Full-length properly gamma-carboxylated mouse recombinant PS was prepared and characterized as we described (Fernandez et al., 2009
). Human plasma-derived PS was purchased from Enzyme Research Laboratories (South Bend, IN). LY294002 and U0126 were purchased from Cell Signaling Technology (Danvers, MA). Pifithrin-α (PFT-α) and caspase inhibitors, Ac-DEVD-CHO (caspase-3), z-IETD-fmk (caspase-8) and z-LEHD (caspase-9), were purchased from Sigma-Aldrich (St Louis, MO).
Primary mouse cortical cells were isolated from mouse brain as described (Bonfoco et al., 1995
; Guo et al., 2004
). Briefly, cerebral cortex was dissected from fetal mice at exactly 16 days of gestation, treated with trypsin for 10 min at 37°C and dissociated by trituration. Dissociated cell suspensions were plated at 5 × 105
cells per well on 12-well tissue culture plates coated with poly-D-lysine, in serum-free Neurobasal medium plus B27 supplement. As we reported, astrocyte growth was suppressed between 0.3% and 1% (Guo et al., 2004
; Guo et al., 2009a
; Guo et al., 2009b
). Cultures were maintained in a humidified 5% CO2
incubator at 37°C for 7 days in vitro
(DIV7) or for 21 days in vitro
(DIV21) to allow neurons to mature, as reported (Zhong et al., 1994
; Lesuisse and Martin, 2002a
; Lesuisse and Martin, 2002b
NMDA injury model
Neuronal cultures were treated for 10 min with 300 μM NMDA/5 μM glycine in Mg2+-free Hank’s balanced salt solution (HBSS) or HBSS alone (controls) followed by incubation with different concentrations of PS (i.e., from 5 to 100 nM) for up to 24 h in serum-free Neurobasal medium plus B27 supplement. NMDA was purchased from Sigma-Aldrich.
Neuritic beading was assessed after incubation of NMDA-treated neurons with or without PS with Cell Tracker Green CMFDA (Invitrogen, Carlsbad, CA) for 30 min at 37°C. Cells were fixed with 4% paraformaldehyde for 10 min followed by immunostaining with mouse monoclonal anti-bovine Map2 antibody which cross reacts with mouse Map2 (1: 500, Chemicon, Billerica, MA). AlexaFluor 568 donkey anti-mouse IgG (1:150; Invitrogen, Carlsbad, CA) was used as a secondary antibody. Images were scanned using a Zeiss 510 meta confocal microscope with a 488 nm Argon laser to detect Cell Tracker Green and a 543 nm HeNe laser to detect AlexaFluor 568 for Map2.
Mitochondrial membrane potential
We used a MitoTracker Red CMXROs (Invitrogen, Carlsbad, CA) to assess mitochondrial membrane potential. Neurons were incubated with 20 nM MitoTracker for 30 min at 37°C. Cells were fixed with 4% paraformaldehyde for 10 min. Images were scanned using a Zeiss 510 meta confocal microscope with a 543 nm HeNe laser. Fluorescent signal intensity was quantified with MetaMorph software (Molecular Devices, Downingtown, PA). Relative signal intensity was expressed as a percentage of control.
We used a luminometric assay (APOSENSERTM Cell Viability Assay Kit, BioVision, Mountain View, CA) to assess intracellular ATP levels as described (Takeuchi et al., 2005
). Briefly, the NMDA-treated neurons with or without PS were lysed and incubated with 100 μL of the Nuclear Releasing Reagent at room temperature for 5 min. 1 μL ATP Monitoring Enzyme was added to the cell lysate for 1 min and then the samples were read in a luminometer (PerkinElmer, Waltham, MA). ATP concentration at each time point was calculated as a percentage of the non-treated control.
Neuronal viability was detected by WST-8 assay (Dojindo Molecular Technologies, Gaithersburg, Maryland), which is a tetrazolium-based assay measuring the activity of the dehydrogenases in cells. The amount of the water-soluble formazan dye generated in the assay is directly proportional to the number of living cells. The cell survival rate was expressed as the viability percentage of the vehicle-treated cells.
