Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors

Abstract

Energy expenditure and metabolism in the vertebrate retina are under circadian control, as we previously reported that the overall retinal ATP content and various signaling molecules related to metabolism display daily or circadian rhythms. Changes in the fission and fusion process of mitochondria, the major organelles producing ATP, in retinal photoreceptors are largely dependent on light exposure, but whether mitochondrial dynamics in photoreceptors and retinal neurons are under circadian control is not clear. Herein, we investigated the possible roles of circadian oscillators in regulating mitochondrial dynamics, mitophagy, and redox states in the chicken retina and mammalian photoreceptors. After entrainment to 12:12-h light-dark (LD) cycles for several days followed by free-running in constant darkness (DD), chicken embryonic retinas and cone-derived 661W cells were collected in either LD or DD at 6 different zeitgeber time (ZT) or circadian time (CT) points. The protein expression of mitochondrial dynamin-related protein 1 (DRP1), mitofusin 2 (MFN2), and PTEN-induced putative kinase 1 (PINK1) displayed daily rhythms, but only DRP1 was under circadian control in the chicken retinas and cultured 661W cells. In addition, cultured chicken retinal cells responded to acute oxidative stress differently from 661W cells. Using pMitoTimer as a mitochondrial redox indicator, we found that the mitochondrial redox states were more affected by light exposure than regulated by circadian oscillators. Thus, this study demonstrates that the influence of cyclic lights might outweigh the circadian regulation of complex mitochondrial dynamics in light-sensing retinal cells.

Keywords

mitochondria photoreceptor fission fusion redox

Circadian oscillators are present in various cell types of the vertebrate retina (Liu et al., 2012), including the photoreceptors (Besharse and Iuvone, 1983; Cahill and Besharse, 1995), bipolar cells (Chong et al., 1998; Hull et al., 2006), amacrine cells (Chong et al., 1998; Gabriel et al., 2001; Dorenbos et al., 2007), ganglion cells (Liang et al., 2004; Ruan et al., 2006; Liu et al., 2012), and Müller glial cells (Ruan et al., 2006; Xu et al., 2016). These oscillators provide a mechanism for the retina to anticipate and adapt to daily changes in ambient illumination over 10 to 12 orders of magnitude (Green and Besharse, 2004; Ruan et al., 2006). We previously showed that the physiology of the chicken retina is under circadian control through complex signaling networks (Ko et al., 2007; Ko et al., 2009a; Huang et al., 2012; Huang et al., 2013; Ko et al., 2013; Huang et al., 2015). In particular, the overall retinal adenosine triphosphate (ATP) content displays a daily rhythm, and the activation of the ATP-sensing kinase, AMP-activated protein kinase (AMPK), is rhythmic and is anti-phase to the ATP rhythm (Huang et al., 2015). The major ATP-generating organelle in a cell is the mitochondrion. Mitochondria are mobile organelles that constantly undergo morphological changes with frequent cycles of fission and fusion, the opposing processes that achieve equilibrium when cells are healthy (Sesaki and Jensen, 1999; Scott and Youle, 2010). Dynamin-related protein 1 (DRP1) is a GTPase that mediates mitochondrial fission, whereas mitofusin 2 (MFN2) coordinates with MFN1 to regulate mitochondrial fusion (Chen et al., 2003; Lee et al., 2004). Healthy and mildly damaged mitochondria can fuse to generate new organelles to compensate for damaged ones (Neuspiel et al., 2005). However, severely damaged mitochondria divide and become fragmented and depolarized. Once the mitochondria are depolarized, a programmed autophagy known as mitophagy is triggered to eliminate the damaged mitochondria. The lysosomes selectively degrade the damaged or dysfunctional mitochondria, recycle the mitochondrial contents, and retain the appropriate number of mitochondria in order to maintain homeostasis and proper metabolism (Jin and Youle, 2012; Esteban-Martinez et al., 2017). Impaired autophagy leads to the accumulation of reactive oxygen species (ROS) and cell degeneration (Kunchithapautham and Rohrer, 2007a; Kunchithapautham and Rohrer, 2007b). Thus, mitochondria are highly dynamic, and their numbers and shape within a cell are tightly associated with cellular metabolism (Dietrich et al., 2013).

