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. 2008 Jul 10;59(1):70-83.
doi: 10.1016/j.neuron.2008.05.023.

Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD

Sungjin Park  1 , Joo Min Park, Sangmok Kim, Jin-Ah Kim, Jason D Shepherd, Constance L Smith-Hicks, Shoaib Chowdhury, Walter Kaufmann, Dietmar Kuhl, Alexey G Ryazanov, Richard L Huganir, David J Linden, Paul F Worley
Affiliations

Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD

Sungjin Park et al. Neuron. .

Abstract

Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

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Figures

Figure 1
Figure 1. Arc/Arg3.1 Is Required for Hippocampal mGluR-LTD
Field excitatory postsynaptic potentials (fEPSPs) were recorded in the hippocampal Schaffer collateral-CA1 synapses derived from Arc/Arg3.1 KO mice and compared to WT littermate controls. (A) Average time course of the change in fEPSP slope induced by the group I mGluR agonist (R,S)-DHPG (50 μM, for 5 min). LTD of WT mice was 72.8% ± 2.0% of baseline at t = 70 min (n = 10). In Arc/Arg3.1 KO, fEPSPs were 92.1% ± 3.7% of the baseline at t = 70 min (n = 9). p < 0.001 when compared to littermate WT. Error bars indicate the standard error of the mean. Measurements correspond to the time points indicated on the time course graph in this and all subsequent figures. (B) Time course of the change in fEPSP slope produced by paired-pulse low-frequency stimulation (PP-LFS: at 1 Hz, 50 ms interstimulus interval, for 15 min) in the presence of the NMDA receptor antagonist D-APV (50 μM). LTD of WT mice was 79.9% ± 2.1% of baseline at t = 80 min (n = 12). In Arc/Arg3.1 KO mice, fEPSPs were 94.3% ± 2.1% of the baseline at t = 80 min (n = 13). p < 0.0001. Scale bars, 0.5 mV/10 ms. (C) Five minutes of DHPG application resulted in a loss of surface GluR1 at 15 min (n = 20, ***p < 0.005) and 60 min (n = 19, *p < 0.05) after DHPG application, compared to untreated controls in WT hippocampal cultures. Arc/Arg3.1 KO neurons did not exhibit any changes in surface GluR1 levels after DHPG treatment. Representative pictures of cultures are shown using an LUT scale where white is high intensity and dark red is low intensity. CTL, control. (D) Five minutes of DHPG application resulted in an increase of internalized GluR1 at 15 min (n = 20, *p < 0.05) compared with untreated cultures. Arc/Arg3.1 KO neurons did not exhibit changes in internalized GluR1 levels after DHPG treatment. Error bars indicate SEM in this and all subsequent figures.
Figure 2
Figure 2. Arc/Arg3.1 Protein Is Rapidly Synthesized by Group I mGluR Activation
(A) Stimulation of hippocampal neurons with DHPG (50 μM) for 5 min increased Arc/Arg3.1 immunoreactivity in both cell body (1.34 ± 0.063 of untreated soma, n = 13) and dendrites (1.58 ± 0.095 of untreated dendrites, n = 38). The rapid increase of Arc/Arg3.1 was blocked by the protein synthesis inhibitor emetine (10 ng/ml, 10 min). (B) High-dose cycloheximide (CHX, 50 μM, total 10 min: 5 min pretreatment and 5 min with or without DHPG) blocked the induction of Arc/Arg3.1 by DHPG (5 min). (C) Transcription inhibitor actinomycin D (ActD: 10 μM, 5 min pretreatment and 5 min with or without DHPG) did not block the induction of Arc/Arg3.1 by DHPG (5 min). (D) Low-dose CHX increased the level of