Dopamine 2 Receptor Activation Entrains Circadian Clocks in Mouse Retinal Pigment Epithelium

Many of the physiological, cellular, and molecular rhythms that are present within the eye are under the control of circadian clocks. Experimental evidence suggests that the retinal circadian clock, or its output signals (e.g., dopamine and melatonin), may contribute to eye disease and pathology. We recently developed a retinal pigment ephithelium (RPE)-choroid preparation to monitor the circadian clock using PERIOD2 (PER2)::LUC knock-in mouse. In this study we report that dopamine, but not melatonin, is responsible for entrainment of the PER2::LUC bioluminescence rhythm in mouse RPE-choroid. Dopamine induced phase-advances of the PER2::LUC bioluminescence rhythm during the subjective day and phase-delays in the late subjective night. We found that dopamine acts exclusively through Dopamine 2 Receptors to entrain the circadian rhythm in PER2::LUC bioluminescence. Finallly, we found that DA-induced expression of core circadian clock genes Period1 and Period2 accompanied both phase advances and phase delays of the RPE-choroid clock, thus suggesting that – as in other tissues – the rapid induction of these circadian clock genes drives the resetting process. Since the RPE cells persist for the entire lifespan of an organism, we believe that RPE-choroid preparation may represent a new and unique tool to study the effects of circadian disruption during aging.


Results
Dopamine, but not melatonin, phase-shifts the circadian rhythm in PER2::LUC bioluminescence. To determine whether DA and/or MLT can entrain RPE rhythms, we applied either compound to PER2::LUC RPE-choroid cultures at various times and measured the resultant shift in rhythm phase. We found that, in contrast to the cornea clock 8 , the RPE clock was not reset by 100 nM MLT (Fig. 1A-C) when applied at any time of day. In contrast, 100 μM DA shifts the clock forward (advance) or backward (delay) by up to 8-12 hours, depending on the time it was administered ( Fig. 1D-F). To aid in analysis, we binned individual phase-shifts Blue circles indicate cultures treated with Veh and red circles indicate culture treated with either MLT (B) or DA (E). Data were divided into 6 bins at 4-hour intervals for statistical analysis. Data were then used to calculate the phase change of MLT or DA versus their vehicle controls. Bars show the mean amount of phase change from controls and error bars show ±SEM for experimental groups. Error bars from x axis show ±SEM for control groups. MLT did not phase-shift the RPE-choroid PER2::LUC bioluminescence rhythm (n = 4-14 for each bin, Two way ANOVA, p > 0.1, (C) whereas DA significantly phase-shifted PER2::LUC rhythm (Two way ANOVA following Tukey tests, *p < 0.05, **p < 0.01, n = 6-8 for each bin, (F). into 4-hour phase intervals for statistical analysis. Two-way ANOVA revealed a significant interaction between treatment (vehicle or drug) and time of treatment in the DA treated cultures (p < 0.001; Fig. 1F), but not in the MLT treated cultures (p > 0.05; Fig. 1C). Overall, DA phase-delayed the RPE clocks by ~6.4 hours when given in an 8-hour window centered on a circadian "dawn" (CT0), phase advanced the RPE clock by approximately 5 hours when given throughout the rest of the circadian "day" (CT [4][5][6][7][8][9][10][11][12], and produced little effect on phase when given during the first 8 hours of the circadian "night" (CT 12-20) (Fig. 1F). Overall, these results suggest that DA, and not MLT, may be a circadian entraining signal for RPE in the retinal circadian system. Activation of D 2 R Signaling in the RPE Phase-shifts the Circadian Rhythm in PER2::LUC Bioluminescence. We then investigated which of the different DA receptors were responsible for phase-shift of the PER2::LUC circadian rhythm. There are five DA receptors in mammals, which are classified into D 1 -like (D 1 R, D 5 R) or D 2 -like (D 2 R, D 3 R, D 4 R) based on similar pharmacological profiles and coupling to second-messenger cascades 14,15 . We found that all but the D 3 R s are detectably expressed in RPE (Fig. 2). We therefore investigated whether general D 1 -like or D 2 -like receptor agonists (SKF38393 or quinpirole, respectively) produced phase-response curves comparable to DA (Fig. 1F). We found that SKF38393 could not induce a phase-shift, regardless of when it was given ( Fig. 3A and B). However, quinpirole administration phase-shifted the rhythm in a manner that was similar to that of DA (Fig. 3C). Quinpirole significantly phase-delayed PER2::LUC rhythms by 2.05 ± 0.65 hrs. when applied at CT 20-24 h (p < 0.05) and significantly phase-advanced PER2::LUC rhythms by 4.60 ± 0.54 hrs. and 3.16 ± 0.55 hrs when applied at CT 0-4 h and CT 4-8 h, respectively (p < 0.05; Fig. 3C,D). Overall, these data suggest that DA acting on D 2 Rs is sufficient to reset RPE rhythms.
We next investigated which of the D 2 -like receptor subtypes where responsible for the observed phenomenon. Since D 3 R mRNA was undetectable in the RPE (Fig. 2), we focused on discriminating between D 2 R and D 4 R. Administration of Sumanirole, a D 2 R specific agonist, at various times of day shifted the clock in a manner similar to DA and quinpirole ( Fig. 4A and B), whereas administering PD168077, D 4 R receptor specific agonist, did not phase-shift PER2::LUC rhythms in RPE-choroid ( Fig. 4C and D). Thus, it appears that activation of D 2 Rs is sufficient to reset the RPE clock.
Removal of D 2 R Signaling Prevents DA-induced Phase-shift of PER2::LUC Bioluminescence Rhythm in RPE. We next tested if D 2 R was required for DA's action on the RPE-clock by determining if DA could reset the clock of RPE-choroid from D 2 R-deficient PER2::LUC (Drd2 −/− ; PER2::LUC) mice. The RPE-choroid tissues obtained from D 2 R −/− ; PER2::LUC mice showed robust circadian rhythms (Fig. 5A) that were comparable in phase and periods to wild-type PER2::LUC controls (phases: 16.54 ± 0.20 hrs vs. 15.67 ± 0.53 hrs, periods: 23.88 ± 0.12 hrs vs. 23.68 ± 0.11 hrs p > 0.05, t-test, control vs. D 2 R −/− respectively). We then treated RPE-choroid with 100 μM of DA at circadian times when DA induces phase advances or delays. As expected, D 2 R-deficiency eliminated DA-induced phase-shifts of RPE-choroid bioluminescence rhythms ( Fig. 5A and B), confirming that DA is signaling RPE clocks exclusively via the D 2 R.

