Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

The nestin-Cre transgenic mice were kindly provided by Ulrich Mueller at the Scripps Research Institute (24), whereas the PDK1 L155E conditional knock-in mice and the genotyping procedures were previously described (25). Animal maintenance conditions and experimental research were in accordance with publication 2010/63/UE regarding the care and use of animals for experimental procedures. The study complies with the ARRIVE guidelines developed by the NC3Rs (26).

Neuronal primary cultures were established from embryonic day 15.5 (E15.5) embryos as previously described (22). Cortical cells were plated at a density of 20 × 10⁴ cells/ml on plates coated with 50 μg/ml of poly-d-lysine and maintained for 6 days in vitro (DIV) before treatments, whereas hippocampal cells were plated at a density of 7.5 × 10⁴ cells/ml on 12-mm glass coverslips coated with 150 μg/ml poly-d-lysine and placed in 24-well plates for 4 days in vitro.

Adult mice were terminated, their brains were dissected, and the right hemispheres were kept at −80°C for biochemical analysis. The left hemispheres were fixed for 2 h in 4% paraformaldehyde, preserved in 70% ethanol solution at 4°C, and embedded in 1.5% agarose in phosphate-buffered saline (PBS). 1H magnetic resonance imaging (MRI) studies were performed in a 7T Bruker BioSpec 70/30 USR spectrometer equipped with a mini-imaging gradient set (400 mT/m), a 72-mm inner-diameter circular polarized linear transmitter volume coil, and a received-only mouse head surface coil. Images were acquired using a multislice fast low-angle shot (FLASH) sequence from the Bruker Paravision 5.1 library (repetition time, 450 ms; echo time, 5.4 ms; excitation flip angle, 40°) in 33 contiguous 0.5-mm-thick slices, an acquisition matrix of 256 by 256, and a field of view of 19.2 mm by 19.2 mm, giving a voxel resolution of 0.0028125 mm³. The imaging data were Fourier transformed in ParaVision and then visualized using ImageJ software.

Organ volume was determined using the Cavalieri method (27) applied to either MRI data sheets of the adult brain or physical sections of embryonic brain samples. MRI images were displayed in ImageJ and outlined, and the total number of pixels was multiplied by the voxel resolution and by a factor of 2 to obtain the adult brain volume, which was assumed to be twice the volume of one hemisphere. Embryonic brain paraffin-embedded sections 5 μm thick were collected at systematically spaced locations (where the distance between sections [k] was 96 μm) from a random starting position and photographed with a Nikon SMZ800 stereomicroscope at ×1 magnification using a digital camera. A square lattice grid of 0.9149 mm² (d²) was then overlaid on the photograph using the program Photoshop vCS5.1, and the number of intersections (P) hitting either the whole head or the brain was scored. The volume was estimated by using the formula ΣP × d² × k. The number and size of the cells were determined on E15.5 dissociated cortex tissues with a Scepter 2.0 handheld automated cell counter (Millipore).

For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro, washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone). Cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, whereas the percentage of apoptosis was determined after Hoechst staining by scoring the number of cells with fragmented or condensed nuclei, as described previously (22). For the proliferation and apoptosis studies, E15.5 cortical neurons were cultured in complete neurobasal medium supplemented with B27 for the indicated number of days in vitro and then processed for immunocytochemistry with antibodies recognizing the Ki67 proliferation marker and the active caspase 3 apoptotic marker.

Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature; rinsed twice with PBS; permeabilized with 0.02% saponin diluted in PBS for 7 min at room temperature; and blocked with 5% bovine serum albumin (BSA), 0.01% saponin, 10 mM glycine in PBS for 1 h at room temperature. Primary antibodies diluted in PBS with 0.01% saponin and 1% normal goat serum were incubated overnight at 4°C. The cells were then incubated with the appropriate secondary antibodies diluted 1:400 in the same solution for 90 min and counterstained with 1 μg/ml of Hoechst 33342 (ThermoFisher no. H1399) for 30 min. Coverslips were mounted on microscope slides with FluorSave (Calbiochem no. 345789) reagent.

