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. 2011 Mar 1;20(5):988-99.
doi: 10.1093/hmg/ddq544. Epub 2010 Dec 15.

A novel Akt3 mutation associated with enhanced kinase activity and seizure susceptibility in mice

Satoko Tokuda  1 , Connie L Mahaffey, Bobby Monks, Christian R Faulkner, Morris J Birnbaum, Steve C Danzer, Wayne N Frankel
Affiliations

A novel Akt3 mutation associated with enhanced kinase activity and seizure susceptibility in mice

Satoko Tokuda et al. Hum Mol Genet. .

Abstract

In a phenotype-driven mutagenesis screen, a novel, dominant mouse mutation, Nmf350, caused low seizure threshold, sporadic tonic-clonic seizures, brain enlargement and ectopic neurons in the dentate hilus and molecular layer of the hippocampus. Genetic mapping implicated Akt3, one of four candidates within the critical interval. Sequencing analysis revealed that mutants have a missense mutation in Akt3 (encoding one of three AKT/protein kinase B molecules), leading to a non-synonymous amino acid substitution in the highly conserved protein kinase domain. Previous knockout studies showed that Akt3 is pivotal in postnatal brain development, including a smaller brain, although seizures were not observed. In contrast to Akt3(Nmf350), we find that Akt3 null mice exhibit an elevated seizure threshold. An in vitro kinase assay revealed that Akt3(Nmf350) confers higher enzymatic activity, suggesting that Akt3(Nmf350) might enhance AKT signaling in the brain. In the dentate gyrus of Akt3(Nmf350) homozygotes, we also observed a modest increase in immunoreactivity of phosphorylated ribosomal protein S6, an AKT pathway downstream target. Together these findings suggest that Akt3(Nmf350) confers an increase of AKT3 activity in specific neuronal populations in the brain, and a unique dominant phenotype. Akt3(Nmf350) mice provide a new tool for studying physiological roles of AKT signaling in the brain, and potentially novel mechanisms for epilepsy.

