Regulation of eukaryotic initiation factor 4E by converging signaling pathways during metabotropic glutamate receptor-dependent long-term depression

Abstract

Long-term depression (LTD) is an activity-dependent decrease in synaptic efficacy that can be induced in hippocampal area CA1 by pharmacological application of the selective group I metabotropic glutamate receptor (mGluR) agonist 3,5-diyhroxyphenylglycine (DHPG). Recent work has demonstrated that DHPG-induced LTD recruits at least two signal transduction pathways known to couple to translation, the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway and the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. However, it remains unclear which translation factors are engaged by these two signaling pathways during mGluR-LTD. In this study, we investigated whether the group I mGluRs couple to the cap-dependent translation proteins: Mnk1, eIF4E, and 4E-BP. We found that both the MEK-ERK and PI3K-mTOR signaling pathways are critical for the DHPG-induced regulation of these translation factors. Furthermore, we demonstrate that increasing eIF4F complex availability via the genetic elimination of 4E-BP2 can enhance the degree of LTD achieved by DHPG application in an ERK-dependent manner. Our results provide direct evidence that cap-dependent translation is engaged during mGluR-LTD and demonstrate that the MEK-ERK and PI3K-mTOR signaling pathways converge to regulate eIF4E activity after induction of DHPG-LTD.

Activation of group I mGluRs results in activation of ERK. A, DHPG application (50 μm, 5 min) increases pp-ERK2 immunoreactivity. B, Both mGluR1 (LY367385: 100 μm, 20 min) and mGluR5 (MPEP: 10 μm, 20 min) antaogonists inhibited maximum DHPG-induced ERK activation. C, DHPG-induced ERK activation was preserved in synaptoneurosomes and was blocked by preincubation with U0126 (20 μm, 30 min). The inset shows PSD-95 (postsynaptic density-95) enrichment in the synaptoneurosome preparation (H, total homogenate; S, synaptoneurosome). D, DHPG application did not result in an increase in p38 kinase phosphorylation (n = 4; p = 0.23). Representative Western blots are shown in each panel. The cumulative data represent mean + SEM, and the number is indicated in the bars unless noted otherwise. *p < 0.05, statistical significance determined by Student’s t test. CTL, Control; LY, LY367385.

Activation of group I mGluRs results in Mnk1 activation and increased eIF4E phosphorylation via ERK. A, DHPG-induced (50 μm, 5 min) increases in p-Mnk1 and p-eIF4E immunoreactivity in both whole homogenates and synaptoneurosomes were ERK dependent (U0126: 20 μm, 30 min). B, Representative micrograph of DHPG-induced increase in p-Mnk1 immunoreactivity in area CA1. s.p., Stratum pyramidal; s.r., stratum radiatum. C, Preincubation with p38 inhibitor SB203580 (1 μm, 1 h) reduced basal Mnk1 and eIF4E phosphorylation but did not significantly attenuate DHPG-induced Mnk1 activation and eIF4E phosphorylation (n = 4). Representative Western blots are shown in each panel. The cumulative data represent mean + SEM. *p < 0.05, statistical significance determined by Student’s t test. CTL, Control.

Activation of group I mGluRs results in increased 4E-BP phosphorylation via PI3K. A, Representative micrographs demonstrating that DHPG application (50 μm, 5 min) resulted in an increase in pp-4E-BP immunoreactivity in area CA1. The DHPG-induced increase in 4E-BP phosphorylation was blocked by preincubation with LY294002 (50 μm, 30 min) but not U0126 (20 μm, 30 min) (n = 4 slices; 4 mice per condition). s.p., Stratum pyramidal; s.r., stratum radiatum. B, Bar graph showing quantification of the immunocytochemical experiments in A. The cumulative data represent mean + SEM, and the number is indicated in the bars. *p < 0.05, statistical significance determined by Student’s t test. CTL, Control; LY, LY294002.

DHPG-induced regulation of eIF4F requires PI3K/mTOR signaling. A, The PI3K inhibitors did not significantly alter the DHPG-induced activation of either ERK2 or Mnk1. B, The mTOR inhibitor rapamycin (20 nm, 30 min) did not significantly alter the DHPG-induced activation of either ERK2 or Mnk1. C, The PI3K inhibitors LY294002 (50 μm, 30 min) and wortmannin (100 nm, 30 min) blocked the DHPG-induced increase in eIF4E phosphorylation. D, The mTOR inhibitor rapamycin (20 nm, 30 min) blocked the DHPG-induced increase in eIF4E phosphorylation. E, DHPG-induced increase in eIF4E phosphorylation occurs in 4E-BP2 knock-out mice but is not sensitive to rapamycin (20 nm, 30 min). Representative Western blots are shown in each panel. The cumulative data represent mean + SEM, and the number is indicated in the bars. *p < 0.05, statistical significance determined by Student’s t test. CTL, Control; LY, LY294002; wort, wortmannin; rap, rapamycin.

DHPG-induced mGluR-LTD is enhanced in 4E-BP2 knock-out mice. A, DHPG application (50 μm, 10 min) induced LTD in wild-type slices that was sensitive to preincubation with U0126 (20 μm, 1 h) (ANOVA; p < 0.0001) but not the inactive analog U0124 (20 μm, 1 h) (n = 5 slices; 5 mice per condition). B, DHPG application (50 μm, 10 min) induced LTD in 4E-BP2 knock-out slices that was sensitive to preincubation with U0126 (20 μm, 1 h) (ANOVA; p < 0.0001) but not the inactive analog U0124 (20 μm, 1 h) (n = 5 slices; 5 mice per condition). C, DHPG-induced LTD is enhanced in 4E-BP2 knock-out slices. The cumulative data are mean + SEM recapitulated from A and B for the indicated time periods. *p < 0.05, statistical significance determined by Student’s t test. D, DHPG-induced LTD in 4E-BP2 knock-out mice is insensitive to rapamycin (n = 4 slices; 4 mice per condition). WT, wild type; KO, knock-out.

eIF4F complex formation is upregulated during DHPG-induced mGluR-LTD. Basal coimmunoprecipitation of eIF4E with eIF4G was elevated in 4E-BP2 knock-out (KO) slices (n = 7 slices; 7 mice per genotype). mGluR-LTD is associated with increased coimmunoprecipitation of eIF4E with eIF4G in wild-type (WT) slices but not in 4E-BP2 knock-out slices 10 min after DHPG application (n = 7 slices; 7 mice per genotype). Rapamycin (rap) inhibited the DHPG-induced eIF4G–eIF4E association in wild-type but not in 4E-BP2 knock-out slices (n = 3 slices; 3 mice per genotype). Representative eIF4G and eIF4E Western blots are shown for each condition along with bead alone (-Ab_WT) control. The cumulative data represent mean + SEM. *p < 0.05, statistical significance determined by Student’s t test. CTL, Control.