University Of California-Irvine

NIH Research Projects · FY 2025 · 2024-01

Project Summary/Abstract Synthetic lethality screens hold great promise for cancer therapy since they enable the identification of chemical compounds that selectively target tumor cells while sparing normal tissue. This approach presumes that cancer cells harboring a specific mutation will be more sensitive to manipulation of certain pathways than normal cells that lack the mutation. The von Hippel-Lindau (VHL) tumor-suppressor gene is functionally lost in about 90% of Clear Cell Renal Cell Carcinomas (ccRCCs). Because VHL-deficient ccRCCs resist current therapies and frequently metastasize, identifying effective new therapies will be crucial for achieving long- lasting responses. This project’s long-term goal is to therapeutically target VHL-deficient ccRCCs using a synthetic lethality approach, and to move the most promising therapeutics to clinical trials. Here we propose to investigate cyclin-dependent kinase (CDK) inhibitors as synthetic lethality-based therapeutics for targeting ccRCC. We have validated a multi-CDK inhibitor—Dinaciclib—as a therapeutic candidate with potent in vivo activity in a patient-derived xenograft (PDX)-based orthotopic mouse model of ccRCC. Our preliminary data indicate that several specific CDK9 inhibitors (CDK9i)—AZD4573, NVP-2, and KB-0742—mimic Dinaciclib- induced death of VHL-deficient cells in vitro. CDK9 represents a promising therapeutic target since: (1) it is a non-canonical CDK regulating RNA polymerase II-mediated transcription; (2) multiple CDK9i have been developed, with several undergoing clinical trials in cancers other than ccRCC. Accordingly, our goal is to evaluate AZD4573 as a potential therapeutic for ccRCC. Our objectives are to dissect the mechanism of synthetic lethality in vitro, and assess AZD4573 efficacy in vivo, including its effects on anti-tumor immunity. We propose the following Specific Aims: 1) Dissect the mechanism of synthetic lethality of CDK9 inhibition with VHL loss in vitro, where we will test the hypothesis that there is a cross-talk between VHL and CDK9 pathways, affecting each other’s activity. We will also test the involvement of several candidate pathways downstream of CDK9 (MCL-1 and Bfl-1 pro-survival proteins) and VHL (HIF-1 and HIF-2) in synthetic lethality to determine the biomarkers of tumor response; and 2) Evaluate CDK inhibitor AZD4573 as a potential therapeutic in an orthotopic PDX-based and immunocompetent models of ccRCC, the latter conducted in combination with immune checkpoint inhibitor (ICI)—anti-PD1. ICIs represent a current front-line treatment for ccRCC, and literature indicates that CDK inhibition by Dinaciclib and a specific CDK9i induce anti-tumor immunity, potentiating their anti-tumor effects. Accordingly, our working hypothesis is that CDK9i will slow the tumor growth in immunodeficient model and cause tumor regression in immunocompetent model, especially when combined with ICI. Successful completion of the experimental plan will provide an efficient therapeutic strategy for VHL-deficient ccRCC—CDK9i + ICI combination treatment. Dissecting the mechanism of synthetic lethality will allow for patient stratification into potential responders and non-responders.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT The search for a cure or treatments of Alzheimer's disease (AD) has been largely unsuccessful, which has compelled several neuroscience fields to develop novel methods to understand and treat the condition. One such approach is the discovery of new unconventional animal models that naturally present AD-like traits. As a member of Dr. Xiangmin Xu's lab, I have been able to contribute to these efforts by conducting neuropathological characterizations of the Octodon degus (degu), a long-lived rodent native to Chile. This proposal focuses on the outbred degu that, in a subset of their adult population, presents cognitive deficits accompanied by spontaneous neuropathologies similar to those seen in human AD. I propose to create neuropathological, cytoarchitectural, and spatial transcriptome maps that will be integrated with behavioral datasets to comprehensively assess AD- like profiles in the outbred degu. I hypothesize that cognitively impaired degus will manifest neuropathology, altered cytoarchitecture, and maladaptive spatial transcriptomic maps reminiscent of human AD that will reveal novel therapeutic gene targets for the disease. The overall objective of this proposal is to create a comprehensive analysis of the outbred degu's AD- like features. This will shed light on the overlap between degu-AD and human-AD, which will lay the grounds for the degu's role in future AD research. I will divide degus into two groups based on their performance in an ethologically relevant burrowing behavior test: cognitively impaired (AD-like) and cognitively healthy (Non-AD) groups. The first aim of this proposal is to conduct a neuropathological and cytoarchitectural analysis (using immunofluorescence and western blot protein quantification) in degu prefrontal cortex (PFC) and dorsal hippocampus (dHIP). This will be complemented by the production of single-cell spatial transcriptomic maps in PFC & dHIP using MERFISH (multiplexed error-robust fluorescence in situ hybridization) in aim 2. This will provide a spatially resolved look at the maladaptive gene expression profiles of the cognitively impaired degu. The proposal culminates with the integration of the data from aim 1 and aim 2 with behavioral datasets (burrowing behavior, Y-maze, and open field tests). This will result in a multi-layered analysis of the degu from the transcriptomic to the behavioral level featuring spatially correlated data. I plan to analyze these integrated datasets to identify genes highly correlated with AD-like features in the degu that could yield therapeutic targets to treat the disease. Dr. Xu and I have fleshed out a training plan to progress the development of my technical, writing, mentorship, and leadership skills. I plan to perfect my skills conducting MERFISH experiments, analyzing -omic data, writing manuscripts/grants, mentoring, and presenting research. All these will contribute towards my long- term goal of becoming an independent scientist and an eventual professor at a research university.

