Amyloid-β and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-β (Aβ) and α-synuclein (αS), associated with Alzheimer’s and Parkinson’s diseases, respectively, on the Cu2+-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu2+ is bound to Aβ or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aβ or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu2+ and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.

formation of aggregates was verified by fluorescence spectroscopy after mixing small aliquots of the samples with ThT at different times. Subsequent dilution with buffer, ascorbate, etc., resulted in working concentrations of aggregated Aβ corresponding to 20 µM or 10 µM monomer.
αS wild-type monomer, 4 αS Δ2-9 variant monomer, 5 and their respective oligomeric and fibrillar species 2 were prepared as described previously. The design of the αS Δ2-9 variant monomer was based on previous studies which reported the main residues involved in copper metal binding at physiological pH. 6,7 Briefly, the αS Δ2-9 primer was prepared from pT7-7 αS WT by inverse PCR using 5'-phosphorylated primer pairs: (Forward) 5'-AAG GCC AAG GAG GGA GTT GTG-3'; and (Reverse) 5'-CAT ATG TAT ATC TCC TTC TTA AAG TTC CC-3'. Self-circularisation was performed by blunt-end ligation with T4 ligase and truncation positive clones were confirmed by DNA sequencing. Both wild-type and Δ2-9 variant human αS were over expressed and purified as a monomeric fraction from E.coli. In order to produce a sample enriched in pure αS oligomeric species, αS (wild-type and the Δ2-9 variant) monomer was dialysed into Milli-Q water, lyophilized in 6 mg aliquots and subsequently resuspended in PBS, pH 7.4, to give a final concentration of ca. 800 µM (12 mg/mL). The solution was then passed through a 0.22 µm cutoff filter prior to incubation at 37 °C for 20 -24 h at quiescent conditions. After this time, low levels of fibrillar species present in the sample were removed by ultracentrifugation for 1h at 90,000 rpm (using a TLA-120.2 Beckman rotor, 288,000 × g), while residual monomeric protein and small oligomers were removed by multiple filtration steps using 100-kDa cutoff filter membranes. αS wild-type and variant fibrils were prepared by incubating the respective monomeric αS at 70 µM (1mg/mL) in PBS, pH 7.4 containing 0.02% NaN 3 (to prevent bacterial growth during aggregation) at 37 °C, under constant agitation (200 rpm) for 4 -7 days. αS fibrils-Cu was prepared in the same way with the addition of 0.5 eq. Cu 2+ in PBS. Each sample was then centrifuged (15 min, 13,200 rpm) and the resultant fibrillar pellet washed twice with PBS before being resuspended into an appropriate volume of PBS. The fibrils of αS and αSΔ2-9 prepared by this method are 43% and 54% saturated with Cu, respectively (Tabel S2). The final concentration of fibrils (~100 µM) was estimated by measuring the absorbance at 275nm using a molar extinction coefficient of 5600 M -1 cm -1 , after disaggregating an aliquot by the addition of guanidinium chloride to a final concentration of 4 M.

Coumarin-3-carboxylic acid assay
The oxidation of 3-CCA to 7-hydroxy-coumarin-3-carboxylic acid (7-OH-CCA) was followed as the increase in fluorescence intensity at 450±20 nm upon excitation at 395±15 nm. Fluorescence measurements were done in black 96-well optical bottom plates (Corning) using a FLUOstar Omega plate reader (BMG Labtech GmbH) or Clariostar plate reader (BMG Labtech) at 25 °C. Samples contained 300 µM ascorbate or 100 µM ascorbate, 100 µM 3-CCA, and where necessary, 1 µM DFO to chelate any trace metals. 1 The final protein concentration was 10 µM or 20 µM, with S4 5 µM or 10 µM Cu 2+ respectively. Buffer with the appropriate concentration of ascorbate was used as background fluorescence and subtracted from the data. Control experiments without Cu 2+ , and without ascorbate were also done. Each set of conditions was replicated at least in duplicates or higher.

Ascorbate oxidation assay
The ascorbate concentration was measured at 265 nm in a 10 mm quartz cuvette using a Shimadzu UV-3600 spectrophotometer (Shimadzu Scientific Instruments), or in 96-Well UV transparent plates (Nunc) using a Tecan M200 NanoQuant plate reader or Clariostar plate reader (BMG Labtech). There was no significant difference in data between the measurements done in the cuvette and in the plates. The experiment temperature was 25 °C. Samples contained 100 µM ascorbate and where necessary, 1 µM DFO. The final protein concentration is 10 µM or 20 µM, with 5 µM or 10 µM Cu 2+ respectively. Samples without ascorbate were used as background absorbance and subtracted from measurements. Experiments were run in duplicate or higher.

