【作者】 程忻；

【导师】 董强； 申勇； 汪寅； 王亮；

【作者基本信息】 复旦大学 ， 神经病学， 2009， 博士

【摘要】 脑淀粉样血管病（cerebral amyloid angiopathy,CAA）是一种与老龄相关的脑部小血管疾病,其病理特点是β淀粉样蛋白（amyloidβ-protein,Aβ）沉积在皮层和软脑膜中小动脉管壁的中层及外层。CAA并发的原发性非外伤性颅内出血（CAA-related hemorrhage,CAAH）是老年人自发性脑出血的常见病因,但其发生机制尚不清楚,也缺乏有效的防治手段。β-分泌酶（β-site of APP-cleavingenzyme,BACE1）是酶切淀粉样前体蛋白（amyloid precursor protein,APP）,生成Aβ的关键酶。散发性阿尔茨海默病（Alzheimer’s disease,AD）病人脑内BACE1的蛋白表达和活性水平明显升高,表明BACE1在淀粉样病变的发病机制中发挥重要作用。本研究首先采用人软脑膜和大脑皮层样本,以及不同的血管细胞株明确BACE1在脑血管壁上是否能表达及其确切的表达部位。然后采用快速尸检、病理明确诊断的CAA病变的人软脑膜血管样本,定量测定BACE1的蛋白表达和酶活性水平,同时以紧密连接（tight junctions,TJs）蛋白作为血脑屏障（blood-brain barrier,BBB）的评价指标,定量测定TJs的蛋白表达水平。最后,通过CAA的转基因动物模型——APP23小鼠和细胞模型——脑微血管内皮细胞株（bEnd.3）,探索BACE1在CAAH发病机制中的作用,以期为CAA和CAAH的防治提供新途径。第一部分BACE1在脑血管壁上的表达目的:BACE1在脑内主要表达于神经元。为了研究BACE1在CAA中的作用,首先需要明确BACE1在脑血管壁上是否能表达及其确切的表达部位。方法:采用快速尸检的正常老年人软脑膜血管和大脑皮层血管样本、人主动脉平滑肌细胞、人脐静脉内皮细胞以及脑微血管内皮细胞株（bEnd.3）等不同的组织和细胞,运用Western-blot、免疫组织化学和免疫细胞化学的方法,采用不同的BACE1抗体,检测BACE1的表达。结果:正常老年人的软脑膜血管和皮层血管壁上能够表达BACE1,与神经元表达的BACE1蛋白分子量相同,但其表达量明显低于神经元。血管内皮细胞（脐静脉内皮细胞和脑微血管内皮细胞）和平滑肌细胞均能表达BACE1,表达部位为胞浆。结论:BACE1不仅表达于神经元,还广泛分布于脑血管内皮细胞和平滑肌细胞中。在人脑微血管壁上,尤其是脑微血管内皮细胞中检测到BACE1的表达,为进一步研究BACE1在CAA和CAAH发病机制中的作用奠定了基础。第二部分BACE1在CAA病变的脑内和脑血管壁上的蛋白表达和活性目的:作为Aβ生成的关键酶,BACE1的蛋白表达和活性水平在AD病人脑内显著升高,并且第一部分已经证实BACE1在人脑微血管壁上能够表达。此部分拟明确BACE1在在有大量Aβ沉积的脑血管壁,即CAA病变的脑内和脑血管壁上的蛋白表达和活性情况。方法:随机选取病理明确诊断的6例AD合并有中重度CAA病变的患者（AD+CAA组）、6例AD患者（AD组）和6例非AD个体（对照组,Con组）,采用其快速尸检的人大脑皮层和软脑膜血管样本,Western-blot检测BACE1的蛋白表达水平以及APP经BACE1酶切后的产物——C99片段的蛋白表达水平,测定BACE1活性水平,并且定性（免疫组织化学）和定量（ELISA）观察脑血管壁上Aβ的沉积量。结果:三组研究对象在性别、年龄、死后尸检时间方面均匹配。AD+CAA组仅1例患者发生CAA并发的脑叶出血。AD+CAA组中有1例死于突发的心血管事件,1例死于急性脑叶出血,其余死于AD并发症（脱水/恶病质,肺炎,心、肾功能衰竭）,AD组患者均死于AD并发症,对照组均死于心或肾功能衰竭。脑内,AD组和AD+CAA组BACE1的蛋白表达和活性水平均显著高于对照组,而AD组与AD+CAA组之间无明显差异;软脑膜血管壁上,AD+CAA组BACE1的蛋白表达水平显著高于AD组和对照组,而AD组和对照组之间无明显差异,AD组和AD+CAA组BACE1的活性水平显著高于对照组,而AD组和AD+CAA组之间无明显差异,C99蛋白表达水平在AD+CAA组显著高于AD组和对照组。AD+CAA组的皮层血管壁上可见大量Aβ的沉积,包括Aβ₄₀和Aβ₄₂,同时伴有血管平滑肌细胞和内皮细胞的破坏和数量减少。Aβ₄₀和Aβ₄₂ ELISA定量测定的结果显示AD+CAA组软脑膜血管壁上Aβ₄₀和Aβ₄₂的沉积量显著高于对照组和AD组。结论:在有中重度CAA病变的软脑膜血管壁上,BACE1的蛋白表达和活性水平显著升高,而AD合并有中重度CAA与AD患者相比,脑内BACE1蛋白表达和活性水平无明显改变。软脑膜血管壁上BACE1的高表达和高活性,可能使APP酶解产生更多的Aβ,并最终形成CAA的病理改变。上述结果也间接提示脑血管壁可以原位产生Aβ,并且脑血管壁原位产生的Aβ是CAA病变中Aβ来源的重要组成部分。第三部分BACE1在CAA相关性脑出血中的作用研究目的:中枢神经系统微血管中相邻内皮细胞通过TJs相互连接,TJs也是BBB的重要组成成份,BBB的破坏可能是CAAH的重要原因之一。此部分拟明确CAA病变的脑血管壁上TJs的蛋白表达情况,并探索BACE1在其中发挥的作用。方法:采用与第二部分相同的快速尸检的人大脑皮层和软脑膜血管样本,Western-blot测定3组研究对象软脑膜血管壁上TJs蛋白（occludin和ZO-1）的表达水平,免疫荧光染色观察皮层血管壁上ZO-1的表达。采用CAA的动物模型——APP23转基因小鼠,Western-blot测定12月龄和24月龄小鼠皮层内occludin和ZO-1的蛋白表达水平,Perl氏普鲁士蓝染色观察脑血管周围微出血。最后,转染pcDNA-BACE1至脑微血管内皮细胞（bEnd.3）,观察BACE1对occludin的影响,并给予BACE1活性抑制剂明确BACE1作用的特异性。结果:AD+CAA组软脑膜血管壁上,occludin和ZO-1的蛋白表达水平显著低于AD组和对照组,AD组occludin和ZO-1的蛋白表达水平显著低于与对照组。AD+CAA组皮层血管壁上ZO-1的表达水平明显低于对照组,并伴有内皮细胞标记——vWF的表达量减少。12月龄的APP23小鼠皮层内ZO-1的蛋白表达水平较野生型（wild type,WT）小鼠显著下降,而此时occludin的蛋白表达水平无明显差异。24月龄时,APP23和WT小鼠皮层内ZO-1的蛋白表达水平均较低,并且与WT小鼠相比,occludin的蛋白表达水平在APP23小鼠皮层内表达显著下降。24月龄APP23小鼠皮层血管周围存在微出血。bEnd.3细胞株过表达BACE1,可使occludin的蛋白表达水平下降,给予BACE1活性抑制剂干预后,occludin的蛋白表达水平能较明显地恢复。结论:AD以及AD合并有中重度CAA病人的脑血管壁TJs存在不同程度的表达量降低,以后者更明显。APP23转基因小鼠是合适的CAA动物模型,从12月龄起出现TJs的破坏,最终可产生微出血。CAA病变的脑血管壁上高表达和高活性的BACE1可使TJs的表达下调,BBB破坏,可能是CAAH发生的重要原因之一,而BACE1可能是新的防治靶点。结论1.CAA病变的脑血管壁上BACE1的蛋白表达和活性水平明显升高,可能在脑血管壁原位生成Aβ的过程中发挥重要作用。2.CAA病变的脑血管壁上高表达和高活性的BACE1可使TJs的表达下调,BBB破坏,可能是CAAH发生的重要原因之一,BACE1可能是新的防治靶点。更多还原

