Vanderbilt University

NIH Research Projects · FY 2026 · 2016-09

PROJECT SUMMARY Dynamic remodeling of the microtubule cytoskeleton is crucial for a variety of cellular processes, including cell division, cell motility and differentiation. Microtubule cytoskeleton reorganization relies on the control of individual microtubule polymers, which switch between phases of growth and shrinkage through a process known as microtubule dynamic instability. Although dynamic instability was discovered decades ago, the molecular mechanisms that underlie microtubule catastrophe and rescue, the transitions between phases of growth and shrinkage, and their control through collective effects of a myriad of regulators are still being unraveled. The goal of this project is to elucidate the fundamental mechanisms underlying microtubule dynamics. Our central hypothesis is that conditions experienced at the time of growth have long-term effects on subsequent microtubule behavior, including catastrophe, shrinkage and rescue. To test this hypothesis, we will employ highly-controlled in vitro reconstitution experiments, combining purified protein components, microfluidics and high spatiotemporal resolution light-microscopy approaches. We will determine the different impacts of distinct growth conditions at the two microtubule ends, giving rise to their unique dynamic behaviors. We will elucidate individual and combined effects of microtubule regulators and their underlying mechanisms. We will particularly focus on microtubule regulators that bind both soluble and polymeric form of tubulin. At the plus end, we will investigate TOG-domain proteins XMAP215 and CLASP to elucidate the similarities and differences in their mechanisms underlying their differential effects on plus-end dynamics. At the minus end, we will investigate the interplay of stabilizing regulators, including Kinesin-14 HSET, and destabilizing regulators, including tubulin-sequestering protein Op18/Stathmin and a poorly-studied microtubule severing protein Fidgetin. Since every one of these microtubule regulators has been implicated in human disease, particularly cancer and neurodevelopmental disorders, revealing their mechanisms of action is of direct health relevance. Our quantitative in vitro measurements will enable us to develop mathematical and computational models reconciling the dynamics of both microtubule ends, and encompassing the collective effects of regulators at each end. We will directly test the models developed based on our in vitro and in silico findings in physiologically-relevant contexts using state-of-the-art fast super- resolution quantitative live cell imaging. Beyond uncovering the fundamental mechanisms underlying microtubule dynamics in cells, we will expand our cellular studies with a focus on the role of CLASP in cell migration and neuronal development. Our cellular investigations will invariably yield new hypotheses to be tested by controlled in vitro and in silico experiments. The continuous feedback between in vitro and cellular approaches will ultimately provide fundamental insights into microtubule cytoskeleton dynamics, bearing critical relevance to both basic science and human health.

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY The long-term mission of my lab is to decipher the critical pathways that sense nucleic acids as a danger, and the post-transcriptional gene regulatory components that facilitate the subsequent cellular response - with the goal that such knowledge significantly contributes to our scientific understanding of innate immunity in human health and disease. Research on innate immunity has traditionally focused on understanding the initiating triggers that ultimately modulate the transcriptional expression of a central set of interferons and cytokines. Much less is known about the post-transcriptional gene regulatory layer, which acts to refine innate immune activation at the RNA level – shaping gene expression to allow for a robust but finite host response while simultaneously preventing aberrant or pathogen- associated gene expression. This remains a striking gap in our understanding given that many aspects of host-pathogen interactions have at its core the detection and suppression of foreign nucleic acids, particularly from viruses. RNA-binding proteins (RBPs) can pre-program the sensitivity of cells to immunogenic stimuli, as well as being essential factors in the anti-viral response. Towards these interests, and in line with our outlined goals of the first cycle of our R35 funding period, we made substantial progress in our investigations on: 1)The characterization of essential and immune-relevant RBPs including ELAVL1 and the YTHDF protein family, 2) the discovery of widespread viral-host RBP interactions through our development of VIR-CLASP and characterization of their impact on viral replication, and 3) the identification and characterization of a novel small molecule catalytic inhibitor of the cGAS-STING pathway, given that we also model dynamic post-transcriptional regulation by examining how this primary cytosolic DNA sensor initiates a generalized innate immune response to perceived foreign nucleic acids. Taken together, my laboratory has grown in expertise in RNA/DNA binding protein biochemistry and -omic scale biology aimed directly at characterizing the essential roles of host RBPs and components of the cGAS pathway in conferring innate immunity. For this research renewal, we aim to expand our understanding within our established research program by pursuing the following major biological questions: 1)How do non-canonical cellular RBPs regulate and maintain host gene expression, especially during viral infection? 2)What are the unique host RBP-viral genome interactions that impact replication and virulence of related viruses within different intracellular environments during early infection? 3) What are the novel components that regulate and facilitate a non-canonical cGAS-dependent pathway promoting innate immune transcriptional activation and programmed DNA damage.

NIH Research Projects · FY 2026 · 2016-08

Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking the G protein coupling site. Second, arrestins can support G protein independent signaling. The best studied arrestin-mediated signaling pathways include the activation of mitogen activated protein (MAP) kinases and Src family kinases. Arrestins also have complex functions beyond the modulation of GPCR signaling. An increasing body of evidence suggests that arrestins may interact with some receptor tyrosine kinases (RTKs) and may also link RTKs to GPCR during some types of transactivation. In our last grant cycle, we expanded what is known about the function of arrestin in signaling, developed working assays for following arrestin functions in cells, and we developed a range of biochemical tools that can help to decipher how arrestins might link signaling cascades. We leverage these findings, tools, and assays with three aims that explore the influence of arrestins on linking signal cascades, in two aims. In Aim 1, we combine in vitro structural and biophysical techniques (array assays, X-ray crystallography, and binding measurements) with functional measurements in cells to investigate how arrestins interact with RTKs. In Aim 2, we combine in cell assays with mutagenesis and functional analysis to reveal the requirements for arrestin to link RTKs with GPCRs during transactivation.

