Direct interaction between GluR2 and GAPDH regulates AMPAR-mediated excitotoxicity
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 10 March 2012
Accepted: 26 April 2012
Published: 26 April 2012
Over-activation of AMPARs (α−amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors) is implicated in excitotoxic neuronal death associated with acute brain insults, such as ischemic stroke. However, the specific molecular mechanism by which AMPARs, especially the calcium-impermeable AMPARs, induce neuronal death remains poorly understood. Here we report the identification of a previously unrecognized molecular pathway involving a direct protein-protein interaction that underlies GluR2-containing AMPAR-mediated excitotoxicity. Agonist stimulation of AMPARs promotes GluR2/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) complex formation and subsequent internalization. Disruption of GluR2/GAPDH interaction by administration of an interfering peptide prevents AMPAR-mediated excitotoxicity and protects against damage induced by oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia.
Glutamate is the principal excitatory neurotransmitter in the brain and is involved in numerous physiological functions including neuronal circuit development, learning and memory . Glutamate-induced neurotoxicity is implicated in neuropathological disorders such as stroke and epilepsy . The effects of glutamate are mediated via two major subfamilies of ligand-gated ion channels: NMDAR (N-methyl-D-aspartate receptor) and AMPAR . AMPAR mediates fast synaptic transmission at excitatory synapses, while NMDAR is critical in producing a number of different forms of synaptic plasticity . In neurons, mature AMPA receptors are found as tetramers consisting of various combinations of GluR1 to GluR4 subunits , each of which has the same topology: three transmembrane domains and one membrain re-entrant loop. All subunits are permeable to both Na+ and Ca2+ ions with the exception of GluR2, which is uniquely impermeable to Ca2+. The majority of AMPA receptors in vivo contain GluR2 subunits whose ion selectivity is dominant over other subunits .
The accumulation of glutamate, which occurs immediately after ischemia, results in excessive stimulation of glutamate receptors and leads to neurotoxicity [6, 7]. NMDAR-mediated neurotoxicity is dependent upon extracellular Ca2+ and is likely mediated by Ca2+ influx directly through receptor-gated ion channels [6, 7]. AMPAR is also tightly associated with a selective pattern of neuronal loss in certain brain areas following both global and focal ischemia [8–20]. Similar to what is reported for NMDAR, excitotoxicity mediated by AMPAR lacking the GluR2 subunit is thought to be dependent on ion influx (Ca2+, Zn2+) through AMPAR channels following agonist stimulation [19–21]. However, as most native AMPARs in the hippocampus contain the GluR2 subunit and therefore are likely impermeable to Ca2+[22–26], it is still unclear how activation of the GluR2-containing AMPAR leads to neuronal cell death.
Protein-protein interactions with the AMPAR have been reported to affect function of AMPAR, among which the best characterized ones, such as GRIP (glutamate receptor interacting protein), ABP (AMPAR-binding protein), SAP97 (synapse-associated protein-97), PICK1 (protein interacting with C kinase-1), stargazin, NSF (N-ethylmaleimide-sensitive factor) and AP2 (adaptor protein-2) [27–34], bind to the intracellular carboxyl terminus of AMPAR. They regulate AMPAR function in a variety of ways, including modulation of AMPAR subcellular localization, clustering and/or trafficking. Recent studies have demonstrated that NARP (neuronal activity-regulated pentraxin) and N-cadherin interact with the amino terminus (NT) of AMPAR subunits and play an important role in AMPAR clustering  as well as dendritic spine formation . In the present study, we have identified a new AMPAR-interacting partner, GAPDH. We show that secreted GAPDH binds specifically to the extracellular NT domain of the GluR2 subunit, a process which is promoted by AMPAR activation. Disruption of GluR2/GAPDH interaction prevents AMPAR-mediated excitotoxicity and protects against damage in OGD model.
