University Of Iowa

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Inhalation of airborne particles, especially fine and ultrafine particulate matter (PM) can lead to pulmonary inflammation, which if not resolved, can cause lung injury and subsequent development of several chronic diseases. Pulmonary cells release signaling molecules to orchestrate inflammatory responses via cell-cell communication. One of the essential cell-cell communication mechanisms is via extracellular vesicles (EVs) and their enclosed cargoes (e.g. microRNAs). Compared to the extracellular signaling molecules, EVs carry the advantages of protecting the messengers better with their membrane structures and enhancing their effective concentrations within the vesicular compartment. Thus, identifying the key EV populations responsible for inflammation regulation and even resolution could greatly help development of therapeutics to alleviate the damage from the airborne particle-induced inflammation. However, it is difficult to pinpoint the exact types of EVs and their cargos responsible for inflammation resolution. We hypothesize that by tracing the EVs derived from pulmonary cells with the special focus on exosomes (Exos) at various time points during inflammation development, we can identify the specific EV sub-groups responsible for inflammation resolution. Hence, we proposed to identify Exos and their miRNA cargos in bronchoalveolar lavage (BAL) fluid and lung tissue in acute and sub-chronic models of pulmonary inflammation (Aim1) and employ NanOstirBar-EnabLed Single EV Analysis (NOBEL-SEA) to analyze cell specific Exos and enclosed miRNAs (Aim 2). NOBEL- SEA is a highly innovative advanced analytical technique developed in Dr. Zhong’s group. This technique enables detection of single EVs and their enclosed miRNA cargos with low sample consumption, high sensitivity and specificity, and short turn-around time. We will examine the kinetic secretion profiles of Exos in two inflammation models induced by two nanoparticles that have shown in our previous work to cause either resolving or persistent inflammation. We will first profile miRNAs from isolated exosomes in BAL fluid and lung tissue and then apply NOBEL-SEA for analyses of cell-specific Exos and miRNAs. Utilizing two inflammation models will allow us to study differences in Exos and miRNAs secretion during inflammation initiation and resolution. Monitoring the dynamic of Exo secretion from different cells and revealing their enclosed miRNAs will help achieve better understanding on how this EV subtype mediates communication between pulmonary cells and contributes to the transformation from pro- to anti-inflammatory states. It will pave the way for our long-term goals in exploring the functions of EVs for alleviation of inflammatory lung diseases induced by exposure to ultrafine airborne particles.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY According to the CDC, drowning is the leading cause of unintentional death among 1-to-4 year-olds and is second only to vehicle accidents among 5-to-14 year-olds. Moreover, non-fatal drownings which often lead to permanent brain damage and other long-term deficits occur even more frequently and across all age groups. A recent NIH Notice of Special Interest (NOT-HD-21-048) was issued “to encourage and facilitate scientific discovery for drowning prevention.” This proposal is an answer to that call. The majority of drownings occur in swimming pools and despite a widespread belief that swimmers are safe when lifeguards are present, drownings occur year after year in guarded swimming pools across the country. The majority of drownings at guarded facilities occur because lifeguards fail to detect and recognize emergencies for what they are (e.g., a body at the bottom of the pool) rather than because they lack sufficient lifesaving skills or are somehow negligent. Several years ago, our group published a review of the basic-research literature on visual perception and cognition in which we identified multiple properties of human perception and attention that are relevant to the surveillance component of lifeguarding. That work led to the general hypothesis that surveillance failures of lifeguards are often rooted in fundamental limitations of human information processing and that a better understanding of the specific ways in which those limitations impact performance could inform the development of targeted training and operational procedures aimed at reducing preventable drownings. A barrier to testing these ideas, however, has been an inability to achieve an effective balance between external validity of in-the-field conditions—which is critical for a real-life task like lifeguarding—and experimental control—which is critical for testing hypotheses. On the one hand, conducting experimental work in field settings (e.g., at swimming pools or water parks) is untenable. Drowning events cannot be experimentally manipulated for both ethical and practical reasons. On the other hand, model laboratory tasks have proven insufficiently similar to actual lifeguard surveillance to generalize to in-the-field surveillance. This R21 project addresses this barrier. It is to develop a virtual reality aquatics environment that simulates many of the immersive aspects of the surveillance component of lifeguarding while also allowing for control and manipulation of relevant factors. The environment will be validated by linking it to well-established and robust effects in the basic literature on visual attention, while systematically building in many of the complexities of the lifeguarding task. Success of this project will open a wide range of opportunities for research and training focused on reducing the number of preventable drownings that occur each year and, in addition, it will provide a path for conducting research on other real-life surveillance tasks.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY This K08 proposal outlines the plan for Kale Bongers, M.D. Ph.D.’s to complete training toward his long-term goal of improving our understanding of post-sepsis muscle atrophy and weakness through investigation of the microbiome. Dr. Bongers is a physician-scientist in Pulmonary and Critical Care Medicine at the University of Michigan with an established record of success in skeletal muscle biology and microbiome studies. This proposal builds on Dr. Bongers’ previously acquired skillset in skeletal muscle biology, critical illness pathophysiology, and microbial ecology with new training in metabolomics and metagenomics. These skills will be integrated to improve our understanding of the role of the gut microbiome in mediating skeletal muscle atrophy after sepsis. This research will be conducted under the guidance of mentor Robert Dickson, M.D., co- mentor Kathleen Stringer, Pharm.D., and an advisory board of renowned investigators with experience in metabolomics, microbial ecology, microbiome studies, skeletal muscle in critical illness, and sepsis pathophysiology. The five-year plan includes intensive mentorship, formal didactic coursework, professional development, and increasingly independent research, with milestones to encourage productive research output and transition to independence. Sepsis is a common and deadly condition of organ dysfunction and immune dysregulation secondary to infection that often leaves its survivors severely debilitated due to muscle atrophy. Studies have suggested that the gut microbiome plays a critical role in the regulation of skeletal muscle size and strength, but to date no study has evaluated the role of the gut microbiome in the pathogenesis of post- sepsis skeletal muscle atrophy. This proposal tests the hypothesis that the gut microbiome plays a critical role in post-sepsis skeletal muscle atrophy via two specific Aims. Aim 1 will identify the key bacterial taxa that contribute to or prevent muscle atrophy in multiple models of murine sepsis, while Aim 2 will identify key bacterially-derived metabolites that mediate these changes in muscle. To accomplish these Aims, Dr. Bongers will leverage both in vivo and in vitro models of skeletal muscle with cutting-edge techniques in metabolomics and metagenomics to identify and mechanistically interrogate how gut bacteria influence skeletal muscle size and function during sepsis. This will lay crucial groundwork for future R01 proposals to 1) determine whether supplementation of key bacteria or key bacterially-derived metabolites can prevent skeletal muscle atrophy and 2) translate these findings to humans using morphomics and pre-existing bacterial swabs. In addition to building a strong line of research to understand the role of the microbiome in muscle atrophy, this proposal will provide Dr. Bongers with new research skills applicable to microbiome and metabolomics research. This K08 award will enable Dr. Bongers to establish himself as an independent physician-scientist and rising leader in this important field.

