Co-expression of PADI isoforms during progenitor differentiation enables functional diversity.

Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted Padi3α and Padi3ß isoforms in hair follicles and describe expression of Padi2ß, a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. Padi2ß transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2ß. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao et al. 2019 Cell Rep. 27, 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2ß inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize Padi3α and Padi3ß to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed in vitro, PADI3α and PADI3ß are enriched in the cytoplasm and precipitate together. Whereas PADI3ß protein stability is low and PADI3ß fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3ß. We propose that PADI3ß modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of Padi2 and Padi3 isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

A human in vitro neuronal model for studying homeostatic plasticity at the network level.

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.

Epigenome-wide DNA methylation in externalizing behaviours: A review and combined analysis.

DNA methylation (DNAm) is one of the most frequently studied epigenetic mechanisms facilitating the interplay of genomic and environmental factors, which can contribute to externalizing behaviours and related psychiatric disorders. Previous epigenome-wide association studies (EWAS) for externalizing behaviours have been limited in sample size, and, therefore, candidate genes and biomarkers with robust evidence are still lacking. We 1) performed a systematic literature review of EWAS of attention-deficit/hyperactivity disorder (ADHD)- and aggression-related behaviours conducted in peripheral tissue and cord blood and 2) combined the most strongly associated DNAm sites observed in individual studies (p < 10-3) to identify candidate genes and biological systems for ADHD and aggressive behaviours. We observed enrichment for neuronal processes and neuronal cell marker genes for ADHD. Astrocyte and granulocytes cell markers among genes annotated to DNAm sites were relevant for both ADHD and aggression-related behaviours. Only 1 % of the most significant epigenetic findings for ADHD/ADHD symptoms were likely to be directly explained by genetic factors involved in ADHD. Finally, we discuss how the field would greatly benefit from larger sample sizes and harmonization of assessment instruments.

Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

Opposite effects of stress on effortful motivation in high and low anxiety are mediated by CRHR1 in the VTA.

Individuals frequently differ in their behavioral and cognitive responses to stress. However, whether motivation is differently affected by acute stress in different individuals remains to be established. By exploiting natural variation in trait anxiety in outbred Wistar rats, we show that acute stress facilitates effort-related motivation in low anxious animals, while dampening effort in high anxious ones. This model allowed us to address the mechanisms underlying acute stress-induced differences in motivated behavior. We show that CRHR1 expression levels in dopamine neurons of the ventral tegmental area (VTA)-a neuronal type implicated in the regulation of motivation-depend on animals' anxiety, and these differences in CRHR1 expression levels explain the divergent effects of stress on both effortful behavior and the functioning of mesolimbic DA neurons. These findings highlight CRHR1 in VTA DA neurons-whose levels vary with individuals' anxiety-as a switching mechanism determining whether acute stress facilitates or dampens motivation.

Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility.

Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-γ) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-γ leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological-based therapies for MS.

Stress-induced depressive-like behavior in male rats is associated with microglial activation and inflammation dysregulation in the hippocampus in adulthood.

Neuroinflammation is increasingly recognized as playing a critical role in depression. Early-life stress exposure and constitutive differences in glucocorticoid responsiveness to stressors are two key risk factors for depression, but their impacts on the inflammatory status of the brain is still uncertain. Moreover, there is a need to identify specific molecules involved in these processes with the potential to be used as alternative therapeutic targets in inflammation-related depression. Here, we studied how peripubertal stress (PPS) combined with differential corticosterone (CORT)-stress responsiveness (CSR) influences depressive-like behaviors and brain inflammatory markers in male rats in adulthood, and how these alterations relate to microglia activation and miR-342 expression. We found that high-CORT stress-responsive (H-CSR) male rats that underwent PPS exhibited increased anhedonia and passive coping responses in adulthood. Also, animals exposed to PPS showed increased hippocampal TNF-α expression, which positively correlated with passive coping responses. In addition, PPS caused long-term effects on hippocampal microglia, particularly in H-CSR rats, with increased hippocampal IBA-1 expression and morphological alterations compatible with a higher degree of activation. H-CSR animals also showed upregulation of hippocampal miR-342, a mediator of TNF-α-driven microglial activation, and its expression was positively correlated with TNF-α expression, microglial activation and passive coping responses. Our findings indicate that individuals with constitutive H-CSR are particularly sensitive to developing protracted depression-like behaviors following PPS exposure. In addition, they show neuro-immunological alterations in adulthood, such as increased hippocampal TNF-α expression, microglial activation and miR-342 expression. Our work highlights miR-342 as a potential therapeutic target in inflammation-related depression.

Cell-type specialization is encoded by specific chromatin topologies.

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Molecular characterization of the stress network in individuals at risk for schizophrenia.

