阿尔茨海默病重要分泌酶BACE1及其抑制剂

阿尔茨海默病（AD）是一种最为普遍的痴呆。其病理特征是神经细胞外不溶性淀粉样蛋白Aβ以及胞内由过磷酸化tau形成的纤维缠结。这种胞外聚合物主要由Aβ4两组成,它是由淀粉样前体蛋白APP依次经β分泌酶（β-secretase）和γ分泌酶（γ～secretase）剪切所产生的。因此,通过抑制此两种酶很有可能能够减少Aβ的产生从而延缓病程。由于γ分泌酶有众多重要的生理功能,被抑制后会产生很多较为严重的副作用,因此β分泌酶的抑制剂可能是对抗AD药物研发的更有效切入点。本文主要对影响BACE1的表达及活性的相关因素和最新研制成功的β分泌酶抑制剂做简要综述。     

糖基化终末产物对人神经母细胞瘤细胞APP表达及Aβ生成的影响

目的通过研究糖基化终末产物（AGEs-BSA）以及阻断其与特异性受体RAGE的结合对培养的人神经母细胞瘤细胞（SH-SY5Y）淀粉样前体蛋白（amyloid precursor protein,APP）的表达以及β淀粉样蛋白（β-amyloid,Aβ）生成的影响。方法以培养的SH-SY5Y细胞为模型,将细胞随机分为4组,空白对照组、BSA组、AGEs-BSA组、AGEs-BSA＋抗RAGE中和抗体组。用MTT法观察细胞形态以及确定AGEs-BSA的最佳干预时间及浓度。用免疫细胞化学方法及免疫印迹方法来检测各组细胞内APP、RAGE表达和Aβ生成情况。结果不同蛋白浓度的BSA处理细胞24、48、72h,与空白对照组比较细胞MTT代谢率,APP、RAGE及Aβ的表达水平没有明显差异,（P〉0.05）;不同蛋白浓度的AGEs-BSA（〉50μg/ml）处理细胞与BSA组比较,细胞MTT代谢率明显降低,并随蛋白浓度升高差异越明显,APP、RAGE、Aβ的表达水平较BSA组明显增加（P〈0.05）,预先用抗RAGE中和抗体（1∶100）1h后再加入AGEs-BSA,APP、Aβ的表达水平较AGEs-BSA组明显减少（P〈0.05）,但仍高于BSA组（P〈0.05）。结论糖基化终末产物能够促使SH-SY5Y细胞中APP、RAGE、Aβ的表达和生成增加。通过阻断其与特异性受体RAGE的结合可以部分减少APP、Aβ的表达和生成。     

Aβ3-10重复片段质粒免疫接种AD鼠后对脑内BACE1的影响

目的探讨Aβ3-10重复片段质粒免疫接种3月龄APPswe/PSEN1双转基因(AD)鼠脑内BACE1的影响。方法应用Aβ3-10重复片段质粒免疫3月龄APPswe/PSEN1双转基因鼠,同等剂量的PBS免疫对照组及C57BL/6J组小鼠,各组小鼠均在免疫后2、4、6次后通过RT-PCR法检测BACE1 m RNA水平,Western blotting法检测BACE1/GFAP/NF-κB蛋白水平,免疫组化法观察Aβ1-42脑内分布情况。水迷宫检测其行为学改变。结果在免疫2次后BACE1 m RNA表达水平C57BL/6J组Aβ3-10组对照组(F=4.649,P=0.021);免疫4次Aβ3-10组C57BL/6J组对照组(F=115.683,P=0.001);免疫6次C57BL/6J组Aβ3-10组对照组(F=86.600,P0.001)。BACE1的蛋白表达水平在免疫2、4、6次后C57BL/6J组Aβ3-10组对照组(F=432.843,P0.001;F=57.673,P0.001;F=26.550,P=0.001),NF-κB的蛋白表达水平在免疫2次后C57BL/6J组Aβ3-10组对照组(F=109.127,P0.001);免疫4,6次后Aβ3-10组C57BL/6J组对照组(F=30.301,P0.001;F=129.967,P0.001)。GFAP的蛋白表达水平在免疫2、4、6次后C57BL/6J组Aβ3-10组对照组(F=27.782,P=0.001;F=26.132,P=0.001;F=26.450,P=0.001);Aβ1-42在脑内的分布C57BL/6J组Aβ3-10组对照组(皮质:F=5.395,P=0.021;F=47.135,P=0.000;F=25.306,P=0.000,海马:F=11.023,P=0.002;F=14.936,P=0.001;F=50.132,P=0.000)。总潜伏期C57BL/6J组Aβ3-10组对照组(F=8.938,P=0.016;F=5.745,P=0.04;F=7.073,P=0.017)。结论 Aβ3-10重复片段质粒可以影响脑内BACE1的表达,影响Aβ产生,改善空间记忆能力。     

