Abstract
Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity.
Introduction
How reactive oxygen species (ROS) affect aging is a fundamental issue in biology (Back et al., 2012a; Krause, 2007; Hekimi et al., 2011; Ristow, 2014; Ristow and Schmeisser, 2014; Kawagishi and Finkel, 2014; Riera and Dillin, 2015; Durieux et al., 2011; Balaban et al., 2005; Melov, 2002; Shadel, 2014; Sena and Chandel, 2012). Chronic exposure to ROS leads to cellular damage and age-associated diseases, including Alzheimer’s disease, Parkinson’s disease, cancer, diabetes, cardiovascular diseases, and chronic inflammation. By contrast, low or acute ROS exposure mobilizes protective mechanisms and increases lifespan in S. cerevisiae (Pan et al., 2011; Mesquita et al., 2010; Schroeder et al., 2013), D. melanogaster (Albrecht et al., 2011), C. elegans (Schulz et al., 2007; Doonan et al., 2008; Yang and Hekimi, 2010; Schmeisser et al., 2013; Lee et al., 2010), and rodents (Lapointe and Hekimi, 2008; Liu et al., 2005), and has been associated with health benefits in humans (Ristow et al., 2009, 2014). This phenomenon is conceptualized as mitochondrial hormesis or mitohormesis (Ristow and Schmeisser, 2014).
Hormesis is defined as the induction of protective mechanisms under exposure to low doses of stressful agents, which at higher or prolonged exposures are harmful. Ninety percent of cellular ROS arise as a by-product of mitochondrial oxidative phosphorylation (Halliwell and Gutteridge, 2007). Moderate mitochondrial dysfunction leads to elevation of mitochondrial-derived ROS, activating protective mechanisms (mitohormesis) and promoting longevity (Ristow and Schmeisser, 2014). Several longevity interventions, such as dietary restriction or reduced insulin/IGF-1 signaling, are associated with an increase in mitochondrial ROS levels, which acts as a retrograde signal to increase lifespan (Schulz et al., 2007; Weimer et al., 2014; Zarse et al., 2012).
One mechanism by which ROS affect cellular signaling is by specifically and reversibly reducing/oxidizing reactive thiol-groups on cysteine residues, thereby modifying protein functions, which is also known as redox signaling. For instance, ROS promote receptor tyrosine kinase (RTK) signaling by oxidizing a cysteine residue in protein-tyrosine phosphatase 1 (PTP1), thereby transiently inactivating its phosphatase activity (Salmeen et al., 2003; Goldstein et al., 2005) and potentiating the activity of its partner RTK. Localized ROS that act as signals do so at short range (~5–20 µm; [Winterbourn, 2008]) with a half-life of ~1 ms (D’Autréaux and Toledano, 2007), in part due to high intracellular concentration of the antioxidant glutathione (1–10 mM; [Meister, 1988]), which keeps the cytosol in a reduced environment (Gilbert, 1990; Romero-Aristizabal et al., 2014; Lambeth and Neish, 2014). Hence, pools of localized ROS must be rapidly generated for redox signaling to occur.
In addition to ROS derived as a by-product of mitochondrial oxidative phosphorylation, cells have membrane-associated enzymes that generate ROS, using nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor to produce a local ROS micro-environment (Bedard and Krause, 2007). In general, NADPH oxidases form complexes with subunits required for their stability and activation (Bedard and Krause, 2007). For instance, upon stimulation of cell surface receptors, guanosine-trisphospate (GTP) bound Rho-guanosine-triphosphatase (GTPase) family members and p21-activated kinase-1 (PAK1) must be recruited to the NADPH oxidase complex to generate ROS (Hurd et al., 2012).
Mammals have seven NADPH oxidase family members, which have been found in almost every tissue and are localized at cellular membranes and within intracellular compartments, such as endosomes and endoplasmic reticulum (ER) (Bedard and Krause, 2007; Krause, 2007). Mammalian NADPH oxidases have been implicated in a wide range of normal physiological functions, (Bedard and Krause, 2007; Krause, 2007), as well as in diseases that include cancer (Truong and Carroll, 2012).
NADPH oxidase-generated ROS have been shown to act as a second messenger to regulate migration of metastasis-committed-cancer cells and as a chemoattractant for immune cells during wound healing (Stanley et al., 2014; Hurd et al., 2012). Mediator of ErbB2 driven cell motility (Memo1) has been shown to play an important role in migration of breast cancer cells and is needed for robust metastatic dissemination from primary tumors to lungs (Marone et al., 2004; MacDonald et al., 2014). During the migratory process Memo1 interacts with Rho GTPase to dynamically reorganize actin and microtubule fibers (Zaoui et al., 2008), and has also been linked to NADPH oxidase activity in breast cancer cells (MacDonald et al., 2014). However, whether NADPH oxidase generated ROS have a biological function during aging is unknown. Here, we used the model organism Caenorhabditis elegans to investigate the role of NADPH oxidase generated ROS in aging. The nematode C. elegans provides the advantages of genetic tractability, and of being transparent that allows in vivo non-invasive visualization of transgenic fluorescent probes that measure ROS levels (Back et al., 2012b). Moreover, C. elegans has a short lifespan, making it ideal to gain mechanistic insights into the aging process. We found that loss of the C. elegans memo-1/C37C3.8 leads to elevated ROS levels generated by BLI-3/NADPH oxidase, which activates an adaptive detoxification system regulated by the transcription factor SKN-1/Nrf1,2,3 in promoting organismal-wide oxidative stress resistance and longevity.
