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BackgroundAs the primary innate immune cells of the brain microglia respond to damage and disease through pro-inflammatory release of cytokines and neuroinflammatory molecules. Histone acetylation is an activating transcriptional mark that regulates gene expression, which is altered in states of disease. Inhibition of histone deacetylase 3 (Hdac3) has been utilized in pre-clinical models of disease to dampen inflammation, but the molecular mechanisms underlying Hdac3’s regulation of inflammatory gene expression in microglia is not well understood.MethodsFunctional changes in immortalized microglia were characterized using a Hdac3 specific inhibitor RGFP966 in response to an immune challenge lipopolysaccharide (LPS). Flow cytometry and cleavage under tags & release using nucleases (CUT & RUN) were used to investigate global and promoter-specific histone acetylation changes, resulting in altered gene expression.ResultsHdac3 inhibition enhanced neuroprotective functions of microglia in response to LPS through reduced nitric oxide release and increased baseline phagocytosis. Inhibition of Hdac3 enhanced histone acetylation globally and at specific gene loci, resulting in the release of gene repression at baseline and enhanced responses to LPS.ConclusionThe findings suggest Hdac3 serves as a negative regulator of microglial gene expression, and that inhibition of Hdac3 facilitates the microglial response to inflammation and its subsequent resolution. Together, this work provides new mechanistic insights into therapeutic applications of Hdac3 inhibition which mediate reduced neuroinflammatory insults through microglial response.

Microglia, the resident macrophages of the central nervous system, play important roles in maintaining brain homeostasis and facilitating the brain’s innate immune responses. Following immune challenges microglia also retain immune memories, which can alter responses to secondary inflammatory challenges. Microglia have two main memory states, training and tolerance, which are associated with increased and attenuated expression of inflammatory cytokines, respectively. However, the mechanisms differentiating these two distinct states are not well understood. We investigated mechanisms underlying training versus tolerance memory paradigms in vitro in BV2 cells using B-cell-activating factor (BAFF) or bacterial lipopolysaccharide (LPS) as a priming stimulus followed by LPS as a second stimulus. BAFF followed by LPS showed enhanced responses indicative of priming, whereas LPS followed by LPS as the second stimulus caused reduced responses suggestive of tolerance. The main difference between the BAFF versus the LPS stimulus was the induction of aerobic glycolysis by LPS. Inhibiting aerobic glycolysis during the priming stimulus using sodium oxamate prevented the establishment of the tolerized memory state. In addition, tolerized microglia were unable to induce aerobic glycolysis upon LPS restimulus. Therefore, we conclude that aerobic glycolysis triggered by the first LPS stimulus was a critical step in the induction of innate immune tolerance.

OBJECTIVE: To determine if closed-loop optogenetic seizure intervention, previously shown to reduce seizure duration in a well-established mouse model chronic temporal lobe epilepsy (TLE), also improves the associated comorbidity of impaired spatial memory. METHODS: Mice with chronic, spontaneous seizures in the unilateral intrahippocampal kainic acid model of TLE, expressing channelrhodopsin in parvalbumin-expressing interneurons were implanted with optical fibers and electrodes, and tested for response to closed-loop light intervention of seizures. Animals that responded to closed-loop optogenetic curtailment of seizures were tested in the object location memory test and then given closed-loop optogenetic intervention on all detected seizures for two weeks. Following this, they were tested with a second object location memory test, with different objects and contexts than used previously, to assess if seizure suppression can improve deficits in spatial memory. RESULTS: Animals that received closed-loop optogenetic intervention performed significantly better in the second object location memory test compared to the first test. Epileptic controls with no intervention showed stable frequency and duration of seizures, as well as stable spatial memory deficits, for several months after the precipitating insult. SIGNIFICANCE: Many currently available treatments for epilepsy target seizures but not the associated comorbidities, which therefore are often not improved through these treatments. There is a need to investigate new potential therapies that may be able to improve both seizure burden and associated comorbidities of epilepsy. In this study, we showed that optogenetic intervention may be able to both shorten seizure duration and improve cognitive outcomes of spatial memory.