NIH Research Projects · FY 2025 · 2023-11

ABSTRACT Forced displacement is on the rise, particularly in areas with high HIV prevalence. Disruption of social networks following forced displacement can amplify the risks of substance use disorders (SUDs) and enable HIV transmission. HIV transmission within and between forcibly displaced and local communities in situations of forced displacement can be facilitated by delayed HIV diagnosis and less viral suppression due to financial, logistical and administrative barriers in access to HIV care encountered by displaced people. Forcibly displaced men who have sex with men (fdMSM) can experience additional barriers due to stigmatization of their sexual orientation (MSM stigma), particularly in countries with high prevalence of MSM stigma such as Ukraine. Since Ukraine has one of the largest HIV epidemics in Europe and is experiencing a war that resulted in internal displacement of an estimated 5.9 million Ukrainians, fdMSM in Ukraine face unique challenges with respect to access to HIV care. We propose a study of 1,200 MSM (600 local and 600 fdMSM) in Ukraine: 800 MSM (400 local and 400 fdMSM) in Kyiv, the capital of Ukraine, and 400 (200 local and 200 fdMSM) in Lviv, a city in Western Ukraine; both cities are hosting large numbers of internally displaced people. Modified respondent-driven sampling will be used to recruit participants and collect social network and behavioral data. Blood samples will be collected from participants living with HIV for viral load testing, HIV genetic sequencing and subsequent phylogenetic analysis. All MSM living with HIV will be invited for follow-up interviews after 6- and 12-months postbaseline recruitment. Our overarching hypothesis is that fdMSM will have higher prevalence of SUDs, HIV, risk-related sexual behaviors, and be more likely to appear in recent HIV transmission clusters compared to local MSM, and that fdMSM in Lviv will have more stigmatizing social network ties that originated after displacement compared to fdMSM in Kyiv. We will address the following Specific Aims: 1) Determine the prevalence of HIV, SUDs, risk-related sexual behaviors, MSM stigma, and HIV testing among local and fdMSM in Kyiv and Lviv, Ukraine; 2) Characterize changes in social network composition since February 2022 and the impact of these changes on SUDs, MSM stigma, HIV risk behaviors, HIV testing, and viral suppression (among MSM living with HIV) in both local and fdMSM; 3) Estimate the impact of MSM stigma and social support disruption on HIV transmission dynamics between and within local MSM and fdMSM using phylodynamic analysis.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Enhancers are short DNA sequences that regulate complex patterns of gene expression during development. Misregulation of enhancer activity is associated with a wide range of pathologies, from congenital disorders to cancer. However, many properties of enhancers are not well understood, particularly how enhancers act over long genomic distances. While some enhancers are located proximally to their target genes, others are located distally, activating gene expression over thousands or even millions of base pairs of genomic distance. Most in vivo techniques that assess enhancer activity make use of a transgene system that places the enhancer directly upstream of a reporter. While transgenesis is a powerful tool, it cannot assess the role of genomic distance in enhancer activity. To address this, our group developed a novel in vivo mouse enhancer reporter assay to characterize the distal activity of enhancers. Using this method, we identified a novel cis-regulatory sequence, the RC element, that is necessary and sufficient for distal enhancer activity. The overall goal of this proposal is to characterize this newly identified RC element and dissect its role in mediating distal enhancer activity. To address this goal, this proposal outlines a plan to visualize the impact of the RC element on enhancer-promoter interaction (Aim 1) and to determine what factors are critical regulators of RC element function (Aim 2). For the former, both fluorescence in situ hybridization and chromosome capture based techniques will be employed to carefully analyze enhancer-promoter co-localization and overall nuclear organization in the presence and absence of the RC element. For the latter, mutagenesis of the RC element will be used to identify critical motifs. In parallel, knockout of candidate transcription factor regulators will be used to evaluate their importance in RC element-dependent enhancer activity. Findings resulting from this proposal will help elucidate a novel mechanism coordinating enhancer activity, adding to our overall understanding of gene regulation. My sponsor (Dr. Evgeny Kvon) and co-sponsor (Dr. Ken Cho) are experts in the field of developmental genetics, and, with their guidance, I have designed a training plan to help guide my transition to independent research. My plan focuses on developing my abilities in five key areas: technical skills, scientific communication, mentoring, and career development.

NIH Research Projects · FY 2026 · 2023-09

Individuals with sickle cell disease (SCD) often endure multiple acute pain crises throughout their lives. The cumulative impact of these crises and physical complications contribute to the emergence of chronic pain syndromes, mental health conditions, and impaired health-related quality of life (HRQoL) in early adolescence that persist through adulthood. In their 2020 pain management guidelines for SCD, the American Society of Hematology acknowledged the limited effectiveness of pharmacologic approaches and the potential benefits of nonpharmacologic integrative approaches such as music therapy (MT). These guidelines identified research priorities including determining which nonpharmacologic therapies are most acceptable and developing manualized, accessible, and developmentally appropriate interventions for chronic SCD pain. In our prior single-site feasibility study, 24 adults with SCD and chronic pain were randomized to receive either an in-person 6-session MT intervention (n = 12) or waitlist control (WLC) (n = 12). We developed procedures for screening, recruitment, retention, and electronic data collection within this randomized controlled trial (RCT). The enrollment rate was 89%, all study measures were completed, and MT participants demonstrated 100% attendance as well as improvements in self-efficacy and HRQoL compared to WLC participants. While results from this pilot RCT are promising, to scale an intervention in preparation for a future, multi-site, pragmatic, definitive RCT, further steps are needed. Furthermore, with MT being increasingly adapted for virtual delivery, including among adults with SCD, trials are needed to investigate the feasibility of hybrid (i.e., one in-person and five virtual sessions) approaches to MT delivery. In this R01, we propose to examine the feasibility of a multi-site study enrolling (1) in-person MT (n=30), (2) hybrid MT (n=30), and (3) hybrid Health Education (HealthEd) (n=30). Our proposal includes the MULTI-MUSIQOLS Data Coordinating Center at University of California Irvine and two recruiting sites: University Hospitals/Case Western Reserve University and Prisma Health/University of South Carolina. A total of 90 subjects will be randomized to either in-person MT, hybrid MT, or hybrid Health Education (HealthED) (1:1:1 allocation) with a focus on feasibility of data collection, participant recruitment, delivery of interventions, and home practice. Our Specific Aims are: AIM 1) conduct a feasibility RCT to examine the data collection processes and intervention (in-person MT, hybrid MT, and hybrid HealthED) implementation overall and across 2 sites; and AIM 2) evaluate the implementation of the in-person MT, hybrid MT, and hybrid HealthED interventions using both quantitative data (study records, stakeholder surveys) and qualitative data (interviews). Successful conduct of the proposed, feasibility RCT will provide the necessary framework for conducting a future, multi-site, pragmatic, RCT of MT compared with HealthED. Completion of the proposed R01 and the subsequent RCT (UG3/UH3) could provide critical evidence to support inclusion of MT as a chronic pain intervention for individuals with SCD and other similar populations.