Preparation and Characterization of Neurotoxic Tau Oligomers
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Preparation and Characterization of Neurotoxic Tau Oligomers Cristian A. Lasagna-Reeves, Diana L. Castillo-Carranza, Marcos J. Guerrero-Muñoz, George R. Jackson, and Rakez Kayed
Supporting Information Preparation and characterization of tau oligomers: Recombinant tau protein tau-441 (2N4R; M.Wt 45.9 kDa) was expressed and purified as described (1, 2). Recombinant tau was treated with 8 M urea to obtain monomeric tau, followed by overnight dialysis against 1X phosphate-buffered saline (PBS), pH 7.4, and normalization to 1 mg/mL with PBS using BCA protein assay (Pierce). Aliquots of tau monomer in PBS were kept at −20 ºC. For preparation of oligomers, 300 µL of the tau stock (1 mg/mL) was added to 700 µL of 1X PBS, final concentration 0.3 mg/mL. Seven microliters of Aβ42 or α-synuclein oligomers (0.3 mg/mL) prepared using the methods previously described (3-7), for thorough protocols see below, was added as seeds, and the sample was mixed by pipetting for 1 min. The samples were then incubated at room temperature for 1 h on an orbital shaker for oligomers. The resulting tau oligomers were purified by fast protein liquid chromatography (FPLC), and used to seed a fresh sample of monomeric tau; after three rounds of seeding with purified tau oligomers, Aβ or α-synuclein oligomer seeds were undetectable. The samples had no Aβ as tested by either enzyme-linked immunosorbent assay (ELISA) or Western blotting using 4G8. This is not surprising since we used a 1/140 Aβ/tau ratio in the first round, less than the standard used in the literature as seeds to promote aggregation. After three rounds, there was estimated to be less than 1/2,470,000 Aβ/Tau, which is below the detection limits of the methods available. To prepare the fibrils, tau oligomers were allowed to mix 1–2 days on the orbital shaker. Similar results were obtained when 10 mM HEPES buffer, pH 7.4, was used instead of 1X PBS. Results were more consistent when monomeric tau was stirred overnight at 500 rpm using a Teflon-coated micro stir bar before the seeds were added. Tau oligomers 1
2
and monomer were purified by size-exclusion chromatography, using an LC-6AD Shimadsu high-performance liquid chromatography (HPLC) system fitted with a TSK-GEL G3000 SWXL (30 cm x 7.8 mm) column, Supelco-808541. PBS, pH 7.4, was used as mobile phase, flow rate 0.5 mL/min. Gel filtration standard (Biorad 51-1901) was used for calibrations. Tau preparations were characterized by atomic force microscopy (AFM). Using a non-contact tapping method (ScanAsyst Air) and a Multimode 8 AFM machine (Veeco) and electron microscopy (EM), 2-4 µL of the sample was adsorbed onto 200-mesh carbon- and Formvar-coated nickel grids, air dried, and washed for 1 min in distilled water. The samples were negatively stained with 3 µL of 2% uranyl acetate for 2 min and viewed with a Zeiss 10CR microscope (80 kV). Preparation of Aβ and α-synuclein oligomer seeds: All oligomer seeds used were A 11 positive (3-7). To make sure that the cross seeding described here is reproducible, we used two different methods to prepare the oligomer seeds. Aβ and α-synuclein oligomers ability to induce tau aggregation was not affected by the method of preparation. Aβ oligomers (HFIP method): Aβ 42 lyophilized peptide 1 mg resuspended in 1.5 mL of 50% acetonitrile/water mixture. Divided into 3 eppendorf tubes each contain 500 µl (0.3 mg) and relyophilized. Soluble oligomers were prepared by dissolving 0.3 mg of the peptide in 200µl hexafluoroisopropanol (HFIP) and incubating it for 10–20 min at room temperature. The resulting solution was added to 700 µl dd H2O in a siliconized eppendorf tube, a cap with holes placed on top to allow the evaporation of HFIP. The samples were then stirred at 500 RPM using a Teflon-coated micro stir bar for 36 h in the fume hood at room temperature. α-synuclein oligomers (HFIP method): Protein dialyzed overnight against water and lyophilized. 1 mg resuspended in 1.0 mL of 50% acetonitrile/water mixture. Divided into 2 eppendorf tubes each contain 500 µl (0.5 mg) and relyophilized. Oligomers were prepared by dissolving 0.5 mg of the protein in 200µl hexafluoroisopropanol (HFIP) and incubating it for 10–20 min at room temperature. The resulting solution was added to 700 µl dd H2O in a siliconized eppendorf tube, a cap with holes placed on top to allow the evaporation
