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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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In vivo analysis of astrocyte ribosome-associated mRNA after traumatic spinal cord injury

ABSTRACT: Analysis of gene expression by astrocytes or non-astrocyte cells in spinal cord injury (SCI) lesions may lead to the identification of molecules that impact on axon regrowth. We conducted genome-wide RNA sequencing of (i) immunoprecipitated astrocyte-specific ribosome-associated RNA (ramRNA) from WT or STAT3-CKO astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples 14 days following SCI. DOI: 10.1038/nature17623 Young adult female mGFAP-Cre-RiboTag or mGFAP-Cre-RiboTag-STAT3-LoxP mice underwent severe crush SCI at thoracic level 10. 14 days following SCI, the central 3mm of the SCI lesion was extracted, homogenized and (i) astrocyte-specific ribosome-associated RNA (ramRNA) precipitated via a hemagglutinin (HA) tag targeted to either WT (n=4) or STAT3-CKO (n=3) astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples. Sex and age-matched mGFAP-Cre-RiboTag mice served as uninjured controls (n=4).

ORGANISM(S): Mus musculus

SUBMITTER: Riki Kawaguchi

PROVIDER: E-GEOD-76097 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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In vivo analysis of astrocyte ribosome-associated mRNA after traumatic spinal cord injury

Project description:Analysis of gene expression by astrocytes or non-astrocyte cells in spinal cord injury (SCI) lesions may lead to the identification of molecules that impact on axon regrowth. We conducted genome-wide RNA sequencing of (i) immunoprecipitated astrocyte-specific ribosome-associated RNA (ramRNA) from WT or STAT3-CKO astrocytes, and (ii) the non-precipitated (flow-through) RNA deriving from non-astrocyte cells in the same tissue samples 14 days following SCI. DOI: 10.1038/nature17623

2016-03-30 | GSE76097 | GEO

In vivo analysis of spinal cord astrocyte and non-astrocyte gene expression after traumatic spinal cord injury, with or without intraspinal treatment with hydrogel depot containing FGF+EGF

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.

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Divergent transcriptional regulation of astrocyte reactivity across disorders [RNA-seq]

Project description:RNA sequencing was applied to examine changes in gene expression in reactive astrocytes following traumatic spinal cord injury (SCI) or neuroinflammatory insult by systemic administration of lipopolysaccharide (LPS). Transgenic Ribotag technology was used to isolate astrocyte ribosome-associated mRNAs from the spinal cord of wild type mice and mice with astrocyte-specific conditional gene deletion (cKO) of test-case transcriptional regulators of reactivity, SMARCA4 (Smarca4-astro-cKO) or STAT3 knockout (Stat3-astro-cKO). Examination of differentially expressed gene (DEG) profiles by upstream transcriptional regulator enrichment analysis (TREA) was used identify transcriptional regulators of reactivity in each condition. Together, findings from this study show that transcriptional changes associated with astrocyte reactivity are exquisitely heterogeneous and are customized from vast numbers of potential DEGs via context-specific combinatorial TR interactions. These data have also been made accessible in a open-access, searchable database: http://tr.astrocytereactivity.com

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Divergent transcriptional regulation of astrocyte reactivity across disorders [TR_ATAC_SCI]

Project description:Single-nuclei Assay for Transposase-Accessible Chromatin with sequencing (snATACseq) was applied to examine chromatin landscape changes and transcriptional regulator (TR) DNA motif accessibility in reactive astrocytes following traumatic spinal cord injury (SCI). Astrocyte nuclei were isolated from the spinal cord of wild type mice and mice with astrocyte-specific conditional gene deletion (cKO) of test-case TRs, SMARCA4 (Smarca4-astro-cKO) or STAT3 knockout (Stat3-astro-cKO). Comparison of differential chromatin accessibility revealed substantial remodeling during astrocyte reactivity, with more chromatin opening than closing. Marked alterations in access to SCI reactivity-associated TR motifs were also detected.

