Differential Regulation of Syngap1 Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity

SYNGAP1, a Synaptic Ras-GTPase activating protein, regulates synapse maturation during a critical developmental window. Heterozygous mutation in SYNGAP1 (SYNGAP1-/+) has been shown to cause Intellectual Disability (ID) in children. Recent studies have provided evidence for altered neuronal protein synthesis in a mouse model of Syngap1-/+. However, the molecular mechanism behind the same is unclear. Here, we report the reduced expression of a known translation regulator, FMRP, during a specific developmental period in Syngap1-/+ mice. Our results demonstrate that FMRP interacts with and regulates the translation of Syngap1 mRNA. We further show reduced Fmr1 translation leads to decreased FMRP level during development in Syngap1-/+ which results in an increase in Syngap1 translation. These developmental changes are reflected in the altered response of eEF2 phosphorylation downstream of NMDA Receptor (NMDAR)-mediated signaling. In this study, we propose a cross-talk between FMRP and SYNGAP1 mediated signaling which can also explain the compensatory effect of impaired signaling observed in Syngap1-/+ mice.

Studies using a mouse model have shown that Syngap1 −/+ causes early maturation of dendritic spines in the hippocampus (Clement et al., 2012), and altered critical period of development in thalamocortical synapses (Clement et al., 2013). These studies have shown abnormal dendritic spine activity, and morphology coincided with an increased AMPAR/NMDAR-mediated currents during Post-Natal Day (PND)14-16 and 4-5 in the hippocampus, and thalamocortical neurons, respectively, that led to an altered critical period of plasticity in Syngap1 −/+ mice. Consistent with its molecular function, studies from human patients have shown that lossof-function mutations in SYNGAP1 resulted in Intellectual Disability (ID), Autism Spectrum Disorder (ASD), and epilepsy (Hamdan et al., 2009(Hamdan et al., , 2011Rauch et al., 2012). All these studies suggest that SYNGAP1 is crucial for the development of neuronal connections during the critical period of development (Jeyabalan and Clement, 2016).
Recent studies using Syngap1 −/+ mice and Syngap1 knockdown in rat cultured cortical neurons demonstrated increased levels of basal protein synthesis in Syngap1 −/+ as compared to WT (Wang et al., 2013;Barnes et al., 2015). The studies also suggested that SYNGAP1 modulates insertion of AMPARs at the post-synaptic membrane, thereby, regulating synaptic plasticity through protein synthesis (Rumbaugh et al., 2006;Wang et al., 2013). However, the molecular mechanisms for SYNGAP1-mediated regulation of protein synthesis, particularly during development, are unclear.
To regulate synaptic protein synthesis, SYNGAP1 may crosstalk with other translation regulators. One such potential candidate to consider is Fragile X Mental Retardation Protein (FMRP). Similar to Syngap1 −/+ mice, Fmr1 knock-out (KO) resulted in excessive levels of basal protein synthesis and altered dendritic spine structure and function (Huber et al., 2002). Additionally, a recent report showed exaggerated protein synthesis-independent mGluR-LTD (Metabotropic glutamate receptor-dependent long-term depression) in Syngap1 −/+ (Barnes et al., 2015), which is another hallmark phenotype of FMRP associated synaptic deficits (Huber et al., 2002). Based on these findings, we hypothesized a possible cross-talk between SYNGAP1 and FMRP in regulating activity-mediated protein synthesis at the synapse. In this study, we have shown that FMRP level was altered during development, especially at PND21-23, in Syngap1 −/+ . Besides, FMRP interacts with and regulates the translation of Syngap1 mRNA, and, thus, compensates for Syngap1 translation in Syngap1 −/+ . These results may explain the impaired NMDAR-mediated signaling observed in Syngap1 −/+ .

