Friedreich’s Ataxia (FRDA), as well as many other disorders associated with mitochondrial dysfunction, is a chronic, degenerative disease that often becomes clinically apparent later in life. It is probable that protective pathways are activated in the pre-symptomatic phase of FRDA to adapt and cope with dysfunctional mitochondria, thus delaying its clinical appearance. Similar pathways may be induced in C. elegans by a mild decrease of the frataxin protein (Ventura et al., 2006). We have now shown that protective stress-response genes, such as heat shock proteins and antioxidants, are strongly induced in worms in response to mild frataxin silencing. In addition, we found that the tumor suppressor protein p53 has a key role in modulating the phenotypic effects resulting from mitochondrial disruption. Our findings hence invoke new players in the etiology of human mitochondrial associated diseases such as FRDA and Leigh Syndrome.
Protective responses to mitochondrial dysfunction
Hormetic responses to mild heat shock or oxidative stress have been associated with prolonged longevity in C. elegans. When we looked at the contribution of daf-16 and skn-1 (which encode transcription factors that modulate heat shock and oxidative stress responses, respectively), to the longevity increase induced by frh-1 RNAi, we found that loss of neither transcription factor prevented life extension. Our results indicate that oxidative stress response, at least that controlled by these two transcription factors, is not the limiting factor regulating Mit mutant lifespan. Importantly, if, as we have previously hypothesized (Ventura et al., 2006), similar pathways counteract mitochondrial dysfunction in both long-lived animals and in the pre-symptomatic phase of FRDA, our result imply that oxidative stress responses may also play a role in the human disease, but that it might not be the only player in eliciting protective pathways to help delay the established pathology.
Caloric Restriction (CR) extends C. elegans longevity and increases respiratory rate in a SIR2.1 and SKN-1-dependent manner (Wang & Tissenbaum, 2006; Bishop & Guarente, 2007). We found that frh-1 RNAi increased C. elegans lifespan independently of sir2.1 and, as mentioned, skn-1. Our findings do not, however, discount a role for increased mitochondrial respiration resulting from a compensatory induction of mitochondrial biogenesis in response to frh-1 RNAi. Indeed, induction of mitochondrial biogenesis in response to mild mitochondrial dysfunction in the pre-symptomatic phase of FRDA could help sustain cellular viability and thus act to clinically delay disease onset. In humans, p53 is known to regulate both mitochondrial biogenesis and mitochondrial respiration (Donahue et al., 2001; Matoba et al., 2006) and we have now found that p53/cep-1 is required for life extension in the Mit mutants. Derry et al. have reported that loss of cep-1, per se, causes significant transcriptional alterations (Derry et al., 2007), although these findings were not confirmed in a subsequent study (Greiss et al., 2008). Nevertheless, among the purportedly altered genes of the former analysis, there were many encoding for mitochondrial proteins (listed in Table S1). We have previously shown that mitochondrial ETC protein expression is pliant and responds uniquely in different Mit mutants (Ventura & Rea, 2007). Additional work is warranted in order to understand the role played by p53 in the regulation of mitochondrial biogenesis, mitochondrial proteins composition and its relationship with longevity specification and disease prevention in response to mitochondrial dysfunction.
The Janus-faced role of p53
Considerable evidence supports a dual role for p53 in eliciting different responses depending on level of cellular stress (Vousden & Lane, 2007). Mild or transient cellular damage, such as mild increase in free radical production, minor DNA-damage, and transitory glucose or ATP depletion, induce protective p53 pathways that improve antioxidant defenses and DNA repair mechanisms, and fulfill energy requirements. In these instances, p53 temporarily arrests the cell-cycle until the stress has been resolved. More severe oxidative stress, irreparable DNA-damage, or complete ATP deprivation leads to p53-dependent cell death or irreversible cell-cycle arrest (replicative cell senescence). Both p53-dependent responses account for its tumor suppressor activity in humans. Consistent with the Janus-faced activity of p53 (Vousden & Lane, 2007), and with p53’s role as a sensor and mediator of mitochondrial metabolism (Jones et al., 2005; Mandal et al., 2005), we have shown that cep-1, the C. elegans p53 ortholog, modulates Mit mutant lifespan in an opposite manner, depending on the level of mitochondrial stress experienced. cep-1 is required for increased longevity under mild mitochondrial disruption, and for mediating the detrimental effect on lifespan when mitochondrial damage is more severe.
