Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity

Abstract

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176^−/− animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176^−/− mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu^−/−Nms^−/−Gpr176^−/− mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

INTRODUCTION

Circadian clocks have evolved to predict and coordinate physiologic processes with daily recurring changes in the environment.^1–3) In mammals, the central circadian pacemaker is localized in the suprachiasmatic nucleus (SCN) of the hypothalamus.⁴⁾ Light is the major external stimulus that entrains circadian rhythms to the external 24-h light/dark (LD) cycle.⁵⁾ Ambient light information is perceived by the retina and transmitted to the SCN neurons through the retino-hypothalamic tract. A light pulse in the night causes phase shifts in a phase-dependent manner, with delays dominating the early subjective night and advances dominating the late subjective night. Importantly, in mammals, the magnitude of phase shifts is also tightly controlled, with the maximum amount of phase shifts caused by a light pulse being approximately 2 h.^6–8)

G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and central to many fundamental cellular signaling pathways. We previously identified Gpr176 as an SCN-enriched orphan GPCR, whose genetic deletion leads to shortened circadian period length in locomotor activity rhythm.^9–11) However, Gpr176 is required for the period maintenance but not required for maintaining proper light-induced phase shifts in mice because Gpr176-deficient (Gpr176^−/−) mice exhibit phase-dependent phase shifts almost identical to those observed in wild-type (WT) mice.¹¹⁾ These data suggest that either Gpr176 is simply unrelated to the modulation of light-induced phase shift or a compensatory mechanism(s) mitigates the potential effects of Gpr176 deficiency.

In the present study, we describe the identification of upregulated expression of neuromedin U (Nmu) and neuromedin S (Nms) mRNA in the SCN of Gpr176^−/− mice. Prompted by this finding, we generated C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice. We noted that the mutant mice show an increased magnitude of phase delay in response to early subjective night light, which is a previously undetected phenotype in studying either Gpr176 deficient mice¹¹⁾ or Nmu/Nms double deficient mice.

MATERIALS AND METHODS

Mouse Strains and Behavioral Activity Monitoring

Nmu^−/−Nms^−/− mice were generated by crossing Nmu^−/− mice¹²⁾ and Nms^−/− mice (BRC No. RBRC04550: https://knowledge.brc.riken.jp/resource/animal/card?__lang__=en&brc_no=RBRC04550). The double knockout mice were further mated with Gpr176^−/− mice¹¹⁾ to produce Nmu^−/−Nms^−/−Gpr176^−/− mice. All mice bred in this study were of the C57BL/6J background. WT mice that we compared with C57BL/6J-background Nmu^−/−Nms^−/−Gpr176^−/− (TKO) mice were inbred C57BL/6J WT mice. Gpr176^+/+ and Gpr176^−/− mice that we used for SCN microarray analysis were siblings that were generated from C57BL/6J-backcrossed Gpr176^+/− mice.¹¹⁾ For behavioral activity monitoring, single-caged adult male mice (7- to 10-week old) were housed individually in light-tight, ventilated closets within a temperature- and humidity-controlled facility. Locomotor activity was detected with passive (pyroelectric) IR sensors (FA-05 F5B; Omron) and data were analyzed with ClockLab software (Actimetrics) developed on MatLab (Mathworks).^13,14) Free-running circadian period was determined with a linear regression line method based on a 14-d interval taken 3 d after the start of continuous dark (DD) condition. For phase shift experiments, mice were exposed to a 15-min light pulse at either circadian time (CT) 14 or CT22 (CT12 corresponds to locomotor activity onset) with a light intensity of 200 lx. Phase shifts were quantified as the time difference between regression lines of activity onsets before and after the light stimulation, using ClockLab software. Behavioral studies were performed using the same experimental conditions as those described in our previous report.¹¹⁾ All animal experiments were conducted in compliance with ethical regulations in Kyoto University and performed under protocols approved by the Animal Care and Experimentation Committee of Kyoto University and the Kurume University Animal Care and Treatment Committee.

