Structural dynamics of the amyloid β-protein monomer folding nucleus

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.     

Large size fibrillar bundles of the Alzheimer amyloid β-protein

Self-assembly of amyloid β-protein (Aβ) and its deposition into senile plaques are distinctive features of Alzheimer’s disease. Aβ forms typical linear aggregates known as amyloid fibrils, with a diameter of a few tens of nanometers and a length spanning from hundreds of nanometers to micrometers. Fibrils eventually assemble into large size clusters and precipitate in vivo in the brain deposits. Here, we study the late stage of aggregation of Aβ(1–40) in vitro at pH 3.1. We characterize the structure of fibrillar aggregates by a combined use of different experimental techniques. Small angle light scattering, heterodyne near field scattering, large angle light scattering, ultra small angle X-ray scattering and small angle X-ray scattering measurements have been performed to highlight the structural features of amyloid bundles over several lengthscales, from nanometers to tens of micrometers. Phase contrast optical microscopy has been used to complement scattering measurements and directly visualize some morphological details. We show that elongated fibrils of Aβ with a diameter of a few nanometers are packed into large size compact bundles having a typical size of tens of micrometers. The linear morphology of fibrils is reflected in the elongated shape of bundles. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Cross-seeding effects of amyloid β-protein and α-synuclein

J. Neurochem. (2012) 122, 883-890. ABSTRACT: Amyloid β-protein (Aβ) and α-synuclein (αS) are the primary components of amyloid plaques and Lewy bodies (LBs), respectively. Previous in vitro and in vivo studies have suggested that interactions between Aβ and αS are involved in the pathogenesis of Alzheimer's disease and LB diseases. However, the seeding effects of their aggregates on their aggregation pathways are not completely clear. To investigate the cross-seeding effects of Aβ and αS, we examined how sonicated fibrils or cross-linked oligomers of Aβ40, Aβ42, and αS affected their aggregation pathways using thioflavin T(S) assay and electron microscopy. Fibrils and oligomers of Aβ40, Aβ42, and αS acted as seeds, and affected the aggregation pathways within and among species. The seeding effects of αS fibrils were higher than those of Aβ40 and Aβ42 fibrils in the Aβ40 and Aβ42 aggregation pathways, respectively. We showed that Aβ and αS acted as seeds and affected each other's aggregation pathways in vitro, which may contribute to our understanding of the molecular mechanisms of interactions between Alzheimer's disease and LB diseases pathologies.     

Effects of amyloid β-protein on hippocampal long-term potentiation

The accumulation of amyloid β-protein (Aβ) plaques is identified as a major pathological feature of Alzheimer's disease (AD). Recent studies show that soluble species of Aβ are involved in the early memory dysfunction long before neurodegenerative changes. However, the mechanism underlying the neurotoxicity of soluble Aβ is still unclear. Long-term potentiation (LTP) has been thought as an important cellular model of synaptic plasticity for many years. The studies on the hippocampal LTP and Aβ, especially those using AD transgenic models, provided more evidence for the Aβ-induced dysfunction of learning and memory. Based on the recent researches on AD, this article reviewed the effects of Aβ, especially soluble Aβ and its active fragments, on the hippocampal LTP. The possible mechanisms by which Aβ impairs hippocampal LTP are also discussed.     

Exogenous amyloidogenic proteins function as seeds in amyloid β-protein aggregation

Amyloid β-protein (Aβ) aggregation is considered to be a critical step in the neurodegeneration of Alzheimer's disease (AD). In addition to Aβ, many proteins aggregate into the amyloid state, in which they form elongated fibers with spines comprising stranded β-sheets. However, the cross-seeding effects of other protein aggregates on Aβ aggregation pathways are not completely clear. To investigate the cross-seeding effects of exogenous and human non-CNS amyloidogenic proteins on Aβ aggregation pathways, we examined whether and how sonicated fibrils of casein, fibroin, sericin, actin, and islet amyloid polypeptide affected Aβ40 and Aβ42 aggregation pathways using the thioflavin T assay and electron microscopy. Interestingly, the fibrillar seeds of all amyloidogenic proteins functioned as seeds. The cross-seeding effect of actin was stronger but that of fibroin was weaker than that of other proteins. Furthermore, our nuclear magnetic resonance spectroscopic studies identified the binding sites of Aβ with the amyloidogenic proteins. Our results indicate that the amyloidogenic proteins, including those contained in foods and cosmetics, contribute to Aβ aggregation by binding to Aβ, suggesting their possible roles in the propagation of Aβ amyloidosis.     

