RESUMO
Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
Assuntos
Fator de Transcrição GATA4/genética , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Cromatina , Elementos Facilitadores Genéticos , Feminino , Coração/crescimento & desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genéticaRESUMO
BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. METHODS: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. RESULTS: We found that a combination of the transforming growth factor-ß inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-ß and WNT inhibition and was achieved most efficiently with GMT plus myocardin. CONCLUSIONS: Transforming growth factor-ß and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.
Assuntos
Benzamidas/farmacologia , Reprogramação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fatores de Transcrição/metabolismo , Animais , Benzamidas/uso terapêutico , Células Cultivadas , Dioxóis/uso terapêutico , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Coração/diagnóstico por imagem , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismoRESUMO
Across species, spatial memory declines with age, possibly reflecting altered hippocampal and medial entorhinal cortex (MEC) function. However, the integrity of cellular and network-level spatial coding in aged MEC is unknown. Here, we leveraged in vivo electrophysiology to assess MEC function in young, middle-aged, and aged mice navigating virtual environments. In aged grid cells, we observed impaired stabilization of context-specific spatial firing, correlated with spatial memory deficits. Additionally, aged grid networks shifted firing patterns often but with poor alignment to context changes. Aged spatial firing was also unstable in an unchanging environment. In these same mice, we identified 458 genes differentially expressed with age in MEC, 61 of which had expression correlated with spatial firing stability. These genes were enriched among interneurons and related to synaptic transmission. Together, these findings identify coordinated transcriptomic, cellular, and network changes in MEC implicated in impaired spatial memory in aging.
RESUMO
Loss of cognitive function with age is devastating. EGL-30/GNAQ and Gαq signaling pathways are highly conserved between C. elegans and mammals, and murine Gnaq is enriched in hippocampal neurons and declines with age. We found that activation of EGL-30 in aged worms triples memory span, and GNAQ gain of function significantly improved memory in aged mice: GNAQ(gf) in hippocampal neurons of 24-month-old mice (equivalent to 70- to 80-year-old humans) rescued age-related impairments in well-being and memory. Single-nucleus RNA sequencing revealed increased expression of genes regulating synaptic function, axon guidance, and memory in GNAQ-treated mice, and worm orthologs of these genes were required for long-term memory extension in worms. These experiments demonstrate that C. elegans is a powerful model to identify mammalian regulators of memory, leading to the identification of a pathway that improves memory in extremely old mice. To our knowledge, this is the oldest age at which an intervention has improved age-related cognitive decline.
Assuntos
Caenorhabditis elegans , Cognição , Humanos , Animais , Camundongos , Idoso , Pré-Escolar , Idoso de 80 Anos ou mais , Caenorhabditis elegans/metabolismo , Cognição/fisiologia , Transdução de Sinais/fisiologia , Neurônios/metabolismo , Memória/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Hipocampo/metabolismo , Envelhecimento/metabolismo , Mamíferos/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismoRESUMO
DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.
Assuntos
Dioxigenases , Proteínas Proto-Oncogênicas , Animais , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina/metabolismo , Dioxigenases/genética , Metilação de DNA/genética , Cognição , Neurônios/metabolismo , Hipocampo/metabolismoRESUMO
Parvalbumin-positive (PV+) interneurons play a critical role in maintaining circuit rhythm in the brain, and their reduction is implicated in autism spectrum disorders. Animal studies demonstrate that maternal immune activation (MIA) leads to reduced PV+ interneurons in the somatosensory cortex and autism-like behaviors. However, the underlying molecular mechanisms remain largely unknown. Here, we show that MIA down-regulates microglial Gpr56 expression in fetal brains in an interleukin-17a-dependent manner and that conditional deletion of microglial Gpr56 [Gpr56 conditional knockout (cKO)] mimics MIA-induced PV+ interneuron defects and autism-like behaviors in offspring. We further demonstrate that elevated microglial tumor necrosis factor-α expression is the underlying mechanism by which MIA and Gpr56 cKO impair interneuron generation. Genetically restoring Gpr56 expression in microglia ameliorates PV+ interneuron deficits and autism-like behaviors in MIA offspring. Together, our study demonstrates that microglial GPR56 plays an important role in PV+ interneuron development and serves as a salient target of MIA-induced neurodevelopmental disorders.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Modelos Animais de Doenças , Interneurônios/metabolismo , Microglia/metabolismo , Parvalbuminas/metabolismoRESUMO
Remyelination declines in the aging central nervous system due to oligodendrocyte precursor cell (OPC) dysfunction. In the latest issue of Cell Stem Cell, Neumann et al. (2019) demonstrate that aged OPCs are amenable to functional rejuvenation by systemic interventions involving alternate-day fasting or treatment with the fasting mimetic metformin.
Assuntos
Metformina , Remielinização , Diferenciação Celular , Dieta , Oligodendroglia , Células-TroncoRESUMO
Mounting evidence in animal models indicates potential for rejuvenation of cellular and cognitive functions in the aging brain. However, the ability to utilize this potential is predicated on identifying molecular targets that reverse the effects of aging in vulnerable regions of the brain, such as the hippocampus. The dynamic post-translational modification O-linked N-Acetylglucosamine (O-GlcNAc) has emerged as an attractive target for regulating aging-specific synaptic alterations as well as neurodegeneration. While speculation exists about the role of O-GlcNAc in neurodegenerative conditions, such as Alzheimer's disease, its role in physiological brain aging remains largely unexplored. Here, we report that countering age-related decreased O-GlcNAc transferase (OGT) expression and O-GlcNAcylation ameliorates cognitive impairments in aged mice. Mimicking an aged condition in young adults by abrogating OGT, using a temporally controlled neuron-specific conditional knockout mouse model, recapitulated cellular and cognitive features of brain aging. Conversely, overexpressing OGT in mature hippocampal neurons using a viral-mediated approach enhanced associative fear memory in young adult mice. Excitingly, in aged mice overexpressing neuronal OGT in the aged hippocampus rescued in part age-related impairments in spatial learning and memory as well as associative fear memory. Our data identify O-GlcNAcylaton as a key molecular mediator promoting cognitive rejuvenation.
Assuntos
Acetilglucosamina/metabolismo , Envelhecimento/fisiologia , Cognição/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , Acilação , Animais , Masculino , Camundongos , Camundongos KnockoutRESUMO
Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell's identity. To determine the molecular mechanisms by which Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF's binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.