University Of Colorado Denver

NIH Research Projects · FY 2026 · 2025-06

Project Summary The complex organization. A human cell efficiently yet correctly distributes intracellular content detailed understanding of the transport mechanism is vital to understand despite its large size and neurodevelopmental and neurodegenerative pathologies. Dedicated kinesin and dynein motor protein complexes walk along microtubules for directional transport of cargoes such as organelles and vesicles, kinetochore, or ribonucleoproteins. involved i structura sufficient Many functionally relevant segments of proteins n this transport are structurally disordered regions (IDRs) such that static average l models provided by cryoEM or artificial intelligence-based structure prediction are not to understand the protein function.Our overarching mission is to tap into unexplored aspects of our understanding of the structure/function relationships in cargo transport in human cells. In this proposal, we ask: spectrum? 1) How can a single cytoplasmic dynein cover its entire functional There is only one major form of cytoplasmic dynein. Itsspecificities are partly due to a variety of cargo-specific motor adaptors, all of which contain IDRs, and the regulatory role of phosphorylation. Dyneinmis-regulation has been implicated in various nervous system disorders and colon cancer. We will reveal the mechanistic basis and functional implications of dynein specificity factors with a special focus on the dynein light intermediate chain (LIC), another IDR. We will also elucidate the role of the cell regulator Pin1, shown to have high interdomain flexibility, in interactions between LIC and the dynein adaptors. Olduvai 2) How does kinesin KIF5A regulation by protein impact human brain development? Olduvai domains are encoded by Neuroblastoma Breaking Point Family proteins and show the largest human-specific increase in copy number of any protein coding region in the genome. This increase is linked to brain size growth, but also to schizophrenia and autism. To date, the molecular mechanisms underlying these functions are entirely unknown. We will show how Olduvai domains, while being IDRs, act through modulation of kinesin KIF5A-driven transport, which occurs predominantly in neurons. For these studies, our tools are nuclear magnetic resonance (NMR), uniquely suited to study IDRs, supplemented with other biophysical methods to probe structure, dynamics, and interactions of proteins involved in transport, and in vitro motility and cell-based assays to study resulting functions. We will build on our 20+ years of NMR expertise, previous work on LIC- adaptor interaction and on allostery in Pin1, and foundational work on Olduvai domains. We anticipate that our work will inspire new questions about regulation of dynein transport and establish the molecular link between intracellular transport, Olduvai copy surge and brain disease.

NIH Research Projects · FY 2025 · 2025-06

Project summary/abstract We are seeking partial support through this proposal to cover the expenses for the 10th International Meeting of the Middle Ear Mechanics in Research and Otology (MEMRO) 2025 conference, scheduled for June 2025 at the KU Leuven, in Belgium. In alignment with the objectives established at the inaugural MEMRO meeting in 1996, the purpose of this triennial event is to bring together experts in middle ear science and engineering with clinical otologists to facilitate an exchange of knowledge and ideas between these typically independent groups. Previous MEMRO meetings in Shanghai (2018) and Denmark (2015) each attracted approximately 200 participants from over 20 countries. The 2022 meeting had slightly fewer participants since scientists from many countries, including China, Japan, Australia, and others, that would normally attend the MEMRO meetings were still precluded from travel due to COVID restrictions. We expect the 10th edition to have a higher attendance. The 2025 meeting aims to continue promoting the free exchange of ideas between basic and clinical scientists and to establish a framework for innovative collaborative efforts to enhance our understanding of middle ear function, dysfunction, and repair. A primary goal of this meeting is to increase the diversity of attendees, with a particular focus on engaging otology residents and faculty by hosting a hands-on workshop on methods and technologies the day prior to the formal conference. Also many efforts have been made to increase the presence of underrepresented countries. We have promoted the conference on multiple occasions on other world conferences. We have representatives from all continents in the scientific committee. The 2025 MEMRO meeting is being organized by KU Leuven, Belgium's largest and highest-ranked university and is an excellent center for education, research and Innovation. Leuven is a vibrant city only 15 minutes by train from Brussels Airport allowing easy travel, and is rich in art, history, and architecture, offering attendees ample opportunities to experience this dynamic international city.

NIH Research Projects · FY 2026 · 2025-06

Project Oligodendrocytes nervous propagation. involves proliferation development. developing chemogenetically preliminary precursor development of also maturation. early Here elucidate glutamate Summary (OLs) produce myelin, a lipid rich membrane that wraps neuronal axons in the central system to provide them with metabolic and trophic support and allow for faster action potential Developmental myelination requires precise spatial and temporal regulation that likely communication between OLs and neurons. In the mature brain, neuronal activity promotes OL and differentiation, but less i s known about how oligodendrocytes mature in early brain To test how glutamatergic neuronal activity modulates OL differentiation in the early brain, we use designer receptors exclusively activated by designer drugs (DREADDs) to inhibit or activate activity in cortica pyramidal neurons in mouse pups. Our data show that r educing neuronal activity n early development unexpectedly causes OL cells (OPCs) to differentiate prematurely, whereas increasing neuronal activity in early inhibits OPC differentiation. Here we propose to investigate the long-term consequences early developmental dysregulation of oligodendrocyte differentiation on later development. We will investigate the impact of deprivation of sensory-evoked neuronal activity on oligodendrocyte Additionally, we identified oligodendroglial glutamate receptors as potential regulators of OPC maturation in response to neuronal glutamatergic activity by single cell RNA sequencing. we urther investigate he role of glutamatergic activity on oligodendrocyte differentiation and the mechanisms of glutamatergic signaling to oligodendroglial ionotropic and metabotropic receptors ex vivo. l i f t

