Recently, several lines of evidence have demonstrated the health effects of extra virgin olive oil (EVOO) and have suggested that its consumption ameliorates age‐related memory decline and decrease the risk of AD onset (Farr et al., 2012 ; Feart, Samieri, Alles, & Barberger‐Gateau, 2013 ; Pitozzi et al., 2010 ). While most of the experimental studies have been focusing on the ability of EVOO to enhance Aβ clearance and reduce its deposition (Abuznait, Qosa, Busnena, El Sayed, & Kaddoumi, 2013 ; Qosa et al., 2015 ), only fewer research groups have been investigating the tau‐modifying ability of EVOO. On this regard, studies reported that some natural phenolic derivatives from olives, as well as oleocanthal, can prevent tau fibrillization in vitro (Daccache et al., 2011 ; Monti, Margarucci, Tosco, Riccio, & Casapullo, 2011 ). More recently, we have shown that chronic administration of an EVOO‐rich diet modulates positively several aspects of the AD‐like pathology in a transgenic mouse model with plaques and tangles, the 3×Tg mice (Lauretti Iuliano, & Praticò, 2017 ; Lauretti, Li, Di Meco, & Praticò, 2017 ). However, since this model manifests both aspects of the AD pathological phenotype, whether EVOO can modulate tau neuropathology independently from Aβ remains to be fully investigated. This is an important biological question with relevance not only for AD but also for primary tauopathies, such as progressive supranuclear palsy and Pick's disease, which are characterized by the presence of only tau pathology. With this goal in mind, in the present paper we evaluated the effect of chronic EVOO administration in tau transgenic mice (hTau) in which the mouse tau gene is replaced by normal human tau (Andorfer et al., 2003 ).

Remarkably, tau pathology strictly correlates with the severity of dementia (Arriagada, Growdon, Hedley‐Whyte, & Hyman, 1992 ; Riley, Snowdon, & Markesbery, 2002 ). Tau toxicity has been mostly attributed to its loss of function as a microtubule stabilizer, which leads to impaired neuronal transport, altered mitochondria function, and synaptic transmission (Lasagna‐Reeves et al., 2012 ). Tau phosphorylation and other post‐translational modifications promote tau self‐aggregation initially into oligomers and eventually into neurofibrillary tangles. Tau oligomers are often found at both pre‐ and postsynaptic sites in AD, supporting the notion that they contribute to synaptic transmission dysfunction (Guerrero‐Muñoz, Gerson, & Castillo‐Carranza, 2015 ; Tai et al., 2012 ).

Alzheimer's disease (AD) and related tauopathies are neurodegenerative disorders characterized by the presence of highly phosphorylated and misfolded forms of the microtubule‐associated tau protein, which accumulate as neurofibrillary or gliofibrillary tangles in the human brain (Di Meco, 2019 ). Currently, the mechanisms and cellular pathways leading to aberrant tau phosphorylation and progressive accumulation are not fully understood (Arendt, 2016 ) and no therapy is available to cure or halt the progression of these pathologies. The most striking symptoms common to all these diseases are the loss of memory and deficits in cognitive function. In fact, the brain of tauopathy patients reveals severe synaptic degeneration, neuronal dysfunction, and ultimately neuronal loss. Studies in animal models of the disease have shown that the early stages of tauopathy are characterized by alterations in both pre‐ and postsynaptic turnover rates, reduction of synaptic strength and dendritic spine size, and decrease in spontaneous and evoked cortical neuronal activity (Busche et al., 2019 ; Jackson et al., 2017 ).

Give that EVOO is known to be rich in polyphenols, potent antioxidant, and anti‐inflammatory agents (Bayram et al., 2012 ; Rigacci, Stefani, Segura‐Carretero, & Gómez Caravaca, 2016 ), we also investigated two well‐established markers of neuroinflammation: IBA1, marker of microglia activation, and GFAP, marker of astrocytes activation. As shown in Figure 5 c and d under our experimental conditions, compared with controls the cellular inflammatory response in the central nervous system of these mice was not influenced by the dietary regimen.