Mitochondrial morphological changes (Uchiyama et al., 1981), gene expression (Young et al., 2001), and redox states (O’Neill and Reddy, 2011) are under circadian control. Mutations of clock genes such as Bmal1 or Clock in mice show altered mitochondrial dynamics, energy metabolism, and oxidative damage in the heart (Durgan et al., 2005; Bray et al., 2008; Kohsaka et al., 2014) and the liver (Peek et al., 2013; Jacobi et al., 2015; Manoogian and Panda, 2016). Mitochondria are highly abundant in retinal photoreceptors (Young and Droz, 1968; Hoang et al., 2002; Kooragayala et al., 2015), and their autophagy in the retina also displays a daily rhythm, which peaks in the light phase (Reme and Sulser, 1977; Reme et al., 1985), but this daily rhythm might be a response to damage elicited by prolonged light exposure. Thus far, it is not clear whether there is a circadian regulation of mitochondrial dynamics in the retina that is not illumination dependent.

Herein, we investigated whether the mitochondrial dynamics, mitophagy, and redox states of the retina and photoreceptors were under circadian regulation or were affected only by light. Since we previously reported that the ATP content in the chicken retina is under circadian control (Huang et al., 2015), we determined whether the protein expression of mitochondrial dynamic proteins (DRP1, MFN2, and phosphatase and tensin homolog [PTEN]-induced putative kinase 1 [PINK1]) displayed daily or circadian rhythms. Since the chicken retina is cone-dominant with 86% of photoreceptors being cones (Morris, 1970), we also compared the mitochondrial dynamics in the chicken retina to cultured mammalian 661W cells. Mammalian 661W cells are originally derived from a mouse retinal tumor and characterized as a cone-photoreceptor cell line, since they express cone-specific opsins, transducin, and arrestin (al-Ubaidi et al., 1992; Tan et al., 2004). As 661W cells respond to light (Kanan et al., 2007), they could possibly be entrained by cycling lights, which we tested in this study. We examined whether the mitochondrial dynamics were under circadian control in the chicken retina and 661W cells, since both were “cone” dominant. We further determined whether the responses to oxidative stress were also under circadian regulation in both light-sensing cells.

Materials and Methods

Circadian Entrainment of Chicken Embryos

Fertilized eggs (Gallus gallus) were obtained from the Poultry Science Department, Texas A&M University (College Station, Texas). Chicken embryos from embryonic day 11 (E11) were entrained in 12:12-h light-dark (LD) cycles for 7 days as previously described (Ko et al., 2009a; Huang et al., 2015). Zeitgeber time zero (ZT 0) was designated as the time when the lights turned on, and ZT 12 was the time when the lights went off. At 6 different ZT points (ZT 0, 4, 8, 12, 16, and 20), retinas from E18 embryos were harvested for Western blot analyses. Some LD-entrained embryos were kept in constant darkness (DD) for another 24 h, after which retinas were excised at 6 different circadian time (CT) points (CT 0, 4, 8, 12, 16, and 20) on the second day of DD for Western blot analyses.

Cell Cultures

After LD entrainment for 7 days, chicken retinas from E18 embryos were dissociated and cultured in an incubator at 39 °C and 5% CO₂ in DD for another 24 h. The culture medium consisted of Eagle’s minimal essential medium (EMEM; BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated horse serum (BioWhittaker), 2 mM Glutamax (Gibco/Invitrogen, Carlsbad, CA), 100 µg/mL penicillin and 100 µg/mL streptomycin (Life Technologies, Grand Island, NY), and 20 ng/mL ciliary neurotrophic factor (CNTF; R&D Systems, Minneapolis, MN). On the second day of DD, retinal cells were treated with 250 µM hydrogen peroxide (H₂O₂) for 2 h at CT 2 and CT 14 prior to harvest for Western blot analyses.

The 661W cells (Tan et al., 2004) were originally obtained from Dr. Al-Ubaidi (originally at the University of Oklahoma, now at the University of Houston). The 661W cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Lonza, Portsmouth, NH) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA), 2 mM Glutamax (Gibco/Invitrogen), 100 µg/mL penicillin and 100 µg/mL streptomycin (Life Technologies), and 1 mM sodium pyruvate (Life Technologies) at 37 °C and 5% CO₂. The incubators for 661W cells were equipped with lights and timers for the entrainment to 12:12-h LD cycles in vitro. After 5 days in cultures under LD, 661W cells were collected at 6 ZT points for further analyses on the sixth day. Some 661W cells were first entrained in LD for 4 days and kept in DD for 2 days, after which the 661W cells were harvested at 6 CT points for Western blots analyses on the second day of DD; in contrast, some 661W cells under this condition were treated with 250 µM hydrogen peroxide (H₂O₂) for 2 h at CT 2 and CT 14 prior to harvest for Western blot analyses.

