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

摘要 脑微血管在神经血管偶联中起着重要作用。线粒体就是能量 细胞感受器和线粒体呼吸功能受损启动对NVC有害的关键信号 导致与阿尔茨海默病(AD)相关的认知功能受损。我们最新的技术 利用安捷伦赛马XFe细胞外流量分析仪的突破开发出线粒体呼吸 在BMVS中进行检测。使用这种新的方法，我们观察到线粒体呼吸的年龄依赖性损害。 以及来自雄性和雌性C57BL/6小鼠的BMV的生物能量学。值得注意的是，我们发现来自APP NL-G-F的宝马 敲入AD模型表现为线粒体呼吸功能受损，衰老加速。此外，我们 观察到幼龄和老年雌性小鼠在微血管能量学上表现出性别依赖性差异 与氧化磷酸化和糖酵解对总能量生产的相对贡献有关。 最后，我们发现，过氧亚硝酸盐清除剂(FeTMPyP)处理增强了非线粒体呼吸 但幼年雌性小鼠增加了幼年雄性小鼠的质子泄漏，表明差异过亚硝酸根 活动是由性别决定的。因此，我们假设过氧亚硝酸盐对微血管有不同的调节作用。 线粒体功能与性别相关，是年龄相关性损伤加重的分子决定因素 阿尔茨海默病患者的NVC和认知功能。我们将使用8个月和20个月的雄性和雌性AD和C57BL/6小鼠 几个月大。目标1将确定过氧亚硝酸盐对生物能量学和 体外培养的BMV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 过氧亚硝酸盐对体内NVC对双光子晶须偏转响应的依赖差分影响 清醒小鼠的激发显微镜。目标3将确定FeTMPyP的性别依赖性差异影响 使用新的纹理辨别力评估胡须依赖知觉学习的认知功能 任务。这项提案的结果将挑战现有的教条，并将展示针对性别的 过氧亚硝酸盐在微血管生物能量学和神经血管单位调节中的生理作用。 此外，我们的结果将坚定地确立微血管过氧亚硝酸盐作为性行为的潜在治疗靶点。 依赖阿尔茨海默病和其他神经退行性疾病的脆弱性和严重性。

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.