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NIGMS - National Institute of General Medical Sciences

Project Summary Mobile genetic elements use strategies that closely resemble those of viruses to replicate, spread, and persist. The long interspersed element-1 (LINE-1, L1) retrotransposon is the only autonomously active retrotransposon in humans and accounts for nearly one-fifth of the genome. L1 replicates through an RNA intermediate and encodes two proteins—ORF1p and ORF2p—that assemble into a ribonucleoprotein (RNP) complex to reverse transcribe the encoding RNA into DNA and insert a new L1 copy into the genome. Despite existing among hundreds of thousands of defective relatives and competing with parasitic elements such as Alu, a small number of full-length L1s continue to replicate. This success relies on cis preference, in which L1 proteins preferentially act on the RNA that encodes them. Similarly, cis preference is observed to varying degrees in viruses. Hepatitis B virus polymerase exhibits a translation-coupled cis preference when interacting with its pregenomic RNA via the epsilon stem-loop, a crucial step in viral genome packaging and reverse transcription. RNA viruses, such as alphaviruses and flaviviruses, exhibit a related bias, with evidence of localized, co-translational coupling between viral proteins and replication templates. The central hypothesis of this proposal is that L1 cis preference is enforced co-translationally, through ribosome-linked mechanisms and/or co-assembly of RNP complexes, analogous to strategies employed by viruses. To test this, we have developed a novel RNA launch system that initiates L1 replication directly from synthetic RNA, with distinct advantages over classical DNA-based retrotransposition assays. Using this platform, we have established a quantitative barcoded assay for L1 cis preference. We will extend the system to study Alu retrotransposition and employ a retron-based surrogate system to probe the generalizability of our findings. Aim 1 will quantify L1 cis preference and determine how Alu subverts it, addressing the dynamics of competition between autonomous and parasitic elements. Aim 2 will dissect the molecular basis of ORF1p’s cis preference using in vitro translation, ribosome profiling, and deep mutational scanning. Aim 3 will define the molecular basis of ORF2p’s cis preference and test whether cis preference can be transferred to a heterologous reverse transcriptase using a bacterial retron system. The impact of this project is to resolve a long-standing mystery in retroelement biology: how cis preference is achieved. By combining innovative RNA-based tools with high-throughput discovery methods, this work will reveal general principles of viral and retroelement replication, illuminate strategies by which selfish genetic elements maintain a fitness advantage in competitive environments, and provide new entry points for biotechnology and therapeutic development.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY Fetal Alcohol Spectrum Disorders (FASD) represent a major public health concern, significantly contributing to intellectual disabilities with a prevalence rate of 1-5% in the United States and an estimated annual economic burden of $5 billion. Despite its substantial impact, there is currently no cure for FASD. Existing research, including our own, indicates that various brain cells and regions exhibit differential vulnerability to alcohol-induced injury, with underlying mechanisms remaining poorly understood. For instance, neurons are particularly susceptible to alcohol stress, often undergoing acute apoptosis, and those that survive exhibit impaired functionality. Our primary goal is to elucidate the brain region- and cell-specific mechanisms underlying alcohol- induced developmental neurotoxicity (AIDN) and develop cell-specific precision interventions. By addressing the following critical questions, we aim to uncover novel targets for intervention: 1) Why are neurons more vulnerable to alcohol-induced injury? What neuron-specific signaling pathways contribute to this vulnerability? What are the brain region- and cell-specific targets for neuroprotection? To achieve these objectives, we will employ cutting- edge, multidisciplinary approaches including virus-based cell-specific gene modification, spatial transcriptomics, various imaging systems (e.g., multiphoton imaging), and various behavioral tests. Our research will utilize complementary models, including human induced pluripotent stem cell-derived 3D mini brains and mouse models, to investigate the mechanisms of AIDN at molecular, cellular, tissue, and animal levels. Additionally, we will assess the neuroprotective effects of neuron-specific and mitochondria-targeted interventions in AIDN. This study aims to provide a comprehensive understanding of the mechanisms driving alcohol-induced cognitive and behavioral impairments. By mapping acute and long-term brain region- and cell-type specific gene expression profiles following developmental alcohol exposure, our research offers promising strategies for early, brain cell type-targeted precision interventions and treatments for FASD patients. Moreover, the inclusion of human stem cell-based mini brain models will enhance the translational potential of our findings, potentially offering insights applicable to other neurodevelopmental disorders.

NCI - National Cancer Institute

Project Summary Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Despite wellcharacterized genetic drivers (APC, KRAS, TP53, and SMAD4), recurrence remains high, and the 5-year survival rate for advanced CRC is only ~15%. A major challenge is the persistence of cellular plasticity that allows nonstem tumor cells to reacquire stemness following therapy, driving tumor regrowth and relapse. My research addresses a central unresolved question: how stem cell transformation is epigenetically regulated during CRC initiation and progression. My preliminary studies reveal that the repressive histone modification H3K27me3 establishes an epigenetic "gatekeeping" barrier that restricts stemness programs to bona fide intestinal stem cells. During APC-driven transformation, this barrier is eroded, unleashing stemness and fetal transcriptional programs, activating onco-enhancers, and promoting tumor initiation. Genetic or chemical modulation of this barrier alters tumor growth, indicating that sustaining this repressive landscape can suppress transformation. The central hypothesis is that the H3K27me3-based epigenetic barrier tightly regulates stemness, and its erosion drives stem cell transformation and CRC progression. Restoring or maintaining this barrier will limit oncogenic plasticity and suppress tumor initiation. In the K99 phase, I will (1) define how strengthening or weakening the H3K27me3 barrier affects stem cell transformation and tumor growth in vivo (Aim 1), and (2) determine how this barrier evolves across sequential APC, KRAS, and TP53 mutations in engineered human colonoids (Aim 2). In the R00 phase, I will investigate how distinct founding mutations (APC versus BRAF) rewire H3K27me3 and DNA methylation programs to shape tumor evolution using single-cell DNA methylation profiling (Aim 3). The majority of this project uses engineered human colonoids to investigate cell-intrinsic epigenetic mechanisms of CRC progression (Aims 2 and 3). Complementary genetic mouse models are needed only for Aim 1 because the proposed studies address biological questions that cannot be answered using human colonoids or in silico approaches. Human colonoids cannot recapitulate native crypt architecture, physiological signaling gradients, or the longitudinal process of adenoma formation within intact tissue. Therefore, complementary genetic mouse models are required to test whether manipulation of the H3K27me3 epigenetic barrier alters stem cell transformation and tumor initiation within the native intestinal environment and to provide the in vivo context needed to interpret the mechanistic and cell-intrinsic findings from Aims 2 and 3. Highly sensitive low-input genomic and epigenomic assays developed in my laboratory further reduce animal use by maximizing the molecular information obtained from each specimen, enabling comprehensive multi-omic analyses from limited tissue without compromising statistical rigor. Collectively, my integrated approach will elucidate how genetic and epigenetic mechanisms cooperate to control stem cell transformation, uncovering therapeutic strategies to block cellular plasticity and prevent CRC recurrence.