DA Induces Period1 and Period2 Gene Expression in the RPE. Previous studies have shown that
acute induction of Period1 (Per1) and Period2 (Per2) gene expression mediates phase-shifting of the circadian clock [16][17][18] . Hence, we decided to measure acute induction of Per1 and Per2 expression in cultured RPE-choroids after either one hour or three hours of DA treatment. DA applied at CT 6 to cause a phase advance ( Fig. 1) significantly induced Per1 expression (Fig. 6A, t-test, p < 0.05). Interestingly, Per1 expression was only elevated at 1 hour after the DA treatment, returning to baseline 3 hours after the DA treatment (Fig. 6B). DA applied at CT 6 did not significantly alter Per2 or Bmal1 mRNA levels (Fig. 6B, t-test, p > 0.05 for both cases). DA administered at CT23, when it induces phase-delays ( Fig. 1), did not significantly change levels of Per1, Per2 and Bmal1 mRNAs at 1-hr ( Fig. 6C, t-test, p > 0.05), but significantly induced Per1 and Per2 mRNA 3-hrs after the DA pulse ( Fig. 6D; t-test, p < 0.05). No changes were observed in Bmal1 mRNA levels (Fig. 6C,D; t-test, p > 0.05). Thus, taken together, our results suggest that D 2 R activation mediates clock reset by acutely inducing expression of Per1 and Per2.

