Aβ Amyloid Formation Research Articles

Intrinsic Disorder and Chaperon-Like Activity of Different Caseins

Casein is the best characterized milk protein and constitutes over 70-80% of total bovine milk protein. In milk, casein exists as large micelle-like particles that comprise four unrelated proteins (αs1-, αs2-, β-, and κ-casein) and calcium phosphate. Although αs1-, αs2-, β-, and κ-casein present important structural differences, all of them adopt extremely open and flexible conformations, enough to be defined intrinsically disordered proteins (IDPs). Caseins are able to inhibiting protein aggregation and amyloid fibrils formation and this chaperon-like activity could be largely due to their structural disorder. In the present study we discuss the meaning of “disorder” in the case of three caseins α, β and k that have similar unordered structure and different sequence. We correlate the different type and disorder degree to the capability of preventing protein aggregation and amyloid formation. The physical-chemical parameters of α, β and k caseins were compared to those of intrinsically unfolded and ideally globular proteins. Moreover, caseins sequences were analyzed by several publicly available disorder-oriented predictors two metaservers, MeDor and metaPRDOS, and by a neural network algorithm (PONDR). We observed that α, β and k caseins have different degree and type of disorder, depending on the parameters under analysis and criteria used by the different predictors. These data were correlated to experimental results (ThT fluorescence, CD) on the caseins effect on 1-40 β-amyloid peptide fibrillogenesis. Experiments showed that k-casein forms ordered aggregates and that it is able to significantly increase lag-time and reduce fibril amount in Aβ amyloid formation. Our results contribute to clear the role of intrinsically disordered proteins and their mechanism of action by functional order/disorder transitions, and offer insight in the field of prevention and therapy in Alzheimer diseases, and, in general, of amyloid pathologies.

The protective effect of crocin on the amyloid fibril formation of Aβ42 peptide in vitro.

Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ40, and the less common but more amyloidogenic form, Aβ42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ42 amyloids. To further investigate the relationship between crocin and Aβ42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

Suppression of γ‐secretase activity increases ganglioside levels at neuritic terminals of differentiated PC12

Mutations in presenilin genes cause the dominant type of familial Alzheimer disease. Presenilin mutations have recently been argued to work as loss‐of‐function; however, their pathological significance remains obscure. In this study, we investigated whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids by treatment of neuronally differentiated PC12 with DAPT, an inhibitor of presenilin‐dependent γ‐secretase. Levels of membrane lipids in neuritic terminals and neuronal somata were analyzed using electrospray ionization tandem mass spectrometry. Through DAPT treatment, whereas cholesterol level stayed unchanged, the levels of phosphatidylcholine and sulfatide increased in both neuritic terminals and neuronal somata. Remarkably, the levels of gangliosides increased in neuritic terminals but decreased in neuronal somata by DAPT treatment concomitant with similar alteration for sphingomyelin. Considering the previous findings that accumulation of gangliosides and sphingomyelin at neuritic terminals induces Aβ amyloid formation, these results suggest that a decrease in γ‐secretase activity affects membrane lipids environment as leading to facilitation of Aβ amyloid formation in the brain. This study was supported by a Grant‐in‐Aid for Research Activity start‐up and for Scientific Research (B) from the Japan Society for the Promotion of Science.

Intrinsic Determinants of Aβ12–24 pH-Dependent Self-Assembly Revealed by Combined Computational and Experimental Studies

The propensity of amyloid- (A) peptide to self-assemble into highly ordered amyloid structures lies at the core of their accumulation in the brain during Alzheimer's disease. By using all-atom explicit solvent replica exchange molecular dynamics simulations, we elucidated at the atomic level the intrinsic determinants of the pH-dependent dimerization of the central hydrophobic segment A and related these with the propensity to form amyloid fibrils measured by experimental tools such as atomic force microscopy and fluorescence. The process of A dimerization was evaluated in terms of free energy landscape, side-chain two-dimensional contact probability maps, -sheet registries, potential mean force as a function of inter-chain distances, secondary structure development and radial solvation distributions. We showed that dimerization is a key event in A amyloid formation; it is highly prompted in the order of pH 5.02.98.4 and determines further amyloid growth. The dimerization is governed by a dynamic interplay of hydrophobic, electrostatic and solvation interactions permitting some variability of -sheets at each pH. These results provide atomistic insight into the complex process of molecular recognition detrimental for amyloid growth and pave the way for better understanding of the molecular basis of amyloid diseases.

