Washington University

NIH Research Projects · FY 2024 · 2019-09

Deep brain stimulation of the subthalamic nucleus (STN DBS) can provide substantial motor benefit yet occasional mood and cognitive side effects in Parkinson disease (PD). Current literature hypothesizes that downstream network level effects are a critical mechanism of STN DBS’s influence on motor and non-motor behavior, however our ability to test this hypothesis has been limited. Common imaging modalities either do not have the temporal resolution necessary to discern resting state functional connectivity of cortical networks or are not suitable or safe for patients with implanted DBS. We have developed a novel high-density diffuse optical tomography (HD-DOT) system for measuring brain hemodynamics which can accurately map the functional connectivity of cortical resting state networks (RSN) or task-evoked responses within the first ~1cm of cortex. HD-DOT has comparable temporal and spatial resolution to fMRI, greater comfort than MRI or PET, no radiation exposure, no electrical artifacts, no metal artifacts and no contraindications or safety concerns for DBS patients. We have strong preliminary data showing the validity and feasibility of assessing cortical RSNs and task-induced responses in STN DBS patients. With our novel HD-DOT system, careful experimental design and rigorous analyses, this study will determine the nature of cortical RSN-level modulation induced by STN DBS and its relationship to DBS-induced motor and cognitive change. Controls and individuals with PD will be enrolled pre-surgically and scanned with HD-DOT and MRI (resting state BOLD, structural]). After implantation and optimization of DBS, PD individuals will be scanned with HD-DOT in several conditions. With these data, we will test hypotheses about networks that are responsive to important characteristics of STN DBS (e.g.location) and their relationship to motor and non-motor function. This information ultimately could provide methods for faster optimization of DBS parameters and help identify cortical nodes or networks involved in STN DBS-induced benefits or side effects that would provide future targets for less invasive neuromodulation. Finally, this work could reveal fundamental properties of cortical network physiology such as the capacity for plasticity in response to up-stream perturbations.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY. The eye offers a unique and relatively unexplored area to study Alzheimer’s disease and related dementias (ADRD). The overarching goal of the Eye Adult Changes in Thought (Eye ACT) study is to further scientific understanding of the aging brain by characterizing aging eyes in exquisite detail with prospective collection of non-invasive visual function and retinal imaging data while analyzing decades of extant eye clinical data. Eye ACT leverages tremendous infrastructure and resources from the parent ACT study that has enrolled and biennially followed dementia-free older adults since 1994. As of 03/2020, total enrollment is 5,763 with ~22,000 study visits, 45,000 person-years of follow-up, 1,350 incident dementia cases, and 1,100 incident AD cases. Eye ACT’s recruitment rate from ACT has been >95% to date. Importantly, the ACT study consents for autopsy (30% agree), and nearly 1,000 autopsies have been completed, making this one of the largest autopsy cohorts in the world. Thus, the parent ACT represents an incredibly well characterized cohort that combines research quality evaluations of cognition, ADRD, and autopsy with an unprecedented amount of eye clinical data. In this renewal, Eye ACT will expand the ophthalmic database and cohort size by leveraging parent ACT’s initiative to expand from 2000 to 3000 active participants. Eye ACT will focus on vascular disease and visual impairment (VI) as potential mechanisms for previously reported strong associations between AD and eye diseases (age-related macular degeneration, diabetic retinopathy, and glaucoma). In the first cycle, we extracted clinical eye data from >4,900 participants with manual extraction of clinical paper records through 2003 and in-house natural language processing algorithms for extracting nearly 80,000 clinic visits from the cohort since 2003. We also extracted >18,000 clinically obtained retinal images. We used these data to develop lifetime eye disease severity models that we will leverage in this funding cycle. In Aim 1, we will begin longitudinal evaluation of extensive retinal imaging and visual function data from current ACT participants while fully integrating home visits. We will determine whether structural and vascular retinal features are associated with cognitive decline. We will investigate whether the association between eye disease and AD dementia risk is mediated by VI. In Aim 2, we will investigate relationships between eye health, ADRD neuropathology, and dementia. We will further characterize and harmonize ACT neuropathology data on brain vascular disease, in particular microinfarcts, an important dementia risk factor. We will evaluate eye disease severity trajectories as predictors of microinfarcts and other ADRD neuropathology findings. We will evaluate whether eye diseases or VI modify the relationship between ADRD neuropathology and ADRD dementia impacting cognitive resilience. In Aim 3, we will develop a novel retinal imaging data curation and sharing platform and build needed tools to make large ophthalmic imaging data findable, accessible, interoperable and reusable (FAIR), propelling future artificial intelligence/machine learning research.

NIH Research Projects · FY 2025 · 2019-08

Spinal cord injury (SCI) often results in motor impairments and neuropathic pain. These conditions are related to changes in neural activity in regions of the spinal cord that control motor output and sensory processing. Generally, there is too little neural transmission in spinal motor pathways below the lesion, whereas there is excessive, inappropriate neural transmission in pain pathways below the lesion. It has previously been shown that delivery of small amounts of electrical current directly to motor regions of the spinal cord can increase neural transmission in motor pathways. This type of spinal stimulation is called therapeutic intraspinal microstimulation. Over time, intraspinal microstimulation can enhance motor recovery after SCI. The overall hypothesis of this proposal is that intraspinal microstimulation for motor rehabilitation can also be designed to reduce transmission in spinal pain pathways. If confirmed, this could lead to development of neuroprosthetic therapies for multimodal rehabilitation after SCI. The primary goal of this proposal is to determine the extent to which intraspinal microstimulation can reduce neural transmission in spinal pain pathways. These effects have not previously been characterized. It is known that intraspinal microstimulation activates a widespread network of non-pain-related sensory pathways that bridge the motor regions of the spinal cord and the pain-processing regions of the spinal cord. Activation of this network by peripheral nerve or spinal surface stimulation can reduce transmission in spinal pain pathways. This proposal will determine the extent to which intraspinal microstimulation can reduce transmission in pain pathways through activation of this network. This proposal will also determine whether intraspinal microstimulation promotes release and/or use of a class of natural neurotransmitters known as the monoamines. Monoamines have the unique ability to simultaneously reduce transmission in spinal pain pathways while increasing transmission in spinal motor pathways. Neurons that utilize monoamines have terminations throughout the network of neurons activated by intraspinal microstimulation. All hypotheses will be tested in vivo in anesthetized rats with chronic, motor- incomplete SCI and in rats without neurological injury. Rats of both sexes will be included. The approach includes electrophysiological, computational, pharmacological and biochemical analyses of neural activity. This proposal will advance the understanding of how intraspinal microstimulation impacts highly interconnected spinal sensorimotor networks. If support for the hypotheses is obtained, this proposal will have also identified a new strategy for neuroprosthetic therapies to deliver multimodal rehabilitation benefits. This would address two critical unmet needs of the SCI population: non-opioid treatments for SCI-related neuropathic pain and multimodal rehabilitation. It would also overcome a key limitation of clinically available spinal stimulators, which are parameterized for treatment of motor or sensory impairments alone.

