Grape skin extract improves muscle function and extends lifespan of a Drosophila model of Parkinson’s disease through activation of mitophagy

Abstract

Recent studies suggest that moderate red wine consumption may confer several health benefits, including protection against heart disease, certain cancers and multiple age-related neurological diseases such as Alzheimer’s disease. These health benefits are assumed to come from a compound from grape skin called resveratrol, which has been proposed to be a pro-longevity agent. Whether resveratrol accounts for all the health benefits of grape-derived nutrients and the molecular and cellular mechanisms underlying the beneficial effects of such nutrients are not well understood. Here we investigated the effect of supplementing grape skin extract (GSE) left from red wine-production process to the daily food intake of a Drosophila melanogaster model of Parkinson’s disease (PD) associated with PTEN-induced kinase 1 (PINK1) loss-of-function. Consumption of GSE resulted in rescue of mitochondrial morphological defects, improvement of indirect flight muscle function and health-span, and prolonged lifespan of the PINK1 mutant flies. Further biochemical and genetic studies revealed a link between activation of mitophagy and the beneficial effects of GSE. Our results indicate that GSE can promote autophagy activation, preserve mitochondria function, and protect against PD pathogenesis, and that the beneficial effect of GSE in mitophagy activation is not accounted for by resveratrol alone.

1. Introduction

Red wine has been consumed for thousands of years, and interestingly, the health benefits of drinking red wine are widely reported in recent years. Studies have suggested that red wine possesses a diverse range of biological actions and may be beneficial in protecting against heart diseases and cancer, reducing the risk of Alzheimer’s disease (AD), and even prolonging lifespan (Bertelli and Das, 2009; Artero et al., 2015; Granzotto and Zatta, 2014). These health benefits have been attributed to the intake of polyphenols from the red wine, in particular, a compound called resveratrol, which is highly concentrated in the dark-skinned grapes used to make red wine (Yu et al., 2012; Lekli et al., 2010; Saiko et al., 2008). Epidemiological studies have supported the notion that red wine as a diet supplement might benefit cardiovascular function. Interestingly, accumulating evidence also suggested the potential of red wine consumption in protecting against age-related neurodegenerative disease, such as AD and PD (Caruana et al., 2016). Different mechanisms may be responsible for these beneficial effects, such as antioxidant and anti-inflammatory responses, as well as protection against neurotoxic protein aggregation (Yu et al., 2012; Sun et al., 2010). For example, resveratrol was reported to prevent neurodegeneration in AD via inhibition of beta-amyloid plaque formation (Marambaud et al., 2005).

Among the age-related neurodegenerative diseases, PD is the second most common after AD. The primary symptoms of the disease include muscular rigidity, resting tremor, and bradykinesia. It is the most common movement disorder, characterized by age-dependent degeneration of dopaminergic neurons in the substantia nigra of the mid-brain. An estimated 10 million people are living with PD worldwide, with more than one million living the US (Pringsheim et al., 2014). Current treatment options only offer symptomatic modification, and limited success has been achieved so far with efforts to slow down disease progression. Increasing evidence suggests that diet and dietary components might significantly delay the occurrence of AD and PD (Albarracin et al., 2012). An example is the traditional mediterranean diet that is characterized by a high consumption of plant foods, olive oil, fish and low intake of meat, combined low-to-moderate consumption of wine during meals. In particular, the neuroprotective benefit of the mediterranean diet has been attributed to a moderate consumption of red wine, which contains nutrients especially polyphenols derived from grape skin during the production process. Besides polyphenols, grape skin/seed extract contains anti-oxidative components like proanthocyanidine, quarcetin, etc. and these active components may also provide protection against oxidative stress and free radical-mediated tissue injury (Basli et al., 2012).

