Ut Southwestern Medical Center

NIH Research Projects · FY 2024 · 2020-04

The cGAS-STING pathway is a crucial signaling pathway of the innate immune system. The STING signaling mechanisms in myeloid cells are well studied. However, STING expression in healthy and diseased tissue and STING signaling activities in other cell types are much less understood. In the previous funding period, we generated the StingS365A mouse, which allowed us to uncover many IFN-independent activities of STING in T cells and their physiological significance in diseases. In this renewal application, we created a Sting reporter mouse and discovered interesting dynamics of STING expression in T cells from development to maturity. We have also created additional transgenic mice that allow us to modulate STING expression and signaling in specific tissues or cell types. Our main hypothesis is that dynamic regulation of STING expression and signaling is essential for maintaining T cell development, homeostasis, and functionality in response to cancer. Aim 1 will define the regulatory mechanism of STING expression in T cells. Aim 2 will determine how STING signaling regulates T cell development and function. Aim 3 will define the role of STING in thymopoiesis and T cell immunity in cancer. Through these studies, we hope to uncover a new facet of STING biology that uniquely connects innate and adaptive immunity. The potential influence of STING signaling on thymopoiesis also has immediate clinical implications for the successful development of STING agonists as cancer immunotherapy.

NIH Research Projects · FY 2025 · 2020-04

Project Summary/Abstract: Over the previous funding periods, it was shown that acetyl-CoA and SAM are key signals of cellular metabolic and nutritional state. Acetyl-CoA is an activated carrier of two carbon units, while S-adenosylmethionine is the biological methyl donor in many reactions critical for life – these are two key metabolic currencies used by cells. This application proposes to continue investigating the mechanisms by which acetyl-CoA and SAM function to regulate important cellular pathways in balancing cell growth versus survival in response to metabolic and nutritional state. A combination of genetics, cell biology, and biochemistry will be utilized to elucidate how these sentinel metabolites are compartmentalized intracellularly and influence multiple aspects of the central dogma of molecular biology. Insights from these studies will be informative as to the role of these sentinel metabolites in aging and age-related diseases.

NIH Research Projects · FY 2026 · 2020-04

Project Summary/Abstract The goal of this project is to understand at the molecular level how cholesterol exerts negative feedback on its own biosynthesis in mammalian cells. Cholesterol is an essential lipid, both serving as a basic building block of cellular membranes and acting as a precursor for steroid hormones. We seek to understand the atomic structures and mechanism of the membrane proteins Scap and Insig, which control SREBP processing and thereby regulate cholesterol biosynthesis. Scap binds to the SREBP transcription factors and controls their maturation in a cholesterol-dependent manner. Insig binds to Scap when cholesterol levels are high and helps to retain Scap and SREBPs in the endoplasmic reticulum (ER), whereas the COPII adapter proteins Sec23/24 binds to Scap when cholesterol levels are low and promotes trafficking to the Golgi apparatus. We use structural biology and biophysical tools to understand at the atomic level how Scap and Insig respond to membrane cholesterol and regulate SREBPs. Our previous work exploited the chicken orthologs of Scap and Insig, as well as a mutation that locks Scap into a cholesterol bound-like state. In these studies, we elucidated a major conformational rearrangement of Scap's transmembrane architecture and ER luminal loop that is stabilized by Insig binding, and therefore presumed to be promoted by high cholesterol. However there are still no structures of Scap/Insig bound by cholesterol itself, to explain how the lipid influences Scap's conformational equilibrium. We will use single-particle cryo-EM methods to elucidate the high-resolution structure of the mammalian Scap/Insig complex bound to cholesterol. Mutagenesis and SREBP functional studies will be used to probe and validate the importance of different bound cholesterol molecules in SREBP regulation. Further we will carry out purification and cryo-EM structure determination of the Scap/Insig complex in the presence of different small molecules that have been proposed as Scap inhibitors. These structures will show how drugs can interact with this ER membrane protein complex to modulate SREBP signaling, and elucidate whether their binding modes overlap with the natural ligand cholesterol. When membrane cholesterol is low, Scap/SREBP complexes are trafficked to the ER through Scap's interaction with COPII. Determining the structure of the Scap/COPII complex is challenging due to the low affinity of this interaction, and no such cargo/COPII complexes have yet been structurally characterized. We will use yeast display selections to isolate nanobodies that stabilize the Scap/COPII complex, and use these reagents to purify the complex in detergent for single-particle cryo-EM analysis. The structure of this complex will show how COPII recognizes Scap's cytosolic surface under low- cholesterol conditions, and further indicate how the interaction depends on the low-cholesterol conformation of Scap. Overall, our molecular dissection of the Scap/SREBP signaling system will show how cholesterol homeostasis is maintained at the atomic level, and provide a framework for future therapeutic development targeting this pathway in metabolic diseases.

NIH Research Projects · FY 2025 · 2019-09

Overall Core Abstract The overall goal of this NEI Core Center Grant for Vision Research is to enhance the capabilities of vision research scientists by facilitating easy access to equipment, training, and technical support that will expand experimental capabilities, add rigor and reproducibility, result in more cost-effective and time-efficient research, and open up new research directions. This Center will serve vision science researchers across the UT Southwestern Medical Center (UTSW) campus. It will also facilitate new research collaborations within the institution, regionally, and nationally. Our proposed NEI Core Center consists of three Resource/Service Modules, each directed by a faculty member with experience and expertise in their unit’s focus area: 1) Stem Cell, Organoid and Cell Phenotyping (Directed by Dr. Robertson), which will support the development of induced pluripotent stem cells (iPSCs) and organoids relevant to the eye, and provide services for immunometabolic profiling; 2) Gene Editing and Virus Production (Directed by Dr. Park), which will generate DNA/RNA constructs for use in mammalian cultures and systems, and purify and concentrate both lentivirus and adeno-associated virus (AAV) for subsequent use in immortalized, primary cultures and in animals in vivo; and 3) Microscopy and Animal Phenotyping (Directed by Dr. Petroll and Dr. Wert), which will provide equipment, infrastructural support, expertise and technical assistance to enable quantitative imaging of cells and tissues, as well as structural and functional assessment of the visual system in vivo. In addition, the Administrative Module will develop and implement programs to promote and foster collaborative projects between UTSW scientists and other researchers across campus, within the region, and throughout the nation. The Specific Aims of the Core Center are to enhance, streamline, and add scientific rigor to the research activities of the participating vision scientists by: 1) Supporting the operation of the Resource/Service Modules by managing usage and operations, providing skilled technical assistance, equipment maintenance, and the purchase of supplies; 2) expanding the number of collaborative research projects between investigators both within UTSW and between UTSW and other institutions; 3) creating a rich environment for developing vision research programs of junior faculty members, and for new vision researchers recruited to the University; and 4) providing resources and training to assist graduate students, medical students, and fellows with their research. Overall, we anticipate that the Core Grant for Vision Research will impact the research programs of participating vision scientists by: 1) expanding their research into new areas through the addition of novel experimental techniques; 2) stimulating new collaborations with other potential Core Center users across campus, regionally, and nationally; 3) improving research rigor, productivity and efficiency through our Core services; and 4) enhancing the training of UTSW students, and fellows interested in vision science.

