Project description:Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS) playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs differentiation into astrocytes is unexplored yet. Here, we address this gap by profiling glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor-alpha (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. Notably, in agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb and Nfkb2, highlighting the interplay between signaling pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of the astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

Project description:Astrocytes arise from multipotent neural stem cells (NSCs) and represent the most abundant cell type of the central nervous system (CNS), playing key roles in the developing and adult brain. Since the differentiation of NSCs towards a gliogenic fate is a precisely timed and regulated process, its perturbation gives rise to dysfunctional astrocytic phenotypes. Inflammation, which often underlies neurological disorders, including neurodevelopmental disorders and brain tumors, disrupts the accurate developmental process of NSCs. However, the specific consequences of an inflammatory environment on the epigenetic and transcriptional programs underlying NSCs' differentiation into astrocytes is unexplored. Here, we address this gap by profiling in mice glial precursors from neural tissue derived from early embryonic stages along their astrocytic differentiation trajectory in the presence or absence of tumor necrosis factor (TNF), a master pro-inflammatory cytokine. By using a combination of RNA- and ATAC-sequencing approaches, together with footprint and integrated gene regulatory network analyses, we here identify key differences during the differentiation of NSCs into astrocytes under physiological and inflammatory settings. In agreement with its role to turn cells resistant to inflammatory challenges, we detect Nrf2 as a master transcription factor supporting the astrocytic differentiation under TNF exposure. Further, under these conditions, we unravel additional transcriptional regulatory hubs, including Stat3, Smad3, Cebpb, and Nfkb2, highlighting the interplay among pathways underlying physiological astrocytic developmental processes and those involved in inflammatory responses, resulting in discrete astrocytic phenotypes. Overall, our study reports key transcriptional and epigenetic changes leading to the identification of molecular regulators of astrocytic differentiation. Furthermore, our analyses provide a valuable resource for understanding inflammation-induced astrocytic phenotypes that might contribute to the development and progression of CNS disorders with an inflammatory component.

Project description:The capacity of astrocytes to adapt their biochemical and functional features upon physiological and pathological stimuli is a fundamental property at the basis of their ability to regulate the homeostasis of the central nervous system (CNS). It is well known that in primary cultured astrocytes, the expression of plasma membrane ion channels and transporters involved in homeostatic tasks does not closely reflect the pattern observed in vivo. The individuation of culture conditions that promote the expression of the ion channel array found in vivo is crucial when aiming at investigating the mechanisms underlying their dynamics upon various physiological and pathological stimuli. A chemically defined medium containing growth factors and hormones (G5) was previously shown to induce the growth, differentiation, and maturation of primary cultured astrocytes. Here we report that under these culture conditions, rat cortical astrocytes undergo robust morphological changes acquiring a multi-branched phenotype, which develops gradually during the 2-week period of culturing. The shape changes were paralleled by variations in passive membrane properties and background conductance owing to the differential temporal development of inwardly rectifying chloride (Cl-) and potassium (K+) currents. Confocal and immunoblot analyses showed that morphologically differentiated astrocytes displayed a large increase in the expression of the inward rectifier Cl- and K+ channels ClC-2 and Kir4.1, respectively, which are relevant ion channels in vivo. Finally, they exhibited a large diminution of the intermediate filaments glial fibrillary acidic protein (GFAP) and vimentin which are upregulated in reactive astrocytes in vivo. Taken together the data indicate that long-term culturing of cortical astrocytes in this chemical-defined medium promotes a quiescent functional phenotype. This culture model could aid to address the regulation of ion channel expression involved in CNS homeostasis in response to physiological and pathological challenges.

			Action	DRS
	SRR23572520_1.fastq.gz	Fastqsanger.gz
	SRR23572520_2.fastq.gz	Fastqsanger.gz
	SRR23572521_1.fastq.gz	Fastqsanger.gz
	SRR23572521_2.fastq.gz	Fastqsanger.gz
	SRR23572522_1.fastq.gz	Fastqsanger.gz

Dataset Information

Transcriptional and chromatin accessibility profiling of neural stem cells differentiating into astrocytes reveal dynamic signatures under homeostatic and inflammatory conditions [ATAC-Seq]

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