Calmodulin regulates the non-amyloidogenic metabolism of amyloid precursor protein in platelets

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP.     

Ellagic acid promotes Aβ42 fibrillization and inhibits Aβ42-induced neurotoxicity

Smaller, soluble oligomers of β-amyloid (Aβ) play a critical role in the pathogenesis of Alzheimer’s disease (AD). Selective inhibition of Aβ oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against Aβ neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on Aβ42 aggregation and neurotoxicity in vitro. EA promoted Aβ fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited Aβ aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in Aβ42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic Aβ aggregates to render them harmless, our MTT results showed that EA could significantly reduce Aβ42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.     

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

The strong pH dependence of A beta oligomerization could arise from favorable intermolecular charge-charge interactions between His and carboxylate groups, or, alternatively, by mutual electrostatic repulsion of peptide molecules. To test between these two possibilities, the pH dependence of the oligomerization of A beta and three charge substitution variants with Asp, Glu and His substituted by Ala is measured. All four peptides oligomerize, as detected by thioflavin T fluorescence, turbidity, and amyloid fibril formation; therefore, specific charge-charge interactions are nonessential for oligomerization. The strong negative correlation between net charge and oligomerization indicates that electrostatic repulsion between A beta monomers impedes their association.     

Rigidity and flexibility of dipeptidyl peptidase IV: crystal structures of and docking experiments with DPIV

Dipeptidyl peptidase IV (DPIV) is an alpha,beta-hydrolase-like serine exopeptidase, which removes dipeptides, preferentially with a C-terminal l-Pro residue, from the N terminus of longer peptide substrates. Previously, we determined the tetrameric 1.8A crystal structure of native porcine DPIV. Each monomer is composed of a beta-propeller and a catalytic domain, which together embrace an internal cavity housing the active centre. This cavity is connected to the bulk solvent by a "propeller opening" and a "side opening". Here, we analyse DPIV complexes with a t-butyl-Gly-Pro-Ile tripeptide, Pro-boroPro, a piperazine purine compound, and aminoethyl phenyl sulfonylfluoride. The latter two compounds bind to the active-site groove in a compact and a quite bulky manner, respectively, causing considerable shifts of the catalytic Ser630 side-chain and of the Tyr547 phenolic group, which forms the oxyanion hole. The tripeptide, mimicking a peptide substrate, is clamped to the active site through tight interactions via its N-terminal alpha-ammonium group, the P2 carbonyl group, the P1-l-Pro side-chain, the C-terminal carboxylate group, and the stable orthoacid ester amide formed between the scissile peptide carbonyl group and Ser630 O(gamma). This stable trapping of the tripeptide could be due to stabilization of the protonated His740 imidazolium cation by the adjacent negatively charged C-terminal carboxylate group, preventing proton transfer to the leaving group nitrogen atom. Docking experiments with the compact rigid 58 residue protein aprotinin, which had been shown to be processed by DPIV, indicate that the Arg1-Pro2 N terminus can access the DPIV active site only upon widening of its side openings, probably by separation of the first and the last propeller blades, and/or of the catalytic and the propeller domain.     

Transgenic potato expressing Abeta reduce Abeta burden in Alzheimer's disease mouse model

Beta amyloid (Abeta) is believed one of the major pathogens of Alzheimer's disease (AD), and the reduction of Abeta is considered a primary therapeutic target. Immunization with Abeta can reduce Abeta burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Abeta1-42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Abeta burden in the brain. Thus, Abeta tandem repeats can be expressed in transgenic potato plants to form immunologically functional Abeta, and these potatoes has a potential to be used for the prevention and treatment of AD.     

Identification of an amyloidogenic region on keratoepithelin via synthetic peptides

Mutations of keratoepithelin (KE) gene in human chromosome 5q31 have been linked with corneal epithelial or stromal dystrophies characterized by the abnormal deposits of amyloid fibrils and/or non-amyloid aggregations in corneal tissue. We report herein that synthetic peptide containing amino acid (a.a.) residues of 515-532 of native KE protein can readily form beta-sheet-containing amyloid fibrils in vitro. Amyloid fibrils formed in various conditions from short synthetic peptides (containing a.a. 515-532 and 515-525, respectively) were characterized by thioflavin T (ThT) fluorescence assay, Congo red staining, electron microscopy (EM) and circular dichroism (CD). Triple-N-methylation of the synthetic peptides prevented the beta-sheet polymerization and related amyloid fibril formation. Comparison study with ThT fluorescence further demonstrated that synthetic peptides containing corneal dystrophy-related mutations within this region formed amyloid fibrils to various extents. Our results suggest that each individual dystrophy-related mutation by itself does not necessarily potentiate amyloid fibril formation of KE. Roles of these intrinsically amyloidogenic foci in abnormal KE aggregations and amyloid deposits of stromal corneal dystrophies await further investigation.     

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL⁻¹) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L⁻¹ h⁻¹).     

Clathrin adaptor AP-1–mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments

One of the hallmarks of Alzheimer’s disease is the accumulation of toxic amyloid-β (Aβ) peptides in extracellular plaques. The direct precursor of Aβ is the carboxyl-terminal fragment β (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer’s disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of Aβ. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and Aβ release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.     

The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.     

Oligomerization and toxicity of Aβ fusion proteins

This study has found that the Maltose binding protein Aβ42 fusion protein (MBP-Aβ42) forms soluble oligomers while the shorter MBP-Aβ16 fusion and control MBP did not. MBP-Aβ42, but neither MBP-Aβ16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aβ42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aβ42 characterization.     

