Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.,ACS Chemical Neuroscience

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Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2020-08-03 , DOI: 10.1021/acschemneuro.0c00342
Saranya Radhakrishnan , Jacob Norley , Stefan Wendt ₁ , Nathan LeRoy , Hana Hall , Stevie Norcross , Sara Doan , Jordan Snaider , Brian A MacVicar ₁ , Vikki M Weake , Libai Huang , Mathew Tantama

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Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

中文翻译：

第二代红移比率传感器揭示了神经元活动依赖的氧化还原区室。

氧化应激是几种与衰老和创伤相关的神经系统疾病的标志，但神经元活动改变如何引起亚细胞氧化还原变化的确切细节仍然难以解决。目前的氧化还原敏感染料和荧光蛋白可以量化活性氧水平的空间明显变化，但需要多色探针来准确分析单细胞中特定的氧化还原动态，而这些动态可能被群体平均所掩盖。我们之前使用福斯特共振能量转移中继策略设计了基因编码的红移氧化还原敏感荧光蛋白传感器。在这里，我们开发了一种名为 rogRFP2 的第二代激发比率传感器，具有改进的红光发射功能，可用于定量活细胞成像。使用该传感器测量单个培养神经元中活性依赖性氧化还原变化，我们观察到一种反相关性，其中线粒体氧化伴随着胞质溶胶的同时减少。这种行为取决于线粒体电子传递链复合物 I 的活性，并且可以通过共培养的星形胶质细胞的存在进行调节。我们还证明，红色荧光 rogRFP2 有助于大鼠脑切片和果蝇视网膜中的比率一光子和双光子氧化还原成像。总体而言，此处报告的概念验证研究表明，这种新型 rogRFP2 氧化还原传感器可以成为了解模型生物体外和体内氧化还原生物学的强大工具。

更新日期：2020-09-02

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