Hippocampal cells fixed at different days in vitro were immunostained with the dendritic marker MAP2 and the axonal marker Tau-1 and counterstained with 1 μg/ml of the nuclear dye Hoechst 33342. Images for the green, red, and blue channels were taken simultaneously with an epifluorescence microscope (Nikon Eclipse 90i) interfaced with a DXM 1200F camera at ×20 magnification. The numbers and lengths of axons, dendrites, and dendritic branches and the soma diameter were measured with the NeuronJ plugin and scored with the cell counter from ImageJ 1.42q (Wayne Rasband, National Institutes of Health).

E15.5 cortical neurons cultured for 6 days in vitro in complete neurobasal medium supplemented with B27 were starved for 4 h in neurobasal medium without B27 and subsequently stimulated with 50 ng/ml BDNF for 15 min. Cells were scraped from the wells in 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%, [wt/vol] Triton X-100, 0.1% [vol/vol] 2-mercaptoethanol, and a 1:100 dilution of protease inhibitor cocktail; Sigma). Tissue extracts were prepared by homogenizing the frozen tissues on ice in a 10-fold volume excess of ice-cold lysis buffer using a Polytron (10674; Kinematika GmbH). The lysates were centrifuged at 4°C for 10 min at 13,000 rpm, and the supernatants were aliquoted and preserved at −20°C. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard. The activation states of the different pathways were assessed by immunoblotting the extracts (10 μg) with the indicated antibodies, which were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies. Membranes were incubated with the enhanced chemiluminescence (ECL) reagent and then either exposed to Super RX Fujifilm and developed, or detected using a ChemiDoc MP imaging system (Bio-Rad), and then quantified using ImageJ software.

Streptavidin-Sepharose (10 μl; GE Healthcare) was conjugated to 0.5 μg of biotinylated PIF peptide (biotin-C₆ spacer-REPRILSEEEQEMFRDFAYIADWC) and incubated with 0.3 mg of precleared tissue lysates at 4°C overnight on a shaking platform. The pulldown products were washed twice with 1 ml of lysis buffer supplemented with 150 mM NaCl, resuspended in sample loading buffer, electrophoresed, and then immunoblotted for PDK1.

The Akt and PRAS40 total antibodies were kindly provided by Dario Alessi, University of Dundee. The PDK1 (no. 3062), TrkB Tyr706/707-P (no. 4621), TrkB (no. 4603), Akt Thr308-P (no. 4056), Akt Ser473-P (no. 4060), PRAS40 Thr246-P (no. 2997), pan-PDK1 site PKCγ Thr514-P (no. 9379), S6K Thr389-P (no. 9234), S6K (no. 9202), S6 protein Ser235/236-P (no. 4858), S6 protein (no. 2217), ERK1/2 (no. 9102), RSK Ser380-P (no. 9335), and RSK 1/2/3 (no. 9355) antibodies were purchased from Cell Signaling Technology. The RSK Ser227-P (no. 12445) antibody was obtained from Santa Cruz Biotechnology, the SGK1 antibody (no. S5188) from Sigma, and the PKCα antibody (no. P16520) from Transduction Laboratories. Secondary antibodies were from Pierce.

For immunofluorescence experiments, we used the Tau-1 (no. MAB3420), GAD67 (no. MAB5406), parvoalbumin (no. MAB1572), and NeuN (no. MAB377) antibodies from Millipore; the pan-axonal neurofilament (no. SMI-312R) antibody from Covance; the rabbit MAP2 (no. M3696) antibody from Sigma; the CUX1 (no. 13024) antibody from Santa Cruz Biotechnology; the Ki67 (no. ab156956) and doublecortin (no. ab18723) antibodies from Abcam; and the caspase 3 cleaved (no. 9661) antibody from Cell Signaling Technology. Alexa Fluor 594-conjugated goat anti-rabbit (no. A11072), Alexa Fluor 488-conjugated goat anti-mouse (no. A11017), and Alexa Fluor 594-conjugated goat anti-rat (no. A11007) secondary antibodies were obtained from Molecular Probes (Life Technologies).

