Single-cell RNA-seq analysis of p53 knockout primary astrocytes in vitro.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE196408 | GEO | 2023/02/23
REPOSITORIES: GEO
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Project description:To investigate the mechansims that underlie astrocyte dedifferentiation, we performed single cell RNA sequencing analysis of primary astrocytes isolated from adult mouse cortex after p53 loss and exposure to mitogens, EGF and and FGF. Primary astrocytes were isolated from cortices of 2-3month old inducible p53 knockout mice (GFAP-CreERT2;p53flox/flox;LSL-tdTomato) using Miltenyi Adult Brain Dissociation kit and ACSA-2 beads. Treatment with 4-hydroxytamoxifen (4OHT) induces p53 loss and tdTomato labelling in GFAP+ astrocytes. Astrocytes were treated with 4OHT in media supplemented with EGF and FGF to induce recombination and astrocyte dedifferentiation in vitro.
2023-02-23 | GSE220477 | GEO
Project description:To investigate the mechanisms that underlie astrocyte dedifferentiation, we performed single cell RNA sequencing analysis of tdTomato+ astrocyte derived cells approx 1 year after stab-wound injury. 2-3 month old GFAP-CreERT2;p53flox/flox;LSL-tdTomato (p53Gfap-icKO) mice were subjected to intracortical endoxifen injection to enable deletion of p53 gene and simultaneous expression of tdTomato for fate-mapping specifically in astrocytes (GFAP+) in the context of a stab-wound injury. GFAP-CreERT2;p53wt/wt;LSL-tdTomato (p53Gfapwt/wt) mice were used as control whereby p53 remains fate-mapped tdTomato+ astrocytes remain p53 wildtype after endoxifen injection. Approx 10-12 months after injury, mice were sacrificed and the peri-wound region was dissected. Mice of the same genotypes were pooled (3 p53Gfap-icKO and 2 p53Gfapwt/wt) and digested using the Miltenyi Adult brain dissociation kit. Single cell suspension was FACS sorted for tdTomato+ cells into 384 well plates and then Smartseq3 libraries were generated.
2023-02-23 | GSE220479 | GEO
Project description:Comparison of genomic data from astrocytes and non-astrocyte cells from mice with or without FGF+EGF after SCI. We conducted genome-wide RNA sequencing of (i) immunoprecipitated astrocyte-specific ribosome-associated RNA (ramRNA) and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells, from spinal cord tissue of mice recieving i) SCI alone, ii) SCI+hydrogel depot containing FGF+EGF, or iii) SCI+empty hydrogel depot.
2018-09-20 | GSE111529 | GEO
Project description:Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. We used SILAC and quantitative proteomics to characterise primary murine telencephalic progenitor cells from FOXG1 (forkhead box G1)-cre driven Tgfbr2 (transforming growth factor beta receptor 2) knockout mice and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFβ-signalling. Impaired TGFβ-signalling affected numbers of GFAP-astrocytes in the ventral telencephalon. In contrast, TGFβ reduced MFGE8 expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells.
2022-03-02 | PXD005072 | Pride
Project description:GFAP and vimentin deficiency alters gene expression in astrocytes and microglia in wild-type mice and changes the transcriptional response of reactive glia in mouse model for Alzheimer's disease. Reactive astrocytes with an increased expression of intermediate filament (IF) proteins Glial Fibrillary Acidic Protein (GFAP) and Vimentin (VIM) surround amyloid plaques in Alzheimer's disease (AD). The functional consequences of this upregulation are unclear. To identify molecular pathways coupled to IF regulation in reactive astrocytes, and to study the interaction with microglia, we examined WT and APPswe/PS1dE9 (AD) mice lacking either GFAP, or both VIM and GFAP, and determined the transcriptome of cortical astrocytes and microglia from 15- to 18-month-old mice. Genes involved in lysosomal degradation (including several cathepsins) and in inflammatory response (including Cxcl5, Tlr6, Tnf, Il1b) exhibited a higher AD-induced increase when GFAP, or VIM and GFAP, were absent. The expression of Aqp4 and Gja1 displayed the same pattern. The downregulation of neuronal support genes in astrocytes from AD mice was absent in GFAP/VIM null mice. In contrast, the absence of IFs did not affect the transcriptional alterations induced by AD in microglia, nor was the cortical plaque load altered. Visualizing astrocyte morphology in GFAP-eGFP mice showed no clear structural differences in GFAP/VIM null mice, but did show diminished interaction of astrocyte processes with plaques. Microglial proliferation increased similarly in all AD groups. In conclusion, absence of GFAP, or both GFAP and VIM, alters AD-induced changes in gene expression profile of astrocytes, showing a compensation of the decrease of neuronal support genes and a trend for a slightly higher inflammatory expression profile. However, this has no consequences for the development of plaque load, microglial proliferation, or microglial activation. 2 cell types from 6 conditions: cortical microglia and cortical astrocytes from 15-18 month old APPswe/PS1dE9 mice compared to wildtype littermates. Biological replicates: microglia from APPswe/PS1dE9, N=7, microglia from WT, N=7, astrocytes from APPswe/PS1dE9, N=4, microglia from WT, N=4
2015-11-04 | E-GEOD-74614 | biostudies-arrayexpress
Project description:Chromosomes and genes are non-randomly arranged within the mammalian cell nucleus. Clustering of genes is of great significance in transcriptional regulation. However, the relevance of gene clustering in their expression during differentiation of neural precursor cells (NPCs) into astrocytes remains unclear. We performed a genome-wide enhanced circular chromosomal conformation capture (e4C) to screen genes associated with an astrocyte-specific gene, glial fibrillary acidic protein (Gfap), during astrocyte differentiation. We identified 13 genes that were specifically associated with Gfap and expressed in NPC-derived astrocytes. These results provide evidence for functional significance of gene clustering in transcriptional regulation during NPCs differentiation. comparison of NPCs vs LIF+ vs LIF- cells.
