Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans
Highlights
► Substantial fratxin protein decrease is required to extend lifespan in C. elegans ► Substantial fratxin protein decrease is required to trigger autophagy ► Life-extension correlates with reduced fat content and antioxidants induction ► Lifespan and fat content are inversely modulated by p53-regulated autophagy ► Autophagy induction is an evolutionary conserved response to frataxin deficiency
Introduction
Mutations in genes that directly or indirectly affect the functionality of the mitochondrial respiratory chain (MRC) lead to a variety of devastating disorders in humans (Wallace, 2005). Friedreich's ataxia (FRDA), the most frequently inherited recessive ataxia, is one such disease and it is ascribed to severe deficiency of frataxin, a nuclear-encoded mitochondrial protein involved in the biogenesis of iron–sulfur cluster (ISC) containing proteins (Campuzano et al., 1996, Puccio et al., 2001). Residual levels of frataxin are critical for survival and inversely correlate with disease onset, progression and severity (McDaniel et al., 2001). Symptoms only appear when levels of frataxin are severely decreased, and non-pathological levels of frataxin deficiency are associated with alterations in gene expression profiles (Haugen et al., 2010, Huang et al., 2009). These observations suggest that animals attempt to cope with partial frataxin deficiency by inducing adaptive responses, which, if characterized, may reveal novel therapeutic strategies to prevent or postpone the established disease in humans.
In the nematode Caenorhabditis elegans (C. elegans), complete knock-out of the frataxin ortholog (frh-1) or severe deficiency of other nuclear-encoded MRC proteins leads to pathological phenotypes such as arrested development or short lifespan (Rea et al., 2007, Ventura and Rea, 2007). On the other hand, partial suppression of the same genes, including frh-1 (Ventura et al., 2005), consistent with the induction of beneficial adaptive responses under these conditions, translates into life extension (Ni and Lee, 2010). Nevertheless, after these initial findings, contrasting results were published on lifespan regulation following frh-1 silencing in C. elegans (Vazquez-Manrique et al., 2006, Zarse et al., 2007). Although some explanations (Ventura et al., 2006) could be evoked to reconcile the opposite lifespan outcomes observed in response to frataxin suppression, these conflicting data still await compelling experimental clarification, which are required to gain insight into the biology of aging and to help establish an appropriate C. elegans model to study Friedreich's ataxia.
The extended longevity of animals with reduced expression of genes directly or indirectly involved in regulating MRC functionality is associated with the induction of different stress responses and it is regulated among others by the p53 C. elegans homolog cep-1 (Torgovnick et al., 2010, Ventura et al., 2009). P53 integrates several intrinsic and extrinsic stress signals to modulate intracellular responses and its activation is impaired in frataxin-deficient mammalian cells (Guccini et al., 2011, Palomo et al., 2011). Besides its classical function in response to DNA damage, p53 controls mitochondrial energy metabolism, antioxidant defenses, and autophagy (Maddocks and Vousden, 2011). Macroautophagy (hereafter referred to as autophagy) is a fundamental housekeeping program responsible for recycling of cellular components and necessary for tissue homeostasis; it is required for normal growth, development, proper energy metabolism and aging, and can be stimulated in response to a variety of different stressors, such as oxidative stress, energy deprivation, hypoxia, and mitochondria or DNA damage (Kroemer et al., 2010). The autophagic process is finely regulated by several proteins, which cooperate to coordinate the nucleation, elongation and degradation of autophagosomes in the lysosomes (Klionsky et al., 2010), and are very well conserved between humans and nematodes. Excessive or hindered activation of this finely regulated program can lead to cell death and has been associated with many diseases in humans, including neurodegenerative disorders (Levine and Kroemer, 2008).
Here we show that a substantial amount of frataxin protein expression must be reduced to trigger autophagy, in turn extending lifespan and reducing lipid content in C. elegans. Interestingly, we found that a reciprocal interplay exists in regulating autophagy between levels of frataxin expression and p53/cep-1. Collectively, we demonstrate a causal connection between induction of autophagy and lifespan extension following reduced frataxin expression, and provide a rationale for investigating autophagy in the pathogenesis and treatment of FRDA and possibly other human mitochondrial-associated disorders (HMAD).
