Fight Aging! Newsletter, July 23rd 2018

Authors have recently reported that infection with a different herpes virus, herpes simplex virus type 1 (HSV1), leads to a similarly increased risk of later developing senile dementia (SD). Further, when the authors looked at patients treated aggressively with antiherpetic medications at the time, the relative risk of SD was reduced by a factor of 10. It should be stressed that no investigations were made on subjects already suffering from SD, and that those treated were the few rare cases severely affected by HSV. Nonetheless, antiherpetic medication prevented later SD development in 90% of their study group. These articles provide the first population evidence for a causal link between herpes virus infection and senile dementia.

Investigators have long suspected that pathogenic microbes might contribute to the onset and progression of Alzheimer's disease (AD) although definitive evidence has not been presented. Whether such findings represent a causal contribution, or reflect opportunistic passengers of neurodegeneration, is also difficult to resolve. We constructed multiscale networks of the AD-associated virome, integrating genomic, transcriptomic, proteomic, and histopathological data across four brain regions from human post-mortem tissue. We observed increased human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) from subjects with AD compared with controls. These results were replicated in two additional, independent and geographically dispersed cohorts. We observed regulatory relationships linking viral abundance and modulators of APP metabolism.

Amyloid-β peptide (Aβ) fibrilization and deposition as β-amyloid are hallmarks of Alzheimer's disease (AD) pathology. We recently reported Aβ is an innate immune protein that protects against fungal and bacterial infections. Fibrilization pathways mediate Aβ antimicrobial activities. Thus, infection can seed and dramatically accelerate β-amyloid deposition. Here, we show Aβ oligomers bind herpesvirus surface glycoproteins, accelerating β-amyloid deposition and leading to protective viral entrapment activity in mouse and human neural cell culture infection models against neurotropic herpes simplex virus 1 (HSV1) and human herpesvirus 6A and B. Herpesviridae are linked to AD, but it has been unclear how viruses may induce β-amyloidosis in brain. These data support the notion that Aβ might play a protective role in central nervous system innate immunity, and suggest an AD etiological mechanism in which herpesviridae infection may directly promote Aβ amyloidosis.

Aging is associated with an increased prevalence of multiple comorbidities, including infectious and malignant diseases. Many of these disorders are thought to stem from old-age-related immune decline. Increasing efforts to characterize the immune system of elderly people in recent years have revealed that most immunocompetent cell compartments present profound quantitative as well as qualitative impairments. The cause of these impairments can vary, and is often related to the exhaustion of the cells or their functions over time in inflammatory settings.

The majority of mature blood cell compartments need, therefore, to be continuously replenished or replaced, which is the role of hematopoietic progenitor cells (HPCs) and, ultimately, hematopoietic stem cells (HSCs). While the self-renewal and differentiation potential of stem cells, along with their blood cell reconstitution capacity, have long been considered as infinite, increasing evidence indicates that this is not the case. Under conditions of stress, HSCs eventually exhibit several functional defects, including a diminished regenerative and self-renewal potential. Loss of stem cell activity is therefore a likely mechanism of impairment common to many mature cell types, thus representing a central cause of immune-competence decline.

Most studies on HSC aging have been carried out in mouse models, and have highlighted extrinsic and intrinsic factors affecting the function of HSCs. A recent study reveals that loss of autophagy in most HSCs from aged mice causes an activated metabolic state, which is associated with accelerated myeloid differentiation, and impairs HSC self-renewal activity and regenerative potential. In humans, much less information is available on the aged HSCs, due to the limited and challenging access to bone marrow samples of elderly humans, the niche of HSCs. Reduced transplantation success in patients receiving HSCs isolated from older (45 years and above) donor bone marrows indicates that human HSC regenerative capacity also declines with aging.

We performed here a comprehensive study of blood HPCs, as an alternative to bone marrow HSCs, to overcome the constraint of sample availability from elderly adults. Based on phenotypic analyses, in vitro T lymphocyte differentiation assays, and gene expression profiling of circulating HPCs from aged subjects, we demonstrate impaired lymphopoiesis and active cell cycling of HPCs with aging, and provide insights into their functional impairments. Our findings reveal that, while mobilized, elderly HPCs present evidence of cellular senescence and increased cell death by pyroptosis. Reduced telomere length and telomerase activity in old HPCs may affect the properties of their progeny, such as mature T lymphocytes. This pre-senescent profile is characteristic of the multiple intrinsic and extrinsic factors affecting HPCs in elderly individuals and represents a major obstacle in terms of immune reconstitution and efficacy with advanced age.

