University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2025 · 2024-08

Abstract Choroidal capillaries are normally kept behind the retina by the retinal pigment epithelium (RPE), a monolayer of cells located between the retinal parenchyma and choroidal plexus. In humans, RPE integrity may deteriorate with aging, especially in a small central area of the retina known as the macula. As a result, the normally avascular photoreceptor tissue may become invaded by leaky choroidal capillaries through choroidal neovascularization (CNV), which is a hallmark of the wet form of age-related macular degeneration (AMD) and a leading cause of blindness among the senior population. Despite the short-term benefits of anti-VEGF therapy, there is still no effective long-term treatment. To promote the development of novel therapies with long-term benefits, we propose to create a chronic CNV model in which CNV occurs due to a set of pathophysiological conditions mimicking those associated with wet AMD, including genetic susceptibility, stressful environment, and aging. We will refer to the novel mouse model as age-related CNV (ArCNV), following the style of “age-related macular degeneration”, although aging is not the only trigger in both cases. In preliminary studies, genetic susceptibility was introduced into mice by the knockin of a Cre-dependent transgene that is designed to knock down 5 specific mRNA targets (through the expression of 5 siRNAs) and overexpress two proteins. The candidate genes were selected because their human homologs are associated with increased risk of wet AMD. To simulate environmental risks, we designed a novel treatment regime consisting of oxidative diet, smoking product, and light (oxDSL). When carried out separately, Cre-activation of the transgene or treatment of wild-type C57BL/6J mice with oxDSL alone led to pre-CNV phenotypes such as RPE degeneration, but not outright CNV. In combination, however, global transgene activation and treatment with oxDSL led to CNV by 7 months of age, but not at much younger ages. Thus, the ArCNV model is age- dependent. On other hand, the occurrence of CNV at the relatively early phase of aging will accelerate research progress. In proposed studies, we will systematically characterize CNV onset and progression, along with related phenotypes and molecular changes (Aim 1). Given the central role of the RPE barrier, we will also activate the transgene with RPE-specific Cre and investigate whether such mice will develop CNV when treated with the oxDSL regime (Aim 2). At the completion of this project, we will have systematically characterized this novel mouse model, thus providing a highly valuable tool to the CNV research community.

NIH Research Projects · FY 2024 · 2024-07

SUMMARY Despite the apparent uniform cytoarchitecture of the cerebellar cortex, imaging studies have revealed discrete functional domains within it. We have recently identified numerous Purkinje cell (PC) subtypes that occupy discrete domains of the developing mouse cerebellar cortex, providing a basis for the functional parcellation of the cerebellar cortex. Climbing fibers, which originate from individual inferior olive (IO) nuclei, specifically target PCs within different domains of the cerebellar cortex. A significant knowledge gap exists regarding the molecular heterogeneity of IO neurons and the mechanisms governing their diversification. Preliminary data suggest the crucial role of transcription factor Foxp2 in diversifying PCs and IO neurons, leading us to hypothesize analogous heterogeneity in climbing fiber neurons. To test this hypothesis, we propose an innovative strategy integrating single-cell RNA-seq and single-cell resolved spatial protein profiling to develop a three-dimensional molecular atlas of the developing IO, under normal conditions and in the absence of Foxp2. Aim 1 is centered on constructing a 3D molecular atlas of the developing IO, while Aim 2 investigates the impact of Foxp2 loss on IO development. We anticipate that the completion of these aims will yield a detailed spatial molecular atlas of the IO and will provide insights into the molecular regulation of IO nucleus formation by Foxp2. This research will not only bridge the existing knowledge gap regarding IO neurons but also enhance our understanding of the discrete connectivity and function of different domains within the cerebellar cortex.