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY/ABSTRACT Self-injurious thoughts and behaviors (SITBs) are a growing and underrecognized problem in preadolescents. About 15% of preadolescents report having some experience with suicidal thoughts; 10% have engaged in self-injury; and 1 in 100 report a suicide attempt. Youth who experience SITBs at a younger age are more likely to progress to more frequent and dangerous forms of self-injury and be hospitalized. Despite the importance of early intervention, there are currently no outpatient, research-supported treatments designed for preadolescents with SITBs. Developing and testing treatments specifically for preadolescents is crucially important, as risk factors differ in this age group. Treatment must also be developmentally appropriate and incorporate caregivers in ways that address the unique needs of preadolescents and their families. Emotion dysregulation and executive functioning (EF) are modifiable, transdiagnostic processes that underlie risk factors for SITBs in preadolescents, are sensitive to treatment change, and have been identified as key targets in recent calls for increased focus on prevention and early intervention of preadolescent SITBs. Dr. Sarah Kennedy (PI) and Dr. Laura Anthony (Co-I) have co-developed and evaluated transdiagnostic interventions targeting these key risk factors. The goal of the proposed R34 is to integrate and adapt these existing interventions to develop SURE-Kids, the first outpatient treatment specifically for preadolescents with SITBs to address emotion dysregulation and EF within the family context. The aims of this project are to: 1) iteratively develop a transdiagnostic intervention for preadolescent SITBs addressing emotion dysregulation and EF, with guidance from experts and end users to maximize implementation potential; 2) evaluate engagement (primary outcome), feasibility, acceptability, and implementation outcomes, using a pilot randomized controlled trial design with a treatment as usual (TAU) comparison condition; and 3) collect preliminary evidence to explore superiority of SURE-Kids to TAU in reducing SITBs and engaging target mechanisms. Trial participants (N = 52) will be recruited from preadolescents ages 7-12 admitted to the emergency department at a large pediatric hospital in Colorado for SITB-related concerns. Participants will complete assessments at baseline, 6, 12, and 24-weeks post- randomization. Engagement will be measured by evaluating whether SURE-Kids participants attend more sessions than TAU participants. Predetermined benchmarks will be used to evaluate feasibility and acceptability, and implementation outcomes will be evaluated both quantitatively and qualitatively. Based on pilot trial results, adaptations to the intervention will be finalized with guidance from an Adaptation and Implementation Team. The end-product will be an iteratively adapted intervention for preadolescent SITBs and refined study procedures, which will be used in a larger, fully-powered, hybrid type I effectiveness- implementation trial.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY While bacteria broadly secrete multiple virulence factors during infection to modulate the host cell machinery for infection, S. aureus is unique in that most strains encode for a distinct protease family of up to six members, broadly referred to as serine proteases-like A-F (SplA-F). Spl family members do not respond to standard serine protease inhibitors, highlighting their unique active siteS. For example, the structures of Spl family members alone reveal such shallow active sites that they have been proposed to be reliant on conformational changes for target recognition. Furthermore, a novel conformational “switch” that allosterically couples the N-termini of Spl family members to their active sites has been proposed to underlie their zymogen activation {Pustelny, 2014 #3042}. Thus, conformational changes and allostery underlie Spl function through unknown mechanisms. Our lab has developed NMR-based methods to specifically identify the role of conformational changes and allostery in enzyme function. Based on our preliminary biological studies that have identified SplB as the most highly expressed Spl family member, we have begun identifying SplB host targets and applying NMR-based methods to elucidate its target interactions and underlying allosteric mechanism of activation. We hypothesize that S. aureus SplB targets multiple host proteins (Aim 1), which induce target- dependent conformational changes (Aim 2). Activation of the SplB zymogen via N-terminal cleavage leads to both global structural and dynamic changes that facilitate active site opening and target entry (Aim 3). This hypothesis will be tested through the following specific aims: Aim 1) Identify host protein targets of S. aureus SplB. Rational-based approaches together with unbiased methods will be used to broadly identify SplB host targets and their cleavage sites will be identified by mass spectrometry (MS). Target sequences will be biochemically validated by inserting them as linkers within a fusion protein. Aim 2) Elucidate the conformational changes required for SplB engagement with host targets. High- resolution NMR structural methods will be used to determine how SplB targets are engaged and whether there are target-specific conformational changes. Aim 3) Identify the allosteric mechanism of SplB activation and the role of dynamics in SplB function. High-resolution NMR structural methods will be used to determine the structural basis of SplB zymogen activation, i.e., the allosteric “switch,” while NMR relaxation methods will be used to determine the dynamic basis of activation and the role of dynamics in function.

NIH Research Projects · FY 2026 · 2025-05

Project Summary/Abstract Mouse forelimb movements are widely studied in systems neuroscience to study healthy and injured motor networks (e.g., after stroke or cerebellar disease). This proposal builds on our novel musculoskeletal model of the mouse forelimb to develop free and open-source tools for the neuroscience community to extract biomechanical features that are difficult or impossible to measure experimentally and to investigate hypothesized control architectures with artificial neural networks and physics-based simulations. In Aim 1, we will develop a computational model based on optimal control theory to estimate muscle activity from kinematics and optional electromyography as state-of-the-art experimental methods can only measure from 3-4 muscles simultaneously out of the 25+ forelimb muscles. We will also develop a tool to predict mouse reaching kinematics and muscle activity when there is a change in the task or limb biomechanics, which could help reduce the number of experiments with mice. The computational models will be validated with experiments of mice reaching to different pellet locations and with different weights placed on their limbs. In Aim 2, we will demonstrate how to use the musculoskeletal model and physics engine to extract biomechanical features and correlate them with neural activity in motor cortex and the cerebellum. This will allow to us to test hypotheses in motor control such as whether interaction torques are represented in Purkinje cells' activity or whether motor cortex drives an abstract representation of the limb or a detailed biomechanical model during reaching. We will develop in silica networks and compare and contrast them with analogous neural structures. We will show that by modeling these artificial networks with different assumptions and architectures, we will be able to dissociate between hypotheses in motor control, such as whether the cerebellum implements a forward model or a control policy during motor adaptation or whether both sensory input and motor cortex exhibit rotational dynamics during three-dimensional reaching to different pellet locations. These artificial neural networks will be validated with empirical neural data. We will synthesize feedback controllers using these artificial neural networks with deep reinforcement learning so that researchers can implement hypothesized control architectures and observe the effects of neural manipulations and disturbances on the predicted kinematics and muscle activity. We will refine our tools with end-users to ensure their ease-of-use and extensibility. A web-based interface will be implemented allowing users to develop their code, visualize the simulations and run optimizations on the cloud or using their own computers or servers. We will disseminate these tools with extensive documentation, example code, online video tutorials and maintaining a forum for end-user questions and requests.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT Chronic thromboembolic pulmonary hypertension (CTEPH) is a deadly sequela following acute pulmonary em- bolism (PE). In CTEPH, the acute PE does not resorb but develops into organized “thrombi,” causing pulmo- nary vascular remodeling, leading to progressive right heart failure and death. The gold standard treatment for CTEPH is surgery, but many patients are not surgical candidates due to comorbidities or surgically inaccessi- ble disease. For these patients, there are only limited options that have only been shown to improve short-term symptoms. Thus, developing novel medical therapies for CTEPH is a significant unmet need. Drug develop- ment has been limited by the significant knowledge gap on how different cell types and signaling pathways within chronic thrombus contribute to pulmonary vascular remodeling and pulmonary hypertension (PH). This puts into context our preliminary studies, using single-cell RNA sequencing (scRNAseq) and ex-vivo culturing of patient thrombus-derived cells, we identified upregulated inflammatory and proliferative signaling pathways that may contribute to chronic thrombus development and pulmonary vascular remodeling. We showed that a phenotypically-modulated smooth muscle cell (SMC) population expresses a high level of PAR1, a receptor that promotes SMC proliferation and migration and propagates IL-17-mediated inflammation. Our data also revealed that SMCs, macrophages, and T cells are enriched for IL-17 signaling, which has been implicated in numerous inflammatory diseases and promotes venous and arterial thrombosis, suggesting a role for the PAR1/IL-17 signaling axis in CTEPH pathogenesis. Thus, our central hypothesis is that the PAR1/IL-17 sig- nal axis propagates abnormal chronic thrombus development and pulmonary vascular remodeling in CTEPH, linking inflammation and pulmonary vascular remodeling in this disease. The overall objective of this applica- tion is to determine the contribution of PAR1/IL-17 signals to the development of abnormal cellular phenotypes in chronic thrombus, initiation, and progression of CTEPH using human samples and animal models. Aim 1 will determine if the PAR1/IL-17 signaling synergistically promotes abnormal thrombus-derived mesenchymal cell phenotypes. In Aim 2, we will evaluate the activity of the PAR1 and IL-17 signaling axes in different immune and mesenchymal cells in human CTEPH using flow cytometry and single-cell technologies. In Aim 3, we will determine whether inhibiting the PAR1/IL-17 signaling axis prevents and reverses CTEPH in vivo using a ro- bust small animal model of CTEPH that we have developed. The rationale for these studies is that defining the mechanisms contributing to inflammation and proliferation will aid the development of novel CTEPH medical therapies. The expected outcome of this project will be to determine whether the PAR1/IL-17 signaling axis promotes disease progression in CTEPH and could serve as a disease target. This will lay the groundwork for future studies on novel CTEPH treatments targeting SMC and immune cell activation. These studies will have a significant positive impact as they will aid in developing new CTEPH therapies.

