Pennsylvania State Univ Hershey Med Ctr

NIH Research Projects · FY 2025 · 2025-09

Abstract Aging is accompanied by complex, cell type-specific epigenetic alterations, yet the impact of these changes on the dysregulation of cellular transcriptomes and functional decline remains poorly understood. Among key epigenetic modulators, Polycomb Repressive Complex 1 (PRC1) maintains gene silencing by establishing a repressive chromatin environment. Previous studies demonstrated that PRC1 complexes inhibit cellular senescence, indicating a potential role of PRC1 in aging. Mechanistically, PRC1 antagonizes senescence by repressing its target gene, CDKN2A, which encodes the CDK inhibitors p16Ink4a and p14Arf. Later research revealed reduced expression of Pcgf4, a key component of PRC1, in the aged brains of both humans and mice. Mice with a heterozygous deletion of Pcgf4 develop normally, but they show cognitive deficits and neurodegeneration during aging. Although PRC1 loss strongly correlates with brain aging, the extent to which PRC1 exerts cell type-specific effects is not fully understood. A significant barrier to understanding the role of PRC1 in aging is the cellular heterogeneity within brain tissues, which complicates in vivo studies of protein- protein and protein-chromatin interactions. To address this challenge, we have recently developed a biotinylation-mediated lineage-specific capture (BLINC) technique, enabling us to define PRC1 composition and function in defined brain cell types. Our overarching goal is to elucidate how PRC1 regulates chromatin environments and maintains cellular transcriptomes during brain aging. We will address this through two specific aims. Aim 1: Using BLINC, we will identify PRC1 composition and chromatin modifications across various brain cell types in young and aged mice. Leveraging BLINC with Affinity purification, mass spectrometry, and deep sequencing, we will reveal how PRC1-mediated chromatin changes contribute to age-related alterations in gene regulation. Aim 2: We will examine the functional consequences of PRC1 loss in glutamatergic cortical neurons using conditional knockout models. By assessing chromatin and transcriptomic changes in these neurons, we aim to uncover mechanisms by which PRC1 prevents age-associated shifts toward a pro-senescence state. By combining BLINC with proteomic and genomic analysis, our study will provide novel insights into PRC1 regulation on chromatin dynamics during aging, which may lead to novel therapeutic targets for age-related conditions.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT A research gap exists for projects focusing on the leading risk factors for death and disability, especially within populations affected by health disparities. To address this gap, The Office of Disease Prevention (ODP), the National Institute on Minority Health and Health Disparities (NIMHD), and the Office of Data Science Strategy (ODSS) at the National Institutes of Health announced their intention to establish the Multi-Sectoral Preventive Interventions (MSPI) Research Network. The ODP has initiated a new trans-NIH research effort, titled ADVANCE (ADvancing preVentive intervention reseArch in populatioNs that experienCe health disparitiEs) Initiative, of which the MSPI Research Network will be a component. The Multi-Sectoral Preventive Interventions (MSPI) Research Network, including the Coordinating Center (CC) and up to 10 Research Projects, will work collaboratively with the NIH to test preventive interventions addressing social determinants of health (SDOH) and risk factors for chronic or acute health conditions, especially in populations that experience health disparities. The Pennsylvania State University College of Medicine (PSUCOM) proposes to serve as the CC for the MSPI Research Network. The CC will provide overarching support and guidance to the Network through three specific aims: 1) Administration, coordination, and communication 2) Methodology, data, and analytic support and consultation 3) Community and other collaborator engagement and dissemination. Our investigative team is highly experienced in biostatistics and leading coordinating centers, health disparities, prevention research, diabetes, obesity, cancer prevention, implementation and team science, community engagement, family-level interventions, community-level interventions, and other expertise across the NIMHD research framework levels and domains of influence. The investigative team will provide innovative support and guidance to the Network. Throughout all specific aims, the CC will focus the Network’s efforts around community engagement and team science best practices to ensure community members are full partners in the research projects. The CC will schedule and document all Network activities, establish and facilitate the Network Steering Committee and working groups, develop governing policies, and build communication platforms and public and private websites to support collaboration; all while considering ease of use for community partners. To ensure the utmost data integrity, the CC will provide methodological and statistical consultation to the Research Projects, provide consultation on SDOH measures, assist with compilation of publicly available data, support data harmonization and archival, and provide all technical assistance needed. The CC will create and support Network-wide community advisory boards, lead information sharing sessions and develop tool kits to exchange best practices for engagement and dissemination across all communities. Further, we will create public-facing communication materials such as social media posts, videos, newsletters, and lay briefs that will both promote the Network and share key research findings for translation into the community to address health disparities.

NIH Research Projects · FY 2025 · 2025-08

Project Summary The zebrafish is a vertebrate model organism with significant genetic, cellular, and physiological similarities with humans. Its powerful genetic and imaging tools have allowed us to decipher gene function, embryonic development, and disease mechanisms. Its small size and recent advances in imaging allow the unbiased visualization of phenotypes across all cell types and tissues through adulthood. This creates a unique opportunity for an integrative atlas resource that will allow the interpretation of multi-omic data in the context of the whole organism. The labeled 3D whole-animal lifespan atlas will be based on microCT customized for histopathology, histotomography that will, in turn, anchor 2D histological and developmental gene expression data to facilitate understanding of normal and abnormal phenotypes. Histology, the study of the microscopic structure of tissues, provides critical understanding of the organization and function of tissue structure across organ systems. Clinical phenotypes are associated with altered gene expression in specific cell types. Single-cell transcriptomic data are best interpreted in the context of the microanatomy of the whole animal. Zebrafish is ideal for testing these principles based on the ability to image the entire organism in 3D at cellular resolution. The integrative zebrafish 3D microanatomical atlas will provide facile access to the first gene expression data anchored on whole-organism, developmental stages, at subcellular resolution. Atlas integration will involve a multi-disciplinary team of researchers with expertise in various fields, including histology, microscopy, imaging, and genomics, and in interdisciplinary research. To ensure the accuracy and completeness of the atlas, we will consult with experts in the field and incorporate feedback from the scientific community. The 2D histology component of the atlases will include vector-based annotation of histology and histology like virtual slices from 3D high-resolution images of the organisms' cells, tissues, and organs. The 3D micro-CT component will consist of cell-resolution 3D reconstructions of key structures, allowing researchers to interrogate spatial relationships between different structures and to manipulate and explore the anatomy of the model organisms in virtual environments. The gene expression component will involve generating both 2D spatial transcriptomic gene expression data (2D spRNA-seq) and single-cell RNA-seq data for a zebrafish, integrating the two datasets to assign cell types likely to be present in each 2D spRNA-seq spot, and performing in situ hybridization experiments to validate the assignment of clusters in three dimensions. A primary goal of this project is to maximize the impact of open-access multimodal, submicron resolution atlasing by facilitating the dissemination of that knowledge across the scientific community. This atlas will comprise a foundation for the long-term goal of applying this approach across model systems and humans that will, in turn, accelerate a broad range of model organism research dedicated to the enhancement of human health.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Neuroblastoma (NB) is the most common extracranial solid pediatric cancer and is one of the leading causes of cancer-related deaths in children. Despite the current multi-modal treatment regimens, majority of patients with advanced-stage NBs develop therapeutic resistance and relapse, leading to poor disease outcomes. There is a large body of knowledge on pathophysiological role of small extracellular vesicles (EVs) in progression and metastasis of multiple cancer types, however, the importance of EVs in NB was until recently not well understood. Our recently published studies along with new preliminary data have demonstrated the involvement of EVs in various aspects of NB pathogenesis, including pre-metastatic niche (PMN) formation, metastasis, and immune responses. Tumor-derived EVs (TEVs), being important regulators of NB progression, thus could become efficient targets for developing novel therapies for NB treatment. We have demonstrated that Tipifarnib (a potent, selective, and orally bioavailable inhibitor of farnesyltransferase that can inhibit the secretion of EVs from cancer cells) prevents the immunosuppressive effects of TEVs and sensitizes high-risk NB to therapy. We have also recently reported that SEMA3A mediates the pro-metastatic effects of NB TEV, that depends on SEMA3A receptor-NRP1. These data support an overarching hypothesis that targeting biogenesis and function of NB- derived TEV should elicit potent anti-metastatic effects and improve the efficacy of anti-cancer therapy. We will test this hypothesis in the following Specific Aims: Aim 1: To define the mechanism by which Tipifarnib suppresses TEV production in NB. We will analyze the stages at which Tipifarnib impedes TEV production and identify the molecular targets of Tipifarnib involved in the regulation of TEV biogenesis or secretion. Aim 2: To determine the role and mechanisms of TEV-mediated SEMA3A signaling in NB progression. We will use genetic, molecular, and pharmacologic approaches to determine TEV-mediated function of SEMA3A in NB pathogenesis. Mechanisms of TEV-mediated SEMA3A actions will be investigated. Aim 3: Targeting TEV production and function for NB therapeutics. We will determine the anti-metastatic effects of Tipifarnib and anti-NRP1 antibodies as mono-agents and in combination to suppress spontaneous or chemotherapy-induced NB metastasis. Completion of these studies should gain the insight on the importance of TEV in NB metastasis, characterize the mechanisms of TEV regulation and function in NB, and develop novel strategies to suppress TEV-mediated NB metastasis.

