Thomas Rando - US grants

DESCRIPTION: Necrotic cell death is the initial pathologic change in muscle of patients with muscular dystrophy. Even for the most common forms of muscular dystrophy, those due to defects in the dystrophin gene, the cause of muscle necrosis remains unknown despite a wealth of information on the dystrophin gene, its message, and its protein product. The broad, long-term objectives of this work are: 1) to understand the pathogenetic mechanisms that lead to cell death in muscular dystrophies; and 2) to study therapeutic approaches to intervene in those biochemical pathways to prevent muscle degeneration. The primary hypothesis of this proposal (the free radical hypothesis) is that dystrophin deficiency renders muscle more susceptible to oxidative injury, and thus normal levels of oxidative stress result in membrane damage sufficient to cause muscle fiber necrosis. The principle investigator proposes to test this hypothesis, in vitro and in vivo, using a mouse strain with dystrophin deficiency (the mdx strain). The specific aims of this proposal are: 1) to examine dystrophin-deficient muscle in vivo for signs of oxidative damage: studies of the pre-necrotic state; (oxidative injury and cellular responses to that injury will be measured in mdx mouse muscle just prior to the onset of necrosis); 2) to study, in vitro, the susceptibilities of normal and dystrophin-deficient muscle to oxidative stress and the effect of different forms of dystrophin in protecting muscle cells against oxidative injury (myotube cultures of normal and dystrophin-deficient muscle will be studied in assays of cell injury and cell death); and 3) to test the effects of enhanced antioxidant engineered by breeding with transgenic strains to express higher levels of an antioxidant enzyme, or mdx mice will be treated with pharmacologic antioxidant agents, and disease progression will be assessed histologically and biochemically.

Atrophy of skeletal muscle is part of the aging process and leads to significant morbidity and mortality in the elderly population. Although the anatomical and physiological changes of aging muscle have been well described, the cellular and molecular bases of those changes remain elusive. The primary hypothesis of this proposal, based on the free radical theory of aging, is that oxidative injury leads to age-related muscle atrophy. We will test this hypothesis in mouse, since rodents have been shown to be good models of human muscle aging, and because using mouse models provides two distinct advantages: genetic analysis and in vitro analysis. As such, the Specific Aims of this proposal are: 1) To test the role of oxidative stress in age-related muscle atrophy;: biochemical and genetic studies; and 2) To study the susceptibility of young and old muscle to oxidative injury: in vitro studies. For Aim 1, we will analyze specific muscles of adult (9 mos) and old (24 mos) mice to obtain quantitative histologic assessments of age-related muscle atrophy. We will test the free radical hypothesis by examining the muscles for biochemical and molecular changes indicative of oxidative stress: lipid peroxidation will be measured as a direct indication of oxidative damage, and induction of antioxidant genes will be assessed as an indirect measure of oxidative stress. For the genetic studies, we will perform identical analyses in strains that have genetic variations in free radical metabolic pathways. We will focus on two strains that we believe may be most informative: 1) a transgenic strain that overexpresses the Cu,Zn superoxide dismutase (SOD-1) gene, since SOD overexpression has been shown to inhibit age-related changes in other species; and 2) a knockout strain that has a deletion in the nitric oxide synthase (NOS) gene expressed in muscle, since nitric oxide (which is produced by NOS) has been shown to mediate oxidative injury and muscle wasting. For Aim 2, we will use differentiated cultures of muscle cells isolated from young and old mice. Myotube cultures will be compared in assays of cell injury to determine the relative susceptibilities to oxidative injury as a test of the free radical hypothesis. In addition, oxidative injury and antioxidant capacities will be compared in young and old myotube cultures. These multifaceted studies should provide a broad test of the free radical hypothesis to investigate basic molecular and cellular mechanisms underlying age- related muscle atrophy.

