NMDAR-mediated protein synthesis is regulated by calcium signals generated by distinct sources

Previously, we have reported that stimulation of NMDARs in primary cortical neurons generates a unique biphasic protein synthesis profile involving initial translation inhibition followed by a phase of translation activation (30). Stimulation of NMDARs is also known to generate a distinct spatio-temporal calcium response in neurons involving NMDARs, L-VGCCs, and calcium release from Ryanodine Receptors (RyRs) on the ER (5, 26, 27, 46, 47). To assess this further, rat primary cortical neurons (DIV15) were used for measuring the calcium response on NMDAR stimulation in the presence or absence of L-VGCC antagonist nifedipine and RyR antagonist dantrolene (Fig 1A). Blocking L-VGCCs using nifedipine significantly reduced the NMDAR-mediated calcium influx by close to 60% at the first, second, third, fourth, and fifth minute of NMDA addition (Fig 1B–G). Blocking RyRs using dantrolene reduced NMDAR-mediated calcium influx by 50% at the first and fifth minute after NMDA addition (Fig 1B–G), though the trend of decrease persisted along the second, third, and fourth minute time-points as well (Fig 1B–G). Thus, cytosolic calcium increase on NMDAR stimulation involves calcium influx from external sources (NMDARs and L-VGCCs), as well as calcium release from ER (RyR-mediated) with distinct temporal contributions.

To study the effect of different calcium sources on the translation response, we investigated the individual contribution of L-VGCCs and RyRs in the background of NMDAR stimulation. We stimulated the neurons with NMDA in the presence or absence of nifedipine or dantrolene, and measured the protein synthesis response using FUNCAT. In addition, to study the effect of IP3Rs and SOCE on NMDAR-mediated translation, we used 2-APB, which acts as a blocker for both IP3Rs and SOCE (Fig S1A). Pre-incubation of neurons with nifedipine, dantrolene, and 2-APB did not show a change in FUNCAT signal compared with the control condition, indicating that the drugs do not affect protein synthesis on their own (Fig S1B and C). Because we used methionine-free DMEM in the FUNCAT assay, we used puromycin incorporation as another readout to confirm that Met-free DMEM does not affect protein synthesis on its own. The change of media to Met-free DMEM or Neurobasal does not affect protein synthesis on its own compared with no medium change condition (Fig S1D). Thus, after these validations, we used FUNCAT as the primary readout for protein synthesis.

As reported previously, NMDAR stimulation for 5 min caused a decrease in the FUNCAT signal indicating translation inhibition (Fig 1H), which shifted to the phase of translation activation by 20 min, as observed by the increase in the FUNCAT signal (Fig 1H). In the presence of nifedipine (L-VGCC antagonist), we observed a decrease in FUNCAT signal at both 5- and 20-min time points of NMDAR stimulation compared with the basal condition (Fig 1I). Similarly, even in the presence of dantrolene (RyR antagonist), the FUNCAT signal was decreased at 5- and 20-min time points on NMDAR stimulation compared with the basal condition (Fig 1J). NMDAR stimulation in the presence of 2-APB caused a reduction in the FUNCAT signal at 5 min and no change in the FUNCAT signal at the 20-min time point compared with the basal condition (Fig S1E). Thus, the temporal profile of NMDAR-mediated protein synthesis, particularly the translation activation at 20 min, was dependent on calcium from L-VGCCs, RyRs, and SOCE (Figs 1K and S1F).

NMDAR-mediated changes in phosphorylation of eukaryotic translation elongation factor 2 (eEF2) are differentially regulated by calcium sources

Activity-mediated protein synthesis in neurons is tightly coordinated through translation inhibition and activation pathways. To obtain mechanistic insights into these two components, we chose to investigate translation inhibition and activation using separate readouts. eEF2, an essential and obligatory factor for ribosome translation on mRNA, is phosphorylated by eEF2 kinase in a calcium-dependent manner (48, 49). The increase in the phosphorylation of eEF2 (p-eEF2) decreases translation elongation and acts as a reliable marker for global protein synthesis inhibition (Fig 2A). As reported previously (25, 30), NMDAR stimulation of primary neurons increased eEF2 phosphorylation at 1 min, after which the phospho-eEF2 levels decreased (Figs 2B and D and S2A and B). As observed in our previous studies (25, 30), at 5-min NMDAR stimulation, eEF2 phosphorylation seemed lower than 1 min and higher compared with untreated conditions, indicating a net translation inhibition (Figs 2B and D and S2A and B). The p-eEF2 data at 5-min NMDAR stimulation correlates with the FUNCAT data indicating a decrease in protein synthesis (Fig 1H). At 20-min NMDAR stimulation, we observed a decrease in eEF2 phosphorylation compared with the basal condition, resulting in a further reduction in translation inhibition and a net increase in protein synthesis (Figs 2B and D and S2A and B), which was also captured by the FUNCAT data (Fig 1H). In the presence of the NMDAR channel antagonist MK801, the eEF2 phosphorylation profile was completely blocked, indicating that the response was specific and initiated by calcium through NMDARs (Fig S2A). Similarly, the changes in eEF2 phosphorylation were abolished in the absence of extracellular calcium, showing that the calcium influx from an external source was the trigger for NMDAR-mediated translation response (Fig S2B).

