GENETIC MODULATION OF NOTCH SIGNALING AND LYSOSOMAL FUNCTION IN DROSOPHILA MELANOGASTER

In the first part of my thesis, I study novel genes that might regulate the Notch signaling pathway. The evolutionarily conserved Notch signaling pathway controls cell proliferation and differentiation decisions that shape development and homeostasis of multiple tissues in metazoans. Notch mutants were discovered in the fruit fly Drosophila melanogaster more than one century ago. Since then, extensive investigation in flies led to a superior understanding of how Notch regulates signaling, and how core cellular processes such as endo-lysosomal trafficking might control Notch signaling. The study of Notch during wing development in Drosophila pioneered the uncovering of an ever-increasing Notch interaction network, that is far from being fully elucidated. My work started by downregulating a set of previously identified putative Notch regulators in the developing Drosophila wing. We identified RNAi against Cam and Trpm, encoding the fly calmodulin homolog and a channel permeable to divalent ions, respectively, as regulators of wing vein patterning, a process controlled by Notch. Further genetic and tissue biology analysis indicated that while RNAi against Cam might prevent Notch activation, RNAi against Trpm might increase it. Indeed, RNAi against Trpm during Drosophila wing development correlates with strong alterations that phenocopy Notch gain of function mutations. Moreover, we found that well-known Notch signaling components genetically interact with RNAi against Trpm. Finally, we observed that RNAi against Trpm could reduce Notch cis-inhibition, a phenomenon that restricts signaling, and strongly interacted with mutants of genes that control endo-lysosomal trafficking of Notch. Altogether, our results could provide new entryways into modulation of the Notch signaling pathway. In the second part of my thesis, I investigate the function of Cathepsin F (CtsF), encoding a lysosomal cysteine protease, in Drosophila. Homozygous inactivating mutations in human CTSF cause Type B Kufs disease, an ultra-rare adult-onset neuronal ceroid lipofuscinosis (NCL) characterized by premature accumulation of lipopigments, neurodegeneration, cognitive decline, dementia, motor dysfunction, ataxia and premature death. I characterized a novel Drosophila model of Type B Kufs disease by taking advantage of CRISPR/Cas9 knock-out CtsF mutants previously generated in the lab. In particular, I assessed whether CtsF mutants could recapitulate features of the disease. I have found that CtsF mutant flies present a very strong motoneuron impairment, which manifests early during adulthood. Motoneuron dysfunction correlated with a trend towards increased brain autofluorescence, an established readout of the accumulation of lipopigments. In addition, CtsF mutant flies displayed increased brain vacuolization, a sign of neurodegeneration in flies. Finally, we observe that CtsF mutants present an alteration of autophagy, which is often associated with accumulation of toxic lysosomal storage or alterations of lysosomal function. Overall, our work indicates that fly CtsF mutants could be a promising model to study type B Kufs disease, thus allowing to clarify pathogenesis in humans.