NINDS - National Institute of Neurological Disorders and Stroke

SUMMARY Axonal degeneration of cortical projection neurons underlies several debilitating neurodegenerative disorders including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. HSPs are a large heterogeneous group of inherited diseases characterized by length-dependent degeneration of corticospinal motor neuron axons, leading to spasticity and weakness of lower limb muscles. SPG11 and SPG15, two common autosomal recessive forms of HSP, are caused by mutations in the SPG11 and ZFYVE26 that encode spatacsin and spastizin protein, respectively. Spatacsin and spastizin are mediators for autophagy lysosomal reformation that is critical for maintaining lysosome homeostasis. However, how this impairment results in axonal degeneration and how this pathway can be targeted to rescue nerve degeneration in HSP remain unknown. Using patient induced pluripotent stem cell (iPSC)-based models of SPG11 and SPG15, our previous work has identified impaired mitochondrial dynamics in these patient stem cell-derived neurons. We further found aberrant autophagy influx and reduced lysosome transport in these neurons, implying their involvement in HSP. The goal of this proposed study is to dissect the interplays between these pathological processes and to determine their roles in axonal degeneration in HSP neurons. Based on strong preliminary data, we hypothesize that perturbed spatacsin and spastizin result in autophagy lysosomal defects and impaired mitochondrial dynamics, which interact with each other to impair cytoskeleton organization and axonal transport, leading to axonal degeneration in SPG11 and SPG15. This hypothesis will be tested by pursuing the following three aims: 1) to identify the role of spatacsin and spastizin in axonal and autophagy lysosomal defects of patient cortical projection neurons; 2) to determine the interplay between autophagy lysosomal and mitochondrial defects in axonal degeneration of SPG11 and SPG15 cortical neurons; and 3) to rescue axonal degeneration by targeting autophagy lysosomal and mitochondrial defects in vitro and in vivo. By regulating autophagy lysosomal and mitochondrial pathways both genetically and pharmacologically, this study will delineate their roles in axonal degeneration in HSP. The efficacy of targeting these pathways in rescuing axonal defects will be evaluated both in vitro using iPSC models and in vivo using HSP mouse models. Thus, the combination of iPSC model, gene targeting, and HSP animal model in this study provides unique opportunities to identify novel targets and develop potential therapeutics to effectively rescue axonal degeneration in HSP.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Brugada syndrome (BrS) is a heritable arrhythmogenic disease associated with sudden cardiac death, and de- spite decades of research there are no preventative treatments. Although some BrS patients have pathogenic variants in the SCN5A gene, the genetic and mechanistic cause of BrS for the majority patients remains elusive. The rationale for our project is that extensive clinical studies have identified the epicardial right ventricular outflow track (RVOT) as the site of arrhythmogenesis in BrS, but no robust human in vitro models specifically for right ventricular (RV) and RVOT arrhythmic diseases exist. We have strong preliminary data demonstrating a novel protocol to generate iPSC-derived cardiomyocytes with an RVOT-like phenotype (iPSC-rvotCMs). We will har- ness our long-standing expertise with iPSCs and electrophysiology to fill these knowledge gaps. Furthermore, our published studies developing and testing suppressor tRNAs in heterologous systems and other tissues pro- vide impetus to transform the mechanistic studies of BrS into a testbed for this novel treatment approach for nonsense mutations in SCN5A. Our major objectives are: 1) to discriminate the mechanistic causes of BrS aris- ing from pathogenic SCN5A missense and nonsense mutations as well as genotype negative patients using iPSC-rvotCMs and cardiac tissue chips (CTCs) and 2) test anti-codon engineered tRNAs therapy for nonsense mutation BrS. Our central hypotheses are that BrS CTCs will exhibit reduced and heterogeneous conduction with iPSC-rvotCMs showing genotype-dependent reduction in INa, increase in Ito, and Nav1.5/Cx43 mislocaliza- tion. Secondly, we hypothesize that engineered anti-codon tRNAs will partially rescue nonsense SCN5A variants’ Nav1.5 expression and function. Our hypotheses will be tested in 3 specific aims: 1) Identify the role of hypoxia in generation of iPSC-rvCMs and iPSC-rvotCMs and test for developmental defects in BrS; 2) Develop predictive models of BrS using iPSCs to provide mechanistic insights and therapeutic testbeds; 3) Quantify codon-edited tRNA correction of NaV1.5 expression and off-target effects in PTC-related BrS iPSC-rvotCMs. In Aim 1, hypoxia will be used to promote the differentiation of second heart field progenitors to iPSC-rvCMs and iPSC-rvotCMs. Apoptosis of BrS iPSC-rvotCMs in response to hypoxia, a known trigger for RVOT remodeling during develop- ment, will be evaluated. An AI assistant (SteMy) will be designed to improve the reproducibility of the differenti- ation protocols. For Aim 2 we will advance the CTC model to include iPSC-derived epicardial cells to generate Epi-rvot CTCs to investigate arrhythmia mechanisms for BrS using optical mapping, cellular electrophysiology, immunolabeling, and scRNA-seq transcriptomics. Aim 3 will develop codon edited tRNAs to rescue a range of PTCs identified in BrS patients using iPSC-rvotCMs and measure Nav1.5 expression, INa and conduction velocity in CTCs. This project is innovative in the development of novel protocols to produce human iPSC-rvotCMs, introduction of an AI assistant for stem cell culture, generation of CTCs, and development of anti-codon tRNAs to advance the first disease modifying therapy available for BrS.

NIGMS - National Institute of General Medical Sciences

SUMMARY Cell-cell communication is essential for coordinating complex biological processes, and extracellular vesicles (EVs) have emerged as critical mediators of intercellular signaling. This project focuses on a unique class of large EVs called midbody remnants (MBRs), which are released during cell division. MBRs represent a novel mode of intercellular communication with potential implications in various physiological and pathological contexts. The overarching goal is to elucidate the mechanisms by which MBRs facilitate the transfer of information between cells. Specific goals over the next five years are to: 1) investigate how the informational content of MBRs varies in different biological contexts by identifying and characterizing conserved and unique molecular cargo (RNAs, small RNAs, and cell surface proteins) of MBRs from different cell types, including cancer, stem, and differentiated cells, and 2) elucidating the mechanisms by which recipient cells recognize and internalize MBRs. In addition, 3) we will investigate the potential hijacking of the MBR pathway by viruses for transmission, by examining viral RNA localization, factors required for viral RNA targeting to MBRs, and the ability of virus-infected MBRs to induce infections. Lastly, we will begin to: 4) investigate the role of MBRs in neurodevelopment and neurodevelopmental disorders, like autism spectrum disorder, by profiling changes in MBR informational content during neural progenitor cell differentiation and mechanistically testing genes necessary for cell fate and proliferative function that we find altered or loss in diseased states. The research design involves isolating MBRs from diverse cell types, performing transcriptomic and cell surface proteomic analyses, functional perturbation studies, live-cell imaging, and utilizing cellular and biochemical tools. This interdisciplinary approach will provide mechanistic insights from the genome- wide scale to sub-micron resolution. The findings from this project have significant implications for public health, as they could unravel the roles of MBRs in cell proliferation, RNA signaling, and EV biology, which are crucial in cancer, stem cell biology, and diseases associated with aberrant cell division and proliferation. Furthermore, understanding MBR function may identify novel therapeutic targets and establish MBRs as potential delivery vehicles for treating various diseases.