The biological mechanisms underlying inter-individual differences in human stress reactivity remain poorly understood. We aimed to identify the molecular underpinning of aberrant neural stress sensitivity in individuals at risk for schizophrenia. Linking mRNA expression data from the Allen Human Brain Atlas to task-based fMRI revealed 201 differentially expressed genes in cortex-specific brain regions differentially activated by stress in individuals with low (healthy siblings of schizophrenia patients) or high (healthy controls) stress sensitivity. These genes are associated with stress-related psychiatric disorders (e.g. schizophrenia and anxiety) and include markers for specific neuronal populations (e.g. ADCYAP1, GABRB1, SSTR1, and TNFRSF12A), neurotransmitter receptors (e.g. GRIN3A, SSTR1, GABRB1, and HTR1E), and signaling factors that interact with the corticosteroid receptor and hypothalamic-pituitary-adrenal axis (e.g. ADCYAP1, IGSF11, and PKIA). Overall, the identified genes potentially underlie altered stress reactivity in individuals at risk for schizophrenia and other psychiatric disorders and play a role in mounting an adaptive stress response in at-risk individuals, making them potentially druggable targets for stress-related diseases.

DNA methylation signatures of aggression and closely related constructs: A meta-analysis of epigenome-wide studies across the lifespan.

DNA methylation profiles of aggressive behavior may capture lifetime cumulative effects of genetic, stochastic, and environmental influences associated with aggression. Here, we report the first large meta-analysis of epigenome-wide association studies (EWAS) of aggressive behavior (N = 15,324 participants). In peripheral blood samples of 14,434 participants from 18 cohorts with mean ages ranging from 7 to 68 years, 13 methylation sites were significantly associated with aggression (alpha = 1.2 × 10-7; Bonferroni correction). In cord blood samples of 2425 children from five cohorts with aggression assessed at mean ages ranging from 4 to 7 years, 83% of these sites showed the same direction of association with childhood aggression (r = 0.74, p = 0.006) but no epigenome-wide significant sites were found. Top-sites (48 at a false discovery rate of 5% in the peripheral blood meta-analysis or in a combined meta-analysis of peripheral blood and cord blood) have been associated with chemical exposures, smoking, cognition, metabolic traits, and genetic variation (mQTLs). Three genes whose expression levels were associated with top-sites were previously linked to schizophrenia and general risk tolerance. At six CpGs, DNA methylation variation in blood mirrors variation in the brain. On average 44% (range = 3-82%) of the aggression-methylation association was explained by current and former smoking and BMI. These findings point at loci that are sensitive to chemical exposures with potential implications for neuronal functions. We hope these results to be a starting point for studies leading to applications as peripheral biomarkers and to reveal causal relationships with aggression and related traits.

DNA methylation associated with persistent ADHD suggests TARBP1 as novel candidate.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by age-inappropriate symptoms of inattention and/or hyperactivity and impulsivity. ADHD is highly prevalent in childhood and often persists into adulthood. Both genetic variants and environmental factors play a role in the onset and persistence of ADHD, and epigenetic changes, such as DNA methylation are considered as a link for their interplay. To investigate this, we studied DNA methylation in 37 candidate genes by performing targeted bisulfite sequencing of DNA isolated from whole blood of N = 88 individuals diagnosed with adult ADHD and N = 91 unaffected individuals (mean age 34.2 years). Differentially methylated sites were assessed by generalized linear models testing ADHD status and ADHD symptoms, accounting for a methylation-based smoking score, age, sex, and blood cell count. DNA methylation of single sites within DRD4 and KLDR1 was associated with adult ADHD status, and multiple DNA methylation sites within TARBP1 were associated with ADHD symptoms in adulthood and childhood. Awaiting replication, findings of this pilot study point to TARBP1 as a new candidate gene for ADHD symptoms. Our work also stresses the need for research to further examine the effects of environmental factors, such as nicotine exposure, on epigenetic modifications associated with psychiatric traits.