The muscular dystrophies are devastating diseases of progressive weakness due to apoptotic and necrotic death of muscle cells. The normal cellular mechanisms regulating cell survival that are disrupted in these diseases are not well understood. Several forms of muscular dystrophy are due to abnormalities of membrane proteins and protein complexes, such as integrins and caveolins, that are known to regulate cellular signaling pathways in general, and cell survival signaling in particular, in different cell types. Others, such as those due to dystrophin mutations, are due to abnormalities of protein complexes that are postulated to transduce signals from the extracellular matrix into the cell. We will focus on three proteins/protein complexes that cause muscular dystrophies when a component of the complex is deficient or defective - alpha5beta1 integrin, caveolin-3, and the dystrophin-glycoprotein complex (DGC). The experiments of this proposal are designed to explore the cellular signaling processes the promote cell survival via these membrane protein complexes, and the mechanisms of cell death when these complexes are disrupted. For studies of integrin signaling, we will use cells genetically deficient in alpha5 integrin to test which isoforms of protein kinase C are important in alpha5 integrin mediated muscle cell survival (based on our previous finding of the importance of protein kinase C in this process). We will explore how alpha5 integrin deficiency leads to muscle cell death by testing for dysregulation of cell survival/cell death pathways involving the Bcl family of proteins, cytochrome c release from mitochondria, and activation of the caspase cascade. We will also examine the role of activation of the PI3 kinase/Akt pathway in alpha5 integrin- mediated muscle cell survival. For studies of the DGC, we will investigate how disruption (genetically, by antibody inhibition, or by antisense expression) of the association of the complex with the extracellular matrix may lead to cell death. In these studies, we will also examine cells for dysregulation of cell survival mechanisms involving Bcl family proteins since apoptosis has been shown to be the earliest change in muscle associated with dystrophin deficiency. For studies of dystrophies due to caveolin-3 mutations, we will render muscle cells functionally deficient in caveolin-3 using both antisense methods and dominant negative inhibitors. We will study the mechanisms by which caveolin-3 deficiency lead to muscle cell death, and we will test whether these mechanisms involve the disruption of either normal integrin signaling or signaling through the DGC.

[unreadable] DESCRIPTION (provided by applicant): This application seeks partial support for the upcoming Federation of American Societies of Experimental Biology (FASEB) conference entitled, Skeletal muscle satellite and stem cells, that will be held on June 16-21, 2007 in Indian Wells, CA. The overall objective of this meeting is to highlight recent advances pertaining to the regulatory mechanisms of myogenic stem cell and progenitor cell populations (such as satellite cells) and their role in development, regeneration, aging and myopathic states. This will be the fifth conference focused on satellite cells/stem cells and muscle regeneration. The increased participation from the first to the fourth meetings has demonstrated the need for a conference dedicated to satellite and stem cells. It is clear from the successes of the previous four meetings that this topic is of high interest and this meeting represents the only conference that directly addresses muscle satellite and stem cell populations. The biennial timing of this multidisciplinary conference has facilitated the presentation of new unpublished data that has generated considerable discussion and promoted collaborative interactions. This forum will focus on the identification of the progenitor/stem cell populations that are resident in adult skeletal muscle with a particular focus on their participation in development, hypertrophy, regeneration, aging and disease. Discussion will be directed toward platforms for cell based therapies. The sessions of this conference include: the developmental origins of satellite cells, satellite cell heterogeneity, satellite cell quiescence and activation, the molecular control of myogenic lineage progression, the muscle stem cell niche, satellite cell self-renewal, pluripotentiality of muscle stem cells, muscle progenitor cell differentiation and fusion, cellular therapies in disease and atrophy, the aging of muscle stem cells, and muscle progenitors in growth and hypertrophy. In addition, there will be a stem cell therapy workshop. Importantly, the participants of this conference will represent interdisciplinary groups that will provide a comprehensive analysis and integration of recent discoveries for the field. The conference will further promote collaborative interactions in an attempt to promote future advancements and translational initiatives directed toward the treatment and cure of patients with myopathic diseases. [unreadable] [unreadable] The overall purpose of a meeting on skeletal muscle satellite and stem cells is to bring together investigators in diverse areas of muscle biology and investigators outside the field to discuss the current research in the field and to consider the potential to translate scientific progress into clinical treatments. The field of muscle stem cell therapy, using stem cells derived from adult tissue, is at the forefront of this area of regenerative medicine. [unreadable] [unreadable] We fully anticipate that progress in this field will pave the way for stem cell therapies for a variety of degenerative, inflammatory, and age-related disorders. [unreadable] [unreadable] [unreadable]

