Supplementation of Spirulina (Arthrospira platensis

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Journal of Dietary Supplements

ISSN: 1939-0211 (Print) 1939-022X (Online) Journal homepage: http://www.tandfonline.com/loi/ijds20

Supplementation of Spirulina (Arthrospira platensis) Improves Lifespan and Locomotor ∆93

Activity in Paraquat-Sensitive DJ-1β Flies, a Parkinson's Disease Model in Drosophila melanogaster Ajay Kumar MSc, Pearl K. Christian MSc, Komal Panchal MSc, B. R. Guruprasad PhD & Anand K. Tiwari PhD To cite this article: Ajay Kumar MSc, Pearl K. Christian MSc, Komal Panchal MSc, B. R. Guruprasad PhD & Anand K. Tiwari PhD (2017): Supplementation of Spirulina (Arthrospira ∆93

platensis) Improves Lifespan and Locomotor Activity in Paraquat-Sensitive DJ-1β Flies, a Parkinson's Disease Model in Drosophila melanogaster, Journal of Dietary Supplements, DOI: 10.1080/19390211.2016.1275917 To link to this article: http://dx.doi.org/10.1080/19390211.2016.1275917

Published online: 06 Feb 2017.

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Date: 06 February 2017, At: 20:07

JOURNAL OF DIETARY SUPPLEMENTS http://dx.doi.org/./..

ARTICLE

Supplementation of Spirulina (Arthrospira platensis) Improves Lifespan and Locomotor Activity in Paraquat-Sensitive DJ-β Flies, a Parkinson’s Disease Model in Drosophila melanogaster Ajay Kumar, MSca,∗ , Pearl K. Christian, MSca,∗ , Komal Panchal, MSca , B. R. Guruprasad, PhDb , and Anand K. Tiwari, PhDa a

Genetics & Developmental Biology Laboratory, School of Biological Sciences & Biotechnology, Indian Institute of Advanced Research, Gandhinagar, India; b Department of Zoology, Regional Institute of Education (NCERT), Mysore, India

ABSTRACT

KEYWORDS

Spirulina (Arthrospira platensis) is a cyanobacterium (blue-green alga) consumed by humans and other animals because of its nutritional values and pharmacological properties. Apart from high protein contents, it also contains high levels of antioxidant and anti-inflammatory compounds, such as carotenoids, β-carotene, phycocyanin, and phycocyanobilin, indicating its possible pharmaco-therapeutic utility. In the present study using DJ-1β 93 flies, a Parkinson’s disease model in Drosophila, we have demonstrated the therapeutic effect of spirulina and its active component C-phycocyanin (C-PC) in the improvement of lifespan and locomotor behavior. Our findings indicate that dietary supplementation of spirulina significantly improves the lifespan and locomotor activity of paraquat-fed DJ-1β 93 flies. Furthermore, supplementation of spirulina and C-PC individually and independently reduced the cellular stress marked by deregulating the expression of heat shock protein 70 and Jun-N-terminal kinase signaling in DJ-1β 93 flies. A significant decrease in superoxide dismutase and catalase activities in spirulina-fed DJ-1β 93 flies tends to indicate the involvement of antioxidant properties associated with spirulina in the modulation of stress-induced signaling and improvement in lifespan and locomotor activity in Drosophila DJ-1β 93 flies. Our results suggest that antioxidant boosting properties of spirulina can be used as a nutritional supplement for improving the lifespan and locomotor behavior in Parkinson’s disease.

C-phycocyanin; Drosophila melanogaster; paraquat; Parkinson’s disease; reactive oxygen species; spirulina

Introduction Spirulina (Arthrospira platensis) is a cynobacterium commonly used as a food source since ancient times (Karkos et al., 2011). In recent years, it has been popularized as a “wonder food” due to its highly rich protein contents, presence of beta-carotene, unsaturated fatty acids, vitamin B, and phycobiliprotein, such as C-phycocyanin (C-PC) and phycocyanobilin (Liu et al., 2016; Seo et al., 2013; Riss et al., 2007). C-phycocyanin is one of the key pigments CONTACT Anand K. Tiwari [email protected] Genetics & Developmental Biology Laboratory, School of Biological Sciences & Biotechnology, Indian Institute of Advanced Research/UIAR, Koba Institutional Area, Gandhinagar-, Gujarat, India. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ijds. ∗ Contributed equally. ©  Taylor & Francis Group, LLC

