Resveratrol Acts Not through Anti-Aggregative Pathways but Mainly via Its Scavenging Properties against Aβ and Aβ-Metal Complexes Toxicity

It has been recently suggested that resveratrol can be effective in slowing down Alzheimer's disease (AD) development. As reported in many biochemical studies, resveratrol seems to exert its neuro-protective role through inhibition of β-amyloid aggregation (Aβ), by scavenging oxidants and exerting anti-inflammatory activities. In this paper, we demonstrate that resveratrol is cytoprotective in human neuroblastoma cells exposed to Aβ and or to Aβ-metal complex. Our findings suggest that resveratrol acts not through anti-aggregative pathways but mainly via its scavenging properties.


Introduction
Alzheimer's disease (AD) is one of the most common form of dementia worldwide. Although the underlying causes of AD are still debated, two pathological hallmarks have been identified: senile plaques (SPs) and neurofibrillary tangles (NTFs). The latter are formed by hyperphosphorilation and abnormal deposition of tau (t) protein. SPs consist of deposits of b-amyloid protein (Ab) mainly. Ab derives from proteolitical cleavage of the amyloid precursor protein (APP) by three enzymes: a-, band c-secretase. When APP is metabolized by band c-secretase, Ab 1-40 and the more toxic form Ab 1-42 are produced; a phenomenon that is known as the ''amyloidogenic pathway'' [1]. An imbalance between production and clearance of these aggregative prone peptides triggers the formation of SPs [2]. Even though SPs are the most evident AD hallmark, recent reports highlight that Ab oligomers, because of their potent synaptotoxicity, play a crucial role in AD onset and development [3][4][5].
This scenario is further complicated by a huge amount of variables that can influence Ab aggregation pathway and toxicity, such as the dyshomeostasis of brain metal ions [6][7][8]. As a matter of fact brain metal dismetabolism has been widely demonstrated in AD patients and it has been proposed as a potential etiological co-factor [9][10][11]. Accordingly to this idea, metals accumulation in the elderly could be seen as a risk factor for AD onset and development. The unbalanced presence of metal ions in the brain can easily exacerbate the oxidative properties of Ab [12][13][14] and its toxicity [15,16].
A mechanism used by Ab, in the presence of metal ions, to exert its toxicity is the production of reactive oxygen species (ROSs).
Several natural compounds have been proposed to date to reduce the oxidative stress found in AD brains [17][18][19]. Among these compounds, resveratrol provoked great interest. Resveratrol is a natural polyphenol widely present in plants and in particular in the skin of red grapes and in wine; resveratrol antioxidant properties have been well demonstrated [20], with a wide range of biological effects [21], and fortunately, the compound is free of adverse effects [22]. In addition, recent papers underline its Ab anti-aggregative properties [23][24][25][26]. Despite all these positive effects, a major constraint holding back the use of resveratrol is its poor bioavailability when taken as dietary supplement [27].
The aim of this study is to test whether resveratrol might have anti-amyloidogenic and fibril-destabilizing properties, not only just against Ab but also against Ab-metal complexes and to assess whether the compound can act as a neuroprotectant. To that aim, we employed neuroblastoma cell cultures treated with Ab complexes in presence or absence of resveratrol.

Congo Red assay
Congo Red (CR) is largely used in histochemical studies to detect Ab fibril deposits. Accordingly to Nilsson (2004) [28], CR can be also used to investigate amyloid fibrillization in vitro. As shown in Fig. 1, with the exception of the Ab-Cu metal complex, resveratrol had no effect on the fibrillization of other Ab metal complexes. On the contrary, the presence of resveratrol seemed to enhance the propensity of the Ab-Cu complex to form fibrils. Strikingly, Ab-Al and Ab-Fe showed no or little propensity to aggregate compared with Ab alone and its complexes with Cu and Zn. These results are not surprising, as we have previously hypothesized, and recently largely confirmed, that Al is able to ''freeze'' Ab in its oligomeric state, stabilizing this assembly [29,30].

Turbidity measurements
To define a possible interaction between metal ions and resveratrol, turbidity measurements of resveratrol in the presence of metal ions (Al, Fe, Cu and Zn) were performed. An increase in absorbance value at 405 nm is indicative of the ability of resveratrol to form aggregates. Absorbance values over time are shown in Fig. 2. As reported in literature, resveratrol shows a strong propensity to form complexes with Cu in vitro [20]; in agreement with these data, solutions containing resveratrol and Cu showed a significant increase in absorbance. Our data also confirm the ability of resveratrol to form complexes with Fe and to a lesser extent with Al and Zn.