NIH Research Projects · FY 2026 · 2024-04

In the United States, Spanish-English bilingual children with speech sound disorders do not have access to the most efficient treatment options. This is a significant barrier to optimal outcomes for a large population in this country, considering that nearly 1 in 4 children in the US lives in a household where a language other than English is spoken. When children enter kindergarten with delayed speech development, they are more likely to struggle with language, reading, and academic achievement. This can have long-term effects on their psychosocial well-being and future employment outcomes. By improving the efficiency of treatment for speech sound disorders, we have the potential to greatly improve outcomes for these children and also reduce strain on healthcare and special education resources. Previous studies have shown that teaching English-speaking children more difficult speech sounds, like consonant sequences, such as "fr" in "free," leads to broader improvements in their speech. They not only learn the targeted sounds but also simpler sounds like "ch," "th," "v," "g," and "k." This suggests that teaching more complex sounds can have an efficient impact on the entire sound system, even on sounds that were not directly practiced. However, in order to apply this approach to Spanish-English bilingual children, we must answer two important questions. First, we need to determine if teaching complex sounds results in similar overall improvements in speech when bilingual children receive treatment in either Spanish or English. Second, because bilingual children are simultaneously developing two languages, we need to understand if teaching more complex speech sounds in one language has a broader impact, not only on the targeted language but also on the untreated language. To address these questions, we will conduct a Phase I Clinical Trial using single-subject experimental design with a) complex and b) simple treatment conditions. This design allows us to observe, for bilingual speech treatment, the extent of system-wide improvement and across-language transfer resulting from complex treatment targets. The treatment will be provided in Spanish (Aim 1) and English (Aim 2). In Aim 3, we will analyze the data gathered from Aims 1 and 2 to explore how specific Spanish or English sounds interact with different treatment targets. This analysis will help us to understand the mechanisms of broad phonological change across different languages. The implications for this research are potentially far-reaching. By uncovering the cross-linguistic mechanisms involved in broad-based phonological learning, we can better understand how complexity influences the transfer of skills across languages. This research would establish a foundation from which to develop more efficient speech treatment procedures that are appropriate for monolingual and bilingual children, thereby better meeting the needs of the entire population of children with speech sound disorders.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Patients who require intensive care unit (ICU) level of care frequently develop hospital-acquired functional decline, a new or worsening loss of ability to perform self-care activities that is associated with prolonged periods in immobility. This morbidity is potentially preventable through initiating mobility interventions in the ICU using a multidisciplinary, evidence-based intervention to maintain functional status. While guidelines for ICU patients' physical activity exist, timely identification of patients suitable for activity interventions is an ongoing challenge due to the dynamic nature of critical illness and the number of locations in the electronic health record (EHR) that clinicians need to click in and out of to synthesize patient data. There is, therefore, a critical need to develop an effective clinical decision support system (CDSS) interface in the EHR for efficient identification of patients appropriate for physical activity interventions and coordination of patient-specific activity plans within the ICU team. Our long- term goal is to accelerate the development and implementation of clinically useful CDSS to prevent hospital-acquired functional decline. Our overall objective is to develop a CDSS interface for consistent patient-specific translation of evidence-based physical activity interventions for ICU settings, and evaluate its usability, usefulness, cognitive workload, acceptability, feasibility, and effectiveness on decision-making outcomes. The proposed project has two phases and four specific aims. In Phase 1 (R21), we will develop a useable, useful, and acceptable CDSS prototype by conducting clinician interviews and a user-centered design approach (R21 Aim 1), and identify clinical workflow considerations, potential barriers, and implementation strategies in preparation for evaluating the feasibility and effectiveness of the CDSS (R21 Aim 2). In Phase 2 (R33), we will evaluate the CDSS usability, cognitive workload, acceptability, and effectiveness for activity guideline adoption in a simulated EHR environment (R33 Aim 3) and on two ICU units in one tertiary care hospital (R33 Aim 4). The results are expected to have an important positive impact by providing strong justification for a subsequent multi-site pragmatic R01-level clinical trial to scale the concurrent use of patient data with guideline recommendations at the point of care to deliver evidence-based interventions to reduce hospital-acquired functional decline at its negative, costly outcomes.

NIH Research Projects · FY 2026 · 2024-04

Project Summary / Abstract HER2-positive breast cancers are highly aggressive and associated with poor prognosis. HER2-targeted therapy is the preferred treatment for these cancers, but drug resistance is a major problem. Our previous studies demonstrated that dysregulation of G protein coupled receptors, in particular, a subgroup of GPCRs that couple to G protein αi/o subunits (Gi/o-GPCRs), contributes to HER2-induced breast cancer initiation and progression. Targeting Gi/o-GPCR signaling blocks tumor progression and enhances the efficacy of HER2-targeted therapy. This proposal aims to delineate how dysregulated Gi/o-GPCR signaling may control HER2+ breast cancer progression through a poorly studied CTLH E3 ubiquitin ligase complex. The CTLH (carboxy-terminal to LisH domain) complex is a mammalian ortholog of the yeast GID (glucose-induced-degradation-deficient) complex that contains multiple subunits, including an adaptor/scaffold protein, WDR26, which may be required for CTLH complex assembly and recruitment of specific substrates for ubiquitination and degradation. The CTLH complex was implicated in tumorigenesis but its exact functions in tumor development remain largely unknown. We previously found WDR26 is a scaffolding protein that regulates GPCR signaling and is highly upregulated in all molecular subtypes of invasive breast carcinoma and associated with worse prognosis. In preliminary studies, we tested WDR26 gene deletion in a Neu transgenic mouse model of HER2+ breast cancer and showed mammary-specific WDR26 gene deletion recapitulated a Gi/o-GPCR-signaling blockade: both inhibited tumor initiation, growth, and lung metastasis. WDR26 likely promotes both G protein signaling and controls CTLH- ubiquitin-ligase-driven degradation of SNF5 (an epigenetic tumor suppressor) in tumor cells. Based on these exciting preliminary data, we hypothesize that, in HER2-driven breast tumors, WDR26 upregulation promotes G-protein signaling and facilitates nuclear CTLH-complex assembly and E3 ubiquitin ligase activity, leading to ubiquitination and proteasomal degradation of SNF5; and this SNF5 depletion activates oncogenic transcriptional programs, in part, by upregulating EZH2 (enhancer of zeste homology 2), ultimately promoting tumor growth and metastasis and resistance to HER2-targeted therapy. In this study, using a combination of cell lines, several newly developed genetic and patient-derived xenograft mouse models, we will determine 1) how WDR26 manifests dysregulated Gi/o-GPCR signaling to drive breast cancer progression via the CTLH complex; 2) how the CTLH complex regulates breast cancer development by targeting SNF5 for proteasomal degradation; and 3) whether targeting the CTLH and SNF5 function improves the efficacy of HER2-targeted therapy. The results of our studies should fundamentally advance understanding of how the poorly studied CTLH E3 ubiquitin ligase complex targets the epigenetic tumor suppressor SNF5 to drive tumor progression and drug resistance. This knowledge should help us identify new strategies for augmenting HER2-targeted therapy in breast cancer.

