The generation of the PDK1^K465E/K465E knock-in mice and the genotyping procedures were previously described (Bayascas et al., 2008). The 3xTg-AD mice colony was established by Dr. Giménez-Llort from progenitors kindly provided by Prof. Frank M. LaFerla (Oddo et al., 2003), whereas the brain samples from mice overexpressing APP (K670N:M671L) together with PS1 (M146L), termed APP/PS1 mice, line 6.2, were provided by Dr. Arancio at Columbia University (Holcomb et al., 1998). Transgenic male and female mice and matched non-transgenic wild type littermates were used in all the experiments. Mice were housed in pathogen-free facilities, 5–6 per cage, maintained on a 12-h light/dark cycle (lights on at 07:00) with ad libitum access to food and water. All animal procedures were performed in accordance with 2010/63/UE regarding the care and use of animals for experimental procedures following the recommendations of the Universitat Autònoma de Barcelona Ethical Committee for Human and Animal Experimentation. The protocols were approved by the Ethical Committee for Human and Animal Experimentation, Universitat Autònoma de Barcelona, and performed under a Generalitat de Catalunya Project License. The study complies with the ARRIVE guidelines developed by the NC3Rs (Kilkenny et al., 2010).

Neuronal primary cultures were established from embryonic day 15,5 (E15,5) embryos as previously described (Zurashvili et al., 2013). Cortical cells were seeded at a density of 5 × 10⁴ cells/cm² onto 50 μg/ml of poly-D-Lysine-coated 24-well plates for cell viability studies, or 6-well plates for western blot analysis, and grown for 6 days in vitro in a 5% CO₂ incubator. The hippocampal cells were plated at a density of 2 × 10⁴ cells/cm² on 24-well plates containing 12-mm glass coverslips coated with 150 μg/ml of poly-D-Lysine for 4 days in vitro in a 5% CO₂ incubator. Stock solutions of tunicamycin and Akti-1/2 dissolved in dimethyl sulfoxide, or TNFα and Aβ dissolved in PBS, were added to cultures as indicated. Cell survival was determined by the MTT reduction assay as described previously (Zurashvili et al., 2013). For the quantification of apoptosis, cells were fixed in 2% paraformaldehyde, stained with 1 μg/ml of the DNA dye Hoechst 33342, and then visualized under the fluorescence microscope. Apoptosis was quantified at each condition point by scoring the percentage of cells exhibiting fragmented or condensed nuclei. At least 300 cells per well from 6 systematically collected fields starting from a random position were manually counted by two participants, with blinding to the treatment conditions.

Tissue extracts were prepared by homogenizing the frozen tissue in a 10-fold excess volume of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium-glycerophosphate, 0.27 M sucrose, 1% w/v Triton X-100, 0.1% v/v 2-mercaptoethanol, and a 1:100 dilution of protease inhibitor cocktail, (Sigma, Merck KGaA, Darmstadt, Germany) using the Polytron homogenizer; cultured cortical neurons were scrapped from wells in the same ice-cold lysis buffer. Lysates were centrifuged at 4°C for 15 min at 15,000 × g and supernatants aliquoted and preserved at −20°C. Protein concentrations were determined by the Bradford method using bovine serum albumin as standard. The activation state of the different pathways was assessed by loading 20 μg of protein extracts onto 6–10% polyacrylamide, 0.25M Tris-HCl pH 8.5, 0.1% (w/v) SDS gels casted in 10-well/1 mm-thick mini-PAGE (polyacrylamide gel electrophoresis) system (Bio-Rad, Hercules, California, USA), then electrophoresed on 25 mM Tris-HCl pH 8.5, 192 mM Glycine, 0.1% (w/v) SDS running buffer at 160 V for 60 min, and finally transferred to nitrocellulose membranes (Whatman, Maidstone, UK) on a 25 mM Tris-HCl pH 8.5, 192 mM Glycine, 20% methanol transfer buffer wet system at 100 V for 90 min. Samples were blocked with 10% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20 for 1 h at room temperature, then immunoblotted over night at 4°C with 1 μg/ml of the indicated primary antibodies diluted in either 5% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20 (total protein antibodies) or 0.5% BSA, 1xTBS, 0.2% (w/v) Tween 20 (phospho-antibodies), which were detected with a 1:5,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibodies diluted in 5% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20. Membranes were incubated with the enhanced chemiluminescence (ECL) reagent, then either exposed to Super RX Fujifilm and developed, or detected using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, California, USA), and quantified using the ImageJ software.