Laser Microdissection and RNA Extraction

Animals were sacrificed by cervical dislocation under a safety red light at the indicated time points in DD. Coronal brain section (30-µm thick) containing the SCN was prepared using a cryostat microtome (CM3050S, Leica) and mounted on POL-membrane slides (Leica). Sections were fixed in ice-cold ethanol-acetic acid mixture (19 : 1) for 2 min and stained with 0.05% toluidine blue. SCN were then excised using a LMD7000 device (Leica) and lysed into Trizol reagent (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

DNA Microarray

Total RNA from the SCN was purified using the RNeasy micro kit (Qiagen, Venlo, the Netherlands) and integrity was assessed by analyzing aliquots on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, U.S.A.). For each sample, 100 ng of total RNA was amplified into cRNA and made into cDNA using the Ambion WT Expression Kit (Thermo Fisher Scientific), and 5.5 µg of the cDNA was fragmented and labelled using the Affymetrix GeneChip WT Terminal Labeling kit. The labeled sense strand DNA was hybridized to the GeneChip Mouse Gene 1.0 ST Array according to the manufacturer’s protocol using a GeneChip Hybridization Oven 640. The staining, washing and scanning of the arrays were performed on the Affymetrix GeneChip Fluidics Station 450, the Affymetrix GeneChip Scanner 3000 7G, and the GeneChip Command Console Software (AGCC). Data were analyzed with the GeneSpring GX software (version 11.5.1, Agilent) in the following settings: Summarization Algorithm, RMA16; Confidence level, All; Baseline Options, Do not perform baseline transformation. DNA microarray data have been deposited at GEO under accession number GSE184440. For the rank order analysis and scatter plot comparison between samples, genes whose signal intensities differed by more than 3 SD between biological duplicate samples were omitted.

Radioisotopic in Situ Hybridization

Radioisotopic in situ hybridization was performed as described⁸⁾ with the following gene-specific probes: for Nmu (nucleotides 28–413, NM_019515) and for Nms (nucleotides 19–338, NM_001011684). Free-floating brain sections (30-µm thick) containing the SCN were hybridized to anti-sense ³³P-labeled cRNA probes. Quantification of expression strength was performed by densitometric analysis of autoradiograph films. We quantified Nms mRNA expression under a normal LD cycle as it is reported that in the rat SCN, Nms exhibits rhythmic expression under LD but not DD.¹⁵⁾

Quantitative (q)RT-PCR

Purified total RNA was converted to cDNA using SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific). For measuring light-induced Per1 and cFos mRNA expression, qPCR was performed as described¹⁰⁾ using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan) on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The primer sets used were following: Per1, Fw: 5′-tgg ctc aag tgg caa tga gtc-3′, Rv: 5′-ggc tcg agc tga ctg ttc act-3′; cFos, Fw: 5′-aga gcg gga atg gtg aag ac-3′, Rv: 5′-ggt ggg ctg cca aaa taa ac-3′; Rplp0, Fw: 5′-ctc act gag att cgg gat atg-3′, Rv: 5′-ctc cca cct tgt ctc cag tc-3′. Melt curve analysis was performed to confirm each of the final PCR product. The data were normalized with Rplp0. As for the profiling of a series of clock genes and clock-related genes in the SCN, qPCR was performed on a BioMark HD System (Fluidigm, South San Francisco, CA, U.S.A.) with a 48.48 Fluidigm BioMark Dynamic Array chip (Fluidigm) as described previously.¹⁶⁾ The primers and probes used in Fluidigm qPCR were also identical to those we reported¹⁶⁾ and are available upon request.

