The phosphoinositide 3-kinase (PI3K) signaling pathway regulates cell survival, proliferation, growth, and motility, as well as metabolism, in response to extracellular signals. Class I PI3Ks phosphorylate the membrane phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2
] to generate the phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3
] second messenger (1
). In neurons, stimulation of PI3K by neurotrophic factors, neurotransmitters, or guidance cues results in the activation of protein kinase B (PKB) (also termed Akt), the most studied downstream effector of this signaling pathway. Akt phosphorylates and inactivates a number of cellular substrates controlling different aspects of neuronal development. They include PRAS40 and TSC2, leading to mammalian TORC1 (mTORC1) activation, which in turn promotes the synthesis of selected sets of proteins involved in the differentiation program (3
); glycogen synthase kinase 3 (GSK3), which regulates cytoskeleton dynamics and participates in the establishment and maintenance of neuronal polarity (4
); and FOXO, which promotes the expression of genes inhibiting apoptosis (6
). Genetic analysis in mice has uncovered the functional significance of PI3K for brain morphology and physiology (7–10
), whereas deregulation of this signaling pathway has pathophysiological consequences in human neurodevelopmental disorders, such as schizophrenia (11–13
) and autism (14
3-Phosphoinositide-dependent protein kinase 1 (PDK1) transduces many agonist-induced cellular responses by activating an entire set of AGC kinase family members, in addition to Akt (16
). They include S6K, SGK, RSK, and protein kinase C (PKC) isoforms. Upon cell stimulation, PDK1 is enabled to phosphorylate the T loops of all these AGC kinases, resulting in their activation (17
Since PDK1 is constitutively active in cells, the previous phosphorylation of a second activating residue located in a C-terminal conserved hydrophobic motif becomes rate limiting for PDK1 to bind and activate most substrates. The phosphorylated hydrophobic motif acts in this manner as a substrate-docking site recognized by a small groove within the PDK1 catalytic domain termed the PIF pocket (19
). In contrast, phosphorylation of Akt at the hydrophobic motif is not required for PDK1 to activate the kinase. The exclusive presence in both PDK1 and Akt of pleckstrin homology (PH) domains able to specifically interact with PtdIns(3,4,5)P3
results in their colocalization at the plasma membrane, where PDK1 can phosphorylate and activate Akt (16
The final demonstration that these two mechanisms operate in vivo
came from analysis of two single-amino-acid, rationally designed PDK1 mutations abrogating the function of either the PH domain or the PIF pocket motif (21
). We recently reported how in PDK1K465E/K465E
knock-in mice, which express a mutant form of PDK1 incapable of phosphoinositide binding, activation of Akt was selectively affected. As a consequence, the ability of hippocampal and cortical embryonic neurons to differentiate was markedly impaired (22
Full knock-in mice expressing a mutant form of PDK1 in which the leucine residue at position 155 within the PIF pocket was replaced by glutamic acid (L155E) were previously generated and died at midgestation. In the PDK1L155E/L155E
mice, activation of all the PDK1-regulated substrates with the exception of Akt was totally abolished (23
). To define the contribution of PDK1 signaling beyond Akt to neurodevelopmental regulation, here we targeted the expression of the PDK1 L155E mutant protein to the developing brain. The PDK1 mutant mice were microcephalic, with neuronal polarization and axonal elongation significantly inhibited in the mutant neurons. As a consequence, the patterning of the brain was dramatically perturbed, as demonstrated by cortical layering alterations and reduced circuitry. This resulted in impaired cognition, along with abnormal behavior, which further highlights the importance of PDK1 targets different from Akt in mediating signaling responses that are key to brain development.
MATERIALS AND METHODS
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.
Determination of organ volume and cell size.
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
) 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
. 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).
Evaluation of neuronal proliferation, apoptosis, and survival.
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.
Evaluation of differentiation.
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).
Generation of protein extracts and Western blot analysis.
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.
Affinity purification of PDK1.
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
A sample of 12-month-old female mice (six PDK1fl/fl
mutants and six PDK1+/fl
matched controls) were confronted with a standardized battery of behavioral tests (28
) 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).