Aβ1—40海马注射对大鼠脑内一氧化氮合酶表达的影响

目的探讨一氧化氮合酶(NOS)在β淀粉样蛋白(Aβ)神经毒性及阿尔茨海默病(AD)病理机制中的作用.方法应用免疫组化方法,观察大鼠海马齿状回Aβ1-40注射后神经元型一氧化氮合酶(nNOS)和诱导型一氧化氮合酶(iNOS)表达变化.结果正常大鼠海马齿状回区含nNOS神经元计数为8.96±0.35个/视野;生理盐水注射后局部含nNOS神经元无明显变化(8.97±0.29个/视野);Aβ1-40注射后,注射区周围含nNOS神经元数目显著减少(2.98±0.24个/视野).正常及生理盐水注射组脑内未见iNOS表达;Aβ1-40注射后2d、10d和30d,注射区持续出现大量含iNOS的胶质细胞(主要为星形胶质细胞),反应面积分别为0.905±0.082、0.962±0.161、0.935±0.125mm2.结论Aβ1-40海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达,NOS在Aβ神经毒性和AD发病中有重要作用.     

冈田酸对拟AD模型大鼠海马CA1区Aβ1-40和nNOS表达的影响

目的观察冈田酸(OA)对大鼠海马CA1区Aβ1-40和nNOS表达的影响,建立更接近临床表现的拟AD大鼠模型。方法在大鼠海马CA1区多次微量注射OA,水迷宫实验观测大鼠行为学改变;Bielschowsky染色观察海马CA1区神经原纤维缠结(NFT)和老年斑(SP)等特征性病理变化;免疫组化方法观察海马CA1区Aβ1-40和nNOS的表达。结果模型组学习记忆减退;海马CA1区出现NFT(P<0.05)和SP特征性病理变化,及Aβ1-40的高表达(P<0.05)和nNOS的低表达(P<0.05)。结论冈田酸海马CA1区微量多次注射,可建立更接近AD临床表现和病理特征的大鼠模型,大鼠海马CA1区SP和NFT的形成,以及Aβ1-40表达增加、nNOS表达减少,是该模型大鼠学习记忆能力减退的可能机制。     

能量限制对APP/PS1双转基因大鼠脑内Aβ的影响

目的探讨能量限制(CR)对APP/PS1双转基因大鼠脑内Aβ的影响,并探讨其可能的机制。方法将24只5月龄APP/PS1双转基因痴呆大鼠随机分为对照组、CR-1组、CR-2组,分别给予自由进食、正常进食量的80%及70%饲养4w,通过Morris水迷宫测试认知能力及免疫组织化学染色观察脑内Aβ阳性细胞数。结果在Morris水迷宫测试在定位航行实验第5天,CR-2组大鼠的逃避潜伏期较对照组明显缩短(P0.05),穿越平台次数明显增多(P0.05);而CR-1组较对照组无显著差异(P0.05)。CR-2组海马区的Aβ阳性细胞数低于对照组,差异有统计学意义(P0.05);而CR-1组较对照组差异无统计学意义(P0.05)。结论 70%的CR可以改善APP/PS1双转基因大鼠的认知能力,其机制可能与减少大鼠脑内Aβ的沉积有关。     

