Rodolfo Pinto-Almazán, Julia J. Segura-Uribe, Marvin A. Soriano-Ursúa, Eunice D. Farfán-García, Juan M. Gallardo,Christian Guerra-Araiza

1 Unidad de Investigación Hospital Regional de Alta Especialidad Ixtapaluca, Carretera Federal México-Puebla km 34.5, C.P. 56530. Ixtapaluca,State of Mexico, Mexico

2 Institute for the Developing Mind, Children’s Hospital Los Angeles, Los Angeles, CA, USA

3 Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

4 Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Av. Cuauhtémoc 330 Col. Doctores. C. P. 06720. Mexico City, Mexico

5 Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón, Col. Casco de Santo Tomás. C. P. 11340. Mexico City, Mexico

6 Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Av. Cuauhtémoc 330 Col. Doctores. C. P. 06720. Mexico City, Mexico

7 Unidad de Investigación Médica en Farmacología, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Av. Cuauhtémoc 330 Col. Doctores. C. P. 06720. Mexico City, Mexico

Introduction

Tau is a microtubule-associated protein that modulates the rate of microtubule assembly and in fluences cell growth and shape depending on the degree of its phosphorylation (Drubin and Kirschner, 1986; Johnson and Stoothoff, 2004; Iqbal et al., 2005; Stoothoff and Johnson, 2005).

It is widely suggested that conformational changes such as truncation, cleavage, and hyperphosphorylation of Tau contribute to its abnormal processing and aggregation into bundles. Known as paired helical filaments (PHF), these structures form the aberrant neuro fibrillary tangles (NFTs)in Alzheimer’s disease (AD) and a family of related neurodegenerative diseases called tauopathies (Buée et al., 2000;Sánchez et al., 2001; Binder et al., 2005; Iqbal et al., 2005;Pevalova et al., 2006).

The different states of Tau phosphorylation result from the speci fic activity of kinases and phosphatases (Buée et al.,2000; Johnson and Stoothoff, 2004; Ferrer et al., 2005; Zhou et al., 2009; Braithwaite et al., 2012). Glycogen synthase kinase-3β (GSK3β) is one of the principal kinases associated with physiological and pathological Tau phosphorylation(Zhou et al., 2009; Avila et al., 2012). On the contrary, Tau dephosphorylation has been associated directly with a preponderance of protein phosphatases-2A (PP2A) activity and indirectly with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity compared to other phosphatases (Kerr et al., 2006; Zhang et al., 2006; Qian et al.,2010; Martin et al., 2013b).

It has been suggested that the unbalance between these kinase and phosphatase activities can be the origin of Tau hyperphosphorylation and aggregation (Campos-Peña et al., 2009; Martin et al., 2013a). Also, the disruption of GSK3β, PP2A, and PTEN has been described in AD postmortem brain material (Avila et al., 2012; Martin et al.,2013a, b).

Previously, Tau phosphorylation and the formation of NFTs were thought to be prominent early events during AD pathogenesis and induced by oxidative stress (OS)(Mondragón-Rodríguez et al., 2008; Su et al., 2010). Currently, it is known that the effects of OS on the phosphorylation of Tau protein are dose- and exposure time-dependent (Mattson et al., 1997; Olivieri et al., 2000, 2001;Lopresti and Konat, 2001; Gomez-Ramos et al., 2003; Zambrano et al., 2004; Lovell et al., 2004; Poppek et al., 2006;Dang et al., 2010; Su et al., 2010; Taga et al., 2011; Petroni et al., 2012).

Different models have been proposed to study the effects of OS in neurodegeneration. It has been well established that chronic O3exposure increases OS markers in the hippocampus of male rats (Rivas-Arancibia et al., 2011).When inhaled, O3increases reactive oxygen species (ROS)that produce OS in the central nervous system (CNS) (Rivas-Arancibia et al., 1998, 2010; Dorado-Martínez et al.,2001; Santiago-López et al., 2010). OS induced by O3causes neuronal damage, neurodegeneration, and cell death, as well as biochemical changes as lipid peroxidation (LPO) in CNS regions such as the hippocampus (Avila-Costa et al., 1999;Dorado-Martínez et al., 2001; Farfán-García et al., 2014;Pinto-Almazán et al., 2014). Some compounds with antioxidant activity have been tested on the chronic OS induced by O3inhalation model, and it has been observed that they improve short- and long-term memory (Guerrero et al., 1999;Rivas-Arancibia et al., 2000; Guevara-Guzmán et al., 2009;Farfán-García et al., 2014; Pinto-Almazán et al., 2014).

Tibolone (TIB) is a steroid prescribed for the treatment of climacteric symptoms and osteoporosis that can exert antioxidant activity and diminish OS in vitro and in vivo (Vural et al., 2005; de Aguiar et al., 2008; Farfán-García et al., 2014;Pinto-Almazán et al., 2014; Stark et al., 2015). In addition to its antioxidant properties, TIB has other neuroprotective effects (Pinto-Almazán et al., 2017). Two studies had been performed to evaluate the effects of TIB on Tau phosphorylation (Pinto-Almazán et al., 2012; Neri-Gómez et al., 2017).Chronic administration of TIB decreased Tau phosphorylation through the increase in the protein content of phosphorylated GSK3β (Ser9), which in turn decreased the active site availability in the hippocampus of young ovariectomized (OVX) rats (Pinto-Almazán et al., 2012). In contrast,a recent report indicates that PHF-1 (hyperphosphorylated Tau epitope in Ser396/404) content decreased with TIB administration, without changes in the phosphorylation of GSK3β in Ser9or PI3K, and decreased phosphorylated and total Akt levels in the hippocampus of aged male mice (Neri-Gómez et al., 2017).

