Tibolone modulates neuronal plasticity through regulating Tau, GSK3β/Akt/PI3K pathway and CDK5 p35/p25 complexes in the hippocampus of aged male mice

Teresa Neri-Gómez, Judith Espinosa-raya, sofía Díaz-Cintra, Julia segura-Uribe, sandra Orozco-suárez, Juan Manuel Gallardo, Christian Guerra-Araiza

1 Unidad de Investigación Médica en Farmacología, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México

2 Laboratorio de Farmacología Conductual, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón Col. Sto. Tomás, Ciudad de México, México

3 Departamento de Neurobiología del Desarrollo y Neurof i siología, Instituto de Neurobiología, Universidad Nacional Autónoma de México Campus Juriquilla, Querétaro, Querétaro, México

4 Enfermedades Neurológicas (Neurological Diseases), Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México

5 Enfermedades Nefrológicas (Kidney Diseases), Hospital de Especialidades, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de Mexico, Mexico

Tibolone modulates neuronal plasticity through regulating Tau, GSK3β/Akt/PI3K pathway and CDK5 p35/p25 complexes in the hippocampus of aged male mice

Teresa Neri-Gómez1,2, Judith Espinosa-raya2, sofía Díaz-Cintra3, Julia segura-Uribe4, sandra Orozco-suárez4, Juan Manuel Gallardo5, Christian Guerra-Araiza1,*

1 Unidad de Investigación Médica en Farmacología, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México

2 Laboratorio de Farmacología Conductual, Escuela Superior de Medicina, Instituto Politécnico Nacional, Plan de San Luis y Díaz Mirón Col. Sto. Tomás, Ciudad de México, México

3 Departamento de Neurobiología del Desarrollo y Neurof i siología, Instituto de Neurobiología, Universidad Nacional Autónoma de México Campus Juriquilla, Querétaro, Querétaro, México

4 Enfermedades Neurológicas (Neurological Diseases), Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México

5 Enfermedades Nefrológicas (Kidney Diseases), Hospital de Especialidades, Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de Mexico, Mexico

How to cite this article: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(4):588-595.

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 the identical terms.

Funding:This study was supported by FIS/IMSS project No. FIS/IMSS/PROT/G13/1216, COFAA, SIP-IPN and by DGAPA-UNAM IN203616.

Aging is a key risk factor for cognitive decline and age-related neurodegenerative disorders. Also, an age-related decrease in sex steroid hormones may have a negative impact on the formation of neurof i brillary tangles (NFTs); these hormones can regulate Tau phosphorylation and the principal kinase GSK3β involved in this process. Hormone replacement therapy decreases NFTs, but it increases the risk of some types of cancer. However, other synthetic hormones such as tibolone (TIB) have been used for hormone replacement therapy.e aim of this work was to evaluate the long-term ef f ects of TIB (0.01 mg/kg and 1 mg/kg, intragastrically for 12 weeks) on the content of total and hyperphosphorylated Tau (PHF-1) proteins and the regulation of GSK3β/Akt/PI3K pathway and CDK5/p35/p25 complexes in the hippocampus of aged male mice. We observed that the content of PHF-1 decreased with TIB administration. In contrast, no changes were observed in the active form of GSK3β or PI3K. TIB decreased the expression of the total and phosphorylated form of Akt while increased that of p110 and p85.e content of CDK5 was differentially modified with TIB: it was increased at low doses and decreased at high doses. When we analyzed the content of CDK5 activators, an increase was found on p35; however, the content of p25 decreased with administration of low dose of TIB. Our results suggest a possible mechanism of action of TIB in the hippocampus of aged male mice.rough the regulation of Tau and GSK3β/Akt/PI3K pathway, and CDK5/p35/ p25 complexes, TIB may modulate neuronal plasticity and regulate learning and memory processes.

nerve regeneration; Tibolone; hippocampus; aged mice; sex steroids; Akt; GSK3β; PI3K; neural plasticity; Tau; neurof i brillary tangles; neural regeneration

Introduction

The association between low testosterone levels and increased risk for Alzheimer’s disease (AD) in aged men may reflect the loss of beneficial androgen-mediated actions in the brain (Rosario et al., 2010).ere are several androgen actions potentially relevant to AD, including promotion of neuron viability (Pike, 2001; Ramsden et al., 2003), synaptic plasticity (Leranth et al., 2003), selected aspects of cognition (Cherrier et al., 2005; Mof f at, 2005; Janowsky, 2006), and reduction of Tau phosphorylation (Papasozomenos, 1997).

