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Vous êtes ici : Accueil / Équipes / Mort Cellulaire Régulée et Génétique de la Neurodégénerescence - B. Mollereau / Research projects / A recessive-lethal screen to identify regulators of neurodegeneration

A recessive-lethal screen to identify regulators of neurodegeneration

Dominant modifier screens are a powerful means to isolate regulators of apoptosis. However, it can only yield candidates for which gene dosage is important. To circumvent this issue, we set up a new screening technique to perform a recessive screen for genes affecting cell survival in adult photoreceptor cells (Gambis A, Dourlen P, et al 2011).  We combined the FLP/FRT mitotic recombination system and the cornea neutralisation technique, which enabled us to directly visualize  PRs expressing fluorescent proteins (GFP or tomato-RFP) in the living flies immoblized under an immersion objective..  To conduct the screen, we marked the homozygous and heterozygous wild-type PRs by expressing both the GFP and tomato RFP, and the mutant PRs with GFP alone. This new double-labeling system allowed us to easily distinguish the mutant tissues from the wild-type ones. We validated the results of the screen by the analyses of known mutations. From a collection of 450 FRT-recombined lines with lethal recessive mutations, we identified 141 mutations that compromise photoreceptor survival. Next, we performed a secondary screen in the context of p35-induced PR degeneration. We recovered nine candidates that cause retinal degeneration in a caspase-dependent manner. We will characterize the role of the candidate genes on the regulation of cell death in PRs. Using physiological reporters and sensors available in the lab, we will test whether these mutants affect pathways such as UPR, autophagy or development.

Using this approach we  characterized a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance (Dourlen et al. 2012).