Brain-derived Neurotrophic Factor Regulates Surface Expression of α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic Acid Receptors by Enhancing the N-Ethylmaleimide-sensitive Factor/GluR2 Interaction in Developing Neocortical Neurons
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m202158200
ISSN1083-351X
AutoresMako Narisawa‐Saito, Yuriko Iwakura, Meiko Kawamura, Kazuaki Araki, Shunji Kozaki, Nobuyuki Takei, Hiroyuki Nawa,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoIn hippocampal neurons, the exocytotic process of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation ofN-methyl-d-aspartate channels and its resultant Ca2+ influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [3H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca2+ and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, andN-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 withN-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction. In hippocampal neurons, the exocytotic process of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation ofN-methyl-d-aspartate channels and its resultant Ca2+ influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [3H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca2+ and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, andN-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 withN-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction. The neurotrophins, nerve growth factor, brain-derived neurotrophic factor (BDNF), 1The abbreviations used are: BDNF, brain-derived neurotrophic factor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid; NMDA, N-methyl-d-aspartate; NEM, N-ethylmaleimide; αCAMKII, α Ca2+/calmodulin-dependent kinase II; NSF, N-ethylmaleimide-sensitive factor; HEK, human embryonic kidney; PKC, protein kinase C; aa, amino acid(s); FITC, fluorescein isothiocyanate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; ATPγS, adenosine 5′-O-(thiotriphosphate). neurotrophin-3, and neurotrophin-4/5, are implicated as important regulators of synaptic development and plasticity in both central and peripheral nervous systems. In particular, activity-dependent synthesis and release of neurotrophins are suggested to be key modulators of neuronal development and synaptic plasticity. BDNF can exert strong neurotrophic activities on immature brain neurons with a subchronic time scale of days. BDNF enhances the dendritic growth, induces synaptogenesis (1McAllister A.K. Katz L.C. Lo D.C. Neuron. 1997; 18: 767-778Google Scholar, 2Lom B. Cohen-Cory S. J. Neurosci. 1999; 19: 9928-9938Google Scholar), and induces various neurochemical phenotypes of developing excitatory and inhibitory neurons (3Xiong H. Yamada K. Han D. Nabeshima T. Enikolopov G. Carnahan J. Nawa H. Eur. J. Neurosci. 1999; 11: 1567-1576Google Scholar, 4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar, 5Mizuno K. Carnahan J. Nawa H. Dev. Biol. 1994; 165: 243-256Google Scholar, 6Sato M. Suzuki K. Nakanishi S. J. Neurosci. 2001; 21: 3797-3805Google Scholar). The biological activity of neurotrophins on the induction of postsynaptic components and/or development of postsynaptic neurons needs to be evaluated, and their underlying molecular mechanisms remain to be characterized in various types of neurons. In addition to such chronic activity of BDNF for days, application of BDNF to cultured neurons enhances excitatory glutamatergic synaptic transmission with increased efficacy of glutamate release from the presynaptic neuron (7Takei N. Numakawa T. Kozaki S. Sakai N. Endo Y. Takahashi M. Hatanaka H. J. Biol. Chem. 1998; 273: 27620-27624Google Scholar, 8Schinder A.F. Berninger B. Poo M. Neuron. 2000; 25: 151-163Google Scholar, 9Jovanovic J.N. Czernik A.J. Fienberg A.A. Greengard P. Sihra T.S. Nat. Neurosci. 2000; 3: 323-329Google Scholar, 10Xu B. Gottschalk W. Chow A. Wilson R.I. Schnell E. Zang K. Wang D. Nicoll R.A., Lu, B. Reichardt L.F. J. Neurosci. 