NMDAR stimulation in the presence of nifedipine (L-VGCC antagonist) did not affect the increase in eEF2 phosphorylation at 1 min (Fig 2B). However, in the presence of nifedipine, the phospho-eEF2 levels did not decrease after 1 min and continued to persist at a higher level until 20 min (Fig 2B). Thus, blocking L-VGCCs led to increased eEF2 phosphorylation at 5 and 20 min as compared to NMDAR stimulation in the absence of nifedipine (Fig 2B). These results were further validated through immunostaining for p-eEF2. NMDAR stimulation for 5 min caused an increase in p-eEF2 levels in MAP2-positive neurons (Fig 2C), and the presence of nifedipine further increased the p-eEF2 as compared to NMDAR stimulation alone (Fig 2C). In the presence of dantrolene (RyR antagonist), NMDAR-mediated eEF2 phosphorylation was not affected at 1- and 5-min time points (Fig 2D). At 20-min time point, although NMDAR stimulation alone led to a decrease in eEF2 phosphorylation, it remained significantly higher in the presence of dantrolene (Fig 2D). Interestingly, immunostaining experiments showed that 5-min NMDAR stimulation in the presence of dantrolene caused a significant increase in p-eEF2 levels as compared to NMDAR stimulation alone (Fig 2E). The use of 2-APB (SOCE blocker) affected the NMDAR-mediated eEF2 phosphorylation mainly at 5 min, where p-eEF2 levels were higher in the presence of 2-APB (Fig S2C and D). However, eEF2 phosphorylation returned to basal levels at 20-min NMDAR stimulation in the presence of 2-APB (Fig S2C). In summary, calcium through L-VGCCs, RyRs, and SOCE are important contributors in regulating eEF2 phosphorylation at 5-min NMDAR stimulation (Figs 2F and S2E). However, L-VGCCs and RyRs seem more important for the reduction in eEF2 phosphorylation and increased translation at 20 min of NMDAR stimulation (Figs 2F and S2E).

NMDAR-mediated changes in ribosomal protein S6 (RPS6) phosphorylation are regulated by different calcium sources

We investigated the phosphorylation status of RPS6 as the readout for translation activation (Fig 3A). Although mTOR is the well-known upstream regulator of RPS6 phosphorylation, reports suggest that S6 kinase activity and RPS6 phosphorylation can be regulated in an Akt- and a calcium-dependent manner as well (50, 51, 52, 53, 54), thus making it an interesting readout for NMDAR-mediated translation activation. NMDAR stimulation of primary cortical neurons showed an increase in RPS6 phosphorylation primarily at 20-min time point indicating translation activation (Fig 3B and D). This was in correlation with the increased FUNCAT signal (Fig 1) and decreased eEF2 phosphorylation (Fig 2) at 20-min NMDAR stimulation. At 5-min NMDAR stimulation, p-RPS6 levels showed a robust increase in MAP2-positive neurons through immunostaining experiments (Fig 3C and E), though this was captured only as a trend of increase in Western blotting (Fig 3B and D). Thus, 5-min NMDAR stimulation correlates with active reduction in translation inhibition (indicated through eEF2 phosphorylation), as well as potential translation activation.

The pre-treatment of neurons with nifedipine (L-VGCC antagonist) prevented the NMDAR-mediated increase in RPS6 phosphorylation at the 20-min time point (Fig 3B). Immunostaining experiments indicated that nifedipine blocked the NMDAR-mediated p-RPS6 increase at 5-min time point as well (Fig 3C). Similar results were observed with pre-treatment of dantrolene (RyR antagonist) and 2-APB (SOCE blocker). Dantrolene and 2-APB blocked the NMDAR-mediated p-RPS6 increase at 20 min as indicated by immunoblotting (Figs 3D and S3A) and 5 min as indicated by immunostaining (Figs 3E and S3B). These results confirm that calcium through L-VGCCs, RyRs, and SOCE was important for increased RPS6 phosphorylation and translation activation on NMDAR stimulation (Figs 3F and S3C).

