Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimer's disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.     

Curcumin Decreases Amyloid-β Peptide Levels by Attenuating the Maturation of Amyloid-β Precursor Protein

Alzheimer disease (AD) is a devastating neurodegenerative disease with no cure. The pathogenesis of AD is believed to be driven primarily by amyloid-β (Aβ), the principal component of senile plaques. Aβ is an ∼4-kDa peptide generated via cleavage of the amyloid-β precursor protein (APP). Curcumin is a compound in the widely used culinary spice, turmeric, which possesses potent and broad biological activities, including anti-inflammatory and antioxidant activities, chemopreventative effects, and effects on protein trafficking. Recent in vivo studies indicate that curcumin is able to reduce Aβ-related pathology in transgenic AD mouse models via unknown molecular mechanisms. Here, we investigated the effects of curcumin on Aβ levels and APP processing in various cell lines and mouse primary cortical neurons. We show for the first time that curcumin potently lowers Aβ levels by attenuating the maturation of APP in the secretory pathway. These data provide a mechanism of action for the ability of curcumin to attenuate amyloid-β pathology.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation

Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1–40 and Aβ1–42 are the dominant forms. The fibril architectures of Aβ1–40 and Aβ1–42 differ and Aβ1–42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1–42 can be cross-templated and incorporated into the ends of Aβ1–40 fibrils, while incorporation of Aβ1–40 monomers into Aβ1–42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1–40 to incorporate into the ends of Aβ1–42 fibrils and the capacity of Aβ1–42 monomers to adopt the properties of Aβ1–40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1–42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1–40 from adopting the fibrillar properties of Aβ1–42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.     

Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer

Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.     

Amyloid-β oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons

Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer''s disease (AD). Soluble amyloid-β oligomers (AβOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AβOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AβOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3β, downstream targets of CaN, prevents BDNF transport defects induced by AβOs. We further show that AβOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AβO-induced BDNF transport disruption.     

Racemization of the Aspartic Acid Residue of Amyloid-β Peptide by a Radical Reaction

An E. coli cell extract containing thermostable α-amylase and maltogenic amylase expressed from the clone pTMA322 was used to produce an anomalously linked oligosaccharides (Alo) mixture from starch. The liquefaction and saccharification of starch were done by one-step procedure using the above cell extract. The resulted Alo mixture contained over 40% oligosaccharides having DP 3 or more including branched forms.     

Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

Background

Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer''s disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-β peptide (QDAβ) and its advanced applications.

Methodology/Principal Findings

The QDAβ construct retained Aβ oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAβ coaggregation with intact Aβ42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Aβ antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAβ more significantly than oligomeric QDAβ, and the ingested QDAβ was mainly accumulated in the lysosome.

Conclusions/Significance

These data demonstrate that QDAβ is a novel nanoprobe for studying Aβ oligomerization and fibrillization in multiple modalities and may be applicable for high-throughput drug screening systems.     

Resveratrol Abrogates Hypoxia-Induced Up-Regulation of Exosomal Amyloid-β Partially by Inhibiting CD147

Hypoxia promotes both total extracellular and exosomal amyloid-β (Aβ) production and aggravates Alzheimer’s disease (AD). Resveratrol (RSV) has been proved to be neuroprotective in AD models, and down-regulated the expression of CD147, an additional subunit of γ-secretase. In this study, we aimed to explore the role and mechanisms of RSV in hypoxia-induced upregulation of Aβ, especially exosomal Aβ. SH-SY5Y cells and HEK293 cells overexpressing amyloid precursor protein (APP) as well as C57BL/6 mice were treated with RSV and exposed to hypoxic conditions. The expression of SIRT1 or CD147 was modulated by transfection of specific siRNAs or plasmid. Aβ1-40 and Aβ1-42 levels were determined by ELISA. Hypoxia increased the levels of both Aβ1-40 and Aβ1-42 in the hippocampal lysates and serum-derived exosomes of mice. Hypoxia also increased both Aβ1-40 and Aβ1-42 levels in the total culture medium (CM), cell-derived exosomal lysates, and exosome-free CM of both cell lines. Treatment with RSV abrogated these changes in Aβ expression, inhibited the hypoxia-induced down-regulation of SIRT1 and up-regulation of CD147. Knockdown of SIRT1 promote total Aβ level but has no effect on exosomal Aβs expression. Knockdown of CD147 inhibits both total and exosomal Aβs expression. Furthermore, overexpressing CD147 in cells exposed to hypoxia facilitated the production of Aβ1-40 and Aβ1-42, while application of RSV reduced the CD147 expression as well as Aβ levels in both exosomes and exosome-free CM. These results suggested that RSV abrogated hypoxia-induced up-regulation of total and exosomal Aβ partially by inhibiting CD147.

     

Degradation of Alzheimer’s Amyloid-β by a Catalytically Inactive Insulin-Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer’s amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE’s catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE’s function.     