NIH Research Projects · FY 2026 · 2016-06

PROJECT SUMMARY/ABSTRACT Over the past several decades, biomedical science has achieved dramatic breakthroughs within laboratory research. The ability to translate those discoveries as well as to make new discoveries within human investigations has been a challenge and is commonly characterized as the bottleneck of clinical research. Added to this context have been the dramatic changes to healthcare organizational environments, constraints on delivery, efficiency, and reimbursements, including recent major changes in public sentiment regarding the investment in healthcare research, both federally and industrially. Lastly, as a result of dramatic systemic changes in healthcare funding structure, the support of higher educational graduates’ careers, specifically doctoral graduates, is experiencing a considerable contraction. Within that changing dynamic landscape, we hypothesize that the constraints in clinical translational research can be dramatically loosened and the visibility to the public can be enhanced with the training of engineers intimately familiar with human treatment and trained in the inception of novel technology-based platforms. The purpose of this training program is to create a new cadre of researchers capable of creating, developing, implementing, clinically evaluating, and translating methods, devices, algorithms, and systems designed with a clear focus on one particular application of medicine, namely, to facilitate surgical/interventional processes and their outcomes. Thematically, our trainees and training program will have a central focus – innovative platform technologies for treatment and discovery. While this focus addresses the pressing problem of facilitating clinical translation research in human investigative systems, the program also provides an exceptional career experience with clinical immersion that accommodates both academic and industrial pathways. Briefly described, the training program is a year 2, 3 program that centers on a novel dual-course clinical immersion sequence (a first course that is a context heavy experience with physicians introducing their specialty and clinical realities, and a second course that is an intensively immersive environment with students embedded within the clinical team). In both courses, students are required to engage in expository writing associated with disease and therapeutic analysis, provocative question solutions, clinical outcome analysis and reviews, and mock grant applications. Apart from this sequence, training continues with additional coursework in areas associated with surgical/interventional guidance and delivery, interventional imaging, medical image processing and analysis, robotics and medical device design, modeling & simulation, interventional therapeutics, and artificial intelligence (AI) and machine learning (ML). Of particular note this cycle is an extended emphasis on medical robotics and AI/ML, as well as new ‘public facing’ training opportunities. This all takes place in the most strategically collocated environments for engineering, surgery, and intervention in the world. This is a Training Program for Innovative Engineering Research in Surgery and Intervention.

NIH Research Projects · FY 2026 · 2016-06

PROJECT SUMMARY Faithful replication of DNA and response to encounters with aberrant DNA are essential to cell propagation and survival. Our long-term goal is to understand the action of multi-protein DNA replication and damage response machinery at eukaryotic replication forks. Our strategy is to elucidate the structural mechanisms using an integrative structural biology approach, coupled to biochemical/biophysical characterization and collaborations to define functional implications. This proposal focuses on critical unsolved questions about the initiation of daughter strand synthesis in replication, and the stalling and remodeling of replication forks upon encountering aberrant DNA. In DNA replication, the processive polymerases δ and ε require a short primer strand on the template to function, which is generated by DNA polymerase a-primase (pol-prim). Although 3D structures have been determined for all components of pol-prim and even the intact heterotetramer, these have provided only limited mechanistic insights because structures of the full-length protein with relevant substrates and essential co-factors are lacking. To address this critical gap in knowledge, we propose to determine the relevant structures using Cryo-EM. We also propose to continue working on characterizing the structure, biochemical properties and functional roles of 4Fe-4S clusters in pol-prim. We will test and refine our hypotheses about the role of: (i) the primase 4Fe-4S cluster redox in modulating DNA binding activity; (ii) the role of the cluster in pol α in driving the transition from RNA synthesis by primase to DNA synthesis by pol α. Together, these studies will solve the fundamental questions about how pol-prim counts the length of the primer at each step and how the substrate hand-offs occur from primase to pol α and then from pol α to pols δ or ε. Our second project addresses two critical gaps in knowledge about replication fork encounters with aberrant DNA. RPA and Rad51 are two highly abundant ssDNA binding proteins that have critical roles in the stalling, reversal and stabilization of stalled forks. RPA-coated ssDNA is the key initiating signal for multiple damage response pathways and plays several additional roles, including recruiting and directing the fork reversal activity of the ATP motor protein SMARCAL1. We propose to elucidate the mechanisms that drive this important aspect of fork remodeling by determining the structure of the RPA and SMARCAL1 on a model fork substrate complex using Cyro-EM. Rad51 plays an essential role in the stabilization of stalled replication forks. Collaborative studies with David Cortez led to the discovery and characterization of RADX, a new DNA damage response protein involved in regulating the activity of Rad51 at stalled forks. We recently discovered RADX also interacts physically with RPA, suggesting there is a RPA-RADX-Rad51 network operating at stalled forks. We propose combined structural, biophysical and functional analyses of RADX and its interactions with DNA, Rad51 and RPA to clarify the roles of RADX at stalled replication forks. Together, our two projects will greatly enhance understanding of how DNA is processed at eukaryotic replication forks and genomes are maintained and propagated.

NIH Research Projects · FY 2025 · 2016-04

Exosomes are small extracellular vesicles (EVs) that drive cancer metastasis through paracrine and autocrine signaling. While the role of specific exosome cargoes in this process is not well understood, integrin adhesion receptors have been shown to be involved in the choice of metastatic site. How this occurs is not entirely clear, but could involve binding of exosome- carried integrins to cognate extracellular matrix (ECM) ligands in distant tissues. Another non- exclusive possibility is that exosomal integrins and other adhesion receptors directly initiate assembly of ECM to allow cancer cell survival and invasion at primary tumor sites and colonization of distant metastatic sites. We will investigate these possible functions of exosomes in breast cancer metastasis. In the prior grant cycle, we identified fundamental mechanisms by which both cancer- and stromal-derived exosomes control tumor aggressiveness, including: 1) autocrine promotion of cancer cell migration; 2) fibroblast exosomes are necessary and sufficient to induce assembly of fibronectin and other stromal matrix proteins, both in vitro and in vivo; 3) and fibroblast- secreted exosomes promote both growth and metastasis of primary breast tumors. In addition, we find that adhesion receptors are enriched on small EVs from both cancer cells and fibroblasts. Based on these other findings, we propose the central hypothesis that adhesion molecules carried by breast cancer and fibroblast exosomes drive multiple steps of the metastatic cascade. To test this hypothesis, we will: 1) Test the hypothesis that clustering of integrins and syndecans within exosomes mediates fibronectin (FN) assembly; 2) Test the hypothesis that breast cancer cell exosomes carry unique adhesion molecules that can induce assembly of epithelial-type ECM; 3) Determine the role of exosomal adhesion receptors in promoting breast cancer metastasis.

