Seizure Phenotype and Underlying Cellular Defects inDrosophilaKnock-In Models of DS (R1648C) and GEFS+ (R1648H)SCN1AEpilepsy

Abstract

Seizure phenotype and underlying cellular defects in Drosophila knock-in models of DS (R1648C) and GEFS+ (R1648H) SCN1A epilepsy ABSTRACT Mutations in the voltage-gated sodium channel gene SCN1A are associated with human epilepsy disorders, but how most of these mutations alter channel properties and result in seizures is unknown. This study focuses on two different mutations occurring at one position within SCN1A. R1648C is associated with the severe disorder Dravet Syndrome, and R1648H, with the milder disorder GEFS+. To explore how these different mutations contribute to distinct seizure disorders, Drosophila lines with the R-C or R-H mutation, or R-R control substitution in the fly sodium channel gene para were generated by CRISPR-Cas9 gene editing. The R-C and R-H mutations are homozygous lethal. Animals heterozygous for R-C or R-H mutations displayed reduced lifespans and spontaneous and temperature-induced seizures not observed in R-R controls.Electrophysiological recordings from adult GABAergic neurons in R-C and R-H mutants revealed appearance of sustained neuronal depolarizations and altered firing frequency that were exacerbated at elevated temperature. The only significant change observed in underlying sodium currents in both R-C and R-H mutants was a hyperpolarized deactivation threshold at room and elevated temperature compared to R-R controls.Since this change is constitutive it is likely to interact with heat-induced changes in other cellular properties to result in the heat-induced increase in sustained depolarizations and seizure activity. Further, the similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines, suggests that disease symptoms of different severity 3 associated with these mutations in humans could be due in large part to differences in genetic background. SIGNIFICANCE STATEMENT: Knock-in Drosophila lines were generated by CRISPR-Cas9 gene editing to explore how the R1648C and R1648H mutations in the SCN1A sodium channel gene contribute to distinct epilepsy disorders, Dravet syndrome and GEFS+ respectively. Drosophila heterozygous for the R-C or R-H mutation displayed spontaneous seizures exacerbated at high temperature. GABAergic neurons in both mutants exhibited sustained depolarizations, interrupting neuronal firing, that increased in incidence at elevated temperature. A hyperpolarized deactivation threshold in R-C and R-H sodium currents was constitutive implicating interaction with other heatsensitive processes to alter firing properties. The similarity of the behavioral and cellular phenotypes in the R-C and R-H fly lines suggests that disorders of different severity in humans could be due in large part to differences in genetic background.

Drosophila melanogaster as a versatile model organism to study genetic epilepsies: An overview

“…Furthermore, a hyperpolarized deactivation threshold in para R-C and para R-H sodium currents was observed, which was present at both room temperature and elevated temperature. Taken together, the similar results from behavioral and electrophysiological studies indicate that the different phenotypes observed in humans may be mainly due to differences in genetic background rather than distinct changes in sodium channel function ( Roemmich et al, 2021 ).…”

“…Seizures are often refractory to treatment and patients display several other clinical features, such as cognitive, behavioral, and motor impairments ( Mei et al, 2019 ; Zuberi et al, 2022 ). To investigate the underlying disease mechanisms of these both disorders, several GEFS+ and DS mutations have been knocked into the orthologous Drosophila gene para at corresponding positions ( Sun et al, 2012 ; Schutte et al, 2014 ; Roemmich et al, 2021 ). The first variant to be explored was the GEFS+ mutation K1270T in the transmembrane segment 2 of homology domain III ( Sun et al, 2012 ).…”

“…Another study focused on two different SCN1A variants that occur at the same position in segment 4 of homology domain IV ( Roemmich et al, 2021 ). While the R1648H (R-H) variant is associated with GEFS+, the R1648C (R-C) variant causes DS.…”

“…The fruit fly Drosophila melanogaster has emerged as an increasingly attractive model system due to its small size, fast generation time, low maintenance costs and ease of genetic manipulation ( Rosch et al, 2019 ). Recent developments in genome editing techniques have facilitated the generation and characterization of humanized flies, carrying the human epilepsy-causing mutation in the corresponding fly gene ( Sun et al, 2012 ; Schutte et al, 2014 ; Roemmich et al, 2021 ). These disease models offer a unique opportunity to unravel the molecular mechanisms underlying genetic epilepsies and to explore potential therapeutic targets.…”

Functional characterization of a KCNAB3 genetic epilepsy with febrile seizures plus adult mouse model

“…This mutation was thought to affect protein function by the bioinformatics tools. For patients with genetic epilepsy, the connection between a specific mutation and their behavioral symptoms is largely unknown (11). To clarify the relationship between the KCNAB3 gene mutation (H258R) and GEFS+, wild and mutant plasmids were constructed and transfected into human embryonic kidney 293 (HEK293) cells for patchclamp detection.…”