Cortex tissues were homogenized in 10 volumes of cold homogenization buffer (50 mM Tris-HCl pH 7.4, 0.27 M sucrose, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium-glycerophosphate, and a 1:100 dilution of protease inhibitor cocktail (Sigma, Merck KGaA, Darmstadt, Germany) 50 strokes using a glass potter homogenizer. Homogenates were centrifuged at 1,000 × g for 15 min at 4°C to remove cellular debris. The cleared homogenates were centrifuged at 20,000 × g for 1 h at 4°C and the resultant supernatant collected as the cytosolic fraction; the pellet was resuspended and washed in the same volume of homogenization buffer at 20,000 × g for 1 h at 4°C. The supernatant was discarded and the pellet containing the membrane fraction resuspended in 1% SDS homogenization buffer.

Aβ oligomers were prepared as shown previously (Manterola et al., 2013). One mg of synthetic amyloid-β peptides (1-42) from amino acid 672–713 in the human amyloid precursor protein sequence (catalog number H-1368.1000, Bachem, Torrance, USA) was monomerized in hexafluor-2-propanol and then evaporated peptides resuspended in 20 μl of fresh anhydrous DMSO, vortexed 20 s, incubated 5 min at room temperature, and diluted with 424 μl of PBS to a final concentration of 500 μM. The solution was again vortexed, incubated at 4°C for 24 h, and then centrifuged at 14,000 × g 4°C for 10 min to remove insoluble pellets. The supernatant containing a mixture of soluble Aβ₁₋₄₂ oligomers was collected and biochemically analyzed by Bicine/Tris/Urea SDS-PAGE (Figure 9A). 1.6M Tris-HCl pH 8.1, 8M urea, 0.4M H₂SO₄ resolving and 0.8M BisTris pH 6.7, 0.2M H₂SO₄ stacking gels were casted in 10-well/1 mm-thick mini PAGE system (Bio-Rad, Hercules, California, USA), then electrophoresed on 0.2M Bicine pH 8.2, 0.1M NaOH, 0.25% (w/v) SDS cathode and 0.2M Tris-HCl pH8.1, 0.05M H₂SO₄ anode running buffers at 100 V for 100 min, finally transferred to PVDF membranes (Whatman, Maidstone, UK) on a 10 mM CAPS pH 11, 10% methanol transfer buffer wet system at 200 mA for 90 min, and then immunoblotted as described.

1 μg of anti-TACE antibody was conjugated with 10 μl of Protein G Sepharose (GE Healthcare, Chicago, Illinois, USA) and incubated with 0.2 mg of pre-cleared tissue lysates overnight at 4°C on a shaking platform. The beads were washed three times with 1 ml of lysis buffer supplemented with 150 mM NaCl, resuspended in protein sample loading buffer, electrophoresed and then immunoblotted with the indicated antibodies. TACE activity was determined using the SensoLyte 520 TACE (α-Secretase) Activity Assay Kit (#AS-72085, AnaSpec, Inc., Fremont, USA) following the manufacturer's instructions. Reaction was started by adding 40 mM of the fluorophoric QXL520/5FAM FRET substrate and was allowed to proceed for 60 min. 30 μg of cortex and hippocampus, or 20 μg of cell lysate proteins were used. Fluorescence intensity was quantified in a fluorescence microplate reader (FLx800, BIO-TEK Instruments, Winooski, USA) at an excitation of 490 nm and emission of 520 nm. Results are expressed as the percentage of fold-change in fluorescence intensity compared to control samples.

