Nitrogen metabolism in sleep homeostasis and pathology

睡眠稳态和病理学中的氮代谢

• 批准号: 10039794
• 负责人: JOSEPH L BEDONT
• 金额: $ 9.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10039794
• 关键词: Acute; Age; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Alzheimer' s disease pathology; Ammonia; Amyloid beta-Protein; Animals; Avena sativa; Back; Behavior; Behavioral; Biochemical; Biological Assay; Brain; Cell Death; Chronic; Coupled; Data; Disease; Dose; Drosophila genus; Electroencephalography; Enzymes; Exhibits; Fatigue; Genetic Engineering; Head; Homeostasis; Human; Individual; Institution; Isotope Labeling; Knowledge; Life; Link; Mass Spectrum Analysis; Measures; Mediating; Memory; Mentors; Mentorship; Metabolic; Motion; Mus; Nerve Degeneration; Neurons; Nitric Oxide Synthase; Nitrogen; Ornithine; Pathologic; Pathology; Pathway interactions; Pharmacology; Phase; Phenocopy; Phenotype; Physiological; Polyamines; Population; Prevention; Production; Proteins; Protocols documentation; Putrescine; RNA Interference; Recovery; Regulation; Research; Research Personnel; Research Project Grants; Rodent Model; Role; Running; Shapes; Sleep; Sleep Deprivation; Sleep Disorders; Sleep disturbances; Stress; Supplementation; Testing; Training; Urea; Urea Nitrogen; Wild Type Mouse; arginase; career; environmental enrichment for laboratory animals; enzyme activity; experimental study; fly; gamma-Aminobutyric Acid; human model; improved; insight; knock-down; metabolome; metabolomics; mutant; neural circuit; neurotoxicity; nitrogen metabolism; relating to nervous system; response; sensor; skills; sleep behavior; tau Proteins; urea cycle

Sleep is essential for life, and chronic sleep deprivation (SD) is associated with pathology including Alzheimer's disease in humans. Urea cycle abnormalities have been observed by others in animal SD paradigms and human sleep and fatigue disorders. I found that polyamines (PAs), coupled to urea cycle by the metabolite ornithine, are elevated in Drosophila sleep mutants, especially acetylated PAs and putrescine. I hypothesize that nitrogen diversion from urea cycle to PA synthesis drives sleep when SD is acute and neurodegeneration when SD is chronic. Mentored Aim 1 will test what mechanisms link SD to nitrogen metabolism by using sleep mutants for 13C-ornithine mass spectrometry to identify the metabolites that ornithine is channeled toward, and enzyme assays to assess what catalytic differences shape the nitrogen metabolome under chronic SD. Mass spectrometry will also be conducted under more acute SD to determine if this mirrors chronic SD in PA profile. Mentored Aim 2 will test how PA supplements, and broadly expressed RNAi that promote putrescine synthesis, both promote sleep in wild-type flies. Subpopulation RNAi sleep experiments will assess what neural circuits are involved in PA sleep responses. Broadly expressed RNAi sleep experiments will determine whether PAs generally are required for rebound sleep following SD, and whether production of putrescine specifically is required for PA supplement sleep increases. Independent Aim 3 will test whether PA increases contribute to the well-documented worsening of Alzheimer's pathology by SD. Mass spectrometry will test whether PA increases observed in mouse Alzheimer's models by others, which are very similar to my fly sleep mutants, carry over to multiple fly Alzheimer's models. I will also test whether broad PA synthesis RNAi that blunts production of acetyl-PAs and putrescine can block the worsening effects of SD in multiple fly Alzheimer's models. Measures of protein pathology, cell death, lethality, and memory will be used as metrics. Independent Aim 4 will test whether PA synthesis is sleep-regulated and sleep-promoting in mouse brain. Mass spectrometry under chronic environmental enrichment SD will test whether mice exhibit similar PA changes to what I observe in my fly sleep mutants. Motion-sensing and EEG sleep metrics will be used to assess whether both PA supplementation and pharmacological depletion of PAs alter sleep in mice. Any one of these four aims has the potential to launch a long-running research project if successful, enhancing my ability to launch an independent career from this proposal. My training plan builds on my core mouse and fly skillsets, adding important complementary skills in genetic engineering, fly memory behavior, specialized mouse sleep behavior protocols, and isotopic labeling metabolomics. My training plan will also enhance my theoretical knowledge of neurodegeneration and enhance my mentorship skills. This proposal will give me the data and skills I need to succeed as an independent investigator at a research institution, building out a lab of my own focused on biochemical regulation of homeostasis, and linkages to neural pathology.

睡眠对于生命至关重要，慢性睡眠剥夺 (SD) 与人类阿尔茨海默病等病理学相关。其他人在动物 SD 范式以及人类睡眠和疲劳障碍中观察到尿素循环异常。我发现果蝇睡眠突变体中通过代谢物鸟氨酸与尿素循环耦合的多胺 (PA) 升高，尤其是乙酰化 PA 和腐胺。我假设，当 SD 为急性时，氮从尿素循环到 PA 合成的转移会驱动睡眠，而当 SD 为慢性时，氮会导致神经退行性变。指导目标 1 将通过使用睡眠突变体进行 13C-鸟氨酸质谱分析来测试 SD 与氮代谢之间的联系机制，以识别鸟氨酸所引导的代谢物，并使用酶测定来评估哪些催化差异塑造了慢性 SD 下的氮代谢组。质谱分析还将在更急性的 SD 下进行，以确定这是否反映了 PA 特征中的慢性 SD。 Mentored Aim 2 将测试 PA 补充剂和促进腐胺合成的广泛表达的 RNAi 如何促进野生型果蝇的睡眠。亚群 RNAi 睡眠实验将评估哪些神经回路参与 PA 睡眠反应。广泛表达的 RNAi 睡眠实验将确定 PA 是否通常是 SD 后反弹睡眠所必需的，以及腐胺的产生是否是 PA 补充睡眠增加所必需的。独立目标 3 将测试 PA 增加是否会导致 SD 导致的阿尔茨海默病病理恶化。质谱法将测试其他人在小鼠阿尔茨海默氏症模型中观察到的 PA 增加是否会延续到多个果蝇阿尔茨海默氏症模型中，这与我的果蝇睡眠突变体非常相似。我还将测试广泛的 PA 合成 RNAi 是否可以抑制乙酰 PA 和腐胺的产生，从而阻止 SD 在多个苍蝇阿尔茨海默病模型中恶化的影响。蛋白质病理学、细胞死亡、致死率和记忆的测量将用作指标。独立目标 4 将测试小鼠大脑中 PA 合成是否具有睡眠调节和促进睡眠的作用。慢性环境富集 SD 下的质谱分析将测试小鼠是否表现出与我在果蝇睡眠突变体中观察到的类似的 PA 变化。运动感应和脑电图睡眠指标将用于评估补充 PA 和 PA 的药理消耗是否会改变小鼠的睡眠。如果成功，这四个目标中的任何一个都有可能启动一个长期的研究项目，从而增强我从该提案中开展独立职业的能力。我的培训计划以我的核心小鼠和果蝇技能为基础，增加了基因工程、果蝇记忆行为、专门的小鼠睡眠行为方案和同位素标记代谢组学方面的重要补充技能。我的培训计划还将增强我的神经退行性疾病理论知识并提高我的指导技能。该提案将为我提供作为研究机构的独立研究者取得成功所需的数据和技能，建立我自己的实验室，专注于体内平衡的生化调节以及与神经病理学的联系。