Results from earlier sections established that L-VGCCs were critical for both reducing translation inhibition (eEF2 phosphorylation) and increasing translation activation (RPS6 phosphorylation). To further validate these findings, we treated primary neurons with BayK8644, an agonist of L-VGCCs, for 5 and 20 min. We validated the activity of BayK8644 by showing that it causes calcium influx in neurons (Fig S3D). A 5-min treatment of BayK8644 resulted in an increase in eEF2 phosphorylation in neurons, which was significantly reduced by 20 min (Fig S3E). Interestingly, BayK8644 treatment also caused an increase in RPS6 phosphorylation at 20 min (Fig S3F). Thus, calcium through L-VGCCs can independently cause the translation inhibition at 5 min, which changes to global translation activation by 20 min.

IP3-mediated calcium release and SOCE are necessary for translation activation on mGluR stimulation

To further validate the role of calcium in regulating neuronal protein synthesis, we employed an independent paradigm of mGluR stimulation. mGluRs being G protein–coupled receptors, their stimulation generates IP3, resulting in cytosolic calcium increase mediated by IP3Rs and SOCE (28, 29, 55) (Fig 4A). Stimulating the neurons with DHPG (Group 1 mGluR agonist) caused a sustained increase in cytosolic calcium for 5 min, which was completely abolished in the presence of 2-APB (IP3R and SOCE blocker) (Fig 4B and C). Activation of mGluRs is also known to cause an up-regulation of neuronal protein synthesis (25, 31, 32, 56). As reported previously, mGluR stimulation using DHPG caused an increase in FUNCAT signal at 5- and 20-min time points (Fig 4D). However, in the presence of 2-APB, mGluR stimulation failed to elicit an increase in the FUNCAT signal (Fig 4E), indicating that calcium through IP3Rs and SOCE was necessary for mGluR-mediated translation activation. We observed an increase in the MAP2 levels at 5 and 20 min of mGluR stimulation (Fig S4A), and hence did not normalize FUNCAT signals to MAP2 in these experiments. The increased translation of MAP2 on mGluR stimulation has been reported previously as well (57). The mGluR-mediated increase in MAP2 levels was also abolished in the presence of 2-APB (Fig S4B), further validating the role of calcium in mGluR mediated translation up-regulation.

Furthermore, we investigated the eEF2 and RPS6 phosphorylation responses on mGluR stimulation at 1, 5, and 20 min. DHPG caused a significant reduction in eEF2 phosphorylation at 5 and 20 min (Fig S4C) while resulting in an increase in RPS6 phosphorylation at the 5-min time point (Fig S4D). We used 2-APB to test the response at the 5-min time point through immunostaining. 5-min mGluR stimulation led to a decrease in p-eEF2 levels in MAP2-positive neurons, which was prevented in the presence of 2-APB (Fig 4F). Similarly, mGluR-mediated increase in p-RPS6 levels was also abolished in the presence of 2-APB (Fig 4G). Hence, mGluR stimulation led to an up-regulation of protein synthesis (decrease in translation inhibition and increased activation), which was completely dependent on the cytosolic calcium increase through IP3Rs and SOCE (Fig 4H).

Dysregulation of NMDAR-mediated calcium and protein synthesis response in Alzheimer’s disease neurons

So far, our results have shown that the calcium signal has an important role in regulating protein synthesis on NMDAR and mGluR stimulation. Furthermore, we aimed to understand the relevance of this link in the context of Alzheimer’s disease (AD) (58). Several studies have indicated that dysregulation of calcium homeostasis is an early phenotype in AD and a key factor in accelerating the pathological changes in AD (34, 35, 36). Defective calcium signaling involving NMDARs, L-VGCCs, RyRs, and SOCE has been implicated in AD (34, 35, 36). Considering this, we investigated the NMDAR-mediated calcium response in AD neurons and measured its effects on protein synthesis.

