Ut Southwestern Medical Center

NIH Research Projects · FY 2024 · 2020-09

Project Summary Humans are under increasing threat from viruses that spill over from animal reservoirs. Most of these viral diseases lack targeted treatments. We speculate that this unmet medical need might be addressable by first understanding the evolutionary principles underlying antiviral immune responses. A major component of innate immunity in mammals is the interferon response. Interferon induces hundreds of genes, many of which encode effector proteins that suppress viral infection. In mammals, these antiviral effectors are rapidly evolving, most likely in response to the genetic "arms race" continually occurring between virus and host. Further, certain mammalian orders, such as primates, rodents, bats, and carnivores, are particularly rich in viruses that have the potential to spill over into humans. We hypothesize that the genomes of these viral zoonotic reservoirs encode unique antiviral effectors that may be harnessed to combat human viral pathogens. The goals of this project are to identify, characterize, and validate the efficacy of novel antiviral proteins from diverse non-model mammalian species. State-of-the-art genetic screening platforms will be used to discover antiviral genes in primary cell cultures obtained from multiples species in the following mammalian orders: Primate, Rodentia, Chiroptera, and Carnivora. Validated effectors will be characterized mechanistically with a suite of virological, biochemical, molecular, and cell biological approaches. The potential for these effectors to suppress human viruses will be tested in murine models of human viral disease. Outcomes will include the creation of the first comprehensive Mammalian Antiviral Protein Atlas, the discovery of new genetically-encoded antiviral mechanisms, and proof-of- concept that human viral disease can be thwarted by naturally occurring proteins from other species. The impact of this proposal will be the development of a new area of biomedical research at the intersection of virology, immunology, and evolutionary biology. Long term prospects include harnessing the results of these studies to inspire alternative approaches to antiviral drug development.

NIH Research Projects · FY 2024 · 2020-09

Abstract Metabolic dysfunction is one of the major factors that impact lifespan in all systems. Mitochondrial defects are known to contribute to tissue dysfunction during aging. Alterations in carbohydrate metabolism, lipid oxidation, and redox metabolism have all been shown to play significant roles in many processes that can help dictate lifespan. One major factor that can dictate human lifespan and aging is the onset of metabolic syndrome. Over the past 30 years there has been a dramatic rise in the prevalence of metabolic disease and currently 1/3 of people world-wide suffer from metabolic syndrome. While genetics, environment, and nutrition play important roles in metabolic disfunction and lifespan, many recent studies have shown that disruptions in maternal metabolism can have a profound impact on progeny physiology and aging. While many studies have examined chromatin state and small RNAs to explain the heritability that maternal metabolism has on progeny disease these studies, in fact, support the idea that other factors contribute to the heritability of metabolic syndrome. Unlike sperm, that only contribute DNA to the early embryo, the oocyte provides a complex stockpile of metabolites, stored nutrients, and mitochondria to the progeny. Our research exploits the Drosophila oogenesis system as a tool to isolate large amounts of staged oocytes and embryos to conduct in-depth biochemical and metabolomics studies of the mechanisms that regulate oocyte physiology and metabolism. These tools combined with the speed and power of Drosophila genetics allow us to identify and characterize biochemical mechanisms in the oocyte that impact progeny metabolism. In this proposal we will examine how changes in systemic metabolism in aged mothers impact the reprogramming of progeny physiology and metabolism. In addition, we will examine whether reprogrammed progeny exhibit alterations to the metabolic shifts that occur normally during aging. We will test whether insulin-mediated changes in oocyte redox metabolism provides a signal that reprograms progeny physiology. We will also define the changes in chromatin landscape that underlie the transcriptional shift we observed in reprogrammed progeny. Our long-term goal is to use these studies to provide a mechanistic platform to study metabolic reprogramming in other systems, such as mice and mammalian cell models, and how it impacts progeny physiology and aging. Overall, this proposal challenges the dogmatic ideas we all have about the heritability of disease and explores the novel concept that changes in oocyte metabolism can reprogram progeny physiology and metabolism during aging.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Placenta Accreta Spectrum (PAS) disorder is a condition in which the placenta fails to separate partially or totally from the uterine wall, resulting in significant intrapartum maternal morbidity and mortality at delivery. Depending on the extent of the placental invasion into surrounding tissue, hysterectomy may be lifesaving. Assessment of PAS severity prior to delivery is crucial for multidisciplinary surgical planning and patient counseling. However, there is an unmet clinical need for quantitative, objective measures of abnormal placentation that can serve as predictors of risk of invasion and hysterectomy. The overall goal of the proposed project is to prospectively assess PAS in high-risk women across gestation using quantitative multi-parametric magnetic resonance (MR) imaging and automated textural radiomics and to correlate findings with surgical outcomes. Coming from a highly interdisciplinary background of MR chemistry, biology, and physics, the candidate's long-term career goal is to become an independent investigator with in-depth knowledge and skills to conduct translational placental imaging research. The candidate's short-term goal is to complete a pilot study under the mentorship of an interdisciplinary advisory committee to evaluate PAS in women with high-risk pregnancies using multi-parametric MRI and textural radiomics. To meet these goals, the following research aims are proposed: 1) Evaluate placental invasion across gestation using multi-parametric MR imaging and deep learning based novel quantification tools in PAS high- risk pregnancies, and 2) Extract the radiomic pattern across gestation of high-risk PAS placenta and determine its association with clinical surgical outcome. These research aims will serve as the platform for the career development plan and training aims which include: 1) Gain knowledge in translational prospective research design, 2) Improve technical skills in advanced clinical MR acquisition development, 3) Acquire applied statistical and data analysis skills, and 4) Develop mentorship and leadership skills while under training. Together, the research and training aims will provide the training, experience and preliminary data for an R01 application. The future R01 will develop a clinical and surgical outcome prediction model for high-risk PAS pregnancies, potentially improving maternal and fetal outcomes. The training will position the candidate to become a leader in translational placental imaging research.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Alzheimer disease (AD) and related tauopathies are major diagnostic, therapeutic, and clinical challenges. Our previous work suggest that different tauopathies are based on unique tau aggregate conformations, or “strains,” and that strain identity predicts specific disease progression patterns in animal models. We hypothesize that distinct, self-propagating tau strains can move between cells of the brain to propagate pathology in humans. Recent publications from our group indicate that tau monomer in fact encodes strains. However this has not been rigorously tested across tauopathies. We will test this idea first by determining whether tau monomer encodes the same sets of strains encoded by larger assemblies in human brain (Aim 1). Next we will use multiplex biosensors to classify monomer from human tauopathies, and determine whether monomer can classify individuals in correlation with neuropathology with the same fidelity as larger assemblies. We will also test the fidelity of strain propagation in human neurons vs. HEK293 cells in comparison those strains isolated directly from human brain (Aim 2). Finally, we use structural biology approaches to determine the conformations of seed-competent monomer. In particular, we will determine whether local structures in tau predict its subsequent assembly into fibrils. This will be based on crosslinking mass spectrometry, molecular modeling, cryoEM, and microcrystallography. We predict that tau monomer will encode strain composition across tauopathies. If we are successful, we anticipate ultimately that it will be possible to classify tauopathies based on the conformation with tau monomer. This could have important implications for accurate diagnosis and personalized therapy.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Circadian rhythms are fundamental to living organisms. The mammalian circadian system, which controls daily rhythms of behavior and physiology, is a multi-oscillatory system composed of a master pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) and many other oscillators in peripheral organs. However, the SCN is not the only pacemaker. When arousing stimuli are present, circadian behavior rhythms are observed even when circadian clocks in the SCN and peripheral tissues are disabled. The molecular mechanisms, anatomical loci, and functional significance of these extra-SCN pacemakers are not known. We have assembled molecular and imaging toolsets and technologies to identify the neural circuitry and physiological outputs of those extra-SCN pacemakers. The proposed studies will uncover the functional significance of the enigmatic extra-SCN circadian pacemakers in mammals. The discovery of the loci and physiological roles of the extra-SCN pacemakers will expand our understanding of the molecular and physiological processes of the circadian system. Importantly, our studies will investigate how the SCN and extra-SCN pacemakers interact to regulate feeding and sleep. Disruption of circadian rhythms and sleep by shiftwork and exposure to artificial light at night increases the risk of human diseases. In addition, sleep and circadian rhythms are impaired in several neurological disorders and in persons with drug addictions. Therefore, understanding how extra-SCN pacemakers control circadian rhythms will elucidate novel processes that could be manipulated to manage circadian disruption in humans.

