RESUMO
The DNA-binding protein SATB2 is genetically linked to human intelligence. We studied its influence on the three-dimensional (3D) epigenome by mapping chromatin interactions and accessibility in control versus SATB2-deficient cortical neurons. We find that SATB2 affects the chromatin looping between enhancers and promoters of neuronal-activity-regulated genes, thus influencing their expression. It also alters A/B compartments, topologically associating domains, and frequently interacting regions. Genes linked to SATB2-dependent 3D genome changes are implicated in highly specialized neuronal functions and contribute to cognitive ability and risk for neuropsychiatric and neurodevelopmental disorders. Non-coding DNA regions with a SATB2-dependent structure are enriched for common variants associated with educational attainment, intelligence, and schizophrenia. Our data establish SATB2 as a cell-type-specific 3D genome modulator, which operates both independently and in cooperation with CCCTC-binding factor (CTCF) to set up the chromatin landscape of pyramidal neurons for cognitive processes.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neurônios/metabolismo , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Genoma , Cognição , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismoRESUMO
The induction of lineage-specific gene programs are strongly influenced by alterations in local chromatin architecture. However, key players that impact this genome reorganization remain largely unknown. Here, we report that the removal of the special AT-rich binding protein 2 (SATB2), a nuclear protein known to bind matrix attachment regions, is a key event in initiating myogenic differentiation. The deletion of myoblast SATB2 in vitro initiates chromatin remodeling and accelerates differentiation, which is dependent on the caspase 7-mediated cleavage of SATB2. A genome-wide analysis indicates that SATB2 binding within chromatin loops and near anchor points influences both loop and sub-TAD domain formation. Consequently, the chromatin changes that occur with the removal of SATB2 lead to the derepression of differentiation-inducing factors while also limiting the expression of genes that inhibit this cell fate change. Taken together, this study demonstrates that the temporal control of the SATB2 protein is critical in shaping the chromatin environment and coordinating the myogenic differentiation program.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Caspases , Cromatina , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Mioblastos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Encéfalo/citologia , Células/classificação , Montagem e Desmontagem da Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformação Molecular , Animais , Sítios de Ligação , Células/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Família Multigênica/genética , Neurônios/classificação , Neurônios/metabolismo , Desnaturação de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Social interaction in an alternative context can be beneficial against drugs of abuse. Stress is known to be a risk factor that can exacerbate the effects of addictive drugs. In this study, we investigated whether the positive effects of social interaction are mediated through a decrease in stress levels. For that purpose, rats were trained to express cocaine or social interaction conditioned place preference (CPP). Behavioural, hormonal, and molecular stress markers were evaluated. We found that social CPP decreased the percentage of incorrect transitions of grooming and corticosterone to the level of naïve untreated rats. In addition, corticotropin-releasing factor (CRF) was increased in the bed nucleus of stria terminalis after cocaine CPP. In order to study the modulation of social CPP by the CRF system, rats received intracerebroventricular CRF or alpha-helical CRF, a nonselective antagonist of CRF receptors. The subsequent effects on CPP to cocaine or social interaction were observed. CRF injections increased cocaine CPP, whereas alpha-helical CRF injections decreased cocaine CPP. However, alpha-helical CRF injections potentiated social CPP. When social interaction was made available in an alternative context, CRF-induced increase of cocaine preference was reversed completely to the level of rats receiving cocaine paired with alpha-helical CRF. This reversal of cocaine preference was also paralleled by a reversal in CRF-induced increase of p38 MAPK expression in the nucleus accumbens shell. These findings suggest that social interaction could contribute as a valuable component in treatment of substance use disorders by reducing stress levels.