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available in spirulina (0.21 mg/mL), has great antioxidant property, and is commonly used by humans as a nutrient supplement, and in pharmaceuticals (Moraes et al., 2011; Sivasankari, 2014). In addition, spirulina also possesses other nutrients, i.e., iron, manganese, zinc, copper, selenium, and chromium (Deng & Chow, 2010; Moorhead et al., 1993). These nutrients help to fight against free radicals, cell-damaging molecules absorbed by the body through pollution, poor diet, injury, or stress, and also improve the immune system to fight against cancer and cellular degeneration (Hwang et al., 2011; Karkos et al., 2011; Angélica et al., 2012). Several studies have suggested the therapeutic effects of spirulina because supplementation of spirulina prevents certain inflammatory diseases, allergies, cancers, viral infections, cardiovascular disease, diabetes, and other metabolic disorders (Barakat et al., 2015). It has been demonstrated that reactive oxygen species (ROS) and oxidative stress play a causal role in the progression of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Trushina & McMurray, 2007). Although there is no permanent cure, drugs and proper care can slow the progression of these diseases. Many herbs have been reported to improve the brain function but details are still missing as to whether these can be used for neurodegenerative diseases such as AD and PD. It is hypothesized that diets enriched in antioxidants and anti-inflammatory factors may modulate neurodegeneration (Havsteen, 2002). Owing to the bioethical limitations of carrying out studies in humans, a model organism, such as fruit fly Drosophila melanogaster, has gained importance in the search for therapeutic targets and the identification of medicinal herbs/plants to prolong/treatment of these diseases (Munoz-Soriano & Paricio, 2011; Bilen & Bonini, 2005; Marsh & Thompson., 2006). The fruit fly Drosophila melanogaster is an invertebrate model organism with ∼75% homology of disease-causing genes in humans, and has similarity in many basic biological, physiological, and neurological processes as those in mammals (Pandey & Nichols, 2011). Owing to ease in handling, less genome complexity, amenable genetics, short life cycle, and high fecundity, behavior analysis available make the fly a popular model organism for the study of neurodegenerative diseases such as AD and PD. In recent years, it has been used as a highthroughput animal model for drug screening (Akasaka & Ocorr, 2009) and for the screening of therapeutic activities of several medicinal plants and their extracts (Kim SI et al., 2011). The effect of plant/plant extract/chemicals/drug can be easily examined by feeding Drosophila larvae/adult flies on fly food mixed with desired components, and its effect can be examined by observing the physiological changes during development such as lifespan, fecundity, fertility, locomotion, vision, and enzymatic/biochemical assay (Kumar et al., 2013; Tiwari et al., 2011). Thus, in the present study, we utilized DJ-1β 93 flies, a PD model in Drosophila, to explore the effect of spirulina supplementation on the lifespan and locomotor behavior in Drosophila. DJ-1 is a highly conserved protein belonging to the protein super family, including DJ-1, ThiJ, and PfpI (Kahle et al., 2009), and plays an important role in protection from oxidative stress induced by paraquat (PQ) or H2 O2 (Bonifati et al., 2003; Mitsumoto & Nakagawa, 2001). Drosophila possesses two homologs of human DJ-1: DJ-1α and DJ-1β. DJ-1α is predominantly expressed in the testis and in the adult head (Aradska et al., 2015; Menzies et al., 2005), while the DJ-1β is ubiquitously found in most tissues, resembling the pattern of human DJ-1 (Meulener et al., 2005). It has been shown that loss of DJ-1 function in Drosophila makes flies highly sensitive to PQ-induced oxidative stress, showing reduced lifespan and motor deficits (Lavara-Culebras & Paricio, 2007; Ortega-Arellano, et al. 2011). Thus, the present study was designed with the aim to study the effect of spirulina (Arthrospira platensis) and its active component C-phycocyanin in DJ-1β 93 flies in Drosophila melanogaster.

