Selected Abstracts from the American Aging Association 47th Annual Meeting “Improving Resilience to Delay Aging” Philadelphia, 2018

Tis11 and Cth1 deficiency extends Saccharomyces cerevisiae lifespan via metabolic reprogramming

Carine Beaupere,¹ Brian Wasko,² Matt Kaeberlein, ² and Vyacheslav M. Labunskyy ¹

¹ Department of Dermatology, Boston University School of Medicine, Boston, MA; ²Department of Pathology, University of Washington, Seattle, WA

Iron (Fe) serves as a cofactor for many enzymes involved in metabolism and mitochondrial function. Dysregulation of Fe homeostasis has been implicated in the pathogenesis of numerous age-related human diseases. However, the role of Fe bioavailability in regulation of lifespan and how aging affects Fe homeostasis remain poorly understood. In the present study, we investigated the role of Tis11 and Cth1, two mRNA-binding proteins (RBP) involved in the cellular response to Fe deficiency, in lifespan regulation using yeast model. Our data demonstrate that deletion of TIS11 and CTH1 significantly extends replicative lifespan. Using a combination of RNA sequencing (RNA-Seq) and ribosome profiling (Ribo-Seq) we quantitatively analyzed translational changes in these deletion mutants. We show that increased lifespan in the tis11Δ and cth1Δ mutants is associated with activation of genes involved in regulation of Fe transport as well as Fe-containing proteins involved in mitochondrial respiration and amino acid metabolism. Moreover, we found that lifespan extension in the cth1Δ deletion mutant is dependent on the Gcn4 transcription factor, which was not observed in the tis11Δ cells. Together, our data suggest that inactivation of Tis11 and Cth1 mRNA-binding proteins extends replicative lifespan via metabolic reprogramming by increasing respiration due to elevated levels of mRNAs that are normally targeted by Tis11 and Cth1 for degradation, and that the mechanisms of lifespan extension in these mutants are at least partially distinct.

Funding: This work was supported by the NIH grants AG040191 and AG054566 (to VML), and P30AG013280 (to MK)

Therapeutic Potential of Dietary Restriction Mimetics to Treat Mitochondrial Disease. Alessandro Bitto¹, Herman Tung¹, Nicole Tatom¹, Alvine Ngouonga¹, Silvan Urfer¹, Sophia Stone¹, Daniel L. Smith², Gene P. Ables³, Ernst-Kayser Bernhard⁴, Margaret Sedensky⁴, Phil Morgan⁴, and Matt Kaeberlein¹.

¹Department of Pathology, University Washington Medical Center; ²Department of Nutrition Science, University of Alabama at Birmingham; ³Orentreich Foundation for the Advancement of Science, ⁴ Seattle Children’s Research Institute.

Dietary Restriction (DR), defined as reduced caloric intake without malnutrition, is generally regarded as the most effective intervention to increase lifespan and delay the onset of age-related impairments across multiple species. Yet, response to DR is greatly influenced by individual differences in genotype, as evinced from studies in yeast and mice.

Mitochondrial mutants are amongst the best responders to DR-mediated lifespan extension in yeast. Based on this observation, we have been testing the efficacy of several DR mimetics at rescuing disease phenotypes in a mouse model of mitochondrial disease caused by depletion of the NADH-Ubiquinone Oxidoreductase Complex (Ndufs4^-/-). This model recapitulates human Leigh syndrome, a childhood mitochondrial disease. Our results indicate that two DR mimetics, rapamycin and acarbose, increase lifespan and delay the onset of neurological symptoms in Ndufs4^-/- mice. Importantly, these interventions do not restore the activity of the mitochondrial electron transport chain in Ndufs4^-/- mice. Instead, both rapamycin and acarbose appear to restore the NAD⁺/NADH ratio by significantly altering carbon metabolism in the brain of mutant animals. Notably, supplementation with NAD⁺ precursors failed to recapitulate the effects of rapamycin and acarbose in Ndufs4^-/‑ mice, while inhibition of glycolysis with glucosamine partially increase lifespan in mutant animals. We are currently investigating the mechanisms by which rapamycin and acarbose rewire nutrient metabolism in mutant mice. Understanding how these treatments increase the lifespan of mitochondrial disease mutants will allow the identification of additional treatments for mitochondrial disease and interventions for similar metabolic alterations that occur during normative aging.

Funding: United Mitochondrial Disease Foundation Post-Doctoral Fellowship, National Institute of Neurological Disorders and Stroke 1 R01 NS98329

WormBot: An Open-Source Robotics Platform for C. elegans Survival Assays.

Ben Blue,¹ Jason N. Pitt,¹ Nolan Strait,¹ Josh Russell,¹ Anuj Vaid¹, Christina Tran¹, Brock J. Johnson¹, Brendon Davis¹, Keong Mu Lim¹ and Matt Kaeberlein¹ Department of Pathology, University of Washington, Seattle, WA, USA

C. elegans is an ideal organism for aging research owing to its brief lifespan, small size, and extensive genetic and reverse genetic resources. Here we describe WormBot, an open-source image capture platform that allows for high-throughput survival analysis. The WormBot can conduct 144 separate C. elegans survival assays per device, for a total of over 5,500 individuals assayed simultaneously. We have hand-validated the accuracy of the device and WormBot replicates the results of manual assays but at a much lower labor cost. Additionally, the open-source nature of the hardware and software will allow for users to extend the platform and implement new software features. While the intended use of the WormBot is to perform survival analysis, the ability to customize the capture settings and software has allowed us to investigate the effects of Aβ, pathogenic bacteria, and hypoxia on worm behavior. The WormBot is constructed from commercially available robotics hardware and a fully operable system can be built for less than $600 and only requires an additional linux workstation to operate. The WormBot is driven by a web-based interface allowing for control and monitoring of experiments from any internet connected device. The device’s extensibility coupled with the low cost and simplicity of the system will allow for automation and increased throughput of C. elegans survival analysis even in small laboratory settings. See http://wormbot.org for more information

Funding: This work was supported by NIA grants P50AG005136 and P30AG013280 to MK. JNP and BB were supported by NIA Grant T32AG000057.

Single-cell transcriptional signatures of the aging nonhuman primate brain Kenneth L. Chiou,¹ Alex R. DeCasien,^2,3 Michael J. Montague,⁴ Chet C. Sherwood,⁵ Michael L. Platt,^4,6,7 and Noah Snyder-Mackler^{1,8 1}Department of Psychology, University of Washington; ²Department of Anthropology, New York University; ³New York Consortium in Evolutionary Primatology; ⁴Department of Neuroscience, University of Pennsylvania; ⁵Department of Anthropology, The George Washington University; ⁶Department of Psychology, University of Pennsylvania; ⁷Department of Marketing, University of Pennsylvania; ⁸Center for Studies in Demography & Ecology, University of Washington

The human brain is capable of rapidly and reliably processing complex stimuli. These abilities decline with age, sometimes manifesting through neurodegenerative diseases such as Alzheimer's. Yet little is understood about how aging influences the distribution and function of individual neurons, data critical for understanding the heterogeneity of the aging process across cells and individuals. Here, we characterize gene expression in single brain cells from rhesus macaques, a model primate organism closely related to humans that shows similar age-related declines in sensory, motor, and cognitive function. We sampled cells from the prefrontal cortex due to its role in higher cognitive functions and known changes during healthy or neurodegenerative aging. We characterized gene expression using sci-RNA-seq, which combines flow cytometry with combinatorial indexing to profile gene expression at single-cell resolution. First, we used sci-RNA-seq to recover transcriptional signatures from 1,473 macaque brain cells sequenced to an average depth of ~10,000 reads per cell. We then used unsupervised clustering to classify cell types based on transcriptional patterns. Cells formed four distinct clusters sufficient for distinguishing neurons and several glial types. We then expanded our sci-RNA-seq experiment to characterize gene expression in 30 free-ranging macaques of varying ages. These data allowed us to identify changes in cell-type composition and single-cell gene expression associated with aging. Future studies will determine the impact of social and environmental factors on changes in gene expression related to aging and will serve as a resource for the development of medical interventions for age-associated diseases in humans.

Funding: This study was funded by the National Institutes of Health (R00-AG051764 and R01-MH108627). K.L.C. is supported by NIH T32-AG000057.

Eldercare Research ProjectAllen D. Colby, Jon Bowen PhD, and Jennifer Pratt University of Maine at Augusta.

The experience of undergraduate students who are engaged in providing informal caregiving to elders in their home or in their community in rural Maine has direct effects on the student’s ability to perform at any level of academia. The Eldercare Research Project aims at understanding the direct correlations to health, academia, family/social relations, and other factors that affect student’s performance through methods such as interviews (i.e., focus groups; lunch and learns, etc.) and system wide surveys that are still being analyzed for important data correlations and conclusions.

This is research that has never been done before and the team who undertakes the tasks of research, data collection, and analysis are very excited to break new ground and share findings. We are currently still transcribing focus group results and transferring those findings to our data sheets, however, the data thus far shows that students who engage in undergraduate studies and give care informally are greatly affected by both experiences and could benefit from other strategically placed services.

In our conclusions, it is evident that as the number of hours a student spends on caregiving, the higher incidence of complications arises in numerous realms of their living – and academic performance is one of them. Further analysis is ongoing.

