自发性阿尔茨海默病恒河猴模型的发现与鉴定

Major differences exist between induced nonhuman primate models of Alzheimer’s disease (AD) and human AD. However, as some individual rhesus monkeys age, they spontaneously demonstrate certain features that are highly similar to characteristics typically observed in patients with AD, suggesting that these particular animals may be useful as a spontaneous rhesus monkey model of AD. Unfortunately, because the features in these animals characteristic of AD are often difficult to recognize, a spontaneous rhesus monkey model of AD has not yet been established. Using the quantitative protein misfolding cyclic amplification technique we previously published in Nature Methods, we recently detected the candidate biomarker of AD misfolded β-amyloid (Aβ seeds) in the plasma collected from 3 of 82 rhesus monkeys. In two of the three monkeys positive for Aβ seeds, quantification in cerebrospinal fluid (CSF) showed a low level of Aβ42 and high levels of tau and p-tau, consistent with results from humans with AD. Analyses of the results from structural magnetic resonance imaging (sMRI) and histochemical staining along with those from their performance on delayed response tasks determined that these two animals presented with classic characteristics of AD in humans, including brain atrophy, Aβ deposition in the brain, and memory loss. In the proposed project, we will expand the number of animals used for plasma sample collection to 800 and screen only those rhesus monkeys positive for Aβ seeds using the strategy described above, including neuroimaging, learning and memory tasks, and pathological assessments, to identify animals that display characteristics consistent with those from human patients with AD. Thus, we plan to establish and support the future expansion of a population of animals that may be used as a spontaneous rhesus monkey model of AD.

诱发性阿尔茨海默病（AD）非人灵长类动物模型与人AD差异大。先前报道有些恒河猴会随年龄增长自发出现高度类似AD的特征，提示可作为自发性AD动物模型。然而AD特征在动物群中难以被发现，使得建立自发性AD恒河猴模型及种群面临瓶颈。利用我们先前在Nature Methods报道的定量蛋白质错误折叠循环扩增技术原理，以血浆中错误折叠的Aβ晶种为潜在AD标志物，我们从82例老年恒河猴血浆中检测出3例为阳性，采集阳性动物的脑脊液发现其中2例的生化指标（Aβ42 降低、tau和p-tau升高）符合疑似AD的参考标准。结构磁共振、延迟反应实验和组织化学分析显示其出现大脑萎缩、记忆下降、脑内Aβ聚集等AD典型特征。本项目拟扩大血浆样本至800例，利用上述策略筛选疑似AD恒河猴，从神经影像、学习记忆、病理等多方面对其进行鉴定，以期建立自发性AD恒河猴模型，为将来扩大该模型种群提供支撑和保障。

恒河猴和食蟹猴等灵长动物是与人亲缘关系最近的常用实验动物，其大脑的解剖结构和生理生化特征与人大脑高度相似，且与阿尔茨海默病（AD）致病关键分子Aβ的氨基酸序列相同。前期我们建立了可检测错误折叠Aβ（Aβ晶种）的定量蛋白质错误折叠循环扩增（qPMCA）技术，本项目利用qPMCA技术从800例猴血浆中检测出其中26例的血浆Aβ晶种为阳性。采集该26例血浆Aβ晶种为阳性猴的脑脊液（CSF）并检测其AD生化标志物（Aβ42、tau 和p-tau）浓度，CSF中Aβ42浓度降低、tau及p-tau浓度增高者为疑似AD猴，其中22例为疑似AD猴。通过延迟反应试验证明该22例疑似AD猴学习记忆能力比对照猴显著降低（P < 0.05），取其中3例疑似AD猴大脑海马进行免疫组织化学和免疫荧光染色分析，发现有明显的Aβ沉积、Tau病变、神经元突触丢失、小胶质细胞和星形胶质细胞增生等神经病变，与AD患者大脑核心病变高度相似，而同龄对照猴则未见相应AD样病变。通过上述策略可筛选自发性AD猴模型，并初步建立了用于筛选和鉴定自发性AD猴模型的参考指标，为下一步扩大样本建立标准化可用的AD灵长动物模型种群奠定扎实基础。