The neuroprotective properties of a novel variety of passion fruit

Journal of Functional Foods 23 (2016) 359–369

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The neuroprotective properties of a novel variety of passion fruit Yael Tal a, Sarit Anavi a, Merav Reisman a, Alon Samach b, Oren Tirosh a, Aron M. Troen a,* a

Institute of Biochemistry Food and Nutrition Science, The Robert H. Smith Faculty of Agriculture Food & Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel b Institute of Plant Sciences and Genetics, The Robert H. Smith Faculty of Agriculture Food & Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel

A R T I C L E

I N F O

A B S T R A C T

Article history:

Passion fruit is a commercially important crop. The neuroprotective activity of fruit ex-

Received 17 November 2015

tracts from two hybrid lines of antioxidant ester thiol-rich Passiflora edulis Sims, the commercial

Received in revised form 20

“Passion Dream” and novel cultivar 428 (“Dena”) line were studied. Crude extracts from line

February 2016

428 displayed the strongest dose-dependent neuroprotective activity, preventing gluta-

Accepted 22 February 2016

mate induced cell-death, mitochondrial depolarization and glutathione depletion, when added

Available online

to the medium of cultured HT4 neurons (p < 0.05). Supplementing diet of mice with the 428 fruit-extract improved survival of dopaminergic neurons by 60% in mice injected with the

Keywords:

neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MTPT) compared to control-fed

Passiflora

MPTP-injected mice (p < 0.05). The neuroprotection conferred by passion fruit extracts in vivo

Neuroprotection

and in vitro shows promise for further research into their bioactive potential for medical

MPTP

exploitation.

Glutamate-toxicity

© 2016 Elsevier Ltd. All rights reserved.

Antioxidant

1.

Introduction

Neurodegenerative diseases, notably Alzheimer’s Disease and Parkinson’s Disease whose main risk-factor is ageing are increasingly prevalent (Joseph, Cole, Head, & Ingram, 2009; Mandel, Amit, Weinreb, & Youdim, 2011) due to increased life expectancy around the world. Symptomatic treatments and

disease-modifying interventions that aim to target the specific hallmark pathologies in each disease (e.g. the plaques and tangles in Alzheimer’s Disease or Lewy bodies in Parkinson’s Disease) do not address the early age-related processes which initiate the disease and put the brain at risk. New preventive therapies are needed because the downstream neurodegenerative pathology is often irreversible by the time the clinical symptoms of the disease appears. Here, we present our

Chemical compounds: 3-Mercaptohexyl acetate (PubChem CID: 518810); 3-Mercaptohexyl butyrate (PubChem CID: 537754); 3-Mercaptohexyl hexanoate (PubChem CID: 518810). * Corresponding author. The Nutrition and Brain Health Laboratory, Institute of Biochemistry Food and Nutrition Science, The Robert H. Smith Faculty of Agriculture Food & Environment, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot, 76100, Israel. Tel.: +972 8 9489476; fax: +972 9489822. E-mail address: [email protected] (A.M. Troen). Abbreviations: 428, “Dena” line of passion fruit; CMFDA, chloromethylfluorescein diacetate; H2DCF, 2’,7’-dichlorodihydrofluorescein diacetate; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Passion Dream; PF, passion fruit; PI, propidium iodide; ROS, reactive oxygen species; SOD, superoxide dismutase; TH, tyrosine hydroxylase http://dx.doi.org/10.1016/j.jff.2016.02.039 1756-4646/© 2016 Elsevier Ltd. All rights reserved.

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Journal of Functional Foods 23 (2016) 359–369

