Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation.

A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases.

Chromatin domain alterations linked to 3D genome organization in a large cohort of schizophrenia and bipolar disorder brains.

Chromosomal organization, scaling from the 147-base pair (bp) nucleosome to megabase-ranging domains encompassing multiple transcriptional units, including heritability loci for psychiatric traits, remains largely unexplored in the human brain. In this study, we constructed promoter- and enhancer-enriched nucleosomal histone modification landscapes for adult prefrontal cortex from H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from 388 controls and 351 individuals diagnosed with schizophrenia (SCZ) or bipolar disorder (BD) (n = 739). We mapped thousands of cis-regulatory domains (CRDs), revealing fine-grained, 104-106-bp chromosomal organization, firmly integrated into Hi-C topologically associating domain stratification by open/repressive chromosomal environments and nuclear topography. Large clusters of hyper-acetylated CRDs were enriched for SCZ heritability, with prominent representation of regulatory sequences governing fetal development and glutamatergic neuron signaling. Therefore, SCZ and BD brains show coordinated dysregulation of risk-associated regulatory sequences assembled into kilobase- to megabase-scaling chromosomal domains.

Neuronal and glial 3D chromatin architecture informs the cellular etiology of brain disorders.

Cellular heterogeneity in the human brain obscures the identification of robust cellular regulatory networks, which is necessary to understand the function of non-coding elements and the impact of non-coding genetic variation. Here we integrate genome-wide chromosome conformation data from purified neurons and glia with transcriptomic and enhancer profiles, to characterize the gene regulatory landscape of two major cell classes in the human brain. We then leverage cell-type-specific regulatory landscapes to gain insight into the cellular etiology of several brain disorders. We find that Alzheimer's disease (AD)-associated epigenetic dysregulation is linked to neurons and oligodendrocytes, whereas genetic risk factors for AD highlighted microglia, suggesting that different cell types may contribute to disease risk, via different mechanisms. Moreover, integration of glutamatergic and GABAergic regulatory maps with genetic risk factors for schizophrenia (SCZ) and bipolar disorder (BD) identifies shared (parvalbumin-expressing interneurons) and distinct cellular etiologies (upper layer neurons for BD, and deeper layer projection neurons for SCZ). Collectively, these findings shed new light on cell-type-specific gene regulatory networks in brain disorders.

Common schizophrenia risk variants are enriched in open chromatin regions of human glutamatergic neurons.

The chromatin landscape of human brain cells encompasses key information to understanding brain function. Here we use ATAC-seq to profile the chromatin structure in four distinct populations of cells (glutamatergic neurons, GABAergic neurons, oligodendrocytes, and microglia/astrocytes) from three different brain regions (anterior cingulate cortex, dorsolateral prefrontal cortex, and primary visual cortex) in human postmortem brain samples. We find that chromatin accessibility varies greatly by cell type and, more moderately, by brain region, with glutamatergic neurons showing the largest regional variability. Transcription factor footprinting implicates cell-specific transcriptional regulators and infers cell-specific regulation of protein-coding genes, long intergenic noncoding RNAs and microRNAs. In vivo transgenic mouse experiments validate the cell type specificity of several of these human-derived regulatory sequences. We find that open chromatin regions in glutamatergic neurons are enriched for neuropsychiatric risk variants, particularly those associated with schizophrenia. Integration of cell-specific chromatin data with a bulk tissue study of schizophrenia brains increases statistical power and confirms that glutamatergic neurons are most affected. These findings illustrate the utility of studying the cell-type-specific epigenome in complex tissues like the human brain, and the potential of such approaches to better understand the genetic basis of human brain function.

Evolution of regulatory signatures in primate cortical neurons at cell-type resolution.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Molecular windows into the human brain for psychiatric disorders.

Delineating the pathophysiology of psychiatric disorders has been extremely challenging but technological advances in recent decades have facilitated a deeper interrogation of molecular processes in the human brain. Initial candidate gene expression studies of the postmortem brain have evolved into genome wide profiling of the transcriptome and the epigenome, a critical regulator of gene expression. Here, we review the potential and challenges of direct molecular characterization of the postmortem human brain, and provide a brief overview of recent transcriptional and epigenetic studies with respect to neuropsychiatric disorders. Such information can now be leveraged and integrated with the growing number of genome-wide association databases to provide a functional context of trait-associated genetic variants linked to psychiatric illnesses and related phenotypes. While it is clear that the field is still developing and challenges remain to be surmounted, these recent advances nevertheless hold tremendous promise for delineating the neurobiological underpinnings of mental diseases and accelerating the development of novel medication strategies.

