Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase (original) (raw)

Uracil-DNA glycosylase targeting construct. We isolated an EcoRI restriction fragment that contained a large portion of the ung gene. The EcoRI restriction fragment was shown to contain exons 1 to 5 and the mitochondria-specific exon 1a by use of a combination of PCR and Southern hybridization techniques. The Neo or hyg-tk targeting vectors were constructed by replacing the _NcoI_-ApaI fragment with a neomycin-resistance cassette or a hygromycin-thymidine kinase–resistance cassette, thereby deleting exons 1, 2, and 3 and part of exon 4 of the ung gene. Homologous recombination of the neo targeting vector in murine 129/SvJae ES cell (J1 ES cells) resulted in the generation of Ung+/neo ES cells, which were used for injection in Balb/c blastocysts. To confirm homologous targeting in ES cells, isolated genomic DNAs were digested with SacI, separated, and blotted on Zetabind nylon membrane (CUNO, Meriden, Connecticut, USA). The blotted DNA fragments were hybridized to radioactive-labeled outside probe from exon 6. Outside probe from exon 6 was amplified from genomic DNA (5′-AAGCGTCACCATGTCCTGCAG-3′ and 5′-GAAATCTCTAACTCTAAAGGATAAAGCGG-3′). Radioactive-labeled probes were synthesized by random labeling (Prime-It-II; Stratagene). WT allele produced a band of 8.2 kb, whereas the neo allele and hyg-tk allele produce bands of 5.2 kb and 4.1 kb, respectively. Only gender-matched littermates from a mixed 129/SvJae ∞ Balb/c background were used for experiments reported in this study.

Cell culture and transfection. WT ES cells (J1 ES cells) (28), Ung+/– mutant ES, and Ung–/– mutant ES cells were cultivated on irradiated MEFs as described (29) or without MEFs by use of a high concentration of leukemia inhibitory factor (LIF) (1,000 U/ml). Transfection of plasmids (Neo or hyg-tk targeting vectors) was performed by the application of the cationic liposome reagent DOTAP from Boehringer Mannheim/Roche Bioscience (Mannheim, Germany). MEFs were cultured in HEPES-buffered Dulbecco’s modified Eagle medium with high glucose (Gibco-BRL, Langley, Oklahoma, USA); 10% heat-inactivated fetal calf serum (HyClone, South Logan, Utah, USA); 0.1 mM nonessential amino acids (Gibco-BRL); 0.1 mM β-mercaptoethanol (Sigma-Aldrich, Deisenhofen, Germany); LIF; and the antibiotics penicillin and streptomycin.

Uracil-DNA glycosylase activity assay (cell culture). ES cells were grown without MEF. Shock-frozen cells or tissues were ground with mortar and pestle and resuspended in activity buffer (20 mM Tris-HCl, pH 7.8; 100 mM KCl; 2 mM EDTA; 1 mM EGTA; 5 mM β-mercaptoethanol; and proteinase inhibitors). Protein concentrations were normalized. Uracil excision activity in lysates was measured by the ability to excise uracil from a [γ-32P]-ATP end-labeled oligonucleotide (5′-GCTTGCATGCCTGCAGGTCGAUTCTAGAGGATCCCCGGGTACCGAGCTCGA-3′) that was hybridized before to a complementary oligonucleotide (5′-TCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGC-3′) to create a G:U mismatch base pair substrate. Apyrimidinic sites were cleaved by basic pH and high temperature (90°C for 3 minutes). The cleaved and uncleaved oligonucleotides were separated on a 15% TBE-urea-acrylamide gel, and bands were quantified by densitometry.

Ung reverse-transcriptase PCR analysis. Total RNA was isolated from WT or Ungneo/hygtk ES cells by use of the Qiagen RNeasy kit (Qiagen, Hilden, Germany) as per the manufacturer’s protocol. Oligo-(dT) directed cDNA was prepared as follows: 10 ∝g total RNA and 1 ∝g oligo-(dT) (Gibco-BRL) were incubated at 75°C for 10 minutes and quick chilled on ice. First-strand cDNA synthesis was then carried out with MuLV reverse-transcriptase (RT) (Superscript II; Gibco-BRL) at 37°C for 60 minutes in the buffer provided by the manufacturer, with the enzyme supplemented by 250 ∝M of each dNTP. Gene-specific primers are indicated below. PCR amplification was performed in a final volume of 50 ∝l by utilizing the reaction buffer provided by the manufacturer (PerkinElmer, Wellesley, Massachusetts, USA) supplemented by 200 ∝M of each dNTP, 20 pmol of each forward and reverse primer, 2 ∝l of the cDNA reaction mix, and 2.5 units of AmpliTaq Gold DNA polymerase (PerkinElmer). The reaction mixture was subjected to 30 cycles in an MJ Research PT-200 (MJ Research Inc., Waltham, Massachusetts, USA). The denaturation, annealing, and extension conditions were as indicated below for each primer set, followed by a single primer extension step at 72°C for 10 minutes. RT-PCR analyses included the genes that expressed UNG, AAG, TDG, OGG1 AP endonuclease (APE), and actin. Details for the RT-PCR are given in Table 4.

