University Of Southern California

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY This project will develop volumetric real-time magnetic resonance imaging (RT-MRI) technology on a novel 0.55 Tesla platform to provide improved assessment of the heart and airway in motion. Rationale: Volumetric real- time imaging by computed tomography and ultrasound have had a profound impact on our ability to understand and evaluate disorders involving movement and of moving organs. These modalities each have limitations that include ionizing radiation, obstructed visualization angles, and limited contrast. MRI is currently capable of slice- by-slice RT-MRI and a leap to volumetric would enable improved assessment of a wide range of heart and airway disorders. Innovation: NIH has developed a 0.55 Tesla MRI instrument, that we argue will enable a breakthrough in RT-MRI performance. This is due to substantially reduced off-resonance effects, substantially reduced tissue heating, and a leap in scan efficiency due to increased flexibility in the MRI pulse sequence. We propose an innovative intramural-extramural partnership and interdisciplinary team to explore this potential. Approach: The objective of this project is to develop and translate low-latency volumetric RT-MRI methods that provide unprecedented spatio-temporal resolution and spatial coverage and will benefit several heart and airway applications. Specifically, we will: 1- develop and technically validate volumetric RT-MRI data acquisition at the 0.55T field strength, 2- develop and technically validate low-latency volumetric RT-MRI reconstruction, artifact mitigation, and segmentation, and 3- clinically evaluate volumetric RT-MRI in two unique patient cohorts at the NIH Clinical Center where it is likely to make an immediate impact on care—patients with tracheomalacia and relapsing polychondritis (N=20), and patients undergoing MRI-guided invasive cardiac catheterization (N=20). Broader Impact: The proposed volumetric RT-MRI technology is generalizable and could benefit many additional applications, such as cardiac function assessment in arrhythmia, and upper airway assessment in obstructive sleep apnea. The intramural and extramural labs each offer unique and complementary expertise to achieve this ambitious technical development. Creation of this new intramural-extramural collaboration will benefit both research programs and lead to new synergies that include basic research, clinical translation, and training of the next generation of scientists and engineers.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Mutations in the muscle-specific splicing factor RBM20 are a recently identified cause of aggressive dilated cardiomyopathy (DCM) characterized by severe arrhythmias. However, the underlying mechanisms are still unclear, and thus no therapies are available. Our group recently discovered a nuclear “splicing factory” involving RBM20 hotspots, which brings into proximity multiple co-regulated loci from different chromosomes. Formation of this three-dimensional (3D) chromatin structure relies the nucleation of RBM20 foci by its main splicing target, the pre-mRNA encoding for the giant protein titin (TTN). Ablating TTN transcription disrupts the RBM20 splicing factory and dysregulates the alternative splicing of genes involved in calcium handling, including the L-type calcium channel (CACNA1C) and calcium/calmodulin-dependent protein kinase II delta (CAMK2D). Thus, the central hypothesis tested in this proposal is that RBM20 assembles membraneless macromolecular condensates that control alternative splicing to centrally regulate cardiac development and disease. Our specific aims are: (1) Identify the functional consequences of dysregulating the RBM20 splicing factory; (2) Define the biophysical properties that drive assembly of the RBM20 splicing factory; (3) Define the key components of the RBM20 splicing factory. In Aims 1 and 2, we will perform cellular experiments to clarify the mechanisms and disease relevance of RBM20 focus formation, as well as functional studies using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Complementing hPSC-CM monolayer cultures, we will characterize 3D engineered heart tissues (3D-EHTs) and human myocardial grafts. We will utilize CRISPR/Cas9 genome editing to study the effect of RBM20 DCM mutations, as well as to generate fluorescent reporter lines to study focus dynamics in physiologically relevant models. We will characterize disease- associated changes in 3D chromatin topology within the RBM20 splicing factory using a combination of established sequencing- and imaging-based methods. We will then synthesize these biophysical, topological, and biochemical changes with functional genome-wide alterations in alternative splicing, to understand the dysregulation of cardiac electrophysiological and contractile properties. Aiming to elucidate the disease mechanism, we will probe the pathogenic role of specific RBM20-regulated splicing isoforms for CACNA1C and CAMK2D. In Aim 3, we will apply a novel interaction-discovery method developed in the Shechner laboratory, oligonucleotide-directed biotinylation (ODB), to perform an unbiased “multi-omic” analysis of the composition of the splicing factory. We will then determine the role of the newly identified splicing targets in RBM20 DCM, and of the putative RBM20 cofactors in the regulation of RBM20 puncta architecture and of cardiac genes' alternative splicing. Collectively, these experiments will elucidate the mechanisms by which LLPS and subnuclear architecture collaborate to drive alternative splicing in DCM, potentially revealing novel therapeutic targets.

NIH Research Projects · FY 2026 · 2022-12

SUMMARY This study aims to investigate a radically new paradigm of glomerular immune cell homing and to uncover the underlying cell and molecular mechanisms and their role in physiological and renal inflammatory disease conditions including diabetic kidney disease (DKD) and lupus nephritis (LN). As an additional translational aim, these studies will inform the repurposing of current treatments for kidney disease and the development of new anti-inflammatory therapies. Circulating and kidney resident immune cells are known to preferentially home in and around the glomerulus compared to other vascular beds in both normal and inflammatory states, suggesting the presence of unique immunomodulatory mechanisms in the glomerular microcirculation that protect the kidney filter. However, the underlying cell and molecular mechanisms have been largely unknown. The focus of the proposed studies is a new function of the cells of the macula densa (MD) which are strategically positioned at the glomerular vascular entrance and traditionally known to regulate renal and glomerular hemodynamics. Our laboratory recently identified that MD cells have the highest rate of protein synthesis among all kidney cell types and they secrete a variety of paracrine-acting angiogenic, cell growth and patterning, and extracellular matrix signaling proteins. Preliminary work identified the high MD-enrichment of several pathways and genes in the inflammatory response and cellular infiltration by leukocytes (e.g., Cxcl12, Cxcl14, Ptgs2, Ptges, Anxa1, Ccn1, Mif) and observed the MD-centric glomerular density and migration of CD44+ and CD8+ T cells in inflammatory diseases including lupus nephritis. Our central hypothesis is that MD cells drive the preferential glomerular homing of immune cells by the release of paracrine-acting pro-inflammatory factors including chemokines and cytokines. This project will use comprehensive experimental approaches including new transgenic mice with conditional, inducible and optogenetic tools (MD-mTORgof/lof, MD-Ai27, MD-Ai39, CCN1-knockout (KO), CCN1- GFP mice), models of renal and systemic inflammation (NTS, STZ-DKD, NZM.2328 LN models), in vivo MPM imaging, MD transcriptome analysis, in vitro cell culture and proteomics, and pre-clinical therapeutic translation. The specific aims are to (1) Examine the global immunomodulatory role and mechanisms of MD cells under physiological and renal inflammatory disease conditions, (2) Elucidate the renal anti-inflammatory role of CCN1, and (3) Test the anti-inflammatory effects of SGLT2 inhibitors (SGLT2i). These newly identified MD mechanisms may be targeted and will inform the development of future anti-inflammatory therapeutic strategies.