NIH Research Projects · FY 2026 · 2019-08

Project Summary/Abstract Basal bodies are large protein complexes that template cilia and organize the cytoskeleton. Defects in basal bodies contribute to both cancer and ciliopathies. Ciliopathies are human syndromic diseases that include brain malformations, mental retardation, polydactyl, blindness, obesity, and defective kidneys. Cilia are key for multiple functions that include respiratory function, fertility, and establishment of laterality. Our long-term goal is to understand the mechanisms that are altered to cause these diseases. We will study events needed for ciliary function and structure and for assembling basal bodies. From our studies using single particle cryo-EM, we will explore the function of microtubule inner proteins (MIPs) that are localized in the lumen of doublet microtubules of the cilia. To understand the roles of MIPs, we will use genetic, biochemical, proteomic, and quantitative imaging approaches. We have found that at least four MIPs are needed for the symmetric waveform that is used in sperm and nodal cilia and variants in these genes are associated with infertility and laterality defects in humans. These MIPs are likely to act via the outer dynein arms and tektin, a protein first described for its insolubility in cilia. We will also study a class of MIPs called SAXO proteins that have a Mn-motif, and test if they play a role in the stability of cilia. To examine basal body assembly, we will study delta, epsilon, and zeta tubulin. Delta-tubulin mutants assemble doublet microtubules and epsilon-tubulin mutants assemble singlet microtubules. Suppressors of ciliary mutants provided great insight into the regulation of motility. We will take advantage of suppressors of a missense epsilon-tubulin mutant to identify proteins that interact with this tubulin isoform. We will use 4x-expansion microscopy for studying the assembly pathway for triplet microtubules using genes identified by suppressors. Tubulin undergoes many post-translational modifications. We are studying a new post-translational modification on α-tubulin that is the addition of the β-sulfonic amino acid, taurine. The role of taurine-tubulin (τ-tub) is unknown, but taurine itself has been implicated in aging and cancer. Our early characterization shows that τ-tub is present in cilia, basal bodies and cytoplasmic microtubules in some tissues. We will use genetics and 4x-expansion microscopy to understand the role of τ-tub in ciliary and basal body function. τ-tub is likely to have consequence on other known post-translational modification of tubulin; we will tease these interactions apart. Our studies will provide excellent training experiences for undergraduate and graduate students as well as postdoctoral researchers and professional research assistants. We will continue our established collaborations with labs that use electron microscopy and mechanics to expand the impact of our studies.

NIH Research Projects · FY 2025 · 2019-08

The Long Life Family Study (LLFS) has enrolled 4,953 participants in 539 pedigrees in the USA and Denmark that are enriched for exceptional longevity, and has measured them longitudinally in three extensive in-person visits measuring key healthy aging phenotypes in all of the major domains of the aging process. We have demonstrated through many publications that selecting on longevity in the first (proband) generation, results in the second (offspring) generation being much healthier than average in many key phenotypes. Analysis of Danish Medical Registry data suggested that the protection persists into this third generation. In Visit 3, we expanded our LLFS pedigrees to include grandchildren. However, LLFS pedigrees are heterogeneous by phenotype, with different families showing familial clustering of protection in cognition, grip strength, pulmonary function, blood pressure, etc. Further linkage analysis identified extremely strong genetic linkage peaks for cross-sectional as well as longitudinal trajectory rates of change phenotypes for a wide variety of healthy aging domains such as exceptional cognitive performance and resilience to Alzheimer's Disease and Related Dementias (ADRD). Pedigree specific LODs and Whole Genome Sequencing (WGS) suggests that these peaks are driven by rare, protective variants running in selected pedigrees. Through linkage and genomc-wide analysis, we found many new rare and protective variants for healthy aging phenotypes (HAPs), including a new gene for AD, MTUS2. On a subset of the cohort (pedigrees with strong linkage evidence), we generated extensive longitudinal OMICs (transcriptomics, methylomics and metablomics). Despite our success, LLFS is almost all European Ancestry (EA). Adding African-Ancestry (AA) pedigrees will allow us to find new protective variants that are unlikely to be found in EAs (because of genetic drift), and to better resolve the causal variants common to both (because the haplotype blocks of AAs are only half as large as EAs reducing the “driver vs. passenger problem”). Visit 1 was nearly 20 years ago. Since then, there have been many medical advances and healthcare changes. We need to add a modest number of concurrent EA families, to contrast with the new AA ones, to avoid the confounding of secular trends with ancestry differences. The offspring are now approaching 80 years old on average, when expected disease incidence increases. Extending longitudinal follow-up of the original pedigrees will better define HAP trajectories, giving greater power to find protective variants for EL and HAPs. We will extend characterization of LLFS to additional domains microbiome, somatic mutation (CHIP), proteomics. To examine associated haplotypes and structural variants, long-read sequencing is crucial. 70% of human structural variation found in long-read WGS is missed by short-read WGS. Extending the omics to the full cohort will help us understand mechanisms of action of associated variants to protective EL and HAPs. Finally, a more sophisticated systems biology approach, including using state of the art Artificial Intelligence approaches, are needed to fully understand the role of genes working in networks to produce HAPs and EL.

NIH Research Projects · FY 2025 · 2019-08

ABSTRACT Women with dense breasts on mammogram have a 4-6-fold increased risk of breast cancer. A sizeable proportion of premenopausal breast cancer cases (29%) are attributable to having dense breasts. Observational and clinical trial data have shown that a decrease in breast density translates to a reduction in breast cancer incidence. Hence, interventions to reduce breast density could prevent breast cancer. However, adult dietary and lifestyle modifications have not been shown to reduce mammographic density. Therefore, identifying a pathway that can be targeted to reduce breast density and breast cancer incidence is crucial. The receptor activator of nuclear factor-κB (RANK) pathway regulates the development of the lobulo-alveolar mammary structures, activates downstream signaling cascades involved in breast cancer and is the major mediator of progesterone-driven expansion of mammary stem cells. The RANK pathway is associated with mammographic density and breast cancer risk. This has led to a strong interest in inhibiting RANK ligand (RANKL) signaling to prevent breast cancer. Nevertheless, clinical trial data providing definitive evidence that would allow the adoption of RANKL inhibition in reducing dense breasts and prevent breast cancer are not yet available. We, thereby, propose to (i) perform a randomized clinical trial to quantify the effect of RANKL inhibition with denosumab on mammographic density in high-risk premenopausal women with dense breasts (Primary Aim); (ii) determine the effect of RANKL inhibition on breast tissue gene expression, metabolome, and biomarkers associated with breast cancer (Secondary Aim). Approach: Study participants will be randomized (1:1) to an intervention (N=105) or placebo arm (N=105). Intervention: The intervention arm will receive two subcutaneous injections of denosumab (60 mg), one at baseline, and a second at 6 months. The placebo arm will receive two subcutaneous placebo injections at baseline, and 6 months. We will use Volpara software to assess mammographic density at baseline, and 12 months. Volpara quantifies volumetric measures of density; volumetric percent density (VPD) allowing us to test differences in change in mammographic density at 12 months among women assigned to intervention vs. placebo. Study population: 210 women undergoing annual screening mammography at the Siteman Cancer Center (SCC), St. Louis, MO. Inclusion criteria: (i) premenopausal; (ii) ≥40 years of age; (iii) dense breasts (BI-RADS Category C and D – equivalent to volumetric percent density ≥7.5% on Volpara. Target population: premenopausal women with dense breasts aged ≥40 years undergo screening mammogram at the Joanne Knight Breast Health Center at Siteman Cancer Center and affiliated hospitals. Impact: Study findings could open up additional therapeutic approaches in primary breast cancer prevention for high-risk premenopausal women with dense breasts, who do not have dominant genetic predisposition.