Arc/Arg3.1 protein. Neurons were treated with vehicle or various doses of CHX for 10 min. Total protein synthesis was measured by counting the incorporation of 35S methionine and cysteine in TCA precipitant. (E) Statistical analysis of western blots. Five minute treatment of DHPG significantly increased the level of Arc/Arg3.1. Inhibition of new protein synthesis by high dose of cycloheximide not only blocked the induction of Arc/Arg3.1 protein but also slightly decreased the level of Arc/Arg3.1 upon stimulation with DHPG. Inhibition of transcription by actinomycin D did not affect the level of Arc/Arg3.1. Low-dose CHX (50–100 nM, 5 min pretreatment and 5 min with or without DHPG) increased the level of Arc/Arg3.1, which was not further induced by DHPG. *p < 0.05, **p < 0.01. (F) The level of Arc/Arg3.1 mRNA was measured using real-time RT-PCR. Stimulation of neurons with BDNF (10 ng/ml) and forskolin (50 μM) induced the level of Arc/Arg3.1 mRNA 40 min and 20 min after stimulation, respectively. DHPG slightly increased the level of Arc/Arg3.1 mRNA at 20 and 40 min after stimulation. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 3
Figure 3. eEF2K Binds Homer and mGluR1/5
(A) Schematic diagram of eEF2K. The N terminus of eEF2K contains a Ca2+/calmodulin (CaM) binding motif and an α-kinase domain. The C-terminal eEF2 targeting domain, which recruits the substrate, eEF2, is linked to the hyperphosphorylated internal region. Putative Homer binding site is shown above the diagram. (B) Coimmunoprecipitation (co-IP) of eEF2K and Homer. HA-tagged (HA-) eEF2K was coexpressed with myc-tagged WT, W27A, or G91N Homer3 in HEK293T cells, and IP was performed with antimyc antibody. HA-eEF2K co-IPed with WT or W27A Homer 3 was coexpressed, but not with G91N Homer. (C) mGluR5 increases the interaction of eEF2K and Homer. HA-eEF2K was transfected with or without HA-mGluR5. IP was performed by anti-Homer2 antibody, which IPed endogenous Homer2 protein. Western blot was performed using anti-HA antibody. Co-IP of HA-eEF2K was increased when mGluR5 was coexpressed. (D) eEF2K co-IPs with mGluR5. HEK293T cells were transfected with HA-eEF2K with or without HA-mGluR5, and lysates were IPed with anti-mGluR5 antibody and blotted with anti-HA antibody. eEF2K co-IPed only when mGluR was coexpressed. Samples were boiled before loading to aggregate and separate mGluR5 monomer from eEF2K. (E) mGluR1 co-IPs with eEF2K. HEK293T cells were transfected with mGluR1 and eEF2K, and lysates were IPed with mycAb. Samples were not boiled to show the monomer of mGluRs. (F and G) mGluR2 and mGluR4 do not co-IP with eEF2K.
Figure 4
Figure 4. Dynamic Interaction of eEF2K and mGluR5
(A) Calcium dissociates eEF2K from mGluR5. HEK293T cells were transfected with HA-eEF2K with or without myc-mGluR5, and cells were harvested with lysis buffer without calcium or containing various concentrations of free calcium. Calmodulin (CaM) (25 μg/ml) was also added to the lysis buffer as indicated. Binding was decreased at [Ca2+] higher than 10 μM. (B) Phospho-eEF2 was not detected in the hippocampus of eEF2K KO, while the level of total eEF2, GluR1, Glur2/3, mGluR5, α-CaMKII, Arc/Arg3.1, and actin was not altered in eEF2K KO mice compared to WT littermate controls. (C) Synaptoneurosomes, prepared from the forebrain of eEF2K KO and WT mice, were stimulated with vehicle or DHPG for 20 min. Synaptoneurosomes were then lysed and immunoprecipitated with anti-eEF2K antibody. mGluR5 co-IPed with eEF2K only in WT samples. Stimulation of synaptoneurosomes with DHPG decreased the co-IP of mGluR5.
