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Figures
- Fig. 1.
Targeting strategy and confirmation of genotype. (A) The genomic organization of the Pkbγ wild-type allele (top) was disrupted using a targeting vector with an IRES-lacZ-Neo-cassette (middle). Targeting of the wild-type allele leads to disruption of exon 4 of the Pkbγ gene (bottom). Arrowheads indicate the localization of the primers for the PCR reaction. (B) The genotype of mice was determined by PCR. Representative results from Pkbγ+/+, Pkbγ+/- and Pkbγ-/- mice are shown. (C) The levels of PKBγ in the brains of Pkbγ+/+, Pkbγ+/- and Pkbγ-/- mice were determined by western blot analysis using a PKBγ-specific antibody. (D) The levels of PKBα and PKBβ, respectively, were determined in brain lysates from Pkbγ+/+, Pkbγ+/- and Pkbγ-/- mice using PKBα- and PKBβ-specific antibodies.
- Fig. 2.
Tissue distribution of PKBγ and levels of PKBα and PKBβ in Pkbγ mutant mice. (A) The mRNA level of PKBγ was determined in 15 different organs from adult Pkbγ+/+ mice. Total RNA was isolated from three adult mice and the levels were normalized to the level of PKBγ in the brain (100%). MG, mammary gland. Error bars represent s.d. mRNA levels of (B) PKBα and (C) PKBβ were determined using total RNA from six different organs of adult Pkbγ wild-type (n=3; black bars) and mutant mice (n=3; white bars). mRNA levels of PKBα and PKBβ were normalized to the level of PKBα in wild-type brain (100%). Error bars represent s.d. (D) Western blot analysis of PKBα, β and γ within ten different brain regions using isoform specific antibodies.
- Fig. 3.
Phospho-western blot analysis of brains from Pkbγ mutant mice. (A) Brains of three wild-type and three mutant mice were analysed for phosphorylation status of proteins involved in PKB signalling. p, indicates phosphorylated protein. (B) Western blot quantification. Levels of p-Ser473 and p27 were normalized to the level of actin, all phosphorylated proteins were normalized to the level of unphosphorylated protein. *P<0.05. Error bars represent s.d.
- Fig. 4.
Dispensable role of PKBγ for body weight and glucose metabolism. (A) Body weights of male Pkbγ+/+ (white bars) and Pkbγ-/- (black bars) mice were measured at different points in time (n=5-8 animals per genotype). Error bars represent s.d. NB, newborn. (B) Blood glucose concentrations from random-fed (RF) and overnight fasted (ONF) mice (n=8; 6 months old; Pkbγ+/+ white bars and Pkbγ-/- black bars). Error bars represent s.d. (C) Insulin tolerance test. Animals (n=6; 6 months old; Pkbγ+/+ white triangles and Pkbγ-/- black circles) were fasted overnight and insulin (1 U/kg) was administered by intraperitoneal injection. (C) Glucose concentrations were determined at indicated time points from whole blood collected from tail veins. Values were normalized to the starting glucose concentration at the administration of insulin. Error bars represent s.d. (D) Glucose tolerance test. Animals (n=6; 5-6 months old; Pkbγ+/+ white triangles and Pkbγ-/- black circles) were fasted overnight and glucose (2 g/kg) was orally administered. Blood glucose concentrations were sampled at the indicated times. Values were normalized to the starting glucose concentration at the administration of glucose. Error bars represent s.d.
- Fig. 5.
Reduced brain weight and size of Pkbγ mutant mice. (A) Weight of freshly dissected brains of male Pkbγ+/+ (white bars) and Pkbγ-/- (black bars) mice were measured at different points in time (n=5-8 animals per genotype). *P<0.05. Error bars represent s.d. NB, newborn. (B) Cranial, (C) caudal and (D) lateral views of brains from adult Pkbγ wild-type (left side) and knockout (right side) mice. Co, cortex; CE, cerebellum; OB, olfactory bulb; BS, brainstem; HY, hypothalamus. (E-J) Representative sections from T2- weighted 3D MRI data sets acquired in vivo from the brains of Pkbγ+/+ (E,G,I) and Pkbγ-/- mice (F,H,J) from Pkbγ+/- matings in sagittal (E,F), horizontal (G,H) and coronal (I,J) orientation. CC, corpus callosum; AC, anterior commissure; PC, posterior commissure. Scale bar: 1 mm.
- Fig. 6.
Histology of brains from Pkbγ mutant mice. Representative sections (HE staining) of the cortex (A,D), hippocampus (B,E) and cerebellum (C,F) from adult Pkbγ+/+ (A-C) and Pkbγ-/- (D-F) mice in parasagittal orientation. Representative sections stained for myelin (Luxol-Fast Blue/Eosin) of whole brain (G,J), corpus callosum (H,K) and anterior commissure (I,L) from Pkbγ+/+ (G-I) and Pkbγ-/- mice (J-L) in coronal orientation. White matter structures are labelled as follows: CC, corpus callosum; AC, anterior commissure.
- Fig. 7.
