680 - Eif4a2 is a critical regulator of zebrafish interneuron development

Publication Number: 2663.680

Anna R. Duncan, Mass General Brigham for Children, Charlestown, MA, United States; Diya Shinglot, MassGeneral Hospital for Children, Boston, MA, United States; Annapurna Poduri, Boston Children's Hospital, Chestnut Hill, MA, United States

Background: GABAergic interneurons are essential for neural transmission and connectivity. Dysregulation of interneurons occurs in many neurodevelopmental disorders (NDD), with common and rare etiologies that include preterm birth, hypoxia exposure, and neurogenetic disorders. Our prior human genetics research demonstrates that genetic variants in EIF4A2 lead to an early onset NDD characterized by epilepsy, hypotonia, and intellectual disability. Recently, we have observed that loss of eif4a2 in a zebrafish model leads to reduced numbers of interneurons. EIF4A2’s role in interneuron development, however, remains largely unknown.

Objective: To define the role of eif4a2 in interneuron development using the zebrafish model system.

Design/Methods: CRISPR/Cas9 gene editing was used to create eif4a2+/- and eif4a2-/- zebrafish models. Seizure-like hyperexcitability and motor function were assessed by high-throughput zebrafish behavioral assays (Ramona Kestrel system). Interneurons were quantified in a transgenic Dlx2-GFP zebrafish model, and protein expression evaluated by western blots.

Results: Motor defects are present in embryos and larvae with tail coiling at 20-24 hours post-fertilization (hpf) and swimming at 5 days post fertilization (dpf) reduced in eif4a2+/- and eif4a2-/- zebrafish compared to WT (P < 0.001 for both). We observed an increase in spontaneous seizures at 7 dpf in both eif4a2+/- and eif4a2-/- larvae vs. WT with increased whirlpool, convulsion, and posture loss events (P < 0.01). We also observed, in transgenic Dlx2-GFP zebrafish, reduced GABAergic interneurons in crispants vs. WT larvae (p < 0.05). Preliminary western blot analyses indicate reduction in proteins critical during interneuron development, including Dlx2 and Gad1b, as well as a reduction in parvalbumin (PV) protein expression in eif4a2-/- vs. WT zebrafish, suggesting that the PV subtype of interneurons are reduced with loss of eif4a2 expression.

Conclusion(s): Zebrafish eif4a2+/- and eif4a2-/- larvae recapitulate the seizures and motor impairments observed in human EIF4A2-related NDD. GABAergic interneurons are decreased in number in these models, in parallel with a decrease in proteins critical for multiple stages of interneuron development. Consistent with studies of preterm brain, PV expression was also decreased, suggesting the PV subtype of interneurons is reduced with loss of eif4a2 expression. Ongoing experiments will determine the mechanism(s) by which eif4a2 impacts interneuron protein expression. This study highlights how rare disorders can elucidate critical pathways important for common neurodevelopmental disorders.