NIH Research Projects · FY 2024 · 2024-07

Spiny projection neurons (SPNs) carry much of the weight of the basal ganglia (BG) information processing. One cell type (dSPNs) contributes to the Direct BG, while the other cell type (iSPNs) projects to the Indirect BG pathway. The imbalance across these two neural pathways is associated with either hypokinetic disorders such as Parkinson's disease (PD), or hyperkinetic disorders such as Huntington's and tics. We will test two hypotheses. >>Hypothesis-1 is grounded in intracellular recordings obtained from the cell bodies of dSPNs and iSPNs, revealing consistent physiological disparities between the two cell types. Our hypothesis posits that these observed physiological distinctions in cell bodies stem from underlying differences in dendritic properties. To validate this hypothesis, we plan to conduct recordings of dendritic electrical signals, including synaptic and AP waveforms, and dendritic regenerative potentials (dendritic spikes), within primary, secondary, and tertiary branches of individual neurons belonging to either the dSPN or iSPN subtype. >>Hypothesis-2 is rooted in the findings of several translational studies demonstrating the protective effects of specific drugs, namely K+ channel blockers, on dopaminergic (DA) neurons in animal models of PD. Our hypothesis posits that the systemic administration of these drugs not only shields DA neurons but also impacts the dendrites and axons of striatal SPNs, which project directly or indirectly to DA cells. We propose that the experimental use of K+ channel blockers in PD therapies significantly alters the electrical signaling within striatal dendrites. To explore this, we pose crucial questions: Do these drugs facilitate or impede the generation of local dendritic NMDA spikes and complex spikes (involving both dendritic and axonal spikes)? Does their pharmacological impact vary between different subtypes of SPNs? If so, it implies that drugs safeguarding DA neurons in PD models also influence the balance between the Direct and Indirect BG pathways. The adjustment of the balance between the “GO” and “NOGO” pathways is a primary objective in experimental therapies for BG disorders. Our research aims to elucidate whether these protective K+ channel blockers could be employed to modulate the Direct and Indirect pathways. If substantiated, this discovery could potentially establish them as supplementary therapies, complementing approved treatments such as levodopa. Our study not only promises a physiological understanding of the functioning of these protective treatments, but also explores the potential of channel modulators in balancing the D/I pathways. Additionally, our proposal marks the pioneering effort in capturing dendritic electrical signaling in striatum through voltage imaging, bridging significant gaps in our comprehension of electrical signal processing within the principal projection neurons of the two BG pathways.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Neuropsychiatric disorders often cause lifelong disruptions of patients’ lives and lack satisfactory pharmacological interventions. Symptoms in these disorders are likely associated with disruption in synapses resulting from dysfunctional synaptic adhesion molecules (SAM), but the mechanisms by which SAMs may contribute to synaptic dysfunction have not been fully elucidated. Complement C1q like 3 (C1QL3), is a promising SAM for potential therapeutics for neuropsychiatric disorders because it selectively regulates excitatory synapses and may therefore constitute a target for correction of the excitatory/inhibitory synaptic imbalances associated with several neuropsychiatric disorders. To contribute to the future development of novel therapeutics, I will elucidate the molecular mechanisms contributing to C1QL3’s roles in regulating excitatory synapses. My preliminary data suggest that C1QL3 associates with the adhesion G protein-coupled receptor B3 and neuronal pentraxin 1 at synapses in mouse primary neuron cultures, consistent with our previous data suggesting that these proteins promote cell-cell adhesion in heterologous cells in a C1QL3-dependent manner. In Aim 1, I will test whether this hypothesized trans-synaptic adhesion complex truly forms at synapses in neuron cultures, using super resolution microscopy. I will characterize the synaptic nano-architecture of this complex, then determine the extent to which it is dependent on C1QL3 by knocking out C1QL3. I will further elucidate the molecular interactions contributing to trans-synaptic adhesion through structure-function analysis using C1QL3 mutants that are not conducive to complex formation. This will elucidate the specific binding interactions that contribute to C1QL3’s role in promoting synapse architecture and maintenance. In Aim 2, I will study how a novel C1QL3 binding protein contributes to synaptic maintenance, potentially through its interactions with C1QL3. My preliminary data suggest this novel protein increases excitatory synapse density in a C1QL3-dependent manner. I will therefore determine this novel protein’s mechanism of synaptic regulation and the extent to which it modulates the hypothesized C1QL3-mediated trans-synaptic adhesion complex. This study will provide insight into the components of the molecular web of SAMs that regulate synapses and may highlight novel targets for repairing synaptic dysfunction. Through this project I will develop and hone many skills that are essential for my goal of becoming an independent physician-scientist at an academic institution. I am well supported in the M.D./Ph.D. program at UConn Health by my sponsors and clinical mentors that will facilitate the successful completion of this project and development into a promising physician-scientist.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Metastasis is responsible for more than 90% of prostate cancer-related mortality and remains a considerable challenge in developing effective and durable therapies. Interestingly, 80% of all patients with metastatic prostate cancer (PC) develop bone metastases, dropping their 5-year survival to 26-30%, which underscores the need to reveal, understand, and exploit the unique cellular pathways, mechanisms, and oncogenic events that drive the initiation, formation, and maintenance of PC bone metastases. Essential to the development and preclinical screening of novel therapeutic technologies, there is an urgent need for a reliable and convenient in vitro/in vivo cellular model that recapitulates the unique PC bone metastatic environment. Prostate-Specific Membrane Antigen (PSMA) is expressed on the epithelium of nearly all PCs and increases with progression to castration resistance and metastatic disease. Tumor vascularity has a major impact on tumor growth and drug responsiveness with respect to tumor oxygenation and permeability of chemotherapeutics. PC cell-vascular endothelial