α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Subtype Glutamate Receptor (AMPAR) Endocytosis Is Essential for N-Methyl-D-aspartate-induced Neuronal Apoptosis

Abstract

Excessive activation of the N-methyl-d-aspartate subtype glutamate receptor (NMDAR) is thought to be involved in mediating programmed cell death (apoptosis) in numerous central nervous diseases. However, the underlying mechanisms remain unknown. We report here that stimulation of NMDARs activates intracellular signaling cascades leading to apoptosis and facilitates clathrin-dependent endocytosis of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors (AMPARs). Both broad spectrum inhibitors of clathrin-dependent endocytotic processes and a specific inhibitor of AMPAR endocytosis selectively inhibit NMDA-induced apoptosis without affecting apoptosis produced by staurosporine. These results demonstrate that clathrin-dependent endocytosis of AMPARs is an essential step in NMDAR-mediated neuronal apoptosis. Our study not only identifies a previously unsuspected step in NMDA-induced apoptosis but also demonstrates that AMPAR endocytosis, in addition to attenuating synaptic strength as previously demonstrated in models of synaptic plasticity, may play a critical role in mediating other important intracellular pathways. Excessive activation of the N-methyl-d-aspartate subtype glutamate receptor (NMDAR) is thought to be involved in mediating programmed cell death (apoptosis) in numerous central nervous diseases. However, the underlying mechanisms remain unknown. We report here that stimulation of NMDARs activates intracellular signaling cascades leading to apoptosis and facilitates clathrin-dependent endocytosis of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors (AMPARs). Both broad spectrum inhibitors of clathrin-dependent endocytotic processes and a specific inhibitor of AMPAR endocytosis selectively inhibit NMDA-induced apoptosis without affecting apoptosis produced by staurosporine. These results demonstrate that clathrin-dependent endocytosis of AMPARs is an essential step in NMDAR-mediated neuronal apoptosis. Our study not only identifies a previously unsuspected step in NMDA-induced apoptosis but also demonstrates that AMPAR endocytosis, in addition to attenuating synaptic strength as previously demonstrated in models of synaptic plasticity, may play a critical role in mediating other important intracellular pathways. Neuronal apoptosis induced by activation of NMDARs 1The abbreviations used are: NMDA, N-methyl-d-aspartate; NMDAR, NMDA subtype glutamate receptor; AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor; PI, propidium iodide; ELISA, enzyme-linked immunosorbent assay; myr-Dyn, dynamin-derived, myristoylated peptide; STS, staurosporine; APV, 2-amino-5-phosphonovalerate; DNQX, 6,7-dinitroquinoxaline-2,3-dione.1The abbreviations used are: NMDA, N-methyl-d-aspartate; NMDAR, NMDA subtype glutamate receptor; AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptor; PI, propidium iodide; ELISA, enzyme-linked immunosorbent assay; myr-Dyn, dynamin-derived, myristoylated peptide; STS, staurosporine; APV, 2-amino-5-phosphonovalerate; DNQX, 6,7-dinitroquinoxaline-2,3-dione. has been postulated to underlie the loss of neurons and neuronal function in many central nervous system disorders (1Mattson M.P. Nat. Rev. Mol. Cell. Biol. 2000; 1: 120-129Crossref PubMed Scopus (1253) Google Scholar, 2Graham S.H. Chen J. J. Cereb. Blood Flow Metab. 2001; 21: 99-109Crossref PubMed Scopus (439) Google Scholar, 3Yu S.P. Canzoniero L.M. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (332) Google Scholar, 4Nicotera P. Lipton S.A. J. Cereb. Blood Flow Metab. 1999; 19: 583-591Crossref PubMed Scopus (142) Google Scholar, 5Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1333) Google Scholar). However, the mechanisms linking NMDAR activation to neuronal apoptosis remain unclear, although excessive calcium influx has been proposed as a primary underlying processes (1Mattson M.P. Nat. Rev. Mol. Cell. Biol. 2000; 1: 120-129Crossref PubMed Scopus (1253) Google Scholar, 3Yu S.P. Canzoniero L.M. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (332) Google Scholar, 4Nicotera P. Lipton S.A. J. Cereb. Blood Flow Metab. 1999; 19: 583-591Crossref PubMed Scopus (142) Google Scholar). Interestingly, several recent studies have indicated that NMDAR activation can also lead to significant facilitation of clathrin-mediated endocytosis of AMPARs, leading to long term depression of AMPAR-mediated synaptic transmission (6Man H.Y. Ju W. Ahmadian G. Wang Y.T. Cell. Mol. Life Sci. 2000; 57: 1526-1534Crossref PubMed Scopus (64) Google Scholar, 7Malinow R. Malenka R.C. Annu. Rev. Neurosci. 