DNA G-Quadruplex Is a Transcriptional Control Device That Regulates Memory.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation.One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

Synapse-Enriched m6A-Modified Malat1 Interacts with the Novel m6A Reader, DPYSL2, and Is Required for Fear-Extinction Memory.

The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.

Fundamental Neurochemistry Review: At the intersection between the brain and the immune system: Non-coding RNAs spanning learning, memory and adaptive immunity.

Non-coding RNAs (ncRNAs) are highly plastic RNA molecules that can sequester cellular proteins and other RNAs, serve as transporters of cellular cargo and provide spatiotemporal feedback to the genome. Mounting evidence indicates that ncRNAs are central to biology, and are critical for neuronal development, metabolism and intra- and intercellular communication in the brain. Their plasticity arises from state-dependent dynamic structure states that can be influenced by cell type and subcellular environment, which can subsequently enable the same ncRNA with discrete functions in different contexts. Here, we highlight different classes of brain-enriched ncRNAs, including microRNA, long non-coding RNA and other enigmatic ncRNAs, that are functionally important for both learning and memory and adaptive immunity, and describe how they may promote cross-talk between these two evolutionarily ancient biological systems.

Localised Cdr1as activity is required for fear extinction memory.

Circular RNAs (circRNAs) comprise a novel class of regulatory RNAs that are abundant in the brain, particularly within synapses. They are highly stable, dynamically regulated, and display a range of functions, including serving as decoys for microRNAs and proteins and, in some cases, circRNAs also undergo translation. Early work in animal models revealed an association between circRNAs and neurodegenerative and neuropsychiatric disorders; however, little is known about the link between circRNA function and memory. To address this, we examined circRNA in synaptosomes derived from the medial prefrontal cortex of fear extinction-trained male C57BL/6J mice and found 12,837 circRNAs that were enriched at the synapse, including cerebellar degeneration-related protein 1 antisense RNA (Cdr1as). Targeted knockdown of Cdr1as in the neural processes of the infralimbic cortex led to impaired fear extinction memory. These findings highlight the involvement of localised circRNA activity at the synapse in memory formation.

From Circuits to Chromatin: The Emerging Role of Epigenetics in Mental Health.

A central goal of neuroscience research is to understand how experiences modify brain circuits to guide future adaptive behavior. In response to environmental stimuli, neural circuit activity engages gene regulatory mechanisms within each cell. This activity-dependent gene expression is governed, in part, by epigenetic processes that can produce persistent changes in both neural circuits and the epigenome itself. The complex interplay between circuit activity and neuronal gene regulation is vital to learning and memory, and, when disrupted, is linked to debilitating psychiatric conditions, such as substance use disorder. To develop clinical treatments, it is paramount to advance our understanding of how neural circuits and the epigenome cooperate to produce behavioral adaptation. Here, we discuss how new genetic tools, used to manipulate neural circuits and chromatin, have enabled the discovery of epigenetic processes that bring about long-lasting changes in behavior relevant to mental health and disease.

On the functional relevance of spatiotemporally-specific patterns of experience-dependent long noncoding RNA expression in the brain.

The majority of transcriptionally active RNA derived from the mammalian genome does not code for protein. Long noncoding RNA (lncRNA) is the most abundant form of noncoding RNA found in the brain and is involved in many aspects of cellular metabolism. Beyond their fundamental role in the nucleus as decoys for RNA-binding proteins associated with alternative splicing or as guides for the epigenetic regulation of protein-coding gene expression, recent findings indicate that activity-induced lncRNAs also regulate neural plasticity. In this review, we discuss how lncRNAs may exert molecular control over brain function beyond their known roles in the nucleus. We propose that subcellular localization is a critical feature of experience-dependent lncRNA activity in the brain, and that lncRNA-mediated control over RNA metabolism at the synapse serves to regulate local mRNA stability and translation, thereby influencing neuronal function, learning and memory.

Adolescent chronic intermittent toluene inhalation dynamically regulates the transcriptome and neuronal methylome within the rat medial prefrontal cortex.