Western Blot Analysis

Samples for Western blots were collected, prepared, and analyzed as described previously (Ko et al., 2009a; Huang et al., 2015). Briefly, chicken retinas were collected and homogenized in a Tris lysis buffer including (in mM) 50 Tris, 1 EGTA, 150 NaCl, 1% Triton X-100, 1% β-mercaptoethanol, 50 NaF, and 1 Na₃VO₄ (pH 7.5). The 661W cells were harvested and lysed in the same Tris lysis buffer. Samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gels by electrophoresis and transferred to nitrocellulose membranes. The primary antibodies used in this study were DRP1 (1:1000; Cell Signaling Technology, Danvers, MA), MFN2 (1:1000; Abcam, Cambridge, MA), PINK1 (1:1000; Abcam), ERK (loading control; 1:1000; Santa Cruz Biochemicals, Santa Cruz, CA), and actin (loading control; 1:1000; Cell Signaling Technology). Blots were visualized using appropriate secondary antibodies (anti-mouse/anti-rabbit; Cell Signaling Technology) at 1:1000 conjugated to horseradish peroxidase and an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL). Band intensities were quantified by densitometry using Scion Image (NIH, Bethesda, MD). Previously, we showed that there is a circadian rhythm of phosphorylated ERK, while total ERK remains constant throughout the course of a day in cultured chicken photoreceptors and chicken retina (Ko et al., 2001; Ko et al., 2007). Thus, total ERK served as the loading control for chicken retina, and actin served as the loading control for 661W cells. Relative protein expression for all proteins involved in this study is reported as a ratio to total ERK or actin. The relative values were further normalized to the first time point (ZT 0 or CT 0), which was arbitrarily set at 1.

Cell Transfection with pMitoTimer and Image Analysis

The 661W cells were cultured on coverglass chambered slides (Nunc Lab-Tek; Thermo Fisher Scientific) with the same medium described above and kept in dual-incubators equipped with lights and timers, in which the incubator lights were 12 h anti-phase to each other. Cells were first kept at 37 °C with 5% CO₂ in LD cycles for 3 days. On the third day, cells were transfected with pMitoTimer (Laker et al., 2014) (Addgene, Cambridge, MA) and continuously cultured for another 48 h in LD cycles. Briefly, cells were incubated with a mixture of 2 µg of plasmid DNA containing pMitoTimer and 2 µL of Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM medium (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM Glutamax (Gibco/Invitrogen), 100 µg/mL penicillin and 100 µg/mL streptomycin (Life Technologies), and 1 mM sodium pyruvate (Life Technologies) at 37 °C in 5% CO₂. On the fifth day at ZT 4 and ZT 16, fluorescent images were obtained using a Stallion microscope (Carl Zeiss AG, Oberkochen, Germany). Some 661W cells were transferred to DD on the fifth day, and images were obtained at CT 4 and CT 16 on the second day of DD. Each fluorescent image was taken under identical settings, including light intensity, exposure time, and magnification (Kim et al., 2017). The averaged fluorescent intensity per pixel for each image was quantified without any modification using the luminosity channel of the histogram function in the Photoshop 6.0 software (Adobe Systems, San Jose, CA), and the green or red fluorescence intensities were measured on a scale of 0 to 40 for brightness levels. A total of 11 to 14 cell images from each group were analyzed in 3 different sets of experiments.

Quantitative Real-time RT-PCR (Q-PCR)

The 661W cells were cultured and entrained under LD cycles as described above. Cells were collected at 6 ZT or CT points for Q-PCR. The method used for Q-PCR analysis was described previously (Ko et al., 2007; Ko et al., 2010). The total RNA from each sample was prepared by using a commercially available purification kit (RNeasy; Qiagen, Germantown, MD). From each sample, 500 ng of total RNA was used to quantify the mRNAs of Period 1 (Per1) and β-actin (loading control) by Q-PCR using a 1-step RT-PCR kit and the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The primers and probes of Per1 (Assay ID: Mm00501813_m1) and β-actin (Assay ID: Mm02619580_g1) were purchased from TaqMan Gene Expression Assays (Thermo Fisher Scientific). All measurements were repeated 6 times. For each individual experiment, a standard curve was generated with known quantities of Per1 or β-actin mRNA loaded in curved dilutions (i.e., 0.5, 1, 2, 4, 8, 16×). The cycle values, corresponding to the log values of the standard curve quantities, were used to generate a linear regression formula. The cycle values from the sample RNAs were fit into the formula, and the mRNA quantities of the samples were obtained. The mRNA values of Per1 were then divided by the values of β-actin mRNA to give “relative Per1 mRNA.”

Statistical Analyses

All data are presented as mean ± standard error of mean (SEM). The Student t test, 1-way analysis of variance (ANOVA), and 2-way ANOVA followed by Tukey’s post hoc test were used for statistical analyses. P values less than 0.05 were regarded as significant.

