Nitrogen metabolism in sleep homeostasis and pathology

睡眠对生命至关重要，慢性睡眠剥夺（SD）与人类阿尔茨海默病等病理有关。尿素循环异常已在动物SD范式和人类睡眠和疲劳障碍中被观察到。我发现，通过代谢产物鸟氨酸与尿素循环偶联的多胺（PAs）在果蝇睡眠突变体中升高，尤其是乙酰化PAs和腐胺。我假设，当SD是急性时，氮从尿素循环转移到PA合成驱动睡眠，当SD是慢性时，神经变性驱动睡眠。指导的Aim 1将通过使用睡眠突变体13c -鸟氨酸质谱法来确定鸟氨酸的代谢产物，并通过酶分析来评估慢性SD下氮代谢组的催化差异，从而测试SD与氮代谢之间的联系机制。质谱分析也将在更急性的SD下进行，以确定这是否反映了PA谱中的慢性SD。受指导的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了PA睡眠反应。广泛表达的RNAi睡眠实验将确定SD后反弹睡眠是否普遍需要PA，以及PA补充睡眠增加是否特别需要腐胺的产生。独立的Aim 3将测试PA的增加是否会导致SD导致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。我还将在多个果蝇阿尔茨海默病模型中测试广泛的PA合成RNAi是否可以抑制乙酰- pas和腐胺的产生，从而阻止SD的恶化效应。测量蛋白质病理，细胞死亡，致命性和记忆将被用作指标。独立Aim 4将测试小鼠脑内PA合成是否调节和促进睡眠。在慢性环境富集SD下的质谱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相似的PA变化。运动感应和脑电图睡眠指标将用于评估PA补充和PA药物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，提高我从这个提案开始独立事业的能力。我的训练计划以我的核心老鼠和苍蝇技能为基础，在基因工程、苍蝇记忆行为、专门的老鼠睡眠行为协议和同位素标记代谢组学方面增加了重要的互补技能。我的培训计划也将增强我在神经退行性疾病方面的理论知识，提高我的指导技能。这一提议将给我所需的数据和技能，我需要成功地作为一个独立的研究者在一个研究机构，建立一个我自己的实验室，专注于生物化学调节的稳态，并与神经病理学的联系。

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.