Transgenerational inheritance of the response to calorie restriction Clara Di Germanio,¹ Andrea Di Francesco¹, Sam Hamilton¹ , Supriyo De², Yongqing Zhang², Sarah Mitchell¹, Michel Bernier¹, and Rafael de Cabo¹. ¹Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, NIH, ²Laboratory of Genetics, Intramural Research Program, National Institute on Aging, NIH

The global increase of human lifespan is becoming one of the main social transformation of the last decades. It is estimated that the number of persons 60 years and over will double by 2050 and even triple by 2100 (UN, 2017). This will also pose a medical emergency, as older people are more susceptible to diseases and the overall medical cost will be impacted. Therefore, there is an urgent need to improve and extend the quality of life. Calorie restriction (CR) is one of the dietary interventions that has consistently demonstrated to delay the onset of age related diseases and preserve function until late in life. However, CR is not universal as previously thought, and its effects on health and survival vary depending on sex, strain and degree of restriction. In particular, DBA/2J (D2) and C57BL/6J (B6) showed a very different response to different degrees of CR, with extremes represented by D2 females having the best longevity compared to B6 females (Mitchell et al., 2016; PMID: 27304509). In this study, we wanted to investigate how genetic and epigenetic factors interact to determine the individual response to CR. To this aim, we assessed lifespan and healthspan in female and male offspring of ‘DBA/2J X C57BL/6J’ (D2B6) and ‘C57BL/6J X DBA/2J’ (B6D2) crosses under ad libitum feeding and two levels of CR. Although the male and female (F1) progeny had the same genotype, the D2B6 and B6D2 hybrid mice showed differential response to CR. First, as expected all F1 progeny had a longer maximum lifespan than parental D2 mice regardless of the feeding regimen and was found comparable to the B6 mice. Furthermore, D2B6 mice of both sexes had extended lifespan under CR, while B6D2 animals barely showed any improvement. Genomic analysis suggested that the reason for these differences may reside in imprinted genes, mitochondrial DNA and epigenetic remodeling. Understanding how calorie restriction, genetics, and environmental factors impact healthspan and/or lifespan will ultimately lead to personalized intervention in humans, tailored to the individual based on their predicted response or risk of disease.

Funding: Intramural Research program - NIH

β-GPA Alters Mitochondrial Energetics to Improve Markers of Health and Function Jonathan Dorigatti,^1,2 Kevin Thyne,^1,2 Michael Bene,^1,2 Yuhong Liu,^1,2 and Adam Salmon^{1,2 1}University of Texas Health Science Center at San Antonio; ²The Sam and Ann Barshop Institute for Longevity and Aging Studies

Metabolism and mitochondrial function have been implicated in the development of age related physiological declines as well as the aging process per se. It is therefore thought that interventions targeting aspects of these systems may delay the development of multiple age related pathologies and enhance overall longevity. Previous studies have suggested that β-GPA, a naturally occurring analog of creatine, improves glucose metabolism, increases mitochondrial biogenesis, and enhances exercise tolerance in young rodents. These outcomes appear to be partially mediated by activation of AMP-activated protein kinase (AMPK) signaling and alterations to mitochondrial fuel preference. This has lead us to hypothesize that β-GPA will reduce age-related physiological declines in function by altering mitochondrial energetics to preserve cellular function. We set out to test this hypothesis using both mammalian (HET3 Mice) and invertebrate (Drosophila) models to identify both physiological outcomes and molecular mechanisms of β-GPA intervention during aging. To investigate the effects of β-GPA when delivered late in life, we administered 1% β-GPA in chow to both male and female HET-3 mice beginning at 20 months of age. We observed that β-GPA treatment prevented age related declines in male but not female body weight, an effect driven primarily by increased adiposity. However, in contrast to reports in young animals, we did not detect significant increases in either exercise tolerance or glucose metabolism. Similarly high-resolution respirometry performed on the soleus did not reveal significant changes in O₂ flux, though flux control ratios were typically higher in β-GPA treated animals. In Drosophila, previous reports indicate that β-GPA delivered in food increases median lifespan of both males and females. However, we have found that doses previously suggested to increase lifespan instead cause significant decreases in median lifespan for both male and female flies. Notably, we have identified lower dosages of β-GPA that can increase median survival of flies subjected to environmental stresses including exposure to high heat (37^c) and oxidative stress (15% H₂O₂). Together, these results suggest that β-GPA can both alter stress resistance and potentially delay or prevent the development of some age related pathologies, though the timing of administration may be critical to these effects.

Funding: NIA Biology of Aging Training Grant (5T32AG021890-13)

Comprehensive Evaluation of Physical Function in Older Mice

Ted G. Graber,¹ Christopher S. Fry,¹ Blake B. Rasmussen¹

¹University of Texas Medical Branch, Department of Nutrition and Metabolism

Sarcopenia and frailty are progressive and incurable. In older adults, both produce a downward trajectory of decreased ability to perform activities of daily living, increased incidence of falls, and eventual loss of independence, with increased mortality. To study the basic mechanisms underlying these diseases, and to assess potential therapies, we need preclinical animal models. Since, in humans, loss of function with age is an important aspect of these diseases, the ability to comprehensively evaluate physical ability in mice is a necessary step prior to translation.

We hypothesized that when compared to adult mice (6/7-month old, n=30), many older mice (24/25-month old, n=26) would display functional deficits; but that others would be well preserved. To test this hypothesis, we used well-validated assays to measure functional ability in adult and older C57BL/6 mice, including, but not limited to: inverted cling (overall strength), grip test (forelimb grip strength), treadmill (endurance), rotarod (overall motor function), and voluntary wheel running (volitional exercise and activity rate). We then developed a composite scoring system comprised of the individual functional tests. In addition, in a subset of mice, we determined maximum dorsiflexion torque with in vivo contractile physiology methodology.

Overall, we determined age-related functional decrements, adjusting for body mass with univariate ANCOVAs, in: inverted cling (-27%, p<0.001), grip test (-20%, p=0.001), rotarod (-29%, p<0.001), and voluntary wheel running (-71%, p<0.001). In older mice (n=8), maximum dorsiflexion torque per gram of body mass was -18% (p=0.003) compared to the adult mice (n=7). Endurance, however, was relatively well maintained in older mice (treadmill, -5%, p=0.077). The composite scoring system determined that older mice had much lower functional aptitude than adult mice (p<0.001), however older mice in the upper quartile (n=6) were not significantly different from the average adult mouse (p=0.325).

In all functional tests, other than the treadmill, the older age group demonstrated significantly lower function, on average, than the adult mice. In conclusion, we propose that our composite functional test for mice will be a repeatable, powerful, non-invasive tool to assess physical function in mice in future longitudinal and cross-sectional studies of therapeutics and basic science discovery in preclinical aging research.

Funding: NIA P30 AG024832 - Pepper Center Pilot Project (TGG)

Programming of cortisol across the life-course in IUGR baboons: evidence for accelerated aging Hillary F Huber,¹ Shanshan Yang,^1,2 Cun Li,^1,3 and Peter W Nathanielsz^{1,3 1}Texas Pregnancy & Life-course Health Research Center, Department of Animal Science, University of Wyoming, Laramie, WY; ²Department of Neurology, the First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China; ³Southwest National Primate Research Center and Texas Biomedical Research Institute, San Antonio, TX

Introduction: The hypothalamo-pituitary-adrenal (HPA) axis plays a central role in balancing growth and differentiation, regulating the stress response, and influencing aging. The HPA axis is sensitive to environmental perturbations. We measured circulating cortisol and its receptor, glucocorticoid receptor (GR), for evidence of developmental programming by intrauterine growth restriction (IUGR). Since baboon cortisol declines with age, programmed alteration in cortisol may affect the relationship between biological and chronological age. We hypothesized elevated cortisol and GR in IUGR from fetal life through early adulthood, as well as a fall in cortisol and frontal cortex (FC) GR across the normal life-course (NLC).

Methods: Pregnant baboons ate ad lib (CTR) or 70% ad lib in pregnancy and lactation producing IUGR offspring (~12% decreased birth weight). Cortisol in IUGR and age-matched CTR males (M) and females (F) was measured at 0.9 gestation (G) (N=14M, 13F CTR; 7M, 7F IUGR), at postnatal months 1-36 (5-12 CTR, 3-8 IUGR), and at adult age ~9 years (y) (N=8 per group). FC grey matter GR was quantified in CTR (12M, 13F) and IUGR (8M, 8F) at 0.9G. NLC regression formulas from 1-23y were developed for cortisol (30M, 24F) and GR (6M, 23F). Fasting morning plasma cortisol was measured by chemiluminescent immunoassay, GR protein by immunohistochemistry (IHC). Data M ± SEM, analyzed by t-tests and linear regression.

Results: At 0.9G, there were no cortisol differences between males and females so data were pooled; cortisol was elevated in IUGR (26.1 ± 2.5 ug/dl) vs. CTR (20.3 ± 1.3; p=0.03). IUGR and CTR cortisol were similar from 1-36 postnatal months. At 9y (human equivalent ~32y), female IUGR cortisol (49.7 ± 3.26) increased vs. CTR (41.2 ± 2.86, p=0.03). Male IUGR (35.4 ± 2.81) and CTR (37.0 ± 2.85) were similar. NLC cortisol fell linearly with age: cortisol = -24.7 x age in years + 735 (p<0.001). IHC in FC at 0.9G indicated GR was elevated in IUGR females (5.8% area stained) vs CTR (2.8%, p=0.04), while in males IUGR (4.8%) and CTR (3.0%) GR were similar (p=0.1). NLC GR fell linearly with age: GR = -1.96 x age in years ± 29.6 (p=0.07).

Conclusion: High cortisol levels are detrimental to tissues and may thus explain adverse cardiovascular and nervous system programming outcomes reported in IUGR. We hypothesize the NLC cortisol fall slows the rate of aging; thus, age-related changes will be accelerated by elevated cortisol. In the NLC, the fall in GR would decrease cortisol's ability to stimulate tissues as animals age, increasing the biological effects of the decreasing cortisol.