RESULTS

Comparison of Gene Expression Profiles between Gpr176^+/+ and Gpr176^−/− Mouse SCN

We performed microarray transcriptome analysis to seek for a potential difference in gene expression between Gpr176^+/+ and Gpr176^−/− mouse SCN (Fig. 1). C57BL/6J-backcrossed Gpr176^+/+ and Gpr176^−/− mice were entrained on a 12-h light : 12-h dark (LD) cycle for two weeks and released into constant darkness (DD). Mice were sacrificed at two independent circadian time (CT) points, CT6 and CT18 (CT12 was defined as the time at which an animal began its daily locomotor activity). These time points were chosen to see potential effects of Gpr176 deletion on gene expression during the subjective day (CT6) and during the subjective night (CT18). SCN tissue samples were isolated by laser microdissection and pooled from 10 individual animals per microarray replicate. For the respective time points, two independent biological samples were prepared from two genotypes and compared using DNA microarray (a full set of all analytical data is provided in Supplementary Data 1). We first rank-ordered Gpr176^+/+ versus Gpr176^−/− mouse SCN mean gene transcript changes from the most upregulated to the most downregulated gene (Fig. 1A). This analysis revealed that among 27,568 genes examined, the gene encoding neuromedin U (Nmu) represents the most upregulated gene in the SCN of Gpr176^−/− mice (Fig. 1A). When compared between CT6 and CT18, the upregulating effect of Gpr176 depletion on Nmu expression was more profound at CT6, a time of peak expression of Nmu in the SCN,^17,18) than CT18 (Figs. 1B, C). Bglap-rs1, Lcn2, C4b, and Ndn were also identified as upregulated genes. Bglap-rs1 was more upregulated at CT18 than CT6. Lcn2, C4b, and Ndn were almost equally upregulated at both CT6 and CT18 (Fig. 1C). Not surprisingly, DNA microarray hybridization signals of Gpr176 were downregulated in all SCN samples (Figs. 1A, D). Except that, Eif2ak4, Mid1, Tmem185b, and Pgap1 were identified as downregulated genes, while the relative degrees of their respective downregulation were within 0.288-fold in the SCN of Gpr176^−/− mice (Fig. 1D). In contrast, expression profiles of >99.9% of the genes analyzed, including all known core clock genes such as Per1, Per2, Clock, and Bmal1, were essentially unimpaired at both CT6 and CT18 in the SCN of Gpr176^−/− mice (Fig. 1E, Supplementary Fig. 1): among 27,568 genes, only 6 genes were upregulated and 9 downregulated by more than 1.25-fold (Fig. 1B, Supplementary Data 1). These data indicate that only a handful of genes were affected by the deletion of Gpr176 gene.

Fig. 1. Microarray Analysis of Gene Expression Comparing Gpr176^+/+ and Gpr176^−/− SCN

(A) Microarray probes in rank order. Values of log₂ fold change in gene expression in Gpr176^−/− SCN relative to Gpr176^+/+ were plotted. The data were calculated using the average microarray value of CT6 and CT18. The dot colored in blue and red indicates Gpr176 and Nmu, respectively. (B) Scatter plots showing the difference in gene expression between Gpr176^+/+ and Gpr176^−/− SCN at CT6 (left) and CT18 (right). Values (log₂) are the mean of two biological replicates. (C–D) The top 5 upregulated (C) and downregulated (D) genes in the Gpr176^−/− SCN. The line graphs below the list show the expression profiles of individual genes. Values are mean ± variation of two biological replicates. (E) Unaffected expression profiles of representative clock genes: Per1, Per2, Bmal1, Clock, and Dbp. Microarray data of other examined clock genes are available in Supplementary Fig. 1.

Upregulated mRNA Expression of Nmu in Gpr176^−/− SCN

Follow-up quantification by qPCR verified the levels of Nmu mRNA expression in the SCN that are higher at CT6 than CT18 and upregulated in the SCN of Gpr176^−/− mice (Fig. 2A, p < 0.05 for genotype effect, two-way ANOVA, Gpr176^+/+ vs. Gpr176^−/−). As expected, Gpr176 transcript expression was undetectable in the SCN of Gpr176^−/− mice (Fig. 2A). The circadian clock genes Per1, Per2, Bmal1, Clock, and Dbp underwent expected circadian variation and exhibited nearly identical mRNA expression levels in the SCN of Gpr176^+/+ and Gpr176^−/− mice at both CT6 and CT18 (Fig. 2A) (see Supplementary Fig. 2 for other clock genes).