Genome-Wide DNA Methylation Patterns in Persistent Attention-Deficit/Hyperactivity Disorder and in Association With Impulsive and Callous Traits.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists into adulthood. ADHD and related personality traits, such as impulsivity and callousness, are caused by genetic and environmental factors and their interplay. Epigenetic modifications of DNA, including methylation, are thought to mediate between such factors and behavior and may behave as biomarkers for disorders. Here, we set out to study DNA methylation in persistent ADHD and related traits. We performed epigenome-wide association studies (EWASs) on peripheral whole blood from participants in the NeuroIMAGE study (age range 12-23 years). We compared participants with persistent ADHD (n = 35) with healthy controls (n = 19) and with participants with remittent ADHD (n = 19). Additionally, we performed EWASs of impulsive and callous traits derived from the Conners Parent Rating Scale and the Callous-Unemotional Inventory, respectively, across all participants. For every EWAS, the linear regression model analyzed included covariates for age, sex, smoking scores, and surrogate variables reflecting blood cell type composition and genetic background. We observed no epigenome-wide significant differences in single CpG site methylation between participants with persistent ADHD and healthy controls or participants with remittent ADHD. However, epigenome-wide analysis of differentially methylated regions provided significant findings showing that hypermethylated regions in the APOB and LPAR5 genes were associated with ADHD persistence compared to ADHD remittance (p = 1.68 * 10-24 and p = 9.06 * 10-7, respectively); both genes are involved in cholesterol signaling. Both findings appeared to be linked to genetic variation in cis. We found neither significant epigenome-wide single CpG sites nor regions associated with impulsive and callous traits; the top-hits from these analyses were annotated to genes involved in neurotransmitter release and the regulation of the biological clock. No link to genetic variation was observed for these findings, which thus might reflect environmental influences. In conclusion, in this pilot study with a small sample size, we observed several DNA-methylation-disorder/trait associations of potential significance for ADHD and the related behavioral traits. Although we do not wish to draw conclusions before replication in larger, independent samples, cholesterol signaling and metabolism may be of relevance for the onset and/or persistence of ADHD.

PAD2-Mediated Citrullination Contributes to Efficient Oligodendrocyte Differentiation and Myelination.

Citrullination, the deimination of peptidylarginine residues into peptidylcitrulline, has been implicated in the etiology of several diseases. In multiple sclerosis, citrullination is thought to be a major driver of pathology through hypercitrullination and destabilization of myelin. As such, inhibition of citrullination has been suggested as a therapeutic strategy for MS. Here, in contrast, we show that citrullination by peptidylarginine deiminase 2 (PAD2) contributes to normal oligodendrocyte differentiation, myelination, and motor function. We identify several targets for PAD2, including myelin and chromatin-related proteins, implicating PAD2 in epigenomic regulation. Accordingly, we observe that PAD2 inhibition and its knockdown affect chromatin accessibility and prevent the upregulation of oligodendrocyte differentiation genes. Moreover, mice lacking PAD2 display motor dysfunction and a decreased number of myelinated axons in the corpus callosum. We conclude that citrullination contributes to proper oligodendrocyte lineage progression and myelination.

Effects of chronic caffeine consumption on sleep and the sleep electroencephalogram in mice.

BACKGROUND: Caffeine is one of the most widely consumed psychostimulants, and it impacts sleep and circadian physiology. AIM: Caffeine is generally used chronically on a daily basis. Therefore, in the current study, we investigated the chronic effect of caffeine on sleep in mice. METHODS: We recorded the electroencephalogram and electromyogram on a control day, on the first day of caffeine consumption (acute), and following two weeks of continuous caffeine consumption (chronic). In the latter condition, a period of six-hour sleep deprivation was conducted during the light period. Control mice, which received normal drinking water, were also recorded and sleep deprived. RESULTS: We found that caffeine induced differential effects following acute and chronic consumption. Over 24 h, waking increased following acute caffeine whereas no changes were found in the chronic condition. The daily amplitude of sleep-wake states increased in both acute and chronic conditions, with the highest amplitude in the chronic condition, showing an increase in sleep during the light and an increase in waking during the dark. Furthermore, electroencephalogram slow-wave-activity in non-rapid eye-movement sleep was increased, compared with both control conditions, during the first half of the light period in the chronic condition. It was particularly challenging to keep the animals awake during the sleep deprivation period under chronic caffeine. CONCLUSIONS: Together the data suggest an increased sleep pressure under chronic caffeine. In contrast to the traditional conception on the impact on sleep, chronic caffeine intake seems to increase the daily sleep-wake cycle amplitude and increase sleep pressure in mice.

Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis.

Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS.

Transcriptional Convergence of Oligodendrocyte Lineage Progenitors during Development.

Pdgfra+ oligodendrocyte precursor cells (OPCs) arise in distinct specification waves during embryogenesis in the central nervous system (CNS). It is unclear whether there is a correlation between these waves and different oligodendrocyte (OL) states at adult stages. Here, we present bulk and single-cell transcriptomics resources providing insights on how transitions between these states occur. We found that post-natal OPCs from brain and spinal cord present similar transcriptional signatures. Moreover, post-natal OPC progeny of E13.5 Pdgfra+ cells present electrophysiological and transcriptional profiles similar to OPCs derived from subsequent specification waves, indicating that Pdgfra+ pre-OPCs rewire their transcriptional network during development. Single-cell RNA-seq and lineage tracing indicates that a subset of E13.5 Pdgfra+ cells originates cells of the pericyte lineage. Thus, our results indicate that embryonic Pdgfra+ cells in the CNS give rise to distinct post-natal cell lineages, including OPCs with convergent transcriptional profiles in different CNS regions.