Stanford University

NIH Research Projects · FY 2025 · 2014-05

Abstract: Despite the recent advances in immunotherapy, such as checkpoint blockade, radical new approaches are needed to both improve the efficacy of existing treatments, and to offer entirely new therapeutic modalities. In the previous term of this award we developed an immunotherapeutic agent that exploited the myeloid branch of the immune system by blocking the SIRPa/CD47 axis, potentiating macrophage attack on tumors. We engineered a unique, high-affinity SIRPa antagonist of CD47 that greatly potentiated the anti-tumor efficacy of several clinically approved anti-tumor antibodies, yet offered the advantage of being tumor selective and non- toxic, which is a limitation of most current anti-CD47 mAbs. This molecule is now in clinical trials for several cancer types, thus completing the cycle of bench to bedside in one term of the award. In this renewal application, we request support to develop a new paradigm for receptor inhibition in oncology. We present a new approach to immune checkpoint blockade (ICB), and antagonizing the CD47/SIRPa axis, by exploiting an untapped natural biological mechanism for dampening immune receptor signaling. We have found that ITIM/ITAM/ITSM/ITTM-containing immunoreceptors, such as the checkpoint receptors PD1 and CTLA4, tonically signal in the absence of ligand engagement. As a result, blocking PD-L1 binding with anti-PD1 antibodies, does not fully “take the brakes off” of T cell activation: ligand-independent PD1 tonic signaling significantly blunts T cell activation. We have devised a strategy reduce or eliminate tonic signaling, termed Receptor Inhibition by Phosphatase Recruitment (RIPR), that relies on the cis-ligation of kinases-mediated signaling receptors to a cell surface phosphatase, such as CD45, to achieve complete signal inhibition through intracellular dephosphorylation of the target receptor intracellular ITAM/ITIM/ITSM/ITTM domains. RIPR molecules are bi- specific antibodies that compel dimerization of a target receptor to a cell-surface phosphatase, inhibiting both ligand binding and tonic signaling. Using the PD1 system as our first target, we find that we achieve significantly greater inhibition of checkpoint blockade using a RIPR that dimerizes PD1 with the T cell phosphatase CD45, versus ligand blocking by anti-PD1 antibodies. Furthermore, we see enhanced therapeutic efficacy over anti- PD1 in several mouse tumor models. In this proposal we seek support to better understand the mechanism of RIPR at the biochemical and cellular level, to assess its therapeutic efficacy in a range of mouse tumor models both alone and in combination with therapeutic antibodies, and to explore RIPR applications beyond checkpoint inhibition to other receptors, such as CTLA4 and SIRPa, that are oncology drug targets.

NIH Research Projects · FY 2024 · 2014-01

SUMMARY Central functions in DNA biology and biotechnology are carried out by nucleoprotein machines. In these dynamic macromolecular assemblies, the DNA duplex is bound and distorted in complex with protein and sometimes RNA. Biophysical measurements and models are needed to understand the mechanisms of these machines, in which coordinated conformational changes in protein and nucleic acid components are coupled with chemical steps such as backbone cleavage or nucleotide hydrolysis. This is a renewal application for a grant in which we previously developed high-resolution and multimodal single-molecule approaches and applied them to elucidate mechanochemical coupling in the ATP-dependent supercoiling motor DNA gyrase from E. coli. Here, we propose to leverage our methods and insights to dissect the dynamics and mechanics of additional nucleoprotein machines, focusing on the RNA-guided nucleases Cas9 and Cas12a and comparing DNA gyrase motors across species. We will characterize substeps in DNA interrogation and DNA supercoiling, molecular determinants of energy landscapes and kinetics, and the effects of mechanical strains experienced in the genome. If successful, the project will determine the physical mechanisms of DNA interrogation by RNA-guided nucleases in dynamic and mechanical detail, providing a quantitative description of the target search process for enzymes that are currently being exploited for gene editing and for a rapidly expanding set of other applications involving specific targeting of activities to sites in the genome. New DNA gyrase measurements will further elucidate biophysical specializations, structural properties, and mechanical regulation of enzymes that are important targets for antibacterial drugs. Finally, single-molecule methods development driven by these biophysical questions will have broad applications in systems ranging from transcription to nucleosome remodeling.

NIH Research Projects · FY 2026 · 2013-12

Project Summary Mitochondrial dysfunction and protein homeostasis (proteostasis) failure are common among diverse brain disorders, from neurodegenerative disease and stroke/traumatic brain injury to brain tumor and psychiatric diseases. It is not clear whether these seemingly disparate pathological features are mechanistically interconnected. Mitochondria play important roles in bioenergetics as well as other essential aspects of cellular physiology, such as calcium buffering, intermediary metabolism, and apoptosis. Maintaining mitochondrial quality and quantity is essential for tissue homeostasis, especially the highly energy-demanding neuromuscular tissues. Pten-induced kinase 1 (PINK1) and Parkin (encoding an E3 ubiquitin ligase), two genes associated with familial Parkinson's disease (PD), constitute a genetic pathway important for maintaining mitochondrial health and neuromuscular integrity. Identification of this pathway offers a much-needed entry point to decipher the relationship between mitochondrial dysfunction and other pathological hallmarks of disease. Our studies in previous funding cycles revealed that PINK1/Parkin directs an interconnected mitochondrial quality control process important for neuromuscular tissue integrity. This process encompasses biogenesis of respiratory chain, mitochondrial fission/fusion dynamics, transport, and mitophagy, and is regulated by mechanistic target of rapamycin complexes (mTORC1 and mTORC2). Importantly, in the last funding cycle, we found that ribosome-associated quality control (RQC), a recently recognized protein quality control mechanism that surveys the translating ribosomes for faulty translation products to safeguard proteostasis, is an important player in PINK1/Parkin-directed mitochondrial homeostasis. These new findings thus provide a mechanistic link between mitochondrial function and cellular proteostasis. We found that a highly conserved RQC factor responds to mitochondrial stress and PINK1 dysfunction and is required for stress adaptation. Activation of this RQC factor effectively rescued PINK1 mutant phenotypes. Moreover, our preliminary studies identified several upstream factors that regulate the post-translational modification (PTM) of this RQC factor. These results laid the foundation for understanding the normal physiological function of this key RQC factor and the upstream signaling mechanisms that regulate its activity in RQC and cellular homeostasis. We will use a powerful combination of molecular genetics, genomics, cell biology, and biochemistry approaches, and move between in vivo fly models, mouse models, and human iPSC-derived cell culture models to seek common mechanisms. We will test the hypothesis that this RQC factor directs the quality control of the translation of mitochondrial outer membrane-associated cytosolic mRNAs to promote mitochondria and neuromuscular tissue homeostasis under stress, and that it provides a signaling node for integrating diverse upstream regulatory inputs to ensure the efficiency and fidelity of this translation process.