NIH Research Projects · FY 2024 · 2020-09

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, and currently has no FDA-approved therapies for treatment. While the pathophysiology of NAFLD is complex, mounting evidence supports a central role for chronic inflammation in disrupting insulin signaling and normal liver metabolic function. Our understanding, however, of the transcriptional mechanisms by which inflammation triggers metabolic dysfunction remains incomplete. Interferon regulatory factors (IRFs) are transcription factors that have been implicated in nearly all aspect of immune function. Our laboratory has identified and characterized numerous points of crosstalk between IRFs and the metabolic effects of overnutrition, including the discovery that IRF3 controls adipose tissue inflammation and thermogenesis. Here, we show that high fat diet activates liver IRF3, and that whole body deficiency in IRF3 protects against both hepatic insulin resistance and steatosis. By generating mice carrying a floxed or constitutively active Irf3 allele, we demonstrate that obesity-induced liver IRF3 activation operates in a 2-cell model, where hepatocyte IRF3 establishes insulin resistance and macrophage IRF3 promotes steatosis. We hypothesize that IRF3 sits at a critical junction between metabolic and inflammatory responses in the liver, and plays a causal role as a transcriptional regulator in the development of hepatic insulin resistance and steatosis in NAFLD. The scientific aims of this K08 are to 1) identify how hepatocyte IRF3 regulates insulin signaling, and 2) to define the precise role of Kupffer cell IRF3 in promoting steatosis. The long-term goal of these studies is to improve the metabolic health of patients by manipulating inflammatory pathways and transcriptional programs in the liver.

NIH Research Projects · FY 2025 · 2020-09

Project Summary: Epidural/spinal administration of analgesics such as opioids, ziconotide and local anesthetics have profound efficacy in some of the most intractable pain conditions such as severe neuropathic pain after failed back surgery, cancer pain and post-operative pain after major abdominal/thoracic surgeries. Despite their profound efficacy, their use is limited primarily because of the side effects such as tolerance, granuloma, psychosis and motor block. Discovery and validation of new spinal analgesic targets for development of therapeutics is urgently needed. Here we propose to validate a novel spinal analgesic target, neurotensin receptor 2 (NTSR2), based upon our mechanistic studies of Contulakin-G (CGX), that has shown preliminary efficacy in humans suffering from one of the hardest to treat neuropathic pain condition-spinal cord injury associated pain. CGX is a snail venom derived peptide that has homology with mammalian neurotensin and was shown to be safe in humans. A small, pilot Phase1A study demonstrated analgesic effect in some patients with spinal cord injury-associated pain. Although, CGX does not have favorable pharmacokinetic properties, these studies suggested a possibility of a novel, non-opioid, analgesic mechanism that is active in humans. Our preliminary studies suggest CGX produces its analgesic actions via activation of spinal neurotensin receptor 2 (NTSR2) and subsequent inhibition of voltage-gated calcium channels. NTSR2 is highly expressed in small/medium size sensory neurons in rodents and co-expressed with voltage gated calcium channels. Transcriptomics confirmed NTSR2 expression in human dorsal root ganglia sensory neurons. Importantly, our pilot studies show that NTSR2 activation by CGX produces profound analgesia and is not associated with unwarranted side effects such as rapid tolerance or motor blockade. Preliminary data thus support a role of spinal NTSR2 in pain modulation, but validation of this receptor as an analgesic target has not been done. In this project, we propose to perform a robust validation of spinal NTSR2 as an analgesic target utilizing three species of both sexes (rat, mice and human), two models (neuropathic pain and post-surgical pain), pharmacological (SA1) and state of the art genetic tools such as CRISPR-Cas9 editing (SA2) and assessment of both sensory and affective measures of pain. Moreover, we propose a rigorous, two-site parallel confirmation study (SA3) designed after multisite clinical trials to further authenticate spinal NTSR2 as an analgesic target. If successful, proposed studies could lead to a development of non-opioid spinal analgesic that has high translational potential.