Cortex and hippocampus BDNF levels were measured with the BDNF Quantikine ELISA Kit (#DBD00, R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer instructions. The levels of the cleaved soluble TNFR1 fragment were measured on either cortex and hippocampus protein extracts or cell culture medium by using the mouse sTNFR1 Quantikine ELISA Kit (#MRT10, R&D Systems, Inc., Minneapolis, USA) according to the manufacturer's instructions. Optical density of each well was measured using an automated microplate reader (GloMax-Multi Microplate Reader, Promega, Madison, Wisconsin, USA).

Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTOMAG-70K-PMX, Millipore, Burlington, Massachusetts, USA) was employed according to manufacturer's instructions to assess the serum concentration of 24 different cytokines and chemokines. The data was obtained with the Xponent software and the cytokine/chemokine factor concentrations calculated based on the corresponding standard curves. All samples were analyzed in duplicate.

Three 21-months old PDK1^+/+ and PDK1^K465E/K465E matched mice were sacrificed by cervical dislocation and brains were immediately collected, frozen in isopentane cooled down to −80°C and stored at −80°C. Brains were embedded in O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and sliced into 10 μm thick sagittal sections. Frozen sections were fixed in 4% paraformaldehyde for 40 min at 4°C. For antigen retrieval, sections were boiled 10 min in 10 mM sodium citrate pH 6, cooled down for 30 min on ice and then washed three times with Tris-buffered saline (TBS; 25 mM Tris pH 7.5, 150 mM NaCl). Samples were blocked with buffer containing 5% goat serum and 0.02% Triton in TBS for 30 min and incubated overnight at 4°C with primary antibodies diluted 1:100 in the same blocking buffer. Sections were washed three times with TBS buffer, incubated with the corresponding Alexa488 conjugated secondary antibodies diluted 1:400 in blocking buffer for 1.5 h at room temperature, and counterstained with 1 μg/ml Hoechst 33342 before mounting in Fluorsave reagent (Southern Biotec, Birmingham, USA). Immunostained sections were photographed with a Nikon Eclipse 90i epifluorescence microscope, the captured images were processed and the staining intensity was measured automatically along a grid line by using ImageJ 1.42q (Wayne Rasband, National Institutes of Health, USA) and Fiji (https://imagej.net/Fiji) softwares in 5 selected cortex or hippocampus sections per mouse using a sum projection of five Z-sections (1 μm/section), whereas Hoechst staining was used to determine the nuclear area for normalization.

Dissociated cortical and hippocampal cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed three times with PBS for 5 min with slow agitation, permeabilized with 0.02% saponin in PBS for 7 min at room temperature, incubated 15 min in 0.01% saponin, 10 mM glycine in PBS, and then blocked in 0.01% saponin, 5% BSA, 10 mM glycine in PBS for 1 h. Primary antibodies were diluted 1:100 in PBS supplemented with 0.01% saponin-1% normal goat serum and incubated overnight at 4°C. Cells were then washed four times, corresponding secondary antibodies conjugated to Alexa488 fluorescent dye were used at a concentration of 1:400, and nuclei were stained with 1 μg/ml Hoechst 33342 for 90 min at room temperature. Cover slips were then mounted with FluoroSave Reagent on microscope slides to measure the intensity of the immunostaining signals as described in the immunohistochemistry section.