Assuntos
Recompensa , Interação Social , Estresse Psicológico/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Hormônio Liberador da Corticotropina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Masculino , Núcleo Accumbens/efeitos dos fármacos , Ratos , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
SATB2 is a schizophrenia risk gene and is genetically associated with human intelligence. How it affects cognition at molecular level is currently unknown. Here, we show that interactions between SATB2, a chromosomal scaffolding protein, and the inner nuclear membrane protein LEMD2 orchestrate the response of pyramidal neurons to neuronal activation. Exposure to novel environment in vivo causes changes in nuclear shape of CA1 hippocampal neurons via a SATB2-dependent mechanism. The activity-driven plasticity of the nuclear envelope requires not only SATB2, but also its protein interactor LEMD2 and the ESCRT-III/VPS4 membrane-remodeling complex. Furthermore, LEMD2 depletion in cortical neurons, similar to SATB2 ablation, affects neuronal activity-dependent regulation of multiple rapid and delayed primary response genes. In human genetic data, LEMD2-regulated genes are enriched for de novo mutations reported in intellectual disability and schizophrenia and are, like SATB2-regulated genes, enriched for common variants associated with schizophrenia and cognitive function. Hence, interactions between SATB2 and the inner nuclear membrane protein LEMD2 influence gene expression programs in pyramidal neurons that are linked to cognitive ability and psychiatric disorder etiology.
Assuntos
Redes Reguladoras de Genes , Hipocampo/citologia , Deficiência Intelectual/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Esquizofrenia/genética , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Núcleo Celular/metabolismo , Plasticidade Celular , Células Cultivadas , Cognição , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/química , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Esquizofrenia/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Evidence suggests that PKA activity in the nucleus accumbens (NAc) plays an essential role in reward-related learning. In this study, we investigated whether PKA is differentially involved in the expression of learning produced by either natural reinforcers or psychostimulants. For that purpose, we inhibited PKA through a bilateral infusion of Rp-cAMPS, a specific PKA inhibitor, directly into the NAc. The effects of PKA inhibition in the NAc on the expression of concurrent conditioned place preference (CPP) for cocaine (drug) and social interaction (natural reward) in rats were evaluated. We found that PKA inhibition increased the expression of cocaine preference. This effect was not due to altered stress levels or decreased social reward. PKA inhibition did not affect the expression of natural reward as intra-NAc Rp-cAMPS infusion did not affect expression of social preference. When rats were trained to express cocaine or social interaction CPP and tested for eventual persisting preference 7 and 14 days after CPP expression, cocaine preference was persistent, but social preference was abolished after the first test. These results suggest that PKA in the NAc is involved in drug reward learning that might lead to addiction and that only drug, but not natural, reward is persistent.
Assuntos
Cocaína/farmacologia , Condicionamento Operante/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Recompensa , Interação Social , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , AMP Cíclico/metabolismo , Masculino , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
During CNS development, the nuclear protein SATB2 is expressed in superficial cortical layers and determines projection neuron identity. In the adult CNS, SATB2 is expressed in pyramidal neurons of all cortical layers and is a regulator of synaptic plasticity and long-term memory. Common variation in SATB2 locus confers risk of schizophrenia, whereas rare, de novo structural and single nucleotide variants cause severe intellectual disability and absent or limited speech. To characterize differences in SATB2 molecular function in developing vs adult neocortex, we isolated SATB2 protein interactomes at the two ontogenetic stages and identified multiple novel SATB2 interactors. SATB2 interactomes are highly enriched for proteins that stabilize de novo chromatin loops. The comparison between the neonatal and adult SATB2 protein complexes indicates a developmental shift in SATB2 molecular function, from transcriptional repression towards organization of chromosomal superstructure. Accordingly, gene sets regulated by SATB2 in the neocortex of neonatal and adult mice show limited overlap. Genes encoding SATB2 protein interactors were grouped for gene set analysis of human GWAS data. Common variants associated with human cognitive ability are enriched within the genes encoding adult but not neonatal SATB2 interactors. Our data support a shift in the function of SATB2 in cortex over lifetime and indicate that regulation of spatial chromatin architecture by the SATB2 interactome contributes to cognitive function in the general population.
