Brian K. Kennedy - US grants

DESCRIPTION (provided by applicant): The use of invertebrate organisms has become a mainstay of aging research, leading to the identification of hundreds of aging genes. Emphasizing the utility of these studies, at least some of these genes and pathways have conserved effects on longevity in mammals. In taking stock of the progress, it is clear that a new, more system wide approach is required to effective move forward. As we see it, there are two main questions that need to be answered: (1) Given that there are hundreds of aging genes, in which altered expression is associated with lifespan extension, in an organism, how many pathways do they represent and how can they be delineated?; (2) what are the mechanisms that drive aging in invertebrate aging models and are they conserved? This latter question has remained stubbornly refractory to a variety of approaches in the aging research field. With an eye toward answering these two questions, in this proposal three research groups with complementary expertise have joined forces to develop a comprehensive understanding of replicative aging in Saccharomyces cerevisiae using a combination of high throughput and state-of-the-art approaches. Dr. Kennedy (in collaboration with Dr. Matt Kaeberlein at the University of Washington) has just completed a genome-wide screen of yeast ORF knockouts for enhanced replicative lifespan. In Aim 1, we will develop the largest epistasis network of aging using the high-throughput capacity of the Kennedy lab to functionally assess which downstream pathways are required for lifespan extension in a set of representative yeast aging genes. In Aim 2, Dr. Li's research group will use a newly developed microfluidic system to determine the state of pathways purported to be involved in aging in the context of long-lived yeast mutants and in Aim 3, Dr. Brem's group will use RNA sequencing to develop a comprehensive gene expression analysis dataset in a range of long-lived mutants. These latter two approaches will help determine the cellular consequences of longevity mutants and by combing those with the epistasis studies in Aim 1, we will generate a comprehensive understanding of replicative aging, identifying the pathways involved and moving toward a mechanistic understanding of longevity.

Project Summary/Abstract Many signaling pathways linked to aging are linked to the regulation of metabolic signaling and stress response pathways. For instance, the target of rapamycin (TOR) pathway is an evolutionarily-conserved nutrient-sensing protein kinase that regulates growth and metabolism in all eukaryotic cells. Two complexes, mTORC1 and 2, have overlapping upstream regulators and downstream effectors. Reduced mTOR signaling, either by genetic intervention or with the clinically approved drug rapamycin, extends longevity in mice (as well as yeast, worms and flies) and delays many pathologies of aging. Given its central role in aging and metabolism, it is critical to understand how different perturbations of the mTOR pathway impact aging and metabolism. Here, we use mouse models and cell culture studies to test one of the major downstream targets of mTORC1, 4E-BP1. Justifying the emphasis on 4E-BP1, enhanced 4E-BP activity is associated with lifespan extension in worms and flies, and we find that transgenic mice overexpressing 4E-BP1 are resistant to high fat diet-induced metabolic dysfunction. We will employ multiple mouse models of 4E-BP1 overexpression in both overnutrition and aging studies. Preliminary data indicates that an inflammation-induced loss of 4E-BP1 expression in the context of a high fat diet underlies the specific propensity of males to become glucose intolerant and insulin resistant. We will determine the mechanisms underlying this gender dimorphism and its impact on the mTOR pathway. Gender-specific responses to diet and aging are rampant in mouse models and in humans, but the underlying causes of gender-specificity is largely unknown. In addition, we will determine why muscle specific activation of 4E-BP1 preserves both skeletal muscle function and brown fat content during overnutrition and aging, focusing on recent findings linking the benefits to skeletal muscle production and secretion of the myokine, FGF21. Together, these studies will yield several new insights regarding the specifics of mTOR signaling in the context of aging and metabolism, providing a better understanding about how this pathway modulates aging and linking modulation of the mTOR pathway to other pharmacologic interventions in aging.

