Navigating the Landscape of Translational Geroscience in Canada: A Comprehensive Evaluation of Current Progress and Future Directions.

The inaugural Canadian Conferences on Translational Geroscience were held as two complementary sessions in October and November 2023. The conferences explored the profound interplay between the biology of aging, social determinants of health, the potential societal impact of geroscience and the maintenance of health in aging individuals. Although topics such as cellular senescence, molecular and genetic determinants of aging and prevention of chronic disease were addressed, the conferences went on to emphasize practical applications for enhancing older people's quality of life. This manuscript summarizes the proceeding and underscores the synergy between clinical and fundamental studies. Future directions highlight national and global collaborations and the crucial integration of early-career investigators. This work charts a course for a national framework for continued innovation and advancement in translational geroscience in Canada.

The glyoxylate shunt protein ICL-1 protects from mitochondrial superoxide stress through activation of the mitochondrial unfolded protein response.

Disrupting mitochondrial superoxide dismutase (SOD) causes neonatal lethality in mice and death of flies within 24 h after eclosion. Deletion of mitochondrial sod genes in C. elegans impairs fertility as well, but surprisingly is not detrimental to survival of progeny generated. The comparison of metabolic pathways among mouse, flies and nematodes reveals that mice and flies lack the glyoxylate shunt, a shortcut that bypasses part of the tricarboxylic acid (TCA) cycle. Here we show that ICL-1, the sole protein that catalyzes the glyoxylate shunt, is critical for protection against embryonic lethality resulting from elevated levels of mitochondrial superoxide. In exploring the mechanism by which ICL-1 protects against ROS-mediated embryonic lethality, we find that ICL-1 is required for the efficient activation of mitochondrial unfolded protein response (UPRmt) and that ATFS-1, a key UPRmt transcription factor and an activator of icl-1 gene expression, is essential to limit embryonic/neonatal lethality in animals lacking mitochondrial SOD. In sum, we identify a biochemical pathway that highlights a molecular strategy for combating toxic mitochondrial superoxide consequences in cells.

Endosomal trafficking protein TBC-2 is required for the longevity of long-lived mitochondrial mutants.

Mutations that result in a mild impairment of mitochondrial function can extend longevity. Previous studies have shown that the increase in lifespan is dependent on stress responsive transcription factors, including DAF-16/FOXO, which exhibits increased nuclear localization in long-lived mitochondrial mutants. We recently found that the localization of DAF-16 within the cell is dependent on the endosomal trafficking protein TBC-2. Based on the important role of DAF-16 in both longevity and resistance to stress, we examined the effect of disrupting tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants nuo-6 and isp-1 in Caenorhabditis elegans. Loss of tbc-2 markedly reduced the long lifespans of both mitochondrial mutants. Disruption of tbc-2 also decreased resistance to chronic oxidative stress in nuo-6 and isp-1 mutants but had little or no detrimental effect on resistance to other stressors. In contrast, tbc-2 inhibition had no effect on oxidative stress resistance or lifespan in isp-1 worms when DAF-16 is absent, suggesting that the effect of tbc-2 on mitochondrial mutant lifespan may be mediated by mislocalization of DAF-16. However, this result is complicated by the fact that deletion of daf-16 markedly decreases both phenotypes in isp-1 worms, which could result in a floor effect. In exploring the contribution of DAF-16 further, we found that disruption of tbc-2 did not affect the nuclear localization of DAF-16 in isp-1 worms or prevent the upregulation of DAF-16 target genes in the long-lived mitochondrial mutants. This suggests the possibility that the effect of tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants is at least partially independent of its effects on DAF-16 localization. Overall, this work demonstrates the importance of endosomal trafficking for the extended longevity and enhanced stress resistance resulting from mild impairment of mitochondrial function.

RNA Sequencing of Pooled Samples Effectively Identifies Differentially Expressed Genes.

