小鼠脑内 CDK5 和 mtor 的磷酸化程度增加, 且 CREB 的活性受到抑制, 这一过程受到两种钙相关蛋白激酶 CaMKII 和 calpain1 的调控我们利用原子吸收光谱法对小鼠脑内钙含量进行了检测结果显示, T2DM 能够显著增加 AD 小鼠脑内钙含量, 引起钙超载以上结果提示,

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1 博士学位论文公示材料学生姓名张帅学号二级学科生物医学工程导师姓名王占友论文题目论文研究方向论文关键词瘦素基因敲除促进阿尔茨海默病小鼠的神经病理改变及其机制研究阿尔茨海默病发病机制研究阿尔茨海默, 糖尿病,Aβ,tau, 钙超载论文摘要 ( 中文 ) 阿尔茨海默病 (Alzheimer s disease, AD) 是一种常见于老年人以进行性痴呆为临床特征的神经退行性疾病, 其主要的病理特征包括神经元间 β- 淀粉样蛋白 (β-amyloid protein, Aβ) 的沉积形成老年斑神经元内 tau 蛋白过度磷酸化形成神经原纤维缠结 (Neurofibrillary tangles, NFTs) 以及突触和特定脑区选择性胆碱能神经元丢失糖尿病 (Diabetes mellitus, DM) 是一种以高血糖为特征的代谢疾病, 按照发病原因可以分为一型糖尿病 (Type 1 diabetes mellitus, T1DM) 和二型糖尿病 (Type 2 diabetes mellitus, T2DM) 大量的临床流行病学研究发现,T1DM 和 T2DM 均会对患者的认知能力和记忆能力造成损伤有 DM 阳性病史的人群, 其 AD 的发病率也明显增高, 并且 DM 的发病越早罹患 AD 的风险越高此外, 尸检结果显示, 部分 DM 患者胰腺和脑内均检测到 Aβ 沉积和 tau 蛋白过度磷酸化等病理, 且同时患有 DM 和 AD 的患者其病变更为明显因此, 近期有学者将 AD 称为 3 型糖尿病, 以此来强调二者之间的内在关系然而, 由于缺乏可靠的动物模型, 关于胰岛素抵抗和高血糖对 Aβ 老年斑的形成及对 tau 蛋白过度磷酸化的影响尚有矛盾的认识, 亦缺乏直接的证据 DM 和 AD 之间是否存在互作关系有待深入探讨本文利用瘦素基因敲除 (ob/ob +/- ) 小鼠与 APP/PS1 转基因小鼠进行杂交, 获得 WT ob/ob APP/PS1 和 APP/PS1-ob/ob 四种基因型小鼠其中,APP/PS1-ob/ob 小鼠为 T2DM 和 AD 的双重病症动物模型采用行为学形态学和分子生物学等手段, 探讨胰岛素抵抗所致 T2DM 促进小鼠脑内 Aβ 沉积 tau 蛋白过度磷酸化, 神经元丢失, 以及降低小鼠学习记忆能力的机制, 并分析脑内胰岛素抵抗介导的炎症性免疫反应与 AD 样大脑病理性改变的关系本项目的实施对指导 T2DM 人群预防 AD 的发生和发展, 以及寻找 AD 防治的新靶点具有重要的理论意义既往的研究证实, 瘦素基因敲除小鼠能够作为 T2DM 的可靠动物模型, 其主要表现为由胰岛素抵抗引起的慢性高血糖症状为了证实该模型小鼠的 T2DM 特征, 本文对 3 月龄各基因型小鼠体内葡萄糖和胰岛素代谢进行了检测血糖监测结果显示, 瘦素基因敲除小鼠表现出明显的高血糖症状葡萄糖耐量实验 (Glucose tolerance test, GTT) 和胰岛素耐量试验 (Insulin tolerance test, ITT) 结果说明, APP/PS1 小鼠敲除瘦素基因后表现出明显的胰岛素抵抗的二型糖尿病症状体内胰岛素检测结果显示, 瘦素基因敲除后, 小鼠血清和大脑中的胰岛素水平增加以上结果说明,APP/PS1-ob/ob 小鼠能够表现出明显的 T2DM 症状, 可以作为探讨 T2DM 和 AD 关系的重要动物模型为了探讨 T2DM 对于 APP/PS1 小鼠记忆及认知功能的影响, 本研究利用 Morris 水迷宫 (Morris water maze, MWM) 和筑巢实验对 6 月龄小鼠的行为表现进行了评价 MWM 结果显示, 瘦素基因敲除后, APP/PS1 小鼠逃避潜伏期显著增长, 逃脱距离增加筑巢实验中,APP/PS1 小鼠在 6 月龄时尚未表现出明显的认知功能障碍,APP/PS1-ob/ob 小鼠的筑巢能力明显低于其他各组小鼠行为学实验提示, 瘦素基因敲除所致糖尿病能够加重 APP/PS1 小鼠的记忆及认知损伤为了进一步探讨 T2DM 对于 AD 小鼠脑内 APP 代谢的影响, 我们利用形态学和免疫印迹的方法对 APP 代谢酶及其代谢产物和 Aβ 沉积进行了检测研究发现,APP/PS1-ob/ob 小鼠脑内老年斑的数量和面积均显著高于 APP/PS1 小鼠此外,Western blot 结果显示, T2DM 能够诱导 APP 表达增加,α- 分泌酶及其剪切产物 sappα 和 CTFα 表达下降以上结果说明,T2DM 可能够促进了 APP 代谢的淀粉样生成途径, 加重 Aβ 产生和沉积 tau 蛋白过度磷酸化和突触损伤是 AD 早期病理的主要表现为了更深入探讨慢性 T2DM 对 AD 小鼠脑内神经原纤维缠结和突触损伤的影响, 本研究利用免疫组织化学和免疫印迹法对小鼠脑内多个 tau 磷酸化位点和突触表达进行了检测结果显示, 瘦素基因敲除能够诱导 APP/PS1 小鼠脑内 Ser396 Ser202 和 Thr231 位点的 tau 磷酸化水平增加, 从而介导突触标志蛋白 SYP NMDAR2B 和 PSD95 的表达下降, 引起突触功能损伤在对 tau 磷酸化和突触损伤机制的研究中发现, 相比于 APP/PS1 小鼠,APP/PS1-ob/ob 1

