成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究
Summary
本协议描述了单体 amyloid-β42肽在成年斑马鱼中产生淀粉样毒性的合成、表征和注射, 以建立阿尔茨海默病模型, 随后进行组织学分析和检测聚合.
Abstract
阿尔茨海默氏病 (AD) 是一种衰弱的神经退行性疾病, 其中有毒 amyloid-β42 (Aβ42) 肽的积累导致突触变性、炎症、神经元死亡和学习缺陷。由于神经干/祖细胞 (NSPCs) 的增殖能力受损, 神经元发生减少, 在 AD 的情况下, 人类无法再生丢失的神经细胞。因此, 有效的再生疗法也应提高 NSPCs 的增殖和神经源能力. 斑马鱼 (斑马鱼) 是一种再生有机体, 我们可以学习基本的分子程序, 我们可以设计处理 AD 的治疗方法。因此, 在斑马鱼中生成类似广告的模型是必要的。利用我们的方法, 我们可以引入 Aβ42肽的合成衍生物, 将组织穿透能力纳入成年斑马鱼脑, 并分析疾病病理和再生反应。比现有的方法或动物模型的优势是, 斑马鱼可以教我们如何一个脊椎动物的大脑可以自然再生, 从而帮助我们更好地治疗人类神经退行性疾病的目标内生 NSPCs。因此, 在成年斑马鱼脑中建立的淀粉样毒性模型可能为神经科学和临床医学领域的研究开辟新的途径。此外, 这种方法的简单执行, 允许 cost-effective 和有效的实验评估。这份手稿描述了 Aβ42肽的合成和注射到斑马鱼脑。
Introduction
AD 是一种慢性渐进性疾病, 其特点是大脑皮层神经元和突触的丢失1,2,3,4,5。经典的病理标志的广告是淀粉样肽的沉积和形成的纤维缠结 (NFTs)6。老年斑块, 也称为淀粉样斑块, 是由淀粉样β (a) 肽组成的β褶结构在脑实质5。Aβ42在 AD 患者中的积累在疾病进展中具有早期和关键性的作用。AD 触发一连串的事件导致突触功能障碍, 可塑性受损, 神经元丢失7,8,9,10。
硬斑马鱼的成年大脑是研究干细胞可塑性调控的优良模型 11,12,13,141516、17、18、19、20以及中枢神经系统中的各种疾病 (包括 AD21、22、23 ,24。由于大量可用的实验方法 19,20,25,26,27,28,2930,31, 这些研究是翔实和可行的。斑马鱼可以补充中枢神经系统13,15,32,33,34,35,36,37, 38, 部分使用在神经元丢失后激活的分子程序19,39,40,4142,43, 44。因此, 在斑马鱼中建立一个神经退行性疾病模型可以帮助解决脊椎动物大脑再生能力和干细胞生物学方面的新问题。
最近, 我们通过注射合成 Aβ42肽 (表 1)39在成年斑马鱼脑中建立了淀粉样毒性模型。这种注射引起了神经表型的人脑病理学 (例如, 细胞死亡, 胶质活化, 突触变性, 和记忆缺陷), 表明斑马鱼可用于诱导神经在斑马鱼脑中, Aβ42肽可通过免疫组化染色检测, 并可识别成年斑马鱼中枢神经系统再生的分子机制39。在本协议中, 我们演示了使用脑室注射液 (CVMI) 方法27,39,45,46 , 将合成的淀粉样肽注射到斑马鱼脑中。模拟淀粉样蛋白沉积 (图 1)。CVMI 提供了一种新的方式提供的多肽, 其聚合后注射作为β板结构和施加毒性。这些肽分布在整个大脑中, 以整个 rostro 尾轴的心室面积为目标45。此外, 该方法还可以分析 NSPCs 在成年斑马鱼脑内淀粉样蛋白包裹体中的形态和分子反应。这些研究将为我们提供一个成功的哺乳动物大脑修复的洞察力。我们的方法可以用来理解的必要分子机制, 成功的再生反应后, AD 样症状, 以诱导补充的丢失神经元和功能恢复。
Protocol
本议定书是欧盟准则 (2010/63) 和欧洲生物和医学鱼类模型协会 (EuFishBioMed) 在卡尔斯鲁厄中国科学院技术 (KIT) 中建议的标准程序。所有在这里描述的方法都得到了道德委员会的批准 (Landesdirektion 德累斯顿; 文档编号 TVV-52/2015). 1. 制备 #946; 42 肽 合成多肽 (参见 表 1 ) 使用标准的 9-芴 (甲氧羰基) 化学与 2-(1 h-benzotriazol-1-基)-11, 33-tetramethyluronoiumhexafluorphosphate…
Representative Results
采用高效液相色谱法纯化了合成的多肽, 并利用质谱法对纯化的淀粉样β肽进行了表征。将 HPLC 柱加热至50° c, 以改善 a 肽的分离, 并收集所有的分数。为了识别正确的合成肽, 对所有的分数进行了质谱分析。UPLC 色谱显示了化合物的纯度。在 UPLC 上产生一个峰值的 HPLC 分数 (即, 所需的淀粉样β肽的正确质量比) 进一步处理实验 (图 2)。 <p class="…
Discussion
淀粉样肽可以被修改, 包括序列变化或各种标签。例如, 可以生成一个被炒的淀粉样肽, 多肽可以在肽末端的 N 端标上荧光标记, 或用载体肽39标记。同样, 在这个协议中, 载体肽是细胞穿透肽 TR, 因为它的有效运输货物深入到脑组织39。此外, 我们的方法允许注射和分析各种多肽, 可能导致有毒的聚合50,51。因此, 我们的系…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 DZNE 和亥姆霍兹协会 (VH-NG-1021)、》、TU 德累斯顿 (FZ-111、043_261518) 和 DFG (KI1524/6) (酸) 的支持;由莱布尼茨协会 (SAW-2011-IPF-2) 和 BMBF (BioLithoMorphie 03Z2E512) (Y.Z.)。我们还要感谢乌莉霍夫曼肽合成, 南 Asokan, Prayag Murawala, 和艾利在拍摄过程中帮助田中。
