The role of pcdh19 during early embryonic development and its implications in pediatric epilepsy
Developmental Neurobiology
Final Report Abstract
The goal of this research project was to further our understanding of the PCDH19 related disorder by generating a small vertebrate animal model through CRISPR/Cas9 mediated gene editing in order to identify new treatment options for patients. PCDH19 related disorder is characterized by seizures and neuropsychiatric comorbidities in heterozygous females in which X-inactivation results in a cellular mosaicism of wildtype and mutated cells within the brain, while seemingly sparring carrier males suggesting that mosaic PCDH19 expression is required to produce epilepsy. There is still no effective treatment for these patients and the exact function of the PCDH19 protein remains unclear. Since mouse models had only limited success in uncovering mechanisms of epileptogenesis or in proposing novel and innovative therapies, I have proposed to model this disorder in zebrafish because they are very well suited for whole organism, high throughput drug testing. I have planned to introduce various missense or truncating mutations based on the PCDH19 registry into the zebrafish genome but due to technical difficulties of knocking these mutations in and generating stable and mosaic mutants that way, I limited the phenotypic characterization to heterozygous, homozygous and mosaic loss of function mutants for this study. Because pcdh19 is not present on a sex chromosome in zebrafish as it is in humans, I generated knockout (KO) mutants through stable and heritable germline frameshift mutations and mosaic mutants through acute sgRNA injections in the one cell stage which results in cellular mosaicism of WT and mutant cells most closely approximating the mosaic expression that occurs in heterozygous female patients. My work demonstrated that mosaic and non-mosaic pcdh19 mutant zebrafish recapitulate the core feature of PCDH19 disorder, the neuronal hyperexcitability through electrophysiological recordings, that correlates with the network dysfunction resulting in epilepsy in human patients. This finding indicates that Pcdh19 cellular mosaicism is not required for network hyperexcitability and prompts us to start evaluating not only affected heterozygous girls but also carrier males. Furthermore, I provided evidence suggesting that the mechanism for hyperexcitability, at least in non-mosaic Pcdh19 mutants may involve abnormal inhibitory neuron development. I showed reduced density of only inhibitory neurons in the brain of pcdh19 mutant fish together with transcriptional downregulation of key genes involved in the function of the inhibitory synapse. Understanding the molecular basis of brain dysfunction that arises from mosaic and non-mosaic mutant Pcdh19 expression provides a fundamental step towards understanding PCDH19 related disease mechanisms. This study encourages future studies to further dissect the dysfunction of the inhibitory system in the brains of animals with heterozygous pcdh19 mutations and to develop mechanism-based strategies to treat symptoms in humans. Unexpectedly, my zebrafish model did not have a strong seizure phenotype in the high throughput seizure and anxiety assays (using behavioral seizure assays in an unprovoking and provoking setting, calcium imaging and the wall hugging behavioral test), making drug screens, which I was hoping to do impossible. Future studies could focus on drug screens using electrophysiology, however, using this low throughput technique defeats the purpose of using zebrafish and it may be more meaningful to use pcdh19+/- mice instead. Based on my findings, the lack of a robust phenotype and the lack of an X chromosome, I conclude that zebrafish isn’t an adequate animal model to study PCDH19 related disorder. However, zebrafish are still useful to model other (not X-linked and mosaic) monogenic disorders such as Dravet syndrome and other developmental epileptic encephalopathies. Using the assay pipeline and analysis methods that are now fully established in the Poduri lab, allows other scientists in our lab to screen other zebrafish models for seizures to allow gene identification and drug testing and to assess which ones are promising for subsequent studies.
Publications
- Mosaic and non-mosaic pcdh19 mutation leads to neuronal hyperexcitability in zebrafish
Robens, Yang, McGraw, Turner, Robens, Thyme, Rotenberg, Poduri
(See online at https://doi.org/10.1101/2021.09.03.458732)
