Purification and cloning of amyloid precursor protein beta-secretase from human brain

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.     

A specific amyloid-beta protein assembly in the brain impairs memory

Memory function often declines with age, and is believed to deteriorate initially because of changes in synaptic function rather than loss of neurons. Some individuals then go on to develop Alzheimer's disease with neurodegeneration. Here we use Tg2576 mice, which express a human amyloid-beta precursor protein (APP) variant linked to Alzheimer's disease, to investigate the cause of memory decline in the absence of neurodegeneration or amyloid-beta protein amyloidosis. Young Tg2576 mice (< 6 months old) have normal memory and lack neuropathology, middle-aged mice (6-14 months old) develop memory deficits without neuronal loss, and old mice (> 14 months old) form abundant neuritic plaques containing amyloid-beta (refs 3-6). We found that memory deficits in middle-aged Tg2576 mice are caused by the extracellular accumulation of a 56-kDa soluble amyloid-beta assembly, which we term Abeta*56 (Abeta star 56). Abeta*56 purified from the brains of impaired Tg2576 mice disrupts memory when administered to young rats. We propose that Abeta*56 impairs memory independently of plaques or neuronal loss, and may contribute to cognitive deficits associated with Alzheimer's disease.     

Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells

Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr?231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr?231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.     

A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease

Vaccinations with amyloid-beta peptide (A beta) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimer's disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of A beta vaccination in a different transgenic model for Alzheimer's disease in which mice develop learning deficits as amyloid accumulates. Here we show that vaccination with A beta protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimer's disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the A beta-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The A beta-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimer's dementia.     

Amyloid plaques, neurofibrillary tangles and neuronal loss in brains of transgenic mice overexpressing a C-terminal fragment of human amyloid precursor protein.

Alzheimer's disease (AD) affects more than 30% of people over 80 years of age. The aetiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated histopathologically with amyloid plaques, neurofibrillary tangles and neuronal loss primarily in the temporal lobe and neocortex of the brain. The core of the extracellular plaque is a derivative of the amyloid precursor protein (APP), referred to as beta/A4, and contains the amino-acid residues 29-42 that are normally embedded in the membrane-spanning region of the precursor. The cellular source of APP and the relationship of its deposition to the neuropathology of AD is unknown. To investigate the relationship between APP overexpression and amyloidogenesis, we have developed a vector to drive expression specifically in neurons of a C-terminal fragment of APP that contains the beta/A4 region, and have used a transgenic mouse system to insert and express this construct. We report here that overexpression of this APP transgene in neurons is sufficient to produce extracellular dense-core amyloid plaques, neurofibrillary tangles and neuronal degeneration similar to that in the AD brain.     

Protease nexin-II, a potent antichymotrypsin, shows identity to amyloid beta-protein precursor

Protease nexin-II (PN-II) is a protease inhibitor that forms SDS-resistant inhibitory complexes with the epidermal growth factor (EGF)-binding protein, the gamma-subunit of nerve growth factor, and trypsin. The properties of PN-II indicate that it has a role in the regulation of certain proteases in the extracellular environment. Here we describe more of the amino-acid sequence of PN-II and its identity to the deduced sequence of the amyloid beta-protein precursor (APP). Amyloid beta-protein is present in neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down's syndrome. A monoclonal antibody against PN-II (designated mAbP2-1) recognized PN-II in immunoblots of serum-free culture medium from human glioblastoma cells and neuroblastoma cells, as well as in homogenates of normal and Alzheimer's disease brains. In addition, mAbP2-1 stained neuritic plaques in Alzheimer's disease brain. PN-II was a potent inhibitor of chymotrypsin with an inhibition constant Ki of 6 x 10(-10)M. Together, these data demonstrate that PN-II and APP are probably the same protein. The regulation of extracellular proteolysis by PN-II and the deposition of at least parts of the molecule in senile plaques is consistent with previous reports that implicate altered proteolysis in the pathogenesis of Alzheimer's disease.     

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease

Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.     

Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.

Amyloid-beta peptide (Abeta) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Abeta42), which is the predominant form found in the amyloid plaques of Alzheimer's disease. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. In the present study, transgenic animals were immunized with Abeta42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-beta deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of beta-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-beta may be effective in preventing and treating Alzheimer's disease.     

Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity

Mutations in the gene encoding the amyloid protein precursor (APP) cause autosomal dominant Alzheimer's disease. Cleavage of APP by unidentified proteases, referred to as beta- and gamma-secretases, generates the amyloid beta-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate its cleavage. Here we identify a new membrane-bound aspartyl protease (Asp2) with beta-secretase activity. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid beta-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by beta-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the beta-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid beta-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.     

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production

Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.     

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

A locus segregating with familial Alzheimer's disease (AD) has been mapped to chromosome 21, close to the amyloid precursor protein (APP) gene. Recombinants between the APP gene and the AD locus have been reported which seemed to exclude it as the site of the mutation causing familial AD. But recent genetic analysis of a large number of AD families has demonstrated that the disease is heterogeneous. Families with late-onset AD do not show linkage to chromosome 21 markers. Some families with early-onset AD show linkage to chromosome 21 markers, but some do not. This has led to the suggestion that there is non-allelic genetic heterogeneity even within early onset familial AD. To avoid the problems that heterogeneity poses for genetic analysis, we have examined the cosegregation of AD and markers along the long arm of chromosome 21 in a single family with AD confirmed by autopsy. Here we demonstrate that in this kindred, which shows linkage to chromosome 21 markers, there is a point mutation in the APP gene. This mutation causes an amino-acid substitution (Val----Ile) close to the carboxy terminus of the beta-amyloid peptide. Screening other cases of familial AD revealed a second unrelated family in which this variant occurs. This suggests that some cases of AD could be caused by mutations in the APP gene.     