NIH Research Projects · FY 2025 · 2025-08

Abstract Membrane geometry is generated and maintained by the interplay of protein-lipid and lipid-lipid interactions. Detection and remodeling of membrane shapes are part of many essential cellular processes. Furthermore, an increasing number of proteins have been found to depend on specific membrane geometry for their function (e.g. ArfGAP1 and Atg3) and localization (e.g. SpoVM). We are investigating the structural and molecular mechanisms for the recognition of membrane geometry and for the translation of membrane geometry into protein function. While pursuing these goals, we are developing innovative tools for the study of geometry-sensitive molecules in native-like environments using spherical nanoparticle supported lipid bilayers and membrane vesicles directly isolated from cells with an in situ NMR spectroscopy. Autophagy is a conserved stress-response pathway in eukaryotes. De novo formation of autophagosomes to engulf cargos targeted for degradation is the hallmark of autophagy. This process is membrane-remodeling intensive and requires over 30 proteins, but how their actions are spatially and temporally coordinated is a major knowledge gap. Atg3 catalyzes a direct covalent conjugation of LC3 to the amino group of phosphatidyl- ethanolamine (PE) lipid. The conjugate LC3–PE triggers phagophore expansion and acts as an adaptor for sequestering cargos for breakdown. Our study has revealed that selective binding of human Atg3 (hAtg3) to highly curved membranes is tightly linked to its conjugase activity via a multifaceted membrane association mechanism. In this system, the highly curved membrane functions like a classical E3 ligase by bringing substrates (Atg3–LC3 and PE lipids) into proximity and priming the active site of Atg3 for catalysis. These results have made major contributions to establishing and advancing the concept that the distinct membrane structure of the cup-like phagophore spatiotemporally regulates autophagosome biogenesis. We are determining the structural and molecular basis of how the multifaceted membrane association of hAtg3 coordinates its curvature sensitivity and conjugase activity to promote LC3–PE conjugation only on the target membrane in the context of the putative E3-like Atg12-Atg5/Atg16 complex. My collaborator, Dr. Wang, has recently discovered that phagophore closure requires a subset of ESCRT units including VPS37A, CHMP2A, and Vps4. ESCRTs mediate membrane remodeling and scission throughout the cell. We have demonstrated that recognition of highly curved membranes by two hydrophobic motifs in the VPS37A is essential for its phagophore localization and autophagosome closure. We are investigating how the VPS37A UEVL domain, in conjunction with the two membrane binding motifs, orchestrates the spatiotemporal process of autophagosome closure. Results from our study will provide the necessary foundation for continued analyzing the poorly understood biogenesis of the autophagosome during autophagy and, moreover, shed lights on the interplay between membrane structure and protein function.

NIH Research Projects · FY 2025 · 2025-08

Abstract: Disease progression/ metastasis is responsible for over 90% or 100,000 deaths in patients diagnosed with lung adenocarcinoma (LAD). Metastasis suppressor 1 (MTSS1) is a metastasis suppressor protein critical for maintaining cell cytoskeletal integrity. Its role in lung adenocarcinoma pathogenesis is largely unknown. We have shown that loss of MTSS1 expression is correlated with poor survival in lung adenocarcinoma patients and that re-expression of MTSS1 inhibits in-vivo metastasis. Our recent investigation has demonstrated MTSS1 expression is critical for disease progression in SMARCA4-mutant LAD, an aggressive subtype of lung adenocarcinoma with no targeted therapies. In this proposal, we will characterize a novel mechanism and targetable pathway mediated by MTSS1 in SMARCA4-mutant lung adenocarcinoma. Using whole transcriptome sequencing/pathway analysis, we have discovered MTSS1 expression decreases myeloid cell tumor microenvironment pathway gene expression in SMARCA4-mutant LAD. We have discovered a novel mechanism by which MTSS1 inhibits NF-κB activity in SMARCA4-mutant LAD. ICAM1 was the most significantly downregulated protein by MTSS1 in SMARCA4-mutant LAD and is an important surface marker of the metastasis phenotype in SMARCA4-mutant LAD. Our preliminary in-vivo studies showed that anti-ICAM1 blocking antibody reduces disease progression in SMARCA4-mutant LAD and also upregulated migratory dendritic cell population in the tumor microenvironment. Migratory dendritic cells are robust tumor antigen presenting cells that also upregulates PD-L1 expression in the tumor microenvironment. Aim 1 will evaluate the synergistic effect of anti-ICAM1 and anti-PD-L1 therapy on SMARCA4-mutant LAD using an immunocompetent CD34+ humanized orthotopic mouse model. Aim 2 will further characterize the alterations of myeloid cells within the tumor microenvironment as well as the single cell gene transcriptional changes following this novel combined therapeutic approach in SMARCA4-mutant LAD. The novel treatment strategy proposed could provide an effective therapy for a lung adenocarcinoma subtype which is resistant to currently available treatments.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Hidradenitis Suppurativa (HS) is a chronic, debilitating inflammatory skin disease that affects the pilosebaceous units in skin folds in 1-4% of the population. It often leads to disfiguring and permanent scarring. Histologically, lesions exhibit epidermal hyperplasia, hair follicle abnormalities, and inflammation. HS skin lesions exhibit a diverse immune cell infiltrate and complex cytokine profile, which evolves with disease severity. Current FDA-approved therapies targeting TNF and IL-17A are effective in only 30-40% of patients, leaving many patients without an effective approved therapeutic option. Recently, JAK inhibitors (JAKi) have been successful in clinical trials, but these drugs carry a black box warning, with increased risks of developing serious infections and cardiovascular events. RNA-seq studies demonstrate enhanced action of JAK/STAT- mediated type I interferons (T1-IFNs) in HS lesions. However, the induction, signaling mechanisms, and functional effects of the HS T1-IFN response are unexplored. Our preliminary data reveal for the first time that a single, non-classical T1-IFN subtype, IFN, is selectively induced in HS skin lesions. While signaling by different T1-IFN subtypes is often viewed as redundant, T1-IFN signaling is remarkably plastic, leading to distinct, context-dependent functional effects, like the effects mediated by multiple EGFR ligands on epithelial cells. IFN expression was first identified in other epithelial tissues including the female reproductive tract, nasopharyngeal, lung, and intestinal tissues, but not yet described in skin under any condition. In this proposal, we will identify the unique mechanisms of IFN induction, signaling, and function in HS pathophysiology, which can inform novel therapeutics that better target HS pathophysiology and reduce adverse events (e.g. infection). Specifically, in Specific Aim 1 we will investigate how IFN, versus other T1-IFNs such as IFNs/, is preferentially produced and signals in HS. In Specific Aim 2 we will investigate how IFN can uniquely produce functional effects in keratinocytes and fibroblasts that are critical to HS pathophysiology. Successful completion of this project will elucidate the mechanisms by which a non-classical T1-IFN contributes to the pathogenic cycle of inflammation and tissue disruption in HS. This new knowledge is critical to our overall understanding of HS pathophysiology. The study of non-classical IFNs, like IFN, propels innovative research into understanding the complexities of the T1-IFN response, which has previously focused on IFNs, IFN, and more recently IFN. Considering individual T1-IFNs as distinct and plastic signaling molecules will significantly advance our understanding of their subtype specific skin functions and how IFN uniquely influences HS pathophysiology. This will lay the foundation for potential selective therapeutic interventions. Future therapeutic approaches will result from our efforts to discover new HS pathomechanisms in support of NIAMS goals to improve the treatment for this debilitating disease.