Neurofibrillary tangles (NFTs) are predominantly composed of post-translationally modified forms of the microtubule-associated protein Tau and can become widespread in hippocampal, neocortical, and limbic neurons in AD (Yankner et al., 2008).

Tau modulates the extent and rate of microtubule assembly and plays an essential role in morphogenetic processes, such as axonal growth (Drechsel et al., 1992). It also increases the rate of tubulin polymerization, decreases the rate of transit into the shrinking phase, and inhibits the rate of depolymerization (Drechsel et al., 1992).

In several diseases, hyperphosphorylation of Tau at multiple sites is related with its dissociation from microtubules and aggregation into oligomeric and fi brillar forms.is hyperphosphorylation may destabilize microtubules and impair anterograde axonal transport of essential macromolecules and organelles to synaptic endings (Skovronsky et al., 2006).

It has been shown that several kinases regulate Tau phosphorylation, but only two have been co-immunoprecipitated with microtubules, GSK3β and CDK5 (Ishiguro et al., 1992; Flaherty et al., 2000). CDK5 is a member of the cyclin-dependent protein kinase family. Whereas CDK5 does not have any role in the cell cycle, it participates in neuronal development and survival, phosphorylation of cytoskeletal proteins, and synaptic plasticity (Malumbres and Barbacid, 2005; Shukla et al., 2012).

Some evidence has suggested that GSK3β levels are increased in AD brains (Baum et al., 1996; Imahori and Uchida, 1997), and that the imbalance in GSK3β activation could compromise neurotrophin signaling.e abnormal Tau phosphorylation that results from increased activity of GSK3β could lead to sequestration of Tau, inhibition of microtubule formation, destabilization of the existing cytoskeleton, and promotion of assembly of large Tau-containing aggregates, and these would inhibit both anterograde and retrograde microtubule-based transport (Niewiadomska et al., 2006).

Some gonadal hormones such as estrogens, progestogens, and androgens can modulate the activation of Tau and GSK3β in female rats (Pinto-Almazán et al., 2012). In a mouse model of AD, dihydrotestosterone can prevent an increase of hyperphosphorylation of Tau as well (Rosario et al., 2010). In addition, different doses of estrogens have been shown to exert different ef f ects (Espinosa-Raya et al., 2011).

Tibolone (TIB), a synthetic steroid, is widely prescribed to treat menopausal symptoms. TIB is metabolized into three biologically active metabolites: 3α-hydroxy and 3β-hydroxy (estrogenic), and Δ-4 keto isomer, which displays progestogenic and androgenic effects (Kloosterboer, 2001; Campisi and Marengo, 2007). TIB metabolites circulate as inactive forms, and its conversion to bioactive forms depends on tissue-specif i c desulfation (Verheuland Kloosterboer, 2006). TIB also exhibited neuroprotective ef f ects when prevented oxidative stress neurodegeneration in male rats (Pinto-Almazán et al., 2014). TIB also exhibits different ef f ects on memory and learning in a dose-dependent manner (Espinosa-Raya et al., 2012; Farfán-García et al., 2014). However, the ef f ects of TIB on the activation of Tau protein and its signaling cascade in the brain of aged males are unknown.e aim of this study was to evaluate the long-term effects of two (low and high) doses of TIB on Tau hyperphosphorylation and PI3K/Akt/ GSK3β signaling pathway, and CDK5/p35/p25 complexes.

Materials and Methods

Animals

Adult male aged (18 months) 129/C57BL/6 mice, weighing 25 ± 5 g, were housed fi ve per cage, under a 12-hour light/ dark cycle (lights on at 9:00 p.m.) and provided water and foodad libitum. All procedures were performed by the Mexican Guidelines for Animal Care and Handling (NOM-062-ZOO-1999). All ef f orts were made to minimize animal discomfort and reduce the number of animals used.