NIH Research Projects · FY 2026 · 2025-04

Project Summary Inflammatory bowel disease (IBD) represents a group of chronic mucosal inflammatory disorders, including Crohn's disease and ulcerative colitis. Many IBD patients have limited therapeutic options. Oxygen metabolism and hypoxia play prominent roles in healthy intestinal tissue and energetic shifts during active inflammation. Hypoxia triggers the temporal stabilization of the transcription factors hypoxia-inducible factors HIF1α and HIF2α, which are essential in maintaining intestinal homeostasis. HIF-1/2α are regulated by a family of prolyl hydroxylases (PHDs). Inhibition of PHDs results in transient stabilization of HIF-1/2α and thus, PHD inhibitors (PHDi) have become attractive targets for the therapeutic treatment of IBD. We have investigated the chemical knockdown of PHDs by “hijacking the proteasome system” using proteolysis targeting chimeras (PROTACs), heterobifunctional compounds that degrade proteins of interest. PROTACs advantages include enhanced specificity, higher potency, and sustained inhibition. In ongoing studies, we have produced a panel of PHD targeting PROTACs. In vitro, we observe significant PHD2 degradation and modest HIF stabilization at nanomolar concentrations, a 2-3 log increase in potency compared to PHDi. Importantly, the wound healing capacity of epithelial cells is significantly enhanced. These results warrant further investigation as there is vast room for improvement in developing fine-tuned PROTACs. The intent of this proposal is to design HIF stabilizing PROTACs and elucidate their role in barrier formation and wound healing as supported by metabolic shifts. The end goal is to reveal the therapeutic potential of PROTAC-HIF stabilization in murine models of IBD. The applicant, Dr. Ornelas Sanchez, has established a scientific niche in which to build a foundation for independent research on the development of PROTACs that influence inflammatory pathways. Dr. Ornelas Sanchez and his mentor, Dr. Colgan, assembled a mentorship committee to regularly meet as a group to provide feedback, critique the research plans, monitor publications, and provide career advice. Of note, he will train under the supervision of Dr. Reigan to learn molecular modeling of PROTACs. Furthermore, we have identified coursework to expand his knowledge in computational modeling, fundamental molecular biology, and cellular biology. Dr. Ornelas Sanchez will submit his work to present at conferences specific to his research to share his results, network with colleagues and potential collaborators, and get valuable feedback. His development will benefit from continued participation in the Mucosal Inflammation Program (MIP), a multi-disciplinary, multi- departmental program initiated to study mechanisms of mucosal inflammation and resolution. The MIP fosters a unique environment for collaboration between physician and research scientists and establishes an environment for young investigators to flourish and develop. The facilities and resources available to and development plan built for Dr. Ornelas provide an ideal environment and path towards his successful transition to independence.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Chikungunya (CHIKV) and Ross River (RRV) viruses are mosquito-transmitted RNA viruses that cause explosive epidemics of debilitating acute and chronic polyarthralgia/polyarthritis. Although CD8+ T cells recognize and eliminate virus-infected cells, their role in arthritogenic alphavirus infection has remained enigmatic. Remarkably, in mouse models of CHIKV and RRV infection, viral burden in joint tissues is equivalent in congenic wild-type and CD8α-/- mice, and priming of CD8+ T cells after CHIKV and RRV infection is delayed and dampened. The mechanistic basis for how arthritogenic alphaviruses evade CD8+ T cell immunity is unknown. scRNAseq analysis of CD8+ T cells in the draining lymph node of RRV-infected mice revealed reduced maturation and proliferation in contrast to CD8+ T cells from LCMV-infected mice, which show a rapid conversion to effector T cells. The impaired CD8+ T cell priming was reversed by loss of type I IFN signaling in DCs, which increased viral infection and antigen in DCs; this result suggests that type I IFN paradoxically impairs CD8+ T cell priming by limiting alphavirus infection of key antigen (Ag)-presenting cells (APCs). Separately, we found that viral Ag presentation by CHIKV-infected or nsP2-transfected joint tissue fibroblasts was inefficient. Primary ankle fibroblasts infected ex vivo failed to activate Ag-specific CD8+ T cells, and mutations in the CHIKV nsP2 protein restored Ag presentation and CD8+ T cell activation. Finally, using intravital microscopy (IVM), we observed that viral epitope-specific CD8+ T cells do not durably engage or kill RRV-infected cells. Our primary goal is to define mechanisms by which arthritogenic alphaviruses evade CD8+ T cell-mediated clearance. In Aim 1, we hypothesize that type I IFN restricts infection of myeloid cells which limits direct antigen presentation and the timely priming of anti-alphavirus CD8+ T cells. We will determine the phenotype and infection status of APCs that intrinsically can or cannot respond to type I IFN during alphavirus infection versus immunization. In addition, we will determine how type I IFN signaling directly affects CD8+ T cell responses during alphavirus infection and test if improved priming enhances clearance of alphavirus infection. In Aim 2, we hypothesize that alphavirus- infected cells in joint tissue are inefficiently targeted by CD8+ T cells because the viral nsP2 protein disrupts MHC-I Ag presentation. We will determine mechanisms by which CHIKV infection and nsP2 protein impair MHC-I Ag presentation and define the extent to which this is a generalizable alphavirus immune evasion mechanism. In Aim 3, we hypothesize that virus-infected cells in joint-associated tissues are inefficiently targeted by CD8+ T cells because the priming environment generates CD8+ T cells that poorly engage virus-infected cells. We will define how the intrinsic absence of type I IFN signaling in APCs regulates the differentiation and maturation of CD8+ T cells during alphavirus infection and immunization. We also will use IVM to determine the impact of APC- and CD8+ T cell-intrinsic type I IFN signaling on viral antigen-specific CD8+ T cell migration and homing. We will define the frequency, duration, and outcome of interactions between CD8+ T cells and virus-infected cells.

