RESUMEN
Midbrain dopamine neurons (DNs) respond to a first exposure to addictive drugs and play key roles in chronic drug usage1-3. As the synaptic and transcriptional changes that follow an acute cocaine exposure are mostly resolved within a few days4,5, the molecular changes that encode the long-term cellular memory of the exposure within DNs remain unknown. To investigate whether a single cocaine exposure induces long-term changes in the 3D genome structure of DNs, we applied Genome Architecture Mapping and single nucleus transcriptomic analyses in the mouse midbrain. We found extensive rewiring of 3D genome architecture at 24 hours past exposure which remains or worsens by 14 days, outlasting transcriptional responses. The cocaine-induced chromatin rewiring occurs at all genomic scales and affects genes with major roles in cocaine-induced synaptic changes. A single cocaine exposure triggers extensive long-lasting changes in chromatin condensation in post-synaptic and post-transcriptional regulatory genes, for example the unfolding of Rbfox1 which becomes most prominent 14 days post exposure. Finally, structurally remodeled genes are most expressed in a specific DN sub-type characterized by low expression of the dopamine auto-receptor Drd2, a key feature of highly cocaine-sensitive cells. These results reveal an important role for long-lasting 3D genome remodelling in the cellular memory of a single cocaine exposure, providing new hypotheses for understanding the inception of drug addiction and 3D genome plasticity.
RESUMEN
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.
Asunto(s)
Encéfalo/citología , Células/clasificación , Ensamble y Desensamble de Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformación Molecular , Animales , Sitios de Unión , Células/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Familia de Multigenes/genética , Neuronas/clasificación , Neuronas/metabolismo , Desnaturalización de Ácido Nucleico , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Dementia is a syndrome that comprises many differing pathologies, including Alzheimer's disease dementia (ADD), vascular dementia (VaD) and frontotemporal dementia (FTD). People may benefit from knowing the type of dementia they live with, as this could inform prognosis and may allow for tailored treatment. Beta-amyloid (1-42) (ABeta42) is a protein which decreases in both the plasma and cerebrospinal fluid (CSF) of people living with ADD, when compared to people with no dementia. However, it is not clear if changes in ABeta42 are specific to ADD or if they are also seen in other types of dementia. It is possible that ABeta42 could help differentiate ADD from other dementia subtypes. OBJECTIVES: To determine the accuracy of plasma and CSF ABeta42 for distinguishing ADD from other dementia subtypes in people who meet the criteria for a dementia syndrome. SEARCH METHODS: We searched MEDLINE, and nine other databases up to 18 February 2020. We checked reference lists of any relevant systematic reviews to identify additional studies. SELECTION CRITERIA: We considered cross-sectional studies that differentiated people with ADD from other dementia subtypes. Eligible studies required measurement of participant plasma or CSF ABeta42 levels and clinical assessment for dementia subtype. DATA COLLECTION AND ANALYSIS: Seven review authors working independently screened the titles and abstracts generated by the searches. We collected data on study characteristics and test accuracy. We used the second version of the 'Quality Assessment of Diagnostic Accuracy Studies' (QUADAS-2) tool to assess internal and external validity of results. We extracted data into 2 x 2 tables, cross-tabulating index test results (ABeta42) with the reference standard (diagnostic criteria for each dementia subtype). We performed meta-analyses using bivariate, random-effects models. We calculated pooled estimates of sensitivity, specificity, positive predictive values, positive and negative likelihood ratios, and corresponding 95% confidence intervals (CIs). In the primary analysis, we assessed accuracy of plasma or CSF ABeta42 for distinguishing ADD from other mixed dementia types (non-ADD). We then assessed accuracy of ABeta42 for differentiating ADD from specific dementia types: VaD, FTD, dementia with Lewy bodies (DLB), alcohol-related cognitive disorder (ARCD), Creutzfeldt-Jakob disease (CJD) and normal pressure hydrocephalus (NPH). To determine test-positive cases, we used the ABeta42 thresholds employed in the respective primary studies. We then performed sensitivity analyses restricted to those studies that used common thresholds for ABeta42. MAIN RESULTS: We identified 39 studies (5000 participants) that used CSF ABeta42 levels to differentiate ADD from other subtypes of dementia. No studies of plasma ABeta42 met the inclusion criteria. No studies were rated as low risk of bias across all QUADAS-2 domains. High risk of bias was found predominantly in the domains of patient selection (28 studies) and index test (25 studies). The pooled estimates for differentiating ADD from other dementia subtypes were as follows: ADD from non-ADD: sensitivity 79% (95% CI 0.73 to 0.85), specificity 60% (95% CI 0.52 to 0.67), 13 studies, 1704 participants, 880 participants with ADD; ADD from VaD: sensitivity 79% (95% CI 0.75 to 0.83), specificity 69% (95% CI 0.55 to 0.81), 11 studies, 1151 participants, 941 participants with ADD; ADD from FTD: sensitivity 85% (95% CI 0.79 to 0.89), specificity 72% (95% CI 0.55 to 0.84), 17 studies, 1948 participants, 1371 participants with ADD; ADD from DLB: sensitivity 76% (95% CI 0.69 to 0.82), specificity 67% (95% CI 0.52 to 0.79), nine studies, 1929 participants, 1521 participants with ADD. Across all dementia subtypes, sensitivity was greater than specificity, and the balance of sensitivity and specificity was dependent on the threshold used to define test positivity. AUTHORS' CONCLUSIONS: Our review indicates that measuring ABeta42 levels in CSF may help differentiate ADD from other dementia subtypes, but the test is imperfect and tends to misdiagnose those with non-ADD as having ADD. We would caution against the use of CSF ABeta42 alone for dementia classification. However, ABeta42 may have value as an adjunct to a full clinical assessment, to aid dementia diagnosis.