A unique role for DNA (hydroxy)methylation in epigenetic regulation of human inhibitory neurons.

Brain function depends on interaction of diverse cell types whose gene expression and identity are defined, in part, by epigenetic mechanisms. Neuronal DNA contains two major epigenetic modifications, methylcytosine (mC) and hydroxymethylcytosine (hmC), yet their cell type-specific landscapes and relationship with gene expression are poorly understood. We report high-resolution (h)mC analyses, together with transcriptome and histone modification profiling, in three major cell types in human prefrontal cortex: glutamatergic excitatory neurons, medial ganglionic eminence-derived γ-aminobutyric acid (GABA)ergic inhibitory neurons, and oligodendrocytes. We detected a unique association between hmC and gene expression in inhibitory neurons that differed significantly from the pattern in excitatory neurons and oligodendrocytes. We also found that risk loci associated with neuropsychiatric diseases were enriched near regions of reduced hmC in excitatory neurons and reduced mC in inhibitory neurons. Our findings indicate differential roles for mC and hmC in regulation of gene expression in different brain cell types, with implications for the etiology of human brain diseases.

Brain Cell Type Specific Gene Expression and Co-expression Network Architectures.

Elucidating brain cell type specific gene expression patterns is critical towards a better understanding of how cell-cell communications may influence brain functions and dysfunctions. We set out to compare and contrast five human and murine cell type-specific transcriptome-wide RNA expression data sets that were generated within the past several years. We defined three measures of brain cell type-relative expression including specificity, enrichment, and absolute expression and identified corresponding consensus brain cell "signatures," which were well conserved across data sets. We validated that the relative expression of top cell type markers are associated with proxies for cell type proportions in bulk RNA expression data from postmortem human brain samples. We further validated novel marker genes using an orthogonal ATAC-seq dataset. We performed multiscale coexpression network analysis of the single cell data sets and identified robust cell-specific gene modules. To facilitate the use of the cell type-specific genes for cell type proportion estimation and deconvolution from bulk brain gene expression data, we developed an R package, BRETIGEA. In summary, we identified a set of novel brain cell consensus signatures and robust networks from the integration of multiple datasets and therefore transcend limitations related to technical issues characteristic of each individual study.

A Novel Method for Rapid Molecular Subgrouping of Medulloblastoma.

Purpose: The classification of medulloblastoma into WNT, SHH, group 3, and group 4 subgroups has become of critical importance for patient risk stratification and subgroup-tailored clinical trials. Here, we aimed to develop a simplified, clinically applicable classification approach that can be implemented in the majority of centers treating patients with medulloblastoma.Experimental Design: We analyzed 1,577 samples comprising previously published DNA methylation microarray data (913 medulloblastomas, 457 non-medulloblastoma tumors, 85 normal tissues), and 122 frozen and formalin-fixed paraffin-embedded medulloblastoma samples. Biomarkers were identified applying stringent selection filters and Linear Discriminant Analysis (LDA) method, and validated using DNA methylation microarray data, bisulfite pyrosequencing, and direct-bisulfite sequencing.Results: Using a LDA-based approach, we developed and validated a prediction method (EpiWNT-SHH classifier) based on six epigenetic biomarkers that allowed for rapid classification of medulloblastoma into the clinically relevant subgroups WNT, SHH, and non-WNT/non-SHH with excellent concordance (>99%) with current gold-standard methods, DNA methylation microarray, and gene signature profiling analysis. The EpiWNT-SHH classifier showed high prediction capacity using both frozen and formalin-fixed material, as well as diverse DNA methylation detection methods. Similarly, we developed a classifier specific for group 3 and group 4 tumors, based on five biomarkers (EpiG3-G4) with good discriminatory capacity, allowing for correct assignment of more than 92% of tumors. EpiWNT-SHH and EpiG3-G4 methylation profiles remained stable across tumor primary, metastasis, and relapse samples.Conclusions: The EpiWNT-SHH and EpiG3-G4 classifiers represent a new simplified approach for accurate, rapid, and cost-effective molecular classification of single medulloblastoma DNA samples, using clinically applicable DNA methylation detection methods. Clin Cancer Res; 24(6); 1355-63. ©2018 AACR.

Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects.

Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT2A-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment.

DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age.

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.

Genetic Tracing of Cav3.2 T-Type Calcium Channel Expression in the Peripheral Nervous System.

