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.
Reviewing Editor
- Kenton Jon Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
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.
Version history
- Received: March 5, 2018
- Accepted: July 31, 2018
- Accepted Manuscript published: August 3, 2018 (version 1)
- Version of Record published: September 14, 2018 (version 2)
- Version of Record updated: September 19, 2018 (version 3)
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.
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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
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- Immunology and Inflammation
- Medicine
Background:
Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin.
Methods:
Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors.
Results:
We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01–2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 μg/mL, p=0.004).
Conclusions:
Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin.
Funding:
LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust).
Clinical trial number:
-
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