NIH Research Projects · FY 2025 · 2025-08

Project Summary This F31 application focuses on investigating the mechanisms of EXO1 regulation as a potential target for a novel chemotherapeutic. I am requesting support for further developing a project which I have previously published a manuscript. This proposal addresses a fundamental gap in knowledge within the DNA repair field and could significantly impact cancer treatment efficacies and overall patient survival. Additionally, the resources provided by this application would greatly benefit my development as a graduate student and allow me to reach my long-term goal of becoming an independent scientist at a research-intensive academic institution. This proposal would grant me the opportunity to learn new in vitro laboratory techniques while also applying techniques I have previously learned to new aspects of my project. Furthermore, I propose pharmacogenomic screens to identify novel genes that are synthetic lethal with EXO1 and increase cisplatin sensitivity. As I have not previously worked with CRISPR screening methods, this fellowship would allow me to further develop my laboratory skills. Receiving this award would provide me with a complex mix of critical skills that will be important for my development into a well-rounded scientist, and for which are highly attainable due to the support and resources provided by my mentorship team and at the Penn State College of Medicine. In this proposal, I focus on elucidating the mechanisms behind PCNA- and MRE11-mediated regulation of EXO1 as well as identifying other unknown genes that impact EXO1-mediated ssDNA gap processing and ultimately chemosensitivity. My recent work showed that EXO1 binds to nascent DNA after the formation of PRIMPOL- generated gaps and performs ssDNA gap expansion (Nusawardhana, et al, 2024). Moreover, I discovered that loss of USP1, a PCNA deubiquitinase, suppresses EXO1 recruitment to ssDNA gaps, indicating a role for ubiquitinated PCNA in regulation of EXO1. Here, I aim to expand on my previous work to further define the mechanism behind this DNA damage tolerance pathway, reduce this gap in knowledge, and gain information on how EXO1 can be targeted mechanistically for future chemotherapies. To investigate the regulation of EXO1 in genome stability, I will explore two aims: 1) determine how PCNA and MRE11 regulate EXO1 in genomic stability, and 2) identify genes that increase EXO1-mediated cisplatin sensitivity using CRISPR screens. Achieving these aims will expand my knowledge in the DNA repair field and uncover the mechanisms of EXO1 regulation. Not only will this work advance my personal goals, but it will significantly impact chemotherapeutic strategies by providing novel information about EXO1 as a potential cancer drug target.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT The cGAS-STING pathway is essential for DNA sensing and plays vital roles in host immunity to pathogens and anti-tumor responses. Activation of cGAS-STING needs to be tightly controlled to promote immunity to pathogens and tumors but prevent unrestrained or chronic inflammation that could be detrimental to host fitness. Given that dysregulated or aberrant activation of cGAS-STING by host DNA has been linked to autoimmunity, autoinflammatory diseases, neurodegenerative diseases, and aging-related chronic inflammation, understanding the mechanisms of the negative regulation of cGAS-STING is of critical importance. After binding to its ligand 2’3’-cGAMP, STING traffics from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. At the trans-Golgi, STING activates the kinase TBK1 and the transcription factor IRF3 to induce type I interferon (IFN). To ensure transient cGAS-STING activation, STING is sorted post-Golgi by the adaptor protein complex 1 (AP-1) into clathrin-coated vesicles which become encapsulated by lysosomes via ESCRT-mediated microautophagy triggering STING degradation. However, the precise mechanisms controlling COPII-dependent STING trafficking and its negative regulation are largely unknown. In preliminary studies for this exploratory proposal, we provide evidence that the endosomal RING-domain E3 ubiquitin ligase RNF11 functions as a negative regulator of STING activation. RNF11-deficient bone marrow-derived macrophages (BMDMs) exhibit more rapid STING degradation and enhanced STING-induced type I IFN and proinflammatory cytokines. Because RNF11 localizes to early and recycling endosomes and can regulate vesicular trafficking of proteins through COPII vesicles, we hypothesize that RNF11 negatively regulates COPII-mediated STING trafficking from the ER to the Golgi. The goals of this project are to determine the mechanisms of RNF11 regulation of STING (Aim 1), and the role of RNF11 in inhibiting a STING-induced inflammatory response (Aim 2). We anticipate that completion of these studies will provide new insight into the negative regulation of the cGAS-STING pathway which could advance cancer immunotherapies and lead to new treatment strategies for autoimmune, autoinflammatory, and neurodegenerative diseases.