Animals
C57/BL6 Wild-type (WT) and Syngap1 −/+ mice were obtained from The Jacksons Laboratory 1 (Kim et al., 2003) and bred and maintained in the Animal Facility, JNCASR, under 12-h dark and light cycle. This study was carried out in accordance with the principles of the Basel Declaration and recommendations of the Institutional Animal Ethics Committee (IAEC; Prof. Anuranjan Anand, Chairman). The protocol was approved by the Committee for Control and Supervision of Experiments on Animals (CPCSEA; Dr. K. T. Sampath, CPCSEA Nominee). 1 http://www.jax.org/strain/008890s Preparation of Hippocampal Slices Acute brain slices were prepared from PND > 90 male and female WT and Syngap1 −/+ mice. Mice were brought from the animal house and sacrificed by cervical dislocation, and the brain was dissected out. The brain was kept in ice-cold sucrose based artificial cerebrospinal fluid (aCSF; cutting solution) comprising of: 189 mM Sucrose (S9378, Sigma Aldrich), 10 mM D-Glucose (G8270, Sigma Aldrich), 26 mM NaHCO 3 (5761, Sigma Aldrich), 3 mM KCl (P5405, Sigma Aldrich), 10 mM MgSO 4 .7H 2 O (M2773, Sigma Aldrich), 1.25 mM NaH 2 PO 4 (8282, Sigma Aldrich) and 0.1 mM CaCl 2 (21115, Sigma Aldrich). The brain was taken out of cutting solution and glued to the brain holder of the vibratome (Leica #VT1200), and 350 µm thick horizontal slices were prepared. Cortex and CA3 regions of the hippocampus were dissected out from each slice. All the slices were kept in slice chamber containing aCSF comprising: 124 mM NaCl (6191, Sigma Aldrich), 3 mM KCl (P5405, Sigma Aldrich), 1 mM MgSO 4 .7H 2 O (M2773, Sigma Aldrich), 1.25 mM NaH 2 PO 4 (8282, Sigma Aldrich), 10 mM D-Glucose (G8270, Sigma Aldrich), 24 mM NaHCO 3 (5761, Sigma Aldrich), and 2 mM CaCl 2 (21115, Sigma Aldrich), in water bath (2842, Thermo Fisher Scientific) at 37 • C for 45 min. Following recovery, slices were kept at room temperature (RT, 25 • C) till the experiment completed. Post-dissection, every step was carried out in the presence of constant bubbling with carbogen (2-5% CO 2 and 95% O 2 ; Chemix, India). All measurements were performed by an experimenter blind to the experimental conditions.

Extracellular Field Recordings
One slice at a time was placed on a bath chamber (Scientifica, United Kingdom) perfused with aCSF, and the temperature in the bath chamber was maintained at 33 • C using in-line solution heaters (Warner Instruments, United States). Field excitatory post-synaptic potential (fEPSP) were elicited from pyramidal cells of CA1 area of stratum radiatum by placing concentric bipolar stimulating electrode (CBARC75, FHC, United States) connected to a constant current isolator stimulator unit (Digitimer, United Kingdom) at Schaffer-Collateral commissural pathway, and recorded from stratum radiatum of CA1 area of the hippocampus with 3-5 M resistance glass pipette (ID: 0.69 mm, OD: 1.2 mm, Harvard Apparatus) filled with aCSF. Signals were amplified using Axon Multiclamp 700B amplifier (Molecular Devices), digitized using an Axon Digidata 1440A (Molecular Devices), and stored on a personal computer. Online recordings and analysis were performed using pClamp10.7 software (Molecular Devices). Stimulation frequency was set at 0.05 Hz. mGluR-LTD was induced by 5 min bath application of the Group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; Cat# 0805, Tocris, United Kingdom).
Tissue was homogenized using Lysis buffer containing Tris-Hcl (50 mM, Tris: 15965, Thermo Fisher Scientific; HCl: HC301585, Merck), NaCl (150 mM, S6191, Sigma Aldrich), MgCl 2 (5 mM, M8266, Sigma Aldrich), Dithiothreitol (DTT, 1 mM, 3483-12-3, Sigma Aldrich), NP40 (1%), RNase I (100 U/µl; Invitrogen, AM2294) and 1X Protease Inhibitor cocktail (P5726, Sigma Aldrich). All reagents were dissolved in Diethylpyrocarbonate (DEPC, D5758, Sigma) treated autoclaved water. Immunoprecipitation was done using anti-FMRP (F4055, Sigma Aldrich), Rabbit IgG (40159050MG, Millipore), and protein-G Dyna beads (10003D, Invitrogen). 30 µl of Dynabeads were equilibrated with lysis buffer, and further 200 µl of lysis buffer containing 5 µg of antibody was added to Dynabeads and incubated at RT for 1 h on a rotor at a slow speed. Afterward, the antibody solution was removed from the beads by placing the tube in the magnetic stand. Tissue lysate was added to the antibody bound beads and was incubated for 1-h at RT. The lysate was given five washes with lysis buffer. After the last wash, IP buffer was removed entirely, and the sample was eluted in either 1X Laemmli buffer (for protein detection) or Trizol (for RNA isolation). For the mRNA enrichment, mRNA copy number in the pellet was divided by mRNA copy number in the supernatant, unless otherwise mentioned.