The following antibodies were raised in sheep and affinity purified on the appropriate antigen. The Akt total antibody was raised against the sequence RPHFPQFSYSASGTA, corresponding to residues 466–480 of rat Akt; the total TSC2 antibody was raised against a sequence encompassing residues 1719–1814 of mouse TSC2; the total PRAS40 antibody was raised against the peptide DLPRPRLNTSDFQKLKRKY, corresponding to residues 238–256 of human PRAS40; total TrkB (catalog number 4603), total ERK (catalog number 9102), phospho-Akt Thr308 (catalog number 9275), phospho-Akt Ser473 (catalog number 9271), phospho-GSK3α/β Ser21/9 (catalog number 9331), phospho-FOXO1 Ser256 (catalog number 9461), total FOXO1 (catalog number 2880), phospho-TSC2 Thr1462 (catalog number 3611), phospho-PRAS40 Thr246 (catalog number 2997), phospho-PDK1 Ser241 (catalog number 3061), total PDK1 (catalog number 3062), phospho-Threonine (catalog number 9381), phospho-PERK Thr980 (catalog number 3179), total PERK (catalog number 5683), phospho-eIF2α Ser51 (catalog number 3597), BiP (catalog number 3177), IRE1α (catalog number 3294), CHOP (catalog number 2895) and PDI (catalog number 3501) antibodies were purchased from Cell Signaling Technology, Danvers, Massachusetts, USA. Total GSK3α/β (catalog number sc-7291) antibody was purchased from Santa Cruz Biotechnology, Dallas, Texas, USA. TACE antibody was purchased from QED Bioscience, San Diego, California, USA (catalog number QEB-1131). TNFR1 antibody was obtained from MBL International, Woburn, Massachusetts, USA (catalog number JM-3125). GAPDH antibody was from Abcam, Cambridge, UK (catalog number ab8245). Aβ (6E10) monoclonal antibody was obtain from Biolegend, San Diego, California, USA (catalog number Sig-39345-200). APP antibody was purchased from Sigma, Merck KGaA, Darmstadt, Germany (catalog number 8717). For immunoblot analysis, appropriate secondary antibodies coupled to horseradish peroxidase were obtained from Thermo, Waltham, Massachusetts, USA. For immunohistochemistry and immunocytochemistry analysis, the Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody was obtained from Invitrogen, Waltham, Massachusetts, USA (catalog number A11008).

Data was analyzed using GraphPad software (GraphPad Software Inc., La Jolla, CA, USA). Statistical significance of the difference between two groups was determined by using the unpaired, two tailed Student's t-test analysis. Multiple groups were assessed by One-way analysis of variance (ANOVA), and the statistical significance of the differences between categories analyzed by Post-hoc using Tukey test. Results are presented as the mean ± standard error of the mean. P-values of < 0.05 and < 0.005 between categories were considered significant and are indicated in the figures.

AD is an age-related neurodegenerative disorder disturbing the function of particular brain regions such as cortex and hippocampus (Huang and Mucke, 2012). To investigate the importance of the PDK1/Akt signaling in AD, we took advantage of the PDK1^K465E/K465E mice, in which we showed that BDNF-mediated activation of Akt was impaired in a time and dose-dependent manner (Zurashvili et al., 2013). As expected, mutation of the PDK1 phosphoinositide binding site in the PDK1^K465E/K465E mice caused a reduction in the levels of Akt activity along the whole adulthood both in the cortex (Figure 1A) and the hippocampus (Figure 1B). This is denoted by the decrease in the levels of phosphorylation of the Akt substrates PRAS40 at the Thr246 and TSC2 at the Thr1462 specific Akt sites, which originated from the decreased phosphorylation of Akt at the Thr308 residue within the activation loop by PDK1, but not the Ser473 within the hydrophobic motif domain that is phosphorylated by mTORC2. Of note, the deficits in Akt signaling tend to be attenuated in the aged 16–21 months-old mice brain, which correlated with elevated levels of BDNF both in the cortex (Figure 1C) and the hippocampus (Figure 1D) mutant tissue extracts.

