Supplementary Information

Supplementary Data.

In vitro testing of the functionality of the epitope-tagged GS and EAAT2 transgenes

We assessed GS activity in HEK293 cell lysates that had been transfected with AAV expression plasmids expressing HA-tagged GS under the control of the strong constitutive CAG promoter or the GFAP promoter. The GFAP promoter had minimal transcriptional activity in this cell line, with GS activity levels similar to that found in non-transfected cells or cell transfected with a destabilised yellow fluorescent protein (dYFP) reporter plasmid (Suppl. Fig a). However high levels of GS activity were evident when the same transgene was placed under control of the CAG promoter (One way ANOVA, F3,8=12062, P<0.001, Tukeys post-hoc analysis CAG-GS versus all treatment groups P<0.0001) (Suppl. Fig. a). These results are in line with the protein expression levels observed by immunocytochemistry using anti-HA and GS antibodies on transfected cells (Suppl. Fig b).

We confirmed the functionality of the EAAT2 transgene using an in vitro rat C6 glioma cell model to quantify [3H]-glutamic acid uptake in cells overexpressing EAAT2 relative to control-treated cells. AAV9 vectors transduced this cell line with low efficiency thus masking any detection in difference in glutamate transport between the two treatment groups (results not shown). We therefore transfected C6 glioma cells with the EAAT2 plasmid used for AAV vector packaging and measured [3H]-glutamic acid uptake at 48 h after transfection. We found a 1.5-fold increase in the rate of [3H]-glutamic acid uptake in EAAT2-transfected cells compared to control-treated cells confirming functionality of the FLAG-tagged transgenic EAAT2 (Suppl. Fig c).

Supplementary Figure legend

(a) GS activity in non-transfected HEK293 cells and cells transfected with HA-tagged GS and dYFP plasmids; (b) HA and GS immunoreactivity in transfected HEK293 cells; (c) Glutamate uptake in C6 glioma cells transfected with pAM-GFAP-EAAT2 or pAM empty plasmid. Bars represent the mean + SEM for mean data over 3 independent experiments. Unpaired t-test, *P = 0.03.

Supplementary methods and materials

Western blot analysis

For the ADK Western blot analyses, 30 ug of protein was separated per lane of a 12% SDS-polyacrylamide gel followed by transfer to nitrocellulose. The membrane was blocked with 5% (w/v) skim milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and incubated with rabbit anti-ADK antibody (1:500, sc-32908, Santa Cruz Biotechnology, Dallas TX) overnight at 4 oC. Following washes in TBS-T, horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000, sc-2004, Santa Cruz) was applied for 2 h before detection of immunoreactive bands with ECL reagent (Amersham). Membranes were then incubated sequentially with mouse anti-GAPDH antibody (1:50,000, ab8245, Abcam) and horseradish peroxidase conjugated anti-mouse antibody (1:5,000, sc-2005, Santa Cruz). Bands were visualized using a LAS-4000 Image reader (Fuji) and density measurements of the ADK band relative to GAPDH conducted to obtain a measure of the extent of knockdown of ADK in each injected hippocampus.

For Western blot analysis of GS and EAAT2 levels, 10 ug of hippocampal or HEK293 cell lysate (as prepared for the GS assay) was resolved by 10% SDS-PAGE, transferred to Hybond-ECL membrane (GE Healthcare), and blocked and probed as described above. Primary antibodies used were rabbit anti-glutamine synthetase (1:10,000, ab49873, Abcam), mouse anti-GAPDH (1:50,000, ab8245, Abcam), rabbit anti-GFP (1:5,000, ab290, Abcam), rabbit anti-HA (1:2,000, ab9110, Abcam), rabbit anti-luciferase (1:1,000, 70C-CR2029RAP, Fitzgerald Industries), mouse anti-FLAG (1:1,000, F3165, Sigma), and goat anti-EAAT2 (1:500, sc-7760, Santa Cruz). Following washes in TBS-T, membranes were incubated in the appropriate horseradish peroxidase-conjugated goat secondary antibody (1:5,000, Santa Cruz). HRP labeled proteins were detected using ECL prime detection reagent (GE Healthcare Life Sciences). For semi quantitation of EAAT2, blots were sequentially probed with GAPDH following EAAT2 chemiluminescent detection. For fluorescent detection of transgenic and endogenous GS, a mixture of mouse anti-HA (1:500, MMS-101P, Covance, Princeton, NJ) and rabbit anti-GS (1:10,000, ab49873, Abcam) were applied and HA and GS labeling detected simultaneously following the application of a mixture of Cy2-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse (both 1:5,000, Jackson ImmunoResearch) secondary antibodies. Multiplex fluorescent detection was used to quantify the level of GS protein by Western blot. Rabbit anti-glutamine synthetase (1:10,000) and mouse anti-GAPDH (1:10,000) were applied simultaneously and proteins detected following the application of a mixture of Cy2-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse (both 1:5,000, Jackson ImmunoResearch) secondary antibodies. Intensity of the GS or EAAT2 band was normalized to the intensity of GAPDH for each sample (GS/EAAT2 intensity:GAPDH intensity) and expressed as a percentage of the ipsilateral control samples. The average value for each sample was determined from three or four Western blot experiments. Imaging and analysis was completed using the ChemiDoc MP imaging system with Image Lab 4.1 software (Bio-Rad)

