Johan Auwerx

TRANSCRIPTIONAL REGULATION OF APOLIPOPROTEIN A-I GENE EXPRESSION BY THE NUCLEAR RECEPTOR RORALPHA

Since elevated concentrations of plasma high density lipoprotein (HDL) and its major apolipoprotein (apo), apoA-I, confer protection against atherosclerosis, considerable research efforts have focussed on the identification of factors regulating apoA-I gene expression in an attempt to increase its production. Nuclear receptors are interesting candidates because they are transcription factors whose activity is ligand-dependent. In the present study we identified the orphan receptor RORα1 as an activator of apoA-I gene transcription. In apoA-I-expressing intestinal Caco-2 cells, overexpression of the RORα1, but not the RORα2 or RORα3 isoforms, increased rat apoA-I gene transcription. Deletion and site-directed mutagenesis experiments identified a functional ROR-responsive element (RORE) in the rat and mouse apoA-I gene promoters, which overlaps with the TATA box. Gel shift experiments indicated that this RORE binds the RORα1 isoform, but not the RORα2 or RORα3 isoforms. Furthermore, compared with wild type mice, apoA-I mRNA levels were significantly lower in small intestines of staggerer mice homozygous for a deletion in the RORα gene. In addition, reverse transcriptase-polymerase chain reaction analysis revealed the expression of RORα in small intestinal epithelium and in Caco-2 cells. These data indicate a novel, physiological role for RORα1 in the regulation of genes involved in lipid and lipoprotein metabolism and possibly in the development of metabolic diseases, such as atherosclerosis.

A microfluidic array for high-content screening at whole-organism resolution

A main step for the development and the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Among the organisms of choice, Caenorhabditis elegansis a soil worm which catches the interest of researchers who study systemic physiopathology (e.g. metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared to rodents, for which (3) the costs and (4) the ethical concerns are substantial.C. elegansis therefore well suited for large screens, dose-response analysis and target-discovery involving an entire organism. We have developed and tested a microfluidic array for high-content screening, enabling the selection of small populations of its first larval stage in many separated chambers divided into channels for multiplexed screens. With automated protocols for feeding, drug administration and image acquisition, our chip enables the study of the nematodes throughout their entire lifespan. By using a paralyzing agent and a mitochondrial-stress inducer as case studies, we have demonstrated large field-of-view motility analysis, and worm-segmentation/signal-detection for mode-of-action quantification with genetically-encoded fluorescence reporters.