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。     

线粒体相关性β淀粉样前体蛋白及线粒体相关性β淀粉样蛋白针对阿尔茨海默病致病机制的研究

线粒体相关性β淀粉样前体蛋白(APP)和线粒体相关性β淀粉样蛋白(Aβ)正逐渐成为研究阿尔茨海默病(AD)病理生理学改变的热点。线粒体相关性APP和线粒体相关性Aβ是指以线粒体为特异性靶点,存在于线粒体膜上或沉积于线粒体基质内的APP和Aβ。文中将分别介绍线粒体相关性APP和线粒体相关性Aβ的来源、结构、功能等方面的研究进展,揭示其与AD发病的密切关系。     

信号转导在淀粉样前体蛋白代谢中的研究进展

淀粉样前体蛋白(APP)裂解主要经过α和β分泌酶两条途径,激活β分泌酶途径可增加β-淀粉样蛋白(Aβ)在脑组织的生成量和沉积量,而激活α分泌酶途径可增加可溶性淀粉样前体蛋白α(sAPPα)的生成,sAPPα具有保护神经元促进轴突生长并且相对地降低Aβ的生成量。体内许多神经递质等可通过相应受体偶联的G蛋白或酪氨酸激酶等激活胞浆内的各种信号蛋白来影响α或β分泌酶的活性,从而影响sAPPα或Aβ的生成量。因此,寻找APP信号转导通路上的阻断剂或激动剂以选择性增加sAPPα的生成而减少Aβ的生成量将为寻找AD治疗药物开辟一条新的途径。     

β-淀粉样蛋白代谢异常与阿尔茨海默病

阿尔茨海默病（AD）临床常见进行性认知功能下降,行为异常,最终出现痴呆。神经病理 可见 tau 蛋白神经纤维缠结、β-淀粉样蛋白（Aβ）蓄积形成老年斑,以及小胶质细胞增生和神经元、白 质、突触的大量缺失。由于淀粉样蛋白病理可能早于 tau 蛋白病理,早于 AD 脑萎缩及临床症状的出现, 现阐述 Aβ 代谢异常与 AD 的相关性。     

多光子激光扫描成像技术对活细胞内淀粉样前体蛋白裂解和β-淀粉样蛋白生成的观察

目的在活细胞内探究淀粉样前体蛋白（amyloidprecursorprotein,APP）的裂解和β-淀粉样蛋白（amyloidbeta,AB）的生成机制。方法利用PCR扩增CFP（编码蓝色荧光蛋白）,YFP（编码黄色荧光蛋白）和C99（编码APP最后99个氨基酸）三片段。含有54个碱基（编码APP中间18个氨基酸）的片段54bp由生物公司合成。CFP,YFP,54bp和C99四个片段连入载体质粒pcDNA3．0得到重组质粒pcDNA3．0-CFP-54bp-YFP和pcDNA3．0-CFP-54bp-YFP-C99。将这两个重组质粒分别瞬时转染SH-SY5Y细胞。利用多光子激光扫描显微镜观察融合基因的表达,荧光共振能量转移（fluorescenceresonanceenergytransfer,FRET）检测APP的B裂解。免疫细胞化学和多光子激光扫描显微镜观察AB的生成。MTT检测转染细胞活性。结果（1）限制性内切酶双酶切消化和测序分析鉴定重组质粒序列完全正确。（2）转染细胞内可以观察到蓝色和黄色荧光。（3）在pcDNA3．0-CFP-54bp-YFP转染细胞中存在FRET现象;在pcDNA3．0-CFP-54bp-YFP-C99转染细胞中观察不到FRET现象。（4）免疫细胞化学和多光子激光扫描显微镜观察到在pcDNA3．0-CFP．54bp-YFP—C99转染的细胞中有AD的生成。（5）AD的沉积广泛分布于细胞内。（6）随着AB在细胞内的产生和聚集,细胞的活性逐步下降。结论C99对于APP的β裂解非常重要。早期Aβ首先在细胞内生成并在细胞内形成广泛沉积。细胞内聚集的Aβ会影响细胞活性,带来不利结果。     