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of HFIP. The samples were then stirred at 500 RPM using a Teflon-coated micro stir bar for 48 h in the fume hood at room temperature. Aβ oligomers (NaOH method): Alternatively Aβ oligomers were obtained by dissolving the relyophilized peptide (0.3mg) in 30µl of 100 mM NaOH, followed by water bath sonication for 30s. The solution was transferred to an eppendorf containing 700 µl 1X PBS, pH=7.4, 0.02 % sodium azide. The solution we oligomerized by shaking on a rocker for 2 days at room temperature. α-synuclein oligomers (PBS method): Oligomers were prepared by dissolving relyophilized protein (0.5 mg) in 700 µl 1X PBS, pH=7.4, 0.02 % sodium azide. The resulting solution was stirred in a closed eppendorf at 500 RPM using a Teflon-coated micro stir bar for 48 h at room temperature. The samples were diluted to 0.3 mg/mL in 1X PBS before use. Western blot analysis: Tau samples mixed with 6µL 4X sample buffer were loaded to the gel (without boiling) and separated by SDS-PAGE using NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen), then transferred onto nitrocellulose membrane 0.45 µm (Bio-Rad 162-0115). After blocking with nonfat dried milk for 1 h, membranes were washed 3 times with Tris-buffered saline (TBS-T), then probed with a tau-5 antibody (1:2000) for 2 h. Antibody immunoreactivity was detected with horseradish peroxidase–conjugated anti-mouse IgG (1:3000, Jackson laboratory) for 1 h, followed by western blotting detection using ECL Plus reagents (Amersham RPN2132). Circular dichroism measurements: Circular dichroism (CD) spectra were recorded at room temperature using a J-720 spectropolarimeter (JASCO) equipped with a temperature controller. Spectra were measured at 0.20-nm intervals, spectral bandwidth of 1 nm, scan speed of 20 nm/min, response time of 8 s. Each spectrum represents the average of three scans in the range of 195 to 350 nm at 0.3 mg/mL protein in PBS. The quartz cells had path lengths of 0.1 mm and were washed with 30% HCl in EtOH, water, and MeOH before use. Bis-ANS fluorescence: 1,1'-bis(anilino)-4-,4'-bis(naphthalene)-8,8'-disulfonate (Sigma), bis-ANS binding was measured using an RF-5301PC spectrofluorometer from SHIMADZU. The excitation wavelength was 360 nm, 3
4
and emission was recorded between 400 and 575 nm. Three hundred microliters of 0.3 mg/mL tau oligomers, fibrils or monomer added to 170 µL of 10 mM bis-ANS in sodium phosphate buffer. The emission spectrum of bis-ANS alone was subtracted. Toxicity assays: MTS assay: SH-SY5Y human neuroblastoma cells were maintained in Dulbecco`s modified Eagle’s medium (DMEM) and grown to confluence in 96-well plates. Cells (≈10,000 cells /well) were treated with 1.0 µM (final concentration) tau oligomers, tau monomer, or tau fibrils for 4 h, all measurements performed in triplicate. Cell viability was assessed using a colorimetric Tetrazolium-based MTS method (Promega). The experiments were repeated using different preparations of tau oligomers; no statistical differences were observed between experiments using different preparations. Absorbance was measured at 490 nm. The percentage of cells affected was calculated as ((ODuntreated control-ODtreated)/ ODuntreated control) x 100. Statistical analysis was based on Two-Way ANOVA test, performed using Origin-8 software (Origin Lab). AlamarBlue assay: SH-SY5Y human neuroblastoma cells were grown to confluence in 96-well plates. Cells were treated with different concentrations of tau oligomers or with 1X PBS for untreated controls.
