Unless otherwise noted, all data are representative of 3 individual experiments.
Chemicals, antibodies, plasmids, overexpression and RNAi-virus
Unless otherwise noted, chemicals were purchased from Sigma. Protein G and A agaroses were purchased from Oncogene. Antibodies were obtained from the following companies: anti-GAPDH monoclonal antibody from Biogenesis; anti-histone H2B antibody and anti-actin antibody from Upstate; anti-HA antibody from Babco; anti-Myc antibody from Calbiochem; anti-nNOS antibody from BD Pharmingen anti-HA antibody from Covance and anti-Siah antibody from Santa Cruz; anti-GFP antibody, anti-mouse Ig conjugated to FITC, and anti-rabbit Ig conjugated to rhodamine from Molecular Probes. Polyclonal antibody against GOSPEL was generated with the following procedures: GST-tagged GOSPEL was produced in E. coli
, purified through glutathione-sepharose beads (Pharmacia Biotech, Piscataway, NJ), and used for antigen. Antisera were produced by a service of Research Genetics, and the sera were immunopurified. Constructs expressing site-directed or deletion mutant proteins were prepared according to the published protocol (Ho et al., 1989
GOSPEL RNAi mediated by a lentiviral system (target sequence: 5′-AAGGCTCTGATTGAGCAGAAG-3′) was generated as follows: the target sequences were cloned in the pFUGW vector, and this construct was transfected in HEK293FT cells along with two other helper/packaging vectors (Δ8.9 and VSVg constructs) to produce lentiviral particles. For the production of control lentivirus, FUGW vector was transfected into HEK293FT cells along with two other helper/packaging constructs. Viral production was allowed to take place for 48-72 h inside HEK293 FT cells, and the supernatant was collected and ultracentrifuged to bring down viral particles, which were resuspended into OPTI-MEM. About 106-107 pfu/ml viruses were infected in primary neurons for 24 h. Viral expression was confirmed by observing GFP encoded in pFUGW backbone, 5 days after infection. Wild-type HA-GOSPEL and HA-GOSPEL-ΔN200 expression by a lentiviral system was prepared as follows: both inserts were cloned in pENTR/D-TOPO vector (invitrogen), and then inserted in pLenti4/TO/V5-DEST vector (invitrogen) individually by homologous recombination (according to the manufacturer's protocol). The final constructs were transfected in HEK293T cells along with two other helper/packaging vectors (Δ8.9 and VSVg constructs), and the supernatant was collected and ultracentrifuged to bring down viral particles, which were resuspended into OPTI-MEM. About 107-108 pfu/ml viruses were infected in primary neurons for 24 h. Viral expression was confirmed by doing Western blotting with an HA-antibody 5 days after infection.
Yeast two-hybrid and cDNA library screenings
The N-terminal (amino acids 1-150) of rat GAPDH open reading frame were cloned into yeast expression vector pPC97, containing the Gal4 DNA binding domain. These constructs were used for the screening of a rat hippocampal cDNA library cloned into pPC86, containing the Gal4 transactivation domain. An adult rat brain cDNA library in lambda ZAPII vector (Stratagene) was screened using the yeast two-hybrid fragment for a portion of GOSPEL open reading frame, labeled with [32P]dCTP using a random priming DNA labeling kit (Boehringer-Mannheim). A total of 1 × 106 clones were screened, yielding 61 overlapping clones.
Northern Blotting and in situ hybridization
cDNA fragment utilized as a probe was labeled with [32
P]dCTP using the High Prime DNA-labeling system (Roche Diagnostics), and Northern hybridization was performed at 68°C for 2 h in QuikHyb solution (Stratagene) with a tissue mRNA blot membrane (CLONTECH) (Burnett et al., 1998
binding experiments were performed according to the published protocol with glutathione-sepharose (Bae et al., 2005
; Kamiya et al., 2005
). Co-immunoprecipitation and Western blotting were conducted as published previously (Bae et al., 2005
; Kamiya et al., 2005
). Subcellular fractionation was carried out with Nuclear/Cytosol Fractionation Kit (BioVision). S
-nitrosylation biotin switch assay was conducted as published previously (Forrester et al., 2007
; Jaffrey and Snyder, 2001
). Trans-nitrosylation assay was performed as follows: purified wild-type GAPDH (1.5 μg) purchased from Sigma was incubated with 200 μM GSNO at 37°C for 1 h. The whole reaction mixture was then passed through a desalting column (Pierce) to remove free GSNO. S
-nitrosylation of GAPDH was confirmed by biotin switch assay. For the trans-nitrosylation reaction, 0.1, 1.0 or 2.5 μg of GST-GOSPEL were incubated with S
-nitrosylated GAPDH in 4°C for 1 h with mild rotation in 1 ml of buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1 mg/ml BSA, and 0.1% CHAPS). The whole solution was used for the biotin switch assay to detect S
-nitrosylation of GOSPEL protein.
