Lacoursiere, C. | Search bioRxiv

bioRxiv2025

Early death and neuronal abnormalities in depdc5 loss-of-function mosaic zebrafish models

DEPDC5 (DEP domain-containing protein 5) encodes a repressor of the mTORC1 signaling pathway. Variants in DEPDC5 are associated with a range of focal epilepsies, including mosaic variants associated with focal cortical dysplasia (FCD) and other focal brain malformations with brain-only somatic mosaic variants. To investigate the role of DEPDC5 in human epilepsy related to mosaic variants, we have generated mosaic depdc5 loss-of-function zebrafish models using homology-based constructs acutely targeting depdc5 and labeled with tdTomato to allow for visualization of the degree of mosaicism. The resulting mosaic depdc5 CRISPants demonstrated early larval death, with [~]50% of CRISPants (vs. 10% of controls) dead by 7 days post fertilization (dpf), analogous to the early death sometimes associated with human DEPDC5-related epilepsy. We compared depdc5 CRISPants with uninjected and scrambled controls from the same clutches. Body and head size were reduced in the depdc5 CRISPants. Analysis of swimming behavior showed a striking reduction in distance traveled and maximum velocity in the depdc5 CRISPants vs. controls. Based on visual confirmation of mutational load, we categorized CRISPants into depdc5+ vs. depdc5++, reflecting weak vs. strong tdTomato fluorescence. We observed that depdc5++ CRISPants had increased episodes of posture loss, suggesting increased seizure-like behavior related to higher percentages of mutant cells. Local field potential recordings revealed increased neuronal hyperexcitability in depdc5 CRISPants vs. controls. Acridine orange staining demonstrated early apoptosis in the CRISPants vs. controls. Our mosaic depdc5 CRISPants provide a clinically relevant model to study the role of mosaic DEPDC5-related epilepsy and early death.

neuroscience↗

bioRxiv2024

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability

Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2200 candidate epilepsy-associated genes, of which 81 were determined suitable for the generation of loss-of-function zebrafish models via CRISPR/Cas9 gene editing. Of those 81 crispants, 48 were successfully established as stable mutant lines and assessed for seizure-like swim patterns in a primary F2 screen. Evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, wnt8b) of the 48 mutant lines assessed. Further characterization of those 5 lines provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Furthermore, RNAseq revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes. HighlightsO_LIZebrafish models generated by CRISPR/Cas9 gene editing display seizure-like swim patterns in five candidate human epilepsy genes: arfgef1, kcnd2, kcnv1, ubr5, wnt8b. C_LIO_LILocal field potential abnormalities recorded from kcnd2 and wnt8b crispants provide additional evidence of hyperexcitability. C_LIO_LIArfgef1 and wnt8b mutant larvae have fewer inhibitory interneurons than wild type in the optic tectum. C_LIO_LICRISPR-generated mutants of epilepsy genes displayed convergent transcriptional dysregulation, consistent with developmental abnormalities and their hyperexcitability phenotype. C_LI

genetics↗