Post-Translational Chemical Modification of Amyloid-ß Peptides by 4-Hydroxy-2-Nonenal.

BACKGROUND: The extraction and quantification of amyloid-ß (Aß) peptides in brain tissue commonly uses formic acid (FA) to disaggregate Aß fibrils. However, it is not clear whether FA can disaggregate post-translationally modified Aß peptides, or whether it induces artifact by covalent modification during disaggregation. Of particular interest are Aß peptides that have been covalently modified by 4-hydroxy-2-nonenal (HNE), an oxidative lipid degradation product produced in the vicinity of amyloid plaques that dramatically accelerates the aggregation of Aß peptides. OBJECTIVE: Test the ability of FA to disaggregate Aß peptides modified by HNE and to induce covalent artifacts. METHODS: Quantitative liquid-chromatography-tandem-mass spectrometry of monomeric Aß peptides and identify covalently modified forms. RESULTS: FA disaggregated ordinary Aß fibrils but also induced the time-dependent formylation of at least 2 residue side chains in Aß peptides, as well as oxidation of its methionine side chain. FA was unable to disaggregate Aß peptides that had been covalently modified by HNE. CONCLUSION: The inability of FA to disaggregate Aß peptides modified by HNE prevents FA-based approaches from quantifying a pool of HNE-modified Aß peptides in brain tissue that may have pathological significance.

Free Radical Chain Reactions and Polyunsaturated Fatty Acids in Brain Lipids.

Polyunsaturated fatty acyl chains (PUFAs) concentrate in the brain and give rise to numerous oxidative chemical degradation products. It is widely assumed that these products are the result of free radical chain reactions, and reactions of this type have been demonstrated in preparations where a single PUFA substrate species predominates. However, it is unclear whether such reactions can occur in the biologically complex milieu of lipid membranes where PUFA substrates are a minority species, and where diverse free radical scavengers or other quenching mechanisms are present. It is of particular interest to know whether they occur in brain, where PUFAs are concentrated and where PUFA oxidation products have been implicated in the pathogenesis of neurodegenerative disorders. To ascertain whether free radical chain reactions can occur in a complex brain lipid mixture, mouse brain lipids were extracted, formed into vesicles, and treated with a fixed number of hydroxyl radicals under conditions wherein the concentrations and types of PUFA-containing phospholipids were varied. Specific phospholipid species in the mixture were assayed by tandem mass spectrometry to quantify the oxidative losses of endogenous PUFA-containing phospholipids. Results reveal crosstalk between the oxidative degradation of ω3 and ω6 PUFAs that can only be explained by the occurrence of free radical chain reactions. These results demonstrate that PUFAs in a complex brain lipid mixture can participate in free radical chain reactions wherein the extent of oxidative degradation is not limited by the number of reactive oxygen species available to initiate such reactions. These reactions may help explain otherwise puzzling in vivo interactions between ω3 and ω6 PUFAs in mouse brain.

Deuterated docosahexaenoic acid protects against oxidative stress and geographic atrophy-like retinal degeneration in a mouse model with iron overload.

Oxidative stress plays a central role in age-related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis-allylic sites of DHA in photoreceptor membranes could prevent iron-induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation-prone positions (D-DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D-DHA caused a dose-dependent increase in D-DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D-DHA provided nearly complete protection against iron-induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D-DHA. Quantitative PCR results were consistent with iron-induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D-DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron-induced retinal degeneration. They also provide preclinical evidence that dosing with D-DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress.

A new synthetic medium for the optimization of docosahexaenoic acid production in Crypthecodinium cohnii.

The heterotrophic microalgae Crypthecodinium cohnii was usually cultivated in complex medium containing glucose, yeast extract and sea salt. For the preparation of DHA with highest purity, a new defined medium without the yeast extract was developed. Different inoculated densities, C/N ratios, temperatures, culture volumes and glucose additions were investigated to optimize the algal growth rate and DHA production. The growth period in C. cohnii was shortened from 12-14 days to 7-8 days, the OD600 was enhanced from 2.0 to 3.0, the glucose consumption was accelerated and used up on day 3-4, and the DHA content in culture were increased from 10 to 45 nmoles/300 µl batch. It was found that C. cohnii had optimal growth and DHA accumulation in 25 °C, 0.2 inoculated density, 5-10 C/N ratio, 5:1 air/culture volume ratio. This is the first time DHA production using C.cohnii has been optimized in synthetic medium. This allows preparation of uniformly radiolabeled 13C- and 14C-DHA.

