NMDAR mediated translation at the synapse is regulated by MOV10 and FMRP
Protein synthesis is crucial for maintaining synaptic plasticity and synaptic signalling. Here we have attempted to understand the role of RNA binding proteins, Fragile X Mental Retardation Protein (FMRP) and Moloney Leukemia Virus 10 (MOV10) protein in N-Methyl-D-Aspartate Receptor (NMDAR) mediated translation regulation. We show that FMRP is required for translation downstream of NMDAR stimulation and MOV10 is the key specificity factor in this process. In rat cortical synaptoneurosomes, MOV10 in association with FMRP and Argonaute 2 (AGO2) forms the inhibitory complex on a subset of NMDAR responsive mRNAs. On NMDAR stimulation, MOV10 dissociates from AGO2 and promotes the translation of its target mRNAs. FMRP is required to form MOV10-AGO2 inhibitory complex and to promote translation of MOV10 associated mRNAs. Phosphorylation of FMRP appears to be the potential switch for NMDAR mediated translation and in the absence of FMRP, the distinct translation response to NMDAR stimulation is lost. Thus, FMRP and MOV10 have an important regulatory role in NMDAR mediated translation at the synapse.
KeywordsFMRP/MOV10/NMDAR mediated translation
Analysis Of Variance
Activity regulated cytoskeleton associated protein
Autism Spectrum Disorder
Calcium/calmodulin dependent kinase II alpha
days in vitro
Embryonic day 18
fragile X mental retardation 1
Fragile X Mental Retardation Protein
Fragile X Syndrome
Glutamate receptor ionic epsilon 1
Human antigen R
metabotropic Glutamate Receptor
miRNA Induced Silencing Complex
Moloney Leukemia Virus 10
postnatal day 30
Protein Kinase B
postsynaptic density 95
phosphatase tensin homolog
RNA Binding Protein
Ribosomal Protein Lateral stalk subunit P0
small interfering RNA
In mature neurons, protein synthesis in the dendrites and spines outweigh that of the cell body due to their sheer volume . Protein synthesis at dendrites and spines is regulated by the activation of many different neurotransmitter receptors such as glutamate, dopamine, and serotonin [2, 3, 4] also termed as activity mediated protein synthesis. Thus, it is important to decipher the specificity of translational response to a given neurotransmitter receptor stimulation. This task has gained significance since the dysregulation of protein synthesis is thought to be a common cause for multiple neurodevelopmental disorders . Glutamate is the major excitatory neurotransmitter in the mammalian brain and NMDAR and the group I metabotropic Glutamate Receptor (mGluR) are two of its primary receptors that mediate synaptic plasticity. Both NMDAR and mGluR regulate protein synthesis, group I mGluR leading to global translation activation and NMDAR to translation inhibition shown through metabolic labelling of proteins [6, 7, 8, 9]. At transcriptome level, both group I mGluR and NMDAR stimulation leads to translation activation of specific subset of mRNAs. Group I mGluR stimulation leads to translation of mRNAs such as Fragile X mental retardation 1 (Fmr1), postsynaptic density 95 (Psd-95), activity regulated cytoskeleton associated protein (Arc) [10, 11, 12] and NMDAR stimulation leading to translation of β-actin, Glutamate receptor ionic epsilon 1 (Grin2a), Fmr1, Calcium/calmodulin dependent kinase II alpha (camk2a) and Arc mRNAs [9, 13, 14, 15, 16, 17]. Group I mGluR mediated translation activation is well studied, however, the mechanistic insight of NMDAR mediated translation is poorly explored [18, 19]. In the current study, we tried to elucidate NMDAR mediated control over the translation machinery by determining the factors involved in it.
