Spatial transcriptomics of the achilles tendon harvested from the injury site with and without TrkA neuronal inhibition
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE245097 | GEO | 2023/11/27
REPOSITORIES: GEO
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Project description:Peripheral afferent neurons terminate at the surfaces of tendons, yet their role after injury beyond nociceptive functions remain unclear. Using transgenic animal models, sensory neurons were found to sprout after Achilles tendon injury in domains of Nerve growth factor (NGF) expression. Conditional deletion of Ngf in either myeloid or mesenchymal cell types led to tendon repair defects – findings phenocopied by inactivation of TrkA (Tropomyosin receptor kinase A) using a knockin mouse model. We sought to identify the signals modulated with neural TrkA inhibition. A combination of single cell and spatial transcriptomic analysis was performed on the Achilles injury site of TrkAF592A animals and compared them to TrkAWT.
2023-11-27 | GSE244921 | GEO
Project description:Here, we investigated the effect of conditioned media (CM) obtained from cultured mouse DRG neurons on Tppp3+ cells, mainly on proliferation and differentiation potential. Peripheral afferent neurons terminate at the surfaces of tendons, yet their role after injury beyond nociceptive functions remain unclear. Using transgenic animal models, sensory neurons were found to sprout after Achilles tendon injury in domains of Nerve growth factor (NGF) expression. Conditional deletion of Ngf in either myeloid or mesenchymal cell types led to tendon repair defects – findings phenocopied by inactivation of TrkA (Tropomyosin receptor kinase A) using a knockin mouse model. A combination of spatial and single cell transcriptomics revealed dysregulated inflammatory and TGF signaling upon lack of neural input. Utilizing sural nerve transection in reporter animals, we identified that neural input is required for proper expansion of Tppp3+ tendon sheath progenitor cells (TSPCs) after injury, and in vitro approaches implicated TGF signaling activation in Tppp3+ TSPC neural response. Finally, in a translational approach, activation of TrkA+ sensory neurons using a partial agonist led to a significant increase in TGF signaling, TSPC expansion, and improved tendon repair. Collectively, these data implicate peripheral afferent neural networks in the coordinated acute tendon injury response, and identify candidate molecules to speed the reparative process.
2023-11-27 | GSE221035 | GEO
Project description:Nerve growth factor (NGF) is a neurotrophin that plays an important role in regulating the survival, growth, and differentiation of sympathetic neurons. Many in vitro studies indicate that Egr transcription factors are coupled to NGF signaling and are essential signaling mediators of NGF-dependent differentiation of sympathetic neurons, such as neuroblastoma cells and pheochromocytoma cells. Mice that are deficient for both Egr1 and Egr3 have profound sympathetic nerve system defects, including abnormal neuron degeneration and impaired differentiation (unpublished observations). To further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. The results will be helpful in understanding the pathobiology of those diseases related to aberrant sympathetic neuron differentiation, such as neuroblastoma and dysautonomias, and may provide new insights into therapies for these refractory diseases. To identify NGF-mediated Egr-dependent target genes in human SH-SY5Y/TrkA neuroblastoma cells: Many potential Egr target genes have been described over the years. However, very few have been characterized to be involved in NGF-mediated sympathetic neuron differentiation. In order to further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. Egr1 and Egr3 are rapidly induced after NGF treatment and Egr1 is involved in activation of the differentiation marker gene NPY in SH-SY5Y/TrkA cells. Therefore, SH-SY5Y/TtrkA cells appear to be an excellent model system to study the role of Egr transcription factors in sympathetic neuron differentiation in vitro. A dominant negative Egr molecule that specifically blocks transcriptional activity mediated by Egr transcription factors will be used in this study to identify Egr-dependent target genes. Egr1 and Egr3 are rapidly induced after NGF treatment in human SH-SY5Y/TrkA neuroblastoma cells, which in turn differentiate into sympathetic-like neurons. We hypothesize that Egr transcription factors are involved in activating downstream signaling pathways during NGF mediated differentiation of SH-SY5Y/TrkA cells. Moreover, we hypothesize that by using a dominant negative Egr (dnEgr) molecule that blocks all Egr mediated gene transcription and Affymetrix microarray analysis, it will be possible to identify NGF-mediated Egr transcription dependent