Recently, two Mit mutations that increase lifespan, isp-1(qm150) and clk-1(qm30), were shown to protect against tumor growth in the C. elegans gld-1 tumor-like mutant, in a cep-1-dependent fashion (Pinkston et al., 2006). In conjunction with our present findings, these results imply that modulating mitochondrial function, by way of p53, can both regulate longevity and concurrently serve as an anti-tumor strategy. A similar situation seems to exist in mice where increased, but otherwise normally-regulated, levels of p53 lead to both tumor suppression and anti-aging effects (Matheu et al., 2007).
p53-activating stressors can lead to transient cell-cycle arrest or apoptosis. While we cannot rule out the possibility that apoptosis via CEP-1 independent pathways play a role in the appearance of Mit mutant phenotypes, our results with egl-1 and atm-1 knock out strains, suggest that apoptosis is not the main mechanism through which CEP-1 is extending longevity in the Mit mutants. On the other hand, deregulated induction of a CEP-1-dependent apoptotic pathway may explain the short lifespan induced by severe mitochondrial stress (this study and (Senoo-Matsuda et al., 2003). Independent of its pro-apoptotic activity, EGL-1 is known to also induce mitochondrial fragmentation (Delivani et al., 2006), a process that may contributes to its lifespan shortening effect (Chan & Mattson, 1999).
In worms the pro-apoptotic and anti-proliferative effects of cep-1 can follow different pathways (Derry et al., 2007). Moreover, in the gld-1 mutant, the two long-lived Mit mutants, isp-1 (qm150) and clk-1(qm30), reduce germ cell hyperproliferation without inducing germ cell apoptosis (Pinkston et al., 2006). Furthermore, aak-2 is an AMP kinase recently shown to be required for isp-1 and clk-1 Mit mutant longevity (Curtis et al., 2006). This same kinase is also necessary for the longevity increase induced by mild treatment of atp-3 RNAi (our unpublished observation). Two papers have shown that, in response to mitochondrial dysfunction and energy deprivation, p53 induces cell-cycle arrest via AMP kinase-dependent phosphorylation (Jones et al., 2005; Mandal et al., 2005). These observation suggest that an AMPK/CEP-1 metabolic checkpoint may play an integral role in specifying Mit mutant longevity.
Another role of p53, which is Janus-faced and therefore potentially relevant to mitochondrial stress, is the control of autophagy (Maiuri et al., 2007; Levine & Abrams, 2008). Autophagy is a form of organelle and cellular digestion that can be either necessary or detrimental for tissue homeostasis and organismal survival. It has been associated both with prevention and causation of diseases in human, including neurodegenerative disorders (Vellai et al., 2007; Levine & Kroemer, 2008). Autophagic genes are required during normal development in C. elegans and also for the increased lifespan observed in several mutant backgrounds, including a p53/cep-1 knock out strain (Melendez et al., 2003; Tavernarakis et al., 2008; Toth et al., 2008). Moreover, gain-of function mutation of the sole C. elegans BH3-only protein EGL-1, induces autophagy, while deletion of EGL-1 compromises starvation-induced autophagy (Maiuri et al., 2007). Based on our results it will be interesting to determine whether autophagy is associated and/or necessary to specify longevity resulting from different degrees of mitochondrial dysfunction.
A developmental checkpoint role for cep-1: the whole p53 family in one gene?
In our studies we found that the absence of cep-1 partially rescued the developmental arrest induced by severe mitochondrial damage. Importantly, we showed that cep-1 mutants also prevented the developmental arrest induced by treatment with EtBr, ruling out the possibility that lack of cep-1 is simply modulating RNAi potency rather then the response to mitochondrial stress. A developmental role of cep-1 in response to stress is consistent with a cep-1-dependent metabolic checkpoint acting during C. elegans development (Rea et al., 2007) and/or by cep-1 functions reminiscent of other p53 family members, such as those of p63 and p73 in differentiation and development (D’Erchia et al., 2006; Ou et al., 2007). Under normal, well-fed conditions, cep-1 appears to be dispensable for C. elegans development. Only under conditions of increased energy and nucleotide demand, when its function may be required for mitochondrial biogenesis, we observed a requirement for cep-1. These conditions may exist when cells are rapidly proliferating, such as during embryonic C. elegans development or at the transition between L3–L4 stages, the point of gonad formation. The regulation of animal lifespan through a mitochondrial checkpoint in proliferating cells during animal development is an interesting possibility.