Effect of chronic administration of EVOO‐rich diet on synaptic integrity and neuroinflammation. (a) Representative Western blot analyses of synaptophysin (SYP) and postsynaptic density protein 95 (PSD95) in brain cortex homogenates of control (CTR) and EVOO‐treated (EVOO) hTau mice ( n = 6 per group). (b) Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. (c) Representative Western blot analyses of GFAP and IBA1 in brain cortex homogenates of CTR and EVOO mice ( n = 6 per group). (d) Densitometric analyses of the immunoreactivities to the antibodies shown in the previous panel. Values represent mean ± SEM

Since tau phosphorylation and tau oligomers are known to target synapse, next we investigated whether overall synaptic integrity of the hTau mice was affected by the EVOO‐rich diet. To this end, we assayed the steady‐state levels of synaptophysin (SYP) indices of presynaptic integrity and the postsynaptic density protein 95 (PSD‐95) in cortices of the two groups of mice. As shown in Figure 5 a, b, no significant differences were observed between the two groups when steady‐state levels of these proteins were assessed.

To fully characterize tau pathology, we also looked at two additional toxic forms of tau protein: the formic acid‐soluble tau aggregates and the soluble aggregates known as tau oligomers, as recognized by tau oligomer‐specific antibody T22 (Lasagna‐Reeves et al., 2012 ). As shown in Figure 4 g and h, although there was no change in the formic acid‐soluble form of tau, EVOO‐treated mice presented a significant reduction in tau oligomers levels when compared with controls.

To identify potential mechanisms that may be responsible for the observed changes in tau phosphorylation in the EVOO group, we measured the levels of the principal kinases involved in these tau post‐translational modifications. When comparing control and EVOO mice, immunoblot analysis showed no change for total glycogen synthase kinase 3‐β (GSK3‐β) but a significant increase in the inhibitory Ser9 phosphorylation of GSK3‐β (Figure 4 d,e). By contrast, no differences between the two groups were observed when steady‐state levels of CDK5, P35, and total or phosphorylated P38 were assessed (Figure 4 d,e). Since EVOO‐rich diet treated mice displayed higher total soluble tau protein levels, to determine whether this was resulting from an effect on transcription of the MAPT gene, we assessed tau mRNA levels. As shown in Figure 4 f, RT‐qPCR analysis revealed no significant changes in tau mRNA levels between the two groups.

EVOO‐rich diet regulates tau phosphorylation and tau oligomers accumulation in the brain of hTau mice. (a) Representative Western blot analyses for soluble tau (HT7) and phosphorylated tau at residues S202/T205 (AT8), T181 (AT270), T231 (AT180), S396/S404 (PHF1), and S396 (PHF13) in brain cortex homogenates from control (CTR) and EVOO‐treated mice (EVOO). (b) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (* p < .05, n = 6 per group). (c) Densitometric analyses of the phospho‐tau/total tau ratio for the different epitope immunoreactivities shown in panel A (* p < .05, n = 6 per group). (d) Representative Western blot analyses for cyclin‐dependent kinase CDK5, P35, glycogen synthase kinase (GSK3β, p‐GSK3β), P38 MAPK kinase, and pP38 MAPK protein levels in brain cortex homogenates from CTR and EVOO mice. (e) Densitometric analyses of the immunoreactivities to the antibodies from panel D (* p < .05) (f) MAPT gene expression level in brain cortex homogenates from CTR and EVOO mice. The mRNA expression levels of tau (MAPT) were determined using qRT–PCR analysis ( n = 6 per group). (g) Representative Western blot analyses for formic acid‐soluble tau (HT7) in brain cortex homogenates from CTR and EVOO mice and densitometry analysis ( n = 6 per group). (h) Representative Western blot analyses for tau oligomers as recognized by T22 antibody in brain cortex homogenates from CTR and EVOO mice and densitometry analysis (* p < .05, n = 6 per group). Values are means ± SEM

