Self-Assembly of Aβ40, Aβ42 and Aβ43 Peptides in Aqueous Mixtures of Fluorinated Alcohols

Fluorinated alcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have the ability to promote α-helix and β-hairpin structure in proteins and peptides. HFIP has been used extensively to dissolve various amyloidogenic proteins and peptides including Aβ, in order to ensure their monomeric status. In this paper, we have investigated the self-assembly of Aβ40, Aβ42, and Aβ43 in aqueous mixtures of fluorinated alcohols from freshly dissolved stock solutions in HFIP. We have observed that formation of fibrillar and non-fibrillar structures are dependent on the solvent composition. Peptides form fibrils with ease when reconstituted in deionized water from freshly dissolved HFIP stocks. In aqueous mixtures of fluorinated alcohols, either predominant fibrillar structures or clustered aggregates were observed. Aqueous mixtures of 20% HFIP are more favourable for Aβ fibril formation as compared to 20% TFE. When Aβ40, Aβ42, and Aβ43 stocks in HFIP are diluted in 50% aqueous mixtures in phosphate buffer or deionized water followed by slow evaporation of HFIP, Aβ peptides form fibrils in phosphate buffer and deionized water. The clustered structures could be off-pathway aggregates. Aβ40, Aβ42, and Aβ43 showed significant α-helical content in freshly dissolved HFIP stocks. The α-helical conformational intermediate in Aβ40, Aβ42, and Aβ43 could favour the formation of both fibrillar and non-fibrillar aggregates depending on solvent conditions and rate of α-helical to β-sheet transition.     

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36–Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31–Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31–35 and residues 38–42. In contrast, Aβ(31–40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val–Val36Pro–Gly38Val (“VPV”) facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution d-Pro36–l-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36–Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

Ellagic acid promotes Aβ42 fibrillization and inhibits Aβ42-induced neurotoxicity

Smaller, soluble oligomers of β-amyloid (Aβ) play a critical role in the pathogenesis of Alzheimer’s disease (AD). Selective inhibition of Aβ oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against Aβ neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on Aβ42 aggregation and neurotoxicity in vitro. EA promoted Aβ fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited Aβ aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in Aβ42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic Aβ aggregates to render them harmless, our MTT results showed that EA could significantly reduce Aβ42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.     

Porcineβ-lactoglobulin A and C

Summary The occurrence of the dominant ‘whey’ protein in samples of milk from 1180 sows is examined. It exhibits genetic polymorphism with some unusual features. Although immunologically different from bovine β-lactoglobulin, it is shown by chemical studies of the isolated protein to be a β-lactoglobulin. Two homozygous genetic variants, designated porcine β-lactoglobulin A and C, are isolated and their amino acid compositions and peptide maps compared. It is shown that the C variant has +1 His, −1 Gln, and +1 Asp, −1 Glu, with respect to the A variant. These variants, containingca. 162 residues per molecule, are considered in relationship to porcine β-lactoglobulins isolated by other workers. The sequence of the first 50 residues is determined and compared with sequence of the bovine protein. The sequences ofca. 70% of the remaining residues is proposed on the basis of the composition of tryptic peptides and assumed homology.     

Amyloid-beta (Aβ) D7H mutation increases oligomeric Aβ42 and alters properties of Aβ-zinc/copper assemblies

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (Aβ(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn(2+) or Cu(2+), Aβ(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.     

Aβ40(L17A/F19A) mutant diminishes the aggregation and neurotoxicity of Aβ40

Aggregated β-amyloid peptides (Aβ) are neurotoxic and responsible for neuronal death both in vitro and in vivo. From the structural point of view, Aβ self-aggregation involves a conformational change in the peptide. Here, we investigated the relationship between conformational changes and amino acid residues of Aβ₄₀. Urea unfolding in combination with NMR spectroscopy was applied to probe the stabilization of Aβ₄₀ conformation. L17 and F19 residues were found more sensitive to environmental changes than the other residues. Replacement of these two residues with alanine could stabilize the conformation of Aβ₄₀. Further analysis indicated that the Aβ₄₀(L17A/F19A) mutant could diminish the aggregation and reduce the neurotoxicity. These results suggest that L17 and F19 are the critical residues responsible for conformational changes which may trigger neurotoxic cascade of Aβ₄₀.     