All
measurements were performed in triplicate. Cytotoxicity was measured using an AlamarBlue assay kit (Serotec), which measures metabolic reduction of the AlamarBlue reagent as an indicator of cell survival and proliferation. AlamarBlue reagent was added in an amount equal to 10% of the culture volume, and cultures were returned to the incubator. Fluorescence was measured 4 h later with an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a POLARstar Omega fluorescence microplate reader. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Margittai, M., and Langen, R. (2004), Proc Natl Acad Sci U S A 101, 10278-10283. Margittai, M., and Langen, R. (2006), J Biol Chem 281, 37820-37827. Kayed, R., and Glabe, C. G. (2006), Methods Enzymol 413, 326-344. Kayed, R., Sokolov, Y., Edmonds, B., McIntire, T. M., Milton, S. C., Hall, J. E., and Glabe, C. G. (2004), J Biol Chem 279, 46363-46366. Necula, M., Kayed, R., Milton, S., and Glabe, C. G. (2007), J Biol Chem 282, 10311-10324. Demuro, A., Mina, E., Kayed, R., Milton, S. C., Parker, I., and Glabe, C. G. (2005), J Biol Chem 280, 17294-17300. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003), Science 300, 486-489.
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Figure S1: Upon longer incubation with continuous shaking, tau oligomers prepared by cross-seeding form mature tau fibrils. (a) Tau oligomers prepared by seeding will Aβ42 oligomers, same electron microscopic image as in Figure 1c. (b). Fibrils start to form after 24 h of continuous shaking. (c) After 48 h, most of the tau oligomers have converted to fibrils. (d) EM image of tau oligomers prepared by seeding with α-synuclein oligomers. (e) After 48 h, most of tau oligomers converted to fibrils.
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Figure S2 Toxicity of tau oligomers and monomer at different concentrations: Neuroblastoma SH-SY5Y cells were treated for 4 h with various concentrations of tau oligomers (black) and monomer (white), and toxicity was assayed using AlamarBlue. Treatment with 0.5 µM, 1 µM, and 2 µM tau oligomers produced significant toxicity (p
Preparation and Characterization of Neurotoxic Tau Oligomers Cristian A. Lasagna-Reeves, Diana L. Castillo-Carranza, Marcos J. Guerrero-Muñoz, George R. Jackson, and Rakez Kayed
Supporting Information Preparation and characterization of tau oligomers: Recombinant tau protein tau-441 (2N4R; M.Wt 45.9 kDa) was expressed and purified as described (1, 2). Recombinant tau was treated with 8 M urea to obtain monomeric tau, followed by overnight dialysis against 1X phosphate-buffered saline (PBS), pH 7.4, and normalization to 1 mg/mL with PBS using BCA protein assay (Pierce). Aliquots of tau monomer in PBS were kept at −20 ºC. For preparation of oligomers, 300 µL of the tau stock (1 mg/mL) was added to 700 µL of 1X PBS, final concentration 0.3 mg/mL. Seven microliters of Aβ42 or α-synuclein oligomers (0.3 mg/mL) prepared using the methods previously described (3-7), for thorough protocols see below, was added as seeds, and the sample was mixed by pipetting for 1 min. The samples were then incubated at room temperature for 1 h on an orbital shaker for oligomers. The resulting tau oligomers were purified by fast protein liquid chromatography (FPLC), and used to seed a fresh sample of monomeric tau; after three rounds of seeding with purified tau oligomers, Aβ or α-synuclein oligomer seeds were undetectable. The samples had no Aβ as tested by either enzyme-linked immunosorbent assay (ELISA) or Western blotting using 4G8. This is not surprising since we used a 1/140 Aβ/tau ratio in the first round, less than the standard used in the literature as seeds to promote aggregation. After three rounds, there was estimated to be less than 1/2,470,000 Aβ/Tau, which is below the detection limits of the methods available. To prepare the fibrils, tau oligomers were allowed to mix 1–2 days on the orbital shaker. Similar results were obtained when 10 mM HEPES buffer, pH 7.4, was used instead of 1X PBS. Results were more consistent when monomeric tau was stirred overnight at 500 rpm using a Teflon-coated micro stir bar before the seeds were added. Tau oligomers 1
