**1. Introduction**

Neurodegenerative diseases are becoming increasingly prevalent in the ageing populations of industrialized nations, going hand in hand with the increase in life expectancy. These disorders, which include Alzheimer's disease and Parkinson's disease, share a common feature: the accumulation of misfolded proteins in pathological inclusions [1].

Parkinson's disease (PD) is a chronic, age-related and adult-onset neurodegenerative disorder characterized by the loss of dopaminergic neurons in an area of the midbrain called substantia nigra (SN) along with intraneuronal inclusions known as Lewy bodies, which contain amyloid aggregates of misfolded α-synuclein [2–4]. PD is considered today as the most common movement disorder that affects 1–2 per 1000 of the population and since the prevalence is increasing with age, PD affects 1% of the population above 60 years [5]. There are dozens of PD-related symptoms and signs but the most typical are motor deficits including tremors, muscle rigidity, bradykinesia, and impaired gait. Among non-motor symptoms, the most common are olfactory dysfunction, cognitive impairment, psychiatric symptoms, and autonomic dysfunction [6]. PD is a multifactorial disorder and the majority of PD cases are sporadic with unknown aetiology possibly caused by an association of genetic and environmental risk factors. At least 23 loci and 19 disease-causing genes for PD have been identified and designated as PD-causing genes [7].

Several hypotheses have been proposed regarding the cause of loss of dopaminergic neurons in PD, whereas oxidative stress, in particular, is strongly associated with the development of PD [8,9]. Other studies have shown defects in the mitochondrial complex-I of neurons, which lead to impaired energy metabolism and cell death [10]. Furthermore, the proteolytic hypothesis describes nigral neuron loss in PD as a result of toxic accumulation of aggregates of misfolded proteins, notably α-synuclein, resulting in neuro-inflammation [11]. Dopaminergic neurons of substantia nigra pars compacta appear particularly vulnerable to the harmful effects of α-synuclein aggregates [12]. Since ageing is a major risk for PD, it has been hypothesized that PD could be, at least in part, a type of segmental ageing, in which the viability of dopaminergic (DA) neurons is impaired by so far unknown localized and accelerated ageing mechanisms [13].

Neurodegenerative disorders are associated with high morbidity and mortality, and few effective options are available for their treatment [1]. Thus, many studies have been conducted focusing on natural compounds present in food as important molecules against neurodegenerative diseases such as PD [14–17]. Several lines of evidence support the beneficial effect of the Mediterranean diet (MD) in preventing neurodegeneration, possibly due to its richness in polyphenols [18,19]. Natural polyphenols exert numerous biological activities, like antioxidant, anti-inflammatory, antiviral, antibacterial, antiproliferative, and anticarcinogenic capacities (reviewed in Stevanovic, et al. [20]), as well as cellular redox state modulation activities through direct action on enzymes, proteins and receptors [21,22]. In addition, in patients affected by osteoarthritis or cardiovascular diseases, beneficial epigenetic chromatin modifications were also caused by polyphenols [23–25].

One possible mode of action of natural polyphenols is the hormesis effect. The biological processes underlying hormetic dose–response, recently, focused attention in the field of neuroprotection, which was mainly elucidated through the exploitation of bioactive polyphenols against the main age-related diseases, particularly in PD [13]. In this light, low levels of exogenous and endogenous stressors have been reported to display hormetic characteristics that induce neurophysiological mechanisms of maintenance and repair, including heat shock, the application of pro-oxidants, as well as the application of polyphenols from plants [26]. Recently, it has been postulated that the MD exerts healthy effects through hormetic mechanisms, as specific olive oil polyphenols (e.g., oleuropein and hydroxytyrosol) likely counteract the effects of neuro-inflammatory stimuli by acting as modulators of stress responsive mechanisms, which result in adaptive stress resistance [27]. Moreover, in vivo studies suggest that a diet rich in phytochemicals may enhance neuroplasticity and stress resistance to neuro-inflammation, mitigating or preventing neurodegenerative changes in the brain that are typical in a number of age-related disorders, including PD [28–30].

It has been hypothesized that extra virgin olive oil polyphenols could be among the main determinants of the beneficial effect of the MD [31–33]. Extra virgin olive oil (EVOO) contains approximately 36 phenolic compounds [21], which represent the main group of antioxidants found in virgin olive oil. The main phenolic subclasses present in olive oil are phenolic alcohols, phenolic acids, flavonoids, lignans, and secoiridoids [34], whereas the latter represent the largest quantity in the EVOO. The main secoiridoids in olive oil are oleuropein aglycone and ligstroside, which undergo transformation to hydroxytyrosol or tyrosol, respectively through two enzymes (beta glucosidase and esterase) in the gastrointestinal tract [35].

