The Post-Ischemic Increase in GluA 2 Ser 880 Phosphorylation Involves NADPH Oxidase

Most 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs) expressed on adult hippocampal pyramidal neurons contain the edited form of GluA2 (Q607R) and are thus impermeable to Ca2+/ Zn2+entry. Following ischemic injury, these receptors undergo a subunit composition change, switching from a GluA2containing Ca2+/Zn2+-impermeable AMPAR to a GluA2-lacking Ca2+/Zn2+-permeable AMPAR. Recent studies indicate that an oxidative stress signaling pathway is responsible for the I/R-induced changes in AMPAR subunit composition. Studies suggest that nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), a superoxide generator, is the source that initiates the oxidative stress-signaling cascade during post-ischemic reperfusion. The objective of the present study was to determine if suppression of NADPH oxidase activity prevents the increase in phosphorylation and subsequent internalization of the GluA2 AMPAR subunit during reperfusion of post-ischemic hippocampal slices. In this study, we demonstrated that exposure of adult rat hippocampal slices to oxygen glucose deprivation/reperfusion (OGD/R) results in an increase in Ser880 phosphorylation of the GluA2 subunit. The increase in Ser880 phosphorylation resulted in the dissociation of GluA2 from the scaffolding proteins Glutamate receptor-interacting protein 1 (GRIP1) and AMPAR binding protein (ABP), thus enabling the association of GluA2 with protein interacting with C kinase 1 (PICK1). OGD/R also resulted in an increase in the association of activated protein kinase C α (PKCα) with PICK1. We have found that pharmacological inhibition of NADPH oxidase with apocynin diminishes the OGD/R-induced increase in activated PKCα association with PICK1 and subsequent Ser880 phosphorylation of GluA2. Suppression of NADPH oxidase activity also blunted OGD/R-induced decreased association of GluA2 with the scaffolding proteins GRIP1 and ABP. Protein phosphatase 2A (PP2A), which regulates PKCα activity by dephosphorylating the kinase, was inactivated by OGD/R-induced increase in tyrosine phosphorylation of the phosphatase (Y307). Inhibition of NADPH oxidase activity ameliorated OGD/R-induced PP2A phosphorylation and inactivation. Our findings are consistent with a model of OGD/R-induced Ser880 phosphorylation of GluA2 that implicates NADPH oxidase mediated inactivation of PP2A and sustained PKCα phosphorylation of GluA2.


Introduction
AMPARs, which arise from 4 genes (GLUA1-GLUA4) to form a functional receptor tetramer, mediate the majority of fast excitatory synaptic transmission in the CNS.While all AMPAR subunits share a large degree of homology, the presence or absence of a GluA2 subunit contained within the functional AMPAR tetramer is important in determining channel properties and ionic permeability.Most AMPARs expressed on adult hippocampal pyramidal neurons contain the edited form of GluA2 (Q607R), thereby rendering GluA2-containing AMPARs impermeable to divalent cation (Ca 2+ and Zn 2+ ) conductance [1], [2].Following ischemia/reperfusion however, AMPARs undergo a subunit composition switch from GluA2 containing Ca 2+ /Zn 2+ -impermeable AMPA receptors to GluA2-lacking Ca 2+ /Zn 2+ -permeable AMPARs, thus allowing the AMPAR to conduct Ca 2+ / Zn 2+ and contributes to delayed neuronal death [3].This change in AMPAR expression pattern is influenced by both the removal of the GluA2-containing receptors and the insertion of the GluA2-lacking receptors to the membrane surface.
Under physiological conditions, GluA2 endocytosis is mediated by PKCα dependent phosphorylation of serine residue 880 (Ser880) [4].PKCα, when activated by calcium influx, is trafficked to the plasma membrane by PICK1 [5], where it binds the GluA2 scaffolding protein ABP and subsequently phosphorylates GluA2 [6].Phosphorylation of GluA2 promotes the dissociation of GluA2 with the scaffolding protein GRIP1, an interaction that is important in anchoring AMPARs to the plasma membrane [5].Additionally, Ser880 phosphorylation of GluA2 enables the increased binding of the subunit to PICK1 [4], an interaction that has been shown to be important in decreasing surface GluA2 levels [7], [8].As a result, the population of GluA2-containing AMPARs is reduced, ultimately allowing for an increase in calcium permeable GluA2-lacking AMPARs, a shift important for synaptic strengthening under physiological conditions [9], but ultimately deleterious if uncontrolled under pathological conditions such as stroke [10].
In this study, we show that the treatment of acute adult rat hippocampal slices to oxygen-glucose deprivation/ reperfusion (OGD/R) results in a sustained phosphorylation of GluA2, leading to the dissociation of GluA2 from GRIP and ABP, both of which favor the association of GluA2 with PICK1.Additionally, the inhibition of NADPH oxidase prevented the OGD/R-induced increase in Ser880 phosphorylation of GluA2, consequently attenuating the loss of GRIP1/ABP-GluA2 interaction and preventing GluA2/PICK1 complex formation.Further, inhibition of NADPH oxidase dampened the increase in association of the activated form of PCKα with PICK1, a necessary step prior to phosphorylation of GluA2, as well as diminished the OGD/R induced inactivation of protein phosphatase 2A (PP2A).While the exact mechanism through which NADPH oxidase influences GluA2 Ser880 phosphorylation has yet to be elucidated, these findings identify this ROS generator as a key participant in the OGD/R induced Ser880 phosphorylation of hippocampal GluA2 subunits and diminished membrane surface expression of GluA2containing AMPARs.

