Dnmt3a2: a hub for enhancing cognitive functions.

The mechanisms responsible for fear memory formation and extinction are far from being understood. Uncovering the molecules and mechanisms regulating these processes is vital for identifying molecular targets for the development of novel therapeutic strategies for anxiety and fear disorders. Cognitive abilities require the activation of gene expression necessary to the consolidation of lasting changes in neuronal function. In this study we established a key role for an epigenetic factor, the de novo DNA methyltransferase, Dnmt3a2, in memory formation and extinction. We found that Dnmt3a2 overexpression in the hippocampus of young adult mice induced memory enhancements in a variety of situations; it converted a weak learning experience into long-term memory, enhanced fear memory formation and facilitated fear memory extinction. Dnmt3a2 overexpression was also associated with the increased expression of plasticity-related genes. Furthermore, the knockdown of Dnmt3a2 expression impaired the animals' ability to extinguish memories, identifying Dnmt3a2 as a key player in extinction. Thus, Dnmt3a2 is at the core of memory processes and represents a novel target for cognition-enhancing therapies to ameliorate anxiety and fear disorders and boost memory consolidation.

Hypoxic/ischemic conditions induce expression of the putative pro-death gene Clca1 via activation of extrasynaptic N-methyl-D-aspartate receptors.

The stimulation of extrasynaptic N-methyl-D-aspartate (NMDA) receptors triggers cell death pathways and has been suggested to play a key role in cell degeneration and neuron loss associated with glutamate-induced excitotoxicity. In contrast, synaptic NMDA receptors promote neuronal survival. One mechanism through which extrasynaptic NMDA receptors damage neurons may involve Clca1, which encodes a putative calcium-activated chloride channel. Here we show that Clca1 expression is induced in cultured rat hippocampal neurons exposed to oxygen/glucose-free media; this induction is mediated by a signaling pathway activated by extrasynaptic NMDA receptors. Clca1 mRNA levels also increased in the gerbil hippocampus following a transient forebrain ischemia caused by bilateral carotid occlusion. Microelectrode array recordings revealed that oxygen-glucose deprivation enhances hippocampal network firing rates, which induces c-fos transcription through a signaling pathway that, in contrast to Clca1, is activated by synaptic but not extrasynaptic NMDA receptors. Thus, conditions of low oxygen/glucose lead to the activation of both extrasynaptic and synaptic NMDA receptors that regulate distinct target genes. Clca1 may be part of the genomic death program triggered by extrasynaptic NMDA receptors; it could be a marker for ischemic brain damage and a possible target for therapeutic interventions.

Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.

Here we report that synaptic and extrasynaptic NMDA (N-methyl-D-aspartate) receptors have opposite effects on CREB (cAMP response element binding protein) function, gene regulation and neuron survival. Calcium entry through synaptic NMDA receptors induced CREB activity and brain-derived neurotrophic factor (BDNF) gene expression as strongly as did stimulation of L-type calcium channels. In contrast, calcium entry through extrasynaptic NMDA receptors, triggered by bath glutamate exposure or hypoxic/ischemic conditions, activated a general and dominant CREB shut-off pathway that blocked induction of BDNF expression. Synaptic NMDA receptors have anti-apoptotic activity, whereas stimulation of extrasynaptic NMDA receptors caused loss of mitochondrial membrane potential (an early marker for glutamate-induced neuronal damage) and cell death. Specific blockade of extrasynaptic NMDA receptors may effectively prevent neuron loss following stroke and other neuropathological conditions associated with glutamate toxicity.

CREB/CBP and SRE-interacting transcriptional regulators are fast on-off switches: duration of calcium transients specifies the magnitude of transcriptional responses.

Transient increases in the intracellular calcium concentration, which are associated with electrical activation of neurones, control synapse-to-nucleus communication. Calcium signals differ in time and space but it is unclear exactly how this translates into stimulus-specific gene expression. Analysis of transcription induced by calcium transients with defined durations revealed that the evoked genomic responses, unlike those following neurotrophin exposure, are not all-or-none but graded events. The CRE-binding protein CREB, its coactivator CREB-binding protein (CBP), and SRE-interacting transcriptional regulators are fast on-off switches: their activities are induced by short-lasting calcium signals, remain active for the duration of the signal and are rapidly shut-off after calcium concentrations have returned to basal levels. CREB is switched on by a fast, nuclear calmodulin (CaM) kinase-dependent mechanism that mediates CREB phosphorylation on serine 133 within 30 s of calcium entry. The second calcium-activated route to CREB involves the MAP kinase/extracellular signal-regulated kinase (ERK1/2) cascade. This pathway can be triggered by brief, 30-60 s calcium transients. ERK1/2 activity peaks several minutes after calcium entry and can outlast the calcium transient. The shut-off of CREB and ERK1/2 involves rapid dephosphorylation of their activator sites. These properties of transcription factors and their regulating kinases and phosphatases provide a mechanism through which the duration of calcium signals specifies the magnitude of the transcriptional response. The decoding of temporal features of calcium transients is likely to contribute to impulse-specific gene expression.

