Heterozygosity for the Mood Disorder-Associated Variant Gln460Arg Alters P2X7 Receptor Function and Sleep Quality

Abstract

A single nucleotide polymorphism substitution from glutamine (Gln, Q) to arginine (Arg, R) at codon 460 of the purinergic P2X7 receptor (P2X7R) has repeatedly been associated with mood disorders. The P2X7R-Gln460Arg variant per se is not compromised in its function. However, heterologous expression of P2X7R-Gln460Arg together with wild-type P2X7R has recently been demonstrated to impair receptor function. Here we show that this also applies to humanized mice coexpressing both human P2X7R variants. Primary hippocampal cells derived from heterozygous mice showed an attenuated calcium uptake upon agonist stimulation. While humanized mice were unaffected in their behavioral repertoire under basal housing conditions, mice that harbor both P2X7R variants showed alterations in their sleep quality resembling signs of a prodromal disease stage. Also healthy heterozygous human subjects showed mild changes in sleep parameters. These results indicate that heterozygosity for the wild-type P2X7R and its mood disorder-associated variant P2X7R-Gln460Arg represents a genetic risk factor, which is potentially able to convey susceptibility to mood disorders.SIGNIFICANCE STATEMENT Depression and bipolar disorder are the most common mood disorders. The P2X7 receptor (P2X7R) regulates many cellular functions. Its polymorphic variant Gln460Arg has repeatedly been associated with mood disorders. Genetically engineered mice, with human P2X7R, revealed that heterozygous mice (i.e., they coexpress the disease-associated Gln460Arg variant together with its normal version) have impaired receptor function and showed sleep disturbances. Human participants with the heterozygote genotype also had subtle alterations in their sleep profile. Our findings suggest that altered P2X7R function in heterozygote individuals disturbs sleep and might increase the risk for developing mood disorders.

Keywords: P2X7 receptor; humanized mouse model; mood disorder; purinergic signaling; sleep; stress.

Figure 1.

Establishment of humanized P2X7R mice (hP2RX7). A, Targeting strategy for knockin of the human P2RX7 cDNA into the mouse P2rx7 locus. Partial restriction maps [only relevant BamHI (B) sites are depicted] of the wild-type P2rx7 locus, targeting vector, and mutant locus following homologous recombination and loxP-flanked humanized locus after Flp recombinase-mediated deletion of the neomycin selection marker (pA; 4 × polyadenylation signal). B, Southern blot analysis of genomic DNA from an embryonic stem cell clone targeted with the human P2X7R-Q460R construct, which was used to generate P2rx7^Q460R mice. The targeted allele is indicated by the presence of an additional 7.4 kb fragment. C, General strategy to substitute the murine P2X7R by the human WT receptor or its P2X7R-Q460R variant. D, The mRNA expression of human P2X7R in knock-in mice fully recapitulates endogenous expression of murine P2X7R, as demonstrated by in situ hybridization on coronal brain sections. Depicted are photomicrographs of the hippocampus (scale bar, 100 μm) with magnifications of the hippocampal CA3 region shown below. E, Human P2X7R mRNA is expressed at identical levels in the cortex (Ctx), hippocampus (Hip), and cerebellum (Cb) of heterozygous P2rx7^+/hWT and P2rx7^+/Q460R mice (n = 6). E, Human P2X7R mRNA is expressed at identical levels in the cortex (Ctx), hippocampus (Hip), and cerebellum (Cb) of heterozygous P2rx7^+/hWT and P2rx7^+/Q460R mice (n = 6). F, Validation of hP2X7R functionality in P2rx7^hWT and P2rx7^hQ460R mice as determined by IL-1β release. Peritoneal macrophages of P2rx7^hWT and of P2rx7^hQ460R mice are able to secrete IL-1β in response to LPS stimulation and subsequent treatment with the P2X7R agonist BzATP (n = 4). Data are expressed as the mean ± SEM.

Figure 2.

