University Of California Los Angeles

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract The chemical synthesis of small molecules continues to be a vital area of research. Such efforts can lead to the development of new and improved methods, provide access to interesting or important scaffolds, and provide fundamental insight into reactivity and synthetic design that, in turn, can enable future research endeavors. This proposal is focused on achieving the total synthesis of the complex molecule dodecahedrane via the development of carbon–carbon bond forming reaction cascades. In Aim 1, a dimeric norbornyl scaffold will be prepared and utilized in an olefin metathesis isomerization reaction. This endeavor will provide access to a late-stage intermediate, while enabling the investigation and development of a unique variant of olefin methathesis for the assembly of molecular complexity. In Aim 2, a symmetrical penta-ene substrate will be synthesized and used in a unique and ambitious poly-ene cyclization, involving all five alkenes, to access dodecahedrane in the final synthetic step.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Society faces increasing threats from infectious diseases, from growing antimicrobial resistance to emerging novel pathogens to pervasive misinformation about vaccines. A robust response including infectious diseases physician-scientists is critical to meet these challenges, requiring diverse expertise from bench research to social science and health policy research with translational relevance to infectious diseases. Our goal is to train such individuals through the infectious diseases fellowship programs at the David Geffen School of Medicine at UCLA. This T32 program will support MD or MD-PhD trainees who are pursuing subspecialty fellowships in adult or pediatric Infectious Diseases seeking training for an independent research career. It will provide dedicated mentorship and protected time to foster a pathway to independence. Required clinical training will be front-loaded in the first year of the fellowship, leaving at least 85% protected time for two additional years of dedicated research training and career development. Each fellow will select a mentorship team from any of the broad and deep departments and research faculty at UCLA and affiliated academic institutions. A primary research mentor will directly oversee research activities, and the team will provide complementary guidance on the scientific project as well as the skills required for academic advancement including publishing, competing for grant funding, and navigating career advancement in academia. This program will include four yearly slots (two fellows each for two years of training) for MD or MD-PhD postdoctoral trainees. The key trainee outcomes include producing publications/abstracts, obtaining career development funding (NIH K award or equivalent), and eventual transitioning to an independent career as a physician-scientist.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY/ABSTRACT Radiation therapy (RT) is an integral part of the standard-of-care against glioblastoma (GBM). While the addition of RT to surgery over surgery alone significantly prolongs patient survival, all patients with GBM ultimately succumb to the disease and survival times are unacceptably low. So far, the addition of targeted or non-targeted therapies to surgery and RT have had limited success and indicate the need for novel strategies against this disease. There is ample evidence supporting the heterogeneity of GBM and the existence of a small population of glioblastoma stem cells (GSCs)), relatively resistant to RT and chemotherapy, and able to regrow the tumor. Together, treatment resistant GSCs, dispersion of cancer cells beyond the visible tumor, large areas of hypoxia and the blood-brain-barrier (BBB) add to the challenge GBM presents to cancer therapy. We have previously demonstrated that radiation reactivates stem cell programs causing cellular multipotency and plasticity, followed by the acquisition of an induced GSC state. Importantly, we showed that this process could be targeted and that preventing the induction of GSCs led to improved survival in animal models of GBM. We have now developed novel compounds that cross the BBB and prevent the radiation-induced generation of GSCs. Furthermore, our preliminary data indicate that a radiation-induced multipotent state can be utilized to allow for terminal differentiation of GBM cells. The studies proposed in this application build on these findings and take the research program into this exciting new direction to utilize this induced multipotent state for altering the radiation responses of GBM.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Heart failure secondary to acute myocardial infarction (AMI) remains the leading cause of death and disability. As a societal cost burden, heart failure care will be nearing $160 billion by 2030. Although emergent strategies to re-establish coronary blood flow have substantially improved mortality, morbidity remains high. Paradoxically, successful reperfusion may contribute to unintended microvascular injury with subsequent intramyocardial hemorrhage. In 40-50% of patients with ST-elevation myocardial infarction, intramyocardial hemorrhage can occur, leading to adverse effects on the myocardial microstructure. Significant structure-function consequences include persistent cardiac dysfunction, fatal arrhythmias, and heart failure. Of the panoply of imaging biomarkers, intramyocardial hemorrhage and the left ventricular ejection fraction (a marker of cardiac function) have the strongest predictive value for poor cardiovascular outcomes including heart failure. Cardiac magnetic resonance (CMR) offers high spatial resolution and is the preferred modality for the characterization of post-infarct myocardial tissue heterogeneity. However, current CMR approaches to detect myocardial bleeding lack sensitivity and specificity whereas methods to simultaneously detect myocardial bleeding and provide cardiac function assessment in a single fast scan are lacking. We propose to develop a fast, free-breathing, whole-heart cine CMR framework that employs a pure intravascular tracer to track and spatially demarcate acute myocardial bleeding while simultaneously providing information about cardiac function. We will rigorously test our technique in swine models and patients with hemorrhagic AMI by sampling from a large and diverse patient pool. We will integrate high-quality, multiscale, multidimensional data to construct a patient-adaptive disease model for the prediction of post-AMI adverse remodeling and heart failure. We expect our proposed approach to spur therapeutic innovations for the management of hemorrhagic transformation as a complementary pathway for mitigating downstream heart failure. Examples may include controlled reperfusion or adjunctive therapy during interventions. Successful completion of our proposed work will shift the focus from delayed detection of hemoglobin degradation metabolites to early simultaneous depiction of intramyocardial hemorrhage and quantification of cardiac function. Our findings will provide a foundation for further development of a patient- adaptive, image-guided approach based on structure-function relationships to improve heart failure outcomes.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a condition of aging, primarily afflicting adults 65 and older, and is the most common cause of dementia worldwide. The histopathological hallmarks of AD are amyloid plaques and neurofibrillary tangles (NFTs), formed by aggregation of proteins amyloid-β and tau, respectively. Studies have shown NFT formation is correlated with brain atrophy and cognitive decline. Therefore, halting tau aggregation at early stages to prevent disease progression is a potential therapeutic avenue for AD. Previous studies in our group determined the micro-electron diffraction structure of 305SVQIVY310, an aggregation-prone segment of tau. We designed a peptide-based inhibitor to target its aggregating interfaces, referred to as WIW, which has been demonstrated to halt tau aggregation in vitro and in cell models. To transport WIW across the blood-brain barrier (BBB), I have linked WIW to a peptide tag that enters the brain via receptor-mediated endocytosis by binding to low-density lipoprotein receptor-related protein 1. In Aim 1, I will probe the mechanism of action of WIW by solving a co-crystal structure of WIW with its target peptide, SVQIVY. In Aim 2, I will modify WIW to optimize its CNS delivery and plasma stability. In Aim 3, I will determine whether WIW or an optimized analog can inhibit tau aggregation in PS19 mice, a mouse model of tauopathy. Although WIW has been demonstrated to halt tau aggregation in vitro and in cell models, these proposed investigations will be the first structural characterization of the mechanism of action of WIW and the first characterization of its ability to halt tau aggregation in vivo.