Biomarkers of aging for the identification and evaluation of longevity interventions.

With the rapid expansion of aging biology research, the identification and evaluation of longevity interventions in humans have become key goals of this field. Biomarkers of aging are critically important tools in achieving these objectives over realistic time frames. However, the current lack of standards and consensus on the properties of a reliable aging biomarker hinders their further development and validation for clinical applications. Here, we advance a framework for the terminology and characterization of biomarkers of aging, including classification and potential clinical use cases. We discuss validation steps and highlight ongoing challenges as potential areas in need of future research. This framework sets the stage for the development of valid biomarkers of aging and their ultimate utilization in clinical trials and practice.

Challenges in developing Geroscience trials.

Geroscience is becoming a major hope for preventing age-related diseases and loss of function by targeting biological mechanisms of aging. This unprecedented paradigm shift requires optimizing the design of future clinical studies related to aging in humans. Researchers will face a number of challenges, including ideal populations to study, which lifestyle and Gerotherapeutic interventions to test initially, selecting key primary and secondary outcomes of such clinical trials, and which age-related biomarkers are most valuable for both selecting interventions and predicting or monitoring clinical responses ("Gerodiagnostics"). This article reports the main results of a Task Force of experts in Geroscience.

Targeting the biology of aging with mTOR inhibitors.

Inhibition of the protein kinase mechanistic target of rapamycin (mTOR) with the Food and Drug Administration (FDA)-approved therapeutic rapamycin promotes health and longevity in diverse model organisms. More recently, specific inhibition of mTORC1 to treat aging-related conditions has become the goal of basic and translational scientists, clinicians and biotechnology companies. Here, we review the effects of rapamycin on the longevity and survival of both wild-type mice and mouse models of human diseases. We discuss recent clinical trials that have explored whether existing mTOR inhibitors can safely prevent, delay or treat multiple diseases of aging. Finally, we discuss how new molecules may provide routes to the safer and more selective inhibition of mTOR complex 1 (mTORC1) in the decade ahead. We conclude by discussing what work remains to be done and the questions that will need to be addressed to make mTOR inhibitors part of the standard of care for diseases of aging.

Control of tissue oxygenation by S-nitrosohemoglobin in human subjects.

S-Nitrosohemoglobin (SNO-Hb) is unique among vasodilators in coupling blood flow to tissue oxygen requirements, thus fulfilling an essential function of the microcirculation. However, this essential physiology has not been tested clinically. Reactive hyperemia following limb ischemia/occlusion is a standard clinical test of microcirculatory function, which has been ascribed to endothelial nitric oxide (NO). However, endothelial NO does not control blood flow governing tissue oxygenation, presenting a major quandary. Here we show in mice and humans that reactive hyperemic responses (i.e., reoxygenation rates following brief ischemia/occlusion) are in fact dependent on SNO-Hb. First, mice deficient in SNO-Hb (i.e., carrying C93A mutant Hb refractory to S-nitrosylation) showed blunted muscle reoxygenation rates and persistent limb ischemia during reactive hyperemia testing. Second, in a diverse group of humans-including healthy subjects and patients with various microcirculatory disorders-strong correlations were found between limb reoxygenation rates following occlusion and both arterial SNO-Hb levels (n = 25; P = 0.042) and SNO-Hb/total HbNO ratios (n = 25; P = 0.009). Secondary analyses showed that patients with peripheral artery disease had significantly reduced SNO-Hb levels and blunted limb reoxygenation rates compared with healthy controls (n = 8 to 11/group; P < 0.05). Low SNO-Hb levels were also observed in sickle cell disease, where occlusive hyperemic testing was deemed contraindicated. Altogether, our findings provide both genetic and clinical support for the role of red blood cells in a standard test of microvascular function. Our results also suggest that SNO-Hb is a biomarker and mediator of blood flow governing tissue oxygenation. Thus, increases in SNO-Hb may improve tissue oxygenation in patients with microcirculatory disorders.

Extending human healthspan and longevity: a symposium report.

For many years, it was believed that the aging process was inevitable and that age-related diseases could not be prevented or reversed. The geroscience hypothesis, however, posits that aging is, in fact, malleable and, by targeting the hallmarks of biological aging, it is indeed possible to alleviate age-related diseases and dysfunction and extend longevity. This field of geroscience thus aims to prevent the development of multiple disorders with age, thereby extending healthspan, with the reduction of morbidity toward the end of life. Experts in the field have made remarkable advancements in understanding the mechanisms underlying biological aging and identified ways to target aging pathways using both novel agents and repurposed therapies. While geroscience researchers currently face significant barriers in bringing therapies through clinical development, proof-of-concept studies, as well as early-stage clinical trials, are underway to assess the feasibility of drug evaluation and lay a regulatory foundation for future FDA approvals in the future.