瑞香素对大鼠前脑缺血后β-APP表达的影响

目的讨论β-淀粉样蛋白前体（β-APP）脑缺血后变化规律并观察瑞香素对其影响。方法wistar大鼠45只平分为三组：缺血再灌注组（简称对照组）分离双侧颈总动脉,无创微动脉夹夹闭双侧颈总动脉15min,然后恢复血流再灌注60min;再夹闭15min,松开动脉夹,恢复血流,缝合切口,术后每日用生理盐水灌胃;假手术组只分离双侧颈总动脉,不予夹闭,余同对照组;缺血再灌注瑞香素治疗组（简称治疗组）手术操作同对照组,每日用瑞香素灌胃。在2周,4周,6周不同的时间点取脑制作β-APP免疫组织化学切片,对β-APP表达阳性细胞计数。结果在2周,4周,6周不同的时间点,治疗组的阳性细胞数高于假手术组,低于对照组,不同时间点阳性细胞数呈递减趋势。假手术组的β-APP阳性细胞数无明显变化。对照组β-APP阳性细胞数最多。结论瑞香素对阻止β-APP的过度表达有一定的作用。     

Aβ_(1-40)海马注射对大鼠脑内一氧化氮合酶表达的影响

目的　探讨一氧化氮合酶 (NOS)在 β淀粉样蛋白 (Aβ)神经毒性及阿尔茨海默病 (AD)病理机制中的作用。方法　应用免疫组化方法 ,观察大鼠海马齿状回Aβ1 4 0 注射后神经元型一氧化氮合酶 (nNOS)和诱导型一氧化氮合酶 (iNOS)表达变化。结果　正常大鼠海马齿状回区含nNOS神经元计数为 8 96± 0 35个 /视野 ;生理盐水注射后局部含nNOS神经元无明显变化 (8 97± 0 2 9个 /视野 ) ;Aβ1 4 0 注射后 ,注射区周围含nNOS神经元数目显著减少 (2 98± 0 2 4个 /视野 )。正常及生理盐水注射组脑内未见iNOS表达 ;Aβ1 4 0 注射后 2d、10d和 30d ,注射区持续出现大量含iNOS的胶质细胞 (主要为星形胶质细胞 ) ,反应面积分别为 0 90 5± 0 0 82、0 96 2± 0 16 1、0 935± 0 12 5mm2 。结论　Aβ1 4 0 海马注射可损伤局部含nNOS神经元及诱导胶质细胞iNOS表达 ,NOS在Aβ神经毒性和AD发病中有重要作用。     

降低细胞胆固醇水平对β-淀粉样肽生成的影响

目的观察降低细胞胆固醇水平对β-淀粉样肽(β-amyloid,Aβ)生成的影响,初步探讨胆固醇和阿尔茨海默病(Alzheimer's disease,AD)的相关性。方法以稳定表达人野生型淀粉样前体蛋白(amyloid precursor protein,APP)的神经母细胞瘤SH-SY5Y细胞为模型,分别给予β-甲基环糊精或洛伐他汀对细胞系进行处理;胆固醇定量试剂盒测定细胞内胆固醇水平的变化,放射免疫法测定细胞培养液中Aβ的含量,Western Blot方法半定量检测全长型APP和可溶性APPα的水平。结果β-甲基环糊精和洛伐他汀处理组分别降低细胞胆固醇水平67.5%和49.5%(P<0.05),细胞培养液中Aβ含量分别降低39.5%和25.7%(P<0.05),sAPPα含量分别增加3.5倍和2.0倍(P<0.05)。结论降低细胞胆固醇水平使细胞外Aβ含量减少,sAPPα含量增加,这提示胆固醇可能通过影响APP代谢途径而参与AD的病理过程。     

Aβ对原代培养海马神经元NO、NOS和LDH影响及bFGF保护作用

目的　探讨β-淀粉样蛋白 (amyloid-beta protein,Aβ)对原代培养海马神经元一氧化氮 (NO)、一氧化氮合酶 (NOS)和乳酸脱氢酶 (LDH)的影响及碱性成纤维细胞生长因子 (b FGF)的保护作用。方法　通过测定原代培养新生大鼠海马神经元培养液中 NO、NOS及 LDH的含量变化 ,观察 Aβ1 -40对神经元的损伤及 b FGF的保护作用。结果　Aβ1 -40作用于海马神经元 2 4h后 ,培养上清液中 NO、NOS含量增加 ,LDH活性升高 ,与对照组比较差异有显著性 (P<0 .0 5 ,P<0 .0 1 )。经 b FGF预处理的海马神经元暴露于 Aβ1 -40 2 4h后 ,NO、NOS含量及 LDH活性均降低 ,且与损伤组比较差异有显著性 (P <0 .0 5 )。结论　 b FGF对 Aβ诱导海马神经元损伤具有一定的保护作用。     