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Materials and methods Fly strains and culture conditions Oregon R+ strain was used as a wild-type strain of Drosophila and DJ-1β 93 (Bloomington stock No. #33601), a transgenic model of PD in Drosophila used for all experimental studies carried out in this paper. These flies are highly sensitive to any kind of chemically-induced oxidative stress. The flies were maintained at 22 ± 1°C in bottles containing agar–agar (SRL, 0140186, India), corn meal, sugar, dried yeast, and water. Nepagin (HIMEDIA, RM 1291, India) and propionic acid (HIMEDIA, RM 3658, India) were added as antifungal and antibacterial agents, respectively. Preparation of spirulina (Arthrospira platensis)-mixed food The spirulina powder was obtained from Krishco Gramin Vikas Sanstha (KGVS, Udaipur, Rajasthan, India). Spirulina-mixed food was prepared by dissolving dried spirulina (Arthrospira platensis) powder (5% and 10% w/v) in Drosophila food media and poured into fly culture vials/bottles, and allowed to dry till further use. Survival assay This was done according to the method described in Nazir et al. (2001) with slight modification. The survivals of adult flies were studied from the day of eclosion. For this, flies were transferred to fresh vials every alternate day and the number of dead flies was recorded until the last fly was dead. The median survival of each group was determined by the Kaplan–Meier survival test using GraphPad PRISM 5.01 . We put “1” for death event and “0” as censored in survival table for each subject during each observation. For each set, 50 flies were taken and divided into 10 flies per vial.

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Locomotor assay This was performed as described previously (Chaudhuri et al. 2007; Feany & Bender. 2000) with slight modifications. Ten flies were placed in a vertical glass tube (30 × 1.5 cm). After 10 min of resting period, the flies were tapped to the bottom of the tube and the number of flies crossing 15 cm/10 s was recorded. Three readings at an interval of 1 min were taken and the mean score of three trials was considered. Polymerase chain reaction Total RNA isolated from 20 flies was homogenized using TRIzol reagent (Ambion, Austin, TX, USA) following the manufacturer’s instructions. The tissues were used for cDNA synthesis using Revert AidTM H Minus first strand cDNA synthesis kit (Fermentas, MD, USA) following the manufacturer’s protocol. Each reaction mixture consisted of total RNA (3 μg), 0.5 μg/μ Loligo (dT)18 primer (1 μL), 5X reaction buffer (4 μL), 20 U Ribolock TM ribonuclease inhibitor, 10 mmol/L deoxyribonucleotide triphosphate mixture (2 μL), 200 U Revert AidTM H Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT), and nuclease-free water to make the final volume of 20 μL. The synthesized cDNA (100 ng/μL)

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Table . Primer sequences. Gene hsp rp

Primer sequence F R F R

 -GAACGGGCCAAGCGCACACTCTC-  -TCCTGGATCTTGCCGCTCTGGTCT-  -AATCTCCTTGCGCTTCTT-  -AGTATCTGATGCCCAACATC-

was used for polymerized chain reaction (PCR) amplification in a thermo cycler (S1000TM Thermal cycler, Bio-Rad, CA, USA) using specific primer for heat shock protein (Hsp) 70 gene (Table 1). The amplicons were separated on 2% agarose gel containing ethidium bromide at 5 V/cm and visualized under fusion gel doc imaging system model SL 3500 x-Press (VilberLourmat, France). Ribosomal protein 49 (rp49)-specific primers were used as internal control. Enzymatic assay This was done to examine the effect of spirulina supplementation on antioxidant enzymatic activity (superoxide dismutase (SOD) and catalase (CAT)) in DJ-1β 93 flies. Preparation of protein sample The protein samples were prepared by homogenizing adult flies fed with normal diet and diet mixed with 5% and 10% spirulina in 100 mM potassium phosphate buffer (KPB). The sample was centrifuged at 8,000 rpm at 4°C for 20 min. The supernatants were transferred into 1.5-mL Eppendorf tube, and optical density (OD) was measured at 280 nm by using spectrophotometer (Eppendorf, Germany). Crude protein samples were further diluted (1:10) for SOD and CAT enzymatic activity test, and were stored at −20°C till further use. Superoxide Dismutase (SOD) enzymatic assay The SOD assay was performed as described in Rukmini et al. (2004) with slight modification. The reaction mixture contained 20 μL of 250 mM methionine (SRL, Cat #19305, India), 5 μL of 10 mM Nitro Blue Tetrazolium (NBT; SRL, Cat #11207), 25 μL of 1 M KPB, 0.5 μL of 100 mM EDTA (SRL, Cat #54960, India), 25 μL of 1 M Na2 CO3 , and 380 μL of distilled water with 40.5 μL of desired diluted protein samples. Riboflavin, 1 mM, 2 μL (SRL, Cat #34392, India) was added in the last and the total volume of the reaction mixture was 500 μL. It was kept under white light for 8 min. The purple color was formed and OD was measured at 560 nm using spectrophotometer (Eppendorf, Germany). Catalase (CAT) assay Catalase enzymatic (EC 1.11.1.6) activity was measured according to the method described in Rukmini et al. (2004) with little modification. The assay is based on the consumption of H2 O2 in the presence of enzymatic source at 25°C. Here, the reaction mixture contained 30% H2 O2 , 50 mM KPB, and 34 μL of diluted protein samples from flies fed with normal diet and 5 and 10% spirulina-mixed diet. The OD (240 nm) of the reaction mixture was taken immediately at 0 min and after 5 min by using spectrophotometer. One unit of enzyme is required to convert 1 mol of H2 O2 to the product in 1 s. Enzymatic activity was expressed as units per milligram of protein.

