University Of Minnesota

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS), a heterogenous group of clonal hematopoietic stem cell disorders, are an acquired bone marrow failure syndrome. MDS is characterized by ineffective hematopoiesis resulting in peripheral blood cytopenia and progenitor expansion. Genes encoding for RNA splicing factors (U2AF1, SF3B1, SRSF2, and ZRSR2) are frequently mutated and occur in the founding clones of MDS, representing a unique class of genetic vulnerability for targeted therapy. However, despite the prevalence of spliceosome mutations, how such mutations impact different cellular mechanisms are largely unclear. Recent studies by us and others suggest that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single- stranded DNA, are a source of genomic instability induced by different spliceosome mutants. In the preliminary studies leading to this application, we find that PARP1 is activated by R-loops and it plays a key role in suppressing R-loop-associated DNA damage. Furthermore, we show that MDS-associated RNA splicing factor mutations promote R-loop accumulation and render cells sensitive to PARP inhibition. These exciting findings lead us to hypothesize that PARP1 is a key sensor of R-loops and a critical suppressor of R-loop-associated DNA damage. Furthermore, aberrant R-loop accumulation represents a new targetable vulnerability in MDS- associated splicing factor mutant cells, making PARP inhibition an attractive way to target R-loop vulnerability in MDS. Finally, since PARP inhibitors achieved limited FDA approval in different diseases, repurposing PARP inhibitors to treat MDS patients harboring RNA splicing factor mutations may provide the fastest route to translate our findings to the clinics. To test these hypotheses, in Aim 1, we will elucidate mechanisms by which PARP1 is activated by R-loops. In Aim 2, we will identify global PARP1 substrates and R-loop distribution landscape in U2AF1-mutant cells, providing a proteomic and genomic view of how PARP1 regulates R-loops. In Aim 3, we will evaluate whether PARP inhibitor, olaparib, can selectively eliminate MDS-associated splicing mutant cells in vitro and in vivo. Together, these studies will mechanistically explain how R-loops are sensed by PARP1 in splicing mutant cells, reveal how PARP1 guards cells against R-loop-associated genomic instability, and address whether R-loop-associated vulnerability in spliceosome-mutant MDS cells can be exploited by PARP inhibitors as targeted MDS therapy. The combined expertise in R-loops, PARP1, DNA damage response and spliceosome mutations in MDS (Nguyen laboratory), PARP regulation by proteomic approach (Leung laboratory, co-I), and MDS GEMM mouse models of U2AF1 and SRSF2 mutations (Lee laboratory, co-I) provides us the unique opportunity to characterize PARP1 function in cells expressing MDS-associated mutations. These studies will not only significantly advance our understanding of R-loop biology and PARP1 signaling, but also repurpose the use of FDA-approved PARP inhibitors in targeted therapy for MDS patients harboring RNA splicing factor mutations by exploiting R-loop-associated vulnerability.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Reversible serine/threonine phosphorylation of proteins plays an essential regulatory function in numerous cellular processes. Type 2C protein phosphatases (PP2Cs) comprise a major class of Ser/Thr phosphatases (PPases), and defects in several human PP2Cs have been implicated in cancer, diabetes, cardiovascular disease, neural disorders, and stress signaling. However, major gaps exist in understanding how PP2C enzyme activity is regulated and what specific proteins and processes are under PP2C control. In plants, PP2C.D PPases inhibit organ growth by repressing cell expansion. In part, this is accomplished by dephosphorylation of a key regulatory phosphosite of plasma membrane (PM) H+-ATPases. The growth hormone auxin stimulates cell expansion by inducing expression of Small Auxin Up RNA (SAUR) genes, which encode novel proteins that bind to PP2C.D PPases to inhibit enzymatic activity. The long-term goal of this project is to thoroughly understand the molecular mechanisms underlying auxin-mediated control of plant growth and development. More specifically, the work described in this proposal will identify regulators and downstream effectors of SAUR-PP2C.D signaling hubs involved in auxin-mediated cell expansion and the integration of volumetric changes with diverse cellular processes to yield a coordinated growth response. Phosphoproteomic studies have identified >140 proteins exhibiting altered phosphorylation in response to auxin. This dataset overlaps substantially with phosphoproteins affected by SAUR overexpression, implicating SAUR-PP2C.D modules as major regulators of the auxin phosphorylome during cell expansion. Using the powerful genetic system of the model plant Arabidopsis, the proposed studies will investigate the functional roles of select phosphoprotein candidates in auxin-induced growth and their regulatory interactions with SAUR- PP2C.D modules. Detailed analysis of auxin’s regulation of PM H+-ATPase activity will also be conducted. Auxin both inhibits H+-ATPase dephosphorylation via SAUR repression of PP2C.D activity and stimulates activation by promoting ATPase phosphorylation by TMK1 and additional kinases, including orthologs of WNK and SPAK/OSR kinases implicated in mammalian cell size control. All of these kinases interact with one another and PP2C.D PPases, and research will address how kinase and phosphatase activities are coordinated and mutually regulated. This work will elucidate PP2C functions and regulatory mechanisms, identify PP2C.D effectors that modulate cell expansion, and illuminate how auxin coordinates diverse cellular processes to control cell size. Given the conservation of PP2C function across kingdoms and the universal process of cell size control, project findings will have broad impact, including implications into human development and disease. Further, as humans depend on plants for sources of food, fiber, and pharmaceuticals, the proposed studies will elucidate plant growth control by SAUR-PP2C.D regulatory modules and facilitate novel strategies for manipulating plant growth to benefit human health.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT Social isolation (SI) during childhood increases the susceptibility to neuropsychiatric disorders, including anxiety disorders, depression, and cognitive impairments. Limited treatment for these disorders highlights the importance of identifying new therapeutic targets. Recent evidence has underscored the role of the cerebellum in early-life stress. For example, the neonatal cerebellum contains the highest level of glucocorticoid receptor (GR) in the entire brain, indicating that the cerebellum is enriched in the molecular machinery for processing the stress response. The cerebellum is extensively connected to brain networks that are sensitive to psychological stress. However, whether and how SI stress regulates gene expression in the cerebellum to result in cerebellar dysfunction and maladaptive behaviors remain elusive. To address the knowledge gap, we isolated experimental mice in singly housed cages. They displayed behavioral changes reminiscent of high anxiety, depression, and social memory loss. Moreover, we found that SI impaired intrinsic excitability of Purkinje cells (PCs), the sole output neurons in the cerebellar cortex. And cerebellar gene expression was highly responsive to stress stimuli such as an elevation of corticosterone, a stress hormone, in rodents. These findings fuel our central hypothesis that SI impairs the cerebellar output activity by specifically affecting the intrinsic excitability of PCs; and restoring PC excitability rectifies SI-caused behavioral deficits via the cerebello-cortical networks. To test the hypothesis, we propose a multidisciplinary approach with three specific aims: (1) Determine the molecular basis of reduced PC intrinsic excitability by SI. We will employ two genome-wide RNA sequencing techniques to obtain an unbiased view of transcriptional signatures and epigenetic modifications of SI as well as to identify SI-responsive ion channels in PCs, e.g., Kv1.5. PC-specific knockout of GR will uncover the GR-dependent genomic reprogramming by SI. (2) Define the significance of PC activity in systemic response to SI. Using viral gene transfer, we will gain precise spatiotemporal control of PC excitability to test the necessity and sufficiency of cerebellar activity in mediating the system-wide response to SI. (3) Specify the cerebellum-cortex gateways underlying maladaptive behaviors of SI. Our efforts will be focused on dissecting the neural circuits that connect the cerebellum to the downstream sub/cortical areas and their contributions to the behavioral phenotypes of SI. Completion of this work will advance our understanding of the molecular, cellular and circuitry mechanisms underpinning the non-conventional role of the cerebellum in the stress response, and the results will ultimately help develop novel therapeutic strategies to improve mental health.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Local sources of Shiga toxin-producing Escherichia coli (STEC) contribute significantly to disease risk; however, inability to differentiate local from non-local cases has precluded full characterization of local transmission systems. The long-term goal of this research is to develop targeted public health interventions using systems epidemiology to elucidate the pathways and mechanisms of STEC maintenance and transmission. In pursuit of this goal, the overall objective of the current study is to identify characteristics of pathogen, host, and environment associated with local STEC transmission. The central hypothesis is that STEC cases infected from local sources are significantly different than those infected by strains from outside the case’s local area. The central hypothesis will be tested by pursuing three specific aims: 1) differentiate and characterize locally transmitted STEC strains, both O157 and non-O157, 2) identify host characteristics associated with acquiring local vs. non-local STEC strains, and 3) identify environmental characteristics associated with local transmission. In aim 1, a structured coalescent phylodynamic model will be used to generate a phylogeny of STEC strains isolated from cases reported to the Minnesota Department of Health (MDH) since 2016 compared to strains isolated outside MN and available on NCBI. The inferred location of tree nodes will be used to classify STEC strains as local or non-local. A generalized linear model will be used to integrate strain characteristics into the tree and determine their influence on the local MN STEC effective population size and migration rates. The second aim will assess the association of host characteristics, including age, sex, and potential exposures, with local vs. non-local STEC. In aim 3, the association between characteristics of the physical and social environment and local STEC transmission will be estimated accounting for spatial correlation. To accomplish these aims, PI Dr. Gillian Tarr will obtain advanced training in bioinformatics and phylodynamic modeling. Dr. Tarr will also enhance her knowledge of food production and distribution systems and further develop her research management skills. With a long history of food safety research and collaboration with MDH, the University of Minnesota provides the optimal environment for this research. The mentorship team has expertise in bioinformatics and applied phylogenetic modeling and includes STEC and food systems subject matter experts. The proposed research is innovative, in the applicants’ opinions, because it will 1) characterize local transmission systems without restriction to isolated outbreaks or use of proxies such as recent travel, and 2) employ a structured coalescent model that has not been applied for this purpose in any comparable disease system. Differentiating local transmission from imported cases and identifying the host, pathogen, and environment characteristics of local transmission is a significant contribution, because it enables specific hypotheses to be developed and tested for local reservoirs and transmission pathways, which can then be targeted by tailored interventions.