NHLBI - National Heart Lung and Blood Institute

Project Summary/Abstract The predominant myosin heavy chain expressed in human heart, beta-MyHC, is encoded by the MYH7 gene. MYH7 variants are well described in hypertrophic cardiomyopathy and less frequently seen in dilated cardiomyopathy. A recent series of publications link variants in the 5’ end of the MYH7 gene as implicated in left ventricular noncompaction cardiomyopathy, often in the setting of a dilated ventricle with impaired function. Importantly, premature truncations as well as missense variation within the MYH7 gene has been linked to LVNC in both population studies and in individuals and families. We now generated a heterozygous premature truncation in MYH7 in human induced pluripotent stem cells (hiPSCs). When differentiated into engineered human heart tissues, we observe the heterozygous premature truncation in MYH7 produces a phenotype consistent with excess proliferation and reduced function, which are key features thought to underlie the development of LVNC in vivo. We hypothesize that truncations and missense variants identified in LVNC are associated with reduced contractility, rather than hyperdynamic MYH7 variants seen in hypertrophic cardiomyopathy. Additionally, many missense variants in MYH7 are considered variants of uncertain significance and methods such as those being used here may help adjudicate variants of risk. Through this training program under the K99 phase, Dr. Monroe will evaluate missense MYH7 variants associated with LVNC and evaluate their performance in engineered heart tissues. In his second aim, he will expand the search for LVNC-associated MYH7 variation to the population scale using linked cardiac imaging and genotype data in the in population datasets. As Dr. Monroe transitions to his independent phase, he will build from work performed earlier in his train implicating the Hippo pathway in proliferation and specification. In Aim 3, he will detail new disease relevance for the Yes-associated protein (YAP) in MYH7-associated LVNC using the models already in hand and further developed under his K99 training. Finally, in Aim 4, Dr. Monroe uses unbiased approaches to characterize human cardiomyocyte heterogeneity in healthy and LVNC engineered heart tissues in order to better delineate the range of differentiation and identify additional downstream pathways that will fuel future investigations. To promote his career development, Dr. Monroe will draw on the strengths of his mentoring committee and primary mentor which will focus on expanding his management and his own mentoring skills.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cell transplantation (HSCT) is a potentially lifesaving treatment for patients with hematological and immune disorders but poses significant risk due to prolonged immunocompromised states before donor stem cell engraftment. Successful engraftment, where hematopoietic stem and progenitor cells (HSPCs) establish themselves in the recipient's bone marrow, requires that circulating donor HSPCs transmigrate the bone marrow endothelium to reach their supportive stem cell niche. Enhancing the transendothelial migration of HSPCs offers a promising strategy to speed engraftment and boost post- transplant immune system reconstitution. The full complement of receptors and intracellular machinery within the bone marrow niche endothelial cells (ECs) that promote HSPC migration remain incompletely understood. In mammals, the transendothelial migration of HSPCs occurs deep inside opaque bones, which are largely inaccessible to imaging and experimentation in live animals. The transparent zebrafish embryo, by contrast, which has a blood and vascular system highly similar to that of humans, enables direct visualization of HSPC migration in vivo. We previously identified a conserved gene expression signature unique to the hematopoietic niche ECs that regulate HSPC transmigration. This expression program includes genes with cell adhesion and endocytosis/vesicle trafficking functions. In my preliminary studies I disrupted candidate factors from the niche EC signature, which blocked endocytosis in the niche ECs and disrupted the niche migration of HSPCs. My data suggest that a combination of specific cell adhesion and endocytosis/vesicle trafficking machinery supports the endothelial transmigration of HSPCs, a hypothesis I will test with my proposed aims. In Aim 1, I use high-resolution in vivo imaging in the zebrafish paired with human cell culture systems to determine how a candidate adhesion molecule promotes HSPC migration. In Aim 2, I will use a rapid F0 zebrafish CRISPR knock-out platform to test whether different niche EC-expressed vesicle trafficking factors are required for HSPC migration. In parallel, I will use a transcription factor reprogramming approach to assess whether induction of the niche EC signature in different human cell types can trigger the transendothelial migration of HSPCs. Together, these proposed experiments will provide key insight into how niche EC-expressed factors promote the migration of HSPCs. Results from my proposed studies could inform methods to enhance bone marrow engraftment or to direct donor HSPCs to new and/or additional niches to improve HSCT outcomes.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Craniofacial bones, unlike other bones in the skeleton, originate from both neural crest and mesoderm. Studies have indicated that bones derived from neural crest regenerate faster than those from mesoderm, but the mechanisms behind this remain poorly understood. We discovered a novel network of p75 neurotrophin receptor-positive (p75-NTR+) cells within the periosteum of neural crest-derived bones that are crucial for the rapid regeneration of these skeletal sites. My central hypothesis is that these cells represent a distinct subset of neural crest-derived stem cells that are developmentally programmed to accelerate craniofacial regeneration through two independent mechanisms: (1) direct osteolineage differentiation and (2) the establishment of a pro-regenerative immune niche. My long-term goal as a dentist-scientist is to identify endogenous mechanisms of accelerated skeletal regeneration and to translate these findings into novel therapeutics that can promote cranial and dentoalveolar bone regeneration. The proposed K99/R00 research and training is designed to help me to achieve this goal. The K99 mentored phase of the project will be carried out with Dr. Erica Scheller and Dr. Farshid Guilak (primary co-mentors) at Washington University in St. Louis. The goal of the K99 phase is to provide additional training and mentoring in three areas: 1) Lab Management, 2) Bioinformatics and in silico New Approach Methodologies (NAMs), and 3) Inflammation and Immunology. A strong Scientific Advisory Committee with members from Washington University in St. Louis, Johns Hopkins University, and the University of Pittsburg has been established with diverse expertise in bone biology, skeletal regeneration and imaging, immunology, and bioinformatics/NAMs to oversee a training plan that complements and expands my scientific background, preparing me for a transition to independence during the R00 phase as faculty at a research-focused institution. To further dissect the regenerative potential of the novel p75-NTR+ cells and their contribution to the acceleration of neural crest-derived bone healing, I am proposing two research aims in this application as follows: Aim 1. define the dual osteogenic and immunomodulatory roles of p75-NTR+ periosteal cells in craniofacial bone regeneration; and Aim 2. explore the targeting of p75-NTR+ cells as a therapeutic strategy to accelerate craniofacial regeneration. Overall, the proposed experiments aim to identify the intrinsic biological pathways that accelerate bone healing while considering the implications of these results for clinical management of skeletal disease. This research holds significant translational therapeutic potential for patients with skeletal defects and is expected to ultimately improve patient outcomes and quality of life.