【Abstract】 Cerebral amyloid angiopathy（CAA） is an age-associated condition pathologically characterized by the deposition of amyloidβ-protein（Aβ） in the medial layer of primarily small and medium-sized arteries of the cerebral cortex and leptomeninges.One of its common complications is primary non-traumatic intracerebral hemorrhage,which is also a significant cause of spontaneous intracerebral hemorrhage in the elderly.While studies demonstrated that the pathologies of CAA are associated with Alzheimer’s disease（AD）,mechanisms of Aβdeposition and CAA-related hemorrhage（CAAH） remain unclear with no effective prevention and therapeutic strategy.The generation of Aβfrom the amyloid precursor protein（APP） is initiated byβ-secretase（β-site of APP-cleaving enzyme,BACE1）.It is reported that BACE1 protein expression and enzyme activities are increased in sporadic AD brains,indicating an important role of BACE1 in the pathogenesis of AD. Here,we propose the hypothesis that BACE1 may also play an important role in CAA pathogenesis.We first utilized human leptomeningeal vessel and cortex samples,and various vessel cell lines to determine whether BACE1 can be detected on cerebral vessels.We have access to a large brain bank from which we can choose age-matched samples from tissue obtained within 3hrs after death.Through this advantage,we examined BACE1 protein expression and enzymatic activities in the leptomeninges from autopsy-confirmed AD,AD with CAA（AD+CAA）,and non AD control individuals.Besides,we also detected tight junctions（TJs） protein expression in the above leptomeningeal vessels to evaluate blood-brain barrier（BBB） function. Furthermore,we used APP23 transgenic mice,a recognized CAA mice model,and cerebral microvessel endothelial cell line to study the role of BACE1 in the pathogenesis of CAAH,expecting to provide a promising therapeutic target for CAA and CAAH.PartⅠThe expression of BACE1 on cerebral vesselsObjectives:BACE1 is regarded as a neuron-specific protein,mainly expressed in neurons in brain.Whether BACE1 is expressed on cerebral vessels needs to be determined first,in order to study roles of BACE1 in the pathogenesis of CAA. Methods:The quickly-autopsied leptomeningeal vessel and cortex vessel samples from a normal aging individual,and human aortic smooth muscle cell line （HA-VSMC）,human umbilical vein endothelial cell line（HUVEC） and mouse cerebral cortex endothelioma cell line（bEnd.3） were used to detect BACE1 expression by Western-blot,immunohistochemistry and immunocytochemistry.Both polyclonal and monoclonal BACE1 antibodies were used to confirm the findings. Results:The same BACE1 can be detected on the leptomeningeal vessels and cortex vessels of normal aging as in neurons,however its expression level in vessels is much lower than that in neurons.Vascular endothelial cells,including cerebral microvessel endothelial