In the present study, we characterized the Akt-independent roles that the PDK1 signaling pathway plays during brain development. Disrupting the interaction of PDK1 with its substrates in the PDK1L155E/L155E
full knock-in mice resulted in an embryonic-lethal phenotype, with embryos dying at E12 and exhibiting severe retardation and a major reduction in forebrain size, highlighting the essential roles that the Akt-independent branch of the PDK1 signaling network plays during embryonic development (23
). To circumvent this lethal period, we targeted the expression of the PDK1 L155E mutant protein to neuronal tissues by conditional knock-in methodologies. The PDK1fl/fl
genotype was observed at birth at a 3-fold-reduced Mendelian frequency. In contrast, the PDK1fl/fl
mice were born at the expected frequency, indicating that low levels of PDK1 wild-type protein were sufficient for the PDK1fl/fl
mice to complete embryonic development and that the further and specific ablation of the wild-type PDK1 sequence in the nervous system was responsible for the lethality in the PDK1fl/fl
mutant embryos. The hypomorphic nature of the PDK1 L155E conditional knock-in allele, which can drive the expression of as much as 60 to 70% of PDK1 L155E mutant protein without compromising the viability of these mice, makes the PDK1fl/fl
conditional knock-in mice an excellent genetic tool to study the function of PDK1, beyond Akt, in a more physiological context. At the same time, these mice could represent an appropriate experimental model to validate the effects of new allosteric modulators targeting the PDK1 PIF pocket that are now being developed and that have enormous therapeutic potential (41
Activation of S6K, RSK, SGK, and PKC but not Akt was selectively decreased in the PDK1fl/fl
mouse cortical neurons compared to the PDK1+/fl
controls and further abolished in the PDK1fl/fl
neurons and tissues. We and others recently described how, in the absence of PtdIns(3,4,5)P3
binding, PDK1 can still activate Akt by binding to the phosphorylated-Akt hydrophobic motif (42
). In agreement with that, in the PDK1K465E/K465E
knock-in mice expressing a mutant form of PDK1 incapable of phosphoinositide binding, activation of Akt was selectively affected but not fully abolished (22
). In contrast, in the PDK1fl/fl
neurons and tissues, Akt was normally activated, demonstrating that under physiological conditions, Akt activation by PDK1 relies mostly on the phosphoinositide-mediated colocalization of both kinases at PtdIns(3,4,5)P3
-rich cellular membranes rather than on the docking-site interaction.
The reduced phosphorylation of S6K and RSK at their activation loops by PDK1 arises from the inability of the PDK1 L155E mutated PIF pocket to interact with the phospho-hydrophobic-motif docking site in S6K and RSK. Unexpectedly, the level of phosphorylation of S6K at the Thr389 and of RSK at the Ser380 hydrophobic-motif sites was also decreased to the same extent as that of the activation loops. The AGC kinases possess a hydrophobic groove in the small lobe of their kinase domains similar to the PDK1 PIF pocket. This hydrophobic pocket establishes intramolecular interactions with the phosphorylated hydrophobic motif that are fundamental for the transition of the enzyme to the active conformation (44
). In the absence of activation loop phosphorylation, the active, closed conformation is not favored, which may allow the exposure of the hydrophobic motif phosphorylation site to the action of phosphatases (23
PDK1 plays a major role in the processing and maturation of several PKC isoforms. Newly synthesized PKC polypeptides are first phosphorylated at their hydrophobic motifs, allowing PDK1 to interact with and phosphorylate the T loops. These first phosphorylation events do not depend on apical agonist stimulation and stabilize the enzyme in a conformation that is catalytically inactive but at the same time competent to respond to the diacylglycerol and calcium second messengers (45
). In agreement with that notion, both the levels of phosphorylation and the levels of expression of PKCα were reduced in the PDK1fl/fl
