Addition Of Aβ Research Articles

Amyloid β-Peptide Effects on Glucose Regulation Are Dependent on Apolipoprotein E Genotype.

The apolipoprotein E gene (APOE) confers the greatest genetic risk factor for Alzheimer's disease (AD), wherein the ε4 allele confers an elevated risk compared with the ε3 allele. Biological mechanisms that differ across these alleles have been explored in mouse models wherein the murine Apoe gene has undergone targeted replacement with sequences encoding human ApoE3 or ApoE4 (ApoE-TR mice). Such models have indicated that the two variants of ApoE produce differential effects on energy metabolism, including metabolic syndrome. However, glucose regulation has not been compared in ApoE-TR mice with and without amyloid β-peptide (Aβ) accumulation. We crossed ApoE3-TR and ApoE4-TR mice with a transgenic line that accumulates human Aβ1-42 In male ApoE3-TR mice, introduction of Aβ caused aberrations in glucose tolerance and in membrane translocation of astrocytic glucose transporter 1 (GLUT1). Phosphorylation of Tau at AD-relevant sites was correlated with glucose intolerance. These effects appeared independent of insulin dysregulation and were not observed in females. In ApoE4-TR mice, the addition of Aβ had no significant effects because of a trend toward perturbation of the baseline values.

Investigation of α-Synuclein and Amyloid-β(42)-E22Δ Oligomers Using SiN Nanopore Functionalized with L-Dopa.

Solid-state nanopores are an emerging technology used as a high-throughput, label-free analytical method for the characterization of protein aggregation in an aqueous solution. In this work, we used Levodopamine to coat a silicon nitride nanopore surface that was fabricated through a dielectric breakdown in order to reduce the unspecific adsorption. The coating of inner nanopore wall by investigation of the translocation of heparin. The functionalized nanopore was used to investigate the aggregation of amyloid-β and α-synuclein, two biomarkers of degenerative diseases. In the first application, we demonstrate that the α-synuclein WT is more prone to form dimers than the variant A53T. In the second one, we show for the Aβ(42)-E22Δ (Osaka mutant) that the addition of Aβ(42)-WT monomers increases the polymorphism of oligomers, while the incubation with Aβ(42)-WT fibrils generates larger aggregates.

Structural and Functional Impairments of Reconstituted High-Density Lipoprotein by Incorporation of Recombinant β-Amyloid42.

Beta (β)-amyloid (Aβ) is a causative protein of Alzheimer’s disease (AD). In the pathogenesis of AD, the apolipoprotein (apo) A-I and high-density lipoprotein (HDL) metabolism is essential for the clearance of Aβ. In this study, recombinant Aβ42 was expressed and purified via the pET-30a expression vector and E.coli production system to elucidate the physiological effects of Aβ on HDL metabolism. The recombinant human Aβ protein (51 aa) was purified to at least 95% purity and characterized in either the lipid-free and lipid-bound states with apoA-I. Aβ was incorporated into the reconstituted HDL (rHDL) (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I) with various apoA-I:Aβ ratios from 1:0 to 1:0.5, 1:1 and 1:2. With an increasing molar ratio of Aβ, the α-helicity of apoA-I was decreased from 62% to 36% with a red shift of the Trp wavelength maximum fluorescence from 337 to 340 nm in apoA-I. The glycation reaction of apoA-I was accelerated further by the addition of Aβ. The treatment of fructose and Aβ caused more multimerization of apoA-I in the lipid-free state and in HDL. The phospholipid-binding ability of apoA-I was impaired severely by the addition of Aβ in a dose-dependent manner. The phagocytosis of LDL into macrophages was accelerated more by the presence of Aβ with the production of more oxidized species. Aβ severely impaired tissue regeneration, and a microinjection of Aβ enhanced embryotoxicity. In conclusion, the beneficial functions of apoA-I and HDL were severely impaired by the addition of Aβ via its detrimental effect on secondary structure. The impairment of HDL functionality occurred more synergistically by means of the co-addition of fructose and Aβ.

Amyloid cross-sequence interaction between Aβ(1‐40) and αA(66–80) in relation to the pathogenesis of cataract

Alzheimer's disease (AD) and cataract represent two common protein misfolding diseases closely associated with aging. Growing evidence suggests that these two diseases may be interrelated with each other through cross-sequence interactions between β-amyloid (Aβ) peptide and the short aggregating peptides derived from proteolytic breakdown of α-crystallin. αΑ(66–80) is one of several peptides produced by the proteolytic breakdown of α-crystallin in aged eye lens. Although it is evident that the Aβ(1–40) and αΑ(66–80) coexist in aged eye lenses and both the peptides are known to form macromolecular assemblies, their cross-sequence interaction and the seeding behavior are not known. In this study, the aggregation behavior of αΑ(66–80) has been examined in the presence of Aβ(1–40) on using thioflavin T (ThT) based aggregation kinetics. The presence of monomeric Aβ(1–40) augmented the aggregation kinetics of αΑ(66–80) and reduced the lag time of αΑ(66–80) aggregation. However, the addition of Aβ(1–40) or αΑ(66–80) fibrils (seeds) didn't result in any change in the rate of αΑ(66–80) aggregation. In this in vitro study, we could show that the presence Aβ(1–40) has substantial effect on the aggregation of αΑ(66–80), which suggests a possible interaction between AD and cataract pathologies.

