Journal of Biological Engineering BioMed Central

Background: Self-assembly of the amyloid- peptide (A) has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, synthetic molecules capable of inhibiting A self-assembly could serve as therapeutic agents and endogenous molecules that modulate A self-assembly may influence disease progression. However, increasing evidence implicating a principal pathogenic role for small soluble A aggregates warns that inhibition at intermediate stages of A self-assembly may prove detrimental. Here, we explore the inhibition of A1–40 self-assembly by serum albumin, the most abundant plasma protein, and the influence of this inhibition on A1–40 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 A1–40 aggregates from monomeric peptide and the ongoing growth of A1–40 fibrils. Inhibition of fibrillar A1–40 aggregate growth is observed at substoichiometric concentrations, suggesting that serum albumin recognizes aggregated forms of the peptide to prevent monomer addition. Inhibition of A1–40 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 A1–40 aggregation leads to an increase in the ability of resulting aggregates to activate endothelial cells for adhesion of monocytes. In contrast, A1–40 activation of endothelial cells for monocyte adhesion is reduced when more complete inhibition is observed. Conclusion: These results demonstrate that inhibitors of A 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 A aggregation inhibitors on aggregate size. Published: 27 April 2009 Journal of Biological Engineering 2009, 3:5 doi:10.1186/1754-1611-3-5 Received: 29 December 2008 Accepted: 27 April 2009 This article is available from: http://www.jbioleng.org/content/3/1/5 © 2009 Barcelo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background
Alzheimer's disease (AD) is the leading cause of dementia in the elderly, afflicting a new victim every 71 seconds [1]. AD is characterized by the accumulation of amyloid plaques in the brain parenchyma and cerebral microvasculature. These plaques are comprised primarily of fibrils formed via self-association of the amyloid-β peptide (Aβ). The assembly of monomeric Aβ into fibrillar form has been implicated in the pathogenesis of AD, a premise formally set forth in the amyloid hypothesis [2,3]. In particular, genetic mutations associated with early onset AD promote Aβ assembly by either elevating total Aβ production or increasing the relative amount of the longer, more fibrillogenic form of the peptide. Overexpression of these mutations in transgenic animal models results in an agedependent development of amyloid plaques as well as deficits in reference and working memory [4]. Consequently, inhibition of Aβ self-assembly is under investigation as a therapeutic strategy for AD. Similarly, endogenous molecules that are able to regulate Aβ assembly can impact disease progression.
Recent revisions to the amyloid hypothesis implicate a principal role for soluble Aβ aggregates, including Aβderived diffusible ligands (ADDLs), oligomers, and protofibrils, in the pathogenesis of AD. Soluble Aβ aggregates are capable of eliciting a number of responses in neuronal cell systems, including the impairment of long-term potentiation, the initiation of synaptic loss, and the alteration of memory function [reviewed in [2,5]]. In addition, soluble Aβ aggregates have been demonstrated to selectively elicit changes in brain endothelial cells associated with the increased immune response observed in AD brain, including the activation of endothelial monolayers for increased adhesion and subsequent transmigration of monocyte cells [6] and the stimulation of increases in endothelial monolayer permeability [7]. Here, a specific role for small soluble aggregates was implicated by an inverse relationship between endothelial response and aggregate size.
Plasma proteins have the potential to mediate AD-linked inflammatory responses observed in the brain endothelium. A number of plasma proteins are known to bind various isoforms of Aβ, including Aβ 1-40 and Aβ 1-42 [reviewed in [8]]. In fact, > 95% of circulating Aβ is bound by carrier proteins in blood plasma [9,10], with the majority of Aβ bound to serum albumin [10]. Several plasma proteins, including serum albumin, are also associated with amyloid plaques deposited in the brain [reviewed in [11]].
Interactions between Aβ and plasma proteins have been observed to inhibit Aβ assembly [reviewed in [8]]. Sixty percent of the inhibitory activity present within human plasma has been ascribed to serum albumin [12], and this inhibitory activity has been suggested to account for the lack of Aβ fibril deposition in the periphery [12,13]. Serum albumin has been shown to inhibit the aggregation of Aβ 1-40 and Aβ 1-42 monomer [12] as well as the incorporation of monomeric peptide into Aβ 1-40 and Aβ 1-42 fibrils [12], Aβ 12-28 oligomers [13], and tissue sections isolated from AD brain [12]. Some studies suggest that serum albumin preferentially binds small Aβ aggregates [12,13]. Trapping these small soluble aggregates has the potential to increase Aβ stimulation of brain endothelium.
In the current study, we examine the relationship between the in vitro inhibitory activity of serum albumin and the activation of endothelial monolayers by inhibited Aβ 1-40 aggregate preparations. An inhibitory role for bovine serum albumin (BSA) in both the formation and subsequent growth of Aβ 1-40 aggregates is confirmed and shown to be dose-dependent. The stoichiometry observed for BSA inhibition of aggregate growth suggests that BSA is capable of binding Aβ 1-40 aggregates. Furthermore, the characterization of Aβ 1-40 aggregates via dynamic light scattering (DLS) demonstrates that BSA inhibition of Aβ 1-40 aggregate formation from monomeric peptide can increase populations of small soluble aggregate species. Inhibition of Aβ 1-40 aggregate formation from monomeric peptide is not paralleled by a dose-dependent decrease in Aβ-induced activation of endothelial cells for monocyte adhesion. Instead, endothelial monolayer activation is enhanced when intermediate levels of inhibitor activity lead to an increase in the number of small soluble Aβ 1-40 aggregates. These results demonstrate that soluble Aβ aggregates trapped by inhibitors of Aβ self-assembly can enhance physiological responses associated with AD.