We used primary cortical neurons (DIV15) prepared from C57BL/6 WT and APPswe/PS1dE9 (APP/PS1) double transgenic AD mice (Fig 5A). Stimulation of NMDARs in WT mouse neurons resulted in a robust increase in cytosolic calcium (Fig 5B). However, NMDAR-mediated calcium influx was clearly reduced in APP/PS1 neurons (Fig 5B), indicating the defective calcium response in AD. Likewise, the NMDAR-mediated protein synthesis response was preserved in WT mouse neurons. WT neurons showed the biphasic translation response on NMDAR stimulation, demonstrated by the decrease in the FUNCAT signal at 5 min and an increase in the FUNCAT signal at 20 min compared with the basal condition (Fig 5C). However, the NMDAR-mediated changes in protein synthesis were completely abolished in AD, as APP/PS1 neurons showed no change in FUNCAT signal at 5 and 20 min compared with the basal condition (Fig 5C). Interestingly, the basal FUNCAT signal was significantly lower in APP/PS1 neurons compared with WT neurons (Fig 5C), displaying both basal and activity-mediated protein synthesis defects in AD.

Furthermore, we measured the NMDAR-mediated changes in eEF2 and RPS6 phosphorylation at 1, 5, and 20-min time points in the context of AD. As shown previously, the neurons from WT mice responded to NMDAR stimulation, showing an increase in eEF2 phosphorylation at 1 and 5 min, which decreased by the 20-min time point (Fig S5A). Accordingly, the RPS6 phosphorylation increased at 20-min NMDAR stimulation in WT neurons (Fig S5B), depicting the translation activation at the 20-min time point. However, the NMDAR-mediated changes in eEF2 and RPS6 phosphorylation were completely dysregulated in AD neurons (Fig S5C and D). APP/PS1 neurons showed a decrease in eEF2 phosphorylation at 1-min NMDAR stimulation, which remained low until the 20-min time point (Fig S5C), whereas RPS6 phosphorylation showed no changes with 1, 5, and 20 min of stimulation (Fig S5D). Thus, the correlation and crosstalk between eEF2 phosphorylation, RPS6 phosphorylation, and protein synthesis (FUNCAT) were perturbed in the AD neurons, indicating an overall dysregulation of basal and NMDAR-mediated translation response (Fig S5E).

Finally, we validated our findings in a human stem cell–derived neuron model of AD. We used iPSCs generated from familial AD patients carrying PSEN1 L150P mutation and its corresponding gene-corrected isogenic control line PSEN1 L150P GC (Fig 5D). The mutant and control iPSCs were characterized for the expression of stem cell markers OCT4, NANOG, SSEA4, and TRA-160 (Fig S5F and G). The iPSCs were differentiated into forebrain glutamatergic neurons (Fig 5D). The intermediate neural stem cell (NSC) stage was characterized using Nestin and Pax6 (Fig S5H and I), whereas the neurons were characterized for dendritic marker MAP2 and axonal marker Tau (Fig S5J and K). The differentiated neurons were subjected to calcium imaging on NMDAR stimulation. Control (PSEN1 L150P GC) neurons showed a robust increase in cytosolic calcium on NMDAR stimulation (Fig 5E), whereas the mutant (PSEN1 L150P) neurons completely lacked the calcium response on the addition of NMDA (Fig 5E). Accordingly, the NMDAR-mediated changes in protein synthesis were also affected in AD neurons. Although the control (PSEN1 L150P GC) neurons showed an increase in eEF2 phosphorylation at 5-min NMDAR stimulation (Fig 5F), the mutant (PSEN1 L150P) neurons showed no changes in eEF2 phosphorylation (Fig 5G). Thus, our results show that calcium is an important regulator of protein synthesis on neuronal activity. AD neurons show perturbation of both calcium and translation response on NMDAR stimulation.

Ethics statement

All animal work was carried out following the Institutional Guidelines for the Care and Use of Laboratory Animals under the approval of the Institutional Animal Ethics Committee and the Institutional Biosafety Committee (IBSC), Centre for Brain Research, Indian Institute of Science Campus, Bangalore, India. The rats used in the study were Sprague Dawley (SD) rats. The mouse work was done with C57BL/6J mice. APPswe/PS1dE9 double transgenic mice were procured from the Jackson Laboratory (stock #34832; MMRRC) (77). APPswe/PSEN1dE9 mice carry two transgenes with AD-linked mutations: a chimeric mouse/human APP with the Swedish mutation (K670N, M671L; deletion–insertion mutation) and human PSEN1 lacking exon 9 (dE9), both under the control of the mouse prion protein promoter. The animals were housed and maintained under pathogen-free conditions in a temperature-controlled room on a 12-h light/12-h dark cycle with ad-libitum access to food and water.

