University Of California Los Angeles

NIH Research Projects · FY 2025 · 2021-08

Project Summary Mitochondria control cell metabolism by converting nutrients into an electrochemical gradient of protons (H+) across the inner mitochondrial membrane (IMM) to generate ATP, the currency of the cell, and heat (called mitochondrial thermogenesis). A precise balance in the distribution of H+ between the two forms of energy production, ATP and heat, defines the metabolic homeostasis of the cell. Brown fat and beige fat mitochondria specialize in the production of heat via the uncoupling protein 1 (UCP1). However, even in other tissues, mitochondrial thermogenesis accounts for 25% of total mitochondrial energy production and can therefore have a considerable impact on the physiology of the entire body. Mitochondrial thermogenesis is not only essential for maintaining core body temperature, it is also the process by which excess calories are burned to prevent diet- induced obesity. In addition, it reduces the production of reactive oxygen species (ROS) by the mitochondria to protect cells from oxidative damage. In addition, chemical uncouplers such as 2,4-dinitrophenol (DNP), which are believed to increase H+ leak independently of proteins, are the most effective anti-obesity drugs to date. Thus, mitochondrial thermogenesis is a powerful regulator of cellular metabolism, and a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction, including metabolic syndrome and age-related disorders. Unfortunately, the precise molecular mechanisms that control the acute activation of thermogenesis in the mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for direct measurement of H+ currents across the IMM. The development of a methodology based on the patch-clamp technique allows for the first time the direct study of H+ leak through the IMM of each tissue and the first biophysical characterization of mitochondrial transporters, such as UCP1 and the ADP/ATP transporter (AAC), which are the mediators of this H+ leak. This unique approach now provides an unprecedented high- resolution direct functional analysis of 1) the mitochondrial ion channels and transporters responsible for mitochondrial thermogenesis and 2) the mechanisms of action of chemical uncouplers such as DNP. Using the new mitochondrial patch-clamp assay combined with modern cellular and molecular techniques, this research project will provide new insights into the mechanisms that control the thermogenic capacity of the mitochondria and how they can be targeted for therapeutic purposes.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT Integrating Volumetric Light-Field with Computational Fluid Dynamics to Study Myocardial Trabeculation and Function Non-compaction cardiomyopathy (NCC) is a disease of endomyocardial trabeculation or known as spongy myocardium. NCC carries a high risk of malignant arrhythmias, thromboembolic events, and ventricular dysfunction in association with congenital heart defects or skeletal myopathy. Studies have linked left ventricular non-compaction with autosomal dominant inherited disorders, and mutations in Notch pathways are implicated in defective trabeculation and ventricular NCC. Biomechanical force is intimately connected with mechanotransduction and cardiac morphogenesis. During development, the myocardium differentiates into an outer compact zone and an inner trabeculated zone. Notch receptor- ligand interaction induces EphrinB2-Nrg-ErbB2 signaling to initiate trabecular formation. Our in silico analysis (Alison Marsden, Stanford) revealed elevated oscillatory shear index (OSI) in trabecular ridges, leading to increased viscous dissipation, which was associated with changes in ventricular contractile function and remodeling. However, uncoupling myocardial contraction from intracardiac flow dynamics to elucidate Notch-mediated trabecular organization and subsequent associated changes in local hemodynamics remains an unmet biomechanical challenge. In this context, we hypothesize that hemodynamic shear and myocardial contractile forces coordinate trabecular organization needed to preserve the ventricular structure and contractile efficiency. In combination of laser light-sheet and light- field for super resolution and volumetric imaging, we simultaneously captured myocardial contraction and intracardiac flow dynamics. In collaboration with Stanford Cardiac Mechanics, we integrated fluid structure interaction (FSI) with super resolution imaging to demonstrate 4-D endocardial shear stress in the trabecular ridges and grooves as possible developmental modulator. To test our hypothesis, we propose three specific aims. In Aim 1, we will demonstrate that intracardiac shear stress activates endocardial Delta-Notch signaling to promote trabecular ridge formation. In Aim 2, we will demonstrate that ventricular contraction activates myocardial Jagged-Notch signaling to organize trabecular groove formation. In Aim 3, we will demonstrate that the combination of trabecular ridge and groove formation leads to optimal local hemodynamics and ventricular energetics. The integration of advanced imaging, fluid structure interaction, and zebrafish genetics is uniquely suitable to unveil trabecular organization in relation to kinetic energy dissipation. Our multi-disciplinary approach provides new biomechanical insights into non-compaction cardiomyopathy with pathophysiological significance to ventricular remodeling and function.