Inhalants containing the volatile solvent toluene are misused to induce euphoria or intoxication. Inhalant abuse is most common during adolescence and can result in cognitive impairments during an important maturational period. Despite evidence suggesting that epigenetic modifications may underpin the cognitive effects of inhalants, no studies to date have thoroughly investigated toluene-induced regulation of the transcriptome or discrete epigenetic modifications within the brain. To address this, we investigated effects of adolescent chronic intermittent toluene (CIT) inhalation on gene expression and DNA methylation profiles within the rat medial prefrontal cortex (mPFC), which undergoes maturation throughout adolescence and has been implicated in toluene-induced cognitive deficits. Employing both RNA-seq and genome-wide Methyl CpG Binding Domain (MBD) Ultra-seq analysis, we demonstrate that adolescent CIT inhalation (10 000 ppm for 1 h/day, 3 days/week for 4 weeks) induces both transient and persistent changes to the transcriptome and DNA methylome within the rat mPFC for at least 2 weeks following toluene exposure. We demonstrate for the first time that adolescent CIT exposure results in dynamic regulation of the mPFC transcriptome likely relating to acute inflammatory responses and persistent deficits in synaptic plasticity. These adaptations may contribute to the cognitive deficits associated with chronic toluene exposure and provide novel molecular targets for preventing long-term neurophysiological abnormalities following chronic toluene inhalation.

The DNA Repair-Associated Protein Gadd45γ Regulates the Temporal Coding of Immediate Early Gene Expression within the Prelimbic Prefrontal Cortex and Is Required for the Consolidation of Associative Fear Memory.

We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45γ, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45γ temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45γ and active DNA demethylation at the same site, which is necessary for memory consolidation.SIGNIFICANCE STATEMENT How does the pattern of immediate early gene transcription in the brain relate to the storage and accession of information, and what controls these patterns? This paper explores how Gadd45γ, a gene that is known to be involved with DNA modification and repair, regulates the temporal coding of IEGs underlying associative learning and memory. We reveal that, during fear learning, Gadd45γ serves to act as a coordinator of IEG expression and subsequent memory consolidation by directing temporally specific changes in active DNA demethylation at the promoter of plasticity-related IEGs.

MicroRNA-mediated disruption of dendritogenesis during a critical period of development influences cognitive capacity later in life.

The prenatal period of cortical development is important for the establishment of neural circuitry and functional connectivity of the brain; however, the molecular mechanisms underlying this process remain unclear. Here we report that disruption of the actin-cytoskeletal network in the developing mouse prefrontal cortex alters dendritic morphogenesis and synapse formation, leading to enhanced formation of fear-related memory in adulthood. These effects are mediated by a brain-enriched microRNA, miR-9, through its negative regulation of diaphanous homologous protein 1 (Diap1), a key organizer of the actin cytoskeletal assembly. Our findings not only revealed important regulation of dendritogenesis and synaptogenesis during early brain development but also demonstrated a tight link between these early developmental events and cognitive functions later in life.

Disrupting the hippocampal Piwi pathway enhances contextual fear memory in mice.

The Piwi pathway is a conserved gene regulatory mechanism comprised of Piwi-like proteins and Piwi-interacting RNAs, which modulates gene expression via RNA interference and through interaction with epigenetic mechanisms. The mammalian Piwi pathway has been defined by its role in transposon control during spermatogenesis; however, despite an increasing number of studies demonstrating its expression in the nervous system, relatively little is known about its function in neurons or potential contribution to behavioural regulation. We have discovered that all three Piwi-like genes are expressed in the adult mouse brain, and that viral-mediated knockdown of the Piwi-like genes Piwil1 and Piwil2 in the dorsal hippocampus leads to enhanced contextual fear memory without affecting generalised anxiety. These results implicate the Piwi pathway in behavioural regulation in the adult mammalian brain, likely through modulation of plasticity-related gene expression.

MotifMap-RNA: a genome-wide map of RBP binding sites.

