Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY/SUMMARY The Weill Cornell Medicine Clinical and Translational Science Center’s (CTSC) future strategies are built on the considerable accomplishments achieved over the last 14 years, providing the translation of research and the generation of research ideas that pave the way for future innovations despite the effect of the pandemic on the medical school, with the city in lockdown for over 14 months. The plans for the next five years are devised to continue to develop the existing infrastructure and its initiatives, to add new ones, to enhance networking. In so doing, we will continue to advance translational science to improve community health. With our multi-institutional hub of distinguished partners firmly in place, the strategic plans for the CTSC are geared to rapidly advance translational science discovery by: 1) Enhancing informatics to increase resource adoption, reduce roadblocks and streamline workflow through training, novel software, and new collaborations; 2) Developing strong translational research engagement with communities, and preparing for health crises; 3) Continuing to develop our highly successful clinical and translational education programs through innovative and entrepreneurial multifaceted initiatives including a new cross-hub KL2/TL1 collaboration with the Georgetown/Howard CTSA; and 4) Creating Team Science initiatives that promote innovative didactic opportunities and seminars on innovation and entrepreneurship, the newest of which will be a collaborate with eLab on “Unmet Needs for Stimulating Device Development,” a five-part series which is designed to stimulate device development. The Pilot Translational and Clinical Studies program will be enhanced to target projects in device and therapeutics development, precision medicine and research in special populations while promoting collaborations between the community, community organizations and CTSC Investigators. The CTSC also offers opportunities that reach the broader community such as our Teaching Ethics Through Art program. The Special Population Network (SPN) will expand to include community outreach studies and continue clinical research studies with a focus on the following populations: children, the elderly, and those with disabilities. Additionally, the Network Capacity Component will do more outreach to encourage local investigators to initiate multi-center clinical trials. The CTSC has also been extremely successful ensuring the availability of biostatisticians from all partners with expertise in multiple disease systems and with adequate computational resources to support the design and conduct of research studies performed within the CTSC. All of these initiatives allow the CTSC to accomplish its goal of translating science to improve public health more effectively and efficiently.

NIH Research Projects · FY 2025 · 2017-09

The fundamental goal of the Weill Cornell Clinical and Translational Science Center TL1 Training Core Program is to equip predoctoral and early postdoctoral trainees from multiple disciplines with the skills necessary to pursue transformative clinical and translational research before their career paths are established by offering distinct educational tracks culminating in advanced degrees that allow trainees to make an immediate impact in the clinic or in industry. The TL1 Training Program has trained highly successful predoctoral medical students, surgical residents and early graduate students, nurses and fellows recruited from all partner institutions. The TL1 trainees are fully integrated into CTSC activities and have access to all services provided by CTSC partners. Building on our successful training program developed over the last 14 years, we will introduce new courses, technologies, and workshops geared towards team-based research, creativity, leadership, innovation, entrepreneurship, informatics, and precision medicine. The TL1 program offers a panoply of didactic programs available to TL1 Scholars including an Advanced Certificate in Clinical/Translational (C/T) Investigation intended for trainees who are interested in learning the fundamentals of Clinical and Translational Research; a Master’s Program in Clinical and Translational Investigation, the foundation of which is a Mentored Research Capstone Project in C/T research that is conducted in a team environment; an - MD/MS, program, a Summer Fellowship in C/T research; and innovative seminars, workshops, and courses offered throughout the year. Other groundbreaking training includes mentored research and externships/mini-sabbaticals with biopharmaceutical companies that will enrich trainees’ exposure to various career options in clinical and translational research. The highly skilled TL1 faculty and mentors are expertly trained to offer support for trainees throughout the program in order to help them pursue successful translational research careers. The CTSC has also established multiple collaborative resources to recruit and retain scholars The enhanced TL1 will leverage the trainees to more rapidly advance in the field of Clinical and Translational Science. The planned duration of appointments is two years and, the projected number of trainees is 10 including 5 predoctoral, 5 postdoctoral trainees.

NIH Research Projects · FY 2024 · 2017-09

ABSTRACT Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to understand how binding of cyclic nucleotides gates (opens/closes) the channels and how other factors such as lipids and proline isomerization modulate this gating. We will accomplish this by combining state-of-the-art techniques: single-particle cryo electron microscopy (cryo-EM) with atomic force microscopy force spectroscopy (AFM-FS), native mass spectrometry (MS), and functional assays like single-channel electrophysiology and stopped flow fluorescence assays of channels incorporated in liposomes. We will employ SthK, a model prokaryotic cyclic nucleotide-modulated channel, and also eukaryotic HCN1 and HCN2 for select sub-aims. Our first aim is to determine the molecular mechanisms for partial agonism and ligand selectivity in SthK. We will determine the structures of specific voltage-sensor SthK mutants that display increased open probability and correlate class averages with the single-channel electrophysiology. To determine the molecular mechanism for ligand selectivity we will use AFM-FS to determine at the single-molecule level the binding kinetics of cAMP and cGMP to either the SthK cyclic nucleotide binding domain alone or in the context of the full-length channel. This will yield the energetics of binding of both cyclic nucleotides and will isolate the contribution of the pore to the binding. This aim will shed light on why cAMP binding does not fully open the SthK channel and why cGMP is an antagonist, although its binding modality to the binding pocket is similar to that of cAMP. Our second aim is to understand how lipids modulate channel activity. We will systematically test the effect of lipids on SthK activity using stopped-flow fluorescence assays and single-channel electrophysiology where channels are in liposomes of controlled composition. We will determine the lipids tightly bound to the channels (both SthK and HCN1) using native MS and determine the mechanism of how they increase activity by perturbing the residues that appear to coordinate these lipid-protein interactions with functional assays. The third aim is to characterize functionally and structurally the regulation of SthK as well as potentially HCN channels by a newly discovered modality: prolyl isomerization of a conserved proline in the cyclic nucleotide binding domain, which appears to be responsible for SthK’s biphasic activation with cAMP. This can be highly impactful, as proline isomerization may turn out to be yet another means to regulate pacemaking activity in the heart and brain. All aims are geared towards unravelling the molecular mechanisms of cyclic nucleotide-modulated channels’ synergistic regulation by ligands, lipids and enzymes, which integrate to yield the channel activation levels required by the physiology of the cell.

