Figure 4.

Sequestration to endosome causes mTORC1 inactivation linked with increased miRNA-Ago2 interaction in glial cells

(A and B) Lowering of p-mTOR in Aβ_1–42 oligomer-treated C6 glioblastoma cells. Western blot data showing the levels of cellular p-mTOR levels along with downstream substrate of mTOR p-S6K levels in C6 glioblastoma cells (A) and in primary astrocytes (B) treated with Aβ_1–42. (C and D) Decreased lysosome targeting of mTOR after Aβ_1–42 oligomer treatment. Confocal images depicting localization of mTOR to lysosome in control and treated cells are shown in (C). Endogenous mTOR was visualized indirectly with immunofluorescence (green), and lysosomes are labeled with LysoTracker (red) in C6 glioblastoma cells. A Pearson’s coefficient of colocalization was used to measure the amount of mTOR translocating to lysosomes in control and Aβ_1–42-treated C6 glioblastoma cells (D). (E and F) Increased endosome-mTOR localization after Aβ_1–42 oligomer treatment. Confocal images show localization of endosomes and mTOR. Endosomes were tagged with YFP-Endo (green), and endogenous mTOR (red) was detected by indirect immunofluorescence. Colocalization between endosomes and mTOR was visualized as yellow (E). A Pearson’s coefficient of colocalization of early endosomes and mTOR in Aβ_1–42 oligomer-treated C6 glioblastoma cells is shown in (F). (G and H) Effect of Rab5-CA on mTOR localization in C6 glioblastoma cells. Colocalization of mTOR in C6 cells expressing YFP-Endo upon Rab5-CA expression was done. The cells with YFP-Endo (green) and mTOR (red) were visualized in control and Rab5-CA-expressing cells (G). A Pearson’s coefficient of colocalization of mTOR and early endosome is shown in (H). (I) Alteration of miRNA activity and levels in mammalian cells defective for endosome maturation due to expression of the constitutively active form of Rab5 protein. A drop in cellular activity of let-7a miRNA in C6 cells upon expression of constitutively activated Rab5 mutant Rab5-CA is shown. The RL reporter with three let-7a miRNA binding sites was used to score the effect of Rab5-CA expression on miRNA repressive activity. (J and K) Levels of cellular and Ago2-associated miR-146a upon Rab5-CA expression in C6 glioblastoma cells. The cellular miR-146 level was normalized against the U6 snRNA level (J). The amount of Ago2 immunoprecipitated from control and Rab5-CA-expressing cells were used for normalization of the amount of miRNAs associated with Ago2 (K). For statistical significance, a minimum of three independent experiments were considered in each case unless otherwise mentioned, and error bars represent means ± SEM. p values were calculated by utilizing a Student’s t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001.Scale bar represents 10μm in panels C, G, E.