NIH Research Projects · FY 2026 · 2025-06

Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) comprise a spectrum disorder characterized by progressive neuronal degeneration. A pathological hallmark of ALS/FTD is the presence of cytoplasmic aggregates containing the RNA binding protein TDP-43 (97% of ALS and 45% of FTD cases). To elucidate the molecular underpinnings of ALS/FTD, we developed Drosophila models of TDP-43 proteinopathy based on overexpression of human TDP-43 in motor neurons (ALS) or mushroom body (MB) neurons (FTD). These models exhibit remarkable similarities to the human disease, including cytoplasmic aggregates (ALS/FTD), neuromuscular deficits accompanied by locomotor dysfunction and reduced lifespan (ALS) as well as memory and sleep deficits (FTD). Using ALS fly models we identified phenotypes that were subsequently validated in patient derived iPSC motor neurons (iPSC MNs) or spinal cords, including alterations in the microtubule associated protein Futsch/MAP1B and the synaptic vesicle chaperone Hsc70-4/HSPA8. These findings support the ribostasis hypothesis, which posits that in disease, TDP-43 mis-localizes to the cytoplasm where it sequesters RNAs rendering them unavailable to ribosomes for translation. To further probe this hypothesis we used a combination of RNA ImmunoPrecipitations (RIP) and Tagged Ribosome Affinity Purifications (TRAP) in fly models of TDP-43 proteinopathy. Functional annotation tools highlight multiple targets and pathways with altered ribostasis in TDP-43 proteinopathy, including dally-like protein (Dlp/GPC6), a regulator of trans-synaptic signaling and plasticity at the neuromuscular junction (NMJ) and a risk factor for Alzheimer’s Disease. We found that Dlp/GPC6 is reduced at the fly NMJ but accumulates in the ventral nerve cord in flies, and ALS patient iPSC MNs and spinal cords. Interestingly, Dlp is significantly reduced in Drosophila MBs in an age dependent manner. Taken together, our published work and these findings led us to hypothesize that TDP- 43 toxicity in neurons is caused in part by translation dysregulation of specific mRNA targets including Dlp/GPC6. To test this hypothesis we will use genetic and molecular approaches in flies to elucidate the mechanism by which Dlp/GPC6 contributes to TDP-43 dependent, neuronal and synaptic dysfunction in ALS. These findings will be validated in patient derived iPSC MNs, NMJs and tissues (Aim 1). We will also establish the contribution of Dlp/GPC6 to FTD using flies and patient derived cortical neurons (CNs), organoids and tissues (Aim 2). Finally, we will use Non-Canonical Aminoacid Tagging (NCAT) in vivo, in flies to identify specific, TDP- 43 dependent changes in new protein synthesis that will be validated in iPSC NMJs and organoids. These studies in ALS and FTD relevant models are expected to fill a critical gap of knowledge, on TDP-43’s role in translation, to mechanistically determine the basis of axonal and synaptic dysfunction in TDP-43 proteinopathy, to provide an opportunity for probing neuronal specific vulnerabilities and may uncover novel molecular targets and pathways with therapeutic potential for ALS/FTD.

NIH Research Projects · FY 2026 · 2025-06

PRORJECT ABSTRACT: JC Polyomavirus (JCPyV) is a ubiquitous asymptomatic virus that infects up to 90% of humans. In immunocompromised individuals, JCPyV can cause progressive multifocal leukoencephalopathy (PML), an often-lethal demyelinating neurodegenerative disease. Because polyomaviruses (PyV) are species-specific, mechanisms of PML pathogenesis are incompletely understood. The Lukacher group studies Mouse Polyomavirus (MuPyV), a natural murine virus, to define virologic and immunologic factors involved in PyV CNS infection and disease. Published work from the Lukacher group has shown that Type I Interferons (IFNs) directly inhibit MuPyV replication and are necessary to mitigate MuPyV encephalitis. The pathway(s) of Type I IFN induction during PyV infection in vivo is unknown. In vitro, cyclic GMP-AMP Synthase (cGAS) and Stimulator of IFN Genes (STING) have been implicated in limiting viral infections by a number of viruses with double-stranded (ds) genomes, including JCPyV and MuPyV. Our preliminary data using STING-/- cells confirms the importance of STING in inducing Type I IFN upon MuPyV infection in tissue culture. In this way, STING is the central adapter protein responsible for the first wave of Type I IFNs. Signal Transducer and Activator of Transcription (STAT) 1 is a downstream target of Type I IFNs. Signaling via STAT1 potently inhibits MuPyV replication. How STAT1 acts as a mediator of antiviral activity in MuPyV infected cells in vivo is unknown. The goal of this proposal is to determine whether cGAS-STING (Aim 1) and/or STAT1 (Aim 2) act in MuPyV-infected neuroglia in vivo to promote the antiviral Type I IFN axis and control virus- induced neurovirulence.

NIH Research Projects · FY 2025 · 2025-06

FTD is the second most common neurodegenerative dementia after Alzheimer’s disease (AD) in adults under the age of 65. Pathologically, 45% of FTD cases are characterized by cytoplasmic protein aggregates containing TAR DNA-binding protein 43 (TDP-43). Genetically, up to 43% of FTD patients have a family history of dementia or related neurodegenerative diseases, with mutations in the C9orf72 (C9) gene representing the most common genetic abnormality in FTD (10-30% of FTD cases). C9 mutations are expansions of GGGGCC (G4C2) hexanucleotide repeats (HR) within its non-coding first intron. When affected by dementia, individuals with C9 FTD most frequently show clinical features of behavioral variant (bv) FTD accompanied by memory impairment. To begin addressing the mechanism of cognitive impairments in C9 FTD/ALS patients, the Sattler group has recently used single nuclei RNA seq (snRNA seq) and, by comparing the transcriptomes of neuronal nuclei containing TDP-43 associated cryptic exons (CEs) to those of nuclei containing canonical splice junctions, identified significant changes in gene expression linked to TDP-43 pathology. Independently, using RNA immunoprecipitations in a Drosophila model of dementia based on TDP-43 overexpression in mushroom bodies (MBs), a neuronal circuit controlling dementia relevant behaviors, the Zarnescu group identified a subset of TDP-43 candidate mRNA targets overlapping with differentially expressed genes in neuronal nuclei with TDP-43 pathology in FTD patients, referred herein as “C9 FTD-TDP targets”. These findings led us to hypothesize that “C9 FTD-TDP targets” mediate dementia relevant phenotypes in Drosophila models of C9orf72 FTD and exhibit altered expression in FTD patient derived neurons and tissues. To test this hypothesis we will cross-validate our C9 FTD-TDP targets between novel Drosophila models of dementia based on G4C2 HRE expression in MBs and human experimental models including iPSC cortical neurons and post-mortem tissues. First, we will use molecular and genetic approaches to identify C9 FTD-TDP targets that are altered in Drosophila models of C9orf72 FTD and modify C9orf72 dependent axonal degeneration, polyGR accumulation, working memory, sleep and lifespan. Second, we will validate C9 FTD-TDP targets in patient cortical neurons and post-mortem tissues. These cross-validation experiments will identify physiologically significant targets of G4C2 HR expansions in neurons and will highlight potentially novel therapeutic approaches for C9 FTD.

