Ohio State University

NIH Research Projects · FY 2024 · 2020-07

Abstract Acquired infections after spinal cord injury (SCI) are prevalent, constitute the main cause of death in patients and have been identified as a modifiable risk factor associated with poor neurological and functional recovery. Infection treatment by orthodox antibiotics is complicated by i) spectrum/efficacy gaps, ii) early development of antibiotic resistancies, and iii) failure of antibiotics to prevent infections when applied early in patients with acute CNS lesions. Moreover, new evidence indicates that antibiotics can impair neurological recovery, due to their ability to cause gut dysbiosis. Improved anti-infective treatment strategies are critically needed, which take the immune-compromised status after SCI into account. Recent data have identified unregulated sympathetic tone originating from below the spinal cord injury site (spinally generated sympathetic nerve activity, SNA) as a major driver of the systemic spinal cord injury immune deficiency syndrome (SCI-IDS). SNA has been identified and independently confirmed as a mechanistic target to restore immune function in vivo. Three aims are proposed to answer one main and novel question: Can immune function and host-defense against pneumonia be re-established with SNA-targeting immunotrophic pharmacological neuromodulation (IPN)? Experiments in Aim 1 will use a novel combination of FDA-approved drugs selected to promote immune system function and reduce susceptibility to bacterial pneumonia acutely or at 4 weeks post SCI. Aim 2 will assess the capacity of IPN to normalize different cellular SCI-IDS characteristics and the sympathetic neuroendocrine reflex. Irrespective of SCI, infections are well-known causes of dysautonomia, which is a pathophysiological conduit for septic conversions. Aim 3 addresses whether IPN can blunt infection-associated autonomic dysfunction and neutralize a sepsis risk factor. If successful, data from these experiments will directly inform effective non-antibiotic anti-infective strategies for SCI patients to reduce infection-associated mortality and disability.

NIH Research Projects · FY 2024 · 2020-07

Project Description Dilated cardiomyopathy of unknown cause (DCM) is a major public health problem affecting more than a million people in the U.S. Most DCM is now known to have an underlying genetic basis. First-degree relatives (FDRs) of an individual with DCM are considered to be genetically at risk, particularly if they carry variants classified as pathogenic (P), likely pathogenic (LP) or uncertain significance (VUS) in DCM genes. Practice guidelines recommend that these FDRs undergo serial imaging because prompt intervention may avert advanced disease. While tissue damage is already well underway when DCM is manifest, myocardial tissue changes, termed “pre- DCM” herein, are known to precede adverse changes in myocardial structure and function. Our central hypothesis states that cardiac magnetic resonance (CMR) imaging may detect pre-DCM in individuals with increased genetic risk by identifying myocardial tissue changes prior to myocardial structural and functional changes. CMR measures of myocardial tissue characteristics, including late gadolinium enhancement and myocardial T1 mapping, have been histopathologically validated and have established diagnostic and prognostic value in DCM. Thus, our specific hypotheses state that adverse CMR-based myocardial tissue characteristics will be associated with (1) A higher burden (number) of relevant variants (P, LP, VUS) in established DCM genes; and (2) Subsequent adverse changes in measures of cardiac structure and function. We propose to leverage the DCM Precision Medicine Study, a multisite DCM Consortium study now completing the enrollment of 1300 DCM patients (probands), balanced for race and sex, and their FDRs, most with no history of DCM. FDRs are cascade tested for relevant variants (P, LP, VUS) in DCM genes identified in probands. We aim to (1) Estimate the associations between CMR-based myocardial tissue characteristics and the number (burden) of the proband's variants in DCM genes in at-risk FDRs. In 650 FDRs of probands with LP/P variants and/or VUSs, CMR scans will be completed at 9 participating DCM Consortium sites. The association between CMR-based myocardial tissue characteristics and the number of the proband's variants of each class (LP/P, VUS) carried by an at-risk FDR in a particular age group will be evaluated, adjusting for biologically relevant covariates. We will also (2) Estimate the association between CMR-based myocardial tissue characteristics and subsequent changes in measures of cardiac structure and function in FDRs with normal baseline left-ventricular size and function. FDRs examined in Aim 1 with normal left ventricular size and systolic function will receive a second CMR exam 2.5 years after their baseline exam. We will estimate the covariate-adjusted associations between baseline myocardial tissue characteristics and subsequent changes in CMR-derived measures of cardiac structure and function in groups defined by the most deleterious of the proband's variants carried (none, VUS, or LP/P). This study will validate a CMR-derived “pre-DCM” phenotype for FDRs who carry P or LP variants (established risk), and also provide preliminary evidence that some VUSs are biologically relevant.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Inflammation and vascular leak are common findings across several pathologies associated with arrhythmias. These include atrial fibrillation (AF), which affects up to 3% of the US population. AF progressively worsens, and increases risk of stroke and cardiovascular disease. Thus, we urgently need novel, mechanistically-driven therapies for AF. Vascular leak in AF patients results from elevated serum levels of inflammatory cytokines such as vascular endothelial growth factor (VEGF). While the thrombogenic impact of vascular leak in AF is widely recognized, its role in arrhythmogenesis remains unclear. One possible link between vascular leak and arrhythmia may be myocardial edema. Recent work by the PI demonstrated that edema disrupts sodium channel (NaV1.5) –rich nanodomains within the intercalated disk (ID), slowing cardiac impulse propagation, and prompting arrhythmias. In preliminary studies, VEGF (at levels found in the serum of AF patients) elevated AF inducibility ex vivo and in vivo mouse experiments within 30 minutes. Therefore, we hypothesize that cytokine-induced vascular leak promotes cardiac edema, and contributes to atrial arrhythmias by disrupting NaV1.5-rich ID nanodomains. In this venture, we will employ cutting edge tools including super-resolution microscopy, 3D electron microscopy, and smart patch clamp to investigate the structural and functional impact of vascular leak on the structure and function of atrial IDs. Furthermore, we will utilize an innovative strategy peptide mimetics of adhesion domains will be used to selectively modulate the structure of different ID nanodomains. Aim 1 will use these peptides to investigate how different ID nanodomains contribute to atrial conduction, and probe fundamental mechanisms underlying these structure-function relationships. In new preliminary data, we demonstrate that VEGF- induced vascular leak induces swelling of ID nanodomains and translocation of NaV1.5 from these sites within 30 minutes. Aim 2 will investigate the acute structural and functional impacts of VEGF- induced vascular leak. Aim 3 will use ex vivo and in vivo models to test the efficacy of preserving the vascular barrier and/or ID nanodomains in preventing AF.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Glioblastoma (GBM) is the most common primary brain tumor and one of the most lethal of all cancers. The main challenge in treating GBM is the quickly developing resistance to all kinds of treatments by tumor cells. Whereas our partial understanding of GBM biology is a major roadblock to elucidate the underlying resistance mechanisms. Our laboratory recently uncovered that GBM greatly alters lipid metabolism to gain sufficient lipids for its rapid growth. We identified that sterol regulatory element-binding protein-1 (SREBP-1), a master transcription factor that controls fatty acid synthesis, is highly expressed in GBM and is essential for tumor growth. Our findings were recently validated by multiple groups showing that SREBP-1 is also elevated in various other cancers. However, whether the dramatically altered lipid metabolism facilitates tumor resistance is completely unknown. Moreover, the mechanism that upregulates SREBP-1 expression in cancer cells remains elusive. Interestingly, we recently found that all-trans retinoic acid (ATRA) and 13-cis-RA could significantly reduce the expression of SREBP-1 and lipogenic enzymes in a dose-dependent manner in GBM cells. These retinoic acids are effective drugs in treating acute promyelocytic leukemia and have also been used to treat GBM and other solid tumors, but tumor resistance has been very challenging. To date, both their antitumor and resistance mechanisms remain poorly understood. We examined the expression of their binding partner, retinoic acid nuclear receptor α (RARα) in GBM patient tissues and found it to be highly expressed in tumor tissues and positively correlated with SREBP-1 expression, while inversely associated with poor patient survival. Interestingly, our data further show that 13-cis-RA and ATRA treatment significantly increased the expression of carnitine palmitoyltransferase 1A (CPT1A), a key enzyme shuttling fatty acids into mitochondria for β-oxidation and energy production. Pharmacological inhibition of CPT1A combined with retinoic acid treatment resulted in marked GBM cell death. Together, these novel preliminary data strongly support the hypothesis that 13-cis-RA or ATRA can significantly alter lipid metabolism in GBM and promote fatty acid oxidation to support tumor cell survival and resistance. We further hypothesize that retinoic acid treatment in combination with suppression of SREBP-1 activation or fatty acid oxidation will effectively inhibit GBM growth and overcome tumor resistance. The goal of this study is to identify the previously unreported roles and mechanisms of retinoic acids and RARα in lipid metabolism regulation and GBM growth (Aim 1), and to develop effective combination approaches to target GBM (Aim 2). Completion of this study will uncover the underlying mechanism upregulating SREBP-1 expression and lipogenesis in GBM, provide great insights into understanding the antitumor and resistance mechanisms of retinoic acids, and identify novel strategies to target GBM and overcome retinoic acid resistance.