NINDS - National Institute of Neurological Disorders and Stroke

Project summary The computational power of the brain relies on the accuracy of synapses to transmit information. At presynaptic terminals, this accuracy depends on the coordinated function between two protein machineries. First, the active zone generates exocytic sites where neurotransmitters are released from synaptic vesicles. Second, the endocytic apparatus restores vesicles for subsequent rounds of neurotransmission. These machineries are assembled in very close proximity, with the endocytic apparatus localized at the plasma membrane region that surrounds the active zone, called periactive zone. This spatial organization, a hallmark of synaptic architecture, ensures sustained neurotransmission. Genetic studies have linked components of both machineries to multiple brain disorders, highlighting their relevance. Our goal is to understand the mechanisms that direct the coordinated assembly of the active zone and the periactive zone, a process that remains poorly understood. We have generated three lines of evidence that indicate that the large GTPase Dynamin is an organizer of both the active zone and the periactive zone. First, deletion of all Dynamin proteins from neurons results in a selective loss of the active zone proteins Munc13-1 and RIM, and decreased neurotransmitter release, which indicates Dynamin roles in active zone assembly. Second, our previous work showed that several endocytic proteins are constitutively deployed to the periactive zone. We have now observed in pilot experiments that these proteins are strictly segregated into different clusters, in line with a disassembled machinery, and that this strict spatial segregation is lost in Dynamin mutants. Third, preliminary data show that Dynamin-1 is localized both at the periactive and at the active zone, consistent with roles bridging compartments. Here, we will dissect mechanisms through which Dynamin organizes the active zone and the periactive zone. We will use neurons cultured from Dynamin-1/2/3 triple mutant mice and human stem cell-derived neurons as models, and further integrate 10X Expansion Microscopy, electrophysiological and live imaging analyses. In Aim-1, we will focus on the active zone. We will define the roles that each of the three Dynamin proteins play in active zone assembly and their nanoscale localization at synaptic terminals. Next, through structure-function rescue experiments, we will identify the sequences that determine the localization of Dynamin proteins and mediate their role in active zone assembly. In Aim-2, we will focus on the periactive zone. We will first characterize the molecular organization of this compartment and how it is modified by synaptic activity. Next, we will establish the role of Dynamin proteins in the organization of the periactive zone and whether impaired membrane fission cause periactive zone disorganization. Finally, we will also test whether disease-associated missense variants of Dynamin-1 result in disrupted active zone or periactive zone organization. Overall, this work will provide insight into the organization of presynaptic protein machinery whose dysfunction is connected to brain disorders, advancing our understanding of synaptic function in health and disease.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Our understanding of immunity largely stems from models of infection with pathogenic microbes. However, the vast majority of microbial-immune encounters occur as a symbiotic relationship with the commensal microbiota. Recently, the contribution of commensal-specific T cells to host physiology has received significant attention. These commensal-specific responses not only control microbiota containment but also promote immune tolerance within the gastrointestinal tract. While commensal-specific CD4+ T cell responses in the lamina propria have dominated models of mucosal immune regulation, these are vastly outnumbered by CD8+ intraepithelial lymphocytes within the epithelium. How CD8+ T cell responses to gut microbiota are primed, differentiate and function under homeostasis has not been addressed. Conversely, aberrant immunity to commensal microbes has been proposed to underlie pathologies of barrier tissues, including inflammatory bowel disease (IBD), where commensal-specific T cells accumulate in blood and intestinal tissues of afflicted patients. A better understanding of the properties and functions of commensal-specific T cell responses is therefore fundamental to studies of tissue immunity in health and disease. Our long term goal is to better understand how commensal-specific T cell responses contribute to barrier tissue homeostasis, and the objective in this application is to investigate the mechanisms regulating induction of commensal-specific CD8+ T cells in homeostasis and how they become dysregulated in IBD. Our rationale for the proposed work is that uncovering these mechanisms has the potential to translate into new therapeutic approaches. Our central hypothesis is that commensal-specific CD8+ T cells develop as functionally restrained intraepithelial lymphocytes (IEL) under homeostasis, but that perturbation of local immune regulation within the intestinal epithelium, in the case of patients with ulcerative colitis, by autoantibody-mediated blockade of integrin avb6 results in aberrant CD8+ effector T cell responses in IBD. Based on strong preliminary data, we will test three specific aims: (1) Determine key antigen-presenting cells (APC) priming SFB-specific CD8⍺β+ IEL. (2) Identify how cell-intrinsic pathways drive differentiation, maintenance and restraint of SFB-specific CD8⍺β+ pIEL. (3) Determine how pathogenic KLRG1+Eomes+ CD8+ T cells arise and contribute to inflammation in murine models of ulcerative colitis Our approach is innovative as it investigates new mechanisms of immunity unique to commensal-specific CD8+ T cell responses. The proposed work is significant because it will establish new insights into the interaction and communication between commensal microbes and immune cells in the gut environment and identify potential targets for therapeutic intervention in conditions of chronic intestinal inflammation.

NIA - National Institute on Aging

Abstract Protein synthesis is a vital biological process, important for neuronal development, synaptic plasticity, and cognitive functions such as learning and memory. In contrast, dysregulated translation is a feature of many neurodegenerative disorders, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Pathogenic changes to the microtubule associated protein tau are thought to cause neurotoxicity and dysfunction in both AD and FTD in part by disrupting several molecular processes, including protein synthesis. However, the molecular mechanisms by which pathogenic tau disrupts protein synthesis remain elusive. In this application, we will determine how FTD-associated heterozygous mutations in tau impact protein synthesis in human neurons. We will use human induced pluripotent stem cell (iPSC)-derived neurons carrying FTD-associated tau mutations as a model. The iPSCs will be differentiated into neurons using Neurogenin-2, a master transcription factor capable of inducing differentiation into excitatory neurons in under two weeks. Using this platform, in the first aim we will determine the impact of FTD-associated tau mutations on translation elongation rates and will perform ribosome profiling to determine the translatome and translational efficiency associated with the FTD- associated tau mutations. In the second aim, we will determine whether the tau mutations alter tau- ribosome interactions and if they cause ribosome collisions. These studies will provide insight concerning the mechanisms by which FTD-associated mutations in tau alters protein synthesis, as well as the biology and subsequent pathobiology of tau in tauopathies such AD and FTD.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ ABSTRACT Blood vessels are the earliest organs formed during embryonic development, and the embryonic cardiovas- cular system is required for nutrient exchange and organogenesis. As early as mouse embryonic day (E) 7.5 in the extraembryonic yolk sac blood islands and embryos, mesodermal precursors undergo the first cell fate de- cision to form the endothelial cell (EC) lineage. ECs further differentiate into arterial, venous, and lymphatic subtypes that are necessary to build the vascular system. However, the clonal relationships and spatial origins of EC subtypes that form the vasculature are poorly defined, and the mechanisms of EC specification and dif- ferentiation in vivo remain incompletely understood, posing a barrier to vascular regenerative medicine. Etv2, an ETS-family transcription factor, is a master regulator of EC specification. Etv2 is transiently ex- pressed in mesoderm progenitors in the yolk sac and developing mouse embryo from E7.5 - E9.5. Mice lacking Etv2 fail to develop blood or vasculature, and Etv2 is responsible for endothelial and hematopoietic lineage specification. Conversely, forced expression of Etv2 induces EC reprogramming. We have carefully interro- gated mechanisms by which ETV2 promotes EC specification in mesoderm progenitors derived from human induced pluripotent stem cells (iPSCs), using scRNA-seq, scATAC-seq, CUT&RUN, and functional CRISPR screens. Novel observations included: (1) the strength or timing of ETV2 expression influenced arteriovenous EC differentiation; (2) in addition to stimulating EC specification, ETV2 also suppressed specification of other mesodermal lineages; and (3) ETV2 suppression of alternative fates required its recruitment of the transcrip- tional repressor REST. These observations lead to our central hypothesis that ETV2 drives EC specification and arteriovenous differentiation, with REST cooperating with ETV2 to restrict alternative lineages during em- bryonic vasculogenesis. To track cell state transitions and fate of Etv2-expressing cells during vasculogenesis, we will integrate cut- ting edge single cell, spatial transcriptomics, and barcoded lineage tracing approaches to track the progeny of Etv2-expressing progenitors. To focus our efforts, in Aim 1 we will study the initial steps of vessel formation in the yolk sac. We will perform spatial and clonal analysis of Etv2-lineage cells during early yolk sac vasculogen- esis and late vascular plexus remodeling. To determine the role of Rest in Etv2-directed EC specification, in Aim 2 we will determine the effect of Rest inactivation on vasculogenesis and Etv2-lineage diversification, un- cover the Rest-Etv2 transcriptional regulatory network, and identify Rest-regulated genes and TFs in Etv2-line- age progenitors required for EC specification. The work will produce a high-resolution phylogenetic tree of vas- cular development originating from individual Etv2+ progenitors and bring new insights into the molecular mechanisms that direct EC subtype differentiation, informing future efforts in vascular regeneration.

