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

Abstract

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.

Fig. 1.

Photoreceptor cell death in norpA. Cross-sections (0.5 μm) of retinas of 7-d dark-raised norpA flies (A), norpA flies exposed to 4 d of continuous light (B), norpA flies after 7 d of light exposure (C), and norpA GMRp35 flies exposed to 7 d of constant light (D) are shown. (Scale bar, 17.5 μm.) (E) Ratio of the total number of discernable rhabdomeres to the total number of ommatidia was used as a measure of photoreceptor cell death progression. Errors bars indicate SEM. Data from 7-d light-exposed WT (w¹¹¹⁸) flies are included as a reference in the blue histogram bar. The number of ommatidia was counted for w¹¹¹⁸ (light), 162 (n = 3 flies); for norpA (dark), 137 (n = 3 flies); for norpA (4 d of light), 107 (n = 4 flies); for norpA (7 d of light), 150 (n = 7 flies); and for norpA p35 (7 d of light), 134 (n = 6 flies).

Fig. 2.

Photoreceptor death requires dredd. Cross-sections (0.5 μm) of retinas of ydredd^B118w flies raised in darkness for 6 d (A), ydredd^B118w flies exposed to light for 6 d (B), dredd^B118wnorpA mutants raised in the dark for 6 d (C), ydredd^B118wnorpA flies exposed to 6 d of light (D), and ywnorpA flies exposed to 6 d of constant light (E) are shown. (Scale bar, 17.5 μm.) (F) Ratio of the total number of discernable rhabdomeres to the total number of ommatidia was used as a measure of photoreceptor viability in the indicated genotypes. Data from 6-d light-exposed yw flies are included as a reference in the yellow histogram bar to compare the effects of genetic background. Ratios for ydredd^B118w (dark), 6.73 ± 0.047 (110 ommatidia, 4 flies); for ydredd^B118w (light), 5.4 ± 0.34 (66 ommatidia, 3 flies); for ydredd^B118wnorpA (dark), 5.19 ± 0.65 (58 ommatidia, 3 flies); for ydredd^B118wnorpA (light), 6.68 ± 0.08 (114 ommatidia, 5 flies); and for ywnorpA (light), 1.09 ± 0.07 (92 ommatidia, 6 flies) are shown. Data are the mean ± SEM.

Fig. 3.

(A and B) Retinal cross sections (0.5 μm) of (A) norpA;; rel^E20 and (B) norpA rel^E20/rel^E38 (B) flies exposed to 7 d of constant light. (C and D) Retinal sections from 7 d light-treated (C) norpA; key¹ and (D) norpA; key¹/key⁴ flies reveal rescue of photoreceptor cells in these backgrounds. (Scale bars, 17.5 μm.) (E) Ratio of total number of discernable rhabdomeres to the total number of ommatidia was used as measure of photoreceptor viability in indicated genotypes. Ratio for norpA;;rel^E20, 6.25 ± 0.16 (82 ommatidia, 5 flies); for norpA;;rel^E20/rel^E38, 6.62 ± 0.144 (47 ommatidia, 3 flies); for norpA;key¹, 6.82 ± 0.80 (70 ommatidia, 4 flies); for norpA;key¹/key⁴, 6.68 ± 0.08 (77 ommatidia, 4 flies). Data are Mean ± SEM. (F and G) Rh1 accumulates in the cytoplasm of (F) norpA and (G) norpA rel^E20 retina in a light pulse-chase experiment. (Scale bar, 5 μm.)

Fig. 4.

Relish is activated in norpA. (A) Data show fold change in the dipt transcript level in 2-d light-exposed norpA eyes relative to dark-reared norpA retinas examined by quantitative RT-PCR assay. The dipt mRNA levels in light-exposed norpA rel flies are not significantly different from those in their dark-reared siblings. (B) Kinetics of dipt expression in norpA and norpA rel flies. (C) Quantitative RT-PCR analysis shows the relative change in dipt expression in WT (w¹¹¹⁸) flies. (D) Relative dipt expression in norpA key¹ flies after light exposure. Error bars indicate SEM.

Fig. 5.

(A–E) Eye morphology of (A) GMR-GAL4/+ control flies and those expressing (B) full-length Relish (C), C-terminal domain (D) or the N-terminal domain using the GMR-GAL4 driver. (E) Eye depicting the effect of increased dosage of NTD in the photoreceptors, accomplished by two copies of the UAS-NTD transgene using the GMR-GAL4 driver. (Scale bar, 150 μm.) (F) MS1096-GAL4-driven expression of full-length Relish, CTD or the NTD in the developing wing. Wing of MS1096-GAL4/+ driver line included as a control. (Scale bar, 1 mm.) (G–J) Effect of NTD expression in adult photoreceptors. (G) UAS-NTD tubGAL80(ts) flies were reared at 30 °C for 4 wk and served as a control in this experiment. (H) Retinal degeneration was evident after 1 wk at 30 °C in UAS-NTD GAL80(ts)/LGMR-GAL4 flies. Extending the time period to (I) 2 wk or (J) 4 wk exacerbated the retinal degeneration. (Scale bar, 20 μm.)

Fig. P1.

Drosophila NF-κB Relish as a pro–cell-death molecule. (A) Tangential section of a wild-type (nonmutated) fly raised in darkness showing viable photoreceptor cells, as demonstrated by the dark-staining rhabdomeres (visual centers). (B) Exposure of norpA flies to light leads to photoreceptor cell death, as shown by the loss of rhabdomeres. (C) Mutations in the relish gene rescue photoreceptors from light-induced cell death. (D) Eye morphology of a wild-type (Oregon R) fly. (E–F) Expression of the transcriptionally active, N-terminal domain of Relish is toxic to the eye (E) and the wing (F, Lower wing).