findings on a new variety of passion fruit that may serve this role. A substantial body of evidence indicates that in these diseases, neurodegeneration is accelerated by mitochondrial dysfunction and oxidative stress (Emerit, Edeas, & Bricaire, 2004; Rao & Balachandran, 2002) and that under conditions of heightened oxidative stress, reactive oxygen species (ROS) can induce cellular dysfunction and even death. The brain is generally susceptible to such oxidative damage due to its high metabolic activity in a lipid rich environment with relatively low levels of endogenous antioxidant defenses. However, certain neuronal populations may be particularly vulnerable due to their specific metabolic and neurochemical profiles. For example, an excess of the neurotransmitter dopamine can generate cytotoxic ROS due to dopamine’s normal metabolism by monoamine oxidase (MAO) or through autoxidation. This phenomenon has been linked to the vulnerability of dopaminergic neurons in the substantia nigra in Parkinson’s Disease (Blesa, Trigo-Damas, Quiroga-Varela, & Jackson-Lewis, 2015; Zucca et al., 2014). Pathologic conditions can also elicit ROS production and excitotoxicity as a result of hyper-stimulation of glutamatergic neurons. (Coyle & Puttfarcken, 1993; Gilgun-Sherki, Melamed, & Offen, 2001). This process in striatal neurons has also been implicated in Parkinson’s Disease (Ambrosi, Cerri, & Blandini, 2014; Van Laar et al., 2015). Chronic oxidative stress in the brain can overwhelm the endogenous antioxidant defenses including the enzymes CuZn- and Mn-superoxide dismutase (SOD), glutathione (GSH) peroxidase and catalase, and deplete antioxidant small molecules such as glutathione (Schulz, Lindenau, Seyfried, & Dichgans, 2000) (Halliwell & Gutteridge, 1985). Under such conditions, delivery of antioxidant to the CNS by increasing the intake of dietary antioxidants may be beneficial (Gilgun-Sherki et al., 2001). Thus, the identification of novel antioxidants as potential therapeutics is a challenging area of neuroscience research (Kelsey, Wilkins, & Linseman, 2010) and a variety of plant-derived compounds have shown neuroprotective benefit in cell and animal models of Parkinson’s Disease (Kim et al., 2010; Lu et al., 2014; McGuire et al., 2006; Nataraj, Manivasagam, Justin Thenmozhi, & Essa, 2015; Rajeswari, 2006; Strathearn et al., 2014; Tseng, Chang, & Lo, 2014). Recent research suggests that consumption of botanical extracts or of the whole fruit, which comprises multiple compounds, may be more beneficial than individual compounds, due to synergistic effects (Joseph et al., 2010). Moreover, superior therapeutic benefit may result if the combination of compounds in a whole fruit targets multiple pathological pathways (polypharmacology) (Mandel et al., 2011). Fruits and especially berries are of special interest for neuroprotection because they contain a wide variety of antioxidants, are palatable and habitually consumed in the diet (Habib & Ibrahim, 2011; Holt et al., 2009; Scalbert, Johnson, & Saltmarsh, 2005). The present study examined the neuroprotective effects, in vitro and in vivo, of a novel cultivar of passion fruit (Passiflora edulis Sims), a member of genus Passiflora, whose vines yield juicy fruit with a distinctive aroma and flavor. There are two described forma within the species: flavicarpa (yellow peel) and edulis (purple peel). The majority of varieties grown commercially are of the flavicarpa forma, with the fruit used primarily as a source for concentrate juice. Hybrids between the two

forma are also grown commercially, mostly for fresh fruit consumption. One such hybrid, ‘Passion Dream’ (PD), is commercially grown in Israel. Ripening attributes and chemical composition of fruit from this cultivar, and from an additional cultivar termed ‘Ripens during summer’ (previously named 428, cultivar name ‘Dena’), an F2 progeny from selffertilized PD (F1), were recently described (Goldenberg, Feygenberg, Samach, & Pesis, 2012). They are of interest here because edible pulp from both cultivars contains relatively high levels of the ester thiol volatiles 3-mercaptohexyl acetate, 3-mercaptohexyl butanoate and 3-mercaptohexyl hexanoate (Goldenberg, 2012; Goldenberg et al., 2012). These thiols are potential antioxidants, scavengers of many ROS species that may interact with cysteine residues in proteins to help keep them in the reduced state. In addition, given their size and structure (low molecular weight, small surface area, hydrophobic compounds with little capacity to form hydrogen bonds) these thiol esters have the ideal traits to cross the blood brain barrier (Atlas, Melamed, & Ofen, 1999; Bahat-Stroomza et al., 2005; Serlin, Shelef, Knyazer, & Friedman, 2015; Talcott, Percival, Pittet-Moore, & Celoria, 2003). Extracts prepared from flowers of a different species, Passiflora incarnata, are reported to be anxiolytic (Miroddi, Calapai, Navarra, Minciullo, & Gangemi, 2013) (Sampath, Holbik, Krenn, & Butterweck, 2011). Since most of the commercial P. edulis fruit is used for juice production, factories collect huge quantities of fruit peel (rind, the fruits’ pericarp) (Canteri et al., 2010). The potential neuroprotective properties of the fruit pulp, that is normally consumed, have not yet been explored, however, several studies suggest that in addition to being a source for industrial pectin extraction, rind extracts have significant anti-inflammatory and antioxidant health benefits (Chilakapati, Serasanambati, Manikonda, Chilakapati, & Watson, 2014). The current study explored the neuroprotective potential of the commercial PD and line 428 fruit extracts using a well-established cell culture model in which oxidative stress and mitochondrial dysfunction are induced by glutamate toxicity. Because the line 428 extract gave the most promising results in cultured neurons, we then tested its capacity to protect against neuronal-cell death in a well-established animal model of neurodegeneration that mimics Parkinson’s disease by selective death of dopaminergic neurons in the brain substantia nigra due to acute administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MTPT).

2.

Materials and methods

2.1.