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Schizophrenia (SZ) is a severe mental health disorder currently treated with antipsychotic drugs which often have serious side effects, are ineffective in ~30% of patients, and are not useful as a preventive treatment. With its burden estimated at $343.2 billion in the US alone, there is a pressing need to improve early identification strategies for SZ and treatments that improve functioning. A better understanding of the underlying neurobiology is key to achieving these goals. Advances in global, large-scale, collaborative neuroimaging efforts are able to generate replicable findings on brain abnormalities in SZ and assess contributions of clinical and confounding variables. Our prior work within the ENIGMA (Enhancing Neuroimaging Genetics through Meta-Analysis) SZ working group identified predominantly gray matter deficiencies such as smaller subcortical volumes and thinner cortex in SZ. However, our findings also highlighted the importance of confounding variables on such clinical neuroimaging data, such as antipsychotic medication or disease chronicity. In contrast, the ENIGMA Schizotypy (SZT) working group, studying well-functioning healthy individuals who self-report subclinical psychotic traits, recently found that higher SZT was associated with thicker cortex. Moreover, the cortical thickness profile in SZT was inversely related with the profile of cortical thinning in SZ. Based on these findings, we posit that a comprehensive characterization of neural abnormalities at the extremes of the SZ spectrum continuum may not only further our understanding of the low liability (SZT) and high liability (SZ) ends of the spectrum but may also have wider implications for the prediction of risk and resilience in SZ (functioning) and in individuals at clinical high-risk for psychosis. This innovative global initiative will be first to integrate data across ENIGMA SZT and SZ cohorts with structural MRI (sMRI), detailed diffusion tensor imaging (DTI), and resting-state functional MRI (rsfMRI) to address the following key questions in SZ research: what are the functional and structural connectivity signatures of SZT and SZ? How are these signatures related to symptom severity, and global functioning? Can label-noise reduced dimensional measures of SZT and SZ liability based on these multimodal neuroimaging measures predict poor functioning? Leveraging global data and expert teams from minimally 24 to possibly well over 100 cohorts, we will tackle imaging, clinical, and predictive questions about the SZ spectrum with unprecedented power. This project will employ standardized image analyses, quality assurance, and statistical analysis procedures across cohorts with multimodal neuroimaging and clinical data from the same individuals. This will yield replicated FC and SC signatures of SZT and SZ, determine relationships between neuroimaging markers of SZT and SZ, and relationships with symptom dimensions and functioning. It will deliver machine- learning based models that can be used to generate imaging-based, dimensional, biomarkers that may serve as treatment targets, predictors of clinical outcome, or predictors of conversion to psychosis in at-risk populations.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Cancer-related cognitive impairment (CRCI), often referred as “chemobrain”, is prevalent up to 75% of all breast cancer survivors. These impairments cause significant distress and reduce the quality for life for survivors. Despite growing realization of the long-term clinical problem of CRCI in millions of cancer survivors, there is a conspicuous absence of clinical recourse available. Therefore, regenerative strategies to restore cognition and normal brain function in the cancer patients and survivors are clearly needed. Our past clinical and pre-clinical studies have established that doxorubicin, which is commonly used in breast cancer, can lead to a significant decline in the blood (human) and brain (rodent) levels of brain derived neurotrophic factor (BDNF). BDNF is abundantly expressed in the prefrontal cortex and hippocampus and plays important roles in neuronal repair and survival, dendritic and axonal growth, long-term potentiation, and neural stem cell maintenance. In our human studies, pathological reductions of BDNF were linked to higher risk of cognitive toxicity. Similarly, we have shown that chronic chemotherapy significantly impaired performance on the hippocampus and cortex-dependent cognitive tasks in the rodents. These deficits were linked with reduced neurogenesis, elevated neuroinflammation, and significant damage to the newly born and mature neuronal architecture, dendrites, spines, and synaptic integrity. To mitigate these deficits, our preliminary rodent studies involving mice receiving doxorubicin with riluzole, an orally active glutamate-modulating medication, has prevented the reduction of hippocampus BDNF levels. Thus, we hypothesize that: i) chemotherapy-induced reduction of BDNF leads to the long-term neurodegenerative consequences culminating into cognitive impairments and, ii) augmentation of BDNF in vivo will restore cognitive function in brains exposed to chemotherapy and will provide neuroprotection against CRCI. We will test our hypothesis with three specific aims. In Aim 1, we will systematically examine brain and plasma BDNF levels to link its trajectory with CRCI and neurobiological underpinnings in a mouse model of breast cancer chemotherapy. In Aim 2, we will determine the neuroprotective impact of enhancing BDNF in vivo to reverse CRCI. In Aim 3, we will evaluate the neuroprotective effect of BDNF-enhancing riluzole to ameliorate CRCI. This study will link the neurobiological underpinnings of chemotherapy and neuroprotective effects of BDNF against CRCI. If demonstrated to be successful, our translationally feasible pharmacological approach will provide basis for future studies to repurpose riluzole as a therapeutic option for mitigating CRCI.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract Failure to form and store long-term memories is a feature of cognitive decline in aging and neurodegeneration. Experts predict that the prevalence of cognitive impairment, ranging from mild to severe dementia, will increase alongside the rapidly growing U.S. population of older adults aged 65 and older, creating new challenges to provide resources and care for older adults. There is a need to understand the epigenetic and molecular mechanisms of memory formation in the aging brain to develop early intervention strategies and preserve cognitive function in old age. As observed in our lab and others, histone deacetylase 3 (HDAC3) is a powerful epigenetic regulator of memory formation and synaptic plasticity. However, mechanisms regulating HDAC3 in the aging brain with regards to memory remain undefined. Emerging data suggesting that HDAC3 may be regulated in cancer cells by upstream kinases and phosphatases led me to hypothesize that the phosphorylation state of HDAC3 determines the ability of HDAC3 to regulate memory formation and synaptic plasticity. Furthermore, that the mechanism of HDAC3 phosphorylation becomes dysregulated during memory consolidation in the aging brain, contributing to age-related memory impairments. Preliminary data in this proposal reveals that baseline levels of phospho-HDAC3 are reduced in the hippocampus of aging mice (18-mo) compared to young adult mice (3-mo). Additionally, I developed HDAC3 mutant viral constructs to test the function of phosphorylated HDAC3 (phospho-mimic) and de-phosphorylated HDAC3 (phospho-null) in memory formation and synaptic plasticity in the young adult and aging brain. Preliminary results demonstrate that viral expression of the HDAC3 phospho-mimic impairs memory formation and synaptic plasticity in young adult mice. However, expression of the HDAC3 phospho-null in aging mice ameliorated age-related impairments in memory formation and synaptic plasticity. Together, these findings suggest that HDAC3 phosphorylation is a mechanism that can dynamically regulate long-term memory and synaptic function. Therefore, this proposal will focus on continuing to investigate the epigenetic regulation of HDAC3 phosphorylation on memory formation in the adult and aging male and female brain. Specific aims within this proposal will determine the following: Aim 1, determine the role of HDAC3 phosphorylation in the young adult brain; Aim 2, determine the role of HDAC3 phosphorylation in the aging brain; Aim 3, determine the mechanism by which HDAC3 phosphorylation regulates memory formation in the young adult and aging brain. Findings from this project will potentially elucidate a novel mechanism of HDAC3 epigenetic regulation in memory that can have a fundamental impact for all aging individuals with cognitive impairments. This training fellowship will allow for development of molecular, physiology and bioinformatics expertise. With the guidance of Dr. Wood and the research and professional environment at UCI, this fellowship will provide a foundation for successful career as an independent investigator focused on understanding the epigenetic mechanisms underlying learning and memory in the aging brain.