Funding: R24OD010916, R24 RR021367, R24 RR025866, HD21350

Non-cell autonomous rescue of brain protein aggregation in a Drosophila model of glucocerebrosidase deficiency Kathryn A. Jewett¹, Marie Y. Davis^2,3, Evelyn S. Vincow¹, Ruth E. Thomas¹, and Leo J. Pallanck^{1 1}Department of Genome Sciences, University of Washington, Seattle, WA; ²Department of Neurology, University of Washington, Seattle, WA; ³VA Puget Sound Healthcare System, Seattle, WA

Brain protein aggregation is a common feature of aging and neurodegenerative disease. While traditionally assumed to arise through a cell-autonomous mechanism, increasing evidence indicates non-cell autonomous processes play important roles in regulating the abundance and spread of protein aggregates. Our recent work indicates that the GBA1 gene, encoding the lipid-modifying enzyme glucocerebrosidase, plays an important role in this process. Mutations in GBA1 are associated with dementia with Lewy bodies and are the strongest genetic risk factor for Parkinson’s disease (PD), both of which are characterized by the accumulation of protein aggregates in the brain. Despite the prevalence and devastating effects of these diseases, our understanding of pathogenesis remains limited and the mechanism by which GBA1 mutations increase susceptibility is unknown.

To study GBA1-dependent disease mechanisms, we use a GBA1-null Drosophila melanogaster model which recapitulates many of the symptoms of PD including shortened lifespan, motor and memory deficits, neurodegeneration, and accelerated accumulation of protein aggregation. Our recent work indicates that at least some of these phenotypes can be rescued through a non-cell autonomous mechanism. In particular, we found that ubiquitinated protein aggregation in the brains of GBA1-null flies, measured through western-blotting and immunohistochemistry, can be rescued by ectopic expression of GBA1 in peripheral tissues such as muscle and gut. Our proteomic studies revealed that the half-lives and abundance of proteins associated with extracellular vesicles (EVs) were disproportionately affected in GBA1-null flies, suggesting a possible mechanism for these non-cell autonomous effects. EVs are membrane-surrounded structures released from cells for the purpose of intercellular communication or trafficking of mRNAs, proteins, and lipids. Correspondingly, we found elevated ubiquitinated protein aggregates in EVs isolated from the hemolymph of GBA1-null flies. Finally, ectopic expression of GBA1 in muscle partially rescued the shortened lifespan and climbing deficit of GBA1-null flies though we’ve yet to elucidate the mechanisms responsible.

Our results suggest that GBA1 mutations promote the spread of protein aggregates through EVs and additional work to explore how GBA1 influences the metabolism or trafficking of EVs is on-going. Our findings of a non-cell autonomous mechanism regulating brain protein aggregation has not only important implications regarding preventative or disease-modifying treatment of diseases associated with GBA1 but also for other neurodegenerative diseases associated with protein aggregation such as Alzheimer’s disease.

Funding: NIH/NIA T32 Genetic Approaches to Aging Training Grant AG000057 to K.A.J. & NIH R01 NS094252 to L.J.P.

A Proposed Biomarker Strategy for a Multi-Center Geroscience-Guided Clinical Trial Jamie N. Justice,¹ Nir Barzilai,² Stephen B. Kritchevsky,¹ George A. Kuchel,^{3 1}Wake Forest School of Medicine; ²Albert Einstein College of Medicine; ³University of Connecticut Health Center;

Major advances indicate that biological aging processes may be modifiable; this has led to geroscience-guided interventions such as the proposed multi-center clinical trial Targeting Aging with MEtformin (TAME): A Geroscience-Guided Trial Targeting Multimorbidity and Functional Outcomes. Biomarkers provide an important exploratory outcome for trials like TAME to provide evidence that hypothesized clinical improvements are reflected in the underlying biology, and to supply a crucial resource for emerging science and discovery. However a defined set of biomarkers of aging for clinical trials does not yet exist.

We convened expert panels and conducted comprehensive reviews to identify 258 candidate biomarkers of aging and age-related disease. We next derived selection criteria to identify blood-based biomarkers that were: 1) reliably measured and feasible; 2) relevant to aging; 3) consistently predictive of all-cause mortality, and clinical/functional outcomes; 4) responsive to intervention. We conservatively applied these selection criteria using established literature, and arrived at a short list of blood-based biomarkers: measures of the investigational drug (metformin levels, markers of glycemic effects), biomarkers of aging hallmarks (IL-6, TNFα-Receptor, CRP, GDF15, insulin, IGF1), and markers of chronic disease (cystatin-C, NT-proBNP, clinical labs).

This work reveals the scarcity of sufficiently mechanistic blood-based biomarkers for large multi-center clinical trials on aging, but has relevance for sample collection and biomarker evaluation in pilot trials of geroscience-identified therapies. We propose a biomarker strategy that combines well-justified biomarkers with data-intensive discovery-based platforms, and curation of resources and biorepository to drive future research in the biology of aging and the next geroscience-guided trial.

Funding: American Federation for Aging Research (AFAR); the Glenn Center for the Biology of Human Aging (Paul Glenn Foundation for Medical Research); National Institutes of Health: P30 AG021332 (SKB, JNJ); AG048023, AG052608, GM124922 (GAK); P30AG038072 (NB)

Title: Spermidine: the next Rapamycin?

Chen-Yu Liao¹ and Brian K. Kennedy¹

¹The Buck Institute

Autophagy, a cellular process that recycles damaged molecules in the cell, has emerged as a central regulator of lifespan. Multiple studies suggest that enhanced autophagy promotes longevity and delays age-related phenotypes. The natural polyamine, spermidine, is a pharmacological activator of autophagy and increases lifespan via an autophagy-dependent mechanism. A recent study showed that spermidine extends lifespan in normal mice by improving cardiovascular functions. To explore the molecular mechanism underlying this pro-longevity, we evaluated the role of spermidine in regulating metabolism in aged mice as well as in mice reared under high-fat diet (HFD), specifically in the liver and adipose tissues. First, we found that spermidine reduced body weight and body fat in aged mice as well as mice under HFD. Spermidine also improved glucose metabolism in mice under HFD as evaluated by glucose tolerance test and insulin tolerance test. Spermidine also increased energy expenditure in mice possibly due to the increased behavioral activity and elevated lipolysis in adipose tissue. These beneficial effects are likely due to the induction of autophagy as well as starvation hormone, fibroblast growth factor 21 (FGF21), by spermidine.

Funding: NIA RO1 AG050441

Ovarian-Dependent, Germ Cell-Independent Extension of Longevity and Abatement of Inflammaging in Post-Reproductive Female Mice Jeffrey B. Mason,¹ Tracy Habermehl,¹ Kyleigh Tyler,¹ McKenna Walters,¹ Kate Parkinson,¹ Yuji Ikeno,² and Björn Schumacher^{3 1}Department of Animal, Dairy and Veterinary Sciences, Center for Integrated BioSystems, School of Veterinary Medicine, Utah State University; ²Department of Pathology, Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center; ³The Institute for Genome Stability in Ageing and Disease, Cologne Cluster of Excellence in Cellular Stress Responses in Aging-associated Diseases Research Center, University of Cologne.

Text of abstract.

The influence of reproduction on health and life span is often thought of as being restricted only to the period of reproductive competency. However, reproductive status influences health throughout all phases of the chronological life span. Evidence produced over the past decade indicate that an individual's reproductive status is correlated with the presence or absence of chronic health conditions, particularly in women. Cardiovascular disease is rare in premenopausal women, but increases sharply at menopause and in young women with premature ovarian failure. Insulin resistance and bone loss also increase at menopause. Two-thirds of Americans with Alzheimer’s disease are women.

The beneficial effects of dietary restriction on glucose and lipid metabolism in intact female rodents do not appear in ovariectomized rodents, supporting a central role for the ovary in female metabolic health. In addition, naturally-menopausal women possess a health advantage over surgically menopausal women, suggesting that even the senescent ovary provides a health advantage, independent of active germ cells. In worms and flies, depletion of gonadal germ cells can significantly extend longevity and health. However, these effects are dependent on the retention of the somatic gonad. We propose that gonadal somatic cells strive to facilitate germline transmission by preserving the somatic health of the organism, thereby positively influencing health and longevity.

Well-defined changes in ovarian signaling mark the end of the traditional reproductive life span. Ovarian transplantation is an efficient experimental method to separate the influence of the reproductive life span or reproductive aging from chronological aging per se. Previous work in our laboratory demonstrated that replacement of the senescent ovaries in postreproductive female mice with young, actively-cycling ovaries can restore many health benefits, including an increase in life span and an improvement in immune function. However, the factors responsible for this ovary-dependent enhancement of health remain unknown. We hypothesized that this phenomenon was driven by germ cell-stimulated ovarian hormone production from the new ovaries. The well-established supportive role for ovarian hormones in many aspects of female health implicates the loss of hormone production from actively cycling germ cells, as the principal cause of increased disease risks at menopause. While the value of ovarian hormones in female health is unquestionable, replacing these hormones in peri- and postmenopausal women does not universally restore the health benefits enjoyed by young women with young ovaries. Our objective here was to determine if removal of germ cells from the young ovaries prior to transplantation would alter the ovarian-dependent extension of life and health span.

