Washington University

NIH Research Projects · FY 2025 · 2024-07

Warfarin is an anticoagulant that prevents venous and arterial clots but doubles the risk of major hemorrhage. Over the past decade, warfarin use has caused more medication-related emergency department visits among older Americans than any other drug. Our long-term goal is to improve the safety and effectiveness of antithrombotic therapy. To advance this goal, we have developed clinical and pharmacogenetic (PGx) dosing algorithms to guide days 1–5 of warfarin dosing and placed them on a non-profit web application (WarfarinDosing.org) that has been accessed more than 1.8 million times. This success provides the rationale for the proposed study: that algorithm-based dosing of warfarin reduces the risks of overdose and iatrogenic hemorrhage compared to trial-and-error dosing. Because the rate of warfarin overdose is highest between approximately 6-28 days of therapy, we will use penalized regression and machine learning (ML) to develop warfarin-dosing algorithms for this interval. To promote use of the new algorithms, we will configure the electronic health record (EHR) used at 23 medical centers in MO and IL to export data into WarfarinDosing.org, seamlessly providing clinicians with clinical decision support to guide warfarin initiation. Aim 1: To use linear regression to develop clinical and PGx dosing models for days 6–28 of warfarin therapy. To balance accuracy and parsimony, we will use penalized regression to elucidate relationships between the therapeutic dose and anthropomorphic, clinical, demographic, laboratory, and genetic variables. We have data collected from 3107 participants in 3 randomized clinical trials. Aim 2: To use ML to develop clinical and PGx dosing models for days 6–28 of warfarin therapy. Aim 3: To validate the models developed in Aims 1 and 2. We will quantify the accuracy of the models developed in Aims 1 and 2 in a set-aside 20% testing sample using mean absolute error (MAE) and secondary metrics (e.g. R2). We hypothesize that the best clinical and PGx models developed in Aims 1 or 2 will predict the therapeutic warfarin dose with MAEs < 1.0 mg/d in the testing sample. Aim 4: To update and expand WarfarinDosing.org to provide clinical decision support based on the best clinical and PGx models validated in Aim 3. WarfarinDosing.org will be revised to interface with Epic (Epic Systems Corp, WI), the EHR used across our 23 medical centers in MO and IL. We hypothesize that integrating its use with Epic will decrease the rate of the composite outcome of a warfarin overdose or a hemorrhage as compared to historic rates among patients starting warfarin. The proposed research is innovative and significant because it uses penalized regression and ML to derive novel PGx and clinical algorithms that will reduce the risk of overdose and iatrogenic hemorrhage from warfarin initiation. The integration of Epic and WarfarinDosing.org will be a sustainable and scalable intervention to improve the safety of anticoagulant therapy.

NIH Research Projects · FY 2024 · 2024-07

Modeling human adult-onset neurodegenerative diseases has been historically difficult, with current induced pluripotent stem cell (iPSC) models often showing mild phenotypes. While iPSC systems capture disease-associated genetic variants, generating these cells erases cellular age – a critical component of many neurodegenerative diseases. An alternative approach is the direct conversion of fibroblasts into neurons, which preserves the epigenetic age of the starting cells. As such, this approach has been highly successful in modeling pathologies of various neurodegenerative diseases including tauopathies, Huntington’s disease, and Alzheimer’s disease. In the proposed research project, we will employ direct neuronal conversion as a new tool to investigate amyotrophic lateral sclerosis (ALS) and associated frontotemporal dementia (FTD). We will initially study familial ALS cells that have been converted into induced motor neurons and subsequently probe for neurodegenerative phenotypes. We have already observed stress-induced degeneration in the context of multiple ALS-associated mutations. Removal of epigenetic age via iPSC conversion and antisense oligonucleotide knockdown of mutant proteins will establish the specificity of this system. We will further expand our studies to sporadic ALS lines, investigating neurodegeneration-associated features including nuclear pore deficits. Finally, cells from ALS/FTD patients will be converted into a cortical identity to provide a novel model for FTD. With these models, we can interrogate disease mechanisms in ALS/FTD. Further, the development of a tractable, patient-derived in vitro model of familial and sporadic ALS would represent a considerable breakthrough for the ALS research community, facilitating the eventual development of novel therapeutic agents.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Alzheimer’s disease (AD) is a devastating and complex neurodegenerative disorder with a crushing social and economic burden currently affecting more than 6 million patients in the US. With the number of affected individu- als projected to increase, identifying disease-modifying treatments and interventions for AD remains one of the most critical undertakings in modern biomedical research. A critical step to the development of these interven- tions involves characterizing the genes and pathways mediating AD risk and resilience. The objective of this proposal is to explore the modulation of AD genetic risk by age and examine how caloric restriction (CR), a lifespan-increasing intervention, can be harnessed to counter AD risk. The proposal will utilize state-of- the-art single-nuclei multi-omic functional genomic analyses of healthy and AD human brain tissue, as well as transgenic AD mouse models, to perform deep molecular characterization of AD risk and resilience across the genetic, epigenomic, and transcriptomic layers. Aim 1 will dissect non-coding AD genetic risk through intensive multi-omic molecular profiling of healthy and AD brains, allowing the nomination of causal variants, cell types, and pathways mediating risk. Aim 2 will identify protective mechanisms in cognitively healthy, long-lived individ- uals, including centenarians, and determine whether AD resilience manifests by maintaining a “young” state or by compensating for detrimental age-related effects and AD genetic risk. This will involve comparison across a broad age spectrum, including molecular profiles of young controls (aged 18-35) and AD patients. Aim 3 will characterize the interplay between CR and neurodegeneration in genetically diverse AD mouse models. This will provide insight into the pathways mediating lifespan-increasing interventions in the brain, their interaction with high-risk and resilient genetic backgrounds, and their overlap with AD risk and resilience pathways in hu- mans. Importantly, this proposal will directly address the connections between lifespan and AD resilience to provide a more nuanced understanding of the potential of CR and its mimetics as therapeutic strategies for AD. This proposal employs an innovative research approach combining observational and interventional study de- signs with state-of-the-art computational techniques. This approach facilitates concurrent human and mouse data analysis, expediting the discovery of molecular pathways mediating AD risk and resilience, with potential implications for developing novel AD prevention strategies. Under the mentorship of Dr. Harari and co-mentors Dr. Goate, Kaczorowski, Karch, and Lee, I will follow a rigorous training program to meet the aims of this K99/R00 award and transition into independent research leadership in the domain of AD and aging. This will include a focus on neurobiology, the biology of aging, mouse models of AD, bioinformatics, and professional development, achieved through courses, workshops, conferences, and advisory committee feedback. In summary, this pro- posal addresses a critical gap in our understanding of AD and aging, with the additional skills acquired during this award laying a solid foundation for my future independence in the molecular biology of AD.