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Figures

Figure 1.
Figure 1.
A single nucleotide substitution in Akt3. (A) Map position of Nmf350 on chromosome (Chr) 1. N2 (n = 58) mice were genotyped with the indicated SSLP markers and phenotyped by ECT tests. The highest linkage was obtained between D1Frk9 and D1Mit115 with a peak LOD score of 14.929, and the critical interval was refined further by progeny testing. The panel below the LOD graph shows genotypes of informative N2 recombinants (I–III) between D1Frk9 and D1Mit115, which were used for progeny testing: gray, B6J and BALB heterozygous; white, BALB homozygous. The number in parenthesis indicates the number of N3 progeny inheriting the same genotype and showing the same phenotype as their N2 parents. (B) Physical map of the Nmf350 locus. The Nmf350 locus corresponded to a 1 Mb segment on the distal Chr 1, containing four candidate genes. (C) The A to T substitution found in exon 8 in the Akt3 gene by genomic sequencing.
Figure 2.
Figure 2.
The missense mutation found in Nmf350 mice is located in the protein kinase domain of AKT3. (A) Schematic figure of the protein structure of AKT3. AKT3 has two phosphorylation sites at Threonine 305 and Serine 472. The mutation resulted in a non-synonymous amino acid substitution from aspartic acid (Asp) to valine (Val) at the amino acid position of 219 in the protein kinase domain. PH, pleckstrin homology domain. (B) Amino acid sequence alignment among protein kinase family. The amino acid at the Nmf350 mutation is highly conserved among protein kinase family and various species. Arrow indicates an amino acid position 219 of mouse AKT3. (C) No gross change of AKT3 protein levels in the brain from Nmf350 mice. Brains from two wild-type (+/+), Nmf350 heterozygote (mt/+) and homozygote (mt/mt), were examined for the expression of AKT3 (60–70 kDa) by western blot analysis. The brain from Akt3 knockout (KO) mouse was used for a negative control.
Figure 3.
Figure 3.
ECT for generalized, tonic–clonic seizures in Nmf350, Akt3 null and congenic control mice. ECT was measured in B6J-Akt3Nmf350,B6J.129-Akt3tm1Mbb (null) mice and B6J.129-Fcrg2btm1Rav congenic control adult mice (age 6–13 weeks). The mean ECT (±1SD) of Akt3Nmf350 homozygous (hom) and heterozygous (het) mice was significantly lower than that of wild-type (wt) animals, while Akt3 null homozygotes showed higher mean ECT than wild-type littermates. The mean ECT of Akt3 null heterozygotes was slightly lower than in the wild-type, but this same effect is also observed in the congenic control mice which also carry 129 strain-derived alleles in the interval. **P < 0.01, *P < 0.01 Tukey HSD test.
Figure 4.
Figure 4.
Increased brain size and ectopic hippocampal neurons in Akt3Nmf350 mice. (A) Brains were dissected from 35-week-old wild-type (WT) and Akt3Nmf350 homozygous (Nmf350) mice (n = 3). The mutant brain was clearly larger than that of the wild-type. (B) Hematoxylin and eosin (H&E) staining (top) or immunofluorescence for DCX (red) of sagittal brain sections derived from wild-type (WT) and Akt3Nmf350 homozygous (Nmf350) mice also suggested that the hippocampus was larger. (C) Confocal maximum projections of DCX (red) immunostaining in the hippocampus of 12-week-old wild-type and Nmf350 mice are shown in the top row. Images are from the dentate hilus (H; boxed region in inset). Hilar ectopic granule cells were rare in wild-type animals and common in Nmf350 mice (blue arrows). The middle and bottom rows show confocal projections of DCX (red) and KI67 (green) immunostaining from a younger, 6-week-old, Nmf350 mouse. In this animal, DCX immunoreactive hilar ectopic cells were more numerous relative to older animals (middle row, blue arrows) and proliferating KI67-positive cells were present in the hilus (light blue arrowhead). Ectopic granule cells (blue arrows) were also evident in the dentate molecular layer (ml, see inset for orientation). Scale bar = 50 μm.
Figure 5.
Figure 5.
Western blot analysis of AKT signaling in brains from Akt3Nmf350 mice. The phosphorylation status of proteins involved in AKT signaling was investigated in cerebrum from adult wild-type mice (+/+) and Akt3Nmf350 heterozygous (mt/+) and homozygous (mt/mt) mutants (n = 2 in each group). No gross change was observed in Akt3Nmf350 mice. p- indicates phosphorylated protein.
Figure 6.
Figure 6.
Immunoprecipitation kinase assay with recombinant AKT3 and mutant AKT3 (D219V). HA-tagged wild-type AKT3 or mutant AKT3 (D219V) protein was transiently expressed in HEK293 cells and purified with the HA antibody for the kinase assay. The recombinant GSK3 protein was used as a substrate. (A) Representative results from five separate in vitro kinase assays. The amount of both phosphorylated substrates and existent HA-tagged AKT3 proteins were measured by western blot analysis. Arrow indicates the specific band of p-GSK3α/β at 27 kDa. (B) The mean kinase activity of wt AKT3 and mt AKT3. Kinase activity was assessed as a relative band intensity of p-GSK to HA and compared between wt and mt AKT3 by a paired t-test (wt Akt3, 0.63 ± 0.18; mt Akt3, 1.15 ± 0.52, n = 5 in each group, P = 0.0410).
Figure 7.
Figure 7.
Increased expression of phosphorylated S6 protein in the Akt3Nmf350 hippocampus. Sections of adult wild-type (wt) and Akt3Nmf350 homozygous (Nmf350) mice were immunostained with the antibodies against phospho-S6 and NeuN. Elevated levels of phosphorylated S6 were seen in the dentate gyrus of Nmf350 mice when compared with wild-type animals (A). Those cells were also NeuN positive. Phospho-S6-positive neurons were occasionally or broadly seen in the CA1 (B) or cerebral cortex (C) area in both Akt3Nmf350 and wild-type animals with the same level, respectively. Scale bars indicate 50 μm.
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References

    1. Gardiner R.M. Impact of our understanding of the genetic aetiology of epilepsy. J. Neurol. 2000;247:327–334. doi:10.1007/s004150050598. - DOI - PubMed
    1. Lucarini N., Verrotti A., Napolioni V., Bosco G., Curatolo P. Genetic polymorphisms and idiopathic generalized epilepsies. Pediatr. Neurol. 2007;37:157–164. doi:10.1016/j.pediatrneurol.2007.06.001. - DOI - PubMed
    1. Buchner D.A., Seburn K.L., Frankel W.N., Meisler M.H. Three ENU-induced neurological mutations in the pore loop of sodium channel Scn8a (Na(v)1.6) and a genetically linked retinal mutation, rd13. Mamm. Genome. 2004;15:344–351. doi:10.1007/s00335-004-2332-1. - DOI - PubMed
    1. Jablonski M.M., Dalke C., Wang X., Lu L., Manly K.F., Pretsch W., Favor J., Pardue M.T., Rinchik E.M., Williams R.W., et al. An ENU-induced mutation in Rs1h causes disruption of retinal structure and function. Mol. Vis. 2005;11:569–581. - PubMed
    1. Keays D.A., Tian G., Poirier K., Huang G.J., Siebold C., Cleak J., Oliver P.L., Fray M., Harvey R.J., Molnar Z., et al. Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans. Cell. 2007;128:45–57. doi:10.1016/j.cell.2006.12.017. - DOI - PMC - PubMed

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