Cultures and Transfection
Cortical astrocyte cultures were prepared from P2−4 mouse pups of BAC GLT1 eGFP transgenic mice. Cortical neuronal cultures were prepared from E14−16 mouse embryos of wild type mice. Co-culture of astrocytes and neurons was performed by plating neurons directly on the top of the confluent astrocyte cultures.
Immunostaining and Microscopy
Cultures were directly fixed in culture dishes with 4% paraformaldehyde (30 min). After three rinses in PBS, cells were treated with blocking buffer (0.4% BSA, 5% goat-serum, and 0.2% Triton-X 100 in PBS) for 20 min at room temperature. Primary antibodies GFAP (mouse, 1:1,000) were incubated overnight at 4°C in blocking buffer. Anti-mouse Alexa 555 conjugated antibody (1:1,000) was added for 90 min at room temperature. Nuclear counterstain was performed with hoechst33342; eGFP+ cells collected from FACS were also examined by direct brightfield visualization at the time of fluorescent microscopy.
Preparation of Cell Suspension from In Vitro Cultures or from Brain Tissue
For FACS of in vivo brain tissue, adult brains from BAC GLT1 eGFP transgenic reporter mice (8- to 12-weeks old) were used. Mice were anesthetized with pentobarbital (50 mg/kg, i.p.), perfused with cold Hanks buffer (Invitrogen, Carlsbad, CA), and decapitated. The brain was immediately dissected in cold Hanks buffer containing glutamate receptor antagonists, 3 mM DNQX and 100 mM APV (Sigma, St. Louis, MO), and cut into small pieces. Cell suspension was prepared as described in the neural tissue dissociation kit (Miltenyi biotech, Auburn, CA). Briefly, small pieces of tissue were treated with papain enzymatic mix (37°C, 15 min) and then digested with DNase I (37°C, 10 min), followed by careful trituration. Cell mixtures were then filtered through a cell strainer (70 mm) and resuspended in cold HBSS solution (5−10 × 106 cells/mL) for FACS. The whole cell suspension procedure was completed in 1−2 h. For cell suspension preparations of in vitro astrocyte cultures or neuron astrocyte co-cultures, cultured cells were gently scraped and spun down (1,000g, 5 min). The cell pellet was then resuspended in cold HBSS solution.
FAC Sorting of Cell Suspension
Cells were sorted using MoFlo MLS high-speed cell sorter (Beckman coulter) with Summit version 4.3 software. GFP and Propidium iodide (PI) were all excited by a 488 nm laser, and emissions were collected by 530/30 nm and 575/26 nm discrimination filters, respectively. The signals were manually compensated, and cells were sorted into cold HBSS. The whole FAC sorting process was completed within 2−3 h.
RNA Isolation and QRT-PCR
Total RNA from FAC sorted cells (0.5−1 × 106) was prepared by using Absolutely RNA Miniprep Kit from Stratagene (Stratagene, La Jolla, CA). Total RNA were then converted to cDNA using high archive cDNA synthesis kit (Applied Biosystems, Foster city, CA). The relative abundance of GLT1, GFAP, and βIII-tubulin mRNA was determined by using TaqMan premade GLT1, GFAP, and βIII-tubulin probes (Applied Biosystems, Foster city, CA). Ribosomal 18s rRNA was used as endogenous control for the normalization of RNA quantity.
Preparation of Site-Specific Methylated GLT1 Promoter and Luciferase Reporter Assay
Mouse GLT1 promoter (1kb) was amplified from genomic DNA by using primers: F: 5′-atatatatctcgag tgctcgccctcggggaa-3′; R: 5′-atatatataagctttgcggggatcctg cacc-3′. GLT1 promoter was then cloned into pGL4.14 luciferase reporter at Xho I/Hind III sites and named as pGL4.14-PGLT1. Primers with methyl group addition on selective CpG sites on GLT1 promoter were then used to amplify linear GLT1 promoter and luciferase reporter fragment from pGL4.14-PGLT1. Primer sequences are summarized in . Purified linear GLT1 promoter luciferase reporter together with beta galactosidase was transfected into HEK cells. Luciferase and β-gal assay was performed 48h after transfection.