Passion fruit extracts

Passion Dream and PF428 fruit were grown on the experimental farm of the Robert H. Smith Faculty of Agriculture, Food and Environment of the Hebrew University of Jerusalem. The juice, pulp and seeds were removed from the ripe fruits, freezedried, powdered and stored at −80 °C until used in cell culture and in vivo studies. For cell culture experiments, stock extracts were prepared by suspending the freeze-dried powder in phosphate buffered saline (PBS) at an initial concentration of 20% by weight. The suspension was centrifuged at 3400g for 20 min at room temperature (RT) and the supernatant was

Journal of Functional Foods 23 (2016) 359–369

filtered through a 45 µm membrane. The resulting stock extracts were then frozen and stored at −80 °C until thawed for use in cell culture studies. For the in vivo study, animal diets were supplemented with 20g freeze-dried fruit powder / kg diet (2% by weight).

2.2.

Cell culture experiments

2.2.1.

Cell culture

Mouse hippocampal HT4 neuronal cells were kindly provided by D.E. Koshland, Jr., University of California at Berkeley. Cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1× glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. Semiconfluent cells were trypsinized and seeded on six-well plates at a concentration of 50,000 cells/ml. All experiments were performed 24 h after seeding the cells, following the same general protocol. Medium was replaced and passion fruit extracts were added to medium at the desired final concentration. Ten minutes later, the cells were challenged by adding glutamate to a final concentration of 10 mM, according to a model of glutamate-induced cytotoxicity (Tirosh, Sen, Roy, & Packer, 2000), followed by the measurement of pre-specified measures of cytotoxicity and oxidative stress as described below.

2.2.2. Cell viability and dose-dependent neuroprotection by passion fruit extract Cell viability was assessed by propidium iodide (PI) staining, which indicates a loss of cell-membrane integrity and cell death. Cells were subjected to the glutamate challenge as described above. To obtain a dose-response curve, PBS, 428 and PD extracts were added to the cultures at the following final concentrationsof 0, 0.4, 0.6, 0.8, 1.6, 2, and 2.4%. Twelve hours after the glutamate challenge (10 mM), cells were trypsinized, centrifuged (1000 g, 5 min), and re-suspended in phosphatebuffered saline (PBS). Cells were filtered through a 90-µm mesh grid, stained with PI (2 µg/ml) and applied to flow cytometry (FACSort, BD, Franklin Lakes, NJ, USA). Fluorescent emission was measured at 575 nm with excitation set at 488 nm. Data were collected from 10,000 cells for each of 6 replicates at each concentration.

2.2.3.

Intracellular ROS production

Intracellular ROS production was measured by 2’,7’dichlorodihydrofluorescein diacetate (H2DCF) with flow cytometry. Six hours after challenging the cells with 10 mM glutamate in the presence of either 2% (v/v) of either PBS, PD or 428 fruit extract, cells were trypsinized, centrifuged (1000g, 5 min), and re-suspended in PBS. Cells were filtered through a 90-µm mesh grid and incubated at 37 °C with H2DCF (25 µM) for 30 min. Fluorescent emission was measured at 530 nm with excitation at 488 nm. Data were collected from 10,000 cells for each of 6 replicates per condition.

2.2.4.

Assessment of mitochondrial membrane potential

Mitochondrial membrane potential (Δψm) was measured using the mitochondria-directed, ratio- metric fluorescent probe JC1. Reversible changes in mitochondrial membrane potential are

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detected as a shift in emitted light from the monomeric form of JC-1 (which emits at 529 nm) to the aggregated form (which emits at 590 nm), indicating sustained membrane potential. The ratio of red (FL2) to green (FL1) fluoresces can be determined by flow cytometry. Six hours after challenging the cells with 10 mM glutamate in the presence of either 2% (v/v) of either PBS, PD or 428 fruit extract, cells were incubated with JC-1 (2 µg/ ml) for 30 min at 37 °C with 5% CO2. The cells were then washed once with PBS and analysed by flow cytometry. Data were collected from 10,000 cells for each of 9 replicates per condition.

2.2.5. Reduced glutathione (GSH) 5 chloromethylfluorescein diacetate (CMFDA) assay Cellular GSH levels were assessed using 5 chloromethylfluorescein diacetate (CMFDA) with flow cytometery. Six hours after challenging the cells with 10 mM glutamate in the presence of either 2% (v/v) of either PBS, PD or 428 fruit extract, cells were washed twice with PBS and then incubated for 15 min with 4 µM CMFDA in DMEM medium without FCS at 37 °C. After 15 min, the medium with CMFDA was replaced with DMEM only without FCS. Thirty minutes later, the cells were trypsinized, centrifuged (1000 g, 5 min at 4 °C), and re-suspended in phosphate-buffered saline (PBS). In addition to CMFDA, cells were also stained with PI (2 µg/ml) to distinguish living from dead cells. Cells were kept on ice until fluorescence was measured by flow cytometry. Fluorescent emission was measured at 530 nm with excitation set at 488 nm. Data were collected from 10,000 cells for each of 3 replicates per condition.

