The role of NOX4 and its interaction with mitochondria and Nrf2 in hyperexcitability-induced neurodegeneration

过度兴奋是许多中枢神经系统(CNS)疾病导致神经退行性变的常见机制。它发生在癫痫和多发性硬化症(MS)中，在这些疾病中，活性氧物种(ROS)已被证明发挥了作用。尽管有压倒性的证据表明它们在神经退化中起着关键作用，但靶向ROS一直具有挑战性，一旦ROS被生产出来，旨在清除ROS的临床试验基本上都失败了。一种解释是，用直接抗氧化剂清除ROS是徒劳的，因为需要大剂量和持续的抗氧化剂供应，这本身可能会产生其他不利影响。因此，以RO的来源为目标，而不是一旦RO已经产生，就是一个有吸引力的战略。这反过来需要仔细描述ROS产生的来源，因为一些可能对细胞有益。我们之前已经证明，烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH)氧化酶是产生ROS的高度专门化的酶，在超兴奋性诱导的神经退行性变中发挥关键作用。NADPH氧化酶有七种不同的亚型(NOX1-5和DUOX1和2)。在初步实验中，我们最近发现NOX4在大脑驻留细胞中高表达，并且NOX4与线粒体和核因子红系2相关因子2(NRF2)相互作用。一方面，这些相互作用是重要的，因为能量耗尽，因此线粒体作为“细胞的动力库”，在超兴奋性诱导的神经退行性变中起主导作用。另一方面，转录因子Nrf2在激活时驱动抗氧化蛋白的转录，也被证明在过度兴奋时导致神经退化。这意味着靶向NOX4可能是对抗超兴奋性诱导的神经退行性变的一种有前途的策略。我们建议首先研究NOX4在超兴奋性诱导的神经退行性变中的影响，以及在超兴奋性过程中NOX4与线粒体、NOX4与Nrf2之间的相互作用。除了对NOX4缺乏的小鼠进行分子生物学分析外，还将利用不同脑部驻留细胞(星形胶质细胞、神经元、内皮细胞和小胶质细胞)的活细胞成像技术来完成这项工作。接下来，我们将使用过度兴奋诱导的神经变性的小鼠模型来研究NOX4的作用，利用全球和组织特异性的NOX4缺陷动物，即内皮或神经元组织中NOX4缺乏的小鼠(NOX4endo-/-和NOX4nero-/-)，以及脑片中的活细胞成像分析，以及电生理学和免疫组织化学。最后，我们将通过对患有多发性硬化症的患者的死后人脑组织进行免疫组织化学分析来证实动物实验获得的数据。多发性硬化症是一种以过度兴奋诱导神经退化为特征的疾病。

Hyperexcitability is a common mechanism leading to neurodegeneration in a plethora of central nervous system (CNS) diseases. It occurs amongst others in epilepsy and multiple sclerosis (MS) where reactive oxygen species (ROS) have been shown to play a role. Despite overwhelming evidence of their critical role in neurodegeneration, targeting ROS has been challenging, and clinical trials aimed at scavenging ROS, once they have been produced, have largely failed. One explanation for this is that scavenging of ROS with direct antioxidants is futile since large doses and a sustained supply of antioxidants are needed, which by themselves might have other adverse effects. Thus, targeting sources of ROS, rather than ROS once they have already been produced, represents an appealing strategy. This in turn requires a careful characterization of the sources of ROS production, as some may be beneficial to the cell.We have previously shown that nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidases, which are enzymes highly specialized in ROS production, play a pivotal role in hyperexcitability-induced neurodegeneration. Seven different isoforms of NADPH oxidases exist (NOX1-5, and DUOX1 and 2). In preliminary experiments, we recently found high expression of NOX4 in brain resident cells, and that NOX4 interacts both with mitochondria and nuclear factor erythroid 2–related factor 2 (Nrf2). On the one hand, these interactions are important since energy depletion and thus mitochondria, as the “powerhouses of the cell”, play a lead role in hyperexcitability-induced neurodegeneration. On the other hand, the transcription factor Nrf2, which upon activation drives transcription of antioxidant proteins, has also been shown to contribute to neurodegeneration during hyperexcitability. This implies that targeting NOX4 is likely a promising strategy to combat hyperexcitability-induced neurodegeneration.We here propose to first study the impact of NOX4 on hyperexcitability-induced neurodegeneration and the interaction between NOX4 and mitochondria and NOX4 and Nrf2 during hyperexcitability. This will be done with live cell imaging techniques in different brain resident cells (astrocytes, neurons, endothelial cells and microglia) in addition to molecular biology analyses in NOX4-deficient mice. Next, we will use a mouse model of hyperexcitability-induced neurodegeneration to study the role of NOX4, taking advantage of global and tissue-specific NOX4-deficient animals, i.e. mice with a NOX4 deficiency in endothelial or neuronal tissue (NOX4endo -/- and NOX4neuro -/-) and live-cell imaging analyses in brain slices in addition to electrophysiology and immunohistochemistry. Finally, we will corroborate data obtained from animal experiments by analysing post mortem human brain tissues of patients suffering from MS, a disease characterized by hyperexcitability-induced neurodegeneration, using immunohistochemistry.