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Immunostaining in adult fly brain Immunostaining in adult brain of Drosophila was performed by dissecting the brain tissue in 1x PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBT (1x PBS, 0.1% Triton X-100) thrice for 10 min. The tissues were blocked in blocking solution (1x PBS, 0.1% Triton X-100, 0.1% bovine serum albumin, 10% fetal calf serum, and 0.02% thiomersal as antifungal agent) for 2 h at room temperature followed by overnight incubation in primary antibody at 4°C. The tissues were washed twice in PBT and incubated in secondary antibody for 2 h at room temperature, washed thrice in PBT, and mounted in DABCO (antifade, Sigma, USA). The primary antibodies used were mouse anti-Hsp70 (1:200, Thermo Scientific, USA) and rabbit anti-phospho-Jun-N-terminal kinase (JNK; 1:500; Promega, USA). Secondary antibodies used were Alexa Fluor 488 conjugated with anti-mouse IgG (1:150, Molecular Probes, Oregon, USA) and anti-rabbit IgG conjugated with Cy3 (1:100, Sigma, USA). Scanning of fluorescent images was done using Leica TCS SP5II laser scanning confocal microscope (Leica Microsystems, Germany), and images were assembled using Adobe Photoshop 7.0 .

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Statistical analyses All data were expressed as the mean values ± SE. Difference between different groups was evaluated with one-way ANOVA (SOD/CAT activity) followed by Tukey’s comparison of all pairs of columns, two-way ANOVA (for locomotor assay), and the Kaplan–Meier test (for survival). The results were considered statistically significant when p < 0.05). The test was performed using the statistical software Graph Pad Prism 5.01 .

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Results To evaluate the effect of dietary supplementation of spirulina and its active component Cphycocyanin on DJ-1β 93 flies, survival assay, locomotor assay, endogenous antioxidant enzymatic activity (SOD/CAT), Hsp70 expression, and JNK signaling were examined. We found the following results. Supplementation of spirulina (5 and 10% w/v) increased lifespan in paraquat -fed DJ-1β93 flies Reduced lifespan is a key feature associated with ectopic induction of oxidative stress in flies as well as in humans (Rascon & Harrison, 2010; Wright et al., 2004). It has been shown that PQ, an herbicide, induces oxidative stress by altering the endogenous antioxidant defense mechanism, including SOD, CAT, and glutathione peroxidase (Gemma et al., 2007; Poon et al., 2004). Thus, to study the effect of spirulina supplementation on DJ-1β 93 flies, we fed DJ-1β 93 flies with 10, 20, and 30 mM PQ and also with PQ and spirulina-mixed diet (5 and 10%). A group of DJ-1β 93 flies were also fed with normal diet as an experimental control. It was observed that DJ-1β 93 flies fed with PQ-mixed diet showed progressively reduced survival in a dose-dependent manner (10 > 20 > 30 mM), while DJ-1β 93 flies fed with spirulina and PQ-mixed diet showed significant increase in their lifespan as compared with the flies fed with PQ alone (Figure 1). The maximum lifespan of DJ-1β 93 flies fed with control food was 68 days; life span was 19 days when fed with 10 mM PQ, 12 days when fed with 20 mM

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Figure . Effect of spirulina on survival of DJ-β  flies. Survival curves of DJ-β  flies fed with standard Drosophila diet + % sucrose (control) and Drosophila diet mixed with , , and  mM PQ alone and in combination with % and % spirulina. A total of  flies from each group were used for the survival assay. The median survival of each group was determined by the Kaplan–Meier survival test; p < ..

