Eva Benito

CREB's control of intrinsic and synaptic plasticity: implications for CREB-dependent memory models

The activation of cAMP-response element binding protein (CREB)-dependent gene expression seems a crucial step in the molecular cascade that mediates the formation of long-lasting memories. This view is based both on correlative evidence and on functional assays that demonstrate, through loss- and gain-of-function experiments, the impact of CREB manipulation on memory performance. Mechanistically, CREBs role in memory is thought to be a consequence of its participation in long-term forms of synaptic plasticity. Recent studies demonstrate that CREB, in addition to synaptic plasticity, also modulates the intrinsic excitability of the neuron. This discovery reveals new intriguing connections between intrinsic and synaptic plasticity and is likely to have a significant impact on our understanding of the role of CREB in memory formation.

Genomic targets, and histone acetylation and gene expression profiling of neural HDAC inhibition.

Histone deacetylase inhibitors (HDACis) have been shown to potentiate hippocampal-dependent memory and synaptic plasticity and to ameliorate cognitive deficits and degeneration in animal models for different neuropsychiatric conditions. However, the impact of these drugs on hippocampal histone acetylation and gene expression profiles at the genomic level, and the molecular mechanisms that underlie their specificity and beneficial effects in neural tissue, remains obscure. Here, we mapped four relevant histone marks (H3K4me3, AcH3K9,14, AcH4K12 and pan-AcH2B) in hippocampal chromatin and investigated at the whole-genome level the impact of HDAC inhibition on acetylation profiles and basal and activity-driven gene expression. HDAC inhibition caused a dramatic histone hyperacetylation that was largely restricted to active loci pre-marked with H3K4me3 and AcH3K9,14. In addition, the comparison of Chromatin immunoprecipitation sequencing and gene expression profiles indicated that Trichostatin A-induced histone hyperacetylation, like histone hypoacetylation induced by histone acetyltransferase deficiency, had a modest impact on hippocampal gene expression and did not affect the transient transcriptional response to novelty exposure. However, HDAC inhibition caused the rapid induction of a homeostatic gene program related to chromatin deacetylation. These results illuminate both the relationship between hippocampal gene expression and histone acetylation and the mechanism of action of these important neuropsychiatric drugs.

cAMP Response Element-Binding Protein Is a Primary Hub of Activity-Driven Neuronal Gene Expression

Long-lasting forms of neuronal plasticity require de novo gene expression, but relatively little is known about the events that occur genome-wide in response to activity in a neuronal network. Here, we unveil the gene expression programs initiated in mouse hippocampal neurons in response to different stimuli and explore the contribution of four prominent plasticity-related transcription factors (CREB, SRF, EGR1, and FOS) to these programs. Our study provides a comprehensive view of the intricate genetic networks and interactions elicited by neuronal stimulation identifying hundreds of novel downstream targets, including novel stimulus-associated miRNAs and candidate genes that may be differentially regulated at the exon/promoter level. Our analyses indicate that these four transcription factors impinge on similar biological processes through primarily non-overlapping gene-expression programs. Meta-analysis of the datasets generated in our study and comparison with publicly available transcriptomics data revealed the individual and collective contribution of these transcription factors to different activity-driven genetic programs. In addition, both gain- and loss-of-function experiments support a pivotal role for CREB in membrane-to-nucleus signal transduction in neurons. Our data provide a novel resource for researchers wanting to explore the genetic pathways associated with activity-regulated neuronal functions.

Chronic enhancement of CREB activity in the hippocampus interferes with the retrieval of spatial information

The activation of cAMP-responsive element-binding protein (CREB)-dependent gene expression is thought to be critical for the formation of different types of long-term memory. To explore the consequences of chronic enhancement of CREB function on spatial memory in mammals, we examined spatial navigation in bitransgenic mice that express in a regulated and restricted manner a constitutively active form of CREB, VP16-CREB, in forebrain neurons. We found that chronic enhancement of CREB activity delayed the acquisition of an allocentric strategy to solve the hidden platform task. The ability to turn on and off transgene expression allowed us to dissect the role of CREB in dissociable memory processes. In mice in which transgene expression was turned on during memory acquisition, turning off the transgene re-established the access to the memory trace, whereas in mice in which transgene expression was turned off during acquisition, turning on the transgene impaired memory expression in a reversible manner, indicating that CREB enhancement specifically interfered with the retrieval of spatial information. The defects on spatial navigation in mice with chronic enhancement of CREB function were not corrected by conditions that increased further CREB-dependent activation of hippocampal memory systems, such as housing in an enriched environment. These results along with previous findings in CREB-deficient mutants indicate that the relationship of CREB-mediated plasticity to spatial memory is an inverted-U function, and that optimal learning in the water maze requires accurate regulation of this pathway.