In the model of OS induced by O3exposure, the administration of TIB decreases LPO, prevents neuronal death,ameliorates cholinergic deficit, and reduces cognitive and motor impairment (Farfán-García et al., 2014; Pinto-Almazán et al., 2014). Hence, these findings suggest that TIB can regulate synaptic and neuronal plasticity, axonal growth,and decrease Tau phosphorylation by GSK3β inhibition and probably by its antioxidant properties.

The purpose of this study was to assess the effects of TIB on the expression and phosphorylation of Tau, GSK3β, Akt,and phosphatases PP2A and PTEN in the hippocampus of rats exposed to O3.

Materials and methods

Animals

Sixty male adult Wistar rats aged 8 weeks old, weighing 250–300 g, from the vivarium of the Hospital de Especia-Iidades, CMN SXXI, Instituto Mexicano del Seguro Social were housed, five animals per cage, in acrylic boxes with free access to water and food (Purina, Minnetonka, MN,USA) and kept in a clear air room maintained under an arti ficial 12 hour light/dark cycle (lights on at 08:00 hours).Animals were treated by the guidelines and requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23,revised 1985) and those of the Ethical Committee of the Scienti fic Research Coordination at the Instituto Mexicano del Seguro Social (approval number R-2011-785-057). All efforts were made to minimize animal suffering and the protocol was designed to keeping the number of animals used to a minimum.

Treatments

Animals were randomly divided into ten experimental groups (n = 6). Each group received one of the following treatments: control [C] (exposed to ozone-free air plus vehicle(300 μL of pure water) by oral gavage for 60 days); control +TIB [C + TIB] (exposed to ozone-free air plus 1 mg/kg of TIB by oral gavage for 60 days); 7, 15, 30, and 60 days of ozone exposure [O3] (daily exposure to 0.25 ppm of monitored ozone for 4 hours; Poppek et al., 2006); and 7, 15, 30, and 60 days of 1 mg/kg of TIB (Schering Plough México, Mexico City, Mexico) by oral gavage before ozone exposure [O3+ TIB] (Farfán-García et al., 2014; Pinto-Almazán et al., 2014). Previous experience has shown that the nasogastric administration of pure water for 7–60 days does not produce relevant changes in the proteins analyzed during this study (Pinto-Almazán et al., 2012). Vehicle as well as TIB were administered 5 minutes before placing the rats into the chamber.

Ozone exposure

Animals were daily placed in a chamber coupled to an air diffuser connected to a variable- flux ozone generator (5 L/s)(Omnialva, Mexico City, Mexico) and exposed to 0.25 ppm per hour for 4 hours. The same chamber was used to treat C group with air flow free of ozone and the groups exposed to monitored ozone. The procedure used has been described elsewhere (Rivas-Arancibia et al., 2010).

At the end of the treatment for each group (7, 15, 30 or 60 days), animals were decapitated. Brains were dissected and the hippocampus was divided: one section for western blot analysis and the other for analysis of OS markers.

Western blot analysis

Hippocampus samples were processed as previously described (Pinto-Almazán et al., 2012). The following primary antibodies were used: rabbit anti-Tau polyclonal antibody(H-150, Santa Cruz Biotechnology, Santa Cruz, CA, USA;diluted 1:1,000), which recognizes six Tau isoforms of 46-80 kDa molecular weight (total Tau) and mouse anti-Tau monoclonal antibody that recognizes phosphorylation on the Ser396 (PHF-13, Santa Cruz Biotechnology; PHF-1;diluted 1:1,000). Mouse anti-GSK3β monoclonal antibody(total GSK3β; BD Biosciences Pharmingen, San Diego, CA,USA; diluted 1:1,000); mouse anti-phosphorylated GSK3β monoclonal antibody (pSer9 GSK3β, Sigma, St. Louis, MO,USA, diluted 1:5,000); rabbit anti-Akt1/2/3 (H-136) polyclonal antibody (total Akt), rabbit anti-pAkt1/2/3 (Ser 473)polyclonal antibody (p-Akt); rabbit anti-PP2A-Cα polyclonal antibody Clone 46 (RUO) for total PP2A-Cα ; anti-goat p-PP2A-Cα/β polyclonal antibody (Tyr 307) (sc-12615)for phoshporylated PP2A-Cα; mouse anti-PTEN (A2B1)monoclonal antibody (sc-7974) for total PTEN; rabbit polyclonal antibody anti-PTEN (Ser 380) for phosphorylated PTEN; rabbit polyclonal antibody against nitrotyrosine (NT;Abcam ab42789) and mouse anti-β3 tubulin (2G10) (Santa Cruz Biotechnology; diluted 1:1,000).

After incubation with the primary antibody, membranes were washed and incubated with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology, diluted 1:10,000) were used. Highly precise detection of proteins from western blots was performed using an enhanced chemiluminescence system (Millipore, Billerica, MA,USA), and their intensity was quantified by densitometry(Hewlett Packard, Palo Alto, CA, USA) and densitometric analysis by KODAK 1D Image Analysis Software (Eastmond Kodak, Rochester, NY, USA). The density of each band of different primary antibodies was normalized to its loading control (tubulin).