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Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Alcoholismo/complicaciones , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/líquido cefalorraquídeo , Sesgo , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Disfunción Cognitiva/sangre , Disfunción Cognitiva/líquido cefalorraquídeo , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/etiología , Intervalos de Confianza , Síndrome de Creutzfeldt-Jakob/sangre , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquídeo , Síndrome de Creutzfeldt-Jakob/diagnóstico , Demencia Vascular/sangre , Demencia Vascular/líquido cefalorraquídeo , Demencia Vascular/diagnóstico , Diagnóstico Diferencial , Demencia Frontotemporal/sangre , Demencia Frontotemporal/líquido cefalorraquídeo , Demencia Frontotemporal/diagnóstico , Humanos , Hidrocéfalo Normotenso/sangre , Hidrocéfalo Normotenso/líquido cefalorraquídeo , Hidrocéfalo Normotenso/diagnóstico , Enfermedad por Cuerpos de Lewy/sangre , Enfermedad por Cuerpos de Lewy/líquido cefalorraquídeo , Enfermedad por Cuerpos de Lewy/diagnóstico , Funciones de Verosimilitud , Sensibilidad y EspecificidadRESUMEN
Patients with schizophrenia show increased striatal dopamine synthesis capacity in imaging studies. The mechanism underlying this is unclear but may be due to N-methyl-D-aspartate receptor (NMDAR) hypofunction and parvalbumin (PV) neuronal dysfunction leading to disinhibition of mesostriatal dopamine neurons. Here, we develop a translational mouse model of the dopamine pathophysiology seen in schizophrenia and test approaches to reverse the dopamine changes. Mice were treated with sub-chronic ketamine (30 mg/kg) or saline and then received in vivo positron emission tomography of striatal dopamine synthesis capacity, analogous to measures used in patients. Locomotor activity was measured using the open-field test. In vivo cell-type-specific chemogenetic approaches and pharmacological interventions were used to manipulate neuronal excitability. Immunohistochemistry and RNA sequencing were used to investigate molecular mechanisms. Sub-chronic ketamine increased striatal dopamine synthesis capacity (Cohen's d = 2.5) and locomotor activity. These effects were countered by inhibition of midbrain dopamine neurons, and by activation of PV interneurons in pre-limbic cortex and ventral subiculum of the hippocampus. Sub-chronic ketamine reduced PV expression in these cortical and hippocampal regions. Pharmacological intervention with SEP-363856, a novel psychotropic agent with agonism at trace amine receptor 1 (TAAR1) and 5-HT1A receptors but no appreciable action at dopamine D2 receptors, significantly reduced the ketamine-induced increase in dopamine synthesis capacity. These results show that sub-chronic ketamine treatment in mice mimics the dopaminergic alterations in patients with psychosis, that this requires activation of midbrain dopamine neurons, and can be ameliorated by activating PV interneurons and by a TAAR1/5-HT1A agonist. This identifies novel therapeutic approaches for targeting presynaptic dopamine dysfunction in patients with schizophrenia and effects of ketamine relevant to its therapeutic use for treating major depression.
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Ketamina , Esquizofrenia , Animales , Dopamina , Humanos , Ketamina/farmacología , Ratones , Piranos , Receptores de N-Metil-D-Aspartato , Esquizofrenia/tratamiento farmacológicoRESUMEN
γ-Aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide local inhibitory control of dopamine neuron activity and send long-range projections to several target regions including the nucleus accumbens. They play diverse roles in reward and aversion, suggesting that they be comprised of several functionally distinct sub-groups, but our understanding of this diversity has been limited by a lack of molecular markers that might provide genetic entry points for cell type-specific investigations. To address this, we conducted transcriptional profiling of GABA neurons and dopamine neurons using immunoprecipitation of tagged polyribosomes (RiboTag) and RNAseq. First, we directly compared these two transcriptomes in order to obtain a list of genes enriched in GABA neurons compared with dopamine neurons. Next, we created a novel bioinformatic approach, that used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) gene ontology database and VTA gene expression data from the Allen Mouse Brain Atlas, from which we obtained 6 candidate genes: Cbln4, Rxfp3, Rora, Gpr101, Trh and Nrp2. As a final step, we verified the selective expression of these candidate genes in sub-groups of GABA neurons in the VTA (and neighbouring substantia nigra pars compacta) using immunolabelling. Taken together, our study provides a valuable toolbox for the future investigation of GABA neuron sub-groups in the VTA.
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Neuronas GABAérgicas/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/fisiología , Área Tegmental Ventral/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Animales , Neuronas GABAérgicas/química , Expresión Génica , Ratones , Ratones Transgénicos , Área Tegmental Ventral/química , Ácido gamma-Aminobutírico/genéticaRESUMEN
GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.
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Óxido Nítrico Sintasa de Tipo I/metabolismo , Porción Compacta de la Sustancia Negra/metabolismo , Sustancia Negra/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Neuronas GABAérgicas/metabolismo , Ratones Endogámicos C57BL , Núcleo Accumbens/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.