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

Randomization of Amyloid‐β‐Peptide(1‐42) Conformation by Sulfonated and Sulfated Nanoparticles Reduces Aggregation and Cytotoxicity

The amyloid-β peptide (Aβ) plays a central role in the mechanism of Alzheimer's disease, being the main constituent of the plaque deposits found in AD brains. Aβ amyloid formation and deposition are due to a conformational switching to a β-enriched secondary structure. Our strategy to inhibit Aβ aggregation involves the re-conversion of Aβ conformation by adsorption to nanoparticles. NPs were synthesized by sulfonation and sulfation of polystyrene, leading to microgels and latexes. Both polymeric nanostructures affect the conformation of Aβ inducing an unordered state. Oligomerization was delayed and cytotoxicity reduced. The proper balance between hydrophilic moieties and hydrophobic chains seems to be an essential feature of effective NPs.

Switching Amyloid β-Peptides Oligomerization and Cytotoxicity with Nanoparticles

The amyloid-β peptide (Aβ) plays a central role in the mechanism of Alzheimer's disease (AD), being the main constituent of the plaque deposits found in AD brains. Aβ amyloid formation is due to a conformational switching to a β-enriched structure. However, the relevance of the plaques to the pathogenesis is unclear and evidences indicate that Aβ toxicity is mediated by soluble oligomers. Our strategy to inhibit aggregation involves re-conversion of Aβ conformation by adsorption to nanoparticles (NPs). We have shown that fluorinated NPs induce α-helical rich structures on Aβ and inhibit fibrillogenesis, whereas their hydrogenated analogues lead to aggregation (Figure 1). In order to test the influence of the zeta potential of NPs on the peptide structure, NPs without fluorine were synthesized by sulfonation and sulfation of polystyrene, leading to microgels and latexes. Studies about the conformational behaviour and oligomerization/cytotoxicity of Aβ in the presence of polymeric nanostructures were performed. Cytotoxicity assays confirmed our hypothesis that the conformational conversion of Aβ has an antiapoptotic activity, increasing the viability of cells treated with oligomeric species. The proper balance between hydrophilic moieties and hydrophobic chains seems to be an essential feature of effective NPs.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Hsp104 Targets Multiple Intermediates on the Amyloid Pathway and Suppresses the Seeding Capacity of Aβ Fibrils and Protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.

Inhibition of β-amyloid peptide aggregation and neurotoxicity by α- d-mannosylglycerate, a natural extremolyte

The aggregation of soluble β-amyloid (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The use of naturally occurring small molecules for inhibiting protein aggregation has recently attracted many interests due to their effectiveness for treating protein folding diseases such as AD, Parkinson's, Huntington's disease, and other amyloidosis diseases. α- d-Mannosylglycerate (MG), a natural extremolyte identified in microorganisms growing under extremely high temperatures up to 100 °C, had been shown to protect proteins against various stress conditions such as heat, freezing, thawing, and drying. Here, we report the effectiveness of MG on the suppression of Alzheimer's Aβ aggregation and neurotoxicity to human neuroblastoma cells. According to our study – carried out by using thioflavin-T induced fluorescence, atomic force microscopy, and cell viability assay – MG had significant inhibitory effect against Aβ amyloid formation and could reduce the toxicity of amyloid aggregates to human neuroblastoma cells while MG itself was innocuous to cells. On the other hand, the structural analogs of MG such as α- d-mannosylglyceramide, mannose, methylmannoside, glycerol, showed negligible effect on Aβ aggregate formation. The results suggest that MG could be a potential drug candidate for treating Alzheimer's disease.

