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Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H2O2). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H2O2. Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H2O2, and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.
Although oxidative stress is commonly associated with aging (1, 2), patients with Alzheimer disease (AD)3 often exhibit increased oxidative damage (3,–10) and subsequent neuronal loss in β-amyloid (Aβ)-rich regions of the brain. The molecular mechanisms by which Aβ contributes to oxidative damage remain unclear (11,–19). Understanding these mechanisms, however, is critical for developing effective methods to manage the disease. One mechanism for Aβ-induced cellular oxidative stress proposes that Aβ peptides interact directly with cellular enzymes responsible for maintaining low physiological levels of reactive oxygen species (ROS) (20,–23). Two potential outcomes from such pathological protein-amyloid interactions are: 1) increased production of ROS, or 2) reduced degradation of ROS.
The major ROS in cells are superoxide and the more reactive hydrogen peroxide (H2O2)-derived hydroxyl radical (24, 25). Both superoxide and H2O2 are primarily produced in the mitochondria (26,–28). Aβ peptides have been shown to accumulate in the mitochondria (29, 30), and, therefore, could exert their detrimental effects through interaction with mitochondrial proteins (23, 31,–34). Superoxide is produced by several enzyme-catalyzed reactions in the mitochondria (25). Behl et al. (11) however, showed that a broad range of inhibitors of several of these enzymes had no effect on Αβ toxicity in clonal and primary neuronal cell cultures. Furthermore, Zhang et al. (35) reported that superoxide levels in cells were not substantially elevated upon exposure to Αβ. These findings suggest that superoxide is not a dominant contributor to Αβ toxicity.
On the other hand, Αβ-induced cellular increase in H2O2 or its metabolites is strongly correlated with Αβ toxicity (11, 13, 36). H2O2 can be generated by several mitochondrial enzymes including monoamine oxidases, superoxide dismutase, and xanthine oxidase (25). Behl et al. (11) showed that inhibitors of monoamine oxidases and xanthine oxidase had no effect on Αβ-induced H2O2 accumulation or Αβ toxicity. Gsell et al. (37) also found that the activity of superoxide dismutase was unaltered in the brains of AD patients. Additionally, Rensink et al. (38) reported that the Dutch mutation of Αβ peptides (HCHWA-D Αβ) did not bind directly to superoxide dismutase and Kaminsky et al. (39) reported only a relatively small effect of Αβ on superoxide dismutase activity and H2O2 production upon chronic exposure of rat brains to Αβ. These results suggest that Αβ peptides do not significantly affect production of H2O2 in cells. Consequently, these findings imply that Αβ-induced oxidative stress may arise from reduced degradation of H2O2 in Αβ-challenged cells.
Degradation of H2O2 in cells is primarily achieved by the enzymes catalase and glutathione peroxidase (GPx), both inside and outside of the mitochondria (25). Sagara et al. (40) found that cells resistant to Αβ toxicity had elevated levels of catalase and GPx. The activity of both enzymes was reduced in rat brains exposed to Αβ. In particular, Pappolla et al. and Lovell et al. found that catalase was associated with senile plaques in human brain sections from patients with AD (6, 41). Several groups have shown that transfection of cells with catalase (40) or addition of catalase to the extracellular environment of cultured cells protected cells from Αβ toxicity (11, 38). This cytoprotective effect was attributed to a catalase-induced reduction of the H2O2 concentration outside and inside the cells, because H2O2 readily diffuses across cell membranes (25, 43). These observations are consistent with the hypothesis that H2O2, or its metabolites, but not superoxide, play a dominant role in Αβ-induced oxidative stress. Moreover, Milton et al. (21) and our previous work (55) revealed that Αβ binds directly to catalase in cell free assays, whereas Αβ does not bind to GPx (supplemental Fig. S12). The binding of Αβ to catalase leads to deactivation of the H2O2-degrading activity of catalase in solution (21). Evidence for direct catalase-amyloid interactions and their resulting detrimental effects within living cells, however, has not yet been reported. Testing the hypothesis that intracellular catalase-Αβ interactions play a significant role in Αβ-induced increases in cellular H2O2 may, therefore, provide a critical and missing mechanistic link between Αβ accumulation and oxidative stress in AD.
