Upstate Medical University

NIH Research Projects · FY 2026 · 2026-06

Project Summary. The Orthoflavivirus genus in the family Flaviviridae contains several pathogenic arthropod- borne viruses, including West Nile virus (WNV) a mosquito-borne orthoflavivirus in the Japanese encephalitis (JE) sero-/genetic complex. The United States has reported >27,000 cases of WN neuroinvasive disease and >2,900 deaths since the introduction of WNV in 1999, making WN vaccine development a public health priority. Live-attenuated vaccines (LAVs) have successfully controlled mosquito-borne orthoflaviviruses, yellow fever and JE virus. Thus, development of a WN LAV is realistic. The development of an attenuation strategy built on multigenic mutations will support the rational design of a candidate WN LAV. To date, attenuating mutations have been identified in 3 virally encoded nonstructural (NS) proteins, but few attenuating mutations have been identified in the structural proteins, including the envelope (E) protein. Because the E protein not only mediates the binding and membrane fusion with the cell but is the primary target of neutralizing antibodies, a correlate of protection for WNV, mutations in the E protein of a candidate WN LAV must not compromise immunogenicity. This project aims to identify attenuating mutations in the E protein to be incorporated into the rational design of a candidate WN LAV alongside those in the NS proteins. The E protein is a class II fusion protein that forms a fusogenic trimer to mediate the membrane fusion process, which is the fundamental viral entry mechanism and is crucial for WNV neurotropism in vertebrate hosts and transmission by mosquitoes. The formation of the E protein trimer requires the rearrangement of its 3 domains (EDI, EDII, and EDIII). The 4 motifs between the EDI and EDII exert a hinge effect for the EDI-EDII interdomain movement to initiate the formation of the E protein trimer, hence the name EDI-EDII hinge region. Site-directed mutagenesis of the EDI-EDII hinge region in WNV strain NY99 rescued from an infectious clone (WNV-NY99ic) demonstrated that the E-A54 and E-Y201 residues each control the formation of the E protein trimer. WNV-NY99ic retained 13 alternative amino acid (aa) substitutions of the respective residues. The E-A54I and E-Y201P mutations each fully attenuated the neuroinvasive phenotype of WNV-NY99ic in outbred Swiss mice, while retaining induction of serum neutralizing antibodies. Built on the attenuated phenotype and immunogenicity of the E-A54I and E-Y201P mutants, the central hypothesis is that impairment of the membrane fusion process by mutations of either the E-A54 residue or the E-Y201 residue will attenuate murine neurotropism and mosquito transmission of WNV- NY99ic without compromising antibody-mediated neutralization. This project will select the lead mutation of the respective residues based on 1) attenuation of the mouse neuroinvasive phenotype and neurovirulence, 2) impairment of transmission by Culex species mosquitoes that are WN vectors, and 3) immunogenicity that elicits serum neutralizing antibody responses conferring passive protection to identify the E protein mutation(s) suited for the rational design of a candidate WN LAV.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury (ALI), characterized by uncontrolled inflammation, excessive pro-inflammatory cytokine expression, neutrophil infiltration, and damage to the alveolar-capillary barrier. Among the causes of ARDS, sepsis is the most prevalent, accounting for 32% of cases. Current management primarily relies on protective mechanical ventilation and supportive critical care. Despite advances, ARDS remains a leading cause of mortality, underscoring the urgent need for effective therapeutic interventions. Corticosteroids are widely used but are often accompanied by significant adverse effects. Alternative approaches have been explored, with IL-10 standing out as a potent pleiotropic cytokine that plays a critical role in modulating inflammation and maintaining immune homeostasis. IL -10 effectively suppresses LPS- and bacteria-induced pro-inflammatory cytokines. It showed promising results in ALI/ARDS treatment. However, its clinical translation has been hindered by suboptimal pharmacokinetics, instability, and non-specific distribution. To address these challenges, we propose developing a lung-targeted IL-10 mRNA therapy using our novel lung-targeting sulfonium lipid nanoparticle (sLNP) technology. Our hypothesis is that delivering IL-10 mRNA specifically to the lungs and inducing its expression at the injury site will enhance therapeutic efficacy while minimizing the risk of systemic side effects. Our pilot studies have demonstrated the efficacy and safety of IL-10 mRNA/sLNP in treating ALI/ARDS in mouse models. In this project, we aim to evaluate the safety, tissue distribution, and therapeutic potential of IL-10 mRNA/sLNP in a more clinically relevant swine model of sepsis-induced ARDS. The success of this study could transform ALI/ARDS management and pave the way for future innovations in targeted mRNA therapeutics for respiratory care.

NIH Research Projects · FY 2026 · 2026-03

Abstract Influenza A virus (IAV) disproportionally affects adults aged ≥ 65, resulting in significant morbidity and mortality due to impaired type-2 immune responses mediated by innate lymphoid cells group- 2 (ILC-2). Our data revealed a critical role of β-catenin in modulating ILC-2 function, which declines with age. Using innovative mouse models, we demonstrated that β-catenin stabilization enhances ILC-2 survival and function, leading to improved resistance against IAV infection. Our study utilizes both loss-of-function (LOF) and gain-of-function (GOF) mouse models to detail the effects of β-catenin levels on ILC-2 numbers and their effector functions, specifically GATA3 and T-bet expression. Results indicate that β-catenin stabilization in GOF models results in significant increases in ILC-2 counts, enhanced Th1 effector immunity, and reduced lung damage upon IAV challenge, whereas LOF models show detrimental outcomes. The proposed proposal aims to elucidate the mechanisms by which β-catenin influences ILC-2 dynamics and its interaction with key transcriptional regulators such as GATA3 and TCF-1, potentially through the canonical Wnt signaling pathway. We will examine the effects of a novel β-catenin-stabilizing small molecule, A124, on ILC-2 populations in mouse and human lung tissues. This will include examining β-catenin's role in modulating immune responses without causing systemic toxicity. Our multidisciplinary team combines expertise in ILC-2 biology, β-catenin signaling, and clinical insights from thoracic surgery and pulmonology to translate these findings into potential therapeutic strategies for enhancing immune protection in the elderly. This research is pivotal for developing targeted interventions against IAV and possibly other respiratory pathogens in vulnerable populations.