Aβ 1-40 monomer aggregation assay
Aβ 1-40 monomer was diluted to 20 μM in 40 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and incubated in the absence (control) or presence of 20-80 μM BSA at 25°C and under vigorous agitation (vortex, 500 rpm). Reaction progress was monitored via thioflavin T fluorescence following dilution of an aliquot into 10 μM thioflavin T, and results are reported as the change in thioflavin T fluorescence (ΔF) with time. Subsequent to the final fluorescence measurement, aggregate size was assessed for undiluted samples via hydrodynamic radius (R H ) measurements. Experiments in which thioflavin T fluorescence and R H were measured for BSA subjected to aggregation conditions in the absence of Aβ 1-40 monomer served as a negative control and ascertained the negligible contribution of BSA to these signals.
Determination of Aβ 1-40 aggregate size using DLS As described previously [6], aggregate size was assessed via R H measurements using a DynaPro MSX DLS instrument (Wyatt Technology, Santa Barbara, CA) to assimilate autocorrelated light intensity data for calculation of translational diffusion coefficients that could be converted to R H using the Stokes-Einstein equation. R H was assessed from the intensity-weighted histogram calculated via data regulation with Dynamics software (Wyatt Technology).

Aβ 1-40 fibril elongation assay
Aβ 1-40 fibrils were diluted in 40 mM Tris-HCl (pH 8.0) containing thioflavin T and incubated for 15 min in the absence (control) or presence of BSA. To initiate growth, Aβ 1-40 monomer was added for final concentrations of 40 μM monomer, 2 μM fibril, 0-10 μM BSA, and 10 μM thioflavin T. Reactions were incubated at 25°C without agitation, and the incorporation of monomer into growing fibrils was monitored via thioflavin T fluorescence. Experiments in which thioflavin T fluorescence of 40 μM Aβ 1-40 monomer was monitored in the absence of fibrils confirmed that monomer self-assembly did not occur under these experimental conditions. In addition, experiments in which thioflavin T fluorescence of 2 μM Aβ 1-40 fibrils was monitored without addition of monomer served as negative controls and reflected the stability of fibrils. Results are reported as the change in thioflavin T fluorescence (ΔF) with time, and elongation rates were determined by linear regression of this data.

Adhesion assay
Confluent HBMVEC monolayers were treated for 24 h with Aβ 1-40 monomer aggregated in the absence (positive control) or presence of BSA. Reaction products were diluted into culture medium containing hydrocortisone for a final Aβ 1-40 concentration of 5 μM. Parallel treatment with an equivalent dilution of BSA or buffer served as negative controls. As described previously for adhesion assays [6], activated monolayers were washed and incubated with Calcein-labelled THP-1 cells (2 × 10 4 cells/well) for 30 min (37°C, 5% CO 2 ). Nonadherent cells were removed by gentle washing (D-PBS). The number of adherent cells was assessed by Calcein fluorescence employing a Synergy 2 microplate reader (BioTek Instruments, Inc., Winooski, VT) equipped with an excitation filter of 485 ± 10 nm and an emission filter of 528 ± 10 nm and using baseline (D-PBS) subtraction. Results are reported as the percentage of adherent cells [(100%)·(Cells adherent /Cells initial )].

Statistical analysis
Statistical analysis was performed with a one-way ANOVA using GraphPad Prism 5 software (San Diego, CA). Dunnett's test was used for multiple comparisons. P < 0.05 was considered significant. Linear regression was assessed using the coefficient of determination, r 2 .