NIH Research Projects · FY 2026 · 2016-04

Project Summary Charcot-Marie-Tooth Disease (CMT) is a common debilitating hereditary peripheral neuropathy. The hallmark of CMT pathology is severely defective PNS myelin. There is currently no treatment or cure for this disease. Roughly 75% of all cases of CMT are caused by Schwann cell overexpression of wild type (WT) peripheral myelin protein (PMP22) due to trisomy (type 1A CMT), underexpression of PMP22 (for the WT/null case), or genetically dominant heterozygous (WT/mutant) mutations that alter the PMP22 protein sequence (type E CMT or the more severe Dejerine-Sottas Syndrome--DSS). Important questions remain regarding the molecular mechanisms by which these different classes of defects in PMP22 result in different forms of CMT. Here we propose to address key outstanding questions regarding the molecular bases for the different forms of CMT. Aim 1. How does the trafficking and mis-trafficking of WT PMP22 under CMT1A WT overexpression conditions differ from a folding-defective DSS mutant form of PMP22? We will test the hypothesis that misfolded WT PMP22 ends up in the cytosol, whereas DSS mutants such as L16P PMP22 are trapped in the ER or ER-to-Golgi intermediate compartment (ERGIC). Aim 2. Determine the role of the BAG6 complex (BAG6/GET4/UBL4A) in the cytosolic trafficking of WT PMP22. Our preliminary data suggests that WT PMP22, but not DSS mutant forms of PMP22 interacts with the cytosolic BAG6 complex. This aim will test the hypothesis that the BAG6 complex serves as a holdase for misfolded WT PMP22 to facilitate its healthy shuttling to the proteasome, but that excess PMP22 in CMT1A overwhelms the proteasome, resulting in formation of aggresomes, which are eventually degraded by BAG6-induced autophagy. Aim 3. Determine if excess WT PMP22 inhibits specific proteasome complexes and/or induces phosphorylation of eIF2α in myelinating SCs. Prompted by our preliminary data, this Aim will test the hypothesis that overexpression of WT PMP22 in SCs inhibits the 26S proteasome and activates an integrated stress response (ISR), resulting in phosphorylation of eIF2α to suppress protein translation.

NIH Research Projects · FY 2026 · 2016-03

Summary Islet β cells secrete insulin on stimuli to regulate whole-body glucose homeostasis. Deregulating this function results in type 2 diabetes (T2D) or hypoglycemia. To avoid pathology, β cells need proper numbers of insulin granules (IGs) positioned to their secretion sites at the cell membrane. How IG availability for glucose- stimulated insulin secretion (GSIS) is regulated is largely unclear. In this proposal, we hypothesize that microtubules (MTs) and MT-dependent molecular motors actively communicate to optimize IG delivery. MTs are long tubulin polymers with plus- and minus-ends. Molecular motors attach to cargos while associating with MTs to drive ATP-dependent transport of cargos along the MT tracks, either from the minus to the plus MT end (e.g. kinesin-1) or in a reversed direction (e.g. dynein). In addition to facilitating transport, motor activities can reorganize MT tracks, providing a feedback loop for finer cargo delivery. In most cells with MT arrays having a net directionality, selective cargo binding by dynein or kinesins will achieve directional cargo delivery. However, we and others have shown that the β-cell MT arrays are overall non-directional so that IGs are moved by motors without a net direction. Therefore, a major challenge for β cells is to tune local MT configuration at the sites of secretion for directional IG delivery and assign distinct motors to accomplish this precise delivery. This is particularly daunting for building different subpopulations of β cells for their heterogeneous function under different physiological stimuli. The major working model proposed here is that different levels/types of motor proteins in individual β cells result in different configurations of MTs and different numbers of IG delivery and removal from the section sites, promoting heterogenous secretory function. Our findings over the past funding cycle (April 2021-present) strongly support this model: (1) MTs govern the assembly of subdomains at the cell membrane which allow for higher levels of GSIS than others (i.e. secretory hot spots); (2) variation in MT stability of each β cell contributes to their secretory heterogeneity; (3) the overall MT stability in islets anti- correlates with GSIS in single islets; (4) the expression levels of several motor genes are higher in β-cell subsets with higher secretory function. Thus, our findings indicate that the MT network constitutes a signaling hub that supports the heterogenous β-cell secretory function at three levels: (1) subcellular assembly of secretion hot spots, (2) different GSIS from individual β cells, and (3) differential GSIS from individual islets. In this application, we will combine the complementary expertise of three PIs to employ high-end microscopy and computational modeling to test this model in mouse and human islets. The three aims are to determine: (1) Roles of molecular motors in peripheral MT organization for heterogeneous GSIS; (2) Role of MT plus-end- directed kinesins in IG transport; and (3) Role of MT minus-end-directed transport by cytoplasmic dynein in GSIS. We expect to unravel a novel MT-based mechanism that impacts the β-cell functional mass and integrates nutritional, hormonal, and neuronal inputs to ensure precise GSIS and glucose homeostasis.

NIH Research Projects · FY 2025 · 2016-02

PROJECT SUMMARY MYC is an oncoprotein transcription factor that features prominently in cancer. As a transcription factor, the ability of MYC to recognize its target genes is paramount to its activity. The long-standing paradigm for how MYC selects its targets is that it dimerizes with MAX, forming a module that binds specific DNA sequences in the regulatory elements of target genes. In recent years, however, it has become clear that target gene recognition by MYC can also depend on additional chromatin-resident proteins that act through avidity to direct MYC:MAX dimers to specific sites in the genome. This 'facilitated recruitment' process is poorly understood, although it likely influences a majority of MYC binding to chromatin in cancer cells. Detailed mechanistic studies of facilitated recruitment are needed to understand this most basic aspect of MYC activity, and are timely because, unlike MYC, these recruiters may be amenable to drug discovery, unlocking new ways to target MYC in the clinic. This project explores the mechanisms and significance of facilitated recruitment of MYC to chromatin by WDR5, a conserved nuclear protein that is an active target for drug discovery by numerous groups. WDR5 recruits MYC to chromatin at genes vital for protein synthesis, including a collection of ribosomal protein genes and genes encoding translation factors and nucleolar RNAs. Disrupting interaction of MYC with WDR5 in a preexisting malignancy promotes rapid and irreversible tumor collapse, indicating that the MYC–WDR5 nexus can be pharmacologically inhibited as a treatment for MYC-driven cancers. Aim 1 of this project uses a combination of high resolution genetic, genomic, and proteomic approaches to characterize the fundamental mechanisms that bring MYC and WDR5 together on chromatin, and to reveal the extent to which facilitated recruitment by WDR5—and other recruiters—determines the genes that are controlled by MYC in cancer cells. Aim 2 blends genetic, genomic, and in vivo studies to probe the importance of the MYC–WDR5 connection in a diverse set of cancer contexts, and to reveal precisely how targeting this connection promotes tumor regression. These studies will lead to a new and robust paradigm for the mechanisms of target gene selection by MYC, identify novel and targetable vulnerabilities in the MYC network, and show how gene-selective recruiters such as WDR5 can be exploited to therapeutically inhibit MYC. Importantly, these studies will also lay the biological groundwork for the implementation of WDR5 inhibitors in the clinic as anti-MYC agents.

