Distinct Regulation of Bioenergetics and Translation by Gp I mGluR and NMDAR (2020)
Figure 3

Figure 3:

mGluR and NMDAR alter global translation by regulating eEF2 phosphorylation with distinct kinetics.

(A) Schematic depicting the experimental workflow and the stimulation protocol to visualize and quantify newly synthesized proteins through metabolic labeling at various time points following DHPG or NMDA treatment in cortical neurons DIV 15 plated at low-density.

(B) Representative images showing newly synthesized proteins on the basal condition visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(C) Representative images showing newly synthesized proteins visualized through FUNCAT metabolic labeling (pseudo-colored) in cortical neurons at various time points following DHPG and NMDA treatment. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(D) Line graph showing the change in the average normalized FUNCAT intensity representing the quantity of newly synthesized proteins at various time points following DHPG and NMDA treatments. Data presented as mean +/- SEM. Data points in all groups were normalized to the average of the basal group. ***p<0.001 n= 21-54 neurons per group from 3 independent platings. Two-way ANOVA followed by Bonferroni's multiple comparison test.

(E) Representative images showing phospho-eEF2 immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(F) Box plot showing phospho-eEF2 normalized intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes and on NMDA treatment for 5 minutes. Data points in all groups were normalized to the average of the basal group. The box extends from 25th to 75th percentile with the middlemost line representing the median of the dataset. Whiskers range from minimum to maximum data point. *p<0.05, **p<0.01, n=31-49 cells per group from 3 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.

(G) Representative immunoblots describing changes in the phospho-eEF2 and total-eEF2 levels at various time points after DHPG and NMDA treatment in cortical synaptoneurosomes. Note in each case phospho and total eEF2 levels were normalized individually to their respective tubulin levels for calculating the phospho/total eEF2 ratio.

(H) Line graph showing the average value of the synaptic phospho/total ratio of eEF2 at various time points after DHPG and NMDA treatment until 5 minutes of recovery. Data presented as mean +/- SEM, **p<0.01. Values in all other groups were normalized to the 0 min group for the corresponding experiment and all the data points are represented as a fraction of the initial time point. ***p<0.001, ****p<0.0001, n≥ 5 animals per group. Two-way ANOVA followed by Bonferroni's multiple comparison test.