A muscle-specific p38 MAPK/Mef2/MnSOD pathway regulates stress, motor function, and life span in Drosophila

Abstract

Molecular mechanisms that concordantly regulate stress, life span, and aging remain incompletely understood. Here, we demonstrate that in Drosophila, a p38 MAP kinase (p38K)/Mef2/MnSOD pathway is a coregulator of stress and life span. Hence, overexpression of p38K extends life span in a MnSOD-dependent manner, whereas inhibition of p38K causes early lethality and precipitates age-related motor dysfunction and stress sensitivity, that is rescued through muscle-restricted (but not neuronal) add-back of p38K. Additionally, mutations in p38K are associated with increased protein carbonylation and Nrf2-dependent transcription, while adversely affecting metabolic response to hypoxia. Mechanistically, p38K modulates expression of the mitochondrial MnSOD enzyme through the transcription factor Mef2, and predictably, perturbations in MnSOD modify p38K-dependent phenotypes. Thus, our results uncover a muscle-restricted p38K-Mef2-MnSOD signaling module that influences life span and stress, distinct from the insulin/JNK/FOXO pathway. We propose that potentiating p38K might be instrumental in restoring the mitochondrial detoxification machinery and combating stress-induced aging.

Figure 1. p38Kb is expressed widely in adult Drosophila

A) Schematic of the p38Kb genomic region depicting three transposon (KG01337) excision induced deletion mutations. B) Western analysis of head and thorax protein in p38Ka and p38Kb mutants probed with anti-phospho-p38K and anti-total p38K antibodies (β-tubulin is used as a loading control). C) Western blot with anti-total p38K antibodies shows muscle overexpression of p38Kb using MHC-GAL4, Mef2-GAL4 or 24B-GAL4 (control is below detection). D) Quantification of qRT-PCR experiments to show abundance of p38Ka and p38Kb mRNA in p38K mutants. E) RNA in situ experiments in the larval brain to detect p38Kb transcript in control and mutant animals. F) p38b-GAL4 expression in both adult brain and flight muscles visualized through the expression of a nuclear-GFP transgene (middle column and green in merged image). Brains are either counter-stained with an antibody to Elav (top row) or an antibody to the active zone protein Brp (middle row). Muscles are counter-stained with fluorescently conjugated Phalloidin to label actin bands. Scale bar for middle row is 50μm and for bottom row is 20μm. Error bars in all figures denotes SEM. See also Figures S1 and S2.

Figure 2. Muscle p38 MAP Kinase activity controls lifespan in Drosophila

A) Lethality profiles of p38K mutants as compared to controls. Dotted lines represent 50% lethality. B) Reduced lifespan in the p38K-DKO animals is rescued through add back of p38Kb in muscles (Mef2-GAL4) but not in neurons (elav^C155-GAL4). C, D and E) Lifelong expression of wild type p38Kb in muscles, using the MHC-GAL4, Mef2-GAL4 and 24B-GAL4 respectively, significantly extends lifespan in a control wild type genetic background. F) Pan-neuronal expression of p38Kb in a wild type genetic background fails to extend lifespan as compared to GAL4-only or UAS-only controls. G, H) Expression of p38K in adult muscle using the GAL4 lines DJ757 and DJ694 also extends lifespan. I) A similar phenotype is observed when p38K is expressed in adult muscles using the TARGET system to limit expression from the MHC-GAL4 line post-eclosion. J) Adult only expression of p38K in the nervous system does not impact lifespan. Males and females were tested independently with similar outcomes. (Mean lifespan in days: Oregon-R controls = 48; yw controls = 37; p38K-DKO = 5; p38Ka^del = 29; p38Kb^Δ45 = 20; p38K-DKO-Mef2-GAL4 = 12; p38K-DKO-Mef2-GAL4-UAS-p38Kb[WT] = 30; p38K-DKO-elav^C155-GAL4 = 10; p38K-DKO-elav^C155-GAL4-UAS-p38Kb[WT] = 14; p<0.01 in each case, Log Rank test). Female only data shown. See also Table S1.

Figure 3. p38K protects against age-dependent motor deficits

A) Graph plotting the percentage of flies that successfully complete a simple negative geotaxis test (climbing assay) and the variation in their performance with age (1, 3 and 15 day old flies). B) Altered gait and walking patterns in single and p38K-DKO mutants (traces depict “footprints” made by flies on carbon coated glass plates). C) Videographic analysis of exploratory walking in open field tests of individual flies from control and p38K mutant or transgenic animals. D) Representative tracks made by individual flies of particular genotypes. p<0.01 for all significant differences denoted by asterisks, one-way ANOVA. Only female data shown. See also Figures S3, and movies S1, S2, S3, and S4.

