RESUMO
To explore genome organization and function in the HIV-infected brain, we applied single-nuclei transcriptomics, cell-type-specific chromosomal conformation mapping, and viral integration site sequencing (IS-seq) to frontal cortex from individuals with encephalitis (HIVE) and without (HIV+). Derepressive changes in 3D genomic compartment structures in HIVE microglia were linked to the transcriptional activation of interferon (IFN) signaling and cell migratory pathways, while transcriptional downregulation and repressive compartmentalization of neuronal health and signaling genes occurred in both HIVE and HIV+ microglia. IS-seq recovered 1,221 brain integration sites showing distinct genomic patterns compared with peripheral lymphocytes, with enrichment for sequences newly mobilized into a permissive chromatin environment after infection. Viral transcription occurred in a subset of highly activated microglia comprising 0.33% of all nuclei in HIVE brain. Our findings point to disrupted microglia-neuronal interactions in HIV and link retroviral integration to remodeling of the microglial 3D genome during infection.
Assuntos
Infecções por HIV , Microglia , Humanos , Microglia/metabolismo , Encéfalo , Ativação de Macrófagos , Macrófagos , Infecções por HIV/genéticaRESUMO
IMPORTANCE: Our mouse model is a powerful tool for investigating the genetic mechanisms governing central nervous system (CNS) human immunodeficiency virus type-1 (HIV-1) infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses induced pluripotent stem cell-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be explored in vivo, which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.
Assuntos
Infecções por HIV , HIV-1 , Células-Tronco Pluripotentes Induzidas , Microglia , Animais , Humanos , Camundongos , Sistema Nervoso Central , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/fisiologia , Microglia/virologia , Latência Viral , XenoenxertosRESUMO
The lysine-63 deubiquitinase cylindromatosis (CYLD) is long recognized as a tumor suppressor in immunity and inflammation, and its loss-of-function mutations lead to familial cylindromatosis. However, recent studies reveal that CYLD is enriched in mammalian brain postsynaptic densities, and a gain-of-function mutation causes frontotemporal dementia (FTD), suggesting critical roles at excitatory synapses. Here we report that CYLD drives synapse elimination and weakening by acting on the Akt-mTOR-autophagy axis. Mice lacking CYLD display abnormal sociability, anxiety- and depression-like behaviors, and cognitive inflexibility. These behavioral impairments are accompanied by excessive synapse numbers, increased postsynaptic efficacy, augmented synaptic summation, and impaired NMDA receptor-dependent hippocampal long-term depression (LTD). Exogenous expression of CYLD results in removal of established dendritic spines from mature neurons in a deubiquitinase activity-dependent manner. In search of underlying molecular mechanisms, we find that CYLD knockout mice display marked overactivation of Akt and mTOR and reduced autophagic flux, and conversely, CYLD overexpression potently suppresses Akt and mTOR activity and promotes autophagy. Consequently, abrogating the Akt-mTOR-autophagy signaling pathway abolishes CYLD-induced spine loss, whereas enhancing autophagy in vivo by the mTOR inhibitor rapamycin rescues the synaptic pruning and LTD deficits in mutant mice. Our findings establish CYLD, via Akt-mTOR signaling, as a synaptic autophagy activator that exerts critical modulations on synapse maintenance, function, and plasticity.
Assuntos
Macroautofagia , Proteínas Proto-Oncogênicas c-akt , Animais , Enzimas Desubiquitinantes/metabolismo , Mamíferos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Large-scale genetic studies have revealed that the most prominent genes disrupted in autism are chromatin regulators mediating histone methylation/demethylation, suggesting the central role of epigenetic dysfunction in this disorder. Here, we show that histone lysine 4 dimethylation (H3K4me2), a histone mark linked to gene activation, is significantly decreased in the prefrontal cortex (PFC) of autistic human patients and mutant mice with the deficiency of top-ranking autism risk factor Shank3 or Cul3. A brief treatment of the autism models with highly potent and selective inhibitors of the H3K4me2 demethylase LSD1 (KDM1A) leads to the robust rescue of core symptoms of autism, including social deficits and repetitive behaviors. Concomitantly, LSD1 inhibition restores NMDA receptor function in PFC and AMPA receptor-mediated currents in striatum of Shank3-deficient mice. Genome-wide RNAseq and ChIPseq reveal that treatment of Shank3-deficient mice with the LSD1 inhibitor restores the expression and H3K4me2 occupancy of downregulated genes enriched in synaptic signaling and developmental processes. The immediate early gene tightly linked to neuronal plasticity, Egr1, is on the top list of rescued genes. The diminished transcription of Egr1 is recapitulated in PFC of autistic human patients. Overexpression of Egr1 in PFC of Shank3-deficient mice ameliorates social preference deficits. These results have for the first time revealed an important role of H3K4me2 abnormality in ASD pathophysiology, and the therapeutic potential of targeting H3K4me2 demethylase LSD1 or the downstream molecule Egr1 for ASD.