cell (EC) crosstalk induces expression of PSMA on the surface of tumor vasculature in PC and in renal cell carcinoma and breast, lung, gastric, colorectal, pancreatic, and bladder cancers . Consequently, PSMA-targeted therapies (radiotherapeutics as well as small-molecule and antibody drug-conjugates) are actively being pursued and are anticipated to modulate PC tumor vasculature and diseases involving pathological angiogenesis. Our long-term goal is to develop a flexible 3D bioprinted tumor microenvironment model that can serve as a preclinical screening platform to enhance the development of novel therapeutic agents for various cancers. This study aims to develop a well-defined in vitro model that mimics the molecular, cellular, and metabolic interplay in the bone-tumor microenvironment of metastatic PC and confirm that it is similarly responsive as the clinical condition is to novel targeted diagnostic and therapeutic agents. The rationale for undertaking the proposed research is that developing a reproducible predictive PC tumor-bone model will accelerate therapeutic development for PC and minimize clinical failures.

NIH Research Projects · FY 2026 · 2024-07

Abstract Placenta accreta is a serious maternal condition affecting more than 1 in 300 women in the US, and rapidly increasing in frequency. Accreta is characterized by the placenta invading too deeply into the uterus, necessitating hysterectomy upon parturition. The major correlate of accreta (or increta, or percreta) is the presence of previous uterine scar, mostly due to caesarian surgery, although the disease could manifest without previous scars. Very little is understood about the cellular and molecular mechanisms underlying this disease, leaving limited therapeutic avenues to pursue. Our overall hypothesis states that while normal placental invasion is predicated on a fine balance of pro- and anti-invasable signaling at the maternal-fetal interface, placenta accreta is a response of scar induced dysregulation in this signaling, leading to excessive and un- controlled invasion. Specifically, we posit that the dysregulated biomechanical microenvironment at the scar transforms decidual fibroblasts into an inflammatory state, producing chemoattractive cytokines recruiting extravillous trophoblasts towards the scar. We have developed a framework termed ELI (Evolved Levels of Invasability) that explains the highly invasive nature of placentation in certain mammals as primarily a stromal characteristic. Based on ELI framework, and our preliminary findings, we posit that aberrant matrix cues from scar dysregulate decidual defense, resulting in uncontrolled trophoblast invasion leading to accreta. Using a bioengineered model of scar decidua (high rigidity, high collagen density, anisotropic nanotopography), we observed dysregulated mechanoresponsive signaling in decidual fibroblasts (dESFs), resulting in chemotactic recruitment of extravillous trophoblasts (EVTs) towards scar, as well as promoting EVT invasion by altered dESF contractility. Specifically, scar results in high activation of Protein Kinase C by Piezo-1 mechanosensitive Ca2+ channel. Increased PKC activation phosphorylates NFkB, a master regulator of inflammatory response, resulting in production of IL-8 and G-CSF, potent chemoattractants for EVTs. In this proposal, we will elucidate the missing links in these mechanisms using a combination of spatially defined exploration at transcriptional, molecular and tissue level scales. We have developed many technological platforms specifically for understanding stromal invasion of trophoblasts: a model of scar decidua that recapitulates accreta phenotype, a nano-fabricated platform for quantification of stromal invasion, cellular force measurement at EVT-dESF interface, and a microfluidic platform to measure paracrine cross-talk between EVTs and dESFs. As accreta is a spatially defined pathology, we will use spatial transcriptomics in human accreta samples to identify changes in decidual state proximal to scar. Finally, we will profile, and establish the causality of paracrine cross-talk between dESFs and EVTs in accreta manifestation. This integrated study combining systems and mechanistic approaches will shed light to elucidate the biology of scar induced uncontrolled placental invasion, and identify targets to therapeutically limit accreta pathogenesis.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Cardiac fibrosis is a hallmark of nearly all forms of heart disease, the leading cause of morbidity and mortality worldwide. However, despite the intensive research, there is no effective therapy for fibrosis, highlighting an urgent need to identify better therapeutic targets. As cardiac fibroblasts are the principal mediators for TGFβ induced fibrosis in heart diseases, targeting a fibroblast-specific molecule which is involved in TGFβ induced fibrosis would be an ideal target. However, fibroblast specific molecules are yet to be discovered. We previously demonstrated that TRPM7 is a major functional Ca2+ permeable channel in cardiac fibroblasts and plays a key role in TGF induced fibrogenesis cascade in cultured fibroblasts. TRPM7 is a unique Ca2+-permeable channel which possess a kinase domain. We have recently discovered that deletion of Trpm7 effectively reduces cardiac fibrosis thereby protecting hearts against pressure-overload induced heart failure. Interestingly, we found that deletion of Trpm7 in fibroblasts is necessary to produce the protective effects, indicating that Trpm7 deletion produces protective effects in a fibroblast-dependent manner. As targeting fibroblasts to inhibit fibrosis has recently been defined as a novel therapeutic strategy for cardiac fibrosis, we propose that TRPM7 may serve as a promising therapeutic target for fibrogenesis. Since TRPM7 has both channel and kinase function, one critical question is whether the channel function or kinase function should be the target for mitigating cardiac fibrosis. We propose two specific aims to investigate whether TRPM7 channel and/or kinase functions contribute to TRPM7 mediated fibrogenesis, and how inhibiting channel and/or kinase functions will protect hearts against fibrosis associated heart diseases. We will apply multidisciplinary approaches including mouse transgenic and knockout models, molecular biology, biochemistry, patch-clamp, Ca2+ imaging, and various in vitro and in vivo disease models, as well as fibroblasts from heart failure patients. The results of this proposal will not only answer the critical questions regarding whether the channel function or kinase activity should be the therapeutic target for fibrogenesis, but also will reveal novel mechanisms of TRPM7-mediated fibrogenesis in fibrosis associated heart diseases. More importantly, this proposal will provide translational insights into therapeutic potential of TRPM7 for pressure overload and ischemic injury induced heart failure.