2000; 20: 6888-6897Google Scholar). Neurotrophins also acutely enhance postsynaptic responsiveness ofN-methyl-d-aspartate (NMDA)-type glutamate receptors in mature synapses (11Suen P.C., Wu, K. Levine E.S. Mount H.T., Xu, J.L. Lin S.Y. Black I.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8191-8195Google Scholar). In addition, endogenous neuronal BDNF is required for various forms of synaptic plasticity (12Kang H. Schuman E.M. Science. 1995; 267: 1658-1662Google Scholar, 13Patterson S.L. Abel T. Deuel T.A. Martin K.C. Rose J.C. Kandel E.R. Neuron. 1996; 16: 1137-1145Google Scholar, 14Figurov A. Pozzo-Miller L.D. Olafsson P. Wang T. Lu B. Nature. 1996; 381: 706-709Google Scholar). However, the latest studies demonstrate that BDNF is released from nerve terminals in an activity-dependent manner and act on target neurons or postsynaptic sites (15Griesbeck O. Canossa M. Campana G. Gartner A. Hoener M.C. Nawa H. Kolbeck R. Thoenen H. Microsc. Res. Tech. 1999; 45: 262-275Google Scholar, 16Kohara K. Kitamura A. Morishima M. Tsumoto T. Science. 2001; 291: 2419-2423Google Scholar). Accordingly, anterograde neurotrophic functions of BDNF have now been established (17Nawa H. Takei N. Trends Neurosci. 2001; 24: 683-684Google Scholar). A large variety of molecules interacting with α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits have been identified and characterized; SAP97 associates with the COOH termini of GluR1 subunits, whereas GRIP/ABP and Pick1 bind to those of GluR2 and GluR3 subunits (18Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Google Scholar, 19Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Google Scholar, 20Dev K.K. Nishimune A. Henley J.M. Nakanishi S. Neuropharmacology. 1999; 38: 635-644Google Scholar, 21Xia J. Zhang X. Staudinger J. Huganir R.L. Neuron. 1999; 22: 179-187Google Scholar). The intracellular domain of GluR1 protein associates with various signal transducers as well, such as Gi protein, and that of GluR2 with Lyn andN-ethyl-maleimide-sensitive factor (NSF) (22Hayashi T. Umemori H. Mishina M. Yamamoto T. Nature. 1999; 397: 72-76Google Scholar, 23Wang Y. Small D.L. Stanimirovic D.B. Morley P. Durkin J.P. Nature. 1997; 389: 502-504Google Scholar, 24Nishimune A. Isaac J.T. Molnar E. Noel J. Nash S.R. Tagaya M. Collingridge G.L. Nakanishi S. Henley J.M. Neuron. 1998; 21: 87-97Google Scholar, 25Osten P. Srivastava S. Inman G.J. Vilim F.S. Khatri L. Lee L.M. States B.A. Einheber S. Milner T.A. Hanson P.I. Ziff E.B. Neuron. 1998; 21: 99-110Google Scholar). The interacting proteins carrying PDZ domains are suggested to cooperate to regulate the dynamics, targeting, and clustering of AMPA receptors at the postsynaptic membrane of the hippocampal neurons (19Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Google Scholar, 26O'Brien R.J. Lau L.F. Huganir R.L. Curr. Opin. Neurobiol. 1998; 8: 364-369Google Scholar, 27Hayashi Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Science. 2000; 287: 2262-2267Google Scholar, 28Carroll R.C. Beattie E.C. von Zastrow M. Malenka R.C. Nat. Rev. Neurosci. 2001; 2: 315-324Google Scholar). Similarly, the NSF-GluR2 interaction is also suggested to have some roles in the subcellular dynamics of AMPA receptors in such excitatory neurons. NSF might contribute to synaptic plasticity as evidenced by electrophysiological studies: Disruption of the interaction between GluR2 and NSF with a decoy peptide influences hippocampal long term depression (29Noel J. Ralph G.S. Pickard L. Williams J. Molnar E. Uney J.B. Collingridge G.L. Henley J.M. Neuron. 1999; 23: 365-376Google Scholar, 30Luthi A. Chittajallu R. Duprat F. Palmer M.J. Benke T.A. Kidd F.L. Henley J.M. Isaac J.T. Collingridge G.L. Neuron. 