a. Mutations of spr-1, spr-3 and spr-4 reduce survival in the presence of oxidative stress. Worms were continuously incubated with the superoxide-generating agent paraquat (5 mM). Shown are representative time courses of survival, and quantification of reduced mean lifespan relative to wild-type in worms incubated with paraquat. Shown are mutants in spr-1 and spr-3, two different mutants in spr-4, and a double spr-3/spr-4 mutant [spr-4(by105);spr-3(ok2525)]. The spr-4(by105);spr-3(ok2525) mutant showed similar survival to wild-type in the absence of paraquat. Values represent the decrease in mean lifespan as % relative to wild-type and represent the mean ± S.D., n=3. *P<0.05 by log-rank test. b. Depletion of spr-4 by RNAi increases sensitivity to oxidative stress and phenocopies the spr-4(by105) mutation. Worms were fed RNAi against the indicated genes or an empty vector control and then transferred to plates seeded with standard OP50 bacteria and containing 5 mM paraquat. Shown is the percent decrease in mean survival relative to the empty vector control from 3 independent experiments. Two spr-4 RNAi-expressing bacterial strains were used (Methods). In addition to N2, we also utilized a C. elegans strain (TU3270) with enhanced dsRNA uptake in neurons (Methods). One of the spr-4 RNAi strains generated a significantly greater reduction in survival in the TU3270 background compared with N2. The paraquat sensitivity of worms fed RNAi against the antioxidant gene sod-1 was used as a positive control. Values are the mean ± S.D., n=3. *P<0.05 by log-rank test. c. A stably integrated SPR4::GFP construct under the control of the endogenous spr-4 promoter is expressed predominantly in neurons in adult worms, as indicated by co-localization with the neuronal marker prab-3::mCherry. Upper panels: pharyngeal ring neurons; lower panels: tail neurons. Scale bar: 20 μm. d. Treatment of adult worms with paraquat (+PQ) from day 1 to day 4 induces expression of SPR4::GFP. Untreated worms (−PQ). Upper panels show confocal imaging of a representative strain; the lower panel graph shows quantitative analysis of SPR4::GFP expression in 3 separate strains. Horizontal bars indicate the median; boxed areas represent the second and third quartiles. n=3 (15 worms each); ***P<0.001 by unpaired t-test. e. REST represses expression of the presenilin hop-1 in C. elegans. Expression of hop-1 mRNA was measured by qRT-PCR in 24-hour post L4 worms of the indicated genotypes. For each replicate, transcript values were normalized to cdc-42. Note that hop-1 mRNA expression is increased in the spr-4(by105) mutant, and that repression is partially restored by wild-type spr-4 or human REST (REST). Values represent fold change relative to the wild-type control (1) and represent the mean ± S.D., n=3. *P<0.05 by Student’s t-test. Scale bars, 20μm.