RNA Extraction and qPCR
Total RNA was extracted from the polysome fractions by Trizol (15596026, Thermo Fisher Scientific) method (For each sample three times the volume of Trizol was added) and the mRNAs were converted to cDNA using iScript cDNA synthesis kit (1708891, Bio-Rad). qPCR was performed for Syngap1, Fmr1, and β-actin using CFX384.
Real-Time System from Bio-Rad. Primers were designed and obtained from Sigma Aldrich, India. SYBR green was obtained from Bio-Rad (1725122). Ct values obtained from the reactions were converted to the copy number of the mRNA (Muddashetty et al., 2007(Muddashetty et al., , 2011, and the percentage of these copy numbers in each fraction was plotted for polysome experiments. mRNA copy number was derived using the Ct values from the standard curve. The equation for the standard curve was y = −1.44x+31.699; Here, y = average Ct value and EXP(x) was the copy number. List of primers used is mentioned below.

Cell Culture and Transfection
HeLa cells were maintained in DMEM containing 10% FBS at 37 • C in a 5% CO 2 environment passaged using 0.05% trypsin-EDTA solution. Transfections were performed using lipofectamine 2000 (11668027, Thermo Fisher Scientific) as per the manufacturer's protocol.
Tissue was homogenized using Lysis buffer containing Tris-Hcl (200 mM, Tris: 15965, Thermo Fisher Scientific; HCl: HC301585, Merck), KCl (100 mM, P5405, Sigma Aldrich), MgCl 2 (5 mM, M8266, Sigma Aldrich), Dithiothreitol (DTT, 1 mM, 3483-12-3, Sigma Aldrich), NP40 (1%), and 1X Protease Inhibitor cocktail (P5726, Sigma Aldrich). All reagents were dissolved in Diethylpyrocarbonate (DEPC, D5758, Sigma Aldrich) treated autoclaved water. Samples were aliquoted into two equal parts and treated with either of the protein synthesis inhibitors: Cycloheximide (CHX, 10 µg/ml, C7698, Sigma Aldrich) or Puromycin (1 mM, P9620, Sigma Aldrich). The lysates were kept at 37 • C for 30 min and centrifuged at 4 • C for 30 min at 18213 RCF. The supernatant was further loaded carefully on to the sucrose gradient prepared in polysome tubes. Sucrose (84097, Sigma Aldrich) gradient tubes were prepared 1-day before the day of the experiment. 15 to 45% gradients were made, and stored at −80 • C. The supernatant was gently added to each polysome tubes (331372, BECKMAN COULTER), and ultracentrifuged (Beckman, OptimaXL 100K) at 4 • C at 39000 RPM for 1 h and 40 min. The tubes were then transferred to UV Visible spectrophotometer [Model: Type 11 Optical unit with reference Flowcell/No bracket, Serial No: 213K20162 at National Centre for Biological Sciences (NCBS)], and fractions were collected at A 254 spectra using Fraction collector instrument (from TELEDYNE ISCO at NCBS). The bottom of the tube was pierced using a syringe attached to a pipe containing 60% sucrose, and the fractions were collected in 1.5 ml tubes. Total of 11 fractions was collected from each polysome tube, and these fractions were treated with SDS loading dye containing β-Mercaptoethanol (MB041, HIMEDIA) for immunoblotting or Trizol for RNA extraction and qPCR. SDS-PAGE was done for these fractions and immunoblotted for RPLP0 and FMRP.

Statistics
All graphs were plotted using Graph Pad Prism 7 and Microsoft Excel 2016. Extracellular field recordings were performed and analyzed using Clampfit 10.7. Time course data shown in Figure 1A were plotted by averaging every 2 min. Example traces were those recorded for 1-2 min around the time point indicated.
Error bars correspond to ± SEM (Standard Error of Mean). Unpaired Student's t-test and 2-way ANOVA were performed to test for difference between groups and different age unless otherwise stated.