Northern blots. Total RNAs were extracted from brain and other tissues by use of a RNAzol kit (Tel-Test, Friendswood, Texas, USA). The products were resolved on an agarose gel, transferred to nylon membrane, and hybridized in Church buffer (0.5 M NaPO4; pH 7.5; 7% SDS; 2 mM EDTA) with a radioactive-labeled probe for 8 to 16 hours at 63°C. Radioactive-labeled probes were synthesized with a random labeling. Final wash was 0.1 ∞ SSC, 0.1% SDS at 62°C. Intensities of hybridized bands were measured by densitometry and normalized to hybridization with β-actin.

Mutation assays in mouse cells. A small number of ES cells (<50) were seeded and independently expanded in the absence of mouse embryonic feeder cells to the desired cell number. The expanded final cell population was trypsinized, counted, and plated in medium containing 2 ∝M of ganciclovir to select for thymidine kinase (TK) mutants. The selection was performed at a density not greater than 107 cells per 150-mm dish in the absence of mouse embryonic feeder cells. Drug-resistant medium was changed frequently. After 14 days of selection, drug-resistant colonies were counted, picked, and further expanded for analysis. All BigBlue cII mutation assays were performed as described previously (30). Briefly, genomic DNA was extracted from the cells (5 to 10 ∞ 106 cells per sample) using Recoverase DNA Isolation Kits (Stratagene) and stored at 4°C. λ-LIZ (LacI/Z) shuttle vectors were rescued from genomic DNA with Transpack packaging extract according to the manufacturer’s protocol (Stratagene). Plaques with mutations in the cII gene were determined essentially as described (30). Mutant frequencies were determined using genomic DNA isolated from at least three separate cell samples. For each DNA sample, plaques were analyzed from at least three packaging reactions to ensure an accurate mutant frequency determination.

SNP exposure and hypoxia. Approximately 200,000 Ung+/+ or Ung–/– MEFs were exposed to different conditions of SNP (45 ∝M to 1.2 mM). After SNP exposure, cells were cultivated for additional 24 hours in normal media with DMEM with 10% FBS. For hypoxia experiments, approximately 200,000 _Ung+/+_or Ung–/– MEF were placed in a chamber and exposed to a hypoxic condition (<5% O2) for 20 hours, followed by cultivation in normal atmosphere for additional 12 hours. At the end of the respective experiments, the final cell population was trypsinized, stained with trypan blue, and counted for vital cells.

Reversed-phase HPLC. DNA was extracted using a Qiagen genomic DNA purification kit (Qiagen) (31). Thirty micrograms of DNA were dissolved in 100 ∝l P1-buffer (30 mM sodium acetate and 0.1 mM zinc sulfate, pH 5.3) and digested with nuclease P1 (ICN, Eschwege, Germany). Then, nucleotides were subjected to bacterial alkaline phosphatase (Sigma-Aldrich) in AP-buffer (50 mM Tris-HCl and 100 ∝M EDTA, pH 8.0) at 37°C for 8 hours. The resulting nucleosides were separated by gradient reversed–phase, high-pressure liquid chromatography (HPLC-UV system with a Class-LC10 assembly; Shimadzu, Berlin, Germany). The mobile phase was 20 mM ammonium acetate with 2% (solution A) or 80% (solution B) acetonitrile (pH 4.3; flow rate 2 ml/min). The solid phase was a 4.6 mm ∞ 25 cm Supelcosil LC-18S column plus precolumn filter (Supelco, Bellefonte, Pennsylvania, USA; 5-∝m particle size). Each run was 12 minutes total (100% solution A from minutes 1 to 4, 15% solution B from minutes 5 to 8.5, 100% solution B from minutes 8.5 to 9, and 100% solution A from minutes 9 to 12). Nucleosides were detected by their distinct UV absorption maxima on an SPD-M10A UV-detector (Shimadzu), and the area under each peak was converted to a mass equivalent by integrating the respective extinction into a equilibration curve. Standard nucleosides (Sigma-Aldrich) were used for identification and quantification.

