An assessment of the rescue action of resveratrol in parkin loss of function-induced oxidative stress in Drosophila melanogaster

Loss-of-function mutations in parkin is associated with onset of juvenile Parkinson’s disease (PD). Resveratrol is a polyphenolic stilbene with neuroprotective activity. Here, we evaluated the rescue action of resveratrol in parkin mutant D. melanogaster. The control flies (w1118) received diet-containing 2% ethanol (vehicle), while the PD flies received diets-containing resveratrol (15, 30 and 60 mg/kg diet) for 21 days to assess survival rate. Consequently, similar treatments were carried out for 10 days to evaluate locomotor activity, oxidative stress and antioxidant markers. We also determined mRNA levels of Superoxide dismutase 1 (Sod1, an antioxidant gene) and ple, which encodes tyrosine hydroxylase, the rate-limiting step in dopamine synthesis. Our data showed that resveratrol improved survival rate and climbing activity of PD flies compared to untreated PD flies. Additionally, resveratrol protected against decreased activities of acetylcholinesterase and catalase and levels of non-protein thiols and total thiols displayed by PD flies. Moreover, resveratrol mitigated against parkin mutant-induced accumulations of hydrogen peroxide, nitric oxide and malondialdehyde. Resveratrol attenuated downregulation of ple and Sod1 and reduction in mitochondrial fluorescence intensity displayed by PD flies. Overall, resveratrol alleviated oxidative stress and locomotor deficit associated with parkin loss-of-function mutation and therefore might be useful for the management of PD.


Results
Resveratrol improves survival rate of parkin mutant D. melanogaster. After 21 days,untreated parkin mutant (PD) flies had a 75% decrease in survival rate compared with the wild type w 1118 flies (Control, Fig. 1). However, the PD flies fed with diet-containing resveratrol (15,30 and 60 mg/kg diet) had significant increases in survival rates compared with the untreated PD flies.
Resveratrol improves climbing activity of parkin mutant D. melanogaster. A significant reduction in climbing rate was observed in PD flies compared with PD flies treated with resveratrol. The PD flies exposed to resveratrol showed a significant increase in climbing activity when compared with untreated PD flies (p < 0.05; Fig. 1B). Also, a significant reduction in the activity of acetylcholinesterase (AChE) by 3.4-fold was observed in untreated PD flies compared with control. However, the treatment of PD flies with resveratrol improved AChE activity compared with untreated PD flies (approx. 2.1, 2.4 and 2.5 folds for 15, 30 and 60 mg/ kg diet of resveratrol, respectively p < 0.05), but not up to the level of the control flies (Fig. 1C).  www.nature.com/scientificreports/ Resveratrol prevents down-regulation of ple and Sod1 genes in parkin mutant D. melanogaster. In order to understand the action of resveratrol at the molecular level, we evaluated its effects on ple and Sod1. The ple encodes tyrosine hydroxylase, the enzyme that catalyzes the first-rate limiting step in dopamine biosynthesis 31 . The Sod1 encodes superoxide dismutase1 (SOD1) 32 , the enzyme that catalyzes the dismutation of superoxide radical to hydrogen peroxide. The effects of resveratrol (15,30  Histology of control and parkin mutant fly brains. The histology data in Fig. 6 indicated no detectable lesion in the brains of control as well as PD flies treated with diets containing resveratrol after 10 days of treatment (Fig. 6).

Action of resveratrol on mitochondrial fluorescence intensity in parkin mutant D. melanogaster.
To understand the action of resveratrol on mitochondrial abundance, the brains were stained with MitoTracker Green (Invitrogen™) as shown in Fig. 7. MitoTracker is a mitochondrial selective probe that covalently binds to mitochondrial proteins via reaction with cysteine residues. This assay is independent of mitochondrial membrane potential and mainly used to represent mitochondrial mass 33 . We found that mitochondrial mass, indicated by fluorescence intensity of MitoTracker Green, was markedly reduced in the brain of PD flies (Fig. 7). However, the treatment of PD flies with resveratrol (30 and 60 mg/kg diet) showed significant increase in mitochondrial fluorescence intensity compared with untreated PD flies (Fig. 7).