NIH Research Projects · FY 2026 · 2019-08

Summary Single-stranded (ss) DNA gaps have emerged as key determinants of genome stability and PARP inhibitor (PARPi) sensitivity, particularly in the context of cancers with mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. During this funding period, we unveiled a central role for PRIMPOL-repriming in generating ssDNA gaps in cells treated with platinum-based compounds and PARPi. This work was enabled by the unique combination of electron microscopy (EM) and single-molecule DNA fiber approaches available in our lab. Using these technologies, we provided the first molecular insight into the mechanisms that repair PRIMPOL-dependent gaps. We also showed that these mechanisms are impaired in BRCA1-deficient cells, explaining why gaps accumulate in these cells. However, we found that gap repair can be restored in BRCA1-deficient cells by inhibiting MRE11 nuclease activity, indicating that nuclease processing plays a central role in ssDNA gap repair. This renewal builds on new EM data showing that the ssDNA gaps in BRCA1-deficient cells treated with PARPi are significantly larger than the ssDNA gaps of BRCA1-proficient cells, and on new biochemical data showing that MRE11 efficiently resects ssDNA gaps in vitro. Based on these findings, we hypothesize that MRE11 plays a key role in processing ssDNA gaps, and that over-resection of the ssDNA gaps by MRE11 in BRCA1-deficient cells prevents gap repair. The first Aim will define the mechanism(s) by which ssDNA gaps are processed by MRE11, in concert with other long-resection nucleases. While processing of double-stranded breaks has been widely studied, the same molecular details are not available for ssDNA gaps. These studies will establish a new paradigm for the roles of nucleases in ssDNA gap resection and repair at DNA replication forks, as well as elucidate the mechanisms controlling nuclease activity and how these are deregulated in BRCA-deficient tumors. We discovered that inhibiting MRE11 activity not only reinstates gap repair in BRCA1-deficient cells, but also changes the pathway of gap repair in BRCA1-proficient cells. The second Aim will determine the mechanisms by which gaps are repaired in PARPi cells and test the innovative hypothesis that MRE11 processing is the key step that governs the choice between template switching and more error-prone translesion synthesis mechanisms of gap repair. Finally, we observed that, unlike the PARPi-sensitive BRCA1-deficient cells, ssDNA gaps are efficiently repaired when the same cells become PARPi-resistant. Thus, we will determine how MRE11 inhibition restores gap repair in BRCA1-deficient cells and whether targeting these repair pathways represents a novel strategy to overcome PARPi resistance in BRCA1-deficient cells. This knowledge is crucial for basic research to inform how nucleases control the balance between error-free and mutagenic mechanisms of ssDNA gap repair and how targeting these mechanisms modulates chemotherapy response in BRCA1-deficient tumors.

NIH Research Projects · FY 2024 · 2019-08

ABSTRACT Mechanical allodynia is a hallmark symptom of chronic pain characterized by painful responses to innocuous stimuli. However, little is known about the cellular and molecular regulation of this process. Recently, the mechanically activated Piezo2 channels were identified as key players of mechanical allodynia in mice and humans, but the molecules and proteins responsible for the sensitization of Piezo2 channels upon injury are still poorly understood. Recent data from our lab show that activation of Gi-protein coupled receptors induces a long-lasting potentiation of Piezo2 currents in Dorsal Root Ganglion (DRG) neurons and HEK293 cells. The potentiation of Piezo2 currents was abolished by inhibiting the activity of Gβγ. Surprisingly, the inhibition of Gβγ-downstream kinases, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), also abolished the potentiation of Piezo2 current suggesting an indirect effect of Gβγ on Piezo2 channels. Therefore, for aim 1 (Ph.D. progress), we described a novel mechanism of regulation of Piezo2 currents by Gi- protein coupled receptors. On the other hand, our lab has also shown that activation of Transient Receptor Potential Vanilloids 1 (TRPV1) channels by capsaicin leads to robust inhibition of Piezo2 currents in DRG neurons and heterologous systems. This inhibition is abolished by removing Ca2+ from the extracellular solution, confirming a pivotal role of Ca2+ on Piezo2 channels. Preliminary data in our lab using total internal reflection fluorescence (TIRF) show that Piezo2 channels are internalized upon activation of TRPV1 channels in HEK293 cells, but whether Piezo2 channels are internalized via endocytosis is not known. For aim 2 (F99 phase), we hypothesize that activation of TRPV1 induces a Ca2+-triggered endocytosis that orchestrate the inhibition of Piezo2 currents. We aim to identify molecules and proteins that regulate the activity of the mechanically activated Piezo2 channels. We hope these novel findings could help us understand the process of tactile allodynia and investigate the changes that affect the periphery and influence the central nervous systems (aim 3-K00 phase) with the ultimate goal of providing new avenues for treatments of mechanical-pain syndromes.

NIH Research Projects · FY 2025 · 2019-07

PROJECT SUMMARY/ABSTRACT The major OBJECTIVES in this application are to expand understanding of mediators of fibrogenic injury and HCC development associated with impaired VLDL secretion in both mice and humans. Our proposal is SIGNIFICANT because of the unmet need to identify subsets of patients with NAFLD whose disease will progress and where a more tailored approach might inform therapeutic strategies for prevention and reversal of NASH/fibrosis and the development of HCC. The BACKGROUND is that genetic defects (APOB, MTTP, TM6SF2) that impair hepatic VLDL secretion cause hepatic steatosis and progress to NASH with fibrosis and HCC, even without obesity or insulin resistance. In addition, VLDL secretion is relatively (ie paradoxically) impaired in a subset of insulin-resistant, NAFLD patients. Accordingly, our overall SCIENTIFIC PREMISE is that identifying shared pathways in genetically modified mouse models and in metabolic subset of patients with NASH and impaired hepatic VLDL secretion will identify druggable targets for fibrosis reversal and prevention of HCC. Our proposal is supported by KEY PRELIMINARY DATA including: (AIM 1) Liver-specific Tm6sf2 knockout mice (Tm6-LKO), phenocopies the loss-of-function human TM6SF2 (E167K) variant rs58542926 with defective VLDL secretion, hepatic steatosis, fibrosis and increased HCC. We generated Tm6sf2 variant knockin (K167K) mice to examine hepatic VLDL and intestinal chylomicron secretion. We generated variant knockin HepG2 cells and iPS-derived hepatocyte-like cell lines to study the loss-of-function variant in human VLDL assembly. (AIM 2). Liver-specific microsomal triglyceride transfer protein knockout mice (Mttp-LKO) mice crossed into Fabp1–/– mice (ML-DKO) mice exhibit reduced steatosis and decreased fibrosis, yet paradoxically increased HCC. By contrast Mttp-LKO mice crossed into liver-specific DGAT2 knockout mice are protected against HCC. We identified a metabolomic signature in patients with NAFLD/NASH that aligns with impaired VLDL secretion and will examine patients with NASH-HCC. The AIMS of this proposal are: AIM 1. How does how the Tm6sf2 variant alter APOB lipidation and regulate hepatic and intestinal VLDL/chylomicron assembly and secretion? How does the K167K variant influence the development of fibrosis and HCC? How are these pathways in mouse VLDL assembly and secretion replicated in representative human hepatocyte-like cell lines (HepG2, iPS-derived hepatocyte-like cells)? AIM 2. How do modifiers of DNL and LD turnover modify signaling pathways in mice with impaired VLDL secretion (Mttp-LKO) that either promote (Fabp1-dependent) or mitigate (Dgat2-dependent) HCC development? We will examine how pathways identified in mice with impaired VLDL secretion and either increased or decreased HCC susceptibility align with transcriptomic signatures in liver of patients with NASH-HCC and a metabotype of impaired VLDL secretion. Taken together, we address a CRITICAL KNOWLEDGE GAP by exploring novel pathways of fibrogenic injury and HCC, focusing on defective VLDL secretion as a nexus point directly relevant to a subset of genetic and acquired etiologies of NAFLD/NASH.

