Initiation, propagation and molecular integration of physiological and pathological redox signals in neurons

以氧化还原为基础的信号正在作为新的第二信使途径出现，它调节身体许多部位的细胞行为，包括神经系统。然而，发生在神经元及其轴突和突触中的氧化还原信号的机制基础，以及这些信号如何影响神经元的功能和存活，目前尚不清楚。基于最近开发的基因编码的氧化还原指示物的转基因表达，结合体内显微镜技术，我们现在已经开发出在完整的神经系统中对神经元氧化还原信号进行成像的新方法。这些方法使我们能够(1)在外周和中枢神经系统中以高时间和空间分辨率追踪单个轴突线粒体的氧化还原信号；(2)利用多参数成像将氧化还原信号与线粒体膜电位和pH的变化以及轴突和线粒体钙水平相关联；(3)采用有针对性的药理学和遗传操作来剖析这些信号背后的分子机制。这种体内成像表明，轴突线粒体中的高动态氧化还原信号既可以由生理挑战(如神经元活性增加)诱导，也可以由病理挑战(如挤压或挫伤损伤)诱导。在拟议的项目中，我们现在希望使用活动依赖(Aim1)和损伤诱导(AIM2)氧化还原信号的多参数体内成像来更好地了解：(1)神经元氧化还原信号何时何地启动，以及它们如何沿着轴突传播；(2)这些氧化还原信号如何与平行的生理和病理钙信号流整合，以调节轴突功能和存活；(3)生理和病理氧化还原变化的机制分析是否可以识别允许靶向病理性氧化还原改变的硫醇开关，同时保持生理氧化还原信号的完整性。我们认为，对于疾病相关的氧化还原信号何时、何地以及如何出现，这种精细的理解对于设计量身定制的“氧化还原调节”策略是必要的，这些策略可以限制在神经系统创伤、炎症和退行性疾病条件下氧化还原失调造成的神经元损伤。

Redox-based signals are emerging as new second messenger pathways that regulate cellular behaviour in many parts of the body, including in the nervous system. Still the mechanistic basis of the redox signals that occur in neurons and their axons and synapses, and how these signals influence neuronal function and survival is currently not well understood. We have now developed novel approaches for imaging neuronal redox signals in the intact nervous system based on the transgenic expression of recently developed genetically encoded redox indicators combined with in vivo microscopy techniques. These approaches allow us (1) to follow redox signals in individual axonal mitochondria with high temporal and spatial resolution both in the peripheral and central nervous systems; (2) to use multi-parametric imaging to correlate redox signals with changes in mitochondrial membrane potential and pH, as well as with axonal and mitochondrial calcium levels; (3) to employ targeted pharmacological and genetic manipulations to dissect the molecular mechanisms that underlie these signals. Such in vivo imaging has revealed that highly dynamic redox signals can be induced in axonal mitochondria both by physiological challenges, such as increased neuronal activity, as well as by pathological challenges, such as crush or contusion lesions. In the proposed project we now want to use multi-parametric in vivo imaging of activity-dependent (Aim1) and injury-induced (Aim2) redox signals to better understand: (1) when and where neuronal redox signals are initiated and how they travel along axons; (2) how such redox signals are integrated with parallel streams of physiological and pathological calcium signals to modulate axonal function and survival; and (3) whether the mechanistic analysis of physiological and pathological redox changes can identify thiol switches that allow targeting pathological redox alterations, while leaving physiological redox signalling intact. We believe that such a refined understanding of when, where and how disease-related redox signals arise is required for the design of tailored "redox-modifying" strategies that can limit neuronal damage caused by redox dysregulation in traumatic, inflammatory and degenerative conditions of the nervous system.