Oregon Health & Science University

NIH Research Projects · FY 2025 · 2025-08

SUMMARY/ABSTRACT The Assisted Reproductive Technologies (ART) Core at the Oregon National Primate Research Center (ONPRC) supports a diverse research portfolio of studies conducted by internal investigators, foundation missions, and academic and industry partners. The ART Core provides advanced technology and exceptional expertise on nonhuman primate (NHP) gamete and ovarian follicle function, contraception, fertilization, early embryogenesis, implantation, fetal development, reproductive toxicology, and customization of NHP genomes for biomedical model development. NHP models of human disease are in high demand as the translational data obtained from studies can potentially revolutionize drug discovery and disease therapy development, supporting the many disciplines of the overall research community. Of particular relevance to this funding opportunity, the ART Core is home to a unique and nationally recognized repository, funded by an R24 mechanism, that contains cryopreserved biospecimens from multiple NHP species across multiple sources, which are accessible on demand to investigators. Currently, these biospecimens are stored in liquid nitrogen (LN2) across multiple free- standing open access containers that must be manually and diligently monitored by ART Core staff with LN2 refilled daily. Sample tracking is maintained through manual cataloguing and data entry. This type of manual storage system makes the samples susceptible to variations in temperature, spontaneous release of the vacuum, and samples being misidentified or lost. We therefore seek funding for a self-contained, automated cryogenic storage system that has multiple superior features including automatic and redundant LN2 monitoring and fill, temperature monitoring with alerts, automated sample placement and retrieval, inventory management and documentation, barcoding of samples, and partitioning of inventory. Such a system would allow the ART Core to ensure sample security and long-term preservation for the ONPRC/OHSU community and the larger biomedical research community, particularly in our mission to safeguard rare and valuable biospecimens to continue support for emerging, existing and future research programs.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Colorectal cancer (CRC) is the second leading cause of cancer death and is predicted to cause nearly 10% of cancer deaths worldwide in 2024. Rectal cancer (RC) accounts for more than one-third of CRC cases, where surgical resection is a standard primary treatment. However, surgeons must balance two main goals: RC cure and nerve preservation to prevent both mortality and lifelong morbidity, respectively. With little visual acuity for nerve plexus recognition and conclusive cancer delineation, ideal outcomes for patients continue to challenge even experienced surgeons. Positive surgical margins and nerve damage occur in up to 8 and 30% of patients, respectively, resulting in poor cancer control as well as incontinence and sexual dysfunction, severely affecting post-operative quality of life. No clinically approved methods exist to enhance direct RC or nerve plexus visualization intraoperatively, where improved delineation of either tissue could substantially improve surgical outcomes. Herein, we will address this unmet clinical need by enabling real-time nerve visualization. Fluorescence-guided surgery (FGS) has demonstrated clinical efficacy in improving surgical outcomes for cancer patients through the use of tumor-specific, molecularly targeted fluorescent contrast agents in conjunction with clinically approved imaging systems. FGS systems operate almost exclusively in the near-infrared (NIR, 650- 900 nm) region, where tissue chromophore absorbance, autofluorescence and scatter all fall to local minima, allowing high contrast imaging at up to centimeter depths. However, to date, there are no clinically approved nerve-specific fluorescent contrast agents with an emission profile compatible with current NIR FGS systems. In preliminary work, our team has developed a library of >400 novel oxazine-based small molecule fluorophores with excitation and emission wavelengths spanning the NIR region. This library enabled identification of the first nerve-specific contrast agent, LGW08-35, with an emission wavelength compatible with current NIR FGS systems that are designed to image FDA-approved indocyanine green (ICG). To enable clinical translation for intraoperative use, we have also developed a clinically relevant formulation for LGW08-35 that can be directly/topically applied as a liquid and rapidly gels upon tissue contact. Hydrogel-formulated LGW08-35 permits nerve-trajectory mapping within minutes of low-dose LGW08-35 application, providing surgeons with new intraoperative on-the-spot decision-making opportunities during crucial moments of the procedure when patients are at high risk of poor postoperative outcomes due to unexpected challenges with delineation between cancer and nerve tissues. In this proposed study, Drs. Gibbs and Alani (MPI) will utilize their complementary expertise in nerve contrast agent development and clinically relevant formulation strategies, respectively, in partnership with Dr. Vahrmeijer, who has complementary expertise in FGS trials. Our team is poised to translate a clinically relevant NIR nerve imaging solution to first in human (FIH) clinical trials. Herein, we will develop GMP-compliant LGW08-35 drug product and complete GLP-grade pharmacology and toxicology testing and FIH Phase I trials.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Powered mobility (PM) interventions offer young children with motor disabilities, such as cerebral palsy, Gross Motor Function Classification System (GMFCS) Levels IV-V, the opportunity to independently explore their environments, which enhances their development and participation. PM experiences in early childhood (12-36 months) are crucial, as this period represents a critical window for development in the areas of cognition, social abilities, motor skills, and daily participation. Despite these proven benefits, PM remains underutilized in early intervention settings due to barriers, such as therapist attitudes, service delivery models, and insufficient training and resources. Further complicating matters, no standardized approaches exist for PM interventions, leaving a critical gap in both research and clinical practice. Early Childhood settings are the ideal setting for implementing PM, as it targets a pivotal developmental stage, allowing for timely access to mobility solutions that can foster independence and participation in daily activities. This project aims to address these barriers by partnering with Early Childhood settings to develop therapist training for PM interventions tailored for children with cerebral palsy, GMFCS Levels IV-V. Using an implementation science and community- engaged research approach, this mixed-methods proposal will focus on three specific aims: Aim 1 involves identifying the needs and barriers faced by early intervention therapists in utilizing PM interventions through surveys and focus groups; Aim 2 aims to co-design a comprehensive therapist training with a Community Advisory Board, which will inform and refine the development of therapist training by reviewing identified needs, evaluating an existing PM intervention’s components, and providing feedback to ensure the training’s relevance; and Aim 3 consists of conducting feasibility trial of the therapist training and PM intervention implementation, assessing the trainings knowledge transfer, confidence, self-efficacy, as well as and the intervention's acceptability, appropriateness, feasibility, fidelity, and pertinent child performance outcomes. My career development goals include enriching my knowledge in pediatric PM intervention design, acquiring skills in implementation science and community-engaged research approaches, enhancing my scientific leadership, and improving my scientific writing skills. My mentors and I have developed a structured 4-year plan encompassing didactics, experiential learning, mentored research, and measurable milestones, aligning with my research aims and maximizing my potential as an independent investigator in pediatric PM intervention research. This proposal is responsive to PA-24-184, which seeks to ‘explore or understand the mechanism of action of an intervention’ and ‘increase the pool of clinical researchers who can conduct patient- oriented studies, capitalizing on the discoveries of biomedical research and translating them to clinical settings.’ My ultimate goal is to bridge the gap between PM evidence and clinical practice, expanding access to essential developmental and motor interventions for children with cerebral palsy.