NIH Research Projects · FY 2026 · 2013-12

Project Abstract: The T cell receptor interaction with peptide-MHC is unique in that each receptor has the capacity to recognize and differentially respond to functionally distinct ligands. Understanding the molecular basis for these properties is important not only for mechanistic insight but for clinical translation to antigen-specific immunotherapies. In this renewal proposal we continue our focus on the structure of the TCR/pMHC interface, its role in the specificity of both the recognition and triggering phases of TCR signal initiation, and exploitation of this information to engineer three different, complementary antigen-specific immunotherapeutic strategies. In the prior term of this proposal, we applied a technology developed in the P.I.’s lab, yeast peptide-MHC display, to identify peptide ligands of ‘orphan’ TCRs in the natural immune system (e.g. Treg) or from pathogenic systems (e.g. cancer, autoimmunity, infectious disease). We deployed this technology effectively on several systems to discover new ligands. In this renewal application we wish to continue leveraging basic mechanisms of TCR/pMHC structure and recognition, to engineer molecules for immune modulation. In our first engineering strategy in Aim #1, we wish to continue this effort on new TCR systems using greatly improved versions of the pMHC display technology that will increase the success of screening and help solve the problem of MHC restriction ambiguity by TCRs. To make the best use of new ligands from Aim #1, in Aim #2 we wish to generate high-affinity “TCR mimic” antibodies and artificial intelligence designed pMHC binders that specifically recognize pMHC ligands and enables us to track tissue expression and inducing selective killing of cells expressing these antigens. In our third engineering strategy in Aim #3, we wish to leverage our surprising mechanistic finding about TCR triggering by the formation of “catch bonds” in the TCR/pMHC interface. We have developed a new approach called ‘catch bond engineering,’ for modifying TCRs to exhibit improved target killing potency in a way that bypasses the dangers of affinity-matured TCRs in adoptive cell therapy. Collectively, this proposal aims to exploit first principles of TCR/pMHC binding structure and chemistry along three different engineering fronts that have direct translational impact.

NIH Research Projects · FY 2026 · 2013-09

Neural stem cell (NSC) homeostasis represents a state of delicate equilibrium between self-renewal, differentiation, and survival. It is fundamental to the development, growth, and regeneration of the nervous system. Defects in NSC homeostasis underlie broad neurodevelopmental, psychiatric, and neurodegenerative disorders. The mechanisms underlying the control of NSC homeostasis remain incompletely understood. Drosophila has been instrumental in discovering signaling molecules such as Notch and Numb and cellular mechanisms such as asymmetric cell division that are centrally involved in NSC homeostasis. The Drosophila larval brain type II neuroblasts (NBs) have served as an excellent model for studying NSC homeostasis. Similar to mammalian NSCs in lineage hierarchy, the type II NB lineages in the Drosophila larval brain contain transit-amplifying intermediate progenitors (IPs), which can generate a vast number of differentiated progenies. Notch signaling is critical for maintaining the homeostasis of type II NB lineages. Inhibition of Notch signaling results in NB not being properly maintained, whereas Notch hyperactivation causes ectopic NB formation and brain tumorigenesis. Notch signaling also regulates the homeostasis of mammalian NSCs, with deregulated Notch signaling having been linked to brain cancer. The molecular mechanisms by which Notch signaling regulates NSC homeostasis, however, are not well delineated. Previous studies have focused heavily on canonical Notch signaling mediated by transcription factors acting in the nucleus. We have found that a non- canonical Notch signaling (NNS) pathway operating in the cytosol and involving mitochondria critically mediates Notch function in NSC homeostasis. The clinical significance of this NNS pathway is underscored by our observation that tumor-initiating cancer stem cells (CSCs) are particularly sensitive to perturbation of this pathway. Moreover, we found that this NNS pathway exerts co-translational quality control over a master regulator of cell growth. The goal of this proposal is to move away from the status quo of transcriptional control of NSC behavior by Notch and focus on the newly discovered co-translational quality control mechanism by NNS signaling. Our central hypothesis is that non- canonical N signaling regulates NSC homeostasis through a signaling cascade emanating from mitochondria and impinging on the ribosome-associated quality control (RQC) of a master regulator of cell growth. To test this hypothesis, we propose three integrated Specific Aims. Aim 1 will characterize features of the translation of the growth regulator that necessitates RQC. Aim 2 will examine the functional relationship between a key RQC factor and this growth regulator in NSCs and CSCs. Aim 3 will dissect the molecular mechanism by which mitochondrial activity signals to the RQC machinery to regulate the translation of the growth regulator. Upon successful completion of these Aims, we will have generated new mechanistic insights into the control of NSC homeostasis by Notch. We anticipate that this will open entirely new directions for studying the fundamental roles of Notch in NSC and cancer biology.

NIH Research Projects · FY 2025 · 2013-07

Project Summary/Abstract: This competing application seeks an additional five years (Years 11-15) of support for a postdoctoral research training program in pain and substance use disorders (SUDs). The magnitude of these two problems in the United States is astounding. Over 100 million Americans have pain that persists for weeks to years, and over 20 million meet the diagnostic criteria for a SUD, costing our country over $1 trillion annually. Contemporary neurobiological, psychological, and epidemiologic research, as well as the tragic experience of the opioid addiction epidemic show a clear intersection of pain and SUDs. Two of the National Academy of Medicine Relieving Pain in America committee’s recommendations for improving research at a national level are to (1) increase support for interdisciplinary research in pain and (2) increase the training of pain researchers. Similarly, the NIH Helping to End Addiction Long-term Initiative has called for increased training of multidisciplinary researchers in SUDs, pain, and their intersection. Our proposal describes a collaborative, interdisciplinary postdoctoral (PhD, MD/PhD, and MD) training program that will produce diverse scientists with a rigorous grounding in pain and SUD research. We request support for 10 postdoctoral fellows. The fellowship typically lasts 2 years for PhDs and 3 years for MDs seeking a robust research foundation. The 23 accomplished faculty mentors are committed to interdisciplinary collaboration and team-based mentorship across their substantive areas of expertise, which range from cells to society. Specific faculty expertise and training opportunities include molecular and cellular biology, optogenetics, electrophysiology, genetics, cognitive neurosciences, psychology, neuroimaging, data sciences, epidemiology, health policy, and economics of pain and SUDs. The training program, housed in the Division of Pain Medicine, Department of Anesthesiology, Stanford University School of Medicine, develops postdoctoral trainees’ skills to prepare them to become independent investigators in the fields of pain and SUDs. The training program includes required and elective coursework, mentored research experiences, an individual integrated research project, seminars, and professional development, including grant and manuscript writing. The training program will continue to be led by Sean Mackey, MD, PhD, in collaboration with a steering committee comprising senior scientists/mentors and a faculty leader in diversity, equity, and inclusion. This team will oversee the program's recruitment, training, scientific, and administrative aspects, including a rigorous internal and external evaluation process. In summary, this training program will bring together a diverse and talented group of postdoctoral trainees, an accomplished team of interdisciplinary mentors, an effective administrative structure, and a world-class research environment at Stanford University. The combination of talent and environment will launch the next generation of independent investigators dedicated to pain and SUD discovery research and clinical translation.