Attenuated cytotoxicity but enhanced betafibril of a mutant amyloid beta-peptide with a methionine to cysteine substitution

Amyloid-beta peptide (Abeta), the major constituent of senile plaques in the Alzheimer's disease (AD) brain, is the main source of oxidative stress leading to neurodegeneration. The methionine residue in this peptide is reported to be responsible for neurotoxicity. Structurally similar substitution with methionine 35 replaced by cysteine in Abeta(40) was synthesized, and this result in enhanced beta-sheet structures according to both circular dichroism (CD) spectra and beta-fibril specific fluorescence assay but attenuated cytotoxicity whether in the presence of copper or not. These findings may provide further evidence on disclosing the connection between amyloid beta-aggregation and Abeta-induced neurotoxicity.     

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

Abstract

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic α-helical structure from Lys² to Leu¹⁴. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp⁹ are in close contact with methylene protons of SDS micelles. T₁ relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp⁹ with Ala⁹, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp⁹ into the membrane, as well as the amphiphilic α-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

The influence of simvastatin, atorvastatin and high-cholesterol diet on acetylcholinesterase activity, amyloid beta and cholesterol synthesis in rat brain

OBJECTIVE: There is evidence to suppose that cholesterol-lowering medicine might confer protection against dementia, probably via modulation of cholesterol synthesis in the brain. The aim of the present study was to investigate the potential influence of statins and cholesterol diet on selected parameters relevant to Alzheimer's disease pathophysiology. METHODS: For 15 days, rats were orally administered simvastatin (10 or 20mg/kg b.wt.), atorvastatin (10 or 20mg/kg b.wt.), or aqua (control group); and one group was fed high-cholesterol (2%) diet. At the end of experiments brain (and plasma) cholesterol, lathosterol, hydroxymethylglutaryl-coenzyme A reductase protein, acetylcholinesterase activity, amyloid beta (40 and 42) and cholesterol synthesis rate (using the incorporation of deuterium from deuterated water) were determined and statistically compared to those of aqua. RESULTS: Both statins were able to lower cholesterol in the plasma, but none elicited an effect on total brain cholesterol. Significant reductions of brain lathosterol and cholesterol synthesis rate were observed after simvastatin and atorvastatin treatment. Acetylcholinesterase activity, amyloid beta and hydroxymethylglutaryl-coenzyme A reductase levels remained unaffected by the two drugs. CONCLUSIONS: This study brings additional evidence of a role for statins in cholesterol synthesis in the brain. Our data question the relationship between amyloid beta, acetylcholinesterase activity and cholesterol synthesis in the rat brain as well as the assumption about no exchange between peripheral and brain cholesterol pools.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid β-peptide (Aβ) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Aβ amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 μM Aβ in the presence or absence of air–water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Aβ concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Aβ concentrations (10 and 20 μM). Importantly, when the 5 μM Aβ solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 μM Aβ solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 μM Aβ solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.     

Interaction of the amyloid imaging tracer FDDNP with hallmark Alzheimer's disease pathologies

The distinctive cortical uptake of the tracer ¹⁸F-FDDNP (2-(1-{6-[(2-fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile) in Alzheimer's disease (AD) is believed to be because of its binding to both neurofibrillary tangles (NFTs) and highly fibrillar senile plaques. We therefore investigated the binding of a tracer concentration of ³H-FDDNP to brain sections containing AD hallmark pathologies. Semi-adjacent sections were labelled with ³H-PIB (Pittsburgh compound-B, 2-[4'-(methylamino)phenyl]-6-hydroxybenzothiazole) and ¹⁴C-SB13 (4- N -methylamino-4'-hydroxystilbene) for comparison. Neocortical sections containing widespread senile plaques and cerebrovascular amyloid angiopathy, produced a sparse and weak labelling following incubation with ³H-FDDNP. Furthermore, in sections containing NFTs, there was no overt labelling of the pathology by ³H-FDDNP. In contrast, sections labelled with ³H-PIB displayed extensive labelling of diffuse plaques, classical plaques, cerebrovascular amyloid angiopathy and NFTs. ¹⁴C-SB13 produced a broadly similar binding pattern to PIB. Radioligand binding assays employing in vitro generated amyloid-β peptide fibrils demonstrated a ∼10-fold reduced affinity for ³H-FDDNP (85.0 ± 2.0 nM) compared with ³H-PIB (8.5 ± 1.3 nM). These data provide an alternative mechanistic explanation for the observed low cortical uptake of ¹⁸F-FDDNP in AD; in that the ligand is only weakly retained by the hallmark neuropathology because of its low affinity for amyloid structures.     

Assembly of hereditary amyloid beta-protein variants in the presence of favorable gangliosides

The mechanisms underlying regional amyloid beta-protein (Abeta) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch- and Italian-type Abetas, and Flemish-type Abeta was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch- and Italian-type Abetas deposit, exclusively express GM3 whereas GD3 is upregulated in the co-culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish-type Abeta deposition. Our results suggest that regional Abeta deposition is induced by the local gangliosides in the brain.     

Diethylamine extraction of proteins and peptides isolated with a mono-phasic solution of phenol and guanidine isothiocyanate

Representative extraction of both RNA and protein from a single biological sample is required for reliable assessment of coordinated changes in gene and protein expression. Such a simultaneous extraction can be performed by using Trizol Reagent. Here, we demonstrate that, as an alternative to SDS, 2% diethylamine is an effective solvent, which can be conveniently used in extraction of Trizol-isolated proteins from various tissues. Diethylamine provides efficient extraction of proteins and compatibility with a variety of common downstream analytical applications.