Univ Of Arkansas For Med Scis

NIH Research Projects · FY 2025 · 2024-06

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever. Q fever presents most often as an acute flu-like illness but can present as fatal chronic endocarditis. Unfortunately, treatment options for Q fever endocarditis are highly ineffective and require many months of antibiotic administration. We recently discovered numerous host-directed drugs that suppress C. burnetii replication within the pathogen’s target cell, the human macrophage. These drugs, which are typically used to treat psychosis- or mood-related disorders by inhibiting neurotransmitter (NTM) system signaling, potentially represent novel anti-Q fever therapies. The current proposal will define the impact of NTM-acting drugs on the ability of C. burnetii to replicate within alveolar macrophages and regulate the macrophage innate immune response to infection. Aim 1 will define the specific NTM machinery involved in C. burnetii replication in macrophages. Aim 2 will define the impact of host-directed drugs on the pathogen’s ability to dampen the cytokine response and shift alveolar macrophages to a pro-replication, anti-inflammatory phenotype. Throughout the proposal, we will use primary human alveolar macrophages and human precision-cut lung slices to provide disease relevance. Collectively, the current proposal will lay the groundwork to characterize novel anti-C. burnetii treatments.

NIH Research Projects · FY 2024 · 2024-06

Abstract The clinical landscape of metastatic melanoma has been revolutionized by immune checkpoint blockade (ICB); however, despite these advancements, the effectiveness of ICB for metastatic melanoma is limited to approximately half of patients1. The development of acquired resistance, in part through the loss of MHC-I antigen presentation, leaves most patients to experience disease progression2–5. Recent advancements have highlighted the use of adoptive cell therapy (ACT) for treatment of ICB resistant melanoma (e.g., CAR T cells)6– 8. However, the solid tumor microenvironment (TME) presents a critical barrier for success of T cell-based therapies largely due to the direct competition between tumor-infiltrating lymphocytes (TILs) and cancer cells for metabolic resources, rendering T cells dysfunctional and exhausted9,10. There is an urgent clinical need for the development of new strategies to engineer adoptive T cells with the ability to sustain function in the harsh solid TME, particularly if these approaches can be combined with other therapies (e.g., small molecule targeted therapy) to improve clinical outcomes. Our group and others have recently reported that TME-induced environmental stress leads to epigenome remodeling events within TILs, including loss of the histone methyltransferase EZH211. EZH2, a key component of the Polycomb Repressive Complex 2 (PRC2), induces the repression of gene transcription via methylation of lysine 27 of Histone H3 (H3K27me3)12. Loss of EZH2, and thereby H3K27me3, in activated T cells leads to transcriptional reprogramming that results in an exhausted T cell phenotype. Our Preliminary Data suggests that protecting EZH2 activity in activated T cells circumvents this exhaustion and produces a T cell with durable activity in the harsh TME. Preliminary Data presented herein, and evidence previously reported by independent groups, support the premise of our proposal that protection of T cell EZH2 expression and/or activity will create a more durable and potent ACT. In this innovative proposal, we will rigorously test the hypothesis that preserving EZH2 function in T cells will enable adoptive T cells to persist and function in the melanoma tumor environment. To test our hypothesis, we will pursue the following Specific Aims: (1) Demonstrate enhanced EZH2 activity can protect T cell function in the solid tumor microenvironment, and (2) Engineer EZH2i resistant CAR T cells for combinatorial treatment of metastatic melanoma.

