D1 Dopamine Receptor Activation Induces Neuronal eEF2 Pathway-Dependent Protein Synthesis

Dopamine, alongside other neuromodulators, defines brain and neuronal states, inter alia through regulation of global and local mRNA translation. Yet, the signaling pathways underlying the effects of dopamine on mRNA translation and psychiatric disorders are not clear. In order to examine the molecular pathways downstream of dopamine receptors, we used genetic, pharmacologic, biochemical, and imaging methods, and found that activation of dopamine receptor D1 but not D2 leads to rapid dephosphorylation of eEF2 at Thr56 but not eIF2α in cortical primary neuronal culture in a time-dependent manner. NMDA receptor, mTOR, and ERK pathways are upstream of the D1 receptor-dependent eEF2 dephosphorylation and essential for it. Furthermore, D1 receptor activation resulted in a major reduction in dendritic eEF2 phosphorylation levels. D1-dependent eEF2 dephosphorylation results in an increase of BDNF and synapsin2b expression which was followed by a small yet significant increase in general protein synthesis. These results reveal the role of dopamine D1 receptor in the regulation of eEF2 pathway translation in neurons and present eEF2 as a promising therapeutic target for addiction and depression as well as other psychiatric disorders.


INTRODUCTION
The dopaminergic system (DS) plays an important role in reward predictions (Schultz, 1998), motivational arousal, and responsiveness to conditioned incentive stimuli (Salamone et al., 2003). In accordance, its dysfunction is associated with disruption of motivation to seek out pleasure experiences, as described in individuals diagnosed with mood disorders such as depression (Sherdell et al., 2012). Dopamine acts via two different metabotropic receptors, D1 and D2, that induce different signal transduction and cellular phenotypes (Boyd and Mailman, 2012). Indeed, recently, Hare et al. (2019) demonstrated that D1 receptors (D1R) in the medial prefrontal cortex (mPFC) might contribute to the antidepressant-like effects of ketamine (Hare et al., 2017;Hare et al., 2019). While it is established that one mechanism through which dopamine affects brain and neuronal states is through the regulation of mRNA translation, the signaling pathways mediating this regulation are not known. Since we and others have previously shown that the fast antidepressant effect of ketamine is mediated by the reduction in eEF2 phosphorylation (Duman and Voleti, 2012;Adaikkan et al., 2018), here, we tested the hypothesis that dopamine regulates mRNA translation regulation through the eukaryotic elongation factor 2 (eEF2).
The eEF2 translation factor and its sole known kinase (eEF2K) (Kenney et al., 2015) play a central role in the regulation of the elongation phase of mRNA translation (Montanaro et al., 1976). This regulation is mediated by eEF2K, which phosphorylates eEF2 on Thr 56 and thereby inactivates it, leading to reduction in the rate of mRNA translation. Since eEF2K activity is regulated by Ca 2+ /calmodulin, elevation in intracellular calcium by synaptic activation receptors such as NMDA or G-coupled receptors (e.g. metabotropic glutamate receptors) results in induced neuronal activity-dependent phosphorylation of eEF2 (Sutton et al., 2007;Barrera et al., 2008;Im et al., 2009;Proud, 2015;Heise et al., 2017). Furthermore, general translation is reduced in dendrites due to eEF2K activity, while certain synaptic proteins are selectively translated at the synapse (Heise et al., 2014). eEF2K can be deactivated by other kinases such as p70S6 kinase (S6K) or p90 ribosomal kinase (p90 RSK), which are activated in response to changes in mammalian target of rapamycin-signaling (mTOR) or extracellular-regulated kinase (ERK) signaling, respectively. These kinases inactivate eEF2K by phosphorylation on its Ser366 residue (Redpath et al., 1993;Wang et al., 2001;Browne et al., 2004).
In this work, we show for the first time that indeed dopamine regulates mRNA translation through its effect on the eEF2 pathway in neurons. Specifically, we found that D1 but not D2 receptor activation increases protein synthesis by eEF2 dephosphorylation. This increase in protein synthesis in cortical neurons is mediated by the D1 receptor and requires NMDA receptor-dependent activation of the MEK/mTOR signaling pathways that lead to inactivation of eEF2K by phosphorylation on Ser 366 residue resulting in eEF2 dephosphorylation. Furthermore, using eEF2K knockout mice, we show that eEF2K/eEF2 is the main pathway for D1-dependent increase in protein synthesis.