NIH Research Projects · FY 2025 · 2022-05

Project Summary Spinocerebellar ataxia type 1 (SCA1) is one of nine fatal inherited neurodegenerative diseases caused by expansion of an inframe CAG trinucleotide repeat. Each repeat tract encodes a stretch of glutamine residues in the affected protein, in the case of SCA1 the protein is ataxin-1 (ATXN1). Symptoms of SCA1 include loss of motor coordination and balance, slurred speech, swallowing difficulty, spasticity, and some cognitive impairment. A characteristic feature of SCA1 pathology is atrophy and eventual loss of Purkinje cells from the cerebellar cortex. Like many neurodegenerative disorders, SCA1 is typically a late onset disease suggesting that physiological changes due to aging contribute to the onset of the disease. There is currently no effective treatment. Identifying signaling pathways and cellular mediators of SCA1 pathogenesis in the cerebellum leading to ataxia and in the brainstem that underlie lethality are critical in the search for therapeutics and are the focus of the research outlined in this application for continued support.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Effective cell-based immunity depends on two major systems: (i) the ability to generate a diverse TCR repertoire capable of recognizing antigens from previously unencountered pathogens, and (ii) the ability to discriminate between self- and non-self-antigens and prevent autoimmunity. Inappropriate balance between these two systems causes a variety of disease states including ineffective tumor immune surveillance and poor pathogen clearance due to gaps in the TCR repertoire, or the onset of autoimmunity and off target immunopathology during infection. Autoimmunity can be curtailed by deleting autoreactive TCRs or diverting them into the Treg lineage in the thymus. However, given the high degree of cross-reactivity of TCRs this process cannot be truly efficient at removing all self-reactive TCRs as this would remove much of the potential diversity of the conventional TCR repertoire, thereby impairing immunity to pathogens. Thus, a key biological question is how thymic selection functions to balance protection against autoimmunity while providing effective immunity against pathogens. We hypothesize that a unique population of thymic recirculating Treg cells (RT-Treg) act as a rheostat to decrease the stringency of selection once peripheral Treg cell tolerance is established, thereby allowing for a more diverse and pathogen-reactive conventional immune system. This will be examined in two specific aims: Aim 1: Define RT-Treg heterogeneity and functional consequences of RT-Treg accumulation. Aim 2: Identify the mechanisms by which RT-Treg cells affect immune repertoires and mTEC numbers. The proposed experiments will elucidate the role that RT-Treg play in governing the stringency of central tolerance, both promoting effector cell diversity and ensuring maintenance of self-tolerance by Treg cells.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Prostate cancer (PCa) is the second leading cause of cancer related death in American men. Typically, treatment for PCa involves blocking androgen synthesis or androgen receptor (AR) signaling known as androgen deprivation therapy (ADT). Although initial response rates are promising, all men eventually progress on ADT and develop castration resistant prostate cancer (CRPC) concomitant with metastatic burden. Metastatic CRPC is incurable and an increasing number of men are developing a highly lethal variant of CRPC known as aggressive variant prostate cancer (AVPC). AR signaling is lost in AVPC rendering the existing hormone targeting treatments ineffective. About a third of AVPC tumors also express neuroendocrine (NE) genes and are classified as neuroendocrine prostate cancer (NEPC), which is the focus of our studies. Very few therapies exist and only offer minimal survival benefits in this setting. Hence, functional assessment of other protein drug targets is needed to effectively treat NEPC. We have previously shown that kinase signaling pathways may be promising therapeutic alternatives in CRPC. The goal of our research is to understand the mechanisms regulating increased kinase gene expression, leading to kinase pathway activation, and how to effectively target these kinases in NEPC. Our hypothesis is that RET mRNA and protein up-regulation is driven by ASCL1, a master neural transcriptional regulator, and that RET kinase-mediated activity serves as a therapeutic vulnerability in NEPC. It is known that RET mutations are key drivers and therapeutic targets in other cancers with NE features such as papillary thyroid carcinoma and small cell lung cancer. Importantly, the contribution of RET kinase signaling in NEPC viability is not entirely elucidated. Our preliminary data shows that RET kinase is overexpressed via activation of a neuronal differentiation transcription factor, ASCL1, and enzymatically activated in mouse models and organoids of NEPC, in human cell lines, and clinical NEPC tumors. Our work also shows that RET kinase inhibition reduces in vivo xenograft tumor growth and in in vitro mouse organoid models of NEPC. The goals of this project are to: 1) confirm ASCL1 as a direct transcriptional regulator of RET, 2) define a RET activity signature and assess the association of this signature to treatment resistance and NEPC in clinical samples, and 3) optimize co-targeting strategies with novel RET inhibitors in NEPC model systems. These goals are collectively designed to investigate the mechanism of RET kinase as a key therapeutic target in NEPC, an incurable variant of PCa. While kinase inhibitors are approved for treatment of several epithelial cancers, clinical trials of kinase inhibitors in PCa have been disappointing. Our data indicate this is likely due to lack of appropriate patient stratification, administered in the wrong clinical context, and improper combination therapies. Hence, the outcomes of the proposed work will provide new insights into select combination therapies for treating NEPC, using re-purposed kinase inhibitors that may be implemented quickly in clinical trials of patients with this subset of lethal PCa.