NIH Research Projects · FY 2025 · 2025-08

Human cytomegalovirus (HCMV) is a ubiquitous, usually benign virus. Nevertheless, HCMV frequently contributes to rejection of organs in immunosuppressed transplant patients and fetusinfections of the can causepost-encephalitic impairment of the infant brain. Among the important cells that HCMV infects in vivo are epithelial and endothelial cells. Three HCMV glycoproteins: trimer, gH/gL/gO, the pentamer gH/gL/UL128-131, and the viral fusion protein gB mediate entry of virus particles into biologically relevant epithelial and endothelial cells. Our model for how HCMV enters epithelial and endothelial cells suggets that HCMV trimers bind to cell surface receptors then viruses are internalized and pentamer acts in endosomes to trigger gB- mediated fusion of the virion envelope with cellular membranes. Given their importance in virus entry, trimer and pentamer are also important targets of antibodies and are considered key players in the design of HCMV vaccines. It was shown that trimer binds to platelet growth factor receptor-alpha (PGFR) to mediate entry into fibroblasts, but PDGFRa is not expressed in epitheial and endothelial cells. Thus, there are, as yet unidentfied, trimer receptors in these two cell types. It is also known that pentamer binds neuropilin-2 (NRP-2) , which is expressed in epithlial and endothelial cells, in a process that appears to allow HCMV to escape endosomes. However, pentamer binds NRP-2 with relatively low affinity, so that other pentamer receptors may exist. We utilized a novel receptor capture technology, which allowed covalent coupling of trimer or pentamer onto epithelial and endothelial cell surface proteins. Proteins bound by trimer and pentamer were identified by mass spectrometry. These studies identified a trimer-binding protein, Transforming Growth Factor  Receptor-3 (TGFR-3) and we showed that soluble form of TGFR-3 inhibited HCMV entry. Four interesting pentamer binding proteins were also identified and characterized including: thrombomodulin (TBHD) and a soluble form of TBHD blocked HCMV entry and calsyntensin-1, a protein that promotes entry of hepatitis C virus. Another pentamer-binding proteins calsyntensin-1 blocked HCMV cell-to-spread but not entry. We will further characterize these novel trimer and pentamer binding proteins in order to detemine if these proteins are important for HCMV entry into epithelial and endothelial cells. These studies will involve: producing soluble forms of these proteins, silencing the receptors, and characterizing anti-receptor antibodies that might block virus entry. High resolution deconvolution imaging, super resolution 3D imaging, and live cell imaging experiments, coupled with HCMV recombinants expressing fluorescent proteins, will be used characterize entry pathways. Whether these receptors act to sort virus particles during virus entry or whether the receptors signal into cells to alter cytoskeleton or activate signaling pathways will also be determined. These receptor candidates, combined with better characterized proteins such as NRP-2, will be extremely useful handles to better understand the cell biology of HCMV entry into biologically relevant epithelial and endothelial cells.

NIH Research Projects · FY 2025 · 2025-08

Modified Project Summary/Abstract Section Exacerbated by HIV, tuberculosis (TB) remains a leading cause of infectious disease mortality worldwide. TB cannot be reasonably eliminated in the absence of a vaccine. While CD4 and CD8 T cells and their associated proinflammatory and cytolytic capacity have been associated with protection in various animal models, correlates of protective immunity in humans remain enigmatic. Recently, prospective human studies have been used to define TCR families associated with immunity to TB progression. Here, TCR families associated with protection from progression to disease were previously identified in Interferon Gamma Release Assay (IGRA) positive South African adolescents (ACS). While M. Musvovsi et al., were able to define three antigens associated with these TCR families, we note that the majority remain unsolved. Furthermore, in our ongoing work with Cascade IMPAcTB using samples that were previously collected, we will define TCR families associated with protection, within a household contact (HHC; Stellenbosch) study. Specifically, to delineate lung inflammation, we are using fluorodeoxyglucose (FDG) Positron Emission Tomography and Computed Tomography (PET-CT), a technique that uses high resolution anatomic imaging (CT) in conjunction with functional imaging that includes FDG as a marker of glucose uptake. HHC with a negative FDG PET-CT, representing protection (adaptive immune control), can be compared those with a positive FDG PET-CT, representing subclinical TB. In this study, TCR families and transcriptional profiling will be available from the lung and periphery. Finally, we will have the opportunity to evaluate the function of T cells recognizing identified protection-associated antigens in an independent cohort of active and latently infected adults in Portland, OR. Key questions to be addressed: 1. What are the TCR families associated with protection? 2. What are the antigens and epitopes recognized by protection-associated TCRs? 3. What is the relative affinity of these TCRs for their cognate peptide? 4. What is the relative affinity of these TCRs for the Mtb-infected cell? 5. What is the functional profile of these cells? In this application, we will simultaneously test multiple mechanisms through which these protective TCRs mediate control: 1) specific TCRs could confer protection by targeting specific antigens, 2) protective TCRs could have higher relative affinity for their cognate antigen(s), which leads to enhanced recognition of the Mtb-infected cell, and 3) T cells expressing these protective TCRs may possess enhanced effector function.

NIH Research Projects · FY 2025 · 2025-08

The contributions of the placenta to normal pregnancy physiology, as well as the pathophysiology of preterm birth, fetal growth disruption, and preeclampsia, are well-recognized, but we cannot predict, prevent, or adequately treat these common complications that affect approximately 10% of human pregnancies. A major challenge to understanding placental development and function using in vivo real-time assessments across gestation (e.g. goal of the Human Placental Project and PAR-22-236) is limited access to longitudinal tissue samples throughout human pregnancy. Others have shown that placental extracellular vesicle (EV) concentrations and contents in maternal plasma may be related to placental development and function, which suggests that placental EVs may be a promising biological resource for in vivo studies. EVs include a range of submicron particles, including small exosomes, larger microvesicles, and cell fragments undergoing necrosis and apoptosis. EVs contain a variety of RNA species (including microRNAs) that provide insights into their source and potential function (e.g. regulating angiogenesis). Methods like density gradient ultracentrifugation, size exclusion chromatography, and affinity capture provide a heterogeneous mixture of EVs from a variety of cell sources and a variety of EV sizes. These methodological shortcomings have limited characterization of placental-specific EVs by failing to precisely characterize their source and cargo. As a result, there is an immediate need for a novel approach to image, count, and isolate cell- and size- specific EVs from maternal blood across gestation to monitor placental development and function. In collaboration with BD Biosciences, we have developed a multiplex high resolution nanoscale flow cytometry method to image, count, isolate and validate placental cell- and size-specific EVs. Importantly, this novel technology enables us to study placental EVs from syncytiotrophoblasts, extravillous trophoblasts, and endovascular trophoblasts that likely have different contents and functions during pregnancy. Moreover, we can compare the content of these sorted EVs with sorted 100nm liposomes spiked into the same plasma to control for background contamination inherent to any EV isolation method. We have also developed a novel 242 monoclonal antibody multiplex design with 3-4 antibodies (3/4-colors)/well in a 96-well plate design to address the entire maternal EV-biome throughout gestation. We hypothesize that nanoFACS sorting technology and our new EV-biome design will bring much needed precision and accuracy to the imaging, counting, and isolation of cell- and size-specific placental EV analysis across gestation, which will help us to better understand the role of EVs in placental development and function in vivo. In this project, we will test for differences in cell- and size-specific EV profiles in maternal plasma, which have already been banked from uncomplicated and complicated pregnancies collected at 12, 24, and 32 weeks’ gestation that are linked to published study subject metrics and unique characterization of uteroplacental blood flow by T2*MRI.