NIH Research Projects · FY 2026 · 2021-08

PROJECT SUMMARY/ABSTRACT Respiratory failure after spinal cord injury (SCI) impairs the health of the injured patients, and respiratory failure is the leading cause of death in patients with SCI. Treatment of respiratory failure consists of mechanical ventilation, in which a mechanical pump is used to facilitate air exchange with the lungs. Mechanical ventilation is invasive, costly, limiting, and carries with it a high risk of complications and death. Mechanical ventilation provides an unvarying pattern of ventilation that is not responsive to physiological demands; it does not recapitulate normal breathing. Normal breathing is a complex behavior under both voluntary and involuntary neural control; it is responsive (in milliseconds or less) to the physiological state of the patient. Restoration of fully integrated, naturalistic breathing would represent a significant advance in the treatment of respiratory failure following SCI. The main hurdle to accessing the neural network for breathing for therapeutic purposes is that the neural mechanisms controlling respiration reside deep in the brainstem, which is dangerous to access surgically. Recently, we elucidated a novel breathing pathway in the spinal cord that can be modulated by electrical stimulation of the cervical spine, an area that is surgically accessible. We have compiled significant data that stimulating the cervical spine can restore or augment breathing. Clinically approved epidural spinal cord stimulators exist to treat pain, and these stimulators can also be used to stimulate the cervical spine to restore respiratory function. The main objective of this project is to provide proof of the concept that cervical epidural stimulation can improve respiratory function in ventilator-dependent patients with SCI and define the stimulation parameters that most effectively restore more normal breathing. The deliverables for this 5-year project include establishing the safety and feasibility of epidural stimulation for respiratory rehabilitation in SCI and providing an algorithm to select the optimum stimulation variables to augment respiratory activity in each patient (e.g., stimulation site, dose, and timing). If successful, we anticipate using epidural stimulation to partially or completely wean each patient with SCI off mechanical ventilation, which would have immediate benefits — increased independence, improved quality of life, and decreased costs and risks associated with mechanical ventilation. Conventional thinking is that once the spinal cord is injured, little or no functional recovery is possible. This dire sense of irrreversibility is at odds with our research in spinal cord neuromodulation, which has shown that substantial recovery of voluntary hand and upper extremity function can result from epidural spinal cord stimulation. A similar neurmodulatory strategy may be used to augment or restore respiratory function in patients with SCI. If successful, this neuromodulatory strategy to restore respiratory function may usher in a new era of respiratory neurorehabiltitation for SCI, and potentially other neurological disorders, and transform our understanding of neural circuits governing respiration and the plasticity of injured states of the spine.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Synaptic connections determine how neural circuits process information. Understanding how the strength and specificity of these connections is established is a central challenge in neurobiology. In many parts of the developing mammalian brain, stereotyped patterns of stimulus-independent neuronal activity precede sensory- driven responses. Whether and how this developmental activity guides synapse assembly at the level of defined cell types and circuits is not well-understood. Here, much of the challenge is due to the size and complexity of the mammalian brain itself: Even in the retina, where developmental activity is best characterized, the technical barriers to pursuing synapse level questions are significant. We recently discovered analogous patterned, stimulus-independent neural activity (PSINA, pronounced `see-nah') in the developing Drosophila brain. With the ever-growing knowledge of its neurobiology, spanning the connectome to behavior, the fly is unmatched in its promise for cell type- and circuit- level studies. PSINA is globally coordinated with brain-wide, periodic active and silent phases. In the visual system, each cell type participates in PSINA with distinct and stereotyped spatio-temporal patterns of activity. These developmental activity patterns are correlated between pairs of neurons known to be synaptic partners in the adult. Our long term goal is to test the hypothesis that the cell-type-specific activity patterns of PSINA refine the emerging connectome to generate wild-type synaptic strength and specificity. Here, we will work toward this goal by leveraging a new genetic handle on PSINA: Trpγ, a cation channel with a weak preference for Ca2+, is required for wild-type PSINA. In trpγ mutants, the amplitude of activity is reduced by >50% across the whole brain, and cell-type-specific activity patterns and synapse numbers are altered. Trpγ is expressed in <1.5% of the neurons in the brain. Notably, silencing only these neurons by overexpressing a hyperpolarizing channel attenuates PSINA by >90%. This indicates that some or all of this diverse group of ~1,700 Trpγ-expressing (i.e. Trpγ+) neurons are critical to coordinating PSINA in the developing brain. We hypothesize that Trpγ+ neurons are the source of the cell-type-specific activity patterns. In Aim 1, we will identify individual Trpγ+ neurons that innervate the visual system and test if these neurons specify the activity patterns of their post-synaptic partners. Determining the origin of these patterns will allow us to ask whether they are the cause or consequence of synapse and circuit maturation. In Aim 2, we will focus on a specific neuron that is part of the well-studied motion detection circuit and ask if the strength of its post-synaptic contacts are altered in trpγ mutants. Identifying the cellular origin of the activity patterns and understanding the effect of PSINA on synaptic development will allow us to reversibly silence, alter, or possibly re-program PSINA. With this knowledge, we will be able to define the contribution of developmental activity to the structural and functional maturation of synapses and circuits, to sensory processing, to learning, memory, and behavior.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Perturbations in metabolic pathways form the epicenter of some of the most devasting threats to mankind, including cardiovascular disease, obesity and NASH. Proper maintenance of metabolic homeostasis requires precise and synchronized control of gene regulation. Recently, the discovery that thousands of mammalian RNAs undergo chemical modifications that powerfully impacts transcript dynamics expands our understanding of gene regulatory mechanisms. N6-methyladenosine methylation (m6A) is the most common internal RNA modification. Multiple lines of evidence suggest that m6A plays a critical role in organismal biology, including stem cell renewal, however, the impact of chemical modifications on RNA in metabolic control is less well understood. The objective of this proposal is to define the physiologic contribution and mechanisms of RNA modifications in metabolism. Capitalizing on our preliminary studies showing that the hepatic m6A landscape is altered in response to diet and strongly enriches lipogenic RNAs, we hypothesize that dynamic RNA modifications are essential for tight regulation of hepatic lipid metabolism. Reinforcing this premise, our studies show that liver-specific knockout of m6A installing machinery leads to increased lipogenesis and alterations in hepatic lipid composition. In aim1, we investigate the function of m6A in hepatic lipid metabolism and fatty liver disease as well as explore opportunities for RNA modification based therapeutic strategies in metabolic disease. In aim2, we define how m6A modifications impact lipogenesis and decipher the hierarchical and cooperative relationship between m6A modifying enzymes and canonical metabolic transcriptional modulators. Our proposed studies are expected to shed fundamental insight into novel mechanisms involved in metabolic control and a model by which RNA modifications can impact health and disease states. In summary, our studies identify a new pathway for lipid degradation and in this application, we propose a series of molecular, cell biological, and animal studies to extend our preliminary observations and test out hypothesis.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Homologous recombination (HR) is important for repairing DNA double strand breaks (DSB), and thus is an essential process in embryonic development, meiosis and suppressing tumorigenesis. HR can also be a double- edged sword: unrepaired breaks lead to cell death; errors by HR, particularly the crossover type, can cause extensive genome rearrangements. While the biochemical process of HR in the repair of an induced DSB has been elucidated by various methods, two critical gaps remain regarding spontaneous HR: 1) the lesions driving spontaneous mitotic HR (e.g. in addition to DSBs, template switching in replication initiated by single-strand nicks can also promote HR); and 2) HR partner choice that determines whether HR is error-free or not. The two gaps are inter-related as HR partner choice could depend on the types of initiating lesions. What determines whether HR is error-free or not is a fundamental question of what governs genome integrity. A major technical roadblock is the lack of scalable, genome-wide tools for studying rare spontaneous HR events in many mutants. To scale up genome-wide HR mapping efforts, I developed sci-L3, which enables linear amplification of single-cell genomes that scales to 1M cells, enabling generating hundreds of single-cell global HR maps per mutant for thousands of mutants. Moreover, genetic assays require detecting “scars” in the genome as traces of repair (e.g. in cancer mutational signature studies), which miss 99% of error-free HR between identical sister chromatids. There is thus a critical need for unbiased global assays that detect both mutational and error-free HR. I recently addressed this gap by developing sci-L3-Strand-seq as the first scalable mapping tool for HR between identical sister chromatids. Our central vision is to determine how spontaneous mitotic crossovers cause genome rearrangements by scalable single-cell assays. In Area1, we will use sci-L3 to explore the full mutant space in hybrid yeast diploids. By generating HR maps in all the single mutants in a pooled manner (160 single-cell HR maps/mutant for 6,000 mutants), we can simultaneously test and generate thousands of hypotheses regarding different lesions and pathways that drive different types of genome instability events genome-wide. In Area2, we focus on a deciding factor for whether HR is error-free or not: HR partner choice of allelic sister chromatid, allelic homolog and non-allelic repeats. With sci-L3-Strand-seq, we propose to map all the seven classes of crossover outcomes in two systems: mammalian cell lines and mouse embryos. In cell lines, we will investigate genome- wide distributions of both error-free and mutational HR outcomes in the wild-type as well as hundreds of perturbations of HR-related genes to determine factors affecting HR partner choice including (epi)genomic contexts, 3D genome organization and HR gene knockdown. We will also develop in vivo sci-L3-Strand-seq/RNA co-assay to dissect cell-type variation in HR partner choice in mouse embryos. In sum, this proposal pursues cost-effective, massively parallel and genome-wide mapping of mitotic crossover outcomes as functions of genetic perturbations and cell types by developing and applying advanced single-cell multi-omics tools.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Steroid hormones in humans and other animals coordinate physiological and behavioral processes underlying optimal responses to the social environment. The brain is a major site of steroid hormone action; however, our knowledge of the role of steroid hormones in regulating gene expression and neuroplasticity in the brain is in its infancy. It has been a challenge to disentangle the role of steroid hormones on brain function because they broadly influence physiology and behavior, making it difficult to characterize direct versus indirect effects. Thus, researchers wishing to use animal models of the hormonal modulation of the brain should have the ability to study separately the physiological and behavioral effects of steroid hormones. My research program aims to uncover the connections between steroid hormones, gene expression in the brain, and neuroplasticity using Astatotilapia burtoni, a cichlid fish that exhibits sophisticated social dynamics. In the wild as in the laboratory, male A. burtoni stratify along a social hierarchy where dominant males possess bright coloration, aggressively defend a territory, and mate with females, while non-dominant males do not. Female A. burtoni do not form a social hierarchy but behave aggressively towards one another for mating opportunities. Social rank is in flux, as dominant and non-dominant males can change their status depending on the social milieu. These complex social interactions are tightly linked to levels of a class of steroid hormones called androgens. My research program will leverage the social dynamics of A. burtoni in the laboratory to discover the role of androgens in controlling genes in the brain and neuroplasticity. We will tackle these questions using cutting- edge techniques such as single-cell genomics, whole-brain imaging, and rich social behavior paradigms. For these experiments, I have used CRISPR/Cas9 gene editing technology to genetically delete distinct androgen receptors (ARs) in A. burtoni. These mutant A. burtoni lack functional genes for ARα, ARβ, or both. Findings in these mutants reveal ARα and ARβ are required for distinct physiological and behavioral aspects of social status, making them ideal for the proposed projects. For example, ARα mutant males do not perform dominant social behaviors but have large testes and bright coloration, while ARβ mutant males perform dominant social behaviors but possess small testes and drab coloration. Males mutant for both receptors lack all of these traits and actually perform female-typical behaviors. As no other laboratory in existence possesses these AR mutants, my research program is highly innovative and in a unique position for addressing these questions. These experiments will be performed in both males and females, yielding novel results about the role of steroid hormones in regulating fundamental brain and behavioral functions. With foundational data from AR mutant A. burtoni, we will be able to address fundamental questions regarding the hormonal control of the brain and social behavior. Indeed, these questions may connect naturally to those on the hormonal control of social behavior in other species such as humans and how social systems emerge throughout evolution.

