High throughput assay for mitochondrial respiration in aged brain microvessels

衰老脑微血管线粒体呼吸的高通量测定

• 批准号: 9980261
• 负责人: DAVID W BUSIJA
• 金额: $ 20.29万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980261
• 关键词: Address; Affect; Age; Age of Onset; Age-Months; Aging; Alzheimer' s Disease; Arteries; Blood Circulation; Blood Vessels; Brain; Caliber; Cells; Cephalic; Cerebrovascular Circulation; Cerebrovascular Disorders; Cerebrovascular system; Cerebrum; Characteristics; Data; Dementia; Development; Disease; Endothelium; Estradiol; Event; Female; Frequencies; Genetic; Glycolysis; Harvest; Health; Hormonal; Impaired cognition; Label; Laboratories; Link; Location; Metabolic; Methods; Microcirculation; Mitochondria; Modality; Morphology; Mus; Neurologic; Non-Insulin-Dependent Diabetes Mellitus; Ovariectomy; Oxidative Phosphorylation; Pathology; Pathway interactions; Physiological; Play; Preparation; Process; Production; Proteins; Proteomics; Publishing; Rattus; Reactive Oxygen Species; Replacement Therapy; Respiration; Respiratory Failure; Rhodamine; Role; Severities; Severity of illness; Signal Transduction; Site; Source; Stroke; Structure; Testing; Therapeutic; Time; Vascular Dementia; Vascular Diseases; Western Blotting; Woman; age effect; age related; aged; aging brain; cerebral artery; cerebral microvasculature; cerebrovascular; critical period; density; high throughput screening; improved; in vivo; male; meetings; men; mouse model; multiphoton microscopy; negative affect; normal aging; novel; novel strategies; post stroke; preservation; prevent; respiratory; response to injury; sex; targeted treatment; therapeutic target; transcriptome sequencing

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.

衰老是脑血管疾病和随后的神经系统后遗症的主要原因。这 这些疾病的发生频率和严重程度、发病年龄以及潜在机制在男性和女性之间存在差异。 女性的患病率更高，而且还因 2 型糖尿病 (T2D) 等与年龄相关的疾病而增加。内皮细胞，一个 微循环的主要组成部分，对衰老或 T2D 的负面影响特别敏感， 线粒体似乎发挥着关键作用。阐明负面潜在机制的进展 由于无能力，影响内皮线粒体和开发有益的疗法受到阻碍 在衰老过程中实时跟踪大脑微循环中的线粒体特征 和与年龄相关的疾病。为了解决这一缺陷，我们将使用三种新方法。首先，我们将使用 我们开发的小鼠模型仅在内皮细胞中使用 Dendra2 进行线粒体基因标记 荧光蛋白（mitoDendr2 FP）。其次，我们将使用我们开发的一种新颖的高通量方法 允许通过新鲜收获的线粒体呼吸和糖酵解来确定能量产生 来自小鼠的脑微血管。糖酵解是内皮和细胞中主要的能量产生过程 氧化磷酸化 (OXPHOS) 和糖酵解随着衰老和 T2D 变化的相对重要性 不清楚。我们还将检查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。我们的初步和 已发表的数据得出了这样的总体假设：线粒体在不利变化中发挥关键作用 衰老和 T2D 期间的脑微循环，并且针对线粒体的疗法是 保护的。因此，我们推测微血管中的线粒体比线粒体中受到的不利影响更多、更早。 衰老期间的大动脉，T2D 加剧微血管中线粒体的变化，糖酵解成为 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，并且线粒体代表了 保护微血管的有用治疗靶点。我们有两个目标。目标 1：阐明机制 衰老过程中线粒体和血管的变化。我们将： a) 确定线粒体和血管 雄性和雌性老年小鼠的体内特征，b) 确定衰老对糖酵解的影响和 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，d) 探索治疗方式。目标 2：确定线粒体和 血管系统在衰老和 T2D 过程中发生变化。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) 确定衰老对糖酵解和 OXPHOS、线粒体燃料和 mitoROS，c) 阐明参与线粒体变化的机制 和血管动力学，d) 探索改善线粒体和血管功能的治疗方法。