The onset and progression of PD are determined by both genetic backgrounds and environmental factors. Human genetic studies have identified PINK1 and PARKIN as disease genes associated with autosomal recessive forms of PD (Valente et al., 2004; Kitada et al., 1998). Recent studies have revealed that PINK1 and PARKIN are directly involved in regulating mitochondrial function and integrity (Park et al., 2006; Clark et al., 2006; Yang et al., 2006; Wang et al., 2006). The PINK1 gene encodes a serine-threonine kinase with a mitochondria-targeting signal at the N-terminus, while PARKIN encodes an E3 ubiquitin ligase that is largely cytosolic under normal conditions. PINK1 has been reported to regulate mitochondrial quality control by promoting mitochondrial outer membrane associated translation, sensing mitochondrial damage, and recruiting PARKIN and the autophagy machinery to damaged mitochondria to promote their clearance in a process called mitophagy (Narendra et al., 2008; Narendra et al., 2010; Vives-Bauza et al., 2010; Matsuda et al., 2010; Gehrke et al., 2015). In Drosophila melanogaster, loss of PINK1 function results in energy depletion, degeneration of select indirect flight muscles and dopaminergic neurons, and shortened lifespan. The muscle degeneration pathology was preceded by mitochondrial enlargement and disintegration (Park et al., 2006; Clark et al., 2006; Yang et al., 2006), supporting the primary role of mitochondrial dysfunction in the pathogenesis of this PD model.

Here we utilized the Drosophila PINK1 model to investigate the potential of GSE in protecting against the detrimental effects of mitochondrial dysfunction, by incorporating GSE powder into the daily food intake of PINK1 mutants. We observed significant improvements of flight muscle function, health-span, and lifespan, which are accompanied by activation of autophagy and removal of aberrant mitochondria. Further mechanistic studies suggested that GSE exerts protective effects in a manner consistent with enhancement of mitophagy, as we observed induction of autophagy receptor p62 expression and increased autophagic flux by GSE, and inhibiting autophagy activation blocked the observed beneficial effects of GSE. Intriguingly, the beneficial effects of GSE could be recaptured only partially by resveratrol, suggesting that other components of GSE may also offer beneficial effects, especially in the activation of mitophagy. Our data provide new insight into the health benefits of grape and grape-derived products such as red wine and suggest their addition to PD patients’ daily diet to help modify disease courses.

2. Materials and methods

2.1. Drosophila (fly) strains

WT control (w/Y; MHC-Gal4/+) or PINK1^B9 mutant (PINK1^B9/Y; MHC-Gal4/+) flies were raised according to standard procedures in the normal corn meal food (NF) at 25 °C and newly hatched adults were collected on the same day and aged at 29 °C for further testing. PINK1 null mutant (PINK1^B9) was a gift from Dr. Jongkeong Chung, UAS-mitoGFP line was a gift from Dr. William Saxton, PINK1 RNAi flies were generated in our lab (Yang et al., 2006). UAS-S6K and rictorΔ2 mutant flies were described before (Liu and Lu, 2010; Wu et al., 2013). ATG1 RNAi flies were obtained from VDRC (Vienna Drosophila Resource Center) and other general stocks were obtained from Bloomington Drosophila Stock Center.

2.2. Grape skin extract (GSE)

The pomace after fermentation was collected from a winery in Sonoma County, Northern California. It was air-dried for about 24 h, dehydrated with low heat (50 °C). Grape skins were separated from seeds and ground into fine powder with a high-speed grinder. The powder was then packaged, vacuum-sealed and stored at room temperature in a dry area. To make GSE-supplemental fly food, we added the 4%, 8% and 16% w/v of GSE powder to the fly food (Yeast 11.9 g, corn meal 35.4 g, agar 5.8 g, H₂O 0.81 L, corn syrup 20 ml, malt 0.1 lb., Tegosept 0.7 g (in 6.8 ml 100% Ethanol) and propionic acid 5.1 ml per liter) before curing. Resveratrol (Sigma-Aldrich) was dissolved in 100% Ethanol and mixed with fly food at indicated concentrations.

To monitor the effect of GSE on food intake, we fed flies on sucrose solution soaked in Whatman paper for 2 h and then starved the animals for 10 h with water only. A blue dye, brilliant blue FCF (C₃₇H₃₄O₉SNa) was mixed with different GSE or Resveratrol foods and used as internal markers of intake, as it was neither toxic nor metabolized at the test conditions (Wu et al., 2012). About 25 flies were put to one vial and fed for 12 h at 25 °C; At least 80 flies were tested in GSE or resveratrol containing foods. The numbers of flies with different intensities of abdominal colors were scored and compared via X² tests. For pair-wise comparisons, we used Student’s t-test. For comparing multiple groups as in Fig. 2C, D, and E, we used one-way ANOVA test followed by Student–Newman–Keuls (SNK) - test plus Bonferroni correction (multiple hypotheses correction).