Structure and Dynamics of Amyloid-β Segmental Polymorphisms

It is believed that amyloid-beta (Aβ) aggregates play a role in the pathogenesis of Alzheimer's disease. Aβ molecules form β-sheet structures with multiple interaction sites. This polymorphism gives rise to differences in morphology, physico-chemical property and level of cellular toxicity. We have investigated the conformational stability of various segmental polymorphisms using molecular dynamics simulations and find that the segmental polymorphic models of Aβ retain a U-shaped architecture. Our results demonstrate the importance of inter-sheet side chain-side chain contacts, hydrophobic contacts among the strands (β1 and β2) and of salt bridges in stabilizing the aggregates. Residues in β-sheet regions have smaller fluctuation while those at the edge and loop region are more mobile. The inter-peptide salt bridges between Asp23 and Lys28 are strong compared to intra-chain salt bridge and there is an exchange of the inter-chain salt-bridge with intra-chain salt bridge. As our results suggest that Aβ exists under physiological conditions as an ensemble of distinct segmental polymorphs, it may be necessary to account in the development of therapeutics for Alzheimer's disease the differences in structural stability and aggregation behavior of the various Aβ polymorphic forms.     

Decreased Amyloid-β Pathologies by Intracerebral Loading of Glycosphingolipid-enriched Exosomes in Alzheimer Model Mice

Elevated levels of amyloid-β peptide (Aβ) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aβ can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aβ metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aβ and with the associated Aβ were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-β precursor protein transgenic mice resulted in marked reductions in Aβ levels, amyloid depositions, and Aβ-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aβ binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aβ by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aβ clearance in the central nervous system. Improving Aβ clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.     

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.     

Hyperphosphorylation of Tau Induced by Naturally Secreted Amyloid-β at Nanomolar Concentrations Is Modulated by Insulin-dependent Akt-GSK3β Signaling Pathway

Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.     

Transient formation of intermediate conformational states of amyloid-β peptide revealed by heteronuclear magnetic resonance spectroscopy

A detailed analysis of the NMR spectra of amyloid-β (Aβ) peptide revealed a decrease in signal intensity at higher temperature, due to a reversible conformational change of the molecule. Although peak intensity did not depend on peptide concentrations, the intensity in the region from D23 to A30 depended significantly on temperature. During the early stages of Aβ aggregation, each molecule might adopt transiently a turn conformation at around D23-A30, which converts mutually with a random coil. Stabilization of a turn by further conformational change and/or molecular association would lead to the formation of a "nucleus" for amyloid fibrils.     

Disintegration of amyloid fibrils of α-synuclein by dequalinium

α-Synuclein is the major amyloidogenic component observed in the Lewy bodies of Parkinson's disease. Amyloid fibrils of α-synuclein prepared in vitro were instantaneously disintegrated by dequalinium (DQ). Double-headed cationic amphipathic structure of DQ with two aminoquinaldinium rings at both ends turned out to be crucial to exert the disintegration activity. The defibrillation activity was shown to be selective toward the fibrils of α-synuclein and Aβ40 while the other β2-microglobulin amyloid fibrils were not susceptible so much. Besides the common cross β-sheet conformation of amyloid fibrils, therefore, additional specific molecular interactions with the target amyloidogenic proteins have been expected to be involved for DQ to exhibit its defibrillation activity. The disintegrating activity of DQ was also evaluated in vivo with the yeast system overexpressing α-synuclein-GFP. With the DQ treatment, the intracellular green inclusions turned into green smears, which resulted in the enhanced cell death. Based on the data, the previous observation that DQ led to the predominant protofibril formation of α-synuclein could be explained by the dual function of DQ showing both the facilitated self-oligomerization of α-synuclein and the instantaneous defibrillation of its amyloid fibrils. In addition, amyloidosis-related cytotoxicity has been demonstrated to be amplified by the fragmentation of mature amyloid fibrils by DQ.     

The Off-rate of Monomers Dissociating from Amyloid-β Protofibrils

The interconversion of monomers, oligomers, and amyloid fibrils of the amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease. The determination of the kinetics of the individual association and dissociation reactions is hampered by the fact that forward and reverse reactions to/from different aggregation states occur simultaneously. Here, we report the kinetics of dissociation of Aβ monomers from protofibrils, prefibrillar high molecular weight oligomers previously shown to possess pronounced neurotoxicity. An engineered binding protein sequestering specifically monomeric Aβ was employed to follow protofibril dissociation by tryptophan fluorescence, precluding confounding effects of reverse or competing reactions. Aβ protofibril dissociation into monomers follows exponential decay kinetics with a time constant of ∼2 h at 25 °C and an activation energy of 80 kJ/mol, values typical for high affinity biomolecular interactions. This study demonstrates the high kinetic stability of Aβ protofibrils toward dissociation into monomers and supports the delineation of the Aβ folding and assembly energy landscape.