NIH Research Projects · FY 2025 · 2024-06

Synthetic chemical toxins developed for warfare continue to be an enigma for homeland security should they be deployed to cause mass casualties. Sulfur mustard, a powerful ocular vesicant causes irreversible corneal damage affecting the epithelium and stroma, as well as the abundant network of corneal sensory nerves. Despite many studies that have examined damaging mechanisms to the epithelium and stroma caused by vesicants, little is known regarding how corneal nerves degenerate in this injury paradigm. This has led to a critical unmet medical need as no therapeutic is available to protect or regenerate the vulnerable corneal nerves from such injuries. Evidence from both human exposures and animal models of vesicant-injury reveal acute ocular pain and chronic loss of corneal sensation manifest but their mechanisms have not been investigated. Corneal axons are ensheathed by Schwann cells (SCs), a glial cell type that provides trophic support to axons. In animal models of injury conducted in other peripheral organs, SCs can actively regenerate and support the regeneration of damaged axons. Here we hypothesize that a similar axonal-supportive role may be anticipated for corneal SCs (cSCs) in vesicant injury. This gives an opportunity to pursue an innovative strategy to identify novel druggable targets in cSCs. We interrogated molecular and biological pathway information gained from our cSC-single cell RNA seq analysis and identified novel targets and biomarkers. Using a genetically labeled SC mouse model subjected to injury experiments, we have also gained important insight into the dynamic relationships of cSC and axons over acute and chronic stages of injury. This model and targeting the candidate target in cSCs have provided us preliminary data to support the idea that cSC could be targeted for axonal regeneration. In the proposed study, we now choose FDA approved drugs for testing to illuminate the most effective method of use that could support rapid and widespread application in any urgency. Because current information is lacking whether these drug candidates have therapeutic efficacy in the paradigm axonal regeneration over acute and chronic stages of vesicant injury, we include studies of extended use. Lastly, in this innovative exploratory grant proposal we will define a new therapeutic countermeasure against vesicant injury that exploits a combination treatment regimen that can restore corneal refractive function and also help to regenerate corneal sensation.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT The human herpesviruses are responsible for lifelong debilitating and congenital infections, and some members of this family are associated with human cancers. HSV causes significant disease during acute infection and establishes persistent latent infections in sensory neurons for the life of the host leading to reactivation and recurrent disease. This proposal is based on a series of observations about the interplay between HSV infection and the intrinsically antiviral cellular protein, PML. In response to interferon treatment, oxidative stress, DNA damage, and viral infection PML oligomerizes to form a mesh-like spherical shell around heterogeneous, phase- separated nuclear condensates, called PML nuclear bodies (PML-NBs). PML oligomerization leads to the activation of its SUMO ligase activity, which in turn induces the recruitment of dozens of cellular proteins that contain SUMO-interacting motifs (SIMs) including repressors of gene expression into PML-NBs via SUMO/SIM interactions. PML-NBs are anti-viral, however, the exact mechanism of their action remains poorly understood. PML-NBs size and number increase in response to HSV infection, and they are actively targeted for degradation by HSV1 as a mechanism to counteract their antiviral effects. During HSV infection PML-NBs have often been observed adjacent to the sites of viral DNA replication and have recently been shown to entrap the entire viral genome. Despite a rudimentary understanding of these processes, many questions remain about how PML-NB formation is triggered by HSV infection and how they are recruited to viral genomes. Oligomerization and sumoylation of PML have been shown to be essential for the effective formation of PML-NBs in response to stress. In Aim 1 we will identify PML domains that are necessary for its SUMO-E3 ligase activity and required for formation of the PML-NBs. One of the most surprising aspects of PML-NB formation is their ability to form around and entrap viral DNA, resulting in genome silencing. The properties of PML that promote this unusual ability to recognize viral DNA are not understood. In Aim 2 we will test the hypothesis that PML contains a DNA-binding domain necessary for recruitment of PML-NBs to viral genomes. Together, these aims will probe the fundamental mechanisms behind the two key events that trigger the formation of PML-NBs in response to HSV infection.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Antibiotic treatment failure is one of the greatest global public health challenges of our generation. Antimicrobial- resistant bacteria directly account for over 1.2 million deaths annually worldwide. Microorganisms that are refractory to a new antibiotic often emerge shortly after the drug is introduced in the clinic. Besides becoming heritably resistant to antibiotics, some bacteria in clonal cultures—coined persisters—can reversibly reprogram their phenotypes and become transiently tolerant to a given drug. This can lead to infection relapse after a course of antibiotics, rendering the treatment ineffective. Evidence further suggests that persisters have a higher likelihood of acquiring resistance-conferring mutations. As such, the development of anti-persistence/resistance therapeutic strategies can increase the success of antimicrobial therapy. In this proposal, we focus on antibiotic persistence and resistance development in Escherichia coli and Pseudomonas aeruginosa cells in slow/non-growing cultures, which are less responsive to antibiotics and more difficult to eradicate than their growing counterparts. We aim to discover strategies to potentiate the activity of existing and new topoisomerase inhibitors against these gram-negative pathogens. We recently found that metabolic stimulation and loss of efflux pump action during topoisomerase inhibitor treatment reduce persistence and resistance in non-growing E. coli. To build upon these findings, we will execute the following aims: Aim 1: Discover metabolites that are abundant at infection sites that can modulate persistence and resistance development of E. coli and P. aeruginosa toward first-in-class non-fluoroquinolone topoisomerase inhibitors that are in clinical trials. Aim 2: Investigate the impact of metabolic stimulation and loss of efflux pump action on bacterial metabolism, DNA integrity, and viability during topoisomerase inhibitor treatment. Aim 3: Deduce the effects of metabolic stimulation and loss of efflux pump action during topoisomerase inhibitor treatment on the coordination of molecular events that are important for persister repair and resuscitation after topoisomerase treatment terminates. We envision that the successful completion of this project will expand our knowledge of persister survival strategies and vulnerabilities. This will enable us to develop methods to enhance the activities of existing antibiotics and preserve the efficacy of new drugs in development before their introduction to the clinic.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY/ABSTRACT Synovial joints are essential for full range of motion and quality of life. Unfortunately, the joints -and articular cartilage in particular- are highly susceptible to congenital-, injury- and age-related diseases that lead to degeneration, a reflection of poor intrinsic cartilage repair capacity. Current clinical interventions do not meet the wide range of demands on articular cartilage due, in large part, to lack of crucial knowledge on the cellular mechanisms that govern normal functions of articular cartilage such as lubrication and tissue maintenance growing adolescents and young adults. In order to advance these strategies, more information is needed on basic mechanisms of articular cartilage development and adaptation/response to environmental changes in vivo. The superficial most layer of articular cartilage is responsible for secreting proteoglycans (lubricants) into the joint capsule that allow for frictionless movement. Many studies have focused on this as their primary function, and increasing lubrication has shown promise for disease treatment. This project will take a broader approach to clearly define unique characteristics and other potential functions for these cells that could be targeted for therapeutic approaches. In particular, this study will focus on interactions of superficial zone cells with underlying articular chondrocytes. Developmental studies by my sponsor's lab and preliminary data I have gathered from adult mice provide strong evidence that the superficial zone does not function as a progenitor population for underlying articular chondrocytes, and instead suggests that superficial cells are unique from articular chondrocytes. Thus, I hypothesize that superficial zone cells are maintained distinctly in articular cartilage, but that their coordinated functions with underlying articular chondrocytes promote sustained, functional organization of articular cartilage. To test this hypothesis, in Aim 1 I will characterize the unique properties of superficial zone cells during growth and during their response to damage compared to articular chondrocytes. In Aim 2, I will directly test the requirement of the superficial zone cells in adult animals. In Aim 3, I will explore mechanisms of coordinated functions between superficial cells and articular chondrocytes to maintain mature articular cartilage structure. I will use multiple analytical tools including histomorphometry, confocal imaging, and nano-scale mechanical testing in combination with RNA sequencing and in situ hybridization. Conditional mouse models, including our transgenic Prg4CreER allele to target superficial zone cells, will be examined at adult stages and following a traumatic injury (DMM-model) that significantly alters mechanical loading in the joint. The proposed studies will provide essential knowledge on mechanisms that underlie superficial zone cell functions and responses to damage/altered mechanical load. The data and insights from the project will prove essential to envision and test future therapeutic joint strategies that target superficial zone cells, providing broad relevance and importance to the project and offering a solid platform on which to establish my independent career in biomedical and translational medicine research.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT While relapsing-remitting multiple sclerosis (RRMS) can effectively be managed by a pantheon of disease modifying drugs with anti-inflammatory activities, the transition to secondary progressive MS (SPMS) is met with refractoriness to therapeutics and a downward spiral of neurodegeneration. This dire scenario has called into question the role of inflammation in SPMS, and cast SPMS treatment as a pressing unmet clinical need! However, meningeal ectopic lymphoid tissues (mELTs), which appear during SPMS in pathognomonic fashion, could drive atypical inflammatory activity and provide novel targets for therapeutic intervention. These indeterminate structures are B cell-rich aggregates that accumulate in the subarachnoid space of the meninges, and resemble tertiary lymphoid organs (TLOs) observed elsewhere in the body during various autoimmune and infectious conditions. In both MS patients and animal models, mELTs are conspicuously located over activated sub-pial microglia in areas of extensive cortical demyelination and neuronal loss, raising speculation they rain down as yet unknown incendiary factors that spark “compartmentalized inflammation” and neurodegeneration. Their role in SPMS etiopathogenesis nevertheless remains ambiguous, as the composition, compartmentalization, and gene expression of the varied cell types comprising mELTs have not been evaluated in relation to disease status and locale along the CNS axis – factors that can significantly impact the meninges and, thus, potentially mELTs. Technical hurdles had precluded examining these issues, but methodological advances by this group now allow for interrogation of mELTs – histologically and transcriptionally – within their native meningeal habitat. Two broad Aims are thus proposed to fill these major gaps, and test the hypothesis The cellular composition, organization and gene expression of meningeal Ectopic Lymphoid Tissues (mELTs) depend on CNS locale and disease status. Aim 1 will analyze animals at early and late stages of secondary progressive disease, and characterize the cell composition of mELTs in brain and spinal cord. Emphasis will be placed on identifying immune, stromal cell and vascular cell types found in known TLOs, and detailing their 3D organization with respect to each other and meningeal elements. Attention to meninges will be critical, as meningeal trabeculae might nucleate mELT assembly and/or segregate mELT cells into functional domains. High-resolution, 3D fluorescence microscopy and immuno-scanning electron microscopy will be used to view mELT cell organization at the light microscopic and ultrastructural levels, respectively, and imaging mass cytometry to reveal the full repertoire of cells in context with meningeal structures. Aim 2 will dissect the respective transcriptomes of the different mELT cell populations in brain and spinal cord in situ during early and late disease, using laser capture microdissection coupled to RNA-seq to preserve context-dependent gene expression patterns. This deliberative and methodical spatiotemporal analysis will yield foundational information necessary for resolving the contribution of mELTs to SPMS and highlighting new therapeutic prospects.

