The curvature of gold nanoparticles influences the exposure of amyloid-ß and modulates its aggregation process.

Gold nanoparticles (GNP) are tunable nanomaterials that can be used to develop rational therapeutic inhibitors against the formation of pathological aggregates of proteins. In the case of the pathological aggregation of the amyloid-ß protein (Aß), the shape of the GNP can slow down or accelerate its aggregation kinetics. However, there is a lack of elementary knowledge about how the curvature of GNP alters the interaction with the Aß peptide and how this interaction modifies key molecular steps of fibril formation. In this study, we analysed the effect of flat gold nanoprisms (GNPr) and curved gold nanospheres (GNS) on in vitro Aß42 fibril formation kinetics by using the thioflavin-based kinetic assay and global fitting analysis, with several models of aggregation. Whereas GNPr accelerate the aggregation process and maintain the molecular mechanism of aggregation, GNS slow down this process and modify the molecular mechanism to one of fragmentation/secondary nucleation, with respect to controls. These results can be explained by a differential interaction between the Aß peptide and GNP observed by Raman spectroscopy. While flat GNPr expose key hydrophobic residues involved in the Aß peptide aggregation, curved GNS hide these residues from the solvent. Thus, this study provides mechanistic insights to improve the rational design of GNP nanomaterials for biomedical applications in the field of amyloid-related aggregation.

Somatostatin, an In Vivo Binder to Aß Oligomers, Binds to ßPFOAß(1-42) Tetramers.

Somatostatin (SST14) is strongly related to Alzheimer's disease (AD), as its levels decline during aging, it regulates the proteolytic degradation of the amyloid beta peptide (Aß), and it binds to Aß oligomers in vivo. Recently, the 3D structure of a membrane-associated ß-sheet pore-forming tetramer (ßPFOAß(1-42) tetramer) has been reported. Here, we show that SST14 binds selectively to the ßPFOAß(1-42) tetramer with a KD value of ∼40 µM without binding to monomeric Aß(1-42). Specific NMR chemical shift perturbations, observed during titration of SST14, define a binding site in the ßPFOAß(1-42) tetramer and are in agreement with a 2:1 stoichiometry determined by both native mass spectroscopy and isothermal titration calorimetry. These results enabled us to perform driven docking and model the binding mode for the interaction. The present study provides additional evidence on the relation between SST14 and the amyloid cascade and positions the ßPFOAß(1-42) tetramer as a relevant aggregation form of Aß and as a potential target for AD.

Aß(1-42) tetramer and octamer structures reveal edge conductivity pores as a mechanism for membrane damage.

Formation of amyloid-beta (Aß) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimer's disease (AD). Here, we present the three-dimensional structure of an Aß oligomer formed in a membrane mimicking environment, namely an Aß(1-42) tetramer, which comprises a six stranded ß-sheet core. The two faces of the ß-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aß(1-42) in the sample, Aß(1-42) octamers are also formed, made by two Aß(1-42) tetramers facing each other forming a ß-sandwich structure. Notably, Aß(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core ß-sheets edges of the oligomers.

Preparation of a Well-Defined and Stable ß-Barrel Pore-Forming Aß42 Oligomer.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered a crucial process that underlies neurotoxicity in Alzheimer's disease (AD). To obtain structural information on this type of oligomers, we were inspired by membrane protein approaches used to stabilize, characterize, and analyze the function of such proteins. Using these approaches, we developed conditions under which Aß42, the Aß variant most strongly linked to the aetiology of AD, assembles into an oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a ß-barrel arrangement. We named this oligomer ß-barrel Pore-Forming Aß42 Oligomer (ßPFOAß42). Here, we describe detailed protocols for its preparation and characterization. We expect ßPFOAß42 to be useful in establishing the involvement of membrane-associated Aß oligomers in AD.

Stabilization of a Membrane-Associated Amyloid-ß Oligomer for Its Validation in Alzheimer's Disease.