NIH Research Projects · FY 2024 · 2019-07

PROJECT SUMMARY/ABSTRACT Many rural communities are medically underserved and experience persistently elevated rates of colorectal cancer (CRC) incidence and mortality relative to declining national rates. Routine screening reduces population CRC mortality, yet its impact is reduced because many adults who have an abnormal screening result with fecal testing do not receive diagnostic follow-up with colonoscopy. Rural residents and healthcare providers face unique barriers to screening follow-up including fewer providers who offer colonoscopy and longer travel distances to obtain healthcare. Rural Southern Illinois is a region with high poverty, slow economic growth, isolated households, widely dispersed medical care, and high CRC mortality. To reduce disparities in CRC mortality in rural areas where fecal immunochemical testing (FIT) is a common first-line screening strategy, we must identify effective, sustainable, and disseminable strategies to improve follow-up of positive screening tests. Researchers at Washington University School of Medicine have collaborated with Southern Illinois Healthcare, a rural not-for-profit health system, since 2015 to identify cancer prevention and control priorities and reduce disparities. From 2017 to 2018, we conducted a formal pre-implementation assessment of CRC screening and follow-up processes to identify feasible and promising evidence-based interventions and strategies for improvement. Based on our substantial and specific preliminary data, we propose the following Aims: Aim 1. Implement a multilevel intervention of follow-up of abnormal colon cancer screening tests in primary care clinics across rural Southern Illinois. Using a stepped wedge trial design and cluster randomization, we will implement the multi-level intervention in 18 clinics. We will intervene at three levels (patients, providers/clinical teams, clinics) and evaluate implementation outcomes per Proctor's evaluation model using interviews, surveys, and field notes. Aim 2. Evaluate the impact of the multilevel intervention on follow-up of abnormal screening test results in rural primary care settings in Southern Illinois. Our stepped-wedge design will allow us to test the impact of the multi-level intervention on rates of screening follow-up. We measure outcomes at three levels. Patient: After positive FIT, receipt of referral and completion of colonoscopy. Primary Care Provider: Receipt of positive FIT results and referral for follow-up. Clinic-level: Patients with positive FIT complete colonoscopy. We will assess change in CRC screening rates and investigate interactions between and across levels. Data for primary outcomes will come from the healthcare system's ongoing patient registry that draws from electronic medical records and lab records. The co- construction of this proposal between university researchers and health system stakeholders enhances the potential for significant and sustainable change for effective and efficient screening and early detection. There is a critical need for real-world strategies that can function within rural community health systems to improve health and reduce disparities.

NIH Research Projects · FY 2024 · 2019-07

ABSTRACT Inflamed bone fracture poses a significant clinical problem. In the United States, approximately 1.6 million bone fractures encounter prolonged healing or non-union each year, among which, the major population bearing with these clinical complications are patients with inflammatory conditions, e.g, elder patients, smoking, diabetic or rheumatoid arthritis (RA) patients. In these patients, the fracture risk is increased due to the poor bone quality, highlighting the potential deleterious role of chronic systemic inflammation in fracture repair. The overarching hypothesis of this proposal is that under inflammatory conditions, NF-κB, the principal mediator of inflammation, induces Rbpjκ expression through downregulating Dnmt3b and its DNA methylation activity. We further hypothesize that Dnmt3b GOF or Rbpjκ inhibition restores MPC differentiation and chondrocyte maturation that are reduced by inflammation during fracture repair. This hypothesis is supported by our preliminary data wherein we show that Dnmt3b is highly expressed in fracture callus during fracture repair and Dnmt3b is the major DNA methyltransferase (Dnmt) responsive to cytokine in MPCs and chondrocytes. Relevant to our proposal, we provide evidence that inflammatory signals inhibit Dnmt3b in MPCs and chondrocytes in an NF-κB-dependent manner. Consistently, mice with Dnmt3b loss-of-function (LOF) in MPCs and chondrocytes display delayed fracture repair; and Dnmt3b gain-of-function (GOF) in MPCs or chondrocytes shows protective effect from inflammation in vitro and accelerates fracture repair in mice. Mechanistically, MPC differentiation defect mediated by inflammation and Dnmt3b LOF coincide with upregulation of Rbpjκ in MPCs and Rbpjκ inhibition can restore differentiation capacity in vitro. In vitro mechanistic studies and in vivo LOF and GOF approaches will be used to modulate IKK2, Dnmt3b and Rbpjκ expression in MPCs and chondrocytes to dissect its effects during fracture repair process. Three main Specific Aims are proposed. Specific Aim 1 will delineate the effect of constitutively active NF-κB signaling (IKK2ca), as the principal molecular driver of inflammation, on Dnmt3b expression and fracture repair. Specific Aim 2 will establish the effect of Dnmt3b GOF in MPCs and chondrocytes on accelerating fracture repair. Specific Aim 3 will delineate the mechanism by which Dnmt3b regulates downstream target, Rbpjκ, during fracture repair. This work will enhance our understanding of mechanisms by which systemic inflammation (via the NF-κB pathway) affects the fracture healing process through Dnmt3b and identify downstream targets of Dnmt3b (such as Rbpjκ) as novel candidates for therapeutic intervention.

NIH Research Projects · FY 2024 · 2019-07

Project Summary Primary prevention of HPV-related urogenital and oropharyngeal cancers could significantly reduce morbidity, mortality, and healthcare costs associated with these commonly occurring cancers. The vaccine targeting oncogenic strains of the human papilloma virus (HPV) can prevent HPV-related cancers if given to girls and boys before exposure to these sexually transmitted viruses. Yet, in the United States (U.S.), more than 10 years after introduction of the vaccine, fewer than half of the target population is vaccinated. The objective of this application is to evaluate the effectiveness of an implementation strategy (the intervention) to improve optimal use of the HPV vaccine. Optimal use requires completion of the 2-dose series before sexual debut. To this end, national recommendations from the Centers for Disease Control and Prevention (CDC) are to complete the 2-dose HPV vaccine series by age 13. The theory-based, multi- component intervention was developed in collaboration with primary care providers and includes: 1) an educational video to increase the provider’s knowledge about guideline recommendations and patient and practice benefits of vaccination by age 13; 2) audit and feedback of vaccine coverage to increase motivation to engage in practice change; 3) a communication strategy to improve the provider’s communication skills and their self-efficacy to address parental hesitation; and 4) practice facilitation to support practice change to develop a sustainable HPV vaccine delivery system. The intervention will be delivered through a series of brief practice visits with the facilitator that occur every 1-4 weeks over 2 years. In a pilot study, the intervention was well-accepted and resulted in a clinically meaningful improvement in vaccine initiation of 19 percentage points over 15-months. This promising intervention will be evaluated in a cluster-randomized trial in 20 primary care pediatric practices in Missouri, a state with low HPV vaccine coverage. Study objectives are to 1) determine if the intervention will increase initiation (primary outcome) and completion (secondary outcome) of the 2-dose HPV vaccine series for preteens before their 13th birthday; 2) determine if provider motivation and improved communication skills mediate the effectiveness of the intervention; and 3) assess factors important for scaling including fidelity, acceptance by primary care providers and parents, and cost. Vaccine use in preteens will be assessed at baseline, at 24 months to assess effectiveness, and at 36 months to assess if change is sustained. For the primary analyses, aggregates of patient-level data will be used to estimate provider behavior, extracting data from the practices’ electronic medical record.