NIH Research Projects · FY 2025 · 2024-04

Cognitive deficits in schizophrenia and bipolar disorder are associated with altered cerebellar activity and this altered activity is thought to play a causal role in the cognitive deficits observed in these disorders. However, the neural mechanisms underlying cerebellar contributions to intact and impaired cognition have not been fully elucidated. We will address this gap in knowledge by examining the neural mechanisms underlying cerebellar communication with forebrain systems during interval timing – a cognitive task requiring attention, working memory, and precise response timing. The primary emphasis of the previous research on the role of the cerebellum in interval timing has been on the ‘ascending’ cerebello-thamo-frontal cortical pathway. However, the medial frontal cortex also projects back to the cerebellum via the pontine nucleus. This ‘descending’ pathway consists of the medial frontal cortical projection to the rostral pontine nucleus and its mossy fiber projection to the cerebellum. A major goal of the current project is to determine the role(s) of the descending pathway in interval timing. The descending pathway could plausibly modulate cerebellar activity with ramping activity or a tonic increase in activity. We hypothesize that both tonic and ramping activity from the descending pathway modulates activity in the cerebellum by increasing the overall firing rate and precision of ramping, respectively. We also hypothesize that sensory information from the start cues in interval timing combines with information from the medial frontal cortex to support cue-specific and interval-specific ramping of cerebellar output. We will test these hypotheses in two aims that examine the pathways for cue information (Aim 1) and mFC timing information (Aim 2) using optogenetics and electrophysiology in rats during performance of a dual- interval timing task. A broad impact of this project is that we will be able to establish whether there are common mechanisms for cerebellar organization and function in motor learning and cognition. The current proposal is a basic research project, but the findings may have important implications for translational research on the role of the cerebellum in schizophrenia and bipolar disorder. Studies of schizophrenia and bipolar disorder have generally focused on how altered cerebellar function affects frontal cortical function, but it is plausible that the altered activity originates in the cortex (or in both areas) and affects the cerebellum via the descending frontal cortical-pontine-cerebellum pathway. The findings of our project could form the foundation for translational research into how descending pathway abnormalities relate to cognitive symptoms in schizophrenia and bipolar disorder.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Dysregulation of central monoamine (dopamine, noradrenaline, and serotonin) signaling is implicated widely in neuropsychiatric and substance use disorders, but there is a fundamental knowledge gap in how monoamines signal at the level of ion channels and individual synapses. It is well-established that serotonin-releasing neurons in the dorsal raphe nucleus are driven to fire action potentials and release serotonin by noradrenergic input and activation of metabotropic alpha1-adrenergic receptors. Recently, we discovered that alpha1-adrenergic receptors depolarize serotonin neurons via coupling to the ‘orphan’ ionotropic delta 1 glutamate receptor (GluD1R) ion channels. Intriguingly, prior research showed that metabotropic glutamate receptors also couple to GluD1R in midbrain dopamine neurons and contribute to burst firing of action potentials. The signaling mechanism between these metabotropic receptors and GluD1R ion channels requires G protein signaling but what occurs downstream to activate GluD1R is not yet known. In addition to this G protein-dependent GluD1R current, we found that GluD1R channels carry a G protein-independent tonic current that depolarizes the resting membrane potential to promote action potential firing. The etiology of the tonic GluD1R current is still unresolved. Together, these findings demonstrate that ionic current carried by GluD1R plays an unexpected and critical role in potentiating the excitability of monoamine neurons. We propose to use mouse brain slice electrophysiology, fluorescence imaging, and pharmacological manipulation to record GluD1R currents in serotonin neurons to (1) delineate the postsynaptic signal transduction mechanisms between alpha1-adrenergic receptors and GluD1R; and (2) determine whether GluD1R current is sensitive to naturally occurring cysteine-modifying agents like the small transition metal zinc and the antioxidant ascorbate, in a manner similar to NMDAR current. Lastly, (3) in midbrain dopamine neurons, we will determine the role of GluD1R in metabotropic receptor-dependent AMPAR synaptic plasticity in physiological conditions and after in vivo exposure to cocaine. We will evaluate the in vivo relevance of GluD1R current using a combination of imaging and behavioral assays in wild-type mice in comparison with mice that lack expression of GluD1R and mice that express a mutated GluD1R that is ‘pore- dead’ or non-conducting. The proposed studies are expected to be significant since they will identify key molecular regulators of serotonin and dopamine neuron excitability through action on GluD1R and the mechanism through which G protein-coupled receptors act upon GluD1R to modulate their ionic current. Our preliminary data suggest these studies may reveal a metabotropic receptor-initiated signal cascade that has not yet been observed in the central nervous system. Further, we anticipate gaining a significant understanding of the contribution of GluD1R current in metabotropic receptor-dependent depolarization, induction and maintenance of synaptic plasticity, and cocaine-related behavior.

NIH Research Projects · FY 2025 · 2024-04

Candida glabrata is the second most common cause of candidemia. Many studies have found that patients with C. glabrata bloodstream infection have higher mortality than those infected with other Candida spp. One potential cause of this increased mortality is the relative ease with which C. glabrata can acquire resistance to antifungal agents, including the major antifungal drug fluconazole (FLC). In vitro studies with a limited number of clinical isolates have led to the conclusion that that C. glabrata develops FLC resistance by one of two mechanisms, petite mutants with mitochondrial defects or gain-of-function mutations in the Pdr1 transcription factor, which result in overexpression of genes encoding drug efflux pumps. These conclusions are based on the analysis of a relatively small number of C. glabrata isolates that were performed in vitro. We analyzed 19 different C. glabrata clinical isolates with elevated FLC minimal inhibitory concentrations (MICs) by whole genome sequencing and Western blotting for proteins involved in FLC resistance. While a majority of these strains do contain alterations in their PDR1 sequence, most of these mutations have not been associated with FLC resistance previously. Additionally, we found 4 unrelated strains with changes in ERG11, and 3 of these strains had the same mutation, strongly suggesting that mutations in this gene may also reduce FLC susceptibility. We also analyzed the transcriptional response of C. glabrata to FLC in the mouse model of disseminated infection and found that the organism responds significantly different to this drug during mammalian infection relative to growth in vitro. We will explore these compelling data by 1) performing functional analysis of the genomes of clinical C. glabrata isolates with elevated FLC MICs, and 2) analyzing the effects of FLC treatment on the C. glabrata transcriptome during mammalian infection. Successful completion of these two aims will important new insight into the molecular basis of FLC resistance in C. glabrata as it infects the mammalian host. This information will serve as the basis for developing new strategies to combat antifungal resistance in this increasingly prevalent pathogen.

NIH Research Projects · FY 2026 · 2024-04

Project Summary / Abstract Sodium plays a vital role in essential physiological processes, including growth, development, and regulation of extracellular fluid volume, making it critical for survival. However, excess sodium intake is associated with an increased risk of hypertension and cardiovascular disorders and, despite efforts by the World Health Organization, average daily salt intake worldwide is approximately twice the recommended level. Due to sodium’s ability to regulate the extracellular fluid volume, and thus impact cardiovascular function, its excretion and intake are tightly regulated with the steroid hormone aldosterone (ALDO) playing a major role. In the adult mouse brain, a single population of neurons located in the nucleus tractus solitarius (NTS) are capable of sensing ALDO due to their co-expression of MR and 11β-hydroxysteroid dehydrogenase type 2 (HSD2), the latter prevents glucocorticoid stimulation of MR. These “NTSHSD2 neurons” have been shown to drive sodium appetite when activated by chemo- or optogenetic tools. Furthermore, chronic treatment with ALDO increases the intrinsic firing rate of NTSHSD2 neurons, though the molecular mechanism is unknown. The objective of this proposal is to investigate the basis for ALDO-induced NTSHSD2 neuron activity and sodium appetite. We hypothesize that ALDO signaling coordinates a transcriptional program that promotes NTSHSD2 neurons activity, ultimately driving sodium appetite. To test this, Aim 1 will determine the importance of ALDO/MR signaling in NTSHSD2 neurons for sodium appetite and investigate the effects of ALDO on NTSHSD2 neurons in vivo. Aim 2 will employ transcriptomics and epigenomics to identify candidate ALDO/MR regulated genes in NTSHSD2 neurons during ALDO treatment that could mediate increased spontaneous activity. Aim 3 will test the role of genes found to be regulated by sodium deficiency or ALDO/MR signaling for their impact on NTSHSD2 neuron activity and sodium appetite. Understanding the regulatory mechanisms of NTSHSD2 neuron activation is crucial to our understanding of sodium appetite and data from this proposal will identify potential therapeutic targets for reducing excess sodium intake.