NIH Research Projects · FY 2025 · 2025-08

Myelination of axons by oligodendrocytes is critical for proper central nervous system function. The transition from oligodendrocyte precursor cells (OPCs) to mature, myelinating oligodendrocytes is tightly regulated, but the molecular mechanisms governing this process remain poorly understood. This proposal aims to elucidate the role of Cyclin Y-like 1 (CCNYL1) in oligodendrocyte differentiation and myelination. Building on preliminary data demonstrating CCNYL1's crucial role in oligodendrocyte differentiation in zebrafish, this research aims to elucidate the molecular mechanisms governing these processes through three specific aims: 1) define CCNYL1's role in developmental oligodendrogenesis using zebrafish and mouse models, 2) investigate pathways regulated by CCNYL1 in differentiating oligodendrocytes with a focus on Wnt signaling, and 3) determine CCNYL1's influence on experience-dependent myelination and remyelination in adult mice. This research employs cutting-edge techniques including in vivo imaging, CRISPR-mediated gene editing, proteomics, and behavioral studies, with the ability to uncover novel molecular pathways controlling oligodendrocyte formation. Understanding these processes is essential for developing targeted therapies for demyelinating diseases like multiple sclerosis, where impaired oligodendrocyte differentiation contributes to remyelination failure. By elucidating CCNYL1's role in oligodendrocyte biology across different contexts—from development to adulthood and in disease states—this work may uncover novel therapeutic targets to enhance myelination and promote repair in the central nervous system. This research will take place at OHSU’s Vollum Institute, a hotspot of glial biologists and myelin researchers, including Drs. Kelly Monk, Ben Emery, and Marc Freeman, who will all advise the progress of this research and provide support and training in biochemistry and proteomics. OHSU’s state of the art equipment and core facilities will streamline the proposed experiments and provide crucial technical support in experimental design and analysis. During the training period, the applicant will master rigorous experimental design and statistical analysis and develop skills in mentorship, teaching, and lab management with an emphasis on supporting underrepresented groups in science. This comprehensive career development plan, combined with the candidate's strong background and supportive mentoring team, positions the applicant well for a successful transition to independence as a faculty member leading a research program in glial cell biology and neuroscience.

NIH Research Projects · FY 2025 · 2025-08

Project Summary This project seeks to unravel the crucial role of short-term plasticity (STP), specifically synaptic facilitation, in cognitive flexibility and the neural representations underlying it. STP, a significant modulator of neurotransmitter release, influences synaptic strength on short timescales, allowing for rapid, experience- dependent changes in neural circuit connectivity. Despite its proposed importance in cognitive functions and potential involvement in various neurological disorders such as schizophrenia, autism spectrum disorder, and Alzheimer's disease, the direct contribution of STP forms, like facilitation, to cognitive behaviors remains underexplored due to past methodological limitations. Our recent breakthroughs have identified key proteins responsible for STP, enabling the genetic manipulation of STP in vivo. Focusing on the protein synaptotagmin- 7 (SYT7), crucial for synaptic facilitation, we have found that mice lacking SYT7 (Syt7-/- mice) exhibit normal learning but impaired behavioral adaptation to changing reward conditions in a frontal cortex-dependent task. Preliminary in vivo electrophysiological evidence suggests altered prefrontal cortex activity in Syt7-/- mice during task learning, suggesting that SYT7-driven facilitation is integral to behavioral flexibility and relevant neural representations. To further explore this, this proposal is structured around three aims: 1) Characterize behavioral flexibility in Syt7-/- mice through cognitive operant assays to pinpoint the aspects of decision-making affected by facilitation deficits; 2) Examine frontal cortical activity in Syt7-/- mice during contingency changes in operant tasks, utilizing in vivo electrophysiology to identify how impaired facilitation impacts cortical representations of behaviorally relevant information; and 3) Model the impact of SYT7-driven synaptic facilitation in simulated neural networks, employing a multi-level modeling approach to delineate the role of synaptic facilitation in network dynamics and cognitive task learning flexibility. This comprehensive approach, combining behavioral assays, in vivo electrophysiology, and computational modeling, promises to offer unprecedented insights into the role of synaptic facilitation in cognitive flexibility and neural circuit function. By elucidating the mechanisms through which STP influences cognitive processes and their dysfunction in disease states, this project aims to lay the groundwork for novel therapeutic strategies targeting STP modulation. The expected outcomes have profound implications for our understanding of neural plasticity, cognitive function, and the treatment of cognitive impairments in neurological diseases, potentially revolutionizing approaches to enhancing cognitive resilience and flexibility.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract The regenerative capacity of the central nervous system (CNS) is severely limited in humans and other mammals, such that neural injury is often irreparable. Thus, damage to the CNS is a catastrophic event and often results in permanent disability, as regenerative medicine lacks an effective strategy to overcome neural loss. To potentially overcome this damage, new neurons are ultimately made from stem cells, yet the genetic programs that stem cells use to regenerate lost neurons remains unknown. Understanding how damaged cells are replaced and how new cells functionally integrate into the existing tissue is of fundamental importance in understanding neural plasticity, and neural injury or degeneration. This proposal utilizes zebrafish and planarians, both powerful models of CNS regeneration. First, I will use larval zebrafish to identify the factors that define neural stem cells (NSCs) in the spinal cord that are activated in response to neural injury. I used a single-cell RNA-sequencing approach; over 40,000 cells were sequenced in total from spinal tissue spanning a week of recovery after injury. This dataset serves as an important guide for the experiments proposed here because neural cells can be isolated using known markers and I have identified injury-enriched neural cell clusters. In Aim 1, I seek to validate transcriptomic data with in vivo gene expression analysis to determine the molecular heterogeneity of NSCs and to perform clonal analysis, using a multicolor cell labeling technique, to understand if heterogeneous NSC populations give rise to diverse cell types. I will also use a conditional cell ablation strategy to determine how each stem cell population is involved in spinal cord regeneration. Experiments described in Aim 2 will investigate one candidate gene whose expression is upregulated in SCI: cytokine receptor-like factor 1a (crlf1a). I have found that crlf1a labels a unique population of NSCs that are potentially de-differentiated ERGs, activated in response to injury. I will determine what stem cell factors are co-expressed in crlf1a+ cells, and I will use a conditional mutagenesis strategy to remove crlf1a from NSCs to determine a functional role during spinal cord regeneration. Aim 3 will determine if cytokine signaling through the ancestral receptor unit, GP130, is required for whole-brain regeneration in planarians. The findings generated from this research will elucidate the complicated biology of stem cell lineage development in the CNS and, in turn, impact understanding of how the neural stem cell state can instruct proper neurogenesis following neural injury. Neural cell types are highly conserved, so we expect these findings to be broadly applicable to human neural injuries, diseases, and therapies. The experiments, lab environment, and additional training opportunities outlined in this proposal will present a phenomenal training experience that will enhance my technical skillset, further develop my knowledge of stem cell biology, and allow me to carve out a research area to lead as an independent investigator with my own laboratory.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Nervous systems are initially assembled with excess neurons and synaptic connections, but these are later refined whereby exuberant neurons, individual neurites, or synapses are eliminated, which is believed to drive optimization of neuronal circuitry for mature brain function. Failures in proper neuronal remodeling can result in significant neurodevelopmental disorders, including Autism Spectrum Disorders, schizophrenia, and epilepsy. Glial cells play a critical role in the circuitry refinement, helping neurons execute remodeling by removing neuronal debris, although the mechanisms mediating these processes are not well understood. To address this, I will explore the role of a novel pathway in Drosophilia, Remoulade (Remo), ortholog of the mammalian orphan adhesion GPCRs Gpr124 and Gpr125. Our preliminary data has shown loss of Remo in astrocytes results in a failure of neurite refinement in a specific subclass of neurons, termed Beat-VaM neurons, which undergo local pruning (i.e. elimination of axon/dendrites and synapses followed by regrowth of the pruned parent neuron). In Aim 1 I will perform a detailed genetic analysis of Remo in the activation of pruning and elimination of neuronal debris in Beat-VaM neurons. In Aim 2, by examining live and fixed tissue samples, I will define the precise role for Remo in the transformation of astrocytes into phagocytes, recognition, internalization and processing of neural debris for elimination; and define the localization of Remo throughout this process. Finally, in Aim 3, I will explore the role of Remo in other glial phagocytic events, including remodeling of other neurons during development, or after axonal injury (i.e. axotomy). Together, these studies will provide exciting new insight into the mechanisms by which glia help activate and execute neuronal remodeling and responses to injury in vivo. My goals as a postdoctoral fellow are to a) develop technical and intellectual approaches needed to start my own independent research lab studying neurodevelopment and b) learn the tools and techniques needed to utilize Drosophila to understand the contributions of glia to neurodevelopment in deep molecular terms. My proposed work has excellent training potential—I will learn a new genetic model system, become an expert in neurodevelopmental studies in Drosophila, and learn cutting edge imaging and genome engineering approaches for use with this system. The additional training activities proposed during my fellowship will also enhance my quantitative and analytical skills, improve my ability to communicate my work, and engage in effective mentorship. Collelectively, this fellowship will help prepare me to successfully transition towards a career as an independent investigator.