NIH Research Projects · FY 2024 · 2020-09

Project Summary: Many human diseases are caused by a dysregulation of lipid metabolism, including atherosclerosis, cancer, neurodegeneration, diabetes, and fatty liver. The development of effective treatments for lipid related disorders is hinder by a lack of modern in vivo biochemistry techniques for studying lipid metabolism. The overall goal of this proposal is to develop tools and protocol to measure the rates of lipid biosynthesis and remodeling by stable isotope labeling with sensitivity comparable to radio-isotope tracing with the specificity and broad coverage of modern mass spectrometry based lipidomics. This is enabled by an ultra-high resolution orbitrap mass spectrometer I developed in collaboration of Thermo Scientific, now commercially available as the Lumos 1M. This instrument has sufficient resolution to resolve the natural abundance 13C from a tracer isotope, for example 2H, in intact lipid ions. By resolving the dominant natural abundance ions from tracer isotopes will improve the signal to noise ratio by at least 2 orders of magnitude (1:1 vs >1:100) and increase the dynamic range. This advancement will allow in vivo analysis of lipid metabolism to study a variety of disease, and will ultimately lead to lipid fluxomics analysis that is translatable to human studies. By measuring lipid flux in patients we will be able to directly studying the progression of metabolic syndrome, potentially circumventing the need for animal models, and measure the effectiveness of therapies and interventions. To facilitate the development and widespread implementation of this technology, I will address the fundamental roadblocks to adapting this technology. Firstly, the commercial instrument is engineered for proteomics applications, in particular the electrospray ionization source. By working with the manufacturer and translating my lipidomics experience to this new platform I will overcome these issue. Secondly, I will develop novel data collection approaches for both chromatography and direct infusion based applications to accommodate the long transient time and coalescence issues associated with ultra-high resolution resonance based mass spectrometry. Thirdly, software tools will be developed to extract ultra-high resolution data in a time efficient manner, convert the data to physically interpretable parameters, and map data onto biochemical pathways. Lastly, I will develop protocols and platforms for stable isotope labeling by deuterium labeled water (D2O) and other isotope labeled metabolic tracers in mouse models of metabolic syndrome relevant to my lab’s research program studying the mechanism for fat accumulation. By accomplishing these aims this technology will be accessible to the biomedical research community. My multi-disciplinary training in engineering, physical, analytical and biochemistry, and mouse genetics makes me well-suited to develop this technology and the lipid centric research environment at UT Southwestern is the ideal location for the initial application.

NIH Research Projects · FY 2025 · 2020-09

The 2019 U.S. practice guidelines recommend direct oral anticoagulants (DOACs) such as dabigatran, rivarox- aban, and apixaban over warfarin for stroke prevention in high-risk patients with atrial fibrillation (AF). However, the selection of a DOAC can be challenging for older ischemic stroke patients. The risk-benefit ratios in second- ary stroke prevention differ substantially from those in primary prevention. Not only are older stroke survivors at increased risk for recurrent ischemic events, older age and history of ischemic stroke are also major risk factors for bleeding complications. While it is known that drug selection should be individualized, it remains unknown how to tailor anticoagulant therapy according to the effectiveness/safety of the agents and patient unique char- acteristics. The long-term goal is to use comparative effectiveness research to improve quality of care and out- comes in patients with cardiovascular disease and stroke. Leveraging the American Heart Association (AHA) Get With The Guidelines-Stroke Registry (GWTG-Stroke) and Medicare inpatient and Part D database, the over- all objective of this application is to develop evidence-based strategies to improve appropriate anticoagulant therapy for secondary prevention in older ischemic stroke patients with AF. The central hypothesis is that differ- ences exist between DOACs in terms of effectiveness and safety, which have direct implications for therapeutic selection. Once the relative effectiveness/safety is known, the selection of an anticoagulant can be made based on patient risk profiles, making treatment safer and more effective. Guided by strong preliminary data, this hy- pothesis will be tested by pursuing two specific aims: 1) Determine the long-term clinical effectiveness and safety of different DOACs for secondary prevention in older ischemic stroke patients with AF; 2) Investigate DOACs dosing patterns and evaluate the potential impact of underdosing or overdosing on long-term outcomes. The proposed research is innovative in four key ways: 1) A patient-centered approach is used to address a real-life decisional dilemma facing stroke survivors and clinicians; 2) It shifts focus from selected samples in clinical trials to a nationwide representative stroke population, including traditionally underrepresented subgroups in commu- nity practice; 3) A novel propensity score inverse probability weighting method using generalized boosted models (a machine learning technique) will be employed to mimic a trial-type multi-treatment design, uncover treatment heterogeneity, and minimize selection bias in observational data; 4) Beyond traditional mechanisms of scientific publications, the evidence generated from this study will be disseminated to stroke survivors, clinicians, and relevant stakeholders through the AHA GWTG-led national quality initiatives and patient-led efforts to ensure a rapid translation of seminal findings into clinical practice. The proposed research is significant because it is expected to help guide personalized anticoagulant therapy in older ischemic stroke survivors that could best meet their needs and lead to better outcomes most meaningful to patients. Ultimately, such knowledge has the potential to inform evidence-based treatment decisions in stroke that now afflicts more than 7.2 million Americans.

NIH Research Projects · FY 2024 · 2020-09

Project Summary While survival rates for critically ill patients during their acute illness continues to improve, the long-term outcomes for these patients are dismal. When ICU survivors progress to a state of chronic critical illness (≥14 days in the ICU), the majority develop significant disability and prolonged recovery times fraught with multiple complications. Ultimately, about 50% eventually succumb to their illness at one year. Unfortunately, once patients progress to a state of chronic critical illness, there is nothing more than supportive care. Given that the healthcare costs required to manage these patients is estimated to be over $20 billion per year, chronic critical illness is creating a new health care crisis. As an ICU physician and basic science researcher, I have the unique ability to both manage these patients at the bedside and actively investigate new ways to improve their care. With all the effort invested in these patients to help them survive their acute illness, it seems like a failure that we are not able to help them long term. The greatest risk factor for the progression to chronic critical illness is the profound immunosuppression that can occur. In the normal response to injury or infection, both the innate and adaptive immune systems deactivate once the inflammatory insult is cleared. However, with a significant insult, such as trauma or sepsis, deactivated immune cells are replaced by more immature cells from the bone marrow that have decreased or suppressive activity and may be unable to effectively eradicate the source. Many have been able to show this immunosuppressed state in critically ill patients, but no one has been able to reverse this response. This proposal synergizes both human and animal studies to better understand the diversities on how individuals respond to a significant injury or infection and attempt to reverse any immunosuppression that may occur. In the first project, four different murine models of injury or infection are utilized and the immune profile of each individual mouse is identified. If they display any type of suppression in their innate or adaptive immune system, or both, specific reversal agents are administered to determine if we can improve their immune response. In the second project, blood samples from critically ill patients admitted to the Surgical ICU are analyzed to determine their immune profiles over the first two weeks of admission. Univariate and multivariate regression analyses will then be performed to determine which immune phenotypes are associated with worse outcomes, with the primary outcome being chronic critical illness. Successful completion of these studies will significantly advance the field of immune phenotypes and set the groundwork for developing individualized immune therapies for a variety of critically ill patients.