Figure 4. Muscle p38Kb is necessary and sufficient for resistance to oxidative stress

A) p38Kb^Δ45 mutants and p38K-DKO animals are hyper-sensitive to Hydrogen Peroxide exposure through continuous feeding. B) Peroxide sensitivity in p38K-DKO animals can be rescued significantly by the expression of wild type p38Kb in muscles (using Mef2-GAL4). C) Expression of wild type p38Kb in wild type animals confers additional resistance to Peroxide. A similar effect is seen when p38Kb is expressed in a spatio-temporal domain specified by the p38Kb-GAL4 line (D). p values = control vs p38Kb^Δ45 <0.05 at 48 hrs and <0.01 for 64 hrs to 104 hrs; control vs p38K-DKO <0.05 at 32 hrs and 112 hrs and <0.01 40 hrs to 104 hrs; p38K-DKO vs Mef2 rescue <0.05 at 24 hrs to 40 hrs and <0.01 for 48 hrs to 72 hrs and 88 hrs and 96 hrs; p38K-DKO vs C155 rescue <0.05 at 32, 56, 64, 88, 104, and 112 hrs, <0.01 at 120 hrs; UAS-p38 wt/Mef2 vs OR/Mef2 <0.05 at 108, 120 and 156 hrs, <0.01 at 132 and 144 hrs; UAS-p38 wt/C155 vs OR/C155 <0.05 at 96 and 156 hrs, <0.01 from 108 to 144 hrs. See also Figure S4.

Figure 5. Cellular markers of oxidative stress are upregulated in p38K mutants

A) Oxyblot analysis measuring total protein carbonylation in neuronal and muscle tissue (head and thorax) of age-matched control, p38Kb^Δ45, and p38K-DKO animals. B) Quantitative comparison of total protein carbonylation between 3 day old control and p38K-DKO animals. (Actin is used as a loading control). C) Paraquat feeding increases protein carbonylation in control animals and to a greater extent, in p38K-DKO animals (“+” denotes Paraquat feeding for a 4 hour period). (D, E, F) Quantitation of GFP expression from an in vivo ARE (anti-oxidant response element) dependent reporter of GST-D1 transcription in the whole fly supports a model in which p38K normally functions to inhibit Nrf2 activity (G). See also Figure S5.

Figure 6. p38K controls MnSOD expression in muscles to regulate lifespan

A) Western blot to measure MnSOD expression in different genotypes. B) Quantification of western blots in A. C) Quantification of viability in p38K mutants and the effect of manipulating MnSOD, CuZnSOD and Catalase in a p38K mutant background. D) Lifespan measurements in p38K mutants and the effect of supplementing MnSOD. E) Lifespan profile of animals overexpressing p38Kb with simultaneous knockdown of MnSOD in muscle. F) Dominant genetic interaction between p38Kb^Δ45 and MnSODⁿ²⁸³ mutant alleles in lifespan regulation. G) Western analysis of MnSOD protein levels following manipulations of MnSOD and p38Kb.

Figure 7. p38 Kinase regulates muscle MnSOD through the transcription factor Mef2

A) Biochemical assay for mitochondrial and cytoplasmic aconitase in age-matched control and p38K-DKO animals. B) Partial reduction of p38K activity renders flies highly sensitive to hypoxia stress and results in smaller adult body size (hypoxia is 5% oxygen). C) Quantification of a mitochondrial protein Drp1 in p38K mutant animals. D) Semi-quantitative RT-PCR and western blotting to measure Mef-2 mRNA and protein expression and SOD protein expression in DfMef-2^X1/+ animals. E) Comparison of MnSOD mRNA levels between thorax and head in control and DfMef-2^X1/+ animals. F) Competitive EMSA (Electrophoretic mobility shift assay) to test the binding potential of one of the five Mef2 binding sites identified by in silico scans using Drosophila nuclear extracts. G) Western blots to measure MnSOD levels following inhibition of Mef2-dependent transcription in muscle tissue using either the MHC-GAL4 or Mef2-GAL4 driver line. These results are quantified in (H). I) Comparison of signaling pathways that regulate stress and lifespan. See also Figure S6.