The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.     

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.     

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.     

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.     

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.     

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.     

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.     

Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene

A mutation at codon 717 of the beta-amyloid precursor protein gene has been found to cosegregate with familial Alzheimer's disease in a single family. This mutation has been reported in a further five out of approximately 100 families multiply affected by Alzheimer's disease. We have identified another family, F19, in which we have detected linkage between the beta-amyloid precursor protein gene and Alzheimer's disease. Direct sequencing of exon 17 in affected individuals from this family has revealed a base change producing a Val----Gly substitution, also at codon 717. The occurrence of a second allelic variant at codon 717 linked to the Alzheimer's phenotype supports the hypothesis that they are pathogenic mutations.     

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.     

依达拉奉对脑缺血再灌注后Aβ及其前体表达干预

为探讨缺血再灌注对大鼠海马区神经元细胞的损伤机制及依达拉奉的干预作用,利用大脑中动脉线栓法制备大鼠脑缺血再灌模型,缺血2 h后再灌注22 h(术后24 h),按照Zea Longa 5级评分法,对大鼠进行神经行为学评分;通过苏木精-伊红染色法(HE)染色大鼠脑组织,观察其病理形态学的改变;通过免疫组织化学,图像分析及Western Blot的方法检测大鼠海马区β淀粉样蛋白(Aβ)及其前体(APP)的表达。结果显示,模型组大鼠表现出明显的神经功能缺损症状,与之相比,6和10 mg/kg的依达拉奉可不同程度改善损伤模型大鼠的神经缺损症状,尤其是10 mg/kg依达拉奉组的大鼠症状改善更为明显(P0.01);HE染色结果显示,模型组大鼠海马区神经元细胞脱失明显,而治疗组可减轻这种形态学改变;免疫组织化学及Western Blot分析结果提示,在模型组中Aβ、APP表达明显高于假手术组(P0.01),而在不同质量分数依达拉奉组中,Aβ、APP含量均减弱(P0.05)。由此得出,缺血再灌注可能通过上调淀粉样蛋白Aβ及其前体APP而引起神经元细胞损伤,而依达拉奉可能通过对它们的抑制起到保护神经元细胞的作用。     

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.     

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.     

Formation of amyloid-like fibrils in COS cells overexpressing part of 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 the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.     

Expression and inhibitory activity analysis of a 25-kD Bowman-Birk protease inhibitor in rice

Bowman-Birk protease inhibitors (BBI), belonging to serine protease inhibitors, are well-studied storage and defense proteins widely existing in both dicoty- ledonous and monocotyledonous plants[1―3]. In dicoty- ledonous plants, the BBIs are about 8-kD p…     

Recent progress in the study of intracellular toxicity of amyloid P peptide in Alzheimer's disease

Amyloidβ(Aβ) deposition is one of the major pathological markers of Alzheimer's disease (AD). Extracellular Afi toxi-city has been studied for a long time in AD research field. However, controversial data show that extracellular Aβload does not correlate with the dementia levels of AD patients and extracellular Aβonly induces significant cell death at non-physiological high concentrations. With the evolvement of Afi hypothesis, considerable attention has been devoted to the study of intracellular Aβtoxicity recently. Intracellular Aβinduces dramatic cell loss in AD transgenic models and in human primary neurons (at pM concentrations) through p53, Bax and caspase-6 pathways. Here, we review the generation, toxicity and possible pathways of intracellular Aβtoxicity, and discuss the implication and current knowledge of intracellular Aβin neuronal cell loss in neurodegenerative diseases.     

Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.     

The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene

Amyloid beta-protein (AP) is a peptide of relative molecular mass (Mr) 42,000 found in the senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles of patients with Alzheimer's disease and Down's syndrome (trisomy 21). Recent molecular genetic evidence has indicated that AP is encoded as part of a larger protein by a gene on chromosome 21 (refs 5-7). The defect in the inherited autosomal dominant form of Alzheimer's disease, familial Alzheimer's disease (FAD), has been mapped to the same approximate region of chromosome 21 by genetic linkage to anonymous DNA markers, raising the possibility that this gene product, which could be important in the pathogenesis of Alzheimer's disease, is also the site of the inherited defect in FAD (ref. 5). We have determined the pattern of segregation of the AP gene in FAD pedigrees using restriction fragment length polymorphisms. The detection of several recombination events with FAD suggests that the AP gene is not the site of the inherited defect underlying this disorder.     

小说界革命的先声——论薛福成的文言小说创作

薛福成的文言小说创作是为其改良主义政治观念做宣传的,在《庸盦笔记》中作者从军事、吏治、教育、民生等几个方面表现他的政治思想,该小说集的刊刻早于梁启超"小说界革命"的提出,故薛福成的小说创作可视为小说界革命的先声,应当引起足够的重视。