In the present paper, we focus on health effects of hydroxytyrosol (HT; Figure 1B) and oleuropein aglycone (OLE; Figure 1A) as neuroprotective agents against PD in the light of recent studies indicating that OLE and HT can be beneficial against PD by stabilizing the monomeric state of α-synuclein, thus, favouring the growth of aggregates devoid of toxicity [36,37]. Furthermore, previous studies have shown that these molecules are strongly protective against neurodegeneration in different transgenic models of Aβ deposition [38–40]. Moreover, we have to consider that hydroxytyrosol is a by-product of dopamine oxidative metabolism [41] and in the last years the gold standard therapy against PD has relied on restoring the optimum level of dopamine [42].

**Figure 1.** Chemical structure of (**A**) oleuropein aglycone and (**B**) hydroxytyrosol.

Tests with mammalian models are very powerful, but are expensive, long-lasting and cause ethical concerns. Therefore, the widely used model organism *Caenorhabditis elegans* (*C. elegans*) was applied for this study. Despite its simple structure, *C. elegans* features several tissues and organs in alignment to higher animals, like muscles, a nervous system, an epidermis, a gastrointestinal tract, and gonads [43]. Furthermore, about 50% of the human protein-coding genome has recognizable worm orthologs [44]. Last but not least, neurological pathways are highly conserved between invertebrates and mammals, and numerous neurodegenerative disease-related transgenic and mutant strains are available in the nematodes [45]. In addition to several neuro-protective substance screenings [45], numerous polyphenols were also successfully tested for their general health and lifespan benefits in *C. elegans* [46–48], making this nematode an optimal model to study the neuroprotective effects of olive polyphenols.

It was hypothesized that the polyphenol OLE and its main metabolite HT increase the mean lifespan of *C. elegans* in the presence and in the absence of stress conditions. Furthermore, it is assumed that they are able to counteract the age-related decline of general health parameters, which were assessed by determining the swim behaviour as a measure of overall body fitness as well as the intestinal autofluorescence, being one of the most popular ageing biomarkers [49]. Furthermore, numerous cell culture studies were already successfully performed to verify the anti-PD effects of olive ingredients, as summarized in Angeloni, Malaguti, Barbalace, and Hrelia [22], however, in vivo studies are hardly present. Therefore, by using a chemically induced and two transgenic PD models of *C. elegans*, the polyphenolic treatments were tested for their anti-PD effects in vivo. Although *C. elegans* is not able to develop PD, the PD models feature characteristic attributes related to PD. Besides the swim performance, neuronal degeneration as well as α-synuclein accumulation were taken into account to assess the anti-PD potential.

#### **2. Results**

#### *2.1. Oleuropein Aglycone and Hydroxytyrosol Extended the Survival of Wild-type* C. elegans *after Heat Stress*

To find the optimal concentration for this study, the treatments with polyphenols were initially tested at different concentrations in a heat stress-resistance test. Due to the relatively fast and simple execution, heat stress resistance is frequently measured to screen treatments for potential health and lifespan benefits [50–52]. Since stress resistance abilities are strongly correlated to ageing, ageing-associated diseases and lifespan [53–56], this test was also used as a pre-test in the current study. The monitored survival after stress exposure revealed an improved stress resistance in OLE-treated nematodes (Figure 2A). Two-hundred-and-fifty μg/mL and 500 μg/mL OLE provoked statistically significant changes in all biological repeats performed. The mean lifespan after heat stress increased by 15% and 22% in the OLE 250 μg/mL and OLE 500 μg/mL treated group, respectively. However, the survival differences between OLE 250 μg/mL and OLE 500 μg/mL treated nematodes were not significant. Furthermore, no significant survival benefits were observed with 30 and 100 μg/mL compared to control. Due to the highest percentage benefit, 500 μg/mL OLE was chosen for subsequent experiments.

**Figure 2.** Heat stress survival in presence of different OLE and HT concentrations. Survival is expressed as a percentage of the initial population combined with three biological replications. At the third day of adulthood (day 0), wild type nematodes were exposed to heat stress at 37 ◦C for 3 h prior monitoring survival. (**A**) Survival curves during OLE treatment with *n* (control) = 70, *n* (30 μg/mL) = 74, *n* (100 μg/mL) = 44, *n* (250 μg/mL) = 68, and *n* (500 μg/mL) = 64; (**B**) Survival curves during HT treatment with *n* (control) = 90, *n* (30 μg/mL) = 50, *n* (100 μg/mL) = 52, *n* (250 μg/mL) = 67, and *n* (500 μg/mL) = 79. Statistical significance was calculated by log-rank test including Bonferroni correction. Differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). *n*: number of tested nematodes.

Similar results were achieved by using HT (Figure 2B). The mean lifespan after heat stress was increased by about 11% by treatment with 100 μg/mL HT, by 22% with 250 μg/mL HT and by 14% with 500 μg/mL HT. There was no significant difference between the survival curves of these treatment groups among each other. Again, 30 μg/mL was not sufficient to improve the survival. Based on these results, 250 μg/mL HT was applied for further experiments. None of the tested concentrations and compounds exerted a harmful effect on the survival of the nematodes after stress exposure.