NIH Research Projects · FY 2026 · 2013-06

Project Summary/Abstract The structural basis for pathway-selective signaling by the µ-opioid receptor. For many patients experiencing acute and chronic pain, opioids like codeine, morphine, and fentanyl have improved their quality of life. Unfortunately, these benefits can be offset by dose-limiting liabilities, like addiction and the respiratory depression responsible for opioid overdose deaths. The µ-opioid receptor (µOR) is responsible for mediating the beneficial and adverse effects of most opioid analgesics. The µOR has complex signaling behavior, activating six different G proteins subtypes (Gi1, Gi2, Gi3, GoA, GoB, Gz) and two arrestin subtypes. Most of the opioid agonists currently used to treat pain activate all of these signaling pathways. Yet there is a growing body of evidence that only a subset of these signaling pathways mediate analgesia, while a different subset may be responsible for the adverse effects. Moreover, it is possible that the Gi/o/z subtypes responsible for alleviating acute pain are different from the subtypes responsible for alleviating chronic pain. We have identified several agonists that preferentially activate subsets of Gi/o/z proteins that retain analgesic efficacy but have fewer adverse effects. The overall goal of the proposal is to determine the structural basis for this pathway-selective G protein signaling. This information will facilitate the development of more pathway- selective agonists. These agonists will provide useful tools for understanding the complex signaling behavior of the µOR and the role of specific pathways in mediating the therapeutic and adverse effects of opioid agonists. The Specific Aims of the overall proposal are: Aim 1A. Determine the structural basis for subtype-selective signaling to Gi/o/z proteins. We will use cryo- electron microscopy to determine structures of the µOR bound to different Gi/o/z subtypes and different pathway selective agonists. Aim1B. Structure-guided synthesis of novel pathway-selective µOR agonists. Aim 2. Characterize the steady-state conformational changes in the cytoplasmic surface of the µOR stabilized by agonists with distinct signaling profiles. We will use double electron-electron resonance (DEER) spectroscopy to map conformational changes in the cytoplasmic surface of the µOR bound to pathway selective and non-selective agonists. Aim 3. Characterize the dynamics of TM6, ICL2, TM7, and the C-terminus in response to the binding of agonists with distinct signaling profiles in the presence and absence of specific G proteins and arrestins. We will use single-molecule Förster Resonance Energy Transfer to monitor conformational changes of these cytoplasmic domains in real-time.

NIH Research Projects · FY 2026 · 2013-06

During neuronal development, stereotyped axonal and dendritic arbor shapes are often achieved through dynamic and stochastic growth, branching and retraction. The shape of arbors are also determined by guidance receptors and ligands. How guidance receptor and ligands interact to achieve the proper shape through a dynamic process is unknown. Our previous work funded by this grant identified that extracellular ligand SAX-7/LICAM dictates the shape of the PVD sensory neuron through its binding to the dendritic guidance receptor DMA-1. Our current unpublished results argue against the popular view in the field and support a new model. Unexpectedly, we find that ligand-free DMA-1 is sufficient to promote robust, stochastic dendrite growth. Our data also suggest that ligand binding might inhibit growth, prevents retraction, and specifies arbor shape. We propose that DMA-1 needs to be endocytosed and subsequently reinserted onto the plasma membrane via recycling endosomes, to generate a pool of ligand-free DMA-1 which is critical to promote dendritic growth. Therefore, ligand-free guidance receptor mediates intrinsic, stochastic dendritic growth, while extracellular ligands instruct dendrite shape by inhibiting growth. Specifically, I propose to test this new model of how ligand-receptor interactions mediate the dynamic process of dendrite development characterized by stochastic growth and retraction but ultimately gives rise to arbors with stereotyped shape with three aims. In Aim 1, we will design specific experiments to dissect the function of the ligand free DMA-1 and ligand bound DMA-1. In Aim 2, we will understand how the cell biology of dendrite guidance receptor is regulated to achieve its function. The size of the dendrite is also determined by its interaction with a neighboring neuron’s dendritic arbor. In Aim 3, we will understand the molecular mechanisms of dendritic tiling by identify the morphogenetic factor and the dendrite-dendrite interaction that limits the size of PVD dendrite. In summary, this proposal will systematically address the molecular and cell biological mechanisms that specify the dendritic arbors.

NIH Research Projects · FY 2026 · 2013-06

The vertebrate retina is comprised of ~100 different cell types that process visual scenes in complex ways. The functions of most of these cells under natural visual processing are unknown, as are the effects of diseases when those cell types are disrupted. Understanding the specific visual functions of retinal circuits has typically proceeded from an identification of an important visual phenomenon such as motion processing, adaptation or prediction, followed by ad hoc experiments that often rely on fortuitous knowledge of pharmacology, physiology, or the random selection of neurons, and necessarily require the use of artificially structured stimuli that have an uncertain relationship to natural scenes. We have developed a highly integrated experimental and computational approach that begins with experiments using natural scenes to create a generalizable and interpretable computational model, and automatically proceeds to testable hypotheses for specific cell types and visual computations under natural scenes. Our approach relies on interpretable neural network models that both capture natural scene responses and a broad range of phenomena of ethological visual processing, and have internal components that carry predictions about the responses and actions of real interneurons. We focus here on a predictive phenomenon that we have previously identified in mammalian and non-mammalian retina known as sensitization, whereby strongly stimulated ganglion cells increase their sensitivity to stimuli that are more likely to occur. In the mouse retina we will test the hypothesis that under natural scenes, sensitization is generated by a diverse set of inhibitory amacrine cells, each of which sensitizes a particular visual feature sensed by ganglion cells. A second goal relates to the critical function of the retina to distinguish between different visual stimuli. Although there has been a great deal of focus on changes in visual sensitivity, the true function of the retina is to support behavior by allowing the higher brain to discriminate between stimuli, from simple determinations of motion direction and object location to complex object recognition. Stimulus discriminability not only depends on the sensitivity of neurons, but also on their stochasticity or noise. We have created neural network models that capture both ganglion cell responses to natural scenes and their stochasticity including noise correlations, allowing us to calculate discriminability for any stimulus condition including natural scenes, and to test experimentally how the actions of interneurons influence discriminability. We study how specific amacrine and ganglion cells influence discriminability. Understanding how visual processing under natural scenes is generated by retinal interneurons is critical to our understanding of retinal diseases involving the degeneration of retinal circuitry. In addition, the computational descriptions of retinal responses under natural scenes will be directly useful in the design of electronic retinal prosthesis systems to best support diverse visual behaviors.