NIH Research Projects · FY 2026 · 2022-12

Project Summary As a major hub of the central auditory system for hearing, the inferior colliculus (IC) receives both bottom-up input from auditory brainstem nuclei, as well as feed-back input from the auditory cortex (AC). IC contributes importantly to the processing of essential features of sounds for communication and localization. Impaired IC processing has been associated with various hearing deficits. However, despite extensive studies of central auditory processing in IC, our understanding of cell-type-specific circuit mechanisms underlying the functional roles of the IC remains limited. In particular, how different types of neurons in the three anatomical and functional subdivisions of IC, the central nucleus (ICc), dorsal cortex (ICd), and external cortex (ICe), interact with each other and contribute differentially to the auditory processing of IC remains largely unclear. Addressing this question requires identification of molecular markers for cell types specifically located in each of the subdivisions. With 10X Genomics single-nucleus RNA sequencing (snRNAseq), we made initial efforts in screening molecular markers for different neuronal populations of IC. By identifying and verifying specific markers for IC subdivisions and exploiting corresponding transgenic Cre mouse lines for the selected marker genes, we will then characterize the anatomical connections and auditory response properties of the selected cell type and its functional role in auditory processing functions. Our preliminary snRNAseq results suggest two potential molecular markers specifically labeling subpopulations of excitatory neurons in the IC cortex. Their verification will endow us with unique opportunities to investigate the specific and diverse functional roles of ICd in auditory perception and behavior. Cutting-edge approaches in electrophysiology, anatomy, and optogenetics coupled with intersectional or projection-based tagging will be applied to address the differential functional contribution of diverse neuronal types in ICd as well as the distinct underlying circuitry mechanisms.

NIH Research Projects · FY 2026 · 2022-12

Project Summary Neuroinflammation is a hallmark of brain aging that may contribute to declines in function and neurodegenerative diseases. As the resident macrophage of the brain, microglial are crucial to brain maintenance but have been demonstrated to take on pro-inflammatory phenotypes with aging. The proposed research will examine the role of microglia-specific epigenetic mechanisms in aging and determine the effects of obesity and aging interactions on microglia phenotypic heterogeneity. We will determine whether aberrant age-related microglia-specific epigenetic patterns can be reversed by heterochronic plasma approaches. Additionally, we will determine how microglia subpopulations differ with age and obesity at a single-cell level with special interests in lipid-droplet- accumulating microglia (LDAM). The goals of the training are to: 1) obtain a strong knowledge and foundation in animal and nutritional models of anti-aging and obesity 2) gain hands-on experience in technologies and bioinformatic skills needed for various omics approaches in specific hippocampal cells and at a single-cell level 3) apply these skills to achieve the research aims outlined in this proposal and 5) gain experience and improve oral and presentation skills, manuscript and grant writing to enable a transition to independence. In Aim 1, we hypothesize that exposure of old mice to young plasma will reverse age-related microglia-specific DNA modifications to restore ‘youthful’ epigenomic patterns. This is based on the premise that DNA modifications are key regulators of the diverse phenotypes required for the fulfillment of microglia functions which are disrupted in aging. We will determine whether the administration of plasma from young into old mice can reverse age-related microglial-specific hippocampal epigenetic and transcriptomic changes in Cx3cr1:NuTRAP mice using whole genome oxidative bisulfite sequencing (WGoxBS) and RNA sequencing, respectively. These studies will identify specific genomic sites amenable to the rejuvenating interventions and serve as targets for future epigenome editing studies. In Aim 2, we will determine how the interaction of obesity and aging affect microglia heterogeneity. We hypothesize that high-fat diet leads to expansion of pathological LDAM resulting in impaired microglial function, which can be reversed by late-life calorie restriction. We will use single-cell transcriptomic analysis, bulk transcriptomics and microglia functional assays (phagocytic uptake and cytokine/chemokine analysis) to determine changes in microglia subpopulations in dietary-induced obese mice with age. In addition, we will determine whether starting calorie restriction at 12 months in dietary-induced obese mice can mitigate the effects of obesity on microglia heterogeneity. This will help identify mechanistic insights into microglia heterogeneity, LDAM and obesity-associated neuroinflammation. In total, the training goals and objectives will provide the needed skills and expertise to pursue a research career focused on developing anti-aging interventions.