Aging is a variety of distinct processes, damages, and errors; therefore, simply treating aging in clinical terms is not a viable endpoint. For a clinical trial to be conducted, it requires a verifiable indication, and aging is too general for the FDA and EMA to classify it as a disease. However, the majority of damage repair therapies, if not all, could be developed as therapies for diseases with accepted indications and verifiable endpoints, which should satisfy bodies such as the FDA and EMA. Therefore, whether regulatory agencies perceive aging as a disease or not is of no consequence to the development of rejuvenation biotechnologies that address the aging processes.

Even though classifying aging as a disease is unnecessary, significant reform in the regulatory system is still needed in order to encourage investors and companies to put the time and money into researching and developing rejuvenation therapies. One area in need of reform is the establishment of aging biomarkers, which indicate the repair or removal of age-related damage, as acceptable endpoints for rejuvenation therapies. Studies that use these biomarkers would also need to include long-term follow-up studies to ascertain the effects of a therapy over a longer period of time. Another area where regulatory bodies have struggled is keeping up with the rapid march of technology and medicine. Technologies such as gene therapies have struggled to gain traction due to an antiquated regulatory framework struggling to cope with them. Thankfully, this is also being acknowledged.

Aging not being classified as a disease by the FDA, EMA, etc. is not a major issue; the real need is for policy changes that make developing drugs and therapies that target the aging processes easier and more financially viable. It is good that changes are being made to current frameworks and that progress will almost certainly continue in these areas. Meanwhile, we can continue to support the development of repair-based approaches to aging knowing that such therapies, if they work, will be approved even in the current regulatory landscape.

Antoxerene, Inc., a portfolio company of Ichor Therapeutics, Inc., focused on small molecule drug discovery for pathways of aging, announced today the launch of a joint venture with Juvenescence Limited. The joint venture, called FoxBio, Inc., will develop Antoxerene's collection of small molecules that target senescent cells. Juvenescence will provide the initiative 10 million in equity financing and drug development expertise.

"There has been a lot of interest surrounding the therapeutic applications of senolytic drugs - compounds that clear toxic senescent cells - particularly with respect to age-associated disease. As molecular pathways unique to senescent cells have begun to be identified, we can now develop drugs to target these pathways. We are eager to work with the Juvenescence team, whose experience in drug development, technical depth, and visionary leadership will help us to deliver on the immense potential of this field."

CEO of Juvenescence Dr. Bailey said, "this is one of the main focuses of Juvenescence - to modify aging through the clearance of senescent cells. FoxBio plays to both companies' strengths, which is why we are excited about working with the Antoxerene team. We are fascinated with the Antoxerene platform and its ability to discover intriguing compounds in the area of senescence. This is a great fit with Juvenescence's track record in drug development, so FoxBio is a very exciting new company in the area of longevity."

Despite its importance for health, most epidemiological research considers aging merely as a confounder, a nuance dimension to be accounted for and then discarded, under the assumption that aging is unavoidable and unchangeable. This view is now changed. As the intrinsic biological mechanism of aging is slowly revealed, there is hope that interventions that slow aging and prevent or delay the onset of chronic disease and functional impairments can be discovered.

A critical goal in the field of aging biomarkers is to identify molecular changes that show robust patterns of change with normal aging, with the assumption that departures from this "signature" pattern provide not only information regarding future risk of pathology and functional decline but also clues on compensatory mechanisms by which our organism counteracts the effects of aging. Such a signature could be used both to identify individuals in the trajectory of accelerated aging and to track the effectiveness of interventions designed to slowdown biological aging.

The "epigenetic clock," a biomarker index that combines weighted information of a subset of DNA methylation sites raised great interest because it is strongly associated with chronological age and predicts multiple health outcomes, including cardiovascular disease, cancer, and mortality. These findings suggest that aging is associated with stereotyped and reproducible molecular changes that can potentially be used to identify individuals who are aging faster or slower than the average population. However, the underpinnings of these molecular changes have not been fully elucidated, at least in part because the effect of methylation on DNA function remains unclear.

