Conformation of Pyroglutamated Amyloid β (3–40) and (11–40) Fibrils – Extended or Hairpin?

Amyloid β (Aβ) is a hallmark protein of Alzheimer‘s disease. One physiologically important Aβ variant is formed by initial N-terminal truncation at a glutamic acid position (either E3 or E11), which is subsequently cyclized to a pyroglutamate (either pE3 or pE11). Both forms have been found in high concentrations in the core of amyloid plaques and are likely of high importance in the pathology of Alzheimer’s disease. However, the molecular structure of the fibrils of these variants is not entirely clear. Solid-state NMR spectroscopy studies have reported a molecular contact between Gly25 and Ile31, which would disagree with the conventional hairpin model of wildtype (WT-)Aβ1–40 fibrils, most often described in the literature. We investigated the conformation of the monomeric unit of pE3-Aβ3–40 and pE11-Aβ11–40 (and for comparison also wildtype (WT)-Aβ1–40) fibrils to find out whether the hairpin or a newly suggested extended structure dominates the structure of the Aβ monomers in these fibrils. To this end, solid-state NMR spectroscopy was applied probing the inter-residual contacts between Phe19/Leu34, Ala21/Leu34, and especially Gly25/Ile31 using suitable isotopic labeling schemes. In the second part, the flexible turn of the Aβ40 peptides was replaced by a (3-(3-aminomethyl)phenylazo)phenylacetic acid (AMPP)-based photoswitch, which can predefine the peptide conformation to either an extended (trans) or hairpin (cis) conformation. This enables simultaneous spectroscopic assessment of the conformation of the AMPP-photoswitch, allowing in situ structural investigations during fibrillation in contrast to structural techniques such as NMR spectroscopy or cryo-EM, which can only be applied to stable conformers. Both methods confirm an extended structure for the peptidic monomers in fibrils of all investigated Aβ variants. Especially the Gly25/Ile31 contact is a decisive indicator for the extended structure along with the characteristic absorption spectra of trans-AMPP-Aβ

Evidence for FITC-albumin leakage across disintegrated endothelium in the early stroke phase.

<p>Finally, the same pattern of extravasation and endothelial damage was found at 5 hours after ischemia induction (A–D), as shown at 25 hours before. While the tight junctions remain, the rest of the endothelial cells may be constituted of debris, only (A and B). Often, the vascular basement membrane is exposed to the vascular lumen (B). Furthermore, electron dense vesicles carrying FITC-albumin can often be observed in the endothelium and the adjacent basement membrane (C) where the content is found to be deployed (D). E = endothelial cells; L = vascular lumen; arrow heads = DAB-filled vesicles; arrow = tight junction.</p

Evidence for leakage across structurally altered endothelium.

<p>In addition to the observation of a dramatic increase of the vesicle density, we often observed a disintegration of the whole endothelial layer. In areas exhibiting BBB breakdown the cellular surface of the endothelium was frequently found to be ruffled or discontinuous. Therefore, the vascular wall was often shown to consist of endothelial debris and basement membranes, only (brackets). Thus, in contradiction to a variety of studies our data strongly suggest a transendothelial leakage pattern of affected vessels. DAB grains indicating extravasation of FITC-albumin are demarked by arrow heads. E = endothelial cells; L = vascular lumen; asterisk = cellular debris in the lumen of the vessel; arrow heads = DAB grains; arrow = tight junction.</p

Ultrastructural evidence for transcellular, not paracellular leakage.

<p>(A) Vessels in control areas on the contralateral hemisphere do not show signs of a transcellular, vesicle-mediated extravasation of FITC-albumin. (B–D) In contrast to alterations within the belts of tight junctions, ultrastructural examination regularly revealed signs for a transcellular leakage of the tracer. The endothelial cytoplasm exhibits a remarkable increase in vesicle density (white arrows) across the whole vascular circumference. Again, tight junctions (black arrow) are found to be intact. DAB grains are indicated by arrow heads. Control = contralateral hemisphere, E = endothelial cells; L = vascular lumen.</p

Ratio of occludin-positive vessels in areas of FITC-albumin leakage.

<p>(A) Quantitative analysis of differences in the expression of occludin in areas of FITC-albumin extravasation and their corresponding control areas was performed using low power (10× objective) magnification. Here, laminin-immunolabeling revealed the total number of vessels, whereas occludin-immunoreactivity visualized a critical tight junction constituent. The ratio of occludin-positive vessels to the total number of vessels was determined in 5 animals. (B) The ratio of occludin-positive vessels to the total number of vessels did not differ significantly in areas of FITC-albumin leakage and their corresponding control areas. Bars represent means and added lines indicate standard errors.</p

Expression of claudin-5 and occludin in areas of FITC-albumin extravasation.

<p>Double fluorescence labeling of claudin-5 (blue) and occludin (red), both being transmembrane proteins critical for tight junction formation, demonstrates extravasation of FITC-albumin (green) in the vicinity of vessels expressing both markers. Please also note the presence of discontinuities in the staining pattern of control vessels with an ‘intact’ endothelial barrier (arrow heads).</p

Vascular leakage in areas of ultrastructurally intact tight junctions.

