Regulation of Heme Oxygenase and Its Cross-Talks with Apoptosis and Autophagy under Different Conditions in Drosophila

Heme oxygenase (HO) is one of the cytoprotective enzymes that can mitigate the effects of oxidative stress. Here, we found that the ho mRNA level oscillates in the brain of Drosophila melanogaster with two minima at the beginning of the day and night. This rhythm was partly masked by light as its pattern changed in constant darkness (DD). It followed a similar trend in the clock mutant per01 under light/dark regime (LD12:12); however, differences between time points were not statistically significant. In older flies (20 days old), the rhythm was vanished; however, 15 days of curcumin feeding restored this rhythm with an elevated ho mRNA level at all time points studied. In addition, flies exposed to paraquat had higher ho expression in the brain, but only at a specific time of the day which can be a protective response of the brain against stress. These findings suggest that the expression of ho in the fly’s brain is regulated by the circadian clock, light, age, exposure to stress, and the presence of exogenous antioxidants. We also found that HO cross-talks with apoptosis and autophagy under different conditions. Induction of neuronal ho was accompanied by increased transcription of apoptosis and autophagy-related genes. However, this trend changed after exposure to curcumin and paraquat. Our results suggest that HO is involved in the control of apoptotic and autophagic key processes protecting the brain against oxidative damage.


Introduction
The brain houses complex groups of neuronal and glial cell types that work collaboratively or independently in coordinating several processes in the organism's body [1]. This sophisticated function requires continuous high energy supply [2], particularly for synaptic transmission [3], which is correlated with the overproduction of reactive oxygen species (ROS). Excessive accumulation of oxygen free radicals may damage DNA and proteins [4][5][6] that, as a consequence, disturb cellular processes [7]. This condition is regarded as a state of oxidative stress that negatively affects the normal functioning of the brain. This cellular threat is prevalent during aging, as aged brains have been reported to produce high levels of oxidative stress-induced mutations in the mitochondrial DNA [8][9][10][11]. Neurodegenerative disorders are also associated with cellular damage incurred by oxidative stress [12][13][14]. Frequent exposures to adverse conditions, such as radiation, UV light, air pollution, and toxic substances, can also contribute to ROS generation [7].
Neutralizing ROS includes the generation of protective enzymes, e.g., superoxide dismutases [SODs], glutathione peroxidases [GPxs], and catalases [CATs] as well as nonenzymatic endogenous antioxidant molecules such as glutathione (GSH) [15][16][17]. Behavior also plays an important role in regulating the effects of accumulating ROS-not excluding the fact that highly mobile animals have more chances of being exposed to toxic conditions,

Animals
The following strains of D. melanogaster were used: wild-type Canton S (CS) strain; per 01 (null mutant of the period gene); elav-GAL4 (a strain that expresses GAL4 under the control of the embryonic lethal abnormal vision (elav) promoter; pan-neuronal cell marker); Upstream Activating Sequence or UAS-hoRNAi (a strain which expresses dsRNA for ho gene under the control of UAS sequence); UAS-ho (a strain which expresses ho under the control of UAS sequence); UAS-Valium10 (control for RNAi strains); and repo-GAL4 (a strain that expresses GAL4 under the control of the reverse polarity (repo) promoter; panglial cell marker). Flies were maintained under 12 h of light followed by 12 h of darkness (LD12: 12) or in constant darkness (DD) at a constant temperature of 24 • C.

Experimental Groups
To detect cyclic expression of ho in the brain, 10-days-old CS or per 01 males were sacrificed at six time points in LD12:12 or DD conditions. Wild-type (CS) flies, 10-daysold, were used as control groups for the succeeding experiments particularly on aging and paraquat exposure. To study whether aging influences ho expression as well as the autophagy-related gene in the brain, 20-days-old CS males were sacrificed at six time points under LD12:12.
To understand the effects of HO on apoptosis, autophagy and DNA repair gene expression, the GAL4/UAS system was exploited to produce flies with overexpressed or silenced ho expression in all neurons or in all glia.

