Dual Oxidase, a Hydrogen-Peroxide-Producing Enzyme, Regulates Neuronal Oxidative Damage and Animal Lifespan in Drosophila melanogaster

Reducing the oxidative stress in neurons extends lifespan in Drosophila melanogaster, highlighting the crucial role of neuronal oxidative damage in lifespan determination. However, the source of the reactive oxygen species (ROS) that provoke oxidative stress in neurons is not clearly defined. Here, we identify dual oxidase (duox), a calcium-activated ROS-producing enzyme, as a lifespan determinant. Due to the lethality of duox homozygous mutants, we employed a duox heterozygote that exhibited normal appearance and movement. We found that duox heterozygous male flies, which were isogenized with control flies, demonstrated extended lifespan. Neuronal knockdown experiments further suggested that duox is crucial to oxidative stress in neurons. Our findings suggest duox to be a source of neuronal oxidative stress associated with animal lifespan.


Introduction
Hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species (ROS) that functions in signaling pathways by specifically oxidizing redox-sensitive proteins in diverse biological processes [1][2][3][4]. However, the upregulated or dysregulated production of H 2 O 2 in combination with diminished anti-ROS activity confers oxidative stress that abrogates redox-dependent signaling or nonspecifically and irreversibly damages macromolecules, in turn accelerating the aging process and decreasing lifespan [5][6][7][8][9]. Neurons are particularly susceptible to oxidative stress, and the elevation of the antioxidant power in neurons has been shown to extend lifespan [10][11][12][13][14]. Various anti-ROS enzymes and anti-ROS transcriptional networks are well known, yet the source of ROS production that provokes oxidative stress in neurons is not yet completely understood. ROS production is inducible by members of the NADPH oxidase (NOX) family; such induced production is distinct from the steady-state ROS production that occurs through oxidative respiration in mitochondria [15]. Dual oxidase (Duox) is a member of the Nox family, with an EF-hand motif that is activated by intracellular calcium to produce H 2 O 2 [1,16,17]. It is widely expressed in the nervous system in rats, zebrafish, and Drosophila [18][19][20][21]. Inflammation, wounds, and various signals that increase intracellular calcium ion concentration all activate duox [22][23][24][25] and, in particular, UV irradiation, ROS, and p38 MAPK signaling increase its expression [18,[26][27][28][29][30]. Duox is known to mediate pain hypersensitivity due to UV-induced damage [31] and, in the CNS, axon regeneration after wounds [32,33]. However, the full role of duox in the nervous system still remains largely unknown. In Drosophila, there is only a single duox gene, in contrast to the two genes present in mammals [21]. Here, we show that through its generation of H 2 O 2 , duox contributes to oxidative stress in neurons and is a determinant of Drosophila melanogaster lifespan.

Lifespan and Oxidative Stress Assays
For lifespan assays, 20 flies per vial were transferred to fresh food vials every two days. Dead flies were counted every day. For survival under oxidative stress, 5-day-old flies were starved for 6 h at 25 • C in vials containing water-soaked tissues and then transferred to vials containing either normal food with 5 mM methyl viologen hydrate (paraquat) or 5% sucrose-agar medium with 5% hydrogen peroxide [34]. Dead flies were counted every 12 h.

Locomotive Activity
Negative geotactic movement assays were performed as described previously [35]. Briefly, a vial (9.5 cm × 2 cm) containing 10 flies was tapped to send the flies to the bottom. Flies that crossed the 8-centimeter line from the bottom within 10 s were scored. In total, 100 male flies were assessed in 10 vials (10 flies per vial).