MATERIALS AND METHODS
Animals eEF2K-KO mice, in which coding exons 7, 8, 9, and 10 of eEF2K were deleted, were generated by the laboratory of Christopher G. Proud. We derived eEF2K wild-type (WT) and KO littermates by crossing heterozygous mice as previously described (8), eEF2K mice were bred from colonies maintained at the University of Haifa. C57BL/6 mice were obtained from local vendors (Envigo RMS, Jerusalem, Israel) and after acclimation to the facility were used for experiments. Animals were provided ad libitum with standard food and water and were maintained on a 12/12 h light/dark cycle. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Haifa, and adequate measures were taken in order to minimize pain, in accordance with the guidelines laid down by the European Union and United States NIH regarding the care and use of animals in experiments.

Immunocytochemistry Quantification
Signal intensity quantification of phosphorylated eEF2 (peEF2) in the cell soma or dendrites was done by NIS Element Advanced Research (Ar) 4.5 (Nikon Japan) software in MAP2 labeled neurons. Confocal images were acquired using a Nikon 63X immersion oil objective at a resolution of 1024 × 1024 pixels. Each image was a Z series projection of 7 to 10 images, taken at depth intervals of 0.5 µm. To define the region of interest for quantification, cell bodies were identified using the DAPI nuclei stain and dendrites 10 µm away from del cell body were manually traced using NIS Element AR software on the MAP2 channel. peEF2 signal intensity (mean pixel intensity) was estimated as the peEF2 integrated fluorescence intensity divided by the area marked by the MAP2 signal.

Puromycin Immunocytochemistry and Quantification
Cells for immunofluorescence were plated on coverslips coated with Poly-L-ornithine and laminin coating. After 14 DIV cells were pre-treated with U0126 (20 µM) or vehicle (DMSO) for 30 min and then treated with SKF38393 (25 µM) for 4 h. After SKF38393 treatment, cells were further incubated with 10 µg/ml puromycin for 10 min in the same medium and fixed in 4% paraformaldehyde. Cells were stained with anti-puromycin (1:1000, clone 4G11, EMD Millipore) and MAP2 (1:1000) antibodies, following the same procedure for peEF2Thr56 immunocytochemistry. Images were taken as Z series projection of 5 to 9 images at depth intervals of 0.25 µm at x60 magnification with an Olympus IX81 microscope using Olympus cellSens1.16 software. Quantification of puromycin incorporation was done by selecting neurons randomly in MAP2 labeled neurons and estimating the puromycin signal as mean intensity divided by the area marked by MAP2 signal using ImageJ 1.51J software. Quantification was done in a blind manner based on three independent experiments for each condition.

RNA Extraction
Tri Reagent was added directly to primary culture plates. Cells were scrapped and transferred to 1.5 ml tubes, 1-bromo, 3-chloropropane was then added at a tenth of the volume of Tri Reagent and mixed thoroughly. After phase separation by centrifugation of 15 min at 12,000 rcf, 2-propanol was added in equal volume to the RNA phase that was separated from the rest of the sample and placed in new tubes. Following 30 min of centrifugation at 12,000 rcf, the pelleted RNA was then washed once with 70% cold ethanol and centrifuged for 10 min at 7,600 rcf, the pellet was air dried and eluted in ultra pure water (Biological Industries, Beit Haemek, Israel). All reagents used were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), unless otherwise stated.

Reverse Transcription and qPCR
RNA samples were copied to cDNA using Applied Biosystems (Thermo Fisher, Waltham, Massachusetts, United States) High Capacity cDNA Reverse Transcription kit. The resultant cDNA was then used in TaqMan gene expression assays. The target primers used were BDNF (Mm04230607_s1), Syn2 (Mm00449780_m1), which were measured against GAPDH (Mm99999915_g1). Both reactions were carried out as per the manufacturer's protocols. Relative quantitation was done using delta-delta ct of the target genes. One way ANOVA was carried out and post hoc Tukey's test comparing each experimental group with the control was done, using GraphPad Prism software.