Biosynthesis of uniformly carbon isotope-labeled docosahexaenoic acid in Crypthecodinium cohnii.

Docosahexaenoic acid (DHA) enriched in brain can yield many important degradation products after the attack of hydroxyl radicals, which is known to serve as a nutraceutical and neuroprotective effects. Oxidative stress is a commonly observed feature of Alzheimer's disease (AD). Therefore, uniformly radiolabeled DHA plays an important role in studying the oxidative fate of DHA in vivo and vitro. However, carbon isotope labeled DHA isn't commercially available now. The heterotrophic microalgae Crypthecodinium cohnii (C. cohnii) has been identified as a prolific producer of DHA. In this study, the growth rate and DHA production in C. cohnii were optimized in a new defined media, and the biosynthesis of U-13C-DHA from U-13C-glucose and U-14C-DHA from U-14C-glucose were analyzed by HPLC-MS/MS. Approximately 40 nmoles of U-13C-DHA with higher isotopic purity of 96.8% was produced in a 300 µL batch, and ~ 0.23 µCi of U-14C-DHA with significant specific activity of 5-6 Ci/mol was produced in a 300 µL batch. It was found that C. cohnii had the optimal growth and DHA accumulation at 25 °C in this defined media (C/N = 10). An efficient protocol for the biosynthesis of U-13C-DHA and U-14C-DHA were set up firstly, which provides the basic support for the analysis of oxidative degradation products of DHA in AD.

Quantitative Mass Spectrometric Assay of Whole and CNBr-Cleaved Amyloid-ß Peptides in Human Brain.

Various amyloid-ß (Aß) peptides accumulate in brain in Alzheimer's disease, and the amounts of specific peptide variants may have pathological significance. The quantitative determination of these variants is challenging because losses inevitably occur during tissue processing and analysis. This report describes the use of stable-isotope-labeled Aß peptides as internal standards for quantitative mass spectrometric assays, and the use of cyanogen bromide (CNBr) to remove C-terminal residues beyond Met35. The removal of residues beyond Met35 reduces losses due to aggregation, and facilitates the detection of post-translationally modified Aß peptides. Results from 8 human brain samples suggest that the tissue concentrations of the 42-residue Aß peptide tend to be similar in different patients. Concentrations of the 40-residue Aß peptide are more variable, and may be greater or lesser than the 42-residue peptide. The concentration of the CNBr cleavage product closely matches the sum of the 40-residue and 42-residue peptide concentrations, indicating that these two Aß peptides account for most of the C-terminal variants in these patients. CNBr treatment facilitated the detection of post-translational modifications such as pyroglutamyl and hexose-modified Aß peptides.

Non-Equilibrium Mass Exchange in AOT Reverse Micelles.

Reverse micelles (RMs) composed of water and sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane have a remarkably narrow size distribution around a mean value determined by the water loading ratio of the system. It has been proposed that RMs establish this equilibrium size distribution either by the diffusion of individual components through the isooctane phase or by cycles of fusion and fission. To examine these mechanisms, a 24 µs all-atom molecular dynamics simulation of a system containing one small RM and one large RM was performed. Results show that the net movement of water from the small RM to the large RM occurred in a direction that made the small RM smaller and the large RM larger-according to water loading ratios that would have been appropriate for their size. Changes in AOT number that would bring the water loading ratio of each RM closer to that of the overall system only occurred via cycles of RM fusion and fission. These behaviors are most likely driven by the electrostatics of sodium AOT and the dielectric effects of water.

Identifying Polymorphs of Amyloid-ß (1-40) Fibrils Using High-Resolution Atomic Force Microscopy.

Many amyloid-ß fibril preparations are highly polymorphic, and the conditions under which they are formed determine their morphology. This report describes the application of high-resolution atomic force microscopy (HR-AFM), combined with volume-per-length analysis, to define, identify, and quantify the structural components of polymorphic Aß fibril preparations. Volume-per-length analysis confirms that they are composed of discrete cross-ß filaments, and the analysis of HR-AFM images yields the number of striations in each fibril. Compared to mass-per-length analysis by electron microscopy, HR-AFM analysis yields narrower distributions, facilitating rapid and label-free quantitative morphological characterization of Aß fibril preparations.

The effects of omega-3 fatty acid deficiency during development on oxidative fatty acid degradation during maturity in a mouse model of Alzheimer's disease.