With age, there is gradual decline in muscle regenerative potential, resulting in incomplete regeneration and fibrosis. Studies from our laboratory have shown that the age-dependent decline in regenerative potential is due to a loss of functionality of the resident muscle stem cells known as satellite cells (SCs). The major goals of the studies of this proposal are to explore molecular pathways implicated in aging of SCs and to understand the mechanisms underiying the age-related decline in SC functionality. Our published and ongoing studies have focused on the role of Wnt signaling in the age-related suppression of SC function, at least in part by inducing a conversion of myogenic stem cells to a fibrogenic lineage. As such, the Specific Aims of this proposal are: 1) to analyze the mechanisms by which Wnt signaling and transcriptional readouts lead to age-related suppression of SC functionality; 2) to analyze how regulators of the Wnt pathway account for specific transcriptional responses and changes with age; and 3) to study the epigenetic profiles and determinants of young and old SC function. We will examine transcriptional programs and regulators in adult and aged SCs, testing for changes in cellular responses with age. A recent area of interest is the role of the co-activator of the Wnt pathway, BCL9, in SC aging and in determining the transcriptional response to Wnt by acting as a histone decoder. Therefore, a major focus of these studies will be the characterization and regulation of the epigenetic status of adult and aged SCs and the regulation of both the Wnt pathway and the epigenetic state of aged SCs by BCL9. Using ultra-high-throughput sequencing, we will examine transcription factor targets in the Wnt/p-catenin pathway, interactions between this pathway and signaling via TERT (in collaboration with Project 2) and Foxo3 (in collaboration with Project 3), and global epigenetic profiles of adult and aged SCs. We will directly examine SC aging in cohorts of mice in which BCL9 is genetically deleted in the SC compartment. These studies will form the basis of a comprehensive analysis of the molecular basis of age-related functional changes of a stem cell population and how those changes both contribute to and are regulated by aging of the tissue and organism. RELEVANCE (See instructions): This Project will investigate basic mechanisms of aging of stem cells in skeletal muscle, a process which renders them less able to participate in muscle maintenance and repair after injury. Our research focuses on biochemical pathways that we believe can be modulated to enhance the functional properties of aged muscle stem cells, thereby reducing muscle atrophy and improving muscle repair after injury or disuse.

The studies outlined in this proposal address fundamental issues of the biology of aging, specifically focused on the molecular characteristics, mechanisms and consequences of stem cell aging. Based on mutual interests and complementary areas of expertise, the three laboratories of this Program form a cohesive, collaborative team to address these basic questions. With expertise in stem cells in skeletal muscle (Rando laboratory, Project 1), epithelial stem cells (Artandi laboratory. Project 2), and neural stem cells (Brunet laboratory. Project 3), the Program has the following Specific Aims: 1) to understand the underlying molecular mechanisms that regulate stem cell function and that are responsible for declining stem cell function with age; 2) to characterize the transcriptional networks and epigenetic profiles in stem cells from different tissues; and 3) elucidate the age-related changes that are common among different stem cell populations and changes that are unique to stem cells in particular tissues. These Overall Aims are reflected in the Specific Aims of each Project, with emphases on the role of signaling pathways that regulate stem cell function. The major pathways to be explored in multiple Projects are the Wnt signaling pathway which has recently been shown to contribute to tissue aging by suppression of stem cell functionality, Telomerase and novel functions of the protein component (TERT) in regulating stem cell function and regulating Wnt signaling, and the Foxo transcription factors which regulate organismal aging. All of these will be examined in all three of the Projects, exploring mechanisms of co-regulation of genes that regulate stem cell function, and focusing on the causes and consequences of changes in transcriptional programs that determine age related changes in stem cell functionality. Of particular interest is the identification of mechanisms of stem cell aging that are shared among different stem cell populations. Within that context, all Projects will examine transcriptional networks and regulatory mechanisms, including epigenetic processes, that govern stem cell function and that drive changes with age. These broad goals will be supported by an Administrative Core and two Scientific Cores - a Mouse Aging Core and a Genomics and Ultra-High-Throughput Sequencing Core. The collaborative and coordinated efforts of the three laboratories participating in this Program are uniquely situated within the outstanding research environment of Stanford University to work at the interface between stem cell biology and the biology of aging to create a highly effective and synergistic research program focused on the molecular mechanisms of stem cell aging.