NIH Research Projects · FY 2024 · 2021-07

ABSTRACT Pulmonary arterial hypertension (PAH) is a chronic lung disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) hypertrophy, RV failure and death. The incidence of PAH is much higher in female patients (4:1 ratio). While previous studies investigating sex differences in PAH have focused extensively on the role of gonadal hormones, in particular estrogen, we are the first lab to investigate the role of sex chromosomes in PAH. Our recent published work using innovative mouse models demonstrated in the absence of sex hormones, the Y chromosome (ChrY) protects against experimental pulmonary hypertension (PH), indicating that gene(s) encoded on ChrY can protect against PH. Only 4 genes on ChrY are expressed in the lungs Uty, Kdm5d, Eif2s3y, and Ddx3y. Our preliminary data identified Uty as the top candidate gene responsible for ChrY protection against PH. Additionally, we demonstrate that Uty expression is reduced in male patients with PAH and multiple animal models of PH. Our RNAseq analysis on the lungs of PH wildtype (WT) and Uty-KD male mice revealed a few promising targets including the proinflammatory cytokines Cxcl9 and Cxcl10. Our preliminary data shows Uty co-localizes with Cxcl9/10 in lung macrophages, and expression of Cxcl9/10 is significantly increased in bone marrow derived macrophages isolated from Uty global KO mice compared to WT. More importantly, our pilot study shows that blocking the shared Cxcl9/10 receptor, Cxcr3, using AMG487 can reduce PH severity in female rats with PH. Our bioinformatics analysis also identified Endothelin-2 (ET-2) is up-regulated in the lung as a result of Uty-KD. The role of ET-2 is currently unknown in PAH. For the first time, we show that increased ET-2 expression may contribute to worsening PH by inhibiting angiogenesis and promoting SMC proliferation in the lung. Our working hypotheses are: (1) ChrY gene Uty protects against PH development; (2) loss or absence of Uty results in more severe PH through increased expression of Cxcl9/10 and ET-2 resulting in vascular EC death, SMC proliferation and pathological angiogenesis; and (3) Blocking Cxcl9/10 alone or together with blocking ET-2 activity reduces the severity of PH in a sex-specific manner. Aim 1. To examine whether knockdown of Uty in the lungs, in the presence and absence of hormones, abolishes the protective role of ChrY in experimental PH; Aim 2. Investigate the mechanistic role of the Uty/Cxcl9/10 and Uty/ET-2 axes in PH pathogenesis; Aim 3. Determine if blocking the activity of downstream Uty genes Cxcl9/10 alone or together with blocking ET-2 rescues PH development by reducing EC apoptosis and SMC proliferation and promoting angiogenesis in male and female rats.