Statistical Analysis
Graphs were prepared using GraphPad Prism 6.01, InStat Software (GraphPad Software, CA, United States). Data are expressed as mean ± SEM. Statistical analysis was performed using SPSS version 24. Each experiment was normalized to its own control (non-treated, NT). In each experiment, 2 replicates of NT cells were used in order to verify the cultures were homogeneous. An average of the NT samples was calculated and values obtained for other samples were divided by it. The average of the control was always calculated based on samples electrophoresed in the same gel. Each type of pharmacological experiment had 5 biological replicates (5 independent cultures). Statistical significance was determined with one-way ANOVA followed by Tukey's post hoc test used for analysis of the different pharmacological manipulation experiments. Mann-Whitney analysis was used in the immunocytochemistry experiments in order to examine the differences in eEF2 phosphorylation between NT cells and SKF-treated cells in both soma and dendrites.
eEF2 Dephosphorylation by D1 Receptor Activation Is Dependent on the NMDA Receptor, MEK, and mTOR Following the correlation found between ERK2 activation, as indicated by its phosphorylation state, and eEF2 dephosphorylation (Supplementary Figure S1C), and since ERK is downstream of both dopamine and glutamate receptor activation (Kaphzan et al., 2006;David et al., 2014), we further asked whether the NMDA receptor plays a role in D1 receptordependent dephosphorylation of eEF2. To test this, primary cortical neuronal cultures were pretreated for 30 min with APV, a NMDA receptor antagonist, followed by 15 or 60 min incubation with D1 receptor agonist SKF38393. Treatment of the cells with APV prevented the dephosphorylation of eEF2 following SKF38393 treatment, both after 15 and 60 min [ Figure 2A; one-way ANOVA, F(5,29) = 7.503, p < 0.001; post hoc test, NT vs. SKF 15 min: p = 0.02; NT vs. SKF 60 min: p = 0.02; SKF  Since eEF2K inhibits eEF2 activity and is negatively regulated by phosphorylation on Ser 366 , we further asked whether treatment with SKF38393 increases eEF2K phosphorylation on Ser 366 at the same time points in which we observed eEF2 dephosphorylation. Indeed, treatment of primary cultures with SKF38393 clearly induced phosphorylation of eEF2K on Ser 366 (Figure 2B; one-way ANOVA, F(5,36) = 20.66, p < 0.001; post hoc test, NT vs. SKF 15 min: p < 0.001; NT vs. SKF 60 min: p < 0.001), which was blocked by pre-incubation with NMDA receptor antagonist, APV (SKF 15 min vs. SKF 15 min +APV: p < 0.001; SKF 60 min vs. SKF 60 min +APV: p < 0.001). As expected, APV pre-treatment also abolished ERK1/2 activation, consistent with previous studies from our lab (Kaphzan et al., 2006;David et al., 2014) (Supplementary Figure S2A).
Recent reports establish that inhibition of CaMKII concurrent with eEF2K-dependent increase in protein synthesis is an essential step in the manifestation of antidepressant effects of ketamine (Adaikkan et al., 2018). Since dopamine can also activate CaMKII signaling (Hasbi et al., 2009;Adaikkan et al., 2018), we examined the role of the pathway in D1 receptor-dependent eEF2 dephosphorylation. Surprisingly, treatment of cultures with SKF38393 in the presence of the CaMKII specific inhibitor TatCN21 peptide (Wong et al., 2019) demonstrated a mild contribution of CaMKII to the D1 receptor-dependent dephosphorylation of eEF2 (Supplementary Figure S2B).
Interestingly, incubation of the cells with rapamycin alone resulted in enhancement of eEF2 phosphorylation (Supplementary Figure S2D). As expected, rapamycin alone blocked S6K phosphorylation as well as the D1-dependent phosphorylation after 15 min of incubation. However, no effect was found on ERK (Supplementary Figure S2D). These findings imply that the MEK-ERK pathway is upstream of the mTOR pathway following D1 receptor stimulation (Figure 2E).