TUNEL and Hoechst staining
Apoptosis was assessed by TUNEL (DeadEndTM Fluorometric TUNEL System, Promega, Madison, Wisconsin) and Hoechst (33,342; Molecular Probes, Eugene, OR) staining using acetone-fixed cells. Images were observed using a Zeiss 510 meta confocal microscope. The number of apoptotic cells was expressed as the percentage of TUNEL-positive cells of the total number of nuclei determined by Hoechst staining.
Caspase-9, caspase-8 and caspase-3 activities
The caspase-9, caspase-8 and caspase-3 activities in neuronal cell lysates were determined using caspase-9, caspase-8 and caspase-3 Colormetric Assay Kits (BioVision, Mountain View, CA). Approximately 200 μg of protein was incubated with DEVD-pNA (for caspase-3; 200 μM), IETD-pNA (for caspase-8; 200 μM) or LEHD-pNA (for caspase-9; 200 μM) and 10 mM DTT at 37°C for 2 hr. Substrate hydrolysis was determined as absorbance change at 405 nm in a microplate reader. Enzymatic activity was expressed in arbitrary units (optical density, OD) per mg protein.
Neuronal cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) with protease inhibitors. Nuclear proteins were extracted using NE-PER nuclear extraction reagent (Pierce Biotechnology, Rockford, IL). Proteins (20–50 μg) were analyzed by 4–15% Tris-HCL gel and transferred to nitrocellulose membranes (0.45 μm, Bio-Rad Laboratories, Hercules, CA), that were then blocked by 5% non-fat milk or 5% BSA in TBS for 1 h. The membranes were incubated overnight with primary antibodies diluted in 5% non-fat milk or 5% BSA in TBS, and then washed and incubated with a HRP-secondary antibody for 1 h. Immunoreactivity was detected using the ECL detection system (Amersham, Piscataway, NJ). We used the following primary antibodies: rabbit polyclonal anti-human AIF antibody which cross reacts with mouse AIF (1:1000, Cell Signaling Technology); rabbit polyclonal anti-mouse Phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology); rabbit polyclonal anti-mouse Akt antibody (1:1000, Cell Signaling Technology); rabbit polyclonal anti-mouse Phospho-Bad (Ser136) antibody (1:200, Cell Signaling Technology); rabbit polyclonal anti-mouse Bad antibody (1:1000, Cell Signaling Technology); mouse monoclonal anti-mouse Bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, California); rabbit polyclonal anti-mouse Bcl-XL antibody (Sigma-Aldrich); rabbit polyclonal anti-human phospho-Mdm2 (Ser166) antibody which cross reacts with mouse phospho-Mdm2 (Ser166) (1:1000, Cell Signaling Technology); rabbit polyclonal anti-human Mdm2 antibody which cross reacts with mouse Mdm2 (1:500, Abcam); rabbit polyclonal anti-human p53 antibody which cross reacts with mouse p53 (1:1000, Cell Signaling Technology); mouse monoclonal anti-mouse Bax (1:100, Santa Cruz Biotechnology); goat polyclonal anti-human β-actin antibody which cross reacts with mouse β-actin (1:1000, Santa Cruz Biotechnology); sheep polyclonal anti-human histone 1 antibody which cross reacts with mouse histone 1 (1:1000; United States Biological, Swampscott, MA). The relative abundance of proteins was determined by scanning densitometry and expressed relative to control groups that were arbitrarily assigned as 1.