NIH Research Projects · FY 2024 · 2019-07

Project Abstract Degeneration of the intervertebral disc (IVD) is a leading contributor towards back pain, an epidemic that costs billions of dollars in the US. The IVD consists of a proteoglycan(PG)-rich nucleus pulposus (NP) surrounded by a collagenous annulus fibrosus (AF) that together provide support and transmit complex loads. The IVD degenerative cascade involves a multifactorial progression of biological, biochemical, and structural changes that lead to the collapse of the disc structure and to compromised mechanical function. Despite its significant public health impact, the pathophysiology of disc degeneration remains unclear. The accumulation of Advanced Glycation End-products (AGEs) is associated with aging and diabetes. Increased AGEs has also been associated with IVD degeneration. AGEs form through nonenzymatic glycation, where extracellular sugars undergo Maillard rearrangement with amino acids to become protein adducts and crosslinks. AGES are known to impair the mechanical function of matrix proteins. Beyond matrix modifications, AGEs activate the cellular Receptor for Advanced Glycation Endproducts (RAGE), and RAGE signaling perpetuates immune and inflammatory responses. Because the IVD is avascular and has relatively low tissue remodeling, IVD tissues are susceptible to accumulate AGEs. Despite these observations, it is not known whether AGEs or RAGE signaling have a causal role in IVD degeneration. In this R01 application, we will determine the AGEs- and RAGE-mediated events as disease mechanisms for IVD degeneration. Specifically, we will identify the role of AGEs in altering IVD structure and function and define the necessity of RAGE-signaling in AGEs-mediated degeneration. If our hypotheses are supported, this will provide the putative targets to alleviate the degenerative cascade. The combination of in vivo and ex vivo- in vitro approaches will enable us to carefully dissect the systemic effects of high AGE-loads from tissue- specific effects of AGEs. We also will further develop the in vivo contrast-enhanced microCT of the intervertebral disc as a key technological innovation. This approach provides a resolution that significantly advances the current state-of-the-art compared to microMRIs. We believe that the successful completion of the proposed aims will significantly advance our knowledge of intervertebral disc biology and intervertebral disc imaging.

NIH Research Projects · FY 2025 · 2019-07

Project Summary This application will investigate the molecular and cellular mechanisms underlying antigen cross-presentation by dendritic cells (DCs), with a major focus on type 1 classical DCs (cDC1) which play a major role in priming CD8 T cell responses that are critical for cancer immunotherapy. Cross-presentation involves the presentation of exogenous antigens by MHC-I molecules, which is predominantly facilitated by cDC1 in vivo. This application will address critical gaps in understanding the cross-presentation process and its implications for immune responses, leveraging several novel in vivo models that we have produced to elucidate the specific roles and mechanisms of cDC1 and cDC2 in antigen processing and presentation. A central facet of our proposal is the investigation of WDFY4, which we identified as critically important for cross-presentation by cDC1. Our preliminary data reveal a pivotal role in mediating CD8 T cell responses against viruses and tumors, and implicating it in autoimmunity contexts such as type I diabetes. The proposal aims to define the molecular basis of cross-presentation, challenging alternative models by providing rigorous data supporting vacuolar pathways for antigen processing that involve WDFY4. Specifically, our proposal challenges the models that rely on a cytosolic route, by providing both an explanation for the apparent dependence on the TAP transporter and evidence for TAP-independent cross-presentation. Aims of this proposal are twofold: firstly, we will rigorously test the hypothesis that WDFY4-dependent cross-presentation in cDC1 employs a vacuolar pathway, contrasting it with the cytosolic route and assessing the role of the TAP transporter in this process. Secondly, we will define the molecular interactions of WDFY4 with proteins that mediate its actions and determine whether they are involved in regulating vesicular trafficking, cargo sorting, or cytoskeletal dynamics, which are crucial for the cross-presentation of antigens. This will involve identifying and validating interacting proteins, determining their structural relationships with WDFY4, and elucidating their functional relevance to cross-presentation in vitro and in vivo. By challenging existing paradigms and employing cutting-edge genetic and molecular tools, this research promises to significantly advance our understanding of dendritic cell biology, particularly the cellular and molecular mechanisms of antigen processing and presentation. The outcomes are expected to enhance our understanding of immune activation and to have broad implications for the design of more effective cancer immunotherapies and interventions for autoimmune diseases.

NIH Research Projects · FY 2026 · 2019-06

Project Summary Children with sickle cell disease (SCD) often face significant hurdles in their cognitive development and educational achievements. Despite the availability of evidence-based practices (EBPs) established through clinical trials and guidelines, there is still a substantial gap in their real-world application. This gap can impact the educational and cognitive development of children with SCD, making it crucial to address these barriers effectively. As a board-certified pediatric hematologist/oncologist and Professor at Washington University School of Medicine, my career has been dedicated to improving the lives of young patients with SCD. With formal training in clinical research methods, public health, education, and implementation science, I work to bridge the gaps between communities, schools, and healthcare providers to ensure comprehensive care for children with SCD. Since my first K24 was funded, I have served as a mentor on ten K-awards, including two international K43s. Ten of my mentees have earned foundation-funded career development awards. Since 2019, my mentees and I have published 65 manuscripts together. I am committed to continuing to mentor trainees and junior faculty, focusing on SCD and implementation science. With the support of ongoing and funded independent awards, my K24 award renewal proposal aims to further this mission through three key objectives: 1) Develop and test sustainable stroke prevention toolkits for low- and middle-income countries (LMICs): This aim focuses on creating user-friendly, adaptable educational materials to train healthcare providers and engage communities in stroke prevention for children with SCD. 2) Create tailored communication strategies for disseminating crucial information about SCD to students, parents, educators, and healthcare providers: These strategies will use user-centered design and multimedia platforms to ensure the effective transfer of knowledge and the establishment of supportive educational environments. 3) Conduct a systematic review to identify the most effective dissemination and implementation strategies for early childhood developmental screening in SCD: This aim will guide best practices for integrating developmental screening into pediatric hematology practices, thereby improving early intervention and outcomes. Each aim will offer valuable opportunities for my mentees' training and research experience. By developing skills in user-centered design, dissemination science, and systematic reviews, I aim to enhance both my mentees' and my abilities to produce impactful research proposals. This K24 award renewal will provide the crucial protected time I need to focus on high-impact research and mentoring junior investigators. By advancing our understanding and application of implementation science methods, we can significantly improve the healthcare and educational outcomes for children with SCD, fostering a brighter future for those with the condition.