Reduced FMRP Level During Development in Syngap1 −/+
Studies have shown that Group I mGluR and NMDA receptors interact via Homer-Shank, thereby, regulating protein synthesis (Tu et al., 1999;Bertaso et al., 2010). To determine whether Group I mGluR activation in Syngap1 −/+ resulted in altered protein synthesis and hippocampal pathophysiology similar to Fmr1 −/y , Group I mGluR-mediated LTD (mGluR-LTD) was induced in the Schaffer-Collateral pathway in adult mice by bath applying 50 µm (S)-DHPG, Group I mGluR agonist, for 5 min. We observed significantly increased mGluR-LTD in Syngap1 −/+ mice (Syngap1 −/+ referred as HET in Figures; 47 ± 4% LTD) as compared to their WT littermate controls (61 ± 3% LTD; p = 0.012; Figure 1A). This result suggests that mGluR-LTD in Syngap1 −/+ is similar to Fmr1 −/y at PND25-32 as shown earlier by Barnes et al. (2015). Our data further showed that abnormal signaling during early stages of development, in fact, continues throughout adulthood (PND90) that may explain the impaired cognitive and social behavior observed in adults. Therefore, we hypothesized that expression of FMRP might be altered during different neurodevelopment stages, including adulthood. We studied the expression of FMRP in the hippocampus of WT and Syngap1 −/+ mice during different stages of development, starting from PND7-9 to 2-5 months of age. Using quantitative immunoblotting, we observed that FMRP level (normalized to β-ACTIN) was reduced in Syngap1 −/+ mice (0.775 ± 0.06) as compared to WT in PND21-23 (1.00 ± 0.07; p = 0.033; Figure 1B) but not in other age groups. FMRP expression profile in WT shows that FMRP level decreases as age increases (Supplementary Figure S1A). Previous studies have shown that reduced SYNGAP1 expression during development led to altered synaptic transmission in Syngap1 −/+ mice (Vazquez et al., 2004;Clement et al., 2012). To study whether reduced level of FMRP is compensating for the altered SYNGAP1 level in Syngap1 −/+ mice, expression of SYNGAP1 in WT and Syngap1 −/+ mice was quantified as shown in Figure 1C and Supplementary Figure S1B (Genotype: p < 0.0001). Upon further analysis, we found that the SYNGAP1 level was increased during PND21-23 (1.12 ± 0.09) compared to PND14-16 in Syngap1 −/+ (0.83 ± 0.05; p = 0.0236; Figure 1C), and no statistical difference was observed in adults (>PND60). In contrast, the level of SYNGAP1 was not altered significantly between PND21-23 (1.82 ± 0.06) and PND14-16 (1.33 ± 0.08) in WT mice (p = 0.0863; Figure 1C). In our study, we considered β-ACTIN as an internal control for normalization. However, β-ACTIN polymerisationdepolymerisation could be modulated by FMRP. Thus, we validated our results using GAPDH as a loading control that showed an expression profile for FMRP, and SYNGAP1 in WT similar to quantification performed with β-ACTIN (Supplementary Figures S1A,B).

