Aggregation of Aβ40/42 chains in the presence of cyclic neuropeptides investigated by molecular dynamics simulations

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST₁₄) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ₄₂ aggregates. Furthermore, SST₁₄’s presence was also found to promote the formation of toxic Aβ₄₂ oligomers in vitro. In order to elucidate how SST₁₄ influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ₄₂ or Aβ₄₀ peptides with SST₁₄ molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ₄₂ in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ₄₂–SST₁₄, Aβ₄₂–AVP, and Aβ₄₀–SST₁₄. The Aβ₄₂–SST₁₄ mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST₁₄ or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST₁₄ was found to impede aggregation in the Aβ₄₂–SST₁₄ system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ₄₂–SST₁₄ oligomerization.     

Preventive effect of Ninjin-yoei-to,a Kampo medicine,on amyloid β1-42-induced neurodegeneration via intracellular Zn2+ toxicity in the dentate gyrus

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer’s disease, here we examined the effect of NYT on amyloid β_1-42 (Aβ_1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ_1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ_1-42-induced increase in intracellular Zn²⁺ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ_1-42-induced Zn²⁺ toxicity in the dentate gyrus. As a matter of fact, Aβ_1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn²⁺-binding proteins, which can capture Zn²⁺ from Zn-Aβ_1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ_1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn²⁺ toxicity induced by Aβ_1-42.     

Overexpression of Heparanase Lowers the Amyloid Burden in Amyloid-β Precursor Protein Transgenic Mice

Heparan sulfate (HS) and HS proteoglycans (HSPGs) colocalize with amyloid-β (Aβ) deposits in Alzheimer disease brain and in Aβ precursor protein (AβPP) transgenic mouse models. Heparanase is an endoglycosidase that specifically degrades the unbranched glycosaminoglycan side chains of HSPGs. The aim of this study was to test the hypothesis that HS and HSPGs are active participators of Aβ pathogenesis in vivo. We therefore generated a double-transgenic mouse model overexpressing both human heparanase and human AβPP harboring the Swedish mutation (tgHpa*Swe). Overexpression of heparanase did not affect AβPP processing because the steady-state levels of Aβ_1–40, Aβ_1–42, and soluble AβPP β were the same in 2- to 3-month-old double-transgenic tgHpa*Swe and single-transgenic tgSwe mice. In contrast, the Congo red-positive amyloid burden was significantly lower in 15-month-old tgHpa*Swe brain than in tgSwe brain. Likewise, the Aβ burden, measured by Aβ_x-40 and Aβ_x-42 immunohistochemistry, was reduced significantly in tgHpa*Swe brain. The intensity of HS-stained plaques correlated with the Aβ_x-42 burden and was reduced in tgHpa*Swe mice. Moreover, the HS-like molecule heparin facilitated Aβ_1–42-aggregation in an in vitro Thioflavin T assay. The findings suggest that HSPGs contribute to amyloid deposition in tgSwe mice by increasing Aβ fibril formation because heparanase-induced fragmentation of HS led to a reduced amyloid burden. Therefore, drugs interfering with Aβ-HSPG interactions might be a potential strategy for Alzheimer disease treatment.     

The chaperone domain BRICHOS prevents CNS toxicity of amyloid-β peptide in Drosophila melanogaster

Aggregation of the amyloid-β peptide (Aβ) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer’s disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays Aβ42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the Aβ42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of Aβ42 and BRICHOS resulted in delayed Aβ42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing Aβ42 alone. Moreover, BRICHOS increased the ratio of soluble:insoluble Aβ42 and bound to deposits of Aβ42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of Aβ42, although significant Aβ42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.KEY WORDS: Amyloid, Alzheimer’s disease, Protein misfolding, Chaperone     

Anti-Aggregating Effect of the Naturally Occurring Dipeptide Carnosine on Aβ1-42 Fibril Formation

Carnosine is an endogenous dipeptide abundant in the central nervous system, where by acting as intracellular pH buffering molecule, Zn/Cu ion chelator, antioxidant and anti-crosslinking agent, it exerts a well-recognized multi-protective homeostatic function for neuronal and non-neuronal cells. Carnosine seems to counteract proteotoxicity and protein accumulation in neurodegenerative conditions, such as Alzheimer’s Disease (AD). However, its direct impact on the dynamics of AD-related fibril formation remains uninvestigated. We considered the effects of carnosine on the formation of fibrils/aggregates of the amyloidogenic peptide fragment Aβ1-42, a major hallmark of AD injury. Atomic force microscopy and thioflavin T assays showed inhibition of Aβ1-42 fibrillogenesis in vitro and differences in the aggregation state of Aβ1-42 small pre-fibrillar structures (monomers and small oligomers) in the presence of carnosine. in silico molecular docking supported the experimental data, calculating possible conformational carnosine/Aβ1-42 interactions. Overall, our results suggest an effective role of carnosine against Aβ1-42 aggregation.     