NIH Research Projects · FY 2024 · 2020-09

SPRINT, and its accompanying cognitive-focused substudy SPRINT-MIND recently showed that blood pressure control (goal SBP<120 mm Hg) reduced incident mild cognitive impairment as well as a combined outcome of MCI and dementia. Divergence in opinion among experts and professional societies about BP goals still remain due to the concerns of potential harms. There is a clear need to implement new practical approaches to control blood pressure in clinical practice and test their effectiveness. Pragmatic clinical trials embedded in health systems (ePCTs) offer a unique opportunity to study the effectiveness of implementation of evidence- based interventions in real-world clinical settings. Our team is currently conducting ICD-Pieces (NCT02587936), the largest ePCT in patients with the coexistent chronic conditions of chronic kidney disease, hypertension and diabetes as part of a demonstration project in the NIH Health Care Systems Research Collaboratory. We now propose the Preventing Cognitive Decline by Reducing BP Target Trial (PCOT), to examine the effects of lowering BP to less than 130/80 upon the incidence of cognitive decline. Our main hypothesis is that patients who receive care with a collaboratory model that combines clinical decision support applied to home BPs and team-based care delivered in primary care practices will have better blood pressure control and a lower incidence of mild cognitive impairment and dementia than patients receiving usual medical care. In this ePCT (1) Will compare the effects of intensive BP control between the intervention and usual care arm on the rate of cognitive decline measuring the change in TICS-m per year. We will recruit 4,000 patients over 70 years of age with BP >130/80 mmHg from 2 diverse health systems and randomize patients within each health system to usual care or to a combination of care with clinical decision support using home BPs and practice facilitators and Pharm Ds to lower home BP to < 130/80 mmHg. The primary outcome will be development cognitive decline as determined by a decrease in TICS-m scores from baseline; (2) Determine the potential harms of intensive lowering BP home BP below 130/80 mmHg with usual care on hospitalizations, emergency department visits, cardiovascular events, deaths, syncope, falls, fractures, hypotension, electrolyte abnormalities and acute kidney injury and; (3) Determine the impact of intensive BP management on QOL with scores obtained using the PROMIS Scale v1.2 - Global Health instrument annually This trial is pragmatic, with broad inclusion criteria and evidence-based interventions informed by patients and delivered in primary care settings by the clinical teams in health systems serving ethnically and socioeconomically diverse population. Lessons from this trial should provide valuable insights to guide clinical practices in BP control and cognitive assessments in real-world settings as well as design and implementation of future pragmatic trials. 1

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Anorexia of acute illness has traditionally been considered a maladaptive response in the face of a presumed hyper-catabolic state. Surprisingly, we found that anorexia is protective in bacterial sepsis. Glucose supplementation during the period of anorexia induced by bacterial sepsis is detrimental and promotes mortality, even in the absence of live pathogen as in the mouse model of lipopolysaccharide (LPS) sepsis. Core fasting metabolic pathways activated in LPS sepsis, including liberation of free fatty acids, ketogenesis, and production of fibroblast growth factor-21 (FGF21), an endocrine FGF hormone that mediates adaptive responses to metabolic stresses such as starvation, are suppressed by glucose supplementation. Knockout mice that are deficient in FGF21 or in peroxisome proliferator-activated receptor alpha, which cannot produce FGF21 or ketones, are more susceptible to bacterial sepsis. We have also found that similar to normal fasting responses, lipid droplets accumulate in the liver and kidney during bacterial sepsis. Emerging evidence suggests that lipid droplets may in fact reflect protective mechanisms against cellular stress rather than lipotoxicity. Based on our preliminary data, we hypothesize that components of fasting metabolism are integral protective mechanisms that support survival and tissue protection during bacterial sepsis. Over the next five years, key goals for the Huen laboratory are to determine whether and how components of fasting metabolism: 1) FGF21, 2) ketogenesis, and 3) lipid droplet formation, are protective in bacterial sepsis. Proposed studies include using pharmacologic targeting and genetic mouse models of tissue-specific deletion of key components of these metabolic processes. Interdisciplinary methods will be used to investigate the interactive physiology between the innate immune system and metabolic organs, in order to elucidate the complex interactions between multiple organ systems including the brain, liver, kidney and heart as part of the adaptive response to bacterial sepsis. The overarching objectives of our proposed studies aim to differentiate between pathologic and protective metabolic pathways in bacterial sepsis, interrogate the beneficial aspects of fasting metabolism that support survival, and elucidate the mechanisms of action.

NIH Research Projects · FY 2024 · 2020-09

A major challenge of treating traumatic brain injury (TBI) patients is the simultaneously occurring complex secondary injury processes following the primary injury. The secondary events such as cerebral hyperglycolysis and mitochondrial failure develop over minutes to months after the primary injury, providing a potential window of opportunity for therapeutic intervention. Given early, this intervention may prevent or reduce secondary brain damage, directly impacting long-term patient outcome. Therefore, the noninvasive detection and characterization of pathophysiology in TBI patients during the acute and early sub-acute stages, will have critical clinical implications for the early diagnosis of individuals with the highest risk of poor neurological outcomes and will be vital for identifying and developing effective therapies. While a number of pathological alternations in TBI are potential biomarkers, no current clinical imaging modalities are sensitive enough to be routinely used to detect the details of metabolic shifts in brain sub-regions with secondary injury. Magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C-labeled substrates provides unique noninvasive measurements of critical in vivo dynamic metabolic processes. In particular, pyruvate occupies a key nodal point in cerebral energy metabolism, among the fates of [1-13C]pyruvate are reduction to lactate as the end product of glycolysis, conversion in mitochondria to form acetyl-CoA and CO2 (detected as HCO3–) via pyruvate dehydrogenase (PDH) flux or anaplerotic pyruvate carboxylase (PC) pathway for oxidative phosphorylation. [2-13C]pyruvate, on the other hand, directly assess the tricarboxylic acid (TCA) cycle by detecting [5-13C]glutamate production. While our preliminary data demonstrated increased lactate and decreased HCO3– (bicarbonate) production from hyperpolarized [1-13C]pyruvate in a rat TBI model and acute TBI patients, however, the role of [13C]HCO3– as a TCA cycle marker needs further verification due to the high pyruvate carboxylation. Another key metabolic alteration following TBI is increased acetate oxidation in astrocytes, playing a neuro-protective role. The increased acetate metabolism tightly interacts with pyruvate metabolism, and thus, should be considered together when interpreting [13C]pyruvate metabolism. The fundamental goal of this project is to understand how TBI influences the in vivo cellular metabolism in the brain using hyperpolarized 13C MRSI as a step towards personalizing therapy for TBI patients. In this proposal, a comprehensive analysis of TBI metabolism will be performed using a rat TBI model by comparing the in vivo imaging results with ex vivo tissue analysis. First, we will develop hyperpolarized [2-13C]pyruvate as a probe to directly measure the altered TCA cycle activity in TBI (aim 1). Second, we will assess the contribution of increased acetate metabolism to pyruvate oxidation in a rat TBI model (aim 2). The longitudinal in vivo imaging data (aims 1&2) will be validated by cross-sectional ex vivo NMR isotopomer analysis of freeze-clamped brain tissues. Finally, we will translate the technique to assess metabolic changes in acute mild TBI patients (aim 3).