Metabolic conditions during brain development may have long-term consequences on brain metabolism, thereby influencing the risk of neurodegenerative disease in later life. To ascertain the long-term consequences of omega-3 (ω3) fatty acid deficiency during brain development on oxidative fatty acid degradation in the brain and the development of Alzheimer-like pathology, wild-type (WT) female mice were fed diets that were either replete or deficient in ω3 fatty acids for 5 weeks. These females were then mated with hemizygous 5xFAD male transgenic (TG) mouse models of Alzheimer's disease, and the progeny were continued on diets that were either ω3-replete or ω3-deficient. When the progeny were 6 months of age, they received radiolabeled arachidonic acid (ARA) by intracerebroventricular injection. Five days after these injections, the brains were harvested and oxidative degradation of the radiolabeled ARA was characterized. Among the progeny of female mice on an ω3-replete diet, TG progeny had lower PSD-95 expression and higher oxidative ARA degradation than WT progeny. Progeny on an ω3-deficient diet, however, had no significant differences in PSD-95 expression between TG and WT mice, or in the extent of ARA degradation. In TG mice, an ω3-deficient diet reduced oxidative ARA degradation to a greater extent than in WT mice. The reductions in oxidative ARA degradation occurred even if the progeny of female mice on an ω3-deficient diet resumed an ω3-replete diet immediately on weaning. These results demonstrate that dietary ω3 fatty acid deficiency during development can cause long-term changes in the expression of a synaptic marker and long-term reductions in the rate of ARA degradation in the WT brain, which are not completely alleviated by an ω3-replete diet after weaning. The elimination of differences between TG and WT mice by an ω3-deficient diet suggests that mechanisms regulating PSD-95 expression and the oxidative degradation of ARA are related and that the timing of dietary ω3 intake during development may influence Alzheimer's disease-related pathological changes later in life.

Analysis of eicosanoid oxidation products in Alzheimer brain by LC-MS with uniformly 13C-labeled internal standards.

The quantitative analysis of polyunsaturated fatty acyl (PUFA) chain oxidation products in tissue samples by mass spectrometry is hindered by the lack of durable internal standards for the large number of possible products. To address this problem in a study of oxidative PUFA degradation in Alzheimer's disease (AD) brain, uniformly 13C-labeled arachidonic acid (ARA) was produced biosynthetically, and allowed to oxidize under controlled conditions into a mixture of U-13C-labeled ARA oxidation products. The components of this mixture were characterized with respect to their partitioning behavior during lipid extraction, their durability during saponification, trends in mouse brain tissue concentrations during post mortem intervals, and their overall suitability as internal standards for multiple-reaction monitoring tandem mass spectrometry. This mixture has now been used as a set of internal standards to determine the relative abundance of ARA and 54 non-stereospecific oxidation products in milligram samples of brain tissue. Many of these oxidation products were recovered from both healthy mouse and healthy human brain, although some of them were unique to each source, and some have not heretofore been described. The list of oxidation products detected in AD brain tissue was the same as in healthy human brain, although simple hydroxy-eicosanoids were significantly increased in AD brain. while more complex oxidation products were not. These results are consistent with an increased level of chemically-mediated oxidative ARA degradation in Alzheimer's disease. However, they also point to the existence of processes that selectively produce or eliminate specific oxidation products, and those processes may account for some of the inconsistencies in previously reported results.

Mass Exchange and Equilibration Processes in AOT Reverse Micelles.

Reverse micelles (RMs) made with sodium bis(2-ethylhexyl)sulfosuccinate suspended in isooctane are commonly used experimental models of aqueous microenvironments. However, there are important unanswered questions about the very characteristic that makes them of interest, namely their size. To explore the factors that determine the size of RMs, all-atom molecular dynamics simulations of RMs with different sizes but the same water-loading ratio were performed. An Anton 2 machine was used so that systems of the necessary size could be extended into the microsecond timescale, and mass exchange processes could be observed. Contrary to hypothesis, there were no net gains or losses of water by diffusion between RMs of different size. However, gains and losses did occur following fusion events. RM fusion followed RM contact only when waters were present among the hydrophobic surfactant chains at the point of contact. The presence of an encapsulated 40-residue amyloid beta peptide did not directly promote RM fusion, but it quickly and efficiently terminated each fusion event. Before fusion terminated, however, the size of the peptide-containing RM increased without a corresponding change in its water-loading ratio. We conclude that the mass transfer between RMs is most likely accomplished through transient fusion events, rather than through the diffusion of component molecules through the organic phase. The behavior of the amyloid beta peptide in this system underscores its propensity to embed in, and fold in response to, multiple interactions with the surfactant layer.

Amyloid Beta Peptide Folding in Reverse Micelles.