1999; 24: 389-399Google Scholar). In contrast to the above information about AMPA receptors in hippocampal neurons, the characteristics of the AMPA receptor dynamics in other types of neurons are largely unknown. We recently reported that chronic BDNF treatment increases and maintains total protein levels of AMPA receptor subunits in neocortical cultures without influencing their mRNA expression (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar, 31Narisawa-Saito M. Silva A.J. Yamaguchi T. Hayashi T. Yamamoto T. Nawa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2461-2466Google Scholar). Consistent with this finding, there is impaired synaptic AMPA receptor expression in BDNF knockout mice (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar). This process presumably depends on a post-translational mechanism and requires chronic stimulation of BDNF for days. In the present study, we found that BDNF triggers dynamic subcellular movement of AMPA receptors in cultured neocortical neurons. Using primary cultured neurons as well as the heterologous system of human embryonic kidney (HEK) cells, we explored the molecular mechanism underlying the rapid BDNF effects on surface translocation of AMPA receptor subunits. We characterized this cellular phenomenon, paying attention to the difference between this BDNF-dependent process and the NMDA receptor-dependent plasticity reported previously. The potential contribution of the individual subunit regulation to the dynamics of the whole receptor complexes is also examined and discussed. Whole cerebral neocortices of embryonic day 18 rats or neonatal rats were mechanically dissociated and plated onto poly-d-lysine-coated dishes at a low density (400–600 cells/mm2). Cortical neurons were grown in serum-free condition or in serum-containing medium as described previously (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar, 31Narisawa-Saito M. Silva A.J. Yamaguchi T. Hayashi T. Yamamoto T. Nawa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2461-2466Google Scholar,32Takei N. Sasaoka K. Inoue K. Takahashi M. Endo Y. Hatanaka H. J. Neurochem. 1997; 68: 370-375Google Scholar). Purified human recombinant BDNF (50 ng/ml) was added 4–5 days after plating. HEK293 cells were transfected with a vector carrying the mouse TrkB cDNA in pRc/CMV (provided by Dr. S. Koizumi) with Ca2+ phosphate method. Cells were selected in medium containing 400 μg/ml Geneticin, and the drug-resistant colonies were isolated and expanded (HEK-TrkB cells). HEK-TrkB cells were transiently transfected with cDNA for GluR1, GluR2, or its derivatives using the lipofection method, and their expression and subcellular dynamics were analyzed using Western blotting and immunostaining methods, respectively. Prior to the BDNF treatment, some cultures were pre-incubated with the reagents known to influence exocytosis or endocytosis, brefeldin A (5 μg/ml; Wako Chemicals, Osaka, Japan), NEM (1 mm; Sigma), botulinum toxin B (100 nm) (7Takei N. Numakawa T. Kozaki S. Sakai N. Endo Y. Takahashi M. Hatanaka H. J. Biol. Chem. 1998; 273: 27620-27624Google Scholar), and bafilomycin A (1 μm; Wako Chemicals). Alternatively, cultures were supplemented with various kinase inhibitors (10 μmU73122 (Calbiochem, San Diego, CA), 25 μm PD98059 (Calbiochem), 1 μm calphostin C (Sigma), and 0.5 μm KN93 (Wako Chemicals)) or with translation inhibitors (2 ng/ml cycloheximide (Sigma) and 30 μm anisomycin (Sigma)). Neocortical cultures or HEK-TrkB cells were treated with 100 ng/ml BDNF for 30 min to 24 h and then incubated with 1 mmsulfo-NHS-LC-biotin (Pierce) in phosphate-buffered saline containing 1 mm CaCl2 and 1 mm MgCl2for 15 min on ice (33Mammen A.L. Huganir R.L. O'Brien R.J. J. Neurosci. 