Oxygen-glucose deprivation/reperfusion of hippocampal slices
Following equilibration in oxygenated aCSF, slices to be treated with OGD/R were rinsed with aCSF without glucose (aCSF with 10 mM Mannitol substituted for 10 mM Glucose, pH 7.4) to remove glucose from the slices, and incubated for 40 minutes in a hypoxic glove box (Coy Laboratories, Grass Lake, MI, USA) containing a gas mixture of 0% O 2 , 95% N 2 , 5% CO 2 with aCSF minus glucose.The aCSF minus glucose media used for OGD was placed in 0% O 2 overnight to ensure complete anoxia.Following OGD, slices were removed from the hypoxic glove box and transferred back to oxygenated aCSF containing glucose for the time points indicated.Normoxic controls were left in aCSF containing glucose throughout the entire experiment and were time matched to the last of the OGD/R time points.

Lysate preparation
Slices were removed from aCSF at the indicated time point and rinsed with ice cold PBS (pH 7.4).For total protein level detection, slices were places in tubes containing lysis buffer (250 mM Sucrose, 20 mM HEPES, 2 mM EDTA, 5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM AEBSF, 1% protease and phosphatase inhibitor cocktail (Thermo, Rockland, IL, USA), 1% Triton X-100, 0.01% saponin, pH 7.4) and lysed immediately via sonication for a 3 separate 5 second bursts at 25% power output with a VirTis Ultrasonic Cell Disrupter 100 (Gardiner, NY, USA).Samples were then spun at 1,000 x g to remove nuclei and cellular debris and a bicinchoninic acid assay (BCA, Thermo) assay was performed to determine protein content.Samples were then denatured in Laemmli buffer and heat (100°C, 10 minutes) and resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Samples were then transferred to a nitrocellulose membrane (Bio-Rad, Berkeley, CA, USA) for subsequent detection via immunoblotting.

Statistical analysis
Indicated statistical tests were performed using GraphPad Prism Software (La Jolla, CA, USA).

Oxygen-glucose deprivation/reperfusion results in an increase in GluA2 Ser880 phosphorylation and interaction between activated PKCα, GluA2 with PICK1
Aberrant endocytic trafficking of GluA2 AMPA receptor subunit is preceded by and dependent on PKCα -PICK1induced phosphorylation of the GluA2 subunit following cerebral trauma [11], [12].We sought to determine whether OGD/R exposure of adult rat hippocampal slices lead to an increase in GluA2 Ser880 phosphorylation.Secondly, we examined whether OGD/R exposure increased association between GluA2, and activated PKCα (phosphThr497) with PICK1.We observed in slices subjected to 40-minutes of OGD incubation, an increase in GluA2 Ser880 phosphorylation that was maximally significant at 30-minutes of reperfusion (Figure 1A and  1B).OGD alone at 40-minutes or at 60-minutes did not lead to an increase in GluA2 Ser880 phosphorylation.Increase phosphorylation at serine residue 880 on GluA2 enables interaction of GluA2 with PICK1, an event demonstrated to reduce surface expression of GluA2 [7], [8].We next sought to determine whether the OGD/ R-induced increase in Ser880 phosphorylation of GluA2 observed in rat hippocampal slices (Figure 1A and 1B) functionally lead to an increase in GluA2-PICK1 association.Temporally consistent with the OGD/R-induced increase in GluA2 Ser880 phosphorylation status, our results indicate a significant increase in GluA2-PICK1 association during reperfusion of OGD-treated slices (Figure 2A and  2B).These results serve as a correlate with the increase in GluA2 Ser880 observed during the reperfusion of OGDtreated slices, and suggest a prevented recycling of GluA2 via PICK1-mediated membrane exclusion.PICK1 functions as a transport and targeting protein that directs the PKCα-dependent GluA2 Ser880 phosphorylation [5], [6].However, it has not been directly demonstrated whether OGD/R exposure increases interaction between activated PKCα and PICK1.Therefore, we next sought to determine whether OGD/R exposure leads to an increase association between activated PKCα.There was a significant increase in the interaction between activated PKCα and PICK1 following OGD/reperfusion (Figure 3A).Interestingly, OGD alone did not result in a significant PKCα-PICK1 association, but the reperfusion of OGD-treated slices did, ultimately resulting in a rapid and sustained phospho-PKCα-PICK1 interaction upon reperfusion.Collectively, these initial results are consistent with OGD/R promoting the activation of PKCα, which associates with PICK1 to promote the Ser880 phosphorylation of the AMPAR