PQ, and 7 days when fed with 30 mM PQ. The lifespan of DJ-1β 93 flies fed with 10 mM PQ + 5% spirulina was increased to 25 days; life span increased up to 43 days when fed with 10 mM PQ + 10% spirulina, increased up to 18 days with 20 mM PQ + 5% spirulina, increased up to 34 days with 10% spirulina, up to 11 days with 30 mM PQ + 5% spirulina, and up to 25 days with 20 mM PQ + 10% spirulina. Thus, our observation suggested a significant increase in the lifespan of DJ-1β 93 flies fed with 5 and 10% spirulina-mixed diet. Supplementation of spirulina improved locomotor activity and reduced antioxidant enzymatic activity in DJ-1β93 flies Locomotor impairment is one of the key features of PD (Van Kampen et al., 2015). Thus, to study the effect of spirulina supplementation in Oregon R+ (wild-type control) and DJ-1β 93 flies, locomotor assays were performed in male and female flies from both the groups fed with 1% sucrose (suc; control), 1% sucrose + PQ (10 and 20 mM) and 1% sucrose + PQ (10 and 20 mM) + 5 and 10% spirulina-mixed diet. Locomotor assay in Oregon R+ flies showed that ∼92.75% male flies fed with 1% sucrose (control diet) travels 15 cm/10 s, while the number was reduced by 69.9 and 53.5% when flies were fed with 10 and 20 mM PQ, respectively. In the combination of 5 and 10% spirulina, the locomotor activity increased by 74.2% (fed with 10 mM PQ + 5% spirulina), 85.4% (fed with 10 mM PQ + 10% spirulina), 68.6% (fed with 20 mM PQ + 5% spirulina, and 79% (fed with 20 mM + 10% spirulina; Figure 2A). Similarly ∼92.75% Oregon R+ female flies fed with 1% sucrose diet traveled 15 cm/10 s, and this percentage of flies was reduced by 60.45% and 40% when flies were fed with 10 mM and 20 mM PQ, respectively. While the locomotor activity of Oregon R+ female flies, fed with PQ-mixed diet in combination with 5 and 10% spirulina, was increased by ∼64.18% (fed with 10 mM PQ + 5% spirulina), 72.33% (fed with 10 mM PQ + 10% spirulina), 55.49% (fed with 20 mM PQ + 5% spirulina), and 65.61% (fed with 20 mM PQ + 10% spirulina; Figure 2A). Thus, the data suggest a significant improvement in the locomotor behavior of Oregon R+ flies fed with spirulina-mixed food. In the case of DJ-1β 93 flies, ∼84% of male flies fed with 1% sucrose traveled 15 cm/10 s, while this number was reduced to 66.01% and 50.33% when flies were fed with 10 and 20 mM PQ, respectively. In combination of 5 and 10% spirulina, the locomotor activity increased to 72.21% (fed with 10 mM PQ + 5% spirulina), 80.35% (fed with 10 mM PQ + 10% spirulina), 61.55% (fed with 20 mM + 5% spirulina), and 76.52% (fed with 20 mM PQ + 10% spirulina; Figure 2B). Similarly, ∼81% control (1% sucrose-fed) DJ-1β 93 female flies traveled 15 cm/10 s, and this number was reduced to 44.48% and 30.85% in flies fed with 10 and 20 mM PQ, respectively. When DJ-1β 93 flies were fed with PQ-mixed diet in combination of 5

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Figure . Locomotor assay in Oregon R+ and DJ-β  flies fed with control, PQ, and PQ + spirulina-mixed diet. (A) Graph showing the percentage locomotor activity in Oregon R+ flies (males and females) fed with Drosophila diet + % sucrose (control), diet mixed with  and  mM PQ separately, and PQ in combination with % and % spirulina, respectively. (B) The percentage locomotor activity in DJ-β  flies (males and females) fed with control diet, diet mixed with  and  mM PQ separately, and PQ in combination of % and % spirulina, respectively. A total of  flies were used for locomotor assay. For (A) and (B), p < . by two-way ANOVA.

