Washington University

NIH Research Projects · FY 2024 · 2016-08

ABSTRACT Membrane proteins are responsible for controlling the passage of nutrients, waste, energy and information in and out of cells. They are critical players in physiology and key targets for pharmacological regulation, however, we lack a fundamental understanding of why these proteins are thermodynamically stable in cell membranes. Simply put, why does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to assemble faithfully into its stable, native structure? In order to investigate this question, the Robertson laboratory has developed a robust and rigorous model system to study equilibrium protein association in membranes based on the reversible dimerization of the CLC-ec1 Cl-/H+ antiporter. Through the development of fluorescence and single-molecule microscopy approaches, it is now possible to experimentally conduct a full thermodynamic analysis of the CLC dimerization reaction in lipid bilayers, yielding the free energy of association (ΔG°) and free energy changes (ΔΔG) due to mutations or different lipid conditions. Recent advances have allowed us to study CLC equilibrium as a function of temperature, enabling a van 't Hoff analysis to dissect the thermodynamic changes in enthalpy (ΔH°) and entropy (ΔS°) upon dimerization. In addition, we have developed methods for measuring equilibrium kinetics of CLC dimerization in a tractable manner - in the membrane and in real-time. With this foundation in place, the Robertson lab is primed to build a full molecular model of CLC dimerization in membranes. In the next phase of this project, we will investigate the hypothesis that CLC subunits are driven to associate due to differential solvent dependent driving forces in the associated and dissociated states, and that the transition state involves a critical solvation/de-solvation step. We will investigate this along three distinct aims, by building a theoretical model of the CLC dimerization free energy in membranes using computational approaches (Aim 1), connecting the CLC sequence and structure to dimerization through experimental measurements of thermodynamics and kinetics (Aim 2), and developing a guest-host approach to experimentally and theoretically quantify the impact of mixed lipid composition on protein association equilibria in membranes (Aim 3). Throughout each of these studies, we integrate experiments and theory hand-in-hand, enabling us to make robust connections between physical driving forces and molecular mechanisms. Regardless of the validity of our hypothesis, our studies will provide meaningful quantitative information to the study of membrane proteins in membranes. Ultimately, we expect that the results from these studies will provide a foundation for the field to build new strategies for targeting membrane protein stability and mis-folding based on fundamental physical principles.

NIH Research Projects · FY 2025 · 2016-07

Overall Project Summary/Abstract: Molecular mechanisms of filoviral-host interactions The family Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), are zoonotic pathogens that cause outbreaks of severe human disease and require biosafety level 4 (BSL4) containment for study. Recent approval of a vaccine and antibody-based therapies against an EBOV represent progress towards medical countermeasures. However, the family is comprised of multiple antigenically distinct species, making identification of pan-filoviral therapeutic approaches desirable. Furthermore, the molecular mechanisms required for replication and pathogenesis are incompletely understood. Defining key filovirus-host interactions and the mechanisms by which they promote viral growth and disease will provide important insight into viral biology and suggest new therapeutic approaches. Existing data, including our own, have identified key host-viral interactions that likely play important roles in the pathogenesis of filovirus disease. Our overarching goal is to address this gap in knowledge by building and expanding upon the strong foundational knowledge on EBOV to define molecular mechanisms at the host-pathogen interface and to identify EBOV-specific and pan-filoviral interactions that contribute to pathogenesis. To achieve our goals, we have assembled a highly accomplished team with track records of effective synergistic collaboration and expertise ranging from molecular biochemistry, structural biology and mass spectrometry to cell biology, virology, and work at BSL4. In the current funding period, we identified multiple host pathways that impact EBOV infection and defined key interactions at the viral-host interface. In our proposed studies, we use a reductionist approach to define molecular mechanisms by biochemical and structural methods (Project 1; RP01), determine the cellular impact and contributions of viral proteins such as VP30 and VP24 in immune response, viral replication, assembly and egress (Project 2; RP02), and evaluate the impact of specific interactions with EBOV and MARV virus in cell culture and in vivo, including specific subnetworks that regulate filoviral entry and replication (Project 3; RP03). Recognizing the complexity of the data being generated we have recruited new expertise in proteomics and genetic network analysis to provide a deeper understanding of host-virus protein connectivity and interaction. These efforts will be further supported by two scientific cores, the Antibody and Reagent Development Core B and the BSL4/ABSL4 laboratory Core C. This work will be guided by an active Administrative Core A that will receive critical input from the Core A Advisory Group (CAAG) and the External Advisory Board (EAB). Each is comprised of preeminent scientists in academia and industry with strong productivity in emerging infectious diseases and immunology and significant advisory experience. Building on our productive initial work, we are poised to define a comprehensive host interaction network, validate regulatory mechanisms that drive viral infection, and identify targets for therapeutic intervention. Our unique innovative experimental framework and highly interactive scientific approach provides a blueprint to tackle other emerging and reemerging pathogens.

NIH Research Projects · FY 2025 · 2016-07

The complexities of biological, behavioral, social, and environmental risk factors for obesity and cardiovascular disease (CVD) necessitate team science, capable of crossing the boundaries of disciplinary-specific silos to conduct and evaluate research from a transdisciplinary approach to prevent and treat obesity and CVD across the lifespan. Efforts to facilitate greater collaboration among scientists trained across many fields and levels of training are not only valuable but also essential to solving such complex problems. Thus, we propose to continue an innovative, transdisciplinary pre- and postdoctoral training program in obesity and CVD at Washington University in St. Louis (WUSTL). WUSTL is an excellent research institution promoting transdisciplinary, translational research through its unique, collegial, scientific environment across campuses and departments. This program: 1) recruits and trains talented transdisciplinary pre- and postdoctoral trainees; 2) provides trainees with collaborative, transdisciplinary mentorship teams; and 3) provides training in the ethical and socially responsible conduct of obesity/CVD research. International authorities on obesity, Denise Wilfley, PhD (Director) and Samuel Klein, MD (Co-Director), lead the program, supported by highly- qualified, primary and co-mentors spanning 12 departments in the biomedical, cognitive and behavioral, and population health sciences. Our mentors are well-funded and have strong collaborative ties to facilitate the training of 4 pre- and 4 postdoctoral trainees each year. Combining pre- and postdoctoral trainees from diverse backgrounds (e.g., genetics, molecular cell biology, psychology, social work, public health, and neurosciences) has created and will continue to create a uniquely dynamic training environment. The WUSTL Nutrition Obesity Research Center, Institute of Clinical and Translational Sciences, Institute for Public Health, and Center for Diabetes Translation Research provide infrastructure and state-of-the-art resources to support trainees’ engagement in transformative, translational science. Program evaluations completed by trainees, program administration, and collaborative mentorship teams ensure trainees achieve short- and long-term indicators of productivity positioning them for success in obtaining academic positions and independent grants. Indeed, postdoctoral graduates of the program have been highly successful in obtaining academic positions and grant funding, and in the case of our predoctoral trainees, continuing their research training in prestigious positions. Not only is our training program innovative in its design but using a team science approach guided by stellar mentors, our trainees are prepared to create and contribute to transdisciplinary scientific approaches that are more likely to yield innovative solutions to the complex problems of obesity and CVD than research previously conducted by individual scientists within a single disciplinary field.