Molecular Plasticity Regulates Oligomerization and Cytotoxicity of the Multipeptide-length Amyloid-β Peptide Pool

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ_1–38 and Aβ_1–40 at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ_1–38 and Aβ_1–43 in addition to Aβ_1–40 and Aβ_1–42, in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ_1–38 and Aβ_1–43 aggregate similar to Aβ_1–40 and Aβ_1–42, respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ_1–38 and Aβ_1–43 significantly affect the behaviors of Aβ_1–40 and Aβ_1–42. The short timescale conformational flexibility of Aβ_1–38 is suggested to be responsible for enhancing toxicity of Aβ_1–40 while exerting a cyto-protective effect on Aβ_1–42. Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.     

Interactions between Aβ and Mutated Tau Lead to Polymorphism and Induce Aggregation of Aβ-Mutated Tau Oligomeric Complexes

One of the main hallmarks of the fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) is the accumulation of neurofibrillary tangles in the brain as an outcome of the aggregation of mutated tau protein. This process occurs due to a number of genetic mutations in the MAPT gene. One of these mutations is the ∆K280 mutation in the tau R2 repeat domain, which promotes the aggregation vis-à-vis that for the wild-type tau. Experimental studies have shown that in Alzheimer’s disease Aβ peptide forms aggregates both with itself and with wild-type tau. By analogy, in FTDP-17, it is likely that there are interactions between Aβ and mutated tau, but the molecular mechanisms underlying such interactions remain to be elucidated. Thus, to investigate the interactions between Aβ and mutated tau, we constructed fourteen ∆K280 mutated tau-Aβ_17-42 oligomeric complexes. In seven of the mutated tau-Aβ_17-42 oligoemric complexes the mutated tau oligomers exhibited hydrophobic interactions in their core domain, and in the other seven mutated tau-Aβ_17-42 oligoemric complexes the mutated tau oligomers exhibited salt-bridge interactions in their core domain. We considered two types of interactions between mutated tau oligomers and Aβ oligomers: interactions of one monomer of the Aβ oligomer with one monomer of the mutated tau oligomer to form a single-layer conformation, and interactions of the entire Aβ oligomer with the entire mutated tau oligomer to form a double-layer conformation. We also considered parallel arrangements of Aβ trimers alternating with mutated tau trimers in a single-layer conformation. Our results demonstrate that in the interactions of Aβ and mutated tau oligomers, polymorphic mutated tau-Aβ_17-42 oligomeric complexes were observed, with a slight preference for the double-layer conformation. Aβ trimers alternating with mutated tau trimers constituted a structurally stable confined β-structure, albeit one that was energetically less stable than all the other constructed models.     

Cross-seeding of WT amyloid-β with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease

Alzheimer’s disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-β (Aβ). Inherited early-onset AD can be caused by single point mutations within the Aβ sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aβ isoform expression. It is therefore important to understand how these mixtures of Aβ isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aβ42 and Aβ40. We confirm that Aβ40(WT) does not readily cross-seed Aβ42(WT) fibril formation. In contrast, fibril formation of Aβ40(Arctic) is hugely accelerated by Aβ42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aβ40(Arctic) and Aβ42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aβ42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aβ40/42 isoforms, which can impact the rate and structure of fibril assembly.     

The calcium-free form of atorvastatin inhibits amyloid-β(1–42) aggregation in vitro

Alzheimer''s disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aβ) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aβ. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer''s Disease Assessment Scale—Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aβ42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aβ42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO₃-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aβ by at least a factor of 2. The ¹H–¹⁵N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the ¹⁶KLVF¹⁹ binding site on the Aβ peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aβ from an α-helix-dominant to a β-sheet-dominant structure.     