MicroRNAs and microRNA induced silencing complex (miRISC) are thought to play an important role in regulating activity mediated protein synthesis. MicroRNA-AGO2 mediated translation inhibition can be reversed by dissociation of miRISC from the mRNA and promoting its translation [11, 20, 21]. This reversibility of miRISC is of particular interest in the context of synaptic plasticity as it can inhibit translation until an appropriate stimulus relieves the inhibition. Under these conditions, microRNAs provide the sequence specificity while several RNA binding proteins (RBP) which are not part of miRISC core complex will act as a molecular switch through their dynamic interaction with AGO2. FMRP is one such RBP which has a significant role in synaptic protein synthesis. Previously, it was shown that FMRP along with AGO2 regulate translation in response to the group I mGluR stimulation at the synapse . While FMRP is also reported to regulate translation through multiple mechanisms [22, 23], its role in reversibility of miRISC mediated inhibition is likely to be of relevance for synaptic translation. The loss of FMRP and the subsequent synaptic dysfunction is the hallmark of Fragile X Syndrome (FXS) . Interestingly, FMRP is reported to interact with a large number of mRNAs  and thus potentially regulates translation beyond mGluR signalling. Another RBP known to regulate translation downstream of synaptic signalling is MOV10 and is also known to interact with both FMRP and AGO2 [19, 26]. Since both NMDAR and group I mGluR mediated plasticity involve protein synthesis, it is also essential to study the role of FMRP and MOV10 in NMDAR mediated protein synthesis at the synapse.
In the current study, we show that the dynamic interaction between AGO2-MOV10-FMRP determines the translation response to NMDAR stimulation. This study highlights the involvement of FMRP and its phosphorylation status in NMDAR mediated signalling and provides a molecular mechanism to explain the specificity of translation on NMDAR stimulation.
MOV10 dissociates from AGO2 and moves to polysomes on NMDAR stimulation
To understand the role of MOV10 on synaptic translation, we looked at the association of MOV10 protein with polysomes in synaptoneurosomes. In synaptoneurosomes, on puromycin (PURO) treatment, actively translating polysomes shift to lighter fractions compared to cycloheximide (CHX) treatment, as shown by ribosomal protein lateral stalk subunit P0 (RPLP0) (Fig. 1b). MOV10 was present in polysomal fractions but puromycin treatment led to significant reduction of MOV10 from heavy polysomes and a shift to lighter fractions (Fig. 1c, d and Additional file 1: Figure S2A) indicating that MOV10 is associated with actively translating polysomes. MOV10 distribution in polysomes was further validated using a sucrose step gradient method  in Neuro 2a cells (Additional file 1: Figures S2C-S2E). Thus, we found that MOV10 associates with AGO2 as well as with puromycin sensitive polysomes. Interestingly, in synaptoneurosomes, the percentage of MOV10 in translating polysomes (puromycin-sensitive) was significantly increased on NMDAR stimulation compared to basal condition (Fig. 1e, f, Additional file 1: Figures S2B and S2F). These results show that on NMDAR stimulation MOV10 dissociates from inhibitory protein AGO2 and moves into translating polysomes.
FMRP is required for the translation response downstream of NMDAR stimulation
FMRP knockdown (Fmr1-siRNA) in Neuro 2a cells resulted in a significantly reduced association of MOV10 with polysomes (Fig. 2c). In the Fmr1-KO synaptoneurosomes, we could detect MOV10 only in the lighter fractions (fractions 1–5) of the linear sucrose gradient and absent in the polysomes (Fig. 2d) while there was no change in the distribution of ribosomes (based on RPLP0 western blot) (Fig. 2d). Earlier we showed that when cortical synaptoneurosomes were stimulated with NMDA, there was a significant increase in the percentage of MOV10 in the heavy polysomes (Fig. 1e and f). This shift of MOV10 to polysomes on NMDAR stimulation was absent in the Fmr1-KO synaptoneurosomes (Fig. 2e). Further, we also studied the role of AGO2 in the distribution of MOV10 in polysomes (Additional file 1: Figure S3D). In the absence of AGO2, the presence of MOV10 in polysomes was not affected (Additional file 1: Figures S3E). These results confirm that FMRP is not only required for the association of MOV10 with AGO2 and translating polysomes at basal state but also for the shift of MOV10 from AGO2 to polysomes in response to NMDAR stimulation.