NIH Research Projects · FY 2026 · 2017-08

Summary/Abstract Mammalian sperm acquire fertilization capacity as they transit through the reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern and become competent to undergo an acrosome reaction and fertilize an oocyte. These capacitation-associated processes require energy, and sperm are dependent on nutrients in their environment to complete them. Sperm are exposed to different nutrients in their surroundings as they pass from being stored in the cauda epididymis to the fallopian tube where they will meet and fertilize the oocyte. In the previous funding period, we demonstrated that sperm supplied with exogenous glucose generate ATP via glycolysis and oxidative phosphorylation, and glucose utilization via both pathways increases in capacitating sperm. In addition, we found that capacitating sperm alter activity through the pentose phosphate pathway and sperm deprived of exogenous nutrients can metabolize endogenous energy sources. We now propose to apply modern metabolite profiling combined with metabolic flux analyses to identify how sperm coordinately regulate their metabolic pathways and leverage different exogenous nutrients as they mature through distinct activation states. Soluble adenylyl cyclase (sAC) is essential for the molecular changes observed during capacitation, and we will use our unique tools for modulating its activity to test the hypothesis that sAC regulates the metabolic changes induced by capacitation.

NIH Research Projects · FY 2025 · 2017-07

Project Summary/Abstract Pancreatic adenocarcinoma (PDAC) ranks among the most lethal cancers due to a late diagnosis and ineffective treatments. Extracellular vesicles and particles (EVPs) are secreted by most cells, including tumor cells, and package selective molecules, including proteins, lipids, nucleic acids, and metabolites. EVPs are actively released into the circulation and mounting evidence suggests that circulating EVPs can serve as biomarkers for early cancer detection. Mass spectrometry (MS) has been extensively utilized for biomarker discovery in liquid biopsies, including EVP protein characterization. However, the scope and depth of the information obtained is limited by (i) the sensitivity and resolution of the analytic technologies and (ii) the proteomic complexity resulting from highly abundant serum-derived contaminants. The objective of this study is to apply an optimized reproducible EVP isolation method in conjunction with asymmetric-flow field-flow fractionation (AF4) technology to isolate EVP subsets with significantly improved purity and to employ three novel analytic technologies, including extremely sensitive timsTOF 4D proteomic MS, super-resolution dSTORM imaging analysis of single EVPs, and photocatalytic proximity labeling-proteomics (µMap) technology, to discover and validate novel, circulating EVP protein biomarkers for early detection of PDAC. In Aim 1, by employing the label-free timsTOF MS and using samples (blood plasma and tumor tissues) collected from patients with newly diagnosed PDAC, we will identify novel circulating EVP protein biomarkers that correlate with early stage disease. Top-ranked candidates will be further validated by robust absolute quantitation assays employing targeted parallel reaction monitoring (PRM) MS. In Aim 2, we will determine the percent representation and structural location of specific EVP biomarker proteins identified in Aim 1 at the single EVP level by utilizing the super-resolution dSTORM imaging analysis. The performance of single EVP analysis will be compared to the bulk analysis of individual protein targets via western blotting and/or ELISA analysis. We will further explore the potential to apply this analytic tool directly to plasma samples without prior EVP isolation. Lastly, in Aim 3, we will define protein-protein interactions (PPIs) of individual biomarkers by employing our recently developed photocatalytic proximity labeling-proteomics (µMap) technology. Three potential biomarker candidates identified in our previous study and novel candidates discovered in this study Aim 1 and Aim 2 will be subjected to the PPI analysis. These interactions will be further validated by super-resolution imaging analysis at the single-EVP, single-molecule level. We will establish if the presence or absence of these interactions provides a more robust approach for early cancer detection. We predict that combined application of these technologies will greatly facilitate novel biomarker discovery for early detection of pancreatic cancer. It also explores EVP PPIs as a new category of biomarkers and provide a rationale for developing therapies targeting these interactive networks in the future.