A promising alternative to current methods may be to construct a similar aging biomarker clock based on circulating proteins. Proteins are attractive because they directly affect phenotypes and provide direct information on biological pathways that can be involved in many of the physiological and pathological manifestations of aging. We conducted proteomic analyses that measured 1,301 proteins in 240 adults aged 22-93 years, free of major chronic diseases, cognitive, and functional impairment. The goal was to identify proteins associated with chronological age avoiding as much as possible the effect of clinically detectable disease, examine their association with several clinical characteristics, and further compare our results to previous proteomic profile analyses that used the same technology.

We found 197 proteins were positively associated, and 20 proteins were negatively associated with age. The functional pathways enriched in the 217 age-associated proteins included blood coagulation, chemokine and inflammatory pathways, axon guidance, peptidase activity, and apoptosis. We created a proteomic signature of age based on relative concentrations of 76 proteins that highly correlated with chronological age. However, the generalizability of our findings needs replication in an independent cohort.

resTORbio today announced newly published data from a Phase 2a clinical trial demonstrating that target of rapamycin complex 1 (TORC1) inhibitor treatment improved immune function and decreased incidence of all infections, including respiratory tract infections (RTIs), in people aged 65 years and older. RTIs in particular are a significant health risk for the elderly with life-threatening consequences and few treatment options.

"Inhibition of TORC1 has extended both lifespan and healthspan in multiple pre-clinical species. The results of this Phase 2a trial raise the possibility that TORC1 inhibition also has health benefits in older humans. In the Phase 2a trial, TORC1 inhibitor treatment was associated with a clinically meaningful reduction in the incidence of infections in people aged 65 years and older and an enhancement in the function of the aging immune system as assessed by influenza vaccination response and antiviral gene expression. The results need to be validated in additional clinical trials, but may have broad implications for the treatment of diseases of aging that we are actively investigating with our TORC1 inhibitor program."

The data for this publication were gathered in a randomized, double-blinded, placebo-controlled Phase 2a study of 264 elderly volunteers at least 65 years of age without unstable medical conditions. Subjects were treated for 6 weeks with study drug and after a 2-week drug-free interval, were given a seasonal influenza vaccine. The incidence of infections was assessed for one year after initiation of study drug treatment. In the RTB101 monotherapy and RTB101+everolimus combination treatment arms, statistically significant and clinically meaningful reductions in the annual rate of infections of 33% and 38%, respectively, compared to placebo, were observed.

Fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by excessive production and persistent accumulation of collagens and other extracellular matrix (ECM) molecules in skin, lungs, and other internal organs. The process underlies a large number of fibrotic diseases that, in aggregate, account for a considerable proportion of deaths worldwide. With no effective therapy to date, fibrosis therefore represents a significant unmet global health need.

TLRs and related pattern-recognition receptors represent the first line of host defense against microbial pathogens. Cell surface receptors such as TLR4 and endosomal receptors such as TLR3 recognize extrinsic pathogen-associated molecule patterns (PAMPs) such as LPS and virus-derived nucleic acids. Significantly, TLRs also recognize damage-associated molecule patterns (DAMPs) that arise endogenously during various forms of noninfectious tissue injury. Regulated PAMP sensing by TLR4 has a unique requirement of myeloid differentiation 2 (MD2), an accessory receptor that interacts with TLR4 to form the signaling-competent receptor. The requirement for MD2 as an accessory pattern-recognition receptor for PAMPs appears to be unique for TLR4.

We recently demonstrated that particular DAMPs are markedly upregulated in fibrotic skin and lungs in patients with SSc and largely colocalize with TLR4-expressing myofibroblasts. In mice, genetic ablation of either of two DAMPs prominently associated with SSc resulted in markedly attenuated skin and lung fibrosis and enhanced fibrosis resolution, suggesting a fundamental pathogenic role for DAMP-TLR4 signaling in driving persistent organ fibrosis.

We developed a small molecule that selectively blocks MD2, which is uniquely required for TLR4 signaling. Targeting MD2/TLR4 abrogated inducible and constitutive myofibroblast transformation and matrix remodeling in fibroblast monolayers, as well as in 3-D scleroderma skin equivalents and human skin explants. Moreover, the selective TLR4 inhibitor prevented organ fibrosis in several preclinical disease models and mouse strains, and it reversed preexisting fibrosis.

Our cells tend to lose their shape as we grow older, contributing to many of the effects we experience as aging. This poses particular problems for the heart, where aging can disrupt the protein network within muscle cells that move blood around the body. Researchers discovered that maintaining high levels of the protein vinculin - which sticks heart muscle cells to one another - confers health benefits to fruit flies. Their work shows that fruit flies bred to produce 50 percent more vinculin enjoyed better cardiovascular health and lived a third of their average life span longer.