NEI - National Eye Institute

Optic neuropathies including glaucoma are the world’s leading cause of irreversible blindness. Though existing treatment for some progressive optic neuropathies slow vision loss, no treatments exist to restore sight lost to these neurodegenerative conditions. This shortcoming relates to the death of retinal ganglion cells (RGCs), which transmit visual information from the eye to the brain via their axons running through the optic nerves and tracts. Though some cold-blooded vertebrates are capable of RGC regeneration, mammals (including humans) lack this capacity. Development of regenerative therapies to repopulate RGCs would improve the quality of life for tens of millions of people suffering from optic neuropathy. Though transplantation of stem cell-derived RGCs has been long considered, challenges to durable graft survival and functional retinal integration have hampered progress. Recently, we overcame these challenges by developing methods to disrupt the retina’s internal limiting membrane (ILM), which dramatically improves the survival, engraftment, and synaptic connectivity of human RGCs in the retina of multiple species. To achieve functional vision restoration, donor RGCs must receive presynaptic input from host bipolar and amacrine cells so that they can encode light stimuli in a manner that is interpretable by the brain. The goal of this project is to augment long-term donor RGC survival and establish physiologic connectivity to functionally relevant inner retinal circuits at translationally relevant levels by transiently suppressing a key cell-intrinsic pathway involved in the transition of neurons from an immature plastic state to a mature non-regenerative state. Our preliminary data show that deletion of the PTEN gene dramatically increases donor RGC survival and dendritogenesis early following transplantation. However, PTEN expression is necessary for neuronal maturation and stability of synapses & neurocircuits. Further, PTEN is a tumor suppressor, and sustained deletion may increase the risk of transformation and tumorigenesis. Therefore, we will leverage inducible CRISPR inhibition to generate human stem cell lines wherein PTEN activity can be suppressed for a specified duration. Using a rat model of glaucoma, we will transplant human RGCs intravitreally and compare the effects of multiple durations of transient PTEN suppression at key early stages of the engraftment process. Using a combination of 1) in vivo adaptive optics scanning laser ophthalmoscopy and 2) volumetric confocal microscopy with AI-based 3D segmentation of donor RGC dendrites within the inner plexiform layer, we will quantify RGC survival, engraftment, and dendritic architecture & localization for up to 16 weeks post-transplantation. Using 3) optical electrophysiology (calcium imaging), 4) transsynaptic circuit tracing, and 5) spatial transcriptomics, we will assess the evolution in and eventual stability of retinal circuit integration, light response properties, and RGC maturation following PTEN re-expression. Successful completion of this project will advance RGC repopulation by determining how transient PTEN suppression of various durations sustains transplant survival & promotes physiologic retinal connectivity to restore vision in optic neuropathy.