NIH Research Projects · FY 2026 · 2016-01

The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates active I- transport into the thyroid, the first step in the biosynthesis of the thyroid hormones. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. NIS activity is electrogenic, with a 2 Na+ : 1 I- stoichiometry. Our group isolated the cDNA that encodes NIS. We have characterized the transporter extensively. We have demonstrated that NIS also transports oxyanions, such as the environmental pollutant perchlorate, but it does so electroneutrally (1 Na+ : 1 I-), showing that NIS translocates different substrates with different stoichiometries. The mechanism of transport by NIS is a topic of great inherent interest that also has significant translational applications. A key part of our progress was that we determined three structures of NIS by single-particle cryo-EM: one with no substrates bound, one with 2 Na+ and 1 I- bound, and one with 1 Na+ and the oxyanion perrhenate (ReO4-) bound. Based on these structures, we have proposed a mechanism of transport that describes the conformations the molecule must adopt during the transport cycle: inwardly open, occluded, and outwardly open. The structure with no substrates bound is in an inwardly open conformation, and that with 2 Na+ and 1 I- bound is in an occluded conformation. To fully understand the NIS transport mechanism, we need to obtain structural and dynamic information on other conformations NIS adopts, particularly its outwardly open conformation. NIS is also the molecule at the center of the treatment for thyroid cancer based on radioiodide (131I-), the most effective internal radiation cancer therapy devised to date. The availability of the NIS cDNA and now of the NIS structures raises the possibility of generating specially designed NIS mutants, with a particular substrate specificity, that can be transfected into extrathyroidal cancers, thus rendering them amenable to treatment with radioactive substrates other than 131I-, to protect the patients’ thyroids. We have engineered the Q72A-NIS mutant and the L253P/V254F double mutant (PF-NIS), both of which selectively transport oxyanions (electrogenically) but no I-. PF-NIS, because of its better kinetic properties, is the kind of engineered NIS molecule that could usher in a new era of gene transfer cancer therapies. We will use a combination of whole-cell-based biochemical experiments, electrophysiological studies, determination of the structures of NIS mutants by cryo-EM, double electron–electron resonance (DEER) spectroscopy, and atomic- level computational analysis, including statistical thermodynamics (ST) and atomistic molecular dynamics (MD) simulations, to pursue the following Specific aims: 1. Dynamics of NIS transport: a) Elucidating transport by WT- NIS and engineered NIS mutants with remarkable substrate selectivity and its statistical dynamics in atomistic detail. b) Do lipids influence the conformational states of NIS? 2. Experimentally ascertaining the conformational landscape of WT-NIS and NIS mutants when the substrates bind and are released, and when NIS transitions from one conformation to the next. 3. Determining the structure of NIS in an outwardly open (OO) conformation.

NIH Research Projects · FY 2024 · 2015-09

PROJECT SUMMARY Alzheimer’s Disease and related dementia are a growing public health crisis affecting 5.8 million Americans, yet there are only four FDA-approved medications for Alzheimer’s Disease, none of which are disease-modifying. Hence, early detection and diagnosis are key to successful patient management and biomarkers are needed for evaluating new therapies in clinical trials. White matter changes are increasingly implicated in early Alzheimer’s Disease progression, and diffusion weighted magnetic resonance imaging (DW-MRI) has been included in many national-scale studies. Yet, quantitative investigation of DW-MRI data is hindered by a lack of consistency due to variation in acquisition protocols, sites, and scanners. DW-MRI enables quantification of brain microstructure and facilitates structural connectivity mapping. Substantial recent progress has been made with calibration and harmonization to reduce inter-subject variance and improve interpretability of computed measures. Yet, the fundamental challenge remains that clinical application of DW-MRI (as currently implemented) is confounded by inter-scanner and inter-site effects. To improve understanding of structural changes in Alzheimer’s Disease, we will construct and evaluate three separate analysis strategies to characterize, calibrate, and optimize DW-MRI for single-subject biomarker development for Alzheimer’s Disease. We will integrate and optimize our strategies using large retrospective multi-site studies and validate the approaches on two distinct prospective cohorts. Specifically, we aim to: Aim 1: Optimize data-driven techniques for stability across sessions, scanners/sites, and field strengths Impact: Harmonized DW-MRI methods will increase sensitivity to Alzheimer’s Disease and its prodromal stages. Aim 2: Translate innovations in microstructural harmonization to structural connectivity (tractography) Impact: Harmonizing structural connectivity will improve understanding of white matter in Alzheimer’s Disease. Aim 3: Advance statistical tools for single-subject inference through normative database construction Impact: Data-driven resources for uncertainty estimation will enable robust single-single subject inference. Relevance and Impact on Healthcare: The proposed research will advance understanding of Alzheimer’s Disease through (1) quantitative harmonization of DW-MRI biomarkers, (2) protocols for harmonization of retrospective and prospective DW-MRI studies, and (3) new tools for single subject inference targeting older cohorts. We will organize workshops/challenges to maximize the translational impact on clinical science. The long-term goal of our research is to (1) provide a well-validated strategy to quantitatively evaluate DW-MRI data across sites, (2) enhance DW-MRI biomarkers for Alzheimer’s Disease, and (3) advance patient care. Our research strategy will transform the manner in which DW-MRI data are interpreted and enable single-subject machine learning to interpret brain properties. The resources, software, and visualization tools will be made freely available in open source through DIPY to facilitate continued innovation.

NIH Research Projects · FY 2025 · 2015-08

PROJECT SUMMARY Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Multiple repair and tolerance responses to replication stress act to complete DNA synthesis while minimizing errors. In this project we seek to understand these mechanisms with a focus on replication fork reversal. Replication fork reversal is thought to stabilize stalled forks, to place DNA lesions back into the context of duplex DNA where it can be removed through excision repair, and to provide an opportunity for template switching. Reversal is catalyzed by ATP-dependent DNA translocases and the RAD51 recombinase. It is also highly regulated, which is needed to prevent inappropriate slowing of replication and nuclease- dependent processing of replication forks. For example, we recently discovered RADX as a single-stranded DNA binding protein that regulates reversal by directly binding RAD51. In this funding period we seek to understand how RADX functions, how replication resumes following a challenge, and to rigorously test the hypothesis that fork reversal is an error-free mechanism of DNA damage tolerance. We will test and refine new conceptual models utilizing a combination of biochemical, genetic, and cell biological approaches. Our many years of accumulated knowledge and reagents and strong team of collaborators uniquely position us to complete these studies. Given the importance of replication stress responses to preventing diseases including cancer, these studies will generate discoveries that could be translated in the future to benefit human health.

NIH Research Projects · FY 2025 · 2015-06

SUMMARY The rapidly increasing number of antibacterial-resistant pathogens and the emergence of new infectious threats underscore the desperate need for novel antimicrobial therapeutics. Academic research institutions have begun to meet this need, but success requires scientists who are trained to apply the techniques of chemical biology to the study of microbial pathogenesis. The Chemical Biology of Infectious Diseases (CBID) Training Program exploits Vanderbilt’s strengths in chemical biology and microbiology and prepares scientists for the challenges of antimicrobial target identification and drug discovery. Twenty-seven faculty preceptors from eight departments in both Vanderbilt University and Vanderbilt University Medical Center serve as mentors for the Program. Four predoctoral students will be selected each year to receive two years of support during their second and third year of study in the five-year program. Highlights of the training program include formal coursework in both microbial pathogenesis and chemical biology, elective courses for specialized training, a “mini-sabbatical” research opportunity in a collaborator’s laboratory, research and professional development workshops, an interactive seminar series in chemical biology, an annual research symposium in chemical biology, participation in the infectious disease case conference and antibiotic stewardship program, an in-depth laboratory research experience, and an external internship at a major pharmaceutical company. Research projects emphasize a multi-disciplinary approach that applies chemical biology to the study of microbiology, and students are advised by at least two CBID preceptors. As a result, students who complete the CBID Training Program are well- grounded in a core discipline and sufficiently well-trained in complementary fields to allow them to work effectively in an interdisciplinary environment.

