Declining signal dependence of Nrf2-MafS-regulated gene expression correlates with aging phenotypes - PubMed (original) (raw)

Declining signal dependence of Nrf2-MafS-regulated gene expression correlates with aging phenotypes

Mohammed Mahidur Rahman et al. Aging Cell. 2013 Aug.

Abstract

Aging is a degenerative process characterized by declining molecular, cell and organ functions, and accompanied by the progressive accumulation of oxidatively damaged macromolecules. This increased oxidative damage may be causally related to an age-associated dysfunction of defense mechanisms, which effectively protect young individuals from oxidative insults. Consistently, older organisms are more sensitive to acute oxidative stress exposures than young ones. In studies on the Drosophila Nrf2 transcription factor CncC, we have investigated possible causes for this loss of stress resistance and its connection to the aging process. Nrf2 is a master regulator of antioxidant and stress defense gene expression with established functions in the control of longevity. Here, we show that the expression of protective Nrf2/CncC target genes in unstressed conditions does not generally decrease in older flies. However, aging flies progressively lose the ability to activate Nrf2 targets in response to acute stress exposure. We propose that the resulting inability to dynamically adjust the expression of Nrf2 target genes to the organism's internal and external conditions contributes to age-related loss of homeostasis and fitness. In support of this hypothesis, we find the Drosophila small Maf protein, MafS, an Nrf2 dimerization partner, to be critical to maintain responsiveness of the Nrf2 system: overexpression of MafS in older flies preserves Nrf2/CncC signaling competence and antagonizes age-associated functional decline. The maintenance of acute stress resistance, motor function, and heart performance in aging flies overexpressing MafS supports a critical role for signal responsiveness of Nrf2 function in promoting youthful phenotypes.

Keywords: Drosophila; Keap1; Nrf2; aging; oxidative stress; small Maf.

© 2013 John Wiley & Sons Ltd and the Anatomical Society.

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Figures

Figure 1

Figure 1. Physical and genetic interactions between MafS and CncC

A. The CncC pathway in Drosophila. In non-stress conditions the Drosophila ortholog of the Nrf2 transcription factor, CncC, is sequestered in the cytoplasm and targeted for ubiquitin-dependent proteasomal degradation by its negative regulator Keap1. Oxidative stress, electrophiles, or drugs like oltipraz cause inhibition of Keap1-mediated CncC degradation. As a result, CncC accumulates in the nucleus and heterodimerizes with the small Maf protein, MafS. MafS/CncC dimers bind to antioxidant response elements (AREs) and activate the transcription of stress response genes. B. MafS and CncC associate in S2 cells. The cartoon shows a schematic representation of CncC and MafS proteins; the positions of their respective bZIP dimerization domains are indicated. The DLG and ETGE motifs in CncC resemble Keap1 interaction domains that have been characterized in vertebrate Nrf2. Epitope-tagged versions of MafS and CncC (3xHA and 3xFlag, respectively) were expressed in S2 cells as indicated. Western blots (WB) with the denoted antibodies demonstrate that MafS was co-immunoprecipitated with CncC from a lysate of cells in which the two proteins were expressed together. C. MafS can bind to CncC target gene promoters. 3xHA-tagged MafS was expressed in transgenic flies under the control of the ubiquitously active_arm-Gal4_ driver. Chromatin from larvae of this genotype (arm-Gal4 / UAS-mafS)and corresponding controls (arm-Gal4/+) was processed for chromatin immunoprecipitation (ChIP) using anti-HA antibody. Semi-quantitative PCR analyses were conducted to assess MafS binding to ARE-containing sequences from the promoters of the CncC target genes,gstD1, keap1 and gclC. The lanes labeled “input” show PCR assays on total genomic DNA. The promoter region of the hsp26_gene, which does not harbor ARE consensus sequences, serves as negative control. In control experiments omitting the anti-HA antibody, no MafS binding was detected. D. MafS can interact genetically with CncC. Transgenic Drosophila lines carrying UAS-cncC, UAS-mafS, UAS-mafSRNAi_or the photoreceptor-specific_sep-Gal4 driver display normal eye morphology (panels 1–4). Similarly, ectopic expression of MafS or mafS_RNAi with sep-Gal4 does not cause any morphological defect in fly eyes (panels 5 and 6). In contrast, ectopic expression of CncC causes a rough eye phenotype, as previously reported (Sykiotis and Bohmann, 2008). This abnormal eye phenotype isenhanced by co-expression of MafS (panel 8) and is suppressed by co-expression of a MafS-targeting dsRNA (panel 9). The images shown in these panels are representative of phenotypes that for a broadly uniform and consistent for a given genotype.

Figure 2

Figure 2. MafS is required for CncC-target gene expression and oxidative stress resistance