The association of beta-site APP cleaving enzyme (BACE) C786G polymorphism with Alzheimer's disease

The deposition of amyloid beta-peptide (Abeta) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Beta-site APP cleaving enzyme (BACE) is the rate-limiting enzyme in the Abeta formation. BACE mutations/polymorphisms may be associated with AD. We searched the BACE coding region mutations/polymorphisms of cDNA in 25 AD patients and 100 healthy controls by single-strand conformational polymorphism. A polymorphism at BACE coding region was identified and confirmed to be 786C/G polymorphism by nucleotide sequencing. Based on these findings, we investigated the association of this polymorphism with the occurrence of AD by PCR-RFLP. A total of 98 AD patients along with 138 controls were recruited in the present study. The allele frequencies of the 786C/G polymorphism were 0.622 for C and 0.378 for G in AD. In controls, the C and G allele frequencies were 0.691 and 0.309, respectively. No significant association of this polymorphism with the occurrence of AD can be established. Larger sample size may be necessary to identify other potential mutations/polymorphisms among BACE gene.     

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that - in addition to its role in APP trafficking - LRP1 affects APP processing by competing for cleavage by BACE1.     

Modulation of APP processing and secretion by okadaic acid in primary guinea pig neurons

Summary. Primary cultures of guinea pig neurons were used as a model system to study the influence of the protein phosphatase inhibitor okadaic acid (OA) on the secretion, processing and phosphorylation of the amyloid precursor protein (APP). This primary cell culture system mimics more closely than other cell culture systems the human in vivo condition, as guinea pig APP is 98% homologous to human APP at the protein level, identical regarding the Aβ sequence and is processed in a similar manner as human APP. Both intracellular and secreted APP was upregulated by OA treatment (0.3 nM–10 nM) of 14 days old cultures in a concentration dependent manner while the amount of Aβ in the medium was decreased. OA treatment did not affect cell membrane integrity of primary neurons but induced DNA fragmentation. Phosphorylation of APP was unchanged by the low OA concentration used. These results show that OA treatment of guinea pig primary cultures might be used as a model to study the effects of modulation of signal transduction on secretion and processing of APP. Received June 29, 1999; accepted September 6, 1999     

Activation of caspase-3 in β-amyloid-induced apoptosis of cultured rat cortical neurons

Amyloid β protein (Aβ) has been thought to participate in the neurodegeneration associated with Alzheimer's disease. We here report on caspase-3 activation by Aβ-treatment of cultured neurons. Treatment of rat primary cortical culture with Aβ 25–35, an active fragment of Aβ, induced neuronal death as determined by a decrease in neuron-specific microtubule-associated protein 2 (MAP2)-like immunoreactivity and by the release of cellular lactate dehydrogenase (LDH). Aβ 25–35 also induced elevation of caspase-3-like Ac-DEVD-MCA cleavage activity in advance of neuronal death with similar concentration-dependency for neuronal death. Inhibitor sensitivity of the Aβ-induced proteolytic activity was similar to that of human recombinant caspase-3. Cleavage of pro-caspase-3 and cleavage of its endogenous substrates, poly (ADP-ribose) polymerase (PARP) and α-fodrin, were produced by Aβ-treatment. A caspase-3 inhibitor, Ac-DEVD-CHO, prevented Aβ-induced DNA fragmentation and cleavage of α-fodrin, but not of PARP. Caspase inhibitor of broad specificity, Z-VAD-CH₂-DCB, additionally prevented Aβ-induced cleavage of PARP and some early loss of cell membrane integrity measured by LDH release. However, Aβ-induced condensation of nuclear chromatin and most of the late disintegration of cell membranes were not prevented in the presence of these caspase inhibitors. These results suggest that activation of both caspase-3 and caspase(s) other than caspase-3 play distinct roles in Aβ-induced apoptosis of rat cortical neurons. Furthermore, in the presence of caspase inhibitors, Aβ-induced neuronal death still occurred with different morphological features.     

Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: Effects of the swedish Alzheimer's disease APP 670/671 mutation on binding levels

Summary We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [³H] quinuclidinyl benzilate ([³H]QNB) and the M2/M4 selective antagonist [³H](±)-5,11-dihydro-11-{[(2-[(di-propylamino)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one ([³H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [³H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine>himbacine>methoctramine>(±)-p-fluoro-hexahydro-sila-difenidol hydrochloride>pirenzepine>muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25–35% lower levels of muscarinic receptors labelled with [³H]QNB, [³H]N-methyl scopolamine and [³H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.     

Docosahexaenoic acid stimulates non-amyloidogenic APP processing resulting in reduced Abeta levels in cellular models of Alzheimer's disease

Epidemiological studies suggest that a high intake of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), is associated with a reduced risk of Alzheimer's disease. Here, we examined the effects of DHA on amyloid precursor protein (APP) processing in cellular models of Alzheimer's disease by analysing levels of different APP fragments, including amyloid-beta (Abeta). DHA administration stimulated non-amyloidogenic APP processing and reduced levels of Abeta, providing a mechanism for the reported beneficial effects of DHA in vivo. However, an increased level of APP intracellular domain was also observed, highlighting the need to increase our knowledge about the relevance of this fragment in Alzheimer's disease pathogenesis. In conclusion, our results suggest that the proposed protective role of DHA in Alzheimer's disease pathogenesis might be mediated by altered APP processing and Abeta production.     

尼莫的平对Alzheimer病大鼠海马和额叶细胞外液氨基酸的影响

目的 研究尼莫的平对Ａｌｚｈｅｉｍｅｒ病大鼠海马和额叶兴奋性氨基酸的影响。方法 在立体定位下于迈内特基底核（ＮＢＭ）注入β淀粉样蛋白（β－ＡＰ）１０μｇ建立Ａｌｚｈｅｉｍｅｒ病模型,对照组在ＮＢＭ注入生理盐水,治疗组在建立模型后腹腔注射尼莫的平,每天注射１次至实验结束,模型组不给任何药物。脑内微透析技术采集大鼠海马、额叶细胞外液、反相高效液相色谱技术测定氨基酸类神经递质。结果 模型组海马和额叶细胞外液４种氨基酸浓度均较对照组升高,谷氨酸最明显。尼莫的平治疗后谷氨酸明显降低。结论 由β－ＡＰ产生的Ａｌｚｈｅｉｍｅｒ病大鼠痴呆与兴奋毒性有关,其作用可能是由钙离子介导。     

Platelets and Alzheimer&rsquo;s disease: Potential of APP as a biomarker

Platelets are the first peripheral source of amyloid precursor protein (APP). They possess the proteolytic machinery to produce Aβ and fragments similar to those produced in neurons, and thus offer an ex-vivo model to study APP processing and changes associated with Alzheimer’s disease (AD). Platelet process APP mostly through the α-secretase pathway to release soluble APP (sAPP). They produce small amounts of Aβ, predominantly Aβ₄₀ over Aβ₄₂. sAPP and Aβ are stored in α-granules and are released upon platelet activation by thrombin and collagen, and agents inducing platelet degranulation. A small proportion of full-length APP is present at the platelet surface and this increases by 3-fold upon platelet activation. Immunoblotting of platelet lysates detects APP as isoforms of 130 kDa and 106-110 kDa. The ratio of these of APP isoforms is significantly lower in patients with AD and mild cognitive impairment (MCI) than in healthy controls. This ratio follows a decrease that parallels cognitive decline and can predict conversion from MCI to AD. Alterations in the levels of α-secretase ADAM10 and in the enzymatic activities of α- and β-secretase observed in platelets of patients with AD are consistent with increased processing through the amyloidogenic pathway. β-APP cleaving enzyme activity is increased by 24% in platelet membranes of patients with MCI and by 17% in those with AD. Reports of changes in platelet APP expression with MCI and AD have been promising so far and merit further investigation as the search for blood biomarkers in AD, in particular at the prodromal stage, remains a priority and a challenge.     

Deposition of mouse amyloid beta in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.