Aβ Association Inhibition by Transferrin

The iron-transport glycoprotein transferrin has recently been shown to serve as a potent inhibitor of Aβ self-association. Although this novel, to our knowledge, inhibitory function of transferrin is of potential therapeutic interest for the treatment of Alzheimer’s disease, the underlying mechanism is still not fully understood. Although it has been shown that the Fe(III) sequestration by transferrin reduces oxidative damage and Aβ aggregation, it is not clear whether transferrin is also able to inhibit Aβ self-association through direct binding of Aβ. Here, using saturation transfer and off-resonance relaxation NMR spectroscopy, we show that transferrin inhibits Aβ aggregation also by preferentially binding Aβ oligomers and outcompeting Aβ monomers that would otherwise cause the growth of the Aβ oligomers into larger assemblies. This inhibitory mechanism is different from the iron-sequestration model, but it is qualitatively similar to a mechanism previously proposed for the inhibition of Aβ self-association by another plasma and cerebrospinal fluid protein, i.e., human serum albumin. These results suggest that Aβ monomer competition through direct Aβ oligomer binding might be a general strategy adopted by proteins in plasma and cerebrospinal fluid to prevent Aβ aggregation.Abbreviations used: AD, Alzheimer’s disease, CNS, central nervous system, CSF, cerebrospinal fluid, HSA, human serum albumin, ICP, inductively coupled plasma, ORR, off-resonance relaxation, SL, spin-lock, STD, saturation transfer difference, STR, saturation transfer reference, Tf, transferrin, WG, watergate water-suppression NMR technique     

Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles

Alzheimer''s disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.     

Vitamin D-binding protein interacts with Aβ and suppresses Aβ-mediated pathology

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer''s disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aβ), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aβ aggregation in vitro. DBP also prevented Aβ-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aβ-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aβ into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aβ by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.     

Alzheimer’s Protective A2T Mutation Changes the Conformational Landscape of the Aβ1–42 Monomer Differently Than Does the A2V Mutation

The aggregation of amyloid-β (Aβ) peptides plays a crucial role in the etiology of Alzheimer’s disease (AD). Recently, it has been reported that an A2T mutation in Aβ can protect against AD. Interestingly, a nonpolar A2V mutation also has been found to offer protection against AD in the heterozygous state, although it causes early-onset AD in homozygous carriers. Since the conformational landscape of the Aβ monomer is known to directly contribute to the early-stage aggregation mechanism, it is important to characterize the effects of the A2T and A2V mutations on Aβ_1–42 monomer structure. Here, we have performed extensive atomistic replica-exchange molecular dynamics simulations of the solvated wild-type (WT), A2V, and A2T Aβ_1–42 monomers. Our simulations reveal that although all three variants remain as collapsed coils in solution, there exist significant structural differences among them at shorter timescales. A2V exhibits an enhanced double-hairpin population in comparison to the WT, similar to those reported in toxic WT Aβ_1–42 oligomers. Such double-hairpin formation is caused by hydrophobic clustering between the N-terminus and the central and C-terminal hydrophobic patches. In contrast, the A2T mutation causes the N-terminus to engage in unusual electrostatic interactions with distant residues, such as K16 and E22, resulting in a unique population comprising only the C-terminal hairpin. These findings imply that a single A2X (where X = V or T) mutation in the primarily disordered N-terminus of the Aβ_1–42 monomer can dramatically alter the β-hairpin population and switch the equilibrium toward alternative structures. The atomistically detailed, comparative view of the structural landscapes of A2V and A2T variant monomers obtained in this study can enhance our understanding of the mechanistic differences in their early-stage aggregation.     