NIH Research Projects · FY 2025 · 2022-12

Post mortem and neuroimaging data suggest an intact locus coeruleus helps to maintain cognitive performance in older adults. However, the basic mechanisms of this relationship are still unknown. The locus coeruleus is the source of most of the brain's noradrenaline but it is not yet clear how age-related decline in the locus coeruleus affects noradrenergic activity, and in turn, how these changes in noradrenergic activity modulate cognitive behavior. For instance, it is unknown whether age-related deficiencies in neuromodulation and processing result from decreased or increased locus coeruleus activity. Our long-term objectives are to determine the role of noradrenergic activity in modulating cognitive impairments and to identify means of preserving stable autonomic function to prevent or delay the progression of neurodegenerative diseases. The specific research goals of this proposal are to identify the mechanisms by which noradrenergic activity modulates distractibility by salient but irrelevant distractors in older and young adults. Both specific aims will utilize resting state recordings and an oculomotor search task that measures attentional inhibition of eye movements to determine whether tonic noradrenergic discharge directly modulates inhibitory mechanisms of attentional control. In Aim 1, we will investigate whether elevating tonic noradrenergic activity in older and young adults using threat of unpredictable shock induces arousal by pupillometric and electrophysiological measures. Furthermore, we will examine whether these changes will influence attentional control by modulating the magnitude of attentional inhibition of eye movements and phasic noradrenergic responses. We predict that older adults' distractibility will be less modulated by threat of shock than that of younger adults given our hypothesis that older adults already sustain higher levels of tonic noradrenergic release and hyperactivity. In Aim 2, we will investigate whether reducing tonic noradrenergic activity following slow-paced breathing and meditative practices improves attentional focus in older adults and young adults with high basal sympathetic activity. The goal of this aim is to determine the functional benefits of managing noradrenergic activity for attentional control as well as for measures of general arousal and phasic noradrenergic discharge. We predict that the benefits of the intervention in Aim 2 will be greater in older adults than that in young adults. The overall findings from this proposal will address a critical gap in knowledge connecting the mechanisms of neuromodulation on age-related declines to behavior, with theoretical implications in neuroprotection and preservation during aging. In addition, the training goals and objectives builds on the applicant's research expertise in psychophysiological research and fMRI with research expertise in the field of aging, training in EEG methodologies and analyses tools that are used in the field, and the professional development necessary to transition into an independent academic investigator. The research and mentorship team comprised of Drs. Mara Mather, Rael Cahn, and Markus Werkle-Bergner are the ideal fit to train the applicant in accomplishing his research and training goals throughout this fellowship.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Synapses are fundamental units of communication in the nervous system, where immense diversity in structure and function serve to tune and calibrate information transfer. Defects in the ability of synapses to properly diversify contribute to the etiology of a variety of neurodevelopmental, psychiatric, and neurodegenerative diseases. One means of generating diversity in synaptic function is through molecular heterogeneity, where combinations of distinct genes are expressed at individual synapses to enable specific functional properties. However, it has become increasingly clear, though difficult to resolve, that remarkable synaptic diversity can be achieved from a limited set of molecular machinery. In principle, the Drosophila neuromuscular junction (NMJ) is a uniquely powerful model to address how synaptic diversity is generated given the sophisticated genetic, electrophysiological, and imaging approaches. In this system, two distinct motor neurons converge to co-innervate individual muscle targets, where transmission from a strong and weak input together drive muscle contraction in the motor circuit. However, an inability to selectively isolate transmission from either input has been a major limitation towards understanding synaptic diversity in this system. Here, we propose to use expression of a unique Botulinum NeuroToxin (BoNT) to selectively silence transmission at strong or weak synaptic inputs. Preliminary data suggests that while each neuron is largely composed of the same molecular machinery at active zones, one core component, previously thought to function universally at all active zones, actually subserves dramatically different roles at strong vs weak synapses. We will use BoNT silencing, super resolution imaging, and the latest calcium reporters targeted to release sites to illuminate differences in active zone nanostructure and function between strong and weak synapses. We will also leverage new innovations in CRISPR mutagenesis to dissect the specialized functions of eight core active zone components at strong vs weak synapses. Finally, we will interrogate how these core active zone components are uniquely targeted for modulation and remodeling at strong vs weak synapses in the context of homeostatic synaptic plasticity. Together, these approaches will unlock fundamental insights into how glutamatergic synaptic diversity is established and adaptively modified through plasticity. Ultimately, this understanding will illuminate key mechanisms through which heterogeneous functional properties at glutamatergic release sites are enabled by a limited molecular toolkit.

NIH Research Projects · FY 2026 · 2022-11

Project Summary Although IL-17-producing T helper (Th17) cells are a major pathogenic contributor to autoimmune diseases, functional diversity undermines their potential as a prospective target for treating autoimmunity. Th17 cells are currently classified into homeostatic and inflammatory subpopulations. It was noted that inflammatory Th17 cells can be beneficial (anti-infection) and pathogenic (pro-autoimmunity). The distinction between homeostatic and inflammatory Th17 cells has been extensively studied. However, how to dissect the anti-infection and pro- autoimmunity functions of inflammatory Th17 cells is largely unknown. There is a critical need for this knowledge given serious infections and fatal outcomes observed in patients receiving general inhibitors of inflammatory Th17 cells (e.g. antibodies against IL-23). Herein, autoimmune and anti-infection subsets of inflammatory Th17 cells are for the first time distinguished experimentally. The objective of this grant is to elucidate the metabolic regulation discriminating autoimmune and anti-infection Th17 cells derived from murine models and human patients. The central hypothesis is that autoimmune and anti-infection Th17 subsets adopt distinct serine metabolic programming. The rationale is that determining the differences between autoimmune and anti-infection Th17 subsets will offer opportunities for novel therapeutics with substantially improved selectivity than the current regimens. The central hypothesis will be tested by pursuing three specific aims: 1) to determine the mechanism for regulating serine metabolism in the autoimmune Th17 subset; 2) to determine the mechanism by which serine regulates pathogenicity of the autoimmune Th17 subset; and 3) to determine the transcriptional and metabolic programming of Th17 cells from patients with inflammatory bowel disease (IBD). Under the first aim, autoimmune Th17 cells recovered from murine models will be used to measure intracellular serine and indicators of autoimmune pathogenicity with modulation of serine metabolic enzymes. For the second aim, biochemical approaches and murine models will be employed to evaluate the relationship between serine-induced intracellular methylation and pathogenic potential of Th17 cells. In the third aim, RNA-seq analysis will be performed to evaluate transcription profiles of Th17 cells from IBD patients. The research proposed in this application is innovative, because it focuses on the immunometabolic regulation discriminating anti-infection and autoimmune Th17 subsets, a heretofore-unexamined mechanism. The proposed research is significant because it is expected to provide novel opportunities to develop autoimmune Th17-selective therapeutics for autoimmune diseases. This would be extraordinarily important for patients that have been infected or exposed to certain pathogens, such as tuberculosis, since existing Th17 inhibitors cannot be used due to the risks of decreasing the patients’ control of the infection.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT To fully understand the basic biology that underlies human aging, and accurately time potential treatments that are aimed at preventing age-related pathology, it will be of vital importance to determine when the events that precipitate human aging occur. One of the processes that drives human aging is mitochondrial mutagenesis. Mitochondrial DNA (mtDNA) mutations accumulate as we grow older, which accelerates the natural aging process and contributes to various age-related diseases, including cancer, muscle wasting and neurodegeneration. However, due to the multiplicity of mitochondrial genomes in a cell, de novo mtDNA mutations are initially harmless. MtDNA mutations need to clonally expand to cause disease. Because this expansion process takes time, we hypothesize that the mutations that precipitate age-related pathology arise relatively early in life, and that the pace of mitochondrial aging is set long before pathology becomes apparent. We propose to test this hypothesis with a new mouse model of DNA polymerase gamma (PolgA), the enzyme that replicates the mitochondrial genome. This model expresses an error prone version of DNA polymerase gamma that can be replaced with a WT allele at will. Accordingly, we can turn off mitochondrial mutagenesis at any time during the animal’s lifespan and determine how mutations that occur early in life affect pathology later in life. In addition, we will use various mutation detection techniques, including random mutation capture, single cell sequencing and duplex sequencing to track the fate of these mutations over the lifespan of our mice. These experiments will describe the natural history of every possible mtDNA mutation in various tissues, effectively dissecting the parameters that control the impact of mtDNA mutations on human health. Finally, we propose to rejuvenate somatic cells and aging mice by manipulating mitochondrial fusion and mitophagy, in an attempt to cure them from deleterious mtDNA mutations. If successful, this strategy could provide a potential treatment for multiple pediatric mtDNA diseases, as well as the mitochondrial component of age-related diseases. Accordingly, our experiments have the potential to revolutionize our understanding of the relationship between mitochondrial mutagenesis and aging, and provide powerful new tools to combat both inherited and age-related diseases that are associated with mtDNA mutations.