FMRP Interacts With Syngap1 mRNA and Regulates Its Translation
FMRP is a known regulator of synaptic translation (Osterweil et al., 2010). A previous study using HITS-CLIP has reported Syngap1 as one of the mRNAs regulated by FMRP (Darnell et al., 2011;Darnell and Klann, 2013). G-quadruplexes are one of the structures present in RNA which could be recognized by FMRP (Darnell et al., 2001). Bioinformatics analysis using Quadruplex forming G-Rich Sequences (QGRS) Mapper predicted the presence of multiple G-quadruplexes structures with high G-Score in Syngap1 mRNA (Supplementary Figure S1C). Besides, G-quadruplex forming residues were found to be conserved among mice, rat, and human Syngap1 mRNA (Supplementary Figure S1C). To further confirm the interaction of FMRP with Syngap1 mRNA, we performed FMRP immunoprecipitation from mouse hippocampal lysates to investigate the enrichment of Syngap1 mRNA by qPCR. We observed a ∼5-fold enrichment of Syngap1 mRNA relative to β-actin mRNA in FMRP-IP pellet over supernatant (5.15 ± 0.43, p = 0.0009; Figure 2A, Supplementary Figure S2A). Psd-95 mRNA, a known FMRP target mRNA (Muddashetty et al., 2011) showed a significant 4.5-fold enrichment compared to β-actin mRNA (4.77 ± 0.09; p = 0.0001; Figure 2A and Supplementary Figure S2A), which we used as a positive control. These results demonstrated that FMRP interacts with Syngap1 mRNA.
We further asked whether the interaction between FMRP and Syngap1 mRNA is altered during development, especially in PND14-16 and 21-23. We did not find a statistical significance in PND14-16 (p = 0.28; Syngap1 −/+ = 2.3 ± 0.66; WT = 1.0 ± 0.1; Figure 2B). Whereas, the interaction between FMRP and Syngap1 mRNA was significantly decreased in Syngap1 −/+ at PND21-23 (p = 0.045; 0.63 ± 0.04; Figure 2B) compared to WT (1.0 ± 0.13). We did not observe any change in the interaction of Psd-95 mRNA with FMRP at any of these age groups (PND14-16:   Supplementary Figures S2C-E). We have shown that a reduction in FMRP led to an increase in GFP-SYNGAP1 (p = 0.01; Scr siRNA 0.58 ± 0.05; FMR1 SiRNA 0.82 ± 0.067; Figure 2C). These results demonstrated that FMRP not only interacts with Syngap1 mRNA but also regulates its translation. On the basis of our data, we speculate that reduced interaction between FMRP and Syngap1 mRNA in Syngap1 −/+ at PND21-23 might lead to increased SYNGAP1 level as observed earlier.

Altered NMDAR-Mediated Translation Response in Syngap1 −/+
Previous studies have shown increased levels of basal protein synthesis in Syngap1 −/+ (Wang et al., 2013;Barnes et al., 2015). SYNGAP1 regulates synaptic maturation during a critical time window, and our results demonstrated altered expression of FMRP during a specific developmental stage in Syngap1 −/+ . Based on this, we hypothesized that the translational status could be different at these developmental stages. To study that, the phosphorylation status of eukaryotic Elongation Factor 2 (eEF2) was used as a read-out of translation response. Phosphorylation of eEF2 has been shown to repress global translation (Scheetz et al., 2000). We analyzed phospho/total-eEF2 in response to NMDAR stimulation from WT and Syngap1 −/+ hippocampal synaptoneurosomes at PND14-16 and 21-23 using immunoblotting analysis. Hippocampal synaptoneurosome preparation was evaluated by validating the enrichment of PSD-95 as shown by Muddashetty et al., 2007 (Supplementary Figure S6A). As a proof of principle, we demonstrated that NMDAR stimulation of synaptoneurosomes from WT mice showed ∼1.5-fold increase in phospho/total-eEF2 1-min post-stimulation (Basal = 0.84 ± 0.11; Stimulated = 1.3 ± 0.12; p = 0.0376; Supplementary Figure  S5A). To validate that the phosphorylation response of eEF2 is indeed resulting from NMDAR stimulation, we pre-treated the synaptoneurosomes with AP-5, a potent antagonist of NMDAR. The NMDAR-mediated phosphorylation was lost on AP-5 pre-treatment, showing the specificity of our assay (Supplementary Figure S6B).