Glycation exacerbates the neuronal toxicity of β-amyloid

Accumulation evidence shows that β-amyloid (Aβ) is a neurotoxic and accumulation of Aβ is responsible for the pathology of Alzheimer''s disease (AD). However, it is currently not fully understood what makes Aβ toxic and accumulated. Previous studies demonstrate that Aβ is a suitable substrate for glycation, producing one form of the advanced glycation endproducts (AGEs). We speculated that Aβ-AGE formation may exacerbate the neurotoxicity. To explore whether the Aβ-AGE is more toxic than the authentic Aβ and to understand the molecular mechanisms, we synthesized glycated Aβ by incubating Aβ with methylglyoxal (MG) in vitro and identified the formation of glycated Aβ by fluorescence spectrophotometer. Then, we treated the primary hippocampal neurons cultured 8 days in vitro with Aβ-AGE or Aβ for 24 h. We observed that glycation exacerbated neurotoxicity of Aβ with upregulation of receptor for AGE (RAGE) and activation of glycogen synthase kinase-3 (GSK-3), whereas simultaneous application of RAGE antibody or GSK-3 inhibitor reversed the neuronal damages aggravated by glycated Aβ. Thereafter, we found that Aβ is also glycated with an age-dependent elevation of AGEs in Tg2576 mice, whereas inhibition of Aβ-AGE formation by subcutaneously infusion of aminoguanidine for 3 months significantly rescued the early cognitive deficit in mice. Our data reveal for the first time that the glycated Aβ is more toxic. We propose that the glycated Aβ with the altered secondary structure may be a more suitable ligand than Aβ for RAGE and subsequent activation of GSK-3 that can lead to cascade pathologies of AD, therefore glycated Aβ may be a new therapeutic target for AD.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Triptolide treatment reduces Alzheimer’s disease (AD)-like pathology through inhibition of BACE1 in a transgenic mouse model of AD

The complex pathogenesis of Alzheimer’s disease (AD) involves multiple contributing factors, including amyloid β (Aβ) peptide accumulation, inflammation and oxidative stress. Effective therapeutic strategies for AD are still urgently needed. Triptolide is the major active compound extracted from Tripterygium wilfordii Hook.f., a traditional Chinese medicinal herb that is commonly used to treat inflammatory diseases. The 5-month-old 5XFAD mice, which carry five familial AD mutations in the β-amyloid precursor protein (APP) and presenilin-1 (PS1) genes, were treated with triptolide for 8 weeks. We observed enhanced spatial learning performances, and attenuated Aβ production and deposition in the brain. Triptolide also inhibited the processing of amyloidogenic APP, as well as the expression of βAPP-cleaving enzyme-1 (BACE1) both in vivo and in vitro. In addition, triptolide exerted anti-inflammatory and anti-oxidative effects on the transgenic mouse brain. Triptolide therefore confers protection against the effects of AD in our mouse model and is emerging as a promising therapeutic candidate drug for AD.KEY WORDS: Alzheimer’s disease, Amyloid β, 5XFAD mice, BACE1, Inflammation, Triptolide     

Mapping the Interactions between the Alzheimer’s Aβ-Peptide and Human Serum Albumin beyond Domain Resolution

Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer’s disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494–515) region and aligns with the central hydrophobic core of Aβ. The HSA (495–515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association.Abbreviations: AD, Alzheimer’s Disease, BBB, Blood Brain Barrier, CNS, Central Nervous System, CSF, Cerebrospinal Fluid, FA, Fatty Acid, HSA, Human Serum Albumin, ICP, Inductively Coupled Plasma, MA, Myristic Acid, SL, Spin-Lock, RC, Random Coil, STD, Saturation Transfer Difference, STR, Saturation Transfer Reference, WG, Watergate water-suppression NMR technique     

MicroRNA-149 is downregulated in Alzheimer’s disease and inhibits β-amyloid accumulation and ameliorates neuronal viability through targeting BACE1

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a critical role in Alzheimer’s disease (AD) pathogenesis. This study aimed to investigate the relationship between microRNA-149 (miR-149) and BACE1, and evaluate the clinical significance and biological function of miR-149 in AD progression. Bioinformatics analysis and a luciferase reporter assay were used to confirm the interaction between miR-149 and BACE1. Expression of miR-149 and BACE1 was estimated using quantitative real-time PCR. The clinical significance of miR-149 in AD diagnosis and severity determination was evaluated using ROC analysis. The effect of miR-149 on Aβ accumulation and neuronal viability was analyzed in Aβ-treated SH-SY5Y cells. miR-149 was found directly binding the 3’-UTR of BACE1 and was negatively correlated with BACE1 in AD patients and cell model. Serum miR-149 expression was downregulated in AD patients and served as a potential diagnostic biomarker. The overexpression of miR-149 in Aβ-treated SH-SY5Y cells resulted in inhibited Aβ accumulation and enhanced neuronal viability. This study demonstrated that serum miR-149 is decreased in AD patients and serves as a candidate diagnostic biomarker, and that the overexpression of miR-149 may suppress Aβ accumulation and promote neuronal viability by targeting BACE1 in AD model cells. Keywords: MicroRNA-149, Alzheimer’s disease, BACE1, diagnosis, APP     