Adult mice were intraperitoneally anesthetized with 0.4 mg/g body weight of pentobarbital, and then, an intracardiac injection of 70 heparin units was administered before perfusion with 0.9% NaCl, followed by 4% buffered paraformaldehyde. Brains were extracted, postfixed for 2 h in 4% paraformaldehyde, and preserved in 70% ethanol solution at 4°C. Embryos were dissected from E15.5 plug-tested pregnant females and decapitated, and the whole heads were fixed for 2 h in 4% paraformaldehyde and then preserved in 70% ethanol solution at 4°C. In both cases, samples from three littermates of different genotypes were embedded in the same paraffin block, with three as the final number of blocks, which were then sliced into 5-μm-thick coronal sections with a Leica RM2255 microtome. The sections were dry heated for 2 h at 60°C; rehydrated; boiled for 10 min in 10 mM sodium citrate, pH 6, for antigen retrieval; cooled for 30 min on ice; and washed three times with Tris-buffered saline (25 mM Tris, pH 7.5, 150 mM NaCl) (TBS). Samples were blocked in TBS containing 0.02% Triton and 5% goat serum for 30 min and incubated overnight at 4°C with primary antibodies diluted in the same blocking solution. The sections were rinsed with TBS buffer, incubated for 1.5 h at room temperature with the corresponding secondary antibodies diluted 1:400 in TBS, and counterstained with 1 μg/ml Hoechst 33342 before mounting in FluorSave reagent. Immunostained sections were photographed with a Nikon Eclipse 90i epifluorescence microscope, and the captured images were processed and analyzed with ImageJ 1.42q (Wayne Rasband, National Institutes of Health) and Fiji (http://pacific.mpi-cbg.de/wiki/index.php/Main_Page) software.

A sample of 12-month-old female mice (six PDK1^fl/fl CRE⁺ mutants and six PDK1^+/fl CRE⁻ matched controls) were confronted with a standardized battery of behavioral tests (28, 29) administered over 10 consecutive days for preliminary screening of the behavioral phenotypes of the mutant mice. On day 1, observation of undisturbed behavior in the home cage, including sociability, barbering, and sleeping in a group pattern, was immediately followed by the assessment of several sensorimotor tasks. Motor coordination and equilibrium were assessed in the wooden and wire rod tests by the distance covered and the latency to falling off a horizontal 1.3-cm-wide wooden rod and a 1-cm-diameter wire rod in two consecutive 20-s trials. Prehensility and motor coordination were measured as the distance covered on the wire hang test, where the animals were allowed to cling with their forepaws for two trials of 5 s and a third, 60-s trial. Muscle strength was measured as the time needed to fall off the wire in the 60-s trial. Nesting behavior using paper towels was measured in isolation (housing conditions on day 10), according to Deacon's 5-point scale (30). The secondary and tertiary screens addressed neuropsychiatric-like deficits by assessing spontaneous exploratory behavior, anxiety-like behavior, circadian activity, and cognition in a series of tests involving different degrees of complexity. Neophobia in the corner test was recorded on day 2 in a new home cage by the horizontal (number of visited corners) and vertical (number and latency of rearings) activity during a period of 30 s. Immediately after, exploratory activity and anxiety-like behaviors in a standard open-field test were measured for 10 min. Horizontal (centimeters) and vertical (rearings) locomotor activities were recorded for each minute of the test. The following items of behavior were recorded: freezing (latency of initial movement), thigmotaxis (latency of leaving the central square and that of entering the peripheral ring 10 cm from the walls), and self-grooming behavior (latency, number, and duration of groomings). Defecation and urination were also measured. On day 3, a marble-burying test was performed in standard cages containing 10 glass marbles (1 by 1 by 1 cm) evenly spaced on a 5-cm-thick layer of sawdust. Latency to contacting a marble was measured, and at the end of the 30-min test, the number of marbles was determined as follows: intact (the number of unmanipulated marbles), change in position (the number of marbles at least half buried in sawdust and rotated 90° or 180°), and buried (the number of marbles 100% buried in sawdust) (30). On the next 3 days (days 4 to 6), perceptual visual learning and spatial learning and memory were assessed in a 3-day water maze. The animals were trained to a criterion (90% escaping in under 60 s) in a series of cued visible-platform trials (7-cm-diameter platform 1 cm above the water surface, with the position of the platform indicated by a visible 5- by 8-cm striped flag; 20-min intertrial time) in a pool (Intex Recreation Corp., CA; 91-cm diameter; 40 cm deep; 25°C opaque water). This required four platform trials (cued visible-platform trial 1 [CUE1] to CUE4). The last visible-platform trial of any animal was considered to be its posthabituation baseline and was designated CUE4. Mice that failed to find the platform within 60 s were manually guided to the platform and placed on it for 5 to 10 s, the same period as successful animals. Twenty-four hours after the last cued platform trial, the animals were tested in a series of four hidden-platform trials (PT1 to PT4, 20 min apart). In these place-learning tasks, the hidden platform (1.5 cm below the water surface) was located in a new position opposite the one used for cue learning. Escape latencies were measured with a stopwatch. Episodes of immobility (flotation) were measured. On days 7 to 10, circadian motor activity was tested for 84 consecutive hours in a home cage equipped with a running wheel (31).