Increased susceptibility to glutamate and staurosporine induced cell death. (A-D) Primary hippocampal neurons were established from E16.5 embryos and kept in culture for 28 days. Immunocytochemistry was performed using antibodies against dendritic (Map2C) or axonal (tau) proteins. (E) PKBα, PKBβ and PKBγ expression in Pkbγ wild-type and mutant hippocampal neurons. (F) Seven-day-old cultures were treated with glutamate (15 mM/24 hours) or with staurosporine (50 nM/12 hours) or were left untreated (Pkbγ+/+ white bars and Pkbγ-/- black bars). Apoptotic cells were identified by TUNEL-assay and at least 200 cells per culture were counted (n=5 cultures per treatment and genotype). *P<0.05. Error bars represent s.d.
Tables
- Table 1.
Cell number in the brain of Pkbγwild-type and mutant mice
Age Newborns One month old Pkbγ+/+ Pkbγ−/− Pkbγ+/+ Pkbγ−/− Body weight (g) 1.21±0.23 1.31±0.14 (107%) 14.1±2.5 13.0±2.3 (92%) Brain weight (g) 0.071±0.001 0.069±0.006 (98%) 0.41±0.03 0.32±0.03 (78%)* Brain/body weight ratio 0.059±0.011 0.054±0.008 (90%) 0.030±0.004 0.025±0.003 (85%)* DNA/brain (mg) 0.59±0.04 0.56±0.12 (96%) 1.38±0.03 1.29±0.07 (93%)* DNA/g of tissue (mg) 8.53±1.64 8.11±1.29 (95%) 3.38±0.20 4.04±0.47 (119%)* -
The cell number in whole brains of Pkbγ+/+ and Pkbγ−/− mice was determined in newborns and 1-month-old animals. Brain weight, brain/body weight and DNA content (parameter of cell number) per brain were significantly reduced in Pkbγ−/− mice at 1 month, but not in newborns. The DNA content per gram of tissue, a parameter of cell density, was significantly increased in Pkbγ mutant mice at 1 month, but not in newborns (*P<0.05).
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- Table 2.
Microarray analysis of Pkbγ mutant brain
Fold change in expression Gene name GenBank Accession Number Day 1 Day 7 Day 30 Function RIO kinase 3 (yeast) AK004748 5.47* 0.59 1.52* Chromatin condensation Kinesin family member 5B BF099632 2.24* 0.72 1.03 Microtubule based transport Small EDRK-rich factor 1 AA709993 1.69* 1.14 1.06 CD24a antigen BB560574 1.57* 1.33 1.12 Cell proliferation SRp20, splicing factor, arginine/serine-rich 3 AV135383 1.53* 1.32 0.91 mRNA splicing WNK1 (protein kinase, lysine deficient 1) BI692255 1.53* 1.00 0.93 Blood pressure ATPase, Na+/K+ transporting, alpha 2 polypeptide BQ175915 2.23* 2.93* 1.71* Nt uptake, K+ uptake SRY-box containing gene 11 BG072739 0.96 1.82* 0.95 Transcription Histone 1, H2af W91024 1.04 1.60* 1.16 Nucleosome structure Upstream transcription factor 1 AF479773 1.53* 1.55* 1.45 Transcription Early B-cell factor 3 AK014058 1.13 1.54* 0.76 Transcription RNA-binding region (RNP1, RRM) containing 2 BB436856 1.40 1.53* 1.46 Transcription CDC28 protein kinase regulatory subunit 2 NM_025415 1.09 1.53* 1.20 Cell cycle Procollagen, type III, alpha 1 BG968894 1.08 1.09 2.10* Cell adhesion Surfeit gene 4 AI788623 1.20 1.23 1.83* Membrane protein GABAA receptor, subunit alpha 6 NM_008068 1.06 0.57* 0.92 Synaptic transmission ATPase, Na+/K+ transporting, alpha 2 polypeptide AI845177 0.72 0.66* 0.56* Nt uptake, K+ uptake Kinesin family member 5A NM_008447 1.04 0.80 0.53* Microtubule based transport ADP-ribosylation factor 3 NM_007478 1.05 0.80 0.56* Protein transport Contactin associated protein 1 NM_016782 0.96 0.96 0.57* Cell adhesion Fibroblast growth factor 1 BM932451 0.86 0.95 0.59* Angiogenesis, cell proliferation Growth arrest specific 7 NM_008088 0.89 0.89 0.59* Neurite outgrowth Ca2+/calmodulin-dependent protein kinase II alpha X14836 1.04 0.69 0.59* Cell cycle, synaptic plasticity K+/Cl− co-transporter (KCC2/SLC12A5) AF332064 1.13 0.90 0.62* Synaptic transmission Glutamate receptor, ionotropic, NMDA1 (ζ1) NM_008169 0.96 0.80 0.63* Synaptic transmission Synaptotagmin 2 AF257304 1.02 1.00 0.64* Synaptic transmission Fascin homolog 1, actin bundling protein BE952057 1.06 0.86 0.64* Actin binding Potassium voltage-gated channel, beta member 2 L48983 0.87 0.86 0.64* Ion transport Protein tyrosine phosphatase, receptor type, F NM_011213 0.98 1.03 0.65* Cell adhesion Coronin, actin binding protein 1C AW548837 0.97 0.94 0.65* Actin assembly Chapsyn-110 [discs, large homolog 2 (DLG2)] BB622767 1.29 0.80 0.65* Synaptic transmission -
Genes with altered expression in male Pkbγ−/− mice (n=3) compared with Pkbγ+/+ animals at different time points during postnatal brain development.
Nt, neurotransmitter.
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↵* Significant changes (1.5 fold).
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