NIH Research Projects · FY 2025 · 2024-03

Abstract Improved approaches are needed to advance the field of toxicology by discovering specific targets and mechanisms through which legacy and emerging environmental contaminants act. G protein-coupled receptors (GPCRs) are critical mediators of physiological processes and have been identified as targets that mediate the harmful effects of a small number of contaminants. However, the current research on contaminant activity toward the human GPCRome is scarce and only investigates specific contaminants and receptors in detail. Our proposed project focuses on the gap in the research concerning environmental contaminant activity on GPCRs. We will leverage contemporary high throughput screening (HTS) and receptor pharmacology techniques to identify novel contaminant-GPCR pairs, giving insight into molecular mechanisms and biological consequences. To test our approach, we trialed a small, preliminary study, which yielded promising results by revealing novel and unpredictable contaminant interactions with specific GPCRs, thus supporting this research's direction. From both literature precedent and our findings, we propose to expand the effort more comprehensively and rigorously with the following research objectives: (1) Interrogate per- and polyfluoroalkyl substances (PFAS) against a focused set of GPCRs to identify PFAS-GPCR relationships that mediate effects of PFAS exposure, (2) Conduct a comprehensive screen of various contaminants against the 'druggable' GPCRome, to discover unpredictable contaminant-GPCR pairs, and finally (3) characterize the newly identified contaminant-GPCR pairs in detail regarding their impact on receptor pharmacology, and cellular consequences. To achieve success in our research objectives, the methods of our approach will draw upon the recently developed PRESTO-TANGO and TRUPATH technologies, in addition to traditional receptor pharmacology techniques. Findings generated from this proposal will include (1) the identification of GPCR-mediated effects of contaminants, (2) the discovery of specific targets acted on by environmental contaminants, and (3) characterizing molecular mechanisms of contaminant exposure in the relevant context of cellular consequences. Furthermore, the significance of this work will extend beyond the insight gathered from the tested compounds. As a 'first-in-class' study, this project would support a new framework for investigating the biological activity of xenobiotic compounds - proving valuable for current research initiatives such as the exposome and future work investigating emerging contaminants.

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT Niemann-Pick disease type C is a fatal, autosomal recessive lipid storage disease affecting all ages. Before neurological defects appear, ~85% of patients develop hepatomegaly which can progress into hepatic steatosis, cirrhosis, hepatocellular carcinoma, and liver failure. Liver defects are particularly detrimental in patients with neonatal-onset, 10% of whom die from liver failure by 6 months of age. Although the liver is a significant contributor to disease, few Niemann-Pick C liver therapeutics are being developed. Niemann-Pick C is commonly caused by loss-of-function mutations in the NPC1 gene (95% of cases), encoding a multipass transmembrane glycoprotein required for exporting unesterified cholesterol from late endosomes and lysosomes. The most common disease-causing mutation (~20% of cases) is an isoleucine to threonine substitution at position 1061 (I1061T). I1061T-NPC1 misfolds in the endoplasmic reticulum (ER) and is rapidly degraded by the proteasome and ER-autophagy. Importantly, I1061T-NPC1 is functional if trafficked to the lysosome. This observation spurred interest in understanding NPC1 degradation for the development of brain proteostasis modulators. However, it is unknown if neuronal NPC1 proteostasis modulators will work in the liver due to the limited understanding of liver NPC1 regulation. Furthermore, our strong preliminary data indicate that liver NPC1 is more heavily glycosylated than brain NPC1. Given the critical role of glycans in protein folding, trafficking, function, and degradation there is a need to understand the role of NPC1 liver- specific glycosylation. The next step in addressing this need is to pursue the overall objective of this application: (i) produce pilot data for an RO1 which demonstrate that liver specific glycan sites are critical for NPC1 proteostasis. Here we will test the central hypothesis that liver-specific glycans alter NPC1 proteostasis. We will test our hypothesis using LC-MS/MS to identify NPC1 glycan sites in human liver and brain. Next, we will take advantage of NPC1 null iPSC derived hepatocytes and add back constructs containing WT, WT without liver specific glycans, I1061T, and I1061T without liver specific glycans. We will leverage biochemical and genetic assays to establish the extent to which liver-specific glycan sites impact NPC1 proteostasis (Aim 1). The rationale for this project is that defining the location and influence of tissue-specific NPC1 glycans will provide deeper insight into NPC1 proteostasis and a strong scientific framework for the development of new Niemann-Pick C liver proteostatic therapeutics. In addition to moving my research into a highly understudied research topic, we anticipate this work will provide a strong scientific framework to investigate he how liver- specific NPC1 glycans regulate proteostasis as a future aim in an R01 grant from NIDDK.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Background and Objectives: Neurons and their synapses require high amounts of energy to sustain normal levels of activity. Mitochondria are the main energy source, producing ATP via oxidative phosphorylation. In turn, oxidative phosphorylation proceeds through the action of large protein complexes, like Mitochondrial Complex I (MCI). But much work shows that mitochondrial components in neurons and at synapses also do far more than generate ATP. Mitochondria buffer calcium, drive Reactive Oxygen Species (ROS) signaling, and influence cell survival. Using the Drosophila melanogaster neuromuscular junction (NMJ) as a model synapse, we found loss of MCI components impact distinct synaptic tissues in profoundly different ways. The objective of this proposal is to use this model to understand exactly how mitochondria impact discrete parts of the synapse. Specific Aims and Research Design: We found that MCI impairment causes profound cytological abnormali- ties at both the pre- and postsynaptic NMJ. But important differences emerged upon examination of individual tissues. NMJ activity is dampened when MCI is impaired in the postsynaptic muscle. NMJ activity can also be dampened when MCI is impaired in neurons – but curiously, this only happens when it is combined with other insults. This project has two specific aims. Aim 1 is to understand how loss of MCI in the muscle causes a diminishment of NMJ function. This phenotype is noteworthy for multiple reasons. First, the NMJ displays phe- notypes reminiscent of neurodegeneration when MCI is lost in muscle. Second, the NMJ is known to employ numerous muscle-to-nerve signaling paradigms to sustain normal activity. We can test if any of those retrograde signals are occluded. Aim 2 is to define how the presynaptic neuron eludes dysfunction when MCI is lost. Com- bining genetics, pharmacology, electrophysiology, and imaging we will test if known homeostatic signaling com- ponents and modalities are co-opted to maintain normal activity. The expected outcome of our work is new understanding of how mitochondrial function and dysfunction impinge upon discrete synaptic compartments. Health Relatedness: Impairment of mitochondria is associated with neurodegenerative conditions like Parkin- son’s Disease, neuromuscular conditions like Leigh Syndrome, as well as forms of epilepsy and ataxia. For any genetic disease or disorder caused by mitochondrial dysfunction, we require better cell-specific models to eluci- date what is happening on the levels of synapses and circuits. Based on our data, the tractable Drosophila NMJ is a good way to define how synapses react to normal mitochondrial function or dysfunction. In turn, this kind of foundational information from the NMJ model could edify downstream investigations into neurological conditions caused by mitochondrial dysfunction, like forms of Parkinson’s disease, epilepsy, and ataxia.

