监测果蝇中朊病毒样蛋白聚集物的细胞间传输
Summary
积累的证据支持这种观点, 即与神经退行性疾病相关的致病蛋白聚集物在细胞间扩散, 与朊病毒类似。在这里, 我们描述了一种方法, 使可视化的细胞到细胞传播的朊病毒样聚集在模型有机体,果蝇。
Abstract
蛋白质聚集是大多数神经退行性疾病的中心特征, 包括阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)。蛋白质团聚体与这些疾病中的神经病理学密切相关, 尽管不知道异常蛋白质聚集扰乱正常细胞稳态的确切机制。新出现的数据有力地支持了这种假说, 即 AD、PD、HD 和 ALS 中的致病性聚集物与普利子有许多相似之处, 它们是负责传染性海绵状性脑病的仅蛋白感染性药物。普利子自复制通过模板化同一蛋白质本机折叠的版本的转换, 导致聚集表型的传播。在 AD、PD、HD 和 ALS 中, 普利子和朊病毒样蛋白是如何从一个细胞移动到另一单元的, 目前是一个激烈调查的领域。在这里, 一个果蝇模型, 允许监测朊病毒样, 细胞对细胞的传播与 HD 相关的突变杭丁顿蛋白 (Htt) 聚集。该模型利用强大的工具来处理许多不同的果蝇组织中的转基因表达, 并利用荧光标记的细胞质蛋白直接报告突变 Htt 聚合体朊病毒样转移。重要的是, 我们在这里描述的方法可以用来确定新的基因和途径, 在不同的细胞类型之间, 介导蛋白质聚合体的传播在体内。从这些研究中获得的信息将扩大对神经退行性疾病的致病机制的有限理解, 并揭示治疗干预的新机会。
Introduction
朊病毒假说指出, 负责传染性海绵状性脑病(例如, 人克雅氏病, 羊瘙痒病绵羊, 鹿和麋鹿的慢性浪费疾病, 和牛的疯牛病) 的传染性剂) 完全由蛋白质组成, 缺乏核酸1。在朊病毒疾病中, 细胞朊蛋白 (prpC) 假设一个非本机、稳定的折叠 (prpSc), 它具有高度 beta 版, 可以通过将单体 PrPC分子转换和招募到稳定的淀粉样中进行自我传播。聚合.PrPSc聚合使用此自复制机制在有机体中的不同单元格之间进行传播, 甚至在单个有机体之间进行扩展2。
蛋白质错误折叠和聚集也是大多数神经退行性疾病 (阿尔茨海默病 (AD)、帕金森病 (PD)、亨廷顿氏病 (HD) 和肌萎缩侧索硬化症 (ALS)) 的中心特征 3.这些疾病中或超细胞内聚集蛋白的形成与细胞毒性4密切相关, 随着时间的推移, 通过大脑的高度可重现性和疾病特定的路径进展5,6. 这些传播模式表明, 与这些疾病相关的致病性聚集物具有类似朊病毒的性质。现在有强有力的支持, 因为朊病毒样的传播与 AD、PD、HD 和 ALS 有关的聚合体-它们从细胞到细胞和模板在以前未受影响的细胞中单体形式的构象变化7, 8。
迄今为止, 大多数研究都是利用哺乳动物细胞培养模型进行的, 调查蛋白质聚集蛋白样的扩散, 从细胞外空间或从另一细胞中转移到天真细胞的细胞质中。细胞质9,10,11,12,13,14,15, 或将含骨料材料注入鼠标大脑和监测聚合外观在注入站点的外部16,17,18,19,20,21,22, 23. 最近, 转基因动物被用来证明胞内聚集物扩散到完整大脑中的其他细胞中24,25,26,27, 28,29,30。在这里, 我们描述了一个方法, 直接可视化的总转移之间的单个细胞在完整的大脑中的果蝇。果蝇模型的 HD/polyglutamine (polyQ) 疾病的首次开发近两年前的31,32 , 并提供了许多宝贵的洞察力的致病机制, 这些疾病的根源33. HD 是一种遗传性神经退行性疾病, 由常染色体显性突变引起, 该基因的编码为蛋白杭丁顿蛋白 (Htt) 34.这种突变导致在 Htt 的 N 终点附近的 polyQ 伸展扩展超过 37 glutamines 的致病阈值, 导致蛋白质 misfold 和聚合35,36。野生型 Htt 蛋白含有 < 37 glutamines 在这个拉伸达到他们的本机褶皱, 但可以诱导聚合后, 直接物理接触 Htt 聚合 "种子"12,27,37。我们利用这一着, 有核聚集的野生型 Htt 作为一个读数的朊病毒样转移和细胞质进入的突变 Htt 聚合体起源于其他细胞。
确定像朊病毒样的聚集物在细胞间穿梭的机制, 可以为无法治愈的神经退行性疾病确定新的治疗靶点。我们利用快速生命周期, 易用性和基因驯良的果蝇, 以确定分子机制的细胞对细胞扩散的突变 Htt 集。我们的实验策略采用了两个二进制表达系统, 在果蝇, 建立良好的 Gal4-specific 上游激活序列 (Gal4-UAS) 系统38和最近开发的 QUAS 系统39。耦合这两个独立的系统允许限制突变体和野生型 Htt 转基因在同一飞行中的不同细胞群的表达40。利用这一方法, 我们通过监测胞质野生型 Htt 从其通常弥漫性、可溶性状态到聚集状态的分布, 对突变体的 Htt 的传播进行检查, 这是与预先形成的突变体物理接触的直接后果 Htt聚合 “种子”。利用突变体 Htt 的生化或生物物理技术, 可以证实野生型 Htt 的转化, 如荧光共振能量转移 (烦恼)9,27,41.