NIH Research Projects · FY 2025 · 2020-05

Project Summary Biologically available sulfur is essential for the synthesis of methionine (Met) and its derivative, S-adenosyl-L- methionine (SAM). SAM is used for diverse metabolic purposes, serving primarily as a methyl donor for DNA and protein methylation, as a 5’-deoxyadenosyl radical donor for radical-SAM reactions, as an aminopropyl donor for polyamine synthesis and volved in the synthesis of acyl-homoserine lactone quorum sensing molecules in bacteria. As a consequence of this metabolism, a dead-end, sulfur-containing byproduct, 5’-methylthioadenosine (MTA) is formed. MTA is a product inhibitor of polyamine synthesis and MTA accumulation is thought to be toxic. Since the assimilation of inorganic sulfur is energetically costly and many organisms encounter sulfur-poor environments, maintaining or salvaging appropriate cellular organic sulfur pools is critical. Moreover, disruption or reduced functioning of methionine salvage pathways (MSPs) has many health-related consequences including influences on cancer cell growth and liver cirrhosis; intermediates of the pathway have also been shown to influence apoptopic processes, while analogs of these intermediates are promising therapeutic agents. Newly discovered MTA pathways from our laboratory, the DHAP-ethylene and methanethiol shunts, were recently described, the genes of which appear to be widespread and selectively found among several pathogenic species. Nonpathogenic species from these genera do not contain these genes. Thus, the hypothesis is that the shunt genes/enzymes hold some special significance to metabolism of these pathogenic species. Moreover, the same novel genes and enzymes were recently found to participate in radical SAM reactions to generate and metabolize 5’-deoxyadenosine (5dAdo), a structurally similar byproduct to MTA, which could potentially be recycled for carbon salvage. The long-term goal will thus be to determine the role and physiological significance of the DHAP/MTA/5dAdo pathways for sulfur and carbon salvage, and the potential of these pathways to influence the successful metabolism of extraintestinal pathogenic Escherichia coli (ExPEC), including uropathogenic (UPEC) strains which contain these genes on a specific pathogenesis island. A specific aim (Aim 1) will be to determine the precise role of these genes and encoded enzymes and resolve further metabolic steps in sulfur/carbon salvage via whole cell feeding experiments using radio-labeled (14C) and 13C MTA and 5dAdo metabolites in wild type and mutant strains. These in vivo studies will be supplemented by in vitro analyses with specific enzymes. The second aim (Aim 2) will involve resolving how these genes are genetically regulated, an important facet of sulfur/carbon salvage in these organisms. Resolution of the specific aims of this project have considerable health relevance as ExPEC/UPEC strains cause major health problems and infect millions of people. It is conceivable that the identification and resolution of a specific sulfur/carbon salvage pathway essential for pathogenesis/fitness will open the way to design specific targets to inhibit infections caused by these organisms.

NIH Research Projects · FY 2025 · 2020-04

The cause of premature ventricular contraction (PVC)-mediated cardiomyopathy is unknown. The estimated prevalence of PVCs is high in the general population with reported 40-75% on ambulatory monitoring. PVCs are often considered benign, but may be associated with increased risk of sudden death or associated with chest pain, syncope, or heart failure, especially when structural heart disease is present. A long-term study on patients with relatively low PVC burden showed no difference in survival. However, high PVC burden is associated with lower left ventricular function in patients with and without structural disease. Unfortunately, suppressing PVCs by ablation or antiarrhythmic medications do not always improve ventricular function in patients. Animal models have showed that bigeminy pacing near the apex could induce a cardiomyopathy that is reversible without myocardial structural changes. However, these models do not explain the lack of cardiomyopathy development in some human subjects with frequent PVCs such as bigeminy. We propose that frequent pathologic PVCs lead to PVC- induced cardiomyopathy in normal hearts and PVC-worsened cardiomyopathy in hearts with abnormal tissue characteristics. The pathologic versus benign PVCs can be determined by LV function during PVC, which is quantitatively assessed using advanced real-time cardiac magnetic resonance imaging (MRI) techniques. LV function during PVC is likely a result of a combination of factors including PVC site of origin, coupling interval, and conduction time through the myocardium. Together with tissue characterization of the ventricular myocardium by cardiac MRI and electroanatomic voltage mapping, this proposal seeks to identify hemodynamic and structural features in PVC-induced or worsened cardiomyopathy in order to better select patients for treatment.