In cell-free binding assay we have purified GAPDH, GOSPEL, and Siah protein separately. GAPDH and GOSPEL were incubated separately with 200 μM GSNO at 37°C for 1 h. The whole reaction mixture was then passed through a desalting column (Pierce) to remove free GSNO. Then SNO-GAPDH and SNO-GOSPEL were mixed 1:1 molar ratio. On top of this mixture, GST-Siah was added in a concentration dependent manner (0-500 nM) and kept at 37°C for 1 h. In another set of experiments we incubated SNO-GADH and Siah along with different concentration of GST-GOSPEL (0-100 nM) at 37°C for 1 h. Then we conducted GST-pull down assays and western blots for GAPDH. After measuring the optical density of GAPDH, we calculated IC50 values for inhibition of binding between GAPDH and GOSPEL by Siah as well as binding between GAPDH and Siah by GOSPEL.
For quantitative PCR total RNA was isolated from granule neuron with the RNeasy mini kit (Qiagen) and was reversely transcribed with the Omniscript RT kit (Qiagen). Real-time PCR was performed using SYBR green PCR master mix for OAS1
. The primers used for OAS1 amplification are as follows: forward primer for OAS1 is 5′-CCATCCTCAAGTGGACAAGAACTG-3′; reverse primer for OAS1 is 5′-TTGGGCTTTGGGCACCTTC-3′ (Bridge et al., 2003
Enzymatic assay for GAPDH
GAPDH (10 nM) or S-nitrosylated GAPDH were preincubated with either GOSPEL (100 nM) or Siah (500 nM) at 37 °C for 30 min. S-nitrosylated GOSPEL (100 nM) was also preincubated separately with 10 nM GAPDH or S-nitrosylated GAPDH in the presence or absence of Siah at 37 °C for 30 min. Then “assay mixture” was added in each tube and the incubation continued at 37 °C for 10 min. The assay mixture contained 10 mM sodium pyrophosphate (pH 8.5), 20 mM sodium arsenite, 2 mM NAD+, and 2 mM glyceraldehyde 3-phosphate. Optical density was measured at 340 nm. All values are normalized to control and expressed as arbitary units.
Cell culture and staining
HEK293 cells and HEK293FT cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS and 2 mM L-glutamine at 37°C with a 5% CO2
atmosphere in a humidified incubator. PC12 cells were maintained in DMEM with 10% FBS, 5% horse serum, 2 mM L-glutamine, and 100 U/ml PS in the same environment. Primary cortical neurons were prepared from fetal mice (14-day gestation) derived from wild-type and nNOS knockout mice, and cultured in Neurobasal media supplemented with B27, 2 mM L-glutamine, and 100 U/ml PS at 37°C with 5% CO2
atmosphere in a humidified incubator. Cerebellar granule neurons were prepared from neonatal mice and cultured in Basal Eagle Medium (BEM) containing 10% FBS, 25 mM KCl, and 2 mM L-glutamine. Immunocytochemistry using confocal microscopy was performed as described (Sawa et al., 1999
). Confocal microscropy images were obtained using a Zeiss Meta Confocal System. For neuron cultures anti-GFP antibody was used to enhance the signal from transfected cells.
Measurement of NMDA-induced neurotoxicity in primary neurons
To activate NMDA receptors in neurons, 8-10-day-old cultures (DIC 8-10) were treated with Mg2+ free Earle's Balanced Salt Solution (EBSS) containing 300 μM NMDA and 5 μM glycine for 10 min.