The Neuronal Activity-Driven Transcriptome

Activity-driven transcription is a key event associated with long-lasting forms of neuronal plasticity. Despite the efforts to investigate the regulatory mechanisms that control this complex process and the important advances in the knowledge of the function of many activity-induced genes in neurons, as well as the specific contribution of activity-regulated transcription factors, our understanding of how activity-driven transcription operates at the systems biology level is still very limited. This review focuses on the research of neuronal activity-driven transcription from an “omics” perspective. We will discuss the different high-throughput approaches undertaken to characterize the gene programs downstream of specific activity-regulated transcription factors, including CREB, SRF, MeCP2, Fos, Npas4, and others, and the interplay between epigenetic and transcriptional mechanisms underlying neuronal plasticity changes. Although basic questions remain unanswered and important challenges still lie ahead, the refinement of genome-wide techniques for investigating the neuronal transcriptome and epigenome promises great advances.

Enhanced cAMP response element-binding protein activity increases neuronal excitability, hippocampal long-term potentiation, and classical eyeblink conditioning in alert behaving mice

The activity-regulated transcription factor cAMP response element-binding protein (CREB) is an essential component of the molecular switch that controls the conversion of short-term into long-term forms of plasticity, including those underlying long-term memory. Previous research in acute brain slices of transgenic animals expressing constitutively active CREB variants has revealed that enhancing CREB activity increases the intrinsic excitability of neurons and facilitates the late phase of long-term potentiation (LTP) in the Schaffer collateral pathway. Here, we report similar changes in plasticity at the Schaffer collateral pathway in alert behaving mice. Forebrain expression of a strong constitutively active CREB variant, VP16–CREB, enhanced in vivo LTP evoked in the Schaffer collateral pathway and caused significant changes in the input/output curve and paired-pulse facilitation in CA3–CA1 synapses, which could be explained by the increased excitability of hippocampal pyramidal neurons. In addition, classical eyeblink conditioning in transgenic mice and control littermates showed larger conditioned responses in mutant mice that were associated to a transient increase in the acquisition rate and in the concomitant learning-dependent change in synaptic strength. The sustained chronic activation of CREB activity, however, impaired the performance in this task. Our experiments demonstrate that the sustained enhancement of CREB function alters the physiology and plasticity of hippocampal circuits in behaving animals and that these changes have important consequences in associative learning.

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 CBPs 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.

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 axa0repressor 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.

Hunting for Synaptic Tagging and Capture in Memory Formation

Long-term potentiation (LTP) and some forms of long-term memory share a number of properties, such as associativity, durability, and protein synthesis dependence. These similarities suggest that LTP could be the cellular substrate for memory ([Bliss and Collingridge, 1993][1]). The recently reported

Fine-tuned SRF activity controls asymmetrical neuronal outgrowth: implications for cortical migration, neural tissue lamination and circuit assembly

The stimulus-regulated transcription factor Serum Response Factor (SRF) plays an important role in diverse neurodevelopmental processes related to structural plasticity and motile functions, although its precise mechanism of action has not yet been established. To further define the role of SRF in neural development and distinguish between cell-autonomous and non cell-autonomous effects, we bidirectionally manipulated SRF activity through gene transduction assays that allow the visualization of individual neurons and their comparison with neighboring control cells. In vitro assays showed that SRF promotes survival and filopodia formation and is required for normal asymmetric neurite outgrowth, indicating that its activation favors dendrite enlargement versus branching. In turn, in vivo experiments demonstrated that SRF-dependent regulation of neuronal morphology has important consequences in the developing cortex and retina, affecting neuronal migration, dendritic and axonal arborization and cell positioning in these laminated tissues. Overall, our results show that the controlled and timely activation of SRF is essential for the coordinated growth of neuronal processes, suggesting that this event regulates the switch between neuronal growth and branching during developmental processes.