NIH Research Projects · FY 2025 · 2022-05

Kinases are a central hub for signaling in multiple disease settings, including cancer, autoimmune disease, heart disease, and beyond. There is significant need for technologies that could streamline multiplexed measurement of kinase activities in live cells using high throughput screening and clinical lab-compatible read-outs for future translation. We develop these kinds of cell-based assay approaches, designing novel substrates tuned to particular read-out technologies and characterize them in vitro and in cell-based assays. Through prior work, we prototyped an in silico pipeline called KINATEST-ID, in which kinase substrate preferences are identified, cross- checked against other “off-target” kinases, then novel peptides are designed based on predicted compatibility with a read-out technique and tested empirically. Our initial iterations of this pipeline have focused on tyrosine kinases and in vitro lanthanide fluorescence assay read-outs, which have limited multiplexability. Mass spectrometry detection of cell-deliverable kinase substrates that report kinase activity in live cells would provide far higher multiplexability, but in prior work we ran up against a fundamental physiochemical limitation of our design pipeline: selecting sequences for Tb3+ chelation produces substrates biased towards acidic amino acids (e.g. D, E) that ionize very poorly in standard MS analyses. We also found that while we could predict biochemical efficiency for kinases that had high-quality preference data available, prediction of selectivity is still inadequate because most kinases lack such data for cross-referencing against each other. These barriers have limited further progress on developing substrates for cell-based kinase profiling assays, particularly with multiplexed MS detection. Further, in order to be more robust for higher throughput analyses, the workflows for the assay, sample processing, and MS detection need to be simplified. In Aim 1, we will expand the KINATEST-ID platform functionality with a focus on MS detection. In Aim 2, we will develop multiplexed cell-based deliverable substrate kinase assays using targeted parallel reaction monitoring (PRM) MS methods in collaboration with colleagues at Cedars Sinai who are developing cutting edge, high throughput proteomics methods that are clinical lab- compatible. This work will produce an optimized platform for developing and implementing MS-compatible, cell-based assays enabling kinase activity profiling in live cells, as well as a path to new tools for understudied kinases. Overall, these tools will have high potential to impact both basic research for rapid profiling of signaling activities, and kinase inhibitor drug discovery with eventual translation to the clinic.

NIH Research Projects · FY 2026 · 2022-04

Alphaviruses, a group of enveloped RNA viruses, cause persistent arthritis and encephalitis among horses and humans. Currently, no licensed vaccine or antiviral therapy is available to prevent or cure the disease. A critical step in enveloped virus infection is membrane fusion between the viral and the host cellular membranes, a process leading to cytoplasmic delivery of the viral genome. Alphaviruses enter the host cell via receptor- mediated endocytosis. The viral E1 protein mediates membrane fusion through conformational changes upon acidification in the early endosomes. The other viral envelope protein, E2, functions as a molecular chaperon for the E1 protein during virus assembly and plays a role in virus capsid core uptake at the budding site and release after membrane fusion. We have obtained exciting, unprecedented images that captured robust alphavirus membrane fusion events with target liposomes by cryo-electron microscopy. Using coordinated biochemical, biophysical, cryo-electron microscopy, and cryo-electron tomography methods, we will investigate the molecular structures of the E1 and E2 proteins in a prototype alphavirus, Sindbis virus, at specific fusion stages. We will test specific predictions about the alphavirus membrane fusion mechanism based on our structural results by generating and characterizing mutants of the glycoproteins. We will test the fusion function of key mutations in the context of recombinant viruses. Completing this research will advance our understanding about how alphavirus glycoproteins promote membrane fusion.