2002; 25: 103-126Crossref PubMed Scopus (2047) Google Scholar). Given the evidence that endocytosis of EGF receptors, in addition to attenuating receptor activation on the plasma membrane, is essential to some signaling pathways mediated by EGF receptors (8Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (822) Google Scholar), we set out to investigate whether the stimulated endocytosis of AMPARs constitutes an essential step in the signaling pathway leading to NMDA-induced neuronal apoptosis. Primary Culture of Hippocampal Neurons—Hippocampal cultures were prepared from embryonic E18 Sprague-Dawley rats and grown in culture medium (Neurobasal medium containing B-27 supplement and 0.5 mm glutamine (Invitrogen)). The medium from mature 14-day in vitro neurons was removed and replaced with 100 μm NMDA plus 10 μm glycine in extracellular solution (10 mm HEPES, pH 7.35, 140 mm NaCl, 25 mm glucose, 5.4 mm KCl, 1.3 mm CaCl2, osmolarity: 310–320 mosm) for 1 h at 37 °C prior to restoring neurons to the culture medium. Twenty-four hours after NMDA/glycine application, neurons were processed using cell death assays. NMDA-induced [Ca2+]i responses were evoked and measured using methods described previously (9Wang Y.T. Salter M.W. Nature. 1994; 369: 233-235Crossref PubMed Scopus (594) Google Scholar). Apoptosis Assays—NMDA-induced apoptosis was quantified using either a Cell Death Detection Elisa Plus Kit (Roche Applied Sciences) or using TdT-mediated addition of biotinylated 11-dUTP to the free 3′-OH ends of DNA (Chemicon). Absorbance readings for both assays were determined using a spectrophotometric microplate reader. Data analysis and expression relative to appropriate controls were performed according to the manufacturer's instructions for each kit. Propidium Iodide (PI) Staining of Nuclei—After the induction of apoptosis, cells were fixed with 4% paraformaldehyde, 4% sucrose for 10 min followed by ice-cold acetone for 1 min, and then stained with 20 mg/ml PI in Dulbecco's phosphate-buffered saline for 30 min and viewed with a Leica fluorescence microscope to identify condensed nuclei. Cells with condensed nuclei were counted as apoptotic, and the percentage of apoptotic cells to the total number of cells was calculated to give a semiquantitative analysis, expressed as percentage of apoptosis. Peptide Treatments—Synthetic peptides, GluR23Y or GluR23A, were incubated with a carrier protein (Pep-1) (10Morris M.C. Depollier J. Mery J. Heitz F. Divita G. Nat. Biotechnol. 2001; 19: 1173-1176Crossref PubMed Scopus (874) Google Scholar) at a ratio of 1:20 in Dulbecco's modified Eagle's medium (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2 for 30 min to allow the formation of GluR2/Pep-1 complexes. Hippocampal neurons (12–14 days in vitro) were then overlaid with the preformed complex to reach a final GluR2 peptide concentration of 1 μm and further incubated for 1 h before experiments commenced. ELISA Cell-surface Receptor Assay—Quantification of cell-surface AMPA or NMDARs was performed by a colorimetric cell-ELISA assay essentially as described previously (11Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), using monoclonal antibodies against the extracellular domains of GluR2 or GluR1 or NR1 (Chemicon, 1 μg/ml). Data are expressed as absorbance measurement under non-permeabilized conditions/absorbance measurement under permeabilized conditions. Transferrin Receptor Endocytosis Assay—Hippocampal neurons were incubated with 2 mg/ml Alexa-A488-conjugated transferrin (Molecular Probes) for 30 min at 37 °C in the presence or absence of endocytosis inhibitors. Internalized receptors were then visualized with a Leica fluorescence microscope. NMDA treatment (100 μm plus 10 μm glycine, 1 h) induced a time-dependent increase in caspase-3 activity, a biochemical indicator of neuronal apoptosis (12Budd S.L. Tenneti L. Lishnak T. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6161-6166Crossref PubMed Scopus (248) Google Scholar), as detected by ELISA assay of DEVD-p-nitroanilide cleavage (Fig. 1A). The increase peaked 12–24 h after the treatment, at which time the majority of neurons were either dying or dead, exhibiting the hallmarks of apoptotic cell death, including DNA laddering as demonstrated by gel electrophoresis of extracted DNA (Fig. 1B), and nuclear condensation with disintegrating processes shown by nucleus staining with PI (Fig. 1C) or the intercalating DNA dye, Hoechst 33258 (bisbenzimide, data not shown). The degree of neuronal apoptosis was also quantified by measuring internucleosomal cleavage of DNA with both 11-dUTP (Fig. 1E) and histone biotinylation assays (Fig. 1F). Furthermore, the NMDA-induced apoptosis was a result of specific activation of NMDARs, as it was fully blocked by the NMDAR antagonist, APV (50 μm; Fig. 1D), but not inhibited by blocking AMPARs with DNQX (10 μm; Fig. 1D). Therefore, in accordance with previous reports (12Budd S.L. Tenneti L. Lishnak T. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6161-6166Crossref PubMed Scopus (248) Google Scholar), the NMDA treatment paradigm used in the present study produced neuronal apoptosis. To investigate the role of NMDA-induced endocytosis in mediating neuronal apoptosis, we first examined the effect of hypertonic sucrose, a well characterized inhibitor of clathrin-dependent endocytosis that prevents the assembly of clathrin-coated pits (11Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 13Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (297) Google Scholar). As shown in Fig. 1E, sucrose treatment (400 mm) dramatically reduced NMDA-induced apoptosis. To more specifically target clathrin-dependent endocytosis, we employed a dynamin-derived, myristoylated peptide (myr-Dyn). This peptide is membrane-permeable and effectively inhibits clathrin-mediated endocytosis (14Marks B. McMahon H.T. Curr. Biol. 1998; 8: 740-749Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 15Nong Y. Huang Y.Q. Ju W. Kalia L.V. Ahmadian G. Wang Y.T. Salter M.W. Nature. 2003; 422: 302-307Crossref PubMed Scopus (352) Google Scholar) by blocking the recruitment of dynamin to clathrin-coated pits by amphiphysin (16Grabs D. Slepnev V.I. Songyang Z. David C. Lynch M. Cantley L.C. De Camilli P. J. Biol. Chem. 1997; 272: 13419-13425Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). We found that myr-Dyn (10 μm) was as effective as hypertonic sucrose in reducing NMDA-induced apoptosis (Fig. 1, E and F). In contrast, control Dyn peptides, both non-myristoylated (membrane-impermeable) Dyn (Dyn; Fig. 1E) and scrambled myr-Dyn (s-myr-Dyn; Fig. 1F), had little effect. To determine whether endocytosis is a general requirement for neuronal apoptosis, we next tested the effect of these inhibitors on a well characterized model of neuronal apoptosis induced by treating neurons with the kinase inhibitor, staurosporine (STS; 100 nm, 1 h) (12Budd S.L. Tenneti L. Lishnak T. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6161-6166Crossref PubMed Scopus (248) Google Scholar). As shown in Fig. 1E, both endocytosis inhibitors failed to significantly alter STS-induced neuronal apoptosis. These results demonstrate that a clathrin-dependent endocytotic process is necessary for NMDAR-mediated, but not STS-mediated, apoptosis. Ca2+ overload following NMDAR activation has been suspected as a primary causal factor leading to neuronal apoptosis (1Mattson M.P. Nat. Rev. Mol. Cell. Biol. 2000; 1: 120-129Crossref PubMed Scopus (1253) Google Scholar, 3Yu S.P. Canzoniero L.M. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (332) Google Scholar, 4Nicotera P. Lipton S.A. J. Cereb. Blood Flow Metab. 1999; 19: 583-591Crossref PubMed Scopus (142) Google Scholar). However, as summarized in Fig. 2A, sucrose at concentrations that inhibited endocytosis and apoptosis did not significantly alter NMDA-evoked transient [Ca2+]i responses measured with the Ca2+ dye, Fura-2. The fact that inhibition of endocytosis blocked NMDA-induced apoptosis without affecting its [Ca2+]i responses strongly suggests that intracellular increases in Ca2+ concentrations, although necessary (3Yu S.P. Canzoniero L.M. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (332) Google Scholar, 4Nicotera P. Lipton S.A. J. Cereb. Blood Flow Metab. 1999; 19: 583-591Crossref PubMed Scopus (142) Google Scholar), may not be sufficient to produce NMDA-induced apoptosis. Activation of certain forms of caspases, such as caspase-3 and caspase-7 (17Okamoto S.S. Li Z. Ju C. Scholzke M.N. Mathews E. Cui J. Salvesen G.S. Bossy-Wetzel E. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3974-3979Crossref PubMed Scopus (117) Google Scholar) (also see Fig. 1A) has been implicated in NMDA-induced neuronal apoptosis. We therefore investigated the effects of inhibiting endocytosis on NMDA dependent activation of caspase-3. NMDA treatment dramatically increased the level of the activated form of caspase-3, as demonstrated by Western blots using an antibody that specifically recognizes activated/cleaved caspase-3, and this effect was efficiently inhibited by the membrane-permeable myr-Dyn (Fig. 2B). The serine/threonine kinase Akt/PKB has previously been shown to be critically involved in protecting neurons from apoptotic cell death (18Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2215) Google Scholar), and inhibition of this kinase activity has previously been suspected of being involved in NMDAR-mediated apoptosis (19Chalecka-Franaszek E. Chuang D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8745-8750Crossref PubMed Scopus (499) Google Scholar). We therefore investigated whether the endocytosis process plays a critical role in the inhibition of Akt activity by determining the level of Akt phosphorylation at serine 473, a residue whose phosphorylation is required for full activation of Akt (20Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (967) Google Scholar). As shown in Fig. 2C, treatment of neurons with NMDA resulted in a significant reduction in Ser473-phosphorylated Akt and hence Akt activity without altering the levels of total Akt. This reduction in Akt activity was largely prevented by the inhibition of endocytosis with hypertonic sucrose. In striking contrast, sucrose treatment had no effect on the reduction of Akt phosphorylation following STS treatment (Fig. 2C). Thus, stimulated endocytosis appears to be an obligatory step that is downstream of rising [Ca2+]i and upstream of caspase activation and Akt inhibition in NMDA-induced neuronal apoptosis. Several recent studies in the synaptic plasticity field have demonstrated that NMDAR activation can selectively stimulate the clathrin-mediated endocytosis of AMPARs (6Man H.Y. Ju W. Ahmadian G. Wang Y.T. Cell. Mol. Life Sci. 2000; 57: 1526-1534Crossref PubMed Scopus (64) Google Scholar, 7Malinow R. Malenka R.C. Annu. Rev. Neurosci. 2002; 25: 103-126Crossref PubMed Scopus (2047) Google Scholar, 21Lin J.W. Ju W. Foster K. Lee S.H. Ahmadian G. Wyszynski M. Wang Y.T. Sheng M. Nat. Neurosci. 2000; 3: 1282-1290Crossref PubMed Scopus (404) Google Scholar, 22Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar, 23Beattie E.C. Carroll R.C. Yu X. Morishita W. Yasuda H. von Zastrow M. Malenka R.C. Nat. Neurosci. 2000; 3: 1291-1300Crossref PubMed Scopus (611) Google Scholar). Consistent with these previous studies, we observed a significant reduction of cell-surface AMPA, but not NMDA, receptors following the NMDA paradigm used here. Since the reduction in AMPARs was observed by measuring cell-surface expression of both the GluR2 subunit (Fig. 3, A and B) and the GluR1 subunit (64 ± 2 and 51 ± 1% of the total receptors on the cell surface for control and NMDA-treated neurons, respectively; n = 5, p < 0.001), it appears to be a decrease in total cell-surface AMPARs and not a replacement of GluR2-containing with GluR2-lacking receptors as a result of GluR2-dependent endocytosis. The AMPAR reduction was blocked by the endocytosis inhibitor myr-Dyn but not the control peptide, Dyn (Fig. 3A), indicating the involvement of facilitated clathrin-dependent endocytosis. We have demonstrated previously that stimulated AMPAR endocytosis requires the GluR2 subunit (11Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar), more specifically the short amino acid sequence between residues tyrosine 869 and glutamic acid 879 within the carboxyl-terminal region (21Lin J.W. Ju W. Foster K. Lee S.H. Ahmadian G. Wyszynski M. Wang Y.T. Sheng M. Nat. Neurosci. 2000; 3: 1282-1290Crossref PubMed Scopus (404) Google Scholar, 24Ahmadian G. Ju W. Liu L. Wyszynski M. Lee S.H. Dunah A.W. Taghibiglou C. Wang Y. Lu J. Wong T.P. Sheng M. Wang Y.T. EMBO J. 2004; 23: 1040-1050Crossref PubMed Scopus (233) Google Scholar). We have therefore reasoned that a short peptide, which comprises the required sequence YKEGYNVYGIE (termed GluR23Y), may function as a dominant inhibitor of NMDAR-mediated AMPAR endocytosis (24Ahmadian G. Ju W. Liu L. Wyszynski M. Lee S.H. Dunah A.W. Taghibiglou C. Wang Y. Lu J. Wong T.P. Sheng M. Wang Y.T. EMBO J. 2004; 23: 1040-1050Crossref PubMed Scopus (233) Google Scholar). As predicted, when the GluR23Y peptide (1 μm) was delivered into cultured neurons by mixing it with a carrier peptide (Pep-1) (10Morris M.C. Depollier J. Mery J. Heitz F. Divita G. Nat. Biotechnol. 