1997; 17: 7351-7358Google Scholar). Cell lysate was incubated with ImmunoPure immobilized streptavidin-beaded agarose (Pierce) overnight at 4 °C. Biotinylated proteins were eluted with 2% sodium dodecyl sulfate (SDS) buffer at 95 °C and processed for Western blotting analysis. Cells were lysed in Laemmli sample loading buffer (10% glycerol, 2% SDS, 65 mm Tris). After centrifugation at 10,000 × g for 10 min, protein in supernatants was denatured at 95 °C in the presence of 0.5m 2-mercaptoethanol and protein samples (15 μg/lane) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) with 1-mm-thick, 8% polyacrylamide slab gels. Protein was transferred to a nitrocellulose membrane by electrophoresis. Primary antibodies were diluted with blocking solution and incubated with the membrane at 4 °C overnight. Immunoreactivity was detected with goat anti-rabbit immunoglobulin conjugated to peroxidase (1:10,000) followed by chemiluminescence reaction combined with film exposure (ECL kit;Amersham Biosciences, Tokyo, Japan). The following primary antibodies were used: affinity-purified rabbit anti-GluR1 COOH terminus (Chemicon International, Temecula, CA), mouse monoclonal antibody against GluR2 NH2 terminus (Chemicon International), affinity-purified rabbit anti-GluR2 intracellular domain (made to a 50-amino acid peptide corresponding to the COOH terminus of GluR2) (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar), rabbit antisera to NSF (a gift of Dr. M. Takahashi, 0.5 μg/ml), and mouse monoclonal pan-cadherin antibody (Sigma). The immunoreactivity of the bands was quantified by densitometric analysis. Cultured neurons or HEK-TrkB cells were rinsed with Tris buffer, pH 7.4, containing 100 μm sodium acetate, 2.5 mm CaCl2, and 5 g/liter glucose. [3H]AMPA (1,561 GBq/mmol, 37 MBq/ml, PerkinElmer Life Sciences) was adjusted to 50 nm with the Tris buffer and diluted with 0 to 800 nm cold AMPA (Sigma). Cells were incubated with these AMPA solutions on ice for 60 min as described previously (34Iwakura Y. Nagano T. Kawamura M. Horikawa H. Ibaraki K. Takei N. Nawa H. J. Biol. Chem. 2001; 276: 40025-40032Google Scholar). An excess amount of cold AMPA (100 μm) was applied to culture to estimate nonspecific [3H]AMPA binding to cells. After rapid washing with cold Tris buffer, cells were lysed with 0.5 n NaOH and the radioactivity of cell lysates was counted using a LSC-3050 liquid scintillation system (Aloka, Tokyo, Japan). Cell sorting was performed using a laser scanning cytometer (LSC2, Olympus). Co-transfected HEK293 cells in live culture were immunostained with anti-GluR1 NH2-terminal antibodies (30 μg/ml) (35Ibaraki K. Otsu Y. Nawa H. J. Neurochem. 1999; 73: 408-417Google Scholar) followed by the FITC-conjugated secondary anti-rabbit immunoglobulin antibody (Jackson Laboratories, Bar Harbor, ME). After fixation, cells were incubated with an anti-GluR2 NH2-terminal mouse monoclonal antibody (20 μg/ml; monoclonal antibody 397, Chemicon International) and the rabbit polyclonal antibody against the anti-GluR2/3 intracellular domain (10 μg/m) (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar). Total immunoreactivity for GluR1 and GluR2 was visualized with the Cy5-labeled secondary anti-rabbit immunoglobulin antibody (2.5 μg/ml; Amersham Biosciences) and the R-phycoerythrin-labeled secondary anti-mouse immunoglobulin antibody (2.5 μg/ml; PharMingen, San Diego, CA), respectively. An argon laser (488 nm) was used to excite FITC and R-phycoerythrin, and a helium/neon laser (633 nm) was used for Cy-5. The COOH-terminal five amino acids (aa 858–862) and 20 amino acids (aa 843–862) of mouse GluR2 were deleted, using the pCI plasmid as a template. The deletion of the NSF binding region (aa 823–832; KRMKVAKNAQ) was performed with a site-directed mutagenesis kit (Clontech, Palo Alto, CA) according to the instructions from the manufacturer. Nucleotide deletion was confirmed by DNA sequencing, and the expression of the GluR2 mutant proteins was verified by Western blotting analysis. HEK-TrkB cells were harvested in immunoprecipitation buffer containing 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5 mm ATPγS (Roche Molecular Biochemicals, Mannheim, Germany), 2 mm EDTA, 1 mm EGTA, and 1% sodium deoxycholate. Cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was used for co-immunoprecipitation (36Song I. Kamboj S. Xia J. Dong H. Liao D. Huganir R.L. Neuron. 