2015-02-20 | E-GEOD-66097 | biostudies-arrayexpress
Project description:Primary cultures of astrocytes from rat optic nerve heads were treated with EGFR ligand, EGF. Two cell lines from two different rat donors were used. The sister cell cultures were set as control and EGF treated groups. Experiment Overall Design: experiment #1: compared control astrocyte cultures to sister cultures treated with EGF for 4 hours. Experiment Overall Design: experiment #2: compared control astrocyte cultures to sister cultures treated with EGF for 12 hours
2007-11-29 | E-GEOD-5282 | biostudies-arrayexpress
Project description:GFAP and vimentin deficiency alters gene expression in astrocytes and microglia in wild-type mice and changes the transcriptional response of reactive glia in mouse model for Alzheimer's disease. Reactive astrocytes with an increased expression of intermediate filament (IF) proteins Glial Fibrillary Acidic Protein (GFAP) and Vimentin (VIM) surround amyloid plaques in Alzheimer's disease (AD). The functional consequences of this upregulation are unclear. To identify molecular pathways coupled to IF regulation in reactive astrocytes, and to study the interaction with microglia, we examined WT and APPswe/PS1dE9 (AD) mice lacking either GFAP, or both VIM and GFAP, and determined the transcriptome of cortical astrocytes and microglia from 15- to 18-month-old mice. Genes involved in lysosomal degradation (including several cathepsins) and in inflammatory response (including Cxcl5, Tlr6, Tnf, Il1b) exhibited a higher AD-induced increase when GFAP, or VIM and GFAP, were absent. The expression of Aqp4 and Gja1 displayed the same pattern. The downregulation of neuronal support genes in astrocytes from AD mice was absent in GFAP/VIM null mice. In contrast, the absence of IFs did not affect the transcriptional alterations induced by AD in microglia, nor was the cortical plaque load altered. Visualizing astrocyte morphology in GFAP-eGFP mice showed no clear structural differences in GFAP/VIM null mice, but did show diminished interaction of astrocyte processes with plaques. Microglial proliferation increased similarly in all AD groups. In conclusion, absence of GFAP, or both GFAP and VIM, alters AD-induced changes in gene expression profile of astrocytes, showing a compensation of the decrease of neuronal support genes and a trend for a slightly higher inflammatory expression profile. However, this has no consequences for the development of plaque load, microglial proliferation, or microglial activation.
2015-11-04 | GSE74614 | GEO
Project description:Brain inflammation, a common feature in neurodegenerative diseases, is a complex series of events, which can be detrimental and even lead to neuronal death. Nonetheless, several studies suggest that inflammatory signals are also positively influencing neural cell proliferation, survival, migration and differentiation. Recently, correlative studies suggested that astrocytes are able to dedifferentiate upon injury, and may thereby re-acquire neural stem cells (NSC) potential. However, the mechanism underlying this dedifferentiation process upon injury remains unclear. In this study, we find that during the early response of reactive gliosis, inflammation induces a conversion of mature astrocytes into neural progenitors. A TNF treatment induces the decrease of specific astrocyte markers, such as GFAP or genes related to glycogen metabolism, while a subset of these cells re-express immaturity markers, such as CD44, Musashi-1 and Oct4. Thus, TNF treatment results in the appearance of cells that exhibit a neural progenitor phenotype and are able to proliferate and differentiate into neurons and/or astrocytes. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway.
2015-09-16 | GSE73022 | GEO
Project description:Astrocyte dysfunction impacts their normal function, including neuronal support, thereby contributing to neurodegenerative pathologies including Alzheimer's disease (AD). Therefore to understand the role of astrocytes in the pathogenesis of age-related disorders we analysed the gene expression profile of astrocytes with respect to Alzheimer-type pathology. The aim of the present study was to combine immuno-LCM and microarray analysis to characterise the astrocyte transcriptome at different Braak stages, and with respect to ApoE genotype, in post-mortem human temporal cortex sampled dervied from the Medical Research Council Cognitive Function and Ageing Study (MRC-CFAS). GFAP-positive astrocytes were isolated from age, sex and brain pH-matched cases derived from the MRC-CFAS cohort, using immuno laser capture microdissection. The extracted RNA was amplified and applied to HGU133 Plus 2.0 Affymetrix gene arrays. Genes were considered differentially expressed if they showed a minimum 1.5 fold change at p<0.02 in all 6 individual cases in each stage of pathology [18 cases in total: 6 cases Braak stages I-II (3 cases carried at least one ApoE epsilon4 allele and 3 were ApoEe4 negative); 6 cases Braak stages III-IV (3 ApoEe4 positive and 3 ApoEe4 negative; 6 cases Braak stages V-VI (3 ApoEe4 positive and 3 ApoEe4 negative)]. keywords: human GFAP-positive astrocytes
2011-06-01 | E-GEOD-29652 | biostudies-arrayexpress