NIH Research Projects · FY 2025 · 2019-09

Project abstract How molecular organization and activity leads to tissue level outcomes is an open question in biology and biomedical research. Addressing this question is technically challenging because we cannot observe with molecular precision at the tissue scale. While optical microscopy is the method of choice to observe architecture and dynamics within living cells and organisms, it has severe limitations in spatiotemporal resolution and volumetric coverage. Thus, our most detailed observations of cellular dynamics and ultrastructure have been limited to single cells on coverslips, which were far removed from their physiological context. Here I propose to extend the capabilities of light-sheet fluorescence microscopy (LSFM), a technology that provides gentle and efficient 3D imaging, but only moderate resolution. We will combine LSFM with super-resolution methods to allow rapid imaging of subcellular dynamics away from coverslips. We will further explore ways to increase the volumetric acquisition rate of LSFM such that large samples can be rapidly explored. These new developments will be tied together by smart sampling strategies, which will enable autonomous exploration of large samples while applying the highest resolution only locally. This way, we expect that rare cellular events can be studied in subcellular detail in entire model organisms or organs that were rendered transparent through clearing. The proposed rapid volumetric imaging capabilities will enable imaging of all neurons in a small model organism such as C.elegans or Zebrafish embryos with up to 100 Hz volume rate. Such rapid pan-neuronal imaging may shed light on how a brain functions. While we expect that the potential for discovery with such microscope technology is immense, access to such advanced instrumentation is often limited. To address this, we will develop a modular light-sheet platform that can be adapted to a wide range of imaging tasks. We will further explore ways to simplify the microscope architecture and design less expensive variants to aid dissemination. The resulting new microscope technology will enable large volume imaging experiments that have been prohibited by either lack of spatial resolution or acquisition speed, and as such accelerate biological and biomedical research.

NIH Research Projects · FY 2024 · 2019-09

Abstract Ferroptosis is a recently recognized form of regulated cell death driven by lipid peroxidation. It is an emerging field in cancer biology, and the detailed molecular regulators of ferroptosis are largely unknown. We recently demonstrated that upregulation of metallothionein (MT)-1G expression contributes to ferroptosis resistance in human hepatocellular carcinoma cells, whereas inhibition of MT-1G expression enhances ferroptosis sensitivity in vitro and in vivo (Hepatology. 2016 63(1):173-184.; Hepatology. 2016 64(2):488-500.). These exciting findings raise several important questions regarding the previously unidentified role of MT-1G in the regulation of ferroptotic cancer cell death. Our central hypothesis is that expression and release of MT-1G limits ferroptotic cancer cell death. To test this hypothesis, we will exploit molecular, cellular, and animal models to pursue the following aims. Aim 1: Define the mechanism responsible for transcriptional regulation of MT-1G expression in ferroptosis. Aim 2: Define the mechanism responsible for phosphorylation modification of MT-1G function in ferroptosis. Aim 3: Define the mechanism responsible for extracellular activity of MT-1G in ferroptosis. The completion of these exciting studies will improve our understanding of the cancer molecular pathobiology of ferroptosis and guide future development of novel MT-1G-based anticancer therapeutic strategies.

NIH Research Projects · FY 2023 · 2019-09

PROJECT SUMMARY Delivery of holistic care to persons with spina bifida/myelomeningocele remains a complex challenge to clinicians in the field, researchers in academic settings, and public health agencies charged with assuring best practice standards. The National Spina Bifida Patient Registry is uniquely positioned to directly support these complex challenges. The leadership, site participants, and individuals who comprise the entirety of the Registry are experts in the field; the structure is well defined, rigorous, yet adequately flexible for ready change when shifts are needed for quality purposes; and there are continuing outcome products (publications, reports, etc.) that reflect the value of the enterprise. The Spina Bifida Program at Texas Scottish Rite Hospital for Children (TSRH) in Dallas, TX, an active participant in the Registry, continues to expand both in numbers of families served and in the broad spectrum of services and programs available for pediatric patients. For over 25 years, there have been – at any time along the way – about 850 to 900 families actively engaged in the Spina Bifida Program Clinics at TSRH. While the majority of these patients come from about a 100-mile radius from the hospital, the Program serves children throughout Texas: El Paso to Dallas; Lubbock to Austin. The diversity of the population is reflective of that in the North Texas region: a slight majority of non-Hispanic white, a near- majority with Hispanic background, and about 10% Asian or Black background. A slight majority carry commercial insurance; a near majority have Medicaid; and a minority carry both. The parents represent a wide socio-economic and educational diversity. The diverse demographic population allows for large, balanced cohorts for both clinical research and reports. Because of the historically unique system of financial support to families by the Hospital and the nature of the services provided, the longitudinal follow-up of patients from birth to adulthood remains remarkably high. Willingness to participate in clinical studies remains high as well. This has allowed the Program to conduct longitudinal research in addition to robust cross-sectional studies. To this point, the Hospital has been able to support to some degree the activities specific to the demands of the Registry. The TSRH Spina Bifida Program can better maximize its potential as a Registry participant, expand the activities of the Program, and advance new initiatives with funding support from the Registry. We are presently involved in two specific initiatives for outcomes studies; funding support should allow greater time and focus of our present research coordinator for added research projects. Through this project we propose the following aims: 1) Continue to collect longitudinal data on individuals with spina bifida in order to identify variation among the NSBPR clinics, 2) Participate in and implement data quality strategies in order to ensure reliability across clinics, and 3) Collaborate with other sites to develop research projects addressing scientific gaps in the spina bifida population.