Determination of Total ROS Levels
Total ROS was measured as previously described [36]. Briefly, 10 flies or the heads of 50 flies were homogenized in 200 µL of 50 mM Tris-HCl, pH 7.4, on ice. Extracts were centrifuged at 13,200 rpm for 10 min at 4 • C. Homogenates were incubated with H 2 DCFDA (Life Technologies, Carlsbad, CA, USA) at 5 µM in a 200-microliter reaction volume at 37 • C for 15 min in darkness, carried out on 96-well plates. Florescence intensity was monitored on a Gemini XPS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) set for 488 nm excitation and 520 nm emission. Florescence intensity was normalized by protein amount measured by Bradford method [37]. All experiments were carried out with three biological replicates.

Determination of H 2 O 2 Levels
H 2 O 2 was measured as previously described [36]. Briefly, 10 flies were homogenized in phosphate saline buffer (50 mM, pH 7.4) containing aminotriazol (2 mg/mL) and incubated at 4 • C for 15 min. The extracts were centrifuged at 13,200 rpm for 10 min at 4 • C to obtain supernatants. H 2 O 2 levels were measured using a hydrogen peroxide assay kit (Cayman Chemical, Ann Arbor, MI, USA) on a microplate reader (VersaMax; Molecular Devices) set to 590 nm. H 2 O 2 levels were normalized according to protein amount measured by Bradford method [37]. All experiments were carried out with three biological replicates.

Determination of Protein Oxidation
Carbonyl groups in proteins were detected using an oxyblot protein oxidation detection kit (Milipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, proteins were extracted from 10 flies or~50 fly heads using RIPA buffer, reacted with 2,4dinitophenylhydrazine (5 mM) at 25 • C for 100 min, and then subjected to SDS/PAGE and Western blotting using an antibody to the dinitrophenyl moiety. The blots were developed with ECL (Amersham, Buckinghamshire, UK) and the images were captured with a Vilber Lourmat Fusion FX (Vilber Lourmat, Eberhardzell, Baden-Württemberg, Germany). Band intensities were determined with the Fusion program (Vilber Lourmat). Finally, blots were stripped with a stripping buffer (Thermo Fisher scientific, Waltham, MA, USA), and then by a separate Western blot carried out using β-actin antibody (Santa Cruz, Dallas, TX, USA), which was used as loading control. All experiments were carried out with three biological replicates.

Determination of DNA Oxidation
The level of 8-hydroxydeomyguanosine (8-OHdG) was measured using the OxiSelect TM Oxidative DNA Damage ELISA kit (Cell Biolabs, San Diego, CA, USA). Briefly, genomic DNA from 10 flies or~50 fly heads was extracted using PureLink TM genomic DNA kits (Invitrogen, Carlsbad, CA, USA), treated at 95 • C for 5 min, and digested with nuclease P1 (20 mM sodium acetate, pH 5.2) and alkaline phosphatase (100 mM Tris, pH 7.5). Samples were centrifuged for 5 min at 6000× g and the supernatants were used for ELISA assays following the manufacturer's instructions. The amount of 8-OHdG in each sample was calculated based on an 8-OHdG standard curve.

TUNEL Labeling
Adult fly brains were dissected in M3 medium (Merck, Boston, MA, USA), fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, 50 mM, pH 7.4) for 20 min at room temperature, washed with PBS, and permeabilized by 2 min incubation in PBS containing Triton X-100 (0.1%) on ice. After washing with PBS three times, the samples were first incubated in Na citrate (0.1 M, pH 6.0) for 10 min at 65 • C, followed by incubation in Tris-HCl buffer (0.1 M, pH 7.5) containing 3% BSA and 20% bovine serum albumin for 30 min. After washing three times with PBS, the samples were incubated in TUNEL assay solution (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland) for 1 h at 37 • C, in accordance with the manufacturer's recommendations. After washing three times with PBS, the samples were mounted in mounting solution (Prolong Gold Antifade reagent; Life Technologies) and examined by confocal microscopy (Leica TCS SP5; Leica Microsystems, Morrisville, NC, USA).

