Wnt dose escalation during the exit from pluripotency identifies tranilast as a regulator of cardiac mesoderm.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation.

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.

Fear extinction is regulated by the activity of long noncoding RNAs at the synapse.

Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.

Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila.

Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow-wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.

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.

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.

Experimentally induced active and quiet sleep engage non-overlapping transcriptional programs in Drosophila.

Sleep in mammals can be broadly classified into two different physiological categories: rapid eye movement (REM) sleep and slow wave sleep (SWS), and accordingly REM and SWS are thought to achieve a different set of functions. The fruit fly Drosophila melanogaster is increasingly being used as a model to understand sleep functions, although it remains unclear if the fly brain also engages in different kinds of sleep as well. Here, we compare two commonly used approaches for studying sleep experimentally in Drosophila: optogenetic activation of sleep-promoting neurons and provision of a sleep-promoting drug, Gaboxadol. We find that these different sleep-induction methods have similar effects on increasing sleep duration, but divergent effects on brain activity. Transcriptomic analysis reveals that drug-induced deep sleep ('quiet' sleep) mostly downregulates metabolism genes, whereas optogenetic 'active' sleep upregulates a wide range of genes relevant to normal waking functions. This suggests that optogenetics and pharmacological induction of sleep in Drosophila promote different features of sleep, which engage different sets of genes to achieve their respective functions.

ADRAM is an experience-dependent long noncoding RNA that drives fear extinction through a direct interaction with the chaperone protein 14-3-3.

Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

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.

Publisher Correction: Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide microRNA profiling in brain and blood samples in a mouse model of epileptogenesis.

OBJECTIVES: This study profiled circulating and hippocampal microRNAs (miRNAs) to identify alterations associated with the risk of epileptogenesis in a mouse temporal lobe epilepsy model. METHODS: Next-generation sequencing was performed to examine the changes in miRNA expression 24 h after pilocarpine-induced status epilepticus (SE) in C57BL/6NCrl mice using both blood and hippocampus samples. Differentially expressed miRNAs were identified from SE animals and matched controls that failed to develop SE after receiving equal doses of pilocarpine (NS animals). Blood and brain miRNA profiles were then compared to identify circulating miRNA alterations reflecting the changes in the brain. RESULTS: We identified 3 miRNAs that were significantly up-regulated and 4 miRNAs that were significantly down-regulated in the blood of SE animals compared with NS animals. When hippocampal miRNAs of SE animals and NS animals were compared, 5 miRNAs were up-regulated and 4 were down-regulated. Of these, miR-434-3p and miR-133a-3p were observed to have greatest changes in both blood and brain of SE animals. SIGNIFICANCE: This study extends current knowledge of changes in miRNAs associated with epileptogenesis by profiling miRNAs in SE and NS animals in an experimental temporal lobe epilepsy model. The study was designed to allow non-specific changes due to the activation of muscarinic cholinergic receptors in peripheral organs by pilocarpine to be ruled out. Significantly altered circulating miRNAs that reflect changes in the brain during epileptogenesis after SE have the potential to be developed as prognostic biomarkers for epileptogenesis.

Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction.

DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state-effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.

Monozygotic twins and triplets discordant for amyotrophic lateral sclerosis display differential methylation and gene expression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons. ALS exhibits high phenotypic variability including age and site of onset, and disease duration. To uncover epigenetic and transcriptomic factors that may modify an ALS phenotype, we used a cohort of Australian monozygotic twins (n = 3 pairs) and triplets (n = 1 set) that are discordant for ALS and represent sporadic ALS and the two most common types of familial ALS, linked to C9orf72 and SOD1. Illumina Infinium HumanMethylation450K BeadChip, EpiTYPER and RNA-Seq analyses in these ALS-discordant twins/triplets and control twins (n = 2 pairs), implicated genes with consistent longitudinal differential DNA methylation and/or gene expression. Two identified genes, RAD9B and C8orf46, showed significant differential methylation in an extended cohort of >1000 ALS cases and controls. Combined longitudinal methylation-transcription analysis within a single twin set implicated CCNF, DPP6, RAMP3, and CCS, which have been previously associated with ALS. Longitudinal transcriptome data showed an 8-fold enrichment of immune function genes and under-representation of transcription and protein modification genes in ALS. Examination of these changes in a large Australian sporadic ALS cohort suggest a broader role in ALS. Furthermore, we observe that increased methylation age is a signature of ALS in older patients.

The DNA modification N6-methyl-2'-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction.

DNA modification is known to regulate experience-dependent gene expression. However, beyond cytosine methylation and its oxidated derivatives, very little is known about the functional importance of chemical modifications on other nucleobases in the brain. Here we report that in adult mice trained in fear extinction, the DNA modification N6-methyl-2'-deoxyadenosine (m6dA) accumulates along promoters and coding sequences in activated prefrontal cortical neurons. The deposition of m6dA is associated with increased genome-wide occupancy of the mammalian m6dA methyltransferase, N6amt1, and this correlates with extinction-induced gene expression. The accumulation of m6dA is associated with transcriptional activation at the brain-derived neurotrophic factor (Bdnf) P4 promoter, which is required for Bdnf exon IV messenger RNA expression and for the extinction of conditioned fear. These results expand the scope of DNA modifications in the adult brain and highlight changes in m6dA as an epigenetic mechanism associated with activity-induced gene expression and the formation of fear extinction memory.