重要的是, 我们还可以访问大量的基因工具在果蝇, 以确定基因和/或途径, 介导朊病毒样的传播蛋白质聚集。最近, 我们使用这种方法来揭开细胞表面清道夫受体, 德雷柏42,43的关键作用, 将突变 Htt 聚合体从神经元轴突转移到果蝇中心附近的吞噬细胞中。神经系统 (中枢神经系统)27。因此, 我们在这里描述的基于遗传和影像学的方法可以揭示在简单使用但功能强大的模型有机体 (果蝇) 中有关疾病相关现象的重要基本生物学信息。
Protocol
Representative Results
Discussion
随着神经退行性疾病患者的数量继续增加, 迫切需要增加对这些疾病分子发病机制的了解, 以便更好地开发治疗方法。在这里, 我们描述的方法, 可以监测病毒样的传播致病蛋白聚集在不同的细胞类型之间的模型有机体,果蝇。我们最近使用这种方法来演示突变体 Htt 集的病毒样传播在体内, 并确定一种吞噬性受体, 介导这些聚合体从神经元扩散到胶质细胞27。我们的方?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Kopito, 罗和皮尔斯实验室的成员在这些方法开发过程中的许多有益的讨论。我们还感谢布赖恩 Temsamrit 对这篇手稿的批判性阅读。这项工作得到了科学大学和水史密斯慈善信托基金的资助。
Materials
| Phosphate buffered saline (PBS), 10X, pH 7.4 | ThermoFisher Scientific | AM9625 | Dilute to 1X |
| Triton X-100 | Sigma-Aldrich | T9284-1L | |
| Kimwipes | Thomas Scientific | 2904F24 | |
| 20% paraformaldehyde (PFA) | Electron Microscopy Sciences | 15713-S | |
| Normal Goat Serum (NGS), filtered | Lampire Biological Laboratories | 7332500 | Aliquot and freeze upon receipt |
| Chicken anti-GFP | Aves Labs | GFP-1020 | Use at 1:500 dilution |
| Rabbit anti-DsRed | Clontech | 632496 | Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.) |
| Mouse anti-Bruchpilot | Developmental Studies Hybridoma Bank | nc82 | Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain |
| FITC anti-chicken | ThermoFisher Scientific | SA1-7200 | Use at 1:250 dilution |
| Alexa Fluor 568 anti-rabbit | Life Technologies | A11011 | Use at 1:250 dilution |
| Alexa Fluor 647 anti-mouse antibody | Life Technologies | A21235 | Use at 1:250 dilution |
| Slowfade Gold Antifade Reagent | Life Technologies | S36936 | |
| Microscope Slides (25 x 75 x 1.0 mm) | Fisher Scientific | 12-550-143 | |
| Cover Glass (22 x 22 mm) | Globe Scientific | 1404-15 | |
| Dumont Biology Grade Forceps, Style 3 | Ted Pella | 503 | use in non-dominant hand |
| Dumont Biology Grade Forceps, Style 5 | Ted Pella | 505 | use in dominant hand |
| LAS X image analysis software | Leica | ||
| Imaris image analysis software | Bitplane |
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