NIH Research Projects · FY 2026 · 2022-04

ABSTRACT Systemic and structural racism is a public health crisis. However, little is known about the impact of structural racism and discrimination (SRD) on the health and emotional well-being of individuals across the life course. While prior studies have shown associations between discrimination and negative health outcomes in adults (e.g., cardiometabolic disease, depression), these studies have been cross-sectional and primarily examined individual-level sources of racism and discrimination. Much more research is needed to fill gaps in our understanding about the relationship between SRD and health disparities before interventions can be developed. To significantly advance the field regarding SRD and health equity, studies need to include: (1) multi-level measures of SRD including individual (both intrapersonal and interpersonal), neighborhood, institutional, and societal/policy levels; (2) rigorous mixed-methods designs (e.g., ecological momentary assessment (EMA), biological measures, geographic information system (GIS) data, surveys); (3) multi-site samples with urban and rural participants; (4) a life course approach; (5) whole-person outcome measures (i.e., mental, physical, behavioral health); and (6) longitudinal study designs. Including these study elements will allow for comprehensively examining the relationships between SRD and health and emotional well-being to identify mechanisms to target in interventions to mitigate SRD. The main objective of the proposed study is to examine multiple levels (i.e., individual, neighborhood, institutional, societal/policy) of SRD and associations with mental, physical, and behavioral health outcomes across the life course to identify intervention targets to promote health equity. The proposed study is built on a prospective longitudinal cohort study of 627 racially/ethnically diverse families (i.e., African American, Hispanic, Native American, Immigrant/Refugee, White) across the life course (childhood, adolescence, adulthood/parenthood) from urban settings (i.e., Minneapolis, St. Paul). The parent R01 already has three time-points of mixed-methods data (i.e., EMA, GIS, survey) that includes discrimination and neighborhood segregation measures and physical and behavioral health outcomes carried out using a community-based participatory approach. For the proposed study, a sample of 300 racially/ethnically diverse families from rural Georgia (i.e., Athens) will be added to compare SRD experiences in urban versus rural settings. In addition, cardiometabolic and stress biomarker data (i.e., heart rate, blood pressure, waist circumference, lipids, HbA1C, cytokines) and multi-level measures of structural racism (i.e., individual, neighborhood, institutional, societal/policy) will be added at two time points, 18 months apart. The proposed study will be one of the first to prospectively measure multiple levels of SRD using mixed-methods across two sites and associations with mental, physical, and behavioral health disparities across the life course in diverse families. Results of the study will inform the development of an intervention targeting multi-level SRDs to promote health equity.

NIH Research Projects · FY 2025 · 2022-04

PROJECT ABSTRACT Executive function is a multifaceted construct that includes higher-order cognitive processes such as response inhibition, working memory, and goal selection. Executive abilities improve throughout adolescence, and deficits of executive function, or executive dysfunction, are a transdiagnostic feature of many psychiatric disorders that emerge during this period of development. Understanding the underlying neurodevelopmental mechanisms that contribute to executive dysfunction is a critical prerequisite for targeted interventions. Iron deficiency is the most common nutrient deficiency in the world and is a known source of executive dysfunction during the vulnerable windows of early childhood and adolescence. However, despite its prevalence and impact, the underlying neurobiological mechanisms are not fully understood. This proposal focuses on a potentially critical but under-explored mechanism linking iron deficiency to transdiagnostic executive dysfunction: brain iron deficiency. Aim 1 will define how brain iron deficiency during adolescence mediates the effect of peripheral iron deficiency on executive dysfunction. We will first investigate this relationship in a large community-based sample (n=9,500 with peripheral iron and cognitive assessment, n=1,601 with neuroimaging) and then replicate and extend the model to a sample that is enriched for individuals with psychiatric disorders. Aim 2 will use a prospectively collected sample of adolescents with and without a history of iron deficiency in routine screenings at 9-18monts of age to determine how iron deficiency across childhood and adolescence impacts brain iron deficiency and executive dysfunction in adolescence. Critically, this aim will inform a multi-hit model whereby iron deficiency across childhood and adolescence will be associated with greater brain iron deficiency and thus more severe executive function than having iron deficiency in only one of the two time periods. Finally, Aim 3 will examine how sex differences in peripheral iron deficiency impact sex differences in brain iron after the onset of puberty. Together, these aims will identify both the timing of vulnerability and the neurobiological mechanisms underlying executive dysfunction, thus informing translational models for targeted treatments and interventions. The support of this K99/R00 Pathway to Independence award will provide the applicant with the training necessary to achieve these aims, including training in developmental neuropsychiatry, cognitive assessment, quantitative magnetic resonance imaging, and advanced biostatistics. These training objectives will be accomplished with the support of an outstanding mentorship team, Drs. Satterthwaite, Gur, Witschey, Shinohara, Wehrli, and Georgieff, and the world class technical and intellectual resources of the University of Pennsylvania. Together, the proposed scientific aims and training objectives will form the foundation for an independent research program aimed at uncovering the neurodevelopmental mechanisms for executive dysfunction in youth with mental illness.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY Breast cancer growth and progression require complex interactions between tumor cells and their surrounding environment. Increased numbers of infiltrating inflammatory cells, in particular macrophages, correlate with poor patient prognosis in breast cancer. Our studies have focused on identifying key macrophage subpopulations that contribute to mammary tumor growth and progression. Based on studies in the normal mammary gland, we have identified a stromal macrophage population that is associated with remodeling hyaluronan in the extracellular matrix and is capable of promoting tumor cell invasion. These macrophages are localized specifically to hyaluronan-enriched regions in the peri-tumoral stroma. Based on our preliminary results, we hypothesize that this macrophage subpopulation represents a distinct tissue resident-derived tumor associated macrophage population that contributes to breast cancer progression through binding to and remodeling the hyaluronan-containing extracellular matrix. Studies proposed in Specific Aim 1 will define the localization and source of this macrophage subpopulation using mouse models of breast cancer. Studies proposed in Specific Aim 2 will delineate the key mechanisms through which these macrophages contribute to extracellular matrix remodeling and drive tumor cell invasion. Finally, studies proposed in Specific Aim 3 will define the localization of this macrophage subpopulation in human breast cancers and use spatial transcriptomics to identify the local environment surrounding these macrophages. Recent studies have highlighted the extensive functional diversity of macrophages in both normal homeostasis and in various diseases, including cancer. Delineating the specific mechanisms that contribute to macrophage heterogeneity and defining their functional contributions within the tumor microenvironment are critical for developing strategies to effectively target this diverse population of cells.

