Poster Abstract 30
Age-related loss of hepatic Nrf2 protein homeostasis is attributed to heightened expression of miR-146a
 
Nicholas O. Thomas, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Kate P. Shay, Linus Pauling Institute, Oregon State University
Eric J. Smith, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Judy A. Butler, Linus Pauling Institute, Oregon State University
Liam F. Finlay, Linus Pauling Institute, Oregon State University
Tory M. Hagen, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

 

Nrf2 regulates the expression of numerous anti-oxidant, anti-inflammatory, and metabolic genes. Paradoxically, we observed that Nrf2 protein levels decline in the livers of aged rats despite the inflammatory environment evident in that organ. To investigate the cause(s) of this loss, we examined the age-related changes in Nrf2 protein homeostasis and activation in cultured hepatocytes from young (4-6 months) and old (24-28 months) Fischer 344 rats. While no age-dependent change in Nrf2 mRNA levels was observed (p>0.05), Nrf2 protein content, as well as the basal and inducible expression of Nrf2-dependent genes were attenuated with age. Conversely, overexpression of Nrf2 in cells from old animals reinstated gene induction. Treatment with Nrf2-inducer, anetholetrithione (A3T), along with bortezomib to inhibit degradation of existing protein, caused Nrf2 to accumulate significantly in cells from young animals (p<0.05), but not old. This indicated a lack of new Nrf2 synthesis. We hypothesized that the loss of synthesis with age may partly stem from an increase in microRNA inhibition of Nrf2 translation. Microarray analysis revealed that six microRNAs significantly increased with age (>2-fold, p<0.05). One of these, miRNA-146a, is predicted to bind with high complementarity to Nrf2 mRNA. Transfection of hepatocytes from young rats with a miRNA-146a mimic caused a 55% attenuation of Nrf2 translation (p<0.05) that paralleled the age-related loss of Nrf2. Overall, these results provide novel insights for the age-related decline in Nrf2 and identify new targets to maintain Nrf2-dependent detoxification with age.

 
Poster Abstract 31
The role of proteostasis in protein aggregation in long-lived species
 
Bharath Sunchu, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Zhen Yu, Linus Pauling Institute, Oregon State University
Kelsey Caples, Department of Biochemistry and Biophysics, Oregon State University
Viviana I. Perez, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

 

Research over the past decade supports the hypothesis that proteotoxicity, the accumulation of misfolded proteins and aberrant protein aggregates, plays a major role in aging and age-related diseases. This view is based on observations such as: 1) mechanisms that maintain protein structure (proteostasis) decline with age in several animal models and in senescent human fibroblasts, 2) increasing proteostasis leads to a decrease in proteotoxicity and an increased lifespan and healthspan in C. elegans. However, almost all of the data on the relationship between longevity and proteotoxicity comes from studies done in short-lived animals, e.g., primarily C. elegans and mice. It is not clear that these observations will translate to species with a long lifespan, such as humans, and because of the difference in time scales, it is possible that the mechanisms used by long-lived species to increase lifespan might differ from mechanisms used by invertebrates or even mice. In this work we are using a comparative biology approach to determine whether enhanced proteostasis reduce proteotoxicity in long-livedspecies. Our previous data showed that fibroblasts from long-lived species have higher activity of proteostatic mechanisms when compared to short-lived ones. This led us to investigate the handling of proteotoxicity and protein aggregation by these species. Based on our data we hypothesized that the enhanced proteostasis mechanisms in long-lived species will confer more protection against toxic misfolded proteins. To test our hypothesis we used a fluorescence based aggregation model (polyQ82-YFP) in skin fibroblasts from long-lived and short-lived species.
The aims of this study are to: 1) Determine if naked mole rat (NMR) fibroblasts handle proteotoxicity induced by polyQ82-YFP better than mouse fibroblasts, and 2) Delineate the mechanism(s) through which NMR fibroblasts manage polyQ82-YFP induced proteotoxicity

 
Poster Abstract 32
Dietary restriction improves cognitive performance and maintains hepatic Sirt7 levels in Nothobranchius guentheri , a short-lived model of aging
 
Kate Petersen Shay, Linus Pauling Institute, Oregon State University
Heather M. Forsythe, Department of Biochemistry and Biophysics, Oregon State University
Nicholas O. Thomas, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Viviana I. Perez, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Tory M. Hagen, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

 