BSA inhibits aggregation of Aβ 1-40 monomer
To assess the effect of BSA on Aβ 1-40 fibril assembly from monomeric peptide, monomer aggregation was induced by continuous agitation in the presence of ratios of BSA to Aβ 1-40 monomer ranging from equimolar to 4-fold excess.
The formation of aggregated β-sheet structure was monitored using thioflavin T fluorescence. To ensure that the presence of BSA did not impede detection of Aβ aggregates, it was confirmed that the thioflavin T fluorescence of pre-formed Aβ 1-40 fibrils was not reduced in the presence of BSA. These results indicate that BSA does not compete with thioflavin T for binding to fibrils nor does it sequester thioflavin T to prevent its binding to fibrils.
As shown in Figure 1, 20 μM Aβ 1-40 monomer incubated in the absence of BSA exhibited the characteristic lag, indicative of nucleus formation, followed by rapid aggregate growth and concluding with a plateau as equilibrium was reached. BSA-induced changes in Aβ 1-40 monomer aggregation were assessed by evaluating both extension of the lag time and reduction of the plateau fluorescence level. When BSA was present at equimolar concentrations with Aβ 1-40 monomer, the lag time was nearly doubled ( Figure 1, Table 1), suggesting that BSA can intervene at early points along the self-assembly pathway. As the concentration of BSA was increased to a level 2-fold in excess of Aβ 1-40 monomer, the lag time was further extended by almost 2.5-fold and the equilibrium plateau was reduced, indicating that at higher concentrations BSA can reduce the quantity of aggregated Aβ. When Aβ 1-40 monomer was agitated in the presence of a 4-fold excess of BSA, nearly complete inhibition was observed over the 4.5 h period. BSA subjected to aggregation conditions in the absence of Aβ 1-40 monomer exhibited negligible change in thioflavin T fluorescence. These results illustrate the ability of BSA to abrogate Aβ 1-40 monomer aggregation at concentrations in excess of Aβ 1-40 monomer and further demonstrate the dose-dependent nature of this inhibition as BSA concentrations approach stoichiometric ratios with Aβ 1-40 .

BSA inhibits growth of Aβ 1-40 fibrils via monomer addition
To determine whether BSA is capable of interacting with pre-formed Aβ 1-40 aggregates to inhibit later stages of Aβ 1-40 assembly, the growth of Aβ 1-40 fibrils was assessed fol-lowing pre-incubation in the presence of BSA at ratios of BSA to Aβ 1-40 fibril ranging from substoichiometric to 5fold excess, where fibril concentrations are expressed in monomer units. Fibril growth was induced via addition of Aβ 1-40 monomer, and the incorporation of monomer into growing fibrils was monitored as the change in thioflavin T fluorescence. Thioflavin T may itself act as both an indicator and an inhibitor of Aβ self-assembly. However, at stoichiometric ratios that exceed those included within fibril elongation reactions thioflavin T has been observed to have no effect upon the formation of Aβ fibrils from monomeric peptide [14]. In addition, the absence of inhibitory activity by thioflavin T within the Aβ fibril elongation assay has been experimentally confirmed for the conditions employed (data not shown).
As shown in Figure 2, steady growth was observed when 2 μM Aβ 1-40 fibril was incubated with 40 μM Aβ 1-40 monomer ( Figure 2). This uninhibited rate of fibril growth was compared to the rate of growth observed in the presence of BSA to determine the extent of inhibition. Aβ 1-40 fibrils pre-incubated in the presence of BSA at a level 5-fold in excess of monomeric units within Aβ 1-40 fibrils exhibited a much slower rate of growth ( Figure 2) and therefore significant inhibition (Table 1), confirming that BSA is able to prevent the addition of Aβ 1-40 monomer to pre-formed Aβ 1-40 fibrils. Pronounced inhibition was also evident when BSA was present at concentrations equimolar to