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The promise of human stem cell-derived cardiomyocytes (hSC-CMs) opens doors towards the feasibility of personalized medicine against cardiac diseases and for performing more accurate drug discovery studies. Moreover, hSC-CMs overcome the issue of species differences when using animal models for high throughput screening studies. However, one of the bottlenecks for scaling up the use of hSC-CMs is their ability to accurately reflect the native structure and function of adult human cardiomyocytes. Current efforts to address this critical challenge involve maturation protocols that use biophysical cues such as electrical stimulation or substrate- induced tissue alignment. Methods for electrical stimulation often utilize electrode contacts for field stimulation, bulky instrumentation for combining electrical stimuli delivery with mechanical or sustained chemical stimulation, or genetically modifying cells to be light-responsive. Although we have seen successes through these induction and stimulation approaches, the field would benefit from a stimulation approach with minimal culture contact to reduce risk of infection during long-term cultures, as well as a light-based approach with higher spatiotemporal resolution than electrode-based stimulation. Here, we propose a new paradigm for stimulating hSC-CMs towards maturation by interfacing these cells with peptide-based substrates that can induce tissue anisotropy and are engineered to convert light to stimulatory cues. Our team will develop peptides functionalized with chromophore units and cell-binding epitopes as materials that can be used for light-based stimulation of hSC- CMs, in combination with induction of tissue alignment, towards maturation. The long-term goal of this project is to establish light stimulation via engineered peptides as a viable method to stimulate cardiomyocytes and promote hSC-CM maturation in an electrodeless and non-genetic manner. We hypothesize that transient charging and other associated light-induced processes at the cardiomyocyte-biomaterial interface can influence extracellular potential, resulting in the photostimulation of hSC-CMs towards maturation. Our rationale for proposing a materials-based approach for stimulating hSC-CMs stems from previous reports of conjugated polymers being used as a photoactive substrate for triggering action potentials of other excitable cells. To test our hypothesis, we propose the following specific aims: (1) establishing design parameters for engineered peptide substrates with optimal photostimulation efficiency; (2) test the cellular- and tissue-level impact of peptide-mediated photostimulation in combination with anisotropic cues; and (3) elucidate the effect of the proposed photostimulation method, along with anisotropy cues, on hSC-CM maturation. By establishing the design rules for the proposed photoexcitable peptides for eliciting combinatorial cues to stimulate hSC-CMs and ensure their capability to excite cardiac cells, this innovative approach offers a new strategy for a “wireless” stimulation of cardiac tissues towards maturation, and can therefore significantly contribute towards addressing the grand challenge of immaturity of stem cell-derived cardiomyocytes.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/ Abstract Inflammatory skin diseases are often multifactorial, with influences from genetics and environmental factors that cause immune dysregulation and impair skin barrier integrity. The epidermis is the outermost layer of the skin and is fundamental to maintaining skin barrier integrity. Also, epidermal skin cells play a primary role in the recruitment and regulation of immune cells upon disruption of homeostasis. The mechanisms that govern the crosstalk between the epidermis and the immune system have yet to be fully elucidated. Current strategies in treating inflammatory skin disease include the administration of corticosteroids and retinoids, which may have unwanted side effects with prolonged use. Therefore, there is a need for a better mechanistic understanding of the crosstalk between the epidermis and the immune system to develop more targeted approaches to treat inflammatory skin disease. Recent reports have implicated SNPs in genes that may play a role in inflammatory skin diseases. One such gene is Ovol1. Ovol1 is a transcriptional repressor of immune cell recruitment and is necessary for maintaining skin homeostasis and regulating proper embryonic epidermis development. Ablation of Ovol1 in psoriasis-like and atopic dermatitis animal models exacerbates disease with an increase of infiltrating neutrophils. Ablation of neutrophils improves disease outcomes. Neutrophils are the principal innate immune responders forming the first line of defense against pathogenic invaders or sterile damages. Neutrophils have an arsenal of effector functions, including the engulfment of pathogens, release of proteases, extracellular trap formation, and release of reactive oxygen species. Appropriately, neutrophils have a destructive potential that requires stringent regulation of recruitment to avoid excess collateral damage to the host. Our preliminary data show that in the absence of epidermal Ovol1, the recruitment of neutrophils to the injured skin increases in minutes. This observation suggests that Ovol1 has a gatekeeping function and prevents the excessive production of neutrophil chemoattractants after skin injury. My central hypothesis is that Ovol1 regulates the production of leukotrienes as well as the expression of chemokines before or minutes after wounding. The absence of epidermal Ovol1 primes the skin for neutrophil infiltration, in turn exacerbating inflammatory skin disease. In this proposal, I will address two aims to support my hypothesis: 1) Evaluate the role of leukotrienes in the excessive recruitment of neutrophils in the absence of epidermal Ovol1, 2) Identify and block chemokines expressed early after skin injury when Ovol1 is absent. The proposed research will untangle the early crosstalk between the epidermis and the innate immune system, potentially revealing better pharmacological targets to alleviate inflammatory skin disease.

NIH Research Projects · FY 2026 · 2023-09

The ability to seamlessly move between global map perspectives (GMPs), such as looking at a map or taking in a scene from a high vantage point, and first-person perspectives (FPPs), such as being on the ground immersed in the scene, has obvious importance for navigation and, more broadly, flexible perspective-taking. Using a multidisciplinary approach, we will investigate the neural architectures, environmental features, and spatial representations that support these transformations. We will develop neural network models that make predictions to be tested in experiments with the rodent and human, and use that experimental data to inform the model.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Amyotrophic Lateral Sclerosis (ALS) is the most common form of motor neuron disease (MND). Several reports have highlighted a link between some disease-causing genes and the immune system. However, whether and how immune cells contribute to MND in specific forms of ALS is yet to be defined. Amyotrophic Lateral Sclerosis 4 (ALS4) is a rare and juvenile MND caused by mutations in the gene SENATAXIN (SETX), which encodes for a nuclear helicase ubiquitously expressed that our group identified as a transcriptional regulator of inflammatory response. By using a mouse model of ALS4, we found that the hematopoietic system contributes to the neurodegenerative process. Additionally, we discovered that CD8 T cells in the CNS and the peripheral blood of disease-affected mice and patients are aberrantly activated. Our goal is to define the role of CD8 T cells in disease initiation and progression. We will determine the function of disease-associated CD8 T cells by transfer and depletion experiments and immune-histological characterization in mice (Aim 1). We will assess molecular pathways of neuro-immune interactions during disease by network modeling of transcriptomic and correlation-structure analyses (Aim 2). Finally, we will validate the presence of activated CD8 T cells in mouse models and patients with other forms of ALS (Aim 3). Our contribution of results will provide a deeper knowledge of the of CD8 T cells in ALS and open potential avenues for their use as immune biomarkers disease progression and as novel therapeutic target.