Vinculin works at the intercalated disks that glue together heart muscle cells, called cardiomyocytes. As we age, cardiomyocytes make less vinculin. Vinculin organizes the heart's contractile proteins, so as vinculin levels fall our heartbeats become disorganized and less efficient. By breeding flies with complementary genes, researchers created a genetic switch that turned on extra copies of the vinculin-coding gene. To ensure that only cardiomyocytes were producing the protein, the group used the same activation machinery as a heart development gene called Tinman.

While typical fruit flies live for roughly six weeks, flies that made more vinculin survived up to nine weeks. Additionally, flies with a vinculin boost were more active and able to climb the walls of their enclosures, a test of fruit fly athletic ability. Researchers were surprised how much improving cardiac function also helped the flies maintain a healthier metabolism. To measure this improvement, researchers fed the flies a special form of glucose and detected how the flies modified and used the sugar. Flies with more vinculin broke down more glucose than their counterparts. The team concluded higher vinculin levels in the flies' hearts enabled other organs to efficiently get the nutrients they needed in the breakdown process.

Waste removal from the central nervous system is essential for maintaining brain homeostasis across the lifespan. Two interconnected, dynamic networks were recently uncovered, which may provide new information concerning the conundrum of how the brain manages waste removal in the absence of authentic lymphatic vessels (LVs). The glymphatic system serves as the brain's "front end" waste drainage pathway that includes a perivascular network for cerebrospinal fluid (CSF) transport, which is connected to a downstream authentic lymphatic network associated with the meninges, cranial nerves, and large vessels exiting the skull. The anatomical and functional components of the two systems are complex, and the processes by which they physically interconnect are only partly understood.

The first pioneering studies documented that soluble amyloid beta (Aβ) protein and tau oligomers - metabolic waste products whose buildup is associated with Alzheimer's disease (AD) - were transported from the interstitial fluid (ISF) space and out of the brain via the glymphatic system. This information was followed by another hallmark study reporting that slow wave sleep enhanced glymphatic Aβ clearance from brain when compared to wakefulness. Collectively, this information was met with excitement in the neuroscience and clinical communities because maintaining efficient brain waste drainage across the lifespan - possibly by preserving normal sleep architecture - emerged as a novel therapeutic target for preventing cognitive dysfunction and decline.

The idea of maximizing brain "waste drainage" as a new preventive or therapeutic target for neurodegenerative disease states was further strengthened by animal studies providing evidence of declining glymphatic transport efficiency in healthy aging, AD models, traumatic brain injury, cerebral hemorrhage, and stroke. Considering the novelty of the glymphatic system concept, along with the rapidly emerging literature associating key physiological processes (e.g., vascular pulsatility, and sleep) with glymphatic transport function and waste solute outflow from brain, we decided it was timely to review this information cohesively. Hence, the goal of this mini-review is to provide a broad overview of the current data, controversies, and gaps in knowledge of the glymphatic system and waste drainage from the brain, while addressing potential consequences of aging as well as critically reviewing evidence for its existence in the human brain.

It doesn't seem like as many people in Europe talk about aging as in the U.S. Is being in Europe instead of the U.S. better or worse for your research?

As usual, the U.S. innovates, China imitates, and Europe hesitates. I returned to Europe for personal reasons, but I have been talking to American investors who want to explore Europe a bit more, and there are possibilities. People here do acknowledge aging as a problem, and the Undoing Aging conference in Berlin was a success. The downside with Silicon Valley is that there are big budgets and a great spirit, but we also need a style of research, which is, in every city, a little bit different. In Europe, the focus is very much molecular. I would like to combine the great vision and budget of Silicon Valley with European quality and maybe a bit of skepticism. We never publish anything unless we are really convinced. In that sense, I like Europe because people are interested in aging here; you just have to talk to the right people, and many people are skeptical. When I talk to scientists about what we do, they also get excited.

Are the regulations regarding trials more stringent than in the U.S.?

Yes, that's true, especially for animal work. There's a lot of societal pressure not to do animal work. We have to deal with these hurdles, but there's good money in science here, certainly in Western Europe, and we can make do quite well. This is generalizing, but we tend to talk less and do more.