2 小鼠脑内 CDK5 和 mtor 的磷酸化程度增加, 且 CREB 的活性受到抑制, 这一过程受到两种钙相关蛋白激酶 CaMKII 和 calpain1 的调控我们利用原子吸收光谱法对小鼠脑内钙含量进行了检测结果显示, T2DM 能够显著增加 AD 小鼠脑内钙含量, 引起钙超载以上结果提示, 慢性胰岛素抵抗可能通过诱导线粒体功能紊乱引起钙稳态失衡, 从而激活 calpain1/cdk5 和 CAMKII/mTOR 通路, 促进 tau 蛋白磷酸化和突触损伤增加为了进一步探究脑内胰岛素抵抗对 AD 小鼠脑内炎症性免疫反应的影响, 我们对小鼠脑内的 ROS 及炎症相关通路进行了检测研究发现, 瘦素基因敲除后,APP/PS1 小鼠脑内 AGEs/RAGE 及下游 ERK/NFκB 通路被进一步激活, 诱导 ROS 产生增加此外,Western Blot 及 RT-PCR 结果显示,T2DM 能够显著增加 AD 小鼠脑内 TNFα IL-1β 等炎症因子的表达 Aβ/GFAP 和 Aβ/IBA1 荧光共定位实验进一步证实了以上结果该实验结果提示, 慢性胰岛素抵抗可能通过激活 AGEs/RAGE 及下游 ERK/NFκB 通路, 诱导 AD 小鼠脑内的炎症反应进一步加剧综上所述, 瘦素基因敲除所致 T2DM 能够促进 APP/PS1 小鼠脑内的淀粉样生成途径, 诱导 Aβ 产生和积累增加 ; 同时,T2DM 能够诱导 APP/PS1 小鼠脑内钙含量升高, 激活钙相关蛋白激酶 calpain1 和 CAMKII, 从而促进 tau 蛋白过度磷酸化和突触损伤 ; 此外, 慢性胰岛素抵抗能够激活 APP/PS1 小鼠脑内 AGEs/RAGE 及其下游 ERK/NFκB 通路, 介导炎症和细胞凋亡加剧 2

