Experimental Control and Characterization of Autophagy in Drosophila

Insects such as the fruit fly Drosophila melanogaster, which fundamentally reorganize their body plan during metamorphosis, make extensive use of autophagy for their normal development and physiology. In the fruit fly, the hepatic/adipose organ known as the fat body accumulates nutrient stores during the larval feeding stage. Upon entering metamorphosis, as well as in response to starvation, these nutrients are mobilized through a massive induction of autophagy, providing support to other tissues and organs during periods of nutrient deprivation. High levels of autophagy are also observed in larval tissues destined for elimination, such as the salivary glands and larval gut. Drosophila is emerging as an important system for studying the functions and regulation of autophagy in an in vivo setting. In this chapter we describe reagents and methods for monitoring autophagy in Drosophila, focusing on the larval fat body. We also describe methods for experimentally activating and inhibiting autophagy in this system and discuss the potential for genetic analysis in Drosophila to identify novel genes involved in autophagy

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila

How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we fi nd evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease

Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy

Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atgl or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development. © 2009 Landes Bioscience

The evolution of nervous system centralization

It is yet unknown when and in what form the central nervous system in Bilateria first came into place and how it further evolved in the different bilaterian phyla. To find out, a series of recent molecular studies have compared neurodevelopment in slow-evolving deuterostome and protostome invertebrates, such as the enteropneust hemichordate Saccoglossus and the polychaete annelid Platynereis. These studies focus on the spatially different activation and, when accessible, function of genes that set up the molecular anatomy of the neuroectoderm and specify neuron types that emerge from distinct molecular coordinates. Complex similarities are detected, which reveal aspects of neurodevelopment that most likely occurred already in a similar manner in the last common ancestor of the bilaterians, Urbilateria. This way, different aspects of the molecular architecture of the urbilaterian nervous system are reconstructed and yield insight into the degree of centralization that was in place in the bilaterian ancestors

Information-Preserving Techniques Improve Chemosensitivity Prediction of Tumours Based on Expression Profiles

Part 19: Computational Intelligence Applications in Bioinformatics (CIAB) WorkshopInternational audiencePrior work has shown that the sensitivity of a tumour to a specific drug can be predicted from a molecular signature of gene expressions. This is an important finding for improving drug efficacy and personalizing drug use. In this paper, we present an analysis strategy that, compared to prior work, maintains more information and leads to improved chemosensitivity prediction. Specifically we show (a) that prediction is improved when the GI50 value of a drug is estimated by all available measurements and fitting a sigmoid curve and (b) application of regression techniques often results in more accurate models compared to classification techniques. In addition, we show that (c) modern variable selection techniques, such as MMPC result in better predictive performance than simple univariate filtering. We demonstrate the strategy on 59 tumor cell lines after treatment with 118 fully characterized drugs obtained by the National Cancer Institute (NCI 60 screening) and biologically comment on the identified molecular signatures of the best predicted drugs