2022-05-26 | GSE199481 | GEO

Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution

Project description:Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord tissue. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+, S100a4+, Gpr84+, C3+/G0s2+, GFAP+/Tm4sf1+, and Gss+/Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphological and physiologically distinct for each astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understanding the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair.

2021-11-21 | GSE189070 | GEO

Systematic analysis of purified astrocytes after spinal cord injury unveils lncRNA Zeb2os as a novel molecular target for astrogliosis [RNA-Seq]

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2021-02-08 | GSE153720 | GEO

Systematic analysis of purified astrocytes after spinal cord injury unveils lncRNA Zeb2os as a novel molecular target for astrogliosis [ChIP-Seq]

Project description:Astrocytes are the predominant component of the scar and play crucial roles in spinal cord injury (SCI) at acute injury stage. Understanding the complex reactive astroglial contributions to SCI pathophysiologies is important for developing therapeutic strategies. To understand the mechanisms of astrogliosis in SCI, and to identify novel molecular targets to improve the injury environment and axonal regeneration, we have studied gene expression changes in SCI epicenter tissue using RNA-Seq at chronic stages (1 month and 3 months) in mouse moderate contusive injury models. Importantly, we have also FACS purified cells successfully from adult spinal cord using transgenic mice and generated RNA-Seq results for both acute (7 days) and chronic (1 month and 3 months) injury stages. The SCI RNA-Seq analysis provided valuable information on the gene expression in injury environment and astrocytes after SCI. In addition to protein coding genes, we were the first to systematically analyze the expression profiles of Long Noncoding RNAs (lncRNAs) in SCI and purified astrocytes. We have tested a number of candidates and found gene of interest Zeb2os which is a highly conserved lncRNA in human. Zeb2os expression has high correlation with an essential transcription factor (TF) in astrogliosis, Stat3, and its antisense protein coding gene Zeb2. Furthermore, our ChIP-seq experiment showed STAT3 bound to Zeb2 promoter region, thus Zeb2os may regulate Zeb2 and Stat3 directly or indirectly and STAT3 may regulate zeb2 expression. Moreover, we have demonstrated for the first time by shRNA gene knockdown (KD) and functional assays that Zeb2os plays an important functional role in astrogliosis. We found Zeb2os KD in primary astrocytes affected a number of downstream genes by RNA-Seq, for example, Gfap, Zeb2, and Stat3 level decreased and reduced astrocyte proliferation.

2021-02-08 | GSE153085 | GEO

New transgenic mouse lines for selectively targeting astrocytes and for studying calcium signals in astrocyte processes in situ and in vivo

Project description:The Khakh laboratory developed and characterized a new Aldh1l1-Cre/ERT2 transgenic mouse line to selectively target astrocytes in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and analyses of the cortical astrocyte transcriptome from adults.

2016-11-07 | GSE84540 | GEO

TGFβ-signalling and FOXG1 expression hallmark astrocyte lineage diversity in the murine ventral and dorsal forebrain

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

Astrocyte-enriched pan-brain aging transcriptome

Project description:Normal brain aging is marked by a cognitive decline, spurred by changes in cellular metabolism and homeostatic dysregulation, as well as modifications in synapses and neuronal connectivity. Astrocytes are well positioned as an effector of these changes, although how properties of astrocytes change with age remains unclear. Here, we address this question by profiling astrocytic gene expression from multiple brain regions of adult (4 month-old) and aged (2 years-old) mice. We isolated mRNA from transgenic mice where ribosomes within astrocytes were genetically tagged (GFAP-cre x RPL22-HA, astrocyte ribotag). We then used RNA sequencing to identify and quantify the astrocyte-enriched mRNA, analyzing 4 different brain regions: visual cortex, somatomotor cortex, hypothalamus, and cerebellum. Overall, we find that astrocyte expression of inflammatory and immune response factors increase with age, as well as changes in genes associated with metabolism, cholesterol synthesis, and synaptogenesis. Going forward, these data contribute to an understanding of astrocyte diversity and provide insight into the role of astrocytes in normal aging.

2017-12-11 | GSE99791 | GEO

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