Here, we investigated the effect of catalase-amyloid interactions on Aβ-induced increases in H2O2 and on the resulting toxicity in live cells. In these studies, we exposed human neuroblastoma cells to a mixture of aggregated Aβ(1–42) peptides (containing soluble oligomers and protofibrils) to mimic the heterogeneous nature of aggregated Aβ species found in the brains of AD patients (44,–46). Because the identity of the most toxic aggregated form of Aβ remains highly debated (47,–53), we chose to use this mixture of different species of aggregated Aβ in these studies in order to increase the likelihood that as many toxic species as possible were included in all of our experiments. To assess whether catalase-amyloid interactions lead to Αβ-induced increases in H2O2 in cells, we designed and synthesized molecular probes with the following six characteristics: 1) capability of generating protein-resistant surface coatings on aggregated Αβ peptides (54,–56) (to inhibit catalase-amyloid interactions in cells), 2) lack of toxicity, 3) cell permeability, 4) capability of accessing the same subcellular compartments of cells as Αβ, 5) intrinsic fluorescence properties (to visualize the intracellular location of the molecules), and 6) chemical stability in oxidizing environments. We achieved these characteristics with two oligo(ethylene glycol) derivatives of 6-methylbenzothiazole aniline (BTA-EG4 and BTA-EG6) (55); BTA analogs have well-known, high affinity binding properties to both Aβ oligomers and fibrils (57, 58). These molecules made it possible to challenge neuroblastoma cells with Aβ and to evaluate the effects of coating aggregated Aβ peptides with a protein-resistant surface on the viability of cells.
A detailed list of materials used in this research can be found in the supplemental information.
Aggregated Aβ(1–42) was prepared by incubation in deionized water (100 μm) at 37 °C for 72 h and characterized as described in the supporting information. For cellular experiments using aggregated Aβ, a stock solution of aggregated Aβ prepared in sterile water was diluted with an equivalent volume of 2× complete culture medium to create the desired concentration of aggregated Aβ dissolved in 1× complete medium. For cellular experiments using aggregated Aβ in the presence of the BTA-EGx molecules, aggregated Αβ (in sterile water) was preincubated for 12 h with various concentrations of BTA-EG4 or BTA-EG6, followed by addition of an equal volume of 2× medium to create the desired solution of aggregated Aβ and BTA-EGx dissolved in 1× medium.
SH-SY5Y cells were plated at a density of 50,000 cells/well in 100 μl of complete medium consisting of a 1:1 mixture of Eagle's Minimum Essential Medium (EMEM) and Ham's F12, supplemented with 10% fetal bovine serum (FBS). Cells were incubated overnight before treatment with 100 μl of various sample solutions. Cells were exposed to solutions containing a final concentration of 25 μm Aβ and various concentrations of the BTA-EGx molecules (0–40 μm) for 24 h at 37 °C. The MTT reagent (20 μl of the solution from the commercial kit) was added, and the cells were incubated for 3 additional hours. The cells were subsequently solubilized with detergent reagent (100 μl of the solution from the commercial kit) and incubated at room temperature overnight. The cell viability was determined by measuring the absorbance at 570 nm using a Spectramax 190 microplate reader (Molecular Devices). All results were expressed as percent reduction of MTT relative to untreated controls (defined as 100% viability), and the average absorbance value for each treatment was blanked with the absorbance reading of wells containing only medium, MTT reagent, and detergent reagent.
Cells were plated in 50 μl of Dulbecco's modified Eagle's medium (DMEM) without phenol red, supplemented with 10% FBS and 4 mm l-glutamine. Cells were incubated overnight before dosing with sample solutions. The solutions of Aβ aggregates in the presence or in the absence of the BTA-EGx molecules were incubated with the cells for 24 h. Cellular H2O2 release was determined by adding 20 μl/well of a solution containing 250 μm Amplex red reagent and 0.5 units/ml horseradish peroxidase (HRP) dissolved in complete medium. The absorbance at 560 nm was subsequently measured 30 min after addition of the Amplex red/HRP solution (59). For control experiments, cells were incubated for 24 h with 40 μm BTA-EGx, 20 mm 3AT, or 20 mm 3AT with 40 μm BTA-EGx, followed by measurement of H2O2 released. All absorbance measurements for each treatment were measured in triplicate and the average absorbance values of control wells containing medium alone were subtracted.