Results

Mitochondrial Fission and Fusion Dynamics Display Daily Rhythms, but Only Mitochondrial Fission Is under Circadian Control

Mitochondria have three major interrelated functions: producing ATP as energy for cells, controlling cellular metabolism and ROS production, and regulating apoptosis (Kaden and Li, 2013). Since we previously demonstrated that the overall retinal ATP content displays a daily rhythm in avian retinas, and given that the circadian rhythm of phosphorylated AMPK (pAMPK) is anti-phase to the ATP rhythm (Huang et al., 2015), we further examined the possible daily or circadian regulation of mitochondrial dynamics by determining the protein expression of DRP1 (mitochondrial fission) and MFN2 (mitochondrial fusion) in the chicken retina and mammalian 661W photoreceptors.

We found that both DRP1 and MFN2 displayed daily rhythms in chicken photoreceptors and were anti-phase to each other, with DRP1 higher during the light phase and MFN2 higher during the dark phase (Fig. 1A). In 661W cells, only MFN2 displayed a daily rhythm with its peak during the light phase (Fig. 1B). Interestingly, even though both chicken retina and 661W cells were under LD cycles, the apparent light-dependent mitochondrial dynamics in the chicken retina were opposite from the dynamics in 661W cells. While the levels of DRP1 (fission) were higher during the light phase in the chicken retina (Fig. 1A), the levels of MFN2 (fusion) were higher in the light phase in 661W cells (Fig. 1B). Thus, we next determined whether the mitochondrial fission and fusion processes were under circadian control.

Figure 1.

The daily rhythms of proteins responsible for mitochondrial fission (DRP1) and fusion (MFN2) in avian retina and mammalian (661W) photoreceptors. (A) Both DRP1 and MFN2 display daily rhythms in the chicken retina. Chicken embryos were entrained in 12:12-h light-dark (LD) cycles for 7 days (E11-18). At E18, retinas were harvested at 6 ZT points (ZT 0, 4, 8, 12, 16, and 20) and processed for Western blots. Representative blots are presented in the top panel. For DRP1, the protein levels are significantly higher at ZT 0 and 4 compared with the level at ZT 20 (n = 7). For MFN2, the protein level at ZT 16 is significantly higher than that at ZT 0 (n = 7). Total ERK serves as the loading control. (B) Only MFN2 displays a daily rhythm in cultured 661W cells. The 661W photoreceptor cells were cultured and entrained under LD cycles for 5 days and reached 80% confluency. There is no daily rhythm of DRP1 (n = 7), but MFN2 protein displayed a daily rhythm with a peak at ZT 4, which is significantly higher than that at ZT 16 and 20 (n = 7). The error bars are SEM. One-way ANOVA: *p < 0.05.

On the second day of DD (E19) after 7 days of LD entrainment in ovo, as well as on the next day of DD following 4 days under LD cycles in vitro, chicken retina and cultured 661W cells were harvested at 6 CT points and processed for Western blots, respectively. We found that under DD, DRP1 displayed a circadian rhythm with its peak at CT 20 in the chicken retina, and it appeared to be anti-phase to MFN2 in its expression pattern, even though we did not find the protein levels of MFN2 to have a significant circadian rhythm (Fig. 2A). Interestingly, DRP1 also displayed a circadian rhythm in 661W cells with its peak in the subjective dark phase similar to that of the chicken retina, while MFN2 did not display any circadian rhythmicity in 661W cells (Fig. 2B). Thus, DRP1, the mitochondrial fission mediator, but not MFN2, the fusion protein, was driven by the circadian oscillators, even though light exposure significantly affected both mitochondrial fission and fusion processes.

Figure 2.

The circadian rhythms of DRP1 (mitochondrial fission) and MFN2 (mitochondrial fusion) in the avian retina and 661W photoreceptors. (A) DRP1, but not MFN2, displays a circadian rhythm in the chicken retina. Chicken embryos at E11 were entrained in LD cycles for 7 days and then kept in DD. On the second day of DD (E19), retinas were excised at 6 CT points (CT 0, 4, 8, 12, 16, and 20) and processed for Western blots. For DRP1, the protein level is significantly higher at CT 20 compared with that at CT 0, 4, and 8 (n = 5). (B) DRP1 but not MFN2 displays a circadian rhythm in cultured 661W cells. The 661W photoreceptor cells were cultured and entrained under LD cycles for 4 days and then kept in DD for 2 days. On the second day of DD, 661W cells were harvested at 6 CT points and processed for Western blots. For DRP1, the protein level is significantly higher at CT 16 than at CT 0 (n = 7). The error bars are SEM. One-way ANOVA: *p < 0.05.

