Inhibition of amyloid-ß aggregation by coumarin analogs can be manipulated by functionalization of the aromatic center.

Aggregation of the amyloid-ß protein (Aß) plays a pathogenic role in the progression of Alzheimer's disease, and small molecules that attenuate Aß aggregation have been identified toward a therapeutic strategy that targets the disease's underlying cause. Compounds containing aromatic structures have been repeatedly reported as effective inhibitors of Aß aggregation, but the functional groups that influence inhibition by these aromatic centers have been less frequently explored. The current study identifies analogs of naturally occurring coumarin as novel inhibitors of Aß aggregation. Derivatization of the coumarin structure is shown to affect inhibitory capabilities and to influence the point at which an inhibitor intervenes within the nucleation dependent Aß aggregation pathway. In particular, functional groups found within amyloid binding dyes, such as benzothiazole and triazole, can improve inhibition efficacy. Furthermore, inhibitor intervention at early or late stages within the amyloid aggregation pathway is shown to correlate with the ability of these functional groups to recognize and bind amyloid species that appear either early or late within the aggregation pathway. These results demonstrate that functionalization of small aromatic molecules with recognition elements can be used in the rational design of Aß aggregation inhibitors to not only enhance inhibition but to also manipulate the inhibition mechanism.

Activation of brain endothelium by soluble aggregates of the amyloid-ß protein involves nuclear factor-κB.

Cerebrovascular accumulation of amyloid-ß protein (Aß) aggregates in Alzheimer's disease (AD) is proposed to contribute to disease progression and brain inflammation as a result of Aß-induced increases in endothelial monolayer permeability and stimulation of the endothelium for cellular adhesion and transmigration. These deficiencies facilitate the entry of serum proteins and monocyte-derived microglia into the brain. In the current study, a role for nuclear factor-κB (NF-κB) in the activation of cerebral microvascular endothelial cells by Aß is explored.Quantitative immunocytochemistry is employed to demonstrate that Aß(1-40) preparations containing isolated soluble aggregates elicit the most pronounced activation and nuclear translocation of NF-κB. This rapid and transient response is observed down to physiological Aß concentrations and parallels phenotypic changes in endothelial monolayers that are selectively elicited by soluble Aß(1-40) aggregates. While monomeric and fibrillar preparations of Aß(1-40) also activated NF-κB, this response was less pronounced, limited to a small cell population, and not coupled with phenotypic changes. Soluble Aß(1-40) aggregate stimulation of endothelial monolayers for adhesion and subsequent transmigration of monocytes as well as increases in permeability were abrogated by inhibition of NF-κB activation. Together, these results provide additional evidence indicating a role for soluble Aß aggregates in the activation of the cerebral microvascular endothelium and implicate the involvement of NF-κB signaling pathways in Aß stimulation of endothelial dysfunction associated with AD.

Comparative study of inhibition at multiple stages of amyloid-beta self-assembly provides mechanistic insight.

The "amyloid cascade hypothesis," linking self-assembly of the amyloid-beta protein (Abeta) to the pathogenesis of Alzheimer's disease, has led to the emergence of inhibition of Abeta self-assembly as a prime therapeutic strategy for this currently unpreventable and devastating disease. The complexity of Abeta self-assembly, which involves multiple reaction intermediates related by nonlinear and interconnected nucleation and growth mechanisms, provides multiple points for inhibitor intervention. Although a number of small-molecule inhibitors of Abeta self-assembly have been identified, little insight has been garnered concerning the point at which these inhibitors intervene within the Abeta assembly process. In the current study, a julolidine derivative is identified as an inhibitor of Abeta self-assembly. To gain insight into the mechanistic action of this inhibitor, the inhibition of fibril formation from monomeric protein is assessed quantitatively and compared with the inhibition of two distinct mechanisms of growth for soluble Abeta aggregation intermediates. This compound is observed to significantly inhibit soluble aggregate growth by lateral association while having little effect on soluble aggregate elongation via monomer addition. In addition, inhibition of soluble Abeta aggregate association exhibits an IC(50) with a somewhat lower stoichiometric ratio than the IC(50) determined for inhibition of fibril formation from monomeric Abeta. This quantitative comparison of inhibition within multiple Abeta self-assembly assays suggests that this compound binds the lateral surface of on-pathway intermediates exhibiting a range of sizes to prevent their association with other aggregates, which is required for further assembly into mature fibrils.