cells,and vascular smooth muscle ceils both express BACE1.BACE1 is located in the cytoplasm of those cells.Conclusions:BACE1 is not only expressed in neurons,but widely expressed in cerebral endothelial and smooth muscle cells as well.The detection of BACE1 in leptomenigeal vessels,especially cerebral microvessel endothelial cells,makes it possible to further study roles of BACE1 in the pathogenesis of CAA and CAAH.PartⅡBACE1 protein expression and enzyme activities in CAA brains and leptomeningeal vesselsObjectives:As the initial and rate-limiting enzyme in the production of Aβ,BACE1 protein expression and enzyme activities are increased in AD brains,and we found that BACE1 can be detected on leptomeningeal vessels in PartⅠ.Therefore,we aim to examine BACE1 protein expression and enzyme activities in CAA brains and leptomeningeal vessels in this part.Methods:We randomly chose autopsy-confirmed 6 AD with moderate and severe CAA（AD+CAA group） patients,6 AD（AD group） patients and 6 non AD control （control group） individuals.We detected BACE1 and C99 protein expression levels by Western-blot,BACE1 enzyme activities in brains and leptomeningeal vessels of those subjects.Meanwhile,we examined Aβdeposition on leptomeningeal vessels by immunohistochemistry and ELISA.Results:Three groups of subjects were well matched on gender,age,and postmortem interval.Only one patient in AD+CAA group had CAA related lobar hemorrhage.In AD+CAA group,one patient died of acute cardiovascular event,and one died of acute cerebral hemorrhage,while the others were all of AD complications （dehydration/cachexia,pneumonia,heart and renal failure）.The cause of death for subjects in AD group was all AD complications,while for subjects in control group was heart or renal failure.In brain samples,BACE1 protein expression and enzyme activities were significantly increased in AD and AD+CAA groups compared to control group,with no significant difference between AD and AD+CAA groups.As for leptomeningeal vessel samples,BACE1 protein expression were significantly elevated in AD+CAA group compared to AD and control groups,with no significant difference between AD and control groups;BACE1 enzyme activities were significantly increased in AD and AD+CAA groups compared to control group,with no significant difference between AD and AD+CAA groups.Besides,we found a significant elevation of C99 protein levels in leptomeningeal vessels of AD+CAA and AD patients compared to that in control individuals.We also found abundant deposition of Aβ₄₀ and Aβ₄₂ on cortex vessels in AD+CAA patients with loss of cerebral smooth muscle cells and endothelial cells.Aβ₄₀ and Aβ₄₂ ELISA analysis revealed significant deposition of Aβ₄₀ and Aβ₄₂ in leptomeningeal vessels of AD+CAA patients compared to that in AD and control individuals.Conclusions:BACE1 protein expression and enzyme activities are significantly increased in CAA affected leptomeningeal vessels,which may trigger more generation of Aβfrom APP and eventually result in pathologies of CAA.Elevated expression level and activity