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Characterization of a P2X7 BAC reporter mouse model and investigation of the P2X4/P2X7 interaction in mouse lung tissue
Characterization of a P2X7 BAC reporter mouse model and investigation of the P2X4/P2X7 interaction in mouse lung tissue
P2X receptors (P2XR) comprise a family of ATP-gated, non-selective cation channels. Out of the seven members of the P2XR family (P2X1R-P2X7R), P2X7R presents important characteristics that make it unique. These are a) the low affinity for the ligand ATP, b) its long, intracellular C-terminus and c) the ability to induce the formation of a macropore under sustained stimulation that allows the passage of large molecules and that may eventually lead to cell death. The best characterized function of the P2X7R is the induction of NLRP3 inflammasome assembly for the activation of caspase-1 and release of pro-inflammatory cytokines IL-1beta and IL-18. The P2X7R is encoded by the P2rx7 gene and is widely expressed in immune cells, where it has proved relevance as a drug target due to its role in inflammation and its demonstrated modulatory effects in various diseases. For example, P2X7R involvement has been found in infectious diseases, autoimmune disorders, and cancer. In the central nervous system, the P2X7R was shown to modulate neuroinflammation. However, the cell type-specific localization and functions of the P2X7R in the central nervous system are incompletely understood. While its large intracellular C-terminus has been proposed to serve as a platform for the interaction with mediators of signaling pathways, details about the identity of these interaction partners, the signaling mechanisms and functional relevance of such interactions remain largely unknown. A physical interaction of P2X7R with the P2X4R subtype has been suggested, although proof of this interaction is mostly restricted to heterologous overexpression systems and evidence for this interaction in vivo is insufficient. In an attempt to address these questions, two transgenic P2X7R reporter mouse models have been generated that express EGFP under the control of the endogenous P2rx7 promoter. One of them expresses a soluble EGFP (sEGFP mouse) and the other one expresses a P2X7-EGFP fusion protein (P2X7-EGFP mouse). However, preliminary data revealed divergent distribution of the reporter proteins in the mouse brain. In this project, a detailed characterization and comparison of both BAC transgenic mouse models was performed. Biochemical and histochemical approaches have been applied to compare the P2X4 and P2X7 expression levels in both mouse models and determine the cell-specific distribution of the EGFP reporter. In the P2X7-EGFP mouse model, this study confirmed that the endogenous P2X7 and P2X4 expression levels were unaffected and that the overexpressed fusion protein is found in microglia and oligodendrocytes. However, analysis of P2X4 and P2X7 levels in the sEGFP mouse model revealed an unexpected overexpression of both proteins, which is explained by the use of a different BAC clone and the respective recombination strategy. Immunohistochemistry experiments demonstrated divergent expression patterns of P2X7 and the sEGFP reporter and revealed its predominant localization in neurons. In conclusion, these results unveiled and explained inconsistencies in the reporter expression of the sEGFP mouse model. In a second part of this thesis, I investigated the reported interaction between P2X7R and P2X4R using the transgenic P2X7-EGFP mouse model. In a biochemical approach the interaction of both receptors was evaluated by pull-down via EGFP-tag. Control experiments were performed upon expression of both subunits in Xenopus laevis oo-cytes and HEK293 cells. Our results confirmed a previously shown P2X4R-P2X7R interaction in X. laevis oocytes but revealed no proof of such an interaction in mouse lung and HEK293 cells. The possibility of a functional interrelation between P2X4R and P2X7R was further explored by comparison of the expression levels in both subunits in wildtype, P2X7-EGFP overexpressing mice and P2rx4-/- and P2rx7-/- knockout mouse models. Together, these data did not support an interaction or mutual interrelation of both receptors in the mouse lung. Lastly, a preliminary comparative proteomic study was performed with wildtype, P2rx7-/- and P2X7-EGFP mice to study P2X7R signaling. To this aim, protocols for sample preparation were tested and samples from mouse hippocampus were submitted to liquid chromatography coupled to mass spectrometry (LC-MS) analysis. However, statistical analysis did not identify significantly enriched proteins. Therefore, a cell-specific approach was considered more promising and a protocol for microglia isolation from adult mouse brain was tested for future proteomic studies. In summary, this study provided the first detailed characterization of the sEGFP P2X7 reporter mouse model and revealed important caveats for its use in basic research. It further showed evidence against the proposed interaction between P2X7R and P2X4R and finally describes a comparative proteomics approach to investigate P2X7R signaling cascades.