NIH Research Projects · FY 2025 · 2024-07

Project summary/abstract Dr. Lou is a cardiothoracic anesthesiologist with a long-term career goal to develop, implement, and disseminate intelligent clinical decision support interventions that improve the care of surgical patients, with a current focus on perioperative blood management. Her prior research training has allowed her to develop expertise in data science and predictive model development using artificial intelligence (AI) techniques. This proposal builds on Dr. Lou’s prior experience by providing the protected time, mentorship, and training necessary to develop skills in the implementation of AI models as clinical decision support (CDS) tools, and in the evaluation and dissemination of such tools. The proposed career development plan will involve mentorship, didactic, and practical research training in the conduct of pragmatic clinical trials, the design and evaluation of implementation strategies for CDS tools, governance for CDS interventions, and foundational training in clinical research. She has brought together a multi-disciplinary group of mentors and advisors, including Thomas Kannampallil, PhD, Michael Avidan, MBBCh, and Sachin Kheterpal, MD, MBA, each of whom have expertise in clinical trials and the implementation of health technology innovations. Washington University provides the ideal environment for Dr. Lou’s training given its infrastructure and strengths in informatics, clinical trials, and implementation science. The proposed research plan provides the framework for Dr. Lou to acquire the skills she needs to achieve her career goals. Presurgical testing and preparation for surgical transfusion is essential for patient safety during surgery, yet excessive preparation is costly and contributes to blood product waste. Given recent blood shortages, presurgical blood orders should be placed only for patients who need it; there is an acute public health need for tools that accurately estimate the risk of transfusion to guide clinical decision- making. In preliminary work, Dr. Lou has developed a personalized AI model, named S-PATH, to estimate surgical transfusion risk, and demonstrated its validity across local and national datasets. The objective of this proposal is to evaluate the feasibility and preliminary effectiveness of S-PATH as a CDS system embedded within the Electronic Health Record (EHR) using a pragmatic cluster-randomized clinical trial. The expected outcome is a generalizable, EHR-agnostic personalized CDS system to guide presurgical blood orders that is feasible to deploy within preoperative workflow. This proposal is significant in its potential to change clinical practice, with considerable public health impact for patient safety, blood conservation, and reduced healthcare costs. The proposed research and training will provide Dr. Lou with the skills needed to launch an independent research program to improve the delivery science for AI innovations in healthcare.

NSF Awards · FY 2024 · 2024-07

This research project will develop innovative solutions for quantile regression analysis of big data. Big data has become prevalent in modern society due to the exponential growth of digital information. Quantile regression is a powerful statistical tool that goes beyond the average relationship provided by traditional regression. However, big data poses fundamental challenges for quantile regression, both statistically and computationally. This project will address those challenges by developing methods that achieve computational efficiency without losing statistical efficiency. The project will contribute to statistical research on both quantile regression and statistical computation. The new methods will be of value for applications in a wide range of fields, including economics, finance, the social sciences, and healthcare. A graduate student will be involved in the conduct of the research. Educational materials and open-source software will be created for the broader research community. This research project will develop an efficient computational and inferential framework for supporting quantile regression analysis in both massive data streams and static big data. To overcome the significant challenges brought by big data's massive scale and complexity, online processing and distributed computing methods become essential for their cost-effectiveness, scalability, and real-time processing capability. However, traditional statistical procedures for quantile regression usually do not scale well to data size and frequently are infeasible when dealing with massive data. The project has three research aims. First, the investigator will develop a fast online quantile regression analysis framework for data streams to bridge the gap between computational and statistical efficiency. Statistical theory for dealing with the non-smooth objective in quantile regression will be studied. Second, a distributed computational and inferential framework for quantile regression analysis in big data with missing data for causal inference will be developed. The new methods will be applied to electronic health record data. Finally, the investigator will develop open-source software in R or Python to implement the advances from this project. Quantile regression is used in a wide range of applications when understanding the effect of variables across the distribution is important. The developed statistical theory and inferential tools will provide new foundations for quantile regression analysis in big data and hence benefit all related application fields. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Neuropathic pain (NeuP) affects 6-8% in the adult population. It leads to substantial functional impairment and has deleterious impact on patients’ quality of life. Patients with NeuP continue to have inadequate response to currently available pharmacotherapy options, with less than 25% achieving meaningful improvement. Moreover, major safety concerns are associated with the use of analgesics such as opioids. There is clearly an unmet need for new effective and safe approaches for treating neuropathic pain. 5-HT3 receptors have emerged as a promising target in NeuP. Studies demonstrated that local intrathecal delivery of 5-HT3 antagonists such as ondansetron alleviates mechanical and thermal hypersensitivity in animal models of nerve injury. These compounds target 5-HT3 receptors in the CNS, which are overexpressed after nerve injury, contributing to pain facilitation. The major challenge in achieving efficacy with systemic administration of currently available 5-HT3 antagonists is P-glycoprotein (Pgp)-mediated efflux that limits their penetration to the CNS, and consequently limits their therapeutic effect in NeuP. With adequate CNS penetration, these drugs are expected to be efficacious in relieving NeuP. We hypothesize that identification of CNS-active lead candidates combined with the current understanding of mechanisms of 5-HT3 