中国科学院昆明动物研究所机构知识库

第一部分：白藜芦醇对阿尔兹海默病的保护治疗效应研究

近年来研究表明甲醛诱导的神经毒性在阿尔兹海默病（Alzheimer’s disease, AD）病理形成过程中起着重要的作用，异常升高的甲醛水平与记忆损伤及老年 斑、神经纤维缠结形成、神经元丢失等 AD 特征性病理症状紧密相关。白藜芦醇 （Resveratrol, Res）， 作为一种多酚类抗氧化剂，已经证明其在 AD 治疗中有潜 在的应用前景。然而， Res 介导的神经保护作用能否对抗甲醛诱导的 AD 病理 相关神经损伤还有待进一步阐明。为了解决这一问题，我们在 N2a 细胞系中研究 了甲醛处理前 Res 预孵育、Res 与甲醛同时处理及甲醛处理后 Res 孵育三种处理 方式对 N2a 细胞的保护效应。研究结果表明，Res 预孵育显著地降低了甲醛诱导 的细胞毒性、细胞凋亡率，同时剂量依赖地降低 tau 蛋白苏氨酸 181 位（pT181） 的磷酸化水平。进一步的研究表明，Res 的这种神经保护效应是通过调节两种 tau 蛋白磷酸化的关键激酶来实现的，即降低糖原合酶激酶 3β（Glycogen synthase kinase 3β，GSK-3β）活性形式（GSK-3β-Y216）的表达，同时降低钙调蛋白依赖 性蛋白激酶 II（Calmodulin-dependent protein kinase II， CaMKII）的表达水平。 此外，研究发现，Res 预孵育后参与调节 tau 蛋白脱磷酸化的蛋白磷酸酶 2A （Phosphoseryl/phosphothreonyl protein phosphatase-2A， PP2A）的表达水平显著 升高。 综上，这些研究结果表明 Res 可以保护 N2a 细胞不受甲醛诱导的损伤， 且这种保护效应是通过下调激酶 GSK-3β 和 CaMKII 的表达，同时上调磷酸酶 PP2A 的表达来分别对抗 tau 蛋白超磷化和调节 tau 蛋白脱磷酸化。这种 Res 对抗 甲醛的可能的神经保护效应机制为 AD 治疗提供了另外一种视角。

第二部分：阿尔兹海默病（AD）猕猴自发模型的筛选鉴定

阿尔兹海默病（AD）是一种多发于老人的以记忆障碍为主要特征的神经退 行性疾病，至今其病因和疾病机制仍不清楚，目前尚无有效诊疗方法。AD 疾病 机制研究和有效预防治疗药物研发已经成为全球性的公共卫生问题。为了解决这 一难题，建立能很好模拟人类 AD 发病过程和临床特点的自发猕猴 AD 模型是一 个重要途径。猕猴是非常好的研究老年化相关疾病的动物模型，与常用的啮齿类 实验动物相比，它们在系统发生上和生理学上都与人类非常相似。目前研究发现： 目前研究发现：所有的老年非人灵长类都会出现与人类 AD 相似的老年斑。但是， 猕猴能否随着老年化自发产生 AD 还不清楚。因此，为了回答这一问题，本研究 在已建立甲醛诱导的 AD 猕猴的基础上，利用中国科学院昆明动物研究所丰富的 老年猕猴资源和成熟的病理学平台和行为学训练设备和范式，采用多种技术手段 综合对老年化猕猴进行筛选：首先通过脑脊液 AD 标志物（Aβ1-40/42，总 tau 蛋白 和磷酸化 tau 蛋白等）检测及脑影像学检测初步筛选出一批潜在的 AD 猕猴，然 后进一步对这些初筛出来的老年猴进行认知能力、AD 病理指标检测，最终从老 年猕猴中筛选出自然发生的猕猴 AD 模型。实验结果表明：猕猴脑脊液中 Aβ42 的 含量随年龄升高降低，老年猕猴脑脊液中 Aβ42 水平显著低于年轻猴和中年猴， 但是 Aβ40 水平、总 tau 蛋白和磷酸化 tau 蛋白水平没有显著变化，表明老年猕猴 脑内可能形成胞内 Aβ 沉积和/或老年斑，导致脑脊液中 Aβ42 含量降低；此外， 老年猕猴脑脊液中甲醛含量显著高于年轻猴和中年猴。免疫染色结果表明，自然 死亡老年猕猴脑片中存在大量胞内 Aβ 沉积和老年斑（Aβ plaque）。这些结果与 AD 病人中发生的病理改变相似，AD 病人脑脊液 Aβ42 水平降低，脑内可观察到 老年斑。对这些初步筛选出来的潜在 AD 猕猴进行认知能力测试，CANTAB 触 屏快速认知能力测试和 WGTA 空间延迟任务测试结果均表明老年猕猴认知能力 下降，且 CANTAB 触屏认知测试评分与脑脊液 Aβ42 水平、Aβ40/ Aβ42 比例和甲 醛含量显著相关。脑组织微量活检结果表明，检测的 6 只老年猕猴中，有 4 只猕 猴前额叶中存在胞内 Aβ 沉积。这些结果提示我们，随着老年化，猕猴很有可能 自发产生类似于人类 AD 的病理及行为改变。AD 病人的最终确诊是通过病人尸 检病理结果，包括 AD 典型的 SP 和 NFTs 及大量的神经元丢失。根据前期筛选 摘要 III 结果，我们将牺牲一到两只潜在 AD 猕猴，进行 AD 相关病理指标全面检测，以 进一步验证目前获得的结果。这一研究的完成不仅将回答猕猴是否存在自然 AD 这一重要科学问题，同时有可能在国内外首次筛选出一个最接近于人类 AD 疾病 过程的自然 AD 猕猴模型，为老年痴呆症机理研究、治疗和预防提供新的平台， 推动我国 AD 研究的发展。

