Hareeri, Rawan
(2020)
Mechanisms of reactive gliosis in cortical and spinal cord astrocytes at different developmental stages.
PhD thesis, University of Nottingham.
Abstract
Astrocytes represent a major class of glial cells responsible for maintaining the microenvironment of the central nervous system. They are thought to play an important role in the development of many diseases, such as stroke, neurodegenerative disease and pain. It is apparent that astrocytes become reactive during the onset of these diseases to form foci of reactive gliosis; however, the mechanisms of reactive gliosis are poorly characterised. This thesis therefore aims to investigate the mechanisms underlying reactive gliosis astrocytes in different CNS regions and during development in vitro.
To investigate the mechanisms of gliosis, we compared the responses of primary cultures of spinal cord astrocytes and cortical astrocytes as a model. We then, compared the response of cortical astrocytes at two developmental stages, before and after birth (E18 and P2, respectively), as a second model to further elucidate the mechanisms of gliosis relative to astrocyte phenotype during development. To induce gliosis, astrocytes were treated with pharmacological stimuli (forskolin, LPS, TNFα, noradrenaline, histamine and substance P). The reactive astrogliosis was quantified by calculating the percentage of stellate cells, quantifying the sensitivity of cells to the P2Y14 receptor agonist UDP-glucose, as this receptor is up-regulated in reactive astrocytes, and measuring the distribution of p-STAT3 between cytoplasm and nucleus.
It was found that neonatal cortical astrocytes demonstrated time-dependent stellation in response to forskolin and noradrenaline. In contrast, primary cultures of other astrocytes exhibited a less reliable and more heterogeneous response to the stimuli, suggesting there are significant differences in the sensitivity of astrocytes to these stimuli or their ability to undergo stellation, depending on the site and age. Further investigation showed that cAMP levels were higher in neonatal cortical cultures compared to the neonatal spinal and embryonic cortical cultures. In addition, the intensity of p-STAT3 (considered a master regulator of reactive gliosis) was different in the different region and during development, both
of which were consistent with the changing of astrocyte morphology.
To investigate the role of STAT3 in stellation, it was found that inhibition of p-STAT3 can prevent astrocytes from undergoing stellation in the presence of forskolin or noradrenaline and decrease the tonic level of STAT3, suggesting the involvement of p-STAT3 in stellation. IL-6 STAT3 activator cannot induce stellation; the inhibitors significantly block stellation in the presence of a cAMP
analog, and forskolin or noradrenaline are unable to activate STAT3, implying a cooperative effect of STAT3 and cAMP to cause stellation. None of the stimuli
induced STAT3 translocation to the nucleus, thus the transcription role of STAT3 was not enabled. This indicates that cytoplasmic STAT3 induces stellation through a non-genomic role.
Together, these data provide evidence of age- and region-related differences in astrocytes stellation, which are correlated with cAMP and STAT3 expression and their activities. These findings help elucidate the mechanisms of stellation and may explain the actions of STAT3 on morphology that are independent of its nuclear transcriptional activity.
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