Neuronal cell cultures. Primary neuronal cultures of cerebral cortex were obtained from mouse embryos (E16 to E18) as described (32, 33). Cortices were dissected, trypsinized, dissociated, and plated in 24-well or 6-well plates precoated with poly-L-lysin (0.5% w/v in PBS) and collagen (0.03% w/v) in a density of 200,000 cells/cm2 (for details see ref. 33). Cultures were kept at 36.5°C and 5% CO2 and were fed, beginning from 4 days in vitro (DIV 4), with cultivating medium (starter medium without glutamate) by replacing half of the medium twice a week. Experiments were performed after DIV 10 to DIV 12.

Oxygen-glucose deprivation, mitochondrial function, and cell death. For OGD, the medium was removed from cultures and preserved. Cultures were subjected to OGD for 120 minutes (severe) or 80 minutes (mild) in a balanced salt solution at PO2 < 1 mmHg, followed by replacement of the preserved medium as described previously (33). For control, cells were placed in a balanced salt solution with 20 mM D-glucose for 120 or 80 minutes in normoxic atmosphere with 5% CO2. Staining with fluorescein diacetate (FD) and propodium iodide (PI) was performed for assessment of cell viability (1 ∝g/ml for 20 minutes at 37°C). Pictures of three randomly chosen high-power fields from different cultures were taken and counted by a naive researcher as viable (FD-positive), dead (PI-positive), or early-stage cell death (double). At various time points (1, 3, 9, and 24 hours), aliquots of the medium were saved for analysis of LDH activity as described (33). MTT assay was performed, which measures the amount of blue formazan produced by viable mitochondria from MTT (Sigma-Aldrich) as described (33). In addition, TMRE (Molecular Probes, Leiden, The Netherlands) was used to assess mitochondrial membrane potential as described (34). After experiments, TMRE was added (100 nM, 40 min) and its mitochondrial uptake was assessed by fluorescence microscopy in the multiwell fluorescence reader, and pictures were taken with a fluorescence microscope and a digital camera.

Mouse model for focal cerebral ischemia. All animal experiments were conducted in accordance with institutional and international guidelines and approved by local authorities (#0054/99; Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit [LAGetSi], Berlin, Germany). Mice (18 to 23 g) were anesthetized with 1.5% halothane (induction) and maintained on 1.0% halothane in 70% nitrous oxide and 30% oxygen. Regional cerebral blood flow (rCBF) was measured by laser Doppler flowmetry (35). In randomly selected animals, the left femoral artery was cannulated for arterial blood pressure and blood gas determination as described (35). Arterial blood samples (50 μl) were analyzed for pH, PaO2, and PaCO2 on a blood gas/pH analyzer (Corning 178; Ciba-Corning Diagnostics, Medford, Massachusetts, USA). MCAo was induced with a silicone-coated, 8-0 monofilament. Thirty minutes later, the filament was completely withdrawn to allow reperfusion. Core temperature was maintained at approximately 37°C ± 0.5°C during the monitoring period.

Infarct volumes and neuronal survival. Seventy-two hours after reperfusion, animals were killed and the brains were snap-frozen in isopentane on dry ice. Lesion areas were quantified with an image analysis system (M4; St. Catharines, Ontario, Canada) on 20 μm-hematoxylin and eosin-stained cryostat sections. Volumes were calculated by summing the volumes of each section directly. NeuN immunocytochemistry was performed on cryostat sections by use of mouse monoclonal anti-NeuN antibodies (1:100; Chemicon, Hofheim, Germany) and diaminobenzidine as chromogene. Only cells with a clear nuclear signal were considered NeuN+ and counted in five randomly selected high-power fields (∞400 magnification) within the ischemic striatum at the level of the anterior commissure (bregma 0.14) and expressed as number of NeuN+ cells/mm2.

Neurological deficits. Mice were tested for neurological deficits by a naive observer 72 hours after MCAo and rated on a scale from 0 (no observable deficit) to 3 (severe) as described (36).

Carbon black. Deeply anesthetized mice were transcardially perfused with carbon black (in an equal volume of 20% gelatin in distilled water). Animals were decapitated and heads stored in 3.7% formaldehyde for 3 hours at 4°C. Brains were carefully removed, and the development of left and right posterior communicating arteries (PComAs) was scored as follows: 0, absent; 1, present but poorly developed (hypoplastic); and 2, well formed. A single PcomA development score was calculated by averaging the left and right scores (13). Anastomoses on the dorsal surface of the brain were localized by tracing the peripheral branches of the anterior and middle cerebral artery as described. Adjacent anastomosis points were connected, and the distance from the midline to the line of anastomoses was measured on photographs taken at coronal planes 2 mm, 4 mm, and 6 mm from the frontal pole (14).