NIH Research Projects · FY 2025 · 2020-03

Summary HIV-1 packages two copies of unspliced viral RNA as a dimer into newly budding virions. The unspliced viral RNA also serves as an mRNA template for translation of viral proteins. Recent studies showed that the fate of the viral RNA (genome vs. mRNA) is determined at the level of transcription. Host RNA polymerase II uses heterogeneous transcription start sites (TSSs) to generate major transcripts that differ in only two guanosines at the 5ʹ end (referred to here as 1G and 3G RNA). Remarkably, this two-nucleotide (nt) difference is sufficient to alter the structure of the 5ʹ-untranslated region (UTR) and generate two pools of RNA with distinct functions; 1G RNA is selectively packaged by the viral Gag protein and functions as the genome, whereas 3G RNA is preferentially translated by the ribosome. The presence of both RNA species is needed for optimal viral replication and fitness, but gaps remain in our understanding of the mechanism by which different RNA structures dictate viral and host factor interactions and thereby impact function. Our long-term goal is to understand how TSS choice influences HIV-1 RNA interactions and function. We previously showed that the stability of the “polyA” hairpin in the 5′UTR is a key regulator of the conformational equilibria, resulting in exposed (1G) versus sequestered (3G) dimerization and Gag binding sites. Tuning polyA hairpin stability allowed us to alter the conformational landscape and packaging selectivity. HIV variants that only express 1G or 3G major transcripts are in hand and will serve as key tools that will enable us to address exciting open questions. The proposed aims will test our major hypothesis that the different RNA structures formed due to TSS heterogeneity significantly impact viral and host factor interactions, RNA 5ʹ-modification, and ribosome loading and scanning. We will: (1) identify and characterize the HIV-1 1G and 3G interactomes in vitro and in cells; (2) determine how HIV-1 TSS choice affects RNA 5ʹ-end modification and dynamics; and (3) determine how HIV-1 TSS choice affects translation in vitro and in cells. The results will help guide the design of novel therapeutic agents that target the 5′UTR and interfere with its essential conformational plasticity.

NIH Research Projects · FY 2025 · 2019-12

SUMMARY The vertebrate innate immunity relies upon a complex set of cytosolic pattern recognition receptors (PRR) to detect pathogen-derived, evolutionarily conserved molecules such as DNA. The sensing of cytosolic DNA by cyclic-GMP-AMP synthase (cGAS) activates the enzymatic synthesis of cyclic guanosine monophosphate- adenosine monophosphate (cGAMP), a cyclic dinucleotide (CDN) second messenger. cGAMP signals via its high affinity receptor protein STING, which subsequently recruits TANK-binding kinase 1 (TBK1) and interferon (IFN) regulatory factor 3 (IRF3) to stimulate the induction of type I IFNs. Although originally identified as a cytosolic sensor of foreign DNA, cGAS is also recruited to and activated by fragments of chromatin from damaged genomic DNA in the cytosol or micronuclei. Multiple convergent studies have recently highlighted the significance of cGAS in the DNA damage–induced inflammatory response and its implications for cellular senescence, tumorigenesis, and metastasis. Nothing, however, is known about whether cGAS activation in these contexts directly contributes to the maintenance of genome integrity. Recent studies in our laboratory have discovered that cGAS/cGAMP signaling triggers DNA damage response (DDR), independently of its well-characterized type I interferon pathway. These studies revealed that cGAMP-induced DDR activates cell cycle checkpoint responses that lead to G1 arrest and subsequent suppression of homology directed repair (HDR) of double-strand DNA breaks (DSB) in CRISPR/Cas9-edited mouse embryos and human and mouse cells. Interestingly, the cGAMP-induced DDR was also demonstrable in invertebrate species (oysters and starlet sea anemone) lacking interferon-based immune system, suggesting that the DNA damage surveillance mechanism of cGAMP predates its more well- known IFN-mediated immune function. The studies proposed here aim to advance these novel findings by elucidating the molecular mechanism of cGAS/cGAMP-induced DDR induction via three thematically integrated, yet independent Aims: (1) Decipher the critical signaling pathways involved in cGAMP-induced ATM activation; (2) Define the molecular mechanism of cGAS-cGAMP-induced suppression of HDR; and (3) Elucidate whether cGAMP-induced DDR potentiates the cGAS-initiated innate immunity. In summary, this work will illuminate novel aspects of the molecular and biochemical basis of cGAMP-induced activation of the apical DDR signaling kinase ATM, and increase understanding of the relationship between cGAMP-induced DDR signaling and the traditional immune function of cGAS.

NIH Research Projects · FY 2026 · 2019-06

Men are at higher risk of coronary heart disease (CHD) than age- and BMI-matched women, especially early in life when androgens are high. However, androgens are needed for health in men, particularly in muscle where they promote glucose metabolism and fatty acid utilization. Gap: we don’t know the mechanisms for the disconnect between the protective functions of androgens and the higher risk of CHD in men. Additionally, although men and women share risk factors for CHD (hypertension, high LDL cholesterol and others), there are likely also sex-specific risk factors for the development of diabetes, fatty liver, and cardiovascular disease in both males and females. We don’t know the therapeutic significance of targeting these sex-specific pathways. In the last funding cycle of this project, our group helped define mechanisms for numerous species differences in the risk of metabolic and CHD with obesity. In humans and rodents, endogenous estrogens and androgens have beneficial effects on glucose metabolism. The increased risk of CHD in men is unique to humans. One key species difference is that humans express cholesteryl ester transfer protein (CETP), which mice naturally lack. Using mice transgenic for CETP we “humanized” this pathway and discovered that CETP transduces unique estrogenic pathways in the liver of females that are beneficial. We discovered that CETP also transduces unique effects of androgens in liver of the males, but that these are largely harmful for triglyceride and cholesterol metabolism; thus “humanizing” these aspects of sex-specific CVD risk. Our overarching hypothesis is that hepatic CETP expression drives sex-specific risk of dyslipidemia and CHD by creating gain-of-function androgen signaling and estrogen signaling in in the liver. We propose 3 AIMS to reduce the harmful lipid effects of androgens in males and preserve the beneficial glucose effects. Additionally, we will identify sex-specific risk factors in both males and females and help translate them to therapeutic targets. In AIM1 we will knock-down hepatic androgen receptor, or over-express hepatic CETP, to define if the CETP- androgen interaction that causes dyslipidemia is a direct effect of androgens and CETP in the liver. We will use metabolic tracers to simultaneously define glucose and fatty acid metabolism in both liver and muscle. We found that CETP alters androgen-regulation of ~3900 liver mRNAs, including gain-of-function for cholesterol and triglyceride metabolism. In AIM2 we will target two key factors that are uniquely up-regulated by androgens when CETP is present, LXR and ChREBP. We expect to preserve androgen benefits on muscle glucose and fatty acid metabolism but block the CETP-androgen axis with regard to dyslipidemia and CHD risk. In AIM3 we will take approaches to reduce the key translational barriers in the mouse with regard to lipid metabolism: 1) the absence of CETP; 2) increased metabolic rate; and 3) different bile acid composition. We will use approaches for each to make them more human-like, then with this model the demonstrate proof-of-principle therapeutic value of a liver-targeted estrogen-glucagon receptor co-agonist in both males and females.

NIH Research Projects · FY 2025 · 2019-06

Title: Laboratory Capacity FDA CVM Vet-LIRN Veterinary Diagnostic Laboratory Program Project Summary/Abstract Veterinary diagnostic laboratories (VDLs) are pivotal not only in preventing and controlling animal diseases but also in monitoring zoonotic agents. Additionally, they play a vital role in detecting contamination in feed from microbiological or chemical agents. VDLs collaborate with public health laboratories, food diagnostic laboratories, and other agencies in monitoring trends in antimicrobial resistance among bacteria isolated from animals. The early and accurate detection of zoonotic and foodborne pathogens, along with the analysis of antimicrobial resistance, constitutes a top priority for the Center of Veterinary Medicine-FDA. The primary objective of this application is to secure funding through a cooperative agreement to procure equipment, supplies, training, and proficiency testing required by the FDA Vet-LIRN annually over at least five years. While the Clinical Diagnostic Laboratory (CDL) at the College of Veterinary Medicine, Ohio State University, will benefit from this agreement, the FDA CVM Vet-LRN Veterinary Diagnostic Laboratory Program will continue building national laboratory capacity. The specific aims of this cooperative agreement include: 1) strengthening coordination with veterinary diagnostic laboratories through the Vet-LIRN Program, participating in three key project areas (sample analysis, providing data, additional projects/method validation), 2) training, equipping, and proficiency evaluating technicians to conduct necessary testing for ensuring food supply safety and accurate diagnosis of pathogens in clinical samples, and 3) participating in the Vet LIRN Whole Genome Sequencing Project by supplying bacterial isolates as a Source Laboratory.