Would you say that a potential side effect of the drug, if not used at the proper dose, could be excessive lysis?

The honest answer is "We don't know." I've seen in mice that you can go too far; if you look at the cell data, it's tenfold more potent against senescent cells. That sounds like a lot, but if you want to treat relatively healthy people with this, if one in ten cells that will be destroyed is basically a healthy cell, I find it very risky. So, you need to have a perfect dose or a perfect range.

With mice, we could scale it and we could say if it's too much or not, but for humans, it's more difficult. What we're doing now is trying to optimize this to make it tenfold more selective. This is version 4, and the published paper is on version 3; the first two were generated in the US in 2012, and they were not so effective. The first step was very short and had a very poor solubility, the second step lasted much longer, and the third peptide we made in D-amino acid is the one we published now. Now, we're making the fourth one because we know where the critical amino acids and the non-important and important ones are in the interaction domain of the two proteins, FOXO4 and p53. We plan on giving number 4 to a team of drug development experts to get it to a hundredfold selectivity, and then it should be much safer for use.

How long would you say it's going to be for this safe version four to be optimal?

That's the fun part. It took me ten years to come to this third version because in academia, we always have 20 other things that are also interesting. Now, we actually teamed up with a company, Cleara, that we founded just recently. The team has 20 people, with 10 structural experts, and they're going crazy on this. Every week, we have a meeting at which they have made some more progress, and it is super fast. We gave ourselves four months for a library screen on the first version, and then it's another ten rounds of optimizations. Once we have a lead candidate, we will start doing all the things that academia never wants to look at, like a liver update and all the stuff that scientists aren't interested in but is important to have. I want to do ten rounds of that, and it's three weeks per round, then we'll know roughly where the weak spots are in our current version, and we can go back and add heavy metal toxicity, etc. We gave ourselves a year for optimization, but I hope sooner.

How well does this treatment compare to other treatment options, such as fasting?

With fasting, you don't kill, you just delay the secretions from senescent cells. It's like rapamycin and aspirin; it just blocks the secretion profile. Fasting offers a transient benefit for sure, but a week later, you eat again, and they're just there again. It's just making them dormant. We have not seen evidence that senescent cells are removed by fasting, in mice or in cells.

Have you looked at other senolytics?

We tried a lot, and the BCL inhibitors look the most promising. What we saw when comparing them to FOXO4-DRI is that they are toxic at low levels and should not be given to healthy people. That's the downside of these drugs. In vitro, if you do low-level navitoclax on healthy cells, you get 10, 20 percent cell death. That's relatively stable. That's a decrease in viability because you're affecting some cells that are apparently sensitive to BCL inhibition. We did not see that with FOXO4, and that's what's reported in our paper. As for quercetin and dasatinib, I'm absolutely not a fan of those. We've tried a couple of experiments; we've never seen a good result.

How often do you think people would need senolytic treatments, will they be for older or younger people?

In mice, over a year; we did it once a month. It seemed to be enough, and I think we can actually reduce that frequency. But, I still have to do the experiment. If we do it once in a while, once every three months, once every half a year in mice, it might actually be sufficient. I don't think they accumulate that fast. Maybe later in life, you'll do it a bit faster. Early in life, there's really no reason to do it so often. It's like a car. If it's only a couple of years old, you don't go to the mechanic as often.

Successful aging has been defined as not suffering from chronic diseases, having optimal social engagement and mental health, and a lack of physical disability. Numerous studies have found that physical activity decreases the risk of many chronic diseases and increases longevity. However, the association between physical activity and successful aging has shown heterogeneity across studies. Some studies have shown either a lack of or a weak independent association between physical activity and successful aging; however, other cohort studies as well as systematic reviews have shown that higher levels of physical activity was associated with aging successfully.

Therefore, in our cohort study of 1,584 adults aged 49+ years at baseline we aimed to investigate whether total physical activity is independently associated with successful aging, which was defined as not experiencing disability and chronic disease (coronary artery disease, stroke, diabetes, cancer), having good mental health and functional independence, and reporting optimal physical, respiratory, and cognitive function during 10 years of follow-up. Participants provided information on the performance of moderate or vigorous activities and walking exercise and this was used to determine total metabolic equivalents (METs) minutes of activity per week.