Determination of OS markers

Hippocampus samples (subiculum, Ammon’s horn and the dentate gyrus) were homogenized and processed as previously described (Farfán-García et al., 2014) at the end of the treatment in each group. Superoxide dismutase (SOD)activity was measured by the Marklund method (Marklund and Marklund, 1974). Lipid peroxidation end product malondialdehyde (MDA) was estimated using thiobarbituric acid reactive substances assay (TBARS) (Hong et al., 2000).The levels of nitrotyrosine (NT) were determined by western blot assay (Pinto-Almazán et al., 2014).

Statistical analysis

Western blot and OS data were analyzed by one-way analysis of variance with a Tukey’s post hoc test. Prism 5.1 program (Prisma Graph Pad, La Jolla, CA, USA) was used for calculating probability values, considering values of P ＜ 0.05 as statistically signi ficant.

Results

Tau and GSK3β expression

The expression of total Tau in the hippocampus was determined by western blot assay. No changes were observed in the expression of total Tau neither in C and C + TIB nor in O3or O3+ TIB groups (Figure 1A). However, a signi ficant reduction of hyperphosphorylated Tau PHF-1 was observed in the C + TIB group in comparison with the C group (P ＜0.05). Chronic exposure to O3for over 30 days resulted in a signi ficant increase in the expression of PHF-1 (P ＜ 0.05)(Figure 1A), while pretreatment with TIB diminished the expression of hyperphosphorylated Tau induced by chronic exposure to O3. Furthermore, the ratio of hyperphosphorylated versus total Tau showed a signi ficant increase in the hippocampus of rats exposed to O3for 30 and 60 days (P ＜0.05), without any effect at 7 and 15 days of exposure (Figure1B). In contrast, O3chronic exposure did not increase the ratio of hyperphosphorylated versus total Tau in any of the O3+ TIB groups (Figure 1B).

Total and phosphorylated GSK3β (Ser9) levels were assessed to determine if the changes in Tau were associated with their expression. The expression of total GSK3β in the hippocampus was not affected in any of the O3and O3+TIB groups (Figure 2A). Furthermore, we observed that the expression of the phosphorylated GSK3β form in C + TIB group increased when compared to C group. In contrast,the expression of the phosphorylated form of the protein decreased with respect to the time of exposure to O3. This decrease was statistically significant over 15 days of exposure (P ＜ 0.05) (Figure 2A). However, the expression of the phosphorylated form of GSK3β in O3+ TIB groups was similar to that of C and C + TIB groups (Figure 2A). The ratio of phosphorylated versus total GSK3β showed a decrease in O3groups from day 15 to day 60 of exposure (P ＜ 0.05)(Figure 2B). Moreover, none of the O3+ TIB groups ratios showed any significant difference compared to C and C +TIB groups but showed statistical differences when com-pared to the groups with the same time of O3exposure from day 15 (Figure 2B).

Akt expression

The expression of total Akt was not modi fied in any of the studied groups (Figure 3A). When the expression of phosphorylated Akt (pSer473) was analyzed to determine if changes in GSK3β phosphorylation were due to Akt activity, it was noticed that the content of phosphorylated Akt decreased significantly in the O3groups since day 15 of O3exposure compared to C and C + TIB groups (P ＜ 0.05). The expression of the phosphorylated form of Akt was unaltered in the O3+ TIB groups, being similar to C and C + TIB groups(Figure 3A). Therefore, the ratio of phosphorylated versus total Akt diminished gradually from day 15 to day 60 of O3exposure (P ＜ 0.05). This decrease was prevented by TIB treatment in every O3+ TIB groups. Statistical signi ficance was observed when compared with both the ratio of control groups and the ratio of the same exposure times of O3+ TIB groups (P ＜ 0.05) (Figure 3B).

Expression of PP2A and PTEN

The expression of the total protein was not affected in C, C+ TIB, O3or O3+ TIB groups for either PP2A (Figure 4A)or PTEN (Figure 5A). In contrast, phosphorylated PP2A levels were reduced from day 15 up to day 60 (P ＜ 0.05)(Figure 4A) and phosphorylated PTEN from day 30 to day 60 of O3exposure (P ＜ 0.05) (Figure 5A). In O3+ TIB groups, both phosphorylated PP2A (Figure 4A) and PTEN(Figure 5A) levels were mainteined when compared with C and C + TIB groups. Consequently, the ratios of phosphorylated PP2A and PTEN forms versus total protein indicated a statistically significant decrease (P ＜ 0.05) in O3groups when compared to controls and O3+ TIB groups (Figures4Band5B).

OS markers

OS markers were determined in the hippocampus of rats exposed to O3and treated with vehicle or TIB (Table 1).No statistical differences were found in the analysis of OS markers between C and C + TIB groups. However, MDA and NT levels increased signi ficantly from 15 to 60 days of exposure to O3in comparison to group C (P ＜ 0.05). Conversely, SOD activity decreased from 15 to 60 days of O3exposure (P ＜ 0.05). The administration of TIB (1 mg/kg)limited the changes in the levels of these markers induced by O3exposure: SOD activity increased while MDA and NT levels decreased since day 15 and were maintained until 60 days in the O3+ TIB groups, unlike the continuous increase in the O3groups. The values between O3and O3+ TIB groups were signi ficantly different for the same periods of O3exposure (P ＜ 0.05).

