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Abstract Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.

Abstract Dynamic interactions of neurons and glia in the ventral midbrain mediate reward and addiction behavior. We studied gene expression in 212,713 ventral midbrain single nuclei from 95 individuals with history of opioid misuse, and individuals without drug exposure. Chronic exposure to opioids was not associated with change in proportions of glial and neuronal subtypes, however glial transcriptomes were broadly altered, involving 9.5 − 6.2% of expressed genes within microglia, oligodendrocytes, and astrocytes. Genes associated with activation of the immune response including interferon, NFkB signaling, and cell motility pathways were upregulated, contrasting with down-regulated expression of synaptic signaling and plasticity genes in ventral midbrain non-dopaminergic neurons. Ventral midbrain transcriptomic reprogramming in the context of chronic opioid exposure included 325 genes that previous genome-wide studies had linked to risk of substance use traits in the broader population, thereby pointing to heritable risk architectures in the genomic organization of the brain’s reward circuitry.

Knockout of Mir125b-2 in mice leads to defects in hippocampus-related behaviors and affects hippocampal expression of Grin2a and NMDAR-mediated currents.

Abstract The pathophysiology of epilepsy underlies a complex network dysfunction between neurons and glia, the molecular cell type-specific contributions of which remain poorly defined in the human disease. In this study, we validated a method that simultaneously isolates neuronal (NEUN +), astrocyte (PAX6 + NEUN–), and oligodendroglial progenitor (OPC) (OLIG2 + NEUN–) enriched nuclei populations from non-diseased, fresh-frozen human neocortex and then applied it to characterize the distinct transcriptomes of such populations isolated from electrode-mapped temporal lobe epilepsy (TLE) surgical samples. Nuclear RNA-seq confirmed cell type specificity and informed both common and distinct pathways associated with TLE in astrocytes, OPCs, and neurons. Compared to postmortem control, the transcriptome of epilepsy astrocytes showed downregulation of mature astrocyte functions and upregulation of development-related genes. To gain further insight into glial heterogeneity in TLE, we performed single cell transcriptomics (scRNA-seq) on four additional human TLE samples. Analysis of the integrated TLE dataset uncovered a prominent subpopulation of glia that express a hybrid signature of both reactive astrocyte and OPC markers, including many cells with a mixed GFAP + OLIG2 + phenotype. A further integrated analysis of this TLE scRNA-seq dataset and a previously published normal human temporal lobe scRNA-seq dataset confirmed the unique presence of hybrid glia only in TLE. Pseudotime analysis revealed cell transition trajectories stemming from this hybrid population towards both OPCs and reactive astrocytes. Immunofluorescence studies in human TLE samples confirmed the rare presence of GFAP + OLIG2 + glia, including some cells with proliferative activity, and functional analysis of cells isolated directly from these samples disclosed abnormal neurosphere formation in vitro. Overall, cell type-specific isolation of glia from surgical epilepsy samples combined with transcriptomic analyses uncovered abnormal glial subpopulations with de-differentiated phenotype, motivating further studies into the dysfunctional role of reactive glia in temporal lobe epilepsy.

Abstract Recent efforts have identified genetic loci that are associated with coronavirus disease 2019 (COVID-19) infection rates and disease outcome severity. Translating these genetic findings into druggable genes that reduce COVID-19 host susceptibility is a critical next step. Using a translational genomics approach that integrates COVID-19 genetic susceptibility variants, multi-tissue genetically regulated gene expression (GReX), and perturbagen signatures, we identified IL10RB as the top candidate gene target for COVID-19 host susceptibility. In a series of validation steps, we show that predicted GReX upregulation of IL10RB and higher IL10RB expression in COVID-19 patient blood is associated with worse COVID-19 outcomes and that in vitro IL10RB overexpression is associated with increased viral load and activation of disease-relevant molecular pathways.

Repeat-rich sequences play a role in 3D genome architecture in higher eukaryotes; however, this remains unexplored in brain cells. Here, the authors show that upregulation of endogenous retroviral (ERV) sequences is linked to changes in the 3D structure in the brain, which is also observed by comparison of mouse strains with recent retrotransposon expansion.