NIH Research Projects · FY 2026 · 2025-09

Project Summary Systemic amyloidosis (SA) is a group of serious disorders caused by misfolding of ~20 circulating proteins and their subsequent fibrillization and deposition in various organs. The most common forms of SA involve misfolding of immunoglobin light chain (LC), transthyretin (TTR), and leukocyte cell-derived chemotaxin-2 (LECT2), and aggregation of these proteins causes kidney and heart dysfunction. Some of the factors that contribute to the misfolding of these proteins are known or suspected, such as destabilizing mutations and loss of bound cofactors, but the underlying molecular mechanisms of SA remain undefined. This proposal pursues the hypothesis that laminar flow shear in the blood, in combination with known conditions that destabilize the affected proteins, is a major driver of protein misfolding in the circulatory system and development of SA. The principal innovation is the introduction of microfluidic devices that recapitulate the dimensions and highly branched structures of the human kidney vasculature, through which ~1 L of one’s blood passes each minute. These structures create shear stress conditions that are extensive and unique in the body. These devices will be used to subject LC, TTR, and LECT2 to the form of physio-mechanical stress that they experience in circulation, to induce specific local unfolding events that bring about their aggregation into amyloid fibrils. The project employs cryo-EM, fluorescence, and biophysical experiments to characterize the structures and properties of misfolded forms generated by the microfluidic devices, including liquid-liquid phase separated condensates and mature amyloid fibrils. Amyloid structures will be compared to those previously obtained from diseased human kidney and heart to test the hypothesis that flow shear reproduces the ex vivo structures. Finally, small binding domains that recognize misfolded forms of LC and TTR will be generated, and fluorescent and luminescent biosensors will be created by plugging these domains into the PI’s existing modular sensor designs. Biosensors will be tested using patient serum and urine. This aim addresses the critical need for an early diagnostic test that can be performed at point-of-care, to detect SA prior to the onset of organ damage.

NIH Research Projects · FY 2025 · 2025-09

Project Summary: Form is inextricably linked with function in the eye, where the topology of the retina in part determines what incident light can be detected; this is true for both the camera eye of vertebrates and for the compound eyes of invertebrates, such as the fly, Drosophila melanogaster. In both cases, retinal development and function is promoted through its association with a support tissue – retinal pigmented epithelium (RPE, vertebrates) or peripodial epithelium (PE, fly). While much study has been focused on the molecular mechanisms underlying retinal specification, photoreceptor differentiation and on vision-associated diseases, the contribution of support tissues to the development and morphogenesis of the adult eye structure has received significantly less attention. Mutations in YAP1 and MITF are associated with congenital malformation of the human eye, resulting in the optic fissure closure defect coloboma (YAP1 and MITF) or microphthalmia (MITF). In neither context are the factors or processes upstream or downstream of YAP1 or MITF known or understood. I have recently described a Retinal Displacement (RDis) paradigm in fly, where developmental misalignment of Retina and PE tissues leads to congenital malformation of the compound eye. This powerful genetic model has led us to identify the conserved fly orthologs of YAP1 (Yki) and MITF (Mitf) as critical regulators of ocular epithelial topology. Furthermore, this model has allowed us to discover how the Hippo signaling cascade regulates Yki in the developing eye. Our preliminary data suggest that another critical factor in fly eye morphogenesis is the attachment of support tissue cells to the extracellular matrix (ECM), and recent work in vertebrate models has highlighted the importance of ECM in maintaining the Retina-RPE tissue boundary. The proposed research will explore the relationships among ECM, Yki and Mitf, in the genetically tractable fly model. In Aim 1 of this proposal, we will perform the first systematic investigation into how ECM factors contribute to ocular tissue morphogenesis, and we will investigate how ECM and PE-ECM connectivity are disrupted in RDis. In Aim 2, we will uncover the transcriptional targets of Yki and Mitf in the developing eye and test the hypothesis that they directly regulate ECM factor expression to maintain ocular epithelium topology. Our enhanced understanding of PE-ECM attachment and its molecular regulation will serve as a springboard for future detailed studies of the interplay among cell and epithelial properties that control eye organogenesis.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The homeodomain transcription factor Crx is expressed at the final mitosis of photoreceptor precursors, is involved in epigenetic remodeling, binds to promoters to activate photoreceptor- specific genes and silence other genes. In this proposal, we will investigate target search dynamics in live photoreceptors – how it transits the complex nuclear environment to find regulatory DNA target elements within the mass of non-specific DNA in chromatin. We will develop quantitative single-particle tracking super-resolution imaging (sptPALM) to characterize Crx dynamics in a new model. We have created a knock-in mouse line in which the coding region of a next-generation photoswitchable fluorescent protein, mEos4b, was inserted into the Crx gene. Since mEos4b is in the endogenous Crx locus, expression levels will be similar to native concentrations throughout development. We will answer the following three fundamental questions: 1) What mobility modes are employed by Crx in photoreceptor nuclei? 2) How long is Crx bound to chromatin? 3) Are Crx dynamics different in rods, cones, photoreceptor precursors, or bipolar cells? Our model will give us unprecedented access to nuclear dynamics of Crx throughout the lifespan of photoreceptors.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY Lipid nanoparticles (LNPs) are the most clinically advanced system for mRNA delivery, as highlighted by the FDA approvals of Spikevax® and Comirnaty®. However, there is no one-fit-all lipid design; lipids that perform well for one application may not work effectively across different cell types or medical conditions. This underscores the need to expand the lipid molecular toolbox to address evolving therapeutic challenges. By introducing sulfonium as a charge-carrying unit to replace traditional amines, we first demonstrated the safety and feasibility of using unconventional, non-amine-based sulfonium lipids for mRNA delivery. Building on these promising results, we propose to further develop sulfonium lipid nanoparticles (sLNPs) as a new platform for mRNA delivery. We will expand the chemical diversity of sulfonium lipids by incorporating novel building blocks, laying the groundwork for structure-activity relationship studies. Similar to amine-based lipids, variations in the head, linker, and tail structures of sulfonium lipids will likely affect self-assembly, cargo delivery, and biocompatibility. A detailed mechanistic investigation will uncover the unique physicochemical and biological properties of sLNPs. Additionally, we will explore the therapeutic potential of organ-targeting mRNA/sLNP formulations for treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in clinically relevant small animal models, providing robust preclinical evidence to pave the way for future translational research of sLNP technology.

