【作者】 蔡艳；

【导师】 罗学港； 严小新；

【作者基本信息】 中南大学 ， 神经生物学， 2010， 博士

【摘要】 第一部分BACE-1在AD病脑组织中的表达目的：观察BACE-1在正常及阿尔茨海默病(Alzheimer’s disease, AD)时的空间表达模式。方法：收集AD或痴呆病史超过3年的病人17例,非痴呆病人14例,不同年龄(22-34岁)恒河猴7只。首先运用尼氏染色、bielschowsky银染、免疫组织化学及western blot等方法对所收集材料进行病理诊断及分组。进而对各组脑进行p-位点APP剪切酶-1(β-site APP cleavage enzyme-1, BACE-1)免疫组织化学检测,观察BACE-1在正常和异常脑内的空间表达模式。结果：尼氏染色结果显示为结构清晰的人脑组织,老年斑和p-Tau同时存在者为AD实验组,二者均不存在者为非AD对照组。按以上标准分别将恒河猴分为老年组及正常组。分组结果显示AD实验组和非AD对照组分别有7例,恒河猴老年组3例,正常组4例。Western blot结果显示,AD实验组p-Tau、淀粉样前体蛋白(amyloid precursor protein, APP)、不同分子量BACE-1及其β位点剪切产物的蛋白表达相对于非AD对照组均有明显增加；恒河猴老年组脑内不同分子量BACE-1蛋白含量显著高于正常组。BACE-1免疫组织化学染色结果显示,非AD对照组及恒河猴正常组BACE-1的表达模式与正常小鼠相似,即最强表达在嗅球及海马苔藓纤维,脑皮质内则主要分布在神经毡内,表现为弱的类似背景染色。AD实验组及恒河猴老年组脑内BACE-1表达出现异常上调,表现为大小不一、形状各异的斑块样结构。结论：BACE-1的异常表达增加可能参与了AD及老年恒河猴脑内淀粉斑的形成第二部分BACE-1上调与Aβ沉积以及失营养性轴突病变之间的关系目的：观察AD病人及AD样恒河猴脑内BACE-1上调与Aβ沉积以及失营养性轴突病变之间的关系,揭示AD时可能的老年斑形成过程。方法：对各组邻片进行免疫组织化学DAB双标及免疫荧光双标观察其脑内BACE-1标记的失营养性神经突起与β淀粉样蛋白(β-amyloid, Aβ), APP、早老素(presenilin-1, PS1)等AD相关分子之间的时空关系。另通过BACE-1与突触囊泡素(synaptophysin, SYN)及微管相关蛋白-2(microtubule-associated protein-2, MAP-2)的免疫双标确定失营养神经突起的性质。结果：AD实验组免疫组织化学邻片结果显示脑内斑块少的样本BACE-1、Aβ空间上完全匹配,而斑块多的样本Aβ表达量明显高于BACE-1;恒河猴老年组免疫荧光双标结果显示异常表达增加的BACE-1定位于失营养性轴突末梢(BACE-1与SYN免疫共存),且与APP (BACE-1剪切底物)、PS1 (Aβ在γ位点的剪切酶)、Aβ等AD相关分子有免疫共存。肿胀轴突内表达上调的BACE-1与Aβ完全共存,轴突周围没有Aβ免疫阳性产物存在。随着肿胀轴突的进-步芽生长大,一些体积小的BACE-1免疫阳性神经突起簇周围可出现少量Aβ阳性产物,并随着神经突起簇的体积增大而增多。当BACE-1标记的失营养性神经突起形成大的玫瑰花瓣样结构时,其周围已有大量致密的Aβ阳性产物存在。结论：1.AD时过量产生的Aβ最初可能来源于失营养神经突起中的轴突成分；2. BACE-1可能参与调节失营养性轴突产生过量Aβ。第三部分BACE-1上调及淀粉斑形成的实验性机制探讨目的：探讨神经元功能降低对Tg2576小鼠脑内BACE-1及老年斑形成的影响。方法：随机获取6月龄Tg2576小鼠28只,制备左侧嗅觉功能剥夺模型以模拟AD时脑内低代谢状态,未剥夺侧(右侧)嗅球作为自身对照,存活至7、8、9、12、18、24个月。运用细胞色素C氧化酶(cytochrome c oxidase, CO)组织化学染色、免疫组织化学、western blot、ELISA等形态学、分子生物学方法观察功能剥夺对Tg2576小鼠嗅觉中枢内BACE-1及淀粉斑形成的影响。结果：邻片对比显示单侧嗅觉剥夺Tg2576小鼠嗅球及梨状皮质内BACE-1与CO表达相反,嗅觉剥夺侧相对于未剥夺侧BACE-1表达增加,CO则表达下降,提示模型制备成功。Western blot、β位点APP剪切酶活性测定和Aβ40. Aβ42的ELISA测定结果显示剥夺侧BACE-1、β位点APP剪切酶C末端剪切产物、Aβ40、Aβ42表达均高于未剥夺侧。BACE-1及Ap免疫组织化学染色邻片对比显示：随着年龄的增长Tg2576小鼠嗅球及梨状皮质内老年斑形成逐渐增多,且斑块最先出现在梨状皮质,然后累及嗅球。BACE-1与Aβ等免疫荧光双标结果显示Tg2576小鼠嗅球及梨状皮质内BACE-1表达在失营养性轴突末梢(BACE-1与SYN免疫共存),表达增加的BACE-1与Ap免疫共存,斑块形成过程类似于第二部分。结论：神经元的代谢功能降低可能是引起BACE-1上调继而形成老年斑的上游机制之一。更多还原