Statistical Analysis
The statistical significance of differences in standard error of the mean (SEM) values was obtained using Student's t-test (two-tailed), analysis of variance (ANOVA) with post hoc analysis (Dunnett's tests, two-tailed). Kaplan-Meier survival curves were used for lifespan and survival analysis with log-rank tests. Prism software (GraphPad version 6.0, San Diego, CA, USA) was used to calculate p-values. Results were considered statistically significant at levels * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Results
To find genes involved in lifespan extension, we carried out a small-scale screening of mutants with extended lifespan, which identified duox heterozygous (duox KG07745/+ ) male flies of interest. In the transgenic allele, the P-element KG07745 was inserted into the second intron of the duox locus, which reduced the duox transcripts to~half the level in control flies ( Figure 1A-C). As the insertion was downstream of the initiation codon, the Duox protein structure was disrupted, suggesting that the duox KG07745 allele was probably null or severely hypomorphic with regards to protein function The. Duox KG07745 homozygotes were lethal, while the duox KG07745 heterozygotes were normal in both appearance and movement ( Figure S1). second intron of the duox locus, which reduced the duox transcripts to ~half the level in control flies ( Figure 1A-C). As the insertion was downstream of the initiation codon, the Duox protein structure was disrupted, suggesting that the duox KG07745 allele was probably null or severely hypomorphic with regards to protein function The. Duox KG07745 homozygotes were lethal, while the duox KG07745 heterozygotes were normal in both appearance and movement ( Figure S1). Twenty flies/vial, total twenty vials. Log-rank test, **** p< 0.0001. duox +/+ denotes w 1118 flies. duox +/− denotes duox +/kG07745 flies, which were isogenized ten times with control flies (w 1118 ). (E) Lifespans of elav-Gal4>UAS-duox RNAi flies. elav/#38907 and elav/#32903 denote elav-Gal4>UAS-duox RNAi #38907, elav-Gal4/+; UAS-duox RNAi #32903/+. Twenty flies/vial, total ten vials, male flies. Log-rank test, **** p < 0.0001. UAS-duox RNAi (#38907, 32903) flies were isogenized with control w 1118 flies ten times to reduce genetic background differences.
Initial screening involved a small number (n = 40) of unisogenized duox heterozygous males. To ensure rigor, we isogenized the duox heterozygous flies with the control flies (w 1118 ) ten times to reduce genetic background differences, and increased the population to 400 flies, with 20 flies per vial given fresh food every two days. We focused on the duox heterozygous male flies due to the small effect of the allele on the lifespans of the heterozygous female flies ( Figure S2A). The control male flies (w 1118 ) lived for a median of 70 days; meanwhile, the isogenized duox heterozygous males had an extended median lifespan of 81 days, which was an increase of 15% ( Figure 1D, Table S1). Next, we employed the bipartite Gal4/UAS expression system to knock down the duox function across the whole body, including in both muscle and neurons. Rigorously, we isogenized the UAS-duox RNAi (#38907, 32903) flies with w 1118 flies ten times to reduce the genetic background differences between the Gal4 > UAS-duox RNAi flies and the control Gal4 > w 1118 flies. Driving the whole-body duox RNAi (#38907 and #32903) expression with the whole-body Gal4 (tub-Gal4) was lethal. A second experiment using muscle-only Gal4 (mef-Gal4) did not demonstrate  Figure S2C,D). However, driving the duox RNAi with the pan-neuronal Gal4 (elav-Gal4) extended the male lifespan by 10% and 20%, respectively, for the duox RNAi #38907 and #32903 relative to the elav-Gal4/+ control ( Figure 1E, Table S1); the lifespan extension in the females was not pronounced ( Figure S2B).
As Duox is a ROS-producing enzyme, the duox heterozygotes (duox +/− ) would be expected to produce less ROS than the control flies (duox +/+ ). The global ROS was measured using the non-fluorescent compound 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA), which reacted with the ROS to produce fluorescent dichlorofluorescein (DCF). The ROS-dependent DCF fluorescence intensity was normalized to the protein concentration in each extract. The whole-fly extracts from the duox heterozygotes produced less ROS than those from the controls (Figure 2A). The head extracts from the flies with the neuronal knockdown of the duox also exhibited less ROS than the controls ( Figure 2B). Similarly, the assays of the H 2 O 2 production revealed that the duox heterozygote whole-fly extracts produced less H 2 O 2 than those from the controls ( Figure 2C). The same was true of the head extracts from the flies with the neuronal knockdown of the duox ( Figure 2D). ROS mediates the oxidation of amino acid residues in proteins and of the bases in DNA [38][39][40], and this oxidation was decreased in the whole-body extracts of the duox heterozygous male flies ( Figure 3A,C). Similarly, the neuronal knockdown of the duox via RNAi resulted in reduced protein and DNA oxidation in the head extracts ( Figure 3B,D).   Given that the duox heterozygous males experienced less oxidative damage, next, we examined whether these males exhibited increased survival when provided with ROSproducing food. When given food-containing paraquat, an agent that produces cellular ROS, the duox KG07745 heterozygous males exhibited increased survival; they also showed increased survival on H 2 O 2 -containing food ( Figure 4A,B). Oxidative stress is relevant to apoptotic cell death and neurodegeneration, suggesting that apoptotic cells might be reduced by duox reduction. Indeed, the apoptotic cells in the brain were reduced by the duox heterozygosity and by its neuronal knockdown ( Figure S3). The observed ROS decrease could have been due to increased levels of anti-ROS enzymes. However, the levels of the anti-ROS enzyme catalase, dSOD1, and dSOD2 were constant with age regardless of the duox status ( Figure S4), suggesting that the lower ROS level in the duox heterozygous flies was not due to anti-ROS enzymes, but to duox heterozygosity.  . n=200 (20 flies/vial, total 10 vials). Log-rank test, **** p < 0.0001. duox +/+ denotes w 1118 flies. duox +/− denotes duox +/kG07745 flies, which were isogenized ten times with control flies (w 1118 ).