Dopamine D1 Receptor Dephosphorylates eEF2 Thr56 in Neuronal Dendrites and Somata
Regulation of eEF2K activity and reduction of eEF2 phosphorylation by synaptic receptors such as the NMDA receptor serves as one possible way to translate mRNA in dendrites (Autry et al., 2011). To examine whether dopaminergic transmission can also regulate dendritic eEF2 phosphorylation, we performed immunocytochemical analysis of cortical neurons from wild-type mice (n = 5 independent cultures) treated with SKF38393 for 15 min. The results revealed a reduction in phospho-eEF2 immunoreactivity in most of the neurons analyzed. The effect was seen in both neuronal soma (Figure 3A; U = 8839, Z = −8.994, p < 0.0001; Mann-Whitney test) and dendrites ( Figure 3B; U = 3975, Z = −6.489, p < 0.0001; Mann-Whitney test). Phospho-eEF2 antibody specificity was tested in primary cultures derived from eEF2K-KO mice. No immunoreactivity was detected in cortical neurons from eEF2K-KO cultures (Supplementary Figure S3). Moreover, although our cultures are mixed, containing both neurons and glia, the effect of D1 receptor activation on eEF2 occurred specifically in neurons, since no changes were found in glia cells (Supplementary Figure S4). These results provide evidence that D1 receptor activation reduces eEF2 phosphorylation in both dendrites and soma.

Dopamine D1 Receptor Activation Enhances Protein Synthesis in Cultured Cortical Neurons
In light of our immunocytochemistry results, we further asked whether the dopamine D1 receptor-dependent eEF2 dephosphorylation coincides with enhanced protein synthesis. Treatment with SKF38393 resulted in rapid de-phosphorylation of eEF2 which returned to baseline after 2 h (Figure 4A; (Schmidt et al., 2009). Time-course experiments showed that puromycin incorporation was significantly increased only FIGURE 3 | D1 receptor activation induces eEF2 dephosphorylation in dendrites more than in cell soma. (A) Immunofluorescence and mean intensity quantification of phospho-eEF2 (Thr 56 , red) in neuronal somata mean intensity in primary cortical cultures from C57BL/6 mice were treated with SKF38393 (25 µM) for 15 min. Scale bar, 20 µm. Images represent 15 to 20 neurons (labeled with MAP2, green) from four independent cultures. (B) Immunofluorescence and mean intensity quantification of phospho-eEF2 (red) in dendrites in primary cortical cultures treated with SKF38393 (25 µM) for 15 min. Scale bar, 10 µm. Images represent 15 to 20 neurons (labeled with MAP2, green) from four independent cultures. Means ± SEM are shown in all graphs. * * * p < 0.0001. after 1.5 and 4 h of incubation with SKF38393 ( Figure 4B; One way ANOVA F(4,31) = 11.89, p < 0.001. Post hoc test, NT vs. SKF1.5 h: p = 0.002; NT vs. SKF4 h: p < 0.001). Similar increase in protein synthesis was reported after ketamine administration, which leads to a reduction in phosphorylation levels of eEF2 via the inhibition of its kinase and CaMKII (Adaikkan et al., 2018).