Curcumin Treatment
CS males, 5-days-old, were fed with a standard diet ((yeast-cornmeal-agar) supplemented with curcumin at 1 mg/mL of the medium for 15 days. Curcumin (EMD Millipore Corp., Darmstadt, Germany) was dissolved in 1% ethanol and mixed in the standard medium. This concentration has been shown to increase SOD and CAT activity [35]. Control groups were fed with the standard medium with 1% ethanol or with the standard medium only. Longer feeding time (15 days) of curcumin supplementation was implemented as it showed a significant increase in ho mRNA level compared to shorter ones. We initially found a strong induction of ho expression in the brain at ZT20 and we used this time point in comparing ho mRNA levels in the brain at different feeding times (5, 10, and 15 days) of curcumin, which was introduced to 5 days old flies (Supplementary Figure S1).

Paraquat Treatment
CS males, 10-days-old, were fed with a 10 mM concentration of paraquat dichloride (synonymous to methyl viologen dichloride hydrate, Sigma-Aldrich, Steinheim, Germany) mixed in the fly's standard diet at six different time points for 24 h. Control flies were fed only with the standard culture medium. This exposure time and concentration induced mortality and upregulated SOD activity [36].

RNA Isolation, cDNA Synthesis and Quantitative PCR
Males, 10 or 20 days old, were decapitated at ZT1, ZT4, ZT8, ZT13, ZT16, and ZT20 (ZT Zeitgeber Time, ZT0 indicates lights-on and ZT12 lights-off) in LD12:12 conditions or at CT1, CT4, CT8, CT13, CT16, and CT20 (CT Circadian Time, CT0 and CT12 stand for the beginning of the subjective day and the beginning of the subjective night, respectively) in DD conditions. These time points were selected in our previous study, in which significant differences in the expression of ho and many clock-controlled genes in the retina were shown [27,37,38]. Heads were fixed in 100% ethanol for 2 h, and brains were isolated. Approximately 20 flies were used for every time point, and each experiment was repeated at least three times.
Total RNA isolation was performed using TriReagent (MRC Inc., Irvine, CA, USA) according to the manufacturer's protocol. cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Vilnus, Lithuania) with random primers according to the provider's instruction. Gene expression was examined using StepOnePlus Real-Time PCR System and SYBR Green Master Mix (KAPA Biosystems, Cape Town, South Africa) in the presence of primer sequences for the genes used in this study which are listed in Table 1. Table 1. Primer sequences of the genes which were used in this study.

Gene
Sequence Accession Number Product specificity was assessed by melting curve analysis, and selected samples were run on 1% agarose gels for size assessment. A standard curve was used to calculate gene expression level. The number of target gene copies was normalized to the geometric mean of the rpl32 gene, previously identified as the housekeeping gene.

Statistical Analysis
Statistical analysis was performed using non-parametric analysis of variance (ANOVA), a Kruskal-Wallis test, and a Conover-Iman's test to check the rhythm of ho expression and a non-parametric Mann-Whitney test to compare between experimental and control groups. All data analyses were performed with the R freeware statistical package version 3.6.2 (http://www.R-project.org/ (accessed on 9 April 2021)) and GraphPad Prism 7.05 software (La Jolla, CA, USA).

Expression of ho in the Brain under LD12:12 and DD
The examination of the ho mRNA level at six time points during 24 h in LD12:12 conditions showed that ho expression cycles in the fly's brain. The mRNA levels of ho in the brain were very low and significant differences were only observed between ZT1 and ZT13 when compared with ZT16 and ZT20 (Figure 1a). This pattern was changed in DD, but the rhythm was maintained (Figure 1b). In addition, the ho mRNA level over the course of the day and night was examined in arrhythmic per 01 mutant (Figure 1c), which did not result in statistically significant differences between all time points studied. However, the expression pattern of ho in per 01 mutant had a similar peak and trough with wild-type flies in the same condition (LD12:12), which implies that light can generate a weak exogenous rhythm of ho mRNA. did not result in statistically significant differences between all time points studied. However, the expression pattern of ho in per 01 mutant had a similar peak and trough with wildtype flies in the same condition (LD12:12), which implies that light can generate a weak exogenous rhythm of ho mRNA.