DESCRIPTION (provided by applicant): The age-related decline in regenerative capacity of many tissues constitutes a poorly understood problem that limits human healthspan. As studies in vertebrate systems point to both stem cell (SC) autonomous and non- autonomous causes for this age-related decline, analyzing SC function in an in vivo context is required, preferentially i short-lived, genetically accessible model systems. In recent years, Dr. Jasper's lab has established the fly intestine as a model to explore somatic SC aging, and to identify interventions that modulate SCs to preserve tissue homeostasis and extend lifespan. The Drosophila posterior midgut epithelium is regenerated by intestinal SCs (ISCs), is experimentally accessible, and of sufficient complexity to model the regenerative activity of similar tissues in vertebrates. Studies in the fly midgut have not only discovered mechanisms that promote the age-related regenerative decline in this tissue, but have also established that improving ISC proliferative homeostasis extends lifespan. Dr. Kennedy's lab, in turn, has performed groundbreaking work on aging and progeroid diseases for many years, with a specific recent focus on nutrient-responsive signaling pathways in the control of aging in mice. Here, Dr. Jasper and Dr. Kennedy propose to combine the strength of the fly system with genetic studies in mice to uncover evolutionarily conserved mechanisms of SC aging. Specifically, it will be tested whether the control of SC maintenance by nutrient-responsive signaling, which the applicants have characterized in the ISC lineage, is conserved in the mouse tracheal epithelial SC (Basal Cell, BC) lineage. The BC lineage has significant similarity with the fly ISC lineage, and serves as an accessible model for insight into epithelial regeneration in vertebrates that is likely to impact a major disease of aging: chronic obstructive pulmonary disease (COPD). The proposed work will address the role of TSC/Tor signaling, a conserved regulator of lifespan, on SC maintenance in flies and mice. Based on preliminary results, the applicants propose a conserved role for the negative regulator of Tor, TSC1/2, in shielding somatic SCs from dietary fluctuations, thus preserving SC identity and regenerative capacity in aging tissues. This model will be tested using genetic and pharmacological approaches to perturb the Tor pathway and to assess SC maintenance and regenerative capacity in aging animals. The study, which includes genetic work in mice and flies, as well as pharmacological interventions with new TorC1-specific inhibitors, will be performed in close collaboration between the Kennedy and Jasper labs, making the multi-PI mechanism optimal. The TSC/Tor pathway has emerged as an evolutionarily conserved nutrient sensor that influences life- and healthspan. Characterizing the biological consequences of long-term Tor repression is critical to develop specific intervention protocols that can promote tissue homeostasis and maintain regenerative capacity. The proposed studies will seek to achieve this important goal.

DESCRIPTION (provided by applicant): The age-related decline in regenerative capacity of many tissues constitutes a poorly understood problem that limits human healthspan. As studies in vertebrate systems point to both stem cell (SC) autonomous and non- autonomous causes for this age-related decline, analyzing SC function in an in vivo context is required, preferentially i short-lived, genetically accessible model systems. In recent years, Dr. Jasper's lab has established the fly intestine as a model to explore somatic SC aging, and to identify interventions that modulate SCs to preserve tissue homeostasis and extend lifespan. The Drosophila posterior midgut epithelium is regenerated by intestinal SCs (ISCs), is experimentally accessible, and of sufficient complexity to model the regenerative activity of similar tissues in vertebrates. Studies in the fly midgut have not only discovered mechanisms that promote the age-related regenerative decline in this tissue, but have also established that improving ISC proliferative homeostasis extends lifespan. Dr. Kennedy's lab, in turn, has performed groundbreaking work on aging and progeroid diseases for many years, with a specific recent focus on nutrient-responsive signaling pathways in the control of aging in mice. Here, Dr. Jasper and Dr. Kennedy propose to combine the strength of the fly system with genetic studies in mice to uncover evolutionarily conserved mechanisms of SC aging. Specifically, it will be tested whether the control of SC maintenance by nutrient-responsive signaling, which the applicants have characterized in the ISC lineage, is conserved in the mouse tracheal epithelial SC (Basal Cell, BC) lineage. The BC lineage has significant similarity with the fly ISC lineage, and serves as an accessible model for insight into epithelial regeneration in vertebrates that is likely to impact a major disease of aging: chronic obstructive pulmonary disease (COPD). The proposed work will address the role of TSC/Tor signaling, a conserved regulator of lifespan, on SC maintenance in flies and mice. Based on preliminary results, the applicants propose a conserved role for the negative regulator of Tor, TSC1/2, in shielding somatic SCs from dietary fluctuations, thus preserving SC identity and regenerative capacity in aging tissues. This model will be tested using genetic and pharmacological approaches to perturb the Tor pathway and to assess SC maintenance and regenerative capacity in aging animals. The study, which includes genetic work in mice and flies, as well as pharmacological interventions with new TorC1-specific inhibitors, will be performed in close collaboration between the Kennedy and Jasper labs, making the multi-PI mechanism optimal. The TSC/Tor pathway has emerged as an evolutionarily conserved nutrient sensor that influences life- and healthspan. Characterizing the biological consequences of long-term Tor repression is critical to develop specific intervention protocols that can promote tissue homeostasis and maintain regenerative capacity. The proposed studies will seek to achieve this important goal.