Soluble aggregates of the amyloid-beta peptide are trapped by serum albumin to enhance amyloid-beta activation of endothelial cells.

BACKGROUND: Self-assembly of the amyloid-beta peptide (Abeta) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting Abeta self-assembly could serve as therapeutic agents and endogenous molecules that modulate Abeta self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble Abeta aggregates warns that inhibition at intermediate stages of Abeta self-assembly may prove detrimental. Here, we explore the inhibition of Abeta1-40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on Abeta1-40 activation of endothelial cells for monocyte adhesion. RESULTS: It is demonstrated that serum albumin is capable of inhibiting in a dose-dependent manner both the formation of Abeta1-40 aggregates from monomeric peptide and the ongoing growth of Abeta1-40 fibrils. Inhibition of fibrillar Abeta1-40 aggregate growth is observed at substoichiometric concentrations, suggesting that serum albumin recognizes aggregated forms of the peptide to prevent monomer addition. Inhibition of Abeta1-40 monomer aggregation is observed down to stoichiometric ratios with partial inhibition leading to an increase in the population of small soluble aggregates. Such partial inhibition of Abeta1-40 aggregation leads to an increase in the ability of resulting aggregates to activate endothelial cells for adhesion of monocytes. In contrast, Abeta1-40 activation of endothelial cells for monocyte adhesion is reduced when more complete inhibition is observed. CONCLUSION: These results demonstrate that inhibitors of Abeta self-assembly have the potential to trap small soluble aggregates resulting in an elevation rather than a reduction of cellular responses. These findings provide further support that small soluble aggregates possess high levels of physiological activity and underscore the importance of resolving the effect of Abeta aggregation inhibitors on aggregate size.

Soluble aggregates of the amyloid-beta protein selectively stimulate permeability in human brain microvascular endothelial monolayers.

Cerebral amyloid angiopathy associated with Alzheimer's disease is characterized by cerebrovascular deposition of the amyloid-beta protein (Abeta). Abeta elicits a number of morphological and biochemical alterations in the cerebral microvasculature, which culminate in hemorrhagic stroke. Among these changes, compromise of the blood-brain barrier has been described in Alzheimer's disease brain, transgenic animal models of Alzheimer's disease, and cell culture experiments. In the current study, presented data illustrates that isolated soluble Abeta(1-40) aggregates, but not unaggregated monomer or mature fibril, enhance permeability in human brain microvascular endothelial monolayers. Abeta(1-40)-induced changes in permeability are paralleled by both a decrease in transendothelial electrical resistance and a re-localization of the tight junction-associated protein zonula occludin-1 away from cell borders and into the cytoplasm. Small soluble Abeta(1-40) aggregates are confirmed to be the most potent stimulators of endothelial monolayer permeability by establishing an inverse relationship between average aggregate size and stimulated changes in diffusional permeability coefficients. These results support previous findings demonstrating that small soluble Abeta(1-40) aggregates are also primarily responsible for endothelial activation, suggesting that these same species may elicit other changes in the cerebrovasculature associated with cerebral amyloid angiopathy and Alzheimer's disease.

Soluble aggregates of the amyloid-beta protein activate endothelial monolayers for adhesion and subsequent transmigration of monocyte cells.

Increasing evidence suggests that the deposition of amyloid plaques, composed primarily of the amyloid-beta protein (Abeta), within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly, the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells, ranging from alteration of protein expression to cell death, the Abeta species accountable for these responses remains unexplored. In the current study, we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast, unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates, including Abeta-derived diffusible ligands, oligomers, and protofibrils, and further show that soluble aggregates also selectively exhibit activity in a vascular cell model.