NIH Research Projects · FY 2025 · 2025-04

PROJECT SUMMARY/ABSTRACT During K23 funding, I will become an independent investigator by adapting and evaluating a Spanish-language version of the evidence-based executive functioning (EF) intervention Unstuck and On Target with children with autism and/or ADHD and their families in multiple contexts. This project is critical because EF difficulties have long-term impacts, evidence-based services to address them are limited, and effective intervention can be applied regardless of diagnosis. Quantitative and qualitative data will be collected on feasibility, acceptability, and effectiveness to guide adaptations while maintaining fidelity to core components. My research background in interventions for children with autism and ADHD and their families, along with my clinical and research training specialization in Spanish-language services, has led to my current focus on evaluating and scaling evidence-based interventions. At the University of Colorado Anschutz Medical Campus, I have significant support and resources to train to independence utilizing the K23 mechanism. My mentorship team is led by Dr. Laura Anthony, an expert in EF intervention, and includes Dr. Mónica Pérez Jolles, an expert in dissemination and implementation science, Dr. Lauren Haack, an expert on running clinical trials, and Dr. Susan Mikulich Gilbertson, an expert in clinical trial and statistical modeling/ analysis. My training objectives include deepening my understanding of: 1) dissemination and implementation science; 2) clinical trials and statistical modeling; 3) qualitative and mixed-methods analyses; and 4) responsible conduct of research in international contexts. I will accomplish these training objectives through coursework and seminars, guidance from mentors/consultants, and completion of the proposed research.

NIH Research Projects · FY 2026 · 2025-04

Summary: Small molecule splicing modulators have emerged recently as exciting new approaches to efficiently modulate protein levels in genetic diseases that affected the CNS. One such compound, risdiplam, is orally bioavailable with excellent CNS distribution and has been approved by the FDA in 2020 for the treatment of Spinal Muscular Atrophy (SMA). This approval has generated tremendous excitement about using small molecule splicing modulators as a new way to reduce huntingtin (HTT) protein levels in Huntington’s disease (HD) by inducing the inclusion of a “poison” exon in HTT, leading to non-sense mediated decay of its mRNA. Although branaplam, a splicing modulator developed by Novartis, has recently been suspended in phase 2 clinical trial for HD due to peripheral neuropathy, PTC Therapeutics was able to identify a different class of splicing modulator that induces the same “poison” exon inclusion but enters the CNS much better, potentially avoiding peripheral neuropathy. An analog in this class is currently in phase 2 clinical trial. These results highlight the potential of splicing modulators for HD therapy, as well as the critical need for understanding the mechanism of action of these modulators to facilitate compound optimization and overcome the many hurdles in the journey of these compounds in becoming an FDA-approved drug. Despite the spectacular success of these splicing modulators in treating CNS disease such as SMA, the mechanism of action of these compounds remains unknown. We propose to evaluate the feasibility of determining the mechanism of action of these compounds using a combination of cyro-EM structural determination and biochemical approaches. Support from this R21 grant will allow us to work out the challenges and develop a tool to understand the mechanism of action of these modulators. This will not only be a huge facilitator for the development of these modulators into HD therapeutics, but also will potentially benefit the development of these compounds for many other neurological diseases in the future.

NIH Research Projects · FY 2026 · 2025-04

Significant improvement in lung squamous cell carcinoma (SCC) mortality requires preventive medical inter- vention at the premalignant lesion (PML) stage to prevent progression to tumors. Historically, phase III lung cancer prevention agent clinical trials were based on population data and the agents tested had no effect or were even harmful to patients. More recently, phase I/II clinical trials of prevention agents supported instead by strong preclinical evidence have generated promising results. This highlights a critical need for preclinical mod- els to support translation of prevention discoveries to clinical trials. However, previous mouse models of lung SCC require intense time and effort and do not include human-relevant exposures. The main objectives of this proposal are to validate the human relevance of a novel lung SCC mouse model and use the model in a sys- tem for screening and testing lung cancer prevention agents. Preclinical prevention studies for lung SCC have been limited to using N-nitroso-tris-cholorethylurea (NTCU) for eight months to induce SCC lesions. We im- proved this model by including a human-relevant exposure, combining NTCU with cigarette smoke (NTCU/S) and inducing PMLs in only three months. However, the NTCU/S model is not credentialed for translationally relevant questions, so we propose to validate the model with previously generated human data. We will then incorporate tissue from the NTCU/S model into our bioengineered Precision Cut Lung Slice (PCLS) model, which maintains the complex microarchitecture, cellular diversity, and functional response of mouse tissue for six weeks in ex vivo culture. We hypothesize that the NTCU/S model will generate data relevant to humans and can be successfully employed in an innovative ex vivo-to-in vivo prevention agent testing strategy. To achieve our main objectives, we propose three aims: 1) Define the human relevance of the in vivo NTCU/S mouse model, 2) Validate an ex vivo PCLS approach to screening new prevention agents for the NTCU/S, 3) Use the NTCU/S ex vivo to in vivo testing strategy to determine the preventive efficacy of combined PD-1 and MEK inhibition. The expected outcome from this project is validation of the NTCU/S model of lung SCC as a robust representation of human biology, advancing the preclinical models available for translational lung can- cer questions. We will establish a novel ex vivo-to-in vivo strategy for rigorously screening and testing preven- tion agents and investigate a new prevention agent combination approach. This work represents a paradigm- shift from the historical path of prevention agent development by leveraging human-relevant preclinical models to boost research efficiency and advance only the most effective agents to clinical studies. Our interdisciplinary team is well-positioned to translate preclinical data to the clinic to achieve our long-term goal of delivering ef- fective prevention agents to patients. Generating human-relevant preclinical models of lung PMLs and preven- tion addresses critical needs of the NCI Division of Cancer Prevention and PAR-23-281, “Research Projects to Enhance the Applicability of Mammalian Models for Translational Research”.

NIH Research Projects · FY 2025 · 2025-04

Project Summary/Abstract Recent developments in super-resolution imaging have revealed that proteins involved in neurotransmitter release and detection are non-uniformly distributed and assemble into regions of high density called nanoclusters (NCs). Pre- and post-synaptic NCs often align across the synapse to form transsynaptic nanocolumns, which are thought to be required for efficient synaptic communication. Transsynaptic cell-adhesion molecules, which span the synaptic cleft and are capable of participating in bidirectional signaling via intracellular and extracellular sequences, are essential for the nanoarchitecture of synapses. Neurexins (Nrxns) are a family of evolutionarily conserved presynaptic adhesion molecules. We recently performed the first 3D dSTORM super-resolution imaging of endogenous neurexins using our novel double epitope-tag mouse line that permits the immunolabeling of Nrxn1 and Nrxn3. We found that Nrxn1 and Nrxn3 each form spatially discrete and non- overlapping NCs. Additionally, Nrxn1 NCs and Nrxn3 NCs preferentially align transsynaptically with GluD1 and LRRTM2, respectively. We proposed that this spatially discrete nano-organization of neurexins and their ligands results in parallel signaling, which explains how neurexin-1 and neurexin-3, despite sharing high sequence homology, control distinct and non-overlapping properties of synapse function. A fundamentally important question is: how are the intrinsically monomeric neurexins organized into discrete and homogeneous NCs? We hypothesize that differential interactions with cytoplasmic proteins establish the discrete nano-organization of Nrxn1 and Nrxn3 NCs. To test our hypothesis, we used purified hippocampal synaptosomes from our double epitope-tag mouse line to perform the first co-immunoprecipitation mass spectrometry of endogenous neurexins and identified cytoplasmic proteins that exclusively co-immunoprecipitated with Nrxn1 or Nrxn3. Importantly, due to limitations circumvented by our novel mouse line, no new neurexin-interacting cytoplasmic proteins have been identified in over twenty years. The top candidate is p130Cas, which exhibits exclusive co-immunoprecipitation with Nrxn1. This proposal builds on our published results and compelling preliminary data that indicate that p130Cas controls the surface expression of neurexin-1, the volume of Nrxn1 NCs, and presynaptic release probability at excitatory synapses. We will use orthogonal 3D STED and 3D dSTORM imaging to dissect how p130Cas regulates neurexin-1 and the nanoarchitecture excitatory synapses in Aim 1. In Aim 2, we will use classic structure-function approaches to define the sequences necessary and sufficient for p130Cas and neurexin-1 binding, and systematically assess the functional implications of Nrxn1 – p130Cas interactions using electrophysiology. Here, we will provide the first critical insight into how neurexin NCs are established, start to define how individual Nrxns differentially control intracellular signaling and, importantly, create a methodological framework to interrogate our other candidates.