The Administrative Core will coordinate the activities of the three Projects and three Cores of this Program Project. The Specific Aims of the Administrative Core are: 1) to provide administrative assistance and fiscal oversight to all Projects and Cores B and C; 2) to facilitate communication both within the Program and between the Program members and other investigators, centers, programs and institutions; and 3) to establish and maintain an Executive Committee, an external Scientific Advisory Committee, and an Internal Advisory Committee. The services offered by the Core will include administrative and secretarial support (ordering of supplies, financial reports, assistance with budget management), organization of regular meetings and of the annual meeting with the Scientific Advisory Committee, and preparation of progress reports and other documents. The Director will oversee and coordinate all activities of the Core. He will meet regularly with the Administrator and with other Project Leaders/Core Directors to discuss administrative and managerial issues to insure that the Program is working effectively and efficiently. The Administrator and the Administrative Assistant will assist the investigators with financial matters, the organization of meetings and conferences, and general needs for the proper functioning of the Program. RELEVANCE (See instructions): This Program Project will investigate basic mechanisms of aging of tissues, focusing on the stem cells in those tissues. This research holds promise for discovering mechanisms to help aged tissues heal more effectively by enhancing the ability of the stem cells to participate in tissue repair and regeneration. This Administrative Core will provide much needed support for the progress of the research of this Program.

? DESCRIPTION (provided by applicant): The development of regenerative medicine technologies holds great potential to drive progress in the prevention and treatment of individuals with a host of acute and chronic pathologies resulting from injury, disease or aging. As we approach this new era of technological advancements, rehabilitation specialists must work closely with regenerative medicine scientists in the development of clinical protocols to optimize functional recovery. While there are many important and superb congresses on the topic of stem cell biology and regenerative medicine, these meetings are rarely attended by those in the rehabilitation field. Similarly, few regenerative biologists are exposed to protocols and methodologies commonly employed in the clinic by rehabilitation professionals, protocols which serve as potent stimuli to drive functional tissue restoration. Nor are most regenerative biologists exposed to the diverse fields of rehabilitation science. There is, therefore, a great need for an established forum in which individuals from the fields of rehabilitative and regenerative medicine may interact such that, as technologies are developed and as understanding of regenerative biology progresses, advances may be smoothly and efficiently translated to the clinic. This R13 proposal seeks funds to help offset conference expenses in order to support the participation of students and young investigators in the Annual Symposium on Regenerative Rehabilitation. This series has enjoyed tremendous growth since its inauguration in 2011 and now comprises academic partnerships from across the country. With this in mind, the meeting rotates locations annually as a means to broaden exposure to and participation by a national audience. The specific aims of this symposium series are: 1. to promote the clinical translation of regenerative and rehabilitation medicine scientific discoveries by communicating and disseminating research findings that demonstrate the synergistic relationship between regenerative medicine and rehabilitation; 2. to provide a forum at which scientists and rehabilitation clinicians may interact, exchange ideas, and identify novel research directions relating to the field of regenerative rehabilitation; and 3. to introduce the concept of regenerative rehabilitation to graduate students, post-doctoral fellows, medical students and medical residents in the rehabilitation field. To achieve these aims, Course Directors and Advisory Board members work to carefully design a highly multidisciplinary and translational 2-day program that includes thematically linked presentations highlighting the importance of mechanical stimulation for tissue regeneration and functional restoration. The support derived from this R13 application will help us keep registration costs affordable in order to attract a larger number of clinical and scientific students, fellows and junior investigators to this unique forum designed to highlight the emerging field of regenerative rehabilitation.

