Research reportBDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities
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.
References (50)
- et al.
Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB
Neuron
(1991) - et al.
Relationship between N-methyl-d-aspartate receptor NR1 splice variants and NR2 subunits
J. Biol. Chem.
(1996) Neurotrophin receptors: a window into neuronal differentiation
Neuron
(1992)- et al.
Molecular characterization of N-methyl-d-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule
J. Biol. Chem.
(1994) - et al.
An improved method detects differential NGF and BDNF gene expression in response to depolarization in cultured hippocampal neurons
Mol. Brain Res.
(1994) - et al.
Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis
Neuron
(1991) - et al.
Nerve growth factor mediates signal transduction through trk homodimer receptors
Neuron
(1992) - et al.
trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3
Cell
(1991) - et al.
Differential tyrosine phosphorylation of N-methyl-d-aspartate receptor subunits
J. Biol. Chem.
(1995) - et al.
Selective role for trkB neurotrophin receptors in rapid modulation of hippocampal synaptic transmission
Mol. Brain Res.
(1996)
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice
Neuron
Neurotrophin expression in rat hippocampal slices: a stimulus paradigm inducing LTP in CA1 evokes increases in BDNF and NT-3 mRNAs
Neuron
The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor
Cell
trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor
Cell
The changing scene of neurotrophic factors
Trends Neurosci.
Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex
Brain Res.
Functional trkB neurotrophin receptors are intrinsic components of the adult brain postsynaptic density
Mol. Brain Res.
Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex
J. Neurosci.
The trk family of neurotrophin receptors
J. Neurobiol.
A synaptic model of memory: long-term potentiation in the hippocampus
Nature
Long-lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path
J. Physiol. (London)
Functional interactions of neurotrophins and neurotrophin receptors
Ann. Rev. Neurosci.
The role of neurotrophins in the developing nervous system
J. Neurobiol.
Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus
Nature
Cited by (246)
Stress Diminishes BDNF-stimulated TrkB Signaling, TrkB-NMDA Receptor Linkage and Neuronal Activity in the Rat Brain
2021, NeuroscienceCitation Excerpt :Included are effects on brain-derived neurotropic factor (BDNF) and its cognate receptor, tyrosine receptor kinase B (TrkB). Activated TrkB influences several downstream signaling cascades (e.g., PLC-PKC, PI3K-Akt, Raf-Erk) and interacts with membrane bound glutamatergic NMDA receptors (Soppet et al., 1991; Suen et al., 1997; Levine et al., 1998; Lin et al., 1998; Minichiello, 2009; Gupta et al., 2013). Furthermore, activation of TrkB and/or NMDA receptors in the hippocampus (HIPPO) and prefrontal cortex (PFC) leads to expression of immediate early genes, including activity-regulated cytoskeleton-associated protein (Arc) (Link et al., 1995; Lyford et al., 1995; Yin et al., 2002; Okuno, 2011; Hunter et al., 2017).
Receptors | Neurotrophin receptor signaling
2021, Encyclopedia of Biological Chemistry: Third EditionA model of negative emotional contagion between male-female rat dyads: Effects of voluntary exercise on stress-induced behavior and BDNF-TrkB signaling
2021, Physiology and BehaviorCitation Excerpt :Phosphorylated TrkB145 (pY-TrkB145) recruits other signaling proteins (e.g., neuronal Src homology and collagen protein (N-Shc) and phospholipase C-g1 (PLC)) which influence cellular activity, including regulation of transcription. In addition, TrkB145 activation can also lead to phosphorylation of other membrane-bound proteins including subunits of glutamatergic NMDA receptors (e.g., NR1 and NR2 subunits) [84,85,86], perhaps through TrkB-NMDA receptor linkage [87-89]. In the hippocampus, this association has been shown to influence the expression of plasticity-related proteins, (such as activity-regulated cytoskeleton-associated protein (Arc)), which promote neuronal stability and are involved in synaptic plasticity [90-92].
- 1
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.