Characterizations of distinct amyloidogenic conformations of the Aβ (1–40) and (1–42) peptides

Major constituents of the amyloid plaques found in the brain of Alzheimer’s patients are the 39–43 residue β-amyloid (Aβ) peptides. Extensive in vitro as well as in vivo biochemical studies have shown that the 40- and 42-residue Aβ peptides play major roles in the neurodegenerative pathology of Alzheimer’s disease. Although the two Aβ peptides share common aggregation properties, the 42-residue peptide is more amyloidogenic and more strongly associated with amyloid pathology. Thus, characterizations of the two Aβ peptides are of critical importance in understanding the molecular mechanism of Aβ amyloid formation. In this report, we present combined CD and NMR studies of the monomeric states of the two peptides under both non-amyloidogenic (<5 °C) and amyloid-forming conditions (>5 °C) at physiological pH. Our CD studies of the Aβ peptides showed that initially unfolded Aβ peptides at low temperature (<5 °C) gradually underwent conformational changes to more β-sheet-like monomeric intermediate states at stronger amyloidogenic conditions (higher temperatures). Detailed residue-specific information on the structural transition was obtained by using NMR spectroscopy. Residues in the N-terminal (3–12) and 20–22 regions underwent conformational changes to more extended structures at the stronger amyloidogenic conditions. Almost identical structural transitions of those residues were observed in the two Aβ peptides, suggesting a similar amyloidogenic intermediate for the two peptides. The 42-residue Aβ (1–42) peptide was, however, more significantly structured at the C-terminal region (39–42), which may lead to the different aggregation propensity of the two peptides.

P4-046: Aβ(1-28) amyloid formation depends on membrane lipid composition

P4-046: Aβ(1-28) amyloid formation depends on membrane lipid composition

Dimerisation of N-acetyl-l-tyrosine ethyl ester and Aβ peptides via formation of dityrosine

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid β-peptide (Aβ). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H2O2). The generation of H2O2 has been linked with Aβ neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Aβ. This residue has also been shown to be important to Aβ aggregation and amyloid formation. It is possible that the formation of an Aβ tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Aβ aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Aβ dimers containing dityrosine. Here we report the use of horseradish peroxidase and H2O2 to dimerise N-acetyl-l-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Aβ peptides. We also report a simple fluorescent plate reader method for monitoring Aβ dimerisation via dityrosine formation.

Metal Ion-dependent Effects of Clioquinol on the Fibril Growth of an Amyloid β Peptide

Although metal ions such as Cu(2+), Zn(2+), and Fe(3+) are implicated to play a key role in Alzheimer disease, their role is rather complex, and comprehensive understanding is not yet obtained. We show that Cu(2+) and Zn(2+) but not Fe(3+) renders the amyloid beta peptide, Abeta(1-40), nonfibrillogenic in nature. However, preformed fibrils of Abeta(1-40) were stable when treated with these metal ions. Consequently, fibril growth of Abeta(1-40) could be switched on/off by switching the molecule between its apo- and holo-forms. Clioquinol, a potential drug for Alzheimer disease, induced resumption of the Cu(2+)-suppressed but not the Zn(2+)-suppressed fibril growth of Abeta(1-40). The observed synergistic effect of clioquinol and Zn(2+) suggests that Zn(2+)-clioquinol complex effectively retards fibril growth. Thus, clioquinol has dual effects; although it disaggregates the metal ion-induced aggregates of Abeta(1-40) through metal chelation, it further retards the fibril growth along with Zn(2+). These results indicate the mechanism of metal ions in suppressing Abeta amyloid formation, as well as providing information toward the use of metal ion chelators, particularly clioquinol, as potential drugs for Alzheimer disease.

Hsp20, a novel α‐crystallin, prevents Aβ fibril formation and toxicity

Abstractβ‐Amyloid (Aβ) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Aβ responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Aβ aggregation still holds promise as a means to reduce Aβ neurotoxicity. In research presented here, we show that Hsp20, a novel α‐crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Aβ amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Aβ of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Aβ fibril formation. Hsp20 was also able to significantly reduce Aβ toxicity to both SH‐SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Aβ toxicity.

