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1.
EMBO J ; 40(21): e107532, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34549820

RESUMO

Astrocytes regulate brain-wide functions and also show region-specific differences, but little is known about how general and region-specific functions are aligned at the single-cell level. To explore this, we isolated adult mouse diencephalic astrocytes by ACSA-2-mediated magnetic-activated cell sorting (MACS). Single-cell RNA-seq revealed 7 gene expression clusters of astrocytes, with 4 forming a supercluster. Within the supercluster, cells differed by gene expression related to ion homeostasis or metabolism, with the former sharing gene expression with other regions and the latter being restricted to specific regions. All clusters showed expression of proliferation-related genes, and proliferation of diencephalic astrocytes was confirmed by immunostaining. Clonal analysis demonstrated low level of astrogenesis in the adult diencephalon, but not in cerebral cortex grey matter. This led to the identification of Smad4 as a key regulator of diencephalic astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocytes show diverse gene expression states related to distinct functions with some subsets being more widespread while others are more regionally restricted. However, all share low-level proliferation revealing the novel concept of adult astrogenesis in the diencephalon.


Assuntos
Astrócitos/metabolismo , Linhagem da Célula/genética , Diencéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Proteína Smad4/genética , Animais , Astrócitos/classificação , Astrócitos/citologia , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Diencéfalo/citologia , Diencéfalo/crescimento & desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Substância Cinzenta/citologia , Substância Cinzenta/crescimento & desenvolvimento , Substância Cinzenta/metabolismo , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anotação de Sequência Molecular , Família Multigênica , Transdução de Sinais , Proteína Smad4/metabolismo
2.
Nat Commun ; 15(1): 2866, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38570482

RESUMO

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.


Assuntos
Lesões Encefálicas , Ferimentos Perfurantes , Animais , Camundongos , Masculino , Proteína Glial Fibrilar Ácida/metabolismo , Neuroglia/metabolismo , Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Ferimentos Perfurantes/complicações , Ferimentos Perfurantes/metabolismo
3.
Nat Protoc ; 6(12): 1847-59, 2011 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-22051798

RESUMO

A comprehensive understanding of the cell biology of adult neural stem cells (aNSCs) requires direct observation of aNSC division and lineage progression in the absence of niche-dependent signals. Here we describe a culture preparation of the adult mouse subependymal zone (SEZ), which allows for continuous single-cell tracking of aNSC behavior. The protocol involves the isolation (approximately 3 h) and culture of cells from the adult SEZ at low density in the absence of mitogenic growth factors in chemically defined medium and subsequent live imaging using time-lapse video microscopy (5-7 d); these steps are followed by postimaging immunocytochemistry to identify progeny (approximately 7 h). This protocol enables the observation of the progression from slow-dividing aNSCs of radial/astroglial identity up to the neuroblast stage, involving asymmetric and symmetric cell divisions of distinct fast-dividing precursors. This culture provides an experimental system for studying instructive or permissive effects of signal molecules on aNSC modes of cell division and lineage progression.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Neurais/citologia , Animais , Encéfalo/citologia , Adesão Celular , Divisão Celular , Linhagem da Célula , Genes Reporter , Camundongos , Camundongos Endogâmicos C57BL , Software , Imagem com Lapso de Tempo , Gravação em Vídeo
4.
Nat Protoc ; 6(2): 214-28, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21293461

RESUMO

Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7-21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.


Assuntos
Astrócitos/citologia , Diferenciação Celular , Córtex Cerebral/citologia , Neurônios/citologia , Animais , Astrócitos/virologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Técnicas de Cultura de Células , Linhagem da Célula , Meios de Cultura , Eletrofisiologia , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Medicina Regenerativa/métodos , Retroviridae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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