NIH Research Projects · FY 2025 · 2025-08

Program Summary (Abstract) This proposal seeks to fund a new training program in Molecular and Translational Visual Sciences (MTVS) at Upstate Medical University (UMU). The goal of the program is to train young research scientists in understanding and investigating important questions related to the biology and pathobiology of the visual system. The foci for the MTVS will be: 1) molecular and cellular mechanisms of normal retinal and ocular tissues; 2) molecular basis of the pathology of the visual system; 3) strategies for retinal and ocular tissue repair and regeneration. Trainees will be 3 pre-doctoral single degree (PhD) students and 1 dual-degree (MD/PhD) student. The current enrollment in our CVR labs, is 20 predoctoral students; the funding request represents support for 20% of current enrollment. Trainees will be drawn from 3 doctoral programs in the College of Graduate Studies (CoGS): Neuroscience; Cell & Developmental Biology; and Biochemistry & Molecular Biology. The sixteen (16) faculty of the Center for Vision Research will serve as preceptors (mentors) for the program. Those faculty are funded by NEI, RPB, BrightFocus and the VA with approximately $6 million in annual, extramural support. Our inclusion criteria for mentors/preceptors will be faculty actively funded, or seeking funding, for vision-related projects. Members of the CVR faculty have key positions in the Neuroscience program (including chair of department and director of the program). However, to provide our trainees with the broadest breadth of expertise, we have invited key leaders from the programs in Cell & Developmental Biology and Biochemistry & Molecular Biology to serve as co-mentors (see Table I). Thus, our trainees will have unsurpassed mentorship in vision sciences and the underlying scientific disciplines. MTVS trainees will be cross-trained in the important and pressing issues in both basic and translational science. While our training program is based in a clinical department (Ophthalmology & Visual Sciences), the research faculty have an outstanding basic (discovery) science focus. Thus, the program integrates discovery science with fundamental questions of clinical relevance. The curriculum and training experience involves both didactic and problem- based training experiences. Basic (discovery) science faculty will ground trainees in the rigors of modern science, whereas the clinical faculty will root trainees in translational needs and goals. Preceptors with projects that are translationally relevant will bridge the gap between the discovery and clinical science. As the CVR is a proven training environment that combines the strengths of the component CoGS programs, it is recognized by predoctoral students as the most intellectually stimulating training site on the campus. The six (6) new students, joining the CVR from the current first year class of 31 candidates, is witness to that excitement. Thus, this program is assured to be successful.

NIH Research Projects · FY 2025 · 2025-07

Four serotypes of dengue virus (DENV1-4) cause 100 million symptomatic dengue cases annually. While the majority of DENV infections resolve without the need for medical intervention, dengue can quickly progress in some patients to potentially fatal dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The risk factors associated with progressing to severe dengue are complex and incompletely understood. However, the risk of developing severe disease increases significantly in individuals experiencing secondary/heterologous infections. The leading mechanistic explanation for this phenomenon is Antibody-Dependent Enhancement (ADE), wherein DENV-specific IgG antibodies elicited by a prior heterotypic DENV infection opsonize DENV without neutralizing infectivity, facilitating uptake by FcγR-bearing phagocytes such as monocytes and macrophages. However, analysis of samples from individuals experiencing acute DENV infection reveals that B cells are the largest reservoir of infected circulating cells, representing a disconnect in our understanding of immune-mediated DENV tropism. While B cells are known to be phagocytic, their antigen-specific receptors provide an additional means of internalizing extracellular antigens. Our team has recently demonstrated that the expression of a DENV-specific B cell receptor (BCR) renders cells highly susceptible to DENV infection. In addition, we have demonstrated that the frequency of DENV-infectable B cell is influenced by flavivirus immune status, with the frequency of DENV-infectible B cells increasing significant in previously flavivirus-naïve individuals after a primary DENV infection. We posit a new paradigm where cross-reactive non-neutralizing antibodies, when expressed in their transmembrane form as B cell receptors (BCR) on DENV-specific B cells are capable of mediating BCR-dependent enhancement (BDE) DENV infection, replication, and immunopathogenesis. Our long-term goal is to understand how B cells themselves alongside their antibody products can contribute to protection from or susceptibility to DENV infection and disease. Accordingly, our objective with this submission is to define the mechanistic requirements underpinning the process of BDE and to understand how risk of BDE is modulated by DENV infection and/or vaccination.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Dengue viruses (DENV) cause a significant and unchecked burden of human death and disease, with vaccine development hindered by critical gaps in our understanding of how multi-serotypic protection against DENV is generated, sustained, and subsequently identified in immunological assays. As the greatest risk for severe dengue illness occurs with secondary infection, DENV vaccines will need to generate protection against at least two serotypes simultaneously to maximize efficacy and safety. Our prior studies have demonstrated that durable, multi-typic immunity can be achieved naturally, through sequential exposures accumulated over time in hyperendemic areas for DENV transmission. Accordingly, our objective is to define the impacts of a child’s earliest flavivirus exposures in shaping DENV humoral immune phenotypes and clinical outcomes of subse- quent DENV exposures, generating important benchmarks for immune correlates of protection. To address this objective, we will leverage an ongoing long-term multigenerational family cohort study for DENV transmission in Kamphaeng Phet, Thailand. The cohort was established in 2015, leveraging NIH P01 and US DOD funds, and has enrolled over 3000 individuals within 500 families. 432 primary DENV infections have been identified among 814 DENV-naïve children to date, with more to be identified by the end of the study period in 2028 and marking 13 years of continuous surveillance. Incident infections are identified through quarterly sampling to detect seroconversions and through active surveillance for acute dengue illnesses. We will relate levels of maternally-transferred immunity, through placental transfer and breastfeeding, to risks of dengue illness with primary DENV infection in 750 mother-infant dyads (including 500 previously-enrolled and 250 newly-enrolled dyads) (Aim 1). Next, we will continue our long-term follow-up of DENV-naïve children and identify isotype- and antigen-specific DENV antibody phenotypes associated with protection from illness with post-primary DENV infection (Aim 2). Finally, we will relate non-DENV flavivirus exposures (Japanese enceph- alitis virus [JEV] vaccination, Zika virus infection, JEV infection) to risks of subsequent dengue illness, defining effects of time since exposure, pre-infection antibody phenotypes, and JEV vaccine type (Aim 3). These activities are consistent with NIAID’s mission to better understand, treat, and ultimately prevent infec- tious diseases. The application is innovative in using a custom multiplex panel for profiling DENV antibodies in saliva, permitting frequent longitudinal sampling, and in using advanced modeling techniques to reconstruct immune kinetics and identify subclinical infections. Successful completion of study aims will represent an im- portant advancement towards identifying immune correlates of durable, multi-serotypic protection against den- gue illness, providing critical benchmarks for diagnostics, triage, and DENV vaccines and immuno-therapies.

