淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透
Summary
这里提供了一个协议，用于研究从不同组织和大脑不同区域分离的来自不同组织和不同区域的线粒体的不同肽和蛋白质的原生形态、前纤维和成熟的淀粉样纤维之间的相互作用。
Abstract
越来越多的证据表明，膜渗透，包括线粒体等内膜，是神经退行性疾病淀粉样体性引起的毒性的常见特征和主要机制。然而，大多数描述膜中断机制的报告都基于磷脂模型系统，而直接针对生物膜水平发生的事件的研究很少见。本文介绍了一个模型，用于研究膜层淀粉样蛋白毒性机制。对于线粒体隔离，密度梯度培养基用于获得以最小的骨髓污染获得制剂。线粒体膜完整性确认后，研究β-核糖核酸、牛胰岛素和母鸡蛋白溶解酶（HEWL）与大鼠脑线粒体（作为体外生物模型）产生的淀粉样纤维蛋白的相互作用。结果表明，用纤维束体处理脑线粒体可引起不同程度的膜渗透和ROS含量增强。这表明淀粉样纤维蛋白和线粒体膜之间的结构相关相互作用。建议淀粉样纤维的生物物理特性及其与线粒体膜的具体结合，可以解释其中一些观测结果。
Introduction
淀粉样蛋白相关疾病，称为淀粉样蛋白，构成由不同组织和器官中不溶性蛋白质沉积的出现所定义的一大群疾病1，2。其中，神经退行性疾病是蛋白质聚集出现在中枢或周围神经系统2中最常见的形式。虽然已经提出一些机制参与淀粉样粒体的毒性3，越来越多的证据表明细胞膜中断和渗透作为淀粉样蛋白病理学的主要机制4， 5.除血浆膜外，内部细胞器（即线粒体）也可能受到影响。
有趣的是，新的证据表明线粒体功能障碍在神经退行性疾病的发病机制中起着关键作用，包括阿尔茨海默氏症和帕金森病6，7。根据这个问题，许多报告表明淀粉样蛋白β-肽、β-核糖核酸、亨廷顿蛋白和ALS相关突变SOD1蛋白与线粒体8、9、10的结合和积累。 11.淀粉样粒体渗透膜的机制被认为是通过离散通道（孔）的形成和/或通过非特异性洗涤剂状机制5、12、 13.值得注意的是，这些结论大多基于涉及磷脂模型系统的报告，而直接针对生物膜中发生的事件的研究很少见。显然，这些人造脂质双层不一定反映生物膜的内在特性，包括线粒体，线粒体是异质结构，由各种各样的磷脂和蛋白质组成。
在本研究中，从大鼠大脑中分离的线粒体被用作体外生物模型，以检查β-核糖核酸（作为淀粉样蛋白）、牛胰岛素（作为模型肽显示的淀粉样纤维素）的破坏性影响。与注射局部淀粉样蛋白病有关的人类胰岛素的重要结构同源性，以及母鸡蛋白液化酶（HEWL;作为研究淀粉样蛋白聚集的常见模型蛋白）。然后通过观察线粒体麻化脱氢酶（MDH）（位于线粒体基质）和线粒体活性氧的释放，研究淀粉样纤维诱导的线粒体膜的相互作用和可能的损害物种（ROS）增强。
Protocol
所有动物实验均按照德黑兰大学医学研究所动物护理和使用委员会（IACUC）进行。通过锐化断头刀刃和采用果断和迅速的叶片运动，尽最大努力尽量减少对大鼠的痛苦和有害影响。 1. 大脑均质化和线粒体隔离 注：所有线粒体隔离试剂均根据西姆斯和安德森14号制备。 为线粒体隔离准备缓冲液 准备 100 mM Tris-HCl 溶液：重量为 …
Representative Results
该协议描述了一个模型，用于研究淀粉样纤维蛋白与大鼠脑线粒体作为体外生物模型的相互作用。对于线粒体制剂，15%（v/v）密度梯度培养基用于去除骨髓，作为脑组织的主要污染14。如图1A所示，在30，700 x g处离心产生了两个不同的物质带，即骨髓（作为波段1的主要成分）和带2，它们含有丰富的线粒体分数。 <p class=…
Discussion
大量的实验结果支持了纤维细胞聚集体细胞毒性与其与生物膜4、5相互作用和渗透的能力显著相关的假设。然而，大多数数据都基于人工脂质双层，不一定反映生物膜的内在特性，生物膜是具有多种磷脂和蛋白质的异构结构。本文以脑线粒体作为体外生物膜，介绍了一个在膜水平上研究纤维集体细胞毒性的模型。
在实验中，重要的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了伊朗赞詹基础科学高级研究所研究理事会的资助。
Materials
| 2′,7′-Dichlorodihydrofluorescein diacetate | Sigma | 35845 | |
| Ammonium sulfate | Merck | 1012171000 | |
| Black 96-well plate | Corning | ||
| Black Clear-bottomed 96-well plate | Corning | ||
| Bovine insulin | Sigma | I6634 | |
| Bovine Serum Albumin (BSA) | Sigma | A2153 | |
| BSA essentially fatty acid-free | Sigma | A6003 | |
| Centrifuge | Sigma | ||
| Crystal clear sealing tape | Corning | ||
| CuSO4 | Sigma | 451657 | |
| Dialysis bag (cut off 2 KDa) | Sigma | D2272 | |
| Dounce homogenizer | Potter Elvehjem | ||
| EDTA | Sigma | E9884 | |
| Fluorescence plate reader | BioTek | ||
| Fluorescence spectrophotometer | Cary Eclipse VARIAN | ||
| Folin | Merck | F9252 | |
| Glycine | Sigma | G7126 | |
| Guillotine | Made in Iran | ||
| HCl | Merck | H1758 | |
| Hen Egg White Lysozyme (HEWL) | Sigma | L6876 | |
| Na2CO3 | Sigma | S7795 | |
| NaH2PO4 | Sigma | S7907 | |
| NaOH | Merck | S8045 | |
| Oxaloacetate | Sigma | O4126 | |
| Percoll | GE Healthcare | ||
| Phosphate Buffer Saline (PBS) | Sigma | CS0030 | |
| PMSF | Sigma | P7626 | |
| Potassium sodium tartrate | Sigma | 217255 | |
| Quartz cuvette | Sigma | ||
| Spectrophotometer | analytik jena | SPEKOL 2000 model | |
| Succinate | Sigma | S2378 | |
| Sucrose | Merck | 1076871000 | |
| Thermomixer | Eppendorph | ||
| Thioflavin T | Sigma | T3516 | |
| Tris-HCl | Merck | 1082191000 | |
| Triton X-100 | Sigma | T9284 | |
| Tryptone | QUELAB | ||
| Water bath | Memmert | ||
| Yeast Extract | QUELAB | ||
| β-NADH | Sigma | N8129 |
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Cite This Article
Zadali, R., Ghareghozloo, E. R., Ramezani, M., Hassani, V., Rafiei, Y., Chiyaneh, S. M., Meratan, A. A. Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils. J. Vis. Exp. (151), e59883, doi:10.3791/59883 (2019).