Cell viability assay
Before testing the effect of resveratrol on neuroblastoma cells exposed to Ab and or its metal complexes, a toxicity profile of the compound was performed and cell death evaluated by MTT assay. The resveratrol concentration needed to inhibit 50% (IC 50 ) of cell viability was 100 mM (Fig. 3A). Resveratrol at 15 mM proved to be largely non toxic and was thereby chosen for the experiments in which neuroblastoma cells were exposed to Ab, metal ions, or Ab-metal complexes.
As shown in Fig. 3B, resveratrol was not toxic when mixed with metals (15 mM for resveratrol and 5 mM for metal ions).
As largely demonstrated by this laboratory [15,30], Ab-Al was the most effective in reducing cell viability on neuroblastoma cells when compared with Ab alone or other Ab-metal complexes. In the presence of resveratrol there was a significant decline in cells mortality. Treatment with resveratrol resulted in significant neuroprotection against Ab and Ab-metal induced toxicity. Resveratrol drastically reduced the toxicity triggered by Ab-Fe and Ab-Zn (p,0.01), while seemed less effective on Ab, Ab-Al and Ab-Cu-induced cell death (p,0.05; Fig. 3C). These results have some limitations due to the use of a trasformed cell line (neuroblastoma, SH-SY5Y). Despite this, data obtained could be helpful to fix the basis for a follow up study.

TEM
With TEM investigated and compared the morphology of Ab and the Ab metal aggregates with resveratrol after 24 hours of incubation. The results are consistent with the CR assay. Ab retained the ability to form mature protofibrils in the presence of resveratrol (Fig. 4A), likewise Ab-Al retained its oligomeric structure (Fig. 4B). It is worth noting that the CR assay confirmed what we have previously shown with other biophysical techniques (ThT fluorescence and TEM) [15,30]. Ab-Cu and Ab-Zn formed unstructured aggregates both in the presence and absence of resveratrol ( Fig. 4D and 4E respectively). Unlike the results obtained during the CR assay, Ab-Fe showed the propensity to form unstructured fibrils similar to those found in the case of Ab-Cu and Ab-Zn (Fig. 4C).

SOD assay
Finally, we tested the effect of Ab and Ab-metal complexes on SOD activity in the presence or absence of resveratrol. 24 h treatments with Ab-Fe, Ab-Cu and Ab-Zn caused a significant increase in SOD activity (p,0.05), while Ab and Ab-Al showed negligible effects, even though Ab-Al complex seemed to reduce SOD activity but not in a statistically significant manner. Resveratrol was able to revert this process as we observed a decrease in SOD activity in samples containing Ab-Fe, Ab-Cu or Ab-Zn, suggesting that the compound has anti-oxidant properties. As for Ab and Ab-Al we did not observe significant changes in SOD expression in the presence of resveratrol (Fig. 5B). SOD activity was also tested after treating neuroblastoma cells with metal ions alone (with or without resveratrol), to rule out that the SOD increase was merely due to the presence of transition metals. A large excess of Al, Fe, Cu and Zn ions was used (5 mM) in these control experiments and we did not observed significant and reproducible changes (data not shown).