Oxidative stress induces macroautophagy of amyloid β-protein and ensuing apoptosis

There is increasing evidence for the toxicity of intracellular amyloid β-protein (Aβ) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Aβ in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Aβ that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Aβ-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Aβ content (Vector < APPwt < APPswe). Furthermore, the degree of apoptosis was positively correlated with lysosomal membrane permeabilization, whereas inhibitors of macroautophagy and lysosomal function decreased oxidant-induced apoptosis and diminished the differences in apoptotic response between different cell lines. These results suggest that oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization, which may help explain the mechanisms behind neuronal loss in AD.     

Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.     

Evidence for lysosomal processing of amyloid β-protein precursor in cultured cells

Amyloid -protein precursor (ABPP) of Alzheimer's disease (AD) represents a family of proteins which includes the parent protein which generates a small (4 kD) fragment that self-assembles to form amyloid fibrils in AD. Thus, the normal and abnormal proteolysis of ABPP may be directly relevant to AD pathogenesis. We have examined the accumulation of ABPP in cultured rodent and human neuronal cell lines in the presence and absence of a battery of protease inhibitors using immunohistochemistry and Western blot analysis. Here we present evidence for a lysosomal pathway for the turnover of ABPP and discuss the relevance of these results to plaque pathology and abnormal ABPP immunostaining in AD.Special issue dedicated to Dr. Paola S. Timiras     

Surprising toxicity and assembly behaviour of amyloid β-protein oxidized to sulfone

Aβ (amyloid β-peptide) is believed to cause AD (Alzheimer's disease). Aβ42 (Aβ comprising 42 amino acids) is substantially more neurotoxic than Aβ40 (Aβ comprising 40 amino acids), and this increased toxicity correlates with the existence of unique Aβ42 oligomers. Met3? oxidation to sulfoxide or sulfone eliminates the differences in early oligomerization between Aβ40 and Aβ42. Met3? oxidation to sulfoxide has been reported to decrease Aβ assembly kinetics and neurotoxicity, whereas oxidation to sulfone has rarely been studied. Based on these data, we expected that oxidation of Aβ to sulfone would also decrease its toxicity and assembly kinetics. To test this hypothesis, we compared systematically the effect of the wild-type, sulfoxide and sulfone forms of Aβ40 and Aβ42 on neuronal viability, dendritic spine morphology and macroscopic Ca2(+) currents in primary neurons, and correlated the data with assembly kinetics. Surprisingly, we found that, in contrast with Aβ-sulfoxide, Aβ-sulfone was as toxic and aggregated as fast, as wild-type Aβ. Thus, although Aβ-sulfone is similar to Aβ-sulfoxide in its dipole moment and oligomer size distribution, it behaves similarly to wild-type Aβ in its aggregation kinetics and neurotoxicity. These surprising data decouple the toxicity of oxidized Aβ from its initial oligomerization, and suggest that our current understanding of the effect of methionine oxidation in Aβ is limited.     

Inhibitors of amyloid β-protein aggregation mediated by GM1-containing raft-like membranes

The aggregation (fibril formation) of amyloid β-protein (Aβ) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Aβ aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Aβ aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Aβ aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Aβ to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Aβ in solution, thus at least partly preventing fibrils from forming. Coincubation of Aβ with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Aβ-induced toxicity in PC12 cells by inhibiting Aβ accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Aβ suggested that the two aggregation processes are not identical.     

Phenolic compounds prevent amyloid β-protein oligomerization and synaptic dysfunction by site-specific binding

Cerebral deposition of amyloid β protein (Aβ) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aβ aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aβ aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aβ oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aβ. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aβ oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aβ through different binding.     