Intracellular Ca2+ measurement
The intracellular calcium [Ca2+
levels in mouse cortical neurons during NMDA stimulation were measured using a calcium-sensitive fluorescent dye Fura-2 AM (Invitrogen) using a similar method as we previously described in brain endothelial cells (Domotor et al., 2003
). Briefly, neurons plated on poly-L-lysine coated coverslips were incubated with 2 μM Fura-2 AM for 20 min in Mg2+
-free HBSS at room temperature, then rinsed and incubated for 30 min at 37°C in Mg2+
-free HBSS. The coverslips were transferred to a Warner RC-25F perfusion chamber fitted to a stage of an inverted Nikon Eclipse Ti microscope and perfused with Mg2+
-free HBSS for 5 min. All reagents were infused via a multi-tube perfusion system. [Ca2+
was measured by digital image fluorescence microscopy (objective, Fluor 40/1.3; Nikon) using Vision 4.0 software from T.I.L.L. Photonics. Excitation wavelengths were 340 nm and 380 nm generated by a polychromator illumination system (T.I.L.L Photonics). Fluorescence emission was collected at 510 nm. A fluorescence ratio image (340 nm/380 nm) was acquired every 2 seconds with a CCD camera (T.I.L.L. Photonics) before, during and after HBSS infusion with vehicle, 100 nM PS, 300 μM NMDA or 300 μM NMDA with 100 nM PS. The images were analyzed with the NIH Image J software integrated density measurement tool. Three to eight individual cells in the image field were analyzed per coverslip and averaged. Six coverslips were analyzed per group. Using the Fura-2 Calcium Imaging Calibration Kit (Invitrogen) a standard curve was generated to convert the Fura-2 fluorescence values obtained from experimental samples into free Ca2+
concentrations using radiometric analysis according to manufactures instructions using the following formula: [Ca2+
=Kd × [(R−Rmin
−R)] × (F380max
) Where R is the ration of 510 nm emission intensity at 340 nm to 380 nm excitation; Rmin
is the ratio at zero free Ca2+
is the ratio at saturating Ca2+
(39 μM); F380max
is the fluorescent intensity using 380 nm excitation at zero free Ca2+
; and F380min
is the fluorescent intensity using 380 nm excitation at saturating free Ca2+
. Kd was calculated from the x-intercept of the plot of [Ca2+
] free on the x-axis versus [(R−Rmin
−R)] × (F380max
) on y-axis acquired from the calibration kit The free Ca2+
for experimental samples were then calculated from the corresponding R values. Calibrated data were pooled and plotted as means ± SEM.
Glutamate release from cultured cortical neurons was measured using an Amplex Red Glutamic Acid/Glutamate Oxidate assay kit (Invitrogen) (Nakatsu et al., 2006
; Kajimoto et al., 2007
). Cortical neurons were maintained for 7 days in vitro
and treated with NMDA with or without PS. The medium was collected and analyzed for glutamate content according to the manufacturer’s instructions. The resulting increase in fluorescence was measured at an excitation of 540 nm and emission of 590 nm using a fluorescence microplate reader (PerkinElmer).
Akt kinase activity assay
To assess Akt kinase activity, cells were washed twice in cold PBS, and lysed in Cell Lysis Buffer (supplied in the Akt Kinase Activity Kit, Cell Signaling Technology) with protease inhibitors. Immunoprecipitation was carried out for 18 h using the immobilized anti-Akt1G1 mAb (supplied with the kit) cross-linked to agarose. Immunoprecipitates were washed three times with lysis buffer and twice with Akt kinase buffer (supplied with the kit). Kinase assays were performed for 30 min at 30 °C under continuous agitation in kinase buffer containing 200 μM ATP, 1 μg of GSK-3 fusion protein (supplied with the kit), according to the manufacturer’s instructions for the non-radioactive Akt kinase assay. Samples were analyzed by Western blotting using phospho-GSK-3α/β (Ser21/9) antibody (supplied with the kit) as the primary antibody and a HRP-conjugated goat-anti rabbit IgG antibody (DAKO) as the secondary antibody.
The kinase-inactive AktK179A
construct (Crowder et al., 1998
) was cloned into a GFP-containing adenoviral vector using AdEasyTM XL system (Stratagene, Cedar Creek, Texas). The Adenoviral product containing AktK179A
was proliferated in HEC 293A cells purchased from ATCC (Manassas, Virginia) and purified using ViraKitTM (Virapur, San Diego, CA). Cortical neurons were transduced with adenoviral constructs (200 MOI) 24 h before studies. The transduction efficiency was determined by GFP signal and Immunoblotting analysis of total Akt.
