Elsevier

Neuroscience Letters

Volume 531, Issue 1, 30 November 2012, Pages 14-19
Neuroscience Letters

c-Jun N-terminal phosphorylation is essential for hippocampal synaptic plasticity

https://doi.org/10.1016/j.neulet.2012.09.048Get rights and content

Abstract

c-Jun N-terminal kinase (JNK), a member of the MAPK family, is an important regulatory factor of synaptic plasticity as well as neuronal differentiation and cell death. Recently, JNK has been reported to modulate synaptic plasticity by the direct phosphorylation of synaptic proteins. The specific role of c-Jun phosphorylation in JNK mediated synaptic plasticity, however, remains unclear. In this study, we investigated the effects of c-Jun phosphorylation on synaptic structure and function by using c-Jun mutant mice, c-JunAA, in which the active phosphorylation sites at serines 63 and 73 were replaced by alanines. The gross hippocampal anatomy and number of spines on hippocampal pyramidal neurons were normal in c-JunAA mice. Basal synaptic transmission, input–output ratios, and paired-pulse facilitation (PPF) were also no different in c-JunAA compared with wild-type mice. Notably, however, the induction of long-term potentiation (LTP) at hippocampal CA3–CA1 synapses in c-JunAA mice was impaired, whereas induction of long-term depression (LTD) was normal. These data suggest that phosphorylation of the c-Jun N-terminus is required for LTP formation in the hippocampus, and may help to better characterize JNK-mediated modulation of synaptic plasticity.

Highlights

► The synaptic role of c-Jun phosphorylation was studied in c-Jun alanine mutant mice, c-JunAA. ► c-JunAA animals show normal hippocampal structure and basal synaptic transmission. ► LTP induction was impaired in c-JunAA mice, whereas LTD induction was normal.

Introduction

c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is a signaling molecule belonging to the mitogen-activated protein kinase (MAPK) family, which also includes proteins such as p38, ERK1, and ERK2 [29]. Three isoforms of JNK exist, each of which modulate neuronal cell fate by inducing neuronal differentiation or cell death [27]. The most widely studied downstream factor in the JNK signaling pathway is c-Jun, a transcription factor that forms activating protein-1 (AP-1) with Fos and ATF [25]. c-Jun activity is regulated by posttranslational modifications including phosphorylation and ubiquitination. Phosphorylation of the N-terminal Ser63 and Ser73 residues of c-Jun decreases ubiquitination, and increases stability [18], [19], transcriptional activity [11], [20], and binding affinity with the transcriptional co-activator CBP (CREB-binding protein) [16].

Recently, the role of JNK in modulating synaptic structure and function has received significant public attention. Knockout animals lacking JNK2 or JNK3 have been reported to have normal anatomical features without any structural anomalies [15], [30], whereas adult JNK1 knockout mice show an abnormal anterior commissure formation [2]. JNK1/2 double knockout exhibits abnormal neural tube formation and embryonic death [14], [15]. Interestingly, hippocampal JNK activity affects the induction of LTP [3], [28], NMDA-dependent LTD [5], and mGluR-LTD [17]. In addition, JNK has been found to be related to pathological changes in Alzheimer's and Parkinson's diseases [4], [27].

JNK-mediated regulation of synaptic plasticity is reportedly mediated by the direct phosphorylation of synaptic target proteins. JNK directly phosphorylates the Ser295 residue of PSD95, an abundant synaptic scaffolding protein, which increases the synaptic accumulation of PSD95 and reduces NMDA-induced AMPA receptor internalization [13]. JNK also phosphorylates the Ser447 residue of delta-catenin, which decreases dendritic branching [7]. JNK has been shown to expedite AMPA receptor recycling by direct phosphorylation of GluA2L and GluA4 [26], [34]. The presence of GRASP1, another synapse enriched scaffolding protein, enables more efficient synaptic JNK function [32]. These signaling pathways allow Wnt and TGF-β to modulate synaptic plasticity via regulation of JNK and its synaptic protein targets [8].

It is still unclear, however, whether synaptic plasticity is modulated directly by c-Jun N-terminal phosphorylation, which is a key component of the JNK signaling pathway. Although JNK knockout mice have been used to study effects on synaptic plasticity, it is difficult to determine the effects of c-Jun phosphorylation alone given that JNK knockouts lack both c-Jun phosphorylation dependent and independent pathways.

In order to determine the effects of c-Jun phosphorylation alone, we used c-JunAA mice in which Ser63 and Ser73 were replaced with alanines. The expression of c-Jun is normal but the phosphorylation of c-Jun N-terminal is blocked in c-JunAA mouse [1]. Interestingly, c-JunAA mice undergo normal development, whereas c-Jun knockout mice show embryonic lethality [1], [12]. Using c-JunAA mice, we monitored synaptic changes in the hippocampus to determine the effect of c-Jun phosphorylation on synaptic structure and function.

Section snippets

Materials

All chemicals were purchased from Sigma (St. Louis, MO, USA). Glass micropipettes (GC100T-01) were obtained from Harvard Apparatus (Holliston, MA, USA), and concentric bipolar electrodes (CB-BRC75) were obtained from Frederick Haer Co. (Bowdoinham, ME, USA).

Animals

c-JunAA mice and wild type mice were obtained by breeding heterozygote male and female mice, and the genotype of offspring was identified by PCR as described previously [2]. They were housed at 23 ± 2 °C with a 12 h light–dark cycle, and fed

Results

In order to visualize the structural changes in c-JunAA mice (Fig. 1A), we monitored the gross morphology of the hippocampus using cresyl violet staining. The c-JunAA mice exhibited normal hippocampal structure (Fig. 1B), suggesting that c-JunAA does not affect the global anatomy of the hippocampus. Dendritic spine structure could be altered, however, given that c-Jun phosphorylation affects actin remodeling-related cytoskeleton proteins such as PAK [10]. Thus, we conducted morphological

Discussion

Although c-Jun N-terminal phosphorylation is a core downstream signaling mechanism of JNK activation, the effects of JNK activation and c-Jun phosphorylation are quite different. Deletion of JNK1/2 induces defects in neural tube formation and embryonic cell death, while c-JunAA mutants show an increase in cell number without neural tube abnormalities [21]. To date, a decrease in kainate-mediated excitotoxic cell death [1] and changes in differentiation-mediated neurite outgrowth [6] have been

Acknowledgements

This work was supported by the Neuroscience Program 2009-0081468 (to SYC) and 2011-0027667 (to SJL) of Korean Ministry of Education, Science, and Technology. The authors have declared that no competing interests exist.

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