Mitophagy Has Daily Regulation in Cultured Photoreceptors

Mitophagy is a targeted removal of dysfunctional mitochondria and their toxic contents, thereby protecting the cells and serving as an alternative to apoptosis (Narendra et al., 2008). As such, mitophagy and mitochondrial fission and fusion are essential for a cell to maintain a homeostatic state (Kaden and Li, 2013). PINK1 is a mitochondrial serine/threonine kinase that promotes mitochondrial fission and mitophagy (Buhlman et al., 2014). Both Parkin (an E3 ubiquitin ligase whose defects are associated with Parkinson’s disease) and DRP1 are co-recruited to the mitochondrial outer membrane in proximity to PINK1, which leads to PINK1/Parkin-mediated mitochondrial clearance (Buhlman et al., 2014). In addition, PINK1 is able to phosphorylate MFN2 and instantaneously convert it from a fusion protein to a Parkin binding protein and cause PINK1/Parkin-dependent mitophagy (Shirihai et al., 2015). Since DRP1 and MFN2 displayed circadian and daily rhythms, respectively, we next examined whether the expression of PINK1 was under circadian control. Under LD cycles, PINK1 peaked at ZT 16 in the chicken retinas (Fig. 3A) but peaked at ZT 4 in 661W cells (Fig. 3B). The daily rhythms of PINK1 were similar to those of MFN2 (Fig. 1). However, there was no circadian rhythm of PINK1 in either chicken retinas or 661W cells (Fig. 3). Hence, light has a more significant impact on PINK1-dependent mitophagy than do circadian oscillators.

Figure 3.

The mitophagy regulatory protein PINK1 displays daily rhythms but not circadian rhythms in both avian retina and 661W photoreceptors. (A) The protein level of PINK1 is significantly higher at ZT 16 compared with that at ZT 0 (n = 8) in chicken retinas. (B) In cultured and entrained 661W cells, the protein level of PINK1 is significantly higher at ZT 4 compared with that at ZT 16 and 20 (n = 7). (C and D) PINK1 does not display a circadian rhythm in (C) chicken retina (n = 5) or (D) 661W cells (n = 6). The arrows indicate the bands of PINK1 at ~66 kDa. The error bars are SEM. One-way ANOVA: *p < 0.05.

Avian Retinal Cells Respond to Oxidative Stress Differently Than 661W Cells

Mitochondrial fission and fusion dynamics are influenced by oxidative stress, which results in mitochondrial fragmentation in cancer cells (Wu et al., 2011). Prolonged exposure to oxidative stress causes apoptosis and autophagy in cultured 661W photoreceptors (Kunchithapautham and Rohrer, 2007a). It was not clear whether brief oxidative stress might also alter the fission and fusion dynamics in cultured photoreceptors. Since exposure to light causes oxidative stress in the retina (Yamashita et al., 1992) and cultured 661W cells (Kuse et al., 2014; Rapp et al., 2014; Corso et al., 2016; Hiromoto et al., 2016; Natoli et al., 2016; Espinoza and Mercado-Uribe, 2017; Kuse et al., 2017), we examined the effects of exogenous oxidative stress in DD after cultured chicken retinal cells and 661W cells were entrained to LD cycles to avoid the light effect. On the second day of DD at CT 2 and CT 14, cultured chicken retinal cells or 661W cells were treated with 250 µM H₂O₂ for 2 h and then harvested for Western blots at CT 4 and CT 16. In chicken retinal cells (Fig. 4A), the protein level of DRP1 was significantly higher at CT 16 than CT 4, and while treatment with H₂O₂ significantly increased DRP1 at both CT 4 and CT 16, it did not abolish the circadian rhythm of DRP1. Treatments with H₂O₂ significantly decreased MFN2 at both CT 4 and CT 16 (Fig. 4A). Unexpectedly, treatment with H₂O₂ in 661W cells did not elevate DRP1 or decrease MFN2 in the same way as it did in chicken photoreceptors (Fig. 4B). Treatments with 250 µM H₂O₂ for 2 h did not cause detectable cell death in cultures. Thus, chicken retinal cells might be more sensitive to oxidative stress than 661W cells, which led us to further examine the mitochondrial redox state in cultured 661W cells.

Figure 4.

Chicken retinal cells respond to oxidative stress differently than 661W cells. Retinal cells were dissociated and cultured in DD after the chick embryos were entrained in LD for 7 days. The 661W cells were entrained in LD for 4 days and kept in DD for 2 days. On the second day of DD, cultured retinal cells and 661W cells were treated with H₂O₂ for 2 h at CT 2 and CT 14 and harvested for Western blot analyses. (A) A significant difference (^&p < 0.05) was found in the protein levels of DRP1 between CT 4 and CT 16 in both control and H₂O₂-treated groups in cultured chicken retinal cells. Treatment with H₂O₂ significantly increased the protein levels of DRP1 but decreased MFN2 at both CT 4 (^#p < 0.05) and CT 16 (^#p < 0.05) compared with control. (B) In cultured 661W cells, no statistical difference was found in the protein levels of DRP1 and MFN2 between CT 4 and CT 16 in both control and H₂O₂ treated groups. Treatment with H₂O₂ did not significantly change DRP1 or MFN2 in these cells. The error bars are SEM. Two-way ANOVA: ^#p < 0.05, ^&p < 0.05.