Effects of Aging on ho and Autophagy Gene Expression in the Brain
The effect of aging on ho expression in the brain was examined in 20 days old wildtype flies at six different time points in LD12:12 and compared with younger flies (10 days old) (Figure 2a). We found that ho is not cyclically expressed in the brain of older flies, and the mRNA level is decreased in the late evening. Loss of the daily rhythm and significant reduction of mRNA level of atg5 were also detected in 20 days old flies (Figure 2b).

Figure 2.
Effects of aging on the daily mRNA expression of heme oxygenase (ho) (a) and autophagy-related 5 (atg5) (b) in the brain of adult male Drosophila melanogaster. Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (10-d old males vs. 20-d old males: (*) p < 0.05 or (**) p < 0.01 at ZT16 and ZT20 for ho expression and ZT1, ZT8, ZT13, ZT16 and ZT20 for atg5 expression).

Effects of Overexpressing and Silencing of ho on Apoptosis, Autophagy, and DNA Repair
The differences in daily mRNA levels of the genes encoding apoptosis, autophagy and DNA repair proteins were analyzed between the controls and the flies with pan-neuronal overexpression or silencing of ho in Drosophila at two different time points (ZT1 and

Effects of Aging on ho and Autophagy Gene Expression in the Brain
The effect of aging on ho expression in the brain was examined in 20 days old wildtype flies at six different time points in LD12:12 and compared with younger flies (10 days old) (Figure 2a). We found that ho is not cyclically expressed in the brain of older flies, and the mRNA level is decreased in the late evening. Loss of the daily rhythm and significant reduction of mRNA level of atg5 were also detected in 20 days old flies ( Figure 2b). did not result in statistically significant differences between all time points studied. However, the expression pattern of ho in per 01 mutant had a similar peak and trough with wildtype flies in the same condition (LD12:12), which implies that light can generate a weak exogenous rhythm of ho mRNA.

Effects of Aging on ho and Autophagy Gene Expression in the Brain
The effect of aging on ho expression in the brain was examined in 20 days old wildtype flies at six different time points in LD12:12 and compared with younger flies (10 days old) (Figure 2a). We found that ho is not cyclically expressed in the brain of older flies, and the mRNA level is decreased in the late evening. Loss of the daily rhythm and significant reduction of mRNA level of atg5 were also detected in 20 days old flies ( Figure 2b).

Figure 2.
Effects of aging on the daily mRNA expression of heme oxygenase (ho) (a) and autophagy-related 5 (atg5) (b) in the brain of adult male Drosophila melanogaster. Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (10-d old males vs. 20-d old males: (*) p < 0.05 or (**) p < 0.01 at ZT16 and ZT20 for ho expression and ZT1, ZT8, ZT13, ZT16 and ZT20 for atg5 expression).

Effects of Overexpressing and Silencing of ho on Apoptosis, Autophagy, and DNA Repair
The differences in daily mRNA levels of the genes encoding apoptosis, autophagy and DNA repair proteins were analyzed between the controls and the flies with pan-neuronal overexpression or silencing of ho in Drosophila at two different time points (ZT1 and Figure 2. Effects of aging on the daily mRNA expression of heme oxygenase (ho) (a) and autophagy-related 5 (atg5) (b) in the brain of adult male Drosophila melanogaster. Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (10-d old males vs. 20-d old males: (*) p < 0.05 or (**) p < 0.01 at ZT16 and ZT20 for ho expression and ZT1, ZT8, ZT13, ZT16 and ZT20 for atg5 expression).

Effects of Overexpressing and Silencing of ho on Apoptosis, Autophagy, and DNA Repair
The differences in daily mRNA levels of the genes encoding apoptosis, autophagy and DNA repair proteins were analyzed between the controls and the flies with pan-neuronal overexpression or silencing of ho in Drosophila at two different time points (ZT1 and ZT16). The expression of the apoptosis activator gene head involution defect (hid) significantly increased at night in flies with overexpressed ho in all neurons (Figure 3a). This is a timedependent effect because elevating ho transcription did not change hid expression in the morning. In addition, silencing of ho expression in all neurons also showed a higher level of hid mRNA (Figure 3b). This result implies that changes in ho expression in neurons can strongly affect apoptosis. ZT16). The expression of the apoptosis activator gene head involution defect (hid) significantly increased at night in flies with overexpressed ho in all neurons (Figure 3a). This is a time-dependent effect because elevating ho transcription did not change hid expression in the morning. In addition, silencing of ho expression in all neurons also showed a higher level of hid mRNA (Figure 3b). This result implies that changes in ho expression in neurons can strongly affect apoptosis.
Increasing ho expression in all neurons was also accompanied by the induction of atg5 transcripts at night (Figure 3c). The atg5 and hid mRNA expression seems to be regulated by high transcription of ho at night since decreasing ho expression did not affect the atg5 transcript levels (Figure 3d).

