Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

Identification of new Nerve Growth Factor-responsive immediate-early genes.

Stimulation of the PC12 pheochromocytoma cell line with the prototypical neurotrophin Nerve Growth Factor (NGF) induces a cellular response of neuronal differentiation and is therefore a widely used model to gain molecular insight into this process. Classically, the transcriptional response to extracellular stimuli such as NGF is divided in genes that require no protein synthesis prior to their induction (immediate-early genes) and genes that do (delayed-response genes). Because an increasing number of studies have reported important roles for immediate-early genes (IEGs) in neuronal differentiation, the goal of the present study was to identify previously unrecognized NGF-responsive IEGs. Stimulation with NGF for 15, 30, 60 and 120 min resulted in a typical transient induction of many known NGF-responsive IEGs. To identify candidate new genes, we analyzed 27000 measured expression profiles and selected 10 genes for further study. Five genes, including Cbp/p300-interacting transactivator 2 (Cited2), Kruppel-like factor 4 (Klf4), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein F (Maff), Kruppel-like factor 10 (Klf10 or Tieg) and Activating transcription factor 3 (Atf3) were selected and positively validated by qPCR. NGF-induced activation of all five genes seems to be mediated by MAPK and PI3K-mediated pathways. Additionally, we tested translation-independent induction and showed that NGF induced upregulation of these genes in both the subclonal Neuroscreen-1 PC12 and parental PC12 cell line. These 5 transcription factors have not been previously reported as NGF-responsive IEGs, however have previously been reported as important regulators of cell differentiation and proliferation in different systems. These observations may therefore provide important new information on the molecular mechanisms underlying NGF-induced differentiation.

Neuroanatomical distribution and colocalisation of nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) in rat brain.

The two structurally related nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid receptors (SMRT) proteins have been found to differentially affect the transcriptional activity of numerous nuclear receptors, such as thyroid hormone, retinoic acid and steroid receptors. Because of the numerous effects mediated by nuclear receptors in brain, it is of interest to extend these in vitro data and to explore the cellular distribution of both corepressors in brain tissue. We therefore examined, using in situ hybridisation, whether the relative abundance of these two functionally distinct corepressors differed in rat brain and pituitary. We find that although both N-CoR and SMRT transcripts are ubiquitously expressed in brain, striking differences in their respective levels of expression could be observed in discrete areas of brain stem, thalamus, hypothalamus and hippocampus. Using dual-label immunofluorescence, we examined in selected glucocorticoid sensitive areas involved in the regulation of the hypothalamus-pituitary-adrenal axis activity, the respective protein abundance of N-CoR and SMRT. Protein abundance was largely concurrent with the mRNA expression levels, with SMRT relatively more abundant in hypothalamus and brain stem areas. Colocalisation of N-CoR and SMRT was demonstrated by confocal microscopy in most areas studied. Taken together, these findings are consistent with the idea that the uneven neuroanatomical distribution of N-CoR and SMRT protein may contribute to the site-specific effects exerted by hormones, such as glucocorticoids, in the brain.

The genetic background modifies the spontaneous and X-ray-induced tumor spectrum in the Apc1638N mouse model.

The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.

Differentially expressed transcripts in X-ray-induced lymphomas identified by dioxygenin-labeled differential display.

Differential display is an easily applied method for comparing gene expression in a variety of systems. We used a nonradioactive differential display technique to analyze X-ray-induced lymphomas derived from Emu-pim-1 transgenic and nontransgenic mice. Fragments of 11 differentially regulated genes were identified, three of which are novel sequences. One of the cloned fragments contained sequences of a mouse VL30 retroelement that was significantly overexpressed in a subset of lymphomas as compared with non-lymphomatous tissue. Interestingly, these lymphomas also displayed high levels of c-myc transcripts. An altered expression pattern of a glutathione S-transferase homologue was identified in several lymphomas. Moreover, a cytotoxic T-lymphocyte lipase appeared to be overexpressed specifically in lymphoma-containing spleen tissue, and the results suggest that it may be related to the endogenous immune response against lymphoma development.

X-ray-induced lymphomagenesis in E mu-pim-1 transgenic mice: an investigation of the co-operating molecular events.

Transgenic mice overexpressing the pim-1 oncogene in their lymphoid compartment display a low incidence of spontaneous T-cell lymphomas, but are highly susceptible to point mutation-inducing genotoxic carcinogens. We show here that total body X-irradiation, which causes mainly chromosomal deletions, rearrangements and amplifications, significantly enhances lymphoma development in E mu-pim-1 transgenic mice. The X-ray-induced E mu-pim-1 and non-transgenic lymphomas have a comparable high cell turnover as shown by a relatively high S-phase fraction and a high apoptotic activity. Consistent with previous observations, in 75% of all lymphomas c-myc mRNA levels are 5- to 20-fold higher than in control, non-lymphomatous spleen/thymus. The expression of other oncogenes, which have previously found to be activated in combination with pim-1 in lymphomagenesis, such as gfi-1/pal-1, frat-1 and tiam-1, and also of the mdm-2 and mdm-x oncogenes, appeared not to be affected. Deletions and/or rearrangements of the p16INK4A and p15INK4B tumor suppressor genes were seldom observed (in three out of 92 X-ray-induced lymphomas). Strikingly, in addition to the high mRNA levels of the pim-1 transgene, the levels of the endogenous pim-1 transcripts were elevated significantly in 16% of the X-ray-induced E mu-pim-1 lymphomas compared with control spleen, even surpassing the level of the pim-1 transgene mRNA by 3- to 5-fold. In combination with previous results, which showed that the lymphoma incidence increased concordantly with higher levels of pim-1, this supports the notion that pim-1 can contribute to lymphomagenesis in a dose-dependent manner.