SH-SY5Y cells were cultured on poly-l-lysine coated glass cover slips and incubated overnight in a 1:1 mixture of EMEM and Ham's F12 supplemented with 10% FBS. The growth medium was removed and solutions of aggregated fluorescently labeled Aβ (5 μm dissolved in medium), or fluorescently labeled Aβ (5 μm) that had been preincubated with BTA-EGx (40 μm), were added to the different glass cover slips. The cells were incubated for 12 h. To visualize co-localization of aggregated Aβ with catalase, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) and permeabilized with 0.25% Triton-X in PBS, followed by incubation with a rabbit anti-catalase polyclonal antibody on a shaker at 4 °C overnight. To detect the primary antibody, a fluorescently labeled goat anti-rabbit antibody (diluted 1:1000) was added to the glass slides and incubated in the dark, on a shaker at room temperature for 2 h. Coverslips were mounted using Hydromount mounting medium (product no. HS-106) from National Diagnostics (Atlanta, GA) according to the manufacturers guidelines and allowed to dry for 48 h in the dark at 4 °C before imaging with a Delta Vision Deconvolution Microscope System (Applied Precision, Issaquah, WA) equipped with a Nikon TE-200 inverted light microscope, with infinity corrected lenses, and with a mercury arc lamp as the illumination source. The fluorescence of the HiLyte Fluor 488-labeled Aβ (Abs/Em: 503/528 nm) was detected using an Ex/bp: 490/20 nm excitation filter and an Em/bp: 528/38 nm emission filter. The fluorescence of the Alexa Fluor 647 goat anti-rabbit antibody (Abs/Em: 650/668) was monitored using an Ex/bp: 640/20 nm excitation filter and an Em/bp: 685/40 nm emission filter. The co-localization was visualized and Pearson's correlation coefficient of the entire three-dimensional volume of the cell was determined using softWoRx image analysis software (Applied Precision, Issaquah, WA). The images shown in Fig. 3 are fluorescence micrographs of representative z-slices within cells.
SH-SY5Y cells were grown to confluence on 10-cm tissue culture-treated dishes. The medium was removed, and solutions of aggregated Aβ(1–42) (25 μm, in medium), Aβ (25 μm) that had been preincubated with BTA-EGx (40 μm) or fresh medium were added to the different dishes and incubated overnight. The solutions were removed, and the cells were carefully washed three times with ice-cold PBS. The cells were incubated with 1 ml of ice-cold lysis buffer (from Cell Signaling Technology, Inc, Danvers, MA, Product No: 9803) containing a protease inhibitor mixture (Sigma-Aldrich, Product No: P2714) and 1 mm phenylmethylsulfonyl fluoride and placed on a shaker for 30 min at 4 °C. The cell lysate was collected, centrifuged, and the supernatant of the sample was precleared with 50 μl of protein G-agarose beads (Roche Diagnostics Co., Indianapolis, IN, Product No: 11719416001) to remove proteins that bind nonspecifically to protein G. The precleared cell lysate was subjected to immunoprecipitation with a goat polyclonal to catalase antibody (Abcam Inc., Cambridge, MA, Product No: ab50434) and protein G-agarose beads were used to extract the anti-catalase antibody and catalase from the lysate. The samples were subjected to 4–12% SDS-polyacrylamide gel electrophoresis. Catalase (from human erythrocytes) and synthetic aggregated Aβ(1–42) were also separated on the same gel as the immunoprecipitated proteins for comparison purposes. Western blot analyses using rabbit polyclonal anti-catalase antibody (Abcam Inc., Product No: ab1877) and 6E10 monoclonal anti-Aβ(1–16) antibody (Covance) were performed. Chemiluminescence was used to detect the proteins.
SH-SY5Y cells were plated in DMEM without phenol red (supplemented with 10% FBS and 4 mm l-glutamine) on 35 mm glass bottom dishes (MatTek Co., Ashland, MA) and incubated overnight. The growth medium was removed, and solutions of pre-aggregated fluorescently labeled Aβ (5 μm dissolved in medium) with or without BTA-EGx (40 μm dissolved in medium) were added to the cells and allowed to incubate for 12 h before imaging. The cells were washed with DMEM (without phenol red or FBS) immediately prior to imaging with a Delta Vision Deconvolution Microscope System (Applied Precision, Issaquah, WA) equipped with a Nikon TE-200 inverted light microscope, with infinity corrected lenses, and with a mercury arc lamp as the illumination source. The co-localization was determined using softWoRx image analysis software (Applied Precision, Issaquah, WA). The intrinsic fluorescence of the BTA-EGx molecules (Abs/Em: 360/440 nm (60)) was observed by excitation with bandpass (bp) filtered light (Ex/bp: 360/40 nm) and the emission monitored at Em/bp: 457/50 nm. The fluorescence of the HiLyte Fluor 488-labeled Aβ (Abs/Em: 503/528 nm) was detected using an Ex/bp: 490/20 nm excitation filter and an Em/bp: 528/38 nm emission filter. The images shown in Fig. 5 are fluorescence micrographs of representative z-slices within cells.