NIH Research Projects · FY 2025 · 2014-09

PROJECT SUMMARY/ABSTRACT Nonhealing skin wounds are a major source of morbidity worldwide and becoming more of a burden due to an increase in health care costs, an aging population, and growing incidence of diabetes. Non-healing skin wounds occur in nearly 25% of diabetic patients, and ~6% are admitted to the hospital for wound-related treatment, which if not successful, can lead to limb amputation or death. More advanced treatments such as synthetic, resorbable dressings that are placed into the wound to provide a scaffolding for cell infiltration and new tissue formation have started to gain clinical impact. However, currently available polymeric biomaterial wound dressings degrade by hydrolysis, releasing acidic byproducts, creating an autocatalytic degradation process. This can lead to inconsistent degradation rate over time and poor matching between the timeline of cell infiltration / new tissue formation and the timeline of polymeric scaffold resorption. The overall goal of the current project is to develop and apply a next generation cellular reactive oxygen species (ROS) degradable, fully synthetic foam wound dressing. These scaffolds are formed by the reaction of ROS-degradable polythioketal (PTK) diols with isocyanate-containing compounds in the presence of a small quantity of water. This generates a crosslinked polyurethane (resultant bond from reaction of isocyanate and hydroxyl) network that is highly porous in nature due to CO2 generation via the blowing reaction between isocyanates and water. The properties of the resultant polythioketal urethane (PTK-UR) foams can be tuned based on the composition of the PTK diol crosslinker which makes up the bulk of the foam. We propose to develop a library of PTK diols with controlled variation in degree of hydrophilicity, consistent density of thioketal bonds in the backbone, and low potential for immunogenicity. This plan is based on highly- promising preliminary data that PTK-UR scaffold hydrophilicity is a critical factor in the wound healing response to the PTK-UR biomaterials. The proposed polymer series will fill previous gaps in our previous work by yielding a well-controlled, highly-scalable chemistry for better fine tuning of PTK-UR hydrophobic/hydrophilic balance across a broad range with diol chemistries that are not based on potentially immunogenic PEG. The aims of the project will involve synthesis and screening of this new class of thioketal diols, benchmarking of the leading PTK- UR formulations against clinical products for wound healing efficacy, and application of lead PTK-UR formulations for cargo delivery to promote healing in the context of pathological (infected and diabetic) wounds. Our multidisciplinary team includes bioengineers, polymer and polyurethane material chemists, preclinical wound healing model and histopathology expertise, expertise in immunology of wound healing / skin wound infection, and clinical wound care. This group is poised to achieve the proposed goals toward establishing a new clinically impactful, cell-resorbable, synthetic polymer-based foam wound dressing.

NIH Research Projects · FY 2025 · 2014-08

Diabetes is a complex disease with multiple risk factors/causes, and a broad spectrum of complications. The rational development of novel clinical treatments for diabetes will require interdisciplinary approaches to integrate multiple data sources, build predictive models, and develop new technologies that will together reveal the multifactorial underpinnings of the disease. Such approaches are well-developed in engineering, but they have not been widely applied to diabetes, in part because of a lack of people skilled in both engineering and biomedical research. To develop such an interdisciplinary workforce, we propose to continue a novel Integrated Training in Engineering and Diabetes (ITED) program to attract both predoctoral students and postdoctoral fellows with engineering and quantitative physical science backgrounds to work on research problems at the interface of engineering and diabetes. Vanderbilt University is an ideal location for this program because of the close integration and proximity of the Schools of Engineering and Medicine, the outstanding environment for diabetes research, and the long-standing tradition of collegiality and collaboration across the Vanderbilt campus. The overall goal of the program is to provide talented individuals from engineering backgrounds with state-of-the-art training in diabetes research, which will give them a broad perspective of both basic biomedical research and associated clinical challenges. Fourty-six faculty preceptors, evenly distributed between those with an engineering background and a diabetes research focus, will work together to offer a wide variety of research opportunities in engineering and diabetes research. Our program includes: a) a novel dual mentor plan where each trainee performs interdisciplinary research in two labs, b) an efficient didactic program that provides trainees with the necessary knowledge base in a compact time-frame that promotes timely completion of their degrees, and c) interactions with other trainees, faculty, and visiting scientists. Two experienced mentors, one from engineering and one from a diabetes research background, will supervise each trainee. This interdisciplinary approach will require the trainees to spend significant time in each of the mentors’ labs. A broad educational program for predoctoral trainees will leverage existing courses in the Schools of Engineering and Medicine, supplemented by research seminars, biweekly data clubs, and an annual ITED Retreat. Postdoctoral trainees with engineering or physical science degrees will be specifically recruited to this program and will participate in all ITED activities. We do not expect all postdoctoral trainees to have a background in diabetes research, so they will achieve competency through didactic instruction to fill existing knowledge gaps. Support for 4 predoctoral trainees and 2 postdoctoral trainees is requested based on the research resources of the faculty preceptors. A Steering Committee that represents the breadth of participating investigators will select trainees based on their academic and research performance, and their promise as independent investigators. The program will provide training in RCR and Methods for Enhancing Reproducibility.