receptor inhibition gained from recent structural studies and using state-of-the-art computational drug design approaches, will allow the design/development of novel 5-HT3 antagonists with CNS activity and competitive intellectual property (IP) position. The goal of this R61 planning grant is to establish a multidisciplinary and collaborative team of scientists in medicinal and computational chemistry, structural biology, pharmacology, pharmacokinetics, pain biology and clinical/translational pain research to develop and characterize lead candidates and establish the models and feasibility of determining their preclinical efficacy in models of neuropathic pain. We will use molecular dynamics simulations, structure-based virtual screening and drug design to identify and synthesize novel lead 5-HT3 antagonists (Aim 1), characterize in vitro pharmacology and in vivo pharmacokinetics of the novel ligands (Aim 2), and determine the analgesic efficacy and abuse liability of these ligands in rodent models (Aim 3). We expect to establish the multidisciplinary team and its workflow for drug development and characterization, to generate the initial data for novel CNS-penetrant 5-HT3 antagonist development in the subsequent U19 or UG3/UH3 grant mechanism.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Poly (ADP-ribose) polymerase inhibitors (PARPi) are a mainstay for the chemotherapeutic regimen of BRCA mutant ovarian and breast cancers. Despite initial positive responses, long -term clinical success with PARPi therapy is limited owing to the inevitable emergence of resistance and the side effects associated with the current dosage. We and others recently reported that loss of a nucleosome sliding enzyme, Amplified in Liver Cancer 1, (ALC1), hypersensitizes BRCA mutant cancer cells to PARPi. Notably, ALC1 loss permits killing of BRCA mutant cancer cells at sub-nanomolar PARPi dosage and restores PARPi sensitivity across various engineered models of chemoresistance. Based on these observations, our overarching goal is to employ ALC1-deficient BRCA mutant cancer cells to define the cellular and biochemical mechanisms that can be exploited to circumvent clinical hurdles associated with PARPi. Our preliminary data highlight a role of ALC1 mediated nucleosome sliding in promoting the repair of base damage lesions called abasic sites. However, it is unclear how ALC1 loss generates abasic sites and how this contributes to PARPi hypersensitivity in BRCA mutant cancer cells. The proposal addresses this knowledge gap via the following Specific Aims. In Specific Aim 1, we will integrate in vitro reconstitution, genetic complementation analysis and DNA repair and replication assays to define the mechanism(s) that lead to increased abasic sites on the chromatin upon the loss of ALC1. These experiments will provide new mechanistic insights into how perturbing chromatin remodeling involved in base damage repair can be leveraged for augmenting PARPi sensitivity in BRCA mutant cancers. In Aim 2, we will use single-molecule replication tract labeling assays, electron microscopy and CRISPR-based genetic editing of patient-derived primary cells to determine how abasic sites results in remodeling of replication forks and generation of lesions that accentuate PARPi sensitivity. These experiments will uncover how the communication between base damage repair and replication forks can be exploited to enhance the therapeutic potential of PARPi. Our studies will provide the foundation to develop new approaches to improve the efficacy and toxicity profile of clinically used PARPi while simultaneously highlighting new biomarkers that can effectively predict PARPi responses in BRCA mutant patients.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Granuloma formation is a feature of tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb) infection. Alveolar macrophages (alvMF) are central to this process as the first cells infected by Mtb and likely as drivers of early granuloma development. The initial events when alvMFs encounter Mtb and the cell-cell interactions occurring in mature granulomas are extensively investigated but less is known about what happens between the initial infection event and later stages of disease. These early events have implications for the trajectory of TB and some individuals restrict Mtb at this stage and never develop disease despite multiple exposures whereas other individuals cannot restrict Mtb replication and progress to granuloma formation. Our lack of knowledge of the molecular and cellular circuits underlying early granuloma formation and what differentiates outcomes at this stage is a fundamental gap that limits the development of therapeutic interventions for TB. This gap is especially acute for people infected with human immunodeficiency virus (HIV), which significantly increases risks of poor outcomes from TB, and addressing it will have significant public health value. Critical unanswered questions in the acute response to Mtb infection include (1) how do interactions between immune cells immediately after alvMF infection contribute to granuloma formation, (2) which components of these interactions can be perturbed to bias disease dynamics to improve control over Mtb replication, and (3) what interactions are altered by HIV infection that increase susceptibility to active TB? We discovered that infected MF secrete interferons (IFNs), cytokines, and chemokines within minutes to hours after infection that drive the inflammatory responses that support granuloma formation. Moreover, we have shown that type I IFN expression and neutrophil recruitment support Mtb replication and we are investigating how these responses contribute to infection outcomes. Recently, we showed that type I IFN drives release of neutrophil extracellular traps that promote Mtb replication instead of restricting it and are associated with the development of necrotic granulomas. Based on these data, we hypothesize that early interactions between macrophages and neutrophils are regulated by a self-propagating cycle of type I IFN and damage-associated molecular pattern (DAMP) signaling that culminates in granuloma formation. Moreover, our work showing that I IFN-conditioned immune cells are less able to control Mtb infection leads us to hypothesize that HIV-induced type I IFN signaling amplifies this type I IFN–DAMP circuit and promotes an environment that supports Mtb replication, tissue damage, and TB susceptibility. We predict that breaking the type I IFN–DAMP cycle therapeutically will push early responses toward protective functions. To test our hypothesis, we propose to 1) Define type 1 IFN and DAMP signaling effects in innate immune cells following initial Mtb infection, 2) Dissect the molecular basis of the type I IFN- DAMP circuit to identify vulnerabilities where therapeutic interventions can promote control of Mtb, and 3) Determine how HIV affects the type 1 IFN-DAMP circuit propagated by Mtb infection.

