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.

The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of the reprogramming strategy. Over 60 murine iPSC lines were derived from different fibroblast origins (embryonic, cardiac, tail tip) via lentiviral integration and doxycycline-induced transgene expression. Despite apparent equivalency according to established iPSC histologic and cytomorphologic criteria, clustering of clonal variability in pluripotency-related gene expression identified transcriptional outliers that highlighted cell lines with unpredictable cardiogenic propensity. Following selection according to a standardized gene expression profile calibrated by embryonic stem cells, the influence of somatic origin on iPSC methylation and transcriptional patterns was negated. Furthermore, doxycycline-induced iPSCs consistently demonstrated earlier differentiation than lentiviral-reprogrammed lines using contractile cardiac tissue as a measure of functional differentiation. Moreover, delayed cardiac differentiation was predominately associated with up-regulation in pluripotency-related gene expression upon differentiation. Starting from a standardized pool of iPSCs, relative expression levels of two pluripotency genes, Oct4 and Zfp42, statistically correlated with enhanced cardiogenicity independent of somatic origin or reprogramming strategy (R2=0.85). These studies demonstrate that predictable iPSC differentiation is independent of somatic origin with standardized gene expression selection criteria, while the residual impact of reprogramming strategy greatly influences predictable output of tissue-specification required for comparative genotype/phenotype analyses.

Cost-effective and efficient, the fruit fly (Drosophila melanogaster) has been used to make many key discoveries in the field of neuroscience and to model a number of neurological disorders. Great strides in understanding have been made using sophisticated molecular genetic tools and behavioral assays. Functional analysis of neural activity was initially limited to the neuromuscular junction (NMJ) and in the central nervous system (CNS) of embryos and larvae. Elucidating the cellular mechanisms underlying neurological processes and disorders in the mature nervous system have been more challenging due to difficulty in recording from neurons in adult brains. To this aim we developed an ex vivo preparation in which a whole brain is isolated from the head capsule of an adult fly and placed in a recording chamber. With this preparation, whole cell recording of identified neurons in the adult brain can be combined with genetic, pharmacological and environmental manipulations to explore cellular mechanisms of neuronal function and dysfunction. It also serves as an important platform for evaluating the mechanism of action of new therapies identified through behavioral assays for treating neurological diseases. Here we present our protocol for ex vivo preparations and whole-cell recordings in the adult Drosophila brain.