Region Of Amyloid Precursor Protein Research Articles

MiR-15b-5p targeting amyloid precursor protein is involved in the anti-amyloid eflect of curcumin in swAPP695-HEK293 cells.

Curcumin exerts a neuroprotective effect on Alzheimer's disease; however, it is not known whether microRNAs are involved in this protective effect. This study was conducted using swAPP695-HEK293 cells as an Alzheimer's disease cell model. swAPP695-HEK293 cells were treated with 0, 0.5, 1, 2, 5, and 10 μM curcumin for 24 hours. The changes in miR-15b-5p, miR-19a-3p, miR-195-5p, miR-101-3p, miR-216b-5p, miR-16-5p and miR-185-5p expression were assessed by real-time quantitative polymerase chain reaction. The mRNA and protein levels of amyloid precursor protein, amyloid-β40 and amyloid-β42 were evaluated by quantitative real-time polymerase chain reaction, western blot assays and enzyme-linked immunosorbent assays. swAPP695-HEK293 cells were transfected with miR-15b-5p mimic, or treated with 1 μM curcumin 24 hours before miR-15b-5p inhibitor transfection. The effects of curcumin on amyloid precursor protein, amyloid-β40 and amyloid-β42 levels were evaluated by western blot assays and enzyme-linked immunosorbent assay. Luciferase assays were used to analyze the interaction between miR-15b-5p and the 3'-untranslated region of amyloid precursor protein. The results show that amyloid precursor protein and amyloid-β expression were enhanced in swAPP695-HEK293 cells compared with HEK293 parental cells. Curcumin suppressed the expression of amyloid precursor protein and amyloid-β and up-regulated the expression of miR-15b-5p in swAPP695-HEK293 cells. In addition, we found a negative association of miR-15b-5p expression with amyloid precursor protein and amyloid-β levels in the curcumin-treated cells. Luciferase assays revealed that miR-15b-5p impaired the luciferase activity of the plasmid harboring the 3'-untranslated region of amyloid precursor protein. These findings indicate that curcumin down-regulates the expression of amyloid precursor protein and amyloid-β in swAPP695-HEK293 cells, which was partially mediated by miR-15b-5p via targeting of the 3'-untranslated region of amyloid precursor protein.

Association between serum aluminium level and methylation of amyloid precursor protein gene in workers engaged in aluminium electrolysis

To investigate the association between serum aluminium level and methylation of the promoter region of amyloid precursor protein (APP)gene in workers engaged in aluminium electrolysis. In 2012, 366 electrolysis workers in an aluminium factory were enrolled as exposure group (working years >10 and age >40 years)and divided into low-exposure group and high-exposure group based on the median serum aluminium level. Meanwhile, 102 workers in a cement plant not exposed to aluminium were enrolled as control group. Graphite furnace atomic absorption spectrometry was used to measure serum aluminium level, methylation specific PCR was used to measure the methylation rate of the promoter region of APP gene, and ELI-SA was used to measure the protein expression of APP in lymphocytes in peripheral blood. The exposure group had a significantly higher serum aluminium level than the control group (45.07 μg/L vs 30.51 μg/L, P< 0.01). The exposure group had a significantly lower methylation rate of the promoter region of APP gene than the control group (18.85% vs 25.49%, P=0.025), and the high-exposure group had a significantly lower methylation rate of the promoter region of APP gene than the low-exposure group (15.84% vs 21.85%, P<0.05). The exposure group had a significantly higher protein expression of APP in lymphocytes in peripheral blood than the control group (66.73 ng/ml vs 54.17 ng/ml, P<0.05); compared with the low-exposure group (65.39 ng/ml), the high-exposure group showed an increase in the protein expression of APP in lymphocytes in peripheral blood (67.22 ng/ml), but there was no significant difference between these two groups (P>0.05). The multivariate logistic regression analysis showed that with reference to the control group, low aluminium exposure (OR=1.86, 95% CI 1.67~3.52)and high aluminium exposure (OR=2.98, 95% CI 1.97~4.15)were risk factors for a reduced methylation rate of the promoter region of APP gene. Reduced methylation of the promoter region of APP gene may be associated with increased serum aluminium level, and downregulated methylation of the promoter region of APP gene may accelerate APP gene transcription.