NSF Awards · FY 2024 · 2024-07

Unexpected “shocks,” or abrupt deviations from periods of stability naturally occur in time-dependent data-generating mechanisms across a variety of disciplines. Examples include crashes in stock markets, flurries of activity on social media following news events, and changes in animal migratory patterns, among countless others. Reliable detection and statistical analysis of shock events is crucial in applications, as shock inference can provide scientists deeper understanding of large systems of time-dependent variables, helping to mitigate risk and manage uncertainty. When large systems of time-dependent variables are observed at high sampling frequencies, information at fine timescales can reveal hidden connections and provide insights into the collective uncertainty shared by an entire system. High-frequency observations of such systems appear in econometrics, climatology, statistical physics, and many other areas of empirical science that can benefit from reliable inference of shock events. This project will develop new statistical techniques for both the detection and analysis of shocks in large systems of time-dependent variables observed at high temporal sampling frequencies. The project will also involve mentoring students and organizing workshops. The investigators will study shock inference problems in a variety of settings in high dimensions. Special focus will be paid to semi-parametric high-frequency models that display a factor structure. Detection based on time-localized principal component analysis and related techniques will be explored, with a goal towards accounting for shock events that impact a large number of component series in a possibly asynchronous manner. Time-localized bootstrapping methods will also be considered for feasible testing frameworks for quantifying the system-level impact of shocks. Complimentary lines of inquiry will concern estimation of jump behavior in high-frequency models in multivariate contexts and time-localized clustering methods. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY / ABSTRACT This K08 application proposes a five-year career development and research program focused on elucidat- ing the transcriptional determinants and molecular heterogeneity of Barrett’s esophagus (BE). The applicant, Ramon Jin, M.D., Ph.D., is a physician-scientist and an Instructor in Medicine in the Section of Hematology and Oncology, Department of Medicine, at Baylor College of Medicine (BCM). Since completing his hematology and oncology fellowship as a chief fellow, Dr. Jin has worked in the laboratory of Dr. Jason Mills, where he has applied his foundation in molecular cell biology to study Barrett’s esophagus and upper gastrointestinal (GI) metaplasias through development of genetic mouse models and establishment of human patient-derived organoids. From a GI developmental perspective, Barrett’s metaplasia reflects a “caudalization” process with loss of esophageal and gain in both gastric and intestinal traits. Specifically, two pioneering transcription factors involved in GI tract patterning, SOX2 and CDX2, are likely to involved, and the proposed research is based on the hypothesis that SOX2 decrease and CDX2 gain are critical steps during BE development and maintenance. To address this hypothesis, Dr. Jin proposes to carry out two specific aims: 1) Characterize the effects of SOX2 and CDX2 dis- ruption on foregut homeostasis with and without bile acid treatment, and 2) Determine the effects of SOX2 and CDX2 restoration on BE maintenance and the molecular heterogeneity of BE. Continued mentorship and protected research time provided by this grant will ensure the support needed to complete these aims and allow Dr. Jin sufficient time to establish an independent research program. Specif- ically, the current award would provide an important opportunity to train in multi-omics techniques and bioinfor- matic analyses, while expanding a unique set of tools including genetically modified mouse models and human BE and gastroesophageal cancer organoids. Dr. Jin’s career development will be nurtured through continued mentorship by Dr. Jason Mills and his mentorship committee. Drs. Cristian Ciorfa and Bing Zhang are both es- tablished computational scientists specializing in bioinformatic integration of multi-omic data; Dr. Kunal Rai is an expert in epigenomic regulation of development and oncogenesis; and Dr. Sarah Blutt is a leader in advanced GI organoid culturing techniques. Dr. Jin’s career development will be supplemented by continued growth in grant- manship, mentorship, leadership, and research community integration through courses and programs offered at BCM; involvement in ad hoc publication, abstract, and grant reviews; active participation in local and national research meetings; and preparation of manuscripts and grants. The guidance provided by his highly experienced research mentor and mentorship committee, the knowledge and technical skills derived from the proposed ex- periments, and the completion of the carefully outlined career development plan will allow Dr. Jin to successfully establish an independent research program that will be highly competitive for R01 funding.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Steroid hormones enrich behavior through actions on sensory and motor circuits, but it remains unclear how these parallel actions are coordinated to generate a coherent behavioral phenotype. This proposal will investigate the effects of steroids on neurons that convey predictive motor signals, termed corollary discharges (CD), which modify sensory processing to account for the sensory feedback predicted to arise from an animal’s own actions, termed reafference. CDs are ubiquitous in sensorimotor systems, and their disruption is thought to underlie some behavioral and sensory deficits in psychiatric conditions including schizophrenia and autism. Hormonal regulation of CD is especially intriguing given that steroid hormone levels also appear to be dysregulated in these conditions. However, the mechanisms by which steroids regulate CD circuitry in nonpathological states remain unknown. The central hypothesis of this proposal is that steroid hormones directly alter the physiology of corollary discharge neurons to match internal motor representations to altered reafferent feedback. This hypothesis will be tested using a well-characterized CD that modulates sensory processing in mormyrid electric fish. Mormyrids communicate using a stereotyped electric pulse known as an electric organ discharge (EOD). Self- generated EODs and EODs of nearby fish activate electroreceptors in the skin, which project to a dedicated communication pathway. To ensure that this pathway only responds to EODs of other fish, a CD signals the timing of EOD production to selectively inhibit responses to self-generated EODs. EODs are elongated in response to increases in testosterone (T) levels in male mormyrids during the breeding season. This T-induced EOD elongation delays electroreceptor activation, shifting the timing of reafferent sensory feedback. To match these shifts in sensory feedback, T concurrently acts in the brain to delay and elongate CD inhibition. Preliminary data from extracellular recordings reveal the locus in the CD pathway whereby activity is delayed and elongated by T. Importantly, the hormonal pathways and physiological mechanisms underlying timing shifts at this site are still unknown. The experiments in Aim 1 will identify the steroid receptors that drive CD plasticity and determine whether these receptors act directly in CD neurons. The experiments in Aim 2 will reveal the physiological mechanisms by which T shifts CD timing in affected neurons. By focusing on a hormone-sensitive circuit that conveys a simple internal model, the timing of EOD production, these investigations will yield mechanistic insight into the seasonal effects of steroid hormones on gene expression and neural excitability in the context of a quantifiable behavioral change. Further, the results of these experiments promise to offer insight into the neural basis of multiple psychiatric conditions in which steroid hormone levels and CD appear to be dysregulated. With the support of this fellowship and his sponsorship team, the fellow will receive exceptional training in electrophysiological and molecular techniques to reveal novel mechanisms of hormonal plasticity. Sustained investment in the fellow’s professional development will complement these research activities and ensure that he can communicate science effectively and rigorously. This proposal details essential steps for the fellow’s development into an independent neuroscientist, in service of his long-term goal of running a healthy and impactful research program.