Discussion
Mutations in parkin have been established as one of the causes of PD 34 . Parkin is an E3 ubiquitin-protein ligase and a member of the quality control protein system. Available PD treatments, only provide symptomatic reliefs, but could not reduce, reverse or halt the neurodegenerative process 35 . Since oxidative stress is linked with the pathogenesis of PD 36 , any agent that can maintain the redox and oxidative stress-antioxidant balance might be a promising therapeutic drug for PD 37 . Resveratrol (3,5,4'-trihydroxystilbene) is a naturally existing polyphenolic compound found in nuts and fruits such grapes 38,39 . It has been reported to possess both anti-inflammatory and antioxidative properties 40 . It also possesses neuroprotective capacity as previously reported 27 . Here, we carried out different experiments to ascertain if resveratrol could alleviate oxidative stress associated with parkin lossof-function in D. melanogaster. In this study, resveratrol improved the survival rate of parkin mutant flies. Different authors have reported the life span-prolonging effect of resveratrol in animal models, including vertebrates (rodents), yeast, and invertebrates (nematodes and insects) 41 . Indeed, resveratrol has been shown to act via different targets such as mediation of autophagocytic death, decreased oxidative stress 41 , activation of sirtuins 42 , reduced inflammation 43 and  www.nature.com/scientificreports/ lipofuscin formation 44 . The observation that resveratrol increased the survival rate of parkin mutant flies aligns with our previous study where resveratrol extended the lifespan of D. melanogaste 27 , and that of Valenzano et al. 45 . Acetylcholine plays important roles in movement and balance 46 . Thus, reduction in its activity may impair acetylcholine function which includes movement. The PD flies displayed reduced locomotor (climbing) activity and acetylcholinesterase activity, which imply dysfunctional cholinergic transmission system. Further, the fact that resveratrol improved locomotor activity and acetylcholinesterase activity in PD flies further indicated its neuroprotective property.
Phase II drug metabolizing enzymes are the major detoxification enzymes that perform important roles in cellular defense. Glutathione-S-transferase (GST), a phase II drug metabolizing enzyme, participates in the detoxification of xenobiotics via conjugation with glutathione 47 . Indeed, overexpression of GST activity has been shown to be protective in parkin mutant fly model 48 . In this study, parkin mutant flies showed increased GST activity, which might be an adaptive response to parkin mutation. In addition, the levels of non-protein thiols and total thiols were reduced in parkin mutant flies. Interestingly, these markers were improved when PD flies were fed with diet-containing resveratrol. The thiol system consists of glutathione and other cysteine-containing molecules 28 that function to maintain the redox balance in biological system 29 . Glutathione acts as an antioxidant and it also acts as a vital factor in the detoxification of xenobiotics together with GST. Thus, the reduction in the levels of these thiols suggest redox imbalance elicited by parkin mutation in D. melanogaster. The observed increase in the level of non-protein thiols in PD flies treated with resveratrol (15 and 30 mg/kg diet) might be an adaptive protective response due to parkin mutation.
Catalase is a first-line antioxidant defence enzyme that catalyzes the conversion of H 2 O 2 to water and molecular oxygen. Since catalase is required for the breakdown of H 2 O 2 , an inhibition of its activity would be expected to result in increased accumulation of H 2 O 2 . The loss of function of Parkin protein caused an overload of reactive oxygen species, including H 2 O 2 , as a result of defective mitophagy. In PD, oxidative stress is deleterious due to accumulation of H 2 O 2 49 . Our results showed an increased level of H 2 O 2 in the parkin mutant flies, indicating oxidative stress. The observation that resveratrol reduced H 2 O 2 level, implies that oxidative stress in the flies was minimised.
Nitric oxide is a gaseous and easily diffusible molecule that acts as neurotransmitter in synapses 50 . Above physiological level, it can act as a reactive nitrogen species and induce oxidative stress in tissues. It forms secondary intermediates called peroxynitrites, which react with macromolecules, causing nitrosative damage. Since the brain has the highest amounts of saturated fats, it is a target for oxidation, nitration and peroxidation 51 . S-nitrosylation of Parkin produces a loss of function of the protein contributing to PD 52 . An elevation of nitrates/nitrites with increased lipid peroxidation as observed in our study, suggests oxidative damage. The observation that resveratrol ameliorated increased nitric oxide level displayed in PD flies further confirms its antioxidative activity.
In D. melanogaster, ple encodes tyrosine hydroxylase, the rate-limiting step in Dopamine (DA) synthesis 31 . In mammals, dopamine is mainly associated with motor control and reward. In flies, dopamine modulates a range of behaviours such as locomotion, learning, sleep and courtship 53 . As found in mammals, flies synthesize  32 . It offers protection against ROS by breaking down superoxide radicals to oxygen and H 2 O 2 , thereby making the latter susceptible to catalase degradation. This consequently prevents oxidative stress that can predispose to different diseases such as PD 55 . Thus, a reduction in the mRNA level of Sod1 noted in the PD flies further implies that oxidative oxidative stress accompanied parkin mutation. This further explains the reason for the accumulation of H 2 O 2 observed in this study. Conversely, treatment of PD flies with resveratrol (15 mg/ kg diet) restored mRNA level of Sod1, which might imply increase in the activity of SOD1 compared with PD flies, leading to the breakdown of excess superoxide radicals in the flies.
There was no detectable histological lesion in the brains of PD flies treated with resveratrol. Thus, we evaluated mitochondrial abundance in the brains of flies using MitoTracker Green, which is commonly used to stain mitochondria 56 . It binds covalently to sulfhydryl groups of mitochondrial proteins 33 . There was reduction in mitochondrial intensity in the brains of PD flies compared with control flies. Interestingly, resveratrol significantly increased mitochondria intensity in the brains of PD flies.
Taken together, resveratrol improved survival rate and locomotor performance of PD flies. It maintains oxidant-antioxidant homeostasis and prevented down regulation of ple and Sod1 genes in PD flies (Fig. 8). We did not investigate the reason(s) for the apparent lack of concentration-dependent correlations of resveratrol in the levels of thiols, activities of catalase and GST as well as mRNA levels of ple and Sod1 genes in this study. Nevertheless, this study indicated that appropriate concentrations of resveratrol can reduce oxidative stress associated with parkin loss-of-function mutation and therefore might be harnessed for the management of PD. It would be interesting to understand if resveratrol could rescue other forms of genetic-induced models of PD.