Fig. 2.

GSE supplementation suppressed the abnormal wing posture phenotype and improved health-span in PINK1 mutant flies. (A) Quantification of colored food intake by PINK1 mutant flies fed with the indicated foods. (B) Typical abnormal wing postures of PINK1 mutant flies with held-up or droopy wings, and quantification of abnormal wing posture in the population of PINK1 mutant flies raised on fly food supplemented with different concentrations of GSE after 7 days of aging at 25 °C. * or #, p < 0.05 compared to NF in 7 days or 15 days records. (C-E) Analysis of PINK1 mutant fly health-span after feeding with GSE- or resveratrol-supplemented food. Parameters measured are jump/flight ability (C), ATP production (D), and ROS production (E). * indicates p < 0.05 in multiple sample comparisons in one-way ANOVA test followed by Student - Newman - Keuls (SNK) test plus Bonferroni correction (multiple hypotheses correction).

2.3. Lifespan analysis

WT or PINK1 mutant flies with the same age were separated randomly into different vials (40 flies per group, 4 groups per experiment, total 160 flies per GSE dosage) containing either standard corn meal fly food (NF) or standard food supplemented with GSE powder or resveratrol (Sigma-Aldrich). Flies were transferred to fresh medium every 3 days to maintain a healthy culture environment and the number of surviving flies was recorded. Statistical analysis was performed by Kaplan-Meier analysis; p < 0.005 is considered as statistically significant.

2.4. Abnormal wing posture analysis

PINK1 mutant flies were allowed to lay eggs on standard fly food or fly food supplemented with GSE or resveratrol. The progenies were kept at 25 °C, and the newly eclosed flies were collected and transferred to vials containing corresponding fresh food. The vials were changed every 2–3 days. After 7 days of culturing at 29 °C, the number of flies with normal wing posture or drooped/held-up abnormal wing posture was scored. The abnormal wing posture penetrance was calculated as the percentage of flies with either held-up or drooped wing posture from the total number of flies scored. For each experiment, around 20 male flies were collected for each group and 4–5 groups were scored per GSE dosage. Statistical analysis was performed by student t-test, p < 0.01 was considered statistically significant.

2.5. Mitochondrial morphology analysis in indirect flight muscle tissue and DA neurons

Mhc-Gal4 driven PINK1 RNAi flies expressing a mitochondrially-targeted mito-GFP reporter were fed with different foods for one week at 29 °C. Fly thorax was collected by dissection in PBS, and indirect fly muscle was examined by live imaging to detect mitoGFP following previously reported procedure (Liu and Lu, 2010), using a Leica TCS SP8 confocal microscope (Leica). To examine the effect of GSE on mitochondrial morphology and DA neuron integrity in PINK1 mutant flies, PINK1^B9 mutant flies expressing a mitochondrially-targeted mito-GFP reporter were fed with different foods for two weeks at 29 °C, and brain samples were processed for whole-mount immunostaining of mito-GFP and the DA neuron marker TH according to previously published procedures (Liu and Lu, 2010; Wu et al., 2013).

2.6. Western blot analysis

MHC-Gal4 > PINK1 RNAi flies were fed with standard fly food or fly food supplemented with GSE powder or resveratrol for one week at 29 °C, and 10 thoraces were isolated for each treatment and homogenized in the tissue lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 10% Glycerol, Protease inhibitor (Roche), pH 7.5). Protein extract mixed with 2 × SDS sample buffer (Biorad) was subjected to SDS-PAGE gel electrophoresis with MOPS running buffer (Invitrogen). Antibodies used in the study were rabbit anti-Ref2P (dP62) (Abcam, ab178440), rabbit anti-ATG8 (Millipore, ABC974), rabbit anti-actin (Sigma-Aldrich, A2066), mouse anti-C-I30 (Abcam, ab14711). Secondary goat anti-rabbit and goat anti-mouse antibodies were from Santa Cruz Biotechnology and goat anti-Rat antibody was from Jackson Laboratory.

2.7. Jumping/flight ability assay

For jumping/flight ability tests, 10 flies were placed in each vial and the jumping/flight events were counted for 2 min while the vial was gently rolled to initiate the jumping/flight events. Results were averaged to represent the jumping ability of 10 individuals. Each analysis was repeated at least 3 times.