ABSTRACT Alzheimer?s disease (AD) is a devastating dementia, afflicting about 5.4 million individuals in the US alone. Its prevalence is rapidly rising due to increases in lifespan and the absence of even a single significant disease-modifying therapy. Genetic variants in the apolipoprotein E (ApoE) locus are associated with AD, as well as other chronic diseases and aging itself. In particular individuals with the ApoE2 allele are enriched among nonagenarians and centenarians in a range of genetic studies, whereas ApoE4 is associated with higher age-associated mortality. Similarly, the ApoE2 allele is protective for AD, whereas ApoE4 is the largest genetic risk factor. Of course, aging is the biggest risk for AD and it remains largely unknown how ApoE plays a role in connecting aging to AD. These findings alone warrant thorough analysis of how ApoE contributes to AD and/or aging, and specifically an analysis of the protective effects of ApoE2. By combining induced pluripotent stem cells (iPSC) and precise CRISPR/cas9 gene editing technology, it is now feasible to generate otherwise isogenic iPSC lines with ApoE alleles. We have made progress toward the generation of these lines and in this proposal will create iPSC lines with combinations of ApoE alleles, differentiate them into neurons and astrocytes, and assess cellular phenotypes associated with AD. We will compare and contrast the effects of ApoE2 and ApoE4, looking for specific mechanisms by which these alleles are protective or sensitizing to AD, respectively. In addition, we will seek to modify the severity of cellular phenotypes associated with neurodegeneration by modifying pathways linked to aging. Since we are working on human differentiated cells in culture, we have the capacity to interrogate several pathways linked to accelerated and delayed aging. This will be accomplished either (1) genetically, for example by expressing genes that accelerate or decelerate organismal aging such as progerin, a mutation in the LMNA gene associated with Hutchinson-Gilford progeria syndrome, or (2) pharmacologically, for example with rapamycin, a drug associated with longevity. The specific hypothesis is that by interrogating multiple aging-related pathways, we will define specific phenotypic interactions between individual pathways, ApoE2 or ApoE4 functions and AD phenotypes that can be assessed in differentiated cells. This unique approach offers the possibility of understanding how aging modifies AD risk at a mechanistic level, identifying potential prognostic and diagnostic markers, and developing and refining therapeutic targets for AD, as well as potentially other age- related cognitive disorders.

Protein synthesis governs if, how fast, and how many times cells divide. Yet how protein synthesis is linked molecularly with cell division is unknown. We use budding yeast as a model system to answer this problem. Because yeast has unique properties, suited for genetic and biochemical experiments. New methodologies can identify transcripts that engage with the protein synthesis machinery, the ribosomes, in the process of translation. For the first time in the field, we applied this ribosome profiling methodology in synchronously dividing cells that maintained the physiological coupling of protein synthesis with their division. In this collaborative proposal, we will leverage these findings to tackle the long-standing problem of protein synthesis requirements for cell divisions. In Aim 1, we will determine how translational control of lipogenic enzymes regulates the remodeling of cellular membranes during cell division. Furthermore, we will determine how protein synthesis adjusts the production of proteins that trigger duplication of the spindle pole body, an essential part of the machinery of chromosome segregation. We will also identify translationally regulated mRNAs under dietary restriction, which changes the size of cells and increases the number of times cells divide before they die. In Aim 2, we will extend ribosome profiling to settings of specific ribosomal protein mutants that delay cell division and increase lifespan. These genetic interventions will enable us to identify mRNA targets of translational control that underpin cell division and replicative longevity when protein synthesis is limited. Knowing how translational control affects the timing and number of cell divisions will reveal fundamental links between cell growth, protein synthesis, cell division and aging, enabling novel therapeutic interventions in proliferative diseases.

Project Summary Aging can be characterized as the progressive loss of homeostasis starting at maturity and ending with senescence and death. Discoveries of the molecular mechanisms of aging can enable the development of new therapies to block age-associated disease and extend healthspan, the healthy years of life. This goal has been elusive even in the simplest model eukaryote, single-celled budding yeast. Yeast longevity has been associated with each of hundreds of genes. Such complexity suggests that yeast aging is controlled by interactions between many molecules and organelles, with any link in this network potentially subject to compromise during a given cell division. But the chain of molecular events by which homeostasis is gradually lost has been obscure to date, in part due to the reliance of the field on bulk-culture measurements in the study of the genomics of aging. The premise of the current proposal is that dissecting the breakdown of the network in many single cells ? observing its many facets over time, perturbing them, and analyzing their response, is critical for a molecular understanding of the phenomenon of aging. Toward this end, we propose to analyze yeast molecular aging trajectories via comprehensive single-cell profiling of protein reporters (Aim 1), to connect lifespan extending mutations to their downstream effectors through systematic epistasis analysis (Aim 2), and to test whether perturbing critical genes at a particular point in life (just-in-time interventions) can decrease mortality rates early in life and/or extend lifespan (Aim 3). Together, these experiments will shed light on the molecular events of the breakdown of homeostasis with age in yeast, identify dynamic interventions for rejuvenation, and reveal novel aging genes and mechanisms to serve as prime candidates for testing in metazoans.

PROJECT SUMMARY No Changes from the originally submitted Project Summary