Discussion
Accumulating evidence indicates that disruption of circadian rhythms due to genetic mutations or environmental factors contributes to the development of many diseases and premature aging. Indeed circadian disruption has been associated with numerous immune, inflammatory, and metabolic disorders [19][20][21] . Experimental evidence also suggests that the retinal circadian clock, or its output signals (e.g., DA and MLT), may contribute to eye disease and pathology. For example, diabetes is associated with reduced clock gene expression in the retina 22 , and Expression of dopamine receptors in the brain, retina, and RPE. Agarose gel electrophoresis of PCR amplicons specific to D 1 R, D 2 R, D 3 R, D 4 R or D 5 R transcripts in brain, retina and RPE. D 3 R mRNA was present in the brain (positive control), but was not amplified in the retina (negative control) or RPE. The electrophoresis bands matched the expected amplicon size.
circadian disruption recapitulates diabetic retinopathy in mice 23 . Removal of Bmal1 from the neural retina alters inner retinal function 24 and a recent paper reported that mice lacking Per1 and Per2 show significant alteration in the distribution of cone photoreceptors 25 . Finally a series of studies have implicated the clock genes Rev-erbα and Rora in retinal functioning 26,27 and age-related macular degeneration 28 . Similarly, disruption of DA or MLT signaling in the mouse retina greatly affects retinal physiology and retinal cell viability [11][12][13][29][30][31][32] .
Disruption of the daily rhythm of RPE phagocytosis impairs retinal and/or RPE functions. RPE of mice lacking αvβ5 integrin (β5 −/− mice) fail to show a circadian burst of phagocytic activity one of two hours after light onset. Also, during the aging process, β5 −/− mice lose both cone and rod photoreceptors faster than control mice 33 . The mechanism controlling the daily rhythm in RPE phagocytosis appears to be located in the RPE 34 and is likely to be directly controlled by the circadian clock located in this tissue 9 .
Previous studies have also reported that DA receptors are involved in the regulation of rhythmic RPE functions. For example, inhibition of DA synthesis during the early part of the light phase induced a significant reduction of disk shedding and phagocytosis 35 . In addition, mice whose dopaminergic neurons have been destroyed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) accumulate a large number of residual bodies in the RPE 36 . It has also been reported that dopamine and a D 1 -like activation decrease phagocytosis by RPE cells 37 . However, the presence of dopamine receptors in the mammalian RPE is still controversial 38 .
Our data indicated that DA can phase-shift in a time dependent manner the circadian rhythm in PER2::LUC bioluminescence (Fig. 1), and that D 1 R, D 2 R, D 4 R and D 5 R mRNAs are present in the mouse RPE (Fig. 2), albeit only in pharmacological activation of D 2 R induced a phase-shift in the PER2::LUC bioluminescence rhythm (Figs 3 and 4). The timing of quinpirole induced phase shifts was advanced compared to DA or Sumanirole. However, this is probably due to a difference in affinity of these ligands for D 2 R 39-41 and not due to involvement of other DA receptors as removing the D 2 R receptor completely abolished any resetting responses of the RPE clock in response to DA (Fig. 5). A number of studies support a model in which the rapid induction of the circadian clock genes Per1 and Per2 drives the resetting process 42,43 . The induction of Per1 mRNA in the suprachiasmatic nucleus of the hypothalamus following a photic stimulus is thought to be driven by activation of cAMP response element-binding protein (CREB) located in the promoter region of the Per1 gene 44,45 . D 2 -like receptors are negatively coupled to adenylyl cyclase and thereby lead to a decrease of cAMP levels. Thus, it is unlikely that D 2 R activation induces Per1 mRNA via the cAMP signaling pathway. Surprisingly our data (Fig. 6) indicates that Per1 mRNA levels were significantly increased 1-hr after DA treatment at CT 6 when DA phase-advanced the PER2::LUC rhythm. In comparison, Per1 and Per2 expression was induced 3 hrs after the treatment at CT 23 when DA phase-delayed the PER2::LUC rhythm. These data agree well with previous studies in the mouse in which it was reported that mice lacking Per1 did not show any phase-advance after a pulse of light, whereas mice lacking Per2 did not show any delays after a pulse of light 43 . Our results are also consistent with the findings of another investigation reporting that a light pulse during the delay zone of the PRC induces the expression of Per1 and Per2 genes, whereas a light pulse during the advance zone of PRC increases only Per1 46 . Thus experimental evidence agrees well with our results and supports the hypothesis that DA -via D 2 R activation -can induce Per1 and Per2 expression. The molecular mechanism by which DA via D 2 R activation induces Per1 and Per2 is unknown and further studies will be required to address this important issue.
Finally, it is worth mentioning that the RPE is composed of a single cell type and persists for the entire lifespan of an organism. Thus, a RPE-choroid preparation may represent a new and unique tool to study the impact of circadian clock function and disruption on cellular biology and longevity over a full lifespan.

Methods
Animals. This study used PER2::LUC mice (C57Bl/6) of 3 to 6 months in age. PER2::LUC mice were crossed with Dopamine 2 Receptor knock-out (D 2 R −/− ) to produce D 2 R −/− PER2::LUC mice. The original D 2 R −/− were purchased from Jackson Laboratory (strain Drd2 tm1Low /J). All mice used in this study were raised at Morehouse  culture dishes were returned to the incubator. At 1 hour or 3 hours after the dopamine treatment, culture tissues were collected from dish and subjected for RNA extraction using Trizol (Thermo fisher scientific, MA). Q-PCR was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using iQ SYBR Green Supermix (Bio-Rad Laboratories). All data for individual genes were normalized to 18S, and are plotted relative to average levels in vehicle treated control samples collected in parallel (n = 6 cultures for DA groups, n = 3 cultures for vehicle controls).
Analysis of Phase-shifts. Bioluminescence recordings emitted from RPE-choroid cultures were detrended by a 24-hour moving average subtraction method and smoothed by a 2-hour moving average. The circadian time (CT) of bioluminescence recordings were determined by the projection of the light cycle to which the mice were Figure 6. DA treatment increases Per1 and Per2 mRNA in RPE-choroid. RPE-choroid cultures were prepared and treated with DA or vehicle at ZT6 (A, advance) or ZT 23 (B, delay) as indicated above, followed by collection for Q-PCR analysis of the indicated mRNAs at 1 or 3 hour intervals. Expression data were normalized using 18S, and are plotted relative to vehicle controls. Blue bars indicate mean ± SEM of vehicle control. Red bars indicate mean ± SEM of DA treated. *Indicates p < 0.05 (t-test) compared to vehicle controls (n = 3-6 in each group).
Scientific RepoRts | 7: 5103 | DOI:10.1038/s41598-017-05394-x exposed (CT 12 = lights off). The circadian peak phase was determined as the highest point of the curve picked by Origin ® (Origin Lab, Northampton, MA) software. The amount of phase-shift (in hours.) was calculated by comparing the regression lines fitted to the circadian peaks before and after treatment. Phase-shifts in an individual culture dish were plotted as the phase response curve. Data were then averaged in 4 hr bins: CT 0-4, CT 4-8, CT 8-12, CT 12-16, CT 16-20, CT 20-24 and normalized with respective vehicle control groups 6 . Two-way ANOVA with a post hoc Tukey test was performed to compare the difference between experimental groups and time points.