In Silico Analysis of the Apolipoprotein E and the Amyloid β Peptide Interaction: Misfolding Induced by Frustration of the Salt Bridge Network

The relationship between Apolipoprotein E (ApoE) and the aggregation processes of the amyloid β (Aβ) peptide has been shown to be crucial for Alzheimer''s disease (AD). The presence of the ApoE4 isoform is considered to be a contributing risk factor for AD. However, the detailed molecular properties of ApoE4 interacting with the Aβ peptide are unknown, although various mechanisms have been proposed to explain the physiological and pathological role of this relationship. Here, computer simulations have been used to investigate the process of Aβ interaction with the N-terminal domain of the human ApoE isoforms (ApoE2, ApoE3 and ApoE4). Molecular docking combined with molecular dynamics simulations have been undertaken to determine the Aβ peptide binding sites and the relative stability of binding to each of the ApoE isoforms. Our results show that from the several ApoE isoforms investigated, only ApoE4 presents a misfolded intermediate when bound to Aβ. Moreover, the initial α-helix used as the Aβ peptide model structure also becomes unstructured due to the interaction with ApoE4. These structural changes appear to be related to a rearrangement of the salt bridge network in ApoE4, for which we propose a model. It seems plausible that ApoE4 in its partially unfolded state is incapable of performing the clearance of Aβ, thereby promoting amyloid forming processes. Hence, the proposed model can be used to identify potential drug binding sites in the ApoE4-Aβ complex, where the interaction between the two molecules can be inhibited.     

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     

Single-Molecule Imaging Reveals Aβ42:Aβ40 Ratio-Dependent Oligomer Growth on Neuronal Processes

Soluble oligomers of the amyloid-β peptide have been implicated as proximal neurotoxins in Alzheimer’s disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aβ oligomers, and the wide range of observed interactions between Aβ and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aβ binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-β(1–40) as well as amyloid-β(1–42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aβ40 oligomers do not grow over the course of 48 h, Aβ42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aβ oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aβ42:Aβ40 ratios. These results are intriguing in light of the increased Aβ42:Aβ40 ratios shown to correlate with familial Alzheimer’s disease mutations.     

