*3.2. Hormesis Is Involved in Neuroprotective E*ff*ects from HD*

Mild stress-induced hormesis represents a promising strategy to improve longevity and healthy aging. A recent paper demonstrated that hormesis leads to ageing-deceleration and highlighted its beneficial effects on lifespan, overall healthspan and especially stress resistance by activation of *daf-16*, *sod-3*, *ctl-1*, *hsp-16.2* and *sir-2.1* longevity genes in *C. elegans* [69]. Other recent studies also indicated the anti-aging effect of hormesis and considered it as an overcompensation stress response to the disruption in homeostasis via HSF-1 and SKN-1/Nrf2 signalling pathways [70,71]. The transcription factor SKN-1, the *C. elegans* orthologue of mammalian Nrf2 protein, is a well-known longevity factor

that plays an essential role in oxidative stress response. It has also been reported that activation of SKN-1 induces the suppression of DAF-16, another well-known longevity gene, leading to detrimental effects on stress resistance and lifespan in *C. elegans*. In the same study, it has been demonstrated that oleic acid induces protective actions by regulating DAF-16 to promote lifespan extension and health in ageing worms [72]. In addition, a recent study reported that a chaperone complex mediates longevity response between HSF-1 and DAF-16/FOXO by reducing insulin/IGF-1 signaling to increase the lifespan in *C. elegans* [73]. The 90-kDa heat-shock protein (HSP-90) is an essential, evolutionarily conserved eukaryotic molecular chaperone [74]. Consistent with this concept, a recent study showed that DAF-21/HSP-90 is required for *C. elegans* longevity and provides a functional crosstalk between the proteostasis and nutrient signaling networks by ensuring DAF-16/FOXO isoform A activity [75]. Interestingly, the sirtuin family, named after mammalian Sirtuin 1 (SIRT1), features a high number of sirtuin-orthologs in several organisms, such as SIR-2.1 in *C. elegans*. Recently, it has been demonstrated that HSP-90/Hsp90 modulates lifespan via SIR-2.1/SIRT1 in *C. elegans* and in mammalian cells [76].

The Brunetti et al. [15] findings were supportive of a hormetic dose response for hydroxytyrosol with the optimal concentration at 250 μg/mL, the same concentration employed in the present study within a similar preconditioning experimental system. These findings are consistent with a substantial body of research that shows that preconditioning experiments which employed an adequate number of conditioning doses typically demonstrate a hormetic dose response [77,78], which is characterized by a low dose stimulation with the maximum response typically about 30–60% greater than the control response, similar to what was reported here. Thus, a hormetic background mechanisms for the action of HD seems plausible. Hidrox® has an excellent safety profile, with no adverse effects even at a very high dose [41]. Consistent with this notion, in vitro studies have evaluated HT as a non-genotoxic and non-mutagenic compound with NOAEL (No Observed Adverse Effects Level) classification, suitable for long-term consumption [79]. Thus, the safety profile of Hidrox® makes it an excellent food supplement.

#### *3.3. Implications in PD Pathogenesis*

Delaying aging, e.g., as seen after HD treatment, is a neuroprotective mechanism that may provide potential prevention in worm models of PD [80]. Parkinson's disease (PD) is a neurodegenerative disorder characterized by a severe depletion in number of dopaminergic cells of the *substantia nigra* (SN). As an effect of this reduction in dopaminergic neurons, a significant fall in brain dopamine (DA) levels occurs [81]. Although several hypotheses have been raised, including (i) defective DNA repair mechanisms, (ii) specific genetic defects, (iii) mitochondrial dysfunction, or (iv) toxic compounds in the environment, none of these, alone, completely explains the cascade of events responsible for the onset and progression of PD [81–84]. A large body of evidence demonstrates that free radicals play a key role in the pathogenesis of PD. In fact, there is a 10-fold increase in hydroperoxide levels in SN in PD, and dopaminergic neurons produce hydrogen peroxide either enzymatically, through the activity of mono-amine oxidase-A (MAO-A), or non-enzymatically via the intracellular autoxidation of DA [81,85]. Once formed, hydrogen peroxide by reacting with the reduced form of transition metals, such as Fe (II) and Cu(I), gives rise to the powerful oxidant hydroxyl radical and oxidative damage to nigral membrane lipids, proteins, and DNA ensues [86]. Reduced glutathione (GSH) significantly contributes to the detoxification of hydroxyl radical; in fact it reacts with the free thiol group of GSH which is oxidized to GSSG [86,87]. Unfortunately, SN has very low levels of GSH compared with other brain areas and this contributes to the triggering of PD pathogenesis by free radicals [88].

