Elsevier

Food and Chemical Toxicology

Volume 120, October 2018, Pages 41-49
Food and Chemical Toxicology

Protective effects of hydroxytyrosol against α-synuclein toxicity on PC12 cells and fibril formation

https://doi.org/10.1016/j.fct.2018.06.059Get rights and content

Highlights

  • HT is a potent inhibitor of α-syn fibril formation.

  • α-Syn fibrils can be effectively destabilised by HT.

  • HT completely counteracts the neurotoxicity induced by α-syn.

  • HT is a promising compound for developing strategies to tackle PD.

Introduction

Parkinson's Disease (PD) is the most common neurodegenerative movement disorder. Currently incurable, PD is characterised by the loss of dopaminergic neurons in the brain's substantia nigra. To explain this process, several mechanisms are involved in the pathogenesis of PD. These include oxidative stress, mitochondrial dysfunction, inflammation and abnormal protein aggregation (Poewe et al., 2017). The principal PD pathological hallmark is the presence of Lewy Bodies, extracellular spherical deposits formed mainly by α-synuclein protein (αsyn). In the case of abnormal aggregation, αsyn gives rise to oligomers, protofibrils and insoluble fibrils which trigger toxicity and lead to neuronal death (Volles and Lansbury, 2003). Therefore, it is generally admitted that a decrease in the formation of these toxic species exerts a protective role against PD. Lately, the reduction of developing mild cognitive impairment, the enhancement of cognitive function, and the reduction of the risk of AD and PD has been related to a close adherence to the Mediterranean Diet (Di Giovanni, 2009; Scarmeas et al., 2009; Alcalay et al., 2012). This diet is characterised by a daily consumption of extra-virgin olive oil in amounts ranging from 25 to 50 g/day (López-Miranda et al., 2010) and the moderate intake of red wine (1–2 glasses (<20 g/day alcohol in women and <40 g/day alcohol in men)). However, there is a need to ascertain a plausible biological mechanism explaining how bioactive food components affect the hallmarks of this disease.

Hydroxytyrosol (3,4-dihydroxyphenylethanol) (HT) and tyrosol (p-hydroxyphenylethanol) (TYR) are the major phenolic compounds present both in olive oil and table olives (Ryan and Robards, 1998; Mateos et al., 2001; Tuck and Hayball, 2002; Romero et al., 2002). Both possess demonstrated antioxidant, cardioprotective, anti-inflammatory, antimicrobial, antidiabetic and antiatherogenic properties (Fernández-Marín et al., 2012; Khalatbary, 2013; Casamenti and Stefani, 2017). Indeed, based on the positive scientific opinion of the European Food Safety Authority (EFSA) (EFSA, 2011), the European Commission has authorised a health claim for olive oil containing HT and its derivatives (at least 5 mg/20 g of olive oil) (Commission Regulation EU 432/2012) regarding the protection of blood lipids from oxidative stress.

Moreover, HT and TYR have also been identified in wine (Piñeiro et al., 2011). It is accepted that yeast produces HT and TYR as secondary metabolites from tyrosine during alcoholic fermentation (Dickinson et al., 2003; Álvarez-Fernández et al., 2018). Concentrations of HT in wine range from 1.50 to 25 mg/L and of TYR from 4.25 to 45 mg/L (Di Tommaso et al., 1998; Proestos et al., 2005; Boselli et al., 2006; Minuti et al., 2006; Dudley et al., 2008).

In addition, oleuropein, another important glycosidic phenolic component present in olive leaves, seed, pulp and in the peel of unripe olives (Ghanbari et al., 2012), undergoes hydrolysis during the maturation of the fruit, the production of olive oil and the preparation of table olives, as well as under gastric conditions yielding different products, including HT and TYR (Vissers et al., 2002). Corona and his co-workers (2006) revealed that the breakdown of oleuropein after a gastric biotransformation effectively increases the relative amount of HT and TYR in human Caco-2 cell monolayers and segments of rat jejunum and ileum.

Additionally, microbiota has been proposed for forming HT from oleuropein. Thus, HT has been reported as one of the most principal products formed from oleuropein and HT acetate after in vitro and in vivo colonic fermentations (Mosele et al., 2014). Furthermore, another endogenous source of HT has been described by oxidative dopamine metabolism (Hashimoto et al., 2004).