We next monitored the degree of activation of this signaling pathway in brain tissues derived from 3xTg-AD and APP/PS1 transgenic mice and their corresponding matched control littermates at different ages of life. Strikingly, the levels of autophosphorylation of PDK1 at the Ser241 activation loop site, and those of Akt at both Thr308 and Ser473 activating residues were 50% higher both in the cortex (Figure 2A) and the hippocampus (Figure 2B) of the 3xTg-AD transgenic mice, which was accompanied by similar increase in the phosphorylation of the Akt substrate PRAS40 at Thr246 and, to a lesser extent, of GSK3β at Ser9 and FOXO1 at Ser256, their specific Akt sites. This biochemical alteration was already detectable in the 3xTg-AD transgenic mice at pre-pathological stages of the disease (6 months of age) and became accentuated by 12 months of age when the symptoms are overt (Oddo et al., 2008). By contrast, the PDK1/Akt signaling was found slightly attenuated in the cortex of the APP/PS1 mice at 6 months of age (Supplementary Figure 1A), and up to 2-fold increased by 12 months of age (Supplementary Figure 1B).

Aging is the most prominent non-genetic risk factor for AD and is characterized by a progressive deterioration in the homeostasis of the neuro-immuno-endocrine systems. In particular, the increase in the secretion of inflammatory cytokines may play a vital role in the occurrence and development of AD (Wang et al., 2015). We therefore profiled the serum levels of a number of cytokines in the PDK1^K465E/K465E mice compared to PDK1^+/+ control littermate mice at different ages of life. Interestingly, the levels of IL-2, IL-9, and TNFα, three cytokines commonly elevated in AD, were also 10-, 4- and 2-fold increased respectively in the PDK1^+/+ control aged mice, but not in the PDK1^K465E/K465E mutant mice (Supplementary Figure 2).

We focused our attention on TNFα, an inflammatory cytokine acting through two specific transmembrane receptors, termed TNFR1 and 2, which have been reportedly shown to contribute to AD-related brain inflammation and to modulate neuronal viability (Yang et al., 2002; Li et al., 2004). TACE mediates the shedding of TNF-α and the two TNF-α receptors TNFR1 and TNFR2 (Gooz, 2010), as well as the α-secretase-mediated cleavage of APP (Buxbaum et al., 1998). We first analyzed by western blot the levels of expression of TACE, TNFR1 and APP in cortical (Figure 3A) and hippocampal (Figure 3B) total protein extracts derived from PDK1^K465E/K465E mutant and PDK1^+/+ control littermate mice at different ages of life, and found no differences between genotypes along the entire adulthood. Likewise, unaltered TACE and TNFR1 protein levels were also observed in the cortex and the hippocampus of 3xTg-AD (Figure 2) and in the cortex of the APP/PS1 (Supplementary Figure 1) transgenic mice at either 6 or 12 months of age.

We next determined the TACE α-secretase catalytic activity levels in cortical and hippocampal protein extracts, which were around 20% higher in the cortex of the 16- and 21-month old and at 12- and 16-month in the hippocampus of the aged PDK1^K465E/K465E mice compared to the young mutant mice samples or the PDK1^+/+ control littermate mice (Figures 3C,E). In agreement with that, the soluble TNFR1 levels were accordingly increased both in the cortex and the hippocampus of the PDK1^K465E/K465E knock-in mice compared to the controls (Figures 3D,F).