NIH Research Projects · FY 2025 · 2019-05

The singular goal of most spinal cord injury (SCI) research programs is to repair the injured spinal cord and restore locomotor function. Unfortunately, restoration of walking is a low priority for most SCI individuals 1. In addition to impaired mobility, SCI causes slow and steady pathological changes in organ systems throughout the body. Failure to recognize and treat multi- organ system pathology as a standard of care may explain why survival rates have not improved for SCI patients (relative to able-bodied individuals) over the past 30 years 2. Emerging data indicate that after SCI, the loss of sympathetic tone and the development of aberrant spinal autonomic reflexes that control immune organs (e.g., spleen) and the major organs that control metabolism (e.g., liver, adrenal gland, muscle, adipose tissue and gut) cause immune dysfunction and multi-organ pathology. Thus, mitigating the onset and downstream consequences of post-injury dysautonomia could improve immune and metabolic homeostasis. Since immune and metabolic processes are normally tightly coupled and are essential for life 3,4, it is likely that most, if not all, co-morbidities that affect SCI individuals (e.g., spontaneous infections in lung or skin, impaired wound healing, non-alcoholic fatty liver disease (NAFLD), chronic depression, atherosclerosis, type 2 diabetes, fatigue and anxiety), can be explained by impaired immunometabolism. Experiments in this proposal are designed to study SCI as a disease of the entire body and will test the overall hypothesis that post-injury dysautonomia breaks neuro-immune homeostasis creating a state of “neurogenic meta-inflammation”. This proposal is an integration of currently funded NINDS R01 grants and new ideas. All experiments and concepts will build on my lab's past successes using both “macroscopic” (systems and networks) and “microscopic” (cells to molecules) tools to study the pathophysiological significance of neuro-immune interactions. Just as recent NIH initiatives have emphasized that cures for human brain disease are likely to arise from better understanding of brain networks or circuits, rather than defects in a single brain region, a cure for SCI is unlikely to originate from a focus only on repairing the injured spinal cord.

NIH Research Projects · FY 2026 · 2019-05

PROJECT SUMMARY Signal transduction is a universal biological process vital to all organisms. Due to their central role in disease, signal transduction systems in humans and in bacterial pathogens are the primary targets for drug design. Our long-term goal is to understand how cells detect, transmit, and adapt to signals. The main focus of our research is on bacterial signal transduction systems, with the goal of revealing their fundamental properties and mechanisms at the molecular level. The main unanswered questions that we propose to address are: (1) Which small molecule ligands are recognized by bacteria; (2) How did signaling networks evolve; and 3) Can we use the knowledge obtained with bacterial systems to advance our understanding of their counterparts in humans, especially with respect to disease. We propose to build on our previous findings and capitalize on our tools and innovative approaches to obtain and capture this knowledge as predictive models. These models will be stored in public databases, such as InterPro, NCBI Conserved Domain database, and our own Microbial Signal Transduction (MiST) database. Current MiST capabilities will be further enhanced to better serve the scientific community.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY/ABSTRACT This proposal organizes the resources and experience of the Cooperative Human Tissue Network Midwestern Division (CHTN MWD) for participation as an Adult Division of the NCI CHTN. Our CHTN MWD group of pathologists is equipped to efficiently provide remnant patient consented biospecimens for basic and early translational cancer research as well as for biomarker research studies to support personalized medicine. Biospecimens will also be collected under a waiver of consent for non-human subjects determined research. The Ohio State University (OSU) Hospital and James Cancer Hospital in Columbus, OH will function as the Coordinating/Procurement Center while University Hospitals Cleveland Medical Center, Cleveland, OH, and University of Pittsburgh Medical Center Shadyside, Pittsburgh, PA, and associated hospitals will function as geographically distributed Procurement/Biorepository sites. The OSU Investigator Management Service will manage the investigator applications and approvals and investigator biospecimen request networking. The OSU Biospecimen Management Service will handle the tissue Quality Control, biospecimen storage and shipping and CHTN fee billing, collections, and monthly accounting. OSU Tissue Procurement Services supports custom biospecimen collection including investigator provided novel preservation fluids or methods. Available informatics support the collection of data related to donor consents, biospecimens, investigators and billing. Shipped biospecimens include a redacted Pathology and QC Report for each biospecimen shipped. Pathology reports at OSU for freshly procured tissues include clinically appropriate molecular testing. Investigators may receive, if IRB approved, extensive clinical data available from the OSU Total Cancer Care Protocol (TCCP) Honest Broker utilizing the Information Warehouse (IW) platform and additional laboratory testing such as immunohistochemistry, tissue microarray construction, digital microscopy or digital morphometrics. Biospecimen donor privacy and confidentiality are essential elements of the CHTN MWD biospecimen management proposal so that all procured biospecimens are patient consented, unless collected under waiver of consent, and coded. Delinking is available for special tissues that were not consented at procurement. The OSU procurement program joined the OSU Comprehensive Cancer Center (OSUCCC) in deploying the TCCP in 2016 to provide comprehensive donor consent. Donors who consent give full use for future research of all their past, present and future remnant biomaterials to IRB approved researchers. Procurement/Biorepository subsites have similar consent programs in place. Scientific or technical knowledge or discoveries acquired in the conduct of our tissue procurement/preservation program will be reported at national and international scientific and technical meetings to contribute to the growth of biospecimen science, to recruit new investigators who have research interests best served by human biospecimen research and to inform the research community and public.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY/ABSTRACT Traumatic Brain Injury (TBI) has emerged as a significant risk factor for Alzheimer's Disease (AD) and related dementias. Epidemiological studies indicate that mild TBI more than doubles the risk for dementia; however, the mechanisms underlying this association remain poorly understood. Given the widespread prevalence of mild TBI, which constitutes the majority of all head injuries, its connection to neurodegenerative diseases represents a significant public health concern. Our work in the previous funding period shows that mild TBI confers risk for adverse AD-related outcomes when combined with additional risk factors such as genetic risk for AD. In this renewal project, we aim to build on these findings by identifying the specific genetic and epigenetic pathways that interact with TBI to exacerbate AD-related outcomes. Several biological pathways are implicated in AD, including the regulation of amyloid-beta (Aβ) formation, tau protein binding, protein-lipid complexes, and immune response activation. These pathways may vary in their interaction with TBI. Therefore, in Aim 1 we will examine polygenic risk scores (PRSs) for each AD-associated pathway to identify those that most significantly interact with TBI in relation to cortical thickness, blood biomarkers, and cognitive function. This will help us pinpoint which biochemical pathways to target following TBI. The goal of Aim 2 is to improve detection of AD pathology biomarkers post-TBI by combining multiple polygenic risk scores to increase the explained variance in AD biomarkers. Finally, in Aim 3, we will explore genetic pathways associated with cognitive resilience, particularly those linked to episodic memory, as indicated by our pilot data. This project is significant because identifying the specific biochemical pathways affected by TBI will enhance our ability to predict early AD pathology in individuals with TBI, facilitating targeted interventions and the identification of candidates for clinical trials. The project is conceptually and technically innovative because it leverages longitudinal and multi-omic data, including genetics, blood-based biomarkers, MRI, and neurocognitive data, from a deeply phenotyped sample of over 1000 war veterans spanning young, middle-aged, and older adulthood. Ultimately, this knowledge has the potential to inform clinical judgements regarding dementia treatments and help resolve long-standing debates regarding the relationship between TBI and development of AD.