NIH Research Projects · FY 2025 · 2021-07

Project Summary (Abstract) Developing three-dimensional antisense oligonucleotide drugs against COVID-19 The culprit of coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), has a very large RNA genome that encodes the proteins and RNA elements required for all aspects of viral infection and replication. This property makes the virus vulnerable to a new class of drugs called antisense oligonucleotide (ASO). ASOs are single-stranded synthetic nucleic acids that achieve therapeutic effects by binding to viral or other target RNAs via Watson-Crick base pairing, the very interaction that defines molecular biology and the foundation of life. The first ASO drug approved by the U.S. Food and Drug Administration is an antiviral against cytomegalovirus. A major challenge of developing ASO antiviral drugs is the strong tendency of RNA to fold into structures that interfere with ASO hybridization. Current ASO design methods do not adequately address this problem. We have developed a structure-based ASO design technology platform that takes advantage of three- dimensional structures of target RNAs. Our “3D-ASOs” recognize not only the sequences but also the shapes of SARS-CoV-2 RNAs. Compared to conventional designs, 3D-ASOs contact viral RNAs more extensively and therefore can achieve greater affinity and specificity. Our technology platform includes four design templates and a 3D-ASO drug development workflow that employs an innovative RNA structure determination method. In a preliminary study, we designed and tested several 3D-ASOs against SARS-CoV-2 viral RNA and identified two lead sequences that strongly inhibit viral replication in cultured human cells to a much greater extent than previously reported sequences. In the proposed research, we will optimize the lead 3D-ASOs by altering their backbone modifications and bases for tighter binding and better fit to the viral RNAs and for stronger inhibition to their functions. We will also cast our net wide by designing and testing additional anti-SARS-CoV-2 3D-ASOs. Finally, the most potent 3D-ASOs will be tested in an animal model. If successful, the project will provide ASO drug candidates for clinical trials. These drugs may be given as nasal sprays or via intravenous injection, as treatments or for prevention. The structure-based design technology we will refine is generally applicable to ASO drug development. Therefore, this research has the potential to turn tide on the battlefield against COVID-19 and in our fight with many other diseases.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY / ABSTRACT Circuits in the healthy central nervous system (CNS) have the capacity for reorganization and remapping of functionality. Growing evidence suggests that circuit remapping may contribute to a number of neurologic diseases as well. For example, it has been widely hypothesized that remapping of circuits underlies recovery after a focal lesion of the CNS, such as stroke. However, how specific changes in neuronal circuits mediate improvement in function and recovery after cortical injury remains a major gap in our understanding. Here, Dr. Zeiger will utilize advanced techniques for imaging and manipulating circuits in vivo to define the local and global changes in neural circuits that occur following a lesion of the somatosensory cortex in mice. In Aim 1, Dr. Zeiger will investigate the role of GABAergic parvalbumin (PV) cells in peri-lesional remapping of somatosensory function after small lesions to the cortex. PV cells shape cortical sensory representations and regulate experience-dependent plasticity. Dr. Zeiger hypothesizes that PV cells in peri-lesional cortex play a critical role in functional remapping. He will test this hypothesis by 1) recording sensory-evoked responses from PV and pyramidal cells throughout recovery using in vivo two-photon (2P) calcium imaging and 2) modulating PV cell activity using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) and measuring the effects on circuit remapping. In Aim 2, Dr. Zeiger will identify novel candidate brain regions for remapping of lost functionalities that mediate behavioral recovery after large cortical lesions. He hypothesizes that remapping after large lesions involves distributed networks of neurons across multiple brain regions. He will test this by generating a quantitative atlas of all remapped whisker-responsive neurons following recovery, allowing identification of novel candidate regions important for remapping. He will then measure changes in circuit function in these sites over time during recovery and confirm the roles of these regions by manipulating neuronal activity with DREADDs and testing the effect on recovery of somatosensory function. Dr. Zeiger is currently an Assistant Professor in Neurology at the University of California – Los Angeles (UCLA). His long-term career goal is to work as a physician-scientist investigating mechanisms of circuit dysfunction contributing to neurologic disease. As part of this proposal he will carry out a detailed career development plan focusing on gaining technical skills in advanced neuroscience methods for investigating neuronal circuits, expanding his knowledge of how circuit dysfunction contributes to movement disorders, and transitioning to an independent career. This work will be carried out at UCLA, a renowned research institution with an extensive community of investigators in neuroscience and neurology and supported by numerous institutional resources such as the UCLA Clinical and Translational Science Institute. Dr. Zeiger’s career development will be guided by a team of mentors including his primary mentor Dr. Carlos Portera-Cailliau and co-mentors Dr. Jeff Bronstein and Dr. S. Thomas Carmichael.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT This K23 Career Development Award will provide early career support for the investigation of behavioral and biological risk factors for HIV/STI transmission caused by methamphetamine (MA) use among men who have sex with men (MSM). This K23 award will provide support for the candidate to develop expertise in the following areas: 1) Biological impacts of MA use and addiction medicine; 2) Clinical trials methods and biobehavioral interventions; 3) Applied immunology; 4) Professional development; and 5) Responsible conduct of research. Dr. Blair will be mentored by a multidisciplinary team with expertise in addiction, infectious diseases, immunology, and statistics. Dr. Steven Shoptaw has an extensive track record in addiction research and training of future independent investigators. Dr. Jesse Clark will provide mentorship in clinical trial methods, operations, and safety procedures; Dr. Grace Aldrovandi will provide mentorship in applied immunology, with an emphasis on mucosal immunology; and Dr. Robert Weiss will provide mentoring in advanced statistical methods. MA use is an important driver of HIV transmission and the burgeoning STI epidemic among MSM. Understanding the interaction of biological and behavioral risk factors for HIV/STI transmission caused by MA use is imperative for effective HIV/STI interventions. Dr. Blair proposes to investigate the joint effects of MA use, HIV, sexual risk behavior, and rectal gonorrhea/chlamydia (GC/CT) on systemic and rectal inflammation. Stored plasma specimens and behavioral data obtained every 6 months over 2 years from 140 MSM will be used to assess the joint effects of HIV and MA use on systemic inflammation and risk behavior using a 2x2 factorial design stratified by HIV serostatus (70 positive; 70 negative) and results of urine MA screening (70 with MA use; 70 without MA use). 40 HIV-negative MA-using MSM (20 with rectal GC/CT; 20 without rectal GC/CT) will be recruited separately from a community-based university research clinic. MA exposure will be manipulated using contingency management (CM) to evaluate the effects of a decline in MA use on biological markers of inflammation (e.g., cytokines). Following initiation of CM, sexual risk behaviors will be assessed weekly for 8 weeks. Inflammatory rectal cytokines will be measured weekly with rectal swabs and linked with biomarkers of MA exposure over 8 weeks. These activities will accomplish the following aims: 1) Measure the joint effects of HIV and MA use on systemic cytokine concentrations and risk behavior; 2) Identify the effects of MA exposure and concomitant rectal GC/CT on rectal cytokine concentrations; and 3) Evaluate the association of MA use frequency with sexual risk behavior in the setting of rectal inflammation. Through this K23 Career Development Award, Dr. Blair will establish herself as an independent clinician-investigator with expertise in intersectional research on the biological and behavioral impacts of MA and other drugs on HIV/STI transmission dynamics.

