Identification of a murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter segment sufficient for driving reporter gene expression in neurons in vitro and in vivo.

In this study, we characterize the functional properties of a segment of the murine methyl cytosine phosphate guanine binding domain-containing factor 3 (MBD3) promoter region. Transient transfection of a chimera consisting of a 1072 base pair region extending upstream from the MBD3 initiation codon fused to a luciferase complementary DNA (cDNA) confirmed the presence of a functional promoter unit. Primer extension analysis failed to identify a single predominant transcription initiation site, but rather detected multiple transcription initiation sites in both brain tissue and cultured neuroblastomaxglioma cell line (NG108-15) and rat pheochromocytoma cell line (PC12) cells. Reporter gene assays revealed that this 1072 base pair fragment efficiently drives expression in transfected NG108-15 cells, PC12 cells, cultured primary neurons, and in neurons of a transgenic mouse brain. Deletion analysis mapped the critical region for promoter activity to a segment of approximately 518 base pairs, located from positions -585 to -68 relative to the translational start codon. Taken together, these data indicate that a 1072 base pair fragment of the MBD3 promoter is sufficient to drive expression in cell lines and primary cultured neurons, and is able to direct transgene expression in the mouse brain in a pattern with spatial similarity to that of native MBD3.

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes.

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3-24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22-q31.1, and GPR23 to chromosome X, region q13-q21.1.

Characterization of a human gene related to genes encoding somatostatin receptors.

We report the identification of a gene, named SLC-1(1), encoding a novel G protein-coupled receptor (GPCR). A customized search procedure of a database of expressed sequence tags (dbEST) retrieved a human cDNA sequence that partially encoded a GPCR. A genomic DNA fragment identical to the cDNA was obtained and used to screen a library to isolate the full-length coding region of the gene. This gene was intronless in its open reading frame, and encoded a receptor of 402 amino acids, and shared -40% amino acid identity in the transmembrane (TM) regions to the five known human somatostatin receptors. Northern blot analysis revealed that SLC-1 is expressed in human brain regions, including the forebrain and hypothalamus. Expression in the rat was highest in brain, followed by heart, kidney, and ovary. Expression of SLC-1 in COS-7 cells failed to show specific binding to radiolabelled Tyr1-somatostatin-14, naloxone, bremazocine, 1,3-di(2-tolyl)-guanidine (DTG), or haloperidol. A repeat polymorphism of the form (CA)n was discovered in the 5'-untranslated region (UTR) of the gene and SLC-1 was mapped to chromosome 22, q13.3.

Transient forebrain ischemia alters the mRNA expression of methyl DNA-binding factors in the adult rat hippocampus.

We have examined how transient cerebral ischemia affects the mRNA expression of a family of methyl CpG-binding domain (MBD)-containing factors in the rat hippocampus. Our results show that each member of this family is affected by cerebral ischemia challenge, but with differing patterns of responsiveness. At 3, 6 and 12 h following reperfusion, MeCP2 and MBD1 expression is maintained at control levels throughout the hippocampus. At 24 h, MeCP2 and MBD1 are induced in both the CA1 and CA3 subfields. This delayed pattern of induction is in contrast to the responses of MBD2 and MBD3. Both MBD2 and MBD3 display significant changes in expression at early times following reperfusion, although their changes are opposite in direction. MBD2 expression is induced throughout the hippocampal formation at 6 h, and remains elevated at 12 and 24 h. MBD3 expression decreases as early as 3 h following insult in the CA3 and dentate gyrus, and the decreased expression remains in the vulnerable CA1 subfield at 6, 12, and 24 h. Taken together, these results are the first to illustrate that the expression of methyl DNA-binding factors are affected by challenges to the brain, and they also illustrate that each methyl DNA-binding factor responds differently to cerebral ischemic challenge. As each of these family members is associated either directly or indirectly with the inhibition of gene transcription, our results suggest that following cerebral ischemia the normal pattern of transcriptional inhibition provided by these factors may be altered in the hippocampus.

Expression and function of A1 adenosine receptors in the rat hippocampus following transient forebrain ischemia.

We investigated how transient cerebral ischemia affects the gene expression, immunoreactive protein levels, and the function of the A1 subtype of adenosine receptor in the rat hippocampus at different times following reperfusion. A1 receptor mRNA levels were altered significantly in different hippocampal subfields as early as 6 h following insult. However, these changes in mRNA levels were not paralleled at the protein level, as western blotting with A1 receptor-specific antibodies revealed that hippocampal A1 adenosine receptor prevalence did not differ from sham control at either 6 or 24 h following insult. The lack of change in A1 receptor prevalence was consistent with functional examinations, as only marginal changes were observed in the ability of A1 receptors to attenuate excitatory post-synaptic potentials in the CA1 subfield at 24 h following reperfusion. These data illustrate that although the mRNA expression levels of the A1 adenosine receptor are altered by transient cerebral ischemia, the immunoreactive prevalence and function of this receptor are maintained in the post-ischemic hippocampus at times preceding the death of the vulnerable neurons.

Perforant pathway kindling transiently induces the mRNA expression of GABA-B receptor subtypes R1A and R2 in the adult rat hippocampus.

We examined the gene expression responses of GABA-B R1A, R1B and R2 receptor subtypes in the hippocampus of perforant pathway-kindled rats at 24 h and 28 days after 15 consecutive daily stimulations. We found R1A expression, but not R1B expression, to be significantly induced in the dentate gyrus at 24 h. No change in the expression of R1A or R1B was observed at 28 days. R2 expression was induced throughout the hippocampus at 24 h, but also returned to control levels by 28 days. Thus, our results show that kindling induces a transient increase in GABA-B receptor mRNA in the hippocampus.

Somatostatin type 2 receptor expression in the rat hippocampus following cerebral ischemia.

We examined how transient cerebral ischemia affects the mRNA expression, and the immunoreactive distribution, of the somatostatin type 2 (sst2) receptor in the adult rat hippocampus. Following reperfusion, sst2 mRNA levels increased significantly in the CA1 region by 3 h, and were also increased in the CA3 and CA4/hilus subfields at 6 and 12 h. At 24 h, however, sst2 receptor mRNA levels returned to baseline throughout the hippocampus. At the protein level, we found the regional immunoreactivity of the sst2a receptor was maintained, or slightly elevated, throughout the hippocampus at 6 h, but not different from control at 24 h. These results suggest that sst2 receptors maintain their normal distribution and prevalence in the post-ischemic hippocampus before the deterioration of the vulnerable CA1 neurons. Thus, they represent attractive targets for neuroprotective interventions.

Discovery of a novel human G protein-coupled receptor gene (GPR25) located on chromosome 1.

We amplified human genomic DNA by the polymerase chain reaction (PCR) using oligonucleotides based on the primary sequence of the genes encoding the somatostatin receptors (SSTR) and the somatostatin-like receptor gene SLC-1. One resultant DNA fragment was used to screen a genomic DNA library resulting in the isolation of a gene, GPR25, encoding an additional member of the G protein-coupled receptor family (GPCR). GPR25 is intronless throughout its open reading frame (ORF) and encodes a protein of 360 amino acids. The receptor encoded by GPR25 shares highest identity to the receptor encoded by GPR15, angiotensin II type 1A receptor, and somatostatin receptor 5. Northern analysis found no transcripts expressed in liver or any of the 12 brain regions analyzed. Fluorescence in situ hybridization analysis localized GPR25 to chromosome 1q32.1.