NIH Research Projects · FY 2025 · 2024-06

Project Summary Plasmodium, the causative agent of malaria, remains one of the most prominent public health challenges today. The combined efforts of the innate and adaptive immune system lead to the control of parasite and disease burdens after infection. Macrophages and monocytes are key effector cells in the killing and removal of blood- stage parasites. The production of IFN-g promotes the recruitment and activation of monocytes in the spleen during the height of the T-cell mediated immune response against Plasmodium, leading to control of parasite burden. However, less is known about the contribution of tissue-resident macrophages to the control of parasite burden during the initial stages of the innate response before activation of the adaptive immune response. Nor do we fully understand the transcription factors that regulate their activity. Here we confirm that myeloid cell populations are essential for controlling early parasite growth in response to infection with the lethal murine P. yoelii 17XL strain. Furthermore, we provide preliminary data showing that the transcription factor Bhlhe40 is required to control early parasite burden after P. yoelii 17XL infection, as Bhlhe40-/- mice succumb to this infection at a similar time as macrophage-depleted mice. Moreover, we provide evidence that splenic macrophages and monocytes express Bhlhe40 after infection. Hence, we will test the hypothesis that Bhlhe40 regulates a transcriptional program in these myeloid cells that is required for controlling parasite burden in response to P. yoelii 17XL infection. We will test this hypothesis as part of two aims proposed here. Aim 1 will determine whether Bhlhe40 expression in macrophages and monocytes is required to promote parasite control after infection with P. yoelii 17XL. Aim 2 will determine how the loss of Bhlhe40 expression impacts the function of macrophages and monocytes in response to infection with P. yoelii 17XL. Together these studies will elucidate a role for Bhlhe40 expression in splenic macrophages and monocytes and determine how modulation of gene expression by this transcription factor impacts the host immune response to infection.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Chlamydia trachomatis is the obligate intracellular bacterium that causes the most prevalent STI worldwide. It is estimated that over 1.6 million of new cases of Chlamydia occurred in 2021 in the United States. Although most Chlamydia infections are asymptomatic, “silent” infections can lead to severe sequelae such as pelvic inflammatory disease, ectopic pregnancy and infertility. Current consensus is that prophylactic measures, such as a vaccine, are needed to flatten the ever-rising infection curve of Chlamydia. To date, there is no licensed human Chlamydia vaccine available, and only one subunit vaccine candidate completed a phase I clinical trial for safety. Understanding how protective immunity is developed in the female reproductive tract (FRT) against Chlamydia is essential for developing a vaccine. Prior research has established the essential role of CD4 T cells in protective adaptive immunity against Chlamydia. In contrast, the early innate defense mechanisms in the FRT are much less well understood in vivo. Our recent studies suggested that innate IFNg production during early Chlamydia infection is essential for preventing systemic bacterial dissemination. Moreover, increasing evidence suggest that innate immune cells can acquire immune memory characteristics through proper “training”. In this application, we propose to (i) explore the landscape of early immune responses to Chlamydia in the FRT and characterize the heterogenous Thy1+ cell population essential for protection; and (ii) characterize the myeloid cell infiltrates and determine whether trained immunity induced by BCG contribute to protective immunity and/or immunopathology during Chlamydia infection. We anticipate that identifying cellular populations and their effector functions responsible for protective immunity vs pathologic outcomes in the FRT will provide rational support for developing vaccines for STIs.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Loss of estrogens at menopause is a major cause of osteoporosis due to an increase in number and activity of the cells that resorb bone called osteoclasts. Estrogens inhibit bone resorption in part via direct effects on osteoclasts. However, the cellular and molecular mechanisms responsible for the direct effects of estrogens on osteoclasts remain unclear. The work proposed in this application seeks to identify the molecular mechanisms by which estrogens directly control osteoclast number, function, or both. We found that estrogens prevent an early stimulatory effect of RANKL – the indispensable cytokine for osteoclast differentiation – on mitochondria respiration and ATP production in osteoclast progenitors. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a mitochondria complex I-associated protein that regulates immune responses in macrophages in response to inflammatory signals. We found that ECSIT is essential for RANKL-induced stimulation of mitochondria activity and ATP production and a target for the anti-osteoclastogenic effects of estrogens. Estrogens also decrease NAD+ levels and NAD+/NADH ratio and promote the mitochondria pathway of apoptosis. The above observations form the foundation of the hypothesis that estrogens reduce osteoclast number and bone resorption by inhibiting RANKL stimulation of mitochondria, decreasing NAD+, and promoting mitochondria-mediated apoptosis. The work proposed will elucidate the independent contribution of ECSIT, NAD, and apoptosis to osteoclastogenesis and the effects of estrogens. We will also identify possible interactions among these pathways. To test the hypothesis, we will determine whether ECSIT stimulation of mitochondria activity is required for the bone loss caused by estrogen deficiency. This will be accomplished using mice lacking ECSIT in osteoclast lineage cells. In addition, we will examine the contribution of NAD to the estrogen deficiency-dependent increase in bone resorption using mice with loss-of- function of Nampt – an essential enzyme of the NAD salvage pathway – in osteoclast lineage cells. We will also overexpress mitochondria-targeted water-forming NADH oxidase from Lactobacillus brevis (LbNOX) in osteoclast cultures to examine the contribution of altered NAD+/NADH redox ratio to the effects of estrogen. Finally, we will investigate whether estrogens decrease osteoclast number by promoting mitochondria- mediated cell death using mice with simultaneous deletion of Bak and Bax – proteins required for mitochondria mediated apoptosis – in osteoclast lineage cells. To distinguish effects of estrogen in progenitors versus mature osteoclasts, we will examine changes in number and gene expression profiles of osteoclast lineage cells in vivo and in vitro using single cell RNA sequencing. Successful completion of this work should establish novel mechanisms that contribute to osteoclast development and its regulation by estrogens.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT The major life-saving treatment for severe acute liver injury (ALI) leading to acute liver failure (ALF) is a liver transplant. However, there is an enduring shortage of healthy donor livers. Moreover, even when a suitable liver can be obtained, transplant recipients face myriad life-threatening risks like graft rejection, infection, renal failure, and vascular problems, leading to death in 10-20% within a year post-transplant. Clearly, better treatments are needed. However, poor understanding of the mechanisms of regeneration specifically in ALI is a major obstacle to achieving that goal. While prior work in tissue regeneration has focused on the roles of proteins like growth factors and cytokines, our laboratory is interested in the underexplored effects of lipid second messengers. In particular, our work has revealed that the glycerophospholipid phosphatidic acid (PA) is a critical regeneration signal in the mouse model of acetaminophen (APAP) overdose, which is the most common cause of ALF in the US. Our data show that PA mediates regeneration signaling by inhibiting glycogen synthase kinase 3β (GSK3β), which normally suppresses hepatocyte proliferation and therefore tissue repair. Now, our objective in this R01 project is to improve understanding of the regeneration process by determining exactly how PA regulates GSK3β. We hypothesize that PA accumulates specifically in lipid membranes – especially in the endoplasmic reticulum – after ALI and coordinately recruits GSK3β and liver kinase b 1 (LKB1) so that LKB1 phosphorylates GSK3β Ser9 and thereby inhibits GSK3β activity, which removes the brakes on regeneration. We will test our hypothesis in two specific aims. Specific Aim 1: Detail the temporal, zonal, and subcellular localization and physical interactions of PA and GSK3β. Our prior work shows that PA deactivates GSK3β during regeneration after acetaminophen (APAP)-induced ALI. In addition, our preliminary data indicate that GSK3β can directly bind to PA. Here, we will use mass spectrometry imaging (MSI), triple quadrupole mass spectrometry, immunohistochemistry, and lipid-binding tests to characterize the location and binding of PA and GSK3β in hepatocytes in vitro and after APAP hepatotoxicity in mice. Specific Aim 2: Define the role of LKB1 in GSK3β phosphorylation and liver regeneration in the mouse model of APAP-induced ALI. Our data show that LKB1 co-precipitates with GSK3β. Furthermore, other groups have demonstrated that LKB1 binds to PA. Thus, it is highly plausible that binding to PA brings LKB1 and GSK3β together. We will use similar methods from Specific Aim 1 to describe the distribution of LKB1 during regeneration. We will then use genetic approaches to disrupt LKB1 signaling in mice and explore the effect on both GSK3β Ser9 phosphorylation and liver regeneration in APAP-induced ALI. Finally, we will assess the relative contributions of PA signaling and conventional Wnt signaling for regulation of β-catenin. Overall, the critical mechanistic insights produced in this project will be valuable to develop new therapeutic approaches for ALF, as well as other high-priority diseases involving GSK3β.

NIH Research Projects · FY 2025 · 2024-03

The clinical experience with anti-PD-1 immune checkpoint inhibition in ovarian cancer has been disappointing. The poor outcomes may at least in part be due to the highly immunosuppressive tumor microenvironment (TME) and the low tumor mutational burden in ovarian cancer, suggesting relatively limited immunogenicity and anti-tumor T cell responses. These barriers may be overcome by clinically relevant combinatorial treatments that (i) stimulate anti-tumor T cell immunity and (ii) alleviate immune suppression in the tumor microenvironment. Favorable clinical results from a recent trial of Th17-inducing dendritic cell (Th17-DC) vaccination in patients with stage III/IV ovarian cancer provided a strong foundation for Th17-DC vaccine- based combinatorial approaches to immunotherapy in ovarian cancer. Indeed, follow-up studies in a mouse model of ovarian cancer showed that Th17-DC vaccination could dramatically improve responses to anti-PD- 1 immune checkpoint inhibition. Several lines of evidence have pointed to a key role for B cell responses in the anti-tumor activity of Th17-DC vaccination. In this proposal, we will test the hypothesis that the efficacy of anti-PD-1/Th17-DC vaccination is dependent on host B cell responses in the following Specific Aims: Aim 1) Determine whether the efficacy of anti-PD-1/Th17-DC vaccination combinatorial immunotherapy is dependent on B cells We will evaluate the role of B cells in anti-PD-1/Th17-DC vaccine-induced antitumor immunity by in vivo depletion of B cells in the ID8 p53-/- and the ID8 p53-/- BRCA2-/- mouse models of ovarian cancer. Aim 2) Determine whether the efficacy of anti-PD-1/Th17-DC vaccination combinatorial immunotherapy is associated with the formation of tertiary lymphoid structures (TLS) We will correlate the prevalence and morphology of TLS in ID8 p53-/- and ID8 p53-/- BRCA2-/- ovarian tumors with therapeutic responses to anti-PD-1/Th17-DC vaccination. We will also identify TLS gene signatures associated with therapeutic responses. The potential synergy between Th17-DC vaccination and anti-PD-1 may have considerable impact for the future use of immune checkpoint inhibitors for treatment of ovarian cancer. The novel concept that the efficacy of Th17-DC vaccination (itself an innovation), either as monotherapy or combined with anti-PD-1 immune checkpoint inhibition, is ultimately dependent on B cell responses also has high impact for the field.