NIH Research Projects · FY 2024 · 2020-09

Neuron loss is a frequent result of neural injury or degeneration. A fundamental but unresolved challenge is how to restore the lost neurons and repair the neural circuits in the adult central nervous system. Stem cell-based transplantation has limitations on immune compatibility, neuronal survival, and functional integration; and it has the potential for tumorigenesis. The long-term goal of this proposal is to define innovative regenerative strategies by using a patient’s own scar-forming cells without cell transplantation. In response to injury or neural degeneration, glial cells become reactive, proliferate, and form scars. Scar formation is initially beneficial by restricting damage but ultimately detrimental to neural regeneration through acting as a physical and chemical barrier to axonal regeneration and growth. Our prior research showed that reactive astrocytes can be in vivo reprogrammed to expandable neural progenitors, which can further produce mature neurons in the adult central nervous system. The current proposal will focus on NG2 glia, another major component of the glial scar. Our preliminary data indicate that they can be in vivo reprogrammed to become neurogenic. We here propose to determine the molecular and cellular mechanisms underlying the reprogramming process of NG2 glia in adult mouse. We will also examine the excitability and synaptic integrations of the induced neurons from NG2 glia. Finally, we will investigate the biological function of these newly generated neurons in a mouse model of neural injury. Results from this work may lead to paradigm-shifting regeneration-based therapeutic strategies for neural injury and degeneration.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Mitochondria are double membrane-bound organelles that perform many crucial cellular functions, including nucleotide and amino acid metabolism, cellular Ca2+ homeostasis, and their most well-known function, generation of cellular energy via oxidative phosphorylation. To perform these diverse functions, the mitochondrial interior is elaborately physically organized. Mitochondria are also distributed throughout cells as part of a dynamic, semi-continuous network that divides and fuses in part to distribute the mitochondrial genome. Correct mitochondrial organization and distribution are critical for cellular function, and defects in processes that maintain mitochondrial architecture can result in a large number of diseases, including neurodegeneration and diabetes. Despite the complicated nature of mitochondrial structure, we have a minimal mechanistic understanding of how cellular metabolic needs are communicated to the interior of the organelle and how mitochondrial organization is dynamically modulated to meet this demand. In the next project period, our goal is to address this deficit by studying three distinct aspects of mitochondrial communication and internal remodeling. We will examine how mitochondrial structure is coordinated across the two mitochondrial membranes during mitochondrial fission (Direction 1), how mitochondria internal organization is remodeled to support local subcellular metabolic demand (Direction 2), and how the interior of the organelle can sense and respond to acute localized stress and communicate this dysfunction to external quality control machinery (Direction 3). This work will lead to insight into the spatial organization and form-function relationship of mitochondria and give us molecular understanding of the disorganization and dysfunction of mitochondrial membranes that frequently occurs in human disease.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Alcohol abuse and related diseases exact a staggering health and economic toll, and thus there is a dire need for novel therapeutic approaches. The hormone FGF21, which is rapidly and robustly induced in liver in response to ethanol exposure, is an exciting new pharmaceutical candidate. FGF21 acts on the brain to suppress alcohol consumption and to stimulate water drinking. In this application, we propose that FGF21 mediates these protective effects by acting directly on neurons expressing the neuropeptide, neurotensin, which is a well-established regulator of ethanol and water intake. We further propose that liver-derived FGF21 also protects against ethanol-induced hypothermia and liver injury by activating the sympathetic nervous system and stimulating thermogenesis in brown adipose tissue. We will test these hypotheses in a series of experiments that employ our unique collection of genetically-engineered mouse models, including mice selectively lacking FGF21’s obligate co-receptor -Klotho in neurotensin neurons. We anticipate that these studies will provide important insights into the tissues and underlying mechanisms whereby FGF21 protects against ethanol-induced toxicity. Moreover, since FGF21 is already in clinical trials for metabolic disease-related indications, these studies will aid in determining whether FGF21 can be repurposed pharmaceutically for treating alcohol abuse and its associated pathologies.