<p>(A) Ultrastructural analysis of a control area located on the contralateral hemisphere shows a smooth endothelial layer (E) with an intact tight junction complex (arrow). The surrounding basement membrane is clearly visible and the adjacent neuropil does not show any structural alterations. (B–D) In areas of FITC-albumin extravasation the tight junction complexes (arrows) regularly appear to be established. The adjacent neuropil often displays cellular edema and cellular debris. Extravasated FITC-albumin and its product of conversion (black DAB grains, arrow heads) can constantly be found in the adjacent brain parenchyma proper. E = endothelial cells; L = vascular lumen.</p

Detection of FITC-albumin leakage following experimental ischemic stroke.

<p>(A) Double fluorescence labeling of laminin (color-coded in blue) and GFAP (red) in combination with applied FITC-albumin (green) reveals areas of ischemia-related BBB breakdown. By application of both, an antibody detecting astrocytes (GFAP, red) and an antibody for pan-laminin (blue) which visualizes vascular as well as glial basement membranes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056419#pone.0056419-Sixt1" target="_blank">[47]</a> the observed leakage can clearly be demonstrated to reach the brain parenchyma proper. Therefore, FITC-albumin is detectable within all the three compartments of the neurovascular unit, represented by the vascular wall (1^st compartment), the perivascular space (2^nd compartment) and the adjacent neuropil (3^rd compartment), delineated by astocytic endfeet (red) forming the glia limitans <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056419#pone.0056419-Bechmann1" target="_blank">[54]</a>. (B) To prove specificity of the applied reagent used for conversion of extravasated FITC-albumin into a permanent labeling by DAB for ultrastructural analysis, we exemplarily performed control stainings on vibratome sections, which were not further processed for electron microscopy. The low magnification of a coronary section clearly demonstrates the specificity of the applied reagent and its reaction product (DAB, brown). These sections were counterstained with hemalaun (blue). Areas of FITC-albumin leakage are clearly confined to the striatum. Higher magnification reveals a general leakage of FITC-albumin into the neuropil (brown background). In perivascular and juxtavascular areas the DAB staining appears to be more intense. (C) To confine ultrastructural analysis to areas with BBB breakdown, we identified areas of FITC-albumin extravasation after embedding in resin on coated microscope slides. These areas were selectively processed for ultrastructural analysis by electron microscopy. In corresponding control areas no FITC-albumin extravasation was observed. Please note, the general brown tissue background is a consequence of the embedding procedure using osmium tetroxide and uranyl acetate, which clearly can be distinguished from DAB staining as indicated on the left.</p

Amyloid β (1–40) Toxicity Depends on the Molecular Contact between Phenylalanine 19 and Leucine 34

The formation of the hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) of amyloid β (1–40) (Aβ(1–40)) is known to be an important step in the fibrillation of Aβ(1–40) peptides. Mutations of this putatively early molecular contact were shown to strongly influence the toxicity of Aβ(1–40) (Das et al. (2015) ACS Chem. Neurosci. 6, 1290−1295). Any mutation of residue F19 completely abolished the toxicity of Aβ(1–40), suggesting that a proper F19–L34 contact is crucial also for the formation of transient oligomers. In this work, we investigate a series of isomeric substitutions of L34, namely, d-leucine, isoleucine, and valine, to study further details of this molecular contact. These replacements represent very minor alterations in the Aβ(1–40) structure posing the question how these alterations challenge the fibrillation kinetics, structure, dynamics, and toxicity of the Aβ(1–40) aggregates. Our work involves kinetic studies using thioflavin T, transmission electron microscopy, X-ray diffraction for the analysis of the fibril morphology, and nuclear magnetic resonance experiments for local structure and molecular dynamics investigations. Combined with cell toxicity assays of the mutated Aβ(1–40) peptides, the physicochemical and biological importance of the early folding contact between F19 and L34 in Aβ(1–40) is underlined. This implies that the F19–L34 contact influences a broad range of different processes including the initiation of fibrillation, oligomer stability, fibril elongation, local fibril structure, and dynamics and cellular toxicity. These processes do not only cover a broad range of diverse mechanisms, but also proved to be highly sensitive to minor modulations of this crucial contact. Furthermore, our work shows that the contact is not simply mediated by general hydrophobic interactions, but also depends on stereospecific mechanisms

Stereoisomers Probe Steric Zippers in Amyloid‑β

Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such “steric zipper” interactions in amyloid-β (Aβ₄₀), whose aggregation is linked to Alzheimer’s disease, by replacing natural residues by their stereoisomers. Such mutations are expected to specifically destabilize the shape sensitive “packing” interactions, which may potentially increase their solubility and change other properties. We study the stereomutants DF19 and DL34 and also the DA2/DF4/DH6/DS8 mutant of Aβ₄₀. F19–L34 is a critical contact in a tightly packed region of Aβ, while residues 1–9 are known to be disordered. While both DF19 and DL34 slow down the kinetics of aggregation and form amyloid fibrils efficiently, only DL34 increases the final solubility. DF19 gives rise to additional off-pathway aggregation which results in large, kinetically stable aggregates, and has lower net solubility. DA2/DF4/DH6/DS8 does not have an effect on the kinetics or the solubility. Notably, both DF19 and DL34 oligomers have a significantly lower level of interactions with lipid vesicles and live cells. We conclude that stereoisomers can cause complex site dependent changes in amyloid properties, and provide an effective tool to determine the role of individual residues in shaping the packed interiors of amyloid aggregates