Understanding the Role of Serine/Threonine Kinase 32a (stk32a) in Regulating Zebrafish Sleep - PROJECT SUMMARY Sleep disorders affect millions of people in the U.S. each year, and efficient sleep is vital for human health and cognitive functions. Developing an understanding of the molecular and genetic pathways that regulate this highly conserved behavior is essential for developing therapies to address these disorders. Human genome-wide association studies (GWAS) have identified many genomic loci that contain genetic variants associated with sleep phenotypes. However, because most of these variants are in non-coding regions, it is unclear which gene within each locus is responsible for the associated sleep phenotype. Thus, these screens have identified many candidate human sleep disorder genes, but their potential contributions to sleep must to be tested in an animal model. In order to address this need, the Prober lab has used CRISPR-Cas9 to mutate and test the zebrafish orthologs of many GWAS-identified human candidate sleep disorder genes. Zebrafish are a useful model system for this purpose because many mutant lines can be quickly and inexpensively generated using CRISPR-Cas9, and the amenability of zebrafish to high-throughput sleep assays makes it possible to efficiently test many different mutants for sleep phenotypes. Additionally, zebrafish are diurnal vertebrates, like humans, whose demonstrated genetic and neuronal mechanisms that regulate sleep are broadly conserved with those of mammals. This project focuses on one gene identified in this screen, the largely uncharacterized protein kinase serine threonine kinase 32a (stk32a). In preliminary studies, we found that stk32a mutant zebrafish exhibit increased nighttime sleep. I will exploit advantageous features of zebrafish to determine the mechanisms through which stk32a acts to influence sleep. First, based on the night-specificity of the mutant phenotype, I will assess whether stk32a regulates dark-dependent, circadian-dependent, or homeostatic mechanisms of sleep regulation. I will do so by performing behavioral experiments using different lighting conditions, by monitoring rebound sleep after sleep deprivation, and by testing for interactions with genes that regulate each of the above mechanisms of sleep control. Second, based on my preliminary gene expression and behavioral data, I will test the hypothesis that the stk32a mutant phenotype is caused by disrupted detection of sensory stimuli by lateral line hair cells. To do so, I will assess the effects of stk32a mutation on lateral line hair cell structure and function, test the effect of disrupting the lateral line on sleep, and test whether rescuing stk32a function in hair cells is sufficient to rescue the stk32a mutant sleep phenotype. The results of these experiments will help to determine the molecular and genetic mechanisms through which stk32a regulates sleep. Together with the NIH’s “Illuminating the Druggable Genome” program, where work is being done to identify stk32a small molecule inhibitors, these studies may eventually lead to novel therapies for sleep disorders based on inhibition of stk32a function.
