P2X7 Receptor-Related Genetic Mouse Models – Tools for Translational Research in Psychiatry

Depression is a common psychiatric disorder and the leading cause of disability worldwide. Although treatments are available, only about 60% of treated patients experience a significant improvement in disease symptoms. Numerous clinical and rodent studies have identified the purinergic P2X7 receptor (P2X7R) as one of the genetic factors potentially contributing to the disease risk. In this respect, genetically engineered mouse models targeting the P2X7R have become increasingly important in studying designated immunological features and subtypes of depression in vivo. This review provides an overview of the P2X7R -related mouse lines currently available for translational psychiatric research and discusses their strengths, weaknesses, and potentials.

In the past three decades, P2X7R -related genetically engineered mouse models have become increasingly relevant in the study of the connection between P2X7R signaling and depression in vivo. In this review, we briefly outline the general properties of the P2X7R and provide a comprehensive overview of currently available transgenic mouse lines.

GENERAL CHARACTERISTICS OF P2X7R
Purinergic signaling is an evolutionarily ancient and ubiquitous cell-to-cell communication, which is involved in tissue homeostasis and multiple pathophysiological conditions, including mood disorders (Verkhratsky and Burnstock, 2014;Ribeiro et al., 2019;Bartoli et al., 2020). Although ATP is well known for its role as an energy carrier in cell metabolism, as well as a relevant cell-to-cell signaling molecule in diverse cell types under physiological conditions, it additionally acts as an extracellular messenger in the context of cell trauma (Burnstock, 2006).
The P2X7R belongs to the P2X family (P2X1-7), which comprises trimeric ligand-gated ion channels responsive to extracellular ATP (Di Virgilio et al., 2017). In the central nervous system (CNS), P2X7R is mainly expressed in microglia, astrocytes, and oligodendrocytes as well as in other immune cells (Jacobson and Müller, 2016;Adinolfi et al., 2018). Each P2X7R monomer has two transmembrane domains with intracellular C-and N-termini and a large ectodomain, where the ATPbinding sites are found at the interface between the monomers (Hattori and Gouaux, 2012;Karasawa and Kawate, 2016;McCarthy et al., 2019).

COMPARING HUMAN AND MURINE P2X7R PROPERTIES
Human and mouse P2X7R s possess species-specific characteristics. The human receptor has a higher affinity for BzATP and ATP (EC 50 : 20 µM and 100 µM, respectively), compared to the murine receptor (EC 50 : 295 µM and 850 µM, respectively) (Moore and MacKenzie, 2008;Khakh and North, 2012). Similarly, the binding affinity for various pharmacological agents varies between different mammalian P2X7R s (Donnelly-Roberts et al., 2009). Furthermore, the human P2X7R has a comparatively higher deactivation speed, which likely depends on the C-terminal region of the receptor (Bartlett et al., 2014). Both human and murine P2X7R s undergo post-translational modifications, including N-linked glycosylation and palmitoylation. Due to the lack of specific ADP-ribosyl transferases, only the murine P2X7R is subject to ADP ribosylation at Arg 125 , which is located in close vicinity to the ATP binding region. This process leads to the ATPindependent activation of the receptor in the presence of NAD + (Sluyter, 2017).
The genes encoding the murine and the human receptors are localized on chromosome 5 and 12, respectively, in a region of conserved synteny. The P2RX7 gene undergoes species-specific alternative splicing. To date, 13 human (P2X7R -A to K, P2X7-V3 and nf P2X7) and five mouse (P2X7R -A, B, C, D, and K) splice variants have been described, with P2X7R -A being the main functional variant in both species (Sluyter and Stokes, 2011;Pegoraro et al., 2021). Studies in mice have shown that some of these alternative variants, P2X7R -K and P2X7R -C in particular, can produce functional receptors that might play a specific role in processes such as cell growth, cell death and inflammation (Cheewatrakoolpong et al., 2005;Feng et al., 2006;Adinolfi et al., 2010;Stokes et al., 2010;Bartlett et al., 2014).