DESCRIPTION (provided by applicant): This is a new application for a Stanford Training Program in Aging Research (TPAR) to initiate a training program that will support graduate students and postdoctoral fellows to pursue research in aging and to become future leaders in the field. This program will build upon the remarkable growth of exciting aging research at Stanford and the recent formation of a broader Program in Aging. While the number of positions requested is modest, we would expect that the training program would grow and expand in conjunction with the broader program over time and based upon a track record of success. In addition, the establishment of TPAR will serve as focal point for the recruitment of the best and brightest students with an interest in aging research, thereby increasing the percentage of Stanford trainees working in the aging field. Along with the three Co-Directors, TPAR consists of an exceptional group of 23 faculty members from 16 different departments and programs to serve as the core affiliated faculty whose interests and areas of expertise span a range of crucial topics within the biology of aging. We have outlined five Tracks (stem cells, genetics, neuroscience, cancer, and immunology) that focus on aging in these specific contexts and that students and fellows can choose to pursue. TPAR aims to provide a solid foundation of training in aging across sub-disciplines by formal didactics, a Frontiers in Aging seminar series, an annual Symposium on Aging, a weekly Trainee Research Talk program, and a designated mentoring program. An innovative aspect of the Program includes an exposure to clinical geriatrics. Receiving broad institutional support, TPAR is poised to be immediately integrated into the established infrastructure of graduate education and postdoctoral training at Stanford University. TPAR will benefit immensely from the continuing efforts and success of Stanford University programs to recruit and retain applicants from diverse background. The population of the country is aging, with a much higher percentage of individuals over 70 years of age than ever before and with an ever increasing percentage affected by devastating diseases of aging such as heart disease, cancer, and neurodegenerative diseases. The emphasis of TPAR will be to provide a broad, interdisciplinary education to a wide range to trainees, to serve to coordinate aging research and training activities across the entire campus, and to help disseminate basic and translational aging research by preparing trainees for the next steps in their careers. In doing so, we would hope that TPAR would serve not only the individual trainees but also the field of aging research and society at large.

? DESCRIPTION (provided by applicant): The advancement of regenerative medicine principles and technologies holds great potential to drive progress in the prevention and treatment of individuals with a host of pathologies resulting from injury, disease or aging. The long-term goal of regenerative medicine is to promote the repair, replacement, or regeneration of tissues. Likewise, rehabilitation seeks to harness the body's innate regenerative potential in order to maximize function. Both fields hold great potential to drive progress in the treatment of a host of acute and chronic pathologies. We propose that these two fields are inextricably intertwined; an intersection of disciplines known as Regenerative Rehabilitation. To fully realize the tremendous potential of Regenerative Rehabilitation, we must promote the interaction of basic scientists with rehabilitation specialists. We must also train rehabilitation clinicians who can help oversee the quality, safety, and validity of these innovative Regenerative Rehabilitation technologies. The overarching goal of the Alliance for Regenerative Rehabilitation Research & Training (AR3T) is to establish a national network that will expand scientific knowledge, expertise and methodologies across the domains of regenerative medicine and rehabilitation. AR3T will provide didactic training that exposes rehabilitation researchers to cutting-edge investigations and state-of-the-art technologies in the field of regenerative medicine (Specific Aim 1). AR3T will drive the science underlying Regenerative Rehabilitation by: cultivating collaborative opportunities among rehabilitation researchers and internationally renowned investigators in the field of regenerative medicine (Specific Aim 2); launching a pilot funding program to support novel lines of Regenerative Rehabilitation investigations (Specific Aim 3); developing and validating technologies to elucidate the stem cell response to extrinsic mechanical signals (Specific Aim 4). Careful monitoring and evaluation of the effectiveness of our program, as outlined in Specific Aim 5, will ensure that these aims achieve the greatest success possible. These five aims have been organized to parallel each of the P2C grant proposal components.