Preparation of extracts from brain tissue. Mice underwent 30 minutes of MCAo and reperfusion. Mice without ischemia (control) or with a filament inserted into the carotid artery without further advancing it (sham) were prepared. Cortex and striatum of ischemic and corresponding tissue from the contralateral hemisphere were isolated, homogenized (Wheaton homogenizer) in PBS (Gibco), and put on ice. After two washes with 1 ml PBS (1,000 g for 10 minutes at 0°C), samples were incubated in 1 ml hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM disodium-EDTA, 0.1 mM EGTA, 1 mM DTT; all from Sigma-Aldrich) with 5 ∝l of 50 mM PMSF (Sigma-Aldrich) for 10 minutes on ice. Samples were incubated with 30 ∝l Igepal CA-630 (Sigma-Aldrich) for 5 minutes, centrifuged at 1,000 g for 10 minutes at 0°C, and supernatants containing cytoplasmatic extracts were stored at –80°C. Pellets (washed once with 500 ∝l hypotonic buffer) were dissolved in 250 ∝l nuclear extract buffer (20 mM HEPES, pH 7.9; 20% glycerol; 420 mM Nacl; 1.5 mM MgCl2; 0.5 mM disodium-EDTA; and 1 mM DTT). Then, 2.5 ∝l of 50 mM PMSF, 2.5 ∝l aprotinin (1 mg/ml), 2.5 ∝l leupeptin (1 mg/ml), and 2.5 ∝l pepstatin (1 mg/ml) (all from Sigma-Aldrich) were added, and samples were placed on ice for 30 minutes. Cellular debris was removed by centrifugation (13,000 g for 10 minutes), and supernatants that contained nuclear extracts were stored at –80°C. Protein concentrations were determined by use of Bradford solution and adjusted to 1 mg/ml.

Uracil-DNA glycosylase activity assay (brain extracts). A Cy5-labeled uracil–containing oligoI (Cy5-5′-AGTCATGCCATTGGCCACATUGTGTCAGCTAGGATT-3′) and complementary oligoII (5′-CAATCCTAGCTGACACGATGTGGCGAATGGCATGAC-3′) were provided by MWG Co (Ebersberg, Germany). A 37-bp oligoduplex formation was allowed by incubating 1.25 pmol of each oligo (I and II) in TE at 95°C for 1 minute and slowly cooling down at 1°C/min to 4°C in a Hybaid (Heidelberg, Germany) thermocycler. For uracil excision, 2.5 pmol of the oligoduplex was incubated with 10 ∝l cytoplasmatic or nuclear extract, respectively, in 27.5 ∝l base excision buffer (1 mM NaCl; 2 mM Tris-HCl, pH 7.5; 0.2 mM MgCl2; and 0.1 mM DTT) for 20 minutes at 37°C in a Trio Thermoblock (Biometra, Göttingen, Germany). Electrophoresis on denaturating 19% polyacrylamide gel (30 ml of 30% Rotiphorese gel [Carl Roth GmbH, Karlsruhe, Germany], 12 ml 5∞ TBE [54 g Tris; 27.5 g boric acid (Carl Roth GmbH); and 20 ml of 0.5 M EDTA, pH 8.0 (Sigma-Aldrich) per 1 l], 15 g urea, [Carl Roth GmbH]) for 110 minutes allowed strand breakage of former uarcil-containing AP sites, which produced a 20-bp Cy5-containing single-stranded fragment. Occurrence of 20-bp fragments was detected by use of an Alf-sequencing machine (55°C, 1500 V, 60 mA, 30W; Pharmacia Biotech, Erlangen, Germany) and the proportion of 20-bp fragments to 37-bp oligoduplex peaks was calculated in terms of percentage (37).

Data analysis. Data are presented as mean ± standard error (SEM). Differences between groups were evaluated by ANOVA followed by Tukey’s or Scheffe’s test; NeuN+ cell counts were compared using the Mann-Whitney U test; neurological sensory-motor deficits scores and PcomA patency scores were analyzed by nonparametric ANOVA on ranks (Kruskal Wallis) followed by Dunn’s test. P values of less than 0.05 were considered of statistical significance.