Materials and methods
Chemicals. All chemicals used in this study were commercial analytical grade products. Reveratrol 98% (HPLC) was purchased from AK Scientific, 30,023 Ahern Ave, Union City, CA 94587, USA.  Male  www.nature.com/scientificreports/ the adult stage, and the pupae without tubby body were noted. Following eclosion, the w; parkin 25 /parkin 25 (PD) flies were separated, reared under controlled environmental conditions and monitored for 72 h before further studies. Thereafter, they were carefully selected, under brief CO 2 anesthesia, 3 days after eclosion. We used the w 1118 flies with white eyes (the strain from which the parkin 25  Behavioural assay. This was carried out manually using negative geotaxis/locomotor assay 58 . Briefly, ten flies from control and PD groups were briefly anaesthetized using ice and placed in glass column (15 cm length and 1.5 cm in diameter). After recovery from anaesthesia, they were gently tapped to the bottom of the column. Then, the number of flies that climbed to the 6 cm mark were recorded. The data were then expressed as percentage of flies that crossed up to and beyond the 6 cm mark of the column.

Generation of parkin mutant (PD) D. melanogaster.
Determination of total thiols and non-protein thiol content. Total thiol content was assayed by the method of Ellman 59 . Briefly, the reaction mixture contained 170 μL of 0.1 M potassium phosphate buffer (pH 7.4), 20 μL of sample, and 10 μL of 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB). After incubation for 30 min at room temperature, the absorbance was measured at 412 nm using SpectraMax microplate reader (Molecular Devices). For non-protein thiol, the sample was precipitated with 4% sulphosalicyclic acid (4%) in the ratio of 1:1. The samples were kept at 4 °C for 1 h and then subjected to centrifugation at 5000 rpm for 10 min at 4 °C. The assay mixture consisted of 170 µl of 0.1 M phosphate buffer, 20 µl of supernatant and 10 µl of DTNB. The reaction was allowed to incubate for 30 min at room temperature, and the absorbance was read at 412 nm using SpectraMax microplate reader. For both total thiols and non-protein thiols, reduced Glutathione (GSH) was used as standard, and the data were expressed as in μmol/mg of protein.

