Natural phenolic substances are secondary plant metabolites, a major group of over 8000 plant compounds chemically characterized by the presence of one or more aromatic rings with one or more hydroxyl Substituent's [15]. Plant polyphenols belong to the phyto alexins family, including molecules synthesized by plants, often in leaves, to combat pathogens (bacteria, fungi) or to discourage leaf-eating insects. In the family of Oleaceae, some polyphenols are highly concentrated also in fruits before ripening, discouraging animals to eat fruits before their maturation for seed dispersal. The olive tree (Olea europaea) produces a specific battery of polyphenols including flavonols, lignans and glycosides. The glycosides are comprised in the iridoids class; these molecules result from a cyclopentane ring fused to a six atom-membered hetero cycle containing oxygen; when the cyclopentane ring is broken the molecule is referred to as a secoiridoid (Figure 1).
Olive tree polyphenols are found in the lipid and water (as minute droplets) fractions (in the latter case as glycones) of olive oil. They include the phenolic alcohols hydroxytyrosol (HT, 3,4-dihydroxyphenylethanol, 3,4-DHPEA) and tyrosol (p-hydroxyphenylethanol, p-HPEA) and their precursors; the HT ester of elenolic acid, known as oleuropein (OLE), is the main responsible for the strong bitter taste of olive leaves and drupes. Less represented polyphenols are the dialdehydic derivative of decarboxymethyl elenolic acid bound to either HT (3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, 3,4-DHPEA-EDA, oleacein) or to tyrosol (p-hydroxyphenylethanolelenolic acid dialdehyde, p-HPEA-EDA, oleocanthal). The latter is responsible for the burning sensation occurring in the throat following EVOO ingestion [27]. Other olive polyphenols include verbascoside, the caffeoylrhamnosylglucoside of HT, the lignans 1-acetoxypinoresinol and pinoresinol. Among these polyphenols, the most represented in olive oil are oleuropein, both in the glycated and in the aglycone forms, its main metabolite, hydroxytyrosol, and oleocanthal. The content of polyphenols in EVOO can vary largely and can reach levels exceeding 60 mg/100 g; it depends on the olive cultivar, the ripening stage of the fruit, environmental factors, the extraction conditions, the systems to separate oil from olive pastes and the storage conditions and time. The latter affect spontaneous oxidation and the deposition of suspended particles.
Molecular Mechanisms Underlying Protection of Olive Polyphenols against NeurodegenerationModulation of the mTOR/AMPK pathwaysMost beneficial properties of EVOO polyphenols, notably OLE, have been associated, in addition to their well-known antioxidant power, to the modulation of several types of signaling pathways involving AMP-activated protein kinase (AMPK) and mammalian target of rapamycin mTOR, whose inhibition favors longevity and reduces inflammatory states [7,28]. As far as AMPK is concerned, it is well known its key role in AD pathogenesis [29]. mTOR is a central regulator of cell metabolism that finely tunes the balance between anabolism and catabolism; it is also one of the most potent upstream inhibitors of autophagy, and this inhibition is removed following phosphorylation by AMPK [30]. However, the modulation by OLE of other neuronal signaling pathways, as it is the case of resveratrol [31-33], cannot be excluded.
Modulation of cell proteostasisPolyphenols, including the olive ones, are involved in regulation of
cell proteostasis, including the ubiquitin/proteasome pathway and the
autophagic flux, the latter under the control, among others, of the
mTOR/AMPK machinery (see above) [34]. The proteasome is the major
proteolytic complex, and increasing evidence indicates that proteasomal
function declines significantly with aging and in aging-related
pathologies [35]. Plant polyphenols, including oleuropein, can increase
proteasome activity, with increased life span [36], together with the
expression of proteasomal subunits and of protein chaperones [37].
Autophagy, notably macroautophagy, is a highly conserved cellular program devoid to degradation of chemically modified, misfolded and aggregated proteins as well as aged or damaged organelles, particularly mitochondria (mitophagy). Autophagy is reduced in aged people and it appears severely impaired in people affected by neurodegenerative pathologies [38] including Alzheimer's and Parkinson diseases [39,40]; accordingly, molecules able to foster autophagy appear as good candidates to combat aging-associated neurodegeneration.
