Apolipoprotein E associated with reconstituted high-density lipoprotein-like particles is protected from aggregation.

Apolipoprotein E (APOE) genotype determines Alzheimer's disease (AD) susceptibility, with the APOE ε4 allele being an established risk factor for late-onset AD. The ApoE lipidation status has been reported to impact amyloid-beta (Aß) peptide metabolism. The details of how lipidation affects ApoE behavior remain to be elucidated. In this study, we prepared lipid-free and lipid-bound ApoE particles, mimicking the high-density lipoprotein particles found in vivo, for all three isoforms (ApoE2, ApoE3, and ApoE4) and biophysically characterized them. We find that lipid-free ApoE in solution has the tendency to aggregate in vitro in an isoform-dependent manner under near-physiological conditions and that aggregation is impeded by lipidation of ApoE.

Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach.

Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer's disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.

Characterization of insulin-degrading enzyme-mediated cleavage of Aß in distinct aggregation states.

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aß) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aß monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aß sequence and show that Aß fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aß clearance in AD patients by modulating IDE activity.

Could ecosystem management provide a new framework for Alzheimer's disease?

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that involves a plethora of molecular pathways. In the context of therapeutic treatment and biomarker profiling, the amyloid-beta (Aß) peptide constitutes an interesting research avenue that involves interactions within a complex mixture of Aß alloforms and other disease-modifying factors. Here, we explore the potential of an ecosystem paradigm as a novel way to consider AD and Aß dynamics in particular. We discuss the example that the complexity of the Aß network not only exhibits interesting parallels with the functioning of complex systems such as ecosystems but that this analogy can also provide novel insights into the neurobiological phenomena in AD and serve as a communication tool. We propose that combining network medicine with general ecosystem management principles could be a new and holistic approach to understand AD pathology and design novel therapies.

Two distinct ß-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aß) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aß mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased ß-amyloid accumulation, and Aß deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aß1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aß both form fibrils characterized by the cross-ß architecture, but with distinct ß-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aß1-42 oligomers and fibrils both display an antiparallel ß-sheet structure, in comparison with the parallel ß-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aß fibrils in a pH-dependent manner, in terms of their underlying ß-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel ß-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.

New mimetic peptides inhibitors of Αß aggregation. Molecular guidance for rational drug design.

A new series of mimetic peptides possessing a significant Aß aggregation modulating effect was reported here. These compounds were obtained based on a molecular modelling study which allowed us to perform a structural-based virtual selection. Monitoring Aß aggregation by thioflavin T fluorescence and transmission electron microscopy revealed that fibril formation was significantly decreased upon prolonged incubation in presence of the active compounds. Dot blot analysis suggested a decrease of soluble oligomers strongly associated with cognitive decline in Alzheimer's disease. For the molecular dynamics simulations, we used an Aß42 pentameric model where the compounds were docked using a blind docking technique. To analyze the dynamic behaviour of the complexes, extensive molecular dynamics simulations were carried out in explicit water. We also measured parameters or descriptors that allowed us to quantify the effect of these compounds as potential inhibitors of Aß aggregation. Thus, significant alterations in the structure of our Aß42 protofibril model were identified. Among others we observed the destruction of the regular helical twist, the loss of a stabilizing salt bridge and the loss of a stabilizing hydrophobic interaction in the ß1 region. Our results may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules and might provide a guide in the design of new aggregation modulating ligands.

Chemical cross-linking/mass spectrometry maps the amyloid ß peptide binding region on both apolipoprotein E domains.

Apolipoprotein E (apoE) binds the amyloid ß peptide (Aß), one of the major culprits in Alzheimer's disease development. The formation of apoE:Aß complexes is implicated in both Aß clearance and fibrillization. However, the binding interface between apoE and Aß is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer's disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3, and E4) for their interaction with Aß1-42 in an early stage of aggregation and at near physiological conditions. Using electron microscopy and Western blots, we showed that all three isoforms are able to prevent Aß fibrillization and form a noncovalent complex, with one molecule of Aß bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aß. Multiple high-confidence intermolecular apoE2/3/4:Aß cross-links confirmed that Lys16 is located in the region of Aß binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aß. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aß at the apoE domain-domain interaction interface, which would explain how apoE is able to stabilize Aß and thereby prevent its subsequent aggregation.

A comparative analysis of the aggregation behavior of amyloid-ß peptide variants.

Aggregated forms of the amyloid-ß peptide are hypothesized to act as the prime toxic agents in Alzheimer disease (AD). The in vivo amyloid-ß peptide pool consists of both C- and N-terminally truncated or mutated peptides, and the composition thereof significantly determines AD risk. Other variations, such as biotinylation, are introduced as molecular tools to aid the understanding of disease mechanisms. Since these modifications have the potential to alter key aggregation properties of the amyloid-ß peptide, we present a comparative study of the aggregation of a substantial set of the most common in vivo identified and in vitro produced amyloid-ß peptides.