Overexpression of Prokineticin 2 in Transgenic Mice Leads to Reduced Circadian Behavioral Rhythmicity and Altered Molecular Rhythms in the Suprachiasmatic Clock

A plasmid with the mouse PK2 cDNA under the control of 5.7 kb PKR2 promoter was constructed. The plasmid DNA was linearized and injected into fertilized eggs of wild type mice by microinjection. Founder mice with the incorporation of the plasmid DNA and confirmed PK2 expression at ZT18 in the SCN were identified. PK2 expression in normal mice was essentially undetectable at ZT18 [4]. Ectopic expression of PK2 in non-SCN sites was also detected in PKR2-positive sites. The founder mice were bred with wild type mice to generate the PKR2-PK2 transgenic mice and non-transgenic littermate controls. All animal procedures were approved by the university’s institutional animal care and use committee.

Monitoring of the circadian locomotor rhythm activity was carried out as described [17,18]. Mice were individually housed with free access to food and water. Mouse cages were equipped with infrared motion sensors for monitoring spontaneous activity and were recorded electronically. Mice were entrained to an initial 12h Light: 12h Dark (LD) cycle (light intensity ~150 lux, lights on at ZT0 7:00 a.m., and lights off at ZT12 7:00 p.m.). After about 2 weeks of activity recording in LD conditions, the mice were placed in constant darkness (DD) with a dim red light (<5 lux) for ~3 weeks. The locomotor activities were calculated as counts per 5-min interval. The free-run period and Fast Fourier Transformation (FFT) were analyzed using ClockLab software (Actimetrics, Evanston, IL) in the MatLab environment. The free-running periods were measured by a χ² periodogram under DD. FFT values representing the peak relative amplitudes in the circadian range were calculated. Statistical analyses were performed by using GraphPad Prism Software Version 5.0 (San Diego, CA).

For the molecular rhythmicity analysis, mutant mice and same number of control mice were sampled every three hours (N = 3 for each time point) under constant darkness for 48 hrs after entrained in 12L:12D for two weeks. In situ hybridization was carried out as described [6]. Brains were quickly removed and frozen in isopentane at 20°C for 30 sec. Twenty-micrometer coronal sections were cut on a cryostat and serial sections collected. The following antisense and sense riboprobes were used: PK2 (GenBank accession number AF487280↗; residues 1–528), mPer1 (340–761 nt of accession number AF022992↗) or mPer2 (9–489 nt of AF035830↗), Bmal1 (864–1362 nt of AB015203↗), mCry1 (1081–1793 nt of AB000777↗) and mCry2 (981–1765 nt of AB003433↗). The riboprobes were generated by T7 or SP6 RNA polymerases and radioactively labeled with ³⁵S-UTP. Probes were used at concentration of 1 × 10⁷ cpm/ml. Tissue sections were hybridized as follows. Briefly, tissue sections were pretreated with proteinase K, hybridized for 18 hours at 60°C, followed by RNase digestion, high stringency washes and dehydration. Tissue sections were then be exposed to BioMax film (Kodak) for 3–4 days. Specific hybridization signals were quantitatively analyzed by comparing to ¹⁴C-standards of known radioactivity using a video-based computer image analysis system (MCID, Imaging Research, St. Catharine’s, Ontario, Canada). A calibration curve of optical density versus radioactivity (dpm/mg tissue wet weight) was constructed using ¹⁴C-standards. Specific hybridization signals in SCN were obtained by subtracting background values obtained from adjacent brain areas that have no hybridization signal. Autoradiographic images were captured using MCID taken under the transillumination microscope (BX50, Olympus) by using Spot camera software version 2.2.2 (Diagnostic Instruments, Sterling Heights, MI).

Fourteen-micron coronal sections were mounted onto Fisherbrand Superfrost Plus slide (Thermo Fisher Scientific, Pittsburgh, PA). The PK2 cDNA fragment-containing vector was linearized with restriction enzyme and used as template to synthesize anti-sense complementary RNA probes, using a digoxigenin (DIG) RNA labeling mix (Roche Applied Sciences, Indianapolis, IN). In situ hybridization and immunological detection of DIG-labeled hybrids was carried out as described previously [38].

To investigate the likely effect of perturbed PK2 signaling on circadian rhythms, a plasmid in which the mouse PK2 cDNA under the control of the 5.7 kb PKR2 promoter (PKR2-PK2) was constructed. As PK2 is a secreted molecule that signal through the receptor PKR2 [20], we reasoned expression of PK2 in the PKR2-positive cells would perturb the normal signaling. In vitro study with luciferase reporter gene indicated the activity of this fragment of the PKR2 promoter was active in the cultured cells that express PKR2 endogenously (data not shown). The plasmid DNA was linearized and microinjected into fertilized eggs of CD1 mice to produce the transgenic mice.