NIH Research Projects · FY 2025 · 2019-09

Project Summary Emerging findings by us and others have revealed critical non-lethal functions of caspases across diverse animal phyla. Our published and unpublished findings indicate marked roles for caspases in ensuring multiple aspects of development including cell-cell communication, cell migration, protein homeostasis, and regulating stress responses. Moreover, our data indicate that a caspase target expressed in the same cell at the same time as the caspase may not be acted upon by the caspase until a specific developmental time point suggesting additional layers of regulation. It is not known how specific non-lethal caspase functions are mediated. Based on our recent findings, it is likely that caspases require other components, such as E3 ligases, to execute these non-lethal functions. We hypothesize that caspases function in complexes with other proteins that confer non-apoptotic specificity according to developmental stage, tissue type, and environmental status. Over the next five years, the critical goals for my lab are to: 1) identify how caspase modulates p38 MAPK-mediated signaling network to support neuronal integrity, 2) understand how UBR E3 ligases coordinately regulate cell fate, and 3) identify novel caspase functions in gene expression and metabolic regulation. Our proposed cross-disciplinary studies include genetic screens, biochemical analyses, translatomics, proteomics, metabolomics, and structure-function studies. The objective of the proposed studies is to understand how proteolytic mechanisms mediate diverse cellular regulatory functions in response to developmental or environmental cues.

NIH Research Projects · FY 2025 · 2019-08

Abstract/Project Summary Accelerating the translation of fundamental scientific discoveries into tangible advancements in human health stands as a foremost national priority. Regrettably, conventional graduate education in biomedical sciences primarily emphasizes didactic knowledge and hands-on research experience. Insufficient emphasis is placed on cultivating the skillset required to propel basic scientific breakthroughs from the laboratory to practical applications at the patient's bedside. Furthermore, once students complete their graduate studies, the opportunities to formally acquire the indispensable knowledge for conducting and propelling translational research become even scarcer. The UT Southwestern Medical Center's Mechanisms of Disease and Translational Science program (MoDTS) was created as a supplementary graduate student track to complement the traditional training regimen for PhD candidates and to close the gap between basic and translational research training. The MoDTS initiative pairs PhD candidates with qualified, passionate clinical preceptors specializing in medical fields pertinent to the student's thesis. The overarching aim revolves around acquainting students with clinical practices and fostering connections that facilitate translational research endeavors. The program initiates in the third year of graduate school and continues until graduation. It encompasses a series of obligatory and elective avenues, including continuous clinical shadowing, medical-student level courses, seminars, nanocourses, and internships. Trainees committed to translational research will be funded for one-year periods to facilitate clinical collaborations and experiential learning opportunities. These avenues provide atypical yet invaluable instruction across an array of subjects, such as crafting IRB protocols, technology transfer and development, patent procedures, and the design and execution of clinical trials. We are requesting 8 positions to allow trainees to commit to extracurricular, translational training, including experiential training and internships. Most trainees will be appointed for one year, however second and even third years of support will be provided under exceptional situations. Ultimately, our program's objective centers on furnishing PhD candidates with the proficiencies requisite for propelling fundamental scientific discoveries out of the laboratory and into the hands of healthcare providers. Our expected outcomes are to train students to be actively involved in translational research through a variety of career options.

NIH Research Projects · FY 2024 · 2019-08

PROJECT SUMMARY/ABSTRACT Recent FDA approval of the anti-beta-amyloid (Ab) antibodies Aducanumab and Lecanemab as the first mechanism-based Alzheimer's disease (AD) therapies has strengthened the central role of the Ab protein in AD pathogenesis. However, these imperfect medications have only shown modest cognitive benefits, as AD clinical symptoms often occur decades after amyloid formation and deposition, and the molecular mechanisms leading to the demise of neurons in AD brains remain poorly understood. Nevertheless, the success of anti-Aβ therapeutic antibodies and identification of an APP mutation that protects against AD attest to the existence of amyloid-related molecular mechanisms that restrain Ab toxicity and confer resilience to AD. Meanwhile, cryo- electron microscopy structural studies have revealed several types of Aβ filaments isolated from AD brains, and they differ from those assembled in vitro. These observations suggest that in vivo assembly of amyloid filaments may require additional cellular factors that control progression of amyloid pathology and connect with non- amyloid AD pathways. Proteomic studies identified three prominent groups of AD pathology-associated proteins (ADPs). The current project seeks to test the hypothesis that Midkine and Netrin proteins, top-ranked human ADPs regulate Ab pathology and connect non-amyloid pathways in AD protein networks. The choice of Midkine and Netrin was informed by unbiased proteomic screening of hundreds of human brains, by validation studies that establish functional and physical links to amyloid pathology, and by their potential connections to UNC5C and ApoE/LRP pathways. The project is organized into three specific aims: 1) to determine the pathophysiological roles and AD protein networks mediated by Midkine and Pleiotrophin; 2) to map Midkine interactome and its physical interactions with Aβ filaments; 3) to elucidate the synergistic roles of Midkine, Pleiotrophin, and Netrin-1 in AD pathogenesis. These studies will reveal the impact of select AD pathology- associated proteins on disease progression, uncover non-amyloid pathways mediated by these ADPs, and elucidate the biochemical mechanisms by which Midkine and Netrin family ADPs regulate assembly of Ab filaments to potentially confer resilience to amyloid toxicity. Success of this project will inform the development of more robust experimental models and more specific and sensitive therapeutic and imaging agents for AD.

