Simultaneous Ablation of the Catalytic AMPK α-Subunit SNF1 and Mitochondrial Matrix Protease CLPP Results in Pronounced Lifespan Extension

Abstract

Organismic aging is known to be controlled by genetic and environmental traits. Pathways involved in the control of cellular metabolism play a crucial role. Previously, we identified a role of PaCLPP, a mitochondrial matrix protease, in the control of the mitochondrial energy metabolism, aging, and lifespan of the fungal aging model Podospora anserina. Most surprisingly, we made the counterintuitive observation that the ablation of this component of the mitochondrial quality control network leads to lifespan extension. In the current study, we investigated the role of energy metabolism of P. anserina. An age-dependent metabolome analysis of the wild type and a PaClpP deletion strain verified differences and changes of various metabolites in cultures of the PaClpP mutant and the wild type. Based on these data, we generated and analyzed a PaSnf1 deletion mutant and a ΔPaSnf1/ΔPaClpP double mutant. In both mutants PaSNF1, the catalytic α-subunit of AMP-activated protein kinase (AMPK) is ablated. PaSNF1 was found to be required for the development of fruiting bodies and ascospores and the progeny of sexual reproduction of this ascomycete and impact mitochondrial dynamics and autophagy. Most interestingly, while the single PaSnf1 deletion mutant is characterized by a slight lifespan increase, simultaneous deletion of PaSnf1 and PaClpP leads to a pronounced lifespan extension. This synergistic effect is strongly reinforced in the presence of the mating-type "minus"-linked allele of the rmp1 gene. Compared to the wild type, culture temperature of 35°C instead of the standard laboratory temperature of 27°C leads to a short-lived phenotype of the ΔPaSnf1/ΔPaClpP double mutant. Overall, our study provides novel evidence for complex interactions of different molecular pathways involved in mitochondrial quality control, gene expression, and energy metabolism in the control of organismic aging.

Keywords: AMPK; CLP protease; Podospora anserina; RMP1; SNF1; aging; development.

FIGURE 1

Deletion of PaClpP affects the energy metabolism. Color-coded heat map shows the fold change log(2) of 5-day-old ΔPaClpP vs. wild-type strains, 20-day-old wild-type vs. 5-day-old wild-type strains, and 20-day-old ΔPaClpP vs. 5-day-old ΔPaClpP strains of four biological replicates, each. For better visualization, log2-fold changes lower than 1 (indicated by ↓) are also presented in darkest blue. In Supplementary Table 1, the exact values are provided. “‡”: In at least one biological replicate, this metabolite is at the detection limit. Abbreviations: citrate/isocitrate, citrate or isocitrate; 2-oxo/ketoglutarate, 2-oxoglutarate or 2-ketoglutarate; 6-P hexose, hexose 6-phosphate; 1/6-P hexose, hexose 1,6-bisphosphate, GAP: glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 2-/3-PG, 2- or 3-phosphoglycerate; PEP, phosphoenolpyruvate; 5-P ribose/ribulose, ribose 5-phosphate or ribulose 5-phosphate; 7-P SH, sedoheptulose 7-phosphate; 6-PGluc, 6-phosphogluconate; PPP, pentose phosphate pathway.