Assuntos
Transtorno Autístico , Histonas , Humanos , Camundongos , Animais , Histonas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Cromatina , Modelos Animais de Doenças , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Large-scale consortia including the Psychiatric Genomics Consortium, the Common Minds Consortium, BrainSeq and PsychENCODE, and many other studies taken together provide increasingly detailed insights into the genetic and epigenetic risk architectures of schizophrenia (SCZ) and offer vast amounts of molecular information, but with largely unexplored therapeutic potential. Here we discuss how epigenomic studies in human brain could guide animal work to test the impact of disease-associated alterations in chromatin structure and function on cognition and behavior. For example, transcription factors such as MYOCYTE-SPECIFIC ENHANCER FACTOR 2C (MEF2C), or multiple regulators of the open chromatin mark, methyl-histone H3-lysine 4, are associated with the genetic risk architectures of common psychiatric disease and alterations in chromatin structure and function in diseased brain tissue. Importantly, these molecules also affect cognition and behavior in genetically engineered mice, including virus-mediated expression changes in prefrontal cortex (PFC) and other key nodes in the circuitry underlying psychosis. Therefore, preclinical and small laboratory animal work could target genomic sequences affected by chromatin alterations in SCZ. To this end, in vivo editing of enhancer and other regulatory non-coding DNA by RNA-guided nucleases including CRISPR-Cas, and designer transcription factors, could be expected to deliver pipelines for novel therapeutic approaches aimed at improving cognitive dysfunction and other core symptoms of SCZ.
Assuntos
Epigenômica , Esquizofrenia/genética , Animais , CamundongosRESUMO
Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Memória de Curto Prazo/fisiologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/fisiologia , Células Piramidais/fisiologiaRESUMO
DNA cytosine methylation has been documented as a potential epigenetic mechanism of transcriptional regulation underlying opioid use disorder. However, methylation of RNA cytosine residues, which would drive another level of biological influence as an epitranscriptomic mechanism of gene and protein regulation has not been studied in the context of addiction. Here, we probed whether chronic morphine exposure could alter tRNA cytosine methylation (m5C) and resulting expression levels in the medial prefrontal cortex (mPFC), a brain region crucial for reward processing and executive function that exhibits opioid-induced molecular restructuring. We identified dynamic changes in glycine tRNA (tRNAGlyGCC) cytosine methylation, corresponding to altered expression levels of this tRNA at multiple timepoints following 15 days of daily morphine. Additionally, a robust increase in methylation, coupled with decreased expression, was present after 30 days of withdrawal, suggesting that repeated opioid administration produces changes to the tRNA regulome long after discontinuation. Furthermore, forebrain-wide knockout of neuronal Nsun2, a tRNA methyltransferase, was associated with disruption of opioid conditioned place preference, and this effect was recapitulated by regional mPFC Nsun2 knockout. Taken together, these studies provide a foundational link between the regulation of tRNA cytosine methylation and opioid reward and highlight the tRNA machinery as a potential therapeutic target in addiction.
Assuntos
Camundongos Endogâmicos C57BL , Morfina , Córtex Pré-Frontal , RNA de Transferência , Recompensa , Síndrome de Abstinência a Substâncias , Animais , Masculino , Camundongos , Morfina/farmacologia , Morfina/administração & dosagem , Morfina/efeitos adversos , Síndrome de Abstinência a Substâncias/genética , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , RNA de Transferência/genética , Epigênese Genética/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Analgésicos Opioides/administração & dosagem , Camundongos Knockout , Comportamento de Procura de Droga/efeitos dos fármacos , Comportamento de Procura de Droga/fisiologiaRESUMO
The central nervous system (CNS) is a major human immunodeficiency virus type 1 reservoir. Microglia are the primary target cell of HIV-1 infection in the CNS. Current models have not allowed the precise molecular pathways of acute and chronic CNS microglial infection to be tested with in vivo genetic methods. Here, we describe a novel humanized mouse model utilizing human-induced pluripotent stem cell-derived microglia to xenograft into murine hosts. These mice are additionally engrafted with human peripheral blood mononuclear cells that served as a medium to establish a peripheral infection that then spread to the CNS microglia xenograft, modeling a trans-blood-brain barrier route of acute CNS HIV-1 infection with human target cells. The approach is compatible with iPSC genetic engineering, including inserting targeted transgenic reporter cassettes to track the xenografted human cells, enabling the testing of novel treatment and viral tracking strategies in a comparatively simple and cost-effective way vivo model for neuroHIV.
RESUMO
Here, we construct genome-scale maps for R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in the proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix, with preferential formation at promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations and defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, R-loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.