Silencing through RNA interference
Small interfering RNA (siRNA) targeting mouse Mdm2, Bad, S1P1, Tyro3 and negative control siRNAs were purchased from Invitrogen. siRNAs were delivered to the mouse cortical neurons by using Lipofectamine provided by Invitrogen. After 48 hours of transfection, neurons were verified for target gene knock-down by immunoblotting analysis and subjected to NMDA treatment. The following pooled sequences of siRNA oligonucleotides were used for targeted gene knockdown: Bad, GACGACGUG UCUCAUGGCAGAGUUU and AAACUCUGCCAUGAGACACGUCGUC; Mdm2, AGGCUUGGAUGUG CCUGAUGGCAAA and UUUGCCAUCAGGCACAUCCAAGCCU; S1P1, GGCAUGGAAUUUAGCCGCAGCAAAU and AUUUGCUGCGGCUAAAUUCCAUGCC; Tyro3, GCAGACGCCAUAUGCUGGCAUUGAA and UUCAAUGCCAGCAUAU GGCGUCUGC.
Bad/Bcl-2 and Bad/Bcl-XL complexes
Neurons were lysed in the Immunoprecipitation Kit Lysis Buffer (Roche, Pleasanton, CA), sonicated at 4°C for 30 min and centrifuged at 20,000 × g for 20 min. The supernatants were incubated for 2 h at 4°C with a rabbit polyclonal anti-Bad antibody (Cell Signaling Technology) to immunoprecipitate Bad and its complexes. Non-immune IgG was used as a negative control. Protein A beads were added to the mixture and incubated overnight at 4°C. Immunoprecpitated proteins were analyzed by 4–15% Tris-HCL gel electrophoresis. To assess the presence of Bad/Bcl-2 and Bad/Bcl-XL complexes, a mouse monoclonal Bcl-2 antibody (Santa Cruz Biotechnology), a rabbit polyclonal Bcl-XL antibody (Sigma-Aldrich) and a rabbit polyclonal Bad antibody (Cell Signaling Technology) were used for immunoblotting. Donkey anti-goat or donkey anti-rabbit HRP-conjugated antibodies (Santa Cruz Biotechnology) were used as secondary antibodies.
Immunostaining for Tyro3, Axl and Mer
Cultured neurons were fixed with 4% paraformaldehyde (PFA) for 10 min and incubated overnight at 4oC with goat polyclonal anti-mouse Tyro3 (1:50, R&D systems, Minneapolis, MN), goat polyclonal anti-mouse Axl (1:50, R&D systems), goat polyclonal anti-mouse Mer (1:50, R&D systems) and mouse monoclonal anti-bovine Map2 (1:500, Chemicon) antibodies. The following day the sections were incubated with fluorescently conjugated secondary antibodies diluted 1:200 in PBS as follows: AlexaFluor 488-conjugated donkey anti-goat IgG (1:150; Invitrogen, Carlsbad, CA) to detect Tyro3, Axl, or Mer and AlexaFluor 568-conjugated goat anti-mouse IgG (1:150; Invitrogen) to detect Map2. Images were obtained using a Zeiss 510 meta confocal microscope. A 488 nm argon laser to excite AlexaFluor 488 and the emission was collected through a 500–550 bp filter, and a 543 nm HeNe laser was used to excite Alex Fluor 568 and the emission was collected through a 560–615 bp filter.
Immunoblotting for Tyro3, Axl and Mer
Thirty μg of neuronal lysate protein was subjected to 4–12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) gel electrophoresis and transferred to nitrocellulose membranes (BioRad). Membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight with the following primary antibodies: Tyro3 (1:100, R&D systems), Axl (1:100 R&D systems) and Mer (1:100, R&D systems). The membranes were washed and incubated with a HRP-conjugated secondary antibody for 1 h. Immunoreactivity was detected using SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL).
transgenic mice were originally on C57Bl6/129 background (Lu et al., 1999
; Lu et al., 2001
). These mice were backcrossed for several generations (> 8) to attain the C57Bl6 background and were generated by null-null breeding. The C57Bl6 mice were used as wild type controls for the TAM null mice, as described in a previous publication (Rothlin et al., 2007
) and on the Jackson Lab website (http://jaxmice.jax.org/strain/007937.html
). Mice were studied at 2–3 months of age. The breeding pairs were provided by Dr. G. Lemke from the Salk Institute.