NIH Research Projects · FY 2025 · 2022-09

Abstract An estimated 43% of children under age 5 in low- and middle-income countries (LMICs) experience compromised development due to poverty, poor nutrition, and inadequate psychosocial stimulation. Numerous early childhood development (ECD) parenting interventions have been shown to be effective at improving ECD outcomes, at least in the short-term, but they are a) still too expensive to implement at scale in low-resource and rural settings, and b) their early impacts tend to fade over time in the absence of continued support. New ways to deliver effective ECD parenting interventions are sorely needed that are both low-cost to be potentially scalable, while also able to sustain impacts long-term. The rapid growth and low cost of mobile communications in LMIC settings presents a potentially promising solution to the competing problems of scalability and sustainability. Yet there is no rigorous research on mobile-health (mHealth) interventions for ECD outcomes in LMIC settings. We recently showed that an 8- month ECD parenting intervention featuring fortnightly group meetings delivered by Community Health Workers (CHWs) from Kenya's rural health care system significantly improved child cognitive, language, and socioemotional development as well as parenting practices, and our group-based delivery model was more cost-effective than previous ECD interventions. Yet it is still too expensive for scaling in a rural LMIC setting such as ours, particularly if we need interventions that can be extended for longer periods of time to increase their ability to sustain impacts. Our proposed study experimentally tests a traditional in-person delivery model for an ECD parenting intervention against two mHealth-based delivery models that partially or almost fully substitute remote delivery for in-person meetings. Kenya is an ideal setting for testing mHealth programs given its high penetration of mobile phones (94%). We will assess the relative effectiveness and costs of these mHealth delivery models against a purely in-person model, and extend the interventions over two years to increase their ability to sustain changes in child outcomes longer term. Our evaluation design is a non- inferiority clustered Randomized Control Trial across 60 CHWs and 1200 households in which we will use an adaptive trial design to allow for midcourse review and feedback on the remote delivery models. By testing three interventions that vary in how much in-person delivery is substituted by remote-delivery, we can assess the degree of substitutability or complementarity to inform the design of more scalable and sustainable interventions. Our goal is to determine the best model to maximize the intervention's reach and sustained impacts to improve child outcomes. By integrating delivery into the ongoing operations of local CHWs within Kenya's rural health care system, utilizing new low-cost technology, and involving local ECD policymakers and stakeholders as key collaborators from the project's inception, our project has the potential to make important contributions towards discovering potentially scalable, sustainable solutions for resource-limited settings.

NIH Research Projects · FY 2025 · 2022-09

Abstract Type II diabetes mellitus (T2DM) increases the risk for developing Alzheimer’s Disease (AD), but the mechanisms are not fully understood. To reduce the risk for cognitive decline in those with T2DM and to identify possible intervention targets, it is imperative to understand how T2DM affects the brain and cognition. Our overall goal is to gain an understanding of how metabolic and Alzheimer’s risk relate to brain measures and cognition in 200 Northern American Latino middle-aged adults, an understudied group that is at higher risk for both T2DM and AD compared with non-Latino Whites. We will: 1) Evaluate regional brain integrity (structural, functional, and vascular) in those with and without T2DM and as related to metabolic blood markers and AD pathology risk as indicated by plasma ptau181 level 2) Evaluate the changes in cerebral blood flow, vascular reactivity, and functional connectivity between fasting baseline and 2 hours after 75 grams of glucose ingestion and relate those changes to changes in metabolic markers to gain a mechanistic understanding of how metabolic risk affects the brain’s functional and vascular response to glucose ingestion, and 3) Identify brain measures of neurovascular function (cerebrovascular reactivity, functional connectivity, cerebral blood flow, and blood brain barrier permeability) that predict cognitive decline and brain deficits over 2 years and investigate their relationship to metabolic function and AD pathology. There are ongoing efforts to repurpose diabetes medications into cognition clinical trials. Achieving our aims will provide insights into the mechanisms underlying cognitive decline in T2DM patients with and without AD pathology, and provide brain imaging biomarkers to guide potential interventions.

