Mechanisms of epileptogenesis in animal models of developmental brain lesions

Abstract

Developmental brain lesions, including glioneuronal tumors (GNT) and malformations of cortical development (MCD), are among the most common causes for pharmaco-resistant epilepsy. Despite substantial differences, their shared major hallmark are dysplastic neurons characterized by abnormal dendritic arborization. However, the exact molecular mechanisms underlying dysplastic neurons and epileptogenesis in these devastating disorders are still largely unknown. This lack in our knowledge significantly hinders the development of novel therapeutic approaches. Therefore, the goal of this study was to gain more insight into various aspects of the pathogenesis of tuberous sclerosis (TSC), focal cortical dysplasia type IIb (FCDIIb) and gangliogliomas (GG) with an emphasis on the mechanisms underlying the emergence of dysplastic neurons. We generated a mouse model for developmental brain tumors by embryonic, focal p53 knockout as well as BRAF-V600E and Akt expression. Our first finding is that a compromised progenitor cell population is sufficient to induce GG resembling tumors harboring dysplastic neurons. As of yet, there were no GG animal models available, thus our mouse model provides an excellent basis for future in depth analysis of these complex tumors as well as potential drug testing. In a second experimental setup we analyzed the so far unclear pathogenetic mechanisms underlying the formation of cortical tubers, a hallmark of TSC. These are composed of large dysplastic neurons and giant cells that are histomorphologically virtually indistinguishable from abnormal cells found in FCDIIb lesions. We found that developmental expression of TSC-associated mutations, but not the FCDIIb-associated allelic variants, of the tuberous sclerosis complex1 (TSC1) gene results in the formation of dysplastic neurons in mice. Respective TSC1-silenced cortical neurons expressing the mutated TSC1 gene product hamartinR692X or hamartinR786X showed cellular features reminiscent of dysplastic neurons, stressing their pathogenic potential. Intraventricular in utero electroporation (IUE) of TSC1-R692X and varying concentrations of wildtype TSC1 revealed that already minimal amounts of functional hamartin are sufficient to prevent the emergence of dysplastic neurons from tuber-like lesions. This strong rescuing capacity stresses substantial silencing of both TSC1 alleles as critical pathogenetic mechanism for the manifestation of cortical tubers and provides a promising basis for gene therapy approaches. <br /> Our next finding suggests loss of the cytoskeleton regulating protein Ste20 like kinase (SLK) as a common pathological event in the emergence of dysplastic neurons across distinct glioneuronal lesions. We find reduced SLK protein levels in dysplastic neurons of highly epileptogenic FCDIIb and GGs. Accordingly, shRNA mediated SLK silencing in developing cortical neurons results in impaired neurite growth and arborization of higher order dendrites together with a progressive and selective loss of inhibitory synapses in mice older than 15 days. A functional impairment of neuronal inhibition was reflected by a higher propensity for chemically evoked epileptic seizures. These results indicate SLK loss as a key factor underlying the development of dysplastic neurons and, hence, as a contributor to the emergence of hyperexcitability in epileptogenic brain lesions. Our data suggest, that the SLK mechanistic pathway is in part regulated by the upstream LIM-domain binding (LDB) proteins, transcriptional co-activators with reduced expression in the abnormal neuronal component of GGs. LDB1 and LDB2 knockdown in developing mouse neurons results in a reduction of neuronal arbor morphology, reminiscent of dysplastic neurons, that is rescued by SLK overexpression but not vice versa. <br /> The present study provides novel insights into the mechanisms of epileptogenesis and the emergence of dysplastic neurons in glioneuronal brain lesions, setting the stage for the development of targeted treatment methods and drug testing.