NIH Research Projects · FY 2025 · 2019-06

SUMMARY Alphaviruses are among the most important disease-causing, arthropod-borne viruses for humans. This genus includes viruses (e.g., chikungunya (CHIKV), Mayaro (MAYV), O’nyong’nyong (ONNV), and Ross River (RRV) viruses) that cause debilitating acute and chronic polyarthritis, have affected millions of people globally, and are emerging and re-emerging threats. Despite their epidemic potential, there are no specific therapies for alphaviruses. We previously used a CRISPR/Cas9-based screen to identify MXRA8 as a novel receptor for multiple arthritogenic alphaviruses including CHIKV, RRV, MAYV, and ONNV, a finding reproduced by several other laboratories. The identification of a receptor enabled us to generate soluble MXRA8- Fc decoy proteins that mitigate infection and disease in mice caused by multiple arthritogenic alphaviruses. In this renewal application, our collaborative team will continue to study the fundamental role of MXRA8 in alphavirus entry and pathogenesis. We will use cryo-electron microscopy to obtain a series of high resolution (< 3.2 Å) reconstructions of MXRA8 bound to multiple arthritogenic alphaviruses and use this information along with directed evolution to design MXRA8 decoy molecules that bind with greater affinity and neutralize infection more potently. We also will perform infection studies in conditional knockout mice and non-human primates (NHPs) with recombinant CHIKV strains that can or cannot bind to MXRA8 to further define its role in pathogenesis of acute and persistent disease. Finally, we will validate and perform structure- function analysis on new candidate MXRA8-independent entry factors for arthritogenic alphaviruses that are being identified through novel CRISPR-Cas9 gain-of-function and loss-of- function genetic screens. The innovative experiments in this renewal application will define fundamental aspects of alphavirus biology that enhance our understanding of how infection, tissue targeting, and disease occurs. This information also may facilitate the development of countermeasures that disrupt MXRA8 interaction with alphavirus envelope proteins, which could form the basis of therapeutics that ameliorate disease of multiple alphaviruses.

NIH Research Projects · FY 2024 · 2019-05

The central nervous system (CNS) changes throughout life, and its interactions with the world produce activity- dependent plasticity that enables it to acquire and maintain useful behaviors. Recent scientific and technical advances support the development of systems that create novel interactions with the CNS that can induce and guide beneficial plasticity. These systems, called adaptive neurotechnologies, measure signals from the CNS and concurrent behavior, derive from those signals the state of the CNS, and adaptively provide real-time feedback that can restore, replace, enhance, supplement or improve CNS functions impaired by injury or disease. Thus, they can provide important new therapies for neurological disorders. The development and use of adaptive neurotechnologies is an inherently multidisciplinary endeavor. The integration of knowledge and ideas from these diverse areas, and their implementation by highly sophisticated software/hardware systems, requires substantial time, effort, and multidisciplinary expertise. This slows progress in research and development of adaptive neurotechnologies and limits the number of groups that are successful in realizing these technologies. The present proposal seeks to address these two major problems. Over the past 18 years, we have developed and disseminated a software platform, called BCI2000, that supports interactions with the CNS and can implement a wide range of adaptive neurotechnologies. To date, we have provided it to more than 6,000 users worldwide who have used it to support experiments described in over 1,200 peer-reviewed publications. Despite this demonstrated value, BCI2000 adoption is limited by the requirements of substantial programming expertise and in-depth understanding of BCI2000 concepts needed to adapt and integrate BCI2000 into the specific experimental protocols and hardware technologies of a particular laboratory. Thus, most research groups cannot take advantage of the advantages provided by BCI2000. We propose to address this deficiency by simplifying the task of configuring BCI2000 for all major classes of adaptive neurotechnology experiments (Aim 1), and by providing a succinct introductory course and on-site training for scientists, engineers, and clinicians (Aim 2). We hypothesize that this work will greatly accelerate realization of adaptive neurotechnologies that will reduce the devastating impact of neurological disorders. Achieving these two aims will create and disseminate a major new software resource for adaptive neurotechnol- ogy research. Its unique utility should enable more scientists, engineers, and clinicians to engage in this exciting work. Furthermore, this common, interoperable, readily adopted, and easily adapted platform should foster a collaborative environment that enables diverse investigators to work together and complement each other. In sum, we expect that the work of this proposal will accelerate realization of novel adaptive neurotechnologies that will hasten scientific investigations to improve treatment for many devastating neurological disorders.

NIH Research Projects · FY 2025 · 2019-05

Direct examination of presynaptic processes has historically been limited by the resolution constraints of conventional light microscopy. As a result, much of what we know about vesicle movement, fusion, and recycling relies on inferences from indirect electrophysiological and/or biochemical assays, or from electron micrographs that reflect a single instant of a dynamic system. The long-term goal of my research program is to understand the fundamental mechanisms of synaptic transmission at central synapses, including details of spatiotemporal dynamics under normal conditions, and what disruptions lead to disease states. Current projects in the lab address two central knowledge gaps. First, we directly probe and track dynamic presynaptic processes in living tissue by applying our own novel, nanoscale resolution imaging technology. Using this approach, we will, for the first time, visualize these processes at the level of single synaptic vesicles within identified synapses. We have already made significant contributions using this approach, including the discovery that synaptic vesicle dynamics are active, not passive, and are controlled by actin cytoskeleton and myosin motors. The second major knowledge gap we address is the contribution of presynaptic deficits to pathophysiology of Fragile X syndrome (FXS). FXS is the most common known cause of heritable intellectual disability and autism. Our recent findings have triggered a necessary shift in the field towards considering the contributions of presynaptic mechanisms in addition to postsynaptic mechanisms, thus creating an entirely new array of diagnostic and therapeutic possibilities. Continuing work in this area will focus on linking presynaptic defects with abnormalities at the circuit level and the implications of these abnormalities for behavior and cognition. Sustained funding through this R35 mechanism will support a multipronged approach to these important neurobiological questions that will maximize the potential for synergy and translational impact.

NIH Research Projects · FY 2024 · 2019-05

PROJECT SUMMARY – This application addresses critical needs and deficiencies in prostate cancer (PCa) patient management. The five year survival rates for localized primary PCa are excellent, but sadly fall to below 1 in 3 for those with metastatic disease. The deluge of academic and clinical efforts to harness targeted agents for imaging of Prostate Specific Membrane Antigen (PSMA), widely overexpressed on prostate cancer tissues, for improved detection represents a sea change in how malignant disease will be monitored. Advancing close behind is a systematic evaluation of therapeutic variants of these agents that deliver an ionizing radiation dose to PSMA- expressing sites of disease. There is considerable interest in alpha particle (α-particle) emitting radionuclides for this targeted radiotherapy as the high linear energy transfer imparts 5-8 MeV in a dense track that is only several cell diameters in length. Unfortunately, widespread background-organ expression of PSMA results in untoward side-effects of absorbed dose to normal tissues. Off-target toxicity places limitations on the activity dose which may be administered; the patient population eligible for the treatment; the requirements for involved long term care of comorbidities; and ultimately the overall impact this treatment will have in the clinic. Here, we propose a strategy that enables organ specific reduction in absorbed dose without affecting tumor targeted uptake. We focus on the salivary glands and kidneys; radiosensitive organs that demonstrate intense PSMA-ligand targeting in pre- and clinical imaging and treatment studies. We have developed and acquired significant insight into a novel prodrug, Tris-POC-2-PMPA that is preferentially deliverd to the kidneys and salivary, and selectively cleaved in these organs, to release the high affinity PSMA inhibitor, 2-PMPA. Our Preliminary Data demonstrate the potential to ensure that tumor specific ablation without toxicity can be achieved while sparing kidney and salivary tissue. Taking advantage of a hybrid imaging and therapy approach, we will define the optimal treatment course required for tumor control, without normal organ toxicity, in multiple small animal xenograft and in an advanced genetically engineered model that most closely recapitulates human disease. This application is being undertaken by a multidisciplinary team composed of experts in radiochemistry, medical physics, pathology, drug development and clinical molecular imaging. This group of investigators and the strength of our data addressing key issues in alpha particle emitting radiopharmaceutical development demonstrate that this application has the potential to realize the transformative capabilities of molecularly targeted radiotherapy for cancer patients.