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Early life interactions between the microbiome and its human host are responsible for an array of immune functions and future health outcomes. It is essential to study this period during early life when the oral microbiome changes from highly dynamic infant microbiota towards a more stable adult-like microbiota. The oral microbiome plays an essential role in oral and systemic health. As such, the study of the oral microbiome may serve as a harbinger for children’s health. Understanding the establishment and early development of the oral microbiome, and its association with the early childhood caries (ECC), may provide innovative solutions for personalized, predictive, and preventive pediatric dentistry. This Mentored Research Development Award K01 study seeks to understand the dynamic changes of the oral microbiome during the first three years of life among children who were born along the United States (US)-Mexico border. These children experience a high risk of dental caries and other oral diseases; therefore, providing a complex environment for the microbiome. The study aims are: (1) to understand the establishment of newborn oral microbiome among Latinx/Hispanic children living along the US-Mexico border by using salivary samples at prenatal (from mother), 4-, 9-, 12-, 24-, 36-months to characterize their oral microbiome establishment from birth to a full set of twenty primary teeth; (2) to compare children’s oral microbiome composition between control and treatment groups (mothers received therapeutic three-month regimen of chlorhexidine mouth rinse to reduce the bacteria that causes tooth decay when their children were between the ages of four to six months) while both groups received oral health counseling; and (3) to develop statistical expertise in modeling the changes of oral microbiota and their association with ECC controlling for confounding factors. This proposed research will use three recently developed Bayesian-based ML algorithms to model the temporal patterns of the oral microbiome and evaluate their ability to predict ECC. The K01 will support Dr. Yan Wang to develop expertise in: (1) oral microbiome development in early childhood, (2) statistical analysis for oral microbiome data, and (3) biomarker discoveries using saliva samples. To achieve these training goals and research activities, Dr. Wang has assembled a highly qualified mentorship team with substantial experience in mentoring early-career investigators and with expertise in oral microbiome research among children. Dr. Wang’s primary mentors at UCLA, Dr. Grace Aldrovandi (School of Medicine) and Dr. David Wong (School of Dentistry), are highly experienced NIH-funded investigators. Co-mentor Dr. Francisco Ramos-Gomez (School of Dentistry) is a leading expert in pediatric oral health, especially among Latinx/Hispanic children. The proposed research and training aims will build strong research capacities and collaborations for Dr. Yan Wang. In addition, this work will advance Dr. Yan Wang’s research career in analytical and translational oral microbiome research.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Hepatocellular carcinoma (HCC) accounts for 80-85% of primary liver cancers, and mainly occurs in patients with liver cirrhosis or chronic hepatitis B virus (HBV) infection. Prognosis of HCC is dismal primarily due to advanced stage of disease at diagnosis. Current clinical practice guidelines recommend HCC surveillance by biannual liver ultrasound with/without serum alpha-fetoprotein (AFP) for at-risk patients to achieve the goal of detecting HCC at a curative stage. However, their accuracy remains relevantly low with sensitivity between 60- 70% at a specificity of 90%. As such, novel biomarkers for early detection of HCC are still desperately needed. Extracellular vesicles (EVs) are a heterogeneous group of lipid nanoparticles that are released by all types of cells, and even more so by tumor cells and those cells within tumor microenvironment. Tumor-associated EVs are present in circulation at relatively early stages of disease and are readily accessible across all disease stages. Since the surface proteins of tumor-associated EVs could mirror those of the parental tumor cells and those cells within tumor microenvironment, exploiting the diagnostic potential of HCC-associated EVs’ surface protein signatures as a novel biomarker for early detection of HCC holds great promise to significantly augment the ability of current diagnostic modalities. We propose an HCC EV Surface Protein Assay (SPA) to quantify subpopulations of HCC-associated EVs for detecting early-stage HCC. The proposed HCC EV SPA couples two powerful technologies: Click Chemistry- mediated EV Click Beads for isolating different subsets of HCC-associated EVs, and downstream 4-plex real- time immuno-PCR for quantification of the isolated subsets of HCC EVs. One of the major challenges emerging in the field of EV utilization for clinical use is the lack of robust and reproducible methods for the isolation of subpopulations of tumor-associated EVs. Conventional methods for isolating EVs, such as ultracentrifugation, filtration, and precipitation, are incapable of isolating subpopulation of tumor-associated EVs from total EVs. New research efforts have been devoted to exploring immunoaffinity-based capture techniques for enriching tumor- associated EVs from the plasma samples of patients with different solid tumors. However, there are challenges identified for the single antibody-mediated tumor-derived EV enriching approaches, such as limited sensitivity/specificity and a need for multiple capture antibodies to overcome the tumor heterogeneity. In order to address these concerns, our research team developed HCC EV SPA, which combines a click chemistry- mediated tumor-associated EV isolation, and downstream 4-plex real-time immuno-PCR. HCC EV SPA is capable of highly sensitive and specific quantification of 32 subpopulations of HCC EVs in patients’ plasma samples, based on the combined use of 8 different HCC-associated surface protein markers and four EV surface markers. The long-term goal of this R01 proposal is to develop, refine, and validate the HCC EV SPA for detecting early-stage HCC from at-risk liver cirrhotic patients by quantifying subpopulations of HCC EVs.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY When making a decision we can use our understanding of available action-outcome relationships to prospectively evaluate the consequences of our potential actions and choose the one that is currently most beneficial. This goal- directed strategy is, thus, quite flexible, allowing us to readily adapt our behavior when circumstances change. But we don’t always think about the consequences of our behavior. Usually this is fine. Such habits are a way for our brain to efficiently execute routine behaviors. A balance between goal-directed and habitual control allows behavior to be adaptive when needed, but efficient when appropriate. But disrupted goal-directed learning and overreliance on habit can cause inadequate consideration of consequences, inflexibility, a lower threshold for compulsivity, and disrupted decision making. This can contribute to aspects of numerous diseases, including substance use disorder (SUD), obsessive-compulsive disorder, obesity, schizophrenia, depression, anxiety, and autism. An obstacle to mechanistic understanding is the dearth of information on the neuronal circuits that support action-outcome and habit learning. Thus, our broad goal is to expose neuronal pathways regulating both forms of learning. Midbrain dopamine neurons have long been implicated in learning. Canonically, they have been thought to signal the reward prediction errors that support habit learning. But emerging evidence indicates that midbrain dopamine has a much broader role in learning than originally thought, including contributions to goal-directed learning. How can dopamine support both these, often opposing, forms of learning? Our solution is simple: dopamine’s different functions in learning are achieved through its different projections. We will reveal the pathways that enable dopamine function in both habit formation and action-outcome learning. Dopamine may mediate action-outcome and habit learning via projections to the amygdala. The basolateral amygdala (BLA) is critical for action-outcome learning. By contrast, the central nucleus of the amygdala (CeA) mediates habit learning. Midbrain dopamine projections to the BLA and CeA have long been known to exist, but little is known of their function. We will reveal a function for each pathway in instrumental learning here. Our working hypothesis is that midbrain dopamine projections to the BLA support action-outcome learning and dopamine projections to the CeA support habit learning. We will test this hypothesis in two aims using a suite of modern systems neuroscience tools including fluorescent sensor-based dopamine monitoring and cell-type and pathway- specific, bidirectional, optogenetic manipulation coupled with theory-driven behavioral assessment of action- outcome and habit learning with outcome-specific devaluation and omission contingency tests. This will provide a critical basic science foundation for our long-term goal of mechanistic understanding of the causes of disrupted decision making and maladaptive habits in pathological states.