Comparative properties of Aβ1–42, Aβ11–42, and [Pyr11]Aβ11–42 generated from O-acyl isopeptides

The use of water-soluble O-acyl isopeptides enabled us to investigate the biochemical properties of Aβ11–42 species, by preparing highly concentrated stock solutions after a pretreatment. Aβ11–42 and [Pyr¹¹]Aβ11–42 showed comparable aggregation capability and cytotoxicity, suggesting that the pyroglutamate modification at Glu¹¹ does not have a crucial role in these events. However, given that Aβ11–42 is converted to [Pyr¹¹]Aβ11–42 by a glutamyl cyclase in vivo, the potential aggregative and cytotoxic nature of [Pyr¹¹]Aβ11–42 that was observed in the present study provides valuable insights into the pathological functions of pyroglutamate-modified Aβ species in Alzheimer’s disease.     

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1^−/− growth plate cartilage but not in ChGn-2^−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1^−/−, and ChGn-2^−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.     

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.     

Protection of TGF-β1 against Neuroinflammation and Neurodegeneration in Aβ1–42-Induced Alzheimer’s Disease Model Rats

Neuroinflammation has been reported to be associated with Alzheimer’s disease (AD) pathogenesis. Neuroinflammation is generally considered as an outcome of glial activation; however, we recently demonstrated that T helper (Th)17 cells, a subpopulation of proinflammatory CD4+ T cells, are also involved in AD pathogenesis. Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, can be immunosuppressive, but its effects on lymphocyte-mediated neuroinflammation in AD pathogenesis have not been well addressed. In the current study we administered TGF-β1 via intracerebroventricle (ICV) and intranasal (IN) routes in AD model rats to investigate its antiinflammatory and neuroprotective effects. The AD rat model was prepared by bilateral hippocampal injection of amyloid-β (Aβ)_1–42. TGF-β1 was administered via ICV one hour prior to Aβ_1–42 injection or via both nares seven days after Aβ_1–42 injection. ICV administration of TGF-β1 before Aβ_1–42 injection remarkably ameliorated Aβ_1–42-induced neurodegeneration and prevented Aβ_1–42-induced increases in glia-derived proinflammatory mediators (TNF-α, IL-1β and iNOS), as well as T cell-derived proinflammatory cytokines (IFN-γ, IL-2, IL-17 and IL-22), in the hypothalamus, serum or cerebrospinal fluid (CSF) in a concentration-dependent manner. TGF-β1 pretreatment also prevented Aβ_1–42-induced decreases in the neurotrophic factors, IGF-1, GDNF and BDNF, and in the antiinflammatory cytokine, IL-10. Similarly, IN administration of TGF-β1 after Aβ_1–42 injection reduced neurodegeneration, elevation of proinflammatory mediators and cytokines, and reduction of neurotrophic and antiinflammatory factors, in the hypothalamus, serum or CSF. These findings suggest that TGF-β1 suppresses glial and T cell-mediated neuroinflammation and thereby alleviates AD-related neurodegeneration. The effectiveness of IN administered TGF-β1 in reducing Aβ_1–42 neurotoxicity suggests a possible therapeutic approach in patients with AD.     

Tau pathogenesis is promoted by Aβ1-42 but not Aβ1-40

Background

The relationship between the pathogenic amyloid β-peptide species Aβ1–42 and tau pathology has been well studied and suggests that Aβ1–42 can accelerate tau pathology in vitro and in vivo. The manners if any in which Aβ1–40 interacts with tau remains poorly understood. In order to answer this question, we used cell-based system, transgenic fly and transgenic mice as models to study the interaction between Aβ1–42 and Aβ1–40.

Results

In our established cellular model, live cell imaging (using confocal microscopy) combined with biochemical data showed that exposure to Aβ1–42 induced cleavage, phosphorylation and aggregation of wild-type/full length tau while exposure to Aβ1–40 didn’t. Functional studies with Aβ1–40 were carried out in tau-GFP transgenic flies and showed that Aβ1–42, as previously reported, disrupted cytoskeletal structure while Aβ1–40 had no effect at same dose. To further explore how Aβ1–40 affects tau pathology in vivo, P301S mice (tau transgenic mice) were injected intracerebrally with either Aβ1–42 or Aβ1–40. We found that treatment with Aβ1–42 induced tau phosphorylation, cleavage and aggregation of tau in P301S mice. By contrast, Aβ1–40 injection didn’t alter total tau, phospho-tau (recognized by PHF-1) or cleavage of tau, but interestingly, phosphorylation at Ser²⁶² was shown to be significantly decreased after direct inject of Aβ1–40 into the entorhinal cortex of P301S mice.

