Aβ Conformation Research Articles

2-Arachidonoylglycerol metabolism is differently modulated by oligomeric and fibrillar conformations of amyloid beta in synaptic terminals

Alzheimer’s disease (AD) is the most prevalent disorder of senile dementia mainly characterized by amyloid-beta peptide (Aβ) deposits in the brain. Cannabinoids are relevant to AD as they exert several beneficial effects in many models of this disease. Still, whether the endocannabinoid system is either up- or down-regulated in AD has not yet been fully elucidated. Thus, the aim of the present paper was to analyze endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in cerebral cortex synaptosomes incubated with Aβ oligomers or fibrils. These Aβ conformations were obtained by “aging” the 1-40 fragment of the peptide under different agitation and time conditions. A diminished availability of 2-AG resulting from a significant decrease in diacylglycerol lipase (DAGL) activity was observed in the presence of large Aβ1-40 oligomers along with synaptosomal membrane damage, as judged by transmission electron microscopy and LDH release. Conversely, a high availability of 2-AG resulting from an increase in DAGL and lysophosphatidic acid phosphohydrolase activities occurred in the presence of Aβ1-40 fibrils although synaptosomal membrane disruption was also observed. Interestingly, neither synaptosomal mitochondrial viability assayed by MTT reduction nor membrane lipid peroxidation assayed by TBARS formation measurements were altered by Aβ1-40 oligomers or fibrils. These results show a differential effect of Aβ1-40 peptide on 2-AG metabolism depending on its conformation.

Amyloid-β Derived from the Brain of the Alzheimer's Disease Transgenic Mouse Is Resistant to Proteolytic Digestion Due to Its Conformation.

The main pathological feature of Alzheimer's disease (AD) is the formation of abundant amyloid-β (Aβ) plaques in the human brain. Studies have reported that Aβ from the AD brain is resistant to proteolytic digestion, which may explain why Aβ cannot be readily eliminated from this organ. However, there are only a few studies that address this important question. We used the AD transgenic mouse (APP/PS1) model to show that Aβ derived from the brain of the old mouse is resistant to proteolytic digestion. This was in contrast to the proteinase K-sensitive human Aβ peptide, whose amino acid sequence was identical to that of AD mouse-derived Aβ but whose conformation was different (i.e., the native protein, but not the peptide, folded into a three-dimensional conformation). To address this question, we denatured AD mouse-derived Aβ with urea and found that Aβ became proteinase K-sensitive. This phenomenon was concentration-dependent, and these results were confirmed by another protein denaturant, guanidinium hydrochloride. We recovered the conformation of the denatured AD mouse-derived Aβ by eliminating urea and adding the human Aβ peptide, and we found that human Aβ was converted to the proteinase K-resistant form in the presence of partially undenatured AD mouse-derived Aβ. However, upon the addition of the rat Aβ peptide, there were no Aβ proteinase K-resistant fragments. Our results show that the resistance of AD mouse-derived Aβ to proteolytic digestion is dependent on the three-dimensional conformation of Aβ. In summary, this study provides new insights on why Aβ plaques fail to be degraded in the human brain.

Impact of a discordant helix on β-amyloid structure, aggregation ability and toxicity

According to amyloid cascade hypothesis, the deposit of amyloid-β (Aβ) peptide is the main cause of Alzheimer's disease (AD). The aggregation ability and toxicity of Aβ peptides are highly associated with the sequence and conformation. A discordant helix is a helical segment with a tendency to form a β-strand conformation and has been found in many amyloid-like proteins or peptides. In this review, we summarize the current knowledge of the properties of a Aβ discordant helix and its impact on the Aβ structure, aggregation ability and cytotoxicity. In an Aβ sequence, a discordant helical region located at residue 15-26 has been proposed. This discordant helix plays a vital role in Aβ conformation, aggregation ability and cytotoxicity. Any factors which can stabilize the structure of the discordant helix may lead to the prevention of aggregation and toxicity of Aβ. This makes the discordant helix an attractive target for the design of new drugs for the treatment of AD.