NIH Research Projects · FY 2024 · 2019-08

PROJECT SUMMARY/ABSTRACT Gram-negative pathogens are becoming increasingly resistant to many antimicrobials. Furthermore, the pipeline for new antibiotics is small and new therapies are urgently needed. This can be especially problematic in patients who suffer from chronic infections or are immunocompromised. Multidrug-resistant Pseudomonas aeruginosa has been identified by the Centers for Disease Control and Prevention as a serious threat. P. aeruginosa causes healthcare associated infections in a variety of clinical settings and hosts, but is particularly devastating in patients with cystic fibrosis (CF). We have been interested in using antisense molecules called PPMOs as potential therapeutics in these infections. These molecules block messenger RNA and prevent the formation of the target protein. We have demonstrated that PPMOs can be used to target genes that are essential for Pseudomonas to grow, such as acpP, lpxC or rpsJ. We showed that blocking these proteins are essential for Pseudomonas to grow in vitro. We also showed that these PPMOs improve survival in mice that were infected with Pseudomonas. For this project, we propose to further characterize our lead PPMOs in a larger collection of Pseudomonas isolates, both antibiotic-sensitive and multidrug-resistant. In addition, efficacy studies will be performed in both models of pneumonia and bloodstream infection. This process will result in 2-4 PPMOs that will undergo further pre-clinical testing including toxicity, resistance, pharmacodynamic and pharmacokinetic studies. By the end of the proposed project, a lead PPMO will have undergone the needed pre-clinical testing for IND submission to the FDA. This innovative approach to developing novel antibiotics, particularly for P. aeruginosa, could help expand the increasingly shrinking classes of effective antibiotics that are used to treat these severe, life-threatening infections.

NIH Research Projects · FY 2026 · 2019-07

The proper function of organisms, their organs, and their tissues requires them to have specific shape. How this shape is specified and maintained is a fundamental question in biology. In animals, the shape is determined through the process of morphogenesis, a concerted sequence of tissue remodeling events leading up to the final body plan. Despite a long-standing effort to understand the physical mechanisms that underlie morphogenesis, these mechanisms still remain largely unknown. Basic considerations from physics imply that in order to completely determine the mechanism of a morphogenetic change, two pieces of information are absolutely required: (1) material/mechanical properties of the tissue, and (2) the active forces that drive tissue deformation. Using Drosophila gastrulation as a model, and by combining biophysical, molecular, and modeling methods, we propose an approach sufficient to determine both. Recent work from our group and others has begun the process of quantifying the mechanical properties of tissues as whole. However, it has become clear that mechanical properties vary in different cellular regions (apical vs basal etc.), and that understanding these differences is crucial for correctly understanding and accurately predicting morphogenesis. In Aim 1, we expand upon our previously established techniques for measuring tissue mechanics, and apply them to directly measure viscous and elastic properties of the apical, lateral, and basal cellular compartments separately. In Aim 2, we will incorporate these measurements into a comprehensive computational model to explain large-scale tissue behaviors based on these microscopic measurements. From this model, we will also be able to extract the spatial and temporal force profiles driving tissue morphogenesis in the early embryo. In Aim 3 we will assess the predictive power of our model to predict mutant phenotypes, and we will also begin identifying the molecular players that contribute to specific mechanical features such as elasticity and mechanical memory. In summary, successful completion of the project will for the first time establish a comprehensive biophysical mechanism of a key model system. Both the techniques and general approach developed here will be applicable to a wide variety of tissue morphogenesis processes.

NIH Research Projects · FY 2025 · 2019-07

The University of Texas Southwestern Medical Center (UTSW) Molecular Biophysics T32 Training Program (MB-T32) aims to develop a robust cohort of PhD students immersed in cutting-edge biophysical concepts and methodologies, for successful biomedical research careers. Combining group initiatives and individualized programming that emphasizes deep understanding, rigorous research practices, safety, and ethics, MB-T32 is designed to nurture emerging and ethical scientists with advanced biophysical research skills. Building upon successful elements of the current program (coursework, training activities, Responsible Conduct of Research training and career development support), we will add new training elements to capitalize on emerging opportunities and address local challenges. The field of biophysics is undergoing rapid change, posing a challenge to stay current in educating and training new biophysicists, especially with the UTSW biophysics community distributed across two campuses and thirteen departments. MB-T32 will meet this challenge by becoming the central, driving force for innovating biophysics education and training across UTSW to ensure trainees receive an up-to-date foundational biophysics education prior to MB-T32 training that will expose them to more cutting-edge topics. MB-T32 coursework will deliver tailored education to each trainee based on their specific biophysical and biomedical interests while activities will promote cohesion and breadth of knowledge. Recruiting is another challenge that MB-T32 addresses in this application: the relatively small population of quantitative biophysicists at UTSW is not meeting the growing demand. By spearheading recruitment of talented biophysics students, MB-T32 will shape the future of this field on campus and more broadly. MB-T32 is committed to cultivating a supportive training environment, showing respect for our trainees and empowering them to develop agency and independence through collaborative leadership opportunities. New activities will facilitate personalized monitoring of trainee progress and interventions, expand the mentoring network, and address challenges related to differences in academic preparations in quantitative sciences, rapid advancements in biophysics, and the lingering effects of the pandemic on social cohesion. MB-T32 is poised to empower the next generation of biophysicists to confront the most pressing questions and challenges in biomedical research.

NIH Research Projects · FY 2025 · 2019-06

Summary Malignant melanoma in advanced stages can be treated with immunotherapeutic antibodies that target CTL-4, PD1, and PDL1, resulting in enhanced survival rates. However, a large proportion of patients still do not respond to such therapies due to the presence of immunosuppressive signaling in the tumor microenvironment (TME). To generate an activated immune microenvironment, our laboratory has developed a liposome-based nanoparticle (NP) that upregulates calreticulin (CRT) in melanoma TME. Our in vitro and in vivo data in immunologically cold B16F10 melanoma suggest that intratumoral in-situ vaccination (ISV) with CRT-NP and its local combination with focused ultrasound (FUS) induce local and systemic immune priming, thereby resulting in a superior anti-tumor immunity. This is highly promising, but most preclinical studies including ours typically employ lean mice to investigate immunotherapeutic mechanisms. Risk factors like excess body weight can exacerbate tumor immunosuppression, but little is known about how CRT-NP efficacy is influenced by this mechanism, and whether the body mass index of patients should be taken into consideration for designing the phase I clinical trial. Herein, we will determine the CRT-NP, FUS, and CRT-NP+FUS (CFUS)-based ISV outcomes in the lean and obese murine models of melanoma. To test our objective, we will first compare the CFUS local efficacy and immune signaling mechanisms in the B16F10 model (Aim 1). Next, we will combine the mono- and CFUS therapies with immune checkpoint inhibitors in a clinically relevant YUMM1.7 murine melanoma (Aim2). The investigation of therapeutic and immune effects in lean and obese mice will enable the successful optimization of local CFUS in liberating tumors from their immune-suppressive state regardless of the physiological and metabolic status of the patients, thereby improving remission rates independent of cancer complexity. If successful, this method will provide a promising new avenue for treating melanoma and other types of solid tumors (e.g., breast, prostate) by significantly overcoming current immunotherapy barriers.