NIH Research Projects · FY 2025 · 2024-07

Project Abstract The question of why animals sleep has puzzled scientists for over a century. Contemporary research suggests that sleep serves as a compensatory mechanism for brain plasticity, restoring the network to a homeostatic set- point known as criticality. However, the precise mechanisms between sleep and neural plasticity are still subjects of intense debate, and the information processing aspect of this problem hasn’t been addressed experimentally. This proposal aims to address this gap by positing the hypothesis that experience-dependent plasticity in neuronal circuitry progressively undermines indices of an optimal computational regime, and proportionally increases the likelihood of sleep. I will adopt a two-pronged approach, leveraging the diverse expertise of my mentorship team. The first objective is to develop a machine learning algorithm that predicts the mechanisms of neural criticality and its homeostasis. This model will challenge existing machine learning approaches, which often lack explanatory power, by extracting dynamical rules from existing datasets using cutting-edge modeling and optimization techniques. The second objective involves empirical testing to ascertain whether a plasticity threshold exists at the network level that triggers sleep. This will be conducted through continuous neuronal recordings in freely behaving mice, coupled with methods to induce plastic change in the network. The third objective is to construct a biophysically grounded mathematical model to identify mechanisms that stabilize network information processing in the face of plastic changes. This model will incorporate methods from control theory and dynamical systems and will be validated against the rules derived in the first objective and the data collected in the second objective. This interdisciplinary project is groundbreaking in its attempt to offer a dynamical and mathematical explanation for why plasticity requires sleep. Furthermore, in contrast to many other works that focus on either theory or experiment, here I will support my theoretical work with empirical data. Overall, the project promises to make significant contributions to both theoretical and applied neuroscience. It seeks to develop a predictive model for sleep/wake behavior based on fundamental rules and to elucidate the functional mechanisms by which sleep serves the healthy brain. By directly testing a dynamical/mathematical explanation of why plasticity requires sleep, the project stands to resolve a long- standing question in neuroscience. Furthermore, uncovering these mechanisms will fill the important gap of adaptation for a possible fundamental framework of brain dynamics.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Leigh Syndrome (LS) is an inherited mitochondrial disease that presents with prominent neurologic symptoms and death in childhood. There are currently no effective therapies for this devastating disease, highlighting an urgent need to identify novel biological pathways that can be targeted therapeutically to treat LS and other inherited mitochondrial diseases. Over the past 15 years, an emerging body of research, including recent studies by us (Brestoff et al., Cell Metabolism, 2021 ), indicates that many cell types export their mitochondria for delivery to neighboring cells, including neurons, in vivo in a process called interce/lular mitochondria transfer. Our laboratory also recently reported that administering purified mitochondria to Ndufs4-1- mice with LS completely restores the cell-intrinsic defects in mitochondrial metabolism of macrophages (Borcherding et al., Cell Metabolism, 2022). However, it is unknown whether intercellular mitochondria transfer can be harnessed therapeutically to treat LS. In new preliminary studies, we found that weekly systemic administration of purified wildtype (WT) mitochondria to Ndufs4-1- mice increased their lifespan and markedly reduced their neurologic morbidity and neurodegeneration. These data provoke our hypothesis that administering exogenous mitochondria can rescue the metabolism of recipient cells and may be a previously unknown therapeutic strategy for LS. Exogenous mitochondria appear to be captured first by macrophages, which are mobile cells that can later export captured mitochondria to neighboring cells. Based on this finding, we transplanted WT bone marrow into Ndufs4-1 - hosts and found that this intervention led to delivery of healthy donor cell-derived mitochondria to host cells, ameliorated LS severity, and extended their lifespan. These data suggest that engrafted immune cells from bone marrow transplantation can provide a systemically distributed, self-renewing supply of healthy mitochondria to diseased cells in vivo. Collectively. these data provoke our central hypothesis that providing sources of healthy mitochondria postnatally may ameliorate LS by rescuing cell-intrinsic metabolic defects. We will address this hypothesis in two aims. In Aim 1, we will determine how administering exogenous mitochondria improves the morbidity and mortality of LS. In Aim 2, we will investigate how bone marrow transplantation improves LS by providing a durable, systemically distributed source of healthy mitochondria. This highly innovative project will leverage our expertise in intercellular mitochondria transfer and new cutting-edge technologies to establish the efficacy of and identify the mechanisms of these two novel therapeutic strategies to treat fatal inherited mitochondrial diseases such as LS.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Psychiatric disorders are the leading cause of disabilities worldwide, and cost over $300B in the U.S alone. Despite the rise in psychiatric medication prescriptions, approximately 30% of patients are treatment resistant. Clinical trials indicate that psilocybin elicits rapid and sustained outcomes for mood and substance use disorders, sparking interest in the therapeutic potential of psychedelics. These effects are believed to be dependent on the serotonin-2A receptor subtype. However, serotonin exerts potent vasoactive effects, which becomes particularly relevant when employing functional magnetic resonance imaging (fMRI) to study the influence of psychedelics on neurophysiology. fMRI indirectly indexes neuronal activity via blood-oxygen-dependent signals which depend on neurovascular coupling (NVC). If NVC is altered, hemodynamic measures of brain activity may inaccurately reflect neuronal activity. Accurate assessment of the neurophysiological effects of psychedelics requires simultaneous mapping of neuronal and hemodynamic activity. That psychedelics alter brain structure and function is supported by molecular evidence demonstrating altered synaptic plasticity. Further, psychedelics can reactivate social critical periods in adult mice in a manner that aligns with the length of human-reported acute subjective effects (intoxication). These observations imply that psychedelics having longer versus shorter intoxication duration may differentially affect neuroplasticity. However, the potential for psychedelics to reopen critical periods beyond social reward circuitry is unknown, as is the impact of psychedelic duration on these processes. The central hypothesis of this proposal is that psychedelics give rise to differential reports of neuronal versus hemodynamic measures of neurophysiology and sensitize neuroplasticity in an intoxication-duration- dependent manner. I have been testing this hypothesis during my PhD research using wide-field optical imaging in awake mice under the influence of the psychedelic 2,5-Dimethoxy-4-iodoamphetamine (DOI). My preliminary results (Aim 1a) reveal that DOI significantly alters NVC, and that calcium (neuronal) and hemodynamic signaling differentially report how DOI alters brain network organization. For the remainder of my PhD, I will determine whether short- versus long-duration psychedelics differentially affect neurophysiology (Aims 1b, F99 Phase). For my postdoctoral research, I will determine whether psilocybin and DMT affect neuroplasticity in the visual system (Aim 2, K00 Phase). Specifically, I will test the hypothesis that psychedelics accelerate functional brain reorganization induced by monocular deprivation in a psychedelic intoxication-duration manner. Results from the proposed experiments will establish network-level neuronal signatures of psychedelics and determine whether psychedelics broadly enhance neuroplasticity. To guide my training, I have assembled an interdisciplinary team of mentors and collaborators at Washington University in St. Louis with diverse, complementary expertise. The outlined research and training plan will allow me to achieve my career goal of becoming an independent investigator designing novel treatment strategies for patients resistant to existing psychiatric therapies.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Our long-term objective is to understand how neural circuits in orbitofrontal cortex (OFC) support adaptive choice behavior and produce choice biases, particularly those pertinent to drug abuse disorders. Our central hypothesis is that distinct OFC output neuron types encode unique decision variables and contribute to specific choice biases. This project has two main goals: to map the OFC neurons projecting to subcortical areas and then to study their role in decision-making by monitoring and manipulating their activity. In Aim 1, we will examine whether functional clusters of OFC neurons correspond to specific decision-making processes and whether their representations are stable over time and across different tasks. In Aim 2, we will comprehensively map OFC subcortical projections, and test if neurons projecting different regions are anatomically and molecularly distinct. Then we will focus on OFC projection neurons to the ventral striatum and the ventral tegmental area and use projection-targeted miniscope imaging to identify their roles in decision-making algorithms. In Aim 3, we will manipulate these projections to test whether they mediate specific choice biases based on past outcomes and reward size, respectively. Upon completing these aims, we expect to provide a blueprint for how decision variables are computed within the OFC and transmitted to its subcortical targets in a projection-specific manner and drive different choice biases.

NIH Research Projects · FY 2024 · 2024-07

For nearly a century, sleep in the brain has been defined by electrical waves that travel slowly across multiple centimeters of the isocortex. In stark contrast, the fast neuronal spike-patterns known to encode sensory information exist in milliseconds of microcircuit activity. My recent pre-print demonstrates that state, too, is reliably encoded in fast, non-oscillatory spike-patterning within diverse individual microcircuits throughout the brain. However, despite revealing a novel, fast and local foundation of sleep and wake, my initial study may underestimate the true minimal scale of the encoding. I hypothesize that the minimal encoding of state in neural activity is spike patterns, not at the level of the microcircuit, but the individual neuron. First, I will verify whether individual neurons encode sleep and wake states in their spike patterns, using previously conducted microelectrode recordings. Statistical comparisons of spike patterns betweens states will lay a reproducible foundation in connection with prior literature. My preliminary results reveal that, while individual neurons encode state in their activity, the general principles require further elucidation. Generally, many encodings are structured by the hierarchical organization of the brain, and this may also be true for sleep/wake encodings. I propose that machine learning can disentangle the general influences of the animal and region from encodings of state that are unique to each individual neuron. Unique neuron- level encodings in spike patterning represent a new minimal scale of state, embedding this information on the scale of neuronal computation. Neuron-level states could provide new insight into brain function and, specifically, animal behavior. My recent pre-print revealed that microcircuit spiking patterns “flicker” between sleep and wake independently of the animal’s overall state. Further, flickers emerge as a function of sleep pressure and drive behavioral discontinuities (twitching, pausing). A new, neuron-level state encoding can reveal whether individual neurons exhibit similar state discontinuities, and whether this explains complex interactions between state and behavior. This work has great potential to disentangle brain states on the scale of brain computation (neurons). Such fundamental knowledge about the basis of sleep and its connection to behavior is highly important to public health. Biomedically, sleep dysfunction is widely implicated in nearly all neurological disorders- from epilepsy to autism to Alzheimer’s. Further, localized forms of sleep amidst wake are increasingly observed in conjunction with attentional disorders and stroke victims. These aims facilitate excellent training, particularly in developing novel machine-learning architectures and performing sophisticated electrophysiological recordings. My highly-supportive co-sponsors are excellent investigators in computational neuroscience and sleep from renowned institutions. Completion of this proposal will help prepare me to one day develop my own systems neuroscience research program.