Fig. 4 Methylation induced functional change of the GLT1 promoter. (A) Sequence analysis of putative transcriptional factors in Me-0 region of GLT1 promoter and human EAAT promoter. CDXA, chicken homeodomain protein; HEN1, Hua enhancer 1; AP4, activating enhancer (more ...)
Luciferase and β-gal Assay
Astrocytes/tissues were collected and lysated in 1× cell culture lysis buffer (Promega, Madison, WI). Lysates (50 μL for luciferase and 50 μL for β-gal) were transfered to a 96-well assay plate and substrate (100 μL for luciferase and 50 μL for β-gal) was added to the assay plate and the activity measured in a FluoSTAR OPTIMA luminometer (BMG labtech, Durham, NC).
Isolation of Genomic DNA and Bisulfite Sequencing Analysis
For genomic DNA isolation, cell pellet (0.5−1 × 106) or homogenized human tissue (0.5 mg) was resuspended in 200−600 μL of nuclear lysis buffer (10 mM Tris/HCl pH 8.0, 400 mM NaCl, and 10 mM EDTA) and was disrupted by supplementing SDS to a final 1%. Proteinase K (4 mg/mL) was then added, and incubated for 3 h at 37°C, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Bisulfite conversion of genomic DNA was performed as described in EZ-DNA methylation-gold kit (Zymo research, Orange, CA). Briefly, 200−400 ng of genomic DNA was mixed with freshly prepared bisulfite reagent and was incubated for 3 h in programmed temperature cycles. After the bisulfite treatment, converted genomic DNA was column purified. Region of interest was amplified by specific bisulfite sequencing primers and was cloned into TOPO TA vector (Invitrogen, Carlsbad, CA) for sequencing analysis. Individual bacteria clones were then selected for the preparation of the plasmid and subsequent sequencing. The frequency of the various methylation patterns recovered in the bacterial clones will provide a close approximation of the overall frequency of the methylation patterns in the native samples.
Promoter CpG Analysis and Design of the Bisulfate-Sequencing Primers
The GC content, distribution of CpG sites, and predicted bisulfite converted sequence on the full genomic promoter of EAAT2 or GLT1 was analyzed by the Web-based program MethPrimer (http://www.urogene.org/methprimer
). On the basis of the distribution of the CpG sites and predicted bisulfite converted sequence, bisulfite-sequencing primers were designed in Vector NTI (Invitrogen, Carlsbad, CA) to amplify each region of interest.
Electrophoretic Mobility Shift Assay (EMSA)
Oligos (45mer) from −833 to −788 of GLT1 promoter were directly synthesized with biotin modification added to both 5′ ends. Cytosine of selective CpG site was also directly methylated during synthesis. Nuclear extracts were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Pierce Biotechnology, Rockford, IL) from freshly dissected cortex of adult wild type mice. Gel shift was performed with procedures described in Lightshift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, IL). In each reaction, 2 mL (50 fmol/mL) biotin labelled oligos and 5 mg nuclear extract were added.
Western Blot Analysis
FAC sorted cells were lysed in a buffer containing 62.5 mM Tris, 2% SDS, 10% sucrose, and protease inhibitor cocktail (Roche). Homogenates were briefly sonicated and were incubated at 37°C for 30 min. Postmortem human tissue (0.5 mg) was homogenized in 1 mL of homogenization buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail) using a electric homogenizer, and the homogenate was sonicated and subsequently centrifuged at 14,000g for 10 min. The supernatants were separated by 4−12% gradient PAGE gel and transferred to PVDF membrane. Blots were placed in blocking solution (5% nonfat milk in TBST) for 1 h at room temperature and then incubated with EAAT2/GLT1 antibody (C-terminal polyclonal, 1:5,000) or anti-actin (1:2,000, Sigma) diluted in TBST overnight at 4°C. After extensive washing, the blots were incubated with horseradish peroxidase conjugated antibodies (1:5,000, Sigma) for 1 h and washed again. Immunoreactive bands were detected using the ECL procedure.