DESCRIPTION (provided by applicant): Proper turnover of organelles and proteins is essential for normal cell function during life. Damaged or altered cytosolic proteins are cleared by the proteasome and autophagy. Importantly, autophagy has the additional role of providing nutrients to cells under stress conditions such as starvation, and is thus essential for energy balance. Not surprisingly, autophagy declines with age, associated with the accumulation of altered, defective components in all model organisms including mice and Drosophila melanogaster. During macroautophagy (MA), membrane structures in the cytoplasm engulf bulk-regions of cytoplasm including organelles in a double membrane vesicle (autophagosome). Autophagosomes fuse to lysosomes leading to the degradation of the engulfed cytoplasm. Most autophagy related genes were originally identified in cellular stress-related genetic screens in yeast and are conserved through the animal kingdom including in Drosophila and humans. Recent genetic screens in C. elegans and Drosophila discovered novel macroautophagy genes not present in yeast but conserved in higher eukaryotes, underscoring the importance of using multicellular organisms to study autophagy. In contrast to MA, less is known about Chaperone Mediated Autophagy (CMA). CMA specifically degrades single proteins that contain a CMA targeting motif (KFERQ related sequences). Substrates containing a KFERQ targeting motif are recognized by the cytoplasmic Hsc70 (and co-chaperones), which then interacts with the lysosomal receptor LAMP-2A, the limiting component of CMA in mammalian cells. To date, LAMP-2A has been identified only in birds and mammalian species including rat, mouse and humans. Due to the inability to identify LAMP-2A by bioinformatics, studies to address whether or not CMA or a CMA-like process exists in other species including zebrafish and invertebrates have not been performed yet. On the other hand, Hsc70, the obligate cytoplasmic chaperone recognizing the KFERQ targeting motif, as well as KFERQ targeting motifs of known CMA substrates are conserved between Drosophila and humans, thus supporting the possibility a CMA-like pathway exists in invertebrates. Consistent with the natural decrease of MA and CMA with age, experimental reduction of autophagy in animal models such as mice or flies leads to reduced organ function and shortened life span. Conversely, overexpressing the central component of CMA, LAMP-2A, prevents natural, age related reduction of CMA and concomitantly rescues the decrease in liver function normally observed in old mice, demonstrating the significance of CMA mediated autophagic clearance of damaged material at old age. The aims of this proposal thus are to functionally test the existence of CMA or a CMA-like process in Drosophila in vivo and to assess its change over the lifespan of flies. To this end we generated transgenic flies expressing an artificial CMA target (KFERQ-PA-GFP). Furthermore, we will systematically knock-down kinases and phosphatases in the Drosophila fat body to identify novel components regulating a potential CMA-like process in vivo, an approach feasible only in a genetically tractable organism with a short life span.
|Effective start/end date||9/1/14 → 5/31/17|
- National Institute on Aging: $208,750.00
- National Institute on Aging: $191,589.00
- National Institute on Aging: $51,396.00