Abstract

We have developed the adult Drosophila secondary cell (SC) as a new in vivo model to study protein aggregation into dense-core granules (DCGs) in real-time. To do this, we have generated an extensive genetic toolbox, producing SCs in which membrane-bound compartments, DCGs and vesicles are fluorescently marked and can be genetically manipulated to knock down or overexpress genes in an SC-specific way. Exploiting these tools and the SC’s uniquely large DCG compartments, we found that the Drosophila homologues of APP and TGFBI co-operate together to trigger protein aggregation in DCGs, a novel cell biological role for these amyloidogenic molecules. Expressing defective forms of these proteins alters DCG assembly and/or disassembly, providing a new model to study pathological amyloidogenesis. We now propose to use these methodologies to: 1. Characterise how APP and TGFBI normally work together to control protein aggregation in DCGs, using gene knockdown and overexpression approaches, and how this process and disassembly of secreted DCGs are disrupted by amyloidogenic versions of these proteins; 2. Determine how physiological protein aggregation in DCGs is affected by intracellular trafficking and exosome biogenesis, and whether amyloidogenic versions of APP/A-beta and TGFBI alter this response; 3. Test whether knockdown of candidate genes implicated in Alzheimer’s Disease affects DCG protein aggregation in the presence of normal and amyloidogenic forms of APP and TGFBI, and characterise the precise roles of genes that we identify. These studies will determine the normal cell biological functions of APP and TGFBI in triggering protein aggregation, and reveal how protein assembly and disassembly is disrupted by amyloidogenic forms of these proteins. By identifying genetic modifiers of this process, we will highlight candidate genes and mechanisms that can be tested in mammalian models and human cells as potential therapeutic targets in amyloid disease.