Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

Astrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture

SummaryAstrocytes have diverse functions in brain homeostasis. Many of these functions are acquired during late stages of differentiation when astrocytes become fully mature. The mechanisms underlying astrocyte maturation are not well understood. Here we identified extensive transcriptional changes that occur during astrocyte maturation and are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lacked expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induced distinct sets of mature astrocytes-specific transcripts. Culturing astrocytes with FGF2 in a three-dimensional gel induced expression of Rorb, Dbx2 and Lhx2 and improved their maturity based on transcriptional and chromatin profiles. Therefore extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

Vimentin Phosphorylation Is Required for Normal Cell Division of Immature Astrocytes

Vimentin (VIM) is an intermediate filament (nanofilament) protein expressed in multiple cell types, including astrocytes. Mice with VIM mutations of serine sites phosphorylated during mitosis (VIMSA/SA) show cytokinetic failure in fibroblasts and lens epithelial cells, chromosomal instability, facilitated cell senescence, and increased neuronal differentiation of neural progenitor cells. Here we report that in vitro immature VIMSA/SA astrocytes exhibit cytokinetic failure and contain vimentin accumulations that co-localize with mitochondria. This phenotype is transient and disappears with VIMSA/SA astrocyte maturation and expression of glial fibrillary acidic protein (GFAP); it is also alleviated by the inhibition of cell proliferation. To test the hypothesis that GFAP compensates for the effect of VIMSA/SA in astrocytes, we crossed the VIMSA/SA and GFAP−/− mice. Surprisingly, the fraction of VIMSA/SA immature astrocytes with abundant vimentin accumulations was reduced when on GFAP−/− background. This indicates that the disappearance of vimentin accumulations and cytokinetic failure in mature astrocyte cultures are independent of GFAP expression. Both VIMSA/SA and VIMSA/SAGFAP−/− astrocytes showed normal mitochondrial membrane potential and vulnerability to H2O2, oxygen/glucose deprivation, and chemical ischemia. Thus, mutation of mitotic phosphorylation sites in vimentin triggers formation of vimentin accumulations and cytokinetic failure in immature astrocytes without altering their vulnerability to oxidative stress.

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1

Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF’s receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

Transcription factors NFIA and NFIB induce cellular differentiation in high-grade astrocytoma

Astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation and therefore could be effectors of cellular differentiation in glioblastoma. We analysed expression of family membersNFIAandNFIBusing high-grade astrocytoma mRNA expression datasets, and with immunofluorescence co-staining. Their expression is reduced in glioblastomas and is associated with differentiated and mature astrocyte-like cells at a cellular level. Furthermore, induction of NFI expression is sufficient to promote cellular differentiation in patient-derived glioblastoma xenografts. Our findings indicate that NFI proteins may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal development. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.

BDNF/TrkB.T1 signaling is a novel mechanism for astrocyte morphological maturation

Brain derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of neurons, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF’s receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1 which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1 in vivo revealed morphologically immature astrocytes with significantly reduced volume and branching, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte functions, including synaptogenic genes. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

Development of Gap Junctions in Hippocampal Astrocytes: Evidence That Whole Cell Electrophysiological Phenotype Is an Intrinsic Property of the Individual Cell

Gap junction communication between astrocytes is prevalent and has been proposed to be involved in several astrocyte functions. One such proposal involves gap junctions in potassium spatial buffering. However, little is known about the developmental time course of gap junction coupling and how much the syncytium affects whole cell measurements of ion currents. Our previous work described three types of hippocampal astrocyte, each with a distinct electrophysiological profile when recorded in whole cell voltage-clamp mode. In the current study we correlated post–whole cell recording immunohistochemistry for GLAST and the spread of injected dye from the recorded cell with the measured electrophysiological phenotype to quantify cell coupling of astrocytes and the type of astrocyte coupled, in the rat hippocampus. We found that passive astrocytes, which predominate after 3 wk postnatally, have much lower membrane resistances ( Rm) and are more frequently dye coupled and to more cells, than outwardly and variably rectifying astrocytes that predominate in early postnatal development. Dye coupling in GLAST(+) cells was first detected in the first postnatal week and the degree of coupling peaked before the complete transition to the low Rm, passive electrophysiological type. Also, the degree of dye coupling did not correlate with the passive electrophysiological phenotype. Passive cells were also detected after pretreatment with a gap junction inhibitor. Further evidence that cell coupling does not contribute to the mature astrocyte electrophysiological phenotype came from recording of excised membrane patches, which predominantly corresponded to the ion channel expression profiles of their cells of origin. These findings imply that in the hippocampus, interastrocyte cell coupling likely contributes little to the overall whole cell current profile of diverse glia, and the electrophysiological passivity reflects the intrinsic ion channel expression of the mature astrocyte.