NIH Research Projects · FY 2026 · 2025-08

The prevalence of gestational diabetes (GDM) is increasing in association with more adverse pregnancy outcomes and greater risk of development of type 2 diabetes in later life. GDM is associated with many fetal and newborn complications and programming of offspring for development of obesity and diabetes. A male fetus is at greater risk of adverse outcome than a female fetus perhaps related to the sexual dimorphism in placental gene expression, placental inflammatory, hypoxia and apoptotic responses, antioxidant defenses, miRNA expression and mitochondrial dysfunction. Placental mitochondria are the main source of ATP required to power peptide hormone production, nutrient uptake and transfer and are central to generation of oxidative stress and inflammatory responses. Placental mitochondrial respiration is reduced with the hyperglycemia, hyperinsulinemia and hyperlipidemia of obesity and GDM together with altered fuel flexibility where male but not female trophopblast lose flexibility to switch between use of glucose, fatty acids and glutamine. Lipidomic analysis showed male but not female placentae from GDM had a significantly lower proportion of docosahexaenoic acid (DHA)-containing triglycerides (storage lipids) vs lean or obese placenta. DHA is an essential fatty acid crucial for fetal brain development but also important for membrane structure/function, reducing oxidative stress and inflammation and regulating mitochondrial function, hence there is a need to define DHA actions in the male vs female placenta and if these are affected with obesity and GDM. Using proteomic analysis, GDM male placentae showed significant downregulation vs lean male placentae of pathways related to protein synthesis and upregulation of inflammatory pathways. In contrast female GDM placentae showed downregulation, vs lean and obese, of pathways related to glucose metabolism. Hence, we have a strong premise to determine the mechanisms underlying the sexual dimorphism of cellular response to the GDM milieu in placenta and particularly if DHA has a central role in this. Our previous `omic' work was on whole placental villous tissue, obscuring the spatial localization of signals. We have developed a metabolome-informed proteome imaging (MIPI) workflow where MALDI-MSI allows multi-modal imaging of 12μm placental villous tissue sections, showing regions of differing metabolic activity and complementary proteome profiling of mapped regions captures region-specific enzymes and highlights functional differences between syncytiotrophoblast and villous core compartments. We will employ the MIPI technology on tissue sections and also employ trophoblast cultures of male and female placentas of lean and obese women each with or without type A2GDM pregnancies to test the hypothesis that there is sexual dimorphism in the setting of maternal obesity and GDM in cellular pathways and mechanisms involving trophoblast mitochondrial function, protein homoeostasis, oxidative stress, inflammation and insulin resistance regulated by the essential fatty acid DHA, This technically and conceptually innovative work may lead to establishment of fetal sex-specific therapeutic approaches in such pregnancies.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Stroke is the leading cause of disability in the United States and around the world. The improved stroke care has increased the number of stroke survivors. White matter injury (WMI) underlies the majority of clinical deficits observed in stroke patients. Investigating the mechanisms of WMI is challenging in rodent brains due to the smaller volume of WM compared to human brains. Because conventional animal stroke models mainly affect gray matter, sparing the corpus callosum, WM protection has not been a primary target in many proposed studies. A scientific gap therefore remains in the research into preserving WM function, which requires a combination of an in vivo WMI rodent model with a clinically relevant approach to preserve WM integrity after stroke. To address this scientific gap, we employed a consistent and reliable in vivo selective subcortical WMI model that can be quantified histologically and with behavioral tests, and longitudinal imaging studies using MRI. We previously showed that ischemia upregulates Casein Kinase 2 (CK2) causing WMI via Cdk5 and AKT/GSK3β pathways. CX-4945, an FDA-approved selective and specific CK2 inhibitor that crosses the blood-brain barrier, promotes axon function recovery by conserving mitochondria in WM when applied after ischemia. Since ischemia activates NADPH oxidase (NOX) in neurons to increase oxidative stress causing mitochondrial dysfunction, we propose a novel mechanism whereby CK2 activates NOX causing mitochondrial dysfunction during ischemia in WM. WM mitochondrial dynamics and axon function show a sexually dimorphic age-dependent recovery after an ischemic episode, because young female axons have better functional recovery with less interruption in mitochondrial motility than young male axons, yet this difference is not observed within aging populations. These findings warrant further investigation of post-ischemic benefits of CK2 inhibition in WM. Our preliminary data show that the selective focal WM injury causes behavioral impairments indicated by loss of bilateral paw use in the cylinder test and paw dexterity in the pasta-eating test. These deficits correspond with persistent edema formation in scans obtained by using MRI modalities. Administration of CX-4945 at 6 hours after stroke preserves WM integrity, alleviates behavioral deficits, and improves MRI modalities. Furthermore, impaired mitochondrial motility following oxygen and glucose deprivation correlates with lower mitochondrial respiratory function in live isolated mitochondria from WM. Because ischemia upregulates NOX enzyme activity in in vitro WM injury and post-ischemic CX-4945 application attenuates NOX activity, we propose that CK2 inhibition after stroke confers WM protection and improves behavioral outcomes by regulating NOX activity to conserve mitochondrial dynamics. We will test our hypothesis in both young and aging male and female WM in an animal model of selective focal WMI.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Opioid use disorder is a significant public health problem driven by complex biological and sociopolitical factors. People with opioid use disorder often cite the development of tolerance, a set of physiological adaptations that decrease response to opioids, as a reason for continued, escalating use. Given the vast role of tolerance in opioid use disorder, understanding how tolerance develops is of great interest. One factor that contributes to tolerance development is adaptations in regulation of opioid signaling at the level of a single cell. Opioids signal through G protein-coupled receptors (GPCR) called mu opioid receptors (MOR). Cells have evolved mechanisms for regulating GPCR signaling and returning to homeostasis in order to respond to additional signaling events. A major aspect of GPCR regulation is through trafficking: activated GPCRs are selectively internalized into the cell, where they travel through the endosome to the lysosome for degradation. However, some receptors, including MORs, can be rescued from a degradative fate and recycled to the plasma membrane as functional receptors, quickly re-establishing the cell’s ability to continue responding to opioids. The intracellular tail of MOR contains a specific amino acid sequence that is necessary for endosomal recycling. Recently, it was discovered that recycling of MOR through its recycling motif requires an endosomal sorting protein complex known as Retromer. However, the mechanism by which Retromer recycles MOR, and the effects of this process on MOR signaling are still unknown. This proposal seeks to identify the mechanisms by which Retromer-dependent MOR recycling occurs and assess the effects of MOR recycling on MOR signaling. The experiments in this proposal will determine how Retromer interacts with the MOR recycling motif to select MORs for recycling and define the subcellular route that Retromer uses to recycle MORs. This proposal will also test the hypothesis that Retromer is involved in MOR signaling regulation by limiting endosomal signaling using classical electrophysiological techniques and novel location-specific sensors of G protein activation. Overall, this work will uncover the mechanisms behind Retromer-dependent MOR trafficking and its functional consequences for MOR signaling. The long-term goal is to understand how Retromer and MOR recycling contribute to the development of pharmacological tolerance in vivo. The training goals of this proposal are supported by a world- class mentorship team of scientists, physicians, and physician-scientists and include rigorous training in hypothesis generation, advanced cellular and chemical biology techniques, oral and written communication to a wide variety of audiences, addiction medicine from a variety of clinical perspectives, and the professional and leadership skills required for a successful career as a physician-scientist.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Amblyopia is a common pediatric eye problem impacting 2-4% of the population. This vision threatening disease can be inexpensively and effectively treated but proves quite challenging due to adherence problems. Studying treatment, adherence, and outcomes using real world electronic health record (EHR) data is limited due to inconsistent documentation and documentation of these concepts only in free-text clinical notes. Large language models (LLM) have the potential to extract these concepts from these notes automatically and at scale, enabling more research as well as the development of clinical decision support tools that use this data in clinical care. This project will develop, evaluate, and validate models using a single site’s data as well as multiple sites through the Sight Outcomes Research Collaborative (SOURCE) consortium.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY There has been a steady increase in grandparent caregiving in the US. This acceleration is due to recent trends in domestic violence, parents’ incarceration, parents’ death due to the COVID-19 pandemic, and opioid overuse that increase familial instability and leave more grandchildren to be cared for by custodial grandparents. These grandparent caregivers face ongoing stressors as they navigate legal custody arrangements, cope with loss of their own child(ren), or face social isolation. A higher prevalence in an array of social determinants of health often put them at higher risk for cardiovascular disease (CVD): belonging to a racial/ethnic minority, living below the poverty line, living in single-caregiver households, and having lower levels of educational attainment. Additionally, custodial grandparents appear to have higher rates of adverse childhood experiences, another known risk for CVD. This unique mixture of stressors manifests in custodial grandparents’ health, where they are at higher risk for CVD compared to their non-caregiving peers. However, there is a dearth of intervention addressing custodial grandparents’ CVD risk. To address this gap and optimize healthy aging and cardiovascular health among this growing at-risk population, we will implement a virtually delivered, evidence-based CVD risk reduction intervention - The Rural Caregiver Heart Health Education [RICHH] Intervention. The RICHH intervention was originally targeted for adult caregivers of adult family members with a chronic illness, and has not been tested in the context of custodial grandparents’ CVD risk. We propose a 3-year planning project to determine the feasibility, acceptability, and initial effect of the RICHH intervention conducted with custodial grandparents. We will employ a mixed method design and conduct a 2-arm randomized controlled trial with 70 custodial grandparents in Oregon. The specific aims are: 1) modify the RICHH intervention to target custodial grandparents from a variety of backgrounds; 2) employ the re-designed intervention and assess its feasibility and acceptability; and 3) evaluate and refine the RICHH protocol among our team members and explore the initial effect of the intervention based on measures of CVD risk, self- management behaviors, and depressive symptoms at 4-months and at 6-months, compared to baseline. Expected outcomes are to complete the sufficient and scientifically necessary groundwork to support a future clinical trial that will test the effectiveness of the RICHH intervention with a longer follow-up and increased inclusivity of participants from marginalized populations, with the objectives of: a) preventing CVD-related morbidity and mortality and overall physical decline, b) improving psychological well-being in these grandparents, and c) fostering a more heart-healthy environment in grandfamilies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Symptoms of depression and anxiety demonstrate a substantial increase in prevalence during adolescence and high estimated comorbidity. The consequences of adolescent-onset internalizing disorder symptoms are considerable, including an increased risk of suicide, which is already a leading cause of death for youth. Given modest remission rates and treatment responses for both depressive and anxiety disorders, interventions based on neurobiology and behavior during adolescence, a critical period of sustained neuroplasticity, may improve early intervention efforts and minimize long-term harm. Research has aimed to elucidate adolescent-emergent internalizing disorders by identifying brain biomarkers of transdiagnostic features, due to the overlap of many depression- and anxiety-related psychiatric diagnoses. Dysfunctional emotion regulation (ER) is one such shared feature, and literature has shown it is exacerbated by negative life events (NLEs), life changes that are challenging (but not necessarily traumatic), particularly during adolescence when emotional reactivity is elevated. NLEs are thought to promote emotion dysregulation and risk for internalizing disorders via prefrontal cortex and amygdala development and connectivity disruption. Stress (broadly defined), internalizing pathology, and ER dysfunction have also been linked to perturbations in resting state functional connectivity (rsFC) of brain networks governing emotion-related higher-order cognition. While self-reported ER dysfunction has been shown to partially mediate the NLEs-internalizing problem association in youth, more well-powered work is needed to understand underlying neurobiological mechanisms, including whether changes in neural functioning provide explanatory power and if neurobiological mediation persists over time. The goal of this study is to investigate the extent to which ER trajectories, behaviorally and neurobiologically, mediate the relationship between NLEs and changes in internalizing symptoms, while accounting for social risk and protective factors, in the large, diverse Adolescent Brain Cognitive Development (ABCD) Study sample. Based on preliminary analyses, I hypothesize that the trajectory of ER behavior and associated brain functioning will (1) at least partly mediate the positive association between NLEs and internalizing symptoms over time; and (2) this mediation will be stronger for those with low social support. Thus, this proposal aims to examine the trajectories of: (1) ER behavior, (2) neural correlates of emotion-influenced cognition (as measured by Emotional N-back Task fMRI), and (3) rsFC of emotion- and higher-order cognition-related brain networks as mediators of the link between NLEs and internalizing symptoms across early adolescence, while evaluating social supports as moderators. Results from this proposal may help to provide insights for more personalized treatments based on neurobiology, behavior, and social context. Additionally, the robust and interdisciplinary training plan within a prominent academic medical and research institution proposed in this application will allow me to establish a strong foundation for a career as an academic physician-scientist at the intersection of psychiatry and translational neuroscience.