NIH Research Projects · FY 2025 · 2016-06

Chronic kidney disease (CKD) affects almost 15% of Americans, and renal injury often targets the renal tubule epithelia. How these tubules respond can determine whether the kidney undergoes repair or tubulointerstitial fibrosis (TIF), the common hallmark of progressive CKD. This proposal focuses on understanding how chronic renal injury induces changes in the renal tubular cell cycle and metabolism and how these changes affect tubular survival and the development of TIF. It is well known that cell cycle, metabolism, and mitochondrial function are all closely coordinated processes, but it is not clear how epithelial G1 to S cell cycle progression affects metabolism in the CKD kidney. Preliminary data suggests that reducing cell cycle progression from G1 to S phase in renal tubules protects against fibrosis in rodent CKD models and decreases tubular apoptosis. In addition, reducing G1 to S progression increased glucose oxidation, the metabolism of glucose to pyruvate which is then oxidized in the mitochondria through the citric acid cycle and electron transport chain. This proposal will test the hypothesis that reducing epithelial G1 to S phase progression in CKD protects against epithelial injury and fibrosis through altered metabolism. To test this, Aim 1 will use either a pharmacologic (palbociclib) or a genetic (conditionally delete cyclin D1 in renal tubules) approach to reduce G1 to S cell cycle progression in mice. We hypothesize that decreasing G1 progression to S phase in epithelial cells is protective in CKD models by reducing tubular injury and fibrosis. Our preliminary data show that reducing cell cycle progression in both injured kidney tissue and in isolated tubule cells also suppresses signaling pathways and inflammatory cytokines associated with kidney injury. This aim investigates how reducing cell cycle progression may alter these signaling pathways to reduce tubule injury and myofibroblast activation by autocrine and paracrine signaling, respectively. The second aim investigates the metabolic changes that occur in injured tubules with reduced G1 to S phase progression using the Seahorse bioflux analyzer, 14C-pyruvate oxidation studies ex vivo, and stable isotopic metabolomics. We hypothesize that reducing epithelial cell cycle progression increases glucose oxidation leading to better epithelial survival and less fibrosis, in part, through the AMP-activated protein kinase pathway. We will also investigate how glucose oxidation in renal tubules, independent of metabolism, affects the response to chronic injury. The impact of cell cycle progression on mitochondrial function and structure will also be defined using Oroboros and super- resolution microscopy. These studies should provide novel information about how changes in epithelial cell cycle and metabolism affect the response to chronic renal injury with the potential identification of novel therapeutic targets to treat CKD.

NIH Research Projects · FY 2025 · 2016-06

DESCRIPTION: This proposal is centered on studies of the mechanism of DNA replication in eukaryotic cells (1) and on the consequences of replication dysfunction with regard to spontaneous and damage- induced mutagenesis (2), and to cell cycle checkpoint activation (3). Our primary approaches combine biochemical and biophysical analysis with genetic analysis in the yeast Saccharomyces cerevisiae, to gain insight in each of these three broadly defined pathways and their interconnectivity. Our studies are augmented with structural studies of the complex machineries that function in these processes. Proposed DNA replication studies are based on a strong record of progress in defining mechanisms of lagging strand DNA replication and Okazaki fragment maturation. Since Okazaki fragments represent by far the most frequent DNA discontinuities in all cells, it is imperative to understand the different layers of regulation of this process. Okazaki fragment maturation has primarily been studied in well-defined biochemical systems, in isolation from other events that occur at the replication fork. We propose to expand our studies within the context of a complete replication fork, which has been assembled at a yeast replication origin. Our mutagenesis studies will center on the main actors, DNA polymerase z and Rev1. On the one hand, Rev1 promotes DNA lesion bypass by Pol z; on the other hand, it limits the extent of mutagenesis by inhibiting Pol z-dependent DNA synthesis outside the narrow environment of the lesion. We will unravel the mechanism that underlies this dual regulatory function of Rev1. The primary focus of our checkpoint studies is on the two sensor protein kinases Mec1 and Tel1, the orthologs of human ATR and ATM, respectively. Biochemical and genetic studies will be combined with cryo-EM studies to understand how the basal activities of these unique protein kinases are activated. Furthermore, the advantage of having an efficient DNA replication system available will allow us to begin addressing the coupling between replication arrest and the downstream response pathways. Finally, in keeping with the MIRA principle, we will pursue other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.

NIH Research Projects · FY 2024 · 2016-05

Project Summary Healthy adipose tissue expansion is necessary for maintaining metabolic health in the setting of over-nutrition – a situation that is increasingly relevant in the US, as the incidence of obesity is estimated at 33% of the US population. Therefore, elucidating the fundamental nutrient sensing mechanisms that regulate adipocyte expansion is critical for understanding, and ultimately treating, the negative metabolic consequences of obesity. We previously identified Leucine Rich Repeat Containing 8A (LRRC8a or SWELL1), a newly discovered essential component of the volume-regulated anion channel (VRAC), as a novel volume-sensing regulator of both insulin sensitivity and insulin secretion. We and others find that SWELL1 activity and protein expression is reduced in metabolically unhealthy obese mice and humans – suggesting that reduced multi-organ SWELL1 activity/expression contributes to obesity-induced metabolic syndrome. Our group has biochemical, patch- clamp and imaging evidence that SWELL1 channel complexes are also expressed and functional in lysosomes. Given that lysosomes are signaling hubs that integrate nutrient sensing and AKT-mTOR signaling, we hypothesize that lysosomal SWELL1-LRRC8 channels participate in cellular nutrient sensing by activating in response to increases in intraluminal lysosomal leucine, and that this signaling mechanism is dysregulated in the setting of obesity-induced diabetes and insulin resistance. To test this hypothesis, we combine unique reagents and innovative methods from the Diwan (lysosomal signaling), Xu (lysosomal patch-clamp), and Held (mass spectrometry) laboratories, with our expertise in SWELL1 signaling, and access to human adipose tissue samples from highly phenotyped metabolically healthy and unhealthy humans (Klein laboratory). Our objective is to understand the mechanisms of plasma membrane and lysosomal SWELL1 (Lyso- SWELL1) nutrient sensing and how it is dysregulated in disease states, including obesity-induced glucose intolerance and insulin resistance. The rationale for these studies is that delineating the contribution of SWELL1 to lysosomal nutrient sensing and mTORC1 activation will advance our understanding of a fundamental cellular signaling mechanism and guide innovative therapeutic approaches for patients with prediabetes and diabetes. We propose the following AIMs: · AIM#1: Delineate the mechanisms of plasma membrane versus lysosomal SWELL1 signaling to AKT- AMPK-mTOR signaling in adipocytes · AIM#2: Examine the contribution of SWELL1 signaling in vivo in the setting of obesity in mice and humans The knowledge gained from these studies will delineate a novel lysosomal ion channel signaling pathway that regulates adipocyte growth and systemic dysglycemia in obesity, and inform therapeutic strategies currently underway to modulate SWELL1 signaling for the treatment of obesity-induced metabolic syndrome.