【Abstract】 PART I Expression of BACE-1 in Alzheimer’s diseaseObjective Determine the pattern of BACE-1 in the normal and AD samples.Methods Cortical samples totally of 17 patients with clinically diagnosed as AD or with a history of dementia (>3 years) and 14 nondemented patients were available for the present study. There were 7 rhesus monkeys with different ages (22-34 years-old). Nissl staining, bielschowsky staining, immunohistochemistry and western-blot methods were used for initial neuropathological screening and to make a pathologic diagnosis and experimental grouping. Furthermore, the normal and abnormal pattern of BACE-1 were detected with the immunohistochemistry.Results Those human cases with well preserved cortical cytoarchitecture in Nissl staining were sorted, and the cases selected for the present study met the following conditions:AD cases (n=7) showed both senile plaques and p-Tau pathology, whereas control cases (n=7) were free of either lesion. Aged group (n=3) and normal group (n=4) of monkey were evaluated using the same criterions as human samples. Western blot showed the elevations of some AD-related protein such as p-Tau, APP, BACE-1 and theβ-site cleavage APP C-terminal fragment (β-CTF) in the diseased brains relative to controls. Levels of the different molecules of BACE-1 proteins are elevated in the aged monkey cortices with cerebral plaques relative to plaques-free cases. BACE-1 immunohistochemistry staining showed that the pattern of BACE-1 was similar to the normal pattern in mouse. The strongest and distinct BACE-1 labeling was expressed in the olfactory bulb and hippocampus fibers and diffuse and in background-like neuropil reactivity occurred in the cortex. The elevation of BACE-1-ir plaques-like profile occurred in the AD and aged rhesus monkeys, appearing as different size and various shape. Conclusion Elevation of BACE-1 maybe promote amyloid plaques formation in the AD and old rhesus monkey PART II The relationship among elevation of BACE-1,Aβdeposition and dystrophic neuritesObjective To explore the relationship among elevation of BACE-1, A(3 deposition and dystrophic neuritis in the AD patients and AD-like rhesus monkey and reveal the potential process of amyloid pathogenesis in AD.Methods To investigate the time-space relationship of the BACE-1-ir dystrophic neuritis with some AD-related protein such asβ-amyloid and presenilin-1 in the adjacent tissues with immunohistochemistry. In addition, the nature of the dystrophic neuritis was determined with double staining of BACE-1 with synaptophysin or microtubule-associated protein-2 (MAP-2) immunofluoresence double staining.Results In adjacent sections from AD cases, BACE-1 labeled profiles appear to match site-specifically with plaques in the cortex with relatively mild plaque load. While in AD cortex with heavier amyloid load, BACE-1 labeled profiles appeared to be less numerous than Aβplaques. Double immunofluoresence from aged monkey case showed that elevated BACE-1 localized in the dystrophic axons (BACE-1 co-localized with SYN)and co-localized with some AD related protein such as APP (substrate of BACE-1), PS 1 (γ-secretase of APP) and Aβ. In the swollen axon, elevated BACE-1 can co-exist locally with A(3 completely and there was no any clear extracellular Aβdeposits surrounded. Among the small plaques, Aβ-ir was relatively weak and turned stronger with the continuous swelling and sprouting dystrophic neurites. When the BACE-1-labeled dystrophic neurite arranged in a rosette-like fashion, there were plenty of densed extracelluar deposition existed.Conclusion1. The onset of the Aβoverproduction maybe derived from the dystrophic axons 2. BACE-1 maybe modulate the dystrophic neurites and contribute to the over-load Aβ PART III The experimental exploration about the BACE-1 elevation and amyloid plaques formationObjective To determine reduced neuronal activity promote BACE-1 and the amyloid plaques formation in Tg2576 mouse olfactory centersMethods Adult Tg2576 mice (n=28) were subjected to the unilateral naris-occlusion on 6 month-old, occluded animals were allowed to survive until 7,8,9,12,18,24 months-old. CO staining, immunohistochemistry, western blot and ELISA were used for detecting the effect of the functional deprivation on the expression of BACE-1 and amyloid plaques in Tg2576 mouse olfactory centers.Results Adjacent sections revealed an inverse modulation between BACE-land CO reactivities in the olfactory bulb and piriform cortex. BACE-1 immunoreactivity is clearly stronger while CO reactivity is clearly lower on the deprived side, which meaned the animal model is successful. Elevations of BACE-1 proteins, enzymatic activity and immediate and end products ofβ-cleavage pathway of APP can be seen in the deprived olfactory bulbs relative to counterparts. The adjacent sections of BACE-1 and A(3 immunohistochemistry staining showed that the amyloid pathogenesis in Tg2576 mouse olfactory bulbs and piriform cortex is age-dependency. BACE-1 labeled neuritis and A(3 deposition occurred firstly in the piriform cortex and then in the bulb. Double immunofluoresence staining of BACE-land Aβrevealed that BACE-1 localized in the dystrophic axons (BACE-1 and SYN colocalization) in Tg2576 mouse olfactory bulbs and piriform cortex. Elevation of BACE-1 colocalized with A(3 and the amyloid plaques formation was similar to Part II.ConclusionLower neuronal activity/hypometablolism maybe one of the upstream mechanisms of BACE-1 elevation and amyloid plaques formation更多还原