Abstract Background Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has been associated with neurological and neuropsychiatric illness in many individuals. We sought to further our understanding of the relationship between brain tropism, neuro-inflammation, and host immune response in acute COVID-19 cases. Methods Three brain regions (dorsolateral prefrontal cortex, medulla oblongata, and choroid plexus) from 5 patients with severe COVID-19 and 4 controls were examined. The presence of the virus was assessed by western blot against viral spike protein, as well as viral transcriptome analysis covering > 99% of SARS-CoV-2 genome and all potential serotypes. Droplet-based single-nucleus RNA sequencing (snRNA-seq) was performed in the same samples to examine the impact of COVID-19 on transcription in individual cells of the brain. Results Quantification of viral spike S1 protein and viral transcripts did not detect SARS-CoV-2 in the postmortem brain tissue. However, analysis of 68,557 single-nucleus transcriptomes from three distinct regions of the brain identified an increased proportion of stromal cells, monocytes, and macrophages in the choroid plexus of COVID-19 patients. Furthermore, differential gene expression, pseudo-temporal trajectory, and gene regulatory network analyses revealed transcriptional changes in the cortical microglia associated with a range of biological processes, including cellular activation, mobility, and phagocytosis. Conclusions Despite the absence of detectable SARS-CoV-2 in the brain at the time of death, the findings suggest significant and persistent neuroinflammation in patients with acute COVID-19.

The cellular heterogeneity in brain obscures the identification of robust cellular regulatory networks. Here the authors integrate genome-wide chromosome conformation data from sorted neurons and glia, with transcriptomic and enhancer profiles, to characterize cell-type-specific gene regulatory landscapes in the human brain, and provide insights into cell-type-specific gene regulatory networks in brain disorders.

Abstract Background Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Results Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Conclusions This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

Isolation during critical periods of development prevents development of normal social behaviours in mice, and this is thought to involve the prefrontal cortex. Here, the authors identify an activation pattern in parvalbumin-positive interneurons in the dorsal medial prefrontal cortex that when activated promotes sociability behaviours in mice.

Abstract Background Midbrain dopaminergic neurons (MDN) represent 0.0005% of the brain’s neuronal population and mediate cognition, food intake, and metabolism. MDN are also posited to underlay the neurobiological dysfunction of schizophrenia (SCZ), a severe neuropsychiatric disorder that is characterized by psychosis as well as multifactorial medical co-morbidities, including metabolic disease, contributing to markedly increased morbidity and mortality. Paradoxically, however, the genetic risk sequences of psychosis and traits associated with metabolic disease, such as body mass, show very limited overlap. Methods We investigated the genomic interaction of SCZ with medical conditions and traits, including body mass index (BMI), by exploring the MDN’s “spatial genome,” including chromosomal contact landscapes as a critical layer of cell type-specific epigenomic regulation. Low-input Hi-C protocols were applied to 5–10 × 103 dopaminergic and other cell-specific nuclei collected by fluorescence-activated nuclei sorting from the adult human midbrain. Results The Hi-C-reconstructed MDN spatial genome revealed 11 “Euclidean hot spots” of clustered chromatin domains harboring risk sequences for SCZ and elevated BMI. Inter- and intra-chromosomal contacts interconnecting SCZ and BMI risk sequences showed massive enrichment for brain-specific expression quantitative trait loci (eQTL), with gene ontologies, regulatory motifs and proteomic interactions related to adipogenesis and lipid regulation, dopaminergic neurogenesis and neuronal connectivity, and reward- and addiction-related pathways. Conclusions We uncovered shared nuclear topographies of cognitive and metabolic risk variants. More broadly, our PsychENCODE sponsored Hi-C study offers a novel genomic approach for the study of psychiatric and medical co-morbidities constrained by limited overlap of their respective genetic risk architectures on the linear genome.

Although many neuropsychiatric risk genes are known to contribute to epigenetic regulation of gene expression, very little is known about specific chromatin-associated mechanisms that govern the formation and maintenance of neuronal connectivity. Here, the authors report that transcallosal connectivity is critically dependent on C11orf46/ARL14EP, a nuclear protein encoded in the chromosome 11p13 WAGR risk locus, and that RNA-guided epigenetic editing of hyperexpressed Sema6a gene promoters in C11orf46-knockdown neurons resulted in normalization of expression and rescue of transcallosal dysconnectivity via repressive chromatin remodeling.