SH-SY5Y cells were grown to confluence on 35 mm tissue culture-treated dishes. The medium was removed, and solutions of aggregated fluorescently labeled Aβ (5 μm dissolved in medium), fluorescently labeled Aβ (5 μm) that had been preincubated with BTA-EGx (40 μm), or fresh medium only were added to different dishes, and incubated overnight. The solutions were removed, and the cells were carefully washed with PBS. The cells were dissociated from the tissue culture dishes with solutions of 0.25% trypsin-EDTA, collected, and counted. The dead cells in each sample were stained using Live/Dead fixable far red stain (Invitrogen Co., Product No: L10120) according to the manufacturer's protocol, and all cell samples were fixed with 4% paraformaldehyde in PBS (pH 7.4). The flow cytometry data were acquired using a FACSCanto flow cytometer (Becton Dickinson, San Jose, CA) equipped with an argon 488 nm laser and a 530/30 band-pass filter to analyze the fluorescently labeled Aβ as well as a helium-neon 633 nm laser and a 660/20 band-pass filter to analyze the far red dye. FACSDiva software (Becton Dickinson) was used to control the setup, acquisition, and analysis of flow cytometry data from the FACSCanto flow cytometer. The samples were gated to only include single cell populations, excluding larger cell aggregates and cellular debris. This population contained more than 95% viable cells (>20,000 cells/sample). The live cells were analyzed for the presence of fluorescently labeled Aβ, and the cellular fluorescence from this cell population was presented as histograms.
The wells of 96-well plates were charged with 48-μl solutions containing final concentrations of 25 μm aggregated Aβ (in water) and 0–100 μm BTA-EGx (in 1% DMSO/1% BSA in 50 mm sodium phosphate buffer). After shaking for 12 h, 15 μl of a catalase solution was added to give a final concentration of 25 nm catalase. The mixture was placed on a shaker for 2 additional hours. The activity of catalase was determined by adding 15 μl of a solution containing 5 μm HRP, 435 μm 4-aminoantipyrine (AP) and 435 μm 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) (HRP, AP, and DHBS collectively are hereby defined as the colorimetric reagents) to the wells, immediately followed by 15 μl of 3 mm H2O2 (in 50 mm sodium phosphate buffer, pH 7.4). Catalase activity was monitored 10 min after the addition of H2O2 by measuring the absorbance at 505 nm (61, 62). The average absorbance values for each concentration of BTA-EGx were subtracted with the absorbance value of blanks consisting of 25 μm Aβ, the corresponding concentration of the BTA-EGx molecules, 25 nm catalase, the colorimetric reagents, and 50 mm sodium phosphate buffer (instead of a solution of H2O2). The results were expressed relative to absorbance values measured when catalase was not exposed to Aβ (defined as 100% catalase activity), and the absorbance measured when the assay was run in the absence of catalase (defined as 0% catalase activity).
We hypothesized that small molecules (e.g. BTA-EGx, Fig. 1A) with the capability of forming protein-resistant surface coatings on aggregated Aβ peptides (55) could protect cells from Aβ toxicity. To test this hypothesis, we determined the viability (63, 64) of human SH-SY5Y neuroblastoma cells after exposure to aggregated Aβ peptides in the presence or in the absence of these small molecules. For these experiments, we mimicked the heterogeneous population of aggregated Aβ species in AD brains (44,–46) by using a preparation of Aβ peptides that contained ~15% small oligomers (MW ~15 kDa corresponding to trimers), ~25% medium-sized oligomers (MW 20–65 kDa corresponding to 5–15 mers), and ~60% soluble protofibrils (MW > 150 kDa corresponding to > 30 mers) (see supplemental Fig. S1). Fig. 1B shows that exposure of cells to 25 μm concentrations of this Aβ preparation reduced their viability to ~27% compared with control cells that were cultured in the absence of Aβ.
Before exploring the effect of the BTA-EGx molecules on reducing the cytotoxicity of Aβ, we tested the tolerance of cells to these BTA-EGx molecules by themselves. These experiments revealed that the BTA-EGx molecules were not toxic at concentrations up to 40 μm4 (Fig. 1B and supplemental Fig. S2). Remarkably, the BTA-EGx molecules were, however, capable of reducing the toxicity of aggregated Aβ, causing an increase in cell viability to ~72% (Fig. 1C).5 Because we used a mixture of aggregated Aβ species in these experiments, the results shown in Fig. 1C suggest that the BTA-EGx molecules could simultaneously protect cells from several potentially toxic forms of aggregated Aβ peptides that are present in this mixture (47,–53). In all of the work shown here, we describe results using both of the structurally related BTA-EG4 and BTA-EG6 molecules because testing two molecules provides 2-fold evidence that this class of molecules can protect cells from Aβ-related injury.