Fisetin for COVID-19 in skilled nursing facilities: Senolytic trials in the COVID era.

The burden of senescent cells (SnCs), which do not divide but are metabolically active and resistant to death by apoptosis, is increased in older adults and those with chronic diseases. These individuals are also at the greatest risk for morbidity and mortality from SARS-CoV-2 infection. SARS-CoV-2 complications include cytokine storm and multiorgan failure mediated by the same factors as often produced by SnCs through their senescence-associated secretory phenotype (SASP). The SASP can be amplified by infection-related pathogen-associated molecular profile factors. Senolytic agents, such as Fisetin, selectively eliminate SnCs and delay, prevent, or alleviate multiple disorders in aged experimental animals and animal models of human chronic diseases, including obesity, diabetes, and respiratory diseases. Senolytics are now in clinical trials for multiple conditions linked to SnCs, including frailty; obesity/diabetes; osteoporosis; and cardiovascular, kidney, and lung diseases, which are also risk factors for SARS-CoV-2 morbidity and mortality. A clinical trial is underway to test if senolytics decrease SARS-CoV-2 progression and morbidity in hospitalized older adults. We describe here a National Institutes of Health-funded, multicenter, placebo-controlled clinical trial of Fisetin for older adult skilled nursing facility (SNF) residents who have been, or become, SARS-CoV-2 rtPCR-positive, including the rationale for targeting fundamental aging mechanisms in such patients. We consider logistic challenges of conducting trials in long-term care settings in the SARS-CoV-2 era, including restricted access, consent procedures, methods for obtaining biospecimens and clinical data, staffing, investigational product administration issues, and potential solutions for these challenges. We propose developing a national network of SNFs engaged in interventional clinical trials.

Targeting the biology of ageing with mTOR inhibitors to improve immune function in older adults: phase 2b and phase 3 randomised trials.

BACKGROUND: The COVID-19 pandemic highlights the need for therapies that improve immune function in older adults, including interferon (IFN)-induced antiviral immunity that declines with age. In a previous phase 2a trial, RTB101 (previously known as BEZ235), an oral mechanistic target of rapamycin (mTOR) inhibitor, was observed to increase IFN-induced antiviral gene expression and decrease the incidence of respiratory tract infections (RTIs) in older adults. Therefore, we aimed to investigate whether oral RTB101 upregulated IFN-induced antiviral responses and decreased the incidence of viral RTIs when given once daily for 16 weeks during winter cold and flu season. METHODS: We did a phase 2b and a phase 3 double-blind, randomised, placebo-controlled trial in adults aged at least 65 years enrolled in New Zealand, Australia, and the USA at 54 sites. In the phase 2b trial, patients were aged 65-85 years, with asthma, type 2 diabetes, chronic obstructive pulmonary disease (COPD), congestive heart failure, were current smokers, or had an emergency room or hospitalisation for an RTI within the past 12 months. In the phase 3 trial, patients were aged at least 65 years, did not have COPD, and were not current smokers. In the phase 2b trial, patients were randomly assigned to using a validated automated randomisation system to oral RTB101 5 mg, RTB101 10 mg once daily, or placebo in part 1 and RTB101 10 mg once daily, RTB101 10 mg twice daily, RTB101 10 mg plus everolimus once daily, or matching placebo in part 2. In the phase 3 trial, patients were randomly assigned to RTB101 10mg once daily or matching placebo. The phase 2b primary outcome was the incidence of laboratory-confirmed RTIs during 16 weeks of winter cold and influenza season and the phase 3 primary outcome was the incidence of clinically symptomatic respiratory illness defined as symptoms consistent with an RTI, irrespective of whether an infection was laboratory-confirmed. Patients, investigators, and sponsor were masked to treatment assignments. All patients who received at least part of one dose of study drug were included in the primary and safety analyses. The phase 2b trial was registered with ANZCTR, ACTRN12617000468325, ClinicalTrials.gov, NCT03373903, and the phase 3 trial was registered with ANZCTR, ACTRN12619000628145. FINDINGS: In the phase 2b trial, we recruited 652 participants in total between May 16, 2017, and Jan 10, 2018, 179 participants to part 1 of the study (randomly assigned 1:1:1 to RTB101 5 mg once daily [61 participants], RTB101 10 mg once daily [58 participants], or matching placebo [60 participants]) and 473 patients to part 2 (randomly assigned 1:1:1:1 to RTB101 10 mg once daily [118 participants], RTB101 10 mg twice daily [120 participants], RTB101 10 mg in combination with everolimus 0·1 mg daily [115 participants] or matching placebo [120 participants]). In our first prespecified statistical analysis of the primary efficacy endpoint for part 2 of the phase 2b trial efficacy of RTB101 10 mg in combination with everolimus 0·1 mg once daily compared with placebo did not meet statistical significance but, in our second prespecified analysis, which included data from part 1 and part 2, we found a statistically significant reduction in the proportion of patients who had one or more laboratory-confirmed RTIs in the RTB101 10 mg once daily treatment group (34 [19%] of 176) compared with the pooled placebo group (50 [28%] of 180; odds ratio [OR] 0·601 [90% CI 0·391-0·922]; p=0·02). In the phase 3 trial, we enrolled 1024 patients between May 7, 2018, and July 19, 2019. 513 (50·1%) participants were randomly assigned to RTB101 10 mg once daily and 510 (49·9%) to placebo. In the full analysis set of the phase 3 trial, RTB101 did not reduce the proportion of patients with clinically symptomatic respiratory illness (134 [26%] of 511 patients in the RTB101 treatment group vs 125 [25%] 510 patients in the placebo treatment group; OR 1·07 [90% CI 0·80-1·42]; p=0·65). In both trials, significantly more IFN-induced antiviral genes were upregulated in patients treated with RTB101 as compared with placebo. The study drug was found to be safe and well-tolerated across trials and treatment groups. Only one patient in the placebo group in the phase 3 trial had serious adverse events (nausea, fatigue, hyponatraemia, and arthralgia) which were considered related to study drug treatment. Three patients died in the phase 2b trial and one in the phase 3 trial but no deaths were considered related to study treatment. INTERPRETATION: The combined results indicate that low doses of the mTOR inhibitor RTB101 are well tolerated and upregulate IFN-induced antiviral responses in older adults. Further refinement of clinical trial endpoints and patient populations might be required to identify whether upregulation of IFN responses by mTOR inhibitors consistently decreases the incidence or severity of viral infections in older adults. FUNDING: resTORbio and the National Institute on Aging.