Conclusions

These results demonstrate that Aβ1–40 plays different role in tau pathogenesis compared to Aβ1–42. Aβ1–40 may have a protective role in tau pathogenesis by reducing phosphorylation at Ser²⁶², which has been shown to be neurotoxic.

     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

Structural basis for Aβ1–42 toxicity inhibition by Aβ C-terminal fragments: discrete molecular dynamics study

Amyloid β-protein (Aβ) is central to the pathology of Alzheimer's disease. Of the two predominant Aβ alloforms, Aβ(1-40) and Aβ(1-42), the latter forms more toxic oligomers. C-terminal fragments (CTFs) of Aβ were recently shown to inhibit Aβ(1-42) toxicity in vitro. Here, we studied Aβ(1-42) assembly in the presence of three effective CTF inhibitors and an ineffective fragment, Aβ(21-30). Using a discrete molecular dynamics approach that recently was shown to capture key differences between Aβ(1-40) and Aβ(1-42) oligomerization, we compared Aβ(1-42) oligomer formation in the absence and presence of CTFs or Aβ(21-30) and identified structural elements of Aβ(1-42) that correlated with Aβ(1-42) toxicity. CTFs co-assembled with Aβ(1-42) into large heterooligomers containing multiple Aβ(1-42) and inhibitor fragments. In contrast, Aβ(21-30) co-assembled with Aβ(1-42) into heterooligomers containing mostly a single Aβ(1-42) and multiple Aβ(21-30) fragments. The CTFs, but not Aβ(21-30), decreased the β-strand propensity of Aβ(1-42) in a concentration-dependent manner. CTFs and Aβ(21-30) had a high binding propensity to the hydrophobic regions of Aβ(1-42), but only CTFs were found to bind the Aβ(1-42) region A2-F4. Consequently, only CTFs but not Aβ(21-30) reduced the solvent accessibility of Aβ(1-42) in region D1-R5. The reduced solvent accessibility of Aβ(1-42) in the presence of CTFs was comparable to the solvent accessibility of Aβ(1-40) oligomers formed in the absence of Aβ fragments. These findings suggest that region D1-R5, which was more exposed to the solvent in Aβ(1-42) than in Aβ(1-40) oligomers, is involved in mediating Aβ(1-42) oligomer neurotoxicity.     

A routine method for cloning,expressing and purifying Aβ(1–42) for structural NMR studies

Nuclear magnetic resonance (NMR) is a key technology in the biophysicist’s toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-β peptide (Aβ) of Alzheimer’s disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aβ. Furthermore, the notorious propensity of Aβ to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched ¹⁵N and ¹³C¹⁵N Aβ(1–42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer’s disease, and perhaps misfolding diseases in general.     

Accumulation of amyloid-like Aβ1–42 in AEL (autophagy–endosomal–lysosomal) vesicles: potential implications for plaque biogenesis

Abnormal accumulation of Aβ (amyloid β) within AEL (autophagy–endosomal–lysosomal) vesicles is a prominent neuropathological feature of AD (Alzheimer''s disease), but the mechanism of accumulation within vesicles is not clear. We express secretory forms of human Aβ_1–40 or Aβ_1–42 in Drosophila neurons and observe preferential localization of Aβ_1–42 within AEL vesicles. In young animals, Aβ_1–42 appears to associate with plasma membrane, whereas Aβ_1–40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Aβ_1–40, in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Aβ_1–42 accumulation. In samples from older animals, Aβ_1–42 is broadly distributed within neurons, but only the Aβ_1–42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Aβ_1–42-containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. We propose that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Aβ_1–42-expressing Drosophila with potential relevance for AD.     

