KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain.

The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions.

CBP and SRF co-regulate dendritic growth and synaptic maturation.

The CREB-binding protein (CBP) exerts tight control of developmental processes. Here, we investigated the consequences of its selective ablation in newborn neurons. Mice in which CBP was eliminated during neuronal differentiation showed perinatal death and defective diaphragm innervation. Adult-born neurons also showed impaired growth and maturation after inducible and restricted CBP loss in dentate gyrus neuroprogenitors. Consistent with these in vivo findings, cultured neurons displayed impaired outgrowth, immature spines, and deficient activity-dependent synaptic remodeling after CBP ablation. These deficits coincided with broad transcriptional changes affecting genes involved in neuronal growth and plasticity. The affected gene set included many predicted targets of both CBP and the serum response factor (SRF), an activity-regulated transcription factor involved in structural plasticity. Notably, increasing SRF activity in a CBP-independent manner ameliorated the transcriptional, synaptic, and growth defects. These results underscore the relevance of CBP-SRF interactions during neuronal outgrowth and synaptic maturation, and demonstrate that CBP plays an essential role in supporting the gene program underlying the last steps of neuronal differentiation, both during development and in the adult brain.

The Epigenetic Factor CBP Is Required for the Differentiation and Function of Medial Ganglionic Eminence-Derived Interneurons.

The development of inhibitory circuits depends on the action of a network of transcription factors and epigenetic regulators that are critical for interneuron specification and differentiation. Although the identity of many of these transcription factors is well established, much less is known about the specific contribution of the chromatin-modifying enzymes that sculpt the interneuron epigenome. Here, we generated a mouse model in which the lysine acetyltransferase CBP is specifically removed from neural progenitors at the median ganglionic eminence (MGE), the structure where the most abundant types of cortical interneurons are born. Ablation of CBP interfered with the development of MGE-derived interneurons in both sexes, causing a reduction in the number of functionally mature interneurons in the adult forebrain. Genetic fate mapping experiments not only demonstrated that CBP ablation impacts on different interneuron classes, but also unveiled a compensatory increment of interneurons that escaped recombination and cushion the excitatory-inhibitory imbalance. Consistent with having a reduced number of interneurons, CBP-deficient mice exhibited a high incidence of spontaneous epileptic seizures, and alterations in brain rhythms and enhanced low gamma activity during status epilepticus. These perturbations led to abnormal behavior including hyperlocomotion, increased anxiety and cognitive impairments. Overall, our study demonstrates that CBP is essential for interneuron development and the proper functioning of inhibitory circuitry in vivo.

Early alteration of epigenetic-related transcription in Huntington's disease mouse models.

Transcriptional dysregulation in Huntington's disease (HD) affects the expression of genes involved in survival and neuronal functions throughout the progression of the pathology. In recent years, extensive research has focused on epigenetic and chromatin-modifying factors as a causative explanation for such dysregulation, offering attractive targets for pharmacological therapies. In this work, we extensively examined the gene expression profiles in the cortex, striatum, hippocampus and cerebellum of juvenile R6/1 and N171-82Q mice, models of rapidly progressive HD, to retrieve the early transcriptional signatures associated with this pathology. These profiles were largely consistent across HD datasets, contained tissular and neuronal-specific genes and showed significant correspondence with the transcriptional changes in mouse strains deficient for epigenetic regulatory genes. The most prominent cases were the conditional knockout of the lysine acetyltransferase CBP in post-mitotic forebrain neurons, the double knockout of the histone methyltransferases Ezh1 and Ezh2, components of the polycomb repressor complex 2 (PRC2), and the conditional mutants of the histone methyltransferases G9a (Ehmt2) and GLP (Ehmt1). Based on these observations, we propose that the neuronal epigenetic status is compromised in the prodromal stages of HD, leading to an altered transcriptional programme that is prominently involved in neuronal identity.

Loss of Kdm5c Causes Spurious Transcription and Prevents the Fine-Tuning of Activity-Regulated Enhancers in Neurons.

During development, chromatin-modifying enzymes regulate both the timely establishment of cell-type-specific gene programs and the coordinated repression of alternative cell fates. To dissect the role of one such enzyme, the intellectual-disability-linked lysine demethylase 5C (Kdm5c), in the developing and adult brain, we conducted parallel behavioral, transcriptomic, and epigenomic studies in Kdm5c-null and forebrain-restricted inducible knockout mice. Together, genomic analyses and functional assays demonstrate that Kdm5c plays a critical role as a repressor responsible for the developmental silencing of germline genes during cellular differentiation and in fine-tuning activity-regulated enhancers during neuronal maturation. Although the importance of these functions declines after birth, Kdm5c retains an important genome surveillance role preventing the incorrect activation of non-neuronal and cryptic promoters in adult neurons.

Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction.