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。     

阿尔茨海默病患者血小板淀粉样前体蛋白代谢的改变

目的 通过观察血小板活化后β淀粉样蛋白(Aβ)水平变化,探讨阿尔茨海默病(Alzheimer disease,AD)患者血小板β-淀粉样前体蛋白(APP)代谢特点.方法 分离36例AD患者和30名健康对照的血小板,用免疫印迹检测凝血酶作用后的可溶性APP水平;同时用放射免疫法测定Aβ含量.结果 凝血酶作用后,血小板上清中可以检测到可溶性APP和Aβ.活化后AD患者血小板上清中可溶性APP水平较对照组水平下降31.0%(P＜0.05).凝血酶活化后,AD组血小板上清Aβ水平从(3.1±2.7)ng/L增加至(5.8±3.2)ng/L(P＜0.01),对照组Aβ水平由(6.1±4.4)ng/L增加为(11.5±5.9)ng/L(P＜0.01).AD组Aβ平均增加(2.8±2.1)ng/L,低于对照组增加水平(5.5±3.6)ng/L(P＜0.01).结论 血小板中含有APP的代谢产物可溶性APP和Aβ.AD血小板可能存在APP代谢异常.     

糖尿病加剧大鼠脑缺血损伤及皮层淀粉样蛋白的生成

目的探讨糖尿病对缺血所致脑损伤和痴呆相关蛋白表达的影响。方法通过高脂饮食结合小剂量链脲佐菌素（STZ）诱导大鼠糖尿病模型;用大鼠大脑中动脉栓塞（MCAO）模型诱导脑卒中。再灌注1周后,采用免疫组织化学染色观察糖尿病对脑缺血后梗塞灶体积以及皮层神经细胞内淀粉样蛋白（Aβ）及其生成关键酶β-分泌酶（BACE）表达的影响。结果脑缺血后,与正常对照组相比,糖尿病组实验动物梗塞灶体积增大,且皮层存在大量神经元样及活化的胶质细胞样细胞内表达Aβ和BACE。结论糖尿病加剧脑缺血损伤及皮层淀粉样蛋白的表达,可能与糖尿病患者卒中后更为严重的认知功能障碍产生有关。     

尼古丁对Aβ25～35细胞毒性的拮抗作用及与β-淀粉样前体蛋白代谢的关系

目的研究尼古丁对β-淀粉样蛋白（Aβ）细胞毒性的拮抗作用及与β-淀粉样前体蛋白（APP）代谢之间的关系。方法不同浓度的尼古丁分别单独或与Aβ25～35同时作用于PC12细胞24h,然后采用MTT法检测细胞活力;WesternBlot法检测PC12细胞上清液中的可溶性β-淀粉样前体蛋白α片段（sAPPα）和细胞内胰岛素降解酶的表达水平。结果（1）尼古丁浓度于0.10～500μmol/L时,对PC12细胞无明显毒性作用（均P〉0.05）,当浓度升至1000μmol/L时则产生一定的毒性作用（P≤0.05）;Aβ25～35浓度于1～100μmol/L时对PC12细胞具有明显毒性作用（均P≤0.01）,Aβ25～35浓度降至0.10μmol/L时则失去其毒性作用（P〉0.05）。（2）尼古丁浓度于0.10～1000μmol/L时,对Aβ25～35诱导的细胞毒性呈现不同的作用：尼古丁浓度于0.10～100μmol/L时可部分拮抗Aβ25～35诱导的细胞毒性作用（均P〈0.01）,但不能使PC12细胞活力恢复至正常细胞水平（均P〈0.01）;而当尼古丁浓度于500μmol/L和1000μmol/L时则不产生拮抗Aβ25～35诱导的细胞毒性作用（均P〉0.05）。（3）Aβ25～35（20μmol/L）和尼古丁（100μmol/L,1000μmol/L）单独或联合作用均可引起PC12细胞可溶性β-淀粉样前体蛋白α片段分泌水平升高（均P〈0.01）,其中以尼古丁浓度为100μmol/L时可溶性β-淀粉样前体蛋白α片段的分泌水平最高。（4）浓度为20μmol/L的Aβ25～35可导致胰岛素降解酶表达水平降低（P〈0.01）,而浓度为100μmol/L的尼古丁可明显拮抗这种作用（P〈0.01）,1000μmol/L的尼古丁则无明显拮抗作用（P〉0.05）;将浓度为100μmol/L和1000μmol/L的尼古丁分别单独作用于PC12细胞时,胰岛素降解酶表达水平明显升高,与正常对照组相比差异有统计学意义（P〈0.01）。结论尼古丁对Aβ25～35诱导的细胞毒性的拮抗作用与可溶性β-淀粉样前体蛋白α片段的分泌水平并无直接关系,而可能与胰岛素降解酶的表达水平相关。     