Figure 5
Figure 5. Rapid Induction of Arc/Arg3.1 by Group I mGluRs Is Dependent on eEF2K
(A) Hippocampal slices were prepared from WT and eEF2K KO mice and were stimulated with DHPG for 5 min. phospho-eEF2 (p-eEF2, red) in area CA1 was increased by DHPG within 5 min and declined by 30 min following washout. Specificity of phospho-eEF2 was confirmed by staining of eEF2K KO slices. s.p., stratum pyramidal; s.r., stratum radiatum. (B) Cultured hippocampal neurons were treated with DHPG for 5 min and stained with phospho-eEF2 (red) and PSD95 (green) antibodies on DIV14. phospho-eEF2 showed punctal distribution in dendritic spines and dendritic shafts. phospho-eEF2 in spines colocalized with PSD95 (arrows). (B2), (B3), and (B4) are enlarged images of the rectangular region of (B1). (C and D) mGluR-dependent rapid synthesis of Arc/Arg3.1 is absent in eEF2K KO neurons. Neurons from the forebrains of WT or eEF2K KO mice were cultured for DIV14 and treated with DHPG (50 μM, 5 min). Phosphorylation of eEF2 was undetectable in eEF2K KO neurons. No difference in the level of mGluR5 was observed between WT and eEF2K KO neurons. An arrowhead indicates a non-specific band. p values were obtained by paired t test comparing basal and drug-treated levels. p values for comparison of WT and eEF2K KO mice were obtained by Student’s t test. *p < 0.05, **p < 0.01, n = 8. Error bars are SEM. (E) Arc/Arg3.1 mRNA expression is not altered in eEF2K KO neurons. The level of Arc/Arg3.1 mRNA was measured in WT and eEF2K KO neurons following the stimulation with DHPG. (F) Low-dose cycloheximide (CHX) increases Arc/Arg3.1 protein expression. Cultured eEF2K KO neurons were treated with indicated doses of CHX for 10 min. *p < 0.05, n = 8.
Figure 6
Figure 6. mGluR-LTD Is Impaired in Hippocampal Slices Derived from eEF2K KO Mice
fEPSPs were recorded in the hippocampal CA1 region of slices derived from eEF2K KO mice and compared to WT littermate controls. (A) Time course of the change in fEPSP slope produced by paired-pulse low-frequency stimulation (PP-LFS: at 1 Hz, 50 ms interstimulus interval, for 15 min) in the presence of D-APV (50 μM). LTD of WT mice was 77.0% ± 2.1% of baseline at t = 75 min (n = 13). In eEF2K KO mice, fEPSPs were 97.5% ± 2.4% of baseline t = 75 min (n = 15) (p < 0.0001). (B) Time course of the change in fEPSP slope by low-frequency stimulation (LFS: 1 Hz for 15 min). This form of NMDAR-dependent LTD was not altered in eEF2K KO hippocampal slices (72.7% ± 2.2% of baseline at t = 75 min, n = 9) compared to WT (73.1% ± 3.4% of baseline at t = 75 min, n = 7) (p > 0.5). (C) Late-phase of LTP was induced by four stimulus trains (100 Hz each) with an intertrain interval of 3 s. In WT, fEPSPs were increased to 171.5% ± 13.4% of baseline immediately after stimulation (t = 30 min) and were sustained at the level of 138.4% ± 7.7% of baseline at t = 175 min (n = 6). However, in eEF2K KO, the initial LTP (204.6% ± 8.9% of baseline at t = 30 min) was maintained for 3 hr after stimulation (200.1% ± 