NIH Research Projects · FY 2026 · 2024-04

T cell responses rely on the T cell receptor (TCR) for antigen specificity, CD28 for a second signal, and a cadre of additional signals including cytokines and certain TNF superfamily costimulatory receptors. The latter includes CD134 (OX40) and CD137 (4-1BB), which provide a powerful boost to clonal expansion of effector CD8 T cells, robust cytokine production, metabolic fitness, and many other attributes including potent anti- tumor cytotoxicity. The molecular mechanism of costimulation-based programming in effector T cells is largely unknown. In particular, a unique gene signature for this process, beyond generalized factors such as NFKB and NFAT, is unclear suggesting that other critical processes have gone undetected. Our recent published data showed only a small number of transcriptional changes after costimulation, which could not explain the power behind costimulation. A major increase, however, in the spliceosome pathway was evident, but whether this process could have a specific impact on effector T cells was unknown. Analysis showed that the spliceosome pathway contained a group of RNA binding proteins involved in alternative RNA splicing, which should not be confused with steady state core intron splicing factors. Alternative RNA splicing can mediate exon skipping, intron retention and other processes that generate multiple mRNA isoforms from a single gene, and thereby greatly expand the proteome of cells. We demonstrated that the RNA binding protein Tardbp played a key role in effector T cell function. These changes in T cell function correlated with Tardbp-dependent exon skipping in the IL-2 repressor IKAROS (Ikzf1) mRNA, and a series of other RNA splicing events that remain to be studied. Aim 1 will examine how Tardbp programs effector CD8 T cell function by addressing effector activities that include target cell cytotoxicity, responses to infection, and a role for specific costimulators. Our results will be integrated with the identification of the direct mRNA isoforms generated by Tardbp, and their potential function. Aim 2 will examine a second RNA binding protein, Tra2b, for its novel role in controlling T cell responses through the action of an ultra-conserved ‘poison exon’ contained within the Tra2b transcript. Using a combination of innovative approaches, we will test if Tra2b’s poison exon impacts TCR sensitivity in CD8 and CD4 T cells during tumor immunity. For Aim 3, the RNA binding protein network in exhausted T cells will be edited to optimize function and translatability. Our proposal uses a combination of in silico and cutting-edge methods that will break new ground in understanding how alternative splicing through the action of RNA binding proteins can control effector T cell programming. In sum, a new understanding of effector T cells and their potential in clinical translation will be gained from this research.

NIH Research Projects · FY 2026 · 2024-04

Myostatin (MSTN, GDF-8) is a secreted signaling protein that we discovered many years ago as a transforming growth factor-ß (TGF-ß) family member that normally acts to limit skeletal muscle growth. A major focus of my laboratory has been to elucidate the molecular, cellular, and physiological mechanisms underlying MSTN activity, with the goal of developing strategies to target this pathway for clinical applications. Biochemical studies carried out by us and by others have identified many key regulatory and signaling components, which has led to the model that MSTN, along with activin A, regulates muscle growth and function by binding first to the type 2 receptors, ACVR2 and ACVR2B, and then engaging the type 1 receptors, ALK4 and ALK5. The formation of this receptor complex leads to activation of SMAD2 and SMAD3, which are the key mediators of the canonical signaling pathway for these ligands. To determine their roles in vivo, we have generated mice carrying flox alleles for each of these components and examined the effect of targeting them either individually or in combination specifically in skeletal myofibers. These studies have led to unexpected findings that have revealed major gaps in our understanding of how this system operates in vivo. One unexpected finding was that simultaneously targeting SMAD2 and SMAD3 led to only modest increases in muscle mass, implying that MSTN/activin A utilize both SMAD-dependent and SMAD-independent pathways in vivo. A second unexpected finding was that targeting the two type 1 receptors for MSTN/activin A has a substantially greater effect on muscle mass than targeting the two type 2 receptors. Our new findings, taken together with studies by other groups showing that muscle growth is regulated by other TGF-ß family members, specifically BMPs, that act antagonistically to MSTN and activin A, have led us to propose a new model that competition between MSTN/activin A and BMPs occurs primarily at the level of the type 2 receptors. The overall goal of this project will be to use a combination of genetic and pharmacological approaches to elucidate further how all of these regulatory components interrelate in muscle, specifically to determine whether different components of this regulatory system have distinct roles in regulating muscle growth and function, particularly with respect to metabolic function. The Specific Aims of this project will be to elucidate SMAD-dependent versus SMAD-independent effects in skeletal myofibers; to assess the effect of blocking SMAD-dependent and SMAD-independent pathways in skeletal muscle in mouse models of metabolic dysfunction and muscle wasting; and to elucidate how MSTN/activin A signaling and BMP signaling interrelate in myofibers. We believe that the studies outlined in this proposal will provide key insights into the mechanisms by which this complex network of regulatory and signaling components function in an integrated manner to regulate muscle homeostasis and will inform drug development efforts to maximize the anabolic potential of targeting this pathway in muscle to treat patients with muscle and metabolic diseases.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Keloids are dermal lesions that grow beyond the margin of the original wound in a tumor-like manner. Keloids can expand for several years, grow to large size and be painful, itchy and inflamed. Despite of the severe physical, psychological and social impact on patients, keloids are understudied and the treatment is unsatisfactory with high recurrence rates. Keloids also contribute to racial disparity in research and health care because prevalence is highest in populations of African ancestry. There is strong evidence for a genetically diverse basis for keloids. However, gene mutations that make patients susceptible and initiate keloids have not been identified. Identifying such causative genetic variants is fundamental for the understanding of keloid pathoetiology and for the identification of potential therapeutic targets. We previously identified several keloid candidate variants by linkage analysis and whole exome sequencing in a large cohort of 103 ethnic Yoruba families from Nigeria with heritable keloid formation. We prioritized four candidate genes variants that segregate in families, are expressed in skin and associated with other fibroproliferative disorders or are involved in wound healing. In this application we aim to determine whether the downstream effects of these four candidate gene variants induce a keloid-like phenotype in skin cells or in organotypic skin equivalents. We will investigate pathways by which the candidate variants act on skin cells. In Aim 1, we will study the roles of keloid candidate gene variants on individual skin cell types derived from human isogenic induced pluripotent stem cells (hiPSCs) carrying the respective gene mutations. We minimize background noise from biological variability by comparing isogenic mutant and wild type cells that differ only by the variant to be investigated. In Aim 2, we examine interactions between mutant fibroblasts, keratinocytes and macrophages that lead to paracrine effects guiding epithelial-mesenchymal transition and fibrosis during wound healing, using co-cultures, organotypic skin equivalents and xenotransplant mouse models. We expect that investigating the role of genes that have not been investigated in keloids will bridge knowledge gaps in keloid and wound healing research and will identify novel factors that put individuals at risk for keloid development.

NIH Research Projects · FY 2026 · 2023-12

ABSTRACT Stroke is a leading cause of death and disability worldwide. Over the past few decades, there have been extensive investigations and enormous research efforts to find better therapies for stroke patients. However, many strategies that are effective in animal models have proven ineffective in clinical trials. Thus, tissue plasminogen activator (tPA) is still the only FDA-approved drug for stroke. The lack of successful bench-to-clinic translation is mainly due to two reasons, with one being the complexity of the pathology of stroke, which includes a multiplex of signaling pathways, various cell types, and both local and systemic inflammatory responses. Secondly, the prevailing clinical conditions in patients, such as hyperlipidemia, are not included in animal models of stroke. We propose that a pleiotropic target which can integrate multiple critical components in various cell types will be the key to developing effective stroke therapies by using clinically relevant animal models. In this proposal, we propose that the oxidative stress activated Ca2+-permeable TRPM2 can serve as this pleiotropic target for developing effective stroke therapies. The goal of this proposal is to investigate whether (Aim 1) and how (Aim2) TRPM2 plays a vital role in ischemic injury cascade, and to determine whether TRPM2 may serve as a pleiotropic target for ischemic stroke using the classical stroke model (Aim 1) and a clinically relevant stroke model (Aim 3). We will use state-of-the-art multidisciplinary approaches to test our hypothesis. Successful completion of this proposal will not only prove the concept that a pleiotropic target represents a new strategy for developing effective therapies for ischemic stroke, but also will provide profound insights into translational implications of developing new stroke treatment by targeting TRPM2.

