Translational control of depression-like behavior via phosphorylation of eukaryotic translation initiation factor 4E

Abstract

Translation of mRNA into protein has a fundamental role in neurodevelopment, plasticity, and memory formation; however, its contribution in the pathophysiology of depressive disorders is not fully understood. We investigated the involvement of MNK1/2 (MAPK-interacting serine/threonine-protein kinase 1 and 2) and their target, eIF4E (eukaryotic initiation factor 4E), in depression-like behavior in mice. Mice carrying a mutation in eIF4E for the MNK1/2 phosphorylation site (Ser209Ala, Eif4e ki/ki), the Mnk1/2 double knockout mice (Mnk1/2^-/-), or mice treated with the MNK1/2 inhibitor, cercosporamide, displayed anxiety- and depression-like behaviors, impaired serotonin-induced excitatory synaptic activity in the prefrontal cortex, and diminished firing of the dorsal raphe neurons. In Eif4e ki/ki mice, brain IκBα, was decreased, while the NF-κB target, TNFα was elevated. TNFα inhibition in Eif4e ki/ki mice rescued, whereas TNFα administration to wild-type mice mimicked the depression-like behaviors and 5-HT synaptic deficits. We conclude that eIF4E phosphorylation modulates depression-like behavior through regulation of inflammatory responses.

Fig. 1

Genetic inhibition of eIF4E phosphorylation induced anxiety- and depression-like behaviors. a Male (M) and female (F) mice mutant for the genes encoding MNK1 and MNK2 (Mnk1/2^−/− mice) and their wild-type littermates (Mnk1/2^+/+) were assessed for despair-like behavior in the forced swim test (FST) (M, n = 8 Mnk1/2^+/+, n = 9 Mnk1/2^−/−; F, n = 3 Mnk1/2^+/+, n = 9 Mnk1/2^−/−). b Male and female mice bearing a mutation in the Eif4e gene (Ser209 was substituted to Ala, Eif4e ki/ki) were also tested in the FST (M, n = 19 Eif4e^+/+, n = 19 Eif4e ki/ki; F, n = 7 Eif4e+/+, n = 8 Eif4e ki/ki). c Novelty suppressed feeding (NSF) was assessed in Mnk1/2^−/− (M, n = 9; F, n = 10) and wild-type mice (M, n = 8; F, n = 10). d NSF was also assayed in Eif4e^+/+ and ki/ki mice (M, n = 9 Eif4e^+/+, n = 9 Eif4e ki/ki; F, n = 8 Eif4e^+/+, n = 9 Eif4e ki/ki). e Latency to feed in the home cage (home cage feeding, HCF) was determined in the Mnk1/2^−/− and f Eif4e ki/ki mice and their littermates. g Time spent in the center of an open field (OF) arena was measured in Mnk1/2^−/− (M, n = 12 Mnk1/2^+/+, n = 8 Mnk1/2^−/−; F, n = 7 Mnk1/2^+/+, n = 13 Mnk1/2^−/−) and h Eif4e ki/ki mice and their littermates (M, n = 17 Eif4e^+/+, n = 18 Eif4e ki/ki; F, n = 11 Eif4e^+/+, n = 19 Eif4e ki/ki). i Locomotion was also measured in Mnk1/2^−/− and j Eif4e ki/ki mice. ** p < 0.01, *** p < 0.001 vs. wild-type littermates (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 2

