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1.
bioRxiv ; 2024 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-39131318

RESUMO

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

2.
bioRxiv ; 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38948843

RESUMO

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

3.
Elife ; 122024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38319699

RESUMO

Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to map circuits at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNA-seq, and 4130 retrogradely labeled cells and 2914 transsynaptically labeled cells in situ. We found that the transcriptomic identities of rabies virus-infected cells can be robustly identified using both single-cell RNA-seq and in situ sequencing. By associating gene expression with connectivity inferred from barcode sequencing, we distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.


In the brain, messages are relayed from one cell to the next through intricate networks of axons and dendrites that physically interact at junctions known as synapses. Mapping out this synaptic connectivity ­ that is, exactly which neurons are connected via synapses ­ remains a major challenge. Monosynaptic tracing is a powerful approach that allows neuroscientists to explore neural networks by harnessing viruses which spread between neurons via synapses, in particular the rabies virus. This pathogen travels exclusively from 'postsynaptic' to 'presynaptic' neurons ­ from the cell that receives a message at a synapse, back to the one that sends it. A modified variant of the rabies virus can therefore be used to reveal the presynaptic cells connecting to a population of neurons in which it has been originally introduced. However, this method does not allow scientists to identify the exact postsynaptic neuron that each presynaptic cell is connected to. One way to bypass this issue is to combine monosynaptic tracing with RNA barcoding to create distinct versions of the modified rabies virus, which are then introduced into separate populations of neurons. Tracking the spread of each version allows neuroscientists to spot exactly which presynaptic cells signal to each postsynaptic neuron. So far, this approach has been used to examine synaptic connectivity in neurons grown in the laboratory, but it remains difficult to apply it to neurons in the brain. In response, Zhang, Jin et al. aimed to demonstrate how monosynaptic tracing that relies on barcoded rabies viruses could be used to dissect neural networks in the mouse brain. First, they confirmed that it was possible to accurately detect which version of the virus had spread to presynaptic neurons using both in situ and single-cell RNA sequencing. Next, they described how this information could be analysed to build models of potential neural networks, and what type of additional experiments are required for this work. Finally, they used the approach to identify neurons that tend to connect to the same postsynaptic cells and then investigated what these have in common, showing how the technique enables a finer understanding of neural circuits. Overall, the work by Zhang, Jin et al. provides a comprehensive review of the requirements and limitations associated with monosynaptic tracing experiments based on barcoded rabies viruses, as well as how the approach could be optimized in the future. This information will be of broad interest to scientists interested in mapping neural networks in the brain.


Assuntos
Vírus da Raiva , Animais , Camundongos , Vírus da Raiva/genética , Neuroanatomia , Neurônios , Análise de Sequência de RNA , RNA
4.
Nature ; 624(7991): 403-414, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38092914

RESUMO

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.


Assuntos
Encéfalo , Perfilação da Expressão Gênica , Vias Neurais , Neurônios , Medula Espinal , Animais , Camundongos , Hipotálamo , Neurônios/metabolismo , Neuropeptídeos , Medula Espinal/citologia , Medula Espinal/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Neurotransmissores , Mesencéfalo/citologia , Formação Reticular/citologia , Eletrofisiologia , Cerebelo/citologia , Córtex Cerebral/citologia
5.
Nature ; 624(7991): 317-332, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38092916

RESUMO

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.


Assuntos
Encéfalo , Perfilação da Expressão Gênica , Transcriptoma , Animais , Camundongos , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/metabolismo , Conjuntos de Dados como Assunto , Hibridização in Situ Fluorescente , Vias Neurais , Neurônios/classificação , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , RNA/análise , Análise da Expressão Gênica de Célula Única , Fatores de Transcrição/metabolismo , Transcriptoma/genética
6.
bioRxiv ; 2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-37034735

RESUMO

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single-cell RNA-sequencing (scRNA-seq) dataset of ~7 million cells profiled, and a spatially resolved transcriptomic dataset of ~4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