2.2.6.

Protein extraction and western blot analyses

Whole-cell lysates were prepared in RIPA buffer, and protein concentration was determined using the Bradford protein assay. Samples (40 µg, n = 6 per condition) were subjected to western blot analysis followed by chemiluminescence detection (Amersham) and densitometric analysis. The following primary antibodies were used: mouse anti-β-actin mouse anti MnSOD (monoclonal, BD Biosciences, NJ, USA) and rabbit antiCu/Zn SOD (polyclonal, Santa Cruz Biotechnology). Goat anti-mouse and goat anti-rabbit were used as secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

2.3.

In vivo experiment

2.3.1.

Animals and diets

All experiments were approved by the Hebrew University Institutional Animal Care and Use Committee. Eight-week-old male C57BL/6 mice were purchased from Harlan laboratories (Jerusalem, Israel). Mice were allocated by systematic-random allocation to four groups of equal mean body weight (20.9 ± 2.2 g; mean ± SD); housed 5–6 mice per cage; provided with free access to food and water; and acclimated to a 12-h dark / 12-h light reverse light schedule. Following acclimation, each group was allocated to receive one of the following experimental treatments: Control diet with saline injection (N = 10); Control diet with MPTP injection (N = 10); PF428 supplemented diet with saline injection (N = 16; PF428 supplemented diet with MPTP injection (N = 15). A control AIN 93M casein-based powder diet was purchased from Harlan Teklad (Madison WI, USA; basal-diet mix TD 03595 and vitamin mix TD 94047). For the

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Journal of Functional Foods 23 (2016) 359–369

PF428 supplemented diet, the freeze-dried fruit powder was added to the control diet at a concentration of 2% by weight and mixed thoroughly (20g freeze-dried fruit powder / kg control diet). Experimental diets were given to the animals beginning 14 days prior to MPTP administration, and food was replaced daily.

2.3.2. Acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration Mice allocated to receive MPTP injections and fed either control or PF428 supplemented diets were administered four i.p. injections of MPTP-HCl (Sigma Aldrich, Rehovot, Israel) in saline at a dose of 16 mg MPTP/kg body weight, and at 2-hour intervals between each injection, in accordance with standard protocols (Jackson-Lewis & Przedborski, 2007). Control mice from each diet group received saline only. This regimen causes striatal dopamine depletion and non-apoptotic dopaminergic cell death in the substantia nigra within 7 days of the last injection. Cages were examined daily after the injections and any deaths were noted, because acute MPTP intoxication causes death in some animals due to peripheral cardiovascular effects in the early days after administration, unrelated to neurodegeneration in the brain. MPTP handling and safety measures were in accordance with published guidelines (Jackson-Lewis & Przedborski, 2007).

2.3.3.

Tissue collection and processing

Mice were decapitated under isoflurane anaesthesia, and brains were rapidly removed and transferred to ice-cold 4% neutralbuffered paraformaldehyde (pH 7.4) for 24 hours, transferred to PBS containing 0.05% sodium azide and held at 4 °C until sectioning. Five brains per group were randomly selected for analysis. Prior to sectioning fixed brains were embedded in gelatin blocks by submerging and infiltrating the tissue with 5% gelatin at 37 °C for 24 hours followed by submersion in 10% gelatin at 37 °C for an additional 24 hours. The gelatin and infiltrated tissue were then transferred to a mold, chilled to 4 °C, and the blocks were hardened by submersion in 4% neutral-buffered paraformaldehyde overnight. Freefloating, 50 µm-thick, serial, coronal-sections were then cut through the striatum and substantia nigra on a vibrating microtome. The sections were collected in 24-well plates in PBS containing 0.05% sodium azide and held at 4 °C until immunohistochemistry.

2.3.4.

overnight at 4 °C with rabbit anti-tyrosine hydroxylase primary antibody (NB-300-109, Novus Biologicals, Littleton, CO, USA) diluted 1:500 in PBST/FBS, rinsed twice in PBST and incubated for 2h at room temperature with a secondary biotinstreptavidin-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA) diluted 1:200 in PBST. Sections were then rinsed twice in 0.1M PBS, and tyrosine hydroxylase immunolabeling was visualized with 3’3’diaminobenzidine according to the manufacturer’s instructions (SK-4100 DAB kit, Vector Laboratories, Burlingame, CA, USA). Sections from the different treatment groups were processed and analysed in parallel to minimize any potential processing bias.