Discussion

Our results suggest that chronic O3exposure induces OS,which in turn may produce hyperphosphorylation of Tau correlated with the decrease in the phosphorylation of Akt/GSK3β kinases and PP2A and PTEN phosphatases in the hippocampus of male rats. These effects can be prevented by the treatment with TIB (Figure 6).

Tau hyperphosphorylation induces destabilization of microtubules, compromises axonal transport, and contributes to neuronal degeneration by the formation of NFTs (Buée et al., 2000; Sánchez et al., 2001; Binder et al., 2005; Iqbal et al.,2005; Pevalova et al., 2006). The effects of OS on the phosphorylation of Tau protein appear to be dose- and exposure time-dependent. At low concentrations of H2O2(＜ 0.1 mM)(Zambrano et al., 2004) and acute conditions of OS (Lopresti and Konat, 2001; Olivieri et al., 2001; Taga et al., 2011), OS could act as a neuroprotective mechanism through transient Tau dephosphorylation. Conversely, some reports indicate that chronic in vitro OS, products of OS such as 4-hydroxynonenal (4-HNE) (Mattson et al., 1997) and acrolein (Gomez-Ramos et al., 2003; Dang et al., 2010), as well as concentrations of toxic substances that exert OS, such as mercury (Hg) (＜ 200 nM) (Olivieri et al., 2000; Petroni et al.,2012), β-amyloid 1—42 and toxic β-amyloid 25—35 peptides(Olivieri et al., 2001), induce hyperphosphorylation of Tau in different epitopes (PS396, PHF-1, TG3 and MC1), which are associated with the formation of NFTs.

Under the last premise, we assessed the effects of chronic O3exposure on Tau phosphorylation. As in previous reports, we observed that chronic O3exposure increases OS markers (MDA and NT) in this model, with the reduction of SOD activity in the hippocampus of male rats (Rivas-Arancibia et al., 2010). In the present study, we report that chronic O3exposure for 30 and 60 days resulted in a signi ficant increase in the expression of hyperphosphorylated Tau.

As stated before, hyperphosphorylation of Tau could be attributed to the unbalance between the activity of kinases and phosphatases. Physiological phosphorylation of Tau by Akt/GSK3β, direct dephosphorylation by phosphatase PP2A, and indirect dephosphorylation by PTEN may be involved in the regulation of microtubule dynamics, neuritic growth, neuronal cell proliferation, synaptogenesis, synaptic plasticity, and synaptic neurotransmission (Hall et al., 2000;Zhou et al., 2009; Martin et al., 2013a, b)

GSK3β regulates Tau hyperphosphorylation at Ser198/Ser199/Ser202and Ser396/Ser404sites (Haque et al., 1999) and,while active in resting cells, it is downregulated by the Ser9phosphorylation as a result of the activation of Akt, first onThr308and then on Ser473, which produce Akt full activation(Piguet and Dufour, 2011; Kitagishi et al., 2012; Matsuda et al., 2013).

It was observed that chronic O3exposure reduced the phosphorylation of GSK3β in Ser9, which correlated with the decrease of Akt phosphorylation of Ser473after 30 days of exposure. These results are consistent with the increase in Tau phosphorylation at this time of exposure and suggest that GSK3β shows longer periods of activity without inhibition by Akt, causing the increase in Tau phosphorylation in the hippocampus of rats exposed to O3.

Figure 1 Effects of TIB on the hyperphosphorylation of Tau protein induced by O3 exposure.

Figure 2 Effects of TIB on the phosphorylation of GSK3β protein induced by O3 exposure.

Figure 3 Effects of TIB on the phosphorylation of AKT protein induced by O3 exposure.

Figure 4 Effects of TIB on the phosphorylation of PP2A protein induced by O3 exposure.

Table 1 Oxidative stress markers in the hippocampus of adult male rats exposed to ozone treated with vehicle or tibolone.

Figure 5 Effects of TIB on the phosphorylation of PTEN protein induced by O3 exposure.

Figure 6 Neuroprotective effects of tibolone on the expression and phosphorylation of proteins involved in the formation of neuro fibrillary tangles in an in vivo O3 exposure model.

For a better understanding of our results, it is important to consider that Akt is a redox-sensitive protein. At physiological pH, both cysteine residues in the activation loop(Cys297and Cys311) are shaped as thiols, which allow its biological effects. However, OS can produce the formation of a disul fide bond between Cys297and Cys311or other oxidized forms, which may reduce Thr308and Ser473phosphorylation in Akt, and consequently decrease its activity (Trachootham et al., 2008; Durgadoss et al., 2012; Wang et al., 2012; Tan et al., 2015). This situation could explain the decrease in Akt phosphorylation of Ser473observed in our study.

PP2A is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. PP2A dephosphorylates Akt at Thr308and Ser473residues (particularly at Thr308), GSK3β at Ser9, and Tau at several phosphorylation sites with different efficiency (Qian et al., 2010; Hers et al., 2011; Martin et al., 2013a). PTEN is a lipid phosphatase which dephosphorylates PI(3,4,5)P3 at the 3-position of the inositol ring and decreases Akt activity (Piguet and Dufour,2011; Martin et al., 2013a).

