Drosophila Hormone Receptor 96 (DHR96) Regulates Cellular Cholesterol Homeostasis

• Author / Creator

  Gopalakrishnan, Akila

• Cholesterol is well known for its adverse cardiovascular effects, however it has crucial cellular roles. For instance, cholesterol is a key component of eukaryotic cell membranes and constitutes the principle steroid hormone precursor in most animals. Cellular cholesterol concentrations have to be strictly controlled, since too much or too little can be fatal. Hence it is necessary to understand the molecular players that maintain cellular cholesterol homeostasis. Insects are sterol auxotrophs and thus obtain cholesterol or other suitable sterols exclusively from the diet. My data indicates that the nuclear receptor DHR96 functions as a cellular cholesterol sensor to regulate cholesterol metabolism. While DHR96 mutants are phenotypically normal on standard media, they arrest development as second instar larvae on diets with low, but sufficient amount of cholesterol to sustain normal development of wild type populations. I utilized DHR96 mutants as a tool to characterize Drosophila sterol requirements, and carried out rescue experiments on lipid-depleted diets supplemented with different sterols to either entirely replace or partly substitute, for the principal functions of cholesterol in insects. My results suggest a novel unidentified function for cholesterol in insects, and that the prohormone alpha-ecdysone has a biological role in addition to its requirement for 20-hydroxyecdysone synthesis. I identified that DHR96 regulates the expression of several genes with predicted roles in cholesterol uptake, metabolism and transport. My data suggests that DHR96 is required for the appropriate regulation of Niemann-Pick type C-2c (Npc2c), at least in part through its function in the midgut. The Drosophila genome harbors eight Npc2-like genes. Mutations in the single human NPC2 gene cause the fatal neurodegenerative Niemann-Pick Type C disease, characterized by cytotoxic cholesterol accumulation within organelles of nearly all tissues. I report the first observation that Drosophila Npc2c is transcriptionally regulated in a cholesterol- iii and DHR96-dependent manner. Ubiquitous expression of Npc2c-RNAi cause developmental arrest phenotypes that cannot be rescued by cholesterol or the steroid hormone ecdysone. I triggered Npc2c-RNAi in a range of tissues and found that Npc2c function in the prothoracic gland (PG) and the midgut is necessary for viability. In the PG, loss-of-Npc2c function results in a dramatic downregulation of ecdysone biosynthetic enzymes, suggesting that Npc2c is vital for ensuring that cellular sterol levels are available for ecdysone synthesis. These data provide the first evidence to link a nuclear receptor in Drosophila to cholesterol homeostasis and to demonstrate that cholesterol regulates gene expression in Drosophila.

• Subjects / Keywords

  • Nuclear receptor
  • Drosophila
  • Cholesterol

• Graduation date

  Spring 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3T14V22C

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.