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Translational control of mGluR-dependent long-term depression and object-place learning by eIF2α

Abstract

At hippocampal synapses, activation of group I metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD), which requires new protein synthesis. However, the underlying mechanism remains elusive. Here we describe the translational program that underlies mGluR-LTD and identify the translation factor eIF2α as its master effector. Genetically reducing eIF2α phosphorylation, or specifically blocking the translation controlled by eIF2α phosphorylation, prevented mGluR-LTD and the internalization of surface AMPA receptors (AMPARs). Conversely, direct phosphorylation of eIF2α, bypassing mGluR activation, triggered a sustained LTD and removal of surface AMPARs. Combining polysome profiling and RNA sequencing, we identified the mRNAs translationally upregulated during mGluR-LTD. Translation of one of these mRNAs, oligophrenin-1, mediates the LTD induced by eIF2α phosphorylation. Mice deficient in phospho-eIF2α–mediated translation are impaired in object-place learning, a behavioral task that induces hippocampal mGluR-LTD in vivo. Our findings identify a new model of mGluR-LTD, which promises to be of value in the treatment of mGluR-LTD-linked cognitive disorders.

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Figure 1: Deficient eIF2α phosphorylation selectively prevents protein synthesis-dependent mGluR-LTD.
Figure 2: Direct stimulation of eIF2α phosphorylation induces LTD.
Figure 3: mGluR-LTD requires eIF2α-mediated translational control.
Figure 4: Activation of mGluR selectively promotes translation of Ophn1 mRNA by eIF2α phosphorylation.
Figure 5: eIF2α phosphorylation selectively mediates the translation of OPHN1 required for mGluR-LTD.
Figure 6: At hippocampal synapses phosphorylation of eIF2α and surface GluR1 immunoreactivity are negatively correlated.
Figure 7: Increased eIF2α-mediated translational control is needed for successful learning of novel object-space configuration.

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Acknowledgements

We thank K. Nakazawa and L. Van Aelst for the fPKR frozen embryos and Ophn1 shRNA, respectively. This work was supported by grants from the US National Institutes of Health to M.C.-M. (NIMH 096816, NINDS 076708) and R.J.K. (DK042394, DK088227, HL052173), the Intellectual Disability Research Center (P30HD024064) and Dan L. Duncan Cancer Center (P30CA125123) Genomic and RNA Profiling Cores, and the Cancer Prevention and Research Institute of Texas (CPRIT) RP100861.

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Authors

Contributions

M.C.-M., G.V.D.P., W.H. and S.A.B. designed the experiments and wrote the manuscript; G.V.D.P. conducted electrophysiology and behavioral experiments and analyzed data; W.H. conducted behavioral, polysome profiling, qRT-PCR and immunoblotting experiments and analyzed data; S.A.B. conducted neuron culture, immunostaining, SUnSET and immunoblotting experiments and analyzed data; C.-C.H. performed firefly luciferase reporter experiments; P.B. analyzed RNA-seq data; A.P. contributed to the discussion of the electrophysiological experiments; C.S. and R.K. contributed to the characterization of ISRIB and the generation of Eif2s1A/A;ftg mice, respectively; K.K. and P.W. contributed to in-depth discussion of the project and editing of the manuscript.

Corresponding author

Correspondence to Mauro Costa-Mattioli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Reduced eIF2α phosphorylation in the hippocampus of Eif2s1S/A mice, breeding strategy used to generate Eif2s1A/A;ftg mice and demonstration that DHPG elicited a normal mGluR-LTD in GFP-positive neurons expressed in WT mice.

a) Western blots show that compared to CA1 from WT Eif2s1S/S mice, in Eif2s1S/A mice, p-eIF2α is significantly reduced (n=3 mice, t=3.9, p=0.02). Results are displayed as mean ± SEM. Statistical significance was assessed by unpaired Student's t-test. b) Eif2s1S/S;ftg were first crossed to Eif2s1S/A mice. The resulting heterozygous transgenic (Eif2s1S/A;ftg) were then bred with Eif2s1S/A mice, which resulted in the generation of Eif2s1A/A;ftg. c) Application of DHPG (100mM, 5 min) elicited LTD in AAV-Cre-GFP+ neurons from WT mice (n=8 cells from 4 mice, t=11.38, p=9X10−6), indicating that the presence of GFP has no effect on LTD. Calibrations: 10 ms, 40 pA. Statistical significance was assessed by paired Student's t-test. Results are displayed as mean ± SEM.