NIH Research Projects · FY 2026 · 2016-05

ABSTRACT During vertebrate development, early inductive processes controlled by maternal and zygotic gene products establish embryonic polarity and form three germ layers: ectoderm, endoderm and mesoderm. Convergence and extension (C&E) gastrulation movements elongate germ layers down the anteroposterior axis and narrow them dorsoventrally into nascent head, trunk and tail. The dorsal Spemann-Mangold organizer (SMO) plays the lead role in vertebrate gastrulation by instructing cell fates and morphogenesis. One conserved mechanism entails secreted antagonists of Bone Morphogenetic Proteins (BMP) to establish a ventral to dorsal signaling gradient that instructs cell fate specification. How does the SMO direct gastrulation movements, and how the inductive and morphogenetic processes shaping germ layers are coordinated is still poorly understood. We address these fundamental questions by leveraging experimental strengths of the zebrafish model. We previously discovered that mesoderm C&E occurs in five domains of distinct cell movement behaviors along the dorsoventral gastrula axis and showed they are specified by the BMP signaling gradient, with the highest BMP levels inhibiting C&E and directing cells to the tailbud. Exploiting optogenetic control of BMP signaling we learned that BMP activation can rapidly redirect dorsally converging mesodermal and endodermal cells in a largely coordinated manner and alter marker gene expression. We hypothesize that BMP signaling regulates C&E by first specifying domains of cell identity with distinct C&E behaviors. We will determine the minimal time of BMP signaling stimulation required to activate its Smad5 signal transducer, redirect cell movements and alter gene expression, and whether these processes are concurrent. We will examine if BMP controls endoderm C&E indirectly by regulating mesodermal expression of Cxcl12b ligand implicated in endoderm movements, and/or directly influences endoderm transcriptome and movements. To test if gastrulation requires overlapping and redundant maternal and zygotic gene expression, we have been conducting an unbiased genetic screen. We found novel phenotypes manifested only when both maternal and zygotic gene functions are mutated. bassethoundstl472 mutants exhibit unique combination of defects, including shorter body, otic placode convergence, coalescence, and movement away from the neural tube. We gathered evidence that stl472 is a regulatory mutation of integrin1b and we aim to define its molecular basis. Transplantations and transgenic approaches for targeted rescue will define tissues in which integrinb1b functions. We will test whether Integrin1b acts with Integrin5 as a receptor for Fibronectin and/or Laminin. We will also study additional mutants found in our screen. Together, these investigations will advance our understanding how cell fate specification and gastrulation morphogenesis are coordinated during vertebrate development. As gastrulation anomalies cause miscarriages and birth defects, our findings can advance their understanding, diagnosis and inform development of therapeutics.

NIH Research Projects · FY 2025 · 2016-04

Abstract The calcium and voltage regulated BK(or SLO1)-type K+ channel is a widely expressed ion channel impacting on regulation of excitability in a variety of both excitable and inexcitable tissues. SLO1 is encoded by the kcnma1(or slo1) gene, one of four SLO family members. The ability of SLO family channels to be regulated by specific cytosolic ions arises from a large cytosolic regulatory domain, containing specific ion binding sites, that is connected to the pore-forming part of the subunits. The ability of SLO family channels to respond to changes in the cytosolic milieu makes them uniquely adapted to play negative feedback roles following activity that leads to alterations in the cytosolic ions. The Ca-regulated BK channel is particularly fascinating, since despite being encoded by a single gene, it is expressed in a wide variety of cells in each case playing very distinct physiological roles. A central tenet of the work in this laboratory is that the functional diversity and the associated broad scope of physiological roles played by BK channels arises from associated with regulatory subunits. For BK channels, tissue-specific expression of up to four different regulatory β subunits (β1-β4) and four γ subunits can define BK function and physiology. Our understanding of the loci of expression, channel composition in particular cells, and the impact of particular regulatory subunits on function and physiology remains rudimentary. β1 and β4 subunits have been implicated in hypertension and epilepsy, respectively, and other indications suggest that BK channels may be therapeutic targets in stroke, hypertension, epilepsy, and tumor growth regulation. To address the gaps in understanding of the roles of BK channels of particular subunit composition, this lab combines methods ranging from biophysical analysis of channel properties to the use of genetic knock-out (KO) of specific regulatory subunits. This permits evaluation not only of the biophysical and functional properties of channels of different auxiliary subunit composition in native cells, but also how these channels contribute to physiological roles. Furthermore, we continue to probe questions of BK channel function pertinent to channel inactivation mechanisms and stoichiometry, guided by available structural information. Recent work on animal models developed in this lab have established important roles of γ1 and γ2 subunits in defining BK channel functions in secretory epithelial cells and inner hair cells, respectively, while β-containing BK channels influence action potential firing rates and burst behavior. Future work will further probe our existing models, e.g., the role of γ1-containing BK channels in colonic epithelium. In addition, a major focus will be the development of animal models that will allow examination of β3-containing BK currents in native cells. β3-containing BK channels remain the least understood of all BK channel subunits and we seek to remedy that deficit. This project is expected to provide new insight into the physiological roles of β3 and γ1 auxiliary subunits, and the roles of BK channels containing such subunits.

NIH Research Projects · FY 2025 · 2016-04

Project Summary During pregnancy, the uterus gradually transitions from a quiescent state characterized by weak, asynchronous, regional contractions to an activated state in which contractions increase in force, frequency, and synchrony to expel the fetus at term. A major driver of this transition is gradual depolarization of the myometrial smooth muscle cell (MSMC) membrane potential. As the inside of the membrane becomes less negatively charged, the myometrium becomes more excitable. However, the molecular pathways controlling this transition are unknown, hampering our ability to develop strategies to regulate uterine contractility to prevent pre- or post-term labor. Here, we propose to test the central hypothesis that a sodium (Na+) signaling complex formed by the Na+- activated potassium (K+) channel SLO2.1 and the Na+ leak channel NALCN regulates this transition. This hypothesis is founded on published and preliminary data we obtained with funding from our previous R01. In primary human MSMCs isolated at term, we showed that Na+ entry through NALCN activated K+ efflux through SLO2.1 and hyperpolarized the membrane. Next, we showed that activation of this complex reduced tension in uterine strips. Finally, we reported that inhibiting this complex induced MSMC depolarization, triggering calcium (Ca2+) entry through voltage-dependent Ca2+ channels and promoting contractility. Together, these data indicate that the NALCN/SLO2.1 complex is a strong candidate to control the MSMC membrane potential. However, because we used human tissues, we could not determine the role of this complex in the gradual depolarization of the MSMC membrane potential over pregnancy. To address this limitation and fully test our hypothesis, our objective is to define the function and regulation of the NALCN/SLO2.1 complex across pregnancy in mouse MSMCs. The goals of this project are to: 1) Define NALCN/SLO2.1 complex activity across pregnancy, 2) Assess the effects of NALCN/SLO2.1 complex activity on intracellular Ca2+ and uterine contractility and 3) Identify additional members of the NALCN/SLO2.1 complex in MSMCs and determine their effects on functionality of the complex. In completing these aims, we will define the main regulators of MSMC membrane potential and how they change as pregnancy progresses. This work will facilitate future efforts aimed at developing therapeutics to inhibit the NALCN/SLO2.1 complex to promote labor or to activate the complex to promote quiescence and prevent preterm labor.