1998; 21: 393-400Google Scholar). Neocortices from young rats (postnatal day 3–4) were homogenized in immunoprecipitation buffer without deoxycholate, containing 5 μg/ml affinity-purified chicken anti-BDNF antibodies (4Narisawa-Saito M. Carnahan J. Araki K. Yamaguchi T. Nawa H. Neuroscience. 1999; 88: 1009-1014Google Scholar), and homogenization was performed on ice, using a Handy Sonic tissue grinder (Tomy Inc., Tokyo, Japan). The lysate was treated with 200 ng/ml BDNF for 30 min at 37 °C and centrifuged at 10,000 × g for 60 min at 4 °C, and the pellet was solubilized in immunoprecipitation buffer containing 1% sodium deoxycholate. All the cleared supernatants were pretreated with excess amounts of protein A-Sepharose and were then incubated with anti-GluR2 NH2-terminal antibodies (4 μg/ml; monoclonal antibody 397, Chemicon International) at 4 °C for 2 h. The immunocomplex was precipitated with protein A-Sepharose and washed five times with immunoprecipitation buffer, boiled in 2× SDS buffer, and subjected to SDS-PAGE followed by Western blotting with anti-NSF antibody. Statistical analysis was performed using one-way analysis of variance followed by the Bonferroni test (for multiple factors). Alternatively, Student's t test was applied to data with multiple variables and one factor. For data from cell sorting, the Mann-Whitney U test was applied. Results were obtained with multiple sister cultures or independent cultures and expressed as means ± S.E. (n = 3–6 cultures). To identify whether BDNF stimulation can influence functional AMPA receptor on the cell surface or not, [3H]AMPA binding assay was performed with cultured neocortical neurons grown for 5 days and then treated with or without BDNF (50 ng/ml). After a 30-min incubation with BDNF, various concentrations of [3H]AMPA were applied to cultures and the binding of the radioactive ligand to neurons was measured. The values of bound [3H]AMPA were subjected to the Scatchard plot analysis, and the total number (B max) and the dissociation constant (K d) of the functional AMPA receptors were determined on the cell surface (Fig.1 A). TheB max of the AMPA receptors on the cell surface increased from 3.88 ± 0.05 to 4.96 ± 0.27 pmol/mg (n = 4, p < 0.05). The dissociation constant of the AMPA receptors (K d) increased from 192 ± 18 to 286 ± 26 nm. The time course of the maximum ligand binding activity of the AMPA receptors (B max) was assessed by stimulating neurons with BDNF for various periods of time and then determined by exposing them to a submaximal concentration of [3H]AMPA (250 nm; Fig. 1 B). The AMPA binding to neocortical culture became evident after a 30-min incubation with BDNF and further increased until 24 h of incubation (277 ± 10% of control,n = 4, p < 0.0001). These results reveal that BDNF triggers rapid subcellular redistribution of AMPA receptors, leading to the functional AMPA receptor increase on the cell surface. To determine the mechanism of this rapid redistribution of AMPA receptors, several reagents known to influence cellular processes for exocytosis (brefeldin A, botulinum toxin B, and NEM) or endocytosis (bafilomycin A) were applied to primary cultures of neocortical neurons prior to BDNF treatment (7Takei N. Numakawa T. Kozaki S. Sakai N. Endo Y. Takahashi M. Hatanaka H. J. Biol. Chem. 1998; 273: 27620-27624Google Scholar) (Table I, part A). After BDNF stimulation, the number of AMPA receptors on the cell surface was estimated by ligand-binding assay with [3H]AMPA. Bafilomycin A, which blocks trans-Golgi transport to inhibit endocytosis, increased