Treatments

Aged intact male mice were randomly divided into three groups (n= 6 per group). According to previous reports, mice in these groups were intregastrically administered vehicle (Veh), 0.01 mg/kg (low dose), or 1.0 mg/kg (high dose) of TIB (Sigma-Aldrich, San Luis, MO, USA), respectively once a day for 12 successive weeks (Espinosa-Raya et al., 2012; Farfán-García et al., 2014).

Western blot analysis

The abovementioned membranes were incubated overnight at 4°C with the primary antibody, then washed and incubated with anti-mouse or anti-rabbit secondary antibody as appropriate for 2 hours at room temperature (Santa Cruz Biotechnology, diluted 1:15,000). Immunoreactive bands were detected using an enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA). Subsequently, the membranes were stripped with a commercial solution (Chemicon, Billerica, MA, USA) and reprobed with anti-GAPDH monoclonal antibody detected by the system mentioned above.e intensity of protein bands was quantified and analyzed by densitometry (KODAK 1D Image Analysis Soware).e density of each band was normalized to its respective loading control (GAPDH). Samples from all animal groups were processed concurrently and under the same conditions in each experiment.

Figure 1 Changes in the content of total Tau and PHF-1 in the hippocampus of aged mice treated with tibolone (TIB).

Figure 2 Changes in the content of total and phosphorylated GSK3β in the hippocampus of aged mice treated with tibolone (TIB).

Statistical analysis

All data are presented as the mean ± standard error (SE) and analyzed by one-way analysis of variance to compare group means for each variable. Tukey’spost-hoctest was used to determine differences between means. Prism 5.0 program (GraphPad, La Jolla, CA, USA) was used to calculate probability values.Pvalues ＜ 0.05 were considered statistically signif i cant.

Results

Ef f ect of TIB on the content of Tau and GSK3β

Western blot was used to analyze the effect of TIB on the content of PHF-1 and total Tau in hippocampal samples of aged mice. The expression of PHF-1 decreased with TIB treatment at both doses (Figure 1A); in contrast, the expression of total Tau was unaffected by TIB (Figure 1B). Furthermore, the ratio of hyperphosphorylated Tauvs. total Tauwas signif i cantly increased (P＜ 0.05) aer TIB treatment at either dose (Figure 1C).

Figure 3 Changes in the content of total and phosphorylated Akt in the hippocampus of aged mice treated with tibolone (TIB).

Figure 4 Changes in the content of the catalytic subunit PI3K p110 in the hippocampus of aged mice treated with tibolone (TIB).

Tau phosphorylation is associated with the modification of GSK3β phosphorylation (Ser9) (Haque et al., 1999). Therefore, we analyzed the expression of total GSK3β, and the levels of phosphorylated GSK3β (Ser9) were assessed to determine if the expression of total GSK3β (Figure 2) was decreased in the hippocampus with the low-dose TIB treatment. However, TIB did not change the expression of phosphorylated GSK3β (Ser9).erefore, there was no difference in the ratio of phosphorylatedvs. total GSK3β.

Ef f ect of TIB on the content of PI3K/Akt pathway

PI3K/Akt pathway is involved in the activation of GSK3β (Woodgett, 1990), so we investigated if there were any changes in Akt content. A decrease in the total content of Akt aer treatment with TIB at both doses was observed (Figure 3A). Phosphorylated Akt content decreased only with the low-dose TIB treatment (Figure 3B). Figure 3C shows that the ratio of phosphorylated Akt and total Akt increased with the low-dose TIB while it decreased with treatment with high-dose TIB.

