The long-term stabilization of memory traces or engram involves the rapid formation of cortical engrams during encoding that mature functionally over time guided by the activity of the hippocampus. The molecular mechanisms that regulate this process remain largely unknown. Here, we found that hippocampal DNA methylation converts short-lasting into long-lasting memories by promoting systems consolidation and the stabilization of cortical engrams.### Competing Interest StatementThe authors have declared no competing interest.

Epigenetic factors are well-established players in memory formation. Specifically, DNA methylation is necessary for the formation of long-term memory in multiple brain regions including the hippocampus. Despite the demonstrated role of DNA methyltransferases (Dnmts) in memory formation, it is unclear whether individual Dnmts have unique or redundant functions in long-term memory formation. Furthermore, the downstream processes controlled by Dnmts during memory consolidation have not been investigated. In this study, we demonstrated that Dnmt3a1, the predominant Dnmt in the adult brain, is required for long-term spatial object recognition and contextual fear memory. Using RNA sequencing, we identified an activity-regulated Dnmt3a1-dependent genomic program in which several genes were associated with functional and structural plasticity. Furthermore, we found that some of the identified genes are selectively dependent on Dnmt3a1, but not its isoform Dnmt3a2. Specifically, we identified Neuropilin 1 (Nrp1) as a downstream target of Dnmt3a1 and further demonstrated the involvement of Nrp1 in hippocampus-dependent memory formation. Importantly, we found that Dnmt3a1 regulates hippocampus-dependent memory via Nrp1. In contrast, Nrp1 overexpression did not rescue memory impairments triggered by reduced Dnmt3a2 levels. Taken together, our study uncovered a Dnmt3a-isoform-specific mechanism in memory formation, identified a novel regulator of memory, and further highlighted the complex and highly regulated functions of distinct epigenetic regulators in brain function.

Neurons respond rapidly to extracellular stimuli by activating signaling pathways that modulate the function of already synthetized proteins. Alternatively, signal transduction to the cell nucleus induces de novo synthesis of proteins required for long-lasting adaptations. These complementary strategies are necessary for neuronal plasticity processes that underlie, among other functions, the formation of memories. Nonetheless, it is still not fully understood how the coupling between different stimuli and the activity of constitutively and/or de novo expressed proteins gate neuronal plasticity. Here, we discuss the molecular functions of the Growth Arrest and DNA Damage 45 (Gadd45) family of proteins in neuronal adaptation. We highlight recent findings that indicate that Gadd45 family members regulate this function through multiple cellular processes (e.g., DNA demethylation, gene expression, RNA stability, MAPK signaling). We then summarize the regulation of Gadd45 expression in neurons and put forward the hypothesis that the constitutive and neuronal activity-induced pools of Gadd45 proteins have distinct and complementary roles in modulating neuronal plasticity. Therefore, we propose that Gadd45 proteins are essential for brain function and their dysfunction might underlie pathophysiological conditions such as neuropsychiatric disorders.

Understanding how we learn and remember has been a long-standing question in neuroscience. Technological developments of the past 15 years have allowed for dramatically increased access to the neurons that hold the physical representation of memory, also known as a memory trace or engram. Such developments have tremendously facilitated advancement of the memory field, since they made possible interrogation of the cellular and molecular mechanisms underlying memory formation with unprecedented cellular specificity. Here, we discuss the studies that have investigated rules governing neuronal recruitment to a particular memory engram. Furthermore, we provide an overview of the evidence that functional and structural changes associated with memory consolidation occur in engram neurons. Moreover, we summarize the expanding literature showing that transcriptional regulatory factors such as transcription factors and epigenetic mechanisms play an important role in the maintained allocation of behaviorally-selected neurons to an engram. Together, these studies have begun elucidating how neuronal networks are selected and modified in order to support memory formation and storage.