NIH Research Projects · FY 2026 · 2016-03

Project Summary: This project investigates the cochlear AMPA-type glutamate receptors (AMPARs) that are necessary for hearing, overactivation of which leads to excitotoxic synapse loss and hearing disorders. Each cochlear afferent synapse expresses many hundreds to a few thousand of these AMPARs, of both the Ca2+-permeable subtype (CP-AMPARs, lacking subunit GluA2) and the Ca2+-impermeable subtype (CI-AMPARs, containing subunit GluA2). The combination of pore-forming GluA subunits and auxiliary subunits of the AMPAR complex, influenced by transsynaptic adhesion factors, determine its physiological properties and pharmacological sensitivities. The cochlear AMPAR complex has properties that make it unique in the nervous system, for example, the absence of GluA1. However, the precise complement of cochlear AMPAR subunits in not known. This proposal uses mouse genetics, in vivo and ex vivo cochlear electrophysiology, proteomics, and ultrastructural molecular anatomy to investigate the subunit composition, pharmacological sensitivity, and functional significance of the cochlear AMPAR complex. We will determine the influence of auxiliary subunit TARP-2 (Stargazin) on cochlear function, synaptic transmission, and GluA subunit expression. We will test the hypothesis that synaptopathy in GluA3KO mice results from an increase in Ca2+-permeability of the AMPAR complex. We will determine how Neuroligin1 and 3 affect AMPAR subunit expression and auditory nerve fiber physiology. We will determine the influence of GluA3, TARP-2, Nlgn1, and Nlgn3 on the intrasynaptic distribution of AMPAR subunits. With recombinant expression of different combinations of GluA pore-forming and auxiliary subunits in HEK cells (with or without GluA3, with or without TARP-2), we will challenge our understanding of the cochlear AMPAR complex by comparing changes in pharmacological sensitivity with those changes observed for the native cochlear synapses (GluA3WT vs GluA3KO, TARP-2WT vs TARP-2KO). The gain of this basic knowledge will inform design of small molecules to target cochlear AMPARs. With chronic systemic administration of the tool compound (CP-AMPAR blocker IEM-1925), we will measure synaptic adaptation and resistance to noise-induced synaptopathy. With acute systemic dosing, we will ask if noise trauma can be prevented if IEM-1925 is given only during, not before, the noise exposure and if IEM- 1925 + antioxidant combination therapy can protect cochlear function from more intense noise exposures. The long-term goal of this line of investigation is to develop systemic drugs to target CP-AMPARs of the inner ear while allowing hearing function to be maintained through CI-AMPARs, and while avoiding unwanted CNS side effects. The successful completion of this collaborative project will determine the precise subunit composition of the cochlear AMPAR complex and its influence on pharmacological sensitivity.

NIH Research Projects · FY 2026 · 2016-03

PROJECT ABSTRACT Osteoarthritis (OA) affects the entire joint. There is growing evidence that cartilage catabolism and synovial inflammation are interdependent and act as primary drivers of OA. In this competitive renewal, we build upon our findings in the first funding period, and explore the central hypothesis that cartilage and synovium are key therapeutic targets for the protective effect of 4-aminobutyrate aminotransferase (ABAT) loss-of-function (LOF) against OA. Important discoveries made during the first funding period defined completely novel targets in the pathogenesis of OA and significantly advanced the field. We discovered that DNA methyltransferase 3b (Dnmt3b) expression is reduced in murine and human OA chondrocytes, and drives the disease process. In rigorous genetic mouse models, we established that Dnmt3b LOF (loss-of function) is catabolic in chondrocytes and accelerates OA, whereas Dnmt3b GOF (gain of function) is anabolic and protects against OA. Integrated analysis of DNA methyl-seq and RNA-Seq identified Abat as a key downstream target of Dnmt3b. Dnmt3b was found to methylate the Abat promoter and suppress its expression, while suppression of Dnmt3b reduced promoter methylation and increased Abat expression, thereby establishing a reciprocal Dnmtb3b-Abat axis in articular chondrocytes. Using lentiviral approaches, we showed that Abat over-expression is catabolic and accelerates OA, while Abat suppression is anabolic and protects against OA. Vigabatrin, a small-molecule inhibitor of Abat, also blocked the development of OA and synovial inflammation in mice with knee injury. To i) definitively establish the Dnmt3b-Abat axis in vivo in cartilage; and ii) determine if Abat LOF protects against OA by acting on cartilage and/or synovium, we created innovative conditional Abat GOF and LOF mice. Specific Aim 1 will use conditional Abat GOF/LOF in combination with Dnmt3b GOF/LOF mouse models to determine whether Abat is the key downstream mediator of Dnmt3b in regulating cartilage homeostasis. Specific Aim 2 will use tissue specific conditional gene deletions in cartilage (Aim 2A), in synovial fibroblasts (Aim 2B), and in synovial myeloid cells (Aim 2C) to determine if the protective effect of Abat LOF against OA is dependent on both cartilage and synovium. Specific Aim 3 will determine whether siAbat gene therapy using a peptide nanoparticle delivery platform confers protection against OA in mice. These findings will definitively show that Abat is the key signal downstream of Dnmt3b, define the relative role of Abat on cartilage and synovium in protection against OA and pain, and establish delivery of siAbat nanoparticles as a novel translational approach for the treatment of OA and joint pain.

NIH Research Projects · FY 2025 · 2016-03

Project Summary/Abstract Non-traumatic lower extremity amputations (NLEA) in people with diabetic peripheral neuropathy (DPN) have devastating consequences, most notable a 3-year mortality rate of up to 71%. Data from our previous award cycle support a foot bone and vascular pathway culminating in NLEA in people with DPN. Bone and vascular deterioration were significantly related to key risk factors for NLEA in the insensate foot; foot deformity, fracture, and delayed wound healing. However, the current bone and vascular pathway does not explain why the highest rate of NLEA occurs in individuals with DPN and end-stage chronic kidney disease (CKD). We believe that CKD-mineral bone disorder (CKD-MBD) is the missing link in the foot bone and vascular pathway to DPN-associated NLEA. CKD-MBD is recognized, in the hip and coronary/aortic vessels, to induce a bone-vascular paradox with loss of skeletal bone volume and strength and vascular calcification. However, the contribution of CKD-MBD to DPN-associated NLEA is unknown. Thus, this renewal application will extend our discoveries and test the central hypothesis that CKD-MBD synergistically combines with DPN in the foot bone and vascular deterioration pathway to place the DPN foot at the highest risk for NLEA. This application will employ a multi-site, cross-sectional (n=216) and longitudinal (n=114), study of people with Type 2 DPN across all stages of CKD: stage 1(No CKD) to 5. Through this project we aim to: 1) quantify the effect of CKD severity and progression on pedal bone quality & quantity and vessel calcification (Aim 1), 2) determine the effect of CKD, mediated through bone quantity and quality and foot vessel calcification, on clinically relevant foot outcomes (Aim 2), 3) explore the ability of CKD, bone quality and quantity, and vascular calcification variables to predict risk for clinically relevant poor foot outcomes. The investigative team from Washington University in St. Louis, High Point University, and University of California San Francisco represents a unique and powerful combination of collective and individual clinical research expertise in DPN tissue deterioration, imaging, and CKD-MBD. This project uses highly innovative cutting-edge technology and processing to measure individual foot bone quantity & quality and pedal vessel calcification with computed tomography and hidden changes in foot bone cortical and trabecular micro- architecture that are not reflected in global measures of BMD using high resolution peripheral quantitative computed tomography (HR-pQCT). Finally, in our NLEA risk prediction aim we include a CKD-MBD serum marker (sclerostin), a critical step in translating CKD-MBD research from bench to bedside. Understanding the pathway to NLEA will improve preventative care and management of people with DPN to reduce the risk of NLEA by making CKD prevention and treatment indispensable, identifying pharmaceutical targets, and identifying risk factors of NLEA, allowing early intervention.