To test this hypothesis, we chemically depleted the germ cells from young ovaries prior to transplantation. At 28 days of age, ovary-donor mice received daily intraperitoneal injections of 160mg/kg 4-vinylcyclohexene diepoxide for 15 days to deplete germ cell-containing follicles. At 60 days of age, germ cell-depleted ovaries (n=10) and intact, germ cell-containing ovaries (n=10) were collected from donor mice and transplanted to 17-month-old virgin CBA/J mice. The mean life span for CBA/J female mice is 644 days. Seventeen-month-old intact ovary recipients lived 798 days. Seventeen-month-old germ cell-depleted ovary recipients lived 880 days. This was 29% longer past the time of surgery, than mice that received germ cell-containing ovaries. The oldest germ cell-depleted ovary recipient was euthanized at 1046 days of age.

In a parallel experiment, germ cell-containing young ovaries (n=5) were transplanted to 13-month-old mice. These mice were collected at 17 months of age (4 months post-transplantation). Pathological analysis revealed that mice that received new ovaries at 13 months displayed decreased severity of inflammation and decreased severity of glomerulonephritis at 17 months of age, compared with age-matched controls. Pathological analysis also revealed that among mice that received new ovaries at 17 months, there was no difference in the severity of inflammation or glomerulonephritis between mice that received germ cell-depleted ovaries or germ cell-containing ovaries. These results suggest that the germ cells were not responsible for the positive, inflammation-associated benefits of the transplanted ovaries.

From 13 months of age to collection at 17 months, our age-matched control group displayed decreased circulating levels of inflammation-associated cytokines (both pro- and anti-inflammatory). Ovarian transplantation prevented this drop. Circulating levels of inflammation-associated cytokines in 17-month-old transplant recipients were preserved/restored to the levels seen previously at 13 months of age.

In the current experiments, germ cell depletion enhanced the longevity-extending effects of young, transplanted ovaries and, as with germ cell-containing ovaries, decreased the severity of chronic inflammation. Taken together, the current work suggests the presence of a potentially evolutionarily conserved, germ cell-independent molecular mechanism that contributes to the ovarian tissue-dependent extension of health and life span in a mammalian model. This research was supported by Utah State University, School of Veterinary Medicine, Department of Animal, Dairy and Veterinary Sciences and by a generous gift of aged CBA/J female mice from Nancy Nadon at the National Institute on Aging.

Therapeutic applications of Nicotinamide riboside to treat liver injury

Mukherjee S, Chellappa K, Mo J and Baur JA

Department of Physiology and Institute for Diabetes, Obesity and Metabolism,

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

Abstract:

Liver injury in humans occurs in response to a variety of insults including toxins and infections. It has been reported approximately 75% of idiosyncratic drug reactions result in liver transplantation or death, and drug induced acute liver failure accounts for 50% of cases in the United States¹. To allow survival, the liver must repair or replace the lost/injured cells. However, the ability to regenerate in response to acute and chronic liver injury are not well understood. Several studies suggest that aging compromises hepatic regeneration by influencing metabolic and signaling pathways, particularly nicotinamide adenine dinucleotide (NAD) metabolism. NAD levels markedly decline with age and in addition its concentration falls during liver regeneration following partial hepatectomy (PHx), possibly due to metabolic competition for common precursors required for DNA synthesis. Our recent study has shown that restoring NAD by supplementation with nicotinamide riboside (NR), a metabolic precursor promotes liver regeneration and attenuates the hepatic steatosis that normally accompanies liver injury². Hence, NR supplementation may be a novel therapeutic strategy to improve metabolic function and regrowth in liver injury models. However, the optimal dosing and mechanism of action remain to be established. Therefore, we propose to address several outstanding questions: 1) Is the protective effect of NR applicable to other types of liver injury and/or effective when initiated post-injury, rather than given prophylactically? 2) What is the effect of NR supplementation on metabolites related to nucleic acid and lipid synthesis? 3) Is the protective effect of NR mediated by sirtuin 1 (SIRT1), an NAD-dependent deacetylase that has previously been implicated in several models of liver failure? Successful completion of this study will provide potential therapeutic applications of NR in acute and chronic liver injury.

Reference:

1. Ostapowicz, G. et al. Results of a prospective study of acute liver failure at 17 tertiary care centers in the United States. Ann Intern Med 137, 947-954 (2002).

2. Mukherjee, S. et al. Nicotinamide dinucleotide biosynthesis promotes liver regeneration. Hepatology 65(2), 616-630 (2017).

Funding:

Supported by grants from the National Institutes of Health (R01 AG043483 and R01 DK098656, to J.A.B.)

Maf1-dependent transcriptional regulation of tRNAs prevents genomic instability to extend lifespan

Eishi Noguchi,¹ Mihir Shetty,¹ Chiaki Noguchi,¹ Sydney Wilson,¹ Esteban Martinez,¹ Kazuhiro Shiozaki,^2,3 and Christian Sell⁴

¹Department of Biochemistry and Molecular Biology, Drexel University College of Medicine; ²Graduate School of Biological Sciences, Nara Institute of Science and Technology; ³Department of Microbiology and Molecular Genetics, University of California; and ⁴Department of Pathology, Drexel University College of Medicine

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is regulated via phosphorylation by the mTOR (TOR in yeast) pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1’s function in tRNA repression is inhibited by mTOR-dependent phosphorylation, while Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with tRNA levels. Protein synthesis is elevated in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by Rad52 DNA damage foci formation and Rad52 recruitment at tRNA genes. Loss of Rad52 DNA repair protein further exacerbates the short lifespan of maf1∆ cells. Strikingly, PP4 deletion, which leads to Maf1 inactivation and elevated tRNA synthesis, alleviates DNA damage and rescues the short lifespan of maf1∆ cells, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.

Funding: National Institute of Health (GM077604 to EN), 2016 Pennsylvania Department of Health Formula Grant (to EN), and the Aging Initiative at Drexel University College of Medicine (to EN)

Tau-induced astrocyte senescence as a driver of neuronal dysfunction in AD Angela Olson,^1,2 Stacy Hussong,^1,2,3 Veronica Galvan^{1,2,3 1}Department of Cellular & Integrative Physiology, University of Texas Health at San Antonio, TX; ²Barshop Institute for Longevity and Aging Studies, University of Texas Health at San Antonio, TX; ³South Texas Veterans Heath Care System

The accumulation of molecular damage in somatic cells can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the “senescent-associated secretory phenotype” (SASP). During the pathogenesis of Alzheimer’s disease (AD) and other tauopathies, the microtubule-stabilizing factor tau is phosphorylated, becomes misfolded, and detaches from microtubules, destabilizing the microtubule cytoskeleton. Misfolded tau forms soluble aggregates that are released extracellularly and are transmitted trans-neuronally, promoting native tau phosphorylation and aggregation in target cells. We recently showed that, in addition to neurons, soluble extracellular tau aggregates propagate to brain microvascular endothelial cells, where microtubule destabilization triggers senescence/SASP. Neuronally-originated extracellular tau can also reach nearby astrocytes, which express abundant tau. Markers of cellular senescence and inflammation are increased in AD brain, including in astrocytes; further, we found that astrocytes in AD models display markers of senescence. In the present study, we tested the central hypothesis that soluble aggregated tau propagates to astrocytes and induces astrocyte senescence/SASP. We found that soluble aggregated tau propagates into primary human astrocytes. Tau transmission into astrocytes triggered endogenous tau phosphorylation and microtubule destabilization, followed by upregulation of markers of permanent cell cycle arrest and the acquisition of SASP. These data provide the first evidence that soluble aggregated tau propagates to astrocytes, and indicate that aggregate propagation triggers astrocyte senescence/SASP. Cocultures of soluble aggregated tau-pretreated senescent astrocytes with primary neurons increased senescent markers and SASP in neurons, suggesting that senescent astrocytes may be a critical mediator of neuronal dysfunction in AD. Because drugs that eliminate senescent cells are FDA-approved and antibody-based approaches to remove tau from brain are in clinical trials, our studies may have rapid translational potential for AD and potentially other tauopathies.

Funding: NIA T32 AG021890; NIA R01 AG057964-01

Aerobic Exercise Improves Redox Capacity in Inactive Older Adults:

A Randomized (Crossover) Control Trial

Ethan Ostrom^1*, Savannah Berry¹, Aaron Done², Derek Sonderegger³, and Tinna Traustadóttir¹

¹Department of Biological Sciences, Northern Arizona University

²University of Arizona College of Medicine

³Department of Mathematics & Statistics, Northern Arizona University

Background. Aging is associated with a loss of redox capacity resulting in increased risk of disease and dysfunction. Physically fit individuals show better outcomes in response to stress, but the extent to which previously inactive individuals can improve their redox capacity after an exercise intervention is unclear. This study tested the hypothesis that an 8-week aerobic exercise intervention would improve redox capacity as measured by the response to a non-exercise oxidative challenge: forearm ischemia/reperfusion (I/R) trial.

Methods. Inactive men and women (62-77yo) were randomized in a 3:2 ratio to an eight-week aerobic exercise intervention (EX; n=13) or a non-exercise (sedentary) control group (CON; n=8). Aerobic exercise was completed 3 days/week at moderate-to-high intensity. Individuals randomized to CON were given the option to cross over into the EX group after completing the control arm. The effectiveness of the exercise intervention was measured by changes in maximal oxygen consumption (VO₂ max). Redox capacity was measured by plasma F₂-isoprostane response to the I/R trial.

Results. Eleven individuals completed the EX intervention and 7 completed the control group, 5 of which crossed over to the EX group. There were no significant baseline differences between groups for age, blood pressure, body composition, VO₂ max, or F₂-isoprostanes. The I/R trial induced a significant response in the whole cohort (P=0.01). The exercise intervention improved VO₂ max by 17% (P<0.05) while CON did not change. The I/R response was significantly lower in EX compared to CON after the 8-week intervention (P=0.05).