Two-way analysis of variance and Student's t test analysis were applied to compare differences among categories, as depicted in the figures. Data analysis was done using Prism software (GraphPad Software, La Jolla, CA).

We followed the minigene conditional knock-in approach to limit the expression of the PDK1 mutant protein to the neuronal lineage (25). In this method, a conditional allele was constructed in which the wild-type protein was expressed from the endogenous locus through the first two PDK1 exons, whereas expression of the remaining coding region occurred through a LoxP site-flanked minigene DNA cassette consisting of the wild-type PDK1 open reading frame from exons 3 to 14 followed by the natural transcriptional termination and polyadenylation signals. The construct was designed so that upon CRE recombinase-mediated excision of the floxed minigene, a mutated version of exon 4 coding for glutamic acid at position 155 of the mouse PDK1 protein would be transcribed (Fig. 1A).

A potential drawback of this floxed allele is its reported inability to efficiently use the termination signals of the minigene cassette to finish transcription, which continued into the knock-in region of the gene even in the absence of CRE and produced significant amounts of mRNA for mutant PDK1 (25). However, we reasoned that this attribute, after the appropriate genetic crosses, would give rise to a valuable allelic series with increasing expression of the mutant PDK1 protein. Hence, mice homozygous for the PDK1 conditional transgene were crossed with nestin-CRE mice, which express the CRE recombinase under the control of the neuron-specific enhancer of the nestin promoter in precursors of both neurons and glia starting at E10.5 (24). The PDK1 L155E conditional floxed allele is referred to here as PDK1^fl for simplicity. PDK1^fl/fl CRE⁺ mice, but not PDK1^+/fl CRE⁻, PDK1^+/fl CRE⁺, or PDK1^fl/fl CRE⁻ mice, were born in a reduced Mendelian distribution (Fig. 1B). We confirmed that the PDK1 protein was expressed at similar levels in samples of the different genotypes analyzed (Fig. 1C). We next incubated protein extracts from either brain or liver with Sepharose conjugated to PIF-tide, a synthetic polypeptide corresponding to the amino acid sequence of the PDK1 substrate-docking site. PIF-tide interacts strongly with the PIF pocket of wild-type PDK1 but cannot interact with the PDK1 L155E mutant protein (32, 33). PDK1 could be affinity purified with high efficiency from PDK1^+/fl CRE⁻, PDK1^+/fl CRE⁺, and, to a lesser extent, PDK1^fl/fl CRE⁻ brain extracts but not from PDK1^fl/fl CRE⁺ mutant mouse brain extracts, thereby biochemically confirming the expected gradation in the expression of the mutant PDK1 L155E protein rather than the wild-type protein. As a control, we demonstrated that in liver tissues, which do not express CRE, PDK1 could be affinity purified with the same reduced efficiency from PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ mice, which are homozygous for the conditional leaky allele, in comparison to PDK1^+/fl CRE⁻ and PDK1^+/fl CRE⁺ controls, which retain a wild-type copy of the PDK1 gene (Fig. 1C).

The resultant PDK1^fl/fl CRE⁺ mutant mice, as well as the PDK1^fl/fl CRE⁻ mice, which also express high levels of the PDK1 L155E mutant protein in nonneuronal tissues (Fig. 1C), were viable and fertile and displayed a 25% reduction in body weight compared to PDK1^+/fl CRE⁻ mice, which retain a wild-type copy of the PDK1 gene (Fig. 2B).