Genome-wide analysis of Saccharomyces cerevisiae identifies cellular processes affecting intracellular aggregation of Alzheimer's amyloid-β42: importance of lipid homeostasis

Amyloid-β (Aβ)–containing plaques are a major neuropathological feature of Alzheimer''s disease (AD). The two major isoforms of Aβ peptide associated with AD are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in AD progression. Improved understanding of cellular processes affecting Aβ42 aggregation may have implications for development of therapeutic strategies. Aβ42 fused to green fluorescent protein (Aβ42-GFP) was expressed in ∼4600 mutants of a Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes affecting intracellular Aβ42 aggregation by assessing the fluorescence of Aβ42-GFP. This screening identified 110 mutants exhibiting intense Aβ42-GFP–associated fluorescence. Four major cellular processes were overrepresented in the data set, including phospholipid homeostasis. Disruption of phosphatidylcholine, phosphatidylserine, and/or phosphatidylethanolamine metabolism had a major effect on intracellular Aβ42 aggregation and localization. Confocal microscopy indicated that Aβ42-GFP localization in the phospholipid mutants was juxtaposed to the nucleus, most likely associated with the endoplasmic reticulum (ER)/ER membrane. These data provide a genome-wide indication of cellular processes that affect intracellular Aβ42-GFP aggregation and may have important implications for understanding cellular mechanisms affecting intracellular Aβ42 aggregation and AD disease progression.     

Oridonin Attenuates Aβ1–42-Induced Neuroinflammation and Inhibits NF-κB Pathway

Neuroinflammation induced by beta-amyloid (Aβ) plays a critical role in the pathogenesis of Alzheimer’s disease (AD), and inhibiting Aβ-induced neuroinflammation serves as a potential strategy for the treatment of AD. Oridonin (Ori), a compound of Rabdosia rubescens, has been shown to exert anti-inflammatory effects. In this study, we demonstrated that Ori inhibited glial activation and decreased the release of inflammatory cytokines in the hippocampus of Aβ_1–42-induced AD mice. In addition, Ori inhibited the NF-κB pathway and Aβ_1–42-induced apoptosis. Furthermore, Ori could attenuate memory deficits in Aβ_1–42-induced AD mice. In conclusion, our study demonstrated that Ori inhibited the neuroinflammation and attenuated memory deficits induced by Aβ_1–42, suggesting that Ori might be a promising candidate for AD treatment.     

Molecular insights into the primary nucleation of polymorphic amyloid β dimers in DOPC lipid bilayer membrane

Alzheimer''s disease (AD) pathology is characterized by loss of memory cognitive and behavioral deterioration. One of the hallmarks of AD is amyloid β (Aβ) plaques in the brain that consists of Aβ oligomers and fibrils. It is accepted that oligomers, particularly dimers, are toxic species that are produced extracellularly and intracellularly in membranes. It is believed that the disruption of membranes by polymorphic Aβ oligomers is the key for the pathology of AD. This is a first study that investigate the effect of polymorphic “α‐helix/random coil” and “fibril‐like” Aβ dimers on 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) membrane. It has been found that the DOPC membrane promotes Aβ_1–42 “fibril‐like” dimers and impedes Aβ_1–42 “α‐helix/random coil” dimers. The N‐termini domains within Aβ_1–42 dimers play a role in Aβ aggregation in membrane milieus. In addition, the aromatic π–π interactions (involving residues F19 and F20 in Aβ_1–42) are the driving forces for the hydrophobic interactions that initiate the primary nucleation of polymorphic Aβ_1–42 dimers within DOPC membrane. Finally, the DOPC bilayer membrane thickness is locally decreased, and it is disrupted by an embedded distinct Aβ_1–42 dimer, due to relatively large contacts between Aβ_1–42 monomers and the DOPC membrane. This study reveals insights into the molecular mechanisms by which polymorphic early‐stage Aβ_1–42 dimers have distinct impacts on DOPC membrane.     

Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-β Variants and Structural Analysis by Solution NMR Spectroscopy

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEAβ from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-¹⁵N]- or [U-¹³C, ¹⁵N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEAβ40 and pEAβ42 for further physiological and biochemical studies.     

Effects of Secondary Metabolite Extract from Phomopsis occulta on β-Amyloid Aggregation