Fig. 2. Nmu mRNA Is Up-Regulated in Gpr176^−/− SCN

(A) Relative mRNA levels of Nmu, Gpr176, Per1, Per2, Bmal1, Clock, and Dbp in the SCN of Gpr176^+/+ (black line) and Gpr176^−/− (pink line) mice at CT6 and CT18, determined by qRT-PCR. Values are mean ± variation of two biological replicates. Either of the values of CT6 or CT18 of Gpr176^+/+ SCN samples are set to 1. n.d., not detectable. (B) Representative brain coronal sections of Gpr176^+/+ and Gpr176^−/− mice hybridized to anti-sense ³³P-labelled Nmu riboprobe. Arrows indicate the position of the SCN. Scale bars, 1 mm. (C) Nmu mRNA abundance in the SCN of Gpr176^+/+ and Gpr176^−/− mice at CT6 and CT18, determined by in situ hybridization autoradiography. Values are presented as the mean ± standard error of the mean (S.E.M.) (n = 4). **** p < 0.0001, *** p < 0.001, two-way ANOVA followed by Holm-Sidak’s post hoc test. Representative autoradiographs are shown on the top. Scale bars, 200 µm.

To further confirm genotype-dependent difference in Nmu expression and examine its SCN-enriched expression, we performed in situ hybridization using a radioisotope-labeled probe for Nmu. Expression of Nmu mRNA was exclusively restricted to the SCN in the coronal brain section containing the SCN in both Gpr176^+/+ and Gpr176^−/− mice (Fig. 2B). The levels of Nmu transcript expressed in the SCN were significantly higher at CT6 than CT18 in both Gpr176^+/+ and Gpr176^−/− mice and significantly upregulated in Gpr176^−/− mice at both CT6 and CT18, as determined by autoradiography (CT6: 0.262 ± 0.004 kBq/g for Gpr176^+/+, 0.408 ± 0.009 kBq/g for Gpr176^−/−, CT18: 0.049 ± 0.004 kBq/g for Gpr176^+/+, 0.100 ± 0.010 kBq/g for Gpr176^−/−, for time effect (CT6 vs. CT18), **** p < 0.0001 for both Gpr176^+/+ and Gpr176^−/−, for genotype effect (Gpr176^+/+ vs. Gpr176^−/−), **** p < 0.0001 for CT6 and *** p < 0.001 for CT18, two-way ANOVA with Holm-Sidak’s post hoc test, Fig. 2C).

Nms, a Homolog of Nmu, Is Also Upregulated in Gpr176^−/− SCN

Neuromedin S (Nms) is a homolog of Nmu and also predominantly expressed in the SCN.¹⁵⁾ Microarray values of Nms were upregulated in the SCN of Gpr176^−/− mice by approximately 1.2-fold, which was the 20th largest fold-increase in the list of 27568 genes (Supplementary Data 1). Differently from Nmu, the levels of Nms mRNA expression showed no remarkable variation between CT6 and CT18 for both genotypes. In addition, we noticed that microarray values of Nms were considerably higher than those of Nmu (compare mean hybridization intensity of Nmu and Nms: in Gpr176^+/+, Nmu, 153, Nms, 1891; in Gpr176^−/−, Nmu, 223, Nms, 2308), suggesting an abundant expression of Nms in the SCN (Figs. 1C, 3A).