embryonic-brain samples, as well as cortical cell extracts, but not in the PDK1fl/fl
samples compared to the PDK1+/fl
controls (Fig. 3
), indicating that the reduced levels of expression of the wild-type PDK1 protein in PDK1fl/fl
mice were sufficient to regulate phosphorylation and stability of PKC isoforms, which was unchanged upon BDNF stimulation of cortical neurons (Fig. 3A
One salient finding of these studies is that disrupting the interaction of PDK1 with its substrates in the PDK1fl/fl
mutant mice had no consequences for neuronal survival. This is consistent with the fact that the PKB/Akt kinase, which is the only PDK1 substrate that is not affected by the PDK1 L155E mutation, is thought to be the critical downstream effector of this pathway in promoting survival by antagonizing programmed cell death (47
). However, in PDK1K465E/K465E
knock-in mice, reduced activation of Akt isoforms was also sufficient to dictate neuronal survival (22
). We recently demonstrated how the inhibition of mTORC2, the Akt and SGK hydrophobic-motif kinase, modestly compromised the viability of PDK1 wild-type neurons at doses that did not affect Akt activation, and this was further aggravated in the PDK1K465E/K465E
mutant neurons (43
). Altogether, these results point to a synergistic role for both Akt and SGK in controlling neuronal survival by coordinately regulating the phosphorylation of some substrates that are relevant to the control of apoptosis. In contrast, both cell proliferation and cell growth were reduced in the PDK1fl/fl
mutant mice, which might be due to the inability of PDK1 to activate S6K despite unaffected PI3K/Akt/mTORC1 signaling. These results implicate S6K as the most critical downstream effector of this signaling pathway in instructing cell and organism growth.
The observation that both the neuronal polarization and axonal elongation responses of hippocampal neurons from PDK1fl/fl
mice were inhibited by the PDK1 PIF pocket mutation is particularly relevant, since the establishment of the axon-dendrite axis is an essential morphogenetic mechanism for the appropriate assembly of neurons into functional neuronal circuits. This process is triggered by different extracellular signals acting through a number of intracellular signaling pathways. Among them, the interplay between the PI3K/Akt signaling pathway (48
), the Par3-Par6-atypical PKC complex (4
), and the LKB1/BRSK axis (49
) is fundamental in controlling axon specification and growth. In agreement with this, in PDK1K465E/K465E
mice, the PI3K/Akt/mTORC1-dependent regulation of the BRSK protein levels represents an integration point for both the PI3K and LKB1 signaling pathways in axonal morphogenesis (22
). In these mice, the hypomorphic reduction of Akt signaling toward the PRAS40/TSC2/mTORC1/S6K axis caused mild phenotypes that could be mimicked ex vivo
with the Akti-1/2 inhibitor, which reduced Akt activity by inhibiting Akt1 and Akt2 but not the Akt3 isoform, and aggravated by using the mTORC1 inhibitor rapamycin. Interestingly, in PDK1fl/fl
mice, in which the PI3K/Akt/mTORC1 axis is normally activated, the defects in both the polarity and axonal-elongation processes were more severe than those reported in PDK1K465E/K465E
mice, which phenocopied the severe differentiation defects previously observed with the mTORC1 inhibitor (22
). Altogether, these observations place a PIF pocket-dependent kinase, namely, S6K, as a key effector of the PI3K/Akt/mTORC1 axis in controlling the synthesis of proteins that are relevant for neuronal morphogenesis. Moreover, while in PDK1K465E/K465E
mice the transient alterations in the timing of the differentiation program did not translate into gross abnormalities in the patterning of the adult brain (22
), the genetic ablation of S6K activation with intact Akt signaling in PDK1fl/fl
adult mice caused microcephaly with profound structural and molecular brain alterations, ruling out the implication of Akt or mTORC1 substrates other than S6K in these phenotypes.
