Irina Conboy - US grants

Muscle atrophy, accompanied by the loss of regenerative capacity, is a debilitating consequence of aging, which remains only marginally understood and is recalcitrant to a therapeutic intervention. This work will characterize muscle precursor/progenitor cells derived from mice of different age in an approach to understand why old muscle tissue regenerates worse than young. Primary myoblasts will be compared in their ability to proliferate, differentiate, and respond to IGF-1. Our preliminary data show age-related differences in the intrinsic properties of these cells. Muscle lineage will be characterized in an adult organism, and potential age-related changes in the relative abundance of muscle precursor cells and in their regenerative capacity will be determined. Role of Notch in the cell fate determination in an adult muscle, and potential age- related changes in Notch presence/activity will be studied. This work will also address the diminished responsiveness of old muscle to IGF-1, by testing whether physiologic signaling by IGF- 1 in primary murine myoblasts requires Cn activity, and whether this pathway changes with age. These aims are designed to reveal key mechanistic differences in regenerative capacity between young/adult and old muscle, and to provide an understanding of muscle atrophy with age. Obtained data may provide a therapeutic opportunity to restore regenerative capacity and growth factor responsiveness in old muscle; and to generate an antibody panel defining adult myogenic lineage proactively.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The candidate plans to complement her achievements in the field of stem cell biology by developing new skills and a profound understanding of the complex science of DNA repair. To achieve this goal, the candidate will use help of Dr. Phil Hanawalt, renown in the field of DNA repair and a dynamic research environment provided by Dr. Tom Rando. The knowledge, skills and collaborations obtained during the initial mentored period will facilitate the career of an independent investigator in the new field of science. Scientifically, this award will give insight into the biology of progenitor cells in normal and aged tissues and provide novel practical solutions in tissue repair. This work will characterize the molecular mechanisms regulating regeneration of skeletal muscle and will investigate why repair deteriorates with age. Adult muscle regenerates due to the activity of satellite cells. With age, the regeneration capacity and muscle strength diminish and inflammatory-related pathologies increase. Biology of satellite cells is controlled, based on preliminary data, by the Notch and Wnt pathways that are active in young muscle cells, but not in old. The proposed work will study why Notch and Wnt signaling that are required for muscle regeneration are lacking after injury in aged muscle. The hypothesis is that in the adult muscle (comprised of terminally differentiated myotubes and quiescent satellite cells), many genes remain silent, and since DNA repair is not efficient in non-transcribed loci, DNA damage accumulates with time in these genes. When these previously silent loci are activated in response to muscle injury, the accumulated DNA damage interferes with transcription and results in the loss of cell function. We will investigate if muscle cells accumulate DNA damage with age and will test a potential molecular link between a quiescence- or differentiation-related decline in DNA repair and the injury-induced expression of Notch and Wnt pathway members. Complimenting this main research goal, we will perform a young-versus-old gene array analysis of genes activated by muscle injury in order to provide more candidates for the DNA repair studies and to identify novel genes regulating muscle regeneration. This work will help to understand postnatal myogenesis and is likely to have therapeutic value for the enhancement of regeneration in adult tissues. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): While many studies of the past years have focused on the derivation, propagation and in vitro differentiation of human embryonic stem cells (hESC), little is known about the self-renewal and pluripotency of hESC in the aged, as opposed to the young, systemic and local organ environments. If the therapeutic hope for these cells and their progeny is to contribute to the ailing tissues in older individuals, but their regenerative capacity is adversely affected by the aged milieu, then their therapeutic value becomes significantly diminished. Unless, of course, the age-related changes affecting stem cell regenerative capacity are understood and countered. Our data strongly suggest that the aged niche has indeed a pronounced inhibitory influence on the regenerative capacity of hESC and that extrinsic cues regulating activation of stem cells become altered with age. It is, therefore, quite possible that the age-related changes in the systemic and organ environments would also preclude a productive repair of old ailing tissues by the transplanted hESC or their progeny. This work will test the specific hypothesis that the ability of hESC to regenerate skeletal muscle is, to a large extent, dependent on the age of their extrinsic environment, will define the changes in hESC regenerative and myogenic potential that are caused by the age-related alterations of their extrinsic niche, will compare the gene expression profile of hESC exposed to "young" versus "old" milieu and will provide initial molecular characterization of the inhibitory components affecting regenerative potential of stem cells in aged tissues. We will use the Federally approved hESC lines WA07 and UC06 for this proposed work. The data will help to understand the molecular mechanism(s) by which local and systemic environments control behavior of stem cells in young versus old organisms. The outcome of these studies is expected to be fundamentally important for deciphering key molecular determinants of aging, for understanding the pathways regulating hESC proliferation and cell-fate determination, and for enhancing the therapeutic value of hESC in the context of aged organs and tissues. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Adult skeletal muscle robustly regenerates throughout adult life but fails to do so in old age. The reason for such a decline in the regenerative potential is not well understood and the relative roles of the changes in muscle cells versus the alterations in their aged environment have not been defined. Recently is has been shown that a young systemic milieu restores the activity of the regeneration-specific Notch pathway and enhances repair of old muscle, suggesting that largely intact regenerative potential of aged satellite cells is not properly triggered in the aged environment. Our most recent data suggest that it is not simply the lack of "positive" systemic factors that causes impaired organ stem cell activation and tissue repair in the old, but that the aged systemic and muscle niches actually inhibit Notch activation and the regenerative potential of both old and young satellite cells. Namely, satellite cells isolated from old muscle or exposed to aged mouse serum in vitro are inhibited in their regenerative capacity and lack Notch activation. Moreover, our preliminary data identifies a molecular mechanism of this age-related inhibition by demonstrating that the reduced regenerative potential of satellite cells in the aged environments stems from excessive TGF-beta/pSmad signaling induced in these cells by their aged niches, which in turn is a result of the elevated levels of TGF- beta-family ligands in aged circulation and muscle tissue. Very importantly, our preliminary results suggest that both myogenic potential and Notch activation can be rejuvenated by attenuation of TGF-beta/pSmad signaling in satellite cells. These studies emphasize high therapeutic relevance of understanding the regulation of adult myogenesis by TGF-beta super-family and of designing the approaches for tunable calibration of TGF-beta/pSmad signaling in muscle stem cells. In order to decipher the age-related role of TGF-beta/pSmad signaling in muscle repair and to progress toward therapeutic applications, it is needed to determine 1) what are the "youthful" positive versus "aged" negative levels of TGF-beta-superfamily ligands in circulation and muscle tissue;2) at what age-related levels TGF-beta/pSmad signaling becomes excessive and "negative" for satellite cell regenerative capacity and 3) how to design a tunable system for a precise "youthful" recalibration of TGF-beta/pSmad signaling in satellite cells. These goals will be approached here in the proposed Specific Aims.

DESCRIPTION (provided by applicant): Accommodating an aging world will pose significant economic and social challenges and will ultimately call for both paradigm-shift in our understanding, and biomedical interventions into the aging process. Extensive data demonstrate that when the systemic niches of organ stem cells are biochemically rejuvenated, stem cells endogenous to multiple old organs engage in productive tissue regeneration. Importantly, the old circulatory milieu rapidly and significantly inhibits the regenerative performance of endogenous stem cells in young organs. This proposal uses what is known about the role of the circulatory milieu in the rejuvenation of aged tissue repair, with the goal o determining the key molecular mechanisms that are responsible for the phenomena of heterochronic parabiosis. Until recently, it was technologically impossible to specifically label and interrogate the proteome of one animal in the setting of heterochronic parabiosis; however the development of cell-selective metabolic labeling of proteins with non-canonical amino acids via expression of mutant aminoacyl-tRNA synthetases in mammalian cells by the co-PI (David Tirrell) enabled this paradigm shifting approach. This high-risk high-reward project becomes feasible due to the united efforts, areas of expertise and experimental models of the Conboy and Tirrell research groups. The Conboy team has proficiency in studies of heterochronic parabiosis and characterization of the effects of defined factors on tissue regenerative capacity, whereas the Tirrell laboratory has pioneered and time-resolved and cell-selective proteomics. The paradigm changing outcomes of these studies are many fold: (1) revealing the youthful pro-regenerative and aged inhibitory proteomes of blood serum, (2) uncovering the mechanisms by which the circulation influences the regenerative performance of organ stem cells in muscle and brain/hippocampus; (3) generating a comprehensive data-base that is required for understanding the genetics of aging and importantly, (4) identifying novel ways to rejuvenate multiple organs and extend healthy life span via systemic administration of defined molecules that emulate the physiologically young blood circulation.