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract The long-term objective of this study is to develop gene therapies that treat Duchenne muscular dystrophy (DMD) cardiomyopathy. DMD cardiomyopathy, characterized by ventricular chamber enlargement and thinning of the ventricular wall, ultimately leads to heart failure. Pathogenic features of DMD cardiomyocytes include contractile dysfunction, poor calcium handling, elevated reactive oxygen species, telomere shortening, and premature cell death. When a large number of cells die in the heart, scar tissue forms, increasing the stiffness of the heart. Although there are treatments available to alleviate symptoms of dilated cardiomyopathy, there are currently no therapies to prevent or delay the onset of this disease. Smaller versions of dystrophin amenable to gene therapy have shown promise to treat DMD-associated severe skeletal muscle wasting; however, surprisingly little is known about their effects in treating heart failure. This research plan will leverage bioengineered hydrogels of tunable stiffness, human induced pluripotent stem cells (iPSCs) with dystrophin mutations, and biochemical techniques to determine if full-length dystrophin can rescue DMD cardiomyocytes from their pathogenic demise. During the K01 award period, Dr. Asuka Eguchi will train under the mentorship of Dr. Helen Blau, an expert on DMD. By engineering hydrogels that mimic stiff, diseased heart tissue, Dr. Eguchi will be able to measure parameters of contraction in cardiomyocytes differentiated from DMD iPSCs. Aim 1 will test if full-length dystrophin can rescue DMD cardiomyocytes from contractile deficits, aberrant calcium handling, and premature cell death. Aim 2 will determine if split vector or lipid nanoparticle approaches can deliver full-length dystrophin to cardiomyocytes. Aim 3 will test whether this gene therapy strategy to deliver full-length dystrophin can delay the onset of DMD cardiomyopathy in a mouse model. Gene therapy approaches targeting the root cause of disease, the lack of dystrophin, is critical for extending lifespan and improving the quality of life of DMD patients. The career development plan is designed to enable Dr. Eguchi to successfully transition to a career as independent investigator. Her scientific advisory committee consist of Dr. Beth Pruitt, a bioengineer with expertise in traction force microscopy, Dr. Joseph Wu, an expert on cardiovascular disease modeling, and Dr. Daniel Bernstein, a pediatric cardiologist. Collectively, these collaborators will help Dr. Eguchi develop skills at the interface of bioengineering, cell biology, and biochemistry to launch an independent research program in cardiovascular research.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY / ABSTRACT Vaccines have been very effective at protecting against infectious diseases that pose serious threats to human health. However, prophylactic vaccines can also be limited, particularly if antigenic drift occurs to create variants of the pathogen; this can result in vaccines losing potency over time, needing boosters to confer protection, and lower neutralization efficacy on emerging viral variants – consequences which are observed in the current COVID-19 pandemic. Recent studies have shown that the release kinetics of vaccines can be important in establishing lasting and efficacious immunity. In particular, extending the exposure to antigens can result in higher antibody titers and increased diversity of neutralizing antibodies that target a more diverse set of epitopes, relative to immune responses from conventional bolus vaccination. Furthermore, vaccines made from protein-based nanoparticles can elicit increased antibody production, broader antigen cross-reactivity, and a more balanced Th1/Th2 response. This study tests the hypothesis that the synergy in combining the effects of nanoparticle vaccines for effective antigen presentation, together with a slower release to give a longer exposure to the vaccine, will elicit increased durability of the immune response and a broader cross-reactivity for emerging viral variants. To test this hypothesis, we propose to encapsulate protein nanoparticle vaccines with a biodegradable PLGA-PEG-PLGA (PPP)-based polymer to modulate the kinetics of its release from an in vivo vaccine depot. This extended-release vaccine strategy will then be applied towards SARS-CoV-2. We will evaluate the durability of the proposed vaccine strategy’s potency and the breadth of cross-reactive immune responses toward the variants of SARS-CoV-2 and other types of coronaviruses. Our specific aims are to: (1) create controlled-release nanoparticle depot vaccines against SARS-CoV-2, and (2) determine the efficacy and immunological responses to these vaccine nanoparticles that are encapsulated by the polymeric depot. Because the design of these vaccines is modular and different antigens can be exchanged in a relatively straightforward approach, the successful implementation of this proposed strategy for coronavirus antigens could have broader applicability towards the development of vaccines for other infectious pathogens.

NIH Research Projects · FY 2026 · 2023-09

Project Summary To date, no therapy exists to restore vision to the over 64 million people worldwide who are legally blind from diseases that damage the optic nerve. Although current approaches for regenerating the optic nerve have successfully directed long distance axon regeneration, these strategies are still limited by the fact that 1) efficacy has generally been demonstrated when therapies were initiated before axon injury, which has limited clinical application; 2) they may carry a risk of neoplastic conversion and thus may not be readily deployed prophylactically; and 3) incidences of off target axon regeneration have been reported, indicating a need not just for signals that promote but also ones that direct axon growth to intended targets. Our multi-disciplinary collaboration between electrical engineers, neuroscientists, electrophysiologists, and neurosurgeons has enabled the collection of compelling preliminary data demonstrating that exogenously applied electric fields (EFs) control the direction of retinal ganglion cell (RGC) axon growth, in-vitro. In vivo, stimulation with asymmetric waveforms was found to be effective at directing full-length optic nerve regeneration, without evidence of aberrant targeting, and restoring partial visual function (local field potential recordings in the superior colliculus and pattern electroretinogram) after crush injury. Interestingly, stimulation with symmetric waveforms was more effective at promoting RGC survival than asymmetric waveforms. The discovery that different waveforms (asymmetric versus symmetric) may activate distinct signaling pathways that control different cellular behaviors provides a unique opportunity to assess for synergistic effects from combined stimulation. Here, we propose to compare the efficacy of combined symmetric and asymmetric EF stimulation on restoring visual function over either treatment alone. Additionally, although EF stimulation with asymmetric waveforms was successful at restoring partial visual function, local field potential amplitudes within the superior colliculus were lower and latencies longer than in normal controls. Whether this dysfunction stems from ineffective RGC synaptic transmission (spatial summation) or absence of myelin (temporal summation) or both is unknown. Here, we propose to employ immunohistochemical, transsynaptic viral labeling techniques, and electrophysiology experiments to interrogate the source of this signaling deficiency. If successful, experiments proposed here have the potential to advance EF application into a strategy that, when applied synergistically with other approaches for RGC axon regeneration, can help regenerate the optic nerve and restore visual function of patients blinded by optic neuropathies.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Support is requested for four positions in a new training grant to continue and expand our successful Immunology Training Program at the University of California, Irvine (UCI). We have a total of 36 mentors that include junior, mid-level and senior training faculty comprising 14 women and 3 URM faculty. UCI immunology researchers are working on: a) host defense and vaccine development; b) tumor immunology and immunotherapy; c) Neurogenerative disease and microglia biology; d) chronic diseases and autoimmunity; and d) synthetic immunology. The pool of immunology graduate students comes primarily from the Cellular and Molecular Bology (CMB) intake program. In 2022, 101 students were accepted to CMB, including 39 URM (38.6%), and their mean GPA was 3.86 (which was similar to 2020 and 2021). There is substantial institutional support from the Office of Research and from the Graduate Division at UCI, which will provide full stipend and tuition for a 5th student. Our prior T32 from 2016-2021 supported 15 students, most of whom have graduated and are in research positions in industry or academia. Dr. Eric Pearlman is the Director of the UCI Institute for Immunology and was the Prinicipal Investigator of the previous T32 training grant. In the current submission, Dr. Pearlman will be PI/Director, and a new faculty recruit, Dr. Ivan Marazzi, will be co-Director. Dr. Pearlman has extensive experience in training graduate students and running T32 grants, and Dr. Marazzi has a very exciting and well funded research program in autoimmunity chronic neurodegenerative diseases and in molecular virology, including influenza and COVID-19. Given the outsanding training record of faculty and the quality of training grant elibile and URM students in the program, we fully anticipate that the UCI Immunology training program will continue to provide outstanding educational and career opportunities for the next generation of immunology researchers.