To assess the effect of EVOO‐rich diet on tau phosphorylation and pathology, we measured levels of total soluble tau protein and its phosphorylated isoforms in brain cortex from both groups. As shown in Figure 4 a, b, we found that while levels of total soluble tau were significantly increased, tau phosphorylated at Ser202/Thr205, as recognized by the AT8 antibody, was significantly reduced when EVOO‐rich diet group was compared to control. By contrast, no significant changes were observed between the two groups of mice when tau phosphorylated at Thr231, Thr181, Ser396/Ser404, and Ser396 as recognized by the antibodies AT180, AT270, PHF1, and PHF13 were assessed, respectively (Figure 4 a,b). However, when we analyzed the phospho‐tau/total tau ratio for the different epitopes we found a significant reduction also for tau phosphorylation at the specific Ser396 epitope, as recognized by PHF13 antibody (Figure 4 c).

Effect of chronic administration of EVOO‐rich diet on hippocampal pre‐ and postsynaptic protein expression in hTau mice. (a) Representative Western blot analyses for SNAP25, syntaxin 1A, synaptobrevin, complexin 1/2, and synaptophysin (SYP) in hippocampus homogenates from CTR and EVOO mice. (b) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (** p < .01; n = 6 per group) . (c) Representative Western blot analyses for the AMPAR subunit glutamate receptor 1 (GluR1), AMPAR subunit glutamate receptor 2 (GluR2), and their phosphorylated forms, pGluR1 (S831), pGluR1 (S845), and pGluR2 (S880), in hippocampus homogenates from CTR and EVOO mice. (d) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A ( n = 6 per group) . Values are mean ± SEM

To elucidate the mechanisms involved in the modulation of basal hippocampal synaptic activity and STP by EVOO‐rich diet, next we measured levels of pre‐ and postsynaptic proteins including SNARE complex, complexin 1/2, and synaptophysin that are critical for a proper function of the presynaptic vesicle release machinery, as well as postsynaptic AMPA receptor subunits phosphorylation and protein levels which are responsible for basal synaptic activity (Neeliyath et al., 2012 ; Trimbuch & Rosenmund, 2016 ). Among all the tested proteins, we found that only complexin‐1 levels were significantly increased in the EVOO‐treated hTau mice when compared with controls (Figure 3 a,b). In addition, the same group had a trend toward an increase in the steady‐state levels of synaptobrevin (Figure 3 b), and a decrease in the levels of AMPA receptor subunit GluA1 phosphorylated at serine 831 (Figure 3 c, d). However, in both cases the differences did not reach statistical significance.

Effect of EVOO‐rich diet on basal synaptic activity, short‐term and long‐term plasticity in hTau mice. (a) Input–output curve of fEPSP slope vs injected current in hTau (CTR) and hTau on EVOO diet (EVOO) mice. The upper inset shows representative voltage traces of fEPSP induced with stimulus intensity of 500 µA. (b) Time dependence of paired‐pulse facilitation of fEPSP slope as a ratio of fEPSP slope at pulse 1 to fEPSP slope at pulse 2 for different time points. The upper inset shows representative voltage traces of paired‐pulse fEPSP at 25 msec, 50 msec, 100 msec, and 200 msec inter‐pulse intervals. (c) Long‐term potentiation induced with 3 consecutive pulses of high‐frequency stimulation of 100 Hz and 1 s inter‐pulse interval. The fEPSP slope values are presented as a percentage of response after LTP induction to averaged response of baseline. The upper insets show representative voltage traces of fEPSP 1 min after LTP induction (left inset) and 60 min after LTP induction (right inset). (CTR = 7, EVOO = 7 number of slices each). * p < .05, ** p < .01