The role of putative intragenic control elements in c-fos regulation by calcium and growth factor signalling pathways.

Sequences in the transcribed region of the c-fos gene have been suggested to control c-fos induction following exposure of cells to mitogens or stimuli that increase intracellular calcium concentrations. Using a mutational analysis we show that putative regulatory elements present in the first intron of the human c-fos gene and the fos-intragenic-regulatory-element (FIRE) are not required for c-fos regulation by growth factor and calcium signalling pathways in AtT20 and PC12 cells. Removal of the c-fos first intron and the FIRE did not increase the basal level of c-fos mRNA and only moderately reduced the magnitude of calcium-induced transcription mediated by either the entire c-fos promoter or the cAMP response element (CRE). Intragenic mutations did not affect serum response element (SRE)-dependent gene expression induced by calcium signals but caused a superinduction of c-fos expression in nerve growth factor-stimulated PC12 cells. These results indicate that c-fos promoter elements, rather than intragenic sequences, are the principal targets of transcription-regulating signalling pathways. This suggests that CRE- and SRE-bound activators of transcription initiation may also enhance, in a signal-dependent manner, c-fos transcript elongation beyond promoter-proximal pause sites.

Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity.

Information storage in the nervous system requires transcription triggered by synaptically evoked calcium signals. It has been suggested that translocation of calmodulin into the nucleus, initiated by submembranous calcium transients, relays synaptic signals to CREB. Here we show that in hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus. The nucleus is particularly suited to integrate neuronal firing patterns, and specifies the transcriptional outputs through a burst frequency-to-nuclear calcium amplitude conversion. Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors. Pharmacological or genetic modulation of nuclear calcium may directly affect transcription-dependent memory and cognitive functions.

Transcription-dependent neuronal plasticity: The nuclear calcium hypothesis.

In neurons, calcium ions control gene transcription induced by synaptic activity. The states and histories of neuronal activity are represented by a calcium code that comprises the site of calcium entry, and the amplitude, duration and spatial properties of signal-evoked calcium transients. The calcium code is used to transform specific firing patterns into qualitatively and quantitatively distinct transcriptional responses. The following hypothesis is proposed: electrical activity causes long-lasting, transcription-dependent changes in neuronal functions when synaptically evoked calcium transients associated with the stimulation propagate to the nucleus; gene transcription activated by dendritic calcium signals only is insufficient to consolidate functional alterations long-term. Similar to enduring increases in synaptic efficacy, nuclear calcium transients are induced by high-frequency firing patterns or by weak synaptic inputs coinciding with backpropagating dendritic action potentials. Nuclear calcium stimulates CREB-mediated transcription and, through inducing the activity of the transcriptional coactivator CREB-binding protein (CBP), may modulate the expression of numerous genes including neurotransmitter receptors and scaffolding proteins. Increases in the transcription rate of target genes are predicted to be transient and in many cases small, however, they collectively contribute to the maintenance of changes in synaptic efficacy. Nuclear calcium may be the common regulator of diverse transcription-dependent forms of neuronal plasticity.

Calcium-regulated protein kinase cascades and their transcription factor targets.

In the nervous system, calcium signals associated with electrical activation of neurons induce gene transcription that may be important for long-lasting adaptation. The type of transcriptional response is determined by the properties of the calcium signal that include subcellular localisation, amplitude, duration and the physical site of entry. Here we review calcium-regulated protein kinase cascades and discuss potential mechanisms through which they propagate calcium signals to and within the nucleus and control the activity of transcription factors and transcriptional co-activators.

Nuclear calcium-activated gene expression: possible roles in neuronal plasticity and epileptogenesis.

Nuclear calcium signals associated with electrical activation of neurons are critical regulators of gene expression and may cause changes in neuronal structure and function. Recent studies have identified a key component of the transcriptional machinery, the coactivator CREB binding protein (CBP), as a target for a nuclear calcium signalling pathway. Because the regulation of many genes involves transcription factors that function through their interaction with CBP, this mechanism, termed 'the coactivator control model', may modulate the expression of a large number of genes. During normal working of the brain, nuclear calcium increases may be transient and initiate transcriptional responses that are important for learning and memory. However, more intense or sustained stimulations of neurons (for example those used in the kindling model) may overactivate nuclear calcium-regulated processes. This may initiate inappropriate gene expression responses and could lead to the formation of epileptic neuronal circuits and disorders of neuronal excitability.

