Project description:Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the longstanding hypothesis that chronic GC exposure promotes brain aging/Alzheimer's disease. Here, we adrenalectomized male F344 rats at 15-months-of-age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid-receptor (GR)-activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between Intermediate- and Low-CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT-upregulated genes included learning/plasticity, differentiation, glucose metabolism and cholesterol biosynthesis, whereas processes overrepresented by CORT-downregulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC-transcriptome with a previously-defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same-direction, the majority were shifted in opposite directions by CORT and aging (e.g., glial inflammatory genes downregulated by CORT are upregulated with aging). These results contradict the hypothesis that GCs simply promote brain aging, and also suggest that the opposite-direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways while GC overstimulation develops in others, together generating much of the brain aging phenotype. Forty male Fischer 344 rats aged 14-15 months (late mid-age) were either sham operated (n=10) or adrenalectomized (n=30) at Harlan, Inc. (Indianapolis) and shipped to University of Kentucky 7-10 d after surgery. Animals were allowed to acclimate for 2 weeks after receipt prior to pellet-implant and were provided with food and water ad lib. Adrenalectomized (ADX) animals were maintained on drinking water containing isotonic saline with 1% sucrose, and also given supplemental feed mash (standard chow mixed with water and warmed) and apple chunks, as well as subcutaneous injections of 1.25 mg corticosterone sulfate (Solu-DeltaCort, Pfizer). During pellet-implant surgery, subjects were anesthetized with isoflurane, placed on a warming pad, and a 3 cm2 area on the animal's flank was shaved and sterilized. A 1-cm incision was made through the skin to form a subcutaneous pocket. Each sham animal received an inert pellet. ADX animals were divided into two groups, low-dose animals (n = 20) received one 25 mg CORT pellet and intermediate-dose animals (n = 10) received one 200 mg CORT pellet (slow release pellets, Innovative Research of America, Sarasota, FL). Incisions were closed with wound clips (removed after 7-10 days). Following implant surgery, subjects were housed singly for 3 months prior to euthanasia at age 18 months. Low-dose animals continued to receive saline/ sucrose drinking water, but other supplementation (food mash, Solu-Delta CORT, apple chunks) was discontinued. One ADX animal was removed from the study for declining health. During the twelfth week, just prior to study termination, half of the ADX low-dose CORT subjects were given a short-term, high-dose regimen consisting of 4 daily IP injections of 5mg/d CORT (considered a relatively high dose- Makino et al., 1995). All animals were killed between 8 and 10 AM on the last two days of the study. After deep CO2 anesthesia and decapitation, trunk blood was collected for CORT radioimmunoassay and hippocampal CA1 brain tissue was dissected as described previously (Blalock et al., 2003) for microarray analysis (see below). Final treatment groups consisted of: 1) Sham (intact) control, 2) Low-CORT (ADX), 3) Intermediate-CORT (ADX), and 4) Low-CORT (ADX) + short-term high-dose injected (n=9-10/group).

Project description:Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the longstanding hypothesis that chronic GC exposure promotes brain aging/Alzheimer's disease. Here, we adrenalectomized male F344 rats at 15-months-of-age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid-receptor (GR)-activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between Intermediate- and Low-CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT-upregulated genes included learning/plasticity, differentiation, glucose metabolism and cholesterol biosynthesis, whereas processes overrepresented by CORT-downregulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC-transcriptome with a previously-defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same-direction, the majority were shifted in opposite directions by CORT and aging (e.g., glial inflammatory genes downregulated by CORT are upregulated with aging). These results contradict the hypothesis that GCs simply promote brain aging, and also suggest that the opposite-direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways while GC overstimulation develops in others, together generating much of the brain aging phenotype.

Project description:In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 μg/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 μg/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome. - See more at: http://press.endocrine.org/doi/10.1210/en.2012-2187?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#sthash.LqK088DP.dpuf

Dataset Information

Characterization of Glucocorticoid-Induced Changes in Gene Expression in the Embryonic Pituitary Gland.

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