DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in C. elegans.

Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension.

CBP gene transfer increases BDNF levels and ameliorates learning and memory deficits in a mouse model of Alzheimer's disease.

Cognitive dysfunction and memory loss are common features of Alzheimer's disease (AD). Abnormalities in the expression profile of immediate early genes that play a critical role in memory formation, such as the cAMP-response element binding protein (CREB), have been reported in the brains of AD patients. Here we show that amyloid-ß (Aß) accumulation, which plays a primary role in the cognitive deficits of AD, interferes with CREB activity. We further show that restoring CREB function via brain viral delivery of the CREB-binding protein (CBP) improves learning and memory deficits in an animal model of AD. Notably, such improvements occur without changes in Aß and tau pathology, and instead are linked to an increased level of brain-derived neurotrophic factor. The resulting data suggest that Aß-induced learning and memory deficits are mediated by alterations in CREB function, based on the finding that restoring CREB activity by directly modulating CBP levels in the brains of adult mice is sufficient to ameliorate learning and memory. Therefore, increasing CBP expression in adult brains may be a valid therapeutic approach not only for AD, but also for various brain disorders characterized by alterations in immediate early genes, further supporting the concept that viral vector delivery may be a viable therapeutic approach in neurodegenerative diseases.

Subtyping Social Determinants of Health in All of Us: Network Analysis and Visualization Approach.

Background: Social determinants of health (SDoH), such as financial resources and housing stability, account for between 30-55% of people's health outcomes. While many studies have identified strong associations among specific SDoH and health outcomes, most people experience multiple SDoH that impact their daily lives. Analysis of this complexity requires the integration of personal, clinical, social, and environmental information from a large cohort of individuals that have been traditionally underrepresented in research, which is only recently being made available through the All of Us research program. However, little is known about the range and response of SDoH in All of Us, and how they co-occur to form subtypes, which are critical for designing targeted interventions. Objective: To address two research questions: (1) What is the range and response to survey questions related to SDoH in the All of Us dataset? (2) How do SDoH co-occur to form subtypes, and what are their risk for adverse health outcomes? Methods: For Question-1, an expert panel analyzed the range of SDoH questions across the surveys with respect to the 5 domains in Healthy People 2030 (HP-30), and analyzed their responses across the full All of Us data (n=372,397, V6). For Question-2, we used the following steps: (1) due to the missingness across the surveys, selected all participants with valid and complete SDoH data, and used inverse probability weighting to adjust their imbalance in demographics compared to the full data; (2) an expert panel grouped the SDoH questions into SDoH factors for enabling a more consistent granularity; (3) used bipartite modularity maximization to identify SDoH biclusters, their significance, and their replicability; (4) measured the association of each bicluster to three outcomes (depression, delayed medical care, emergency room visits in the last year) using multiple data types (surveys, electronic health records, and zip codes mapped to Medicaid expansion states); and (5) the expert panel inferred the subtype labels, potential mechanisms that precipitate adverse health outcomes, and interventions to prevent them. Results: For Question-1, we identified 110 SDoH questions across 4 surveys, which covered all 5 domains in HP-30. However, the results also revealed a large degree of missingness in survey responses (1.76%-84.56%), with later surveys having significantly fewer responses compared to earlier ones, and significant differences in race, ethnicity, and age of participants of those that completed the surveys with SDoH questions, compared to those in the full All of Us dataset. Furthermore, as the SDoH questions varied in granularity, they were categorized by an expert panel into 18 SDoH factors. For Question-2, the subtype analysis (n=12,913, d=18) identified 4 biclusters with significant biclusteredness (Q=0.13, random-Q=0.11, z=7.5, P<0.001), and significant replication (Real-RI=0.88, Random-RI=0.62, P<.001). Furthermore, there were statistically significant associations between specific subtypes and the outcomes, and with Medicaid expansion, each with meaningful interpretations and potential targeted interventions. For example, the subtype Socioeconomic Barriers included the SDoH factors not employed, food insecurity, housing insecurity, low income, low literacy, and low educational attainment, and had a significantly higher odds ratio (OR=4.2, CI=3.5-5.1, P-corr<.001) for depression, when compared to the subtype Sociocultural Barriers. Individuals that match this subtype profile could be screened early for depression and referred to social services for addressing combinations of SDoH such as housing insecurity and low income. Finally, the identified subtypes spanned one or more HP-30 domains revealing the difference between the current knowledge-based SDoH domains, and the data-driven subtypes. Conclusions: The results revealed that the SDoH subtypes not only had statistically significant clustering and replicability, but also had significant associations with critical adverse health outcomes, which had translational implications for designing targeted SDoH interventions, decision-support systems to alert clinicians of potential risks, and for public policies. Furthermore, these SDoH subtypes spanned multiple SDoH domains defined by HP-30 revealing the complexity of SDoH in the real-world, and aligning with influential SDoH conceptual models such as by Dahlgren-Whitehead. However, the high-degree of missingness warrants repeating the analysis as the data becomes more complete. Consequently we designed our machine learning code to be generalizable and scalable, and made it available on the All of Us workbench, which can be used to periodically rerun the analysis as the dataset grows for analyzing subtypes related to SDoH, and beyond.

