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. 2022 Jul 4;145(7):2313–2331. doi: 10.1093/brain/awac082

Figure 3.

Figure 3

Loss of PI3K-C2β causes mTORC1 hyperactivity in cultured neurons and in mouse brain. (A) Immunohistochemical detection of p-S6-positive cells in hippocampal neurons (DIV 14) from WT and Pik3c2b KO mice left untreated or treated with the mTORC1 inhibitor rapamycin (200 nM). Scale bar = 50 μm. Rapamycin treatment essentially eliminates p-S6 staining. (B) Semi-automated intensity quantification of representative data shown in A. One-way ANOVA, **P < 0.005, ***P < 0.001, n = 5 independent cultures. Error bars represent SEM. (C) Quantification of neuronal soma size. Unpaired t-test; n = 5 independent cultures. Soma area is unaffected (P = 0.5813). (D and E) Dose-dependent loss of PI3K-C2β activates mTORC1 signalling. (D) Representative immunoblot of PI3K-C2β, p-S6, S6 and vinculin content in whole brain lysates from 8-week-old WT, HET and KO mice. (E) Quantification of the p-S6/S6 ratio as shown in D. One-way ANOVA, *P < 0.05. n = 4 independent experiments. Error bars represent SEM. (F and G) mTOR hyperactivation in mouse brain is rescued by acute everolimus treatment. (F) Representative immunoblot of PI3K-C2β, p-S6, S6 and vinculin content in whole brain lysates from 8-week-old WT and KO mice, untreated or pretreated with 10 mg/kg of everolimus. (G) Quantification of the p-S6/S6 ratio as shown in F. Everolimus treatment potently downmodulates mTORC1 signalling in both WT and KO mice. One-way ANOVA, *P < 0.05, ***P < 0.001. n = 6 independent experiments. Error bars represent SEM.