NIH Research Projects · FY 2025 · 2016-02

ABSTRACT In 2015, we reported the discovery of the tetracycline destructases (TDases), a family of flavoenzymes capable of inactivating tetracycline (Tet) antibiotics by enzymatic degradation, distinguishing them from canonical mech- anisms of Tet resistance. Since that report we have expanded the pool of known TDases to >100 functionally identified enzymes, reported crystal structures of numerous TDases, and proposed a class of small molecule inhibitors to combat these enzymes. TDases are now widely recognized as a clinically-relevant resistance mech- anism. The central motivation for this proposal is to better understand the molecular mechanisms, evolutionary origins, and structural features of TDases in order to rationally design better diagnostics and inhibitors to restore efficacy of a vital class of antibiotics as TDases continue to disseminate and become a widespread cause of morbidity and mortality. Our collaborative effort has yielded impactful scientific results, and we are ideally equipped to carry out our three independent yet complementary specific aims: 1) Elucidate the mechanism of Tet inactivation by the TDases, 2) Understand the evolution of TDases at genetic and population levels, and 3) Develop inhibitors and diagnostic agents for TDases. The first aim will test the hypothesis that diverse sub- strate-binding modes and FAD cofactor orientations determine the atomic site of Tet oxidation and reg- ulate the catalytic cycle of the TDases. We propose that we can correlate observed enzymatic degradation products with respective binding modes using X-ray crystallography, photoaffinity crosslinking, enzyme kinetics, and isotopic labeling studies with a variety of TDases and substrates. The second aim examines the sequence determinants of flavin monooxygenase evolution toward Tet inactivation as well as the selective ad- vantage that TDases provide in the context of bacterial populations expressing different mechanisms of Tet resistance. We will identify novel enzymes through iterative sequence-based predictions and phenotypic validation, identify structural elements required for activity using saturation mutagenesis and DNA shuffling, and examine the population-level fitness advantages of TDases using high-throughput reporter assays. The third aim will determine whether anhydrotetracycline (aTC) analogs can be optimized to inhibit TDases by controlling ligand binding mode and whether chromogenic Tets can serve as diagnostic agents for TDase expression in pathogens. We will use robust semi-synthetic methods developed by us for modification of the Tet and aTC scaffolds and study the resulting novel compounds with rigorous biochemical assays, X-ray crystallography, and phenotypic whole-cell studies. The proposed research is significant because antibiotic re- sistance is a public health crisis, and TDases that degrade all known tetracyclines are widely distributed in di- verse environmental and pathogenic bacteria. The proposed research is impactful because it combines funda- mental understanding of enzyme evolution and mechanism with the development of co-therapeutic and diag- nostic agents that have the potential to mitigate the emerging threat posed by enzymatic Tet inactivation.

NIH Research Projects · FY 2025 · 2015-09

Abstract Young investigators are essential to the maintenance of a vibrant Neurology/Neurosurgery physician-scientist workforce, but are facing increasing clinical demands, diminishing research time, and limited funding for scientific training. Moreover, the rapid pace of basic neuroscience advancement and the emergence of new enabling technologies add to the challenges facing these future physician-neuroscientists. To meet this pressing need, we have built upon the successes of our prior Neurology/Neurosurgery R25 program to establish a comprehensive UE5 training program that begins during early residency training that aims to recruit and prepare the next generation of Neurology/Neurosurgery physician-neuroscientists to successfully compete for the best junior faculty positions in the country, secure extramural career development funding, including K-series grants from the National Institutes of Health, and ultimately become independent principal investigators. The structure of the Washington University Physician Neuroscientist Training Pipeline (PNTP) pathway improves upon our prior R25 program in several fundamental ways: (1) we established a more formalized pipeline beginning early in residency training to guide and nurture future physician-neuroscientists in neurosurgery and neurology, (2) we created a resident-led workshop series for Neurology and Neurosurgery trainees that exposes them to faculty research, career-relevant topics relevant to the transition to independence, and cross-disciplinary research centers and core facilities at Washington University, (3) we established an annual symposium, pairing R25/UE5 fellow research presentations with an invited distinguished physician-neuroscientist keynote lecture, (4) we established two advisory groups, an external advisory committee and an operations guidance team, to provide programmatic oversight and input, and (5) we expanded our training program to now accept neuroscience research-focused residents from other departments. The Departments of Neurosurgery and Neurology at Washington University each provide excellent institutional environments for basic, translational, and clinical neuroscience research and education, with long and distinguished track records of training academic physician- neuroscientists spanning many decades. In the past decade, over 60% of Neurosurgery graduates have entered academic practice and nearly 20% of graduates have become successful NIH-funded neurosurgeon-scientists, while ~80% of Neurology graduates have taken positions at academic medical centers, and 23% have received independent fellowship grants, K awards, and/or R01 grants to date. Taken together, the proposed PNTP provide an enhanced and distinct research training pipeline, specifically for neurosurgeons and neurologists, but also other cognate subspecialists, interested in academic careers as independent neuroscience researchers, with the overall long-term goal of ensuring that highly-trained physician-scientists will be available to make future advances to reduce the burden of neurological disease across the lifespan.

NIH Research Projects · FY 2026 · 2015-09

ABSTRACT This grant renewal continues our long-standing research program on the role of the innate immune system in the onset and progression of Alzheimer's disease (AD), with a particular focus on microglia and the TREM2-DAP12 receptor complex. AD is the most prevalent form of dementia, and recent research highlights the critical role of microglia in disease onset and progression. Microglia respond to amyloid-beta (Aβ) plaques by adopting a disease-associated microglia (DAM) phenotype, which helps contain pathology and delay disease progression. The recent introduction of anti-Aβ antibodies, which promote Aβ clearance by microglia as a treatment for AD, has underscored the potential of leveraging microglia to remove Aβ plaques and restore tissue homeostasis. Similarly, the microglial receptor complex TREM2-DAP12 has gained attention as a target for enhancing microglial responses to Aβ plaques and addressing other aspects of AD pathology. However, targeting microglia with a monoclonal antibody that activates TREM2 has not demonstrated significant clinical improvements in AD, underscoring the need for more refined approaches. Specifically, monoclonal antibody-mediated engagement of TREM2 may not fully recapitulate the complex intracellular signaling pathways downstream of TREM2 that convert microglia into protective DAM. This proposal aims to address the limitations of current TREM2 therapies by investigating the complex signaling mechanisms downstream of TREM2. In Specific Aim 1, we will explore how key signaling molecules downstream of TREM2 and other activating receptors, such as DAP10, LAT2, and VAV1, contribute to microglial function and control of AD pathology. In Specific Aim 2, we will evaluate a small-molecule TREM2 agonist that enhances signaling by strengthening the TREM2-DAP12 interaction without disrupting normal ligand binding, offering a potential alternative to antibody-based therapies. In Specific Aim 3, we will study the human TREM2 mutation S162R, which selectively impairs TREM2 signaling, likely by affecting the TREM2-DAP12 interaction. This will allow us to define the molecular underpinnings of the TREM2-DAP12 interaction and assess how a small-molecule agonist can enhance this interaction. By deepening our understanding of TREM2 signaling, this proposal seeks to identify novel therapeutic strategies to enhance microglial activation through the TREM2-DAP12 signaling pathway and improve AD outcomes.

