Nitrogen metabolism in sleep homeostasis and pathology

睡眠是生命所必需的，而慢性睡眠剥夺（SD）与包括人类阿尔茨海默病在内的病理学有关。其他人在动物SD范例和人类睡眠和疲劳障碍中观察到尿素循环异常。我发现多胺（PAs），通过代谢产物鸟氨酸与尿素循环偶联，在果蝇睡眠突变体中升高，特别是乙酰化PAs和腐胺。我假设，当SD是急性时，从尿素循环到PA合成的氮转移驱动睡眠，当SD是慢性时，驱动神经退行性变。指导目标1将通过使用睡眠突变体进行13 C-鸟氨酸质谱分析来确定鸟氨酸被引导的代谢物，并通过酶测定来评估慢性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。还将在更急性SD下进行质谱分析，以确定这是否反映PA特征中的慢性SD。指导目标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后反弹睡眠所需的，以及PA补充睡眠增加是否需要特异性腐胺的产生。独立目标3将测试PA增加是否有助于SD的阿尔茨海默病病理学的充分记录的恶化。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增加是否与我的果蝇睡眠突变体非常相似，是否会延续到多个果蝇阿尔茨海默氏症模型中。我还将测试是否广泛的PA合成RNAi，钝化生产的乙酰-PA和腐胺可以阻止SD在多个苍蝇阿尔茨海默氏症模型的恶化的影响。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PA合成是否在小鼠大脑中是睡眠调节和睡眠促进的。在慢性环境富集SD下的质谱法将测试小鼠是否表现出与我在我的果蝇睡眠突变体中观察到的类似的PA变化。运动传感和EEG睡眠指标将用于评估PA补充和PA的药理学消耗是否会改变小鼠的睡眠。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，如果成功的话，提高我从这个建议中开始独立职业生涯的能力。我的训练计划建立在我的核心老鼠和苍蝇技能基础上，增加了基因工程、苍蝇记忆行为、专门的老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。我的培训计划也将提高我对神经退行性疾病的理论知识，并提高我的指导技能。这个建议将给我的数据和技能，我需要成功地作为一个独立的研究机构的调查员，建立了一个我自己的实验室，专注于生化调节的稳态，和神经病理学的联系。

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.