Department of Biomedical Genetics, University of Rochester Medical Center.
Department of Biomedical Genetics, University of Rochester Medical Center;
J Vis Exp. 2021 Sep 7(175). doi: 10.3791/61100.
The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington's disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.
在正常衰老过程中，蛋白质组（蛋白质的内稳态）保持适当功能和折叠的能力会下降，从而促进越来越多的与年龄相关的疾病的发生。例如，具有多聚谷氨酰胺扩展的蛋白质易于聚集，亨廷顿蛋白就是一个例子，同时伴随着亨廷顿病的发生。通过使用表达多聚 Q 重复序列融合到黄色荧光蛋白 (YFP) 的转基因秀丽隐杆线虫，广泛研究了蛋白质组与年龄相关的恶化。这种多聚 Q::YFP 转基因动物模型通过成像荧光焦点（即蛋白质聚集体）的渐进形成以及随后由于蛋白质组崩溃而导致的运动缺陷的发生，直接量化了与年龄相关的蛋白质组的下降。此外，多聚 Q::YFP 转基因的表达可以由组织特异性启动子驱动，从而允许在完整多细胞生物体的背景下评估跨组织的蛋白质稳定性。该模型非常适合遗传分析，因此提供了一种与寿命测定互补的定量老化的方法。我们描述了如何在衰老过程中准确测量神经元或体壁肌肉中的多聚 Q::YFP 焦点形成，以及随后行为缺陷的发生。接下来，我们强调了如何适应更高的通量，以及使用其他新兴的秀丽隐杆线虫遗传分析策略的潜在未来应用。