The interior of the neuronal cell nucleus is a highly organized three-dimensional (3D) structure where regions of the genome that are linearly millions of bases apart establish sub-structures with specialized functions. To investigate neuronal chromatin organization and dynamics in vivo, we generated bitransgenic mice expressing GFP-tagged histone H2B in principal neurons of the forebrain. Surprisingly, the expression of this chimeric histone in mature neurons caused chromocenter declustering and disrupted the association of heterochromatin with the nuclear lamina. The loss of these structures did not affect neuronal viability but was associated with specific transcriptional and behavioural deficits related to serotonergic dysfunction. Overall, our results demonstrate that the 3D organization of chromatin within neuronal cells provides an additional level of epigenetic regulation of gene expression that critically impacts neuronal function. This in turn suggests that some loci associated with neuropsychiatric disorders may be particularly sensitive to changes in chromatin architecture.

Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)-binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

Plasmalemmal sodium-calcium exchanger shapes the calcium and exocytotic signals of chromaffin cells at physiological temperature.

The activity of the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) is highly sensitive to temperature. We took advantage of this fact to explore here the effects of the NCX blocker KB-R7943 (KBR) at 22 and 37°C on the kinetics of Ca(2+) currents (ICa), cytosolic Ca(2+) ([Ca(2+)]c) transients, and catecholamine release from bovine chromaffin cells (BCCs) stimulated with high K(+), caffeine, or histamine. At 22°C, the effects of KBR on those parameters were meager or nil. However, at 37°C whereby the NCX is moving Ca(2+) at a rate fivefold higher than at 22°C, various of the effects of KBR were pronounced, namely: 1) no effects on ICa; 2) reduction of the [Ca(2+)]c transient amplitude and slowing down of its rate of clearance; 3) blockade of the K(+)-elicited quantal release of catecholamine; 4) blockade of burst catecholamine release elicited by K(+); 5) no effect on catecholamine release elicited by short K(+) pulses (1-2 s) and blockade of the responses produced by longer K(+) pulses (3-5 s); and 6) potentiation of secretion elicited by histamine or caffeine. Furthermore, the more selective NCX blocker SEA0400 also potentiated the secretory responses to caffeine. The results suggest that at physiological temperature the NCX substantially contributes to shaping the kinetics of [Ca(2+)]c transients and the exocytotic responses elicited by Ca(2+) entry through Ca(2+) channels as well as by Ca(2+) release from the endoplasmic reticulum.

Ablation of CBP in forebrain principal neurons causes modest memory and transcriptional defects and a dramatic reduction of histone acetylation but does not affect cell viability.

Rubinstein-Taybi syndrome (RSTS) is an inheritable disease associated with mutations in the gene encoding the CREB (cAMP response element-binding protein)-binding protein (CBP) and characterized by growth impairment, learning disabilities, and distinctive facial and skeletal features. Studies in mouse models for RSTS first suggested a direct role for CBP and histone acetylation in cognition and memory. Here, we took advantage of the genetic tools for generating mice in which the CBP gene is specifically deleted in postmitotic principal neurons of the forebrain to investigate the consequences of the loss of CBP in the adult brain. In contrast to the conventional CBP knock-out mice, which exhibit very early embryonic lethality, postnatal forebrain-restricted CBP mutants were viable and displayed no overt abnormalities. We identified the dimer of histones H2A and H2B as the preferred substrate of the histone acetyltransferase domain of CBP. Surprisingly, the loss of CBP and subsequent histone hypoacetylation had a very modest impact in the expression of a number of immediate early genes and did not affect neuronal viability. In addition, the behavioral characterization of these mice dissociated embryonic and postnatal deficits caused by impaired CBP function, narrowed down the anatomical substrate of specific behavioral defects, and confirmed the special sensitivity of object recognition memory to CBP deficiency. Overall, our study provides novel insights into RSTS etiology and clarifies some of the standing questions concerning the role of CBP and histone acetylation in activity-driven gene expression, memory formation, and neurodegeneration.

Syndromic features and mild cognitive impairment in mice with genetic reduction on p300 activity: Differential contribution of p300 and CBP to Rubinstein-Taybi syndrome etiology.

Rubinstein-Taybi syndrome (RSTS) is a complex autosomal-dominant disease characterized by mental and growth retardation and skeletal abnormalities. A majority of the individuals diagnosed with RSTS carry heterozygous mutation in the gene CREBBP, but a small percentage of cases are caused by mutations in EP300. To investigate the contribution of p300 to RSTS pathoetiology, we carried out a comprehensive and multidisciplinary characterization of p300(+/-) mice. These mice exhibited facial abnormalities and impaired growth, two traits associated to RSTS in humans. We also observed abnormal gait, reduced swimming speed, enhanced anxiety in the elevated plus maze, and mild cognitive impairment during the transfer task in the water maze. These analyses demonstrate that p300(+/-) mice exhibit phenotypes that are reminiscent of neurological traits observed in RSTS patients, but their comparison with previous studies on CBP deficient strains also indicates that, in agreement with the most recent findings in human patients, the activity of p300 in cognition is likely less relevant or more susceptible to compensation than the activity of CBP.