Mitochondrial Redox State Displays a Daily Rhythm in 661W Cells

Mitochondria generate ROS as a byproduct of respiration (Murphy, 2009). To control the ROS within a range compatible with normal cellular function, there are mitochondrial enzymes, such as superoxide dismutases (converting ROS to H₂O₂), peroxidases (reducing H₂O₂ to H₂O and O₂), and other antioxidant enzymes, that covert and remove ROS (Okado-Matsumoto and Fridovich, 2001; Salvi et al., 2007). Thus, normal mitochondrial function is affected by redox-dependent processes (Rasbach and Schnellmann, 2007; Handy et al., 2009). We employed a novel method using p MitoTimer to determine whether the mitochondrial redox state was under circadian control or mainly affected by the LD cycles. MitoTimer is a reporter gene that encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized (Laker et al., 2014). The MitoTimer fluorescence spectrum is determined by a balance between the newly synthesized protein (green) and the oxidized one (red). Since the MitoTimer protein is expressed under control of constitutively active promoters, an overall increase of the red/green fluorescence ratio of the mitochondrial network is likely due to increased oxidation of the protein rather than altered transcription/translation (Laker et al., 2014).

After 2 days of LD entrainment, cultured 661W cells were transfected with pMitoTimer at ZT 4 or ZT 16 and then returned to LD cycles for another 48 h. The images were taken at ZT 4 or ZT 16. To determine whether the mitochondrial redox state was under circadian control, 24 h after transfection, cells from ZT 4 or ZT 16 were kept in DD for another 24 h, and the fluorescence was determined on the second day of DD at CT 4 or CT 16. We found that 661W cells were more oxidized at ZT 16 than at ZT 4, as the red/green fluorescence ratio was higher at ZT 16 (Fig. 5A). However, we found no difference in the red/green fluorescence ratio between cells examined at CT 4 and CT 16 (Fig. 5B). Thus, our results (Fig. 5) indicate that the mitochondrial redox states might be affected by light exposure more significantly than the circadian oscillators in 661W cells.

Figure 5.

The redox state of mitochondria displays a daily but not circadian rhythm in 661W photoreceptors. (A) Cultured and entrained 661W cells were transfected with pMitoTimer and maintained under LD for 48 h, followed by fluorescent imaging under a confocal microscope at ZT 4 and ZT 16. The green fluorescent proteins (represented as the green bars) are newly synthesized mitochondrial proteins, and the red fluorescent proteins (represented as the red bars) are irreversibly oxidized mitochondrial proteins. A significant increase in mitochondrial oxidation (represented as red/green fluorescence ratio) is noted at ZT 16 (n = 12) compared with ZT 4 (n = 14). (B) After transfection with pMitoTimer, these 661W cells remained under LD for 24 h and then were kept in DD for 2 days. On the second day of DD, the fluorescent images were taken at CT 4 and CT 16. No statistical difference is noted in mitochondrial oxidation between cells imaged at CT 4 (n = 11) and CT 16 (n = 11). Scale bar is 10 µm. The error bars are SEM. Student t test: *p < 0.05.

Discussion

In this report, we showed that mitochondrial dynamics displayed daily and circadian rhythms in the chicken retina and mammalian photoreceptors. In the chicken retina, the mitochondrial fission protein DRP1 was high during the light phase whereas fusion protein MFN2 was high during the dark phase when the embryos were maintained under LD cycles. In DD, only DRP1 displayed a circadian rhythm with a peak during the subjective dark phase. In cultured 661W cells, only MFN2 displayed a significant daily rhythm, but in DD, DRP1 was significantly higher during the subjective dark phase. The mitophagy regulator PINK1 displayed significant daily rhythms but not circadian rhythms in both chicken retinas and 661W cells.

