Cell type specificity of PI3K signaling in Pdk1- and Pten-deficient brains

Abstract

Loss of PTEN causes unregulated activation of downstream components of phosphatidylinositol 3-kinase (PI3K) signaling, including PDK1, and disrupts normal nervous system development and homeostasis. We tested the contribution of Pdk1 to the abnormalities induced by Pten deletion in the brain. Conditional deletion of Pdk1 caused microcephaly. Combined deletion of Pdk1 and Pten rescued hypertrophy, but not migration defects of Pten-deficient neurons. Pdk1 inactivation induced strikingly different effects on the regulation of phosphorylated Akt in glia versus neurons. Our results show Pdk1-dependent and Pdk1-independent abnormalities in Pten-deficient brains, and demonstrate cell type specific differences in feedback regulation of the ubiquitous PI3K pathway.

Figure 1.

Pdk1 is indispensible for brain size regulation. Sagittal H&E-stained sections from mice with the indicated genotypes, in which hp-cre drives conditional deletion. The dramatic hypertrophy of Pten cKO was rescued in Pten;Pdk1 dKO brains, which show a similar size reduction to Pdk1-deficient brains when compared with control. Bar, 2 mm. Mice were 10 wk old.

Figure 2.

Pdk1 is required for cell-autonomous hypertrophy of Pten-deficient neurons. (A) IHC for p-S6 (Ser 235/236) in the dentate gyrus from the indicated genotypes combined with hp-cre. Elevated levels of p-S6 in Pten cKO neurons were abrogated in Pten;Pdk1 dKO, consistent with deletion of Pdk1. Bar, 100 μm. (B) The nuclear diameter of granule neurons was measured using Pten IHC to distinguish between Pten-positive and Pten-negative neurons. The mean ± SEM of 50 measured nuclei per brain section from six to eight mice is shown. P values ([***] P < 0.0001; [ns] not significant, P > 0.05) were determined by a Student's unpaired t-test. Mice were 10 wk old.

Figure 3.

Pdk1 deficiency did not rescue the migration defect in Pten cKO cerebellar granule neurons. IHC for p-S6 (Ser 235/236) on cerebella from the indicated genotypes combined with cb-cre. Pten cKO cerebella contained ectopic neurons that were hypertrophic and contained elevated p-S6. Ectopic granule neurons persist in Pten;Pdk1 dKO cerebella, but they do not show hypertrophy or elevated p-S6, consistent with deletion of Pdk1. Insets show IHC for GABA_A Rα6 demonstrating that ectopic cells are cerebellar granule neurons. Bar, 50 μm. Mice were 8 wk old.

Figure 4.

Pdk1 deficiency blocked phosphorylation of downstream effectors of the PI3K pathway and induced up-regulation of p-Akt S473. Western blot analysis of protein lysates from hippocampi of hp-cre mice. (Lane 1) Pten cKO. (Lane 2) Pten;Pdk1 dKO. (Lane 3) Pdk1 cKO. (Lane 4) Control. Pten cKO mice showed increased phosphorylation of Akt at S473 and T308, and increased phosphorylation of downstream substrates GSK3β, S6, and Fkhr. (Lane 2) This enhanced phosphorylation of Akt T308 and substrates was blocked by Pdk1 deletion. (Lane 3) Note the high levels of p-Akt S473 in Pdk1 cKO. β-Actin is shown as a loading control. The arrow denotes a specific band. Mice were 10 wk old.

Figure 5.

Pdk1 deficiency caused elevation of p-Akt S473 selectively in glia. IHC with p-Akt S473 antibody on the hippocampus (left) and cortex (center). (Right) X-Gal histochemistry followed by p-Akt IHC revealed β-galactosidase-positive glia. The p-Akt-positive neurons in Pten cKO do not overlap with X-gal, while high levels of p-Akt are present in X-gal-positive glia within Pten;Pdk1 dKO and Pdk1 cKO cerebral cortices compared with control. Bars: left and center panels, 400 μm; right panel, 100 μm. Mice were 10 wk (left and center panels), and 2 wk old (right panel).