NIH Research Projects · FY 2025 · 2024-03

Abstract Immune checkpoint inhibition (ICI) can control intracranial disease in approximately 50% of patients with melanoma brain metastases (MBM), which is the leading cause of death for these cancer patients - implicating that immune microenvironment (IM) modulation can be therapeutic in the brain. There has been a paradigm shift in clinical management wherein immunotherapy can be considered rather than traditional surgery and/or radiation. However, there is a lack of preclinical models available that model the IM in patient MBM, which are needed to test and improve immune therapies (IT). Our goal for this R03 proposal is to develop and test a patient derived organoid model that mimics the IM found in patient MBM, which will have future applications in the development and optimization of IT strategies. The development of this model will provide foundational data to study cellular cross-talk in the IM of MBM, which will be the focus of a larger NCI R01. Our laboratory has experience using ex vivo patient derived brain organoids from surgical samples for precision medicine assays and drug screening. Our preliminary data derived from single cell RNA sequencing (scRNA-seq) of patient MBM samples demonstrates that CD8+ T cells in the parent tumor express exhaustion markers (i.e., PDCD1, CTLA4, LAG3, and TIGIT) – supporting therapeutic strategies targeting T cell activation. Further, cell-cell communication analyses of our scRNA-seq data suggests that cancer associated fibroblasts (CAFs) are the dominant cells receiving T cell crosstalk in MBM - indicating that T cells modulate the extracellular matrix. We hypothesize that patient derived organoid models need ECM to retain T cell characteristics of patient MBM and that an organoid with ECM will provide an optimal model for testing IT strategies. The aims of this study are: 1.) To identify the T cell phenotype in immunocompetent MBM organoids with and without ECM. 2.) To identify T cell phenotype following ICI treatment in MBM organoids. The development of a preclinical, immunocompetent, ex vivo model of MBM has yet to be accomplished and will facilitate the following innovations: i) establishment of biospecimen workflow that will allow for preservation of tumor stroma and immune cells in brain metastasis organoids, ii) identification of T cell-tumor cell interactions that can be targeted in the future, and iii) identification of biomarkers of ICI efficacy in the brain. The innovations gained by this R03study will contribute to the lives of patients with end stage melanoma as brain metastases is the leading cause of death in these patients. Our data will provide a basis for a future NCI R01 application.

NIH Research Projects · FY 2026 · 2024-03

Summary Cancer affects the health of millions of Americans. Studying molecular mechanisms that endow cells with malignant properties is an essential component of advancing pre-clinical studies and a key part of efforts to improve patient outcomes. The purpose of this COBRE submission is to establish the Center for Molecular Interactions in Cancer (CMIC) at the University of Arkansas for Medical Sciences (UAMS). The mission of the CMIC is to study molecular features and functional properties of biomolecules that drive cancer. The unifying theme of research among Center members is the coupling of structural biology and high-resolution imaging with precise, quantitative analysis of biochemical and cellular processes to understand how molecular interactions govern the initiation, progression and treatment of cancer. In Phase 1 of this COBRE, we will pursue the following aims: (Aim 1) establish the CMICcreating an organizational framework that ensures the success of junior investigators as independent scientists and strengthens the research infrastructure at UAMS and its affiliate institutions, (Aim 2) build a thematic, multidisciplinary COBRE center with unique collaborative opportunities and strong research programs focused on understanding the molecular basis of cancer, and (Aim 3) implement a plan for long-term sustainability of the CMIC. The organization of the CMIC will include 5 Research Projects, an Administrative Core, and 2 new research coresthe Structural Biology Core and the Biomolecular Interactions Core. The Administrative Core will oversee operation of the Center, including a Pilot Project Program to fuel the pipeline of replacement Research Project Leaders (RPLs) and a formalized Faculty Development Plan that includes individualized mentoring. The Structural Biology Core will help RPLs plan and execute structure-function studies by ensuring high-quality sample preparation and providing access to world-class instrumentation and computational resources required for high-resolution structural studies. The Biomolecular Interactions Core will enable researchers to perform quantitative analysis of macromolecular interactions and dynamics down to the level of single-molecules. Both research cores will assist users in experimental design, data analysis and interpretation of results, while also providing training and educational outreach. The CMIC will address an institutional priority at UAMS and contribute to the mission of the Winthrop P. Rockefeller Cancer Institute by creating a critical mass of investigators who are able to gain molecular-level insights into mechanisms that are crucial for a deep understanding of cancer biology. Our long-term goal is to leverage faculty mentoring, strategic recruitment, and cutting-edge core resources to develop a critical mass of investigators that will support a self- sustaining center in which research advances our knowledge of cancer through precise and comprehensive analyses of molecular events that impact malignant pathogenesis.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT The low number of college graduates from UR groups in Arkansas, together with the small number of UR students nationally who graduate in the biological sciences, results in a small pool of UR students entering fields of biomedical research in Arkansas. To address this shortfall, the University of Arkansas for Medical Sciences (UAMS) created the Initiative for Maximizing Student Development (IMSD) Program was created in 2009 to specifically provide a comprehensive start-to-finish mentoring and training program that includes 2 years of salary/tuition/fees support for UR students pursuing biomedical PhD degrees. The overall goals and objectives of the UAMS IMSD Program are (1) to enhance our success in retaining UR students and (2) to refine strategies to recruit additional UR students. Our specific, measurable objectives are to matriculate 6 UR doctoral students each year and to graduate 90% of these students. Specific Aim 1 is to matriculate 6 new UR doctoral students each year. In the year prior to implementation of the IMSD Program (2008), 10 of 91 (11%) domestic PhD applications were UR students. In the fall of 2022, 24.7% of domestic PhD applications to GPIBS and Pharmaceutical Sciences at UAMS were from UR students; this shows a significant increase from previous years. Specific Aim 2 is to retain and graduate 90% of the students in the UAMS IMSD Program. We will work to retain and graduate IMSD students by providing the preparation and support necessary for student success. We will provide support in numerous ways. First, we will enhance our PhD Summer Transition Program for matriculated IMSD doctoral students. The PhD Summer Transition Program will include new training in biomedical informatics and additional career and professional development workshops, along with workshops on mentoring, cultural competency, implicit bias, mindfulness, resilience, and health disparities research. We will create activities to enhance the communication skills of students, implement an Individual Development Plan (IDP), enhance our team-mentoring plan, and use IMSD students as mentors in a Near- Peer Mentoring Program. We will continue the peer mentoring program, faculty research seminar series, and student training on academic portfolios already implemented in the current IMSD program. Lastly, we will partner with UAMS and other T32 programs to expose IMSD students to training grant opportunities. Specific Aim 3 is to identify and further improve the program components that contributed to past program success, and disseminate the results. Program evaluation will be through a logic model, and results will be disseminated to the UAMS campus and other schools through the website, newsletters, presentations, and publications. Together, our proposed aims will contribute to the global recruitment, retention, and graduation of UR students at UAMS, ultimately helping to boost the number of UR faculty and investigators in the biomedical sciences serving Arkansas and the nation.

