High throughput assay for mitochondrial respiration in aged brain microvessels

衰老是脑血管疾病和随后的神经后遗症的主要因素。这个 这些疾病的频率和严重程度、发病年龄和潜在机制在男性和 与年龄有关的疾病，如2型糖尿病(T2D)，也会增加女性的患病风险。内皮细胞，一种 微循环的主要成分，对衰老或T2D的负面影响特别敏感 线粒体似乎扮演着关键角色。负向阐明潜在机制的研究进展 影响内皮细胞线粒体和开发有益的治疗方法因无法 在衰老发展过程中实时跟踪脑微循环中线粒体的特征 以及与年龄相关的疾病。为了解决这一不足，我们将使用三种新方法。首先，我们将使用 我们用Dendra2基因标记血管内皮细胞线粒体的小鼠模型 荧光蛋白(MitoDendr2 FP)。其次，我们将使用我们开发的一种新的高通量方法 允许通过线粒体呼吸和糖酵解测定新鲜收获的 取自小鼠的脑微血管。糖酵解是内皮细胞的主要能量产生过程， 氧化磷酸化(OXPHOS)和糖酵解的相对重要性随年龄和T2D而变化 不清楚。我们还将研究线粒体燃料是否发生变化。第三，我们将使用RNA测序和 蛋白质组学，探索涉及能量产生途径的潜在机制。我们的初选和 已发表的数据导致了线粒体在不利变化中发挥关键作用的总体假设 在衰老和T2D期间的大脑微循环中，针对线粒体的治疗是 防护性的。因此，我们推测，微血管中的线粒体受到不利影响的时间比 大动脉在衰老过程中，T2D使微血管中的线粒体发生变化，糖酵解成为一种 老化和T2D期间更重要的ATP来源，OXPHOS的替代燃料来源变得重要 在衰老和T2D期间，女性微血管中的线粒体更具弹性，线粒体代表 有效的治疗靶点，保护微血管。我们有两个目标。目的1：阐明心绞痛的发病机制 线粒体和血管在衰老过程中的变化。我们将：a)确定线粒体和血管 雄性和雌性老龄小鼠的体内特征，b)决定衰老对糖酵解和 OXPHOS、线粒体燃料和mitoROS，c)阐明影响线粒体和糖酵解的机制 动态，以及d)探索治疗方式。目的2：确定线粒体和线粒体的作用机制 在衰老和T2D期间血管系统的变化。我们将：a)确定线粒体和血管 老年雄性和雌性小鼠在T2D期间的特征，b)决定了衰老对糖酵解和 OXPHOS、线粒体燃料和mitoROS，c)阐明线粒体变化的机制 以及d)探索改善线粒体和血管功能的治疗方法。

Aging is a major contributor to cerebrovascular disease and subsequent neurological sequelae. The frequency and severity of these diseases, age of onset, and underlying mechanisms differ between men and women and are augmented by age-related diseases such as type 2 diabetes (T2D). The endothelium, a principle component of the microcirculation, is particularly sensitive to the negative effects of aging or T2D and mitochondria appear to play a pivotal role. Progress in elucidating the underlying mechanisms negatively affecting endothelial mitochondria and developing beneficial therapies has been obstructed due to the inability to follow mitochondrial characteristics in the brain microcirculation in real time during the development of aging and age-associated diseases. To address this deficiency, we will use three new approaches. First, we will use a mouse model which we developed with genetic labeling of mitochondria only in endothelium with Dendra2 fluorescent protein (mitoDendr2 FP). Second, we will use a novel, high throughput method we developed that allows the determination of energy production by mitochondrial respiration and glycolysis in freshly harvested brain microvessel from the mouse. Glycolysis is a major energy producing process in the endothelium and the relative importance of oxidative phosphorylation (OXPHOS) and glycolysis changes with aging and T2D is unclear. We also will examine whether mitochondrial fuels change. Third, we will use RNA Sequencing and Proteomics to explore underlying mechanisms involving energy producing pathways. Our preliminary and published data have led to the overall hypothesis that mitochondria play key roles in adverse changes in the cerebral microcirculation during aging and T2D and that therapies targeting mitochondria are protective. Thus, we speculate that mitochondria in microvessels are adversely affected more and earlier than large arteries during aging, T2D acerbates changes in mitochondria in microvessels, glycolysis becomes a more important source of ATP during aging and T2D, alternative fuel sources for OXPHOS become important during aging and T2D, mitochondria are more resilient in female microvessels, and mitochondria represent a useful therapeutic target to protect microvessels. We have 2 aims. Aim 1: Elucidate mechanisms of mitochondrial and vascular changes during aging. We will: a) determine mitochondrial and vascular characteristics in vivo in male and female aged mice, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms affecting mitochondrial and glycolytic dynamics, and d) explore treatment modalities. Aim 2: Determine mechanisms of mitochondrial and vasculature changes during aging and T2D. We will: a) determine mitochondrial and vascular characteristics in aged male and female mice during T2D, b) determine effects of aging on glycolysis and OXPHOS, mitochondrial fuel, and mitoROS, c) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and d) explore therapeutic approaches to improve mitochondrial and vascular function.