To assess whether the BTA-EGx molecules can minimize Aβ-induced increases in cellular H2O2 levels, we measured the amount of H2O2 released by SH-SY5Y cells in the presence of aggregated Aβ peptides. Fig. 2A demonstrates that cells incubated with 25 μm Aβ for 1 day released 3-fold more H2O2 than control cells that were not exposed to Aβ. Moreover, Fig. 2B shows that concentrations of BTA-EGx above 10 μm significantly inhibited Aβ-induced elevations in H2O2 levels in these cells. The presence of the BTA-EGx molecules together with Aβ resulted in low H2O2 levels that were indistinguishable from control cells. Additional control experiments showed that 40 μm solutions of the BTA-EGx molecules by themselves (i.e. in the absence of Aβ) did not affect cellular H2O2 levels compared with cells that were not exposed to these small molecules (Fig. 2A).
To evaluate if the BTA-EGx molecules exerted their action by either specifically inhibiting Aβ-induced increases in cellular H2O2 levels or by reacting chemically with H2O2 or other ROS, we inhibited catalase with a known inhibitor (3-amino-1,2,4-triazole, 3AT (25, 65)). We expected this treatment to increase cellular levels of H2O2. Fig. 2A shows that a 20 mm solution of 3AT caused a 3-fold increase in H2O2 released by cells compared with untreated control cells. This increase in H2O2 levels was not affected by incubating cells with 3AT in the presence of 40 μm BTA-EGx (Fig. 2A), demonstrating that the anti-oxidant properties of the BTA-EGx molecules are specific to Aβ-mediated oxidative stress. Mass spectrometry analysis provided additional evidence that the BTA-EGx molecules did not react directly with H2O2 in solution (see supplemental information). The BTA-EGx molecules, therefore, exerted their activity by a mechanism that is different from a chemical anti-oxidant mechanism.
To test the hypothesis that intracellular protein-amyloid interactions contribute to Aβ-induced increases in H2O2 levels and to Aβ toxicity in live cells, we exposed SH-SY5Y cells to a solution containing N-terminal, fluorescently labeled Αβ peptides. This experiment made it possible to determine the location of these peptides inside of cells. Fig. 3, A–D and supplemental Figs. S3 and S4 illustrate that aggregated Aβ(1–42) peptides readily enter live cells and distribute to multiple subcellular locations such as the lysosomes (supplemental Fig. S3) and mitochondria (supplemental Fig. S4). Because catalase is the primary enzyme responsible for degradation of H2O2 in cells (25, 71), we investigated whether the interaction of Aβ peptides with intracellular catalase (72) could contribute to the observed Aβ-induced increase in H2O2 levels. Fig. 3, A–D and supplemental Fig. S5 show that a significant fraction of the aggregated Aβ(1–42) peptides co-localized with catalase in the peroxisomes and in the cytosol; this observation has not been reported previously in live cells (30, 33, 66,–70). Fig. 3 also shows that BTA-EG4 (Fig. 3H) and BTA-EG6 (Fig. 3L) significantly reduced the intracellular co-localization of Aβ and catalase. Although Fig. 3 shows only representative z-slice fluorescence micrographs of these cells, we quantified the degree of co-localization of Aβ and catalase within the entire three-dimensional volume of cells with the Pearson's correlation coefficient (73). Before addition of the BTA-EGx molecules, this coefficient was 0.46, whereas it was 0.10 or 0.29 when cells were introduced to Aβ together with BTA-EG4 or BTA-EG6, respectively. The molecular association between Aβ(1–42) peptides and catalase was further supported by exposing SH-SY5Y cells to aggregated Aβ(1–42) peptides and immunoprecipitating catalase with an anti-catalase antibody. Western blot analysis revealed a protein band of ~4.5 kDa in the lysates (corresponding to the MW of Aβ monomers), which was immunoreactive to an anti-Aβ antibody and co-immunoprecipitated with catalase (one subunit ~60 kDa) (Fig. 4). This ~4.5 kDa band was not observed in the Western blots from control cells that were not treated with Aβ. Additionally, immunoprecipitation of catalase in the presence of the BTA-EGx molecules reduced the co-immunoprecipitation of Aβ peptides with the catalase from the cell lysate (Fig. 4). Interestingly, the fluorescence microscopy experiments and the co-immunoprecipitation experiments revealed a consistent trend for the slightly better performance of BTA-EG4 for reducing the interaction of Aβ with catalase in cells compared with the BTA-EG6. We attribute this apparent enhanced performance of BTA-EG4 to its increased lipophilicity compared with BTA-EG6 (55), which may facilitate internalization in cells.