Assuntos
Cognição/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Neocórtex/fisiologia , Fatores de Transcrição/genética , Adulto , Animais , Humanos , Memória de Longo Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Transcrição Gênica/genéticaRESUMO
SATB2 is a DNA binding protein that specifically binds the nuclear matrix attachment region and functions as a regulator of the transcription of large chromatin domains. Unlike its well addressed role during brain development, the role of SATB2 in adult brain is under-investigated. It has been shown that deletion of SATB2 from the forebrain of adult mice significantly impaired long-term memory for contextual fear and object recognition memory. The aim of the present study was to investigate the effects of appetitive stimuli such as cocaine and social interaction (SI) on SATB2 expression in the adult rat brain. For that, we performed conditioned place preference (CPP) to cocaine (15 mg/kg) and to SI, then assessed SATB2 expression in the brain 1 h (24 h after the last conditioning) and 24 h (48 h after the last conditioning) after the CPP test. We found that SATB2 expression in the paraventricular thalamus of rats was increased 1 h after the cocaine CPP test. This increase was selective for the cocaine-paired environment since the SI-paired environment did not increase SATB2 expression in the paraventricular thalamus. Also, the cocaine paired environment-induced increase of SATB2 levels in the paraventricular thalamus was due to cocaine conditioning as the unpaired cocaine group did not show an increase of SATB2 in the paraventricular thalamus. These results suggest that SATB2 in the paraventricular thalamus appears to be involved in the association between cocaine effects and environmental context. Further studies are needed to address the functional role of SATB2 in cocaine conditioning.
RESUMO
SATB2 is associated with schizophrenia and is an important transcription factor regulating neocortical organization and circuitry. Rare mutations in SATB2 cause a syndrome that includes developmental delay, and mouse studies identify an important role for SATB2 in learning and memory. Interacting partners BCL11B and GATAD2A are also schizophrenia risk genes indicating that other genes interacting with or are regulated by SATB2 are making a contribution to schizophrenia and cognition. We used data from Satb2 mouse models to generate three gene-sets that contain genes either functionally related to SATB2 or targeted by SATB2 at different stages of development. Each was tested for enrichment using the largest available genome-wide association studies (GWAS) datasets for schizophrenia and educational attainment (EA) and enrichment analysis was also performed for schizophrenia and other neurodevelopmental disorders using data from rare variant sequencing studies. These SATB2 gene-sets were enriched for genes containing common variants associated with schizophrenia and EA, and were enriched for genes containing rare variants reported in studies of schizophrenia, autism and intellectual disability. In the developing cortex, genes targeted by SATB2 based on ChIP-seq data, and functionally affected when SATB2 is not expressed based on differential expression analysis using RNA-seq data, show strong enrichment for genes associated with EA. For genes expressed in the hippocampus or at the synapse, those targeted by SATB2 are more strongly enriched for genes associated EA than gene-sets not targeted by SATB2. This study demonstrates that single gene findings from GWAS can provide important insights to pathobiological processes. In this case we find evidence that genes influenced by SATB2 and involved in synaptic transmission, axon guidance and formation of the corpus callosum are contributing to schizophrenia and cognition.
Assuntos
Cognição , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Transtornos do Neurodesenvolvimento/genética , Esquizofrenia/genética , Fatores de Transcrição/metabolismo , Sucesso Acadêmico , Animais , Orientação de Axônios/genética , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/metabolismo , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Genômica/métodos , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Mutação , Transtornos do Neurodesenvolvimento/patologia , Esquizofrenia/patologia , Transmissão Sináptica/genética , Fatores de Transcrição/genéticaRESUMO
Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the CACNA1A gene. As one of the few bicistronic genes discovered in the human genome, CACNA1A encodes not only the α1A subunit of the P/Q type voltage-gated Ca2+ channel CaV2.1 but also the α1ACT protein, a 75 kDa transcription factor sharing the sequence of the cytoplasmic C-terminal tail of the α1A subunit. Isoforms of both proteins contain the polyglutamine (polyQ) domain that is expanded in SCA6 patients. Although certain SCA6 phenotypes appear to be specific for Purkinje neurons, other pathogenic effects of the SCA6 polyQ mutation can affect a broad spectrum of central nervous system (CNS) neuronal subtypes. We investigated the expression and function of CACNA1A gene products in human neurons derived from induced pluripotent stem cells from two SCA6 patients. Expression levels of CACNA1A encoding α1A subunit were similar between SCA6 and control neurons, and no differences were found in the subcellular distribution of CaV2.1 channel protein. The α1ACT immunoreactivity was detected in the majority of cell nuclei of SCA6 and control neurons. Although no SCA6 genotype-dependent differences in CaV2.1 channel function were observed, they were found in the expression levels of the α1ACT target gene Granulin (GRN) and in glutamate-induced cell vulnerability.