Serotonergic dysfunction in Eif4e ki/ki mice. a Representative whole cell recordings of spontaneous excitatory post-synaptic currents (sEPSCs) from layer V pyramidal neurons of the medial prefrontal cortex (mPFC) of wild-type and Eif4e ki/ki mice. Traces represent consecutive recordings of synaptic currents before (baseline) and after 5-HT (20 μM) treatment from single pyramidal cells in each group. b Frequency of sEPSCs from pyramidal neurons of wild-type and Eif4e ki/ki mice before (−) and after 5-HT at 20 μM (n = 7 cells from 5 Eif4e^+/+; n = 8 cells from 7 Eif4e ki/ki), 50 μM (n = 4 cells from 3 Eif4e^+/+; n = 5 cells from 3 Eif4e ki/ki) or 100 μM (n = 5 cells from 3 Eif4e^+/+; n = 6 cells from 3 Eif4e ki/ki). c In vivo single-unit extracellular recordings of dorsal raphe neurons were performed in anesthetized Eif4e^+/+and ki/ki mice. d Firing rate of dorsal raphe 5-HT neurons, measured in Eif4e^+/+and ki/ki mice (n = 56 single unit recordings from 8 Eif4e^+/+mice; n = 43 single unit recordings from 7 Eif4e ki/ki mice). e 5-HT neurons recorded per descent (track) in the dorsal raphe of wild-type and Eif4e ki/ki mice (n = 8 Eif4e^+/+mice; n = 7 Eif4e ki/ki mice). f Firing rate of non-5-HT neurons in the DR (n = 27 single unit recordings from 6 Eif4e^+/+mice; n = 16 single unit recordings from 5 Eif4e ki/ki mice). g Number of non-5-HT neurons per descent in the DR (n = 6 Eif4e^+/+mice; n = 5 Eif4e ki/ki mice). h Number of NeuN + cells in the DR nuclei, which was subdivided into DR dorsal part (DRD), DR lateral part (DRL), posterodorsal raphe nucleus (PRN), and DR ventral part (DRV). Data are means of 4 slides per animal (n = 4 Eif4e^+/+mice; n = 5 Eif4e ki/ki mice). i Number of tryptophan hydroxylase 2 (TPH2) positive cells in the DRN. j Representative images of the DR IHC; blue: nuclear DAPI staining; red: TPH2; green: NeuN. Scale bar is 100 μM. * p < 0.05, *** p < 0.001 vs. wild-type littermates (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 3

Normal response to Fluoxetine in Eif4e ki/ki mice. a Male wild-type and Mnk1/2^−/− mice were treated with saline or fluoxetine (IP, 3 mg kg⁻¹) and tested in the FST after 30 min (n = 7 Mnk1/2^+/+Saline, n = 9 Mnk1/2^−/− Saline; n = 10 Mnk1/2^+/+Fluoxetine, n = 10 Mnk1/2^−/− Fluoxetine). b Male Eif4e^+/+ and ki/ki were also treated with saline or fluoxetine (IP, 3 mg kg⁻¹) and performed the FST after 30 min (n = 9 Eif4e^+/+Saline, n = 8 Eif4e ki/ki Saline; n = 6 Eif4e^+/+Fluoxetine, n = 9 Eif4e ki/ki Fluoxetine). c eIF4E phosphorylation in the mPFC/HPC of male wild-type mice treated with saline, 1 dose of fluoxetine (0.5 h, IP, 3 mg kg⁻¹), chronic fluoxetine (14 × , IP, 10 mg kg⁻¹) or repeated citalopram (3 × , IP, 10 mg kg⁻¹) (n = 6/group). d Total eIF4E levels in the same samples as in (d). e Representative western blots for mice treated with vehicle or SSRIs. ** p < 0.01, *** p < 0.001 vs wild-type (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 4

Pharmacological inhibition of MNK1/2 induces despair-like behavior and 5-HT synaptic impairments. a Cercosporamide, an MNK1 and MNK2 inhibitor, was administered for 5 consecutive days to wild-type mice (IP, 20 mg kg⁻¹). b Representative western blot images for phosphorylated eIF4E (p-eIF4E), total eIF4E and GAPDH in the cortex-hippocampus of mice treated with vehicle or cercosporamide. c Average levels of p-eIF4E in vehicle and cercosporamide-treated wild-type mice (vehicle (0 mg kg⁻¹), n = 7; cercosporamide, n = 7). d Cercosporamide administration induced increased immobility in the Forced Swim Test (FST) in wild-type mice (vehicle (0 mg kg⁻¹), n = 8; cercosporamide, n = 7). e Representative sEPSC traces from recordings in layer V mPFC from wild-type or Eif4e ki/ki mice treated with vehicle or cercosporamide. f Layer V mPFC sEPSC frequency of wild-type (left panel, vehicle n = 5 neurons from three mice, cercosporamide n = 6 neurons from two mice) or Eif4e ki/ki mice (right panel, vehicle n = 5 neurons from 2 mice, cercosporamide n = 7 neurons from 3 mice) treated with vehicle or cercosporamide. g Firing rate of 5-HT DR neurons in wild-type and Eif4e ki/ki mice treated with vehicle or cercosporamide (Eif4e^+/+: Vehicle 33 neurons from 6 mice, cercosporamide 22 neurons from 10 mice; Eif4e ki/ki: vehicle 22 neurons from 4 mice, cercosporamide 18 neurons from 4 mice). * p < 0.05, ** p < 0.01, ***p < 0.001 vs vehicle-treated wild-type mice; & p < 0.05 vs. saline-treated Eif4e^+/+ (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 5