1P211 ラフト中でのアミロイド前駆体タンパク質の膜貫通部位の二量化にコレステロールが与える影響(13B. 生体膜・人工膜:ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

1P211 ラフト中でのアミロイド前駆体タンパク質の膜貫通部位の二量化にコレステロールが与える影響(13B. 生体膜・人工膜:ダイナミクス,ポスター,第52回日本生物物理学会年会(2014年度))

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin-binding disulfide-constrained loop region of human ß-amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C-terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL-37, these peptides efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop-structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein.

Decreased Aβ secretion by cells expressing familial Alzheimer's disease-linked mutant presenilin 1

Presenilin (PS) is a catalytic subunit of the γ-secretase complex that cleaves the intramembranous region of amyloid precursor protein (APP), producing amyloid β (Aβ) peptide. Familial Alzheimer's disease (FAD) results from PS mutations, which may alter γ-secretase activity to enhance the production of highly aggregable Aβ42. The precise pathogenic effects of mutant PS remain unclear. To exclude the effects of endogenous PS, we established cell lines from PS1/PS2-deficient (PS−/−) fibroblasts capable of stably expressing either wild-type (wt) PS1 or different mutant PS1s. Although both wt PS1 and mutant PS1 formed γ-secretase complexes of the same size and containing the same components, the amount of Aβ secreted by FAD mutant PS1-expressing cells was significantly reduced. The ratio of Aβ42 to Aβ40 (Aβ42/Aβ40) secreted by these cells, however, was significantly higher than that secreted by cells expressing wt PS1, which corroborated findings from a previous report. The elevated Aβ42/Aβ40 ratio observed with mutant PS1-expressing cells may be due to reduced Aβ40 production not increased Aβ42 production.

Proteomic identification of less oxidized brain proteins in aged senescence-accelerated mice following administration of antisense oligonucleotide directed at the Aβ region of amyloid precursor protein

Amyloid β-peptide (Aβ) is the major constituent of senile plaques, a pathological hallmark of Alzheimer's disease (AD) brain. It is generally accepted that Aβ plays a central role in the pathophysiology of AD. Aβ is released from cells under entirely normal cellular conditions during the internalization and endosomal processing of amyloid precursor protein (APP). However, accumulation of Aβ can induce neurotoxicity. Our previous reports showed that decreasing the production of Aβ by giving an intracerebroventricular injection of a 42-mer phosphorothiolated antisense oligonucleotide (AO) directed at the Aβ region of the APP gene reduces lipid peroxidation and protein oxidation and improves cognitive deficits in aged senescence-accelerated mice prone 8 (SAMP8) mice. In order to investigate how Aβ level reduction improves learning and memory performance of SAMP8 mice through reduction of oxidative stress in brains, we used proteomics to identify the proteins that are less oxidized in 12-month-old SAMP8 mice brains treated with AO against the Aβ region of APP (12 mA) compared to that of the age-control SAMP8 mice. We found that the specific protein carbonyl levels of aldoase 3 (Aldo3), coronin 1a (Coro1a) and peroxiredoxin 2 (Prdx2) are significantly decreased in the brains of 12 mA SAMP8 mice compared to the age-controlled SAMP8 treated with random AO (12 mR). We also found that the expression level of α-ATP synthase (Atp5a1) was significantly decreased, whereas the expression of profilin 2 (Pro-2) was significantly increased in brains from 12 mA SAMP8 mice. Our results suggest that decreasing Aβ levels in aged brain in aged accelerated mice may contribute to the mechanism of restoring the learning and memory improvement in aged SAMP8 mice and may provide insight into the role of Aβ in the memory and cognitive deficits in AD.