NIH Research Projects · FY 2025 · 2014-08

SUMMARY Clinical genetic testing has become standard-of-care for many diseases including the congenital long-QT syndrome (LQTS). However, interpreting genetic test results is often confounded by the discovery of ‘variants of uncertain significance’ (VUS) for which there are insufficient data to determine whether a particular variant is pathogenic or benign. The goal of this project is to use a novel paradigm for distinguishing pathogenic from benign variants in LQTS with a focus on KCNQ1, the most common cause of LQTS. During the prior periods of support, we implemented high throughput strategies to determine the functional consequences of ~180 KCNQ1 variants, the functional consequences of all known disease-associated KCNE1 variants, and assessed the stability, structure, and cell surface expression of several dozen KCNQ1 variants. We then integrated data on KCNQ1 structure, function and sequence conservation with machine learning tools to build a gene-specific algorithm in a web-based format to predict the likelihood that specific KCNQ1 variants are deleterious. In the next funding period, we propose to continue this powerful and productive multidisciplinary paradigm to extend our research. We used our machine learning approach incorporating an artificial neural network model to predict the functional consequences of 136 KCNQ1 VUS from ClinVar. In Aim 1, we will experimentally evaluate these predictions by determining functional consequences of the variants using automated patch clamp recording. In separate experiments, we will perform deep mutational scanning (DMS) of major regions of KCNQ1 (pore and voltage-sensing domains, C-terminus) to identify all possible single nucleotide variants that cause impaired trafficking of the channel to the plasma membrane. In Aim 2, we will use our quantitative flow cytometry-based method to evaluate cell surface expression of disease-associated KCNQ1 variants in regions of the channel (pore domain, C-terminus) that not been fully investigated for trafficking, and to determine if dysfunctional KCNE1 variants interfere with KCNQ1 trafficking. We will also employ biophysical methods (nano-differential scanning fluorimetry, cellular thermal shift assay) to evaluate the stability of trafficking- impaired KCNQ1 variants in the context of purified channel protein consisting of the voltage-sensor and pore domains. In Aim 3, we will evolve our machine learning algorithm as a deep neural network and enhance algorithm performance by using structural channel models built with a custom version of AlphaFold2.0, computed free energy, and outputs from molecular dynamics simulations of KCNQ1-KCNE1 channels. Our study will yield a large and unprecedented database of functional, structural, and biochemical properties of hundreds of KCNQ1 and KCNE1 variants, along with an advanced, data-trained computational prediction algorithm capable of accurately discriminating deleterious from benign variants. These results will contribute to improving genetic test interpretation and medical decision-making for LQTS.

NIH Research Projects · FY 2026 · 2013-09

SUMMARY Differentiating enterocytes build ~3000 microvilli on their apical surface and organize these protrusions into an array referred to as the brush border (BB). BB microvilli exhibit perfect tight packing and this unique morphology is critical for maximizing the number of protrusions, the holding capacity for membrane associated transporters and channels, and in turn, the functional capacity of the cell. The physiological importance of the BB is underscored by the fact that numerous intestinal diseases that are linked to the destruction and/or malformation of microvilli. Our group has made fundamental discoveries on mechanisms that enterocytes use to organize microvilli into functional, ordered BB arrays. Groundbreaking studies from our group identified two BB-specific protocadherins, CDHR2 and CDHR5, which form a heterophilic intermicrovillar adhesion complex (IMAC) that links the tips of adjacent microvilli and promotes ordered packing on mature villus enterocytes. Importantly, our work with CDHR2 KO mice established that loss of IMAC function leads to ~35% fewer microvilli on the apical surface and a corresponding growth rate reduction at the whole animal level. How IMACs drive the accumulation of thousands of microvilli over time during enterocyte differentiation remains the critical open question that we will tackle in this proposal. In exciting preliminary ultrastructural studies, we discovered that crypt microvilli initially exhibit robust accumulation at cell margins, which implies the existence of a mechanism for anchoring nascent protrusions at these sites. We also observed similar marginal accumulation of microvilli on the surface of differentiating intestinal and kidney epithelial cell lines. In all models examined, microvilli extending from one cell span intercellular space to make physical contact with microvilli on a neighboring cell. Remarkably, super- resolution microscopy of native tissue and epithelial culture models showed that microvilli in these contacts contain both CDHR2 and CDHR5, suggesting they represent transjunctional IMACs, a novel form of epithelial cell-cell contact. Using a cell mixing approach to drive the formation of transjunctional IMACs, photobleaching measurements revealed that transjunctional IMACs are much longer lived vs. medial IMACs. Finally, we learned that CDHR2 loss-of-function models, which are unable to form IMACs, exhibit defects in tight junctions. These initial findings lead us to propose the following hypothesis: transjunctional IMACs drive the accumulation of nascent microvilli into a mature BB while promoting the integrity of canonical cell junctions. Using state-of-the- art microscopy and novel epithelial model systems, we will: (Aim 1) define the subcellular mechanism of IMAC formation, (Aim 2) determine if transjunctional IMACs promote microvillus accumulation, and (Aim 3) determine if transjunctional IMACs promote the integrity of canonical cell junctions. These studies will reveal how the enterocyte surface becomes maximally packed with microvilli over the full time-course of differentiation and in doing so, offer new paradigms for understanding apical morphogenesis.

NIH Research Projects · FY 2025 · 2011-07

Much of our knowledge of islet physiology stems from rodent studies. However, we now know that marked discrepancies exist between rodent and human islets in regard to (for example) architecture, hormone secretion, and islet cell transcription factor (TF) expression, demonstrating potential limitations in assuming that rodent models entirely mimic human islet physiology and disease. An example of such a discrepancy lies in the islet β cell enriched MAFA TF, a fundamentally important protein to these cells in postnatal rodents. Thus, the MAFA protein is barely detectable in human islet β cells until ~9 years of age, whereas this TF is first detected developmentally and then produced throughout the lifespan of rodent insulin+ cells. We propose that humans have at least two postnatal, age-dependent MAFA-producing β cell populations capable of maintaining euglycemia: juvenile β cells (i.e. <~9 years old) with little MAFA (i.e. MAFALow) and post-juvenile β cells with robust MAFA (MAFAHigh). In fact, independent studies have established distinct molecular and functional properties of these human cell populations. Here we will examine the impact of MAFA on human β cells and hypothesize that this TF plays an essential role in regulating insulin secretion in adults. Our analysis in the first aim will be conducted using the β-like cells produced from both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) (collectively termed human pluripotent stem cells, or hPSCs). Here we will determine how knockout and over-expression of MAFA influences β cell maturation and function, appreciating that their greatly improved glucose-stimulated insulin secretion properties parallel enhanced MAFA expression after transplantation into immunocompromised NSG mice. In addition, we will investigate how a pathogenic variant of MAFA (Serine (S) 64 -> Phenylalanine (F)) that prominently increases its protein stability affects human β cell activity. Notably, MAFAS64F predisposes subjects to either adult-onset diabetes or insulinomatosis (i.e. non-syndromic insulin-producing β cell tumors) in a gender-biased manner. We generated a mouse model harboring this mutation in the endogenous MafA gene, and our results show glucose intolerance in males and improved glucose clearance in females, mimicking the findings in human subjects. Significantly, dysfunction in male mice was associated with premature cellular aging and senescence. Recently, independent reports have linked pathologic, senescent β cell populations to type 1 diabetes and type 2 diabetes islet dysfunction. Our experimentation in the second aim will define the molecular and functional consequences of MAFAS64F in human β cells. Our overall focus on human islet cells is viewed as innovative, as well as the combined use of human stem cell derived β-like cells and human islets for obtaining novel, physiologically relevant mechanistic insights.