NIH Research Projects · FY 2024 · 2022-04

PROJECT SUMMARY Temozolomide (TMZ) chemotherapy is a key component of treatment for patients with newly diagnosed glioblastoma (GBM) and provides clinically meaningful survival benefits. Cytotoxicity from TMZ results from failure to repair TMZ-induced DNA methylation adducts. During replication, these lesions ultimately result in replication fork collapse associated with DNA double strand breaks that are critically repaired by homologous recombination (HR). In this context, we recently discovered that retinoblastoma binging protein 4 (RBBP4) functions in a complex with histone acetyltransferase p300 (p300) as a epigenetic writer to key DNA repair processes including six key HR genes (RAD50, BRCA1, BARD1 BRIP1 FIGNL1, and RAD51) that play different roles in HR pathway. Specifically, knockdown of either RBBP4 or p300 in glioma cell lines and GBM patient-derived xenograft (PDX) models results in marked suppression of these six gene products, impaired HR activity associated with enhanced sensitivity to PARP inhibitors, and dramatically enhanced sensitivity to TMZ in animal models. Downstream of RBBP4/p300 complex, bromodomain and extraterminal domain (BET) family members (BRD2, BRD3, BRD4) function as key readers of p300-mediated acetylation marks to drive gene expression. Based on this and our preliminary data, we hypothesize that the RBBP4/p300/BRD axis is a key regulator of HR efficiency and is a promising pharmacologic strategy for developing a robust, novel, TMZ- sensitizing strategy. There are both p300- and dual p300/BET-inhibitors now entering clinical testing in oncology, which highlight the importance of fully understanding how this complex functions to regulate DNA repair. We will explore this concept in a series of three specific aims: Aim 1: Define the role of RBBP4/p300 in regulation of HR genes. We will extend our initial observations in GBM43 to define the regulation of HR genes across multiple GBM models by this complex. Aim 2: Evaluate the impact of RBBP4/p300 on DNA repair proficiency. We hypothesize that coordinated suppression of multiple HR genes associated with disruption of RBBP4/p300/BET function results in profound HR suppression and TMZ sensitizing effects as compared to modulation of only one component of HR. Aim 3: Define the impact of targeting the p300/BRD4 axis on therapy response in GBM We will test the efficacy of p300 and p300/BET inhibitors alone and in combination with TMZ in PDX models. Ultimately, these studies are designed to provide a strong rationale to pursue these inhibitors in clinical trials for GBM.

NIH Research Projects · FY 2026 · 2022-04

Alzheimer’s Disease (AD) and AD-Related Dementias (ADRD) are projected to affect 115 million people worldwide by 2050. Vascular contributions to cognitive impairment and dementia (VCID)—a primary type of ADRD—is a major research focus for the NIH, which has called for the need for studies of prevention across the lifespan and in diverse populations (ADRD summit 2019). Underrecognized but relevant to VCID, prior research indicates that arrhythmias are associated with lower cognitive function and greater risk of AD/ADRD. However, current knowledge of arrhythmias’ neurocognitive impact is lacking in the fastest growing ethnic group in the USA—Hispanics/Latinos—who are anticipated to contribute disproportionately to the projected increase in prevalence of mild cognitive impairment (MCI) and ADRD. Thus, robust and comprehensive data on prevalence and neurocognitive impact of arrhythmias in Hispanics/Latinos are critically needed. Furthermore, from a prevention standpoint, although prior research shows that modifiable lifestyle and cardiovascular disease (CVD) risk factors are related to ADRD risk and arrhythmia risk and burden, it is unknown whether optimizing lifestyle/CVD risk factors can mitigate the arrhythmia-ADRD relationship. To address the foregoing knowledge gaps, we will apply the ZioXT Patch—a non-invasive ambulatory continuous ECG monitor—to ~5,000 participants (age ≥45 years) in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) in 2022-24 (concurrent with Visit 3) and leverage the rich data in HCHS/SOL and related ancillary studies: neurocognitive assessment, adjudicated MCI and ADRD, brain MRI, cardiac MRI, lifestyle/CVD risk factors including 7-day accelerometer-based physical activity, adiposity measures, and blood pressure. We hypothesize that in Hispanic/Latino adults, higher arrhythmia frequency will be associated with higher incidence of MCI or ADRD, greater cognitive decline, more brain infarcts and small vessel ischemic disease, and lower brain volumes. These associations will be independent of CVD and cardiac structure/function and will be more evident in those with less optimal (vs. more optimal) lifestyle/CVD risk factor trajectories. We will evaluate these aims: Aim 1: Define the prevalence and frequency of arrhythmias in Hispanic/Latino adults using ambulatory heart rhythm monitoring, Aim 2: Evaluate the association of arrhythmias with change in cognitive function and incident MCI or ADRD, Aim 3: Assess the association of arrhythmias with markers of vascular brain injury based on brain MRI. Importantly, for each Aim, we will examine the consistency of associations by antecedent 10-year lifestyle/CVD risk factor trajectories. This project will combine exceptional and complementary scientific expertise, wearable and imaging technology, rigorous epidemiological and analytical methods, and leverage an existing NIH cohort to refine the VCID paradigm and overcome critical barriers in AD/ADRD and minority health research, thus achieving a sustained and powerful impact on public health and clinical practice.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY The ultimate objective of this K01 proposal is to enable Dr. Ida Fonkoue to become an independent research investigator by 1) developing expertise in vascular, hormonal and sleep measures in humans; 2) acquiring scientific growth through a rigorous training plan, within an outstanding scientific environment that has a long tradition in translational vascular research; and 3) generating sufficient preliminary results to support an NIH R01 application. The candidate’s long-term goal is to build an NIH-funded research program in clinical and translational research in women’s health, studying derangements of vascular, neural and hormonal control, that contribute to the high rates of hypertension and cardiovascular disease (CVD) in women living with chronic stress exposure such as those with post-traumatic stress disorder (PTSD), generalized anxiety disorder or panic disorder. Over 7 million U.S. adults have PTSD, a disorder associated with a greater risk for hypertension and CVD. While healthy premenopausal women are relatively protected from CVD compared to men, a diagnosis of PTSD increases CVD risk in women by up to 3-fold. Understanding the mechanisms underlying CVD risk in women with PTSD is of paramount importance to develop intervention strategies aiming at protecting the future health of this vulnerable population. Based on our preliminary data, the working hypothesis of this project is that: PTSD inhibits nitric oxide bioavailability, resulting in decreased endothelial function, increased arterial stiffness and increased sympathetic activation; and that these changes are exacerbated by low estradiol levels and sleep disturbances. Aim 1 will identify alterations in SNS activity in premenopausal women with PTSD and determine if these alterations are a function of low E2 levels and sleep disturbances. Aim 2 will Identify alterations in vascular function in premenopausal women with PTSD and determine if these alterations are a function of low E2 levels and sleep disturbances. Emory University, where the PI’s entire mentoring team is located, boasts an intellectually rich research environment whose resources will be used to carry out the proposed research, including an NIH-funded Georgia Clinical and Translational Science Alliance (GA CTSA). During this K01 award, the PI will devote 75% effort to this project and career development-related activities as highlighted in her four years training and research plans. She will complete a Master of Science in Clinical Research offered by the GA CTSA and prepare future career development grant submission. This research project, combined with multidisciplinary mentorship, didactic education, and practical experience, will provide Dr. Fonkoue with the training and skills to become a successful independent investigator.