BSA inhibition of Aβ 1-40 aggregate assembly modulates physiological activity
To assess the effect of BSA upon the physiological activity of Aβ 1-40 aggregates, brain endothelial cells, which are in direct and continuous contact with circulating serum proteins, were selected. Aβ 1-40 has been previously observed to stimulate HBMVECs for increased adhesion of both THP-1 monocytes [6,15,16] and primary human peripheral blood monocytes [6,16] via a mechanism that involves Aβ recognition of the receptor for advanced glycation end products [15,16]. The ability of BSA to attenuate this Aβ-induced adhesion in HBMVECs was explored.
When HBMVEC monolayers were treated with 5 μM Aβ 1-40 aggregates formed in the absence of BSA, a 2.3-fold increase in adhesion of THP-1 monocytes relative to untreated control monolayers was observed (Figure 3). This result is similar to that reported previously [6]. Aβ 1-40 aggregates formed in the presence of a 2-fold excess of BSA stimulated an even more pronounced 2.9-fold increase in adhesion of THP-1 monocytes, despite the decreased thioflavin T fluorescence observed under these aggregation conditions (Figure 1). In contrast, when Aβ 1-40 aggregates were formed in the presence of a 4-fold excess of BSA, a smaller increase in THP-1 adhesion of only 1.5-fold was observed. BSA treatment of endothelial monolayers in the absence of Aβ made an insignificant contribution to the observed changes in adhesion. These results demonstrate that inhibition of in vitro Aβ 1-40 monomer aggregation by BSA is not paralleled by a dosedependent decrease in physiological activity.

Physiological activity of Aβ 1-40 aggregates correlates with aggregate size
Previous studies revealed that the size of Aβ 1-40 aggregates determines their ability to stimulate endothelial monolayers for monocyte adhesion. The most pronounced increases in adhesion were observed for small soluble aggregates exhibiting R H values of 20-40 nm. Physiological activity decreased as aggregate size increased to 100 Effect of BSA on Aβ 1-40 fibril growth via monomer addition nm, and aggregates exceeding 100 nm increased endothelial adhesion only modestly [6]. A similar inverse correlation was also observed for Aβ 1-40 stimulation of endothelial permeability [7]. Thus, it was speculated that the differential effect of BSA on the physiological activity of Aβ 1-40 aggregate preparations might be related to differences in the resulting aggregate size.
DLS was employed to evaluate R H for aggregates formed in the presence and absence of BSA as well as BSA subjected to aggregation conditions in the absence of Aβ 1-40 monomer. Aβ 1-40 monomer incubated in the absence of BSA yielded aggregates with an R H of 140 nm ( Figure 4B) that produced, as expected, a moderate but pronounced increase in endothelial adhesion (Figure 3). While addition of BSA at a concentration 2-fold in excess of Aβ 1-40 monomer yielded less aggregated peptide (Figure 1), these aggregates exhibited a smaller R H of 34 nm ( Figure 4C) and were thus capable of eliciting higher physiological activity ( Figure 3). In contrast, Aβ 1-40 aggregations performed in the presence of a 4-fold excess of BSA yielded little aggregated peptide (Figure 1) and exhibited a hydrodynamic radius of 3.4 nm ( Figure 4D). This peak may be ascribed to BSA, as an identical peak was observed when BSA was subjected to aggregation conditions in the absence of Aβ 1-40 monomer ( Figure 4A). Due to the exponential relationship between scattered light and aggregate size, this BSA peak would obscure detection of Aβ monomer or aggregates exhibiting R H smaller than 3.4 nm. Thus, it may be deduced that following aggregation in the presence of a 4-fold excess of BSA, Aβ 1-40 exists primarily as monomeric or oligomeric structures. However, a lower intensity peak at 22 nm ( Figure 4D) indicates the presence of a small population of soluble aggregates with high physiological activity, which likely accounts for the modest increase in adhesion observed for this Aβ 1-40 aggregate preparation ( Figure 3). Together, these results demonstrate that the enhanced physiological activity observed for partially inhibited monomer aggregations correlates with an increase in the population of small soluble Aβ 1-40 aggregates.