Characterizing the distinct functions of the T-type ion channel subunits Cav3.1, 3.2 or 3.3 has proven difficult due to their highly conserved amino-acid sequences and the lack of pharmacological blockers specific for each subunit. To precisely determine the expression pattern of the Cav3.2 channel in the nervous system we generated two knock-in mouse strains that express EGFP or Cre recombinase under the control of the Cav3.2 gene promoter. We show that in the brains of these animals, the Cav3.2 channel is predominantly expressed in the dentate gyrus of the hippocampus. In the peripheral nervous system, the activation of the promoter starts at E9.5 in neural crest cells that will give rise to dorsal root ganglia (DRG) neurons, but not sympathetic neurons. As development progresses the number of DRG cells expressing the Cav3.2 channel reaches around 7% of the DRG at E16.5, and remains constant until E18.5. Characterization of sensory neuron subpopulations at E18.5 showed that EGFP+ cells are a heterogeneous population consisting mainly of TrkB+ and TrkC+ cells, while only a small percentage of DRG cells were TrkA+. Genetic tracing of the sensory nerve end-organ innervation of the skin showed that the activity of the Cav3.2 channel promoter in sensory progenitors marks many mechanoreceptor and nociceptor endings, but spares slowly adapting mechanoreceptors with endings associated with Merkel cells. Our genetic analysis reveals for the first time that progenitors that express the Cav3.2 T-type calcium channel, defines a sensory specific lineage that populates a large proportion of the DRG. Using our Cav3.2-Cre mice together with AAV viruses containing a conditional fluorescent reporter (tdTomato) we could also show that Cre expression is largely restricted to two functionally distinct sensory neuron types in the adult ganglia. Cav3.2 positive neurons innervating the skin were found to only form lanceolate endings on hair follicles and are probably identical to D-hair receptors. A second population of nociceptive sensory neurons expressing the Cav3.2 gene was found to be positive for the calcitonin-gene related peptide but these neurons are deep tissue nociceptors that do not innervate the skin.

Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum.

DNA methylation (DNAm) levels lend themselves for defining an epigenetic biomarker of aging known as the 'epigenetic clock'. Our genome-wide association study (GWAS) of cerebellar epigenetic age acceleration identifies five significant (P<5.0 × 10(-8)) SNPs in two loci: 2p22.1 (inside gene DHX57) and 16p13.3 near gene MLST8 (a subunit of mTOR complex 1 and 2). We find that the SNP in 16p13.3 has a cis-acting effect on the expression levels of MLST8 (P=6.9 × 10(-18)) in most brain regions. In cerebellar samples, the SNP in 2p22.1 has a cis-effect on DHX57 (P=4.4 × 10(-5)). Gene sets found by our GWAS analysis of cerebellar age acceleration exhibit significant overlap with those of Alzheimer's disease (P=4.4 × 10(-15)), age-related macular degeneration (P=6.4 × 10(-6)), and Parkinson's disease (P=2.6 × 10(-4)). Overall, our results demonstrate the utility of a new paradigm for understanding aging and age-related diseases: it will be fruitful to use epigenetic tissue age as endophenotype in GWAS.

NeuN+ neuronal nuclei in non-human primate prefrontal cortex and subcortical white matter after clozapine exposure.

Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved. We exposed 26 young adult macaque monkeys for 6 months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts. After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups. Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted.

Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.

Substantial DNA methylation differences between two major neuronal subtypes in human brain.

The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons--GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.

Decrease of mRNA Editing after Spinal Cord Injury is Caused by Down-regulation of ADAR2 that is Triggered by Inflammatory Response.

We recently showed that spinal cord injury (SCI) leads to a decrease in mRNA editing of serotonin receptor 2C (5-HT2CR) contributing to post-SCI spasticity. Here we study post-SCI mRNA editing and global gene expression using massively parallel sequencing. Evidence is presented that the decrease in 5-HT2CR editing is caused by down-regulation of adenosine deaminase ADAR2 and that editing of at least one other ADAR2 target, potassium channel Kv1.1, is decreased after SCI. Bayesian network analysis of genome-wide transcriptome data indicates that down-regulation of ADAR2 (1) is triggered by persistent inflammatory response to SCI that is associated with activation of microglia and (2) results in changes in neuronal gene expression that are likely to contribute both to post-SCI restoration of neuronal excitability and muscle spasms. These findings have broad implications for other diseases of the Central Nervous System and could open new avenues for developing efficacious antispastic treatments.