Discussion
Neurons are vulnerable to oxidative stress, which is related to neuronal aging, neuropathies, and lifespan. Considerable attention has been paid to antioxidant defense systems, but the ROS sources that oxidize macromolecules in neurons are not yet well defined. Here, we identify Duox, a calcium-activated NADPH oxidase, as a determinant of neuronal oxidative stress and lifespan. We found that neuronal oxidative damage was reduced and lifespan was extended by the neuronal knockdown of duox in Drosophila melanogaster. Our findings suggest that duox is a source of ROS production in neurons, which affects lifespan.
It is intriguing that oxidative stress, particularly stress in neurons, should impact lifespan. It was previously shown that lifespan can be extended by enhancing anti-oxidative power in neurons, specifically through the neuronal overexpression of Peroxiredoxin 4 (prx4), an anti-ROS protein, and of glutamate-cysteine ligase, an enzyme that catalyzes the biosynthesis of the reducing agent, glutathione [13,41]. Similarly, the neuronal knockdown of Kelch-like ECH-associated protein 1 (keap1) increases longevity [8,12]; this gene encodes an inhibitor of the cap 'n' collar (CncC) transcription factor, the Drosophila homolog of the mammalian nuclear factor E2-related factor 2 (NRF2), which provides a major anti-ROS cellular system by directly inducing expression of anti-ROS genes. Thus, our findings are in line with previous reports that reducing oxidative stress in neurons is beneficial to lifespan.
We further observed that lifespan extension is pronounced in duox heterozygous males but less so in heterozygous females, and a similar trend was seen with the neuronal knockdown of the duox. Such sexual dimorphism in longevity has previously been observed in other contexts. For example, increased reducing power in neurons via the increased activation of CncC due to keap1 mutation results in extended lifespan for males, . Log-rank test, **** p < 0.0001. duox +/+ denotes w 1118 flies. duox +/− denotes duox +/kG07745 flies, which were isogenized ten times with control flies (w 1118 ).

