Abstracts

Rationale: Forward genetics screens are a potentially powerful approach to discover, in an unbiased manner, new molecules involved in epilepsy. A novel ethylnitrosourea-induced mouse mutation, Nmf350, causes a low seizure threshold inherited in a dominant manner, and occasional spontaneous convulsions. In this study we mapped the underlying gene and further investigated their phenotype to ultimately explore the mechanisms underlying seizure sensitivity in this new model. Methods: Nmf350 mice were detected by virtue of their low threshold to acute electroshock seizures on the C57BL/6J (B6) inbred strain, which is relatively seizure-resistant. To map the mutation, B6-Nmf350 mice were backcrossed to BALB/cByJ, the electroconvulsive sensitivity of 42 backcross progeny was assessed by the minimal forebrain clonic seizure endpoint at the 3% response level (CC3) for parental strains and a genome scan was done using microsatellite markers. The expression of a candidate gene was examined by RT-PCR and western blotting. Body and brain weight of adult mutant mice was measured to compare with those of wild-type littermates. Adult brains of NMF350 were dissected following 4% PFA fixation. The tissue slides were observed under light microscopy. Results: Segregation analysis of B6-Nmf350 showed that half of progeny obtained by crossing affected animals to wild-type B6 easily reach the minimal forebrain clonic seizure endpoint at CC3, with some mice rapidly progressing to the more severe tonic hindlimb extension endpoint. Occasional sporadic seizures were also observed in B6-Nmf350 mice. Similarly, almost half of all backcross animals were susceptible to electroconvulsion, suggesting that the seizure phenotype of Nmf350 is most likely under the control of a single major gene. A genome scan and subsequent fine mapping implicated a 1 Mb segment of Chr 1 containing four candidate genes: Cep170, Sdccag8, Akt3 and Zfp238, three of which are expressed in the brain. We examined Akt3 first, since knockout mice previously showed that Akt3 is pivotal in postnatal brain development although seizures were not described. Sequencing analysis revealed that Nmf350 mice have a missense mutation in Akt3, leading to nonsynonymous amino acid substitution in a highly conserved segment of the protein kinase domain. Both mRNA and protein levels were normal, as was the phosphorylation of total brain AKT. Unlike the knockout mice, the brain/body weight ratio of Nmf350 and neuron size was normal. The only morphological abnormality similar between Nmf350 and knockout brain was that some Nmf350 mice had agenesis of the corpus callosum. Conclusions: We identified Nmf350 as a missense mutation in a gene encoding Akt3. Transcript and protein levels and overall phosphorylation was unchanged. Unlike known Akt3 knockout mice, neither the reduction of brain size nor obvious morphological dysfunction was observed. Those results suggest that the Akt3-Nmf350 mutation is unique. Further investigation may provide new insight into the normal functions of Akt3 and its potential role in epilepsy.