The α-secretase activity of TACE is mainly regulated by controlling its localization to the plasma membrane (Edwards et al., 2009). Phosphorylation of TACE at the C-terminus cytosolic domain by different upstream kinases modulate TACE trafficking (Gooz, 2010). Among them, PDK1 has been proposed to promote TACE phosphorylation and internalization (Pietri et al., 2013). Given the increased cortical TACE α-secretase activity observed in the PDK1^K465E/K465E mutant samples in which the Akt kinase activity is reduced, we sought to determine the consequences of this signaling lesion for TACE subcellular localization. TACE protein levels were three times more abundant in the membrane fractions of the PDK1^K465E/K465E 21-months old mice brain when compared to their corresponding controls, which was accompanied by a nearly absence of TNFR1 protein in those subcellular fractions (Figure 4A). This was corroborated by immunohistochemical analysis, which revealed a 40% increase in TACE surface staining and a 20% decreased detection of TNFR1 in the 21-months old PDK1^K465E/K465E mice brain cortical and hippocampal sections compared to the controls (Figure 4C). Of note, the levels of phosphorylation of TACE, as detected with antibodies raised against phosphorylated threonine residues, were nearly 2-fold reduced in the PDK1^K465E/K465E TACE immunoprecipitates compared to the controls, thereby raising the possibility of Akt being a major upstream kinase downstream of PDK1 governing the phosphorylation and shuttling of TACE (Figure 4B).

TNFα is synthesized as a membrane-bound pro-protein which is processed by TACE to generate the mature cytokine. Since TNFα can elicit pro-apoptotic signals acting through TNFR1, we next aimed to determine whether the increased TNFR1 shedding observed in the PDK1 mutant mice brain could be instrumental in protecting primary cultures of cortical neurons against TNFα toxicity. Immunocytochemical analysis of cortical neurons exposed to 100 ng/ml TNFα for 24 h revealed a marked 40% decrease in the levels of TACE and a clear 40% increase in the levels of membrane-bound TNFR1 in the wild type samples, but not in the mutant cultures (Figure 5A). Accordingly, similar reduction both in the α-secretase activity of TACE as well as in the concentration of soluble TNFR1 released to the culture media was observed in the wild type cells, which was significantly attenuated in the mutant neurons (Figures 5B,C). In spite of these data, TNFα failed to compromise cell viability both in the wild type and the mutant cultures (Figure 5D). Neurons can overcome the pro-apoptotic signals dictated by TNFα by expressing the anti-apoptotic Bcl-2 family member Bcl-XL (Gozzelino et al., 2008). Treatment of the cells with TNFα in the presence of the broad RNA synthesis inhibitor Actinomycin-D drastically reduced the cell viability of the wild type cultures, as denoted by a 70% decrease in the MTT assay values, but to a lesser extent in the mutant cells, which exhibited a significant 50% decrease instead.

The accumulation of misfolded proteins is a common characteristic of neurodegenerative syndromes such as AD, and alterations in the ability of the endoplasmic reticulum (ER) to counteract this stress through the unfolding protein response (UPR) could represent a molecular connection among these neurodegenerative diseases (Mercado et al., 2013). This adaptive response consists on a complex signaling network restoring homeostasis or triggering apoptotic cell dead depending on the intensity of the insult. We monitored the UPR by western blot analysis with specific antibodies against different elements on this signaling cascade. Among them, we consistently found up to 5-fold decreased levels of phosphorylation of the PERK kinase at Thr980 within the activation segment, which was accompanied by a 2-fold reduction in the levels of phosphorylation of the eIF2α substrate at the specific Ser51 PERK site, in the cortex of the PDK1^K465E/K465E aged mice compared to either the wild type or the young mice (Figure 6A). In sharp contrast, the levels of phosphorylation of both PERK at Thr980 and eIF2α at Ser51 were increased by 2-fold in the cortex of the 12-months old 3xTg-AD mice (Figure 6B), whereas the protein levels of other components of the UPR analyzed such as BIP, PDI, IRE1α or CHOP remained unchanged between genotypes.

