Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2024-04

Project Summary: Posterior fossa group A (PFA) ependymoma is a type of central nervous system tumor primarily found in the brain or spinal cord of infants and toddlers. PFA ependymoma is thought to be an epigenetically driven tumor characterized by a global loss of H3K27me3 levels, which promotes cancer cell proliferation. Polycomb repressive complex 2 (PRC2) is the sole enzyme responsible for histone H3K27 methylation in mammalian cells. Low H3K27me3 in PFA ependymoma is dictated by at least two mechanisms, including PRC2 inhibition and metabolic rewiring. EZH1 and EZH2 are the catalytic subunits of PRC2. The EZH1/2 inhibitory protein EZHIP (a.k.a. CXorf67) is normally expressed in gonads. EZHIP is abnormally expressed in PFA ependymoma, where it inhibits PRC2 enzymatic activity using a protein sequence mimicking the H3K27M oncohistone. The H3K27M-like sequence occupies the lysine-binding channel at the active site of PRC2 and thereby precludes histone substrate binding. FPA ependymoma cells are addicted to H3K27 hypomethylation, which underlies enhanced expression of glycolysis and TCA cycle metabolism genes that help fuel cancer cells. PRC2 inhibition by EZHIP ultimately depends on their physical interaction. Using a hybrid approach combining AlphaFold structural modeling, biochemistry, structural biology, and crosslinking mass spectrometry, we will clarify an important missing link between EZHIP and PRC2, which we found critical for PRC2 inhibition in the preliminary study. Specifically, in Aim 1, we will build an atomic model of a previously uncharacterized EZHIP motif bound to PRC2. We will also show that the newly identified binding interface between EZHIP and PRC2 can be specifically mutated or targeted to relieve PRC2 inhibition. Finally, we will generate a structural model of full-length EZHIP engaged with a PRC2 holo complex by integrating spatial constraints from crosslinking mass spectrometry into AlphaFold structural modeling. In Aim 2, we will be focused on the direct competition between EZHIP and EPOP, a naturally occurring accessory subunit of PRC2, in PRC2 binding. This is an exciting mechanistic model strongly supported by our preliminary data showing that EPOP competes off EZHIP from PRC2 and restores H3K27me3 levels in EZHIP-expressing cells. Importantly, we will study the molecular mechanism of how EPOP may impact the epigenetic and metabolic pathways by disrupting the EZHIP–PRC2 complex in PFA ependymoma cell models. In addition, EPOP-based protein inhibitors of the EZHIP–PRC2 interaction will be optimized and tested both alone and in synergy with metabolism-targeting drugs in the treatment of PFA ependymoma.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The functions of human spermatogonial stem cells (SSCs), including self-renewal and differentiation, are required for the constant production of male gametes over a long reproductive lifespan; imbalances in this process directly contribute to infertility or germ cell-derived cancers. Despite this wide range of healthcare implications, the cellular and molecular regulation of human SSCs is poorly understood, in particular with respect to the microenvironment in which SSCs reside, termed the niche. Despite research efforts, a major gap in knowledge remains, i.e., how does the SSC niche influence the functions of human SSCs? Our long-term goal is to understand the molecular mechanisms that regulate human SSCs. Unlike rodents, for which genetic models are routinely used to evaluate the roles of the niche in regulating SSC functions in vivo, functional dissection of the human SSC niche has been challenging largely due to a lack of experimental tools. Single cell omics has enabled a molecular catalogue of human testicular cell types but falls short on providing insights into the spatial and functional interactions between SSCs and the niche due to tissue disassociation. We have recently established a spatial transcriptomics (ST) approach that successfully recapitulates many aspects of the mouse and human spermatogenesis. This approach quantifies genome-wide gene expression of individual testicular cells within intact tissue sections. Using this ST approach, coupled with a series of functional, cellular, and molecular analyses, we will test our central hypothesis that human SSCs are functionally regulated by the niche through a selective set of ligand-receptor (LR) interactions. First, we will perform a systematic characterization of LR interactions at the human SSC niche. Second, we will comparatively study the human and mouse SSC niches under the hypothesis that human specific LR pairs may be crucial to human SSC functions. Finally, we will examine the contribution of germline cells to the functions of human SSC niche. The successful completion of the proposed work will significantly enhance our mechanistic understanding of the functional role of the niche in regulating human SSC activities. It will also provide much needed insights into the etiology of male infertility, fertility preservation for cancer patients, and the successful establishment of in vitro spermatogenesis.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Biomolecular condensates are diverse and abundant membrane-less organelles that partition the intracellular environment into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates participate in various essential housekeeping, stress-response, or cell type-specific processes. Furthermore, an increasing number of studies have linked the physiological state of a cell or the spatial locations of condensates in a cell with the composition of the condensates. Thus, to understand the functions of biomolecular condensates, it is important to know how the molecular content in the condensates change in health vs. disease or in different spatial locations. In this proposal, we aim to develop a novel spatial genomics platform that will enable multi-modal molecular characterization of biomolecular condensates at the sub-micron scale. This will be revolutionary for studying the functional roles of biomolecular condensates in cellular processes under homeostatic and pathological conditions. Our tools will enable us to look at the genome, epigenome, and transcriptome within the condensates in a spatially resolved manner all under native cell and tissue context. We will ensure these novel genomic tools are easy to use and adopt. The successful completion of this work will lead to a comprehensive toolset for understanding cell and tissue biology from the perspective of biomolecular condensates.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Triple-negative breast cancer (TNBC) is highly aggressive and uniformly lethal in the metastatic setting despite recent approvals of immuno-therapeutics for this disease. Outcomes could potentially be improved by closing gaps in our understanding of immunological control of TNBC. A subset of human TNBCs (6-20%) strongly overexpress the immune receptor VISTA (V-domain immunoglobulin suppressor of T cell activation), which bears close homology to the drug target PD-L1. Clinical trials in humans are now evaluating the safety and efficacy of targeting VISTA’s extracellular domain for cancer. However, we lack a mechanistic understanding of how VISTA signals to the interior of the cell. Preliminary data show that VISTA+ TNBCs have immunologically “cold” tumor microenvironments and decreased proliferative index. These effects are controlled by both the extracellular and intracellular domains of VISTA. By purifying proteins bound to the intracellular domain, we have established that VISTA can recruit and sequester cytoplasmic clathrin-adaptor proteins, causing defects in plasma membrane receptor endocytosis and trafficking. Consequently, important immunological and cancer growth receptors like TLR4 and EGFR do not function effectively when VISTA is expressed. This leads to the hypothesis that VISTA’s intracellular domain is a cell-intrinsic repressor of membrane receptor trafficking and that targeting both VISTA’s extracellular and intracellular domains is critical to improve VISTA therapeutics. This hypothesis will be tested in three specific aims: (1) To mechanistically dissect how VISTA blocks receptor trafficking, we will characterize how proteins bind to VISTA’s intracellular domain; (2) Define the mechanistic roles of VISTA’s intracellular domain binding partners in receptor trafficking and activity; and (3) Evaluate therapeutic strategies for VISTA+ triple-negative breast cancers. These aims will be accomplished through biochemical analysis of purified proteins and cell lines with engineered mutations to disrupt clathrin adaptor binding to VISTA’s intracellular domain. Analysis will include the effects of VISTA in both tumor cells that overexpress VISTA to model human TNBCs and immune cells that naturally express VISTA for normal physiology. Combination regimens to explore the effects of blocking both extracellular and intracellular functions of VISTA will be tested in immunocompetent mouse models of triple-negative breast cancer. This work should address the need to develop novel immunomodulatory agents to improve outcomes for TNBC. Although VISTA-targeted therapeutics are currently in clinical development, all current approaches exclusively target VISTA’s extracellular domain. Our studies have discovered that VISTA’s intracellular domain is a critical determinant of receptor function that we propose to extensively characterize to catalyze improved VISTA-targeted therapies.