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Worldwide, more than 1 in 10 babies are born premature. Prematurity is not only the leading cause of death in children under 5 years of age, but also has serious long-term consequences for those preterm infants who survive. As such, there is a growing population of infants at risk for retinopathy of prematurity (ROP), visual impairment and neurodevelopmental impairment. Understanding how the eye is altered due to preterm birth, while accounting for postnatal factors such as oxygen use and anti-vascular endothelial growth factor (anti- VEGF) treatment, is key to determining those infants at highest risk for visual and neurodevelopmental impairment. Historically, this vulnerable population has been difficult to study, as invasive testing is both difficult to perform as well as potentially harmful. Our study will utilize non-invasive eye imaging, using ocular coherence tomography and angiography (OCT/OCTA), in preterm and term infants requiring intensive care. These images will be analyzed using established measures, to identify how eye development differs in newborn infants exposed to various systemic and local treatments such as oxygen and anti-VEGF intravitreal injections. Moreover, we will identify retinal biomarkers from OCT/OCTA imaging that predict a number of key visual and development outcomes, including treatment-requiring ROP shortly after birth and visual and neurodevelopmental impairment at 2-3 years of age. Results from these studies will broaden and deepen our understanding of retinal blood vessel development in preterm infants, the eye’s response to oxygen and anti-VEGF. In addition, developing non- invasive retinal biomarkers as predictors for visual and neurodevelopmental impairment will augment identification of at-risk infants early in the postnatal period will help to initiate individualized therapies during a period of critical neurodevelopment which will optimize their long-term outcomes.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT/SUMMARY The functional limitations and logistical challenges of using patient-derived (autologous) products for adoptive T cell therapy has prompted the exploration of a universal source of “off-the-shelf” T cells generated from self- renewing PSCs which can be readily genetically engineered to enhance function and expanded without limit. However current PSC differentiation systems are characterized by low T cell output and concurrent production of innate lymphoid cells (ILCs). Our preliminary studies suggest that the earliest stages of T cell specification and commitment seen during PSC differentiation do not fully recapitulate either normal human thymopoiesis or in vitro models that use definitive hematopoietic stem and progenitor cells (HSPC) to initiate T cell development. The goal of this proposal is to understand the cellular and molecular differences between normal and PSC-derived T cell development, with a focus on the role of the transcription factor BCL11B. T cells are generated in the thymus after notch signaling from the microenvironment triggers a series of transcriptional events that initiate the T-lineage program in HSPCs; these events first produce early thymic progenitors (ETPs) (T lineage specification) and then extinguish alternative (non-T) lineage programs in multipotent ETPs (T lineage commitment). BCL11B is a critical regulator of both of these processes. Our published and preliminary data show that, in contrast to the mouse model, BCL11B is essential for T cell specification during human thymopoiesis and initiates the expression of several T-cell genes. Moreover, when BCL11B is overexpressed in cord blood HSPCs, the T cell program is launched more rapidly and efficiently, even in the absence of notch signaling. Surprisingly little is known about how the T cell lineage is generated from PSCs. Through scRNA-Seq analysis we have identified candidate ETPs and their immediate progeny as they emerge from PSC-derived hematopoiesis. We hypothesize that the rare PSC-derived ETPs in which the T cell program is launched are functionally and transcriptionally different from ETPs in the thymus, and that these intrinsic differences are detrimental for the generation of conventional T cells from PSCs. Further, we propose that chromatin remodeling induced by BCL11B mediates both T lineage specification and the fate decisions between the conventional T cell and innate lymphoid pathways. Specifically we will: 1. Define the earliest T lineage progenitors generated during PSC differentiation; 2. Determine the epigenetic underpinnings of T-cell specification in PSC-ATOs and in primary thymopoiesis; and 3. Define how BCL11B affects conventional T and innate lineage fate choices. These studies will yield new mechanistic insights about T-cell differentiation that are critical for the development of PSC-derived T-cell immunotherapies.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Studies of the thymic microenvironment have focused largely on the central role of thymic epithelial cells (TECs) during development, disease and aging. However recent evidence suggests that the thymic vascular compartment also plays an active and complex role within the thymus beyond that of a simple conduit for hematopoietic cells. This role is particularly evident during the neonatal period when robust thymopoiesis is accompanied by a profound though transient phase of angiogenesis, followed by rapid maturation of TECs. This research proposal builds on the recent and novel finding that the vasculature of the neonatal thymus is in fact dominated by lymphatic endothelial cells (LECs) rather than blood endothelial cells (BECs), the latter becoming dominant by early adulthood. The application of classical LEC-associated cell surface markers is problematic in the thymus in which these markers are either undetectable or expressed on other cell types. The few reports describing the existence of lymphatics in the thymus have studied the adult organ in which LECs are very rare. As a result, the existence of the lymphatic compartment of the thymus has remained largely ignored and the function of LECs and the vasculature they form is unknown. The recent development of key technical advances now allows us to explore this poorly understood cellular compartment of the thymus. Reliable mouse models developed to study LECs in other organs, will now be applied for the first time to identify and manipulate LECs in the thymus. Single cell transcriptomics, including spatial gene expression and proteomics, will allow the detailed simultaneous analysis of LECs, BECs and the other compartments of the thymus during development and after in vivo perturbation of LECs. By combining these powerful technologies, we will test the hypothesis that the lymphatic vasculature of the neonatal period plays a critical role in both the remodeling and maturation of the murine thymic architecture and the differentiation and egress of T cells during the same period. The transcriptional signature of thymic LECs so developed in the mouse will allow direct comparative transcriptional studies of the LECs in the human thymus that would be impossible by standard assays. Specifically, we aim to: 1. Define the lymphatic compartment of the thymus during development at the cellular and transcriptional level 2. Determine if inhibition of lymphangiogenesis affects development of the thymic microenvironment 3. Define the role of lymphangiogenesis in T cell differentiation and egress from the developing thymus In summary we will use the unique window of the murine neonatal period to define the factors that control the growth and regression of the lymphatic endothelium in the thymus, as well as the role of LECs in the dramatic concurrent changes seen in the other thymic compartments. The ultimate goal of these studies is to uncover novel pathways that may be explored for enhancing thymic function and regeneration to improve immunity and health.

