【作者】 唐军；

【导师】 白云；

【作者基本信息】 第三军医大学 ， 免疫学， 2009， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease , AD)是一种中枢神经系统进行性变性疾病,临床症状可表现为进行性的认知损伤、注意力下降、记忆障碍、空间记忆缺失以及情绪失衡,生活自理能力丧失,严重危害着老年人的身体健康和生活质量。目前认为,AD是由遗传学因素、环境因素和代谢因素等多种因素,共同作用所引起的一种病理过程。其特征性病理改变为细胞外的老年斑(senile plaques,SP)沉积和神经元细胞轴突内的神经原纤维缠结,两者分别由β淀粉样蛋白(β-amyloid peptide, Aβ)和过度磷酸化的Tau蛋白组成。大量的研究证实,神经末梢的Aβ异常聚集可导致突触损伤和神经元的退行性病变。海马作为与学习记忆、空间认知等高级功能密切关联的重要脑区,也是AD病程中受累及的主要脑区之一。对海马病变的病理机制的深入研究必将有助于我们全面认识AD并为治疗提供有力线索。近年来,成年神经发生已在鸟类、爬行类、啮齿类、灵长类包括人类的不同种属间得到证实。一般认为,成年哺乳动物脑内的神经发生限于侧脑室的室下区和海马齿回(dentate gyrus,DG)的颗粒下区(subgranular zone,SGZ)。SGZ新生的神经元能迁移到颗粒细胞层并分化为成熟的颗粒神经元。海马是公认的与学习记忆相关的结构,而DG是海马回路的重要通路,与海马相关的学习任务训练能增加成年大鼠新生神经元数量。以学习记忆、认知功能障碍等为特征的AD病变中,成年海马神经发生是否异常以及其机制研究,这是近年来的研究热点。以往在不同AD病人和动物模型的研究中,观察到的海马成年神经发生出现增强或减弱的两种截然相反的报道。我们认为这种不一致性主要是由于AD患病人群的种群差异,家族性早发性AD与散发性AD的相关基因的表达差异,以及动物模型的遗传背景、动物品系等差异。目前对成年海马神经发生的调控机制的研究表明,局部微环境或者壁龛(Niche)控制神经发生。对神经干细胞(neural stem cells,NSCs)微环境的认识,在理解NSCs如何增殖、分化,最终整合到成熟的神经环路中有重要提示。从分子机制的角度看,有几个重要的微环境成分被提出。其中骨形成蛋白(bone morphogenetic protein,BMPs)和拮抗蛋白Noggin,已被证实是NSCs微环境的关键组成成分。BMP4是转化生长因子β蛋白家族中的一员,BMPs和它们的调节基因与早期胚胎发生相关,在细胞分化中起重要的的定向诱导作用。BMP4和noggin在出生后和成年中枢神经系统表达,提示BMP4和Noggin是NSCs正常分化所需微环境的关键成分,并可能在成年神经发生中也发挥重要的调控作用。已有的研究证实,Noggin与BMPs共同调控成年脑内室下区NSCs的分化。室下区室管膜细胞表达Noggin,拮抗BMPs信号作用而促进神经元产生。由此推测,室管膜通过产生Noggin来封闭内源性BMPs信号,为邻近的室下区干细胞提供有利于神经发生的微环境。Noggin和BMP4在出生后大鼠海马SGZ增殖区有表达。对于APPswe/PS1dE9转基因小鼠,成年海马齿状回NSCs的增殖与分化是否正常,神经发生异常与组织病变有无关联,微环境中有哪些重要信号分子参与成年海马神经发生,目前尚缺乏相关报道。另外,老年斑内Aβ1-42蛋白与活化的星形胶质细胞同样作为AD脑内微环境的重要组分之一,是否也参与了海马NSCs的“命运”转归的调控?机制怎样?我们推测,BMP4/Noggin、Aβ、星形胶质细胞在分子水平与细胞水平参与AD海马NSCs的异常成年神经发生的调节。本研究主要的实验内容:1. APPswe/PS1dE9小鼠成年海马神经发生水平下降。运用BrdU标记方法来观察APPswe/PS1dE9小鼠海马DG区神经干细胞的神经发生情况,并进一步观察APPswe/PS1dE9小鼠Morris水迷宫空间学习记忆行为及其特征性脑组织病理学变化。探讨APPswe/PS1dE9小鼠成年神经发生异常及其病理学、行为学的关联。结果显示:APPswe/PS1dE9小鼠在成年及老年期的海马神经发生水平下降。能够模拟AD主要病理特征的Aβ沉积、星形胶质细胞活化的老年斑结构,但无显著神经元丢失。老年期存在空间学习记忆能力障碍。结果提示,APPswe/PS1dE9小鼠成年海马神经发生下降与海马区Aβ淀粉样斑块的不断聚集存在显著的相关性。Aβ淀粉样蛋白可能参与了AD成年神经发生的异常。2. BMP4、Noggin分子参与调节APPswe/PS1dE9小鼠成年海马神经发生。采用原位杂交与免疫组化方法,观察APPswe/PS1dE9小鼠海马区BMP4和Noggin在mRNA和蛋白水平的表达情况,并且运用侧脑室注射Noggin蛋白来进一步观察海马区NSCs的增殖与分化的改变。探讨BMP4和Noggin在APPswe/ PS1dE9小鼠海马区的表达及其与成年海马神经发生的潜在联系。结果显示:BMP4 mRNA和蛋白质的高表达,Noggin的mRNA和蛋白质的低表达,都具有年龄依赖性,且二者之间具有显著的相关性。首次发现APPswe/PS1dE9小鼠海马BMP4的高表达与海马齿状回NSCs增殖能力的下降具有正相关性。Noggin的低表达与海马齿状回NSCs增殖能力的下降具有负相关性。并且运用Noggin嵌合蛋白拮抗BMP4作用,可促进APPswe/PS1dE9小鼠海马NSCs的增殖与分化。结果提示BMP4与Noggin可能协同参与海马神经发生的调控。原代分离纯化小鼠海马神经干细胞,添加不同浓度BMP4细胞因子,观察BMP4对NSCs的增殖与分化的影响。结果显示BMP4能抑制海马NSCs增殖,抑制其向神经元分化。进一步采用Noggin重组腺病毒感染神经干细胞,再加入BMP4细胞因子,以观察Noggin对NSCs的增殖和分化的影响。结果显示Noggin通过拮抗BMP4的作用,能有效促进海马NSCs的增殖,并促进其向神经元分化。3. Aβ、星形胶质细胞参与对海马神经干细胞增殖与分化的调控。成功分离原代星形胶质细胞和原代小鼠海马神经干细胞,利用Transwell共培养体系,分别观察Aβ、星形胶质细胞对NSCs的增殖与分化的影响。结果显示:在体外培养条件下,Aβ42蛋白和星形胶质细胞均可有效的诱导NSCs的细胞迁移,显著促进NSCs的细胞增殖与神经分化,但是以胶质分化为主。结果提示Aβ、星形胶质细胞可能在AD某一病程阶段,发挥正性作用,参与神经干细胞增殖与分化的调节。综上所述,本研究首次发现APPswe/PS1dE9小鼠的成年海马神经发生下降,这种异常发生状态与BMP4、Noggin在成年海马区的异常表达以及协同作用机制有密切关联。并经体外实验证实老化聚集的Aβ蛋白和活化的星形胶质细胞可以激发神经干细胞的增值与分化。这些结果提示了成年海马神经发生的下降可能是导致AD病变发生发展的重要机制之一,微环境因素也积极参与了干细胞巢神经发生的调控。本研究不仅更加深入的认识了AD成年海马神经发生的可能机制,而且为我们这些调控环节中寻求新的AD治疗靶点提供了更多理论知识。更多还原