NIH Research Projects · FY 2026 · 2024-03

SUMMARY Left ventricular (LV) pressure overload caused by conditions such as chronic hypertension or aortic stenosis is a significant risk factor for the development of heart failure. A decline in microbial diversity caused by certain antibiotics also increases the risk of heart failure, suggesting that the gut harbors cardioprotective microbes. However, our current knowledge of which microbes are cardioprotective and which have deleterious cardiac effects is inadequate, not allowing us to leverage gut microbial manipulation with antibiotics to treat or mitigate heart failure. microRNA-204-5p (miR-204) is a noncoding RNA well-expressed in mouse and human hearts. We recently reported that the miR-204 inhibits mouse myocardial hypertrophy induced by experimental pressure overload, and its expression in many tissues, including the heart, is sensitive to changes in the gut microbiome. Our preliminary data show that glycopeptide antibiotic vancomycin alters the mouse gut microbial landscape, leading to the enrichment of Lactobacillus sp that confers protection from pressure overload-induced myocardial hypertrophy and LV dysfunction. Moreover, vancomycin increases the abundance of the bacterial metabolite tryptamine in the feces and serum, which inhibits myocardial hypertrophy and LV dysfunction. Both, vancomycin and tryptamine stimulate cardiac miR-204 expression, which is essential for their cardioprotective effects. Based on these data, we hypothesize that vancomycin-induced reshaping of the gut microbiome enriches Lactobacillus sp. that promotes tryptophan's metabolism to tryptamine, leading to upregulation of cardiac miR-204 that inhibits pressure overload-induced myocardial dysfunction. This application will leverage unique reagents and tools to connect vancomycin-induced reshaping of the gut microbiome to cardiac miR-204-regulated cardioprotection. The application will determine whether vancomycin-induced enrichment of Lactobacillus sp. in the gut stimulates bacterial tryptophan metabolism to confer protection from myocardial hypertrophy and LV dysfunction. Next, we will explore the role of cardiac miR-204 in mediating the cardioprotective effect of vancomycin on pressure overload-induced myocardial hypertrophy and LV dysfunction. We expect our study to identify specific microbes (e.g., Lactobacillus murinus), metabolites (e.g., tryptamine), and signaling intermediaries (e.g., miR-204) that mediate the cardioprotective effects of vancomycin. There is a crucial knowledge gap in our understanding of how the gut microbiome modulates cardiac contractile function. This gap is a hurdle to using gut microbiome manipulation as a therapeutic strategy for heart failure. This application will narrow this gap by exploring a novel nexus between gut bacteria, bacterial metabolites, and cardiac miR-204.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Orofacial clefts (OFCs), specifically cleft lip with or without cleft palate, are among the most common class of birth defects and they contribute to a significant health and financial burden. The development of OFCs is influenced by complex interactions between genetic and environmental factors. Developing a comprehensive understanding of the cellular pathways and molecular regulators of palatogenesis will be essential to enhancing treatment options and disease intervention. My overall objective of the proposed project is to identify molecular pathways that modulate palatal development. Genetic studies in the human identified over 50 loci associated with OFC. However, mutations in very few of these genes can cause isolated OFC with high penetrance, making them well-suited for mechanistic studies and better candidates for clinical interventions. One such gene is Rho GTPase activating protein 29 (ARHGAP29). ARHGAP29 contributes to cyclic regulation of the small GTPase RhoA, inactivating it. To explore the role of ARHGAP29 during craniofacial development, Arhgap29 was previously deleted in the mouse. Although Arhgap29 knockout embryos were found to die around embryonic day e8.5, i.e., before craniofacial structures develop, heterozygous loss-of-function embryos were viable and displayed intraoral adhesions, a phenotype associated with OFCs. In the craniofacial region, ARHGAP29 is expressed in cell lineages derived from both the ectoderm (periderm and epithelial cell layers) and the neural crest (mesenchymal cells). To tease apart the contributions of ARHGAP29 to each cell lineage during palatogenesis, I initiated a tissue-specific knockout strategy using the Cre-Lox system. My preliminary results from these animals show that the loss of ARHGAP29 in either ectoderm- or neural crest-derived lineages results in a delay in palatogenesis (apparent at e14.5). However, only the loss of ARHGAP29 in ectoderm-derived cells results in a cleft palate at e18.5. These findings are consistent with ARHGAP29 playing a tissue-specific role during palatogenesis. My central hypothesis is that ARHGAP29 in ectoderm- and neural crest-derived cells of the palatal shelves is required for proper palatogenesis because it promotes remodeling of adherens junctions and force transduction across the palatal shelves as they elevate. The premise for this hypothesis is that ARHGAP29 is a modulator of RhoA, which promotes the actomyosin contractility that is required for both the remodeling of cell-cell junctions and cellular contractility that are required for palatal shelf elevation and fusion. In Aim 1 I will identify the tissue(s) in which ARHGAP29 is required to promote proper palatogenesis in vivo, using a series of tissue-specific Cre recombinase driver alleles and characterizing embryos at time points critical to palate development. In Aim 2 I will define the molecular mechanisms by which ARHGAP29 regulates the epithelial shape and adhesions required during palatogenesis, using an in vitro culture system in conjunction with biochemical and confocal microscopy-based analyses. This project will provide me with in-depth training in all these techniques, as well as in the scientific communication skills that I will need to become a successful independent investigator.

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT. Type 1 Diabetes (T1D) results from autoimmune-mediated destruction of pancreatic β-cells. Currently, no long- term treatments have been successful in preventing disease, and β-cell contribution to early pathology is poorly understood. Early defects in β-cell secretory function, such as mis-trafficking of secretory proteins, are evident prior to symptomatic onset. These defects are suggested to play a role in early disease progression, possibly by neoantigen formation, yet underlying mechanisms are poorly understood. Our recent work demonstrates that proinflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), substantially disrupt β-cell Golgi structure and function, which may explain early defects in β-cell secretory function. The purpose of this proposal is to investigate how cytokine exposure drives alterations to Golgi structure, and the implications this has on Golgi functions. To this point, we demonstrate treatment with a chemical NO donor can recapitulate altered β-cell Golgi structure and function. Our preliminary data further demonstrates knockdown (KD) of GRASP55, a key Golgi structural protein, can substantially block Golgi fragmentation during cytokine stress, which may be regulated via post-translational modification. Our central hypothesis is that proinflammatory cytokines drive NO-dependent, GRASP55- mediated Golgi remodeling, leading to dysfunctional protein sorting and glycosylation. We will test this hypothesis through two aims. Aim 1) will investigate the role of iNOS and NO in altered Golgi structure and cell-surface glycosylation through genetic and pharmacological models of iNOS inhibition, as well as flow cytometry to measure cell surface glycosylation. Aim 2) will investigate PTM regulation of GRASP55 in modulation of Golgi structure and improper secretion of CtsD through GRASP55 KD models and expression of PTM-mutants in a GRASP55 knockout (KO) cell line. Pharmacological inhibition of Golgi export will also be used to investigate how GRASP55-dependent Golgi remodeling drives improper CtsD secretion. This proposed work will advance understanding of how proinflammatory cytokines affect β-cell Golgi structure and function, providing novel insight to β-cell contributions to disease pathology early in T1D and potential targets for new therapeutics.