2.8. ATP measurement

Measurements of ATP contents in thoracic muscle were performed as described previously (Gehrke et al., 2015), using a luciferase based bioluminescence assay (ATP Bioluminescence Assay Kit HS II, Roche Applied Science). Three thoraces were used for one assay and 3–4 assays were performed for each genotype.

2.9. ROS assay

Fly mitochondria were isolated using the standard method as described previously (Gehrke et al., 2015). ROS levels in fly mitochondria were assayed with the DCFH (Dichlorfluorescein) dye (Wu et al., 2012), which is sensitive to H₂O₂ and its derivative, such as ·OH. Signals were monitored using Fluoroskan Ascent Microplate Fluorometer and Luminometer (Thermo Electron, Corp., USA).

3. Results

3.1. GSE supplementation extended the lifespan of the Drosophila PINK1 PD model

As a first test of the beneficial effect of GSE against the pathogenesis of PD, we measured the lifespan of WT and PINK1 mutant flies raised on standard corn meal fly food or fly food supplemented with GSE powder. As described before (Yang et al., 2006), PINK1 loss of function resulted in a dramatically shortened lifespan (Fig. 1A). GSE significantly extended the lifespan of PINK1 mutant flies in a dosage-dependent manner (4% GSE: median lifespan of 28 days; 8% GSE: median lifespan of 33 days) (Fig. 1B, F). In contrast to PINK1 mutant, WT flies did not show much change of lifespan after intake of GSE (Fig. 1C, F). At high dose (16%) GSE, we did not observe obvious effect on lifespan in either PINK1 mutant flies or WT flies (data not shown). When fed with food supplemented with resveratrol, only very high dose of resveratrol (1 mM) produced a partial extension of lifespan of PINK1 mutant flies compared to GSE (Fig. 1D, F), while no difference was observed in WT flies fed with resveratrol (Fig. 1E, F). No effect on food intake by GSE or resveratrol supplementation, as measured by quantifying the accumulation in fly gut of a blue dye mixed in fly food, was observed (Fig. 2A). These results suggest that GSE positively extends the lifespan of PINK1 mutant, with resveratrol contributing only part of the GSE effect.

Fig. 1.

Effects of GSE and resveratrol on the lifespan of WT and PINK1 mutant flies. (A) Lifespan of WT and PINK1 mutant flies raised on normal fly food (NF) (p < 0.001). (B) Lifespan of PINK1 mutant flies fed with NF or NF supplemented with GSE (2%, n.s: non-significant; 4%, p < 0.001; 8%, p < 0.001). (C) Lifespan of WT flies fed with NF with or without GSE supplementation (2%, n.s.; 4%, n.s.; 8%, n.s.). (D) Lifespan of PINK1 mutant flies fed with NF supplemented with resveratrol (Resv) compared to feeding with NF (High: 1 mM Resv, p < 0.001; Low: 0.2 mM Resv, n.s.). (E) Lifespan of WT flies fed with NF supplemented with Resv compared to feeding with NF only (High: 1 mM Resv, n.s.; Low: 0.2 mM Resv, n.s.). (F) Statistical analysis of the median lifespan of WT and PINK1 mutant flies subjected to the various treatment conditions shown in A-E. * or #, indicates p < 0.05 compared to NF in PINK1 mutant or WT groups.

3.2. GSE rescued the abnormal wing posture of the Drosophila PINK1 model

One of the prominent features of the PINK1 mutant is the observable abnormal wing posture (either held-up or droopy positions) caused by indirect flight muscle degeneration (Fig. 2B). In order to examine whether GSE was able to slow down the progressive muscle degeneration of PINK1 mutant, we treated the newly eclosed PINK1 mutant flies with different concentrations of GSE. After aging for one week at 25 °C, about 50% of the newly born PINK1 mutant flies fed with standard fly food had developed abnormal wing posture, whereas those fed with high dose of GSE (8% and 16%) or high dose of resveratrol (1 mM) showed significantly reduced occurrence of abnormal wing posture (Fig. 2B), suggesting suppression of indirect flight muscle degeneration by GSE.

