Nitrogen metabolism in sleep homeostasis and pathology

睡眠是生命所必需的，而慢性睡眠剥夺(SD)与包括阿尔茨海默病在内的人类病理有关。其他人在动物SD范例和人类睡眠和疲劳障碍中也观察到了尿素循环异常。我发现，在果蝇睡眠突变体中，多胺(PAS)通过代谢产物鸟氨酸与尿素循环结合，特别是乙酰化PAS和腐胺。我假设，从尿素循环到PA合成的氮素转移会在SD急性发作时导致睡眠，在SD慢性发作时导致神经退化。指导目标1将测试将SD与氮代谢联系起来的机制，方法是使用睡眠突变体的13C-鸟氨酸质谱学来确定鸟氨酸被引导到的代谢物，并通过酶分析来评估慢性SD下的氮代谢体的催化差异。还将在更严重的SD下进行质谱分析，以确定这是否反映了PA图谱中的慢性SD。指导的Aim 2将测试PA补充剂和广泛表达的促进腐胺合成的RNAi如何促进野生型果蝇的睡眠。亚群RNAi睡眠实验将评估PA睡眠反应涉及哪些神经回路。广泛表达的RNAi睡眠实验将确定SD后的反弹睡眠是否通常需要PA，以及PA补充睡眠增加是否特别需要腐胺的产生。独立目标3将测试PA的增加是否有助于SD对阿尔茨海默氏症病理恶化的充分证明。质谱仪将测试其他人在小鼠阿尔茨海默病模型中观察到的PA增加是否会延续到多个果蝇阿尔茨海默氏症模型中，这些模型与我的苍蝇睡眠突变非常相似。我还将测试广泛的PA合成RNAi能否阻止SD在多种果蝇阿尔茨海默病模型中的恶化作用。蛋白质病理、细胞死亡、致命性和记忆力的测量将被用作衡量标准。独立目标4将测试小鼠大脑中PA的合成是否受睡眠调节和促进睡眠。在长期环境浓缩的情况下，SD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类似的PA变化。运动感知和脑电睡眠指标将被用来评估补充PA和药物耗尽PA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，增强我从这个提议中独立开始职业生涯的能力。我的训练计划建立在我的核心小鼠和苍蝇技能基础上，增加了基因工程、苍蝇记忆行为、专门的小鼠睡眠行为协议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对神经退行性变的理论知识，并增强我的指导技能。这项提议将为我提供作为一家研究机构的独立研究员取得成功所需的数据和技能，建立一个我自己的实验室，专注于动态平衡的生化调节，以及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.