神經(jīng)退行性疾病指那些進(jìn)展緩慢且無法治愈、嚴(yán)重影響患者身體和精神的衰弱性疾病。癡呆癥是神經(jīng)退行性疾病最常見的癥狀，其特征是記憶喪失和認(rèn)知及語言能力下降。根據(jù)全球腦健康研究所和世界衛(wèi)生組織的數(shù)據(jù)，到2030年，全球癡呆癥的預(yù)計治療費(fèi)用將增加到2萬億美元，到2050年，癡呆癥患者的數(shù)量預(yù)計將增加兩倍，達(dá)到1.5億。由于疏于預(yù)防并缺乏有效的治療，神經(jīng)退行是一個迫在眉睫的全球健康問題。

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.

本文轉(zhuǎn)載自:?The-Scientist

"revolutionizing Neurodegeneration Research with Cryo-EM"

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神經(jīng)退行性疾病中的蛋白質(zhì)聚集體

Protein Aggregates in Neurodegeneration

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阿茨海默癥、帕金森癥和亨廷頓癥是常見的神經(jīng)退行性疾病，他們具有神經(jīng)元死亡加速和大腦功能連接喪失的特點。這些神經(jīng)退行性疾病共同的組織病理學(xué)標(biāo)志是由特定的毒性蛋白產(chǎn)生的長纖維和斑塊，它們在患者大腦中形成大的蛋白聚集體。例如，淀粉樣斑塊和濤蛋白絲是阿茨海默病患者大腦中的獨特結(jié)構(gòu)，其中異常的淀粉樣?蛋白在細(xì)胞外空間聚集，濤蛋白在神經(jīng)元上聚集。在亨廷頓病患者中，突變的亨廷頓蛋白形成復(fù)雜的聚集物，而在帕金森病患者中，ɑ-synuclein聚集體在神經(jīng)元中聚集并形成路易體。

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 ? 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.

蛋白質(zhì)聚集和神經(jīng)退行之間的聯(lián)系仍然不清楚，且受到爭議。一般來說，蛋白質(zhì)的結(jié)構(gòu)和功能是相關(guān)的。通過了解這些蛋白質(zhì)聚集物的結(jié)構(gòu)特征，科學(xué)家們可以了解它們?nèi)绾涡纬?，如何與細(xì)胞環(huán)境相互作用，以及如何改變大腦功能。利用結(jié)構(gòu)生物學(xué)工具，研究人員開始以原子分辨率解開蛋白質(zhì)聚集體的結(jié)構(gòu)特征，以確定它們在腦組織中的病理學(xué)。

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.

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神經(jīng)退行性疾病的結(jié)構(gòu)生物學(xué)研究

Structural Biology for Neurodegenerative Diseases

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目前有幾種結(jié)構(gòu)生物學(xué)技術(shù)可以解決三維蛋白質(zhì)結(jié)構(gòu)：如X射線晶體學(xué)、核磁共振和冷凍電鏡（cryo-EM）。通過這些技術(shù)，結(jié)構(gòu)生物學(xué)家能夠以原子級分辨率對分子進(jìn)行成像，突破傳統(tǒng)方法的分辨率極限。

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年首次使用以來，X射線晶體學(xué)一直是結(jié)構(gòu)生物學(xué)研究中的一個主流方法。研究人員對目的蛋白進(jìn)行純化和結(jié)晶，并使用X射線生成原子級分辨率的蛋白質(zhì)分子圖像。這些圖像提供了諸如界面結(jié)構(gòu)、化學(xué)鍵、結(jié)合點和蛋白質(zhì)的相互作用等信息。然而，生成高質(zhì)量的晶體是具有挑戰(zhàn)性的，而且目前無法對絲狀或纏結(jié)形式的復(fù)雜蛋白質(zhì)聚合體進(jìn)行結(jié)晶。在這種情況下，核磁共振波譜法為不適合結(jié)晶的蛋白質(zhì)結(jié)構(gòu)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，核磁共振方法僅限于分子量低于50kDa的蛋白質(zhì)，所以它并不適合解析大分子量的蛋白質(zhì)聚集體，如在神經(jīng)退行性疾病中發(fā)現(xiàn)的那些：例如，研究人員采用核磁共振波譜來確定濤蛋白的結(jié)構(gòu)，但該方法并不適用于分子量大且結(jié)構(gòu)復(fù)雜的濤蛋白纏結(jié)。最近，冷凍電鏡的發(fā)展促進(jìn)了對大分子蛋白質(zhì)的結(jié)構(gòu)解析。使用冷凍電鏡的科學(xué)家們在成像前無需對蛋白質(zhì)進(jìn)行結(jié)晶，而是將蛋白質(zhì)溶液快速冷凍成玻璃狀的水合狀態(tài)。在樣品進(jìn)入電鏡后，一束加速的電子就會擊中蛋白質(zhì)樣品。這時，蛋白質(zhì)分子會散射電子，從而產(chǎn)生顯示蛋白質(zhì)的原子組成和排列的圖像。

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.