We also examined the effects of GSE and resveratrol on health-span. We found that moderate to high (8% and 16%) GSE supplementation or high (1 mM) resveratrol supplementation significantly improved the locomotor activity and muscle ATP production in PINK1 mutant flies (Fig. 2C, D). Our measurement of ROS production using DCFH fluorescent probe showed that both GSE and resveratrol significantly reduced ROS production at the high and low concentrations used (Fig. 2E).

3.3. GSE removed aberrant mitochondrial aggregates from PINK1 mutant tissues

PINK1 RNAi flies exhibit mitochondrial abnormality and aggregation in the indirect flight muscles that control wing posture, with mitochondrial abnormality occurring prior to flight muscle degeneration (Yang et al., 2006). In order to directly observe the effect of GSE on mitochondrial pathology, we used the genetically-encoded mitoGFP reporter to visualize mitochondrial morphology in the fly thoracic muscle tissue. PINK1 RNAi flies showed strong mitochondrial aggregation, as reported before (Yang et al., 2006; Liu and Lu, 2010). We found that while low dosage of GSE (2–4%) had no observable effect, moderate to high GSE doses (8% and 16%) were able to effectively remove the mitochondrial aggregation (Fig. 3A, B), indicating an effect of GSE in promoting mitochondrial quality control. In DA neurons, we also observed that moderate to high GSE doses (8% and 16%) were able to effectively remove the mitochondrial aggregates (Fig. 3C), and that 16% GSE was effective in preventing DA neuron loss in PINK1 mutant flies (Fig. 3D).

Fig. 3.

GSE supplementation partially removed the mitochondrial aggregation in the flight muscle of PINK1 RNAi flies. Mitochondria are monitored with a mitochondria-targeted GFP (mito-GFP) marker. The strongly accumulated green signals indicate aggregated mitochondria in flight muscle. Scale bar, 50 μm. (B) Quantification of data shown in A. Mitochondrial aggregates larger than 5 μm and 10 μm were scored in a 200 μm × 200 μm region from 3–4 independent areas. * or #, p < 0.05 compared to NF group when measuring mitochondria > 5 μm or 10 μm diameters. (C, D) Analysis of the effect of GSE supplementation on mitochondrial morphology (C) and neuronal maintenance (D) in DA neurons of PINK1 mutant flies expressing mito-GFP reporter. *, p < 0.05 compared to NF group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. GSE promotes mitophagy in PINK1 loss-of-function flies

Mitophagy has been proposed to play an essential role in mediating PINK1 function in mitochondrial quality control by removing dysfunctional or damaged mitochondria (Durcan and Fon, 2015). We therefore examined the effect of GSE on mitophagy in PINK1 RNAi flies. Drosophila has a single autophagy receptor p62 and a single LC3 homolog ATG8. Reduction of p62 protein level and conversion of LC3-I to LC3-II, a lipidated form of LC3, are indicators of autophagy induction (Tanida et al., 2008). Interestingly, in GSE-treated PINK1 RNAi flies, we found reduced p62 accumulation and increased LC3-II/LC3-I ratio. In addition, we found normalized C-I 30 protein level, indicating restoration of mitochondrial integrity (Fig. 4A). In WT flies treated with GSE, we observed that while LC3-II/LC3-I ratio was not changed, there was dose-dependent increase of p62 and total LC3 expression (Fig. 4A). We assume that in wild type condition, autophagic flux is operating normally and p62 is not rate limiting, such that GSE-induced p62 can accumulate to a higher level. Thus, GSE ingredients may exert multimodal actions in the mitophagy process, including induction of autophagy receptor expression or recruitment, and increase of autophagic influx. The restoration of mitophagy in PINK1 mutant by GSE through enhanced autophagic influx may result in lysosomal degradation of p62, including both p62 accumulated due to mitophagy block in mutant condition and those induced by GSE. However, such activation of mitophagy was not visible in resveratrol treated flies (Fig. 4B). Instead, we saw more accumulation of p62 protein and no change of C-I 30 protein level or LC3-II/LC3-I ratio (Fig. 4B), suggesting that resveratrol behaves differently from GSE with regard to mitophagy induction in PINK1 loss-of-function flies. Although resveratrol treatment has been shown to promote autophagy and lead to reduction in p62 levels (Zhang et al., 2015), these studies were carried out in cells with relatively normal PINK1/Parkin activity. Here we are using PINK1 mutant flies, which are defective in mitophagy. We assume that the accumulation of p62 upon resveratrol treatment observed here reflects the autophagy-inducing effect of resveratrol. However, due to the block of mitophagy by PINK1 mutation, this accumulated p62 was not removed by autophagic flux, resulting in increased steady state p62 level. To corroborate the western blot data, we performed immunostaining of the muscle tissue of GSE-treated PINK1 RNAi flies. We observed reduced p62 (Fig. 4C) and increased LC3/ATG8 (Fig. 4D) signals co-localizing with mitochondria.

