Identification of autophagy mediators through screening transgenic animal library

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

? DESCRIPTION (provided by applicant): Autophagy is a physiological process critical for maintaining tissue homeostasis by preventing protein aggregate formation, fat droplet accumulation, hyperplastic cell growth and/or mitochondrial dysfunction. Using Drosophila and mouse models, we discovered that Sestrin- and target of rapamycin (TOR)-regulated autophagy is critical for metabolic homeostasis and mitochondria quality control, which are essential for preventing oxidative damage and age-associated tissue degeneration. Autophagy-regulated 1 (Atg1, Ulk1 and Ulk2 in mammals), an autophagy-initiating protein kinase that is inhibited by TOR, was shown to be critical for this process. However, we currently have very limited information about how Atg1 initiates autophagic processes and which downstream components mediate its autophagy-inducing functions. The proposed research tackles this important problem through the utilization of recently established transgenic dsRNA Drosophila library which allows for highly efficient tissue-specific gene silencing with genome-wide coverage. For the primary screening of Atg1 mediators, we constructed a Drosophila line that stably expresses functionally activated Atg1 in the eye, which subsequently exhibits strong eye degeneration phenotype due to excessive autophagy induction. We will set up single pair crosses between this starter line and each individual line from the library that will express dsRNA specific for eah target gene. In the F1 progeny, only the flies expressing dsRNA that targets essential mediators of Atg1-induced autophagy will be protected from the eye degeneration. Therefore, through simple examination of the external eye phenotype of the F1 progenies, we will be able to identify many new autophagy-mediating genetic components downstream of Atg1. We expect that these components will construct a novel signaling pathway that links diverse extracellular/intracellular stimuli to autophagic regulation. In addition, we have set up a streamlined secondary and tertiary screening regimen that can effectively eliminate false-positive genes from the candidate pool. The final candidates will then be analyzed simultaneously in three independent model systems - Drosophila, cultured cells and mice - for its autophagy-controlling physiological roles in maintaining tissue homeostasis. These new components will be mechanistically connected to the known autophagy regulators, which will together establish the comprehensive big picture of physiological signaling network on autophagy regulation. This understanding is especially important for improving human health because autophagy deregulation is associated with diverse human diseases such as neurodegeneration, cancer, muscular dystrophy, cardiac malfunction, metabolic derangements and defective immune responses.