Long-term memory formation is supported by functional and structural changes of neuronal networks, which rely on de novo gene transcription and protein synthesis. The modulation of the neuronal transcriptome in response to learning depends on transcriptional and post-transcriptional mechanisms. DNA methylation writers and readers regulate the activity-dependent genomic program required for memory consolidation. The most abundant DNA methylation reader, the Methyl CpG binding domain protein 2 (MeCP2), has been shown to regulate alternative splicing, but whether it establishes splicing events important for memory consolidation has not been investigated. In this study, we identified the alternative splicing profile of the mouse hippocampus in basal conditions and after a spatial learning experience, and investigated the requirement of MeCP2 for these processes. We observed that spatial learning triggers a wide-range of alternative splicing events in transcripts associated with structural and functional remodeling and that virus-mediated knockdown of MeCP2 impairs learning-dependent post-transcriptional responses of mature hippocampal neurons. Furthermore, we found that MeCP2 preferentially affected the splicing modalities intron retention and exon skipping and guided the alternative splicing of distinct set of genes in baseline conditions and after learning. Lastly, comparative analysis of the MeCP2-regulated transcriptome with the alternatively spliced mRNA pool, revealed that MeCP2 disruption alters the relative abundance of alternatively spliced isoforms without affecting the overall mRNA levels. Taken together, our findings reveal that adult hippocampal MeCP2 is required to finetune alternative splicing events in basal conditions, as well as in response to spatial learning. This study provides new insight into how MeCP2 regulates brain function, particularly cognitive abilities, and sheds light onto the pathophysiological mechanisms of Rett syndrome, that is characterized by intellectual disability and caused by mutations in the Mecp2 gene.

Additional file 10: Table S7. GO analysis and DEG/DAS overlap for baseline and learning state conditions. This file contains gene ontology (GO) analysis and overlap for DEGs and DAS for baseline and learning conditions.

Additional file 4: Table S3. ΔPSI for Learning-induced DAS. This file contains ΔPSI values for the differential alternative splicing (DAS) events per subtype for learning-induced conditions.

Memories are encoded by memory traces or engrams, represented within subsets of neurons that are synchronously activated during learning. However, the molecular mechanisms that drive engram stabilization during consolidation and consequently ensure its reactivation by memory recall are not fully understood. In this study we manipulate, during memory consolidation, the levels of the de novo DNA methyltransferase 3a2 (Dnmt3a2) selectively within dentate gyrus neurons activated by fear conditioning. We found that Dnmt3a2 upregulation enhances memory performance in mice and improves the fidelity of reconstitution of the original neuronal ensemble upon memory retrieval. Moreover, similar manipulation in a sparse, non-engram subset of neurons does not bias engram allocation or modulate memory strength. We further show that neuronal Dnmt3a2 overexpression changes the DNA methylation profile of synaptic plasticity-related genes. Our data implicates DNA methylation selectively within neuronal ensembles as a mechanism of stabilizing engrams during consolidation that supports successful memory retrieval.

5 1 Department of Neurobiology, Interdisciplinary Centre for Neurosciences (IZN), Heidelberg 6 University, INF 366, 69120 Heidelberg, Germany 7 2 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina de Lisboa, 8 Universidade de Lisboa, 1649-028 Lisbon, Portugal 9 3 Present address: Department of Cognitive Neuroscience, Donders Institute for Brain, 10 Cognition and Behaviour, Radboud University Medical Center Kapittelweg 29, 6525 EN 11 Nijmegen, The Netherlands 12 4 Present address: Directors' Research, European Molecular Biology Laboratory (EMBL), 13 Meyerhofstrasse 1, 69117 Heidelberg, Germany 14

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

Age-related memory loss is observed across multiple mammalian species and preferentially affects hippocampus-dependent memory. Memory impairments are characterized by accelerated decay of spatial memories. Nevertheless, the molecular mechanisms underlying these deficits are still largely unknown. Here, we investigated the expression and function of the growth arrest DNA damage (Gadd45) family during aging and cognition, respectively. We report that aging impairs the expression of Gadd45γ in the hippocampus of cognitively impaired male mice. Mimicking this decrease in young adult male mice led to age-like memory deficits in hippocampus-dependent memory tasks. Gadd45γ reduction impaired the activity of key components of the mitogen-activated protein kinase (MAPK) pathway (p38 and JNK) in mouse hippocampal cultures. Furthermore, we found that activation of downstream targets, such as ATF-2, c-Jun, and CREB (cAMP response element-binding protein), was disrupted. Finally, we showed that Gadd45γ is required for induction of key early- and late-response genes that have been associated with aging. Together, these findings indicate that Gadd45γ expression regulates cognitive abilities and synapse-to-nucleus communication and suggest Gadd45γ dysfunction as a potential mechanism contributing to age-related cognitive impairments.SIGNIFICANCE STATEMENT A high percentage of subjects experience age-related memory loss that burdens daily performance. Although many advances have been made, the precise changes in the brain governing these deficits are unclear. Identifying molecular processes that are required for cognition and are altered during old age is crucial to develop preventive or therapeutic strategies. Here, we show that baseline and learning-induced expression of the growth arrest DNA damage (Gadd45) γ is selectively impaired in the hippocampus of aged mice with cognitive deficits. Next, we show that modeling this impairment in young adult mice with normal cognitive performance disrupts long- and short-term memories in an age-like manner. Finally, we demonstrate that Gadd45γ regulates synapse-to-nucleus communication processes that are needed for plasticity-associated gene expression.