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Ramírez Fernández, Antonio
2023
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Ramírez Fernández, Antonio (2023): Characterization of a P2X7 BAC reporter mouse model and investigation of the P2X4/P2X7 interaction in mouse lung tissue. Dissertation, LMU München: Faculty of Medicine
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Abstract

P2X receptors (P2XR) comprise a family of ATP-gated, non-selective cation channels. Out of the seven members of the P2XR family (P2X1R-P2X7R), P2X7R presents important characteristics that make it unique. These are a) the low affinity for the ligand ATP, b) its long, intracellular C-terminus and c) the ability to induce the formation of a macropore under sustained stimulation that allows the passage of large molecules and that may eventually lead to cell death. The best characterized function of the P2X7R is the induction of NLRP3 inflammasome assembly for the activation of caspase-1 and release of pro-inflammatory cytokines IL-1beta and IL-18. The P2X7R is encoded by the P2rx7 gene and is widely expressed in immune cells, where it has proved relevance as a drug target due to its role in inflammation and its demonstrated modulatory effects in various diseases. For example, P2X7R involvement has been found in infectious diseases, autoimmune disorders, and cancer. In the central nervous system, the P2X7R was shown to modulate neuroinflammation. However, the cell type-specific localization and functions of the P2X7R in the central nervous system are incompletely understood. While its large intracellular C-terminus has been proposed to serve as a platform for the interaction with mediators of signaling pathways, details about the identity of these interaction partners, the signaling mechanisms and functional relevance of such interactions remain largely unknown. A physical interaction of P2X7R with the P2X4R subtype has been suggested, although proof of this interaction is mostly restricted to heterologous overexpression systems and evidence for this interaction in vivo is insufficient. In an attempt to address these questions, two transgenic P2X7R reporter mouse models have been generated that express EGFP under the control of the endogenous P2rx7 promoter. One of them expresses a soluble EGFP (sEGFP mouse) and the other one expresses a P2X7-EGFP fusion protein (P2X7-EGFP mouse). However, preliminary data revealed divergent distribution of the reporter proteins in the mouse brain. In this project, a detailed characterization and comparison of both BAC transgenic mouse models was performed. Biochemical and histochemical approaches have been applied to compare the P2X4 and P2X7 expression levels in both mouse models and determine the cell-specific distribution of the EGFP reporter. In the P2X7-EGFP mouse model, this study confirmed that the endogenous P2X7 and P2X4 expression levels were unaffected and that the overexpressed fusion protein is found in microglia and oligodendrocytes. However, analysis of P2X4 and P2X7 levels in the sEGFP mouse model revealed an unexpected overexpression of both proteins, which is explained by the use of a different BAC clone and the respective recombination strategy. Immunohistochemistry experiments demonstrated divergent expression patterns of P2X7 and the sEGFP reporter and revealed its predominant localization in neurons. In conclusion, these results unveiled and explained inconsistencies in the reporter expression of the sEGFP mouse model. In a second part of this thesis, I investigated the reported interaction between P2X7R and P2X4R using the transgenic P2X7-EGFP mouse model. In a biochemical approach the interaction of both receptors was evaluated by pull-down via EGFP-tag. Control experiments were performed upon expression of both subunits in Xenopus laevis oo-cytes and HEK293 cells. Our results confirmed a previously shown P2X4R-P2X7R interaction in X. laevis oocytes but revealed no proof of such an interaction in mouse lung and HEK293 cells. The possibility of a functional interrelation between P2X4R and P2X7R was further explored by comparison of the expression levels in both subunits in wildtype, P2X7-EGFP overexpressing mice and P2rx4-/- and P2rx7-/- knockout mouse models. Together, these data did not support an interaction or mutual interrelation of both receptors in the mouse lung. Lastly, a preliminary comparative proteomic study was performed with wildtype, P2rx7-/- and P2X7-EGFP mice to study P2X7R signaling. To this aim, protocols for sample preparation were tested and samples from mouse hippocampus were submitted to liquid chromatography coupled to mass spectrometry (LC-MS) analysis. However, statistical analysis did not identify significantly enriched proteins. Therefore, a cell-specific approach was considered more promising and a protocol for microglia isolation from adult mouse brain was tested for future proteomic studies. In summary, this study provided the first detailed characterization of the sEGFP P2X7 reporter mouse model and revealed important caveats for its use in basic research. It further showed evidence against the proposed interaction between P2X7R and P2X4R and finally describes a comparative proteomics approach to investigate P2X7R signaling cascades.