NIH Research Projects · FY 2026 · 2019-06

Project Summary Exposure to new sensory experiences activates new gene transcription programs in neurons and the products of these genes mediate the development of lasting adaptive behaviors. Defects in neuronal activity-dependent transcription programs manifest in neurodevelopmental diseases, intellectual disability, and autism spectrum disorders. Understanding how neuronal activity-dependent transcription is regulated is therefore significant. An intriguing finding in this regard is that neuronal activity induces the DNA topoisomerase, Top2B, to form DNA double strand breaks (DSBs) within the promoters of key early response genes (ERGs), such as Fos, Npas4, and Egr1, and that DSB formation in this manner facilitates the rapid transcription of these ERGs. Yet precisely how neuronal activity-induced DSBs are orchestrated to occur at specific positions, how they facilitate the transcription of associated genes, and how activity-induced DSBs are repaired are all poorly understood. To address these issues, various signaling pathways that affect synapse-to-nucleus communication in cultured mouse cortical neurons were perturbed. These efforts identified that activity-induced DSB formation is controlled by the phosphatase, calcineurin, which dephosphorylates Top2B and induces it to form DSBs upon neuronal stimulation. These signaling events are spatially compartmentalized to occur at the nuclear periphery and sites that incur activity-induced DSBs also preferentially localize to the nuclear periphery. Based on this, proposed experiments will employ high-resolution imaging and biochemical methods to determine whether similar molecular events also govern DSB formation in the hippocampus following relevant physiological stimulation and how radial gene position affects the recruitment of neurons into functional ensembles. Preliminary data suggest that while calcineurin regulates Top2B at the nuclear periphery, additional mechanisms preclude Top2B from forming DSBs at ectopic sites. Proposed experiments will decipher these mechanisms. Chromosome conformation capture experiments indicate that DSBs are necessary and sufficient to stimulate enhancer- promoter contacts at ERGs. Moreover, recurrent DSB formation progressively potentiate ERG transcription by pruning interactions between ERG promoters and heterochromatin while relatively stabilizing interactions with enhancers. Recent reports suggest that DSB repair mechanisms drive chromatin reorganization at DSB sites, yet exactly how activity-induced DSBs are repaired is unknown. Preliminary data suggest that the enzyme, TDP2, catalyzes the repair of activity-induced DSBs and the proposed experiments will investigate how TDP2 loss affects activity-dependent gene transcription and chromosome organization during recurrent stimulation of hippocampal neurons in vivo. Mutations in TDP2 cause the disease, SCAR23, which is characterized by intellectual disability and seizures, but the underlying mechanisms are unknown. Ablating Tdp2 increased the duration of UP states in acute cortical slices and the proposed experiments will use electrophysiology and assess intrinsic neuronal excitability and synaptic function to determine how TDP2 loss affects neuronal function.

NIH Research Projects · FY 2026 · 2019-05

The University of Texas Southwestern Medical Center (UT Southwestern) has consistently provided key leadership within the National Clinical Trials Network (NCTN). During the current UG1 Lead Academic Participation Site (LAPS) funding period (March 2019-February 2025), we enrolled 817 patients in NCTN trials spanning 10 disease categories. During this time, UT Southwestern faculty authored or co-authored 52 publications directly related to NCTN trials, and chaired, co-chaired, or served as cooperative group committee chairs on 12 NCTN trials. Our research led to major, NCTN-relevant policy changes, including modification of FDA trial eligibility guidance (2020 and 2024) and allowing full participation of advanced practice providers in Cancer Therapy Evaluation Program (CTEP) trials (2021). Ongoing, substantial growth positions this LAPS to expand our accrual and scientific contributions. Reflecting a 30% increase in new adult cancer cases seen, new outpatient cancer care facilities have opened at our safety-net site (2021), main university campus (2022), and in South Dallas (designated a “medical desert,” 2022). The new O’Donnell School of Public Health (2022) and a new Department of Biomedical Engineering (2021) have expanded faculty expertise. Our vibrant therapeutic discovery program is supported by Specialized Program of Research Excellence (SPORE) grants in lung cancer and kidney cancer, a Program of Excellence in Intelligent Medicine (focused on developing and deploying Artificial Intelligence technologies), an Advanced Imaging Research Center, a Clinical Pharmacology Core, and a Clinical and Translational Science Award (CTSA). Because the UT Southwestern Simmons Cancer Center remains the only NCI-designated cancer center in the fastest growing metro area in the U.S. (Dallas-Ft. Worth, 2024 population 8.5 million), this LAPS reaches a large population that otherwise would not have access to NCTN trials. Our Specific Aims are the following: Aim 1. Contribute to NCTN accrual. With our unique clinical trials navigation program, we will enroll patients on therapeutic and non-therapeutic trials. Aim 2. Contribute to NCTN scientific direction. We will bring UT Southwestern basic, clinical, and population research to hypothesis-driven clinical trials and correlative studies. Aim 3. Provide NCTN leadership. UT Southwestern faculty will continue to chair and co-chair NCTN clinical trials and committees and will participate in NCI activities and initiatives. Aim 4. Promote career development of junior faculty and trainees. Through a new LAPS Junior Investigator Program and the new Simmons Cancer Center K12 award, we will provide NCTNfocused mentoring and guidance. Together, these Aims position UT Southwestern meet and overcome the persistent challenges of clinical cancer research, helping lead and expand NCTN efforts.