Inhibition of β-amyloid (Aβ) aggregation is an attractive therapeutic and preventive strategy for the discovery of disease-modifying agents in Alzheimer''s disease (AD). Phomopsis occulta is a new, salt-tolerant fungus isolated from mangrove Pongamia pinnata (L.) Pierre. We report here the inhibitory effects of secondary metabolites from Ph. occulta on the aggregation of Aβ42. It was found that mycelia extracts (MEs) from Ph. occulta cultured with 0, 2, and 3 M NaCl exhibited inhibitory activity in an E. coli model of Aβ aggregation. A water-soluble fraction, ME0-W-F1, composed of mainly small peptides, was able to reduce aggregation of an Aβ42-EGFP fusion protein and an early onset familial mutation Aβ42E22G-mCherry fusion protein in transfected HEK293 cells. ME0-W-F1 also antagonized the cytotoxicity of Aβ42 in the neural cell line SH-SY5Y in dose-dependent manner. Moreover, SDS-PAGE and FT-IR analysis confirmed an inhibitory effect of ME0-W-F1 on the aggregation of Aβ42 in vitro. ME0-W-F1 blocked the conformational transition of Aβ42 from α-helix/random coil to β-sheet, and thereby inhibited formation of Aβ42 tetramers and high molecular weight oligomers. ME0-W-F1 and other water-soluble secondary metabolites from Ph. occulta therefore represent new candidate natural products against aggregation of Aβ42, and illustrate the potential of salt tolerant fungi from mangrove as resources for the treatment of AD and other diseases.     

Aβ42 Mutants with Different Aggregation Profiles Induce Distinct Pathologies in Drosophila

Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer''s disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.     

Molecular Mechanisms of Alzheimer Disease Protection by the A673T Allele of Amyloid Precursor Protein

Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96–99). The Ala-673 residue lies within the β-secretase recognition sequence and is part of the amyloid-β (Aβ) peptide cleavage product (position 2 of Aβ). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V_max) of APP by the BACE1 enzyme, without affecting the affinity (K_m) of the APP substrate for BACE1. We also show a reduced level of Aβ(1–42) aggregation with A2T Aβ peptides, an observation not conserved in Aβ(1–40) peptides. When combined in a ratio of 1:9 Aβ(1–42)/Aβ(1–40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aβ(1–42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aβ peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aβ aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.     

Cytotoxic effects of GM1 ganglioside and amyloid β-peptide on mouse embryonic neural stem cells

AD (Alzheimer’s disease) is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides) in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between G_M1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells), but the precise effects of G_M1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of G_M1 and Aβ-(1–40) on mouse NECs (neuroepithelial cells), which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either G_M1 or Aβ-(1–40). On the contrary, a decreased number of NECs were cultured in the presence of a combination of G_M1 and Aβ-(1–40). The exogenously added G_M1 and Aβ-(1–40) were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase) pathway, important for cell proliferation, was intact in NECs simultaneously treated with G_M1 and Aβ-(1–40), but caspase 3 was activated. NECs treated with G_M1 and Aβ-(1–40) were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, an indicator of cell death. It was found that G_M1 and Aβ-(1–40) interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40) and G_M1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.     

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Aβ) peptide by using a small engineered binding protein (Z_Aβ3) that binds with nanomolar affinity to Aβ, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z_Aβ3 in the brains of Drosophila melanogaster expressing either Aβ₄₂ or the aggressive familial associated E22G variant of Aβ₄₂ abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Aβ binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Aβ aggregation and reveal that Z_Aβ3 not only inhibits the initial association of Aβ monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.     

Cu2+ Affects Amyloid-β (1–42) Aggregation by Increasing Peptide-Peptide Binding Forces

The link between metals, Alzheimer''s disease (AD) and its implicated protein, amyloid-β (Aβ), is complex and highly studied. AD is believed to occur as a result of the misfolding and aggregation of Aβ. The dyshomeostasis of metal ions and their propensity to interact with Aβ has also been implicated in AD. In this work, we use single molecule atomic force spectroscopy to measure the rupture force required to dissociate two Aβ (1–42) peptides in the presence of copper ions, Cu²⁺. In addition, we use atomic force microscopy to resolve the aggregation of Aβ formed. Previous research has shown that metal ions decrease the lag time associated with Aβ aggregation. We show that with the addition of copper ions the unbinding force increases notably. This suggests that the reduction of lag time associated with Aβ aggregation occurs on a single molecule level as a result of an increase in binding forces during the very initial interactions between two Aβ peptides. We attribute these results to copper ions acting as a bridge between the two peptide molecules, increasing the stability of the peptide-peptide complex.     

3,6'‐disinapoyl sucrose attenuates Aβ1‐42‐ induced neurotoxicity in Caenorhabditis elegans by enhancing antioxidation and regulating autophagy

The aggregation of β‐amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer''s disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6''‐Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti‐depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ_1‐42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg‐laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ_1‐42 transgenic C. elegans. Gene analysis showed that DISS could up‐regulate the mRNA expression of sod‐3, gst‐4, daf‐16, bec‐1 and lgg‐1, while down‐regulate the mRNA expression of daf‐2 and daf‐15 in Aβ_1‐42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ_1‐42‐induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.