Analysis of gene expression changes across the genome provides a powerful, unbiased tool for gaining insight into molecular mechanisms. We have effectively used RNA sequencing to identify differentially expressed genes in long-lived genetic mutants in C. elegans to advance our understanding of the genetic pathways that control longevity. Although RNA sequencing costs have come down, cost remains a barrier to examining multiple strains and time points with a sufficient number of biological replicates. To circumvent this, we have examined the efficacy of identifying differentially expressed genes by sequencing a pooled RNA sample from long-lived isp-1 mitochondrial mutant worms. We found that sequencing a pooled RNA sample could effectively identify genes that were found to be significantly upregulated in the two individually sequenced RNA-seq experiments. Finally, we compared the genes significantly upregulated in the two individually sequenced RNA-seq experiments to two previous microarray experiments to come up with a high-confidence list of modulated genes in long-lived isp-1 mutant worms. Overall, this work demonstrates that RNA sequencing of pooled RNA samples can be used to identify differentially expressed genes.

Huntingtin Decreases Susceptibility to a Spontaneous Seizure Disorder in FVN/B Mice.

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT in humans or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in approximately 28% of FVB/N mice, which we have called FVB/N Seizure Disorder with SUDEP (FSDS). These abnormal FVB/N mice demonstrate the cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, mice heterozygous for the targeted inactivation of Htt (Htt+/- mice) exhibit an increased frequency of this disorder (71% FSDS phenotype), while over-expression of either full length wild-type HTT in YAC18 mice or full length mutant HTT in YAC128 mice completely prevents it (0% FSDS phenotype). Examination of the mechanism underlying huntingtin's ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by decreasing huntingtin levels have ramifications for huntingtin-lowering therapies that are being developed to treat HD.

Biological resilience and aging: Activation of stress response pathways contributes to lifespan extension.

While aging was traditionally viewed as a stochastic process of damage accumulation, it is now clear that aging is strongly influenced by genetics. The identification and characterization of long-lived genetic mutants in model organisms has provided insights into the genetic pathways and molecular mechanisms involved in extending longevity. Long-lived genetic mutants exhibit activation of multiple stress response pathways leading to enhanced resistance to exogenous stressors. As a result, lifespan exhibits a significant, positive correlation with resistance to stress. Disruption of stress response pathways inhibits lifespan extension in multiple long-lived mutants representing different pathways of lifespan extension and can also reduce the lifespan of wild-type animals. Combined, this suggests that activation of stress response pathways is a key mechanism by which long-lived mutants achieve their extended longevity and that many of these pathways are also required for normal lifespan. These results highlight an important role for stress response pathways in determining the lifespan of an organism.

Evolutionarily Conserved Role of Thioredoxin Systems in Determining Longevity.

Thioredoxin and thioredoxin reductase are evolutionarily conserved antioxidant enzymes that protect organisms from oxidative stress. These proteins also play roles in redox signaling and can act as a redox-independent cellular chaperone. In most organisms, there is a cytoplasmic and mitochondrial thioredoxin system. A number of studies have examined the role of thioredoxin and thioredoxin reductase in determining longevity. Disruption of either thioredoxin or thioredoxin reductase is sufficient to shorten lifespan in model organisms including yeast, worms, flies and mice, thereby indicating conservation across species. Similarly, increasing the expression of thioredoxin or thioredoxin reductase can extend longevity in multiple model organisms. In humans, there is an association between a specific genetic variant of thioredoxin reductase and lifespan. Overall, the cytoplasmic and mitochondrial thioredoxin systems are both important for longevity.

Endosomal trafficking protein TBC-2 modulates stress resistance and lifespan through DAF-16-dependent and independent mechanisms.