Part 1：Study on the Neuroprotective Effect of Resveratrol on Alzheimer's Disease

Recent studies have demonstrated that formaldehyde (FA) - induced neurotoxicity is important in the pathogenesis of Alzheimer’s disease (AD). Elevated levels of FA have been associated to memory impairments and the main hallmarks of AD pathology, including β-amyloid plaques, tau protein hyperphosphorylation and neuronal loss. Resveratrol (Res), as a polyphenol anti-oxidant, has been considered to have therapeutic potential for the treatment of AD. However, it has not been elucidated whether Res can exert its neuroprotective effects against FA-induced neuronal damages related to AD pathology. To answer this question, the effects of Res were investigated on Neuro-2a (N2a) cells prior to and after FA exposure. The experiments found that pretreatment with Res significantly decreased FA-induced cytotoxicity, cell apoptosis rates and inhibited the hyperphosphorylation of tau protein at Thr181 in a dose-dependent manner. Further tests revealed that this effect was associated to the suppression of glycogen synthase kinase (GSK-3β) activity and decreased expression of calmodulindependent protein kinase II (CaMKII), both of which are important kinases for tau protein hyperphosphorylation. In addition, Res was found to increase the expression of phosphoseryl/phosphothreonyl protein phosphatase-2A (PP2A). In summary, these findings provide evidence that Res protects N2a cells from FA-induced damages and suggests that inhibition of GSK-3β and CaMKII and the activation of PP2A by Res protect against the hyperphosphorylation and/or mediates the dephosphorylation of tau protein respectively. These possible mechanisms underlying the neuroprotective effects of Res against FA-induced damages provide another perspective on AD treatment via inhibition of tau protein hyperhosphorylation.

Part 2：Screening and Identification of a Spontaneous Rhesus Monkey Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is regarded as the most common neurodegenerative disease in human, characterized by progressive patterns of cognitive and functional impairments. The molecular mechanism of AD is poorly understood and currently there is no effective treatment. With an aging population and the associated increased social and economic burden on society, China great needs urgently to better understand and effectively treat Alzheimer’s disease. Obviously the development of proper AD models is a key element in the study of AD, especially a spontaneous model that could mimic the natural physiopathologic aspects of human AD. Rhesus monkeys are useful for the study of age-associated changes in the brain as a model that is phylogenetically and physiologically highly similar to the human. Previous studies revealed that all nonhuman primates analyzed to date could develop Aβ plaques with age as seen in humans. Nevertheless, it is still unclear if rhesus monkeys could spontaneously develop AD with aging like human do. Thus, to specifically address this issues, in this study, we attempts to screen naturally occurring (spontaneous) AD monkeys as a model of human AD, using a variety of techniques based on the successfully established new formaldehydeinduced AD macaque model and the rich resources of elderly rhesus monkeys in Kunming institute of Zoology. Cerebrospinal fluid (CSF) levels of AD biomarkers (Aβ1-40/42，total tau and phosphorylated tau etc.) as well as brain imaging will be employed to screen potential AD monkeys. Then cognitive ability (working memory) will be further assessed with CANTAB and WGTA. Finally, postmortem histological evaluations will be used to validate the AD pathologic changes of the monkeys. The result from the present study revealed that significant age-associated declines in CSF Aβ42 level, but not in Aβ40 and total tau protein and phosphorylated tau levels. The Aβ42 level of the aged monkeys was significantly lower than in the young and mid-aged subjects, while the Aβ40, total-tau and phospho-tau did not significantly differ between the groups. In the meanwhile, the formaldehyde level of the aged monkeys was also ABSTRACT VII significantly higher than in the young and mid-aged monkeys. Furthermore, massive intracellular Aβ depositions and extracellular plaques were observed in the brain of natural died old macaques using immunostaining. These findings appear to parallel changes seen with human aging, in which decreased levels of Aβ42 and increased formaldehyde levels as well as Aβ plaques can be seen in normal older adults and AD patients. The cognitive ability of potential AD monkeys were further assessed with CANTAB and WGTA. Both the CANTAB touch screen rapid cognitive ability test and spatial delayed response task showed a declined performance of old monkeys. In addition, the linear regression results showed that Aβ42 levels and formaldehyde levels were significantly correlated with performance in the CANTAB touch screen rapid cognitive ability. Immunohistology result of brain tissue biopsy showed that 4 of 6 tested aged monkeys had intracellular Aβ deposites in the frontal cortex. These results together indicate that, as evolutionary neighbor of human, monkeys may spontaneously develop AD-like pathology and behavior changes during aging, and supporting that aged rhesus monkeys would be a useful model for studying AD. The clinical diagnosis of AD can only be confirmed by histologicol examination of brain tissue. In this study, one or two potential AD macaques will be sacrificed to further validate the results obtained at present. The completion of this study will not only answer whether monkeys could spontaneously develop AD, but also will likely screen and identify a spontaneous AD monkey model that could mimic the natural disease process of human AD for the first time both at home and abroad. Which will establish a new platform for the study of mechanism, treatment and prevention of AD and promote the development of AD research in China.