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First published online 1 June 2005
doi: 10.1242/dev.01864

Development 132, 2943-2954 (2005)
Published by The Company of Biologists 2005
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Essential role of protein kinase B{gamma} (PKB{gamma}/Akt3) in postnatal brain development but not in glucose homeostasis

Oliver Tschopp1, Zhong-Zhou Yang1, Daniela Brodbeck1, Bettina A. Dummler1, Maja Hemmings-Mieszczak2, Takashi Watanabe3, Thomas Michaelis3, Jens Frahm3 and Brian A. Hemmings1,*

1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
2 Novartis Pharma AG, Lichtstrasse 35, CH-4056, Basel, Switzerland
3 Biomedizinische NMR Forschungs GmbH am Max-Planck-Institut für biophysikalische Chemie, 37070 Göttingen, Germany



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  		Fig. 1. Targeting strategy and confirmation of genotype. (A) The genomic organization of the Pkb{gamma} 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 Pkb{gamma} 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 Pkb{gamma}+/+, Pkb{gamma}+/- and Pkb{gamma}-/- mice are shown. (C) The levels of PKB{gamma} in the brains of Pkb{gamma}+/+, Pkb{gamma}+/- and Pkb{gamma}-/- mice were determined by western blot analysis using a PKB{gamma}-specific antibody. (D) The levels of PKB{alpha} and PKBß, respectively, were determined in brain lysates from Pkb{gamma}+/+, Pkb{gamma}+/- and Pkb{gamma}-/- mice using PKB{alpha}- and PKBß-specific antibodies.

 


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  		Fig. 2. Tissue distribution of PKB{gamma} and levels of PKB{alpha} and PKBß in Pkb{gamma} mutant mice. (A) The mRNA level of PKB{gamma} was determined in 15 different organs from adult Pkb{gamma}+/+ mice. Total RNA was isolated from three adult mice and the levels were normalized to the level of PKB{gamma} in the brain (100%). MG, mammary gland. Error bars represent s.d. mRNA levels of (B) PKB{alpha} and (C) PKBß were determined using total RNA from six different organs of adult Pkb{gamma} wild-type (n=3; black bars) and mutant mice (n=3; white bars). mRNA levels of PKB{alpha} and PKBß were normalized to the level of PKB{alpha} in wild-type brain (100%). Error bars represent s.d. (D) Western blot analysis of PKB{alpha}, ß and {gamma} within ten different brain regions using isoform specific antibodies.

 


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  		Fig. 3. Phospho-western blot analysis of brains from Pkb{gamma} 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.

 


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  		Fig. 4. Dispensable role of PKB{gamma} for body weight and glucose metabolism. (A) Body weights of male Pkb{gamma}+/+ (white bars) and Pkb{gamma}-/- (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; Pkb{gamma}+/+ white bars and Pkb{gamma}-/- black bars). Error bars represent s.d. (C) Insulin tolerance test. Animals (n=6; 6 months old; Pkb{gamma}+/+ white triangles and Pkb{gamma}-/- 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; Pkb{gamma}+/+ white triangles and Pkb{gamma}-/- 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.

 


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  		Fig. 5. Reduced brain weight and size of Pkb{gamma} mutant mice. (A) Weight of freshly dissected brains of male Pkb{gamma}+/+ (white bars) and Pkb{gamma}-/- (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 Pkb{gamma} 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 Pkb{gamma}+/+ (E,G,I) and Pkb{gamma}-/- mice (F,H,J) from Pkb{gamma}+/- 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.

 


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  		Fig. 6. Histology of brains from Pkb{gamma} mutant mice. Representative sections (HE staining) of the cortex (A,D), hippocampus (B,E) and cerebellum (C,F) from adult Pkb{gamma}+/+ (A-C) and Pkb{gamma}-/- (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 Pkb{gamma}+/+ (G-I) and Pkb{gamma}-/- mice (J-L) in coronal orientation. White matter structures are labelled as follows: CC, corpus callosum; AC, anterior commissure.

 


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  		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) PKB{alpha}, PKBß and PKB{gamma} expression in Pkb{gamma} 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 (Pkb{gamma}+/+ white bars and Pkb{gamma}-/- 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.

 

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© The Company of Biologists Ltd 2005