Further experiments must be undertaken in the future to elucidate the connection between the impaired PDK1 PIF pocket-dependent signaling in mutant mice and the altered population of interneurons evidenced by the GAD67 and parvoalbumin marker defects. In this regard, defects in the translation machinery in neurons caused by elongator subunit Elp3 deletion have been recently related to impaired neuronal differentiation and unbalanced neurogenesis, leading to microcephaly resulting from the premature differentiation of neurons (51
). In an analog form, the S6K PIF pocket-dependent kinase is indispensable for preinitiation complex (PIC) formation and protein translation (53
). Abrogation of S6K activation in PDK1 L155E mutant mice could have impaired the normal protein translation in neurons, leading to unbalanced neurogenesis, thereby generating a potential molecular connection between the PIF pocket mutation and the described brain defects associated with microcephaly. Moreover, the deficient polarization and axonal outgrowth of the PDK1 L155E mutant neurons might have caused altered migration patterns during cortex lamination, contributing to the decreased density of axonal fibers in the cortex and hippocampus, increased density of glutamatergic neurons with normal numbers of cells in cortical layers II to IV, decreased GAD67 expression levels, and mislocalized parvalbumin-positive interneurons, which were mostly excluded from layer IV. Intriguingly, these alterations are commonly observed in human mental diseases, such as schizophrenia. They included reduced axonal density in the cortex and the hippocampus (55
); elevated neuronal density in cortical layer IV (56
); GAD67 deficits in the upper cortical layers, especially in the cingulated cortex (57
); and abnormal localization of the parvalbumin-positive interneurons (58
). The current hypotheses for the etiology of schizophrenia state that this molecular and structural scenario is thought to result in deregulated GABAergic interneuron-mediated inhibition of glutamatergic neurons, resulting in overactivation of the neuronal circuits and thus contributing to the characteristic symptoms of the disease.
mice, the behavioral disturbances included a wide array of impairments, ranging from sensorimotor and basic species executive functions to both cognitive and noncognitive behavioral alterations. The different tests assessed point to relevant and persistent bizarre behavior, severe reduction of exploratory activity, increased freezing, diminished motivation, and impaired short-term working memory. Much future work needs to be performed to determine whether the presence of exacerbated disruptive behavior, suggesting hypersensitivity to stressful situations, but reduced activity and diminished motivation have validity as negative symptoms that characterize the clinical features of the schizophrenia spectrum (59
). Moreover, cognitive impairment affecting learning and memory, but also attention and executive functions, would also be compatible with cognitive clinical symptoms of schizophrenia.
Human schizophrenia might arise from an inherited genetic predisposition causing brain developmental alterations combined with environmental factors that can influence brain maturation during childhood. A number of genes that have been associated with familiar forms of schizophrenia converge on the same signaling pathway. They include the neuregulin 1 (NRG1) growth factor gene (60
), the dystrobrevin-binding protein 1 (DTNBP1) gene (12
), the DISC1 scaffolding protein gene (13
), and the Akt1 gene (11
), which together participate in modulation of Akt signaling outputs. To our knowledge, this is the first report showing the involvement of PDK1 downstream effectors other than Akt in mouse neuropsychiatric-like disorders, with potential face and construct validity for negative and cognitive symptoms of schizophrenia. Our results point to a prominent function for PIF pocket-dependent kinases as major effectors of this signaling hub downstream of Akt in the etiopathogenesis of schizophrenia that might provide construct validity to the PDK1 L155E mutants.
We thank Dario Alessi (MRC Protein Phosphorylation Unit, Dundee, United Kingdom) for providing the mice and some of the antibodies; Cristina Gutierrez, Mar Castillo, and Núria Barba (Cell Culture, Histology and Microscopy Services of the Institut de Neurociències) for technical assistance; Jessica Pairada and Núria Moix (Estabulari de Rosegadors of the Universitat de Lleida) for animal care; and the joint nuclear magnetic resonance facility of the Universitat Autònoma de Barcelona and Centro de Investigación Biomédica en Red-Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN) (Cerdanyola del Vallès, Spain) for time allocated. We express our gratitude to Alfredo Miñano and Montse Solé for helpful discussion and to Arnaldo Parra, Lilian Enríquez, and Carles Saura for their expert advice on mouse brain development.
L.C.-B., J.R.B., J.M.L., and E.C. designed the experiments, analyzed the data, and wrote the manuscript. L.C.-B. performed most of the biochemical, cell biology, survival, differentiation, and brain anatomopathological experiments. S.P.-G. conducted the stereological analysis, as well as the proliferation and apoptosis studies. S.Y. and S.P.-G. carried out the signaling analysis. C.N. helped with the differentiation studies. S.L.-P. assisted with the MRI analysis. L.G.-L. carried out the behavioral characterization. J.R.B. managed and genotyped the mouse colonies.
We have no relevant conflicts of interest to disclose.