Tau is a microtubule-associated protein that is abundant in the central nervous system (CNS) and expressed mainly in axons. In the human brain, Tau proteins constitute a family of six isoforms, ranging from 352 to 441 amino acids, all derived from single gene by alternative splicing (Goedert et al., 1989). The amino-terminal region of Tau, also called the "projection domain," project from the microtubule (MT) space to interact with the plasma membrane and is essential for determining axonal diameter (Chen et al., 1992). The carboxy-terminal region is characterized by the presence of 3 or 4 repeats that mediate the properties of Tau to stabilize MT and promote their polymerization (Weingarten et al., 1975; Cleveland et al., 1977a,b; Himmler et al., 1989; Butner and Kirschner, 1991; Gustke et al., 1994). These functions are negatively regulated by phosphorylation at multiple sites in, and around, the MT binding domain (for review, see Buée et al., 2000; Avila et al., 2004). Indeed, at least 30 Ser/Thr potential Tau phosphorylation sites have been described on Tau (Sergeant et al., 2008). Tau phosphorylation is regulated by numerous Ser/Thr kinases and phosphatases; among them glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are considered to be the major Tau kinase and phosphatase *in vivo*, respectively (Planel et al., 2002; Tian and Wang, 2002).

Intracellular aggregates of abnormally hyperphosphorylated Tau characterize a group of neurodegenerative diseases called "Tauopathies" (for review, see Buée et al., 2000). Tau hyperphosphorylation may lead to its aggregation *in vitro* (Alonso et al., 2001; Sato et al., 2002) and is thought to induce NFT formation and neurodegeneration in AD brains (Trojanowski and Lee, 1994).

#### **INTERACTION BETWEEN TAU AND AMYLOID PATHOLOGY**

The second major neuropathological hallmark of AD are amyloid plaques, mainly composed of amyloid-β (Aβ), a peptide of 39–43 amino acids generated *in vivo* by specific proteolytic cleavage of the APP, a transmembrane glycoprotein (Hardy and Higgins, 1992). It appears that soluble oligomeric Aβ forms, rather than amyloid plaques, contribute to the cellular pathology of the AD and correlate with the severity of cognitive impairment in humans (Lue et al., 1999; and for review, see Laferla et al., 2007).

To date, the interaction between Tau pathology and amyloid plaques in AD remains unresolved. Several murine animal models combining the two types of lesions have been investigated to better address this issue.

However, results have shown different possible types of interaction between the two pathologies. Thus, it was reported that injection of Aβ42 peptide potentiates Tau pathology in a mouse model of Tauopathy overexpressing a mutant form of the protein (Gotz et al., 2001). Similarly, double transgenic APP/Tau mice show early Tau pathology in comparison to Tau transgenic mice (Lewis et al., 2001; Perez et al., 2005; Ribe et al., 2005; Terwel et al., 2008). Interestingly, cultured neurons obtained from Tau−*/*− mice seem to be protected against neuronal death and cytotoxicity induced by Aβ, suggesting that Tau is crucial for Aβ-induced neurotoxicity (Rapoport et al., 2002). These results were further supported *in vivo*, in animal studies combining both Tau−*/*− and APP transgenic mice and showing that Tau reduction is protective against neurological and behavioral deficits induced by Aβ (Palop et al., 2006; Roberson et al., 2007). More importantly, this hypothesis is further supported by numerous *post-mortem* neuropathological studies showing that the pathway of Tau pathology is progressive, sequential and hierarchical (Bierer et al., 1995; Braak et al., 1999; Delacourte and Buée, 2000), whereas amyloid deposition is diffuse, widespread and extremely heterogeneous (Braak et al., 1999; Delacourte et al., 1999). Moreover, NFT formation was found to be strongly correlated with the degree of dementia and memory loss in AD brains, whereas Aβ was not (Braak and Braak, 1991, 1997; Delacourte et al., 1999). These observations have led some authors to the hypothesis that Tau pathology could be the major cause of cognitive decline in humans (Wilcock and Esiri, 1982; Delaere et al., 1989; Arriagada et al., 1992; Duyckaerts et al., 1997, 1998; Gomez-Isla et al., 1997; Delacourte et al., 2002; Giannakopoulos et al., 2003; Guillozet et al., 2003; Bretteville and Planel, 2008). These findings are in accordance with numerous animal studies showing significant impairment of cognitive functions, synaptic dysfunctions, as well as altered hippocampal synaptic plasticity in different transgenic mouse models of Tauopathies (Polydoro et al., 2009; Hoover et al., 2010; Van Der Jeugd et al., 2011; Burnouf et al., 2013).

# **INSULIN DYSFUNCTION**

#### **BASICS OF INSULIN SIGNALING**

The insulin receptor (IR) is a hetero-tetrameric receptor composed of two extracellular α-subunits that bind insulin, and two transmembrane β-subunits that have intracellular tyrosine kinase activity. The binding of insulin to the α-subunits of IR leads to the activation of the β subunit via auto-phosphorylation. When activated, IR phosphorylates insulin receptor substrate (IRS) proteins, which then activate phosphoinositide-3 kinase (PI3K). Two major signaling pathways are further activated by PI3K: the mitogen-activated protein kinase (MAPK) pathway and the Akt pathway (for review, see White, 1997; Taha and Klip, 1999). MAPK pathway is mainly involved in cell differentiation, cell proliferation and cell death, whereas Akt signaling is implicated in cell proliferation, cell growth, and protein synthesis (for review, see Brazil and Hemmings, 2001; Le Roith and Zick, 2001; Tremblay and Giguere, 2008). Akt further induces the phosphorylation of GSK-3β, rendering it inactive, which results in increase of glycogen synthesis in the periphery. Thus, disruption of Akt signaling leads to the dephosphorylation of GSK-3β, and hence to its activation; (for review, see Taha and Klip, 1999).

In insulin resistance that occurs in T2DM, the capacity of insulin to induce all these phosphorylation cascades is significantly decreased. Muscle biopsies of insulin resistant subjects revealed a reduction in the enzymatic activity and phosphorylation state of kinases and substrates involved in the IR signaling pathway (for review, see Schulingkamp et al., 2000; White, 2003; Pirola et al., 2004; Youngren, 2007). However, the site(s) of the initial perturbation of signal transduction are far from clear.

#### **INSULIN IN THE CNS**

In the past, the brain was described as "an insulin insensitive organ" (Goodner and Berrie, 1977). However, it is currently well known that insulin and its receptors are widely present in the CNS (Baskin et al., 1988; Lerorrtt et al., 1988; for review, see Schulingkamp et al., 2000). Although the origin of brain insulin is still a matter of debate, several evidence suggest that central insulin is actively transported from periphery across the bloodbrain barrier (BBB) (Banks et al., 1997a,b), and might be also produced locally by neurons in the brain (Devaskar et al., 1994).

The initial evidence of *de novo* insulin synthesis in the CNS came from a report showing insulin immunoreactivity in adult rat brain (Havrankova et al., 1978b). This evidence was later confirmed by other studies showing insulin gene expression and synthesis in cultured mammalian neurons (Schechter et al., 1988, 1990; Devaskar et al., 1994).

Interestingly, it was shown that high peripheral insulin concentrations acutely increase the levels of insulin in the brain and the cerebrospinal fluid (CSF), whereas prolonged peripheral hyperinsulinemia leads to a down-regulation of IRs in the BBB and to a reduction in insulin transport into the brain (Wallum et al., 1987).

IRs are selectively distributed in the brain.   Differences between peripheral and central IRs are not well understood, although some structural differences, mainly including the molecular weight and antigenicity, have been noted (Heidenreich et al., 1983; Heidenreich and Gilmore, 1985).

The expression of IRs in specific brain regions, particularly the hippocampus and medial temporal cortex suggests that insulin is implicated in memory processes (Singh et al., 1997; Zhao et al., 1999; Duarte et al., 2012; De Felice, 2013). For example, it was reported that both mRNA and protein levels of IRs are increased in the rat brain following a spatial memory task, suggesting that insulin might regulate normal memory function (Zhao et al., 1999).

By contrast, several findings have showed that insulin dysfunction, e.g., chronic hyperinsulinemia, DM or insulin resistance have a negative impact on memory process and cognitive function (Roriz-Filho et al., 2009; Sims-Robinson et al., 2010; Benedict et al., 2012; Crane et al., 2013b).

Mechanisms by which insulin might influence memory are reviewed elsewhere (Zhao and Alkon, 2001; Craft and Watson, 2004; Gerozissis, 2008; Freiherr et al., 2013). In brief, insulin signaling was shown to regulate neuronal survival, neurotransmission and synaptic activities (Zhao and Alkon, 2001). Insulin might also regulate synaptic plasticity by modulating long-term potentiation (LTP) (Zhao et al., 2010; Nistico et al., 2012), and promoting long-term depression (LTD) (Van Der Heide et al., 2005; Labouèbe et al., 2013), two major mechanisms involved in learning and memory.

Interestingly, it was recently established that administration of intranasal insulin is a non-invasive technique that leads to an increase of insulin levels specifically in the CNS, without affecting peripheral glucose and insulin levels (Reger et al., 2008a). Thus, several clinical studies have demonstrated a beneficial effect of intranasal insulin on memory and cognitive function in AD patients (Reger et al., 2008a,b; Craft et al., 2012).

Therefore, although emerging evidence support a role of brain insulin signaling in several complex CNS functions, additional mechanistic studies are warranted to better elucidate the contribution of insulin signaling in normal and diseased brains.

# **DIABETES MELLITUS**

Diabetes mellitus (DM) is a chronic disease, marked by high levels of blood glucose. It occurs in two main forms: type 1 and type 2 diabetes, that respectively represent about 5–10% and 90–95%, of the total diabetes cases (CDC, 2008).

### *Type 1 diabetes mellitus*

Type 1 diabetes mellitus (T1DM) was previously known as Insulin-dependent DM (IDDM) or juvenile diabetes to distinguish it from type 2 diabetes mellitus (T2DM), which generally has a later onset; however, the majority of new-onset type 1 diabetes is seen in adults (Naik and Palmer, 2007).

It is due to autoimmune destruction of the insulin-producing β-cells resulting in low or no production of insulin hormone. Thus, insulin therapy is absolutely required in patients with T1DM (McCrimmon and Sherwin, 2010). However, one major feared side effect of insulin therapy is iatrogenic hypoglycemia, which might occur in almost 90% of all insulin treated patients (Cryer, 1997, 2001, 2004; Cryer et al., 2003).

### *Type 2 diabetes mellitus*

Type 2 diabetes mellitus (T2DM), previously known as noninsulin dependent DM (NIDDM), was usually associated with aging. About 40% of the population over 65 and 50% over 80 years of age have T2DM or impaired glucose tolerance, and half of diabetic people are not diagnosed (Lamberts et al., 1997). However, T2DM is not a disease exclusively associated with old age, as it is more and more diagnosed in young people as the number of cases has risen in parallel with obesity. Today, there are 382 million people with T2DM, and it is estimated that this number will rise to 592 million by 2035 (IDF, 2013). In T2DM, pancreas produces insulin, but, for reasons that remain unclear, cells are unable to use it, a characteristic of insulin resistance etiology. Thus, in a positive feedback loop, pancreas overproduces insulin to overcome the resistance, leading to a hyperinsulinemia in early phases of T2DM (Festa et al., 2006). Over time, the suffering pancreas becomes unable to produce enough insulin, leading to hyperglycemia and insulin deficiency that accompany T2DM in the end of its natural history (Defronzo, 2004).

# **IMPACT OF INSULIN DYSFUNCTION ON TAU PHOSPHORYLATION**

Over the last decade, there has been considerable interest on the impact of insulin dysfunction and diabetes on Tau pathology. It has been shown that insulin could regulate Tau phosphorylation in neuronal cells (Hong and Lee, 1997; Lesort et al., 1999; Lesort and Johnson, 2000), which was confirmed by observations of hyperphosphorylated Tau in mice showing abnormal insulin levels in the brain (Schubert et al., 2003, 2004; Planel et al., 2004; Schechter et al., 2005). Despite all these data, very little is known about the effects of diabetes on Tau pathogenesis *in vivo*.

#### *IN VITRO* **STUDIES**

Observations from *in vitro* studies strongly suggest that Tau phosphorylation might be regulated by insulin signaling through a biphasic manner. Thus, short insulin treatment (*<*2–3 min), either in rat primary cortical neurons (Lesort and Johnson, 2000), or in SH-SY5Y human neuroblastoma cells (Lesort et al., 1999), leads to a rapid and transient Tau hyperphosphorylation at the AT8 (Ser202/Thr205), AT180 (Thr231), PHF-1 (Ser396/Ser404) and T3P (Ser396) epitopes (Lesort et al., 1999; Lesort and Johnson, 2000). By contrast, prolonged exposure to insulin, in both human neuroblastoma cells (up to 60 min of treatment) (Lesort et al., 1999), and human neuronal NT2N cells (5 min of treatment) (Hong and Lee, 1997), results in a significant decrease in Tau phosphorylation. Interestingly, all these studies have led to the conclusion that the effects of insulin are mediated by GSK-3β activity (Hong and Lee, 1997; Lesort et al., 1999; Lesort and Johnson, 2000).

Therefore, insulin signaling plays an important role in the regulation of Tau phosphorylation, and this effect is correlated with GSK-3β activity, further suggesting a key role of this kinase in Tau pathology.

#### *IN VIVO* **STUDIES**

The specific effects of insulin dysfunction on Tau pathology are not fully understood. Therefore, several animal models are currently used to investigate the impact of both T1DM and T2DM on Tau phosphorylation.

#### *T1DM and Tau phosphorylation*

Models of T1DM used in the literature mainly mimic insulin deficiency that characterizes human T1DM. These models can develop T1DM either spontaneously, due to a specific genetic background, or by treatment with a drug called Streptozotocin (STZ).

STZ (2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-glucopyr-anose)) is a toxin synthesized by *Streptomycetes achromogenes*, that specifically enters pancreatic β cells via the glucose transporter GLUT2, and leads to cell death through DNA alkylation (Delaney et al., 1995). Thus, it is widely used to induce insulin deficiency associated to T1DM (Ganda et al., 1976), both in wild-type background context and AD pathological animal models.