【Abstract】 Alzheimer’s disease (AD), the most common neurodegenerative disorder and cause of dementia and memory loss in the elderly, is neuropathologically marked by the extracellular deposits of beta amyloid (Aβ) plaques,and the intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Neurologically, it is initially manifested as a series of mild cognitive impairments, deficits in short-term memory, loss of spatial memory, and emotional imbalances.Several lines of evidence have suggested that the abnormal accumulation of Aβoligomers at nerve terminals in AD can lead to the synaptic damage and ultimately to neurodegeneration. More recent studies have uncovered evidence that neurodegenerative processes taking place in AD may include interference with neurogenesis in the hippocampal formation. The hippocampus is regarded as one of the most important region associated with the learning and memory,and also as the major damaged region in AD.So,the intensive investigation of the pathogenesis in the hippocampus of AD would help us to know more about AD.Adult hippocampus neurogenesis has been confirmed in birds, reptiles, rodents, and primates, including humans. Adult neurogenesis has consistently been found in the subventricular zone (SVZ) of the lateral ventricle and in the hippocampal subgranular zone. Throughout adulthood, stem cells in the subgranular zone (SGZ) of the dentate gyrus generate neuroblasts that migrate into the inner granule cell layer and differentiate into new granule cells. A critical feature of adult neurogenesis is that stem cell proliferation and cell migration are spatially restricted to a specific permissive environment . Previous reports have suggested that perturbations of neurogenesis can lead to functional changes in the hippocampus. Such findings have focused attention on the status of hippocampal neurogenesis in individuals with AD. Some studies have reported a decline in neurogenesis in AD brain, while others have reported increased neurogenesis; no clear consensus has yet emerged. Moreover, in transgenic animal models of AD, clear evidence has been presented of perturbed neurogenesis with both decreases and increases being reported.The differences in the human population of AD patients,the difference in expression of the AD-related gene between the family early-onset AD and dispersed AD,the differences in the genetic background of the mouse model of AD,all these differences might account for these contradictory results mentioned above.Much effort has been made on the molecular mechanisms that regulate adult hippocampal neurogenesis in recent years. Stem cell self-renewal and progenitor differentiation is regulated by the specialized microenviroment-or“niche”-in which these cells reside. Such niches are composed of soluble factors as well as membrane bound molecules and extracellular matrix (ECM). Adult SVZ cells themselves produce bone morphogenetic proteins (BMPs) and their receptors. Noggin, a secreted BMP antagonist, is strongly expressed in ependymal cells. Noggin encodes a BMP antagonist that binds with high affinity to BMP2/4; it is thought to bind to other BMPs but with lower affinity. Noggin binding to BMPs inhibits their activation of BMP receptors. It has been demonstrated that noggin antagonizes BMP4 to create a niche for