P2X7R AND PSYCHIATRIC DISORDERS
In genetic studies, single nucleotide polymorphisms (SNPs) in the P2RX7 gene haven been linked to brain disorders such as depression, Alzheimer's disease, or bipolar disorder (Lucae et al., 2006;Czamara et al., 2018;Andrejew et al., 2020). In particular, the region of chromosome 12 harboring the P2RX7 gene (12q24) has been linked to psychiatric disorders (Degn et al., 2001;Abkevich et al., 2003;Curtis et al., 2003;McGuffin et al., 2005;Shink et al., 2005). Foremost the SNP rs2230912, which causes the non-synonymous 1405 A > G transition and translates to a Gln460Arg substitution in the C-terminal intracellular domain of the protein Lucae et al., 2006;Andrejew et al., 2020). Although the association between rs2230912 and mood disorders is inconsistent (Green et al., 2009;Grigoroiu-Serbanescu et al., 2009;Viikki et al., 2011), this SNP seems to influence symptom severity of mood disorders. Patients carrying the G allele experience longer disease course and greater symptom severity (Nagy et al., 2008;Hejjas et al., 2009;Soronen et al., 2011). Moreover, carriers of both alleles show subtle alterations in sleep patterns, suggesting a heterozygote disadvantage (Metzger et al., 2017b). Although a meta-analysis from 2014 did not detect an association between rs2230912 and mood disorders (Feng et al., 2014), a more recent meta-analysis, which included more studies, presented a significant association of rs2230912 with mood disorders (Czamara et al., 2018). However, a genome-wide association study, which identified risk variants for depression and bipolar disorder with genomewide significance, did not detect any association related with the P2RX7 gene (Wray et al., 2018;Stahl et al., 2019).
The lack of consistency among studies may be explained by the fact that depression is a heterogeneous and polygenic disorder where many low-impact loci interact with each other and the environment to promote disease development (Peterson et al., 2018). Thus, studying the impact of a single SNP is insufficient to understand the complexity of depression genetics. Haplotype studies go one step further and consider combinations of polymorphisms found in the same region. Specifically, rs2230912 and rs1718119 (Ala348Thr) lead to a gain-of-function variant of P2X7R. In fact, carrying these two SNPs causes an increased production of IL-1β in response to ATP (Stokes et al., 2010) and a higher severity of depression (Vereczkei et al., 2019).
Translational research in rodents has shown that chronic stress causes extracellular ATP increase and P2X7R activation in the brain, which results in depressive-like behavior and impaired neuroplasticity Wohleb et al., 2016;Metzger et al., 2017b;von Muecke-Heim et al., 2021). Vice versa, blockage along the P2X7R -NLRP3-IL-1β cascade has been shown to promote stress resilience with microglia and monocytes playing an important part (Schiweck et al., 2020;von Muecke-Heim et al., 2021). These studies point out P2X7R's signaling role as an important interface between chronic stress and the behavioral features of clinical depression.
In summary, a multitude of clinical and translational studies provide strong evidence that P2X7R may contribute to depression genesis, severity, and treatment response. This highlights the needed to further study the properties and functions of P2X7R in the context of stress-related disorders including depression.

GENETIC MOUSE MODELS TARGETING P2X7R
To elucidate the role of P2X7R signaling in physiological and pathological conditions in vivo, several loss-and gain-of-function as well as reporter mouse lines have been generated in the past few decades.

Constitutive P2X7R Knockout Mouse Lines
Three knockout mouse lines were created by pharmaceutical companies to investigate the consequences of a lack of P2X7R expression. The first P2X7R knockout mouse line was established by GlaxoSmithKline (GSK) by inserting a LacZ-neomycin reporter cassette into exon 1, which results in a fusion transcript comprising the 5 part of exon 1 and LacZ (Figure 1A; Sikora et al., 1999). A second knockout mouse line, generated by Pfizer Inc., (Pfizer), contains a neomycin selection cassette disrupting exon 13, which corresponds to the C-terminal region of the protein (Figure 1A; Solle et al., 2001). Finally, the knockout mouse line established by Lexicon Genetics involves as substitution of exons 2 and 3 by a LacZ-neomycin cassette, producing a fusion transcript by splicing exon 1 to the LacZneomycin construct (Figure 1A; Basso et al., 2009). In the initial studies, the authors analyzed P2X7R mRNA and protein expression and confirmed that P2X7R -dependent release of IL-1β and its pore forming function were impaired in vitro and in vivo. These studies also observed inflammatory hyposensitivity in the knockout animals (Csölle et al., 2013a). Therefore, the functional effects in these loss-of-function mouse lines are consistent with the lack of P2X7R expression.
Studies on constitutive P2X7R knockout mice generally show that genetic inactivation, similar to pharmacological inhibition of P2X7R, leads to a decrease of depressive-like behaviors under baseline (Basso et al., 2009;Csölle et al., 2013a) and stress conditions (Boucher et al., 2011;Csölle et al., 2013b;Yue et al., 2017). Although some studies could not detect an influence of P2X7R inactivation on anxiety-like phenotypes (Basso et al., 2009;Csölle et al., 2013a), others have reported reduced anxietylike behaviors in P2X7R knockout mice even under baseline conditions (Boucher et al., 2011;Yue et al., 2017). However, it is important to note that further studies on these knockout animals have unveiled that P2X7R splice variants, some of which present residual or even altered P2X7R activity, are able to escape gene inactivation in the Pfizer and GSK mice (Nicke et al., 2009;Masin et al., 2012). Therefore, these lines cannot be considered fully deleterious for P2X7R, implying that conclusions from the studies that employed these mice should be drawn with care.
Besides classical targeting strategies, approaches involving transgenic short hairpin RNA-based knock-down (Delic et al., 2008) and CRISPR/Cas9-mediated gene deletion have also been used to disrupt P2X7R expression and function (Gao et al., 2018).

