Preconditioning-Activated AKT Controls Neuronal Tolerance To Ischemia Through The MDM2–p53 PathwayⅡ

3. 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 [33,39–41] using a validated IPC experimental model. 

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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 [3,33,38,42–44]. 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 Ser166, 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 Ser473 prevents ischemia-induced 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 to 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 Ser166, which is located near the nuclear localization signal [52], and this effect is maintained after OGD/R injury. 

Our results show that phosphorylation of MDM2 at Ser166 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 Ser166 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 phosphorylations 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 Ser166, 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]. 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 outcomes. 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 before 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 the IPC-enhanced PI3K/AKT signaling pathway promotes phosphorylation of MDM2 at Ser166, 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.

4. Materials and Methods 

4.1. Primary Cultures of Cortical Neurons 

Neuronal cultures were prepared from C57Bl/6J or p53-null (Tp53−/−, B6.129S2, The Jackson Laboratory) mouse embryo (14.5E) cortices. Neurons were seeded at 1.8 × 105 cells/cm2 in a Neurobasal medium supplemented with 2% B27 and 2 mM glutamine (Invitrogen, Madrid, Spain) and incubated at 37 ◦C in a humidified 5% CO2-containing atmosphere [55].

4.2. Oxygen Glucose Deprivation and Preconditioning Models 

After 9–10 days in vitro (DIV), neurons were exposed to oxygen and glucose deprivation (OGD) by incubating cells at 37 ◦C for 90 min in an incubator equipped with an airlock and continuously gassed with 95% N2/5% CO2. The incubation medium (buffered Hanks’ solution without glucose: 5.26 mM KCl, 0.43 mM KH2PO4, 132.4 mM NaCl, 4.09 mM NaHCO3, 0.33 mM Na2HPO4, 2 mM CaCl2, and 20 mM HEPES, pH 7.4) was previously gassed with 95% N2/5% CO2 for 30 min. Under these conditions, oxygen concentrations in the incubation medium were 6.7 ± 0.5 µM as measured with a Clark-type oxygen electrode [56,57]. 

When indicated, the neurons were exposed to ischemic preconditioning (IPC; short OGD for 20 min followed by 2 h of reoxygenation) before subsequent prolonged ischemia (OGD, 90 min) and 4 h of reoxygenation (IPC + OGD/R) (Figure S1B). In parallel, neurons were incubated in normoxia (Nx) at 37 ◦C in a humidified atmosphere of 95% air/5% CO2 or ischemic preconditioning (IPC). When indicated, neurons were incubated 30 min before IPC in buffered Hanks’ solution (pH 7.4), in the absence or presence of wortmannin (100 nmol/L), as described previously [19].

4.3. Cell Transfections 

Neurons (8 DIV) or HEK-293T cells were transfected with a plasmid vector expressing YFP-tagged Mdm2 from MDM2 human promoter. MDM2p/Mdm2-YFP was a gift from Uri Alon & Galit Lahav (Addgene plasmid # 53962, Watertown, MA, USA) [58]. When required, an empty vector (pYFP) was used as a control in the same conditions. Plasmid transfection was performed using Lipofectamine® LTX (Invitrogen, Carlsbad, MA, USA), according to the manufacturer’s instructions. Cells were transfected with 1.5 µg/µL of the plasmid vectors and used after 24 h. 

AKT knockdown in 6 DIV neurons was achieved by transfection with small interfering double-stranded ribonucleotides (siRNA). Targeted sequences were as follows: 50–CUCAAGUACUCAUUCCAGAtt–30, antisense: 5 0–UCUGGAAUGAGUACUUGAGgg–30 (mouse, s62216, corresponding to nucleotides 1006–1025, GenBank accession number NM_009652) [59]. As a negative control, we used Silencer™ Select Negative Control No. 1 siRNA (siControl). All siRNAs were purchased from Ambion®, Invitrogen®, and Thermo Fischer Scientific (Offenbach, Germany). According to the degree of protein knockdown, the efficiency of transfection of siRNA was estimated to be 70–80% at 3 days post-transfection. For silencing experiments, neurons were transfected with siRNA (10 nM) using Lipofectamine® RNAiMAX (Invitrogen), following the manufacturer’s instructions. Neurons were further incubated in a Neurobasal medium for 72 h before their use.

4.4. 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].

4.5. 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-amino4-methyl coumarin (AMC) moiety. The AMC concentration is calculated using an AMC standard.