DISCUSSION
Many synaptic plasticity mechanisms are dependent on activity mediated local protein synthesis in neurons (Klann et al., 2004;Pfeiffer and Huber, 2006). Protein synthesis is regulated stringently in the synapse. One such crucial regulator of synaptic protein synthesis is FMRP, which is encoded by FMR1 gene, the absence of which leads to Fragile X Syndrome, a monogenic cause of ID similar to SYNGAP1 −/+ (Garber et al., 2008;Hamdan et al., 2009). Our observation of enhanced mGluR-LTD in the CA1 hippocampal region of Syngap1 −/+ complements previous observation of enhanced basal protein synthesis in Syngap1 −/+ prompted us to investigate the role of FMRP in the pathophysiology of Syngap1 −/+ mutation (Wang et al., 2013;Barnes et al., 2015). Till date, only one report has studied interrelation between SYNGAP1 and FMRP (Barnes et al., 2015). They proposed that mutations in Fmr1 and Syngap1 lead to an opposite effect on synapse development, with FMRP deficits resulting in delayed synaptic maturation and deficit in SYNGAP1 causing accelerated maturation of dendritic spines. Considering this, Barnes et al. crossed Fmr1 −/Y with Syngap1 −/+ but failed to rescue the neurophysiological deficits observed in Syngap1 −/+ (Barnes et al., 2015). This study indicates that chronic depletion of these genes may not be a useful measure to rescue the pathophysiology observed in Syngap1 −/+ , as both these genes are essential for normal brain development. Since SYNGAP1 is known to regulate synaptic maturation during a specific developmental window (Clement et al., 2012(Clement et al., , 2013, we hypothesized that the role of FMRP in Syngap1 −/+ could also be developmentally regulated. Hence, we looked at the developmental expression profile of FMRP in the hippocampus of Syngap1 −/+ mice. Our results show reduced expression of FMRP specifically in PND21-23 in Syngap1 −/+ . A study by Darnell et al., have identified Syngap1 mRNA as one of the targets of FMRP by a highthroughput analysis. However, many such targets were not validated (Darnell et al., 2011). Our study is the first to validate the interaction between FMRP and Syngap1 mRNA, FIGURE 5 | Model, illustrating the regulation of FMRP-mediated translation of Syngap1 during development. This model shows that FMRP regulates Syngap1 mRNA translation, which in turn regulates NMDAR-mediated signaling. In WT, NMDAR stimulation in synapse led to increased phosphorylation of eEF2, which resulted in global translation inhibition and the signaling was efficiently regulated by SYNGAP1. Whereas, in Syngap1 −/+ at PND14-16, NMDAR-mediated signaling was impaired as depicted by the loss of phosphorylation response to eEF2 due to a decreased level of SYNGAP1. At PND21-23 in Syngap1 −/+ , FMRP level was low that increased translation of Syngap1 mRNA leading to an increased SYNGAP1 level compared to PND14-16. Thus, an elevated level of SYNGAP1 might recover the NMDAR-mediated signaling via phosphorylation of eEF2. thereby, regulating its translation. Our result suggests that the reduction in FMRP levels, as well as its reduced interaction with Syngap1 mRNA at PND21-23 in Syngap1 −/+ , might lead to the compensatory increase in SYNGAP1 levels via increased Syngap1 mRNA translation. In polysome profiling assay, we did not observe any significant difference in the A 254 traces or the distribution of protein RPLP0 between WT and Syngap1 −/+ animals indicating no difference in the basal translation in the hippocampus from Syngap1 −/+ animals at PND14-16 and PND21-23.
Studies have reported that NMDAR-mediated signaling is dysregulated in Syngap1 −/+ (Komiyama et al., 2002;Rumbaugh et al., 2006;Carlisle et al., 2008). These studies have further shown that SYNGAP1 associates with NR2B (Rockliffe and Gawler, 2006) and negatively regulates NMDAR-mediated ERK activation (Kim et al., 2005) and, hence, regulates insertion of AMPAR in the post-synaptic membrane (Rumbaugh et al., 2006). In line with this, Komiyama et al., have demonstrated increased basal levels of ERK phosphorylation in Syngap1 −/+ (Komiyama et al., 2002) which does not explain the deficits observed in NMDAR-LTP in Syngap1 −/+ mice as NMDAR stimulation resulted in a robust increase in ERK activation in slices from Syngap1 −/+ mice (Komiyama et al., 2002). Thus, to understand the deficits seen in NMDAR-mediated signaling in Syngap1 −/+ mice, we studied NMDAR-mediated translation repression. It has already been reported that NMDAR activation causes a reduction in global translation through phosphorylation of eEF2 (Scheetz et al., 2000). In our study, we measured the basal levels of phosphorylated eEF2 in hippocampal synaptoneurosomes from WT and Syngap1 −/+ at PND14-16 and PND21-23 which showed increased phosphorylation of eEF2 at the basal condition in Syngap1 −/+ . This increase in the basal level of phosphorylation of eEF2 could be due to enhanced excitatory neuronal activity in Syngap1 −/+ which might lead to an increase in Ca 2+ levels and a subsequent increase in eEF2 phosphorylation via Ca 2+ -Calmodulin kinase. We report that, at PND14-16, NMDAR activation fails to cause eEF2 phosphorylation in Syngap1 −/+ animals. Strikingly, even though we observed an increase in basal phospho/total-eEF2 in Syngap1 −/+ synaptoneurosomes at PND21-23, NMDARmediated increase in eEF2 phosphorylation was similar to WT. This observation suggests that NMDAR-mediated translation response at PND21-23 in Syngap1 −/+ may be restored. This change observed in PND21-23 could be due to a compensatory mechanism through increased NMDARmediated signaling. These findings further corroborate with the observations made by Clement et al. in which they have demonstrated increased synaptic transmission and increased AMPAR/NMDAR-mediated currents in PND14-16 but return to normal level in the later age (Clement et al., 2012). Based on our findings, we propose a model in which increased NMDAR-mediated response to protein synthesis is compensating for the loss of SYNGAP1 during development in Syngap1 −/+ . We further propose that fine-tuned downregulation of Fmr1 translation during a specific developmental window in Syngap1 −/+ mice might compensate for the dysregulation in NMDAR-mediated signaling.
These findings are interesting concerning the critical period of maturation of the hippocampus in mice. Early maturation of hippocampal neurons has been shown in Syngap1 −/+ at PND14-16, whereas WT matures at PND21 (Clement et al., 2012). Our findings indicate that these two age groups are crucial for any compensation to occur. Once the window of critical period of development is lost, rescuing the pathophysiology becomes difficult.
Our data based on eEF2 phosphorylation on NMDAR activation is correlative to FMRP downregulation in Syngap1 −/+ at PND21-23. Previous studies have shown dysregulated NMDAR-mediated signaling in the Fmr1 KO mouse model owing to the fact that FMRP plays an essential role in NMDAR-mediated pathway (Toft et al., 2016). Also, whisker stimulation and visual experience that dependent on NMDAR activation led to increased FMRP protein level (Todd et al., 2003;Gabel et al., 2004). Therefore, NMDAR-mediated protein synthesis could be regulated by the level of FMRP as studies have shown that FMRP regulates translation downstream of NMDAR-mediated signaling (Chmielewska et al., 2018). However, regulation of NMDAR-mediated signaling proteins by FMRP in Syngap1 −/+ is unclear. Our study is the first to suggest a potential regulation of NMDARmediated signaling proteins by FMRP. Thus, it is crucial to study FMRP's role in NMDAR-mediated signaling and its regulation by FMRP.

