Elsevier

Molecular Brain Research

Volume 55, Issue 1, 30 March 1998, Pages 20-27
Molecular Brain Research

Research report
BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities

https://doi.org/10.1016/S0169-328X(97)00349-5Get rights and content

Abstract

While neurotrophins are critical for neuronal survival and differentiation, recent work suggests that they acutely regulate synaptic transmission as well. Brain-derived neurotrophic factor (BDNF) enhances excitatory postsynaptic currents in cultured dissociated hippocampal neurons within 2–3 min through postsynaptic, phosphorylation-dependent mechanisms. Moreover, BDNF modulates hippocampal long-term potentiation, in which postsynaptic NMDA (N-methyl-d-aspartate) receptors (NRs) play a key role. We now report that BDNF acutely increases tyrosine phosphorylation of the specific NMDA receptor subunit NR2B, which has recently been shown to play a role in long-term potentiation. Incubation of BDNF with cortical or hippocampal postsynaptic densities for 5 min increased tyrosine phosphorylation of the NR2B subunits in a dose-dependent manner. A maximal increase to 165% of control phosphorylation occurred at a BDNF concentration of 2 ng/ml. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, had no effect on NR2B phosphorylation. Further, BDNF action was selective, since it did not alter tyrosine phosphorylation of NR2A subunits. Our results suggest that tyrosine phosphorylation of NR2B subunits of the NMDA receptor may contribute to neurotrophin modulation of postsynaptic responsiveness and long-term potentiation.

Introduction

Neurotrophins are critical for long-term survival and differentiation of selected neuronal subpopulations 7, 10, 25, 44. The trophins typically elicit changes over the course of hours, or even days. Neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) 3, 7, 8, 16. The neurotrophins bind receptor tyrosine kinases of the trk proto-oncogene family with high affinity. TrkA is the preferred high-affinity receptor for NGF 2, 7, 8, 14, trkB for BDNF and NT-4/5 2, 7, 8, 39, 40as well as NT-3 8, 39, 40, and trkC for NT-3 2, 8, 23.

Besides classical survival actions, emerging evidence suggests that neurotrophins can modulate synaptic transmission 13, 17, 18, 19, 20, 26, 27, 28. Our previous studies revealed that BDNF enhanced synaptic transmission between hippocampal neurons in culture 26, 27. Injection directly into the postsynaptic cell of the trk tyrosine kinase inhibitor, K-252a, blocked BDNF-induced enhancement in synaptic transmission. Conversely, injection of the phosphatase inhibitor, okadaic acid, potentiated the BDNF effect. Consequently, postsynaptic phosphorylation mechanisms are apparently involved in BDNF-enhanced synaptic transmission [26].

More recently, we found that trkB, the high-affinity receptor for BDNF and NT-4, is a functionally active, intrinsic component of the postsynaptic density (PSD) [49], consistent with a postsynaptic locus of BDNF action. The PSD is a disc-shaped proteinaceous structure apposed to the cytoplasmic surface of the postsynaptic membrane. Transmitter receptors and protein kinases are anchored to the PSD [38], suggesting that the structure participates in signal transduction and receptor regulation. In particular, the PSD contains NMDA (N-methyl-d-aspartate) receptors 33, 34, 38, 42, 43, a major glutamate receptor subtype.

NMDA receptor function is regulated by protein tyrosine kinases (PTKs): phosphorylation potentiates NMDA currents in hippocampal neurons [46]. Regulation of NMDA receptor conductance may involve phosphorylation of NR2A or NR2B subunits, since tyrosine phosphorylation of these subunits has been reported 24, 30, 35, 36. NR2A, NR2B and trkB are all intrinsic PSD components 43, 49, raising the possibility that a localized BDNF-induced phosphorylation cascade modulates excitatory NMDA receptors, enhancing synaptic transmission. Native NMDA receptors exist as heteromeric complexes consisting of NR1 and various NR2 subunits 4, 9, 37, 45. NR1 is essential for ion selectivity and agonist binding to the receptor, while NR2 subunits are mainly responsible for regulating channel properties [15]. Indeed, we recently found that BDNF acutely enhances phosphorylation of the hippocampal NMDA receptor subunit, NR1 [41], which is also localized in the PSD, thereby associating receptor phosphorylation and BDNF action. To extend our studies of BDNF synaptic actions, we focused on tyrosine phosphorylation of the NMDA receptor subunits, NR2A and NR2B, in the purified cortical and hippocampal PSD. Our goal was to determine whether BDNF potentially alters NMDA channel properties by altering the phosphorylation of NR2 subunits.

Section snippets

Materials

BDNF was provided by Cephalon (West Chester, PA). NGF was purchased from Boehringer-Mannheim (Indianapolis, IN). Anti-NR2A and anti-NR2B antibodies were supplied by Chemicon International (Temecula, CA). The specificity of anti-NR2A and anti-NR2B antibodies has previously been described and characterized by Blahos and Wenthold [4]. Anti-phosphotyrosine antibody, PY20, was obtained from Transduction Laboratories (Lexington, KY). All other chemicals were purchased from Sigma (St. Louis, MO).

Experimental animals and dissection

Effects of BDNF on tyrosine phosphorylation of NR2B or NR2A in the cortical PSD

We began exploring BDNF-activated postsynaptic signalling mechanisms by using the PSD, which contains trkB [49]. We initially examined the cerebral cortex, which affords abundant tissue for multiple, parallel studies. Our results revealed that BDNF enhanced tyrosine phosphorylation of NR2B subunits in the purified PSD fraction in a dose-dependent manner. Maximal increase to 165% of control values were elicited at 2 ng/ml BDNF (Fig. 1A–C). Higher doses did not further increase the

Discussion

We and others have previously shown that BDNF acutely enhances synaptic transmission in the developing and adult hippocampus 17, 26, 27. This is due, at least in part, to postsynaptic phosphorylation mediated by trkB activation [26]. The present studies explored underlying molecular mechanisms, focusing on BDNF-induced phosphorylation in the mature cortical and hippocampal PSDs, which contain functionally active trkB [49]. BDNF specifically enhanced tyrosine phosphorylation of NR2B subunits of

Acknowledgements

We thank Ms. Betty Wheeler for excellent technical assistance. This research was supported by the National Institutes of Health Grant HD 23315.

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    Present address: Dept. of Dentistry, Children's Hospital, Lin-Kou Medical Center and Dept. of Physiology, Chang Gung College of Medicine and Technology, Kwei-Shan, Tao-Yuan 333, Taiwan.

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