Inhibition of cAMP response element-binding protein reduces neuronal excitability and plasticity, and triggers neurodegeneration.

The cAMP-responsive element-binding protein (CREB) pathway has been involved in 2 major cascades of gene expression regulating neuronal function. The first one presents CREB as a critical component of the molecular switch that controls long-lasting forms of neuronal plasticity and learning. The second one relates CREB to neuronal survival and protection. To investigate the role of CREB-dependent gene expression in neuronal plasticity and survival in vivo, we generated bitransgenic mice expressing A-CREB, an artificial peptide with strong and broad inhibitory effect on the CREB family, in forebrain neurons in a regulatable manner. The expression of A-CREB in hippocampal neurons impaired L-LTP, reduced intrinsic excitability and the susceptibility to induced seizures, and altered both basal and activity-driven gene expression. In the long-term, the chronic inhibition of CREB function caused severe loss of neurons in the CA1 subfield as well as in other brain regions. Our experiments confirmed previous findings in CREB-deficient mutants and revealed new aspects of CREB-dependent gene expression in the hippocampus supporting a dual role for CREB-dependent gene expression regulating intrinsic and synaptic plasticity and promoting neuronal survival.

cAMP response element-binding protein-mediated gene expression increases the intrinsic excitability of CA1 pyramidal neurons.

To investigate the role of CREB-mediated gene expression on the excitability of CA1 pyramidal neurons, we obtained intracellular recordings from pyramidal neurons of transgenic mice expressing a constitutively active form of CREB, VP16-CREB, in a regulated and restricted manner. We found that transgene expression increased the neuronal excitability and inhibited the slow and medium afterhyperpolarization currents. These changes may contribute to the reduced threshold for LTP observed in these mice. When strong transgene expression was turned on for prolonged period of time, these mice also showed a significant loss of hippocampal neurons and sporadic epileptic seizures. These deleterious effects were dose dependent and could be halted, but not reversed by turning off transgene expression. Our experiments reveal a new role for hippocampal CREB-mediated gene expression, identify the slow afterhyperpolarization as a primary target of CREB action, provide a new mouse model to investigate temporal lobe epilepsy and associated neurodegeneration, and illustrate the risks of cell death associated to a sustained manipulation of this pathway. As a result, our study has important implications for both the understanding of the cellular bases of learning and memory and the consideration of therapies targeted to the CREB pathway.

Desensitized nicotinic receptors that, however, afford cytoprotection in bovine chromaffin cells.

Neuronal nicotinic receptors for acetylcholine (nAChRs) are among the ionotropic receptors that suffer the most desensitization upon prolonged exposure to their agonists. This is particularly true for the alpha7 subtype of nAChRs, although alpha3beta4 receptors also suffer quick desensitization. This study was planned to test the hypothesis that even after suffering desensitization, a given nAChR might still afford cell protection against a noxious stimulus. Of the many agonists developed for nAChRs, we selected the poorly desensitizing ligand dimethylphenylpiperazinium (DMPP) (Britt and Brenner, 1997) and the highly desensitizing agent epibatidine (EPB) (Marks et al., 1996). We have measured nAChR currents, catecholamine secretory responses, and changes of [Ca2+]c elicited by stimulation of nAChRs with DMPP or EPB. We have also investigated cytoprotection elicited by DMPP and EPB against the cytotoxic effects of veratridine in bovine chromaffin cells.

Blockade of Ca2+ -activated K+ channels by galantamine can also contribute to the potentiation of catecholamine secretion from chromaffin cells.

Galantamine is a drug in clinical use for the treatment of Alzheimer's disease, but its mechanism(s) of action remains controversial. Here we addressed the question whether galantamine could potentiate neurotransmitter release by inhibiting small conductance Ca2+ -activated K+ channels (KCa2). Galantamine potentiated catecholamine secretory responses induced by 10 s pulses of acetylcholine and high [K+]o applied to fast-superfused bovine adrenal chromaffin cell populations. Catecholamine release was significantly enhanced by galantamine although we did not find concentration dependence in the range 0.1-1 microM. The KCa2 channel blocker apamin (0.3 microM) occluded the potentiating effects of galantamine on acetylcholine-evoked secretion. Like apamin, galantamine also modified the firing of action potentials, but to a lesser extent. In addition, 1 microM galantamine reduced by 41% the KCa2 current without modifying the voltage-dependent Ca2+ currents. These results constitute the first direct evidence that galantamine can potentiate neurotransmitter release by blocking KCa2 channels, in addition to its already demonstrated capacity to mildly block acetylcholinesterase or potentiate allosterically nicotinic receptors.