NIH Research Projects · FY 2025 · 2024-01

ABSTRACT Tumor-associated macrophages are critical regulators of tumor development, progression, and metastasis. Factors in the tumor microenvironment induce tumor-associated macrophages to adopt a tumor-supportive phenotype that suppresses immune responses. The accumulation of tumor-supportive macrophages in tumors is associated with accelerated tumor progression and a poor patient prognosis. Macrophages sense and respond to their environment by expressing pattern-recognition receptors such as Class-A Scavenger Receptors (SR-A). In preliminary data, we show that SR-A binds to glycans expressed on breast cancer cells but not on non-cancer cells. Furthermore, we show that SR-A expression promotes the development of palpable tumors and lung metastases in mouse models that spontaneously develop breast cancer. Importantly, results from gene expression profiling indicate that tumor-associated macrophages adopt an immune-suppressive phenotype in SR-A-expressing mice but not in SR-A knock-out mice. These findings support the novel conclusion that SR-A binding to tumor-specific ligands induces the polarization of tumor-infiltrating macrophages toward an immune- suppressive phenotype that enhances breast cancer growth and metastases. It logically follows that inhibiting SR-A interactions in tumors may provide an innovative approach that repolarizes TAMs to an antitumor phenotype, improves the effectiveness of anti-tumor immune response, and inhibits breast cancer progression. Thus, the goals of the proposed experiments are to i) identify the ligands on breast cancer cells that bind SR-A and induce macrophage polarization toward a protumor phenotype, and ii) demonstrate that antagonizing SR-A repolarizes macrophages toward a tumor-inhibitory phenotype, enhances the immune response, and prolongs survival in preclinical models of breast cancer. Showing that antagonizing SR-A interactions increases anti-tumor immune activity and prolongs survival in animal models will confirm the therapeutic potential of this approach to treat breast cancer. In addition, identifying tumor-associated ligands that interact with SR-A may suggest additional tumor-specific strategies for inhibiting these interactions.

NIH Research Projects · FY 2025 · 2023-09

Arkansas is a rural southern state with a high toll of homicide relative to its total population (AR ranks 6th nationally in firearm homicide rates). Furthermore, most counties in Arkansas are considered rural and have severely limited healthcare services appropriate for survivors of violent assault (e.g. physical therapy, supportive medical care, behavioral health services, etc.). The University of Arkansas for Medical Sciences (UAMS), the lead institution for the proposed research, is the only Level 1 trauma center in the state and provides healthcare to Arkansans in nearly every county, largely through regional satellite clinics and telemedicine services. Importantly, UAMS recently partnered with the City of Little Rock and other community agencies to start up the first and only hospital based violence intervention program (HVIP) in the state of Arkansas. While the focus of this HVIP is on residents of Little Rock, our proposed study will extend the reach of the UAMS program to include counties within the Central Arkansas region, many of which have sizable rural populations impacted by community violence. Importantly, our proposed research extends the reach of our City of Little Rock based efforts and incorporates multiple enhancements such that it is a more comprehensive approach to addressing community violence. In this application, we propose to engage community partners throughout Central Arkansas in a multi-level, multi-faceted intervention to reduce the toll of violent assault on individuals and communities. Our proposal will engage community at every stage such that it will be contextually appropriate, community-informed and community led. We will use an optimization randomized control trial (ORCT) design to test the preliminary efficacy of our multi-level intervention with an explicit focus on secondary violence prevention through hospital-community partnerships to prevent escalation of firearm violence among survivors living in counties in Central Arkansas, the region of the state where violent assault is greatest. We will also incorporate key implementation science metrics to evaluate the feasibility and reach of our proposed intervention.