NIH Research Projects · FY 2024 · 2024-07

Project Summary/Abstract Washington University in St. Louis has a rich tradition of radiation research and is a world leader in the field of radiation oncology for clinical care, as well as physics and biological research. Over the past twenty years, much emphasis has been placed on the development of instrumentation for the non-invasive imaging of biological processes in small animals, thereby providing investigators with a better understanding of the biology and the potential of therapeutic interventions in preclinical models. In this regard, Washington University is a recognized leader, with outstanding programs and instrumentation for preclinical magnetic resonance, nuclear, and optical imaging. Preclinical photon irradiators that can deliver radiation to precise anatomical locations in small animals are an important component of these research efforts. We currently have an XStrahl small animal radiation research platform (SARRP) irradiator, however, it is nearing the end its useful lifetime and the technology is outdated. We therefore request funds to purchase a new, state-of-the-art Precision X-Ray SmART+ Biological Irradiator for small animal radiation delivery. We believe this irradiator is superior to the latest irradiator produced by Xstrahl. The state-of-the-art SmART+, is an image-guided, isocentric (360°rotation, with arc therapy capability), self-shielded research irradiator with integrated safety interlock system. It combines a high resolution (100 µm) cone-beam CT imaging system and a high-dose rate (up to approximately 6.5 Gy/min) therapeutic X- ray source into a single integrated, computer-controlled platform. It comes standard with a 4500W generator and has the highest degree of targeting accuracy 0.05mm about the isocenter. Research projects for seven major users with NIH funding and a critical need for the SmART+ are described, along with two minor users. The PI has managed the current small animal irradiator for the past eight years and has of expertise in preclinical radiation and cancer biology research. He has assembled a strong team to oversee operations and QA of the instrument. In addition, Washington University has made a significant institutional commitment to ensure the successful use of the device after its installation. The irradiator will allow investigators to study basic questions regarding radiation research in an in vivo, preclinical model that will eventually lead to new paradigms in the way we treat disease in the clinical setting.

NSF Awards · FY 2024 · 2024-07

As the ultimate arbiter of crucial legal disputes, the U.S. Supreme Court occupies a pivotal position in American democracy. However, relatively few cases make it to the Court. In any given year, the Court receives thousands of petitions asking it to review lower court decisions, of which less than one hundred are granted review, or writ of certiorari. The discretionary authority to choose its cases endows the Court with substantial influence in shaping national public policy. Given the statistical rarity of a case receiving a formal decision by the U.S. Supreme Court, and the unrepresentative nature of the petitions that justices choose among, it is critical to understand not just the decisions the Court makes on the merit, but also the density and diversity of issues and interests competing for the Court’s consideration. How does the supply of certiorari petitions shape the justices’ behaviors? How do external actors, such as interest groups and the media, shape the Court’s docket? How does the Court’s selection of petitions granted review represent the plurality of public interests? To answer these and related questions, the research project endeavors to collect, categorize and analyze a host of important features in the writ of certiorari process. Recognizing the seminal importance of the agenda-setting stage, the research intends to provide a better understanding of judicial decision-making and judicial processes as well as the essential role of courts in American democracy. The research enhances existing theories of judicial behavior, with a specific focus on the early stage of decision-making in the United States Supreme Court: the decision to grant review, or writ of certiorari. The research collects and analyzes case features from all writ of certiorari petitions— that is, all lower court cases where the losing party, dissatisfied with the outcome, appeals to the US Supreme Court. The study catalogs—for the first time—the geographic origin, temporal distribution, and issue areas of cases the Court is asked to review. It also examines the role played by external actors in shaping the Court’s agenda, including the media and interest groups, as well as the extent to which the Court’s final selection of cases is consistent with the expectations of the plurality of interests in society. This multifaceted investigation inquires into how these factors collectively impact the decision-making process of the justices, leading to both empirical and theoretical contributions in the study of judicial decision-making. First, it provides new data on writs of certiorari petitions that are comprehensive over time and space, and across issues. Second, the project presents a unique perspective on the courts more generally by contributing to theories that are built around informational cues, and by using computational social science methods to test how the selection of cases impacts judicial outcomes. Third, it seeks to refine and extend machine learning algorithms for legal text analysis of cert petitions and external actors, potentially paving the way and setting new standards for data-driven research in judicial politics. Finally, it supplements the quantitative analyses with interviews of former Supreme Court clerks and amicus-filing entities to provide a richer perspective on how the justices sort through the large number of petitions received every year, and how the Court selects the final set of cases to review. Through the research, and, importantly, the creation of a dataset that categorizes a host of features of all cert petitions to the Court, this project will provide judicial, interest group, media scholars and the public with new insights on the workings of the judicial system. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-07

