Regulation and function of P2X7 receptors in the central nervous system under physiological and pathophysiological conditions
Final Report 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 in previous projects P2X7-BAC transgenic mice that overexpress EGFP-tagged P2X7 receptors. The aim of the project described here was the original characterization of these mouse lines and 1) the determination of the precise cellular and subcellular localization of P2X7 receptors in the central and peripheral nervous system, 2) identification of interaction partners of the P2X7 receptor, 3) investigation of receptor expression in models of central and peripheral demyelinating neuropathies, and 4) analysis of the physiological and pathophysiological roles of the P2X7 receptors in the CNS. 1) Comparison of several trangenic lines and extensive characterization of the line with the highest P2X7 overexpression revealed dominant P2X7-EGFP protein expression in microglia and oligodendrocytes, but not in neurons of the central, peripheral, and gut nervous system. These findings were further validated by microglia and oligodendrocyte-specific P2X7 deletion in the cns and a novel specific nanobody against P2X7. 2) To identify interaction partners of the P2X7 receptor, we developed an in situ cross-linking methodology and optimized the purification of cross-linked P2X7-EGFP-containing complexes from lung tissue. Initial analysis of these complexes by mass spectrometry revealed a number of proteins that are involved in leucocyte migration which are currently further confirmed. In addition, the proposed physical interaction and mutual interrelation between P2X7 and P2X4 receptors was investigated in vivo by quantitative PCR, biochemical and microscopic analysis using P2X7R-EGFP BAC transgenic mice and P2X4 and P2X7 knockout mice. The obtained data argue against a significant interaction between P2X4 and P2X7 receptors. 3) An obvious motor phenotype was observed and further analysed in one of the BAC transgenic mouse lines with a high P2X7 overexpression level. Further analysis by fluorescence in situ hybridization revealed a partial trisomy that most likely accounted for the pathological phenotype of these mice. Since no obvious phenotype was observed in the other lines, the originally postulated causative role of P2X7 in demyelinating diseases was not further investigated. 4) In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we obtained preliminary data showing potential consequences of its overexpression in ischemic retina and posttraumatic cerebral cortex grey matter, such as increased microglia numbers in both affected tissue, increased loss of neurons in the stab wound model and increased interleukine 1β production in the ischemic retina. In a further project, consequences of P2X7 overexpression were investigated in the myenteric plexus preparations of the gut where we found a higher number of macrophages and significantly enhanced neuronal damage upon ATP application in transgenic mice. In conclusion, this novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases. Despite the delay in the projects and the fact that we had to establish a variety of novel methods, the most important aims were successfully addressed. The identification of P2X7 interaction partners and its role in diseases of the gut and the brain will be continued in the EU-funded H2020 ITN "Purines DX" and within the SFB1328 "Adenine Nucleotides in Immunity and Inflammation".
Publications
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(2018) Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody. eLife 7 e36217
Kaczmarek-Hajek, Karina; Zhang, Jiong; Kopp, Robin; Grosche, Antje; Rissiek, Björn; Saul, Anika; Bruzzone, Santina; Engel, Tobias; Jooss, Tina; Krautloher, Anna; Schuster, Stefanie; Magnus, Tim; Stadelmann, Christine; Sirko, Swetlana; Koch-Nolte, Friedric
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(2013) Heteromeric assembly of P2X subunits. Front. Cell. Neurosci. 7: 250-250
Saul A, Hausmann R, Kless A, Nicke A
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(2013) The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor. Neuropharmacology 75C: 365-379
Hempel, C, Nörenberg W, Sobottka H, Urban N, Nicke A, Fischer W, Schäfer M
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P2X receptors (2018). P2X Receptors. In A. Bhattacharjee (Ed.), The Oxford Handbook of Neuronal Ion Channels. New York: Oxford University Press
Nicke A, Grutter T, Egan T