NIH Research Projects · FY 2026 · 2015-09

Abstract Acute kidney injury (AKI) has a wide spectrum of outcomes ranging from full recovery to failed repair and transition to chronic kidney disease. According to a report from the CDC examining trends in hospitalizations for acute kidney injury in the US from 2000 to 2014, the rate of AKI hospitalizations increased by 230% over this time frame, going from 3.5 to 11.7 per 1000 persons. Furthermore, it has been reported that Medicare patients aged 66 years and older who were hospitalized for AKI had a 35% cumulative probability of a recurrent AKI hospitalization within one year and 28% were diagnosed as having CKD in the year following an AKI hospitalization. Despite the strong medical need, care of patients with AKI is primarily supportive. Developing treatment options to accelerate successful repair of AKI, and to reduce the AKI to CKD transition, is critically important. Recent work from our laboratory has identified a small proportion of injured proximal tubule (PT) cells that fail to undergo full repair and restoration of a healthy PT phenotype. These so-called failed repair PT (FR-PT, also called "maladaptive PT" in the literature), are characterized by expression of Vcam1 and they take on a proinflammatory, profibrotic, senescent-associated secretory pathway phenotype, thus providing a possible mechanism for AKI to CKD transition. We hypothesize that the FR-PT cell state drives progressive kidney damage, and that inhibiting or reversing transition to the failed repair state would be prevent the AKI to CKD transition. We have also identified a possible role for the transcription factor Nuclear factor of activated T-cells 5 (NFAT5) in driving the FR-PT state. We propose the following investigations to critically test the importance of FR-PT in regulating the proximal tubule injury response and to determine if this can be manipulated to promote repair: Specific Aim 1: Define the spatio-temporal dynamics of FR-PT by genetic lineage tracing using a new mouse line that allows for genetic manipulation specifically in the FR-PT lineage. Specific Aim 2: Investigate the role of NFAT5 expression in regulating the FR-PT state. We will assess the effects of Nfat5 modulation using a variety of approaches including high resolution spatial transcriptomics. Specific Aim 3: Determine the functional contribution of Vcam1+ FR-PT to both successful repair and the AKI to CKD transition through cell ablation analysis. We will test whether genetic ablation of pro-inflammatory FR-PT at late timepoints reduces the AKI to CKD transition and prevent AKI-associated long-term decline in glomerular filtration rate.

NIH Research Projects · FY 2026 · 2015-08

Project Summary/Abstract Motile cilia are essential for airway clearance and their dysfunction leads to lung diseases. Loss of motile cilia may be environmentally influenced in conditions like chronic obstructive lung disease, or due to genetic causes that are collectively known as primary ciliary dyskinesia (PCD). The goal of our studies is to determine the biologic basis of PCD lung disease phenotypes. Pathologic variants (mutations) occur in nearly 60 PCD genes. Variants result in abnormal cilia function due to defects in ciliary protein production, transport, or placement along the axoneme, which is the microtubule skeleton of cilia. While we know many components of the motile cilia machinery, we do not yet understand the assembly process, how it is interrupted and the consequences for disease in patients with PCD. Analysis of PCD patients reveals a wide spectrum of genotype-phenotype differences. Disease severity has been attributed primarily to impaired ciliary motility. Our studies reveal multiple cell pathologies that are independent of motility defects. The discoveries came as we sought to determine why PCD variants, CCDC39 and CCDC40, cause more severe lung disease than in other PCD variants. Using single particle cryo-electron microscopy, we found that CCDC39 and CCDC40 form a heterodimer attached to the axoneme. Proteomic analysis of cilia from CCDC39 variants identified 90 missing proteins from major ciliary structures. We deduced that a CCDC39/CCDC40 scaffold provides the addresses for a set of 14 key proteins that anchor an extensive connectome. In addition, the loss of CCDC39/CCDC40 leads to several downstream consequences on the airway cells that have not been previously described in PCD: the cilia are shortened, the axonemal microtubules lack structural integrity, the periciliary layer is disrupted, proteasomal activity is altered, and the multiciliated cells switch to a mucous cell fate. We hypothesize that loss of the CCDC39/40 scaffold, and its connectome, result in airway periciliary barrier failure and proteostatic stress leading to severe PCD. The mechanism and impact of these motility-independent phenotypes will be investigated in these Aims: (1) Determine how the CCDC39/CCDC40 scaffold provides addresses and anchors for ciliary structures that are affected in patients with PCD; (2) Identify and test the mechanistic role of ciliary structures that are required for maintaining the periciliary layer; (3) Determine how the large number of ciliary connectome proteins that remain in the cytoplasm are processed, which proteasomal machines are used, and how proteotoxic stress may influence cell fate. Completion of these aims will resolve questions related to lung disease severity in patients with PCD and identify new pathways for therapies.

NIH Research Projects · FY 2024 · 2015-08

Abstract: Sickle cell anemia (SCA) affects one in 1000 individuals worldwide, causing multi-organ ischemia, long-term disability, and premature death, with a life expectancy of 42 years. Among its complications, cerebral infarction and cognitive disability are prevalent and increase with age, with > 50% of young adults having silent infarcts. Great headway has been made in pediatric SCA using neuroimaging screening tools to select high-risk children, yet adults remain understudied. As the brain demands disproportionately more oxygen than other organs at ~20% of total blood supply (but only 2% of body weight), low arterial oxygen content (CaO2) due to anemia, places the sickle cell brain at lifelong risk of hypoxia. Thus, to try to meet cerebral oxygen demand (CMRO2), the brain is continually under hemodynamic and metabolic “stress”, marked by elevated cerebral blood flow and oxygen extraction fraction (OEF), respectively. Findings from our previous grant cycle have helped shape a new understanding of ischemic brain injury mechanisms in SCA. Importantly, specificity of both global and regional OEF for stratifying stroke risk, at patient and tissue levels, suggests great promise for the clinical utility of this imaging biomarker. We are now completing follow-up MRIs to determine if OEF longitudinally predicts infarction in pediatric SCA. Two unexpected findings emerged from our results which warrant further investigation. First, we expected that compensatory increases in CBF and OEF in SCA would serve to maintain a normal cerebral oxygen metabolic demand; however, we found that resting CMRO2 is globally elevated in SCA. This increase in oxygen demand parallels an elevation in total body resting energy expenditure in SCA, which is postulated to be due to chronic inflammation. The finding is intriguing as an elevated cerebral oxygen demand may increase ischemic vulnerability. Indeed, sickling and high blood velocity injure the endothelium inducing a variety of leukocyte-endothelial interactions. Therefore, we hypothesize that neuroinflammation may promote ischemia by increasing cerebral oxygen demand. Second, while we find global OEF elevation in adults with SCA compared to controls, regional OEF elevation in the deep white matter is less prominent in adults compared to children, suggesting a decrease in regional OEF with disease duration. It is postulated that capillary flow heterogeneity (CFH) due to change in capillary microarchitecture leads to a reduction in local OEF. This is of great interest in SCA because capillary morphology is disrupted and transit times are short due to anemia. Thus, we hypothesize that progressive microvascular disease in SCA will disrupt capillary flow patterns, decreasing oxygen supply, as an additional ischemic mechanism. In this renewal, we shift focus to adults with SCA, as a growing and understudied population. First, we will determine if cerebral metabolic stress predicts ischemic brain injury and cognitive decline. Next, we will employ novel MR approaches to investigate two mechanisms (neuro- inflammation and CFH) which perturb cerebral oxygen metabolic physiology, to further our understanding of oxygen supply-demand mismatch in SCA, each which can be developed as a novel therapeutic target.

NIH Research Projects · FY 2024 · 2015-08

ABSTRACT Lower urinary tract symptoms (LUTS), including storage, voiding, urinary incontinence symptoms and pain, and their associated diagnoses are common, costly, and negatively impact women's quality of life throughout the life course. Despite their common occurrence and high cost, little research to date has focused on identification of risk and protective factors for LUTS and prevention, and even less has focused on promotion and maintenance of bladder health. To address this gap, the Prevention of Lower Urinary Tract Symptoms (PLUS) Research Consortium (of which our site is a founding Clinical Research Center) was established to develop the scientific foundation for future evidence-based bladder health promotion and LUTS prevention interventions in adolescent and adult women. In this application, we propose to build upon foundational work conducted in PLUS 1 to further this scientific foundation. Specifically, we propose to establish a large, longitudinal, national, population-based, observational cohort study (currently under development and co-led by the Washington University School of Medicine site PI) designed to: a) determine the distribution of and changes in bladder health over time in the general female population; and b) identify new risk and protective factors for LUTS and bladder health amenable to intervention. Per the FOA, we have selected one important risk/protective factor research question for incorporation into the population-based cohort study: does chronic delayed voiding, a common behavior among women across the life course that can be addressed by preventive interventions at multiple levels of social ecology, contribute to bladder health deterioration and the development of LUTS? Guided by an intervention mapping approach and community partner insight, we will build the evidence base for this research question by conducting a series of innovative, multi-method, transdiciplinary studies nested within the population-based cohort study, as well as complementary to this study. Together, this comprehensive set of quantitative and qualitative studies will provide: 1) critical new data to address the causal nature of associations between chronic delayed voiding and bladder health/LUTS (i.e., high-quality, prospective epidemiologic data devoid of temporal biases and supportive biologic, mechanistic data); 2) new findings to inform the optimal focus and mode of delivery of future prevention interventions (i.e., multi-method data on the strongest individual-behavioral, interpersonal, institutional, and community/societal determinants of chronic delayed voiding, the most effective mode of delivery of educational interventions [assuming that future interventions will include an educational component], and initial promising educational messages); and 3) new tools to evaluate future interventions. As such, this complementary set of studies holds the promise to greatly expand the foundation of knowledge for future bladder health promotion and LUTS prevention interventions.

