The proteolytic processing and nuclear function of Protocadherin-19

Mutations in the X-linked gene PCDH19 lead to epilepsy with cognitive impairment in heterozygous females and post-zygotic mosaic males. The disorder phenotype is currently explained by cellular mosaicism of PCDH19 expressing and non-expressing PCDH19 cells in the brain, which leads to defective cell-cell communication and circuits. Although the gene codes for a cell adhesion protein belonging the cadherin superfamily localized at the cell membrane, recent reports have implicated PCDH19 in the regulation of gene expression and have identified the protein in the nucleus. Despite this, the nuclear function of PCDH19 in neurons and the potential proteolytic processing of PCDH19 have not been investigated yet. This thesis focussed on the proteolytic processing of PCDH19 as a potential mechanism of membrane-to-nucleus signalling in neurons. mESC-derived neurons were used to test the involvement of different proteases in the processing of PCDH19 and it was established that PCDH19 can undergo activity-dependent proteolysis. As the cytoplasmic domain of PCDH19 was found to localise to the nucleus, the nuclear function of the generated fragment was investigated. To determine potential transcriptional targets of the PCDH19 cytoplasmic domain, a mouse embryonic stem cell line that overexpresses the cytoplasmic domain of PCDH19 from the Rosa26 locus (PCDH19-CYTO), and an isogenic PCDH19-knockout line (PCDH19-KO) were generated. The transcriptional profile of embryonic stem cell-derived progenitors and neurons was obtained via RNA sequencing. The results of the analysis suggest a role for the cytoplasmic domain of PCDH19 in modulating expression of genes related to neuronal circuit assembly and synaptic function. Further analysis will be necessary to determine the relevance of these findings in the context of PCDH19-epilepsy

Cellular and behavioral characterization of Pcdh19 mutant mice: subtle molecular changes, increased exploratory behavior and an impact of social environment

Mutations in the X-linked cell adhesion protein PCDH19 lead to seizures, cognitive impairment, and other behavioral comorbidities when present in a mosaic pattern. Neither the molecular mechanisms underpinning this disorder nor the function of PCDH19 itself are well understood. By combining RNA in situ hybridization with immunohistochemistry and analyzing single-cell RNA sequencing datasets, we reveal Pcdh19 expression in cortical interneurons and provide a first account of the subtypes of neurons expressing Pcdh19/PCDH19, both in the mouse and the human cortex. Our quantitative analysis of the Pcdh19 mutant mouse exposes subtle changes in cortical layer composition, with no major alterations of the main axonal tracts. In addition, Pcdh19 mutant animals, particularly females, display preweaning behavioral changes, including reduced anxiety and increased exploratory behavior. Importantly, our experiments also reveal an effect of the social environment on the behavior of wild-type littermates of Pcdh19 mutant mice, which show alterations when compared with wild-type animals not housed with mutants