Amplex red assay
Amplex red is a non-fluorescent compound that reacts with H 2 O 2 in the presence of horseradish peroxidase (HRP) to produce the highly fluorescent product, resorufin. Fluorescence of the resorufin product was detected at 590±20 nm upon excitation at 530±15 nm. Fluorescence measurements were done in black 96-well optical bottom plates (Corning) using a Clariostar plate reader (BMG Labtech) at 25 °C. Samples contained 100 µM ascorbate, 50 µM Amplex red, 0.1 U/mL HRP. The final protein concentration is 10 µM or 20 µM, with 5 µM or 10 µM Cu 2+ respectively. Standard calibration curves were obtained using H 2 O 2 solutions with concentrations ranging from 0.25 µM to 25 µM. Buffer with 100 µM ascorbate was used as background fluorescence and subtracted from the data. Control experiments without Cu 2+ , and without ascorbate were also done. Each set of conditions was replicated at least in duplicate or higher.

MALDI-TOF MS MALDI-TOF analysis was performed on a Waters MALDI MicroMX (Waters, UK).
The analyzed samples contained 10 µM protein, 5 µM Cu 2+ and 100 µM ascorbate. After 30 minutes, trifluoroacetic acid was added to a final concentration of 0.2% to quench the reaction. Guanidinium chloride was added to a final concentration of 4 M to all samples so as to disaggregate the aggregated species for subsequent mass determination. Aβ samples were desalted on C18 ziptips, and eluted in HCCA matrix in 50% acetonitrile with 0.2% formic acid. α-synuclein samples were desalted on C18 ziptips, and eluted in 2,5-dihydroxyacetophenone (DHAP) matrix in 50% acetonitrile with 0.2% formic acid. Samples were analyzed in linear mode.

Inductively Coupled Mass Spectrometry (ICP-MS)
The ICP-MS analysis of the Cu content in fibrillated αS and αSΔ2-9 were performed using a Sciex ELAN 6000 mass spectrometer from Perkin-Elmer. The fibrils were dissolved by addition of high purity concentrated HNO 3 . This solution and the supernatants from the washing steps during the fibril preparation were diluted with the 20mM HEPES buffer prior to ICP-MS analysis. The total copper content in the samples was quantified by monitoring the 63 Cu and 65 Cu isotopes and comparing to a matrix-matched calibration curve.
Supporting Table   Table S1. Cu content in fibrillated αS and in the supernatant of the fibrillation reaction (Sup 0) and two successive washes in PBS of the fibrils (Sup 1 and Sup 2). The concentrations in the pellets are relative to resuspending in the same volume as supernatants from the washes.
[Cu] was measured by ICP-MS and [fibril] by absorbance. The difference in [fibril] between the two variants is a consequence of different fibrillation propensities. The data for wt αS is an average of two samples, while the data for αSΔ2-9 is from a single measurement.
[fibril] (µM) [Cu] (µM)  ] only the effect of 20 µM monomeric protein (red) were compared to free Cu 2+ (green). For αSΔ2-9 seven different conditions were compared (red, monomeric protein; black, fibrils formed in the absence of Cu 2+ ; grey, fibrils formed in the presence of Cu 2+ ; blue, oligomers; purple, Cu 2+ alone; yellow, ascorbate alone; green, Cu 2+ and ascorbate alone). All assays were performed at 25 °C. The αSΔ2-9 concentration was 10 µM. Cu 2+ was added to 0.5 molar equivalents relative to the protein concentration and the ascorbate concentration was 100 µM. Figure S3. Initial rates of ROS production in the presence of Aβ 40 or αS in the monomeric (red), oligomeric (blue) and fibrillar (black) states relative to the rate of ROS production for free Cu 2+ (green bar). Fibrils generated in the presence of Cu 2+ were also evaluated (grey bars). The relative initial rates obtained for all the protein species are very consistent across the three spectroscopic assays. All assays were performed at 25 °C. In all assays, the concentration of protein, Cu 2+ and ascorbate were 10 µM, 5 µM and 100 µM, respectively.