Tissue collection and RNA in situ hybridization
Brains, spinal cords and optic nerves at various stages were harvested, fixed in 4% paraformaldehyde at 4°C overnight, infused with 25% sucrose in PBS overnight, embedded in OCT and cryosectioned at 16 µm. Digoxigenin-and/ or fluorescein-labeled riboprobes were used to perform RNA in situ
hybridization, as described previously 21
. For double in situ, the second probe was developed with Tyramide amplification system (PerkinElmer, Inc.). The probes used were: murine GPR17
. Detailed protocols are available upon request.
Northern blot and quantitative real time polymerase chain reaction (qRT-PCR)
Northern blotting was performed as previously described 45
. qRT-PCR was performed using the ABI Prism 7700 Sequence Detector System (Perkin-Elmer Applied Biosystems) as previously described with Gapdh
(glyceraldehyde-3-phosphatase dehydrogenase, TaqMan kit, Applied Biosystems) as an internal control 19
. Primer sequences used for expression analyses are Rat Id2
forward 5'- atggaaatcctgcagcacgtcatc, reverse 5'- acgtttggttctgtccaggtctct; Rat Id4
forward 5'- actgtgcctgcagtgcgatatgaa, reverse 5'- tgcaggatctccactttgctgact; Rat Mag:
forward 5'- acagcgtcctggacatcatcaaca -3', reverse 5'- atgcagctgacctctacttccgtt -3'; Rat Cgt:
forward 5'- agataaagatgggcggccgttact -3', reverse 5'- tatttgaggtgctcctgccggtta -3'; Rat Mbp
forward 5'- ttgactccatcgggcgcttcttta -3', reverse 5'-gctgtgccacatgtacaaggactca -3'; Rat Cnp
forward 5'- tggagaaggacttcctgccacttt -3', reverse 5'- tggaggagctgggaaatcacaaggct -3'; Rat Plp
forward 5'- tctttggcgactacaagaccacca -3', reverse 5'- gttccagaggccaacatcaagctcat -3'; Mouse Mbp
: forward 5'- tcacagaagagaccctcaca-3', reverse 5'- gccgtagtgggtagttcttg -3'; Mouse Gpr17:
forward 5'- acacagttgtctgcctgcaa -3', reverse 5'- tgaccgtggtgatgaatggg -3' ; Mouse Plp:
forward 5'- tgctcggctgtacctgtgtacatt-3', reverse 5'- tacattctggcatcagcgcagaga-3'; Mouse Cnp:
forward 5'- tccacgagtgcaagacgctattca -3', reverse 5'- tgtaagcatcagcggacaccatct -3'; Mouse Cgt
: forward 5'- tgggtaagtggtgctcaggaacat -3', reverse 5'-tcctgccagtttgttggcaatgtc -3'; Mouse Mag
: forward 5'- tggtgtgtggctgagaaccagtat -3', reverse 5'-tgcacagtgtgactccagaaggat -3'.
Generation of GPR17 transgenic mice
An MBP enhancer in a pMG2 vector 46
was replaced with a 4.0-kb CNP1
promoter, which has previously been shown to direct transgene expression specifically in oligodendrocytes in the CNS 47
. The GPR17
coding region with an N-terminal Myc-tag was inserted after the CNP1
promoter. The NotI fragment carrying the transgene was purified by elutip-D columns and injected into fertilized mouse eggs to produce transgenic founders. All viable founder mice appeared to be mosaic and fertile, however, their progeny developed severely myelination deficits. Tissues of progeny were collected at different stages and processed for various assays such as immunocytochemistry, Western blot and electron microscopic analysis. Progeny from three founder mice carrying GPR17
transgene gave rise to the same dysmyelinating phenotype. The data presented are derived from the progeny of a single transgenic line.