NIH Research Projects · FY 2025 · 2022-09

Gains in life expectancy and population aging are driving a sharp rise in Alzheimer’s Disease and Related Dementias (ADRD). In this context, it is crucial to understand what factors can modify ADRD risk and cognitive decline in older ages. Education has been identified as one potential modifier, as higher education is robustly associated with lower ADRD risk. However, little is known about how much of this association reflects a causal effect from education to ADRD risk and how much is driven by common third factors, such as genetics, that may confound and moderate this relationship. In addition, the relevance of factors beyond the quantity of education – in particular the importance of education quality as a driver of ADRD risk, as well as a moderator in the relationship between education and ADRD – are not well understood. Filling these knowledge gaps is essential to the design of effective policies aimed at improving cognitive health and reducing disparities in ADRD risk. In this project, we propose to study how much of the association between education, cognition and ADRD risk in late-life is due to a causal effect running from education quantity and quality to cognition/ADRD risk. To deal with the fact that different people self-select into different types and quantities of schooling, we will use two natural experiments: one school reform that affected education quantity (years of compulsory schooling) and another that affected quality (academic curriculum). We will supplement existing datasets with the construction of polygenic indexes (PGIs) for educational attainment (EA) and for Alzheimer’s Disease (AD), and by linking participants to local historic school quality measures such as pupil/teacher ratios and teacher pay. This will allow us to study the role of genetics and school quality in moderating the effects of both school reforms on ADRD. We will use data from three large international aging cohorts: the UK Biobank (UK), FinnGen (Finland) and Lifelines (the Netherlands). These cohorts allow us to study administrative-based measures of ADRD diagnosis, ADRD risk factors and survey-based measures of cognition. Moreover, the three cohorts were genotyped, allowing us to explore the role of genetics in driving ADRD risk as well as in moderating the relationship between education and ADRD risk. Establishing whether education has a causal effect on late-life cognition and ADRD risk is challenging but essential for identifying clinical and policy interventions. Without causal evidence, policy makers do not know whether education improves individuals’ later-life cognitive health or whether the education-ADRD association reflects differences in the characteristics of individuals who self-select into education. Moreover, it is equally important to know what aspects of education causally affect ADRD risk. What is the relative benefit of increasing the quantity of education versus improving its quality? Are individuals who are predicted to get more education based on their genes protected against genetic risk of AD? The lack of such knowledge limits the design of policies aimed at reducing disparities in ADRD risk. Our project aims to start filling these knowledge gaps.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Although aging is a conserved phenomenon across evolutionary distant species, key aspects of biological process have been found to differ between males and females of the same species during aging. For instance, accumulating evidence suggests that immune cells of male vs. female individuals are clearly distinct throughout life. Despite these clear differences and their potential significance, biomedical research has historically focused exclusively on male individuals. Thus, sex- driven differences, their molecular underpinning and impact on various aspects of adult health, including lifelong immune responses, are still poorly understood. Interestingly, both sex hormones (i.e. androgens vs. estrogens) and sex-chromosomes (i.e. XX vs. XY) have key impact outside of reproduction and gonadal development. Indeed, accumulating evidence supports the notion of widespread sex-dimorphism in biological processes. For example, immune responses differ between biological sexes, with a more robust immune response in females vs. increased susceptibility to infection in males. Neutrophils are a major leukocyte population serving as a “first line of defense” against infections. We have observed strong sex-dimorphism in the transcriptome, metabolome and lipidome of murine neutrophils, as well as changes in neutrophil-mediated immune phenotypes. Together, our results suggest that mechanisms involving gonadal hormones and/or sex chromosomes can regulate neutrophil-based immunity. We hypothesize that mechanisms involving both sex chromosomes and lifelong exposure to gonadal hormones independently modulate neutrophil-based genomic networks and immune phenotypes throughout life. To test our hypothesis, we will investigate how neutrophil-based phenotypes are fine-tuned as a function of sex throughout life. Sex hormones and sex chromosome complement are intimately linked in wild-type animals, complicating the study of determinants of sex-dimorphic phenotypes. To address this shortcoming, we will leverage an innovative model of adult somatic-sex reprogramming (the adult Foxl2-iKO) to assess the impact of hormonal vs. genetic sex on neutrophil phenotypes throughout life. This unique and tractable system will enable us to understand the consequences of adult exposures to higher levels of estrogens vs. androgens on immune responses. Together, our proposed experiments will provide insights on sex-dimorphic mechanisms of immune regulation and reveal how hormonal inputs may exert lifelong impact on immune cells.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT Skeletal muscle has recently arisen as a novel regulators of Central Nervous System (CNS) function and aging, secreting bioactive molecules known as myokines with proteostasis and metabolism-modifying functions in targeted tissues, including the CNS. Myokine secretion is heavily modified by exercise, suggesting that myokine signaling in the periphery may underlie the well documented geroprotective benefits of exercise on the brain. The following studies address muscle proteostasis, a pathway highly activated during exercise, as a potential new regulator of the neurocognitive benefits of exercise. We have recently generated a novel transgenic mouse with enhanced muscle proteostasis via moderate overexpression of Transcription Factor E-B (TFEB), a powerful master regulator of cellular clearance and proteostasis. We have discovered that the resulting enhanced skeletal muscle proteostasis function can significantly ameliorate proteotoxicity in the aging CNS and also improve cognition and memory in aging mice. Enhancing muscle proteostasis also reduced neuroinflammation and accumulation of AD-associated pathological hallmarks in plaque based and a tau- based models of AD. We have also identified previously unreported alterations in the transcriptome of skeletal muscle from patients with AD, as well as potential unique populations of skeletal muscle factors that may be driving these CNS benefits. In this project, we will determine if enhanced skeletal muscle proteostasis promotes neuroprotection against AD-associated phenotypes, and using powerful transfer learning and computational modeling approaches, identify exercise-associated circulating factors as new therapeutic interventions for the preservation of CNS function during AD.

NIH Research Projects · FY 2024 · 2022-09

ABSTRACT The goal of our proposal is to develop a scalable platform for structure-based virtual screening of Giga- and Tera- scale drug-like compound libraries, enabling streamlined discovery of high-quality drug candidates. Availability of protein target structures and Giga-scale REAL Space libraries of virtual compounds (>10 billion) position docking-based virtual screening as a key paradigm for drug discovery. However, the computational cost of Giga- scale screening becomes a major bottleneck limiting further growth of the screening libraries. Recently, we have introduced a highly scalable synthon-based technology, V-SYNTHES, which performs hierarchical structure- based screening of REadily AvaiLable for synthesis (REAL) libraries (Sadybekov et al, Nature accepted). By iteratively screening synthon-scaffold combinations, the V-SYNTHES approach makes possible rapid detection of the best-scoring compounds in the Giga-scale chemical space while performing docking of only a small fraction (~2 million) of the library. First tests of V-SYNTHES demonstrated strong enrichment in computational benchmarks and significantly improved experimental hit rates on cannabinoid receptor CB2 and ROCK1 kinase targets, while requiring 100 times less computational resources than standard virtual screenings. Building upon these preliminary results, our proposal aims to: (1) Further develop a fully automated V- SYNTHES algorithm, optimize its parameters and expand it to Tera-scale REAL libraries. (2) Apply and experimentally validate the V-SYNTHES approach on a set of therapeutic targets of different classes, which includes such challenging targets as nucleotide and lipid binding pockets, allosteric pockets, and orphan receptors (3) Establish portability of the algorithm to an open-source docking platform to further facilitate V- SYNTHES adoption in academic labs. The open-source algorithm will be distributed as a workflow for Linux clusters and computing clouds. Successful completion of this project will establish V-SYNTHES as a robust computational platform for structure-based ligand discovery in most classes of therapeutic targets, scaleable for rapidly growing REAL modular libraries. Most importantly, it will help to make fast virtual screening of the Giga-to-Tera-scale libraries broadly accessible for the whole research community with reasonable computational resources.