and 10% spirulina, the locomotor activity increased by 59.31% (fed with 10 mM PQ + 5% spirulina), 62.43% (fed with 10 mM PQ + 10% spirulina), 42.57% (fed with 20 mM PQ + 5% spirulina), and 51.50% (fed with 20 mM PQ + 10% spirulina; Figure 2B). Thus, the data suggest a significant improvement in the locomotor behavior of DJ-1β 93 flies fed with spirulina-mixed food. A total of 200 flies from each group were used for locomotor assay. As discussed above, supplementation of flies with PQ induces oxidative stress and causes locomotor impairment and cellular stress. Thus, in order to find out whether dietary supplementation with spirulina induces antioxidant defense system, reduces oxidative stress by reducing the activity of free radicals, SOD and CAT enzymatic activity were measured in DJ1β 93 flies fed with 1% sucrose (control), 1% sucrose + 20 mM PQ, and 1% sucrose + 20 mM PQ in combination with 5% and 10% spirulina, respectively. DJ-1β (93) flies fed with 1% sucrose + 20 mM PQ showed an induction of SOD and CAT activity, while SOD/CAT activity was significantly down-regulated when the flies were fed with PQ in combination with 5% or 10% spirulina-mixed diet (Figures 3A and B). These results clearly suggests that spirulina possesses antioxidant activity and it reduces PQ-induced oxidative stress in DJ-1β 93 flies. Supplementation of spirulina (5 and 10%) reduced cellular stress by lowering Hsp70 and JNK signaling in Oregon R+ and DJ-1β93 flies Heat shock proteins 70 are highly conserved proteins that play a key role in cellular defense under stressful conditions. It has been demonstrated that Hsp70 plays a cytoprotective role, and by examining its expression level, one can know the level of cellular stress (Gupta et al., 2005). Thus, to ascertain that rescue obtained by spirulina supplementation was due to reduction in cellular stress, Hsp70 expression level was examined by carrying out RT-PCR for Hsp70 gene. One group of newly enclosed flies were fed with 1% sucrose + PQ (10, 20, and 30 mM) alone, and other groups were fed with 1% sucrose + PQ in combination with 5%

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Figure . Antioxidant enzymes (SOD/CAT) activities in DJ-β  flies. (A) SOD enzymatic assay in DJ-β  flies fed with Drosophila diet + % sucrose (control), % sucrose +  mM PQ, and % sucrose +  mM PQ in combination with % and % spirulina. Error bars represent mean ± SEM (p < .). (B) CAT enzymatic assay in DJ-β  flies fed with % sucrose, % sucrose +  mM PQ, and % sucrose +  mM PQ in combination with  and % spirulina. Error bars represent mean ± SEM (p > .). The antioxidant enzymes assays were performed in triplicate.

and 10% spirulina for 16 h; flies were immediately examined by carrying out PCR. It was determined that flies fed with PQ showed an increase in Hsp70 expression, representing an increase in cellular stress, while showing a significant reduction in Hsp70 expression when flies were fed with PQ + spirulina (5 or 10%)-mixed diet (Figure 4A). Since C-phycocyanin is a known active component in spirulina, we also performed the RT-PCR analysis in flies fed with

Figure . RT-PCR analysis in DJ-β  flies fed with spirulina and C-phycocyanin-mixed diet. (A) Gel images showing the expression of Hsp in DJ-β  flies fed with (left to right) Drosophila diet + % sucrose (control), % sucrose +  mM PQ, % sucrose + // mM PQ in combination with  and % spirulina. (B) Gel images showing the expression of Hsp in DJ-β  male and female flies fed with % sucrose (control), % sucrose +  mM PQ and % sucrose +  mM PQ in combination with  μg/mL C-phycocyanin. rp was used as a loading control.