Bioorthogonal Metabolic Labeling of Nascent RNA in Neurons Improves the Sensitivity of Transcriptome-Wide Profiling.

Transcriptome-wide expression profiling of neurons has provided important insights into the underlying molecular mechanisms and gene expression patterns that transpire during learning and memory formation. However, there is a paucity of tools for profiling stimulus-induced RNA within specific neuronal cell populations. A bioorthogonal method to chemically label nascent (i.e., newly transcribed) RNA in a cell-type-specific and temporally controlled manner, which is also amenable to bioconjugation via click chemistry, was recently developed and optimized within conventional immortalized cell lines. However, its value within a more fragile and complicated cellular system such as neurons, as well as for transcriptome-wide expression profiling, has yet to be demonstrated. Here, we report the visualization and sequencing of activity-dependent nascent RNA derived from neurons using this labeling method. This work has important implications for improving transcriptome-wide expression profiling and visualization of nascent RNA in neurons, which has the potential to provide valuable insights into the mechanisms underlying neural plasticity, learning, and memory.

A Functional Role for the Epigenetic Regulator ING1 in Activity-induced Gene Expression in Primary Cortical Neurons.

Epigenetic regulation of activity-induced gene expression involves multiple levels of molecular interaction, including histone and DNA modifications, as well as mechanisms of DNA repair. Here we demonstrate that the genome-wide deposition of inhibitor of growth family member 1 (ING1), which is a central epigenetic regulatory protein, is dynamically regulated in response to activity in primary cortical neurons. ING1 knockdown leads to decreased expression of genes related to synaptic plasticity, including the regulatory subunit of calcineurin, Ppp3r1. In addition, ING1 binding at a site upstream of the transcription start site (TSS) of Ppp3r1 depends on yet another group of neuroepigenetic regulatory proteins, the Piwi-like family, which are also involved in DNA repair. These findings provide new insight into a novel mode of activity-induced gene expression, which involves the interaction between different epigenetic regulatory mechanisms traditionally associated with gene repression and DNA repair.

Whole-exome sequencing in amyotrophic lateral sclerosis suggests NEK1 is a risk gene in Chinese.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease characterised by the degeneration of motor neurons, which are responsible for voluntary movement. There remains limited understanding of disease aetiology, with median survival of ALS of three years and no effective treatment. Identifying genes that contribute to ALS susceptibility is an important step towards understanding aetiology. The vast majority of published human genetic studies, including for ALS, have used samples of European ancestry. The importance of trans-ethnic studies in human genetic studies is widely recognised, yet a dearth of studies of non-European ancestries remains. Here, we report analyses of novel whole-exome sequencing (WES) data from Chinese ALS and control individuals. METHODS: WES data were generated for 610 ALS cases and 460 controls drawn from Chinese populations. We assessed evidence for an excess of rare damaging mutations at the gene level and the gene set level, considering only singleton variants filtered to have allele frequency less than 5 × 10-5 in reference databases. To meta-analyse our results with a published study of European ancestry, we used a Cochran-Mantel-Haenszel test to compare gene-level variant counts in cases vs controls. RESULTS: No gene passed the genome-wide significance threshold with ALS in Chinese samples alone. Combining rare variant counts in Chinese with those from the largest WES study of European ancestry resulted in three genes surpassing genome-wide significance: TBK1 (p = 8.3 × 10-12), SOD1 (p = 8.9 × 10-9) and NEK1 (p = 1.1 × 10-9). In the Chinese data alone, SOD1 and NEK1 were nominally significantly associated with ALS (p = 0.04 and p = 7 × 10-3, respectively) and the case/control frequencies of rare coding variants in these genes were similar in Chinese and Europeans (SOD1: 1.5%/0.2% vs 0.9%/0.1%, NEK1 1.8%/0.4% vs 1.9%/0.8%). This was also true for TBK1 (1.2%/0.2% vs 1.4%/0.4%), but the association with ALS in Chinese was not significant (p = 0.14). CONCLUSIONS: While SOD1 is already recognised as an ALS-associated gene in Chinese, we provide novel evidence for association of NEK1 with ALS in Chinese, reporting variants in these genes not previously found in Europeans.

Cross-ethnic meta-analysis identifies association of the GPX3-TNIP1 locus with amyotrophic lateral sclerosis.

Cross-ethnic genetic studies can leverage power from differences in disease epidemiology and population-specific genetic architecture. In particular, the differences in linkage disequilibrium and allele frequency patterns across ethnic groups may increase gene-mapping resolution. Here we use cross-ethnic genetic data in sporadic amyotrophic lateral sclerosis (ALS), an adult-onset, rapidly progressing neurodegenerative disease. We report analyses of novel genome-wide association study data of 1,234 ALS cases and 2,850 controls. We find a significant association of rs10463311 spanning GPX3-TNIP1 with ALS (p = 1.3 × 10-8), with replication support from two independent Australian samples (combined 576 cases and 683 controls, p = 1.7 × 10-3). Both GPX3 and TNIP1 interact with other known ALS genes (SOD1 and OPTN, respectively). In addition, GGNBP2 was identified using gene-based analysis and summary statistics-based Mendelian randomization analysis, although further replication is needed to confirm this result. Our results increase our understanding of genetic aetiology of ALS.Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease. Here, Wray and colleagues identify association of the GPX3-TNIP1 locus with ALS using cross-ethnic meta-analyses.