We have recently reported on the preparation of a membrane-associated ß-barrel Pore-Forming Aß42 Oligomer (ßPFOAß42). It corresponds to a stable and homogeneous Aß42 oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a ß-barrel arrangement. As a follow-up of this work, we aim to establish ßPFOAß42's relevance in Alzheimer's disease (AD). However, ßPFOAß42 is formed under dodecyl phosphocholine (DPC) micelle conditions-intended to mimic the hydrophobic environment of membranes-which are dynamic. Consequently, dilution of the ßPFOAß42/DPC complex in a detergent-free buffer leads to dispersion of the DPC molecules from the oligomer surface, leaving the oligomer without the hydrophobic micelle belt that stabilizes it. Since dilution is required for any biological test, transfer of ßPFOAß42 from DPC micelles into another hydrophobic biomimetic membrane environment, that remains associated with ßPFOAß42 even under high dilution conditions, is a requisite for the validation of ßPFOAß42 in AD. Here we describe conditions for exchanging DPC micelles with amphipols (APols), which are amphipathic polymers designed to stabilize membrane proteins in aqueous solutions. APols bind in an irreversible but non-covalent manner to the hydrophobic surface of membrane proteins preserving their structure even under extreme dilution conditions. We tested three types of APols with distinct physical-chemical properties and found that the ßPFOAß42/DPC complex can only be trapped in non-ionic APols (NAPols). The characterization of the resulting ßPFOAß42/NAPol complex by biochemical tools and structural biology techniques allowed us to establish that the oligomer structure is maintained even under high dilution. Based on these findings, this work constitutes a first step towards the in vivo validation of ßPFOAß42 in AD.

Direct Evidence of the Presence of Cross-Linked Aß Dimers in the Brains of Alzheimer's Disease Patients.

Brain-derived amyloid-ß (Aß) dimers are associated with Alzheimer's disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link has been proposed to make brain-derived dimers (brain dimers) more synaptotoxic than Aß monomers and would also make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aß (CL Aß) dimers, obtained by means of the photoinduced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aß dimers. Second, we used these PICUP CL Aß dimers as standards to improve the isolation of brain Aß dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aß dimer samples allowing the detection of the CL [Aß(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aß dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

Aß42 assembles into specific ß-barrel pore-forming oligomers in membrane-mimicking environments.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer's disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aß variants, Aß40 and Aß42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aß in lipid environments-namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aß40 and Aß42 showed different behavior. Aß40 aggregated into amyloid fibrils, whereas Aß42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a ß-barrel arrangement that we named ß-barrel pore-forming Aß42 oligomers (ßPFOsAß42). Because Aß42, relative to Aß40, has a more prominent role in AD, the higher propensity of Aß42 to form ßPFOs constitutes an indication of their relevance in AD. Moreover, because ßPFOsAß42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of ßPFOs and, more generally, membrane-associated Aß oligomers in AD.

SDS-PAGE analysis of Aß oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS.

The characterization of amyloid-beta peptide (Aß) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aß40 and Aß42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aß42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aß oligomer samples.

Hydrogen/deuterium exchange-protected oligomers populated during Aß fibril formation correlate with neuronal cell death.

The aggregation of the amyloid-ß peptide (Aß) to form fibrils and plaques is strongly associated with Alzheimer's disease (AD). Although it is well established that this process generates neurotoxicity, it is also heterogeneous with a variety of species being formed during the conversion process. This heterogeneity makes it difficult to detect and characterize each of the aggregates formed, which precludes establishing the specific features responsible for the neurotoxicity observed. Here we use pulse-labeling hydrogen-deuterium exchange experiments analyzed by electrospray ionization mass spectrometry (PL-HDX-ESI-MS) to distinguish three ensembles populated during the aggregation of the 40 and 42 residue forms of the Aß peptide, Aß40 and Aß42, on the basis of differences in their persistent structure. Noticeably, two of them are more abundant at the beginning and at the end of the lag phase and are therefore not detectable by conventional assays such as Thioflavin T (ThT). The ensembles populated at different stages of the aggregation process have a surprisingly consistent average degree of exchange, indicating that there are definite structural transitions between the different stages of aggregation. To determine whether an ensemble of species with a given hydrogen exchange pattern correlates with neurotoxicity, we combined PL-HDX-ESI-MS experiments with parallel measurements of the neurotoxicity of the samples under study. The results of this dual approach show that the maximum toxicity correlates with the ensemble comprising HDX protected oligomers, indicating that development of persistent structure within Aß oligomers is a determinant of neurotoxicity.

Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer's disease.

Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimer's disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-ß (Aß42) soluble species, delays Aß42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by Aß42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with Aß42 soluble species, delaying Aß plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.

Template-assisted lateral growth of amyloid-ß42 fibrils studied by differential labeling with gold nanoparticles.

Amyloid-ß protein (Aß) aggregation into amyloid fibrils is central to the origin and development of Alzheimer's disease (AD), yet this highly complex process is poorly understood at the molecular level. Extensive studies have shown that Aß fibril growth occurs through fibril elongation, whereby soluble molecules add to the fibril ends. Nevertheless, fibril morphology strongly depends on aggregation conditions. For example, at high ionic strength, Aß fibrils laterally associate into bundles. To further study the mechanisms leading to fibril growth, we developed a single-fibril growth assay based on differential labeling of two Aß42 variants with gold nanoparticles. We used this assay to study Aß42 fibril growth under different conditions and observed that bundle formation is preceded by lateral interaction of soluble Aß42 molecules with pre-existing fibrils. Based on this data, we propose template-assisted lateral fibril growth as an additional mechanism to elongation for Aß42 fibril growth.

Aß40 and Aß42 amyloid fibrils exhibit distinct molecular recycling properties.

A critical aspect to understanding the molecular basis of Alzheimer's disease (AD) is the characterization of the kinetics of interconversion between the different species present during amyloid-ß protein (Aß) aggregation. By monitoring hydrogen/deuterium exchange in Aß fibrils using electrospray ionization mass spectrometry, we demonstrate that the Aß molecules comprising the fibril continuously dissociate and reassociate, resulting in molecular recycling within the fibril population. Investigations on Aß40 and Aß42 amyloid fibrils reveal that molecules making up Aß40 fibrils recycle to a much greater extent than those of Aß42. By examining factors that could influence molecular recycling and by running simulations, we show that the rate constant for dissociation of molecules from the fibril (k(off)) is much greater for Aß40 than that for Aß42. Importantly, the k(off) values obtained for Aß40 and Aß42 reveal that recycling occurs on biologically relevant time scales. These results have implications for understanding the role of Aß fibrils in neurotoxicity and for designing therapeutic strategies against AD.

Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by hydrogen/deuterium exchange.

The aggregation of proteins into amyloid fibrils is a complex and fascinating process associated with debilitating clinical disorders such as Alzheimer's and Parkinson's diseases. The process of aggregation involves a series of steps during which many intermediate aggregation states are populated. Recent evidence points to these intermediate states as the toxic moieties primarily responsible for cell damage or cell death, which are critical steps in the origin and progression of these disorders. To understand the molecular basis of these diseases, it is crucial to investigate and define the details of the aggregation process, and to achieve this objective, researchers need the tools to characterize the structure and kinetics of interconversion of the various species present during amyloid fibril formation. Hydrogen-deuterium (HD) exchange experiments are based on solvent accessibilities and provide one means by which this kind of information may be acquired. In this Account, we describe research based on HD exchange processes that is directed toward better understanding the dynamics and structural reorganizations involved in the formation of amyloid fibrils. Amide hydrogens that normally undergo rapid exchange with solvent hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. The rates of exchange can be monitored by replacing some hydrogens with deuterons. When peptide and protein molecules assemble into amyloid fibrils, the fibrils contain a core region based on repetitive arrays of beta-sheets oriented parallel to the fibril axis. HD experiments have been applied extensively to map such structures in different amyloid fibril systems. By an extension of this approach, we have observed that HD exchange can be governed by a mechanism through which molecules making up the fibrils are continuously dissolving and reforming, revealing that amyloid fibrils are not static but dynamic structures. Under such circumstances, the kinetic parameters that define this "recycling" behavior can be determined, and they contain information that could be of significant value in the design of therapeutic strategies directed against amyloid-related diseases. More recently, to gain insights into the variety of intermediates that are thought to be involved in the aggregation process, we have applied a kinetic pulse labeling HD experiment that is able to characterize such species even if they are only transiently populated. Using this approach, we have been able to obtain structural insights into the different aggregates populated during the process of amyloid fibril formation as well as kinetic and mechanistic information on the structural reorganizations that take place during aggregation. HD exchange experiments, when carefully designed, constitute powerful tools for mapping the core structures of amyloid fibrils, for investigating the recycling of fibril components, and for characterizing the various types of structural reorganization that occur during aggregation. Such information is invaluable for understanding and addressing the molecular origins of the increasingly common and highly debilitating diseases associated with protein misfolding and aggregation.