Project Summary Chronic use of commonly used migraine therapies, such as triptans and opioids, can lead to medication overuse headache (MOH). This is a paradoxical increase in severity of migraine-associated symptoms and headaches which are refractory to other treatments. Currently, the first-line treatment for MOH is drug cessation. However, during this abstinence period, patients continue to suffer from severe migraine, and in the case of opioids from withdrawal; and a majority of MOH patients return to these medications within the first year. Targeted therapies specifically for MOH would result in better headache management and increased patient quality of life. Our lab has recently investigated mechanisms of MOH. Through an unbiased peptidomic screen, our lab identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), as being augmented in preclinical models of chronic migraine and MOH. Our preliminary data suggests that antagonism of PAC1, a high affinity PACAP receptor, inhibits allodynia and aura associated with MOH. However, the PACAPergic system is relatively understudied and there is a lack of selective ligands to the PAC1 receptor. Currently there are only 3 PAC1 inhibitors, and two of them are peptides. Although the small molecule antagonist, PA8, is effective in our models, it has only moderate potency. The objective of this proposal is to develop novel PAC1 receptor antagonists for the treatment of MOH. This grant aims to develop a collaboration between the Pradhan, Majumdar, and Katritch labs. The Pradhan, Katritch and Majumdar labs have expertise in PAC1 pharmacology/behavioral models, computational modeling, medicinal chemistry, pharmacology and structure- based design of drug molecules. In Aim 1, the Katritch group will utilize a combination of virtual ligand screening, and their new platform based on V-SYNTHES on structures of PAC1 to identify novel molecules with antagonistic activity at this receptor. As a second avenue, we also propose to optimize the potency of PA8, using structure- based design, and discover new compounds with improved in vivo activity compared to the parent (Katritch/Majumdar). In Aim 2 we will pharmacologically characterize promising lead compounds. We will evaluate PAC1 antagonists for potency and selectivity in cAMP assays in transfected cell lines (Pradhan) as well as in vitro ADME and pharmacokinetic analysis in plasma and brain (Majumdar). Finally, in Aim 3 we will test the most promising lead PAC1 antagonists in models of MOH and migraine. We will also perform preliminary tests exploring adverse CNS effects of lead compounds. This R61 mechanism will allow us to establish a multidisciplinary and collaborative team to identify and characterize promising lead candidates targeting the PAC1 receptor for the treatment of MOH.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The 5-year survival rate for patients diagnosed with pancreatic ductal adenocarcinomas (PDACs) is 11%. This poor outcome is due to multiple factors, including rapid progression to metastatic disease, poor clinical responses to standard of care therapies, and no effective targeted or immunotherapeutic approaches1. The treatment refractory nature of PDAC is likely due in part to the profoundly fibrotic and immune suppressive tumor microenvironment (TME) that is a hallmark of this disease. Two major drivers of this TME include a dense fibrotic tumor stroma and a robust infiltration of tumor-supportive myeloid cells. PDAC contains phenotypically diverse cancer associated fibroblasts (CAFs) subsets. These subsets include myofibroblasts (myCAF), inflammatory fibroblasts and a small subset of antigen presenting CAFs. Recently, it has been proposed by some investigators that myCAFs may have tumor restraining properties, while other investigators have found myCAFs can promote tumor progression and treatment resistance. What is clear is that tumor restraining or tumor promoting features are likely phenotype and context dependent. We propose herein that cellular senescence may be distinguished by the tumor-promoting and restraining CAF subsets. Our overall hypothesis that: Stromal senescence plays a key role in driving tumor progression by altering tumor immune and ECM properties. To address this hypothesis, we will use state of the art biophysical and immunological techniques in human PDAC specimens, and state of the art genetically engineered mouse models for both PDAC and the study of senescent cells, to evaluate the following aims. Aim 1. Determine how biophysical properties of the extracellular matrix regulate the induction and function of senescent CAFs. Aim 2. Determine the impact of senescent CAFs on myeloid and dendritic cell driven immune surveillance. Aim 3. Determine the organ specific impact of senescent CAFs on metastatic progression. Significance: Understanding how the PDAC TME's regulate tumor immunity is critical to employing stromal modulatory therapy to enhance immunotherapeutics. This concept is central to these studies.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY Mycobacterium tuberculosis (Mtb) causes one of the world’s most deadly infections. Initially, Mtb infects alveolar macrophages (AMs), and over time, it disseminates into monocytes, interstitial and recruited macrophages, and neutrophils. Once an adaptive immune response develops, CD4 T cells promote the ability of macrophages to control Mtb, although the mechanisms by which CD4 T cells confer protection are not fully understood. In addition to secreting diffusible cytokines like IFN-, cognate interactions between CD4 T cells and Mtb-infected macrophages are important for Mtb control. At the same time, Mtb undermines macrophage-T cell interactions. Thus, optimizing macrophage-T cell interactions may be an effective strategy to generate protection. However, until now, there has not been a way to determine which macrophages have presented antigen to CD4 T cells. We discovered that Mtb-infected macrophages induce the cell surface molecule SLAMF1 (SLAM/CD150) in response to cognate interactions with CD4 T cells. We show that SLAMF1 distinguishes macrophages that are infected and have interacted with T cells from uninfected, bystander macrophages that experience the same cytokine milieu, both in vivo in mice and ex vivo in macrophage-T cell co-cultures. Moreover, we found that Slamf1-/- mice have higher Mtb burden in the lungs, enhanced inflammatory cell recruitment, altered cytokine responses, and die earlier from TB compared to WT mice. Macrophage Slamf1 expression also correlates with protection in non-human primates. SLAMF1 is a type I transmembrane receptors that is found exclusively on hematopoietic cells. SLAMF1 mediates cell-cell signaling through homotypic SLAMF1-SLAMF1 interactions, and it can also directly bind bacteria. We hypothesize that when macrophages present antigen to CD4 T cells, macrophages to upregulate SLAMF1, which promotes control of Mtb in the macrophages by activating reactive oxygen species (ROS) and autophagy-related pathways. Activated T cells also express SLAMF1, and we propose that SLAMF1 signaling from macrophages to the T cells promotes resolution of inflammation. Thus, activating SLAMF1 may improve Mtb control while reducing immunopathology. Here, making use of Slamf1fl/fl conditional knock-out mice that we made, we will determine the cell type specific roles of SLAMF1 during TB. Using transwell assays to study macrophage-T cell interactions, we will establish whether macrophage-CD4 T cell interactions and SLAMF1 contribute to Mtb control in human and murine macrophages. We will establish whether CD4 T cells and SLAMF1 drive macrophage ROS production and autophagy, whether this pathway is undermined by Mtb virulence factors, and whether it can be augmented by small molecules and SLAMF1 agonists. Finally, making use of Slamf1 reporter mice that we made, we will elucidate the dynamics of early macrophage-T cell interactions during Mtb infection, and how these interactions are altered by vaccines, trained immunity, and contained infection. These studies will yield mechanistic insight into early events in host immunity to TB and support the development of novel therapies and vaccines to generate sterilizing immunity.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY This proposal will train the candidate to become a leading expert on the role of biological aging in cognitive impairment that can affect persons with HIV (PWH). A detailed training plan has been developed, including advanced coursework and instruction in the biology of aging and the epigenome, machine learning and bioinformatics methods, and career-enhancing training through mentorship, leadership, and grant-writing courses. This plan will both promote the success of the proposed Research Aims and set the candidate on a trajectory toward an independent and productive career. Regular engagement with an experienced, multi- disciplinary mentorship team will ensure the candidate’s success during the award period. The proposed research study will investigate the relationship between HIV-related cognitive impairment and biological aging, including aging of the central nervous system. While combination antiretroviral therapy has improved life expectancy for PWH, cognitive symptoms persist in 20–50% of this population. Growing evidence indicates that elevated biological aging is associated with adverse health outcomes for PWH. However, it remains unclear whether accelerated aging still occurs in PWH with undetectable viral load, especially in diverse populations at greater risk for comorbidities and adverse social determinants of health. It is also unclear how biological aging relates to cognitive function in such populations. These questions are of crucial importance for the health and quality of life of the aging population of PWH in the US. The proposed study will address the central hypothesis that accelerated biological aging in PWH contributes to cognitive impairment, in combination with comorbidities and social risk factors. The study will represent a seminal effort to combine measures of biological aging from DNA methylation and brain magnetic resonance imaging (MRI) with machine learning methods to build and test robust models of cognitive impairment in PWH. This approach will be centered on two relatively novel but well-validated aging biomarkers. Epigenetic age acceleration (EAA) will be measured using DNA methylation clocks, such as GrimAge, which the candidate will derive from DNA methylation microarrays applied to blood samples from a diverse group of PWH and control persons without HIV (PWoH). Analogously, deep neural network models will be used to quantify brain-age acceleration (BAA) using volumetric brain MRI data from the same participants. The ultimate goal is to leverage epigenomics and brain morphology data to produce a predictive computational model of cognitive impairment. The role of non-AIDS HIV-associated comorbidities such as cardiovascular disease and social determinants of health including neighborhood deprivation will be considered in parallel with biological measures. This study will provide insights into the origins of HIV-associated cognitive impairment, a major clinical problem that reduces quality of life for millions of PWH, and launch the applicant on a unique research career in neuroimaging and aging biology.