NIH Research Projects · FY 2025 · 2023-09

The discovery of safe and effective analgesics is an urgent societal need and a centerpiece of NIH’s HEAL initiative. Here, we test the hypothesis that the enzyme N-Acylethanolamine Acid Amidase (NAAA) offers a novel target for analgesic medications devoid of abuse potential. NAAA hydrolyzes palmitoylethanolamide (PEA), a lipid messenger that suppresses nociception by engaging the nuclear receptor PPAR-α. The substantial antinociceptive properties of NAAA inhibitors have been recently recognized by a meta-analysis of the preclinical literature conducted by the International Association for the Study of Pain (IASP), but the cellular substrates underlying such properties are still unknown. We found that Naaa-knock out (ko) mice (i) have markedly reduced nocifensive responses to formalin, compared to wild-type littermates; and (ii) fail to develop persistent sensory abnormalities in the chronic constriction injury (CCI) model. Conversely, Naaa- overexpressing and Ppara-ko mice exhibit robust nocifensive behavior even when given a subthreshold formalin dose. The reduced sensitivity of Naaa-ko mice to pain does not result from developmental compensation, because it can be phenocopied by administration of the NAAA inhibitor ARN19702. Importantly, ARN19702 does not exert positive motivational effects in the mouse conditioned place preference (CPP) test, which is suggestive of a lack of rewarding properties. These data point to NAAA as a promising target for the discovery of non-addictive analgesics. We will test this hypothesis in two specific aims: Aim 1. Validate NAAA as a molecular target for analgesic drug discovery. Three questions will be addressed: (1) What cell types are directly targeted by NAAA inhibitors? We will generate cell-specific NAAA-ko mouse lines and evaluate, in the formalin and CCI models, pain-related behaviors and sensitivity to NAAA blockade. In parallel, we will determine the impact of pathological pain on NAAA-regulated signaling in dorsal root ganglia (DRG) and spinal cord (SC) of wild-type mice. (2) What cell types mediate NAAA-regulated antinociceptive signaling? We will identify PPAR-α-expressing cells involved in NAAA-dependent signaling by creating cell-specific PPAR-α-ko mouse lines and evaluating their pain-related responses and their sensitivity to NAAA inhibitors in the formalin and CCI tests. (3) Is the pharmacodynamic profile of NAAA inhibitors compatible with safe and effective use in pain therapy? We will determine whether ARN19702 (i) alleviates spontaneous nociception, (ii) produces tolerance after repeated administration, and (iii) exhibits rewarding and/or addicting potential. Aim 2. Develop improved NAAA inhibitors and examine their efficacy as analgesic agents. Better NAAA inhibitors are needed to assess NAAA’s possible role in pain therapy. We will combine computational structure-based drug and structure-activity relationship studies to discover novel agents with improved pharmacodynamic and pharmacokinetic properties, robust analgesic activity, and no addictive potential.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive impairment. AD leads to a significant reduction in quality of life, and with a rapidly growing prevalence, there is a dire need for improved therapies. Peroxisome Proliferator-Activated Receptor delta (PPARd) is a ligand activated transcription factor that has emerged as a potential target for neuroprotection. PPARd agonism has been shown to improve disease phenotypes in neurodegenerative disease model mice, and disruption of PPARd function in the central nervous system (CNS) of normal mice has been shown to elicit neurodegeneration. PPARd agonism is also currently in a Phase 2a clinical trial for treatment of mild-to-moderate AD. Of all the brain cells, microglia express PPARd most highly; however, the mechanistic basis of PPARd activity in microglia remains undefined. Microglia are the resident immune cells of the CNS and have been repeatedly implicated in the pathogenesis of AD, so understanding the role of PPARd in microglia will provide insight into its therapeutic value. Preliminary data reveals that PPARd can suppress inflammation in the brains of Huntington’s disease, Parkinson’s disease and tauopathy model mice. Additionally, preliminary RNA-sequencing data on isolated microglia from wild-type mice treated with the PPARd agonist KD3010 shows that PPARd agonism leads to downregulation of inflammatory genes (e.g. Il-1b and SPP1) as well as the downregulation of several AD relevant genes in microglia (e.g. C1QA/C1QB, IL12b and TYROBP). Microglia have been shown to take on aberrant phenotypes in disease settings. These altered phenotypes have been shown to be cell-autonomous in human induced pluripotent stem cell (iPSC)-derived microglia like cells (iMGLs) that harbor mutations relevant to AD. For example, APOE4 microglia have been shown to exhibit reduced uptake of beta-amyloid, have shortened processes, and have increased pro-inflammatory cytokine secretion. iMGLs are powerful models for human microglia, as they are transcriptionally and phenotypically similar. This proposal will uncover the mechanistic basis of PPARd function in microglia and interrogate whether PPARd agonism can attenuate the aberrant phenotypes seen in microglia in the context of AD. I will first assess whether PPARd transactivation of neuroprotective targets is dependent upon phase separation with the transcriptional coactivator Mediator 1 and whether PU.1, which is a genetic risk factor for AD, interferes with PPARd neuroprotection. I will then derive iMGLs from iPSCs that harbor an AD risk allele (APOE4) to assess whether PPARd can attenuate the abnormal phenotypes, focusing on cytokine secretion and phagocytic capacity. Understanding the role that PPARd plays in microglia and how its activation affects microglia in an AD- relevant setting has the potential to provide further support as to why PPARd agonism should continue to be pursued as a therapeutic for AD.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Obesity and the associated insulin resistance are the established risk factors for type 2 diabetes (T2D). Although more than 90% of T2D patients are overweight or obese, only about 30% of obese people develop T2D. One major determining factor for the development of T2D in obese patients is islet β−cell decomposition or failure, resulting in relative insulin deficiency. It is well documented that islet failure has a strong genetic predisposition. Genome-wide association study (GWAS) provides a powerful tool to associate genetic variants (SNPs) with T2D. However, the vast majority of the diabetes risk SNPs from GWAS are found in the non-coding regions, posing significant challenges to identifying the SNP-associated genes for T2D. 3’untranslated regions (3’UTR) are non- coding sequences containing cis-regulatory elements (CRE), such as binding sites for miRNAs and RNA-binding proteins (RBPs) that regulate mRNA fate and protein expression. Alternative polyadenylation (APA) at the 3’UTR is an RNA-processing mechanism that generates mRNA isoforms with significantly different 3’UTR lengths with distinct CREs. SNPs that regulate APA may lengthen or shorten 3’UTR, thereby altering gene expression and function. We recently developed a novel 3’UTR APA quantitative trait loci (3’aQTLs) analysis tool to colocalize disease-associated SNPs with APA. Using the 3’aQTLs tool, we found that the lengthening of 3’UTR of zinc finger CCCH domain-containing protein 13 (ZC3H13) in the pancreas was highly associated with T2D. ZC3H13 is a key epitranscriptomic factor that forms an N6-methyladenosine (m6A) RNA modification writer complex with methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). Both