NIH Research Projects · FY 2025 · 2013-02

Food allergies (FAs) are a world-wide problem, and allergies to multiple foods are particularly problematic. Peanuts or tree nuts cause the majority of these deaths, and a recent survey in the U.S. found that 1.4% of the population is allergic to peanuts or tree nuts and ~30% of patients with FAs have allergies to multiple foods. In peanut allergy (PA), landmark studies by A. Wesley Burks and colleagues have shown that children can be desensitized to peanut via an oral immunotherapy (OIT) protocol. We have replicated these results in adults and children, and also have carried out a pilot study showing that FA patients can be desensitized to multiple food allergens simultaneously (i.e., multi-OIT). Moreover, we found that there were fewer adverse events in the build-up phase of multi-OIT if patients received the anti-IgE antibody, omalizumab, concomitantly with multi-OIT. In an effort to improve understanding of the systemic and local (i.e., GI) immune responses that underlie PA and therapeutic responses to OIT, we have completed a placebo-controlled, randomized, phase 2 clinical trial of OIT in 120 children and adults with PA (the POISED trial), and have applied state-of-the-art human immune monitoring methods to analyze blood and GI tissue specimens of participants in that study. In our current AADCRC U19 program, we are conducting a placebo-controlled, randomized, phase 2 clinical trial of OIT with or without omalizumab or the anti-IL-4Ra antibody, dupilumab, in children and adults with multiple FAs (multi-FAs). We are assessing a broad range of cellular findings in the blood, as well as serologic and clinical findings, in longitudinal samples from multi-FA participants in three treatment arms: omalizumab + OIT (N=50), omalizumab & dupilumab + OIT (N=50), and dupilumab + OIT (N=10). We will use these data to define how key immune parameters change during multi-OIT, and which are most predictive of the nature and durability of patient responses to this therapy. Specifically, we will evaluate whether there are blood-derived biomarkers that predict therapeutic responses to individual allergens, or to all allergens, in multi-OIT. In addition, we will seek to identify immune monitoring parameters, including findings derived from analyses of basophil phenotype and function that can be rapidly performed in a clinical laboratory using small amounts of blood, that could be used to predict the clinical reactivity to offending allergens in multi-FA subjects, to improve the safety and efficacy of OIT protocols, and/or to tailor the OIT protocol to each individual subject.

NIH Research Projects · FY 2025 · 2012-09

Excessive alcohol drinking Initiated during adolescence is known to disturb typical neurodevelopmental patterns, increase the risk of developing alcohol use disorder (AUD), and accelerate involutional processes in adulthood. In response to RFA-AA-21-009, the Data Analysis Resource (DAR) proposes to support the next 5 years' data collection and analysis across a community sample that were recruited in 3 age bands between 12 and 21 years old, were mostly no-to-low drinkers, and tracked over the last 8 years across 5 sites (N=831; 93% retention rate). Monitoring has involved annually-acquired multimodal neuroimaging (MRI, DTI, resting state fMRI, task fMRI) and cognitive, clinical, behavioral, and biological data, collected in person or remotely by computer and our mobile app. These measures will now be complemented with new advanced neuroimaging and sleep and physical activity tracking. This cohort sequential design uniquely positions NCANDA-A to quantify transient or enduring alcohol-related disturbances in specific adolescent and early adult neural system growth trajectories and functional concomitants. NCANDA-A proposes four consortium-wide specific aims and two specialty project aims. In Aim 1, NCANDA-A will investigate the impact of excessive alcohol drinking during adolescence and emerging adulthood on subsequent developmental trajectories of cognitive performance, brain structure and function, and psychopathology. Aim 2 analyses will identify neurodevelopment patterns describing the extent to which alcohol’s effects on brain structure and function resolve or persist during desistance after binge drinking. Aim 3 will deploy data-driven analysis to identify adolescent biological, environmental, and behavioral factors (e.g., age of drinking onset) that forecast excessive drinking during early adulthood. In Aim 4, NCANDA-A will quantify the impact of stressful events on social, emotional, and economic wellbeing and their relations with alcohol use patterns. For each aim, differences in development, alcohol use patterns and history, impact of alcohol use on the brain, and psychosocial factors will be tested. The goal of the DAR is to support hypothesis testing based on five aims. Aim D1 will ensure that procedures for collection and quality control of neuroimaging, neuropsychological, and clinical assessment data are standardized. In Aim D2, the DAR will advance the existing informatics infrastructure for integrating data collected across all sites. Aim D3 will enhance macrostructural, microstructural, and functional neuroimage processing and analysis. In Aim D4, the DAR will create machine (deep) learning frameworks identifying predictive markers of early adulthood drinking. Aim D5 will maintain data sharing and distribution systems for consortium PIs and the scientific public at large. With the longitudinal data collected into early adulthood during this renewal, NCANDAA will provide novel information to the public on the enduring and transient effects of adolescent drinking on adult functioning.

