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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

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2022-05-26 | GSE199481 | GEO

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

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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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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.

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

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

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