In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in young mice constantly receiving novel stimulation. We profiled epigenome and chromatin architecture in whole cortex and sorted neurons by deep-sequencing techniques. Specifically, we studied chromatin accessibility, gene and protein regulation, and 3D genome conformation, combined with predicted enhancer and chromatin interactions. We identified increased chromatin accessibility, transcription factor binding including CTCF-mediated insulation, differential occupancy of H3K36me3 and H3K79me2, and changes in transcriptional programs required for neuronal development. EE stimuli led to local genome re-organization by inducing increased contacts between chromosomes 7 and 17 (inter-chromosomal). Our findings support the notion that EE-induced learning and memory processes are directly associated with the epigenome and genome organization.

Social dominance hierarchies are a common adaptation to group living and exist across a broad range of the animal kingdom. Social dominance is known to rely on the prefrontal cortex (PFC), a brain region that shows a protracted developmental trajectory in mice. However, it is unknown to what extent the social dominance hierarchy is plastic across postnatal development and how it is regulated. Here we identified a sensitive period for experience-dependent social dominance plasticity in adolescent male mice, which is regulated by mechanisms that affect cortical plasticity. We show that social dominance hierarchies in male mice are already formed at weaning and are highly stable into adulthood. However, one experience of forced losing significantly reduces social dominance during the adolescent period but not in adulthood, suggesting adolescence as a sensitive period for experience-dependent social dominance plasticity. Notably, robust adolescent plasticity can be prolonged into adulthood by genetic deletion of Lynx1, a molecular brake that normally limits cortical plasticity through modulation of cortical nicotinic signaling. This plasticity is associated with increased activation of established nodes of the social dominance network including dorsal medial PFC and medial dorsal thalamus evidenced by increased c-Fos. Pharmacologically mediated elevation of cortical plasticity by valproic acid rapidly destabilizes the hierarchy of adult wildtype animals. These findings provide insight into mechanisms through which increased behavioral plasticity may be achieved to improve therapeutic recovery from psychiatric disorders that are associated with social deficits.

Chromatin accessibility may be altered in disease states. Here the authors carry out ATAC-seq on a large number of samples of dorsolateral prefrontal cortex from individuals with schizophrenia, and healthy controls.

Summary: Homeostatic synaptic downscaling reduces neuronal excitability by modulating the number of postsynaptic receptors. Histone modifications and the subsequent chromatin remodeling play critical roles in activity-dependent gene expression. Histone modification codes are recognized by chromatin readers that affect gene expression by altering chromatin structure. We show that L3mbtl1 (lethal 3 malignant brain tumor-like 1), a polycomb chromatin reader, is downregulated by neuronal activity and is essential for synaptic response and downscaling. Genome-scale mapping of L3mbtl1 occupancies identified Ctnnb1 as a key gene downstream of L3mbtl1. Importantly, the occupancy of L3mbtl1 on the Ctnnb1 gene was regulated by neuronal activity. L3mbtl1 knockout neurons exhibited reduced Ctnnb1 expression. Partial knockdown of Ctnnb1 in wild-type neurons reduced excitatory synaptic transmission and abolished homeostatic downscaling, and transfecting Ctnnb1 in L3mbtl1 knockout neurons enhanced synaptic transmission and restored homeostatic downscaling. These results highlight a role for L3mbtl1 in regulating homeostasis of synaptic efficacy. : Synaptic homeostasis is crucial for maintaining proper neuronal excitability and excitatory/inhibitory balance in the brain. Mao et al. report that an activity-dependent chromatin reader protein is required for homeostatic control of synaptic strength through the regulation of downstream target gene Ctnnb1. Keywords: homeostatic plasticity, synaptic scaling, chromatin reader, glutamate receptor, synaptic transmission, hippocampus, neuronal activity

Summary: Modulation of transcription, either synthetic activation or repression, via dCas9-fusion proteins is a relatively new methodology with the potential to facilitate high-throughput up- or downregulation studies of gene function. Genetic studies of neurodevelopmental disorders have identified a growing list of risk variants, including both common single-nucleotide variants and rare copy-number variations, many of which are associated with genes having limited functional annotations. By applying a CRISPR-mediated gene-activation/repression platform to populations of human-induced pluripotent stem cell-derived neural progenitor cells, neurons, and astrocytes, we demonstrate that it is possible to manipulate endogenous expression levels of candidate neuropsychiatric risk genes across these three cell types. Although proof-of-concept studies using catalytically inactive Cas9-fusion proteins to modulate transcription have been reported, here we present a detailed survey of the reproducibility of gRNA positional effects across a variety of neurodevelopmental disorder-relevant risk genes, donors, neural cell types, and dCas9 effectors. : Brennand and colleagues report a survey of the reproducibility of CRISPR-mediated transcriptional modulation across varied neurodevelopmental disorder risk genes, donors, neural cell types, and dCas9 effectors. We report a number of practical limitations that must be considered when designing hiPSC-based studies using this promising new tool. Keywords: CRISPR, human-induced pluripotent stem cell, neural progenitor cell, transcriptional modulation, dCas9-VP64, dCas9-VPR, dCas9-KRAB