2
and monomer were purified by size-exclusion chromatography, using an LC-6AD Shimadsu high-performance liquid chromatography (HPLC) system fitted with a TSK-GEL G3000 SWXL (30 cm x 7.8 mm) column, Supelco-808541. PBS, pH 7.4, was used as mobile phase, flow rate 0.5 mL/min. Gel filtration standard (Biorad 51-1901) was used for calibrations. Tau preparations were characterized by atomic force microscopy (AFM). Using a non-contact tapping method (ScanAsyst Air) and a Multimode 8 AFM machine (Veeco) and electron microscopy (EM), 2-4 µL of the sample was adsorbed onto 200-mesh carbon- and Formvar-coated nickel grids, air dried, and washed for 1 min in distilled water. The samples were negatively stained with 3 µL of 2% uranyl acetate for 2 min and viewed with a Zeiss 10CR microscope (80 kV). Preparation of Aβ and α-synuclein oligomer seeds: All oligomer seeds used were A 11 positive (3-7). To make sure that the cross seeding described here is reproducible, we used two different methods to prepare the oligomer seeds. Aβ and α-synuclein oligomers ability to induce tau aggregation was not affected by the method of preparation. Aβ oligomers (HFIP method): Aβ 42 lyophilized peptide 1 mg resuspended in 1.5 mL of 50% acetonitrile/water mixture. Divided into 3 eppendorf tubes each contain 500 µl (0.3 mg) and relyophilized. Soluble oligomers were prepared by dissolving 0.3 mg of the peptide in 200µl hexafluoroisopropanol (HFIP) and incubating it for 10–20 min at room temperature. The resulting solution was added to 700 µl dd H2O in a siliconized eppendorf tube, a cap with holes placed on top to allow the evaporation of HFIP. The samples were then stirred at 500 RPM using a Teflon-coated micro stir bar for 36 h in the fume hood at room temperature. α-synuclein oligomers (HFIP method): Protein dialyzed overnight against water and lyophilized. 1 mg resuspended in 1.0 mL of 50% acetonitrile/water mixture. Divided into 2 eppendorf tubes each contain 500 µl (0.5 mg) and relyophilized. Oligomers were prepared by dissolving 0.5 mg of the protein in 200µl hexafluoroisopropanol (HFIP) and incubating it for 10–20 min at room temperature. The resulting solution was added to 700 µl dd H2O in a siliconized eppendorf tube, a cap with holes placed on top to allow the evaporation
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of HFIP. The samples were then stirred at 500 RPM using a Teflon-coated micro stir bar for 48 h in the fume hood at room temperature. Aβ oligomers (NaOH method): Alternatively Aβ oligomers were obtained by dissolving the relyophilized peptide (0.3mg) in 30µl of 100 mM NaOH, followed by water bath sonication for 30s. The solution was transferred to an eppendorf containing 700 µl 1X PBS, pH=7.4, 0.02 % sodium azide. The solution we oligomerized by shaking on a rocker for 2 days at room temperature. α-synuclein oligomers (PBS method): Oligomers were prepared by dissolving relyophilized protein (0.5 mg) in 700 µl 1X PBS, pH=7.4, 0.02 % sodium azide. The resulting solution was stirred in a closed eppendorf at 500 RPM using a Teflon-coated micro stir bar for 48 h at room temperature. The samples were diluted to 0.3 mg/mL in 1X PBS before use. Western blot analysis: Tau samples mixed with 6µL 4X sample buffer were loaded to the gel (without boiling) and separated by SDS-PAGE using NuPAGE Novex 4–12% Bis-Tris gel (Invitrogen), then transferred onto nitrocellulose membrane 0.45 µm (Bio-Rad 162-0115). After blocking with nonfat dried milk for 1 h, membranes were washed 3 times with Tris-buffered saline (TBS-T), then probed with a tau-5 antibody (1:2000) for 2 h. Antibody immunoreactivity was detected with horseradish peroxidase–conjugated anti-mouse IgG (1:3000, Jackson laboratory) for 1 h, followed by western blotting detection using ECL Plus reagents (Amersham RPN2132). Circular dichroism measurements: Circular dichroism (CD) spectra were recorded at