NIH Research Projects · FY 2026 · 2024-03

Abstract Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs) and their axons. Damage from glaucoma is permanent and may cause irreversible blindness. Glaucoma is a major public health problem in the United States and worldwide, where it is the leading cause of irreversible blindness. Four classic risk factors have been identified for the most common type of glaucoma, primary open angle glaucoma (POAG): increasing age, race / ethnicity, family history, and increased intraocular pressure (IOP). Currently, all treatments for glaucoma slow or halt disease by targeting one risk factor, increased IOP. However, IOP-lowering therapies fail to prevent vision loss in many patients. Thus, glaucoma patients desperately need new, more effective therapies that target other aspects of glaucoma beyond elevated IOP, such as the mechanisms underlying family history of glaucoma. Unfortunately, most genetic risk associated with glaucoma remains poorly understood. Most cases of POAG have a complex genetic basis and involve many risk factors. To date, >127 risk factor loci have been identified with genome-wide association studies (GWAS's). In each of these loci, several single nucleotide polymorphisms (SNPs), have been associated with risk for POAG and the effect of these SNPs on the nearest gene has been presumed to be the source of risk. However, little progress has been made in precisely defining these loci, including the causative SNP(s), gene(s), or mechanism(s). Very few of the disease-associated SNPs the >127 glaucoma risk loci are in coding sequence. Consequently, we hypothesize that the functional SNPs in each locus confer glaucoma risk by increasing or decreasing transcript levels of effector genes in the locus. To test our hypothesis, we have prioritized three loci for detailed studies (chr 4p14, chr 21q21.3, and chr 17q21.3), which each containing an exemplar candidate for being the effector gene (APBB2, APP, MAPT), which are all expressed in RGCs and all share links with neurodegeneration associated with Alzheimer disease. To test whether these are indeed the effectors and study their mechanisms, we propose a complementary set of experiments using human, mouse, and molecular approaches. In Specific Aim 1 we will use genotyped human donor retinal tissue to identify how transcript and protein levels for the exemplar candidates are altered in association with the high-risk allele at each locus (using scRNAseq, IHC, and ELISA), as well as assess other transcriptomic changes across the locus (and genome). We will also use BiT-STARR-seq, to identify SNPs in each locus capable of changing transcription. In Specific Aim 2 we will use AAV2 constructs in mice to manipulate expression levels of the exemplar candidate genes and study how dysregulation of these genes influences RGC health. With completion of these experiments, we will advance understanding the basic biology of glaucoma risk factors identified by GWAS. We will also identify gene regulation and relevant biological pathways associated with familial glaucoma risk, which are key to the development of new sight-saving therapies.

NIH Research Projects · FY 2026 · 2024-03

Project Summary/Abstract Adolescent brain development is sensitive to exposures and has impacts on neurobehavioral functioning. Insecticide exposure may impact executive and attentional brain circuits which mature during adolescence due to pubertal hormonal influences and these changes may manifest as ADHD-related symptoms. There are gaps in knowledge about how insecticide exposure affects the adolescent brain, when neurobehavioral vulnerability can undermine lifelong relationships, economic attainment, and overall health. While most general populations, including adolescents, are exposed to low levels of insecticides through non- agricultural uses and consuming fruits and vegetables, occupational populations are known to have substantially higher levels of exposure. Therefore, we have worked extensively with a cohort of Egyptian adolescent pesticide- applicators who are occupationally exposed to insecticides (α-cypermethrin and chlorpyrifos). Increased ADHD symptoms and altered neurobehavioral performance in this cohort correlate with α-cypermethrin and chlorpyrifos toxicological burden. However, moderators and mechanisms underlying these associations remain undefined. Our pilot data indicate that combined adolescent exposure leads to altered neurobehavior and dopamine systems in the brain in mice. The effects in pesticide applicators are present many months after exposure. The epigenome is a potential mediator of the relationship between exposures and long-term neurobehavioral effects due to its unique sensitivity to the environment and potential to regulate gene expression throughout the lifespan. Our overall hypothesis is that combined adolescent insecticide exposures impact neurobehavioral outcomes. We additionally hypothesize that effects are modified by testosterone level, act via oxidative stress in dopamine neurons, and have a long-term trajectory via epigenetic change. We will test these with three synergistic aims joining human investigation and animal mechanistic assessments with translation between them, facilitated by a transdisciplinary investigator team, novel approaches, and a well laid-out consortium plan. In Aim 1, we will leverage stored samples and data from adolescent pesticide-applicators to quantify the neurobehavioral impact of combined versus single insecticide exposures, assessing the high- reward, high-risk hypothesis that pesticide-applicator testosterone level moderates this association. In Aim 2, we will model these adolescent insecticide exposures in mice to examine causality and use manipulations to test oxidative stress as a mechanism and testosterone level as a moderator of effects. Cell culture studies will provide convergent results for the role of dopamine neurons. In Aim 3, we will examine DNA methylation across both human and mouse samples collected after completion of peak insecticide exposure, to examine the potential epigenetic mechanisms translationally of long-term neurobehavioral risk. This work is the first step to accelerate translation of scientific research into meaningful improvements in human health— namely improving insecticide exposure policies and developing interventions to protect youth.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Candida albicans is both a component of the human mycobiome as well as one of the most important human fungal pathogens, capable of causing disease in both immunocompetent and immunocompromised people. The pathogenic cycle that leads to invasive C. albicans disease can be divided into three steps: 1) transition from mucosal colonization to sub-epithelial invasion; 2) filamentous morphogenesis within the sub-epithelium leading to tissue damage; and 3) intravascular dissemination to target organs. Infections that stop after the first two steps lead to mucosal diseases such as oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis while those that progress to the third step cause life-threating candidemia and deep organ disease. Although all three steps are required for the development of invasive disease, the third has been the most extensively studied through the widely used tail-vein inoculation mouse model of disseminated infection. In this application, we propose to use our barcoded collections of transcription factor (TF) and protein kinase (PK) mutants in competitive fitness assays to genetically define the global regulatory networks and key downstream effectors required for OPC mucosal infection (Aim 1). This aim will be facilitated by our recent development of an in vivo RNA-seq approach to characterize the transcriptional profile of C. albicans during OPC. In Aim 2, we will characterize the functions of the TF networks and identify the upstream PK signaling pathways that regulate in vivo filamentation. We will identify PK substrates critical for in vivo filament initiation and elongation. The successful execution of these aims will provide an unprecedentedly detailed in vivo profile of the regulatory mechanisms for the first two steps in C. albicans pathogenesis. Since PKs have emerged as important therapeutic targets across multiple areas of medicine, identification of PK networks with critical roles in C. albicans pathogenesis also holds promise to provide new insights with relevance to antifungal drug development.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Allergic disease is a chronic inflammatory state where CD4+ T cells manifest a biased T helper 2 (Th2) cell differentiation and effector phenotype. The polarization and ultimate effector function requires timed and transient intracellular signaling regulated by positive and negative hemostatic factors. One such negative factor we recently identified is the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Both asthma and allergic bronchopulmonary aspergillosis (ABPA) are more common in patients with cystic fibrosis (CF, the disease due to mutations in CFTR) and represent an exuberant CD4+ Th2 cell inflammatory response, suggesting that CD4+ Th2 cells may be important in CF airway disease. We have studied the adaptive inflammatory response to Alternaria alternata, a ubiquitous fungal aeroallergen that is associated with severe asthma in Cftr-/- mice and found that compared to Cftr+/+ mice, Cftr-/- mice produce higher levels of the Th2 cytokines, IL-5 and IL-13. Moreover, Cftr-/- CD4+ T cells have increased in vitro Th2 polarization and cytokine production compared to Cftr+/+ CD4+ T cells, suggesting that the absence of functional CFTR produces an exaggerated Th2 response. Furthermore, we have found that the master regulator of Th2 cell differentiation and function, GATA3, binds to the murine Cftr promoter following CD4+ T cell activation. Based upon these preliminary results we hypothesize that CFTR functions to inhibit CD4+ T cell Th2 polarization and cytokine production, and that CFTR expression is driven by GATA3 transcriptional activity. In Specific Aim 1 we will determine the role of CFTR in negatively regulating CD4+ Th2 polarization and effector function. We will define whether CFTR signaling is sufficient to inhibit Th2 polarization using CFTR overexpression in murine naïve CD4+ T cells and whether endogenous CFTR expression inhibits Th2 cytokine production in Th2 polarized murine CD4+ T cells. Specific Aim 2 will determine the importance on GATA3 binding on CFTR expression. We will delineate whether GATA3 overexpression or transcriptional repression is sufficient or necessary for CFTR expression, respectively. In Specific Aim 3 we will determine if CFTR modulation using recently FDA approved small molecules capable of correcting or potentiating mutant and wild-type CFTR, 1) decreases Th2 polarization and/or effector function in healthy or CF human CD4+ T cells, 2) reduces Alternaria alternata induced adaptive inflammation in a murine model expressing CFTR modulator-sensitive human CFTR, and 3) decreases type 2 inflammatory biomarkers (IgE and eosinophils) in individuals with CF post-CFTR modulator therapy compared to pre-CFTR modulator levels. Together these studies hold promise of elucidating the importance of CFTR in CD4+ T cell biology and may represent a novel therapeutic approach to Th2 mediated allergic disease. Importantly, this award will provide training in immunology from world-renowned immunologists and cellular biologists to further my long-term goal of becoming an independent physician scientist with expertise in pulmonary manifestations of CD4+ T cell mediated allergic disease.