NIH Research Projects · FY 2025 · 2021-07

Abstract Cushing Disease (CD) is a life-threatening “orphan disease” caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma driving excess adrenal cortisol production. There is a large unmet medical need for CD treatment. However, translational research has been greatly hampered due to unavailability of any human pituitary corticotroph tumor cell models. Using single cell RNA-sequencing (scRNAseq) and microarray transcriptome analysis of surgically resected human corticotroph tumors, we observed that loss of pituitary corticotroph tumor ACTH secretion coincided with reduced angiogenesis, survival signals and immune responses in parallel with increased collagen catabolism, cell adhesion and extracellular matrix organization. Guided by these findings, we developed a unique 3-dimensional (3D) pituitary tumor culture system and for the first time, we have been able to generate 3D human corticotroph tumor cultures that secrete ACTH >4 months. We have assembled an experienced multidisciplinary team to complete 3 focused specific aims using this first of its kind resource. Firstly, we will use whole exome sequencing to characterize the genomic landscape of our corticotroph 3D culture biobank and compare genomic and genetic fidelity between the original corticotroph tumor, normal blood and matched 3D corticotroph tumoroid cultures from the same individual patient. ScRNAseq analysis of serial passages of individual patient-derived corticotroph tumor cultures will monitor for transcriptome changes in a temporal fashion over the course of culture. The histopathological structure of our 3D corticotroph cultures at the single cell level will quantify tissue architecture so we can map corticotroph tumoroid cellular composition and distribution. A second aim will employ a miniaturized automated system to conduct a high throughput drug screen in our 3D corticotroph tumor cultures. Compounds will be subjected to rigorous evaluation to define primary “hits” and validated by re-screening in triplicate using 20 concentrations from 100µM to 20pM (2-fold dilution) to reliably calculate an EC50 for each compound. Finally, three complementary approaches, computational cheminformatic profiling, scRNAseq to delineate transcriptomic changes at the single cell level following drug treatment and functional genomics will be employed to explore the MOA of validated hit compounds. This integrated interrogation of our drug screen results and the genetic features of our patient-derived 3D tumor cultures as well as that of the original tumor tissue, will allow us to disentangle an individual drug's mode(s) of action, and directly document drug sensitivity of individualized parental corticotroph tumors. In summary, we will use our unique biobank of comprehensively molecularly characterized pituitary corticotroph tumor tissues and paired derived 3D corticotroph tumor cultures to test libraries of clincially relevant compounds. This pituitary 3D tumor culture system is transformative in the field due to the lack of any human pituitary corticotroph tumor cell models and will pave the path for much needed improved therapy for patients with this dreadfully disabling and often fatal disorder, Cushing disease.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated coronavirus disease (COVID-19) is an evolutionarily unprecedented natural experiment that causes major changes to the host immune system. Several high risk COVID-19 populations have been identified. Older adults, males, persons of color, and those with certain underlying health conditions (e.g., diabetes mellitus, obesity, etc.) are at higher risk for severe disease from COVID-19. While it is too soon to fully understand the impact of COVID-19 on overall health and well-being, there are already several reports of significant sequelae, which appear to correlate with disease severity. There is a clear and urgent need to develop prediction tests for adverse short- and long-term outcomes, especially for high-risk COVID-19 populations. We hypothesize that complementary multi-dimensional information gathered near the time of symptom onset can be used to predict new onset or worsening frailty, organ dysfunction and death within one year after COVID-19 onset. A single parameter provides limited information and is incapable of adequately characterizing the complex biological responses in symptomatic COVID-19 to predict outcome. Since they were designed for other illnesses, it is unlikely that existing clinical tools, such as respiratory, cardiovascular, and other organ function assessment scores, will precisely assess the long-term prognosis of this novel disease. Our extensive experience in biomarker development suggests that integrating molecular and clinical data increases prediction accuracy of long-term outcomes. We have chosen to test our hypothesis in a population reflecting US-demographics that is at increased risk of adverse outcomes from COVID-19. We will enroll patients, broadly reflecting US demographics, from a hospitalized civilian population in one of the country’s largest metropolitan areas and a representative National Veteran’s population. We anticipate that a prediction test that performs well in this hospitalized patient group will: help guide triaging and treatment decisions and, therefore, reduce morbidity and mortality rates, enhance patient quality of life, and improve healthcare cost-effectiveness. More accurate prognostic information will also assist clinicians in framing goals of care discussions in situations of likely futility and assist patients and families in this decision-making process. Finally, it will provide a logical means for allocating resources in short supply, such as ventilators or therapeutics with limited availability.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Aged craniofacial skeleton significantly impairs the repair and regeneration of trauma-induced bony defects. Advanced age is a critical risk factor for many chronic and debilitating skeletal diseases including osteoporosis and periodontitis. Periodontitis is the inflammatory destruction of alveolar bone and periodontal connective tissue, resulting in the loss of tooth support. The disease susceptibility and severity increase dramatically with age, leading to a significant public health concern in the aging society. However, the mechanisms that drive craniofacial skeletal aging and age-related exacerbation of periodontitis remain largely unknown. Cellular senescence, the halting of proliferation for aged and damaged cells, play an important role in age- related chronic diseases including diabetes, osteoporosis and periodontitis. Mesenchymal stem cells (MSCs) possess self-renewal ability and multiple lineage potentials. Exhaustion of the MSC pool through senescence represents one of the hall marks for skeletal aging. Senescent MSCs lose potential for proliferation, self-renewal and osteogenic differentiation, contributing to the impaired bone mass and delayed repair in long-bone. MSC senescence is also associated with age-induced acculumation of oxidative stress, mitochondrial dysfunction and DNA damage. The stress-induced senescence could alter MSC-mediated immunomodulation through senescence-associated secretory phenotype (SASP). The direct evidence on the molecular link between MSC senescence and age-related craniofacial bone loss is lacking. Notably, alveolar bone marrow derived MSCs (aBMSCs), compared to long-bone MSCs, are more suitable for craniofacial repair, but exhibit niche-specific behaviors and responses to environmental stimuli. Peroxisome proliferator-activated receptor γ coactivators 1α (PGC-1α) is a transcriptional coactivator with essential roles in mitochondrial biogenesis and regulation of oxidative stress in various mitochondria-rich tissues. Recently, we found that PGC-1α directly regulates cell fate decisions of MSCs to protect against skeletal aging and osteoporosis. PGC-1α depletion also impaired ROS defense in MSCs, resulting in increased oxidative stress. However, the role of PGC-1α in MSC senescence and craniofacial skeletal tissue is unknown. Based on our preliminary experiments, MSC-specific depletion of PGC-1α significantly exacerbated age-induced trabecular bone loss in the mandible. Global depletion of PGC-1α exacerbated periodontal inflammation and bone loss in murine periodontitis models. Intriguingly, in vitro assays revealed that lack of PGC-1α promoted replicative senescence of aBMSCs. Thus, we hypothesize that PGC-1α modulates aBMSC senescence via regulation of oxidative stress to impact age-related craniofacial and periodontal bone loss. To test our hypothesis, we propose the following aims:1) To determine if PGC-1α regulates senescence of aBMSCs and craniofacial skeletal aging; 2) To determine if PGC-1α regulates cellular senescence to influence age-exacerbated periodontal bone loss; 3) To elucidate the underlying molecular mechanism of how PGC-1α modulates aBMSC senescence.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY / ABSTRACT This career development proposal will support Dr. Enrico Castillo to become an independent researcher focused on serious mental illness (SMI), incarceration, homelessness, and public mental health systems, with expertise in conducting mixed methods studies to improve the capacity of public systems to eradicate the health and social inequities experienced by individuals with SMI. People with SMI experience severe inequities, which are particularly evident within homeless populations and correctional facilities where people with SMI are grossly overrepresented. These national challenges are reflected in Los Angeles County, which has the largest unsheltered population of people with SMI in the US. The Los Angeles County jail is the largest facility in the world for the confinement of people with SMI. In the face of scarce public mental health resources and concentrations of people with mental illness in jails, some have posited that jails may serve important public health functions and have positive mental health effects on individuals with SMI. This raises important scientific questions. Dr. Castillo plans to focus his research on understanding the unmet health and social needs of individuals with SMI by studying the effects of incarceration on subsequent health and social trajectories—the jail-to-homelessness pipeline. Dr. Castillo’s proposal centers on mentored career development and research activities that will develop the skills to achieve these career goals: 1) quantitative analysis of linked administrative data, 2) qualitative research methods, specifically ethnography, archival research, and mixed methods dialogue, 3) criminal justice systems and vulnerable justice-involved populations, and 4) dissemination and translation of health services research findings to policy and practice. Given the challenges of studying individuals after jail release, relatively less is known about the precise relationship between incarceration and subsequent homelessness and its downstream effects on healthcare and social trajectories for people with SMI. Fragmented systems of care and siloed data infrastructures are additional barriers to research, coordination of care, allocation of resources, and public health planning. To address these lacunae, Dr. Castillo’s research project will 1) use an existing linked administrative database of eight public service agencies in Los Angeles County to understand whether jail is disruptive to mental health and social service use and housing stability for people with SMI; 2) conduct archival research and direct ethnographic observations in the Los Angeles County jail to elucidate the jail experiences and services and ascertain the mechanisms underlying post-incarceration trajectories; and 3) prepare for a R01 health services research grant (PAR-17-264). Building on this project’s findings about factors that lead to post-incarceration homelessness, that future multi-site R01 will employ the same mixed methods to investigate the full circuit of institutional recidivism (reincarceration, hospitalization, homelessness), to identify the public service use trajectories, programs, and other mutable factors (individual- to system-level) that prevent or disrupt those outcomes.