NIH Research Projects · FY 2025 · 2025-03

Summary Increasingly liberal cannabis policies in the U.S. have been associated with both “replacement”, whereby cannabis is substituted for alcohol, thus decreasing alcohol use, and “enhancement”, whereby cannabis use increases alcohol use. These conflicting patterns may be partially explained by the fact that the potency of delta-9-tetrahydrocannabinol (THC; the main psychoactive cannabinoid in cannabis) in cannabis in the U.S. varies widely, with legal-market cannabis containing increasingly higher THC potencies. When combined with alcohol, cannabis may confer either synergistic or mitigating effects on craving, impulsivity, cognitive impairment and subsequent drinking, likely depending on several factors, including cannabinoid dose and content. The limited literature in this area has generated conflicting findings; some studies have shown that THC increases alcohol intake and has synergistic effects on the subjective effects of alcohol, and others have shown that THC decreases alcohol intake or has no effects on these outcomes. Emerging work also suggests that alcohol and cannabis exert opposing effects on the digestive, immune, and central nervous systems, collectively known as the microbiota-gut-brain-axis (MGBA). Alcohol is linked to immune dysfunction and disturbances in gut microbial species (microbiota), and these MGBA disruptions have been associated with neurobehavioral AUD symptoms (e.g., craving, impaired control). Conversely, preclinical data suggest that cannabinoids may confer beneficial effects on aspects of the MGBA. The opposing findings regarding the effects of cannabis on alcohol use may be partially due to the differential actions of cannabinoids throughout the MGBA, which need to be better characterized in humans. Thus, the goal of this naturalistic study is to explore effects of legal-market cannabis on acute and daily alcohol consumption, neurobehavioral AUD phenotypes and MGBA function in heavy drinkers. We will recruit N=61 heavy drinking, regular users of legal- market cannabis to complete daily diary measures of alcohol and cannabis use during two 7-day periods (a no- cannabis period and an ad lib cannabis use period) and undergo two lab sessions; Visit A assesses cognitive function, impulsivity, craving, alcohol self-administration, MGBA biomarkers and blood-THC levels in the absence of acute cannabis, and Visit B tests the same outcomes following subjects’ self-administration of their preferred legal-market cannabis in their homes. The study uses the Mobile Cannabinoid Pharmacology Lab (an IRB-approved method developed by our team to test acute effects of legal-market cannabis and quantify blood-THC). The PI, Dr. Karoly, will pursue training aims to broaden her skillset and enable her to develop expertise in 1) MGBA analysis, 2) cannabinoid pharmacology, and 3) biostatistics. To guide her research and training, Dr. Karoly has assembled a premiere mentorship team with expertise spanning these domains. She will be well-supported as she develops the skills and expertise necessary to launch her independent, patient- oriented, translational program of research focused on novel AUD treatment and harm reduction strategies.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Right Ventricular (RV) dysfunction is the primary cause of acute RV failure in pulmonary arterial hypertension (PAH). RV failure in turn is the cause of at least 70% of deaths in PAH patients and is almost universally correlated to poor prognosis. RV function is routinely assessed with echocardiography, which allows for dynamic visualization of RV motion as well as assessment of major cardiac structures. As part of a routine clinical exam, echo-derived global longitudinal strain (GLS) is measured from a two-dimensional echo (2DE) image; strain is a mechanical measure of cardiac deformation during RV contraction. GLS has been incorporated into recent echo guidelines, is emerging as a useful measure of RV systolic and diastolic function and has prognostic significance. GLS tends to be an earlier measure of ventricular dysfunction, with reduced values showing up before global function (as measured by ejection fraction, EF) is significantly reduced. However, the 2DE modality by which GLS is obtained is, by definition, confined to a 2D plane, and thus cannot fully visualize contractions over the asymmetrically shaped RV. We have recently developed an analysis of three-dimensional echo (3DE) image data that overcomes these limitations and provides 3D surface (3DS) strain values upon the entire RV surface. We have shown that these 3DS strains are in reasonable agreement with 2D GLS and further 1) provide additional deformational parameters such as shear strains and two normal principal strains (PS); 2) shown the PS have stronger correlation with EF compared to GLS and display disease-specific changes in direction on the ventricular surface. Below, we demonstrate preliminary evidence that these 3DS parameters have strong associations to one-year composite outcomes. We believe the 3DS strains add truly novel detail regarding how the RV deforms in three dimensions; although this work focuses on pediatric patients, initial work with our adult PAH collaborators suggests this holds true there as well. Overall, 3DS strain should allow for earlier bedside detection of ventricular dysfunction and in turn, earlier management changes for PAH patients. We now have a functional computational pipeline for the processing of strain data from clinical imaging; our secondary analysis goals for this proposal are to further enhance this pipeline to enable the routine spatial and temporal examination of 3D strains via machine learning and the comparison of these strain parameters to disease outcomes. Upon completion of these goals, we will be well-prepared to extend these studies into larger prospective populations, and even potentially transfer these concepts towards better characterization of LV function.