NIH Research Projects · FY 2026 · 2012-08

Project Summary Hedgehog (Hh) signaling specifies the embryonic tissue pattern of many metazoan organs and maintains this tissue pattern post-embryonically by regulating the expression of proliferation- or differentiation-inducing signals that target adult tissue stem or progenitor cells. Drugs developed to block Hh pathway activity, based on our previous work, have received FDA approval for treatment of ectodermally-derived cancers, such as basal cell carcinoma. In pancreatic, bladder, and other cancers of endodermal origin, Hh pathway activity in tumor- associated stroma presents a barrier to tumor growth and progression, thus suggesting pathway activation rather than inhibition as a therapeutic approach. In addition, pathway activation has a beneficial regenerative role in bone and muscle repair, in reducing pathology associated with inflammatory bowel disease, and in preventing or ameliorating injury and breach of the blood-brain-barrier, among other emerging biological activities. On the other hand, chronic low-level elevation of pathway activity in the lung, as is associated with reduced expression of the Hh pathway inhibitor Hhip (Hh-interacting protein), is genetically linked to chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide. During the previous funding period for this project we utilized protein structure determination and biochemical and cell biological approaches to establish the molecular mechanism of Hh signaling, in which Hh binding to its receptor Patched1 (Ptch1) activates the pathway by alleviating Ptch1-mediated suppression of the essential transducer and GPCR family member, Smoothened (Smo). We found that cholesterol is the crucial link between Ptch1 and Smo, that cholesterol in the inner leaflet of the membrane is decreased by Ptch1 transport activity, and that Hh binding to Ptch1 blocks this transport activity. These events critically regulate pathway activity, as conformational switching of Smo to its active state requires entry and binding of a sterol from the inner leaflet of the membrane into a central cavity within the Smoothened seven-transmembrane bundle. We also showed how the Dispatched1 (Disp1) transporter, structurally related to Ptch1, uses Na+ flux to power its export and packaging of the dually lipid modified Sonic hedgehog protein signal (ShhNp), enabling it to move through tissues as a soluble morphogen in complex with its carrier Scube2. We propose here to deepen our understanding of Hh signal transduction and pathway regulation by establishing the energy sources and the step-by-step lipid- handling mechanisms of the Ptch1 and Disp1 transporters. We will determine the structure of the ShhNp:Scube2 morphogen, and the mechanism of its release from Disp1. Finally we plan to elucidate the mechanism of Hh signal antagonism by Hhip, using cryo-EM to determine the high-resolution structure and functionally dissect a membrane-associated tent-like Hhip multimeric complex that occludes all receptor-interacting surfaces of the Hh protein. Our findings may provide a basis for new approaches to therapeutic modulation of Hh pathway activity.

NIH Research Projects · FY 2026 · 2012-07

Project Summary ATP-Dependent Chromatin Remodeling in Cancer Chromatin and epigenetic regulators have emerged as important contributors to human cancer. The subunits of the mSWI/SNF or BAF ATP-dependent chromatin remodeling complex are mutated in over 20% of human cancers. In addition, several other ATP-dependent remodelers make important contributions to the pathogenesis of specific cancers. These complexes often function as genetically dominant tumor suppressors, however the BAF complex also plays oncogenic roles in synovial sarcoma and squamous cell carcinoma. Despite their prevalent roles in human cancer their oncogenic mechanism(s) remain unclear and a detailed molecular understanding, necessary for therapeutic development, has been elusive. One of the most well documented roles of BAF complexes is their opposition to Polycomb Repressive Complexes (PRC) complexes. Indeed, BAF subunits were discovered in flies as suppressors of PRC1 mutations. In addition, inhibition of PRC is therapeutic in some cancers having loss of function mutations in the BAF complex. However, the mechanisms underlying the opposition between BAF chromatin remodeling complexes and Polycomb complexes is still unclear. We will obtain a detailed understanding of the physical interaction between these complexes and explore their oncogenic roles with the goal of identifying potential sites of therapeutic intervention. One possible mechanism underlying the BAF-PRC opposition is the potential of BAF complexes to exchange or evict nucleosomes modified by PRC1 and 2. Measured rates of nucleosome exchange by several techniques and by several groups show that nucleosomes exchange several times per cell cycle. This observation seems inconsistent with the widely held concept that histone and/or nucleosome modifications are the basis of epigenetic and phenotypic stability. Another way of stating this is “why do the 32 SNF2-like ATP-dependent remodelers encoded in the mammalian genome not quickly erase all histone modifications by nucleosome exchange? This conflict would be resolved if nucleosome exchange by ATP- dependent remodelers was selective to a specific remodeler and a specific nucleosomal modification. Thus, we are developing two new techniques that will fill this gap in our knowledge by measuring exchange of specifically modified nucleosomes and attributing them to specific remodelers, including their post translational or oncogenic modifications. These techniques should allow the understanding of the paradox that rates of nucleosome exchange appear to be far faster than the rate of change of histone modifications. We will use these techniques to assign changes in the epigenetic landscape to specific ATP-dependent chromatin regulators and to understand the stability of epigenetic histone modifications in normal and malignant cells. At the conclusion of our studies, we hope to have a deeper and more detailed understanding of both the normal and oncogenic mechanisms related to mutation or dysfunction of the mSWI/SNF or BAF chromatin remodeling complex.

NIH Research Projects · FY 2025 · 2012-05

PROJECT SUMMARY While dilated cardiomyopathy (DCM) is one of the most common hereditary heart diseases, we currently lack effective and targeted treatments. Cardiac fibrosis is an important hallmark of DCM and the presence of excessive fibrotic tissues can severely hamper heart function. However, limited information exists on how DCM mutations can induce cardiac fibrosis in familial DCM patients. This study aims to address this significant knowledge gap using patient-specific human induced pluripotent stem cells (iPSCs) generated from common DCM patients. In Aim 1, we will differentiate iPSCs into cardiac fibroblasts (iPSC-CFs) and cardiomyocytes (iPSC-CMs) to study how cardiomyocyte-specific and non-cardiomyocyte-specific mutations can activate cardiac fibroblasts. In Aim 2, we will examine whether iPSC-CFs induce cardiac dysfunction through secretory factors and/or direct interaction with cardiomyocytes using a 3D engineered heart tissues (EHT) platform. In Aim 3, we will identify gene/mutation-specific druggable targets using state-of-the-art CRISPR/dCas9i (inhibitory) and CRISPR/dCas9a (activating) technologies, and validate these targets in an established DCM mouse model. By completing this study, we expect to identify fibrosis-inducing DCM mutations, understand how they activate cardiac fibroblasts, and potentially discover novel druggable mechanism to manage DCM. Collectively, this multi- PI R01 study has tremendous translational value to aid future precision cardiovascular medicine.

NIH Research Projects · FY 2024 · 2012-02

Project summary A major goal of genetics and evolutionary biology is to understand how changes in genotype affect phenotype. Genetic variants affecting fitness are especially informative for investigating evolution, since natural selection acts exclusively on these variants. By identifying specific variants that influence fitness, we can begin to understand the molecular mechanisms driving the incredible adaptations of all organisms to their environments. We recently developed an approach that allows us to edit genomes with unprecedented efficiency (~100%) and throughput, and precisely measure each edit’s effect on fitness. In our initial screen, we measured the fitness effects of 16,000 natural genetic variants differing between two strains of Saccharomyces cerevisiae. In this pilot experiment, we measured the effects of each variant in isolation, in a single condition. We found that nearly all strong fitness effects were from promoter variants, rather than protein-coding regions, and these were especially enriched at transcription factor binding sites. Here we propose to utilize this powerful system to investigate two concepts of fundamental importance: the role of selection in shaping genetic variation in Aim 1, and gene-by-environment (GxE) interactions in Aim 2. This project will reveal key insights into the evolutionary process that would be unapproachable without our high-throughput precision genome editing technology.