Fig. 4.

GSE but not resveratrol supplementation promoted mitophagy in PINK1 loss-of-function flies. (A, B) Western blots of dP62 (Ref2p), LC3 (ATG8) and C-I 30 in WT or PINK1 mutant flies fed with fly food containing GSE (A) or resveratrol (B); Actin as loading control. NF, normal food; Resv, resveratrol. Values under Actin, p62, and C-I30 blots represent protein levels normalized with NF (in blue). Values under LC3 blot represent LC3-I/LC3-II ratio for each sample. (C, D) Immunostainings of Mhc-Gal4 > dPINK1-RNAi; mito-GFP muscle tissue for GFP in green and p62 (A) or ATG8 (B) in red. Merged images are shown on the right. Arrowheads point to colocalized signals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. GSE depends on autophagy activity to rescue PINK1 mutant phenotypes

To further test the notion that GSE exert its protective effects in the fly PD model by promoting mitophagy, we inhibited mitophagy in PINK1 RNAi flies by knocking-down ATG1, the fly homolog of ULK1 that acts as a key regulator of autophagy induction (Scott et al., 2004). We reasoned that if GSE offers protective effects in the PINK1 PD model by promoting autophagy of abnormal mitochondria, then inhibition of the autophagy process should abolish such protective effects. We found that ATG1 RNAi effectively blocked the rescue of the abnormal wing posture by GSE in the PINK1 mutant (Fig. 5A) or PINK1 RNAi background (Fig. 5B). This result further supported the notion that GSE exerts its protective effects in the PINK1 loss-of-function model through the induction of mitophagy.

Fig. 5.

Assessing the roles of ATG1 and mTOR pathways in mediating the effect of GSE in rescuing PINK1 mutant phenotypes. (A) Bar graph showing the percentage of abnormal wing posture in the population of PINK1 mutant flies after ATG1 RNAi. (B) Bar graph showing the percentage of abnormal wing posture in the population of PINK1 RNAi flies after ATG1 RNAi or genetic manipulations of mTORC2 (rictor mutant) or mTORC1 (S6K OE). Flies were raised on fly food containing different concentrations of GSE and aged at 25 °C for 7 days. *, #, p < 0.05 compared to NF in 7 days or 15 days groups; &, p < 0.05 compared to white RNAi control; n.s: non-significant between different groups.

PINK1 has previous been shown to interact with the mTORC1 and mTORC2 pathways (Liu and Lu, 2010; Wu et al., 2013). To test the involvement of TOR pathway in mediating GSE effects, we tried feeding GSE to Mhc-Gal4 > dPINK1 RNAi; UAS-S6K OE and rictorΔ2/Y; Mhc-Gal4 > dPINK1 RNAi flies. Our previous studies showed that overexpression of S6K of the mTORC1 pathway, or inhibition of the mTORC2 pathway enhanced PINK1 mutant phenotypes (Liu and Lu, 2010; Wu et al., 2013). We did not observe rescue of the abnormal wing posture phenotypes by GSE in these flies (Fig. 5B). It is possible that the TOR pathways are required to mediate the GSE effect; alternatively, the phenotypes of the double mutants might be too strong for GSE to rescue. Further studies are required to distinguish these possibilities.