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Many pathologic or disease conditions can now be ascribed to disrupted osteocyte functions. However, a limited number of osteocyte-enriched genes have been studied in bone disease. Our previous work focused on the transcription factor SP7 and its role in regulating osteocyte dendrite formation. One major goal of this proposal is to further elucidate the osteocytic function of SP7 and decode how SP7 regulates osteocytogenesis. To address this fundamental question, I have developed a comprehensive approach based on in vivo and in vitro methods to define the effect of an osteogenesis imperfecta-causing SP7 R316C mutation in osteocytes. Results from these approaches, in combination with single-cell transcriptomics and complementary bioinformatic analysis, will illuminate the nature of the human R316C mutation in osteocyte development; this includes determining whether this mutation selectively affects the osteocytic function of SP7, identifying direct target genes that are selectively affected by this mutation, and how R316C influences osteocytogenesis by capturing the osteocyte subpopulations that are blocked by this mutation from maturation. To perform transcriptomic profiling of developing osteocytes, I will develop novel laser-assisted microdissection methods to isolate viable matrix embedded cells for single cell RNA-sequencing. Like neurons in the brain, osteocytes in bone communicate with one another through an extensive network of dendritic connections. I will perform bioinformatic analyses to identify genes with restricted expression in neurons and osteocytes, and the functional skeletal roles of candidate shared genes will be tested in vitro and in vivo. Overall, the aims described in this proposal have strong potential to define the role of osteocyte-specific genes (e.g., SP7) in bone, as well as uncover the contribution of osteocyte-specific genes in human skeletal disease. Moreover, this work may lead to identification of new pathways that can be targeted by therapeutics to ensure the osteoblast-to-osteocyte transition. My long-term career goal is to obtain a tenure-track faculty position and successfully establish a lab that is at the forefront of bridging the gap between osteocyte development and bone health. I expect the K99 phase of this proposal, which includes completing the characterization of R316C mutation in mice and the identification of direct targets and novel pathways affected by the R316C mutation, to take 1-2 years and result in at least one high quality publication. The training and mentorship provided during the K99 phase will prepare me with strong background and starting point for my continuing studies and grant applications as an independent investigator. The following R00 phase of the award will then permit me to further explore the regulation of SP7 during osteocytogenesis, as well as to examine the skeletal impact of neuron-osteocyte shared genes in vivo. Together, these data will be used to justify future studies proposed in an R01 grant application that I expect to submit at the beginning of the third year of the independent phase.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Flux through the mevalonate (MVA) pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)- localized HMG CoA reductase (HMGCR), the rate limiting enzyme in MVA pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. One mechanism for this control involves sterol-induced ER-associated degradation (ERAD). Previous studies revealed that ERAD of HMGCR is accelerated by two classes of sterols 1) methylated sterols such as the cholesterol synthesis intermediate 24, 25-dihydrolanosterol (DHL) and 2) oxysterols including 25-hydroxycholesterol (25HC). 25HC, but not DHL, blocks proteolytic activation of sterol regulatory element-binding proteins (SREBPs), another Insig-mediated reaction. HMGCR is the molecular target of statin drugs, long prescribed to lower LDL cholesterol; however, statins block ERAD of HMGCR, causing its accumulation that blunts the drugs’ efficacy. This proposal aims to gain mechanistic understanding for how sterols initiate HMGCR ERAD and provide insights on how targeting the reaction can be harnessed to prevent statin resistance. Studies show the 1,1-bisphosphonate ester SRP3042 mimics sterols in accelerating HMGCR ERAD. Photoactivatable srpDHY, a derivative of SRP3042, specifically crosslinks HMGCR, indicating sterols directly bind the enzyme to initiate ERAD. During the K99 phase of my training, I will use cryo-EM to determine how SRP3042 accelerates HMGCR ERAD. In the R00 phase, I will determine whether SRP3042 administered to mice can be used to block the statin-induced accumulation of hepatic HMGCR by accelerating the protein’s ERAD as a viable strategy to augment statin therapy. Lipid homeostasis is maintained by the SREBP family of transcription factors which modulate expression of enzymes required for lipid synthesis. Preliminary studies show the sterol-like molecule, LY295427 acts through a unique unknown mechanism to block 25HC-mediated events. These include inhibition of transcriptional regulation of LXR, regulation of acyl-coenzyme A cholesterol acyltransferase (ACAT) activity, and SREBP cleavage. However, LY295427 does not reverse 25HC-medated HMGCR ERAD. My R00 studies will identify the molecular target of LY295427 utilizing a variety of techniques including a CRISPR/Cas9 knockout screen, genomic siRNA screen, and a biochemical approach coupling click chemistry and mass spectrometry. My mentor, Dr. DeBose-Boyd (Professor of Molecular Genetics), employs a multi-disciplinary approach to explore regulatory mechanisms, and fully supports collaborative opportunities that will ensure my transition away from his interests. I have assembled a strong, scientifically diverse mentoring committee to aid my transition to independence. Completion of these studies will reveal novel lipid sensing pathways that can be harnessed for development of regulatory molecules for diseases associated with lipid dysregulation. Moreover, they will provide strong preliminary data for a future R01 application that will enhance my transition into an independent scientist.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract The endoplasmic reticulum (ER) plays a central role in many cellular processes, including lipid metabolism. The ER network is formed from a single membrane and yet contains functionally distinct subdomains that allows the ER to regulate lipid metabolism and respond to stress. How this functional diversity is mechanistically achieved is not well understood. One type of ER subdomain are regions of the ER in close contact with other organelles, called membrane contact sites (MCSs). They play important roles in cellular lipid homeostasis, signaling, and stress responses. Other subdomains are regions where some organelles are formed. One is lipid droplets (LDs), which are lipid storage depots that have many roles in cell physiology. Here, I take a multidisciplinary approach to address key gaps in our knowledge about the formation and function of ER subdomains. Central to the projects in this proposal is the question of how cells control the distribution of lipids within the ER and how this contributes to cellular lipid homeostasis. There are three directions in the proposal. 1) Direction one addresses key gaps in our understanding of how LD biogenesis occurs and goes awry in disease. We will leverage a novel in vitro LD biogenesis assay we have developed to investigate mechanism of LD formation and the roles LD biogenesis proteins. We will also determine how disease-causing mutations in these proteins alters their functions. 2) Direction two addresses the mechanism and functions of a recently described new family of tube- like lipid transport proteins that operate MCSs. 3) Direction three addresses a key gap in our knowledge of how the hydrophobic metabolite Coenzyme Q (CoQ) exits mitochondria and reaches the ER and other compartment and how export is regulated in response to oxidative stress. Capitalizing on a novel genetic screen we conducted, we will identify proteins that facilitate and regulate CoQ interorganelle transport. Collectively, these directions will provide mechanistic insights into the formation and functions of ER subdomains and how defects in these processes contribute to various diseases.