Effects of Familial Mutations on the Monomer Structure of Aβ42

Amyloid beta (Aβ) peptide plays an important role in Alzheimer’s disease. A number of mutations in the Aβ sequence lead to familial Alzheimer’s disease, congophilic amyloid angiopathy, or hereditary cerebral hemorrhage with amyloid. Using molecular dynamics simulations of ∼200 μs for each system, we characterize and contrast the consequences of four pathogenic mutations (Italian, Dutch, Arctic, and Iowa) for the structural ensemble of the Aβ monomer. The four familial mutations are found to have distinct consequences for the monomer structure.Amyloid beta (Aβ) peptides have long been thought to play a central role in Alzheimer’s disease (AD). Usually 40 or 42 residues in length, Aβ peptides are proteolytic products of the Aβ precursor protein and they aggregate to form the fibrillar plaques in AD patients’ brains. Besides fibrillar plaques, Aβ oligomers are also neurotoxic. The significance and nature of Aβ oligomerization has recently become a focus of intensive research studies and debates (1,2). Notably, numerous pathogenic mutations have been identified in the Aβ precursor protein sequence and in the enzymes involved in Aβ processing (3). These mutations generally lead to early onset of AD or cerebral amyloid angiopathy. Understanding how the pathogenic mutations alter Aβ oligomerization/aggregation is essential to our understanding of the disease mechanism.Four of these pathogenic mutations (Italian E22K, Dutch E22Q, Arctic E22G, and Iowa D23N) cluster in the region of E22 and D23 in the Aβ sequence (distal from proteolytic cleavage sites) and they have higher neurotoxicity compared to wild-type (WT) Aβ (4). These mutations are thought to modify the physicochemistry of the peptide. For example, kinetic studies (4) show that the E22K and E22Q mutations lead to faster peptide aggregation, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (although the E22G mutation shows increased protofibril formation (5)). Recent solid-state NMR studies also suggest that rather than the in-register β-sheet conformation adopted by WT Aβ, the Iowa D23N mutant forms amyloid fibrils with antiparallel β-sheet structure (6).To understand how the mutations modify the peptide oligomerization/aggregation it is critical to characterize the starting point of the process, the monomers. Unfortunately, investigating the early phase of the oligomerization process experimentally is a challenging task due to the high aggregation propensity of Aβ and its intrinsic disorder. Therefore, a number of computational approaches have been adopted to investigate the consequences of mutations for the monomer structure (7–16). However, due to the high computational demands of explicit-solvent molecular dynamics (MD) simulations to simulate full-length Aβ peptides, most of these computational studies are either on Aβ fragments (to decrease the system size) using explicit-solvent simulations (8–12) or on full-length Aβ using implicit-solvent simulations (which are less computationally demanding and enable longer simulation times, but lack explicit water molecules in the simulations to fully describe water-peptide interactions) (13–15). In a very recent report, explicit-solvent simulations were used to study the effects of the E22Q mutation on full-length Aβ; however, rather limited data (<10 μs) were collected (16). Thus, characterizing full-length Aβ monomers remains quite a daunting task even with simulations.To characterize the effects of mutations on full-length Aβ monomer using explicit-solvent MD simulations, we employed distributed computing (17) to simulate the WT Aβ₄₂, Aβ₄₂-E22K, Aβ₄₂-E22Q, Aβ₄₂-E22G, and Aβ₄₂-D23N monomers. MD simulations of >200 μs were performed for each system and AMBER ff99sb (18) and the tip3p water model (19) were used for force field parameters. Peptide configurations in the MD trajectories were clustered with the root mean-square deviation metric to identify representative conformations (i.e., states) and transitions between these states were counted. Markov state model analysis was then performed where the master equations were solved and the equilibrium population of each state deduced (20). Details of the MD simulation procedures and Markov state model analysis can be found in the Supporting Material.Each of the five Aβ monomer systems exhibits great structural diversity and can only be characterized in an ensemble fashion (rather than described by a handful of representative configurations). This is in accord with the notion that full-length Aβ peptides are intrinsically disordered (21,22). Using the Dictionary of Secondary Structure of Proteins program (23) to assign secondary structure, it is clear that the five Aβ monomer systems are found overall not well structured, although small β-hairpins and α-helices are observed. In Fig. 1 we plot the residue-dependent extended β propensity and α-helix propensity, in the top and bottom panels, respectively, for each Aβ monomer system. Although we are reasonably confident of the convergence behavior of the α-helix propensity, we note that the convergence of the extended β-propensity might be more challenging and demand a much longer sampling time than the current aggregate simulation time of ∼200 μs (24).Open in a separate windowFigure 1Ensemble-averaged %population of β-strand (top) and α-helix (bottom) propensity for all five monomer systems. The sequence of the WT Aβ₄₂ is given on the x axis.We observe in Fig. 1 that all five Aβ monomer systems share a rather similar residue-dependent tendency to form an extended β-structure, although minor differences are present. On the other hand, these pathogenic mutations alter the α-helix propensity quite significantly. The E22K and E22Q mutations increase the α-helix propensity in the region of residues 20–23. All four mutations (E22K, E22Q, E22G, and D23N) decrease the α-helix propensity in the region of residues 33–36.Notably, we find that in all five systems only short stretches of α-helices are formed. That is, when a residue is involved in α-helix formation, it participates in forming mostly short helical segments (consisting of only four helical residues). To provide more insight into the changes of α-helix propensity due to the mutations, in Fig. S1 we plot the tendency of forming short α-helices along the sequence for all five systems. Each data point in Fig. S1 represents the propensity to form an α-helix of four residues in length, ending at the specific residue. For example, in the structural ensemble adopted by the WT peptide, ∼5.5% of the conformations have a short α-helix of size four, involving residues 15–18. We see from Fig. S1 that the E22K and E22Q mutations induce the formation of two short helices in residues 19–22 and 20–23. The higher α-helix propensity in this region for the E22K mutant compared to the WT was previously attributed to the elimination of the electrostatic repulsion between E22 and D23 in the WT by the mutation and the longer aliphatic chain of K22 in the mutant compared to E22 in the WT (9,22). This is consistent with the observation that the E22Q mutation also induces helix formation in this region (by eliminating the electrostatic repulsion between E22 and D23 in the WT) but to a lesser extent, possibly due to the shorter aliphatic chain of Q22 compared to K22.In the E22G mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, glycine is known to be a helix breaker (25), leading to diminished α-helix propensity in the region around residue G22 seen in Fig. S1.In the D23N mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, it does not induce (or rather even slightly decreases) helix formation around residue 23. This may be due to the short aliphatic chain of N23 but it is possible that the mutation induces some nonlocal effects on the peptide structure, disfavoring helix formation in this region.It is worth noting that all four mutations (E22K, E22Q, E22G, and D23N) virtually eliminate the α-helix propensity in the region of residues 33–36. This region is rather far away from the mutation sites in sequence but its α-helix propensity is nonetheless affected. The origin of such a nonlocal effect is less straightforward to explain and further analysis will aid untangling this behavior. Nonetheless, the diminished α-helix propensity in the region of residues 33–36 appears to be a consistent feature across all four mutants.The four mutations studied here (E22K, E22Q, E22G, and D23N) have been thought to modify the physicochemistry of the peptide and alter the oligomerization/aggregation process, leading to higher neurotoxicity. In predicting intrinsic aggregation propensities using peptide sequences, all four mutants are suggested to be more aggregation prone (26). On the other hand, kinetic studies show that only the E22K and E22Q mutants aggregate more quickly, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (4). Our simulation results suggest these pathogenic mutations have complicated effects on the monomer structure—all four mutations decrease helix propensity in residues 33–36, whereas only the E22K and E22Q mutations increase helix propensity in residues 20–23. It is interesting to note that α-helix propensity is generally thought to anticorrelate with aggregation propensity; however, recent studies have suggested an important role of α-helical intermediates in amyloid oligomerization (27–29). Our studies suggest that it would be of great value to investigate how the distinct patterns of α-helix propensity in these five systems may propagate to give rise to different oligomerization kinetics or even mechanisms. The pathogenic mutations studied here have complex effects on the oligomerization of the peptide. The characterization of the monomer structural ensembles reported here should aid understanding of such an important and complicated process.     