NIH Research Projects · FY 2026 · 2019-03

PROJECT SUMMARY The Ohio State University Comprehensive Cancer Center (OSUCCC) has been an NCI National Clinical Trial Network (NCTN) Lead Academic Participating Site (OSU-LAPS) since 2014. OSU-LAPS provides the infrastructure to support a robust participation in the scientific and clinical research activities of the NCTN. OSUCCC faculty are actively participating in NCTN scientific and administrative activities, attend the Operation Center and other related NCI meetings, manage multiple core labs, design studies, and enroll patients in all existing adult NCTN Operation Centers. OSUCCC supports faculty in part with internal resources to leverage NCTN activities. Hence, OSU-LAPS investigators develop and lead studies designed to enroll patients on multi- disciplinary treatment trials, as well as develop and perform correlative science-related research studies by bringing special expertise in hematologic malignancies and solid tumors. Our medical, hematologic, surgical, and radiation oncologists, transplanters, psychiatrists, pathologists, cytogeneticists, translational and basic laboratory scientists, statisticians, epidemiologists, nurses, pharmacists, and clinical research coordinators help to frame the clinical and basic science questions, whose answers contribute to improved patient outcomes and to understanding tumor biology. OSU-LAPS will offer innovative NCI-sponsored trials to the OSUCCC patient population, support scientific discoveries about tumor biology, find better treatments, and augment the quality of life for patients, thus improving outcomes across the spectrum of cancers affecting adults. This comprehensive program aims to: 1) investigate new therapeutic agents and their toxicities in Phase II to III NCI clinical trials; 2) evaluate the efficacy and toxicity of novel combinations based on preclinical data to exploit synergistic combinations more effectively; 3) develop multi-modal approaches using surgical, immunological, and radiotherapeutic therapies in optimal combinations; 4) integrate experts in molecular genetics, biochemistry, pharmacology, immunology, and biostatistics in the design and execution of therapeutic protocols; 5) improve cancer outcomes through discovery and education of pre- and post-doctoral students, nurses, allied medical personnel and physicians, 6) understand and exploit tumor heterogeneity to fully exploit the value of targeted therapies, 7) improve the management of cancer related symptoms, 8) form the next generation of clinical investigators by offering them formal teaching and mentoring within the NCTN, and 9) importantly enroll volunteering patients on cancer trials.

NIH Research Projects · FY 2025 · 2019-01

Pulmonary arterial hypertension (PAH) has a 3-year mortality rate of up to 55%. The survival benefit of vasodilator therapy does not last beyond one year, indicating that pulmonary artery (PA) remodeling, rather than vasoconstriction, predicts long-term prognosis. New anti-proliferative therapies emphasize targeting PA remodeling in PAH. A better understanding is needed of how ECs create an abnormal microenvironment that promotes PA remodeling. This proposal aims to fill this crucial knowledge gap by elucidating a novel mechanism by which ECs profoundly impact other PA mural cells. The discovery that the endosomal GTPase RAB7 is a critical gatekeeper for lung EC and vascular function forms the foundation of this work. However, the molecular regulation of RAB7 expression in ECs in PAH and the mechanism by which endothelial RAB7 deficiency induces pulmonary hypertension (PH) remain unclear. Here, studies are proposed to address these two critical issues. Based on new supportive data, the unique paradigm is presented that hypoxia signaling represses endothelial RAB7 transcription in PAH, promoting PA remodeling and PH via an altered secretome. Exciting new data reveal MEIS2 as a new transcriptional inducer of RAB7 in ECs. Additionally, a previously unknown repression mechanism is shown where hypoxia-inducible factor (HIF)- 2α accelerates MEIS2 mRNA decay via microRNA (miR)-199a. Unbiased screening identified a new interaction between RAB7 and the RNA-binding protein cold shock domain containing E1 (CSDE1) in ECs. The loss of RAB7:CSDE1 interaction upregulated target mRNAs via CSDE1, subsequently increasing their levels in the secretome and promoting PH via the proliferation of PA smooth muscle cells (PASMCs) and PA adventitia fibroblasts (PAAFs). Notably, the FDA-approved CSDE1 modulator Clofoctol reduced experimental PH. These results led to the HYPOTHESIS: In PAH, the transcriptional repression of RAB7 in ECs arises from accelerated decay of MEIS2 mRNA via the HIF-2α→miR-199a pathway and leads to the CSDE1- dependent dysregulation of the endothelial secretome, which contributes to PH. This hypothesis will be tested with the two SPECIFIC AIMS: Aim 1. To determine the MEIS2-driven transcriptional regulation of RAB7 in ECs. Aim 2. To investigate the molecular mechanism of RAB7-dependent PA remodeling via CSDE1 in ECs. In vitro experiments using gene manipulation, co-culture experiments, in vivo rat and mouse experiments, and Omics approaches will be employed to test the hypothesis. This research will establish a new molecular model connecting RAB7 transcriptional regulation via the HIF-2α→miR-199a→MEIS2 axis to EC dysfunction and PAH. This approach will further elucidate for the first time the molecular link between RAB7 deficiency, mRNA stability, PASMC and PAAF growth, and PA remodeling by studying CSDE1 as a key regulator of the endothelial secretome. These results will fundamentally enhance the understanding of EC dysfunction in PAH and provide novel therapeutic opportunities.