α-Helix Mimetics as Modulators of Aβ Self-Assembly

A key molecular species in Alzheimer's disease (AD) is the Aβ42 alloform of Aβ peptide, which is dominant in the amyloid plaques deposited in the brains of AD patients. Recent studies have decisively demonstrated that the prefibrillar soluble oligomers are the neurotoxic culprits and are associated with the pathology of AD. Nascent Aβ42 is predominantly disordered but samples α-helical conformations covering residues 15-24 and 29-35 in the presence of micelles and structure-inducing solvents. In this report, a focused library of oligopyridylamide based α-helical mimetics was designed to target the central α-helix subdomain of Aβ (Aβ13-26). A tripyridylamide, ADH-41, was identified as one of the most potent antagonists of Aβ fibrillation. Amyloid-assembly kinetics, transmission electron microscopy (TEM), and atomic force microscopy (AFM) show that ADH-41 wholly suppresses the aggregation of Aβ at a substoichiometric dose. Dot blot and ELISA assays demonstrate the inhibition of the putative neurotoxic Aβ oligomers. ADH-41 targets Aβ in a sequence and structure-specific manner, as it did not have any effect on the aggregation of islet amyloid polypeptide (IAPP), a peptide which shares sequence similarity with Aβ. Spectroscopic studies using NMR and CD confirm induction of α-helicity in Aβ mediated by ADH-41. Calorimetric and fluorescence titrations yielded binding affinity in the low micromolar range. ADH-41 was also effective at inhibiting the seed-catalyzed aggregation of Aβ probably by modulating the Aβ conformation into a fiber incompetent structure. Overall, we speculate that ADH-41 directs Aβ into off-pathway structures, and thereby alters various solution based functions of Aβ. Cell-based assays to assess the effect of ADH-41 on Aβ are underway and will be presented in due course.

Pre-plaque conformational changes in Alzheimer\u2019s disease-linked A\u03b2 and APP

Reducing levels of the aggregation-prone Aβ peptide that accumulates in the brain with Alzheimer’s disease (AD) has been a major target of experimental therapies. An alternative approach may be to stabilize the physiological conformation of Aβ. To date, the physiological state of Aβ in brain remains unclear, since the available methods used to process brain tissue for determination of Aβ aggregate conformation can in themselves alter the structure and/or composition of the aggregates. Here, using synchrotron-based Fourier transform infrared micro-spectroscopy, non-denaturing gel electrophoresis and conformational specific antibodies we show that the physiological conformations of Aβ and amyloid precursor protein (APP) in brain of transgenic mouse models of AD are altered before formation of amyloid plaques. Furthermore, focal Aβ aggregates in brain that precede amyloid plaque formation localize to synaptic terminals. These changes in the states of Aβ and APP that occur prior to plaque formation may provide novel targets for AD therapy.

Statistical Thermodynamic Modeling of Early Aβ Oligomer Formation

Alzheimer's disease (AD) is the most common neurodegenerative disease. A mounting body of research implicates amyloid-β (Aβ) oligomers as an etiological factor in AD. However, the exact structure and stability of these species remains unclear. We have implemented an approach that uses Aβ conformations obtained from replica exchange molecular dynamics (REMD) simulations in conjunction with a statistical thermodynamic model to derive thermodynamic information on Aβ oligomer formation. Fully atomistic REMD simulations of Aβ were performed using Gromacs, the Charmm 36 force field and the TIP3P water model.

Fibrillation-prone conformations of the amyloid-β-42 peptide at the gold/water interface

Proteins in the proximity of inorganic surfaces and nanoparticles may undergo profound adjustments that trigger biomedically relevant processes, such as protein fibrillation. The mechanisms that govern protein-surface interactions at the molecular level are still poorly understood. In this work, we investigate the adsorption onto a gold surface, in water, of an amyloid-β (Aβ) peptide, which is the amyloidogenic peptide involved in Alzheimer's disease. The entire adsorption process, from the peptide in bulk water to its conformational relaxation on the surface, is explored by large-scale atomistic molecular dynamics (MD) simulations. We start by providing a description of the conformational ensemble of Aβ in solution by a 22 μs temperature replica exchange MD simulation, which is consistent with previous results. Then, we obtain a statistical description of how the peptide approaches the gold surface by multiple MD simulations, identifying the preferential gold-binding sites and giving a kinetic picture of the association process. Finally, relaxation of the Aβ conformations at the gold/water interface is performed by a 19 μs Hamiltonian-temperature replica exchange MD simulation. We find that the conformational ensemble of Aβ is strongly perturbed by the presence of the surface. In particular, at the gold/water interface the population of the conformers akin to amyloid fibrils is significantly enriched, suggesting that this extended contact geometry may promote fibrillation.