Translation of specific mRNAs is affected by the absence of MOV10 and FMRP
NMDAR stimulation leads to translation of FMRP-MOV10 target mRNAs
Among the four candidates that we tested in this assay, we found that Pten and Psd-95 mRNAs showed an increase in translating fractions on NMDAR stimulation and Ank2 and β-actin did not show any change (Fig. 5d-g, with average line graphs in Additional file 1: Figure S6C-S6F). Thus, NMDAR leads to translation activation of Pten and Psd-95 mRNAs which is mediated through both MOV10 and FMRP.
Dephosphorylated FMRP forms the inhibitory complex with MOV10-AGO2 and phosphorylation of FMRP dissociates this complex
Protein synthesis is known to play an important role downstream of both NMDAR and mGluR stimulation . But currently, there is no clear understanding regarding the distinct translational response downstream of these pathways. Though mGluR stimulation is correlated with global translation activation and NMDAR stimulation with translation inhibition, there are many contrasting reports when it comes to individual transcripts [9, 12, 35].
Since there is an overlap of many signalling components between NMDAR and mGluR , we hypothesized that similar to mGluR mediated translation regulation, specificity of NMDAR mediated translation is regulated at the messenger ribonuclearprotein (mRNP)-mRNA level . RNA binding proteins such as FMRP, Human antigen R (HuR), Staufen2, and MOV10 play a crucial role in regulating translation of target mRNAs in a reversible manner. Staufen2 (Stau 2) is required for the transport and translation of microtubule associated protein 1b (Map1b) mRNA downstream of mGluR activation  whereas interaction of HuR with cationic amino acid transporter (CAT-1) mRNA was shown to relieve it from miRISC mediated inhibition in response to stress . MOV10 is the mammalian homolog of Drosophila Armitage protein which is shown co-localize with AGO2 in HEK cells and is a component of miRISC . An earlier study in hippocampal neurons has linked MOV10 to NMDAR mediated translation activation . MOV10 is known to bind to mRNAs  and regulate the translation of CamK2a, lysophospholipase 1 (lypla1) mRNAs via their 3′ untranslated region (3’UTRs) . In the above studies, the RNA binding proteins are shown to influence microRNA mediated inhibition and the translation of specific mRNA in response to particular signalling cues. In this regard, MOV10 was an ideal candidate for NMDAR mediated translation regulation as its role has been established previously [19, 27]. In order to characterise its regulatory role, we looked at MOV10 association with miRISC protein AGO2 and polysomes in synaptoneurosomes.
We found that MOV10 associates with both miRISC (AGO2) and polysomes and thus is involved in both translation inhibition and activation. On NMDAR signaling, the association of MOV10 with AGO2 decreases and it concomitantly increases in translating polysomes. Thus, MOV10 promotes the active translation of its bound mRNAs on NMDAR stimulation. In agreement with this, MOV10 knockdown showed a decrease in translation of its selected target mRNAs shown by polysome profiling. These results indicate that MOV10 not only acts an inhibitory RBP as shown previously [19, 27] but also has a role in translation activation of mRNAs downstream of NMDAR stimulation.
We also show that another RBP, FMRP, has a crucial role in MOV10 mediated translation regulation downstream of NMDAR activation. The role of FMRP as a translational regulator is well established in response to mGluR stimulation , but very little is known about the role of FMRP in the context of NMDAR stimulation. In this study, we show that there is an active translation of a specific subset of mRNAs downstream of NMDAR stimulation and FMRP along with MOV10 is critical for this regulation. Our data shows that FMRP is essential for the formation of MOV10-AGO2 inhibitory complex and for the shift of MOV10 (along with its target mRNAs) to translating polysomes on NMDAR stimulation. Studying the phosphorylation status of FMRP seems to provide the key molecular insight in understanding synaptic translation. In contrast to mGluR stimulation as shown previously , we found that on NMDAR stimulation there is an increase in the phosphorylation of FMRP at S499. Overexpression of phospho-mimetic of FMRP (FMRP-S499D) increases the FMRP-AGO2 complex but leads to the shift of MOV10 from AGO2 to translating polysomes. In contrast, dephospho-mimetic FMRP (FMRP-S499A) leads to an increase in MOV10-FMRP-AGO2 complex and decreased the MOV10 in translating polysomes. Based on these data, we propose a model (Fig. 7e) that indicates a possible mechanism for NMDAR mediated translation activation through FMRP and MOV10. These results indicate that NMDAR mediated FMRP phosphorylation has an effect on MOV10 mediated translation of mRNAs. Phosphorylation of FMRP is the switch downstream of NMDAR that leads to MOV10 moving into polysomes promoting translation of its target mRNAs.
