The Crowded Environment of a Reverse Micelle Induces the Formation of β-Strand Seed Structures for Nucleating Amyloid Fibril Formation

A hallmark of Alzheimer’s disease is the accumulation of insoluble fibrils in the brain composed of amyloid beta (Aβ) proteins with parallel in-register cross-β-sheet structure. It has been suggested that the aggregation of monomeric Aβ proteins into fibrils is promoted by “seeds” that form within compartments of the brain that have limited solvent due to macromolecular crowding. To characterize these seeds, a crowded macromolecular environment was mimicked by encapsulating Aβ40 monomers into reverse micelles. Fourier-transform infrared spectroscopy revealed that monomeric Aβ proteins form extended β-strands in reverse micelles, while an analogue with a scrambled sequence does not. This is a remarkable finding, because the formation of extended β-strands by monomeric Aβ proteins suggests a plausible mechanism whereby the formation of amyloid fibrils may be nucleated in the human brain

ARA, DPA, and DHA content in the brain tissue of animals fed diets with adequate or deficient amounts of ω3-PUFAs.

<p>Fractions correspond to the five normal phase LC fractions described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.ref018" target="_blank">18</a>]. GC/MS determinations were made as described in methods. LC/MS/MS results are the sum of all data in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g003" target="_blank">3</a>. Uncertainties for the five fractions are standard deviations of 6 measurements. Uncertainties for the fraction totals were calculated according to the variance sum law. All units are nmoles/gm tissue wet weight.</p

The concentrations of ARA-containing phospholipid species in the parietal cortex of animals fed a diet with adequate amounts of ω3 PUFAs (black bars) and a diet deficient in ω3 PUFAs (gray bars) in nmoles / gm tissue.

<p>Each result is the mean of 6 different brain extracts, and the error bars represent standard deviations. The absence of a bar for an sn1 chain indicates that the corresponding MRM transition was monitored, but no signal detected. There were no statistically significant differences between groups. See text for information about the nature of quantitative uncertainty in these data.</p

Increased ω6-Containing Phospholipids and Primary ω6 Oxidation Products in the Brain Tissue of Rats on an ω3-Deficient Diet

<div><p>Polyunsaturated fatty acyl (PUFA) chains in both the ω3 and ω6 series are essential for normal animal brain development, and cannot be interconverted to compensate for a dietary deficiency of one or the other. Paradoxically, a dietary ω3-PUFA deficiency leads to the accumulation of docosapentaenoate (DPA, 22:5ω6), an ω6-PUFA chain that is normally scarce in the brain. We applied a high-precision LC/MS method to characterize the distribution of DPA chains across phospholipid headgroup classes, the fatty acyl chains with which they were paired, and the extent to which they were oxidatively damaged in the cortical brain of rats on an ω3-deficient diet. Results indicate that dietary ω3-PUFA deficiency markedly increased the concentrations of phospholipids with DPA chains across all headgroup subclasses, including plasmalogen species. The concentrations of phospholipids containing docosahexaenoate chains (22:6ω3) decreased 20–25%, while the concentrations of phospholipids containing arachidonate chains (20:4ω6) did not change significantly. Although DPA chains are more saturated than DHA chains, a larger fraction of DPA chains were monohydroxylated, particularly among diacyl-phosphatidylethanolamines and plasmalogen phosphatidylethanolamines, suggesting that they were disproportionately subjected to oxidative stress. Differences in the pathological significance of ω3 and ω6 oxidation products suggest that greater oxidative damage among the ω6 PUFAs that increase in response to dietary ω3 deficiency may have pathological significance in Alzheimer’s disease.</p></div

The ratio of +O addition (oxidation) for selected PE species in the parietal cortex of animals fed the diet deficient in ω3 PUFAs, relative to the diet with adequate amounts of ω3 PUFAs.

<p>The three panels represent PE species containing either oxidized (a) ARA, (b) DPA, or (c) DHA. Thin blue bars in each panel represent results for the <i>sn</i>-1 chains indicated, which correspond to the 6 most abundant PE species in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g001" target="_blank">1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164326#pone.0164326.g003" target="_blank">3</a>. The wide red cross-hatched bar in each panel is the average ratio for all 6 species in aggregate, with a standard error of the mean indicated. By 2-tailed T test of unpaired samples with unequal variances, the ratio for 22:5(+O)-PE species in aggregate (1.63) differs from the ratio for 20:4(+O)-PE species (1.16) at P ≤ 0.05, and from the ratio for 22:6(+O)-PE species (1.01) at P ≤ 0.01.</p

Identification of 18:0/22:5(OH)-PE by LC/MS/MS.

<p>(a) Single reaction monitoring chromatogram of the 808.8 → 345.2 transition, with solvent flow reduced to 850 μl/min. 18:0/22:5(+O)-PE eluted 0.3 min later than 18:0/22:5-PE, consistent with the slightly greater polarity of the oxidized species. (b) Product ions produced by the <i>m/z</i> 808.8 parent ion at 12.6 min. Due to the low concentrations of the parent species (a maximum of only 400 transitions/sec observed in panel a), this spectrum was collected with the Q1 resolution set to “unit”, while the Q3 resolution was set to “open”. The position of the oxygen on the 22:5 chain is unknown, and possibly variable. The identity of the <i>m/z</i> 672.9 and 749.5 product ions is not known.</p

A Synchrotron-Based Hydroxyl Radical Footprinting Analysis of Amyloid Fibrils and Prefibrillar Intermediates with Residue-Specific Resolution

Structural models of the fibrils formed by the 40-residue amyloid-β (Aβ40) peptide in Alzheimer’s disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register β-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aβ40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Aβ40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aβ40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aβ40. Combined with mass-per-length measurements performed by dark-field electron microscopy, the results of this study are consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aβ40 fibril (such as 2LMN, 2LMO, 2LMP, and 2LMQ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. The results also demonstrate that individual Aβ40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aβ peptide

Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid β Peptide Fibrils

Amyloid β peptides form fibrils that are commonly assumed to have a dry, homogeneous, and static internal structure. To examine these assumptions, fibrils under various conditions and different ages have been examined with multidimensional infrared spectroscopy. Each peptide in the fibril had a ¹³C¹⁸O label in the backbone of one residue to disinguish the amide I′ absorption due to that residue from the amide I′ absorption of other residues. Fibrils examined soon after they formed, and reexamined after 1 year in the dry state, exhibited spectral changes confirming that structurally significant water molecules were present in the freshly formed fibrils. Results from fibrils incubated in solution for 4 years indicate that water molecules remained trapped within fibrils and mobile over the 4 year time span. These water molecules are structurally significant because they perturb the parallel β-sheet hydrogen bonding pattern at frequent intervals and at multiple points within individual fibrils, creating structural heterogeneity along the length of a fibril. These results show that the interface between β-sheets in an amyloid fibril is not a “dry zipper”, and that the internal structure of a fibril evolves while it remains in a fibrillar state. These features, water trapping, structural heterogeneity, and structural evolution within a fibril over time, must be accommodated in models of amyloid fibril structure and formation