As a control for the fluorescence microscopy co-localization experiment, we also investigated the possible intracellular interaction of aggregated Aβ peptides with superoxide dismutase (SOD). Supplemental Fig. S6 shows results from fluorescence confocal imaging experiments revealing a reduced degree of overlap of aggregated Aβ and SOD throughout the entire volume of the cell (Pearson's coefficient = 0.27) relative to the overlap of Aβ and catalase (Pearson's coefficient = 0.46) under the same conditions. Additionally, the presence of the BTA-EGx molecules did not affect the degree of overlap of Aβ and SOD (Pearson's coefficient = 0.30 for both BTA-EGx molecules) compared with cells that were treated with Aβ alone (supplemental Fig. S6). Finally, we exposed SH-SY5Y cells to aggregated Aβ(1–42) peptides and immunoprecipitated SOD with an anti-SOD antibody. These experiments reveal that SOD does not appear to co-immunoprecipitate with Aβ (supplemental Fig. S7), which is in contrast to the observed co-immunoprecipitation of Aβ with catalase under the same conditions (Fig. 4). These results support that Aβ and SOD do not likely interact significantly within cells.
Fig. 3 and supplemental Fig. S5 clearly illustrates that the BTA-EGx molecules are capable of reducing the co-localization of catalase and Aβ in cells. To test if this effect could be due to the interaction of the BTA-EGx molecules with Aβ, we determined whether the BTA-EGx molecules could also interact with Aβ in the cells. Fig. 5, A–F shows that the intrinsically fluorescent (60) BTA-EGx molecules distributed throughout the cells and can occupy overlapping subcellular regions of the cells that are also occupied by Αβ. Additionally, Fig. 5G shows results from flow cytometry experiments demonstrating that the BTA-EGx molecules (especially BTA-EG4) do not significantly affect the uptake of the aggregated fluorescently labeled Aβ peptides in the cells. These results are consistent with the possibility that the BTA-EGx molecules were capable of entering cells and could, hence, interact with intracellular pools of Aβ peptides. We hypothesize that this interaction between the BTA-EGx molecules and Aβ protected cells from harmful protein-amyloid interactions by forming protein-resistant molecular surface coatings on Aβ (55). Such a mechanism would explain the observed reduction in intracellular co-localization of catalase and Aβ in the presence of the BTA-EGx molecules. To test this hypothesis, we examined whether the BTA-EGx molecules protected the H2O2-degrading activity of catalase in Aβ-rich environments.
We reported previously that BTA-EG4 and BTA-EG6 could inhibit catalase-Aβ binding interactions in solution by forming protein-resistant surface coatings on aggregated Aβ(1–42) peptides (55). In order to determine whether these molecules could also protect the enzymatic activity of catalase in an Aβ-rich environment, we developed an assay for quantifying catalase activity (see supplemental Fig. S8). We designed this assay to be compatible with the presence of the BTA-EGx molecules and with the presence of aggregated Aβ peptides in solution (see supplemental Fig. S9). Fig. 6A shows that micromolar concentrations of Aβ suppressed catalase activity substantially, in agreement with previous reports (21). Importantly, Fig. 6, B and C shows that the presence of BTA-EGx molecules preserved 80–90% of catalase activity despite the presence of Aβ. The concentration of BTA-EGx molecule that resulted in half-maximal preservation of catalase activity was ~8 nm for BTA-EG4 and ~40 nm for BTA-EG6.6 These results are consistent with the hypothesis that the BTA-EGx molecules protect cells from Αβ-induced increases in H2O2 levels and from Αβ toxicity through a mechanism involving the formation of protein-resistant molecular coatings on aggregated Aβ.7
To consider another possible mechanism by which the BTA-EGx molecules could affect Aβ-catalase interactions, for instance, by affecting the aggregation state of Aβ, we performed dynamic light scattering experiments of Aβ preparations (74,–76). We carried out these experiments in the presence and in the absence of the BTA-EGx molecules and found that the aggregation states of Aβ were not altered significantly due to the presence of the BTA-EGx molecules (see supplemental Fig. S10). In contrast, incubation of aggregated Aβ peptides with vanillin, a molecule known to inhibit Aβ aggregation (77), resulted in a population of Aβ monomers.