NIH Research Projects · FY 2026 · 2017-07

Abstract Decades of research have revealed that intestinal bacteria are critical for regulating homeostatic and protective immune responses. However, recent studies suggest that additional players such as fungi and viruses have high potential to influence these processes. While important trans-kingdom relationships between gut fungi (mycobiota) and bacteria have been recently unveiled, how fungi influence intestinal homeostasis, states of inflammation and responses to therapeutic interventions for Inflammatory Bowel Disease (IBD) is still less clear. In prior works, we defined profound effects of gut mycobiota on local and gut distal immunity through interaction with CX3CR1+ mononuclear phagocyte or by shaping host antibody repertoires that influence fungal commensalism. We defined that these processes are affected in IBD. In a multicenter placebo-controlled clinical trial of fecal microbiota transplantation (FMT) in Ulcerative colitis (UC) we recently determined that fungal clearance and blunted immune activation against Candida albicans correlates with a response to therapy. These findings suggest a possible role of gut mycobiota in efficacy of and response to therapeutic interventions. In preliminary studies we demonstrate the presence of rich genetic and phenotypic diversity of opportunistic Candida strains that dominated the colonic mucosa of IBD patients. We found that these isolates differ by their ability to cause host cell damage and are functionally diverse across individuals. In this competitive renewal we propose studies aiming to decipher the processes on inflammation caused by patient-associated strains. We hypothesize that these organisms influence inflammation and response to therapy through the production of factors that are regulated at strain-specific level. The results of this study will map the human gut mycobiota functionally and might provide a basis for targeted novel therapies and co-therapies for inflammatory diseases.

NIH Research Projects · FY 2025 · 2017-07

Understanding how genetic and environmental factors impact drug use and abuse may be critical for addiction prevention and diagnosis, as well as the development of novel effective addiction therapeutics. The objective of this renewal application plans to provide 4 predoctoral training slots (for 1-2 yrs each, starting in the 2nd yr) in the Weill Cornell Graduate School (WCGS) Neuroscience and Pharmacology Programs with the rationale of understanding the impact of genes and environment on drug addiction. A unique feature of this training plan is the faculty expertise in both genetic (e.g. sex, single nucleotide polymorphisms, gene splice variants and epigenetics) and environmental (e.g., HIV positivity, material environment, developmental age) factors that are essential for the emergence of addictive disease. Our faculty is also noteworthy for the breadth of the approaches they bring to addiction science; we have expertise in studying how several major abused drugs (i.e. alcohol, opiates, cocaine, and other psychostimulants) impact neuronal function from the expression and behavior of single molecules to the performance of complex functional systems that regulate the behavior of rodents and humans. In addition to our talented faculty, this training grant will take advantage of the WCGS outstanding research environment, educational resources, and recruiting activities, particularly our history of attracting and training basic and clinical scientists. Particular strengths of the training grant include: 1) the experience of the Director and Co-Director in mentoring, teaching and drug abuse research; 2) the broad scope of multidisciplinary research training provided by the faculty; 3) extensive collaborations and co-mentoring between the faculty; 4) the strong emphasis on “bench-to- bedside” translational research. Training grant faculty will be divided into three groups: 1) Major Sponsors: thesis mentors with NIDA mission supported research programs; 2) Minor Sponsors: individuals with NIDA-mission interests who will collaborate with Major Sponsors and their trainees; 3) Training Sponsors: individuals with extensive experience in drug abuse research who will work closely with Major Sponsors and their trainees. Beyond the laboratory, key activities of the training plan include: 1) courses designed specifically for this T32 (“Addiction and Society” and “Challenges in Pain Management”); 2) drug abuse focused retreat; 3) WCGS developed programs in fellowship preparation; and 4) training experiences in teaching, mentoring, networking and career opportunities. These activities together with the existing coursework and curricula, symposia and lectures, as well as each students individual training plan, will provide a solid foundation for promoting the development of successful transitions of 12-15 PhD students over a period of 5 years into careers in the biomedical workforce.

NIH Research Projects · FY 2026 · 2017-02

Although the increase in hypertension and cardiovascular disease-risk at menopause is well-recognized, the mechanisms of menopausal hypertension are still inadequately understood. Irregular cycles and declining levels of estrogen have been suspected to play a critical role in the emergence of hypertension at the onset of menopause. However, a clear understanding of estrogen’s role in menopausal hypertension has been limited by the confounding effects of variables such as aging in the human literature and the use of models primarily reliant on the use of ovariectomized animals that do not replicate natural menopause in the preclinical literature. Significantly, a mouse model of accelerated ovarian failure (AOF) induced by 4-vinylcyclohexene diepoxide (VCD) can recapitulate early (i.e., peri-AOF) and late (i.e., post-AOF) stages of human peri- and postmenopause, respectively. The AOF model has proven effective in isolating the role of gonadal hormones in blood pressure, particularly with respect to models of neurogenic hypertension involving the hypothalamic paraventricular nucleus (PVN), a brain area critical for coordinating sympathetic and neurohumoral processes important for the regulation of blood pressure. In the last grant award, we found that peri-AOF hypertension induced by slow-pressor angiotensin II (AngII) was associated with a signaling pathway involving estrogen receptor beta (ER) and the NMDA-type glutamate receptor in PVN neurons. It is important to recognize that after perimenopause women transition to postmenopause, however, it is unclear if the mechanisms of hypertension during postmenopause are mediated by similar mechanisms. In pilot data, we show that hypertension at post-AOF is associated with altered signaling involving GluA1-expressing AMPA, but not NMDA receptors. We further show that administration of an agonist of the G-protein coupled estrogen receptor 1 (GPER), but not ER, inhibits AngII hypertension in post-AOF mice. In this proposal, we will test the central hypothesis that post-AOF mice are predisposed to hypertension that is dependent on GluA1 plasticity in the PVN and alleviated by GPER signaling. Two aims will test this hypothesis. Aim 1 tests the sub-hypothesis that alterations in PVN GluA1 signaling contribute to hypertension in post-AOF mice. Aim 2 tests the sub-hypothesis that post-AOF hypertension is associated with GPER signaling in the PVN. These studies will be performed using a combination of approaches including high-resolution anatomical, neurophysiological, in vivo gene targeting, and single-cell RNA sequencing approaches.

