Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Introduction: Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. Methods: To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). Results: We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Discussion: Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes.

Focal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed an in vitro CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes: LRRC4, EIF3A, TSN, HIP1, PIK3R3, and URI1. All six genes increased phosphorylation of S6. However, only two genes, PIK3R3 and HIP1, caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD.

Epilepsy and sudden unexpected death in epilepsy in a mouse model of human SCN1B-linked developmental and epileptic encephalopathy.

Voltage-gated sodium channel ß1 subunits are essential proteins that regulate excitability. They modulate sodium and potassium currents, function as cell adhesion molecules and regulate gene transcription following regulated intramembrane proteolysis. Biallelic pathogenic variants in SCN1B, encoding ß1, are linked to developmental and epileptic encephalopathy 52, with clinical features overlapping Dravet syndrome. A recessive variant, SCN1B-c.265C>T, predicting SCN1B-p.R89C, was homozygous in two children of a non-consanguineous family. One child was diagnosed with Dravet syndrome, while the other had a milder phenotype. We identified an unrelated biallelic SCN1B-c.265C>T patient with a clinically more severe phenotype than Dravet syndrome. We used CRISPR/Cas9 to knock-in SCN1B-p.R89C to the mouse Scn1b locus (Scn1bR89/C89). We then rederived the line on the C57BL/6J background to allow comparisons between Scn1bR89/R89 and Scn1bC89/C89 littermates with Scn1b+/+ and Scn1b-/- mice, which are congenic on C57BL/6J, to determine whether the SCN1B-c.265C>T variant results in loss-of-function. Scn1bC89/C89 mice have normal body weights and ∼20% premature mortality, compared with severely reduced body weight and 100% mortality in Scn1b-/- mice. ß1-p.R89C polypeptides are expressed in brain at comparable levels to wild type. In heterologous cells, ß1-p.R89C localizes to the plasma membrane and undergoes regulated intramembrane proteolysis similar to wild type. Heterologous expression of ß1-p.R89C results in sodium channel α subunit subtype specific effects on sodium current. mRNA abundance of Scn2a, Scn3a, Scn5a and Scn1b was increased in Scn1bC89/C89 somatosensory cortex, with no changes in Scn1a. In contrast, Scn1b-/- mouse somatosensory cortex is haploinsufficient for Scn1a, suggesting an additive mechanism for the severity of the null model via disrupted regulation of another Dravet syndrome gene. Scn1bC89/C89 mice are more susceptible to hyperthermia-induced seizures at post-natal Day 15 compared with Scn1bR89/R89 littermates. EEG recordings detected epileptic discharges in young adult Scn1bC89/C89 mice that coincided with convulsive seizures and myoclonic jerks. We compared seizure frequency and duration in a subset of adult Scn1bC89/C89 mice that had been exposed to hyperthermia at post-natal Day 15 versus a subset that were not hyperthermia exposed. No differences in spontaneous seizures were detected between groups. For both groups, the spontaneous seizure pattern was diurnal, occurring with higher frequency during the dark cycle. This work suggests that the SCN1B-c.265C>T variant does not result in complete loss-of-function. Scn1bC89/C89 mice more accurately model SCN1B-linked variants with incomplete loss-of-function compared with Scn1b-/- mice, which model complete loss-of-function, and thus add to our understanding of disease mechanisms as well as our ability to develop new therapeutic strategies.