Plant, including olive, polyphenols stimulates remarkably the autophagic flux similarly to other well-known autophagy inducers such as lithium, valproic acid and rapamycin [41]. Activation of autophagy appears as one of the ways polyphenols induce most of their beneficial effects against neurodegeneration [23].
Plant polyphenols can increase chaperone-mediated autophagy, a type of autophagy, through their reported effect of cell stressors, with activation of the heath-shock response and increased chaperone levels, as it has been shown for resveratrol [42] and other polyphenols, although Hsp90 imbibition's by oleocanthal has also been reported [43]. In cultured neuronal cells, we have shown that OLE triggers macroautophagy by increasing intracellular calcium levels with ensuing AMPK activation through dependent protein kinase β (CAMKKβ) [44]. A similar mechanism was previously reported for resveratrol [45]. Other authors have shown that resveratrol stimulates AMPK activity through PKA in double transgenic mice [31]. In both cases, the final outcome was mTOR inhibition through phosphorylation of TSC or the mTOR component Raptor [46]. AMPK can also stimulate autophagy directly, by phosphorylation and activation of ULK1, a key component of the autophagic pathway [47]. These data support the idea that autophagy activation by OLE (and presumably other olive polyphenols) proceeds via the Ca2+/CAMKKβ/AMPK signaling pathway coupled with mTOR inhibition, similarly to data reported for other plant polyphenols [48].
Less data on the beneficial effects of other olive polyphenols in biological and animal models are presently available. In the case of oleocanthal, a recent study performed on a human blood brain barrier model (BBA) and on the TgSwDI murine model of AD showed that animals administered for 4 weeks with the molecule displayed enhanced cerebral clearance of Aβ matching significantly reduced plaque load in the hippocampus. The enhanced cerebral clearance resulted both from increased transport across the BBA and from the activation of the ApoE-dependent clearance pathway [23]. As far as HT is concerned, this polyphenols and its acetylated derivative were reported to be protective in rats fed for 7 days with 5 or 10 mg/kg per day of either compound, respectively [49]. Finally, recent experiments reported that HT administration to the C57BL/6 mouse model of AD results in a significant improvement of the Neuro-behavioral dysfunction in terms of spatial reference and working memory, induced by intra cerebral injection of Aβ42 plus ibotenic acid [10].
Modulation of the redox status
Oxidative and nitrosative stress can impair the functionality of brain tissue thus resulting one of the abnormalities underlying neurodegeneration; accordingly, the presence of anti-oxidant molecules, such as olive polyphenols can ameliorate the redox status and hence restore optimal neuronal function. Actually, plant polyphenols, including OLE, have been reported to attenuate the oxidative stress not only due to their well-known antioxidant power but, more remarkably, by restoring mitochondrial function following activation of the nuclear factor (erythroid-derived- 2)-like 2, (Nrf2) pathway. Nrf2 is a key transcription factor and a master regulator of the antioxidant response element; it is involved in cell protection against oxidant molecules [50] and its activity matches strongly mitochondrial function and biogenesis. Oleuropein reduces the oxidative damage in the substantia nigra of senescent rats by increasing the catalytic activity of antioxidant enzymes thus protecting against dopaminergic neuronal loss [51]; it also improves the mitochondrial activity in the paraventricular nucleus of the hypothalamus through fostering the Nrf2 pathway with increased expression of antioxidant enzymes such as hemoxigenase 1 and NAD(P)H dehydrogenase quinone 1 [52].
The ability of plant polyphenols to restore a correct redox status is of particular significance in PD, where the substantia nigra dopaminergic cells are exposed to the prooxidant products of dopamine metabolism. In particular, 3,4-dihydroxyphenylacetaldehyde (DOPAL), the main dopamine metabolite, induces in vivo the loss of dopaminergic neurons in PD thus favoring α-synuclein neurotoxicity [53,54]. Another product of dopamine metabolism is HT, the main metabolite of oleuropein. Due to its strong anti-oxidant power, HT relieves DOPAL cytotoxicity; it also diminishes NO-induced cytotoxicity in murine dissociated brain cells [55] and protects rat brain slices subjected to hypoxia-reoxygenation [49]. HT has also been reported to protect cultured dopaminergic cells against the toxic insult provided by dopamine and 6-hydroxydopamine. Also in this case, it was reported that cell protection resulted from the HT-induced expression of phase II detoxifying enzymes, notably NAD(P)H quinone oxidoreductase [24]. These data suggest the possible efficacy of HT against dopamine metabolism-related dopaminergic dysfunction and hence in PD therapy.