Methyl CpG binding protein 2 (MeCP2) was first identified as a nuclear protein with a transcriptional repressor role that recognizes DNA methylation marks. MeCP2 has a well-established function in neurodevelopment, as evidenced by the severe neurological impairments characteristic of the Rett syndrome (RTT) pathology and the MeCP2 duplication syndrome (MDS), caused by loss or gain of MeCP2 function, respectively. Research aimed at the underlying pathophysiological mechanisms of RTT and MDS has significantly advanced our understanding of MeCP2 functions in the nervous system. It has revealed, however, that MeCP2 has more varied and complex roles than previously thought. Here we review recent insights into the functions of MeCP2 in neurodevelopment and the less explored requirement for MeCP2 in adult brain function. We focus on the emerging view that MeCP2 is a global chromatin organizer. Finally, we discuss how the individual functions of MeCP2 in neurodevelopment and adulthood are linked to its role as a chromatin regulator.

Epigenetic mechanisms have gained increasing attention as regulators of synaptic plasticity and responsiveness to drugs of abuse. In particular, it has been shown that the activity of the DNA methyltransferase 3a (Dnmt3a) mediates certain long-lasting effects of cocaine. Here we examined the role of the Dnmt isoforms, Dnmt3a1 and Dnmt3a2, within the nucleus accumbens (NAc) on transcriptional activity of immediate early genes (IEGs) and acute and long-lasting responsiveness to cocaine and cocaine conditioned cues. Using primary striatal cultures, we show that transcription of Dnmt3a2, but not that of Dnmt3a1, is activated by dopamine D1 receptor signaling and that knockdown of Dnmt3a2 using viral vector-mediated expression of Dnmt3a2-specific shRNAs impairs induction of the IEGs, Arc, FosB, and Egr2 Acute cocaine administration increases expression of Dnmt3a2 but not that of Dnmt3a1 in the NAc shell. In contrast, in the NAc core, expression of Dnmt3a1 and Dnmt3a2 was unaffected by cocaine administration. shRNA-mediated knockdown of Dnmt3a2 in vivo impairs the induction of IEGs, including Egr2 and FosB indicating that Dnmt3a2 regulates cocaine-dependent expression of plasticity genes in the rat NAc shell. Cocaine self-administration experiments in rats revealed that Dnmt3a2 regulates drug cue memories that drive reinstatement of cocaine seeking as well as incubation of this phenomenon within the NAc shell. Dnmt3a2 does not influence the primary reinforcing effects of cocaine. Thus, Dnmt3a2 mediates long-lasting cocaine cue memories within the NAc shell. Targeting Dnmt3a2 expression or function may interfere with cocaine craving and relapse.SIGNIFICANCE STATEMENT In humans, drug craving can occur in response to conditioned cues, even after extended periods of abstinence. In rats, cue-induced cocaine seeking has been shown to increase progressively during the first 2 months of abstinence from drug self-administration. This phenomenon, referred to as incubation of cocaine seeking, is consistent with the hypothesis that in humans craving increases over time and remains high following prolonged abstinence. Those long-lasting behavioral changes are likely to be mediated by epigenetic effects and neuroplastic changes within the mesolimbic brain reward system. Here we show that a specific isoform of DNA-methyltransferases in the NAc shell regulates drug cue memories that drive reinstatement of cocaine seeking after both early abstinence and incubation of cocaine craving.

MeCP2 is required both during postnatal neurodevelopment and throughout the adult life for brain function. Although it is well accepted that MeCP2 in the maturing nervous system is critical for establishing normal development, the functions of MeCP2 during adulthood are poorly understood. Particularly, the requirement of hippocampal MeCP2 for cognitive abilities in the adult is not studied. To characterize the role of MeCP2 in adult neuronal function and cognition, we used a temporal and region-specific disruption of MeCP2 expression in the hippocampus of adult male mice. We found that MeCP2 is required for long-term memory formation and that it controls the learning-induced transcriptional response of hippocampal neurons required for memory consolidation. Furthermore, we uncovered MeCP2 functions in the adult hippocampus that may underlie cognitive integrity. We showed that MeCP2 maintains the developmentally established chromatin configuration and epigenetic landscape of CA1 neurons throughout the adulthood, and that it regulates the expression of neuronal and immune-related genes in the adult hippocampus. Overall, our findings identify MeCP2 as a maintenance factor in the adult hippocampus that preserves signal responsiveness of the genome and allows for integrity of cognitive functions. This study provides new insight into how MeCP2 maintains adult brain functions, but also into the mechanisms underlying the cognitive impairments observed in RTT patients and highlights the understudied role of DNA methylation interpretation in adult cognitive processes.

Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies.SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.

DNA methylation was traditionally viewed as a static mechanism required during cell fate determination. This view has been challenged and it is now accepted that DNA methylation is involved in the regulation of genomic responses in mature neurons, particularly in cognitive functions. The evidence for a role of DNA methylation in memory formation and maintenance comes from the increasing number of studies that have assessed the effects of manipulation of DNA methylation modifiers in the ability to form and maintain memories. Moreover, insights from genome-wide analyses of the hippocampal DNA methylation status after neuronal activity show that DNA methylation is dynamically regulated. Despite all the experimental evidence, we are still far from having a clear picture of how DNA methylation regulates long-term adaptations. This review aims on one hand to describe the findings that led to the confirmation of DNA methylation as an important player in memory formation. On the other hand, it tries to integrate these discoveries into the current views of how memories are formed and maintained.

DNA methylation was traditionally viewed as a static mechanism required during cell fate determination. This view has been challenged and it is now accepted that DNA methylation is involved in the regulation of genomic responses in mature neurons, particularly in cognitive functions. The evidence for a role of DNA methylation in memory formation and maintenance comes from the increasing number of studies that have assessed the effects of manipulation of DNA methylation modifiers in the ability to form and maintain memories. Moreover, insights from genome-wide analyses of the hippocampal DNA methylation status after neuronal activity show that DNA methylation is dynamically regulated. Despite all the experimental evidence, we are still far from having a clear picture of how DNA methylation regulates long-term adaptations. This review aims on one hand to describe the findings that led to the confirmation of DNA methylation as an important player in memory formation. On the other hand, it tries to integrate these discoveries into the current views of how memories are formed and maintained.

A ciencia normal concentra o conhecimento nas Universidades. As novidades sao difundidas meios cientificos tradicionais. A populacao raramente esta incluida no processo de descoberta de novos paradigmas. Com o intuito de democratizar os saberes, o Nucleo de Cultura, Ciencia e Saude, da Secretaria Municipal de Saude e Defesa Civil do Rio de Janeiro oferece ha tres anos uma oficina gratuita sobre saude publica. O objetivo do presente trabalho e descrever a Oficina de Imunologia para o Cidadao 2011: Arte e Ciencia da Saude Humana. Trata-se de um estudo descritivo. A coleta de dados foi realizada no site do Nucleo nccsrio.blogspot.com e pela presenca e participacao durante a oficina. O curso aconteceu no Centro de Artes Calouste Gulbenkian entre 4 de outubro e 22 de novembro, com 2 horas semanais. O coordenador do Nucleo problematizava o tema proposto e realizava uma explicacao, a qual era acrescida por comentarios espontâneos dos interessados, vivenciando a negacao a educacao bancaria. No primeiro encontro, o tema foi o modelo vigente da medicina, baseada no cartesianismo, na separacao entre corpo e mente, entre medico e paciente. Uma vez denunciada a falencia desse modelo, iniciou-se a exposicao das bases teoricas. O segundo tema abordado foi a Biologia do Conhecer e da Linguagem de Humberto Maturana, o qual defende a autopoiese, que o individuo e estruturalmente determinado e condicionado pelo meio. O terceiro encontro contou com a participacao do imunologista Nelson Vaz, esclarecendo que nos parecemos mais com rios e redemoinhos que com estatuas. Tornou-se clara a busca por explicacoes e nao por principios explicativos. Ja num quarto momento, introduziu-se a filosofia spinozana, difundindo a salvacao pelo conhecimento, a necessidade do amor ao imutavel e a felicidade efemera gerada por riqueza, prazer e honra. Nise da Silveira foi a cientista revolucionaria estudada no quinto encontro, tal medica usava o afeto catalisador para a cura. Paulo Freire, autor de Acao Cultural para a Liberdade e Pedagogia da Autonomia, foi o tema do sexto. No penultimo, houve um encontro entre Amir Haddad e Heloisa Helena Costa na Lapa, realizando uma audiencia publica sobre as relacoes horizontais no teatro de rua e educacao patrimonial. Ao final, os participantes foram convidados a unirem teoria e pratica durante a Celebracao da Saude e da Cidadania na Cinelândia. Sendo assim, demonstra-se que ciencia e arte podem atuar juntas para a promocao de saude integral.