Gene expression analysis in the hippocampal formation of tree shrews chronically treated with cortisol.

Adrenal corticosteroids influence the function of the hippocampus, the brain structure in which the highest expression of glucocorticoid receptors is found. Chronic high levels of cortisol elicited by stress or through exogenous administration can cause irreversible damage and cognitive deficits. In this study, we searched for genes expressed in the hippocampal formation after chronic cortisol treatment in male tree shrews. Animals were treated orally with cortisol for 28 days. At the end of the experiments, we generated two subtractive hippocampal hybridization libraries from which we sequenced 2,246 expressed sequenced tags (ESTs) potentially regulated by cortisol. To validate this approach further, we selected some of the candidate clones to measure mRNA expression levels in hippocampus using real-time PCR. We found that 66% of the sequences tested (10 of 15) were differentially represented between cortisol-treated and control animals. The complete set of clones was subjected to a bioinformatic analysis, which allowed classification of the ESTs into four different main categories: 1) known proteins or genes (approximately 28%), 2) ESTs previously published in the database (approximately 16%), 3) novel ESTs matching only the reference human or mouse genome (approximately 5%), and 4) sequences that do not match any public database (50%). Interestingly, the last category was the most abundant. Hybridization assays revealed that several of these clones are indeed expressed in hippocampal tissue from tree shrew, human, and/or rat. Therefore, we discovered an extensive inventory of new molecular targets in the hippocampus that serves as a reference for hippocampal transcriptional responses under various conditions. Finally, a detailed analysis of the genomic localization in human and mouse genomes revealed a survey of putative novel splicing variants for several genes of the nervous system.

Identification of genes regulated by chronic psychosocial stress and antidepressant treatment in the hippocampus.

Analysis of differentially expressed genes in the brain is a promising tool for elucidating pathological mechanisms that lead to central nervous disorders. Stress is known to be involved in the development of psychopathologies such as depression. In the present study, we searched for differentially expressed genes in the hippocampal formation after chronic psychosocial stress and after treatment with the antidepressant clomipramine. Experiments were conducted in male tree shrews, a valid psychosocial stress model in which antidepressant drugs prevent diverse effects of stress. Because many effects of stress have been attributed to the stress-induced elevation in glucocorticoids, we screened two subtractive hippocampal cDNA libraries generated from RNA of chronic cortisol-treated animals. Using real-time PCR to measure mRNA amounts, we identified five sequences whose expression levels differed between stressed animals and controls. Transcript levels of four of them, nerve growth factor (NGF), membrane glycoprotein 6a (M6a), CDC-like kinase 1 (CLK-1) and G-protein alpha q (GNAQ) were reduced by chronic psychosocial stress. Reduced amounts of these genes, which are all related to processes of cell differentiation, is in agreement with previous findings showing a retraction of dendrites and an impairment of neurogenesis in the hippocampal formation after chronic stress. An additional expressed sequence that was also regulated by stress could not be assigned to any known gene. Treatment with the antidepressant clomipramine prevented stress effects on expression of M6a, CLK-1, GNAQ and the novel sequence, but showed no effect on NGF stress-induced down-regulation. These findings support the concept that depressive disorders are accompanied by processes of neuronal dedifferentiation, at least in the hippocampal formation, and that antidepressants prevent these processes.

Differential regulation of polysialyltransferase expression during hippocampus development: Implications for neuronal survival.

Polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST add polysialic acid (PSA) to the neural cell adhesion molecule (NCAM). Surface-located PSA is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. This study was undertaken to investigate the polysialyltransferase regulation pattern during hippocampal development. Polysialyltransferase expression levels analyzed by real-time RT-PCR indicated that ST8SiaII/STX mRNA is markedly down-regulated in vivo, decreasing abruptly at about the first week of postnatal development. ST8SiaII/STX mRNA is also down-regulated in hippocampal cells in culture, accompanying the morphological differentiation of neuronal interconnectivity. In contrast, ST8SiaIV/PST levels remain comparatively low during hippocampus ontogeny. Immunolabeling of primary hippocampal culture assays demonstrated that PSA expression parallels ST8SiaII/STX mRNA levels. In comparison, polysialyltransferase mRNA levels are not regulated in neuroblastoma cells during their proliferation. Sequence analysis of the 3'-untranslated region of ST8SiaII/STX cDNA indicated putative regulatory motifs. This information and the observed changes in mRNA half-life during development suggest that ST8SiaII/STX might be also regulated at the posttranscriptional level. To understand the reasons for the tight control of ST8SiaII/STX expression during development, we overexpressed the enzyme in hippocampal primary cultures by transfection. Overexpression of ST8SiaII/STX wild type as well as of a mutant lacking enzymatic activity affected neuronal viability, leading to cell death. However, this phenomenon was abolished by a double mutation in the ST8SiaII/STX that prevents formation of its three-dimentional structure. Interestingly, the overexpressed polysialyltransferase accumulates not only in the perinuclear region but also in the plasma membrane. Thus, overexpression of an ST8SiaII/STX that conserves its structure leads to abnormal accumulation of the protein, probably on the neuronal surface, affecting cell viability. This result explains the importance of an accurate regulation of polysialyltransferase expression during development.

Analysis of gene expression in the rat hippocampus using Real Time PCR reveals high inter-individual variation in mRNA expression levels.

In mammals, gene transcription is a step subjected to tight regulation mechanisms. In fact, changes in mRNA levels in the central nervous system (CNS) can account for numerous phenotypic differences in brain function. We performed a high-resolution analysis of mRNA expression levels for 37 genes selected from a normal rat hippocampus cDNA library. mRNA amounts were quantified using a Real Time PCR SYBR Green assay. We found that, in general, individuals from an inbred rat population (n = 20) have shown 2-3 times differences in the basal level of expression of the genes analyzed. Up to several fold differences among individuals were observed for certain genes. These inter-individual differences were obtained after correction for the different amounts of mRNA in each sample. Power calculations were performed to determine the number of individuals required to detect reliable differences in expression levels between a control and an experimental group. These data indicated that, depending on the variability of the candidate gene selected, it was necessary to analyze from five to 135 individuals in each group to detect differences of 50% in the levels of mRNA expression between two groups investigated. The comparison of mRNA abundance from different genes revealed a wide range of expression levels for the 37 genes, showing a 26,000-fold difference between the highest and lowest expressed gene.