[Abstract unavailable

Many genes whose expression is restricted to neurons in the brain contain a silencer element (RE1/NRSE) that limits transcription in nonneuronal cells by binding the transcription factor REST (also named NRSF or XBR). Although two independent domains of REST are known to confer repression, the mechanisms of transcriptional repression by REST remain obscure. We provide multiple lines of evidence that the N-terminal domain of REST represses transcription of the GluR2 and type II sodium-channel genes by recruiting the corepressor Sin3A and histone deacetylase (HDAC) to the promoter region in nonneuronal cells. These results identify a general mechanism for controlling the neuronal expression pattern of a specific set of genes via the RE1 silencer element.

Inositol phosphates are well-known signaling molecules, whereas the inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7/IP7) and bis-diphosphoinositol tetrakisphosphate (InsP8/IP8), are less well characterized. We demonstrate physiologic regulation of Dictyostelium chemotaxis by InsP7 mediated by its competition with PtdIns(3,4,5)P3 for binding pleckstrin homology (PH) domain-containing proteins. Chemoattractant stimulation triggers rapid and sustained elevations in InsP7/InsP8 levels. Depletion of InsP7 and InsP8 by deleting the gene for InsP6 kinase (InsP6K/IP6K), which converts inositol hexakisphosphate (InsP6/IP6) to InsP7, causes rapid aggregation of mutant cells and increased sensitivity to cAMP. Chemotaxis is mediated by membrane translocation of certain PH domain-containing proteins via specific binding to PtdIns(3,4,5)P3. InsP7 competes for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo. InsP7 depletion enhances PH domain membrane translocation and augments downstream chemotactic signaling activity.

Inhibition of mTOR by rapamycin has been shown to suppress seizures in TSC/PTEN genetic models. Rapamycin, when applied immediately before or after a neurological insult, also prevents the development of spontaneous recurrent seizures (epileptogenesis) in an acquired model. In the present study, we examined the mTOR pathway in rats that had already developed chronic spontaneous seizures in a pilocarpine model. We found that mTOR is aberrantly activated in brain tissues from rats with chronic seizures. Furthermore, inhibition of mTOR by rapamycin treatment significantly reduces seizure activity. Finally, mTOR inhibition also significantly suppresses mossy fiber sprouting. Our findings suggest the possibility for a much broader window for intervention for some acquired epilepsies by targeting the mTOR pathway.

The mechanisms underlying seizure-induced changes in gene expression are unclear. Using a chromatin immunoprecipitation assay, we found that acetylation of histone H4 in rat hippocampal CA3 neurons was reduced at the glutamate receptor 2 (GluR2; GRIA2) glutamate receptor promoter but increased at brain-derived neurotrophic factor promoter P2 as soon as 3 hr after induction of status epilepticus by pilocarpine. This result indicates that status epilepticus rapidly activates different signal pathways to modulate histone acetylation in a promoter-specific manner. H4 deacetylation preceded seizure-induced GluR2 mRNA downregulation. The histone deacetylase inhibitor trichostatin A prevented and quickly reversed deacetylation of GluR2-associated histones. Trichostatin A also blunted seizure-induced downregulation of GluR2 mRNA in CA3. Thus, rapid gene-specific changes in histone acetylation patterns may be a key early step in the pathological processes triggered by status epilepticus.

Materials and MethodsNeuronal Cell Cultures and Cytotoxicity. Primary cortical or hippocampal neuronal cultures were prepared as previously described (7). To induce excitotoxicity, the cells were prewashed with Tris-buffered control salt solution (CSS; 120 mM NaCl͞5.4 mM KCl͞1.8 mMCaCl 2 ͞25 mM Tris⅐HCl, pH 7.4͞15 mM glucose) and treated with CSS containing 300 M NMDA for 5 min. Toxicity was assayed 20-24 h after NMDA exposure by microscopic examination with computer-assisted cell counting. Total and dead cells were determined by nuclei staining with 100 ng͞ml 4Ј,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) (10 M), respectively. After a 10-min incubation, the cells were examined under a fluorescence microscope (Zeiss) with excitation at 360 nm. Cell death was determined as the ratio of dead to total cells and quantified by counting 1,000 cells. For staining of dead cells by terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL) assay, cells were fixed in 4% paraformaldehyde͞PBS and then stained by using a TUNEL Assay Kit (Molecular Probes) following protocols provided by the manufacturer. The cell death inhibitory effect of various agents was examined essentially as described (7,8).Western blotting was performed essentially as described (9).Focal Cerebral Ischemia Model. C57BL͞6 mice weighing 17-25 g were used for transient focal cerebral ischemia. After a midline neck incision, the left common carotid artery was isolated from the vagus nerve and ligated. The external carotid artery also was ligated, and the internal carotid artery was isolated carefully from the surrounding tissue. An 8-0 nylon filament (Ethicon, Somerville, NJ) was inserted into the common carotid artery through a small incision made in the proximity of the carotid bifurcation and advanced to the proximal part of the anterior cerebral artery to compromise the middle cerebral artery (MCA) flow. The filament was fixed in position by ligature. In sham-operated animals, the above procedures were performed except for the insertion of an intraluminal filament. For histological examinations, mice were perfused transcardially with heparinized PBS followed by 4% paraformaldehyde͞PBS for tissue fixation. Brains were removed and postfixed in 4% paraformaldehyde͞PBS at 4°C overnight. Coronal frozen sections (20 m) were prepared on a cryostat and stored at Ϫ80°C until use. The frozen sections were thawed, washed three times in PBS, permeabilized with 0.1% Triton X-100͞PBS at room temperature for 5 min, and then blocked in 5% skim milk͞3% BSA/PBS for 60 min. Subsequently, they were incubated with primary antibodies (1:200) at 4°C overnight and with secondary antibodies at room temperature for 2 h, and immunoreactivity was visualized by the avidin-biotin complex (ABC) method.Cell Lines and Cell Death Assays. HeLa cells, a human cervical carcinoma-derived cell line, were maintained in DMEM with 10% FBS, 2 mM L-glutamine, and 100 units of penicillin͞ streptomycin at 37°C with a 5% CO 2 atmosphere in a humidified incubator. P...

Prevailing views of neurotrophin action hold that the transcription factor CREB is constitutively bound to target genes with transcriptional activation occurring via CREB phosphorylation. However, we report that within several CRE-containing genes, CREB is not constitutively bound. Upon exposure of neurons to brain-derived neurotrophic factor (BDNF), CREB becomes rapidly bound to DNA coincident with phosphorylation at its transcriptional regulatory site, Ser133. This inducible CREB-DNA binding is independent of CREB Ser133 phosphorylation and is not affected by inhibition of the ERK or PI3K signaling pathways. Instead, BDNF regulates CREB binding by initiating a nitric oxide-dependent signaling pathway that leads to S-nitrosylation of nuclear proteins that associate with CREB target genes. Pharmacological manipulation of neurons in vitro and analysis of mice lacking neuronal nitric oxide synthase (nNOS) suggest that NO mediates BDNF and activity-dependent expression of CREB target genes. Thus, in conjunction with CREB phosphorylation, the NO pathway controls CREB-DNA binding and CRE-mediated gene expression.

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.