Inhibition of NADPH oxidase with apocynin decreases the OGD/R-induced increase in PKCα activation and subsequent Ser880 phosphorylation of GluA2
Apocynin, an inhibitor of NADPH oxidase activity, has been demonstrated by our lab and others to decrease ROS [13], decrease damage to post-synaptic proteins [14], and provide neuroprotection [15] during ischemia/ reperfusion.Oxidative stress has also been implicated in affecting ionic homeostasis during ischemia/reperfusion, through the enhancement of NMDAR currents [16], [17].Phospholipase C mediated mobilization of intracellular calcium stores [18], mitochondrial calcium release [13], [19] and the opening of cation channels [20] such as the transient receptor potential (melastatin) 2 (TRPM2).Therefore, we sought to determine if the reduction in oxidative stress observed with the inhibition of NADPH oxidase during OGD/R resulted in an attenuation of activated PKCα associated with PICK1.Our results illustrate that hippocampal slices pre-treated with apocynin (30 μM) prior to OGD/R showed a significant reduction in the activated form of PKCα associated with PICK1 (Figure 3A and 3B), indicating that NADPH oxidasemediated oxidative stress was involved in the OGD/Rinduced increase in PKCα activity.Additionally, inhibition of NADPH oxidase activity also dampened the OGD/Rinduced increase in GluA2 Ser880 phosphorylation (Figure 3C and 3D).These experiments show that the increase in the oxidant environment following OGD/R resulting from NADPH oxidase activity contributes to the sustained activation of PKCα associated with PICK1 and the subsequent rise in GluA2 Ser880 phosphorylation.

Inhibition of NADPH oxidase prevents the OGD/R-induced increase in GluA2-PICK1 association and OGD/R-induced loss of GluA2-GRIP1 association
On the C-terminal tail of GluA2, the sequence (IESVKI) serves as the PKCα mediated phosphorylation site at Ser880 and also as a PDZ binding domain.When GluA2 is in the dephospho-Ser880 state, it preferentially binds the PDZ binding scaffolding proteins GRIP1 and ABP, which have been demonstrated to be important in retaining GluA2 at the plasma membrane [21], [22].Upon Ser880 phosphorylation, the affinity for GRIP1 and ABP is reduced and the binding of another PDZ binding protein, PICK1 is promoted [4], potentially facilitating the surface removal of GluA2.Therefore, experiments were performed to determine if the decrease in Ser880 GluA2 phosphorylation observed with NADPH oxidase inhibition during OGD/R resulted in maintenance of GluA2-GRIP1/ABP anchoring, as well as a decrease in GluA2-PICK1 association.Consistent with the findings from the previous figure (Figure 3) of an OGD/R-induced increase in GluA2 Ser880 phosphorylation, inhibition of NADPH oxidase with apocynin prevented the disruption of GluA2-GRIP1/ABP anchoring (Figure 4A,4B,4C and 4D).Additionally, the interaction between GluA2-PICK1 was diminished (Figure 4E and 4F), further confirming the role of NADPH oxidase in promoting the OGD/R-induced increase in GluA2 Ser880 phosphorylation and the interaction of GluA2 with its PDZ binding partners GRIP1, ABP, and PICK1.