NIH Research Projects · FY 2025 · 2025-05

Abstract Drosophila Optix belongs to the evolutionarily conserved SIX family of homeobox transcription factors. Family members control cell fate, morphology, proliferation and/or survival in multiple tissues and organs of metazoans, including the eye. In Drosophila, Optix functions as a critical regulator of visual system development. Optix homozygous mutant tissue shows defect in retina neurogenesis and patterning of the ocelli. In addition, Optix is an essential regulator of neuroepithelial maintenance and patterning in the Drosophila brain, specifically in the optic lobe. The vertebrate homologs SIX3 and SIX6 are also required for the development of the mammalian eye and brain, and SIX3/6 mutations in humans are associated with severe eye and brain malformation. Optix, as other SIX family members, functions as a transcriptional regulator together with a variety of protein cofactors (CoFs) that modify Optix activity, resulting in changes in the activation or repression of target genes and reporters. Hence, to understand Optix protein function in specific developmental contexts, we need to identify the discrete Optix-CoF complexes at work, define their properties, and understand their specific roles. We propose here to characterize Optix-CoF complexes in multiple ways (Aim 1), and then to generate Optix protein variants that fail to engage in either one or few critical protein-protein interactions to demonstrate the context-dependent requirement for the affected complex(es) in cell culture (Aim 2). Lastly, we will develop tools for the dissection of the complexes’ biological roles in vivo (Aim 3). The research proposed here will lay the groundwork for detailed studies of SIX-CoF transcription complexes in vivo under physiological conditions and define their specific developmental roles by uncovering the precise biological processes they control through their transcriptional activity and TC-specific transcriptomes. This work aims to further our understanding of the intricate ways in which these transcription factors control fly eye development, and in which their dysfunction may lead to human disease and birth defects.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY/ABSTRACT Elevated intraocular pressure (IOP) is a major risk factor for optic nerve damage in glaucoma. Perplexingly, optic nerve sensitivity to IOP varies widely across individuals. The overarching goal of this proposal is to investigate fundamental mechanisms that drive optic nerve sensitivity to IOP. IOP elevation induces pathologic biomechanical strains on the optic nerve head (ONH). Our previous work identified astrocytes within the ONH as key sensors of IOP-related biomechanical strain. We recently engineered a 3D hydrogel system to precisely quantify the response of ONH astrocytes to pathologic biomechanical strains. Our preliminary data using this innovative system support that pathologic biomechanical strains induce similar ONH astrocyte dysfunction in vitro as IOP elevations do in vivo. They undergo process retraction and dysregulate oxidative phosphorylation and mitophagy pathways. We additionally identified the mechanosensitive channel Piezo1 as a key sensor of biomechanical strain. In the current proposal, we aim to use a combination of cutting-edge 3D bioengineering tools, transgenic animal models, and ex vivo donor ONH tissue, to test our central hypothesis: Transient biomechanical strains prime ONH astrocytes to develop greater glaucomatous morphologic and mitochondrial dysfunction via Piezo1 activation. We propose the following specific aims: Aim 1: Determine to what extent ONH astrocytes integrate transient biomechanical strains to predispose to future glaucomatous dysfunction. Aim 2: Determine if transient biomechanical strains predispose ONH astrocytes to lactate dysregulation and mitochondrial dysfunction. Aim 3: Determine if ONH astrocyte Piezo1 regulates ONH astrocyte sensitization and RGC viability in response to transient biomechanical pre-strains. The proposed experiments will advance our understanding of glaucomatous optic nerve damage by (i) determining if astrocytes within the ONH regulate optic nerve sensitivity to IOP, (ii) exploring a mechanistic link between biomechanical strain, metabolic insufficiency, and ONH susceptibility in glaucoma, and (iii) investigating if inhibiting mechanosensory pathways will prevent ONH astrocyte priming by biomechanical strain.