NIH Research Projects · FY 2025 · 2010-05

PROJECT SUMMARY This renewal application seeks continued support for the T-32, Interdisciplinary Training in Rheumatic Diseases at Vanderbilt University. The program seeks support for 2 predoctoral and 3 postdoctoral positions. The goal of the program is to provide the next generation of investigators with the tools necessary to make critical discoveries and to advance our understanding and treatment of complex rheumatic disorders. Our faculty have created an interactive environment of discovery that bridges both clinical and basic research for pre and post-doctoral trainees. An interdisciplinary approach is coupled with a carefully orchestrated mentoring process so that trainees are provided with a broad perspective and they are exposed to opportunities for clinical translation of problems that cannot be solved by a single laboratory. The preceptors in the program are highly interactive and maintain extensive collaborative efforts between and among the research groups. These interactions create four Interest Groups within the program: Innate and Adaptive Immunity; Vascular Biology and Inflammation; Precision Medicine; and Musculoskeletal Biology. The program is organized to meet the career goals of individuals who want to apply advanced technologies, such as genomics or proteomics to rheumatic disease as well as to support the careers of individuals who want to translate these advances to improve health care. In addition to Departmental and preceptor-specific laboratory instruction, each trainee receives rheumatic disease research training through an interdisciplinary curriculum. Combined research forums, journal clubs and shared core facilities promote interactions beyond individual laboratories or divisions. Trainees in the program are actively mentored by both faculty members and through “peer mentoring” by former students and senior fellows in the program. Degree granting pathways for MSCI and MPH will be available to the program. Significant institutional investment in the Division's facilities and the recruitment of outstanding new faculty further strengthen the program for the future.

NIH Research Projects · FY 2025 · 2008-06

The Integrated Biological Systems Training in Oncology (IBSTO) program was established in 2008, with the objective of providing pre-doctoral students and post-doctoral fellows with cancer-focused training that integrates basic biological processes and translational research through understanding of the approaches and model systems that fuel cancer discovery. The IBSTO philosophy is that future breakthroughs in the diagnosis, prevention, and treatment of cancer will most likely be made by those individuals who are facile in a broad suite of methodologies and approaches, who understand the importance of collaboration and cross-disciplinary research, and who can bridge the divide between basic scientific discovery and clinical implementation. The IBSTO program takes place in a vibrant and inclusive cancer-forward research environment with superb resources, state-of-the-art facilities, top-ranked basic science departments, an outstanding NCI-designated comprehensive cancer center, and world-class basic and translational researchers and physician-scientists. Since its inception, we have recruited an outstanding cohort of more than 60 pre- and post-doctoral trainees, researching in the laboratories of well-funded and productive preceptors in a wide range of areas including cell biology, genetics, biochemistry, structural biology, cancer immunology, computational and systems biology, single cell approaches, omics, and translational cancer research. Productivity and outcomes for IBSTO trainees—both short and long term—are superb. In this third renewal application, we fortify our traditional areas of training strength and introduce a number of exciting and innovative new initiatives that keep this program fresh and effective. Four pre- and four post-doctoral trainees will spend two years each in the program, receiving a foundational training in cancer biology, sharing their discoveries, being exposed to the latest in basic and translational cancer research, and receiving intensive training in grant writing, scientific and communication, and career building. A new career series "Cancer Research Journeys" will provide first-hand exposure to the interplay between scientific discovery and professional development in the cancer realm. A new signature course "Scientific Exposition and Ethics in Cancer Research" will coach skill building through the lens of communication and interactivity, and teach the tools, approaches, and strategies needed for an impactful career that contributes to the amelioration of cancer. A streamlined administrative structure will keep the program nimble. And a new program co-Director will further our efforts, bring new ideas and energy, and position IBSTO for continued success in the next period and beyond.

NIH Research Projects · FY 2026 · 2006-01

Recent clinical and preclinical studies suggest that highly selective positive allosteric modulators (PAMs) for specific muscarinic acetylcholine receptor (mAChR) subtypes have exciting potential as novel treatments for positive symptoms, negative symptoms, and cognitive disturbances in patients suffering from schizophrenia. However, the precise mechanisms by which mAChRs regulate brain circuits that are relevant for the major symptom clusters associated with schizophrenia are unknown. It will be critical to develop a full understanding of the precise cellular and circuit roles of each mAChR subtype that could be relevant for schizophrenia and related brain disorders. Early stages of schizophrenia are associated with hyperactivity of the prefrontal cortex (PFC) and this is important for some of the cognitive and negative symptoms associated with the disease. Interestingly, we have found activation of the M1 subtype of mAChR induces long-term depression (M1-LTD) of transmission at excitatory synapses in the PFC, and also induces a robust increase in inhibitory transmission in this region. These combined actions could contribute to the established ability of highly selective M1 PAMs to reverse cognitive deficits in rodent models that are relevant for schizophrenia. Based on previous and new preliminary studies, we postulate that M1-LTD and M1-induced increases in synaptic inhibition in the PFC are mechanistically distinct and depend on activation of M1 in different neuronal populations. Specifically, we postulate that M1-LTD is mediated by activation phospholipase D (PLD) in PFC pyramidal cells, and that M1 increases inhibitory transmission through direct excitatory effects on specific populations of inhibitory interneurons through a PLD-independent mechanism. We propose a series of studies in which we will selectively delete M1 from specific neuronal populations and use M1 PAMs that differentially regulate coupling of M1 to PLD and phospholipase C (PLC), along with optogenetic silencing of specific neuronal populations, to rigorously evaluate the roles of M1 expressed in each of these major neuronal populations. Specifically, we will test the hypothesis that M1 PAMs induce M1-LTD by actions on M1 expressed in PFC pyramidal cells and do not require activation of M1 in inhibitory neurons (Aim 1). We will then perform a series of studies to test the hypothesis that M1 activation increases inhibitory transmission in the PFC by actions on defined populations of inhibitory interneurons (Aim 2). Finally, we will take advantage of our range of genetic, optogenetic, and pharmacological tools to test the hypothesis that M1 PAMs reverse deficits in specific behavioral measures of cognitive function by actions on different neuronal populations in the PFC (Aim 3). These studies will provide important new insights into the cellular and circuit mechanisms by which M1 PAMs enhance and restore deficits in specific domains of cognitive function that could be critical in advancing novel therapeutic strategies based on targeting these receptors.

NIH Research Projects · FY 2026 · 2006-01

PROJECT SUMMARY The study will investigate the neural mechanisms through which visual information is maintained in working memory. The neural basis of working memory has been a matter of debate in recent years, with competing theories proposing alternative neural correlates for its maintenance. Neurophysiological recordings will target multiple subdivisions of the prefrontal cortex, a brain area implicated in working memory tasks, as well as the anterior cingulate cortex, which has been implicated in learning to perform such tasks. Monkeys will be trained in cognitive tasks that require them to observe and remember the identity of different visual objects, which can be manipulated parametrically. A chronic array of microelectrodes will be implanted, and spike and local field potential recordings will be acquired as the animals are trained to perform the working memory task, and as they learn to perform the task with different stimulus sets. The experiment will allow us to determine the patterns of neuronal activation that determine the behavioral performance in the task and adjudicate between competing models. Analysis will determine changes at the level of single neurons, and neuronal populations, as well as changes evident in the rhythmicity of local field potentials. The experiments will also uncover the nature of changes that take place in the prefrontal cortex during training to perform an object working memory task and uncover what aspects of neural activity are critical for task acquisition. Finally experiments will investigate the flow of information within areas of the prefrontal cortex and between the prefrontal cortex and other cortical areas. Collectively, these experiments will uncover the fundamental mechanisms through which the neural circuits of the prefrontal cortex allow the maintenance of visual object information in working memory.