11.9% of baseline at t = 175 min, n = 5). LTP was significantly greater in slices derived from eEF2K KO mice compared to those from WT mice at this time point (p < 0.005). (D) Average time course of the change in fEPSP slope induced by DHPG (50 μM, for 5 min). LTD of WT mice was 64.7% ± 5.2% of baseline at t = 90 min (n = 7). In eEF2K KO mice, LTD was significantly impaired (108.7% ± 3.6% of baseline at t = 90 min, n = 8). Treatment with low-dose cycloheximide (LD-CHX, 50–75 nM) for 10 min starting from 5 min prior to DHPG restored DHPG-LTD in eEF2K KO (75.7% ± 7.4%, n = 5). In WT mice, treatment with LD-CHX did not alter the expression of LTD (69.0% ± 2.6%, n = 5). p < 0.001 when eEF2K KO DHPG only was compared to eEF2K KO LD-CHX + DHPG, WT DHPG only, or WT LD-CHX + DHPG. Scale bars, 0.5 mV/10 ms.
Figure 7
Figure 7. LTD Is Impaired in Hippocampal Slices Derived from Arc/Arg3.1/Fmr1 Double KO Mice
(A) DIV14 Fmr1 KO neurons were treated with DHPG as indicated in Figure 5C. Rapid but not delayed synthesis of Arc/Arg3.1 was absent in Fmr1 KO. The regulation of phospho-eEF2 was intact in Fmr1 KO neurons. (B) High-dose cycloheximide (60 μM: HD-CHX) did not block DHPG-LTD of Fmr1 KO slices. In the presence of a high dose of cycloheximide, DHPG-LTD of Fmr1 KO was 72.3% ± 4.8% of baseline at t = 105 min (n = 5), while DHPG-LTD in WT (FVB) slices was blocked (fEPSP was 95.5% ± 2.9% of baseline at t = 105 min (n = 4); p < 0.01 when Fmr1 KO was compared to FVB WT). (C) Average time course of fEPSP slope of Arc/Arg3.1/Fmr1 double KO (DKO) mice. mGluR-LTD was induced by DHPG (50 μM, for 5 min). DHPG-LTD of Arc/Arg3.1/Fmr1 DKO was 85.9% ± 4.1% of baseline at t = 75 min (n = 8). In Fmr1 KO, DHPG-LTD was 68.2% ± 2.6% of baseline at t = 75 min (n = 6). In WT, DHPG-LTD was 73.0% ± 6.6% of baseline at t = 75 min (n = 5). p < 0.01 when Arc/Arg3.1/Fmr1 DKO was compared to either WT or Fmr1 KO. fEPSPs of post-DHPG in Fmr1 KO were not significantly different from those in FVB WT. (D) Time course of the change in fEPSP slope by PP-LFS. PP-LFS LTD of Arc/Arg3.1/Fmr1 DKO was 88.3% ± 2.1% of baseline at t = 65 min (n = 6). In Fmr1 KO, PP-LFS LTD was 75.5% ± 3.7% of baseline at t = 65 min (n = 8). In FVB WT, PP-LFS LTD was 80.5% ± 2.6% of baseline at t = 65 min (n = 8). p < 0.05 when Arc/Arg3.1/Fmr1 DKO was compared to either WT or Fmr1 KO. fEPSPs of post-DHPG in Fmr1 KO were not significantly different from those in FVB WT (p = 0.4). Scale bars, 0.5 mV/10 ms.
Figure 8
Figure 8. eEF2K, FMRP, and Rapid De Novo Translation of Arc/Arg3.1 Protein in mGluR-LTD
Group I mGluRs activate eEF2K via calcium-calmodulin (CaM). eEF2K phosphorylates eEF2, which inhibits elongation generally but increases Arc/Arg3.1 translation. Arc/Arg3.1 forms a complex with endophilin2/3 (Endo) and dynamin (Dyn) and induces the internalization of AMPAR (Chowdhury et al., 2006). FMRP inhibits the translation of Arc/Arg3.1 at the basal state. Arc/Arg3.1 induction alone is not sufficient for mGluR-LTD, indicating that mGluR activates another pathway that is required to internalize AMPAR (Cho et al., 2008). In Fmr1 KO mice, the synthesis of Arc/Arg3.1 protein is constitutively derepressed, and de novo synthesis of Arc/Arg3.1 is not required for mGluR-LTD.
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