Epigenetic effects
Plant polyphenols can also counteract aging as well as many of its pathological consequences resulting from aberrant epigenetic mechanisms [56,57]; accordingly, epigenetic issues targeted by diet polyphenols have become an attractive approach for the anti-neurodegeneration power of these substances. In the field of neurodegenerative diseases, the epigenetic modifications have been shown to induce effects similar to those provided by caloric restriction in humans [7]. In this context, we have recently shown that OLE administration down regulates the expression of histone deacetylase 2 (HDAC2), an enzyme known to be up regulated in AD [58], both in the hippocampus and in the cortex of TgCRND8 mice and that such decrease matches a significant increase of histone H3 and H4 acetylation [59]. It must be stressed that histone acetylation has been reported to improve cognitive deficits in animal models of AD and this indication has been proposed as a novel therapeutic strategy against AD [60]. Epigenetic involvement in some of the aforementioned positive effects resulting from OLE administration is further supported by the modifications of the expression levels of effector proteins such as the glutaminyl cyclase, involved in the generation of pyro (3-42) Aβ (pE3-Aβ) a particularly sticky Aβ derivative considered involved in plaque generation, and some autophagic players such as beclin-1 and LC3. Overall, these data agree with the reported ability of hydroxytyrosol to restore the expression of a number of genes involved in the regulation of memory and survival in transgenic mice [10]. They also add to a large wealth of data that support the ability of many plant polyphenols to induce epigenetic modifications mainly in cancer [61], leading to the proposal of the possibility of an epigenetic diet [62]. Thus, in spite of the limited information on the epigenetic effects of olive polyphenols presently available, modulation of epigenetic flaws by natural polyphenols appears as a promising subject for the development of new drugs based on their molecular scaffold especially designed to combat several chronic degenerative pathologies, possibly including aging-associated neurodegeneration [63].
Olive Polyphenols Protect Transgenic Mouse Models Against Neurodegeneration
The TgCRND8 (Tg) mice, encoding the Indiana and the Swedish mutations in the APP gene, are cognitively impaired by the age of 3 months [64] and develop a pattern of Aß deposition recalling several aspects of human AD [21]. Small size Aß42- immunopositive plaques appear in various brain areas, including cortex and hippocampus, by the age of 3 months. As a function of age, they become small-medium to big in size and radiating in shape and acquire a compact core, becoming numerous and reaching the maximum roughly by 7-9 months of age [21]. In addition, in later stages, from 6 to 12 months of age, of their life this Tg strain exhibits a huge amount of pyroglutamylated Aß (pE-Aß) deposits throughout the cortical and hippocampal areas with a pE3-Aß load much higher than that of Aß 42 [59]. We previously showed that dietary supplementation of OLE (50 mg/kg of diet) to TgCRND8 mice results in a significant reduction of both soluble and aggregated Aß and of the astrocyte reaction in AD relevant brain areas with a remarkable attenuation of Aß-mediated cognitive deterioration [21], LTP restoration and reduction of pE3-Aβ. In addition, in aged mice displaying increased pE3-Aß in the brain deposits, we found that OLE is active against glutaminylcyclase-catalyzed pE3-Aß generation by reducing enzyme expression and that it interferes with both Aß42 and pE3-Aß aggregation [59].
Similarly, feeding TgCRND8 mice with a mix of polyphenols from olive mill waste water at the total dose of 50 mg/kg of diet resulted in a remarkable improvement in both non-spatial episodic memory and working memory that correlates with the loss of Aß42 and pE3-Aß load in the brain [65]. Altogether, these data show that olive polyphenols are beneficial either when administered as pure components, OLE, or as a mixture and that their effects are comparable.