Amyloidogenic and anti-amyloidogenic properties of recombinant transthyretin variants

Most transthyretin (TTR) mutations lead to TTR amyloid depositions in patients with familial amyloidotic polyneuropathy and familial amyloidotic cardiomyopathy. However, though an amyloidogenic protein itself, TTR inhibits aggregation of Alzheimer's amyloid beta protein (Aβ) in vitro and in vivo. The pathogenic relationship between two amyloidogenic processes remains unclear. To understand how TTR mutations influence the ability of TTR to inhibit Aβ amyloidosis, forty-seven recombinant TTR variants were produced and analyzed. We showed that all recombinant proteins formed tetramers and were functional in thyroxine binding. Acid denaturation at pH 3.8 resulted in aggregation and fibril formation of all TTR variants. However, only TTR G42 and TTR P55 formed fibrils at pH 6.8. Most TTR variants bound to Aβ and inhibited Aβ aggregation in vitro. TTR variants S64, A71, Q89, V107, H114 and I122 revealed decreased binding to Aβ and decreased inhibition of Aβ aggregation. Only TTR G42 and TTR P55 completely failed to bind Aβ and to inhibit Aβ aggregation. We suggest that TTR variants characterized by decreased binding to Aβ or by decreased inhibition of Aβ aggregation in vitro may contribute to Aβ amyloid formation in vivo. These TTR variants might be important targets for epidemiological studies in Alzheimer's disease.

Protein aging hypothesis of Alzheimer disease

Alzheimer disease (AD), the most common form of aging-related neurodegenerative disorders, is associated with formation of fibrillar deposits of amyloid β-protein (Aβ). While the direct involvement of Aβ in AD has been well documented, the relations between Aβ production, amyloid formation, and neurodegeneration remain unknown. We propose that AD is initiated by a protein aging-related structural transformation in soluble Aβ. We hypothesize that spontaneous chemical modification of aspartyl residues in Aβ to transient succinimide induces a non-native conformation in a fraction of soluble Aβ, rendering it amyloidogenic and neurotoxic. Conformationally altered Aβ is characterized by increased stability in solution and the presence of a non-native β-turn that determines folding of Aβ in solution and the structure of Aβ subunits incorporated into amyloid fibrils. While the soluble ‘non-native’ Aβ is both the factor triggering the neurodegenerative cascade and the precursor of amyloid plaques, these two events...

Primate-like amyloid-β sequence but no cerebral amyloidosis in aged tree shrews

A central pathological feature of Alzheimer’s disease is the profuse deposition of amyloid-β protein (Aβ) in the brain parenchyma and vessel walls. Aβ also forms deposits in the brains of a variety of mammals, including all aged non-human primates studied to date. The sequence of Aβ in these animals is identical to that in humans. No Aβ deposits have been found in the brains of wild-type rats and mice, suggesting that the three amino acid differences between their Aβ and that of amyloid-bearing mammals impedes the fibrillogenicity of Aβ. Analysis of the primary sequence of the β-amyloid precursor protein in tree shrews revealed a 98% similarity and 97% identity with the human protein. Furthermore, the predicted amino acid sequence of Aβ in tree shrews is identical to that in humans. However, immunohistochemical analysis failed to reveal β-amyloid deposits in the neural parenchyma or vasculature of eight aged (7–8 years) tree shrews ( Tupaia belangeri). The lack of correlation between the Aβ sequence and amyloid formation suggests that other factors contribute to cerebral amyloid deposition in aged animals.

522 Abnormal homeostasis of pH and zinc: The prelude for cerebral Aβ amyloid formation?

522 Abnormal homeostasis of pH and zinc: The prelude for cerebral Aβ amyloid formation?

Fibrillogenesis of synthetic amyloid-β peptides is dependent on their initial secondary structure

Synthetic peptides containing the sequence of Alzheimer's amyloid-β peptide (Aβ) spontaneously form amyloid-like fibrils in vitro, and have been extensively used to study the factors that modulate fibrillogenesis. Contradictory observations have been reported regarding the neurotoxicity of A/3 and the influence of some Aβ-binding proteins on in vitro Aβ amyloid formation. In this study, we show that Aβ1–40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties. No differences were detected in the chemical structure or in the initial assembly state by mass spectroscopy, reverse-phase high performance liquid chromatography and denaturating or non-denaturing gel electrophoresis. However, there was a direct correlation between the ability of soluble peptides to form amyloid and their percentage of β-sheet structure, as determined by electron microscopy, fluorescence associated to thioflavine T bound to amyloid, and circular dichroism. The data suggest that the determinant factor of Aβ fibrillogenesis is the secondary structure adopted by the peptide in its soluble state.