NIH Research Projects · FY 2026 · 2025-08

Project summary Nervous system injury activates a complex series of events including axon degeneration, reactive glial responses, and nervous system inflammation. These processes conspire in poorly-defined ways to drive the destruction of axons and synaptic connections, and neuronal death, which ultimately result in neural circuit dysfunction. Our goal is to define molecular pathways that drive neurodegeneration and use that information to block axon and synapse loss in patients. In previous work we identified the TIR domain molecule dSarm/SARM1 (flies/mammals) as a key regulator of axon degeneration after axotomy. In dSarm/SARM1 mutant flies or mice, distal severed axons survive and remain functionally intact for weeks after axotomy without attachment to a cell body. This work identified dSarm/SARM1 as the first “axon death” gene, an endogenous gene whose normal function was to promote axon and synapse destruction. Since this discovery, we and others have shown that the SARM1 pathway is required to drive axon degeneration in a number of preclinical models of human neurological disease. SARM1 is now a therapeutic target for many pharmaceutical companies to treat neurodegenerative disorders, and the first Phase 1 trials with SARM1 inhibitors began in 2022. We sought to identify new molecules that drive neurodegeneration with dSarm/SARM1. A key step in activating dSarm/Sarm1 in both flies and mice is loss of the primary NAD+ biosynthetic enzyme in axons, Nmnat. Nmnat is a labile survival factor that is transported down axons from the cell body. After injury or in neurological disease, loss of Nmnat supplies depletes axonal NAD+ and activates dSarm/SARM1-dependent neurodegeneration. In a screen for genes required for neurodegeneration after depletion of Nmnat, we identified the Drosophila with-no-lysine kinase (dWnk) gene. dWnk is a conserved (WNK1-4 in mammals) kinase that is best known for acting as an osmosensor in kidney, where it activates the downstream kinases Spak/OSR1, and ultimately modulates blood pressure through activation of Na+/K+/Cl- co- transporters. In preliminary work we show that loss of dWnk or fray (fly Spak/OSR1) can suppress neurodegeneration induced by depletion of Nmnat, and provide strong genetic evidence that dWnk/Fray act in parallel with dSarm to drive neurodegeneration through the downstream BTB/Back domain molecule Axundead. In Aim1, we will define the key domains required for dWnk to drive neurodegeneration, explore its signaling relationship with dSarm, assay dynamic changes in localization of dWnk during neurodegeneration, and whether it is required for neurodegeneration in two simple fly models of chemotherapy-induced neuropathay and ALS. In Aim 2 we will perform a similar study with Fray, and explore how its activation is sufficient to bypass the requirements for dSarm in neurodegeneration, as our preliminary data strongly suggests. In Aim 3 we will use candidate gene, forward screens and proximity labeling approaches to identify key downstream signaling molecules required for Fray to promote neurodegeneration. Together, our work will define the role of several new pro-degenerative molecules that work with dSarm in vivo and determine how they drive neurodegeneration. Given the strong conservation of dSarm/Sarm1 and dWnk/WNK signaling, we anticipate our work will also provide fundamental new insights into the mechanisms of axon degeneration in human disease.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY/ABSTRACT Placental dysfunction causes more than half of preventable stillbirths in the United States. Placental dysfunction also causes preeclampsia and fetal growth restriction, both of which can represent near misses for stillbirth. Tools for preventing stillbirth due to placental dysfunction include heightened fetal surveillance and recommending early delivery for known at risk pregnancies. However, these tools are only effective if we can accurately detect which pregnancies are at increased risk of placental dysfunction and stillbirth. Thus, accurately predicting risk of placental dysfunction is key to stillbirth prevention. The current national guidelines to identify high-risk pregnancies are based on presence of individual risk factors and do a poor job of accurately categorizing pregnancies. This is in part due to lack of quantitative assessment of cumulative risk or use of biomarkers of placental health. A predictive model can both calculate cumulative risk from multiple factors and incorporate biomarkers into risk calculations. If we can accurately calculate individual pregnancy risk of placental dysfunction, we will be better able to prevent stillbirths using fetal surveillance and early delivery. Our long-term goal is to improve prediction and prevention of stillbirth. We propose using a predictive model to determine individual pregnancy risk of placental dysfunction. The main objective of this project is to externally validate and optimize our existing clinical predictive model of placental dysfunction to enable individual risk assessment for preventable stillbirth. Aim 1: Validate and update a novel predictive model of individual risk of placental dysfunction. Aim 2: Measure incremental benefit of adding biomarkers of placental health to the predictive model and determine the optimal combination of biomarkers to guide fetal testing and timing of delivery. Aim 3: Conduct cost-effectiveness analysis (CEA) of clinical implementation of predictive models of placental dysfunction, both with and without key biomarkers. Predictive models calculate a predicted probability, or risk, of a specific outcome. With knowledge of individual risk, use of tools to prevent stillbirth can be customized by degree of predicted risk (i.e., recommending the most intense surveillance and earlier delivery to those at highest risk). To test Aims 1 and 2, we will enroll 640 pregnant people and follow them throughout pregnancy, checking biomarkers (ultrasound and blood tests) at three time points. We will then identify the combination of biomarkers and clinical variables that best predict placental dysfunction. We have selected biomarkers that are already clinically available to ease future translation to real world use. To achieve Aim 3, we will build decision analytic models for using predictive models to inform decisions regarding both use of antenatal surveillance and timing of delivery. Models will be prioritized based on performance, cost- effectiveness, and practicality. Upon completion of this project, we will have an updated, optimized predictive model of placental dysfunction including clinically meaningful biomarkers along with CEA to inform the economics of implementation in future projects.