NIH Research Projects · FY 2026 · 2025-01

Condensates play a crucial role in spatially organizing and regulating biochemical reactions within cells. Dynamic condensates arise from phase separation, while insoluble aggregates result from protein misfolding. Although both can manifest as fluorescent foci in bacterial cells, they exhibit distinct biophysical properties and cellular functions. To overcome the challenge of probing the material state of these nanometer-sized foci in bacteria, I developed an experimental framework to assess phase separation during my postdoctoral training. This framework challenges the paradigm that inclusion bodies are exclusively solid aggregates and calls for a thorough re-examination of processes primarily studied using inclusion bodies, such as protein quality control (PQC). In the same study, I demonstrated that the material state of protein complexes influences their association with chaperones. While bacterial inclusion bodies are nucleoid-excluded and localize at the cell poles, functional condensates can be dynamically positioned on the nucleoid, as observed with carboxysomes – bacterial microcompartments responsible for CO2 fixation. The carboxysome positioning system represents the only known system for condensate positioning and the governing mechanism is still unclear. Therefore, this proposal seeks to determine how the material state of protein complexes influences mechanisms of PQC (Aim 1), and to develop minimal positioning systems for studying and manipulating condensates in bacteria (Aim 2). During the K99 phase, I gained expertise in single-molecule tracking and protein purification, advanced construct building for Aim 1, and engineered spatial control for condensates and other major bacterial organelles for Aim 2. In the R00 phase, I aim to elucidate differences in PQC principles governing bacterial condensates and aggregates and to determine the effects of spatial regulation on cargo yield and activity. These contributions are expected to lay a robust foundation for developing therapeutics targeting condensates and tools to spatially organize them. Building on the systems and methods developed in this proposal, my long-term goal is to determine how protein material states affect processes such as cell division and pathogenesis; to study the formation, functions, and spatial regulation of condensates and bacterial microcompartments from uncultivable human-associated bacteria; and to engineer bacterial organelles for metabolic and therapeutic purposes. With support from the R00 phase, my start-up package, and the collegial environment at SUNY Upstate, I am well-positioned to launch a productive independent research program and make significant contributions to the field of condensates and spatial regulation of organelles in bacteria.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Common fragile sites (CFSs) are recurrent “wounds” in every person's genome that predispose the chromosomes to DNA double strand breaks (DSBs) and rearrangements. Known features associated with CFSs include late replication timing, which is further enhanced upon replication stress, and large transcribed genes, which may cause replication-transcription conflict. CFS formation/breakage underlies a wide variety of human diseases, including cancer and neurological disorders. We recently mapped replication stress-induced DNA DSBs in a normal human lymphoblastoid cell line, using Break-seq, a powerful NextGen-sequencing based technique developed in my laboratory. DSBs, with or without replication stress, are associated with late replication timing. However, these DSBs were not enriched inside large transcribing genes, nor are they enriched inside cytologially defined CFS core sequences. We hypothesize that the differences between Break-seq signals and CFS core sequences are attributable to the inherent differences between technological platforms as each is biased toward a partial feature of the human CFS, with Break-seq detecting the DSBs whereas the cytological methods detecting ssDNA gaps. The main objective of our proposal is to directly test this hypothesis by creating an upgraded sequencing technology, Fragile Site (FS)-seq, to simultaneously map ssDNA gaps and DSBs (Aim 1). Moreover, preliminary evidence suggests that the ssDNA inside the CFS core sequences is a consequence of “rogue” DNA initiation events upon high levels of DNA replication stress. Therefore, we will test if alterations in replication timing gives rise to ssDNA at the CFS core regions (Aim 2). The proposed project will bridge the gap in our current understanding of the mechanisms of CFS formation and genome instability.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that impacts cognition and memory, imposing a substantial burden on the aging population. While individuals generally need to display core clinical and biological features to meet diagnostic criteria for AD, there is substantial variability among patients regarding onset and the course of the disease. This poses significant challenges to early diagnosis of AD and impedes the development of effective therapeutic strategies. Molecular subtyping of AD is of high importance due to its potential to identify disease mechanisms and new therapeutic targets. Recent studies have uncovered evidence indicating that molecular subgroups of AD display distinct disease characteristics aligning with key clinical features. The identification of distinct AD subtypes has spurred research regarding their potential to be identified using non-invasive neuroimaging techniques, which holds promise for early detection. However, progress towards this overarching goal has been impeded by studies lacking comprehensive data collected from the same individuals. The goal of this proposal is to enhance our understanding of the biological signatures that distinguish subgroups of patients with AD. We will use state-of-the-art computational approaches to jointly analyze multiple levels of data collected from the same individuals, encompassing a genetic, epigenetic, and neuroimaging information, constituting an unprecedented cohort in which to investigate AD subtypes. The proposed investigation will address the following gaps in the current scientific knowledge: 1) We will determine whether AD subtypes can be identified solely using neuroimaging-derived features. To address this question, we will use using state-of-the-art machine learning algorithms to predict molecular subtype of AD from magnetic resonance imaging (MRI)-derived phenotypes. 2) AD is a multifaceted disorder in which epigenetic regulatory mechanisms play a crucial role. However, prior studies on AD subtypes demonstrated limited or no integration of epigenetic information, thereby restricting knowledge of variability between AD subtypes. To address this critical knowledge gap, we will integrate epigenetic profiles previously associated with AD, encompassing expression levels of small non-coding RNAs (miRNAs) and DNA methylation (DNAm) markers, to determine their contribution to AD subtypes. This multi- omic approach has the potential to uncover new molecular signatures of distinct AD subgroups.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Most studies in the field of feeding regulation have primarily concentrated on the impact of energy homeostasis on feeding behavior, with less attention to the hormones and neural mechanisms governing nutrient-specific feeding. Leveraging the genetic advantages and well-characterized nervous and gut systems of the fruit fly, Drosophila melanogaster, we aim to explore the neuronal mechanisms that underlie nutrient-specific feeding regulation. Our preliminary findings highlight a specific group of Dh31R+/AstC+ neurons as target neurons for the gut-derived peptide Diuretic hormone 31 (Dh31) in regulating protein consumption. Additionally, we have identified another subset of gut cells that are responsive to sucrose intake and the essential role of its receptor in suppressing sucrose feeding. This proposal aims to investigate the molecular and neural mechanisms underlying the regulation of dietary protein and sucrose consumption, with a particular focus on how gut-derived peptide hormones, triggered by specific nutrient intake, activate neural targets in the brain to govern nutrient- specific feeding regulation. Furthermore, we propose identifying the central neural hub where various nutrient- specific feeding circuits converge to modulate food intake. The proposed experiments include characterizing the neural substrate of the gut hormone Dh31 responsible for suppressing protein feeding (Aim 1), elucidating the satiety signal for sucrose and understanding the neural mechanism governing sucrose feeding regulation (Aim 2), and identifying the central hub that integrates signals for both protein and sucrose to regulate overall food intake (Aim 3). The expected mechanistic insights from this research will illuminate the processes through which diverse macronutrient signals influence feeding behavior, highlighting the interplay between gut-derived signals and neural pathways.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY Glioblastoma (GBM) is an aggressive brain cancer type that responds poorly to standard treatment and ultimately develops chemo- and radioresistance. This is due, in large part, to the presence of a sizable and heterogeneous population of GBM stem-like cells (GSCs) that escape conventional therapies and replenish the tumor mass. This project addresses the dire need of attacking these resilient GSCs in order to improve GBM therapy. We will pursue a novel approach focused on targeting cell-scaffolding proteins that are essential for cellular functions. Because GSCs rely on these scaffolding proteins to maintain signaling mechanisms that are critical to radioresistance, we expect that our approach will create an inescapable vulnerability to the effects of radiation, achieving significantly increased tumor lethality. We will focus on a cell-scaffolding protein of the DLG (Disc Large Homologs) family, which are protein-carriers that are critical to keep signaling mechanisms active in their correct locations within the cell. We recently discovered that an unusual member of this family, DLG5, is highly upregulated in GBM and is necessary to maintain the GSC population in the tumor. Our published and preliminary work shows that DLG5 keeps tumor stemness and radioresistance mechanisms, such as Sonic Hedgehog (Shh) and Hippo, in a persistently active state. Accordingly, our central hypothesis is that DLG5 maintains redundant mechanisms that contribute to tumor stemness and the resistance of GSCs to radiation. We predict that targeting of DLG5 will create a non-recoverable vulnerability that can be combined with radiotherapy for improved attack of GBM. To validate this hypothesis, our first Aim is to characterize how the genetic targeting of DLG5 sensitizes GBM cells to radiotherapy. We will investigate the regulation of complementary Shh/Hippo signaling by DLG5 in GSCs and will determine if DLG5 deficiency causes a dominant negative effect that synergizes with radiation to kill these tumor cells. Phenotypic and mechanistic studies will be pursued in GSC cultures, followed by studies in tumor organoids and in vivo orthotopic GBM models. Our second Aim is to validate new agents to disrupt DLG5 functions and radioresistance in GBM. We will engineer cells with DLG5 deletion constructs to identify DLG5 domains that are critical to keep active Shh/Hippo signaling and to maintain the radioresistant features of GSCs. Next, we will focus on our described interaction of DLG5 with the ubiquitin-ligase cullin-3, which is "sequestered" by DLG5 in order to keep stemness pathways in a persistently active state. We will test novel cell-penetrating peptides designed to disrupt the interaction of these two proteins, with the expectation that releasing cullin-3 from DLG5 will result in dominant negative effects on radioresistance. Successful completion of this exploratory project will demonstrate the importance of cell- scaffolding proteins as high-level targets that can be disrupted to create an inescapable vulnerability in the tumor stem cell population, increasing the lethality of radiotherapy and other conventional treatments. Targeting scaffolding proteins can open new avenues to develop medicines with high therapeutic impact against GBM, improving the survival of patients with this aggressive cancer.