α-分泌酶与阿尔茨海默病的治疗

淀粉样前体蛋白(APP)在脑内经β和γ分泌酶的作用生成β淀粉样蛋白(Aβ),也可在α和γ分泌酶的作用下从Aβ序列内部进行降解,生成sAPPα而避免Aβ的生成,sAPPα可对神经细胞产生神经营养和神经保护作用。Aβ在脑内沉积从而对细胞造成毒性作用即Aβ毒性学说被认为是阿尔茨海默病(AD)发病机制之一。目前AD的治疗策略主要集中于抑制β分泌酶和γ分泌酶的研究。新近的研究显示,一类属于解聚素和金属蛋白酶(主要指ADAM10、ADAM17和ADAM9)分子具有α分泌酶的功能,提高α分泌酶的表达可以降低Aβ,有可能在AD的治疗中具有潜在作用。     

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background: During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42. Methods: Cell‐free γ‐secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI‐TOF MS analysis was carried out to detect the N‐termini of AICD species. Aβ and AICD species were measured by ELISA and immunoblotting techniques. Results: Effects on the ε‐cleavage by AD‐associated pathological mutations around the ε‐cleavage sites (i.e., βAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε‐cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aβ42 production. Only AICDε51, though, and not Aβ42 production, dramatically changed with modifications to the cell‐free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. Conclusion: Our current data show that the generation ratio of AICDε51 and Aβ42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aβ42 generation, measurement of the relative level of Aβ42‐related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε‐cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε‐cleavage as a means for decreasing Aβ42 production.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

Altered levels and distribution of amyloid precursor protein and its processing enzymes in Niemann‐Pick type C1‐deficient mouse brains

Niemann‐Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder characterized by intracellular accumulation of cholesterol and glycosphingolipids in many tissues including the brain. The disease is caused by mutations of either NPC1 or NPC2 gene and is accompanied by a severe loss of neurons in the cerebellum, but not in the hippocampus. NPC pathology exhibits some similarities with Alzheimer's disease, including increased levels of amyloid β (Aβ)‐related peptides in vulnerable brain regions, but very little is known about the expression of amyloid precursor protein (APP) or APP secretases in NPC disease. In this article, we evaluated age‐related alterations in the level/distribution of APP and its processing enzymes, β‐ and γ‐secretases, in the hippocampus and cerebellum of Npc1^−/− mice, a well‐established model of NPC pathology. Our results show that levels and expression of APP and β‐secretase are elevated in the cerebellum prior to changes in the hippocampus, whereas γ‐secretase components are enhanced in both brain regions at the same time in Npc1^−/− mice. Interestingly, a subset of reactive astrocytes in Npc1^−/− mouse brains expresses high levels of APP as well as β‐ and γ‐secretase components. Additionally, the activity of β‐secretase is enhanced in both the hippocampus and cerebellum of Npc1^−/− mice at all ages, while the level of C‐terminal APP fragments is increased in the cerebellum of 10‐week‐old Npc1^−/− mice. These results, taken together, suggest that increased level and processing of APP may be associated with the development of pathology and/or degenerative events observed in Npc1^−/− mouse brains. © 2010 Wiley‐Liss, Inc.     