Activated Scavenger Receptor A Promotes Glial Internalization of Aβ

Beta-amyloid (Aβ) aggregates have a pivotal role in pathological processing of Alzheimer’s disease (AD). The clearance of Aβ monomer or aggregates is a causal strategy for AD treatment. Microglia and astrocytes are the main macrophages that exert critical neuroprotective roles in the brain. They may effectively clear the toxic accumulation of Aβ at the initial stage of AD, however, their functions are attenuated because of glial overactivation. In this study, we first showed that heptapeptide XD4 activates the class A scavenger receptor (SR-A) on the glia by increasing the binding of Aβ to SR-A, thereby promoting glial phagocytosis of Aβ oligomer in microglia and astrocytes and triggering intracellular mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, XD4 enhances the internalization of Aβ monomers to microglia and astrocytes through macropinocytosis or SR-A-mediated phagocytosis. Furthermore, XD4 significantly inhibits Aβ oligomer-induced cytotoxicity to glial cells and decreases the production of proinflammatory cytokines, such as TNF-α and IL-1β, in vitro and in vivo. Our findings may provide a novel strategy for AD treatment by activating SR-A.     

Aβ-Cu复合物与Aβ纤丝对小胶质细胞激活作用比较研究

目的:比较具有氧化还原活性的过渡金属离子Cu~(2+)(Cu)诱导形成的Aβ聚集物(Aβ-Cu复合物)与Aβ自聚集形成的纤丝(Fibrillar Aβ,f Aβ)对小胶质细胞激活作用的差异。方法:制备Aβ-Cu复合物和fAβ,利用小鼠BV-2小胶质细胞株,分别以不同浓度的Aβ-Cu复合物和fAβ于37℃刺激24 h,检测细胞上清液中的TNF-α(Tumor necrosis factor-α)、NO(Nitric oxide)以及H_2O_2(Hydrogen Peroxide)的含量。分别收集Aβ-Cu复合物和f Aβ作用24 h后的大鼠原代小胶质细胞条件培养液,通过观察该条件培养液对大鼠原代海马神经元细胞活力的影响,评价小胶质细胞介导的间接神经元毒性。结果:(1)在不引起直接神经毒性剂量(2.5μM)下,Aβ-Cu复合物激活小胶质细胞释放TNF-α(P0.01)、NO(P0.05)以及H_2O_2(P0.05)的作用强于f Aβ。(2)在此剂量下,Aβ-Cu复合物通过激活小胶质细胞引起的间接神经元毒性强于fAβ(P0.05)。结论:与fAβ相比,非神经毒性剂量的Aβ-Cu复合物对于小胶质细胞具有更强的激活作用,并由此引发更为明显的神经元毒性。     

阿尔茨海默病Aβ构象转变机制研究进展

阿尔茨海默病(Alzheimer's disease,AD)是一种以神经系统退行性变为特征的"构象病",淀粉样蛋白Aβ聚集形成富含β折叠的肽类聚合物是其发病机制的核心.不同构象的Aβ肽成分除分子量、溶解性、二级结构等基本性质不同之外,尚有免疫原性、神经细胞毒性等生物学功能的差别.由Aβ构象转变引起的空间结构差异,影响其与生物膜、受体等结合蛋白之间的相互作用,通过不同途径关联到线粒体损伤、神经突触功能异常、早期认知障碍、神经元丢失等病理效应.本文综述了Aβ动态结构特征、Aβ构象转变与神经毒性作用的关系、抑制Aβ构象转变的靶点等,以期为明确构象在AD发病机制中的作用乃至"构象病"的共同机制研究提供新的思路.     

Aβ的外周清除与阿尔茨海默病

脑β-淀粉样蛋白(Amyloid-β,Aβ)沉积是阿尔茨海默病(Alzheimer's disease,AD)发病的核心问题。Aβ的沉积与其生成和清除失衡有关,尤其是清除障碍是导致Aβ沉积的主要原因。Aβ经RAGE,LRP1及P-gp介导跨血脑屏障(blood-brain barrier,BBB)转运至外周和经血管旁淋巴引流途径到达局部淋巴结是脑Aβ转运至外周进而发挥外周清除效应的两条途径,亦是Aβ清除的主要途径。RAGE,LRP1及P-gp表达异常、血管旁淋巴引流途径受阻及外周清除组织功能障碍均会影响Aβ外周清除,促进AD病程的进展。因此干预BBB上转运体的功能及血管旁淋巴引流通路,实现外周组织的清除效应,有利于Aβ外周清除。随着外周Aβ清除,脑Aβ则会源源不断转运至外周,发挥外周降解效应,即外周Sink效应。基于外周Sink效应,将AD治疗重点由促进Aβ脑内清除转为外周清除将是一条有效且相对安全的途径。     