NIH Research Projects · FY 2026 · 2025-03

Breast cancer (BC) is one of the most diagnosed cancers in women (second only to skin cancer) and comprises ~30% of all new female cancers each year. Nearly all BC-related deaths are caused by metastatic dissemination. Triple negative breast cancer (TNBC), which is characterized by the absence of estrogen receptors, progesterone receptors, and amplification of human epidermal growth factor receptor 2 (ER/PR/HER2), is highly metastatic, with poorer short-term survival than other BC subtypes. Because of the lack of actionable targets, this subtype of BC has limited targeted therapies. Recent data suggest that TNBC is more immunogenic than other BCs, due to its higher mutational burden. However, preliminary data from immunotherapy trials have shown only modest responses in TNBC, likely because TNBC is a heterogeneous disease, and can exhibit immunoreactive or immunosuppressive tumor immune microenvironments (TIME). Thus, understanding molecular pathways that specifically contribute to TNBC are critical for developing new therapeutic avenues. In this proposal we study the role of a critical developmental transcription coactivator and phosphatase, Eya3, and its interaction with the B55α subunit of PP2A, in mediating breast cancer metastasis. Eya3 is overexpressed in TNBC when compared to other BC subtypes, and we have previously demonstrated that it enhances BC growth via a Myc-PD-L1 axis that suppresses CD8+ T-cell elimination of tumor cells. Preliminary data in this proposal demonstrate that Eya3 does not only regulate primary tumor growth, but also strongly, and specifically, regulates the TIME in the pre-metastatic niche and increases metastasis. Our preliminary data further show that in addition to stabilizing Myc, Eya3 directly regulates NFkB signaling, which is known to mediate inflammation and promote metastasis. In this proposal we will test the hypothesis that Eya3 mediates tumor progression not only by regulating Myc signaling, but also by directly regulating NFkB signaling through a shared mechanism that requires an interaction between Eya3 and PP2A-B55α and leads to an altered TIME. Understanding the molecular mechanism(s) by which Eya3 regulates PP2A function to alter two critical cancer nodes may in the future lead to novel, less toxic means to target immune suppression and metastasis in TNBC. To test this hypothesis, we will carry out two aims: 1) To determine the molecular and cellular mechanism(s) by which the Eya3-NFkB axis enhances TNBC metastasis, 2) To determine the molecular mechanism(s) by which Eya3 regulates NFkB signaling and whether this axis can be inhibited simultaneously with the Myc axis. This proposal will enable us to understand how Eya3 promotes TNBC via enhancing the tumor promotional, rather than suppressive, actions of PP2A. It will also set the stage for the exciting possibility that direct inhibition of the Eya3/PP2A-B55a interaction may be an effective means to target two major tumor promotional pathways that are difficult to target directly (Myc) or whose targeting leads to significant toxicities (NFkB), as a means to enhance the adaptive and innate immune response to TNBC.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY High temperature requirement A (HtrA) enzymes represent a diverse family of serine proteases found across prokaryotes and eukaryotes that are critical for cellular homeostasis. In pathogenic bacteria, these enzymes are also shed from membranes after their N-terminal anchoring domain to target the host cell machinery for infection. However, the molecular mechanisms of the entire phylogenetic branch of Gram-positive bacteria that include S. pneumoniae HtrA remain largely unexplored. Considering that our biological studies have confirmed that S. pneumoniae HtrA is a virulence factor critical for infection, we propose to elucidate its molecular mechanism for the first time. The exploratory nature of this R21 is ideal for investigating the unique mechanistic features of the first family member from this phylogenetic branch of pathogenic Gram-positive bacteria. Our discovery that S. pneumoniae HtrA is a stable monomer provides a unique opportunity to address fundamental questions. For example, most HtrAs form trimers and some higher order oligomers. HtrAs have been crystallized in both an “open” form that is substrate-accessible and “closed” form where the PDZ blocks the PD active site, prompting questions as to how this occurs. Our preliminary findings of S. pneumoniae HtrA resolve such issues, as we can directly detect a dynamic process of “opened” and “closed” conformational sampling through NMR within the monomer and observe a stepwise oligomer formation used for substrate engagement through EM. Thus, we propose to fully elucidate this dynamic “opening”/“closing” of S. pneumoniae HtrA (Aim 1) and determine how this first family member within this largely unexplored Gram-positive phylogenetic branch oligomerizes for target engagement (Aim 2). We hypothesize that S. pneumoniae HtrA PDZ “opens” and “closes” over the PD active site while oligomerization and target formation involve unique conformational changes specific for this phylogenic branch. Our hypothesis will be addressed through the following specific aims: Aim 1: What are the structural rearrangements and associated dynamics of the S. pneumoniae HtrA monomer? NMR will be used to determine the conformational changes that occur between “opened” and “closed” conformations within the HtrA monomer along with relaxation experiments used to probe the underlying dynamics and cooperativity of such changes. Aim 2: What is the structural basis of S. pneumoniae HtrA oligomerization and substrate engagement? Cryo-EM will be used to elucidate the structural basis of HtrA oligomerization. Specifically, we will determine the structure of HtrA S234A mutant, which allosterically triggers trimer formation, and HtrA in complex with -casein, which induces hexamer formation. These studies will identify the molecular determinants responsible for the hierarchy of oligomerization.

NIH Research Projects · FY 2026 · 2025-03

Abstract Severe bleeding is a major cause of morbidity and mortality worldwide. The inability to stop non- compressible hemorrhage leads to high mortality in low-resource settings. We aim to engineer hemostatic red blood cells targeted to collagen type I and to fibrin clots (t-RBCs). We hypothesize that t-RBCs will adhere to collagen-I and fibrin in the injured blood vessel and, due to their sizeable corpuscular volume and surface area, will “plug” the bleeding site. Slowing or stopping the bleeding will buy precious time for surgical interventions and resuscitation procedures. Our previously published data support the feasibility of RBC modifications with ligands. RBC is the ideal biomaterial for clot formation due to its availability, biocompatibility, large corpuscular volume, and enormous surface area. This approach has never been explored in emergency medicine. This work will focus on synthesizing t-RBCs, testing the ability to enhance clot formation and stability in vitro, and performing a pilot hemostasis experiment in vivo. We propose the following Specific Aims: 1) engineer t-RBCs and test binding efficiency in vitro; 2) perform hemostasis measurements in vitro using thromboelastography and rheometry; 3) determine the circulation time, binding to the clot, and the effect on hemostasis in an in vivo model. This novel project will impact management and survival in non-compressible hemorrhage.

NIH Research Projects · FY 2026 · 2025-03

The work described in this proposal investigates a novel approach to reduce drug accumulation in healthy tissues and improve the efficacy of chemotherapy. More specifically, we take advantage of recent studies demonstrating an innate immune response (epithelial tightening) that limits viral infection, and our preliminary data show that this response can be exploited to reduce the accumulation of nanoparticles in healthy tissues. The key molecule involved in signaling the tightening response is IFN-λ, and we have shown that this cytokine is produced in response to the injection of virus-like particles (lipoplexes). Moreover, pretreatment with this cytokine elicits tightening in healthy tissues without affecting delivery to the tumor. We propose that eliciting this response will permit unimpeded delivery to tumors under conditions where deposition in healthy tissues is significantly reduced. As a demonstration of these effects, tumor-bearing mice pre-treated with IFN-λ exhibited reduced accumulation of liposomal doxorubicin (Doxil®) in healthy organs, reduced toxicity, and improved survival. By characterizing the tightening response in terms of IFN-λ dose, route of administration, and timing as well as its effects on organ and skin toxicity, our experiments will identify conditions that can be used to minimize adverse effects of chemotherapy while enhancing drug accumulation in the tumor. Finally, we will explore the potential of utilizing the reduced accumulation in healthy tissues to more aggressively dose chemotherapy in two syngeneic mouse models for ovarian cancer and conduct immune profiling to assess synergistic effects of IFN-ʎ with Doxil.

