Project Summary/Abstract Our genetic screens in Drosophila previously identified several mutations with strong effects on patterns of sleep. Recently we found that three of these genes, insomniac, cullin3 and nedd8, are expressed in the blood-brain-barrier-forming subperineurial glia of the fly, and that the morphological and biophysical properties of the barrier are altered in inc, cullin3, and nedd8 mutants. In our proposed studies we will test the effects of classical barrier mutants on sleep, evaluate barrier function across the entire range of available sleep mutants and in aged vs young flies, and explore evidence for regulatory interactions among sleep and classical barrier-regulating genes. Most Drosophila sleep mutants severely reduce longevity. We will determine whether mutations that increase longevity also improve barrier function and sleep duration in sleep mutants. In a second branch of our proposed research we will examine the role of circadian clock genes in commonly encountered disorders of human sleep. We recently discovered a mutation of the circadian clock gene CRY1 that is associated with a form of delayed sleep phase disorder (DSPD) that affects ~1 in 100 individuals worldwide. The results of our study suggest a novel approach for exploring the heritability of similarly common sleep disorders: Predictive algorithms will be applied to several large human exome databases to select candidate circadian variants for cellular phenotyping. Prevalent alleles that are associated with altered circadian rhythmicity in cell culture assays also will be studied by behavioral phenotyping of carrier subjects identified by collaborators at Bilkent University in Ankara, Turkey. Disordered sleep is often accompanied by chronic diseases including diabetes, obesity, or certain mood and anxiety disorders. Although causality in such instances has been difficult to establish by traditional approaches, we will employ deep physiological and behavioral phenotyping of individuals sharing specific genetic variations affecting sleep in tests for linkage to specific co-morbidities. These studies may significantly enrich our understanding of biological pathways regulated by circadian clocks in humans as well as fundamental disease etiologies.