CONCLUSION
In conclusion, our study suggests that an altered response to activity-mediated protein synthesis during development is one of the major causes of abnormal neuronal function in Syngap1 −/+ . However, chronic depletion of two genes with common core pathophysiology may not be a useful measure to rescue the deficits observed in either of these mutations, i.e., Fmr1 −/y and Syngap1 −/+ , as both these genes are essential for healthy brain development. Therefore, modulating these proteins at a specific developmental window could be a potential therapeutic strategy for treating ID-related pathophysiology.

ETHICS STATEMENT
This study was carried out in accordance with Institutional Animal Ethics Committee (IAEC), and Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

AUTHOR CONTRIBUTIONS
AP and BN performed all the experiments. JPC did mGluR-LTD in Figure 1A.  SYNGAP1 normalized to β-ACTIN (middle) and normalized to GAPDH (below; only WT) at PND7-9, PND14-16, PND21-23, and PND > 60 (N = 4 for all age groups, samples were run on the same gel). SYNGAP1/ β-ACTIN: Two-way ANOVA followed by Bonferroni's multiple comparison test; WT vs HET at PND7-9: p = 0.23; PND14-16: * * p = 0.0014; PND21-23: * * * p = 0.0006; PND > 60: * * * p = 0.0004. SYNGAP1/GAPDH: One-way ANOVA followed by Tukey's multiple comparison tests; NS, not significant across age. (C) Multiple putative G-quadruplex was detected using QGRS Mapper in the validated sequence available for mouse Syngap1 from NCBI (Gene ID: 240057). Three G-quadruplex sequences having high G-score were highlighted in the red box. All these sequences have been mapped in the Coding Sequence (CDS) (left panel). Multiple sequence alignment of the highest score G-quadruplexes of mouse Syngap1 compared with Human and Rat. G score: 82 showing putative G-quadruplexes conserved among Human, Mouse, and Rat, respectively (right panel).  . Quantified data as histogram shows increased phosphorylation of eEF2 on NMDA treatment is lost when co-treated with AP-5 (below). * p < 0.05, One-way ANOVA followed by Dunnett's multiple comparison tests.