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Aβ and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Aβ_1–42, we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A–I (apoA-I). We show that purified human apoA-I and Aβ form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Aβ. Significantly, Aβ/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Aβ-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Aβ aggregation and Aβ-induced neuronal damage and that the Aβ-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Aβ toxicity.     

Ferrocene tripeptide Gly-Pro-Arg conjugates: Synthesis and inhibitory effects on Alzheimer’s Aβ1–42 fibrillogenesis and Aβ-induced cytotoxicity in vitro

Alzheimer’s disease (AD) is the most common cause of dementia, and currently there is no clinical treatment to cure it or to halt its progression. Aggregation and fibril formation of β-amyloid peptides (Aβ) are central events in the pathogenesis of AD. Many efforts have been spent on the development of effective inhibitors to prevent Aβ fibrillogenesis and cause disaggregation of preformed Aβ fibrils. In this study, the conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, Boc-Gly-Pro-Arg(NO₂)-Fca-OMe (4, GPR–Fca) and Fc-Gly-Pro-Arg-OMe (7, Fc–GPR) (Fc: ferrocene; Fca: ferrocene amino acid) were synthesized by HOBT/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Aβ_1–42 fibrillogenesis and to disaggregate preformed Aβ fibrils. The inhibitory properties of ferrocene GPR conjugates on Aβ_1–42 fibrillogenesis were evaluated by thioflavin T (ThT) fluorescence assay, and confirmed by atomic force microscopy (AFM) analysis. The interaction between the ferrocene GPR conjugates and Aβ_1–42 was monitored by electrochemical means. Our results showed that both GPR and GPR–Fca can significantly inhibit the fibril formation of Aβ_1–42, and cause disaggregation of the preformed fibrils. As expected, GPR–Fca shows stronger inhibitory effect on Aβ_1–42 fibrillogenesis than that of its parent peptide GPR. In contrast, Fc–GPR shows no inhibitory effect on fibrillogenesis of Aβ_1–42. Furthermore, GPR–Fca demonstrates significantly protection against Aβ-induced cytotoxicity and exhibits high resistance to proteolysis and good lipophilicity.     

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1–16 peptide

The binding of metal ions to Aβ peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu²⁺ on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H₂O₂ generation. The oxidation of Aβ1–16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aβ1–16–Cu²⁺ (1:2) complex.     

The Peculiar Role of the A2V Mutation in Amyloid-β (Aβ) 1–42 Molecular Assembly

We recently reported a novel Aβ precursor protein mutation (A673V), corresponding to position 2 of Aβ1–42 peptides (Aβ1–42_A2V), that caused an early onset AD-type dementia in a homozygous individual. The heterozygous relatives were not affected as an indication of autosomal recessive inheritance of this mutation. We investigated the folding kinetics of native unfolded Aβ1–42_A2V in comparison with the wild type sequence (Aβ1–42_WT) and the equimolar solution of both peptides (Aβ1–42_MIX) to characterize the oligomers that are produced in the early phases. We carried out the structural characterization of the three preparations using electron and atomic force microscopy, fluorescence emission, and x-ray diffraction and described the soluble oligomer formation kinetics by laser light scattering. The mutation promoted a peculiar pathway of oligomerization, forming a connected system similar to a polymer network with hydrophobic residues on the external surface. Aβ1–42_MIX generated assemblies very similar to those produced by Aβ1–42_WT, albeit with slower kinetics due to the difficulties of Aβ1–42_WT and Aβ1–42_A2V peptides in building up of stable intermolecular interaction.     

β-Amyloid (1–40) Peptide Interactions with Supported Phospholipid Membranes: A Single-Molecule Study

Recent evidence supports the hypothesis that the oligomers formed by the β-amyloid peptide early in its aggregation process are neurotoxic and may feature in Alzheimer’s disease. Although the mechanism underlying this neurotoxicity remains unclear, interactions of these oligomers with neuronal membranes are believed to be involved. Identifying the neurotoxic species is challenging because β-amyloid peptides form oligomers at very low physiological concentrations (nM), and these oligomers are highly heterogeneous and metastable. Here, we report the use of single-molecule imaging techniques to study the interactions between β-amyloid (1–40) peptides and supported synthetic model anionic lipid membranes. The evolution of the β-amyloid species on the membranes was monitored for up to several days, and the results indicate an initial tight, uniform, binding of β-amyloid (1–40) peptides to the lipid membranes, followed by oligomer formation in the membrane. At these low concentrations, the behavior at early times during the formation of small oligomers is interpreted qualitatively in terms of the two-state model proposed by H. W. Huang for the interaction between amphipathic peptides and membranes. However, the rate of oligomer formation in the membrane and their size are highly dependent on the concentrations of β-amyloid (1–40) peptides in aqueous solution, suggesting two different pathways of oligomer formation, which lead to drastically different species in the membrane and a departure from the two-state model as the concentration increases.     