NIH Research Projects · FY 2026 · 2025-06

Abstract Impulsive aggression and chronic irritability (IACI) often occur together and are some of the most common reasons why children present for behavioral health (BH) care. ADHD is frequently associated with IACI as upwards of 50% of youth with ADHD manifest impairing levels of it. IACI is the most common reason that children with ADHD are prescribed antipsychotics and admitted to inpatient BH units. Systematic dose optimization of CNS stimulants improves levels of IACI, reducing the need for these more intensive and burdensome treatments. However, response varies, with over half of children with ADHD showing meaningful improvement, upwards of 40% receiving minimal benefit and 3 to 10% exhibiting increased IACI levels. Phenotypic and demographic variables are of limited utility for predicting response, suggesting the need to move beyond symptoms in the search for treatment predictors. Youth with ADHD and IACI struggle with multiple aspects reinforcement learning (RL), defined as learning from interactions with the environment to reach a goal. Successful RL efforts tap multiple cognitive functions. In controlled laboratory tasks, youth with IACI and ADHD exhibit abnormal behavioral and neural response to reward (reward responsiveness), difficulty processing environmental cues to adapt behavior (set shifting/goal updating) and impaired attentional allocation when blocked from a goal (frustrative nonreward). Event-related potentials (ERP) exist for each of these research domain criteria (RDOC) that serve as established neural measures offering a child friendly means to assess their contribution to observable levels of IACI. CNS stimulants improve functioning in these specific realms and impact these associated ERPs to the degree that differences between ADHD and non- ADHD youth disappear. In response to NIMH’s Notice of Special Interest on aggression (NOT-MH-22-095), we will examine the capacity of these ERPs to predict levels of IACI in naturalistic settings. We will then assess if interindividual variance in the capacity of CNS stimulants to impact RL associated ERPs accounts for differences in the clinical effects of these medications on IACI at home using a multimethod battery integrating ERPs, guardian report and task performance. Specifically, we will examine variance in the reward positivity (RewP) ERP when receiving reward feedback, the switch positivity (SwP) ERP measuring mental effort when cued to shift set and the change in P3b amplitude measuring attention allocation when transitioning from reward to nonreward on a go-no-go task. To achieve these aims, 136 children with ADHD and elevated IACI will have their CNS stimulant dose optimized over six weeks and then complete a two week within subjects crossover trial of placebo versus optimal dose. ERP collection will be completed within each blinded week. Guardian ratings will be gathered 3x per day including during peak and off-peak times of medication effect to capture variance in IACI levels within the day and disentangle reports of worsening IACI related to loss of previously beneficial medication effects versus those most likely due to a direct adverse response to drug.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY/ABSTRACT Overdose deaths have increased, reaching all-time-highs recently. Harm reduction (HR) strategies can help reduce the number of these fatalities, while connecting individuals to substance use disorder (SUD) treatment when they are ready. HR-focused smart vending machines (sVMs) delivering naloxone, fentanyl test strips, and other public health items, represent a potential non-stigmatizing, low-barrier approach to HR that may increase access and engagement with underserved populations, reduce overdoses and related public health threats, and health disparities in SUD. As HR-focused VMs grow in popularity in the US and are implemented in communities with different characteristics and preferences, there is a need for an implementation science- driven, rigorous approach to implementation and evaluation of VMs’ impact on individuals and communities. This K23-supported study will build the foundation toward implementation and evaluation strategies for sVMs in communities impacted by overdoses and SUD. Using a Type 1 effectiveness-implementation hybrid design, the K23 study will test the hypothesis that application of evidence-based strategies to develop and test research protocols will promote future effective implementation and evaluation of HR-focused sVMs in diverse communities. The K23 will identify implementation barriers and facilitators to refine an early-phase protocol developed during a pilot study, using the Exploration, Preparation, Implementation and Sustainment (EPIS) framework and qualitative input from community partners. The refined protocol will guide sVM placement in a new community setting (Aim 1). The sVM’s reach, utilization, adoption, and acceptability will be evaluated in a convergent mixed methods design, informed by the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework (Aim 2). The sVM’s impact will also be explored using the convergent mixed methods approach and public health data from existing, local databases (Aim 2). Finally, the study will also use an explanatory sequential mixed methods design to assess the utility of sVM’s data to guide community resource allocation. The sVM-collected data on user interest in specific services, and interviews with key community stakeholders will help assess the community’s capacity to meet the expressed demand for specific services, informing resource allocation (Aim 3). By applying evidence-based approaches to HR-focused sVM and its evaluation, this research is a critical first step toward future rigorous implementation and dissemination of sVM as a non-stigmatizing, accessible path for HR and SUD in underserved populations. The mentored research career development award (K23) will also support PI’s in-depth training in addiction medicine, implementation science, mixed methods, community-engaged research, and large dataset analyses that will enable her to become a successful, independent physician-scientist who develops, implements, and evaluates innovative community-based interventions to reduce SUD-related harms and health disparities.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY The goal of this Shared Instrumentation Grant application is to provide high-parameter flow cytometric and image-based cell sorting to the Penn State College of Medicine research community through the well- established Flow Cytometry Core Facility by purchase of the BD FACSDiscover S8 Cell Sorter. Our current high-parameter sorter will soon become obsolete by the manufacturer, creating an urgent need for its replacement to avoid interruptions to current NIH-funded projects. Cell sorting by high-parameter flow cytometry enables isolation of pure target cell populations from a complex mixture of cell types derived from a variety of sources, an essential technology for our NIH-funded users. These cell populations are identified using a strategically developed panel of fluorochrome-tagged biomarkers which are excited by lasers and the resulting emission captured by sensitive photodetectors to yield intensity signals linked to each cell. Through electronic gating of the resulting cell subpopulations, each cell of interest is electrostatically sorted into a receptacle of choice for further experiments. Multiple populations and large numbers of cells can be simultaneously identified and collected in a short time due to processing of thousands of events per second. Our current heavily used BD Biosciences FACSAria instrument will become obsolete by the manufacturer in 2025. This instrument is a workhorse for our users to purify cell populations for a wide array of downstream studies investigating biological and genomic cell properties. In this proposal, we describe the new BD FACSDiscover S8 Cell Sorter and how its state-of-the-art technology is essential for the NIH funded projects of our users. The S8 sorter uses full spectrum fluorescence detection to increase the sensitivity and expand the number of fluorochromes that can be simultaneously detected on single cells. In addition, this instrument provides novel image-based cell sorting, not previously achievable, so dramatically expands the cell sorting capabilities that can be offered to support our investigators. This new instrument will support the funded research of 11 major users and three minor users addressing key questions in medical science that include cancer, infectious diseases, immunology, heart disease and neuroscience. The added benefit of imaging-based quality control combined with full spectrum fluorescence detection is expected to increase the rigor and reproducibility of cell sorting experiments. Penn State College of Medicine provides consistently strong institutional support in the form of a fully staffed Flow Cytometry Core and purchase of yearly service contracts to maintain state-of-the-art instrumentation in top working order. The studies proposed by our investigators match well with the public health goals established by the NIH and Penn State College of Medicine. This instrument will increase the achievable discoveries supported by existing NIH funding and will provide a powerful research tool to support new NIH funded studies in cancer, immunology, virology, genomics, cardiovascular disease and neuroscience.

NIH Research Projects · FY 2025 · 2025-05

ABSTRACT Amyotrophic lateral sclerosis (ALS) is fatal neurodegenerative disease that affects 2/100,000 individuals worldwide. The disease is caused by motor neuron dysfunction and death, which in turn causes paralysis within 2-5 years of diagnosis. A pathological hallmark of ALS is the presence of cytoplasmic aggregates containing the DNA/RNA binding protein TDP-43 in 97% of ALS cases. Furthermore, TDP-43 is associated with cytoplasmic aggregates across a broad range of neuromuscular and neurodegenerative disorders including ALS, inclusion body myositis (IBM) and fronto-temporal dementia (FTD). While a plethora of studies have focused on TDP-43 pathophysiology in neurons and glia, much remains unknown about TDP-43 proteinopathy in muscle, the cell type most visibly affected by ALS as it undergoes rapid atrophy. TDP-43 loss of function has been shown to cause muscle weakness and degeneration in mice, zebrafish and flies. Recent studies have shown that TDP-43 associates with myogranules and plays a role in muscle formation, likely by regulating cytoplasmic mRNAs, including several that encode sarcomeric proteins such as titin and myosin heavy chain. In addition to their physiological role in muscle differentiation and regeneration, TDP-43 containing myogranules are also present in mouse models of IBM, consistent with also having a role in disease pathomechanisms. Furthermore, these findings suggest that TDP-43 containing myogranules provide an opportunity to uncover differences between protective and toxic aggregates.To address the gap in knowledge regarding TDP-43 pathophysiology in muscles, we set out to study TDP-43 dependent, muscle specific alteration in newly synthesized proteins. Preliminary results using Non-Canonical Aminoacid Tagging (NCAT) in vivo, in the context of Drosophila models of TDP-43 proteinopathy show that TDP-43 overexpression in muscles causes a marked reduction in newly synthesized proteins involved in translation itself, myofibril assembly, mitochondria and nuclear pores among others. We hypothesize that similar to its neuronal role, TDP-43 alters the translation of specific mRNA targets in muscles, and this in turn may impact the morphology and/or function of muscles, and contribute to the muscle atrophy observed in patients. We will test this hypothesis by first identifying TDP-43 translation targets in vivo, in Drosophila muscles using both overexpression and loss of function approaches (Aim 1). Next, we will validate candidate targets in fly muscles and patient tissues (Aim 2). These studies are poised to identify novel, muscle specific therapeutic targets of TDP-43 proteinopathies and related neuromuscular disorders.