Abstract Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and causes thousands of deaths each year. There are no effective treatments for DMD and new DMD therapeutics are urgently needed. Cas9 based therapeutics have the potential to revolutionize the treatment of DMD, because they can correct dystrophin mutations, via homology directed DNA repair. However, developing Cas9 based therapeutics for DMD has been challenging because it requires simultaneously delivering Cas9 protein, guide RNA, and donor DNA in vivo and delivery vehicles have not been developed that can accomplish this. The central objective of this proposal is to develop a new family of nanoparticle delivery vehicles, termed CRISPR-Nanoparticles, which are designed to treat DMD by delivering Cas9 protein, guide RNA and donor DNA in vivo. CRISPR-Gold is our first generation CRISPR-Nanoparticle and is composed of gold nanoparticles complexed with Cas9 RNP, donor DNA and the endosomal disruptive polymer PASp(DET). We have been able to demonstrate that CRISPR-Gold, can correct 5% of the dystrophin mutations (via HDR) in muscle fibers after a direct injection in mdx mice, and thus has tremendous potential as a treatment for DMD. A successful DMD therapeutic needs to be able to correct 20% of the dystrophin mutations in muscle tissue, and the experiments in this proposal focus on developing 2nd and 3rd generation CRISPR-Nanoparticles, which can generate a 20% HDR rate in muscle tissue. In particular, the 2nd and 3rd generation CRISPR- Nanoparticles address the key factors preventing CRISPR-Gold from generating a 20% HDR rate in a clinical setting, which are (1) its lack of biodegradability, (2) its toxicity, and (3) the lack of cell division in muscle tissue. The central hypothesis of this proposal is that: Delivery vehicles that complex Cas9 protein, guide RNA, donor DNA and endosomal disruptive polymers will be able to efficiently induce HDR and treat DMD. The central objective of this proposal will be accomplished by completing the following Specific Aims. Specific Aim 1: Develop biodegradable CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 2: Develop biocompatible CRISPR-Nanoparticles that can correct dystrophin mutations Specific Aim 3: Enhance the HDR efficiency of CRISPR-Nanoparticles with FDA approved stimulating agents At the conclusion of this proposal we will have identified a CRISPR-Nanoparticle formulation that can efficiently correct dystrophin mutations in vivo and has the biocompatibility needed for clinical translation. The experiments in this proposal are innovative because CRISPR-Gold is the first example of a delivery vehicle that can simultaneously deliver RNA, DNA and protein in vivo and induce HDR. The experiments in this proposal are significant because they will lead to the development of a new therapeutic for DMD (and other genetic diseases) that has the HDR efficiency and biocompatibility needed to enter into clinical trials.

Heterochronic parabiosis and more recent blood apheresis studies provided a proof of principle that tissue stem cells residing in an old mammal are capable of productive regenerative responses (2-4). In support of idea that youthful calibration of key signaling networks will emulate the positive effects of heterochronic parabiosis, defined approaches for rapid enhancement of aged tissue stem cells as robust as parabiosis have been reported (8), (9), (10, 11). The highest risk of modulating key cell-fate regulatory signaling pathways is that skewing these, up or down, from healthy homeostatic levels produces severe multi-tissue side-effects. We hypothesized that using both Alk5 inhibitor (Alk5i) and oxytocin (OT) will allow lowering the dose of Alk5i, thus avoiding the side-effects known to result from overtly inhibiting TGF-beta signaling (34, 57, 58), and hence broadening the positive effects on multiple tissues as compared to a single prong approach. Our Preliminary results show that when used alone, Alk5i attenuates oxytocin receptor (OXTR), hence becoming less therapeutic, but when used together: Alk5i+OT, maintain the pro-regenerative OXTR. And, while we find that each molecule alone does not improve neurogenesis or learning in old mice, low dose of Alk5i+OT does. Our goal is to confirm and extrapolate in both genders the preliminary findings that the defined Alk5i+OT mix safely enhances regeneration and health of multiple old tissues and broadly attenuates the p16 expression in the old mammals. In Aim 1 we will conduct studies of myogenesis, hippocampal neurogenesis, hepatogenesis and will examine health of these tissues and animal performance following the Alk5i+OT defined pharmacology. Tumor pathologies and the expression of key tumor suppressors and oncogenes will be also examined to confirm the safety of this two-molecule technology. In Aim 2 we will examine in more detail the attenuation of p16 and other CDKIs by the Alk5i+OT, focusing on the levels of regulation (transcription, translation, epigenetic status of the loci). We will also study if Alk5i+OT treatment results in apoptosis of p16high cells. Finally, in Aim3 we will explore the epistatic interactions between the TGF-beta/pSmad and OTR/pERK pathways; and we will further narrow down the optimal doses of Alk5i and OT that do not skew the TGF-beta/pSmad2,3 and OTR/pERK signaling intensities from youthful-healthy ranges, while promoting tissue health and regeneration and attenuating p16 to degree that is similar of young animals. Both genders will be studied, providing critically lacking information on female mammals.