The content of the catalytic subunit of PI3 kinase, p110, and its phosphorylated form (pPI3 kinase), were also ana-lyzed (Figure 4). No changes were observed for total PI3 kinase p110 (Figure 4B), whereas TIB increased the expression of pPI3 kinase p110 (Figure 4C). This effect was obvious when phosphorylated PI3K/PI3K ratio was analyzed (Figure 4D).e levels of the regulatory subunit of PI3 kinase p85 decreased with low-dose TIB treatment and increased with high-dose TIB treatment (Figure 5B). TIB at both doses increased the expression of pPI3 kinase p85 (Figure 5C), and the pPI3Kp85/PI3Kp85 ratio was the highest aer treatment with TIB at 0.01 mg/kg (Figure 5D).

Ef f ect of TIB on the content of CDK5/p35/p25 complexes

Another kinase involved in the regulation of Tau hyperphosphorylation is CDK5. We analyzed the expression of CDK5 and its activators p35 and p25. The content of CDK5 was differentially modified with TIB treatment: it increased with treatment with low dose TIB, and with treatment with high dose TIB, decreased CDK5 content was observed (Figure 6). When we analyzed the content of the activators, an increase was observed in the content of p35. In contrast, the content of p25 decreased with treatment with low dose TIB (Figure 7).

Discussion

Our results suggest that TIB can modulate the content of total and phosphorylated microtubule-associated protein Tau through the PI3K/Akt/GSK3β pathway and CDK5/p35/p25 complexes in the hippocampus of aged male mice.

Our results showed that aging increased the phosphorylation of Tau and TIB administration decreased the phosphorylation of Tau in the hippocampus of the aged mice. These results were in accordance with previous studies showing that aging increased the phosphorylation of Tau in the brain tissue of monkeys and rats (Niewiadomska et al., 2006; Carlyle et al., 2014) and that TIB decreased the phosphorylation of Tau in the hippocampus of ovariectomized rats (Pinto-Almazán et al., 2012)

Tau phosphorylation reduces the af fi nity for microtubules and its ability to promote microtubule assemblyin vitro(Lindwall and Cole, 1984; Biernat et al., 1993; Lu and Wood, 1993). Many of the phosphorylated residues in Tau during the development of paired helical filaments in Alzheimer’s disease are serines and threonines preceding a proline (Mietelska-Porowska et al., 2014). GSK3β, a proline-directed kinase, induces phosphorylation of Tau at least on some of these residues. Co-transfection of Tau with GSK3β elevates GSK3β activity in fibroblast cells, so as to phosphorylate Tau (Lovestone et al., 1994; Sperber et al., 1995). Androgen depletion has been shown to signif i cantly accelerate the development of Alzheimer’s disease-like neuropathology in the triple-transgenic mouse model of AD (3xTg-AD).is ef f ect is prevented by androgen treatment. It was observed that the levels of Tau hyperphosphorylation in sham gonadectomy (GDX) 3xTg-AD male mice were modest and only increased slightly by GDX. In GDX male mice, treatment with testosterone (T) or 17β-estradiol (E2) but not with dihydrotestosterone (DHT) reduced Tau hyperphosphorylation to levels lower than observed in sham animals. Because T is metabolized into both the androgen DHT and the estrogen E2 in the brain, T has been shown to mediate its effects through androgen or estrogen metabolic pathways (Rosario et al., 2010).

Considering that GSK3β plays a significant role in the regulation of Tau phosphorylationin vivo, the understanding of signaling pathways that modulate the activity of GSK3β is of critical importance. It is well known that phosphorylation on the Ser9residue of GSK3β inhibits its activity, while phosphorylation on Tyr216increases it (Jope and Johnson, 2004). GSK3β regulates Tau hyperphosphorylation at Ser198/Ser199/Ser202sites and Ser396/Ser404sites (Haque et al., 1999). Furthermore, as a down-regulator of insulin signaling, GSK3β is regulated by Akt (Beurel et al., 2015). In addition, long-term treatment with insulin or IGF-1 results in Akt activation, which in turn phosphorylates GSK3β on Ser9(Hong and Lee, 1997; Lesort and Johnson, 2000).

