Wild type mice (C57/B6), SOD1 G93A transgenic mice, BAC GLT1 eGFP (previously generated in our lab) (Regan et al. 2007
), BAC NG2 DsRed (kind gift of Dr. Dwight Bergles, Dept. of Neuroscience, Johns Hopkins University), BAC ALDH1L1 eGFP mice (GENSAT project), GFAP eGFP mice (Jackson laboratory) were used for in vivo experiments. The care and treatment of animals in all procedures strictly followed the NIH Guide for the Care and Use of Laboratory Animals and the Guidelines for the Use of Animals in Neuroscience Research and the Johns Hopkins University IACUC. Mice were housed at standard temperature (21°C) and in a light controlled environment with ad libitum access to the food and water. BAC ALDH1L1 eGFP (and GFAP eGFP) mice were crossed with SOD1 G93A mice to obtain double transgenic mice. Littermates were used as control. Mice were sacrificed at designated time point following breeding or experiment. A 30-gauge needle was used to induce an acute lesion in the striatum.
Generation of the EAAT2-tdTomato transgenic mice
EAAT2-tdTomato transgenic mouse was generated by inserting the tdTomato reporter downstream of a 8.3kb EAAT2 promoter fragment. Multiple (4) founders were established following pronuclear injection at the transgenic core laboratory of Johns Hopkins University. The mice will be available upon request.
Primary neuron and astrocyte cultures
Procedures used for culturing primary neuron and astrocytes were described before (Yang et al. 2009
). Briefly, cortical astrocyte cultures were prepared from P2-4 wild type mouse pups and grown in DMEM supplemented with 10% heat-inactivated FBS and 1% of penicillin/streptomycin. Astrocytes were ready for experiments once confluent (5-7 DIV). No dbcAMP was added into the culture medium. For neuronal cultures, cortex was dissected out from E14-16 mouse embryos of wild type mice, cortical neurons were cultured in Neurobasal medium supplemented with N27, penicillin/streptomycin, and glutamine.
Immunohistochemistry and Microscopy
For immunohistochemistry, mice were perfused with 4% paraformaldehyde and brain/spinal cord tissue were collected and cryoprotected in 30% sucrose. Tissue sections (20μm) were then prepared by cryostat sectioning and were treated with blocking buffer (0.4% BSA, 5% goat-serum, and 0.2% Triton-X 100 in PBS) for 30-60 min at room temperature. Primary antibodies for ALDH1L1 (NeuroMab), GFAP (Chemicon), APC (cc1 clone, Calbiochem), PDGFRα (BD Pharmingen), Iba1 (Wako), Olig2 (Millipore), and NeuN (Millipore) were incubated overnight at 4 °C in blocking buffer. After rinse in PBS, anti-mouse Alexa 555 conjugated antibody was added for 90min at room temperature. Cells labelled with either immunostain or transgenic reporters were manually counted in Axiovision from digital images taken by fluorescent microscopy. For some single cell analyses, mean fluorescence intensity (MFI) was determined. Typically, MFI measurements were based upon at least 4000 cells/sample with a minimum of 3 mice for each experiment. Data was plotted in PRISM (GraphPad Software, Inc). One-way ANOVA (Bonferroni posthoc analysis) and Student’s t-test were used for statistical analysis where appropriate (see figure legend for details).
Preparation of cell suspension and FAC sorting of astrocytes
Brains or spinal cords from BAC GLT1 eGFP or BAC ALDH1L1 eGFP transgenic reporter mice (age from P5-P130) 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 (37C, 15min) and then digested with DNase I (37C, 10min), followed by careful trituration. Cell mixtures were then filtered through a cell strainer (40-70mm) and resuspended in cold HBSS solution (5-10 × 106 cells/ml) for FACS. The whole cell suspension procedure was completed in 1-2 h. Cells were sorted by using MoFlo MLS high speed cell sorter (Beckman coulter) with Summit version 4.3 software. Propidium iodide (PI) and eGFP were all excited by a 488 nm laser, and emissions were collected by 575/26 nm and 530/30 nm discrimination filters, respectively. The signals were manually compensated, and cells were sorted into cold HBSS. The whole procedure for cell suspension preparation and FAC sorting process was completed within 2-3h.
RNA isolation and QRT-PCR
Total RNA was prepared from brain or spinal cord tissue by using Absolutely RNA Miniprep Kit from Stratagene (Stratagene, La Jolla, CA). Total RNA were then converted to cDNA by using high archive cDNA synthesis kit (Applied Biosystems, Foster city, CA). The relative abundance of ALDH1L1 mRNA was determined by using a TaqMan pre-made ALDH1L1 probes (Applied Biosystems, Foster city, CA). Ribosomal 18s rRNA was used as endogenous control for the normalization of RNA quantity.
Western blot analysis
Primary cultures or tissue 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 37C for 30min. The supernatants were separated by 4-12% gradient PAGE gel and transferred to PVDF membrane. Blots were placed in blocking solution (5% non-fat milk in TBST) for 1 h at room temperature and then incubated with ALDH1L1 antibody (Neuromab) or β-actin antibody (Sigma) diluted in TBST overnight at 4C. After extensive washing, the blots were incubated with horseradish peroxidase conjugated antibodies (Sigma) for 1h and washed again. Immunoreactive bands were detected using the ECL procedure.
Microarray and data analysis
Total RNA was prepared from FAC sorted astrocytes. Total RNA was lineally amplified and labeled in a Nugene protocol. Sample labeling and hybridization with Mouse Exon 1.0 ST chips (Affymetrix) were performed in the Johns Hopkins University microarray facility. After hybridization, hybridization signals were acquired and normalized with the use of RMAexpress
). Data was further filtered to remove genes with signals less than 80 in all the samples to reduce the noise. Differential gene expression between the different conditions was assessed by statistical linear model analysis using the bioconductor package limma
. The moderated t-statistic p-values derived from the limma
analysis above were further adjusted for multiple testing by Benjamini and Hochberg’s method to control false discovery rate (FDR). The FDR cutoff of <10% was used to obtain the list of differentially expressed genes.