Hepatic response to oxidative injury in long-lived Ames dwarf mice.

Multiple stress resistance pathways were evaluated in the liver of Ames dwarf mice before and after exposure to the oxidative toxin diquat, seeking clues to the exceptional longevity conferred by this mutation. Before diquat treatment, Ames dwarf mice, compared with nonmutant littermate controls, had 2- to 6-fold higher levels of expression of mRNAs for immediate early genes and 2- to 5-fold higher levels of mRNAs for genes dependent on the transcription factor Nrf2. Diquat led to a 2-fold increase in phosphorylation of the stress kinase ERK in control (but not Ames dwarf) mice and to a 50% increase in phosphorylation of the kinase JNK2 in Ames dwarf (but not control) mice. Diquat induction of Nrf2 protein was higher in dwarf mice than in controls. Of 6 Nrf2-responsive genes evaluated, 4 (HMOX, NQO-1, MT-1, and MT-2) remained 2- to 10-fold lower in control than in dwarf liver after diquat, and the other 2 (GCLM and TXNRD) reached levels already seen in dwarf liver at baseline. Thus, livers of Ames dwarf mice differ systematically from controls in multiple stress resistance pathways before and after exposure to diquat, suggesting mechanisms for stress resistance and extended longevity in Ames dwarf mice.

PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. We sought to determine whether peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) would have a beneficial effect on this disease. METHODS: PGC-1α transgenic mice were crossed with SOD1 mutant G93A DL mice. RESULTS: We observed a moderate but non-significant increase in average lifespan in PGC-1α/G93A DL mice, as compared with G93A DL mice (292 ± 3 days vs. 274 ± 7 days). Although the onset of ALS was not altered, progression of the disease was significantly slower (≈34% increase in duration) in the PGC-1α/G93A DL mice. These mice also exhibited markedly improved performance on the rotarod test, and the improved motor activity was associated with a decreased loss of motor neurons and less degeneration of neuromuscular junctions. CONCLUSION: A sustained level of excitatory amino acid transporter protein 2 (EAAT2) in astrocytes of the PGC-1α/G93A DL mice may contribute to neuronal protection.

Is the oxidative stress theory of aging dead?

Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.

Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span.

Methionine sulfoxide reductase A (MsrA) repairs oxidized methionine residues within proteins and may also function as a general antioxidant. Previous reports have suggested that modulation of MsrA in mice and mammalian cell culture can affect the accumulation of oxidized proteins and may regulate resistance to oxidative stress. Thus, under the oxidative stress theory of aging, these results would predict that MsrA regulates the aging process in mammals. We show here that MsrA(-/-) mice are more susceptible to oxidative stress induced by paraquat. Skin-derived fibroblasts do not express MsrA, but fibroblasts cultured from MsrA(-/-) mice were, nevertheless, also more susceptible to killing by various oxidative stresses. In contrast to previous reports, we find no evidence for neuromuscular dysfunction in MsrA(-/-) mice in either young adult or in older animals. Most important, we found no difference between MsrA(-/-) and control mice in either their median or maximum life span. Thus, our results show that MsrA regulates sensitivity to oxidative stress in mice but has no effect on aging, as determined by life span.

