Abstracts

Rationale: Epilepsy is a condition defined by unprovoked seizures, sometimes with other neurodevelopmental consequences. Focal cortical dysplasia (FCD) is a common cause of focal epilepsy. FCDs are associated with genes in the mammalian target of rapamycin (mTOR) pathway, involved in cell growth and signaling. Genomic analysis from our group and others have demonstrated that germline and somatic mosaic variants in the DEPDC5 gene are associated with inherited and non-inherited focal epilepsy and contribute to Sudden Unexpected Death in Epilepsy. Germline, germline mosaic, or brain somatic variants in DEPDC5 are associated with FCD and other focal brain malformations associated with focal epilepsy. Disease-associated loss-of-function variants in DEPDC5 lead to mTORC1 activation in dysmorphic neurons.


Methods: Since existing mouse models with Depdc5 germline variants demonstrate early death, we aimed to design mosaic models of Depdc5. Here, we demonstrate generating a Depdc5 mosaic loss-of-function zebrafish model to understand the role of mosaicism in Depdc5 and related genes. Mature Casper zebrafish were crossed, and the fertilized eggs were microinjected at 1-cell and 2-cell stages with Depdc5 gRNA, Depdc5 dTomato+UFlip construct, and CRISPR/Cas9mRNA (2nl/per embryo). The larvae were sorted under red fluorescence for a successful cut in Depdc5 and studied for survival up to 14dpf (days post fertilization). In parallel, we quantified the morphological abnormalities in these larvae, including smaller head size and total body length when compared to controls. We also studied their abnormal behavioral patterns and neuronal hyperexcitability at dpf5.

Results: The F0 larvae sorted under red fluorescence, an indicator for a successful cut in Depdc5, studied for survival up to 14dpf, demonstrated early death starting at dfp1, recapitulating human DEPDC5-related epilepsy. We observed an abnormal head-to-body ratio in the mutants. We demonstrate that the acute crispants display neuronal hyperexcitability using local field potential recordings, as well as neuronal cell death (early apoptosis).


Conclusions: Our developed DEPDC5 loss of function mosaic models are crucial as they recapitulate the premature death related to DEPDC5-related epilepsy. We additionally are in the process of studying abnormal levels of mTOR pathway downstream proteins. This model is important for establishing high throughput drug screening assays towards therapeutic development.


Funding: Rosamund Stone Zander Translational Neuroscience Center (RSZ TNC) Postdoctoral Fellowship, Boston Children’s Hospital, awarded to Sneham Tiwari (07/2023 to 06/2025)

Cooper Trewin Foundation