Supplementary Figure 2 DHPG reduced surface GluR1 density in cultured hippocampal pyramidal neurons from WT mice but not in neurons from either Eif2s1S/A or Eif2s1A/A mice.

Surface GluR1 levels were detected as described in Methods. a-c) DHPG treatment (100mM, 5 min) reduced surface GluR1 density at 1 h post-treatment onset of cells from WT Eif2s1S/S (a) but not Eif2s1S/A (b) or Eif2s1A/A (c) mice. In contrast, synapsin-1 density was uniform among genotypes and treatment conditions. MAP2 immunostaining revealed that Eif2s1S/A and Eif2s1A/A cells displayed typical neuronal morphology in culture. Boxed areas indicate the dendritic segment shown in the expanded view immediately below the full-neuron, single-channel images. Merged images show combined data from the three individual channels. Scale bar indicates 20mm. d-e) Quantification of surface GluR1 (d) and synapsin-1 (e) density of DHPG-treated neurons normalized to vehicle-treated control neurons from the indicated genotype (n=51 for both Eif2s1S/S and Eif2s1S/A and n=34 for Eif2s1A/A; sGluR1, F(2,12)=19.3, p=0.00018; synapsin-1, F(2,12)=0.14, p=0.87). Statistical significance was assessed by a two-way ANOVA with Bonferroni correction for multiple comparisons. Results are displayed as mean ± SEM.

Supplementary Figure 3 Only when combined, sub-threshold concentrations of Sal003 and DHPG increased eIF2α phosphorylation in WT slices.

Separate applications of low concentrations of DHPG (10 μM, 5 min) or Sal003 (5 μM, 10 min) failed to induce eIF2α phosphorylation; but when at the same low concentrations Sal003 (5 μM, 10 min) immediately followed by DHPG (10 mM, 5 min) the phosphorylation of eIF2α was sharply boosted (n=4 independent experiments, F(3,20)=3.15, p=0.048). Statistical significance was assessed by a one-way ANOVA. Results are displayed as mean ± SEM.

Supplementary Figure 4 At a concentration that triggers phosphorylation of eIF2α, Sal003 elicited a sustained LTD in WT slices that was insensitive to mGluR1 and mGluR5 antagonists.

a) Brief application of Sal003 (20μM, 10 min) leads to a persistent long-lasting depression of evoked EPSCs (n=6 cells from 4 mice, t=7.07, p=0.0008, paired two sided t-test). b-c) The concurrent application (15min) of the mGluR1 blocker LY367385 (100mM) and the mGluR5 blocker MPEP (10 mM) prevents DHPG-induced LTD (b; n=6 cells from 2 mice, t=7.06,p=3.5X10-5, unpaired t-test) but had no effect on Sal003-induced LTD (c cells from 3 mice; n=8, t=0.53, p=0.61, unpaired two sided t-test). Calibrations: 10 ms, 40 pA. Results are displayed as mean ± SEM.

Supplementary Figure 5 Sal003 reduced surface GluR1 expression in control neurons, but not in eIF2α phosphorylation-deficient neurons.

a-c) One hour after starting application of Sal003 (20mM, 10 min), surface GluR1 density was reduced in neurons from WT Eif2s1S/S (a) but not Eif2s1S/A (b) or Eif2s1A/A (c) neurons. In contrast, synapsin-1 density did not differ significantly between genotypes or treatment conditions. MAP2 staining revealed that Eif2s1S/A and Eif2s1A/A cells displayed typical neuronal morphology. Boxed areas indicate the dendritic segment shown in the expanded view immediately below the full-neuron, single-channel images. Merged images show combined data from the three individual channels. Scale bar indicates 20mm. d-e) Quantification of Surface GluR1 (d) and synapsin-1 (e) density of Sal003-treated normalized to vehicle-treated control cultures from the indicated genotypes (n=55 for Eif2s1S/S and Eif2s1S/A and n=38 for Eif2s1A/A; d, p=0.0002; e, p=0.83). Statistical significance was assessed by two-way ANOVA with Bonferroni correction. Results are displayed as mean ± SEM.