NIH Research Projects · FY 2024 · 2019-01

An estimated 51,540 new oropharyngeal cancer cases and 10,030 deaths will occur in U.S. during 2018. Oral squamous cell carcinoma (OSCC) is one of the most challenging to treat human cancers due to the insidious nature of its early disease, dependence on radical surgery for treatment and difficulty achieving locoregional control. Further, even OSCC patients who are cured by surgery must face major esthetic and functional changes of their face and mouth. OSCC arises from malignant transformation of its precursor lesion i.e. oral intraepithelial neoplasia (OIN). While not all OINs progress to OSCC, up to 87% of high-risk lesions transform. Despite refined predictive parameters, we do not yet have the methodology to predict which OIN lesions will progress to OSCC. Further, approximately a third of OIN lesions recur despite microscopically clear surgical margins; findings which imply heritable defects in the keratinocyte stem cell pool. As OSCC's devastating effects are well-recognized, numerous OSCC prevention trials have been conducted. The majority of these studies employed systemic delivery and were largely ineffective. Systemic delivery limitations include drug inactivation during first pass metabolism in the liver which results in difficulty achieving therapeutic levels of active drug at the target site and adverse side effects. In contrast, local delivery formulations provide therapeutic levels directly to the treatment site using appreciably less drug and without adverse side effects. The mouth's visible accessibility facilitates agent placement by patients and clinical monitoring. Our lab has previously conducted a local delivery OSCC chemoprevention trial and obtained strong results including complete OIN resolution in some patients. Not all patients derived chemopreventive benefits which prompted development of a new local delivery formulation. The Specific Aims of this proposal are: 1) identify the clinical lead patch formulation in vivo and characterize the metabolic profile of locally delivered fenretinide (4-HPR), 2) confirm application time in healthy participants then evaluate chemopreventive efficacy in persons with microscopically confirmed OIN lesions. Experimental methodology will include PK analyses, LC-MS, IHC and laser capture microdissection followed by LOH analyses. The trial biomarkers (histologic grade, clinical presentation and LOH events) are all associated with OIN progression. This formulation i.e. a 4-HPR patch is expected to provide more pervasive chemopreventive effects across the trial cohort. Public Heath Relevance: Oral cancer, which arises from the cells lining the inside of the mouth, is a devastating cancer that is managed by aggressive surgery. Even if cured by surgery, patients live with swallowing, eating, talking difficulties and deformities to their face and mouth. Previous oral cancer prevention programs, which used pills that could affect the entire body, were not successful and often caused adverse side effects including very sore mouths and night blindness. In contrast, this project introduces a more efficient and safer approach i.e. application of the cancer preventing agent directly to the precancerous tissue.

NIH Research Projects · FY 2025 · 2018-09

ABSTRACT Over the past two decades, the prognosis for glioblastoma (GBM), the most lethal brain tumor, has remained dismal, with a median survival of only 12-16 months from diagnosis. We recently demonstrated that GBM cells acquire large amounts of fatty acids (FAs) and cholesterol by dramatically upregulating their de novo synthesis and uptake for rapid tumor growth. However, excess FAs and cholesterol can alter membrane dynamics and function, leading to cellular damage. How GBM cells avoid this lipotoxicity to sustain proper lipid levels in different cellular compartments, particularly in the mitochondria, is poorly understood. During the past 5 years of funding, we have made great progress in understanding how GBM controls FA homeostasis. We demonstrated that GBM cells upregulate diacylglycerol acyltransferase 1 (DGAT1), allowing them to store abundant FAs as triacylglycerol-containing lipid droplets (LDs) to prevent excess FA accumulation to induce toxicity. In this renewal proposal, we will address two unanswered critical questions: 1) how is cholesterol homeostasis regulated in GBM cells? and 2) can effective therapeutic approaches be developed for GBM by disrupting lipid homeostasis? We recently found that cholesteryl esters (CEs), which form LDs to store excess cellular cholesterol, are largely present in GBM tissues, and blocking CE synthesis results in dramatic mitochondrial fragmentation in GBM cells. Moreover, our preliminary data showed that cholesterol is transferred from CE- containing LDs (CE-LDs) to the plasma membrane, while inhibition of autophagy blocks this transfer. These data suggest that CE-LDs maintain proper cellular cholesterol levels via autophagy. Our preliminary data further showed that stearoyl-CoA desaturase 1 (SCD1), which has been shown to prevent endoplasmic reticulum (ER) stress and ferroptosis, is upregulated upon DGAT1 inhibition. Finally, preliminary data showed that the expression of multiple antioxidant genes is significantly elevated in response to DGAT1 inhibition. These results strongly suggest that GBM cells can activate defense mechanisms to alleviate the lipotoxicity triggered by disruption of FA storage, possibly leading to tumor resistance to DGAT1 inhibition. Thus, we hypothesize that CE-LDs serve as critical reservoirs for controlling cholesterol homeostasis and mitochondrial function, and that combining disruption of storage or redistribution of cholesterol with interference with mitochondrial cholesterol import, or disruption of FA storage with either inhibition of SCD1 or blockade of antioxidant pathways are effective strategies for targeting GBM. In Aim 1, we will examine the impact of inhibiting cholesterol storage or redistribution from CE-LDs on cholesterol homeostasis and mitochondrial function, and whether such blockade can synergize with interfering in cholesterol import into mitochondria to efficiently kill tumor cells in GBM xenograft models. In Aim 2, we will examine whether inhibiting SCD1 or antioxidant pathways can strongly synergize with DGAT1 inhibition to effectively inhibit GBM growth in vitro and in vivo. Successful completion of this study will provide strong pre-clinical data on potential novel strategies to target GBM.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY/ ABSTRACT Transfer RNAs (tRNAs) are the universal adaptor molecules necessary to convert the nucleic acid-based genetic code into protein sequence during protein synthesis (translation) by the ribosome. This process is universally conserved and fundamental to all life, and, as such, defects in the molecular players of translation, including tRNAs, result in diverse human diseases. Specific chemical modifications such as methylation are common in tRNA, but a detailed understanding of the enzymes that incorporate them and their contributions to tRNA function (and disfunction in disease) have only recently emerged for a few select examples. Since the discovery of the tRNA methyltransferase (Trm10) in Saccharomyces cerevisiae, an accumulating body of evidence, including phenotypes in yeast and a multisymptomatic disease associated with human mutations, has established a significant role for Trm10 in tRNA biology. To better understand the implications of Trm10 modification, the mechanisms by which Trm10 family enzymes specifically recognize and act on their substrate tRNA, and the impact of tRNA modifications on important cellular processes need to be addressed. This project will determine the molecular basis for Trm10 mechanism and function using a multi-disciplinary approach. Genetic, biochemical and molecular enzymology approaches will be combined with structural analyses of enzyme-tRNA complexes using synthetic analogs of the native methyl donor, S-adenosyl-L-methionine, to uniquely identify the role of Trm10 in the maintenance of a high-quality pool of tRNA. A newly developed vertebrate model for Trm10 function will enable investigation of previously challenging questions on Trm10's role in the biological function of multicellular eukaryotes. The studies will be performed in three complementary but independent aims that will: 1) Determine how specific tRNA substrates are selected for modification by yeast and vertebrate Trm10 enzymes using structural, biochemical and genetic approaches; 2) Assess the molecular basis for and biological significance of the uniquely conserved vertebrate m1A9 modification exploiting a new vertebrate model for Trm10 function, and 3) Identify tRNA-specific functions for G9 modification in yeast and zebrafish using complementary genetic approaches in both model species. Collectively, the proposed studies will advance the fields of enzymology, RNA biochemistry, and tRNA biology by providing mechanistic and biological insight into a tRNA modification enzyme that is universally conserved among eukaryotes and is critically important for human health, yet whose molecular mechanism and biological functions are not at all understood. These results will also provide new insight into the dynamic landscape of tRNA modifications in multicellular eukaryotes.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY The Ohio State University Comprehensive Cancer Center (OSUCCC) seeks its first renewal of its “Cancer Prevention and Control Training Program” (CPCTP) for postdoctoral fellows. The philosophy of this program is to foster transdisciplinary team science training through high quality mentorship to reduce the cancer burden in our communities. This is done by providing state-of-the-art research training across the cancer control spectrum from the laboratory to the clinic and community, high quality research infrastructure, and relevant didactic course work. The co-Program Directors (co-PDs) are Drs. Peter Shields and Chyke Doubeni. Dr. Ce Shang is the Associate Program Director (APD). The transdisciplinary training includes an emphasis on dual mentorship, transdisciplinary mentorship and biostatistical/bioinformatics advisors. During the current grant cycle, there were several enhancements of the program including: 1) formalizing the mentoring team structure; 2) enabling early stage investigators to serve as primary mentors with senior mentor support; 3) securing an additional $3000 salary stipend through institutional funding; 4) expansion to Nationwide Children’s Hospital for a lifespan approach including research on adolescents and young adults; and, 5) securing an additional fellow slot with institutional funding. Despite the challenges of the COVID-19 pandemic, all goals were met for training. To date, there have been 9 fellows enrolled in the program, 2 who have completed their training and are now in academic tenure track positions, and 2 completing their training with offers for academic appointments. Five of 9 were recruited from other institutions. In the next cycle, we will: 1) increase the number of slots from 3 per year to 4 per year (plus the one slot funded through institution support, total 5); 2) develop a new course for Cancer Prevention and Control; 3) begin a yearly scientific symposium for current and former fellows; 4) provide funding to fellows for the use of shared resources; and, 5) enhance efforts to recruit and retain fellows. CPCTP has internal and external advisory committees. There are 39 mentors (including 13 early career mentors [ECM]), all of whom have track records for peer-review funding (ECM have career development awards). Particular strengths among the faculty include expertise in carcinogenesis, cancer risk, behavioral interventions, survivorship, nutrition,, and tobacco control. While the OSU has several other training programs, there are essentially none that overlap with the CPCTP. The training program would be administratively managed through the OSUCCC Center for Cancer Mentoring, Education, Leadership, and Oncology-related Training (CAMELOT), a comprehensive resource established in 2018. Plans are in place for ongoing trainee, mentor and the overall program evaluations.