subventricular zone neurogenesis.Precise control of BMP4 signaling in the extracellular space appears to play a critical role in multiple events during development, including neural induction,tissue patterning, epithelial- mesenchymal interactions, and in the creation of stem cell "niches" in developing and adult organs. BMPs and Noggin are known to play important roles in the brain, including the regulation of neural induction and of the stem cell“niche”in adult organs. Previous reports showed that both Noggin and BMP4 are expressed in the proliferative SGZ of postnatal rats; an antisense Noggin oligodeoxynucleotide was found to decrease cell proliferation in the dentate gyrus (DG) of adult rats. Increased BMP4 expression in the hippocampal DG might therefore contribute to the decrease in cell proliferation in the DG of the APP/PS1 transgenic mouse model. Although experimental evidence demonstrates that BMP can modulate dentate neurogenesis under pathological conditions, little is known of the role that Noggin might play in this process. On the other hand,the Aβand activated astrocyte in the senile plaques, also act as the important composition of the microenvironment in the brain of AD.Whether and how are involved in the regulation of neural stem cells in the hippocampus? So,we hypothesis that the BMP4/Noggin,the Aβand astrocytes are involved in the modulation of the adult hippocampal neurogenesis of AD at the molecular and cellular levels.To test this hypothesis , the experiments were designed as followed:1. Decreased adult neurogenesis in hippocampus of the APPswe/PS1dE9 mouse model of Alzheimer’s disease.BrdU labeled technique is applied to observe the status of the adult hippocampal neurogenesis in the APPswe/PS1dE9 mouse model of AD. Then, the capability of spatial learning and memory was tested by the Morris water maze,and the classical pathological changes were also tested.All these research were designed to explore the relationship among the adult hippocampal neurogenesis, the behavior and the pathogenesis.The data showed that adult hippocampal neurogenesis is decreased.The brain of this AD mouse is defined as the Aβdeposition and activated astrocyte,while no neural loss was observed yet.The mouse also showed a deficit of the spatial recognition. These results implied an intensive correlation among the abnormal adult neurogenesis and the progressive deposition of Aβpalques,and the possibility of the Aβpeptides involved the abnormal adult hippocampal neurogenesis.2. Involvement of BMP4 and Noggin in the regulation of the adult hippocampal neurogenesis in the APPswe/PS1dE9 mouse model of AD.The in situ hybridization(ISH) and immonuhistochemistry are used to examine that expression of BMP4 and Noggin in hippocampal DG region at the mRNA and protein levels respectively,and the Noggin/Fc chimera were injected into the lateral ventricle to investigate the role of the Noggin played in the proliferation and differentiation of the neural stem cells(NSCs) in the DG of hippocampus. We planed to explore the possible relationship between the expression of BMP4/Noggin and the adult hippocampal neurogenesis. The results showed that