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Mis-segregated chromosomes entrapped in micronuclei are susceptible to catastrophic shattering through a process termed chromothripsis. The resulting DNA fragments subsequently undergo error-prone re-ligation, in turn generating complex genomic rearrangements that are detected in approximately one-third of diverse tumor types. Chromothripsis drives the punctuated evolution of cancer genomes by rapidly rearranging individual chromosomes within a few cell cycles, which can simultaneously inactivate multiple tumor suppressor genes, generate oncogenic gene fusions, and/or amplify cancer-associated genes as circular extrachromosomal DNA elements. Therefore, a complete mechanistic understanding of the etiology of chromothripsis represents a critical need toward defining how cancer cells acquire complex genomic alterations. Although chromothripsis may be partially triggered by DNA damage within micronuclei during interphase, recent evidence support a second wave of extensive DNA damage that is inflicted upon mitotic entry. The mechanisms underlying such mitotic DNA damage and its contributions to chromothripsis are poorly understood. To address this knowledge gap, we leveraged a chromosome-specific micronucleus and chromothripsis platform to conduct pooled CRISPR/Cas9 screens targeting the mammalian DNA damage response. We unexpectedly identified multiple components of the Fanconi anemia (FA) pathway as a requirement for chromothripsis. Although the FA pathway is normally a genome-protective DNA repair mechanism, our preliminary studies indicate that it functions abnormally during mitotic entry to induce extensive fragmentation of under-replicated chromosomes in micronuclei followed by mitotic DNA synthesis. We hypothesize that pathological activation of the FA pathway triggers chromothripsis during mitosis through the cleavage of incomplete DNA replication intermediates that accumulate within micronuclei. To test this central hypothesis, we propose three complementary aims to define the molecular mechanisms driving the mitotic shattering of micronucleated chromosomes. First, we will comprehensively characterize intrinsic defects associated with micronuclear DNA replication and determine the cell cycle stages during which they arise (Aim 1). Next, we will investigate how the FA pathway functions to shatter micronucleated chromosomes during mitosis, including identifying the structure-specific nuclease(s) involved in inducing widespread mitotic chromosome cleavage (Aim 2). Lastly, we will examine the role of the FA pathway in generating complex genomic rearrangements and extrachromosomal DNAs in experimental models of chromothripsis and in FA patient cancer genomes (Aim 3). Successful completion of these aims will provide a detailed mechanistic understanding of how mitotic errors instigate rapid karyotypic changes in cancer genomes. These studies seek to provide paradigm-shifting insight into how defective DNA replication activates an uncharacterized function of the FA pathway in triggering chromothripsis and cancer genome instability.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT The nature of obesity-associated islet inflammation and its impact on β cell abnormalities remain poorly defined. In this application, we explore the immune cell components of islet inflammation and define their roles in regulating β cell function. Islet inflammation in obese mice is dominated by macrophages. We identified that G protein-coupled receptor 92 (GPR92) is exclusively expressed in islet macrophages and its expression level is regulated by high fat diet (HFD)-feeding in mice. Our computer simulation modeling combined with site-directed mutagenesis of GPR92 has revealed the sites responsible for binding to farnesyl pyrophosphate (FPP) activation, which portend various biological and medicinal functions for GPR92. Although GPR92 agonists have been reported, their physiological function has not yet been studied. In several studies, the GPR92 locus (12q13.31) has been linked by a genome-wide association study (GWAS) to type 1 and type 2 diabetes (T2D). However, the role of GPR92 in this field is largely unknown. Since obesity induces expansion of islet macrophages and increased pro-inflammatory status which impair β cell function, we hypothesize whether islet macrophage GPR92 and its activation affect nearby β cell function in obesity. Our recent publication revealed that GPR92 global knockout mice exhibited reduced insulin+ β cells as well as lower insulin levels, but increased macrophages in the islets compared to those of WT mice in both normal chow diet (NCD)- and HFD-fed groups. In this application, we propose a novel approach to harness islet macrophages via a specific molecular target, GPR92, to regulate intercellular communication with β cells. This strategy proposes to simultaneously regulate islet inflammation and β cell function in a diet-induced obese condition using genetic and pharmacological means to alter GPR92 function and further identify the molecular and cellular mechanism of islet macrophage-β cell communication via GPR92 activation. If successful, this approach will have a significant impact on T2D-mediated islet dysfunction. Furthermore, insulin insufficiency and beta cell failure occur in all forms of diabetes. This macrophage-based β cell regulation strategy may also have a broader application potential for other types of diabetes.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Outcomes for patients with gastric cancer, which is the fourth-most lethal cancer worldwide, are poor and improved therapies are needed. While immune checkpoint inhibitors (ICIs) extend survival for patients suffering from a variety of cancers, most gastric cancer patients do not respond to ICIs. The overall low response of gastric cancers to ICI therapy is not only disappointing in terms of inability to improve overall survival, but it also means that a large portion of patients treated with ICIs suffer the toxicities of the treatment without any clinical benefit. Thus, to improve outcomes for gastric cancer patients, there is a critical need to identify novel biomarkers that guide ICI use and to find new strategies that augment ICI efficacy. The investigators’ long- term goal is to gain a deeper understanding of the gastric cancer tumor microenvironment to refine the use of ICIs. The objective of this proposal is to identify biomarkers that predict ICI response and to discover targets that are actionable to improve ICI efficacy. Based on the investigative team’s published and unpublished results, the central hypothesis of the proposal is that artificial intelligence can analyze transcriptomic, digital pathology, and spatial data to guide ICI use for patients with gastric cancer. To test this hypothesis, a close collaboration between a computational scientist and surgeon-scientist has been established to pursue 3 aims. In Aim 1, we will identify novel transcriptomic, digital pathology, and spatial biomarkers that predict ICI response by comparing tumor samples obtained from gastric cancer patients who did and did not respond to ICIs. In Aim 2, we will use explainable machine learning approaches to analyze multimodal datasets to predict ICI response. In Aim 3, we will perform preclinical testing of therapies that target pathways and cell-cell interactions that are enriched in ICI nonresponders in an effort to increase ICI efficacy. Three candidate targets have already been identified. The conceptual innovations of this proposal are 1) that artificial intelligence can analyze multiple streams of data to discover predictive biomarkers, identify ICI response mechanisms, and predict ICI response, and 2) there are actionable mediators of ICI nonresponse that are identifiable via detailed analysis of the tumor microenvironment. The proposal is also supported by multiple technical innovations that include the use of 1) cutting-edge spatial profiling techniques to simultaneously acquire both spatial and transcriptomic information within the tumor microenvironment, 2) novel artificial intelligence algorithms that identify image-based predictive biomarkers, 3) novel artificial intelligence algorithms to integrate and process high-dimensional datasets and provide practical guidance on the probability of ICI response, and 4) a novel gastric cancer organoid platform to test candidate therapies to improve ICI response. The expected results from the integrated analyses of clinical, computational, and experimental data will be impactful as they will provide novel insights into gastric cancer biology and have the potential to improve both quality of life and survival outcomes for patients with gastric cancer.

