|Data collection method:
TAPI-1, Amyloid Precursor Protein β-Secretase Inhibitor (βSI), and β-Secretase Inhibitor IV (βIV) were purchased from Merck Millipore. DAPT was purchased from Tocris. TAPI, βSI, βIV, and DAPT were made up as 1000x concentrated stocks in DMSO (Sigma).
Plasmids encoding wildtype α-synuclein (RefSeq accession number XM_011532204) and β-synuclein (RefSeq accession number NM_001001502) in a pcDNA 3.1 (+) vector were previously described (1,2). A pCI-neo-APP695 plasmid was kindly donated by Prof. Chris Miller, Kings College London (3). Steap3 plasmid was generated by cloning the human Steap3 sequence (RefSeq accession number NG_042823.1) into a pcDNA 3.1 (+) vector. pFR-Luciferase reporter vector (pLuc) containing firefly (Photinus pyralis) luciferase gene, with a synthetic promoter of yeast Gal4 upstream activation sequence in 5 tandem repeats upstream of a minimal TATA box, was from Promega. phRL thymidine kinase vector (pTK) containing sea pansy (Renilla reniformis) luciferase gene, under control of the herpes simplex virus-TK promoter, was also from Promega. pRC-CMV-APP695-Gal4 (APP-Gal4) and pSec-Tag2-Notch3Gal4 (Notch-Gal4) were kindly provided by Dr Robert J. Williams, University of Bath (4,5). Human BACE1 promoter luciferase reporter, a pGL3-Basic vector containing a 4.3kb fragment of BACE1 promoter from -4372 to -1 of the promoter sequence, was previously described (6).
Cell Cultures and Transfection
SH-SY5Y neuroblastoma cells (obtained directly from ATCC, cat no. CRL-2266) were grown in 1:1 DMEM (high glucose with L-Glutamine, Lonza) and Ham’s F 12 (Lonza), supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma). Growth conditions were maintained at 37°C and 5% CO2 in a humidified incubator. Cells were stably transfected with pcDNA 3.1 (+)-α-synuclein, or mutations thereof. The mutants included truncations Δ2-9, at the extreme N-terminus, and Δ71-82, in the NAC domain of α synuclein. PD-associated point mutations A30P, E46K and A53T were also included. Additionally two substitutions of S129, a phosphorylation site in the C-terminus of α-synuclein. S129A blocks phosphorylation, whereas S129D aims to mimic permanent phosphorylation. Transfection was achieved using FuGene HD lipid reagent (Promega) according to the manufacturer’s instructions. Stable selection was performed with 0.8 mg/ml G418 (Sigma) 24 hours after transfection, and cells maintained in 0.4 mg/ml G418. Successful transfections were assessed by western blotting to ensure over-expression of the protein.
Rabbit monoclonal anti-α-synuclein (MJFR1, Abcam, immunogen human α-synuclein 1-150) was used for human α synuclein detection at a dilution of 1:4000. Mouse monoclonal anti-α-synuclein (610787, BD Biosciences, immunogen rat α-synuclein 15-123) was used for rat α-synuclein detection at a dilution of 1:2000. Mouse monoclonal anti-α tubulin (T5186, Sigma, immunogen acetylated tubulin from Strongylocentrotus purpuratus sperm axonemes) was used at a dilution of 1:10,000. Rabbit monoclonal anti-APP C-terminus (Y188, Abcam immunogen human APP750 C-terminus) was used at a dilution of 1:2000. Rabbit monoclonal anti-BACE1 (D10E5, Cell Signaling Technology immunogen human BACE1 residues around His490) was used at a dilution of 1:1500.
Cells were lysed in 0.5% Igepal CA-630 and ‘complete’ protease inhibitor cocktail (Roche), sonicated 3 x 3 seconds on ice, and centrifuged 10 000 xg for 3 minutes to remove insoluble membranes. Protein concentration was determined with a Bradford protein assay (Bio-Rad), according to the manufacturer’s instructions. Supernatant protein concentrations were normalized and boiled for 5 minutes with 1 x Laemmli SDS-PAGE buffer. To determine levels of α-synuclein, full-length APP, or BACE1: samples were loaded into a 12% acrylamide SDS-PAGE gel, with a buffer of Tris (250 mM) + Glycine (1.92 M) + SDS (0.1% w/v), run at 250V for 45 minutes. To resolve bands of C99 and C83 APP: samples were loaded into a 16% acrylamide + 10% glycerol SDS-PAGE gel, with an anode buffer of Tris-HCl (20 mM, pH 8.9), and a cathode buffer of Tris (100 mM) + Tricine (100 mM) + SDS (1% w/v). The 16% gel was electrophoresed at 100 V, 4 °C, for several hours. Separated proteins were transferred to a PVDF membrane by a semi-dry transfer apparatus, run at 25V for 1.3 hours. Membranes were blocked in 5% w/v non-fat milk powder dissolved in TBS-T (0.05% Tween-20, 10 mM Tris, 100 mM NaCl) for 30 minutes, incubated with primary antibody for 1-2 hours, and washed 3 x 5 minutes in TBS-T. Membranes were blocked again and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. A further 3 x 10 minute washes were performed, and the membranes developed with Luminata Crescendo or Luminata Forte ECL substrate (Thermo Scientific), and imaged with a Fusion SL CCD imaging system (Vilber Lourmat).