All the human stem cell work was carried out as per approval from the Institutional Human Ethics Committee and Institutional Biosafety Committee at the Centre for Brain Research, Indian Institute of Science Campus, Bangalore, India.

Rat primary neuronal cultures

Primary neuronal cultures were prepared from cerebral cortices of Sprague Dawley rat embryos (E18.5) as previously published by our laboratory (25, 30, 78). Briefly, the cortices were trypsinized for 5 min at 37°C using 0.25% trypsin (15050057; Thermo Fisher Scientific). The homogenized and dissociated cells were plated on cell culture dishes, which were pre-coated with poly-L-lysine (P2636; Sigma-Aldrich) solution (0.1 mg/ml) made in borate buffer (pH 8.5) for 5–6 h. Cells were plated at a density of 40,000–50,000 cells/cm² for biochemistry experiments and 30,000–40,000 cells/cm² for imaging-based experiments. Nitric acid–treated coverslips were used for imaging experiments. The neurons were initially plated on MEM (10095080; Thermo Fisher Scientific) supplemented with 10% FBS to aid their attachment. After 3 h, MEM was changed to Neurobasal medium (21103049; Thermo Fisher Scientific) supplemented with B27 (17504044; Thermo Fisher Scientific) and 1X GlutaMAX. The neurons were maintained in culture for 15–17 d at 37°C, with 5% CO₂. The neurons were supplemented with Neurobasal media every 5–6 d.

Genotyping of mouse pups

Genotyping was performed to identify the WT and APPswe/PS1dE9 double transgenic pups (Tg APP/PS1) from the litter. A small piece of the tail was collected from the day-zero pups (P0 pups) in a sterile 1.5-ml tube and 650 μl of STE buffer (sodium chloride–Tris–EDTA buffer) (100 mM Tris, 5 mM EDTA, 200 mM NaCl, 0.2% SDS), and 20 μl of proteinase K (20 mg/ml stock solution) was added to it. The sample was incubated at 55°C for approximately 3 h with periodic vortexing to aid proper digestion. After this, the sample was vortexed for a minute and centrifuged at 14,000g for 10 min in a microcentrifuge. The supernatant was collected, and 600 μl of isopropanol was added to it. The solution was mixed, incubated on ice for 5 min, and centrifuged at 14,000g for 10 min. 600 μl of 70% ethanol was added to the pellet and centrifuged at 14,000g for 10 min. The DNA pellet obtained was dried at 55°C for 15 min, resuspended in 50 μl autoclaved sterile water, and incubated at 55°C for a few minutes to ensure complete dissolution. The concentration of isolated DNA was determined using a NanoDrop. Genomic DNA (50 ng) was subjected to PCR using Prp Forward Primer (0.6 μM) (5′-CCTCTTTGTGACTATGTGGACTGATGTCGG-3′), Prp Reverse Primer (0.8 μM) (5′-GTGGATAACCCCTCCCCCAGCCTAGACC-3′), and APP Forward Primer (0.6 μM) (5′-CCGAGATCTCTGAAGTGAAGATGGATG-3′). The PCR conditions were as follows: initial denaturation at 95°C for 1 min (1×), denaturation at 98°C for 10 s (35×), annealing at 65°C for 1 min 30 s (35×), extension at 72°C for 1 min 30 s (35×), and final extension at 72°C for 5 min (1×). The amplified PCR products were resolved on 1% agarose gel (made and run in 1× TAE buffer) through gel electrophoresis and imaged using Bio-Rad Chemi-imager.

Mouse primary neuronal cultures

Primary neuronal cultures were prepared from cerebral cortices of day-zero pups (P0 pups) from WT (C57BL/6) and transgenic APP/PS1 (Tg APP/PS1) mice. After genotyping, the cortices from the WT and Tg pups were trypsinized for 5 min using 0.25% trypsin–EDTA at RT. The homogenized and dissociated cells were plated on dishes with pre-coated poly-L-lysine (P2636; Sigma-Aldrich). The protocol for coating and density for plating were the same as rat neurons. The neurons were plated in Neurobasal media (21103049; Thermo Fisher Scientific) supplemented with B27 (17504044; Thermo Fisher Scientific) and 1× GlutaMAX. After 4 h, the media was replaced with fresh Neurobasal media. The neurons were maintained in culture for 15–17 d at 37°C with 5% CO_2. The neurons were supplemented with Neurobasal media every 5–6 d.