*STZ-induced wild-type animal models.* Using an extensive panel of phospho-dependent antibodies that detect specific epitopes on Tau protein, several animal studies have investigated the impact of peripheral STZ treatment on Tau phosphorylation in non-transgenic animals (**Table 1**). For example, we have previously reported that STZ administration (200 mg/Kg, i.p.) resulted in a massive Tau hyperphosphorylation at the Tau-1 (Ser195/Ser198/Ser199/Ser202), pS199 (Ser199), PHF-1, AT8, AT180, Ser422, Ser262, Ser356 and Ser400epitopes in the brain of nontransgenic mice (Planel et al., 2007b). Other studies have confirmed our results of increased Tau phosphorylation following STZ treatment either in mice (Clodfelder-Miller et al., 2006; Jolivalt et al., 2008; Ke et al., 2009; Kim et al., 2009), or in rats (Qu et al., 2011), and observed a further increase in Tau phosphorylation at the AT270 (Thr181) and Thr212 epitopes (**Table 1**).

Some of the phosphoepitopes detected are associated to particular functions during Tau pathology. For example, Ser422 is associated with early pre-tangle formation and is characteristic of abnormal, AD-like Tau phosphorylation (Augustinack et al., 2002). AT8 is considered an early marker of Tau dysfunction (Matsuo et al., 1994; Hasegawa et al., 1996; Bussière et al., 1999; Augustinack et al., 2002), whereas PHF-1 is associated with late paired helical filament and NFT formation (Bramblett et al., 1993; Goedert et al., 1993, 1994). In addition, some phosphorylation sites have been linked to specific aspects of Tau pathology such as the sequestration of normal Tau (e.g., Thr231/Ser235) (Alonso et al., 1997, 2004), the inhibition of Tau MT binding (e.g., Ser262) (Biernat et al., 1993; Drewes et al., 1995), and the promotion of Tau aggregation (e.g., Ser396, Ser422) (Gong and Iqbal, 2008).

Interestingly, we have shown that the massive Tau hyperphosphorylation in STZ treated mice was mostly, but not completely, rescued by returning animals to normothermia, suggesting that hypothermia was partially involved in STZ-mediated Tau hyperphosphorylation. We have previously demonstrated that Tau phosphorylation can be induced by hypothermia (Planel et al., 2004, 2007b). Moreover, we have shown that this mechanism is mainly attributable to a direct and rapid inhibition of PP2A activity in the brain (Planel et al., 2004).

In fact, hypothermia is a common outcome in experimental diabetes (Shalaby et al., 1989; Kilgour and Williams, 1996, 1998) and several human diabetic populations (Neil et al., 1986; Scott et al., 1987).

In addition, Tau phosphorylation is known to be exquisitely sensitive to temperature, increasing by 80% per degree Celsius drop under 37◦C in mice (Planel et al., 2007a; Papon et al., 2011).

**1|SummaryofanimalstudiesshowingTauhyperphosphorylationanditsmolecularmechanismsindiabeticanimal**

It has to be mentioned that previous studies done in other laboratories described above did not monitor the temperature of animals and therefore it is likely that hypothermia could contribute to the observed Tau hyperphosphorylation in these studies (Clodfelder-Miller et al., 2006; Ke et al., 2009; Kim et al., 2009; Qu et al., 2011).

Therefore, temperature control during Tau analysis experiments in animal models is crucial to avoid artifactual hypothermic Tau hyperphosphorylation.

*STZ-induced transgenic animal models.* Beside studies performed in a physiological context, several investigators have addressed the impact of STZ treatment on Tau phosphorylation and NFT formation in transgenic mouse models of AD (**Table 1**). For example, Ke et al. have shown that insulin depletion increases more significantly Tau phosphorylation at multiple epitopes in mice that overexpress P301L mutant human Tau, and are prone to develop NFT (Gotz et al., 2001). This suggests that experimental diabetes leads to advanced NFT formation, as well as early neurofibrillar deposition in Tau transgenic mice (Ke et al., 2009). Similarly, insulin deficiency enhances the severity of Tau phosphorylation in the hippocampi of APP transgenic mice, which are characterized by the accumulation of the β-amyloid peptide and high levels of Aβ-immunoreactive plaques (Jolivalt et al., 2010).

Therefore, it seems that STZ-induced T1DM recapitulate NFT formation observed during of AD, and this effect is further worsened when the induction is combined to genetic pre-disposition to AD.

However, it is important to note that several studies from those cited above have used anesthesia prior to sacrifice (Clodfelder-Miller et al., 2006; Jolivalt et al., 2008, 2010; Ke et al., 2009; Kim et al., 2009) (**Table 1**), which is known to enhance Tau phosphorylation (Planel et al., 2007a; Papon et al., 2011; Whittington et al., 2011; Le Freche et al., 2012). This increase can be induced either directly, through a pharmacological effect of the drug itself (Whittington et al., 2011; Le Freche et al., 2012), or indirectly, through anesthesia-induced hypothermia (Planel et al., 2007a).

Thus, further animal studies with temperature control are warranted to separate the impact of hypothermia from that of insulin dysfunction on Tau phosphorylation.

*Animal models of spontaneous T1DM.* Although several studies have reported Tau hyperphosphorylation in STZ-induced animal models, it is important to extend these findings to a model that does not require a drug to induce T1DM, because, as we will see later, if STZ reaches the brain in minute quantities, it can lead to Tau hyperphosphorylation on its own.

Thus, we have investigated Tau hyperphosphorylation in the non-obese diabetic (NOD) mouse, one of the most valuable genetic animal models for T1DM (Leiter, 2001). These mice spontaneously develop T1DM at 13–25 weeks of age as a consequence of selective destruction of insulin-producing β cells (Makino et al., 1980). Interestingly, our data indicated that Tau hyperphosphorylation correlates with the appearance of spontaneous diabetes in adult NOD mice, and this effect was exacerbated when the mice became hypothermic as a consequence of diabetes. Interestingly, even in the absence of any deregulation in

the glucose metabolism, we have observed a slight increase in Tau phosphorylation at Tau-1 and Ser422 epitopes in non-diabetic adult NOD mice. Moreover, the onset of diabetes (the appearance of hyperglycemia and glycosuria) was correlated with an extensive Tau hyperphosphorylation at the AT8, CP13 (Ser202), Ser262 and Ser422 epitopes in comparison to control. Furthermore, the appearance of hypothermia further extended Tau hyperphosphorylation to PHF-1 and TG3 (Thr231) epitopes in diabetic NOD mice (Papon et al., 2013), (**Table 1**).

Of note, Li et al. have also investigated Tau phosphorylation in BB/Wor rats, another model of spontaneous T1DM. Although these authors did not find any significant changes in the levels of Tau phosphorylation at the Ser396 epitope (Li et al., 2007), it should be mentioned that there was no report of the rats temperature and that they were anesthetized, which could have masked a potential elevation in Tau phosphorylation.

Thus, while our study demonstrated Tau hyperphosphorylation in NOD mice, additional studies should be done to our results in other animal models of spontaneous T1DM.

*The insulin knockout animal model.* In addition to genetic and STZ-induced animal models, the insulin knockout mouse model (Insulin−*/*−) might also mimic insulin deficiency that characterizes T1DM.

Interestingly, Schechter and colleagues have investigated the impact of insulin deficiency on Tau phosphorylation in Insulin−*/*− mice. Thus, among all phospho-epitopes investigated, these authors have observed a significant increase in Tau phosphorylation at the AT180 epitope, suggesting that insulin signaling might affect Tau phosphorylation *in vivo* (Schechter et al., 2005), (**Table 1**).

#### *T2DM and Tau phosphorylation*

Models of T2DM used in animal studies mainly reproduce human T2DM features, including obesity, hyperglycemia, hyperinsulinemia, and insulin resistance. These models develop T2DM either spontaneously, or by using a variety of treatments including special diets.

*Animal models of spontaneous T2DM.* Several genetic animal models are used in the literature to address the impact of genetic T2DM on AD pathogenesis (Ellis et al., 1998; Li et al., 2007; Jolivalt et al., 2008; Takeda et al., 2010). These models are mainly characterized by an impairment of leptin signaling, by either lacking leptin (ob/ob mice) (Coleman, 1978), or carrying specific mutations in the leptin receptor (Bio-Breeding Zucker Diabetic Rats/Wor (BBZDR/Wor) rats and db/db mice) (Hummel et al., 1966; Tirabassi et al., 2004). Leptin is an adipocyte-specific hormone that plays important role in satiety and energy expenditure. It acts through the leptin receptor, a single-transmembrane-domain receptor of the cytokine receptor family. However, mechanisms by which leptin signaling leads to the development of T2DM are not well understood.

Several studies have reported Tau hyperphosphorylation in rat (Li et al., 2007; Jung et al., 2011, 2013) and mouse models (Kim et al., 2009; Li et al., 2012a) of spontaneous T2DM. For example, Jung et al. have reported an increase in Tau phosphorylation at the Tau-1, Thr212 (Jung et al., 2011, 2013), Thr231, Ser262 and Ser396 (Jung et al., 2013) epitopes in the chronic Otsuka Long Evans Tokushima Fatty (OLETF) T2DM rat model, and these changes were further increased with age (Jung et al., 2013). In addition, Li and colleagues have also observed Tau hyperphosphorylation at the Ser396 site in the cortex of BBZDR/Wor rat strain (Li et al., 2007). These results were also confirmed in db/db mice that showed Tau hyperphosphorylation at the AT8, Ser199, Ser202, Ser396, and Ser422 epitopes (Kim et al., 2009; Li et al., 2012a) (**Table 1**). However, since these animals were anesthetized prior to sacrifice and their temperature was not reported, it would be interesting to repeat these experiments in controlled conditions.

*Animal models of induced T2DM.*  Moroz et al. have reported that HFD increases body weight, reduces brain weight and leads to brain insulin resistance, since mice fed with HFD showed increased brain insulin gene expression paralleled with decreased IR binding (Moroz et al., 2008). However, levels of both phosphorylated and total Tau were not affected in the brain of non-transgenic animals treated with HFD, although Tau mRNA levels were significantly increased (Moroz et al., 2008). Similarly, levels of total soluble and insoluble Tau, but not phospho-Tau were increased in HFD fed 3xTg-AD transgenic mice, suggesting that effects of HFD are modest on Tau phosphorylation (Julien et al., 2010).

On the other hand, a recent study has demonstrated that HFD leads to Tau phosphorylation in a model of Tauopathy treated with HFD, in a manner independent of insulin resistance, suggesting that, other obesity-related factors, might contribute to Tau pathology (Leboucher et al., 2012).

Interestingly, Bhat and Thirumangalakudi have reported that feeding mice with high-fat/cholesterol (HFC) diet also leads to peripheral insulin resistance. Importantly, these authors showed that HFC results in increased levels of Tau hyperphosphorylation at the PHF-1 and Thr231 epitopes in the mice hippocampi (Bhat and Thirumangalakudi, 2013).

It is difficult to draw conclusion from HFD treatments since published results are so divergent. The reasons for these differences could include the composition of the diet, the duration of the treatment, the background strains, the levels of contamination of the animal facilities and, of course, confounding hypothermia.

*Insulin signaling impairment animal models.* The group of Schubert et al. have generated and explored a brain/neuron insulin receptor knockout (NIRKO) mouse model that exhibit a conditional inactivation of the IR gene in the CNS (Schubert et al., 2004). These mice are mainly characterized by hyperinsulinemia, mild insulin resistance, obesity and reduced fertility (Bruning et al., 2000), and might therefore represent a model of T2DM. Central IR deficiency in these mice has resulted in a significant increase of Tau phosphorylation at the AT180 epitope (Schubert et al., 2004).

Another model of insulin signaling impairment that might represent a model of T2DM is the IRS-2−*/*− mouse model. These mice are insulin resistant and develop diabetes as a result of impairment in survival and function of pancreatic β-cells (Bruning et al., 1998; Withers et al., 1998, 1999; Kulkarni et al., 1999; Michael et al., 2000). Schubert et al. have demonstrated that IRS-2−*/*− mice exhibit Tau hyperphosphorylation at the Ser202epitope (Schubert et al., 2003), (**Table 1**).

Therefore, animal models of insulin signaling dysfunction have provided additional evidence that insulin plays crucial role in the regulation of Tau phosphorylation *in vivo.* However, further studies are needed to better understand mechanisms underlying these effects.

#### *T3DM and Tau phosphorylation*

Data from T1DM and T2DM animal models strongly suggest that insulin signaling plays a key role in modulating AD pathogenesis. However, in almost all animal models cited above, Tau pathology was investigated in a context of peripheral insulin dysfunction. Therefore, animal models exhibiting specific alteration of the central insulin function might be helpful to address the role of brain insulin disruption in AD pathology. Thus, the group of Hoyer and colleagues have suggested that rats treated with intra-cerebroventricular (i.c.v.) STZ represent a sporadic AD animal model characterized by a specific insulin-resistant brain state (Nitsch and Hoyer, 1991; Duelli et al., 1994; Lannert and Hoyer, 1998; Salkovic-Petrisic and Hoyer, 2007). Interestingly, de la Monte et al have later proposed that AD might represent a specific form of brain diabetes and thus proposed the term of "type 3 diabetes" (T3DM) (De La Monte et al., 2006; Lester-Coll et al., 2006).

In induced T1DM animal models, STZ is administrated peripherally at high doses (55–200 mg/Kg). However, in T3DM rats, STZ is injected in the brain ventricles at doses of up to 100 times lower than those used in systemic injections (Nitsch and Hoyer, 1991; Duelli et al., 1994; Lannert and Hoyer, 1998). Although it was shown that pancreatic architecture, as well as levels of both insulin and glucose are not affected following central STZ treatment (De La Monte et al., 2006; Lester-Coll et al., 2006), numerous studies have reported that T3DM rat models exhibit several neurochemical, structural and behavioral changes that are similar to cellular abnormalities observed in AD brains. These changes include brain atrophy, cell loss, neurodegeneration (De La Monte et al., 2006; Lester-Coll et al., 2006), a decrease in glucose utilization notably in the hippocampi and entorhinal cortices of treated rats (Duelli et al., 1994), a reduction of energy metabolism (Lannert and Hoyer, 1998; Prickaerts et al., 2000; Yun et al., 2000), an impairment in learning and memory, as well as a significant increase in oxidative stress (Sharma and Gupta, 2001; Veerendra Kumar and Gupta, 2003; Ishrat et al., 2006; Pathan et al., 2006).

The mechanism of action of central STZ is far from clear, but some observations suggest that it might be similar to that in the periphery. Indeed, GLUT2, the glucose transporter targeted by peripheral STZ, was found to be regionally distributed in the mammalian brain (Brant et al., 1993; Leloup et al., 1994; Ngarmukos et al., 2001). In addition, DNA damage was observed in T3DM rat brains (Nitsch and Hoyer, 1991; Lannert and Hoyer, 1998).