The FOXO transcription factor, DAF-16, plays an integral role in insulin/IGF-1 signaling (IIS) and stress response. In conditions of stress or decreased IIS, DAF-16 moves to the nucleus where it activates genes that promote survival. To gain insight into the role of endosomal trafficking in resistance to stress, we disrupted tbc-2, which encodes a GTPase activating protein that inhibits RAB-5 and RAB-7. We found that tbc-2 mutants have decreased nuclear localization of DAF-16 in response to heat stress, anoxia, and bacterial pathogen stress, but increased nuclear localization of DAF-16 in response to chronic oxidative stress and osmotic stress. tbc-2 mutants also exhibit decreased upregulation of DAF-16 target genes in response to stress. To determine whether the rate of nuclear localization of DAF-16 affected stress resistance in these animals, we examined survival after exposure to multiple exogenous stressors. Disruption of tbc-2 decreased resistance to heat stress, anoxia, and bacterial pathogen stress in both wild-type worms and stress-resistant daf-2 insulin/IGF-1 receptor mutants. Similarly, deletion of tbc-2 decreases lifespan in both wild-type worms and daf-2 mutants. When DAF-16 is absent, the loss of tbc-2 is still able to decrease lifespan but has little or no impact on resistance to most stresses. Combined, this suggests that disruption of tbc-2 affects lifespan through both DAF-16-dependent and DAF-16-independent pathways, while the effect of tbc-2 deletion on resistance to stress is primarily DAF-16-dependent. Overall, this work demonstrates the importance of endosomal trafficking for the proper nuclear localization of DAF-16 during stress and that perturbation of normal endosomal trafficking is sufficient to decrease both stress resistance and lifespan.

Exercise preserves physical fitness during aging through AMPK and mitochondrial dynamics.

Exercise is a nonpharmacological intervention that improves health during aging and a valuable tool in the diagnostics of aging-related diseases. In muscle, exercise transiently alters mitochondrial functionality and metabolism. Mitochondrial fission and fusion are critical effectors of mitochondrial plasticity, which allows a fine-tuned regulation of organelle connectiveness, size, and function. Here we have investigated the role of mitochondrial dynamics during exercise in the model organism Caenorhabditis elegans. We show that in body-wall muscle, a single exercise session induces a cycle of mitochondrial fragmentation followed by fusion after a recovery period, and that daily exercise sessions delay the mitochondrial fragmentation and physical fitness decline that occur with aging. Maintenance of proper mitochondrial dynamics is essential for physical fitness, its enhancement by exercise training, and exercise-induced remodeling of the proteome. Surprisingly, among the long-lived genotypes we analyzed (isp-1,nuo-6, daf-2, eat-2, and CA-AAK-2), constitutive activation of AMP-activated protein kinase (AMPK) uniquely preserves physical fitness during aging, a benefit that is abolished by impairment of mitochondrial fission or fusion. AMPK is also required for physical fitness to be enhanced by exercise, with our findings together suggesting that exercise may enhance muscle function through AMPK regulation of mitochondrial dynamics. Our results indicate that mitochondrial connectivity and the mitochondrial dynamics cycle are essential for maintaining physical fitness and exercise responsiveness during aging and suggest that AMPK activation may recapitulate some exercise benefits. Targeting mechanisms to optimize mitochondrial fission and fusion, as well as AMPK activation, may represent promising strategies for promoting muscle function during aging.

Genetic basis of enhanced stress resistance in long-lived mutants highlights key role of innate immunity in determining longevity.