basal levels of surface AMPA receptors and BDNF further elevated their levels. In contrast, the exocytosis blockers, brefeldin A, botulinum toxin B, and NEM, inhibited or reduced the BDNF effects. The drug sensitivity suggests that the BDNF-triggered increase in AMPA receptors reflects an exocytotic process of the receptors.Table IEffects of Ca2+ chelators, exocytosis blockers, and kinase inhibitors on BDNF-mediated cell surface translocation of AMPA receptorsReagentControl+BDNFA.Control100 ± 15171 ± 12**1 μm bafilomycin A166 ± 16**206 ± 10*5 μg/ml brefeldin A111 ± 15136 ± 111 mm NEM102 ± 24133 ± 17100 nm botulinum toxin89 ± 6101 ± 11B.Control100 ± 3150 ± 6***5 mm BAPTA84 ± 5*132 ± 5**0.1 mm BAPTA-AM85 ± 1795 ± 4C.Control100 ± 2153 ± 2***1 μmcalphostin C87 ± 2*96 ± 610 μmU7312248 ± 1**135 ± 8***25 μmPD9805999 ± 2120 ± 2*0.5 μmKN9353 ± 1**84 ± 5***After a 3-h incubation with BDNF, [3H]AMPA binding assay was performed in cultured cortical neurons with a submaximal concentration of the ligand (250 nm). Nonspecific background binding of [3H]AMPA was estimated in culture with addition of excess amounts of cold 100 μm AMPA and subtracted from raw values of [3H]AMPA binding. [3H]AMPA binding was normalized with protein yields, although they were not significantly influenced by any treatment. Comparison was done between BDNF-treated and untreated pairs in each (right column). Basal effects of blockers and inhibitors were analyzed statistically in comparison with untreated control (middle column). *, p < 0.05; **,p < 0.01; ***, p < 0.001 (n = 4 sister cultures). Open table in a new tab After a 3-h incubation with BDNF, [3H]AMPA binding assay was performed in cultured cortical neurons with a submaximal concentration of the ligand (250 nm). Nonspecific background binding of [3H]AMPA was estimated in culture with addition of excess amounts of cold 100 μm AMPA and subtracted from raw values of [3H]AMPA binding. [3H]AMPA binding was normalized with protein yields, although they were not significantly influenced by any treatment. Comparison was done between BDNF-treated and untreated pairs in each (right column). Basal effects of blockers and inhibitors were analyzed statistically in comparison with untreated control (middle column). *, p < 0.05; **,p < 0.01; ***, p < 0.001 (n = 4 sister cultures). Second, we assessed the involvement of Ca2+ using its chelator, BAPTA, and its derivative. Intracellular Ca2+ was trapped with cell-permeable BAPTA-AM, whereas extracellular Ca2+ was chelated with nonpermeable BAPTA (Table I, part B). Under this condition, cultured neocortical neurons were incubated with BDNF for 3 h. BAPTA-AM inhibited the BDNF-triggered increase in [3H]AMPA binding. In contrast, BAPTA had no significant effect. The above findings suggest that the surface translocation of AMPA receptors by BDNF involves the exocytotic pathway triggered by intracellular Ca2+ (37Kuijpers T.W. Hoogerwerf M. Roos D. J. Immunol. 1992; 148: 72-77Google Scholar). BDNF is known to trigger intracellular signaling cascades of phospholipase C and mitogen-activated protein kinase. In addition, protein kinase C (PKC) and αCAMKII are often implicated in the subcellular dynamics of AMPA receptors (26O'Brien R.J. Lau L.F. Huganir R.L. Curr. Opin. Neurobiol. 1998; 8: 364-369Google Scholar, 27Hayashi Y. Shi S.H. Esteban J.A. Piccini A. Poncer J.C. Malinow R. Science. 2000; 287: 2262-2267Google Scholar, 28Carroll R.C. Beattie E.C. von Zastrow M. Malenka R.C. Nat. Rev. Neurosci. 