GluR2 subunit directly interacts with GAPDH via its Y142-K172 region of N-terminus
Before conducting further experiments, we examined whether GluR2/GAPDH complex exists in vivo. As shown in Figure 1C, the GluR2 antibody was able to co-immunoprecipitate (Co-IP) GAPDH from solubilized proteins extracted from rat hippocampal tissues confirming the in vivo association between GluR2 and GAPDH. In order to smooth the way for the following functional studies, three GluR2NT GST-fusion proteins (GluR2NT1: V22-S271, GluR2NT2: K272-I421, GluR2NT3: L422-E545) were constructed (Figure 1D) and utilized in affinity purification experiments to delineate the region (s) of GluR2NT involved in the interaction with GAPDH. As shown in Figure 1E, GST-GluR2NT1, but not GST-GluR2NT2, GST-GluR2NT3 or GST alone, precipitated GAPDH indicating that the GluR2 subunit interacts with GAPDH through its NT region V22-S271. A series of truncations of the GluR2NT1 region were then created to map the site that interacts with GAPDH (Figure 1D). As shown in Figure 1F and 1G, GST-GluR2NT1-3 (H122-K172) and GST-GluR2NT1-3–2 (Y142-K172) were able to precipitate GAPDH from rat hippocampal tissues.
While these results suggested the existence of the GluR2/GAPDH complex, it did not clarify whether this GluR2/GAPDH complex was formed through either a direct interaction or was mediated indirectly by other accessory binding proteins. Therefore we performed in vitro binding assays to examine whether GAPDH and the GluR2 subunit directly interact with each other. As shown in Figure 1H, in vitro translated [35 S]-GAPDH probe bound with GST-GluR2NT1 but not with GST-GluR2NT2, GST-GluR2NT3 or GST alone, indicating the specificity of the direct protein-protein interaction between GAPDH and GluR2NT1. Consistent with the results from affinity purification experiments, the in vitro translated [35 S]-GAPDH probe only hybridized with GST-GluR2NT1-3 and GST-GluR2NT1-3–2, (Figure 1I, J). Together, these data provided in vitro evidence that GAPDH forms a direct protein-protein interaction with the GluR2 subunit through the Y142-K172 region of the GluR2NT.
Agonist-facilitated GluR2/GAPDH complex formation occurs extracellularly
Furthermore, we examined the effect of the AMPAR activation on the formation of GluR2/GAPDH complex. By conducting Co-IP experiments, we found that AMPAR activation with either 100 μM glutamate in HEK-293 T cells expressing GluR1/2 subunits or 100 μM kainic acid (KA) in hippocampal neurons facilitated the GluR2/GAPDH complex formation by 75 ± 18% and 58 ± 11% (mean ± SEM, n = 3), respectively (Figure 2D, E; top panels). In each Co-IP experiment, 500 μg of protein were incubated in the presence of primary antibodies anti-GluR2 or rabbit IgG, and 50 μg of extracted protein was used as positive control. The level of directly immunoprecipitated GluR2 subunit was not significantly altered by the agonist stimulation (Figure 2D, E; bottom panels). If the GluR2NT1-3–2 region is essential for GluR2 to interact with GAPDH, application of the peptide encoding GluR2NT1-3–2 would disrupt the GluR2/GAPDH interaction by competing with GluR2 for GAPDH. As expected, pre-incubation of the GluR2NT1-3–2 peptide (10 μM, 1 hour), but not the scrambled GluR2NT1-3–2 peptide (GluR2NT1-3-2Scram), significantly inhibited the agonist-induced increase of the GluR2/GAPDH complex formation in transfected HEK-293 T cells (Figure 2D, 65 ± 8% decrease; mean ± SE, n = 3) and in hippocampal neurons (Figure 2E, 46 ± 6% decrease; mean ± SE, n = 3). The fact that extracellular application of the interfering GluR2NT1-3–2 peptide was able to disrupt the GluR2/GAPDH interaction further supports the notion that the GluR2/GAPDH complex formation occurs extracellularly.
Disruption of GluR2/GAPDH interaction inhibits AMPAR-mediated excitotoxicity
AMPAR-mediated toxicity is often considered a contributing, if not an underlying, causative factor in ischemia, which deprives brain cells of glucose and oxygen, causing irreversible brain damage within minutes. Cells in ischemic brain tissue undergo a number of changes: they rapidly lose their energy supplies, their membranes become depolarized, calcium loads are increased, reactive oxygen types are produced and excitotoxic effects are found. These biochemical changes are followed by irreversible changes to cellular structures and cell death. The oxygen glucose deprivation (OGD) cell lesion model represents a valid simulation of the conditions in brain ischemia [45, 46]. Therefore, we assessed the effectiveness of the GluR2NT1-3–2 peptide to rescue cells from neurotoxic stress in the OGD model to verify the implication of the GluR2/GAPDH interaction in ischemia. As shown in Figure 4B, the GluR2NT1-3–2 peptide pretreatment (10 μM, 1 hour) was able to significantly attenuate OGD-induced cell death (30.4% ± 9.5%) in the presence of 10 μM MK-801 and 2 μM nimodipine.
