BioProject

Several recent reports show that the cerebral cortex in humans and animals with altered expressions of Wnt/cadherin network-associate molecules display cytoarchitectural abnormalities reminiscent of cortical dysplasias seen in some (mouse-, rat-, and monkey-based) animal models of prenatal cocaine exposure. Therefore, we employed oligo microarrays followed by real-time RT-PCR to compare expressions of genes involved in Wnt and cadherin systems in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8-18) and drug-naive (saline, s.c.) mice. The pregnant mice chronically treated with cocaine in the above-described manner represent one of the animal models producing offspring with widespread cortical dysplasias. Out of more than 150 relevant genes in the arrays, 32 were upregulated and 9 were downregulated in cocaine-exposed fetuses. The majority of these genes (30 out of 41) were similarly affected in the frontal and occipital regions of the cerebral wall. We also used Western immunoblotting to examine the ability of cocaine to regulate the protein levels of beta-catenin, the key functional component of both Wnt and cadherin systems. While the total cell levels of beta-catenin were increased throughout the cerebral wall of cocaine-exposed fetuses, its nuclear (gene-transcription driving) levels remained unaltered. This suggests a transcription-unrelated role for cocaine-induced upregulation of this protein. Overall, our findings point to an intriguing possibility that that cerebral cortical dysplasias observed in several animal models of prenatal cocaine exposure may be at least in part related to alterations in the Wnt/cadherin molecular network. It has been demonstrated that exposure to cocaine increases cell death in the fetal CNS. To examine the molecular mechanisms of this effect, we employed mouse oligo microarrays followed by real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) to compare expressions of apoptosis-related genes in the cerebral wall of 18-day-old (E18) fetuses from cocaine-treated (20 mg/kg cocaine, s.c., b.i.d., E8th-E18th) and drug-naive (saline, s.c.) mice. Out of approximately 400 relevant genes in the arrays, 53 showed alterations in expression in cocaine-exposed fetuses. Upregulation was observed in 35 proapoptotic and 8 antiapoptotic genes; 4 proapoptotic and 6 antiapoptotic genes were down-regulated. The affected genes encode a wide range of apoptosis-related proteins, including death receptors (NTF-R1, NTF-R2, DR3, DR5, LTbeta-R, GITR, P57 TR-1) and their adaptor and regulatory proteins (MASGE-D1, TRAF-2, SIVA, MET, FLIP, FAIM, IAP1, ATFA), members of transcription regulatory pathways (JNK, NF-kappaB, P53), members of BCL-2 family of proteins (BID, BAD, BAX, BIK, NIP21, NIP3, NIX, BCL-2), DNA damage sensor (PARP-1), caspases and their substrates and regulatory proteins (caspases 8, 4, 9, and 3, ACINUS, CIDE-A, CIDE-B, GAS2), mitochondrially released factors (cytochrome c, AIF, PRG3), specific endoplasmic reticulum- and oxidative stress-associated factors (BACH2, ABL1, ALG2, CHOP), members of cell survival AKT and HSP70 pathways (PIK3GA, PTEN, HSP70, BAG1, BAG2), and others. This suggests that cocaine affects survival of developing cerebral cells via multiple apoptosis-regulating mechanisms. Keywords: Whole genome oligo microarray, Real time RT-PCR, gene expression analysis Overall design: Animals Timed pregnant Swiss Webster (CFW; Charles River Lab. Wilmington, MA) dams were maintained in individual cages in a climate-controlled room on a 12/12-h light/ dark cycle. They were divided into two groups. The first (experimental) group received subcutaneous (at the dorsum of the neck) injections of 20 mg/kg cocaine hydrochloride (Research Technology Branch, National Institute of Drug Abuse, Rockville, MD) dissolved in 200 mkl of 0.9% saline, twice a day (at 8:00 AM and 8:00 PM) from 8th through 18th day of pregnancy (E8–E18). The second (control) group was subjected to the same schedule of 0.9% saline only injections. The cocaine treatment was designed to replicate the one capable of reducing cerebral cortical mass in mouse offspring. Also, the relatively protracted period the chronic treatment was chosen to maximize the changes in the tissue expression of cocaine-regulated genes of interest. Throughout the treatment, all mice were weighed daily, and, from E8, the control and experimental animals were pair-fed, with the daily amount of food (Mouse Chow; Ralston Purina Saint Louis, MO) provided to each control dam being matched to that consumed by the paired experimental dam. Water was available ad libitum. We found that this feeding regiment resulted in similar weight gains from E8 to E18 in both experimental and control