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Title: Investigation of the molecular machinery underlying sleep homeostasis in Drosophila melanogaster
Author: Song, Seoho (Michael)
ISNI:       0000 0004 6499 0197
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Though universal and highly conserved, the biological function of sleep remains largely mysterious. One approach to solving this mystery is to study the neural mechanisms that regulate sleep and waking. There are two such mechanisms: the circadian clock, which synchronizes sleep with periodic changes in the external environment, and the sleep homeostat, which responds to (still largely unknown) internal changes that require sleep to reset. Understanding this second, homeostatic control system will likely offer important clues to the biological function of sleep. In the fruit fly, Drosophila melanogaster, a homeostatic sleep switch has been discovered in a cluster of two dozen neurons with projections to the dorsal fan-shaped body (dFB) in the central brain. The electrical activity of these cells induces sleep and is antagonistically modulated by two potassium currents: Shaker-based A-type currents promote sleep, whereas Sandman-based leak currents inhibit sleep. The potassium conductances are, in turn, regulated by Rho GTPase activating proteins such as Crossveinless-c (Cv-c). How this molecular machinery logs a fly's sleep history and responds by altering the electrical properties of the dFB neuronal membrane is currently unknown. In this study, two molecular mechanisms were identified that signal changes in sleep drive within dFB neurons. The first mechanism involves Rho GTPases and their regulators. Catalytically 'dead' Cv-c was found to be unable to rescue the short-sleeping phenotype of cv-c mutants, implicating the GTPase cycle of a small G protein of the Rho family in sleep regulation. Through behavioural screens, Rho1 was identified as a candidate Cv-c substrate, and Rac1 as a Rho1 antagonist. Targeted ablations of Rho1 and Rac1 in the dFB neurons of cv-c mutants rescued and exacerbated sleep deficits, respectively. Thus, active Rac1 and Rho1 promote sleep and wakefulness in a mutually antagonistic fashion. The second mechanism involves the action of oxidation by-products on Hyperkinetic (Hk), a beta subunit of the potassium channel Shaker whose function in dFB neurons is important for sleep. Hk is a functional aldo-keto reductase and senses cellular redox levels. This redox sensing capacity is required to restore sleep in short-sleeping Hk1 mutants. Mitochondrial respiration is a primary source of reactive oxygen species (ROS). In vivo measurements of mitochondrial ROS in sleep deprived flies revealed that sleep pressure correlated with ROS concentrations in dFB neuron dendrites. Elevating ROS in dFB neurons by overexpressing mutant superoxide dismutase (Sod1) increased sleep, whereas overexpressing wild type Sod1, which neutralizes ROS, decreased sleep. These effects were occluded in the absence of functional Hk, which implicates it as the site of redox modulation of membrane excitability. Thus, ROS acts as a potential link between energy metabolism, oxidation, and sleep.
Supervisor: Miesenböck, Gero Sponsor: Commonwealth Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available