RESUMO
The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.
Assuntos
Córtex Cerebral/metabolismo , Neurônios/metabolismo , Antagonistas do Receptor Purinérgico P2X/administração & dosagem , Receptores Purinérgicos P2X7/genética , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/química , Humanos , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologiaRESUMO
As a result of brain injury, astrocytes become activated and start to proliferate in the vicinity of the injury site. Recently, we had demonstrated that these reactive astrocytes, or glia, can form self-renewing and multipotent neurospheres in vitro. In the present study, we demonstrate that it is only invasive injury, such as stab wounding or cerebral ischemia, and not noninvasive injury conditions, such as chronic amyloidosis or induced neuronal death, that can elicit this increase in plasticity. Furthermore, we find that Sonic hedgehog (SHH) is the signal that acts directly on the astrocytes and is necessary and sufficient to elicit the stem cell response both in vitro and in vivo. These findings provide a molecular basis for how cells with neural stem cell lineage emerge at sites of brain injury and imply that the high levels of SHH known to enter the brain from extraneural sources after invasive injury can trigger this response.
Assuntos
Lesões Encefálicas/patologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Lesões Encefálicas/complicações , Lesões Encefálicas/metabolismo , Morte Celular , Proliferação de Células , Separação Celular , Córtex Cerebral/patologia , Modelos Animais de Doenças , Gliose/complicações , Gliose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologiaRESUMO
The stem cell niche plays an important role for the maintenance and differentiation of neural stem/progenitor cells (NSPCs). It is composed of distinct cell types that influence NSCPs by the release of paracrine factors, and a specialized extracellular matrix that structures the NSPC environment. During the past years, several components of the neural stem cell (NSC) niche could be deciphered on the molecular level. One prominent constituent is the tenascin-C (Tnc) glycoprotein and its isoforms that intervene in NSPC proliferation and differentiation. Distinct chondroitin sulfate proteoglycans (CSPGs) associate with Tnc in the niche territory and we could show that these have functional connotations in the stem cell compartment in their own rights. In this chapter, we give an account of the tools and methods we developed to unravel the structures and functions of CSPGs in the NSC niche.
Assuntos
Encéfalo/citologia , Proteoglicanas de Sulfatos de Condroitina/química , Células-Tronco Neurais/química , Nicho de Células-Tronco/química , Animais , Biomarcadores Tumorais/química , Proliferação de Células , Imuno-Histoquímica , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CNS lesions stimulate adult neurogenic niches. Endogenous neural stem/progenitor cells represent a potential resource for CNS regeneration. Here, we investigate the response to unilateral focal laser-lesions applied to the visual cortex of juvenile rats. Within 3 days post-lesion, an ipsilateral increase of actively cycling cells was observed in cortical layer one and in the callosal white matter within the lesion penumbra. The cells expressed the neural stem/progenitor cell marker Nestin and the 473HD-epitope. Tissue prepared from the lesion area by micro-dissection generated self-renewing, multipotent neurospheres, while cells from the contralateral visual cortex did not. The newly formed neural stem/progenitor cells in the lesion zone might support neurogenesis, as suggested by the expression of Pax6 and Doublecortin, a marker of newborn neurons. We propose that focal laser-lesions may induce the emergence of stem/progenitor cells with neurogenic potential. This could underlie the beneficial effects of laser application in neurosurgery.