Figure 3.
Effects of pan-neuronal overexpression of heme oxygenase (ho) on the expression of the apoptosis activator head involution defect (hid) (a) and autophagy-related 5 (atg5) genes (b) mRNA at ZT1 and ZT16 as well as the effects of panneuronal silencing of ho on the expression of hid (c) and atg5 (d). Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (elav > ho or elav > hoRNAi vs. controls: p < 0.05).
These observations were only detectable in neuronal ho as overexpression of ho in all glia did not produce significant changes in the mRNA levels of apoptosis and autophagyrelated genes. In addition, the protective mechanism of HO in neurons seems to be connected to the regulation of apoptosis as well as with autophagy, and not for DNA repair as mRNA of eIF4a (translation initiation factor gene) and Xpc (recruits nucleotide excision repair gene) did not change after overexpressing or silencing ho in all neurons and specifically elevating ho expression in all glia (Supplementary Figures S2-S5). Effects of pan-neuronal overexpression of heme oxygenase (ho) on the expression of the apoptosis activator head involution defect (hid) (a) and autophagy-related 5 (atg5) genes (b) mRNA at ZT1 and ZT16 as well as the effects of pan-neuronal silencing of ho on the expression of hid (c) and atg5 (d). Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (elav > ho or elav > hoRNAi vs. controls: p < 0.05).
Increasing ho expression in all neurons was also accompanied by the induction of atg5 transcripts at night (Figure 3c). The atg5 and hid mRNA expression seems to be regulated by high transcription of ho at night since decreasing ho expression did not affect the atg5 transcript levels (Figure 3d).
These observations were only detectable in neuronal ho as overexpression of ho in all glia did not produce significant changes in the mRNA levels of apoptosis and autophagyrelated genes. In addition, the protective mechanism of HO in neurons seems to be connected to the regulation of apoptosis as well as with autophagy, and not for DNA repair as mRNA of eIF4a (translation initiation factor gene) and Xpc (recruits nucleotide excision repair gene) did not change after overexpressing or silencing ho in all neurons and specifically elevating ho expression in all glia (Supplementary Figures S2-S5).

Effects of Chronic Curcumin Supplementation on ho, Apoptosis, and Authophagy Gene Expression in the Brain
Chronic supplementation of wild-type flies with curcumin strongly induced ho expression. We detected a significant increase in the ho mRNA level in the brain at all time points studied (Figure 4a). We also found that the supplementation of food with curcumin prevented the loss of rhythm in ho expression in the brain of older flies (20 days old). This increase of ho mRNA level in the brain after consecutive 15 days of curcumin feeding was accompanied by the induction of hid expression at ZT16 and ZT20 (Figure 4b). In the same experiment, we also analyzed the expression of the skl gene, another gene coding apoptotic protein, which also showed an increase in mRNA level during the night: ZT13 and ZT16 (Figure 4c). This time-dependent upregulation of apoptotic genes in the brain after long feeding time (15 days) of curcumin, which had a similar effect when the ho gene was overexpressed in all neurons, confirmed association of ho transcription with apoptosis. However, it was not consistent with atg5 expression in the brain after chronic curcumin supplementation, since we found significant reduction in atg5 and atg10 mRNA levels at all time points studied when ho expression was induced by curcumin (Figure 4d,e).