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Sepsis is a complex, heterogenous disease that results in a dysregulated immune response. It is associated with hyperinflammation, known as “cytokine storm”, causing multiple organ failure and death, as well as simultaneous compensatory anti-inflammatory responses, leading to immune suppression by abundant anti- inflammatory cytokine secretion and immune cell death. Sepsis survivors may suffer chronic critical illness, known as Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PIICS), leading to serious complications and later death. In sepsis triggered by Gram negative bacterial infection, lipopolysaccharide (LPS), an endotoxin, promotes uncontrolled inflammation and cytokine storm through TLR4 activation. It can also trigger significant cell-lytic death of immune cells, known as inflammasome-mediated pyroptosis, which promotes further immune response for pathogen clearance. However, excessive pyroptosis during hyperinflammation results in prolonged immunosuppression and increased organ dysfunction, contributing to the development of PIICS. Currently, there are no available therapies to directly target both excessive cytokine production and immune cell pyroptosis in sepsis, a gap we will fill with our novel immune modulating itaconate- loaded telodendrimer nanoparticles (ITA:TD NP). In Luo lab, we have developed a series of bioactive immune modulating TD nanodrugs, which mimic the molecular pattern of LPS (multivalent charge and fatty acid tails). As a result, the optimized TD nanodrug attenuates LPS-induced inflammation via competitive binding with TLR4. Itaconate, a metabolite produced by activated macrophages, is known inhibit immune cell pyroptosis. Unfortunately, the in vivo application of ITA is limited by the unfavorable pharmacokinetic properties and cytotoxicity. Our LPS-antagonizing TD nanodrug can form a nanocarrier for efficient encapsulation of itaconate, thus to synergize the in vivo application and immune modulation. We hypothesize that concurrent inhibition of LPS signals, inflammasome activation, and membrane pore formation will effectively control both early phase hyperinflammation and pyroptosis-mediated later stage PIICS for improved sepsis treatment. Preliminary results indicate a survival benefit with ITA:TD NP treatment in septic mice induced by LPS and effective inhibition of both inflammation and pyroptosis. Thus, further studies to evaluate ITA:TD NP mechanism of action and efficacy to prevent both acute and late death in sepsis are needed. The aims of this study are: 1) investigate ITA:TD NP-mediated inhibition of hyperinflammation in sepsis, and 2) elucidate the protective effect of ITA:TD NP to prevent PIICS-associated morbidity and mortality. Results from these studies will provide insight to the mechanism of action of ITA:TD NP and its therapeutic potential for concurrent treatment of hyperinflammation and prevention of PIICS in sepsis to reduce mortality and fill this critical gap in patient care.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Loss and changes to central nervous system (CNS) myelin architecture are prevalent in neurological conditions across the lifespan. Incomplete restoration of myelin sheaths, as seen in multiple sclerosis, leads to disability. Our long-term goal is to identify developmental mechanisms controlling CNS myelin formation essential to CNS function, thus informing future therapies that remediate lost and aberrant myelin in neurological diseases. Myelin sheath architecture, particularly length, controls axonal signaling speed. The >10-fold variation in myelin sheath lengths observed in the CNS adjusts neuronal signaling speeds and, therefore, is thought to enable neural network coordination. Considering this pivotal role for myelin sheath length, a critical question arises: how can appropriate myelin sheath lengths be restored after disruption in neurological disorders to faithfully enable neural networks? The objective of this proposal, to determine mechanisms that establish CNS myelin sheath lengths, is paramount to addressing this question. Contrasting the long-held hypothesis that biochemical instruction from neurons is required for CNS myelination, our team made the unprecedented discovery that diameter of synthetic axons (a.k.a. microfibers) is sufficient to control oligodendrocyte myelin sheath length. These findings led to a new model for myelin formation and to our central hypothesis: axon diameter is a primary instructive cue that establishes CNS myelin length, triggering oligodendrocyte mechanotransduction (i.e., how physical cues are translated into molecular responses) mediating myelin growth. Based upon our preliminary data, we propose that oligodendrocytes respond to the physical cue of diameter (1) via a mechanosensitive ion channel, (2) triggering Ca2+ signaling, and (3) that diameter-triggered mechanotransduction coordinates translational machinery essential to promote myelin sheath growth. We experimentally test this model using complementary approaches: primary oligodendrocyte-microfiber cultures, ex vivo slice cultures, and traditional in vivo tissue analysis. We combine our innovative oligodendrocyte-microfiber cultures with live timelapse imaging and photomanipulation methods that allow us to both observe and manipulate signaling within individual myelin sheaths formed by single oligodendrocytes. This uniquely enables us to assess signals triggered solely by diameter during myelin formation with spatial precision—at the level of individual myelin sheaths–and experimentally interrogate current proposed models for how each myelin sheath independently controls sheath growth/elongation. Identifying mechanotransduction signals that establish myelin sheath length in this proposed project will lay the groundwork to directly test the impact of altered myelin lengths on CNS function that will inform strategies for myelin restoration in neurological disorders.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Excess body weight is a significant contributor to mortality rates in the United States, with class 2+ obesity increasing the risk of mortality by up to 91%. Physical activity plays a crucial role in managing prevalent diseases such as obesity, type 2 diabetes, and cardiovascular disease. Recently, exercise-stimulated myokines have been found to play a crucial role in inter-tissue signaling, promoting metabolic health and cardioprotection. However, the mechanism by which myokine expression is activated is poorly understood. Furthermore, therapeutically effective myokines are yet to be identified. Mitochondrial Precursor Overaccumulation Stress (mPOS) is a novel mediator of mitochondria-induced stress signaling, triggered by mitochondrial protein import stress and the accumulation of unimported mitochondrial proteins in the cytosol. In our preliminary study, we found the drastic activation of candidate myokines in a newly established mouse model of mPOS. This is accompanied with a strong lean phenotype and increased cardiac ejection fraction. These observations invited the hypothesis that mPOS may mediate myokine signaling, which ultimately promotes metabolic and cardiac health. In this predoctoral fellowship application, we will test the hypothesis that mitochondrial protein import stress can specifically induce myokine signaling by specific transcriptional factors in a bioenergetic-independent manner (Specific Aim 1). We will also test the hypothesis that mPOS-induced myokine release mediates fat loss and the improvement of cardiac health during aging (Specific Aim 2). Success of the proposed research represents a significant advancement in understanding mitochondria-induced myokine signaling and its potential implications for metabolic and cardiovascular health. Utilizing the murine and cell-based models, this study will uncover the mechanisms underlying myokine signaling and its impact on inter- tissue communication. These findings may lead to the discovery of novel health- promoting myokines and identification of therapeutic targets for metabolic and cardiovascular diseases, offering potential interventions to improve overall health, especially for aging populations and individuals unable to engage in physical exercise.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract Leukocyte migration out of the vasculature into peripheral tissue is crucial for their role in fighting pathogens, promoting tissue repair, and attacking solid tumors. This process is a key control point in the inflammatory response and relies heavily on signaling events downstream of leukocyte integrins and their endothelial ligands. Manipulating integrin signaling could serve as untapped avenue to control immune cell functions in inflammatory diseases and cancer, but hindering efforts on this front is a lack of mechanistic understanding of integrin signaling events. Importantly, integrins are known to be central players in mechanotransduction (converting mechanical information into a biochemical response), yet very little is known about how immune cells use integrins to sense and respond to the mechanical cues on the molecular level. This is surprising, since there is strong evidence that inflammation causes endothelial cells to change their mechanical properties, driving immune cell infiltration. Our preliminary data show that, indeed, T cells can respond to the mechanical properties of their substrate through the integrin LFA-1, kicking off cytoskeletal changes that drive T cell migration. Importantly, we found a signaling scaffold, named CasL, that is mechanically activated and required for T cell migration downstream of LFA-1. Surprisingly, T cells lacking CasL fail to form an actin-rich leading edge, but instead display numerous membrane blebs, suggesting CasL may control cytoskeletal responses and/or cortical integrity. Based on our preliminary data, we hypothesize that CasL is a crucial mechanosensitive signaling hub governing cytoskeletal responses during T cell migration. Specifically, we hypothesize CasL controls cytoskeletal organization by regulating the activity of one or more of the Rho family GTPases, thus coordinating proper cytoskeletal responses and optimum motility. In Aim 1, we will take a systematic approach to determine how CasL regulates cytoskeletal dynamics and cortex-membrane integrity, focusing on the Rho GTPases and their downstream effector proteins, and then test the hypothesis that CasL function is crucial to maintain normal cortex-membrane stiffness. In Aim 2, we will leverage our ability to introduce mutants into primary T cells lacking CasL to undertake a structure/function analysis of CasL in T cell migration. We will define how CasL phosphorylation, as well as how each individual domain, contributes to actin responses, migration, and membrane blebbing. Lastly, in Aim 3, we ask how loss of CasL affects T cell migration into inflamed tissue in vivo. Taken together, these studies will provide fundamental information on the mechanisms of T cell migration and mechanotransduction and lay the foundation for future work aimed and manipulating these pathways in inflammatory diseases and cancer.