Preferential Intercalation of Human Amyloid-β Peptide into Interbilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment.

This study focuses on the interaction of human amyloid β-peptide (Aβ) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aβ is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aβ-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aβ. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aβ under noncrowding conditions. However, the addition of Aβ under crowding conditions induced shape deformation and aggregation to SUV resulting in multilamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aβ peptide into the interbilayer region. This phenomenon was only observed at the gel (Lβ) phase. These results suggest that an intracellular crowding environment promotes Aβ-membrane interaction and a selective accumulation of Aβ peptides into the interbilayer regions.

Pyroglutamate-modified Aβ(3-42) affects aggregation kinetics of Aβ(1-42) by accelerating primary and secondary pathways.

The aggregation into amyloid fibrils of amyloid-β (Aβ) peptides is a hallmark of Alzheimer's disease. A variety of Aβ peptides have been discovered in vivo, with pyroglutamate-modified Aβ (pEAβ) forming a significant proportion. pEAβ is mainly localized in the core of plaques, suggesting a possible role in inducing and facilitating Aβ oligomerization and accumulation. Despite this potential importance, the aggregation mechanism of pEAβ and its influence on the aggregation kinetics of other Aβ variants have not yet been elucidated. Here we show that pEAβ(3-42) forms fibrils much faster than Aβ(1-42) and the critical concentration above which aggregation was observed was drastically decreased by one order of magnitude compared to Aβ(1-42). We elucidated the co-aggregation mechanism of Aβ(1-42) with pEAβ(3-42). At concentrations at which both species do not aggregate as homofibrils, mixtures of pEAβ(3-42) and Aβ(1-42) aggregate, suggesting the formation of mixed nuclei. We show that the presence of pEAβ(3-42) monomers increases the rate of primary nucleation of Aβ(1-42) and that fibrils of pEAβ(3-42) serve as highly efficient templates for elongation and catalytic surfaces for secondary nucleation of Aβ(1-42). On the other hand, the addition of Aβ(1-42) monomers drastically decelerates the primary and secondary nucleation of pEAβ(3-42) while not altering the pEAβ(3-42) elongation rate. In addition, even moderate concentrations of fibrillar Aβ(1-42) prevent pEAβ(3-42) aggregation, likely due to non-reactive binding of pEAβ(3-42) monomers to the surfaces of Aβ(1-42) fibrils. Thus, pEAβ(3-42) accelerates aggregation of Aβ(1-42) by affecting all individual reaction steps of the aggregation process while Aβ(1-42) dramatically slows down the primary and secondary nucleation of pEAβ(3-42).

Neutron Scattering Studies of the Interplay of Amyloid β Peptide(1-40) and An Anionic Lipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol.

The interaction between lipid bilayers and Amyloid β peptide (Aβ) plays a critical role in proliferation of Alzheimer’s disease (AD). AD is expected to affect one in every 85 humans by 2050, and therefore, deciphering the interplay of Aβ and lipid bilayers at the molecular level is of profound importance. In this work, we applied an array of neutron scattering methods to study the structure and dynamics of Aβ(1–40) interacting 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) bilayers. In the structural investigations of lipid bilayer’s response to Aβ binding, Small Angle Neutron Scattering and Neutron Membrane Diffraction revealed that the Aβ anchors firmly to the highly charged DMPG bilayers in the interfacial region between water and hydrocarbon chain, and it doesn’t penetrate deeply into the bilayer. This association mode is substantiated by the dynamics studies with high resolution Quasi-Elastic Neutron Scattering experiments, showing that the addition of Aβ mainly affects the slower lateral motion of lipid molecules, especially in the fluid phase, but not the faster internal motion. The results revealed that Aβ associates with the highly charged membrane in surface with limited impact on the structure, but the altered membrane dynamics could have more influence on other membrane processes.

