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RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush

ABSTRACT: The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors 3 days after sciatic nerve injury that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with axon regeneration. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that the expression of a number of regeneration-associated transcription factors including cJun, Atf3, Sox11 and other are upregulated in Tsc2 mutant neurons in the absence of an injury. Additionally, the target genes of several other normally expressed regeneration-associated transcription factors are differentially expressed in Tsc2 mutant neurons, such as Creb1 and Klf’s. We conclude Tsc2 deletion and consequent mTORC1 activation establishes a pro-regenerative gene expression profile in nociceptors in the absence of an injury.

ORGANISM(S): Mus musculus

PROVIDER: GSE125685 | GEO | 2019/01/26

REPOSITORIES: GEO

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RNA-seq analysis of FACS-sorted control and Tsc2-deleted nociceptors

Project description:The goals of this study are to compare the transcriptomes of control heterozygous and Tuberous sclerosis 2 (Tsc2) homozygous nociceptors that have been enriched by fluorescence-associated cell sorting (FACS) with the aim of identifying differences in genes associated with sensory behavior. Nav1.8-Cre transgenic mice were used to delete Tsc2 as well as express Green fluorescent protein from the Rosa26 locus. Tsc2 deletion constitutively activates mTORC1 signaling cell autonomously. Dissociated dorsal root ganglia from adult mice were FACS-sorted for GFP and analyzed by RNA-seq. We find that a number of sensory behavior associated genes are affected by Tsc2 deletion including Calca, Ntrk1, Mrgpra3, and Nppb. Additionally, profiling of ion channel expression showed a reduction in almost half of expressed sodium, potassium and calcium channel as well as G-protein coupled receptors. Other categories of genes such as transcription factors and ligand-gated ion channels contained an even numbers of up- and downregulated genes with many that did not change as a result of Tsc2 deletion. In addition, expression of most marker genes enriched in Isolectin B4 (IB4) negative neurons were strongly reduced in Tsc2 mutant nociceptors, consistent with reduced sensitivity to heat, which is a modality related to these neurons. We conclude Tsc2 deletion and consequent mTORC1 activation alters gene expression of nociceptors resulting in altered sensory thresholds.

2018-04-01 | GSE112499 | GEO

Proteomics on Nav1.8-positive nociceptors of mouse DRG

Project description:Nociceptors play an essential role in both acute pain and chronic pain conditions. In this study, we examined the proteome of mouse dorsal root ganglia and compared NaV1.8Cre+/-; ROSA26-flox-stop-flox-DTA (Diphtheria toxin fragment A) mutant mice (NaV1.8Cre-DTA), in which NaV1.8-positive neurons (mainly nociceptors) in dorsal root ganglia (DRG) were ablated, with respective littermate wildtype controls.

2022-10-03 | PXD034447 | Pride

RNA-seq analysis of FACS-sorted control and Tsc2-deleted nociceptors

Project description:RNA-seq analysis of FACS-sorted control and Tsc2-deleted nociceptors

| PRJNA447995 | ENA

RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush

Project description:RNA-seq analysis of FACS-sorted control and Tsc2 cKO nociceptors with and without sciatic nerve crush

| PRJNA517164 | ENA

Expression data from FACS purified nociceptor neurons from peripheral sensory ganglia

Project description:The goal of this study was to analyze global gene expression in specific populations of nociceptor sensory neurons, the neurons that detect damaging/noxious stimuli. The dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia are anatomically distinct peripheral sensory ganglia that contain nociceptors which innervate skin, gut, lungs, and other distinct organ tissues. We used flow cytometry to purify nociceptors from these ganglia and profiled their global gene expression signatures to compare gene expression between these different anatomically distinct nociceptors. Nav1.8-Cre were bred with Rosa26-TdTomato to generate Nav1.8-Cre/R26-TdTomato reporter progeny, where all peripheral nociceptor neurons are genetically marked with red fluroescence due to specific expression of the TTX- resistant sodium channel Nav1.8. Lumbar region dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia were dissected from mice (3 mice were pooled/sample). Highly red fluorescent neurons were Facs purified, RNA extracted, and processed for microarray analysis.

2013-08-28 | E-GEOD-46546 | biostudies-arrayexpress

Transcription profiling of mouse dorsal root ganglia after diphtheria-toxin deletion of the Nav1.8-expressing cells using the cre/loxP system

Project description:Comparison of transcript levels after diphtheria-toxin deletion of the Nav1.8-expressing cells using the cre/loxP system.<br> Pain requires input from nociceptors which are specialised peripheral sensory neurons in dorsal root ganglia (DRG). To explore the nociceptor-specific mRNA profiles, we used microarray analysis to examine the altered repertoire of genes expressed in DRG from mice depleted of Nav1.8-expressing neurons which are mainly nociceptors. To generate the animal, a Nav1.8 knock-in Cre-expressing mouse (Nav1.8-Cre) was used to excise a floxed stop upstream of globally expressed diphtheria toxin A-subunit gene (ROSA26-eGFP-DTA). By crossing heterozygous Cre mice with homozygous toxin-expressing floxed mice, experimental toxin-expressing (DTA) mice, in which DTA expressing nociceptors had been deleted, were generated. 6 DTA mice and 6 litter mate controls were used for microarray analysis. 3 Affymetrix Mouse Genome 430 2.0 Array chips were employed for analysis of the gene transcripts of each conditions.

2008-08-02 | E-MEXP-1622 | biostudies-arrayexpress

Transcription profiling by array of mouse immortalized embryonic fibroblasts to identify genes regulated by mTORC1

Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-^Slow/low/high/lowM-^T or M-^SLLHLM-^T, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons

2011-08-02 | E-GEOD-27982 | biostudies-arrayexpress

Identification of Tfeb-dependent genes downstream of mTORC1 in MEFs

Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-bM-^@M-^\low/low/high/lowM-bM-^@M-^] or M-bM-^@M-^\LLHLM-bM-^@M-^]. We found that mTORC1 regulated the expression of, among other lysosomal genes, V-ATPases through the transcription factor EB (TFEB, Tcfeb in the mouse). The knockdown of Tfeb resulted in the 'flattening' of the LLHL pattern and allowed the identification of genes regulated by mTORC1 through Tfeb Mouse embryo fibroblasts (MEFs) wild type or deficient in Tsc2 expressing a Tfeb shRNA or scrambled shRNA vector were treated with 25 nM rapamycin or vehicle (methanol) for 24 h under low serum conditions (0.1% FBS)

2011-08-02 | E-GEOD-28021 | biostudies-arrayexpress

Effect of nociceptors on dendritic cells

Project description:We explored the effects of nociceptors on DC under various conditions by means of in vitro coculture of Flt3L-induced bone marrow-derived DCs and primary mouse nociceptors.

2023-04-01 | GSE217500 | GEO

NGF-induced gene expression in silent nociceptors

Project description:Silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. We had previously shown that mouse silent nociceptors express the nicotinic acetylcholine receptor alpha-3 subunit (CHRNA3) and can thus readily be identified in Tg(Chrna3-EGFP)BZ135Gsat reporter mice (Prato et al. Cell Reports 2017). Most importantly, we had shown that CHRNA3+ slient nociceptors acquire mechanosensitivity upon treatment with the inflammatory mediator nerve growth factor and demonstrated that this process requires de-novo gene transcription. Here, we performed paired-end RNA-sequencing to identify transcripts that might be involved in the acquisition of mechanosenstivity in mouse silent nociceptors.

2022-12-01 | GSE199580 | GEO

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