NIH Research Projects · FY 2025 · 2021-07

While most people with psychosis are not dangerous and most violence is committed by non-psychotic people, people with psychotic disorders are at increased risk for violence, and violence is associated with worse outcomes and increased stigma. Therefore, decreasing violence risk in psychosis is clinically relevant and has important public health implications. Several clinical studies suggest that clozapine is superior to other antipsychotic medications in reducing violence or aggression. However, there were numerous limitations of these studies including that most of them were observational and non-randomized, included small sample sizes, or focused on hostility, non-physical aggression, or self-harm, rather than violent acts. Further, the majority of these trials were not generalizable to outpatient, community settings. No large effectiveness study has examined the effects of clozapine on violent behavior in community settings. We propose a randomized, parallel-group, 24-week, open-label, single (rater)-blind, 7-site clinical trial to examine the effects of treatment with clozapine vs. treatment as usual (TAU) on the risk of violent acts in 280 individuals with schizophrenia at high risk for violence. This trial will be a collaboration of 7 sites, coordinated by the New York State Psychiatric Institute. The 6 additional collaborating sites contribute unique expertise and will ensure an adequate sample size for this trial. Our primary effectiveness outcome is time to violent acts as measured by the MacArthur Community Violence Interview (MCVI). We will also explore the effects of clozapine on the Point Subtraction Aggression Paradigm. While many factors may contribute to violent behavior in individuals with schizophrenia, including positive symptoms, psychopathy, impulsivity, and substance use, evidence suggests that the final common pathway for many of these disparate causal influences likely runs through behaviors captured by the Excitement Factor of the Positive and Negative Syndrome Scale (i.e., a composite of the scores of excitement, uncooperativeness, poor impulse control, and hostility). Importantly, our target (the excitement factor of the PANSS) has been validated to measure excitement-like symptoms in clinical trials in schizophrenia, is sensitive to treatment, has been linked to the neurobiology of violence in spectroscopy and PET studies, and differentiates clozapine from other antipsychotic drugs. We will also explore the effects of clozapine vs. TAU on positive symptoms (e.g., persecutory delusions) and alcohol and substance use, and how these effects influence the risk for violent acts. To enhance the safe implementation of this study in this vulnerable population at risk of violent behaviors, we will implement clinical safety and treatment engagement protocols that rely upon standard personnel and that will be readily generalizable. This trial will provide guidance on the use of clozapine for violence in community settings and will definitively test hypotheses regarding mechanisms of its anti-violence effects. The results will be immediately relevant to practice and will impact public health because there is currently no standard approach for the treatment of violence in schizophrenia.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT In response to PAR-20-056, we propose to reestablish the T90/R90 comprehensive institutional training program at the UCLA School of Dentistry. This application has been built on more than 20 years of our combined research training and career development at UCLA that launched careers of dentist-scientists and oral health-scientists and prepared them for adverse challenges in science, healthcare, and society. The application continues this long-standing tradition and aims to bolster a vigorous and diverse dental, oral and craniofacial research workforce via a rigorous and interdisciplinary training program that offers trainees novel and innovative research training experiences in a highly supportive institutional environment. We have assembled an excellent team of faculty mentors who embody four areas of scientific strengths – (1) Cancer Biology, Oral Cancer & Stem Cells, (2) Craniofacial Biology, Bioengineering & Regenerative Medicine, (3) Microbes – Virulence Mechanisms and Advanced Imaging by CryoEM, and (4) Translational Genetics, Epigenetics, & Genomics. Our interdisciplinary training program links trainees to mentors at the School of Dentistry, School of Medicine, School of Engineering, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, Stem Cell Institute, and California NanoSystem Institute. The integration of these interdepartmental centers and institutes provides a rich training environment with enormous physical resources and centralized core facilities. We offer five training tracks, (i) Dentist-Scientist Trainee Program (dual degree DDS/PhD), (ii) Dentist-PhD Program (predoctoral research training to dentists), (iii) Dentist-Scientists Postdoctoral Fellow (postdoctoral training to dentists), (iv) Predoctoral PhD Trainee (predoctoral training in oral health), and (v) Oral Health Postdoctoral Fellow (postdoctoral training in oral health). Collectively, our proposed training program will train the next generation of dentist-scientists and oral health scientists to conduct basic, translational, and clinical research to improve dental, oral, and craniofacial health.

NIH Research Projects · FY 2025 · 2021-07

In response to PAR-20-056, we propose to reestablish the T90/R90 comprehensive institutional training program at the UCLA School of Dentistry. This application has been built on more than 20 years of our combined research training and career development at UCLA that launched careers of dentist-scientists and oral health-scientists and prepared them for adverse challenges in science, healthcare, and society. The application continues this long-standing tradition and aims to bolster a vigorous and diverse dental, oral and craniofacial research workforce via a rigorous and interdisciplinary training program that offers trainees novel and innovative research training experiences in a highly supportive institutional environment. We have assembled an excellent team of faculty mentors who embody four areas of scientific strengths – (1) Cancer Biology, Oral Cancer & Stem Cells, (2) Craniofacial Biology, Bioengineering & Regenerative Medicine, (3) Microbes – Virulence Mechanisms and Advanced Imaging by CryoEM, and (4) Translational Genetics, Epigenetics, & Genomics. Our interdisciplinary training program links trainees to mentors at the School of Dentistry, School of Medicine, School of Engineering, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, Stem Cell Institute, and California NanoSystem Institute. The integration of these interdepartmental centers and institutes provides a rich training environment with enormous physical resources and centralized core facilities. We offer five training tracks, (i) Dentist-Scientist Trainee Program (dual degree DDS/PhD), (ii) Dentist-PhD Program (predoctoral research training to dentists), (iii) Dentist-Scientists Postdoctoral Fellow (postdoctoral training to dentists), (iv) Predoctoral PhD Trainee (predoctoral training in oral health), and (v) Oral Health Postdoctoral Fellow (postdoctoral training in oral health). Collectively, our proposed training program will train the next generation of dentist-scientists and oral health scientists to conduct basic, translational, and clinical research to improve dental, oral, and craniofacial health.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Food insecurity is highly prevalent in the U.S., affecting 11.1% of households. This high prevalence is significant because food insecurity is associated with metabolic consequences such as obesity, metabolic syndrome (MetS), and chronic diseases such as diabetes. One solution for relieving the health burden of food insecurity is to target those most at risk for its poor outcomes. Therefore, the overarching goal of this project is to use a multidisciplinary, multimethod approach to identify such individuals. The central hypothesis of this project is that those with food insecurity and high levels of the stress hormone cortisol are most at risk for the negative behavioral and health consequences of food insecurity. This hypothesis is based on literature and preliminary data showing that (a) food insecurity can be stressful for many; (b) cortisol is a causal driver of high-fat, -sodium, -sugar, and - carbohydrate (“hyperpalatable”) food consumption and (b) cortisol is associated with poor metabolic outcomes like diabetes and MetS. Further, preliminary data for this project show that cortisol modulates the relationship between experimentally manipulated stressful states and consumption of hyperpalatable foods. Two patterns of cortisol levels can potentially index higher risk: (1) chronically high levels of cortisol and/or (2) high cortisol reactivity to acute in-the-moment stressors. This project examines both by pursuing the following specific aims: AIM 1. Determine the modulating effect of chronic high cortisol levels on associations between food insecurity and (a) hyperpalatable food intake and (b) MetS—Recently collected data from the study team’s NHLBI Growth and Health Study (N = 624; R01 HD073568 ) will test the hypothesis that those with higher chronic cortisol levels indexed in hair will show a stronger relationship between food insecurity with hyperpalatable food intake and MetS, respectively. AIM 2. Determine the modulating effect of experimentally manipulated high cortisol reactivity on the association between food insecurity and objectively measured hyperpalatable food intake—In a laboratory paradigm using within-subjects design, 400 individuals with food insecurity will be exposed to a gold-standard laboratory stressor to measure cortisol reactivity compared to a no-stress session. The hypothesis tested will be that those with greater cortisol reactivity to stress (vs. control) will engage in greater hyperpalatable food intake, measured objectively. In an EXPLORATORY AIM, the project will examine potential roles of perceived stress and psychosocial resilience factors. By successfully achieving these aims and demonstrating the strong biobehavioral drivers of unhealthy diet, federal food programs (updated every 5 years) will have stronger rationale to prioritize nutritious foods over hyperpalatable ones. With screening for food insecurity becoming commonplace in clinical settings, additional resources for stress screening and management could be disseminated. Discovering factors that confer resilience in the face of food insecurity will allow future work testing them as intervention targets. This project, therefore, could ultimately reduce the consequences of food insecurity and improve the nation’s health.