MOTIVATION: RNA plays a critical role in gene expression and its regulation. RNA binding proteins (RBPs), in turn, are important regulators of RNA. Thanks to the availability of large scale data for RBP binding motifs and in vivo binding sites results in the form of eCLIP experiments, it is now possible to computationally predict RBP binding sites across the whole genome. RESULTS: We describe MotifMap-RNA, an extension of MotifMap which predicts binding sites for RBP motifs across human and mouse genomes and allows large scale querying of predicted binding sites. AVAILABILITY AND IMPLEMENTATION: The data and corresponding web server are available from: http://motifmap-rna.ics.uci.edu/ as part of the MotifMap web portal. CONTACT: rspitale@uci.edu or pfbaldi@uci.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Experience-Dependent Accumulation of N6-Methyladenosine in the Prefrontal Cortex Is Associated with Memory Processes in Mice.

UNLABELLED: The RNA modification N(6)-methyladenosine (m(6)A) influences mRNA stability and cell-type-specific developmental programming, and is highly abundant in the adult brain. However, it has not been determined whether m(6)A is dynamically regulated by experience. Based on transcriptome-wide profiling of m(6)A, we report that the level of m(6)A increases in the medial prefrontal cortex (mPFC) of mice in response to behavioral experience. The modulation was enriched near the stop codon of mRNAs, including genes related to neuronal plasticity. In primary cortical neurons, in vitro, modulation of m(6)A by the RNA demethylase FTO influenced the degradation profiles of a subset of transcripts with modulated sites. In vivo, the expression of Fto and the m(6)A methyltransferase, Mettl3 correlated with the observed increase in m(6)A levels post-training. Furthermore, targeted knockdown of FTO in the mPFC led to enhanced consolidation of cued fear memory. Thus, together with its role in early development, the dynamic regulation of m(6)A in the adult brain serves as an important epitranscriptomic mechanism associated with behavioral adaptation. SIGNIFICANCE STATEMENT: N(6)-methyladenosine (m(6)A) is the most prevalent internal modification on RNA, however, its cellular dynamics in vivo remains elusive. Here we provide the first demonstration of m(6)A upregulation in the mouse medial prefrontal cortex (mPFC) following behavioral training. Knocking down the m(6)A demethylase FTO in the mPFC, which increases total m(6)A level, results in enhanced consolidation of fear memory. Our findings suggest that m(6)A is regulated in an activity-dependent manner in the adult brain, and may function to fine-tune mRNA turnover during memory-related processes.

Cell-Selective Bioorthogonal Metabolic Labeling of RNA.

Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue, we developed a chemical-genetic strategy for metabolic labeling of RNA. Cell-specific labeling of RNA can be profiled and imaged using bioorthogonal chemistry. We anticipate that this platform will provide the community with a much-needed chemical toolset for cell-type specific profiling of cell-specific transcriptomes derived from complex biological systems.

Genetic and epigenetic factors underlying sex differences in the regulation of gene expression in the brain.

There are inherent biological differences between males and females that contribute to sex differences in brain function and to many sex-specific illnesses and disorders. Traditionally, it has been thought that such differences are due largely to hormonal regulation; however, there are also genetic and epigenetic effects caused by the inheritance and unequal dosage of genes located on the X and Y chromosomes. Here we discuss the evidence in favor of a genetic and epigenetic basis for sexually dimorphic behavior, as a consequence of underlying differences in the regulation of genes that drive brain function. A better understanding of sex-specific molecular processes in the brain will provide further insight for the development of novel therapeutic approaches for the treatment of neuropsychiatric disorders characterized by sex differences. © 2016 Wiley Periodicals, Inc.

Maternal separation induces hippocampal changes in cadherin-1 (CDH-1) mRNA and recognition memory impairment in adolescent mice.

In rodents, disruption of mother-infant attachment induced by maternal separation (MS) is associated with recognition memory impairment and long-term neurobiological consequences. Particularly stress-induced modifications have been associated to disruption of cadherin (CDH) adhesion function, which plays an important role in remodeling of neuronal connection and synaptic plasticity. This study investigated the sex-dependent effect of MS on recognition memory and mRNA levels of classical type I and type II CDH and the related ß -catenin (ß -Cat) in the hippocampus and prefrontal cortex of late adolescent mice. We provided evidence that the BALB/c mice exposed to MS present deficit in recognition memory, especially females. Postnatal MS induced higher hippocampal CDH-2 and CDH-8 mRNA levels, as well as an upregulation of CDH-1 in the prefrontal cortex in both males and females. MS-reared female mice presented lower CDH-1 mRNA levels in the hippocampus. In addition, hippocampal CDH-1 mRNA levels were positively correlated with recognition memory performance in females. MS-reared male mice exhibited higher ß -Cat mRNA levels in the hippocampus. Considering sex-specific effects on CDH mRNA levels, it has been demonstrated mRNA changes in CDH-1, ß -Cat, and CDH-6 in the hippocampus, as well as CDH-1, CDH-8 and CDH-11 in the prefrontal cortex. Overall, these findings suggest a complex interplay among MS, CDH mRNA expression, and sex differences in the PFC and hippocampus of adolescent mice.

Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation.

5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation.

Running during adolescence rescues a maternal separation-induced memory impairment in female mice: Potential role of differential exon-specific BDNF expression.

Exposure to early life stress has been associated with memory impairments related to changes in brain-derived neurotrophic factor (BDNF) signaling. However, the potential impact of physical exercise to reverse these effects of maternal separation has been under investigated. Mice were subjected to maternal separation during the first 2 weeks of life and then exposed to a 3-week running protocol during adolescence. The spontaneous object recognition task was performed during adolescence followed by analysis of hippocampal expression of exons I, IV, and IX of the BDNF gene. As expected, maternal separation impaired recognition memory and this effect was reversed by exercise. In addition, running increased BDNF exon I expression, but decreased expression of BDNF exon IV in all groups, while exon IX expression increased only in MS animals exposed to exercise. Our data suggest that memory deficits can be attenuated by exercise and specific transcripts of the BDNF gene are dynamically regulated following both MS and exercise.

Sex differences in learned fear expression and extinction involve altered gamma oscillations in medial prefrontal cortex.

Sex differences in learned fear expression and extinction involve the medial prefrontal cortex (mPFC). We recently demonstrated that enhanced learned fear expression during auditory fear extinction and its recall is linked to persistent theta activation in the prelimbic (PL) but not infralimbic (IL) cortex of female rats. Emerging evidence indicates that gamma oscillations in mPFC are also implicated in the expression and extinction of learned fear. Therefore we re-examined our in vivo electrophysiology data and found that females showed persistent PL gamma activation during extinction and a failure of IL gamma activation during extinction recall. Altered prefrontal gamma oscillations thus accompany sex differences in learned fear expression and its extinction. These findings are relevant for understanding the neural basis of post-traumatic stress disorder, which is more prevalent in women and involves impaired extinction and mPFC dysfunction.

Persistent prelimbic cortex activity contributes to enhanced learned fear expression in females.

Anxiety disorders, such as post-traumatic stress, are more prevalent in women and are characterized by impaired inhibition of learned fear and medial prefrontal cortex (mPFC) dysfunction. Here we examined sex differences in fear extinction and mPFC activity in rats. Females showed more learned fear expression during extinction and its recall, but not fear conditioning. They also showed more spontaneous fear recovery and more contextual fear before extinction and its recall. Moreover, enhanced learned fear expression in females was associated with sustained prelimbic (PL) cortex activity. These results suggest that sex differences in learned fear expression may involve persistent PL activation.

Activation-induced cytidine deaminase regulates activity-dependent BDNF expression in post-mitotic cortical neurons.

Activity-dependent gene expression depends, in part, on transcriptional regulation that is coordinated by rapid changes in the chromatin landscape as well as the covalent modification of DNA. Here we demonstrate that the expression of brain-derived neurotrophic factor (BDNF), a gene that is critically involved in neural plasticity and subject to epigenetic regulation, is regulated by the RNA/DNA editing enzyme, activation-induced cytidine deaminase (AID). Similar to previous reports, we observed an activity-dependent induction of BDNF exon IV mRNA expression, which correlated with a reduction in DNA methylation within the BDNF P4 promoter. Lentiviral-mediated knockdown of AID disrupted these effects and inhibited BDNF exon IV mRNA expression, an effect that was associated with decreased cAMP response element-binding protein occupancy within the BDNF P4 promoter. Thus, together with other epigenetic mechanisms, AID plays a key role in regulating activity-dependent BDNF expression in post-mitotic cortical neurons.