NIH Research Projects · FY 2026 · 2025-03

The leading cause of breast cancer deaths is metastasis. Metastatic relapse can occur months to years after the initial diagnosis and treatment of the primary tumor. Cancer cells can disseminate from the primary tumor into different tissues including lungs and remain in a dormant state for years to decades. Awakening of these dormant disseminated cancer cells (DCC) leads to metastasis. Finding factors that trigger the awakening of dormant DCC and developing strategies to reduce the risk of awakening is therefore an unmet need. While it is known that inflammation is a key contributing factor to the awakening of dormant DCC, no studies have investigated whether inflammation triggered by viral respiratory infections (a very common infection worldwide) in the lung can promote the expansion of DCC and lead to the development of metastases. Our recent studies using a mouse model of breast cancer DCC dormancy in the lung have revealed a dramatic increase in DCC awakening and expansion in the lungs following influenza virus infection. Our data support the hypothesis that respiratory viral infections can promote DCC awakening and expansion through two phases: first, through IL-6 dependent DCC awakening and expansion, and second, CD4 T-cell mediated protection from elimination (in part by CD8 cells). We further show that infection with a mouse-adapted SARS-CoV-2 promotes a similar awakening and expansion of DCC in mice. Finally, epidemiological studies reveal how prior infection with SARS-CoV-2 infection increases metastatic progression in lungs and cancer- related deaths for cancer survivors. We propose to determine mechanisms by which acute respiratory viral infections induce the awakening of dormant DCC leading to metastatic disease, whether and how such infections can prime DCC for activation by subsequent exposures, and how CD4 and CD8 cells differentially control the persistence of expanded DCC during influenza virus infection. Impact: Proposed studies to understand how different pulmonary viral infections alter DCC dormancy and host immune responses, to determine the consequences for progression to metastatic disease, and to explore underlying mechanisms, should yield valuable and actionable insight into the key cell types and molecular mediators, informing early detection and prevention strategies for at-risk individuals.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY/ABSTRACT Prenatal cannabis use is prevalent and associated with several adverse consequences for the mother and offspring. Cannabis includes many compounds, including cannabidiol (CBD; a phytocannabinoid of the flower cannabis sativa) and associations between specific cannabis compounds and maternal/child outcomes are lacking. Specifically, little is known about CBD-only product use during pregnancy, despite the increase in availability and acceptability of CBD over the past decade. We address this gap by addressing the following aims: Aim 1: Estimate the prevalence of CBD use overall and by trimester in a clinical sample pregnant women receiving prenatal care at the University of Colorado; Aim 2: Characterize women who use CBD and/or cannabis during pregnancy (socio-demographics, mental health profiles, physical health concerns and cannabinoid use behaviors and risk perception); and Exploratory Aim: Describe the pregnancy- and birth-related outcomes associated with prenatal CBD use. Using an observational study design, we will study pregnant individuals in their third trimester receiving care at University of Colorado perinatal clinics (n=800). All participants will complete an electronically delivered survey, donate a one-time blood sample for analysis of cannabinoids (via liquid chromatography-mass spectrometry), and consent to chart review of electronic medical records (EMR) once their baby is born. Participants will be categorized into one of four groups: a) using CBD-only products, b) using cannabis, c) using both CBD-only products and cannabis, and d) using neither substance during pregnancy. We will assess prevalence of CBD-only product and cannabis use during pregnancy both at any time and by trimester based on both the survey responses and blood sample analysis [Aim 1]. Further, the survey will assess participants’ use patterns, mental and physical health concerns, demographics, and pregnancy-related concerns [Aim 2]. We will review EMRs of all participants after the birth of their children to understand pregnancy- and birth-related outcomes associated with CBD-only product use, whole cannabis use, and CBD-only product plus cannabis use [Exploratory Aim]. Understanding associations between CBD and pregnancy- and birth-related outcomes will be an important step in identifying potential safety concerns related to CBD use in pregnancy. The PI works with an experienced team of mentors (Drs. Joseph Sakai, Susan Mikulich, Ashley Brooks-Russell, M. Camille Hoffman, and Sharon Hunter) and will pursue training relevant to this proposal including: (1) clinical research skills (survey design, study implementation, grant writing), (2) cannabinoids’ effects on early human development (3) assessment of perinatal and infant research outcomes, (4) statistical expertise in longitudinal analysis, causal analysis, (5) research presentation skills, (6) manuscript writing skills, and (7) engagement in professional development opportunities. The training, mentorship, collaborative relationships, and results of this project will serve as important steps to pursue an R01 grant and transition to research independence.

NIH Research Projects · FY 2026 · 2025-02

Project Summary The nanoscale organization of key synaptic proteins is now considered an important contributor to synaptic activity and plasticity. However, there is a lack of research on how such delicate nano-organization might be involved in brain diseases, which often directly impact synaptic function. Alzheimer’s Disease (AD) is associated with brain accumulation of the Amyloid-Beta (Aß) peptide. Furthermore, oligomeric assemblies of the 42 amino acid variant, Aß42, which directly target synapses, have been found especially pernicious toward synaptic function. In preliminary research, I have observed through multiple replicates that a brief, 15-minute application of 500 nM Aß is sufficient to disrupt existing synaptic nanostructure. This manifests as the addition of nanoscale objects of the PSD95 scaffolding protein, and of GluA1 subunit-containing AMPA-type glutamate receptors, a phenomenon we have titled, “fragmentation”. As this observation occurs along the same timescale as Aß- mediated NMDA-type glutamate receptor dysfunction and deficits to LTP (Long-Term Potentiation), I hypothesize that Aß-mediated fragmentation of synaptic nanostructure ultimately leads to deficits in synapse function and plasticity. In this proposal, I aim to thoroughly characterize both the kinetics, and dose-response of Aß-mediated fragmentation, as well as to what extent this fragmentation occurs (i.e., does fragmentation include select synaptic proteins, or completely restructure synaptic nanoarchitecture). I also plan to assess what signaling mechanisms underlie Aß-mediated fragmentation and whether these are similar, or distinct from already established mechanisms causing Aß-mediated synaptic dysfunction. Lastly, my preliminary data show that I can prevent the fragmentation of AMPAR nanostructure at the synapse by crosslinking receptors with a GluA1 antibody prior to Aß exposure. Using this method, as well as others our lab has developed, I will then test whether Aß-mediated synaptic nanostructure fragmentation is responsible for LTP deficits and synapse loss. Importantly, this fellowship proposal provides the opportunity for my extensive training in multiple experimental techniques including: super-resolution microscopy (multicolor and live-cell STED imaging, including preparation of STED-dye conjugated primary antibodies), writing computer based imaging-analyses, receptor signaling pharmacology, molecular cloning (CRISPR-based knockout / knockin strategies, optogenetic / chemogenetic construct design), electrophysiology and Ca2+ imaging, and 2-photon glutamate uncaging.