7.
bioRxiv ; 2023 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-38168182

RESUMO

Biological aging can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function. Aging is a complex and dynamic process which influences distinct cell types in a myriad of ways. The cellular architecture of the mammalian brain is heterogeneous and diverse, making it challenging to identify precise areas and cell types of the brain that are more susceptible to aging than others. Here, we present a high-resolution single-cell RNA sequencing dataset containing ~1.2 million high-quality single-cell transcriptomic profiles of brain cells from young adult and aged mice across both sexes, including areas spanning the forebrain, midbrain, and hindbrain. We find age-associated gene expression signatures across nearly all 130+ neuronal and non-neuronal cell subclasses we identified. We detect the greatest gene expression changes in non-neuronal cell types, suggesting that different cell types in the brain vary in their susceptibility to aging. We identify specific, age-enriched clusters within specific glial, vascular, and immune cell types from both cortical and subcortical regions of the brain, and specific gene expression changes associated with cell senescence, inflammation, decrease in new myelination, and decreased vasculature integrity. We also identify genes with expression changes across multiple cell subclasses, pointing to certain mechanisms of aging that may occur across wide regions or broad cell types of the brain. Finally, we discover the greatest gene expression changes in cell types localized to the third ventricle of the hypothalamus, including tanycytes, ependymal cells, and Tbx3+ neurons found in the arcuate nucleus that are part of the neuronal circuits regulating food intake and energy homeostasis. These findings suggest that the area surrounding the third ventricle in the hypothalamus may be a hub for aging in the mouse brain. Overall, we reveal a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal aging that will serve as a foundation for the investigation of functional changes in the aging process and the interaction of aging and diseases.

8.
Proc Natl Acad Sci U S A ; 119(18): e2115638119, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35476521

RESUMO

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.


Assuntos
Proteínas de Ciclo Celular , Ciclinas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Ciclinas/genética , Ciclinas/metabolismo , Células-Tronco/metabolismo
9.
Cell ; 184(12): 3222-3241.e26, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34004146

RESUMO

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.


Assuntos
Hipocampo/citologia , Neocórtex/citologia , Transcriptoma/genética , Animais , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
10.
Neuron ; 109(9): 1449-1464.e13, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33789083

RESUMO

Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.


Assuntos
Encéfalo/citologia , Neurônios/classificação , Neurônios/citologia , Análise de Célula Única/métodos , Animais , Conjuntos de Dados como Assunto , Dependovirus , Humanos , Camundongos , Camundongos Transgênicos
11.
Neuron ; 109(3): 545-559.e8, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33290731

RESUMO

The evolutionarily conserved default mode network (DMN) is a distributed set of brain regions coactivated during resting states that is vulnerable to brain disorders. How disease affects the DMN is unknown, but detailed anatomical descriptions could provide clues. Mice offer an opportunity to investigate structural connectivity of the DMN across spatial scales with cell-type resolution. We co-registered maps from functional magnetic resonance imaging and axonal tracing experiments into the 3D Allen mouse brain reference atlas. We find that the mouse DMN consists of preferentially interconnected cortical regions. As a population, DMN layer 2/3 (L2/3) neurons project almost exclusively to other DMN regions, whereas L5 neurons project in and out of the DMN. In the retrosplenial cortex, a core DMN region, we identify two L5 projection types differentiated by in- or out-DMN targets, laminar position, and gene expression. These results provide a multi-scale description of the anatomical correlates of the mouse DMN.


Assuntos
Encéfalo/diagnóstico por imagem , Rede de Modo Padrão/diagnóstico por imagem , Rede Nervosa/diagnóstico por imagem , Neurônios/fisiologia , Animais , Encéfalo/citologia , Conectoma , Rede de Modo Padrão/citologia , Imageamento por Ressonância Magnética , Camundongos , Rede Nervosa/citologia , Neurônios/citologia
12.
Science ; 366(6466): 734-738, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31699935

RESUMO

Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3' untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.


Assuntos
Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/metabolismo , Fator de Transcrição PAX3/genética , Poliadenilação , Regiões 3' não Traduzidas , Animais , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Mutantes , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo
13.
Cell Stem Cell ; 24(2): 213-225, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30735649

RESUMO

Stem cells can reside in a state of reversible growth arrest, or quiescence, for prolonged periods of time. Although quiescence has long been viewed as a dormant, low-activity state, increasing evidence suggests that quiescence represents states of poised potential and active restraint, as stem cells "idle" in anticipation of activation, proliferation, and differentiation. Improved understanding of quiescent stem cell dynamics is leading to novel approaches to enhance maintenance and repair of aged or diseased tissues. In this Review, we discuss recent advances in our understanding of stem cell quiescence and techniques enabling more refined analyses of quiescence in vivo.


