Project description:The experiment was made to compare gene expression of human control (H1) ES-derived astrocytes and isogenic astrocytes carrying a mutation causing fragile x syndrome.
Project description:Astrocytes negatively impact neuronal development in a range of neurodevelopmental disorders (NDs), however how they do this, and if mechanisms are shared across multiple disorders, is not known. We developed an in vitro system to ask how astrocyte protein secretion and gene expression change in 3 genetic NDs. This identified disorder specific changes, as well as core proteins that are increased in release in all 3 NDs. ND astrocytes increase release of Igfbp2, and blocking Igfbp2 partially rescues the neurite outgrowth inhibitory effects of Rett Syndrome astrocytes. We identified that ND astrocytes upregulate release of Bmp6, which acts on astrocytes themselves and is upstream of astrocyte secretion changes in NDs. Blocking Bmp signaling in Fragile X Syndrome astrocytes reverses the inhibitory effects of FXS astrocytes on neuronal development. We provide a resource of astrocyte secreted proteins in health and NDs, as well as novel targets for intervention in diverse NDs.
Project description:Primary astrocyte cultures were prepared from wildtype and Gde3 knockout mouse brain. Conditioned media was collected, concentrated and analyzed using TMT-MS.
Project description:Astrocytes negatively impact neuronal development in a range of neurodevelopmental disorders (NDs), however how they do this, and if mechanisms are shared across multiple disorders, is not known. We developed an in vitro system to ask how astrocyte protein secretion and gene expression change in 3 genetic NDs (Fragile X syndrome (FXS), Rett's syndrome (RTT), Down's syndrome (DS)). This identified disorder specific changes, as well as core proteins that are increased in release in all 3 NDs. We provide a resource of astrocyte secreted proteins and gene expression in health and NDs, as well as novel targets for intervention in diverse NDs.
Project description:Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. To identify proteins contributing to protective intracellular neuronal signalling originating from astrocytes, endogenous PI3K was immunoprecipitated from Ht22 cells exposed to primary astrocyte conditioned media (ACM) or cell free media (CFM), followed by iTRAQ-based quantitative proteomic analysis.
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.
Project description:Mutations in the gene encoding the transcriptional modulator methyl-CpG binding protein 2 (MeCP2) are responsible for the neurodevelopmental disorder Rett syndrome that is one of the most frequent sources of intellectual disability in women. It has been shown that mutant astrocytes contribute to neuronal defects, probably as a result of aberrant secretion of soluble factor(s). Based on the hypothesis that this irregular secretion of soluble factor is the result of abnormal transcription of those factors, we have compared the gene expression profiles of wild type and mutant astrocytes from Mecp2 308/y mice by using Affymetrix mouse 2.0 microarrays. P0 newborns from pregnant Mecp2 308/+ heterozygous mice were used for the preparation of dissociated cortical astrocyte cultures. Four wild-type and four mutant confluent astrocytes cultures were used for total RNA extraction with the Qiagen RNeasy kit (Qiagen, Courtaboeuf, France) as described by the manufacturer. The gene expression profiles of wild type and mutant astrocytes from Mecp2 308/y mice have been assayed by using Affymetrix mouse 2.0 microarrays.
Project description:Inflammation features in diverse central nervous system disorders such as stroke, trauma, neurodegeneration, infection and autoimmunity. To better understand how inflammatory mediators may alter astrocyte functions, we examined the effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS) and interferon-gamma (IFNγ) on purified, murine, primary cortical astrocyte cultures. We used microarrays to conduct whole genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS and IFNγ significantly modulated astrocyte expression of over 6,800 genes and resulted in both additive and synergistic changes compared with individual stimuli alone. Bioinformatic analysis revealed that combinatorial treatment significantly and markedly up regulated molecular networks and pathways associated with immune signaling and with regulation of cell compromise, death, growth and proliferation. These findings provide databases of astrocyte transcriptome changes elicited by the inflammatory stimuli, TGF-β1, LPS and IFNγ alone and in combination, and show that these stimuli up regulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling evoked by multiple GPCR. We used microarrays to examine the effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS) and interferon-gamma (IFNγ) on purified, murine, primary cortical astrocyte cultures.