NIGMS - National Institute of General Medical Sciences

Project Summary Incomplete penetrance is a common but poorly understood biological phenomenon in which one person with a genetic mutation develops a disease, while another person with the same mutation does not. This project outlines a trajectory from using zebrafish to model incomplete penetrance in my dissertation work to using patient derived models to study incomplete penetrance in my postdoctoral work. The overall goal of this project is to identify conserved mechanisms of incomplete penetrance that span multiple biological systems and genetic disorders. Specifically, this project focuses on paralogous compensation and DNA methylation of paralogs as shared mechanisms of incomplete penetrance. Paralogous compensation describes a phenomenon in which closely related genes can function in place of each other when one is lost. DNA methylation is a biochemical modification in that controls gene expression. Compensation by paralogs and differences in DNA methylation states have been identified as putative mechanisms of incomplete penetrance in both human diseases and in my system. In this work, both a zebrafish model system and patient-derived induced pluripotent stem cells (iPSCs) will be used to test whether altering paralog expression can be used to rescue a deleterious mutation. One genetic disorder displaying incomplete penetrance is MEF2C haploinsufficiency syndrome. Zebrafish mef2ca mutants can be used to model this disorder and display incomplete penetrance. In my dissertation work I seek to test the hypothesis that heritable changes in DNA methylation and paralog expression underlie incomplete penetrance. Aim 1.1 will use selective breeding to drive high or low phenotype penetrance and evaluate differences in the genome, transcriptome, and methylome that segregate with penetrance. Aim 1.2 will functionally test whether mef2 paralogs can compensate for mef2ca loss using zebrafish transgenic lines. These experiments will elucidate whether paralogs can functionally rescue gene loss, and whether paralog expression is the engine of selection when breeding for incomplete penetrance. To build upon this work and test whether the same mechanisms are conserved across systems, my postdoctoral work will be performed in iPSCs from patients and incompletely penetrant mutation carriers. This work will test the hypothesis that carriers of disease-causing mutations will have higher paralog expression and lower levels of methylation at paralog loci than their penetrant family members. Further work in this system will test whether paralog expression and methylation can be manipulated to replicate an incompletely penetrant condition. This work will provide insight into how we can harness mechanisms of incomplete penetrance to understand and eventually treat mendelian genetic disorders.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Sickle cell disease (SCD) is a devastating inherited hemolytic anemia that affects millions of people worldwide. Over 500,000 infants are born with SCD annually and the majority die before 5 years of age due to spleen dysfunction. In studies conducted with my K23 award, we determined that spleen damage in SCD impacts adaptive immunity. We identified an age-related decline in unswitched memory B cells (UMBC), the peripheral blood equivalent to splenic marginal zone B cells, and a corresponding increase in naïve B cells compared to children without SCD. Children with SCD/very low UMBC had >2-fold lower splenic expression of three genes important for B cell differentiation compared to SCD/low UMBC: IL21R, PF4, and CX3CR1. Expression of genes for ligands that activate B cell differentiation, DLL1 and JAG1, were significantly higher in children with SCD/very low UMBC compared to SCD/low UMBC. These data suggest that IL21R, PF4, CX3CR1, DLL1, and JAG1 have a role in the mechanism of MZB loss, B cell differentiation, and adaptive immunity in SCD. There is a critical gap in knowledge about the mechanisms of adaptive immune dysfunction in the spleen, and how to target these pathways to prevent life-threatening infections and autoimmunity in SCD. Our purpose of this limited R03 award is to prioritize genes important for B cell development in SCD spleen for future clinical and mechanistic studies. Our central hypothesis is that SCD alters expression of key genes important for B cell differentiation, leading to low UMBCs and higher naïve B cell counts. Aim 1. Develop an ex vivo system to investigate the role of IL21R, PF4, and CX3CR1 in B cell development in SCD. We will validate our findings with RNA sequencing of additional spleen samples. We will measure serum IL21, PF4, and fractalkine (the ligand for CX3CR1) by ELISA in SCD and correlate levels with B cell subsets. We will differentiate CD34+ stem cells into B cells to compare expression and activity of IL21R, PF4, and CX3CR1 in SCD- versus non-SCD-derived B cell subsets using flow cytometry and transcriptomic approaches. Aim 2. Determine the role of DLL1 and JAG1 in B cell differentiation in the spleen in SCD. We will use spatial transcriptomics to localize DLL1 and JAG1 signaling in human spleen tissue. We will use imaging flow cytometry to compare interactions between cells that express DLL1 and JAG1 and B cells in spleen tissue from patients with and without UMBC loss. We will validate the Townes SCD mouse model as a tool to examine how inflammatory stimuli influence the expression of Dll1 and Jag1 in the spleen in vivo. Impact: I expect my research will lead to significantly improved outcomes for SCD by identifying novel pathways in adaptive immunity that contribute to complications in SCD. Enhanced understanding of these pathways will yield new targets urgently needed for druggable disease modification. Within two years, I will have necessary tools and preliminary data to apply for independent funding through an R01 or similar mechanism.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT This research proposal is in response to the NOT-OD-24-079 “Notice of Special Interest: Women’s Health Research” focused on health conditions that are female-specific. The ovary is the first organ to age in the human body. Ovarian aging negatively influences lifespan and a broad range of health outcomes in cardiovascular, skeletal, metabolic, immune, and neurocognitive systems in women. Despite these broad impacts, ovarian aging has received limited scientific attention. The biological mechanisms that drive ovarian aging, and how they influence broader healthspan in women, remain poorly understood. The objective of this proposal is to investigate the molecular mechanisms by which NBR2, a long non-coding RNA, contributes to the remarkably complex processes of ovarian aging. By performing genome wide association studies (GWAS) of two reproductive aging- related traits, age of natural menopause (ANM) and reproductive lifespan (RL), we found that genetic variants within a gene-rich haplotype block at the BRCA1 locus are associated with both traits. Our integrative post- GWAS analysis, combined with functional genomic studies in human ovarian cell models, identified a causal non-coding regulatory variant (rs2298862 T>C) associated with both later ANM and longer RL. Although previous ANM GWAS studies identified BRCA1 as the causal gene at this locus, our functional genomic studies experimentally validated that NBR2 at the true target gene. The variant downregulated NBR2 expression, suggesting that NBR2 is a likely driver of the reproductive longevity phenotypes. The major goal of this project is to uncover the mechanisms by which NBR2 modulates female reproductive longevity (Aim 1) and elucidate the mechanisms by which the causal regulatory variant regulates NBR2 expression in diverse ovarian cell types (Aim 2). In Aim 1, I will test the hypothesis that reduced NBR2 expression delays ovarian aging by modulating pro-longevity signaling pathways. I will generate CRISPR/Cas9-mediated knockout NBR2 cell models and perform unbiased RNA-immunoprecipitation followed by mass spectrometry to identify NBR2 interactors and downstream targets. In Aim 2, I will test the hypothesis that the rs2298862 (T>C) variant reduces NBR2 expression by altering long-range chromatin interactions, disrupting transcription factor binding, and modulating enhancer activity. I will generate multiple ovarian cell types from CRISPR-engineered human embryonic stem cells carrying the variant and assess its impact on NBR2 expression, chromatin architecture, transcription factor (TF) binding, and aging-related cellular phenotypes. By identifying pathways and regulatory mechanisms by which NBR2 and its downstream regulators influence ovarian aging, this project will provide a molecular framework for understanding human reproductive longevity and may reveal targets for preserving ovarian function and healthspan in women.