The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling.

Sleep is vital for most animals, yet its mechanism and function remain unclear. We found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. We observed increased BBB permeability in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian-each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep.

Molecular mechanisms and physiological importance of circadian rhythms.

To accommodate daily recurring environmental changes, animals show cyclic variations in behaviour and physiology, which include prominent behavioural states such as sleep-wake cycles but also a host of less conspicuous oscillations in neurological, metabolic, endocrine, cardiovascular and immune functions. Circadian rhythmicity is created endogenously by genetically encoded molecular clocks, whose components cooperate to generate cyclic changes in their own abundance and activity, with a periodicity of about a day. Throughout the body, such molecular clocks convey temporal control to the function of organs and tissues by regulating pertinent downstream programmes. Synchrony between the different circadian oscillators and resonance with the solar day is largely enabled by a neural pacemaker, which is directly responsive to certain environmental cues and able to transmit internal time-of-day representations to the entire body. In this Review, we discuss aspects of the circadian clock in Drosophila melanogaster and mammals, including the components of these molecular oscillators, the function and mechanisms of action of central and peripheral clocks, their synchronization and their relevance to human health.

Studying circadian rhythm and sleep using genetic screens in Drosophila.

The power of Drosophila melanogaster as a model organism lies in its ability to be used for large-scale genetic screens with the capacity to uncover the genetic basis of biological processes. In particular, genetic screens for circadian behavior, which have been performed since 1971, allowed researchers to make groundbreaking discoveries on multiple levels: they discovered that there is a genetic basis for circadian behavior, they identified the so-called core clock genes that govern this process, and they started to paint a detailed picture of the molecular functions of these clock genes and their encoded proteins. Since the discovery that fruit flies sleep in 2000, researchers have successfully been using genetic screening to elucidate the many questions surrounding this basic animal behavior. In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then move on to describe techniques currently employed for mutagenesis and genetic screening in the field. The emphasis lies on comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis, in particular in their application to circadian behavior and sleep. We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab.

Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.

The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. Here we report the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. Our study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as "semiprofessional" phagocytes in the nervous system.

Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide.

Mycobacterium tuberculosis is a facultative intracellular pathogen that inhibits phagosome maturation in macrophages thereby securing survival and growth. Mycobacteria reside in an early endocytic compartment of near-neutral pH where they upregulate production of complex glycolipids such as trehalose dimycolate. Here, we report that trehalose dimycolate coated onto beads increased the bead retention in early phagosomes, i.e. at a similar stage as viable mycobacteria. Thus, a single mycobacterial lipid sufficed to divert phagosome maturation and likely contributes to mycobacterial survival in macrophages. Previous studies showed that activated macrophages promote maturation of mycobacterial phagosomes and eliminate mycobacteria through bactericidal effectors including nitric oxide generated by inducible nitric-oxide synthase. We show that deceleration of bead phagosome maturation by trehalose dimycolate was abolished in immune-activated wild type, but not in activated nitric-oxide synthase-deficient macrophages, nor when hydroxyl groups of trehalose dimycolate were chemically modified by reactive nitrogen intermediates. Thus, specific host defence effectors of activated macrophages directly target a specific virulence function of mycobacteria.