Assuntos
Canais de Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Ataxias Espinocerebelares/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Fatores de Transcrição/metabolismo , Expansão das Repetições de Trinucleotídeos/fisiologiaRESUMO
SATB2 is a risk locus for schizophrenia and encodes a DNA-binding protein that regulates higher-order chromatin configuration. In the adult brain Satb2 is almost exclusively expressed in pyramidal neurons of two brain regions important for memory formation, the cerebral cortex and the CA1-hippocampal field. Here we show that Satb2 is required for key hippocampal functions since deletion of Satb2 from the adult mouse forebrain prevents the stabilization of synaptic long-term potentiation and markedly impairs long-term fear and object discrimination memory. At the molecular level, we find that synaptic activity and BDNF up-regulate Satb2, which itself binds to the promoters of coding and non-coding genes. Satb2 controls the hippocampal levels of a large cohort of miRNAs, many of which are implicated in synaptic plasticity and memory formation. Together, our findings demonstrate that Satb2 is critically involved in long-term plasticity processes in the adult forebrain that underlie the consolidation and stabilization of context-linked memory.
Assuntos
Regulação da Expressão Gênica , Hipocampo/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Memória de Longo Prazo , MicroRNAs/biossíntese , Fatores de Transcrição/metabolismo , Animais , Técnicas de Inativação de Genes , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
The presence of the p75 neurotrophin receptor (p75NTR) in adult basal forebrain cholinergic neurons, precursor cells in the subventricular cell layer and the subgranular cell layer of the hippocampus has been linked to alterations in learning as well as anxiety- and depression- related behaviors. In contrast to previous studies performed in a p75NTR(exon III-/-) model still expressing the short isoform of the p75NTR, we focused on locomotor and anxiety-associated behavior in p75NTR(exon IV-/-) mice lacking both p75NTR isoforms. Comparing p75NTR(exon IV-/-) and wildtype mice for both male and female animals showed an anxiolytic-like behavior as evidenced by increased central activities in the open field paradigm and flex field activity system as well as higher numbers of open arm entries in the elevated plus maze test in female p75NTR knockout mice. Morphometrical analyses of dorsal and ventral hippocampus revealed a reduction of width of the dentate gyrus and the granular cell layer in the dorsal but not ventral hippocampus in male and female p75NTR(exon IV-/-) mice. We conclude that germ-line deletion of p75NTR seems to differentially affect morphometry of dorsal and ventral dentate gyrus and that p75NTR may play a role in anxiety-like behavior, specifically in female mice.
RESUMO
We have previously shown that animals acquired robust conditioned place preference (CPP) to either social interaction alone or cocaine alone. Recently it has been reported that drugs of abuse abnormally activated p38, a member of mitogen-activated protein kinase family, in the nucleus accumbens. In this study, we aimed to investigate the expression of the activated form of p38 (pp38) in the nucleus accumbens shell and core of rats expressing either cocaine CPP or social interaction CPP 1 h, 2 h and 24 h after the CPP test. We hypothesized that cocaine CPP will increase pp38 in the nucleus accumbens shell/core as compared to social interaction CPP. Surprisingly, we found that 24 h after social interaction CPP, pp38 neuronal levels were decreased in the nucleus accumbens shell to the level of naïve rats. Control saline rats that received saline in both compartments of the CPP apparatus and cocaine CPP rats showed similar enhanced p38 activation as compared to naïve and social interaction CPP rats. We also found that the percentage of neurons expressing dopaminergic receptor D2R and pp38 was also decreased in the shell of the nucleus accumbens of social interaction CPP rats as compared to controls. Given the emerging role of p38 in stress/anxiety behaviors, these results suggest that (1) social interaction reward has anti-stress effects; (2) cocaine conditioning per se does not affect p38 activation and that (3) marginal stress is sufficient to induce p38 activation in the shell of the nucleus accumbens.