淀粉样前体蛋白胞内域的功能和机制

Alzheimer's disease (AD),a degenerative neurological disorder,is the most common form of dementia among older people,whose symptoms include gradual memory loss,cognitive impairments and deterioration of language skills.Amyloid precursor protein (APP) is cleaved by serials of secretases and generates Aβ,sAPPα/β and APP intracellular domain (AICD).Aβ forms amyloid plaques,together with neurofibrillary tangles (NFTs) which is comprised with hyperphosphorylated tau,are hallmarks ofAD.Aβ,especially in its oligomeric form,plays important roles in AD,causing cell death,calcium influx,loss of spines and repression of long-term potentiation (LTP)[1].However,recent studies indicate that in addition to Aβ,other fragments of APP after its cleavage,such as AICD,play essential roles in AD as well.In this article,the function of AICD and its underlying mechanisms will be reviewed.     

Effects of cholesterol transport inhibitor U18666A on APP metabolism in rat primary astrocytes

Amyloid β (Aβ) peptides generated from the amyloid precursor protein (APP) play an important role in the degeneration of neurons and development of Alzheimer's disease (AD). Current evidence indicates that high levels of cholesterol—which increase the risk of developing AD—can influence Aβ production in neurons. However, it remains unclear how altered level/subcellular distribution of cholesterol in astrocytes can influence APP metabolism. In this study, we evaluated the effects of cholesterol transport inhibitor U18666A—a class II amphiphile that triggers redistribution of cholesterol within the endosomal–lysosomal (EL) system—on APP levels and metabolism in rat primary cultured astrocytes. Our results revealed that U18666A increased the levels of the APP holoprotein and its cleaved products (α‐/β‐/η‐CTFs) in cultured astrocytes, without altering the total levels of cholesterol or cell viability. The cellular levels of Aβ_1‐40 were also found to be markedly increased, while secretory levels of Aβ_1‐40 were decreased in U18666A‐treated astrocytes. We further report a corresponding increase in the activity of the enzymes regulating APP processing, such as α‐secretase, β‐secretase, and γ‐secretase as a consequence of U18666A treatment. Additionally, APP‐cleaved products are partly accumulated in the lysosomes following cholesterol sequestration within EL system possibly due to decreased clearance. Interestingly, serum delipidation attenuated enhanced levels of APP and its cleaved products following U18666A treatment. Collectively, these results suggest that cholesterol sequestration within the EL system in astrocytes can influence APP metabolism and the accumulation of APP‐cleaved products including Aβ peptides, which can contribute to the development of AD pathology.     

Inhibition of endocytosis activates alternative degradation pathway of βAPP in cultured cells

Background: Alzheimer’s disease associated βAPP is sequentially endoproteolyzed by α/β‐secretase and γ‐cleavage. In the process, extracellular shedding by α‐secretase (ADAM 9/10/17) or β‐secretase (BACE 1/2) at position L¹⁷ or D¹ (Aβ numbering) are prerequisites for the generation of P3 or Aβ, respectively. In addition, several alternative extracellular cleavage sites in βAPP have been reported at position I^?6, V^?3, R⁵, E¹¹, F²⁰, and A²¹. Among these sites, position R⁵ is considered to be cleaved by α‐secretase‐like activity, whereas position E¹¹, F²⁰ and A²¹ are cleaved by β‐secretase. Therefore, extracellular shedding of βAPP is thought to be mediated exclusively by α/β‐secretase activities. However, so far the characteristics of cleavages at position V^?3 and I^?6 are not well understood. The aim of this study is to characterize these two cleavages of βAPP. Methods: We analyzed the conditioned media of βAPP wt or sw expressing cells with or without pharmacological agents. Results: Here, we show that the cleavage at position I^?6 of βAPP has characteristics distinct from that of α/β‐secretase, while the cleavage at V^?3 seems to be mediated by β‐secretase. Although inhibition of endocytosis enhances the cleavages at both V^?3 and I^?6, PMA, an α‐secretase stimulator, treatment enhances neither of these cleavages. Interestingly, a β‐secretase inhibitor, z‐VLL‐CHO, suppressed V^?3 but not I^?6 cleavage. The pathological βAPP Swedish mutant adjacent to the cleavage sites shows similar effects. Conclusions: Our data demonstrate that neither α nor β‐secretase undergoes extracellular shedding at I^?6 of βAPP. Therefore, our data may indicate a novel alternative βAPP degradation pathway which is up‐regulated upon low level of endocytosis.     