Humanized P2X7R Mouse Lines
Although constitutive knockout mice provide valuable insights with respect to the physiology of the murine P2X7R, studying the human receptor in mice would allow for additional insights, for example with respect to the receptor's in vivo pharmacology. To this end, a humanized P2X7R mouse model was generated. The humanized allele [P2rx7 tm1.1(P2RX7)Jde ] involves the knockin of the human P2X7R cDNA (exons 2-13) downstream of exon 1 substituting murine exon 2 ( Figure 1B). This way, the humanized P2X7R presents the same expression pattern as the endogenous murine protein in wild-type mice. Consistent with the previously described EC 50 values of the human and murine receptors (Moore and MacKenzie, 2008;Khakh and North, 2012), the humanized P2X7R showed a 10-fold higher sensitivity to BzATP compared to its murine counterpart. The same strategy was used to generate another humanized mouse line harboring the Q460R polymorphism that had previously been linked to depression [P2rx7 tm2.1(P2RX7 * )Jde ]. Studies on these humanized mouse lines revealed how a specific genetic alteration in the human protein interacting with environmental risk factors can impact depression-and anxiety-related behavior. Similar to FIGURE 1 | Strategies targeting the P2RX7 gene in constitutive and conditional knockout mouse lines. (A) P2X7R constitutive knockout alleles. GSK (P2rx7 tm1Ipch ): a LacZ-neomycin (neo) cassette was inserted in exon 1, which results in an exon 1-LacZ fusion transcript. Lexicon Genetics (P2rx7 tm1Lex ): a LacZ-neomycin cassette was used to substitute exons 2 and 3, which results in an exon 1-LacZ-neo fusion transcript. Pfizer (P2rx7 tm1Gab ): a neomycin cassette was inserted in exon 13, which truncates the transcript in the C-terminal region. " * ": truncated P2X7R exon. Dashed lines indicated the insertion site of the construct. (B) Humanized P2X7R locus. The human cDNA sequence from exon 2-13 substitute's murine exon 2. The cDNA sequence is flanked by LoxP sites. (C) P2X7R conditional knockout locus. Upon Cre recombinase activity, the cDNA fragment is deleted resulting in a disrupted P2RX7 gene. The loss of exon 2 prevents the expression of any functional P2X7R variants. "pA/red box": Polyadenylation signal.
human heterozygous subjects, heterozygous mice suffered from altered sleep architecture and quality. These findings might reflect a prodromal disease state, which translates into higher stress vulnerability under conditions of chronic social stress (Metzger et al., 2017b).

Conditional P2X7R Mouse Lines
Another trait of the humanized P2X7R line is its susceptibility to conditional inactivation. The human cDNA fragment is flanked by loxP sites and thus can be removed by Cre-mediated recombination ( Figure 1B). This results in a truncated murine gene, which lacks the ability to produce a functional receptor ( Figure 1C). Therefore, crossing these mice with respective Cre driver mouse lines enables spatially and temporally controlled P2X7R inactivation. In contrast to constitutive knockout mice, this approach prevents potential developmental or pleiotropic effects of P2X7R inactivation due to its early and relatively broad expression, which naturally entails compensatory mechanisms. The lack of P2X7R following Cre-mediated inactivation was tested functionally, by assessing the capability of peritoneal macrophages to produce IL-1β, as well as lack of Ca 2+ influx after stimulation with BzATP. The deficiency of the known P2X7R variants was also tested demonstrating the absence of any functional transcripts (Metzger et al., 2017a).
In addition, a conditional allele flanking murine exon 2 with loxP sites has been generated by the European Conditional Mouse Mutagenesis (EUCOMM) Program [P2rx7 tm1a(EUCOMM)Wtsi ]. It has been demonstrated that Cremediated deletion of exon 2 results in a complete loss of P2X7R function (Kaczmarek-Hajek et al., 2018;Douguet et al., 2021).

P2X7R Reporter Mouse Lines
There has been a significant advance in the biological targeting of P2X7R, with the recent development of a specific P2X7R nanobody (Kaczmarek-Hajek et al., 2018). However, commercially available P2X7R antibodies still show considerable deficits in functionality and specificity (Anderson and Nedergaard, 2006;Illes et al., 2017). In general, the comparably low expression contributes to the challenge of reliably detecting P2X7R expression in the CNS. Therefore, mouse lines that express fluorescent reporters under the control of the P2X7R promoter or a tagged P2X7R represent valuable tools to study P2X7R expression.