FIGURE 2

Ablation of PaSNF1 affects growth rate and fertility. (A) Phenotypes of ΔPaSnf1, ΔPaClpP, and ΔPaSnf1/ΔPaClpP; the complementation strain ΔPaSnf1/Flag::PaSnf1; and the wild type at the age of 9 days on M2 medium, cultivated at 27°C and in constant light. (B) Growth rate of ΔPaSnf1 (n = 28), ΔPaClpP (n = 22), ΔPaSnf1/ΔPaClpP (n = 25), ΔPaSnf1/Flag::PaSnf1 (n = 21), and wild type (n = 32) on M2 medium at 27°C and in constant light. (C) Content of fruiting bodies from cultures after a 12-day incubation of pairs of opposite mating types (confrontational crosses) of two wild-type strains, of two ΔPaSnf1 strains, of wild type × ΔPaSnf1, or of ΔPaSnf1/Flag::PaSnf1 × ΔPaSnf1, respectively. (C, right) Content of a fruiting body of the cross of two ΔPaSnf1 after two additional days of incubation (14 day). Scale bar of fruiting body content corresponds to 250 μm and the scale bar of ascus pictures corresponds to 100 μm. (D) Quantification of spore morphology. Only mature black spores (10 fruiting bodies of each cross) were evaluated and the relative amount of elliptical spores is indicated. Evaluated ascospores of specific crosses: wild type × wild type n = 1691 spores, ΔPaSnf1 × ΔPaSnf1: n = 760; wild type × ΔPaSnf1: n = 1595; ΔPaSnf1/Flag::PaSnf1 × ΔPaSnf1: n = 1423. (E) Relative amount of fruiting bodies resulting from the spermatization of the wild type (n = 6) and ΔPaSnf1 (n = 6) with different male or female partners. (B,D,E) The values presented are mean values ± SD (^∗∗∗p < 0.001; ^∗∗p < 0.01, two-tailed Student’s t-test).

FIGURE 3

PaSNF1 impacts on lifespan. (A,B) Lifespan analysis at 27°C of ΔPaSnf1 (n = 28, p < 0.001), ΔPaClpP (n = 22, p < 0.001), ΔPaSnf1/ΔPaClpP (n = 25, p < 0.001), and the wild type (n = 32), grown on M2 medium in constant light. (B) Mean lifespan of cultures from panel (A). (C) Lifespan analysis at 27°C of ΔPaSnf1 (mat+) (n = 16, p = 0.0389), ΔPaClpP (mat+) (n = 10, p = 0.0058), ΔPaSnf1/ΔPaClpP (mat+) (n = 13, p < 0.001), and wild type (mat+) (n = 18) on M2 medium in constant light. “(mat+)” represents the mating type “plus” (rmp1-2). (D) Lifespan analysis at 27°C of ΔPaSnf1 (mat–) (n = 12, p < 0.001), ΔPaClpP (mat–) (n = 12, p < 0.001), ΔPaSnf1/ΔPaClpP (mat–) (n = 12, p < 0.001), and wild type (mat–) (n = 14) on M2 medium in constant light. “(mat–)” represents the mating type “minus” (rmp1-1). (E) Lifespan analysis at 35°C of ΔPaSnf1 (n = 17, p = 0.523), ΔPaClpP (n = 28, p = 0.691), ΔPaSnf1/ΔPaClpP (n = 19, p = 0.024), and wild type (n = 25), grown on M2 medium in constant light. (F) Mean lifespan of cultures from panel (E). (A,B,E, and left group in panel F): the data of strains of both mating types are combined (“mat– & mat+”). (A,C–E): p-values of the lifespan curves in comparison to wild type were determined by SPSS with three different statistic tests. A compilation of all p-values is provided in Supplementary Tables 2, 3. (B,F) Shown are mean values ± SEM (^∗∗∗p < 0.001; ^∗∗p < 0.01; *p < 0.05, two-tailed Student’s t-test).

FIGURE 4

Deletion of PaSnf1 leads to impaired autophagy. (A) Western blot analysis of ΔPaSnf1 and wild type expressing the autophagy marker gene PaSod1::Gfp of 7- (PaSod1::Gfp n = 5; ΔPaSnf1/PaSod1::Gfp n = 4; ΔPaSnf1/ΔPaClpP/PaSod1::Gfp n = 3) and 20-day-old (PaSod1::Gfp n = 5; ΔPaSnf1/PaSod1::Gfp n = 5; ΔPaSnf1/ΔPaClpP/PaSod1::Gfp n = 4) strains. 50 μg of total protein extract was separated in a 12% SDS polyacrylamide gel. After transfer on a PVDF membrane, the gel was stained with Coomassie and serves as loading control. The signal of “free GFP” is marked with an arrow. (B) Quantification of the relative amount of “free GFP”/total GFP. The values shown are mean ± SD. (C) Lifespan analysis of ΔPaSnf1 (n = 28, p < 0.001), ΔPaClpP (n = 29, p < 0.001), ΔPaSnf1/ΔPaClpP (n = 9, p < 0.001), and wild type (n = 23), grown on M2 medium without nitrogen and with 1.5-fold glucose at 27°C (constant light). P-values of the lifespan curves in comparison to wild type were determined by SPSS with three different statistic tests. All p-values are provided in Supplementary Tables 2, 3. (D) Mean lifespan of cultures from (C) ± SEM. “mat– & mat+” represents the mean lifespan taking together both mating types; “mat–” shows the mean lifespan of cultures with mating type “minus” (rmp1-1). “mat+” shows the mean lifespan of cultures with mating type “plus” (rmp1-2). (^∗∗∗: p < 0.001; **: p < 0.01; *: p < 0.05, two-tailed Student’s t-test).