The effect of gonadectomy and estradiol on sensitivity to oxidative stress.

The sexual dimorphism of life span and caloric restriction effects in numerous species suggest that estradiol (E2) is protective against oxidative damage. The only direct test of E2's protective effect in mice against in vivo oxidative stress to date may have been confounded by E2's direct chemical action as an antioxidant because it was administered at very high dosages. Therefore, we have identified a low yet physiologically effective dose of E2. We then administered this dose using subcutaneous time-release pellets to ovariectomized mice. Two weeks after E2 pellet implantation, sham-operated, ovariectomized, and ovariectomized E2-supplemented female mice were injected with a lethal dose of paraquat and their survival was followed. It was observed that ovariectomy exacerbates paraquat-induced mortality and is rescued by E2 supplementation. An equivalent experiment was performed on sham-operated, orchidectomized, and E2-supplemented orchidectomized male mice. The survival of male mice was improved by orchidectomy, and E2 gave no further benefit. We interpret the results to mean that E2 is protective against oxidative stress through its regulatory role and that testosterone diminishes protection against oxidative stress.

Patient Engagement and Attitudes Toward Using the Electronic Medical Record for Medical Research: The 2015 Greater Plains Collaborative Health and Medical Research Family Survey.

BACKGROUND: Electronic health records (EHRs) are ubiquitous. Yet little is known about the use of EHRs for prospective research purposes, and even less is known about patient perspectives regarding the use of their EHR for research. OBJECTIVE: This paper reports results from the initial obesity project from the Greater Plains Collaborative that is part of the Patient-Centered Outcomes Research Institute's National Patient-Centered Clinical Research Network (PCORNet). The purpose of the project was to (1) assess the ability to recruit samples of adults of child-rearing age using the EHR; (2) prospectively assess the willingness of adults of child-rearing age to participate in research, and their willingness (if parents) to have their children participate in medical research; and (3) to assess their views regarding the use of their EHRs for research. METHODS: The EHRs of 10 Midwestern academic medical centers were used to select patients. Patients completed a survey that was designed to assess patient willingness to participate in research and their thoughts about the use of their EHR data for research. The survey included questions regarding interest in medical research, as well as basic demographic and health information. A variety of contact methods were used. RESULTS: A cohort of 54,269 patients was created, and 3139 (5.78%) patients responded. Completers were more likely to be female (53.84%) and white (85.84%). These and other factors differed significantly by site. Respondents were overwhelmingly positive (83.9%) about using EHRs for research. CONCLUSIONS: EHRs are an important resource for engaging patients in research, and our respondents concurred. The primary limitation of this work was a very low response rate, which varied by the method of contact, geographic location, and respondent characteristics. The primary strength of this work was the ability to ascertain the clinically observed characteristics of nonrespondents and respondents to determine factors that may contribute to participation, and to allow for the derivation of reliable study estimates for weighting responses and oversampling of difficult-to-reach subpopulations. These data suggest that EHRs are a promising new and effective tool for patient-engaged health research.

A System for an Accountable Data Analysis Process in R.

Efficiently producing transparent analyses may be difficult for beginners or tedious for the experienced. This implies a need for computing systems and environments that can efficiently satisfy reproducibility and accountability standards. To this end, we have developed a system, R package, and R Shiny application called adapr (Accountable Data Analysis Process in R) that is built on the principle of accountable units. An accountable unit is a data file (statistic, table or graphic) that can be associated with a provenance, meaning how it was created, when it was created and who created it, and this is similar to the 'verifiable computational results' (VCR) concept proposed by Gavish and Donoho. Both accountable units and VCRs are version controlled, sharable, and can be incorporated into a collaborative project. However, accountable units use file hashes and do not involve watermarking or public repositories like VCRs. Reproducing collaborative work may be highly complex, requiring repeating computations on multiple systems from multiple authors; however, determining the provenance of each unit is simpler, requiring only a search using file hashes and version control systems.

Estimate risk difference and number needed to treat in survival analysis.