Fig. 3. Nms mRNA Is Up-Regulated in Gpr176^−/− SCN

(A) DNA microarray values of Nms in the SCN of Gpr176^+/+ (black line) and Gpr176^−/− (pink line) mice at CT6 and CT18. Data are mean ± variation of two biological replicates. (B) Representative brain coronal sections of Gpr176^+/+ and Gpr176^−/− mice hybridized to anti-sense ³³P-labelled Nms riboprobe. Arrows indicate the position of the SCN. Scale bars, 1 mm. (C) 24-h profile of Nms expression in the SCN of Gpr176^+/+ and Gpr176^−/− mice, determined by in situ hybridization autoradiography. Values are presented as the mean ± S.E.M. (n = 4). **** p < 0.0001, two-way ANOVA test. Representative autoradiographs are shown on the top. Scale bars, 200 µm.

We next examined Nms expression by quantitative radioactive in situ hybridization using brain samples collected at 4-h intervals for 24 h to explore in more detail whether the SCN Nms is upregulated in the Gpr176^−/− mice throughout the day (Figs. 3B, C). Nms displayed SCN-enriched expression in the coronal brain section containing the SCN of Gpr176^+/+ and Gpr176^−/− mice (Fig. 3B), as detected previously in rats¹⁵⁾ and mice.¹⁹⁾ As expected from microarray values, the intensities of Nms radioactivity in the SCN were more than 10 times higher than those of Nmu (Figs. 2C, 3C, daily mean radioactivity: in Gpr176^+/+, Nmu, 0.16 kBq/g, Nms, 7.70 kBq/g; in Gpr176^−/−, Nmu, 0.25 kBq/g, Nms, 9.11 kBq/g). We found that Nms expression did not show an overt diurnal rhythm in the SCN and that the levels of its transcript were almost constantly upregulated in the SCN of Gpr176^−/− mice compared with those of Gpr176^+/+ mice (Fig. 3C, p < 0.0001 for genotype effect, two-way ANOVA).

Nmu^−/−Nms^−/−Gpr176^−/− Mice Exhibit an Increased Magnitude of Phase Delay to Early Subjective Night Light

Upregulated expression of Nmu and Nms might have a compensatory role to mitigate the genetic effect of Gpr176 deficiency. However, circadian period length was not additionally altered by simultaneous deletion of Nmu, Nms, and Gpr176 (Fig. 4, Nmu^−/−Nms^−/−Gpr176^−/−). A free-running period of locomotor activity rhythm of Nmu/Nms/Gpr176-triple knockout (TKO) mice under constant darkness was slightly diminished when compared with that of WT mice; however, the extent of this shortening in period was not substantially different from that of reported Gpr176 single knockout mice.¹¹⁾ We verified normal (thus, WT-like) circadian period length of Nmu/Nms-double knockout (Nmu^−/−Nms^−/−) mice under constant darkness, as reported previously by Lee et al.¹⁹⁾ (Fig. 4). Differently from the phenotype in period, a distinct alteration in light-induced phase-shifts was observed for the TKO mice (Fig. 5), a phenotype that was not previously described for the Gpr176 single knockout mice.¹¹⁾ A light stimulation can shift the phase of the circadian clock in a phase-dependent manner, with phase-delays in response to light given in the early subjective night and phase-advances to light given in the late subjective night.^7,20) We found that a light pulse stimulation at CT14 (i.e., in the early subjective night) caused greater magnitudes of phase-delay in Nmu^−/−Nms^−/−Gpr176^−/− TKO mice than control WT and Nmu^−/−Nms^−/− mice (Fig. 5, phase-delay, h, TKO, 2.54 ± 0.20, WT, 1.84 ± 0.08, Nmu^−/−Nms^−/−, 1.45 ± 0.12, ** p < 0.01, **** p < 0.0001, one-way ANOVA with Holm–Sidak’s post hoc test). A light stimulation at CT22 (i.e., in the late subjective night) also caused greater magnitudes of phase-advance in Nmu^−/−Nms^−/−Gpr176^−/− mice than Nmu^−/−Nms^−/− mice (Fig. 5, TKO, 1.08 ± 0.20, Nmu^−/−Nms^−/−, 0.44 ± 0.11, * p < 0.05, one-way ANOVA with Holm–Sidak’s post hoc test), although there was no significant difference between WT and Nmu^−/−Nms^−/−Gpr176^−/− mice.