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C-phycocyanin (1 μg/mL) and C-phycocyanin (1 μg/mL) + PQ (10, 20, and 30 mM)-mixed diet. It was observed that flies fed with C-phycocyanin (1 μg/mL)-mixed diet showed considerable decrease in Hsp70 expression level as compared with the DJ-1β 93 flies fed with 1% sucrose or 1% sucrose + PQ (10, 20, and 30 mM; Figure 4B). To further confirm above observations, we performed immunostaining in the brains of Oregon R+ and DJ-1β 93 adult flies by using anti-Hsp70 antibody. Anti-Hsp70 staining in Oregon R+ flies fed with 1% sucrose + 20 mM PQ showed an induction of Hsp70 protein level in medulla (me), lateral deutocerebrum (l deu), and outer medulla neuropils (me o) regions of adult brain (Figure 5B), while the Hsp70 expression level was significantly reduced in adult brains of Oregon R+ flies fed with 1% sucrose + 20 mM PQ along with 5% (Figure 5C) and 10% spirulina-mixed diet (Figure 5D). Similarly, Oregon R+ flies fed with 1% sucrose + 20 mM PQ with 1 and 2 μg/mL C-phycocyanin-mixed diet showed a significant reduction in Hsp70 expression in adult brains (Figures 5E and F, respectively). Anti-Hsp70 staining in DJ-1β 93 flies fed with 1% sucrose + 20 mM PQ showed a strong elevation in Hsp70 protein in adult brain tissues (Figure 5H), while Hsp70 protein level was significantly reduced in DJ-1β 93 flies fed with 1% sucrose + 20 mM PQ with 5% and 10% spirulina-mixed diet (Figures 5I and J, respectively). Further, DJ-1β 93 flies fed with 1% sucrose + 20 mM PQ with 1 μg/mL or 2 μg/mL C-phycocyanin-mixed diet showed a significant reduction in Hsp70 expression (Figures 5K and L, respectively), suggesting reduction in cellular stress. Since JNK signaling is well known in the context of stress (Wang et al., 2003; Wu et al., 2009; Tiwari & Roy, 2009), and as discussed above that exposure of flies to PQ induces oxidative/cellular stress, we also examined the expression level of phosphorylated-JNK (activated JNK) in the adult brain of Oregon R+ and DJ-1β 93 flies fed with PQ and PQ with 1 μg/mL and 2 μg/mL C-phycocyanin (Figure 6). It was determined that Oregon R+ flies fed with 1% sucrose + 20 mM PQ showed an elevation in P-JNK signal in adult brain (Figure 6B), while this expression was significantly reduced when the Oregon R+ flies were fed with 1% sucrose + 20 mM PQ with 5% and 10% spirulina (Figures 6C and D, respectively) and in combination of 1 and 2 μg/mL C-phycocyanin (Figures 6E and F, respectively). In DJ-1β 93 flies, a strong induction in P-JNK was determined (Figure 6H) when fed with 1% sucrose + 20 mM PQ, while the P-JNK expression was significantly reduced when DJ1β 93 flies were fed with 1% sucrose +20 mM PQ + 1 μg/mL C-phycocyanin (Figures 6I and D, respectively) and 1% sucrose + 20 mM PQ + 2 μg/mL C-phycocyanin (Figure 6J). A strong expression seen in 1% sucrose + 20 mM PQ-fed flies was due to DJ-1β 93 mutation in flies. This result clearly suggested that supplementation of spirulina and C-phycocyanin reduces cellular stress by reducing Hsp70 and JNK levels in Drosophila.

Discussion In the present study, using DJ-1β 93 , a fly model of PD in Drosophila, we showed the therapeutic potential associated with spirulina and its active component C-phycocyanin. DJ-1β 93 is a homolog of human PARK7 (Menzies et al., 2005) and its mutation causes increased risk of PD-like symptoms when exposed to an ROS generator (i.e., paraquat) in vitro. Several studies had suggested that free radicals (free radical hypothesis) play a critical role in aging, and increasing the antioxidant level exogenously (diet) maintained oxidative balance, defense against oxidative damage, and improved lifespan in different model organisms (Muller et al., 2007; Perez et al., 2009; Sadowska-Bartosz & Bartosz, 2014). Drosophila melanogaster has been extensively used as a model organism in the search for life extension

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Figure . Anti-Hsp staining in Oregon R+ and DJ-β  adult fly brains. (A)–(F) Anti-Hsp staining in Oregon R+ flies fed with % sucrose (A), % sucrose +  mM PQ (B), % sucrose +  mM PQ + % spirulina (C), % sucrose +  mM PQ + % spirulina (D) % sucrose +  mM PQ +  μg/mL C-phycocyanin (E), and % sucrose +  mM PQ +  μg/mL C-phycocyanin (F). (G)–(L) Anti-Hsp staining in DJ-β  flies adult brain fed with % sucrose (G), % sucrose +  mM PQ (H), % sucrose +  mM PQ + % spirulina (I), % sucrose +  mM PQ + % spirulina (J), % sucrose +  mM PQ +  μg/mL C-phycocyanin (K), and % sucrose +  mM PQ +  μg/mL C-phycocyanin (L). Scale bars represent  μm (A)–(L). me: medulla; l deu: lateral deutocerebrum; me o: outer medulla neuropils.