Human life expectancy has grown over the last century, but the number of years lived relatively healthfully and independently with minimal medical needs, or health span, has not kept pace. This is detrimental not only for the individual, but it places additional strain on an already overburdened health care system. It is essential that we identify strategies for improving human health span. One such strategy, evaluated in laboratory animals, is dietary restriction (DR) of 20-40% of caloric intake. DR is known to lengthen lifespan and delay age-associated disease onset. This strategy is difficult to test in humans due to compliance, and challenging to test in many model species because evaluating age- related effects over the lifetime is resource and labor intensive. To overcome this hurdle, we have developed a novel animal model, Nothobranchius guentheri, a short-lived (< 1 year) fish that experiences rapid aging and can withstand up to 50% DR with no ill effects. Using male N. guentheri, we evaluated the ability of DR to prevent age-related decline in cognitive function. Our results demonstrated that fish whose diet was restricted by 50% performed better in an active avoidance test, and middle aged DR fish showed no decline in performance versus young control fish. We also measured hepatic levels of the nuclear sirtuins, histone deacetylases that regulate the cellular response to nutrient availability. Loss of sirtuin activity is strongly correlated with aging. We found that the expression of SirT7 protein, which is mainly nucleolar, was preserved by DR from early to middle age. SirT7 plays a role in maintaining the rate of protein synthesis, a process that declines with age. We have thus identified a potential DR-regulated molecular target for improving health span.

 
Poster Abstract 33
Long-term supplementation with a novel nutrient blend mimics calorie restriction transcriptomics in multiple tissues of old mice
 
Angela Mastaloudis, Nu Skin Enterprises
Eva Serna, Servicio Análisis Multigénico Unidad Central de Investigación Faculty of Medicine, University of Valencia
Steven M. Wood, Nu Skin Enterprises Shelly Hester, Nu Skin Enterprises
Richard Weindruch, LifeGen Technologies LLC, Department of Medicine, SMPH, University of Wisconsin, Education and Clinical Center, William S. Middleton Memorial Veterans Hospital
Tomas A. Prolla, LifeGen Technologies LLC, Departments of Genetics and Medical Genetics, University of Wisconsin
Jose Vina, Department of Physiology, Faculty of Medicine, University of Valencia, INCLIVA

 

Identification of caloric restriction mimetics (CRMs), compounds that mimic the beneficial effects of caloric restriction (CR) without restriction of dietary energy would be an advancement in anti- aging science. The present study investigated whether the transcriptomic profiles of a putative CRM nutrient blend could mimic that of CR in diverse tissues following long-term feeding. B6C3F1 male mice; n=7 per group. Young Controls (YC; 5 months) and 3 groups treated from 14-30 months of age: Old Controls (OC), Old CR (OCR; 25% CR) and Old Supplemented (OS). Gene expression profiling in cerebral cortex tissue (CCT), skeletal muscle (gastrocnemius) (SKL), heart (HRT) and liver (LVR) was performed using Affymetrix Mouse 2.0ST arrays. Principal component analysis revealed that gene expression profiles of YC and OC were distinct from one another in all tissues. Using differential analysis, genes commonly expressed in OCR and OS groups compared to the OC group were identified in CCT (3,468), SKL (2,386), HRT (3,523) and LVR (1,276). The OS mimicked OCR transcriptomics most dramatically in tissues most relevant to aging and age-associated diseases, CCT, HRT and SKL. These CRM effects, elicited by a mid-life intervention, may have positive implications for healthy human aging or ‘youthspan’ and warrants further investigation.

 
Poster Abstract 34
Rapamycin inhibits the secretory phenotype of senescent cells by an Nrf2-independent mechanism
 
Rong Wang, Linus Pauling Institute, Oregon State University
Zhen Yu, Linus Pauling Institute, Oregon State University
Bharath Sunchu, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
James Shoaf, Department of Biochemistry and Biophysics, Oregon State University
Ivana Dang, Department of Biochemistry and Biophysics, Oregon State University
Stephanie Zhao, Department of Biochemistry and Biophysics, Oregon State University
Kelsey Caples, Department of Biochemistry and Biophysics, Oregon State University
Lynda Bradley, Department of Biochemistry and Biophysics, Oregon State University
Laura M. Beaver, Biological and Population Health Sciences, Linus Pauling Institute, Oregon State University
Emily Ho, Biological and Population Health Sciences, Moore Family Center for Whole Grain Foods, Linus Pauling Institue, Oregon State University
Christiane V Löhr, College of Veterinary Medicine, Linus Pauling Institute, Oregon State University
Viviana I Perez, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

 

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β- gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.