NIH Research Projects · FY 2024 · 2020-08

Project Summary: Proline is a multifunctional imino acid with myriad uses in the cell. Aside from direct incorporation into protein, proline can be metabolized via a process known as the proline cycle. Here, proline is oxidized by proline oxidase (PRODH) to form D1-pyrroline-5-carboxylate (P5C). PRODH is a FAD+ dependent enzyme that donates electrons to complex II of the mitorchondrial electron transport chain thus coupling proline oxidation to ATP synthesis. P5C is converted back into proline by the NADH dependent enzyme pyrroline-5-carboxylate reductase (PYCR) to provide reducing power for glycolysis and the pentose phosphate pathways. It is unknown how osteoblasts obtain proline, how proline uptake is regulated, or if and when the proline cycle is required during differentiation. Osteoblasts express a diverse array of membrane-tethered amino acid transporters to facilitate proline uptake. We have identified the system A neutral amino acid transporter SNAT2 (encoded by Slc38a2) as the most highly expressed putative proline transporter in osteoblasts. Our preliminary data indicates WNT stimulates proline uptake through SNAT2 that is necessary for osteoblast differentiation in vitro. Moreover, mice homozygous for a null allele of Slc38a2 (Slc38a2-/-) have defects in endochondral ossification. In this proposal, we will 1) establish the necessity of proline uptake through Slc38a2/SNAT2 to regulate osteoblast differentiation and bone formation in vivo, 2) determine how SNAT2 activity is regulated by WNT signaling and 3) elucidate the necessity of proline metabolism via the proline cycle in differentiating osteoblasts. Our findings will have broad implications in bone development, maintenance of bone mass, skeletal repair and regeneration.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT The purpose of this K01 proposal is to facilitate the applicant's transition to an independent research career focused on stress and sex hormone biology to understand mechanisms of sex differences in cardiovascular disease and potential disparities among women. Training components for the proposed research will include direct clinical training in stress testing and vascular physiology, new training in sex hormones and measures of ovarian function, faculty career development, active mentoring, and completion of the proposed research. The premise of this research project is based on preliminary data from the applicant's own research which suggest that compared to men, women (and particularly, younger women) with coronary heart disease have distinct vascular mechanisms in relation to stress and myocardial ischemia, and have higher basal and stress-related inflammation. We have also reported that women ≤ 60 years of age are more likely to develop myocardial ischemia during mental stress than men of similar age. The underlying explanation for these sex differences is unknown; however, one potential mechanism involves sex hormone biology among premenopausal women and across the menopausal transition. Stress may impair female reproduction through diminished ovarian reserve and induce earlier age at menopause, which have been associated with cardiovascular risk, possibly through sex hormone-related pathways influencing immune response, vascular, and cardiac function. The overall goal of this project is to understand mechanisms for sex differences in inflammatory and vascular responses to mental stress, and the role of ovarian function and menopausal status among women. The applicant will leverage the infrastructure of the Myocardial Infarction and Mental Stress Study 2 (MIMS2: R01HL109413), which was recently renewed for a new enrollment wave (MIMS3: 2R01HL109413). The two waves will total 300 men and 300 women with a recent myocardial infarction (MI), ≤ 60 years of age. The applicant will add the collection of data on anti-Müllerian Hormone (AMH), a biomarker of ovarian reserve and menopausal status in women and examine AMH levels in relation to stress responses. The applicant will also recruit a sample of healthy women (n = 100) matched for age to the post-MI women, for comparison of AMH levels and age at menopause. The scientific aims of this project are to: 1) Examine sex differences in inflammation and vascular function at baseline and in response to mental stress and the role of gonadal aging by comparing young and middle-aged women and men; 2) Examine and compare inflammatory and vascular profiles and gonadal aging and age at menopause with age-matched control women; and 3) Examine whether psychosocial stressors such as depression, early life adversity, discrimination, and neighborhood disadvantage are associated with gonadal aging (lower AMH levels) in women with a recent MI and age matched controls. The new enrollment of patients and controls will provide key opportunities for training and data collection on gonadal aging and vascular function to inform the development of a future NIH-R01 proposal.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Small cell lung cancer (SCLC) afflicts more than 30,000 patients per year and is rapidly fatal in 95% of cases, with median survival is less than one year. Belying this grim prognosis, treatment-naive SCLC is highly sensitive to chemotherapy. However, relapse is nearly inevitable, and relapsed SCLC presents two obstacles that have been insurmountable for at least 30 years: cross-resistance to chemotherapy, and absence of biomarker-driven targeted therapy. Following relapse, resistance often extends beyond etoposide/platinum (EP) to other DNA damaging agents. Although topotecan is the only approved second-line therapy for SCLC, the NCCN guidelines list 10 agents of roughly equivalent efficacy. None are particularly effective in unselected patients, and a disease that was once highly chemosensitive becomes inexorably progressive. However, the molecular determinants of cross- resistance in SCLC remain unclear. Although critically important, cross-resistance is difficult to study experimentally, as it requires a model system that faithfully reproduces clinical outcomes, and is adequately powered to capture inter-tumoral molecular heterogeneity across a population of patients. We have generated a panel of 44 SCLC patient-derived xenograft models (PDXs) from biopsy specimens and circulating tumor cells (CTCs). Our panel includes successive models from individual patients at time points before and after specific lines of therapy, with detailed information about the corresponding clinical response. For both standard chemotherapy and experimental agents in clinical trial, these models faithfully mirror patient responses. However, unlike the patient experience, multiple strategies can be compared for identical tumors. We propose to use these models to directly compare three clinical strategies that depend on induction of DNA damage: standard first line EP, second line topotecan, anad a promising experimental regimen, olaparib plus temozolomide (OT), currently in a phase I/II trial at MGH. Individually, these PDX population trials are designed to reveal biomarkers of sensitivity and mechanisms of resistance for promising experimental therapies. Collectively, through comparative analysis with reference to the clinical histories of each model, they present a novel opportunity to model cross-resistance, a problem that has beleaguered management of SCLC for over three decades.

NIH Research Projects · FY 2026 · 2020-08

Project Summary The activation of innate antiviral responses after viral infection is critical for the restriction of viral pathogens by eukaryotic hosts. However, viral immune evasion proteins (IEPs) can subvert these host responses, allowing viral pathogens to replicate and cause disease. Thus, identifying and characterizing virus- host interactions within this evolutionary “arms race” is critical for understanding how hosts combat virus infection and the mechanisms viral pathogens use to counter host defenses. Moreover, viral IEPs can be used as tools to discover and probe the cellular machinery they target, thereby revealing fundamental aspects of host biology. We developed innovative approaches to identify viral IEPs that target conserved eukaryotic antiviral machinery and that are capable of tipping the balance between abortive and productive viral infection. We exploit naturally abortive arbovirus infections in lepidopteran (moth and butterfly) cells as a screening tool to identify novel IEPs encoded by mammalian viruses that convert these abortive infections to productive infections by countering host immune responses. By identifying IEPs encoded by mammalian viruses that are immunosuppressive in insect cells, we select for IEPs that target antiviral responses conserved between insect and mammalian hosts. Using this approach, we identified mammalian poxvirus-encoded A51R proteins as novel IEPs that target a previously unappreciated “FACT-ETS-1 Antiviral Response (FEAR)” pathway. This pathway requires the evolutionarily- conserved “FACT” histone chaperone complex and ETS-1 transcription factor, which activate antiviral gene expression programs in cells to block viral replication. Here, we seek to understand how this pathway is both activated and countered by viruses and to identify the FEAR pathway-induced factors required for virus restriction. Our preliminary data suggest that host sensors of pathogen-associated molecular patterns are required for FEAR pathway activation and, using transcriptomics, we identify a core set of FEAR pathway- induced host factors, some of which display antiviral properties. In addition, we identified new FEAR pathway antagonists from disparate mammalian viruses, suggesting this pathway is relevant to a wide array of viral pathogens. Our data suggest these antagonists inhibit the FEAR pathway through multiple, distinct mechanisms. Thus, we aim to characterize these antagonists and use them to interrogate FEAR pathway activation/regulation and to reveal new FEAR pathway factors. Finally, we will further exploit our arbovirus-lepidopteran host system to identify other viral IEPs targeting conserved host factors by screening a newly constructed mammalian virus ORFeome library. Our initial screens have already identified several putative viral IEPs that relieve arbovirus restriction in insect cells, suggesting they target defenses shared between insects and mammals. Our ultimate goal is to identify novel IEPs encoded by mammalian viruses in our unique system and use them to provide mechanistic insights into the antiviral (and normal) functions of the conserved host machinery they manipulate.