NIH Research Projects · FY 2025 · 2025-08

Abstract. Delivering therapeutic drugs specifically to target tissues with minimized off-target accumulation can improve the efficacy and safety of therapies. With this overall motivation, various delivery platforms have been developed in recent years for targeted drug delivery, including antibody-drug conjugates (ADCs) and nanomedicines. These drug delivery platforms typically target an antigen to deliver toxins into diseased cells. However, targeting a specific antigen brings several important limitations such as antigen expression in healthy tissues often causing a narrow therapeutic window, inter- and intra-patient heterogeneity in antigen expression yielding highly variable response only in a subpopulation of patients, and development resistance against therapy due to the post translational modifications or loss of antigen expression. To address these issues with targeted therapies, in this proposal, we will utilize a novel peptide amphiphile (PA) platform that our lab recently developed. These PAs dynamically interact with two types of endogenous lipid-containing biomolecules, lipoproteins and lipid rafts, and exploit them to target a broad range of solid tumors. In the preliminary experiments, we found that our PAs strongly accumulated in ~15 xenografted, syngeneic, and transgenic tumor models in mice and rats, including tumors with sizes <1 mm. We have also shown that our PAs can deliver different small molecule drugs in cells, enabling improved anti-tumor efficacy in mouse models with reduced side effects compared to free drugs. Building upon these results, our goal in this proposal is to develop a platform that can universally deliver a broad range of drugs to human diseases without targeting a specific antigen. Another major goal of this proposal is to perform in-depth mechanistic studies to better understand the biodistribution of PAs and their trafficking in cancer cells. To achieve these goals, we will rationally design a library of PAs using different saturated and unsaturated lipid modifications and evaluate their interactions with plasma components, cell membranes, and subcellular components using in vitro experiments and molecular dynamics simulations in the first Aim. The second Aim will evaluate the effects of PA structure on their biodistribution in wild-type and tumor-bearing mice. We will also use various genetically engineered mouse models to understand how aberrant lipoprotein metabolism, such as high cholesterol and cholesterol medicines, affects the biodistribution of PAs as they are trafficked in vivo on lipid- containing biomolecules. In the final Aim, we will evaluate this platform to deliver a broad range of drugs with varying hydrophobicities and mechanisms of action in mouse models. At the conclusion of this project, we will learn more about the universal cancer-targeting mechanism of our PA platform. In addition, we will optimize its structure further for the precise delivery of various drugs. If successful, our technology will be available to deliver a broad range of drugs and contrast agents to detect and treat almost any type of cancer and potentially other diseases associated with increased lipid metabolism, such as cardiovascular diseases and inflammation.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Defining how hormonal and genetic factors modulate neurodevelopment is key to understanding the origins of sex differences in brain function and behavior, addressing sex biases in neurological and psychiatric disorders, and developing effective treatment for disorders of sex development, and gender-affirming hormone replacement therapies. The zebra finch robust arcopallial (RA) nucleus exhibits extreme sexual dimorphism and offers unique opportunities for defining how hormonal and sex-linked genetic factors mediate sex differences in cortical circuits that subserve complex behaviors of relevance to human cognition. Only males sing, and RA neuronal size and complexity undergo dramatic increases during vocal development in males, while regressing in females. Analogous to the human laryngeal cortex, RA integrates inputs from cortical and basal ganglia vocal-motor areas, provides the sole descending output of the cortical vocal circuit, and plays key roles in the motor encoding and production of vocalizations. Notably, early exposure to estradiol (E2) masculinizes the song circuitry and leads to song emergence in females, indicating high sensitivity to sex steroids, whereas other evidence points to the role of sex-linked genes with sex-biased expression. Our recent cellular, physiological and molecular studies have led us to hypothesize that RA's sexual dimorphism relates to sex differences in cell types, intrinsic excitable and synaptic properties, and sex-linked genes with sex- biased expression during vocal learning, and that the masculinizing action of E2 in females shifts RA's cell composition, transcriptome, and neuronal excitability towards males. To test these hypotheses, our Specific Aims will: SA1- Determine how sex, age and hormonal factors affect cell type composition in RA. We will use single nucleus (sn)RNA-seq to define RA cell types at key stages of vocal development. We predict that RA's unique cell types emerge in males but not females, and that E2 shifts cell types in females towards males. SA2- Determine how masculinization affects neuronal excitable and synaptic properties in female RA. We will use whole-cell patch clamp recordings of single neurons in slices to test if female masculinization by E2 involves modulation of RA excitability and synaptic function. We predict that E2 shifts RA excitability towards males, enabling the high-frequency firing of ultranarrow spikes required for the emergence of song. SA3- Determine the role of sex-biased Z-linked genes in modulation of RA excitability We will use viral-based gene expression to test the prediction that sex chromosome (Z-linked) transcription factors (TCF4, KLF4) and downstream targets (SCN4B/3B, KCNC1) determine sex differences in RA excitability. This overall effort will lead to in-depth understanding of how hormonal vs. genetic factors act to regulate the dimorphism of a cortical circuitry that captures key aspects of human brain function and communication behavior. This mechanistic knowledge will in turn provide key insights into the potential and limitations of hormone-based therapies, and a solid basis for designing therapies for sex development disorders and gender-affirming hormone therapies.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY A major obstacle to HIV-1 eradication is the persistence of infected cells despite suppressive antiretroviral therapy (ART). HIV-1 predominantly resides in immune compartments that are difficult to access through routine sampling in people with HIV (PWH), limiting our understanding of the tissue burden of HIV-1 and the dynamics of viral reservoirs. This also hampers our ability to explore the relationship between active viral reservoirs and anatomical recrudescence following analytical treatment interruption (ATI), as well as the changes in reservoir dynamics during various stages of infection. Non-invasive techniques, such as positron emission tomography (PET) imaging, are urgently needed to directly assess HIV burden in vivo and evaluate potential HIV cure strategies. Our group has pioneered the first-in-human use of PET immunoimaging to target the HIV-1 envelope (Env) protein, enabling the identification of regions where HIV-1 protein persists and continues to exhibit translational activity in PWH, both on and off ART. Additionally, we have developed a second imaging approach using PET tracers targeting CD30, a non-viral biomarker associated with transcriptionally active HIV infection. Our findings show that HIV RNA is highly enriched in CD30+CD4+ T cells under suppressive ART, and in vivo targeting of CD30 with the