NIH Research Projects · FY 2025 · 2011-08

Project Summary/Abstract The goal of our work is to develop a high-resolution electronic epiretinal implant for treating incurable blindness from retinal degeneration. To further this goal, we propose here to develop novel techniques for adaptive, high-resolution, multi-electrode recording and stimulation of retinal ganglion cells (RGCs) in the isolated macaque and human retina as an experimental lab prototype for the future device. The major goals of this project are to (1) adaptively use current steering with electrode triplets to enhance selective targeting of RGCs, (2) adaptively use spatio-temporal dithering and multiplexing to produce naturalistic activity in large RGC populations that can support high-quality visual coding, and (3) test the fidelity of electrically evoked visual signals in RGCs of the central human retina. Tackling these aims will allow us to emulate the neural code for vision, cell-by-cell and spike-by-spike, over a region of the central retina. Our unique technical approach involves large-scale, high-resolution electrical recording and stimulation, combined with novel computational approaches to adjust device function to the complex circuitry in which it is embedded, including the degenerated retina, and thus to optimize vision restoration.

NIH Research Projects · FY 2024 · 2011-04

Each year in the U.S., more than one in 10 children are born preterm (PT). Approximately half of very preterm survivors, born at < 32 weeks’ gestation, develop language-based learning impairments that may be discovered late and put children at substantial risk for poor outcomes throughout their lives. Our previous grant (HD069150) convincingly demonstrated that language processing efficiency, assessed at 18 months in an eye- tracking paradigm, called looking-while-listening (LWL), was more predictive of long-term outcomes than standardized tests and parent reports. PT children who were faster at language processing at 18 months showed advantages in both verbal and non-verbal skills at 54 months. Our next step is to understand early predictors of language processing efficiency in PT children. In this renewal, we enroll PT neonates (n = 140) from two language groups, primarily English- and primarily Spanish families, to increase the diversity of our sample and to improve generalizability. We assess social-environmental predictors at 12 months (infant environment) and 18 months (toddler environment) using day-long audio recordings of the child’s language environment and naturalistic laboratory observations of caregiver-child interactions. We assess neurobiological predictors, focusing on white matter microstructure, in the neonatal period (neonatal scans) and at 12 months (infant scans). We use two complementary types of MRI scans to assess white matter axonal properties and myelin content. At 18 months, the primary outcome measure is language processing speed in the LWL task, the time it takes the child to shift eye gaze to the picture of an object that was just named. Parent reports of vocabulary and scores on a standardized test of language development are secondary measures. Our aims are to: (1) determine if properties of the infant and/or toddler environments predict language processing speed and secondary outcomes in PT children from the two language-groups; (2) determine if properties of white matter pathways, assessed from neonatal and/or infant MRI scans, predict language processing speed and secondary outcomes, after consideration of language group, clinical variables, and other covariates; and (3) investigate the contributions of social-environmental factors and white matter development on language processing speed in this diverse sample of children born PT. Our main hypothesis is that relations between language learning environments and language processing speed are mediated by changes in white matter development, suggesting that supportive learning environments impact language outcomes because learning environments advance the development of white matter microstructure. The demonstration that white matter change mediates the association of social-environmental factors on language outcomes provides a clear example of experience-dependent plasticity in the human brain. This finding would represent a theoretical contribution to models of learning and development in PT children across language groups and would inform clinical practice and early intervention for PT children who are at high risk for poor language outcomes.

NIH Research Projects · FY 2024 · 2010-09

Abstract The goal of this project is to describe the function of synaptic adhesion molecules of the Neuroligin family (Nlgns) in the mouse brain and in human neurons. Recent single cell expression studies have highlighted the obversation that Nlgns are expressed also in non-neuronal cells, in particular oligodendrocyte precursors cells (OPCs) and astrocytes who express Nlgns to even higher levels than neurons. Since little is known about the function of Nlgns in glia and their effect on neurons and neural circuits, we propose to specifically delete Nlgns in OPCs and astrocytes using our triple conditional Nlgn1-3 knock-out strain. Brains will be characterized morphologically, electrophysiologically on the cellular and circuit level, and mutant mice will be characterized by behavior. Next, we will perform an in-depth molecular characterization of the Neuroligin proteins by characterizing the molecular mechanisms underlying the surprising functional diversity of Nlgns. We will map their functional domains in mouse neurons by expressing various domain-mutant proteins in Nlgn1-4 quadruple knock-out cells. We will explore whether Nlgn sequence relates to functional specificity and investigate the notion of a synaptic Neurexin “code” that may determine Nlgn specificity. To complement our mouse studies and explore human-specific Neuroligin function as well as human disease-associated mutations, we will capitalize on our previous human stem cell and reprogramming work in which we have developed human induced neuronal (iN) cells that exhibit all principal functional properties of primary mouse neurons including robust synapse formation. We propose to utilize this system to investigate the so far obscure function of NLGN4Y, a Y chromosomal gene closely related to NLGN4 on the X- chromosome and a member of the family not present in mouse. We will assess subcellular targeting by tagging the endogenous locus and assess the functional consequences of genetic deletion. Another frequently mutated Nlgn gene is NLGN3. Unlike NLGN4 it is better conserved in mice, but almost nothing is known about its function in human cells. In addition to generate loss-of-function alleles, we will study the functional consequences of distinct ASD-associated mutations introduced into the human NLGN3 gene. We will use a conditional mutagenesis approach as we have successfully done in the past, as it allows the generation of a perfect control conidition derived from the identical cell line as the experimental condition. Mutant human neurons and controls will be characterized biochemically, morphologically, by gene expression, and electrophysiologically. Finally, we propose to investigate the role of the proposed Nlgns-modulators MDGAs which are also found mutated in ASD and other neurodevelopmental disorders. We will assess their requirement for proper synapse formation and function by generating loss-of-function alleles in human neurons. We will further probe their function as Neuroligin modulators as competitive Nlgn binding molecules.