NIH Research Projects · FY 2025 · 2021-07

Abstract The biomedical sciences are drowning in big data. Progress in fields such as genomics and medical imaging is being stymied by the lack of ap- propriate computational tools. This grant promotes the development of algorithms, statistical methods, and software for the analysis of the big datasets encountered in the biomedical sciences. The NIH All of Us Pro- gram, the Million Veteran Project (MVP) sponsored by US Department of Veterans Affairs (VA), and the UK Biobank are three prime examples of recent massive datasets. These datasets require terabytes of storage on sample sizes ranging from 105 to 106 and above subjects. The datasets are also dynamic, growing over time in size and complexity. In addition, the datasets are heterogeneous; for example, the UK Biobank offers ge- nomic data, electronic health record (EHR) data, and imaging data on the same study individuals. Finally, as with most real-world data, the data are fraught with missingness and inaccuracy. We propose attacking the issues of parameter estimation and model selection raised by such massive datasets. We will be guided by princi- ples of parsimony and high-dimensional optimization. Most of the specific applications we have in mind involve imaging and genomics, particularly genomewide association discovery. Fortunately, most of the tools and soft- ware we construct will be more generically useful. Our successful algo- rithms will be coded in the modern scientific programming language Julia and posted on publicly available websites. We will focus on constrained and sparse regression, EM and MM algorithms for optimization, variance components models, bootstrapping of linear mixed models, a copula-like model for correlated data, and sensitivity analysis in epidemic models. These are all subjects of paramount importance in modern genomics, bio- statistics and data mining.

NIH Research Projects · FY 2025 · 2021-07

Abstract This clinical research training program emphasizes developing research scientists who focus on schizophrenia and other psychotic disorders. It particularly emphasizes training in cognitive and affective deficits in psychotic disorders, influences on clinical and functional outcomes, and intervention research to improve clinical and functional outcomes. Training will include core didactic coursework and direct experiences in designing and conducting clinical research with patients with psychotic illnesses, including randomized clinical trials to evaluate efficacy of interventions and research into mechanisms of action of interventions. Direct mentoring by clinical investigators and other scientists and statisticians is a key feature of this program, with the focus being on development and implementation of clinical research with individuals with schizophrenia, severe mood disorders, and other psychotic disorders and those at risk for such disorders. This postdoctoral training program will focus on individuals with Ph.D. in clinical psychology and those with an M.D. followed by a psychiatry residency. In addition, individuals with a Ph.D. in cognitive or affective neuroscience and similar fields who focus on research on psychotic disorders will be welcomed. The training program includes (1) extensive hands-on training in the development and implementation of clinical research on cognitive and affective factors in outcomes and on interventions to improve clinical and functional outcomes, (2) a weekly Research Seminar on the Psychoses taught jointly by clinical researchers and other scientists focused on research methods, recent research results, and grant preparation issues, (3) a set of core courses in research design, statistics, and implementation of clinical trials and other clinical research, (4) other coursework, workshops, and laboratory training tailored to individual research interests, (5) an intensive career development retreat and other career development sessions, and (6) training to enhance the responsible conduct and reproducibility of research. The training will be closely interfaced with many clinical research opportunities that allow access to research participants with schizophrenia and other psychotic disorders.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders often affect our most distinguishing cognitive and social capabilities, which are thought to have developed as a result of the expansion of the human neocortex. The unique mechanisms orchestrating cortical neurogenesis and differentiation in the developing human neocortex forming the basis of this expansion are beginning to be described. However, although genetic variation in non-coding gene-regulatory regions, rather than in protein coding genes, drives these evolutionary changes, the cis gene regulatory elements (GREs), including promoters and enhancers, and the transcription factors (TFs) governing cortical neurogenesis remain to be characterized. To begin to investigate this understudied mechanism, we and others have leveraged next generation sequencing approaches to profile chromatin accessibility and interaction in parallel with gene expression to create GRE maps of varying levels of spatiotemporal specificity. We previously identified thousands of developmentally dynamic GREs and their putative gene targets by contrasting GRE activity in progenitor versus neuron-enriched laminae of mid-gestation human neocortex, and functionally validated the role of select GREs in cortical neurogenesis using primary human neural progenitor cells. Further, we found that human-gained enhancers (HGEs), a subset of GREs more active in the human than the macaque or mouse neocortex, regulate genes enriched in outer radial glia (oRG), a neural progenitor with prominent roles in cortical gyrification. This work supports the hypothesis that human developmentally dynamic GREs and HGEs direct gene expression programs controlling the proliferation and differentiation of progenitor pools key to cortical expansion. In this proposal, we seek to test this hypothesis and move from a tissue- and gene-level resolution atlas to a cellular- and gene isoform-level resolution atlas. We will perform single nucleus ATAC-seq to identify cell-specific GREs and leverage a novel single-cell isoform sequencing (scIso-seq) technology to investigate a previously understudied mechanism of gene regulation – alternative promoter usage. This new atlas will inform our work to functionally define the GREs impacting cortical neurogenesis at scale using CRISPR interference (CRISPRi) libraries containing capture tags enabling simultaneous reading of transcriptome and sgRNA at the single-cell level. Finally, we will define and characterize the TFs directing the balance of proliferation versus differentiation of progenitors. These results will enable us identify the cellular basis of genomic variation causing risk for neuropsychiatric disease, and influencing cognition and brain structure. Together this work will create a robust single cell-resolution functional annotation of non-coding GREs and TFs acting in developing human neocortex and elucidate evolutionary mechanisms driving cortical expansion. Broadly, this work will provide a blueprint for scalable approaches to study non-coding genetic variation and cellular diversity.