NIH Research Projects · FY 2025 · 2023-12

Chronic kidney disease (CKD) affects 37 million adults in the United States and results in anemia in more than 5 million patients. Patients with anemia due to CKD often require transfusion or pharmaceutical intervention to combat fatigue and hypoxia. The most common pharmaceutical intervention is injection of Erythropoietin (EPO), a cytokine that is produced in the healthy kidney in response to hypoxia. EPO promotes erythropoiesis by enhancing the survival and proliferation of erythroid-committed progenitors in the bone marrow. However, pharmaceutical EPO administration can cause thrombocytosis with an increased risk of stroke. This off-target consequence highlights an understudied role for EPO in non-erythroid-committed cell types and emphasizes the need for further research to clarify the effect of EPO on hematopoietic progenitors such as the Megakaryocytic-Erythroid bipotent progenitor and Megakaryocytic-committed progenitor. Furthermore, the receptors and downstream signaling pathways activated by EPO have not been fully elucidated in subpopulations of hematopoietic progenitors. Our preliminary results indicate that EPO supports survival and self-renewal of MEP. The research proposed in this application will investigate the cell-autonomous effects of EPO on bipotent human Megakaryocytic-Erythroid progenitors, Megakaryocytic-committed progenitors, and Erythroid- committed progenitors ex vivo by measuring activation of signaling mediators, transcriptional expression, and phenotypic changes. It will also clarify the receptors and targets that are activated in response to EPO signaling in defined hematopoietic subpopulations utilizing novel receptor-specific EPO mimetics. We hypothesize that in response to EPO, Megakaryocytic-Erythroid progenitors expand to increase the pool of progenitors capable of giving rise to erythroid and platelet-producing megakaryocytes, resulting in increased production of both platelets and red blood cells. Successful completion of these studies will resolve the molecular mechanism of the action of EPO on specific populations of cells in the bone marrow, which will aid in the development of more targeted and effective therapies for patients with chronic anemia.

NIH Research Projects · FY 2026 · 2023-12

Project Summary Type 2 diabetes mellitus (T2DM) is a primary cause of the marked increase of ASCVD, accounting for over 30% of symptomatic cases. Accordingly, the ASCVD risk in T2DM patients is 2-4 fold more than in the non-T2DM population. The concomitant increase of T2DM and ASCVD in the US prompted the urgency for accurate ASCVD prediction followed by an effective mitigation strategy. However, recent clinical trials and extensive cohort studies revealed underachievement by controlling conventional lipid/hypertension/glycemic factors, warranting new directions additional to the existing mitigation paradigm. Furthermore, multiple studies comparing existing ASCVD risk prediction models, including those derived from general or T2DM populations, achieved only marginal or underperformance in multiple independent cohorts, endorsing the significance of this significant unmet biomedical challenge. Our proposed study aims to fill this critical scientific and clinical gap in increased atherosclerosis cardiovascular disease risk (ASCVD) in T2DM. Supported by our recent discoveries and development of multiple immune cell annotation tools, we propose to test the overarching hypothesis that the foaming process in circulating monocytes can be directly altered by T2DM towards pathogenic foaming, thus directly contributing to the elevated risk of ASCVD. Application of our newly developed cell function annotation tools uncovered distinct macrophage-derived foam cell development programs, followed by ASCVD causal gene signature identification and proof-of-concept ASCVD predictive modeling by incorporating a selected gene list using the original machine learning program. Our new work further revealed that super-networks governed by three upstream regulators are uniquely altered by T2DM and associated with future ASCVD incidence. Hence, in two aims: (AIM 1) we will determine the impact of suppressed super-networks governed by identified master regulators (SNW1, NCOR2, CITED2) on pathogenic foaming program using established foam cell development system with quantitative methods and single cell transcriptomics; (AIM 2) will assess the efficacy of T2DM- ASCVD signatures on predictive modeling using existing and innovative feature selection and feature extraction tools. Completing this project will provide crucial information to significantly advance our understanding of ASCVD risk in the T2DM population. Our innovative computational programs, derived from novel and in-depth mechanistic investigation, will offer translatable ASCVD prediction tools and molecular targets for drug development to achieve personalized intervention for T2DM patients to mitigate ASCVD risks.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Maternal morbidity and mortality rates are increasing in the United States (US), and extreme racial and geographic disparities persist, despite these outcomes being largely preventable with timely and appropriate care. Most medical responders are not optimally proficient in caring for patients who experience maternal medical emergencies, including maternal cardiac arrest. This gap exists among first responders and across medical specialties, and even for OB-specialists trained in Advanced Cardiovascular Life Support. Leading organizations in women’s health care and resuscitation have all called for efforts to better prepare healthcare workers (HCWs) for maternal medical emergencies, and increasingly federal agencies and state legislatures are incentivizing or requiring hospitals to provide this education and training. Nonetheless, the implementation of evidence-based education for maternal medical emergencies in health systems across the US is inconsistent and national credentialing standards do not exist. The study team developed Obstetric Life SupportTM (OBLSTM), the first of its kind interdisciplinary simulation curriculum to train HCWs across the chain of survival on preventing, recognizing, and managing maternal medical emergencies. Preliminary data from a randomized, cross-over trial shows significant improvement in clinical competencies, knowledge, and confidence for the intervention group compared to the control. However, as this validated simulation training is only just now available and accessible to providers and healthcare organizations, dissemination and implementation best practices do not yet exist. The objective of this project is to evaluate a train-the-trainer approach for implementing OBLSTM in a diversity of hospital and prehospital contexts throughout Arizona. The study will be conducted in partnership with the Arizona Perinatal Trust, the oldest regionalized perinatal healthcare system in the US, and Arizona Emergency Medical Services. A Steering Committee composed of local, regional, and national stakeholders will provide guidance and oversight for all study activities. Specifically, the study aims to: (1) Identify and train HCWs from hospital and prehospital contexts in Arizona to be OBLSTM instructors, prioritizing those that serve maternity care deserts and other marginalized communities at risk of adverse maternal health outcomes; (2) Implement OBLSTM in hospital and prehospital contexts across the state, with the training being led by local instructors; and (3) Evaluate OBLSTM implementation and process outcomes. The study design entails a mixed methods approach, informed by the Consolidated Framework for Implementation Research and the RE-AIM framework. To promote rapid learning, we will conduct two back-to-back OBLSTM instructor training, implementation, and evaluation cycles, whereby the second cycle will take into account feedback and lessons learned from the first. The study’s rigorous pragmatic approach will result in expeditious and relevant findings that will be used to promote national scale-up of this important health care innovation.