Immunocytochemistry

Transfected HEK293 cells were briefly fixed in 10% (w/v) neutral-buffered formalin (Sigma Aldrich, St Louis, MO) for 15 min and incubated in 1% (v/v) H2O2 in 100% methanol for 2 min. Rabbit anti-GS (1:2,500, ab49873, Abcam) or rabbit anti-HA (1:4,000, ab9110, Abcam) diluted in PBS containing 0.2% (v/v) Triton-X100 and 10% (v/v) horse serum was applied and incubated overnight at room temperature. Biotinylated donkey anti-rabbit (1:1000, Jackson ImmunoResearch, West Grove, PA) was then applied for 3 h at room temperature before incubation in ExtrAvidin Peroxidase (1:250, Sigma Aldrich) for 2 h. Sections were washed with PBS containing 0.2% (v/v) Triton-X100 between each incubation step. Immunoreactivity was visualized by incubation with diaminobenzidine, and images were captured on an Nikon TE2000 microsope using a digital camera and Picture Frame image capture software (Optronics, v 2.2).

Transient transfection of HEK293 cells

Human embryonic kidney 293 cells (HEK293) maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% (v/v) foetal bovine serum (FBS), 25 mM HEPES, 0.5 mM L-glutamine, 44 mM NaHCO3, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate were plated into 24-well plate or 10 cm dish format and transfected with AAV plasmids expressing an identical GS transgene under the control of either the cytomegalovirus enhancer/chicken b-actin (pAM-CAG-GS) or GFAP (pAM-GFAP-GS) promoter using the X-tremeGENE HP DNA transfection reagent (Roche). Cells in 24-well plate and 10 cm dish format were transfected with 0.5 µg and 10 µg of plasmid respectively, using a 1:2 (w/v) mixture of plasmid and transfection reagent. Forty eight hours after transfection cells in 24-well plate format were used for immunocytochemistry and cells in 10 cm dishes were harvested for GS assay.

Glutamine synthetase assay

Equal volumes (0.05 mL) of homogenate (transfected HEK293 cell lysate or vector-injected hippocampal samples) and reaction buffer (20 mM MgCl2, 100 mM L-glutamate, 200 mM imidazole-HCl pH 7.4, 20 mM 2-mercaptoethanol, 100 mM hydroxylamine-HCl pH 7.4, 20 mM ATP; pH to 7.4) were incubated at 37°C for 30 min before 0.2 mL of stop solution was added (370 mM FeCl3, 670 mM HCl, 200 mM trichloroacetic acid). Samples were centrifuged at 12,000 g for 3 min at room temperature and absorbance measured at 530nm (BioTek Synergy 2 with Gen5 v. 1.09 software). Absorbance of samples was determined from a standard curve generated by the reaction of 0.05 mL of L-γ-glutamylhydroxamate standard treated with stop solution, and glutamine synthetase activity expressed as mmol L-γ-glutamylhydroxamate/mg protein/hour. Reaction mixtures lacking ATP or L-glutamate, or reactions at zero time, were used as negative controls. Readings at 30 min minus background (zero time) were expressed as the percentage of glutamine synthetase activity of the contralateral non-injected side. Duplicate or triplicate reactions were prepared for each assay and samples assayed in 2-3 independent experiments.

Glutamate uptake assay

Rat C6 glioma cells (50,000 cells/well) in high glucose Dulbecco’s modified Eagle’s medium with HEPES and L-glutamine (Life Technologies) and supplemented with 1 mM sodium pyruvate (Life Technologies) and 10% fetal bovine serum (HyClone, Tauranga, New Zealand) were transfected with 1 μg pAM-GFAP-EAAT2, pAM-GFAP-GFP or pAM-GFAP-empty plasmid (consisting of pAM backbone only) using FuGene HD (Promega, Madison, WI) as per manufacturer’s instructions. Forty hours after transfection, cells were washed twice in HBSS (containing NaCl to measure glutamate uptake or choline chloride to measure non-specific uptake) for 5 min at 37°C in a humidified incubator. HBSS: 140 mM NaCl or choline chloride, 5 mM Tris base, 10 mM HEPES, 2.5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 1.2 mM K2HPO4, 10 mM dextrose; pH 7.4). L-glutamic acid (0.5 μM, Sigma Aldrich) and 5 nM L-[3,4-3H]-glutamic acid (49 Ci/mmol, 1 mCi/mL; Perkin Elmer) in HBSS were added and the cells incubated for zero or 10 min at 37°C in a humidified incubator. Uptake was stopped by rinsing three times with ice-cold HBSS and cells were lysed in 250 μL 0.1 M NaOH for 30 min at room temperature. Lysate was combined with 2.5 mL scintillation fluid (Perkin Elmer Betaplate Scint) and radioactivity measured using a scintillation counter (Perkin Elmer Wallac Trilux 1450 MicroBeta with Wallac 1450 MicroBeta Windows Workstation software v. 4). Protein concentration of parallel transfected wells was determined using a Pierce BCA protein assay (Thermo Scientific) of 0.1 M NaOH lysed cells. Data from duplicate samples and 3 independent experiments was averaged, non-specific uptake by HBSS with choline chloride was subtracted, and protein assay data used to calculate fmol glutamate/mg protein/min. Zero minute time-point data were subtracted, and a ratio of GFAP-EAAT2:empty was calculated.