NIH Research Projects · FY 2025 · 2017-02

Summary The kidneys control extracellular fluid volume and blood pressure by adjusting the excretion of Na to match the dietary Na intake and the overall physiological needs of the organism. Aldosterone is a key hormone that helps to mediate this process. In response to a reduction in extracellular fluid volume the adrenals increase secretion of this steroid, which in turn signals parts of the renal tubule (the so-called aldosterone-sensitive distal nephron) to increase Na reabsorption. This occurs at least in part through stimulation of the uptake of Na from the urine across the apical membrane through epithelial Na channels (ENaC). How this occurs, however, is incompletely understood. In the most prevalent model of this process, aldosterone stimulates the synthesis of a key enzyme, the serum and glucocorticoid induced kinase (SGK1). SGK1 then phosphorylates the ubiquitin ligase Nedd4-2, inhibiting its interaction with ENaC and diminishing the rate of channel internalization. Na reabsorption is then enhanced due to increased residence times of the channels at the apical surface. However, several lines of evidence suggest that this is not the main mechanism through which the hormone operates. First, the effects of inhibiting the binding of Nedd4-2 to ENaC by truncating the C-terminal of the ENaC surface (mimicking Liddle’s syndrome in humans) are synergistic with those of elevated aldosterone levels (5,10). This is not expected if the two manipulations affect the same cellular processes. Second, analysis of ENaC distribution in the cell and the excretion of ENaC protein in urinary exosomes suggests that the major effect of aldosterone is to increase forward trafficking to the apical membrane (18). Finally, measurement of the ubiquitination state of ENaC indicates that when transport is stimulated by aldosterone the number of ubiquitinated subunits increases, whereas the standard model predicts a decrease. In the work described in this application, we will examine a revised model of ENaC trafficking that is more consistent with these data. Here subunit protein is expressed in excess even under basal conditions when the need for transport is minimal. The main hormone-stimulated event is transport of ENaC protein from the ER to the plasma membrane. Arrival at the apical membrane exposes the channels to urinary proteases, which mediate the final proteolytic processing and activation of the channels. This increases the susceptibility of the subunits to ubiquitination, limiting their lifetime at the surface and/or their active states. This model will be tested using a variety of experimental techniques including quantitative Western blots to estimate subunit numbers, immunocytochemistry, in situ biotinylation and Western blotting to assess the cleavage states of ENaC subunits, electrophysiology to measure overall channel function, and ubiquitin assays to assess the modification of channels is subcellular compartments.

NIH Research Projects · FY 2025 · 2016-09

The Cooperative Center of Research Translation entitled “Center for Lupus Research” (CLR) and based at Weill Cornell Medicine and JAX/GM aims i) to advance the knowledge of pathways that contribute to the establishment and amplification of Systemic Lupus Erythematosus (SLE), ii) to identify molecular mechanisms responsible for failure to respond to standard of care (SOC) therapies, and iii) to develop assays and tools to monitor these dysfunctional pathways and stratify patients towards personalized therapies. Understanding major disease pathogenic drivers and identifying biomarkers to follow them in the clinical setting are highly significant goals to advance clinical trial design and ultimately personalized patient care. The proposed studies stem from our previous CORT cycle and build on our work uncovering both molecular heterogeneity and basic mechanisms contributing to the generation of immunostimulatory nucleic acids (NAs) in this disease. We now provide data supporting that erythroid cells are a novel source of mitochondrial NAs giving rise to myeloid cell activation and pro-inflammatory loops in children with SLE. In Project 1, we will dissect the basic mechanisms, pathogenic role and biomarker potential of this novel pathway. In Project 2, we will capitalize on the groundwork that we have developed over the past 4 years of this CORT cycle using next generation single cell (sc) transcriptional and epigenetic profiling as well as on our discoveries of novel gene splice variants expressed in SLE patient immune cells. Both projects will incorporate cutting- edge technologies and build upon our established expertise in immune profiling. The projects will be critically supported by collaborations with Clinicians, Molecular and Cellular Biologists, Computer Scientists and experts in Systems Immunology. The Administrative Core will organize and operate the Center, and assure communication and interactions among all members and collaborators. The Clinical Core will oversee patient enrollment and clinical assessment, clinical data collection and sample storage and distribution. Samples obtained via the Clinical Core will enable the Center scientists to work together and the CLR will ensure integration of clinical and laboratory data associated with samples across Institutions. The specific aims of the projects are i) to identify the upstream mechanisms leading to retention of mitochondria in SLE Red Blood Cells (RBCs); ii) to follow the presence of Mito+ RBCs and their upstream dysfunctional pathways in pediatric and adult SLE patients during flares and remissions to establish their value as biomarkers and a stratification tool; iii) to dissect the contribution of erythroid-derived mitochondrial NAs to SLE inflammatory loops; iv) to identify transcriptional markers of SOC resistance pathways at the single-cell level and to identify isoforms associated with disease severity and response to treatment; v) to define epigenomic signatures of SLE Disease Activity and therapy responsiveness, and vi) to characterize the phenotype of therapy-sensitive and resistant Plasmablasts (PBs) and identify, through functional genomics, the molecules/pathways that confer resistance to therapy.

