**1. Introduction**

Emerging research has recently focused on increasing the life expectancy of humans which is, however, accompanied by a progressively greater prevalence of neurodegenerative disorders, notably Parkinson's disease (PD). PD is the second most prevalent late-age onset neurodegenerative disorder affecting 1% of the population after the age of about 60 years old and 4% of those over 80 years old, causing motor impairments, cognitive dysfunction, sleep difficulties, autonomic dysfunctions, and pain [1]. PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (*SNpc*) of the midbrain area [2]. At the cellular level, the neuropathological hallmarks of PD include intra-cytoplasmic inclusions that contain α-synuclein aggregates, a primary component of intraneuronal Lewy bodies and Lewy neurites in vulnerable neurons of the brain [3]. The loss of dopaminergic neurons results in major motor impairments including resting tremor, muscle rigidity, bradykinesia and postural instability. Since PD affects neurons in the central and peripheral nervous systems, patients typically also exhibit multiple non-motor symptoms including anxiety, depression, memory loss, perturbed proteostasis, mitochondrial dysfunction, oxidative stress, dysregulation of redox homeostasis as well as neurotoxicity [4]. Although the etiology of PD is currently unknown, genetic and environmental triggers are two major factors that play a role in the development of the disease, with the environment accounting for over two-thirds of all cases [5]. Recently, longitudinal studies have identified at least 23 loci and 19 disease-causing genes for familial parkinsonism associated with the progression of non-motor symptoms in PD patients [6,7]. Moreover, several studies have suggested that mitochondrial complex I deficiency in different brain regions is associated with impairment of energy metabolism and neuronal death in PD [8]. It has been postulated that neuroinflammatory processes might play a crucial role in the pathogenesis of PD. The proteomic approach revealed accumulation of neurotoxic misfolded α-synuclein aggregates inducing microglial activation associated to loss of dopaminergic neurons in nigrostriatal system underlying PD pathogenesis [9]. Although treatments are available to alleviate motor symptoms, currently, there are no preventive therapies that can target and lessen PD progression. Recently, epidemiological and clinical studies have supported the idea that Mediterranean diet (MD) is strongly associated with lifespan extension as well as with healthy aging process by reducing the progression of age-related pathologies [10]. The beneficial effects of natural polyphenols and derivatives comprise multi-target activities including the anti-amyloid aggregation, antioxidant, antimicrobial, antihypertensive, hypoglycemic, antiproliferative and vasodilator effects, as well as redox homeostasis activities through a direct modulation of enzymes and proteins involved in stress response pathways [11–14]. Particular attention has been paid recently by our laboratory to the effects of natural olive polyphenols such as hydroxytyrosol (HT) and oleuropein aglycone (OLE), [10,15] known to possess healthspan benefits against α-synuclein aggregation into intracellular Lewy bodies, as found in PD neurons of the mesencephalic substantia nigra [16]. In addition, in vitro and in vivo studies have shown that HT exerts various protective effects, particularly, strong anti-oxidant and radical scavenging activities [17,18]. With regard to mechanism of polyphenol action, the biological concept of hormesis has emerged as a significant dose response model in the field of neuroprotection elicited by low dose of olive polyphenols [19]. Notably, increasing evidence suggests that mild stressors such as HT may offer beneficial effects in a hormetic-like manner by activating Nrf2-stress response pathway and enhancing brain resilience, neuroplasticity as well as lifespan in vitro and in experimental PD models [20,21]. Moreover, HT activates the Nrf2–antioxidant response element (ARE) pathway, leading to the activation of phase II detoxifying enzymes and the protection of dopaminergic neurons exposed to hydrogen peroxide or to 6-hydroxydopamine [22,23]. This is consistent with the idea that neurohormesis may have anti-aging effects due to induction of adaptive pathways triggered to cope with a mild neuronal stress and open novel potential therapeutic strategies for clinical interventions against the onset and/or progression of PD in humans. In these ways, neurohormetic polyphenols might protect neurons against injury and disease by stimulating the production of antioxidant enzymes, neurotrophic factors, protein chaperones and other proteins that help cells to withstand stress [24,25]. Interestingly, our recent in vivo study with olive polyphenols has demonstrated that HT and OLE exert neuroprotective effects, an improved overall healthspan and, in part, longevity in *Caenorhabditis elegans* (*C. elegans*) models of PD and wild type [15].