Mutations that extend lifespan are associated with enhanced resistance to stress. To better understand the molecular mechanisms underlying this relationship, we directly compared lifespan extension, resistance to external stressors, and gene expression in a panel of nine long-lived Caenorhabditis elegans mutants from different pathways of lifespan extension. All of the examined long-lived mutants exhibited increased resistance to one or more types of stress. Resistance to each of the examined types of stress had a significant, positive correlation with lifespan, with bacterial pathogen resistance showing the strongest relationship. Analysis of transcriptional changes indicated that all of the examined long-lived mutants showed a significant upregulation of multiple stress response pathways. Interestingly, there was a very significant overlap between genes highly correlated with stress resistance and genes highly correlated with longevity, suggesting that the same genetic pathways drive both phenotypes. This was especially true for genes correlated with bacterial pathogen resistance, which showed an 84% overlap with genes correlated with lifespan. To further explore the relationship between innate immunity and longevity, we disrupted the p38-mediated innate immune signaling pathway in each of the long-lived mutants and found that this pathway is required for lifespan extension in eight of nine mutants. Overall, our results demonstrate a strong correlation between stress resistance and longevity that results from the high degree of overlap in genes contributing to each phenotype. Moreover, these findings demonstrate the importance of the innate immune system in lifespan determination and indicate that the same underlying genes drive both immunity and longevity.

Huntingtin Overexpression Does Not Alter Overall Survival in Murine Cancer Models.

A reduced incidence of various forms of cancer has been reported in Huntington's disease patients and may be due to pro-apoptotic effects of mutant huntingtin. We tested this hypothesis by assessing the effects of huntingtin protein overexpression on survival in two murine cancer models. We generated YAC HD mice containing human huntingtin transgenes with various CAG tract lengths (YAC18, YAC72, YAC128) on either an Msh2 or p53 null background which have increased cancer incidence. In both mouse models of cancer, the overexpression of either mutant or wild-type huntingtin had no significant effect on overall survival. These results do not support the hypothesis that mutant huntingtin expression is protective against cancer.

Effect of DMSO on lifespan and physiology in C. elegans : Implications for use of DMSO as a solvent for compound delivery.

Dimethyl sulfoxide (DMSO) is a solvent that has been used for basic and medical research based on its ability to dissolve both polar and non-polar compounds. In order to use DMSO to deliver compounds that may impact longevity or neurodegeneration, it is important to first determine the effects of DMSO on aging and physiology. We examined the effect of different concentrations of DMSO on lifespan and development time in C. elegans. We found that DMSO concentrations up to 2% DMSO did not affect longevity in wild-type worms, while concentrations of up to 0.5% DMSO were compatible with normal development times. 0.5% DMSO also had minimal effect on fertility and movement. In summary, our results show that concentrations of DMSO up to 0.5% can be safely used to deliver compounds to C. elegans with little or no modifying effects on lifespan or physiologic rates.

The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan.

FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging.

Mitochondrial thioredoxin system is required for enhanced stress resistance and extended longevity in long-lived mitochondrial mutants.

Mild impairment of mitochondrial function has been shown to increase lifespan in genetic model organisms including worms, flies and mice. To better understand the mechanisms involved, we analyzed RNA sequencing data and found that genes involved in the mitochondrial thioredoxin system, trx-2 and trxr-2, are specifically upregulated in long-lived mitochondrial mutants but not other non-mitochondrial, long-lived mutants. Upregulation of trx-2 and trxr-2 is mediated by activation of the mitochondrial unfolded protein response (mitoUPR). While we decided to focus on the genes of the mitochondrial thioredoxin system for this paper, we identified multiple other antioxidant genes that are upregulated by the mitoUPR in the long-lived mitochondrial mutants including sod-3, prdx-3, gpx-6, gpx-7, gpx-8 and glrx-5. In exploring the role of the mitochondrial thioredoxin system in the long-lived mitochondrial mutants, nuo-6 and isp-1, we found that disruption of either trx-2 or trxr-2 significantly decreases their long lifespan, but has no effect on wild-type lifespan, indicating that the mitochondrial thioredoxin system is specifically required for their longevity. In contrast, disruption of the cytoplasmic thioredoxin gene trx-1 decreases lifespan in nuo-6, isp-1 and wild-type worms, indicating a non-specific detrimental effect on longevity. Disruption of trx-2 or trxr-2 also decreases the enhanced resistance to stress in nuo-6 and isp-1 worms, indicating a role for the mitochondrial thioredoxin system in protecting against exogenous stressors. Overall, this work demonstrates an important role for the mitochondrial thioredoxin system in both stress resistance and lifespan resulting from mild impairment of mitochondrial function.