The vast majority of studies that have investigated Tau phosphorylation following i.c.v. STZ administration (ranging from 1 to 3 mg/Kg) were performed in non-transgenic animal models. Interestingly, all these studies have observed a significant increase in Tau phosphorylation at several epitopes, notably AT8, PHF-1, Ser199, Ser202, Ser396, Ser404, Thr181, Thr205, Thr212, and Tau-1(Chu and Qian, 2005; Salkovic-Petrisic et al., 2006; Grunblatt et al., 2007; Deng et al., 2009; Chen et al., 2011, 2013b; Li et al., 2012b; Du et al., 2013) (**Table 1**).

Interestingly, Chen et al. have recently investigated the impact of T3DM on Tau phosphorylation in the 3xTg-AD mouse model. These authors demonstrated an increase in Tau phosphorylation at the Ser199/Ser202, 12E8 (Ser262/Ser356) and Ser422epitopes (Chen et al., 2013b).

By contrast, Plascke et al. didn't detect any increase in the level of phosphorylated Tau in the brain of Tg2576 mice (Plaschke et al., 2010).

Therefore, T3DM leads to several alterations resembling to those found in human AD patients, further giving consistent evidence that brain insulin resistance might be a central event in AD. However, it should be noted that it remain difficult to conclude from all studies cited above because they are quite heterogeneous, considering differences in the rat strain used, the dose of STZ delivered, the age at STZ treatment and brain regions investigated (for review, see Salkovic-Petrisic et al., 2006).

# **MECHANISMS LINKING INSULIN DYSFUNCTION TO TAU PATHOLOGY**

#### **KINASES ACTIVATION**

The increased phosphorylation of Tau on many residues might be attributable to the activation of multiple kinases. Among the kinases able to phosphorylate Tau *in vitro*, GSK-3β is considered to be the major physiological and pathological Tau kinases (Planel et al., 2002; Hernandez et al., 2013).

Studies that have addressed GSK-3β activation in diabetic animal models (using antibodies detecting the phosphorylation state either at the Ser9 inhibitory site, or the Tyr216 activation site of the kinase) have shown contradictory findings (**Table 1**), thus making it difficult to conclude about the implication of this kinase in the correlation between diabetes and Tau pathogenesis. Of note, we (Planel et al., 2007b), and others (Clodfelder-Miller et al., 2006; Deng et al., 2009), have demonstrated that GSK-3β is inhibited in STZ-induced T1DM mice, and our data demonstrated that this inhibition can be attributed to hypothermia (Planel et al., 2001), as GSK-3β S9 phosphorylation is a constant feature of hypothermia in mouse brain during metabolic stress (Planel et al., 2004) or anesthesia (Planel et al., 2007a). However, it is important to note that, even when inhibited, GSK-3β is still participating to Tau hyperphosphorylation, as we previously demonstrated with lithium, a GSK-3β inhibitor, in mice rendered hypothermic by starvation (Planel et al., 2001).

#### **PHOSPHATASES INHIBITION**

Ser/Thr protein phosphatases (PP) are classified into five types, PP1, PP2A, PP2B, PP2C, and PP5, on the basis of their substrate specificities and sensitivity to specific activators and inhibitors (Liu et al., 2005). Biochemical studies have demonstrated that PP1, PP2A, PP2B (Calcineurin), and PP5 are involved in Tau dephosphorylation (Gong et al., 1994; Tian and Wang, 2002). Importantly, it is believed that PP2A is the major Tau phosphatase *in vivo* (Gong et al., 2000; Planel et al., 2001, 2004, 2007a), with PP2A, PP1, PP5, and PP2B contributing to 71, 11, 10 and 7%, respectively, of the total Tau phosphatase activity in the brain (Liu et al., 2005).

The analysis of different Tau phosphatases in diabetic animal models is less documented in comparison to kinases (**Table 1**). However, although several investigators have observed a decrease only in the expression of PP2A in T2DM mice brains (Schubert et al., 2003; Li et al., 2012a), we (Planel et al., 2007b), and others (Clodfelder-Miller et al., 2006; Qu et al., 2011; Jung et al., 2013), have reported that PP2A activity is inhibited in both T1DM and T2DM animal models.

Interestingly, Deters and colleagues demonstrated that coexpression of P301L mutant human Tau and a dominant negative form of PP2A in the brain of transgenic mice significantly increases Tau hyperphosphorylation and NFT formation, suggesting a crucial role for PP2A in regulating Tau pathology (Deters et al., 2009). This finding is particularly important since PP2A is inhibited in human AD patients and seems to be an important factor in the progression of the disease (Gong et al., 1993, 1995; Vogelsberg-Ragaglia et al., 2001).

Thus, PP2A seems to be a key protein in the link between insulin dysfunction and AD. Extending analyses to other diabetic mouse models or genetically modified mice will facilitate the understanding of molecular mechanisms underlying PP2A function, and thus help for the identification of molecules that may compromise the reversibility of Tau hyperphosphorylation.

#### **INFLAMMATION**

Several studies have shown that insulin dysfunction is associated with inflammation. In the periphery, insulin was shown to modulate many aspects of the inflammatory process. Thus, at low doses, insulin exerts anti-inflammatory effects (Dandona, 2002); whereas, during chronic hyperinsulinemia, insulin may exacerbate inflammatory responses and increase markers of oxidative stress (Krogh-Madsen et al., 2004). Specifically, it was demonstrated that levels of proinflammatory cytokines, including Interleukin-1 (IL-1), IL-6, and C-reactive protein (CRP) are elevated in diabetic patients (Hak et al., 2001; Spranger et al., 2003).

In the CNS, several animal studies have also associated insulin dysfunction to inflammation. For example, a significant increase in the number of glial fibrillary acidic protein (GFAP)-reactive astrocytes (also known as astrogliosis) was reported in the hippocampi of both NOD and STZ-induced mouse models (Saravia et al., 2002).

In addition, T2DM was shown to aggravate vascular inflammation in an AD mouse model, changes that can be related to impaired central insulin signaling (Takeda et al., 2010). Notably, a study by Pistell et al. has established that different diet composition results in various inflammatory reactions in the brain. Thus, these authors have demonstrated that HFD consumption leads to a significant increase in the levels of proinflammatory cytokines, chemokines as well as reactive astrocytosis and microgliosis (Pistell et al., 2010). These data suggest that inflammation might be tightly related to Tau hyperphosphorylation observed during T2DM.

Moreover, brains of i.c.v.-STZ treated rats showed increased levels of IL-1 and tumor necrosis factor-α (TNF-α) (Chen et al., 2013a), as well as pronounced astrogliosis and microglial activation (Prickaerts et al., 1999; Chen et al., 2013a).

On the other hand, there is evidence that Tau pathology is associated with neuroinflammation. Thus, IL-1, IL-6 as well as nitric oxide have been shown to exacerbate Tau pathology and NFT formation *in vitro* (Li et al., 2003; Quintanilla et al., 2004; Saez et al., 2004). Moreover, Shepherd et al. have demonstrated that microglial activation might contribute to Tau hyperphosphorylation and NFT formation in *postmortem* AD brains (Shepherd et al., 2000; for review, see Arnaud et al., 2006). Similarly, administration of lipopolysaccharide (LPS), which is a bacterial endotoxin commonly used in animal studies to induce systemic inflammation (Lien et al., 2000), significantly increases Tau hyperphosphorylation in a triple transgenic mouse model of AD (Kitazawa et al., 2005). In addition, Yoshiyama et al. have demonstrated that microglial neuroinflammation precedes Tau pathology in P301S transgenic mice (Yoshiyama et al., 2007).

Mechanisms by which neuroinflammation might contribute to Tau pathology are not well elucidated. However, it is known that one signaling pathway by which neurons and microglia communicate is fractalkine (CX3CL1) and its cognate receptor (CX3CR1). In the CNS, CX3CL1 is highly expressed in neurons, whereas CX3CR1 is exclusively expressed in microglia cells (Harrison et al., 1998). Therefore, Bhaskar et al. have suggested that the CX3CL1/CX3CR1 pathway could modulate Tau pathology, and that this effect might be dependent upon microglialderived IL-1-p38 MAPK signaling pathway (Bhaskar et al., 2010).

Overall, the link between insulin dysfunction, inflammation (both peripheral and/or central), and Tau hyperphosphorylation is unclear and would benefit from future research focus.

#### **STRESS**

Stress might represent another mechanistic link between insulin dysfunction and Tau hyperphosphorylation. Indeed, it was recently reported that stress leads to both peripheral and central insulin resistance, as well as increased Tau hyperphosphorylation in the mouse brain. Notably, these authors have pointed a central role of JNK (c-Jun N-Terminal Kinase), a major stress signaling pathway (Solas et al., 2013). These results suggest that insulin resistance might mediate Tau phosphorylation through a stress-dependent mechanism. Further studies are required to better understand this issue.

#### **INSULIN SIGNALING: THE CONVERGING ROAD**

#### *Insulin signaling impairment in diabetic animal models*

While impaired peripheral insulin signaling is well known in animal models of diabetes, there is conflicting evidence of central insulin signaling dysfunction.

The phosphorylation levels of IRs were reported to be significantly decreased in mice treated with systemic STZ (Jolivalt et al., 2008, 2010), as well as in the insulin KO mouse model (Schubert et al., 2004) (**Table 1**). By contrast, levels of the β subunit of IR (IRβ) have shown contradictory findings; whereas Jung *et al* have observed that IRβ levels are increased in the brain of the OLETF T2DM rat model (Jung et al., 2011), Li et al. have reported a significant decrease in IRβ levels in the cortex of spontaneous T1DM, but not T2DM rats (Li et al., 2007). Similarly, the insulin KO mouse model has revealed a decrease in the expression of IRβ in the brain (Schechter et al., 2005).

Conflicting results have also been reported for Akt, a key kinase implicated in the insulin-signaling pathway, and also known to phosphorylate Tau *in vitro* (Ksiezak-Reding et al., 2003). Thus, a significant reduction in the phosphorylation levels and the activity of Akt (Li et al., 2007; Qu et al., 2011) was reported in the brain of both T1DM and T2DM animal models (Jolivalt et al., 2008, 2010; Qu et al., 2011; Bhat and Thirumangalakudi, 2013). By contrast, several data showed augmented phosphorylation of Akt and GSK-3β Ser9 (inhibitory phosphorylation) in NOD and STZtreated mice (Clodfelder-Miller et al., 2005, 2006; Planel et al., 2007b). It is interesting to note that the rise in Akt and GSK-3β Ser9 phosphorylation in both NOD and STZ-treated mice is probably due to the inhibition of PP2A, as inhibitors of the phosphatase upregulate the phosphorylation of the two kinases (Andjelkovic et al., 1996; Planel et al., 2001). However, how peripheral insulin dysfunction results in central PP2A inhibition remains to be elucidated.

Moreover, Moroz et al. have demonstrated that HFD-induced T2DM leads to an increase in mRNA levels of IRS-1 and IRS-4 in the brain of non-transgenic mice (Moroz et al., 2008). By contrast, Bhat and Thirumangalakudi have reported a decrease in IRS-1 levels in the brain of HFC-treated mice (Bhat and Thirumangalakudi, 2013).

Interestingly, a recent study has demonstrated that HFD potentiated Tau pathology in a mouse model of Tauopathy, in a manner independent from insulin resistance, suggesting that other factors, probably linked to obesity, might be implicated in Tau pathology during T2DM (Leboucher et al., 2012).

Several investigators have also reported a deregulation in gene and/or protein expression of the insulin-signaling pathway in T3DM animal models. For example, the phosphorylation level of PI3K was significantly decreased in mice treated with central STZ (1.5 mg/Kg, i.c.v.) (Deng et al., 2009). Moreover, Grunblatt et al. have demonstrated that mRNA levels of both insulin and IRs are decreased, whereas levels of phosphorylated IRβ are increased in the hippocampi of T3DM rats (Grunblatt et al., 2007), (**Table 1**).

In summary, the involvement of the central insulin signaling in Tau pathology is still controversial, with divergent results showing a decrease, an increase, or even no significant changes in the insulin signaling proteins in the brains of diabetic animals (**Table 1**). Therefore, further investigations are crucial to get a more definitive picture of the role of central insulin signaling in the progression of Tau pathology.

#### *Implication of insulin signaling on Tau pathology*

Beside its classical function of regulating glucose metabolism, there is increasing evidence supporting a role for the

insulin-signaling pathway in neuronal development as well as in learning and memory, therefore suggesting a crucial role of insulin signaling in AD pathogenesis (Zhao and Alkon, 2001; Craft and Watson, 2004; Gerozissis, 2008).

It has been proposed that insulin could affect Tau phosphorylation through the regulation of GSK-3β, since it is a kinase downstream in the IR signaling pathway (Hong and Lee, 1997). Although insulin administration in STZ treated mice completely (Planel et al., 2007b), or partially (Clodfelder-Miller et al., 2006; Jolivalt et al., 2008) rescued Tau phosphorylation in mice, these findings are in contrast with human studies that report a higher risk of AD in patients treated with insulin (Ott et al., 1999; Luchsinger et al., 2001). However, one should be cautious in making direct conclusions from these epidemiological studies, as it might be because the patients were having a more severe stage of diabetes that they required treatment with insulin. Thus, more controlled studies in animals, as well as in human patients are important to better understand the role of insulin in the brain.

Furthermore, several studies have investigated the impact of rosiglitazone treatment on Tau phosphorylation in animal models. For example, Escribano et al. have observed a significant decrease in Tau phosphorylation in the brain of transgenic hAPP mice following rosiglitazone treatment (Escribano et al., 2010). Similarly, Yoon et al. have demonstrated that this drug reduced Tau phosphorylation both in *vitro*, and in the hippocampi of OLETF T2DM rats. Interestingly, these authors suggest that this effect might be due to a decrease in JNK activity (Yoon et al., 2010).

Thus, although brain insulin signaling might have an important effect on Tau phosphorylation, the molecular mechanisms underlying this effect are far from clear. Better understanding of these mechanisms might help to develop therapeutic strategies aiming to reduce Tau hyperphosphorylation in the brain.

# **CONCLUSION**

Overall, numerous preclinical studies examining the correlation between insulin dysfunction and Tau hyperphosphorylation converge to indicate that both T1DM and T2DM might affect Tau pathology, either directly or indirectly (**Figure 1**). Apart from some emerging evidences, mechanisms by which insulin dysfunction and/or other features of diabetes such as obesity and inflammation contributes to Tau pathology are still not fully elucidated. Therefore, further studies using other animal models (e.g., ob/ob mice. . . ) are required to better understand the contributions of these mechanisms to Tau hyperphosphorylation. However, future studies must carefully report and control physiological parameters such as body temperature, and should avoid using anesthesia for the sacrifice of the animals.