As well as occurring in foods, the bioavailability of HT in humans has been studied in depth. Firstly, HT absorption takes place in the small intestine and colon (Vissers et al., 2002) and reaches a maximum plasmatic concentration 5–10 min after ingestion (Bai et al., 1998). Furthermore, the absorption process depends on the vehicle employed, being most effective in the form of olive oil (Tuck et al., 2001). Moreover, HT in its free form, as well as its derivatives, ortho-methylic derivatives (homovanillic alcohol), glucuronide derivatives (Miró-Casas et al., 2003) and glutathionyl conjugates (Corona et al., 2006), can be found in plasma and in urine. Additionally, a high number of conjugates with glucuronide and sulphate are generated from HT and from the abovementioned metabolites, which are detectable both in plasma and in urine (Tuck and Hayball, 2002). HT and its metabolites have a good distribution in tissues such as muscle, testis, liver, and brain. It is, furthermore, accumulated in kidney and liver (D'Angelo et al., 2001). This widespread distribution is responsible for the health-beneficial properties of HT (Serra et al., 2012).

With specific regard to the brain, it is well-documented that HT is able to cross the Blood Brain Barrier (BBB) (D'Angelo et al., 2001; Wu et al., 2009). Indeed, HT was measured in rat brain after 5 min of intravenous injection (1.5 mg/kg), the brain tissue containing 0.31% of the administrated dose (D'Angelo et al., 2001). Wu et al. (2009) determined in rat brain a Cmax of 2.1 μg/mL after an intravenous dose of 100 mg/kg at Tmax of 15 min and T1/2 of 22.1 min.

This evidence that HT can cross the BBB attracts scientific interest in exploring the mechanisms underlying the putative neuroprotective effects of HT, which might in turn be related to epidemiological evidence of the beneficial properties of extra-virgin olive oil with regard to cognitive function (Scarmeas et al., 2009).

In this context, certain recent studies indicate that one HT mechanism could be the reduction of the toxic effect induced by amyloid-β peptide (Aβ) (the principal pathological hallmark of Alzheimer's Disease (AD)) (St-Laurent-Thibault et al., 2011; Peng et al., 2016). Conversely and, as far as we know, the possible preventive role of HT towards αsyn toxicity remains unexplored. Therefore, since protocatechuic acid, a phenolic acid produced in vivo as a result of anthocyanin metabolism (de Ferrars et al., 2014), shares the substitution on the aromatic ring structurally similar to HT and additionally has demonstrated to prevent αsyn toxicity (Hornedo-Ortega et al., 2016), our hypothesis is that HT could be a potential bioactive as well. The main purpose of this work is to study the neuroprotective capacity of HT, TYR and other tyrosine metabolites against αsyn toxicity and αsyn aggregation and as a consequence, their capacity to combat PD. To this end, the Thioflavin T (ThT) assay, Transmission Electronic Microscopy (TEM), electrophoresis and staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) with HT and related compounds (TYR and hydroxyphenyl acetic acid (HPAA)) have been explored.

Section snippets

Chemicals and reagents

Standards and reagents were purchased from the following suppliers: Sigma Aldrich Steinheim, Germany (Tyrosol (TYR) and hydroxyphenylacetic acid (HPAA), Thioflavin T (ThT), dimethyl sulfoxide (DMSO), Dulbecco's modified Eagle's medium (DMEM)-Glutamax, trypsin−EDTA, MTT, Phosphate Buffered Saline (PBS), l-glutamine, foetal horse serum, foetal bovine serum and penicillin/streptomycin); Alexotech, Umeå, Sweden (α-synuclein protein); Panreac, Castellar del Vallès, Barcelona, Spain (Na2HPO4/NaH2PO4

Inhibitory effect of HT, TYR and HPAA on αsyn fibril formation

To evaluate whether or not HT, TYR and HPAA could inhibit αsyn fibril formation, 100 μM final concentrations of these compounds were incubated with αsyn (70 μM) and ThT assay was performed over 6 days (37 °C, 1000 rpm). The recorded ThT fluorescence data displayed in Fig. 2A clearly shows that HT is the only compound that strongly inhibited the aggregation of αsyn.

In view of these results, HT was selected for further experiments in which different concentrations (25, 50, 100 and 200 μM) were

Discussion

The presence of amyloid aggregates is considered a key feature in the pathological signs of PD that currently requires more research. As a consequence, much effort has been dedicated to search for molecules able to interfere with the amyloidogenic proteins such as αsyn, preventing the appearance of toxic species (oligomers and protofibrils). The identification of novel agents that inhibit αsyn aggregation and toxicity are thought to be of particular interest; small molecules such as phenolic

Acknowledgements

The authors are very grateful to the Spanish Government (Ministerio de Economía y Competitividad MINECO for its financial assistance (Projects MICINN AGL 2013–47300-C3-2-R and MICINN AGL 2016–77505-C3-2-R). We acknowledge Dr. M. Carballo-Álvarez and Dr. C. Vaquero from the Biology and Microscopy services (CITIUS) of the University of Seville for their technical assistance. The authors would also like to thank the VPPI-US for both Ruth Hornedo-Ortega and Ana B. Cerezo's research contracts.

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