NIH Research Projects · FY 2024 · 2018-08

Project Summary The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. One of the best models for studying core mechanisms of fertilization is the marine gastropod abalone, where sperm are highly enriched for only a few proteins. The first identified pair of interacting reproductive proteins were abalone sperm lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that likely form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for these hydrogen bonds, allowing sperm to pass and fuse with the oocyte. These ZP-N repeats are also a principal component of mammalian egg coats, and multiple lines of evidence support the human protein ZP2 as the functional analog of VERL. However, three major outstanding questions are (1) besides lysin and VERL, which sperm and egg proteins are interacting, (2) what sites in these proteins are important for mediating this interaction, and (3) how might mutations in these sites impact protein structure and fertility. To address these fundamental questions of reproduction in both abalone (K99) and humans (R00), two specific aims are proposed that utilize state-of-the- art genomic, proteomic, and structural methods. In aim 1, for the K99 phase, I will combine long read sequencing technology, tests for molecular evolution, and quantitative proteomics to identify potential pairs of interacting reproductive proteins in abalone; for the R00 phase, I will extend these quantitative proteomic methods to identify potentially novel pathways that contribute to male infertility. In aim 2, for the K99 phase, the solution structure of VERL repeat 1 and its interactions with lysin will be determined by NMR, with deep mutational scanning used to identify mutations that interfere lysin-VERL interactions. In the R00 phase, these technologies will be applied to human ZP2 and I will identify mutations that interfere with sperm binding and characterize their structural effects. The proposed research is innovative for its combined use of genomic, proteomic, and structural techniques to characterize the molecular mechanisms underlying the interactions of rapidly evolving reproductive proteins. The results are expected to shed insight into the core processes that mediate egg-sperm interactions, and provide foundational information towards understanding the more complex mammalian system.

NIH Research Projects · FY 2026 · 2018-08

The eukaryotic cell is a highly compartmentalized structure that is subdivided into distinct functional areas by the presence of both membrane-bound and membraneless organelles. These latter compartments have also been referred to as biomolecular condensates. Although most of these membraneless structures have been identified only recently, condensate formation has been found to be important for many essential processes in the cell. It is therefore critical that we develop a thorough understanding of the mechanisms underlying condensate formation and the nature of their biological activities in the cell. We have been addressing these broader issues by studying the biology of one particular condensate, the Processing body, or P-body. This cytoplasmic granule is highly conserved and contains translationally- repressed mRNAs and proteins involved in the processing of these transcripts. Our efforts over the past 15 years have been focused on developing a better understanding of the physiological roles of these granules in the eukaryotic cell. Studies during the prior grant period furthered this understanding by identifying a potential role for P-bodies in the regulation of microtubule dynamics. Specifically, we found that a distinct subtype of P-body granule was induced when the integrity of the microtubule network was disrupted. Moreover, our preliminary data suggest that these granules may be involved in the specific turnover of the TUB mRNAs that encode tubulin monomers. The experiments in this proposal are organized into two aims that will (1) define the assembly pathway for these novel granules and (2) determine how their biological activities are regulated. The studies in Aim 1 will specifically test a model proposing that these novel granules form as a result of select P-body components being recruited to the TUB mRNAs. Interestingly, we have found that this latter decay in S. cerevisiae exhibits the hallmarks of tubulin autoregulation, a process that has been studied for decades in mammals. However, the decay machinery responsible for this mRNA turnover has not yet been identified. Therefore, the studies here could provide resolution for a long-standing question in tubulin biology. In Aim 2, we will examine how the biological activity of a biomolecular condensate can be controlled by specific constituents of that structure. We will specifically address a key question concerning the role of P-bodies with respect to mRNA stability. The primary issue is whether P-bodies are sites of decay or long-term storage for the resident mRNAs. The experiments here will test a model proposing that P-bodies can alternate between these different activities and that the transition from one state to the other is controlled by protein constituents of the granule, like the Hrr25 protein kinase. Finally, we will assess the possibility that the P-body decay machinery is portable and recruited to select messages that are targeted for degradation. In all, the completion of this work should provide us with a better understanding of the diversity and physiological roles of a conserved biomolecular condensate in eukaryotic cells.