In light of the increased incidence of diabetes in the young population, future focus on the correlation between insulin dysfunction and Tau pathology may provide invaluable information for the treatment and prevention of sporadic AD.

#### **ACKNOWLEDGMENTS**

This work was supported by a Biomedical Doctoral Award from the Alzheimer Society of Canada (to Noura B. El Khoury), and grants to Emmanuel Planel from the Canadian Institute of Health Research (MOP-106423, PCN-102993), Fonds de Recherche en Santé du Quebec (16205, 20048) and the Natural Sciences and Engineering Research Council of Canada (354722). The funders had no role in the decision to publish, or preparation of the manuscript.

Noura B. El Khoury and Emmanuel Planel wrote the manuscript. Marie-Amélie Papon, Maud Gratuze, and Alexis Bretteville reviewed the manuscript.

#### **REFERENCES**

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expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats. *J. Biol. Chem.* 274, 34893–34902. doi: 10.1074/jbc.274.49.34893

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 27 November 2013; paper pending published: 11 December 2013; accepted: 16 January 2014; published online: 11 February 2014.*

*Citation: El Khoury NB, Gratuze M, Papon M-A, Bretteville A and Planel E (2014) Insulin dysfunction and Tau pathology. Front. Cell. Neurosci. 8:22. doi: 10.3389/fncel. 2014.00022*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 El Khoury, Gratuze, Papon, Bretteville and Planel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Pin1, a new player in the fate of HIF-1α degradation: an hypothetical mechanism inside vascular damage as Alzheimer's disease risk factor

**Elena Lonati, Anna Brambilla, Chiara Milani, Massimo Masserini, Paola Palestini and Alessandra Bulbarelli\***

Department of Health Science, University of Milano-Bicocca, Monza (MI), Italy

#### **Edited by:**

Ludovic Martin, Université de Nantes, France

#### **Reviewed by:**

Jing-Zhang Wang, Hebei University of Engineering, China Amandine Magnaudeix, University of Limoges, France

#### **\*Correspondence:**

Alessandra Bulbarelli, Department of Health Science, University of Milano-Bicocca, via Cadore 48, 20900 Monza (MI), Italy e-mail<EMAIL_ADDRESS>Aetiology of neurodegenerative mechanisms underlying Alzheimer's disease (AD) are still under elucidation. The contribution of cerebrovascular deficiencies (such as cerebral ischemia/stroke) has been strongly endorsed in recent years. Reduction of blood supply leading to hypoxic condition is known to activate cellular responses mainly controlled by hypoxia-inducible transcription factor-1 (HIF-1). Thus alterations of oxygen responsive HIF-1α subunit in the central nervous system may contribute to the cognitive decline, especially influencing mechanisms associated to amyloid precursor protein (APP) amyloidogenic metabolism. Although HIF-1α protein level is known to be regulated by von Hippel-Lindau (VHL) ubiquitin-proteasome system, it has been recently suggested that glycogen synthase kinase-3β (Gsk-3β) promotes a VHL-independent HIF-1α degradation. Here we provide evidences that in rat primary hippocampal cell cultures, HIF-1α degradation might be mediated by a synergic action of Gsk-3β and peptidyl-prolyl cis/trans isomerase (Pin1). In post-ischemic conditions, such as those mimicked with oxygen glucose deprivation (OGD), HIF-1α protein level increases remaining unexpectedly high for long time after normal condition restoration jointly with the increase of lactate dehydrogenase (LDH) and β-secretase 1 (BACE1) protein expression (70 and 140% respectively). Interestingly the Pin1 activity decreases about 40–60% and Pin1S16 inhibitory phosphorylation significantly increases, indicating that Pin1 binding to its substrate and enzymatic activity are reduced by treatment. Co-immunoprecipitation experiments demonstrate that HIF-1α/Pin1 in normoxia are associated, and that in presence of specific Pin1 and Gsk-3β inhibitors their interaction is reduced in parallel to an increase of HIF-1α protein level. Thus we suggest that in post-OGD neurons the high level of HIF-1α might be due to Pin1 binding ability and activity reduction which affects HIF-1α degradation: an event that may highlight the relevance of ischemia/HIF-1α as a risk factor in AD pathogenesis.

**Keywords: Alzheimer's disease, cerebrovascular deficiencies, hippocampal neurons, Pin1, HIF-1**α**, GSK-3**β**, oxygen glucose deprivation**

#### **INTRODUCTION**

Alzheimer's disease (AD) is a multifactor neurodegenerative pathology affecting the elderly population. The pathogenesis of sporadic late-onset AD has not been identified, but further studies support that cerebral ischemia/stroke significantly increases AD

**Abbreviations:** PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; SEM, standard error media; TBST, Tris-buffered saline-Tween; OGD, oxygen glucose deprivation; Ara-C, 1-β-D arabinofuranosylcytosine; BSS, balanced salt solution; LDS, lauryl-dodecyl sulphate; ECL, enhanced chemiluminescence; DMSO, dimethyl sulfoxide; pNA, *p-*nitroanilide; juglone, 5-Hydroxy-1,4-naphtoquinone; APP, amyloid precursor protein; PI3K, phosphatidylinositol-3 kinase; Pin1, peptidyl-prolyl *cis/trans* isomerase (PPIase); HIF-1α, hypoxia-inducible transcription factor subunit-α; Gsk-3β, glycogen synthase kinase-3β; BACE1, β-secretase 1; LDH, lactate dehydrogenase; VHL, von Hippel-Lindau protein.

risk (Kalaria, 2000; Zhang et al., 2007). Indeed, it has been suggested that cerebral hypoperfusion causing neuronal damage in vulnerable brain areas (Koistinaho and Koistinaho, 2005; Zhang et al., 2007) may serve as a basis for some cases of dementia after stroke (Ogunshola and Antoniou, 2009).

At the molecular level, a large percentage of hypoxic responses are controlled by hypoxia-inducible transcription factor-1 (HIF-1; Webb et al., 2009) whose involvement in neurodegenerative disorders is becoming widely accepted, although its role may greatly depend on whether it is the cause or the consequence in disease progression (Ogunshola and Antoniou, 2009; Bulbarelli et al., 2012).

HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1β subunit and oxygen-regulated HIF-1α subunit (Wang et al., 2006). Under hypoxic conditions HIF-1α is stabilized, and in complex to the constitutive HIF-1β subunit induces the transcription of a plethora of genes (Semenza, 2003), whereas in normoxic conditions it is rapidly subjected to proteasomal degradation (Webb et al., 2009). HIF-1α degradation system displays enormous plasticity since it can be induced by hydroxylation and phosphorylation events either alone or in combination (Flügel et al., 2007). Indeed, although HIF-1α degradation mainly depends on two prolyl hydroxylation allowing the binding with tumor suppressor von Hippel-Lindau protein (VHL; Salceda and Caro, 1997; Sonenberg and Gingras, 1998; Semenza, 2010), evidences show that glycogen synthase kinase-3β (Gsk-3β), by phosphorylation of Ser551, Thr555, and Ser589 residues in HIF-1α oxygen degradation domain (ODD), can promote its ubiquitination and proteasomal degradation in a VHL-independent manner (Flügel et al., 2007).

In addition, during a brief hypoxic event, the HIF-1α protein stabilization needs the Akt-induced inhibitory phosphorylation of Gsk-3β on Ser9 residue (Mottet et al., 2003). A growing number of reports showed that both hypoxic and non hypoxic stimuli appear to promote HIF-1α stabilization by means of PI3K/Akt pathway (Zhong et al., 2000; Zundel et al., 2000; Hirota and Semenza, 2001; Li et al., 2008) in a cell and tissue specific way (such as in cortical neurons; Zhang et al., 2009).

The Gsk-3β-mediated degradation of HIF-1α implies a scenario similar to that of c-myc and cyclin E (Yeh et al., 2004, 2006) which upon phosphorylation by the kinase and isomerasemediated conformational change, are ubiquitinated and degraded in the proteasome. Gsk-3β can indeed cooperate synergistically with the peptidyl-prolyl *cis/trans* isomerase (Pin1) in ubiquitination of a wide range of proteins (Liou et al., 2011). Gsk-3β as a proline-directed kinase can selectively phosphorylate Ser/Thr-Pro residues allowing the Pin1 substrate recognition and their *cis* to *trans* isomerization. The *cis* or *trans* conformation of phospho-Ser/Thr-Pro motif, as recently suggested, could be a crucial determinant in regulating protein degradation (Liou et al., 2011) in view of the fact that the ubiquitin E3 ligase complex might have a structural preference for phosphorylated substrates via a *trans* conformation. Pin1-mediated conformational change in phospho-Ser/Thr-Pro motifs, hence, represents a novel molecular switch in a large number of biological processes. Therefore, Pin1 is tightly regulated by multiple levels (Lu et al., 2002), and alterations in its functionality often lead to several pathologies, included cancer and neurodegeneration (such as AD; Lu et al., 2007). In pathological conditions, the Pin1 ability to interact with downstream substrates is inhibited by phosphorylation of Ser16 residue in its binding domain (Eckerdt et al., 2005; Lonati et al., 2011) while oxidative modification in the catalytic domain can abolish the enzymatic activity of isomerase (Butterfield et al., 2006a,b).

Although recent studies highlight indirect link between HIF-1α regulation/activity and Pin1 overexpression in breast cancer (Kim et al., 2008) and in prostate cancer (Yuan et al., 2011), little is known about the relationship of this two proteins in neuronal tissues, under physiological or pathological conditions. Consistent with that we asked whether Pin1 might participate in HIF-1α modulation under normoxic and post-ischemic conditions, such as those mimicked after oxygen glucose deprivation (OGD) treatment, where HIF-1α protein levels are carefully regulated and Pin1 activity might be altered.

Here we show that, in rat primary hippocampal cultures, Pin1 interacts with HIF-1α, and catalyzing its isomerization plays a central role in Gsk-3β-mediated proteasomal degradation of the transcription factor. Moreover in neurons subjected to OGD, Pin1 binding and activity interestingly are partially inhibited affecting HIF-1α ubiquitination and protein level.

Considering that recent studies performed in the central nervous system highlight the pathophysiological relevance of hypoxia/HIF-1 pathways regulation of β-secretase 1 (BACE1) expression and amyloid precursor protein (APP) amyloidogenic metabolism (Zhang et al., 2007; Bulbarelli et al., 2012), alterations in HIF-1α protein levels/degradation pathway may contribute to the cognitive decline and dementia in AD patients influencing the disease course.

# **MATERIALS AND METHODS MATERIALS**

All commercial chemicals were of the highest available grade: Sprague-Dawley rats were from Charles-River Laboratories (Lecco, Italy). The 5% CO2: 95% N2 gas cylinder was from Sapio, Monza, Italy. Complete protease inhibitor cocktail was from Roche Diagnostics S.p.A (Milano, Italy). Hydroxy-1,4 naphtoquinone (juglone), Lactacystin, 1-β-D arabinofuranosylcytosine (Ara-C), lithium chloride solution, SB-216763 Gsk-3 inhibitor, solutions for electrophoresis were from Sigma Chemical Co. (Milano, Italy). All the stock solutions for cell culture were from Euroclone (Celbio Milano, Italy). Gibco Neurobasal medium (NBM) and B27 supplement, Dynabeads® protein G, sodium dodecyl sulphate (SDS) NuPAGE reagents (4–12% Bis-Tris gel; sample buffer; running buffer), Novex Sharp Protein Standard, anti-Tau was from Life Technologies (Milano, Italy).

Anti-Pin1, anti P-Pin1S16 and anti-Ubiquitin (PD41) were from Cell Signaling (Beverly, USA). Anti-HIF-1α and anti-lactate dehydrogenase (LDH) antibodies were from Abcam (Cambridge Science Park, UK). Anti P-Ser/Thr-Pro (MPM2) and Anti-Pin1 for immunoprecipitation antibodies were from Millipore S.p.A (Milano, Italy). Anti-BACE1 antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary HRPconjugated antibodies and ECL SuperSignal detection kit were from Pierce (Rockford, IL, USA). Anti-Actin and anti P-TauT231 antibodies were from Sigma Chemical Co (Milano, Italy).

### **CELL CULTURE**

Hippocampal neurons cultures were prepared from E18-E19 rat hippocampi as previously described, with minor modifications (Brewer et al., 1993; Bulbarelli et al., 2009). Neurons were plated on polylysine coated dishes (60 mm diameter, 5 × 105 cells/dish). The medium for cell culture was NBM containing 2% B27 supplement and 12.5 nM glutamate. After 72 h in culture, half of the cell medium was replaced with NBM w/o glutamate and supplemented with 1-β-D arabinofuranosylcytosine (Ara-C) (5 µM final concentration) to prevent glial proliferation. Cells were maintained at 37◦C, 5% CO2 for 8 days before treatment. All experiments were carried out in accordance with the guidelines established by the European Community Council and were approved by the Italian Ministry of Health (DL 116/92). Adequate measures were always taken to minimize animal pain or discomfort.

#### **OXYGEN AND GLUCOSE DEPRIVATION TREATMENT**

Primary hippocampal neurons were subjected to OGD as previously described (Bulbarelli et al., 2012). Briefly, culture medium was replaced by through exchange with a glucose-free balanced salt solution (BSS; NaCl 130 mM, KCl 5.5 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 20 mM). Then cells incubated in a hypoxia chamber (Billups–Rothenberg, Del Mar, CA) saturated for 10 min with 5% CO2: 95% N2 were sealed at 37◦C for 3 h. After OGD, cells were supplemented with the restoration solution: glucose (final concentration 5 mM) and B27 (final concentration 2%) in NBM.

Cells were maintained in normal culture conditions (37◦C in a 5% CO2 atmosphere) for different times of restoration: one hour (R 1 h) and overnight (R o/n) after the reestablishment of normoxia. Untreated hippocampal cells were incubated in NBM supplemented with 2% B27 in a 5% CO2 atmosphere.

# **TREATMENTS WITH INHIBITORS**

5-Hydroxy-1,4-naphtoquinone (juglone) was dissolved in dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution and used as Pin1 catalytic activity inhibitor, accordingly to previous studies (Chao et al., 2001; Galas et al., 2006). Hippocampal cells were incubated with a 10 µM juglone final concentration for 8 h as already described (Bulbarelli et al., 2009), or with 1 µM lactacystin, an inhibitor of ubiquitin-proteasome degradation system, for 16 h (Cazzaniga et al., 2007). According to literature Gsk-3 kinase activity was inhibited by means of lithium chloride administrated to hippocampal cells at 10 mM final concentration for 1 h (Stambolic et al., 1996) or SB-216763 administrated at 10 µM (Facci et al., 2003) for 3 h accordingly to experimental indications obtained in our cellular model.