Inhibition of NADPH oxidase prevents the OGD/R-induced inactivation of PP2A
PP2A has been shown to play a role in regulating PKC activity [23].Studies have demonstrated that oxidative stress induced by H 2 O 2 inactivates PP2A, antioxidant treatment prevents this inactivation [24].We demonstrated an OGD/R-induced increase in PCKα activity; this sustained phosphorylation may be mediated by the inactivation of PP2A.Therefore, experiments were performed to determine if PP2A undergoes OGD/ R-induced inactivation, and if so, does reduction of oxidative stress by inhibition of NADPH oxidase prevent PP2A inactivation.We show an OGD/R-induced increase in PP2A Tyr307 phosphorylation (Figure 5A and 5B), which was diminished in the presence of apocynin (Figure 6A and 6B).Our results indicate that NADPH oxidasemediated oxidative stress is involved in the OGD/Rinduced inactivation of PP2A.

Discussion
Multiple lines of evidence have shown that the activation of Ca 2+ /Zn 2+ -permeable AMPARs following ischemic injury significantly contributes to neuronal death [12][25], [26], [27], [28], [29].While it has been demonstrated that ischemia results in a decrease in GluA2 protein levels and an increase in AMPAR-mediated Ca 2+ currents [30], the exact mechanistic signals resulting in GluA2-mediated surface removal have yet to be fully elucidated.
In this study, the data presented demonstrates that OGD/R-induced reduction of surface GluA2 AMPAR subunits involves NADPH oxidase dependent phosphorylation of the subunit.We found that inhibition of NADPH oxidase activity with apocynin during OGD/R not only dampened localized activation of PKCα with PICK1, but also attenuated the OGD/R-induced increase in Ser880 phosphorylation of GluA2.Furthermore, the decrease in Ser880 phosphorylation observed with NADPH oxidase inhibition during OGD/R also resulted in a decreased GluA2 interaction with PICK1, in addition to maintaining association of GluA2 with the membrane anchoring proteins GRIP1/ABP, ultimately rescuing the OGD/R-induced loss of GluA2 protein levels.We found that inhibition of NADPH oxidase activity with apocynin during OGD/R also diminished the OGD/R-induced inactivation of PP2A.
The PKCα-mediated Ser880 phosphorylation of GluA2 [31] has been shown to be important in the surface removal of GluA2 [4].While some studies have indirectly implicated that increased Ser880 phosphorylation of GluA2 occurs during ischemia/reperfusion [12], no studies to date have directly shown that an increase in Ser880 occurs with such treatment.We directly found that Ser880 phosphorylation of GluA2 increased significantly during reperfusion of OGD-exposed slices, but not during OGD without reperfusion.Our data also indicated that the association of activated PKCα with PICK1, a step necessary prior to Ser880 phosphorylation of GluA2 [6], occurred maximally during reperfusion of OGD treated slices and not during OGD alone, data that is consistent with the time course established for the increase in Ser880 phosphorylation of GluA2.
Phosphorylation of GluA2 at Ser880 has been shown to disrupt the GluA2 association with GRIP1/ABP, proteins that are important in the anchoring of AMPARs to the cell surface [32].Dissociation from GRIP1/ABP exposes the PDZ binding domain on GluA2 where the Ser880 residue resides, and when phosphorylated, consequently allows increased association with PICK1 [22], a protein important in restricting GluA2 from the cell surface [7], [8].Ischemia has been shown previously to enhance the interaction of PICK1 with GluA2 and decrease the association of GRIP1/ ABP with GluA2 [12].Consistent with these findings, the data presented in this study illustrates a significant increase in PICK1-GluA2 association as the GRIP1/ABP interaction with GluA2 decreases.Interestingly, inhibition of NADPH oxidase with apocynin maintains GluA2 anchoring to GRIP1/ABP and decreases PICK1-GluA2 association following OGD/R, findings in line with the decrease in GluA2 Ser880 phosphorylation observed with NADPH oxidase inhibition during OGD/R.Additional studies need be performed to determine the precise manner in which NADPH oxidase contributes to the OGD/R-induced increase in PKCα activation and GluA2 Ser880 phosphorylation.The first possibility involves the direct redox-modulation of PKCα via NADPH oxidase derived ROS.Published reports have demonstrated that PKC activity is increased with oxidative stress [33], which can be attenuated with anti-oxidant treatment using α-tocopherol [34] or apocynin treatment [35].However, studies have demonstrated that α-tocopherol reduces PKCα activity through non-antioxidant effects [36], potentially confounding the published results of redoxsensitive PKC activity.However, a reduction in glutathione levels has been reported to result in drastic increases in PKC activity [33] Further research must be completed to fully understand the relationship between ROS and direct PKC activation.
A second possibility for OGD/R-induced NADPH oxidase-mediated increases in PKCα activity includes phosphatase inactivation.Protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) have been demonstrated to be important in regulating PKC activity [23].Studies have shown that H 2 O 2 -induced oxidative stress inactivates both PP1 and PP2A, which is prevented with antioxidant treatment [24].Additionally, PP1 has been identified as the phosphatase responsible for the Ser880 dephosphorylation of GluA2 [37].OGD/R-induced ROSmediated inactivation of this phosphatase may therefore serve as an important mediator in not only regulating PKC activity, but also GluA2 Ser880 phosphorylation.Furthermore, studies have demonstrated that the filamentous actin binding protein neurabin is important in targeting PP1 to the post-synapse for GluA2 Ser880 dephosphorylation [37].Additionally, oxidative stress has been shown to result in the de-polymerization of actin [38], thereby potentially decreasing the synaptic localization of PP1 necessary for GluA2 Ser880 dephosphorylation.Decreased synaptic localization in addition to increased phosphatase inactivation could be important steps involved in the sustained activation of PKCα and phosphorylation of GluA2, events that are attenuated with apocynin by decreasing NADPH oxidasemediated oxidative stress during OGD/R.A third possibility for the mechanism in which OGD/Rinduced NADPH oxidase activity mediates PKCα activation involves enhancing intracellular Ca 2+ concentrations.PKCα activity is positively regulated by increases in Ca 2+ [39], therefore any enhancement in Ca 2+ concentrations will increase PKCα activity.Oxidative stress and NADPH oxidase activity has been linked to effecting Ca 2+ levels through NMDARs [14], phospholipase C-dependent release of intracellular Ca 2+ stores [18], mitochondrial Ca 2+ release [13], [19] as well as the opening of cation channels [20].Therefore, inhibition of NADPH oxidase activity during OGD/R could potentially accomplish the dampening of PKCα activity not only through the maintenance of PP1 and PP2A phosphatase activity, but also through the attenuation of oxidative stressenhanced rises in Ca 2+ entry and intracellular Ca 2+ release necessary for PKCα activation.
Taken together, this study demonstrates the effect of OGD/R-induced NADPH oxidase-mediated oxidative stress on GluA2 Ser880 phosphorylation and subsequent effect on membrane scaffolding.Though these data provide a novel mechanism for the OGD/R-induced loss of GluA2 surface proteins, further studies will be necessary to fully characterize the upstream signaling pathways involved in mediating PKCα activation and the resultant increase in GluA2 Ser880 phosphorylation.