Stereological analysis demonstrated that both the brain and head volumes were proportionally reduced by 20% in PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ E15.5 embryos compared to PDK1^+/fl CRE⁻ controls (Fig. 2A and C). While the PDK1^fl/fl CRE⁻ mice exhibited a 25% reduction in both the volume and mass of the brain compared to the PDK1^+/fl CRE⁻ controls, which is proportional to the reduction in body weight, the PDK1^fl/fl CRE⁺ adult mutant mice exhibited microcephaly, with brains showing a reduction of about 50% in volume and mass compared to those from the PDK1^+/fl CRE⁻ controls (Fig. 2D). Determination of the volume of the neuronal soma and the number of neurons purified at E15.5 from the embryonic cortex suggested that the small brain size of the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ mice might be mostly due to a reduction in the size of the cells rather than to a reduced number of cells (Fig. 2E and F).

To define the importance of the PDK1 substrate-docking site in the ability of PDK1 to activate its different cellular targets, primary cultures of cortical neurons derived from littermate PDK1^+/fl CRE⁻, PDK1^fl/fl CRE⁻, and PDK1^fl/fl CRE⁺ embryos were stimulated with BDNF for 15 min. As a control for stimulation, the activation of the BDNF receptor TrkB was monitored by measuring its phosphorylation at the activation loop residues Tyr706/707, which was very robust in all three genotypes analyzed (Fig. 3A). In contrast, phosphorylation of TrkB was not detectable in whole brain tissue lysates at E15.5, despite the high levels of expression of the receptor (Fig. 3B).

BDNF induced clear activation of Akt to the same extent in all three genotypes analyzed, as judged by the level of phosphorylation of the two activating residues, Thr308 and Ser473. This was corroborated by measuring the phosphorylation levels of the Akt substrate PRAS40 at Thr246, which was not affected by the PDK1 L155E mutation (Fig. 3A). To further validate these data in a more physiological context, the levels of phosphorylation and activation of Akt were also measured in whole protein extracts derived from the E15.5 developing brain, which were similar in all three different genotypes analyzed (Fig. 3B).

In contrast, phosphorylation of S6K at the T loop Thr229 site by PDK1 was markedly reduced in the PDK1^fl/fl CRE⁻ cell and tissue samples compared with the PDK1^+/fl CRE⁻ controls and almost abolished in the PDK1^fl/fl CRE⁺ samples. As a consequence, S6K activation, as monitored by the phosphorylation of the S6 ribosomal protein at Ser235, was similarly affected (Fig. 3).

BDNF also induced robust autophosphorylation of the Ser380 hydrophobic-motif site of RSK and the consequent recognition and phosphorylation of the Ser227 T loop site by PDK1 (34) in the PDK1^+/fl CRE⁻ control cells. In contrast, phosphorylation of RSK at Ser227 by PDK1 was gradually reduced in the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ cells and tissues (Fig. 3).

Activation of SGK1 requires the phosphorylation of the hydrophobic motif at Ser422 by mTORC2, which primes the binding of PDK1 (35). As expected, BDNF-induced phosphorylation of SGK1 at the Thr256 PDK1 site within the T loop was reduced in the PDK1^fl/fl CRE⁻ cortical cultures compared with the PDK1^+/fl CRE⁻ controls and further decreased in the PDK1^fl/fl CRE⁺ samples and tissues (Fig. 3).

PDK1 also controls the stability of PKC isoforms through the phosphorylation of their T loop sites (36). We found that both the levels of phosphorylation and the levels of expression of PKCα were reduced in the PDK1^fl/fl CRE⁺ cells and tissues (Fig. 3) compared to the PDK1^fl/fl CRE⁻ and PDK1^+/fl CRE⁻ samples.

The neuron-specific PDK1 PIF pocket mutation most likely compromised growth responses during brain development, leading to microcephaly in the PDK1^fl/fl CRE⁺ mutant adult mice. It is well known than the PI3K/PDK1/Akt signaling pathway plays critical roles in mediating the responses to survival signals that antagonize the intrinsic programmed cell death in the developing nervous system (6, 37). We recently showed how decreasing the efficiency of PKB/Akt activation by expressing the mutant form of PDK1 K465E, which is incapable of phosphoinositide binding, had no consequences for neuronal survival (22). From that, it was postulated that either the reduced levels of Akt activity attained in the PDK1^K465E/K465E mice were sufficient to preserve normal neuronal viability or that PDK1-activated kinases other than Akt were responsible for transducing the survival program elicited by extracellular signals. To address this question, we took advantage of the PDK1 L155E neuron-specific knock-in mice. Neuronal programmed cell death can be accurately recapitulated in vitro in primary cultures of embryonic cortical neurons, which survive in the presence of particular neurotrophins, such as BDNF, and die through apoptosis upon trophic factor withdrawal. Trophic factor deprivation induced the death of about half of the cells, as evidenced by a 50% decrease in the MTT reduction values (Fig. 4A) and a 5-fold increase in the number of apoptotic cells (Fig. 4B and C) in all four genotypes analyzed. BDNF completely restored the cell viability values (Fig. 4A) while decreasing by 2- to 3-fold the number of cells exhibiting apoptosis (Fig. 4B and C), to the same extent in all four genotypes analyzed. From this, it was concluded that the PDK1 PIF pocket-dependent AGC kinases were not essential for the neuroprotective actions of BDNF, at least in cortical neurons.