NIH Research Projects · FY 2026 · 2025-05

Project Summary. The Problem. In a recent paper entitled “Preaddiction - A Missing Concept for Treating Substance Use Disorders”, McLellan, Koob and Volkow [7] argue that intervention need not be limited to the final stage of the disease and likely would be more successful if administered early in the disease state. Treatment early in the disease of addiction, however, requires early identification of risk. At present, we do not necessarily have the tools to identify risky preaddiction in humans; and treatments for ‘preaddiction’, consequently, have not been well explored. That being said, we have used an animal model that can identify individual vulnerability early in the development of the disease and, then, can be used to study early intervention. The Model: If a cue precedes the delivery of drug, the cue will, with repeated pairings, come to elicit a conditioned behavioral/physiological response that opposes the impact of the coming drug. The valence and magnitude of this conditioned behavioral/physiological response reflects the nature and intensity of the impact of the drug on the organism. Thus, in our model, a saccharin cue predicts the impending availability of cocaine and rats that exhibit the greatest conditioned aversion (i.e., the greatest conditioned withdrawal) toward that saccharin cue ultimately exhibit the greatest drug seeking and taking. Treatment. This finding led us to hypothesize that addiction involves a hi-jacking of not only reward substrates, but of substrates involved in physiological need as well – i.e., in the need for drug. Given this need-state hypothesis, we predict that known satiety agents, such as glucagon-like peptide-1 (GLP-1), will reduce the need for drug as reflected by a reduced aversive conditioned response and reduced drug seeking and taking. Specific Aim 1 will challenge this hypothesis by testing if systemic treatment with a GLP-1 receptor agonist will block the aversive behavioral and physiological conditioned response to the taste cue (i.e., aversive taste reactivity behavior (gapes) and low dopamine in the nucleus accumbens shell) and subsequent cocaine seeking and taking in rats. Specific Aim 2 will measure c-Fos activation patterns in the entire brain to determine whether greater rejection of the cocaine- predicting cue in high taker/seekers is accompanied by activation of substrates involved in aversion/withdrawal and reward/seeking and, importantly, whether these patterns of activation also are reversed by treatment with a GLP-1R agonist. Specific Aim 3 will employ site-specific infusion of the GLP-1R antagonist, Extendin-9, to begin to interrogate the circuits identified in Aim 2. Thus, in the completion of three aims, we will use the conditioned withdrawal elicited by a cocaine-predicting saccharin cue to track the development of addiction in both vulnerable and resistant rats, we will identify neural circuitry associated with this need-state, and we will use a known satiety agent, a GLP-1R agonist, to reverse these patterns in brain and behavior early in the development of the disease – i.e., during ‘preaddiction.’

NIH Research Projects · FY 2026 · 2025-04

Despite the availability of recommended human papillomavirus (HPV) cancer prevention services that can prevent over 37,300 HPV cancers in the US every year, only 62.6% of girls and boys were up-to-date in 2022. Low-quality provider recommendations and time constraints during clinic visits limit parents’ opportunities to discuss and make HPV cancer prevention decisions. Pre-visit education to parents could complement provider communication. There is a critical need to identify better communication strategies to increase uptake of the recommended HPV cancer prevention service, including the use of narrative messaging and existing digital technologies in clinics and at home (patient portal, mobile devices). We propose the Stories to Prevent (StoP) HPV Cancers Study, a randomized controlled trial (RCT), to evaluate the association of a narrative communication intervention delivered through digital and mobile technology before clinic visits on initiation rates of HPV cancer prevention in primary care. Parents of children ages 9-12 will be randomized (1:1) to a brief video intervention showcasing local cancer survivors narrating their experiences with an HPV-associated cancer and recommending prevention or control (placebo video). Our overall objectives are to understand (1) how narrative communication is received in the context of HPV cancer prevention decision-making, including the psychological mechanisms (cognitive and emotional) narratives use to inform parents’ informed decisions, and (2) how digital technologies can support the delivery of this intervention. This project builds on our feasibility study with parents of adolescents, where we found that 52% of the sample said that their decision to get the recommended HPV cancer prevention service was influenced by a narrative video intervention that we delivered through the patient portal before clinic visits. Aim 1 is to evaluate the association of our narrative communication intervention on HPV cancer prevention initiation rates among 9- to 12-year-olds. We will recruit cancer survivors to film their narratives and produce our intervention videos (n=6). RCT participants will be the parents (n=200) of children ages 9-12 who have not yet initiated HPV cancer prevention. Our primary outcome is initiation of the recommended HPV prevention service among children ages 9-12 at the time of the wellness visit. Aim 2 is to explore the effect of narratives on theory-based mediators, including parents’ cognitive (e.g., risk perception) and emotional (e.g., hope, anticipated regret) reactions. Our expected outcome is to demonstrate the efficacy of a highly scalable intervention to educate, engage, and encourage parents to initiate HPV cancer prevention for their children. This study is innovative in (1) maximizing the use of available clinic and mobile technologies to deliver messaging responsive to parents’ information preferences and (2) in developing a greater understanding of the use of narratives in the context of cancer prevention. We expect a significant impact on HPV cancer prevention as we address parents’ communication needs with an intervention that fits the existing technology ecosystem and workflow of primary care clinics.

