Knockdown ofNeuroD2leads to seizure-like behaviour, brain neuronal hyperactivity and a leaky blood-brain barrier in aXenopus laevistadpole model of DEE75

Abstract

AbstractDevelopmental and Epileptic Encephalopathies (DEE) are a genetically diverse group of severe, early onset seizure disorders. DEE are normally identified clinically in the first six months of life by the presence of frequent, difficult to control seizures and accompanying stalling or regression of development. DEE75 results fromde novomutations of theNEUROD2gene that result in loss of activity of the encoded transcription factor, and the seizure phenotype was shown to be recapitulated inXenopus tropicalistadpoles. We used CRISPR/Cas9 to make a DEE75 model inXenopus laevis, to further investigate the developmental aetiology.NeuroD2.SCRISPR/Cas9 edited tadpoles were more active, swam faster on average, and had more unprovoked escape responses (C-starts) than their sibling controls. Live imaging of Ca2+signalling revealed prolongued, strong signals sweeping through the brain, indicative of neuronal hyperactivity. While the resulting tadpole brain appeared grossly normal, the blood-brain barrier was found to be leakier than that of controls. Additionally, the TGFβ antagonist Losartan was shown to have a short-term protective effect, reducing neuronal hyperactivity and reducing permeability of the blood- brain barrier. Severity of the behavioral phenotype correlated with increased with editing efficiency. Our results support a haploinsufficiency model of DEE75 resulting from reduced NeuroD2 activity during vertebrate brain development, and indicate that a leaky blood- brain barrier contributes to epileptogenesis.