gene regulatory networks that may be involved in growth and differentiation of neuroblastoma. An unbiased approach to understanding these gene regulatory networks may lead to new insights relating to NGF signaling involved in neuronal growth and differentiation. Human neuroblastoma SH-SY5Y/TrkA cells will be infected with either dnEgr-expressing adenovirus (SH-SY5Y/TrkA-dnEgr) or with EGFP-expressing control adenovirus (SH-SY5Y/TrkA-EGFP). Equivalent infection efficiency and lack of viral toxicity will be verified by EGFP fluorescence microscopy 24 hours after infection and the cells will be treated with NGF (100 ng/ml). Total RNA will be extracted from SH-SY5Y/TrkA (uninfected), SH-SY5Y/TrkA-dnEgr, and SH-SY5Y/TrkA-EGFP cells treated with NGF for 0, 1 hour and 3 hours. Total RNA will be prepared from all of the samples and a portion subjected to real-time PCR analysis to ensure that NGF mediated Egr gene induction was not altered by the context of viral infection. Pilot experiments demonstrate that Egr genes are still induced in the context of viral infection greater than 100-fold. Egr1 mRNA peak expression is known to occur at 1 hour and decrease by 3 hours after NGF treatment in all of the samples. The peak expression of Egr target genes is expected to occur later than Egr1 peak expression since Egr1 proteins need to be expressed first to initiate the transcription of target promoters. Therefore, the RNA samples from SH-SY5Y/TrkA-dnEgr and SH-SY5Y/TrkA-EGFP treated with NGF for 3 hours will be used to probe Affymetrix high-density human genome U133 Plus 2.0 Arrays to identify differentially expressed genes. RNA amplification for probe synthesis should not be necessary since we will provide 10 ug of intact total RNA for each sample. We will provide three sets of samples to perform the comparative microarray analysis twice from different starting materials and a nine-way comparative analysis of the data will be performed. We expect that cells containing high levels of dnEgr will inhibit NGF mediated Egr-dependent target gene expression and that these gene networks should be identifiable when compared to EGFP infected cells that have normal Egr gene transcriptional activity. Experiment Overall Design: as above
2008-06-12 | E-GEOD-2864 | biostudies-arrayexpress
Project description:Background: Though exosomes, as the by-products of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), it remains unclear through which manner exosomes act. Aim: In order to figure out whether exosomes attenuate lesion size of SCI by the amelioration of neuronal injury triggered by secondary inflammatory storm and induction of inner motivation of neurite outgrowth, we designed and performed this experiment. Methods: We determined absolute contents of all exosomal miRNAs and investigated the potential mechanisms of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and vitro. Results: miR-199a-3p/145-5p, which were relatively top-ranking miRNAs in exosomes, promoted PC12 cells differentiation suppressed by lipopolysaccharide (LPS) in vitro through the modulation of NGF/TrkA pathway. We also demonstrated that Cblb was the direct target of miR-199a-3p and Cbl was the direct target of miR-145-5p. Cblb and Cbl genes knock down revealed that TrkA ubiquitylation level significantly decreased, subsequently, activating the NGF/TrkA downstream pathways Akt and Erk. In return, overexpression of Cblb and Cbl suggested that TrkA ubiquitylation level significantly increased, subsequently, inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and co-immunoprecipitation confirmed the direct interaction between TrkA and Cblb, TrkA and Cbl. In vivo experiment, exosomal miR-199a-3p/145-5p were found to up-regulate TrkA expression in the lesion site and promote locomotor function of SCI rats as well. Conclusion: In summary, our study suggested that hUC-MSCs derived exosomes may treat SCI by transferring miR-199a-3p/145-5p to neurons, and modulating TrkA ubiquitylation, and strengthening the NGF/TrkA signaling pathway in SCI rats.
2020-12-08 | GSE159814 | GEO
Project description:We used quantitative mass spectrometry-based proteomics to unravel global nerve growth factor (NGF)-induced TrkA signaling dynamics at the interactome, phosphoproteome and proteome level. A tetracycline-inducible system for TrkA expression was generated in the human neuroblastoma cell line, SH-SY5Y. TrkA-induced cells were stimulated with NGF for different time points to follow phosphoproteome, interactome and proteome changes on a temporal scale. In a triple SILAC setup (Light: Lys0,Arg0; Medium: Lys4,Arg6; and Heavy: Lys8,Arg10), the samples were stimulated with NGF as indicated. Phosphoproteome: 0, 10, 45 min and 0, 120, 120 min+cycloheximide. Interactome: 0, 5, 10 min. Proteome: 0, 24, 48 h. All experiments were performed as biological replicates.