iPSC maintenance and neuronal differentiation

The iPSCs were generated from the fibroblasts of a familial AD patient containing the PSEN1 L150P mutation (L150P mutant) (79). The L150P mutant iPSCs were subjected to CRISPR/Cas9-based gene editing to correct the mutation and obtain the isogenic L150P gene-corrected lines (L150P GC) (80). The iPSCs were maintained on hESC-qualified Matrigel (354277; Corning) using mTeSR1 complete media (72232; StemCell Technologies) at 37°C, 5% CO₂ conditions. A mixture of 1 mg/ml collagenase IV (17104019; Thermo Fisher Scientific), 0.25% trypsin, 20% knock-out serum (10828028; Thermo Fisher Scientific), and 1 mM calcium chloride (made in PBS) was used to dissociate the iPSCs for passaging.

The protocol for neural differentiation was adapted from Yichen Shi et al (81) and Yu Zhang et al (82) to differentiate iPSCs into forebrain glutamatergic neurons. The Neural Basic Media (NBM) for differentiation contained 50% DMEM/F-12 (21331–020; Thermo Fisher Scientific), 50% Neurobasal medium, 0.1% Pen-Strep, GlutaMAX, N2 (17502–048; Thermo Fisher Scientific), and B27 without vitamin A (12587–010; Thermo Fisher Scientific). Once the iPSCs reached 70–80% confluency, they were subjected to monolayer neural induction by changing the mTeSR1 media to Neural Induction Media (NIM). The NIM is composed of NBM supplemented with small molecules SB431542 (10 μM, an inhibitor of the TGFβ pathway) (72232; StemCell Technologies) and LDN193189 (0.1 μM, an inhibitor of the BMP pathway) (72142; StemCell Technologies). The cells were subjected to neural induction for 12–15 d by changing the media every day until a uniform neuroepithelial layer had formed. After the induction, the monolayer was dissociated using Accutase (A6964; Sigma-Aldrich) and centrifuged at 1,000g for 3 min at RT. The cells were plated overnight in NIM containing 10 μM ROCK inhibitor (Y0503; Sigma-Aldrich) on pre-coated poly-L-ornithine/laminin dishes. Poly-L-ornithine (1:10 dilution in 1X PBS) (P4957; Sigma-Aldrich) coating was performed at 37°C for a minimum of 4 h, and washed thrice with 1X PBS, followed by overnight coating with laminin (5 μg/ml diluted in 1X PBS) (L2020; Sigma-Aldrich) at 37°C. The NSCs were maintained in Neural Expansion Media composed of NBM supplemented with FGF (10 ng/ml) (100-18C; Peprotech) and EGF (10 ng/ml) (AF-100-15; Peprotech). Neuronal maturation and terminal differentiation were achieved by plating the NSCs at a density of 25,000–35,000 cells/cm² in the Neural Maturation Media composed of NBM supplemented with BDNF (20 ng/ml) (450-02; Peprotech), GDNF (10 ng/ml) (450-10; Peprotech), L-ascorbic acid (200 μM) (A4403; Sigma-Aldrich), and db-Camp (50 μM) (D0627; Sigma-Aldrich). The neurons were subjected to maturation for 4–5 wk by supplementing them with Neural Maturation Media every 4–5 d.

FUNCAT (fluorescent non-canonical amino acid tagging)

