Characterization of insulin-degrading enzyme-mediated cleavage of Aβ 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 (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met35-Val36 as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity

Two distinct β-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 (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ 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 β-amyloid accumulation, and Aβ 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 Aβ1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aβ both form fibrils characterized by the cross-β architecture, but with distinct β-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aβ1-42 oligomers and fibrils both display an antiparallel β-sheet structure, in comparison with the parallel β-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aβ fibrils in a pH-dependent manner, in terms of their underlying β-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel β-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

Influence of genetic variability and external regulating factors on amyloid-beta peptide aggregation

Protein aggregation has been associated with a wide range of highly debilitating and increasingly prevalent human diseases, ranging from neurodegenerative disorders to non-neuropathic amyloidoises. One of the most widespread neurodegenerative diseases is Alzheimer’s disease (AD), which is the leading cause of dementia, affecting millions of people worldwide and imposing an enormous economic burden in terms of health and hospice care.\ud \ud Substantial evidence points to the amyloid-beta (Aβ) peptide as a major causative factor in AD. This apparently harmless intrinsically disordered monomeric peptide converts into higher ordered and toxic aggregates, and eventually into amyloid fibrils that deposit into extracellular plaques in the brain. One of the leading hypothesis states that Aβ aggregation initiates a cascade of molecular events culminating in widespread neurodegeneration. Several drug discovery strategies have therefore been directed at interfering with Aβ production, aggregation, clearance or toxicity. However, clinical trials have been unsuccessful up to date due to a lack of efficacy or safety issues. This lack of success reflects the general failure to fully comprehend amyloid deposition and its dynamics.\ud \ud The research presented in this doctoral thesis aims to provide more insight into Aβ dynamics and its implication for AD therapy. First, the dynamic nature of Aβ is illustrated and is defined at the intra- and intermolecular level. Next, the influence of genetic variability and external regulating factors on Aβ dynamics, in particular on the aggregation and structural properties of Aβ, was investigated in vitro using a biophysical approach complemented with cell culture studies. Genetic variability includes different Aβ peptide lengths and mutants, originating from mutations within genes encoding amyloid precursor protein and secretases, and Apolipoprotein E (ApoE), the major lipid transporter in the brain of which the ApoE4 isoform is a major genetic risk factor for AD. External regulating factors that were investigated comprise insulin-degrading enzyme, a well-known Aβ-degrading enzyme, and peptidomimetics capable of interfering with Aβ aggregation. Both single- and potential multi-target AD treatment strategies are considered, and I suggest that combining network medicine with general ecosystem management principles is a new holistic approach to better understand AD mechanisms and potentially design more successful therapies

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 (Aβ) 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

Characterization of insulin-degrading enzyme-mediated cleavage of A beta 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 (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met35–Val36 as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.publisher: Elsevier articletitle: Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states journaltitle: Biochimica et Biophysica Acta (BBA) - General Subjects articlelink: http://dx.doi.org/10.1016/j.bbagen.2016.03.010 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.status: publishe

Key molecular interactions between the amyloid beta peptide and apolipoprotein E isoforms revealed by chemical crosslinking/mass spectrometry

info:eu-repo/semantics/nonPublishe

Chemical crosslinking/mass spectrometry defines key molecular interactions between apolipoprotein E and amyloid beta peptide

info:eu-repo/semantics/nonPublishe

Chemical crosslinking/mass spectrometry to map the amyloid beta peptide binding region on apolipoprotein E isoforms

info:eu-repo/semantics/nonPublishe

Chemical Cross-Linking/Mass Spectrometry Maps the Amyloid β Peptide Binding Region on Both Apolipoprotein E Domains

Apolipoprotein E (apoE) binds the amyloid β peptide (Aβ), one of the major culprits in Alzheimer’s disease development. The formation of apoE:Aβ complexes is implicated in both Aβ clearance and fibrillization. However, the binding interface between apoE and Aβ 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 Aβ_1–42 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 Aβ fibrillization and form a noncovalent complex, with one molecule of Aβ bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aβ. Multiple high-confidence intermolecular apoE2/3/4:Aβ cross-links confirmed that Lys16 is located in the region of Aβ binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aβ. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aβ at the apoE domain–domain interaction interface, which would explain how apoE is able to stabilize Aβ and thereby prevent its subsequent aggregation