Gangliosides smelt nanostructured amyloid Aβ(1–40) fibrils in a membrane lipid environment

Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aβ(1–40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aβ(1–40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aβ(1–40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes.Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy.We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).     

以Aβ4-11为免疫原制备Aβ1-42多克隆抗体

β淀粉样蛋白1-42(βamyloid 1-42,Aβ1-42)是阿尔茨海默病(Alzheimer’s disease,AD)的主要致病蛋白,其抗体一直处于研发当中。现以Aβ4-11耦联KLH免疫2月龄雌兔,ELISA检测雌兔免疫血清中所产生的抗Aβ4-11 IgG滴度,Protein G-琼脂糖亲和层析纯化IgG,Bradford方法进行IgG蛋白含量测定,SDS-PAGE进行IgG纯度测定,最后运用所制备的多克隆抗体采用免疫组化的方法分别对正常小鼠和9月龄APP转基因小鼠进行老年斑的检测,以鉴定此抗体对Aβ1-42的特异性及亲和力。结果显示Aβ4-11具有良好的体液免疫原性,而且以其为免疫原制备的抗体对Aβ1-42表现出良好的特异性及亲和力。结果表明,以Aβ4-11亚单位片段为免疫原制备的Aβ1-42多克隆抗体,可以作为AD的一种有效研究工具,同时也为AD的诊断和治疗提供了一种新的可能。     

A deuterohemin peptide protects a transgenic Caenorhabditis elegans model of Alzheimer’s disease by inhibiting Aβ1–42 aggregation

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disease and is the most common cause of dementia in the elderly. The main hallmark of AD is the deposition of insoluble amyloid (Aβ) outside the neuron, leading to amyloid plaques and neurofibrillary tangles in the brain. Deuterohemin-Ala-His-Thr-Val-Glu-Lys (DhHP-6), a novel porphyrin-peptide, has both microperoxidase activity and cell permeability. In the present study, DhHP-6 efficiently inhibited the aggregation of Aβ and reduced the β-sheet percentage of Aβ from 89.1% to 78.3%. DhHP-6 has a stronger affinity (K_D = 100 ± 12 μM) for binding with Aβ at Phe⁴, Arg⁵, Val¹⁸, Glu¹¹ and Glu²². In addition, DhHP-6 (100 μM) significantly prolonged lifespan, alleviated paralysis and reduced Aβ plaque formation in the Aβ_1–42 transgenic Caenorhabditis elegans CL4176 model of AD. Our results demonstrate that DhHP-6 is a potential drug candidate that efficiently protects a transgenic C. elegans model of Alzheimer’s disease by inhibiting Aβ aggregation.     

NMR structure of the water soluble Aβ17–34 peptide

Alzheimer''s disease is the most common neurodegenerative disorder in the world. Its most significant symptoms are memory loss and decrease in cognition. Alzheimer''s disease is characterized by aggregation of two proteins in the brain namely Aβ (amyloid β) and tau. Recent evidence suggests that the interaction of soluble Aβ with nAChR (nicotinic acetylcholine receptors) contributes to disease progression. In this study, we determine the NMR structure of an Aβ_17–34 peptide solubilized by the addition of two glutamic acids at each terminus. Our results indicate that the Aβ peptide adopts an α-helical structure for residues 19–26 and 28–33. The α-helical structure is broken around residues S26, N27 and K28, which form a kink in the helical conformation. This α-helix was not described earlier in an aqueous solution without organic solvents, and at physiological conditions (pH 7). These data are in agreement with Aβ adopting an α-helical conformation in the membrane before polymerizing into amyloid β-sheets and provide insight into the intermediate state of Aβ in Alzheimer''s disease.