Characterization of Orexin/Hypocretin Kinetics in Alzheimer's Disease

项目摘要 阿尔茨海默病（Alzheimer's disease，AD）是一种以β淀粉样蛋白（amyloid-β，Aβ）沉积为不溶性斑块、tau蛋白聚集为特征的疾病 和过度磷酸化，神经元变性，突触丢失，最终的认知功能障碍， 痴呆可溶性Aβ和tau蛋白是阿尔茨海默病发病机制的关键蛋白， 脑脊液（CSF）与睡眠觉醒活动。CSF Aβ和tau浓度增加， 在小鼠和人类的睡眠期间，根据这些发现，睡眠是 假设是阿尔茨海默病病理学的潜在标志物和/或阿尔茨海默病的可改变的风险因素， 老年痴呆症食欲素-A和食欲素-B（也称为hypocretin-1和hypocretin-2）是两种唤醒食欲的蛋白质。 促进由共同的前体多肽前食欲素原编码的神经肽。食欲素系统 调节睡眠-觉醒活动、进食行为、能量稳态和奖励系统。食欲素 缺乏会导致嗜睡症，这是一种导致白天过度嗜睡的睡眠障碍。大量证据 支持食欲素系统在阿尔茨海默病病理学发展中的作用。敲除 淀粉样蛋白前体蛋白（APP）转基因小鼠中的食欲素基因导致淀粉样蛋白沉积， 大脑中淀粉样病变的显著减少。APP转基因小鼠的研究发现， Almorexant是一种在受体阻断食欲素的药物， 增食欲素增加了它们。此外，延长治疗与almorexant减少淀粉样蛋白沉积。在 在人类中，CSF食欲素-A也与CSF Aβ、tau、磷酸化tau和α-突触核蛋白浓度相关， 患有AD和其他痴呆症的人。发作性睡病患者（即，食欲素缺乏症） 与年龄和性别相比，CSF Aβ、tau、磷酸化tau浓度和淀粉样蛋白PET上的淀粉样蛋白沉积- 匹配的控制。这些发现有力地表明食欲素是一种潜在的阿尔茨海默病 阻断食欲素将调节脑中淀粉样蛋白和潜在的tau病理。 食欲素动力学（例如，产生和清除率）尚不清楚，CSF食欲素仍然很差 在蛋白质结构和同种型水平上表征。测量CSF食欲素-A的金标准是 放射免疫法这项提案的总体目标是充分表征CSF中的食欲素亚型 使用质谱法从患有和不患有AD病理学的人中定量前体蛋白， 食欲素、食欲素-A、食欲素-B和13 C6-亮氨酸标记的和未标记的食欲素，并评估 定量AD神经变性和睡眠-觉醒变化的食欲素浓度和动力学 活动这项研究将有助于确定疾病特异性亚型，用于未来的生物标志物使用，改善 了解睡眠障碍的病理生理学，如原发性失眠，嗜睡症，和其他 中枢起源的嗜睡症，以及允许了解食欲素系统是如何参与 AD病理学和谁可能对睡眠干预有反应。

PROJECT SUMMARY Alzheimer’s disease (AD) is characterized by deposition of amyloid-β (Aβ) as insoluble plaque, tau aggregation and hyperphosphorylation, neuronal degeneration, synaptic loss, and eventual cognitive dysfunction and dementia. Soluble forms of Aβ and tau, proteins critical to Alzheimer’s disease pathogenesis, change in cerebrospinal fluid (CSF) with sleep-wake activity. CSF Aβ and tau concentrations increase during wakefulness and decrease during sleep in both mice and humans. Based on these findings, sleep is hypothesized to be a potential marker for Alzheimer’s disease pathology and/or a modifiable risk factor for Alzheimer’s disease. Orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) are two wake- promoting neuropeptides encoded by a common precursor polypeptide, prepro-orexin. The orexin system regulates sleep-wake activity, feeding behavior, energy homeostasis, and the reward system. Orexin deficiency causes narcolepsy, a sleep disorder resulting in excessive daytime sleepiness. Substantial evidence supports a role for the orexin system in the development of Alzheimer’s disease pathology. Knocking out the orexin gene in amyloid precursor protein (APP) transgenic mice that develop amyloid deposition led to a marked decrease in amyloid pathology in the brain. Studies in APP transgenic mice found that treatment with almorexant, a drug that blocks orexins at their receptors, decreased Aβ concentrations while administration of orexin increased them. Further, prolonged treatment with almorexant decreased amyloid deposition. In humans, CSF orexin-A also correlates with CSF Aβ, tau, phospho-tau, and alpha-synuclein concentrations in individuals with AD and other dementias. Patients with narcolepsy (i.e., with orexin deficiency) have reduced CSF Aβ, tau, phospho-tau concentrations, and amyloid deposition on amyloid PET compared to age- and sex- matched controls. These findings strongly suggest that orexin is a potential Alzheimer’s disease biomarker and that blocking orexin will modulate amyloid and potentially tau pathology in the brain. Orexin kinetics (e.g., production and clearance rates) are not understood and CSF orexin remains poorly characterized at the protein structure and isoform level. The gold standard for measuring CSF orexin-A is a radioimmunoassay. The overall goal of this proposal is to fully characterize orexin isoforms in CSF from humans with and without AD pathology using mass spectrometry in order to quantify prepro- orexin, orexin-A, orexin-B, and 13C6-leucine labeled and unlabeled orexins, and assess the utility of orexin concentrations and kinetics in quantifying AD neurodegeneration and changes in sleep-wake activity. This study will aid in identifying disease specific isoforms for future biomarker use, improve understanding of the pathophysiology of sleep disorders such as primary insomnia, narcolepsy, and other hypersomnias of central origin, as well as allow for an understanding of how the orexin system is involved in AD pathology and who may respond to a sleep intervention.