NIH Research Projects · FY 2026 · 2022-04

Summary As biotechnology advances, biomedical investigations have become more complex due to high-throughput and high-dimensional data collected at a genomic scale. Of paramount importance is unraveling the regulatory roles of genetic variants on genes and gene-to-gene regulatory relationships. On this ground, biomedical researchers can identify causal Single-Nucleotide Polymorphisms (SNPs) and genes for complex traits and neurodegenerative diseases such as Alzheimer's disease (AD) to develop treatment strategies. Given the urgent need to under- stand the progression and etiology of these diseases, particularly AD, the PIs propose to develop statistical and computational tools for accurate estimation and inference of gene regulatory networks, with a focus on AD and other complex traits. The project consists of two major components: estimation and inference of gene regulatory networks with SNPs as instrumental variables (IVs). The main thrust will be on causal network reconstruction and inference with IVs as interventions in the possible presence of invalid IVs and hidden confounders, with particular effort on high-dimensional data, in which the number of variables may exceed the sample size. Concerning causal network reconstruction, the project will develop novel methods of reconstructing gene regulatory networks as directed acyclic graphs describing casual relationships among the SNPs (interventions), genes, and traits such as AD. The project will develop high-dimensional inferential tools based on modified likelihood ratio tests and a data perturbation scheme to account for the uncertainty involved in a discovery process. Moreover, it will focus on hypothesis testing on (1) the directionality and strength of multiple (linear/nonlinear) causal relations and (2) the presence of a pathway of causal relations. Computationally, the project will develop innovative methods and algorithms for large-scale problems. For application, based on the reconstructed gene regulatory networks, we will first identify causal genes for AD and AD's risk factors, such as lipids, then infer which of the risk factors are (putatively) causal to AD.

NIH Research Projects · FY 2026 · 2022-04

ABSTRACT Despite major improvements, significant disparities in healthcare and outcomes exist in type 1 diabetes (T1D) in low vs high income countries. In our recent study of 68 African youth with T1D, patients were treated and educated by trained pediatric endocrinologists, performed self-monitoring of blood glucose (SMBG) an average of 2.1x/day, and had access to sufficient quantities of insulin. Average HbA1c was 11%. Blinded continuous glucose monitoring (CGM) demonstrated extremes of both hyper- and hypoglycemia. Glucose percent time-in- range (TIR, 70-180 mg/dl) was only 30%, and time-in-hypoglycemia (glucose <54 mg/dl) was 7%, with more than 80% of subjects spending ~ 2 hours a day hypoglycemic. Current practices are failing these children, who are at very high risk for diabetes acute and chronic complications. This RCT aims to improve T1D care in East African youth age 4-26 years by testing the hypothesis that enabling patients to continuously monitor glucose levels with flash CGM will improve glucose TIR, and that this therapy will be cost effective in the setting of a low-resource country. All subjects will receive identical monthly diabetes education. For the first 6 months, half of patients (n=90) will be given unblinded flash CGM so they can see their glucose levels in real time, while half (n=90, the control group) will perform ≥3x daily self-monitoring of blood glucose by fingerpoke (SMBG) while wearing a blinded CGM for endpoint measurement. The primary outcome measure, TIR, is assessed at 6 months. After 6 months, the unblinded CGM cohort will continue on this treatment for another 6 months to assess the impact of 1 year of unblinded CGM therapy. The control group will switch to unblinded CGM months 6-12. All patients in this study, including those in the control group, will receive more intensive education, greater attention from the diabetes team, and more test strips than are commonly available today. If this approach results in similar levels of improvement in glucose TIR in control subjects compared to patients who also receive CGM, this study will have performed an important service by demonstrating that there is no need for CGM therapy and that more focus, instead, needs to be placed on patient education and interaction with the medical team. But if CGM leads to significantly greater improvement in diabetes metabolic control by reducing hyper-and hypoglycemia, then the ethical question is not whether to provide this therapy in resource poor settings, but how to make it affordable. Such decisions must be guided by data obtained from children in the specific and unique settings found in low income nations. The goal of this protocol is to obtain these data.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY AND ABSTRACT Many neurological and psychiatric disorders are essentially connectionist disorders: certain sets of neurons have abnormally increased or decreased connectivity with other sets of neurons. Deep brain stimulation (DBS) therapies target small, unique populations of axons and/or cell bodies in order to treat brain disorders and normalize connectivity. Thus, mapping the wiring diagram of the brain is an important goal. Macroscale connectivity has been studied indirectly in humans using noninvasive neuroimaging. In order to develop a much higher resolution connectivity map of the brain, this project will develop depth-resolved polarized light imaging to visualize axons and fiber tracts. Since brain imaging and mapping at microscopic resolution is feasible with intrinsic optical contrasts (e.g. polarization-based) and depth-resolved block-face imaging is desired before histological processing, we have developed the serial optical coherence scanner (SOCS) for large-scale or whole brain imaging with microscopic resolution. SOCS combines a polarization-maintaining fiber based polarization-sensitive optical coherence tomography and a tissue slicer. This project will create a novel SOCS system that can image axonal tracts at the micron scale spatial resolution using unbiased optical contrasts (Aim 1). The approach will be evaluated, refined, and compared in the same brain tissue to neural tract-tracer labeling of tracts associated with DBS targets for brain disorders, in nonhuman animal models (Aim 2). The approach will then be applied to DBS targets in the human brain (Aim 3). The physical scales at which this project investigates the brain microstructure are unique (1-10 μm resolution across centimeters of tissue). This project will pave the way for the foundation of a future human connectome at the micron scale, which is the highest resolution achievable with current optical technology for imaging an entire human brain.

NIH Research Projects · FY 2025 · 2022-03

Project Summary Gene therapy is a promising treatment for many diseases. For gene therapy to become increasingly successful, three hurdles must be overcome: We need viral vectors that are (1) safe, (2) efficient, and (3) cell type specific. Adeno-associated virus (AAV) has emerged as a viral vector that is safe in humans, efficient at delivering transgenes to both dividing and arrested cells, and able to drive long-term expression. Unfortunately, the broad tropism of AAV is detrimental when gene delivery to specific cells (e.g., cancer) is paramount and ectopic expression in healthy cells or tissues poses a risk to the patient’s safety. We recently reported a working prototype of a novel configurable viral gene delivery technology. This technology consists of a capsid that we genetically engineer to express an adapter domain to which we covalently attach monoclonal antibodies to form antibody-AAV composites. AAV tropism is redirected toward the antibody’s cognate receptor, which is expressed on a targeted cell type, but not off-target cell populations. Here, we will take the next critical steps to build on this prototype and broaden the impact of our technology. We will improve composite-AAV formation efficiency and infectivity (Aim 1), comprehensively map additional engineerable capacity across AAV serotypes identify new capsid engineering strategies and enable machine- learning guided AAV design (Aim 2) and, as a proof of concept, determine target specificity and spread of AAV composites in vivo (Aim 3). The outcome of this work will be a validated viral vector platform technology that uses antibodies to target gene delivery to rationally identified cell types. This technology will enable fundamentally new gene therapy paradigms and, in the longer term, lead to new therapeutic approaches for inherited disorders and cancer.