2001; 2: 315-324Google Scholar). To explore intracellular signals leading to the surface AMPA receptor increase, cultured neurons were stimulated with BDNF in the presence of kinase inhibitors, U73122 (phospholipase C), PD98059 (mitogen-activated protein kinase), calphostin C (PKC), and KN93 (αCAMKII) (Table I, part C). Calphostin C almost abolished the BDNF-dependent increase in [3H]AMPA binding. Although U73122 and KN93 themselves markedly reduced the basal [3H]AMPA binding, BDNF treatment still markedly up-regulated the [3H]AMPA binding in the presence of these inhibitors. This observation suggests that PKC kinase may involve the BDNF-dependent translocation of AMPA receptors. As the basal amount of [3H]AMPA binding was modestly influenced by BAPTA and BAPTA-AM, as well as by the other kinase inhibitors, there might be an alternative signaling pathway that maintains the basal surface AMPA receptor levels. To confirm the BDNF effects on subcellular distributions of individual AMPA receptor subunit proteins, cultured neocortical neurons were treated with or without BDNF. Following the treatment, proteins on cell surfaces were biotinylated, collected with streptavidin-coupled beads, and subjected to Western blotting. The biotinylated (cell surface-expressed) GluR1 and GluR2/3 proteins were markedly increased by BDNF treatment compared with nontreated cells (Fig.2 A). In parallel, AMPA receptor protein levels were also increased in total cellular lysates (Fig. 2 B). BDNF is known to trigger glutamate release from cultured neurons (32Takei N. Sasaoka K. Inoue K. Takahashi M. Endo Y. Hatanaka H. J. Neurochem. 1997; 68: 370-375Google Scholar). Is the activation of glutamate receptors involved in the surface increase of AMPA receptors? To examine the contribution of endogenous glutamate to the BDNF effect, both NMDA-type and AMPA-type glutamate receptors were blocked by their inhibitors AP-5 and CNQX in culture, respectively (Fig. 2 C). In the presence of these blockers, BDNF similarly increased the surface expression of the AMPA receptor subunits. These results suggest that BDNF significantly altered the levels of the AMPA receptors on neuronal surface, irrespective of excitatory synaptic activities. To explore the above molecular mechanism for the surface translocation of AMPA receptors, we attempted to reconstitute AMPA receptor translocation in non-neuronal cells. This system presumably excluded the involvement of presynaptic elements or activity-dependent processes. HEK293 cells carrying TrkB receptors were established with permanent integration of human TrkB gene under the cytomegalovirus promoter. By transfecting cDNA for each AMPA receptor subunit into the HEK-TrkB cells, the influences of BDNF on the surface expression of each receptor subunit, GluR1 or GluR2, were evaluated by the cell surface biotinylation assay. BDNF triggered an increase in surface protein levels of GluR2 as determined after a 5-h incubation with BDNF (Fig.3 A). The up-regulation of surface expression was pronounced for GluR2 (392 ± 8% of control, n = 3, p < 0.01). In contrast, surface expression of GluR1 as well as that of cadherins was not significantly influenced (103 ± 15% and 106 ± 7% of control, respectively). This may eliminate the possibility that BDNF simply facilitates general turnover of intracellular membranes. The cell surface distribution of the active AMPA receptor subunits was ascertained by the ligand-binding assay using [3H]AMPA (Fig. 3 B). When these cells were stimulated with BDNF, [3H]AMPA binding activity on the cell surface was markedly increased in the HEK-TrkB cells carrying GluR2 but not GluR1. These results indicate that the effects of BDNF on translocation specifically involve the GluR2 subunit in HEK-TrkB cells. We have found that BDNF can enhance translation rates to increase protein synthesis in cortical neurons (38Takei N. Kawamura M. Hara K. Yonezawa K. Nawa H. J. Biol. Chem. 2001; 276: 42818-42825Google Scholar). Thus, we assessed potential contribution of protein synthesis to the surface AMPA receptor increase both in cortical neurons and HEK293 cells. The total protein increase in GluR1 and GluR2/3 levels lagged behind the rapid surface
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