We next measured proliferation, as well as cell death parameters, in primary cultures of cortical neurons during the first 4 days in vitro by using the Ki67 proliferation marker and the cleaved/active caspase 3 apoptotic marker. At DIV0, the PDK1^+/fl CRE⁻ control cultures reached their highest proliferation rates, above 20%, which were significantly reduced in both the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ cultured cortical neurons (Fig. 5A and B). This proliferation activity, most likely corresponding to the neurogenesis peak of the progenitors that would later populate the upper cortical layers, mostly ceased as the cells progressed to DIV4, which was accompanied by a moderate increase in the apoptotic index to similar levels in all three genotypes analyzed (Fig. 5A and C). Accordingly, coronal brain sections from E15.5 embryos stained with the newborn neuron marker doublecortin revealed a clear reduction of the neuronal progenitor cells in PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ embryos compared to the PDK1^+/fl CRE⁻ controls (Fig. 5D and E). In contrast, no evidence of apoptosis was observed in any of the three genotypes analyzed, as judged by the near absence of active caspase 3 staining (Fig. 5D and F). Since the soma size analysis of hippocampal neurons in culture revealed a genotype-dependent decrease in the diameter of the cellular soma (Fig. 6D), altogether, our data demonstrate a combined reduction in progenitor proliferation and cell growth responses.

The PI3K signaling pathway controls several aspects of neuronal morphogenesis, including neuronal polarization, axonal outgrowth, and dendrite arborization (38). In PDK1^K465E/K465E mice, the Akt/mTORC1 axis regulated PI3K-mediated neuronal polarization and axon outgrowth (22). Since S6K is a major effector of mTORC1 in controlling the translation of proteins that are relevant for neuronal differentiation, we reasoned that the PDK1 L155E mouse model, with normal levels of Akt activity but in which S6K cannot be activated by PDK1, could be instrumental in defining the contribution of S6K to neuronal differentiation. To that end, we analyzed cell polarization and measured axon length during the differentiation of hippocampal primary neurons in culture. In the PDK1^+/fl CRE⁻ control cultures, most of the neurons exhibited a differentiated axon by DIV3, as evidenced by the expression of the axon-specific microtubule-associated protein Tau-1. In contrast, neuronal polarization was severely impaired in both the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ mutant hippocampal cultures, in which only half of the neurons showed axons by DIV3 and -4 (Fig. 6A and B). The complexities of the neuritic processes, as inferred from the number of neurites per cell (Fig. 6C), as well as their lengths and the numbers of ramifications (Fig. 6E), were similar in PDK1^+/fl CRE⁻, PDK1^fl/fl CRE⁻, and PDK1^fl/fl CRE⁺ mouse hippocampal neurons at all the time points analyzed. In contrast, the average length of the axon was reduced by 30% in the PDK1^fl/fl CRE⁻ neurons and as much as 40% in the PDK1^fl/fl CRE⁺ neurons compared to the PDK1^+/fl CRE⁻ control cells at both DIV3 and -4 (Fig. 6E).

We next explored how the deficient differentiation abilities of the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ embryonic neurons detected ex vivo had physiological consequences in the adult brain. Immunostaining of brain sections with antibodies to microtubule-associated proteins that are selectively localized in either axons (SMI312) or dendrites (MAP2) revealed a clear decrease in the density of axonal fibers in various regions of the PDK1^fl/fl CRE⁻ and PDK1^fl/fl CRE⁺ cortex (Fig. 7A) and hippocampus (Fig. 7B), in a genotype dose-dependent manner.