Mutation of the human genePHF8, encoding a histone demethylase, is linked to cognitive defects but its role in development is unclear. Here, the authors show that Phf8deletion in mice causes no overt developmental defects but confers resilience to depression, likely through increased serotonin signalling.

Chromosome conformation is a dynamic process, especially in brain. Here, Mitchell and colleagues devise a method they call NeuroDam that can prospectively tag chromosome conformation in the mouse brain in vivo, and longitudinally assess long range chromosome looping weeks and months later.

Human-induced pluripotent stem cells (hiPSCs) have revolutionized our ability to model neuropsychiatric and neurodegenerative diseases, and recent progress in the field is paving the way for improved therapeutics. In this review, we discuss major advances in generating hiPSC-derived neural cells and cutting-edge techniques that are transforming hiPSC technology, such as three-dimensional “mini-brains” and clustered, regularly interspersed short palindromic repeats (CRISPR)-Cas systems. We examine specific examples of how hiPSC-derived neural cells are being used to uncover the pathophysiology of schizophrenia and Parkinson’s disease, and consider the future of this groundbreaking research.

A large portion of common variant loci associated with genetic risk for schizophrenia reside within noncoding sequence of unknown function. Here, we demonstrate promoter and enhancer enrichment in schizophrenia variants associated with expression quantitative trait loci (eQTL). The enrichment is greater when functional annotations derived from the human brain are used relative to peripheral tissues. Regulatory trait concordance analysis ranked genes within schizophrenia genome-wide significant loci for a potential functional role, based on colocalization of a risk SNP, eQTL, and regulatory element sequence. We identified potential physical interactions of noncontiguous proximal and distal regulatory elements. This was verified in prefrontal cortex and -induced pluripotent stem cell–derived neurons for the L-type calcium channel (CACNA1C) risk locus. Our findings point to a functional link between schizophrenia-associated noncoding SNPs and 3D genome architecture associated with chromosomal loopings and transcriptional regulation in the brain.

Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans.

[This corrects the article DOI: 10.1371/journal.pone.0143563.].

Cortical and subcortical dysfunction in schizophrenia includes altered expression of RNA and proteins involved in neurotransmission, metabolism, myelination and other functions. The molecular mechanisms underlying this type of alteration remain largely unknown. Here, we summarize findings from postmortem brain studies and argue that transcriptional dysregulation, including changes in DNA and histone modifications involved in epigenetic control of gene expression, as well as microRNA-mediated post-transcriptional mechanisms contribute to the neurobiology of schizophrenia.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion of CAG repeats in the Huntingtin (HTT) gene. Previous studies have shown mutant HTT can alter expression of genes associated with dysregulated epigenetic modifications. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls, and its association with gene expression measured by RNA-sequencing. We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down-regulated in HD. Although H3K4me3 signals are expected to be associated with mRNA levels, we found an unexpected discordance between altered H3K4me3 peaks and mRNA levels. Gene ontology (GO) term enrichment analysis of the genes with differential H3K4me3 peaks, revealed statistically significantly enriched GO terms only in the genes with down-regulated signals in HD. The most frequently implicated biological process terms are organ morphogenesis and positive regulation of gene expression. More than 9,000 H3K4me3 peaks were located not near any recognized transcription start sites and approximately 36% of these "distal" peaks co-localized to known enhancer sites. Six transcription factors and chromatin remodelers are differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, PRC2 repressive state was significantly depleted in HD-enriched peaks, suggesting the epigenetic role of PRC2 inhibition associated with up-regulated H3K4me3 in Huntington's disease. In summary, our study provides new insights into transcriptional dysregulation of Huntington's disease by analyzing the differentiation of H3K4me3 enrichment.