中药对阿尔兹海默症APP/PS1转基因小鼠的治疗作用研究进展

阿尔兹海默症(alzheimer’s disease,AD),是一种中枢神经系统退行性疾病,以老年斑块沉积和神经纤维缠结为主要特征,患者表现为认知能力减退、生活不能自理,该病的预防与治疗已经成为国内外研究的热点。在防治AD的实验研究领域,APP/PS1转基因小鼠模型有着广泛的应用,因为其能较好地反映阿尔兹海默症患者的病理特征、临床表现,是一种较理想的AD动物模型。本文综述近五年来中药对APP/PS1转基因AD小鼠治疗作用的研究进展,以期为后续防治AD的研究提供参考。     

Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.

Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.     

A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease

Much evidence indicates that abnormal processing and extracellular deposition of amyloid-beta peptide (A beta), a proteolytic derivative of the beta-amyloid precursor protein (betaAPP), is central to the pathogenesis of Alzheimer's disease (reviewed in ref. 1). In the PDAPP transgenic mouse model of Alzheimer's disease, immunization with A beta causes a marked reduction in burden of the brain amyloid. Evidence that A beta immunization also reduces cognitive dysfunction in murine models of Alzheimer's disease would support the hypothesis that abnormal A beta processing is essential to the pathogenesis of Alzheimer's disease, and would encourage the development of other strategies directed at the 'amyloid cascade'. Here we show that A beta immunization reduces both deposition of cerebral fibrillar A beta and cognitive dysfunction in the TgCRND8 murine model of Alzheimer's disease without, however, altering total levels of A beta in the brain. This implies that either a approximately 50% reduction in dense-cored A beta plaques is sufficient to affect cognition, or that vaccination may modulate the activity/abundance of a small subpopulation of especially toxic A beta species.     

Fe65L2基因存在4种剪接形式

Ｆｅ６５Ｌ２是一种与老年痴呆症关蛋白－淀粉样肽前体蛋白相互作用的蛋白。报道了Ｆｅ６５Ｌ２基因的２种新的剪接形式,并证实了该基因存在４种剪接形式。这４种剪接形式是由同一个内含子中分别为６ｎｔ和２１ｎｔ的２段外显子序列按不同组合拼接而成的。     

老年驾驶人的应激操作反应特性分析

随着年龄的增长,老年驾驶人的感知认知能力、判断决策能力及操作反应能力均出现衰退,进而导致其交通风险感知和应激反应能力发生变化。为了研究风险情景下老年驾驶人的应激操作反应特性,设计并搭建了行人与机动车、非机动车与机动车、机动车与机动车冲突的道路交叉口风险虚拟试验场景;分别招募了15名中青年与老年驾驶人进行了驾驶模拟试验,采集驾驶过程中驾驶人的操作行为及车辆运行数据,选取相关的指标对试验进行分析。结果表明,老年驾驶人在各风险情境下的反应时间与制动踏板行程总体上比中青年驾驶人长;而在高风险驾驶场景下,老年驾驶人的纵向加速度的波动幅度比中青年驾驶人大,其中有10名老年驾驶人发生了碰撞事故,占所有受试者的比例为33.3%。研究结果为交通安全政策制定者的措施制定提供一些提高老年驾驶人驾驶安全性的理论依据。     

Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor.

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of amyloid fibrils is beta/A4 amyloid protein, which can be generated by the aberrant processing of a large membrane-bound glycoprotein, the beta/A4 amyloid protein precursor (APP)3. To test whether overexpression of APP generates abnormally processed derivatives that affect the viability of neurons, we stably transfected full-length human APP complementary DNA into murine embryonal carcinoma P19 cells. These cells differentiate into post-mitotic neurons and astrocytes after exposure to retinoic acid. When differentiation of the APP cDNA-transfected P19 cells was induced, all neurons showed severe degenerative changes and disappeared within a few days. The degenerating neurons contained large amounts of APP derivatives that were truncated at the amino terminus and encompassed the entire beta/A4 domain. These results suggest that post-mitotic neurons are vulnerable to overexpressed APP, which undergoes aberrant processing to generate potentially amyloidogenic fragments.     

帕金森病的认知障碍

 帕金森病是仅次于老年痴呆的第二大神经退行性疾病。随着社会老龄化的加剧,帕金森病的患病率大幅提高。认知障碍等非运动症状是目前影响帕金森病患者生活质量的主要因素。帕金森病认知障碍的生物学机制目前尚不明确,临床上患病率高、识别率低,缺乏有效的治疗手段。本文将围绕帕金森病认知障碍的前沿热点问题,介绍相关神经科学和医学研究的最新进展,涵盖认知障碍的神经化学基础、风险基因、其他非运动症状的影响和生物标记物等。     

Failure of familial Alzheimer's disease to segregate with the A4-amyloid gene in several European families

The gene coding for the amyloid protein, a component of neuritic plaques found in brain tissue from patients with Alzheimer's disease, has been localized to chromosome 21, and neighbouring polymorphic DNA markers segregate with Alzheimer's disease in several large families. These data, and the association of Alzheimer's disease with Down's syndrome, suggest that overproduction of the amyloid protein, or production of an abnormal variant of the protein, may be the underlying pathological change causing Alzheimer's disease. We have identified a restriction fragment length polymorphism of the A4-amyloid gene, and find recombinants in two Alzheimer's disease families between Alzheimer's disease and the A4-amyloid locus. This demonstrates that the gene for plaque core A4-amyloid cannot be the locus of a defect causing Alzheimer's disease in these families. These data indicate that alterations in the plaque core amyloid gene cannot explain the molecular pathology for all cases of Alzheimer's disease.