Conclusion. These data demonstrate that redox capacity can be restored with regular exercise in inactive older individuals. Importantly, the exercise-induced improvements translate to greater protection from oxidative stress produced under non-exercising conditions.

Funding: NIH R15AG055077

mTOR signaling in adipose tissue influences systemic lipid metabolism

Lauren M. Paolella,¹ Cassie Tran,¹ Bruna Bellaver, ¹ Karthikeyani Chellappa, ¹ Sarmistha Mukherjee, ¹ James G. Davis, ¹ William Quinn, ¹ Daniel J. Rader, ¹ and Joseph A. Baur¹

¹University of Pennsylvania

Rapamycin, a fungicidal macrolide, has been shown to increase life span and delay the onset of age related diseases across several model organisms. However, rapamycin treatment leads to metabolic dysregulation including glucose intolerance and hyperlipidemia, which limits its potential utility as an anti-ageing agent. Rapamycin treatment increases serum triglycerides in human patients by 95%, and while it influences the expression of multiple proteins involved in lipid handling in liver and adipose tissue, the precise mechanism by which rapamycin treatment causes hypertriglyceridemia is not known

Rapamycin is an allosteric inhibitor of mTOR (mechanistic target of rapamycin), a serine/threonine protein kinase that functions as a master regulator of cellular growth and metabolism. mTOR exists in two distinct protein complexes, mTORC1 (characterized by the presence of raptor) and mTORC2 (characterized by the presence of rictor), with each complex phosphorylating a distinct set of effector proteins. Rapamycin acutely inhibits mechanistic target of rapamycin complex I (mTORC1), but can also inhibit mTORC2 under chronic conditions. Tissue specific disruption of mTORC1 or mTORC2 has revealed that each complex has different effects on whole body glucose and lipid homeostasis in different organs. Genetic ablation of either mTORC1 or mTORC2 in hepatocytes fails to phenocopy the effects of rapamycin treatment, suggesting that mTOR signaling in another tissue is responsible for rapamycin-induced hypertriglyceridemia.

LPL hydrolyzes the TG in VLDL into FFA for uptake into adipose tissue. In addition, mTORC1 has been shown to inhibit lipolysis and therefore the releases of FFA into plasma. Using adipose specific raptor knockout mice, we show that TG clearance is delayed and LPL gene expression and genes related to LPL processing are altered in the absence of mTORC1 signaling. The decrease in TG clearance can be a result of the decrease in adipose tissue LPL synthesis due to decreased mRNA and/or decreases secretion of active LPL into the blood. This can result in decreased rate of VLDL-TG hydrolysis and slower clearance of plasma TG, leading to a buildup of TG levels in the blood. In addition, upon refeeding, these KO mice have increased serum TG and FFA, suggesting a failure to suppress lipolysis. Based off phosphoproteomics analysis, phosphorylation of ATGL, the rate limiting enzyme in lipolysis, is increased with loss of mTORC1 in adipose tissue, thus increasing activity of ATGL. ATGL-Raptor double knockout mice were generated. The DKO mice, upon refeeding, phenocopy control mice serum TG and FFA. This suggest that with loss of mTORC1 there is an increase in lipolysis in adipose tissue when lipolysis should be inhibited, which leads to increased release of FFA and uptake into the liver. This increase leads to an increase in liver TG for increased VLDL-TG secretion. Decreased LPL activity and increased lipolysis in adipose can both increase plasma TG levels. We hypothesize that inhibition of mTORC1 signaling in adipose tissue is the major driver of hyperlipidemia caused by rapamycin.

Funding: This work is supported by predoctoral fellowship (F31AG057171) and R01AG043483 from the National Instituate on Aging

Mechanisms and Consequences of Reduced Allopregnanolone in Brain Aging Eileen Parks^1,2, William E. Sonntag^1,2,3. ¹Oklahoma Center for Neuroscience, ²Reynolds Oklahoma Center on Aging, ³Department of Geriatric Medicine.

Introduction: Age-related cognitive decline is a growing problem in today’s society that frequently leads to reduced quality of life, dependence on caretakers, and significant health care costs. We have previously shown that the levels of the progesterone metabolite, Allopregnanolone (ALLO), are reduced with age in male C57BL/6 mice and have proposed that a deficiency in this neurosteroid contributes to cognitive impairment. Importantly, a single subcutaneous injection of ALLO improved spatial learning and memory, as well as increased hippocampal neural stem cell activation in aged animals. In this study, we investigate the role of age-related changes in steroidogenic enzyme activity in the decline in ALLO with age and whether inflammatory factors contribute to this process.

Methods: Hippocampal tissue from 3, 12 and 24 month old male C57BL/6 mice were isolated to quantify mRNA expression for various steroidogenic enzymes and microsomes were isolated for enzyme activity assays. ¹⁴C-progesterone was added as the initiating substrate, and enzyme activity was assessed by quantifying neurosteroid metabolite formation. To assess the role of the inflammatory factor, IL-6, primary astrocytes were cultured from post-natal day 3 mouse pups, treated with or without 100 ng/mL interleukin-6 (IL-6), and enzyme activity studied.

Results: Progesterone is metabolized to ALLO, Corticosterone (CORT), and Testosterone (T) through a network of steroidogenic enzymes. The expression of ALLO synthesizing enzymes did not change with age, however, the activity of other enzymes (specifically 21-Hydroxylase and 11β-hydroxysteroid dehydrogenase) that regulate CORT were increased. Furthermore, IL-6 administration to astrocyte cultures led to an upregulation of CORT synthesizing enzymes.

Conclusion: The decline in ALLO with age appears to be the result of enhanced activity of CORT synthesizing enzymes that reduces ALLO substrate availability. This process appears to be regulated by inflammatory factors that are known to increase with age.

Funding: Donald W. Reynolds Predoctoral Fellowship; Oklahoma Nathan Shock Center Award; Grants AG37847 and NS56218 to WES. OCNS seed grant 2017

Roles of the Fanconi anemia DNA repair pathway and acetaldehyde detoxification in preventing genomic instability in esophageal keratinocytes Jasmine D. Peake,¹ Koji Tanaka,² Amber Theriault,¹ Hiroshi Nakagawa,² and Eishi Noguchi¹ 1Drexel University College of Medicine, Department of Biochemistry and Molecular Biology; ²University of Pennsylvania, Department of Gastroenterology

Chronic alcohol consumption is a worldwide health issue, serving as the major cause of 1.8 million deaths per year and 3.6% of all cancers. Alcohol contributes to cellular accumulation of acetaldehyde, a primary metabolite of alcohol and a major human carcinogen. Individuals with a deficiency in acetaldehyde detoxification (aldehyde dehydrogenase ALDH2 deficiency) have an increased risk of esophageal squamous-cell carcinoma (ESCC). Acetaldehyde causes DNA adducts and damage, activating the Fanconi anemia (FA) DNA repair pathway, which is responsible for the removal of DNA interstrand crosslinks (ICLs) induced by DNA damaging agents. Defects in this pathway cause Fanconi anemia, a genetic disorder featuring bone marrow failure and predisposition to malignancies, including ESCC. However, the molecular basis of how acetaldehyde induces genomic instability and ESCC remains elusive.

In order to establish the role of both alcohol intake and the FA pathway in development of ESCC, we investigated acetaldehyde-mediated DNA damage response in esophageal keratinocytes. ALDH2 inhibition via disulfiram or gene knockout sensitized cells to acetaldehyde. Acetaldehyde induced 53BP1 and BRCA1 DNA damage foci in esophageal cells. Mouse esophageal 3D-organoid cultures were grown ex vivo and treated with acetaldehyde, then analyzed for H2AX expression levels. The H2AX level in ALDH2-/- organoids treated with acetaldehyde was significantly higher than that in ALDH2+/+ organoids, indicating that ALDH2-dependent acetaldehyde clearance plays a critical role in preventing DNA damage. Acetaldehyde also caused chk1 activation cell cycle delay in G2/M-phase, suggesting that these cells accumulate unrepaired acetaldehyde-dependent DNA damage, thus not allowing for cells to progess into mitosis. Furthermore, FANCD2, a protein required for replication fork stabilization, was ubiquitinated and formed DNA damage foci in response to acetaldehyde, and FANCD2 depletion sensitized cells to acetaldehyde. Taken together, our results suggest that the FA pathway and acetaldehyde detoxification play important role in preventing genomic instability in esophageal keratinocytes. Thus, our studies are relevant in understanding the role of alcohol intake in the development of ESCC.