Blockade of nicotinic receptors of bovine adrenal chromaffin cells by nanomolar concentrations of atropine.

Nanomolar concentrations of atropine have been considered up to now to be selective for blockade of muscarinic receptors for acetylcholine. A collateral finding indicated to us that these low concentrations of atropine could also target the neuronal nicotinic receptors. We report here a detailed study on this novel property of atropine. Catecholamine release, measured on-line with amperometry in chromaffin cells stimulated with acetylcholine pulses was blocked by atropine in a competitive manner. To corroborate a direct action of atropine on nicotinic receptors, we have employed N,N-dimethyl-N'-phenyl-piperazinium (DMPP), a pure nicotinic receptor agonist; atropine blocked its secretory responses with an IC50 of 2.04 nM. Nicotinic currents, recorded with the whole cell configuration of the patch-clamp technique were blocked by atropine in a concentration-dependent manner (IC50 of 11 nM), also showing a competitive nature. Nicotinic receptor currents in oocytes expressing bovine alpha7 and alpha3beta4 nicotinic receptors were blocked by atropine with an IC50 of 11.2 and 46.8 nM, respectively. Atropine (30 nM) also decreased the increment of the cytosolic calcium concentrations after stimulation with 30 microM DMPP in bovine chromaffin cells. However, action potentials evoked by DMPP were not modified by atropine. Our results demonstrate that nicotinic currents and their downstream consequences (i.e. cytosolic calcium elevations and catecholamine release) were blocked by nanomolar concentrations of atropine; although the blockade was partial, it must be considered when using atropine to study cholinergic neurotransmission, particularly at synapses where both nicotinic and muscarinic receptors are present i.e., the adrenal medulla and autonomic ganglia.

A choline-evoked [Ca2+]c signal causes catecholamine release and hyperpolarization of chromaffin cells.

In bovine chromaffin cells fast-superfused with Krebs-HEPES solution containing 1-2 mM Ca2+, 5 s pulses of choline (1-10 mM), elicited catecholamine secretory responses that were only approximately 10% of those evoked by ACh (0.01-0.1 mM). However, in high-Ca2+ solutions (10-20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine alpha7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing alpha3beta4, however, the inward current elicited by choline amounted to only 4% of the size of alpha7 current. Our results suggest that choline activates the entry of Ca2+ through alpha7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+-dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high-Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.

Antimigraine dotarizine blocks P/Q Ca2+ channels and exocytosis in a voltage-dependent manner in chromaffin cells.

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.

Effect of inositol 1,4,5-trisphosphate receptor stimulation on mitochondrial [Ca2+] and secretion in chromaffin cells.

Ca(2+) uptake by mitochondria is a potentially important buffering system able to control cytosolic [Ca(2+)]. In chromaffin cells, we have shown previously that stimulation of either Ca(2+) entry or Ca(2+) release via ryanodine receptors triggers large increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)) approaching the millimolar range, whose blockade dramatically enhances catecholamine secretion [Montero, Alonso, Carnicero, Cuchillo-Ibañez, Albillos, Garcia, Carcia-Sancho and Alvarez (2000) Nat. Cell Biol. 2, 57-61]. In the present study, we have studied the effect of stimulation of inositol 1,4,5-trisphosphate (InsP(3)) receptors using histamine. We find that histamine produces a heterogeneous increase in [Ca(2+)](M), reaching peak levels at approx. 1 microM in 70% of the mitochondrial space to several hundred micromolar in 2-3% of mitochondria. Intermediate levels were found in the rest of the mitochondrial space. Single-cell imaging experiments with aequorin showed that the heterogeneity had both an intercellular and a subcellular origin. Those mitochondria responding to histamine with increases in [Ca(2+)](M) much greater than 1 microM (30%) were the same as those that also responded with large increases in [Ca(2+)](M) following stimulation with either high-K(+) medium or caffeine. Blocking mitochondrial Ca(2+) uptake with protonophores or mitochondrial inhibitors also enhanced catecholamine secretion induced by histamine. These results suggest that some InsP(3) receptors tightly co-localize with ryanodine receptors and voltage-dependent Ca(2+) channels in defined subplasmalemmal functional units designed to control secretion induced by different stimuli.

Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed.

During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM ryanodine more than halved the responses to ACh but did not affect the responses to K+ responses. In these ER Ca2+-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 microM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1-5 s) whereas responses to K+ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca2+] ([Ca2+](c)) that were substantially smaller than those elicited by K+ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca2+](c) elevations, whereas K+ pulses produced higher [Ca2+](c) transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca2+](c) elicited by ACh (that evokes firing of action potentials) much better than that induced by K+ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca2+](c) changes resulting from the physiological triggering of action potentials by ACh.