NIH Research Projects · FY 2025 · 2016-08

Summary/Abstract My patient-oriented research program focuses on addressing key knowledge gaps related to the problem of later-life pain, a common, morbid, and costly disorder. I seek renewal of my National Institute on Aging (NIA) K24 Award to expand a robust program of mentorship established over the past 4 years and to support my program of patient-oriented research focused on later-life pain and symptom management. Specific aims I intend to accomplish over the next funding period include: 1) Developing, testing, and disseminating non- pharmacologic intervention strategies to improve pain and pain-related outcomes among older adults with a focus on the use of new technologies when appropriate; 2) Expanding an already existing robust pipeline of trainees committed to careers in patient-oriented research; 3) Expanding my research focuses to gain further expertise in and ability to conduct patient-oriented research on: a) cognitively impaired older adult populations with pain; b) the role of family caregivers in improving pain care outcomes; c) non-pharmacologic approaches to pain other than behavioral interventions (e.g., cognitive behavioral therapy) such as non-invasive neuro- stimulation techniques (e.g., transcranial direct current stimulation); and d) the development and evaluation of new pain assessment tools; and finally 4) Obtaining new funding to support research that capitalizes on recently completed and ongoing research, thereby expanding opportunities to attract and train new mentees throughout the award period. My mentorship program consists of structured educational and career development activities A supervised research program tailored to the trainees’ experience, interests, and needs constitutes the core mentorship activity. This training is supplemented by access to a rich array of educational and other career building resources and the development of an individualized career development plan along with regularly scheduled evaluations that help to ensure timely trainee progress and achievement of mutually agreed upon goals. The Cornell environment offers extensive resources to support the candidate’s research and mentoring programs and provides a rich array of trainee pipelines. Cornell’s NIA-funded Edward R. Roybal Center (which I direct) and a postdoctoral training program in behavioral geriatrics research (which I co-direct) constitute major leveraged resources. Evidence of mentorship success since receipt of K24 funding in 2016 includes recruiting, training and fostering the career development of 25 trainees, including 5 minority trainees and 18 women. Evidence of research productivity includes publishing 70 peer-reviewed articles over this period of time, many in high impact journals, as well as successfully launching the T32 training program and obtaining competitive renewal funding for Cornell’s Roybal Center (P30 grant). The focus of my ongoing projects, my proposed career development activities described above, and my continued mentorship of junior and early mid-career investigators, will help to move the field of later-life research forward, leading to improved health and well-being of adults adversely affected by later-life pain.

NIH Research Projects · FY 2025 · 2016-08

Abstract Short-term memory function is commonly supported through persistent activity, the sustained response of populations of neurons following the offset of a memorized stimulus. This form of activity underlies diverse tasks including navigation, motor control, and decision-making. Classic mechanistic theories have idealized such activity through models that assume strongly homogeneous populations of neurons that encode only a single variable and generate perfectly stable patterns of activity. This contrasts with recent work showing that neurons in real biological memory networks exhibit multiplexed encoding of multiple stimulus attributes, temporally varying responses across the population, and context dependence. Here we address the circuit mechanisms and role of this diversity in function through a combined experimental-theoretical approach. Experiments are conducted in a short-term memory circuit of the larval zebrafish gaze control system that contributes to stable vision by precisely maintaining the eyes on a visual target. Taking advantage of the quantitative precision and experimental tractability of this system, we combine whole-circuit, synapse-resolution anatomy with circuit-wide recordings and perturbations of activity at cellular resolution. In Aim 1, we combine these data into a model of the system in which neurons map in a one-to-one manner with experimentally recorded neurons. This enables us to infer the interactions within and between neurons of different anatomical, genotypic, and functional cell classes and form predictions for how these interactions govern circuit function. In Aim 2, we use 3D cellular resolution optical imaging and stimulating perturbations of neuronal activity to refine our model and test model predictions. In Aim 3, we expand our capacity to form precise characterizations of within and between cell-class interactions by developing and applying 3D suppression of neurons across the memory circuit. Altogether, this work promises to greatly expand our understanding of the circuit mechanisms and role of cell type diversity in persistent firing, short-term memory, and motor control.

NIH Research Projects · FY 2026 · 2016-08

Project Summary Dysfunction of gamma-aminobutyric acid (GABA)ergic interneurons is strongly associated with neurological disorders including epilepsy, schizophrenia and autism spectrum disorders. Although recent evidence highlights bewildering subtype diversity of these neurons, the notion that developmental perturbations in subtype function cause defects in the formation of cortical circuits with lasting functional deficits has not been explored in detail. The long-term goal of this research is to uncover how early interneuron dysfunction in the developing postnatal brain leads to lasting neurological pathologies. The objective of this proposal is to reveal how the activity of specific interneuron populations during critical windows of perinatal development shape circuits underlying primary sensory-dependent behavior. To this end, we will use the murine barrel cortex as a well-established model for the study of activity-dependent circuit maturation. We will focus our studies in superficial circuits since our previous work indicates that these circuits are exquisitely sensitive to environmental perturbations in the neonate. In the near term, this proposal is aimed at investigating the role of layer I interneuron subtypes in regulating the emergence of cortical columns (Aim 1). In addition, this project will determine the role of LI interneurons in the emergence of interhemispheric circuits. (Aim 2). Finally, we will assess how developmental defects in early LI interneuron function lead to abnormal brain activity and impaired sensory integration in the adult (Aim 3). With respect to the outcomes, our work is expected to identify basic mechanisms fundamental for the emergency of a healthy excitatory/inhibitory balance. In addition, these results are expected to have a significant translational impact because they will expand our mechanistic knowledge on how early interneuron dysfunction may lead to behavioral abnormalities frequently observed in ASD patients.

