0041 Drug Screening and CRISPR/Cas9 Screening of HCN Channels

Abstract Introduction A growing body of evidence suggests a role for HCN (hyperpolarization-activated cyclic nucleotide-gated) channels in regulation of sleep. We first tested effects of three HCN channel blockers (Corlanor, Zatebradine Hydrochloride and ZD7288) on sleep/wake behavior in wild type zebrafish compared to DMSO control. Then, to more definitively investigate roles of HCN channels, we knocked out genes encoding for HCN channel subunits in zebrafish by utilizing CRISPR/Cas9 technique and we assessed sleep/wake behavior in the HCN channel mutants. Methods The compounds were tested at six concentrations varying between 0.1-30 ◻M (.i.e., 0.1 μM, 0.3 μM, 1.0 μM, 4.5 μM, 10 μM and 30 μM) in zebrafish larvae in our drug screening study. Control groups were administered DMSO. In CRISPR screening, single cell-stage zebrafish embryos were injected with preformed ribonucleoprotein complexes containing Cas9 protein and two crRNAs targeting each homologous gene of human HCN1, HCN2, HCN3, and HCN4. Control groups were negative control crRNA injected embryos. Sleep phenotyping was performed by placing individual larva into the wells of 96 well plates and by recording their activity via commercially available video monitoring equipment. Results Drug screening study showed shorter latency to sleep at 0.1 μM dose of Ivabradine, moderate reductions in average activity at 30 μM dose of Zatebradine Hydrochloride, and increased sleep at 4.5 μM dose of ZD7288. Our CRISPR/Cas9 screening resulted in decreased activity in hcn1 crispants, fewer sleep bouts in daytime in hcn2 crispants, and no difference in sleep parameters in hcn3 crispants. hcn4 crispants had shorter sleep in both day and nighttime, hyperactivity and shorter sleep bouts at night and fewer sleep bouts in daytime. Conclusion Blocking HCN channels decreased wakefulness in our drug screening study. Genetically targeting hcn1 and hcn2 genes resulted in the same direction with the results of drug screening study and knocking out hcn3 didn’t change sleep parameters. Change in sleep parameters were in the opposite direction in hcn4 crispants. HCN channel blockers might be selecting HCN1 and HCN2 however further work is required to confirm this suggestion. Support (if any)