The PI3K signaling pathway also controls the radial migration of newborn cortical neurons during cortex development, resulting in an ordered arrangement of neurons into six different cortical layers. It has been proposed that Akt, but not mTORC1, controls cortical development downstream of PI3K activation (39). Since the neuronal differentiation program is integrated with the program of neuronal migration, we aimed to determine whether the PI3K/PDK1 effectors that are independent of Akt activation were also important for cortical development. We observed that in the PDK1^fl/fl CRE⁻ adult brain, neurons in layer IV of the somatosensory cortex were packed together (Fig. 8B) and further compacted in PDK1^fl/fl CRE⁺ mice (Fig. 8C) compared to the PDK1^+/fl CRE⁻ controls (Fig. 8A), as revealed by immunohistochemical analysis with the NeuN neuronal marker.

Cortical layer IV is mainly composed of pyramidal neurons and GABAergic interneurons. For that reason, we employed antibodies against CUX1, a transcription factor that is expressed in glutamatergic cortical neurons from upper layers II to IV of the somatosensory cortex, and GAD67, the decarboxylase that catalyzes the conversion of glutamate to the GABA-inhibitory neurotransmitter in the GABAergic interneurons. While CUX1 staining confirmed that the neuronal packing arises from an increase in the glutamatergic-neuron density with an unchanged number of cells (Fig. 9A to D), a clear decrease in GAD67 expression in the somatosensory cortex (Fig. 9A and E) and the cingulated cortex (Fig. 9F) was observed, arising from the increase in the PDK1 L155E mutant protein levels across the different genotypes analyzed. Moreover, the parvalbumin-positive interneurons were mostly excluded from layer IV in the PDK1^fl/fl CRE⁺ cortex samples compared with its distribution in the PDK1^fl/fl CRE⁻ and PDK1^+/fl CRE⁻ cortices (Fig. 9B).

A three-stage protocol for behavioral and functional comprehensive phenotype assessment of mutant mice (28, 29) evaluated the physical condition, sensorimotor functions, behavioral and psychological profiles (spontaneous behavior, locomotor and exploratory activities, anxiety-like behaviors, and motivation), and cognition (learning and memory, attention, and executive functions). Somatic growth, as measured by body weight, was reduced, with lower food intake (Fig. 10A). Sensorimotor functions were severely (2-fold) reduced, even in an easy task, such as the wooden rod test (Fig. 10B). A deficiency in a highly preserved daily life ethological activity, such as nesting behavior, evidenced impairment of executive functions (Fig. 10C). The mutant mice showed apparently undisturbed behavior in the home cage, with normal socialization and sleeping in groups but absence of barbering. Disrupted behavior was aroused by handling, with a high incidence of hyperreactivity shown as refusal to be handled (5/6), vocalizations (3/6), bizarre behavior (4/6) (Fig. 10D) (stereotyped stretching), refusal to perform tests like the hanger test (6/6), and diminished motivation to swim in the water maze (Fig. 10G) (flotation). Neophobia in the corner test suggested flight behavior, which in a more anxiogenic environment, such as the open-field test (Fig. 10E), turned into freezing behavior. Overall reduced total vertical exploratory activity was observed, while the thigmotaxis ratio (locomotion in the periphery versus the center) was maintained in the mutants. These results were confirmed in the 84-h continuous circadian activity recordings in a cage equipped with a wheel (Fig. 10H). In that setting, the time course from the first (novelty) to the fourth (habituation) night was strikingly reduced in the mutant mice, mostly within the first two nocturnal activity periods. In the marble-burying test, the mutants exhibited a higher number of marbles changed in position, to the detriment of the number of buried pieces (Fig. 10F). With respect to their cognitive abilities, the two groups solved the first experience in the water maze equally well. However, a poorer acquisition curve was seen in the mutant group, resulting in a worse cognitive capacity at the end of the perceptual visual learning paradigm, which involves attention and motivation (Fig. 10G, CUE1 to -4). Similarly, on the subsequent days, the mutant mice showed cognitive deficits in their spatial-learning and memory abilities in the place-learning task. They performed similarly in the first trial of each day (long-term memory) but did not progress successfully in the trial-by-trial test (short-term and working memory), again with worse learning curves and ratios than the age-matched wild-type group. A higher incidence of periods of immobility (flotation), as a nonsearching behavior indicative of changes in motivation in the mutants, was also shown throughout the test (Fig. 10G).