NIH Research Projects · FY 2025 · 2016-05

Weill Cornell Post-Doctoral Training Program in Behavioral Geriatrics (TPiBG) The TPiBG develops independent investigators capable of conducting patient-oriented research to improve the quality of life and quality of care of older adults. Behavioral Geriatrics is a scientific orientation that integrates social/behavioral approaches with geriatric medicine approaches to study clinically significant and pressing issues of aging (e.g., pain, cognitive impairment, polypharmacy, caregiving, end-of-life medical decision- making, bereavement). The Program, led by Cary Reid, MD, PhD and Holly Prigerson, PhD, accepts both MD and PhD postdoctoral trainees (2/year) seeking careers integrating biomedical and innovative social/behavioral approaches to improve care and care outcomes in older adults. A formal didactic core curriculum covers topics including clinical and psychosocial epidemiology, community-based participatory research, trial design as related to the study of older adults, scientific rigor and reproducibility and role of technology in aging research. Completing the Cornell CTSC Master’s degree or Certificate Program in Clinical Research is mandatory for MD trainees. Trainees participate in monthly “work-in-progress” sessions and a monthly Trainees’ Forum provides instruction in the presentation and publication of results, ethical conduct of research, grant preparation, and helps to build career development skills. Biostatisticians and data entry and management personnel from existing grants are available to assist T32 trainees. The centerpiece of the training is Co-Mentored research in Year 1, culminating in a Year 2 research project for which the Trainee serves as PI under Co-Mentor supervision. Our cadre of experienced and successful core faculty mentors include PI Reid (management of multifactorial pain in later life), Co-PI Prigerson (care of patients and families at end of life), Dr. Ronald Adelman (palliative care), Dr. Mark Lachs (elder abuse), Dr. Sara J. Czaja (aging and technology) and Dr. Monika Safford (healthcare equity), and Drs. Karl Pillemer and Elaine Wethington (social isolation/integration). Trainees are immediately integrated into a large, ”research-ready” network of New York City organizations serving ethnically diverse older adults. Trainee recruitment resources include featuring the program in-person and at annual, research meetings, including the Gerontological Society of America and American Academy of Hospice and Palliative Medicine. Further strengths include: (1) geriatric and social/behavioral science co- mentorship, (2) well-established infrastructure and flexible, tailored mentorship plans; (3) clearly articulated metrics to gauge progress (published papers, national presentations, funded career awards); and (4) membership in a diverse network of behavioral geriatrics researchers. Institutional strengths include an outstanding pipeline of potential trainees and multiple aging-related Center grants and R01s to support Trainees’ research. The Program produces Behavioral Geriatrics researchers trained in the synergistic disciplines of geriatric medicine and social/behavioral science, uniquely capable of addressing one of the largest challenges to public health – the aging of the population.

NIH Research Projects · FY 2026 · 2016-04

PROJECT ABSTRACT The gastrointestinal tract is colonized with trillions of normally beneficial microbes, termed the microbiota, that are essential for human health. However, the pathogenesis of numerous infectious, inflammatory, and metabolic diseases involves a loss of immunologic tolerance to the gut microbiota. This has been demonstrated robustly in basic, translational, and clinical studies of inflammatory bowel disease (IBD). The fundamental focus of our proposed renewal for R01AI123368 is to build on recent paradigm-shifting results from the first funding cycle that detailed a novel pathway that is essential to orchestrate immune tolerance to gut microbiota and restrain intestinal inflammation. Specifically, our prior studies detailed how group 3 innate lymphoid cells (ILC3s) present microbiota-derived antigens to CD4 T cells on MHCII, instruct the differentiation of a unique form of RORgt+ regulatory T cells (Tregs), and prevent spontaneous intestinal inflammation. Further, we find that these cellular interactions are dysregulated in the inflamed intestine of individuals with IBD (see Zhou et al., Nature, 2022 and Lyu et al., Nature, 2022 as the most recent examples). We generated new preliminary data to support this renewal application where we propose to mechanistically advance this paradigm and will explore the therapeutic potential of our key findings. This includes three specific aims, which will: (i) define how ILC3s sense microbiota to initiative antigen presentation, (ii) elucidate key molecular determinants endowing a tolerogenic program in MHCII+ ILC3s, and (iii) identify whether MHCII+ ILC3s can be harnessed to enforce tolerance to diverse microbes or restrain gut inflammation as a pre-clinical therapy. We will employ basic mouse models, as well as translate our findings into human specimens. This research is directly relevant to IBD and many other chronic inflammatory diseases where disruptions to immune tolerance underlie disease pathogenesis, and it is expected that our mechanistic results will provoke new opportunities for preventative, therapeutic or curative treatment strategies.