NIH Research Projects · FY 2025 · 2019-05

PROJECT SUMMARY The growing role of imaging in clinical care and biomedical research has resulted in an acute shortage of well- trained clinician-scientists in Radiology. Only a small fraction of current Radiology residents pursue an academic career and much fewer get funded. Dr. Mattrey has improved upon the successful training model he developed at UC San Diego 20-year ago that he recreated at UT Southwestern (UTSW) in 2016. Applicants are selected only if they added 1+ years of mentored research during medical school while engaged in fundable research rather than rely on recruiting from clinically bound trainees. UTSW is fortunate to receive over 150 T32-qualified such applicants/year to choose from. We interview about 30/year that also performed well in medical school to ensure that they can compete with their highly competitive clinical colleagues. We also improved the UTSW training model since the last cycle by: 1) including the internship year to form a 6- year comprehensive training program, which is now mandatory for recent medical school graduates; 2) Interns will spend the 1st 9 months on clinical rotations and the last 3 months in Radiology to prepare for their research year; 3) trainees will now meet with all program faculty to compare interests rather than a select few and will rotate through a subset of faculty laboratories; 4) we have added and funded the option for trainees to spend up to 7.5 months on research after they pass their comprehensive board examination; 5) we added a mock NIH study section to the workshop run by NIH reviewers of a grant being submitted to give the PI feedback and the trainees a first-hand experience of NIH review; and 6) provided a career path from trainee to faculty for those that excelled in both clinical and research, particularly if they submit a K-award in their last year. The training model immerses trainees in mentored research and clinical training over the entire 6 years guided by both research and career mentors. Trainees are kept engaged with their research team during clinical training by providing them with 6 weeks of dedicated research time per year, and more so in their final year. This provides trainees with 21-28 months of mentored research over the 6 years giving them the opportunity to publish their results and submit grants. Their clinical training is identical and synchronized with their clinical peers making them equally skilled. Using this model our trainees over the past 6 years have published 24 papers with 9 as 1st author, presented 60 oral or poster presentations, received national awards, and were granted 6 RSNA R&E Foundation Resident Research grants 1/year the 1st 2 years and 2/year the last 2 years. UTSW and the UTSW Department of Radiology with their extensive physical and human resources are well suited to train the next generation of imaging scientists. Radiology has outstanding and well-funded mentors 11 of whom are themselves clinician scientists. Our mentors have trained over 280 postdocs in the past 10 years and have 61 current trainees. Each trainee has published on average 5.34 papers. Of the 25 mentors, 18 are currently funded as PIs. Including all 25 mentors, the average support is ~371K/year on 1.8 awards.

NIH Research Projects · FY 2026 · 2019-04

Project Summary Dr. Anand Rohatgi is a Professor in the Department of Internal Medicine/Division of Cardiology at UT Southwestern Medical Center. He has established a successful and independent research program focused on elucidating the role of HDL metabolism in cardiovascular disease. His experiences as an active clinical cardiologist, translational researcher, and mentor have positioned him to be an ideal candidate for renewal of the K24 Mentoring Career Development Award. His clinical focus on preventive cardiology, especially in the South Asian population, have paved the way for a synergistic translational research program focused on HDL metabolism and atherosclerotic CV disease and a passion for mentoring trainees and faculty. Dr. Rohatgi has applied both epidemiologic and translational POR to this objective, resulting in a number of published observations that have moved the field forward with respect to insights into pathophysiology and refinement of HDL-related markers for risk prediction and as targets of therapy. This work has been supported by continuous funding from the NIH/NHLBI (K08>R01>R01), the AHA, and industry. During the K24 funding period, he has mentored 25 trainees (some ongoing), resulting in 8 first-author papers, multiple meeting presentations, two AHA Post-Doctoral Fellowship Awards, a tenure-track faculty position, and several K and R submissions. Overall, sixteen of Dr. Rohatgi's total mentees are women. In addition, during the K24 funding period, Dr. Rohatgi launched a new South Asian Heart Clinic, began a pilot prospective POR study in this population, and is PI on an R01 studying lipid metabolism in South Asians (2022-2027). The K24 renewal award will support Dr. Rohatgi's scientific career activities by allowing him to engage in professional development via coursework/training and execution of novel projects that will enhance the primary aims of his R01s and support future independent funding. In addition to supporting scientific career activities, the K24 renewal award will directly allow Dr. Rohatgi to pursue mentorship/leadership training and enhance his ability to more deeply engage with specific mentees pursuing K and R01 awards and more broadly engage with all mentees in cardiovascular training. In the renewal period, in addition to established mentoring roles, Dr. Rohatgi will be leading R01 grant workshops and provide mentor coaching to faculty applying for K and R grants. The overall scientific aims of this proposal are to: 1) Determine the contribution of genomic factors to variability in HDL function; 2) Identify disease-relevant protein-based HDL subspecies and determine their role in cardiometabolic disease; 3) Determine the association between advanced measures of lipoprotein metabolism and cardiometabolic phenotypes in South Asians.

NIH Research Projects · FY 2026 · 2019-02

Project Summary/Abstract: Normal proteins utilize most or all of life’s 20 amino acids to fold into stable structures responsible for their biological function. Perplexingly, between 10 and 20% of the proteins found in eukaryotic cells are unusual in containing only a subset of the 20 amino acid residues utilized by normal proteins. These unusual proteins are described as being of low sequence complexity and have long been understood to exist in states of intrinsic disorder. Despite constituting no more than 20% of the proteome, upwards of 75% of all forms of post-translational modification have been mapped to low complexity (LC) domains. It is likewise the case than more than 50% of all forms of alternative pre-mRNA splicing map to LC domains. These facts predict that a disproportionate amount of cellular regulation funnels through LC domains. More than a decade ago the group led by Dirk Gorlich at the Max Planck Institute for Biophysical Chemistry in Munich, Germany described the surprising ability of a low complexity domain to become phase separated in the form of a hydrogel. The work of Gorlich and colleagues was focused on the phenylalanine:glycine-rich low complexity (LC) domains of nucleoporin proteins, and their work offered a conceptual framework for understanding how the permeability barrier of the nuclear pore might work in a mechanistic sense. Parallel work by the McKnight lab in the Biochemistry Department of UT Southwestern Medical Center yielded similar findings in studies of the tyrosine:glycine-rich LC domain of the fused-in-sarcoma (FUS) RNA binding protein. In the latter case, self-association by the FUS LC domain was postulated to represent the basis by which RNA-rich membraneless granules form in the cytoplasm of eukaryotic cells. Over the past decade the McKnight laboratory investigated the concept that self-association of LC domains is mediated by the formation of labile cross-β structures poised at the threshold of thermodynamic equilibrium. In collaborative experiments with Robert Tycko at the National Institutes of Health, the McKnight group described the first atomic structure of a labile cross-β structure. This work revealed the chemical basis accounting for both the lability of the FUS structure and the specificity of self-association. It also showed that the structure was invariantly formed from the same, limited segment of the FUS LC domain. The structure-forming region of the FUS LC domain has come to be termed a labile, cross-β core. Similarly labile and invariantly localized cross-β cores have now been discovered within the LC domains of three other RNA binding proteins (hnRNPA2, TDP-43 and ataxin-2), the phenylalanine:glycine repeats of the Nup54 and Nup98 nucleoporin proteins, and the LC domains localized within the amino terminal segments of six different intermediate filament proteins. Extensive evidence has established that cross-β cores nucleate self-association, phase separation and the biological function of LC domains. This labile cross-β concept of LC domain self-association explains how phosphorylation of specific residues can disassemble both RNA granules and intermediate filaments, how methionine oxidation can disassemble labile cellular structures surrounding mitochondria, and how idiosyncratic human mutations within LC domains can aberrantly stabilize otherwise labile assemblies leading to neurological and neurodegenerative diseases.