In order to further confirm the role of GAPDH in the AMPAR-mediated cell death, GAPDH siRNA was transfected into HEK-293 T cells to block the expression of GAPDH, but not the expression of GluR2 (Figure 4C). As shown in Figure 4D-E, AMPAR-mediated cell death was significantly attenuated in the presence of GAPDH siRNA. Together, these data suggest that the GluR2/GAPDH interaction may play a critical role in the GluR2-contaning AMPAR-mediated cell death.
Activation of AMPAR induces AMPAR/GAPDH complex internalization through the GluR2/GAPDH interaction
To further investigate whether the observed GAPDH internalization is dependent on the GluR2 internalization, we tested whether blockade of GluR2 endocytosis will inhibit GAPDH internalization. Previous studies demonstrated that GluR2 endocytosis is dynamin-dependent and that the expression of the dominant-negative dynamin mutant (K44E) was able to block the GluR2 internalization [47, 49]. Thus, after confirming the ability of the K44E mutant to block the GluR2 internalization (Figure 5E), we examined whether the K44E mutant affected cell surface-associated GAPDH internalization in HEK-293 T cells expressing GluR1/2 subunits. As shown in Figure 5F, the K44E mutant significantly inhibited glutamate-induced cell surface-associated GAPDH internalization, indicating that GAPDH internalized through a dynamin-dependent pathway and further confirmed that GAPDH was co-internalize with the GluR2 subunit. Moreover, the K44E mutant also attenuated glutamate-induced cell death in HEK-293 T cells expressing GluR1/2 subunits (Figure 5G), indicating that GluR2/GAPDH complex internalization may play an important role in the GluR2-containing AMPAR-mediated cell death.
AMPAR-mediated excitotoxicity has been implicated in the pathogenesis of neuronal loss associated with a number of brain disorders, including transient forebrain ischemia [8–20]. However, the underlying mechanisms remain unclear. An uncontrollable rise in intracellular Ca2+ and Zn2+, with subsequent activation of diverse downstream cell death signals has been one of the most prominent hypotheses to explain excitotoxic neuronal death [19, 20, 51–55]. Although GluR2-containing AMPARs are calcium impermeable, recent studies have suggested that selective reductions in the expression of GluR2, resulting in an increase in Ca2 + −permeable AMPA receptors, have been associated with an increased vulnerability of neurons to ischemic injury [16, 56–61]. Although the mechanisms involved are not fully understood, it has been suggested that GluR2 internalization may enhance the Ca2+-influx that results in neurotoxicity, either through newly synthesized Ca2+-permeable AMPARs  or by activation of a caspase-dependent apoptotic pathway . Consistent with previous studies, our data has shown that agonist stimulation of AMPAR results in the internalization of GluR2 and promotes extracellular GAPDH internalization via a GluR2/GAPDH coupling-dependent process. This is the first evidence showing that the N-terminal of the GluR2 subunit plays an important role in AMPA receptor-mediated excitotoxicity through regulating AMPAR trafficking. Many studies have shown that agonist-induced GluR2 internalization is a dynamin-dependent process [47, 49]. The observations of our study that mutant dynamin abolishes both GluR2 and GAPDH internalization and the inability of GAPDH to internalize in cells lacking GluR2 suggest that GAPDH internalization is a passive process facilitated by the GluR2/GAPDH interaction and mediated by GluR2 internalization.
Given the fact that GAPDH interacts with the extracellular NT of GluR2, it is likely that the GluR2/GAPDH protein complex may be in an endocytosed vesicle following the agonist-induced internalization. On this basis it would be logical to further ask how the GluR2/GAPDH complex gets out of the vesicle and promotes excitotoxic neuronal death. There are many possibilities for this question. First, the complex may be transported to the nucleus via a retrograde vesicle transport mechanism leading to the fusion of the vesicle with ER or nuclear membranes or via mechanisms recently proposed for the nuclear translocation of another plasma membrane receptor, the EGF receptor [63, 64]. Second, the GluR2/GAPDH complex formation in the vesicle may lead to the activation of lysosome in the vesicle that breaks the vesicle and release the GluR2/GAPDH into the cytoplasm.