NIH Research Projects · FY 2025 · 2016-01

Recent evidence indicates that the presence of conventional dendritic cells type 1 (cDC1) in the tumor microenvironment (TME) is required for response to immune checkpoint blockade (ICB) therapy. In addition to cross- presenting cancer cell-derived antigens to CD8+ and CD4+ T cells, cDC1 promote tumor infiltration by effector T cells, and support their survival and function. Thus, interventions that improve cDC1 recruitment to the TME could enhance patient responses to ICB. Focal radiation therapy (RT) increases responses to ICB therapy, at least in part by inducing type I interferon (IFN-I) and driving cDC1 into the irradiated tumor. We have previously shown that cDC1 are essential for immune-mediated regression of irradiated and synchronous non-irradiated tumors (abscopal effect) in mice treated with RT and ICB. Abscopal responses have also been achieved in metastatic cancer patients treated with RT and ICB, but less reliably than expected, and the determinants of such responses remain unclear. We hypothesize that a previously unexplored barrier to abscopal responses is the limited infiltration of poorly immunogenic tumors by cDC1, which precludes effector T cells generated at the irradiated tumor site from rejecting non-irradiated tumors. Moreover, we hypothesize that activation of a strong IFN-I response in the irradiated tumor is essential for achieving systemic activation of natural killer (NK) cells, which can home to non-irradiated tumors and foster the recruitment of cDC1. This hypothesis is supported by a strong scientific premise which is based on the recent literature and on our extensive published and unpublished data, including the fact that increased serum IFNb post-RT was the top predictor for abscopal responses in metastatic lung cancer patients treated with RT+anti-CTLA4 (Nat Med 2018). To test this hypothesis three independent but related aims that address different mechanistic questions are planned. Aim 1 will investigate the role of RNA:DNA hybrids, which accumulate in the cytosol of irradiated cancer cells and in the cargo of small extracellular vesicles (sEV) they produce, in activating the IFN-I pathway via cGAS/STING in cancer cells and locoregional DCs. The role of RT-induced IFNb in systemic NK cell activation will be confirmed by using IFNAR1-deficient NK cells. Aim 2 will determine the contribution of sEV to RT-induced IFN-I activation in vivo by using Rab27a-deficient cancer cells. Aim 3 will directly address the role of NK cells in driving cDC1 infiltration in abscopal tumors and abscopal responses to RT+ICB. In addition, NK cell functional subsets present in the blood of lung cancer patients with abscopal response to RT+anti- CTLA4 will be investigated by single cell analysis. Results of proposed studies will identify a novel mechanism whereby local IFN-I induction by RT activates a systemic cross-talk between NK cells and cDC1, required for T-cell mediated rejection of abscopal tumors.

NIH Research Projects · FY 2025 · 2015-12

Salt consumption across the world greatly exceeds minimal requirements, and excessive dietary salt has emerged as a powerful risk factor for cognitive impairment and dementia. Increasing evidence indicates that a high salt diet (HSD) is harmful to brain health independently of the increase in blood pressure associated with HSD in salt-sensitive individuals. Unfortunately, public health efforts to curb salt intake have been futile and dietary salt consumption continues to rise worldwide. The long-term goal of this research program is to elucidate the mechanisms by which HSD is injurious to cognitive health and to develop new approaches to counteract it. During the previous funding period, we have demonstrated that HSD in mice leads to a reduction in cerebral blood flow (CBF) and cognitive impairment through suppression of endothelial nitric oxide (NO) production. These effects are mediated by a subclass of T-helper lymphocytes (Th17) in the small intestine that increases circulating levels of the cytokine IL17. IL17, in turn, leads to inhibition of endothelial NO synthase (eNOS) in cerebral endothelial cells. The resulting deficit in endothelial NO induces cognitive impairment through neuronal accumulation of hyperphosphorylated tau, a microtubule associated protein linked to Alzheimer’s disease and related dementias. However, the factors triggering the production IL17 in the gut, the cellular localization of the IL17 receptors inducing eNOS inhibition, and the role of the CBF reduction in tau accumulation remain to be established. This renewal application seeks to advance the mechanistic understanding of the cognitive effects of HSD by testing the following novel hypotheses: (a) HSD triggers distinct innate and adaptive immune responses in the gut through the microbiota, (b) the resulting increase in circulating IL17 acts on cerebral endothelial IL17 receptors to inhibit eNOS through downregulation of the eNOS regulatory protein striatin and, (c) the increased leukocyte adhesion resulting from the NO deficit leads to microvascular occlusions (capillary stalling) which promote tau accumulation in brain by reducing its microvascular clearance into the bloodstream. We will use a well-characterized model of HSD in young and old males and female mice and state-of-the- art approaches to examine gut-brain immune interactions, microvascular function, hyperphosphorylated tau, and cognitive deficits. These studies advance the understanding of the pathobiology of excessive dietary salt at the cellular and molecular levels and may lead to new approaches to mitigate its harmful effects on brain health that lead to cognitive impairment.