NIH Research Projects · FY 2026 · 2025-02

Project Summary The long-term goal of our laboratory is to elucidate the molecular basis for intestinal homeostasis and its dysregulation in intestinal inflammation, and to develop novel approaches for prevention and therapy of inflammatory bowel diseases (IBD). The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a selective barrier between luminal and host environment. Although increased intestinal permeability with loss of intestinal TJ barrier function is associated with intestinal disorders and IBD, the TJ barrier is complex and context dependent, and the physiological need of epithelial paracellular permeation for the appropriate gut immune response is being recognized. The aryl hydrocarbon receptor (AHR), a susceptibility locus for IBD and an environmental sensor, acts as a class I, basic helix-loop-helix transcriptional regulator. Deficiency of AhR increases severity of experimental colitis by perturbing intestinal stem cell homeostasis and differentiation and dysregulating the gut immune responses. On the other hand, activation of the AhR pathway improves colitis outcomes in animal IBD models. Thus, there is a lot of scientific and clinical interest in the regulation of AhR activity. In our present studies, and consistent with published reports, we found that the severity of colitis is increased in intestinal epithelial specific AhR deleted (Ahr∆IEC) mice in several IBD models. Interestingly, we found that the baseline colonic TJ barrier permeability is reduced in Ahr∆IEC mice. This reduction in colonic paracellular permeability was accompanied by increased inflammatory tone (increased pro-inflammatory cytokines and inflammatory phenotype of dendritic cells and macrophages) and impairment of immune tolerance (reduced IL-10, TGF-β, Foxp3, Programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte- associated protein 4 (CTLA4)) in Ahr∆IEC mouse colonic mucosa. Based on these findings, we hypothesize that reduction in the homeostatic paracellular permeability, in the absence of AhR, affects immune tolerance. We will address this hypothesis with the following specific aims: 1) To determine the mechanisms of reduced intestinal epithelial permeability in the absence of AhR and 2) To delineate the role of AhR-regulated paracellular permeability in gut immune tolerance. The proposed studies in Ahr∆IEC mouse model, presents a unique opportunity to study the role of paracellular permeation in maintaining the gut homeostasis. Once completed, this study will provide unique knowledge about the role of intestinal barrier in shaping up the immune homeostasis which can provide a foundation for further investigations to explore pathogenesis-based therapeutic tools for IBD and other immune-dysregulated diseases.

NIH Research Projects · FY 2026 · 2025-02

Pattern recognition receptors (PRRs), including the DNA sensing nucleotidyl transferase cGAS and the endoplasmic reticulum (ER)-localized adaptor STING, detect pathogen-associated molecular patterns (PAMPs), which in turn leads to induction of type I interferons (IFNs) via IRF3 and NF-κB transcription factors. The cGAS- STING DNA sensing pathway can also be aberrantly activated by damage-associated molecular patterns (DAMPs) such as mitochondrial DNA (mtDNA). To maintain mitochondrial quality control and homeostasis, cells have evolved dedicated mechanisms to remove damaged or superfluous mitochondria through mitophagy, an autophagy-mediated lysosomal degradation pathway. Impaired mitophagy leads to the release of mtDNA into the cytoplasm, triggering activation of the cGAS-STING pathway and induction of type I IFN and inflammatory cytokines. Emerging studies have implicated aberrant activation of the cGAS-STING pathway in numerous inflammatory diseases including aging-related chronic inflammation and neurodegeneration. However, the cellular mechanisms that prevent the release of mtDNA into the cytoplasm and subsequent cGAS-STING activation and chronic inflammation are incompletely understood. Our preliminary data indicate that the zinc finger protein and A20 family member ZFAND6 functions as a novel regulator of mitophagy. We have generated Zfand6–/– mice and found that Zfand6–/– bone marrow-derived macrophages (BMDMs) exhibited spontaneous expression of interferon-stimulated genes (ISGs) in a STING-dependent manner. The ISGs were upregulated in Zfand6–/– cells due to the accumulation of damaged mitochondria accompanied by increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential. Zfand6–/– cells exhibited impaired mitophagy basally and after acute mitochondrial damage. ZFAND6 interacts with the E3 ubiquitin ligase TRAF2 and promotes its recruitment to damaged mitochondria suggesting that ZFAND6 may be an adaptor or shuttling protein for TRAF2. Zfand6–/– mice spontaneously accumulate lymphoid aggregates in their lungs and are highly sensitive to inflammatory stimuli, likely through mitochondrial DAMPs priming innate immune sensing pathways such as cGAS-STING and the NLRP3 inflammasome. The central hypothesis driving these investigations is that ZFAND6 serves as an adaptor for TRAF2 that is essential for TRAF2-dependent mitophagy. The goals of this proposal are to determine the mechanisms of: ZFAND6 regulation of mitophagy (Aim 1), ZFAND6 regulation of inflammation (Aim 2) and ZFAND6 regulation of the NLRP3 inflammasome (Aim 3). We anticipate that completion of these studies will provide new insight into the mechanisms underlying the regulation of mitophagy, inflammation and immune homeostasis.

