的很多神经退行性疾病都具有一系列共同特征，那就是基因突变和个体衰老所引起的环境压力最终导致了蛋白聚集。因此，关于这类疾病发生的机制有两个假说：蛋白聚集物本身带来的获得性毒性机制(gain of toxic function)和基因突变导致的关键蛋白功能缺失性细胞毒性(loss of critical physiological function)。我们一方面使用冷冻电镜电子断层扫描技术，结合生化及其他手段研究与ALS相关的蛋白聚集对细胞微环境的影响。与此同时通过单颗粒重构技术解析导致Huntington’s disease的关键蛋白Huntington的结构，为进一步了解其生物学功能提供基础。

水木视界丨iss. 7