Distinct Regulation of Bioenergetics and Translation by Gp I mGluR and NMDAR (2020)
Figure 4

Figure 4:

mGluR modulates AMPK function through a protein synthesis-dependent feedback mechanism:

(A) Representative images showing phospho-AMPK 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.

(B) Box plot showing the normalized phospho-AMPK 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=30-46 cells per group from 3 independent platings. One-way ANOVA followed by Bonferroni's multiple comparison test.

(C) Representative immunoblots describing changes in the phospho-AMPK and total-AMPK levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin. Note in each case phospho and total AMPK levels were normalized individually to their respective tubulin levels before calculating the phospho/total ratio of AMPK.

(D) Line graph showing the normalized average value of the synaptic phospho/total ratio of AMPK at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. 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<0.05, **p<0.01, ****p<0.0001, n= 5 animals per group. Two-way ANOVA followed by Bonferroni's multiple comparison test.

(E) Representative immunoblots depicting the changes in the α-SNAP protein levels at various time points after DHPG treatment in cortical synaptoneurosomes in the presence or absence of anisomycin.

(F) Line graph showing the normalized average value of the synaptic α-SNAP protein levels at various time points after DHPG treatment in the presence or absence of anisomycin. Data presented as mean +/- SEM. *p<0.05. 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<0.01, n≥ 4 animals per group. Two-way ANOVA followed by Bonferroni's multiple comparison test.

(G) Representative images showing the α-SNAP (cyan) and AMPK (magenta) immunolabeling in low-density cortical neurons. Merge shows the colocalization of the two channels both on the basal condition and on DHPG treatment for 5 minutes. Scale bar 10μm. Zoomed in representative images of dendrites showing the merge of both channels on the basal and DHPG treated conditions. Scale bar 5μm.

(H) Box plot depicting the quantification of co-localization through Pearson's correlation coefficient between α-SNAP and AMPK in cortical neurons on the basal condition and on DHPG treatment for 5 minutes. 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<0.01, n≥35 cells per group from 4 independent platings. Unpaired-sample t-test.

(I) Representative images showing the phospho-AMPK immunolabeling (Pseudo-colored) in low-density cortical neurons on the basal condition and on DHPG treatment for 5 minutes in the presence of scrambled siRNA and in the presence of α-SNAP siRNA. MAP2B immunolabeling was used for identifying neurons and intensity was used for normalization. Scale bar 10μm.

(J) Box plot showing the normalized phospho-AMPK intensity distribution across multiple neurons on the basal condition in the presence of scrambled siRNA, on DHPG treatment for 5 minutes in the presence of scrambled siRNA, on the basal condition in the presence of α-SNAP siRNA, on DHPG treatment for 5 minutes in the presence of α-SNAP siRNA. Data points in all groups were normalized to the average of the scrambled siRNA 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, n=29-35 cells per group from 3 independent platings. Kruskal-Wallis test followed by Dunn's multiple comparison test.