NIH Research Projects · FY 2026 · 2025-01

Abstract Social behavior is regulated by a set of brain regions that integrate external stimuli with internal emotional states to generate context-appropriate behavioral responses. Dysfunctional social behavior has been implicated in many brain disorders including neurodevelopmental disorders (e.g., autism), neurological disorders (e.g., epilepsy), and neurodegenerative disorders (e.g., Alzheimer’s disease). Despite its importance, we have very limited knowledge of underlying neurobiological mechanisms about how emotion can impact neural processing to regulate social behavior. Previously, our unbiased brain mapping study identified dense oxytocin receptor (Oxtr) expression in the ventral claustrum-dorsal endopiriform nucleus (vCLA-EPd). Limited previous studies suggest that the vCLA-EPd is involved in multi-sensory processing, emotion, memory, and attention. The dysfunction of the vCLA-EPd has been implicated in many brain disorders, including Alzheimer’s disease, autism, epilepsy, and even depression. We found that vCLA-EPd Oxtr neurons are mostly excitatory neurons with extensive connections to the olfactory, limbic, and many neuromodulatory areas. Moreover, we found strong projections from vCLA-EPd Oxtr neurons to the medial prefrontal cortex (mPFC), a hub region that regulates social behavior. Importantly, our neural recording in awake and behaving mice showed that vCLA-EPd Oxtr neurons have high neural activity during resting and exploratory states, while novel social cues induced long- lasting suppression of their activity. These preliminary data led us to hypothesize that the vCLA-EPd serves as a bottom-up regulator to disinhibit the mPFC to mediate the initial social cognition. To test the hypothesis, we will carry out three complementary experiments to understand the neurobiological mechanisms of the vCLA-EPd. In Aim 1, we will investigate anterograde and mono-synaptic input connectivity of vCLA-EPd Oxtr neurons with high-resolution 3D mapping methods. In Aim 2, we will investigate the detailed molecular characteristics of vCLA- EPd Oxtr neurons by using single-cell transcriptome and spatial transcriptome methods. In Aim 3, we will test our hypothesis that bottom-up disinhibition of the mPFC by the vCLA-EPd is necessary to mediate initial social behavior by using systems neuroscience approaches and electrophysiological methods. Results from the proposal will establish foundational knowledge to understand the anatomy and function of the vCLA-EPd and will critically inform future studies to investigate the vCLA-EPd in various pathological conditions.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT Neurodevelopmental and psychiatric disorders (NPDs) are highly prevalent and devasting neurological conditions without cure. Recent genetic studies on NPDs identified hundreds of high-risk NPD-related genes, yet it remains unclear how these genes individually or collectively affect neurodevelopmental processes to create pathological conditions in the brain. To identify core biological pathways implicated in NPDs, it is critical to understand how the loss of specific NPD genes leads to changes in the molecular and cellular level to alteration in neural circuits, which ultimately result in pathological behavioral changes. Here, we assemble a team of seven investigators with complementary expertise to create an Assay and Data Generation Center (ADGCs) to comprehensively investigate the impact of loss of NPD genes in mice. We aim to investigate NPD genes through Cross-Scale Interrogation of NPD Genes (SING), focusing on gene expression, cellular changes, neural circuits, and behaviors affected by 100 NPD genes in mice. Utilizing Knockout Mouse Project (KOMP) resources, we will examine the effect of NPD gene deletion in mice across developmental stages, employing high-throughput methodologies and integrative data analysis. First, we will utilize single-cell and spatial transcriptome approaches to examine molecular and cellular changes. Second, we will deploy MRI and high-resolution 3D mapping methods to examine altered neural circuit connectivity and brain anatomy. Third, we will employ a high throughput home cage system to automatically track altered behavior. Moreover, we will perform a pilot study to develop CRISPR based viral approaches to rapidly knockout target genes in developing mice and to examine altered neural circuit maturation patterns. We plan to integrate multiscale data and perform machine learning-based analyses to identify shared biological pathways for specific NPDs, and broadly disseminate the data for public access. We expect to generate critical experimental data at an unprecedented scale, to rapidly advance our understanding of key biological pathways of NPDs, which will inform future therapeutic approaches.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Advances in medicine and science have led to improved health in women, yet hypertensive disorders of pregnancy such as pre-eclampsia (PreE) remain one of the reasons that maternal cardiac disease is a leading cause of pregnancy-related death in the United States. In this project we examine the impact of severe PreE on human heart function in women from diverse racial (Non-Hispanic/Black, Non-Hispanic/White, Hispanic, South/Southeast Asian) backgrounds. We will evaluate heart function at time of delivery, and at 6- and 12- months postpartum. Our pilot data demonstrates that women with severe PreE have evidence of persistent heart dysfunction up to 3 months after delivery, and these same women display a significant number of deleterious cardiomyopathic genetic polymorphisms/DCGPs that encode for proteins implicated in cardiomyopathy. In this proposal, (Aim 1) we will describe the cardiomyopathic genetic profile of non-Hispanic Black, non-Hispanic White, Hispanic, and South/Southeast Asian women with severe PreE. Using in-silico analyses, we will characterize the pathogenicity of the identified variants in each racial cohort and determine the potential impact of the variants on peri- and post-partum heart function in each racial group. We predict that founder effect and migration may have an impact on the types of DCGPs present in each racial group, and that disease expression in the form of heart dysfunction, may be unique amongst the groups due to the social construct of race. In Aim 2 we will study the functional impact of specific DCGPs on cardiomyocyte/CM function. Using human iPSC derived CMs we will determine the impact of the mutants on kinetic function at rest and in response to stress. We will also evaluate the relationship of Ca2+ transients to CM function at rest and with stress, to determine the degree to which this may play a role in the observed contractile phenotype, a first step to link mutant status to pathogenicity. We believe that accomplishing the proposed innovative aims will significantly improve our understanding of the impact of race and genetics on heart function in women with severe PreE. This study will be the first human investigation to focus on genetic links and temporal patterns of heart function in a racially diverse cohort with severe PreE, uncovering potential new areas of investigation and future novel therapeutic pathways specific to individual race and cardiomyopathic genetic profile.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Diabetic retinopathy (DR) is a significant ocular complication caused by diabetes that can progress to blindness. Diabetes causes an increased abundance of the stress response protein Regulated in Development and DNA Damage 1 (REDD1) in the retina, which has been implicated in visual function deficits in both preclinical models and diabetic patients. In fact, patients with diabetic macular edema had dose-dependent improvement in best- corrected visual acuity with intravitreal administration of an siRNA targeting the REDD1 mRNA (PF-04523655). However, the approach was abandoned over a decade ago due to its failure to outperform vascular endothelial growth factor (VEGF) blockade. Our laboratory recently made an important discovery that revealed a key weakness in the prior approach used to inhibit REDD1. Specifically, we discovered that formation of a redox- sensitive disulfide bond acts allosterically to prevent the normally rapid degradation of REDD1 protein in the context of diabetes. The observation suggests that REDD1 mRNA knockdown may only be partially effective for reducing REDD1 protein abundance in DR, due to the blockade of REDD1 protein degradation. The objective of this F31 pre-doctoral fellowship is to address important knowledge gaps in the field by characterizing the molecular mechanisms that promote retinal REDD1 protein abundance in response to diabetes and exploring alternative intervention strategies for preventing the retinal complications that are caused by REDD1. The central hypothesis is that diabetes-induced allosteric regulation of REDD1 contributes to increased REDD1 protein abundance, leading to loss of retinal homeostasis and the development of retinal pathology. The proposed studies will employ a newly developed REDD1 CRISPR knockin mouse that expresses a REDD1 variant that continues to be rapidly degraded even after its redox-modification. Aim 1 will characterize the mechanism through which diabetes acts to alter REDD1 proteolysis. The proposed studies will evaluate a role for competitive binding of REDD1 to thioredoxin interacting protein (TXNIP) versus heat shock cognate 70 kDa (HSC70) in the suppressive effect of diabetes on REDD1 proteolysis. To complement this genetic approach, artificial intelligence (AI) molecular docking, Microscale Thermophoresis (MST), and biochemical techniques will also be used to validate small molecule inhibitors of REDD1 allostery. Aim 2 will evaluate a role for REDD1 allosteric regulation in the development of diabetes-induced retinal defects. Through the proposed training plan, I will develop new laboratory skills to evaluate REDD1 protein allostery (e.g., biotin-switch assay, MST) and develop the technical expertise to determine its impact on retinal pathophysiology [e.g., optical coherence tomography (OCT), FITC- BSA perfusion, electroretinography (ERG), behavioral optometry]. This project has significant implications for addressing visual dysfunction in diabetic patients, as it may facilitate the development of improved therapies that more effectively address the specific molecular events that contribute to early disease progression.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Genomic stability is critical for cellular function, however, postmitotic cells such as highly metabolically active neurons face the biggest challenge as it must maintain its genome over organismal lifetime. DNA damage increases with age and is accelerated in Alzheimer’s disease (AD) and multiple neurodegenerative disorders. Hence specialized DNA polymerases have evolved to repair different DNA lesions that destabilize DNA helical structure and obstruct replication and transcription. The Y-family polymerases are unique in bypassing DNA damage and synthesizing DNA past specific lesions, thus recovering replication in dividing cells such as DNA Pol kappa (Polk) that can bypass DNA lesions in both S and G0 phase. Surprisingly, we observed high levels of Polk expression in non-dividing differentiated neuronal nuclei in the mice brain that mislocalizes to lysosomes with age and in mice with AD associated transgenes. Almost nothing is known about Polk in neurons and its role in the protracted aging process. Our long-term goal is to understand the role of Polk in combating the DNA damages caused by cumulative exogenous and endogenous stressors, maintenance of the central nervous system (CNS) genome and its relationship with AD. Here we will investigate Polk's role in neuronal nuclei, and how mislocalization of Polk impacts neuronal maintenance during aging and in AD. This will open a novel line of investigation of specialized Y-family DNA polymerases in age associated neuronal disorders like Alzheimer’s. Our central hypothesis is that Polk assists in multiple DNA damage response pathways to prevent genomic instability and combat constant accumulation of DNA damage in post-mitotic neurons, where its expression is critical in the neuronal nuclei. However, with aging and age-associated neuropathy decline in Polk's expression in the nucleus and concomitant accumulation in the lysosomes results in neuronal genomic instability. We will leverage our proteomics data to identify Polk associated proteins that work together as DNA damage response to sustain neuronal genomic stability. Unbiased proteomics in neuronal nuclei will be performed to identify novel Polk interactors. Overexpression of Polk in aging and AD genotype mice will investigate causal role of Polk in neuronal genomic stability. Specific Aim1 will test the hypothesis that Polk is associated with distinct DNA damage response pathways in neurons and identify novel interactors of Polk Specific Aim2 will test the hypothesis that recovering Polk's expression in neuronal nuclei can rescue genomic stability in aging and AD neurons