2001; 19: 1173-1176Crossref PubMed Scopus (874) Google Scholar) 1 h prior to and during the NMDA treatment, the NMDA-induced reduction of cell-surface AMPARs was completely prevented (Fig. 3B). To be sure that the blockade by this peptide was not due to nonspecific effects on the endocytotic process, we examined its effect on transferrin receptor endocytosis, a well characterized clathrin-mediated receptor endocytosis (13Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (297) Google Scholar). As shown in Fig. 3C, internalization of fluorescently labeled transferrin as a result of transferrin receptor endocytosis was eliminated by hypertonic sucrose, but not by GluR23Y to these neurons 1 h prior to and during the transferrin Thus, GluR23Y not clathrin-mediated endocytosis. NMDA-induced apoptosis requires AMPAR endocytosis, that the GluR23Y peptide be effective in NMDA-induced apoptosis. Consistent with GluR23Y plus significantly reduced NMDA-induced apoptosis as quantified by the histone biotinylation assay (Fig. and by PI nuclear staining (Fig. and GluR23Y plus the control peptide which the tyrosine residues were replaced with (24Ahmadian G. Ju W. Liu L. Wyszynski M. Lee S.H. Dunah A.W. Taghibiglou C. Wang Y. Lu J. Wong T.P. Sheng M. Wang Y.T. EMBO J. 2004; 23: 1040-1050Crossref PubMed Scopus (233) Google had effect on NMDA-induced apoptosis (Fig. to the general blockade of the clathrin-mediated endocytotic process with either sucrose or myr-Dyn, with AMPAR endocytosis by GluR23Y did not alter STS-induced neuronal apoptosis (Fig. results evidence for an obligatory requirement for AMPAR endocytosis in mediating NMDA-induced neuronal apoptosis. In the present study we have found that clathrin-dependent AMPAR endocytosis is specifically required for but not apoptosis of neurons in primary endocytosis has no effect on NMDA-induced Ca2+ responses but prevents both NMDA-induced activation of caspase-3 and inhibition of Akt Since AMPAR endocytosis is to be an intracellular process (11Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 21Lin J.W. Ju W. Foster K. Lee S.H. Ahmadian G. Wyszynski M. Wang Y.T. Sheng M. Nat. Neurosci. 2000; 3: 1282-1290Crossref PubMed Scopus (404) Google Scholar, 23Beattie E.C. Carroll R.C. Yu X. Morishita W. Yasuda H. von Zastrow M. Malenka R.C. Nat. Neurosci. 2000; 3: 1291-1300Crossref PubMed Scopus (611) Google Scholar), it is that Ca2+ influx NMDARs may function as a leading to the facilitation of AMPAR endocytosis, which may in a neuronal apoptosis. Thus, the present study has AMPAR endocytosis as a critical between NMDA-induced overload and intracellular cascades leading to apoptosis. A of evidence has a central role for stimulated clathrin-dependent AMPAR endocytosis in reducing synaptic to the expression of certain forms of well characterized synaptic plasticity including long term depression in both (11Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, C. H. E.C. Carroll R.C. von Zastrow M. Malenka R.C. R.A. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and Y.T. Neuron. 2000; 25: Full Text Full Text PDF PubMed Scopus Google Scholar). The requirement for AMPAR endocytosis in NMDA-induced apoptosis demonstrated in the present study further suggests in addition to the decrease in synaptic AMPARs also play previously unsuspected in intracellular signaling pathways. The of NMDA-induced apoptosis is The mechanisms by which AMPARs to the of downstream apoptotic cascades remain to be However, to the cell apoptosis induced following stimulated endocytosis of A. M. J. R. Neuron. 2000; 28: Full Text Full Text PDF PubMed Scopus Google Scholar, L. P.J. Neuron. 2000; 28: Full Text Full Text PDF PubMed Scopus Google Scholar, Nat. Rev. Neurosci. 2001; PubMed Scopus Google Scholar) and activation of EGF protein kinase signaling by EGF receptors (8Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (822) Google Scholar), it is that AMPARs, in to NMDA may with some of the protein a signaling complex that the apoptotic the present study the first evidence for the involvement of stimulated AMPAR endocytosis in NMDA-induced neuronal apoptosis, a pathway in mediating cell apoptosis. NMDAR-mediated apoptosis is of the primary leading to the loss of neurons in both and and clathrin-dependent AMPAR endocytosis is a process in the control of of synaptic transmission at linking the endocytosis of AMPARs to neuronal apoptosis, study suggests the of the AMPAR endocytotic pathway may be for the of a of We and Li for the of some of the neuronal cultures and for