Effects of Hydroxylated Carbon Nanotubes on the Aggregation of Aβ16–22 Peptides: A Combined Simulation and Experimental Study

The pathogenesis of Alzheimer’s disease (AD) is associated with the aggregation of amyloid-β (Aβ) peptides into toxic aggregates with β-sheet character. In a previous computational study, we showed that pristine single-walled carbon nanotubes (SWCNTs) can inhibit the formation of β-sheet-rich oligomers in the central hydrophobic core fragment of Aβ (Aβ_16–22). However, the poor solubility of SWCNTs in water hinders their use in biomedical applications and nanomedicine. Here, we investigate the influence of hydroxylated SWCNT, a water-soluble SWCNT derivative, on the aggregation of Aβ_16–22 peptides using all-atom explicit-water replica exchange molecular dynamics simulations. Our results show that hydroxylated SWCNTs can significantly inhibit β-sheet formation and shift the conformations of Aβ_16–22 oligomers from ordered β-sheet-rich structures toward disordered coil aggregates. Detailed analyses of the SWCNT-Aβ interaction reveal that the inhibition of β-sheet formation by hydroxylated SWCNTs mainly results from strong electrostatic interactions between the hydroxyl groups of SWCNTs and the positively charged residue K16 of Aβ_16–22 and hydrophobic and aromatic stacking interactions between SWCNTs and F19 and F20. In addition, our atomic force microscopy and thioflavin T fluorescence experiments confirm the inhibitory effect of both pristine and hydroxylated SWCNTs on Aβ_16–22 fibrillization, in support of our previous and present replica exchange molecular dynamics simulation results. These results demonstrate that hydroxylated SWCNTs efficiently inhibit the aggregation of Aβ_16–22; in addition, they offer molecular insight into the inhibition mechanism, thus providing new clues for the design of therapeutic drugs against amyloidosis.     

