Drosophila vitellogenic oocytes as an in vivo model to dissect the regulatory mechanism for low-density lipoprotein receptor-related protein 1

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Project Summary/Abstract Alzheimer's disease (AD) is tightly linked to aberrant lipid metabolism that elicits amyloid plaque formation, neuroinflammation, and synaptic dysfunction, while the lipid transport protein apolipoprotein E4 (APOE4) isoform is the greatest risk factor for late-onset AD. Importantly, Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) and Low-Density Lipoprotein Receptor (LDLR), the integral members of LDL receptor family (LDLRs), play pivotal roles in lipid metabolism, regulate Amyloid Beta (Aβ) production and clearance, and interact with APOE. Thus, a clear understanding of the regulation of LRP1 and LDLR is essential for elucidating the pathogenic mechanisms and finding effective cures for AD. The LDLR family is a group of evolutionarily conserved single-transmembrane proteins in higher eukaryotes, composed of a large extracellular domain involved in ligand binding and a short cytoplasmic tail important for receptor internalization and signaling. One critical regulation of LDLRs is ligand binding-induced endocytosis and subsequent endolysosomal trafficking. For example, upon binding with ligands such as ApoE and Aβ, LRP1 is quickly internalized into early endosomes, followed by endosomal sorting to either recycle back to the plasma membrane or traffic to late endosomes and lysosomes for degradation. However, despite extensive studies in mammalian cell models, the mechanisms underlying this regulation remain murky, primarily due to the difficulties in capturing the highly dynamic and heterogeneous endolysosomal structures as well as the complication from the high degree of gene redundancy for key regulators of membrane trafficking in mammalians. Here, we propose that the vitellogenic oocyte in the classical genetic organism Drosophila is ideal in vivo model to address the above challenges, given its many unique strengths, including the simpler structure and enormous size of the oocyte, its massive receptor-mediated endocytosis of extracellular lipoproteins by a fly LDLR homolog Yolkless (Yl), its large and easily recognizable endolysosomal compartments that are ideal for live- and super-resolution imaging, and the low complexity and redundancy of the fly genome. Further, by creating a novel Yl genome-tagging line that expresses a fully functional Yl-eGFP-3xHA reporter and applying it in a pilot screen in the oocyte, we demonstrated that Yl trafficking follows the canonical endolysosomal trafficking processes and the platform is robust for high- throughput genetic and proteomic studies. Building upon this platform, this study aims to explore the full potential of this vitellogenic oocyte model to identify the key regulators of LDLRs trafficking by integrating the traditional genetic and genome-wide screen tools in Drosophila with advanced imaging and proteomic techniques. The results should lay the groundwork for a comprehensive dissection of the conserved mechanisms that control the intracellular trafficking of an endogenous LDLR protein at a whole organism level under its physiological condition, potentially paving the way for a complete elucidation of the regulatory mechanisms for LRP1 and other LDLR members and for uncovering novel therapeutic targets against AD and other lipid-associated diseases.