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.

A peptide derived from the C-terminal region of acetylcholinesterase modulates extracellular concentrations of acetylcholinesterase in the rat substantia nigra

It is well established that acetylcholinesterase (AChE) has ‘non-classical’ functions independent of cholinergic transmission. A region of AChE distinct from the catalytic site may be responsible for these actions via a 14-residue peptide located between residues 586–599 at the C-terminus of human AChE. This AChE-peptide possesses a high amino acid sequence homology with a region of amyloid precursor protein and shares many biophysical and physiological characteristics. In this study, the effect of AChE-peptide (AEFHRWSSYMVHWK) on the extracellular levels of endogenous AChE was examined in rat substantia nigra in vitro. A chemiluminescent assay was used to continuously measure the soluble AChE concentration from tissue punches of the substantia nigra. Application of NMDA evoked an increase in extracellular AChE levels consistent with previous results obtained from in vivo models. AChE-peptide, when applied alone, had no effect on AChE release: however, when co-applied with NMDA, AChE-peptide reduced the effectiveness of NMDA to evoke release of AChE. These results indicate, in a region of the brain central to the aetiology of Parkinson's disease, that an AChE-peptide fragment derived from AChE displays a bioactivity that could involve regulation of Ca 2+ availability and hence the release of AChE.

Gamma-Secretase: characterization and implication for Alzheimer disease therapy.

gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

A Second Locus for Very-Late-Onset Alzheimer Disease: A Genome Scan Reveals Linkage to 20p and Epistasis between 20p and the Amyloid Precursor Protein Region

We used a covariate-based linkage method to reanalyze genome scan data from affected sibships collected by the Alzheimer Disease (AD) Genetics Initiative of the National Institute of Mental Health. As reported in an earlier article, the amyloid-beta precursor protein (APP) region is strongly linked to affected sib pairs of the oldest current age (i.e., age either at last exam or at death) who lack E4 alleles at the apolipoprotein E (ApoE) locus. We now report that a region on 20p shows the same pattern. A model that includes current age and the number of E2 alleles as covariates gives a LOD score of 4.1. The signal on 20p is near the location of the gene coding for cystatin-C, previously shown to be associated with late-onset AD and to codeposit with APP in the brains of patients with AD. Two-locus analysis provides evidence of strong epistasis between 20p and the APP region, limited to the oldest age group and to those lacking ApoE4 alleles. We speculate that high-risk polymorphisms in both regions produce a biological interaction between these two proteins that increases susceptibility to a very-late-onset form of AD.

Specific binding of Cu2+ ions by a pentapeptide fragment present in the cysteine-rich region of amyloid precursor protein

The βA4 amyloid precursor protein fragment situated in the cysteine-rich region is a very effective binding site for Cu2+ ions due to the presence of three His residues in the His–Xaa–His–Yaa–His sequence.

Up-regulation of Nucleolin mRNA and Protein in Peripheral Blood Mononuclear Cells by Extracellular-regulated Kinase

The signal transduction pathways regulating nucleolin mRNA and protein production have yet to be elucidated. Peripheral blood mononuclear cells treated with phorbol 12-myristate 13-acetate showed steady state levels of nucleolin mRNA that were 2-2.5-fold greater than untreated control cells. The up-regulation of nucleolin mRNA was substantially repressed by U0126, a specific inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores and ionomycin also activated ERK and substantially elevated nucleolin mRNA levels, demonstrating phorbol 12-myristate 13-acetate and calcium signaling converge on ERK. Drugs that affected protein kinase C, protein kinase A, and phospholipase C signal transduction pathways did not alter nucleolin mRNA levels significantly. The half-life of nucleolin mRNA increased from 1.8 h in resting cells to 3.2 h with phorbol ester activation, suggesting ERK-mediated posttranscriptional regulation. Concomitantly, full-length nucleolin protein was increased. The higher levels of nucleolin protein were accompanied by increased binding of a 70-kDa nucleolin fragment to the 29-base instability element in the 3'-untranslated region of amyloid precursor protein (APP) mRNA in gel mobility shift assays. Supplementation of rabbit reticulocyte lysate with nucleolin decreased APP mRNA stability and protein production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production.

Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

PCR amplification in bisulfite methylcytosine mapping in the GC-rich promoter region of amyloid precursor protein gene in autopsy human brain

Alterations in DNA 5-methyldeoxycytidine pattern influence gene expression for certain mammalian genes in development, differentiation, carcinogenesis, and aging. Detection of DNA methylation at the promoter region, which generally represses transcription activity, is one important element in studying changes in molecular expression with aging and age-associated disorders. Bisulfite genomic sequencing is a useful method for mapping methylated cytosines. However, PCR amplification for bisulfite-treated DNA does not yield a sufficient amount of products that have a sufficient level of specificity, especially in the GC-rich sequences usually seen at the promoter regions of house keeping genes. We present a method for increasing the sensitivity and specificity of PCR amplification in bisulfite methylcytosine mapping in an extremely GC-rich promoter region of amyloid precursor protein (APP) gene from the cerebral cortex of human autopsy brain. The PCR used consists of two cycles using the lower primer alone to amplify the sense sequence, and then eight cycles at a theoretical annealing temperature (60°C) and 30 cycles at a lower annealing temperature (50°C) using both the upper and lower primers. The present method likely can also be applied to other GC-rich genomic sequences. Theme: Cellular and molecular biology Topic: Gene structure and function: general.

HnRNP C increases amyloid precursor protein (APP) production by stabilizing APP mRNA.

We have previously shown that heterogeneous nuclear ribonucleoprotein C (hnRNP C) and nucleolin bound specifically to a 29 nt sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. Upon activation of peripheral blood mononuclear cells, hnRNP C and nucleolin acquired APP mRNA binding activity, concurrent with APP mRNA stabilization. These data suggested that the regulated interaction of hnRNP C and nucleolin with APP mRNA controlled its stability. Here we have directly examined the role of the cis element and trans factors in the turnover and translation of APP mRNA in vitro . In a rabbit reticulocyte lysate (RRL) translation system, a mutant APP mRNA lacking the 29 nt element was 3-4-fold more stable and synthesized 2-4-fold more APP as wild-type APP mRNA. Therefore, the 29 nt element functioned as an APP mRNA destabilizer. RNA gel mobility shift assays with the RRL suggested the presence of endogenous nucleolin, but failed to show hnRNP C binding activity. However, wild-type APP mRNA was stabilized and coded for 6-fold more APP when translated in an RRL system supplemented with exogenous active hnRNP C. Control mRNAs lacking the 29 nt element were unaffected by hnRNP C supplementation. Therefore, occupancy of the 29 nt element by hnRNP C stabilized APP mRNA and enhanced its translation.

The Amino-Terminal Region of Amyloid Precursor Protein Is Responsible for Neurite Outgrowth in Rat Neocortical Explant Culture

We have previously shown that secreted forms of β-amyloid precursor protein (APPss) promote neurite outgrowth in embryonic rat neocortical explant culture. To determine the region of APPsresponsible for its biological activity, we produced both amino- and carboxyl-terminal regions of APPsusing a yeast expression system. The purified fragment corresponding to the amino-terminal region (NAPP) enhanced neurite outgrowth of neocortical explants, but the carboxyl-terminal region fragment did not. The neurite-promoting activity of full length APPsand NAPP was blocked by the antibody, 22C11, specific for the amino-terminal region, and the 16-mer peptide of epitope for 22C11 also enhanced neurite outgrowth. However, the 17-mer peptide which contains RERMS sequence did not enhance the neurite outgrowth, but promoted the survival of neocortical neurons in dissociated culture. These findings suggested that the amino-terminal region is responsible for the neurite-promoting activity of APPss.

