Download Supplementary Information (doc 38K)

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 controltreated 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 TBST, 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 antiGAPDH 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 nonessential 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 -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 24well 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 vectorinjected hippocampal samples) and reaction buffer (20 mM MgCl2, 100 mM Lglutamate, 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-γglutamylhydroxamate/mg protein/hour. Reaction mixtures lacking ATP or Lglutamate, 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, pAMGFAP-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-[3,43H]-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 GFAPEAAT2:empty was calculated.