Previous results have shown that TIB treatment increases only the phosphorylation of GSK3β, the inactive form of this protein, in the hippocampus.ese results correlate with the decrease of Tau hyperphosphorylation in female rats (Pinto-Almazán et al., 2012), and suggest that TIB may decrease Tau phosphorylation through the inactivation of GSK3β in females. In contrast, our results indicate that, despite a decrease in the total GSK3β content with the low dose of TIB, this hormone does not modify the content of the active form of GSK3β in the hippocampus of aged male mice. Unlike the previous investigation, where a model with young, ovariectomized female rats was used (Pinto-Almazán et al., 2012), we used aged male mice as a model in the present investigation.e differences in the observed results may be because ovariectomy in female rats modif i es the hypothalamic-pituitary-gonadal axis.

Furthermore, we investigated Akt activation, which can be achieved by the binding of neurotransmitters or growth factors on many specif i c cell-surface receptors, which in turn initiate a cascade of second messengers related to the PI3K pathway (Cardona-Gomez et al., 2001). Moreover, these two signaling pathways converge downstream to GSK3β, which is inhibited by Akt (Woodgett, 1990). PI3K signaling results in the activation of Akt following its phosphorylation on ther308and Ser473residues (Beaulieu, 2010). A previous report showed that in the rat cerebellum, E2 increased the phosphorylation of Akt at 6 and 12 hours aer its administration (Morissette et al., 2008). However, in this study, we observed that TIB decreased the content and phosphorylation of Akt in the hippocampus of aged males. These results, together with those observed for GSK3β, indicate that the GSK3β/ Akt signaling pathway is likely not involved in the regulation of TIB-mediated Tau phosphorylation in the hippocampus of the old mice.e negative regulation of TIB on Akt may have an ef f ect on the decrease in Tau phosphorylationthrough inactivating another kinase involved in this process such as CDK5. Overall, these results indicate the need for the analysis of the ef f ect of TIB on the CDK5 signaling pathway in the old mouse hippocampus.

It has been observed that the activation of PI3K by the phosphorylation of tyrosine residues of its regulatory subunit p85 leads to the activation of the downstream kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) by phosphorylating PDK1 at Ser241(Fyffe and Falasca, 2013). Activated PDK1 then activates Akt by its phosphorylation atr308. Full activation of Akt also requires its phosphorylation at Ser473. A major target of Akt is GSK3.e activity of GSK3 is inhibited when it is phosphorylated at Ser21in GSK3α or at Ser9in GSK3β by Akt, which results in glycogen synthesis. GSK3β is also a major Tau kinase (Takashima, 2006; Avila and Hernández, 2007). We observed that TIB increased the expression of the catalytic p110 and regulatory p85 subunits. Therefore, these modifications may lead to a down-regulation of the Tau phosphorylation pathway.

These differences may be because the regulation of Tau phosphorylation is subject to a balance between the action of kinases and phosphatases. Mitogen-activated protein kinases (MAPKs) and CDK5 are involved in the regulation of Tau phosphorylation in the hippocampus (Hyman et al., 1994; Crespo-Biel et al., 2007), and steroid hormones can modulate its activation (Guerra-Araiza et al., 2009; Harburger et al., 2009). In 2010, Amorim et al. reported that the phosphorylation of protein phosphatase 2A (PP2A) was regulated by hormones. PP2A is also capable of dephosphorylating Akt at Thr308 (Millward et al., 1999), and it regulates Tau phosphorylation directly orviaGSK3β (Qian et al., 2010).

Another kinase involved in the hyperphosphorylation of Tau is CDK5. This kinase works with its co-activators, named p39/p35 and p29/p25. Without these activators, the monomeric form of CDK5 is enzymatically inactive (Gong and Iqbal, 2008).

Under pathological conditions, there is a cleavage of the activator p35 that leads to p25.e complex CDK5/p25 has been shown to induce the hyperphosphorylation of Tau, and also causes neurodegeneration.is complex was found in patients with Alzheimer’s disease and co-localized with Tau aggregates (Imahori and Uchida, 1997). Moreover, the accumulation of this complex is located in several areas, including the frontal cortex, inferior parietal cortex, and hippocampus. Hyperphosphorylation of Tau and neurof i laments have been observed in mice overexpressing human p25, an activator of CDK5 (Tseng et al., 2002; Shelton and Johnson, 2004; Martina et al., 2013).

