Fig 2: Increased TACE and decreased TNFR1 protein levels at the plasma membrane in the PDK1K465E/K465E knock-in mice brain. (A) Brain cytosolic (CYT) and membrane (MEM) subcellular fractions obtained from PDK1+/+ wild type (WT, black bars) and PDK1K465E/K465E mutant (K465E, white bars) 21-months old mice were immunoblotted with the indicated antibodies, where ERK1/2 and TrkB are employed as cytosolic and membrane markers, respectively. (B) TACE was immunoprecipitated (IP TACE) with specific antibodies from total brain protein extracts from PDK1+/+ wild type (WT) and PDK1K465E/K465E mutant (K465E) 21-months old mice and immunoblotted both with TACE and pan-pThr antibodies, as depicted. The levels of TACE in the total cellular lysates and the immunoglobulin controls (IgG) are also shown. (A,B) Representative Western blots of three independent experiments are shown, and the band densitometry quantification expressed as a percentage of the wild type controls is plotted at the bottom. (C) Representative epifluorescence micrographs of cortical and CA3 middle hippocampal sections obtained from PDK1+/+ wild type (WT) and PDK1K465E/K465E mutant (K465E) 21-months old mice brain stained with TACE, TNFR1, and the nuclear Hoechst dye; arrows indicate the specific surface staining; the intensity of the signals were quantified and expressed as a percentage of the wild type controls. The data are represented as the mean ± SEM for at least three different mice per genotype. *p < 0.05 and **p < 0.005 compared with wild types as obtained by the Student's t-test.

Fig 3: Reduced TNFα–induced cell death in the PDK1K465E/K465E neurons. Primary cultures of cortical neurons obtained from PDK1+/+ wild type (WT, black bars) and PDK1K465E/K465E mutant (K465E, white bars) E15,5 embryos were grown for 6 days in vitro and then treated or not with 100 ng/ml of recombinant TNFα and/or1 nM Actinomycin D for 24 h, as indicated. (A) Representative epifluorescence images stained with TACE or TNFR1 and merged with the nuclear Hoechst dye, as indicated; bars, 50 and 30 μm for TACE and TNFR1, respectively; quantification of the cell surface signals is shown below as the mean ± SEM for 200 neurons from four different embryos per condition, and expressed as a percentage of the wild type controls. The TACE α-secretase activity (B) and the levels of soluble TNFR1 in the culture medium (C) were also determined and expressed as the mean ± SEM from four different embryos per condition. (D) Cell viability was determined by the MTT reduction assay and represented as the mean ± SEM for at least five independent embryos per genotype from two separate experiments, with each sample assayed in triplicate. *p < 0.05 and **p < 0.005 compared with wild types as obtained by the Student's t-test. #p < 0.05 and ##p < 0.005 between controls and treatments analyzed by the Tukey test.

Fig 4: Protection of the PDK1K465E/K465E neurons against Aβ–toxicity. Primary cultures of cortical neurons obtained from PDK1+/+ wild type (WT, black bars) and PDK1K465E/K465E mutant (K465E, white bars) E15,5 embryos were grown for 6 days in vitro and then treated the indicated concentration of amyloid-β peptides (Aβ) for 48 h (A), with 5 μM of Aβ for the indicated time points in hours (B,C), or with 5 μM of Aβ for 24 h (D–G). (A) Upper panel, the cell viability was determined on the indicated cortical cultures with the MTT reduction assay and is expressed as a percentage of the untreated cells; bottom panel, the percentage of apoptotic cells was obtained by scoring the number of nuclei exhibiting chromatin fragmentation divided by the total. Data represents the mean ± SEM for at least five independent embryos per genotype from two separate experiments, with each sample assayed in triplicate. (B,C) Protein extracts from matched PDK1 wild-type (WT) and PDK1 mutant knock-in (KI) mice were obtained from the indicated cortical cell samples and then immunoblotted with the depicted antibodies. (D) Representative immunofluorescent labeling of TACE or TNFR1 counterstained with the Hoechst nuclear dye, as indicated; bars, 40 and 30 μm for TACE and TNFR1, respectively. (E) Quantification of the cell surface signals is expressed as the mean ± SEM for 200 neurons from four different embryos per condition, and expressed as a percentage of the wild type controls. The TACE α-secretase activity (F) and the levels of soluble TNFR1 in the culture medium (G) were also determined and expressed as the mean ± SEM from four different embryos per condition. *p < 0.05 or **p < 0.005 compared with the corresponding wild types for each condition as obtained by the Student's t-test. #p < 0.05 and ##p < 0.005 (Tukey test) statistically significant differences between controls an treatments.