SUMMARY The loss of tissue regenerative capacity is an almost ubiquitous aspect of mammalian aging. Underlying this age-related change is a decline in the potential of tissue-specific stem cells to participate in tissue repair and regeneration. In skeletal muscle, this has been amply demonstrated for the resident muscle stem cells (MuSCs), whose response to injury has been shown to decline with age in response to cell-intrinsic changes and to suppressive activity from the aged systemic milieu, the former perhaps arising from the latter. Nevertheless, the molecular basis of the cellular changes that integrate these diverse inputs to render a MuSC less responsive with age remains to be determined. Likewise, the molecular basis for the cellular memory that persists ex vivo and that can be ?reprogrammed? in vivo in response to different environments remain to be elucidated. Understanding these fundamental mechanisms of MuSC aging provide the potential for being able to intervene to restore youthful characteristics to aged stem cells, thus enhancing aged tissue repair and regeneration. Toward these goals, this Project focuses primarily on epigenetic mechanisms based on the hypothesis that it is the epigenome that integrates diverse signals, mediates cellular responses, and is amenable to reprogramming in response to diverse environmental influences. In collaboration with Projects 2 and 3 and with Core C, we will explore the epigenetic features and regulators of young and aged MuSCs, in terms of transcriptome (RNA-seq), epigenome (ChIP-seq), and nucleosome positioning (ATAC-seq). In collaboration with Core B, we and Projects 2 and 3 will explore in more detail the notion of ?epigenetic rejuvenation? by studying the epigenetics of aged MuSCs (and other stem cells) exposed to rejuvenating interventions that have been shown to restore youthful function to aged stem cells. We will examine a specific histone mark, trimethylation of lysine 27 on histone 3 (H3K27me3) what we have shown previously to be strikingly enriched in aged MuSCs. We will examine how that pattern changes in response to rejuvenating strategies and how it is regulated by chromatin modifiers. In collaboration with Project 3, we will explore the role of DNA methylation in MuSC aging, also testing how this epigenetic pattern, and resulting cellular function, changes in response to Core B interventions and to alterations in expression of DNA demethylases. We will also use modified CRISP/cas9 technology to modify DNA methylation in a locus-specific manner. Finally, in collaboration with Project 2, we will explore the population dynamics of MuSC aging by using single cell RNA-seq analysis to evaluate changes in clonal diversity and to test for clonal expansion, a process that we will also explore independently using clonal lineage tracing strategies. We will work closely with all members of this Program to integrate our efforts to lead the field of epigenetics of stem cell aging.

PROJECT SUMMARY In recent years, there has been considerable interest in the possible role that members of the transforming growth factor-ß family may play in regulating tissue aging and the possibility that manipulating their levels of signaling may be a new therapeutic strategy to combat tissue dysfunction in the elderly. Much of this interest has focused on two highly related signaling molecules, myostatin (MSTN, GDF-8) and GDF-11, both of which were originally identified by my laboratory many years ago. We showed that MSTN normally acts to limit muscle growth, and as a result, there has been considerable interest in the possibility that inhibitors of MSTN signaling might be effective in enhancing muscle strength and regeneration; in this respect, there are currently at least 11 phase II or phase III clinical trials being conducted by 7 pharmaceutical and biotechnology companies testing MSTN inhibitors in patients with muscle loss, including in patients who have undergone hip replacement surgery resulting from falls as well as in the elderly with age-related sarcopenia. In the case of GDF-11, recent studies by other groups have suggested that GDF-11 may play an important role in tissue aging. Specifically, several papers reported that circulating GDF-11 levels decrease as a function of age and that systemic administration of purified GDF-11 protein can reverse age-related tissue dysfunction in the heart, skeletal muscle, and nervous system. Other studies, however, reported the opposite, namely, that GDF- 11 circulating levels do not decrease with aging and that administering GDF-11 protein has a detrimental effect on muscle regeneration. Clearly, elucidating the roles of these signaling molecules in regulating adult tissue homeostasis will be critical not only to understanding the control of tissue aging but also to the development of therapeutic strategies for manipulating the activities of these molecules for clinical applications in the elderly. In this project, we will attempt to elucidate the roles of this signaling pathway in aging skeletal muscle by focusing on MSTN, GDF-11, and the related ligand, activin A as well as their inhibitory binding proteins, follistatin (FST), FSTL-3, GASP-1, and GASP-2, all of which circulate in the blood. The overall goal of this project is to determine whether these ligands and binding proteins are pro-geronic or anti-geronic. For this project, we will take advantage of the many genetic and pharmacological tools that we have developed over many years targeting the various components of this regulatory network. The Specific Aims are: to determine how circulating levels of MSTN, GDF-11, and activin A and their inhibitory binding proteins change as a function of age in mice and how their expression patterns in skeletal muscle following injury differ in aged versus young mice; to use mouse lines carrying targeted mutations in genes encoding these ligands and their binding proteins to examine the roles of these proteins in regulating skeletal muscle and other tissues in aged mice; and to use genetic and pharmacological approaches in conjunction with parabiosis studies to examine effects of targeting these ligands and binding proteins on skeletal muscle regeneration in aged mice.