Aβ has been implicated as a possible causative agent for oxidative stress in AD (9, 78). Proposed mechanisms for Aβ-induced oxidative damage in cells include: 1) Aβ peptides interact directly with anti-oxidant enzymes resulting in accumulation of ROS (20,–23); 2) Aβ peptides interact directly with cellular membranes resulting in uncontrolled calcium influx as a consequence of forming transient, ion channel-like pores (79,–87); the resulting disruption of calcium homeostasis can lead to mitrochondrial dysfunction, followed by increased H2O2 production and generation of high levels of ROS (86, 88); and 3) Aβ peptides interact directly with intracellular transition metal ions such as Fe2+ (89,–92), resulting in the catalytic reduction of O2 to H2O2 in cells (17, 18, 89).
Here, we developed a small molecule probe to investigate, in detail, the first mechanism, i.e. the specific contribution of protein-amyloid interactions toward Aβ-induced oxidative stress. The results presented here support that deactivation of catalase by intracellular catalase-amyloid interactions can lead to Aβ-induced increases in H2O2 levels and to subsequent toxicity in cells. We base this conclusion on the following three key observations. First, Aβ internalized in cells and co-localized with catalase. The results from fluorescence imaging experiments (Fig. 3 and supplemental Figs. S3–S6) demonstrate that exogenous aggregated Aβ(1–42) is capable of entering the cells through an endocytotic lysosomal pathway (67, 70, 93, 94) or a non-endocytotic pathway (95). After the initial uptake of Aβ, the Aβ peptides can accumulate in the cytosol, peroxisomes, mitochondria, and other intracellular compartments. Fig. 7 summarizes the results presented here in a graphical illustration that describes the proposed deactivation of catalase as a contributor of Aβ-induced oxidative stress. Because we found a significant fraction of Aβ co-localized with catalase in the peroxisomes and in the cytosol (25, 96) (Fig. 3C) and co-immunoprecipitated with catalase (Fig. 4), it is apparent that Aβ interacts directly with catalase inside of live cells (Fig. 7, pathway 1). Moreover, addition of small molecule inhibitors of catalase- Aβ interactions significantly reduced co-localization of Aβ and catalase inside the cells (Figs. 3, H and L and and7,7, pathway 2) without affecting significantly the uptake of Aβ in cells (Fig. 5G).
Second, binding interactions between catalase and Aβ deactivate catalase inside of live cells, leading to decreased degradation of H2O2 in solution (Fig. 6A) and increased cellular levels of H2O2 (Fig. 2A). The results presented here reveal that small molecule inhibitors of catalase- Aβ interactions are capable of protecting the activity of catalase in Aβ-rich solutions (Fig. 6, B and C) and of attenuating Aβ-induced increases in H2O2 levels in cells (Fig. 2B).
Third, Aβ significantly reduced cell viability (Fig. 1A) at concentrations that led to a 3-fold increase in cellular H2O2 levels (Fig. 2A). These results provide evidence for a correlation between Aβ-induced oxidative stress and Aβ toxicity as proposed by others (11, 13, 36). Remarkably, the BTA-EGx molecules (by inhibiting catalase-amyloid interactions) significantly reduced the toxicity of Aβ peptides in cells (Fig. 1B) at concentrations that also reduced Aβ-induced increases in cellular H2O2 levels (Fig. 2B). This result further supports the important role of catalase-Aβ interactions in Aβ-induced elevations of H2O2 and in Aβ-mediated toxicity.
Although the results presented here clearly demonstrate that intracellular catalase-amyloid interactions induce elevations in cellular H2O2 levels, an important question is whether these interactions are relevant to AD pathology. The accumulation of catalase in senile plaques (41) suggests that in vivo environments do indeed provide evidence for binding of catalase to Aβ. We hypothesize that these in vivo catalase-amyloid interactions lead to deactivation of catalase, resulting in neuronal loss as a consequence of increased levels of H2O2 and its metabolites in the brain. This hypothesis is supported by the strong correlation of deactivation of intracellular catalase by either Aβ (Fig. 6A) or by catalase inhibitor 3AT (97) with the ensuing increased H2O2 levels (Fig. 2A) as well as with reduced viability (Fig. 1B and supplemental Fig. S11) in cells.