of BACE1 in CAA affected leptomeningeal vessels may also contribute to Aβin-situ production in cerebral vessels.PartⅢRoles of BACE1 in the pathogenesis of CAA related hemorrhageObjectives:One of the most important characteristics of the microvasculature of the central nervous system is the cell-to-cell contact between two adjacent endothelial cells of the cerebral capillaries through the TJs,which are also key players in the control of BBB properties.BBB breakdown might be an important cause of CAAH. Therefore,we aim to determine the TJs protein expression levels in CAA leptomeningeal vessels and to study its interaction with BACE1 in this part.Methods:The same cerebral cortex and leptomeningeal vessel samples used in PartⅡwere used here.We examined TJs（occludin and ZO-1） protein expression levels in leptomeningeal vessels of those subjects by Western-blot,and ZO-1 expression in cortex vessels by immunohistochemistry.Besides,APP23 transgenic mice,a recognized AD and CAA mice model,were used to determine TJs protein expression levels by Western-blot and observe microhemorrhage by Perl’s Prussian staining. Furthermore,bEnd.3 cell line overexpressed with pcDNA-BACE1 and BACE1 inhibitor were utilized to determine the effect of BACE1 on cerebral vessel degeneration.Results:Occludin and ZO-1 protein expression levels were significantly reduced in leptomeningeal vessels of AD+CAA group compared to AD and control groups.In AD group,we also found significantly reduced protein expression levels of occludin and ZO-1 compared to that in control group.In cortex vessels,ZO-1 expression was reduced in AD+CAA patients with loss of vWF expression as well.In 12-month-old APP23 mice,ZO-1 protein expression was significantly reduced compared to that in wild type（WT） mice,while no significant difference in occludin expression was detected between APP23 and WT mice at this age.In 24-month-old APP23 and WT mice,both expressed low levels of ZO-1,however,APP23 mice exhibited significantly lower levels of occludin.Importantly,we observed that overexpressing BACE1 in cerebral microvessel endothelial cells resulted in TJs degradation,which was prevented by BACE1 inhibitor.Conclusions:TJs protein expression levels are significantly reduced in AD and AD+CAA patients,which is even more obvious in the latter.APP23 transgenic mice exhibit TJs degradation from 12 month old and eventually results in microhemorrhage, which is a suitable mice model for CAA study.Elevated expression level and activity of BACE1 in CAA affected leptomeningeal vessels contribute to TJs degradation and BBB breakdown,which is an important cause of CAAH,and BACE1 might be a potential therapeutic target.Conclusions1.Elevated expression level and activity of BACE1 in CAA affected leptomeningeal vessels may contribute to Aβin-situ production in cerebral vessels.2.Elevated expression level and activity of BACE1 in CAA affected leptomeningeal vessels contribute to TJs degradation and BBB breakdown,which is an important cause of CAAH,and BACE1 might be a potential therapeutic target.更多还原