NIH Research Projects · FY 2026 · 2024-05

Patients with neovascular disease, including Retinopathy of Prematurity and Familial Exudative Vitreoretinopathy (FEVR), develop insufficiently vascularized, or hypovascular, retinas. We have observed a similar hypovascular phenotype in the Tbx3 conditional knockout retina. Our long-term goal is to understand how retinal neurons and vascular cells form the neurovascular unit. The objective of this proposal is to determine the role Tbx3 plays in retinal angiogenesis as a way to uncover new molecular mechanisms driving retinal vascular disease. In our model, TBX3 is required during three events that are essential for retinal angiogenesis, when 1) retinal ganglion cells become metabolically active, causing retinal astrocytes to proliferate; 2) the embryonic hyaloid vasculature regresses in response to increases in dopamine levels; and 3) astrocytes migrate into the retina to form the astrocytic lattice. Our central hypothesis is that TBX3 regulates signals that affect retinal angiogenesis via its regulation in three separate cell types: first, in dorsal retinal ganglion cells, next, in dopaminergic amacrine cells, and finally, in astrocytes. The rationale underpinning this hypothesis is that key genetic networks controlling neurogenesis and angiogenesis are shared and thereby coordinate neurovascular coupling. We will test the central hypothesis by determining the TBX3-regulated molecular mechanisms that affect angiogenesis in 1) dorsal retinal ganglion cells via Sonic Hedgehog signaling, 2) dopaminergic amacrine cells via regulation of tyrosine hydroxylase, and 3) retinal astrocytes via control of migration. Each of these aims will be pursued using a combination of large-scale transcriptomic studies coupled with genetic manipulation and molecular analysis, which are standard technologies in our lab. Our studies are significant because they will fill a major gap in knowledge about vascular formation and identify new molecular pathways activated during angiogenesis. The expected outcome of this work is that it will add to our fundamental knowledge about retinal angiogenesis. Moreover, these studies will produce a database of dorsal-specific factors that will provide an understanding of the fundamental, cellularly distinct, differences in this region of the retina. In addition, this research fits in well with the NEI’s goal of ‘study[ing]…genetic factors that underlie structure, function, and the biology of retinal diseases.’ Our results will have a positive, immediate impact because they will provide a better understanding of retinal angiogenesis, and long-term impact because we expect our research will identify novel targets for better therapies of vascular disease.