Aβ中枢及外周清除机制

β淀粉样蛋白(amyloid-βpeptide,Aβ)沉积是阿尔兹海默病(Alzheimer's disease,AD)的病理特征之一。Aβ在体内的清除途径包括:蛋白酶的降解作用、细胞的清除作用、血脑屏障的转运作用、脑脊液和组织间液淋巴引流作用和外周细胞及组织的清除作用。结合最新进展,本文综述了Aβ在中枢和外周的清除机制。     

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.

     

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。     

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.     

A monoclonal antibody against synthetic Aβ dimer assemblies neutralizes brain-derived synaptic plasticity-disrupting Aβ

Diverse lines of evidence indicate that pre-fibrillar, diffusible assemblies of the amyloid β-protein (Aβ) play an important role in Alzheimer's disease pathogenesis. Although the precise molecular identity of these soluble toxins remains unsettled, recent experiments suggest that sodium dodecyl sulfate (SDS)-stable Aβ dimers may be the basic building blocks of Alzheimer's disease-associated synaptotoxic assemblies and as such present an attractive target for therapeutic intervention. In the absence of sufficient amounts of highly pure cerebral Aβ dimers, we have used synthetic disulfide cross-linked dimers (free of Aβ monomer or fibrils) to generate conformation-specific monoclonal antibodies. These dimers aggregate to form kinetically trapped protofibrils, but do not readily form fibrils. We identified two antibodies, 3C6 and 4B5, which preferentially bind assemblies formed from covalent Aβ dimers, but do not bind to Aβ monomer, amyloid precursor protein, or aggregates formed by other amyloidogenic proteins. Monoclonal antibody 3C6, but not an IgM isotype-matched control antibody, ameliorated the plasticity-disrupting effects of Aβ extracted from the aqueous phase of Alzheimer's disease brain, thus suggesting that 3C6 targets pathogenically relevant Aβ assemblies. These data prove the usefulness of covalent dimers and their assemblies as immunogens and recommend further investigation of the therapeutic and diagnostic utility of monoclonal antibodies raised to such assemblies.     

WNT5A signaling contributes to Aβ-induced neuroinflammation and neurotoxicity

Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aβ), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aβ-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aβ-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1β and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aβ-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.     

A 1,4-β-D-glucan-synthase system fromDictyostelium discoideum

Particulate membrane preparations have been isolated from culminatingDictyostelium discoideum cells. The preparations incorporated glucose from uridine 5′-diphosphate-glucose into a glucose polymer or polymers. These have been shown to be homopolymers ofβ-linked glucose. A high percentage (78% by methylation analysis) of the linkages formed are 1,4-linkages and a lower percentage (12%) are 1,3-linkages. The glucan-synthase complex present in the particulate membrane preparation has an apparent K_m of 0.28 mM and a V_max of 1.59 nmol·min^?1·(mg protein)^?1. The enzyme system is dependent upon Mg²⁺ and cellobiose for maximal activity, but is inhibited by millimolar levels of Ca²⁺. Particulate membrane preparations were made from cells at various times during a synchronous developmental time course and demonstrated that the glucan-synthase activity appeared at the tight-aggregate stage of development.     

Aβ对阿尔茨海默病影响机制的研究进展

阿尔茨海默病(Alzheimer’s disease, AD)是一种以突触丢失、认知功能衰退和渐进性神经元死亡为特征的退行性神经系统疾病,主要临床表现为认知能力下降和行为障碍。在众多病因学说中,淀粉样蛋白斑块是AD病理的主要标志,在AD的发病过程中起着相当重要的作用。胞外β淀粉样蛋白(amyloidβ-protein, Aβ)在脑内的过度沉积会导致线粒体功能障碍、突触功能障碍和炎症反应等病理过程,严重干扰了大脑对信息的处理。目前该疾病仍缺乏有效的治疗方法。本文结合Aβ的毒性作用及其对AD的主要影响作一综述,以期为揭示AD病理机制及进一步促进AD在基础与应用等方面的深入研究提供一定的资料参考。