Early death and neuronal abnormalities in <i>depdc5</i> loss-of-function mosaic zebrafish models

<i>DEPDC5</i> (DEP domain-containing protein 5) encodes a repressor of the mTORC1 signaling pathway. Variants in <i>DEPDC5</i> 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 <i>DEPDC5</i> in human epilepsy related to mosaic variants, we have generated mosaic <i>depdc5</i> loss-of-function zebrafish models using homology-based constructs acutely targeting <i>depdc5</i> and labeled with tdTomato to allow for visualization of the degree of mosaicism. The resulting mosaic <i>depdc5</i> 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 <i>DEPDC5</i>-related epilepsy. We compared <i>depdc5</i> CRISPants with uninjected and scrambled controls from the same clutches. Body and head size were reduced in the <i>depdc5</i> CRISPants. Analysis of swimming behavior showed a striking reduction in distance traveled and maximum velocity in the <i>depdc5</i> CRISPants vs. controls. Based on visual confirmation of mutational load, we categorized CRISPants into <i>depdc5</i>+ vs. depdc5++, reflecting weak vs. strong tdTomato fluorescence. We observed that <i>depdc5</i>++ 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 <i>depdc5</i> CRISPants vs. controls. Acridine orange staining demonstrated early apoptosis in the CRISPants vs. controls. Our mosaic <i>depdc5</i> CRISPants provide a clinically relevant model to study the role of mosaic DEPDC5-related epilepsy and early death.