Research and resource needs for understanding host immune responses to SARS-CoV-2 and COVID-19 vaccines during aging.

On 16 and 17 March 2021, the National Institute of Allergy and Infectious Diseases and the National Institute of Aging convened a virtual workshop to discuss developments in SARS-CoV-2 research pertaining to immune responses in older adults, COVID-19 vaccines in both aged animals and older individuals, and to gain some perspective on the critical knowledge gaps that need addressing to establish scientific priorities for future research studies.

TORC1 inhibition enhances immune function and reduces infections in the elderly.

Inhibition of the mechanistic target of rapamycin (mTOR) protein kinase extends life span and ameliorates aging-related pathologies including declining immune function in model organisms. The objective of this phase 2a randomized, placebo-controlled clinical trial was to determine whether low-dose mTOR inhibitor therapy enhanced immune function and decreased infection rates in 264 elderly subjects given the study drugs for 6 weeks. A low-dose combination of a catalytic (BEZ235) plus an allosteric (RAD001) mTOR inhibitor that selectively inhibits target of rapamycin complex 1 (TORC1) downstream of mTOR was safe and was associated with a significant (P = 0.001) decrease in the rate of infections reported by elderly subjects for a year after study drug initiation. In addition, we observed an up-regulation of antiviral gene expression and an improvement in the response to influenza vaccination in this treatment group. Thus, selective TORC1 inhibition has the potential to improve immune function and reduce infections in the elderly.

Short-term Low-Dose mTORC1 Inhibition in Aged Rats Counter-Regulates Age-Related Gene Changes and Blocks Age-Related Kidney Pathology.

Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes.

Translational strategies in aging and age-related disease.

Aging is a risk factor for several of the world's most prevalent diseases, including neurodegenerative disorders, cancer, cardiovascular disease and metabolic disease. Although our understanding of the molecular pathways that contribute to the aging process and age-related disease is progressing through the use of model organisms, how to apply this knowledge in the clinic is less clear. In September, Nature Medicine, in collaboration with the Volkswagen Foundation, hosted a conference at the beautiful Herrenhausen Palace in Hannover, Germany with the goal of broadening our understanding of the aging process and its meaning as a 'risk factor' in disease. Here, several of the speakers at that conference answer questions posed by Nature Medicine.

mTOR inhibition improves immune function in the elderly.

Inhibition of the mammalian target of rapamycin (mTOR) pathway extends life span in all species studied to date, and in mice delays the onset of age-related diseases and comorbidities. However, it is unknown if mTOR inhibition affects aging or its consequences in humans. To begin to assess the effects of mTOR inhibition on human aging-related conditions, we evaluated whether the mTOR inhibitor RAD001 ameliorated immunosenescence (the decline in immune function during aging) in elderly volunteers, as assessed by their response to influenza vaccination. RAD001 enhanced the response to the influenza vaccine by about 20% at doses that were relatively well tolerated. RAD001 also reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age. These results raise the possibility that mTOR inhibition may have beneficial effects on immunosenescence in the elderly.

Antibody targeting of TGF-ß in cancer patients.