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Myeloproliferative neoplasms (MPNs) are a clonal expansion of hematopoietic cells, and progression leads to deposition of collagen fibers throughout the blood-forming spaces of the bone marrow. In advanced cases, this causes bony replacement of the marrow cavity, blood maturation outside the bone marrow (extramedullary hematopoiesis), and transformation to fatal acute leukemia. Stromal cells normally form a hematopoietic niche to support hematopoietic stem cells, but in MPNs these cells are reprogrammed to produce overwhelming collagen. We dissected the signals from abnormal hematopoietic cells in MPNs that target bone marrow stromal cells, disrupting their ability to support normal hematopoiesis. We recently reported that transforming growth factor-J3 (TGF-J3) promotes collagen deposition and suppresses expression of key niche factors by bone marrow stromal cells. Moreover, blockade of TGF-J3 signaling in stromal cells can reverse bone marrow fibrosis in mouse models of MPN. However, when TGF-J3 signaling is blocked there is still disruption of the hematopoietic niche and development of extramedullary hematopoiesis. To identify additional signals that disrupt the hematopoietic niche in MPNs, I isolated stromal cells from patient bone marrow samples and identified candidate signaling pathways that alter the bone marrow microenvironment. Based on these data, I hypothesize that tumor necrosis factor-alpha (TNF) and platelet-derived growth factor (PDGF) contribute to loss of niche factors. In Aim 1, I will define the role of PDGF receptor signaling in bone marrow stromal populations in MPNs. Using mouse models to abrogate signaling through the two PDGF receptors in bone marrow stromal cells, I will transplant mouse cells with MPL w515 L or JAK2v61 7 F mutations to model MPNs. I will assess hematopoietic stem/progenitor cells, extramedullary hematopoiesis, niche factor expression, fibrosis, and survival. In Aim 2, I will define the role of TNF receptor signaling in bone marrow stromal populations in MPNs. I will abrogate TNF signaling through the two TNF receptors in stromal cells and use MPN transplant models to assess the impact on hematopoietic stem/progenitor cells, niche factor expression, extramedullary hematopoiesis, and fibrosis. Achieving these aims will improve our understanding of alterations in the bone marrow microenvironment through TNF and PDGF that lead to disease progression in MPNs. These insights will refine therapeutic targeting for more effective interventions to improve outcomes for patients. The overall goal is to establish an independent research laboratory studying contributions of the bone marrow microenvironment to normal and malignant hematopoiesis. This proposal outlines a five-year training plan to acquire advanced skills including innovative approaches in hematopoiesis and MPNs. The primary mentor is Dr. Daniel Link, a distinguished scientist in hematopoiesis, bone marrow microenvironment, and myeloid neoplasms, with a committee of Drs. Tim Ley, Stephen Oh, and Grant Challen for scientific and career development advising. Washington University is an exceptional environment for training with strong collaborations to gain expertise in stem cell biology and MPNs.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Migraine is a highly prevalent and disabling disorder that affects 15% of children and adolescents worldwide. Adolescents with migraine frequently undergo changes in their disease symptoms during puberty, with individuals experiencing increases, decreases, or no change in headache frequency. It is not currently possible to predict if an individual will experience improvement, no change, or worsening of migraine symptoms, and the underlying mechanisms controlling these changes remain uncharacterized. Thus, in the proposed study, we aim to identify the baseline factors determining migraine prognoses in adolescents (Aim 1), determine the hormonal, neural, and psychophysical changes related to migraine prognoses in adolescents (Aim 2a), and identify the temporal relationships between hormonal, neural, and psychophysical changes preceding vs. following changes in headache frequency (Aim 2b). Preliminary data support testosterone levels, conditioned pain modulation (CPM) response, and functional connectivity (FC) of the amygdala as factors that may determine migraine prognosis. Our preliminary data indicate that CPM response and amygdalar FC at baseline may predict the change in headache frequency following a behavioral intervention in adolescents with migraine. In addition, changes in testosterone levels are associated with changes in migraine symptoms. Study participants will be adolescents with episodic migraine (ages 10–13, 50% females, migraine onset > 6 months, headache frequency between 4–15/month, without or with a stable preventative treatment for migraine). Psychophysical, neural, and hormonal factors will be assessed at baseline and at 1- and 2-year follow-ups. Participants will meet with a headache specialist at all study visits to confirm migraine diagnosis and for rigorous characterization of migraine symptoms. Migraine outcomes will be categorized as an increase or decrease (> 30%) or no change (< 30%) in headache frequency from baseline to 2-year follow-up. A healthy control group will complete all study procedures to control for normal pubertal changes. We hypothesize that male sex and greater CPM responses, lower amygdala-prefrontal cortex (PFC) FC, and higher testosterone levels at baseline will be associated with a decrease in headache frequency after two years. We expect that adolescents with increased headache frequency will have a greater reduction in CPM efficiency, increased amygdala-PFC FC, and a smaller increase in testosterone levels compared to adolescents with a decrease or no change in headache frequency and healthy adolescents. We expect smaller increases in testosterone levels to precede increases in headache frequency and greater reductions in CPM efficiency and increases in amygdala-PFC FC to follow increases in headache frequency. Characterizing the mechanisms underlying changes in migraine symptoms is imperative for the development of new migraine treatments. Identification of adolescents who are at risk of worsening migraine symptoms during puberty will facilitate the development of personalized early-preventive strategies, which can reduce patient burden.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY / ABSTRACT Acute kidney injury (AKI) is a common clinical disorder, with a total of more than 13 million people affected globally every year. It has a high associated morbidity and mortality, and there are currently no definitive treatments besides supportive care. One of the most common causes of AKI is ischemia reperfusion injury (IRI), characterized by acute tubular necrosis and intrarenal inflammation. Regulated cell death pathways have been increasingly implicated in the tubular injury and resulting inflammation of AKI, and necroptosis in particular plays a critical role during IRI. There are very few known host factors that regulate necroptosis. Preliminary data in this proposal has identified interferon-stimulated gene 15 (ISG15) as a novel regulator of necroptosis and inflammation. Mice lacking ISG15 display a complete susceptibility to AKI in an IRI model, which can be rescued by also eliminating the executioner of necroptosis, mixed-linage kinase domain like pseudokinase (MLKL). In addition, preliminary data suggest that ISG15 negatively regulates necroptosis in murine primary proximal tubule cells in vitro. The overall hypothesis is that ISG15 acts as a critical factor in the host response to renal IRI by regulating the magnitude and timing of necroptosis in proximal tubule cells and downstream inflammation in the kidney. In Aim 1, damage to the kidney epithelium and the magnitude and timing of necroptosis due to an 18-minute bilateral IRI in vivo will be determined, as well as the host inflammatory response in the kidney. In Aim 2, the cell type in which ISG15 is acting to limit injury will be identified, and the interaction of ISG15 with the key signaling complex of necroptosis will be determined. The overall objective of this proposal is to advance understanding of the role of cell death pathways during renal IRI and elucidate the novel role of ISG15 as a host protective factor regulating cell death. This information is critical to advancing our fundamental understanding of the pathophysiology of AKI and developing effective therapeutics. The proposed research and training plan will facilitate the applicant’s development of key knowledge and skills to become an independent physician-scientist. Dr. Deborah Lenschow, the sponsor of this work, has extensive experience studying disease pathogenesis and the innate immune response to tissue injury, and the co-sponsor Dr. Benjamin Humphreys has deep expertise in kidney biology, genomics, and injury. The institutional environment provides a rigorous and supportive intellectual atmosphere as well as collaborative experts in AKI and kidney injury pathogenesis. This fellowship will support the applicant in becoming an independent investigator and a practicing physician-scientist.