Assuntos
Ciclo Celular , Células-Tronco/citologia , Células-Tronco Adultas/citologia , Animais , Humanos , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Cell Rep ; 21(7): 1994-2004, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141228

RESUMO

Muscle stem cells (MuSCs) persist in a quiescent state and activate in response to specific stimuli. The quiescent state is both actively maintained and dynamically regulated. However, analyses of quiescence have come primarily from cells removed from their niche. Although these cells are still quiescent, biochemical changes certainly occur during the isolation process. Here, we analyze the transcriptome of MuSCs in vivo utilizing MuSC-specific labeling of RNA. Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes. However, using the transcription inhibitor α-amanitin, we show that the ex vivo transcriptome remains largely reflective of the in vivo transcriptome. Together, these data provide perspective on the molecular regulation of the quiescent state at the transcriptional level, demonstrate the utility of these tools for probing transcriptional dynamics in vivo, and provide an invaluable resource for understanding stem cell state transitions.


Assuntos
Perfilação da Expressão Gênica/métodos , Mioblastos/metabolismo , Transcriptoma , Animais , Perfilação da Expressão Gênica/normas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/citologia
15.
Proc Natl Acad Sci U S A ; 114(43): E8996-E9005, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29073096

RESUMO

Tissue regeneration depends on the timely activation of adult stem cells. In skeletal muscle, the adult stem cells maintain a quiescent state and proliferate upon injury. We show that muscle stem cells (MuSCs) use direct translational repression to maintain the quiescent state. High-resolution single-molecule and single-cell analyses demonstrate that quiescent MuSCs express high levels of Myogenic Differentiation 1 (MyoD) transcript in vivo, whereas MyoD protein is absent. RNA pulldowns and costainings show that MyoD mRNA interacts with Staufen1, a potent regulator of mRNA localization, translation, and stability. Staufen1 prevents MyoD translation through its interaction with the MyoD 3'-UTR. MuSCs from Staufen1 heterozygous (Staufen1+/-) mice have increased MyoD protein expression, exit quiescence, and begin proliferating. Conversely, blocking MyoD translation maintains the quiescent phenotype. Collectively, our data show that MuSCs express MyoD mRNA and actively repress its translation to remain quiescent yet primed for activation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína MyoD/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Camundongos , Células Musculares/fisiologia , Proteína MyoD/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
16.
Pediatr Res ; 78(5): 520-6, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26270577

RESUMO

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.


Assuntos
Encéfalo/cirurgia , Hipóxia-Isquemia Encefálica/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Cognição , Modelos Animais de Doenças , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Hipóxia-Isquemia Encefálica/psicologia , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Camundongos Endogâmicos C57BL , Atividade Motora , Proteína Básica da Mielina/metabolismo , Reconhecimento Psicológico , Recuperação de Função Fisiológica , Medição de Risco , Fatores de Tempo
17.
Dev Neurosci ; 37(2): 142-52, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25765537

RESUMO

Neonatal encephalopathy due to perinatal hypoxia-ischemia (HI) is a severe condition, and current treatment options are limited. Expression of endogenous osteopontin (OPN), a multifunction glycoprotein, is strongly upregulated in the brain after neonatal HI. Intracerebrally administered OPN has been shown to be neuroprotective following experimental neonatal HI and adult stroke. In the present study, we determined whether intranasal, intraperitoneal or intracerebral treatment with a smaller TAT-OPN peptide is neuroprotective in neonatal mice with HI brain damage. The TAT-OPN peptide exerts bioactivity as it was as potent as full-length OPN in inducing cell adhesion in an in vitro adhesion assay. Intranasal administration of TAT-OPN peptide immediately after HI (T0) or in a repetitive treatment schedule of T0, 3 h, day (D) 1, 2 and 3 after HI did not protect cerebral gray or white matter after HI. Intraperitoneal TAT-OPN treatment at T0 or in two extended treatment schedules (D5, 7, 9, 11, 13, 15 after HI or T0, D1, 3, 5, 7, 9, 11, 13 and 15 after HI) did not result in neuroprotection either. Moreover, no functional improvement (cylinder rearing test and adhesive removal task) was observed following TAT-OPN treatment in any of the intraperitoneal treatment schedules. We validated that the TAT-OPN peptide reached the brain after intranasal or intraperitoneal administration by using an HIV-TAT staining. Finally, also intracerebral administration of the TAT-OPN peptide 1 h after HI did not reduce cerebral damage. Our data show that administration of the TAT-OPN peptide did not exert neuroprotective effects on neonatal HI-induced brain injury or sensorimotor behavioral deficits.