NIH Research Projects · FY 2024 · 2022-09

Despite a wide-ranging interest in performing clinical research for bioelectronic medicine applications, there are no open-architecture and open-source implantable systems for autonomic nerve stimulation and recording available to researchers. As a result, progress towards bioelectronic clinical therapies is hampered by the significant technical, regulatory, and financial hurdles faced by researchers to gain access to the few commercial implantable neuromodulation technologies for early clinical studies. The few clinical closed-loop implantable neuromodulation systems presently available are not suitable for the many bioelectronic medicine applications envisioned, as they lack key functional modules for accessing the autonomic nerves; moreover, most use closed architectures (e.g., the use of custom ASICs instead of commercial over-the-shelf components) and proprietary software, limiting the ability to adapt such systems for different clinical indications. To address these shortcomings, the overall objective of this HORNET OpenNerve Platform project is to develop and disseminate a fully open-architecture and open-source implantable system for autonomic nerve stimulation and end-organ sensing. In this administrative supplement, we will perform short-term and long-term validation of the OpenNerve Platform functionality in large animals for closed-loop sacral nerve stimulation for treatment of chronic constipation. The OpenNerve Platform includes an external charger and controller, implantable pulse generator, and an assortment of implantable leads for nerve stimulation and internal organ sensing. The use of sacral nerve stimulation for treating chronic constipation was selected based on potential clinical indications that are of interest to the NIH SPARC program.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract. Intrinsically Disordered Proteins (IDPs) represent approximately 40% of the human proteome and are implicated in a large number of human diseases, including neurological disorders and cancer. Therefore, the biological relevance of IDPs has garnered substantial interest over the last decade. IDPs often interact with curved membranes to form structures that are essential to cellular physiology, such as synaptic and endocytic vesicles. These interactions are facilitated by membrane curvature sensing. The PI recently discovered that IDPs are potent sensors of membrane curvature. This discovery is a substantive departure from the predominant structure-function paradigm, as IDPs lack fixed three-dimensional structure and are often incorrectly assumed to also lack biophysical functionality. The curvature sensitivity of IDPs, as well as many other types of proteins, is normally studied at thermodynamic equilibrium. However, membrane-interacting proteins exist in a dynamic equilibrium between their membrane-bound and membrane-unbound states. It can take minutes to achieve thermodynamic equilibrium between proteins and membranes, but cellular processes, such as cell signaling, occur anywhere between milliseconds to seconds. It is clear from this mismatch in timescales that when thermodynamic equilibrium alone is considered, dynamic information that is pertinent to the timescale of cellular processes is omitted. Little to no literature exists that examines this phenomenon, likely owing to the difficulty associated with achieving such experimental measurements. Thus, dynamic interactions between proteins and curved membrane structures are poorly understood. Using their expertise in quantitative fluorescence microscopy and protein engineering, the goal of the PI's laboratory is to develop and apply techniques and strategies that will allow for direct visualization and characterization of dynamic interactions between IDPs and curved membrane structures. The PI's future research program contains 3 overarching research projects. Work in Project 1 will evaluate the extent to which protein structure influences adsorption and desorption kinetics, testing the working hypothesis that curved membranes affect various protein structures differently. Work in Project 2 will evaluate the impact of protein networks on the binding dynamics of IDPs, answering questions about the influence of protein multivalency on dynamic behavior. Work in Project 3 will develop experimental techniques and strategies that mimic the intracellular environment, answering questions about dynamic interactions between IDPs and curved membrane substrates that occur in the presence of two- dimensional, phase separated protein mixtures on the membrane surface or three-dimensional protein aggregates in the bulk solution. As previously mentioned, our current understanding of the interactions between proteins and curved membranes was derived from systems in which the partitioning of proteins was measured after thermodynamic equilibrium was achieved. In contrast, the proposed work will fill a key gap in existing knowledge by directly observing and quantifying dynamic interactions between proteins and curved membranes.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT In this K08 career development award, the principal investigator (PI) aims to determine how DNA-methylation is altered after DNA damage and investigate the impact of these changes on 3D chromatin organization, gene regulation and treatment resistance. The PI studies this process in glioblastoma, a difficult to treat malignant brain tumor. Training and mentoring activities will facilitate meeting not only scientific goals, but also the principal investigator's career development by addressing gaps in knowledge and expanding training through coursework, meetings, networking, and an expert mentoring team consisting of a primary mentor, co-mentor, and four faculty advisors. The research proposed will be addressed through this K08 phase of training and serve as the scientific basis for the applicant's career as an independent investigator. The candidate will acquire skills necessary to complete the aims through selected mentors with non-overlapping expertise and coursework. The central hypothesis is that, after DNA damage repair, the local epigenetic state is not restored correctly, leading to epigenetic alterations, gene expression changes and treatment resistance. This hypothesis is tested using human patient-derived glioblastoma cell cultures as a model system. The rationale for this project is the observation that stochastic DNA methylation alterations can be detected with radiation damage models, and endonuclease damage can alter local DNA methylation states. The mechanism underlying this process and the extent to which it occurs in cancer, however, is not known. This hypothesis is challenging to test using stochastic damage, such as radiation, or traditional endonuclease damage models, which are unable to cut methylated DNA, and have a fixed and limited number of sites. To circumvent this issue, the investigator developed a CRISRP-Cas9 tool to reproducibly induce genome-wide double strand breaks to study DNA methylation alterations and genome organization around sites of DNA damage. The central hypothesis will be tested by two specific aims to (i) test how DNA methylation and genome organizational alterations evolve at damaged DNA loci, and (ii) test if genome re-organization factors can be targeted therapeutically during radiation stress. This training proposal is innovative because it (i) develops tools to map DNA methylation and 3D chromatin organization alterations following DNA damage and (ii) implicates this process in treatment resistance. The significance of this proposed research is that it fills knowledge gaps in epigenetics, DNA-damage repair, and the understanding of the effects of treatment on cancer cells. Successful completion of these studies will provide translatable insight into the interplay between DNA damage, DNA methylation and genome re-organization in glioblastoma.

NIH Research Projects · FY 2025 · 2022-09

Studies suggest an important role for cerebral hypoperfusion and blood brain barrier (BBB) permeability in the onset and progression of cognitive impairment and dementia. Ambient air pollution may differentially impact cognitive health in individuals with underlying cerebrovascular disease. Murine studies demonstrate that particulate matter (PM) exposure and chronic cerebral hypoperfusion (CCH) have supra-additive effects on subcortical white matter injury and neurocognitive deficits. This proposal leverages cell culture studies and a murine bilateral carotid artery stenosis (BCAS) model to assess the effects of Diesel Exhaust Particulate (DEP) in a system that isolates a purely vascular component of cognitive decline and dementia. Potential synergies between axonal growth inhibitors and extravascular fibrinogen deposition are studied as critical regulators of white matter injury and repair. The specific aims of this project are: 1) Examine the role of the Nogo/NgR1 pathway in axonal regeneration and white matter repair following DEP exposure. 2) Establish the roles of BBB permeability and extravascular fibrinogen on axonal regeneration and white matter injury following DEP/CCH exposures and 3) Examine NgR1 and 67KDa Laminin receptor (67 LR) binding/ internalization as a potential therapeutic strategy to mitigate white matter pathology in the setting of DEP/CCH exposures. A factorial design will determine the independent and combined effects of DEP and CCH on white matter toxicity and neurocognition. We expect PM exposure to prime the NgR1 pathway for neurite outgrowth inhibition. We also expect PM exposure to increase plasma fibrinogen levels. Neither of these changes result in substantial white matter injury on their own. We expect CCH to amplify the PM effects on neurite outgrowth inhibition and white matter toxicity. We hypothesize this will occur through 1) decreased cAMP levels secondary to hypoxia that augment DEP-induced axonal growth inhibition and 2) increased BBB permeability that results in extravascular fibrinogen deposition in the subcortical white matter. In addition to causing direct white matter toxicity, fibrinogen can impair axonal regeneration through the integrin b3/ Epidermal Growth Factor Receptor (EGFR) pathway and increased chondroitin sulfate proteoglycan (CSPGs), which can further inhibit white matter repair through the NgR1 pathway. We will assess the impact of epigallocatechin-3-gallate (EGCG) administration to mitigate white matter injury following PM and CCH exposure. We expect this to work through a coordinated strategy of NgR1 and 67LR binding/ internalization and cAMP mediated signaling.

