Arnold Meghan Lee, Cooper Jason, Grant Barth D, Driscoll Monica
Department of Molecular Biology and Biochemistry, Rutgers University.
Department of Molecular Biology and Biochemistry, Rutgers University;
J Vis Exp. 2020 Sep 18(163). doi: 10.3791/61368.
Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called "exophers" and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.
错误折叠蛋白的毒性和线粒体功能障碍是促使跨物种与年龄相关的功能性神经元衰退和神经退行性疾病的关键因素。尽管长期以来这些神经毒性挑战一直被认为是细胞内在的，但现在有大量证据支持，起源于一个神经元的错误折叠的人类疾病蛋白可以出现在邻近细胞中，这一现象被认为会促进人类神经退行性疾病中的病理扩散。表达聚集蛋白的秀丽隐杆线虫成年神经元可以挤出大的（约4微米）被膜包围的囊泡，其中可以包含聚集蛋白、线粒体和溶酶体。这些大囊泡被称为“外排体”，与外泌体不同（外泌体大约小100倍，且生物发生过程不同）。通过外排体排出细胞碎片可能是通过一种保守机制发生的，该机制构成了神经元蛋白质稳态和线粒体质量控制的一个基本但以前未被认识的分支，与聚集物在人类神经退行性疾病中扩散的过程相关。虽然外排体主要是在触觉神经元中表达高拷贝转基因mCherry的动物中进行研究的，但这些方案在使用荧光标记的细胞器或各类神经元中其他感兴趣的蛋白质研究外排体发生方面同样有用。这里描述了秀丽隐杆线虫外排体的物理特征、检测策略、鉴定标准、定量的最佳时机以及控制可调节外排体产生水平的应激的动物生长方案。总之，这里概述的方案细节应有助于建立跨实验室对外排体进行定量分析的标准。本文件旨在为该领域的实验室提供资源，这些实验室试图阐明外排体产生以及邻近和远处细胞对外排体作出反应的分子机制。