Reporter Mice Expressing EGFP-Tagged P2X7R (P2X7R-EGFP)
The P2X7R -EGFP mouse line [Tg(RP24-114E20P2X7451P-Strep-His-EGFP)17Ani] is a bacterial artificial chromosomes (BAC) transgenic mouse line, generated by adding an EGFP sequence at the C-terminus of the P2X7R producing a P2X7R -EGFP fusion protein (Figure 2A). The resulting mouse was tested for receptor functionality as well as for its expression pattern. No significant difference in physiological responses between the native and the fusion protein were found, concluding that the EGFP-tag has no influence on P2X7R function (Kaczmarek-Hajek et al., 2018). However, Southern blot analysis shows that several copies of the P2X7R -EGFP construct are integrated into the genome of these mice, which means that this line is considerably overexpressing P2X7R (Kaczmarek-Hajek et al., 2018).

Reporter Mice Expressing Soluble EGFP
The soluble EGFP (sEGFP) mouse line [Tg(P2rx7-EGFP)FY174Gsat] is a BAC transgenic line generated by the Gene Expression Nervous System Atlas (GENSAT) project by insertion of an EGFP cassette followed by a polyA signal downstream of exon 1 of the P2RX7 gene ( Figure 2B). Therefore, EGFP expression is driven by the P2X7R promoter and supposed to mirror the endogenous P2X7R expression pattern. Different from the P2X7R -EGFP mouse line, the receptor cannot be detected directly, but only approximated based on the extent of the P2X7R promoter activity (Gong et al., 2003). The recent detailed characterization of this mouse line revealed that there is unexpected P2X7R and P2X4R overexpression. Moreover, these mice show an aberrant expression pattern compared to the endogenous P2X7R, questioning their use as a valid reporter line (Ramírez-Fernández et al., 2020).

DISCUSSION
The P2X7R is a relevant target for several disorders of the CNS due to its demonstrated involvement in response to cellular stress. It activates the production of inflammatory mediators, which have been associated with depression, suggesting the possibility that interfering with the physiology of P2X7R may have an impact on depression development and progression. Although P2X7R has been widely studied, there are still many unanswered questions related to it. First, there are studies that link certain P2X7R SNPs to depression, but the underlying consequences of these associations are still not fully understood. In fact, even the expression pattern of P2X7R has not been elucidated yet, and therefore, the physiological and pathophysiological pathways in which it is involved still need to be thoroughly analyzed.
Inactivating P2X7R seems to lead to altered anxiety-and depression-related phenotypes. However, it is now known that the knockout mouse models employed still have active P2X7R splice variants. Furthermore, some studies found significant behavioral effects of P2X7R inactivation under Frontiers in Neural Circuits | www.frontiersin.org baseline conditions, while others only observed them under stress, or did not observe them at all. These facts make apparent the need for a reassessment of anxiety and depressive-like behaviors in a validated full P2X7R knockout mouse line. Overexpression of P2X7R is a phenomenon that has not been exploited in depression research. The P2X7R -EGFP mouse line presents P2X7R overexpression consistent with the endogenous pattern. Therefore, this mouse line could be useful to study the effect of P2X7R overexpression on various phenotypes.
Depression is a complex, multifactorial, heterogeneous disorder, with a relatively high ratio of treatment-resistance (McIntyre et al., 2014). Although it is known that antiinflammatory mechanisms are involved in the treatment response of depression, current therapies are still mainly "one fits all" approaches targeting monoaminergic pathways (van Buel et al., 2015;Otte et al., 2016;Wohleb et al., 2016;Kruse et al., 2018;Binder, 2020;Nettis et al., 2021). To progress toward an effective application of P2X7R -targeting drugs in a clinical setting, there is a need to understand the involvement of P2X7R in psychiatric disorders (Deussing and Arzt, 2018). To that end, it is important to invest in reproducible studies that apply transgenic mouse models to offer novel perspectives on P2X7R signaling. Animal models allow the study of certain depression-related features, such as anxiety, anhedonia, or coping mechanisms. However, other aspects inherent to depression cannot be measured, such as guilt, thoughts of death or hopelessness. Behavioral tests and research frameworks need to evolve past current limitations toward comprehensive and standardized phenotyping reflective of human depression heterogeneity. Gradual and collaborative methodological refinement of behavioral and neurogenetic paradigms in psychiatry research will potentiate the value of animal-derived data in the context of depression and promote the translation of P2X7R evidence into clinical research.

AUTHOR CONTRIBUTIONS
LU-T reviewed the scientific literature and wrote the manuscript. I-AM-H contributed to revision and structuring of the manuscript. JD reviewed and edited the manuscript and provided scientific advice and guidance. All authors contributed to the article and approved the submitted version.