NIH Research Projects · FY 2026 · 2010-09

Population-based studies identifying the genetic variants that affect complex human diseases have relied heavily on population-genetic principles in important tasks such as study design, quality control, and genotype imputation. The dramatic growth of large-scale genotyping and sequencing studies of disease generates new challenges both for modeling the underlying generative population-genetic processes that give rise to evidence of disease association in data sets and for performing statistical analysis to uncover disease variants. These challenges magnify the potential for approaches grounded in population genetics to maximize the return from ongoing investigations. This project builds on productive efforts in two previous funding periods, capitalizing on the study of human population genetics to enhance the design, analysis, and interpretation of genomic studies of disease. It exploits the fundamental principle of human genetics that population-genetic phenomena are responsible for homozygous placement of recessive risk variants, and the recent recognition that accumulations of runs of homozygosity (ROH), and hence, of multiple recessive deleterious variants of small effect in homozygous form, can contribute to disease risk. Particularly for large-scale genotyping and low-coverage sequencing studies, in which rare recessive variants are difficult to analyze, this project uses the population genetics of ROH to enhance discovery. The project expands beyond the setting of rare diseases in small populations, building on observations that ROH and accumulations of recessive deleterious variants of small effect contribute to complex disease risk in outbred groups, including admixed populations. (1) We will construct models of the effects of interacting population-genetic forces on ROH. Such models will make it possible for researchers to attribute ROH patterns to effects of inbreeding, population size history, admixture, and selection against deleterious recessive variants. (2) We will develop powerful new tests that measure effects of ROH on complex disease risk. These tests will employ population-genetic models that incorporate features of genetic architecture and genomic parameters to assess if associations between ROH and disease reflect the likely presence of recessive disease variants. (3) We will differentiate between germline and somatically acquired homozygosity, leveraging signals in intermediate data types and genotype distributions from population genetics, to identify false-positive ROH and to refine detection of chromosomal alterations. (4) We will comprehensively evaluate the impact of ROH on medical traits in multiple disease studies, using the Michigan Genomics Initiative and UK Biobank to test and inform our approaches. The application of association testing between ROH and disease will contribute a phenome-wide association study to identify traits for which ROH variables possess meaningful predictive connections to phenotypes. To facilitate use of our methods, we will produce, test, and distribute new user-friendly software.

NIH Research Projects · FY 2026 · 2010-09

PROJECT SUMMARY Meta-learning refers to the ability to learn to learn. Whereas much progress has been made in understanding the neural mechanisms of learning, the neural mechanisms of meta-learning are a mystery. This project will investigate a candidate synaptic mechanism of meta-learning. Using integrated molecular, cellular, systems, and behavioral neuroscience approaches, the proposed research will test the hypothesis that the timing rules governing synaptic plasticity are themselves learned. Preliminary results suggest that the timing rules for associative synaptic plasticity in the cerebellum can be adaptively tuned though experience to solve what theorists have called the temporal credit assignment problem, precisely compensating for delays in the feedback about behavioral errors, so that only synapses that were active around the time that an error was generated undergo weakening during learning. If confirmed, this would represent a new dimension of the algorithms that neural circuits use to tune their own performance through experience, with broad scientific and clinical implications.

NIH Research Projects · FY 2025 · 2010-07

This proposal is designed to provide circuit-dynamics understanding of anhedonia, a psychiatric symptom domain of enormous clinical significance that is well-suited for study in laboratory animals. This work, alongside our recently-developed methods for obtaining brainwide cellular-resolution activity readout and control, has created a powerful and fortuitous alignment enabling us to bridge local and global neuronal dynamics, and to identify brain-spanning circuitry mediating behavioral drives, conflicts, and resolutions. In Aim 1, we identify single-cell-resolved orbitofrontal (OFC) dynamics underlying distinct consummatory behaviors. We have developed a temporally-precise alternative-choice mouse-behavioral paradigm, crucially designed for compatibility with our wide-field cellular-resolution imaging/recording methods, in which mice select among multiple motivational drives, and adjust action planning in light of internal or external context. We apply this paradigm along with our cellular-resolution readouts and analyses, beginning with addressing both hunger and thirst in OFC. We identify dynamics of motivational drive resolution both in the presence or absence of controlled internal states, and in the presence or absence of external (social) context, using our new methods; we hypothesize from prior work (Jennings et al., Nature 2019) that resolution of these conflicts will depend upon not only the motivational (internal) state of the animal but also the external context. In Aim 2, we map causal global dynamics of motivational drive conflict and resolution, quantifying the high-speed cellular-resolution brainwide circuit dynamics underlying these motivational drive interactions (drives naturally-occurring; or, to leverage our fast electrophysiological readout, instead induced in temporally- precise fashion by optogenetically driving AGRP neurons in the case of hunger, and/or SFO inputs to the MnPO in the case of thirst, using our established models and methods; Allen et al., Science 2019; Jennings et al., Nature 2019; Marshel et al., Science 2019). Identification of novel region-specific dynamics in conditions of varying motivational drive and social context will feed back to inform Aim 1 imaging workflow, already with a firm foundation from our prior work imaging OFC states corresponding to social and thirst drive interaction. In Aim 3, we define cells underlying inter-drive competition and corresponding brainwide dynamics. Multiple single cells identified by natural activity will be optogenetically targeted with our unique wide-field and high-resolution spatial light-guidance technology. We register cellular ensembles observed to be naturally and causally involved, to detailed 3D intact-tissue (STARmap) transcriptomic information from the same cells in the same organism. Alignment with wiring-based anatomy and deep molecular datastreams allow cell-type- resolved and single cell-level insight into, and targeting of, survival drive competition and resolution processes, with both basic significance and relevance to brain disease. Together, the approaches proposed here will integrate novel technology to probe causal underpinnings of key symptom domains in freely-moving mammals.

NIH Research Projects · FY 2026 · 2010-07

The Stanford University School of Medicine is a research-intensive medical school with a rich history of innovation and translation of biomedical science from the bench to the bedside. This is the second competing renewal for the Training Program in Myocardial Biology at Stanford (TIMBS). Our program faculty are drawn from departments at Stanford across many disciplines, and represent world renowned experts in their chosen domains. The cohesion of this group, which includes physiologists, molecular biologists, engineers, geneticists, and cardiologists has been fostered by the principal investigators in an environment which has for decades placed a high value on multi-disciplinary collaboration. Close co-localization of basic science departments and bioengineering with the school of medicine and brand-new adult and pediatric hospitals facilitates great synergy and a culture of innovation and translation. Although still relatively young, the TIMBS program has over the last nine years already established itself as an important venue for the training of post-doctoral fellows. The success of our initial trainees in obtaining grants and faculty positions, even at this early stage, is testament to the talent of the applicants, the training provided, and the priority given the program by the principal investigator mentors and faculty. This renewal application underscores and seeks to build on this proven success. In particular, we reaffirm our commitment to rigorous scientific training by offering a period of up to three years of protected time for research. The commitment begins with a ‘mentored’ recruitment process, continues with a comprehensive approach to learning the scientific method with opportunities ranging from dedicated training seminars to a ‘visiting internship’, extensive mentor-trainee interaction, and a comprehensive series of lectures and courses, including biostatistics, the responsible conduct of research, and rigor and reproducibility, as well as our highly successful grant writing academy specifically targeted to K award applicants. Our commitment extends well beyond the completion of the funded period with programs for career development, assistance and advice to ensure our trainees’ success as they enter into full time academic positions. Our aim is to mentor the next generation of leaders in myocardial biology. Our obligation extends until our trainees fulfill this goal and beyond.