NIH Research Projects · FY 2026 · 2025-02

PROPOSAL SUMMARY (ABSTRACT) Following federal legalization of hemp and cannabinoids in 2018, as defined by <0.3% Δ9-tetrahydrocannabinol (Δ9-THC), use of minor cannabinoids has grown exponentially. Minor cannabinoids are considered cannabis- derived cannabinoids that are not Δ9-THC or cannabidiol (CBD). Currently, the most popular, and in highest abundance in the cannabis plant include cannabinol, cannabigerol, and cannabichromene (CBC). Importantly, these compounds are available in highly purified and concentrated amounts. CBC in particular is gaining in popularity due to anecdotal claims of anti-inflammatory, anti-tumor, and anti-depressant properties. Due to its recent legalization, comprehensive scientific literature investigating these claims and fundamental pharmacokinetic characteristics are lacking, and it is the goal of this proposal to address this gap in knowledge. Knowing Δ9-THC and CBD metabolites remain biologically active, we will characterize the metabolism of CBC and determine subsequent activity of the metabolites. Our preliminary data suggests the major phase I oxidative metabolite of CBC is the result of a cytochrome P450 (CYP)-mediated epoxidation and subsequent intramolecular rearrangement into 2’-hydroxycannabicitran. The kinetics of this reaction are unknown and therefore the reactivity of CBC metabolism requires further investigation. These initial findings support the notion that CBC pharmacology is complex, but by dissecting metabolism, novel therapeutic modalities limiting toxicological impacts will be identified. The goal of this proposal is to define the metabolic profile of CBC and characterize the biologic activity of CBC and its major metabolites to reveal potential therapeutic or toxicological aspects of this minor cannabinoid. It is our working hypothesis that CBC is heavily metabolized into both phase I- and phase II-type metabolites. We further hypothesize that these metabolites are biologically active and have potential to contribute to metabolizing enzyme inhibition and/or physiological effects of CBC. Through the utilization of translationally relevant systems, namely immortalized human hepatocyte/cholangiocyte cocultures (HepaRG cells), we will identify the human metabolites of CBC in vitro (Aim 1). In Aim 2, we will phenotype metabolizing enzymes (recombinant human CYP450s and uridine 5'-diphospho-glucuronosyltransferase (UGTs)) responsible for the production of major metabolites identified from Aim 1. Further, we will determine if CBC can cause drug-drug interactions and hepatocellular toxicity. We will then characterize the activity of the metabolites in the endocannabidome in silico and through in vitro assays (Aim 3). Taken together, these studies will reveal novel CBC metabolites, determine risk for drug-drug interactions, provide a basis for CBC metabolite activity, and impact safety guidelines in the future.

NIH Research Projects · FY 2026 · 2025-02

Project Summary Crohn's disease (CD) is a highly prevalent form of inflammatory bowel disease (IBD) much in need of further understanding. Most patients who are diagnosed with CD will develop complications from intestinal fibrostenosis, yet there are no medical therapies for the treatment of intestinal fibrosis. The lack of therapy is in large part due to our incomplete understanding of the underlying pathophysiological mechanisms of intestinal fibrosis. We have shown that AXL, a tyrosine kinase, is a pro-fibrotic mediator in the intestine. AXL is a promoter of cellular proliferation, migration, epithelial-mesenchymal transition, and has been shown to be involved in the development of fibrosis in other organs such as the liver and kidney. In ongoing work, we have found that in the intestine AXL is primarily expressed by stromal cells, while the primary ligand of AXL (GAS6) is expressed primarily by the epithelial cells. Further, we have discovered that the intestinal epithelium responds to acidosis and hypoxia, which are both key features of an inflammatory microenvironment, by acutely decreasing expression of GAS6. These findings show that AXL signaling can promote intestinal fibrosis, and suggest that GAS6/AXL signaling is a likely mode of epithelial-stromal cross talk in response to the luminal microenvironment. Unraveling the intricacies of this signaling and how it contributes to intestinal responses, and ultimately fibrosis, is the intent of this proposal. The ultimate goal is that this work will lead to novel medical therapies for intestinal fibrosis that target the AXL signaling pathway. The applicant, Dr. Steiner, has established a scientific niche in which to build a foundation for independent research on the cellular and molecular mechanisms of intestinal fibrosis. Dr. Steiner and his mentor, Dr. Colgan, assembled a mentorship committee to regularly meet as a group with Dr. Steiner throughout the duration of the award to provide feedback on the research, critique the research plans, monitor publications, and provide career advice. Drs. Colgan and Steiner have identified coursework relating to each aim to facilitate expansion of Dr. Steiner's foundation of knowledge in fundamental molecular biology, cellular biology, and use of murine model systems. Dr. Steiner will submit his work to present at conferences specific to his research to share his research and network with colleagues and potential collaborators, and get valuable feedback from the scientific community. Dr. Steiner's development will benefit from continued participation in the Mucosal Inflammation Program (MIP), a multi-disciplinary, multi-departmental program initiated to study mechanisms of mucosal inflammation and resolution. The MIP fosters a unique lab environment for collaboration between physician scientists, clinicians, and research scientists, and works to establish an environment for young investigators to flourish and develop. The facilities and resources available to and development plan built for Dr. Steiner provide an ideal environment and path for his successful transition to independence.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY Synaptic inhibition in the brain is critical for controlling neuronal circuit function, and its disruption underlies the pathology of multiple neurological, psychiatric, and neurodevelopment disorders including epilepsy, anxiety, autism, and schizophrenia. GABAergic inhibitory synapses mediate synaptic inhibition and regulate neuronal excitability, cell firing, dendritic integration, and synaptic plasticity. Synaptic plasticity mechanisms drive changes in synaptic strength in response to increased or decreased neuronal activity, which results in long-term alterations responsible for learning, memory, and cognition. In one form of inhibitory synaptic plasticity, inhibitory long-term potentiation (iLTP), GABAA receptors (GABAARs) accumulate at inhibitory synapses and are stabilized, consequently increasing synaptic strength. However, the precise organization of newly recruited synaptic GABAARs during iLTP and how this may contribute to increased synaptic strength is unknown. Advances in super-resolution (SR) imaging techniques have revealed a common nanoscale architecture at synapses in which neurotransmitter receptors, scaffolds, and adhesion molecules cluster into subsynaptic domains (SSDs). Under basal conditions, SSDs of GABAARs require positioning close to sites of presynaptic release for efficient synaptic transmission. This structured architecture is defined as the inhibitory nanocolumn. It remains unclear if newly recruited GABAARs organize into SSDs within nanocolumns during iLTP, and whether alterations to the inhibitory nanocolumn are required for synaptic potentiation. The overall goal of this proposal is to determine how inhibitory synaptic nanostructure contributes to iLTP, and if the inhibitory synaptic nanocolumn is a locus for synaptic plasticity. I hypothesize that precise positioning of GABAARs into SSDs within the inhibitory nanocolumn opposite presynaptic release sites is an important mechanism involved in functional iLTP. Using a range of super-resolution microscopy techniques, optogenetics, and electrophysiology I will: identify changes in the size and density of GABAAR SSDs during iLTP, and importantly their positioning within the inhibitory nanocolumn (Aim 1), determine whether mobile GABAARs are trapped in nanoscale SSDs during iLTP (Aim 2), and investigate if positioning of GABAARs within the inhibitory nanocolumn is required for synaptic potentiation (Aim 3). Together, this approach will determine a detailed understanding of the precise nanoscale organization of GABAARs at inhibitory synapses during iLTP and provide crucial insight into key mechanisms involved in inhibitory plasticity, overall synapse function, and neuronal activity.