NIH Research Projects · FY 2025 · 2023-06

Modified Project Summary/Abstract Section National and global substance misuse challenges require sustainable pipelines of well-trained investigators with a wide range of perspectives and talents. Despite initiatives to increase workforce development programs, there is a deficit of comprehensive training programs to prepare junior scholars for careers in addiction research. Many scholars face multidimensional obstacles that limit their access to the early enrichment, rigorous training, effective mentoring, nourishing environments, and financial support necessary to become scientists. Many junior scholars struggle to meet the publication and funding expectations. These challenges are due in part to a deficit of clinical training opportunities in graduate school and have domino effects throughout one’s career to adversely affect employment, tenure qualifications, and other aspects of academic life. It is essential that we arrest these trends by developing optimal training programs for scholars. To that end, we propose a year-long comprehensive research education program titled “Scientific Training in Addiction Research Techniques (START).” START specifically prepares investigators to access, analyze, and disseminate data from the Adolescent Brain Cognitive Development (ABCD) study through both mentored research experiences and didactic skill-building courses. The ABCD Study is the largest long-term study of brain development and child health in the United States and uses an “open science” framework to make all data publicly available. Trainees will complete coursework to support their professional development and will complete an independent, mentored research project using ABCD data. START scholars will present and/or publish their scientific findings, and a comprehensive program evaluation will monitor trainees, mentors, and the program as a whole. The overarching goal of START is to create a pipeline of substance misuse clinical researchers who are trained in the analysis and dissemination of data from the ABCD study.

NIH Research Projects · FY 2026 · 2023-06

Abstract: The social motivation/reward theory of autism spectrum disorder (ASD) outlines that individuals with autism tend not to pursue, engage or maintain social interactions because they find social interactions less rewarding than individuals without ASD. A major hypothesis is that traditional reward circuits play an important role in social motivation and reward and may be dysfunctional in autism; yet, mechanistic proof for this intriguing hypothesis has been sparse. We have recently discovered that a significant proportion of nucleus accumbens (NAc) D1R neurons, a key node in the reward circuit, are activated during social interaction and that a subset of these neurons maintains a neural population representation of social interaction across days. We have also found that both short- and long-term NAc population representations of social interaction become destabilized in the Cntnap2 -/- mutant animals with social behavioral dysfunction. In addition, we have developed a social reward task where animals are rewarded for specific actions with social contact. VTA dopaminergic projections to NAc are activated and dopamine is released during the performance of this social reward task, again highlighting the importance of NAc for social reward. Furthermore, using Neuropixels recordings in freely behaving animals, we show reciprocal coordination of the prefrontal cortex and NAc during social interaction, suggesting that multiregional synchronization may play a key role in modulating social reward. The Golshani and Hong labs have a strong history of productive collaboration. Together, we will test the overarching hypothesis that unstable long-term NAc D1R social representations, resulting from altered inputs to NAc, drive abnormal social behaviors in models with social behavioral dysfunction. In Aim 1, using calcium imaging with wire-free miniaturized microscopes and selective labeling of D1R-MSN and D2R-MSNs in NAc, followed by decoding analysis, we will test the hypothesis that D1R-MSN neural representations of social interaction and social reward are degraded and less stable across days in the Cntnap2 and Shank3b mutant animals with social behavioral dysfunction. In Aim 2, using retroAAV-mediated labeling of specific VTA and mPFC inputs to NAc, miniaturized microscopy and multi-fiber photometry, we will test the hypothesis that the mPFC and dopaminergic VTA projections to NAc show degraded, unstable and uncoordinated social representations in the two mutant animals with abnormal social behavior. We will also probe fine-scale synchrony between mPFC and NAc using Neuropixels recordings in freely behaving animals. In Aim 3, we will use activity-dependent ChR2 labeling of NAc neurons activated by social interaction and closed-loop reactivation of these neurons to test whether stabilization of activity during social interaction can prolong and increase the probability of social interactions in both mutant animals with abnormal social behavior. These experiments will validate targets for future neuromodulation and find convergent causes for social dysfunction across different genetic models with social behavioral dysfunction.