PP2A and PTEN are highly expressed in the brain, where they act on several substrates in different cell processes(Hall et al., 2000; Martin et al., 2013a). As Akt, the activities of these phosphatases depend on the redox state of the cell; therefore, they are considered as Cys-dependent phosphatases (CDP). At physiological pH, Cys exist as a thiolate anion, which is required for phosphatase activity. In contrast, under OS conditions, S-nitrosylation or the produced disul fide bonds inhibit their activity (Trachootham et al.,2008).

As in previous studies, we observed a gradual decrease in the phosphorylation of both PP2A Tyr307and PTEN Ser380since day 15 of O3exposure. These results may indicate that ROS oxidize the catalytic Cys residues to the sul finic (—SO2H) or sulfonic (—SO3H) acid forms that lead to irreversible phosphatase inactivation. However, further studies are needed to verify this effect.

Antioxidant (Vural et al., 2005; de Aguiar et al., 2008;Farfán-García et al., 2014; Pinto-Almazán et al., 2014; Stark et al., 2015) and neuroprotective (Qiu et al., 2009; Espinosa-Raya et al., 2012; Pinto-Almazán et al., 2012; Avila-Rodriguez et al., 2014; Beltrán-Campos et al., 2014; Neri-Gómez et al., 2017) bene fits of TIB against different models of neuronal damage have been reported (Espinosa-Raya et al., 2012;Pinto-Almazán et al., 2012, 2014; Farfán-García et al., 2014;Neri-Gómez et al., 2017). In this study, we observed that chronic treatment with TIB prevented the hyperphosphorylation of Tau in contrast to the observed in the untreated O3groups. Consistent with our findings, it was previously reported that Tau phosphorylation on PHF-1 epitope was reduced with the chronic treatment of TIB in two other different models: young female OVX rats and aged male mice(Pinto-Almazán et al., 2012; Neri-Gómez et al., 2017).

The expression and the content of phosphorylated GSK3β, Akt, PP2A, and PTEN in the O3+ TIB groups were similar to that observed in control groups. Previous reports have indicated that Tau phosphorylation on PHF-1 epitope was reduced and correlated with the increase in the phosphorylation of GSK3β on Ser9in the hippocampus and cerebellum of adult OVX rats chronically treated(two months) with TIB (Pinto-Almazán et al., 2012). In contrast, Neri-Gómez et al. (2017) reported that although a decrease in the hyperphosphorylation of Tau occurred,this was not produced through the inactivation of GSK3 by Akt phosphorylation but through induced dose-dependent changes on the content of CDK5/p35/p25 complexes. All these studies revealed that TIB can modify different signaling pathways involved in the regulation of neuroprotection mechanisms.

Although the aforementioned mechanism may explain the protective effects of TIB in this study, we propose another hypothesis. As in previous studies, we observed in this model that pretreatment with TIB can ameliorate the damage induced by chronic exposure to O3in the hippocampus of male rats (Farfán-García et al., 2014; Pinto-Almazán et al., 2014). Different studies have analyzed the antioxidant effects of chronic TIB treatment, demonstrating that TIB reduces in vivo OS by increasing the total antioxidant capacity with the increase in antioxidant enzymatic defenses, such as SOD content and activity and vitamin E (Vural et al.,2005; de Aguiar et al., 2008; Farfán-García et al., 2014; Pinto-Almazán et al., 2014). Previously, we have reported that TIB reduced lipid peroxidation and protein oxidation by increasing SOD content and activity in the chronic O3exposure model, preventing memory and motor de ficits as well as the cholinergic system disruption (Farfán-García et al.,2014; Pinto-Almazán et al., 2014). In a recent study, Stark et al. (2015) reported that TIB did not show signi ficant antioxidant capacity, but most of its active metabolites showed antioxidant effects. It has also been reported that TIB is rapidly metabolized after oral administration into its estrogenic metabolites (3α- and 3β-hydroxy tibolone) in rats (Verhoeven et al., 2002). Therefore, TIB metabolites could be those which exert antioxidant effects since these metabolites reach the brain at higher concentrations than TIB. In the present study, it was demonstrated that the chronic oxidative stress induced by ozone exposure was prevented by treatment with TIB, which resulted in the modulation of the activity of the PI3K/Akt/GSK3β signaling cascade and the phosphatases PP2A and PTEN, to finally observe a decrease in the hyperphosphorylation of Tau.

Some limitations of the present study are important to mention. One limitation is that the research was conducted only in male rats according to the OS model designed by Rivas-Arancibia et al. (1998). However, further research is necessary to verify if these results are gender-dependent.According to the design of the experiments, another limitation of our research is that we were not able to identify if the prevention of hyperphosphorylation of Tau induced by O3exposure was due to TIB itself or its metabolites. Therefore, further investigation and appropriate experiments to address this matter are required in this O3exposure model.

In conclusion, this work is a significant contribution to the study of the mechanisms of TIB neuroprotection against Tau neuronal damage in an OS in vivo model. Apparently,TIB can modulate Tau phosphorylation through various mechanisms, such as modulating the activity of kinases(GSK3β/Akt/PI3K pathway and CDK5/p35/p25 complexes)and phosphatases (PP2a and PTEN) or through the antioxidant property of its estrogenic metabolites. This study may allow scholars to develop new strategies to prevent neurodegeneration produced by OS.

Author contributions:RPA, JSU, JMG, and EFG participated in data collection and paper preparation. MSU, RPA, and CGA were responsible for data analysis. CGA participated in study design, study supervision,and manuscript instruction. All authors approved the final version of this paper.