Our results showed that at a low dose, TIB increased the content of CDK5. On the contrary, at a high dose, the content of CDK5 decreased. When we analyzed the content of the p35 and p25 activators, it was observed that low doses of TIB increased the content of p35 and decreased the content of p25. As mentioned above, in mice overexpressing human p25, the hyperphosphorylation of Tau and neurofilaments have been observed in some areas, such as frontal cortex, cerebellum, and amygdala (Ahlijanian et al., 2000).

Our results provide a possible mechanism of action of TIB in the aged hippocampus. Through the regulation of Tau, GSK3β/Akt/PI3K pathway and CDK5 p35/p25 complexes, TIB can modulate neuronal plasticity and participate in the regulation of learning and memory processes. Further studies are needed to determine whether TIB could be used as a neuroprotective agent for the prevention of tauopathies or other neurodegenerative diseases in aged males.

Acknowledgments:This work was submitted in partial fulf i llment of the requirements for the Ph.D. degree of Teresa Neri-Gómez at Doctorado en Investigación en Medicina (ESM/IPN). Christian Guerra-Araiza received Beca de Excelencia en Investigación by Fundación IMSS, A. C.

Author contributions:TNG and JSU performed western blot analysis to detect protein expression levels and wrote the paper. SDC established aging mouse models. JER, SOS, JMG and CGA were responsible for data analysis. CGA designed this study and contributed to paper writing. All authors approved the f i nal version of this paper.

Conf l icts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:This paper was double-blinded and stringently reviewed by international expert reviewers.

Ahlijanian MK, Barrezueta NX, Williams RD, Jakowsk A, Kowsz K, Mc-Carthy S, Coskran T, Carlo A, Seymour PA, Burkhardt JE, Nelson RB, John D, McNeish JD (2000) Hyperphosphorylated tau and neurof i lament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5. Proc Natl Acad Sci U S A 97:2910-2915.

Amorim MA, Guerra-Araiza C, Pernía O, da Cruz e Silva EF, Garcia-Segura LM (2010) Progesterone regulates the phosphorylation of protein phosphatases in the brain. J Neurosci Res 88:2826-2832.

Avila J, Hernández F (2007) GSK-3 inhibitors for Alzheimer’s disease. Expert Rev Neurother 7:1527-1533.

Baum L, Hansen L, Masliah E, Saitoh T (1996) Glycogen synthase kinase 3 alteration in Alzheimer disease is related to neurofibrillary tangle formation. Mol Chem Neuropathol 29:253-261.

Beaulieu JM (2010) A role for Akt and glycogen synthase kinase-3 as integrators of dopamine and serotonin neurotransmission in mental health. J Psychiatry Neurosci 37:7-16.

Beurel E, Grieco SF, Jope RS (2015) Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacoler 148:114-131.

Biernat J, Gustke N, Drewes G, Mandelkow EM, Mandelkow E (1993) Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding. Neuron 11:153-163.

Campisi R, Marengo FD (2007) Cardiovascular effects of tibolone: a selective tissue estrogenic activity regulator. Cardiovasc Drug Rev 25:132-145.

Cardona-Gómez GP, Mendez P, DonCarlos LL, Azcoitia I, Garcia-Segura LM (2001) Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection. Brain Res Brain Res Rev 37:320-334.

Carlyle BC, Nairn AC, Wang M, Yang Y, Jin LE, Simen AA, Ramos BP, Bordner KA, CraGE, Davies P, Pletikos M, Sestan N, Arnsten AFT, Paspalas CD (2014) cAMP-PKA phosphorylation of tau confers risk for degeneration in aging association cortex. Proc Natl Acad Sci U S A 111:5036-5041.

Cherrier MM, Matsumoto AM, Amory JK, Asthana S, Bremner W, Peskind ER, Raskind MA, CraS (2005) Testosterone improves spatial memory in men with Alzheimer disease and mild cognitive impairment. Neurology 64:2063-2068.

Figure 5 Changes in the content of regulatory subunit PI3K p85 in the hippocampus of aged mice treated with tibolone (TIB).