STRUCTURAL BASIS OF UNIQUE SELECTIVITY OF ADUCANUMAB FOR OLIGOMERIC FORMS OF AMYLOID-β

There has been a renewed interest in immunotherapy targeting Aβ peptide for treatment of Alzheimer’s disease (AD) with the intriguing Phase 1b findings showing aducanumab significantly reduces amyloid in patients’ brains. Hypotheses being investigated for positive clinical data are the preferred epitope and selectivity for toxic Aβ aggregates. Aducanumab (BIIB037) is a human monoclonal antibody derived from a healthy donor cohort of elderly subjects lacking signs of cognitive impairment, which is in advanced clinical testing for treatment of AD. Aducanumab is unique among amyloid-targeted antibodies for its exquisite selectivity for both soluble oligomeric and insoluble fibrillar aggregates of Aβ found in brain β-amyloid plaques. To gain further insight into the epitope conformation and selectivity for Aβ aggregates, we investigated the structural details of amino-terminal epitope in Aβ using X-ray crystallography of aducanumab Fab:Aβ complex. Biochemical validation of critical interface residues was confirmed using standard ELISA and PepSpot binding assays. Here we report the crystal structure of a recombinant Fab fragment of aducanumab in complex with Aβ1-11 peptide solved at 2.5Å resolution. The N-terminus of Aβ is bound to aducanumab in an extended conformation with the binding interface composed primarily of Phe 4 and His 6 of Aβ. Comparison with other N-terminal Aβ targeting antibodies reveals that aducanumab has an exceptionally compact epitope, which contributes to its selectivity and high avidity for Aβ oligomers and fibrils, including aggregates composed of N-truncated Aβ variants, without targeting physiologic Aβ monomers. In silico Rosetta-based interface and antibody–flexible Aβ docking analysis further highlight aducanumab’s distinct biophysical interaction with the Aβ monomer that further corroborates its selectivity for Aβ oligomers. Aducanumab selectively targets pathologic Aβ conformations displayed by oligomeric and fibrillary aggregates. Our results provide the structural basis for this unique binding profile that differentiates aducanumab from other N-terminal specific antibodies, both in the conformation and orientation of the bound Aβ peptide, as well as the molecular details that comprise the antibody-peptide interface.

Alzheimer's Amyloid-β Sequesters Caspase-3 in Vitro via Its C-Terminal Tail.

Amyloid-β (Aβ), the main constituent in senile plaques found in the brain of patients with Alzheimer's disease (AD), is considered as a causative factor in AD pathogenesis. The clinical examination of the brains of patients with AD has demonstrated that caspase-3 colocalizes with senile plaques. Cellular studies have shown that Aβ can induce neuronal apoptosis via caspase-3 activation. Here, we performed biochemical and in silico studies to investigate possible direct effect of Aβ on caspase-3 to understand the molecular mechanism of the interaction between Aβ and caspase-3. We found that Aβ conformers can specifically and directly sequester caspase-3 activity in which freshly prepared Aβ42 is the most potent. The inhibition is noncompetitive, and the C-terminal region of Aβ plays an important role in sequestration. The binding of Aβ to caspase-3 was examined by cross-linking and proteolysis and by docking and all-atom molecular dynamic simulations. Experimental and in silico results revealed that Aβ42 exhibits a higher binding affinity than Aβ40 and the hydrophobic C-terminal region plays a key role in the caspase-Aβ interaction. Overall, our study describes a novel mechanism demonstrating that Aβ sequesters caspase-3 activity via direct interaction and facilitates future therapeutic development in AD.

Synthesis and structure–activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of β-amyloid-42 aggregation

It is assumed that amyloid-β aggregation is a crucial event in the pathogenesis of Alzheimer’s disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the β-sheet conformation of Aβ via donor–acceptor–donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aβ. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aβ aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aβ aggregation.