NIH Research Projects · FY 2025 · 2015-08

Project Summary The long-term goal of this project is to develop effective, precision therapies directed against the initiating mutations of Acute Myeloid Leukemia (AML). During the current funding period, we performed a series of genomic and epigenomic studies that have clarified the mechanisms that AML initiating mutations use to "reprogram" hematopoietic stem progenitor cells (HSPCs), increasing their fitness for transformation. In the next funding period, we will use primary human AML samples, induced pluripotent stem cells (iPSCs), and genetically engineered mouse models to further evaluate the molecular mechanisms involved in preleukemic reprogramming, and progression to AML. Four well characterized events (that initiate more than half of AML cases) will be studied in detail. We will continue our work with DNMT3A mutations and PML- RARA, and add the study of Core Binding Factor AML fusions (RUNX1-RUNX1T1 and CBFB-MYH11). The "toolkit" for these studies will involve the analysis of preleukemic and fully transformed hematopoietic cells from these models, using bulk DNA and RNA sequencing, whole genome bisulfite sequencing, ATAC-seq, ChIP- seq and/or CUT&RUN to detect the genomic locations of activating and repressive histone marks (and the fusions themselves), and single cell technologies for RNA, DNA, and ATAC-seq. We will also be performing comprehensive proteomic studies to complete "proteogenomic" datasets for these initiating events, including 1) the identification of the hematopoietic proteins that interact with the initiating proteins listed above, and 2) the development of quantitative deep-scale proteomic and phosphoproteomic datasets broadly representative of all AML subtypes. The integrative analysis of these datasets (and their availability to the AML community) should provide important new insights about AML pathogenesis, and suggest mechanistically targeted therapies. In this proposal, we provide one representative example of this process: using novel methods to identify proteins that interact with DNMT3A, we discovered several mutations that disrupt the normal interaction of DNMT3A with an inactive isoform of DNMT3B (DNMT3B3); these mutations destabilize DNMT3A and decrease its activity. Remarkably, we found that we can restore the activity of many mutant DNMT3A proteins by retrovirally overexpressing DNMT3L, a protein that normally interacts with DNMT3A and 3B in embryonic cells to increase their activity. "Addback" of DNMT3L into hematopoietic cells with the Dnmt3aR878H mutation remethylates DNA, and decreases the growth of AML cells initiated by this mutation. Since DNMT3L is epigenetically silenced in nearly all AMLs, a program to identify drugs and genetic strategies to reactivate DNMT3L in AML cells will be developed. We have already found that Romidepsin, an HDAC1 inhibitor, potently induces DNMT3L expression, and a clinical trial designed to evaluate the activity of this drug in DNMT3A mutant AMLs is planned. Additional approaches for developing mechanistically driven therapies designed to thwart initiating mutations will be developed during the next funding period.

NIH Research Projects · FY 2025 · 2015-08

Heat shock proteins (HSPs) or protein chaperones are essential to maintain cellular protein homeostasis. Mutations of proteins within this network can lead to degenerative diseases including muscular dystrophy. We have now identified mutations in two domains of an HSP40 co-chaperone, DNAJB6, in an autosomal dominantly inherited Limb Girdle Muscular Dystrophy (LGMDD1). Our central hypothesis is that LGMDD1 mutations alter the conformation of DNAJB6 such that the interface between these two domains is disrupted, leading to myofibril disorganization and muscle dysfunction. Based on our results, we further hypothesize that all LGMDD1 mutations in DNAJB6 can be rescued by perturbation of Hsp70 interaction or activity. Thus, our approach is to employ all of the tools we have developed to test these hypotheses. In order to tackle this challenging problem, we will explore DNAJB6 chaperone function and dysfunction utilizing multiple systems and the expertise of two PIs. Aim 1 will explore whether manipulating DNAJB6:HSP70 interactions rescues LGMDD1 phenotypes in mouse models and human cells. Aim 2 will determine the effect of newly identified LGMDD1-associated DNAJB6 J- domain mutations on chaperone activity in cellular models. Aim 3 will determine the effect of LGMDD1 mutations on chaperone and co-chaperone function in vivo and in vitro. Our preliminary data suggest that we have identified additional therapeutic targets that may be better tolerated in patients than global inhibitors of Hsp70. The overarching goal of this work is to leverage our transdisciplinary success to develop therapeutic intervention for patients with LGMDD1.

NIH Research Projects · FY 2024 · 2015-07

PROJECT SUMMARY/ABSTRACT During protein synthesis, the ribosome integrates multiple cues to ensure that the correct protein is made at the right place, the right time and at the right concentration. These cues are the result of signals triggered by varying cellular needs and environmental conditions such as proliferation and stress. In eukaryotes, the integrated-stress response (ISR) responds to stresses through the activation of kinases that act on the initiation factor eIF2. Phosphorylation of eIF2 represses global translation, but also derepresses translation of key pro-survival mRNAs. In yeast, ISR is activated by the eIF2 kinase Gcn2. Recent studies from several groups, including ours, have pointed to a central role for ribosomes and in particular their stalling during the activation of ISR. Interestingly, ribosome stalling also activates ribosome-quality control (RQC), which depends critically on an E3 ligase Hel2. During the previous funding period, we established that Hel2 is activated in response to ribosome collisions and showed that chemical insults that damage RNA trigger RQC. Notably, these very same agents also activate ISR, suggesting that RQC and ISR are tightly be coordinated. In a very recent study, we showed that not only do ribosome collisions activate both processes, but that the activation of one suppresses that of the other. Emerging from these studies is the observation that collided ribosomes are widely used as sensors to trigger an appropriate response, depending on the type and level of stress. Indeed, in preliminary data presented in this proposal, we provide compelling evidence for a role for ribosome collisions in signaling to other nucleic acid damage pathways, particular those involved in DNA-damage repair. This proposal is focused on understanding the molecular rationale by which collided ribosomes can activate these seemingly unrelated processes. Our preliminary data indicate that the A status of the ribosome is important for ISR activation, and in Aim 1 we will probe the conformation of ribosomes under various stress conditions and assess how they impact Gcn2 recruitment. We will expand on these studies by reconstituting ISR and RQC activities to provide a mechanistic understanding for the apparent preferential activation of RQC over ISR. Notably, robust ISR also requires the presence of the highly conserved transcriptional coactivator Mbf1, which we and others showed to bind collided ribosomes. In Aim 2, we will test the hypothesis that stalling activates ISR using a two-pronged mechanism, in which collided ribosomes in addition to activating Gcn2 modulate ISR coactivation by Mbf1. In particular, we will dissect the role of Mbf1 interactions with the ribosome in regulating its function through post- translational modification. Finally, we have a wealth of preliminary data linking RNA-quality control processes with DNA repair. Aim 3 establishing molecular details about how signaling is transduced between the two processes, which is hitherto unexplored. Altogether, we will leverage our expertise in ribosome biochemistry and yeast genetics in combination with resources we accrued over the past funding period to reveal how collided ribosomes provide a structural platform for several conserved signaling processes.