Figure 6 Changes in the content of CDK5 in the hippocampus of aged mice treated with tibolone (TIB).

Figure 7 Changes in the content of p35 and p25 in the hippocampus of aged mice treated with tibolone (TIB).

Crespo-Biel N, Canudas AM, Camins A, Palla’s M (2007) Kainate induces AKT, ERK and cdk5/GSK3beta pathway deregulation, phosphorylates tau protein in mouse hippocampus. Neurochem Int 50:435-442.

Drechsel DN, Hyman AA, Cobb MH, Kirschner MW (1992) Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol Biol Cell 3:1141-1154.

Espinosa-Raya J, Plata-Cruz N, Neri-Gómez T, Camacho-Arroyo I, Picazo O (2011) Effects of short-term hormonal replacement on learning and on basal forebrain ChAT and TrkA content in ovariectomized rats. Brain Res 1375:77-84.

Espinosa-Raya J, Neri-Gómez T, Orozco-Suárez 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 def i cit induced by ozone exposure in the male rat hippocampus. Neurochem Res 39:1776-1786.

Flaherty DB1, Soria JP, Tomasiewicz HG, Wood JG (2000) Phosphorylation of human tau protein by microtubule-associated kinases: GSK-3beta and cdk5 are key participants. J Neurosci Res 62:463-472.

Fyf f e C, Falasca M (2013). 3-Phosphoinositide-dependent protein kinase-1 as an emerging target in the management of breast cancer. Cancer Manag Res 5:271-280.

Gong CX, Iqbal K (2008) Hyperphosphorylation of microtubule-associated protein tau: a promising therapeutic target for Alzheimer disease. Curr Med Chem 15:2321-2328.

Guerra-Araiza C, Amorim MA, Pinto-Almazán R, González-Arenas A, Campos M, Garcia-Segura LM (2009) Regulation of the phosphoinositide-3 kinase and mitogen-activated protein kinase signaling pathways by progesterone and its reduced metabolites in the rat brain. J Neurosci Res 87:470-481.

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.

Harburger LL, Saadi A, Frick KM (2009) Dose-dependent effects of post training estradiol plus progesterone treatment on object memory consolidation and hippocampal extracellular signal-regulated kinase activation in young ovariectomized mice. Neuroscience 160:6-12.

Hong M, Lee VM (1997) Insulin and insulin-like growth factor-1 regulate tau phosphorylation in cultured human neurons. J Biol Chem 272:19547-19553.

Hyman BT, Elvhage TE, Reiter J (1994) Extracellular signal regulated kinases. Localization of protein and mRNA in the human hippocampal formation in Alzheimer’s disease. Am J Pathol 144:565-572.

Imahori K, Uchida T (1997) Physiology and pathology of tau protein kinases in relation to Alzheimer’s disease. J Biochem (Tokyo) 121:179-188.

Ishiguro K1, Omori A, Takamatsu M, Sato K, Arioka M, Uchida T, Imahori K (1992) Phosphorylation sites on tau by tau protein kinase I, a bovine derived kinase generating an epitope of paired helical fi laments. Neurosci Lett 148:202-206.

Kloosterboer HJ (2001) Tibolone: a steroid with a tissue-specif i c mode of action. J Steroid Biochem Mol Biol 76:231-238.

Leranth C, Petnehazy O, MacLusky NJ (2003) Gonadal hormones af f ect spine synaptic density in the CA1 hippocampal subf i eld of male rats. J Neurosci 23:1588-1592.

Lesort M, Johnson GV (2000) Insulin-like growth factor-1 and insulin mediate transient site-selective increases in tau phosphorylation in primary cortical neurons. Neuroscience 99:305-316.

Lindwall G, Cole RD (1984) Phosphorylation affects the ability of Tau protein to promote microtubule assembly. J Biol Chem 259:5301-5305.

Lovestone S, Reynolds CH, Latimer D, Davis DR, Anderton BH, Gallo JM, Hanger D, Mulot S, Marquardt B, Stabel S (1994) Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells. Curr Biol 4:1077-1086.

Lu Q, Wood JG (1993) Functional studies of Alzheimer’s disease tau protein. J Neurosci 13:508-515.