room temperature using a J-720 spectropolarimeter (JASCO) equipped with a temperature controller. Spectra were measured at 0.20-nm intervals, spectral bandwidth of 1 nm, scan speed of 20 nm/min, response time of 8 s. Each spectrum represents the average of three scans in the range of 195 to 350 nm at 0.3 mg/mL protein in PBS. The quartz cells had path lengths of 0.1 mm and were washed with 30% HCl in EtOH, water, and MeOH before use. Bis-ANS fluorescence: 1,1'-bis(anilino)-4-,4'-bis(naphthalene)-8,8'-disulfonate (Sigma), bis-ANS binding was measured using an RF-5301PC spectrofluorometer from SHIMADZU. The excitation wavelength was 360 nm, 3
4
and emission was recorded between 400 and 575 nm. Three hundred microliters of 0.3 mg/mL tau oligomers, fibrils or monomer added to 170 µL of 10 mM bis-ANS in sodium phosphate buffer. The emission spectrum of bis-ANS alone was subtracted. Toxicity assays: MTS assay: SH-SY5Y human neuroblastoma cells were maintained in Dulbecco`s modified Eagle’s medium (DMEM) and grown to confluence in 96-well plates. Cells (≈10,000 cells /well) were treated with 1.0 µM (final concentration) tau oligomers, tau monomer, or tau fibrils for 4 h, all measurements performed in triplicate. Cell viability was assessed using a colorimetric Tetrazolium-based MTS method (Promega). The experiments were repeated using different preparations of tau oligomers; no statistical differences were observed between experiments using different preparations. Absorbance was measured at 490 nm. The percentage of cells affected was calculated as ((ODuntreated control-ODtreated)/ ODuntreated control) x 100. Statistical analysis was based on Two-Way ANOVA test, performed using Origin-8 software (Origin Lab). AlamarBlue assay: SH-SY5Y human neuroblastoma cells were grown to confluence in 96-well plates. Cells were treated with different concentrations of tau oligomers or with 1X PBS for untreated controls.
All
measurements were performed in triplicate. Cytotoxicity was measured using an AlamarBlue assay kit (Serotec), which measures metabolic reduction of the AlamarBlue reagent as an indicator of cell survival and proliferation. AlamarBlue reagent was added in an amount equal to 10% of the culture volume, and cultures were returned to the incubator. Fluorescence was measured 4 h later with an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a POLARstar Omega fluorescence microplate reader. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Margittai, M., and Langen, R. (2004), Proc Natl Acad Sci U S A 101, 10278-10283. Margittai, M., and Langen, R. (2006), J Biol Chem 281, 37820-37827. Kayed, R., and Glabe, C. G. (2006), Methods Enzymol 413, 326-344. Kayed, R., Sokolov, Y., Edmonds, B., McIntire, T. M., Milton, S. C., Hall, J. E., and Glabe, C. G. (2004), J Biol Chem 279, 46363-46366. Necula, M., Kayed, R., Milton, S., and Glabe, C. G. (2007), J Biol Chem 282, 10311-10324. Demuro, A., Mina, E., Kayed, R., Milton, S. C., Parker, I., and Glabe, C. G. (2005), J Biol Chem 280, 17294-17300. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003), Science 300, 486-489.
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Figure S1: Upon longer incubation with continuous shaking, tau oligomers prepared by cross-seeding form mature tau fibrils. (a) Tau oligomers prepared by seeding will Aβ42 oligomers, same electron microscopic image as in Figure 1c. (b). Fibrils start to form after 24 h of continuous shaking. (c) After 48 h, most of the tau oligomers have converted to fibrils. (d) EM image of tau oligomers prepared by seeding with α-synuclein oligomers. (e) After 48 h, most of tau oligomers converted to fibrils.
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Figure S2 Toxicity of tau oligomers and monomer at different concentrations: Neuroblastoma SH-SY5Y cells were treated for 4 h with various concentrations of tau oligomers (black) and monomer (white), and toxicity was assayed using AlamarBlue. Treatment with 0.5 µM, 1 µM, and 2 µM tau oligomers produced significant toxicity (p