We were also able to show that translation of Pten mRNA is upregulated on NMDAR activation and is regulated by FMRP and MOV10. PTEN is a known inhibitor of Protein Kinase B (Akt/PKB) pathway and pten mutations have been linked with autism spectrum disorders (ASD) . We have used polysome profiling and immunoprecipitation along with MOV10 knockdown and FMRP knockout systems to show their role in NMDAR mediated translation but there is a scope to further test the roles of these RBPs in NMDAR signalling. Thus, in summary, this work draws attention to the importance of studying the role of FMRP beyond mGluR stimulation and particularly in NMDAR mediated signalling which will have a clear bearing on the molecular pathology of Fragile X Syndrome (FXS) and autism spectrum disorders (ASD).
Materials and methods
Cell line and primary neuronal culture: Primary neuronal cultures were prepared from cerebral cortices of embryonic day 18 (E18) rats (Sprague-Dawley) according to the established protocol . 2-3 × 106 dissociated cells were plated on poly-L-lysine (0.2 mg/ml in borate buffer, pH 8.5) coated 10 cm culture dishes. Neurons were attached to the substrate in minimal essential medium with FBS (10%) for 3 h, then later grown in defined Neurobasal Medium (Invitrogen) with GlutaMAX™ supplement (Gibco™) and B-27 supplements (Invitrogen). Neurons were cultured for 14d at 37 °C in a 5% CO2 environment. For knockdown studies in neurons, NeuroMag (OZ Biosciences) was used as the transfection reagent. Silencer select siRNAs from Ambion against MOV10 transcript were transfected on days in vitro (DIV) 12 and the neurons were lysed on DIV 14.
Neuro2a cells were maintained in DMEM (Gibco®) with 10% FBS (Sigma) and GlutaMAX™ supplement (Gibco™). For knockdown studies, Silencer Select siRNA from Ambion were used. siRNA transfections were done using Lipofectamine® 2000 transfection reagent. For overexpression studies, phosphomutants of FMRP, FMRP-S499D and FMRP-S499A plasmid constructs  were transfected using Lipofectamine® 2000 and cells were lysed 24 h after the transfection.
Immunoprecipitation was done using anti-EiF2C2 (Abnova H00027161-MO1), anti-FMRP (Sigma-F4055), anti-MOV10 (Abcam-ab80613), Mouse IgG (Abcam-ab37355) and protein G Dyna beads (Invitrogen). Samples were processed for either western blotting or quantitative (real-time) PCR following immunoprecipitation as described previously . The above antibodies including RPLP0 (Abcam-ab101279), α-tubulin (Sigma T9026), FLAG M2 (Sigma Millipore F3165) and β-III tubulin (Tuj1, Sigma T8578), phospho FMRP-S499 (Abcam-ab183319) were used for immunoblotting.
Sucrose step gradient
800 μl of 20% sucrose solution was overlaid on 800 μl of 30% sucrose solution. 400 μl cell lysate was added and the centrifugation was carried out at 40,200 rpm for 2 h in SW 50.1 rotor (Beckman Coulter) . Fractions were then collected and analyzed by qPCR and western blotting. All sucrose solutions were made in gradient buffer (20 mM Tris-Cl pH 7.4, 100 mM KCl, 5 mM MgCl2 0.1 mg/ml cycloheximide, protease inhibitor and RNase inhibitor). The lysis buffer consisted of the gradient buffer with 1% Nonidet P-40 (NP40). For puromycin treatment, 1 mM puromycin was added to Neuro 2a cells or synaptoneurosomes and incubated for 2 h or 30 min respectively at 37 °C before lysis.