NIH Research Projects · FY 2026 · 2024-02

Significant advances in prevention and mitigation of cardiovascular disease have been made, but related morbidity and mortality remain high. Cardiac conditioning is a therapeutic approach to induce endogenous protective adaptive responses in the heart for counteracting myocardial loss. Identification of new, widely applicable, practical, and effective strategies to induce cardiac conditioning continue to be a priority. Exercise is one of the most powerful modifiers of cardiovascular risk, with proven benefits for both healthy and diseased hearts. One key component of exercise is episodic heart rate (HR) acceleration interspersed with periods of rest. The degree and pattern of HR acceleration informs the benefit of physical activity, with a HR increase to 75-85% maximum predicted by age, for 75-150 min/wk, divided over 3-5 d/wk, being a typical recommendation for promoting cardiovascular conditioning. Conversely, an impaired HR response to exercise is associated with adverse clinical outcomes in healthy adults and those with heart disease. Furthermore, our recently published findings indicate that an episodic exercise-similar envelope of HR acceleration can serve as a trigger for myocardial conditioning and ischemic stress resistance. Specifically, in mice sham intervention was compared to exercise-similar episodic HR acceleration, delivered by an atrial pacing protocol with preserved atrioventricular and interventricular synchrony. The episodic exercise-similar pacing envelope improved myocardial ischemic stress tolerance with an effect size similar to that afforded by treadmill exercise or ischemic preconditioning. Hearts from paced mice displayed changes in Ca2+ handling, coupled with changes in transcriptional and posttranslational remodeling associated with a cardioprotective paradigm. In human subjects with already-implanted pacing devices for treatment cardiomyopathies with chronic, medically refractory systolic heart failure, and left ventricular ejection fraction (EF) <=35%, the exercise-similar pacing pattern, delivered through manual programming, was hemodynamically and symptomatically well- tolerated. Furthermore, preliminary results in subjects who underwent pacing intervention 3 days/week over 4 weeks indicate increased 6-min walk distance, as well as trend towards improved EF, vs. sham intervention. Building on these data, the proposed project will further delineate the mechanisms for myocardial conditioning promoted by the exercise HR envelope using two synergistic but independent approaches: 1) Study of wildtype and genetic mouse models to probe, dissect and validate molecular mechanisms underlying cardioprotection driven by an exercise HR pattern, and 2) Leveraging the compatibility of the proposed intervention with existing clinical pacing platforms to establish a mechanistic link between exercise’s HR pattern and cardiac conditioning in human subjects with cardiomyopathies. If successful, the results will lay the groundwork for expanded use of existing pacing hardware as a novel treatment to improve functional status and quality of life, reduce the risk of cardiovascular events, and promote engagement in physical activities.

NIH Research Projects · FY 2025 · 2024-02

Project Summary Relapse to cocaine use, and the underlying failure to inhibit cocaine-seeking behavior, remain significant barriers to the treatment of cocaine use disorder. However, our understanding of the neural pathways and network mechanisms underlying the inhibition of cocaine seeking remain insufficient. Previous studies investigating fear conditioning and drug seeking suggest that, often, the infralimbic cortex (IL) inhibits and the prelimbic cortex (PL) promotes such behaviors. Although downstream projections from these brain regions have been identified in the involvement of regulating fear conditioning and drug seeking, it is unknown what inputs to these regions determine their influence over cocaine-seeking behavior. Evidence from fear conditioning studies suggests that separate subpopulations of basolateral amygdala (BLA) neurons project to the PL vs. IL and oppose each other for control over fear conditioning vs. the extinction thereof. These findings raise the possibility that the BLA is a key region influencing PL and IL activity and determining the degree of cocaine-seeking behavior. Indeed, global manipulations suggest that the BLA promotes cocaine seeking as well as fear conditioning, but more recent reports suggest the BLA may also have a role in extinction and inhibition of drug seeking. However, whether the BLA separately regulates the IL and PL to extinguish or engage in cocaine seeking and how these regions engage in complex network interactions during cocaine seeking has never been investigated. Therefore, the goal of this proposal is to examine BLA→IL and BLA→PL pathways in cocaine extinction learning using optogenetics (Aim 1) and examine network activity in the BLA, IL, and PL simultaneously during cocaine seeking via multi-site electrophysiological recordings (Aim 2). The overall hypothesis is that the BLA-IL system is critical for the extinction of cocaine seeking, whereas the BLA-PL system opposes this effect and that neural activity between these systems during cocaine seeking will reflect the degree of cocaine-seeking behaviors. The findings from these experiments will provide fundamental knowledge regarding the neural systems that regulate cocaine-seeking behaviors. The sponsor (Dr. Ryan LaLumiere) and co-sponsor (Dr. Rainbo Hultman) for this proposal are leaders in the field of drug-seeking behavior and mood disorder, respectively, and have expertise in all techniques needed for the proposed research. Moreover, the University of Iowa and the Psychological and Brain Sciences Graduate Program provide an excellent environment and resources for neuroscience research. The training plan for this work includes training in the technical and conceptual skills for the proposed research along with development of teaching and mentoring skills, networking through neuroscience conferences, scientific writing and presentations. Therefore, the applicant will receive first- rate training that will propel her forward in her career and help to achieve her goals of becoming an independent investigator.