### **IMMUNOPRECIPITATION**

Cells subjected or not to OGD or to inhibitor treatments for different times, were rinsed twice with phosphate-buffered saline (PBS) and harvested by scraping with a rubber policeman after lysis with non denaturising buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) in presence of complete protease inhibitor cocktail and phosphatase inhibitors (2 mM Na orthovanadate, 1 mM Na fluoride, 1 mM Na pyrophosphate). Then, immunoprecipitation of the endogenous HIF-1α, Pin1 or P-Ser/Thr-Pro (MPM2) proteins was performed from 1 mg of total proteins using anti-HIF-1α (1:100), anti-Pin1 (1:100), anti-MPM2 (1:100) and Dynabeads protein G according to the manufacturer's instruction. Immunoprecipitated proteins were eluted in lauryl-dodecyl sulphate (LDS) lysis buffer with reducing agent (Life Technologies) and boiled for 10 min at 70◦C before analysis on NuPAGE Bis-Tris 4–12% polyacrylamide gels.

### **SDS-PAGE ELECTROPHORESIS AND IMMUNOBLOTTING**

Samples were obtained after lysis with denaturising buffer (2% SDS lysis, 50 mM Tris-HCl, pH 6.8 plus protease inhibitor cocktail and phosphatase inhibitors). The total protein amount was evaluated by means of Bicinchoninic acid assay and 25 µg of each sample were subjected to on SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% polyacrylamide gels. For immunoprecipitation, samples obtained were loaded on NuPAGE Bis-Tris 4–12% gels. After SDS-PAGE electrophoresis samples were transferred to a nitrocellulose membrane (Amersham, GE Healthcare Europe GmbH, Milano, Italy) and proteins revealed by Ponceau staining (Sigma Chemical Co., Milano, Italy). Membranes were blocked in TBS-Tween 0.1% buffer containing 5% non-fat milk or TBS-Tween 0.2% buffer containing 3% bovine serum albumin, and probed with specific antibodies in TBS-T buffer containing 5% non-fat milk or 5–3% bovine serum albumin according to manufacturer's instructions. Immunoblotting was performed using anti-Pin1 (1:1000), anti-P-Pin1S16 (1:1000), anti-HIF-1α (1:1000), anti-ubiquitin (PD41) (1:1000), anti LDH (1:2000), anti-BACE1 (1:1000), anti-P-Ser/Thr-Pro (MPM2) (1:1000), anti-P-TauT231 (1:5000), anti-Tau (1:1000) and anti-β-actin (1:1500). Immunoreactive proteins were revealed by enhanced chemiluminescence (ECL) and semi-quantitatively estimated by a KODAK image station 2000R. Normalization was carried out with respect to the amount β-actin in the same sample.

# **PPIase ASSAY**

Pin1 activity was measured according to the methods of Janowski et al. (1997) and Hennig et al. (1998) with slight modifications as follows (Janowski et al., 1997; Hennig et al., 1998). Stock solutions for the protease-free assay was prepared as described: the phosphorylated substrate Ac-AA(pS)PR-pNA was dissolved in 0.47 M LiCl/trifluoroethanol (anhydrous) at 30 mg/ml concentration; trypsin protease was dissolved in 35 mM HEPES, pH 7.8 at 50 mg/ml concentration.

For a typical measurement, to determine the PPIase activity originating from Pin1, the sample buffer (35 mM HEPES, pH 7.5) was incubated in a temperature-controlled cuvette holder at 4◦C. Then Ac-AA(pS)PR-pNA (0.02 mg/ml) was added and the reaction was started by the injection of trypsin (0.1 mg/ml). After the initial burst phase where all trans peptides were cleaved, protein samples obtained after lysis, as described for immunoprecipitation, was added to the reaction. The absorption at 390 nM, which detects the formation of free *p*-nitroanilide (pNA), was monitored using a Beckman Coulter DU 800 spectrophotometer. The enzymatic activity of Pin1 in the sample (unit/mL) was calculated as follows: absorbance (OD) ∗ total volume/9620 M−1 cm−1 (extinction coefficient ) ∗ volume of sample in mL ∗ 1 cm (*d*). Normalization was carried out with respect to total protein in the same sample.

Unit/mL =

 (1Abs/ min) ∗ (*V* tot in mL) . ε ∗ (*V* of sample in mL) ∗ *d*

The measured activity was normalized to the total protein content of the lysate.

### **STATISTICAL ANALYSIS**

All data are expressed as mean ± SEM of three separate experiments performed in triplicate. The differences were calculated by means of Student's *t*-test. A *p* value <0.05 was considered to be statistically significant.

# **RESULTS**

### **EFFECTS OF THE OXYGEN AND GLUCOSE DEPRIVATION ON HIF-1**α **PROTEIN EXPRESSION, Pin1 PHOSPHORYLATION AND ACTIVITY IN HIPPOCAMPAL NEURONS CULTURES**

Hippocampal cells were subjected to OGD for 3 h. Following the treatment, we investigated the HIF-1α protein levels after 1 hour (R 1 h) and overnight (R o/n) of normal oxygen and glucidic conditions restore. As already demonstrated by our group (Bulbarelli et al., 2012), hippocampal neuron viability is not affected by the OGD treatment.

As shown in **Figure 1A**, HIF-1α protein level increased at R 1 h (60%) remaining significantly higher than control up to R o/n. Concomitantly, we detected that HIF-1α ubiquitination state was decreased about 40% at R 1 h almost unvarying at R o/n (**Figure 1B**).

Relatively to HIF-1α expression increase after normal condition restoration (post-OGD) we examined lactate dehydrogenase (LDH) and BACE1 protein expression, two proteins whose *genes* are under HIF-1α transcriptional regulation. We observed that LDH expression increased about 70% already at R 1 h, slightly decreasing during overnight, while BACE1 protein level significantly increased during o/n, reaching 150% of increment (**Figure 1A**).

Contemporary we observed that Pin1 activity was reduced in a time dependent manner of about 40% at R 1 h, decreasing till 60% during overnight (**Figure 2A**). In addition, in the same conditions we assessed Pin1S16 phosphorylation (a state that inhibits Pin1 binding to its substrate): the isomerase was significantly phosphorylated at R 1 h (150%) slightly decreasing at R o/n (110%; **Figure 2B**).

### **PHOSPHORYLATION OF HIF-1**α **Ser/Thr-Pro MOTIFS FOR SPECIFIC Pin1 RECOGNITION AND INTERACTION**

We examined the amino acid HIF-1α protein sequence to evaluate the presence of conserved Ser/Thr-Pro motifs potentially recognizable by Pin1 when phosphorylated (Flügel et al., 2007), by means of the alignment of human (Q16665) (Iyer et al., 1998) and rat (O35800) HIF-1α protein sequence (Kietzmann et al., 2001; Align tool EXPASY). As shown the analyses revealed a number of these motifs in HIF-1α sequence. In particular, the Gsk-3β target residue Ser589 precedes a proline in one of the above mentioned Ser/Thr-Pro motifs (**Figure 3A**).

Therefore in order to find one or more HIF-1α phosphorylated Ser/Thr-Pro motifs (PSer/Thr-Pro), we employed the monoclonal antibody MPM2, which specifically recognizes the PSer/Thr-Pro motifs. Immunoprecipitated HIF-1α protein was identified by MPM2 antibody, confirming that HIF-1α protein contains PSer/Thr-Pro motifs under normal culture conditions. On the other hand, among proteins immunoprecipitated with MPM2, HIF-1α was identified by a specific antibody (**Figure 3B**).

Given that this phosphorylation might allow Pin1 interaction with its hypothetic substrate, we therefore tested in our neuronal model whether HIF-1α associates with Pin1 by

**FIGURE 1 | In post-OGD conditions HIF-1**α **protein levels increases while its ubiquitination state decreases**. Cells were subjected to OGD treatment and restoration of normoxic and glucidic conditions for different times (R 1 h, R o/n). **(A)** Cells were collected and lysed with 2% SDS lysis buffer. Equal amounts of samples (as protein content) were subjected to SDS-PAGE and western blot analysis. HIF-1α, LDH, BACE1 levels were detected by specific antibodies, revealed by enhanced chemiluminescence (ECL) and semi-quantitatively determined by a KODAK image station 2000R. Anti-β-actin antibody was employed to confirm equal protein loading in the different lanes. Results obtained are shown on the bar-graphs. Normalization was carried out with respect to the β-actin amount in the same sample. **(B)** HIF-1α ubiquitination state was evaluated after immunoprecipitation using western blot analysis. Cells were collected and lysed in non denaturising NP40 buffer. Western blot analysis performed on immunoprecipitated proteins showed bands corresponding to ubiquitinated HIF-1α protein (140 kDa), HIF-1α protein (110 kDa). Proteins were detected by specific antibodies (anti-HIF-1α; anti-ubiquitin), revealed by ECL and semi-quantitatively determined by a KODAK image station 2000R and the results obtained are shown on the bar-graphs. IP, immunoprecipitation; WB, western blot. Data represent the mean ± SEM of three separate experiments performed in triplicate. Statistical significance is obtained with Student's t-test in comparison with controls. ∗ p < 0.05, ∗∗ p < 0.01.

co-immunoprecipitation of endogenous HIF-1α or Pin1. As shown in **Figure 3C**, HIF-1α was detected among proteins immunoprecipitated by the anti-Pin1 antibody as well Pin1 was

protein content were subjected to SDS-PAGE and Western blot analysis. P-Pin1S16 and total Pin1 were detected by specific antibodies and revealed by ECL, and semi-quantitatively determined by a KODAK image station 2000R. Anti-β-actin antibody was employed to confirm equal protein loading in the different lanes. Results obtained are shown on the bar-graphs. The amount of P-Pin1S16 correspond to phospho-protein level on Pin1 total protein. Normalization was carried out with respect to the β-actin amount in the same sample. Data represent the mean ± SEM of three separate experiments performed in triplicate. Statistical significance is obtained with

detected in the protein complex immunoprecipitated by the anti HIF-1α antibody suggesting that HIF-1α and Pin1 might interact each other. Immunoprecipitation with LDH antibody was performed as negative control.

Student's t-test in comparison with controls. \* p < 0.05, \*\* p < 0.01.

#### **Pin1 INHIBITION AFFECTS HIF-1**α **DEGRADATION PATHWAY**

In order to understand whether Pin1 plays a role in HIF-1α ubiquitination and the following degradation, hippocampal cells were treated for 8 h with 10 µM juglone, an irreversible inhibitor of Pin1 catalytic activity. As expected, juglone treatment triggered a strong inhibition of Pin1 isomerase activity (almost 75%; **Figure 4A**).

The effect of Pin1 inhibition was firstly studied in the whole lysate. As shown in **Figure 4B**, HIF-1α protein level increased of about 70% with respect to control level. Since it is known that Pin1 promotes Tau dephosphorylation (Bulbarelli et al., 2009), a more convincing evidence of the Pin1 inhibition was obtained evaluating Tau phosphorylation on residue Thr231. As expected we observed an increase in Tau phosphorylation.

In parallel, to evaluate the proteasomal degradation of HIF-1α in our cellular model, cells were incubated for 16 h with 1 µM lactacystin an irreversible proteasome inhibitor, revealing HIF-1α protein increment of about 80%. Data obtained are comparable to ones detected after juglone, suggesting that Pin1 might be engaged in the HIF-1α degradation. Contemporary, immunoprecipitated HIF-1α resulted about 35% less ubiquitinated than in untreated cells (**Figure 4C**).

#### **Gsk-3**β **INHIBITION INFLUENCES Pin1/HIF-1**α **ASSOCIATION**

To assess the involvement of Gsk-3β activity in phosphorylation of Ser/Thr-Pro motifs, hippocampal neurons were incubated in presence of 10 mM LiCl for 1 h, or in presence of 10 µM SB-216763 for 3 h. Then we performed HIF-1α protein immunoprecipitation to analyse phosphorylation state in Ser/Thr-Pro motifs and the amount of Pin1 co-immunoprecipitated.

As shown in **Figure 5A**, phosphorylation of Ser/Thr-Pro motifs was greatly reduced in cells treated with the inhibitors, concomitantly to a considerable decrease in the amount of coimmunoprecipitated Pin1, although total Pin1 amount resulted almost unchanged (**Figure 5B**). In addition, we observed the HIF-1α ubiquitination decrease and a slight enhance of protein level both in the immunoprecipitated fractions (**Figure 5A**) and in the total lysates (**Figure 5B**), indicating a possible reduction in the HIF-1α protein degradation.

In order to confirm the Gsk-3β inhibition, **Figure 5B** shows the increase of Gsk-3β inhibitory phosphorylation on Ser9 after LiCl treatment, accordingly to Martin et al. (2009). Moreover, inhibitory effect on Gsk-3β activity was assessed evaluating Tau phosphorylation on Thr231 residue, a well known target of kinase activity (**Figure 5B**; Lin et al., 2007). As expected, after treatment with both the inhibitors, Tau phosphorylation decreased.

#### **EFFECTS OF THE OXYGEN AND GLUCOSE DEPRIVATION ON Gsk-3**β **PHOSPHORYLATION AND HIF-1**α**/Pin1 ASSOCIATION**

Considering results above, to investigate whether HIF-1α ubiquitination decrease in post-OGD conditions might correlate to Gsk-3β inhibition, the P-Gsk-3β S9 levels were evaluated. As shown in **Figure 6A**, Gsk-3β S9 inhibitory phosphorylation increased about 40% at R 1 h and duplicated at R o/n, while total Gsk-3β protein resulted unchanged during the treatment. Concomitantly we detected a significant decrease in Ser/Thr-Pro motifs phosphorylation, revealed by anti-MPM2, on the immunoprecipitated HIF-1α protein at R 1 h (**Figure 6B**). At R o/n we observed a slightly increase of P-Ser/Thr-Pro motifs that however did not reach control levels (**Figure 6B**). Moreover, we also evaluated the

co-immunoprecipitated Pin1 amount: a considerable decrease in HIF-1α/Pin1 association was revealed at R 1 h, while the Pin1 amount at R o/n was quite equivalent to the control (**Figure 6B**).