A number of worm models of PD have been generated through either exposing worms to a neurotoxic chemical such as 1-methyl-4-phenylpyridium ions (MPP+) or *6*-*hydroxydopamine* (6-OHDA) or by reproducing the genetic defect present in the inherited form of PD [53]. In particular, many recent data suggest a key role played by phenolic components of extra virgin olive oil (EVOO) in counteracting protein misfolding and proteotoxicity, with a particular emphasis on the mechanisms leading to the onset and progression of PD. Notably, HT efficiently neutralizes free radicals and protects

biomolecules from ROS-induced oxidative damage. In this regard, 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 [89–91]. These protective enzymes include NADPH quinone oxidoreductase-1, heme oxygenase-1, glutathione S-transferase, and the modifier subunit of glutamate cysteine ligase, which catalyzes the first step in the synthesis of GSH [10]. It is noteworthy that HT, a product of dopamine metabolism, is present in the brain [92]. Specifically, monoamine oxidase (MAO) catalyzes oxidative deamination of dopamine in a neurotoxic metabolite, DOPAL (3,4-dihydroxyphenylaldehyde) in dopaminergic neurons [93]. The latter can be oxidized by aldehyde dehydrogenase (ALDH) to DOPAC (3,4-dihydroxyphenylacetic acid), the major metabolite of dopamine in the brain or may be reduced to HT by alcohol dehydrogenase (ADH). At the same time, DOPAC reductase can transform DOPAC into HT [94]. DOPAL is a highly reactive metabolite, suggesting that it might be a neurotoxic dopamine metabolite with a role in the pathogenesis of PD.

Several studies reported the neurotoxicity of DOPAL in vivo and strongly suggest its role in PD pathogenesis [95].

The health effects of olive polyphenols, particularly HD and HT on the ageing process in old worms (i.e., increased locomotion and reduced intestinal autofluorescence) have been reported here. Our study reinforces the hypothesis that HD is protective against PD pathogenesis. This is in agreement with other studies showing that the olive fruit derivatives oleuropein and HT, as well as other polyphenols, such as the green tea derivative epigallocatechin 3-gallate (EGCG) inhibit DA-related toxicity and protect against environmental or genetic factors that induce DA neuron degeneration by the modulation of Nrf2-Keap1 and PGC-1α anti-oxidative signaling pathways in vitro and in vivo [96–98]. In addition, another recent study suggested that tyrosol from EVOO is effective in reducing α-synuclein inclusions, resulting in a lower toxicity and extended lifespan of treated transgenic nematodes [99].

Notably, α-synuclein is an aggregation-prone neuronal protein whose cellular function is not well known. As *C. elegans* has no orthologue of this protein, worm models have been generated by overexpression of wild-type or mutant forms of human α-synuclein in different tissues (i.e., either body-wall muscle, pan-neuronal, or only in the dopaminergic neurons). In most cases, overexpression leads to locomotion defects or the degeneration of dopaminergic neurons [26].

Interestingly, both polyphenols mitigated, age-dependently, the build-up of human α-synuclein in the body wall muscle cells of a transgenic *C. elegans* model (strain OW13) and improved swim performance. Our results are relevant to PD pathogenesis, due to the central role of mitochondria in metabolism, ROS regulation, and proteostasis [100]. The extent to which these pathways, including the mitochondrial unfolded protein response (UPR) and mitophagy, are active may predict severity and progression of these disorders, as well as sensitivity to chemical stressors. This holds true when considering, in a PD-like context, that transgenic nematodes express the Lewy body constituent protein α-synuclein. In fact, recent studies suggested that co-expression of α-synuclein and ATFS-1-associated dysregulation of the UPR synergistically potentiate dopaminergic neurotoxicity [101]. Moreover, other evidences have demonstrated that, in *C. elegans*, the inducible transcription factor SKN-1, directly controls UPR signaling and controls the transcription factor genes of XBP-1 and ATF-6 [102].