METTL3 and METTL14- mediated m6A have been shown to be essential for β-cell function. Our preliminary data show that longer ZC3H13 3’UTR reduced ZC3H13 protein expression without changes to mRNA levels. Knocking down ZC3H13 suppressed insulin production in the cultured β-cells. Furthermore, ZC3H13 heterozygous knockout mice exhibited impaired glucose tolerance with reduced insulin levels when challenged with a high-fat diet. We, therefore, hypothesize that the reduced ZC3H13 protein expression from 3’aQTLs-associated 3’UTR lengthening contributes to the genetic predisposition of islet failure in T2D. We propose three aims to study what SNPs cause ZC3H13 3’UTR lengthening, why ZC3H13 3’UTR lengthening reduces protein expression, and how the reduced ZC3H13 protein impairs islet function. Aim 1: To identify the causal SNPs impacting APA of ZC3H13 using functionally informed fine-mapping of 3'aQTLs. Aim 2: To determine the mechanisms by which 3’UTR lengthening reduces ZC3H13 protein expression. Aim 3: To investigate the mechanisms by which reduced ZC3H13 expression impairs insulin production and islet function. Our studies will advance the field by uncovering ZC3H13 APA as a novel genetic risk factor and elucidating ZC3H13-mediated epitranscriptomic regulation as a novel mechanism for the impaired β-cell function in T2D.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Despite increasing diagnoses, gastroenteropancreatic neuroendocrine tumors (GEP-NETs) remain poorly understood, both in relation to their molecular pathogenesis and cells-of-origin. My recent work using transgenic mice demonstrated, for the first time, the involvement of enteric neural crest-derived cells (ENCCs) in neuroendocrine differentiation and tumor development driven by Men1 gene deletion. Consistent with its role in cell fate patterning, Hedgehog signaling was implicated in neuroendocrine reprogramming of ENCCs. Menin, encoded by the MEN1 gene, has been shown to regulate Hedgehog signaling in mouse models of pancreatic NET development, however reciprocity between menin and Hedgehog has yet to be studied in ENCCs. This proposal aims to address whether loss of menin in ENCCs drives acquisition of the neuroendocrine cell fate with lineage commitment being actively modulated by Hedgehog signaling. My goals are to define the cells-of- origin for MEN1 GEP-NETs and to decipher the role of Hedgehog in driving neuroendocrine cell patterning. I will combine my experience using novel transgenic mouse models and ex vivo tissue culture techniques with state-of-the-art single cell and spatial transcriptome profiling to define the contribution of ENCCs to neuroendocrine differentiation and GEP-NET development. I hypothesize that reciprocal signaling by menin and Hedgehog drives ENCC reprogramming and gives rise to hyperplastic neuroendocrine cells with tumorigenic potential. This Research Plan will determine whether MEN1-associated GEP-NETs originate from reprogrammed ENCC populations (Aim 1) and decipher the role of Hedgehog signaling in neuroendocrine reprogramming of MEN1 GEP-NETs (Aim 2). During the mentored K01 award period, I will work closely with my primary mentor Dr. Juanita Merchant and co-mentor Dr. Megha Padi, distinguished experts in gastrointestinal biology and single cell analysis respectively, to develop the skillset to accomplish my research and career objectives. My Career Development Plan will facilitate my goal of becoming a productive independent investigator by combining rigorous didactic training and formal mentorship under a team of faculty who bring established expertise in cell and molecular biology, gastrointestinal physiology, large genomic data analysis, and 3-D organoid systems. Finally, the breadth of career development resources and extensive shared research facilities at the University of Arizona make it an ideal environment for me to carry out the proposed research project and achieve my career objectives. By leveraging innovative GEP-NET models and cutting-edge sequencing methods, this award will enable me to establish a state-of-the-art research program with the long- term goal of defining the cellular signals that govern enteric neuroendocrine cell fate.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Alzheimer's Disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia among older adults, 65 years and older. AD is characterized by the presence of extracellular amyloid β plaques (Aβ), intracellular neurofibrillary tau tangles (NFTs), and brain-wide neuroinflammation. Many current therapeutics targeting either Aβ or tau development have not been successful; therefore, it is imperative to understand the mechanisms in which these pathological hallmarks interact and influence each other. Aβ plaques develop many years before disease onset and do not correlate with clinical symptoms like the spread of tau. Moreover, immunotherapy targeting amyloid has failed to show cognitive improvement despite the significant reduction in Aβ load. While NFT-like tau pathology can develop without the presence of Aβ, the introduction of Aβ fibrils significantly accelerates NFT formation, further highlighting the need to better understand their molecular interactions. Recently, a case study has identified a carrier of the familial AD PSEN1 E280A mutation with a rare mutation in APOE3 (APOE R136S or APOE3ch) that resulted in resistance to neurodegeneration along with reduced NFTs while still exhibiting an elevated amyloid plaque load in the brain. The R136S mutation is located in a region of APOE known to have a role in binding to lipoprotein receptors and heparan sulfate proteoglycan (HSPGs), which have been suggested to promote amyloid-β aggregation and neuronal uptake of extracellular tau. Therefore, the APOE3ch mutation provides a unique interface to study the interaction between Aβ and tau. To that end, we have developed a novel ApoEch mouse model to be crossed with two different mouse models of amyloid and tau, 5xFAD and PS19, respectively. Thus, I propose to 1) Examine the protective effect of the ApoEch mutation in reducing tau pathology and rescuing neurodegeneration in PS19 mice, and 2) Assess the protective effect of the ApoEch mutation in dampening Aβ-associated plaque pathology and inflammation in 5xFAD mice. Collectively, this proposal will elucidate the role of this unique mutation in the development and manifestation of both Aβ and tau pathologies in mice.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Microbes in natural habitats mostly exist in groups arranged in the 3D space of biofilms, and spatial heterogeneity plays a key role in the stabilization, communication, and functions in these multi-species consortiums. However, a proper platform to build a synthetic community of bacteria and systematically vary the spatial parameter to deduce key fundamental knowledge in 3D microbial consortia – cellular phenotype, differentiation, communication, gene expression, and metabolism – is currently lacking. My research program aims to (1) develop a printing platform to construct 3D microbial consortia with well-defined polymers, (2) study the physiology of cells confined in these 3D environments, and (3) engineer cells and their 3D arrangement to yield biomaterials performing useful functions. The outcomes of our research endeavor will be (1) new fundamental knowledge and understanding of microbial consortia with a 3-dimensional spatial context and (2) functional biomaterials directly contributing to human health. Our contributions will be significant and have a long-lasting impact by filling in the critical knowledge gap in our understanding and engineering capability of microbial consortia relevant to biomedical science.