Regulation of IL-1β-Induced Cyclooxygenase-2 Expression by Interactions of Aβ Peptide, Apolipoprotein E and Nitric Oxide in Human Neuroglioma

Alzheimer disease (AD) is characterized by chronic neuroinflammation, which may lead to dysfunction in neuronal circuits. Although reactive microglia are found in association with accumulation of beta amyloid (Aβ) plaques in the AD brain, their contribution to neuronal cell loss remains speculative. A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele, which has been shown to contribute significantly to neurodegeneration in AD. Many studies have documented the ability of Aβ fibrils in vitro to induce microglia to undergo phenotypic activation, which results in the secretion and/or expression of a plethora of free radicals and pro-inflammatory mediators. These mediators, such as reactive nitrogen/oxygen species and IL-1β as well as cyclooxygenase-2 (COX-2) with associated prostaglandin E2 (PGE(2)), are believed to be neurotoxic and to contribute to the underlying cause of AD. We have used the human H4 neuroglioma cells as a model astroglial system to examine the interactions between IL-1β and nitric oxide (NO) as co-stimulators of Aβ(1-40) in enhancing the expression of COX-2 and production of PGE(2) in the presence of recombinant human apolipoprotein E4 (apoE4). Neither Aβ(1-40) nor its reverse sequence analog Aβ(40-1) alone had a significant effect on COX-2 expression and PGE(2) production in the cells. In contrast, after co-incubation with apoE4, Aβ(1-40) increased IL-1β-induced COX-2 expression and PGE(2) production. Aβ(12-28), which binds with high affinity to apoE4, blocked apoE4-mediated effects on Aβ(1-40). Furthermore, (±)-S-Nitroso-N-acetylpenicillamine (SNAP), an agent that releases nitric oxide (NO) in situ, alone did not affect IL-1β-induced COX-2 expression, but increased PGE(2) production only. Addition of Aβ(1-40) preincubated with apoE4 to H4 cells in the presence of SNAP led to an additive IL-1β-induced COX-2 expression and PGE(2) production. These observations indicate that increased PGE(2) resulted from increased nitrosative stress, which is enhanced by apoE4. Thus a molecular understanding of the interactions of apoE4 with Aβ, NO and IL-1β on the regulation of the COX-2/prostaglandin pathway may open new avenues in understanding the mechanism of development of neurodegenerative disease such as AD.

Effects of butylphthalide on the apoptosis of PC-12 cells under the induction of β-amyloid peptide

To study the protective effects of butylphthalide (NBP) on the Aβ(25-35)-induced apoptosis in PC-12 cells. The apoptosis of PC-12 cell was analyzed by MTT assay, transmission electron microscope and PI method at different concentrations of NBP (0.1, 1.0, 10, 100 µmol/L) with the addition of Aβ or not. The expressions of Bcl-2 and cytochrome C (Cyt-C) were detected by Western blot. As demonstrated by the MTT assay, the values of cell viability were 76.5% ± 1.1%, 84.2% ± 1.3%, 89.5% ± 1.3% and 81.9% ± 1.9% at various concentration (0.1, 1.0, 10, 100 µmol/L) of NBP respectively. The model group was 71.7% ± 1.4%. It was revealed that the former could significantly prevent the cell viability under the induction of Aβ(25-35) (P < 0.05). A pretreatment with 10 µmol/L NBP could significantly inhibit the decrease of viability under the induction of Aβ(25-35) (P < 0.05). PI showed that the apoptosis rate of the 10 µmol/L NBP treatment group was significantly lower than that of the model group. Under electron microscope, the characteristics of cell apoptosis were significant in the model group. And the cell morphology of the 10 µmol/L NBP treatment group was normal. The expression rate of Bcl-2 protein in the 10 µmol/L NBP treatment group was obviously higher than that in the model group. Cyt-C was weakly expressed in nerve cells of the normal and the 10 µmol/L NBP groups. But it had a strong expression in the model group. NBP prevents the injury of PC12 cells by Aβ. And the mechanism may be related to the elevated level of Bcl-2 and the inhibited mitochondrial release of Cyt-C.

Amyloid-β protein impairs Ca2+ release and contractility in skeletal muscle

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by β-amyloid underlie the effects observed in muscle, in vitro Ca(2+) release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aβ(1-42). Application of Aβ(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aβ(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered βAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

Effects of ageing and Alzheimer’s disease on mitochondrial function of human platelets

Mitochondrial dysfunction may play an important role in the pathogenesis of ageing and age-neurodegenerative diseases such as Alzheimer’s disease (AD). Platelet mitochondrial membrane potential (reflected by measurement of JC-1 fluorescence ratio) and adenosine 5′-triphosphate (ATP) contents of 24 moderate probable AD patients, 20 age-matched control subjects and 20 young control subjects were measured. Also, a beta-amyloid peptide (Aβ)-induced damage model of platelets was established. After the addition of Aβ, platelet JC-1 fluorescence ratio and ATP content of platelets were measured in 16 AD patients, 20 aged and 20 young control subjects. Young control subjects had higher JC-1 fluorescence ratio than both AD patients and aged control subjects. No significant differences in platelet ATP contents were found among AD patients, aged and young control subjects. After the addition of Aβ, platelet JC-1 fluorescence ratio and ATP content of aged and young control subjects lowered markedly, but no obvious decrease of platelet JC-1 fluorescence ratio of AD patients was found compared with those of aged and young control subjects. Decrease of platelet JC-1 fluorescence ratio of aged control subjects was lower than that of young control subjects following the addition of Aβ. These results indicated that mitochondrial dysfunction may occur during ageing and platelet mitochondria of AD patients and aged subjects showed a tolerance to Aβ-induced damage. Therefore, blood platelets might serve as a biomarker for detection of mitochondrial function and age-related disease.