cytotoxic antibody-drug conjugate (ADC) brentuximab-vedotin (BV) leads to a reduction in both HIV RNA and DNA. Importantly, CD30 expression strongly correlates with HIV RNA levels in lymphoid tissues, suggesting that CD30 may serve as a more sensitive marker for transcriptionally active HIV infection in the context of ART, particularly for PET imaging. The goal of this project is to use multimodal PET immunoimaging with zirconium-89 (89Zr)-labeled SIV Env or CD30 tracers to directly visualize sites of viral persistence and identify early tissue foci of viral rebound in SIVmac239-infected rhesus macaques (RM) undergoing ART. Additionally, we aim to explore whether near-simultaneous PET imaging of T cell activation (using [18F]F-AraG) alongside SIV Env or CD30 expression can help define the spatial relationships between virus persistence and adaptive immune responses. Finally, we will combine PET imaging of T cell activation with SIV Env or CD30 expression to track the dynamics of both viral and T cell responses following mRNA/SIVgag or control vector vaccination during ART and after ATI. To further validate these findings, PET imaging will be paired with targeted biopsies of deeper tissues, enabling a comprehensive evaluation of tracer specificity and immune activity. This approach will provide critical insights to support the clinical development and application of PET immunoimaging in PWH as a tool for HIV eradication therapeutic development.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Individuals with Opioid Use Disorder continue to remain vulnerable to relapse due to craving for the drug during abstinence. I will study oxycodone (Oxy) craving using the incubation of craving model, in which rats show progressive intensification of cue-induced seeking for drugs of abuse including Oxy during the weeks after ending drug self-administration. Craving then remains high for an additional period before declining. Our published behavioral data show that incubated Oxy seeking requires upregulation of Ca2+-permeable AMPA receptors (CP- AMPAR) on medium spiny neurons (MSN) in both Core and Shell subregions of the nucleus accumbens (NAc). Our preliminary data show this occurs on both of the main MSN subtypes: DA D1R-expressing MSN (D1+) and adenosine A2a/DA D2R-expressing MSN (A2a/D2+ MSN; A2a and D2 receptors co-localize). I propose to extend our knowledge of pathways (Aim 1) and MSN subtypes (Aim 2) involved in Oxy incubation. One candidate input originates in the basolateral amygdala (BLA). Our preliminary electrophysiological data show elevated CP- AMPAR levels in both BLA-Core and BLA-Shell pathways after Oxy incubation. Besides our data, there have been no studies of these pathways in Oxy incubation. Therefore, in Aim 1, I will use whole cell patch clamp recordings to measure the contribution of CP-AMPARs to synaptic transmission in the BLA-Core and BLA-Shell pathways onto D1+ or A2a+ MSN. I will use D1-Cre and A2a-Cre rats crossed with TdTomato reporter rats (D1- Td, A2a-Td) to selectively patch D1+ or A2a+ MSN. I hypothesize that CP-AMPAR upregulation will occur on both MSN subtypes. I will then determine if inhibition of these pathways disrupts incubated cue-induced Oxy seeking. The inhibitory DREADD AAV-hM4Di will be infused into BLA, and CNO will be delivered into Core or Shell to inhibit nerve terminals prior to a seeking test. I hypothesize that both BLA-Core and BLA-Shell pathways contribute to Oxy incubation. For Aim 2, it is known that the mu-opioid receptor (MOR) is expressed on a subset of D1+ MSN and A2a+ MSN in NAc. However, the potential role of these MOR+ MSN in incubated Oxy seeking has not been investigated. First, I will determine excitatory synaptic properties of MOR+ MSN compared to MOR- MSN after Oxy incubation. Cre-dependent AAV expressing eYFP under control of the MOR promoter will be infused in Core or Shell of D1-Td or A2a-Td rats, enabling differentiation of MOR+/D1+, MOR-/D1+, MOR+/A2a+ and MOR-/A2a+ MSN. For each, I will determine synaptic properties (paired pulse ratio, AMPA/NMDA, and CP- AMPAR levels). Second, I will determine if MOR+ MSN are necessary for incubated Oxy seeking by infusing AAV-DIO-hM4Di into Core or Shell of MOR-Cre rats. I hypothesize that MOR+ MSN will exhibit distinct plasticity and contribute to Oxy incubation. This project will address a literature gap for understanding incubation of Oxy craving while providing rigorous training in electrophysiology, chemogenetics and experimental design. This proposal will also advance my professional development through writing manuscripts and attending professional conferences, enabling me to become a well-rounded scientist competitive for future PI or staff-scientist positions.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic cancer pathologic subtype, has a 5- year survival rate of only 13%, largely due to its metastatic propensity. Most patients present with metastatic disease, rendering them ineligible for curative intent surgery. Among those that undergo a successful resection, 78% will have metastatic recurrence within 2-years. Despite significant efforts to identify metastasis-specific driver mutations, few targetable pathways have been identified, underscoring the need to explore alternative mechanisms, such as post-transcriptional regulation. Human antigen R (HuR, ELAVL1) is an RNA-binding protein implicated in post-transcriptional regulation of stress-adaptive networks. We propose HuR is essential for PDAC cells to overcome challenges associated with the metastatic cascade. Under stress conditions, HuR stabilizes and enhances translation of mRNAs required for cellular adaptation. Preliminary data from our lab reveal that HuR knockout (KO) in PDAC cells via CRISPR-Cas9 reduces liver metastatic burden in our orthotopic murine metastatic model, highlighting HuR activity as a potential therapeutic vulnerability in metastasis. My central hypothesis is that HuR is a key regulator of PDAC metastasis by selectively stabilizing pro-metastasis mRNA targets. In Aim 1, I will investigate the functional role of HuR in supporting PDAC metastasis. I hypothesize that HuR is most critical for metastasis during the outgrowth phase in the secondary site, when PDAC cells must adapt to unique secondary site microenvironments. To test this, I will rescue HuR activity at distinct stages of metastasis in our HuR KO murine model and monitor metastatic burden. Additionally, I will assess HuR expression levels and activity in matched primary and metastatic tumors from human PDAC patients to determine whether metastatic lesions have a greater reliance on HuR. In Aim 2, I will identify HuR-bound mRNAs that mediate metastatic adaptation. I hypothesize that HuR modulates distinct mRNA targets and pathways depending on site-specific stress conditions. To test this, I will perform RNA immunoprecipitation sequencing (RIP-seq) on primary, liver, and lung metastatic PDAC tumors to comprehensively map HuR mRNA binding targets specific to metastasis biology. These targets will be prioritized using PDAC patient RNA-seq datasets and clinical data from the Brenden-Colson Center for Pancreatic Care (BCCPC) at Oregon Health and Science University (OHSU). I will validate identified HuR-bound mRNA PDAC metastatic drivers as bona fide HuR targets through various molecular techniques, such as independent RIP-qPCR, western blot analysis, and actinomycin D decay assays. Lastly, I will assess the functional relevance of top targets using target site blockers (TSBs) to disrupt HuR–mRNA target interactions and measure the effects on metastasis. Importantly, integrating preclinical and clinical data will enhance the translational impact of these findings. Completion of this proposal will elucidate a novel mechanism by which PDAC cells utilize HuR to successfully metastasize, identifying specific HuR-dependent pathways as potential therapeutic vulnerabilities in PDAC.