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Myotonic Dystrophy Type 1 (DM1) is a progressive neuromuscular disorder affecting 1 in 8,500 individuals. Although primarily characterized by skeletal muscle dysfunction, over 80% of DM1 patients exhibit neurological manifestations, including cognitive impairment, autistic features, ADHD, depression, anxiety, sleep disturbances and excessive daytime sleepiness. DM1 is caused by a CTG repeat expansion in the 3' untranslated region of the DMPK gene. The mutant RNA transcripts containing expanded CUG repeats form nuclear RNA foci and sequester the Muscleblind-Like (MBNL) family of RNA-binding proteins, leading to their functional depletion and the dysregulation of RNA processing. Studies in skeletal muscle have revealed that characteristic DM1 symptoms, including myotonia and muscle weakness, result from mis-splicing of MBNL target genes such as the chloride channel ClC-1 and calcium channel Cav1.1. While knockout of the MBNL paralogs, MBNL1 and MBNL2, reproduces DM1-like neurological phenotypes in mice, the molecular mechanisms underlying DM1 brain pathology remain unclear, particularly whether these symptoms also stem from the dysregulation of alternative splicing or other MBNL-dependent processes such as RNA localization and stability. To investigate the neurological manifestations of DM1, I generated a novel DM1 brain mouse model, CUG960, which expresses 960 interrupted CUG repeats throughout the central nervous system (CNS). This model recapitulates key DM1 features, including nuclear RNA foci, MBNL sequestration, reduced brain weight, and behavioral abnormalities. The CUG960 mouse model is doxycycline-repressible, enabling temporal control of CUG repeat expression for rescue studies. Despite robust physiological and behavioral deficits, CUG960 mice show only modest splicing changes, suggesting additional mechanisms may contribute to the CNS pathology. The goals of this proposal are threefold. First, I will perform behavioral assays and sleep studies on the CUG960 mouse model to determine the short and long-term effects of CUG repeat RNA expression in the CNS and identify brain regions most vulnerable to CUG repeat toxicity. Second, using the CUG960 mouse model, I will identify changes in alternative splicing, gene expression, and RNA localization and determine the relative contributions of nuclear and cytoplasmic MBNL loss-of-function through rescue experiments. Third, I will use the doxycycline-repressible feature of the CUG960 mice to suppress CUG repeat expression at different timepoints to determine if the neurological DM1 symptoms are reversible and define the critical therapeutic time window. Completion of this proposal will establish the direct effects of CUG repeat RNA expression in the CNS, elucidate the molecular mechanisms driving the neurological manifestations in DM1, and determine whether and when these phenotypes can be reversed. These findings will provide crucial insights into the mechanisms underlying DM1 brain disease and inform the development of therapeutic approaches.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Cell therapies are a promising alternative therapy for the treatment of type 1 diabetes, and stem cell-derived insulin secreting beta cells (sBC) have demonstrated the feasibility of restoring insulin independence in clinical models. However, this strategy has limited long-term application due to the requirement of ineffective and toxic immunosuppressive drug regimens and immune rejection, which currently limits the applicable patient pool to high-risk patients. The leading commercial entities clinically testing stem cell-derived islet replacement products are pursuing macroencapsulated cell delivery methods to overcome this critical hurdle to translation. Macroencapsulation devices can blunt immune responses to the graft and confer the safety benefit of cell delivery in a single, retrievable device. However, functional clinical success of these devices has not been demonstrated to date due in part to inevitable antigen shedding resulting in indirect antigen recognition, which results in immune destruction of encapsulated cells. Thus, synergistic immunomodulatory approaches are necessary to fully immunoprotect encapsulated cell grafts, and achieve immunological tolerance in the absence of immune suppression. The fetal-maternal interface is a robust model of immune tolerance toward allogeneic tissue, where placental trophoblasts maintain tolerance by two main approaches: (1) presenting an inert surface to maternal immune cells, a strategy akin to cell encapsulation which blocks direct antigen recognition; and (2) through secretion of tolerogenic factors which induce tolerance toward fetal antigens that escape the placenta. Our preliminary data demonstrates that a macroencapsulated tolerogenic trophoblast cell therapy can evade rejection and delay rejection of bystander macroencapsulated cell grafts in a challenging xenotransplant model. In this proposal, we aim to (1) validate our preliminary studies using translatable cell sources of sBC and trophoblasts, and (2) elucidate the immunological mechanisms of trophoblast cell therapy-induced tolerance in xeno and humanized allogeneic transplantation models. This will be addressed in three Specific Aims: (1) Identify immune mechanisms of trophoblast cell therapy-induced transplant site-dependent graft tolerance, (2) Optimize and characterize tolerogenic cell therapy dose impact on tolerance induction, and (3) Untangle the contributions of antigen-specific and non-specific mechanisms of trophoblast cell therapy tolerance induction. In this work, we expect to identify the immunological mechanisms by which tolerogenic trophoblast cell therapies delay or prevent immune rejection.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Many emerging zoonotic viruses (animal viruses that transmit to humans) are highly pathogenic, having the potential to cause deadly epidemics or even global pandemics. The risks zoonotic viruses pose are highlighted by the emergence of the SARS/MERS coronaviruses, Ebola virus, and HIV-1, all of which are related to animal viruses that were unknown before they caused substantial cases of disease in humans. Given the risk animal viruses pose to humans, many researchers have turned to viral discovery—using genome sequencing tools and metagenomic analyses, researchers hope to identify novel animal viruses before they emerge in humans. We've developed a pipeline that integrates viral surveillance with molecular investigations in the laboratory to identify pre-emergent viruses with epidemic potential. Using this approach, we've provided compelling evidence suggesting that simian arteriviruses (SAVs)—understudied and neglected pathogens of African monkeys—are poised for spillover, posing a threat to human health. We demonstrate key biological features that poise SAVs for zoonosis, including: (1) compatibility with human receptors; (2) high titer propagation in human cells; and (3) potential for evasion of human innate immunity. Further interrogation of the biology of SAV infection is crucial for future epidemic preparedness efforts. The objective of this proposal is to uncover mechanisms of cell entry, immune evasion, and zoonotic potential for these highly concerning viral pathogens. In Aim 1, we employ a series of molecular, biochemical, structural, and functional approaches to define SAV-receptor interactions and establish proof-of-concept strategies for future therapeutics—an essential step in outbreak preparedness. In Aim 2, we will identify SAV proteins that antagonize the human innate immune response, with the goal of revealing vulnerabilities that may help develop safe and effective antiviral approaches. In Aim 3, we will thoroughly evaluate the zoonotic potential of diverse SAVs. This includes: (1) identifying novel SAVs through whole virome sequencing of wild African primate biomaterials; (2) the development and application of non-human primate induced-pluripotent stem cell (iPSC)-derived macrophages to isolate novel SAVs in cell targets from natural host species; and (3) detailed infection studies in human cells to evaluate human compatibility. Further, we will perform the first in-depth serosurvey for SAV exposure history using banked sera from a Ugandan case-control cohort. When taken together, this proposal will lead to a deeper understanding of the molecular biology and pathogenesis of these understudied viruses, as well as a greater appreciation for the zoonotic risk that they pose. It is imperative that we invest in characterizing the biology and pathogenesis of SAVs now so that we may begin to develop platform technologies (i.e., diagnostics, vaccines, therapeutics) in case they do emerge in the future.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Abstract No curative therapy exists for ulcerative colitis (UC) due to a critical gap in knowledge regarding the mechanism(s) driving chronic inflammation in UC. The rationale for this proposal is built upon emerging evidence that (1) CD90+ mesenchymal cells, known as myo-/fibroblasts (MFs), are critical to the pathophysiology of UC, although this has not been studied extensively; (2) JAK/STAT signaling is among the key pathways that drive inflammation in IBD; (3) microbiota/stem cell interplay is suggested to be a potential avenue for therapeutic improvement of inflammatory diseases. An increase in pathological type 2 and 17 immune responses by CD4+T and NKT cells, together with abnormal interferon (IFN) signaling, is a hallmark of the inflammation in UC. Our published and preliminary data show that, under gut homeostasis, MFs act as major immunosuppressors of T/NKT cell responses. By contrast, an increase in the inflammatory population of MFs occurs in UC (UC-MFs), supporting pathological T/NKT responses in UC. Thus, we propose that MFs are among the key cells in the pathogenesis of UC. However, the mechanisms responsible for the generation/activation of inflammatory UC-MFs are unknown. We reported that abnormal differentiation of mesenchymal stem cells (MSCs) to MFs occurs in UC. Our preliminary data demonstrated an increase in JAK2 expression and activity in the population of inflammatory MFs in UC. Our initial data suggest that this abnormally high Jak2 activity is key to the pathological responses of UC-MFs and that upregulation of JAK2 expression in UC-MFs is likely to occur during differentiation from tissue-resident MSCs in response to the dysbiotic microbial ligands. MSC therapy has shown promise for treatment of moderate-to-severe UC, but about 50% of patients relapse within the first five years post therapy; the cause of this relapse is unknown. We found that depletion of dysbiotic microbiota prior to MSC treatment shows improved outcome in a preclinical animal model of UC. Thus, we hypothesize that overexpression of JAK2 is key to the pathological activation of UC-MFs, that MyD88-dependent activation of MF progenitors (MSCs) by dysbiotic microbial ligands is a critical event in the generation of Jak2high UC- MFs, and these processes have potential as therapeutic targets. Three specific aims are proposed: (1) Define mechanism(s) by which overexpression of JAK2 contributes to the inflammatory activation of MFs in UC.; (2) Define the role of microbial ligand-dependent MyD88 signaling in the mechanism(s) of upregulation of JAK2 expression within progenitors of MFs and the generation of Jak2high UC-MFs; (3) Evaluate how microbial dysbiosis impacts MSC therapy effectiveness and MSC-mediated replacement of Jak2high UC-MFs in preclinical animal models of UC. We expect to define the novel mechanisms contributing to the pathological activation of mesenchymal cells in UC and to provide a scientific, preclinical basis for the development of specific pathway-mediated, combined mesenchymal cell/microbiota therapeutic approaches.