Neurobiology of PCDH19-female epilepsy

Abstract

PCDH19-female epilepsy (PCDH19-FE) is a female-limited epilepsy characterised by a spectrum of neurodevelopmental and behavioural problems. It is caused by predicted, loss of function mutations in an X-chromosome gene, Protocadherin 19 (PCDH19). PCDH19-FE presents because of cellular mosaicism, typically in females where heterozygous mutations cause mosaicism resulting from random X-inactivation. Currently, limited cell and molecular biology data is available to explain how mutations in PCDH19 cause this debilitating disorder. PCDH19 shows spatially and temporally regulated expression in the developing and adult brain, with layer-specific expression in the cortex and expression in the neurogenic regions of the developing brain. Thus, PCDH19 is likely to have important functions in both the developing and adult cortex. To investigate the role of PCDH19 in cortical brain development, both mouse and human based in vitro models were developed and employed. Utilising wildtype (WT) and Pcdh19 knockout (KO) mice, neural stem and progenitor cells (NSPCs) were isolated and cultured as neurospheres to investigate NSPCs behaviours and the cellular mosaicism of the PCDH19-FE individuals. In the absence of Pcdh19, increased neuronal migration was observed. This was associated with increased neuronal differentiation at the expense of oligodendrocyte differentiation. The mosaic cultures showed a phenotype intermediate to the Pcdh19 WT and Pcdh19 KO cells in all assays, suggesting cell intrinsic properties were maintained. Genome-wide expression analysis implicated multiple genes and gene networks involved in neuronal development. In particular, genes involved in the regulation of the actin cytoskeleton via Rho GTPases were highlighted, a pathway which could underlie the cellular phenotypes observed. Human induced pluripotent stem cells (hiPSC) provide a unique system to study neurological disorders using disease relevant cells that are otherwise unattainable from the patients. hiPSCs were generated from patient skin fibroblasts with a pathogenic PCDH19 missense mutation. Additionally, an optimised in vitro protocol of human cortical development was developed and shown to be reproducible across multiple pluripotent stem cell lines. Using this protocol with WT and PCDH19 Mutant hiPSCs, PCDH19-FE was modelled by again replicating the cellular mosaicism of the patient brain. PCDH19 was found to be important for the maintenance of NSPC polarity during cortical development, with PCDH19 Mutants being able to form neural rosette structures, but unable to properly maintain these structures as evidenced by a decrease in lumen size and number of polarised neural rosette structures/rosette colony area. A significant increase in the number of neurons at the edge of the rosette colonies was also observed suggesting premature neuronal differentiation. PCDH19 was also shown to regulate axonal extensions with PCDH19 Mutant and Mosaic neurons having an increased primary neurite length. Taken together this study has shown that PCDH19 has important functional roles during the early stages of cortical brain development. This work identifies novel roles for PCDH19 in NSPC polarity, neurogenesis, neuronal migration and neuronal morphology. This study suggests that the PCDH19-FE pathology is attributed to the presence of two differing cell populations (WT and Mutant/KO) resulting in abnormal brain development and neuronal network formation at later stages of cortical development.