Squalestatin protects neurons and reduces the activation of cytoplasmic phospholipase A 2 by Aβ 1–42

Alzheimer's disease is characterised by the loss of neurons and the production of Aβ peptides. We report that the addition of Aβ 1–42 to neurons resulted in activation of cytoplasmic phospholipase A 2 (cPLA 2), the production of prostaglandin E 2, synapse damage and reduced neuronal survival. Pre-treatment with simvastatin, a clinically relevant statin that penetrates the brain, protected against Aβ 1–42 induced synapse damage and neuronal death in vitro. The neuroprotective effects of simvastatin were shared by squalestatin, a squalene synthase inhibitor that reduces neuronal cholesterol production and crucially, does not affect isoprenoid formation. The protective effect of both these drugs was reversed by the addition of exogenous cholesterol. These drugs did not alter the amounts of extracellular Aβ 1–42 ingested by neurons; rather they reduced Aβ 1–42 induced activation of cPLA 2 and prostaglandin E 2 production. Treatment prevented the migration of Aβ 1–42 and cPLA 2 to caveolin-1 containing lipid rafts. We propose that critical concentrations of Aβ 1–42 trigger the amalgamation of individual micro-domains containing signalling molecules to form lipid raft platforms in which sustained activation of cPLA 2 leads to neuronal dysfunction and ultimately neuronal death. This process is dependent on the amounts of cholesterol in neuronal membranes and is susceptible to treatment with squalestatin or simvastatin.

Effects of β-amyloid peptide and estrogen on platelet mitochondrial function of Sprague-Dawley rats

β-Amyloid peptide (Aβ) peptides play a central role in the development of Alzheimer's disease. They are known to induce mitochondrial dysfunction and caspase activation, resulting in apoptosis of neuronal cells. In the present experiment, an Aβ-induced damage model of platelets was established to observe the effects of Aβ, estradiol benzoate (EB) and genistein on platelets and platelet mitochondria. It was found that after the addition of Aβ, platelet number, platelet mitochondrial membrane potential (ΔΨm) and adenosine triphosphate (ATP) content were lowered while no protective effects of EB and genistein had been observed. The platelets could serve as a biomarker for detection of mitochondrial function and age related disease.

Modulation of neutrophil apoptosis by β-amyloid proteins

This study examined the effect of amyloid β peptide (Aβ) and the secretase inhibitors of amyloid precursor proteins (APP) on the spontaneous apoptosis of neutrophils. Aβ(1–40) decreased the apoptotic rate of neutrophils. The delayed apoptosis by Aβ was not blocked by pertussis toxin and N-formyl peptide receptor-like 1 antagonistic peptide, WRWWWW. The inhibitors of phoshoinositide 3-kinase (LY294002), phospholipase C (U73122), or Ca++-dependent protein kinase C (Go6976) abrogated the anti-apoptotic effect of Aβ on neutrophils. Moreover, the Aβ-induced delay of apoptosis was inhibited by a calcium chelator, BAPTA/AM. The amount of the APP protein was reduced in the cultured neutrophils and the APP level in the Aβ or pancaspase-treated neutrophils was lower than that in the cultured neutrophils. However, the reduction in APP level was recovered after treating them with the secretase inhibitors or anti-Fas antibody. The exogenous addition of cell permeable β- and γ-secretase inhibitors resulted in an increase in the rate of the apoptosis. The regulation of neutrophil apoptosis by the addition of Aβ and secretase inhibitors occurred via the caspase -8, -9, -3, and mitochondrial-dependent pathways. This suggests that the intracellular β-amyloid proteins play a role as regulating factor of neutrophil survival and that Aβ-induced delay of apoptosis is mediated by other receptors rather than a seven-transmembrane G protein-coupled receptor(s).

Peptide Blockers of the Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Amyloid β

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.

A Novel Action of Alzheimer's Amyloid β-Protein (Aβ): Oligomeric Aβ Promotes Lipid Release

Interactions between amyloid beta-protein (Abeta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of Abeta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric Abeta, but not monomeric or fibrillar Abeta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of Abeta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these Abeta and lipids formed particles and were recovered from the fractions at densities of approximately 1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by Abeta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-l-cysteine. These Abeta-lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric Abeta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.