Genetically dissecting P2rx7 expression within the central nervous system using conditional humanized mice

Purinergic Signalling (2017) 13:153–170 DOI 10.1007/s11302-016-9546-z ORIGINAL ARTICLE Genetically dissecting P2rx7 expression within the central nervous system using conditional humanized mice Michael W. Metzger 1 & Sandra M. Walser 1 & Fernando Aprile-Garcia 2,3 & Nina Dedic 1 & Alon Chen 1,4 & Florian Holsboer 1,5 & Eduardo Arzt 2 & Wolfgang Wurst 6,7,8,9 & Jan M. Deussing 1 Received: 5 August 2016 / Accepted: 26 October 2016 / Published online: 17 November 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The purinergic P2X7 receptor (P2X7R) has attracted considerable interest as a potential target for various central nervous system (CNS) pathologies including affective and neurodegenerative disorders. To date, the distribution and cellular localization of the P2X7R in the brain are not fully resolved and a matter of debate mainly due to the limitations of existing tools. However, this knowledge should be a prerequisite for understanding the contribution of the P2X7R to brain disease. Here, we generated a genetic mouse model by humanizing the P2X7R in the mouse as mammalian model organism. We demonstrated its functionality and revealed * Jan M. Deussing 1 Max Planck Institute of Psychiatry, 80804 Munich, Germany 2 Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET- Partner Institute of the Max Planck Society, Buenos Aires, Argentina 3 Present address: Max Planck Institute of Immunbiology and Epigenetics, 79108 Freiburg, Germany 4 Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, 85764 Neuherberg, Germany 5 Present address: HMNC Brain Health, 80539 Munich, Germany 6 German Center for Neurodegenerative Diseases (DZNE), Site Munich, 81377 Munich, Germany 7 Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität München, 80336 Munich, Germany 8 Department of Neurobiology, Weizmann Institute of Science, 7610001 Rehovot, Israel 9 Chair of Developmental Genetics c/o Helmholtz Zentrum München, Technische Universität München-Weihenstephan, 85764 Neuherberg, Germany species-specific characteristics of the humanized receptor, compared to the murine ortholog, regarding its receptivity to activation and modulation by 2′,3′-O-(benzoyl-4-benzoyl)adenosine 5′-triphosphate (BzATP) and trifluoperazine (TFP). This humanized P2rx7 allele is accessible to spatially and temporally controlled Cre recombinase-mediated inactivation. In contrast to previously generated knockout (KO) mice, none of the described P2rx7 splice variants evade this null allele. By selective disruption and assessment of human P2RX7 expression in different brain regions and cell types, we were able to demonstrate that the P2X7R is specifically expressed in glutamatergic pyramidal neurons of the hippocampus. Also, P2X7R is expressed in major non-neuronal lineages throughout the brain, i.e., astrocytes, oligodendrocytes, and microglia. In conclusion, this humanized mouse model provides the means for detailed assessment of human P2X7R function in vivo including evaluation of agonists or antagonists. In addition, this conditional allele will enable future loss-of-function studies in conjunction with mouse models for CNS disorders. Keywords P2X7 receptor . P2rx7 gene . Pore formation . Mouse model . Knockout . Gene expression Introduction P2X receptors are trimeric receptors composed of 3 subunits that can form homo- or heteromers. Each subunit consists of an intracellular C- and N-terminal domain as well as 2 transmembrane domains, joined by a cysteine-rich ectodomain that binds adenosine triphosphate (ATP) [1, 2]. Within the P2X family, P2X7 is the largest subunit consisting of 595 amino acids (aa) with a unique intracellular C-terminal domain of 239 aa, which is significantly longer than in the rest of the 154 family [3–5]. This long C-terminal domain comprises several protein and lipid binding motifs as well as a cysteine-rich domain, including a binding domain for lipopolysaccharides (LPS) [6]. In contrast to other family members, P2X7 receptors are predominantly homomers [7]. Despite the fact that human and murine P2X7 share about 80 % sequence homology, differences between the species have been described with regards to receptor sensitivity toward various ligands. The human P2X7R has been shown to be 10–100 times more sensitive to stimulation by the agonist 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate (BzATP) compared to the murine ortholog [4, 8, 9]. Moreover, it has been demonstrated that human and murine receptors show different susceptibility regarding modulation of their activity by various compounds [9–11]. The human P2RX7 gene is located on chromosome 12 and mouse P2rx7 on chromosome 5 in a region of conserved synteny. Both genes comprise 13 exons, which give rise to multiple splice variants. While 13 transcripts have been described for human P2RX7, only 5 alternative transcripts have been described for the mouse so far: P2rx7-a, P2rx7-b, P2rx7c, P2rx7-d, and P2rx7-k [7, 12, 13] (compare Fig. 4a). P2rx7b, P2rx7-c, and P2rx7-d are characterized by a truncated Cterminus. In addition, the first exon in P2rx7-c and P2rx7-d is affected by alternative splicing [14]. It has repeatedly been shown that in particular, the C-terminally truncated isoforms have a negative regulatory effect on receptor function when co-expressed with full-length P2X7 [3, 12, 15, 16]. P2rx7-k is characterized by an alternative exon 1, whereas exons 2–13 are identical to P2rx7-a. This isoform seemingly does not disturb receptor function; on the contrary, it shows higher sensitivity toward the endogenous agonist ATP. Further, it has been shown that different to the most abundant isoform P2rx7-a, P2rx7-k is highly sensitive to activation by extracellular nicotinamide adenine dinucleotide (NAD+) via ADPribosylation [17]. Moreover, these 2 isoforms are differentially expressed; P2rx7-k predominantly occurs in regulatory T cells whereas P2rx7-a is the dominant variant in peritoneal macrophages and skeletal muscle [13]. The P2X7R is broadly expressed in immune cells of the hematopoietic lineage including monocytes, lymphocytes, macrophages, and dendritic cells [18, 19]. Surprisingly, the precise expression of the P2X7R in the brain is still a matter of debate in the field [20]. In particular, neuronal expression of the P2X7 receptor has been controversially discussed and contested [21, 22]. The non-selectivity of available P2X7R antibodies in the brain has been demonstrated using P2X7R KO mouse lines generated by GlaxoSmithKline and Pfizer, respectively [23, 24]. While the loss of P2X7R protein was readily detected in peripheral tissues, the detection of P2X7R in the brain in both KO lines was masked by an unknown protein of similar size [25, 26]. This finding prevented the reliable detection of P2X7R in the brain including mo (...truncated)