Measurement of Aβ40 and Aβ42 by Meso Scale Discovery Assay
Fresh serum-free B-27-supplemented DMEM, ± compounds, was added to SH-SY5Ys a day after seeding in 24-well plates. Conditioned media was collected after 72 hours and immediately assayed without further manipulation, using the V-PLEX Plus Aβ Peptide Panel 1 (6E10) Kit from Meso Scale Discovery, according to the manufacturer’s instructions. The plate was read with a Sector Imager 6000 (Meso Scale Discovery). Peptide concentrations (pg/ml) were calculated by Meso Scale Discovery Workbench software, with reference to a standard curve, and were normalised to the mean concentration for each experiment.
Dual Glo Luciferase Reporter Assay
SH-SY5Ys in 24-well plates were co-transfected with plasmids complexed with 1.5 µl/well FuGene HD (Promega). For the APP-Gal4 reporter assay for amyloidogenic processing, cells were transfected with 50 ng each of APP-Gal4 plasmid, pLuc, and pTK. For measuring human BACE1 promoter activity, cells were transfected with 200 ng of BACE1 promoter luciferase reporter plasmid, and 50 ng pTK. For the Notch-Gal4 reporter assay for γ-secretase activity, cells were transfected with 100 ng of Notch-Gal4 plasmid, and 50 ng each of pLuc and pTK. Luciferase activity was measured 20-22 hours post-transfection using the Promega Dual-Luciferase Reporter Kit according to the manufacturer’s instructions. Raw data was normalised by division with the mean firefly or Renilla luminescence for that experiment. Relative Luciferase Units (RLU) were calculated for each well by division of the firefly signal by the Renilla signal. The average RLU for 3-5 replicate transfections was calculated in each experiment.
Oxidative Stress Assay
Cells were seeded at a density of approximately 1x106 cells/ml onto a poly-D-lysine-coated 48-well plate. After 48 hours, cells were incubated with 10 µM CM-H2DCFDA probe in HEPES-buffered media (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1.2 mM CaCl2, 5.5 mM glucose) for 20 minutes. The probe was removed and 300 µl of HEPES-buffered media added. Fluorescence intensity (Ex/Em= 488/534 nm) was measured every 5-10 minutes for 60 minutes. The linear rate equation was determined from a kinetics plot using MS Excel, and the rate at 60 minutes calculated. For each experiment, rates were normalised to the average.
Viral Vector Production and Titration
Serotype 6 adeno-associated viral (AAV6) vectors were produced and titrated as previously described (7). The number of transducing units (TU) was determined by infecting HEK293T cells. The number of S1-nuclease resistant vector genome copies was measured by real-time PCR at 48 hrs post-infection. The AAV6-α-syn vector encodes expression of full-length wild-type human α-syn under the control of the constitutive mouse pgk-1 promoter. The titre of the vector suspension was 7.9E10 TU/ml.
All procedures were performed in accordance with Swiss legislation and the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. Female adult Sprague-Dawley rats (Charles River Laboratories, France), weighing 180-200 g, were housed in a 12-hour light-dark cycle, with ad libitum access to water and food. Rats were injected with empty or α-synuclein-encoding AAV6 vectors in the right substantia nigra, with the non-injected left acting as an internal control. The total injected dose for each animal was 2.8E7 TU in a volume of 2 µl. The general procedure for vector injection was as previously described (7). We used the following stereotaxic coordinates: 5.2 mm anterior and 1.9 mm lateral to bregma point, 7.9 mm ventral from the skull surface. The injected rats were culled one month after injection. For biochemical analysis, fresh striatal tissue was obtained from each brain hemisphere separately. Neuropathology and motor deficits of the rat model have been previously characterised (7, 8, 9). Additionally, brain α-synuclein expression and aggregation were visualised by immunohistochemistry. For immunohistochemistry, the rats were sacrificed three months post-injection, and the brains were perfused with 4% paraformaldehyde (1.5 h post-fixation), before being transferred to 25% sucrose solution. Immunostaining for tyrosine hydroxylase (Millipore, #AB152; 1:1000), total α-syn (Millipore #AB5334P; 1:1000) and aggregated α-syn (clone 5G4, Millipore #MABN389; 1:1000) were performed on striatal sections (25 µm) from the rat striatum and substantia nigra, using standard procedures.
|Data processing and preparation activities:
||Statistical analysis was performed using MS Excel. With the exception of the rat material, all data were analysed by unpaired two-tailed Student’s t-tests, with an assumption of equal variance. Rat striata were analysed by a paired two-tailed Student’s t-test. Differences were defined as statistically significant when p < 0.05.
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Methodology and documentation files