Antisera to an amino-terminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimer's disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.     

The dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific.

Bacterial fusion proteins, constructed from overlapping fragments of the open reading frame coding for gp86 of human cytomegalovirus (HCMV) strain AD169, were used to localize antigenic regions recognized by antibodies from human convalescent sera. A major domain for binding of conformation-independent antibodies was localized on fusion protein AP86, containing amino acids 15 to 142 of gp86. Human antibodies, affinity purified on AP86, neutralized infectious virus in tissue culture. In addition, a mouse monoclonal antibody (AP86-SA4), raised against AP86, also neutralized HCMV. AP86-SA4 was reactive with viral gp86 in immunoblot assays and showed a plasma membrane staining on intact HCMV-infected fibroblasts late in infection. After exonuclease III deletions of the viral gene, the binding site of neutralizing human as well as mouse antibodies was localized between amino acid residues 34 and 43. The domain has sequence variation between laboratory strains AD169 and Towne, and binding of the antibodies was strain specific. To our knowledge, this is the first characterization of a strain-specific neutralizing epitope on HCMV.     

Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.     

Analysis of mammalian dynein using antibodies against a polypeptides of sea urchin sperm flagellar dynein

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.     

Antibodies to potato virus Y bind the amyloid beta peptide: immunohistochemical and NMR studies

Studies in transgenic mice bearing mutated human Alzheimer disease (AD) genes show that active vaccination with the amyloid beta (Abeta) protein or passive immunization with anti-Abeta antibodies has beneficial effects on the development of disease. Although a trial of Abeta vaccination in humans was halted because of autoimmune meningoencephalitis, favorable effects on Abeta deposition in the brain and on behavior were seen. Conflicting results have been observed concerning the relationship of circulating anti-Abeta antibodies and AD. Although these autoantibodies are thought to arise from exposure to Abeta, it is also possible that homologous proteins may induce antibody synthesis. We propose that the long-standing presence of anti-Abeta antibodies or antibodies to immunogens homologous to the Abeta protein may produce protective effects. The amino acid sequence of the potato virus Y (PVY) nuclear inclusion b protein is highly homologous to the immunogenic N-terminal region of Abeta. PVY infects potatoes and related crops worldwide. Here, we show through immunocytochemistry, enzyme-linked immunosorbent assay, and NMR studies that mice inoculated with PVY develop antibodies that bind to Abeta in both neuritic plaques and neurofibrillary tangles, whereas antibodies to material from uninfected potato leaf show only modest levels of background immunoreactivity. NMR data show that the anti-PVY antibody binds to Abeta within the Phe4-Ser8 and His13-Leu17 regions. Immune responses generated from dietary exposure to proteins homologous to Abeta may induce antibodies that could influence the normal physiological processing of the protein and the development or progression of AD.     

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.     

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimer's Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (A beta PP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175-186), predicted from cDNA sequence for A beta PP, was used. The immunoreactivity of A beta PP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD = 27 and normal = 30) were analyzed for A beta PP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of A beta PP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.     

Comparative studies on [Ca2+]i-level of fibroblasts from Alzheimer patients and control individuals

The accumulation of the -amyloid peptide (AP) in the brain, produced from the ubiquitously expressed amyloid precursor protein (APP) is a defining feature of Alzheimer's disease (AD). Consistent with studies demonstrating the importance of skin biopsy in the diagnosis of neurodegenerative disorders, we investigated whether differences in intracellular free calcium levels ([Ca²⁺]_i) of cultured cutaneous fibroblasts derived from sporadic AD patients and from age-matched control individuals might be present. [Ca²⁺]_i was measured in Fura-2AM-loaded human fibroblasts by dual wavelength spectrofluorimetry. AD cells exhibited lower [Ca²⁺]_i as compared to the control cultures. Exposure of fibroblasts to AP resulted in increased [Ca²⁺]_i of the control cells, but not of AD fibroblasts. Our test could prove useful in supporting the diagnosis of (sporadic) AD in patients suspected of suffering from the disease.     

Bin1 and CD2AP polarise the endocytic generation of beta‐amyloid

The mechanisms driving pathological beta‐amyloid (Aβ) generation in late‐onset Alzheimer's disease (AD) are unclear. Two late‐onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aβ generation in neurons. We identify a novel neuron‐specific polarisation of Aβ generation controlled by Bin1 and CD2AP. We discover that Bin1 and CD2AP control Aβ generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aβ generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aβ in neurons increasing the risk for late‐onset AD.     

The structure and function of the beta 2-adaptin appendage domain

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.     

Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals

During receptor-mediated endocytosis, AP2 complexes act as a bridge between the cargo membrane proteins and the clathrin coat by binding to sorting signals via the mu 2 subunit and to clathrin via the beta subunit. Here we show that binding of AP2 to sorting signals in vitro is regulated by phosphorylation of the mu 2 subunit of AP2. Phosphorylation of mu 2 enhances the binding affinity of AP2 for sorting motifs as much as 25-fold compared with dephosphorylated AP2. The recognition of sorting signals was not affected by the phosphorylation status of the alpha or beta 2 subunit, suggesting that phosphorylation of mu 2 is critical for regulation of AP2 binding to sorting signals. Phosphorylation of mu 2 occurs at a single threonine residue (Thr-156) and is mediated by the newly discovered adaptor-associated kinase, AAK1, which copurifies with AP2. We propose that phosphorylation of the AP2 mu 2 subunit by AAK1 ensures high affinity binding of AP2 to sorting signals of cargo membrane proteins during the initial steps of receptor-mediated endocytosis.     

Lipid peroxidation and protein oxidation in Alzheimer's disease brain: potential causes and consequences involving amyloid beta-peptide-associated free radical oxidative stress

Amyloid beta-peptide (A(beta)) is heavily deposited in the brains of Alzheimer's disease (AD) patients, and free radical oxidative stress, particularly of neuronal lipids and proteins, is extensive. Recent research suggests that these two observations may be linked by A(beta)-induced oxidative stress in AD brain. This review summarizes current knowledge on phospholipid peroxidation and protein oxidation in AD brain, one potential cause of this oxidative stress, and consequences of A(beta)-induced lipid peroxidation and protein oxidation in AD brain.     

Two Monoclonal Antibodies Recognize Alzheimer''s Neurofibrillary Tangles, Neurofilament, and Microtubule-Associated Proteins

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.     

非遗传性老年痴呆的动物模型及行为学,病理学改变

通过毁损Ｗｉｓｔａｒ大鼠迈内特基底核（ｎｂＭ）建立非遗传性老年痴呆（ＡＤ）动物模型。用Ｍｏｒｒｉｓ水迷宫、六胺银染色、ａｃｅｔｙｌｃｈｏｌｉｎｅｓｔｅｒａｓｅ（ＡＣｈＥ）细胞化学、β淀粉蛋白（βＡＰ）和τ蛋白免疫细胞化学及超微结构观察等方法进行行为学及病理学变化研究。结果证明：毁损ｎｂＭ鼠学习、记忆能力下降,乙酰胆碱酯酶ＡＣｈＥ锐减,βＡＰ和τ蛋白样免疫神经元增加,细胞水胞、溶解,溶酶体、微管增加     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Closing in on the amyloid cascade

Accumulation of the amyloid-beta (A beta) peptide in the central nervous system (CNS) is considered by many to be the crucial pathological insult that ultimately leads to the development of Alzheimer's disease (AD). Regulating the production and/or aggregation of A beta could therefore be of considerable benefit to patients afflicted with AD. It has long been known that A beta is derived from the proteolytic processing of the amyloid precursor protein (APP) by two enzymatic activities, beta-secretase and gamma-secretase. Recent breakthroughs have led to the identification of the aspartyl protease BACE (beta-site APP-cleaving enzyme) as beta-secretase and the probable identification of the presenilin proteins as gamma-secretases. This review discusses what is know about BACE and the presenilins, focusing on their capacity as secretases, as well as the options for therapeutic advancement the careful characterization of these proteins will provide. These findings are presented in the context of the "amyloid cascade hypothesis" and its physiological relevance in AD pathogenesis.     

A pool of beta-tubulin is hyperphosphorylated at serine residues in Alzheimer disease brain.

In Alzheimer disease (AD) brain, activities of protein phosphatase (PP)-2A/PP-1 which are known to be associated with microtubules are compromised and are probably a cause of neurofibrillary degeneration through hyperphosphorylation of microtubule proteins. In the present study, an increase of approximately 11 pmol phosphate/microg protein in 100,000 x g pellet from AD compared with age-matched control brains was found. Tau protein, which is hyperphosphorylated in AD can only account for approximately 4 pmol phosphate/microg protein, suggesting the presence of non-tau hyperphosphorylated proteins in the diseased brain. Western blot analysis with phosphoserine antibodies revealed a approximately 54 kDa non-tau protein to be significantly hyperphosphorylated in AD compared with age-matched control cases in the particulate fraction. The approximately 54 kDa protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as beta-tubulin by immunolabeling with specific antibodies, mass spectrometry analysis and by N-terminal amino acid sequencing. The purified protein was hyperphosphorylated at serine residues in AD.