NIH Research Projects · FY 2025 · 2021-07

1 Project Summary 2 3 The overall goal of this project is to develop accurate and reliable prediction tools and pharmacological targets 4 for the prevention of rupture of intracranial aneurysms (IAs). Abnormal hemodynamic stress such as 5 impingement flow with high wall shear and oscillating flow with low wall shear, is intimately linked with the growth 6 and rupture of IAs. However, detailed mechanisms underlying weak IA walls are not yet defined due to (1) the 7 absence of technologies for profiling the spatial distribution of gene expression of endothelial cells (ECs) induced 8 by the complex hemodynamic flow stressors created in IAs, (2) difficulties in collecting sequential clinical images 9 of growing IAs and acquiring human IA tissue samples to validate biologic mechanisms, and (3) the absence of 10 technologies allowing integration of the data from 3D multimodal techniques. To overcome these obstacles, we 11 have built a strong, multidisciplinary team and created a new experimental system that bridges human samples, 12 imaging, and dynamic modeling platforms. In this project, we challenge two fundamental questions regarding 13 hemodynamic stress and induced responses within the IAs. First, does complex abnormal hemodynamic stress 14 within human IAs induce abnormal regulation of EC signaling pathways? Second, what signaling pathways in 15 EC link unstable wall remodeling during IA growth and rupture? To address these questions, we have pioneered 16 a 3D Live EC Aneurysmal Flow Simulator (3D LEAFS) for profiling the spatial distribution of EC responses to 17 complex hemodynamic flow stress created in patient-specific IAs. Preliminary studies demonstrate that abnormal 18 flow in IAs induces abnormal EC morphology, cellular dysfunction and inflammation, and increased permeability. 19 We have developed an extensive database of clinical images of growing IAs and also tissue samples, exploiting 20 integrated flow analysis and 3D histological imaging of human IA tissue scanned with micro-CT and multiphoton 21 microscopy. With this database, we have linked abnormal flow with IAs to growth, wall thinning and weak wall 22 remodeling leading to rupture. By combining these state-of-the-art technologies, we propose to examine 23 fundamental impact of abnormal flow stress on ECs, and identify relationships between EC pathophysiological 24 responses and wall changes leading to fragile walls, growth and rupture. The proposed research is innovative 25 because this will be the first research to answer the above questions by utilizing multimodalities including 26 longitudinal follow-up images, surgical video, micro-CT, multiphoton microscopy, in vitro 3D endothelialized flow 27 simulator, and flow analysis for development of a pipeline for linking flow-induced EC responses to pathologic 28 changes in human IA tissue. The specific aims of this project are: 1) determine the EC signaling pathways 29 associated with unstable wall remodeling, 2) correlate pathological EC responses with IA growth, and 3) 30 determine the EC responses evoked by several characteristic abnormal hemodynamic flow conditions. The 31 proposed research will enhance development of precision medicine strategies that leverage diagnostic imaging 32 with risk prediction and translational therapies.

NIH Research Projects · FY 2025 · 2021-07

Project Summary The overarching research goal of the Park lab is to gain systems-level understanding of metabolism (including its regulation) and rationally engineer mammalian and microbial metabolism for biotechnology and medicine. We are a team of open-minded and hardworking researchers who employ core analytical techniques and ceaselessly innovate (and adopt) new technologies to solve challenging problems associated with various diseases and organisms. Our current research is twofold: microbial conversion of carbon dioxide into value-add products for economic and environmental benefits; and elucidation of thermodynamic and kinetic mode of metabolic control in mammalian gluconeogenesis. One of our goals over the next five years is to develop key technologies to mathematically reconstruct human central carbon metabolism in thermodynamic and kinetic terms. Until recently, characterization of metabolism has relied mainly on comparison of relative metabolite and enzyme levels between control and experimental groups. We will go beyond measuring just the “levels” and quantify rates and energies, which are direct representation of metabolism in action yet difficult to measure because they are substantive yet intangible. To this end, we will employ state-of-the-art liquid chromatography-mass spectrometry, mathematical modeling, and novel isotope tracers that can yield the most thermodynamic and kinetic information in cellular metabolism. We aim to apply these techniques to investigating the two central metabolic pathways: glycolysis and gluconeogenesis. The two pathways largely share a common enzyme set, yet the former converts glucose into cellular energy and biomass precursors while the latter converts non- carbohydrate substrates into glucose. These functionally opposite metabolic pathways support systemic glucose homeostasis in humans and, in microbes, various bioproduct synthesis from a wide range of carbon substrates with varying degrees of oxidation. This project will map kinetic and thermodynamic bottlenecks of the two pathways in mammalian cells and elucidate regulatory mechanisms that enable seamless transitions and coordination between them. As dysregulation of these pathways are implicated in type II diabetes and cancer, we envision that this research program will lead to effective metabolic control and engineering strategies to remedy defective carbon metabolism in diseases. The upshot of successfully completing the proposed research will contribute to advancing therapeutic development for diabetes and cancer.

NIH Research Projects · FY 2025 · 2021-07

Project Summary / Abstract Periodic paralysis and myotonia are ion channelopathies of skeletal muscle with debilitating episodes of severe weakness lasting hours to days and activity-dependent muscle stiffness. The long-term goal of this project is to advance our understanding of disease mechanism in these disorders of muscle excitability and to apply this knowledge in the design and pre-clinical testing of therapeutic interventions. Much progress has been made in establishing a causal relationship between the biophysical defect of a mutant channel and the clinical phenotype. For example, over 80 missense mutations have been identified in the NaV1.4 sodium channel, and we have shown by functional expression studies, coupled with simulations of fiber excitability, that mutations with gain of function changes (e.g. impaired inactivation) cause hyperkalemic periodic paralysis (HyperPP) with myotonia. Alternatively, the NaV1.4 mutations in hypokalemic periodic paralysis (HypoPP) are all R/X substitutions in S4 segments of voltage sensor domains that share a common functional defect - the anomalous gating pore leakage current. In all forms of periodic paralysis, the transient attacks of weakness result from sustained depolarization of 𝑉𝑟𝑒௦௧ and loss of excitability, which are often triggered by stress, diet (carbohydrate, salt content, fasting), cold temperature, or exercise. The mechanisms by which these triggers destabilize 𝑉𝑟𝑒௦௧, in the setting of a static defect for a mutant channel, are fundamental open questions in the field and also represent opportunities for therapeutic intervention. A major impediment to progress has been the scarce availability of affected muscle. We created three knock-in mutant mouse models of PP that have robust phenotypes for HyperPP (NaV1.4-M1592V) or HypoPP (NaV1.4-R669H; CaV1.1-R528H). These mouse models have led to new insights on disease mechanism (e.g. recovery from acidosis is a potent trigger of HypoPP) and have led to novel therapeutic interventions that are now in clinical trials (bumetanide inhibition of the NKCC1 cotransporter prevents HypoPP). We will extend our investigations of periodic paralysis by focusing on the impact of ion gradients. Changes in extracellular [K+]o are established triggers for HypoPP (low) or HyperPP (high), but relatively little is known about Na+ and Cl- shifts in PP. Limited human data suggest an acute rise of [Na+]in during an episode of HyperPP or chronically high [Na+]in for HypoPP. In addition, we showed that reducing Cl- influx completely prevents HypoPP attacks. We have developed improved ion-selective microelectrodes, that in combination with the unique resource of our knock-in mutant mice, will enable us to (1) characterize muscle fiber Na+ and Cl- content at rest and during an attack of PP, (2) define the contribution of specific ion transport systems (mutant NaV1.4, NKCC1, Na/K-ATPase, Cl- exchangers) in setting ion concentrations in muscle channelopathies, (3) define the functional consequences of ion gradient perturbations in PP, based on computational modeling and simulation, and (4) use these insights in the design and pre-clinical testing of disease-modifying interventions. .