Inactivation of glycogen synthase kinase-3beta protects against kainic acid-induced neurotoxicity in vivo.

Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3beta (GSK-3beta), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3beta phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3beta inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3beta using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3beta in cell death in response to excitotoxins in vivo and further confirms that GSK-3beta plays a role in cell death/cell survival pathways.

Estrogen receptor (ER)-mediated transcriptional regulation of the human corticotropin-releasing hormone-binding protein promoter: differential effects of ERalpha and ERbeta.

CRH-binding protein (CRH-BP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis by binding and inhibiting CRH. We investigated for the first time transcriptional regulation of the human CRH-BP promoter using transient transfections. Estrogen receptors (ERs) contributed to ligand-independent constitutive activation of the promoter, whereas in the presence of estradiol ERalpha induced and ERbeta repressed promoter activity in a dose-dependent manner. TNFalpha inhibited promoter induction by ERalpha in the absence and presence of estradiol. Three ERE half-sites in the CRH-BP promoter bound ERalpha and ERbeta in an EMSA, and disruption of ERE half-sites by site-directed mutagenesis abolished ligand-independent induction by ERalpha and ERbeta and promoter enhancement by estradiol-activated ERalpha. Repression by estradiol/ERbeta was unaffected by disruption of ERE half-sites, activating protein 1, cAMP response element, GATA, or nuclear factor kappaB sites, and reversed to promoter induction by estrogen antagonists, tamoxifen and ICI 182,780, suggesting corepressor involvement. In hypothalamic GT1-7 cells, Western blotting demonstrated rapid induction of endogenous CRH-BP expression by estradiol-bound ER, which was inhibited by TNFalpha. We propose a model in which ERs maintain basal CRH-BP expression in pituitary and neurosecretory cells, whereas in the presence of ERalpha estrogen enhances CRH-BP transcription, causing down-regulation of the HPA axis, and nuclear factor kappaB-activating cytokines activate the HPA axis by inhibiting ERalpha.

Inhibition of glycogen synthase kinase 3 beta is involved in the resistance to oxidative stress in neuronal HT22 cells.

Oxidative stress is involved in several neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and ischemic reperfusion injury (stroke). We have established clones of the murine hippocampal neuronal cell line HT22, which are resistant to the oxidative stress-causing agents glutamate and hydrogen peroxide, respectively. These cell clones show a mutual cross-resistance to other oxidative stressors, but not to essentially non-oxidative neurotoxins. We have discovered that the amount of phosphorylated, inactive glycogen synthase kinase (GSK) 3beta is elevated in both resistant clones. Pharmacological inhibition of GSK-3beta with lithium chloride in the sensitive parental neuronal cells results in an increased tolerance to glutamate and hydrogen peroxide, suggesting that GSK-3beta is involved in the control of oxidative stress resistance in these cells.

CB1 cannabinoid receptors and on-demand defense against excitotoxicity.

Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. We generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice,the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.

Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain.

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reaction-based differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex 1 h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 zeta protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 zeta represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid.

Estrogen receptor alpha-mediated silencing of caveolin gene expression in neuronal cells.

Estrogen receptors (ER alpha/ER beta) are expressed in neuronal cells and exhibit a variety of activities in the central nervous system. ER activity is regulated in a ligand-dependent manner and by co-regulatory factors. Caveolin-1 is a recently identified co-activator of ER alpha mediating the ligand-independent activation of this steroid receptor. Here the influence of ERs on caveolin expression in human neuroblastoma SK-N-MC cells as well as in rodent brain was investigated. We found that ectopic expression of ER alpha in SK-N-MC cells (SK-ER alpha) leads to a ligand-independent transcriptional suppression of caveolin-1/-2 genes. This suppression is specifically mediated by ER alpha and not ER beta because ER beta counteracts the observed caveolin-silencing process. Interestingly, decreased caveolin expression in SK-ER alpha is accompanied by changes in the methylation pattern of caveolin promoters. The analysis of selected promoter regions of the human caveolin-1 gene showed that certain CpG dinucleotides were hypermethylated in SK-ER alpha cells, whereas the same sites were unmethylated in control, ER beta-, and ER alpha/beta co-expressing SK-N-MC cells. Inhibition of DNA methylation or histone deacetylation led to partial re-expression of caveolin-1/-2 genes in SK-ER alpha. In vivo analysis revealed a down-regulation of caveolin-1 expression after long term estrogen exposure in certain regions of the mouse brain. In conclusion, we have shown for the first time that ER alpha and not ER beta silences caveolin-1/-2 expression in an epigenetic fashion in neuronal cells. The observed mechanism of gene silencing by ER alpha may have implications for the transcriptional regulation of further ER alpha target genes.