The isolation of RNA and quantitative real-time RT-PCR.
Total RNA was isolated from 25 mg whole flies using Trizol (TRI Reagent ® , Zymol Research) using manufacturer's protocol as previously described 67 . The RNA isolated was resuspended in 50 µl RNase-free water, quantified spectrophotometrically using MaestroNan-oDrop Pro (Maestro Gen) and visualized in 2% agarose gel after gel electrophoresis (BioRad). Total RNA (0.3 µg) was used for cDNA synthesis following the manufacturer's protocol for ProtoScript II kit (New Egland BioLabs) and carried out in BioRad T100 thermal cycler. The primer sequences used in this study (ple and Sod1, Table 1) were obtained from the GeneBank overview (GenBank Overview (nih.gov)) and designed using the primer BLAST tool (https:// www. ncbi. nlm. nih. gov/ tools/ primer-blast/) which were custom synthesized by Invitrogen. Subsequently, qPCR was carried out and the mRNA expression levels were standardized to two genes (Beta Tubulin 86D and RPL32). Luna Universal qPCR Master mix kit was used for qPCR. A total reaction volume of 20 µl was used with 9 ng of cDNA, Luna Universal qPCR Mix, 10 µM forward and reverse primers, and nucleasefree water. The cycling condition was as follows: an initial denaturation at 95 °C for 60 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 60 s followed by a dissociation curve analysis. The SYBR fluorescence was analysed by the SDS 2.1 software (Applied Biosystems, ABI Prisms 7900HT). The reaction was carried out in triplicates of each independent group. Dissociation curves at 60-95 °C was carried out to ascertain the amplification of a single specific product for each reaction. The 2 −ΔΔCT method was used to determine the expression values of each gene.
Histology of fly brains. Adult fly brains were fixed in 10% neutral buffered formalin, deparaffinized and processed for Hematoxylin and Eosin (H/E) histological staining 68 . The slides were viewed using light microscopy and interpreted by Veterinary Pathologists who were blinded to the control and PD fly brains.
Mitochondrial staining in fly brains. Apart from H/E histological study, we carried out additional experiment to evaluate mitochondrial mass/abundance (based on fluorescence intensity of MitoTracker™ Green stain, Invitrogen™) 56 in the brains of control and parkin mutant flies treated with resveratrol for 10 days. Briefly, the fly brains (15/group) were excised, fixed in 4% buffered formalin for 40 min and transferred to 30% sucrose solution in PBS. Thereafter, the whole brains in a group were dehydrated and embedded in paraffin. Then, 5 µm sections of fly brains were prepared from a block and stained on charged slides. The slides were deparaffinised in xylene solutions (10 min each) and rehydrated in decreasing concentrations of ethanol solution in PBS. The slides were then stained with 0.1% MitoTracker™ Green (Invitrogen™) in PBS for 20 min. Nuclei were stained with DAPI (0.1 µg/mL) and slides were mounted in PBS-Glycerol (1:1) solution containing N-propyl gallate. Images were acquired using Zeiss Axioscop fluorescent microscope and signal intensity quantitation as well as background correction were carried out using ImageJ.
Statistical analysis. For statistical analysis, GraphPad Prism 9 was used. The Kaplan-Meier's method was used to analyze the survival rate and comparisons were made with the log-rank tests. For biochemical analyses, statistical significance was evaluated using one way analysis of variance (ANOVA), followed by Dunnett's post  Tubulin  TGG GCC CGT CTG GAC CAC AA  TCG CCG TCA CCG GAG TCC AT   RPL32  CCC AAG ATC GTG AAG AAG CG  TGG GCT TGC GCC ATT TGT G   ple  CAG CAA GGC AAA TGA TTA CGGT AAT CCG GGG TGG TTC ATG TC   Sod1  GGA GTC GGT GAT GTT GAC CT  GTT CGG TGA CAA CAC CAA  www.nature.com/scientificreports/ hoc test. Data points correspond to the mean of independent experiments and error bars (S.E.M); the level of significance was set at p < 0.05 and indicated in the charts.

Data availability
All data that support the findings of this study are available on reasonable request to the corresponding author. The contributing authors declare that all relevant data are included in the paper. www.nature.com/scientificreports/