Section snippets
Nematode strains and maintenance
We employed standard nematode culture conditions (Stiernagle, 2006). All strains were maintained at 20 °C on Nematode Growth Media agar supplemented with Escherichia coli (OP50 or transformed HT115). The complete list of strains utilized in this work can be found in Supplemental Information (SI).
RNAi feeding
The different dsRNA constructs against frh-1 (I, II, III, IV) were generated using pL440 vector through standard molecular cloning techniques and utilized to transform HT115(DE3) E. coli for RNAi
Substantial suppression of frataxin protein expression is required to extend lifespan in C. elegans
Residual levels of frataxin expression are crucial to maintain cell survival in FRDA disease, but how this is achieved is largely unknown. Moreover, contrasting evidence has been reported following frataxin suppression in modulating C. elegans lifespan (Vazquez-Manrique et al., 2006, Ventura et al., 2005, Zarse et al., 2007). To reconcile discrepancies in the field, and to shed light on mechanistic aspects of frh-1-suppression control of longevity, we generated four dsRNA constructs targeting
Discussion
In this paper we unambiguously show that a substantial level of frataxin protein expression (below 50% of its wild-type level) must be reduced to extend C. elegans lifespan and remodel different aspects of animal metabolism. Importantly, we propose that this is achieved by inducing an adaptive response regulated by cep-1, namely autophagy, a fundamental cellular recycling program.
Different experimental conditions coupled with dose-dependent effects may therefore help to reconcile the opposite
Conclusions
In summary, our findings suggest that stimulation of an intact autophagic flux is an evolutionarily conserved mechanism activated in response to severe frataxin deficiency, which is required in C. elegans to extend lifespan and reduce lipid content in a p53-regulated manner. Since frataxin deficiency affects the expression of different MRC subunits (Rotig et al., 1997) and induces same phenotypic features observed in other RNAi-mediated Mit mutants, a similar autophagic control of lipid content
Acknowledgments
We thank all members of the Laboratory of Signal Transduction for constructive discussions and technical advices, and Joyce Chu for technical help. Some nematode strains were provided by the Caenorhabditis Genetics Center, funded by the National Institute of Health (NIH) National Center for Research Resources. We also thank Drs. Chris Link, Tom Johnson, Richard Roy, and Anton Gartner, for providing additional C. elegans strains. Financial support to carry out this study was provided by the
References (47)
- et al.
A pool of extramitochondrial frataxin that promotes cell survival
J. Biol. Chem.
(2006) - et al.
AMP-activated protein kinase induces a p53-dependent metabolic checkpoint
Mol. Cell.
(2005) - et al.
Autophagy and the integrated stress response
Mol. Cell.
(2010) - et al.
Autophagy and lipid metabolism coordinately modulate life span in germline-less C. elegans
Curr. Biol.
(2011) - et al.
Autophagy in the pathogenesis of disease
Cell
(2008) - et al.
Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila
Dev. Cell
(2005) - et al.
Monitoring the role of autophagy in C. elegans aging
Methods Enzymol.
(2008) - et al.
Methods in mammalian autophagy research
Cell
(2010) - et al.
AMPK and p53 help cells through lean times
Cell Metab.
(2005) - et al.
A role for p53 in mitochondrial stress response control of longevity in C. elegans
Exp. Gerontol.
(2010)
Regulation of lifespan by sensory perception in Caenorhabditis elegans
Nature
Long-lived mitochondrial (Mit) mutants of Caenorhabditis elegans utilize a novel metabolism
FASEB J.
Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion
Science
Aging networks in Caenorhabditis elegans: AMP-activated protein kinase (aak-2) links multiple aging and metabolism pathways
Aging Cell
Rates of behavior and aging specified by mitochondrial function during development
Science
Frataxin participates to the hypoxia-induced response in tumors
Cell Death Dis.
A role for autophagy in the extension of lifespan by dietary restriction in C. elegans
PLoS Genet.
Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: cellular model of pathology
PLoS Genet.
Rescue of Caenorhabditis elegans pharyngeal development by a vertebrate heart specification gene
Proc. Natl. Acad. Sci. U. S. A.
Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant
Proc. Natl. Acad. Sci. U. S. A.
Autophagy, mitochondria and cell death in lysosomal storage diseases
Autophagy
A comprehensive glossary of autophagy-related molecules and processes
Autophagy
A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity
Nat. Genet.
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Current address: Pathobiology Program, University of Washington.