Chem. Res. Toxicol. 2004, 17, 1743-1749
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Role of Phenylalanine 20 in Alzheimer’s Amyloid β-Peptide (1-42)-Induced Oxidative Stress and Neurotoxicity Debra Boyd-Kimball,† Hafiz Mohmmad Abdul,† Tanea Reed,† Rukhsana Sultana,† and D. Allan Butterfield*,†,‡ Department of Chemistry, Center for Membrane Sciences, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40506-0055 Received July 26, 2004
Senile plaques are a hallmark of Alzheimer’s disease (AD), a neurodegenerative disease associated with cognitive decline and aging. Aβ(1-42) is the primary component of the senile plaque in AD brain and has been shown to induce protein oxidation in vitro and in vivo. Oxidative stress is extensive in AD brain. As a result, Aβ(1-42) has been proposed to play a central role in the pathogenesis of AD; however, the specific mechanism of neurotoxicity remains unknown. Recently, it has been proposed that long distance electron transfer from methionine 35 to the Cu(II) bound at the N terminus of Aβ(1-42) occurs via phenylalanine 20. Additionally, it was proposed that substitution of phenylalanine 20 of Aβ(1-42) by alanine [Aβ(1-42)F20A] would lessen the neurotoxicity induced by Aβ(1-42). In this study, we evaluate the predictions of this theoretical study by determining the oxidative stress and neurotoxic properties of Aβ(142)F20A relative to Aβ(1-42) in primary neuronal cell culture. Aβ(1-42)F20A induced protein oxidation and lipid peroxidation similar to Aβ(1-42) but to a lesser extent and in a manner inhibited by pretreatment of neurons with vitamin E. Additionally, Aβ(1-42)F20A affected mitochondrial function similar to Aβ(1-42), albeit to a lesser extent. Furthermore, the mutation does not appear to abolish the ability of the native peptide to reduce Cu(II). Aβ(1-42)F20A did not compromise neuronal morphology at 24 h incubation with neurons, but did so after 48 h incubation. Taken together, these results suggest that long distance electron transfer from methionine 35 through phenylalanine 20 may not play a pivotal role in Aβ(1-42)-mediated oxidative stress and neurotoxicity.
Introduction Brain oxidative stress is extensive in Alzheimer’s disease (AD), a neurodegenerative disorder of the elderly associated with loss of cognitive function. Amyloid β-peptide (1-42) [Aβ(1-42)] has been shown to induce lipid peroxidation and protein oxidation in vivo and in vitro (1-9). As a result, Aβ(1-42) has been implicated as a causative agent in AD (10, 11). Although the mechanism of toxicity remains elusive, several hypotheses have been proposed including the aggregation properties of the peptide (12, 13), the role of methionine 35 (7, 14-18), and the role of copper binding and reduction (19-24). NMR studies have shown that Cu(II) complexes to three key histidine residues (residues 6, 13, and 14) in the N terminus of Aβ(1-42). It has been proposed that tyrosine 10 acts as an electron donor for the reduction of the bound Cu(II) to Cu(I) leading to the subsequent production of hydrogen peroxide (22, 24), but recent mutational studies from our laboratory suggest that this may not be the case.1 Consistent with this notion, studies utilizing the fragment Aβ(1-28) containing all of the residues proposed to play a role in copper binding and reduction show no oxidative stress or neurotoxicity in the absence of me* To whom correspondence should be addressed. Tel: 859-257-3184. Fax: 859-257-5876. E-mail:
[email protected]. † Center for Membrane Sciences. ‡ Sanders-Brown Center on Aging.
thionine (22). This finding supports the role of methionine 35 as the primary mechanism of toxicity. Recently, a theoreticical study has proposed a mechanism of toxicity that combines the role of methionine 35 and copper binding and reduction (25). The study suggests that in the process of forming a S-centered sulfuranyl free radical on Met-35, long distance electron transfer might occur from methionine 35 via phenylalanine 20 to the Cu(II) bound within the N terminus of Aβ(1-42). Furthermore, the modeling study suggests that substitution of phenylalanine 20 with alanine would abrogate the long distance electron transfer from methionine 35 to Cu(II). Likewise, it was proposed that mutation of phenylalanine 20 to alanine would reduce the neurotoxicty exhibited by Aβ(1-42). In the current study, we evaluated this prediction based on this prior theoretical study by investigating the effect of the mutation of phenylalanine 20 to alanine [Aβ(1-42)F20A] on the oxidative stress and neurotoxicity exhibited by Aβ(1-42) in primary neuronal culture (Figure 1). We found that the substitution did not abolish the protein oxidation, lipid peroxidation, and loss of 1 Boyd-Kimball, D., Sultana, R., Mohmmad-Abdul, H., and Butterfield, D. A. (2004) Role of methionine 35 versus Cu(II) in Alzheimer’s Aβ(1-42)-mediated oxidative stress and neurotoxicity in primary neuronal cell cultures: A critical comparison. Submitted for publication.