Generation and basal characterization of heterozygous humanized P2X7R mice. A, Scheme illustrating the generation of P2rx7^hWT, P2rx7^hHET, and P2rx7^hQ460R mice. B, Genotyping was performed by PCR and subsequent restriction digestion of the 557 bp PCR product with PvuII. C, Calcium uptake following BzATP treatment of primary hippocampal cells is attenuated in heterozygous hP2rx7^hHET mice. For each primary culture preparation, three independent measurements were performed (RM ANOVA, *p < 0.05, n = 4–7). D–G, Basal behavioral characterization of hP2X7R mice revealed no differences in the total distance traveled of the OF test (D), in the time spent in the open arm of the EPM (E) or lit zone time of the DaLi test (F), and in total immobility during the FST (one-way ANOVA, n = 11–12; G). Data are expressed as the mean ± SEM.

Figure 3.

Heterozygosity for hP2X7R-WT with hP2X7R-Gln460Arg alters sleep parameters in hP2RX7 mice. A, B, No genotype differences were observed in the length of NREMS (A) or REMS (B) across the 24 h recording period (2 h mean ± SEM). C, The architecture of REMS was significantly altered in P2rx7^hHET mice that showed more frequent entries to the REMS epoch during the light period (12 h mean ± SEM; *p < 0.05). D, SWA during NREMS was significantly attenuated in heterozygous mice (2 h mean ± SEM; *p < 0.05, **p < 0.001). E, P2rx7^hHET mice rarely entered SWS₂ across the entire 24 h (2 h mean ± SEM; **p < 0.001). F, Hypnograms and spectrograms of representative animals for each genotype. P2rx7^hHET mice show suppression of EEG power in lower-frequency bands, indicating a lower sleep quality.

Figure 4.

Heterozygosity alters sleep parameters in humans. A, EEG power spectra (log-transformed) during NREMS between homozygous (A/A) and heterozygous (A/G) healthy participants. EEG power density in the heterozygous genotype was significantly higher in the 13, 25, and 26 Hz bins (two-way mixed ANOVA with the between-subject factor genotype and the within-subject factor derivation for F3A2, C3A2, P3A2, and O1A2; *p < 0.05). B, The genotype effect on power density was most prominent in parietal EEG derivation (P3A2) in the 13 Hz bin. C, The 13 Hz frequency in human EEG overlaps with sleep spindles activity, whose frequency usually varies between 12 and 15 Hz. Heterozygous A/G carriers showed a significantly lower mean peak frequency of all sleep spindles detected in parietal (P3A2) EEG derivation within NREMS (two-tailed unpaired t test, **p = 0.001).

Figure 5.

CSDS induces robust physiological and neuroendocrine changes independent of genotype. A, CSDS led to a decrease in fur quality from day 4 onward, depicted by an increase in the fur state index. B, Thymus weights were significantly reduced in stressed animals compared with their littermate controls. C, An increase in relative adrenal weights was observed in all mice subjected to CSDS. D, CSDS led to an enhanced corticosterone response following a forced swim test. Data are expressed as the mean ± SEM. *p < 0.05.

Figure 6.

CSDS induces robust behavioral deficits in hP2RX7 mice. A, B, CSDS induced an overall decrease in locomotion in hP2X7R mice in the OF when assessed for the entire test duration (15 min; A) as well as in the initial 5 min (B). C, Accordingly, immobility in the OF was significantly increased during the first 5 min of the OF test. D, hP2RX7 mice showed an enhanced anxiety response to CSDS in the elevated plus maze. E, CSDS evoked a decreased interaction time with the social target in hP2RX7 mice during the social avoidance paradigm. F, However, hP2RX7 mice also spent significantly less time interacting with the empty wire cage during the first trial of the social avoidance task. G, During the female urine sniffing test, a decrease in time spent sniffing estrus female urine was observed in all stressed hP2RX7 animals compared with nonstressed controls. H, No significant genotype and/or condition differences were detectable in the water trial of the female urine sniffing test. Data are expressed as the mean ± SEM. *p < 0.05.