Geert Depuydt

Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2

Significance Recently it has been suggested that metformin, the most commonly used antidiabetic drug, might also possess general health-promoting properties. Elucidating metformin’s mode of action will vastly increase its application range and will contribute to healthy aging. We reveal a signaling cascade in which metformin is able to extend lifespan by increasing the production of reactive oxygen species (ROS). This allowed us to further work at the crossroads of human disease and aging research, identifying a key molecule that is able to translate the ROS signal into a prolongevity cue: an antioxidant peroxiredoxin is also able to activate a lifespan-promoting signaling cascade, here described in detail. Continued research efforts in this field lead toward a targeted improvement of aging-related complications. The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling.

Exploring real-time in vivo redox biology of developing and aging Caenorhabditis elegans.

Reactive oxygen species (ROS) are no longer considered merely toxic by-products of the oxidative metabolism. Tightly controlled concentrations of ROS and fluctuations in redox potential may be important mediators of signaling processes. Understanding the role of ROS and redox status in physiology, stress response, development, and aging requires their nondisruptive, spatiotemporal, real-time quantification in a living organism. We established Caenorhabditis elegans strains bearing the genetically encoded fluorescent biosensors HyPer and Grx1-roGFP2 for the detection of hydrogen peroxide (H(2)O(2)) and the glutathione redox potential, respectively. Although, given its transparency and genetic tractability, C. elegans is perfectly suitable as a model organism for such approaches, they have never been tried before in this nematode. We found that H(2)O(2) treatment clearly induces a dose-dependent, reversible response of both biosensors in the living worms. The ratio of oxidized to reduced glutathione decreases during postembryonic development. H(2)O(2) levels increase with age and this effect is delayed when life span is extended by dietary restriction. In young adults, we detected several regions with distinct redox properties that may be linked to their biological function. Our findings demonstrate that genetically encoded biosensors can reveal previously unknown details of in vivo redox biology in multicellular organisms.

Reduced Insulin/Insulin-like Growth Factor-1 Signaling and Dietary Restriction Inhibit Translation but Preserve Muscle Mass in Caenorhabditis elegans

Reduced signaling through the C. elegans insulin/insulin-like growth factor-1-like tyrosine kinase receptor daf-2 and dietary restriction via bacterial dilution are two well-characterized lifespan-extending interventions that operate in parallel or through (partially) independent mechanisms. Using accurate mass and time tag LC-MS/MS quantitative proteomics, we detected that the abundance of a large number of ribosomal subunits is decreased in response to dietary restriction, as well as in the daf-2(e1370) insulin/insulin-like growth factor-1-receptor mutant. In addition, general protein synthesis levels in these long-lived worms are repressed. Surprisingly, ribosomal transcript levels were not correlated to actual protein abundance, suggesting that post-transcriptional regulation determines ribosome content. Proteomics also revealed the increased presence of many structural muscle cell components in long-lived worms, which appeared to result from the prioritized preservation of muscle cell volume in nutrient-poor conditions or low insulin-like signaling. Activation of DAF-16, but not diet restriction, stimulates mRNA expression of muscle-related genes to prevent muscle atrophy. Important daf-2-specific proteome changes include overexpression of aerobic metabolism enzymes and general activation of stress-responsive and immune defense systems, whereas the increased abundance of many protein subunits of the proteasome core complex is a dietary-restriction-specific characteristic.

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.

FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

Summary Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditis elegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans.

Significance The hypothalamic neuropeptide TRH (thyrotropin-releasing hormone) is one of the major endocrine factors that regulate vertebrate physiology. For decades the general assumption has been that TRH neuropeptides are not present in protostomes, at least not in ecdysozoans, despite the presence of TRH receptor orthologs in these phyla. Here we identify a TRH-related neuropeptide–receptor pathway in the nematode Caenorhabditis elegans. TRH-like neuropeptides activate the C. elegans TRH receptor ortholog in cell-culture cells. Using RNAi and CRISPR/Cas9 reverse genetics, we discovered that TRH-related signaling in the pharyngeal system promotes C. elegans growth. Our study provides evidence of a functional TRH neuropeptide–receptor pathway in invertebrates, suggesting that TRH signaling had evolved in a bilaterian ancestor more than 700 million years ago. In vertebrates thyrotropin-releasing hormone (TRH) is a highly conserved neuropeptide that exerts the hormonal control of thyroid-stimulating hormone (TSH) levels as well as neuromodulatory functions. However, a functional equivalent in protostomian animals remains unknown, although TRH receptors are conserved in proto- and deuterostomians. Here we identify a TRH-like neuropeptide precursor in Caenorhabditis elegans that belongs to a bilaterian family of TRH precursors. Using CRISPR/Cas9 and RNAi reverse genetics, we show that TRH-like neuropeptides, through the activation of their receptor TRHR-1, promote growth in C. elegans. TRH-like peptides from pharyngeal motor neurons are required for normal body size, and knockdown of their receptor in pharyngeal muscle cells reduces growth. Mutants deficient for TRH signaling have no defects in pharyngeal pumping or isthmus peristalsis rates, but their growth defect depends on the bacterial diet. In addition to the decrease in growth, trh-1 mutants have a reduced number of offspring. Our study suggests that TRH is an evolutionarily ancient neuropeptide, having its origin before the divergence of protostomes and deuterostomes, and may ancestrally have been involved in the control of postembryonic growth and reproduction.

Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant

In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose.

Protein Metabolism and Lifespan in Caenorhabditis elegans

Lifespan of the versatile model system Caenorhabditis elegans can be extended by a decrease of insulin/IGF-1 signaling, TOR signaling, mitochondrial function, protein synthesis and dietary intake. The exact molecular mechanisms by which these modulations confer increased life expectancy are yet to be determined but increased stress resistance and improved protein homeostasis seem to be of major importance. In this chapter, we explore the interactions among several genetic pathways and cellular functions involved in lifespan extension and their relation to protein homeostasis in C. elegans. Several of these processes have been associated, however some relevant data are conflicting and further studies are needed to clarify these interactions. In mammals, protein homeostasis is also implicated in several neurodegenerative diseases, many of which can be modeled in C. elegans.