Assuntos
Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Núcleo Accumbens/enzimologia , Recompensa , Comportamento Social , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Imunofluorescência , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Núcleo Accumbens/efeitos dos fármacos , Testes Psicológicos , Distribuição Aleatória , Ratos Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Comportamento Espacial/efeitos dos fármacos , Comportamento Espacial/fisiologia , Estresse Psicológico/enzimologia , Fatores de TempoRESUMO
Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.
Assuntos
Antígenos de Diferenciação/metabolismo , Embrião de Mamíferos/metabolismo , Crista Neural/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Medula Espinal/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Fator 4 Semelhante a Kruppel , Camundongos , Crista Neural/citologia , Crista Neural/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Medula Espinal/citologia , Medula Espinal/embriologiaRESUMO
We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), differentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. We also investigated the involvement of the lateral accumbens core and the dorsal caudate putamen. The anterior cingulate 1 (Cg1) region served as a negative control. Contrary to our expectations, we found that all regions of the accumbens corridor showed increased expression of the early growth response protein 1 (EGR1, Zif268) in rats 2 h after reacquisition of CPP for cocaine after a history of cocaine CPP acquisition and extinction. Previous counterconditioning with dyadic social interaction inhibited both the reacquisition of cocaine CPP and the activation of the whole accumbens corridor. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less affected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders.
RESUMO
After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration. After sciatic nerve crush, functional recovery in vivo was retarded in SNS-gp130(-/-) mice, which specifically lack gp130 in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin from SNS-gp130(-/-) compared with control mice after nerve crush. Neurite outgrowth and STAT3 activation in vitro were severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained from SNS-gp130(-/-) mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin. Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regeneration in vitro and in vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons.
Assuntos
Receptor gp130 de Citocina/metabolismo , Regeneração Nervosa , Neuritos/metabolismo , Fator de Transcrição STAT3/metabolismo , Nervo Isquiático/fisiologia , Células Receptoras Sensoriais/metabolismo , Animais , Processos de Crescimento Celular , Células Cultivadas , Receptor gp130 de Citocina/genética , Leptina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Fosforilação , Fator de Transcrição STAT3/genética , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologiaRESUMO
The value of human disease models, which are based on induced pluripotent stem cells (iPSCs), depends on the capacity to generate specifically those cell types affected by pathology. We describe a new iPSC-based model of Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disorder with an intronic GAA repeat expansion in the frataxin gene. As the peripheral sensory neurons are particularly susceptible to neurodegeneration in FRDA, we applied a development-based differentiation protocol to generate specifically these cells. FRDA and control iPSC lines were efficiently differentiated toward neural crest progenitors and peripheral sensory neurons. The progress of the cell lines through discrete steps of in vitro differentiation was closely monitored by expression levels of key markers for peripheral neural development. Since it had been suggested that FRDA pathology might start early during ontogenesis, we investigated frataxin expression in our development-related model. A pronounced frataxin deficit was found in FRDA iPSCs and neural crest cells compared to controls. Whereas we identified an upregulation of frataxin expression during sensory specification for control cells, this increase was not observed for FRDA peripheral sensory neurons. This early failure, aggravating frataxin deficiency in a specifically vulnerable human cell population, indicates a developmental component in FRDA.