Retro-enantio N-methylated peptides as beta-amyloid aggregation inhibitors.

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of beta-amyloid protein (Abeta). We applied the retro-enantio concept to design an N-methylated peptidic inhibitor of the Abeta42 aggregation process. This inhibitor, inrD, as well as the corresponding all-L (inL) and all-D (inD) analogues were assayed for inhibition of Abeta42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Abeta42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Abeta42 aggregation more effectively than inL, that inrD decreases Abeta42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro-enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation.

Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.

Redesign of protein domains using one-bead-one-compound combinatorial chemistry.

A novel combinatorial strategy for the redesign of proteins based on the strength and specificity of intra- and interprotein interactions is described. The strategy has been used to redesign the hydrophobic core of the B domain of protein A. Using one-bead-one-compound combinatorial chemistry, 300 analogues of the C-terminal alpha-helix of the B domain, H3x, have been synthesized using a biocompatible resin and the HMFS linker, allowing the screening to occur while the peptides were bound to the resin. The screening was based on the ability of the H3x analogues to interact with the N-terminal helices of the B domain, H1-H2, and retain the native B domain activity, that is binding to IgG. Eight different analogues containing some nonconservative mutations were obtained from the library, the two most frequent of which, H3P1 and H3P2, were studied in detail. CD analysis revealed that the active analogues interact with H1-H2. To validate the redesign strategy the covalent modified domains H1-H2-H3P1 and H1-H2-H3P2 were synthesized and characterized. CD and NMR analysis revealed that they had a unique, stable, and well-defined three-dimensional structure similar to that for the wild-type B domain. This combinatorial strategy allows us to select for redesigned proteins with the desired activity or the desired physicochemical properties provided the right screening test is used. Furthermore, it is rich in potential for the chemical modification of proteins overcoming the drawbacks associated with the total synthesis of large protein domains.

Molecular recycling within amyloid fibrils.

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of beta-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

Hydrogen exchange, core modules, and new designed proteins.

A strategy for design of new proteins that mimic folding properties of native proteins is based on peptides modeled on the slow exchange cores of natural proteins. We have synthesized peptides, called core modules, that correspond to the elements of secondary structure that carry the very slowest exchanging amides in a protein. The expectation is that, if soluble in water, core modules will form conformational ensembles that favor native-like structure. Core modules modeled on natural bovine pancreatic trypsin inhibitor have been shown by NMR studies to meet this expectation. The next step toward production of a native state mimic is to further shift the conformational bias of a core module toward more ordered structure by promoting module-module interactions that are mutually stabilizing. For this, two core modules were incorporated into a single molecule by means of a long cross-link. From a panel of several two-module peptides, one very promising lead emerged; it is called BetaCore. BetaCore is monomeric in water and forms a new fold composed of a four-stranded, antiparallel beta-sheet. The single, dominant conformation of BetaCore is characterized by various NMR experiments. Here we compare the individual core module to the two-module BetaCore and discuss the progressive stabilization of intramodule structure and the formation of new intermodule interactions.

BetaCore, a designed water soluble four-stranded antiparallel beta-sheet protein.

BetaCore is a designed approximately 50-residue protein in which two BPTI-derived core modules, CM I and CM II, are connected by a 22-atom cross-link. At low temperature and pH 3, homo- and heteronuclear NMR data report a dominant folded ('f') conformation with well-dispersed chemical shifts, i, i+1 periodicity, numerous long-range NOEs, and slowed amide hydrogen isotope exchange patterns that is a four-stranded antiparallel beta-sheet with nonsymmetrical and specific association of CM I and CM II. BetaCore 'f' conformations undergo reversible, global, moderately cooperative, non-two-state thermal transitions to an equilibrium ensemble of unfolded 'u' conformations. There is a significant energy barrier between 'f' and 'u' conformations. This is the first designed four-stranded antiparallel beta-sheet that folds in water.