Since 661W cells are murine cone-derived cells, DRP1, MFN2, and PINK1 should have similar daily phases in the 661W cells as in the chicken retina, which is cone-dominant. However, the daily rhythms of DRP1, MFN2, and PINK1 in the chicken retina were not in phases similar to those observed in cultured 661W cells. A few possibilities might explain this outcome: First, we used whole chicken retina in our experiments, such that various retinal cell types were included in our assays. While cones are the dominant photoreceptors in the chicken retina, other retinal cell types in the inner retina might modulate the overall outcomes under LD, even though cultured embryonic cone photoreceptors have circadian oscillators independent from other cell types (Ko et al., 2001). In addition to photoreceptors, horizontal cells, bipolar cells, amacrine cells, and ganglion cells all express circadian genes (Ruan et al., 2006). Under light or during the daytime, retinal amacrine cells release dopamine that is able to inhibit the release of melatonin from the photoreceptors (Zawilska and Iuvone, 1989; Kazula et al., 1993; Thomas et al., 1993; Adachi et al., 1995; Ebihara et al., 1997; Luft et al., 2004; Ivanova et al., 2008). Dopamine also regulates cGMP-gated cation channels in chicken photoreceptors (Ko et al., 2003), so amacrine cells might modulate the photoreceptor daily rhythmicities and light sensitivities. Second, the overall avian circadian clock is a multioscillator system comprising the retina, the pineal gland, and the avian homologs of the suprachiasmatic nuclei, whose mutual interactions ensure coordinated physiological functions, which are in turn synchronized to ambient cyclic lights via encephalic, pineal, and retinal photoreceptors (Cassone and Westneat, 2012). Since we entrained the chicken retina in ovo, under LD, the daily rhythms of DRP1, MFN2, and PINK1 are the summation of all retinal cells in the chicken retina that could be under the influence of the avian-SCN and pineal gland, which would be different from the outcome for stand-alone 661W cells.

Third, while 661W cells respond to light, they might not have functional circadian oscillators that modulate light responses as the oscillators in normal cone photoreceptors do. The naive cone photoreceptors have independent oscillators that regulate ion channels responsible for phototransduction and neurotransmitter release (Ko et al., 2001; Ko et al., 2004; Ko et al., 2007; Liu et al., 2012), and the circadian regulation in these photoreceptors outweighs the short illumination effects on cell signaling (Ko et al., 2009b). However, even though we detected the canonical circadian genes including Period 1 (Per 1), Per 2, and Bmal1 in cultured 661W cells (data not shown), these cells might not possess a functional oscillator, since Per 1 did not display circadian rhythmicity under constant darkness (Suppl. Fig. S1). An alternative explanation is that under DD, the 661W cells were desynchronized, but individual 661W cell might still oscillate. Unlike cultured SCN slices that maintain robust oscillations with cellular synchrony (Yamaguchi et al., 2003), in dissociated SCN neuron cultures, individual SCN neurons but not the whole culture display independent oscillations (Welsh et al., 1995; Herzog et al., 1998; Honma et al., 1998), which is due to the desynchronization among dissociated SCN neurons without proper synaptic connections and gap junctions (Welsh et al., 2010). While 661W cells form synapse-like contacts in cultures (Tan et al., 2004), they are not known to form gap junctions like rods and cones in the intact retina that are critical for maintaining rod-cone coupling and the overall retinal circadian rhythms (Ribelayga and Mangel, 2005; Ribelayga et al., 2008; Jin et al., 2015; Zhang et al., 2015; Jin and Ribelayga, 2016). In addition, we did not observe these entrained 661W cells having individual oscillations on the second day of DD in our MitoTimer study (Fig. 5). Thus, it is unlikely that the lack of circadian rhythms of cultured 661W cells is due to cellular desynchronization. Fourth, the daily rhythms of DRP1, MFN2, and PINK1 in cone photoreceptors could be species-dependent, given that chickens are diurnal animals whereas laboratory mice (origin of 661W cells) are nocturnal. This possibility is highly unlikely, since there is no evidence indicating any differences in cone photoreceptor light-sensing properties across diurnal or nocturnal mammalian species, and we did not directly compare cone photoreceptors derived from a diurnal mammal with 661W cells.

Energy and oxygen consumption in the retina are greatly influenced by light and darkness (Medrano and Fox, 1995; Okawa et al., 2008; Linton et al., 2010). The retinal photoreceptors especially have a higher metabolic activity in the dark (Wong-Riley, 2010), meaning that ATP is hydrolyzed in an accelerated rate to support tonic neurotransmitter release, so mitochondria will have to produce more ATP to sustain photoreceptor activities. As a result, the mitochondrial enzymes that are responsible for ATP production should be more active in darkness, which is supported by Huang et al. (2004), who reported that the mitochondrial enzymes cytochrome C oxidase III and adenosine triphosphatase-6 are down-regulated by higher light intensity. Thus, overall ATP production may be lower in bright light. However, retinal energy metabolism is also under circadian regulation. By measuring the pH changes in the fish retina, Dmitriev and Mangel (2004) demonstrated a circadian regulation of retinal energy metabolism. We previously reported that the chicken retinal ATP content is rhythmic, and the activation and phosphorylation of AMPK, the intracellular energy sensor, are anti-phasic to the ATP rhythm (Huang et al., 2015). Therefore, the retinal energy expenditure and production can be light and darkness driven as a reflection of acute light-dark adaptation, as well as circadian regulated, since the retina anticipates and adapts to the upcoming light changes at dawn and dusk (Huang et al., 2015).