The possible mechanisms that underlie this GluR2/GAPDH related cell death is particularly interesting. It is somewhat surprising to find that the AMPAR-mediated cell death involves GAPDH, a key enzyme involved in glycolysis with a ubiquitous intracellular distribution. However, additional roles for GAPDH have been discovered recently, including membrane fusion/transport, binding to low molecular weight G proteins, regulation of the cytoskeleton, accumulation of glutamate into presynaptic vesicles, and apoptosis [65–71]. Recent studies have shown that GAPDH binds to Siah1 and triggers apoptosis . Moreover GAPDH has also been reported to interact with p53 , a tumor suppressor and transcription factor that has been implicated in glutamate-mediated excitotoxicity [73–75]. Numerous evidence show that activation of p53 can trigger apoptosis (for reviews, see ) under conditions of cellular stress mediated by phosphorylation or acetylation of p53 . Whether Siah1, p53 or other molecules are involved in GluR2/GAPDH-related cell death pathway requires much more additional work for a better understanding of the detailed molecular mechanisms.
Stroke is the second leading cause of death worldwide yet there are very few effective pharmacological treatments for patients suffering ischemic stroke. Thrombolytics such as alteplase and tenecteplase have been a significant advance in the treatment of ischemic stroke. However, thrombolytics must be given soon after a stroke to be effective (within 3 hours of ischemic episode). This short time frame has limited their use in many situations. There continues to be a significant unmet need for acute pharmacological treatments beyond thrombolytics. Advances in recent years include hypothermia [78–80], oxygen therapy , stem cell transplantation  and cerebral plasticity stimulation (trophic factor) strategies . These novel techniques are intriguing, but will require further well-designed prospective trials to assess clinical feasibility, safety, and efficacy . Another approach that has received considerable attention is agents that inhibit ischemia-induced excitotoxicity though directly blocking glutamate receptors. However, all have failed at various stages of development for a variety of reasons. One of the main drawbacks of the glutamate receptor antagonists is that they block normal excitatory neurotransmission necessary for maintaining basic brain functions. For this reason, much research has been directed at identifying drugs and peptides that may be able to selectively target protein-protein interactions that have more narrow function than a certain neurotransmitter receptor. In the present study, we have shown that administration of the interfering GluR2NT1-3–2 peptide to interrupt the GluR2/GAPDH interaction significantly mitigates neuronal cell death in a cell model of ischemia, revealing a previously unappreciated signaling pathway underlying AMPAR-mediated excitotoxicity and it may provide a new avenue for the development of a complementary therapeutics in the treatment of neuropathological disorders, such as stroke and epilepsy.
Materials and methods
Cell culture and transient transfection
HEK293 T cells were cultured in α-MEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and maintained in incubators at 37°C, 5% CO2. HEK293T cells were transiently transfected with plasmid constructs and/or siRNA using lipofectamine 2000 reagents (Invitrogen). Cells were harvested 48 hours post transfection.
Primary hippocampal neuron culture and OGD treatment
Primary cultures from hippocampus were prepared from fetal Wistar rats (embryonic day 17–19) on Cell + (Sarstedt) culture dishes as previously described [85–87]. The cultures were used for experiments on 12–15 days after plating. Hippocampal cultures were pretreated GluR2NT1-3–2 peptides prior to kainic acid treatment. OGD treatment was performed in the presence of MK-801 and nimodipine as previously described .
GST fusion proteins
To construct GST-fusion proteins encoding truncated GluR2 and GAPDH, cDNA fragments were amplified by using PCR method with specific primers. Except where specified, all 5′ and 3′ oligonucleotides incorporated BamH1 site (GGATCC) and Xho1 sites (CTCGAG), respectively, to facilitate subcloning into vector pGEX-4T3 (for GST-fusion protein construction). GST-fusion proteins were prepared from bacterial lysates with Glutathione Sepharose 4B beads as described by the manufacturer (Amersham). To confirm appropriate splice fusion and the absence of spurious PCR generated nucleotide errors, all constructs were resequenced.
Protein affinity purification, in vitro binding, co-immunoprecipitation and western blot
Protein affinity purification, in vitro binding, co-immunoprecipitation and Western blot analyses were performed as previously described [85–87]. Antibodies used for immunoprecipitation, Western blots and cell surface ELISA assays include GAPDH (polyclonal from Abcam, monoclonal from Chemicon), GluR2 (Western blots: Chemicon; immunoprecipitation: Upstate), and α-tubulin (monoclonal, Sigma-Aldrich).
