Alternative pathway of cell death in Drosophila mediated by NF-κB transcription factor Relish.

Photoreceptor cell death accompanying many retinal degenerative disorders results in irreversible loss of vision in humans. However, the precise molecular pathway that executes cell death is not known. Our results from a Drosophila model of retinal degeneration corroborate previously reported findings that the developmental apoptotic pathway is not involved in photoreceptor cell demise. By undertaking a candidate gene approach, we find that players involved in the immune response against gram-negative bacteria are involved in retinal degeneration. Here, we report that the NF-κB transcription factor Relish regulates neuronal cell death. Retinal degeneration is prevented in genetic backgrounds that block Relish activation. We also report that the N-terminal domain of Relish encodes unique toxic functions. These data uncover a unique molecular pathway of retinal degeneration in Drosophila and identify a previously unknown function of NF-κB signaling in cell death.

Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration.

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.

Characterization of two dominant alleles of the major rhodopsin-encoding gene ninaE in Drosophila.

PURPOSE: In this study we investigated the biochemical and cell biologic characteristics of flies expressing two novel dominant alleles of the major rhodopsin encoding gene neither inactivation nor afterpotential E (ninaE) in a heterozygous background. METHODS: Presence of the deep pseudopupil in flies was assayed 5 days post eclosion. For structural analysis, 1-µm-retinal cross sections were obtained from fixed and resin-embedded Drosophila heads. Confocal microscopy was performed on dissected retinas stained with antibodies specific for rhodopsin, NinaA, and F-actin. Rhodopsin levels were determined by western and slot blot analysis. RESULTS: Dominant rhodopsin mutants showed progressive age-dependent and light-independent loss of the deep pseudopupil, without any apparent retinal degeneration at the morphological level. Expression of mutant rhodopsin caused rhodopsin to mislocalize to the cell body and the endoplasmic reticulum compartment. Mutant rhodopsin also caused loss of solubility of wild-type rhodopsin and its accumulation presumably as a high molecular mass complex in the photoreceptor cell body. CONCLUSIONS: In heterozygous mutant flies, there is loss of wild-type rhodopsin immunoreactivity on a western assay but less reduction using slot blot analysis. This suggests that mutant rhodopsin is likely inducing the misfolding and insolubility of wild-type rhodopsin. Localization of rhodopsin revealed that in mutant flies, wild-type rhodopsin is mislocalized to the cell body and the endoplasmic reticulum.

Glycolytic reliance promotes anabolism in photoreceptors.

Vertebrate photoreceptors are among the most metabolically active cells, exhibiting a high rate of ATP consumption. This is coupled with a high anabolic demand, necessitated by the diurnal turnover of a specialized membrane-rich organelle, the outer segment, which is the primary site of phototransduction. How photoreceptors balance their catabolic and anabolic demands is poorly understood. Here, we show that rod photoreceptors in mice rely on glycolysis for their outer segment biogenesis. Genetic perturbations targeting allostery or key regulatory nodes in the glycolytic pathway impacted the size of the outer segments. Fibroblast growth factor signaling was found to regulate glycolysis, with antagonism of this pathway resulting in anabolic deficits. These data demonstrate the cell autonomous role of the glycolytic pathway in outer segment maintenance and provide evidence that aerobic glycolysis is part of a metabolic program that supports the biosynthetic needs of a normal neuronal cell type.