Funding: R01 Diversity Supplement - R01GM106262-04S1

Muscle-Specific Expression of Mitochondrial-Targeted Catalase Does Not Rescue Denervation-Induced ROS Production or Skeletal Muscle Atrophy Gavin Pharaoh,^1,2 Rojina Ranjit,² Kavithalakshmi Sataranatarajan,^,2 Kendra Huseman², and Holly Van Remmen^{1,2,3 1}Physiology Department, University of Oklahoma Health Sciences Center; ²Aging & Metabolism Research Program, Oklahoma Medical Research Foundation; ³Oklahoma City VA Medical Center

Age-related declines in muscle mass and function reduce quality of life. The mechanisms underlying the initiation of these changes are still not defined, but previous studies from our laboratory have shown that defects in neuromuscular innervation (denervation) and increased muscle mitochondrial reactive oxygen species (ROS) generation play key roles. Specifically, we have demonstrated that loss of innervation induces increased muscle mitochondrial ROS generation associated with muscle atrophy. The goal of the current study was to examine whether the increase in muscle mitochondrial ROS production plays a causative role in denervation atrophy. To do this, we utilized a mouse model that has increased expression of mitochondrial-targeted catalase (MCAT) specifically in skeletal muscle mitochondria (skmMCAT mice). We asked whether increased expression of catalase targeted to mitochondria in skmMCAT mice can prevent or reduce denervation induced changes in muscle mitochondrial ROS production and alter muscle atrophy in a surgical denervation model. Our studies were performed in 6-8 month-old female wildtype and MCAT stop flox mice on a C57Bl6 genetic background. MCAT stop-flox mice were crossed with human α-skeletal actin (ACTA)-Cre mice to express MCAT in skeletal muscle (skmMCAT mice). Surprisingly, muscle-specific MCAT mice have reduced lean mass compared to wildtype control mice measured using quantitative magnetic resonance. We performed sciatic nerve transection on the left hindlimb and sham surgery on the right hindlimb of wildtype and skmMCAT mice and euthanized them at 7 days post-transection. Gastrocnemius mass is significantly reduced (~20%) at 7 days post-transection in both wildtype and skmMCAT mice. ROS generation in the gastrocnemius muscle, innervated by the sciatic nerve, was measured in isolated mitochondria (fluorometer) and permeabilized muscle fibers (Oroboros O2K) using the fluorescent probe Amplex Red, which has been proposed to have specificity for hydrogen peroxide. ROS production measured in denervated permeabilized muscle fibers and isolated mitochondria from wild type mice shows increased State 1 ROS generation at 7 days post transection. While MCAT expression decreases baseline muscle mitochondrial ROS production, it does not diminsh the ROS generation in denervated State 1 or glutamate/malate stimulated respiration as measured by Amplex Red. Interestingly, addition of exogenous catalase to isolated mitochondria from both wildtype and MCAT mice decreases the Amplex Red signal in mitochondria from sham but not denervated hindlimb. These results suggest that the ROS signal after denervation is not generated by hydrogen peroxide alone but also includes some other species that react with the Amplex Red probe. Thus, decreasing levels of mitochondrial hydrogen peroxide alone post-denervation does not rescue denervation induced atrophy or the mitochondrial ROS signal. Future studies will focus on identification of the non-hydrogen peroxide signal and experiments to scavenge this target to determine its role in denervation induced atrophy.

Funding: 1R01AG050676-01A1 Defining the Relative Roles of Pre- and Post-Synaptic Events in the Initiation and Progression of Sarcopenia. Oklahoma Medical Research Foundation Pre-doctoral Scholarship and NIA Training Grant (T32AG052363).

Aging is associated with loss of motor neurons and disruption of extracellular matrix homeostasis Katarzyna M Piekarz,¹ Shylesh Bhaskaran,² Kavithalakshmi Sataranatarajan,² Kaitlyn Riddle, ² and Holly Van Remmen ^{1,2 1} Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center; ² Program in Aging and Metabolism, Oklahoma Medical Research Foundation

Loss of muscle mass and function is a universal characteristic of aging and our laboratory has identified loss of innervation and neuromuscular junction disruption as a critical component in this process. The goal of this study is to identify changes in the spinal cord that contribute to the loss of innervation during aging.

Spinal cord sections from young (2-7 mo) and old (24-27 mo) mice were immunostained with anti-NeuN, anti-GFAP and anti-Iba1 antibodies. Spinal cord homogenates were used to measure caspase-3, MBP and MMP-12 levels by immunoblotting. Demyelination was further assessed by electron microscopy of spinal cord white matter, while cytokine protein levels were measured with Mouse Cytokine Array (R&D kit). Blood-spinal cord barrier (BSCB) permeability was evaluated by HRP assay. Also, RNA-seq analysis was used to uncover changes in transcriptome.

There is a 41% loss of motor neurons (MN) in aged spinal cord, and it is accompanied by morphological changes, such as an increase in MN cell body area. Cleaved caspase-3 is increased, indicating an increase in apoptosis that may be related to the MN loss. We observe a slight demyelination, supported by the decrease in MBP protein level, and the loss of myelin visible in electron micrographs. A significant increase (172-fold) in MMP-12 expression and enrichment of inflammation and microglia activation-related genes, as well as microglia and astrocyte activation and increase in sICAM-1, eotaxin, and IL-12p70 protein levels indicate the presence of a para-inflammatory state. BSCB permeability also increases with age.

Spinal cord aging is accompanied by MN loss, increased apoptosis, increase in MN cell body area, and demyelination. There is a para-inflammatory state, while ECM dynamics is altered, due to increase in MMPs levels and changes in ECM components expression. Increased BSCB permeability further contributes to alterations in spinal cord homeostasis.

Funding: This work was funded by the P01 grant 5198-03-00-0 awarded to HVR

Reduced methylation at the TRIP12 promoter is associated with multi-morbidity: Findings from the InCHIANTI Study Shabnam Salimi,¹ Brian Chen,² Elisa Fabbri,³ Toshiko Tanaka,⁴ Stefania Bandinelli,⁵ Jack Guralnik,⁶ Luigi Ferrucci^{7 1,6}University of Maryland Baltimore School of Medicine; ⁵ Geriatric Unit, Azienda Sanitaria di Firenze; ^2,3,4,7 National Institute on Aging

Introduction: Aging is a strong risk factor for many chronic diseases. The large number of older patients with multiple chronic diseases are a challenge to the health care system and to society. Understanding the mechanisms underlying age-related multi-morbidity may lead to new and more effective strategies to manage geriatric patients. Changes in DNA methylation, which have been observed during aging, may offer one possible mechanistic factor in multi-morbidity.

Methods: In 477 individuals with mean age 62 (range 21-91 yrs), multi-morbidity was defined as having ≥2 chronic diseases in Invecchiare in Chianti(InCHIANTI) study. DNA methylation was measured in the whole blood using Illumina 450K arrays. We performed a case/control Methylome-Wide Analysis adjusted for age, sex, batch of samples, and differential peripheral blood cells.

Results: We found that reduced methylation at the TRIP12 promoter was associated with multi-morbidity (standardized beta =-0.07, p<5.0×10^-8). The magnitude of effect increased with increased number of diseases (beta= -0.24). We excluded cancer patients, and the results remained robust. Pathway analysis revealed interactions between TRIP12, P53, and USP7, suggesting that the protein encoded by TRIP12 (Thyroid Hormone Receptor Interactor 12) regulates USP7 and P53, two tumor suppressor genes.

Conclusion: The reduced methylation at the TRIP12 promoter in whole blood was associated with multi-morbidity. We plan to further study collective effects of DNA methylation changes on temporal multi-morbidity phenotype in whole blood and in specific immune cells.

Metabolic effects of 17-alpha estradiol are growth hormone independent and sex specific Silvana Sidhom,¹ Michael B. Stout,² Augusto Schneider,³ Andrzej Bartke⁴, Yimin Fang⁴, Samuel McFadden⁴, Allyson K. Palmer⁵, Frederik J Steyn⁶, Michal M. Masternak¹

¹College of Medicine, Burnett School of Biomedical Sciences, University of Central Florida ²Department of Nutritional Sciences, University of Oklahoma Health Sciences Center ³Faculdade de Nutrição, Universidade Federal de Pelotas ⁴Department of Internal Medicine, Geriatrics Research, Southern Illinois University ⁵Medical Scientist Training Program, Mayo Clinic, Rochester MN ⁶Centre for Clinical Research, the University of Queensland, Herston, Australia

Aging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop new pharmacological interventions that would delay aging and protect from age-related diseases.

17-alpha estradiol (17α-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice. Because 17α-E2 does not induce feminization in males it holds potential as a novel therapeutic in humans for age-related metabolic dysfunction. Importantly, we have previously shown that 17α-E2 causes an increase of circulating and hepatic IGF-1 in aged mice, without any changes in pulsatile GH release in treated animals. Based on this study we propose a new hypothesis that 17α-E2 acts through a novel, GH-independent pathway stimulating production of IGF-1 and positively modulating metabolic function in a sex-specific manner. Here we studied 17α-E2 treated long-lived growth hormone receptor knockout (GHRKO) mice, characterized by severely reduced circulating and hepatic IGF-1 due to GH-resistance. We found an increase in circulating IGF-1 after treatment in normal and GHRKO male mice, with no effects seen in female mice. Analysis of key genes involved in insulin signaling pathways in the liver showed that 17α-E2 upregulated expression of PPARγ, PPARα and IRS1 in normal male mice. However, the intervention did not affect these genes in normal females or in either sex of the long-living GHRKO mice. These findings support our hypothesis that 17α-E2 upregulates IGF-1 independently of GH action. Our data also suggests that there is sex and genetic specific divergence in response to 17α-E2 treatment in regulation of genes involved in insulin signaling.

Age-associated expansion of gamma delta T cells promotes visceral adipose tissue inflammation Whitney L Powell¹, Allison M Steele¹, Beverly K Balasuriya¹, Stephanie F Mori¹, Donald A Cohen², Marlene E Starr^1,3; Departments of ¹Surgery; ²Microbiology, Immunology, and Molecular Genetics; ³Pharmacology and Nutritional Sciences; University of Kentucky

Chronic inflammation originating from adipose tissue, even in the absence of obesity, is strongly linked to the development of cardiometabolic disorders. To date, the majority of research in this area has focused on obesity without regard for aging, despite the fact that aging often poses a larger risk. The objective of this study was to identify age-specific alterations in adipose tissue physiology which may underlie chronic inflammation. Our previous microarray analyses of visceral adipose tissue from young (4-mo) and aged (24-mo) mice showed evidence of increased T cell populations in the aged, including a 6-fold increase in gene expression of T cell receptor gamma (PMC3633415). In the present study using flow cytometry, we found that visceral adipose tissues of aged mice contain 5-fold more gamma delta (γδ)-T cells than that of young mice (p<0.01). This expansion was unique to visceral fat, not being observed in subcutaneous fat, spleen, blood, or skin. Further, the adipose tissue γδ-T cells were CD44^hiCD62^low and positive for CD69, suggesting a tissue-resident memory T cell phenotype (T_RM). Using T cell receptor delta (TCRδ) knockout mice, we found that genetic deficiency of γδ-T cells significantly diminished the inflammatory response (measured by IL-6 production) of aged adipose tissues to ex vivo stimulation (p<0.01). Additionally, adipose tissues from aged TCRδ^-/- mice exhibited reduced expression of senescent cell marker p16^Ink4a compared to age-matched wild-type mice. Collectively, these findings suggest that expansion of a T_RM γδ-T cell population in visceral adipose tissues contributes to age-related chronic inflammation.