To elucidate changes in synaptic activity that may underlie the beneficial effect of EVOO‐rich diet on the observed hippocampal‐associated behavioral performance, we studied basal synaptic activity, STP, and LTP in the CA1 area of hippocampus of control and EVOO‐rich diet treated mice. To this end, we stimulated the Schaffer collaterals as an input and recorded fEPSP as an output response from stratum radiatum dendrites of the CA1 area to probe the activity of CA3‐CA1 synapses. As show in Figure 2 a, we found an enhanced basal synaptic activity as the analysis of input–output curve revealed significant increase in fEPSP slope induced by administration of current at high intensity in the EVOO diet treated mice when compared to controls. Next, we studied the effect of EVOO diet on short‐term plasticity by measuring the time dependency of paired‐pulse facilitation (PPF). For this parameter, the ratio of fEPSP slope at pulse 2 to fEPSP slope at pulse 1 was measured at different inter‐pulse intervals. Compared with controls, we observed that slices from EVOO group had a significant increase in the paired‐pulse ratio at a broad range of inter‐pulse time intervals, thus exerting the potentiating effect on PPF (Figure 2 b). Finally, we tested the effect of EVOO‐rich diet on LTP using the high‐frequency stimulation (HFS) protocol. In this test, we found that right after the LTP induction with HFS, slices from the controls had a significant failure in post‐tetanus potentiation, which is another form of short‐term plasticity, which in contrast was significantly ameliorated in the EVOO‐treated group (Figure 2 c). However, there was no significant difference at the mid‐late phase of LTP recording between the two groups (Figure 2 c).

Compared with regular diet, the EVOO‐rich diet had no effect on total body weight of the mice, since we did not observe any significant difference in this parameter between the two groups throughout the study (Figure 1 d). No significant differences in the results of any of the behavioral tests implemented were observed when males and females were analyzed separately (not shown).

Chronic administration of EVOO‐rich diet ameliorates behavioral impairments in hTau mice. Starting at 6 months of age, hTau mice were randomized to receive regular chow diet (CTR) or diet enriched with EVOO (EVOO) until they were 12 months old. (a) Number of entries and percentage of alternation in the Y‐maze paradigm (* p < .05). (b) Discrimination index in the training and probe phase of novel object recognition task for both groups (CTR n = 8, EVOO, n = 9) (* p < .05). (c) Training phase of Morris water maze (MWM) as measured by latency to reach the platform zone over five consecutive days, number of entries to platform zone, latency to initial platform crossing, and swimming speed for the two groups in the probe trial (CTR n = 8, EVOO, n = 9) (* p < .05). (d) Total body weight of CTR ( n = 8) and EVOO ( n = 9) mice from the beginning until the end of the study. Values represent mean ± SEM

As shown in Figure 1 a, in the Y‐maze, when compared with the control group, mice receiving the EVOO‐rich diet showed a significant increase in the percentage of alternation but no difference in the number of entries, indicating that the while EVOO diet improved working memory it did not affect their normal locomotor activity. Next, mice underwent the novel object recognition task. During the training phase, mice did not display any preference for one side of the cage or for one of the two identical objects. However, in the probe trial, while the control group showed no preference for the novel object, which demonstrates lack of recall or loss of memory, the EVOO group spent significantly more time with the novel object suggesting memory improvement (Figure 1 b). Finally, animals were tested in the Morris water maze paradigm. All mice in each group were able to visually find the platform (data not shown) and to reach the training criterion within 5 days (Figure 1 c). No differences were found during the training session and in their swimming speed between the two groups (Figure 1 c). During the probe test, we observed that compared with controls, mice receiving the EVOO‐rich diet manifested a significant increase of the number of entries in the platform zone (Figure 1 c).

To determine the effect of EVOO‐rich diet on learning ability and memory, animals were tested at the end of the study, after 6 months of diet (at age of 12 months) in three different paradigms: Y‐maze, Morris water maze, and novel object recognition.

3 DISCUSSION

The findings presented in this paper show that chronic administration of a diet enriched in EVOO results in a significant improvement of working memory, spatial and learning memory, amelioration of hippocampal basal synaptic activity and short‐term plasticity, and a significant reduction in tau neuropathology in a relevant mouse model of human primary tauopathy.