3 论文摘要 ( 英文 ) Alzheimer's disease ( AD ) is a neurodegenerative disease that is common in the elderly and characterized by progressive dementia. The main pathological features of AD include the formation of senile plaques induced by Aβ aggregation between neurons, neurofibrillary tangles in neurons caused by tau phosphorylation as well as the loss of synapse and cholinergic neurons in specific brain regions. Diabetes mellitus ( DM ) is a metabolic disorder characterized by hyperglycemia, and it can be classified into type 1 diabetes mellitus ( T1DM ) and type 2 diabetes mellitus ( T2DM ) according to the cause of disease. A large number of clinical epidemiological studies have revealed that both T1DM and T2DM contribute to cognitive and memory impairment in patients. In addition, autopsy results showed that both Aβ deposition and tau protein phosphorylation were detected in the pancreas and brain of patients with DM, and patients with both DM and AD had more pronounced pathological features. In recent years, some scholars have called AD " type 3 diabetes " as an emphasis on the internal relationship between the two diseases. However, due to the lack of reliable animal models, there is a conflicting understanding of the effects with insulin resistance and hyperglycemia on the formation of Aβ senile plaques and tau hyperphosphorylation. Therefore, the interaction between DM and AD remains to be explored. In this study, WT, ob/ob, APP/PS1 and APP/PS1-ob/ob mice, an animal model with T2DM disease and AD, were obtained by hybridization between leptin knockout ( ob/ob +/- ) mice and APP/PS1 transgenic mice. We investigated the mechanisms by which DM, induced by insulin resistance, promoted Aβ deposition, tau phosphorylation, neuronal loss, memory and cognitive impairment in mice by the methods of behavior, morphology and molecular biology. In addition, the relationship between inflammatory immune response mediated by insulin resistance and brain pathological changes of AD like was analyzed. The implementation of this project has important theoretical significance for guiding the DM population to prevent the occurrence and development of AD, and finding new targets for the prevention and treatment of AD. Previous studies have shown that leptin knockout mice can be used as reliable animal models of T2DM, which are characterized by chronic hyperglycemia induced by insulin resistance. In order to confirm the T2DM characteristics of the model mouse, glucose and insulin metabolism in 3 month old mice were examined. The results showed that leptin knockout mice exhibited marked hyperglycemia symptoms. After knockout of leptin gene, APP/PS1 mice exhibited marked symptoms of insulin resistance in ( ITT ) and glucose tolerance test ( GTT ). The results of insulin testing indicated that leptin knockout mice had increased levels of insulin in the serum and brain. The results above suggest that APP/PS1-ob/ob mice exhibit distinct T2DM symptoms and therefore can serve as an important animal model for the study of the relationship between T2DM and AD. To investigate the effects of T2DM on memory and cognitive impairment in APP/PS1 mice, we evaluated the behaviour of 6 month old mice using the Morris water maze ( MWM ) and nesting experiments. The results indicated that the escape latency and escape distance of APP/PS1 mice in the water maze increased significantly after leptin knockout. Although APP/PS1 mice did not exhibit significant cognitive impairment, the ability of nesting in APP/PS1-ob/ob mice was significantly lower than that of other groups at the age of 6 months. Behavioral experiments have suggested that diabetes induced by leptin knockout can aggravate memory and cognitive impairments in APP/PS1 mice. To further investigate the effects of T2DM on APP metabolism in AD mice, we examined APP metabolizing enzymes, metabolites, and Aβ deposition by the methods of morphological and Western Blot. Our research showed that the number and area of senile plaques in the brain of APP/PS1-ob/ob mice were significantly higher than those in APP/PS1 mice. In addition, Western Blot show ed that DM could induce an increased expression of APP and a decreased expression of α-secretase and its shear products, sappα and CTFα. These results suggest that diabetes can contribute to the amyloid pathway of APP metabolism, thereby inducing the Aβ production and deposition. Tau hyperphosphorylation and synaptic damage are the main manifestations of AD's early pathology. To further investigate the effects of chronic diabetes mellitus on neurofibrillary tangles and synaptic damage in the brain of AD mice, multiple tau phosphorylation sites and synaptic expressions were detected by immunohistochemistry and Western blot in this study. The results showed that the levels of tau phosphorylation in Ser396, Ser202 and Thr231 sites were significantly increased in the brain of APP/PS1 mice with leptin gene knockout. Consistent with the immunohistochemical results, the expression levels of NMDAR and PSD95 protein were, PSD95 and NR2B in the brains of APP/PS1-ob/ob mice were significantly decreased when compared with APP/PS1 group, subsequent synaptic damage occurred. Studies of the mechanisms on tau phosphorylation and synaptic damage have showed a significantly increased 3