Three days prior to NMDA receptor activation, cerebellar granule neurons were transfected with plasmids as follows: after pre-incubation of cells with Neurobasal (Gibco BRL) including B27 for 24 h from DIV 5, neurons were transfected with 1 μg of GFP construct and a total of 3 μg of varied combinations of plasmids using lipofectamine 2000. The molar ratio of expression constructs to GFP plasmid was 3:1. Using fluorescent microscopy with a digital camera, we captured images of more than 20 fields per preparation, which were randomly chosen in a blind manner. GFP positive neurons were tallied in each field and added together to determine the percentage of viable cells compared to control.
Three days prior to NMDA receptor activation, RNAi-lentivirus was infected into granule or cortical neurons. Viability of neurons was assayed 12 h or 24 h after exposure to NMDA as follows: cells were washed with PBS and incubated in 1 μg/ml propidium iodide for 10 min (dead cells are stained in red). After extensive washing to remove non-specifically attached propidium iodide to cell debris by PBS, neurons were then fixed in 4% paraformaldehyde in PBS and stained with DAPI to visualize the total cell population. As the majority of neurons were infected, the ratio of propidium iodide-stained cells to DAPI-stained cells was employed to reflect toxicity.
Measurement of nitric oxide species
To measure reactive nitric oxide level inside cells, we used 4,5 diamino fluorescein diacetate (DAF-FM, Molecular probes), a cell permeable derivative of the indicator DAF-2 whose fluorescence reflects the level of endogenous nitric oxide (Choi et al., 2000
; Chvanov et al., 2006
). Briefly, neurons were washed once with Hanks Balanced Salt Solution (HBSS) and then were loaded with 5 μM DAF-FM in HBSS and incubated at 37°C with 5% CO2
atmosphere in a humidified incubator for 60 min. NMDA (300 μM) and glycine (5 μM) were added to the cells and incubated for another 10 min. Cells were washed, harvested in HBSS and fluorescence was measured in a fluorimeter utilizing excitation 485 nm and emission 530 nm. DAF-2 fluorescence was also detected using a laser scanning confocal microscope (Zeiss Meta).
Animals and MPTP Treatment
All experiments were approved and conformed to the guidelines set by the Institutional Animal Care Committee. Eight-week-old male CD1 mice (The Jackson Laboratory) were used. Mice received four i.p. injections of MPTP-HCl (20 mg/kg free base; Sigma) in saline at 2-h intervals and were sacrificed after 16 h and 24 h after the first injection (n
= 5). Control mice received saline only. In each case the striatum was isolated and was used for imunoprecipitation and S
-nitrosylation experiments (Hara et al., 2006b
Stereotaxic injection into intact mice
Adult male C57BL/6 mice (20-25 g; Charles River, Wilmington, MA) were used for NMDA mediated cytotoxicity measurement. Animal protocols, approved by the Institutional Animal Care and Use Committee of Johns Hopkins University, were used in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Animal well being was determined by monitoring the weight and rectal temperature before the surgical procedure. Mice were anesthetized with 3.0% isoflurane, maintained with 1.0% isoflurane and mounted on a stereotaxic frame (Stoelting Co., Wood Dale, IL). The skull was exposed and 5.0 μl (107-108 pfu/ml) of viral particles of either full length GOSPEL (n=8) or delta-N200 GOSPEL (n=9) protein was slowly injected into the cortex (anterior 0.5 mm, lateral 3.5 mm from bregma, and ventral 1.0 mm relative to dura) over a period of 20 min with the help of a 5.0 μl Hamilton® syringe, and the needle was left in place for additional 5 min. Seven days after viral infection, overexpressed full length GOSPEL or delta-N200 GOSPEL in coronal sections were detected by western blot and immunohistochemistry with anti-HA antibody (1:200). On day 8, 15 nmol NMDA was slowly injected at the same location with 1.0 μl Hamilton® syringe. After each injection, mice were placed in a thermoregulated chamber maintained at 31 ±0.5°C and returned to their cages after full recovery from anesthesia. The rectal temperatures were monitored and maintained at 37.0 ± 0.5 °C during the experimental procedure.
NMDA-induced lesion quantification
At 24 h post-NMDA injection, mice were trancardially perfused with 0.1 M PBS and fixed with 4% PFA. Brains were harvested and soaked in 4% PFA overnight, then equilibrated with 30% sucrose. Sequential brain sections of 25 μm obtained on cryostat were stained with cresyl violet to estimate the lesion volume as described previously (Ahmad et al., 2006
; Ahmad et al., 2007
). One-way ANOVA followed by Tukey's post-hoc analysis was used to calculate the difference between the groups.