4. Discussion

PD is the most common neurodegenerative disease affecting locomotor behavior and it exhibits a complex etiology. While current medication for PD treatment aims to prevent the breakdown of dopamine or increase dopaminergic neurotransmission, there is still no effective treatment that targets the root cause of neurodegeneration. Many studies report that complementary use of green tea, red wine, arctic root, and dwarf periwinkle may have certain health benefits (Morgan and Grundmann, 2017). Botanical extracts, and some other forms of complementary and alternative approaches are widely used. However, the health benefit claims of these products, and especially their mechanism of actions, remain largely unclear. Our data clearly indicated that GSE offers beneficial effects in an animal model of PD. We further showed that GSE could help maintain mitochondrial function through induction of mitophagy to remove dysfunctional or damaged mitochondria in PINK1 loss-of-function PD flies. While other researchers have reported that grape skin components may protect mitochondria from oxidative damage in Drosophila PD models (Long et al., 2009), our study revealed a new mechanism underlying grape skin’s protective effect.

In our lifespan study, PINK1 mutants showed a much shorter lifespan than WT flies, which is consistent with earlier reports (Yang et al., 2006). GSE extended the median lifespan of PINK1 mutant flies when supplemented at 4% and 8% concentrations to normal fly food. However, no significant extension was observed for wild type flies under the same condition. Somewhat surprisingly, high dose (16%) of GSE exhibited better rescue in the biochemical and pathological assays; however, that was not the case in lifespan assays. High dose (16%) of GSE had no obvious effect on lifespan in PINK1 mutant or wild type flies. We suspect that this might be due to imbalance of nutrients in high dose GSE food. Compared to the high dose, moderate doses (4% and 8%) of GSE performed better in lifespan extension of PINK1 mutant flies. We speculate that lifespan is a very complicated phenotype, which is sensitive to genetic, nutrient, and environmental factors, making the extension of lifespan in disease models a delicate task than restoration of the biochemical and cellular phenotypes. Similarly, high dose of resveratrol supplementation was able to exert lifespan extension only in PINK1 mutant but not wild type flies. These results suggest that the PINK1 mutant is particularly sensitive to GSE supplement. Resveratrol has been reported to extend lifespan in a variety of organisms, including yeast, nematodes, and Drosophila (Wood et al., 2004). However, the effect of resveratrol on the lifespan of wild type flies is controversial, as other studies failed to observe beneficial effects (Bass et al., 2007). Moreover, both red wine and white wine and their non-resveratrol components are capable of activating longevity genes and promoting cell survival (Mukherjee et al., 2009), supporting that resveratrol may not be the only longevity nutrient in grape products.

PINK1 mutant flies show an easily observable abnormal wing posture, which is caused by indirect flight muscle degeneration during and after metamorphosis. Both females and males exhibited either a drooped or a held-up wing posture, whereas WT flies always held their wings parallel to their body axis. The penetrance of this abnormal wing posture phenotype increases with age. In order to maximize the rescue effects of GSE, we let the PINK1 mutant flies lay eggs in fly food with or without GSE powder and aged them in the same food after their eclosion. Under this condition, high doses of GSE supplement (8% and 16%) but not low dose (2%) were able to significantly reduce the penetrance of abnormal wing posture of PINK1 mutant flies, indicating a strong suppression of indirect flight muscle degeneration. These results, taken together with the lifespan data, suggest that GSE possesses protective effects relevant to PD.

Mitochondrial dysfunction has long been implicated in the pathogenesis of PD (Yang and Lu, 2009). Previous studies have demonstrated that PINK1 helps maintain mitochondrial integrity and function in both flight muscle and dopaminergic neurons, cell types with higher demand for energy and thus mitochondrial function, and inactivation of PINK1 results in the accumulation of mitochondria with abnormal morphology (Liu and Lu, 2010; Wu et al., 2013). Under the fluorescence microscope, while wild type flies showed mitochondrial morphology of relatively uniform sizes, PINK1 mutant flies showed severe mitochondrial aggregations. High doses of GSE (8% and 16%) were able to reduce the amount of mitochondrial aggregates, especially the smaller ones, indicating a partial rescue of the mitochondrial aggregation phenotype. The effect on large mitochondrial aggregates was not as dramatic, suggesting that the rescuing-effect was not complete and that longer treatment duration or stronger induction of autophagy might be needed to remove the larger aggregates.