A. Knock-down of MafS decreases basal and inducible CncC reporter gene activity in adult flies. mafSRNAi was ubiquitously expressed in Drosophila adults under the control of the RU486-inducible driver tubulin Gene Switch Gal4 (tub-GS-Gal4). The flies also carried the CncC-responsive ARE-GFP reporter (Sykiotis and Bohmann, 2008). Animals were treated, as indicated, with oltipraz to stimulate CncC activity and/or with 300 μM RU486 to induce the expression of mafSRNAi. Both basal and induced activities of the CncC reporter were suppressed in the presence of mafSRNAi. In a control experiment flies carrying the tub-GS-Gal4 driver, but not the _UAS-mafSRNA i_transgene showed that RU486 itself has no effect on oltipraz-induced CncC activity (upper panels). A parallel experiment was conducted using a control reporter in which the ARE is mutated to render it unresponsive to CncC (mRE-RFP, red images). The low level of control reporter gene activity arising from this reporter was not affected by mafSRNAi expression, thus ruling out an unspecific effect that might have been caused by general suppression of gene activity or viability. B. MafS knock down increases sensitivity to paraquat. MafSRNAi was expressed in Drosophila adults under the control of actin-GS-Gal4 driver, causing a knockdown of mafS, but no lethality. The presence of 300 μM RU 486 in the diet had no effect on paraquat sensitivity in flies carrying the actin-GS-Gal4 driver only (upper graph). In contrast, flies carrying both actin-GS-Gal4 driver and UAS-mafSRNAi showed increased paraquat sensitivity when the expression of the RNAi construct was induced by RU486. Error bars indicate standard deviations of three biologically independent replicates (each replicate with a cohort of ~25 flies) arising from three independent crosses. _p_-values were calculated by two-way ANOVA using Qi Macros software.

Figure 3

Figure 3. MafS over-expression counteracts the loss of stress resistance, motor function, and heart performance in aging flies

A. Ubiquitous over-expression of MafS increases paraquat resistance in old, but not in young flies. Flies of different ages over-expressing MafS (arm-Gal4 / UAS-mafS) or controls carrying the driver or the UAS construct alone (arm-Gal4/+ and UAS-mafS / +) were treated with paraquat and their survival was recorded over a period of up to 26 hours, as detailed in Experimental Procedures. Percent survival over time is plotted for each genotype. Error bars indicate standard deviations of three biologically independent replicates (each with a cohort of ~25 flies) in which flies were collected from separate crosses. Statistical significance was calculated by two-way ANOVA using Qi Macros software. The two asterisks represent _p_-values less than 0.003.Note that the protective effect of MafS overexpression in old flies is evident and statistically significant relative to both controls, the driver or the UAS construct alone. This indicates that the effect is not influenced by different genetic backgrounds. B. MafS expression delays the loss of motor function in aging flies. 10, 20, 30 and 40 day old flies of the genotypes arm-Gal4/+, arm-Gal4 / UAS-mafS and _UAS-mafS/+_were scored for their climbing ability as described in Experimental Procedures. This assay detected no difference between the three genotypes when young flies were tested. Older arm-Gal4 / UAS-mafS flies, however, performed better than the two control groups of matched ages, with progressively larger differences. Error bars indicate standard deviations of three biologically independent replicates. Statistical significance was calculated using two-way ANOVA. The asterisks represent _p_-values less than 0.002. C. MafS expression ameliorates the age-associated decline in heart performance. Heart rhythmicity was analyzed in young flies (7 days after hatching) or in 35-day-old flies of the indicated genotypes. In control flies (Gal4 drivers or UAS-mafS transgene alone, grey bars) the heartbeats became irregular with age, i.e. the arrhythmia index increased, as previously reported (Ocorr et al., 2007). In contrast, overexpression of MafS, either in the myocardium (GMH5-Gal4 / UAS-mafS), or in the pericardial cells (Dorothy-Gal4 / UAS-mafS) prevented the decline in cardiac performance as manifested by an incremental, not statistically significant increase of age-associated arrhythmias (green bars).

Figure 4

Figure 4. Age-associated decline of CncC reporter gene activation by oltipraz is rescued by MafS over-expression

Control flies (arm-Gal4/+ or UAS-mafS/+) or flies over expressing MafS (arm-Gal4 / UAS-mafS), were maintained on standard food for 10 days (left panels) or 40 days (right panels). All flies carried a CncC-responsive GFP reporter (ARE-GFP, (Chatterjee and Bohmann, 2012). Young flies (10days old, left panels) of all genotypes showed efficient reporter activation after transfer to food containing 1 mM of the Nrf2 inducer oltipraz for 48 hours. This response was lost in 40dayold _UAS-mafS/+and_arm-Gal4 / + flies, which failed to efficiently induce ARE-GFP reporter activity in response to oltipraz feeding (middle). In contrast, 40 day old arm-Gal4 / UAS-mafS flies retained the ability to efficiently induce the reporter in response to oltipraz (middle right panel, central two flies). The lower panels show a parallel experiment using the control reporter which is not responsive to CncC. The mRE reporter was constructed with the DSred.T4 gene which can be visualized by its red fluorescence (mRE-RFP). The activity of this mutant reporter did not change with age or oltipraz exposure. Images of the mRE reporter flies where taken at a higher gain because the activity of the mutant reporter is much lower than that of the intact ARE reporter.

Figure 5

Figure 5. MafS restores the age-associated loss of inducibility of endogenous CncC target genes by oxidative stress

10 day old and 40 day old flies over expressing MafS (arm-Gal4/UAS-mafS) and control flies (arm-Gal4/+ or UAS-mafS/+) of the same ages were exposed to paraquat(red bars) or mock treated (blue bars). After 12 hours of treatment 8 flies from each group were collected for quantitative RT-PCR analysis to measure the mRNA expression levels of the CncC target genes_gstD1_, gclC, and gclM. These genes are known to be inducible by pharmacological or genetic activation of CncC and their promoters harbor CncC binding sites as mapped by the Mod Encode project. The y-axis indicates the quantification of the respective mRNA relative to the internal reference, the mRNA of the rp49 gene, which is not regulated by CncC. The fold induction of each gene’s expression in response to paraquat decreases in the controls flies when they age (top and bottom panels), but the over-expression of MafS restores the inducibility (middle panels). Error bars indicate standard deviations of three biologically independent replicates arising from three independent crosses. Statistical significance (_p-_values) was calculated using Student’s T-Test.

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