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.

睡眠对生活至关重要，慢性睡眠剥夺（SD）与包括人类的阿尔茨海默氏病在内的病理有关。其他人在动物SD范式和人的睡眠和疲劳疾病中观察到尿素周期异常。我发现，在果蝇睡眠突变体，尤其是乙酰化的PA和perrescine中，多胺（PAS）与代谢物鸟氨酸耦合到尿素周期。我假设当SD急性和神经退行性时，当SD慢性时，氮从尿素周期转移到PA的合成会使睡眠。指导的AIM 1将通过使用睡眠突变体用于13C-雌激素质谱法将SD与氮代谢联系起来，以识别鸟氨酸向鸟氨酸引导的代谢产物，并酶测定法评估哪些催化差异在慢性SD下形成氮代谢组。还将在更急性的SD下进行质谱法，以确定这是否反映了PA轮廓中的慢性SD。指导的AIM 2将测试PA补充剂的补充，并广泛表达的RNAi促进腐蚀性合成，都可以促进野生型苍蝇的睡眠。亚群RNAi睡眠实验将评估PA睡眠反应中涉及哪些神经回路。广泛表达的RNAi睡眠实验将确定SD后通常需要PAS来反弹睡眠，以及是否需要专门生产pa pa补充睡眠睡眠。独立AIM 3将测试PA增加是否有助于通过SD对阿尔茨海默氏病的有据可查的恶化。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是否会增加与我的飞式睡眠突变体非常相似的模型，它将延伸到多个阿尔茨海默氏症模型。我还将测试将乙酰基-PAS和腐烂产生的宽阔PA合成RNAi是否可以阻止多个阿尔茨海默氏症模型中SD的恶化影响。蛋白质病理学，细胞死亡，致死性和记忆的度量将被用作指标。独立目标4将测试PA合成是否受到睡眠调节和小鼠脑中的睡眠促进。慢性环境富集SD下的质谱法将测试小鼠是否表现出与我在苍蝇睡眠突变体中观察到的相似的PA变化。运动敏感性和脑电图指标将用于评估PA补充和PA的药理耗竭是否会改变小鼠的睡眠。如果成功的话，这四个目标中的任何一个都有可能启动长期运行的研究项目，从而增强了我从该提案中启动独立职业的能力。我的培训计划以我的核心鼠标和飞行技巧为基础，在基因工程，飞行记忆行为，专业的鼠标睡眠行为方案以及同位素标记代谢组学方面增加了重要的互补技能。我的培训计划还将增强我对神经退行性的理论知识，并提高我的指导能力。该提案将为我提供在研究机构成为独立研究人员的成功所需的数据和技能，建立了我自己专注于生化稳态调节的实验室，并与神经病理学联系。