NIH Research Projects · FY 2024 · 2020-07

We will develop the application of a promising novel vascular disrupting agent (VDA) in combination with leading therapies to enhance treatment of kidney cancer. Renal cell carcinoma (RCC) is usually characterized by inactivation of the von Hippel Lindau (vHL) tumor suppressor protein, promoting accumulation of Hypoxia Inducible Factor (HIF) and consequent development of extensive vasculature. The endothelium of normal blood vessels is largely quiescent, but the invasive neovasculature of tumors is immature, lacks pericyte support, and exhibits increased permeability providing a selective target for cancer therapy. The therapeutic goal of VDAs is to cause rapid widespread disruption of established tumor vasculature leading to regional ischemia, induction of hypoxia and tumor necrosis. We have identified OXi8007 as a new potent, water-soluble VDA prodrug generating protracted vascular disruption, dose dependent tumor growth delay and no apparent systemic toxicity. However, VDA monotherapy generally results in re-growth at the tumor periphery and OXi8007 will likely be most effective in augmenting current lead therapies based on complementary modes of action. We will investigate a small-molecule HIF-2 antagonist, PT2977 developed by Peloton Therapeutics, which represents a new class of chemotherapeutic in early clinical trials. Meanwhile, cabozantinib, the small- molecule kinase inhibitor that targets the c-MET receptor, AXL, and VEGFR-2 was recently approved as a first line treatment option for patients with metastatic RCC. We also recognize the emerging success of immunotherapy and anticipate that OXi8007-induced necrosis will enhance antigen presentation promoting response. Our overarching hypothesis is that combining these therapeutic approaches will achieve robust long term control of RCC. Investigations will benefit from the resources of the UT Southwestern Kidney Cancer SPORE, which has developed a number of new patient derived tumor lines exhibiting differential sensitivity to HIF-2 antagonists. Effective therapy combination will likely depend on timing of administration of the respective agents and non- invasive imaging will reveal the spatial and temporal pharmacodynamics of tumor response. Bioluminescence imaging (BLI) will effectively interrogate luciferase-transfected RENCA cells in immunocompetent mice. In addition, recently available multispectral optoacoustic tomography (MSOT) non-invasively reveals vascular extent and regional oxygenation without the need for exogenous reporter molecules or cell transfection. Complementary cell-based studies are designed to further explore OXi8007 mechanism of action. Effectively combining targeted therapies should enhance treatment and ultimately survivorship of kidney cancer patients. The goal of these investigations is to demonstrate effective combination therapy as a foundation for investigations in large animals and translation to the clinic.

NIH Research Projects · FY 2024 · 2020-07

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms, called schistosomes, that affects hundreds of millions of the world’s poorest people. The pathology of this disease stems from the fact that schistosomes can lay hundreds-to-thousands of eggs per day while living in the blood vessels of their human hosts. Therefore, understanding the mechanisms that control schistosome egg production could present new opportunities to both limit the spread of the disease and abrogate the pathology caused by the parasite. Interestingly, female schistosomes only become sexually mature when they are in constant physical contact with a male worm. Indeed, females grown in the absence of male worms produce no eggs and cause no serious adverse consequences in their hosts. Although the requirement of male worms for female sexual development was described almost 100 years ago, there are few insights into how this process is regulated on a molecular level. A major impediment for addressing this issue is that current culture conditions do not support schistosome sexual development in vitro. To address this issue we have developed a novel media formulation that supports male-induced female sexual development and long-term egg production in vitro. Capitalizing on this medium, we have made two important observations. First, we find that gene expression changes in females immediately upon pairing with a male worm do not occur in the reproductive organs, but rather in the parasite’s somatic tissues including neurons and muscle cells. Second, we identified a transcription factor highly expressed in the nervous system and muscle cells that is essential for female sexual development after pairing with a male worm. Based on these data, we hypothesize that somatic non-reproductive cell types in the female worm are responsible for perceiving the presence of the male worm and in turn regulating the development of the reproductive organs. Defining the cell types directly responsible for perceiving the presence of the male worm could provide important clues about the nature of the male-derived signal that controls female sexual development. Therefore, we will test our central hypothesis in two specific aims. In Specific Aim 1 we will describe the cellular and molecular response of the reproductive and somatic tissues of the female worm to pairing with a male worm using basic developmental biology approaches and single cell RNA sequencing. Specific Aim 2 will utilize RNA interference and single cell RNA sequencing to discover genes and somatic cell types that are essential for controlling female maturation and determine how these genes act in concert to coordinate the female response to pairing with a male. We expect these studies to define the signaling events that stimulate female maturation and hope they will suggest new therapeutic targets against schistosomes.

NIH Research Projects · FY 2024 · 2020-06

U01 Abstract Despite extensive research into cancer immunotherapy, immune-related adverse events (irAE) remain a critical and poorly understood issue. To address this critical need, we have assembled a multidisciplinary research team with broad and relevant expertise. The co-PIs of this proposal have expertise in cancer immunotherapy, immunology, assay development, and bioinformatics. Together, we have assembled a cohort of ~400 cancer patients treated with ICI, collecting longitudinal treatment, efficacy, and toxicity data, as well as blood samples at pre-treatment baseline, throughout therapy, and at time of toxicity. In our real-world data set, over 10 percent of cases have a history of autoimmune disease, providing insight into use of ICI in a population widely excluded from clinical trials yet routinely treated with these therapies off protocol. Our high-quality clinical data annotation—without which correlative studies have little meaning—addresses the reality that irAE may occur months after ICI initiation and are far more complex to detect and characterize than toxicities of conventional chemotherapy or molecularly targeted therapies. Through existing funding mechanisms, we have already completed autoantibody, cytokine, genetic, and functional assays in these cases. However, we do not currently have resources for comprehensive, integrated analysis of these diverse laboratory and clinical data. The overarching goal of this U01 proposal is to determine the optimal balance between ICI efficacy and toxicity, ultimately identifying a set of biomarkers useful for selection of patients, treatment type and duration, and clinical monitoring. We will achieve this through determination of cellular immunity, comprehensive data analysis, and clinical validation. We have three Aims: (1) Determine cellular immunity in patients experiencing irAE and/or achieving beneficial responses from ICI. We will perform mass cytometry (CyTOF) and T-cell receptor sequencing at multiple time-points. (2) Determine genetic, humoral, and cellular factors associated with irAE and/or beneficial responses from ICI. We will develop a database to integrate and analyze the CyTOF and T-cell receptor sequencing data with clinical efficacy and toxicity data, as well as data from the assays already completed through other mechanisms. (3) Perform analytical and clinical validation of emerging biomarkers. We will apply the best classifying phenotypes emerging from our comprehensive and integrated data analysis to a test set of patients from our existing cohort, eventually identifying a subset of biomarkers with potential for clinical application. Together, these Aims directly address the FOA purpose of reducing the incidence and/or severity of irAE while retaining anti- tumor efficacy.