Effective anti-Alzheimer Aβ therapy involves depletion of specific Aβ oligomer subtypes.

Background:Recent studies have implicated specific assembly subtypes of β-amyloid (Aβ) peptide, specifically soluble oligomers (soAβ) as disease-relevant structures that may underlie memory loss in Alzheimer disease. Removing existing soluble and insoluble Aβ assemblies is thought to be essential for any attempt at stabilizing brain function and slowing cognitive decline in Alzheimer disease. IV immunoglobulin (IVIg) therapies have been shown to contain naturally occurring polyclonal antibodies that recognize conformational neoepitopes of soluble or insoluble Aβ assemblies including soAβ. These naturally occurring polyclonal antibodies have been suggested to underlie the apparent clinical benefits of IVIg. However, direct evidence linking anti-Aβ antibodies to the clinical bioactivity of IVIg has been lacking.Methods:Five-month-old female Dutch APP E693Q mice were treated for 3 months with neat IVIg or with IVIg that had been affinity-depleted over immobilized Aβ conformers in 1 of 2 assembly states. Memory was assessed in a battery of tests followed by quantification of brain soAβ levels using standard anti-soAβ antibodies.Results:We provide evidence that NU4-type soAβ (NU4-soAβ) assemblies accumulate in the brains of Dutch APP E693Q mice and are associated with defects in memory, even in the absence of insoluble Aβ plaques. Memory benefits were associated with depletion from APP E693Q mouse brain of NU4-soAβ and A11-soAβ but not OC-type fibrillar Aβ oligomers.Conclusions:We propose that targeting of specific soAβ assembly subtypes may be an important consideration in the therapeutic and/or prophylactic benefit of anti-Aβ antibody drugs.

Molecular architecture of Aβ fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aβ plaque formation

Objectives: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1–40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation.Methods: We have used hydrogen–deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils.Results and conclusions: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (Aβ). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (VH) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic Aβ42 peptide segment (Aβ residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding VH domains. We find that VH expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, Aβ VH domains with negatively charged CDR3 mutations show significant preference for recognizing Aβ fibrils relative to Aβ monomers, whereas the same VH domains with other polar CDR3 mutations recognize both Aβ conformers. We observe similar behavior for a VH domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8-37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.

Membrane-Induced Dichotomous Conformation of Amyloid β with the Disordered N-Terminal Segment Followed by the Stable C-Terminal β Structure.

Various neurodegenerative disorders are ascribed to pathogenic molecular processes involving conformational transitions of amyloidogenic proteins into toxic aggregates characterized by their β structures. Accumulating evidence indicates that neuronal cell membranes provide platforms for such conformational transitions of pathogenic proteins as best exemplified by amyloid β (Aβ). Therefore, membrane-bound Aβ species can be promising targets for the development of novel drugs for Alzheimer’s disease. In the present study, solid-state nuclear magnetic resonance spectroscopy has elucidated the membrane-induced conformation of Aβ, in which the disordered N-terminal segment is followed by the stable C-terminal β strand. The data provides an insight into the molecular processes of the conformational transition of Aβ coupled with its assembly into parallel β structures.

The effect of structural heterogeneity on the conformation and stability of Aβ–tau mixtures

Oligomeric and fibrillar amyloids, which cause neurodegenerative diseases, are typically formed through repetitive fracture and elongation processes involving single homogeneous amyloid monomers.

Curcumin Attenuates Amyloid-β Aggregate Toxicity and Modulates Amyloid-β Aggregation Pathway.

