Authors :
Presenting Author: Sneham Tiwari, PhD – Boston Children's Hospital
Hyunyong (Howard) Koh, MD, PhD – Research Fellow, Neurology, Boston CHildren's Hospital; Christopher LaCoursiere, MS – Lead Research Technologist, Neurology, Boston Children's Hospital; Annapurna Poduri, MD, MPH – Principal Investigator, Director, Epilepsy Genetics Program, Boston Children’s Hospital Co-Director, Program in Neurogenetics, Boston Children’s Hospital Professor of Neurology, Harvard Medical School Diamond Blackfan Chair of Neuroscience Research, Boston Children’s Hospital, Neurology, Boston Children's Hospital
Rationale:
The
DEPDC5 (DEP domain-containing protein 5) gene, encoding a repressor of the mTOR (mammalian target of rapamycin) signaling pathway, has an important role in the regulation of mTORC1 (mammalian target of rapamycin complex 1). Germline, germline mosaic, or brain somatic variants in
DEPDC5 are associated with Focal cortical dysplasia (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. To study the effect of loss of function depdc5, we have generated a mosaic DEPDC5 loss-of-function zebrafish model.
Methods:
We designed a construct based on a GAL
4-VP16/UAS (GAL4-VP16 is a chimeric transcription factor that binds to upstream activation sequences) plasmid and inserted it in the competent cells. DNA extracted from positive colonies after transfection was used in the microinjection. Wildtype male and female Casper fish were crossed, and eggs were harvested post-fertilization and prepared for microinjections. Depdc5 construct along with Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 messenger RNA (CRISPR/Cas9mRNA), depdc5-specific guide RNA (gRNA), and universal single guide RNA (sgRNA) were injected into one-cell stage Zebrafish embryos (2nl/per embryo). At 24 hours post-injection, eggs, with red fluorescence were indicated as successful insertion of the depdc5 plasmid and cutting. Positive larvae were observed for survival until days post-fertilization (dpf) 14. Results:
We observed that the introduction of the
depdc5 construct resulted in the early death of larvae starting by age dpf1 days post fertilization. We also studied the morphology and behavioral patterns of these mosaic larvae and observed abnormal morphology and behavior patterns in the mutants when compared to wild-type controls.
Conclusions:
Our developed
DEPDC5 loss of function mosaic models are crucial as they recapitulate the premature death related to DEPDC5-related epilepsy. Hence, these are crucial models for studying phenotypic and behavioral patterns and in high throughput drug screening and therapeutics 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). Manton Centre Fellowship, Boston Children’s Hospital, awarded to Hyun Yong Koh (7/2021 to 06/2023).