Exchange transfusion (apheresis) is a routine strategy for the management of several diseases, such as sickle cell disease, hemolytic disease of newborns, autoimmune disorders, etc. Interestingly, the body of published work on heterochronic parabiosis, the surgical joining of two animals of different ages, and more recently, our study on heterochronic blood exchange, suggest that blood apheresis can be repositioned and used as a new modality: restoring the circulatory environment of aged mammals back to a productive, young, composition, may help to rapidly and broadly enhance the maintenance and repair of multiple organs, combatting a number of degenerative diseases of brain, muscle, liver, etc. and inflammatory disorders. In contrast to the permanent anastomosis of parabiosis, in our small animal blood exchange system animals are connected and disconnected at will, removing the influence of shared organs, adaptation to being joined, etc. Unlike parabiosis, where joint circulation is stochastically established in ~7-10 days, our procedure is less invasive and accurately controlled, the exchange volumes are easily programmed and measured; and the onset and duration of the effects can be accurately and with ease interrogated. The cells versus plasma heterochronicity can be studied and immuno-affinity modules enable removal of specific inhibitory molecules from the old circulation prior to return of such ?rejuvenated? blood into the old animal; as well as a screen for candidate systemic proteins, removal of which from young circulation inhibits tissue maintenance and repair. We will (1) determine the onset and duration of the effects of heterochronic blood apheresis on health and maintenance of muscle, liver and brain, animal performance (strength and cognition), and systemic cytokine profile, also defining the minimal functional dose of exchanged blood that significantly influence these parameters; (2) engineer the V2 device where, in continuous apheresis between two animals of different age, circulating leukocytes are separated from plasma, studying how a cross-match of the age of cells and plasma influences tissue maintenance and repair; (3) engineer the V3 device with imunoaffinity modules allowing to screen and attenuate to young ? healthy levels candidate systemic proteins, using IL6 and TNF-a as the first candidates, that when elevated with age have negative effects on tissue health and regeneration.

Project Summary The improvement in living standards and the advancement in modern medicine have greatly extended human life expectancy. However, aging-related functional decline and diseases, in particular cognitive impairment and neurodegeneration, also become more prevalent. Studies of heterochronic blood exchange reveal that the aged systemic milieu inhibits neurogenesis and impairs cognitive functions in young animals, suggesting the existence of age-elevated systemic factors detrimental to brain health. In particular, inflammation may become excessive and chronic with aging (?inflammaging?) and impair normal brain functions. Thus proteins involved in inflammatory responses, such as cytokines, are candidates of such systemic factors implicated in brain aging. Building upon published literature and our recent finding, we hypothesize that aging-associated alterations in systemic inflammatory factors activate microglia (resident immune cells in the central nervous system) and lead to microglia-mediated synapse loss; restoring the expression pattern of such factors to the healthy young state rescues synaptic defects and improves cognitive functions. In Aim 1, we will use bio-orthogonal non- canonical amino acid tagging (BONCAT) to determine how treatment with a cocktail of Alk5 inhibitor (Alk5i) and oxytocin (OT, a neurotrophic, anti-inflammatory peptide) or heterochronic blood exchange affects the expression profile and distribution of inflammaging-related systemic factors in the brain and peripheral tissues. Aim 2 examines how Alk5i+OT treatment and heterochronic blood exchange affect neuro-immune interaction in the brain, taking advantage of in vivo two-photon imaging to study microglia-synaptic interactions and their effects on synaptic integrity and dynamics in the cortex. Using Array Tomography, a high-throughput, super- resolution proteomic imaging technique, Aim 3 conducts molecular dissection and reconstruction of large populations of individual synapses and determines the effect of Alk5i+OT treatment and heterochronic blood exchange on synaptic molecular signatures and inflammatory cytokine distribution in the brain. Together, these studies will provide a comprehensive characterization of age-specific effects of blood on the brain proteome and synaptic circuits, and outline candidate mechanism(s) responsible for brain aging.