4.6. Immunoblots and Co-Immunoprecipitation Assay 

Neurons were lysed in buffer containing 1% SDS, 2 mM EDTA, 150 mM NaCl, 12.5 mM Na2HPO4, and 1% Triton X-100 (NP40: 1% NP40, EDTA diK+ 5 mM, Tris pH8 20 mM, NaCl 135 mM, and 10% glycerol) supplemented with phosphatase inhibitors (1 mM Na3VO4 and 50 mM NaF) and protease inhibitors (100 mM phenylmethylsulfonyl fluoride, 50 µg/mL anti-papain, 50 µg/mL pepstatin, 50 µg/mL amastatin, 50 µg/mL leupeptin, 50 µg/mL bestatin, and 50 µg/mL soybean trypsin inhibitor), stored on ice for 30 min and boiled for 5 min. Aliquots of lysed extracts were subjected to SDS polyacrylamide gel (MiniProtean®, Bio-Rad) and blotted with antibodies overnight at 4 ◦C. Antibodies used were anti-AKT (9272), anti-p(Ser473)AKT (9271), anti-cleaved caspase-3 (Asp175, 9661) (Cell Signaling, Danvers, MA, USA), anti-p53 (554157, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti (Ser166)MDM2 (ab131355), anti-GFP (ab290; also detects YFP) (Abcam, Cambridge, UK), anti-LAMIN B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany), and antiGAPDH (Ambion, Cambridge, UK) overnight at 4 ◦C. After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce, Thermo Scientific) or goat anti-mouse IgG (Bio-Rad), membranes were immediately incubated with enhanced chemiluminescence SuperSignal West Dura (Pierce) for 5 min before exposure to Kodak XAR-5 film for 1 to 5 min and the autoradiograms were scanned. Band intensities were quantified using ImageJ 1.48v software, as described previously [60]. 

For the co-immunoprecipitation assay, neurons were lysed in ice-cold buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% NP-40) supplemented with phosphatase inhibitors described above. After clearing debris by centrifugation, neuronal lysates (100 mg) were incubated with 1 mg of the antibody for 24 h at 4 ◦C followed by the addition of 10 mL of protein A-agarose (GE Healthcare Life Sciences) for 2 h at 4 ◦C. Immunoprecipitates were extensively washed with lysis buffer and resolved by SDS-PAGE and immunoblotted with indicated antibodies [61]. The relative protein abundances are shown in Figure S1. Full blots and gel scans are included in Figure S3.

4.7. Immunocytochemistry and Image Analysis 

Neurons were grown on glass coverslips and fixed with 4% (w/v, in PBS) paraformaldehyde for 30 min and immunostained with rabbit anti-phosphoAKT (Ser473; 9271; Cell Signaling, MA, USA), mouse anti-MDM2 (2A10, ab-16895), mouse anti-MAP2 (1:500; M#1406, Sigma-Aldrich, St. Louis, MO, USA) [55], mouse anti-p53 (1:200; 554157, BD Pharmingen, San Diego, CA, USA), and anti-GFP (1:1000; ab290; also validated to detect YFP). Immunolabeling was detected using secondary antibodies anti-rabbit IgG–Cy3 or anti-mouse IgG–Cy2 (1:500; Jackson ImmunoResearch. Cambridge, UK). 

Nuclei were stained with 40,6-diamidino-2 phenylindole (DAPI; D9542, Sigma-Aldrich). Coverslips were washed, mounted in SlowFade light antifade reagent (Invitrogen) on glass slides, and examined using a microscope (Nikon Inverted microscope Eclipse Ti-E, (NY, USA) equipped with 40× objective, a pre-centered fiber illuminator Nikon Intensilight C-HGFI, and a black-and-white charge-coupled device digital camera Hamamatsu ORCAER or a scanning laser confocal microscope (“Spinning Disk” Roper Scientific Olympus IX81, Tokyo, Japan) with three lasers 405, 491, and 561 nm, equipped with 40×, 63×, and 100× PL Apo oil-immersion objective for high-resolution imaging and device digital camera Evolve Photometrics. 

All microscope settings were set to collect fluorescent images below saturation and were kept constant for all images taken in the experiment. Images were analyzed with the ImageJ 1.48v software (National Institutes of Health). The percentage of p(Ser473)AKT+ and p53+ neurons and the quantification of maximal protein fluorescence intensity of p(Ser473)AKT and p53 are shown in Figure S2A. In MDM2-GFP-transfected neurons, the nucleocytoplasmic distribution of MDM2-GFP was calculated as the ratio of the nuclear mean fluorescence to the cytoplasmic mean fluorescence of endogenous MDM2, measured in 24 neurons (six neurons per condition in four different neuronal cultures) (Figure S2B) [62]. 

To quantify the maximal nuclear fluorescence intensity of endogenous MDM2 staining and pSer473AKT, 40 neurons (10 neurons per condition in four different cultures) were measured (Figure S2C), as described previously [44]. In the representative cross-sectional intensity profiles shown in Figure 5B, the percentage of p(Ser473)AKT and MDM2 indicated below each condition was quantified as nuclear mean fluorescence. In all cases, nuclei were identified by DAPI staining. The maximal nuclear MDM2 fluorescence intensity in neurons treated with wortmannin or siAkt is shown in Figure S2D.

4.8. 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).

How does Cistanche protect neurons?

There is some evidence to suggest that Cistanche may protect neurons by reducing apoptosis (programmed cell death) and promoting neuronal survival. Apoptosis is a natural process that occurs in the body to remove damaged or unwanted cells, but it can be harmful when it occurs excessively or inappropriately. Cistanche has been found to inhibit apoptosis in laboratory studies, and this effect may help to protect neurons from damage.

In addition, Cistanche contains several bioactive compounds that have been shown to have neuroprotective effects. For example, it contains echinacoside, which has been shown to protect neurons from oxidative stress and inflammation. It also contains acteoside, which has been found to have anti-inflammatory and antioxidant properties.

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