NIH Research Projects · FY 2026 · 2019-01

Abstract Transmembrane receptors are the major mediators of signaling for cells to communicate with the environment, playing essential roles in many cellular processes, including migration, proliferation and immunity. Malfunction of these receptors are associated with diseases such as cancer and neurological disorders. The long-term goal of this research program is to understand the general mechanisms by which signal is transduced from one to the other side of the membrane through the transmembrane region of receptors, in particular single-pass transmembrane receptors. On the other hand, each receptor has its own unique properties and mechanisms. We also study the individual characteristic features of these receptors, especially larger assemblies beyond the ligand-induced dimeric receptor paradigm. A better understanding of the transmembrane signaling mechanisms of these receptors will lay the foundation for the development of targeted therapies for associated diseases. We use structural approaches, including both cryo-EM and X-ray crystallography, in combination with in vitro biophysical and biochemical analyses and cell-based functional assays. In the past, we had focused mostly on plexin, the largest family of guidance receptors critical for the development of the nervous and cardiovascular systems. Plexin activated by the semaphorin ligand transduces repulsive signal to steer the growth cone of the neuron for the formation of the neuronal network. Plexin is also critical for regulating immunity and wound healing. In the next few years, one major goal of the work on plexin is to understand how the transmembrane regions of plexin and its co-receptor neuropilin couple the extracellular ligand-binding region and the intracellular effector region for precise controlling of signaling across the plasma membrane. In particular, we will analyze novel regulatory mechanisms endowed by large assemblies of semaphorin, plexin and neuropilin. In addition, we will expand our work to other receptors involved in the neuronal, cardiovascular and immune systems. For example, neuropilin also serves as a co- receptor for VEGF receptor (VEGFR), a receptor tyrosine kinase essential for vasculogenesis and angiogenesis. Neuropilin can dramatically increases the potency of VEGF in activating VEGFR. We will analyze the mechanism of the signaling enhancing effect of neuropilin on VEGFR. The functions of semaphorin also extent beyond plexin. Red blood cell-derived Sema7A binds GPIb, a protein complex specifically expressed on platelets, and thereby stimulates thrombo-inflammation in myocardial ischemia- reperfusion injury. GPIb contains transmembrane receptors GPIbα, GPIbβ and GPIX in a 1:2:1 stoichiometry. The best-known function of GPIb is triggering platelet activation in response to VWF (von WilleBrand Factor), vital to hemostasis. We will analyze how Sema7A and VWF bind GPIb and how the binding induces the activation of GPIb.

NIH Research Projects · FY 2026 · 2019-01

Project Summary Recent studies have pointed to roles for the cerebellum beyond its canonical roles in motor coordination, with evidence pointing to prominent roles in the regulation of non-motor behaviors and parallel evidence for the contribution of cerebellar dysfunction to numerous conditions marked by non-motor challenges throughout development1-5. Indeed, our lab has shown that cerebellar function is necessary for social behaviors and for behavioral flexibility6. During the course of our previously funded grant period, we have published multiple studies delineating the presence of critical periods in cerebellar-regulated behaviors, demonstrated a key role for cerebellar CrusI in the regulation of social and repetitive behaviors, and demonstrated an important anatomic circuit between this region and the mPFC in the regulation of these behaviors7-10. Importantly, we also demonstrated that modulation of this circuit is sufficient to improve behaviors in a cerebellar-specific neurodevelopmental model of tuberous sclerosis complex (TSC). However, important questions remain, including whether this circuit has more generalizable importance in models beyond TSC and whether circuit modulation might be sufficient to benefit, not just cerebellar-specific models, but also global, constitutive human disease-relevant models. Moreover, although we have shown that these circuits are necessary for proper performance of these social behaviors, how this circuit communicates and actually contributes to social behaviors remains unknown. In these proposed studies, we hypothesize and show preliminary data to support that these circuits are relevant to both cerebellar-specific and global neurodevelopmental models; that neuronal activity within the cerebellar CrusI-mPFC circuit processes social behaviors; that this communication is necessary for proper social behavior; that disruptions of the mPFC or CrusI impairs this communication; and that social behaviors improve upon improvement of this communication. To evaluate these hypotheses further, we propose the following specific aims: Specific Aim1. Delineate CrusI-mPFC circuit contribution to global/constitutive Cntnap2 mutant mice Specific Aim2. Delineate CrusI-mPFC communication during social behaviors Specific Aim3. Delineate integrity of CrusI-mPFC communication in cerebellar (PC-Tsc1) and constitutive (Cntnap2) mutant mice and evaluate impact of CrusI modulation on this communication In summary, this work will both delineate basic mechanisms underlying the cerebellar and mPFC circuit contributions to social behaviors while also providing a pre-clinical foundation for understanding the impact of circuit-based neuromodulation on these behaviors.

NIH Research Projects · FY 2026 · 2018-09

PROJECT SUMMARY The increasing rates of obesity and diabetes highlight the need to understand the brain circuits and cellular mechanisms regulating energy balance and glucose homeostasis. Prominent among these is the central leptin- melanocortin system, which includes the pro-opiomelanocortin (POMC) neurons, subsets of which express leptin receptors (LEPRs). While enormous strides have been made to understand the role of hypothalamic POMC neurons that produce α-MSH in metabolism, relatively little is known about how the three isoforms of - MSH produced in hypothalamus and pituitary gland POMC-expressing cells respond to metabolic challenges. The current application extends our previous discovery that LEPR-expressing POMC neurons are required for coordinating hepatic glucose production and responding to metabolic challenges. We will determine the roles for -MSH peptides produced from POMC neurons in the regulation of metabolism in dynamic challenges. This project is also significant because it will be the first to combine the power of mouse genetics with advanced mass spectrometry to quantitatively measure and map specific POMC-derived -MSH peptides in hypothalamic and pituitary tissues. We will also correlate -MSH peptide levels with parameters of energy and glucose homeostasis, and deermine which peptides underlie metabolic adaptation. These studies will broaden our understanding of the functional mechanism by which the leptin-melanocortin system regulates endocrine and autonomic functions, particularly at the level of liver and adipose tissues.