Effects of Chronic Curcumin Supplementation on ho, Apoptosis, and Authophagy Gene Expression in the Brain
Chronic supplementation of wild-type flies with curcumin strongly induced ho expression. We detected a significant increase in the ho mRNA level in the brain at all time points studied (Figure 4a). We also found that the supplementation of food with curcumin prevented the loss of rhythm in ho expression in the brain of older flies (20 days old). This increase of ho mRNA level in the brain after consecutive 15 days of curcumin feeding was accompanied by the induction of hid expression at ZT16 and ZT20 (Figure 4b). In the same experiment, we also analyzed the expression of the skl gene, another gene coding apoptotic protein, which also showed an increase in mRNA level during the night: ZT13 and ZT16 (Figure 4c). This time-dependent upregulation of apoptotic genes in the brain after long feeding time (15 days) of curcumin, which had a similar effect when the ho gene was overexpressed in all neurons, confirmed association of ho transcription with apoptosis. However, it was not consistent with atg5 expression in the brain after chronic curcumin supplementation, since we found significant reduction in atg5 and atg10 mRNA levels at all time points studied when ho expression was induced by curcumin (Figure 4d,e).

Effects of Acute Paraquat Exposure on per, atg5, Hid and ho mRNA Level in the Brain
Flies were shown to undergo stress after being exposed to paraquat at 10 mM concentration for 24 h. Firstly, we found a disrupted circadian expression pattern of the per gene in the fly's brain with the highest mRNA level in the middle of the day (ZT4) (Figure 5a). We also detected a reverse pattern in the daily oscillation of atg5 transcripts in the brain, reaching the maximum level at ZT4 (Figure 5b). Paraquat also induced hid expression at ZT4 which resulted in a different cycling pattern of hid transcription during the day (Figure 5c). The examination of ho mRNA level in the brain after acute paraquat exposure showed changes in the daily pattern of ho expression compared to the control (Figure 5d). We found a significant increase in ho mRNA level in the brain after acute paraquat exposure at ZT1 or ZT13, which in normal condition (LD 12:12) had the lowest mRNA levels. This particular response after acute exposure to paraquat may be explained as a time-dependent protection by ho of the fly's brain during stress. High activity of males at the beginning of the day and night is correlated with higher exposure to toxic insults, and ho expression seems to be activated in response to stress. The significant decrease of ho mRNA in the brain at ZT4 by paraquat was accompanied by the increase of per, atg5, and hid mRNA. Effects of chronic curcumin supplementation on the mRNA level of heme oxygenase (ho) (a), apoptosis activator head involution defect (hid) (b) and sickle (skl) (c), and autophagy-related 5 (atg5-(d) and atg10-(e) genes in the brain of adult males of Drosophila melanogaster. Data shown as means ± SD. Statistically significant differences between groups are indicated with asterisks (p < 0.05).

Effects of Acute Paraquat Exposure on per, atg5, Hid and ho mRNA Level in the Brain
Flies were shown to undergo stress after being exposed to paraquat at 10 mM concentration for 24 h. Firstly, we found a disrupted circadian expression pattern of the per gene in the fly's brain with the highest mRNA level in the middle of the day (ZT4) ( Figure  5a). We also detected a reverse pattern in the daily oscillation of atg5 transcripts in the brain, reaching the maximum level at ZT4 (Figure 5b). Paraquat also induced hid expression at ZT4 which resulted in a different cycling pattern of hid transcription during the day (Figure 5c). The examination of ho mRNA level in the brain after acute paraquat exposure showed changes in the daily pattern of ho expression compared to the control (Figure 5d). We found a significant increase in ho mRNA level in the brain after acute paraquat exposure at ZT1 or ZT13, which in normal condition (LD 12:12) had the lowest mRNA levels. This particular response after acute exposure to paraquat may be explained as a time-dependent protection by ho of the fly's brain during stress. High activity of males at the beginning of the day and night is correlated with higher exposure to toxic insults, and ho expression seems to be activated in response to stress. The significant decrease of ho mRNA in the brain at ZT4 by paraquat was accompanied by the increase of per, atg5, and hid mRNA.