NIH Research Projects · FY 2026 · 2023-06

Abstract We use a geroscience framework to advance Alzheimer’s disease-related dementias (ADRD) research by establishing how biological aging affects cognitive and physical decline and defining the role of sleep in these relationships. We use the interface of aging and breast cancer in older women for this purpose because of their unique bi-directional relationships. Biological aging processes increase the risk of developing cancer, so that newly diagnosed breast cancer patients may have accelerated aging prior to therapy. Cancer treatments can further accelerate aging processes. Despite possible inverse relationships between cancer and ADRD, breast cancer therapy is associated with short-term cognitive decline and this may be due to the effects of accelerated biological aging on underlying early ADRD or damage to similar systems as involved in ADRD. As we age, sleep is fundamental to repairing damage to maintain system regulation and homeostasis, including clearance of waste products seen in Alzheimer’s disease (AD). Poor sleep is common in cancer survivors and sleep has been associated with biological aging and/or physical decline and cognitive problems and increased risk for ADRD in non-cancer settings. However, there is very limited longitudinal research testing these relationships in older survivors with control groups to inform research into cognitive aging and early ADRD. Our transdisciplinary team of nationally recognized leaders in aging and cancer, biological aging, sleep, neuroscience, Alzheimer’s disease and geriatrics are uniquely placed to fill this gap. The proposed study leverages an extant cohort to efficiently conduct a novel new study of the effects of biological aging on health. Our primary research questions are: 1) Do breast cancer survivors have more biological aging before systemic treatment than concurrent frequency- matched non-cancer controls?, 2) Do systemic treatments drive further biological aging and lower cognitive and physical function in survivors beyond that seen in non-cancer controls over time? and 3) Does poor sleep lead to more aging and lower function? To address these questions, we begin with breast cancer survivors (N=368) aged 60+ and contemporaneously evaluated non-cancer controls (N=354) frequency-matched to survivors on age, racial group, education level and recruitment site. These women have rich pre-treatment/enrollment neurocognitive, physical and sleep data and blood obtained and banked to specifically test aging markers. We will conduct follow-up out to 48-months and perform assays of several hallmarks of biological aging (epigenetic age, DNA damage, cellular senescence, SASP, and leukocyte telomere length). While accelerated biological aging has been postulated to explain cognitive and functional decline among people with and without cancer, this rigorous project will be the first to our knowledge to definitively evaluate this theory. The population and questions proposed are significant and will only become more important with the aging of the population. This clinical translational project addresses key NIA priority areas and will provide important data to inform future ADRD studies and interventions to improve health and resilience of our aging population.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Increased intraindividual variability (IIV) in task performance is a robust symptom of attention deficit/hyperactivity disorder (ADHD). However, while theoretical frameworks suggest inefficient neural processing as a potential cause of IIV, evidence is limited because IIV is typically measured using summary statistics of behavioral outcomes, such as response time variability (RTV). Not only are such measures limited by sparse temporal sampling, but they also aggregate time, ignoring underlying dynamics, and limiting the specificity and thus the translational value of IIV in ADHD. In this project, we propose that neurophysiological measures of neural processing efficiency can be continuously derived from the oscillatory and signal properties of EEG, to track the contribution of top-down signals (low-frequency power), network efficiency (low-frequency oscillatory small-world index) and network interaction stability (signal complexity). These measures can be used as a continuous, within- subject neurophysiological index of neural processing efficiency, and one that bridges summary statistics derived from reaction times and underlying network dynamics. To test this idea, we revisit three existing datasets (n=514) that include children and adults, with and without ADHD, and that contain EEG and concurrent EEG and fMRI, collected during sustained attention tasks. In each dataset, we compute continuous measures of neural efficiency based on EEG signals to, in Aim 1, differentiate between alternate neurophysiological profiles and mechanisms of IIV in ADHD and test if these predict performance outcomes and individual differences in symptoms. In Aim 2, we additionally test if neural efficiency measures predict, within-subject, aberrant interactions between core attention networks and those previously associated with ADHD – namely fronto-parietal, default-mode, ventral/dorsal attention, visual and fronto-striatal. The goal of the present work is to establish the neurophysiological basis of IIV in ADHD, and thus speak to putative clinical targets of IIV, differentiate between current theories of IIV, as well as to validate EEG-based neural efficiency as an effective intermediate indicator of underlying network dynamics.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Cell reprogramming represents a major advancement in biology, and has wide applications in regenerative medicine, disease modeling and drug screening. Somatic cells such as fibroblasts can be directly converted into induced neuronal (iN) cells via the forced expression of three transcription factors: Ascl1, Brn2 and Myt1l (BAM). However, a major challenge of cell reprogramming, especially iN reprogramming, is the low reprogramming efficiency, which has limited the translation of this technology for biomedical applications. Biophysical factors from the microenvironment have been shown to regulate many aspects of cell functions such as cell growth, migration and differentiation. Recently, we have shown that mechanical deformation of cell nucleus through microfluidic channels can promote open chromatin structure and enhance cell reprogramming, yet the underlying mechanisms are not well understood. Based on our recent findings, we hypothesize that a mechanopriming process such as mechanical squeezing of cell nucleus can induce NL reorganization and a permissive chromatin state to facilitate the activation of neuronal genes in the heterochromatin of fibroblasts, which promotes iN reprogramming and CRISPR- mediated gene editing/activation. To test our hypothesis, we propose three Specific Aims: (1) To investigate how nuclear deformation modulates the epigenetic state to enhance iN reprogramming; (2) To determine the role of nuclear lamina in mediating nuclear deformation-induced LAD dissociation, epigenetic changes and iN reprogramming; (3) To investigate the enhancement of CRISPR-mediated neuronal gene activation by mechanical squeezing. We have assembled a multidisciplinary team with expertise on cell engineering, microdevice fabrication, high-throughput genomic and epigenomic analysis, neuroscience, biosensors and CRISPR gene editing to work together and investigate the mechanical regulation of epigenetic state and cell reprogramming. We propose to optimize a high-throughput microfluidic device, further investigate the causative mechanisms and profile the genome-wide site-specific epigenetic changes induced by nuclear deformation, which can provide a rational basis for the design of site-specific gene editing for cell engineering. Accomplishment of this project will advance our understanding of how biophysical factors regulate cell reprogramming and the epigenetic state, and unravel new mechanisms of cell fate determination, which will have wide applications in gene editing, cell and tissue engineering, disease modeling and drug discovery.