For metabolic labeling, DIV15 neurons were incubated in methionine-free DMEM (21013024; Thermo Fisher Scientific) for 30 min. After this, the neurons were treated with L-azidohomoalanine (AHA, 1 µM) (1066100; Click Chemistry Tools) for 30 min in Met-free DMEM (21013024; Thermo Fisher Scientific). During the last 10 min of the AHA treatment, the drugs nifedipine (50 μM), dantrolene (60 μM), or 2-APB (50 μM) were added (refer to Table 1 for details of the drugs). After the 30 min of AHA treatment, NMDA (20 μM) or DHPG (50 μM) was added for 5 or 20 min to stimulate NMDARs and mGluRs, respectively. After the stimulation, the coverslips were washed with 1X PBS and fixed with 4% PFA for 15 min. After three washes with 1X PBS, the neurons were permeabilized for 10 min with 0.3% Triton X-100 solution prepared in TBS₅₀ (50 mM Tris, 150 mM NaCl, pH 7.6), followed by blocking for 1 h with a mixture of 2% BSA and 2% FBS prepared in TBS₅₀T (TBS50 with 0.1% Triton X-100). After blocking, the neurons were subjected to the FUNCAT reaction for 2 h as per the kit instructions where the AHA-incorporated proteins were tagged with an alkyne-fluorophore Alexa Fluor 555 through click reaction (C10269, CLICK-iT Cell Reaction Buffer Kit; Click Chemistry Tools). After three washes with TBS₅₀t, the neurons were incubated overnight with MAP2 antibody at 4°C. This was followed by secondary antibody incubation for 1 h at RT to visualize MAP2 (refer to Table 2 for details of the antibody). The coverslips were mounted using Mowiol and imaged on an Olympus FV3000 confocal laser scanning upright microscope with a 60X objective. The pinhole was kept at 1 Airy Unit and the optical zoom at 2X to satisfy Nyquist’s sampling criteria in the XY-direction. The objective was moved in Z-direction with a step size of 1 µm (∼8–9 Z-slices) to collect light from the planes above and below the focal plane. The image analysis was performed using FIJI software and in-house written Python code. The MAP2 channel was used to define the region of interest (ROI) of the neurons. The mean fluorescent intensity (total intensity normalized to the area) was measured for the FUNCAT and MAP2 channels for the defined ROIs. The maximum intensity projection of the slices was used for quantification of the mean fluorescent intensities. The mean fluorescent intensity of the FUNCAT channel was normalized to the MAP2 channel for comparison between different treatment conditions.

Immunostaining for p-eEF2 and p-RPS6

Primary neuronal cultures were treated with nifedipine (50 μM), dantrolene (60 μM), or 2-APB (50 μM) for 10 min, followed by NMDAR or mGluR stimulation (20 μM NMDA or 50 μM DHPG) for 5 min, and fixed with 4% PFA for 10 min (refer to Table 1 for details of the drugs). After 10 min, the neurons were washed thrice with 1X PBS, followed by permeabilization with 0.3% Triton X-100 (made in TBS₅₀) for 10 min. This was followed by 1 h of blocking with 2% BSA and 2% FBS prepared in TBS₅₀T (with 0.1% Triton X-100). They were incubated with the primary antibody (prepared in blocking buffer) overnight at 4°C. Table 2 contains the details of dilutions and catalog numbers of all the antibodies used. This was followed by three washes with TSB₅₀T and 1-h incubation with the secondary antibody (prepared in blocking buffer) at RT. Table 2 contains the details of dilutions and catalog numbers of the secondary antibodies used. After three washes with TBS₅₀T, the neurons were mounted using Mowiol. The slides were imaged on an Olympus FV3000 confocal laser scanning upright microscope with a 60X objective. The pinhole was kept at 1 Airy Unit and the optical zoom at 2X to satisfy Nyquist’s sampling criteria in the XY-direction. The objective was moved in Z-direction with a step size of 1 µm (∼8–9 Z-slices) to collect light from the planes above and below the focal plane. The image analysis was performed using FIJI software. The MAP2 channel was used to define the ROI of the neurons. The maximum intensity projection of the slices was used for quantification of the mean fluorescent intensities. The mean fluorescent intensity of the p-eEF2 or p-RPS6 channel was normalized to the MAP2 channel for comparison between different treatment conditions.

Puromycin incorporation assay

DIV 15 rat primary cortical neurons were subjected to puromycin incorporation under three different conditions–conditioned Neurobasal media (no medium change), media changed to fresh Neurobasal, and media changed to methionine-free DMEM. The different media were incubated for 30 min, followed by treatment with puromycin (5 μM) for 10 min. After this, the neurons were subjected to the standard immunostaining protocol with overnight incubation of puromycin and MAP2 antibodies. This was followed by secondary antibody incubation for 3 h. Table 2 contains the details of the dilutions and catalog numbers of the antibodies used. The imaging and analysis were performed using the same conditions as described for p-eEF2 and p-RPS6 imaging.

Treatment of neurons and lysis

Primary neuronal cultures were subjected to NMDAR or mGluR stimulation (20 μM NMDA or 50 μM DHPG, respectively) for 1, 5, and 20 min in the presence or absence of the drugs. The neurons were pre-treated with the drugs nifedipine (50 μM), dantrolene (60 μM), 2-APB (50 μM), or MK801 (50 μM) for 10 min before the stimulation (refer to Table 1 for details of the drugs). In the experiments with no calcium in the media, the neurons were stimulated with NMDA in artificial cerebrospinal fluid (ACSF—120 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 3 mM NaHCO₃, 1.25 mM NaH₂PO₄, 15 mM Hepes, 30 mM glucose, pH 7.4) with or without calcium (2 mM CaCl₂). For stimulation of L-VGCCs, BayK8644 (100 μM) was used for 5 and 20 min. After the treatment, the cells were lysed in a buffer containing 20 mM Tris–HCl, 100 mM KCl, 5 mM MgCl₂, 1% Nonidet P-40 (NP-40), 1 mM dTT, 1X Protease Inhibitor Cocktail, RNase inhibitor, and 1X Phosphatase Inhibitor and centrifuged at 20,000g and 4°C for 20 min. The supernatant was denatured in SDS dye for Western blotting.