Figure 1 :
Figure 1: OGD/R exposure of adult rat hippocampal slices promotes the Ser880 phosphorylation of GluA2.Representative Western blot (A) from 3 independent experiments of hippocampal slices illustrates the time course of GluR2 Ser880 phosphorylation.Quantification (B) of GluR2 Ser880 phosphorylation levels expressed as a ratio of phospho-GluR2(Ser880) to total GluA2 protein levels expressed as fold change over control ± S.D from three separate experiments.(asterisk * in (B) indicates a p <0.05 from normoxic controls; ANOVA with post hoc Bonferroni test).

Figure 2 :
Figure 2: OGD/R promotes the association of PICK1 with GluA2.Lysates were prepared from hippocampal slices subjected to time matched normoxia, OGD alone, or OGD/R and immunoprecipitated with a PICK1 antibody (A).Also shown in (A) is the GluA2 immunoreactive band resulting from the supernatant following immunoprecipitation of PICK1, in addition to total levels of both PICK1 and GluA2.Quantification (B) of the PICK1-GluA2 immunocomplex band expressed as fold change over control ± S.D from three separate experiments.(asterisk * in (B) indicates a p <0.01 from normoxic controls; ANOVA with post hoc Bonferroni test).

Figure 3 :
Figure 3: The OGD/R-induced increase in activated PKCα associated with PICK1 and rise in Ser880 phosphorylation of GluA2 is blunted with inhibition of NADPH oxidase.Representative Western blot (A) from 3 independent experiments of hippocampal slices illustrates the pattern of PKCα (phospho-Thr497)-PICK1 association of slices exposed to OGD/R in the presence of vehicle (1:1000 DMSO) or apocynin (30 μM) pre-treatment.(C) Representative Western blot of the OGD/R-induced increase in GluA2 Ser880 phosphorylation.Quantification (B) of the density of the phospho-PK-Cα (Thr497)-PICK1 immunoreactive band expressed as fold change over control.Quantification (D) of GluA2 Ser880 phosphorylation levels expressed as a ratio of phospho-GluA2 (Ser880) to total GluA2 protein levels.Data represent fold change over control ± S.D from three separate experiments.(asterisk * in (B) and (D) indicates a p <0.05 from normoxic controls; ANOVA with post hoc Bonferroni test).