RESUMO
Regulatory mechanisms associated with repeat-rich sequences and chromosomal conformations in mature neurons remain unexplored. Here, we map cell-type specific chromatin domain organization in adult mouse cerebral cortex and report strong enrichment of Endogenous Retrovirus 2 (ERV2) repeat sequences in the neuron-specific heterochromatic B2NeuN+ megabase-scaling subcompartment. Single molecule long-read sequencing and comparative Hi-C chromosomal contact mapping in wild-derived SPRET/EiJ (Mus spretus) and laboratory inbred C57BL/6J (Mus musculus) reveal neuronal reconfigurations tracking recent ERV2 expansions in the murine germline, with significantly higher B2NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B2NeuN+ disintegration and rewiring with open chromatin domains enriched for cellular stress response genes, along with severe neuroinflammation and proviral assembly with infiltration of dendrites . We conclude that neuronal megabase-scale chromosomal architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, results in transcriptional dysregulation and unleashes ERV2 proviruses with strong neuronal tropism.
Assuntos
Cromossomos/metabolismo , Neurônios/metabolismo , Retroelementos/genética , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/genética , Evolução Molecular , Amplificação de Genes , Inativação Gênica , Genes de Partícula A Intracisternal/genética , Genoma Viral/genética , Gliose/genética , Gliose/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Microglia/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/virologia , Provírus/genética , Vírion/genética , Vírion/metabolismoRESUMO
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.
Assuntos
Encéfalo/metabolismo , COVID-19/imunologia , Perfilação da Expressão Gênica/métodos , Imunidade/genética , Imunidade/imunologia , Transcriptoma , Plexo Corióideo/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Humanos , Inflamação , Microglia , Córtex Pré-Frontal/metabolismo , SARS-CoV-2/genéticaRESUMO
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.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Polimorfismo Genético , Locos de Características Quantitativas , Esquizofrenia/genética , Adipogenia , Animais , Índice de Massa Corporal , Cromossomos/genética , Cognição , Humanos , Metabolismo dos Lipídeos , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese , Esquizofrenia/metabolismo , Esquizofrenia/patologiaRESUMO
To explore the developmental reorganization of the three-dimensional genome of the brain in the context of neuropsychiatric disease, we monitored chromosomal conformations in differentiating neural progenitor cells. Neuronal and glial differentiation was associated with widespread developmental remodeling of the chromosomal contact map and included interactions anchored in common variant sequences that confer heritable risk for schizophrenia. We describe cell type-specific chromosomal connectomes composed of schizophrenia risk variants and their distal targets, which altogether show enrichment for genes that regulate neuronal connectivity and chromatin remodeling, and evidence for coordinated transcriptional regulation and proteomic interaction of the participating genes. Developmentally regulated chromosomal conformation changes at schizophrenia-relevant sequences disproportionally occurred in neurons, highlighting the existence of cell type-specific disease risk vulnerabilities in spatial genome organization.
Assuntos
Cromossomos Humanos/química , Conectoma , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Células-Tronco Neurais/citologia , Neurogênese/genética , Esquizofrenia/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Cromatina/química , Montagem e Desmontagem da Cromatina , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Conformação de Ácido Nucleico , Mapas de Interação de Proteínas/genética , Proteômica , Risco , Transcrição Gênica , TranscriptomaRESUMO
PHF8 is a histone demethylase with specificity for repressive modifications. While mutations of PHF8 have been associated with cognitive defects and cleft lip/palate, its role in mammalian development and physiology remains unexplored. Here, we have generated a Phf8 knockout allele in mice to examine the consequences of Phf8 loss for development and behaviour. Phf8 deficient mice neither display obvious developmental defects nor signs of cognitive impairment. However, we report a striking resiliency to stress-induced anxiety- and depression-like behaviour on loss of Phf8. We further observe misregulation of serotonin signalling within the prefrontal cortex of Phf8 deficient mice and identify the serotonin receptors Htr1a and Htr2a as direct targets of PHF8. Our results clarify the functional role of Phf8 in mammalian development and behaviour and establish a direct link between Phf8 expression and serotonin signalling, identifying this histone demethylase as a potential target for the treatment of anxiety and depression.