PROJECT SUMMARY Alzheimer?s disease (AD) is a complex neurodegenerative condition that affects millions of individuals worldwide and currently has no preventative or disease-modifying therapies. Exciting results from heterochronic parabiosis studies indicate the presence of rejuvenating factors in the circulation that can restore youthful characteristics to aged cells and tissues, including the restoration of aged neural stem activity and cognitive function. Intriguingly, heterochronic parabiosis studies have also shown that exposure of transgenic AD model mice to a young systemic environment results in a marked amelioration of the cognitive deficits and AD-like neuropathology observed in these animals. The beneficial effects of young blood have been observed in multiple transgenic AD models. However, the identities of the blood-borne factors that mediate these effects are still unknown. This proposal is built upon the exciting results emerged from studies funded by R01AG057433, with the goals of identifying and characterizing circulating anti-geronic factors conserved in mammals, including human, bovine, and mouse. We found that systemic administration of one of the top candidate anti-geronic factors, PEDF, significantly improves deficits in cognitive function in aged wild type mice. We hypothesize that conserved circulating anti-geronic factors such as PEDF mediate the rejuvenation effects of young blood in both aged wild type animals and transgenic animal models of AD, and that systemic treatment of these factors to an AD mouse model will improve cognitive function and ameliorate AD-like neuropathology. To test this hypothesis, we will employ the well-studied 5XFAD transgenic mouse model of AD and will investigate whether and to what extent top conserved anti-geronic factors exert beneficial effects on cognitive function and AD-like neuropathology in vivo by measuring changes in amyloid plaque formation, gliosis, synaptic density, cerebrovascular integrity, and cognitive behavior, as compared to vehicle-treated controls. To understand the mechanisms through which these factors exerts protective and restorative effects, we will perform single cell RNA-seq analysis to identify the biological processes and specific cell types mediating their beneficial effects in the brain. Defining the cell type- specific transcriptional programs that are altered in the brain of the 5XFAD mouse model during the progression of AD pathology and how these are affected by treatment with anti-geronic factors will provide mechanistic insights into the protective and restorative effects of youthful blood-borne factors and may identify new therapeutic targets for AD.

OVERALL: ABSTRACT The scope of regenerative medicine encompasses the repair, regeneration, and replacement of defective, injured, and diseased tissues and organs. The success of regenerative therapies is dependent, at least in part, on a favorable microenvironment in which the regenerative processes occur. Technological innovations and a deepened mechanistic understanding of how these microenvironmental signals influence tissue regeneration has drawn attention to the critical importance of the clinical field with foundations in the application of physical, thermal, and electrical stimuli to promote functional restoration?rehabilitation. We propose that the fields of regenerative medicine and rehabilitative science are inextricably intertwined, an intersection of disciplines that we and others have termed Regenerative Rehabilitation. To realize the full potential of Regenerative Rehabilitation, there is a need for formalized mechanisms that promote the interaction of basic scientists with rehabilitation specialists. During the initial funding cycle, the Alliance for Regenerative Rehabilitation Research & Training (AR3T) built a national network of investigators and programs that has helped to expand scientific knowledge, expertise and methodologies across the domains of regenerative medicine and rehabilitation. This proposal seeks funding for AR3T 2.0, in which we will build on successes achieved and lessons learned over the initial period of support with the goal of being even more responsive to the needs of the greater community. Six specific aims define a framework upon which we will achieve our goals. AR3T will provide education and drive the science underlying Regenerative Rehabilitation by: 1) Providing didactic programs that expose rehabilitation researchers to cutting-edge investigations and state-of-the-art technologies in the field of regenerative medicine (Didactic Aim); 2) Cultivating collaborative opportunities between renowned investigators in the fields of regenerative medicine and rehabilitation (Collaborations Aim); 3) Coordinating a pilot funding program to support novel lines of Regenerative Rehabilitation research (Pilot Funding Aim); 4) Developing and validating technologies to advance the measurement and use of the regenerative rehabilitation programs (Technology Aim); 5) Promoting our center?s expertise to a broad community of trainees, investigators, and clinicians (Promotion Aim); 6) Carefully monitoring and evaluating the effectiveness of our program will ensure that we are successful in achieving our goals (Quality Control Aim). Administrative note: In the preparation of this proposal, we made every effort to present a comprehensive and detailed plan for achieving our goals while minimizing redundancy. Therefore, in multiple places, we refer the reader to specific components of the application, rather than repeating text. We appreciate the time and effort the reviewers devote to the evaluation of the proposals. Sincerely, Fabrisia, Tom and Mike