2015-05-06 | PXD001115 | Pride
Project description:The Achilles tendon is the thickest tendon in the human body, and Achilles tendinopathy is its most prevalent disorder, often considered a consequence of overuse. While disruption to the collagen fibers represents a significant manifestation of Achilles tendinopathy, strikingly little is known about the mechanisms by which healthy tendon accumulates damage in vivo.As existing studies of tendon biomechanics and mechanobiology predominantly relied on in vitro or ex vivo experiments on isolated tissues, it is still largely unknown whether and how disruptions occur to the collagen molecules in healthy Achilles tendons following physiological activities. We reported the first RNA-seq analysis reflecting transcriptome changes in healthy rat Achilles tendons following running, providing a resource for future investigations in tendon mechanobiology and sports medicine.
2024-10-07 | GSE269529 | GEO
Project description:Achilles tendinopathy is often thought to be a consequence of overuse of the Cells within the Achilles tendon of healthy rats undergo a series of changes following physiologic levels of mechanical stimulation after running, and we further explored the transcriptome changes in Achilles tendon cells during post-exercise recovery. Our current experiment reveals RNA-seq analysis of the transcriptome of the rat Achilles tendon after 12 hours of rest following running.
2024-10-07 | GSE270400 | GEO
Project description:We have compared the gene expression profile of rat Achilles tendon-derived stem cells in post-natal tendon development. Rat tendon stem/progenitor cells (TSPCs) were isolated at different stages of post-natal development: TSPCs-1d, TSPCs-7d and TSPCs-56d.
2015-07-10 | GSE68485 | GEO
Project description:Melanomas are generated from melanocytes, the neural crest derivatives sharing a neuroectodermal origin with the nervous system. In investigating whether immune privilege of the nervous system might be exploited by melanoma, we found that nerve growth factor (NGF) exerts both melanoma cell-intrinsic and -extrinsic immunosuppression. In melanoma cells, autocrine NGF engages TrkA receptor to desensitize IFN-gammasignaling, leading to T and NK cell exclusion. In effector T cells, which upregulate surface TrkA expression upon T cell receptor (TCR) activation, paracrine NGF dampens TCR signaling and effector function. Targeting NGF genetically or pharmacologically with larotrectinib sensitizes melanoma responsiveness to immune checkpoint blockade (ICB) therapy for tumor eradication and induces durable protection by eliciting robust memory of low-affinity T cells. Together, these findings uncover a comprehensive mechanism through which the NGF-TrkA axis suppresses anti-tumor T cell immunity, thus providing a novel mode of action to repurpose larotrectinib for immune sensitization. Moreover, by enlisting low-affinity tumor-specific T cells, anti-NGF reduces acquired resistance to ICB therapy and prevents melanoma recurrence.
2023-11-02 | GSE243814 | GEO
Project description:Melanomas are generated from melanocytes, the neural crest derivatives sharing a neuroectodermal origin with the nervous system. In investigating whether immune privilege of the nervous system might be exploited by melanoma, we found that nerve growth factor (NGF) exerts both melanoma cell-intrinsic and -extrinsic immunosuppression. In melanoma cells, autocrine NGF engages TrkA receptor to desensitize IFN-gammasignaling, leading to T and NK cell exclusion. In effector T cells, which upregulate surface TrkA expression upon T cell receptor (TCR) activation, paracrine NGF dampens TCR signaling and effector function. Targeting NGF genetically or pharmacologically with larotrectinib sensitizes melanoma responsiveness to immune checkpoint blockade (ICB) therapy for tumor eradication and induces durable protection by eliciting robust memory of low-affinity T cells. Together, these findings uncover a comprehensive mechanism through which the NGF-TrkA axis suppresses anti-tumor T cell immunity, thus providing a novel mode of action to repurpose larotrectinib for immune sensitization. Moreover, by enlisting low-affinity tumor-specific T cells, anti-NGF reduces acquired resistance to ICB therapy and prevents melanoma recurrence.
2023-11-02 | GSE236680 | GEO