Con flicts of interest:Tibolone was used in this study. The authors declare that there are no competing interests in this study.

Financial support:This study was supported by FIS/IMSS project No.FIS/IMSS/PROT/G09/751 and FIS/IMSS/PROT/G11-2/1013. The funding bodies played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, and in the decision to submit the paper for publication.

Research ethics:This study was performed in accordance with the guidelines and requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985) and approved by the Ethical Committee of the Scienti fic Research Coordination at the Instituto Mexicano del Seguro Social(approval number R-2011-785-057).

Data sharing statement:Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check:Checked twice by iThenticate.

Peer review:Externally peer reviewed.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under identical terms.

Open peer reviewer:Ubaldo Armato, University of Verona Medical School, Italy.

Additional file:Open peer reviewer report 1.

Avila J, León-Espinosa G, García E, García-Escudero V, Hernández F,Defelipe J (2012) Tau phosphorylation by GSK3 in different conditions. Int J Alzheimers Dis 2012:578373.

Avila-Costa MR, Colín-Barenque L, Fortoul TI, Machado-Salas P,Espinosa-Villanueva J, Rugerio-Vargas C, Rivas-Arancibia S (1999)Memory deterioration in an oxidative stress model and its correlation with cytological changes on rat hippocampus CA1. Neurosci Lett 270:107-109.

Avila-Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, Barreto GE, Ávila Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, Barreto GE (2014)Tibolone protects T98G cells from glucose deprivation. J Steroid Biochem Mol Biol 144 Pt B:294-303.

Beltrán-Campos V, Díaz-Ruiz A, Padilla-Gómez E, Aguilar Zavala H,Ríos C, Díaz Cintra S (2014) Effect of tibolone on dendritic spine density in the rat hippocampus. Neurologia 30:401-406.

Binder LI, Guillozet-Bongaarts AL, Garcia-Sierra F, Berry RW (2005)Tau, tangles, and Alzheimer’s disease. Biochim Biophys Acta 1739:216-223.

Braithwaite SP, Stock JB, Lombroso PJ, Nairn AC (2012) Protein phosphatases and Alzheimer’s disease. Elsevier Inc.

Buée L, Bussière T, Buée-Scherrer V, Delacourte A, Hof PRR (2000)Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Rev 33:95-130.

Campos-Peña V, Tapia-Ramírez J, Sánchez-Torres C, Meraz-Rios MA(2009) Pathological-like assembly of tau induced by a paired helical filament core expressed at the plasma membrane. J Alzheimers Dis 18:919-933.

Dang TN, Arseneault M, Murthy V, Ramassamy C (2010) Potential role of acrolein in neurodegeneration and in Alzheimer’s disease.Curr Mol Pharmacol 3:66-78.

de Aguiar RB, Dickel OE, Cunha RW, Monserrat JM, Barros DM,Martinez PE (2008) Estradiol valerate and tibolone: Effects upon brain oxidative stress and blood biochemistry during aging in female rats. Biogerontology 9:285-298.

Dorado-Martínez C, Paredes-Carbajal C, Mascher D, Borgonio-Pérez G, Rivas-Arancibia S (2001) Effects of different ozone doses on memory, motor activity and lipid peroxidation levels, in rats. Int J Neurosci 108:149-161.

Drubin DG, Kirschner MW (1986) Tau protein function in living cells. J Cell Biol 103:2739-2746.

Durgadoss L, Nidadavolu P, Valli RK, Saeed U, Mishra M, Seth P,Ravindranath V (2012) Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt. FASEB J 26:1473-1483.

Espinosa-Raya J, Neri-Gomez T, Orozco-Suarez S, Campos MG,Guerra-Araiza C (2012) Chronic administration of tibolone modulates anxiety-like behavior and enhances cognitive performance in ovariectomized rats. Horm Behav 61:76-83.

Farfán-García ED, Castillo-Hernández MC, Pinto-Almazán R, Rivas-Arancibia S, Gallardo JM, Guerra-Araiza C (2014) Tibolone prevents oxidation and ameliorates cholinergic deficit induced by ozone exposure in the male rat hippocampus. Neurochem Res 39:1776-1786.

Ferrer I, Gomez-Isla T, Puig B, Freixes M, Ribé E, Dalfó E, Avila J(2005) Current advances on different kinases involved in tau phosphorylation, and implications in Alzheimer’s disease and tauopathies. Curr Alzheimer Res 2:3-18.

Gomez-Ramos A, Díaz-Nido J, Smith MA, Perry G, Avila J (2003)Effect of the lipid peroxidation product acrolein on Tau phosphorylation in neural cells. J Neurosci Res 71:863-870.

Guerrero AL, Dorado-Martínez C, Rodriguez A, Pedroza-Ríos K,Borgonio-Pérez G, Rivas-Arancibia S (1999) Effects of vitamin E on ozone-induced memory de ficits and lipid peroxidation in rats. Neuroreport 10:1689-1692.

Guevara-Guzmán R, Arriaga V, Kendrick KM, Bernal C, Vega X,Mercado-Gómez OF, Rivas-Arancibia S (2009) Estradiol prevents ozone-induced increases in brain lipid peroxidation and impaired social recognition memory in female rats. Neuroscience 159:940-950.