Discussion
Neurons are vulnerable to oxidative stress, which is related to neuronal aging, neuropathies, and lifespan. Considerable attention has been paid to antioxidant defense systems, but the ROS sources that oxidize macromolecules in neurons are not yet well defined. Here, we identify Duox, a calcium-activated NADPH oxidase, as a determinant of neuronal oxidative stress and lifespan. We found that neuronal oxidative damage was reduced and lifespan was extended by the neuronal knockdown of duox in Drosophila melanogaster. Our findings suggest that duox is a source of ROS production in neurons, which affects lifespan.
It is intriguing that oxidative stress, particularly stress in neurons, should impact lifespan. It was previously shown that lifespan can be extended by enhancing anti-oxidative power in neurons, specifically through the neuronal overexpression of Peroxiredoxin 4 (prx4), an anti-ROS protein, and of glutamate-cysteine ligase, an enzyme that catalyzes the biosynthesis of the reducing agent, glutathione [13,41]. Similarly, the neuronal knockdown of Kelch-like ECH-associated protein 1 (keap1) increases longevity [8,12]; this gene encodes an inhibitor of the cap 'n' collar (CncC) transcription factor, the Drosophila homolog of the mammalian nuclear factor E2-related factor 2 (NRF2), which provides a major anti-ROS cellular system by directly inducing expression of anti-ROS genes. Thus, our findings are in line with previous reports that reducing oxidative stress in neurons is beneficial to lifespan.
We further observed that lifespan extension is pronounced in duox heterozygous males but less so in heterozygous females, and a similar trend was seen with the neuronal knockdown of the duox. Such sexual dimorphism in longevity has previously been observed in other contexts. For example, increased reducing power in neurons via the increased activation of CncC due to keap1 mutation results in extended lifespan for males, but not females [42]. A recent report similarly found that oltipraz, an NRF2-activating drug, extends lifespan more in males than in females [43]. It appears that sexual dimorphism exists in regard to neuronal oxidative-stress-dependent lifespan.
It is worth mentioning the duox-dependent lifespan extension in C. elegans. In this model nematode, lifespan is expectably shortened upon the substantive increase of duox activity [44]. However, and much more unexpectedly, a modest increase in duox results in extended lifespan [44,45]. Why is a low-level increase in duox activity beneficial to lifespan? The underlying mechanism involves the activation of the expression of SKN-1, the C. elegans homolog of NRF2 [44]. NRF2 is a transcription factor that responds to ROS and is known to extend lifespan [9,[45][46][47]. Therefore, extension of lifespan with a low-level increase in duox is achieved by strengthening anti-ROS power via NRF2 activation. Thus, our findings concerning Drosophila duox in combination with other reports regarding C. elegans duox support the notion that oxidative stress regulates lifespan.
Oxidative stress can occur due to an imbalance between ROS production and the cellular antioxidant defense network. In Drosophila, NRF2-dependent anti-ROS power declines with age, suggesting that oxidative stress is more likely to occur in older animals [8,43,48,49]. Duox activity and expression are increased in gut and embryo epithelial cells undergoing apoptotic cell death, in mammalian neurons through the stabilization of duox transcripts by ROS, and in gut epithelial cells by PLCβ-calcium and p38 MAPK signaling [18,24,27,29,30]. In addition, duox is elevated in aged brains and in Drosophila models of Alzheimer's disease [50]. Thus, chronic inflammation, protein-aggregate-induced cell death, and various signals involving PLCβ-calcium or p38 MAK could aberrantly activate duox activity and expression in neurons, which could confer oxidative stress and lifespan determination. It would be informative to examine whether neuronal duox knockdown reduces neuronal inflammation, a hallmark of which is the activation of microglial cells or astrocytes. However, the pursuit of this idea is hindered by the lack of antibodies or markers for activated microglial and astrocytic cells, which is a limitation of these studies.