Administration of olive polyphenols reverts neurodegeneration in the TgCRND8 mouse model
The importance of autophagy derangement in neurodegeneration was confirmed by our studies carried out in the TgCRND8 mouse model showing the importance of autophagy activation as a key effect of OLE protection against neurological impairment. When compared with untreated littermates, we found that OLE administration for 8 weeks to these mice activated
autophagy, reduced the inflammatory response resulting from the accumulation of amyloid aggregates of the Aß peptides and the tissue levels of Aß itself and of its pE3-Aßderivative following down-regulation of glutaminyl cyclase. The OLE-treated mice also displayed a significant reduction of the astrocyte reaction in the affected brain areas, with strong improvement of memory and behavioral performance, that reached the levels recorded in wild-type mice. Finally, the OLE-treated mice displayed increased hippocampal neurogenesis and improved synaptic behavior (as increased LTP) [21,59]. Similar results were reported in C57BL/6 mice, a transgenic model of AD, injected with HT in the cerebral ventriculi. The injected mice showed a remarkable improvement of the neurobehavioral dysfunction, with reversal of dysregulation of several signaling pathways and preservation of the mitochondrial architecture respect to untreated controls [10].
Drawbacks Associated with the Use of Plant Polyphenols
The pharmacokinetics and pharmacodynamics of olive polyphenols together with the minimal effective doses to be taken to ensure health benefits are poorly investigated. In the case of OLE-fed TgCRND8 mice, it was shown that OLE supplementation could be reduced by 80% without a significant loss of protection whereas a 99% reduction was completely ineffective (Casamenti et al. unpublished data).This lack of knowledge matches a problem associated with the use of olive, and other plant, polyphenols, i.e. their reduced bioavailability due both to incomplete intestinal absorption and to rapid biotransformation. However, many studies have clearly shown that OLE is indeed absorbed by animals and humans and distributed, at least in part, to tissues before degradation and excretion [66-68]; moreover, we have shown that some OLE metabolites, notably HT, arising mainly from gastric hydrolysis, are found in the brain of TgCRND8 mice after an acute oral administration of OLE, which supports the ability of OLE and/or some of its derivatives, including HT, to cross the bloodbrain barrier [59]. We have also shown that the protective effects found in TgCRND8 mice fed with OLE were reproduced when the animals were fed with the same amount of total polyphenols present in olive mill waste water, supporting the idea that different olive polyphenols possess, directly or indirectly, similar protective properties in animals and are similarly absorbed [65]. Overall, in spite of the reduced available information on many aspects of plant polyphenols pharmacokinetics, pharmacodynamics and their metabolic modifications in the organism, also including chemical modifications by the gut micro biota, it is possible to consider that OLE and other olive, and more generally, plant polyphenols are absorbed, though in reduced amounts, and distributed to the whole organism, including the brain, before metabolic modification and disposal [16].
Conclusion
The identification of novel effective molecules or molecular scaffolds able to contrast protein/peptide aggregation hindering the formation of the plaque deposits widely considered key responsible for neuronal impairment in neurodegenerative diseases is a major, still unmet, medical need for the coming years. The studies carried out in the last decade have highlighted the beneficial effects of the MD against several aging-associated pathologies including neurodegenerative diseases, thus providing strong value to this alimentary regimen. More recently, an increasing body of evidence has started to unravel the molecular basis of these beneficial effects, assigning a key role to the polyphenols enriched in such a diet, particularly those found in red wine (resveratrol) and in the EVOO, key components of the MD. These data are providing a strong rationale and a convincing scientific basis to the health value of the MD in general and, particularly, to the use of nutraceuticals to supplement the dietary intake of these substances, also in the light of their reduced bioavailability. More knowledge is needed to fully confirm these considerations in humans, mainly for what a deeper insight on the molecular and physiological basis of the claimed effects and a better knowledge of all the factors contributing to the effective bioavailability of these substances are concerned. However, the data presently available can be a strong starting point to definitely ascertain the real efficacy of any polyphenols-based nutraceutical treatment aimed at hindering the appearance of aging-associated neurodegeneration and its clinical signs.
The authors are supported by grants from the University of Florence and the Ente Cassa di Risparmio di Firenze (2015).
Open Access
by Scient Online is licensed.