Hall GF, Chu B, Lee G, Yao J (2000) Human tau filaments induce microtubule and synapse loss in an in vivo model of neuro fibrillary degenerative disease. J Cell Sci 113 (Pt 8):1373-1387.

Haque N, Tanaka T, Iqbal K, Grundke-Iqbal I (1999) Regulation of expression, phosphorylation and biological activity of tau during differentiation in SY5Y cells. Brain Res 838:69-77.

Hers I, Vincent EE, Tavaré JM (2011) Akt signalling in health and disease. Cell Signal 23:1515-1527.

Hong YL, Yeh SL, Chang CY, Hu ML (2000) Total plasma malondialdehyde levels in 16 Taiwanese college students determined by various thiobarbituric acid tests and an improved high-performance liquid chromatography-based method. Clin Biochem 33:619-625.

Iqbal K, Del C. Alonso A, Chen S, Chohan MO, El-Akkad E, Gong C-XX, Khatoon S, Li B, Liu F, Rahman A, Tanimukai H, Grundke-Iqbal I (2005) Tau pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta 1739:198-210.

Johnson GVW, Stoothoff WH (2004) Tau phosphorylation in neuronal cell function and dysfunction. J Cell Sci 117:5721-5729.

Kerr F, Rickle A, Nayeem N, Brandner S, Cowburn RF, Lovestone S(2006) PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3.FEBS Lett 580:3121-3128.

Kitagishi Y, Kobayashi M, Kikuta K, Matsuda S (2012) Roles of PI3K/AKT/GSK3/mTOR pathway in cell signaling of mental illnesses.Depress Res Treat 2012:1-8.

Lopresti P, Konat GW (2001) Hydrogen peroxide induces transient dephosphorylation of tau protein in cultured rat oligodendrocytes.Neurosci Lett 311:142-144.

Lovell MA, Xiong S, Xie C, Davies P, Markesbery WR (2004) Induction of hyperphosphorylated tau in primary rat cortical neuron cultures mediated by oxidative stress and glycogen synthase kinase-3. J Alzheimers Dis 6:659-671.

Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469-474.

Martin L, Latypova X, Wilson CM, Magnaudeix A, Perrin ML, Terro F (2013a) Tau protein phosphatases in Alzheimer’s disease: the leading role of PP2A. Ageing Res Rev 12:39-49.

Martin L, Latypova X, Wilson CM, Magnaudeix A, Perrin ML, Yardin C, Terro F (2013b) Tau protein kinases: involvement in Alzheimer’s disease. Ageing Res Rev 12:289-309.

Matsuda S, Nakanishi A, Wada Y, Kitagishi Y (2013) Roles of PI3K/AKT/PTEN pathway as a target for pharmaceutical therapy. Open Med Chem J 7:23-29.

Mattson MP, Fu W, Waeg G, Uchida K (1997) 4-Hydroxynonenal,a product of lipid peroxidation, inhibits dephosphorylation of the microtubule-associated protein tau. Neuroreport 8:2275-2281.

Mondragón-Rodríguez S, Basurto-Islas G, Santa-Maria I, Mena R,Binder LI, Avila J, Smith M a., Perry G, García-Sierra F (2008)Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer’s disease. Int J Exp Pathol 89:81-90.

Neri-Gómez T, Espinosa-Raya J, Díaz-Cintra S, Segura-Uribe J, Orozco-Suárez S, Gallardo JM, Guerra-Araiza C (2017) Tibolone modulates neuronal plasticity through regulating Tau, GSK3β/Akt/PI3K pathway and CDK5 p35/p25 complexes in the hippocampus of aged male mice. Neural Regen Res 12:588-595.

Olivieri G, Baysang G, Meier F, Müller-Spahn F, Stähelin H, Brockhaus M, Brack C (2001) N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation. J Neurochem 76:224-233.

Olivieri G, Brack C, Müller-Spahn F, Stähelin HB, Herrmann M,Renard P, Brockhaus M, Hock C (2000) Mercury induces cell cytotoxicity and oxidative stress and increases β-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells. J Neurochem 74:231-236.

Petroni D, Tsai J, Agrawal K, Mondal D, George W (2012) Low-dose methylmercury-induced oxidative stress, cytotoxicity, and Tau-hyperphosphorylation in human neuroblastoma (SH-SY5Y) cells. Env Toxicol 27:549-555.

Pevalova M, Filipcik P, Novak M, Avila J, Iqbal K (2006) Post-translational modi fications of tau protein. Bratisl Lek Listy 107:346-353.

Piguet AC, Dufour JF (2011) PI(3)K/PTEN/AKT pathway. J Hepatol 54:1317-1319.

Pinto-Almazán R, Calzada-Mendoza CC, Campos-Lara MG, Guerra-Araiza C (2012) Effect of chronic administration of estradiol,progesterone, and tibolone on the expression and phosphorylation of glycogen synthase kinase-3β and the microtubule-associated protein tau in the hippocampus and cerebellum of female rat. J Neurosci Res 4:878-886.

Pinto-Almazán R, Rivas-Arancibia S, Farfán-García ED, Rodríguez-Martínez E, Guerra-Araiza C (2014) Neuroprotective effects of tibolone against oxidative stress induced by ozone exposure. Rev Neurol 58:441-449.

Pinto-Almazán R, Segura-Uribe JJ, Farfán-García ED, Guerra-Araiza C (2017) Effects of tibolone on the central nervous system: Clinical and experimental approaches. Biomed Res Int 2017:1-9.

