There is growing evidence for the role of DNA methylation (DNAm) quantitative trait loci (mQTLs) in the genetics of complex traits, including psychiatric disorders. However, due to extensive linkage disequilibrium (LD) of the genome, it is challenging to identify causal genetic variations that drive DNAm levels by population-based genetic association studies. This limits the utility of mQTLs for fine-mapping risk loci underlying psychiatric disorders identified by genome-wide association studies (GWAS). Here we present INTERACT, a deep learning model that integrates convolutional neural networks with transformer, to predict effects of genetic variations on DNAm levels at CpG sites in the human brain. We show that INTERACT-derived DNAm regulatory variants are not confounded by LD, are concentrated in regulatory genomic regions in the human brain, and are convergent with mQTL evidence from genetic association analysis. We further demonstrate that predicted DNAm regulatory variants are enriched for heritability of brain-related traits and improve polygenic risk prediction for schizophrenia across diverse ancestry samples. Finally, we applied predicted DNAm regulatory variants for fine-mapping schizophrenia GWAS risk loci to identify potential novel risk genes. Our study shows the power of a deep learning approach to identify functional regulatory variants that may elucidate the genetic basis of complex traits.

Metabotropic glutamate receptor 3 (mGlu3, mGluR3), encoded by GRM3, is a risk gene for schizophrenia and a therapeutic target. It is unclear whether expression of the receptor is altered in the disorder or related to GRM3 risk genotype. Antibodies used to date to assess mGlu3 in schizophrenia have not been well validated.

Organized neuronal firing is crucial for cortical processing and is disrupted in schizophrenia. Using rapid amplification of 5' complementary DNA ends in human brain, we identified a primate-specific isoform (3.1) of the ether-a-go-go-related K(+) channel KCNH2 that modulates neuronal firing. KCNH2-3.1 messenger RNA levels are comparable to full-length KCNH2 (1A) levels in brain but three orders of magnitude lower in heart. In hippocampus from individuals with schizophrenia, KCNH2-3.1 expression is 2.5-fold greater than KCNH2-1A expression. A meta-analysis of five clinical data sets (367 families, 1,158 unrelated cases and 1,704 controls) shows association of single nucleotide polymorphisms in KCNH2 with schizophrenia. Risk-associated alleles predict lower intelligence quotient scores and speed of cognitive processing, altered memory-linked functional magnetic resonance imaging signals and increased KCNH2-3.1 mRNA levels in postmortem hippocampus. KCNH2-3.1 lacks a domain that is crucial for slow channel deactivation. Overexpression of KCNH2-3.1 in primary cortical neurons induces a rapidly deactivating K(+) current and a high-frequency, nonadapting firing pattern. These results identify a previously undescribed KCNH2 channel isoform involved in cortical physiology, cognition and psychosis, providing a potential new therapeutic drug target.

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.

RNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases.

Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iμ and acceptor IH exons, located 5' upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iμ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called "immune privileged" tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

Beyond being one the most widely used psychoactive drugs in the world, cannabis has been identified as an environmental risk factor for psychosis. Though the relationship between cannabis use and psychiatric disorders remains controversial, consistent association between early adolescent cannabis use and the subsequent risk of psychosis suggested adolescence may be a particularly vulnerable period. Previous findings on gene by environment interactions indicated that cannabis use may only increase the risk for psychosis in the subjects who have a specific genetic vulnerability. The type 1 cannabinoid receptor (CB1), encoded by the CNR1 gene, is a key component of the endocannabinoid system. As the primary endocannabinoid receptor in the brain, CB1 is the main molecular target of the endocannabinoid ligand, as well as tetrahydrocannabinol (THC), the principal psychoactive ingredient of cannabis. In this study, we have examined mRNA expression and DNA methylation of CNR1 in human prefrontal cortex (PFC), hippocampus, and caudate samples. The expression of CNR1 is higher in fetal PFC and hippocampus, then drops down dramatically after birth. The lifespan trajectory of CNR1 expression in the DLPFC differentially correlated with age by allelic variation at rs4680, a functional polymorphism in the COMT gene. Compared with COMT methionine158 carriers, Caucasian carriers of the COMT valine158 allele have a stronger negative correlation between the expression of CNR1 in DLPFC and age. In contrast, the methylation level of cg02498983, which is negatively correlated with the expression of CNR1 in PFC, showed the strongest positive correlation with age in PFC of Caucasian carriers of COMT valine158. Additionally, we have observed decreased mRNA expression of CNR1 in the DLPFC of patients with schizophrenia. Further analysis revealed a positive eQTL SNP, rs806368, which predicted the expression of a novel transcript of CNR1 in human DLPFC, hippocampus and caudate. This SNP has been associated with addiction and other psychiatric disorders. THC or ethanol are each significantly associated with dysregulated expression of CNR1 in the PFC of patients with affective disorder, and the expression of CNR1 is significantly upregulated in the PFC of schizophrenia patients who completed suicide. Our results support previous studies that have implicated the endocannabinoid system in the pathology of schizophrenia and provided additional insight into the mechanism of increasing risk for schizophrenia in the adolescent cannabis users.