更大的樣品成像區(qū)域，更好的電子捕獲探測器，以及更快的圖像處理：種種優(yōu)勢表明冷凍電鏡是在神經(jīng)退行性疾病研究中觀察并解析復(fù)雜蛋白質(zhì)結(jié)構(gòu)的理想選擇。結(jié)構(gòu)解析對于了解蛋白質(zhì)如何工作，在疾病中蛋白質(zhì)為何失常，以及后續(xù)的靶點成藥性篩選是非常有用的。神經(jīng)退行性疾病的研究人員受益于冷凍電鏡革命，發(fā)現(xiàn)了許多在神經(jīng)退行性疾病中錯誤折疊的蛋白原子結(jié)構(gòu)，如濤蛋白絲、ɑ-synuclein纖維、淀粉樣?聚合體，以及與這些蛋白聚合體結(jié)合的小分子候選藥物。

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 ? aggregates, as well as small molecule drug candidates that bind to these protein aggregates.

觀察阿茨海默癥的大腦

Peeking into the Alzheimer’s Brain

細(xì)胞外的淀粉樣β蛋白在阿爾茨海默病患者的大腦中形成高度不溶性、密集的絲狀物。這些蛋白質(zhì)聚集成長纖維，形成斑塊的組成部分。斑塊積聚在神經(jīng)元之間，破壞了大腦功能。這些信息有助于解釋阿爾茨海默病的病理生理學(xué)。冷凍電鏡使研究人員能夠了解淀粉樣斑塊的結(jié)構(gòu)特征以及它們是如何形成的。

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.

長按并掃描上方二維碼，下載來自賽默飛世爾科技和The Scientist的完整電子書，了解冷凍電鏡的進(jìn)步如何使研究人員能夠確定蛋白質(zhì)聚合體的原子結(jié)構(gòu)并推進(jìn)神經(jīng)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.

《水木未來x賽默飛聯(lián)合公開課第十一講》

北京大學(xué)PI 郭強(qiáng)博士

公開課簡介：冷凍電子斷層掃描技術(shù)是目前唯一可以在細(xì)胞生理環(huán)境下對生物大分子及亞細(xì)胞結(jié)構(gòu)進(jìn)行分子分辨率(1-10納米)結(jié)構(gòu)分析的技術(shù)手段。世界衛(wèi)生組織(WHO)預(yù)測在2040年神經(jīng)退行性疾病將取代癌癥成為人類第二大致死疾病。至今為止還沒有任何針對這類疾病的有效治療方案，因此了解這類疾病的致病機(jī)理的需求尤為迫切。包括阿茲海默癥(Alzheimer’s disease)、帕金森(Parkinson’s disease)、亨廷頓(Huntington’s disease)、以及肌萎縮側(cè)索硬化癥(ALS)在內(nèi)的很多神經(jīng)退行性疾病都具有一系列共同特征，那就是基因突變和個體衰老所引起的環(huán)境壓力最終導(dǎo)致了蛋白聚集。因此，關(guān)于這類疾病發(fā)生的機(jī)制有兩個假說：蛋白聚集物本身帶來的獲得性毒性機(jī)制(gain of toxic function)和基因突變導(dǎo)致的關(guān)鍵蛋白功能缺失性細(xì)胞毒性(loss of critical physiological function)。我們一方面使用冷凍電鏡電子斷層掃描技術(shù)，結(jié)合生化及其他手段研究與ALS相關(guān)的蛋白聚集對細(xì)胞微環(huán)境的影響。與此同時通過單顆粒重構(gòu)技術(shù)解析導(dǎo)致Huntington’s disease的關(guān)鍵蛋白Huntington的結(jié)構(gòu)，為進(jìn)一步了解其生物學(xué)功能提供基礎(chǔ)。

水木視界丨iss. 7