Translation of Nfkbia mRNA is reduced in Eif4e ki/ki mice. a Representative polysome profile of cortex-hippocampus from Eif4e^+/+and ki/ki mice (1 mouse per genotype). b Distribution of the Nfkbia mRNA in the polysome fractions of wild-type and Eif4e ki/ki mice (n = 4 Eif4e^+/+; n = 5 Eif4e ki/ki). c, Actb mRNA was measured in both mouse strains as a control. d Total mRNA levels of Nfkbia in the cortex-hippocampus of Eif4e^+/+and ki/ki mice. e Average protein levels and representative western blot images of the Nfkbia mRNA product, IκBα, in the mPFC of Eif4e wild-type and ki/ki mice (n = 6/group). f Average protein levels and representative western blot images IκBα, in the mPFC of Mnk1/2^+/+ and ^−/− mice (n = 7 Mnk1/2^+/+; n = 8 Mnk1/2^−/−). g Levels of the cytokine tumor necrosis factor α (TNFα) in the mPFC in wild-type and Eif4e ki/ki mice (n = 9 Eif4e^+/+; n = 10 Eif4e ki/ki). h Total levels of Tnf mRNA in the mPFC in wild-type and Eif4e mutant mice (n = 10 Eif4e^+/+; n = 12 Eif4e ki/ki). i mRNA levels of the microglial marker Iba1 (Aif1) in the mPFC in wild-type and Eif4e mutant mice (n = 6 Eif4e^+/+; n = 5 Eif4e ki/ki). j Number of Iba1 positive cells (Iba1+) per 0.01 mm² of mPFC from wild-type and Eif4e ki/ki mice 48 h after saline or LPS (IP, 2.5 mg kg⁻¹) treatment (n = 3/group). k Representative images of the mPFC of wild-type or Eif4e ki/ki mice 48 h after saline or LPS treatment. l mRNA levels of Il1b in the mPFC of wild-type or Eif4e ki/ki mice 48 h after saline or LPS treatment (n = 6 Eif4e^+/+Saline, n = 6 Eif4e ki/ki Saline, n = 3 Eif4e^+/+LPS; n = 4 Eif4e ki/ki LPS). * p < 0.05, *** p < 0.001 vs. wild-type; &&& p < 0.001 vs. saline-treated Eif4e ki/ki (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 6

TNFα causes behavioral impairments in Eif4e ki/ki mice. a wild-type and Eif4e ki/ki mice were administered (ICV) a dominant negative mutant version of TNFα (DN TNF) or vehicle for 12 days (via an osmotic pump connected to a guide cannula). Male mice were then evaluated in the FST (n = 11 Eif4e^+/+ Saline, n = 7 Eif4e^+/+ DN TNF, n = 10 Eif4e ki/ki Saline, n = 8 Eif4e ki/ki DN TNF). b Mice were also tested in the novelty suppressed feeding task (n = 10 Eif4e^+/+ Saline, n = 8 Eif4e^+/+ DN TNF, n = 8 Eif4e ki/ki Saline, n = 8 Eif4e ki/ki DN TNF). c Latency to feed in their home cage was also measured. d Female mice also administered with vehicle or DN TNF (ICV) were evaluated in the FST (n = 7 Eif4e^+/+ Saline, n = 8 Eif4e^+/+ DN TNF, n = 8 Eif4e ki/ki Saline, n = 8 Eif4e ki/ki DN TNF). e Female mice exploratory behavior was also measured in the center of an open field (OF) (n = 7 Eif4e^+/+ Saline, n = 10 Eif4e^+/+ DN TNF, n = 10 Eif4e ki/ki Saline, n = 9 Eif4e ki/ki DN TNF). f Their locomotion in the OF was also measured. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. wild-type saline; &, p < 0.05, && p < 0.01, &&& p < 0.001 vs. saline-treated Eif4e^+/+or ki/ki (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 7