We used the 10x Genomics Visium platform to define the spatial topography of gene expression in the six-layered human dorsolateral prefrontal cortex. We identified extensive layer-enriched expression signatures and refined associations to previous laminar markers. We overlaid our laminar expression signatures on large-scale single nucleus RNA-sequencing data, enhancing spatial annotation of expression-driven clusters. By integrating neuropsychiatric disorder gene sets, we showed differential layer-enriched expression of genes associated with schizophrenia and autism spectrum disorder, highlighting the clinical relevance of spatially defined expression. We then developed a data-driven framework to define unsupervised clusters in spatial transcriptomics data, which can be applied to other tissues or brain regions in which morphological architecture is not as well defined as cortical laminae. Last, we created a web application for the scientific community to explore these raw and summarized data to augment ongoing neuroscience and spatial transcriptomics research ( http://research.libd.org/spatialLIBD ).

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based "epigenetic age" of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.

The molecular diversity of glia in the human hippocampus and their temporal dynamics over the lifespan remain largely unknown. Here, we performed single-nucleus RNA sequencing to generate a transcriptome atlas of the human hippocampus across the postnatal lifespan. Detailed analyses of astrocytes, oligodendrocyte lineages, and microglia identified subpopulations with distinct molecular signatures and revealed their association with specific physiological functions, age-dependent changes in abundance, and disease relevance. We further characterized spatiotemporal heterogeneity of GFAP-enriched astrocyte subpopulations in the hippocampal formation using immunohistology. Leveraging glial subpopulation classifications as a reference map, we revealed the diversity of glia differentiated from human pluripotent stem cells and identified dysregulated genes and pathological processes in specific glial subpopulations in Alzheimer's disease (AD). Together, our study significantly extends our understanding of human glial diversity, population dynamics across the postnatal lifespan, and dysregulation in AD and provides a reference atlas for stem-cell-based glial differentiation.

The presence of proteinopathy, the accumulation of specific proteins as aggregates in neurons, is an emerging aspect of the pathology of schizophrenia and other major mental illnesses. Among the initial proteins implicated in forming such aggregates in these conditions is Trio and F-actin Binding Protein isoform 1 (TRIOBP-1), a ubiquitously expressed protein involved in the stabilization of the actin cytoskeleton. Here we investigate the insolubility of TRIOBP-1, as an indicator of aggregation, in brain samples from 25 schizophrenia patients, 25 major depressive disorder patients and 50 control individuals (anterior cingulate cortex, BA23). Strikingly, insoluble TRIOBP-1 is considerably more prevalent in both of these conditions than in controls, further implicating TRIOBP-1 aggregation in schizophrenia and indicating a role in major depressive disorder. These results were only seen using a high stringency insolubility assay (previously used to study DISC1 and other proteins), but not a lower stringency assay that would be expected to also detect functional, actin-bound TRIOBP-1. Previously, we have also determined that a region of 25 amino acids in the center of this protein is critical for its ability to form aggregates. Here we attempt to refine this further, through the expression of various truncated mutant TRIOBP-1 vectors in neuroblastoma cells and examining their aggregation. In this way, it was possible to narrow down the aggregation-critical region of TRIOBP-1 to just 8 amino acids (333-340 of the 652 amino acid-long TRIOBP-1). Surprisingly our results suggested that a second section of TRIOBP-1 is also capable of independently inducing aggregation: the optionally expressed 59 amino acids at the extreme N-terminus of the protein. As a result, the 597 amino acid long version of TRIOBP-1 (also referred to as "Tara" or "TAP68") has reduced potential to form aggregates. The presence of insoluble TRIOBP-1 in brain samples from patients, combined with insight into the mechanism of aggregation of TRIOBP-1 and generation of an aggregation-resistant mutant TRIOBP-1 that lacks both these regions, will be of significant use in further investigating the mechanism and consequences of TRIOBP-1 aggregation in major mental illness.