NIH Research Projects · FY 2025 · 2019-04

Overall. The NIH has made substantial research investments in response to the large burden of musculoskeletal diseases in the United States. The Washington University Resource-Based Center for Musculoskeletal Biology and Medicine was formed to enhance NIH-funded, musculoskeletal research on our campus, with a focus on basic and pre-clinical research. The Center has profoundly enhanced the musculoskeletal Research Community at WashU and beyond through state-of-the-art Resource Cores and a multifaceted Enrichment Program. Since 2019, our three Resource Cores have supported the work of 155 principal investigators (PIs), including 15 non-WashU PIs. Their work led to 237 publications that have accrued >3,000 citations. We currently represent a Research Community of 97 PIs. The majority (85) are WashU faculty, representing 15 departments. With this renewal application, we aim to increase the impact of our Center with a new Affiliate Member category targeting investigators in neighboring states who bring added expertise to our Center. Our initial group of Affiliates comprises 12 faculty from six institutions located in five states. In total, the annual extramural funding of our Research Community is $108 million, with $89 million from NIH, of which $13 million is from NIAMS. Our work addresses the most prevalent and costly musculoskeletal disorders – arthritis, back pain, bone and joint trauma, osteoporosis, and metastatic cancer. We have designed the three Resource Cores of the P30 Center to support the critical needs of the Research Community in the development, implementation and evaluation of research models for musculoskeletal biology and medicine. The Administrative Core (A) will provide Center leadership and direct an Enrichment Program. The Musculoskeletal Structure and Strength Core (B) will support access, training and cost-effective utilization for X-ray based imaging methods, and biomechanical methods to quantify mechanical properties of musculoskeletal tissues. The Musculoskeletal Histology and Morphometry Core (C) will perform processing, embedding, sectioning and staining for the range of tissues and techniques required in musculoskeletal research, and support access and training for routine microscopy and histomorphometry, and for advanced microscopy. The Animal Models of Bone & Joint Injury and Disease Core (D) will provide training and hands-on implementation for reproducible mouse models of arthritis (OA, RA) and fracture, and support access and training for state-of-the-art measures of in vivo pain and function. In summary, we propose Cores that will provide essential support for our Research Community to develop, implement, evaluate and interpret research models for musculoskeletal biology and medicine. A common theme for each Core is support for training and instruction to enhance rigor and reproducibility, and for enrichment activities to foster the development of the next generation of musculoskeletal investigators. *

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY/ABSTRACT Recent estimates from the United States suggest that 8% of pregnant women used cannabis in the past month. While evidence remains mixed, emerging research documents an association between prenatal cannabis exposure (PreCE) and offspring birthweight, brain development (e.g., white matter microstructure), and behavior, especially inattention, impulsivity and broader externalizing symptoms. The Cannabis Use During Development and Early Life (CUDDEL; DA046224) study collected data on pregnant women who reported cannabis use during pregnancy (validated using self-report and urine screens; PreCE, n=200) and women who used cannabis prior to but never during pregnancy (NPreCE, n=200). Of these women, 150 PreCE and 100 NPreCE mother- child dyads were recruited to participate in the postpartum arm of the project where neonatal brain imaging was conducted soon after birth, and children were followed up with behavioral assessments at ages 6, 12 and 18 months. In this renewal application, we propose to longitudinally follow this cohort of mother-child dyads until the children are 7 years of age. Children will participate in brain imaging and behavioral assessments at ages 4-5 and 6-7 years providing data that will outline whether PreCE exerts an enduring effect into childhood. As cannabis use becomes more common and rates of second hand exposure increase, second hand cannabis exposure (SCE) will be assessed using maternal self-report and quantification of THC-COOH in biospecimens from mother and child. In addition to studying the longitudinal associations between PreCE, SCE and brain and behavioral development, placental and offspring blood signatures of endogenous cannabinoid (eCB) signaling and immune markers (cytokines, transcriptomics, brain imaging derived) will allow for a comprehensive examination of the role of eCB- mediated immune mechanisms in PreCE and SCE effects on child health. The specific aims of the CUDDELup renewal project are to: (i) examine enduring effects of PreCE (N=150; with N=100 NPreCE) and postnatal second-hand cannabis exposure (SCE) on offspring behavior; (ii) assess contributions of PreCE and SCE to longitudinal alterations in brain structure and resting state functional connectivity and test whether these neural phenotypes shape trajectories of early childhood cognition and behavior; (iii) evaluate whether PreCE and SCE are associated with eCB and immune function; and (iv) identify the multifaceted nature through which biomarkers and neural phenotypes are associated with child behavior. With these data, CUDDELup will be uniquely poised to disarticulate cannabis-specific sequelae and test a plausible eCB-immune mechanism of PreCE effects, thus contributing to our understanding of how cannabis affects the developing child, and adding to knowledge regarding prevention and interventions. CUDDELup is highly aligned with NIH priorities (study of neurodevelopmental conditions, child health, nutrition and exposures, actionable and measurable health outcomes, reproducible science) and with NIDA’s focus on substance-exposure related immune mechanisms.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY Mitochondria are centers of metabolism and signaling, and their functions are essential for all but a few eukaryotic cell types. Despite the widespread importance of these organelles, many aspects of mitochondrial biology remain remarkably obscure. This fact contributes to our poor ability to diagnose mitochondrial diseases and our near complete inability to rectify mitochondrial dysfunction therapeutically. This dysfunction is associated with ~350 rare inborn errors of metabolism and an increasing number of common diseases—including Parkinson’s, Alzheimer’s, various cancers, and type 2 diabetes—often through distinct, yet unclear means. A major bottleneck to further understanding mitochondrial processes and addressing their dysfunction in human disease is that the proteins driving them have often not been identified. Concurrently, the basic biochemical functions of many mitochondrial proteins that may fulfill these roles are undefined, or at best are poorly understood. This reality was made manifest by my efforts to generate MitoCarta, which doubled the number of known mammalian mitochondrial proteins and revealed a striking ~300 lacking any annotated function. Thus, an overarching goal of my research program is to achieve a more comprehensive understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease-related processes. We do so by first devising novel, multi-dimensional analyses designed to make new connections between these proteins and established pathways and processes. These include customized, multiomic analyses of yeast and human cell gene knockouts, deep mutational scanning approaches, large-scale genetic screens, and other mass spectrometry-based investigations. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth, and chemical biology approaches to design small molecules to manipulate their functions in vitro and in vivo. This strategy leads us into diverse and unanticipated new directions; however, we also maintain a persistent focus on the biochemistry, biosynthesis, and transport of coenzyme Q (CoQ). We are driven to define how this remarkable, redox-active, extremely hydrophobic molecule is produced in mitochondria, distributed throughout the cell, and how its cofactor and antioxidant functions empower an ever-growing list of diverse biochemical processes. Overall, these “systems biochemistry” efforts promise to help establish a deep, mechanistic understanding of mitochondrial biochemistry that will motivate novel therapeutic strategies for the vast array of human disorders rooted in mitochondrial dysfunction.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY/ABSTRACT There is a long-standing unmet need for innovative brain drug delivery strategies to solve clinical challenges in the treatment of brain tumors and other central nervous system diseases, which are major public health problems in the United States. Focused ultrasound combined with microbubble-mediated intranasal delivery (FUSIN) can address this unmet need by achieving noninvasive, spatially targeted, and efficient drug delivery to diseased brain sites without jeopardizing healthy brain regions and other organs. FUSIN utilizes the intranasal route for direct nose-to-brain drug administration, bypassing the BBB and minimizing systemic exposure. It also uses transcranial focused ultrasound (FUS) induced microbubble cavitation (i.e., volumetric expansion and contraction of the microbubble) to enhance the delivery of IN-administered agents to the FUS- targeted brain location. We have been supported by NIH/NIBIB (R01EB027223, 4/1/2019–1/31/2023) to develop FUSIN in mice. The objective of this renewal application is to establish the biophysical mechanism of FUSIN and obtain compelling large-animal data to support the clinical translation of FUSIN. Our objective will be achieved by completing the following three specific aims: Aim 1 will establish the biophysical mechanisms of FUSIN using mouse models; Aim 2 will optimize FUSIN for efficient and safe brain drug delivery in a large animal model (pigs); Aim 3 will demonstrate the clinical translation potential of FUSIN in a large animal disease model (pig glioblastoma model). This project is significant because FUSIN has the potential to radically advance the treatment of a broad spectrum of brain diseases by enhancing therapeutic agent delivery to diseased brain sites, substantially reducing systemic toxicity, and eliminating the need for invasive surgery. A multidisciplinary team with expertise in ultrasound engineering, cancer biology, radiochemistry, radiology, and neuro-oncology will advance FUSIN through the research phase and into future clinical trials. This study has three main innovations: (1) it proposes a novel mechanism for FUSIN, which is through microbubble cavitation-enhanced glymphatic transport of intranasal-administered agents; (2) it is the first to scale-up FUSIN from small to large animals; (3) the pig glioblastoma model provides a unique model that is crucial for obtaining unequivocal evidence in support of the clinical translation of FUSIN. The proposed research is expected to have a powerful impact on the research field of brain drug delivery. The outcomes of this project are expected to advance our knowledge of the biophysical mechanisms underlying microbubble-mediated drug transport in the brain, produce a unique platform technology for drug delivery in the brain of large animals, and gather large animal data needed to translate FUSIN into the clinic.