Cortical synaptoneurosomes were prepared by differential filtration method  from Sprague Dawley (SD) WT or fmr1 KO  rats. For stimulation, synaptoneurosome solution was pre-warmed at 37 °C for 5 min and then stimulated with N- Methyl-D-Aspartate (NMDA, Sigma 20 μM) for 5 min at 37 °C with mock stimulation considered as the basal condition.
For PTEN and PSD-95 protein detection, post NMDAR stimulation, the synaptoneurosomes were pelleted, the buffer was replaced with fresh synaptoneurosomes buffer and the synaptoneurosomes were incubated at 37 °C for additional 20 min. Synaptoneurosomes were then lysed and denatured by SDS-denaturing buffer. For AP-5 treatment, the synaptoneurosomes were pre-incubated with AP-5 (100 μM) for 10 min at 37 °C, stimulated with NMDA for 5 min at 37 °C. Post NMDAR stimulation, the synaptoneurosomes were further incubated at 37 °C for 20 min in fresh synaptoneurosome buffer and then lysed and denatured by SDS-denaturing buffer. Anti-PTEN (CST 9552S) and anti-PSD-95 (Abcam 76,115) antibodies were used for western blotting.
Electron microscopy was done from synaptoneurosomes as described earlier . Synaptoneurosomes were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium cacodylate. Fixed samples after washes were then embedded in epoxy resin at 60C for 48h. Blocks were sectioned and imaged using TEM (FEI-Technai biotwin T12) at 100kV.
Polysome assay was done from synaptoneurosome samples after stimulation . In brief, synaptoneurosome/cell lysate was separated on a 15–45% linear sucrose gradient in the presence of cycloheximide or puromycin. 1.0 mL fractions were collected and used for further analysis through western blot and qPCR.
Quantitative analysis for polysome profiling: qPCR data was analysed by the absolute quantification method using a standard curve as mentioned previously . Absolute copy numbers for a particular mRNA were obtained from each of the 11 fractions. These copy numbers were then represented as percentage distribution across the 11 fractions.
Fractions 7 to 11 were considered as translating pool based on sensitivity to puromycin (Fig. 5a).
Translating pool/non-translating pool = sum of the percentage of mRNA from fraction 7 to fraction 11 ÷ sum of the percentage of mRNA from fraction 1 to fraction 6.
For primary neurons (Additional file 1: Figure S3A)
Translating pool/non-translating pool = sum of the percentage of mRNA from fraction 8 to fraction11 ÷ sum of the percentage of mRNA from fraction 1 to fraction 7.
Quantitative PCR primers
For 18S ribosomal RNA (rRNA) quantification, cDNA samples were diluted one thousand times and then used for qPCR.
Forward sequence (5’→3′)
Reverse Sequence (5’→3′)
Group comparisons were made using one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Statistical significance was calculated using paired/unpaired Student’s t-test for biochemical experiments as mentioned. Data are presented as mean ± Standard error of Mean (SEM). p values less than 0.05 were considered statistically significant.
We would like to thank the Shanta Wadhwani Foundation for supporting travel to present this work internationally. We are grateful to the sequencing facility, electron microscopy facility and animal facility of NCBS/inStem for the various technical help.
The work has been funded by Dept. of Biotechnology (BT/PR8723/AGR/36/776/2013), India, and NeuroStem grant (BT/IN/Denmark/07/RSM/2015–2016) to RSM and Centre for Brain Development and Repair (CBDR), Dept. of Biotechnology (BT/MB-CNDS/2013) to SC. Animal work in the NCBS/inStem Animal Care and Resource Center was partially supported by the National Mouse Research Resource (NaMoR) grant# BT/PR5981/MED/31/181/2012;2013-2016 & 102/IFD/SAN/5003/2017-2018 from the Indian Department of Biotechnology.
Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
PMK and RSM designed the experiments. PMK, SR., and NN carried out biochemical experiments. SC provided resources and suggestions. PMK and RSM wrote the manuscript. All authors read and approved the final manuscript.
All the work was done with due approval from the Institutional Animal Ethics committee (IAEC) and the Institutional Biosafety Committee (IBSC), InStem, Bangalore, India.
Consent for publication
The authors declare that they have no competing interests.
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