Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
- Max Planck Institute for Experimental Medicine, Germany
- Ludwig-Maximilians-Universität München, Germany
- University of Regensburg, Germany
- University Hospital Hamburg-Eppendorf, Germany
- University of Genova, Italy
- Royal College of Surgeons in Ireland, Ireland
- Friedrich-Alexander University Erlangen-Nürnberg, Germany
- University Medical Center Göttingen, Germany
Abstract
The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7-BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (Ni 592/4-5)
- Annette Nicke
Deutscher Akademischer Austauschdienst
- Santina Bruzzone
Deutsche Forschungsgemeinschaft (No 310/11-1)
- Friedrich Koch-Nolte
Science Foundation Ireland (13/SIRG/2098)
- Tobias Engel
Horizon 2020 Framework Programme (766124)
- Tobias Engel
- Annette Nicke
Deutsche Forschungsgemeinschaft (SFB 1328)
- Tim Magnus
- Friedrich Koch-Nolte
- Annette Nicke
Science Foundation Ireland (17/CDA/4708)
- Tobias Engel
Deutsche Forschungsgemeinschaft (Ni 592/4-7)
- Annette Nicke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal handling and experimental procedures were performed in accordance with German and European Union guidelines and were approved by the State of Upper Bavaria (stab wound injury (55.2.1.54-2532-171-11), retinal ischemia (TVV 54/12; 55.2 DMS-2532-2-182), transcardial perfusion (55.2-1-54-2532-59-2016)) and Lower Saxony (generation of BAC transgenic mice, transcardial perfusion (33.9-42502-04-12/0863), behavioral experiments (3392 42502-04-13/1123)). Status epilepticus was induced in accordance with the principles of the European Communities Council Directive (86/609/EEC) and procedures reviewed and approved by the Research Ethics Committee of the Royal College of Surgeons in Ireland (REC 205 and 1322) and performed under license from the Department of Health and Children, Ireland. All efforts were made to minimize suffering and number of animals used.
Copyright
© 2018, Kaczmarek-Hajek et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,531
- views
-
- 827
- downloads
-
- 135
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody
eLife 7:e36217.
https://doi.org/10.7554/eLife.36217
Further reading
-
- Immunology and Inflammation
Psoriasis is a multifactorial disorder mediated by IL-17-producing T cells, involving immune cells and skin-constituting cells. Semaphorin 4A (Sema4A), an immune semaphorin, is known to take part in T helper type 1/17 differentiation and activation. However, Sema4A is also crucial for maintaining peripheral tissue homeostasis and its involvement in skin remains unknown. Here, we revealed that while Sema4A expression was pronounced in psoriatic blood lymphocytes and monocytes, it was downregulated in the keratinocytes of both psoriatic lesions and non-lesions compared to controls. Imiquimod application induced more severe dermatitis in Sema4A knockout (KO) mice compared to wild-type (WT) mice. The naïve skin of Sema4A KO mice showed increased T cell infiltration and IL-17A expression along with thicker epidermis and distinct cytokeratin expression compared to WT mice, which are hallmarks of psoriatic non-lesions. Analysis of bone marrow chimeric mice suggested that Sema4A expression in keratinocytes plays a regulatory role in imiquimod-induced dermatitis. The epidermis of psoriatic non-lesion and Sema4A KO mice demonstrated mTOR complex 1 upregulation, and the application of mTOR inhibitors reversed the skewed expression of cytokeratins in Sema4A KO mice. Conclusively, Sema4A-mediated signaling cascades can be triggers for psoriasis and targets in the treatment and prevention of psoriasis.
-
- Immunology and Inflammation
- Medicine
Metabolic abnormalities associated with liver disease have a significant impact on the risk and prognosis of cholecystitis. However, the underlying mechanism remains to be elucidated. Here, we investigated this issue using Wilson’s disease (WD) as a model, which is a genetic disorder characterized by impaired mitochondrial function and copper metabolism. Our retrospective clinical study found that WD patients have a significantly higher incidence of cholecystitis and a poorer prognosis. The hepatic immune cell landscape using single-cell RNA sequencing showed that the tissue immune microenvironment is altered in WD, mainly a major change in the constitution and function of the innate immune system. Exhaustion of natural killer (NK) cells is the fundamental factor, supported by the upregulated expression of inhibitory receptors and the downregulated expression of cytotoxic molecules, which was verified in clinical samples. Further bioinformatic analysis confirmed a positive correlation between NK cell exhaustion and poor prognosis in cholecystitis and other inflammatory diseases. The study demonstrated dysfunction of liver immune cells triggered by specific metabolic abnormalities in WD, with a focus on the correlation between NK cell exhaustion and poor healing of cholecystitis, providing new insights into the improvement of inflammatory diseases by assessing immune cell function.