NIH Research Projects · FY 2025 · 2024-03

Neurodegeneration in Parkinson's disease (PD) is closely associated with the accumulation of α- synuclein (α-syn) protein aggregates known as Lewy bodies. Understanding the diverse conformations adopted by α-syn in both healthy and diseased states is crucial for developing effective interventions. Recent advances in structural biology have provided valuable insights into the structural diversity of α- syn amyloid fibrils. However, the extent to which these in vitro models represent the conformations amplified in biological settings remains uncertain. This uncertainty hinders our ability to develop tools like PET-tracers, small molecules that bind to amyloid fibrils that can be used to localize and monitor disease progression in vivo. This project aims to bridge this gap by determining if the amyloid conformations that can be propagated in vitro can be propagated inside cells. To do so, we will leverage the sensitivity gains of dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy to investigate the conformations of α-syn fibrils amplified inside cells and compare them with in vitro amplified fibrils. By using biosensor cell lines capable of propagating α-syn amyloid conformations, we will examine the structural features of α-syn fibrils in cellular environments. This knowledge will facilitate the optimization of in silico structure-based and chemical screens to identify selective α-syn ligands.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Type II topoisomerases (Top2) alter chromatin topology by forming transient double-strand breaks (DSBs) in the DNA, allowing a second DNA segment to pass through the DSB, and then rejoining the broken strands. Mutations in the topoisomerase Top2B have been linked to neurodevelopmental delay and autism in humans. Studies in mice have shown that Top2B regulates the transcription of developmentally regulated and neuronal activity- dependent genes. A key mechanism by which Top2B regulates gene expression in neurons is by forming long- lasting stimulus-dependent DSBs within the promoters of specific genes. However, how Top2B is regulated is unknown. The proposed research attempts to bridge these knowledge gaps. Recently we published that in response to neuronal stimulation, calcium influx through NMDA receptors activates the phosphatase calcineurin, to dephosphorylate Top2B at residues S1509 and S1511 within its C-terminal domain (CTD), which induces Top2B to form DSBs promoting the transcription of immediate early genes (IEGs). Exposing mice to a fear learning paradigm also triggers DSB formation and Top2B dephosphorylation at S1509 and S1511 in the hippocampus. Additional to these dephosphorylations, we observed other phosphorylation changes at the CTD induced by neuronal activity that we have yet to study. Together, these observations provide the first insights into the regulation of Top2B by its CTD. However, since the CTD is not part of the catalytic domains and is not necessary for Top2B relaxation activity in vitro, it is unknown how the CTD regulates the activity of the catalytic core in response to neuronal activity. It is critical to study how the CTD regulates Top2B because mutations at the CTD cause late-onset sensorineural hearing loss in humans. The proposed research will use CRISPR on the auditory neuroblast cell line US/VOT-N33 (N33) and/or the sensorineural HEI-OC1, to produce cell lines with the mutations reported in patients (D1613H and K1435del) or without the serine residues that undergo dynamic changes in phosphorylation in response to neuronal activity. The cells will be differentiated into sensory hair cells or ganglion neurons and the effects of mutations on the formation of DSBs, and the regulation of IEG transcription will be assessed. Then we will purify Top2B from the differentiated cells to study relaxation, cleavage, and decatenation activity. We will also study the effect of the mutation on protein-protein interaction by coprecipitation and mass spectrometry. The allosteric effects of the CTD on the catalytic core of Top2B will be examined using cross-linking mass spectrometry. Finally, we will assess the distribution by subcellular fractionation and immunocytochemistry, and genome-wide distribution by ChIP of active Top2B. Together, these efforts will illuminate how Top2B is regulated by the CTD in response to stimulation and address how the mutations that cause hearing loss impair this regulation, which is relevant to understand this disease.