NMDAR, MEK, and eEF2K Are Necessary for D1-Dependent Increased Protein Synthesis
The results presented in Figure  1 and Supplementary Figure S5A demonstrate that D1 but not D2 receptor activation can lead to eEF2 dephosphorylation. blocking D1 receptor with its antagonist SCH23390 decreased puromycin labeling, suggesting D1 but not D2 receptor dependence (Figure 5A; one-way ANOVA F(3,26) = 9.75, p < 0.001; post hoc test, NT vs. SCH: p = 0.98; NT vs. SKF 4 h: p < 0.001; NT vs. SKF 4 h +SCH: p = 0.99; SKF 4 h vs. SKF 4 h +SCH: p = 0.001]. Our results also imply the involvement of the NMDA receptor in D1-dependent eEF2 dephosphorylation. In order to examine the necessity of the NMDA receptor for D1 receptor-induced protein synthesis, we measured puromycin incorporation in the presence of NMDA receptor antagonist APV after 4 h of incubation at the time point of a large increase in protein synthesis (Figure 5B, Supplementary Figure S5A). SKF38393 treatment with NMDA receptor blockade abolished the induction of protein synthesis, suggesting a pivotal role for the NMDA receptor in the D1-dependent protein synthesis (Figure 5B; one-way ANOVA F(3,26) = 13.67, p < 0.0001;  To test if the eEF2K/eEF2 pathway is necessary for the D1 receptor increase of protein synthesis, we used primary cultures from eEF2K-KO mice. These mice show no eEF2 phosphorylation, but display normal phosphorylation of other translation factors (Heise et al., 2017;Adaikkan et al., 2018). Cortical cultures from eEF2K-KO mice and their wild-type littermates were treated for 4 h with SKF38393 and global protein synthesis was analyzed by SUnSET. Immunocytochemistry and western blot analyses of puromycin incorporation showed a significant increase in wild type but not in eEF2K-KO cultures (n = 4 independent cultures) following SKF38393 incubation (Figures 5C,D, Supplementary Figures S5B,C). To probe whether the D1 receptor activation-induced increase in translation is dependent on ERK/mTOR/eEF2K/eEF2 signaling, we pre-treated cortical neurons derived from wild type and eEF2-KO mice with or without MEK inhibitor U0126, followed by SKF38393 treatment. We found that the SKF38393-mediated increase in translation was reduced by treatment with U0126 in wild type neurons, while no change in puromycin incorporation was detected in eEF2K-KO mouse-derived cultures treated with U0126 ( Figures 5C,D