Discussion
In the present study, we showed that the gene encoding heme oxygenase (ho) in the brain of Drosophila is regulated by the circadian clock and by light exposure. The ho transcript level in the brain changes during the day and night under LD12:12 conditions, with lowest levels at the beginning of the day (ZT1) and the beginning of the night (ZT13). This rhythm has a similar pattern in per null mutants, which indicates that light may generate a weak exogenous rhythm of ho mRNA in the brain. In the case of DD, the pattern of ho expression was changed, but the rhythm was maintained since the significant difference between CT1 and CT16 was observed. It means that the circadian pattern of ho is masked by light in LD12:12. Some clock-controlled genes (ccgs) like Tor, atg5, and atg7 in the brain were reported to show differences in the expression pattern in LD12:12 and DD [39], like in the present study in case of ho mRNA. The same phenomenon was observed in the rhythm of locomotor activity in Drosophila which is bimodal in LD12:12 but unimodal in DD.
In the mammalian brain, in which HO-1 was also detected, its concentration was very low [40][41][42] in contrast to the retina, which is exposed to intense UV and visible lightinduced oxidative stress [43]. Using the publicly available visualization tool for large-scale single cell RNA sequencing (scRNA) analysis, known as SCope (http://scope.aertslab.org/ (accessed on 15 October 2020)), we verified the presence and distribution of ho mRNA in the brain. The ho gene was detected at various expression levels in different cell clusters in the fly's brain, with the highest expression in the optic lobe, followed by the expression in the central brain and then in glial cells. Higher expression of ho in the optic lobe is expected since the visual system is especially affected by oxidative stress as a result of the phototransduction [44].
It has already been found that ho is a clock-controlled gene (ccg) in the retina [20,27]. The brain is a heterogeneous tissue composed of different cell clusters which have different expression patterns of the ho gene (SCope). This may be the reason why the pattern of ho expression was not the same in the brain as in the retina. Our study also showed that the daily oscillation of ho transcription in the brain differs in the daily pattern compared with whole head homogenates which included retinas [20]. However, the rhythm observed in the Drosophila brain was similar to that detected in isolated glial cells under light/dark regime in our previous study [27]. The expression pattern of ho in glia was strongly rhythmic; however, it was not examined in DD or in per null mutants to verify if this rhythm was clock-controlled.
Aside from light, the transcription of ho in the brain is influenced by other factors which we documented in our study. Firstly, we found that aging affects HO because the rhythm of ho mRNA level in the brain in younger flies was abolished in older flies. We also detected a decline in the ho mRNA level in the brain of older flies, which is a common response in the aging brain since the total RNA level decreases overtime [45]. A decrease of ho mRNA cycling during aging has also been observed in mammals [42,46]. This can be explained by the fact that aging is associated with weakened circadian oscillations [26] that negatively affect clock-dependent processes [47]. Secondly, we observed that curcumin supplementation, an antioxidative compound, leads to upregulation of ho in the brain, regardless at which time of the day it was examined. A similar observation was made in the case of HO-1 since its mRNA and protein levels increased after exposure to curcumin [48][49][50][51][52][53][54][55], which can result from activating the nuclear factor, erythroid 2 like 2 (Nrf2) pathway [54,55]. Nrf2 and its inhibitor Kelch-like ECH associated protein (Keap) 1 regulate the expression of several cytoprotective genes [7]. HO-1 is known as a Nrf2-dependent gene and, when Nrf2 siRNA was used, curcumin-induced HO-1 expression was significantly reduced [54].
Finally, we showed that, under stress, mediated by the ROS inducer paraquat, ho expression in the brain was increased. The same result was observed in mammals in which HO-1 mRNA was induced after the exposure to paraquat [56]. The HO-1 gene was previously reported to be regulated by several factors, including curcumin, paraquat, or aging; however, it has not been studied in Drosophila and similar effects have not been expected. Moreover, we focused on HO at the transcription level since mRNAs are more sensitive to internal and external factors than proteins.
The specific expression of HO during the day in the fly's retina was reported to control several processes, including neuroprotection and the innate immune system. Here, we found that neuronal HO cross-talks with apoptosis and autophagy in a time-dependent manner. Apoptosis is a process of programmed cell death which eliminates unwanted or damaged cells, while autophagy is a cellular degradation and recycling process for cytosolic macromolecules and damaged organelles. Both processes work to maintain cellular homeostasis and are intimately associated with each other. Apoptotic signaling can regulate autophagy and conversely autophagy can regulate apoptosis (not exclusive to other cell death mechanisms) [57]. Notwithstanding, molecular interactions between autophagy and cell death are complicated and may have different contexts. However, it is possible that the same proteins can control both apoptosis and autophagy [57], just like we found in our study in case of HO. The interactions between apoptosis, autophagy, and HO have already been reported in mammals depending on injury. In Drosophila, apoptosis and autophagy genes were downregulated after increasing ho expression [34]. We found, however, an opposite effect when we genetically induced ho expression only in all neurons. The apoptosis activator head involution defect (hid) and autophagy gene atg5 expression were increased at night in whole head homogenates when the ho gene was overexpressed. However, when we silenced ho in neurons, hid expression increased but atg5 remained at the same level as in the control. This implies that any changes of the physiological level of neuronal HO can trigger apoptosis. An association of neuronal HO with autophagy was shown to enhance atg5 transcription when the ho mRNA level was high during the night. These differences may be attributed to the fact that our samples, which we used for analyses of apoptosis or autophagy transcript levels, were from whole heads and not from specific head tissues. There might be additional or compensation effects from other cells of the head after silencing or overexpressing ho. Nevertheless, we did not find any connections of autophagy and apoptosis with glial HO. In addition, we hypothesize that ho expression affects DNA damage repair genes as HO was shown to reduce DNA damage in the retina during phototransduction and/or UV radiation [27,33]. Surprisingly, we did not observe any changes in the expression of genes involved in DNA repair after silencing or overexpressing neuronal ho or overexpressing glial ho. It means that DNA breaks after oxidative stress induce apoptosis activated by HO rather than DNA repair.