Identification of Novel Therapeutic Targets for Polyglutamine Diseases That Target Mitochondrial Fragmentation.

Huntington's disease (HD) is one of at least nine polyglutamine diseases caused by a trinucleotide CAG repeat expansion, all of which lead to age-onset neurodegeneration. Mitochondrial dynamics and function are disrupted in HD and other polyglutamine diseases. While multiple studies have found beneficial effects from decreasing mitochondrial fragmentation in HD models by disrupting the mitochondrial fission protein DRP1, disrupting DRP1 can also have detrimental consequences in wild-type animals and HD models. In this work, we examine the effect of decreasing mitochondrial fragmentation in a neuronal C. elegans model of polyglutamine toxicity called Neur-67Q. We find that Neur-67Q worms exhibit mitochondrial fragmentation in GABAergic neurons and decreased mitochondrial function. Disruption of drp-1 eliminates differences in mitochondrial morphology and rescues deficits in both movement and longevity in Neur-67Q worms. In testing twenty-four RNA interference (RNAi) clones that decrease mitochondrial fragmentation, we identified eleven clones-each targeting a different gene-that increase movement and extend lifespan in Neur-67Q worms. Overall, we show that decreasing mitochondrial fragmentation may be an effective approach to treating polyglutamine diseases and we identify multiple novel genetic targets that circumvent the potential negative side effects of disrupting the primary mitochondrial fission gene drp-1.

Targeting Mitochondrial Network Disorganization is Protective in C. elegans Models of Huntington's Disease.

Huntington's disease (HD) is an adult-onset neurodegenerative disease caused by a trinucleotide CAG repeat expansion in the HTT gene. While the pathogenesis of HD is incompletely understood, mitochondrial dysfunction is thought to be a key contributor. In this work, we used C. elegans models to elucidate the role of mitochondrial dynamics in HD. We found that expression of a disease-length polyglutamine tract in body wall muscle, either with or without exon 1 of huntingtin, results in mitochondrial fragmentation and mitochondrial network disorganization. While mitochondria in young HD worms form elongated tubular networks as in wild-type worms, mitochondrial fragmentation occurs with age as expanded polyglutamine protein forms aggregates. To correct the deficit in mitochondrial morphology, we reduced levels of DRP-1, the GTPase responsible for mitochondrial fission. Surprisingly, we found that disrupting drp-1 can have detrimental effects, which are dependent on how much expression is decreased. To avoid potential negative side effects of disrupting drp-1, we examined whether decreasing mitochondrial fragmentation by targeting other genes could be beneficial. Through this approach, we identified multiple genetic targets that rescue movement deficits in worm models of HD. Three of these genetic targets, pgp-3, F25B5.6 and alh-12, increased movement in the HD worm model and restored mitochondrial morphology to wild-type morphology. This work demonstrates that disrupting the mitochondrial fission gene drp-1 can be detrimental in animal models of HD, but that decreasing mitochondrial fragmentation by targeting other genes can be protective. Overall, this study identifies novel therapeutic targets for HD aimed at improving mitochondrial health.

Mild mitochondrial impairment enhances innate immunity and longevity through ATFS-1 and p38 signaling.