The nematode *C. elegans* is a multicellular model organism that offers several advantages for investigating both aging and neurodegenerative disorders [26]. The biological features of *C. elegans* are multiple and including short life cycle (i.e., about 3.5 days from egg to adult) and a lifespan of only about 20 days, transparent body, conserved gene network and neurological pathways [27,28]. Moreover, comparative proteomics indicates that for 83% of the *C. elegans* proteome human homologous can be identified [29]. Comparative genomic analysis also shows that nearly 53% of the human protein-coding genome has recognizable worm orthologues [30]. Furthermore, there is a tight connection between lifespan extension and resistance to diverse environments [31]. In this regard, several studies indicate that different stressors acting in hormetic-like manner extend lifespan in *C. elegans* [32], and suggest that hormetic effects could be exploited to prevent the onset of neurodegenerative diseases [20]. Most importantly, its complete genomic sequence is available. Therefore, *C. elegans* as a model organism is widely applied for screening natural bioactive compounds [33]. Several polyphenols effectively increase healthspan and lifespan as well as mitochondrial function in *C. elegans* [34–38]. Notably, *C. elegans* represents an excellent model to study the neuroprotective effects of olive polyphenols. In this context, recent research has demonstrated that extracts from olive leaves efficiently scavenged free radicals in vitro and significantly increased the expression of antioxidant enzymes extending lifespan and increased stress resistance in *C. elegans* [39,40].

In the current study we focus on the health-promoting effects of two hydroxytyrosol preparations, pure hydroxytyrosol and Hidrox®. Hidrox®/ Olivenol Plus™ (HD) is a patented freeze-dried hydroxytyrosol-rich formulation obtained from the acidic hydrolysis of olive vegetation water (OVW or olive juice) and where hydroxytyrosol (40–45% at the total water-soluble olive polyphenols) is maintained in its "natural" environment [41]. Olive juice (aqueous fraction) represents 50% of the weight of the olive fruit and is normally discarded as wastewater. Several findings have reported that HD displays different activity than pure or synthetic HT [40]. International in vitro and in vivo studies showed the health benefits and efficacy of HD as anti-inflammatory, anti-oxidant, anti-scavenger as well as anti-aggregating compound, particularly in PD [42].

We hypothesized that the polyphenolic treatments, Hidrox® and pure hydroxytyrosol have the capacity to increase the mean lifespan of *C. elegans* in the presence and absence of thermal stress. Furthermore, it is assumed that they are able to counteract the age-related deterioration of general health parameters, which were assessed by determining the swim performance as a measure of overall body fitness as well as the autofluorescence as one of the most popular ageing biomarkers [43]. Moreover, numerous in vitro studies were already successfully performed to verify the anti-PD effects of olive ingredients [14], however, in vivo studies were rarely conducted. Therefore, by using one chemically-induced and two transgenic PD models of *C. elegans*, the polyphenolic treatments were tested for their anti-PD effects in vivo. Besides the swim performance, neuronal degeneration as well as α-synuclein accumulation were taken into account to assess the anti-PD potential. Finally, it was hypothesized that HD is even more effective in preventing PD- and ageing-related symptoms than HT, which was tested by a direct comparison of the action of HT and HD.

#### **2. Results**

#### *2.1. HD and HT Enhanced the Health and Lifespan of Wild Type Nematodes*

As stress resistance is one of the key features characterizing the health status of an organism [44], heat stress resistance was determined in the presence and absence of HD in different concentrations. This test was also used to find the optimal concentration for further investigations. The results of HT treatment are shown in addition to enable the direct comparison between pure and mixed polyphenol treatments.

We observed the survival of worms starting from the 3rd day of adulthood, which was the day of heat stress exposure, until all worms died. The mean survival, which refers to the time between that stress exposure day until the end of the test, was increased by about 22% (from 2.23 days in

the control group to 2.71 days in the treated group) during 250 μg/mL HT treatment (Figure 1A), whereas 250 μg/mL HD extended the survival by even 40% (from 2.96 in the control to 4.13 days) (Figure 1B). Moreover, the maximum survival, defined as the time point when 90% of the population was dead, increased by 63% during 250 μg/mL HD treatment (*p* ≤ 0.001 with Fisher's Exact Test) and only by 14% during 250 μg/mL HT treatment (*p* ≤ 0.05 with Fisher's Exact Test) compared to the respective control. 250 μg/mL was the most effective concentration for both treatments in terms of mean and maximum survival after stress exposure (Table S1), thus, this concentration was selected for the following experiments.