Materials
| Fmoc-protected amino acids | IRIS Biotech GmbH (Marktredwitz, Germany) | Fmoc-based amino acids for solid phase peptide synthesis (SPPS) | |
| N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) | IRIS Biotech GmbH (Marktredwitz, Germany) | RL-1030 | Activator |
| Oxyma | IRIS Biotech GmbH (Marktredwitz, Germany) | RL-1180 | Racemization supressor |
| N,N-Diisopropylethylamine | IRIS Biotech GmbH (Marktredwitz, Germany) | SOL-003 | Base |
| Dimethylformamide | IRIS Biotech GmbH (Marktredwitz, Germany) | SOL-004 | Solvent |
| N-Methylmorpholine | Thermo Fisher (Kandel) GmbH, Germany | A12158 | Base |
| 1-Hydroxybenzotriazole hydrate (HOBT) | Sigma-Aldrich Co. LLC. (St. Louis, MO, USA) | 157260 ALDRICH | Activator |
| Piperidine | MERCK KGaA (Darmstadt, Germany) | 822299 | Fmoc deprotection reagent |
| Dichlormethane (DCM) | MERCK KGaA (Darmstadt, Germany) | 106050 | Solvent |
| Formic acid (FA) | MERCK KGaA (Darmstadt, Germany) | 100264 | Buffer component for HPLC |
| Trifluoroacetic acid (TFA) | MERCK KGaA (Darmstadt, Germany) | 808260 | Clevage Mixture reagent |
| Triisopropylsilane(TIS) | MERCK KGaA (Darmstadt, Germany) | 233781 ALDRICH | Clevage Mixture reagent |
| Acetonitrile (for UPLC/LCMS) | Sigma-Aldrich Laborchemikalien GmbH | 34967-1L | Solvent |
| Acetonitrile (for HPLC) | VWR International Ltd, England | 83639.320 | Solvent |
| Diethylether | VWR International Ltd, England | 23811.326 | Solvent for peptide precipitation |
| Dithiotritol (DTT) | VWR International Ltd, England | 0281-25G | Clevage Mixture reagent |
| TentaGel S RAM Fmoc rink amide resin | Rapp Polymere GmbH (Tuebingen, Germany) | S30023 | Solid phase for SPPS |
| Peptide synthesis 5 ml syringes with included filters | Intavis AG (Cologne, Germany) | 34.274 | Reaction tube for SPPS and for clevage from the Solid Phase |
| Polytetrafluoroethylene (PTFE) filter | Sartorius Stedtim (Aubagne, France) | 11806-50-N | Filteration of precipitated peptides |
| Polyvinylidenefluoride (PVDF) syringe filter | Carl Roth GmbH + Co. KG Karlsruhe | KC78.1 | Pre-filteration for HPLC |