Fig. 4. Nmu^−/−Nms^−/−Gpr176^−/− Mice Display a Shortened Free-Running Period

(A) Representative double-plotted locomotor activity records of WT, Nmu^−/−Nms^−/−, and Nmu^−/−Nms^−/−Gpr176^−/− mice. Mice were housed in a standard 12-h light:12-h dark cycle (LD) for 6 d and released into constant darkness (DD). Periods of darkness are indicated by grey backgrounds. Data are shown in double-plotted format. Each horizontal line represents 48 h; the second 24-h period is plotted to the right and below the first. (B) Period-length distribution of WT, Nmu^−/−Nms^−/−, and Nmu^−/−Nms^−/−Gpr176^−/− mice. Free-running period was determined with a linear regression line method based on a 14-d interval taken after 3 d of a DD regime. Plots show the period length of individual animals (WT, n = 15; Nmu^−/−Nms^−/−, n = 10; Nmu^−/−Nms^−/−Gpr176^−/−, n = 12). Data indicate the mean ± S.E.M. **** p < 0.0001, one-way ANOVA with Holm–Sidak’s post hoc test. n.s., not significant.

Fig. 5. Nmu^−/−Nms^−/−Gpr176^−/− Mice Exhibit an Increase in the Magnitude of Phase-Delay to Early Subjective Night Light

(A) Representative double-plotted locomotor activity records of WT, Nmu^−/−Nms^−/−, and Nmu^−/−Nms^−/−Gpr176^−/− mice before and after a 15-min light pulse exposure at CT14 or CT22. CT was determined for individual animals based on their free-running period and the onset of locomotor activity (which is defined as CT12). Phase shifts (delay at CT14, advance at CT22) were quantified as the time difference between regression lines of activity onset before and after the light pulse. Triangles indicate the day for light administration. (B) Magnitude of light-induced phase-shifts of WT, Nmu^−/−Nms^−/−, and Nmu^−/−Nms^−/−Gpr176^−/− mice. By convention, delays are negative and advances are positive. Data indicate the mean ± S.E.M. (CT14, WT, n = 14; Nmu^−/−Nms^−/−, n = 7; Nmu^−/−Nms^−/−Gpr176^−/−, n = 12; CT22, WT, n = 9; Nmu^−/−Nms^−/−, n = 7; Nmu^−/−Nms^−/−Gpr176^−/−, n = 12). **** p < 0.0001, ** p < 0.01, * p < 0.05, two-way ANOVA with Holm–Sidak’s post hoc test. n.s., not significant.

The Extent of Light-Induced Transcriptional Activation of Per1 and cFos in the SCN Is Not Increased but Attenuated in the Nmu^−/−Nms^−/−Gpr176^−/− Mice