#### *3.4. HD Is More E*ff*ective in Health Maintenance than Pure HT*

In the present paper, we compared the health effects of Hidrox® and hydroxytyrosol as neuroprotective agents in *C. elegans* wild type and PD models. An important consideration that has emerged from this study relates to the different strength of biological activities delivered by hydroxytyrosol in its "natural" environment versus hydroxytyrosol in its purified, synthetic form. Although HT and HD were formulated to the same concentrations of 250 μg/mL, the two formulations used in the experiments contain dramatically differing hydroxytyrosol concentrations. HD as a raw formulation, indeed, contains a much smaller amount of hydroxytyrosol. 250 μg of HD correspond to 30 μg total polyphenols and only approximately 15 μg hydroxytyrosol. Thus, in comparison to the HT

formulation (100% hydroxytyrosol), HD has 1/17th of the hydroxytyrosol in HT, but provides similar or even higher activity in all the assays so far described.

The difference between a "natural" formulation of olive polyphenols and purified fractions of olive polyphenols is, however, not surprising. Several studies published in the literature [14,21,42] have confirmed that hydroxytyrosol, stripped from its natural environment and/ or cofactors which can be in minute concentrations in the juice of olives, provided a much less and limited range of activities in vitro and in vivo. Therefore, HD activity cannot just be accounted by and attributed solely to hydroxytyrosol. While this study confirms that "natural" HD has a superior range of activities than its purified or synthetic hydroxytyrosol counterpart, further studies are needed to support this conclusion.

Due to its composition (see chromatogram in Figure S1), it is plausible that other polyphenols than HT alone, might be involved in the beneficial action of HD. HD, in fact, contains compounds such as oleuropein, oleuropein aglycone, tyrosol, and gallic acid, to only name a few [41,103]. Previous studies already showed their abilities to improve the life- and healthspan of the nematodes [15,35,40,98], thus, it could be argued that the beneficial action of HD is due to one of those polyphenolic ingredients independent of HT. However, oleuropein, for instance, being the second most abundant polyphenol in HD, is only present in trace amounts. Only about 1% of HD consists of oleuropein so that the final concentration of 250 μg/mL HD delivers only about 2.5 μg/mL oleuropein. In our previous study, we could show that even 30 μg/mL HT or oleuropein aglycone were not sufficient to provide healthspan benefits [15]. Thus, it is unlikely that any single polyphenol included in HD is responsible for the observed effects. More likely, the polyphenols act in concert, whereas HT as the most abundant polyphenol in HD plays a central role. Additional experiments with chemically defined mixtures of polyphenols and other HD ingredients must be performed in future to find the responsible elements for healthspan enhancement by HD.

Our findings are in agreement with other studies indicating a higher cytoprotective activity of HD than that of purified HT. Consistent with this line of evidence, in vitro studies have measured the damage produced to the cell membrane by potent oxidant agents like H2O2 and 15-HPETE in bovine heart endothelial cells, and revealed that HD was extremely active in preventing membrane damage even at concentrations in micromolar range (10−6), while pure HT obtained by HPLC separation was not protective against oxidant insult and damage [104]. On the contrary, high concentrations of purified HT produced a pro-oxidation effect and increased the cytotoxicity of the oxidants [103]. Another in vitro study confirmed that HD had higher antioxidant and anti-inflammatory activity than the same amount of pure HT, as demonstrated following the oxidation of mitochondrial membrane lipids by free radicals applying Electron Spin Resonance (ESR) spectroscopy (using Superoxide, HO radical and NO radicals as toxic agents) [104]. Furthermore, by measuring the effect of HT and HD on atherosclerosis lesions in an animal model, it was shown that the phenolic-enriched olive product (HT), out of its original matrix, could not only be not beneficial but actually harmful. The results suggest that the formulation of functional foods may require maintaining the natural environment in which these molecules are found [105]. In humans, HD administrated orally (1 mL of Olivenol, 2.5 mg HT) significantly increased plasma antioxidant activity and, in addition, bioavailability studies have established that, after ingestion, the absorption of HT from olive oil is dose-dependent, with low adsorption bioavailability after administration of a high amount of pure HT [106].