the higher expression of BMP4 and lower expression of Noggin are proved to be age dependent,and obviously to be correlated with each other. We firstly reported that there existed a significant positive correlation between the higher expression of BMP4 and the decreased expression of the adult neurogenesis,and also a significant negative correlation between the lower expression of Noggin and the decreased expression of the adult neurogenesis.The application of the antagonism of the Noggin to BMP4 would promote the proliferation and differentiation of the hippocampal NSCs.These might imply that BMP4 and Noggin might co-modulate the adult neurogenesis.We isolated and purified the hippocampal NSCs of mouse and added different dosage of the BMP4 into the cultural medium of NSCs to observe the influence of BMP4 on the proliferation and differentiation of the hippocampal NSCs.The data showed that the BMP4 inhibited the proliferation and differentiation of the hippocampal NSCs,and inhibited its differentiation in neurons. The Noggin- recombinated Adenovirus was used to infect the NSCs to prove the effect of Noggin on the proliferation and differentiation of the hippocampal NSCs.Results showed that Noggin could effectively promote the proliferation and differentiation of the hippocampal NSCs,and help the induction of neuronal differentiation.3. Involvement of the Aβand astrocyte in the modulation of the proliferation and differentiation of the hippocampal NSCs.The mouse primary astrocytes were isolated and co-cultured with astrocyte in Transwell system.The aged Aβ1-42 peptides were also used to test the induced migration of the mouse NSCs and the proliferation,neural differentiation of NSCs.The in vitro study demonstrates that the aged Aβ1-42 peptides could induce the significant migration of hippocampal NSCs compared with the NS control group,and the astrocyte combined with the aged Aβ1-42 peptides could promote the percentage of the S phase in the cell cycle. The flow cytometry(FCM) also showed more proliferative NSCs,and enhance the differentiation of NSCs in vitro,although the NSCs-drived astroglia accounted for most of the new born cells. We might hypothesis that the Aβand astrocyte might play a positive role in the regulation of the proliferation,neural differentiation of NSCs under early pathological process of AD.In conclusion, the present study indicates the adult hippocampal neurogenesis in the APPswe/PS1dE9 mouse model of AD is decreased,and this status is significantly associated with the abnormal expression of BMP4 and Noggin in the hippocampal DG region.The in vitro study also proved that the aged- Aβand astrocyte might activate the proliferation and differentiation of NSCs..All results demonstrated that the decreased adult hippocampal neurogenesis might be responsible for the development of pathogenesis of AD.The microenvironmental factors are also involved in the regulation of the adult neurogenesis in the neural stem cell niche. Our study support us with more knowledge about the possible mechanism of the adult hippocampal neurogenesis in AD,and also suggest a prospect for the endogenous NSCs therapy for AD in future.更多还原