NIH Research Projects · FY 2026 · 2022-03

Project Summary The molecular mechanisms by which stem cell proliferation is precisely controlled during the course of regeneration are poorly understood. Namely, how a damaged tissue senses when to terminate the regeneration process, inactivates stem cell mitotic activity, and organizes ECM integrity remain fundamental unanswered questions. Uncontrolled proliferation of stem cells in regenerative tissues can result in tumor formation. The Drosophila midgut intestinal stem cell (ISC) has recently emerged as an attractive model system to study tissue homeostasis and regeneration. This is due to striking similarities in genetic control and cellular composition between the Drosophila and mammalian digestive systems, and powerful genetic tools available in this model. Importantly, Drosophila ISC proliferation is promoted in response to tissue damage to stimulate tissue repair. Using this model system, a number of studies have been conducted to reveal the pathways that activate ISC proliferation. Despite a wealth of knowledge on the activation of proliferative capacity of stem cells, it is largely unknown how the tissue properly down-regulates stem cell proliferation at the end of regeneration and how this process is linked to epithelial remodeling. We recently established the Drosophila ISCs as an excellent model to study the molecular basis for regeneration termination. Using RNA-seq, we identified sets of genes and pathways that are up/down-regulated at different phases of regeneration. Among these, we found that dMOV10, a component of the microRNA (miRNA) gene silencing complex, is required for the proper termination of the regeneration process. Further analyses identified direct target mRNAs of dMOV10-containing miRISC, including baboon (the Type I receptor of activin signaling) and two major non- integrin ECM receptors, Syndecan (a transmembrane heparan sulfate proteoglycan) and Dystroglycan (an integral membrane component of the dystrophin-glycoprotein complex). In addition to the identification of dMOV10 as a termination stage-specific gene, this same RNA-seq analysis showed that key components for septate junctions, and actin regulators are specifically upregulated during regeneration and return to normal level at a late stage. In this proposal, we will define these key molecules in stem cell inactivation and ECM remodeling at the termination stage of Drosophila midgut regeneration through the following Specific Aims. Aim 1. Determine the role of septate junction components in midgut regeneration. Aim 2. Define the role of Sdc and Dg in ECM remodeling. Aim 3. Define the global landscape of the miRNA-mediated network.

NIH Research Projects · FY 2026 · 2022-03

Project Summary The incentive value of drug-associated cues drives several facets of addiction, including escalation of drug use and the propensity to relapse even after long periods of abstinence. Cues with high incentive value can drive reward-seeking behaviors that are disconnected from an individual’s goals and the value of expected outcomes (i.e. rewards or punishments). This may lead to perseverative or compulsive drug use despite adverse consequences. A critical barrier to progress in neuromodulation-based treatments for addiction is lack of knowledge regarding the circuits engaged in cue-driven reward-seeking behavior, and how these circuits are distinct from those involved in goal-directed behavior, which relies on accurate mental representations of expected outcomes and their value. This proposal focuses on the role of the ventral pallidum (VP), a region of the basal forebrain that is critical for both relapse to drug use and positive affect. Our objective is to identify the VP neural populations that encode the incentive value of cues, and the neural circuit mechanisms by which cues drive motivated behavior. Our central hypothesis is that neurons that represent the incentive value of cues are distinct from those that represent the expected value of future outcomes, and that these neurons can be defined based on output pathway. We predict that the activity of GABAergic VP neurons projecting to the ventral tegmental area (VTA) encodes cue-driven reward-seeking and that activity in this population is critical for cue-driven motivated behavior. We will test our hypothesis by pursuing the following aims. In Aim 1 we will examine encoding of the incentive value of cues and expected value of outcomes by individual neurons in VP. Our hypothesis is that separate neurons in VP encode incentive value and expected value. We will use in vivo single unit electrophysiology to measure activity patterns in individual VP neurons during presentations of reward-related cues and determine whether activity in these neurons predicts cue- elicited reward-seeking behavior and/or the current expected value of a predicted outcome. In Aim 2 we will identify the VP output pathway(s) that encode the incentive value of cues. Our hypothesis is that VP neurons that encode incentive value are GABAergic and project to the VTA. We will use fiber photometry to measure calcium signals in VP GABA neurons projecting to the VTA or thalamus during presentations of reward cues and determine whether activity in these populations predicts cue-elicited reward-seeking behavior and/or expected value. In Aim 3 we will test the functional role of activity in these VP output pathways in behavioral responses to cues. Successful completion of this research will characterize the brain mechanisms of incentive value representations and define the downstream circuit targets for the invigoration of motivated behavior by VP incentive value signals. Advancements in our understanding of these circuits will contribute to the refinement of brain-based therapies that target the neural mechanisms underlying compulsive drug use.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Type-2 diabetes mellitus (T2DM) sequelae damage the cerebral microvasculature and augment Alzheimer's pathology by inducing brain insulin resistance characterized by sub-physiological insulin levels and impaired insulin-signaling in the brain. Conversely, soluble amyloid beta (sAβ) peptides that accumulate in the plasma and brain during Alzheimer's progression exacerbate the impact of T2DM and aggravate brain insulin resistance. A critical need exists to identify how T2DM sequelae and sAβ exposure inhibit insulin delivery to the brain and intensify brain insulin resistance. The long-term goal is to elucidate cerebrovascular and metabolic contributions to Alzheimer's disease and facilitate the development of novel therapeutic interventions. The overall objective in this application is to determine the combined effects of T2DM sequelae and sAβ on insulin delivery to the brain and to identify the underlying cellular and molecular mechanisms. The central hypothesis is that T2DM sequelae and sAβ peptides perturb insulin signaling/trafficking at the cerebrovascular endothelium [referred to as the blood brain barrier (BBB)] and reduce insulin delivery to the brain. It is also hypothesized that these effects are further aggravated by the pathological synergism between T2DM sequelae and sAβ. The rationale for the proposed research is that a mechanistic understanding of how sAβ exposure and T2DM sequelae disrupt brain insulin delivery will allow us to develop novel therapeutic strategies to address brain insulin resistance in Alzheimer's disease and T2DM. Guided by preliminary data, the following three specific aims are proposed: 1) Determine the effect of T2DM sequelae on insulin trafficking/signaling at the BBB; 2) Determine the effects of sAβ alone and in conjunction with T2DM sequelae on insulin trafficking/signaling at the BBB; and 3) Identify insulin trafficking pathways at the BBB, vulnerable to sAβ exposure and impaired insulin signaling. Under the first and second aims, dynamic SPECT/CT imaging will be used to characterize insulin uptake kinetics at the BBB in mouse models that exhibit T2DM and Alzheimer's sequelae. Moreover, the dysregulation in insulin signaling at the BBB will be captured by reverse phase protein arrays. For the third aim, flow cytometry and TIRF microscopy will be used to determine the effects of sAβ ± insulin signaling inhibitors on insulin transcytosis in BBB monolayers. The proposed research is potentially innovative because it employs dynamic imaging methods coupled with quantitative modeling techniques to capture changes in insulin trafficking kinetics at the BBB in T2DM and Alzheimer's mouse models. The proposed research is significant because the contribution it is expected to have broad translational importance in repurposing existing drugs to treat brain insulin resistance and in identifying candidate targets to discover novel drugs. Upon completion of the work, the new knowledge generated is expected to have an important positive impact by facilitating the identification of novel therapeutic strategies to combat brain insulin resistance in Alzheimer's patients with T2DM.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY Ultra high field neuroimaging in humans enables unprecedented resolutions that enable, for the first time, the non invasive investigation of directional information processing in vivo. These advances result from the ability of high resolutions to uncover layer specific (feedforward, feedback) activation patterns. These tools however are not validated in translational models that mimic human brain function. The reason for that is primarily due to our current inability to obtain functional magnetic resonance imaging (fMRI) signals at resolutions high enough to resolve layer specific responses in the non human primate. This project aims at developing novel and innovative radiofrequency hardware and using it at the ultra high field strength of 10.5 Tesla to directly test how well whole brain directional connectivity estimates from fMRI correspond with ground truth tract tracing experiments. This development and validation can directly aid us in translating findings from the animal model into future studies of the mesoscopic circuit effects of human mental illness.