Funding: This study was supported by the National Institute of General Medical Science (P20 GM103527) of the National Institutes of Health.

Rapamycin and acarbose prevent fat accumulation in adult mice fed a high-fat diet. Alessandro Bitto¹ Nicole Tatom¹, Jessica Snyder², Mariya Sweetwyne¹, Daniel Larry Smith³, and Matt Kaeberlein¹.

¹Department of Pathology, University Washington Medical Center; ² Department of Comparative Medicine, University of Washington Medical Center; ³Department of Nutrition Science, University of Alabama at Birmingham.

Chronic inhibition of mTORC1 via rapamycin increases lifespan and ameliorates several age-related phenotypes in multiple species, including mice. However, long-term administration of rapamycin has potential detrimental effects on lipid and glucose homeostasis. Lipodystrophy, metabolic syndrome, and type II diabetes are common age-associated metabolic impairments that may be adversely affected by rapamycin treatment.

Acarbose is a FDA-approved drug for the treatment and management of type II diabetes. Acarbose also increases lifespan in mice and prevents fat accumulation in a genetic model of lipodystrophy. Preliminary evidence from our laboratory suggests that both rapamycin and acarbose shift nutrient metabolism away from glycolysis towards catabolism of fats and amino acids via biochemically distinct signaling pathways, implying that the two drugs together could have synergistic effects on metabolism.

To study these effects, 66 nine-month-old mice were fed either a low (11%) or high (66%) fat diet and treated with rapamycin, acarbose, or a combination thereof for 6 weeks. Weights, food consumption, fasting blood glucose, and body composition were measured before onset of treatment and at regular intervals during treatment. At the end of this period, the mice were sacrificed, and the tissues were collected for further analysis. Mice treated with rapamycin gained less weight than those without drug intervention or treated with acarbose alone and did not show any obvious increase in blood glucose levels. The combination of both treatments did not have additive effects on weight gain but did reduce fat mass accumulation more than either treatment alone in female mice. We are further investigating the effects of rapamycin and acarbose in this context by conducting post mortem analyses, including the composition of the caecal microbiome, tissue histopathology, activation of fat metabolism, and mitochondrial uncoupling in white adipose tissue.

Funding: United Mitochondrial Disease Foundation Post-Doctoral Fellowship, National Institute of Neurological Disorders and Stroke 1 R01 NS98329

Sulfur Metabolism in Methionine Restriction Thyne KM,¹ Liu Y,¹ and Salmon AB^{1 1}The Sam and Ann Barshop Institute for Aging and Longevity Studies

Caloric restriction, a form of dietary restriction, is perhaps the most robust and well-studied method for improving lifespan. Another form of dietary restriction involving limiting the essential amino acid methionine has also been shown to improve lifespan and confer benefits similar to caloric restriction. Despite the apparent similarities between caloric and methionine restrictions, they both appear to have their own idiosyncrasies. More specifically, sulfur metabolism has emerged as a major contributor to methionine restriction’s effects. To help elucidate these mechanisms our goal is to examine components of sulfur biochemistry that have remained previously uncharacterized in methionine restriction. Additionally, we address the central role of methionine oxidation in these mechanisms. The sulfur atom of the methionine thioether is sensitive to oxidation, however this damage can be reversed by methionine sulfoxide reductases, such as MsrA, found in eukaryotic cells. Interestingly, some of the metabolic effects of methionine restriction may be mediated by the presence of MsrA. How the oxidation state of methionine connects with the benefits of its restriction is unclear, but these findings suggest a functional requirement for MsrA expression in methionine restriction.

Funding: T32 Training Grant – 5T32AG021890-15

Resistance to Oxidative Stress in Older Men and Women;

Effects of Aerobic Fitness are Sex Dependent

Tinna Traustadóttir,¹ Savannah R. Berry,¹ and Ethan L. Ostrom¹

¹Department of Biological Sciences, Northern Arizona University

Background. Resistance to oxidative stress is reduced with age but there is lack of data regarding sex differences. In general, many sex differences are driven by sex hormones and thus might be expected to be lessened after menopause and at older ages. Aerobic fitness has been shown to increase redox capacity in older adults but whether the adaptations differ between men and women is unclear. The aim of the present study was to investigate sex differences in redox capacity, while including aerobic fitness in the analyses.

Methods. Healthy men (68y ±1, n=20) and postmenopausal women (65y ± 1, n=18) ages 60-86y participated in this study. Maximal oxygen consumption (VO₂ max) was measured with a graded exercise test on a cycle ergometer. Resistance to oxidative stress was measured by F₂-isoprostane response to a forearm ischemia/reperfusion (I/R) trial from blood draws at 7 time points.

Results. The groups did not differ in age, BMI, or VO₂ max. As expected men had a greater waist circumference and achieved a higher maximal workload during the VO₂ max test (p<0.01). The I/R trial elicited a significant F₂-isoP response in the cohort as a whole (p<0.05). Women had higher F₂-isoP levels across time compared to men and an earlier peak (time-by-sex interaction and main effect of sex; p<0.05). There was a trend for women having higher baseline levels (p=0.07) and we therefore analyzed the data as percent change from baseline. The time-by-sex interaction remained significant (p<0.01) but the group difference was no longer significant. Fitness levels were negatively correlated with both the overall F₂-isoP levels (AUC; r=-0.490) and the response with respect to baseline (AURC; r=-0.476) in women (p<0.05) but not in men.

Conclusion. These data demonstrate that postmenopausal women have elevated F₂-isoprostane levels relative to age-matched men. While the magnitude of the response to an oxidative challenge was not different between sexes, the pattern of the response differed. Interestingly, protective effects of fitness on the redox capacity were only seen in women. The lack of significant effect in men may be due to lower levels of F₂-isoPs.

Funding: NIH R15AG055077 and Northern Arizona University Faculty Grant Program (FY2017).

Neuronal Dysfunction Induced by Senescent Astrocytes

Ferit Tüzer¹_, Ankita Patil²_, Peter W. Baas², Claudio Torres¹

¹Drexel University College of Medicine, Department of Pathology and Laboratory Medicine

²Drexel University College of Medicine, Department of Neurobiology and Anatomy

As organisms age, many cell types lose their replicative potential and enter a state known as senescence. Replicative or oxidative stress can cause senescence due to DNA-damage response signaling, which can also lead to apoptosis at higher rates. However, senescent cells upregulate pro-survival genes and therefore resist apoptosis. Senescence not only reduces the regenerative capacity of an organism, but also can disrupt function of nearby cells and tissues by the senescence associated secretory phenotype that is pro-inflammatory and tissue remodeling. In the brain, there is evidence of senescence of multiple cell types, and we have established that in vitro, senescent human astrocytes undergo widespread gene expression changes, which lead to loss of expression of astrocyte-specific genes, and hence of astrocyte function.

The effects of astrocyte senescence in the brain have not been explored. Our results with rat astrocytes and neurons isolated from post-natal and embryonic brains, respectively, show that, culturing neurons with conditioned media from senescent astrocytes significantly decreases their mitochondrial membrane potential compared to culture with media from proliferating cells. As neurons depend heavily on oxidative phosphorylation and the brain uses ca. 20% of the oxygen consumed by the body, loss in their energy producing capability could have severe consequences. Neurons incubated with media from senescent cells have also significantly lowered chymotrypsin-like proteasome activity of the 20S proteasome. The 20S proteasome is involved in degradation of oxidized and disordered proteins, including α-synuclein and tau which are increased in the age-related diseases Parkinson’s disease and Alzheimer’s disease (AD), respectively. Moreover, a link between proteasome inhibition and mitochondrial dysfunction has previously been shown in human fibroblasts. We are investigating whether these events are linked and whether this dysfunction leads to an increase in reactive oxygen species in neurons as well. Overall, these results point to a possible mechanism for neurodegeneration in aging and AD, where the population of senescent astrocytes increases.

Funding: Coins for Alzheimer’s Research Trust - Neuron Senescence as Mediator of Neurodegeneration in Alzheimer’s Disease, NINDS R01NS28785 - Microtubule dynamics and axon growth

The NIA Nonhuman Primate Core Program: Providing Translational Aging Research Support

Kelli L. Vaughan^1,2 & Julie A. Mattison¹

¹NIA Nonhuman Primate Core, NIH Animal Center, Poolesville, MD; ²SoBran, Inc., Burtonsville, MD

The NIA Nonhuman Primate Core was established in 2012 to support the experimental goals of intramural and extramural investigators. We provide support for translational research projects concentrated on the development and application of nonhuman primate models of disease, particularly those related to characterization of and novel treatments for age-associated illnesses. The Core consists of a team of skilled research technicians, experienced in various animal testing techniques including physiological sampling, experimental data collection, and behavioral characterization and training of nonhuman primates. Core staff specialize in safe practices of a wide range of technical procedures required in the collection, analysis, and interpretation of complex nonhuman primate data. The Core assists in the development of translational research methodologies to facilitate the attainment of project specific objectives while reducing the cost burden by sharing resources. Pursuant with project needs, the Core maintains the resources and capabilities to provide research support that is required from protocol development, IACUC submissions and approvals, through the implementation of data collection procedures, and culminating with the organization and storage of resultant tissues and/or data sets. In addition to novel projects, the Core maintains an extensive repository of archived samples, from male and female rhesus monkeys of varying ages and experimental conditions, that have been collected over the past 20 years. These samples are also available for proof of concept projects, assay validation studies, and in vitro analyses.