Poppek D, Keck S, Ermak G, Jung T, Stolzing A, Ullrich O, Davies KJ,Grune T (2006) Phosphorylation inhibits turnover of the tau protein by the proteasome: in fluence of RCAN1 and oxidative stress.Biochem J 400:511-520.

Qian W, Shi J, Yin X, Iqbal K, Grundke-Iqbal I, Gong CX, Liu F. (2010)PP2A regulates Tau phosphorylation directly and also indirectly via activating GSK-3 β. J Alzheimers Dis 19:1221-1229.

Qiu J, Bosch MA, Rønnekleiv OK, Kloosterboer HJ, Kelly MJ (2009)Tibolone rapidly attenuates the GABAB hypothalamic neurones. J Neuroendocr 20:1310-1318.

Rivas-Arancibia S, Dorado-Martínez C, Borgonio-Pérez G, Hiriart-Urdanivia M, Verdugo-Diaz L, Durán-Vázquez A, Colin-Baranque L,Avila-Costa MR (2000) Effects of taurine on ozone-induced memory de ficits and lipid peroxidation levels in brains of young, mature,and old rats. Environ Res 82:7-17.

Rivas-Arancibia S, Gallegos-Ríos C, Gomez-Crisostomo N, Ferreira-Garcidueñas E, Flores-Briseño D, Navarro L, Rodríguez-Martínez E (2011) Oxidative stress and neurodegenerative disease. In: Neurodegenerative Diseases - Processes, Prevention,Protection and Monitoring (Chuen-Chung Chang R, ed), pp 53-88.InTech.

Rivas-Arancibia S, Guevara-Guzmán R, López-Vidal Y, Rodríguez-Martínez E, Zanardo-Gomes M, Angoa-Pérez M, Raisman-Vozari R (2010) Oxidative stress caused by ozone exposure induces loss of brain repair in the hippocampus of adult rats. Toxicol Sci 113:187-197.

Rivas-Arancibia S, Vazquez-Sandoval R, Gonzalez-Kladiano D,Schneider-Rivas S, Lechuga-Guerrero A, Álvaro L-G (1998) Effects of ozone exposure in rats on memory and levels of brain dismutase,pulmonary superoxide. Environ Res 76:33-39.

Sánchez MP, Alvarez-Tallada V, Avila J (2001) The microtubule-associated protein tau in neurodegenerative diseases. Tauopathies. Rev Neurol 33:169-177.

Santiago-López D, Bautista-Martínez JA, Reyes-Hernandez CI, Aguilar-Martínez M, Rivas-Arancibia S (2010) Oxidative stress, progressive damage in the substantia nigra and plasma dopamine oxidation, in rats chronically exposed to ozone. Toxicol Lett 197:193-200.

Stark J, Varbiro S, Sipos M, Tulassay Z, Sara L, Adler I, Dinya E,Magyar Z, Szekacs B, Marczell I, Kloosterboer HJ, Racz K, Bekesi G(2015) Antioxidant effect of the active metabolites of tibolone. Gynecol Endocrinol 31:31-35.

Stoothoff WH, Johnson GV (2005) Tau phosphorylation: physiological and pathological consequences. Biochim Biophys Acta 1739:280-297.

Su B, Wang X, Lee H-G, Tabaton M, Perry G, Smith MA, Zhu X (2010)Chronic oxidative stress causes increased tau phosphorylation in M17 neuroblastoma cells. Neurosci Lett 468:267-271.

Taga M, Mouton-Liger F, Paquet C, Hugon J (2011) Modulation of oxidative stress and tau phosphorylation by the mTOR activator phosphatidic acid in SH-SY5Y cells. FEBS Lett 585:1801-1806.

Tan PL, Shavlakadze T, Grounds MD, Arthur PG (2015) Differential thiol oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle.Int J Biochem Cell Biol 62:72-79.

Trachootham D, Lu W, Ogasawara MA, Nilsa RD, Huang P (2008)Redox regulation of cell survival. Antioxid Redox Signal 10:1343-1374.

Verhoeven CH, Vos RM, Delbressine LP (2002) The in vivo metabolism of tibolone in animal species. Eur J Drug Metab Pharmacokinet 27:1-10.

Vural P, Akgül C, Canbaz M (2005) Effects of menopause and tibolone on antioxidants in postmenopausal women. Ann Clin Biochem 42:220-223.

Wang X, Tao L, Hai CX (2012) Redox-regulating role of insulin: the essence of insulin effect. Mol Cell Endocrinol 349:111-127.

Zambrano CA, Egana JT, Nunez MT, Maccioni RB, Gonzalez-Billault C (2004) Oxidative stress promotes tau dephosphorylation in neuronal cells: the roles of Cdk5 and PP1. Free Radic Biol Med 36:1393-1402.

Zhang X, Li F, Bulloj A, Zhang YW, Tong G, Zhang Z, Liao FF, Xu H(2006) Tumor-suppressor PTEN affects tau phosphorylation, aggregation, and binding to microtubules. FASEB J 20:1272-1274.

Zhou XW, Winblad B, Guan Z, Pei JJ (2009) Interactions between glycogen synthase kinase 3beta, protein kinase B, and protein phosphatase 2A in tau phosphorylation in mouse N2a neuroblastoma cells. J Alzheimers Dis 17:929-937.