PINK1 and PARKIN have been suggested to activate the mitochondrial autophagy or mitophagy pathway and play critical roles in mitochondrial quality control (Yang and Lu, 2009; Durcan and Fon, 2015). Autophagy is an essential cellular process that maintains homeostasis or normal function by removing aberrant proteins or organelles. Defects in autophagy are frequently associated with cancer, neurodegeneration, and aging (Mizushima et al., 2008). Here we used p62 and LC3 as markers to examine the level of autophagy in both WT and PINK1 loss-of-function flies in response to GSE supplementation. In addition, we used respiratory chain complex-I 30 kD subunit (C-I 30) as a marker to indicate removal of dysfunctional mitochondria and restoration of mitochondrial function, as PINK1 is a regulator of C-I 30 mRNA translation and C-I 30 protein level, and C-I30 protein expression is sensitive to mitochondrial health (Gehrke et al., 2015). Compared to the control, 8% and 16% GSE supplement led to a modest increase of LC3-II/LC3-I ratio in the PINK1 RNAi flies, which indicates enhanced autophagic flux. We also found a reduced p62 accumulation, indicating its removal as an autophagy receptor together with cargoes during active autophagy, consistent with increased autophagic flux by GSE. This data corroborated the reduction of aberrant mitochondria in PINK1 mutant flies fed with GSE. Intriguingly, in the wing posture assay, we observed smaller body size when progenies were raised continuously in 8% or 16% GSE food, which might be caused by altered nutrient composition in such food, or a result of autophagy induction. Interestingly, in wild type animals treated GSE we observed increased p62 expression, although LC3-II/LC3-I ratio was not changed. We assume that in wild type condition, autophagic flux is operating normally and p62 is not rate limiting, such that GSE-induced p62 can accumulate to a higher level. Our data also suggested that the GSE effect is mediated by Rictor/mTORC2 signaling. In our previous studies, we showed that mTORC2 activates a downstream kinase Trc to regulate Parkin recruitment and mitophagy in both Drosophila tissues and mammalian cells (Wu et al., 2013). Moreover, recent studies indicate that mTORC2 signaling can directly impinge on autophagy (Vlahakis and Powers, 2014). Thus, although the exact mechanism of GSE effect on mitophagy remains to be further delineated, our data suggest that GSE ingredients exert multimodal actions in the mitophagy process, including induction of autophagy receptor expression or recruitment, increase of autophagic influx, and possible engagement of mTORC2 pathway.

Taken together, our study suggests that pharmacological manipulations of the mitochondrial pathways, such as enhancing mitophagy by GSE may prove beneficial to combat PD. Unexpectedly, the effect of resveratrol in mitophagy induction is not as evident as GSE. We observed effect of high dose resveratrol on lifespan and health-span in PINK1 mutant flies, as would be expected. However, we only saw mild change of C-I 30 protein levels, or autophagic flux as indicated by p62 level or LC3-II/LC3-I ratio after resveratrol supplementation. Our results suggest that resveratrol’s rescue of lifespan of PINK1 mutant flies may come from its effect on other aspects of mitochondrial function, not necessarily mitophagy. Grape skin is rich in polyphenols, including several bioactive chemical classes, such as quercetin, myricetin, catechins, tannins, anthocyanidins, and ferulic acid, in addition to resveratrol, it is possible that there are non-resveratrol components may exert mitophagy promoting effects. A previous study showed that Amurensin G, a compound from wild grape, could induce autophagy and attenuate cellular toxicities in a rotenone model of PD in human cells (Ryu et al., 2013). The various components in GSE may act together in a multi-pronged manner targeting various pathogenic mechanisms of PD, including mitochondrial dysfunction and oxidative damage, to exert their neuroprotective effects. In fact, Long J et al. has reported that grape extract can protect mitochondria from oxidative damage and improve locomotor dysfunction in a Drosophila α-Synuclein overexpression model of PD (Long et al., 2009), although the underlying mechanism was not clear. In the future, separate tests of individual compounds or combinations would be performed to find the most effective way to promote mitophagy and preserve mitochondrial function.

Many pharmaceutical interventions have focused on diverse molecular pathways suspected to underlie PD initiation and progression (Park and Stacy, 2015). Unfortunately, no effective disease-modifying medicine is yet available. Results from our study support the previous report that grape-derived nutrients may offer beneficial effects in PD. We further showed that GSE might exert its neuroprotective effect through rescue of mitochondrial dysfunction via promoting mitophagy. Thus, the combination of conventional medicine with GSE may offer greater therapeutic potential. However, before testing this in humans, it is necessary to perform further validation studies in models of other subtypes of PD and in other species such as rodents.