animal groups (46.41F0.45% and 44.99F0.59% respectively). On E18, 1 h after the morning treatment, the animals were anesthetized by peritoneal injection of 50 mg/kg sodium pentobarbital (Abbott Lab., Abbott Park, IL) and their uteri were removed. The frontal cerebral wall (containing cerebral cortex and underlying transient cortical zones anterior to the striatal level) and the occipital cerebral wall (containing cerebral cortex and underlying transient cortical zones posterior to the level of the hippocampus) were dissected from the fetuses and stored in liquid nitrogen prior to analysis. The animal experimentations used in this study were approved by the University of Maryland Animal Care and Use Committee. Microarray Tissue from two control and two experimental fetuses were used in the processing of a single microarray slide (Part G4121A, Agilent Technol). Five slides were processed per region of the fetal cerebral wall. For a given region, the tissue for each microarray slide was obtained from a separate set of control and experimental litters. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA). The yield of total RNA was determined by absorbance at 260 nm on a DU 640 Spectrophotometer (Beckman Coulter, Fullerton, CA). The 260/280 nm ratios of the samples were >1.8. For each assay, 5 mkg of RNA from control tissue and 5 mkg of RNA from experimental tissue were reverse transcribed using 3DNA Array Detection Kit (Genesphere, Hatfield, PA) with primers containing different dcaptureT sequences for control and experimental samples. SuperScript II Reverse Transcriptase for these reactions was purchased from Gibco BRL (Gaithersburg, MD). The resultant cDNAs were hybridized to microarray slides for 16 h in Agilent Hybridization Buffer (Agilent Technol) at 60 8C. Upon completion of the hybridization, the slides were washed for 10 min with 0.005% Triton in 6xsodium chloride/sodium citrate buffer (pH 7; SSC) at room temperature and then for five more min with 0.005% Triton in 0.1xSSC and 1 more minute in 0.1xSSC (pH 7), both at 4 8C. Washed slides were air-dried for 30 s. For fluorescent labeling of microarray-bound cDNA, the slides were incubated for 3 h at 65 8C with Capture Reagent Hybridization Mixture from 3DNA Array Detection Kit (Genesphere). In this mixture, Alexa 546 fluorochromeincorporating dendrimers contained single-stranded arms complimentary to the dcaptureT sequences used in the reverse transcription of RNA from control tissue, while Alexa 647 fluorochrome-incorporating dendrimers contained arms complimentary to the capture sequences used in reverse transcription of RNA from experimental tissue. The labeling reaction was terminated by washing of the microarray slides at room temperature for 10 min with 0.005% Triton in 2xSSC and for another 10 min with in 0.2xSSC. After that, the slides were air-dried for 30 s. The processed microarray slides were scanned at 10 Am resolution on a GenePix 4100A scanner (Axon Instr., Union City, CA) with the laser excitation at 532 nm [emission filter 575DF35 (green); photomultiplier voltage 550] for Alexa 546 (control samples) and the laser excitation at 635 nm [emission filter 670DF40 (red); photomultiplier voltage 695] for Alexa 647 (experimental samples). The signals were converted into 16-bits-per pixel resolution images, providing color depth of 65,536 levels. The densitometry was performed with GenePix Pro 4.1 software (Axon Instr.). Background was subtracted using the dlocal background correctionT procedure available in the software. Quality control utilized 255 positive controls, 161 negative controls and 646 QC-spots present on Agilent’s arrays. For replicates within a slide, median signal values were calculated. For each array, the data were normalized in Acuity 3.1 software (Axon Instr.) by applying locally weighted scatterplot smoothing (LOWESS) transformation. The between array scale normalization was done using intensity log10 ratio distribution box plots (GeneSight software, BioDiscovery, El Segundo, CA) for verification. Statistical analysis of the normalized data was conducted by the Significant Analyses for Microarray Algorithm (SAM) using Excel macros available at the Stanford SAM website. In the analyses of both frontal and occipital regions of the fetal cerebral wall, the cut-off thresholds were set to identify the maximum number of cocaine exposure-regulated genes at the minimal false discovery rate (FDR) allowed by SAM for a given set of microarray data. For both regions, the FDR rates were <0.5%. The apoptosis and Wnt pathway-related genes were identified with Pathway Assist 2.0 software (Stratagene, La Jolla, CA). This was followed by manual review of literature for each identified gene.