NIH Research Projects · FY 2025 · 2023-09

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Biological Magnetic Resonance Data Bank (BMRB) is the authoritative worldwide resource that provides free access to the wealth of information on biomolecules derived from nuclear magnetic resonance (NMR) spectroscopy. These NMR experimental data underlie the three-dimensional structures and conformations of many proteins and nucleic acids and provide important insights into their dynamics, chemical properties, and molecular interactions. NMR is also useful for characterizing biomolecular interactions, with applications to drug discovery, and in quantifying the components of complex mixtures of biomolecules, including metabolites and natural products. Data archived at BMRB include chemical shifts, peak intensities, scalar couplings, dipolar couplings, and relaxation and cross-relaxation rates. BMRB integrates these NMR data into a unified, global, molecular database of general utility to the broad scientific community. The growing volume of data available from BMRB is catalyzing transformative scientific applications, such as the determination of protein structure and dynamics directly from chemical shifts. BMRB maintains software tools for integrating the retrieval, analysis, and display of NMR data in the context of molecular structure and conformation. As a member of the World Wide Protein Data Bank (wwPDB), BMRB has close ties with the other wwPDB partners: the Research Collaboratory for Structural Bioinformatics (RCSB), the Protein Data Bank in Europe (PDBe), the Electron Microscopy Data Base (EMDB), and the Protein Data Bank of Japan (PDBj). In its role in the wwPDB, BMRB implements standards for NMR data types and maintains software for data deposition and validation. This project supports ongoing operation of BMRB, including maintenance of deposition pipelines and infrastructure to support search and retrieval of NMR data, and to maintain a productive dialogue with users and creators of biomolecular NMR data to increase the impact of the archive, and to maintain BMRB’s role in the wwPDB.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY At this time, alcohol is the top substance used by US emerging adults under the legal drinking age (underage emerging adults; U-EA; ages 18-19). While drinking rates had recently been trending downward, 2021 national data reflect rises in youth drinking across all metrics. This is highly consequential in terms of youth safety and neurodevelopment. One of the central challenges of U-EA alcohol use is that youth are unlikely to seek, receive, or complete indicated alcohol intervention. In turn, it is imperative to find brief, effective interventions to impactfully intervene with U-EA hazardous alcohol use. Additionally, the developmental neuroscience literature robustly reflects that peers hold higher neural salience during this developmental window, as evidenced by youths’ differential neural response in conditions with real and/or simulated peers, even when those peers were not friends, and particularly in the context of alcohol. Furthermore, studies are recognizing that peers concomitantly activate positive (prosocial) neural and behavioral responses in this age group, and this has been observed in conditions of negative and positive peer feedback. The role of negative and positive peer feedback is consequential given that one of most widely-used U-EA intervention service delivery methods are group-based formats. Here, we aim to build upon PI Feldstein Ewing’s 15 year history of continuous NIH funding in youth translational (brain:behavioral) approaches evaluating youth within-session factors, youth neural response, and subsequent behavior change. Across independent samples, our team has found distinct developmental brain response to MI interventions, largely localized to default mode network (DMN) regions [precuneus, posterior central cortex (PCC)]. Further, this observed DMN activation to examined within-session factors (client language; therapist language) was significantly associated with youth post-treatment behavior change. We thus respond to PAR-21-280: “Dyadic Interpersonal Process and Biopsychosocial Outcomes” and propose functional magnetic resonance imaging (fMRI) to evaluate U-EA brain response in the underexamined, but widely-utilized, group MI context. study, we will examine the nature of peer-peer dyadic exchanges in the youth brain during group MI In this interventions (Aim 1) and predictors of behavior change: prospective relationships between youth brain response and behavior change in the novel context of group MI (Aim 2). These data are crucial for guiding improvements in brief behavioral group-based intervention programming alcohol use. for U-EA engaged in hazardous