NIH Research Projects · FY 2024 · 2018-08

Emerging evidence suggests that the recurrent form of Major Depressive Disorder (MDD) tends to worsen, with more severe and longer episodes as people move into adulthood. Yet, we are still a very long way from treatments that can reduce short- and long-term Risk for Depressive Relapse (RDR). Understanding specific mechanisms of RDR is urgently needed to promote relapse prevention (and may have long-term benefits for treatment). The present proposal uses a treatment that has evidence for modifying the ruminative habit, a tendency to approach difficulties and associated negative emotions with a repetitive, passive and abstract mental pattern (habit). In contrast, we teach adolescents to utilize adaptive, concrete and specific mental patterns (e.g., problem-solving, emotion processing). Rumination focused CBT (RFCBT) was developed to specifically target and reduce or modify the ruminative habit. Initial work among adults and our pilot data among adolescents suggests that rumination can be effectively reduced with RFCBT. In addition, work by our group and a few other small studies suggests that the ruminative habit can be measured at the neural level in connectivity of brain nodes in the Default Mode Network (DMN, increased within network connectivity) as well as in patterns of connectivity between the DMN and Cognitive Control Network (CCN). As such, we proposed to use RFBCT to modify the ruminative habit in 160 adolescents (age 14-18) with remitted MDD (via KSAD-PL) and elevated rumination (above T of 50), with the goal of achieving significant clinical change in rumination, and to observe changes in connectivity between key DMN and CCN nodes associated with decline in the ruminative habit. The latter knowledge may provide information on how to use alternative therapies (e.g., neuromodulation) to effectively modify the ruminative habit and the associated neural network pathways, which would lead to greater precision in the targeting of treatment. The RFCBT arm (N=30) is compared to an assessment only (AO) arm (N=30) for the R61, and to an active therapy, Relaxation Therapy (RelaxT) for the R33 (N = 50 in each group), that shares some of the active strategies for change.. For R61, we expect that (Aim 1) RF-CBT will result in (a) significant decreases in rumination and (b) decreased connectivity from LPPC to RIFG, both "Go" criteria for continuation to the R33 phase. For (Aim 2), we expect that degree of homework engagement will relate to degree of change in (a) ruminative habit, (b) in LPCC-RIFG rs-fMRI connectivity, and degree of increased activation during exploratory rumination induction task. Stability of RRS changes will also be evaluated at 12 months. This R61/R33 has the same Go criteria of RRS change (effect size of .5) and LPCC - RIFG connectivity changes (.5 effect size). In addition, the R33 adds prediction of MDD recurrence (Aims 3a (dimensional) and Aim 3b(categorical)) to proceed to the RO1 multisite phase. Our preliminary data suggest that even if our Go criteria are not met, we will make iterative gains in gaining a better understanding neural network targets, both in maintenance of or compensation for the ruminative habit, as well as for RDR.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY Mineralocorticoid receptor (MR) antagonists (MRAs) have long been considered as anti-fibrotic drugs for treatment of heart failure. Although MRAs also have therapeutic benefits on skeletal muscle pathology in mouse models of Duchenne muscular dystrophy (DMD), their effects on skeletal muscle fibroblasts have never been defined. Fibroblasts produce extracellular matrix proteins that constitute fibrosis that replaces muscle tissue that leads to loss of ambulation, respiratory insufficiency and cardiomyopathy in DMD. Both MRAs and genetic deletion of MR in myofibers lead to reduced fibrosis in dystrophic models. In contrast, deletion of MR from myeloid cells increases fibrosis in dystrophic muscles. These data support the overall hypothesis that MR signaling has complex cross-talk between cell types in the injured muscle microenvironment that ultimately regulates fibrosis. The endogenous MR agonist aldosterone increases proliferation and migration of cardiac and renal fibroblasts, which is prevented by MRAs. Recent data support that fibroblasts differ between organs, preventing extrapolating conclusions from one tissue to another. In addition to its role in disease, fibrosis is essential for homeostasis of organ structure and wound healing. MRAs reduce the kinetics of acute injury repair in normal muscle, supporting a normal role for MR signaling in skeletal muscle wound healing. In this application, we will identify fibroblast regulation through non-fibroblast MR signaling and directly through fibroblast MR signaling in muscle wound healing and muscular dystrophy. We will compare molecular signatures, proliferation, migration, and activity between fibroblasts isolated from dystrophic mice treated with MRAs or untreated. We will compare these mechanisms in more fibrotic dystrophic diaphragm with less fibrotic limb muscles. To define mechanisms of MR signaling in essential fibrosis during wound healing versus chronic fibrosis during disease, we will compare fibroblasts isolated from mice with myofiber and myeloid MR knockouts in dystrophic muscles and in acutely injured wild-type muscles. Single-cell RNA-sequencing will be used to identify MRA effects on mononuclear cell populations in acute injury versus dystrophy. We will perform co-culture and conditioned media experiments with MR knockout and control myeloid cells or myotubes with fibroblasts to identify MR-regulated cross talk between these cell types. Direct MR signaling in skeletal muscle fibroblasts, different signaling in resolving fibrosis in wound healing versus chronic fibrosis in dystrophy, between dystrophic muscles with different fibrosis levels, or between young and old muscles have never been studied. Fibroblasts isolated from young and old mdx diaphragms and limb muscles and acutely-injured wild- type muscles will be treated with increasing dosages of aldosterone with or without an MRA and gene expression, proliferation, migration and activity will be compared. A fibroblast specific MR knockout will be generated for in vivo validation. These studies will define direct and indirect effects of MRAs on muscle fibroblasts to optimize MR modulation for DMD, other muscle diseases and possibly for acute muscle injuries.

NIH Research Projects · FY 2025 · 2018-01

Our published data show that genetic ablation and pharmacologic inhibition of NFATc3 in macrophages is beneficial in maintaining alveolar-capillary barrier function, prevents inflammatory cytokine release and neutrophilic inflammation, improved arterial oxygenation and survival in the LPS and cecal ligation puncture mouse models of ARDS. Here, we propose to determine the granular details of the downstream molecular targets of NFATc3 using a 2-hit mouse model, human lung macrophages, and BALF from patients with ARDS. Our team has developed a novel non-toxic cell permeable calcineurin inhibitory (CNI) peptide (CNI103) that blocks activation of NFATc3 in macrophages and mitigates ARDS in mice. We propose to determine the molecular targets of NFATc3 using a pre-clinical 2-hit mouse model, human lung macrophages, and BALF from patients meeting the Berlin criterion for ARDS. Our central hypothesis is that activation of calcineurin-dependent NFAT in macrophages regulates the development of ALI/ARDS, and inhibition of NFAT activation by a novel peptide calcineurin inhibitor (CNI) lessens disease severity. We propose two specific aims: Specific Aim 1: To delineate the downstream molecular targets of NFATc3-Calcineurin activation pathway in pulmonary macrophages during ALI/ARDS. Novel preliminary data show that the lipid content of extracellular vesicles (EVs) in BALF are NFATc3 dependent and mediate disruption of barrier function in lung microvascular endothelial cells (MVEC). In SA1, we will 1) determine the spectrum of NFAT regulated lipids mediators and enzymes involved in lipid metabolism, 2) determine whether these mediators are packaged in extracellular vesicles, 3) assess whether EVs mediate the the cell-to-cell communication that results in permeability pulmonary edema and 4) determine whether blocking NFAT activation prevents lung injury and inflammation in clinically relevant mouse and cellular models of ARDS. Specific Aim 2: To determine the efficacy and safety of an optimized cell permeable calcineurin inhibitor, which prevents NFAT activation, in clinically relevant infectious and non-infectious mouse models of ALI/ARDS. We will test whether cell permeable calcineurin peptide inhibitors prevent and reverse lung injury and inflammation in mouse models of ARDS. We will also assess the pharmacokinetic and pharmacodynamics (PK/PD) properties, cellular selectivity, safety, efficacy, and potency in preventing and reversing lung injury in preclinical mouse models of ARDS. These studies will advance knowledge about the essential role of NFATc3 activation in macrophages and other lung cell types in the pathogenesis of ALI/ARDS. We anticipate that knowledge gained from these studies will establish NFATc3 as a novel therapeutic target that regulates EV-mediated cell-cell interactions by governing the composition of biologically active lipid and protein mediators.