NIH Research Projects · FY 2026 · 2022-03

Project Abstract Opioid addiction is a public health crisis, characterized by compulsive drug seeking and a pervasive vulnerability to relapse, with few therapeutic options available. The nucleus accumbens (NAc) is a central hub within reward circuitry and plays a critical role in the motivational value of drugs and drug-associated cues. Within the heterogeneous neuronal subtypes of the NAc exist the sparsely distributed fast-spiking interneurons (FSIs). The NAc FSIs receive strong excitatory inputs from cortical, thalamic and limbic regions and exert powerful inhibition over the local projection neurons, making them a prime candidate for translational therapeutic strategies to reduce the burden of the opioid abuse epidemic. The overall objectives of this application are to elucidate how NAc FSI circuitry responds to, and mediates, opioid self-administration. Under the primary mentorship of Dr. Patrick Rothwell at the University of Minnesota, Dr. Lefevre has utilized graduate training in molecular and behavioral pharmacology techniques, and post-doctoral training in ex- vivo electrophysiology to study adaptations in NAc circuitry in non-contingent opioid addiction models. This Pathway to Independence Award will provide the opportunity to broaden the candidate’s expertise to include in vivo fiber photometry calcium imaging, and chemogenetics, under the continued mentorship of Dr. Rothwell. To expand career development in addiction research, Dr. Lefevre will receive additional support from co-mentor Dr. Mark Thomas in the implementation of contingent opioid self-administration models in female and male mice. During the mentored (K99) phase of the award, fiber photometry calcium imaging will be used to monitor in vivo activity patterns of NAc FSIs in an Intermittent Access (IntA) fentanyl self-administration protocol to identify how this neural population responds to contingent fentanyl and fentanyl-associated cues. Subsequently, a chemogenetic approach, which will involve targeted expression of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) by NAc FSIs, will be used to test the hypothesis that these neurons play a functional role in addiction-related behavior. Following fentanyl self-administration, DREADDs will be used to excite or inhibit NAc FSIs prior to progressive ratio and cue-induced reinstatement tests. This innovative combination of tools will be used to test the relationship between NAc FSIs and the manifestation of fentanyl self-administration. The R00 phase of the award will distinguish Dr. Lefevre’s independent research from her mentors and broaden to identifying the role of excitatory synaptic inputs to NAc FSIs. Ex-vivo electrophysiology will be utilized to define input-specific excitatory synaptic plasticity adaptations in NAc FSIs induced by fentanyl self- administration. Additionally, the R00 aims will incorporate electrophysiology, optogenetic and calcium imaging expertise to develop stimulation protocols that restore opioid induced shifts in FSI circuitry and addiction-like behavior. Overall, the aims proposed in this award will identify key FSI circuits in opioid self-administration and provide the foundation for Dr. Lefevre’s independent research program studying the neural circuitry of addiction.

NIH Research Projects · FY 2026 · 2022-03

SUMMARY/ABSTRACT Overview: Reproduction relies on a complex series of cell fate decisions, cell cycle transitions, and differentiation events, which coordinate the formation and function of gametes with the process of meiosis. A century of research using many experimental organisms, including the nematode Caenorhabditis elegans, defined key intercellular signals regulating germline development, delineated central players in meiosis, and revealed the importance of post-transcriptional gene regulatory mechanisms. Yet, important mechanistic questions remain about how intercellular communication integrates the underlying molecular mechanisms into a coherent germline developmental program. This research proposal addresses several central questions: •How do soma-germline gap junctions orchestrate germline development, coordinate germline developmental subroutines, and integrate nutritional signals to optimize reproduction? •How do intercellular signals and gametic interactions regulate protein translation to enable fertilization and ensure the vitality of the embryo? •Can we exploit the dynamic nature of germline development in which germ cell nuclei move within the gonadal tube to provide broad insights into the cell biology of diseases affecting the nuclear envelope? Goals: The chief goal of our work is to delineate the regulatory networks governing conserved and essential steps in germline development. Our focus will be on three areas: (1) The role of gap junctions in controlling soma-germline interactions; (2) The regulation of protein translation by intercellular signaling during oocyte meiotic maturation and the oocyte-to-embryo transition; and (3) Studies in the C. elegans germline system that provide insights into the cell biology of a class of human diseases affecting the nuclear envelope. In pursuing these goals, we will fill three key gaps in understanding: (i) the nature of the active biomolecules that transit through gap junctions to mediate their many essential reproductive functions; (ii) the function and regulation of a large translational regulatory machine that lies at the heart of the oocyte meiotic maturation decision and the oocyte-to-embryo transition; and (iii) the involvement of nuclear mechanotransduction and endogenous mechanical forces in the origin of diseases affecting the nuclear envelope. Vision: Using the powerful combination of genetic and modern molecular technologies available in C. elegans, we will advance our understanding of germline development, germline stem cell behavior, and oogenesis. In the course of these investigations, we will discover new molecular principles vital to human health and inform the development of novel therapeutic strategies to treat disease. This fundamental research in a genetic model system will generate foundational knowledge for understanding reproduction and the origin of birth defects.