The abnormal misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet enriched insoluble deposits initiates a cascade of events leading to pathological processes and culminating in cognitive decline in Alzheimer's disease (AD). In particular, soluble oligomeric/prefibrillar Aβ have been shown to be potent neurotoxins. The naturally occurring polyphenol curcumin has been shown to exert a neuroprotective effect against age-related neurodegenerative diseases such as AD. However, its protective mechanism remains unclear. In this study, we investigated the effects of curcumin on the aggregation of Aβ40 as well as Aβ40 aggregate induced neurotoxicity. Our results show that the curcumin does not inhibit Aβ fibril formation, but rather enriches the population of "off-pathway" soluble oligomers and prefibrillar aggregates that were nontoxic. Curcumin also exerted a nonspecific neuroprotective effect, reducing toxicities induced by a range of Aβ conformers, including monomeric, oligomeric, prefibrillar, and fibrillar Aβ. The neuroprotective effect is possibly membrane-mediated, as curcumin reduced the extent of cell membrane permeabilization induced by Aβ aggregates. Taken together, our study shows that curcumin exerts its neuroprotective effect against Aβ induced toxicity through at least two concerted pathways, modifying the Aβ aggregation pathway toward the formation of nontoxic aggregates and ameliorating Aβ-induced toxicity possibly through a nonspecific pathway.

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers.

Opposite effect of Ca2+/Mg2+ ions on the aggregation of native and precursor-derived Aβ42

Work with the Alzheimer’s disease-related synthetic peptide beta-amyloid (Aβ) is a challenging task because of its disadvantageous dissolution properties and high propensity for aggregation. Recently, a new synthetic derivative, iso-Aβ42, has been introduced, which is a precursor of Aβ42, and it offers advantages as concerns its synthesis and use for sample preparation. These two Aβ forms showed high similarity in their biological effects, as well as in their main structural characteristics under well-chosen experimental circumstances. When we changed these conditions, considerable dissimilarities appeared in the aggregation properties of the two peptides. In the present study, the aggregation pathways of native and precursor-derived Aβ42 oligomers were compared in a physiological buffer with and without divalent metal ions (Ca2+/Mg2+). The presence of these ions influenced the Aβ conformations, the morphology as well as formation dynamics of aggregates in a different manner, as it was demonstrated by thioflavin-T-binding experiments, transmission electron microscopy and electronic circular dichroism measurements. Namely, the aggregation of native Aβ42 to fibrils was facilitated, while the aggregation of precursor-derived Aβ42 was hindered by these divalent metal ions. The observed differences in the aggregation had an impact also on the biological efficiency of native and precursor-derived Aβ42 as it was elucidated by viability assays with enhanced sensitivity on primary endothelial cell cultures. Using replica exchange molecular dynamics, we modeled the conformational ensembles of the two investigated Aβ variants evolving during preparation process. We found considerable differences in the probability distribution of the conformers that can explain the observed dissimilarities in their aggregation properties.

Different zinc(II) complex species and binding modes at Aβ N-terminus drive distinct long range cross-talks in the Aβ monomers

The present study addresses the reconstruction of the free-energy landscapes of amyloid-beta1-42 (Aβ42) coordinated respectively with one and two zinc ions, to scrutinize whether different Aβ–zinc complex species, i.e., mononuclear and dinuclear metal complexes, induce different Aβ conformation features.We found a subtle switch of intramolecular interactions, depending both on the zinc coordination environment and on the peptide to zinc stoichiometric ratio. On the one side, hairpin-like structures are predominant in mononuclear complexes, where a salt-bridge that involves Lys28-Glu22 and Lys16-Asp23 is stabilized. On the other side, elongated conformations are instead stabilized in the dinuclear zinc complexes. Experimental studies of atomic force microscopy as well as of zinc–Aβ complex species distribution diagrams provide evidence that the theoretical calculations can be rationalized in terms of the correlation between the increased amount of amorphous aggregates and the Aβ/Zn2+ ratio.

Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway

Aβ(1–40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved.Here we present evidences of the effect of Thioflavin T (ThT), a worldwide used fluorescent dye to monitor amyloid growth, on the Aβ(1–40) conformation, stability and aggregation. By combining experimental information and Molecular Dynamics simulation results, we show that the presence of ThT in solution affects peptide conformation inducing peculiar supramolecular association. In particular ThT interactions with specific Aβ(1–40) residues promote a rigid partially-folded conformation which shifts the balance between different species in solution toward a more aggregation-prone ensemble of peptides, leading to aggregation. Our findings suggest ways for developing strategies to reverse and block aggregation or to stimulate supramolecular assembly and consequently reduce the presence of transient oligomers. This investigation underlines the need of developing label-free techniques for unbiased quantitative studies of Aβ(1–40) aggregation processes.