A protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development.

Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the integrated stress response, an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap Minos-mediated integration cassette insertion in the crc locus generates a Crc-GFP fusion protein that allows visualization of Crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows Crc-GFP induction in a Drosophila model for retinitis pigmentosa. This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival. This article has an associated First Person interview with the first author of the paper.

Drosophila fabp is required for light-dependent Rhodopsin-1 clearance and photoreceptor survival.

Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional Rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.

Periphery signals generated by Piezo-mediated stomach stretch and Neuromedin-mediated glucose load regulate the Drosophila brain nutrient sensor.

Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.

Neuronally expressed anti-tau scFv prevents tauopathy-induced phenotypes in Drosophila models.

We have derived single-chain variable fragments (scFv) from tau antibody hybridomas and previously shown their promise as imaging diagnostic agents. Here, we examined the therapeutic potential of anti-tau scFv in transgenic Drosophila models that express in neurons wild-type (WT) human tau (htau) or the human tauopathy mutation R406W. scFv expressing flies were crossed with the tauopathy flies and analyzed. Overall, the survival curves differed significantly (p < .0001). Control flies not expressing htau survived the longest, whereas R406W expressing flies had the shortest lifespan, which was greatly prolonged by co-expressing the anti-tau scFv (p < .0001). Likewise, htau WT expressing flies had a moderately short lifespan, which was prolonged by co-expressing the anti-tau scFv (p < .01). In addition, the htau expression impaired wing expansion after eclosion (p < .0001), and caused progressive abdomen expansion (p < .0001). These features were more severe in htau R406W flies than in htau WT flies. Importantly, both phenotypes were prevented by co-expression of the anti-tau scFv (p < .01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (p < .05-0.01), and its prevention of tau-mediated neurotoxicity (p < .05-0.001). In summary, these findings support the therapeutic potential of an anti-tau scFv, including as gene therapies, and the use of Drosophila models for such screening.

highroad Is a Carboxypetidase Induced by Retinoids to Clear Mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa Models.

Rhodopsins require retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, knockdown or genetic deletion of highroad blocks the degradation of folding-defective Rhodopsin-1 mutant, ninaEG69D. Moreover, loss of highroad accelerates the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected in ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter, santa maria, impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality-control mechanisms against misfolded Rhodopsin-1 involve regulation of gene expression by retinoids.

The requirement of IRE1 and XBP1 in resolving physiological stress during Drosophila development.

IRE1 mediates the unfolded protein response (UPR) in part by regulating XBP1 mRNA splicing in response to endoplasmic reticulum (ER) stress. In cultured metazoan cells, IRE1 also exhibits XBP1-independent biochemical activities. IRE1 and XBP1 are developmentally essential genes in Drosophila and mammals, but the source of the physiological ER stress and the relative contributions of XBP1 activation versus other IRE1 functions to development remain unknown. Here, we employed Drosophila to address this question. Explicitly, we find that specific regions of the developing alimentary canal, fat body and the male reproductive organ are the sources of physiological stress that require Ire1 and Xbp1 for resolution. In particular, the developmental lethality associated with an Xbp1 null mutation was rescued by transgenic expression of Xbp1 in the alimentary canal. The domains of IRE1 that are involved in detecting unfolded proteins, cleaving RNAs and activating XBP1 splicing were all essential for development. The earlier onset of developmental defects in Ire1 mutant larvae compared to in Xbp1-null flies supports a developmental role for XBP1-independent IRE1 RNase activity, while challenging the importance of RNase-independent effector mechanisms of Drosophila IRE1 function.