We have contributed to research projects in the areas of immunology, microbiome, muscle physiology and peripheral artery disease, macular degeneration, bone aging, Alzheimer’s disease, and diabetes, to name a few. And, we have investigated dietary interventions and pharmacological substances like resveratrol, disulfiram, estradiol, incretins, dpp-4 inhibitors, and resveratrol, among others.

Funding: This project was supported entirely by the Intramural Research Program, National Institute on Aging, NIH.

A Drosophila Model of Genetic Variation in Alzheimer’s Disease Adrienne M. Wang¹, Ming Yang², Natalie Pearlman², Jake Mouser², Irene Cruz Talevara², Alex Zhu², and Daniel Promislow².

¹Department of Biology, Western Washington University; ²Department of Pathology, University of Washington

Age is the primary risk factor for many neurodegenerative diseases, including Alzheimer’s disease (AD). With a rapidly aging population, strategies to mitigate the direct and indirect costs of caring for patients with dementia are paramount. AD exhibits a strong genetic basis, and mutations in three well-established genes have been associated with rare, familial, early-onset AD (EOAD). These mutations alter production of the amyloid beta peptide, the principal component of the amyloid plaques that, along with tau neurofibrillary tangles, are pathological hallmarks of AD. Despite success in identifying causal mutations for EOAD, susceptibility loci associated with the much more common sporadic, or late-onset AD (LOAD) have proven more difficult to identify and validate due to the genetic heterogeneity of the disease, the established environmental effects, and potential epistatic interactions. Evidence from a number of GWAS indicate that the disease is highly polygenic and affected by multiple alleles with small effect size.

Here, we use a Drosophila model of AD that expresses both the toxic Aβ_1-42 peptide as well as full-length Tau in the fly eye. Expression of these proteins in the Drosophila compound eye results in degeneration of the eye that is easily visible and quantifiable as a rough eye phenotype using a semi-automated imaging program. We combine this disease model with the Drosophila Genetic Reference Panel (DGRP), a model of natural variation that has been used to identify and in some cases validate alleles and pathways that affect complex traits and diseases, such as retinitis pigmentosa, diabetes, and lifespan and fecundity, among others. This living library of polymorphisms takes advantage of the high density of common variants, decay in linkage disequilibrium over only hundreds of base pairs, and the ability for repeat measurements in inbred lines that Drosophila affords in order to increase statistical power and the accuracy of phenotypic quantification under strict environmental control. By expressing Aβ_1-42 and Tau across the 200 isogenic and fully sequenced lines of the DGRP, we will be able to leverage the power of Drosophila genetics to understand how genotypic variation maps to phenotypic variation in a Drosophila model of AD.

Funding: NIA 1R21AG056872-01

Combating Cardiac Aging Using Mitochondrial Pharmacotherapeutics: SS-31 and NMN Jeremy A. Whitson,¹ Ying Ann Chiao,¹ Kathryn Mills,² Shin-ichiro Imai², Eric G. Shankland,¹ Kevin E. Conley¹ and Peter S. Rabinovitch¹

¹University of Washington; ²Washington University in St. Louis

Mitochondria are highly abundant in the heart, where they are essential for meeting its unique energetic demands. This makes mitochondria ideal targets for pharmacotherapeutics to improve the health of the aging heart and treat age-related cardiovascular diseases.

We tested the effect of two drugs, the mitochondrial-targeted tetrapeptide SS-31 (also known as elamipretide) and the NAD(P) precursor NMN, over the course of 8 weeks in 2-year old mice. Using a novel in vivo nuclear magnetic resonance approach, energetic and redox dynamics, including NAD(H) and NADP(H) dynamics, were measured in beating mouse hearts and compared to those of 7-month old mice. These data were coupled with heart functional measurements by echocardiography and treadmill performance to determine the full in vivo effect of these drugs from the metabolite level to structural and performance levels. Additionally, shotgun thiolation proteomics of SS-31-treated mouse hearts demonstrated the ability of the drug to restore the proteome thiolation state to closely match the profile of the young mouse heart proteome.

These results provide an integrated picture of the effect of mitochondrial pharmacotherapeutics on the aging heart and are highly informative of the potential these drugs hold in treating human age-related heart impairments and pathologies.

Funding: Genetic Approaches to Aging Training Grant (T32AG000057-40)

Aging and oxidative stress modify the osteocyte microtubule cytoskeleton, a key regulator of skeletal mechanotransduction Katrina M. Williams¹, James S. Lyons¹, Christopher W. Ward,² and Joseph P. Stains¹. ¹Department of Orthopaedics, University of Maryland, School of Medicine; ²Department of Orthopaedics, University of Maryland, School of Nursing

The skeleton responds to biomechanical stimuli to dynamically regulate its material properties. During aging, the skeleton’s ability to properly respond to mechanical stimuli declines, resulting in reduced bone mass. Low bone mass and the associated increased propensity to fracture have become a substantial problem for the expanding elderly population. The multi-factorial mechanisms leading to age-related osteopenia are incompletely understood, and therapeutic opportunities exist in elucidating the mechanisms contributing to loss of bone mass. Sclerostin, a negative effector of bone mass, has been used as a pharmacological target for the treatment of bone loss, including age-dependent osteoporosis. Recently, our lab has identified a role for the osteocyte cytoskeleton in the bone mechanoresponse, which down regulates the sclerostin protein and leads to increased bone mass. Specifically, we showed that a subset of the microtubule network, which is post-translationally modified by detyrosination (deTyr), is required to sense and transduce mechanical load signals to downstream signaling effectors (phospho-Ca2+/calmodulin -dependent protein kinase II (pCAMKII) and sclerostin) via NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS) and opening of the TRPV4 channel. Activation of this pathway, which is dependent upon cytoskeletal stiffness, ultimately leads to reduced sclerostin.

In the present study, we examine the aging dependent changes in the microtubule network and cytoskeleton that would predict mechanoresponsiveness. Using micro-computed tomography (microCT), we show in a cohort of mice throughout the lifespan (ages 4, 8 , 12, 18, and 24 months) that trabecular bone mass in the distal femur significantly decreases as early as 12 months of age, and femoral cortical geometry is altered by 24 months of age. Concomitantly, the deTyr- and total α-tubulin protein begins to increase by 12 months, followed by increases in the microtubule-associated protein (MAP) Tau, MAP1b, MAP4, and an isoform of alpha-tubulin which already lacks the terminal tyrosine, α-Tub4A. These increased PTMs and MAPs suggest a stiffer microtubule network, which would be consistent with a decreased mechanical load response. Our preliminary data during this aging trajectory reveal increases in glutathione reductase (Grx) protein and the Nrf2-activated gene Nqo1, consistent with increased oxidative stress. We predict that oxidative stress may be a driver of changes in cytoskeletal stiffness as cultured osteocytes (Ocy454) exposed to exogenous ROS (H2O2) increase MT PTMs and Tau expression, which would be associated with increased cytoskeletal stiffness. In total, our data suggest that age-dependent oxidative stress may be a driver of microtubule-dependent changes in the cytoskeleton that would be predicted to blunt bone mechanoresponsiveness and lead to low bone mass.

Funding: NIH/NIAMS R01AR071614

Wheel Running Predicts Resilience to Tumors in Old Mice

Lida Zhu,¹ Jorming Goh,¹ Christina Pettan-Brewer,¹ Warren Ladiges¹

¹Department of Comparative medicine, University of Washington

A major gerontological challenge is how to predict healthy aging, and the need for intervention strategies with increasing age. Assessment of physical resilience could be highly informative in this regard, especially for age-related diseases such as cancer. Physical resilience is the ability of an organism to respond to physical stress, specifically, stress that acutely disrupts normal physiological homeostasis. It is the ability to quickly resolve these unexpected or unusual challenges such as environmental, medical or clinical that should be relevant to a better understanding of the underlying health status. Measures of resilience are aimed at various types of stressors. Voluntary wheel running is a mildly stressful physical activity that is easily quantifiable. We addressed the question of whether the motivation and capacity for running, measured by voluntary wheel running, would be predictive of tumor resilience.

Male C57BL/6 mice in cohorts of 4, 12, 20, and 28 months of age were allowed to access a slanted in-cage running wheel for 3 days. After 3 months, mice were injected subcutaneously with B16 melanoma tumor cells and followed for one month before harvesting. The relation between running distance and tumor burden was statistically analyzed by statistical software JMP Pro using bivariate fitting.

Running distance and tumor burden both decreased with increasing age. No observable relation was found between running distance and tumor burden in mice at age 4 or 12 months; however, a linear negative relation was found in mice at ages 20 and 28 months. Moreover, with increasing age, the correlation strength and proportional ratio also increased, which indicated that age increased the relation. The results show that the motivation and ability to run, as defined by a 3-day running event, align with tumor progression in older age mice. These findings suggest that short term exercise capability could be a marker for resilience to tumors, and possibly other age-related disease conditions. This type of non-invasive examination strategy might be helpful in determining the need for aging-intervention approaches in the elderly.

Funding: NIH R01-AG057381