Gene expression analysis is becoming increasingly utilized in neuro-immunology research, and there is a growing need for non-programming scientists to be able to analyze their own genomic data. MGEnrichment is a web application developed both to disseminate to the community our curated database of microglia-relevant gene lists, and to allow non-programming scientists to easily conduct statistical enrichment analysis on their gene expression data. Users can upload their own gene IDs to assess the relevance of their expression data against gene lists from other studies. We include example datasets of differentially expressed genes (DEGs) from human postmortem brain samples from Autism Spectrum Disorder (ASD) and matched controls. We demonstrate how MGEnrichment can be used to expand the interpretations of these DEG lists in terms of regulation of microglial gene expression and provide novel insights into how ASD DEGs may be implicated specifically in microglial development, microbiome responses and relationships to other neuropsychiatric disorders. This tool will be particularly useful for those working in microglia, autism spectrum disorders, and neuro-immune activation research. MGEnrichment is available at https://ciernialab.shinyapps.io/MGEnrichmentApp/ and further online documentation and datasets can be found at https://github.com/ciernialab/MGEnrichmentApp. The app is released under the GNU GPLv3 open source license., Author summary Recent technological and computational advances have produced a massive amount of sequencing data that is often inaccessible to the non-bioinformatician. This is particularly true in multi-disciplinary areas of study such as neuro-immunology, where scientists come from a diversity of background fields. We developed a tool to allow wet-lab scientists without computational skills to utilize previous findings on microglia, the innate immune cells of the brain. Our web hosted tool allows users to compare their genes of interest against a large database of previously published gene lists relevant to microglia and brain disorders. With just a few clicks on the interface, users can upload their genes of interest from mouse or human studies, and query their list by selecting options for running statistical analysis. The application compares the user input to each database list, performs a statistical comparison and returns the results to the user, which can be viewed within the application or downloaded for publication. We have included two example datasets of genes from Autism Spectrum Disorder human brain samples. With these example datasets we demonstrate that this type of analysis can be utilized to identify new biological insights and high priority targets for further study in the lab.

Neurodevelopmental disorders (NDDs) affect 7–14% of all children in developed countries and are one of the leading causes of lifelong disability. Epigenetic modifications are poised at the interface between genes and environment and are predicted to reveal insight into NDD etiology. Whole-genome bisulfite sequencing was used to examine DNA cytosine methylation in 49 human cortex samples from 3 different NDDs (autism spectrum disorder, Rett syndrome, and Dup15q syndrome) and matched controls. Integration of methylation changes across NDDs with relevant genomic and genetic datasets revealed differentially methylated regions (DMRs) unique to each type of NDD but with shared regulatory functions in neurons and microglia. NDD DMRs were enriched within promoter regions and for transcription factor binding sites with identified methylation sensitivity. DMRs from all 3 disorders were enriched for ontologies related to nervous system development and genes with disrupted expression in brain from neurodevelopmental or neuropsychiatric disorders. Genes associated with NDD DMRs showed expression patterns indicating an important role for altered microglial function during brain development. These findings demonstrate an NDD epigenomic signature in human cortex that will aid in defining therapeutic targets and early biomarkers at the interface of genetic and environmental NDD risk factors.

The BTBR T(+)Itpr3(tf)/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge resulting in hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses.

The BTBR T+Itpr3tf/J (BTBR) mouse has been used as a complex genetic model of Autism Spectrum Disorders (ASD). While the specific mechanisms underlying BTBR behavioral phenotypes are poorly understood, prior studies have implicated profound differences in innate immune system control of pro-inflammatory cytokines. Innate immune activation and elevated pro-inflammatory cytokines are also detected in blood of children with ASD. In this study, we examined how underlying BTBR genetic variants correspond to strain-specific changes in chromatin accessibility, resulting in a pro-inflammatory response specifically in BTBR bone marrow derived macrophages (BMDM). In response to repeated lipopolysaccharide (LPS) treatments, C57BL/6J (C57) BMDM exhibited intact endotoxin tolerance. In contrast, BTBR BMDM exhibited hyper-responsive expression of genes that were normally tolerized in C57. This failure in formation of endotoxin tolerance in BTBR was mirrored at the level of chromatin accessibility. Using ATAC-seq, we specifically identified promoter and enhancer regions with strain-specific differential chromatin accessibility both at baseline and in response to LPS. Regions with strain-specific differences in chromatin accessibility were significantly enriched for BTBR genetic variants, such that an average of 22% of the differential chromatin regions had at least one variant. Together, these results demonstrate that BTBR genetic variants contribute to altered chromatin responsiveness to endotoxin challenge and a failure in formation of tolerance, resulting in a hyper-responsive innate immunity in BTBR. These findings provide evidence for an interaction between complex genetic variants and differential epigenetic regulation of innate immune responses. Our findings also provide novel mechanistic insight into the complex genetic architecture and immune abnormalities observed in ASD.

Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and experience-induced expression of the circadian gene Per1 in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampal Per1 is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation., Circadian rhythms are known to modulate memory, but it’s not known whether clock genes in the hippocampus are required for memory consolidation. Here, the authors show that epigenetic regulation of clock gene Period1 in the hippocampus regulates memory and contributes to age-related memory decline, independent of circadian rhythms.

Dysregulation in immune responses during pregnancy increase the risk of a having a child with an autism spectrum disorder (ASD). Asthma is one of the most common chronic diseases among pregnant women, and symptoms often worsen during pregnancy. We recently developed a mouse model of maternal allergic asthma (MAA) that induces changes in sociability, repetitive and perseverative behaviors in the offspring. Since epigenetic changes help a static genome adapt to the maternal environment, activation of the immune system may epigenetically alter fetal microglia, the brain’s resident immune cells. We therefore tested the hypothesis that epigenomic alterations to microglia may be involved in behavioral abnormalities observed in MAA offspring. We used the genome-wide approaches of whole genome bisulfite sequencing to examine DNA methylation and RNA sequencing to examine gene expression in microglia from juvenile MAA offspring. Differentially methylated regions (DMRs) were enriched for immune signaling pathways and important microglial developmental transcription factor binding motifs. Differential expression analysis identified genes involved in controlling microglial sensitivity to the environment and shaping neuronal connections in the developing brain. Differentially expressed associated genes significantly overlapped genes with altered expression in human ASD cortex, supporting a role for microglia in the pathogenesis of ASD.Main Points:Maternal allergic asthma induces changes in DNA methylation and transcription in juvenile offspring microgliaDifferentially methylated regions are enriched for functions and transcription factor binding motifs involved in inflammation and microglial developmentDifferentially expressed genes and differentially methylated regions are enriched for genes dysregulated in Autism Spectrum Disorders

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53b Delta SB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53b Delta SB2 mice in an effort to rescue LTP and memory. BAF53b Delta SB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders.

Author(s): Silverman, Jill L; Berg, Elizabeth L; Patten, Kelley T; Valenzuela, Anthony E; Wallis, Christopher; LaSalle, Janine M; Ciernia, Annie Vogel; Bein, Keith J; Wexler, Anthony S; Lein, Pamela J

Increasing evidence points to a complex interplay between genes and the environment in autism spectrum disorder (ASD), including rare de novo mutations in chromatin genes such as methyl-CpG binding protein 2 (MECP2) in Rett syndrome. Epigenetic mechanisms such as DNA methylation act at this interface, reflecting the plasticity in metabolic and neurodevelopmentally regulated gene pathways. Genome-wide studies of gene sequences, gene pathways and DNA methylation are providing valuable mechanistic insights into ASD. The dynamic developmental landscape of DNA methylation is vulnerable to numerous genetic and environmental insults: therefore, understanding pathways that are central to this ‘perfect storm’ will be crucial to improving the diagnosis and treatment of ASD.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1 deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory, and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio (RER), but decreased food intake compared to wildtype. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulate early and progressive metabolic and motor phenotypes of human RTT.

Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and learning-induced expression of the circadian genePer1in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampalPer1is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation.

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader–Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders., Many genes have oscillating gene expression pattern in circadian centers of the brain. This study shows cortical diurnal DNA methylation oscillation in a mouse model of Prader-Willi syndrome, and describes corresponding changes in gene expression and chromatin compaction.

BackgroundMaternal care during early-life plays a crucial role in the sculpting of the mammalian brain. Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this potent phenotypic change commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood.MethodsHere, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression.ResultsA significant decrease in global DNA methylation was observed in offspring hypothalamus following a week of augmented maternal care, corresponding to differential methylation and expression of thousands of genes. Differentially methylated and expressed genes were enriched for functions in neurotransmission, neurodevelopment, protein synthesis, and oxidative phosphorylation, as well as known stress response genes. Twenty prioritized genes with three lines of evidence (methylation, expression, and altered miRNA target) were identified as highly relevant to the stress resiliency phenotype.ConclusionsThis combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the central epigenomic mechanisms underlying the profound effects of maternal care on the developing brain.

