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. 2013 Feb;48(2):191-201.
doi: 10.1016/j.exger.2012.12.002. Epub 2012 Dec 13.

Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans

Alfonso Schiavi  1 , Alessandro Torgovnick, Alison Kell, Evgenia Megalou, Natascha Castelein, Ilaria Guccini, Laura Marzocchella, Sara Gelino, Malene Hansen, Florence Malisan, Ivano Condò, Roberto Bei, Shane L Rea, Bart P Braeckman, Nektarios Tavernarakis, Roberto Testi, Natascia Ventura
Affiliations

Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans

Alfonso Schiavi et al. Exp Gerontol. 2013 Feb.

Erratum in

  • Exp Gerontol. 2013 Sep;48(9):1001

Abstract

Severe mitochondria deficiency leads to a number of devastating degenerative disorders, yet, mild mitochondrial dysfunction in different species, including the nematode Caenorhabditis elegans, can have pro-longevity effects. This apparent paradox indicates that cellular adaptation to partial mitochondrial stress can induce beneficial responses, but how this is achieved is largely unknown. Complete absence of frataxin, the mitochondrial protein defective in patients with Friedreich's ataxia, is lethal in C. elegans, while its partial deficiency extends animal lifespan in a p53 dependent manner. In this paper we provide further insight into frataxin control of C. elegans longevity by showing that a substantial reduction of frataxin protein expression is required to extend lifespan, affect sensory neurons functionality, remodel lipid metabolism and trigger autophagy. We find that Beclin and p53 genes are required to induce autophagy and concurrently reduce lipid storages and extend animal lifespan in response to frataxin suppression. Reciprocally, frataxin expression modulates autophagy in the absence of p53. Human Friedreich ataxia-derived lymphoblasts also display increased autophagy, indicating an evolutionarily conserved response to reduced frataxin expression. In sum, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, thus providing the rationale for investigating autophagy in the pathogenesis and treatment of Friedreich's ataxia and possibly other human mitochondria-associated disorders.

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Figures

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Fig. 1
Fig. 1
Substantial frataxin suppression is required to extend C. elegans lifespan. A) frh-1 gene and dsRNA interference constructs. One construct against the entire frataxin genomic CDS (frh-1IV), and three additional constructs targeting the three single exons, respectively named frh-1I, frh-1 II and frh-1III, were generated in this work. B) Frataxin protein (FRH-1) expression was quantified in 1-day-old adult wild-type (WT) animals fed bacteria expressing either empty-vector (con) or one of the four different frh-1 dsRNA. In the left panel FRH-1 expression was revealed through western blot with a specific antibody anti Ce-FRH-1 and normalized to tubulin (TUB). In the first lane 10 ng of FRH-1 recombinant protein (rec) were loaded. FRH-1 appears at the expected molecular weight ~ 15 kDa. In the right panel, bars and errors indicate mean FRH-1 expression ± standard error of the mean (SEM) after densitometric analysis from two independent experiments. *p < 0.05, **p < 0.001 t-test. C) Survival analysis of wild-type animals fed bacteria expressing either empty-vector (con) or the different frh-1 dsRNA.
Fig. 2
Fig. 2
Characterization of neuronal features. A) Sensory function (chemotaxis index), B) proprioception (touch test) and C–D) locomotion activity (body bends and speed), in wild-type (1 day-old adults) animals fed empty-vector (con) or frh-1IV dsRNA (frh-1) expressing bacteria. Bars and errors indicate mean values + SEM from three independent replicates. *p < 0.05 vs control, t-test.
Fig. 3
Fig. 3
Characterization of mitochondria and metabolic features. Levels of A) reactive oxygen species (ROS), B) oxidized/reduced glutathione (GSSG/GSH), C) ATP, D) lipid content and E) lysosome-related organelles (LROs) in 1-day-old adult WT animals fed bacteria expressing either empty-vector (con) or life-lengthening frh-1 dsRNA (frh-1). RFU, Relative Fluorescence Unit. RLU, Relative Luminescence Unit. F–I) Transcript expression of key metabolic enzymes in animals treated as in A. Bars and errors indicate mean fold changes and the 95% confidence interval of the mean, respectively, of cumulative data from three independent experiments. *p < 0.05 of each single gene vs control, t-test.
Fig. 4
Fig. 4
Frataxin suppression acts independently of the AMP kinases, in parallel to CR. A–F) Survival analysis of strains carrying mutant alleles of different AMP regulatory kinases fed bacteria expressing either empty-vector (con) or frh-1 dsRNA (frh-1). G–H) Lysosome-related organelles staining and food intake (normalized pumping rate) in WT and eat-2(ad465) mutants fed bacteria expressing either empty-vector (con) or frh-1 dsRNA (frh-1). Bars and errors indicate mean values ± SEM from different replica. *p < 0.05 vs control, t-test.
Fig. 5
Fig. 5
Frataxin suppression induces autophagy. A–B) Number of autophagosomal GFP+ foci in embryos (A) and seam cells of L3 larvae (B) of C. elegans GFP::LGG-1 transgenic animals fed bacteria expressing empty-vector (con) or frh-1 dsRNA (frh-1). C) Total amount of GFP::LGG-1 protein expression in embryos of animals treated as in A. D) Transcriptional changes of autophagy modulator genes in WT animals treated as in A. E) Autophagosome accumulation assessed by increased formation of LC3II in Friedreich's Ataxia patient derived lymphoblast (FRDA) compared to control lymphoblast derived from a healthy brother (Healthy). During the autophagic process the LC3/LGG-1 protein undergoes a posttranslational modification that accelerates its migration on acrylamide gel (from LC3I to LC3II). Fxn, human frataxin. F) Autophagosome accumulation in FRDA lymphoblasts left untreated or treated for two hours with the lysosome inhibitor NH4Cl. In each panel, bars and errors indicate mean values + SEM after densitometric analysis from three independent replicates. *p < 0.05 vs control, t-test.
Fig. 6
Fig. 6
Autophagy is required to extend lifespan and reduce lipid accumulation in frh-1(RNAi) animals. A) Fertility and viability of WT and unc-51 mutants fed bacteria expressing either empty-vector (con) or frh-1 dsRNAi (frh-1). B) Survival curves of WT animals fed bacteria expressing empty-vector (con), an equal mix of empty-vector and frh-1 dsRNA (frh-1), an equal mix of empty-vector and bec-1 dsRNA (bec-1), or an equal mix of both dsRNA (bec-1;frh-1). C) Number of autophagosomal GFP+ foci in embryos of GFP::LGG-1 animals fed as in B. D) Number of autophagosomal GFP+ foci in embryos of GFP::LGG-1 and of GFP::LGG-1;cep-1(lg12501) strains fed bacteria expressing either empty-vector (con) or frh-1 dsRNA (frh-1). E–F) Content of lipid and of lysosomes-related organelles (LROs) were respectively measured by fixing animals and staining them with Oil-Red-O (E, RIU = relative intensity unit), and by feeding animals with the vital dye Nile Red (F, RFU = relative fluorescence unit) in WT animals treated as in B. Bars and errors indicate mean values + SEM from at least three independent replicates. *p < 0.05 vs control, t-test.
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