Supplementary Figure 6 ISRIB, but not its inactive analog ISRIBinact, is a potent inhibitor of ATF4-driven translation.

a) Structures of ISRIB and its inactive analog, ISRIBinact. b) In HEK293T cells the ATF4 luciferase reporter (5'UTR of ATF4 mRNA fused to F-luciferase) was inhibited by ISRIB but not by the inactive analog ISRIBinact. Inhibition is plotted as function of the concentration of ISRIB or ISRIBinact. Inhibition assay was performed as described by Sidrauski et al.25.

Supplementary Figure 7 OPHN1 fluorescence intensity was positively correlated with p-eIF2α levels at individual synapses of cultured hippocampal neurons.

a-d) Representative two-channel immunostaining for p-eIF2α and OPHN1 from a dendritic segment in 14DIV control- (b), DHPG- (c), and Sal003-treated (d) hippocampal neuron cultures fixed 15 min after treatment onset. Framed areas (white squares) in (b) are expanded on the left (a). p-eIF2α and OPHN1 fluorescence intensity (FI) were positively correlated at individual synapses. b-d) As shown in the scatter plots depicting data from a total of 315 synapses from 9 neurons analyzed per condition. Lines in b-d represent the regression lines of the log-transformed population FI data (in AU). Slopes (m) and correlation coefficients (r) of the respective regression lines are indicated. Scale bars indicate 2mm. e-f) Bar graphs of the average FI of synaptic p-eIF2α (e) and OPHN1 (f) normalized to vehicle-treated controls [p-eIF2a: control = 1.0 ± 0.06, DHPG = 2.2 ± 0.1, Sal003 = 2.4 ± 0.07, F(2,24)=19.6, p=9X10-6; OPHN1: control = 1.0 ± 0.05, DHPG = 1.9 ± 0.08, Sal003 = 1.8 ±0.1, F(2,24)=21.0, p=5.4X10-6]. Statistical significance was assessed by one-way ANOVA. Results are displayed as mean ± SEM.

Supplementary Figure 8 OPHN1 and Arc mRNA translation are required for mGluR-LTD in CA1.

a) A specific shRNA against OPHN1 mRNA blocked DHPG-induced LTD (n=6 cells from 5 mice, t=0.88, p=0.42) whereas scrambled control shRNA did not (n=5 cells from 3 mice, t=14.4, p=0.00014; group difference t=6.28, p=0.0002). Viruses expressing GFP together with OPHN1-shRNA or scrambled-control-shRNA were injected into the hippocampus of WT mice. Examples of inset traces from which plots were drawn are illustrated. b) DHPG-induced LTD was suppressed by an antisense oligo (as) that blocks Arc synthesis (n=12 cells from 5 mice, t=0.77, p=0.46) but not by the control mismatch oligo (ms) (n=8 cells from 5 mice, t=6.61, p=0.0003; group difference t=6.28, p=2X10−5). Calibrations: 10 ms, 40 pA. c) Sal003 increased eIF2α phosphorylation, but not Arc levels in hippocampal slices (n=5 independent experiments, t=0.5, p=0.62). Statistical significance was assessed by paired Student's t-test. Results are displayed as mean ± SEM.

Supplementary Figure 9 Spatial recognition increased eIF2α phosphorylation and OPHN1 levels in the hippocampus and the exploratory behavior was similar in all experimental groups.

a,c) Western blots from hippocampal tissue show increased eIF2α phosphorylation (a) and OPHN1 levels (c) after exposure to two objects on day 2 (n=3 mice). b,d) Quantification of normalized p-eIF2α (b, at 10 min t=2.91, p=0.033; at 90 min t=3.37, p=0.020) and OPHN1 (d, at 10 min t=0.287, p=0.79; at 90 min t=2.98, p=0.031). e-g) Raw data for individual animals during training (day 2) or testing (day 3). Plot shows exploration times for individual Eif2s1S/S and Eif2s1S/A mice (e), vehicle-injected and ISRIB-injected mice (f) and control-shRNA-injected and OPHN1-shRNA-injected mice (g) on Day 2 and 3. Statistical significance was assessed by unpaired Student's t-test. Results are displayed as mean ± SEM.

Supplementary Figure 10 Images of full-length blots presented in all figures.

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Supplementary Table 1

mRNAs translationally upregulated by mGluR activation. (XLSX 28 kb)

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Di Prisco, G., Huang, W., Buffington, S. et al. Translational control of mGluR-dependent long-term depression and object-place learning by eIF2α. Nat Neurosci 17, 1073–1082 (2014). https://doi.org/10.1038/nn.3754

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