Tyro3 tyrosine phosphorylation
Neurons were lysed with Radio ImmunoPreciptation Assay (RIPA) Buffer (50 mM Tris, pH 8.0, 150 mM NaCI, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate and Roche protease inhibitor cocktail) and incubated with a rabbit anti-phospho-tyrosine antobody (Abcam, Cambridge, MA) or a control non-immune IgG (Sigma-Aldrich) overnight at 4°C. The samples were then immunoprecipitated using a protein G immunoprecipitation kit (Roche) followed by SDS-PAGE separation and transfer onto nitrocellulose membranes (Millipore Corp). After blocking non-specific sites with 5% milk, the membranes were incubated with a rat monoclonal anti-mouse Tyro3 antibody (R&D systems) or a rabbit anti-mouse vascular endothelial growth factor receptor 2 (VEGFR2) antibody (Millipore, Billerica, MA) for a loading control. Following incubation with an HRP-conjugated donkey-anti goat secondary antibody (Santa Cruz Biotechnology), the immunoreactivity was detected using the SuperSignal® West Pico chemiluminescent substrate (Thermo Scientific). Cells were treated with mouse PS for 15 min.
The thrombin-cleaved PS was prepared as described (Heeb et al., 2002
). Briefly, human plasma-derived PS (1.8 μM) was incubated with immobilized thrombin (50 U/ml) and sampled at different time points (20 min-24 h). Aliquots were resolved in SDS-PAGE under reducing or non-reducing conditions, and applied to silver staining (Silver staining kit, Amersham Pharmacia Biotech, Upsala, Sweden). The reducing gel demonstrates rapid cleavage on Arg49 and non-reducing gel demonstrates slow cleavage on Arg70.
Synthetic human micro-PS containing the Gla domain, the TSR region, and the first EGF domain and exhibits about 30% of PS anticoagulant APC-cofactor activity was prepared as described (Hackeng et al., 2000
The human recombinant rSHBG-like module was a gift by Dr Sophie Gandrille (Univ. of Paris, France). As reported, the recombinant rSHBG module of human PS does not exhibit anticoagulant APC-cofactor activity but appears to retain its native conformation as in full length PS (Saposnik et al., 2003
Activated partial thromboplastin time (aPTT) assay
The anticoagulant activities of PS variants were determined by an aPTT assay, as described (Heeb 2002
), using a ST coagulameter (Diagnostica Stago, Asnieres, France). The anticoagulant APC-cofactor activity of PS variants was expressed as a percentage of wildtype, full length PS whose activity was taken as 100%.
NMDA-induced in vivo brain lesion
We used an NMDA model of excitotoxic lesions in the mouse brain in vivo, as described (Ayata et al., 1997
; Guo et al., 2004
; Guo et al., 2009a
). Male C57BL6 control mice and Tyro3−/−
mutants weighing 26–30 g were used throughout the study. Mice were anesthetized with 1.5% isolfurane (in 70% nitric oxide and 30% oxygen). Animals received micro-infusions into the right striatum (0.5 mm anterior, 2.5 mm lateral, 3.2 mm ventral to the bregma) of either vehicle, NMDA (20 nmol in 0.3 μL of PBS, pH 7.4) or NMDA and PS (0.002, 0.02 and 0.2 μg in 0.3 μL of PBS) or NMDA and Ac-DEVD-CHO (240 μg in 0.3 μl of PBS), z-IETD-fmk (60 μg in 0.3 μl of PBS), z-LEHD (240 μg in 0.3 μl of PBS) or PFT-α (20 nmol in 0.3 μl of PBS). The solutions were infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL). In wt mice, the final concentrations of PS in brain tissue at the site of injection after 2 min infusion ranged from 2.6 to 260 nmol/L as determined from a dilution factor of Evans-blue albumin infused simultaneously with PS in a separate series of experiments, as reported (Kakee et al., 1996
; see below). In TAM mutants and control wild type mice the concentration of PS and PS variants at the site of injection after 2 min infusion was 260 nM. Animals were sacrificed 48 h later. Brains were quickly removed, frozen on dry ice and stored at −80°C until processing. Thirty μm thick coronal sections were prepared using a cryostat. Every fifth section 1 mm anterior and posterior to the site of injection was stained with cresyl violet. The lesion area was identified by the loss of staining as reported (Ayata et al., 1997
; Guo et al., 2004
; Guo et al., 2009a
). The lesion areas were determined using NIH Image J software and integrated to obtain the volume of injury. All studies were performed in a blind fashion. We studied 4–6 mice per group. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Rochester using US National Institutes of Health Guidelines.
