Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans.
Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans.
复制标题
DOI：
10.3791/61368
复制
发表时间：
2020-09-18
期刊：
影响因子：
--
通讯作者：
Driscoll M
中科院分区：
文献类型：
--
作者：
Arnold ML;Cooper J;Grant BD;Driscoll M
错误折叠蛋白的毒性和线粒体功能障碍是跨物种促进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。尽管这些神经毒性挑战一直被认为是细胞固有的，但现在有相当多的证据支持，源自一个神经元的错误折叠的人类疾病蛋白可以出现在邻近的细胞中，这一现象被认为促进了人类神经退行性疾病的病理传播。表达聚集蛋白的秀丽隐杆线虫成体神经元可以挤出大的（~ 4 μm）膜包裹的囊泡，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“外泌体”，与外泌体不同（外泌体约小100倍，具有不同的生物起源）。外显子体内的细胞碎片可能是通过一种保守的机制发生的，这种机制构成了神经元蛋白质平衡和线粒体质量控制的一个基本分支，但以前未被认识到，这与人类神经退行性疾病中聚集体传播的过程有关。虽然外显性主要是在触觉神经元内表达高拷贝转基因mCherry的动物中进行研究，但这些方案同样适用于使用荧光标记细胞器或其他感兴趣的蛋白质在各种类型的神经元中进行外显性研究。本文介绍了秀丽隐杆线虫的物理特征、检测策略、鉴定标准、定量的最佳时机，以及控制可调节外显菌生产水平的应激的动物生长方案。总之，这里概述的协议细节应该有助于建立跨实验室的外显子定量分析的标准。本文件旨在作为实验室在该领域的资源，以寻求详细阐述外显子产生的分子机制以及邻近和远处细胞对外显子的反应。
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.