NIH Research Projects · FY 2025 · 2023-09

Patients with skeletal dysplasias, including osteogenesis imperfecta (OI), often suffer from congenital respiratory problems that have limited therapeutic opportunities, and increased perinatal and early childhood mortality. OI is caused by dominant mutations in COL1A1 and COL1A2 genes or by loss of function of genes involved in COL1 processing (e.g., CRTAP, encoding an essential protein for collagen post-translational modification and folding). The current concept is that the respiratory abnormalities in OI are secondary to the skeletal defects, such as thoracic wall deformities and deviations of the spine curvature (kyphoscoliosis), which do not allow proper expansion and inflation of the lungs leading to restrictive disease. We demonstrated that COL1 production is dysregulated in lung fibroblasts from a mouse model of OI lacking the Crtap gene (CrtapKO). These mice also exhibit defective lung alveolar formation, loss of alveolar epithelial cells, and several changes in genes expression, including decreased myofibroblast markers, as quantified by spatially resolved transcriptomics. In addition, CrtapKO mice and two others mouse models of OI with COL1 mutations (Col1a2G610C/+ and oim/oim) exhibit altered respiratory mechanics at 3 months of age. Based on this evidence and because COL1 is expressed in most tissues including the lung, our central hypothesis is that the respiratory defects in patients with OI and other skeletal dysplasias are due to intrinsic lung dysfunction that, in the future, could be treated and/or corrected independently from the skeletal fragility. The specific goals are 1) dissect the contribution of intrinsic lung defects versus extrinsic skeletal defects to impaired lung functions in OI; and 2) unravel cellular and molecular mechanisms triggered by COL1 defects leading to abnormal lung development and impaired respiratory function. To test our hypothesis, we assembled a team with complementary expertise that is uniquely positioned to accomplish our goals. The specific aims are: to determine the respiratory phenotype of a novel knock-in mouse model expressing a classical Col1a1 OI glycine substitution mutation in the lung but not in the skeleton (Col1a1Flox/+;Tbx4-Cre) and compare it to that of mice expressing this mutation globally (aim 1). To identify the cause of impaired alveolar morphogenesis and key pathways contributing to changes in alveolar mesenchymal-epithelial cell interactions in CrtapKO cell cultures and organoids (aim 2). To identify causes of impaired alveolar morphogenesis and abnormal patterning and function of lung cells in CrtapKO mice in vivo by analyzing the entire transcriptome in 5-7 µm lung sections with 100 µm or better lateral resolution during the critical stage of alveolar formation (aim 3). Together, aims 1-3 will provide mechanistic insights and establish the relationship between the collagen matrix and cellular dysfunction causing lung-intrinsic defects in OI leading to a better understanding of the role of the matrix in the last stage of lung development. Ultimately, this work will provide new insights into more common diseases of collagen dysregulation in the lung such as fibrosis and bronchopulmonary dysplasia.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Three percent of liveborn babies in the US have a major structural birth defect. The emotional, psychological and economic impact of birth defects on families, society and the healthcare system is significant. Inpatient hospitalization costs associated with birth defects for children and adults with birth defects in the US in 2013 was $22.9 billion, which was 3% of all hospitalizations and 5% of total hospital costs. Children who survive infancy with a major birth defect have a high risk of other chronic short- and long-term health conditions and diminished quality of life. Although birth defects pose a significant public health challenge, public heath efforts to prevent most birth defects are limited. About 70% of cases of birth defects have unknown causes but are thought to arise from complex interactions between environmental and genetic factors. Thus, studies are needed to identify risk factors that can be modified to ultimately prevent birth defects. The long-term goal of our research is to determine the etiology birth defects and long-term health effects of teratogens and birth defects on maternal, and child health and development. The overall objective of this proposal is to elucidate the multifactorial etiology of structural birth defects. The rationale for this project is that the Centers for Disease Control and Prevention launched 2 multisite, population-based, case-control studies, the National Birth Defects Prevention Study (NBDPS), which ended in 2013 and the Birth Defects STudy to Evaluate Pregnancy exposureS (BD-STEPS) which began in 2014. The Arkansas Center for Birth Defects Research and Prevention, a site in the NBDPS for 16 years and a site in BD STEPS for 9 years, proposes to attain the objective through 4 specific aims: (1) identify and characterize pregnancies affected by specific birth defects and livebirths without major birth defects to participate as a Center in BD-STEPS; (2) conduct collaborative and local epidemiologic studies using NBDPS, BD-STEPS, and other data sources to identify potentially modifiable risk factors for specific birth defects; (3) conduct collaborative and local genetic and epigenetic studies using genetic data from the NBDPS, BD-STEPS, and other sources to identify genetic or gene-environment factors associated with birth defects; and (4) train and mentor the next generation of research scientists in birth defects epidemiology, implementation, conduct and analysis of population-based research. Our statewide population-based birth defects registry, sole academic health center, sole tertiary pediatric hospital, central health department and diverse high-risk population of reproductive age women, make Arkansas an ideal site for BD-STEPS. Through our continued participation in BD-STEPS, we will maintain a leading role in identifying modifiable risk factors that can be used to develop public health strategies to prevent birth defects.

NIH Research Projects · FY 2025 · 2023-09

Opioid overdose is the leading cause of death among people recently released from incarceration. Yet, while there is growing research on opioid use disorder (OUD) and medications for OUD (MOUD) in jails and prisons, studies find that few people who are referred to post-release community MOUD initiate treatment. Emerging research suggests that therapy for posttraumatic stress disorder (PTSD), a common and deleterious OUD comorbidity, could profoundly increase the likelihood of MOUD engagement; however, this has not been tested in jails and considerable work to first tailor PTSD screening and treatment to jails and to people with OUD/PTSD in this setting would be required. Therefore, this 6-year R61/R33 aims to develop and test innovative approaches to PTSD screening and treatment among people who are eligible for jail-based OUD services. In the R61, we will form a coalition of carceral, community, and academic stakeholders to collaboratively adapt the Screening, Brief Intervention, and Referral to Treatment model to the jail context and to the needs of jailed adults with OUD/PTSD (SBIRT-J). In the R33, we will 1) assess the implementation outcomes of SBIRT-J for linking jailed adults with OUD/PTSD to therapy for PTSD via a summative evaluation guided by the Consolidated Framework for Implementation Research and 2) assess the effectiveness and implementation outcomes of competing models of PTSD therapy timing. To evaluate the latter, we will use a patient-randomized Hybrid type I implementation-effectiveness design in which jailed adults identified as having OUD/PTSD through the SBIRT-J model are randomly assigned to either immediate initiation of therapy for PTSD in jail or initiation of PTSD therapy upon release. Our primary effectiveness outcome will be post- release MOUD initiation at 6-months post-release from jail; secondary outcomes will include MOUD retention, OUD severity, PTSD symptoms, victimization, and additional drug use. Participants in the effectiveness portion of the trial (N = 304; 50% female) will be enrolled from the largest jail in Arkansas. Importantly, our study vision aligns with NIDA's Strategic Plan, as we aim to “support research to scale up the application of evidence- based interventions for SUDs, including in justice settings” (Goal 4.3). Our overall goal is to translate research to practice to increase the provision of high-quality care for justice-involved persons with OUD, consistent with the goals of NIH's HEAL Initiative. Indeed, this study will be the first trial of a treatment for PTSD in jails as a method for improving OUD outcomes, providing foundational information on PTSD as a novel intervention target for meeting the needs of a particularly vulnerable population and providing the implementation data to inform rapid scale-up if effective.

NIH Research Projects · FY 2025 · 2023-09

Abstract Tumor cells in TNBC tissue exhibit enhanced cell plasticity and stemness, which is crucial for cancer metastasis. Metabolic and epigenetic reprogramming plays crucial roles in the regulation of cell plasticity. Succinate is an intermediate metabolite of the tricarboxylic acid (TCA) cycle. Aberrant accumulation of succinate has been detected in many cancers. However, the cellular function and regulation of succinate in breast cancer development and progression have not been well investigated. We showed that the epithelial-to-mesenchymal transition (EMT) was associated with profound changes in metabolites, including elevation of cytoplasmic succinate. Importantly, treatment with the membrane permeable succinate was sufficient to induce mesenchymal phenotypes, enhance cancer cell stemness and colonization, and reduce DNA hydroxymethylation and gene transcription in mammary epithelial cells. Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) catalyzes lysine hydroxylation in endoplasmic reticulum and generates succinate as another product. We found that PLOD2 expression was induced in cancer cells in TNBC tissue, and increased PLOD2 expression correlates with poor prognosis and high chances of cancer metastasis. Silence of PLOD2 in cancer cells significant reduced cancer cell colonization and metastasis. PLOD2 expression was induced during EMT and associated with elevation of cytoplasmic succinate levels. Using gain- and loss-of function approaches, we showed that increased PLOD2 expression is necessary and sufficient to enhance the accumulation of cytoplasmic succinate. Importantly, silencing PLOD2 in breast cancer cells inhibited cancer cell stemness. The overall objective of this project is to define roles of the PLOD2/succinate axis in regulating cancer cell plasticity and stemness, and to evaluate biological activity of the new PLOD2 inhibitor in suppressing TNBC progression and metastasis. The central hypothesis of this proposal is that PLOD2-induced succinate accumulation enhances cancer cell plasticity and metastasis by reducing DNA hydroxymethylation; therefore, targeting the PLOD2/succinate axis is a potential strategy to halt TNBC progression and metastasis. To test this hypothesis, we propose the following aims: Aim 1. Elucidate the molecular and cellular mechanisms by which succinate enhances cancer cell plasticity and stemness. Aim 2. Define roles of the PLOD2/succinate axis in regulating breast cancer progression. Aim 3. Evaluate the potential of targeting PLOD2 to suppress breast cancer metastasis. Successfully completion of the project will reveal novel function of collagen hydroxylation enzymes in regulating succinate-associated epigenetic reprogramming and identify PLOD2 as a potential therapeutic target to suppress breast cancer metastasis.