Discussion
It has been reported that resveratrol can extend the lifespan in several organisms [31][32][33] and therefore the compound has gathered great interest as anti-aging molecule.
In our study, it is demonstrated how resveratrol can reduce the toxicity in neuroblastoma cells exposed to either Ab or Ab-metal complexes. We chose Ab-metal complexes because metal ions (such as Al, Cu, Fe and Zn) greatly potentiate Ab aggregation as well as its intrinsic toxicity [15,30].
Several papers have highlighted that resveratrol can be a potent anti-amyloidogenic and fibril-destabilizing polyphenol [23][24][25][26]. In our opinion this neuroprotective mechanism of action is unsatisfactory for two reasons: 1) in accordance with the observations reported by Hudson et al. [34] resveratrol biases the Thioflavin T fluorescence assay for amyloid fibril detection through nonspectral interferences. 2) In the context of AD, anti-aggregative drugs might exert more harm than as amyloid oligomers are more toxic than fibrils [35,36]. We performed our CR assay to detect the presence of amyloid fibrils in the presence of resveratrol and found (Fig. 1), that the compound does not influence Ab-metal complexes aggregative pathway, except for Ab-Cu where we observed an increase in fibrillization. One possible explanation could be that resveratrol stabilizes Ab-Cu complex in more ordered structures because of its Cu chelating properties (Fig. 2). In agreement with the CR assay, TEM micrographs do not show an anti-amyloidogenic effect of resveratrol.
After excluding an anti-amyloidogenic activity observed during our experimental conditions (see different Ab procedure preparation in [26]), we set to investigate any potential antioxidant  properties. It has been widely demonstrated that the AD brain shows damages caused by ROS [37][38][39]. In AD, ROS are the byproduct of several pathological events, including the production of hydrogen peroxide by Ab [40][41][42] and the accumulation of transition metals (such as Fe 3+ , Cu 2+ and Zn 2+ ) [43][44][45]. Ab-metals complexes cause the coexistence of these two by-products, exacerbating damages due to cellular oxidative stress. In accordance with this hypothesis, we observed an increase in super-oxide dismutase (SOD) activity in neuroblastoma cells treated with Ab-Fe, Ab-Cu and Ab-Zn compared with non treated cells (Fig. 5B). SOD (both as SOD1 and SOD2) act as an antioxidant, protecting cells from being damaged by free radical species [46]; however, their increased level is an indication of cellular oxidative stress due to ROS overproduction. Neuroblastoma cells treated simultaneously with Ab-metal complexes and resveratrol showed negligible differences in SOD activity when compared with relevant controls. Altogether these data suggest that Ab-Fe and Ab-Zn complexes exerted their toxicity mainly through oxidative stress; while Ab-Cu seemed to exert its toxicity through different pathways; in fact this complex was still toxic, even in the presence of resveratrol.
Ab and especially Ab-Al resulted significantly toxic on neuroblastoma cells, a phenomenon that occurred without increasing SOD activity, suggesting that Ab and Ab-Al were not directly involved in free radical species production in our experimental conditions. In this connection, Al 3+ is neither a redox metal nor involved in oxidative stress processes [47][48][49], meanwhile Ab, not complexed with metals, is involved in of H 2 O 2 production but it seems not involved in that of free radical species [40]. Diversely, Ab and Al capacity to produce free radicals is linked exclusively to the presence of transition metals such as Fe and Cu [9,41,45,47,50,51]. Nevertheless, resveratrol promoted neuroprotection also against Ab and Ab-Al-mediated toxicity suggesting that it can act through alternative mechanisms that do not require SOD activity. It is noteworthy to point out that resveratrol plays its neuroprotective role through several activities including: activation of protein kinase C, reduction of malondialdehyde levels, blocking of COX-2 expression, reduction of neuroinflammatory responses and scavenging [20,25,[52][53][54].
Despite these chemopreventive properties, Ab-Al and Ab-Cu retained toxic activity on neuroblastoma cells even after treatment with resveratrol; this suggests that these two Ab-metal complexes exert their toxicity through mechanisms that cannot be prevented by resveratrol. These findings are in agreement with previous data from our lab indicating that Ab-Al can damage cell membranes because of its high superficial hydrophobicity [16]. Similarly, Ab-Cu exerts its toxicity through mechanisms that are not only oxidative stress dependent [55,56].
Collectively, our findings indicate that : 1) in our experimental conditions we did not observe any anti-amyloidogenic and fibrildestibilizing effect played by resveratrol, as proposed by other groups [23][24][25][26]54,57]; 2) resveratrol exerts its neuroprotective activity not only against Ab but also against Ab-metal complexes; 3) resveratrol acts as a ROS scavenger against those generated by Ab-Fe, Ab-Cu and Ab-Zn, thereby reducing their toxicity; and 4) eventually, resveratrol is not sufficient to fully block Ab-Al and Ab-Cu toxicity.

Preparation of Ab-metal complexes
Human Ab was solved in hexafluorisopropanol (HFIP) for 40 min at room temperature. HFIP was removed under vacuum in a Speed Vac (Sc110 Savant Instruments). This treatment was repeated three times (modified protocol from [58]). The Ab metal complexes were prepared by 24-h dialysis against metal solutions 10 mM ([CH 3 CH(OH)COO] 3 Al, FeCl 3 , CuCl 2 , ZnCl 2 ) at T = 4uC using Spectra/PorH Float-A-LyserH tubes (Spectrum Labs) with 100 Molecular Weight Cut Offs (MWCO). Then, Ab metal complexes were dialyzed against bidistilled water (three water changes, pH = 7) for 24 h to remove the excess of metals. The same treatment was also performed with Ab alone. Aliquotes of Ab, Ab-metal complexes were stored at 220uC until used.

Congo Red spectroscopy assay
Congo Red (CR) spectroscopic assay was performed in agreement with Nilsson's protocol (2004) using a 300 mL 96-well plate with Ubottom [28]. Kinetic was followed for 24 h by monitoring the changes in absorbance at 487 nm using a Microplate SPECTRAmaxH reader. The increase in absorbance at this wavelength is indicative of amyloid fibrils formation. The final protein concentration in each well was 1,5 mM, while resveratrol concentration was 45 mM (concentration ratio protein-resveratrol was 1:30). Resveratrol was dissolved in absolute ethanol (final concentration 100 mM) and further diluited as needed. The final ethanol concentration in wells was 2% (v/v). This concentration of ethanol in solution did not change Ab and Ab-metal complexes aggregation kinetics (data not shown). The signals due to the buffer alone was subtracted.
The absorbance of a solution containing resveratrol, metal ions (Al, Fe, Cu and Zn) and CR dye was measured at two wavelengths: 405 nm and 487 nm, to exclude potential artifactual cross-interactions. The first wavelength to exclude the formation of precipitates (turbidity assay), the second to exclude the capability of resveratrol and metal ions to coordinate CR. Concentrations in each well were: as for CR and resveratrol the same used in Ab fibrils detection (70 mg/ml and 15 mM respectively), while for metal ions 3 mM. Results obtained allow us to state that resveratrol and metal ions did not seem to bias CR spectroscopic activity (data not shown).