Assuntos
Diferenciação Celular , Ataxia de Friedreich/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Ligação ao Ferro/biossíntese , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células Receptoras Sensoriais/metabolismo , FrataxinaRESUMO
Dopaminergic neurons derived from pluripotent stem cells are among the best investigated products of in vitro stem cell differentiation owing to their potential use for neurorestorative therapy of Parkinson's disease. However, the classical differentiation protocols for both mouse and human pluripotent stem cells generate a limited percentage of dopaminergic neurons and yield a considerable cellular heterogeneity comprising numerous scarcely characterized cell populations. To improve pluripotent stem cell differentiation protocols for midbrain dopaminergic neurons, we established extensive and strictly quantitative gene expression profiles, including markers for pluripotent cells, neural progenitors, non-neural cells, pan-neuronal and glial cells, neurotransmitter phenotypes, midbrain and nonmidbrain populations, floor plate and basal plate populations, as well as for Hedgehog, Fgf, and Wnt signaling pathways. The profiles were applied to discrete stages of in vitro differentiation of mouse embryonic stem cells toward the dopaminergic lineage and after transplantation into the striatum of 6-hydroxy-dopamine-lesioned rats. The comparison of gene expression in vitro with stages in the developing ventral midbrain between embryonic day 11.5 and 13.5 ex vivo revealed dynamic changes in the expression of transcription factors and signaling molecules. Based on these profiles, we propose quantitative gene expression milestones that predict the efficiency of dopaminergic differentiation achieved at the end point of the protocol, already at earlier stages of differentiation.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Embrionárias/metabolismo , Mesencéfalo/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Neurônios Dopaminérgicos/fisiologia , Neurônios Dopaminérgicos/transplante , Células-Tronco Embrionárias/fisiologia , Expressão Gênica , Genes Controladores do Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Mesencéfalo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Doença de Parkinson Secundária/patologia , Doença de Parkinson Secundária/terapia , Ratos , Ratos Wistar , Transdução de Sinais , TranscriptomaRESUMO
Evidence from carefully conducted open label clinical trials suggested that therapeutic benefit can be achieved by grafting fetal dopaminergic (DAergic) neurons derived from ventral mesencephalon (VM) into the denervated striatum of Parkinson's disease (PD) patients. However, two double-blind trials generated negative results reporting deleterious side effects such as prominent dyskinesias. Heterogeneous composition of VM grafts is likely to account for suboptimal clinical efficacy.We consider that gene expression patterns of the VM tissue needs to be better understood by comparing the genetic signature of the surviving and functioning grafts with the cell suspensions used for transplantation. In addition, it is crucial to assess whether the grafted cells exhibit the DAergic phenotype of adult substantia nigra pars compacta (SNpc). To investigate this further, we used a GFP reporter mouse as source of VM tissue that enabled the detection and dissection of the grafts 6 weeks post implantation. A comparative gene expression analysis of the VM cell suspension and grafts revealed that VM grafts continue to differentiate post-implantation. In addition, implanted grafts showed a mature SNpc-like molecular DAergic phenotype with similar expression levels of TH, Vmat2 and Dat. However, by comparing gene expression of the adult SNpc with dissected grafts we detected a higher expression of progenitor markers in the grafts. Finally, when compared to the VM cell suspension, post-grafting there was a higher expression of markers inherent to glia and other neuronal populations.In summary, our data highlight the dynamic development of distinctive DAergic and non-DAergic gene expression markers associated with the maturation of VM grafts in vivo. The molecular signature of VM grafts and its functional relevance should be further explored in future studies aimed at the optimization of DAergic cell therapy approaches in PD.
Assuntos
Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Oxidopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Adrenérgicos/farmacologia , Anfetaminas/farmacologia , Animais , Transplante de Células/métodos , Galinhas , Discinesias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Marcadores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Neurônios/metabolismo , Fenótipo , Ratos , Ratos Wistar , Células-Tronco/citologia , Substância Negra/embriologia , Substância Negra/metabolismo , Fatores de TempoRESUMO
Positive social interaction could play an essential role in switching the preference of the substance dependent individual away from drug related activities. We have previously shown that conditioned place preference (CPP) for cocaine at the dose of 15 mg/kg and CPP for four 15-min episodes of social interaction were equally strong when rats were concurrently conditioned for place preference by pairing cocaine with one compartment and social interaction with the other. The aim of the present study was to investigate the differential activation of brain regions related to the reward circuitry after acquisition/expression of cocaine CPP or social interaction CPP. Our findings indicate that cocaine CPP and social interaction CPP activated almost the same brain regions. However, the granular insular cortex and the dorsal part of the agranular insular cortex were more activated after cocaine CPP, whereas the prelimbic cortex and the core subregion of the nucleus accumbens were more activated after social interaction CPP. These results suggest that the insular cortex appears to be potently activated after drug conditioning learning while activation of the prelimbic cortex-nucleus accumbens core projection seems to be preferentially involved in the conditioning to non-drug stimuli such as social interaction.