NIH Research Projects · FY 2026 · 2019-03

PROJECT SUMMARY With the identification of hundreds of genes associated with autism spectrum and related neurodevelopmental disorders (ASD/NDD), there is a pressing need to define the molecular pathways these genes contribute to in the nervous system and to dissect how their disruption alters brain function to drive disease. Methylation of cytosines in DNA classically occurs at CG dinucleotides in mammalian cells, serving as an epigenetic mark often associated with gene repression. However, a unique form of non-CG DNA methylation that occurs primarily at CA dinucleotides (mCA) is highly enriched in neurons and has emerged as an essential regulatory modification needed to tune neuronal transcriptomes. Notably, this DNA mark is susceptible to disruption due to mutation of the ASD/NDD gene DNMT3A, as well as the Rett syndrome methyl-DNA-binding protein MeCP2, suggesting that the specialized neuronal DNA methylation pathway may be vulnerable to disruption across additional causes of ASD/NDD. In recent studies, we have uncovered new mechanisms that govern the patterning of mCA in neurons and identified key functional outputs for the neuronal DNA methylation pathway in regulating cell-type-specific transcriptomes. We have shown that the histone H3 lysine 36 dimethyl mark (H3K36me2) is necessary for targeting DNMT3A to deposit mCA and demonstrated that mutation of the ASD/NDD-associated gene, NSD1, disrupts this histone mark, leading to altered neuronal DNA methylation. We have further uncovered evidence that mCA deposited by the NSD1- H3K36me2-DNMT3A cascade is read out by MeCP2 in a cell-type specific manner to control expression of genes that define neuronal subtype-specific transcriptomes. In our proposed studies we will build on these findings to investigate the mechanisms of DNMT3A targeting by H3K36me2 and assess their potential disruption due to additional genetic lesions in ASD/NDD (Aim 1). We will then dissect the cell-type specific epigenetic consequences of perturbing the NSD1-H3K36me2-DNMT3A cascade in NSD1 mutant mice (Aim 2). Finally, we will employ cutting- edge spatial transcriptomic technologies to probe gene dysregulation caused by mutations in the neuronal DNA methylation pathway across more than a hundred subtypes of neuronal and non-neuronal cells with single-cell spatial resolution (Aim 3). Together these studies are significant because they will define new roles for ASD/NDD-associated factors in the neuronal DNA methylation pathway and explore how disruption of these factors can alter neuronal function. Furthermore, our implementation of spatial transcriptomic analysis for the study of ASD/NDD will provide systematic understanding of the transcriptomic impacts caused by disease-associated mutations in this pathway at the highest level of resolution.

NIH Research Projects · FY 2025 · 2019-03

Abstract Airway epithelial cells were originally regarded as an inert barrier to the environment, but are now viewed as key regulators of the response to injury and infection with a critical role in airway repair that mimics lung development. Furthermore, altered behavior of this cell population is central to the pathogenesis of common airway diseases such as asthma and COPD, making it essential to understand the mechanisms responsible to normal and abnormal programming of this cell population. My research program is thematically focused on airway epithelial cell programming with the goal of characterizing the molecular basis of airway epithelial cell function and dysfunction for airway homeostasis versus disease. Our work to date has contributed to new paradigms in airway epithelial cell biology, including the first evidence of an active role for airway epithelial cells in directing the immune response towards airway disease and now the first data for an elusive airway progenitor epithelial cell (APEC) population that can be respiratory-virus activated to orchestrate disease and thereby explain how a transient infection could lead to long-term disease. Building on this work, we will focus going forward on creating a new concept for tissue homeostasis versus disease based on a set of transformative paradigms where progenitor cell reprogramming switches a normal airway epithelial barrier to one dominated by mucus production and the consequent morbidity and mortality of airway disease. We will provide the first definition of the key population of airway progenitor epithelial cells and the first mechanisms for how these cells are switched to disease-producing cells, incorporating unprecedented roles for: (1) endogenous viral, water channel, and nucleokine control of mitotic chromatin in these cells; and (2) an exogenous danger loop from these cells to immune cells and back to drive a distinct progenitor-cell kinase now targeted with structure-based drug design to interrupt mucus production. Translational impact also derives from new mouse and pig models and validation in humans with comparable disease. This substrate is combined with new approaches to cell isolation, 3D manipulation, and transplantation based on targets identified from genomic and proteomic analyses. Each of the individual approaches within the overall Program is charged to investigators in training to integrate scientific career development into the mission for medical research and discovery. In addition, the Program relies on vital and sophisticated input from senior pulmonary scientists for additional mentoring and cutting-edge approaches and innovations. The Program also incorporates the wider University and extramural resources to deploy multidisciplinary technologies with outstanding collaborators. Together, we expect our Program to provide a transformative paradigm for true progenitor epithelial cell programming and its role in cell proliferation and differentiation, including skewing towards mucous cell formation and excess mucus production that is central to airway disease. We also fully expect that our studies will identify the first tractable cellular and molecular targets and corresponding therapeutic intervention to attenuate airway disease, consistent with the mission of NHLBI.