### **DISCUSSION**

Coexistence of ischemic and neurodegenerative pathology seems to have a deep impact on the expression of the dementia, suggesting common mechanisms interactions. Cerebrovascular diseases such as ischemia leading to cerebral blood flows reduction (hypoperfusion) might indeed initiate and/or accelerate the neurodegeneration cascade *via* amyloid deposition and synaptic neuronal dysfunction (Iadecola, 2010; Kalaria and Ihara, 2012). Depletion of oxygen and glucose sources typical of brain ischemia (Brouns and De Deyn, 2009), results in the activation of highly heterogeneous phenomena in which one of the key component is the transcription factor HIF-1. Therefore protein levels of its hypoxia responsive α subunit (HIF-1α) are finely regulated by degradation through ubiquitin-proteasome system that may be induced by hydroxylation (VHL-mediated) and as recently suggested by phosphorylation (Gsk-3β-mediated; Flügel et al., 2007, 2012). Here we propose that, in hippocampal cellular model, the Pin1 plays an important role in Gsk-3β-mediated HIF-1α ubiquitination/degradation pathway.

In cortical neurons, as already demonstrated, HIF-1α protein levels are maintained higher for long time after reperfusion following OGD by PI3K/Akt signaling (Zhang et al., 2009). Since the PI3K/Akt pathway is known to inhibit Gsk-3β and to regulate HIF-1α in a cell type specific manner (Mottet et al., 2003), we speculate that in neuronal cells HIF-1α degradation might be also regulated by the Gsk-3β pathway working in parallel or in alternative to the well known VHL-mediated mechanism.

Gsk-3β regulates the ubiquitination state of several targets in cooperation with Pin1 (Liou et al., 2011), that mediating prolyl *cis/trans* isomerization can influence the stability of its substrates via phosphorylation-dependent ubiquitin-mediated proteolysis both under physiological and pathological conditions; accordingly, Pin1 might interact and catalyze the isomerization of HIF-1α phosphorylated by the kinase under physiological conditions. Interestingly, here we show, for the first time in neurons, that OGD treatment results in a Pin1 activity reduction, an event that in turn leads to HIF-1α protein levels increase. Indeed, although the transcription factor should be immediately degraded in normoxic conditions by VHL-mediated pathway restoring control levels (Semenza, 2010), in hippocampal cells high levels of HIF-1α protein are detectable at R 1 h and maintained for long time (even up to overnight) after normal oxygen and glucidic conditions restore following OGD. Moreover, HIF-1α high levels are accompanied by a significant decrease of protein ubiquitination state, indicating an impairment in degradation pathway.

Concerning to Pin1, OGD triggers a partial inhibition of its enzymatic activity and also increases Ser16 residue

treated or not with juglone 10 µM for 8 h. **(A)** PPIase activity decreases in cells subjected to juglone treatment. Cells were lysed and collected in non denaturising NP40 buffer and Pin1 isomerase activity against Ac-AA(pS)PR-pNA substrate were measured as described in Section materials and methods. The measured activity was normalized to the total protein concentration of the lysate. **(B)** Neurons were treated or not with juglone or lactacystin (lac) 1 µM for 16 h. Equal amounts of total cell lysates (in the presence of 2% SDS) were subjected to SDS-PAGE and western blot analysis. HIF-1a, Tau, P-TauT231 levels were detected by specific antibodies, revealed by ECL and semi-quantitatively determined by a KODAK image station 2000R. HIF-1α normalization was carried out with respect to the β-actin amount in the same sample. **(C)** HIF-1α ubiquitination state decreases after juglone treatment. Western blot analysis showed a 110 kDa and 140 kDa bands corresponding to HIF-1α and to ubiquitinated HIF-1α protein respectively. Proteins were detected by specific antibodies (anti-HIF-1α; anti-ubiquitin), revealed by ECL and semi-quantitatively determined by a KODAK image station 2000R. Results obtained are shown on the bar-graphs. IP, immunoprecipitation; WB, western blot. Data represent the mean ± SEM of 3 separate experiments performed in triplicate. Statistical significance is obtained with Student's t-test in comparison with controls. \* p < 0.05, \*\* p < 0.01.

phosphorylation in the binding domain. Therefore, on one side Ser16 phosphorylation may inhibit Pin1 function in recognizing and binding its substrates, on the other side Pin1 isomerization activity might be diminished by oxidative modification occurring in the catalytic domain (Lu et al., 2002; Lonati et al., 2011). Indeed, as already known oxidative stress might reduce Pin1 function (Butterfield et al., 2006a,b).

Considering data above, we suppose that Pin1 partial inhibition may directly affect the HIF-1α degradation pathway, thus we

antibodies and revealed by ECL. TauT231 phosphoryalation as well known Gsk-3β target was assessed to evaluate the kinase inhibition. Anti-β-actin antibody was employed to confirm equal protein loading in the different lanes. IP, immunoprecipitation; WB, western blot; MPM2, antibody against phosphorylated Ser/Thr-Pro motifs.

evaluated if Pin1/HIF-1α interaction might be required for HIF-1α ubiquitination.

Firstly, after exploring in HIF-1α amino acid sequence the presence of motifs constituted by serine/threonine residues preceding a proline (Ser/Thr-Pro) that, when phosphorylated, allows Pin1 to specifically recognise its substrates, we found these phosphorylated motifs in HIF-1α protein, employing the anti-MPM2 antibody (which specifically recognizes the PSer/Thr-Pro motifs). Given that this evidence pinpoints the transcription factor as an hypothetic target for Pin1 binding, we provided the evidence of HIF-1α/Pin1 association by co-immunoprecipitation, indicating that the transcription factor and the isomerase may interact each other.

Afterwards, we evaluated the HIF-1α level expression, inhibiting Pin1 catalytic activity by means of juglone treatment. Since

**FIGURE 6 | In post-OGD conditions P-Gsk-3**β **S9 levels increases**. Cells were subjected to OGD treatment and restoration of normoxic and glucidic conditions for different times (R 1 h, R o/n). **(A)** Cells were collected and lysed with 2% SDS lysis buffer. Equal amounts of samples (as protein content) were subjected to SDS-PAGE and western blot analysis. Gsk-3β and P-Gsk-3β S9 levels were detected by specific antibodies, revealed by ECL and semi-quantitatively determined by a KODAK image station 2000R. Anti-β-actin antibody was employed to confirm equal protein loading in the different lanes. Results obtained are shown on the bar-graphs. The amount of P-Gsk-3β S9 correspond to phospho-protein level on Gsk-3β total protein. Normalization was carried out with respect to the β-actin amount in the same sample. **(B)** HIF-1α phosphorylation on Ser/Thr-Pro motifs and HIF-1α/Pin1 interaction was evaluated after HIF-1α immunoprecipitation using western blot analysis. Cells were collected and lysed in non denaturising NP40 buffer. Western blot analysis performed on immunoprecipitated proteins showed bands corresponding to Pin1 protein (18 kDa), HIF-1α protein and HIF-1α phosphorylated on Ser/Thr-Pro motifs (110 kDa). Proteins were detected by specific antibodies (anti-HIF-1α; anti-MPM2; anti-Pin1), revealed by ECL and semi-quantitatively determined by a KODAK image station 2000R and the results obtained are shown on the bar-graphs. IP, immunoprecipitation; WB, western blot. Data represent the mean ± SEM of three separate experiments performed in triplicate. Statistical significance is obtained with Student's t-test in comparison with controls. \* p < 0.05, \*\* p < 0.01.

we had already established that juglone-mediated Pin1 inhibition leads to TauT231 phosphorylation increase (Bulbarelli et al., 2009), here we show Tau phosphorylation augment after juglone as positive control of the treatment. Under conditions in which Pin1 enzymatic activity is highly repressed, data obtained showed that HIF-1α levels strongly increase in parallel with a mild decrease of its ubiquitination state, demonstrating that Pin1 affects protein ubiquitination and degradation. Interestingly the HIF-1α levels observed after juglone treatment were comparable to that observed in proteasome inhibited cells suggesting that in our cellular model, Pin1 might play a main role in the fate of the transcription factor. Taking into account that HIF-1α and Pin1 co-immunoprecipitate, it is plausible that the enzyme catalyzes HIF-1α peptydil-prolyl conformational isomerization.

As Liou and colleagues reported (Liou et al., 2011), Pin1 can cooperate synergistically with Gsk-3β in ubiquitination of a wide range of proteins, therefore the role of this kinase in Ser-Thr/Pro phosphorylation and the consequent interaction between Pin1 and HIF-1α have been investigated.

Among the several Gsk-3β phosphorylation consensus motifs in HIF-1α amino acid sequence individuated by Flügel et al. (2007), we observed that the Ser589 is neighboring to a proline constituting a Ser-Thr/Pro motif, which phosphorylated might be the consensus for Pin1 recognition. Therefore we speculate that this sequence could be significant in the HIF-1α stability.

To understand whether decreasing HIF-1α phosphorylation levels might affect Pin1/HIF-1α interaction, we employed two different Gsk-3β activity inhibitors: the well known LiCl and the more specific SB-216763. Since LiCl treatment induces Gsk-3β inhibitory phosphorylation on Ser9 (Martin et al., 2009), here we confirme the kinase inhibition showing an increase in Ser9 phosphorylation. On the contrary, SB-216763 induces the dephosphorylation of Gsk-3β at Ser9 (Jaeger et al., 2013), nevertheless inhibits Gsk-3β activity in an ATP competitive manner (Cross et al., 2001).

Interestingly, data obtained after the treatment with Gsk-3β activity inhibitors showed: (i) a strong reduction of phosphorylated Ser/Thr-Pro motifs of HIF-1α protein; (ii) a considerable decrease of co-immunoprecipitated Pin1 amount; (iii) an higher HIF-1α protein level with respect to control. The reduction of HIF-1α phosphorylated motifs accompanied by protein levels increase, give reason for Pin1 binding decrease, suggesting that Pin1 might be involved in the Gsk-3β/HIF-1α stability regulation independent from the VHL pathway (Flügel et al., 2007). Indeed, Flügel et al. (2007) suggest that the regulation of HIF-1α degradation is not limited by the presence of oxygen, resulting therefore independent of HIF-1α hydroxylation and VHL-E3 ligase complex. Moreover, the F-box protein Fbw7, an E3 ubiquitin ligase, has been recently identified as novel critical component in HIF-1α degradation that is recruited to HIF-1α after its phosphorylation by Gsk-3β (Flügel et al., 2012): in a wider framework we can speculate that Pin1 might be involved in the Gsk-3β/Fbw7 HIF-1α degradation system. Taken together our experiments indicate for the first time that Pin1 interacts with HIF-1α in order to regulate the transcription factor protein levels. Therefore the decrease of Pin1 ability to bind and isomerize PSer/Thr-Pro motif might correlate with high levels of HIF-1α observed in post-OGD conditions (see a schematic representation in **Figure 7**). To deepen the OGD effect on HIF-1α and Pin1 interaction we analyzed Gsk-3β S9, HIF-1α Ser/Thr-Pro motif phosphorylation and HIF-1α/Pin1 association. Interestingly, here we show that, Gsk-3β S9 inhibitory phosphorylation increased at R 1 h, correlating both to the Ser/Thr-Pro motifs phosphorylation decrease and HIF-1α/Pin1 association reduction. *Vice versa* at R o/n we observed a slightly increase of P-Ser/Thr-Pro motifs that however did not reach control levels and the co-immunoprecipitated Pin1 amount was quite equivalent to the control. This observation apparently in contrast with the HIF-1α increase might be explained by the fact that although Pin1 might be able to bind the transcription factor, since Pin1S16 phosphorylation decreased, the isomerase resulted enzymatically inhibited (see the PPIase assay in **Figure 2**). Hence, we hypothesize that in post-OGD hippocampal neurons, Gsk-3β and Pin1 inhibition lead to a decrease of HIF-1α ubiquitination affecting the consequent degradation of the transcription factor. Since, unconventional VHL-independent HIF-1α degradation pathway involving RACK1 has been already described (Isaacs et al., 2002; Semenza, 2010), here we propose that Pin1/Gsk-3βmediated HIF-1α degradation may be a functionally predominant pathway in our cellular model, that instead of VHL requires the involvement of the E3 ubiquitin ligase Fbw7.

Furthermore, it has been recently proposed that Pin1 inhibits Gsk-3β activity (Ma et al., 2012) promoting Ser9 phosphorylation, however in our cellular model subjected to OGD this event

**FIGURE 7 | Model showing HIF-1**α **degradation pathway mediated by Pin1/Gsk-3**β. In healthy hippocampal neurons Gsk-3β phosphorylates the HIF-1α transcription factor on Ser/Thr-Pro motif potentially recognizible by Pin1. After phosphorylation Pin1 is able to recognize and bind the cis form of HIF-1α (circle form), and catalyzes its isomerization to trans conformation (square). The E3 ligase complex having a structural preference for phosphorylated substrates in trans conformation to favors ubiquitination and the consequent degradation of the transcription factor via proteasome. In post-OGD neurons, instead, Gsk-3β is inhibited by phosphorylation on Ser9, therefore HIF-1α is not phosphorylated on Ser/Thr-Pro motif resulting unrecognizable by Pin1. Moreover Pin1 itself is inhibited by phosphorylation on Ser16, and its enzymatic activity is blocked leading to loss of HIF-1α cis/trans isomerization. HIF-1α is not more ubiquitinated and degradated via proteasome, accumulating in its cis conformation.

seems to be independent from Pin1, deserving future experiments to deeply investigate.

For all above mentioned, under condition mimicking an ischemic event influencing Pin1 activity might be extremely detrimental causing intracellular molecular mechanisms deregulation, eventually leading to pathophysiological conditions such as in AD. In fact Pin1 has been identified as a common regulator of both Tau and APP pathologies (Lu and Zhou, 2007), regulating Tau phosphorylation levels and the NFTs formation as well as levels of Aβ peptide production (Liou et al., 2003; Pastorino et al., 2006).

Our group has recently demonstrated that after OGD the amount of peptide Aβ42 increases both in neurons and in cerebrovascular endothelial cells (Bulbarelli et al., 2012), probably related to the increase of HIF-1α transcriptional activity (Zhang et al., 2007).

Notably, in parallel to HIF-1α high levels we observed the increase of BACE1 protein under our experimental conditions. Hence, in our hypothesis, the alteration in Pin1-mediated HIF-1α degradation resulting from an ischemic event, might accelerate APP amyloidogenic metabolism/Aβ42 production in neurons, supporting the theory that cerebral hypoperfusion induces Aβ deposition. As well in AD the Pin1 deregulation might lead to a more rapid HIF-1α stability/activation inducing the expression of genes implicated in pathological intracellular mechanisms involved in vascular diseases and neurodegeneration.

In view of that there could be a pathogenic synergy between the two disease processes, stroke and AD might effectively share common risk factors (Iadecola, 2010): in this scenery the role of Pin1 in HIF-1α isomerization and degradation may outcome as central mechanism in vascular damages *vs.* AD and *vice versa*. Furthermore, considering that it has been recently demonstrated the correlation between Pin1 activity reduction and the essential hypertension (Wang et al., 2013a,b), the isomerase partial loss of function might be detrimental in multiple interrelated disease; hence the role of Pin1 in HIF-1α regulation might contribute to the morbidity of hypertension, becoming in turn a risk factor for stroke and AD.

#### **ACKNOWLEDGMENTS**

The authors wish to thank Dr. Giulio Sancini for critically reading the manuscript. This work was supported by grants from FAR 2009, Far 2010 and "Dote ricercatori" from European Social Fund and Regione Lombardia (ID Project 12-AR).

#### **REFERENCES**

pin1 in breast cancer cells. *Biochem. Biophys. Res. Commun.* 369, 547–553. doi: 10.1016/j.bbrc.2008.02.045

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 September 2013; accepted: 01 January 2014; published online: 17 January 2014.*

*Citation: Lonati E, Brambilla A, Milani C, Masserini M, Palestini P and Bulbarelli A (2014) Pin1, a new player in the fate of HIF-1*α *degradation: an hypothetical mechanism inside vascular damage as Alzheimer's disease risk factor. Front. Cell. Neurosci. 8:1. doi: 10.3389/fncel.2014.00001*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Lonati, Brambilla, Milani, Masserini, Palestini and Bulbarelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The role of extracellular Tau in the spreading of neurofibrillary pathology

# **Miguel Medina1 and Jesús Avila1,2\***

1 Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 2 Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Madrid, Spain

#### **Edited by:**

Ludovic Martin, Université de Nantes, France

#### **Reviewed by:**

Khalid Iqbal, New York State Institute for Basic Research in Developmental Disabilities, USA Amy Pooler, King's College London, UK

#### **\*Correspondence:**

Jesús Avila, Centro de Biología Molecular "Severo Ochoa" CSIC-UAM, Nicolás Cabrera 1, 28049 – Madrid, Spain e-mail<EMAIL_ADDRESS>
The microtubule-associated protein (MAP) tau plays a critical role in the pathogenesis of Alzheimer's disease (AD) and several related disorders collectively known as tauopathies. Development of tau pathology is associated with progressive neuronal loss and cognitive decline. In the brains of AD patients, tau pathology spreads following an anatomically defined pattern. Mounting evidence strongly suggests that accumulation of abnormal tau is mediated through spreading of seeds of the protein from cell to cell and point at the involvement of extracellular tau species as the main agent in the interneuronal propagation of neurofibrillary lesions and spreading of tau toxicity throughout different brain regions in these disorders. That would support the concept that pathology initiates in a very small part of the brain many years before becoming symptomatic, spreading progressively to the whole brain within 10–20 years. Understanding the precise molecular mechanism underlying tau propagation is crucial for the development of therapeutics for this devastating disorder. In this work, we will discuss recent research on the role of extracellular tau in the spreading of tau pathology, through synaptic and non-synaptic mechanisms.

**Keywords: Alzheimer, exosomes, vesicles, neurodegeneration, propagation, spreading, tau, tauopathies**

### **INTRODUCTION**

A common pathological feature of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) or prion diseases, among others, is the abnormal deposition of proteins in the brain. Among these pathological proteins, the MAP tau forms intraneuronal filaments in a spectrum of neurological disorders collectively known as tauopathies.

Tau protein is a MAP that under physiological conditions regulates microtubules (MT) assembly, dynamic behavior, and spatial organization, and has also been shown to regulate the axonal transport of organelles, including mitochondria. The gene encoding tau protein *MAPT* is located on chromosome 17q21.3, spans approximately 150 kb and consists of 16 exons (Pittman et al., 2006) from which six major isoforms are expressed in adult brain through alternative splicing (reviewed in Andreadis, 2012). The interaction between tau and tubulin is mediated by four imperfect repeat domains (encompassing 31–32 residues) encoded by exons 9–12 (Lee et al., 1989). Alternative splicing of exon 10 results in the production of isoforms containing 3 or 4 binding domains (3R and 4R tau) (Himmler et al., 1989).

Adult human brain contains equal amounts of 3R and 4R isoforms whereas foetal brain, however, only expresses 3R tau, demonstrating developmental regulation of exon 10 splicing (Goedert et al., 1989). Different brain regions also differ in the relative levels of 3R and 4R isoforms with granule cells in the hippocampal formation reported to have only 3R tau. Disturbances, usually increases, in the 3R/4R ratio are a common feature in most neurodegenerative tauopathies. Furthermore, morphological differences exist among different diseases or disease types as different tau isoforms are accumulated in diseased brains, namely, six tau isoforms in AD, 3R tau isoforms in Pick's disease, and 4R tau isoforms in profressive supranuclear palsy (PSP) and cortical basal degeneration (CBD; Goedert and Spillantini, 2011). Interestingly, a recent study has shown that the 4R/3R ration may have been underestimated in AD brains when compared with PSP or CBD, presumably due to extensive deamidation at Asn279 (Dan et al., 2013).

Within neurons, tau is predominantly found in axons (Hanger et al., 2009) as a highly soluble phosphoprotein (Iqbal et al., 2009). Phosphorylation is also developmentally regulated, with a high tau phosphorylation level during embryogenesis and early development, when only the shortest of the isoforms is being expressed. By contrast, adult brain expresses all six isoforms with relatively reduced phosphorylation levels compared with the foetal one (Liu et al., 2007).

The key discovery directly involving tau protein in neurodegeneration and dementia came from the finding that highly penetrant, dominant mutations in the *MAPT* gene encoding tau cause an inherited form of frontotemporal dementia and parkinsonism (Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998). A number of neurodegenerative disorders present prominent tau pathology in the CNS, predominantly within the neuronal compartment, but also within glial cells. Because of this shared histopathological feature, they are referred collectively as tauopathies, although they constitute a group of etiologically heterogeneous, clinically and neuropathologically overlapping disease entities (Ballatore et al., 2007; Spillantini and Goedert, 2013). In tauopathies, the intracellular soluble tau forms filamentous structures of aggregated, hyperphosphorylated tau, which are associated with synaptic loss and neuronal death. The occurrence of fibrillar tau inclusions in tauopathies strongly supports a key role in the observed clinical symptoms and pathology.

Further insights into the overlapping pathogenic and etiologic aspects of the discrete diseases will help to design (perhaps common) disease-modifying treatment strategies (Medina, 2011). To achieve that goal however, it is critical to understand the normal biological roles of tau, the specific molecular events that induce tau to become neurotoxic, the biochemical nature of pathogenic tau, the means by which pathogenic tau exerts neurotoxicity, and how tau pathology propagates.

As mentioned, the recognition of the MAP tau as a key player in the pathobiology of human neurodegenerative diseases has led to major efforts to understand its biological and pathological function(s). This has resulted in an improved understanding of tau cellular functions beyond its classical role in stabilizing MT (Morris et al., 2011) to unveil novel physiological tau functions that may also play a role in pathogenesis.  Furthermore, despite lacking an identified nuclear localization signal, tau has also been reported in nuclei in a number of cell lines (Loomis et al., 1990; Wang et al., 1993) and human brain (Brady et al., 1995) where it may play a role in DNA protection (Sultan et al., 2011).

### **EXTRACELLULAR TAU**

It has been over 20 years since the original report that intracellular tau levels are increased in the brains of AD patients when compared to non-demented controls (Barton et al., 1990; Khatoon et al., 1992). This increase in the amount of tau could be toxic to neurons since a reduction in the amount of intracellular tau has indeed a protective effect in mouse models of neurodegeneration (Rapoport et al., 2002; Roberson et al., 2007) and it has been suggested that reducing tau levels may be therapeutically beneficial (Götz et al., 2013). However, we must be cautious since other studies in similar tau-deficient mice point in the opposite direction, suggesting that loss of tau function can actually lead to neurodegeneration (Dawson et al., 2010).

Little is known about how tau synthesis is regulated although some factors such as fibroblast growth factor (Tatebayashi et al., 1999), Dyrk1A (Qian et al., 2013), or the haplotype H1 have been involved in increased synthesis whereas the miRNA-34 family (Dickson et al., 2013) seems to downregulate tau levels.

Conventional wisdom has suggested that the presence of tau in the brain parenchyma or in the cerebrospinal fluid (CSF) is a consequence of tau protein being released from dead cells. However, this has recently been challenged by a number of studies showing extracellular tau being released from cell lines and neurons via multiple pathways, strongly supporting the notion that secretion of tau protein may be an important biological function of tau protein, especially in disease. Despite the fact that tau lacks a signal sequence a number of reports have now shown that tau is released into culture medium from neuroblastoma cells, tau-expressing non-neuronal cells, induced pluripotent stem cell-derived human neurons, and mouse primary neurons (Kim et al., 2010; Shi et al., 2012). Thus, tau has been reported to be secreted unconventionally in naked form (Chai et al., 2012) or associated to exosomes (Saman et al., 2012) and/or other membrane vesicles (Simón et al., 2012a). Since increased tau cellular levels are detrimental, secretion has been proposed as a mechanism to eliminate the excess of tau protein thereby avoiding its toxicity (Simón et al., 2012b). Interestingly, while full length tau has been detected in the extracellular space, C-terminal cleavage of tau has been shown to enhance its secretion (Plouffe et al., 2012) which could have pathological relevance since some truncated tau species appears to be characteristic of particular tauopathies whereas other tau fragments may be common to several tauopathies (Hanger and Wray, 2010; Kovacech et al., 2010).

Extracellular tau has also been detected in the brain interstitial fluid of both wild-type and P301S tau-expressing mice in microdialysis studies (Yamada et al., 2011), as it has also been the case in patients following severe traumatic brain injury (Marklund et al., 2009; Magnoni et al., 2012). Actually, exosomal tau secretion has been suggested to account for the elevated CSF tau levels typically observed in early AD (Saman et al., 2012). Interestingly, tau mutations that are associated with the development of tauopathy appears to reduce tau release (Karch et al., 2012). Interestingly, physiological secretion of endogenous tau by cortical neurons appears to be regulated by neuronal activity, as tau release is enhanced by glutamate receptor stimulation induced by the agonist *S*-AMPA (Pooler et al., 2013). This process is calciumdependent and modulated by phosphorylation and released tau is present in a relatively dephosphorylated state, compared to that of intracellular tau.

Thus, increasing evidence point out to extracellular tau as a physiological process independent of cell death (**Figure 1**), although the precise relationship between tau release under physiological conditions and the propagation of pathology in AD and other tauopathies remains to be determined.

Tau can be toxic when applied extracellularly to cultured cells (Gómez-Ramos et al., 2006; Kopeikina et al., 2012). Several mechanisms for internalization of tau has been proposed, such as internalization of soluble, uncoated ("naked") tau via receptor-mediated endocytosis (Gómez-Ramos et al., 2009), dynamin-driven endocytosis of non-fibrillar, soluble tau aggregates (Wu et al., 2013) or even actin-dependent, proteoglycanmediated macropynocytosis (Holmes et al., 2013). Furthermore, it has been suggested that extracellular tau might provoke a receptor-activated increase in intracellular calcium through M1/M3 muscarinic receptor stimulation (Gómez-Ramos et al., 2008; Díaz-Hernández et al., 2010) and that such receptor activation could lead to endocytosis of extracellular tau. Remarkably,

**FIGURE 1 | Cell-to-Sources of extracellular tau and -cell spreading**. The established view has long considered the presence of tau in the brain parenchyma or in the CSF as a consequence of tau protein being released after cell lysis. However, extracellular tau appears to result from a physiological process independent of cell death, as tau being can be released from cell lines and neurons via multiple pathways, either in naked form or vesicle-associated. In addition, tau misfolding in diseased brain leads to abnormal conformations of tau that can be taken up by surrounding neurons. Thus, pathological progression could involve transmission of tau protein through a prion-like mechanism resulting in neurodegeneration in susceptible brain regions.

tau phosphorylation could inhibit its interaction with M1/M3 receptors and it has been proposed that such alterations might be involved in the transmission of tau pathology (Simón et al., 2013).

The discovery of extracellular tau as a physiological process that is independent of cell death (Pooler et al., 2013), indicates that tau release does not occur only as a result of reduced neuronal viability, and therefore that the increased tau observed in interstitial fluid and CSF in tauopathies may not be due solely to tau release from dying neurons (Yamada et al., 2011; Nedergaard, 2013). It is worth mentioning that tau phosphorylation at threonine 181 and total tau levels in CSF are considered useful biomarkers of neuronal degeneration or injury in the recent National Institute on Aging and Alzheimer Association (NIA-AA) revised criteria for the diagnosis of AD (Jack et al., 2012).

### **PROPAGATION OF TAU PATHOLOGY**

Development of tau pathology is associated with progressive neuronal loss and cognitive decline. In the brains of AD patients, tau pathology propagates following an anatomically defined pattern described by the neuropathological Braak sequential staging (Braak et al., 2011). As a matter of fact, recent NIA-AA guidelines recommend the assessment of Braak and Braak staging of neurofibrillary degeneration as part of the so called "ABC score" for the neuropathological diagnosis of AD (Montine et al., 2012).

The originally staging system (Braak and Braak, 1991) defined six stages based on the presence and density of characteristic argyrophylic inclusions (neurofibrillary tangles (NFT), neuropil threads) in the medial temporal lobe and several brain isocortical regions. This system was subsequently adapted by the authors for routine use in paraffin-embedded tissue based on tau immunohistochemistry (Braak et al., 2006). Stages I–II (transentorhinal) correlate with the lengthy preclinical phase of the disease; whereas stages III–IV (limbic) do so with mild cognitive impairment (loss of episodic memory) or mild dementia; and advanced V–VI stages (isocortical) usually correspond to cases with moderate to severe dementia. Accurate staging of AD-related tau-positive pathology may be particularly important in the classification of preclinical disease and in the identification of atypical AD phenotypes. The above mentioned NIA-AA guidelines allow for standardization for diagnostic and research purposes.

Clinicopathological studies show that tau pathology progression from the entorhinal cortex through the hippocampus and into the limbic and association cortex is the main neuropathological variable that correlates with the clinical cognitive status of the patient (Arriagada et al., 1992; Nelson et al., 2012). Whether that pattern of accumulation reflects cell-to-cell spreading of disease, or simply successive involvement of differentially resistant neuronal populations, has been a matter of debate in recent years. Recent evidence from human studies suggests that tau pathology is actually linked to existing networks of neuronal connectivity. Thus, rather than diffuse, random, or confluent, tau pathology would target specific large-scale distributed networks that in the healthy brain feature convergent intrinsic functional and structural covariance (Seeley et al., 2009). However, the precise molecular and cellular mechanisms by which tau propagates and neuronal networks degenerate are still unknown.

