Genetic alterations in cortical development as a cause of epileptogenic disorders

Resum

ABSTRACT During embryogenesis, the developing cerebral cortex undergoes a dramatic expansion and folding. Alterations in cortical folding cause intellectual impairment and epileptogenic disorders, demonstrating its significant functional relevance. Some of the most common pathologies are polymicrogyria, the formation of multiple small folds; and periventricular nodular heterotopia (PVNH), where neurons accumulate ectopically in the vicinity of the telencephalic ventricles forming nodules, which act as epileptic foci. Mutations in PIK3R2 and FLNA are found causative for these pathologies, respectively. A novel gain-of-function mutation was identified in PIK3R2, a gene coding for a regulatory subunit in the (PI3K)-AKT-mTOR pathway, causing bilateral polymicrogyria in patients. To understand the cellular mechanisms underlying this pathology, we performed functional experiments in developing mice and ferrets by overexpressing PIK3R2 with a mutation found in patients, or its wild-type counterpart. We found that overexpression of PIK3R2 in mouse embryos by in utero electroporation caused an increase in proliferation of Radial Glia (RGCs) and Intermediate Progenitor Cells (IPCs), leading to their greater self-amplification. Migration of cortical neurons was also impaired, resulting in their accumulation of cells in deep cortical layers at embryonic and postnatal stages. Similarly, in ferret visual cortex the overexpression of Pik3r2 causes a delay in neuronal migration, a defect observable in long-term experiments in which juvenile animals showed an increase in cells populating cortical layers V and VI with respect to controls. In contrast, when the manipulation was performed in the parietal cortex, we detect a greater neurogenesis and an ectopic positioning of neurons at embryonic stages, which was consolidated in juvenile ferrets, presenting neuronal periventricular heterotopias and an overmigration defect altering cortical surface. Our findings demonstrate that increased levels of PIK3R2 alters progenitor cell proliferation and neuronal migration during cortical development in an area-specific manner, which suggests a different robustness of cortical areas. Periventricular nodular heterotopia (PVNH) has been associated to mutations in the FLNA gene. Here, we analyzed the expression pattern of FlnA in cortical development of mouse and ferret, and we observed the highest levels of expression in germinal layers in both animal models. Importantly, FlnA expression in ferret was heterogeneous along the Outer Subventricular Zone (OSVZ), being higher in the prospective gyri compared to sulci. Next, we performed overexpression and knock down experiments by in utero electroporation of full length FlnA and FlnA-shRNA, respectively, in mouse embryos. Overexpression of FlnA causes a delay in neuronal migration at embryonic stages. Additionally, this retention is maintained postnatally (P21), when we can find accumulation of neurons resembling periventricular nodules. Upon FlnA knock down, we find that those neurons that finally migrate fail to acquire their proper laminar position, ending misplaced in superficial positions. An alteration in neuronal migration was observed also in postnatal ferrets electroporated with FlnA knock down, but not in overexpression conditions. However, overexpression of FlnA causes a disruption of the apical lamina resulting in premature cell delamination or death, although this do not cause the formation of periventricular nodules. Finally, we analyzed the expression pattern of a gene involved in subcortical band heterotopia, Eml1, during ferret cortical development. We observed that it is expressed in germinal layers and in the cortical plate, but in contrast to the other gene that we have analyzed, the highest expression in the germinal layers starts later in development, contrary to what has been previously observed in mouse. Overexpression experiments performed in ferret visual cortex showed a slight neuronal migration defect at early postnatal stages that was self-corrected later during development.