**Figure 1.** Heat stress resistance in the presence of HT and HD. At the third day of adulthood (day 1) wild type nematodes were exposed to heat stress at 37 ◦C for 3 h prior monitoring survival. The survival is plotted as the percentage of the initial population in the control group as well as in the HT (**A**) and HD (**B**) treated groups. Three biological replicates were combined with a total of ≥52 nematodes per treatment. Statistical significance was calculated by log-rank test. Differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*).

Besides stress resistance, locomotion is one of the most important features that reflects the general fitness and health status of nematodes [45,46] and shows a constant decline during the ageing process [47]. Therefore, the swimming behavior was monitored in three different age classes with and without polyphenol treatment. Three age-dependent motion-parameters were chosen, that is wave initiation rate (often referred to as thrashing speed), activity index, and body wave number. The wave initiation rate is the number of body waves per minute, which indicates the movement-speed, whereas the activity index adds up the number of pixels that are covered by the nematode during the time spent for two strokes as an indicator for the vigorousness of bending over time. Furthermore, the body wave number, which is low in healthy and young worms, determines the waviness of the body at each time point. The data obtained in the current study verify the age-dependence of all selected swim parameters (Figure 2A–C). As previously described in Restif et al. [47] and Ibáñez-Ventoso et al. [48], the wave initiation rate (Figure 2A) and the activity index (Figure 2C) decreased with age, whereas the body wave number (Figure 2B) increased during ageing.

**Figure 2.** Healthspan benefits in wild type nematodes treated with HD and HT. The analysis of locomotion after polyphenol treatments comprises three parameters: the wave initiation rate (**A**), the body wave number (**B**) and the activity index (**C**). In two independent repeats, a total of ≥63 nematodes were analysed per treatment and age. In addition, the autofluorescence was monitored in two biological repeats with a total of ≥37 nematodes per treatment and age (**D**). Data are represented as mean ± SEM and statistical differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). A3, A7, A12: 3rd, 7th and 12th day of adulthood. Finally, an example for the typical appearance of red autofluorescence is shown (**E**) with the respective shot in bright field (**F**) as well as the merged and processed picture for the analysis in CellProfiler (**G**).

Interestingly, the movement speed was not influenced by 250 μg/mL HT in any age group, but 250 μg/mL HD could provoke an increase of 28%, 36%, and 42% in the wave initiation rate at the 3rd, 7th, and 12th day of adulthood, respectively (Figure 2A). Furthermore, HD was also able to enhance healthspan by decreasing the body wave number by at least 27% in all three age groups (Figure 2B) and by increasing the activity index by 30% and 48% at the 7th and 12th day of adulthood (Figure 2C), whereas HT did only positively influence these parameters at the 12th day of adulthood.

In addition, a well-known biomarker was investigated to analyse the beneficial effects of 250 μg/mL HD on the healthspan of *C. elegans*. The amount of autofluorescent material, sometimes referred to as lipofuscin or "age pigment", increases in *C. elegans* during ageing [49]. It was shown that red autofluorescence, which is mainly located in the intestine, reflects the ageing and health status of nematodes in the most reliable way [43]. Therefore, red fluorescence was measured in *C. elegans* during ageing, whereas the total intensity was calculated per worm body as illustrated by Figure 2E–G. Both treatments, HT and HD, were able to decrease the accumulation of the fluorescent material at the 12th day of adulthood by 3%, an effect which was absent in younger worms (Figure 2D).

Finally, the influence of HT and HD on the mortality rate was measured in wild type nematodes under standard laboratory conditions. The treatment with 250 mg/mL HT and HD resulted in an increase of mean lifespan by 14% (Figure 3A) and 12% (Figure 3B), respectively. The maximum lifespan, however, was only slightly (without significance according to the Fisher's Exact Test) increased by 7% after HD-treatment and the biggest increase was visible in the median lifespan (16%). A similar pattern was observed in the HT-treated group.

**Figure 3.** Life prolonging effects of HT and HD in wild type. The survival curves of control and polyphenol treated nematodes are shown. Survival is expressed as a percentage of the initial population per day. The curves represent three independent trials with a total of 250 and 286 nematodes in the control and HT treated nematodes, respectively (**A**) and two independent trials with a total of 172 and 144 nematodes in the control and HD treated nematodes, respectively (**B**). Statistical significance was calculated by log-rank test; differences compared to control were considered significant at *p* < 0.001 (\*\*).