The hyperactivation of the PERK/eIF2α signaling observed in the 3xTg-AD mice cortex is most likely caused by the exposure of the neurons to elevated levels of Aβ peptides, which could contribute to the neuronal loss observed in this model of disease. In order to clarify whether the attenuated levels of PERK/eIF2α phosphorylation observed in the aged PDK1^K465E/K465E mice cortex could have physiological consequences, primary cultures of cortical neurons derived from PDK1^K465E/K465E and PDK1^+/+ mice were treated with tunicamycin, a compound blocking the initial step of glycoprotein biosynthesis that causes the accumulation of unfolded glycoproteins, therefore leading to ER stress. Tunicamycin treatment caused a dramatic up to 90% dose-dependent reduction of cell viability, as assayed with the MTT method, which was followed by a vast up to 5-fold increase in the percentage of cells exhibiting apoptotic nuclear morphology in the control cells. Interestingly, cell viability was two times higher, and the percentage of apoptotic cells 20% reduced, in the PDK1^K465E/K465E neurons compared to the controls (Figure 7A). These differences can be attributed to the attenuated tunicamycin-induction of the UPR observed in the PDK1^K465E/K465E mutant samples, as denoted by the decreased levels of phospho-PERK Thr890 and phospho-eIF2α Ser-51, but not the BIP, PDI or IRE1α protein levels (Figure 7B).

The accumulation of plaques containing misfolded Aβ peptides is a characteristic neuroanatomical hallmark of AD and at the same time a central contributing factor to neurotoxicity. Since the reduced activation of Akt in the PDK1^K465E/K465E neurons protected cells against TNFα and ER stress, two proposed key mediators of Aβ-induced pathology, and the PDK1/Akt signaling pathway is hyperactivated in AD transgenic mice models, we reasoned that the PDK1^K465E/K465E mutant neurons should be also protected from Aβ-induced neurotoxicity. As expected, the exposure of primary cultures of wild type cortical neurons to Aβ oligomers elicited a dramatic dose-dependent reduction of up to 50% in cell viability, which is accompanied by a massive increase in the percentage of apoptotic cells reaching 50% values at the highest doses of Aβ tested. Remarkably, both the loss of cell viability as well as the increase in the percentage of apoptotic cells are clearly attenuated in the PDK1^K465E/K465E neurons, in which a 20% decrease in cell viability and a 30% of apoptotic cells are observed at the highest concentration of Aβ oligomers (Figure 8A). Of note, Aβ treatment did not change the levels of activation of Akt, as judged by the levels of phosphorylation of Akt at the Thr308, PDK1 activation site, as well as the levels of phosphorylation of the Akt substrates PRAS40 at Thr246 and TSC2 at Thr1462, which were as expected consistently reduced in the PDK1^K465E/K465E protein extracts compared to the controls along the whole treatment (Figure 8B). By contrast, the exposure of the neurons to Aβ oligomers induced the phosphorylation of PERK at Thr980 and of eIF2α at Ser51 more potently in the PDK1^+/+ control neurons than in the PDK1^K465E/K465E mutant ones; BIP protein was only induced upon 24 h of treatment and to the same level in the two genotypes analyzed, whereas PDI and IRE1α levels remained unchanged (Figure 8C). Of note, Aβ treatment also induced a 20% decrease in the levels of TACE accompanied by a 2.5-fold increase in the levels of membrane-bound TNFR1 in the control cells, which were less pronounced in the PDK1^K465E/K465E mutant cells (Figures 8D,E). In agreement with that, the TACE α-secretase catalytic activity (Figure 8F) as well as the levels of soluble TNFR1 released to the medium (Figure 8G) were further 10% reduced in the PDK1^+/+ wild type cells when compared to the PDK1^K465E/K465E mutant cultures.

In order to assess the contribution of the Akt signaling to the Aβ-toxicity protection exhibited by the PDK1^K465E/K465E mutant cells, we pre-treated wild type cultures with increasing doses of the Akt1 and Akt2 isoform-specific allosteric inhibitor Akti-1/2 prior to the exposure to Aβ oligomers. Pharmacological inhibition of Akt activity to levels equivalent to those found in the PDK1^K465E/K465E mutant cells protected wild type cortical cells against Aβ-induced toxicity, thereby mimicking the impact of the PDK1 K465E mutation in impeding Akt activation (Figure 9).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AD and handling Editor declared their shared affiliation.