4 phosphorylation of CDK5 and mtor accompanied with CREB inhibition in the brain of APP/PS1-ob/ob mice when compared with APP/PS1 mice. This process is regulated by two calcium related protein kinases, CaMKII and calpain1. We detected the calcium content in mice brain by atomic absorption spectrometry, and it was found that diabetes could significantly increase the calcium content in the brain of AD mice and caused calcium overload. These results suggest that chronic insulin resistance may induce mitochondrial dysfunction, and lead to a disequilibrium of calcium homeostasis. Activation of calpain1/cdk5 and CAMKII/mTOR pathways promotes the phosphorylation of tau and synaptic damage. To further investigate the effects of insulin resistance on the inflammatory response in the brain of AD mice, we examined ROS level and inflammation related pathways in mice brain. Our study found that AGEs/RAGE pathway and its downstream factor ERK/NFκB were further activated in APP/PS1 mice with leptin gene knockout, similar results were found in the detection of ROS. In addition, Western Blot and RT-PCR showed that chronic diabetic conditions significantly increased the expression of inflammatory factors such as TNFα and IL-1β in the brain of AD mice. The above results were further confirmed by fluorescence co-localization of Aβ/GFAP and Aβ/IBA1. In summary, the diabetes induced by leptin gene knockout can promote the amyloid pathway in the brain of APP/PS1 mice and induce Aβ production and accumulation; In addition, diabetes can induce increased calcium content in AD mice brain and activate calcium related protein kinase, such as calpain1 and CAMKII, thereby promoting tau phosphorylation and synaptic damage; Finally, chronic insulin resistance exacerbates inflammation and apoptosis in AD mice brain by activating AGEs/RAGE and its downstream ERK/NFκB pathway. 4

5 论文主要创新点近年来, 随着人口老龄化进程的不断加速,T2DM 与 AD 的发病均呈显著上升的趋势而 T2DM 与 AD 之间的关系日益受到人们的重视, 且临床上已有许多关于 T2DM 患者存在记忆及认知障碍的报道到目前为止, 虽然针对 T2DM 和 AD 的研究已经取得了很大进展, 但对于 T2DM 加重 AD 的具体作用机制尚存在矛盾认识, 亦缺乏直接证据由此, 本课题围绕 T2DM 促进 AD 发病这一关键科学问题, 利用瘦素基因敲除 (ob/ob +/- ) 小鼠与 APP/PS1 转基因小鼠进行杂交, 获得 WT ob/ob APP/PS1 和 APP/PS1-ob/ob 四种基因型小鼠其中,APP/PS1-ob/ob 小鼠为 T2DM 和 AD 的双重病症动物模型采用行为学形态学和分子生物学等手段, 探讨胰岛素抵抗所致 T2DM 促进小鼠脑内 Aβ 沉积 tau 蛋白过度磷酸化, 神经元丢失, 以及降低小鼠学习记忆能力的机制, 并分析脑内胰岛素抵抗介导的炎症性免疫反应与 AD 样大脑病理性改变的关系攻读博士学位期间取得的学术成果注 : 按有关规范填写本人为第一作者或导师是第一作者本人为第二作者, 且第一署名单位为东北大学的研究成果 [1] Zhang S, Chai R, Yang YY, Guo SQ, Wang S, Guo T, Xu SF, Zhang YH, Wang ZY and Guo C. Chronic diabetic states worsen Alzheimer neuropathology and cognitive deficits accompanying disruption of calcium signaling in leptin-deficient APP/PS1 mice. Oncotarget, 2017, 8(27): [2] Guo C*, Zhang S*, Li JY, Ding C, Yang ZH, Chai R, Wang X, Wang ZY. Chronic hyperglycemia induced via the heterozygous knockout of pdx1 worsens neuropathological lesion in an Alzheimer mouse model. Sci Rep, 2016, 6:29396 [3] Guo C, Yang ZH*, Zhang S*, Chai R, Xue H, Zhang YH, Li JY and Wang ZY. Intranasal lactoferrin enhances alpha-secretase-dependent amyloid precursor protein processing via the ERK1/2-CREB and HIF-1alpha pathways in an Alzheimer's Disease mouse model. Neuropsychopharmacology,

Microsoft Word - 360145.doc

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