Calcium as a versatile second messenger in the control of gene expression.

The elevation of intracellular calcium is a major effector of stimulus-induced physiological change in a variety of cell types. Such change is invariably complex and frequently involves the activation of gene expression. Calcium signals are often able to activate different subsets of genes within the same cell, the basis for which has been unclear. Recent studies have revealed that a number of differing properties of the calcium signal are responsible for distinct cellular responses.

Control of recruitment and transcription-activating function of CBP determines gene regulation by NMDA receptors and L-type calcium channels.

Recruitment of the coactivator CBP by signal-regulated transcription factors and stimulation of CBP activity are key regulatory events in the induction of gene transcription following Ca2+ flux through ligand- and/or voltage-gated ion channels in hippocampal neurons. The mode of Ca2+ entry (L-type Ca2+ channels versus NMDA receptors) differentially controls the CBP recruitment step to CREB, providing a molecular basis for the observed Ca2+ channel type-dependent differences in gene expression. In contrast, activation of CBP is triggered irrespective of the route of Ca2+ entry, as is activation of c-Jun, that recruits CBP independently of phosphorylation at major regulatory c-Jun phosphorylation sites, serines 63 and 73. This control of CBP recruitment and activation is likely relevant to other CBP-interacting transcription factors and represents a general mechanism through which Ca2+ signals associated with electrical activity may regulate the expression of many genes.

c-Jun functions as a calcium-regulated transcriptional activator in the absence of JNK/SAPK1 activation.

Calcium is the principal second messenger in the control of gene expression by electrical activity in neurons. Recruitment of the coactivator CREB-binding protein, CBP, by the prototypical calcium-responsive transcription factor, CREB and stimulation of CBP activity by nuclear calcium signals is one mechanism through which calcium influx into excitable cells activates gene expression. Here we show that another CBP-interacting transcription factor, c-Jun, can mediate transcriptional activation upon activation of L-type voltage-gated calcium channels. Calcium-activated transcription mediated by c-Jun functions in the absence of stimulation of the c-Jun N-terminal protein kinase (JNK/SAPK1) signalling pathway and does not require c-Jun amino acid residues Ser63 and Ser73, the two major phosphorylation sites that regulate c-Jun activity in response to stress signals. Similar to CREB-mediated transcription, activation of c-Jun-mediated transcription by calcium signals requires calcium/ calmodulin-dependent protein kinases and is dependent on CBP function. These results identify c-Jun as a calcium-regulated transcriptional activator and suggest that control of coactivator function (i.e. recruitment of CBP and stimulation of CBP activity) is a general mechanism for gene regulation by calcium signals.

CBP: a signal-regulated transcriptional coactivator controlled by nuclear calcium and CaM kinase IV.

Recruitment of the coactivator, CREB binding protein (CBP), by signal-regulated transcription factors, such as CREB [adenosine 3', 5'-monophosphate (cAMP) response element binding protein], is critical for stimulation of gene expression. The mouse pituitary cell line AtT20 was used to show that the CBP recruitment step (CREB phosphorylation on serine-133) can be uncoupled from CREB/CBP-activated transcription. CBP was found to contain a signal-regulated transcriptional activation domain that is controlled by nuclear calcium and calcium/calmodulin-dependent (CaM) protein kinase IV and by cAMP. Cytoplasmic calcium signals that stimulate the Ras mitogen-activated protein kinase signaling cascade or expression of the activated form of Ras provided the CBP recruitment signal but did not increase CBP activity and failed to activate CREB- and CBP-mediated transcription. These results identify CBP as a signal-regulated transcriptional coactivator and define a regulatory role for nuclear calcium and cAMP in CBP-dependent gene expression.

Mechanisms controlling gene expression by nuclear calcium signals.

Nuclear calcium is an important regulator of gene expression following membrane depolarisation of electrically excitable cells. Here we describe nuclear calcium transients in hippocampal neurons following activation of calcium influx through L-type voltage-sensitive calcium channels and N-methyl-D-aspartate (NMDA) receptors, as well as following calcium release from intracellular caffeine-sensitive stores. Increases in nuclear calcium activate gene transcription by a mechanism that is distinct from gene regulation by cytoplasmic calcium signals and involves the cAMP response element (CRE) and the CRE binding protein, CREB. The nuclear calcium/calmodulin dependent (CaM) protein kinase IV, which is expressed in cultured hippocampal neurons and in the mouse pituitary cell line AtT20, may function as a mediator of nuclear calcium-induced transcription.

Nuclear calcium: a key regulator of gene expression.