Generation and genotyping of GPR17 knockout mice
To construct the GPR17 targeting vector, a 10.1 kb SnaB1 genomic fragment was cloned into pKO-915 (Stratagene) vector carrying the DTA gene. In a homologous recombination event, an h2bGFP/neo (neomycin-resistance gene) cassette replaced entire GPR17 coding exon. H2bGFP protein expression is in-frame fused to the GPR17 coding region immediately after the translation start site and is under the control of endogenous GPR17 promoter. This construct was used to target the GPR17 locus in SM-1 (129/Sv) embryonic stem cells. Correctly targeted cells were confirmed by Southern blotting and were injected into blastocysts to generate chimeric mice. Chimeras were crossed to C57Bl/6 mice to generate germ-line transmitted heterozygous mice. GPR17 null mice appear normal and were fertile without obvious motor or behavior abnormalities. Genotypes were determined by PCR of tail DNA (Gpr17-wt: forward 5'- ctgcttctaccttctggacttcatc-3', reverse 5'- aggtgagcatagagaggtcttcga -3'; Gpr17-ko: forward 5'- cttgtgcaacttgctctgcagca-3', reverse 5'- cacgagtgaagtcactgagtgtct -3') and yielded 310-bp wild-type and 380-bp mutant allele products, respectively.
Neural stem/progenitor cell culture and oligodendroglial culture
The adult hippocampal neural stem/progenitors used in this study were originally isolated from adult (8–10 wks old) female Fisher 344 rats and have been characterized previously 26
. These progenitor cells were cultured in N2-FGF growth medium [DMEM:F12 with N2 supplement (Invitrogen) and basic fibroblast growth factor (20 ng/ml FGF-2)] and induced to differentiate into oligodendrocytes in the presence of IGF-1 (100 ng/ml).
For mouse oligodendroglial precursor culture, cortical precursors were isolated from embryos at E15.5 and were grown in the N2-bFGF growth medium to enrich Olig2+ OPCs as previously indicated 32
, then passaged by trypsinization and cultured in oligodendrocyte growth medium (N2 supplemented with 20 ng/ml FGF2 and 10 ng/ml PDGF-AA)33
. An oligodendrocyte differentiation medium [N2 supplemented with 400 ng/ml Triiodothyronine T3 and 20 ng/ml ciliary neurotrophic factor (CNTF)] was used to promote oligodendrocyte differentiation 33, 48
. Primary rat OPCs were isolated from P1-2 Sprague Dawley rats as described previously 33
Transient transfection and immunohistochemistry
Adult rat hippocampus-derived neural progenitor cells (HCN) were seeded and grown in Dulbecco's modified Eagle medium with N2 supplemented with IGF1 (100 ng/ml) before transfection with Amaxa according to the manufacturer's protocol (Roche Applied Science, Indianapolis, IN
) and assayed 72-hour post-transfection for immunocytochemistry. Primary rat OPCs were transfected with either GPR17
-expression or control vector using the NeuroFECT according to the manufacturer's protocol (Genlantis, San Diego, CA). On the next day, OPCs were trypsinized and seeded onto established astrocytes. 3–5 days after seeding, cells were fixed and the progression of transfected OPCs was analyzed by immunocytochemistry. Transfected cells were identified by co-expressed GFP. The immunohistochemical staining procedure was performed as described previously 19
. Antibodies used: Olig1, Olig2 (gift of Chuck Stiles), MBP and Neurofilament SMI312 (Covance), Myc (Covance), O4, CNPase, MAG and MOG (Roche), PDGFRα (BD Bioscience), ID2 (BD Bioscience), ID4, GFAP and Myc (Santa Cruz), CC1 (Oncogene research) and Caspase-3 cleaved form (Upstate). Monoclonal antibody to RIP was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa under the auspices of the National Institute of Child Health and Human Development.
Western blotting and nuclear/cytoplasmic fractionation
For protein blotting analysis, whole cell lysates were prepared from HCN cells using RIPA buffer (Tris-HCl, 50 mM, pH 7.4; NP-40, 1%; Na-deoxycholate, 0.25%; NaCl, 150 mM; EDTA, 1 mM; PMSF, 1 mM; aprotinin, leupeptin, pepstatin, 1 mg/ml; Na3VO4, 1 mM; NaF, 1 mM). For nuclear and cytoplasmic extracts, HCNs were lysed in hypotonic buffer (Hepes, 10 mM, pH8; MgCl2, 1.5 mM; KCl, 10 mM; DTT, 1mM). Nuclei was then isolated and resuspended in the same buffer with NaCl, 420 mM; EDTA, 0.2 mM; glycerol, 25%; NP-40, 1%; PMSF, 1 mM; aprotinin, leupeptin, pepstatin, 1 mg/ml. Protein concentration in centrifugation-clarified cell lysates were measured by the BCA Protein Assay Kit (Pierce) and equal amounts of protein were separated on a 4–12% SDS-PAGE and transferred to Hybond PVDF (Amersham Biosciences). Protein blots were done using the NuPage gel transfer system with 4–20% Tris-Bis gels (Invitrogen). Primary Abs for protein blotting included: mouse anti-GAPDH (1:5000; Chemicon), rabbit anti-ID2 (1:500; Santa Cruz); rabbit anti-CREB (1:100; Cell Signaling). Immunoreactive bands were visualized using HRP-conjugated secondary antibodies, followed by ECL (Amersham Biosciences).