NIH Research Projects · FY 2025 · 2010-07

This Mechanisms and Innovations in Vascular Disease T32 Training Program provides valuable, unique, and innovative scientific training and professional development opportunities to a group of promising early career investigators in vascular disease research. This T32 program supports NHLBI’s mission to develop novel integrative strategies to prevent, diagnose, and treat cardiovascular diseases, while at the same time fulfilling its educational goal to train tomorrow’s leaders in basic, translational, and applied vascular research. This T32 is Stanford's only University-wide effort to develop the next generation of translational vascular investigators. The program goals include foundational training in cardiovascular research; complemented by rigorous training in the responsible conduct of research, methods to enhance rigor and reproducibility, and professional development topics to support trainees’ transition to independence. Trainees develop a focused area of vascular research expertise and are exposed to a wide range of complementary research techniques. Trainees are embedded in our institution’s large and thriving vascular research community. They are supported in their research focus, as well as training in rigorous and responsible research, by their primary research mentor, a co-mentor with complementary expertise, as well as the program directors. Additional training milestones include presentation at institutional and national conferences, authorship of primary research publications, and submission of applications for independent funding. Former trainees have become independent researchers in premier academic institutions and industry; and are emerging leaders in the field of vascular research. The program proposes to continue training 6 postdoctoral fellows in multidisciplinary vascular research. Fellows are appointed to this T32 annually, with a strong encouragement to seek their own funding for additional years as part of the skills imparted by the program. To date, 47 trainees have benefited from this program. Six fellows are currently in training. Evaluations indicate a high degree of satisfaction with the program. The Program is directed by Philip Tsao, PhD and Nicholas Leeper, MD; who have extensive experience in conducting high impact vascular research, mentoring early career scientists, and leading institutional training programs. Administrative and program management support is provided by a dedicated team of educators in the Stanford Cardiovascular Institute. An Internal Advisory Board consisting of senior Stanford faculty from a broad range of disciplines and an External Advisory Board consisting of leading experts in vascular medicine and research in the US play a vital role in monitoring the progress of this training program, providing ongoing support and advice as needed. Our overarching goal is to train the next generation of investigators in vascular research, and to facilitate their transition into productive and successful academic and industry leaders.

NIH Research Projects · FY 2025 · 2010-07

This competing continuation application for the “Stanford Research in Anesthesia Training Program (ReAP)” seeks an additional five years of support for a postdoctoral research program to train leaders in academic anesthesia. We recognize that in order to accomplish this goal, substantial training beyond an MD or PhD is required. ReAP provides the guidance, training, and mentoring critical for the successful initiation of an independent research career and becoming a leader in the broad field of Anesthesiology. Trainees must learn to pose important and well thought out questions, to think critically, and to use cutting edge interdisciplinary tools to answer these questions. Success also requires the development of skills in presentation of results in oral and written format, in preparation of competitive grant proposals, and in the ability to engage in collaboration when this will more effectively advance the research. The training program starts by recruiting the most talented trainees from MD/PhD, MD and, occasionally, PhD applicants interested in pursuing a career in anesthesia research and academic anesthesia. This recruitment is facilitated by our department’s research training continuum featuring both a formal residency research track and, later, comprehensive support in transitioning to a junior faculty position. Once appointed, ReAP trainees select a primary research mentor and, as needed, a secondary mentor to monitor and facilitate their progress. Close interaction with mentors and other accomplished faculty is essential to master critical skills that form the core of our training program. This is supplemented by didactic material, and, in the case of clinical research, may be augmented further by a master’s degree in epidemiology or health services research. Administratively the program consists of a Program Director, Associate Program Director, Steering Committee, External Advisory Committee, Administrative Support and a group of 23 highly skilled and successful training faculty from the anesthesia department and 5 other departments within the medical school. There are already established interactions among many of the faculty members. The faculty is divided into four overarching areas: 1) Perioperative neuroscience & neural mechanisms of drug action, 2) Basic, translational and clinical pain, 3) Outcomes, economics and data science, and 4) Precision Perioperative Medicine & Inflammation. These divisions encompass research areas at the forefront of our field. Our institutionally well-supported program and pipeline of highly qualified candidates will easily support a total of four trainees with two appointed per year anticipating two-year training experiences for most candidates.

NIH Research Projects · FY 2025 · 2010-06

The human gastrointestinal tract houses a complex gut microbiota, or microbiome that is tightly linked with numerous inflammatory and metabolic diseases. The gut microbiota influences local and systemic immune responses, chronic low-grade inflammation, and plays an important role in the pathogenesis of obesity-related insulin resistance, type 2 diabetes, and inflammatory bowel diseases. Diet is a key modulator of gut microbiota metabolism, composition, and functional capacity as well as human health. Despite recent progress, still relatively little is known about the mechanisms that connect specific diets and molecules within diet to the microbiota and human health. Fermented foods have been recently shown to increase human gut microbiota diversity and decrease markers of inflammation, and preliminary data within this application show that fermented vegetable brine (FVB), filter sterilized FVB (sFVB) and lactate (a major metabolite within sFVB) can induce regulatory T cells (Tregs) in mice. This application is focused on investigating molecules within fermented vegetable brine, with a focus on lactate, that are capable of modulating aspects of host biology relevant allergy and inflammation and understanding how cues from gut microbes impact these interactions. The goals of this application include (i) defining the mechanisms by which fermented foods vegetable brine facilitate the engraftment of new strains to increase microbiota diversity, (ii) characterizing the requirement of gut microbes in lactate induced Treg expansion, (iii) determining the host perception pathways that are responsible for lactate-induced Treg expansion in a mouse model. Aim 1 will determine how microbiota diversity acquisition, a feature of gut microbiomes linked to health, is promoted by fermented foods. This aim will reveal to what extent diversity increase is intrinsic to a microbiota versus requires exogenous microbes. Aim 2 will investigate the dependence of lactate induced Treg expansion on components of the gut microbiota. Experiments will identify how specific gut resident microbes impact Treg expansion in the presence of oral lactate and what molecular cues are required for Treg expansion. Aim 3 will employ mice genetically lacking lactate-binding G-protein coupled receptors to establish the basis of lactate perception. This project will advance our understanding of how fermented foods and metabolites within these foods impact the microbiota and host biology for improved health.