HEK-293 T cells transfected with plasmid constructs were treated with 100 μM glutamate or extracellular solution (ECS) before fixing in 4% (W/V) paraformaldehyde for 10 minutes in the absence (non-permeabilized conditions) or presence (permeabilized conditions) of 1% (V/V) Triton X-100. Cells were incubated in 1% (W/V) glycine for 10 minutes at 4°C to recover from the fixing. Cells were then incubated with specific primary antibodies for the purpose of labeling the receptors or proteins on the cell surface under non-permeabilized conditions or the entire receptor pool under permeabilized conditions. After incubation with corresponding HRP-conjugated secondary antibodies (Sigma-Aldrich), the HRP substrate o-phenylenediamine (Sigma-Aldrich) was added to produce a color reaction that was stopped with the equal volume of 3 N HCl. Fluorescence intensity in each well was measured with a plate reader (Victor3; PerkinElmer). The cell surface expression of HA-GluR2 after pre-treatment with glutamate was presented as the ratio of colorimetric readings under non-permeabilized conditions to those under permeabilized conditions, and then normalized to their respective control groups (pretreated with ECS). Afterwards, cells were scrapped from the dishes, and the protein concentration of each dish was measured. The results of cell surface expression of receptors or proteins were calibrated by the protein concentration of each well. Analysis was done using at least 9 separate wells in each group. Cell ELISA using primary hippocampal neurons was performed identically with assays using HEK-293 T cells, with the exception that the anti-GluR2 antibody (MAB397; Chemicon) was used as primary antibody instead of anti-HA.
Quantification of AMPAR-mediated excitotoxicity
HEK-293 T cells transfected with GluR1/2 subunits were exposed to 300 μM glutamate/25 μM cyclothiazide at 37°C for 24 hour. Cells were allowed to recover for 24 hours at 37°C. To quantify AMPAR-mediated cell death, culture medium was replaced by extracellular solution containing 50 μg/ml of propidium iodide (PI) (Invitrogen, Carlsbad, CA). After 30 minutes incubation at 37°C, fluorescence intensity in each well was measured with a plate reader (Victor3; PerkinElmer, Waltham, MA). The fraction of dead cells was normalized to the total cell number. Primary hippocampal neurons were exposed to 100 μM KA/25 μM cyclothiazide in the presence of NMDAR and Ca2+ channel antagonists (10 μM MK-801 and 2 μM nimodipine, respectively) at 37°C for 1 hour.
For cell surface biotinylation, cells were rinsed four times with ice-cold PBS2+ (PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2) after treatment, and incubated twice with 1.0 mg/ml sulfo-NHS-LC-biotin (Pierce, Rockford, IL) for 20 minutes at 4°C. Non-reactive biotin was quenched by 20 minutes incubation at 4°C in ice-cold PBS2+ and 0.1 M glycine. Cells were solubilized in RIPA buffer (10 mM Tris, ph7.4, 150 mM NaCl, 1.0 mM EDTA, 0.1% (W/V) SDS, 1.0% (V/V) Triton X-100 and 1.0% (V/V) Sodium deoxycholate) containing protease inhibitors (1.0 mM PMSF and 1.0 μg/ml protease cocktail). Biotinylated and non-biotinylated proteins were separated from equal amounts of cellular protein by incubation with 50 μl of 50% slurry of immobilized streptavidin-conjugated beads (Pierce, Rockford, IL) overnight with constant mixing at 4°C. Unbound proteins (supernatant) were saved for later co-immunoprecipitation experiment. Proteins bound to streptavidin beads were eluted in biotin elution buffer. Biotinylated and non-biotinylated samples were applied to protein A/G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for co-immunoprecipitation.
We gratefully acknowledge Mr. Brian Vukusic for technical assistance. We thank Dr. Paul Talyor for help with the MS experiments and analysis. We thank Dr. Qi Wan for providing the OGD cell model. F.L. is a recipient of Career Investigator Award of the Heart and Stroke Foundation of Canada. S.P.L. is a recipient of post-doctoral fellowship of the Heart and Stroke Foundation of Canada. The work is supported by operating grant from Heart and Stroke Foundation of Canada (F. L.).
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