Chromatin immunoprecipitation (ChIP)
Chromatin was isolated from 1×107
rat hippocampus neural progenitor cells that were transfected either with empty vector containing Myc-nls-Tag or vector expressing Myc-nls-tag fused Olig1. After immunoprecipitation with 5µg anti-Myc antibody (mouse monoclonal sc-40X, Santa Cruz), chromatin was reverse cross-linked using EZ ChIP kit (Upstate Biotechnology). Proteins were digested with proteinase K and the recovered DNA was purified using QIAGEN QIAquick PCR purification kit and subjected to PCR amplification. PCR primers for ChIP assays were designed to amplify around 200 bp fragments from the upstream of start site of GPR17
containing with or without E-box (CANNTG) consensus sequences for bHLH protein binding. Real-time PCR was carried out using quantitative SYBR green PCR mix (Applied Biosystems, Inc.). The fold-enrichments were determined by the 2−ΔCT
methods as previously described 49
. Primer sequences are for region I: forward 5'-ccaacgtaaagtagccagtcctgg-3', reverse 5'-gctggtcaactctattgtgaccagc -3'; Region II: forward 5'- aggtagctttccaggctctgagtt -3', reverse 5'-aactcgggagaaacatggctgttc-3'.
Induction of EAE
EAE was induced in 6–8 week old C57/BL6 mice (Taconic, NY) by s.c. injection over 2 sites in the flanks with 200 µg MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA, supplemented with M. tuberculosis (4mg/ml) (Difco, Detroit, MI). Pertussis toxin (200 ng/mouse) in PBS was injected i.p. at the time of immunization and 48 h later. Mice were scored on scale of 0 to 6 as previously described 50
: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5 quadriplegia or premoribund state; 6, death. Animal use and studies were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas.
Analysis of human MS samples
Tissue specimens from MS patients were obtained from the Human Brain and Spinal Fluid Resource Center, VA West Los Angeles Healthcare Center, Los Angeles, CA 90073 which is sponsored by NINDS/NIMH, National Multiple Sclerosis Society, and Department of Veterans Affairs. Frozen autopsy samples from 10 MS and 10 normal control cases were processed for total RNA using Qiagen RNeasy, with adjacent sections being processed for histology (H&E). For each MS donor, samples included plaques, adjacent white matter, and nearest grey matter; all samples were from frontal lobe.
Total RNA was assessed for quality by Agilent Bioanalyzer and expression of huGPR17 was measured by quantitative real-time RT-PCR using the ABI PRISM 7900HT sequence detection system (Applied Biosystems, Inc.) and normalized to the expression of a housekeeping gene (HPRT). Two unique qPCR primer/probe sets (5’-3’) were used to determine relative expression of Gpr17 cDNA and confirm expression patterns. All RNA was DNase-treated (DNA-free, Ambion) prior to generation of cDNA using a Taqman reverse transcription kit (Applied Biosystems Inc). Using 20 ng of total RNA equivalent cDNA, samples were subject to qPCR using 2x Taqman Universal Buffer (Applied Biosystems Inc.) with 200 µM forward and reverse primers and 900 µM probe with the following PCR conditions 50°C for 2 min; 95°C for 10 min; 95°C for 15 s; 60°C for 1 min (40x). Each PCR reaction was run in triplicate for each biological sample included in the study.
Results were analyzed for statistical significance using two-tailed Student’s t test and all error bars were expressed as standard deviations (SD). Values of p<0.05 were considered significant. For multiple comparisons, which were done using one-way analysis of variance analysis (ANOVA) with posttest: Newman-Keuls Multiple comparison test.