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity in model organisms. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain results in upregulation of genes involved in innate immunity, which is driven by the mitochondrial unfolded protein response (mitoUPR) but also dependent on the canonical p38-mediated innate immune signaling pathway. Both of these pathways are required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitoUPR act in concert on the same innate immunity genes to promote pathogen resistance and longevity and that input from the mitochondria can extend longevity by signaling through these pathways. This indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

High confidence ATFS-1 target genes for quantifying activation of the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (mitoUPR) is an evolutionarily conserved pathway that restores homeostasis to the mitochondria after various disturbances. This pathway has roles in both resistance to exogenous stressors and longevity. The mitoUPR is mediated by the transcription factor ATFS-1/ATF-5, which modulates the expression of genes involved in protein folding, metabolism and stress resistance. MitoUPR activation in C. elegans is most commonly evaluated through transcriptional reporter strains for the mitochondrial chaperones HSP-6 and HSP-60. In order to obtain a more comprehensive view of transcriptional changes resulting from activation of the mitoUPR, we compared gene expression changes from three different mitoUPR-activating interventions: mutation of nuo-6, RNA interference (RNAi) knockdown of spg-7,and constitutive activation of ATFS-1. We specifically focused on gene expression changes that are dependent on ATFS-1. From this comparison, we identified 61 high confidence target genes that can be used to monitor mitoUPR activation. Notably, neither hsp-6 nor hsp-60 were significantly upregulated under all three mitoUPR activating conditions. We ranked the 61 genes according to the magnitude of upregulation and identify multiple genes that may serve as robust readouts of mitoUPR activation.

Activation of mitochondrial unfolded protein response protects against multiple exogenous stressors.

The mitochondrial unfolded protein response (mitoUPR) is an evolutionarily conserved pathway that responds to mitochondria insults through transcriptional changes, mediated by the transcription factor ATFS-1/ATF-5, which acts to restore mitochondrial homeostasis. In this work, we characterized the role of ATFS-1 in responding to organismal stress. We found that activation of ATFS-1 is sufficient to cause up-regulation of genes involved in multiple stress response pathways including the DAF-16-mediated stress response pathway, the cytosolic unfolded protein response, the endoplasmic reticulum unfolded protein response, the SKN-1-mediated oxidative stress response pathway, the HIF-1-mediated hypoxia response pathway, the p38-mediated innate immune response pathway, and antioxidant genes. Constitutive activation of ATFS-1 increases resistance to multiple acute exogenous stressors, whereas disruption of atfs-1 decreases stress resistance. Although ATFS-1-dependent genes are up-regulated in multiple long-lived mutants, constitutive activation of ATFS-1 decreases lifespan in wild-type animals. Overall, our work demonstrates that ATFS-1 serves a vital role in organismal survival of acute stressors through its ability to activate multiple stress response pathways but that chronic ATFS-1 activation is detrimental for longevity.

Beneficial and Detrimental Effects of Reactive Oxygen Species on Lifespan: A Comprehensive Review of Comparative and Experimental Studies.

Aging is the greatest risk factor for a multitude of diseases including cardiovascular disease, neurodegeneration and cancer. Despite decades of research dedicated to understanding aging, the mechanisms underlying the aging process remain incompletely understood. The widely-accepted free radical theory of aging (FRTA) proposes that the accumulation of oxidative damage caused by reactive oxygen species (ROS) is one of the primary causes of aging. To define the relationship between ROS and aging, there have been two main approaches: comparative studies that measure outcomes related to ROS across species with different lifespans, and experimental studies that modulate ROS levels within a single species using either a genetic or pharmacologic approach. Comparative studies have shown that levels of ROS and oxidative damage are inversely correlated with lifespan. While these studies in general support the FRTA, this type of experiment can only demonstrate correlation, not causation. Experimental studies involving the manipulation of ROS levels in model organisms have generally shown that interventions that increase ROS tend to decrease lifespan, while interventions that decrease ROS tend to increase lifespan. However, there are also multiple examples in which the opposite is observed: increasing ROS levels results in extended longevity, and decreasing ROS levels results in shortened lifespan. While these studies contradict the predictions of the FRTA, these experiments have been performed in a very limited number of species, all of which have a relatively short lifespan. Overall, the data suggest that the relationship between ROS and lifespan is complex, and that ROS can have both beneficial or detrimental effects on longevity depending on the species and conditions. Accordingly, the relationship between ROS and aging is difficult to generalize across the tree of life.