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. A major genetic effect on rhythmic methylation was identified in a mouse Snordll6 deletion model of the imprinted disorder Prader-Willi syndrome (PWS). Of the >23,000 diurnally rhythmic CpGs identified in wild-type cortex, 97% lost rhythmic methylation in PWS cortex. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus was observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of crosstalk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and were enriched for PWS-relevant obesity phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene encoding methyl CpG binding protein 2 (MeCP2) that occur sporadically in 1:10,000 female births. RTT is characterized by a period of largely normal development followed by regression in language and motor skills at 6-18 months of age. Mecp2 mutant mice recapitulate many of the clinical features of RTT, but the majority of behavioral assessments have been conducted in male Mecp2 hemizygous null mice as offspring of heterozygous dams. Given that RTT patients are predominantly female, we conducted a systematic analysis of developmental milestones, sensory abilities, and motor deficits, following the longitudinal decline of function from early postnatal to adult ages in female Mecp2 heterozygotes of the conventional Bird line (Mecp2tm1.1bird-/+), as compared to their female wildtype littermate controls. Further, we assessed the impact of postnatal maternal environment on developmental milestones and behavioral phenotypes. Cross-fostering to CD1 dams accelerated several developmental milestones independent of genotype, and induced earlier onset of weight gain in adult female Mecp2tm1.1bird-/+ mice. Cross-fostering improved the sensitivity of a number of motor behaviors that resulted in observable deficits in Mecp2tm1.1bird-/+ mice at much earlier (6-7 weeks) ages than were previously reported (6-9 months). Our findings indicate that female Mecp2tm1.1bird-/+ mice recapitulate many of the motor aspects of RTT syndrome earlier than previously appreciated. In addition, rearing conditions may impact the phenotypic severity and improve the ability to detect genotype differences in female Mecp2 mutant mice.

Long-term memory formation requires the coordinated regulation of gene expression. Until recently nucleosome remodeling, one of the major epigenetic mechanisms for controlling gene expression, had been largely unexplored in the field of neuroscience. Nucleosome remodeling is carried out by chromatin remodeling complexes (CRCs) that interact with DNA and histones to physically alter chromatin structure and ultimately regulate gene expression. Human exome sequencing and gene wide association studies have linked mutations in CRC subunits to intellectual disability disorders, autism spectrum disorder and schizophrenia. However, how mutations in CRC subunits were related to human cognitive disorders was unknown. There appears to be both developmental and adult specific roles for the neuron specific CRC nBAF (neuronal Brg1/hBrm Associated Factor). nBAF regulates gene expression required for dendritic arborization during development, and in the adult, contributes to long-term potentiation, a form of synaptic plasticity, and long-term memory. We propose that the nBAF complex is a novel epigenetic mechanism for regulating transcription required for long-lasting forms of synaptic plasticity and memory processes and that impaired nBAF function may result in human cognitive disorders.

© 2016 The Author(s) Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes.

Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn–HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn–HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn–HM3D impaired LTP and hSyn–HM4D facilitated LTP. Follow-up experiments indicated that the hSyn–HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn–HM4D-mediated increase of fEPSP is induced by a reduction in GABAAreceptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα–HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα–HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα–HM3D hippocampal slices, whereas slices from CaMKIIα–HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP.SIGNIFICANCE STATEMENTThis study evaluates the efficacy of designer receptors exclusively activated by designer drug (DREADDs) as a means of bidirectionally modulating the hippocampus in not only a hippocampus-dependent task but also in hippocampal synaptic plasticity. This is the first study to evaluate the effects of DREADD-mediated inhibition and excitation in hippocampal long-term potentiation. More specifically, this study evaluates the effect of promoter-specific expression of DREADD viruses in a heterogenic cell population, which revealed surprising effects of different promoters. With chemogenetics becoming a more ubiquitous tool throughout studies investigating circuit-specific function, these data are of broad interest to the neuroscientific community because we have shown that promoter-specific effects can drastically alter synaptic function within a specific region, without parallel changes at the level of behavior.