NIH Research Projects · FY 2026 · 2024-03

The developing spinal cord has long been a classic model for understanding neurodevelopmental mechanisms yet linking developmental cell types to their adult counterparts has proved elusive. Developmental transcription factors are required for the differentiation and specification of cell types during embryogenesis and are expressed in discrete domains in the developing spinal cord. Yet in the adult, expression of these developmental transcription factors is lost making it difficult to connect embryonic progenitor domains to their adult cell types and function. We will address this gap in knowledge by tracking developmental lineages over time using genetic lineage tracing of molecularly-defined progenitor domains and assaying the changes in transcription, epigenome, and anatomical distribution. Comprehensive understanding of the developmental lineages will reveal true structural and functional complexity of spinal cord circuits that are not reflected in single- cell transcriptomics of adult tissue. We will focus on the developing dorsal progenitor domains that give rise to adult cell types of the dorsal to intermediate spinal cord. The dorsal spinal cord is the primary area for integration of somatosensory stimuli from the periphery and consists of the senses of nociception (pain), thermosensation (temperature), mechanosensation (touch), pruriception (itch), and proprioception (limb and body position). The goal of this proposal is to generate three types of mouse atlases. In Aim 1, we will generate a molecular signature developmental atlas that catalogs the transcriptional and epigenomic changes of dorsal progenitor domain lineages using the 10x Genomics Multiome platform. We have identified five CRE-recombinase mouse lines that together will isolate progenitor domains of the developing spinal cord. We will use these lines to assay the molecular signatures of these lineages at three major time points across the lifetime of the animal and along the rostral-caudal axis. In Aim 2, we will generate an anatomical phenotype developmental atlas to understand the distribution of the molecular signatures using standard histological techniques and a multiplexed spatial transcriptomics platform, MERSCOPE. In Aim 3, we will create a molecular lineage 3-dimensional atlas of all five CRE-lineages at adult stages using the TissueCyte platform. Altogether, these three atlases will provide an invaluable resource for the spinal cord and somatosensory neuroscience community. Comprehensive cell profiling of the dorsal spinal cord will catalog spinal cell types in native states that can be compared to injured or diseased states. It will also serve as a reference for the cell type profiling of the spinal cord in other species. Furthermore, by adding on a layer of developmental lineage, we will answer long standing questions about the development of the spinal cord. Understanding this developmental relationship will provide insight into whether a particular adult cell type is constrained by its developmental lineage, how discrete progenitor domains generate the diversity of cell types in the adult spinal cord and lay the necessary foundation for cell-type regeneration and engineering efforts for spinal cord tissue.

NIH Research Projects · FY 2026 · 2024-03

Abstract Retinal ganglion cells (RGCs) are the only projection neurons in the retina that relay visual information to the brain. These neurons, which collectively form the optic nerve, are highly vulnerable when their axons are damaged. Preclinical animal studies have tested several approaches to promote RGC survival following axonal damage. Clinical trials aimed at assessing the neuroprotective effects are currently underway. However, attaining strong neuroprotection for patients suffering from optic neuropathy remains an elusive goal. Long noncoding RNAs (LncRNAs) are a diverse class of transcribed RNAs, defined as transcripts with lengths exceeding 200 nucleotides that do not encode proteins. Although several lncRNAs have been shown to play vital roles in regulating gene expression networks in the central nervous system (CNS), the functional roles of the majority of lncRNAs are unknown. In our recent effort to investigate RGC type-specific gene expression, we performed RNAsequencing (RNA-seq) on distinct RGC populations. Through this work, we identified hundreds of lncRNAs that are uniquely induced in different RGC types after axonal injury. The functions of these lncRNAs remain to be determined. To investigate lncRNAs’ functional roles, and to examine whether modulating the expression of injury-induced lncRNAs affects RGC survival after axonal injury, we generated adeno-associated viruses (AAV) expressing shRNAs against select lncRNAs, and carried out an in vivo screen using optic nerve crush (ONC). Through this work, we find that silencing one lncRNA results in striking neuroprotection for RGCs after ONC. This lncRNA is a long intergenic non-coding RNA (lincRNA) which we name optic nerve injury-induced lncRNA 1 or Onil1. Like the vast majority of lncRNAs that we found to be differentially expressed in RGCs, virtually nothing is known about this lincRNA. Overall, our study provides us with an exciting and unique opportunity to investigate the role of lncRNAs in regulating RGC survival after injury, and elucidate the possible molecular mechanisms underlying this process. In this proposal, we seek to combine in vivo models of optic nerve injury, functional and behavioral assays, and bioinformatics to investigate the contribution of Onil1 and other lncRNAs in regulating RGC survival. To this end, in Aim 1, we will systemically assess the extent of RGC protection conferred by silencing lncRNAs in adult RGCs after ONC. In Aim 2, we will assess whether silencing lncRNA induces RGC protection and functional rescue in mouse models of glaucoma. Lastly, in Aim 3, we will use immunostaining, RNA-seq, ATAC- seq and ChIRP-MS to investigate the mechanisms by which Onil1 regulates RGC survival.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Small cell lungcancer (SCLC) is a pulmonary neuroendocrine cancer with very poor prognosis and limited effective therapeutic options. Chemotherapy has been used for the past 30 years as for the treatment for SCLC. While most SCLCs initially respond to this treatment, nearly all relapse. Recently, immunotherapies have been introduced in SCLC treatment. While these treatments have revolutionized the treatment of non-small cell lung cancer (NSCLC), they are only effective in a very small subset of SCLCs. Thoracic radiation is recommended in the course of both limited stage (LS) and extensive stage (ES) SCLC. Radiation has been shown to induce durable responses by engaging anti-tumor immunity. However, radiation therapy while effective in some individuals, does not always convert immunologically non-responsive “cold” tumors to become immune responsive “hot”. These observations highlight the need to develop new effective treatments for SCLC. In many cancers, the loss of cell cycle checkpoints, together with oncogene activation, leads to cell survival even with high levels of replication stress (RS) and DNA damage. Cellular pathways respond to RS, to ensure that DNA is properly replicated and to prevent cells from prematurely entering mitosis. However, in most advanced solid tumors, there is an increase in a variety of errors during DNA synthesis, disruption of the DNA damage response, and mitotic catastrophe. This continuous and high degree of RS and dependence on DNA repair provides a potential cancer vulnerability and therapeutic opportunity. The vast majority of human tumors including nearly all of SCLCs are dependent on telomerase holoenzyme to bypass replicative senescence resulting from telomere shortening with each cell division and this creates a dependency. We are proposing to utilize a molecule that interferes with the function of telomerase to rapidly stop SCLC growth with minimal or no cytotoxic side effects in normal telomerase silent tissues. In our preliminary studies, we discovered that this molecule not only can kill cancer cells in culture dish, it can also activate the immune system when given intermittently to the mice. We propose to discover how this molecule sensitizes SCLCs to radiation or chemotherapy treatment (Aim 1), Induce immune responses (Aim 2), and to determine whether it can overcome resistance to existing treatments (Aim 3). For this goal, we will utilize fully immunocompetent mouse models. Therapies that activate immune system have been less toxic compared to other treatments and often provide more durable responses in patients. We hope that our studies with this molecule will generate information to develop rationally designed clinical trials which utilize combinations with minimal toxicity and maximum therapeutic efficacy in small cell lung cancer