A growing body of evidence shows the role of the Drosophila ho in oxidative challenges, particularly in the retina [27,[31][32][33][34]. We also found that aging may limit the protection by HO. Aging and age-related diseases are a consequence of free radical-induced damages of cellular macromolecules and inability to counterbalance these changes by endogenous antioxidant defense processes, which decline in aged brains. It has been found that, during aging, ROS level increases, along with lipid peroxidation, neurodegeneration, loss of synapses, and memory retention, while SOD, CAT, GSH, and choline acetyltransferase (ChAT) levels decrease [58]. We observed that chronic supplementation with curcumin has a potential to enhance the antioxidant mechanisms in the brain in the early phase of aging. We found the induction of ho expression at all time points studied as well as the restoration of the daily rhythm of ho mRNA lost in older flies after chronic curcumin supplementation. Furthermore, shorter feeding time with curcumin did not produce a significant increase of ho mRNA at the time when the expression level was observed at its peak (ZT20) after 15 days of feeding with curcumin. We assume that curcumin activates neuroprotective processes dependent on HO. This model of using curcumin as an antioxidant and anti-aging compound in mammals has been extensively exploited in neuroprotection by enhancing the HO-1 related pathways [49]. This is supported by the findings about curcumin protection which depends on HO-1 against H 2 O 2 -mediated cell death, and likely through the generation of CO [52]. Anti-apoptotic function of curcumin required the upregulation of HO-1 protein through the PI3K/Akt signaling pathway [53].
However, there is a need to further verify the extent to which the induction of ho expression after curcumin treatment protects the brain from aging or oxidative stress. We have only checked daily oscillations of ho mRNA levels with the chronic supplementation of curcumin under normal conditions and not combined with any stress factors. Nevertheless, curcumin also induces endoplasmic reticulum stress causing calcium release and destabilization of the mitochondrial compartment, which results in apoptosis. Autophagy in this case failed to rescue all cells and most cells underwent cell death [59,60]. We found the same relationship of apoptosis and autophagy expression in the fly's brain after curcumin feeding. Curcumin induced ho expression at all time points studied which was accompanied by the reduction of atg5 and atg10 expression, while hid and skl mRNA levels increased only at night. It has also been reported that inducing autophagy inhibits curcumin-induced cell death [61]. The neuroprotective effects of curcumin can also be linked through modulating various stages of the autophagy signaling pathway [62,63]. Curcumin, through the modulation of Atg7 and p62, can inhibit differentiation, promote cell survival and inhibit cell cycle progression from G1 to S [62]. Our study was only limited to the mRNA expression of autophagy-related genes atg5 and atg10.
We also propose that HO may play a neuroprotective role against paraquat toxicity in a time-dependent manner. The increase of ho mRNA level in the brain 1 h after lights-on and 1 h after lights-off, which we observed, may imply the neuroprotective response under stress and may be linked with the Drosophila circadian rhythm in behavior. Males are more active in locomotion at the beginning of the day and night and are therefore more exposed to toxic insults. The expression of ho can be increased to defend against stress during these times. However, it was suggested that the induction of HO-1 does not affect paraquat toxicity [64]. Nonetheless, a lesser concentration (50-250 µM) of paraquat was used, and it may be possible that the cells were not stressed at all. The survival after paraquat exposure was higher in flies treated with 5 mM paraquat or lower, and they also survived longer (in days) than those fed with higher concentration (10 mM) of paraquat [65]. The concentration which we used (at 10 mM) was shown to be ingested and lethal after administration in the fly's standard food. It also upregulated SOD, an enzyme that breaks down superoxide anions which are harmful ROS [36].
We also observed a change in the expression pattern of the period gene (per), along with ho transcription, after exposure to paraquat, which confirmed that, in this concentration, flies were under stress. Disrupted ho expression due to oxidative stress may also disturb the circadian clock, or vice versa. The effect of HO, mediated by carbon monoxide (CO), was documented to regulate the circadian clock gene expression [27]. Silencing ho in the retina increases and decreases the expression of the canonical clock genes period (per) and Clock (Clk), respectively, which, as it has been shown, has an opposite effect after increasing ho expression [27]. Clock disruption can negatively affect many cellular processes, including oxidative stress [66]. Paraquat also affected the daily expression of autophagy and apoptosis genes in the brain, which can also be linked to the effect of paraquat on HO. At the time when ho mRNA level was significantly reduced after acute paraquat exposure, we found accumulation of atg5 mRNA in the middle of the day, along with the induction of the apoptosis activator hid transcript levels. For the rest of the day, autophagy gene mRNA was kept at minimum levels by paraquat which was accompanied by insignificant change in the hid expression. This finding confirmed the studies that showed the importance of autophagy in regulating paraquat-induced apoptosis [67,68]. Induction of ho by paraquat at the beginning of the day and night may also play a role in stabilizing apoptosis at the required level.