Alterations in histone lysine methylation and epigenetic regulators of gene expression could play a role in the neurobiology and treatment of patients diagnosed with mood spectrum disorder, including depression and anxiety. Mutations and altered expression of various lysine methyltransferases (KMTs) and demethylases (KDMs) have been linked to changes in motivational and emotional behaviors in preclinical model systems. However, it is not known whether regulators operating downstream of histone lysine methylation could affect mood-related behavior. Malignant Brain Tumor (MBT) domain 'chromatin reader' proteins bind to methylated histone lysine residues and associate with chromatin remodeling complexes to facilitate or repress gene expression. MBT proteins, including the founding member, L3mbtl1, maintain high levels of expression in neurons of the mature brain. Here, we exposed L3mbtl1 null mutant mice to a wide range of tests exploring cognition and mood-relevant behaviors at baseline and in the context of social isolation, as a stressor to elicit depression-related behavior in susceptible mice. L3mbtl1 loss-of-function was associated with significant decreases in depression and and anxiety in some of the behavioral paradigms. This was not associated with a more generalized neurological dysfunction because cognition and memory remained unaltered in comparison to controls. These findings warrant further investigations on the role of MBT chromatin reader proteins in the context of emotional and affective behaviors.

Huntington's Disease (HD) is a devastating neurodegenerative disorder that is caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT) gene. Transcriptional dysregulation in the human HD brain has been documented but is incompletely understood. Here we present a genome-wide analysis of mRNA expression in human prefrontal cortex from 20 HD and 49 neuropathologically normal controls using next generation high-throughput sequencing. Surprisingly, 19% (5,480) of the 28,087 confidently detected genes are differentially expressed (FDR

To explore the role of neurotrophin-3 (NT-3) during cerebral ischemia, NT-3-deficient brains were subjected to transient focal ischemia. Conditional mutant brains produced undetectable amounts of NT-3 mRNA, whereas the expression of the neurotrophin, BDNF, the NT-3 receptor, TrkC, and the nonselective, low-affinity neurotrophin receptor p75NTR, were comparable to wild-type. Baseline absolute blood flow, vascular and neuroanatomical features, as well as physiological measurements were also indistinguishable from wild-type. Interestingly, the absence of NT-3 led to a significantly decreased infarct volume 23 h after middle cerebral artery occlusion. Consistent with this, the addition of NT-3 to primary cortical cell cultures exacerbated neuronal death caused by oxygen-glucose deprivation. Coincubation with the oxygen free radical chelator, trolox, diminished potentiation of neuronal death. NT-3 also enhanced neuronal cell death and the production of reactive oxygen species caused by oxidative damage inducing agents. We conclude that endogenous NT-3 enhanced neuronal injury during acute stroke, possible by increasing oxygen-radical mediated cell death.

Transcriptional dysregulation has long been recognized as central to the pathogenesis of Huntington's disease (HD). MicroRNAs (miRNAs) represent a major system of post-transcriptional regulation, by either preventing translational initiation or by targeting transcripts for storage or for degradation. Using next-generation miRNA sequencing in prefrontal cortex (Brodmann Area 9) of twelve HD and nine controls, we identified five miRNAs (miR-10b-5p, miR-196a-5p, miR-196b-5p, miR-615-3p and miR-1247-5p) up-regulated in HD at genome-wide significance (FDR q-value

Dysfunction of the prefrontal cortex may contribute to the autistic features and mental retardation of Rett syndrome, a neuropsychiatric condition caused by mutations of the gene encoding methyl-CpG-binding protein 2 (MeCP2). Because nothing is known about the expression of MeCP2 and other chromatin-associated factors in primate brain, we studied in monkey prefrontal cortex and murine cerebral cortex expression patterns of MeCP2 and of macrohistone H2A (MacroH2A), which like MeCP2 is associated with transcriptionally silent chromatin. In both species, MeCP2 and MacroH2A appeared to be ubiquitously expressed by cortical neurons, including projection neurons and GABAergic interneurons. In the adult monkey, MeCP2 expression was robust throughout all layers of the prefrontal cortex but it was limited in fetal monkeys at embryonic day 110 to the deeper cortical layers and the subplate. These results suggest that MeCP2 may be important for neuronal maintenance in the developing and in the mature primate prefrontal cortex, consistent with the previously reported phenotype of MeCP2-null mutant mice.

