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

基于氧化还原的信号正在成为新的第二信使途径，调节身体许多部位的细胞行为，包括神经系统。然而，神经元及其轴突和突触中发生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功能和存活，目前还没有很好的理解。我们现在已经开发了新的方法，用于成像神经元氧化还原信号在完整的神经系统的基础上，最近开发的基因编码的氧化还原指标结合在体内显微镜技术的转基因表达。这些方法使我们能够（1）在外周和中枢神经系统中以高的时间和空间分辨率跟踪单个轴突线粒体中的氧化还原信号：（2）使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的变化相关联;（3）采用靶向药理学和遗传学操作来剖析这些信号背后的分子机制。这样的体内成像已经揭示，高度动态的氧化还原信号可以在轴突线粒体中通过生理挑战（例如增加的神经元活性）以及病理挑战（例如挤压或挫伤病变）来诱导。在所提出的项目中，我们现在希望使用活动依赖性（Aim 1）和损伤诱导的（Aim 2）氧化还原信号的多参数体内成像来更好地理解：（1）神经元氧化还原信号何时何地启动以及它们如何沿沿着轴突传播;（2）这些氧化还原信号如何与生理和病理钙信号的平行流整合以调节轴突功能和存活;和（3）生理和病理氧化还原变化的机理分析是否可以鉴定允许靶向病理氧化还原改变的巯基开关，同时保持生理氧化还原信号传导完整。我们相信，这种精细的理解，何时，何地，以及如何出现疾病相关的氧化还原信号是设计定制的“氧化还原修饰”的策略，可以限制神经系统的创伤，炎症和退行性疾病的氧化还原失调引起的神经元损伤所需的。

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.