The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription

Liang-Fu Chen,Michelle R Lyons,Fang Liu,Matthew V Green,Nathan G Hedrick,Ashley B Williams,Arthy Narayanan,Ryohei Yasuda,Anne E West

doi:10.1074/jbc.ra119.010266

Abstract

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Highlights

N-Methyl-D-aspartate type glutamate receptors (NMDARs) are essential for coupling sensory experience with brain development
Knockdown (KD) of GluN3A with either of the two short hairpin RNA (shRNA) had no significant effect on brain-derived neurotrophic factor (Bdnf) IV levels in TTX silenced neurons but resulted in a significant potentiation of Bdnf IV mRNA transcription upon then acutely withdrew TTX (TTX w/d) for each shRNA compared with its paired control infected neurons (Fig. 1, D and E)
The potentiation of Bdnf promoter IV activation in GluN3A KD neurons was selective for transcription induced by NMDARs, because there were no significant differences in the fold change of Bdnf IV mRNA induced in GluN3A KD versus control neurons following potassium chloride (KCl)-mediated membrane depolarization (Fig. S2A), which activates transcription selectively via the opening of Ltype voltage-gated calcium channels (LVGCCs) and does not require the activation of NMDARs [18]

Summary

Introduction

N-Methyl-D-aspartate type glutamate receptors (NMDARs) are essential for coupling sensory experience with brain development. In addition to functioning as ligand-gated ionotropic receptors, NMDARs exert long-lasting effects on neuronal biology by activating intracellular signaling cascades that subsequently impact synapse formation, maturation, and function. These inductive effects of NMDARs are mediated at least in part by the regulation of programs of gene transcription. CaRF knockout mice show enhanced NMDAR-induced transcription of Bdnf both in culture and in vivo in a manner that correlates with reduced GluN3A expression [18], raising the possibility that GluN3A functions to transiently inhibit the NMDAR-dependent induction of Bdnf and other neuronal activity-regulated genes in developing neurons.

Methods

Results

Conclusion