NIH Research Projects · FY 2026 · 2024-02

Substance use disorders affect ~15% of the population, with sex differences in all stages of substance use. Female sex-steroid hormones explain some of the disparity, such as accelerated transition from casual use to addiction, as estradiol produces sex-specific differences in rodent learning and cocaine self-administration. Prolonged cocaine use is known to engage dorsal striatal circuits. Synaptic plasticity in such circuits is critical for a variety of types of reward learning, highlighting the potential role such plasticity could play in substance use disorders. Thus, understanding sex differences in cocaine use requires determining how estradiol impacts molecular signaling and synaptic plasticity in the dorsal striatum. Our preliminary results show that estradiol, acting at estradiol receptor type α, impairs long term potentiation (LTP) in dorsomedial striatum (DMS) in estrous females; however, the cell type in which estradiol acts has yet to be identified. Striatal spiny projection neurons (SPNs) are either direct pathway SPNs, which promote action, or indirect pathway SPNs, which inhibit action, and changes in these SPNs are critical for various behavioral consequences. Thus, it is essential to understand LTP deficits in both direct and indirect pathway SPNs. In this proposal, we will test the hypothesis that estradiol impairs LTP in indirect pathway SPNs in the DMS. LTP in the dorsal striatum critically depends on activation of extracellular regulated kinase (ERK), which also is modified by both cocaine and estradiol. Estradiol enhances cocaine-mediated dopamine release and interacts with metabotropic glutamate receptors to modify ERK activation, but an unbiased approach is needed to determine whether estradiol impacts other signaling pathways. Our preliminary results have identified several signaling pathways that are modified by estradiol; however, a critical question is how cocaine interacts with estradiol to modulate these signaling pathways. We propose cutting-edge molecular and transgenic approaches combined with novel computational modeling to test the hypothesis that estradiol-mediated changes in gene expression impair LTP and to determine how cocaine further modifies the signaling pathways underlying synaptic plasticity. In Specific Aim 1, we perform electrophysiology in transgenic mice to determine whether LTP is impaired by estradiol in one or both SPNs. In Specific Aim 2, we use innovative techniques of single nuclei RNA sequencing, translating ribosome affinity purification followed by RNA sequencing and spatial transcriptomics to identify signaling pathways modified by estradiol and cocaine self-administration in a cell-type specific and spatial manner. In Specific Aim 3, we use innovative, data-driven modeling of signaling pathways followed by model-driven experiments to causally test which interactions between critical signaling pathways produce estradiol-mediated deficits in LTP. Successful completion of the proposed research will delineate how estradiol influences synaptic plasticity in dorsal striatum, including in conjunction with cocaine, and provide a foundation for future work to understand sex differences in reward learning and the consequences of cocaine use.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Differentiating Takotsubo syndrome (TTS) from acute myocardial infarction (AMI) is often challenging in real time. Meanwhile, the poor phenotypic grouping of TTS patients prevents investigation of effective therapeutic strategies for long-term risk reduction. Built on our current study that already yielded an accurate and broadly applicable spatiotemporal deep convolution neural network (DCNN) for echocardiographic diagnosis of TTS, in this proposal, we plan to augment and optimize a DeSeg-TTSD algorithm using large-scale multi-institution and multi-vendor echocardiographic datasets, as well as clinical metadata to increase the robustness, generalizability, and interpretability of deep learning modeling in real-time imaging analysis. We also plan to develop and validate a DeSeg-TTSP model for TTS prognostication and phenomapping by extracting latent spatiotemporal features correlating with TTS pathophysiology and outcome, so as to develop personalized treatment. We hypothesize that, when trained on an echocardiographic video task, a spatiotemporal DCNN can unlock sub-visual predictive information with advanced learning and computational analysis, to discover distinctive myocardial motion patterns and assimilate latent spatiotemporal imaging features to improve the accuracy of diagnosis and prognostication for TTS patients. The proposed research project brings together multiple innovations: It is based on a solid scientific premise, builds on already achieved results and extends state of art of spatiotemporal deep learning modeling in real time imaging, to provide decision support for clinical diagnosis and prognostication using imaging information routinely available in daily practice. Other than our local research team assembling imaging engineering experts, cardiologists and statisticians, an inter-institutional team including 57 board-certified expert human readers will perform human classification, data visualization and evaluate the feasibility of the application and integration into a clinical setting of the established and validated DL model. We will fulfill the following specific aims 1: Develop and validate fully automated spatiotemporal DL diagnosis models from echocardiographic videos that enable discrimination of TTS from AMI in real time. 2: Integrate spatiotemporal DL prognostication into clinical prediction models to endorse long-term TTS prognostication. 3: Perform data visualization on spatiotemporal DL framework and investigate TTS pathophysiology to develop personalized treatment Establishing this spatial-temporal hybridized deep learning framework will become a foundation for the development of additional precision medicine decision support tools for patients with acute cardiovascular disorders to address urgently-needed diagnostic decisions, resolve time-sensitive therapeutic dilemmas and obtain advanced imaging markers to develop specific primary and secondary prevention strategies.

NIH Research Projects · FY 2025 · 2024-01

Abstract Voltage-gated sodium channels (NaVs) maintain the electrical cadence in cardiac muscle tissues by selectively controlling the rapid inward passage of sodium. The NaV complex is comprised of a 260-kDa pore- forming α-subunit (encoded primarily by NaV1.5 in heart) that partners with β -subunits (β1-β4) comprised of a single transmembrane segment and exofacial immunoglobulin (Ig) fold. These β-subunits belong to a larger family of β/MPZ proteins that includes other single pass Ig proteins such as MPZ(P0). Defects in sodium channel function resulting from inherited mutations in either the α or β subunits are established causes of human disease, and are associated with sudden infant death, atrial fibrillation, reperfusion and ischemia injury, arrhythmia in the failing heart, epilepsy, and a variety of pain-causing syndromes. Other forms of heart disease that develop later in life and that are exacerbated by obesity are also characterized by altered sodium channel activity, in particular the inability to quickly and completely inactivate during the course of the cardiac action potential. The β-subunits have been proposed to regulate essentially every aspect of the pore-forming α subunit; including protein complex trafficking and turn-over, voltage-dependent function, and pharmacology. However, the molecular bases for these wide-ranging effects are poorly resolved primarily because the ‘gold standard’ heterologous cells that are used for ion channel characterization and high-throughput screening exhibit near ubiquitous expression of β-subunits, and their near relatives. As such, the variability amongst expression systems has stymied systematic study of β-subunit function. This in turn has prevented translational studies to examine the effect of various β subunit disease-associated mutants as well as efforts to identify drugs that may specifically modify specific α/β NaV complexes. We recently generated a CRISPR-modified human haploid cell line that lacks multiple members of the β/MPZ family including β1-β4. Electrophysiological experiments with these cells have revealed new emergent properties of NaV1.5 in the absence and presence of β subunits. These data provide the proof-of-concept that mammalian cells lacking the β/MPZ family will provide a powerful and needed way to specifically study many aspects of particular NaV α/β complexes, allowing better fundamental structure/function studies, better understanding of how disease-causing mutations in both α and β subunits cause pathology, and more precise tools for drug screening and drug safety profile testing.