Miao Kun, Wei Lu
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Cent Sci. 2020 Apr 22;6(4):478-486. doi: 10.1021/acscentsci.9b01196. Epub 2020 Mar 6.
Polyglutamine (polyQ) diseases are a group of neurodegenerative disorders, involving the deposition of aggregation-prone proteins with long polyQ expansions. However, the cytotoxic roles of these aggregates remain highly controversial, largely due to a lack of proper tools for quantitative and nonperturbative interrogations. Common methods including biochemical, spectroscopic assays, and live-cell fluorescence imaging all suffer from certain limitations. Here, we propose coupling stimulated Raman scattering microscopy with deuterium-labeled glutamine for live-cell imaging, quantification, and spectral analysis of native polyQ aggregates with subcellular resolution. First, through the enrichment of deuterated glutamine in the polyQ sequence of mutant Huntingtin (mHtt) exon1 proteins for Huntington's disease, we achieved sensitive and specific stimulated Raman scattering (SRS) imaging of carbon-deuterium bonds (C-D) from aggregates without GFP labeling, which is commonly employed in fluorescence microscopy. We revealed that these aggregates became 1.8-fold denser compared to those with GFP. Second, we performed ratiometric quantifications, which indicate a surprising dependence of protein compositions on aggregation sizes. Our further calculations, for the first time, reported the absolute concentrations for sequestered mHtt and non-mHtt proteins within the same aggregates. Third, we adopted hyperspectral SRS for Raman spectroscopic studies of aggregate structures. By inducing a cellular heat shock response, a potential therapeutic approach for inhibiting aggregate formation, we found a possible aggregate intermediate state with changed solvation microenvironments. Our method may hence readily unveil new features and mechanistic insight of polyQ aggregates and pave the way for comprehensive investigations.
聚谷氨酰胺（polyQ）疾病是一组神经退行性疾病，涉及具有长聚谷氨酰胺扩展的易于聚集的蛋白质的沉积。然而，这些聚集体的细胞毒性作用仍存在很大争议，主要是由于缺乏用于定量和非侵入性研究的合适工具。包括生化、光谱分析和活细胞荧光成像在内的常用方法都有一定的局限性。在这里，我们提出将受激拉曼散射显微镜与氘标记的谷氨酰胺相结合，用于对天然聚Q聚集体进行活细胞成像、定量分析和具有亚细胞分辨率的光谱分析。首先，通过在亨廷顿舞蹈病突变型亨廷顿蛋白（mHtt）外显子1的聚Q序列中富集氘代谷氨酰胺，我们实现了对聚集体中碳-氘键（C-D）的灵敏且特异的受激拉曼散射（SRS）成像，无需荧光显微镜中常用的绿色荧光蛋白（GFP）标记。我们发现，与带有GFP标记的聚集体相比，这些聚集体的密度增加了1.8倍。其次，我们进行了比率定量分析，结果表明蛋白质组成对聚集大小有着惊人的依赖性。我们进一步的计算首次报告了同一聚集体中隔离的mHtt和非mHtt蛋白的绝对浓度。第三，我们采用高光谱SRS对聚集体结构进行拉曼光谱研究。通过诱导细胞热休克反应（一种抑制聚集体形成的潜在治疗方法），我们发现了一种可能的聚集体中间状态，其溶剂化微环境发生了变化。因此，我们的方法可能很容易揭示聚Q聚集体的新特征和作用机制，并为全面研究铺平道路。