NIH Research Projects · FY 2024 · 2023-08

Shigella spp. are major enteric pathogens, causing acute diarrhea and bacillary dysentery leading to severe mortality and morbidity worldwide. Yet, there is no licensed vaccine to prevent shigellosis. Shigella virulence requires a T3SS and at least 30 secreted effectors that are often functionally redundant, yet required to invade host cells, maintain a replicative niche, minimize alarm signals, and promote colonization. We previously showed that S. flexneri T3SS activity is detected in macrophages by Caspase-1 inflammasomes, resulting in pyroptosis. In the recent mouse shigellosis model, the role of the inflammasome is only focused on gut intestinal epithelial cells (IECs). However, it is generally believed that Shigella initially infect macrophages and takes advantage of pyroptotic cell death to exit the cells and subsequently infect IECs. On the other hand, macrophage pyroptosis is known to generate pore-induced traps (PITs), trapping, and neutralizing intracellular bacterial pathogens. Since macrophages pyroptosis is considered to play dichotomous roles during Shigella infection, we propose to investigate the interaction between macrophages' inflammasomes and S. flexneri. We propose two specific Aims: In Aim1, we will investigate how pyroptotic macrophages from PITs trap intracellular bacteria. In Aim 2. We will define the role of pyroptotic macrophages and PITs during S. flexneri infection in vitro and in vivo. We hope that examining the role of macrophages pyroptosis against S. flexneri infection, will be highly significant and relevant for better understanding immunity and disease pathologies during Shigella infection and thereby providing the basis for developing novel safer, and more effective vaccines.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract The increase in diabetes patients in countries where tuberculosis (TB) is also endemic has led to the re-emerging importance of diabetes as a serious risk factor for TB. There is an urgent need to implement strategies for TB prevention and control among the millions of diabetes patients exposed to Mycobacterium tuberculosis (Mtb), the causative agent of TB. Although diabetes is known to modulate immune responses, most of the studies on TB-diabetes comorbidity have been primarily focused on the altered functions of lymphocytes. Lung macrophages are among the first host cells that respond to Mtb and are recognized as one of the most crucial cell types in determining the consequences of disease. Our previous work has demonstrated that lung macrophage metabolism plays a critical role in promoting or controlling the progression of TB. However, whether the increased susceptibility to TB in diabetes is caused by altered metabolic activities in lung macrophages is virtually unknown, thus representing a significant knowledge gap. The phenotype and functions of tissue-resident macrophages are greatly influenced by the level of nutrients in their environmental niches. Given that diabetes induces chronic hyperglycemia, a key factor that contributes to the development of TB in diabetic conditions, we hypothesize that the altered metabolism in lung macrophages, due to the hyperglycemic environment, leads to increased susceptibility to TB in diabetes. We will test this hypothesis with two aims: Aim 1. Determine the impact of hyperglycemia on the metabolic status and permissiveness of lung AMs during Mtb infection. Aim 2. Interrogate how hyperglycemia influences the heterogeneity of lung macrophages in TB. We will use multi- disciplinary approaches, including metabolism, genomics and microbiology to interrogate the underlying mechanism of TB-diabetes comorbidity from a completely novel perspective.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Lymphedema is a major complication after radiation and/or surgery for breast and gynecological cancers. Advancements in surgical techniques mitigate the risk, but the incidence of lymphedema is still high, and there are no approved medications to prevent or treat it. Doxorubicin (DOX) is a central chemotherapy drug for treating breast and gynecological cancers, but it increases the risk of lymphedema by 3-fold. The mechanism by which DOX contributes to chronic lymphedema is unknown, but we found that clinically relevant concentrations of DOX acutely inhibit lymph vessel (LV) contractions and reduce lymph flow by activating ryanodine receptors (RYRs, intracellular calcium channels) in lymph muscle cells (LMCs), resulting in tonic Ca2+ leak from the sarcoplasmic reticulum (SR) and lymphostasis. Sustained high levels of cytosolic Ca2+ [Ca2+i] can promote lipid peroxidation and cell death pathways, and the increase in intraluminal pressure produced by lymphostasis can damage LV walls and valve leaflets, synergistically causing chronic lymphatic injury. It is unclear whether DOX activates RYRs through a direct interaction or indirectly by mediating the oxidation of RYRs. Indeed, DOX elevates both cytosolic and mitochondrial superoxide (O2•-), which could contribute to RYR oxidation (receptor opening) and subsequent Ca2+ leak. It is also unknown which RYR subtype (RYR1, RYR2, RYR3) is activated by DOX in LMCs; if known, it could serve as a potential therapeutic target to prevent DOX-induced lymphatic dysfunction. We propose RYRs are novel therapeutic targets in LMCs to prevent DOX-induced lymphatic dysfunction and the development of chronic lymphedema. We hypothesize that DOX generates O2•- to acutely oxidize and open RYRs to increase [Ca2+i] in LMCs, inhibiting LV contractions and inducing lymphostasis and lymphatic injury, and added surgical insult potentiates this effect. Accordingly, we will use our well-established rat model to evaluate RYRs as therapeutic targets to prevent DOX-induced lymphatic dysfunction. Three aims will integrate techniques in preclinical studies to explore this hypothesis and will rely on protein and functional analysis of isolated LVs, use optical imaging to assess volumetric lymph flow in vivo in response to DOX and RYR blockade, and investigate the utility of RYR blockers, as a potential therapeutics in a preclinical model of lymphatic insufficiency. Aim 1 will determine whether DOX-induced RYR activation is mediated by O2•- in LMCs. Aim 2 will define the role of RYR subtypes in DOX-induced Ca2+ leak in isolated LVs and in lymph flow in vivo. Aim 3 will investigate the combined impact of DOX ± RYR blocker on lymphatic function, lipid peroxidation, and lymphatic morphology in a preclinical rat model of lymphatic insufficiency. Thus, we plan to explore RYRs as novel therapeutic targets to prevent DOX-related lymphedema and evaluate whether RYR blockers can be utilized as anti-lymphedema agents.