The hazard ratio (HR) is a measure of instantaneous relative risk of an increase in one unit of the covariate of interest, which is widely reported in clinical researches involving time-to-event data. However, the measure fails to capture absolute risk reduction. Other measures such as number needed to treat (NNT) and risk difference (RD) provide another perspective on the effectiveness of an intervention, and can facilitate clinical decision making. The article aims to provide a step-by-step tutorial on how to compute RD and NNT in survival analysis with R. For simplicity, only one measure (RD or NNT) needs to be illustrated, because the other measure is a reverse of the illustrated one (NNT=1/RD). An artificial dataset is composed by using the survsim package. RD and NNT are estimated with Austin method after fitting a Cox-proportional hazard regression model. The confidence intervals can be estimated using bootstrap method. Alternatively, if the standard errors (SEs) of the survival probabilities of the treated and control group are given, confidence intervals can be estimated using algebraic calculations. The pseudo-value model provides another method to estimate RD and NNT. Details of R code and its output are shown and explained in the main text.

Liver specific expression of Cu/ZnSOD extends the lifespan of Sod1 null mice.

Genetic ablation of CuZn-superoxide dismutase (Sod1) in mice (Sod1(-/-) mice) leads to shortened lifespan with a dramatic increase in hepatocellular carcinoma and accelerated aging phenotypes, including early onset sarcopenia. To study the tissue specific effects of oxidative stress in the Sod1(-/-) mice, we generated mice that only express the human SOD1 gene specifically in the liver of Sod1(-/-) mice (Sod1(-/-)/hSOD1(alb) mice). Expression of hSOD1 in the liver of Sod1(-/-) mice improved liver function, reduced oxidative damage in liver, and partially restored the expression of several genes involved in tumorigenesis, which are abnormally expressed in the livers of the Sod1(-/-) mice. However, liver specific expression of hSOD1 did not prevent the loss of body weight and muscle mass and alterations in the structure of neuromuscular junctions. The expression of hSOD1 in the liver of Sod1(-/-) mice significantly improved the lifespan of Sod1(-/-) mice; however, the lifespan of the Sod1(-/-)/hSOD1(alb) mice was still significantly shorter than wild type mice.

Mitochondrial metabolites extend lifespan.

Disruption of mitochondrial respiration in the nematode Caenorhabditis elegans can extend lifespan. We previously showed that long-lived respiratory mutants generate elevated amounts of α-ketoacids. These compounds are structurally related to α-ketoglutarate, suggesting they may be biologically relevant. Here, we show that provision of several such metabolites to wild-type worms is sufficient to extend their life. At least one mode of action is through stabilization of hypoxia-inducible factor-1 (HIF-1). We also find that an α-ketoglutarate mimetic, 2,4-pyridinedicarboxylic acid (2,4-PDA), is alone sufficient to increase the lifespan of wild-type worms and this effect is blocked by removal of HIF-1. HIF-1 is constitutively active in isp-1(qm150) Mit mutants, and accordingly, 2,4-PDA does not further increase their lifespan. Incubation of mouse 3T3-L1 fibroblasts with life-prolonging α-ketoacids also results in HIF-1α stabilization. We propose that metabolites that build up following mitochondrial respiratory dysfunction form a novel mode of cell signaling that acts to regulate lifespan.

Rapamycin Increases Mortality in db/db Mice, a Mouse Model of Type 2 Diabetes.

We examined the effect of rapamycin on the life span of a mouse model of type 2 diabetes, db/db mice. At 4 months of age, male and female C57BLKSJ-lepr (db/db) mice (db/db) were placed on either a control diet, lacking rapamycin or a diet containing rapamycin and maintained on these diets over their life span. Rapamycin was found to reduce the life span of the db/db mice. The median survival of male db/db mice fed the control and rapamycin diets was 349 and 302 days, respectively, and the median survival of female db/db mice fed the control and rapamycin diets was 487 and 411 days, respectively. Adjusting for gender differences, rapamycin increased the mortality risk 1.7-fold in both male and female db/db mice. End-of-life pathological data showed that suppurative inflammation was the main cause of death in the db/db mice, which is enhanced slightly by rapamycin treatment.

Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy.

BACKGROUND: Synucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy. METHODS: We determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches. RESULTS: After 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal. CONCLUSION: Taken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.