Development of prefrontal and other higher-order association cortices is associated with widespread changes in the cortical transcriptome, particularly during the transitions from prenatal to postnatal development, and from early infancy to later stages of childhood and early adulthood. However, the timing and longitudinal trajectories of neuronal gene expression programs during these periods remain unclear in part because of confounding effects of concomitantly occurring shifts in neuron-to-glia ratios. Here, we used cell type-specific chromatin sorting techniques for genome-wide profiling of a histone mark associated with transcriptional regulation--H3 with trimethylated lysine 4 (H3K4me3)--in neuronal chromatin from 31 subjects from the late gestational period to 80 years of age. H3K4me3 landscapes of prefrontal neurons were developmentally regulated at 1,157 loci, including 768 loci that were proximal to transcription start sites. Multiple algorithms consistently revealed that the overwhelming majority and perhaps all of developmentally regulated H3K4me3 peaks were on a unidirectional trajectory defined by either rapid gain or loss of histone methylation during the late prenatal period and the first year after birth, followed by similar changes but with progressively slower kinetics during early and later childhood and only minimal changes later in life. Developmentally downregulated H3K4me3 peaks in prefrontal neurons were enriched for Paired box (Pax) and multiple Signal Transducer and Activator of Transcription (STAT) motifs, which are known to promote glial differentiation. In contrast, H3K4me3 peaks subject to a progressive increase in maturing prefrontal neurons were enriched for activating protein-1 (AP-1) recognition elements that are commonly associated with activity-dependent regulation of neuronal gene expression. We uncovered a developmental program governing the remodeling of neuronal histone methylation landscapes in the prefrontal cortex from the late prenatal period to early adolescence, which is linked to cis-regulatory sequences around transcription start sites.

Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans.

TAR DNA binding protein 43 KD (TDP-43) is an essential gene that regulates gene transcription, mRNA splicing and stability. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal neurodegenerative diseases, TDP-43 is fragmented, generating multiple fragments that include the C-terminal fragment of ∼25 KD. The role of these fragments in the pathogenesis of ALS and FTD is not clear. Here we investigated the aggregation propensity in various polypeptide regions of TDP-43 in mammalian cells and the effect of these fragments on cultured neurons. By expressing the full length and various TDP-43 fragments in motor neuron-derived NSC-34 cells and primary neurons, we found that both N- and C-terminal fragments of TDP-43 are prone to aggregate and the C-terminal end of RRM2 region is required, though not sufficient, for aggregation. The aggregation of the TDP-43 fragments can drive co-aggregation with the full-length TDP-43, consequently reducing the nuclear TDP-43. In addition, the TDP-43 fragments can impair neurite growth during neuronal differentiation. Importantly, overexpression of the full-length TDP-43 rescues the neurite growth phenotype whereas knockdown of the endogenous TDP-43 reproduces this phenotype. These results suggest that TDP-43 fragments, particularly the pathologically relevant C-terminal fragments, can impair neuronal differentiation by dominant-negatively interfering with the function of the full length TDP-43, thus playing a role in pathogenesis in ALS and FTD.

Dysfunction of prefrontal cortex in schizophrenia includes changes in GABAergic mRNAs, including decreased expression of GAD1, encoding the 67 kDa glutamate decarboxylase (GAD67) GABA synthesis enzyme. The underlying molecular mechanisms remain unclear. Alterations in DNA methylation as an epigenetic regulator of gene expression are thought to play a role but this hypothesis is difficult to test because no techniques are available to extract DNA from GAD1 expressing neurons efficiently from human postmortem brain. Here, we present an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of potential gene expression as opposed to repressive or silenced chromatin. Methylation patterns of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined for each of the two chromatin fractions separately, using a case-control design for 14 schizophrenia subjects affected by a decrease in prefrontal GAD1 mRNA levels. In controls, the methylation frequencies at CpG dinucleotides, while overall higher in repressive as compared to open chromatin, did not exceed 5% at the proximal GAD1 promoter and 30% within intron 2. Subjects with schizophrenia showed a significant, on average 8-fold deficit in repressive chromatin-associated DNA methylation at the promoter. These results suggest that chromatin remodeling mechanisms are involved in dysregulated GABAergic gene expression in schizophrenia.

The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5' CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts--defined by chronic neurodegeneration (Alzheimer's) or lack thereof (schizophrenia)--were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK) typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase.