RESUMO
Neuromodulators transform animal behaviors. Recent research has demonstrated the importance of both sustained and transient change in neuromodulators, likely due to tonic and phasic neuromodulator release. However, no method could simultaneously record both types of dynamics. Fluorescence lifetime of optical reporters could offer a solution because it allows high temporal resolution and is impervious to sensor expression differences across chronic periods. Nevertheless, no fluorescence lifetime change across the entire classes of neuromodulator sensors was previously known. Unexpectedly, we find that several intensity-based neuromodulator sensors also exhibit fluorescence lifetime responses. Furthermore, we show that lifetime measures in vivo neuromodulator dynamics both with high temporal resolution and with consistency across animals and time. Thus, we report a method that can simultaneously measure neuromodulator change over transient and chronic time scales, promising to reveal the roles of multi-time scale neuromodulator dynamics in diseases, in response to therapies, and across development and aging.
Assuntos
Envelhecimento , Neurotransmissores , Animais , Neurotransmissores/metabolismoRESUMO
Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.
Assuntos
Processamento de Proteína Pós-Traducional , Transdução de Sinais , Camundongos , Animais , Fosforilação , Neurotransmissores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
Astrocyte-specific ion pump α2-Na+/K+-ATPase plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Here, we test the effect of Atp1a2 mRNA-specific antisense oligonucleotides (ASOs) to induce α2-Na+/K+-ATPase knockdown in the widely used ALS animal model, SOD1*G93A mice. Two ASOs led to efficient Atp1a2 knockdown and significantly reduced SOD1 aggregation in vivo. Although Atp1a2 ASO-treated mice displayed no off-target or systemic toxicity, the ASO-treated mice exhibited an accelerated disease onset and shorter lifespan than control mice. Transcriptomics studies reveal downregulation of genes involved in oxidative response, metabolic pathways, trans-synaptic signaling, and upregulation of genes involved in glutamate receptor signaling and complement activation, suggesting a potential role for these molecular pathways in de-coupling SOD1 aggregation from survival in Atp1a2 ASO-treated mice. Together, these results reveal a role for α2-Na+/K+-ATPase in SOD1 aggregation and highlight the critical effect of temporal modulation of genetically validated therapeutic targets in neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Camundongos , Esclerose Lateral Amiotrófica/patologia , Astrócitos/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios Motores/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive, cannot easily target sparse or hard-to-transfect/infect cell types, and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.
RESUMO
Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.
Assuntos
Astrócitos/metabolismo , Ataxia/genética , Metaboloma/genética , Enxaqueca com Aura/genética , ATPase Trocadora de Sódio-Potássio/genética , Transcriptoma , Animais , Astrócitos/patologia , Ataxia/metabolismo , Ataxia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Neuroimagem Funcional , Glicina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Enxaqueca com Aura/metabolismo , Enxaqueca com Aura/patologia , Teste de Desempenho do Rota-Rod , Serina/metabolismo , ATPase Trocadora de Sódio-Potássio/deficiênciaRESUMO
Neuronal-activity-dependent transcription couples sensory experience to adaptive responses of the brain including learning and memory. Mechanisms of activity-dependent gene expression including alterations of the epigenome have been characterized1-8. However, the fundamental question of whether sensory experience remodels chromatin architecture in the adult brain in vivo to induce neural code transformations and learning and memory remains to be addressed. Here we use in vivo calcium imaging, optogenetics and pharmacological approaches to show that granule neuron activation in the anterior dorsal cerebellar vermis has a crucial role in a delay tactile startle learning paradigm in mice. Of note, using large-scale transcriptome and chromatin profiling, we show that activation of the motor-learning-linked granule neuron circuit reorganizes neuronal chromatin including through long-distance enhancer-promoter and transcriptionally active compartment interactions to orchestrate distinct granule neuron gene expression modules. Conditional CRISPR knockout of the chromatin architecture regulator cohesin in anterior dorsal cerebellar vermis granule neurons in adult mice disrupts enhancer-promoter interactions, activity-dependent transcription and motor learning. These findings define how sensory experience patterns chromatin architecture and neural circuit coding in the brain to drive motor learning.
Assuntos
Retroalimentação Sensorial , Genoma , Aprendizagem/fisiologia , Destreza Motora/fisiologia , Vias Neurais , Plasticidade Neuronal/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Vermis Cerebelar/citologia , Vermis Cerebelar/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Feminino , Masculino , Camundongos , Fibras Musgosas Hipocampais , Regiões Promotoras Genéticas/genética , Células de Purkinje , Reflexo de SobressaltoRESUMO
In this Letter, '≥' should be '≤' in the sentence: "Intra-chromosomal reads were further split into short-range reads (≥1 kb) and long-range reads (>1 kb)". This error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.
Assuntos
Encéfalo/citologia , Proliferação de Células , Cerebelo/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Animais , Comportamento Animal , Piscadela/fisiologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/genética , Cerebelo/citologia , Biologia Computacional , Grânulos Citoplasmáticos/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Genes myc/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/fisiologiaRESUMO
Although ubiquitin ligases have been implicated in autism, their roles and mechanisms in brain development remain incompletely understood. Here, we report that in vivo knockdown or conditional knockout of the autism-linked ubiquitin ligase RNF8 or associated ubiquitin-conjugating enzyme UBC13 in rodent cerebellar granule neurons robustly increases the number of parallel fiber presynaptic boutons and functional parallel fiber/Purkinje cell synapses. In contrast to the role of nuclear RNF8 in proliferating cells, RNF8 operates in the cytoplasm in neurons to suppress synapse differentiation in vivo. Proteomics analyses reveal that neuronal RNF8 interacts with the HECT domain protein HERC2 and scaffold protein NEURL4, and knockdown of HERC2 or NEURL4 phenocopies the inhibition of RNF8/UBC13 signaling on synapse differentiation. In behavior analyses, granule neuron-specific knockout of RNF8 or UBC13 impairs cerebellar-dependent learning. Our study defines RNF8 and UBC13 as components of a novel cytoplasmic ubiquitin-signaling network that suppresses synapse formation in the brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Neurônios/metabolismo , Sinapses/ultraestrutura , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Animais , Cerebelo/citologia , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Microscopia Eletrônica , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Proteômica , Células de Purkinje/metabolismo , Células de Purkinje/ultraestrutura , Ratos , Transdução de Sinais , Sinapses/metabolismoRESUMO
Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating messenger RNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain.
Assuntos
Cerebelo/fisiologia , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Dendritos/fisiologia , Inativação Gênica , Neurônios/fisiologia , Animais , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Epithelial-mesenchymal transition (EMT) is a fundamental cellular process that contributes to epithelial tissue morphogenesis during normal development and in tumor invasiveness and metastasis. The transcriptional regulator SnoN robustly influences EMT in response to the cytokine TGFß, but the mechanisms that regulate the fundamental role of SnoN in TGFß-induced EMT are not completely understood. Here we employ interaction proteomics to uncover the signaling protein TIF1γ as a specific interactor of SnoN1 but not the closely related isoform SnoN2. A 16-amino acid peptide within a unique region of SnoN1 mediates the interaction of SnoN1 with TIF1γ. Strikingly, although TIF1γ is thought to act as a ubiquitin E3 ligase, we find that TIF1γ operates as a small ubiquitin-like modifier (SUMO) E3 ligase that promotes the sumoylation of SnoN1 at distinct lysine residues. Importantly, TIF1γ-induced sumoylation is required for the ability of SnoN1 to suppress TGFß-induced EMT, as assayed by the disruption of the morphogenesis of acini in a physiologically relevant three-dimensional model of normal murine mammary gland (NMuMG) epithelial cells. Collectively, our findings define a novel TIF1γ-SnoN1 sumoylation pathway that plays a critical role in EMT and has important implications for our understanding of TGFß signaling and diverse biological processes in normal development and cancer biology.
Assuntos
Transição Epitelial-Mesenquimal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sumoilação/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.
Assuntos
Axônios/enzimologia , Quinase 3 da Glicogênio Sintase/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Feminino , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Nestina/genética , Neurônios/enzimologia , Neurônios/ultraestrutura , Fosforilação/fisiologia , Pseudópodes/enzimologia , Serina/metabolismo , Fator de Resposta Sérica/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Signal regulatory protein alpha (SIRPα) is a cell surface glycoprotein with inhibitory functions, which may regulate neutrophil transmigration. SIRPα is mobilized to the neutrophil surface from specific granules, gelatinase granules, and secretory vesicles following inflammatory activation in vitro and in vivo. The lack of SIRPα signaling and the ability to upregulate SIRPα to the cell surface promote neutrophil accumulation during inflammation in vivo.
Assuntos
Membrana Celular/metabolismo , Neutrófilos/imunologia , Vesículas Secretórias/metabolismo , Adulto , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Degranulação Celular/genética , Células Cultivadas , Retroalimentação Fisiológica , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Transporte Proteico , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismoRESUMO
Thrombospondin-1 (TSP1) limits the angiogenic and vasodilator activities of NO. This activity of TSP1 can be beneficial in some disease states, but endogenous TSP1 limits recovery of tissue perfusion following fixed ischemic injury in dorsal skin flaps in mice. Using mice lacking the TSP1 receptors CD36 or CD47, we now show that CD47 is the necessary receptor for limiting NO-mediated vascular smooth muscle relaxation and tissue survival following ischemic injury in skin flaps and hindlimbs. We further show that blocking CD47 or TSP1 using monoclonal antibodies and decreasing CD47 expression using an antisense morpholino oligonucleotide are effective therapeutic approaches to dramatically increase survival of soft tissue subjected to fixed ischemia. These treatments facilitate rapid vascular remodeling to restore tissue perfusion and increase skin and muscle viability. Thus, limiting CD47-dependent antagonism of NO-mediated vasodilation and vascular remodeling is a promising therapeutic modality to preserve tissues subject to ischemic stress.