The megencephaly mouse : From gene to neuronal proliferation

Abstract: Megalencephaly, enlarged brain, can be associated with a wide range of symptoms from benign to severe. Severe cases are often associated with mental retardation and seizures. The megencephaly mouse shows a dramatic, progressive increase in size of the hippocampus and ventral cortex. The enlargement is colocalized with expression disturbances of molecules in the insulin-like growth factor system and several neuropeptides. The mceph mutation has previously been mapped to a 3 centi-Morgan interval on distal chromosome 6. The aims of this thesis was to identify the mceph mutation, perform functional studies of the MCEPH protein and to characterize the brain enlargement. Using a positional cloning approach the mceph mutation was identified as an 11 bp deletion in the Shaker-like voltage gated potassium channel Kv1.1. The deletion leads to a frame shift and a premature stop codon. The predicted truncated protein would retain only 230 out of 495 amino acids. Kv1.1 mRNA was upregulated in the brain of mceph/mceph mice. Expression patterns of Kv1.2 and Kv1.3 was disturbed in the hippocampus suggesting an interaction between these proteins and the putative MCEPH protein. The presence of abnormal electrical activity suggests that the mceph/mceph mice are epileptic. Low levels of the truncated MCEPH protein is expressed in the brain of mceph/mceph mice, mainly in the hippocampus and ventral cortex. Glycosylation analysis suggests that MCEPH is retained in the endoplasmic reticulum. Studies in cell culture shows that MCEPH has the ability to assemble with full length Kv 1.1. In addition, it has a dominant negative effect on Kv1.2 and Kv1.3 currents in Xenopus oocytes. However, no interaction could be detected with Kv1.2 in the brain. Compositional analysis had suggested that the underlying cause of the brain enlargement in the mceph/mceph mice was hypertrophy rather than hyperplasia. By applying the optical fractionator method and the Cavalieri principle, the volume and total number of neurons, and astrocytes in the hippocampus of 12 weeks old mceph/mceph and wild type mice was estimated. The number of both neurons (expressing NeuN) and astrocytes (expressing GFAP), as well as structure volume, were increased approximately 2-fold within dentate gyrus, CA3 and hilus of mceph/mceph versus wild type mice. As a first step towards understanding the mechanisms underlying the hyperplasia, cell proliferation was studied within the subgranular zone of the dentate gyrus using BrdU. At three weeks of age, before onset of epileptic symptoms, there was a 3-fold increase in proliferation in mceph/mceph mice compared to control mice. In the severely epileptic 9 weeks old mceph/mceph mice there was a less dramatic increase in proliferation, reaching 1.5-fold more BrdU labeled cells in relation to wild type mice. The Kv1.1 null mouse is epileptic but has not been reported to have an enlarged brain. Since the mceph trait showed a reduced penetrance in intercrosses, genetic background was suspected to be important. To minimize the differences between mceph/mceph and the Kv 1.1 null mice, the Kv 1.1 null alleles were transferred to BAL13/c13y.J. At this genetic background, lack of Kv1.1 was found to induce excessive growth of the hippocampus and the ventral cortex. In summary, the enlarged mceph/mceph hippocampus has an increased rate of proliferation and more neurons as well as astrocytes. The causative mutation is an 11 bp deletion in the gene encoding Kv1. 1. It results in a truncated protein that is functional in vitro. However, lack of functional Kv1.1 in combination with a BAL13/c13y.J genetic background is sufficient for the excessive growth of hippocampus and ventral cortex.