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Figure . Anti-phospho JNK staining in Oregon R+ and DJ-β  adult fly brains. (A)–(F) Images showing anti-phospho JNK (activated JNK) staining in adult brains of Oregon R+ flies fed with % sucrose (A), % sucrose +  mM PQ (B), % sucrose +  mM PQ + % spirulina (C), % sucrose +  mM PQ + % spirulina (D), % sucrose +  mM PQ +  μg/mL C-phycocyanin (E), and % sucrose +  mM PQ +  μg/mL C-phycocyanin (F). (G)–(J) Anti-phospho JNK staining in DJ-β  flies fed with % sucrose (G), % sucrose +  mM PQ (H), % sucrose +  mM PQ +  μg/mL C-phycocyanin (I), and % sucrose +  mM PQ +  μg/mL C-phycocyanin (J). Scale bars represent  μm (A)–(J). me: medulla; l deu: lateral deutocerebrum; me o: outer medulla neuropils.

property and health benefits of several medicinal plants, and it was demonstrated that supplementation of vitamin E, N-acetyl cysteine, wheatgrass, citric acid, curcumin, nectarine, blueberry extract, and grape extract increased lifespan in Drosophila melanogaster (Boyd et al., 2011; Long et al., 2009; Lee et al., 2010; Pant et al., 2013; Panchal et al., 2016; Chandrashekara et al., 2014).

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As discussed above, spirulina contains high levels of active compounds such as C-phycocyanin, phycocyanobilin, and beta carotene, and vitamins that can improve the antioxidant defense system of an individual and increase the lifespan and health span. The lifespan analysis of DJ-1β 93 flies fed with PQ-mixed diet showed reduced survival, while the lifespan was significantly improved when the flies were fed with 5% and 10% spirulina-mixed diet. The increase in lifespan of DJ-1β 93 might be due to the antiaging and antioxidant properties, or the effect of spirulina or its active components on the gene or signaling pathway modulating the lifespan of an organism. Several studies had suggested that dietary intake of food/food supplements rich in antioxidant played a key role in extending the lifespan of an organism (Carey et al., 2008; Lee et al., 2008; Skorupa et al., 2008), thus the increase in lifespan seen in spirulina-fed flies might be due to the antioxidant boosting property associated with spirulina. Further, SOD/CAT enzymatic assay in spirulina-fed flies suggested the antioxidant potential associated with spirulina. Our study has also demonstrated a significant reduction in Hsp70 and JNK expression in spirulina- (5 or 10%) and C-phycocyanin (1 or 2 μg/mL)-fed flies. As discussed above, Hsp70 expression can be used as the first tier indicator of cellular stress and JNK signaling, a conserved signaling pathway known to modulate lifespan in the different model organisms such as yeast, C.elegans, flies, and rodents (Fontana et al., 2010; Kenyon, 2010). Thus, a reduction in Hsp70 and JNK expressions in spirulina- and C-phycocyanin-fed flies suggested a reduction in cellular stress. The increase in lifespan and improvement in locomotor activity of DJ-1β 93 flies fed with spirulina/C-phycocyanin-mixed diet was associated with improved antioxidant defense system and reduction in cellular stress due to the reduction in Hsp70 and JNK signaling in flies (Figures 5–7). In summary, the present study demonstrated that dietary supplementation of spirulina/Cphycocyanin increases lifespan and health benefits in Drosophila melanogaster by boosting antioxidant defense system and lowering the Hsp70 and JNK signaling in Drosophila.

Figure . Schematic diagram showing the possible action of spirulina/C-phycocyanin supplementation in DJ-β  flies.

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Acknowledgments We are grateful to Professor J. K. Roy, Banaras Hindu University, Varanasi, India, for Oregon R+ and DJ-1β 93 fly stocks, and Dr. M. L. Changwal, Udaipur, Rajasthan, India, for spirulina (Arthrospira platensis) powder. The laser scanning confocal microscope facility provided by the Department of Biotechnology (DBT), New Delhi is also acknowledged.

Declaration of interest The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Funding The work was partly supported by grants from the Department of Biotechnology (DBT) for Programme Support Grant and the Puri Foundation for Education in India awarded to Anand K. Tiwari.

About the authors Ajay Kumar, MSc, is interested in the study of Drosophila eye development and neurodegerative disease (AD & PD) mechanism using Drosophila model system. Pearl K. Christian, MSc, is interested in human genetics. Komal Panchal, MSc, is interested in the study of axonal transport defect during neurodegenerative diseases using Drosophila model system. B. R. Guruprasad, PhD, is interested in finding the medicinal properties of plants using Drosophila melanogaster as a model organism. Anand K. Tiwari, PhD, is group leader of Genetics and Developmental Biology Laboratory, Indian Institute of Advanced Research (IIAR), Gandhinagar, interested in the study of Drosophila eye development, neurodegerative disease (AD & PD) mechanism using Drosophila model system, and identification of therapeutic potentials of plant-derived components using AD & PD model in Drosophila.

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