Discussion
Aβ has the opportunity to interact in vivo with endogenous proteins, peptides, and small molecules prior to and during its self-assembly to form fibrillar aggregates which deposit in the brain parenchyma and cerebral microvasculature and become hallmarks of AD. Implications that small soluble aggregation intermediates may play a principal role in disease progression leave open the possibility that endogenous molecules which intervene at intermediate stages of self-assembly could elevate physiological activity. Several serum proteins, including serum albumin, have been observed to bind Aβ and inhibit the formation of fibrils [reviewed in [8]]. These proteins may Effect of BSA inhibition of Aβ 1-40 monomer aggregation on aggregate size distribution influence Aβ activation of endothelial monolayers, a response which is specific for small soluble Aβ aggregates [6,17]. In the current study, we provide evidence that partial inhibition of Aβ 1-40 aggregate assembly by BSA can enhance Aβ 1-40 stimulation of endothelial monolayers for monocyte adhesion as a result of an increase in the number of small soluble aggregates.
Other studies have reported inhibition of Aβ aggregate assembly by serum albumin. Bohrmann et al. observed complete inhibition of Aβ 1-40 monomer aggregation when human serum albumin (HSA) was present at an 11fold excess [12] and a dose-dependent inhibition by both BSA and HSA for the incorporation of Aβ 1-40 or Aβ 1-42 monomer into immobilized fibrils [12]. Using solution NMR to facilitate atomic resolution of Aβ monomer and oligomer, Milojevic et al. demonstrated that the presence of HSA retarded the addition of monomeric Aβ 12-28 to high molecular weight oligomers [13]. In the current study, dose-dependent BSA inhibition of both the formation of Aβ 1-40 aggregates from monomeric peptide and the growth of fibrillar Aβ 1-40 aggregates was examined to further understand the stoichiometry of these interactions. The decreasing inhibitory action observed in monomer aggregation assays as albumin reached a 1:1 ratio with monomeric Aβ 1-40 ( Figure 1,  [12,13], including oligomers [13]. The observed inhibition of Aβ 1-40 fibril growth by monomer addition could likewise result either from the trapping of monomeric Aβ 1-40 by BSA or from the blocking of sites for monomer addition via binding of BSA to Aβ 1-40 fibrils. A dominant contribution of the latter mechanism is implicated by the pronounced inhibition of monomer addition to fibrils when Aβ 1-40 monomer concentrations (40 μM) significantly exceed BSA levels (0.5 μM). Furthermore, the inhibition of Aβ 1-40 fibril growth at substoichiometric ratios of BSA to monomeric units within Aβ 1-40 fibrils ( Figure 2, Table 1) combined with the previously reported micromolar affinity of serum albumin for Aβ [9,18] suggests that the binding of BSA to individual monomer units within Aβ 1-40 fibril structures is not required for the inhibition of Aβ 1-40 fibril elongation. Instead, BSA may bind aggregates at selected sites, among which are those that prevent subsequent addition of monomeric peptide. Hydrophobic interactions have been identified as one of the principal driving forces in the assembly of Aβ aggregates [19]. Serum albumin possesses multiple hydrophobic binding domains [20]. It is therefore reasonable to speculate that BSA may prevent the addition of monomer to fibrils by capping exposed hydrophobic sites. A similar hypothesis was proposed by Milojevic et al. to explain the arrest of monomer-oligomer exchange following the preferential binding of HSA to high molecular weight oligomers [13].
In solutions of newly formed aggregation intermediates, the ensuing effect of such strategic capping may be the trapping of small soluble aggregates that display a high physiological activity. Trapping of small aggregates has been observed for other molecules that inhibit Aβ assembly, including naphthalene sulfonates [21], inositol [22], calmidazolium chloride [23], an antibody recognizing Aβ 1-11 [24], and submicellar concentrations of sodium dodecyl sulfate [25]. In the current study, both thioflavin T fluorescence and DLS were employed to illustrate that the relative ratio of inhibitor to monomeric Aβ 1-40 determines both the quantity ( Figure 1) and size (Figure 4) of aggregates trapped. As expected, uninhibited reactions allowed the formation of significant quantities of large Aβ 1-40 aggregates and complete inhibition yielded primarily monomeric or oligomeric Aβ 1-40 . In contrast, intermediate levels of inhibition by BSA led to the accumulation of soluble aggregation intermediates. Moreover, this accumulation was paralleled by an increase in Aβ stimulation of endothelial monolayers for monocyte adhesion ( Figure  3). Some extent of this observed stimulation might be simply explained by the kinetic reversibility of binding between BSA and Aβ 1-40 aggregates. However, the accumulation of small aggregate species is likely responsible for the augmented stimulation of endothelial monolayers relative to that observed for Aβ 1-40 aggregates prepared in the absence of BSA. Aβ aggregate size has been shown previously to correlate inversely with the ability of aggregates to stimulate endothelial monolayers for monocyte adhesion [6] and permeability [7]. These results further demonstrate that small aggregates formed as a result of trapping by an inhibitor also exhibit a higher physiological activity. Conversely, Pallitto et al. observed a decrease in neurotoxicity when Aβ 1-39 was incubated in the presence of a small peptide capable of accelerating aggregation and increasing aggregate size [26].

Conclusion
Results presented within this study support implications that small soluble Aβ aggregates may play a principal role in AD pathogenesis [reviewed in [2,5]] and emphasize the danger of extrapolating in vitro Aβ inhibition data to reductions in physiological activity. The trapping of small soluble aggregates by both endogenous inhibitors, such as serum albumin, as well as synthetic compounds can lead to an undesired increase in physiological activity. Such potential consequences will be critical to consider in the future design of therapeutic agents targeted at inhibition of Aβ assembly.