NIH Research Projects · FY 2026 · 2024-05

Abstract The overwhelming immune response to systemic infection and traumatic injury may result in hyperinflammation, multiple organ failure, and death in sepsis. Sepsis is a complex and heterogeneous critical illness with multiple dynamic dysregulated inflammatory pathways triggered by systemically released Damage or Pathogen Associated Molecular Patterns (DAMPs/PAMPs). Dysregulated inflammation also contributes to the pathogenesis of many other diseases, e.g. autoimmune diseases, chronic wounds, etc. Clinical challenges: Versatile immune modulation therapy to resolve inflammation in critical illness is still a significant unmet need. Therapeutics targeting a specific inflammatory mediator, e.g. cytokines or DAMPs/PAMPs, have failed to improve sepsis survival in the clinic. On the contrary, anti-inflammatory steroid drugs are associated with immune suppression, resulting in an increased risk of secondary infections. Solutions: I have developed a novel, well-defined linear-dendritic telodendrimer (TD) nanoplatform for structure-based nanocarrier design in therapeutic delivery and immune modulation. In my on-going R01 project, I have developed TD nanotrap (NT) platforms for both systemic injection and hemoperfusion therapy to passively capture a broad spectrum of the overflowing inflammatory signals simultaneously, e.g. LPS, cytokines, and DAMPs/PAMPs, to restore immune regulation. The optimized TD NT resins provide a cure in a severe sepsis mouse model when combined with a moderate dose of antibiotics, as reported in our recent publications in Nature Communications 2020 and Advanced Therapeutics 2022. Recently, we created a series of novel TD constructs with a well-defined and optimal combination of negative charges and hydrophobic moieties that can actively block immune cell inflammation triggered by various immune stimulating molecules, e.g. endotoxins from gram negative bacteria (LPS) and gram positive bacteria (LTA), as well as bacterial cell lysate and even TNF-α. At the same time, these TDs can assemble into nanocarriers for the encapsulation of vital antibiotics for infection control; they can also effectively deliver endogenous potent pyroptosis inhibitors to control hyperinflammation and prevent immune suppression in the later stage of sepsis. In the next five years, I will focus on the development of transformative, next generation multimodal TD nanodrugs for sepsis treatment in three directions: PROJECT- 1: Develop a novel next-generation bioactive TD nanodrug for inflammation control; PROJECT-2: Develop multi-functional TD nanotherapeutics to inhibit pyroptosis in sepsis; PROJECT-3: Develop multimodal TD nanomedicine to control infection and inflammation in sepsis. In addition to sepsis, these TD nanodrugs can also be applied in other inflammatory diseases, e.g., ARDS, chronic wounds, cancer perioperative inflammation, rheumatoid arthritis, and inflammatory bowel diseases, etc. In summary, our innovative TD nanodrugs are poised to provide resolution to inflammation and improve sepsis survival in the clinic by both immune modulation and infection control.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT The trabecular meshwork (TM)/Schlemm’s canal (SC)-interface is critical for normal aqueous humor outflow function and intraocular pressure. Flow across the circumference of the outflow tract is non-uniform or segmental, with low-flow (LF) regions exhibiting higher extracellular matrix stiffness than high-flow (HF) regions. Dysfunction of the outflow tract causes decreased aqueous drainage and consequently increased intraocular pressure that poses a serious threat to normal vision. However, despite the strong association of outflow impairment with development of high-pressure glaucoma, the underlying mechanisms – including contributions from LF/HF regions – are incompletely understood. This largely stems from the inability of current outflow tissue models to precisely simulate the dynamic TM/SC-interface at high resolution necessary for in- depth mechanistic studies. To overcome critical limitations of previous outflow tissue replicas, this work seeks to generate a first-in-class TM/SC-interface-on-a-chip that accurately recapitulates the outflow tissue’s complex microenvironment and segmental elasticity profile. The novel 3D outflow tissue platform allows us to dissect the mechanisms of resistance generation by selectively manipulating individual outflow pathway components in ways otherwise not possible using other models. This will enable quantitative measurements of dynamic changes at the interface in real-time, while also being compatible with critical end-point tests. Based on key preliminary data acquired by our investigative team, we propose the following specific aims: Aim 1: Design and validate a microfluidic chip-based TM/SC-interface to investigate dynamic outflow regulation. Aim 2: Investigate segmental outflow regulation using localized ECM stiffness patterns containing region- specific TM cells.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Neuronal damage in glaucoma occurs following pathologic mechanical strains and fibrosis of the optic nerve head (ONH). Despite an abundance of evidence implicating mechanical strain and matrix fibrosis in glaucomatous damage, it is not known to what extent and how ONH cells signal to each other to produce matrix fibrosis in response to mechanical strains. This is due, in part, to a lack of adequate model systems to test mechanisms of matrix fibrosis following mechanical strain. In this exploratory R21, we propose to overcome limitations of existing models by establishing an in vitro 3D culture system that (a) accounts for native tissue stiffness, (b) permits dynamic matrix remodeling, (c) includes human ONH astrocytes and lamina cribrosa (LC) cells, and (d) allows for targeted manipulation of cell type-specific molecular pathways. We will then use this system to examine fibrotic signaling pathways between ONH astrocytes and LC cells in response to mechanical strain. Specifically, our preliminary data have identified the extra domain A isoform of fibronectin (FN+EDA) – Toll-Like Receptor 4 (TLR4) signaling axis as critical to matrix deposition by ONH cells. We will use our model system to test the role of the FN+EDA-TLR4 signaling axis in mechanical strain-induced matrix fibrosis. The experiments proposed in this R21 will advance our understanding of ONH mechanobiology by (i) investigating whether crosstalk between ONH astrocytes and LC cells is critical to mechanodysfunction, (ii) establishing quantitative models to test mechanisms of mechanical strain-induced matrix fibrosis, and (iii) investigating the potential of ONH matrix proteins to act as key signaling molecules to enhance ONH fibrosis. Our specific aims are: Aim 1, Generate quantitative models of the fibrotic response of human ONH astrocytes and LC cells after mechanical strain, in monocultures and co-cultures. Aim 2, Test whether astrocyte-derived FN+EDA stimulation of LC cell TLR4 promotes mechanical strain-induced matrix fibrosis.