Scott E. Nelson

Age-related changes in the protein expression of subunits of the NMDA receptor.

C57Bl/6 mice show decreased expression of the mRNA for the epsilon2 and zeta1 subunits of the N-methyl-D-aspartate (NMDA) receptor in subregions of the cerebral cortex and hippocampus with increased age. The purpose of this study was to determine the effects of aging on the protein expression of the three major subunits of the NMDA receptor. Semi-quantitative Western blot techniques were applied with the use of antibodies that recognize either the epsilon1 (NR2A), epsilon2 (NR2B) or zeta1 (NR1) subunits of the NMDA receptor or a synaptic terminal protein, synaptophysin. In the cerebral cortex of 30-month-old mice, the level of protein expression of both the epsilon2 and zeta1 subunits were decreased significantly from levels found in the 3- and 10-month-old mice and the protein expression of the epsilon1 subunit showed a significant decline between 10 and 30 months of age. In the hippocampus, the epsilon2 subunit exhibited a higher protein expression level in the 10-month-old mice as compared to both the young and old mice and the zeta1 subunit showed a significant drop in expression in the old mice from both 3- and 10-month-olds. Synaptophysin showed significant declines in protein expression with increasing age. These results demonstrated that changes in the protein expression of the major subunits of the NMDA receptor occur during the aging process and, in some cases, were greater than changes seen previously in mRNA expression. These subunit alterations may explain some of the changes that are seen in NMDA receptor functions during aging.

Cell-specific expression of the mouse gonadotropin-releasing hormone (GnRH) receptor gene is conferred by elements residing within 500 bp of proximal 5' flanking region.

Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by the hypothalamus. Upon binding to specific high-affinity receptors on gonadotrope cells of the anterior pituitary gland, GnRH stimulates the synthesis and secretion of LH. In light of the critical role of GnRH in reproduction much effort has been directed toward understanding the regulation of this hormone and its cognate receptor. The recent availability of genomic clones for the GnRH receptor has facilitated research to address the molecular mechanisms underlying regulation of GnRH receptor gene expression. We have expanded the analysis of the promoter for the mouse GnRH receptor gene and report that in addition to transcriptional start sites located within 100 bp of the translation start codon there is a more distal transcriptional start site approximately 200 bp 5′ of the initiation codon. The initiation of transcription from this more distal site was sufficient to confer cell-specific expression on luciferase. Further, transient expression assays of constructs containing progressive 5′ deletions in the GnRH receptor gene promoter reveal the presence of one or morecis-acting elements located between −500 and −400 (relative to ATG) necessary for transcriptional activity in the gonadotrope-derived αT3 cell line. Finally, αT3 but not COS-7 cell nuclear extract contained protein(s) that bind to at least two separate motifs contained within the −500 to −400 region. We suggest that activation of GnRH receptor gene expression in the αT3 cell line requires the binding of at least two transcriptional regulatory proteins to basal enhancer elements located within a 100 bp region between −500 to −400 relative to the translation start codon in the mouse GnRH receptor gene.

Changes in the expression of the NR2B subunit during aging in macaque monkeys.

Humans, non-human primates and rodents show declines in spatial memory abilities with increased age. Some of these declines in mice are related to changes in the expression of the epsilon2 (epsilon2) (NR2B) subunit of the N-methyl-D-aspartate receptor. The purpose of this study was to determine whether primates show changes during aging in the mRNA expression of the NR2B subunit. In situ hybridization was performed on tissue sections from three different ages of Rhesus monkeys (Macaca mulatta; 6-8, 10-12, and 24-26 years). There was a significant decrease in the mRNA expression of the NR2B subunit overall in the prefrontal cortex and in the caudate nucleus between young and old monkeys. There were no significant changes in NR2B mRNA expression in the hippocampus or the parahippocampal gyrus. The results in the prefrontal cortex, caudate and hippocampus were similar to those seen previously in C57BL/6 mice during aging, which suggests that mice may be useful as a model for primates to further examine the age-related changes in the expression of the NR2B subunit of the NMDA receptor in several important regions of the brain.

Cellular Mechanisms by Which Oxytocin Mediates Ovine Endometrial Prostaglandin F2α Synthesis: Role of Gi Proteins and Mitogen-Activated Protein Kinases

Abstract Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2α synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2α synthesis. The objective of experiment 1 was to determine whether Gi proteins mediate oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of Gi proteins, had no effect on the ability of oxytocin to induce PGF2α synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF2α synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2α synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF2α synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to Gi proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2α synthesis in ovine endometrium.

Development and aging of n-methyl-d-aspartate receptor expression in the prefrontal/frontal cortex of mice

The present study was designed to determine whether the changes that occur during aging in the expression of the N-methyl-D-aspartate (NMDA) receptor and two NMDA receptor subunits, zeta1 and epsilon2, are a continuation of developmental processes and whether protein and mRNA expression patterns of the subunits are similar across the lifespan. The prefrontal/frontal cortex of C57BL/6 mice of eight different ages (7-8, 13-15, 30-32, 49-53, and 70-72 days and 4.5, 11, and 25 months of age) were used to examine NMDA-displaceable [(3)H]glutamate binding and mRNA in tissue sections and mRNA and protein from homogenates. The lateral prefrontal/frontal cortex of C57BL/6 mice showed more significant declines in density of agonist binding to NMDA receptors during both development and aging than the medial cortex. Changes in mRNA expression for the epsilon2 subunit across the lifespan appeared to be related to the changes in NMDA receptor binding in the lateral cortex, even though the protein expression of the epsilon2 subunit did not show the same pattern of expression as the mRNA during development. The changes in epsilon2 subunit mRNA expression during adult aging may be a continuation of developmental processes, but there was also evidence that expression levels plateaued during early adulthood. The developmental expression of the zeta1subunit in the prefrontal/frontal cortex was influenced by gender and there was no significant effect of adult aging on either the protein or mRNA expression of this subunit. Determining how the expression of the NMDA receptor and its subunits change throughout the lifespan can help us to better understand the processes affecting the receptor during aging. These results should be useful for designing interventions into the aging process to repair or prevent changes in the NMDA receptor and its associated functions, such as learning and memory.