NIH Research Projects · FY 2025 · 2022-09

Abstract ILC2s are the dominant innate lymphoid cell population in the lungs at steady state and their release of type-2 cytokines is a central driver in responding eosinophil infiltration, increased airway hyperreactivity and associated lung tissue injury. Previously, our laboratory identified a subset of ILC2s (ILC210s) that actively produce and secrete IL-10, an anti-inflammatory cytokine with the ability to ameliorate allergic lung inflammation signaling (J Allergy Clin Immunol., 2020). Importantly, these results have been confirmed by other groups in a variety of allergic disease models (J Exp Med., 2020, Immunity, 2021). The proposed research plan is motivated by recent preliminary observations demonstrating that key molecular and transcriptional requirements may be required for the induction of IL-10, with the potential for targeted modulation. SA1 is intended to explore the regulation of transcription factors for the induction of IL-10. We propose a series of experiments in acute and chronic models of allergic airway inflammation to assess the involvement of key transcription factors cMaf and Blimp-1 first by expansive, cutting-edge chromatin sequencing methods, and next by retroviral induction and knock-out mouse models. In SA2, we also observed that production of IL-10 relies significantly on key metabolic pathways often utilized by ILC2s. We intend to expand our studies by investigating the role of glycolysis, fatty acid oxidation and signaling protein AMPK, with the aim of identifying mechanistic targets for the potential modulatory therapies for allergic disease. Additionally, mitochondrial regulation of IL-10 production with be explored through in vitro and ex vivo mitochondrial dynamic assays. Finally, the two parts of this project come together to address specific transcriptional and metabolic requirements for the modulation of pathogenic ILC2s with the intention of targeted conversion to ILC210s with the ability to regulate airway hyperreactivity. The results obtained from this study will provide novel insights into an important and understudied role of ILC210s in diseases associated with ILC2s such as allergic lung inflammation and asthma.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in women in the US and globally. Menopausal transition poses remarkably elevated risk of CVD making postmenopausal women a population of special attention. There is an increasing concern about the exposure to environmental chemicals, particularly persistent organic pollutants (POPs), that disrupt human endocrine milieu and adversely affect cardiovascular health. The bioaccumulation of POPs over lifetime induces significant long-term health impact, especially among older population. However, the long-term effect of POPs on subclinical atherosclerosis progression, an early pathological feature of CVD, has not been studied well in postmenopausal women. In addition, the effect of POPs on atherosclerosis progression has not been evaluated in the US population. To our knowledge, no longitudinal study has been conducted to investigate the effects of POP mixtures and atherosclerosis progression. The only longitudinal study reporting an adverse effect of a specific class of POPs, per- and polyfluoroalkyl substances (PFASs), on increased carotid intima-media thickness (IMT, ultrasound) over 10 years was from a Swedish senior cohort. To fill the gaps in our understanding, we will investigate the long-term associations of plasma POPs concentrations and atherosclerosis progression in postmenopausal women in a unique cohort; Early vs Late Intervention Trial with Estradiol (ELITE) with 5-year longitudinal measurements of subclinical carotid atherosclerosis including gold-standard ultrasound measures (IMT, echogenicity, and stiffness) and frozen plasma samples to analyze absolute concentrations of 60 POPs from four main classes (polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorinated pesticides (OCPs), and (PFASs). ELITE is a randomized clinical trial including 596 early (<6years since menopause) and late (≥10years since menopause) postmenopausal women comparing rates of atherosclerosis progression over 5 years between women randomized to hormone therapy (HT) and placebo. Beyond the goal of investigating the effect of POP mixtures on atherosclerosis progression, we will investigate the impact of POP mixtures on risk factors of atherosclerosis including metabolic (lipids, glucose, HbA1c, and insulin resistance) and inflammatory biomarkers. Important covariates including the design factors (HT and placebo, early and late-postmenopausal groups), obesity, smoking, diet, and physical activity will be adjusted for in the analysis. To assess the generalizability of the adverse effect of POP exposure across various subgroups of postmenopausal women, we will evaluate POPs’ associations with atherosclerosis progression in subgroups of women randomized to HT and placebo, as well as early and late postmenopausal groups. This study will provide important evidence on long- term effect of POP mixtures on atherosclerosis progression and related metabolic and inflammatory pathophysiology among postmenopausal women who are at high risk of CVD. Findings from this study will help identify key individual and subgroups of POPs as targets for regulations, remediations, and CVD prevention.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract ALS is a complex disease with diverse genetic etiologies. Although drugs targeting known causal mutations (e.g. SOD1 ASOs) may treat individual forms of ALS, this approach cannot address the vast majority of cases with unknown genetic etiology. Moreover, the large number of causal genes and rarity of each genetic form suggest that treating ALS will require many patient-specific therapies or broadly-effective treatments. Thus, there is a pressing need for new, scalable approaches that identify patient-specific or broadly-effective therapeutic strategies for multiple forms of ALS, particularly those with unknown genetic etiologies. Studies using induced motor neurons (iMNs) from iPSCs indicate that iMNs from most ALS patients, including those without known mutations, display ALS disease phenotypes including rapid degeneration. We performed phenotypic screening on ALS iMNs to identify the most efficacious therapeutic targets. However, iMN drug responses are heterogeneous across patient lines, and probing disease mechanisms and drug responses on a sufficient number of lines is prohibitively expensive and labor intensive. In this transformative project, we will overcome this critical barrier in ALS drug discovery by combining ALS iPSC disease modeling with GENEVA, a novel platform we developed for cancer therapeutics that uses single cell transcriptomics to assess drug effects on dozens of patient lines in one dish. GENEVA uses SNP-based computational demultiplexing of single-cell RNA-seq data to profile responses to therapies across pools of many iPSC lines. We developed computational tools that analyze the high-content readout of scRNA-seq to (i) precisely quantify the sensitivity of every line based on its representation within the population in the case and control arm, (ii) identify the molecular mechanisms that underlie the response to the drug and possible mechanisms of resistance, and (iii) reveal differences in response between subpopulations as a result of heterogeneity within every line. GENEVA will increase the scale of ALS lines in drug discovery by 10-50-fold and reveal disease and drug response mechanisms at single cell resolution, enabling the discovery of new therapeutic targets with either broad efficacy or high patient specificity. Using this “population-in-a-dish” approach, GENEVA-ALS will identify neurodegenerative and drug response mechanisms across ALS patient cohorts at an unprecedented scale, removing a critical bottleneck in ALS drug discovery. The proposed study will 1) establish the GENEVA-ALS population-in-a-dish platform, 2) establish temporal maps of iMN disease processes for 45 ALS lines, 3) validate 3 therapeutic targets with novel mechanisms of action that show broad efficacy across ALS iMN lines, 4) determine if GENEVA-ALS can predict efficacy in ALS patients in a phase 2 clinical trial, and 5) identify new therapeutic targets in a genome-wide CRISPRi screen on 30 ALS lines. Our study seeks to shift current research by overcoming the critical barrier of patient heterogeneity in drug discovery.