FIGURE 5

Ablation of PaSNF1 affects mitochondrial morphology. (A) Confocal laser scanning fluorescence microscopy with stained mitochondria of 7- and 20-day-old ΔPaSnf1, ΔPaClpP, ΔPaSnf1/ΔPaClpP, and the wild type. After aging on M2 medium at 27°C (constant light), pieces of mycelium were used to inoculate microscope slides with a central depression filled with M2 media. After a 1-day incubation at 27°C (constant light), the mycelium was incubated with MitoTracker Red (2 μM, 15 min, in the dark) to visualize mitochondria. The scale bar refers to 10 μm. (B) Western blot analysis of 100 μg total protein extracts of 20-day-old ΔPaSnf1 (n = 3), ΔPaClpP (n = 4), ΔPaSnf1/ΔPaClpP (n = 4), and wild-type strains (n = 3) with an anti-PaDNM1 antibody. (C) Quantification of PaDNM1 (B) after normalization to the Coomassie-stained gel. (D) Western blot analysis of 50 μg mitochondrial protein extracts of 7- and 20-day-old ΔPaSnf1 (n = 3), ΔPaClpP (n = 3), ΔPaSnf1/ΔPaClpP (n = 3), and wild type strains (n = 3) with an anti-PaDNM1 antibody. (E) Quantification of PaDNM1 (D) after normalization to the Coomassie-stained gel. (B,D): 8% SDS polyacrylamide gels were used. After transfer, the gels were stained with Coomassie and served as loading control. The values shown are mean ± SEM (^∗∗∗p < 0.001, two-tailed Student’s t-test).

FIGURE 6

Lack of PaSNF1 does not significantly affect oxygen consumption and impairs growth on glycerol. (A) Oxygen consumption rate (OCR) of mycelium from 7-, 12-, and 20-day-old ΔPaSnf1, ΔPaClpP, ΔPaSnf1/ΔPaClpP, and wild type. [7 days: ΔPaSnf1 (n = 3), ΔPaClpP (n = 6), ΔPaSnf1/ΔPaClpP (n = 4), and wild type (n = 12) // 12 days: ΔPaSnf1 (n = 4), ΔPaClpP (n = 6), ΔPaSnf1/ΔPaClpP (n = 4), and wild type (n = 13) // 20 days: ΔPaSnf1 (n = 3), ΔPaClpP (n = 6), ΔPaSnf1/ΔPaClpP (n = 3), and wild type (n = 10)]. The values presented are mean ± SD. (B) Lifespan analysis of ΔPaSnf1 (n = 19, p < 0.001), ΔPaClpP (n = 18, p < 0.001), ΔPaSnf1/ΔPaClpP (n = 9, p < 0.001), and wild type (n = 10), grown on M2 medium at 27°C with glycerol as carbon source instead of glucose. The p-values of the lifespan curves in comparison to wild type were determined by SPSS with three different statistic tests. A compilation of all p-values can be found in Supplementary Tables 2, 3. (C) Mean lifespan of cultures from panel (B), the values presented are mean ± SEM. “mat– & mat+” represent the combined mean lifespan of both mating types; “mat–” shows the mean lifespan of cultures with mating type “minus” (rmp1-1). “mat+” shows the mean lifespan of cultures with mating type “plus” (rmp1-2) (^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05, two-tailed Student’s t-test).