NIH Research Projects · FY 2024 · 2015-06

PROJECT SUMMARY/ABSTRACT Adolescent idiopathic scoliosis affects up to 3% of children from all ethnicities. African Americans present with larger curves, report greater pain, and have more surgical complications. Healthcare disparities also increase the risk for curve progression and serious, life-threatening neurologic and cardiac complications, many of which can be prevented with early diagnosis. In the initial funding period, we identified several important risk factors for severe scoliosis, including rare variants in fibrillin-1 (FBN1), musculoskeletal collagen genes, and distal chromosome 16p11.2 duplications that confer >10-fold increased risk. However, it is not yet known whether risk factors are generalizable to diverse patient populations. Even when genes are known, precisely determining the pathogenicity of individual human genetic variants remains a bottleneck because of the high frequency of variants of uncertain significance, meaning that there is insufficient clinical or functional data to assign them as either pathogenic or benign. In an era of Precision Medicine, there is an unmet need to change the clinical practice paradigm for scoliosis, however, our ability to interpret genetic data in underserved populations remains limited. Our central hypothesis is that genetic data, when combined with functional analysis, improves the diagnostic precision of variant interpretation for scoliosis and related conditions. To accomplish these goals, we will study a cohort of 1000 African American scoliosis patients recruited during the initial funding period in order to determine whether known risk factors are generalizable to African Americans, and to identify risk variants that can only be identified by studying individuals of African ancestry. Second, we will identify phenotypes associated with scoliosis risk variants in an older population using a gene-first approach that leverages the Geisinger DiscovEHR dataset consisting of >175,000 participants with linked electronic health record and exome sequence data. Finally, to test the hypothesis that knowledge of functional effects improves genetic variant classification, we will utilize high-throughput assays developed in our laboratory for deep mutational scanning. The effects of every possible coding variants in three genes associated with scoliosis and life-threatening aortic aneurysm (COL3A1, SMAD3, and FBN1) will be quantified. Computational classifiers to predict pathogenicity of variant alleles will be built based on our functional data. Variants will be validated in zebrafish models of scoliosis. Renewal of this multicenter study of scoliosis will speed up the pace of gene discovery and its clinical application for patients of all ages and ethnicities. By comprehensively and quantitatively determining the effects of genetic variants on protein function, as well as their impact on diverse individuals across the lifespan, we move closer to the goal of precision medicine for scoliosis.

NIH Research Projects · FY 2026 · 2015-05

PROJECT SUMMARY/ABSTRACT The NCTN’s Alliance for Clinical Trials in Oncology (Alliance) and its associated biorepositories represent an unequaled resource of expertise, institutional infrastructure, and high quality, densely annotated patient biospecimens collected from several decades of cancer therapeutic trials. The scientific mission of the Alliance Biorepository and Biospecimen Resource (ABBR) is to support the activities of the Alliance, the NCTN, and the broader cancer research community through four specific aims, which are: 1) To prospectively support Alliance and NCTN-wide clinical cancer trials with respect to biospecimen procurement, tracking, processing, quality assurance, storage, and distribution. The ABBR implements a number of innovative approaches to streamline biospecimen collection and support the collection of novel biospecimen types for genomic, proteomic, immunoncology, and ‘liquid biopsy’ biomarker studies in the context of therapeutic trials; 2) To provide a resource of high quality, densely annotated biospecimens for secondary correlative science studies directed toward biomarker validation with high clinical impact. The ABBR cooperates with other NCTN biorepositories, the NCTN Biospecimen ‘Front Door Service’, and the NCTN Navigator tool to provide inventories of biospecimens that may be suitable for secondary correlative science studies proposed both within the NCTN groups as well as the broader cancer research community. It also works with other NCTN members and NCI to create an efficient and transparent process for reviewing, approving, and executing such requests so as to speed the translation of candidate biomarkers to cancer diagnostics with clinical utility; 3) To provide scientific leadership to the NCTN biobanking enterprise, by active participation in the NCTN Group Banking Committee (GBC). The ABBR will continue its outstanding commitment to harmonizing and improving biobanking Best Practices by active participation in GBC meetings, subcommittees activities, and work products, and; 4) To provide expertise and infrastructure support for biobanking efforts aligned with and beyond the NCTN, including the Cancer and Immune Monitoring Analysis Centers (CIMACs) and the Community Oncology Research Program (NCORP). Having created a federated infrastructure of biorepository sites and a unifying informatics platform for tracking biospecimens across them, the ABBR continues to be uniquely poised to collaborate with other NCI programs, whenever biorepository or biospecimen resources are required for specific translational cancer research efforts.

NIH Research Projects · FY 2025 · 2015-05

ABSTRACT Economic choice behavior is specifically disrupted in mental disorders such as frontotemporal dementia, major depression and drug addiction. To shed light on these diseases and to pave the way for treatments, it is critical to understand the neural underpinnings of this behavior. In this respect, the past 15 years witnessed very significant progress. Evidence from clinical data, lesion studies, functional imaging and neurophysiology links economic choice to the orbitofrontal cortex (OFC). In particular, work in my lab examined the activity of OFC neurons in monkeys choosing between different juices. We thus identified three populations of cells intimately related to choices: offer value cells encoding individual offer values, chosen juice cells encoding the binary choice outcome, and chosen value cells. In a recent breakthrough, we used electrical stimulation to show that offer values encoded in OFC are causal to choices. Together with work from other labs, our results lay the foundations for a satisfactory understanding of economic choices. However, at least three major questions remain open. (1) It is unclear where in the brain and how value comparisons (i.e., decisions) take place. (2) It is unclear where and how offer values are first computed (or “constructed”). (3) It is unclear whether offer values represented in other brain regions – such as the amygdala – are also causal to choices. The overarching goal of this proposal is to address these fundamental questions. All the experiments will be conducted in non- human primates. Using a combination of behavioral manipulation, neuronal recordings, electrical stimulation and computational techniques, we will pursue two Specific Aims. Under Aim 1, two experiments will examine whether neurons in the OFC participate in value comparison. The cell groups identified in this area represent both the input (offer value) and the output (chosen juice, chosen value) of the decision process, suggesting that they constitute the building blocks of a decision circuit. Exp.1 will assess whether the three cell groups are stable across choice conditions. In parallel, Exp.2 will use electrical stimulation to assess whether neuronal activity in OFC is necessary for value comparison. Under Aim 2, we will examine whether and how three brain regions interconnected with OFC – gustatory cortex (GC), inferotemporal cortex (IT) and basolateral amygdala (BLA) – are involved in the choice process. A working hypothesis is that GC and/or IT might participate in the construction of offer values. Two experiments will test this hypothesis by recording neuronal activity from these two areas. Work conducted in the previous cycle found that neurons in BLA encode the same variables represented in OFC, with some differences. In the last experiment, we will use electrical stimulation to assess (a) whether offer values encoded in BLA are causal to choices and (b) whether BLA participates in the decision process. Fulfilling these Aims will significantly advance our understanding of the neural circuits that underlie economic choices and that malfunction in mental illness. Our track record and substantial preliminary results indicate a high likelihood of success.