NIH Research Projects · FY 2024 · 2023-08

Project Summary The Kaposi's sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining B cell malignancy, primary effusion lymphoma (PEL). KSHV is found in all tumor cells and is tightly latent in >95% of infected cells. Latency is an intricately organized program that highly restricts gene expression to a handful of genes (i.e. latency genes). PEL cell lines require these latency genes to be constitutively expressed while the lytic genes are silenced for survival. Thus, both expression of the latency genes and maintenance of the latency program are critical for PEL cells. However, the contributions of most viral genes to the survival and proliferation of PEL-derived cell lines are unknown. Our long-term goal is to accelerate our understanding of the biology of this AIDS-defining lymphoma by comprehensively analyzing viral factors required for PEL tumor cell survival. We, therefore, performed an RNA-targeting CRISPR/CasRx tiled screen to identify KSHV transcripts required for the survival of PEL cells. Our results reveal that the left-hand origin of lytic replication (OriLytL) encodes a previously unannotated RNA critical for the survival of PEL cell lines. The OriLytL serves as one of two initiation sites crucial for viral DNA replication during reactivation from latency. In this lytic phase, the OriLytL produces two long noncoding RNAs required for lytic viral DNA replication. However, our data suggest that the latency-specific OriLytL (OriLytLlat) RNA is distinct and functions differently from the lytic OriLytL RNAs. Our central hypothesis is that the OriLytLlat RNA acts to maintain the latency program. Since latency is crucial for the survival of tumor cells, the OriLytLlat RNA is a critical oncogenic driver of KSHV-associated cancers. The over-all objective is to characterize this latency-specific OriLytLlat RNA and determine its role in latency maintenance in PEL. In Aim 1, we will characterize the latent viral transcriptome, including the full-length OriLytLlat RNA using long-read RNA sequencing. In Aim 2, we will define the role of the OriLytLlat RNA in the recruitment of chromatin binding factors and resulting epigenetic changes in the viral genome that contribute to establish and maintain the latency program. Together, this work will provide a new model on how viral latency is influenced by a latent RNA from the lytic origin of replication. Importantly, our studies will also shed light on the biology of other KSHV-associated malignancies, including Kaposi’s sarcoma.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Over the past few decades, our knowledge of the mechanisms by which cells interact with drugs and toxins has exploded due to new molecular analysis techniques and the application of genomic methods. Accordingly, the emphasis of graduate education in the disciplines of pharmacology and toxicology needs to shift from a reductionist view to a systems approach in which doctoral students are comprehensively trained so they can formulate a strategy to solve important biological questions not only at the molecular, cellular, and tissue levels but also at the whole-animal level. Systems pharmacology and toxicology describes a field of study that considers the broad view of drug action. A systems approach using in vivo animal models is necessary to establish efficacy, safety and the pharmacodynamic/pharmacokinetic profile of candidate drugs but there is a shortage of students trained in this area. This Training in Systems Pharmacology and Toxicology (T-SPaT) Program is designed for PhD students in their second year of graduate study pursuing dissertation research projects in the pharmacological sciences. Trainees (2/year for up to 2 years of support) and 33 Program faculty are drawn from the Graduate Program in Interdisciplinary Biomedical Sciences (GPIBS) and the MD/PhD Program. The T-SPaT program will train students to use an in vivo approach to answering relevant questions in pharmacology and toxicology with emphasis on metabolism, drug design, pharmacodynamics, pharmacokinetics, and signaling. The rationale for the T-SPaT program is that this type of training will provide students with a much broader perspective on pharmacology and toxicology that will better prepare them to conduct safe, ethical and rigorous research as leaders of multidisciplinary research teams in the pharmacological sciences. We will integrate T- SPaT into PhD training programs already active at our graduate training sites in the Little Rock area that include faculty/scientists in the Colleges of Medicine, Pharmacy, and Public Health on the UAMS, the Arkansas Children’s Hospital campus, and at the Food and Drug Administration-funded National Center for Toxicological Research. The unique focus of T-SPaT is training with in vivo systems pharmacological and toxicological approaches and concepts. The objective of T-SPaT is to provide in vivo pharmacology and toxicology training that complement the cellular and molecular training that students receive in their home programs. The training program consists of didactic training in pharmacology, toxicology, physiology, pharmacokinetics, metabolism, biostatistics, grant writing, and the Responsible Conduct of Research along with laboratory research using an in vivo model of human disease. The T-SPaT program will also provide strong mentoring, extensive networking, and teaching and leadership opportunities for its trainees through its programmatic activities.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Multiple myeloma (MM) is characterized by the growth and accumulation of monoclonal malignant plasma cells in the bone marrow. MM remains incurable due to its high rate of relapse and the development of drug resistance to therapy. Moreover, MM causes a devastating bone disease, increasing fracture risk and decreasing quality of life. Notch signaling, a pathway mediating cell communication in the MM tumor microenvironment (TME), promotes MM proliferation and MM cell survival and stimulates bone destruction. Notch inhibition decreases MM growth and bone disease in preclinical models, but causes unwanted severe, dose-limiting side effects. The long-term goal of this proposal is to examine Notch receptor 3 (Notch3), a membrane-bound Notch receptor integrating cell-to-cell Notch signals from surrounding cells, as a target to treat MM. The premise of these studies is supported by published and preliminary work showing that 1) cells of TME activate Notch signaling and increase Notch3 expression in MM cells, 2) Notch3 signaling in MM cells promotes MM cell proliferation and bone destruction, and 3) Notch3 signaling dictates MM cell responses to Bortezomib-based therapies. The specific goal of this application is to evaluate the efficacy of targeting Notch3 to decrease tumor growth, mitigate bone disease, and improve therapeutic responses to front-line therapies in MM. The central hypothesis of this proposal is that Notch3 in MM cells integrates TME signals leading to tumor growth, bone destruction, and drug resistance. This hypothesis will be tested in two specific aims. Aim 1 will determine the effects of the pharmacological inhibition of Notch3 on MM growth, bone destruction, and its impact on the transcriptome of TME and MM cells. Aim 2 will evaluate the role of Notch3 signaling in MM cells on TME-induced resistance to Bortezomib-based therapies and identify the underlying molecular mechanisms. To complete these aims, I have assembled a mentoring team of leaders in cancer and drug resistance, bone biology, bioinformatics, and imaging that will allow me to receive a unique multidisciplinary and rigorous training, preparing me to pursue a career as an independent academic scientist.