DISCUSSION
In summary, while previous studies have shown that dopamine regulates mRNA translation (Smith et al., 2005), our data show for the first time that this regulation is mediated by the eEF2 pathway. Specifically, we show that dopamine D1 receptor activation in neurons inhibits eEF2K, resulting in reduced eEF2 phosphorylation. Furthermore, we observed a small but significant increase in general protein synthesis in neurons 1 h after D1 activation with increase of specific eEF2K-related proteins such as BDNF and synapsin 2b. The eEF2 rapid dephosphorylation following D1 activation, occurred at the same time with S6K activation, but there was no phosphorylation changes at these time points in other translation factors as 4E-BP or eIF2α. Both dopamine and eIF2α pathway are pivotal for memory consolidation (Belelovsky et al., 2009;Stern et al., 2013;Ounallah-Saad et al., 2014;Segev et al., 2016;Gal-Ben-Ari et al., 2018;Sharma et al., 2018), however, possibly via different molecular and cellular mechanisms. From a signal transduction perspective, NMDA receptor is required for the D1 receptor-dependent induction of MEK/mTOR activity that leads to inactivation of eEF2K.
Our data support the view that dopamine D1 receptor activation regulates neuronal proteostasis, specifically affecting the elongation phase of translation, which mediates the effect of antidepressants in the CNS (Flight, 2011;Adaikkan et al., 2018;Hare et al., 2019). D1 receptor activation has different effects including enhanced learning and memory in various learning paradigms such as CTA, fear conditioning, and object recognition in rodents (Nagai et al., 2007;Nosyreva et al., 2013;Balderas et al., 2013;David et al., 2014;Péczely et al., 2014a,b). Infusion of D1 receptor antagonist SCH23390 into the prefrontal cortex of monkeys or rats impaired spatial working memory, while D2 receptor antagonist showed no effect.
Interestingly, the time frame of eEF2 dephosphorylation after D1 receptor activation proceed the time of increased general protein synthesis. However, they are linked, most probably indirectly, since no increase in puromycin incorporation was detected in the eEF2K-KO mice cultures.
Our data provide further evidence of the well-known relationship between D1 and NMDA receptors and its effect on signal transduction (Dunah and Standaert, 2001;Lee et al., 2002;Pei et al., 2004;Martina and Bergeron, 2008;Stramiello and Wagner, 2008;David et al., 2014). The results indicate that, in addition to the canonical calcium-calmodulin-dependent activation of eEF2K following NMDA receptor stimulation, NMDAR-D1 interaction induces translational changes via ERK and S6K signaling cascades. Moreover, the eEF2K pathway accounts for the increase in protein synthesis following dopamine D1 receptor activation.
Although eEF2K is activated by the Ca 2+ -Calmodulin complex, it is also regulated by phosphorylation. mTOR-and MEK-dependent phosphorylation of eEF2K reduces its activity, while PKA-and AMPK-mediated phosphorylation does the opposite (Wang et al., 2001;Heise et al., 2014;Tan et al., 2014). Given this complex regulation of its function, we propose that eEF2K functions as a pivotal convergence signaling hub, linking synaptic information to regulation of specific protein synthesis. In addition, it was suggested that eEF2 acts as a biochemical sensor to discriminate between evoked action potential and spontaneous miniature synaptic transmission (Sutton et al., 2007).
Our results and previous time-dependent studies suggest the existence of different phases in eEF2 regulation in neurons: The first phase causes a rapid increase in eEF2 phosphorylation, due to synaptic activation and NMDA receptor-dependent high calcium influx (Belelovsky et al., 2009;Gildish et al., 2012;Taha et al., 2013), which leads to inhibition of general protein synthesis and an increase in specific proteins such as c-Fos, Arc (Chotiner et al., 2003;Park et al., 2008), and CaMKII (Scheetz et al., 2000). The second phase is mediated by dopamine D1 receptor-dependent eEF2 dephosphorylation and the molecular pathway described in the study, which accumulates with increased expression of a specific subset of proteins such as BDNF (Figure 6). The third phase with prolong increase in protein synthesis (between 1.5and 4 h) occurs long after eEF2 reduced phsophorylation is back to baseline, most probably in an indirect way.
In a similar manner, certain levels of dopamine D1 receptor stimulation in dendrites of specific neurons can gate out "noise", while high levels, e.g. during stress (Arnsten et al., 2015;Hare et al., 2019), suppress delayed firing. For instance, maintenance of synaptic strength in hippocampal slices treated with low concentrations of dopamine D1 agonist SKF 38393 requires MEK and CaMKII activation, while in slices treated FIGURE 6 | Model of D1 receptor-dependent dephosphorylation of eEF2 in cortical neurons. Dopamine D1 receptors activated in dendrites lead to small calcium influx via the NMDA receptor, which elevates both MEK-ERK and mTOR pathways. Both pathways inhibit eEF2K activity by phosphorylating it on Ser366, leading to eEF2 Thr56 dephosphorylation and increased protein synthesis.
with high concentrations, maintenance of synaptic strength is dependent only on MEK activation (Barcomb et al., 2016). The authors reported that the increase in dopamine levels in the mPFC following ketamine administration increases D1R signaling and contributes to the synaptic actions of ketamine. Our results suggest a mild contribution of CaMKII to eEF2 dephosphorylation following D1 receptor stimulation. This mild dependency could be part of the mechanism underlying the effect of ketamine on CaMKII and eEF2K, resulting in increased protein synthesis and induction of its rapid antidepressant effect (Adaikkan et al., 2018;Chang et al., 2019;Hare et al., 2019). Further investigation will be needed in order to establish a direct correlation between dopamineand ketamine-dependent activation of CaMKII. Nonetheless, assuming eEF2 is a main target for D1 activation, it is not surprising that the dopamine D1 receptor agonist, in a similar way to ketamine, can potentially serve as an antidepressant (D'Aquila et al., 1994).
Our findings establish a link between dopamine D1 receptor activation and eEF2K activity, opening a door to better understanding the molecular mechanism underlying the effect of antidepressants and role of neuromodulators in synaptic plasticity, addiction, and memory formation. Future studies aiming to better understand neurodegenerative diseases and depression-like syndrome, combined with a circuit approach and behavioral paradigms will better link the eEF2 pathway and dopamine to establish the eEF2 pathway as a potential target for therapy.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Haifa.

AUTHOR CONTRIBUTIONS
OD and IB designed and performed the experiments, analyzed the data, and wrote the manuscript. NG contributed data to mRNA analysis. SG-B-A contributed to the primary culture preparations and edited the manuscript. KR designed experiments, supervised the project, and wrote the manuscript.