NIH Research Projects · FY 2025 · 2018-09

Parietal regions, including the posterior cingulate cortex, participate in brain networks critical for recollection, order memory, autobiographical retrieval, and episodic simulation. The importance of regions such as the posterior cingulate to episodic processing has been highlighted by data from animal models, non—invasive imaging studies, brain stimulation experiments, and rare reports that use directly recorded brain activity in humans. However, significant knowledge gaps remain related to the specific neurophysiological processes that occur within the posterior cingulate and how this region integrates with hippocampal memory networks. We propose three highly innovative experiments to address these knowledge gaps. First, we will obtain microelectrode recordings from the posterior cingulate cortex during episodic encoding and retrieval. We will identify time cells, a population of neurons that provide direct representation of temporal contextual information. We will also identify episodic boundary cells, which represent a complementary population of neurons critical for episodic construction. We will identify neuronal assemblies in the MTL and concomitant ripple activity in the PCC. Second, we will use the novel experimental manipulation of administering the anticholinergic agent scopolamine to human intracranial EEG subjects performing an episodic memory task and record simultaneous hippocampal and parietal activity (from the posterior cingulate cortex). Based on our preliminary data using this manipulation in this patient population, we predict that we will observe a decrease in activity in the 2-5 Hz `slow theta’ frequency range, as well as commensurate changes in hippocampal—parietal connectivity in the 5-9 Hz `fast theta’ frequency band. The use of scopolamine has direct relevance for understanding cholinergic modulation in hippocampal memory circuits and implications for understanding how degenerative conditions such as Alzheimer’s Disease impact these circuits. Finally, we will use direct brain stimulation applied to the posterior cingulate cortex and angular gyrus in the same experimental subjects to understand how these regions may differentially modulate hippocampal theta oscillations, building on our published work using this experimental approach. These experiments will take advantage of our unique opportunity to obtain direct brain recordings from hippocampal networks in surgical epilepsy patients. Our expertise in this area, demonstrated in our published findings from the previous funded period, supports our ability to collect these proposed data and generative novel, high value datasets that will allow us to address the knowledge gaps outlined above.

NIH Research Projects · FY 2026 · 2018-08

PROJECT SUMMARY The goal of this grant is to understand how bystander infections with intestinal parasites alter chronic infection with γ-herpesviruses. This is important because herpesviruses infect virtually all people and approximately a quarter of the world’s population is simultaneously infected with a parasite. Herpesvirus infections are chronic, but these viruses do not persistently replicate in a healthy host. Instead, they establish a quiescent infection, termed latency. We discovered previously that co-infection with an intestinal helminth parasite after infection with γ-herpesvirus led to increased virus reactivation from latency. We detailed a mechanism whereby reactivation of the virus depended on sensing host cytokines produced in response to the parasite. A remaining question is whether the timing of the dual infections is important. In this grant we propose to address this question by changing the order of virus-parasite co- infection to determine whether reactivation of the virus is increased by parasite infection when the parasite infection occurs before the γ-herpesvirus infection. We have data to indicate that prior infection with an intestinal parasite increases γ-herpesvirus reactivation, but that the mechanism is different than what we discovered previously when parasite infection occurs after the herpesvirus infection. We find that parasite infected animals have increased resident peritoneal macrophages. We also find that when retinoic acid, which is required for maintenance of resident peritoneal macrophages, is depleted in mice, parasite infection no longer increases virus reactivation. Our hypothesis is that parasite infection, in a retinoic acid dependent manner, alters the tissue composition of resident macrophages. This promotes retention of a population of infected macrophages with enhanced γ-herpesvirus reactivation. In this proposal, we aim to examine the role of resident macrophages and retinoic acid in parasite-induced herpesvirus reactivation. These studies will deliver insight into the mechanisms that drive herpesvirus reactivation during co-infection and will increase our understanding of parasite modulation of tissue resident macrophages. Harnessing the power of mouse model pathogens, these studies will advance our understanding of pathogen co-infection.

NIH Research Projects · FY 2025 · 2018-08

ABSTRACT A leading cause of microbial infections in hospitalized patients is Gram-negative bacteria, which release the cell wall component lipopolysaccharide (LPS) capable of activating innate immune pathways. The aconitate decarboxylase 1 (ACOD1) is an LPS-inducible mitochondrial enzyme that was previously implicated as a negative innate immune regulator through catalyzing the production of anti-inflammatory itaconate. However, we recently demonstrated that the LPS-induced ACOD1 up-regulation also confers a robust pro-inflammation response in monocytes and macrophages in an itaconate-independent manner. Genetic deletion of ACOD1 or its upstream signaling CDK2 in myeloid cells or pharmacological inhibition of CDK2 (with dinaciclib) uniformly attenuated infection-induced cytokine storm and animal lethality in pre-clinical setting. Clinically, the CDK2- ACOD1 axis was similarly up-regulated and positively correlated with the severity of bacterial infections in a cohort of 40 patients. Thus, our findings have suggested a novel role for ACOD1 in promoting dysregulated innate immune responses to lethal infections. Our central hypothesis is that ACOD1 exerts pro-inflammatory action through interacting with other effectors such as GIMAP7. To test this hypothesis, we will exploit a multifaceted strategy to pursue the following integrated aims. Aim 1: Define the adaptor proteins responsible for CDK2-mediated ACOD1 upregulation in monocytes and macrophages. Aim 2: Identify the effectors responsible for ACOD1-mediated pro-inflammatory cytokine production in monocytes and macrophages. Aim 3: Evaluate the efficacy of anticancer drugs in disrupting ACOD1/GIMAP7 interaction and fighting against lethal infections in preclinical settings. The completion of these studies will provide new insights into the intricate mechanism underlying infection-induced innate immune dysfunction and shed light on the development of novel therapeutic strategy for the management of lethal infections.