NIH Research Projects · FY 2025 · 2023-06

Policies to facilitate participation in school nutrition programs could reduce diagnosis rate disparities in behavioral disorders, namely attention-deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and conduct disorder (CD). Food insecurity predisposes children to these conditions and aggravates their symptoms. It impairs child development, contributes to inattention, and is associated with externalizing behaviors. Preliminary data show that two school meal delivery options could make a difference. One is the Community Eligibility Provision (CEP), which enables schools to provide free meals to all enrolled children, regardless of household socio-economic status. The other is making school breakfast available after classes begin (breakfast after the bell). There is growing evidence that these meal-delivery options reduce disciplinary incidents at school, but no studies have incorporated information on diagnosis or utilization of services for behavioral disorders. Our central hypothesis is that better access to school meals reduces the burden of behavioral disorders among children in underserved communities who face an elevated prevalence of food insecurity. There is evidence that exclusionary discipline (i.e., suspension or expulsion) contributes to an escalation of behavioral issues, which may predispose the child to an ODD or CD diagnoses and/or interactions with the juvenile justice system. Universal free meals and breakfast after the bell increase participation in school meals and are feasible meal delivery options for many schools but remain underutilized. Using a novel longitudinal all-payer claims database linked to educational achievement and disciplinary outcomes, three specific aims are proposed: 1. Describe variation in medical diagnoses for ADHD, ODD, and CD across schools by race, ethnicity, sex, age, rurality, and neighborhood characteristics. 2. Quantify the effect of school meal delivery policies and medical diagnoses on disciplinary outcomes and academic achievement by race/ethnicity, rurality, and socioeconomic status. 3. Determine the effect of universal meals and breakfast after the bell, alone and in combination, on the likelihood of diagnosed childhood behavioral disorders and subsequent service utilization overall and by race, ethnicity, and rurality. The study will draw from the population of Arkansas public schoolchildren. Arkansas ranks second in the nation for the percentage of children diagnosed with ADHD, third in the percentage of children taking ADHD medications, and second in the incidence of childhood food insecurity. Understanding whether policies to make meals more accessible prevent the medical diagnosis of behavioral disorders that can escalate into disciplinary events at school will have significant implications for improving health-related quality of life, human capital development, and fewer encounters with the juvenile and adult criminal justice systems.

NIH Research Projects · FY 2026 · 2023-05

We established the pluripotent interleukin-1β (IL-1 β) cytokine as a significant player in the pathogenesis of Alzheimer’s disease as it sets in motion a self-amplifying positive-feedback cycle in which neuronal stress induces synthesis of the neuron's acute phase protein β APP and release of its fragments sAPPa and Aβ. Both these proteins activate microglia and a progressive elevation of IL-1 β, which drives chronically enhanced formation of the hallmark aggregates of AD: A β plaques and, via IL-1 β -induced synthesis and activation of specific kinases, hyperphosphorylation of tau in neurofibrillary tangles in neurons and glial fibrillary acidic protein (GFAP) in astrocytes. Interestingly, both events are dramatically enhanced in AD patients who inherit the Alzheimer gene from both parents (genotype ApoE4,4). Although these IL-1 β and ApoE genotype driven events favor negative outcomes, our progress in drug-development initiatives shows that they are amenable to treatment. Specifically, GFAP-binding chemical agents were shown to inhibit protein aggregation. Further, we discovered a novel function of the APOE£4 gene, which is a toxic gain-of- function exhibited by its protein product, ApoE4. We have demonstrated that ApoE4 competes with Transcriptional Factor EB (TFEB) for binding to the CLEAR DNA motifs, thus, hindering the transcription of three proteins crucial for lysosomal autophagy. We show this to be the case in brain tissues from AD 4,4, but not AD 3,3 patients. Now, importantly, we have identified a lead compound that binds to ApoE4 protein, obviating its interactions with CLEAR DNA and restoring the expression of three autophagy genes, that encode for production of p62, LC3B, and LAMP2 proteins. Now, we are prepared to further elucidate the role of IL-1 β in cellular pathophysiology, establishing its effects on kinases and kinase targets that manifest the modifications which drive predominant aggregate nucleation or propagation events. Our advanced molecular and histological approaches will be applied to confirm predictions of protein-protein interactions derived from proteomics approaches, particularly those involving GFAP. Moreover, these interactions can now be evaluated across brain regions and disease states to test their concordance with known AD parameters. Finally, we will elucidate the mechanisms of action of our identified novel small-molecule drugs targeted to ApoE4 in inhibiting all its pathognomonic interactions with other ApoE4 targeted DNA sequences. Successful completion of our proposed work promises a preventive strategy for foiling the known dramatic role that ApoE4 plays in Alzheimer neuropathogenesis. RELEVANCE: Successful completion of this proposed work will obviate the many pathognomonic aspects of inheritance of the Alzheimer gene (APOEE4) in the 1 in 4 individuals in the US, i.e., 80 million, who inherit one or both copies of this gene. Through the action of our specific drug candidates to inhibit the ApoE4 protein we will, therefore, restore the lysosomal autophagy necessary for efficient clearance of large aggregates, which, as we reported, is repressed in the brains of those who inherit one or both APOE4 genes.

NIH Research Projects · FY 2026 · 2023-04

Alzheimer’s disease (AD) is associated with a decline in the brain’s use of glucose, its most important fuel. Astrocytes play a key role in shuttling glucose from the bloodstream to where it is needed by the neuronal units of activity deeper in the brain tissue. We find evidence of a defect in a key glucose transport molecule of astrocytes in AD and in a mouse line genetically modified to reproduce some aspects of AD. This mouse line, overproducing the β-amyloid peptide (Aβ), exhibits dysregulation of circulating glucose, as well as a decline in brain glucose use. These effects are correlated with poor performance in a test of spatial memory. Further mimicking human AD, the mice show these problems in the absence of obesity, hyperglycemia, disruption of appetite, changes in physical activity, pancreatic abnormality, or insulin resistance. Together, these findings inspire the hypothesis that Aβ, the levels of which begin to rise in the aging brain even without frank AD, perturbs the ability of astrocytes to bring peripheral glucose to neurons, where it is needed for the increased neurological activity associated with memory and other functions. We further propose that the product of a genetic variant of the apolipoprotein E gene known as ApoE4 contributes to the glucose deficiency, likely through its impact on gene regulation. These ideas will be tested through studies of the status and function of glucose transport proteins in the mouse model of AD. First, we will assess the relative roles of astrocytes and other cell types and inflammation in these events. We will also determine the role of ApoE, particularly that of ApoE4’s interaction with specific DNA regulatory elements, in the disruption of glucose transport. Finally, we will utilize on-campus drug-discovery resources in an attempt to reverse the glucose dysregulation pharmacologically. These studies test a novel hypothesis about a specific element of energy utilization in the AD brain and its connection to cognitive impairment. As such, the project may provide innovative strategies for therapeutic intervention. Relevance This project focuses on the impact made on brain energy utilization by risk factors for Alzheimer’s such as inflammation and apolipoprotien E4. Alzheimer’s involves a drop in brain glucose delivery. This study will help us understand details about how Alzheimer risk factors bring about mental deterioration, and it may identify chemical compounds that can be developed into therapeutic agents.