The role of TGF-ß in tumor development and progression is complex. Genetic mutations that disrupt the antiproliferative signaling effects of TGF-ß play a key role in the process of malignant transformation for many types of tumors. Paradoxically, this loss of sensitivity to TGF-ß's inhibitory actions often leads to TGF-ß overexpression by the tumor cells or by normal cells that are recruited to the tumor microenvironment. Elevated concentrations of TGF-ß in the tumor microenvironment have been shown to facilitate tumor growth and metastasis. Numerous published studies have provided evidence that inhibition of TGF-ß using antibodies, soluble receptors and small molecule inhibitors of TGF-ß signal transduction can have beneficial effects in murine models of cancer. Given the pleiotropic nature of TGF-ß and its homeostatic role in numerous biological processes, serious concerns have been expressed regarding the safety of administering TGF-ß antagonists to human patients. Interestingly, the results of numerous animal toxicology studies of TGF-ß antibodies in normal rodents and primates have shown that administration of neutralizing anti-TGF-ß antibodies is well tolerated and any adverse effects were reversible or self-limiting. Likewise, administration of a human anti-TGF-ß antibody (fresolimumab) in three separate human phase 1 clinical trials has also been shown to be well tolerated.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells.

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Measurement of protein S-nitrosylation during cell signaling.

S-Nitrosylation, the modification of a cysteine thiol by a nitric oxide (NO) group, has emerged as an important posttranslational modification of signaling proteins. An impediment to studying the regulation of cell signaling by S-nitrosylation has been the technical challenge of detecting endogenously S-nitrosylated proteins. Detection of S-nitrosylated proteins is difficult because the S-NO bond is labile and therefore can be lost or gained artifactually during sample preparation. Nevertheless, several methods have been developed to measure endogenous protein S-nitrosylation, including the biotin switch assay and the chemical reduction/chemiluminescence assay. This chapter describes these two methods and provides examples of how they have been used successfully to elucidate the role of protein S-nitrosylation in cell physiology and pathophysiology.

S-Nitrosothiol measurements in biological systems.

S-Nitrosothiol (SNO) cysteine modifications are regulated signaling reactions that dramatically affect, and are affected by, protein conformation. The lability of the SNO bond can make SNO-modified proteins cumbersome to measure accurately. Here, we review methodologies for detecting SNO modifications in biology. There are three caveats. (1) Many assays for biological SNOs are used near the limit of detection: standard curves must be in the biologically relevant concentration range. (2) The assays that are most reliable are those that modify SNO protein or peptide chemistry the least. (3) Each result should be quantitatively validated using more than one assay. Improved assays are needed and are in development.

Immunoregulatory and antimicrobial effects of nitrogen oxides.

The therapeutic effects of inhaled nitric oxide (NO) therapy are thought to be restricted to the pulmonary vasculature because of rapid inactivation of NO by hemoglobin in the bloodstream. However, recent data suggest that inhaled NO may not only be scavenged by the heme iron of hemoglobin but also may react with protein thiols in the bloodstream, including cysteine-93 of the hemoglobin B subunit. Reaction of NO with protein or peptide thiols is termed S-nitrosylation and results in the formation of relatively stable protein S-nitrosothiols that carry NO bioactivity to distal organs. Thus, inhaled NO-induced protein S-nitrosylation may allow inhaled NO to have multiple as yet undiscovered physiologic and pathophysiologic effects outside of the lung. Here we review the immunoregulatory and antimicrobial functions of NO and the potential effects of inhaled NO therapy on host defense.

S-nitrosothiol depletion in amyotrophic lateral sclerosis.

Recent data suggest that either excessive or deficient levels of protein S-nitrosylation may contribute to disease. Disruption of S-nitrosothiol (SNO) homeostasis may result not only from altered nitric oxide (NO) synthase activity but also from alterations in the activity of denitrosylases that remove NO groups. A subset of patients with familial amyotrophic lateral sclerosis (ALS) have mutations in superoxide dismutase 1 (SOD1) that increase the denitrosylase activity of SOD1. Here, we show that the increased denitrosylase activity of SOD1 mutants leads to an aberrant decrease in intracellular protein and peptide S-nitrosylation in cell and animal models of ALS. Deficient S-nitrosylation is particularly prominent in the mitochondria of cells expressing SOD1 mutants. Our results suggest that SNO depletion disrupts the function and/or subcellular localization of proteins that are regulated by S-nitrosylation such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thereby contributes to ALS pathogenesis. Repletion of intracellular SNO levels with SNO donor compounds rescues cells from mutant SOD1-induced death. These results suggest that aberrant depletion of intracellular SNOs contributes to motor neuron death in ALS, and raises the possibility that deficient S-nitrosylation is a general mechanism of disease pathogenesis. SNO donor compounds may provide new therapeutic options for diseases such as ALS that are associated with deficient S-nitrosylation.