Assuntos
Hipóxia-Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores , Osteopontina/administração & dosagem , Osteopontina/farmacologia , Administração Intranasal , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Infusões Intralesionais , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
19.
PLoS One ; 9(6): e90584, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24603553

RESUMO

Subarachnoid hemorrhage (SAH) represents a considerable health problem with an incidence of 6-7 per 100.000 individuals per year in Western society. We investigated the long-term consequences of SAH on behavior, neuroinflammation and gray- and white-matter damage using an endovascular puncture model in Wistar rats. Rats were divided into a mild or severe SAH group based on their acute neurological score at 24 h post-SAH. The degree of hemorrhage determined in post-mortem brains at 48 h strongly correlated with the acute neurological score. Severe SAH induced increased TNF-α, IL-1ß, IL-10, MCP-1, MIP2, CINC-1 mRNA expression and cortical neutrophil influx at 48 h post-insult. Neuroinflammation after SAH was very long-lasting and still present at day 21 as determined by Iba-1 staining (microglia/macrophages) and GFAP (astrocytes). Long-term neuroinflammation was strongly associated with the degree of severity of SAH. Cerebral damage to gray- and white-matter was visualized by immunohistochemistry for MAP2 and MBP at 21 days after SAH. Severe SAH induced significant gray- and white-matter damage. MAP2 loss at day 21 correlated significantly with the acute neurological score determined at 24 h post-SAH. Sensorimotor behavior, determined by the adhesive removal task and von Frey test, was affected after severe SAH at day 21. In conclusion, we are the first to show that SAH induces ongoing cortical inflammation. Moreover, SAH induces mainly cortical long-term brain damage, which is associated with long-term sensorimotor damage.


Assuntos
Córtex Cerebral/patologia , Encefalite/fisiopatologia , Hemorragia Subaracnóidea/fisiopatologia , Animais , Dano Encefálico Crônico/imunologia , Córtex Cerebral/imunologia , Encefalite/imunologia , Macrófagos/imunologia , Masculino , Neutrófilos/imunologia , Transtornos Psicomotores/imunologia , Ratos Wistar , Recuperação de Função Fisiológica , Hemorragia Subaracnóidea/imunologia
20.
J Clin Invest ; 123(12): 5023-34, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24231349

RESUMO

Chronic pain is a major clinical problem, yet the mechanisms underlying the transition from acute to chronic pain remain poorly understood. In mice, reduced expression of GPCR kinase 2 (GRK2) in nociceptors promotes cAMP signaling to the guanine nucleotide exchange factor EPAC1 and prolongs the PGE2-induced increase in pain sensitivity (hyperalgesia). Here we hypothesized that reduction of GRK2 or increased EPAC1 in dorsal root ganglion (DRG) neurons would promote the transition to chronic pain. We used 2 mouse models of hyperalgesic priming in which the transition from acute to chronic PGE2-induced hyperalgesia occurs. Hyperalgesic priming with carrageenan induced a sustained decrease in nociceptor GRK2, whereas priming with the PKCε agonist ΨεRACK increased DRG EPAC1. When either GRK2 was increased in vivo by viral-based gene transfer or EPAC1 was decreased in vivo, as was the case for mice heterozygous for Epac1 or mice treated with Epac1 antisense oligodeoxynucleotides, chronic PGE2-induced hyperalgesia development was prevented in the 2 priming models. Using the CFA model of chronic inflammatory pain, we found that increasing GRK2 or decreasing EPAC1 inhibited chronic hyperalgesia. Our data suggest that therapies targeted at balancing nociceptor GRK2 and EPAC1 levels have promise for the prevention and treatment of chronic pain.


Assuntos
Dor Crônica/prevenção & controle , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Hiperalgesia/fisiopatologia , Animais , Carragenina/toxicidade , Bovinos , Dor Crônica/etiologia , Dor Crônica/genética , Dor Crônica/fisiopatologia , AMP Cíclico/fisiologia , Dinoprostona/fisiologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Inativação Gênica , Terapia Genética , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Membro Posterior/inervação , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/terapia , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Nociceptores/enzimologia , Nociceptores/fisiologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Oligopeptídeos/toxicidade , Proteínas Recombinantes de Fusão/genética , Nervo Isquiático/patologia , Sistemas do Segundo Mensageiro , Células Receptoras Sensoriais/enzimologia , Células Receptoras Sensoriais/fisiologia
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