Peroxynitrite is a Molecular Determinant of Impaired Microvascular Energetics in Alzheimer's Disease

总结 脑微血管（BMV）在神经血管耦联（NVC）中起重要作用。线粒体是能量 细胞的传感器和受损的线粒体呼吸功能启动对NVC有害的关键信号传导 导致与阿尔茨海默病（AD）相关的认知功能受损。我们最近的技术 利用Agilent Seahorse XFe细胞外通量分析仪的突破性进展开发了线粒体呼吸 BMV中的含量测定。使用这种新的方法，我们观察到年龄依赖性的线粒体呼吸损伤 和来自雄性和雌性C57 B1/6小鼠的BMV中的生物能量学。值得注意的是，我们发现来自APP NL-G-F的BMV 敲入AD模型显示线粒体呼吸受损和加速衰老。而且我们 观察到年轻和老年雌性小鼠在微血管能量学方面表现出性别依赖性差异， 与氧化磷酸化和糖酵解对总能量产生的相对贡献有关。 最后，我们发现，过氧亚硝酸盐清除剂（FeTMPyP）处理增强非线粒体呼吸 年轻的雌性小鼠，但增强质子泄漏在年轻的雄性小鼠表明，差异过氧亚硝酸盐 活动依赖于性别。因此，我们假设过氧亚硝酸盐差异调节微血管 线粒体的功能依赖于性别，并且是夸大的年龄相关损伤的分子决定因素 NVC与AD患者认知功能的关系我们将使用8个月和20个月的雄性和雌性AD和C57 B1/6小鼠， 月龄。目的1将确定过氧亚硝酸盐对生物能量学的性别依赖性差异影响， 离体BMV中的酶活性（克雷布斯循环、糖酵解和抗氧化剂）。目标2将决定性别 过氧亚硝基阴离子对双光子致须偏转在体NVC反应的依赖性差异影响 兴奋显微镜在清醒的小鼠。目的3将确定FeTMPyP对小鼠的性别依赖性差异影响。 通过使用新的纹理辨别评估胡须依赖的感知学习的认知功能 任务这一建议的结果将挑战现有的教条，并将证明性别特异性 过氧亚硝酸盐在调节微血管生物能学和神经血管单位中的生理作用。 此外，我们的研究结果将坚定地建立微血管过氧亚硝酸盐作为一个潜在的治疗目标，在性- AD和其他神经退行性疾病的依赖性脆弱性和严重性。

Summary Brain microvessels (BMVs) play an important role in the neurovascular coupling (NVC). Mitochondria are energy sensors of cells and impaired mitochondrial respiratory function initiate critical signaling detrimental to NVC leading to impaired cognitive function associated with Alzheimer's disease (AD). Our recent technological breakthrough utilizing Agilent Seahorse XFe extracellular flux analyzer developed a mitochondrial respiration assay in BMVs. Using this novel method, we observed age-dependent impairment of mitochondrial respiration and bioenergetics in BMVs from male and female C57Bl/6 mice. Notably, we found that BMVs from APP NL-G-F Knock-in model of AD display impaired mitochondrial respiration and accelerated senescence. Furthermore, we observed that young and aged female mice display sex-dependent differences in microvascular energetics related to the relative contribution of oxidative phosphorylation and glycolysis to overall energy production. Finally, we found that peroxynitrite scavenger (FeTMPyP) treatment enhanced non-mitochondrial respiration young female mice but enhanced proton leak in young male mice indicating that the differential peroxynitrite activity is sex-dependent. Therefore, we hypothesize that peroxynitrite differentially regulates microvascular mitochondrial function sex-dependently and is the molecular determinant of exaggerated age-related impairment of NVC and cognitive function in AD. We will employ male and female AD and C57Bl/6 mice of 8 months and 20 months age. Aim 1 will determine the sex dependent differential impact of peroxynitrite on the bioenergetics and enzyme activities (Krebs cycle, glycolysis, and antioxidants) in BMVs ex vivo. Aim 2 will determine the sex dependent differential impact of peroxynitrite on in vivo NVC responses to whisker deflections by two-photon excitation microscopy in awake mice. Aim 3 will determine the sex dependent differential impact of FeTMPyP on cognitive function by assessing whisker-dependent perceptual learning using the novel texture discrimination task. The results of this proposal would challenge the existing dogma and will demonstrate the sex-specific physiological role of peroxynitrite in regulating the microvascular bioenergetics and neurovascular unit. Furthermore, our results will firmly establish microvascular peroxynitrite as a potential therapeutic target in sex- dependent vulnerability and severity of AD and other neurodegenerative diseases.