NIH Research Projects · FY 2024 · 2023-08

Primary Open Angle Glaucoma (POAG) is the leading cause of irreversible blindness affecting over 57 million people worldwide. Progressive loss of retinal ganglion cells (RGCs) and degeneration of optic nerve axons is the pathological hallmark of glaucoma. Elevated intraocular pressure (IOP) due to dysfunction of trabecular meshwork (TM) is the most significant and the only known modifiable risk factor for glaucoma. Understanding of the pathological mechanisms of glaucomatous TM dysfunction and neurodegeneration is limited due to lack of robust and faithful mouse model that mimics both TM dysfunction and glaucomatous neurodegeneration. Developing a mouse model of known genetic cause of POAG represents an ideal strategy to understand the pathophysiology of POAG. Mutations in myocilin (MYOC) gene are the most common genetic cause of POAG. Using TARGATT site-specific knockin strategy, we developed a Cre-inducible transgenic mice that expresses DsRed-tagged Y437H mutant of human myocilin (Tg.Cre-MYOCY437H). This technology utilizes serine integrase, PhiC31 (ΦC31) to insert any gene of interest (a single copy) into a preselected intergenic and transcriptionally active genomic locus (Hipp11), which has been engineered with a docking site. This allows stable and site- specific transgene integration. In our preliminary studies, we observed that a single intravitreal injection of helper adenovirus (HAd) 5 expressing Cre selectively induced human mutant myocilin protein in mouse TM. Importantly, Ad5.Cre injection resulted in significant and sustained IOP elevation in Tg.Cre-MYOCY437H mice. We hypothesize that TM-specific expression of mutant myocilin leads significant and pronounced IOP elevation and glaucomatous neurodegeneration in Tg.Cre-MYOCY437H mice. The major goals of this application are to induce mutant myocilin expression in TM using HAd5-cre injections and to characterize glaucoma phenotypes of Tg.Cre-MYOCY437H mice. In Aim 1, we will determine whether HAd5-mediated Cre induces mutant myocilin expression in TM and elevates IOP in Tg.Cre-MYOCY437H mice. In Aim 2, we will determine whether HAd5-Cre- induced IOP elevation leads to glaucomatous neurodegeneration in Tg.Cre-MYOCY437H mice. Our proposal will utilize highly innovative approaches. These include use of efficient and site-specific gene knockin strategy for generation of transgenic mice, a comprehensive investigation of outflow pathway, RGC functional and structural loss, optic nerve damage and damage to the visual centers of the brain. Our proposed studies will develop much needed mouse model of POAG that faithfully replicate all features of glaucoma.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ ABSTRACT: Vitiligo is an autoimmune skin disease characterized by the progressive destruction of melanocytes by autoreactive CD8+ T cells, resulting in disfiguring patches of white depigmented skin that cause significant psychological distress among patients. The gold standard treatment involves narrow band ultraviolet B (NBUVB) therapy, which induces the migration of hair follicle melanocyte precursors, in conjunction with topical steroids to suppress the local immune. Unfortunately, treatment is time consuming and re-pigmentation is often uneven. It is unknown why some depigmented areas persist and others re-pigment within the same lesion. Our recent work using non-invasive multiphoton microscopy (MPM) and single-cell RNA sequencing (scRNA-seq) has demonstrated that a subpopulation of keratinocytes enriched in vitiligo skin is important in disease persistence. We termed these cells ‘stress keratinocytes’ as they upregulate molecules associated with inflammation, wounding and other injuries. In this K08 application, we propose to study how local signals from keratinocytes interact with T cells and melanocytes to drive vitiligo persistence and repigmentation after treatment. Using MPM imaging, scRNA-seq and spatial transcriptomics, we will define if stress keratinocytes signals to T cells and paucity of melanocyte recruiting factors are enriched in treatment resistant areas (specific aim 1). We will also compare test to see whether adding these factors locally is sufficient to promote repigmentation (specific aim 2). Completion of this work will define how local tissue factors shape immune responses and melanocyte homeostasis in the skin has implications beyond vitiligo. This K08 application is designed to provide Dr. Jessica Shiu, MD PhD, the scientific training and professional development necessary to become an independent R01-funded investigator in the field of cutaneous biology. She will be mentored by Dr. Anand Ganesan, a physician scientist and a pigment cell biology expert with expertise on single cell genomics and skin imaging. Her secondary mentors, Drs. Bogi Andersen and Qing Nie, have extensive experience in keratinocyte biology and single-cell RNA sequencing analyses. Additional scientists, Drs. Mihaela Balu and Tinoco will provide further expertise in noninvasive imaging and cutaneous immunity. The work takes place within the outstanding scientific environment at UCI in the Department of Dermatology and Skin Biology Resource Center and has the support of multiple state-of-the-art centers including the NSF-Simons Center for Multiscale Cell Fate Research and Genomics High Throughput Facility. This training plan will help her develop technical skills on noninvasive imaging and spatial transcriptomics as well as quantitative methods needed for the analysis of dynamic populations of keratinocytes so that she will be positioned as a leading physician scientist in the field of cutaneous biology.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY Early-life adversity (ELA) is associated with vulnerability to mental illnesses that involve disruption of the brain’s reward circuits. These vulnerabilities may manifest as anhedonia, a reduction in reward desire or pleasure that is core feature of major depression. However, whether the association of ELA with anhedonia is causal is difficult to establish in humans, and mechanisms underlying this relationship are not understood. Our well-characterized rodent ELA model reliably leads to reward-circuit disruptions in a sex-dependent manner, yet the circuit nodes and pathways that are most affected remain unclear. In searching for ELA-sensitive reward-circuit components, we discovered and are characterizing a novel projection from basolateral amygdala (BLA) to nucleus accumbens (NAc) that expresses the neuropeptide corticotropin releasing hormone (CRH). Neurons expressing CRH are often stress-sensitive, and our preliminary data suggest this is also the case for the novel CRH+BLA-NAc pathway. Building on these robust data, we will determine the functional roles of the projection in mice and test the hypothesis that ELA- induced plasticity in this pathway contributes to sex-dependent effects of ELA on reward pursuit and consumption, significantly advancing our understanding of the origins of mental illness. Aim 1 will test the hypothesis that the novel CRH+BLA-NAc pathway modulates reward pursuit (motivation) and / or consumption in typically reared male and female mice, capitalizing on the temporal resolution of optogenetics and on formal motivation tasks to probe the specific role of the projection in the motivational vs. consummatory aspects of reward. Aim 2 will use the same technologies to determine the role of the CRH+ BLA-NAc projection in aberrant, sex-specific reward behaviors resulting from ELA. Aim 3 will examine the molecular and cellular mechanisms by which aberrant CRH+ BLA-NAc inputs regulate reward behaviors: we will identify the target cells of the projection following ex vivo optogenetic activation of BLA- origin projection fibers in the NAc, and determine the relative roles of GABA neurotransmission vs CRH receptor activation in the effects of projection stimulation on reward behaviors in male and female mice.