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The recent advancement of stereotactic body radiotherapy (SBRT) enables highly focused dose delivery to tumors while sparing surrounding normal tissues. Emerging clinical evidence showing superior tumor control and patient survival suggests that SBRT will play an increasingly critical role in liver tumor management. However, radiation-induced toxicity, especially of the dose-sensitive normal liver tissues, poses a lingering challenge to liver radiotherapy and SBRT. Proton therapy, with its unique ‘Bragg peaks’, allows simultaneous tumor dose escalation and normal tissue sparing, paving the way to boost the efficacy and safety of liver SBRT. Unfortunately, precisely delivering planned proton doses to liver tumors is significantly challenged by the intra- treatment liver motion, especially for the increasingly dominant pencil beam scanning (PBS) technique. Current motion management techniques including abdominal compression, beam re-scanning, and respiratory gating, however, all suffer from various sources of uncertainties and inaccuracy, resulting in limited and case- dependent efficacy. Motion tracking, a technique that tracks the real-time tumor motion to accordingly adjust the beam delivery, can systematically and fundamentally address the motion challenge. However, such a technique is not yet available, and is currently facing three major challenges for the site of the liver: 1). The respiration-induced fast liver motion requires real-time imaging within hundreds of milliseconds, which results in extremely limited sampling that makes 3D deformable motion tracking challenging. 2). The low contrast of liver tumors against surrounding normal liver parenchyma adds another layer of uncertainty to tumor motion tracking. 3). The beam-tracking technique is purely ‘geometry-guided’, which requires fast energy switching that is difficult to achieve in current proton systems. It also fails to consider cumulative tracking errors caused by system latency, motion prediction error, and dose changes due to anatomical deformation. The overarching goal of this project is to develop a real-time imaging and proton plan adaptation (RIPA) system, composed of two sub-systems (MeshBioNet and MAO) to solve real-time 3D deformable motion (MeshBioNet) for simultaneous dose calculation, accumulation, and on-the-fly proton plan adaptation (MAO). MeshBioNet uses Artificial Intelligence-driven methods to solve real-time 3D deformable motion from a single x-ray projection. MAO features the first closed-loop, ‘dosimetry-guided’ framework that actively monitors and adapts the proton dose to ensure its matching with the planned dose without requiring tracking-related fast energy switching. We have three Specific Aims: 1) Develop and optimize the real-time imaging sub-system (MeshBioNet), 2) Develop and optimize the on-the-fly proton plan delivery adaptation sub-system (MAO), and 3). Test RIPA via a pre-clinical motion-enabled phantom study and a retrospective patient study. The successful conduct of the project will result in the first end-to-end system to improve the delivery accuracy of proton therapy for liver, and to unleash the full potential of proton therapy, especially via SBRT, in advancing liver disease care.

NIH Research Projects · FY 2026 · 2024-02

Abstract Interleukin-2 (IL-2) is an immunostimulatory cytokine that plays a central role in antitumor immunity, particularly in the activation and proliferation of T cells and natural killer (NK) cells for tumor eradication. Proleukin® is the first FDA approved immunotherapy drug with curable responses in a subset of renal and skin cancer patients. However, significant immune-related toxicities, short half-life, and inadvertent activation of immune tolerant regulatory T cells (Tregs) restrict the wide clinical adoption of Proleukin® therapy. Pleotropic effect of IL-2 in the activation of both immune suppressive Tregs and tumor killing CD8+ T cells and NK cells drove the development of IL-2 mutein superkines with polarized affinity toward CD8+ T/NK cells. Moreover, tumor acidity has been shown to deactivate wildtype IL-2, which spurred the design of low pH- resistant IL-2 muteins. Long circulating IL-2 has also been developed by fusion of Fc or albumin proteins to IL-2. Despite extensive efforts, most protein-engineered IL-2 still encounter significant systemic toxicities that limit the dose and efficacy in cancer therapy. The long-term goal of this application is to establish tumor- activatable IL-2 mutein superkine nanoparticles as an integrated paradigm for IL-2 therapy. We aim to combine nanoparticle engineering with protein engineering to minimize systemic toxicity while maintaining antitumor efficacy in the acidic tumor microenvironment. In the past decade, our lab invented a library of ultra- pH sensitive (UPS) nanoparticles with cooperative micelle/unimer phase transitions in response to a specific pH threshold. Preliminary data show UPS delivery of wildtype IL-2 Fc greatly reduced systemic toxicities (e.g., >100-fold reduction in interferon- levels) while maintaining the antitumor efficacy of IL-2 Fc. Our collaborator Dr. Tao Yue’s lab recently identified a low pH-resistant IL-2 superkine with 20-fold higher binding to IL-2 receptor  (R) than wildtype IL-2 while maintaining high activity at pH 6.4 whereas wildtype IL-2 lost its activity. In this application, we will test the central hypothesis that tumor-activatable delivery of R-tropic, acidity-resistant IL-2 superkine by the ultra-pH sensitive nanoparticles will effectively mask the systemic toxicity in normal tissues while achieving robust activation of cytotoxic lymphocytes in tumors for efficacious therapy. We will carry out three specific aims. Aim 1: Establish acidity-resistant IL-2 superkine-UPS nanoparticles with stable loading and pH-activatable release. Aim 2: Investigate the safety and antitumor efficacy of IL-2 Fc superkine-UPS nanoparticles in tumor-bearing mice and elucidate tropism of CD8+ T/NK cell activation over Tregs. Aim 3: Investigate the synergy of sodium lactate treatment with IL-2 Fc superkine nanoparticle therapy. We anticipate successful execution of the proposed application will lead to a new paradigm in interleukine-2 therapy with improved safety and antitumor efficacy outcomes.

NIH Research Projects · FY 2020 · 2024-02

Summary/Abstract Functional neuroanatomy in human dentate nucleus (DN) remains largely unmapped. Functional magnetic resonance imaging research has redefined broad categories of functional division in the human brain showing that primary processing, attentional “task positive” processing, and default-mode “task negative” processing are three central poles of neural macro-scale specialization. This new macro-scale understanding of the range and poles of brain function has revealed that not only cerebral cortex, but also thalamus, striatum, and cerebellar cortex contribute to the full spectrum of human neural organization. Whether functional specialization in DN obeys a similar set of macroscale divisions, and whether DN is yet another compartment of full-spectrum representation of human brain function remains unknown. This proposal aims to explore functional territories in human DN. Preliminary results using data-driven gradient-based clustering analysis reveal three functional zones as indexed by high spatio-temporal resolution resting-state MRI, and that these three distinct territories contribute uniquely to default-mode, salience-motor, and visual brain networks. Our goal is to replicate the results in an independent larger sample to provide a systems neuroscience substrate for cerebellar output to influence all broad categories of neural control – namely default-mode, attentional, and multiple unimodal streams of information processing including motor and visual. The overarching aim of this proposal is to apply these functional territories towards clinical translation, specifically in the context of Autism Spectrum Disorder.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT Mitochondrial energetic dysfunction is implicated in many common human diseases, including conditions impacting the liver such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and liver failure. The precise consequences of loss of mitochondrial energetics in the mammalian liver are largely unknown. To date our understanding of the roles of mitochondrial complex V (mCV), or ATP synthase, has arisen primarily from in vitro cultured cells or yeast studies. These seminal results revealed structural and functional insights into mCV’s role, but are unable to clearly delineate the physiologic effects of mitochondrial energy dysfunction. Our lab has developed a new mouse model that conditionally deletes a core subunit of mCV and ablates the ATP synthesis activity of the mitochondria. This model will enable us to study pure mitochondrial energy dysfunction in any organ system, and in particular this proposal aims to determine effects of mitochondrial energy dysfunction in the adult murine liver. To accomplish this, a combination of isotope tracing mass spectrometry, nuclear magnetic resonance, specialized mitochondrial respiratory assays, electron microscopy, next generation sequencing and mouse physiology studies will be used. The specific aims of this proposal intend to 1) understand the mitochondrial structural and functional consequences of mCV disruption in liver and 2) determine the contributions of mitochondrial ATP to liver and whole body homeostasis. The findings of this proposal will provide insights into basic mitochondrial biology and the pathophysiology of common liver diseases.