A simple explanation for the possible underlying mechanism of the enhanced phase-shifting phenotype of Nmu^−/−Nms^−/−Gpr176^−/− mice may involve increased transcriptional activation of Per1⁸⁾ and cFos,²¹⁾ representative light-responsive genes in the SCN. However in reality it was not the case (Fig. 6): quantitative RT-PCR analysis using total RNA purified from laser-microdissected SCN (Fig. 6A) revealed a lower fold-induction ratio of Per1 and cFos mRNA expression in the SCN of Nmu^−/−Nms^−/−Gpr176^−/− mice than WT mice after a light exposure at CT14 (Fig. 6B, fold-induction, Per1: WT, 4.45 ± 0.26, Nmu^−/−Nms^−/−Gpr176^−/−, 3.27 ± 0.33, * p < 0.05; cFos: WT, 19.35 ± 4.05, Nmu^−/−Nms^−/−Gpr176^−/−, 9.56 ± 1.43, * p < 0.05, two-way ANOVA with Holm–Sidak’s post hoc test). This observation cannot explain the enhanced phase-shifting phenotype of Nmu^−/−Nms^−/−Gpr176^−/− mice. In an attempt to gain additional insight into another potential underlying mechanism of the phenotype observed, we examined circadian clock gene expression in these mice (Fig. 6C). A customized panel of 41 SCN genes, which include representative core clock genes, clock-controlled genes, and circadian clock-related neurotransmitters and receptors, were analyzed for this purpose using Fluidigm RT-PCR. Measurement at 4 CT points, CT0, CT6, CT12, and CT18, identified upregulated mRNA expression of Per1, Per2, Prok2, Rgs16, and Rasl11b at CT6 in the SCN of Nmu^−/−Nms^−/−Gpr176^−/− mice compared to WT mice, although it is unclear whether these expressional changes are accountable for the enhanced phase-shifting phenotype (Fig. 6C). Apart from these changes in expression, the core clock genes Cry1, Cry2, Clock, and Bmal1 and other clock-related genes, such as Vip, Vipr2, Avp, and Avpr1a, were virtually unaffected in the SCN of Nmu^−/−Nms^−/−Gpr176^−/− mice, which was consistent with the significant but minor alteration in the length of period of locomotor activity rhythms of these mice.

Fig. 6. Altered Light-Induced Expression of Per1 and cFos mRNA in the SCN of Nmu^−/−Nms^−/−Gpr176^−/− Mice

(A) Micrographs showing a representative SCN tissue section before and after laser microdissection. v, third ventricle; oc, optic chiasm. Toluidine blue staining. Scale bar, 200 µm. (B) Per1 and cFos mRNA expression in the SCN of WT and Nmu^−/−Nms^−/−Gpr176^−/− mice after light exposure at CT14. Mice with or without a 15-min light pulse were sacrificed 1 h after light onset. Relative mRNA levels were determined by qRT-PCR. Data are presented as the mean ± S.E.M. (n = 4–6). * p < 0.05, two-way ANOVA with Holm–Sidak’s post hoc test. n.s., not significant. (C) Circadian expression profiles of representative core clock genes, clock-controlled genes, circadian clock-related neurotransmitters and receptors in the SCN of WT and Nmu^−/−Nms^−/−Gpr176^−/− mice. Relative mRNA levels were determined by qRT-PCR. The mean of the peak values of WT SCN was set to 1. Values are the mean ± S.E.M. (n = 4–5). n.d., not detectable.

DISCUSSION

In the present study, we demonstrated that Nmu/Nms/Gpr176-triple knockout mice exhibit an increased magnitude of phase delay in response to early subjective night light, while Nmu/Nms double knockout mice as well as Gpr176-single knockout mice¹¹⁾ were found normal in the phase shifts induced by light. In our genome-wide transcriptome analysis, both Nmu and Nms were identified among the highly ranked upregulated genes in the SCN of Gpr176-deficient mice; thus these genes may have a compensatory role for the lack of Gpr176. On the consideration of previously reported phase-shift inducing activity of Nmu¹⁷⁾ and Nms,¹⁵⁾ upregulated expression of these genes may help normalize the amplitude of phase shift in Gpr176^−/− mice. Our data suggest that there is a mechanism requiring both Gpr176 and Nmu/Nms to modulate the magnitude of light-induced phase shift in mice.

A light pulse in the night that induces phase shift causes induction of Per1 and cFos mRNA expression in the SCN, and the extent of their induction has been considered to positively correlate with the magnitude of phase shift, in WT mice.^6,8) However, contrary to our naïve speculation, Nmu^−/−Nms^−/−Gpr176^−/− mice showed a diminished light induction of Per1 and cFos mRNA expression in the SCN, despite their behavioral phase shift being increased.

The mechanism behind the above dissociation between light-induced gene expression and the amount of phase shift in Nmu^−/−Nms^−/−Gpr176^−/− mice is still unknown by our study. However, in the literature, such a result is not unprecedented. Given similar results that are reported in other animal models, including Cry2-deficient mice,²²⁾ PACAP-type 1 receptor-deficient mice,²³⁾ and miR-132/212-deficient mice,²⁴⁾ all displaying enhanced phase shifts and decreased induction of Per1 expression, one could argue for a potential underlying mechanism that would affect the size of phase shifts irrespective of the Per1 mRNA change. In this respect, although further studies are required to relate to our current finding, previous studies demonstrated that the extent of phase shift can be modulated by posttranscriptional mechanisms, including those by the protein kinase C α (PKCα), affecting the phase shift by phosphorylating Per2,²⁵⁾ as well as those via the eukaryotic translation initiation factor 4E (eIF4E), which facilitates phase resetting by promoting translation of Per proteins.²⁶⁾

Besides the above possible posttranscriptional mechanism(s) that needs to be examined, we also observed mildly upregulated mRNA expressions for the Per1, Per2, Prok2, Rgs16, and Rasl11b at CT6 in Nmu^−/−Nms^−/−Gpr176^−/− mice. In addition, our DNA microarray analysis examining SCN transcripts in Gpr176-deficient mice revealed Bglap-rs1, Lcn2, C4b, and Ndn as upregulated and Eif2ak4, Mid1, Tmem185b, and Pgap1 as downregulated genes in the SCN of Gpr176^−/− mice. Among these altered genes, Ndn has already been verified for its expression in the SCN and its genetic loss causes a faster re-entrainment to an 8-h advanced jet-lag scheme as compared to WT mice.²⁷⁾ Although clarifying a contribution of the altered expression of the above-mentioned clock genes and Ndn etc. requires additional validation experiments, it is tempting to speculate that these expressional changes may also reflect the potential underlying mechanism of the phenotype of Gpr176^−/−Nmu^−/−Nms^−/− mice.

In our study, a light pulse administration at CT22 (i.e., in the late subjective night) resulted in a slightly larger phase-advance in Nmu^−/−Nms^−/−Gpr176^−/− mice as compared to Nmu^−/−Nms^−/− mice, but no statistically significant difference was observed when compared with WT mice, indicating a less obvious phase-shifting phenotype at CT22, compared to CT14. In this line, it may be worth describing that while a late subjective night light typically leads to a phase advance, several investigators, including ourselves, found the extent of light-induced phase advances to be relatively weak in C57BL/6 congenic mice.^28–30) As such, we cannot negate the possibility that the limited amounts of phase advancing effects at CT22 may influence the phenotype observed. Alternatively, because expression levels of Nmu and Gpr176 in the SCN robustly oscillate and are lowered in the late subjective night,^9,11,17) the deficiency of these genes may lead to a less obvious phenotype at CT22 than CT14. Further investigation is needed to elucidate the mechanism of the phenotype of Nmu^−/−Nms^−/−Gpr176^−/− mice.

Circadian clock dysregulation-associated physiological disturbances are becoming more common due to various factors, including artificial light, jet lag, shiftwork, and global networking around the clock.^1–3,31,32) Simultaneous regulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Acknowledgments

We are grateful to Dr. Masayasu Kojima (Kurume University) for providing Nmu^−/−Nms^−/− mice. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20H05783, 20K21426, 22H04987, 22H00441, 22K06594), the Basis for Supporting Innovative Drug Discovery and Life Science Research program of the Japan Agency for Medical Research and Development (JP22ama121034), the Kobayashi Foundation, and the Kusunoki 125 of Kyoto University 125th Anniversary Fund.

Author Contributions

M.D. conceived the project; M.D. and H.O. designed the research; Y.Y., I.M., and M.T. performed experiments in collaboration with S.D., T.S., R.H., K.K., and T.M.; M.D. and Y.Y. wrote the paper with input from all authors.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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