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.

Ruthenium red colorimetric and birefringent staining of amyloid-ß aggregates in vitro and in Tg2576 mice.

Alzheimer's disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-ß (Aß) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aß fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aß aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aß deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aß plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aß plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aß fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aß fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aß aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Probing and trapping a sensitive conformation: amyloid-ß fibrils, oligomers, and dimers.

Alzheimer's disease (AD) is a devastating neurodegenerative disease with pathological misfolding of amyloid-ß protein (Aß). The recent interest in Aß misfolding intermediates necessitates development of novel detection methods and ability to trap these intermediates. We speculated that two regions of Aß may allow for detection of specific Aß species: the N-terminal and 22-35, both likely important in oligomer interaction and formation. We determined via epitomics, proteomic assays, and electron microscopy that the Aß(42) species (wild type, ΔE22, and MetOx) predominantly formed fibrils, oligomers, or dimers, respectively. The 2H4 antibody to the N-terminal of Aß, in the presence of 2% SDS, primarily detected fibrils, and an antibody to the 22-35 region detected low molecular weight Aß species. Simulated molecular modeling provided insight into these SDS-induced structural changes. We next determined if these methods could be used to screen anti-Aß drugs as well as identify compounds that trap Aß in various conformations. Immunoblot assays determined that taurine, homotaurine (Tramiprosate), myoinositol, methylene blue, and curcumin did not prevent Aß aggregation. However, calmidazolium chloride trapped Aß at oligomers, and berberine reduced oligomer formation. Finally, pretreatment of AD brain tissues with SDS enhanced 2H4 antibody immunostaining of fibrillar Aß. Thus we identified and characterized Aßs that adopt specific predominant conformations (modified Aß or via interactions with compounds), developed a novel assay for aggregated Aß, and applied it to drug screening and immunohistochemistry. In summary, our novel approach facilitates drug screening, increases the probability of success of antibody therapeutics, and improves antibody-based detection and identification of different conformations of Aß.

Amyloid-ß metabolite sensing: biochemical linking of glycation modification and misfolding.

Glycation is the reaction of a reducing sugar with proteins and lipids, resulting in myriads of glycation products, protein modifications, cross-linking, and oxidative stress. Glycation reactions are also elevated during metabolic dysfunction such as in Alzheimer's disease (AD) and Down's syndrome. These reactions increase the misfolding of the proteins such as tau and amyloid-ß (Aß), and colocalize with amyloid plaques in AD. Thus, glycation links metabolic dysfunction and AD and may have a causal role in AD. We have characterized the reaction of Aß with reactive metabolites that are elevated during metabolic dysfunction. One metabolite, glyceraldehyde-3-phosphate, is a normal product of glycolysis, while the others are associated with pathology. Our data demonstrates that lipid oxidation products malondialdehyde, hydroxynonenal, and glycation metabolites (methylglyoxal, glyceraldehyde, and glyceraldehyde-3-phosphate) modify Aß42 and increase misfolding. Using mass spectrometry, modifications primarily occurred at the amino terminus. However, the metabolite methylglyoxal modified Arg5 in the Aß sequence. 4-Hydroxy-2-nonenal modifications were similar to our previous publication. To place such modifications into an in vivo context, we stained AD brain tissue for endproducts of glycation, or advanced glycation endproducts (AGE). Similar to previous findings, AGE colocalized with amyloid plaques. In summary, we demonstrate the glycation of Aß and plaques by metabolic compounds. Thus, glycation potentially links metabolic dysfunction and Aß misfolding in AD, and may contribute to the AD pathogenesis. This association can further be expanded to raise the tantalizing concept that such Aß modification and misfolding can function as a sensor of metabolic dysfunction.

Hydralazine modifies Aß fibril formation and prevents modification by lipids in vitro.

Lipid oxidative damage and amyloid ß (Aß) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aß misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aß production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aß misfolding and (2) Aß protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aß aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aß, and Aß was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aß and also did not prevent Aß aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aß formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aß misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.