Determination of the final concentration of PS and PS variants in brain tissue
The final concentrations of PS and PS variants in brain tissue were determined from the dilution factor of the injected proteins into the brain interstitial fluid (ISF), as reported (Kakee et al., 1996
). Evans blue, a dye that avidly binds albumin (67 kDa) was used to determine the dilution factor from its diffusion volume within the brain ISF. The molecular weight of the Evans blue-albumin complex (68 kDa) is similar to that of PS (69 kDa). In brief, Evans blue (4 mg/ml) was incubated with the equimolar concentration of bovine serum albumin (BSA) in artificial CSF for 3 h at room temperature, filtered using 0.2 μm filter and 0.3 μl microinjected into the striatum over 2 min, as in the NMDA-induced in vivo
brain lesion experiments. After 30 min, the brain was rapidly removed, placed in a Brain Matrix on an ice cold dish and cut into 1 mm thick sections. The Evans blue-stained areas of each section were carefully removed, using a dissecting microscope, and weighed. The diffusion volume of Evans blue was estimated assuming a brain specific density of 1, as reported (Kakee et al., 1996
). The dilution factor (32.8 ± 3.8, n=3) was determined by dividing the diffusion volume by the injected volume (data not shown). The final concentration of PS and PS variants in the brain tissue was determined by dividing their injected concentrations by the dilution factor (data not shown).
Caspase-3 activity in the mouse striatum
Mouse striatum ipsilateral to NMDA lesion was collected 24 h after NMDA (20 nnmol in 0.3 μL of PBS, pH 7.4) or NMDA and PS (0.2 μg in 0.3 μL PBS) micro-infusions. The tissue was lysed using Cell Lysis Buffer (Cell Signaling Technology) with protease inhibitors. Caspase-3 activity was determined using caspase-3 Colorimetric Assay Kit (BioVision), as described above.
Immunobloting analysis in the mouse striatum
Mouse striatum ipsilateral to NMDA lesion was collected 24 h after NMDA (20 nnmol in 0.3 μL of PBS, pH 7.4) or NMDA and PS (0.2 μg in 0.3 μL PBS) micro-infusions. Tissue sdamples were snap-frozen in liquid nitrogen and homogenized using Cell Lysis Buffer (Cell Signaling Technology) with protease inhibitors. Nuclear proteins were extracted using NE-PER nuclear extraction reagents (Pierce Biotechnology, Rockford, IL). Proteins (50 μg) were analyzed by immunoblotting as described above. We used the following primary antibodies: rabbit polyclonal anti-human phospho-Mdm2 (Ser166) antibody which cross reacts with mouse phospho-Mdm2 (Ser166) (1:1000, Cell Signaling Technology); rabbit polyclonal anti-human p53 antibody which cross reacts with mouse p53 (1:1000, Cell Signaling Technology); mouse monoclonal anti-mouse Bax (1:100, Santa Cruz Biotechnology); rabbit polyclonal anti-mouse Phospho-Bad (Ser136) antibody (1:200, Cell Signaling Technology); goat polyclonal anti-human β-actin antibody which cross reacts with mouse β-actin (1:1000, Santa Cruz Biotechnology); sheep polyclonal anti-human histone 1 antibody which cross reacts with mouse histone 1 (1:1000, United States Biological).
We used S-plus 7.0 for statistical calculations. Data were presented as mean ± SEM. One-way or two-way analysis of variance (ANOVA) followed by Tukey post hoc test were used to determine statistically significant differences. P < 0.05 was considered statistically significant.