Two founder mice (TG10 and TG14) with confirmed PK2 expression at ZT 18 in the SCN were identified (Figure 1). PK2 expression in normal mice is essentially undetectable at ZT18 [4]. Also as expected, ectopic PK2 expression was observed in the related brain regions (Figure 1C) that normally express its receptor, PKR2 (Figure 1C), such as the paraventricular nucleus of thalamus. The circadian expression profile of PK2 in the PKR2-PK2 transgenic mice was then examined by sampling every three hours for a continuous 24 hour circle. As shown in Figure 2, the circadian expression profile of PK2 in the SCN of the transgenic mice was altered. Particularly, the amplitude of PK2 oscillation in the SCN of the PKR2-PK2 transgenic mice was significantly reduced, mainly due to the remarkable increase of the PK2 expression during the dark phase (Figure 2).

We next investigated the locomotor rhythms of the PKR2-PK2 transgenic mice. The locomotor activities of mice were continuously monitored by using infrared beam recording in their home cages. As illustrated in Figure 3, both lines of the transgenic mice displayed reduced circadian locomotor rhythmicity. Quantification of circadian locomotor rhythmicity revealed that the PKR2-PK2 transgenic mice had significantly lower relative FFT power under DD, compared to their wild type littermate controls (Figure 4). The PKR2-PK2 transgenic mice also appeared to have reduced consolidation in the activity during subject night phase and display apparent behavioral splitting, especially apparent in the TG14 line (Figure 3). In addition, although not statistically significant, the PKR2-PK2 transgenic mice displayed trend of lengthening of the free-running periods (23.43 +/- 0.01 hr for WT, 23.66 +/- 0.27 hr for TG10, 23.98 +/- 0.27 hr for TG14). These studies indicate that overexpression of PK2 under the control of the PKR2 promoter in transgenic mice led to reduced amplitude of circadian locomotor rhythm.

We then examined the effects of PK2 overexpression on the rhythm of the SCN circadian genes by in situ hybridization (Figure 5). In the SCN of the PKR2-PK2 transgenic mice, the mRNA expression of Bmal1, one of the key core components of the circadian clock, was significantly altered, compared to control mice. The changes in the expression of Cry2, Per1 and Per2 in the SCN of the PKR2-PK2 transgenic mice were also significant, although less remarkable than that of Bmal1 (Figure 5). Subtle alteration of clockwork expression in the SCN and elongated behavioral period had actually previously been noted in PK2-deficient mice [17]. Taken together, these data suggest that PK2 signaling may feedback to affect the expression of the core oscillator genes in the SCN.

In this study, we demonstrated that overexpression of PK2 in transgenic mice under the control of its receptor (PKR2) led to reduced oscillation of PK2 mRNA in the SCN and decreased amplitude of circadian locomotor rhythms. The oscillation of PK2 mRNA in the SCN was reduced, primarily due to the increase PK2 expression in the SCN during the dark phase (Figures 1 and 2). The reduction of circadian locomotor rhythm was observed in two lines of independent transgenic mice (Figures 3 and 4). These observations were consistent with the signaling role of PK2 as an output signaling molecule of the SCN circadian clock. PK2 has been postulated as an SCN output molecule based on several previous findings [4,5,17,25,38]. Particularly, results from knockout mice that lack functional PK2 or its receptor (PKR2) have strongly supported the essential role of PK2 signaling for the maintenance of robust circadian rhythms. In the absence of PK2 signaling, the amplitudes of circadian locomotor parameters were remarkably reduced, with rhythmicity amplitude of wheel-running activity of PK2-deficient mice less than 20% of wild type mice [17,25]. Rhythmicity of other circadian parameters, including sleep–wake cycle, body temperature, circulating glucocorticoid and glucose levels, as well as the expression of peripheral clock genes, was also significantly reduced [17,25]. The current overexpression studies in the transgenic mice have provided corroborative evidence for a signaling role of PK2 in the control of circadian rhythms.

The altered molecular rhythms in the SCN of the PKR2-PK2 transgenic mice indicate that PK2 also appears to regulate the molecular rhythmicity of core clockwork, particularly the amplitude of Bmal 1 (Figure 5). PK2 has been primarily considered as an output signaling molecules of the SCN [4,5,17,25]. However, the receptor for PK2, PKR2, is highly expressed in the SCN [4,6], and SCN neurons are known to form local circuits within the SCN, sending collateral projections within the SCN [1,2,7]. Therefore, in addition to affecting SCN target neurons such as the paraventricular nucleus of thalamus [37], PK2 signaling functions upstream of the SCN, as supported by recent report showing the expression of PK2 in the intrinsically photosensitive retinal ganglion cells that project to SCN neurons [39]. Careful analysis of PK2-deficient mice indicated the free-running periods of residual circadian rhythms in the absence of PK2 signal is slightly lengthened [17]. Subtle alteration in the expression of one clockwork gene was indeed observed in the SCN of PK2-deficient mice [17]. A recent study with mice that have Bmal1 deletion specific to AVP-positive subpopulations of the SCN neurons showed altered circadian expression of genes in the SCN [22], including reduced PK2 expression rhythm. As the PK2 rhythm in the SCN is also reduced in V1a-null mice [18], the two SCN output molecules, PK2 and AVP, may have a similar effect in modulating the molecular rhythm in the SCN. Taken together, these results appear to support the inference that PK2 signaling plays a role in both the input and output of the SCN clock.