In recent years, evidence has been accumulating in support of the beneficial effects of dietary consumption of EVOO, a key component of the Mediterranean diet, in terms of brain health (Farr et al., 2012; Feart et al., 2013; Pitozzi et al., 2010). In particular, EVOO‐rich diet has been associated with lower risk of AD, mild cognitive impairment, and dementia (Peterson & Philippou, 2016). The PREDIMED‐NAVARRA randomized clinical trial involving a cohort of 268 subjects (74.1 ± 5.7 years old) showed that long‐term intervention with an EVOO‐rich Mediterranean diet (6.5 years) results in better cognitive performances, especially across fluency and memory tasks, and less MCI incidence as compared to controls (Martinez‐Lapiscina et al., 2013). Moreover, looking specifically at progression of well‐established AD biomarkers, including β‐amyloid deposition, glucose metabolism, and neuronal loss, a more recent longitudinal study confirmed that, in middle‐aged adults, strong adherence to the Mediterranean diet (MeDi) provide significant protection against AD (Valls‐Pedret et al., 2015). Consistently, another randomized clinical trial showed that in an older population Mediterranean diet supplemented with EVOO (1 L/week) was associated with improved cognitive function after 4.1 years follow‐up (Berti et al., 2018).

Studies in animal models of the disease have revealed that this natural product positively influences many aspects of the AD pathology, including memory, neuroinflammation, and amyloidosis (Abuznait et al., 2013; Qosa et al., 2015). Although much attention has been devoted to the latter aspect of the AD phenotype, the ability of EVOO and its derivatives to potentially modulate also phosphorylation and aggregation of tau protein so far has been evaluated only using in vitro systems (Daccache et al., 2011; Li et al., 2009). Our group recently explored the effect of EVOO on the triple transgenic mouse model of AD, which is known to develop Aβ plaques and tau tangles. Notably, in this model besides an improvement of cognition we found a significant reduction of Aβ peptides and tau phosphorylation (Lauretti, Iuliano, et al., 2017; Lauretti, Li, et al., 2017). However, since in this model Aβ can drive tau phosphorylation and subsequent pathology, the results obtained in that study would not address the important question of whether the observed effect of EVOO on tau was dependent or independent from the effect on Aβ. This is an important biological question with relevance not only for AD pathophysiology, but also for the pathogenesis of other dementing disorders called primary tauopathies, such as progressive supranuclear palsy and Pick's disease, which are characterized by the exclusive presence of tau pathology.

Thus, in order to dissect the effect of EVOO on tau pathology and on tau‐dependent synaptic dysfunction, in the current paper, we used a pure tauopathy mouse model, which expresses wild‐type human tau protein. This model is characterized by age‐dependent impairment in cognitive function, synaptic activity as well as progressive loss in pre‐ and postsynaptic integrity, and for these reasons represents an ideal model system to investigate the biological effect of chronic administration of EVOO (Alldred, Duff, & Ginsberg, 2012; Polydoro, Acker, Duff, Castillo, & Davies, 2009).

At the end of the treatment, compared with hTau mice kept on a regular diet, the hTau EVOO group displayed better working memory, as assessed by the percentage of spontaneous alternations in the Y‐maze paradigm. Moreover, chronic consumption of EVOO ameliorated spatial learning and recognition memory ability as demonstrated by increased number of entries to the platform zone and higher discrimination index, respectively, for the Morris water maze and the novel object recognition test. Importantly, the diet did not alter general motor function of these animals since no significant differences were observed between the two groups when the number of entries in each arm of the Y‐maze and the swim speed in the Morris water maze was assessed.

To prove that these benefits were associated with changes in hippocampal synaptic activity and plasticity, we assessed synaptic function by using field potential recordings in the Schaffer collateral (CA3)‐CA1 synapses. Importantly, this analysis revealed an elevation in basal synaptic activity and short‐term plasticity in the EVOO‐treated mice providing the mechanism and the biological substrate for the observed beneficial effect on cognition. Additionally, we found that EVOO‐rich diet affected in a positive manner also two forms of short‐term plasticity: increased paired‐pulse facilitation and restoration of post‐tetanic potentiation. Taken together, these data support the novel idea that EVOO directly exerts a positive effect on synaptic activity and short‐term plasticity specifically at the presynaptic level.