NIH Research Projects · FY 2026 · 2025-07

Project Abstract Many adult organisms, including humans, turnover a significant number of neurons during life, and many non- human organisms can regenerate substantial neural tissue following injury. New neurons in both cases are ultimately made from adult stem cells (ASCs), yet it is largely unknown how ASCs make the correct diversity of neural cell types at any given time while maintaining proper patterning and function in an otherwise healthy tissue. Understanding, at the single-cell level, how ASCs can access neural fates, make appropriate numbers and types of new cells, and then restore brain function is of fundamental importance in devising regenerative therapies for human neural injuries. However, in order to understand the mechanisms of successful neural regeneration, model organisms capable of both the biology and gene-function analyses must be used. To understand complex biology of adult neural regeneration we use complementary model organisms that can regenerate substantial parts of the nervous system following injury: the freshwater planarian and zebrafish. Planarians have the key advantages of being able to regenerate their entire brain following decapitation, as well as test gene function rapidly by RNAi during the regeneration process in vivo. Zebrafish are the best genetic vertebrate model of neural regeneration and have the advantages of live-imaging and the ability to recover from complete spinal cord transections. Only now do we have the tools in both systems to determine the conserved mechanisms of neural regeneration at the single-stem cell level. Despite much work from our lab and others, we understand little about stem cell heterogeneity or conserved stem cell programs of regenerative neurogenesis in either system. This proposal addresses these unknowns. In Aim 1, we will test four conserved transcription factors that we hypothesize drive the neural stem cell state in planarian brain regeneration. In Aim 2, we will test 57 novel factors that we have discovered in planarians that turn on specifically in neural-fated stem cells during brain regeneration, including two homologs of the conserved musashi family of RNA-binding genes. In Aim 3, we will determine the first roles of the musashi-1b gene in stem cells during spinal cord regeneration in zebrafish using conditional genetic strategies and live-clonal analyses. Similar to planarians, musashi-1b also turns on in zebrafish neural stem cells in response to injury and we will determine the RNA targets of musashi-1b in spinal cord regeneration. In total, this proposal will deliver conserved mechanisms of adult neural regeneration from stem cells that will be of broad relevance to designing regenerative therapies for human neural injuries.

NIH Research Projects · FY 2026 · 2025-07

Project Summary During development, axon growth is initiated by specialized types of neurons called “pioneer neurons”. As their name implies, they are the first to explore the dynamic environment of a developing tissue. After pioneer neurons extend their axons toward a particular target, their axon tracts act as a scaffold for the subsequent axon extension of other neurons, termed “followers”. Pioneer neurons have been well documented in the central and peripheral nervous systems of both vertebrates and invertebrates, but whether their molecular make up is distinct from that of followers is not known. We have recently shown that the neurotrophic factor receptor ret is specifically enriched in sensory pioneer neurons of the zebrafish, suggesting pioneer and follower neurons have different transcriptional profiles. To test this hypothesis, we conducted single-cell-RNA-sequencing (scRNA-seq) of these sensory neurons. Our scRNA-seq demonstrated that ret+ neurons indeed have a distinct transcriptional profile from ret- neurons. Expression analysis revealed 101 differentially expressed (DE) genes between these two populations. Furthermore, single molecule in situ hybridization of DE genes confirmed the presence of two distinct neuronal populations in vivo. To demonstrate that the presumptive pioneer-specific cluster indeed marks pioneer neurons, we knocked in a red fluorescent protein into rpz5 locus (rpz5 is almost exclusively expressed in the ret+ cell population). The reporter labeled pioneer axon terminals, confirming the ret+ population indeed represents pioneer neurons. We also discovered that a pioneer neuron “gene signature” is enriched in both developing peripheral and central nervous systems. Thus, the pioneer cell state we discovered represents a common feature in the nervous system. Based on this and additional preliminary data, we propose three specific aims that will determine: 1) the lineage relationship between pioneers and followers and molecular signals that specify pioneers; 2) the role of DE signaling pathways in differentiation of pioneers and followers; and 3) the molecular basis of distinct physiological properties of pioneers and followers. The successful completion of these aims will identify the mechanisms by which pioneer neurons arise and how they are specified. The core set of cellular functions in pioneers is characteristic of many types of neurons, so the knowledge gleaned from these studies will be relevant to pioneer neurons in other systems. Finally, identifying main factors regulating pioneer neuron fate commitment will create a list of novel therapeutic targets for studies involving nerve injury and regeneration.