2.3.5. Cell counts of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra Dopaminergic neuron survival in the brain of mice subjected to MPTP or saline administration was quantified using the optical fractionator method (West, Slomianka, & Gundersen, 1991) with StereoInvestigator software (MBF Bioscience, Williston, VT, USA). Five mice were used per treatment group. For each mouse, tyrosine hydroxylase positive neurons were counted on 4 sections spanning the substantia nigra. Sections were coded to ensure blind counting. The substansia nigra was defined using specific landmarks to maintain consistency. A grid was placed randomly over the region of interest. At regularly predetermined positions of the grid, cells were counted within three-dimensional optical dissectors. Within each dissector, a 1 µm guard distance from the top and bottom of the section surface was excluded. Section thickness was measured regularly on all collected sections to estimate the mean section thickness for each animal after tissue processing. The total number of neurons N is given as the number of neurons counted in the fraction of the region of interest that was sampled, adjusted for the entire volume of the region of interest, according to the following equation: N = Q × 1/ssf × 1/ asf × 1/hsf, where Q is the number of neurons counted, ssf is the “section sampling fraction” (the number of sections sampled as a fraction of all of the sections cut through the region of interest), “asf “ is the area sampling fraction (the total area of the sampling dissectors as a fraction of the total area of the ROI on the sampled sections), and hsf is the “height sampling fraction” (the height of the sampling dissectors as a fraction of the total section height).

Immunohistochemistry

For each brain, every 5th section was sampled systematically throughout the substantia nigra and striatum beginning with a random starting section. Sections were rinsed for 5 min in 0.1 M PBS (pH 7.4), transferred to 10 mM sodium citrate (pH = 6.0) and heated to 80 °C for 20 min for antigen retrieval. Sections were cooled to room temperature, rinsed twice for 5 min in 0.1 M PBS, and incubated for 10 min in 0.1 M PBS containing 1%H 2 O 2 /10% methanol to inactivate endogenous peroxidases. After washing twice in 0.1 M PBS, sections were permeabilized for 20 min in 0.1 M PBS containing 1% TritonX. Non-specific binding was blocked for 60 min at room temperature in 0.1 M PBS with 0.3% Triton-X (PBST) with 5% Foetal Bovine Serum (FBS). Sections were then incubated

2.3.6. Densitometric assessment of tyrosine hydroxylase expression in striatum Sections were observed under a Zeiss AxioImager M2 motorized microscope equipped with a high resolution colour camera. Images of tyrosine hydroxylase labeling in the striatum were acquired using identical illumination conditions and exposures for all slides. NIH ImageJ software (NIH, Bethesda, MD, USA) was used for denositometric analysis. Colour images were converted to grey scale and inverted so that arbitrary density units ranged from 0–255 (black to white). Image density values were normalized to the mean control density according to the formula (255-mean image density for mouse)/(mean density for controls).

Journal of Functional Foods 23 (2016) 359–369

2.4.

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Statistics

Descriptive statistics, one-way ANOVA with Tukey’s and Dunnet’s post-hoc tests and Kaplan–Meier survival analysis with the Mantel–Cox test were performed with SPSS 18.0 software (IBM, Armonk, NY, USA).

3.

Results

Exposure of HT4 hippocampal neurons to 10 mM glutamate resulted in >90% cell death within 12-15h. The addition of passion fruit extracts to the culture medium resulted in a dosedependent protection against glutamate-induced neurotoxicity. The 428 extract potently prevented cell death in a dosedependent manner and completely restored cell survival to control levels at a final concentration of only 0.4% of the medium. The PD extract also showed dose-dependent neuroprotection but dose dependency was less potent than a similar extract of 428, and only restored complete survival at a final concentration of 2% (Fig. 1A). We next determined the capacity of passion fruit extracts to mitigate the accumulation of reactive oxygen species (ROS) in HT4 cells following the addition of glutamate to the medium. We evaluated intracellular ROS production in the presence of PF extracts by the redox-sensitive H2DCF fluorescence dye. Measurements were taken 6 h after glutamate and before cell death occurred (Tirosh et al., 2000). Exposure to 10 mM glutamate led to a significant 50% increase in intracellular ROS production compared to unexposed controls, (p < 0.05) (Fig. 2). Both 428 and PD pre-treatment prevented glutamate-induced ROS production: Addition of 428 extract to the culture medium without glutamate significantly decreased ROS production by 60% compared to control cells unexposed to either glutamate or fruit extract (p < 0.05). Furthermore, it abolished the glutamate-induced increase in ROS production observed in cells without the extract (p < 0.05). In contrast to 428, the PD extract only marginally reduced ROS production compared to controls (75%, p > 0.05); however, it prevented the glutamate-induced increase in ROS production (p < 0.05) and to a similar extent as the 428 extract. To determine whether these observations were related to mitochondrial dysfunction, we examined the effect of glutamate with and without the extracts on mitochondrial membrane potential. Exposure of control cells to glutamate for 6h resulted in a progressive 20% loss of mitochondrial membrane potential. Treatment with the 428 extract completely abolished this glutamate-induced depolarization of the mitochondrial membrane (Fig. 3). PD extract did not prevent glutamate-induced mitochondrial depolarization. PD extract by itself also caused a significant 17% depolarization (p < 0.05 compared to control cells unexposed to either glutamate or fruit extract) (Fig. 3). The effect of PF extracts on the endogenous cellular antioxidant defense system was evaluated. Concentration of reduced glutathione (GSH) and the expression of the antioxidant enzymes magnesium dependent and copper/zinc dependent superoxide dismutase (also known as SOD1 and SOD2, respectively) were measured. Exposure to 10 mM glu-