Interestingly, our data are in agreement with studies showing that oleuropein, one of the main components of EVOO, facilitates hippocampal synaptic activity and long‐term plasticity (Pu et al., 2005), whereas in the CA1 area of hippocampus of TgCRND8 mice it restores post‐tetanic potentiation and long‐term potentiation (Luccarini et al., 2015).

To further elucidate potential mechanisms by which EVOO modulated synaptic activity in our mice, we tested for different proteins considered key pre‐ and postsynaptic components for hippocampal synaptic function. Among them, we found that complexin 1 (CPLX1) levels were significantly higher in the EVOO‐treated group than in controls. CPLX1 is a presynaptic protein known as SNARE complex‐binding protein which plays an important role in vesicle release promoting synchronization of Ca2+ triggered vesicle fusion and inhibiting spontaneous vesicles release (Lai et al., 2014). Downregulation of CPLX1 is associated with impaired basal synaptic strength and short‐term plasticity (Chang et al., 2015), and more recently decreased CPLX1 levels have been suggested as a significant risk factor associated with AD and Parkinson's disease (Lahut et al., 2017; Ramos‐Miguel et al., 2017). Our results further support the importance of CPLX1 for the maintenance of basal synaptic activity and short‐term plasticity and provide new evidence on the involvement of this protein in neurodegenerative disorders associated with tau pathology. In addition, they highlight CPLX1 as a novel target of EVOO at synaptic level, and represent the first demonstration that chronic consumption of EVOO by driving upregulation of CPLX1 facilitates synaptic function in the hippocampus.

Since progressive accumulation of phosphorylated tau is another feature of the phenotype in hTau mice, next we assessed the effect of chronic administration of EVOO on this aspect of tau neuropathology. In agreement with previous findings, we saw that the EVOO‐rich diet resulted in a significant reduction in tau phosphorylation at specific epitopes. Mechanistically, this effect was secondary to the increase in the inhibitory phosphorylation at Ser9 of GSK3β kinase, which was found in the EVOO group when compared with controls. However, we observed that EVOO‐treated mice had a significant elevation of total soluble tau protein levels, which was not secondary to a similar change at the mRNA levels. Since tau hyper‐phosphorylation precedes oligomers and tangles formation, we also looked at these two additional forms of toxic tau under our experimental conditions and found that although the insoluble tau fraction was not changed, EVOO‐treated mice had a significant decrease in the levels of tau oligomers.

These findings are highly relevant to the current debate in the field on whether the soluble forms of tau rather than the insoluble ones are indeed more prone to alter synaptic and dentritic normal properties (Tracy & Gan, 2018). Interestingly, growing evidence indicates that the presence of tau oligomers at the synapses induces abnormal synaptic plasticity, reduces the number of dendritic spines and impairs LTP (Guerrero‐Muñoz et al., 2015). Thus, the observed increase in nonphosphorylated soluble tau together with the reduction in tau phosphorylated and oligomeric tau species would strongly suggest that neurons from EVOO‐treated mice had more readily available functional tau protein compared to control, which then correlated with better neuronal health and synaptic communication found in the same experimental group.

In conclusion, our paper demonstrates that in tau transgenic mice chronic administration of EVOO‐rich diet results in an amelioration of working memory, spatial learning, basal synaptic activity, and short‐term plasticity together with a significant reduction in the amount of phosphorylated tau and tau oligomers. Collectively, our findings represent a significant step forward in the in vivo research effort on the biological effect of EVOO on tau pathology and synaptic function. They provide strong preclinical evidence in support of the novel concept that EVOO should be considered as a potential and viable multi‐targeting agent not only for AD but also for primary tauopathies.