Reactive astrocytes with inflammatory signature in CCM disease in hypoxia and normoxia conditions
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ABSTRACT: The CCM endothelium is hypersensitive to angiogenesis and can induce a hypoxic program associated with changes in angiogenesis, inflammation, and endothelial-cell metabolism under normoxic conditions. However, the role of active drivers of angiogenesis as CCM disease modifiers in human disease remains unclear. To assess whether CCM reactive astrocytes with neuroinflammatory capacity respond to hypoxia-induced CCM exacerbation, we employed the astrocyte-specific (Aldh1l1-EGFP/Rpl10a) Translational Ribosome Affinity Purification (TRAP) system in combination with a CCM mouse model (Pdcd10BECKO;Aldh1l1-EGFP/Rpl10a). TRAP protocol was performed using Slco1c1-iCreERT2;Pdcd10fl/fl;Aldh1l1-EGFP/Rpl10a mice to isolate ribosomes from astrocytes as previously described. Astrocyte-TRAP mRNAs were from cerebral tissue of mice age P50 exposed to hypoxia or normoxia conditions.
Project description:BAC array purification of hippocampus astroglial ribosome-bound transcriptome in astrocytes from Aldh1l1:Rpl10a eGFP/ Cx43 KO and Aldh1l1:Rpl10a eGFP/ Cx43FL mice
Project description:The CCM endothelium is hypersensitive to angiogenesis and can induce a hypoxic program associated with changes in angiogenesis, inflammation, and endothelial-cell metabolism under normoxic conditions. However, the role of active drivers of angiogenesis as CCM disease modifiers in human disease remains unclear. To examine if hypoxia, a driver of angiogenesis, may contribute to CCM exacerbation, we performed bulk RNA-seq of brain tissue from P50 Pdcd10BECKO mice under normoxia and hypoxia. P50 Pdcd10fl/fl littermate controls under normoxia and hypoxia were used as controls.
Project description:The role astrocytes play in brain development and function has come under increased focus as the diversity of roles they are involved in has become apparent. We have previously shown ethanol exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. Here we utilize the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found large differences in the total RNA pool in comparison to the translating RNA pool while the ethanol response within each pool showed a large degree of overlap. Comparisons to published datasets indicate the in vitro model used here are most similar to PD1-PD7 in vivo cortical astrocytes and the ethanol regulated genes showed significant overlap with models of chronic ethanol exposure in astrocytes and a model of third-trimester ethanol exposure in the hippocampus and cerebellum. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes alter brain development.
Project description:Psychiatric disorders, especially major depressive disorder, are prominent cause of disability worldwide, and in dire need of better diagnostic and therapeutic tools. In order to identify the cellular mechanisms underlying major depressive disorder we sought to understand the role of astrocytes, the most numerous subtype of glia, in this disease utilizing the TRAP gene expression analysis and chronic social defeat mouse model of depression. TRAP translational profiling is a method that allows investigators to genetically characterize specific cell types in complex tissues such as mouse brain. Using this technique we obtained RNA-Seq data from actively translating transcripts present in astrocytes. We generated the molecular profile of astrocytes from multiple brain regions affected by stress-induced depression, the prefrontal cortex, hippocampus, accumbens nucleus and caudate putamen of adult Aldh1l1-EGFP/Rpl10a (JD130) mice after chronic social defeat stress paradigm. The results of this study will further our understanding of depression pathophysiology and will provide possible targets for novel or supplementary therapies.
Project description:Cerebral cavernous malformation (CCM) immunothrombosis is the connection between immune cells, platelet activation, coagulation cascades and astrocyte-CCM endothelium interaction, and its excessive activation may contribute to neurological disabilities in CCM disease. We characterized the spatial organization of CCM immunothrombosis observed in Pdcd10BECKObrains under normoxic conditions using the murine 10X Genomics Visium Spatial Gene Expression platform.
Project description:Background: Fetal Alcohol Spectrum Disorders (FASD) cause life-long cognitive and behavioral impairment and are highly prevalent across the world. The effects of developmental ethanol exposure on astrocyte functions in vivo are poorly understood. To fill this gap, we assessed changes in the astrocyte translatome following in vivo developmental ethanol exposure and mechanistically linked some of these changes to altered neuronal development. Methods: The translating RNA from hippocampal astrocytes of neonatal Aldh1l1-EGFP-Rpl10a mice exposed to ethanol was isolated by Translating Ribosomal Affinity Purification and sequenced; neuronal dendritic morphology was assessed by Golgi-Cox staining and morphometric analysis; chondroitin sulfate glycosaminoglycan (CS-GAG) disaccharides were measured by Liquid Chromatography/Mass Spectrometry; neurite outgrowth was assessed in in vitro hippocampal neurons incubated in the presence of astrocyte-conditioned medium prepared from control and Chpf2-silenced astrocytes. Results: Translatome results suggested that ethanol alters mechanisms of astrocyte-neuron interactions involved in neuronal development. Moreover, ethanol increased dendritic arborization in hippocampal pyramidal neurons, inhibited the astrocyte translation of enzymes involved in the biosynthesis of inhibitory CS-GAGs, and decreased the levels of disaccharides forming CS-GAGs in vivo. Lastly, the silencing of CS-GAG biosynthetic enzyme Chpf2 in astrocyte cultures increased neurite branching of hippocampal neuron in vitro. Conclusions: Translatome analysis revealed novel effects of developmental ethanol exposure on astrocytes and provided mechanistic insights into altered brain development possibly relevant to the pathophysiology of FASD. We also identified a novel mechanism by which ethanol-induced reduction of astrocyte-produced inhibitory extracellular matrix components CS-GAGs increases dendritic arborization.
Project description:Translating ribosome affinity purification (TRAP) was performed on spinal cord dissections pooled from 3-4 mice 21 days post birth that were positive for the eGFP-L10A fusion ribosomal marker protein under the expression of either the Chat promoter (Tg(Chat-EGFP/Rpl10a)DW167Htz) or the Snap25 promoter (Tg(Snap25-EGFP/Rpl10a)JD362Jdd). RNA-sequencing was performed on both TRAP and pre-immunoprecipitation (PreIP) control RNA samples.