Fig. 1 – Survival in glutamate (Glu) treated HT4 cells is improved by Passion fruit extracts in a dose dependent manner. (A) HT4 cells were incubated with increasing concentrations of 428 or PD extracts (0.4, 0.6, 0.8, 1.6, 2 and 2.4% of medium) and with 10 mM glutamate (Glu). Incubation with the extracts began 10 minutes before glutamate was added. 12 hours later, cell viability was determined by flow cytometry with propidium iodide staining. (B) Representative images of cell cultures following 12h incubation with 2% 428 and PD and 10 mM Glu (B). n = 6. Bars represent mean ± SE. *p < 0.05 vs. control group. # p < 0.05 PD vs. 428 at the same dose.

tamate significantly depleted intracellular GSH by nearly 40% (p < 0.05) (Fig. 4). Addition of the 428 extract abrogated glutamate-induced glutathione depletion. In contrast, the PD extract had no protective effect against glutamate-induced depletion of glutathione (Fig. 4). Examination of Mn SOD and Cu/Zn SOD expression by western blot showed that glutamate had no significant effect on either enzyme. Addition of 428 extract to the medium of cells that were not exposed to glutamate, increased Mn SOD expression by approximately 75% (p < 0.05) and Cu/Zn SOD expression by approximately 50% (p < 0.05) compared to unexposed controls. This augmentation was lost upon exposure to glutamate (Fig. 5 A & B). In contrast to 428, the PD extract did not significantly affect Mn SOD expression but it did increase Cu/Zn SOD expression by approximately 40% (p < 0.05). As with the 428 extract, this augmentation was lost upon exposure to glutamate (Fig. 5 A & B). Because the 428 extract was found to be effective in preventing glutamate-induced neurotoxicity in cell culture, we then tested it in an animal model of Parkinson’s disease in which dopaminergic neurons of the substantia nigra are selectively

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Fig. 2 – Passion fruit extracts prevent glutamate-induced ROS production in glutamate (Glu) treated cells. HT4 cells were incubated in medium containing 10% 428 or PD extracts with and without 10 mM glutamate. ROS production was measured at 6h by flow cytometry with H2DCF fluorescence. n = 6 per replicate. Bars represent mean ± SE. Means with different letters in each group differ at p < 0.05.

killed by acute exposure to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine. As with glutamate-induced neurotoxicity, the mechanism of cell death involves mitochondrial damage and associated ROS production. Consuming the passion fruit-supplemented diet did not affect body weight compared to the control diet. In the week following MPTP administration, among the saline-injected mice, none of the mice fed the control diet died and only one of those fed the 428-supplemented diet died. Among the mice injected with MPTP, 7 mice fed the control diet died compared to 4 of those fed the 428-supplemented diet (Fig. 6). Mantel–

Fig. 3 – PF 428 but not PD extract prevents reduction in mitochondrial membrane potential (ΔψM) in glutamate (Glu) treated cells. HT4 cells were incubated in medium containing 10 % 428 or PD extracts for 10 minutes, followed by the addition of 10 mM glutamate. Mitochondrial membrane potential ΔψM was measured at 6 h by flow cytometry with JC-1 fluorescence. n = 9 per replicate. Bars represent mean ± SE. Means with different letters in each group differ at p < 0.05.

Fig. 4 – PF 428 but not PD extract prevents glutathione depletion in GSH treated cells. HT4 cells were incubated in medium containing 10% 428 or PD extracts for 10 minutes, followed by the addition of 10 mM glutamate. Reduced glutathione levels were measured at 6 h by flow cytometry with CMFDA fluoresence. n = 3 per replicate. Bars represent mean ± SE. Groups with different letters differ at p < 0.05.