Previously published experimental studies have suggested that when the 40-residue amyloid beta peptide is encapsulated in a reverse micelle, it folds into a structure that may nucleate amyloid fibril formation (Yeung, P. S.-W.; Axelsen, P. H. J. Am. Chem. Soc. 2012, 134, 6061 ). The factors that induce the formation of this structure have now been identified in a multi-microsecond simulation of the same reverse micelle system that was studied experimentally. Key features of the polypeptide-micelle interaction include the anchoring of a hydrophobic residue cluster into gaps in the reverse micelle surface, the formation of a beta turn at the anchor point that brings N- and C-terminal segments of the polypeptide into proximity, high ionic strength that promotes intramolecular hydrogen bond formation, and deformation of the reverse micelle surface to facilitate interactions with the surface along the entire length of the polypeptide. Together, these features cause the simulation-derived vibrational spectrum to red shift in a manner that reproduces the red-shift previously reported experimentally. On the basis of these findings, a new mechanism is proposed whereby membranes nucleate fibril formation and facilitate the in-register alignment of polypeptide strands that is characteristic of amyloid fibrils.

Biosynthesis of uniformly labeled 13C- and 14C-arachidonic acid in Mortierella alpina.

Arachidonic acid (ARA) is one of the most abundant polyunsaturated fatty acids (PUFAs) in the mammalian brain. Many enzymatically- and nonenzymatically-produced metabolic products have important and potent pharmacological properties. However, uniformly isotope labeled forms of ARA are not commercially available for studying the metabolic fates of ARA. This study describes a simple and efficient protocol for the biosynthesis of U-13C-ARA from U-13C-glucose, and U-14C-ARA from U-14C-glucose by Mortierella alpina. The protocols yield approximately 100nmol quantities of U-13C-ARA with an isotopic purity of 95% from a 500µl batch volume, and approximately 2µCi quantities of U-14C-ARA with an apparent specific activity in excess of 1200Ci/mol from a 250µl batch volume.

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.

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

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.

The Size of AOT Reverse Micelles.

Reverse micelles (RMs) made from water and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) are commonly studied experimentally as models of aqueous microenvironments. They are small enough for individual RMs to also be studied by molecular dynamics (MD) simulation, which yields detailed insight into their structure and properties. Although RM size is determined by the water loading ratio (i.e., the molar ratio of water to AOT), experimental measurements of RM size are imprecise and inconsistent, which is problematic when seeking to understand the relationship between water loading ratio and RM size, and when designing models for study by MD simulation. Therefore, a systematic study of RM size was performed by MD simulation with the aims of determining the size of an RM for a given water loading ratio, and of reconciling the results with experimental measurements. Results for a water loading ratio of 7.5 indicate that the interaction energy between AOT anions and other system components is at a minimum when there are 62 AOT anions in each RM. The minimum is due to a combination of attractive and repulsive electrostatic interactions that vary with RM size and the dielectric effect of available water. Overall, the results agree with a detailed analysis of previously published experimental data over a wide range of water loading ratios, and help reconcile seemingly discrepant experimental results. In addition, water loss and gain from an RM is observed and the mechanism of water exchange is outlined. This kind of RM model, which faithfully reproduces experimental results, is essential for reliable insights into the properties of RM-encapsulated materials.

Amyloid Plaque-Associated Oxidative Degradation of Uniformly Radiolabeled Arachidonic Acid.

Oxidative stress is a frequently observed feature of Alzheimer's disease, but its pathological significance is not understood. To explore the relationship between oxidative stress and amyloid plaques, uniformly radiolabeled arachidonate was introduced into transgenic mouse models of Alzheimer's disease via intracerebroventricular injection. Uniform labeling with carbon-14 is used here for the first time, and made possible meaningful quantification of arachidonate oxidative degradation products. The injected arachidonate entered a fatty acid pool that was subject to oxidative degradation in both transgenic and wild-type animals. However, the extent of its degradation was markedly greater in the hippocampus of transgenic animals where amyloid plaques were abundant. In human Alzheimer's brain, plaque-associated proteins were post-translationally modified by hydroxynonenal, a well-known oxidative degradation product of arachidonate. These results suggest that several recurring themes in Alzheimer's pathogenesis, amyloid ß proteins, transition metal ions, oxidative stress, and apolipoprotein isoforms, may be involved in a common mechanism that has the potential to explain both neuronal loss and fibril formation in this disease.

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-ß (Aß40) 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 ß-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 Aß40 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 Aß40. 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 Aß40 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 Aß40. 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 Aß40 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 Aß40 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 Aß peptide.