NIH Research Projects · FY 2026 · 2005-07

Project Summary In the previous funding period we investigated the mechanism of rapid antidepressant activity of ketamine, an ionotropic glutamatergic n-methyl-d-aspartate (NMDA) receptor antagonist. We demonstrated that Brain- derived neurotrophic factor (BDNF), and its high affinity receptor TrkB, are required for the rapid antidepressant effects of ketamine as these effects are lost in forebrain specific BDNF knockout mice and conditional forebrain specific TrkB knockout mice. We found the antidepressant effects of ketamine require protein translation, but not transcription, resulting in increases in BDNF protein levels in the hippocampus that are critical for the behavioral effect. Ketamine's blockade of spontaneous NMDA receptor mediated neurotransmission inactivates eukaryotic elongation factor 2 kinase (eEF2K) resulting in dephosphorylation of its only known substrate, eukaryotic elongation factor 2, thereby increasing protein translation of target transcripts, including BDNF. In turn, BDNF is postulated to act via eliciting insertion of 3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors at the postsynaptic membrane, which results in potentiation of AMPA receptor-mediated CA3-CA1 field excitatory postsynaptic potentials (fEPSPs) by ketamine. While this potentiation is suggested to be a cellular correlate of rapid antidepressant effects, its properties deviate from classical Hebbian forms of plasticity and rather coincide with homeostatic synaptic scaling seen after activity suppression. These data provide the basis for the novel hypothesis that BDNF-TrkB signaling regulates synaptic scaling in vivo that is critical for the rapid antidepressant effects of ketamine. The objective of this renewal is to specifically test the causal and instructive role of BDNF-TrkB signaling in the hippocampus in ketamine-mediated synaptic scaling and rapid antidepressant effects. Collectively, this information will provide novel information on the synaptic locus, as well as key molecules, necessary for ketamine's rapid antidepressant effects.

NIH Research Projects · FY 2026 · 2003-12

Project-Summary Recent studies have identified a large number of variants in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) machinery components such as SNAP25 that give rise to intractable early childhood brain disorders. In this project, we will use rodent hippocampal neurons as well as human embryonic stem cell derived neurons and a unique SNAP25 variant knock-in mouse model, to fully define the synaptic transmission and plasticity deficits associated with key disease causing SNAP25 variants and evaluate their responsiveness to neurotherapeutics. Importantly, our studies have identified SNAP25 variants that dramatically alter spontaneous release with limited or no effect on properties of action potential evoked neurotransmission. As spontaneous release process is a key determinant of synapses' homeostatic state and responsiveness to certain neurotherapeutics, we expect that the effects of these mutations will go beyond their immediate impact on the release process and alter synaptic plasticity as well as treatment response. Overall, we aim to test the hypothesis that disease causing neurotransmitter release machinery variants elicit crucial downstream signaling defects altering plasticity mechanisms that affect responsiveness to therapeutics via three Specific Aims. The first aim will address synaptic mechanisms adversely affected by SNAP25 variants using electrophysiology, single synapse optical imaging, electron-microscopy and super-resolution imaging. The second aim will focus on examining SNAP25 variant induced synaptic deficits in human neurons. In this system, we will evaluate the impact of experimental therapeutics that elicit homeostatic synaptic plasticity on SNAP25 variant induced deficits. Finally, aim 3 will examine circuit specific synaptic transmission and plasticity deficits in a unique SNAP25 variant mouse model. Information attained from these studies will provide new insight to the synaptic substrates that are affected not only by these disorders but also a number of neuropsychiatric and neurological disorders with synaptic abnormalities including major depressive disorder, autism, schizophrenia and neurodegeneration.

NIH Research Projects · FY 2025 · 2002-07

The goal of this fourth renewal of our multidisciplinary postdoctoral Training Program in Functional Neurogenomics is to continue equipping new investigators with state-of-the-art skills required to link genetic or genomic alterations to normal behavior and/or its dysregulation in brain diseases. Behavior depends on appropriate expression and activity of numerous genes and proteins in the brain. Dysfunction of these processes due to inheritance of specific risk alleles or epigenetic alterations often causes brain disorders. Despite the efforts of numerous scientists, links between (epi)genetic variations, molecular and synaptic defects, and brain circuit abnormalities that underly most behavioral disorders remain poorly understood. To address these gaps in knowledge, neuroscientists need to be facile in both modern molecular genetic approaches using different model systems and cutting-edge molecular bioinformatics analyses. We propose to continue recruiting 2-3 postdoctoral fellows each year to 2-year appointments, building on our substantial success training 70 fellows during prior grant cycles, with almost all alumni in independent faculty positions or other successful research-related careers. Future trainees are anticipated to follow similar career paths, bolstered by a slightly larger pool of training preceptors offering an expanded range of state-of-the-art interdisciplinary technologies to our trainees. Our efforts continue to be supported by substantial institutional investments in the Vanderbilt Brain Institute and training faculty, advanced research core facilities with essential technological support, and innovative educational programs. As a result, trainees will enjoy an academically rich and rigorous environment, in which to gain expertise in a broad range of genetic model systems, translating human genetic findings into construct-valid models, in vivo manipulations of molecules, cells and circuits using advanced molecular genetic approaches, and in the epigenetic, physiological, and behavioral analysis of genetic manipulations. The Program Director, Roger J. Colbran, Ph.D., Professor and Vice-Chair of the Department of Molecular Physiology & Biophysics, has a long-standing research program investigating molecular mechanisms involved in synaptic plasticity and behavior using multi-disciplinary approaches from biochemical structure-function studies to mouse genetics and behavior. The Associate Director, Lisa M. Monteggia, Ph.D., is a Professor of Pharmacology and the Director of the Vanderbilt Brain Institute, and has served on the Board of Scientific Councilors of the NIMH intramural program. Dr. Monteggia has made numerous contributions to understanding the role of BDNF and epigenetic regulation in normal brain function and Rett Syndrome, and continues to pioneer studies investigating the pathophysiology of depression and mechanisms of antidepressant action. The program leadership and the pool of training preceptors have a strong track record of mentoring the next generation of neuroscientists, thereby facilitating the establishment of enduring and productive research careers for their trainees.