Of the cohort, 249 (15.7%) participants had aged successfully 10 years later. Older adults in the highest level of total physical activity (more than 5000 MET minutes/week; n = 71) compared to those in the lowest level of total physical activity (less than 1000 MET minutes/week; n = 934) had 2-fold greater odds of aging successfully than normal aging. Our finding of a positive association between physical activity levels and successful aging is in agreement with the existing literature showing that physical activity might be an important parameter in enabling people to age successfully. Moreover, a systematic review found that the effect sizes for the association of successful or healthy aging with high levels of physical activity ranged from 1.27 to 3.09, which is in line with our observed estimate.

Upon persistent damage or during aging, senescent cells accumulate, probably due to an inefficient clearance by immune cells, and this accumulation may lead to chronic inflammation and fibrosis. Indeed, evidence in mice indicates that the accumulation of senescent cells actively contributes to multiple diseases and aging. In this regard, genetic ablation of senescent cells delays and ameliorates some aging-associated diseases, reverts long-term degenerative processes associated with chemotherapy, and extends longevity. Importantly, senescent cells present vulnerabilities to particular small molecule inhibitors, known as "senolytics", that trigger apoptosis preferentially in senescent cells. These pharmacological treatments reduce the number of senescent cells in vivo and show therapeutic activity against senescence-associated diseases and aging.

Senescent cells in vitro are characterized by high levels of lysosomal β-galactosidase activity, known as senescence-associated β-galactosidase. In addition to β-galactosidase, senescent cells present high levels of most tested lysosomal hydrolases. Indeed, senescent cells show a remarkable accumulation of lysosomes, together with abnormal endosomal traffic and autophagy. Interestingly, damaged or diseased tissues generally contain cells that are positive for SAβGal, while normal healthy tissues are negative for this marker.

Here, we have explored the possibility of using lysosomal β-galactosidase as a vulnerable trait of senescent cells that can be exploited to deliver tracers or drugs preferentially to diseased tissues with high content of senescent cells. Our approach is based on the encapsulation of diagnostic or therapeutic agents with β(1,4)-galacto-oligosaccharides and their delivery to lysosomes via endocytosis. In a model of chemotherapy-induced senescence, encapsulated cytotoxic drugs target senescent tumor cells. Moreover, in a model of pulmonary fibrosis in mice, encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders.

Alterations in microRNA (miRNA) processing have been previously linked to aging. Here we used the small molecule enoxacin to pharmacologically interfere with miRNA biogenesis and study how it affects aging in C. elegans. Enoxacin extended worm lifespan and promoted survival under normal and oxidative stress conditions. Enoxacin-induced longevity required the transcription factor SKN-1/Nrf2 and was blunted by the antioxidant N-acetyl-cysteine, suggesting a prooxidant-mediated mitohormetic response. The longevity effects of enoxacin were also dependent on the miRNA pathway, consistent with changes in miRNA expression elicited by the drug. Among these differentially expressed miRNAs, the widely conserved miR-34-5p was found to play an important role in enoxacin-mediated longevity.

And how does miR-34-5p down-regulation affect lifespan? Mir-34 has been previously associated with lifespan and the onset of age-related diseases in model organisms, but the directionality and the mechanisms underlying its effects have been a matter of debate. A previous study demonstrated that mir-34 loss-of-function significantly extends lifespan through activation of autophagy, but other studies did not see an effect on survival or even found the opposite. Here we show that both enoxacin and mir-34 loss-of-function extend lifespan via a mechanism that requires a prooxidative effect. Different types of food (e.g., dead bacteria here versus live bacteria in the previous studies) and slightly different experimental conditions could have created different thresholds of sensitivity to prooxidant agents or a different redox balance which in turn could explain the apparent discrepancies in reports associating mir-34 with longevity.

Sub-lethal levels of mitochondrial reactive oxygen species (ROS) are usually associated with beneficial effects and lifespan extension, while elevated ROS can be toxic - a phenomenon often referred to as mitohormesis. Mitochondrial ROS requires the transcription factor SKN-1/Nrf2 to increase lifespan and confer their beneficial effects, and so does enoxacin. Together, these results indicate that enoxacin promotes non-toxic levels of ROS through inhibition of miR-34-5p, which in turn activates stress response pathways mediated by SKN-1 and autophagy. In addition to its beneficial outcomes, there is a toxic effect caused by enoxacin treatment. Consistent with this notion, enoxacin-mediated miR-34-5p inhibition confers a lesser lifespan extension than deletion of the mir-34 gene.