The results presented here provide clear evidence for the important role of catalase-amyloid interactions in Aβ-induced oxidative stress in cells, as outlined in Fig. 7. The results, however, do not rule out the possibility that amyloid interactions with proteins other than catalase could also contribute to cellular injury. We previously reported that BTA-EG4 and BTA-EG6 are capable of inhibiting several different Aβ-binding proteins from associating with aggregated Aβ peptides (55). It is, therefore, likely that the BTA-EGx molecules in cells also attenuate harmful effects of Aβ on proteins other than catalase. From a therapeutic point of view, this is, of course, an attractive characteristic. Furthermore, these BTA-EGx molecules exhibit desirable drug-like characteristics (98, 99) because they are: 1) non-toxic and cell permeable; 2) small (MW of BTA-EG4 = 418 and MW of BTA-EG6 = 504) and have beneficial topological polar surface areas (42, 100); 3) low in the number of H-bond donors (i.e. less than 5); and 4) low in the number of H-bond acceptors (i.e. less than 10). Most importantly, as revealed in this work, these molecules inhibit catalase- Aβ interactions, reduce the extent of co-localization of Aβ and catalase in cells, protect the activity of catalase in Aβ-rich environments, inhibit Aβ-induced increases in cellular H2O2 levels, and reduce Aβ toxicity. In conclusion, the formation of protein-resistant surface coatings on aggregated forms of amyloid proteins introduces an attractive and novel strategy for probing the effect of Aβ on cells and potentially for treatment of AD.
We thank Jennifer Meerloo and Dr. Kersi Pestonjamasp for technical assistance. We thank Neal Sekiya of the UCSD Flow Cytometry Research Core for acquiring the flow cytometry data and Valentina Matlachova for preliminary work in developing the catalase activity assay. We acknowledge the UCSD Cancer Center Microscopy Shared Facility supported by Grant P30 CA23100 and the UCSD Neuroscience Microscopy Shared Facility supported by Grant P30 NS047101. We also acknowledge the National Science Foundation for support of the mass spectrometry facility at UCSD (CHE-0116662).
*This work was supported in part by the Hellman Foundation, the Alzheimer Disease Research Center (3P50 AG005131), the Alzheimer Association (NIRG-08-91651), and the National Science Foundation (CAREER Award CHE-0847530, to J. Y.).
4Further examination showed that these molecules are not toxic to this cell line at concentrations below 60 μm (supplemental Fig. S2).
5Interestingly, when SH-SY5Y cells are preincubated with the BTA-EGx molecules for 24 h prior to challenging cells with untreated aggregated Aβ peptides (i.e., aggregated Aβ that was not preincubated with BTA-EGx), we did not observe significant inhibition of Aβ-induced toxicity in the cells. Because the effect of the BTA-EGx molecules on protection of Aβ-induced toxicity in cells depended on whether they were preincubated with the aggregated Aβ prior to exposure of the peptides to the cells, we hypothesize that distribution of the BTA-EGx molecules in cells may be different depending on whether they are introduced to cells alone (i.e., in the absence of Aβ) or whether the BTA-EGx molecules are preincubated and introduced to cells together with Aβ peptides. Preincubation with Aβ may, therefore, improve internalization of the BTA-EGx molecules and facilitate, enhance, or stabilize intracellular interactions between BTA-EGx and Aβ.
6Repeating the experiments used to generate the data shown in Fig. 6, B and C revealed variation of roughly an order of magnitude in the estimation of IC50 values for BTA-EGx, suggesting there is no significant difference in the potency between BTA-EG4 and BTA-EG6 in this cell-free assay.
7We attribute the differences in required concentrations of BTA-EGx, either to protect catalase activity in cell-free assays or to inhibit Aβ-induced increases in H2O2 concentrations in cellular assays, to the numerous proteins, metabolites, and cellular components that may interfere with the binding of the BTA-EGx molecules to Aβ in the cell assays. These differences may translate into the apparent requirement of a higher concentration of the BTA-EGx molecules in the cell experiments in order to exert the same protective effects on catalase activity as seen in the cell-free enzymatic assay. Additionally, the precise intracellular concentration of catalase is not known. Differences in intracellular concentration of catalase and the concentration of catalase used in the enzymatic assay (25 nm) would make it difficult to directly and quantitatively compare the results between the cellular and cell-free experiments. Qualitatively, however, the results from the cellular and cell-free experiments are consistent with one another.
3The abbreviations used are: