Preconditioning-Activated AKT Controls Neuronal Tolerance to Ischemia through the MDM2–p53 Pathway

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2–p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2–p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.


Introduction
In humans, the existence of transient ischemic attack (TIA) has been revealed as an endogenous preconditioned state with benefits of functional outcome in stroke patients [1][2][3]. Endogenous neuroprotection induced by a short, subtoxic ischemic stimulus, known as ischemic preconditioning (IPC), is considered a strategy in the emerging field of neuroprotection against ischemic injury [4][5][6]. Evidence shows that IPC-promoted neuroprotection depends on transcription, translation, and post-translational mechanisms, which alters the function of key proteins after ischemia [6][7][8][9][10][11]. However, the mechanism involved in IPCinduced ischemic tolerance (IT) has not been fully clarified in the human brain [12,13]. The development of new experimental approaches to understand IPC-mediated neuroprotection represents a powerful tool to decipher the endogenous mechanisms underlying brain IT [6], which have the potential to unveil novel therapeutic targets aimed at minimizing brain damage in stroke patients. In the last two decades, apoptotic neuronal cell death has positioned itself as an essential mechanism involved in cerebral ischemic injury [14][15][16]. In this sense, protein kinase B or AKT, a serine/threonine kinase that requires a functional phosphoinositide kinase (PI3K) to be activated, has been considered as an essential target for neuroprotective therapies after ischemia [17,18]. Recently, we demonstrated that the inhibition of the PI3K/AKT signaling pathway increases neuronal susceptibility to excitotoxicity [19]. AKT is involved in a complex anti-apoptotic signaling network [20], whose components may be present at different subcellular locations depending on tissue type [21,22]. In the heart, AKT has been involved in preconditioning-promoted cardioprotection [23]. Evidence also shows that phosphorylated AKT promotes neuronal survival in the onset of cerebral ischemia [24]. Although AKT activation may contribute to the induction of IT in the brain [25], the exact mechanism involving its IPC-mediated activation remains elusive.
In tumor cells, activation of the PI3K/AKT signaling pathway leads to MDM2 phosphorylation at Ser 166/186 , which promotes the nuclear translocation of MDM2 [26,27] and enhances its ubiquitination activity [28]. In the nucleus, MDM2 binds to p53 and promotes its ubiquitination and subsequent proteasomal degradation, which inhibits p53 function [29]. Under stress conditions, p53 could also trigger MDM2 overexpression, which conversely suppresses p53 activation in a negative feedback loop [30]. The inhibition of PI3K prevents AKT activation [31] and MDM2 phosphorylation in the preconditioned heart [32]. In this context, we previously found that in vivo brain preconditioning reduced infarct volume after transient middle cerebral artery occlusion (tMCAO) by increasing MDM2 protein level expression. Consequently, the MDM2-p53 complex attenuated ischemia-induced activation of the p53/PUMA/caspase-3 signaling pathway in primary cortical neurons [33]. Here, we dig into the role of the PI3K/AKT signaling pathway in IPC-mediated neuronal tolerance against a subsequent ischemic injury, as well as the underlying mechanism, and we mainly focus on the potential link between AKT activation and the MDM2-p53 complex.

IPC-Promoted Neuroprotection Is Mediated by Phosphorylation of AKT at Ser 473 , Phosphorylation of MDM2 at Ser 166 , and p53 Destabilization
We previously described the impact of MDM2-p53 interaction on neuronal susceptibility to ischemia [34] and IT [33]. Here, we explored the potential role of AKT on IPC-mediated neuroprotection as a candidate to be involved in the MDM2-p53 pathway. First, we confirmed that ischemia promoted AKT activation in neurons, as evidenced by AKT phosphorylation at Ser 473 . As shown in Figure 1A, short (20 min) OGD significantly induced p(Ser 473 )AKT and MDM2 expression, whereas AKT protein levels remained unaltered ( Figure S1A). However, AKT phosphorylation was not observed when neurons were subjected to prolonged OGD (90 min). Moreover, MDM2 protein levels were lower, which is consistent with the higher expression of p53 protein as shown in Figure 1A.
The time-dependent upregulation of Mdm2 expression after OGD ( Figure 1B) confirms that subacute ischemia may be important to induce mechanisms that prevent p53 stabilization after OGD, as previously described [33]. We used short OGD (20 min) followed by 2 h of reoxygenation as a model of IPC (Figure S1B) [33]; thus, we analyzed neuronal extracts collected at 4 h of reoxygenation after OGD (OGD/R) or after OGD preceded by the IPC protocol (IPC + OGD/R). In parallel, neurons were incubated in normoxia (Nx) or preconditioning (IPC) settings ( Figure S1B).

Figure 1.
IPC promotes phosphorylation of AKT at Ser 473 and MDM2 at Ser 166 and reduces p53 stabilization. Mouse cortical neurons (9-10 DIV) were exposed to a validated in vitro model of IPC and ischemia, as indicated in Figure S1B. (A) Phosphorylation of AKT at Ser 473 , p(Ser 473 )AKT, MDM2, and p53 protein levels as detected by Western blotting after Nx and OGD (20 or 90 min). (B) Mdm2 mRNA levels as quantified by real-time qPCR. (C) AKT, p(Ser 473 )AKT, MDM2, its phosphorylated form at Ser 166 , p(Ser 166 )MDM2, and p53 were analyzed by Western blotting. AKT and GADPH were used as loading control. Relative protein abundance quantification from three or five different neuronal cultures is shown in Figure S1A,C, respectively. MW, molecular weight. (D) Representative images of cortical neurons stained with p(Ser 473 )AKT (red), p53 (green), and DAPI (blue, nuclear marker). Scale bar, 50 μm. Percentage of positive AKT and p53 neurons is quantified in Figure S2A.
As shown in Figure 1C and Figure S1C, IPC induced the early activation of AKT, as revealed by phosphorylation at Ser 473 [35], followed by MDM2 protein stabilization and phosphorylation at Ser 166 . IPC also prevented p53 stabilization induced by OGD/R (Figure 1C). Interestingly, immunofluorescence images shown in Figure 1D revealed that IPC promoted AKT phosphorylation at Ser 473 in neurons, which predominantly accumulated Mouse cortical neurons (9-10 DIV) were exposed to a validated in vitro model of IPC and ischemia, as indicated in Figure S1B. (A) Phosphorylation of AKT at Ser 473 , p(Ser 473 )AKT, MDM2, and p53 protein levels as detected by Western blotting after Nx and OGD (20 or 90 min). (B) Mdm2 mRNA levels as quantified by real-time qPCR. (C) AKT, p(Ser 473 )AKT, MDM2, its phosphorylated form at Ser 166 , p(Ser 166 )MDM2, and p53 were analyzed by Western blotting. AKT and GADPH were used as loading control. Relative protein abundance quantification from three or five different neuronal cultures is shown in Figure S1A,C, respectively. M W , molecular weight. (D) Representative images of cortical neurons stained with p(Ser 473 )AKT (red), p53 (green), and DAPI (blue, nuclear marker). Scale bar, 50 µm. Percentage of positive AKT and p53 neurons is quantified in Figure S2A.
As shown in Figure 1C and Figure S1C, IPC induced the early activation of AKT, as revealed by phosphorylation at Ser 473 [35], followed by MDM2 protein stabilization and phosphorylation at Ser 166 . IPC also prevented p53 stabilization induced by OGD/R ( Figure 1C). Interestingly, immunofluorescence images shown in Figure 1D revealed that IPC promoted AKT phosphorylation at Ser 473 in neurons, which predominantly accumulated in the nucleus, and decreased p53 stabilization after OGD/R (IPC + OGD/R), when compared with non-preconditioned neurons (OGD/R). Consequently, IPC prevented neuronal apoptosis and caspase-3 activation caused by OGD/R, as measured by flow cytometry ( Figure S1D) and fluorimetry assays ( Figure S1E), respectively. To confirm the role of p53 in IPC-mediated neuroprotection, we used neurons expressing (wild-type; wt) or not (knockout; ko) p53 protein. Our results show that neurons lacking p53 ( Figure S1F) were more resistant to OGD-induced apoptosis than p53 wt neurons. Moreover, apoptosis levels in p53KO neurons were similar to those observed in preconditioned (IPC + OGD/R) wt neurons ( Figure S1G), thus confirming the key role of p53 destabilization in IPC-mediated neuroprotection [33]. Our results show that IPC induced neuroprotection against an ischemic insult through a mechanism that involves phosphorylation of AKT at Ser 473 , MDM2 stabilization and phosphorylation at Ser 166 , and p53 destabilization.

IPC Triggers MDM2 Phosphorylation at Ser 166 via the PI3K/AKT Pathway
The PI3K/AKT signaling pathway is involved in neuronal IT both in vitro [36] and in vivo [37]. However, the role of IPC-mediated activation of PI3K/AKT pathway in the regulation of the MDM2-p53 complex remains unexplored. In order to clarify this, neurons were incubated with the irreversible and specific inhibitor of the PI3K/AKT pathway, wortmannin [19]. As shown in Figure 2A, wortmannin abrogated IPC-enhanced (Ser 473 )AKT phosphorylation and p53 destabilization, as shown in Figure 1D. These results suggest a direct link between AKT activation and inhibition of p53-mediated neuronal apoptosis ( Figure S1D) and caspase-3 activation ( Figure S1E) induced after OGD/R. The main regulator of p53 stabilization, MDM2, is a target of AKT [26], which phosphorylates MDM2 at Ser 166 and Ser 186 [26]. PI3K inhibition with wortmannin prevented the phosphorylation of both (Ser 473 )AKT and (Ser 166 )MDM2 induced by IPC ( Figure 2B). The specific AKTmediated phosphorylation of MDM2 at Ser 166 induced by IPC was confirmed by using a small interfering RNA (siRNA) specifically designed against AKT1 protein (siAkt), highly expressed in cortical neurons, and whose activity is essential for neuronal survival after ischemia [38]. As shown in Figure 3, siAkt reduced total AKT and p(Ser 473 )AKT protein levels at day 3 after transfection, both in HEK-293T cells ( Figure 3A) and in cortical neurons ( Figure 3B). Moreover, AKT knockdown (siAkt) prevented (Ser 166 )MDM2 phosphorylation ( Figure 3B). These results demonstrate that the IPC-activated PI3K/AKT signaling pathway promotes MDM2 phosphorylation at Ser 166 , which may be responsible for MDM2 stabilization and consequent p53 destabilization after ischemic injury.

IPC-Activated AKT Triggers Nuclear MDM2 Protein Stabilization after Ischemia
The activation of AKT has been involved in nuclear translocation of MDM2 in tumor cells [26]. Considering the relevance of nuclear MDM2 stabilization for neuronal survival after ischemia [34] and, more specifically, its neuroprotective role in IPC [33], we decided to further investigate the relevance of PI3K/AKT signaling pathway in the regulation of subcellular localization of MDM2 protein. Thus, neurons or HEK-293T cells were transfected with human MDM2-tagged protein (MDM2-GFP). Representative blots of transfected HEK-293T cells and images from neurons ectopically expressing human MDM2 protein after four different experimental conditions (Nx, IPC, OGD/R, and IPC + OGD/R) are shown in Figure 4A and Figure S1H, respectively. Ectopic expression of MDM2-GFP confirmed that IPC promotes MDM2 nuclear accumulation compared with nonpreconditioned ischemic (OGD/R) or normoxic (Nx) neurons ( Figure 4A

IPC-Activated AKT Triggers Nuclear MDM2 Protein Stabilization after Ischemia
The activation of AKT has been involved in nuclear translocation of MDM2 in tumor cells [26]. Considering the relevance of nuclear MDM2 stabilization for neuronal survival after ischemia [34] and, more specifically, its neuroprotective role in IPC [33], we decided to further investigate the relevance of PI3K/AKT signaling pathway in the regulation of subcellular localization of MDM2 protein. Thus, neurons or HEK-293T cells were transfected with human MDM2-tagged protein (MDM2-GFP). Representative blots of transfected HEK-293T cells and images from neurons ectopically expressing human MDM2 protein after four different experimental conditions (Nx, IPC, OGD/R, and IPC + OGD/R) are shown in Figure 4A and Figure S1H, respectively. Ectopic expression of MDM2-GFP confirmed that IPC promotes MDM2 nuclear accumulation compared with non-preconditioned ischemic (OGD/R) or normoxic (Nx) neurons ( Figure 4A,B), as revealed by the quantification of nuclear/cytosolic fluorescence ratio ( Figure S2B) and nuclear fluorescence intensity of MDM2-GFP ( Figure S2C).
Moreover, confocal immunofluorescence analysis revealed that IPC induced the colocalization of p(Ser 473 )AKT and endogenous MDM2 within the nucleus, whereas this effect was not observed under OGD/R condition ( Figure 5A,B). The maximal intensity of endogenous nuclear MDM2 was higher in preconditioned (89.1% IPC and 90.3% IPC + OGD/R) than in non-preconditioned neurons (43.7% Nx and 52.6% OGD/R). Values for p(Ser 473 )AKT protein were also higher in preconditioned neurons (96.5% in IPC and 82.7% in IPC + OGD/R) compared to non-preconditioned neurons (39.8% in Nx and 54.9% in OGD/R) ( Figure S2C). Moreover, confocal immunofluorescence analysis revealed that IPC induced the colocalization of p(Ser 473 )AKT and endogenous MDM2 within the nucleus, whereas this effect was not observed under OGD/R condition ( Figure 5A,B). The maximal intensity of endogenous nuclear MDM2 was higher in preconditioned (89.1% IPC and 90.3% IPC + OGD/R) than in non-preconditioned neurons (43.7% Nx and 52.6% OGD/R). Values for p(Ser 473 )AKT protein were also higher in preconditioned neurons (96.5% in IPC and 82.7% in IPC + OGD/R) compared to non-preconditioned neurons (39.8% in Nx and 54.9% in OGD/R) ( Figure S2C).
Furthermore, neuron treatment with wortmannin or siAkt before the IPC or OGD/R protocol impaired MDM2 nuclear translocation after OGD/R, when compared with control neurons ( Figure 5A and Figure S2D). Indeed, both treatments (wortmannin or siAkt) prevented the IPC-increased mean percentage of nuclear MDM2 maximal fluorescence intensity (39.4% and 45%) and p(Ser 473 )AKT (15.3% and 20%) after OGD/R, respectively ( Figure 5B). Thus, inactivation of the AKT signaling pathway results in MDM2 protein accumulation in the cytoplasm of preconditioned neurons after ischemia. These results highlight the relevance of IPC-enhanced nuclear translocation and stabilization of MDM2 mediated by the activation of the PI3K/AKT pathway, which might confer protection against ischemic injury.  Figure S2B.
Furthermore, neuron treatment with wortmannin or siAkt before the IPC or OGD/R protocol impaired MDM2 nuclear translocation after OGD/R, when compared with control neurons ( Figure 5A and Figure S2D). Indeed, both treatments (wortmannin or siAkt) prevented the IPC-increased mean percentage of nuclear MDM2 maximal fluorescence intensity (39.4% and 45%) and p(Ser 473 )AKT (15.3% and 20%) after OGD/R, respectively ( Figure 5B). Thus, inactivation of the AKT signaling pathway results in MDM2 protein accumulation in the cytoplasm of preconditioned neurons after ischemia. These results highlight the relevance of IPC-enhanced nuclear translocation and stabilization of MDM2 mediated by the activation of the PI3K/AKT pathway, which might confer protection against ischemic injury.  Following demonstration of the role of IPC-enhanced activation of PI3K/AKT pathway in the nuclear stabilization of MDM2, we further investigated whether p(Ser 473 )AKT and MDM2 interacted within the nucleus ( Figure 6A). MDM2 immunoprecipitation from nuclear protein extracts, followed by immunoblotting against MDM2 and p(Ser 473 )AKT, revealed that IPC promoted the interaction between p(Ser 473 )AKT and MDM2, after OGD/R, thus preventing OGD/R-induced nuclear p53 stabilization, as shown in the nucleus input ( Figure 6A).  Figure S2C. In the case of neurons treated with wortmannin and siAkt, the maximal fluorescence intensity of nuclear MDM2 from five different cultures of cortical neurons is shown in Figure S2D.

IPC Promotes p(Ser 473 )AKT and MDM2 Interaction, Which Enhances MDM2 Stabilization in the Nucleus and Reduces Induced Neuronal Apoptosis upon Ischemia
Following demonstration of the role of IPC-enhanced activation of PI3K/AKT pathway in the nuclear stabilization of MDM2, we further investigated whether p(Ser 473 )AKT and MDM2 interacted within the nucleus ( Figure 6A). MDM2 immunopre-  Figure S2C. In the case of neurons treated with wortmannin and siAkt, the maximal fluorescence intensity of nuclear MDM2 from five different cultures of cortical neurons is shown in Figure S2D.
Lastly, we studied the detrimental effect of PI3K/AKT pathway disruption on neuronal apoptosis ( Figure 6B). AKT inhibition counteracted the protective effect of IPC prior to OGD, which confirms the neuroprotective role of AKT-MDM2 in the context of IT. Our results, thus, demonstrate that IPC induces phosphorylation and activation of AKT, which promotes MDM2 phosphorylation at Ser 166 and nuclear translocation, where it interacts with p(Ser 473 )AKT. This mechanism may contribute to enhanced nuclear stabilization of MDM2, which plays an essential role in IPC-induced ischemic tolerance. Figure 6. Nuclear p(Ser 4730 )AKT-MDM2 interaction may be essential to ensure IPC-promoted neuronal tolerance against ischemia. Neurons were exposed to Nx, IPC, OGD/R, and IPC + OGD/R, and the subcellular location of MDM2 and p(Ser 473 )AKT, as well as the possible protein interaction, was analyzed by nucleus-cytosol fractionation followed by coimmunoprecipitation assay. (A) Nuclear neuronal extracts were obtained and immunoprecipitated with anti-MDM2. Expression of Figure 6. Nuclear p(Ser 4730 )AKT-MDM2 interaction may be essential to ensure IPC-promoted neuronal tolerance against ischemia. Neurons were exposed to Nx, IPC, OGD/R, and IPC + OGD/R, and the subcellular location of MDM2 and p(Ser 473 )AKT, as well as the possible protein interaction, was analyzed by nucleus-cytosol fractionation followed by coimmunoprecipitation assay. (A) Nuclear neuronal extracts were obtained and immunoprecipitated with anti-MDM2. Expression of MDM2 and p(Ser 473 )AKT proteins was analyzed by Western blot from neuronal immunoprecipitated (IP) samples, while 10% were loaded on SDS-PAGE as a nucleus input control. AKT and LAMIN B were used as loading control. Relative nuclear protein abundance quantification from three different neuronal cultures is presented. M W , molecular weight. (B) Neuronal apoptosis was analyzed by flow cytometry. Annexin V/APC-stained neurons that were 7AAD-negative were considered to be apoptotic (represented by the percentage of AnnexinV + /7AAD − neurons). Data are means ± SEM from three different cultures of cortical neurons. In all cases, p < 0.05 was considered significant. Statistical analysis of the results was evaluated in Figure 6A by Student's t-test. * p < 0.05 versus Nx; # p < 0.05 versus OGD/R. Statistical analysis of the results was evaluated in Figure 6B by one-way ANOVA followed by Bonferroni post hoc. * p < 0.05 versus control Nx; # p < 0.05 versus control OGD/R. Lastly, we studied the detrimental effect of PI3K/AKT pathway disruption on neuronal apoptosis ( Figure 6B). AKT inhibition counteracted the protective effect of IPC prior to OGD, which confirms the neuroprotective role of AKT-MDM2 in the context of IT. Our results, thus, demonstrate that IPC induces phosphorylation and activation of AKT, which promotes MDM2 phosphorylation at Ser 166 and nuclear translocation, where it interacts with p(Ser 473 )AKT. This mechanism may contribute to enhanced nuclear stabilization of MDM2, which plays an essential role in IPC-induced ischemic tolerance.

Discussion
Our results reveal that IPC-mediated activation of the PI3K/AKT signaling pathway triggers neuronal IT by controlling the MDM2-p53 complex in primary cortical neurons. We first confirmed the efficiency of the preconditioning in terms of neuroprotection [33,[39][40][41] using a validated IPC experimental model. We found that experimental IPC induced by a short (20 min) oxygen and glucose deprivation (OGD) followed by 2 h of reoxygenation resulted in neuroprotection, as shown by the prevention of both neuronal apoptosis and caspase-3 activation induced by prolonged OGD (90 min) followed by 4 h of reoxygenation (OGD/R). We show that IPC reduces caspase-3 activation in cortical neurons, which correlates with less apoptosis after a subsequent and more severe ischemic insult.
The balance between pro-and antiapoptotic signals is fundamental to ensure neuronal survival after ischemia [3,33,38,[42][43][44]. Although the relevance of such events has been shown in both hemorrhagic and ischemic in vivo stroke models [43,45], the mechanisms that regulate these signaling pathways are not yet fully understood in the context of ischemic tolerance.
The role of antiapoptotic AKT and its related pathways have been extensively studied in cancer cells [46,47] and brain tissue [38,48]; however, so far, the role of the AKT/MDM2-p53 signaling pathway in IPC-mediated neuronal tolerance against ischemic injury remains elusive. Here, we found that the activation of the PI3K/AKT signaling pathway caused by IPC promotes phosphorylation of MDM2 at Ser 166 , which triggers its nuclear translocation and protein stabilization, preventing p53-induced apoptosis via caspase-3 activation after ischemia.
The activation of AKT via phosphorylation promotes neuronal survival [24,49] and may contribute to the induction of IT [25,50]. Our results show that the relative abundance of AKT protein is unchanged under ischemic or preconditioning stimuli. Interestingly, we found that early PI3K-mediated phosphorylation of AKT at Ser 473 prevents ischemiainduced p53 stabilization in the preconditioned neurons. The effect was not due to modifications in p53 mRNA levels [33,34,44], but to decreased p53 protein levels due to IPC prior OGD/R. Since MDM2 is the main regulator of p53 stabilization and is also a direct target of AKT, our results point out the role of the AKT/MDM2-p53 signaling pathway in neuronal tolerance to ischemia.
MDM2 mRNA rapidly increases after OGD [34], but MDM2 activity is mainly controlled by post-translational modifications, particularly phosphorylation [51]. In good agreement with this, we found that, once activated by phosphorylation after IPC, AKT in turn phosphorylates MDM2 at residue Ser 166 , which is located in close proximity to the nuclear localization signal [52], and this effect is maintained after OGD/R injury. In fact, our results show that phosphorylation of MDM2 at Ser 166 is sufficient to exert the IPC-mediated neuroprotective effect via p53 destabilization. Thus, herein, we identified a time-dependent activation of AKT/MDM2-p53 pathway after ischemic injury and, indeed, we demonstrate that IPC-activated AKT triggered nuclear translocation of ectopic MDM2, as well as endogenous protein stabilization. Moreover, AKT remains active within the nucleus, where PI3K could also migrate in response to oxidative stress and then account for AKT phosphorylation [53]. The inhibition of PI3K-mediated phosphorylation of AKT or AKT knockdown promotes the retention of MDM2 protein in the cytoplasm, and it prevents Ser 166 phosphorylation of MDM2, as well as IPC-mediated neuroprotection against ischemia-induced neuronal apoptosis. On the contrary, we showed that active AKT binds to nuclear MDM2 protein. As a consequence, active AKT promotes both phosphorylation of MDM2 and its nuclear stabilization, which contribute to IPC-mediated neuroprotection. Our results reveal that IPC-promoted neuroprotection was dependent on PI3K-mediated AKT activation, which phosphorylated MDM2 at Ser 166 , promoting MDM2 nuclear accumulation after an ischemic insult. Accordingly, inhibition of PI3K/AKT by wortmannin or AKT depletion by siRNA abolished IPC-promoted neuroprotection, leading to p53 stabilization and the subsequent neuronal apoptosis after ischemia. Hence, our results help to clarify the essential role of IPC-dependent activation of the AKT-MDM2 pathway in neuronal survival against ischemic injury.
The p53 protein is involved in the control of neuronal death/survival determining prognosis in stroke patients [34,42,54], as well as in TIA patients [3]. In fact, p53 stabilization compromises preconditioning-mediated neuroprotection to ischemia/reperfusion injury [33]. The MDM2-p53 interaction will, therefore, be critical for neuronal survival in this context [34] and for IPC-mediated tolerance against ischemic injury [33]. Thus, the control of such interaction will also have an impact on stroke outcome. In this context, we recently found that a single-nucleotide polymorphism (SNP) 309T>G in the MDM2 promoter determines the expression of MDM2 and, in turn, modulates the recovery of patients suffering from stroke [34]. Additionally, we observed that a Tp53 gene SNP (rs1042522) modulates mitochondrial p53 stabilization and neuronal tolerance to ischemia, while predicting the functional recovery of patients who suffer a TIA prior to stroke [3]. Therefore, the control of p53 apoptotic pathways will be essential to ensure the neuroprotective effect of IPC. These results provide a translational approach to the study that could be implemented in the future for the benefit of patients, and they pose PI3K/AKT-MDM2-p53 signaling pathway as an essential target for the preconditioning-promoted IT strategies in ischemic stroke.
In summary, we demonstrate that IPC-enhanced PI3K/AKT signaling pathway promotes phosphorylation of MDM2 at Ser 166 , leading to MDM2 nuclear translocation and its stabilization, which triggers neuronal IT by promoting p53 destabilization and subsequent inactivation of apoptotic death induced after ischemic insult. Our results highlight the potential benefits of early activation of AKT in IPC-mediated neuronal tolerance, which regulates the MDM2-p53 apoptotic pathway under ischemic injury. These findings highlight an opportunity to understand the mechanisms that regulate neuronal the AKT-MDM2-p53 signaling pathway to develop novel neuroprotective strategies for IT-related disorders.

Flow Cytometric Detection of Apoptotic Cell Death
Neurons were carefully detached from the plates using 1 mM EDTA tetrasodium salt in PBS (pH 7.4) and were stained with annexin V/APC and 7-AAD, performed exactly as previously described [55].

Caspase-3 Activity Assay
Caspase-3 activity was assessed in cell lysates [33] and according to the manufacturer's instructions using the Fluorimetric Assay kit CASP3F from SIGMA and read at emission at a wavelength of 405 nm. The method is based on the release of the fluorescent 7-amino-4-methyl coumarin (AMC) moiety. The AMC concentration is calculated using an AMC standard.

Statistical Analysis
Experimental results were evaluated by one-way analysis of variance, followed by the Bonferroni post hoc test, used to compare values between multiple groups. The results are expressed as means ± SEM. Student's t-test was used for comparisons between two groups of values. In all cases, p < 0.05 was considered significant (* p < 0.05 versus Nx; # p <0.05 versus OGD). Statistical analyses were performed using SPSS Statistics 24.0 for Macintosh (IBM).