TNFα causes serotonergic impairments in Eif4e ki/ki mice. a Representative sEPSC traces from mPFC pyramidal neurons in brain slices of wild-type mice treated with vehicle (saline), DN TNF (200 ng ml⁻¹) or mouse recombinant TNFα (10 ng ml⁻¹). Synaptic currents were recorded before (baseline) and after 5-HT (20 μM) administration. b Representative sEPSC traces from Eif4e ki/ki mice treated with either vehicle or DN TNF (200 ng ml⁻¹), measured before and after 5-HT (20 μM) administration. c Frequency of sEPSCs in mPFC slices of male and female wild-type mice. Slices were directly treated with saline (M: n = 10 cells from 6 mice; F: n = 9 cells from 6 mice), DN TNF (200 ng ml⁻¹; M: n = 8 cells from 5 mice; F: n = 7 cells from 4 mice) or mouse recombinant TNFα (10 ng ml⁻¹; M: n = 9 cells from 6 mice; F: n = 6 cells from 5 mice), pre- and post-5-HT (20 μM) application. d Average sEPSC frequency in Eif4e ki/ki mice before and after 5-HT (20 μM) application; slices were treated with either saline (M: n = 11 cells from 6 mice; F: n = 9 cells from 7 mice) or DN TNF (200 ng ml⁻¹; M: n = 8 cells from 6 mice; F: n = 9 cells from 7 mice). e, Firing rate of DR neurons in wild-type and Eif4e ki/ki mice chronically treated with DN TNF or saline (ICV, 12 days; n = 33 recordings from 6 Eif4e^+/+ Saline mice; n = 31 recordings from 5 Eif4e^+/+ DN TNF mice; n = 16 recordings from Eif4e ki/ki Saline mice; n = 33 recordings from 6 Eif4e ki/ki DN TNF mice). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. wild-type saline; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. saline-treated Eif4e^+/+or ki/ki (see Supplementary Table 2 for detailed results of statistical tests)

Fig. 8

Simultaneous inhibition of mPFC and DR by TNFα underlies despair-like behavior. a Acute recombinant mouse TNFα (ICV, 0.02 pg ml⁻¹) into wild-type mice. b Firing rate of DR neurons in wild-type mice treated with either artificial cerebrospinal fluid (ACSF) (n = 4) or mouse recombinant TNFα (n = 6) averaged in 5 minute bins. c Representative integrated firing rate histograms (spikes per 10 s) showing traces of DR neurons in wild-type mice treated with either saline or mouse recombinant TNFα. d Acute treatment of TNFα (0.1 fg in 0.5 µl ACSF) into the mPFC of wild-type mice or DN TNF (100 pg in 0.5 μl ACSF) to Eif4e ki/ki mice. d Firing rate of DRN 5-HT neurons of wild-type mice treated with ACSF (n = 6) or TNFα (n = 6) into the mPFC. e Immobility time in the FST, 20 min after ACSF (n = 6) or TNFα (n = 7) into the mPFC of wild-type mice. f, Firing rate of DRN 5-HT neurons of Eif4e ki/ki mice treated with ACSF (n = 5) or DN TNF (n = 5) into the mPFC. g FST immobility time 20 min after ACSF (n = 6) or DN TNF (n = 7) into the mPFC of Eif4e ki/ki mice. h Similar treatments were performed into the DRN. i 5-HT DR neuron firing rate after ACSF (n = 7) or TNFα (n = 6) in wild-type mice. j Despair-like behavior in wild-type mice treated with ACSF (n = 6) or TNFα (n = 10). k DRN 5-HT neurons in Eif4e ki/ki treated with ACSF (n = 5) or DN TNF (n = 4). l FST immobility in Eif4e ki/ki treated with ACSF (n = 8) or DN TNF (n = 12). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. wild-type ACSF (see Supplementary Table 2 for detailed results of statistical tests)