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Vascular calcification affects ~60% of adults over 60 years of age and is frequently seen in patients with atherosclerosis. Atherosclerotic calcification is an independent risk factor for cardiovascular morbidity and mortality. Despite decades of research, no medical therapy has been convincingly established to prevent or reverse vascular calcification. The intimal (luminal) endothelium forms the innermost layer of the vasculature. Under disease conditions, the intimal endothelial cells (ECs) are stimulated to transform into osteoprogenitor cells through endothelial-mesenchymal transitions and contribute to vascular calcification. ECs in the adventitia contribute to neoangiogenesis in vascular disease. However, little is known about whether adventitial ECs are associated with vascular calcification. During bone development, a subset of ECs defined by high expression of the EC marker CD31 and the glycoprotein Endomucin (Emcn) are critical for bone formation. Our preliminary study using Matrix Gla Protein (Mgp) knockout (Mgp-/-) mice as a model of vascular calcification revealed two distinct populations of ECs in the calcified aortas, the intimal ECs (i-ECs) defined by CD31+Emcn- and the adventitial ECs (a-ECs) characterized by CD31+Emcn+. These two EC subtypes with distinct Emcn expression levels were also detected in human calcified arteries. Bulk RNA sequencing studies showed i-ECs were enriched in stem cell and osteogenic markers and a-ECs exhibited upregulated Notch expression. Endothelial deletion of the Notch1 gene reduced vascular calcification and increased the survival of the Mgp-/- mice. In this proposal, we hypothesize that a-ECs support the biomineralization in the vascular calcification in animal models and human atherosclerotic lesions, and are closely regulated by Notch signaling. In Aim 1, we will define the molecular signature of the a-EC (CD31+Emcn+) population in vascular calcification using single cell RNA sequencing. We will delineate the developmental trajectories of a-ECs in vascular calcification with correlation to the extent and severity of calcification. In Aim 2, we will determine the contribution of endothelial subtypes (a- ECs and i-ECs) and Notch signaling in atherosclerotic calcification. We will investigate the effect of endothelial- specific deletion of the Notch1 receptor on EC subtypes, calcification and transcriptional profiles in atherosclerotic lesions. The proposed studies will provide novel insight into the fundamental mechanisms of endothelial cell biology in atherosclerotic calcification and may identify potential gene targets for selective therapeutic modulation. Together with the mentored career development plan, the completion of the projects will serve as a foundation to facilitate the candidate to transition into a successful and independent physician scientist in cardiovascular research.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract Macrophages are first responder immune cells present in every tissue. Their responses are mediated by signaling pathways that activate hundreds of immune response genes. Two functional hallmarks characterize the deployment of all macrophage functions: (1) Stimulus-Response Specificity. Immune responses are powerful, and often detrimental for the host. Hence, they must be deployed on an “only-as-needed” basis. However, it remains unknown how specific macrophage responses are, and what mechanisms control Response Specificity. Quantifying the specificity of responses requires single-cell measurements of signaling or gene expression trajectories, and the development of analysis methods to compare distributions, quantify information content, precision of classification and confusion. (2) Context-Dependent Functional States. Macrophage functions adapt to the tissue microenvironment via the cytokine milieu characteristic of the tissue and the prior history of immune responses or pathogen exposure. As monocytes circulate through the body passing through tissues, they are potential biosensors of injury or infection. While prior studies have characterized these states via steady-state molecular profiling of chromatin or transcriptome, single-cell stimulus response data may be more informative of actual functional states. Considering these functional hallmarks of macrophages lead to two hypotheses that this proposal addresses: 1) Quantitative measurements of single cell stimulus responses reveal that macrophage Response Specificity is modulated by physiological and pathological context by affecting the distributions that characterize heterogeneous responses in the population. 2) Quantifying the Response Specificity of individual macrophages allows for a characterization of their Functional States, that is distinct from single-cell transcriptomic profiling. We will address these hypotheses using experimental, math modeling, and computational analysis iteratively. In Aim 1, we will determine which molecular mechanisms that drive cell-to-cell heterogeneity and why the specificity of NFκB stimulus-responses is altered by cytokine polarization states. In Aim 2, we will use a novel model-aided data integration approach to quantify for the first time the Response Specificity of individual macrophages. This will allow us to map the landscape of macrophage states based on stimulus-responses and parameters, and compare it to maps of traditional steady-state scRNA-seq data. In Aim 3, we will study how the stimulus-specificity of immune gene expression responses in single-cells are affected by polarization. With a novel model-aided approach, we are able to reconstruct dynamic trajectories and determine whether Response Specificity is better assessed by mRNA abundances or dynamical features. After finetuning these approaches on in vitro polarized macrophages, we will apply them to macrophages from mouse models of ill-health. Insights may guide future translational studies to assess Innate immune Health.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a gatekeeper that balances anabolic and catabolic processes through sensing nutrients. Dysregulations of this pathway lead to debilitating diseases such as cancer. One of the most abundant essential amino acids in cancer cells is leucine. However, the process of leucine sensing and transport to activate mTORC1 pathway is not clear. The lysosomal associated transmembrane protein (LAPTM4b) is an oncoprotein that is involved in localizing leucine transporter to the lysosomal surface to activate mTORC1 pathway. LAPTM4b also functions as a ceramide transporter and compartmentalizes ceramide to help cancer cells evade apoptosis. Currently, there is no high-resolution structure of this protein to elucidate its mechanism of ceramide transport and complex formation with the leucine transporter. It is paramount to explore the structure and functions of LAPTM4b in detail since more than ~70% of cancers have LAPTM4b upregulation and LAPTM4b has been linked to poor prognosis. This proposal focuses on understanding the structure and functions of LAPTM4b and its protein complexes to lay the foundation on mTORC1 activation through leucine. The structures will be solved using state-of-the-art cryogenic electron microscopy techniques including Microcrystal Electron Diffraction (MicroED) and Single Particle Analysis (SPA). In Aim 1, the mechanism of ceramide binding in LAPTM4b will be elucidated by high resolution structures of LAPTM4b in complex with ceramide using MicroED. The structures solved in this aim will provide insights on the effect of ceramide to the global confirmation of LAPTM4b and provide basis on structure guided drug development. Functional studies of LAPTM4b in complex with the leucine transporter in liposome assays will be investigated in Aim 2. Kinetics of leucine transporter will be evaluated in vitro to provide insights into how LAPTM4b, ceramide, and ceramide analogues affect leucine transport. In Aim 3, the structure of the protein complex of LAPTM4b and the leucine transporter will be solved to understand their interactions and guide drug development to disrupt this complex for mTORC1 inactivation. The applicant’s career goal is to use tools in structural biology combined with biochemistry experiments to answer scientific questions on disease-related proteins. Since there is an urgent public health need to alleviate cancer, the applicant aims to study challenging membrane proteins involved in the mTORC1 pathway. The structure- function relationship of these proteins will guide future research on designing effective cancer therapeutics to disrupt this pathway. The proposed research is an integral step of the applicant’s career path. The applicant will receive intensive training and constructive guidance under a leading researcher in the structural biology field.