Assuntos
Ansiedade/metabolismo , Comportamento Animal , Depressão/metabolismo , Histona Desmetilases/deficiência , Histona Desmetilases/metabolismo , Resiliência Psicológica , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Alelos , Animais , Ansiedade/patologia , Ansiedade/fisiopatologia , Disfunção Cognitiva/patologia , Disfunção Cognitiva/fisiopatologia , Depressão/patologia , Depressão/fisiopatologia , Deleção de Genes , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Receptores de Serotonina/metabolismo , Estresse Psicológico/fisiopatologiaRESUMO
We report locus-specific disintegration of megabase-scale chromosomal conformations in brain after neuronal ablation of Setdb1 (also known as Kmt1e; encodes a histone H3 lysine 9 methyltransferase), including a large topologically associated 1.2-Mb domain conserved in humans and mice that encompasses >70 genes at the clustered protocadherin locus (hereafter referred to as cPcdh). The cPcdh topologically associated domain (TADcPcdh) in neurons from mutant mice showed abnormal accumulation of the transcriptional regulator and three-dimensional (3D) genome organizer CTCF at cryptic binding sites, in conjunction with DNA cytosine hypomethylation, histone hyperacetylation and upregulated expression. Genes encoding stochastically expressed protocadherins were transcribed by increased numbers of cortical neurons, indicating relaxation of single-cell constraint. SETDB1-dependent loop formations bypassed 0.2-1 Mb of linear genome and radiated from the TADcPcdh fringes toward cis-regulatory sequences within the cPcdh locus, counterbalanced shorter-range facilitative promoter-enhancer contacts and carried loop-bound polymorphisms that were associated with genetic risk for schizophrenia. We show that the SETDB1 repressor complex, which involves multiple KRAB zinc finger proteins, shields neuronal genomes from excess CTCF binding and is critically required for structural maintenance of TADcPcdh.
Assuntos
Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neurônios/metabolismo , Animais , Fator de Ligação a CCCTC , Caderinas/genética , Linhagem Celular , Metilação de DNA , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Proteínas Repressoras/metabolismoRESUMO
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.
Assuntos
Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Clozapina/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Substância Branca/efeitos dos fármacos , Administração Oral , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Contagem de Células , Feminino , Citometria de Fluxo , Substância Cinzenta/anatomia & histologia , Substância Cinzenta/efeitos dos fármacos , Substância Cinzenta/metabolismo , Haloperidol/farmacologia , Imuno-Histoquímica , Macaca , Imageamento por Ressonância Magnética , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Tamanho do Órgão , Distribuição Aleatória , Substância Branca/anatomia & histologia , Substância Branca/metabolismoRESUMO
Lysine (K) methyltransferase 2a (Kmt2a) and other regulators of H3 lysine 4 methylation, a histone modification enriched at promoters and enhancers, are widely expressed throughout the brain, but molecular and cellular phenotypes in subcortical areas remain poorly explored. We report that Kmt2a conditional deletion in postnatal forebrain is associated with excessive nocturnal activity and with absent or blunted responses to stimulant and dopaminergic agonist drugs, in conjunction with near-complete loss of spike-timing-dependent long-term potentiation in medium spiny neurons (MSNs). Selective ablation of Kmt2a, but not the ortholog Kmt2b, in adult ventral striatum/nucleus accumbens neurons markedly increased anxiety scores in multiple behavioral paradigms. Striatal transcriptome sequencing in adult mutants identified 262 Kmt2a-sensitive genes, mostly downregulated in Kmt2a-deficient mice. Transcriptional repression includes the 5-Htr2a serotonin receptor, strongly associated with anxiety- and depression-related disorders in human and animal models. Consistent with the role of Kmt2a in promoting gene expression, the transcriptional regulators Bahcc1, Isl1, and Sp9 were downregulated and affected by H3K4 promoter hypomethylation. Therefore, Kmt2a regulates synaptic plasticity in striatal neurons and provides an epigenetic drug target for anxiety and dopamine-mediated behaviors.
Assuntos
Potenciais de Ação/genética , Ansiedade , Dopaminérgicos/farmacologia , Histona-Lisina N-Metiltransferase/deficiência , Proteína de Leucina Linfoide-Mieloide/deficiência , Plasticidade Neuronal/genética , Neurônios/fisiologia , Estriado Ventral/citologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Ansiedade/tratamento farmacológico , Ansiedade/genética , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Modelos Animais de Doenças , Feminino , Histona-Lisina N-Metiltransferase/genética , Locomoção/efeitos dos fármacos , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Neuronal epigenomes, including chromosomal loopings moving distal cis-regulatory elements into proximity of target genes, could serve as molecular proxy linking present-day-behaviour to past exposures. However, longitudinal assessment of chromatin state is challenging, because conventional chromosome conformation capture assays essentially provide single snapshots at a given time point, thus reflecting genome organization at the time of brain harvest and therefore are non-informative about the past. Here we introduce 'NeuroDam' to assess epigenome status retrospectively. Short-term expression of the bacterial DNA adenine methyltransferase Dam, tethered to the Gad1 gene promoter in mouse prefrontal cortex neurons, results in stable G(methyl)ATC tags at Gad1-bound chromosomal contacts. We show by NeuroDam that mice with defective cognition 4 months after pharmacological NMDA receptor blockade already were affected by disrupted chromosomal conformations shortly after drug exposure. Retrospective profiling of neuronal epigenomes is likely to illuminate epigenetic determinants of normal and diseased brain development in longitudinal context.