The role of oxidative damage and stress in aging.

The Free Radical/Oxidative Stress Theory of Aging, which was first proposed in 1956, is currently one of the most popular explanations for how aging occurs at the biochemical/molecular level. However, most of the evidence in support of this theory is correlative, e.g., oxidative damage to various biomolecules increases with age, and caloric restriction, which increases life span and retards aging, reduces the age-related increase in oxidative damage to biomolecules. The most direct test of the Free Radical/Oxidative Stress Theory of Aging is to specifically alter the age-related increase in oxidative damage and determine how this alteration affects life span. For the first time, investigators can use genetically altered animals to test directly the role of oxidative damage in aging. In this manuscript, we critically review the past research in this area and discuss potential future research directions in testing the Free Radical/Oxidative Theory of Aging.

Short-term rapamycin treatment in mice has few effects on the transcriptome of white adipose tissue compared to dietary restriction.

Rapamycin, a drug that has been shown to increase lifespan in mice, inhibits the target of rapamycin (TOR) pathway, a major pathway that regulates cell growth and energy status. It has been hypothesized that rapamycin and dietary restriction (DR) extend lifespan through similar mechanisms/pathways. Using microarray analysis, we compared the transcriptome of white adipose tissue from mice fed rapamycin or DR-diet for 6 months. Multidimensional scaling and heatmap analyses showed that rapamycin had essentially no effect on the transcriptome as compared to DR. For example, only six transcripts were significantly altered by rapamycin while mice fed DR showed a significant change in over 1000 transcripts. Using ingenuity pathway analysis, we found that stearate biosynthesis and circadian rhythm signaling were significantly changed by DR. Our findings showing that DR, but not rapamycin, has an effect on the transcriptome of the adipose tissue, suggesting that these two manipulations increase lifespan through different mechanisms/pathways.

Combined treatment of rapamycin and dietary restriction has a larger effect on the transcriptome and metabolome of liver.

Rapamycin (Rapa) and dietary restriction (DR) have consistently been shown to increase lifespan. To investigate whether Rapa and DR affect similar pathways in mice, we compared the effects of feeding mice ad libitum (AL), Rapa, DR, or a combination of Rapa and DR (Rapa + DR) on the transcriptome and metabolome of the liver. The principal component analysis shows that Rapa and DR are distinct groups. Over 2500 genes are significantly changed with either Rapa or DR when compared with mice fed AL; more than 80% are unique to DR or Rapa. A similar observation was made when genes were grouped into pathways; two-thirds of the pathways were uniquely changed by DR or Rapa. The metabolome shows an even greater difference between Rapa and DR; no metabolites in Rapa-treated mice were changed significantly from AL mice, whereas 173 metabolites were changed in the DR mice. Interestingly, the number of genes significantly changed by Rapa + DR when compared with AL is twice as large as the number of genes significantly altered by either DR or Rapa alone. In summary, the global effects of DR or Rapa on the liver are quite different and a combination of Rapa and DR results in alterations in a large number of genes and metabolites that are not significantly changed by either manipulation alone, suggesting that a combination of DR and Rapa would be more effective in extending longevity than either treatment alone.

Mice fed rapamycin have an increase in lifespan associated with major changes in the liver transcriptome.

Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.

Rapamycin extends life and health in C57BL/6 mice.

Target of rapamycin inhibition by rapamycin feeding has previously been shown to extend life in genetically heterogeneous mice. To examine whether it similarly affected mouse health, we fed encapsulated rapamycin or a control diet to C57BL/6Nia mice of both sexes starting at 19 months of age. We performed a range of health assessments 6 and 12 months later. Rapamycin feeding significantly reduced mTOR activity in most but not all tissues. It also reduced total and resting metabolic rate during the light (inactive) phase of the light:dark cycle in females only but had no effect on spontaneous activity or metabolism during the dark (active) phase of either sex. Males only had less fragmented sleep when fed rapamycin, whereas stride length and rotarod performance were improved in both sexes. Survival was also improved by this late-life rapamycin feeding, and some pathological lesions were delayed. We found no adverse health consequences associated with rapamycin treatment.