NIH Research Projects · FY 2024 · 2023-06

PROJECT SUMMARY/ABSTRACT Cancer treatment accelerates aging. In people over 65, accelerated aging may have far greater consequences than in younger adults. One of the most important consequences of accelerated aging is frailty. Frailty is linked to loss of independence, falls, and death. Older cancer survivors develop frailty 2- to 4-fold more frequently and at an earlier age than age-matched controls. Mechanisms underlying frailty are just starting to be understood. One key aging mechanism driving frailty is cellular senescence – a state of terminal growth arrest. Senescence is the result of both natural aging and cancer treatment; radiation and chemotherapy both generate senescent cells (Sncs). In pilot biomarker studies, I observed that older survivors treated with chemotherapy (vs. no chemotherapy) have increased T-cell expression of P16INK4a (p16). p16 is an established marker of Sncs. I also observed that the percentage of T-cells expressing p16 correlates with clinical frailty. My findings are consistent with published studies linking p16 and frailty in childhood cancer survivors. Together, these data provide the premise for testing clinical interventions to target and eliminate Sncs in older survivors. Recently, drugs have been discovered that selectively eliminate Sncs – senolytics. One such senolytic is fisetin, a natural product flavonoid found in strawberries and other fruits. Because the amount of fisetin varies considerably in food, it is not possible to achieve sufficient levels for eliminating Sncs in a natural diet; however, fisetin is available as a dietary supplement. In preclinical models, fisetin reduces Sncs, inflammation, and frailty. As such, fisetin is now in >10 efficacy trials to alleviate age-related conditions in frail older adults and, so far, has had a favorable safety profile. No trial to date has tested fisetin in frail older cancer survivors. Here, I propose a randomized placebo- controlled trial with multi-modality biomarkers to test the preliminary efficacy, safety, and tolerability of fisetin to improve frailty and reduce Snc burden in frail older cancer survivors. Guided by a firm mechanistic rationale and preliminary data, my overall hypothesis is that fisetin is efficacious (improves frailty), safe, and tolerable in frail older survivors. To test my hypothesis, I will randomize cancer survivors age >65 with diminished gait speed (<0.8 m/s) to a 60-day course of fisetin vs. placebo. The primary endpoint is change in gait speed from day 1 to day 60. Secondary endpoints include changes in p16, inflammatory biomarkers, and frailty measures (Fried’s criteria, frailty index, grip strength). I also will assess safety and tolerability of fisetin and explore longer- term sustainability of efficacy, as measured by gait speed at 150 days. Promising results from this study will provide preliminary evidence for a large multi-center clinical trial (R01) to establish the efficacy of fisetin in older survivors. Additionally, by completing this study, I will fill a gap in my prior training in cancer clinical trials with training in geroscience research. This study will form the basis of my independent research program to develop geroprotective interventions to ensure that older cancer survivors live healthy lives well after cancer treatment.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Chamber specific postnatal growth is the cornerstone of postnatal heart development, however, the underlying molecular mechanisms are almost entirely unexplored. In preliminary studies, we analyzed and compared key intracellular signaling activities between LV and RV in neonatal mouse hearts and discovered that p38 MAP kinase activation displayed a unique chamber-specific and developmental stage specific pattern in RV during neonatal to adolescent transition. Strikingly, cardiomyocyte specific inactivation of p38 activity in the developing mouse heart led to lethal cardiomyopathy associated with RV specific induction of myocyte proliferation and hypertrophy in neonatal mouse heart while the LV was minimally affected. Furthermore, IRE1α- Xbp1 axis is essential downstream signaling in p38 mediated regulation of cardiomyocyte proliferation. Taken together, these findings reveal for the first time that two previously established pathogenic stress-related signaling pathways, p38 MAPK and IRE1α/Xbp1, are also indispensable players in normal chamber specific development in postnatal heart during fetal to adult transition. In this proposal, we aim to explore this novel finding by accomplishing the following three specific aims. Aim 1): Determine the functional and molecular impact of IRE1α/Xbp1 axis in chamber-specific postnatal heart development using novel mouse models with targeted manipulation of IRE1α/Xbp1 activity. 2) Establish the specific contribution IRE1α/Xbp1 axis in p38 mediate regulation of chamber-specific growth during postnatal heart development. 3) Uncover downstream targets underlying chamber specific regulation of p38/IRE1α/Xbp1 signaling in postnatal heart. These studies will establish for the first time an intracellular signaling network for chamber-specific postnatal development in neonatal heart and fill a critical gap in our current knowledge in this important area of cardiac biology.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Memory of a single event can last a lifetime. Certain experiences can guide behavior minutes, months, and years after the event has transpired. For this to happen, the brain must encode the relevant information from the event, retain it for an indeterminate amount of time, then retrieve that memory when given appropriate cues. Previous work has shown that retrieval of a fearful experience is behaviorally similar across recent and remote retrieval times of a memory, yet the neural circuits mediating that memory are importantly different across the memory timeline. In short, behavior remains stable while neural activity is dynamic. Evidence suggests that memories are reorganized across time – increasingly involve a distributed cortical network. Although we have identified several independent cortical regions that participate in remote memory, our understanding of how they interact at the and the nature of these changes is lacking My preliminary data indicates that an uninvestigated medial prefrontal cortex (mPFC) projection coordinates remote but not recent memory via projections to association cortex. Further exploration is needed to determine the specific activity patterns in these circuits during memory retrieval over time. These discoveries would reveal fundamental principles by which the brain organizes and retrieves salient experiences across time. The overall goal of this proposal is to investigate the cell-type specific changes that occur in mPFC ensembles as fear memories reorganize from recent to remote and to determine the nature of this reorganization. Based on published work from my mentor and my preliminary findings, my project will focus on the connection between mPFC and auditory association areas. To address these questions, I will use circuit-specific, optogenetic approaches to establish necessity and sufficiency of a mPFC–cortical association circuit for remote memory. This will address the functional role of these mPFC projection neurons. I will then use freely-moving calcium imaging to record the activity of these cells at both recent and remote memory timepoints in order to examine how these mPFC neurons encode and behavior and task variables across memory timepoints. Finally, with a combined viral-genetic approach, I will label activity-dependent ensembles while employing whole-brain circuit mapping to understand the anatomical relationship between recent and remote ensembles and their downstream connections. Together, these aims will reveal novel insights into the time-dependent changes in memory organization and enhance our understanding of mPFC function in organizing cortical networks, potentially identifying improved circuits to target for treatment of fear disorders (e.g. Post Traumatic Stress Disorder).