NIH Research Projects · FY 2025 · 2022-09

Elder mistreatment (EM) has profound effects on 1 in 10 older Americans, and rates are amongst the highest for people living with dementia (PLWD). Family caregivers most frequently inflict this harm and are typically remorseful for their behavior. This proposal will address this problem through a novel approach that identifies care partners/caregivers (CPG) at primary care medical clinics, whether they are there for their own care or that of the PLWD. During the project’s R61 phase, we will develop and test the feasibility of an evidence-based brief Risk Assessment Screen (RAS) for use in primary care clinics to screen CPGs of PLWD. A 3-component intervention will be developed and feasibility-tested. The first component will direct the CPG during the clinic visit to a website specifically designed to engage them in solution-focused strategies. The second component will provide 1-3 home or technology-assisted visits with a care navigator who provides person-centered guidance to facilitate effective caregiving strategies and alerts CPGs to risks of EM. A third component will educate the clinical care team to address caregiving needs directly with the CPG during the clinic visit and schedule a follow-up visit within 2 months to monitor for change. We will develop an Outcome Tool that includes a compilation of validated measures of modifiable risk factors known to be associated with EM by CPGs which will be used to measure change in risk of EM over time. During the project’s R33 phase, the research team will conduct a cluster randomized controlled trial to test the effectiveness of the RAS and the 3-component intervention. Primary care clinics across Los Angeles County will be randomized to one of the three study arms: control, RAS only, or RAS plus intervention. Analyses will assess the impact of screening and the intervention on participants’ level of risk of EM, as well as other outcomes at the level of the CPG and the PLWD. Additionally, potential harms from the RAS and/or the intervention will be assessed. Finally, we will generate exploratory qualitative data to improve our understanding of the mechanisms of risk and change that may result from our application of the RAS and the intervention.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY 1 out of 7 Americans develop chronic kidney disease (CKD). When kidney function continues to decline, CKD patients may develop end-stage renal disease (ESRD, or kidney failure). 2 out of 1000 adults in the U.S. develop ESRD and these patients must live on dialysis or get a kidney transplant to survive. Each year, more than $49 billion is spent to treat patients with ESRD and ESRD also greatly reduces longevity and quality of life for patients. Compared to dialysis, kidney transplant offers the best chance of survival, but few donor organs are available. Thus, there is an urgent need for innovative solutions that address the shortage of kidneys available for transplantation. Current strategies towards developing a kidney replacement therapy face significant challenges with limited success. Here we propose a radically different approach to generating a transplantable kidney: the synthetic kidney approach. The synthetic kidney is engineered from native progenitor populations to generate a structure similar to the embryonic kidney. The synthetic kidney is then transplanted into the abdomen of the recipient, where it will continue to grow, differentiate, vascularize and functionally mature in situ by following the normal process of kidney organogenesis. The proposed study is based on our solid technological innovations over the course of the past decade. Step by step, we have established systems to generate large quantities of high-quality nephron progenitor cells (NPCs) and ureteric bud progenitor cells (UPCs), the two most important building blocks for a developing kidney. We have also succeeded in using cultured NPCs and UPCs to assemble an in vitro self-organizing synthetic kidney, which shows extensive branching, nephron induction, patterning, and maturation. We are thus uniquely positioned to carry out this innovative synthetic kidney project with the goal of solving the shortage of kidneys available for transplantation. With an interdisciplinary research team covering expertise of stem cell, kidney development, kidney physiology, animal models and bioengineering, we will 1) generate a scaled-up transplantable synthetic kidney by combining stem cell technologies and bioengineering strategies; 2) evaluate the growth, differentiation, vascularization, and functional maturation of the transplanted synthetic kidney; 3) determine the therapeutic potential of the synthetic kidney in CKD and ESRD animal models. If the synthetic kidney approach can halt CKD progression and provide alternative organ transplants for ESRD patients, there will be a dramatic reduction in CKD-related complications and dialysis, thereby improving the patient care and reducing health care costs.

NIH Research Projects · FY 2025 · 2022-09

Project Summary: Technologies for monitoring chemical signaling in neuronal activities have long been desired to understand the mysterious function of the brain, and the unravel underlying mechanisms of neurological disorders such as epilepsy and Alzheimer’s disease. This project creates novel bio- orthogonal nanosensors for in-vitro and in-vivo imaging of physiological ions and small molecule neurotransmitters such as acetylcholine. Physiological ions such as K+, Na+, Cl-, and Ca2+ are key to membrane potential of the neuron, and propagation of action potentials. In-vitro and in- vivo recording of levels of these ions during neuronal communication has been focus of research for decades. The neurotransmitter acetylcholine (ACh) is involved in memory and learning with implications in Alzheimer’s disease and psychiatric disorders. Studying ACh is important for unravelling the pathophysiology of neurodegenerative and understudying the connection between the gut microbiome and brain health. The scope of work proposed in this application has potential to contribute major advances in public health through better understanding of disease pathophysiology. The immediate goal of this proposal to create bio-orthogonal fluorous nanosensors with dual functionalities. To sense ionic neurotransmitters and to release these compounds upon light stimulation. The nanoparticles will be developed using fluorous materials. Fluorous compounds (molecules with high content of fluorine atoms) are extremely non-polar and non-polarizable to the extent that they are not miscible with water and fatty substances. That is, fluorinated compounds are both hydrophobic and lipophobic. As a matter of fact, living systems are made of water and lipophilic compounds, making fluorocarbons bio-orthogonal, meaning that they do not interfere with biology. This feature allows development of stable and nontoxic nanosensors with widespread applications. The scientific questions that this proposal is answering are (i) Can we control the fluorous- aqueous interface and use partially fluorinated voltage sensitive dyes for contact-free readout of interfacial potential? (ii) Can we record chemical signaling in neuronal communication using a platform and modular fluorous nanosensor? (iii) Can we trap fluorinated metastable-photo-acids in superhydrophobic nanoparticles and use blue light for local release of ionic moieties? (iv) Can we use local release of ions to start a dialogue with nerve cells, and mimic the chemical signaling?