NIH Research Projects · FY 2025 · 2024-02

Patients with fatty liver disease have a high risk of progressing to liver cancer, including hepatocellular carcinoma. At the molecular level, two major changes occur in hepatocytes caused by the accumulation of fat in the liver: a switch in energy metabolism, based on mitochondria disfunction and altered TCA cycle flux, and abnormal epigenetic activities, including higher expression of chromatin modifying enzymes. However, the connection between aberrant hepatocyte metabolism and aberrant epigenetic activities in the non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) to hepatocellular carcinoma (HCC) progression has not been defined, nor therapeutically exploited to combat the onset of HCC. We propose to do so in this application. We will focus on establishing metabolic biomarkers and targeting metabolic/epigenetic co-vulnerabilities during HCC development. The rationale for this approach is that oncogenic epigenetic Jumonji histone demethylase enzymes, chromatin modifiers upregulated in HCC which delete histone methylation marks, use TCA metabolite alpha- ketoglutarate (2-OG) as a co-substrate for catalysis. Indeed, not only is 2-OG a co-substrate for these enzymes but succinate and fumarate can compete with 2-OG for binding, and thus act as inhibitors of Jumonji activity. Current Jumonji inhibitors we and other have developed work by competing with 2-OG. This presents an enormous opportunity in the context of fatty liver disease progressing to HCC, since this progression thus establishes new metabolic vulnerabilities with targetable epigenetic consequences. We hypothesize that altered cellular TCA metabolism in fatty livers triggers aberrant Jumonji KDM histone demethylase levels/activity before HCC onset and that this pro-oncogenic event is targetable through small molecule inhibitors of Jumonji enzymes that compete with 2-OG, to prevent HCC development. We will test this hypothesis by measuring 2-OG related metabolites in the liver and the blood during the NAFLD to HCC progression to identify metabolits that correlate with response to Jumonji inhibition (aim 1, biomarker focus) and by evaluating the efficacy of three available Jumonji inhibitors to prevent or slow down the progression of NAFLD to NASH and to HCC in vivo (aim 2, therapeutic focus). We will use DIAMOND mice as our in vivo model since they recapitulate the human NAFLD, NASH to HCC continuum faithfully, are well characterized, and spontaneously develop HCC within a year. Our studies will inform future personalized medicine applications in the prevention and treatment of hepatocellular carcinoma, making this proposal highly clinically relevant, timely, and potentially transformational.

NIH Research Projects · FY 2026 · 2024-02

Project Abstract Light-sheet fluorescence microscopy (LSFM), through its gentle, efficient, and fast 3D imaging capacity, has tremendous potential in biological, biomedical, and translational applications. However, LSFM has fragmented into a myriad of specialized instruments, each optimized for a specific class of samples and imaging regimes. As such, the widespread use and ultimate impact of LSFM has been curtailed by its lack of adaptability. Here we propose a universal LSFM platform that can be adapted to a wide range of applications, ranging from sensitive live cell imaging to imaging organs and tissues that have been rendered transparent by different clearing techniques. Further, we will improve the spatial and temporal resolution, as well as the volumetric coverage of LSFM. These additional improvements will be packaged in modules, which can be integrated into our proposed platform on demand. To demonstrate its biomedical potential, we will image human cardiomyocytes, both live and with super-resolution microscopy, to evaluate the performance of emerging cardiac therapies on a molecular level. In its most sensitive configuration, our LSFM platform will be able to study spatiotemporal patterns of calcium signaling, in health and disease, which we will analyze with statistical times series analysis methods. Overall, our proposal will increase the capabilities and accessibility of LSFM, and as such will spur three-dimensional imaging in biological and biomedical research.