Turbidity measurements
Turbidity assay was performed using a 300 mL 96-well plate with flat bottom. Absorbance at 405 nm was read using a Microplate SPECTRAmaxH reader. The concentrations in the wells were the following: resveratrol 200 mM, metals (Al, Fe, Cu, Zn) 400 mM, ethanol 2%. The absorbance due only to metallic solutions was subtracted. Resveratrol only kinetics is reported to exclude possible hydrophobic interaction between the molecules in solution causing precipitation; resveratrol is sparingly soluble in water (solubility 0.03 g/L). Data reported are not biased by spectroscopic interferences due to resveratrol, its UV spectrum (not shown) shows a maximum at 308 nm [20], while all turbidity measurements were carried out at 405 nm.

Transmission Electron Microscopy (TEM)
All samples at 10 mM protein concentration, after an incubation period of 24 h, were absorbed onto glow-discharged carboncoated butwar films on 400-mesh copper grids. The grids were negatively stained with 1% uranyl acetate and observed at 40,0006 by transmission electron microscopy (TEM) (Tecnai G2, FEI). The samples observed contained Ab and its metal complexes with resveratrol 300 mM in 2% v/v of absolute ethanol.

Neuroblastoma Cells
SH-SY5Y human neuroblastoma cells were purchased from ECACC (European Collection of Cell Culture, Salisbury, UK). The medium in which they were cultured contained DMEM/F12 (Gibco, Carlsbad,CA USA) with 15% (v/v) fetal bovine serum (FBS, Sigma-Aldrich, St. Loius, MO), 100 units/ml penicillin and 100 mg/ml streptomycin (Gibco, Carlsbad, CA USA) and 1% (v/ v) MEM non essential amino acid (NEAA) (Sigma-Aldrich, St. Loius, MO). Cells were stored at 37uC with 5% CO 2 in a humidified atmosphere (90% humidity). Cells were used until passage 25 for both the MTT assay and the SOD assay. The culture medium was replaced every two days.

Cell Viability Assay
Cell viability was determined through MTT reduction assay. SH-SY5Y cells were seeded into 24-well plates at a density of 15610 4 cells per well in 1 ml culture medium. 15% FBS-culture medium containing: Ab, Ab-metal complexes (0.5 mM) with or without resveratrol (15 mM) was added to the cells for 24 hours. Resveratrol was dissolved in absolute ethanol, the final ethanol concentration in the medium was 0,2% (v/v). This ethanol concentration resulted largely non-toxic (data not shown). 100 mL of 5 mg/ml MTT was added to each well and incubated in the dark at 37uC for 3 hours. Then cells were lysed with 1 ml of acidic isopropanol (0.04 M HCl in absolute isopropanol) [59]. Color intensity was measured with a 96-well ELISA plate reader at 550 nm (Microplate SPECTRAmaxH). Toxicity due to metals alone (5 mM) in the presence and in the absence of resveratrol (15 mM) was also tested. All MTT essays were performed three times, in triplicate. Viability was defined as the relative absorbance of treated vs. untreated, expressed as a percentage.

SOD assay
Total cellular superoxide dismutase (SOD) activity was determined with a SOD assay kit (Sigma-Aldrich) by following manufacturer's protocol. 1.0610 6 neuroblastoma cells were seeded into 25 cm 2 flasks. Cells were grown at 80% confluency, then cells were treated with Ab and Ab-metal complexes (0.5 mM in the medium) in the presence and in the absence of resveratrol (15 mM in the medium). After 24 h cells were scraped, washed three times in cold PBS buffer and then disrupted using Cell Extraction Buffer (Invitrogen) containing 1 mM PMSF and 16 protease inhibitor cocktail (Sigma-Aldrich). Whole cell lysates of SH-SY5Y was centrifuged at 10,000 rpm (4uC, 10 minutes). Supernatant was transferred into 0.5 mL Eppendorf tube. Obtained solutions were used to determine protein concentration and SOD activity.

Statistical Analysis
Congo Red spectroscopy, MTT, turbidity and SOD assays were statistically analyzed by Student's t test and one-way analysis of variance. Results were reported as highly statistically significant if P,0,01 and statistically significant if P,0,05. Results are presented as mean 6 standard deviation (SD).