Cox analysis showed that mice injected with MPTP were significantly more likely to die compared to saline injected control-fed mice (p < 0.05 for both diets). Although the consumption of PF428 improved acute survival from 53% to 73% among the MPTP treated mice, we were underpowered to show statistical significance (p = 0.3). The number of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra of the different treatment groups is shown in Fig. 7. Saline-injected mice fed the control and 428-supplemented diets had similar cell counts (14,960 ± 3899 vs. 11,851 ± 801, respectively; Mean N ± SD, p = 0.50 by one-way ANOVA with Dunnet post-hoc test). On average, MPTP administration caused a 69% loss of neurons compared to control-fed saline-injected mice (4741 ± 1509 vs. 14,960 ± 3899, respectively, p = 0.01). Supplementing the diet with 428 diminished the effect of MPTP, resulting in only 51% cell loss compared to control-fed saline-injected mice (7605 ± 440 vs. 14,960 ± 3899, respectively, p = 0.055). In comparison to the MPTP-injected, control-fed group, this represented a significant 60% improvement in cell survival (7605 ± 440 vs. 4741 ± 1509, respectively, p = 0.049). Despite the improved survival of substantia nigra neurons in mice fed the supplemented diet, we did not detect a difference in the optical density of tyrosine hydroxylase staining of nerve terminals in the striatum (online supplemental material Fig. S1).

4.

Discussion

This study provides the first evidence for the neuroprotective benefits of eating passion fruit, particularly cultivar Dena (line 428). The glutamate-induced neurotoxicity model in HT4 neurons allowed us to explore the capacity of passion fruit extracts to mitigate the convergent effects of excitotoxic and

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Fig. 5 – Effect of glutamate and fruit extracts on Mn- and Cu/Zn superoxide dismutase expression in vitro. (A) MnSOD and (B) Cu/Zn SOD expression was evaluated in harvested cells by western blot. Protein expression for each treatment group normalized to the expression in control cells incubated without any extract or glutamate. Glutamate did not induce statistically significant elevations of either SOD enzyme. n = 6 per replicate. Bars represent mean ± SE. Groups with different letters differ at p < 0.05.

excitotoxicity-independent pathways that deplete intracellular GSH and increase ROS production (Aharoni-Simon, Reifen, & Tirosh, 2006; Tirosh et al., 2000). In the excitotoxic pathway, hyper-activation of ionotropic glutamate receptors results in

Saline

an influx of free calcium, mitochondrial stress and the subsequent generation of ROS (Savolainen, Loikkanen, Eerikäinen, & Naarala, 1998). The excitotoxicity-independent process results from an imbalance between oxidants and antioxidants in the

428+saline

MPTP

428+MPTP

100% 90% 80%

SURVIVAL (%)

70% 60% 50% 40% 30% 20% 10% 0%

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DAYS AFTER INJECTION

Fig. 6 – Dietary supplementation with PF428 improves survival of mice subjected to acute MPTP intoxication. Kaplan–Meier survival curves show 7-day survival rates for mice in the four treatment groups: Control mice fed a standard AIN-93M diet and injected with normal saline (closed squares); Control mice fed a standard diet supplemented with 2% (w/w) PF428 freeze dried fruit extract, and injected with normal saline (closed triangles); Mice fed the PF428 supplemented diet and injected with MPTP (black circles); Mice fed the control diet and injected with MPTP (grey circles).

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Fig. 7 – Dietary supplementation with PF428 improves survival of substantia nigra dopaminergic neurons in mice subjected to acute MPTP intoxication. (A) Dot plot shows total number of tyrosine-hydroxylase immunopositive (TH+) dopaminergic neurons in the substantia nigra (SN) by treatment groups as estimated by unbiased stereology (N = 5 mice group). Mean neuronal survival was 50% higher in 428 treated vs. untreated MPTP injected mice (* p < 0.05). Legend: Cont – Control mice fed a standard AIN-93M diet and injected with normal saline; 428 – Control mice fed a standard diet supplemented with 2% (w/w) 428 freeze dried fruit extract, and injected with normal saline; MPTP – Mice fed the control diet and injected with MPTP; 428 + MPTP – Mice fed the PF428 supplemented diet and injected with MPTP. (B) Representative images of tyrosine hydroxylase (TH) labeling in the substantia nigra of each group.

cell (oxidative stress) and inhibition of cystine uptake via the xc-cystine/glutathione antiporter (which also leads to excessive ROS production and the depletion GSH) (Aharoni-Simon et al., 2006). Both PD and the 428 extracts improved cell survival in the face of these insults in a dose-dependent manner without toxicity and both offered similar prevention of ROS production. However, the 428 extract was superior to the PD line in the effective dose needed for survival, and in preventing both mitochondrial dysfunction and GSH depletion. It also augmented the expression of both Mn- and Cu/Zn SOD, whereas the PD extract appeared to upregulate Cu/Zn SOD alone. Although further work will be necessary to provide a detailed molecular account of the mechanisms that yield these effects,

our findings provide evidence that the 428 extract possesses substantial antioxidant capacity and potentially neuroprotective properties. Work in animal models has shown that a chronic, experimentally-induced reduction in brain GSH increases the susceptibility of dopaminergic neurons to neurotoxic insults. For example, intracerebral administration of buthionine sulfoximine in rats caused the depletion of brain GSH followed by loss of striatal dopamine and nigral tyrosine hydroxylase immuno-positive cells (Pileblad, Magnusson, & Fornstedt, 1989; Wüllner et al., 1996). The importance of glutathione in preventing MPTP toxicity is illustrated by studies of GSH peroxidase-deficient mice which suffer significantly