NIH Research Projects · FY 2025 · 2023-09

Project Abstract Alzheimer’s disease and related dementias (ADRD) are the most prevalent, debilitating neuro-degenerative diseases of aging, and current treatments for neuropsychiatric symptoms (NPS) (e.g., agitation, aggression, and depression) of ADRD are mixed. While non-pharmacological behavioral interventions are recommended as first-line treatments for these NPS of ADRD, they require substantial time and resources, and may be less effective for severely agitated, aggressive, or depressed older patients with ADRD. Electroconvulsive therapy (ECT) is an effective and safe treatment for a range of psychiatric disorders, including treatment-resistant depression, schizoaffective disorder, and bipolar disorder. Case series and naturalistic studies support preliminary evidence for the efficacy of ECT to treat severe agitation, aggression, depression, or other NPS in ADRD. To date, associations of ECT with long-term improvement in NPS and geriatric syndromes (e.g., functional declines and frailty), as well as with hospital and nursing home admission rates and all-cause mortality rates are largely unexplored. The proposed nationwide cohort study explicitly addresses these knowledge gaps. Using the Centers for Medicare and Medicaid Services’ 2016-2019 Medicare claims data (Parts A, B, and D) linked with multiple data sources (e.g., Home Health Outcome and Assessment Information Set, Medicare Current Beneficiary Survey, and National Death Index), this proposed study features the following specific aims: 1) to examine incidence and prevalence rates of ECT use and socio-demographic and clinical factors associated with ECT use; 2) to investigate longitudinal associations of ECT with NPS and geriatric syndromes; and 3) to examine differential risks of hospital or nursing home admission rates and all-cause mortality rates by ECT use in older adults with ADRD. Longitudinal data analyses, such as generalized linear mixed modeling and competing-risk regression methods, will be used for Aims 2 and 3. In this nationwide cohort study, we will employ propensity-score matching and instrumental variable techniques to adjust for both observed and unobserved confounders. Intent-to-treat and as-treated analyses will also be conducted. Earlier studies support that ECT is associated with improvement in NPS of ADRD and our analyses show that ECT use with presence of both depression and ADRD was associated with a lower likelihood of all-cause mortality rates; we thus hypothesize that ECT may also be protective against geriatric syndromes. This is the first nationwide, longitudinal cohort study investigating the long-term effectiveness and safety of ECT in adults with ADRD. The proposed study is innovative since it will provide a better understanding of ECT use and its association with NPS, geriatric syndromes, and other health outcomes. Findings from this study will inform clinical guidance on ECT use in older adults with ADRD.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY: Uropathogenic Escherichia coli (UPEC) is one of the major causative agents of urinary tract infections (UTIs). With growing frequency of antibiotic treatment failure and slowing of antibiotic discovery, the rate of recurrent UTIs is poised to continue to increase. Although antibiotic resistant pathogens are of particular concern, treatment failure can often occur by non-genetic mechanisms, without detectable resistance. One of the non- genetic mechanisms of antibiotic treatment failure—persistence—is characterized by antibiotic tolerance in a small group of cells among a susceptible population. Bacterial persisters, which can reversibly exit the antibiotic- tolerant state following drug removal, is able to repopulate an infection and impede success of treatment. Persisters are known to be enriched in slow growing populations, where overall cellular activity and metabolism is reduced; however, there is a lack of knowledge on how the surrounding nutrient environment can affect bacterial physiology, and how these factors affect susceptibility to antibiotics. Our central objective is to determine the impact of carbon source availability on UPEC persistence to fluoroquinolone (FQ) antibiotics, which are bactericidal through the targeting of DNA topoisomerases and the accumulation of DNA damage. Aim 1 aims to address changes to metabolism, biomolecular synthesis, and DNA integrity in response to carbon source availability during FQ treatment. We will assess these factors by performing experiments with fluorescence-based probes, as well as mass spectrometry analysis of metabolites. These data will elucidate the ways by which carbon source availability can impact the susceptibility of cells to FQs, allowing us to explore specific mechanisms as potentiation targets. Aim 2 will enable us to understand how the coordination of DNA damage response changes due to carbon source availability after FQ treatment. We will deploy powerful genetic approaches and RNA sequencing in E. coli to interrogate the timing of gene expression and DNA repair. Completion of this aims will provide great insight into the importance of various DNA repair mechanisms in response to FQ treatment. Overall, the completion of these aims will reveal metabolic, biosynthetic, and expressional changes that underlie FQ persistence in E. coli. This information could illuminate potential genes that can be targeted to potentiate the activity of FQs and reduce resistance development in persister progenies. Additionally, these findings could lead to interesting implications for clinical infections, with a potential connection between host metabolism and antibiotic efficacy. This research could highlight the potential of adding glucose, as well as other metabolites, as supplemented compounds during antibiotic administration to improve the treatment of infections.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the United States. Immune checkpoint inhibitors (ICIs) like anti-PD-1 have increased overall survival in NSCLC, but most patients still do not respond to treatment. Cancer vaccines that target tumor-specific antigens, known as neoantigens, may increase the efficacy of ICIs and other immunotherapies by expanding neoantigen-reactive CD8+ T cells that can recognize and destroy tumor cells. Alternative splicing is a ubiquitous post-transcriptional regulatory process that allows cells to produce different mRNA and protein sequences from the same gene. Alternative splicing is broadly dysregulated in many cancer types including NSCLC and may generate novel peptide sequences absent from normal tissue that can be recognized as neoantigens by CD8+ T cells. To identify alternative splicing- derived neoantigens in NSCLC, we used long-read RNA sequencing to comprehensively map full-length mRNA isoforms in NSCLC tumors and predict the proteins they encode with high accuracy. We found 145,914 predicted peptides that were specific to tumors and shared by up to 70% of NSCLC patients. To identify which of these peptides might be immunogenic, we used immunopeptidomics to directly sequence peptides bound to MHC Class I in three NSCLC cell lines. We identified 21 peptides that are bound to MHC Class I on NSCLC cells and are encoded by tumor-specific alternatively spliced mRNA isoforms. These splicing-derived peptides are potentially shared neoantigens that might represent vaccine targets for NSCLC. Therefore, Aim 1 will test whether any of these 21 splicing-derived peptides can be recognized by CD8+ T cells from NSCLC patients. We will examine whether patient CD8+ T cells can proliferate, secrete cytokines like interferon-gamma, and lyse target cells in response to these peptides. The experiments proposed in Aim 1 will provide crucial insight into the frequency and immunogenicity of alternative splicing-derived neoantigens in NSCLC. Aim 2 will examine which regulators of alternative splicing are driving production of these peptides. To this end, we will leverage publicly available databases to identify splicing factors whose expression in tumors or target binding sites suggest an association with the mRNA isoforms that code for the 21 splicing-derived peptides. We will use targeted genetic approaches to study whether candidate splicing factors directly regulate peptide-coding isoform splicing in vitro. This work will highlight mechanisms that can drive the production of tumor-specific splicing-derived peptides and may reveal novel targets that can be exploited to enhance NSCLC immunogenicity. Altogether, these studies may identify candidates for new immunotherapies, including personalized NSCLC cancer vaccines that can be used to treat multiple patients who share expression of immunogenic splicing-derived neoantigens. This proposal will provide me excellent training that will facilitate my career goals as a physician-scientist who leverages advances in genomics and immunology to improve care for patients with cancer.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Autism spectrum disorder (ASD) is a highly heritable, heterogeneous neurodevelopmental disorder affecting 1 in 53 children in the US. The prefrontal cortex, which mediates social cognition and language, is oddly enlarged in at least 15% of patients with ASD who suffer from severe symptoms. Macrocephaly (large brain) is caused by excessive proliferation of cortical progenitors, and progenitors derived from ASD patients show excessive proliferation. However, the extent to which prefrontal macrocephaly itself contributes to the pathophysiology of ASD is unclear. ASPM (abnormal spindle-like microcephaly-associated) is a neurodevelopmental gene that determines cortical size, and may play a role in macrocephaly, as well as in ASD. ASPM controls cell proliferation, and its loss-of-function mutations are the most common cause of genetic microcephaly (small brain) that are particularly severe in the prefrontal cortex. Importantly, it is expressed in cortical progenitors but not in neurons. Recently, de novo variants in ASPM have been associated with ASD. Our preliminary data show that one such variant increases ASPM protein levels in cultured cells, suggesting gain-of-function mutation. Furthermore, we generated Aspm knock-in mice with the gain-of-function mutation, which show excessive neurogenesis, perinatal macrocephaly, and abnormal social behavior recapitulating ASD-like symptoms. Our long-term goal is to understand the mechanisms by which abnormal cerebral cortical development underlies functional abnormalities in ASD. Our central hypothesis is that excessive embryonic neurogenesis, which results in macrocephaly, is sufficient to elicit some ASD-like behaviors by disturbing cell signaling and composition in the postnatal brain. To test the hypothesis, we will leverage the ASD-associated gain-of-function mutation in ASPM, and examine Aspm knock-in mice in three Specific Aims. Thus, we will (Aim 1) investigate the neurodevelopmental trajectory using immunostaining, (Aim 2) determine changes in cell composition and signaling using single-nucleus RNA sequencing, (Aim 3) social cognitive behaviors in Aspm knock-in mice. Our proposed research is significant as we directly address the pathophysiological role of macrocephaly in ASD. It is innovative as we analyze a novel ASD mouse model with a gain-of-function mutation in the neurodevelopmental gene ASPM using diverse, state-of-the-art techniques. Macrocephaly is observed in some ASD patients with severe symptoms. However, the extent to which macrocephaly itself contributes to ASD is unclear. Our novel Aspm knock-in mice carry an ASD-associated mutation and display perinatal macrocephaly with abnormal social behavior. Exploring dysregulated cell types and signaling pathways in Aspm knock-in mice may provide novel therapeutic interventions for ASD.