Preparation of pure populations of covalently stabilized amyloid β-protein oligomers of specific sizes

Evidence suggests that amyloid β-protein (Aβ) oligomers may be seminal pathogenic agents in Alzheimer's disease (AD). If so, developing oligomer-targeted therapeutics requires an understanding of oligomer structure. This has been difficult due to the instability of these non-covalently associated Aβ assemblies. We previously used rapid, zero-length, in situ chemical cross-linking to stabilize oligomers of Aβ40. These enabled us to isolate pure, stable populations of dimers, trimers, and tetramers and to determine their structure-activity relationships. However, equivalent methods applied to Aβ42 did not produce stable oligomers. We report here that the use of an Aβ42 homologue, [F10, Y42]Aβ42, coupled with sequential denaturation/dissociation and gel electrophoresis procedures, provides the means to produce highly pure, stable populations of oligomers of sizes ranging from dimer through dodecamer that are suitable for structure-activity relationship determination.     

Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid β-protein fibrils

Aggregation of 42-residue amyloid β-protein (Aβ42) plays a pivotal role in the etiology of Alzheimer's disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of Aβ42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the Aβ42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the Aβ42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the Aβ42 fibrils. The observed (13)C-(13)C cross peaks indicated that curcumin interacts with the 12th and 17-21st residues included in the β-sheet structure in the Aβ42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the Aβ42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the Aβ42 fibrils.     

Protein engineering to stabilize soluble amyloid β-protein aggregates for structural and functional studies

The molecular biology underlying protein aggregation and neuronal death in Alzheimer's disease is not yet completely understood, but small soluble nonamyloid aggregates of the amyloid β-protein (Aβ) have been shown to play a fundamental neurotoxic role. The composition and biological action of such aggregates, known as oligomers and protofibrils, are therefore areas of intense study. However, research is complicated by the multitude of different interconverting aggregates that Aβ can form in vitro and in vivo, and by the inhomogeneity and instability of in vitro preparations. Here we review recent studies in which protein engineering, and in particular disulfide engineering, has been applied to stabilize different Aβ aggregates. For example, several techniques now exist to obtain stable and neurotoxic protofibrillar forms of Aβ, and engineered Aβ dimers, or larger aggregates formed by these, have been shown to specifically induce neuronal damage in a way that mimics Alzheimer's disease pathology. Disulfide engineering has also revealed structural properties of neurotoxic aggregates, for instance that Aβ in protofibrils and globular oligomers adopts a β-hairpin conformation that is similar to, but topologically distinct from, the conformation of Aβ in mature amyloid fibrils. Protein engineering is therefore a workable strategy to address many of the outstanding questions relating to the structure, interconversion and biological effects of oligomers and protofibrils of Aβ.     

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.     

Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes

Pathogenesis of Alzheimer’s disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK.     

Decreased levels of soluble amyloid β-protein precursor and β-amyloid protein in cerebrospinal fluid of patients with systemic lupus erythematosus

Symptoms originating from the central nervous system (CNS) frequently occur in patients with systemic lupus erythematosus (SLE). These symptoms are extremely diverse, including a state of dementia. The aim of this study was to examine the cerebrospinal fluid (CSF) content of soluble molecules indicating axonal degeneration and amyloidogenesis. One hundred and fourteen patients with SLE and age-matched controls were evaluated clinically, with magnetic resonance imaging of the brain and CSF analyses. Levels of tau, amyloid precursor protein (APP), beta-amyloid protein (Abeta42), and transforming growth factor beta (TGF-beta) were all determined using sandwich ELISAs.APP and Abeta42 levels were significantly decreased in SLE patients irrespective of their CNS involvement, as compared with healthy controls. Patients with neuropsychiatric SLE who underwent a second lumbar puncture following successful cyclophosphamide treatment showed further decreases of Abeta42. CSF-tau levels were significantly increased in SLE patients showing magnetic resonance imaging-verified brain pathology as compared with SLE patients without such engagement. Importantly, tau levels displayed significant correlation to Abeta42 levels in the CSF. Finally, TGF-beta levels were significantly increased in patients with neuropsychiatric SLE as compared with those without. Low intrathecal levels of Abeta42 found in SLE patients seem to be a direct consequence of a diminished production of APP, probably mediated by heavy anti-inflammatory/immuno-suppressive therapy. Furthermore, our findings suggest that CSF tau can be used as a biochemical marker for neuronal degeneration in SLE. Finally, the increased TGF-beta levels observed may support a notion of an ongoing anti-inflammatory response counteracting tissue injury caused by CNS lupus.     

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.     

Identification of the amyloid β-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.