Stability of Transmembrane Amyloid β-Peptide and Membrane Integrity Tested by Molecular Modeling of Site-Specific Aβ 42 Mutations

Interactions of the amyloid β-protein (Aβ) with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer’s disease. Natural mutations in Aβ ₄₂, such as the Arctic mutation (E22G) have been shown to increase Aβ ₄₂ aggregation and neurotoxicity, leading to the early-onset of Alzheimer’s disease. A correlation between the propensity of Aβ ₄₂ to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ ₄₂ peptide it was further revealed that the aggregation of different Aβ ₄₂ mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ ₄₂ mutation and peptide–membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ ₄₂ variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants) as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ ₄₂ stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ ₄₂ peptide and a decreased stability for D23G compared to wild-type Aβ ₄₂, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.     

Aβ1–42 reduces P-glycoprotein in the blood–brain barrier through RAGE–NF-κB signaling

The reduced clearance of amyloid-β peptide (Aβ) from the brain partly accounts for the neurotoxic accumulation of Aβ in Alzheimer''s disease (AD). Recently, it has been suggested that P-glycoprotein (P-gp), which is an efflux transporter expressed on the luminal membrane of the brain capillary endothelium, is capable of transporting Aβ out of the brain. Although evidence has shown that restoring P-gp reduces brain Aβ in a mouse model of AD, the molecular mechanisms underlying the decrease in P-gp expression in AD is largely unknown. We found that Aβ_1–42 reduced P-gp expression in the murine brain endothelial cell line bEnd.3, which was consistent with our in vivo data that P-gp expression was significantly reduced, especially near amyloid plaques in the brains of five familial AD mutations (5XFAD) mice that are used as an animal model for AD. A neutralizing antibody against the receptor for advanced glycation end products (RAGE) and an inhibitor of nuclear factor-kappa B (NF-κB) signaling prevented the decrease in Aβ_1–42-induced P-gp expression, suggesting that Aβ reduced P-gp expression through NF-κB signaling by interacting with RAGE. In addition, we observed that the P-gp reduction by Aβ was rescued in bEnd.3 cells receiving inductive signals or factors from astrocytes making contacts with endothelial cells (ECs). These results support that alterations of astrocyte–EC contacts were closely associated with P-gp expression. This suggestion was further supported by the observation of a loss of astrocyte polarity in the brains of 5XFAD mice. Taken together, we found that P-gp downregulation by Aβ was mediated through RAGE–NF-κB signaling pathway in ECs and that the contact between astrocytes and ECs was an important factor in the regulation of P-gp expression.Alzheimer''s disease (AD) is a neurodegenerative disorder that is characterized by a progressive loss of cognitive function leading to dementia. The major pathological hallmark of AD is the deposition of neurotoxic amyloid-β peptide (Aβ) within the brain.¹ The amyloid hypothesis proposes that the accumulation of Aβ is caused by an imbalance between Aβ production and clearance.² Although genetic alterations increase the production of Aβ in rare familial AD, reduced Aβ clearance from the brain likely accounts for sporadic AD, which is much more common.³ The mechanisms that are involved in clearing Aβ from the brain include enzymatic degradation, perivascular drainage, and the most significant, active transport across the blood–brain barrier (BBB).⁴The BBB regulates molecular exchanges at the interface between the blood and the brain.⁵ It plays a critical role in maintaining the brain microenvironment.⁶ The BBB, which is formed by cerebral endothelial cells (ECs) and which, interacts with astrocytes, neurons, pericytes, and the extracellular matrix, is organized into a neurovascular unit.^{7, 8} Although the relationship between BBB breakdown and AD pathology is unclear,⁹ it has been proposed that the BBB loses its Aβ clearing capability, thus increasing amyloid deposition in the outer capillary membrane and resulting in the distortion of the neurovascular unit with neuronal loss.¹⁰Recently, it has been suggested that P-glycoprotein (P-gp), which is an ATP-driven efflux transporter that is highly expressed in the luminal membrane of the brain capillary endothelium, is also involved in the clearance of Aβ from the brain.¹¹ P-gp, which is able to transport various kinds of substrates, has been shown to play an important role in clearing toxic substances in the brain and protecting it from harmful molecules in the circulation.¹² Along with other BBB properties, P-gp expression is induced when ECs are in contact with astrocytes in vitro and in vivo.^{13, 14} ECs respond to inductive signals or factors from astrocytes that encircle the capillary endothelium.¹³Several lines of evidence have shown that P-gp plays an important role in Aβ clearance. It has been shown in vitro that P-gp mediates the transport of Aβ and that blocking P-gp function reduces the clearance of Aβ.^{15, 16} In addition, cerebral Aβ deposition in elderly non-demented individuals has been demonstrated to be inversely correlated with brain capillary P-gp expression.¹⁷ Furthermore, in P-gp knockout mice, Aβ deposition is increased by the reduced efflux of Aβ,¹⁸ while it has been shown that restoring P-gp at the BBB reduces brain Aβ in a mouse model of AD.¹⁹ However, the molecular mechanisms underlying the decrease in P-gp expression that is observed in AD have not been identified. We found that Aβ decreased P-gp expression by increasing nuclear factor-kappa B (NF-κB) through an interaction with the receptor for advanced glycation end products (RAGE). Moreover, we observed that the P-gp reduction by Aβ was rescued by inductive signals or factors from astrocytes that made contact with ECs in bEnd.3 cells. These results suggested that alterations in astrocyte–EC contact in AD likely decrease P-gp expression by Aβ. Together, we identified a mechanism by which the Aβ–RAGE interaction mediated the downregulation of P-gp in the BBB by increasing NF-κB signaling in AD and that astrocyte–EC contact played a critical role in maintaining P-gp expression.     