APP intracellular domain is increased and soluble Abeta is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease

Cholesterol is one of multiple factors, other than familial genetic mutations, that can influence amyloid-beta peptide (Abeta) metabolism and accumulation in Alzheimer disease (AD). The effect of a high-cholesterol diet on amyloid precursor protein (APP) processing in brain has not been thoroughly studied. This study was designed to further investigate the role of cholesterol in the production of Abeta and APP intracellular domain (AICD) in 12-month-old Tg2576 transgenic mice. The mice were maintained on a high-cholesterol diet for 6 weeks. We found that diet-induced hypercholesterolemia increased the APP cytosolic fragment AICD and reduced sAPPalpha in the Tg2576 mice compared to the mice on a control basal diet. In addition, the levels of detergent-extracted Abeta40 were reduced, although no change in guanidine-extracted Abeta levels was observed. Full-length APP, alpha/betaC-terminal fragment (alpha/betaCTF), and beta-secretase (BACE) were not different in the cholesterol-fed mice compared to the control diet-fed mice. This study suggests that a high dietary cholesterol in aged mice may not only influence Abeta metabolism, but also regulate the AICD levels. AICD has a proposed role in signal transduction and apoptosis, hence modulation of AICD production could be an alternative mechanism by which cholesterol contributes to AD pathogenesis.     

Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells.

The Alzheimer's amyloid precursor (APP) is cleaved by an unidentified enzyme (APP secretase) to produce soluble APP. Fractionation of PC12 cell homogenates in a detergent-free buffer showed the presence of the Kunitz protease inhibitor (KPI)-containing soluble APP (nexin II) in the particulate fraction. Digitonin or sodium carbonate treatment of this fraction solubilized nexin II suggesting that it is contained in the lumen of vesicles. Nexin II production was not affected by lysosomotropic agents, suggesting that APP secretase is not a lysosomal enzyme. Labelling of cell surface proteins by iodination failed to detect full-length APP on the surface of PC12 cells, suggesting that most of this protein is located intracellularly. Furthermore, pulse-chase experiments showed that nexin II is detected in cell extracts before it appears in the culture medium. Cellular nexin II was detected at zero time of chase after only 5 min of pulse labelling with 35S-sulfate, indicated that APP secretase cleavage takes place immediately after APP is sulfated. Temperature block, pulse-chase, and 35S-sulfate-labelling experiments suggested that APP is cleaved by APP secretase intracellularly in the trans-Golgi network (TGN) or in a post-Golgi compartment.     

Restoring Soluble Amyloid Precursor Protein α Functions as a Potential Treatment for Alzheimer's Disease

Soluble amyloid precursor protein α (sAPPα), a secreted proteolytic fragment of nonamyloidogenic amyloid precursor protein (APP) processing, is known for numerous neuroprotective functions. These functions include but are not limited to proliferation, neuroprotection, synaptic plasticity, memory formation, neurogenesis, and neuritogenesis in cell culture and animal models. In addition, sAPPα influences amyloid‐β (Aβ) production by direct modulation of APP β‐secretase proteolysis as well as Aβ‐related or unrelated tau pathology, hallmark pathologies of Alzheimer's disease (AD). Thus, the restoration of sAPPα levels and functions in the brain by increasing nonamyloidogenic APP processing and/or manipulation of its signaling could reduce AD pathology and cognitive impairment. It is likely that identification and characterization of sAPPα receptors in the brain, downstream effectors, and signaling pathways will pave the way for an attractive therapeutic target for AD prevention or intervention. © 2016 Wiley Periodicals, Inc.