Through the evolution of multicellular organisms, calcium has emerged as the preferred ion for intracellular signalling. It now occupies a pivotal role in many cell types and nowhere is it more important than in neurons, where it mediates both the relaying and long-term storage of information. The latter is a process that enables learning and memory to be formed and requires the activation of gene expression by calcium signals. Evidence from a number of diverse organisms shows that transcription mediated by the transcription factor CREB is critical for learning and memory. Here we review the features of CREB activation by calcium signals in mammalian cells. In contrast to other transcription factors, its regulation is dependent on an elevation of nuclear calcium concentration, potentially placing this spatially distinct pool of calcium as an important mediator of information storage.

Calcium controls gene expression via three distinct pathways that can function independently of the Ras/mitogen-activated protein kinases (ERKs) signaling cascade.

Calcium ions are the principal second messenger in the control of gene expression by electrical activation of neurons. However, the full complexity of calcium-signaling pathways leading to transcriptional activation and the cellular machinery involved are not known. Using the c-fos gene as a model system, we show here that the activity of its complex promoter is controlled by three independently operating signaling mechanisms and that their functional significance is cell type-dependent. The serum response element (SRE), which is composed of a ternary complex factor (TCF) and a serum response factor (SRF) binding site, integrates two calcium-signaling pathways. In PC12 cells, calcium-regulated transcription mediated by the SRE requires the TCF site and is not inhibited by expression of the dominant-negative Ras mutant, RasN17, nor by the MAP kinase kinase 1 inhibitor PD 98059. In contrast, TCF-dependent transcriptional regulation by nerve growth factor or epidermal growth factor is mediated by a Ras/MAP kinases (ERKs) pathway targeting the TCF Elk-1. In AtT20 cells and hippocampal neurons, calcium signals can stimulate transcription via a TCF-independent mechanism that requires the SRF binding site. The cyclic AMP response element (CRE), which cooperates with the TCF site in growth factor-regulated transcription, is a target of a third calcium-regulated pathway that is little affected by the expression of RasN17 or by PD 98059. Thus, calcium can stimulate gene expression via a TCF-, SRF-, and CRE-linked pathway that can operate independently of the Ras/MAP kinases (ERKs) signaling cascade in a cell type-dependent manner.

Gene regulation by nuclear and cytoplasmic calcium signals.

Calcium entry into neuronal cells through N-methyl-D-aspartate (NMDA)-type glutamate receptors or L-type voltage-gated calcium channels is a key event in the control of gene expression following electrical activation. Calcium acts both in the cytoplasm and the nucleus to activate signalling pathways that stimulate gene expression through different DNA regulatory elements. Differential control of transcription by spatially distinct calcium signals provides a mechanism by which a single second messenger can generate diverse transcriptional responses. This may allow for stimulation-specific modulation of gene expression critical for adaptive changes in the nervous system.

Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.

Calcium entry into neuronal cells through voltage or ligand-gated ion channels triggers neuronal activity-dependent gene expression critical for adaptive changes in the nervous system. Cytoplasmic calcium transients are often accompanied by an increase in the concentration of nuclear calcium, but the functional significance of such spatially distinct calcium signals is unknown. Here we show that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses. We used nuclear microinjection of a non-diffusible calcium chelator to block increases in nuclear, but not cytoplasmic, calcium concentrations following activation of L-type voltage-gated calcium channels. We showed that increases in nuclear calcium concentration control calcium-activated gene expression mediated by the cyclic-AMP-response element (CRE), and demonstrated that the CRE-binding protein CREB can function as a nuclear calcium-responsive transcription factor. A second signalling pathway, activating transcription through the serum-response element (SRE), is triggered by a rise in cytoplasmic calcium and does not require an increase in nuclear calcium.

N-methyl-D-aspartate receptors are critical for mediating the effects of glutamate on intracellular calcium concentration and immediate early gene expression in cultured hippocampal neurons.

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, calcium imaging, specific RNA analysis and immunocytochemistry were carried out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2+]i), and determination of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fosB, c-jun, junB, zif/268 (also termed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expression of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-methyl-D-aspartate subtype of glutamate receptor was necessary and sufficient for the transcriptional response. Non-N-methyl-D-aspartate receptors, while present in cultured hippocampal neurons, contributed relatively little to the regulation of transcription. Calcium imaging showed that glutamate-induced changes in [Ca2+]i were almost entirely mediated by N-methyl-D-aspartate receptors, rather than by L-type voltage-sensitive calcium channels. Previous studies have shown that stimulation with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can lead to an increase in [Ca2+]i and c-fos expression. Here we demonstrate that in our hippocampal culture system glutamate controls [Ca2+]i and induces immediate early gene transcription primarily by activating N-methyl-D-aspartate receptors.