 
Poster Abstract 35
Safe and proven daily actions to improve your healthspan
 
Alfred B. “Roc” Ordman, Beloit College
 

There is vast confusion and much disagreement about nutrition and human aging research in the American people. Within the community of geroscientists, however, there is optimism about some new discoveries. This poster documents disagreements even among LPI attendees and proposes possible resolutions. All decisions in this presentation are based on peer-reviewed research supporting choices that are likely to extend most people’s healthspan. Choices for diet, supplements, and various activities are listed. Some of these are organized into a morning and evening routine. There are also a few recipes. The objective is to promote awareness and discussion of choices to improve your own and others’ healthspans. Comments are invited to: ordman@beloit.edu

 
Poster Abstract 36
Cellular senescence contributes to hepatic “Inflamm-aging”
 
Amanda Kelley, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Nicholas O. Thomas, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Aaron Sugiyama, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Kate Petersen-Shay, Linus Pauling Institute, Oregon State University
Judy Butler, Linus Pauling Institute, Oregon State University
Eric Smith, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University
Liam Finlay, Linus Pauling Institute, Oregon State University
Karyn Bird, Department of Biomedical Sciences, College of Veterinary Medicine, Linus Pauling Institute, Oregon State University
Kathy R. Magnusson, Department of Biomedical Science, College of Veterinary Medicine, Linus Pauling Institute, Oregon State University
Tory M. Hagen, Department of Biochemistry and Biophysics, Linus Pauling Institute, Oregon State University

 

Our lab has recently shown that old (24 months) Fischer 344 (F344) rat livers have increased expression of genes related to inflammation and tissue remodeling. These results indicate that livers from old animals are in a state of chronic inflammation, which increases the risk of hepatocarcinogenesis and metabolic dysfunction. The exact cause(s) of this inflammatory phenotype are unknown, other tissues, such as skin fibroblasts, are known to accumulate senescent cells. Senescent cells are identified by cell cycle arrest, lysosome expansion, and the secretion of pro-inflammatory factors (senescence-associated secretory phenotype or SASP) that ultimately result in the perpetuation of senescence. As of now, there is minimal evidence that the liver, a regenerative organ, accumulates senescent cells with age. Thus, the purpose of this study is to examine the amount of cellular senescence in the aging rat liver to gain an understanding of its contribution towards the pro-inflammatory phenotype that our previous work has shown. Livers were harvested from young (3-6 month) and old (24-27 month) F344 rats throughout the study. Histological analysis of immune cell infiltration was used to assess inflammation and tissue remodeling. Using hemotoxylin and eosin staining, histological analysis revealed that livers from old rats had increased bile duct hyperplasia and extensive fibrosis in comparison to young animals. In addition, livers from old animals showed increased immune cell infiltration as assessed by CD45 staining (young = 37.50 ± 5.500; old = 137.500 ± 11.50). Additionally, immune cell infiltration appeared to be highest near bile ducts in old animals. Furthermore, there was an age-related increase in staining for beta-galactosidase, an established marker of cellular senescence. While it is not clear which cells express increased levels of beta-galactosidase in cryo-sectioned tissue, isolated liver parenchymal cells (i.e. hepatocytes) show increased expression of beta-galactosidase. Hepatocytes are integral in detoxification, metabolism of nutrients, and bile acid secretion. Thus, these results suggest that cellular senescence may play a role in the compromised liver function observed in older animals. To quantify the SASP observed in the aging rat liver, whole liver tissue was used for qPCR analysis. Our results show that interleukin-6, an established marker of general inflammation and the SASP, increases over 3-fold with age. Using the ratio of interleukin-6 to interleukin-10, known pro-inflammatory and anti-inflammatory interleukins, respectively, there is a shift towards a pro-inflammatory state. A marker of more advanced cellular senescence, microRNA-146a, increases 2.70-fold in old liver tissue. Additionally, we have found that there is increased secretion of microRNA-146a in exosomes from hepatocytes in old animals, indicating that hepato-senescence may contribute to systemic inflammation in old animals. Overall, our work shows that the aging rat liver has a pro-inflammatory phenotype characteristic of "inflamm-aging", and we have preliminary evidence that cellular senescence possibly plays a key role in its cause.