Malumbres M, Barbacid M (2005) Mammalian cyclin-dependent kinases. Trends Biochem Sci 30:630-641.

Martina L, Latypovac X, Wilsona CM, Magnaudeixa A, Perrina ML, Yardina C, Terroa F (2013) Tau protein kinases: Involvement in Alzheimer’s disease. Ageing Res Rev 12:289-309.

Mietelska-Porowska A, Wasik U, Goras M, Filipek A, Niewiadomska G (2014) Tau protein modif i cations and interactions: their role in function and dysfunction. Int J Mol Sci 15:4671-4713.

Millward TA, Zolnierowicz S, Hemmings BA (1999) Regulation of protein cascades by protein phosphatase 2A. Trends Biochem Sci 24:186-191.

Mof f at SD (2005) Ef f ects of testosterone on cognitive and brain aging in elderly men. Ann N Y Acad Sci 1055:80-92.

Morissette M, Le Saux M, D’Astous M, Jourdain S, Al Sweidi S, Morin N, Estrada-Camarena E, Mendez P, Garcia-Segura LM, Di Paolo T (2008) Contribution of estrogen receptors alpha and beta to the ef f ects of estradiol in the brain. J Steroid Biochem Mol Biol 108:327-338.

Niewiadomska G, Baksalerska-Pazera M, Lenarcik I, Riede G (2006) Compartmental protein expression of Tau, GSK-3b and TrkA in cholinergic neurons of aged rats. J Neural Transm 113:1733-1746.

Pike CJ (2001) Testosterone attenuates beta-amyloid toxicity in cultured hippocampal neurons. Brain Res 919:160-165.

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 90:878-886.

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 58:441-448.

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-3beta. J Alzheimers Dis 9:1221-1229.

Ramsden M, Shin TM, Pike CJ (2003) Androgens modulate neuronal vulnerability to kainate lesion. Neuroscience 122:573-578.

Rosario ER, Carroll J, Pike CJ (2010) Testosterone regulation of Alzheimer-like neuropathology in male 3xTg-AD mice involves both estrogen and androgen pathways. Brain Res 1359:281-290.

Shelton SB, Johnson GV (2004) Cyclin-dependent kinase-5 in neurodegeneration. J Neurochem 88:1313-1326.

Shukla V, Skuntz S, Pant HC (2012) Deregulated cdk5 activity is involved in inducing Alzheimer’s disease. Arch Med Res 43:655-662.

Skovronsky DM, Lee VM, Trojanowski JQ (2006) Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu Rev Pathol 1:151-170.

Sperber BR, Leight S, Goedert M, Lee VM (1995) Glycogen synthase kinase-3 beta phosphorylates tau protein at multiple sites in intact cells. Neurosci Lett 197:149-153.

Takashima A (2006) GSK-3 is essential in the pathogenesis of Alzheimer’s disease. J Alzheimers Dis 9 (Suppl):309-317.

Tseng HC, Zhou Y, Shen Y, Tsai LH (2002) A survey of Cdk5 activator p35 and p25 levels in Alzheimer’s disease brains. FEBS Lett 523:58-62.

Verheul HA, Kloosterboer HJ (2006) Metabolism of exogenous sex steroids and ef f ect on brain functions with a focus on tibolone. J Steroid Biochem Mol Biol 102:195-204.

Woodgett JR (1990) Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 9:2431-2438.

Yankner BA, Lu T, Loerch P (2008)e aging brain. Annu Rev Pathol-Mech 3:41-66.

Zhang Z, Simpkins JW (2010) Okadaic acid induces tau phosphorylation in SH-SY5Y cells in an estrogen-preventable manner. Brain Res 1345:176-181.

Copyedited by Li CH, Song LP, Zhao M

*< class="emphasis_italic">Correspondence to: Christian Guerra-Araiza, christianguerra2001@gmail.com.

Christian Guerra-Araiza, christianguerra2001@gmail.com.

orcid: 0000-0002-7164-4116 (Christian Guerra-Araiza)

10.4103/1673-5374.205098

Accepted: 2017-03-22