Conclusions
Our study showed the expression of the gene encoding HO in the fly's brain cycles during the day and night. This rhythm is generated by the circadian clock but also regulated by light. During aging, HO shows a decline in its neuroprotective function along with autophagy, as mRNA levels of both genes decreased over time and their rhythmic expression patterns were lost in older flies. The loss of rhythmicity in ho expression was prevented by curcumin, and there was a significant increase in the ho mRNA level at all time points. The results obtained in the present study provide evidence that HO in the fly's brain plays an important role in cell survival and protects against paraquat-induced oxidative stress. HO can also be enhanced by curcumin to fight age-related oxidative challenges. Neuronal ho transcription also regulates apoptosis and autophagy under different conditions, which seems to be its protective mechanism in redox homeostasis. The timing, age, exposure to stress, and the presence of antioxidative factors must be considered to fully optimize the neuroprotective function of HO for therapeutic purposes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10111716/s1, Figure S1: Heme oxygenase (ho) expression in the brain of adult male Drosophila melanogaster fed with curcumin for 5, 10, and 15 days at ZT20, Figure S2: Effects of panneuronal overexpression of heme oxygenase (ho) on the expression of the nucleotide excision repair gene Xpc at ZT1 and ZT16 (a) as well as effects of pan-neuronal silencing of ho on Xpc expression at ZT1 and ZT16 (b), Figure S3: Effects of pan-neuronal overexpression and silencing of heme oxygenase (ho) on the expression of the translation factor eIF4a at ZT16, Figure S4: Effects of pan-glial overexpression of heme oxygenase (ho) on the expression of: hid (a), atg5 (b), and Xpc (c) at ZT1 and ZT16, Figure S5: Effects of pan-glial overexpression of heme oxygenase (ho) on the expression of the translation factor eIF4a at ZT16.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the fact that our results are only qPCR data that do not need to be stored in a database.

Conflicts of Interest:
The authors declare no conflict of interest.