Distinct Regulation of Bioenergetics and Translation by Gp I mGluR and NMDAR (2020)
Figure 6

Figure 6:

Perturbation of AMPK function disrupts receptor-specific translation response:

(A) Schematic depicting the experimental workflow for quantifying p-eEF2 levels and FUNCAT intensity changes on DHPG and NMDA treatment in low-density cortical neurons (DIV 15).

(B) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (pseudo-colored) in cortical neurons on the basal condition and on DHPG (50μM) treatment for 5 minutes in the presence or absence of AICAR (1mM). MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(C) Box plot showing the normalized phospho-eEF2 intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. 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, ****p<0.0001, n=17-46 cells per group from 3 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.

(D) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on DHPG treatment for 5 minutes, on AICAR treatment and on DHPG treatment with AICAR. 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<0.0001, n=18-57 cells per group from 3 independent platings. Kruskal-Wallis test followed by Dunn's multiple comparison test.

(E) Representative images showing the phospho-eEF2 immunolabeling (Pseudo-colored) and newly synthesized proteins as FUNCAT signal (Pseudo-colored) in cortical neurons on the basal condition and on NMDA treatment for 5 minutes in the presence or absence of Compound C. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(F) Box plot showing phospho-eEF2 intensity distribution across multiple neurons on basal condition (Vehicle control), on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. 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=21-36 cells per group from 3 independent platings. Kruskal-Wallis test followed by Dunn's multiple comparison test.

(G) Box plot showing the FUNCAT intensity distribution across multiple neurons on the basal condition, on NMDA treatment for 5 minutes, on Compound C treatment and on NMDA treatment with Compound C. 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, ****p<0.0001, n=24-52 cells per group from 3 independent platings. Kruskal-Wallis test followed by Dunn's multiple comparison test.

(H) Model of the receptor-specific regulation of AMPK-eEF2 signaling axis and their subsequent effect on global translation. mGluR stimulation (Left) inactivates AMPK, a necessity for the dephosphorylation of eEF2. This, in turn, leads to an enhanced protein synthesis thereby leading to the consumption of ATP. NMDAR stimulation (Right), however, led to an activation of AMPK by allowing the entry of extracellular Ca2+ through open NMDAR channels. This resulted in an increase in the eEF2 phosphorylation and an inhibition of global protein synthesis having no significant impact on neuronal energetics.

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