10.1021/tx049796w CCC: $27.50 © 2004 American Chemical Society Published on Web 11/24/2004
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Chem. Res. Toxicol., Vol. 17, No. 12, 2004
Figure 1. Sequences of Aβ(1-42) and Aβ(1-42)F20A.
mitochondrial function induced by Aβ(1-42) but did lessen the extent. Moreover, Aβ(1-42)F20A did affect the neuronal morphology at 48 h in contrast to Aβ(1-42) with a 24 h treatment. Additionally, the substitution resulted in a decrease in the ability of the peptide to reduce Cu(II) to Cu(I) as compared to Aβ(1-42) but did not abolish the ability of the peptide to reduce Cu(II). Taken together, these results suggest that long distance electron transfer from methionine 35 via phenylalanine 20 may exert some effects, but such processes likely do not play a pivotal role in Aβ(1-42)-mediated neurotoxicity and oxidative stress but does lessen the extent of the effects. Thus, the findings are consistent with the notion that the theoretical predictions may have some merit, but likely other mechanisms are operative for Aβ(1-42)-induced Cu(II) reduction in ways that involve methionine 35.
Experimental Procedures Chemicals. All chemicals were of the highest purity and were obtained from Sigma (St. Louis, MO) unless otherwise noted. All peptides were purchased from Anaspec (San Jose, CA) with HPLC and MS verification of purity. The peptides were stored in the dry state at -20 °C until use. The OxyBlot protein oxidation detection kit was purchased from Chemicon International (Temecula, CA). anti-4-Hydroxynonenal (HNE) was purchased from Alpha Diagnostic International (San Antonio, TX). Cell Culture Experiments. Neuronal cultures were prepared from 18 day old Sprague-Dawley rat fetuses (25). Aβ peptides were dissolved in sterile water that has been stirred over Chelex-100 resin. The peptides were preincubated for 24 h at 37 °C prior to addition to cultures. The final concentration of the peptides in the cell culture was 10 µM, and the effects of Aβ on the neurons were measured after 24 h of exposure. In some experiments, the neuronal cultures were pretreated for 1 h with SOD1 (100 U/mL) or catalase (100 U/mL) follwed by 10 µM Aβ(1-42) or 10 µM Aβ(1-42) F20A. These antioxidant enzyme concentrations were chosen based on the literature (26, 27). Mitochondrial function was evaluated by the 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide (MTT) reduction assay. Briefly, MTT was added to each well with a final concentration of 1.0 mg/mL and incubated for 1 h. The dark blue formazan crystals formed in intact cells were extracted with 250 µL of dimethyl sulfoxide, and the absorbance was read at 595 nm with a microtiter plate reader (Bio-Tek Instruments, Winooski, VT). Protein Carbonyls. Protein carbonyls are an index of protein oxidation (2). The sample (5 µL) was incubated for 20 min at room temperature with 5 µL of 12% sodium dodecyl sulfate (SDS) and 10 µL of 2,4-dinitrophenylhydrazine that was diluted 10 times with water from a 200 mM stock. The samples were neutralized with 7.5 µL of neutralization solution (2 M Tris in 30% glycerol). The resulting sample (250 ng) was loaded per well in the slot blot apparatus. Samples were loaded onto a nitrocellulose membrane under vacuum pressure. The mem-
Boyd-Kimball et al. brane was blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 (wash blot) for 1 h and incubated with a 1:100 dilution of anti-DNP polyclonal antibody in wash blot for 1 h. Following completion of the primary antibody incubation, the membranes were washed three times in wash blot for 5 min each. An anti-rabbit IgG alkaline phosphatase secondary antibody was diluted 1:8000 in wash blot and added to the membrane for 1 h. The membrane was washed in wash blot three times for 5 min and developed using Sigmafast Tablets (BCIP/NBT substrate). Blots were dried, scanned with Adobe Photoshop (San Jose, CA), and quantitated with Scion Image. HNE. This alkenal is a product of lipid peroxidation (2). The sample (10 µL) was incubated with 10 µL of modified Laemmli buffer containing 0.125 M Tris base, pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol. The resulting sample (250 ng) was loaded per well in the slot blot apparatus. Samples were loaded onto a nitrocellulose membrane under vacuum pressure. The membrane was blocked with 3% (w/v) BSA in wash blot for 1 h and incubated with a 1:5000 dilution of HNE polyclonal antibody in wash blot for 90 min. Following completion of the primary antibody incubation, the membranes were washed three times in wash blot for 5 min each. An anti-rabbit IgG alkaline phosphatase secondary antibody was diluted 1:8000 in wash blot and added to the membrane for 90 min. The membrane was washed in wash blot three times for 5 min and developed using Sigmafast Tablets (BCIP/NBT substrate). Blots were dried, scanned with Adobe Photoshop, and quantitated with Scion Image. Thioflavin T (ThT) Binding Assay. The ThT binding assay was performed according to the method of Levine (28). A solution of 50 mM glycine, pH 8.5, containing 5 µM ThT was added to Aβ peptides incubated in solution for 24 h to a final peptide concentration of 2.5, 5, 7.5, and 10 µM. The resulting fluorescence was measured at λex ) 440 nm and λem ) 485 nm with a SpectraMax GeminiXS (Molecular Devices, Sunnyvale, CA). Copper Reduction. The ability of the Aβ peptides to reduce copper was measured by the BCA method described by Huang et al. (20). Briefly, the peptides (10 µM), copper(II) (25 µM), and the copper(I) indicator BCA (250 µM) were coincubated in PBS, pH 8.0, in a 96 well microtiter plate for 1 h at 37 °C. Controls were conducted with ascorbate (10 µM) in place of the peptide. Absorbance was measured at λ ) 562 nm (Bio-Tek Instruments). The concentration of Cu(I) was calculated based on the known molar absorptivity ( ) 7700 M-1 cm-1). Electron Microscopy. Electron microscopy was used to assess the ability of the Aβ peptides to form fibrils upon incubation in solution for 24 h. Aliquots of 5 µL of the peptide solutions that were used for the cell culture experiments were placed on a copper mesh Formvar carbon-coated grid. After 1-1.5 min of incubation at room temperature, excess liquid was drawn off, and samples were counterstained with 2% uranyl acetate. Air-dried samples were examined in a Philips Tecnai Biotwin 12 transmission electron microscope (FEI, Eindhoven, Netherlands) at 80 kV. Images were captured with a 2K × 2K digital camera (Advanced Microscopy Techniques). Circular Dichroism (CD) Spectroscopy. CD spectra were recorded using a JASCO spectropolarimeter with a quartz cell of 1.0 mm path length at 25 °C. Spectra were measured from 260 to 190 nm, 1 nm bandwidth, 20 accumulations, and 50 nm/ min of scanning speed on a sample containing Aβ(1-42) or Aβ(142)F20A peptides in chelexed water. For each peptide, ellipticity and photomultiplier voltage (H.T.) baselines were measured using 2 mL of chelexed water in a 1 mm path length cuvette. Then, an aliquot of freshly prepared peptide solution was diluted into the chelexed water, to a concentration of approximately 500 µg/mL, and its CD and absorbance spectra were measured. Analysis of DNA Fragmentation. Cultures were rinsed three times in PBS, fixed with 4% paraformaldehyde for 10 min at 37 °C, rinsed, and stained with Hoechst 332584 (1 µg/mL) for 10 min at room temperature. The staining was visualized
Role of Phenylalanine 20 in Alzheimer’s
Figure 2. Neurotoxic properties of Aβ peptides as measured by MTT reduction. The results are shown as means ( SEM of independent measurements. Treatment of neurons with both 10 µM Aβ(1-42) and 10 µM Aβ(1-42)F20A showed a significant decrease in MTT reduction. *p < 0.02, analysis of variance, n ) 4. The statistical comparison was performed between control and Aβ treatment data sets. No significant difference was found between peptide data sets. using a fluorescent microscope with a DAPI filter. The nuclear staining with Hoechst 332584 provided morphological discrimination between normal and apoptotic cells as described by Darzynkiewicz et al. (29). Statistical Analysis. ANOVA was used to determine statistical significance. p values of