The ATP level is critical in regulating mitochondrial dynamics. The mitochondrial fusion process is dampened by a decrease of intracellular ATP (Legros et al., 2002). Low levels of intracellular ATP prevent PINK1 translation and Parkin mitochondrial translocation (Lee et al., 2015). We found that under LD, the levels of fission protein DRP1 were lower at night, with MFN2 and PINK1 being higher in the chicken retina, but these daily rhythms do not follow the overall retinal ATP rhythm, which peaks at ZT 13 (Huang et al., 2015). One explanation is that the previous studies on ATP and mitochondrial dynamics were performed in cultured cell lines, while our results were from whole chicken retinas, which is not a homogenous tissue. Under DD, only the fusion protein DRP1 displayed circadian rhythms in both chicken retinas and 661W cells (with the peaks in the subjective night), while MFN2 and PINK1 were not rhythmic. Since mitochondrial fission and fusion are complex processes, it is necessary to account for multiple players (such as DRP1, MFN2, and PINK1) collectively. Thus, when the rhythms of DRP1, MFN2, and PINK1 are compared under LD and DD, cyclic lights or illumination might have a stronger influence on mitochondrial dynamics than the circadian oscillators in photoreceptors.

In Drosophila, there is a daily rhythm in the sensitivity to oxidative stress, in which exposure to H₂O₂ (88 µM) for 4 h during the day at ZT 8 causes significantly higher mortality than at other times of the day, indicating that flies are most vulnerable to oxidative stress during the midday. If the daily rhythm of flies is abolished either by a null mutation of the clock gene Period (Per) or by keeping these flies in constant light, the mortality rate is equally high regardless of when the flies are exposed to H₂O₂ (Krishnan et al., 2008). Such daily rhythm in the sensitivity to acute H₂O₂-induced oxidative stress is observed also in salmon, with major detoxification enzymes expressed in the liver but not the gills displaying daily rhythms (Vera and Migaud, 2016). While the circadian oscillators certainly play an important role in the sensitivity of oxidative stress in these organisms, these results do not exclude the possibility that longer term (~8 h) light exposure might cause additional stress leading to mortality in Drosophila.

The dynamics of mitochondrial fusion and fission are essential for the physiological integrity of neurons, so perturbation of mitochondrial dynamics inevitably contributes to neurodegenerative diseases (Gunawardena and Goldstein, 2004; Bereiter-Hahn and Jendrach, 2010). In various cell types, mitochondrial fission correlates with oxidative stress, an overproduction of ROS (Wu et al., 2011). Our observation of increased mitochondrial fission by H₂O₂ treatments in cultured chicken retinas is consistent with previous reports (Yu et al., 2006; Fan et al., 2010; Wu et al., 2011). However, we did not find that H₂O₂ caused increased mitochondrial fission or decreased mitochondrial fusion in a time-of-day fashion in cultured chicken retinas. Rather, hours of illumination might have a greater effect on mitochondrial dynamics in the retinas, which was also reflected in the daily but not circadian rhythm of mitochondrial redox states in cultured 661W cells.

In this study, we demonstrated daily rhythms of mitochondrial fission and fusion processes, but only the fission protein displayed circadian oscillations. Because mitochondrial dynamics are complex, entailing multiple players, and since photoreceptors have both light-sensing and circadian machineries, our data presented here suggest that cyclic lights or illumination might have a stronger influence than the circadian oscillators on mitochondrial dynamics in retinal cells.

Supplemental Material

Supplementary Material

Supplemental material, Supp_Figure_(1) for Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors by Janet Ya-An Chang, Liheng Shi, Michael L. Ko, and Gladys Y.-P. Ko in Journal of Biological Rhythms

Footnotes

Acknowledgements

We thank Dr. Rola Barhoumi Mouneimne, associate director of the Image Analysis Laboratory, College of Veterinary Medicine and Biomedical Sciences (CVMBS) at Texas A&M University, for assistance in using the imaging facility. We thank Dr. Al-Ubaidi (originally at the University of Oklahoma, now at the University of Houston) for the 661W cell line. This work was supported in part by a departmental bridge fund to G.Y.P.K. and a graduate research grant from CVMBS to J.Y.A.C.

Conflict of Interest Statement

The author(s) have no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Notes

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