NIH Research Projects · FY 2026 · 2024-01

For an organism to survive, its proteins must adopt a diversity of conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. This situation becomes critical when considering proteins with energetic folding landscapes that permit many conformational states. In these cases, the environment can clearly influence the conformation by favoring one pathway over another. Because the aggregating proteins that are responsible for neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases often have identical sequences in healthy and diseased individuals, differences in cellular environment are responsible for the conformational switch. Yet, despite the importance of the environment for protein folding, structural investigations of biomolecules are typically confined to in vitro systems, which cannot capture important structural features imposed by biological environments. Solid-state NMR spectroscopy is currently undergoing a “sensitivity renaissance” with the development of dynamic nuclear polarization (DNP). Experiments that would require decades of experimental time with traditional ssNMR methods can be collected in a day with DNP NMR. Moreover, while most structural biology approaches require purified samples, NMR spectroscopy does not. Because NMR reports quantitatively on the relative populations - with atomic level precision - it can report on the identity and relative abundance of structural polymorphs. Here, we will capitalize on the methodology for in cell structural biology using DNP- assisted NMR we have developed in our group to determine if and how biological settings influence the conformations of both the highly ordered and intrinsically disordered regions with atomic level precision.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Many children arrive at kindergarten unprepared to learn to read, at-risk of falling more behind, with major inequities linked to race, geography and poverty (rates >50%). These are amplified during disruptions such as COVID, when access to information and resources is perturbed. Low proficiency is strongly linked to adverse school, vocational and health outcomes, with estimated costs >$350 billion/year. As parents are a child’s “first and most important teachers,” home reading routines have a large impact on these outcomes. However, there are wide disparities in these between high- and low-resource families, fueled by household stressors, cultural differences, literacy challenges and other factors. Marginalized families also often face barriers to access of reliable literacy-promoting information, programs and resources, worsening disparities. Given trusted access to families when parenting routines are shaped, health providers are poised to help mitigate these barriers, yet guidance tends to be general, inconsistent and can fade-out at home. The objective of the proposed project is to enhance, “localize” and test a new, free mobile app designed to provide reliable shared reading guidance and resources for parents (Reading Bees; RB) in an efficient, engaging way. The rationale is that no similar approach exists, RB is free and designed to enhance existing programs, and there is evidence that its features will be useful and effective. Content is evidence-based and has been co-developed with input from community stakeholders and families from disadvantaged backgrounds. Core principles are clarity, credibility, flexibility (e.g., parents set their own goals), responsiveness (child age, family concerns, ZIP), engaging content (tips, videos, resources) and positive reinforcement (“LitCoin” awards). The long-term goal of this project is to use RB to help improve reading and literacy outcomes. To achieve this, teams in 3 culturally distinct areas (OH, WV, FL) will collaborate in a 3-year project. Content will first be added to address needs in each community: lists of local reading-related resources curated by area stakeholders and a Spanish language version of RB. Enhanced, “localized” RB will then be tested with parents in each area, first through focus groups to gauge usefulness and guide refinement, and then by providing RB to parents (ages 0-6) during clinic visits and measuring use over the next 2 months. Outcome measures involve feasibility, acceptance and useflness. The central hypothesis is that local stakeholders will be engaged by the opportunity to highlight resources in their area; families will rate RB content as useful and use RB often, especially to earn LitCoin awards; and improved access to information and resources will fuel better reading and literacy outcomes. This work is significant and innovative as it involves a tech-enabled, user-centered approach that is scalable within existing pediatric, library and program infrastructure and empowers parents to read more interactively and access reliable information. The expected outcome is that this work will provide vital enhancements to RB, show feasibility and usefulness and provide a flexible, collaborative model to “localize” and scale use of RB into other areas.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Current ulcerative colitis (UC) treatments only alleviate symptoms and maintain remission of UC. Thus, studies to identify new regulatory factors are required. Long noncoding RNAs (lncRNAs) account for a significant proportion of the human genome and have been implicated in the development of chronic and inflammatory diseases. To date, a small number of studies have indicated that lncRNAs play a role in regulating intestinal epithelial barrier integrity, and lncRNA expression has been shown to correlate with disease progression in UC. Yet, the physiological functions of lncRNAs in the pathogenesis of UC remain unknown. Recent studies indicate that lncRNAs can localize to specific organelles and regulate cellular metabolic pathways. However, there is a significant gap in our knowledge of the physiological function of these subcellular lncRNAs in the tissue microenvironment. During my postdoctoral studies in the laboratory of Dr. Kate Fitzgerald at UMass Chan Medical School, I identified a novel transcript of the lncRNA HOXA11os that translocates to the mitochondria in colonic cells. Through direct interactions with proteins associated with the Krebs cycle and complex I of the electron transfer chain, HOXA11os regulates oxidative phosphorylation and ATP production in the distal colon to restrict intestinal inflammation. This study pioneered our understanding of lncRNA function in colonic tissue and provided the first evidence of a lncRNA that localizes to the mitochondria to regulate its activity. The question remains whether additional lncRNAs localize to other organelles and regulate metabolic pathways. Thus, the overall goal of this proposal is to identify and characterize organelle-residing lncRNAs that regulate tissue homeostasis and inflammation through direct regulation of organelle function and metabolic pathways. This proposal is focused on three Research Areas. The first Research Area will focus on identifying endogenous lncRNAs that localize to organelles and the molecular mechanisms regulating their subcellular trafficking and organelle import. The second Research Area will focus on characterizing the precise molecular mechanisms by which organelle-residing lncRNAs regulate protein activity in metabolic pathways associated with UC. The third Research Area will focus on identifying the physiological role of organelle-residing lncRNAs in the onset and progression of UC to target them for treating UC. Together, these studies will identify novel lncRNAs that regulate the function of metabolic organelles to maintain homeostasis and restrict intestinal inflammation. The goals of this proposal are highly relevant to the mission of the NIAID and will enhance our understanding of distinct lncRNA biology in the colon microenvironment. Ultimately, this work will aid in the design and development of therapeutics for the treatment of UC and will potentiate future studies to assess the role of organelle-residing lncRNAs in other inflammatory diseases.

NIH Research Projects · FY 2025 · 2024-01

Although brain oscillations are a promising target for neuromodulation for cognitive disease, we have a poor understanding of the molecular mechanisms that lead to oscillatory activity. Motivated by this gap in knowledge, the Lega-Konopka collaboration has developed groundbreaking techniques to study the links between gene expression and memory-enhanced brain oscillations. By integrating gene expression data and memory-relevant iEEG signatures of the same human epilepsy patients, the collaboration discovered significant associations between memory-associated oscillations and specific gene expression patterns in the temporal pole. This work identified SMAD3 as a highly promising candidate for future study. We found strong links between SMAD3 gene expression and slow theta oscillations, which are uniquely important for human cognitive processes. We also found evidence that SMAD3 binds the regulatory elements of dozens of other genes connected with slow theta oscillations. Many of these putative SMAD3 gene targets are implicated in neuropsychiatric disorders characterized by cognitive dysfunction. Taken together, this evidence suggests that SMAD3 may coordinate the transcription of many genes that affect brain activity during memory processing. However, the brain-specific transcriptional targets of SMAD3 and the precise impact of SMAD3-mediated gene networks on human neural activity have yet to be definitively identified. The few studies on the functional role of SMAD3 in the brain showed that Smad3 knockout mice have impaired neurogenesis and long-term potentiation. Although animal models generate valuable insight into brain function, the complicated nature of human cognitive processes and related oscillations decreases the translational value of approaches using animals. To overcome these problems, I will use human organotypic slice culture (OSC) to define the function of SMAD3 in the regulation of memory-related gene networks and neural activity in the human brain. In Aim 1, I will enhance SMAD3 activity in human OSC then use snRNA-seq and snATAC-seq to identify SMAD3 gene targets. In Aim 2, I will demonstrate the ability to silence SMAD3 expression in human OSC using lentiviral shRNA constructs. I will then use high-density microelectrode array recordings to understand how the loss of SMAD3 impacts activity at the single neuron and network levels. By increasing our understanding of the mechanisms underlying memory-relevant brain activity, the completion of this project will lead us toward new therapies for cognitive disorders. This would be impossible without the stellar support of Drs. Lega and Konopka, who have expertly guided my development as an aspiring physician-scientist. The training plan in place for this fellowship period will allow me to learn and develop innovative skills for studying human brain tissue in vitro with a combination of electrophysiology, molecular biology, and genomic approaches. Benefitting from the unique combination of expertise from my mentors and the resources provided by UTSW, this training will allow me to grow as a rigorous, passionate physician-scientist with the tools to develop novel methods to study and treat the neurological disorders of my patients.