Snake coiled fibres in rat soleus muscle in chloroquine induced myopathy share immunohistochemical characteristics with amyloid depositions in Alzheimer's disease brain tissue

Pathological and immunopathological studies were carried out on snake coiled fibres (SCF) which occurred in affected soleus muscle in chloroquine treated rats. The SCF began to appear in denervated soleus muscle by 8 days after chloroquine injection. By day 14, typical SCF were observed with an unusual swirling pattern of the myofibrils, presenting a bizarre appearance. By day 21 or later, the SCF became less remarkable, and were fragmented and broken apart to form large vacuoles. Immunopathological studies demonstrated that the amyloid beta (A beta) and N and C-terminal regions of amyloid precursor protein (APP), and the amyloid associated proteins tested, apolipoprotein E (apoE), SP-40,40, alpha 1-antichymotrypsin (alpha 1-ACT), and ubiquitin, which are known to be components of amyloid depositions found in Alzheimer's disease (AD) affected brains, were present in the SCF. ApoE, SP-40,40, alpha 1-ACT, and ubiquitin are induced following certain cell challenges (e.g. heat shock, various drugs and injury). The significance of APP, A beta, and amyloid associated proteins are discussed in respect to snake coiled fibre formations in chloroquine rat myopathy and in the amyloidogenesis of AD.

Induction of connexin43 and gap junctional communication in PC12 cells overexpressing the carboxy terminal region of amyloid precursor protein

Induction of connexin43 and gap junctional communication in PC12 cells overexpressing the carboxy terminal region of amyloid precursor protein

Induction of connexin43 and gap junctional communication in PC12 cells overexpressing the carboxy terminal region of amyloid precursor protein.

Previous studies have shown that PC12 cells overexpressing beta/A4 amyloid peptide display altered morphology characterized by pronounced membrane ruffling and extensive intercellular appositions. Having observed other cell types in which these features accompany increased connexin43 (Cx43) production and gap junctional communication, we examined Cx43 in normal and beta/A4-transfected PC12 cells. Studies of two beta/A4-transfected PC12 clones revealed an induction of Cx43 expression by Western blotting, intracellular and plasma membrane-associated Cx43 in some cells of cultures processed by immunofluorescence, dye-transfer between some cells microinjected with Lucifer Yellow, and gap junctions between cells examined by EM. Normal and vector-transfected PC12 cells exhibited none of these properties. Increased immunofluorescence in some clusters of beta/A4-transfected cells was also observed with a monoclonal antibody against connexin32. The results suggest that beta/A4 amyloid peptide may cause aberrant intercellular communication and gap junction formation through induction or increased expression of connexins in cells that are not normally coupled or only poorly coupled by gap junctions.

Amyloid β protein in rat soleus muscle in chloroquine-induced myopathy using end-specific antibodies for Aβ40 and Aβ42: immunohistochemical evidence for amyloid β protein

Previous immunohistochemical studies from this laboratory demonstrated that monoclonal antibodies raised against various regions of amyloid precursor protein (APP) (i.e., N-terminus, amyloid β protein (Aβ), and C-terminus) strongly labeled vacuoles in chloroquine-induced myopathy-affected muscle in rats. In this study, we used antibodies end specific for the Aβ40 and Aβ42 species, and a monoclonal antibody to A01-9 which reacts with APP and Aβ. Most vacuoles clearly reacted with anti-Aβ1–9, while about half reacted with anti-Aβ42, and only a few reacted with anti-Aβ40. These results demonstrate that vacuoles in chloroquine-induced myopathy-affected muscle contain cleaved Aβ, and that distribution of the two major Aβ species is similar to what is observed in Aβ deposition in Alzheimer's disease (AD)-affected brain. This provides further evidence that chloroquine-induced myopathy in rats provides a suitable model to understand APP processing into Aβ, and the role of APP in terms of the pathogenesis of AD.