NIH Research Projects · FY 2026 · 2020-05

Project Summary Influenza virus genomic RNAs enter the host cell nucleus where they are transcribed into viral mRNAs. These viral mRNAs are then exported from the nucleus to the cytoplasm for translation into viral proteins. Thus, nuclear export of viral mRNAs is an obligatory pathway for viral gene expression, and this is the topic of this application. The intranuclear protein complex termed TREX (TRanscription and EXport) links cellular transcription, mRNA processing and nuclear export. Through the combined action of the TREX complex and other mRNA export factors, the major mRNA export receptor heterodimer NXF1:NXT1 is recruited to the mRNA to mediate nuclear export through the nuclear pore complex to the cytoplasm. We have evidence that the diverse influenza virus mRNAs require specific RNA-Binding proteins (RBPs) to be differentially exported from the nucleus. Here, we will systematically define the RBP roles in divergent nuclear export pathways for the influenza virus mRNAs. In Aim 1, we will determine the role of hnRNP H proteins as new mediators of influenza virus mRNA nuclear export. We have preliminary evidence demonstrating that hnRNP H proteins are involved in differential nuclear export of viral M, NA and HA mRNAs. We have also identified hnRNP H1 interactions with constituents of the mRNA nuclear export machinery, which can serve as starting points for uncovering molecular mechanisms involved in the recruitment of viral mRNAs to the mRNA export pathway. Therefore, we will functionally characterize the interactions between hnRNP H1 and H2 proteins with the mRNA export pathway using biochemical, genetics, and imaging approaches. We will then determine the hnRNP H-binding sites on the viral mRNAs by carrying out in vitro biochemical assays, UV crosslinking, and RNA-affinity pull-down assays. The impact of the hnRNP H- viral mRNA interactions on the virus life cycle will be assessed by comparing wild-type and mutant viruses on the hnRNP H binding sites in viral mRNA nuclear export and protein assays. A mouse mutant on the hnRNP H interacting partner SF3B1, which prevents its interaction with hnRNP H, will be subjected to viral replication and pathogenesis studies to determine the impact of this pathway in vivo. In Aim 2, we will systematically identify and functionally characterize RNA-binding proteins that differentially mediate nuclear export of all influenza virus mRNAs. We have evidence that various RBPs are required for differential nuclear export of influenza virus mRNAs. We will use single-molecule RNA FISH to detect the intracellular distribution of each viral mRNA upon RBP depletion in a focused RBP genetic screen. In parallel, we will test RBPs known to bind the virulence factor NS1 or the cellular mRNA export receptor NXF1, which promote viral mRNA nuclear export. These results will be followed by biochemical approaches described in Aim 1 to determine RBP-viral mRNA interactions. We will then generate mutant viruses on the RBP-binding sites, which we will be tested in viral mRNA nuclear export and protein assays. Knowledge of these functions are key for understanding influenza virus gene expression and could be potentially used for designing new therapeutic strategies against influenza virus infection.

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY/ABSTRACT Cell-cell fusion is critical to the conception, development, and physiology of multicellular organisms, and is involved in a variety of biological processes, such as fertilization, placenta development, bone remodeling, immune response, and skeletal muscle development and regeneration. Failure in cell fusion leads to defects such as infertility, osteopetrosis, immune deficiency, pre-eclampsia, and congenital myopathy and muscular dystrophy. Compared with our understanding of intracellular vesicle fusion and virus-cell fusion, much less is known about the underlying mechanisms of cell fusion. A mechanistic understanding of cell fusion is not only important for fundamental biology but may also provide basis for its manipulation in therapeutic settings. My laboratory uses a multifaceted approach including genetics, molecular biology, biochemistry, biophysics, live imaging, super-resolution microscopy and electron microscopy to study the mechanisms underlying cell-cell fusion. Using Drosophila myoblast fusion as a model initially, we discovered the asymmetric fusogenic synapse, where one cell invades its fusion partner using F-actin-propelled invasive membrane protrusions to promote plasma membrane juxtaposition and fusion pore formation. Building on the insights that we learned about myoblast fusion in vivo, we have reconstituted high-efficiency cell-cell fusion in an otherwise non-fusogenic, non- muscle cell line and uncovered a novel function for invasive membrane protrusions in fusogen engagement across the apposing plasma membranes. Furthermore, we have discovered dynamic mechanosensory responses in the receiving fusion partner and demonstrated that mechanical tension is a driving force for cell- cell fusion. In the last grant period, we have extrapolated the mechanism that we discovered in Drosophila to vertebrate models and demonstrated that myoblast fusion in zebrafish and mouse is also mediated by invasive protrusions at the asymmetric fusogenic synapses. Moreover, we have elucidated the mechanism by which the large GTPase dynamin bundles actin at the fusogenic synapse, uncovered how inter-organ steroid hormone signaling promotes myoblast fusion via direct transcriptional regulation of a single key effector gene, and discovered a novel function for the ABC G1/G4 transporters and free/accessible cholesterol in promote protrusion formation and cell-cell fusion. In the next five years, we will expand our research into three new directions. First, we will perform mechanistic studies to understand the function of the unconventional fusogen, Myomaker, by dissecting the interaction between Myomaker and the actin cytoskeleton and investigating whether Myomaker affects the local lipid composition on plasma membrane. Second, we will investigate how the Hippo-YAP/TAZ-TEAD signaling pathway regulates the cell fusion machinery, by characterizing the actin cytoskeleton phenotypes in TEAD2DN cells, identifying TEAD target genes involved in cell fusion, and pinpointing the TEAD-binding sites in target genes. Third, we will explore whether altered mechanical tension causes myofiber splitting in Duchenne muscular dystrophy, by vvisualizing satellite cell-induced myofiber splitting on micropatterns at the single-fiber level and modulating myofiber stiffness to alleviate fiber splitting. Our research will not only provide novel insights into the fundamental principles of cell-cell fusion, but also have far-reaching impact on a broad range of fields, including membrane biology, cell biology, and development and disease.