SDS–PAGE and Western blotting

For Western blotting analysis, the denatured lysates were run on 10% resolving and 5% stacking acrylamide gels and subjected to overnight transfer onto the PVDF membrane. The blots were subjected to blocking for 1 h at RT using 5% BSA prepared in TBST (TBS with 0.1% Tween-20). This was followed by primary antibody (prepared in blocking buffer) incubation for 2–3 h at RT. HRP-tagged secondary antibodies were used for primary antibody detection. The blots were incubated with the secondary antibodies (prepared in blocking buffer) for 1 h at RT. Three washes of 5–10 min each were given after primary and secondary antibody incubation using TBST solution. The blots were subjected to chemiluminescent-based detection of the HRP-tagged proteins. Table 3 contains the details of dilutions and catalog numbers of all the antibodies used. For the analysis of eEF2 phosphorylation and RPS6 phosphorylation, the samples were run in duplicates where one set was used to probe for the phospho-proteins (p-eEF2 and p-RPS6), and the other set was used to probe for the total proteins (eEF2 and RPS6). In each set, Tuj1 was used as the loading control. In every set, the blots were cut to probe for eEF2 and RPS6 on the same blot. Hence, the Tuj1 used to normalize eEF2 and RPS6 was the same in a given set. Similarly, p-eEF2 and p-RPS6 were probed on the same blot for a given set, using the corresponding Tuj1 to normalize the given set. All the Western blot quantifications were performed using densitometric analysis on ImageJ software.

Calcium imaging

Calcium imaging was done on DIV15 neurons plated on 35-mm dishes at a density of 200,000–250,000 cells per dish. The imaging and washes were performed with ACSF media (120 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 3 mM NaHCO₃, 1.25 mM NaH₂PO₄, 15 mM Hepes, 2 mM CaCl₂, 30 mM glucose, pH 7.4). The cells were washed once with ACSF and incubated at 37°C with 1 ml of freshly prepared Fluo-4 AM dye solution (2 µM Fluo-4 AM and 0.002% pluronic acid in ACSF) (F14217; Thermo Fisher Scientific) for 20 min. They were given two washes and incubated in ACSF at 37°C for 10–20 min before imaging. The drugs nifedipine or dantrolene were added during this incubation step (refer to Table 1 for details of the drugs). The neurons were imaged using an Olympus FV3000 confocal laser scanning upright microscope with a 20X water dipping objective, excited with 488-nm lasers. The neurons were imaged for a total of 7 min at a rate of 3 s per frame (140 frames in total). They were imaged in the basal condition for 1 min (20 frames). After that, they were imaged for 5 min (100 frames) on NMDA addition. Finally, they were imaged for 1 min (20 frames) on the addition of ionomycin solution (10 μM ionomycin with 10 mM CaCl₂) (407950; Sigma-Aldrich). The images obtained were analyzed using the Time-Series Analyzer plug-in on FIJI. Only the ionomycin-responsive neurons were selected for the analysis. Hence, the ionomycin frames were used to define the ROIs of the neurons for each series. The average intensities were obtained for the selected ROIs in each frame. The change in fluorescent intensity at each frame was normalized to the fluorescent intensity of the first frame (F0) for each ROI. The normalized change in fluorescent intensity (ΔF/F0) was plotted along the time axis and used for statistical analysis as well.

Statistical analyses

All statistical analyses were performed using GraphPad Prism software. The normality of the data was checked using the Kolmogorov–Smirnov test. For experiments with less than five data points, parametric statistical tests were applied. Data were represented as the mean ± SEM in all biochemical experiment graphs. FUNCAT and imaging data were represented as boxes and whiskers with all the individual data points. Statistical significance was calculated using an unpaired t test (two-tailed with equal variance) in cases where two groups were being compared. One-way or two-way ANOVA was used for multiple group comparisons, followed by the required multiple comparison test. A P-value less than 0.05 was considered to be statistically significant.