The molecular organization of the human neocortex has been historically studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally-defined spatial domains that move beyond classic cytoarchitecture. Here we used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex (DLPFC). Integration with paired single nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we map the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains. Finally, we provide resources for the scientific community to explore these integrated spatial and single cell datasets at research.libd.org/spatialDLPFC/ .

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and adult onset are unknown. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due, in part, to a partial loss of TAF1 function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from three XDP female carrier individuals for X chromosome inactivation status and identified clonal lines that express either the wild-type X or XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression in neurotypical humans, and found that SVA-F expression decreases after 30 years of age in the brain and that TAF1 is decreased in most female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dimorphic and decreased after adolescence. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum.Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.

Smoking is a leading cause of preventable morbidity and mortality. Smoking is heritable, and genome-wide association studies (GWAS) of smoking behaviors have identified hundreds of significant loci. Most GWAS-identified variants are noncoding with unknown neurobiological effects. We used genome-wide genotype, DNA methylation, and RNA sequencing data in postmortem human nucleus accumbens (NAc) to identify cis-methylation/expression quantitative trait loci (meQTLs/eQTLs), investigate variant-by-cigarette smoking interactions across the genome, and overlay QTL evidence at smoking GWAS-identified loci to evaluate their regulatory potential. Active smokers (N=52) and nonsmokers (N=171) were defined based on cotinine biomarker levels and next-of-kin reporting. We simultaneously tested variant and variant-by-smoking interaction effects on methylation and expression, separately, adjusting for biological and technical covariates and using a two-stage multiple testing approach with eigenMT and Bonferroni corrections. We found >2 million significant meQTL variants (padj<0.05) corresponding to 41,695 unique CpGs. Results were largely driven by main effects; five meQTLs, mapping to NUDT12, FAM53B, RNF39, and ADRA1B, showed a significant interaction with smoking. We found 57,683 significant eQTLs for 958 unique eGenes (padj<0.05) and no smoking interactions. Colocalization analyses identified loci with smoking-associated GWAS variants that overlapped meQTLs/eQTLs, suggesting that these heritable factors may influence smoking behaviors through functional effects on methylation/expression. One locus containing MUSTIN1 and ITIH4 colocalized across all data types (GWAS + meQTL + eQTL). In this first genome-wide meQTL map in the human NAc, the enriched overlap with smoking GWAS-identified genetic loci provides evidence that gene regulation in the brain helps explain the neurobiology of smoking behaviors.

Alcohol use disorder (AUD) has a profound public health impact. However, understanding of the molecular mechanisms underlying the development and progression of AUD remain limited. Here, we interrogate AUD-associated DNA methylation (DNAm) changes within and across addiction-relevant brain regions: the nucleus accumbens (NAc) and dorsolateral prefrontal cortex (DLPFC).

Cigarette smoking during pregnancy is a major public health concern. While there are well-described consequences in early child development, there is very little known about the effects of maternal smoking on human cortical biology during prenatal life. We therefore performed a genome-wide differential gene expression analysis using RNA sequencing (RNA-seq) on prenatal (N = 33; 16 smoking-exposed) as well as adult (N = 207; 57 active smokers) human postmortem prefrontal cortices. Smoking exposure during the prenatal period was directly associated with differential expression of 14 genes; in contrast, during adulthood, despite a much larger sample size, only two genes showed significant differential expression (FDR < 10%). Moreover, 1,315 genes showed significantly different exposure effects between maternal smoking during pregnancy and direct exposure in adulthood (FDR < 10%)-these differences were largely driven by prenatal differences that were enriched for pathways previously implicated in addiction and synaptic function. Furthermore, prenatal and age-dependent differentially expressed genes were enriched for genes implicated in non-syndromic autism spectrum disorder (ASD) and were differentially expressed as a set between patients with ASD and controls in postmortem cortical regions. These results underscore the enhanced sensitivity to the biological effect of smoking exposure in the developing brain and offer insight into how maternal smoking during pregnancy affects gene expression in the prenatal human cortex. They also begin to address the relationship between in utero exposure to smoking and the heightened risks for the subsequent development of neuropsychiatric disorders.

Epigenome-wide association studies of human disease and other quantitative traits are becoming increasingly common. A series of papers reporting age-related changes in DNA methylation profiles in peripheral blood have already been published. However, blood is a heterogeneous collection of different cell types, each with a very different DNA methylation profile.

DNA methylation levels change with age. Recent studies have identified biomarkers of chronological age based on DNA methylation levels. It is not yet known whether DNA methylation age captures aspects of biological age.