NIH Research Projects · FY 2026 · 2023-06

PROJECT ABSTRACT The proposed study (PETAL: Promoting Early intervention Timing and Attention to Language) aims to determine the timing of a parent mediated intervention among infants with Increased Likelihood for Autism (ILA) (at risk for autism by virtue of having an older sibling with autism) on communication and language outcomes at 24 months. ILA infants are likely to experience delays in language with ~ 40% at risk for language delay and/or later diagnosis of ASD. However, interventions for ILA infants remain rare and none of the current interventions explicitly focus on language or include many infants from lower socioeconomic circumstances who may be at even greater risk for language delays. Although enthusiasm is high for very early interventions due to known brain plasticity in the first years of life, we do not know WHEN or with WHAT MEASURES to determine if an intervention is appropriate for ILA infants who are not yet showing signs of autism or delay. The overarching goal of the proposed study is to determine the optimal timing of very early intervention for ILA infants (starting at 9, 12 or 15 months) that explicitly targets communication and language. We will use a battery of brain- and behavioral-based markers to identify the combination of change in language, behaviors and brain measures that predict expressive language outcomes at 24 months. 140 infants beginning at 6 months of age will participate at two sites, Los Angeles and Boston areas with many from traditionally marginalized and minoritized families. All parents will receive infant developmental monitoring beginning at 6 months, and then using a four-phase, sequential multiple assignment randomized trial design, parents will receive augmentation with a specialized language coaching intervention at 9, 12 or 15 months; all infant parent dyads receive coaching by 15 months. A diverse sample of dyads will be recruited; assessments will occur at home at 6-,9-,12-,15-, 18- and 24-months using brain-based EEG measures, social- communication, and language measures. Intervention support will be provided by clinicians remotely. The study addresses key questions of whether earlier intervention is better on primary language and secondary outcomes of social communication behaviors that support language development (e.g., joint engagement, vocalizations, words, object play) and which measures will inform the ideal transition to intervention on language outcomes at 24 months. Moreover, using LENA to capture the home language environment, adult word count and conversational turns will be examined for mediation on language outcome. Results of this study have potential for determining when (9 vs 12 vs 15), and based on which measures (brain, language, or their combination), to augment parental support with a specialized parent-mediated coaching intervention.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY The overarching goal of the research presented in this application is to dissect the genetic, epigenetic, and metabolic programs that encode and transmit a memory of environmental exposure for several generations. In that context, we aim to understand how environmental influences alter the reproductive program of organisms in a transgenerational fashion and what makes the germline a particularly important and sensitive target of exposures. In mammals, in utero ethanol exposure is associated with an array of well-characterized morphological, neurobehavioral, and reproductive issues. However, there is mounting evidence in a variety of model organisms that some adverse reproductive and neurological features are also detectable in the third generation following exposure indicating a transgenerational effect. Alcohol also has a clear epigenetic impact and directly contributes to the modification of the epigenome. Nevertheless, despite the fact that heritable effects of alcohol imply an alteration of the information contained in germ cells, it is unclear how the memory of ethanol exposure is initiated in the germline and then transmitted to future generations. Here, we combine the tractability and conservation of the model system C. elegans with state-of-the-art epigenomic analyses, classical genetic, and cytological approaches to shed light on the mechanisms of memory of ethanol exposure. Our preliminary data shows that ethanol exposure causes strong transgenerational reproductive and behavioral impairments. We also show that, in line with recent mammalian studies, ethanol causes an increase in histone acetylation. Thus, we hypothesize that ethanol exposure causes transgenerational perturbations of germline function by altering the germline epigenome, specifically histone acetylation. Our aims are designed to address the molecular, metabolic and epigenetic requirements for these transgenerational impacts of ethanol. In aim 1, we will build on our preliminary reproduction data to interrogate through classical genetics tools meiotic pathways at the root of ethanol's trans-generational increase in germline apoptosis and embryonic lethality. In aim 2, we will examine via mass spectrometry the modulation in 80 different histone marks stemming from direct ethanol exposure and test whether increased histone acetylation is a required event for the initiation ethanol's transgenerational reproductive effects. Finally, in aim 3, we will test the epigenetic requirement for the transgenerational transmission of ethanol's effects and also map by CUT&RUN the changes in the epigenetic landscape of histone modifications in the germline. At the completion of the aims, we will have identified the molecular underpinnings for the initiation and transmission of ethanol transgenerational reproductive outcomes.

NIH Research Projects · FY 2026 · 2023-05

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease with inflammation, demyelination, axonal damage, glial activation and synaptic loss. There are relapses and permanent disabilities. Despite success of treatments targeting cells in the immune system, there is an unmet need for treatments targeting cells in central nervous system (CNS) to repair disabilities. Four observations provide rationale for a new approach to neurodegeneration in MS: 1) MS patients are heterogenous in their disabilities, and distinct disabilities (walking, vision, cognition, coordination) are served by different CNS regions, 2) Even in healthy brain, a given CNS cell type differs in gene expression from one brain region to another, 3) Being female versus male impacts disability worsening, and 4) Aging aligns with disability progression. Here, we will use a cell-specific, region-specific, and sex-specific approach to discover optimal treatment targets for distinct disabilities in MS women and men. Bedside to Bench to Bedside in MS: Clinical observations of sex differences are investigated at the preclinical level then translated back to the clinic as trials designed for each sex. Preclinical use of female and male mice with experimental autoimmune encephalomyelitis (EAE) entails in vivo MRI for region-specific atrophy, neuropathology of each region, RNA-sequencing of distinct CNS cells in each region, immunohistochemistry validation of top genes in highly differentially expressed pathways, cell-specific conditional knockout (CKO) of target genes to reverse phenotype, and knockdown of target genes with pharmacologic treatment to reverse phenotype. The effect of genetic (CKO vs WT) and/or pharmacologic (treatment vs placebo) interventions on reversal of gene expression is determined in each sex. Human MS data guide preclinical research at three checkpoints: i) MRI in females and males with MS revealing sex differences in substructure atrophy prioritize regions in EAE with atrophy, ii) Single nuclei RNA-seq analyses in females and males with MS revealing gene pathways of interest prioritize gene pathways in EAE, iii) immunohistochemistry in females and males using MS postmortem tissues validate immunohistochemistry in EAE. Substitution of use of female versus male mice (as above) with use of gonadectomized versus gonadally intact mice will reveal activational effects of sex hormones. Use of Four Core Genotype mice will reveal sex chromosome effects versus developmental hormone effects. Use of young versus old mice will reveal the effect of aging. This R35 proposal will: 1) Extend our cell-specific and region-specific transcriptomics in astrocytes and oligodendrocytes to microglia and neurons, with cell:cell interactions revealed in mice double-labelled to show gene expression changes in two distinct cell types in the same region in the same mouse, and 2) Determine if there are effects of sex and/or age on the most differentially expressed cell-specific and region-specific pathways. In summary, this R35 proposal takes our research to the next level: Identifying sex by age interactions in cell-specific and region-specific transcriptomics, neuropathology, and substructure atrophy on MRI.