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Myelodysplastic Syndromes (MDS) is a group of heterogenous bone marrow failure syndromes often seen with advancing age. Mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are the driving genetic alterations in over half of all MDS. These mutations have classically been linked to alternative splicing of oncogenes or tumor suppressors, but recent studies suggest broader defects including disruption of co-transcriptional splicing, which is the close functional coupling of transcription and splicing. Our group has recently shown that mutant SF3B1 impairs spliceosome assembly and slows RNA Polymerase II (Pol II) elongation, resulting in transcription-replication conflicts and replication stress. These changes reorganize chromatin, reducing promoter accessibility and histone marks. Notably, this model can explain the mutual exclusivity of SF mutations: cumulative transcriptional stress from multiple mutations is unsustainable for clonal expansion. In this application, we seek to define the role of HTATSF1, a protein with roles in both splicing and transcription, in transcriptional dysregulation in SF-mutant MDS. Our preliminary results show reduced interaction of HTATSF1 with mutant SF3B1. We hypothesize that this reduced binding of HTATSF1 to mutant SF3B1 impairs its recruitment to Pol II, disrupting the coordination between transcription and splicing. Conventional genome-wide assays lack spatial and temporal resolution to study the complexity of highly dynamic complexes such as spliceosome and Pol II. To overcome this, we will use multi-color, single-molecule imaging to resolve HTATSF1 recruitment and interaction kinetics in real time. We have leveraged CRISPR/Cas9 to introduce tags (degron for acute degradation and HaloTag for high resolution live-cell imaging in primary murine embryonic stem cells. Two aims are proposed to determine HTATSF1’s role in SF-mutant MDS. In the first aim, we will study how impairment of transcription noted in SF-mutant MDS is linked to HTATSF1. Total Internal Reflection Fluorescence (TIRF) microscopy will be used to track endogenously tagged HTATSF1 at a single-molecule resolution. In the second aim, we will determine HTATSF1’s role in altered splicing, a feature of SF-mutant We will utilize single-molecule imaging as well as differential phosphoproteomics in these studies. Ultimately, our findings may inform the development of therapies targeting transcriptional dysregulation, replication stress, and chromatin dysregulation in MDS.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of advanced non-small cell lung cancer (NSCLC), especially Anaplastic Lymphoma Kinase (ALK) inhibitors (ALKi), which have significantly improved progression-free survival in ALK-positive NSCLC patients. Despite these advances, ALKi treatment is associated with adverse cardiac effects, such as bradycardia and sinoatrial node (SAN) dysfunction (SAND), which pose serious risks, including sudden cardiac death. These adverse effects necessitate thorough evaluation of ALKi- induced cardiotoxicity to enhance patient safety and treatment efficacy. This K99/R00 proposal details a five-year research plan designed to uncover the molecular mechanisms of ALKi- induced SAND and to explore therapeutic interventions. The project leverages human induced pluripotent stem cell-derived sinoatrial node-like cells (iSANCs), engineered heart tissues (EHTs), and advanced single-cell omics techniques. Dr. Ren will test the hypothesis that ALKi induces SAND by disrupting Ca2+ and membrane clocks through off-target effects on other kinases and that SAN-specific restoration of disrupted protein kinase may counteract ALKi-SAND. In Aim 1 (K99), Dr. Ren will establish an in vitro model to recapitulate the clinical phenotype of ALKi-SAND at the single-cell level using iSANC. Single-cell RNA sequencing will then be performed on pooled iSANCs to uncover transcriptomic dysregulation associated with ALKi-induced SAND. In Aim 2 (K99), Dr. Ren will assess ALKi-induced electrical remodeling and identify impacts on SAN automaticity by investigating the effects on calcium and membrane clocks as well as exploring protein kinase’s potential to rescue observed phenotypes. In Aim 3 (R00), Dr. Ren will evaluate the therapeutic potential in ALKi-SAND in a more physiologically relevant settings with pacemaker EHTs and a mouse model. With the well-structured research training plan during the K99 phase, the support from esteemed mentors, advisors, and collaborators, as well as the outstanding environment at Stanford University, Dr. Ren will be equipped with advanced knowledge in stem cell biology, cardio-oncology, and bioengineering. The development of iPSC-derived pacemaker disease modeling and a population-scale platform for evaluating the impact of cancer drugs on the SAN (K99), as well as mechanistic insights and therapeutic strategies for TKI-induced SAN dysfunction (K99/R00), will enable Dr. Ren to conduct disease modeling and translational research specifically in cardio-oncology and cardiac pacemaker research. The new skills and experience gained during this K99/R00 career development award, combined with Dr. Ren’s prior expertise in cardiac electrophysiology and pacemaker biology, will facilitate Dr. Ren’s transition to an independent career conducting basic and translational research in cardio-oncology.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Stem cells are required for tissue homeostasis and regeneration. Accomplishing these tasks requires intimate association with a niche, a cellular microenvironment that forms in a specialized tissue location with precise morphology to enable communication with stem cells. Niches are formed by cells that must be specified for niche identity, receive signals directing migration to the appropriate tissue compartment, and respond to those cues with changes in gene expression and cytoskeletal behavior. Studying this has proven challenging, as most niches are established during embryogenesis when the tissue is inaccessible to live imaging. Under previous GM funding, my lab work established an in vivo live imaging method to enable exploration of each of these facets in the assembling Drosophila testis niche, a tractable and conserved model. Foundational studies in the adult testis have repeatedly unveiled concepts that apply to other systems, yet before my work, we did not know how this niche formed. Our method permits direct in vivo visualization, revealing discreet steps of morphogenesis. This application will perform lab work to investigate the underlying mechanisms for each step. We ask (1) How are niche cells specified? (2) How do regulators of the cytoskeleton enable niche morphogenesis? and (3) What signals direct the location of niche assembly? My previous work showed that signals from adjacent visceral muscle (Vm) are required to assemble the testis niche during embryogenesis. In response to signals, niche cells express the transcription factor islet (mammalian ortholog, Isl1), which I found polarizes F-actin and regulates anterior niche assembly. An open question is whether F-actin polarization directly enables niche morphogenesis, or if it is polarized in consequence of niche assembly. This application will harness our in vivo imaging protocol along with an incisive optogenetic approach to test direct contributions of cytoskeletal regulators in each step of niche development. Our unpublished work supported by GM R15 funds has further shown that Vm cues induce Tbx1 ortholog org-1 expression to influence niche establishment. This proposal will define genetic regulatory mechanisms through which Tbx1 regulates niche identity and morphogenesis. Finally, our data reveal that a gonad-intrinsic, non-niche cell population is guiding niche morphogenesis in concert with signals from adjacent visceral muscle. This represents a novel mechanism for niche development, which we will uncover in this application. Our work will combine the power of Drosophila genetics with incisive assays in cellular mechanics, including live in vivo imaging, optogenetics for precise temporal manipulation of the niche cortical cytoskeleton, and laser ablation to define underlying forces driving niche and stem cell behavior. Mechanisms we unveil in this model will reveal mechanics of niche establishment required to form a compartmentalized niche with appropriate cellular architecture to enable tissue function.