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greater loss of dopamine and its metabolites than those seen in wild-type mice (Klivenyi et al., 2000; Schulz et al., 2000). In contrast, mice over expressing Cu/Zn SOD are resistant to the neurotoxic effects of MPTP (Dauer & Przedborski, 2003; Przedborski et al., 1992). As noted above, passion fruit is rich in thiol and thioesther compounds. Line 428 in particular contains several thioesthers (3-mercaptohexyl acetate (PubChem CID: 518810); 3-mercaptohexyl butyrate (PubChem CID: 537754); 3-mercaptohexyl hexanoate (PubChem CID: 518810)). The concentration of these compounds in the juice of fresh fruit was previously found to be 44.5 ηl/l in PD and 61.5 ηl/l in line 428 fruit (Goldenberg, 2012; Goldenberg et al., 2012). Cold storage increases thioester content by ~10% for the PD fruit and more than four-fold for the 428. Total free thiol content of both lines was approximately 400 nM which is comparable to the concentration of free thiols found in mango and guava, more than twice that of tomatoes, and about 20–40 times higher than that found in apples and grapes (Goldenberg, 2012; Goldenberg et al., 2012). Such thiol-compounds can cross the blood brain barrier and have been shown to protect against MPTP induced neurotoxicity (Bahat-Stroomza et al., 2005; Chen, Yin, Hsu, & Liu, 2007). Thus, the bioactive thioester and thiols in the supplemented diets can be expected to augment brain antioxidant defenses and confer neuroprotection. Collectively, these observations, and our in vitro results, offer a plausible explanation for why supplementing diet with line 428 fruit provides partial protection from MPTP neurotoxicity. However, we cannot rule out other mechanisms. The MPTP mouse model was selected for a proof of concept that dietary passion fruit supplementation could mitigate neurodegenerative pathology because it reproduces key pathological and biochemical features of oxidative stress, mitochondrial dysfunction and apoptosis in selective neuronal populations, and is widely used to study neuroprotective effects of candidate therapies (Przedborski & Vila, 2001; Schmidt & Ferger, 2001). In broad terms, the toxicity of MPTP results from the uptake of MPTP by glial and serotonergic cells where it is metabolized by monoamine oxidase (MAO) to MPP+. In this highly toxic form, it is selectively transported via the dopamine transporter into dopaminergic neurons where it has potently inhibits mitochondrial electron transport chain complex I leading to increased ROS production and ATP depletion (Dauer & Przedborski, 2003). Our finding that dietary supplementation with t428 conferred significant neuroprotection against MPTP toxicity is encouraging; however, results should be interpreted with caution. First of all, only partial protection was observed. This could be due to the potency of MPTP toxicity, sub-optimal dosage and duration of the supplementation or a mismatch between some of the protective bioactive fruit components and the specific mechanism of MPTP toxicity. The incomplete protection as gauged from neuronal counts may partly reflect a differential survivor bias between the supplemented and non-supplemented mice. Since a larger number of nonsupplemented mice died before their brain could be examined, it is likely that the surviving mice were more resistant to MPTP. Such a bias would be expected to increase the surviving cell counts and diminish the true differences between groups. The apparent inconsistency between the lack of protection for ty-

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rosine hydroxylase positive nerve endings in the striatum and the significantly higher number of surviving neurons in the substantia nigra, may reflect differences between the acute MPTP model where the toxin is administered systemically and the dynamics of retrograde degeneration along the nigral-striatal pathways that occurs in other models where degeneration follows from genetic manipulation or the infusion of toxins such as 6 hydroxydopamine (6-OHDA) directly into the striatum (Berger, Przedborski, & Cadet, 1991; Nordström et al., 2015).

5.

Conclusions

The encouraging findings of this work suggest the neuroprotective potential of eating certain passion fruit varieties warrants further studies, including work aimed at enhancing their bioactive content, to better define their molecular targets and mechanisms, and to optimize their effectiveness in this and other translational models of neurodegeneration and brain aging.

Author contributions YT carried out all the experiments, analysed the data, and helped draft the manuscript as part of her graduate studies; SA participated in the cell culture experiments; MR participated in the animal experiments; AS developed, cultivated and supplied the passion fruit, OT and AMT designed the study, interpreted the data and wrote the manuscript.

Conflicts of interest AS, OT and AMT have applied for a patent pertaining to the findings reported here. The funding sponsors had no role in the study design, data collection, analysis, or interpretation, in the manuscript preparation writing or in the decision to publish the results. There are no other conflicts of interest to declare.

Acknowledgements This work was supported by an intramural “Yissumit” applied R&D seed grant from the Hebrew University of Jerusalem.

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