Seizures, increased interhemispheric synchrony, altered brain transcriptomics and a leaky blood-brain barrier result from loss of ap3b2 in a CRISPR tadpole model of DEE48

Abstract

Loss-of-function variants in AP3B2, a neuronal adaptor protein required for synaptic vesicle formation, cause a severe early-onset neurodevelopmental epilepsy known as Developmental and Epileptic Encephalopathy 48 (DEE48). To investigate how AP3B2 loss alters brain development, leading to increased seizure susceptibility, we generated a Xenopus laevis model by targeting the orthologous gene using CRISPR/Cas9. Ap3b2.S-/- (mosaic) F0 tadpoles displayed increased locomotor activity with frequent seizure-like episodes when compared to sibling controls. Visualisation of forebrain and midbrain activity using the genetically encoded Ca2+ sensor GCaMP6s detected spontaneous, large amplitude, prolonged and widespread neural activity, alongside increased interhemispheric synchrony of both regions. Comparison of whole-brain transcriptomes from ap3b2 CRISPants and unedited sibling controls detected mainly downregulation of brain expressed genes, with significant over-representation of pathways involved in ion transport, axon formation and guidance, inhibitory (GABA) neurotransmission, and transport across the blood-brain barrier (BBB). In a simple assay for BBB integrity, CRISPant tadpoles were confirmed to have faster leakage of sodium fluoresceinAcute exposure to the angiotensin receptor blocker losartan significantly reduced locomotor hyperactivity, and CRISPant cohorts treated with losartan tended to have lower neural activity, indicating incomplete rescue of the ap3b2.S CRISPant phenotype. These findings demonstrate how AP3B2 loss of function alters brain development and the establishment of the BBB, with the resulting alterations in neurotransmitter pathways predisposing the brain to spontaneous seizures. Our results suggest that traditional anti-seizure medications designed to alter ion transport and GABA metabolism could be augmented with drugs targeting neuroinflammation, as adjunct seizure control options in infants with DEE48.