Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

Glutamate mediates fast synaptic transmission at the majority of excitatory synapses throughout the central nervous system by interacting with different types of receptor channels. Cloning of glutamate receptors has provided evidence for the existence of several structurally related subunit families, each composed of several members. It has been proposed that KA1 and KA2 and GluR-5, GluR-6, and GluR-7 families represent subunit classes of high-affinity kainate receptors and that in vivo different kainate receptor subtypes might be constructed from these subunits in heteromeric assembly. However, despite some indications from autoradiographic studies and binding data in brain membranes, no functional pure kainate receptors have so far been detected in brain cells. We have found that early after culturing, a high percentage of rat hippocampal neurons express functional, kainate-selective glutamate receptors. These kainate receptors show pronounced desensitization with fast onset and very slow recovery and are also activated by quisqualate and domoate, but not by a-amino-3-hydroxy-5-methylisoxazole-4-propionate. Our results provide evidence for the existence of functional glutamate receptors of the kainate type in nerve cells, which are likely to be native homomeric GluR-6 receptors.

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, ␣CREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of ␣CREM is not required for the interaction, but phospho-␣CREM shows higher affinity for DREAM. The interaction with ␣CREM is independent of the Ca 2؉ -binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both ␣CREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca 2؉ .Transcriptional derepression is an important mechanism for the accurate control of gene expression. Transcriptional repressors can bind directly to DNA or act indirectly by interacting with other DNA-associated proteins (23,32). DREAM, a calcium-binding protein, represses basal expression of target genes through specific interaction with downstream regulatory elements (DREs) in the DNA (5, 6). Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca 2ϩ and protein kinase A (PKA) activation (5, 6). Other central players in the nuclear response to cyclic AMP (cAMP) and Ca 2ϩ are activator and repressor basic regionleucine zipper (LZ) transcription factors that bind to cAMPresponsive promoter elements (CREs) (10,15,25). They include proteins encoded by the CREB and CREM genes whose function is tightly regulated via phosphorylation by several kinases, including PKA and Ca 2ϩ -calmodulin-dependent kinases (8,12,13). As such, they represent the convergence point for various signaling cascades.The transcriptional repressor DREAM contains four EF hands, of which three (II, III, and IV) are responsible for the binding of calcium ions. In the absence of stimulated levels of nuclear calcium, DREAM binds with high affinity to the DRE sequence as a tetramer. Upon stimulation and increase in intracellular calcium, DREAM detaches from DNA without disruption of the tetrameric structure (6). The regulation by intracellular Ca 2ϩ of DREAM binding to DRE sites is a general mechanism that depends primarily on the EF-hand domains of DREAM. Mutation of two key amino acids within any of the functional EF hands results in mutated DREAM forms that stay bound to DNA also after calcium stimulation. Since DREAM binds to DRE sites as a tetramer, DREAM mutants insensitive to Ca 2ϩ behave as dominant negative mutants in a background of wild-type DREAM (unpublished observation). Similarly, PKA activation also results in loss of DREAM binding to the DRE and derepression of the target gene prodynorphin in human neuroblastoma cells (5). The molecular mechanism or the domains in DREAM that mediate this derepression by cAMP are unknown, and ...

There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls. Alzheimer disease (AD)3 is characterized by the loss of neurons and the presence of amyloid plaques and neurofibrillary tangles. The plaques are dense deposits of amyloid-␤ peptide and cellular material outside and around neurons, whereas the tangles are aggregates of the microtubule-associated protein tau, which has become hyperphosphorylated and accumulates inside the cells (1). In AD, tau pathology follows a reproducible pattern, in which hyperphosphorylated and aggregated tau first appears in the entorhinal cortex and hippocampus, and from there the disease spreads to the surrounding areas (2). During this process, neuronal loss occurs and tau protein may be found in the extracellular space in monomeric form or in aggregated form, assembled in extracellular ghost tangles. Indeed, an inverse correlation can be found between the number of extracellular tangles and the number of living neurons in the hippocampus (3-5). It has been also suggested that extracellular aggregated tau can promote the aggregation of intracellular tau (6). Moreover, it has been reported that extracellular monomeric tau is toxic for neurons, playing a role in the spreading of AD pathology (7-9). Monomeric tau-dependent toxicity occurs when extracellular tau binds and activates cell membrane receptors, identified as M1 and M3 muscarinic receptors (7).Sluggish disassembly of aggregated tau and slow degradation of its monomeric form in extracellular media provide this protein with a long stay outside the cell. In this location, hyperphosphorylated monomeric tau can be recognized as a substrate of several extracellular enzymes, some of which can remove the phosphates from the protein (10, 11). One of these enzymes is tissue-nonspecific alkaline phosphatas...

The calcium-binding protein DREAM binds speci®c-ally to DRE sites in the DNA and represses transcription of target genes. Derepression at DRE sites following PKA activation depends on a speci®c interaction between aCREM and DREAM. Two leucinecharged residue-rich domains (LCD) located in the kinase-inducible domain (KID) and in the leucine zipper of aCREM and two LCDs in DREAM participate in a two-site interaction that results in the loss of DREAM binding to DRE sites and derepression. Since the LCD motif located within the KID in CREM is also present in CREB, and maps in a region critical for the recruitment of CBP, we investigated whether DREAM may affect CRE-dependent transcription. Here we show that in the absence of Ca 2+ DREAM binds to the LCD in the KID of CREB. As a result, DREAM impairs recruitment of CBP by phospho-CREB and blocks CBP-mediated transactivation at CRE sites in a Ca 2+ -dependent manner. Thus, Ca 2+ -dependent interactions between DREAM and CREB represent a novel point of cross-talk between cAMP and Ca 2+ signalling pathways in the nucleus.

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Downstream Regulatory Element Antagonist Modulator (DREAM) is a Ca2 þ -dependent transcriptional repressor expressed in the brain, thyroid gland and thymus. Here, we analyzed the function of DREAM and the related protein KChIP-2 in the immune system using transgenic (tg) mice expressing a cross-dominant active mutant (EFmDREAM) for DREAM and KChIPs Ca 2 þ -dependent transcriptional derepression. EFmDREAM tg mice showed reduced T-cell proliferation. Tg T cells exhibited decreased interleukin (IL)-2, -4 and interferon (IFN)c production after polyclonal activation and following antigen-specific response. Chromatin immunoprecipitation and transfection assays showed that DREAM binds to and represses transcription from these cytokine promoters. Importantly, specific transient knockdown of DREAM or KChIP-2 induced basal expression of IL-2 and IFNc in wild-type splenocytes. These data propose DREAM and KChIP-2 as Ca 2 þ -dependent repressors of the immune response.

The nucleolus is implicated in sensing and responding to cellular stress by stabilizing p53. The pro-apoptotic effect of p53 is associated with several neurodegenerative disorders, including Huntington's disease (HD), which is characterized by the progressive loss of medium spiny neurons (MSNs) in the striatum. Here we show that disruption of nucleolar integrity and function causes nucleolar stress and is an early event in MSNs of R6/2 mice, a transgenic model of HD. Targeted perturbation of nucleolar function in MSNs by conditional knockout of the RNA polymerase I-specific transcription initiation factor IA (TIF-IA) leads to late progressive striatal degeneration, HD-like motor abnormalities and molecular signatures. Significantly, p53 prolongs neuronal survival in TIF-IA-deficient MSNs by transient upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor that inhibits mammalian target of rapamycin signaling and induces autophagy. The results emphasize the initial role of nucleolar stress in neurodegeneration and uncover a p53/PTEN-dependent neuroprotective response.