Increasing evidence suggests that synaptic dysfunction is a key pathophysiological hallmark in neurodegenerative disorders, including AD which has been indeed considered a synaptopathy (Selkoe, 2002; Sheng et al., 2012), as synapse density best correlates with the cognitive decline observed in patients. Long regarded primarily as an axonal protein, when hyperphosphorylated tau also accumulates in the somatodendritic compartment during AD (Ballatore et al., 2007). Actually, tau mislocation in dendritic spines has been proposed to lead to synaptic dysfunction by various mechanisms, including regulating the amount of glutamate receptors in spines (Hoover et al., 2010), interacting with post-synaptic signaling complexes, targeting of synaptic mitochondria (Pooler et al., 2014) or destabilizing dendritic spines and dendritic arbor (Koleske, 2013). Presence of tau in the synapse in healthy brains suggests a role for tau in regulating normal synaptic function whereas during neurodegeneration, tau synaptotoxicity seems to be related to soluble forms rather than insoluble aggregates (Pooler et al., 2014).

On the other hand, emerging evidence strongly suggests that tau is essential for Aβ-induced synaptotoxicity (Ittner et al., 2010), a process that may involve EphB2, and NMDA receptors (Cissé et al., 2011; Sheng et al., 2012). Furthermore, studies in mouse organotypic hippocampal slice cultures from amyloid precurssor protein transgenics have demonstrated that extrasynaptic NR2Bcontaining NMDA receptors are required for tau-induced neurodegeneration (Tackenberg et al., 2013). Could then tau play a role in the transition between synaptic and extrasynaptic NMDA receptors? Although we do not have a definitive answer to that, the improved NMDA receptor antagonist nitromemantine protects against Aβ-induced synaptic dysfunction (Talantova et al., 2013). Nitromemantine selectively inhibits extrasynaptic over synaptic

NMDA receptor activity (Kaufman et al., 2012), thus preventing the toxic effect of the activation of extrasynaptic NMDA receptors. Aβ binding to synaptic or extrasynaptic receptors may lead to different signaling and consequences (protection or death) (Li et al., 2011). Increase of extrasynaptic receptors with aging and AD may also explain the progression of the disease (**Figure 2**).

Interestingly, recent *in vivo* studies in tauopathy transgenic mouse models expressing human mutant tau specifically in the entorhinal cortex have shown relocation of tau from axons to the somatodendritic compartment as well as propagation of tau pathology to regions outside the entorhinal cortex, strongly suggesting a trans-synaptic mechanism of spreading of pathology through anatomically connected neuronal networks (de Calignon et al., 2012; Liu et al., 2012). These findings have been further supported by more recent neuropathological studies in postmortem brains from argyrophylic grain disease (AGD), a sporadic tauopathy mainly involving the medial temporal lobe and the limbic region (Ferrer et al., 2008). This pathology exhibits a short number of closely related tau-positive inclusions and a highly homogeneous pattern of distribution and progression of pathology along several regions of the medial temporal lobe with known connectivity between them and with extra-temporal areas of involvement, leading to its proposition as a natural model for studying tau propagation in human brain (Rábano et al., 2014).

Recently, release and subsequent uptake of tau fibrils that directly contact native protein in recipient cells have been shown to mediate propagation of tau misfolding among cells, at least *in vitro* (Frost et al., 2009; Kfoury et al., 2012). Remarkably, intracerebral inoculation of synthetic preformed tau fibrils induced NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner (Iba et al., 2013). Furthermore, conformation-specific trans-cellular propagation of tau fibrils after release into the extracellular space and subsequent triggering of aggregation in recipient cells by contacting native protein has been show in co-culture experiments (Kfoury et al., 2012). Thus, newly aggregated intracellular tau can transfer between co-cultured cells (**Figure 1**), thus providing a mechanism for tau-targeted immunotherapies as therapeutic strategy for AD and tauopathies (Gu and Sigurdsson, 2011; Medina, 2011; Golde et al., 2013). Actually, it has been suggested that the most likely mechanism of action for anti-tau antibodies is targeting tau released from cells (Yanamandra et al., 2013). The recent development of imaging-based biomarkers (Maruyama et al., 2013) will enable to track the progression of tau pathology in living patients and greatly facilitate the early phase testing of tau immunotherapy and other tau-based therapeutic strategies.

# **CONCLUSIONS**

In summary, we have highlighted recent developments in tau biology relevant to AD and tauopathies. It has become increasingly clear that, apart from the well-established intracellular functions of tau in microtubule stabilization and axonal transport, intracellular and extracellular tau most likely have important signaling roles that could contribute to the neurodegenerative process in AD and related tauopathies. Furthermore, the presence of tau in synaptic regions of healthy brain suggest that tau may play a role in the regulation of normal synaptic function. In addition, recent studies have suggested that misfolding of tau in diseased brain leads to abnormal conformations of tau that can be taken up by surrounding neurons. Thus, pathological progression could involve transmission of tau protein through a potential prion-like, seeding mechanism resulting in neurodegeneration in susceptible brain regions. However, insufficient evidence exists yet to reliably determine whether there is a direct relationship between the recent identification of a physiological role for extracellular tau and the impairments in tau function associated with disease.

Key questions still remain open, such as the neuronal selectivity, the nature of the extracellular tau species involved, or the precise seeding/templating mechanisms, among many others. More research is needed to identify disease mechanisms driving release and propagation of tau pathology and to determine the impact of extracellular tau on cognitive decline during neurodegeneration. The observation that misfolded tau can be secreted and taken up by adjacent neurons calls for the development of novel strategies to block the propagation of tau pathology in the brain. Despite the fact that the presence of extensive tau pathology is central to the disease, tau-based therapeutic strategies have received little attention until recently (Medina and Avila, 2014). Next few years will certainly bring new insights into the cellular mechanisms underlying tau secretion and uptake, likely identifying novel therapeutic approaches intended to interfere early on in the process of propagation of tau pathology.

#### **REFERENCES**

beta-amyloid-induced neurodegeneration and synaptic loss. *Cell Death Dis.* 4:e608. doi: 10.1038/cddis.2013.129

fluid tau levels in P301S human tau transgenic mice. *J. Neurosci.* 31, 13110– 13117. doi: 10.1523/jneurosci.2569-11.2011

Yanamandra, K., Kfoury, N., Jiang, H., Mahan, T. E., Ma, S., Maloney, S. E., et al. (2013). Anti-tauantibodies that blocktauaggregate seeding in vitro markedly decrease pathology and improve cognition in vivo. *Neuron* 80, 402–414. doi: 10. 1016/j.neuron.2013.07.046

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 February 2014; accepted: 05 April 2014; published online: 23 April 2014*.

*Citation: Medina M and Avila J (2014) The role of extracellular Tau in the spreading of neurofibrillary pathology. Front. Cell. Neurosci. 8:113. doi: 10.3389/fncel.2014.00113 This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2014 Medina and Avila. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

# Multiple effects of β-amyloid on single excitatory synaptic connections in the PFC

# *Yun Wang1,2\*, Thomas H. Zhou2, Zhina Zhi 1, Amey Barakat 2, Lynn Hlatky2 and Henry Querfurth3*

*1 School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China*

*2 Steward St. Elizabeth's Medical Center, Tufts Medical School, Tufts University, Boston, MA, USA*

*3 Department of Neurology, Rhode Island Hospital, Brown University, Providence, RI, USA*

#### *Edited by:*

*Ludovic Martin, Université de Nantes, France*

#### *Reviewed by:*

*Patrick Dutar, INSERM, France Filippo Tempia, University of Turin, Italy*

*Fernando Peña, UNAM, Mexico*

#### *\*Correspondence:*

*Yun Wang, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, 325003, Zhejiang, China e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Prefrontal cortex (PFC) is recognized as an AD-vulnerable region responsible for defects in cognitive functioning. Pyramidal cell (PC) connections are typically facilitating (F) or depressing (D) in PFC. Excitatory post-synaptic potentials (EPSPs) were recorded using patch-clamp from single connections in PFC slices of rats and ferrets in the presence of β-amyloid (Aβ). Synaptic transmission was significantly enhanced or reduced depending on their intrinsic type (facilitating or depressing), Aβ species (Aβ40 or Aβ42) and concentration (1–200 nM vs. 0.3–1 μM). Nanomolar Aβ40 and Aβ42 had opposite effects on F-connections, resulting in fewer or increased EPSP failure rates, strengthening or weakening EPSPs and enhancing or inhibiting short-term potentiation [STP: synaptic augmentation (SA) and post-tetanic potentiation (PTP)], respectively. High Aβ40 concentrations induced inhibition regardless of synaptic type. D-connections were inhibited regardless of Aβ species or concentration. The inhibition induced with bath application was hard to recover by washout, but a complete recovery was obtained with brief local application and prompt washout. Our data suggests that Aβ40 acts on the prefrontal neuronal network by modulating facilitating and depressing synapses. At higher levels, both Aβ40 and Aβ42 inhibit synaptic activity and cause irreversible toxicity once diffusely accumulated in the synaptic environment.

**Keywords: β-amyloid (Aβ), synaptic connection, synaptic dynamics, excitatory post-synaptic potential (EPSP), short term potentiation (STP), synaptic augmentation (SA), post-tetanic potentiation (PTP)**

# **INTRODUCTION**

In patients and animal models of the early stages of Alzheimer's Disease (AD), declines in episodic or spatial memory and cognition are correlated with an increase in brain levels of soluble β-amyloid (Aβ) (Lue et al., 1999; Walsh et al., 2002; Rowan et al., 2003). A causal link between the accumulation of Aβ in a soluble, toxic state and impairment of neuronal mechanisms that support memory was demonstrated in a mature βAPP transgenic mouse model wherein a single systemic injection of an antibody to Aβ eliminated the memory deficit (Dodart et al., 2002). There is a major focus on the synapse as the initial site of damage in AD (Selkoe, 2002; Nimmrich and Ebert, 2009). Synaptic dysfunction as a consequence of diffusible Aβ is also inferred from studies showing reduced basal transmission and altered plasticity (Klyubin et al., 2005; Shankar et al., 2008; Minano-Molina et al., 2011). In anatomic terms, synapse numbers are reduced early in some AD brain regions (Davies et al., 1987), especially in the prefrontal cortex (PFC) and medial temporal lobe (Morris and Baddeley, 1988). Meanwhile, additional studies in the recent decade indicate that low levels of Aβ peptides could be essential for the modulation of synaptic plasticity (Parihar and Brewer, 2010).

In AD, limbic and association cortices are selectively involved while primary cortical areas remain relatively preserved. These regions of neuronal vulnerability in fact correspond to the degree to which neuronal plasticity can be demonstrated in them (Arendt, 2001). The PFC is a critical association region associated with executive type cognitive function. PFC also orchestrates a unique form of short-term memory termed "working memory." Working memory is a limited capacity system that supports nonroutine types of daily activity. It is a temporary storage system for maintaining and rapidly manipulating information, and is closely connected with attention, strategic information flow and action (Goldman-Rakic, 1996). Experimentally, prefrontal cortical neurons are found to remain persistently active during the delay between sensory cue and an executed response task. The ongoing activity, an electrical correlate of working memory, is stable to the interference from distractors (Goldman-Rakic, 1995). Further, fMRI studies confirm the role of PFC in strategic encoding and goal directed control over the retrieval process in episodic type memory processes (Simons and Spiers, 2003). Less appreciated than episodic memory, working memory is also impaired in the early stages of AD, according to clinical and *in vivo* studies (Morris and Baddeley, 1988). Impairment of PFC function may precede the pathological changes of AD in other cortical association areas (Reid et al., 1996). Correspondingly, soluble Aβ accumulates in the PFC to one of the highest and earliest levels across several cortical regions in the pre-tangle stages of AD (Gouras et al., 2000) and in transgenic mice (Zhuo et al., 2008). Tangle formation in the PFC is also highly correlated with the transition to clearly recognizable dementia (Wang and Al, 2001).

In the PFC network, excitatory synaptic connections in layer V show both facilitated (F-connection) and depressed (D-connection) excitatory post-synaptic potentials (EPSPs) in response to short train stimuli (Wang et al., 2006). F-connections are formed predominantly by complex-type pyramidal cells (PCs) which feature dual apical dendrites, a high degree of interconnectivity and of reciprocity in chemical synaptic connections. In contrast, D-connections are typically formed by simple PCs that are common to primary cortices. A computer simulation study revealed that these facilitating synapses play a crucial role in the formation of persistent neuronal activity, consistent with the properties of working memory (Mongillo et al., 2008). The critical role of the PFC in working memory and early involvement in AD make it a suitable region to examine the electrophysiologic effects of Aβ.

Numerous studies of AD-promoting factors (e.g., Aβ) have examined their effects on field electrophysiological characteristics in the hippocampus and cortex. Because of competing synaptic inputs and influences from other local networks, the use of field recordings may account for seemingly contradictory early reports, where Aβ either increased excitability through membrane depolarization and enhancing long term potentiation (LTP) or depressed both synaptic transmission and LTP induction (Wu et al., 1995; Selkoe, 2002; Walsh et al., 2002; Esteban, 2004; Puzzo et al., 2008; Li et al., 2011). Up until now, a study focused on activity-dependent plasticity specific to the association cortex has not been reported. It is therefore timely to carry out an investigation of an *in situ* neural network, especially at the resolution of individual synaptic connections, within an association cortical area such as the PFC.

In this study, using multi-neuron patch clamp recording from PFC slices, we found that synaptic responses of single excitatory synaptic connections were significantly enhanced or reduced depending on their intrinsic type (facilitating or depressing), the tested Aβ species (40 or 42 amino acids) and concentration (low dose 1–200 nM vs. high dose 0.3–1μM). Low-doses of Aβ40 enhanced F-connections and inhibited D-connections in the PFC. In contrast, high-doses of Aβ40 and low-doses of Aβ42 inhibited all types of excitatory synaptic connections. Further, the inhibition induced with bath application was commonly difficult to recover or even became worse by washout. However, direct local and brief application of the peptides by pipette at comparable concentrations produced similar inhibitions with a rapid and complete recovery upon washout. Based on the principles of synaptic dynamics that have been well-studied in our previous computer simulation of synaptic responses of single synaptic connections, the effects of Aβ were considered to be produced via both pre- and post-synaptic mechanisms.

### **MATERIALS AND METHODS**

#### **ELECTROPHYSIOLOGICAL RECORDINGS**