| Peptide Synthesizer | Intavis, Cologne, Germany | ResPep SL | Automated solid-phase peptide synthesizer |
| Water Alliance HPLC | Waters, Milford Massachusetts, USA | Waters 2998, Waters e2695 | Semi-preparative reverse-phase high pressure liquid chromatography (HPLC) |
| PolymerX, bead size 10μm, 250×10 mm | Phenomenex Ltd. Germany | 00G-4328-N0 | Porous polystyrene divinylbenzene HPLC column |
| Milli-Q Advantage A10, with a Milli-Q filter | EMD Millipore Corporation, Billerica, MA, USA | LCPAK0001 | Water purification system |
| Filtration Unit | Sartorius Stedtim (Aubagne, France) | 16307 | Filtration unit for peptide precipitation |
| UPLC Aquity with UV Detector | Waters, Milford Massachusetts, USA | M09UPA 664M | Analytical reverse phase ultra HPLC for LC-MS |
| ACQUITY UPLC BEH C18, bead size 1.7 μm, 50×2.1 mm | Waters, Milford Massachusetts, USA | 186002350 | Analytical C18 column |
| ACQUITY TQ Detector | Waters, Milford Massachusetts, USA | QBB908 | Electrospray ionization mass spectrometry (ESI-MS) |
| CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6 | Martin Christ Gefriertrocknungsanlagen GmbH, Germany | 16706, 101542 | Lyophilizer with vaccum pump |
| Paradigm plate reader | Beckman Coulter | ||
| MESAB (ethyl-m-aminobenzoate methanesulphonate) | Sigma-Aldrich | A5040 | |
| Petri dishes | Sarstedt | 821.472 | |
| Phosphate-buffered saline | Life Technologies, GIBCO | 10010-056 | |
| Needle | Becton-Dickinson | 305178 | |
| Dissecting microscope | Olympus, Leica, Zeiss | Varies with the manufacturer | |
| Dumont Tweezers | World Precision Instruments | 501985 | |
| Gillies Dissecting Forceps | World Precision Instruments | 501265 | |
| Glass injection capillaries | World Precision Instruments | TWF10 | |
| PicoNozzle | World Precision Instruments | 5430-12 | |
| Pneumatic PicoPump | World Precision Instruments | SYS-PV820 | |
| Ring illuminator; Ring Light Guide | Parkland Scientific | ILL-RLG | |
| Cryostat | Leica | CM1950 |
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Citer Cet Article
Bhattarai, P., Thomas, A. K., Cosacak, M. I., Papadimitriou, C., Mashkaryan, V., Zhang, Y., Kizil, C. Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain. J. Vis. Exp. (128), e56014, doi:10.3791/56014 (2017).