Nr4a1 and Nr4a2 are transcription factors and immediate early genes belonging to the nuclear receptor Nr4a family. In this study, we examine their role in long-term memory formation for object location and object recognition. Using siRNA to block expression of either Nr4a1 or Nr4a2, we found that Nr4a2 is necessary for both long-term memory for object location and object recognition. In contrast, Nr4a1 appears to be necessary only for object location. Indeed, their roles in these different types of long-term memory may be dependent on their expression in the brain, as NR4A2 was found to be expressed in hippocampal neurons (associated with object location memory) as well as in the insular and perirhinal cortex (associated with object recognition memory), whereas NR4A1 showed minimal neuronal expression in these cortical areas. These results begin to elucidate how NR4A1 and NR4A2 differentially contribute to object location versus object recognition memory.

SummaryThree-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here we describe activity-regulated long-range loopings bypassing up to 0.5 Mb of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3′ intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a “cargo” of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher-order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition.

The ability to store and recall our life experiences defines a person's identity. Consequently, the loss of long-term memory is a particularly devastating part of a variety of cognitive disorders, diseases and injuries. There is a great need to develop therapeutics to treat memory disorders, and thus a variety of animal models and memory paradigms have been developed. Mouse models have been widely used both to study basic disease mechanisms and to evaluate potential drug targets for therapeutic development. The relative ease of genetic manipulation of Mus musculus has led to a wide variety of genetically altered mice that model cognitive disorders ranging from Alzheimer's disease to autism. Rodents, including mice, are particularly adept at encoding and remembering spatial relationships, and these long-term spatial memories are dependent on the medial temporal lobe of the brain. These brain regions are also some of the first and most heavily impacted in disorders of human memory including Alzheimer's disease. Consequently, some of the simplest and most commonly used tests of long-term memory in mice are those that examine memory for objects and spatial relationships. However, many of these tasks, such as Morris water maze and contextual fear conditioning, are dependent upon the encoding and retrieval of emotionally aversive and inherently stressful training events. While these types of memories are important, they do not reflect the typical day-to-day experiences or memories most commonly affected in human disease. In addition, stress hormone release alone can modulate memory and thus obscure or artificially enhance these types of tasks. To avoid these sorts of confounds, we and many others have utilized tasks testing animals’ memory for object location and novel object recognition. These tasks involve exploiting rodents’ innate preference for novelty, and are inherently not stressful. In this protocol we detail how memory for object location and object identity can be used to evaluate a wide variety of mouse models and treatments.

Significance Transcriptional dysregulation is an early and reproducible feature of Huntington disease (HD); however, mechanisms underlying this dysregulation are unclear. This article describes a unique pattern of the chromatin mark H3K4me3 at transcriptionally repressed promoters in HD mouse and human brain identified by genome-wide analysis. Reducing the levels of the demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression and was neuroprotective in a Drosophila HD model. These results suggest that targeting epigenetic signatures may be an effective strategy to ameliorate the consequences of HD and other neurodegenerative diseases.

Recent exome sequencing studies have implicated polymorphic Brg1-Associated Factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several human intellectual disabilities and cognitive disorders. However, it is currently unknown how mutations in BAF complexes result in impaired cognitive function. Postmitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Mice harboring selective genetic manipulations of BAF53b have severe defects in long-term memory and long-lasting forms of hippocampal synaptic plasticity. We rescued memory impairments in BAF53b mutant mice by reintroducing BAF53b in the adult hippocampus, which suggests a role for BAF53b beyond neuronal development. The defects in BAF53b mutant mice appeared to derive from alterations in gene expression that produce abnormal postsynaptic components, such as spine structure and function, and ultimately lead to deficits in synaptic plasticity. Our results provide new insight into the role of dominant mutations in subunits of BAF complexes in human intellectual and cognitive disorders. © 2013 Nature America, Inc. All rights reserved.

Under basal conditions histone deacetylases (HDACs) and their associated co-repressor complexes serve as molecular ‘brake pads’ to prevent the gene expression required for long-term memory formation. Following a learning event, HDACs and their co-repressor complexes are removed from a subset of specific gene promoters, allowing the histone acetylation and active gene expression required for long-term memory formation. Inhibition of HDACs increases histone acetylation, extends gene expression profiles, and allows for the formation of persistent long-term memories for training events that are otherwise forgotten. We propose that emotionally salient experiences have utilized this system to form strong and persistent memories for behaviorally significant events. Consequently, the presence or absence of HDACs at a selection of specific gene promoters could serve as a critical barrier for permitting the formation of long-term memories.