The Resveratrol Trimer Miyabenol C Inhibits β-Secretase Activity and β-Amyloid Generation

Accumulation and deposition of amyloid-β peptide (Aβ) in the brain is a primary cause of the pathogenesis of Alzheimer’s disease (AD). Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages first by β-secretase and then by γ-secretase. Inhibiting β-secretase activity is believed to be one of the most promising strategies for AD treatment. In the present study, we found that a resveratrol trimer, miyabenol C, isolated from stems and leaves of the small-leaf grape (Vitisthunbergii var. taiwaniana), can markedly reduce Aβ and sAPPβ levels in both cell cultures and the brain of AD model mice. Mechanistic studies revealed that miyabenol C affects neither protein levels of APP, the two major α-secretases ADAM10 and TACE, and the γ-secretase component Presenilin 1, nor γ-secretase-mediated Notch processing and TACE activity. In contrast, although miyabenol C has no effect on altering protein levels of the β-secretase BACE1, it can inhibit both in vitro and in vivo β-secretase activity. Together, our results indicate that miyabenol C is a prominent β-secretase inhibitor and lead compound for AD drug development.     

Phloroglucinol Attenuates the Cognitive Deficits of the 5XFAD Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia among the elderly. Neuritic plaques whose primary component is amyloid beta peptide (Aβ) and neurofibrillary tangles which are composed of hyperphosphorylated tau, are known to be the neuropathological hallmarks of AD. In addition, impaired synaptic plasticity in neuronal networks is thought to be important mechanism underlying for the cognitive deficits observed in AD. Although various causative factors, including excitotoxicity, mitochondrial dysregulation and oxidative damage caused by Aβ, are involved in early onset of AD, fundamental therapeutics that can modify the progression of this disease are not currently available. In the present study, we investigated whether phloroglucinol (1, 3, 5—trihydroxybenzene), a component of phlorotannins, which are plentiful in Ecklonia cava, a marine brown alga species, displays therapeutic activities in AD. We found that phloroglucinol attenuates the increase in reactive oxygen species (ROS) accumulation induced by oligomeric Aβ_1–42 (Aβ_1–42) treatment in HT-22, hippocampal cell line. In addition, phloroglucinol was shown to ameliorate the reduction in dendritic spine density induced by Aβ_1–42 treatment in rat primary hippocampal neuron cultures_. We also found that the administration of phloroglucinol to the hippocampal region attenuated the impairments in cognitive dysfunction observed in 22-week-old 5XFAD (Tg6799) mice, which are used as an AD animal model. These results indicate that phloroglucinol displays therapeutic potential for AD by reducing the cellular ROS levels.