NIH Research Projects · FY 2025 · 2015-09

Despite great strides that have been made in the understanding and treatment of cancer, the number of cancer deaths remains on the rise and cancer remains the 2nd leading cause of death in the United States (US). Not only is the number of people dying of cancer increasing, but the quality of those deaths is alarmingly poor. End-of-life (EoL) care in the US has been deemed a public health crisis by the National Academy of Medicine -- a conclusion bolstered by disturbing findings from my group. My research shows that end-stage cancer patients receive chemotherapy troublingly close to death, that end-stage cancer patients are shockingly uninformed of their prognosis and harms of treatments, and that severe emotional pain and suffering remain largely unchecked. The initial Outstanding Investigator Award (OIA) identified and targeted psychosocial factors to address these problems; the results have proved paradigm-shifting and practice-changing. For example, we showed that: 1) “palliative chemotherapy” does not “palliate” and may actually do more harm than good -> highlighting the need for oncologists to recognize the harms of “overtreatment” and refrain from prescribing chemotherapy to patients they deem close to death; 2) oncologist prognostic communication and therapeutic alliances can improve patient prognostic understanding and lead to more informed, value-concordant EoL care –> our Oncolo-GIST approach as a simple, effective way oncologists can feel comfortable communicating the gist of a patient’s prognosis; 3) that healthcare chaplaincy can address frequently high unmet spiritual care needs of patients with advanced cancer and, thereby, also promote advance care planning (ACP); 4) that psychosocial distress is an important influence on, as much as outcome of, EoL decision-making->our EMPOWER psychosocial intervention targeting “experiential avoidance” to promote caregiver psychosocial adjustment and engagement in ACP. Going forward, this OIA will focus on: 1) oncologist communication; 2) the role of caregivers in promoting better EoL cancer care; 3) the role of healthcare chaplains and 4) psychosocial influences (e.g., therapeutic alliances) in addressing unmet needs of cancer patients at the EoL. I will leverage data, theories, and the clinical and scholarly resources (colleagues and collaborators) developed under the auspices of the current OIA to: improve delivery of EoL cancer care; increase the frequency and effectiveness of their prognostic disclosures; promote cancer patients’ prognostic understanding and therapeutic alliances with their oncologists; and reduce psychosocial distress of patients and caregivers to enhance their mental health and promote their engagement in ACP. Renewal of this OIA has and will enable me to conduct research helping to ensure that dying cancer patients and their caregivers receive the highest quality of EoL cancer care possible

NIH Research Projects · FY 2026 · 2015-07

In the 10 years since our initial award, we have established and sustained CHERISH (Center for Health Economics of Treatment Interventions for Substance Use Disorder, HCV, and HIV) as a National Center of Excellence for health economics research related to substance use disorder (SUD), HCV, and HIV care of people who use substances. Our unique multi-institutional center leverages outstanding researchers in synergistic areas of expertise. In the initial award period, we addressed the needs of integrated healthcare system payers and providers; in the current period, we have expanded our focus to include individual-, system- and community-level concerns. In this renewal, we will emphasize financial sustainability, advanced methods, and knowledge translation, as well as development of the next generation of researchers in our field. Reflecting both the expansion of our network of collaborators and the maturity of our field, Center Specific Aims address service to the research community and advance methods that improve the quality and relevance of economics research on SUD and HCV/HIV care of people who use drugs (PWUD). Aim 1: To facilitate and develop innovative methods for substance use economics research that supports financial sustainability of evidence-based interventions for SUD and HCV/HIV care of PWUD. Aim 2: To apply and advance substance use economics research that uses population-level data and simulation modeling to improve economic outcomes and advanced research methods in SUD, and HCV/HIV care for PWUD. Aim 3: To engage in and improve knowledge translation between health economics researchers and decision makers that enhances evidence-based decision making about SUD and HCV/HIV care for PWUD. Aim 4: To provide resources, training, and mentoring that develop the next generation of investigators to conduct rigorous and impactful SUD and HCV/HIV economics research. Each Center Aim will be led by a specific Core that will develop and maintain research partnerships and cross-Center collaborations to support the aim: Methodology Core (Aim 1), Population Data & Modeling Core (Aim 2), Dissemination & Policy Core (Aim 3), and Pilot Grant, Training & Mentoring Core (Aim 4). The Administrative Core provides the leadership, management, and support structure that ensures productive cross-Core coordination and now will be the home for our highly successful and uniquely national CHERISH Consultation Service and Research Affiliates Program, which draw on and provide expertise across Cores. The Administrative Core also leads evaluation of Center activities in concert with a Center Advisory Board.

NIH Research Projects · FY 2025 · 2014-09

SUMMARY N6-methyladenosine (m6A) is a modified nucleotide in mRNA whose transcriptome-wide expression and distribution are altered in various cancers, including acute myeloid leukemia (AML) cells. m6A has a pivotal role in controlling cell fate decisions especially in the hematopoietic lineage. Our recent work has revealed that this effect of m6A is mediated in part by its ability to regulate “symmetric commitment,” in which a stem cell differentiates into two daughter cells that both adopt the same new cell identity. We recently identified SON, one of the most highly methylated transcripts in hematopoietic stem cells, as a major mediator of m6A-dependent control of symmetric commitment. However, the mechanism by which SON regulates these phenomena is unclear, and moreover it is unclear if deregulated m6A control over SON contributes to the undifferentiated phenotype of AML cells. Inhibitors of METTL3, the m6A biosynthetic enzyme, are a promising approach to target AML, but appear to exhibit diverse effects in cells not seen with METTL3 knockout. Also, unlike METTL3 knockout, METTL3 inhibitors also appear to preferentially target cancer cells. A major goal is to understand the basis for the actions of METTL3 inhibitors compared to METTL3 knockout and to determine if specific cellular pathways can influence whether cells, and ultimately patients, would be more or less sensitive to METTL3 inhibitor treatment. To significantly advance our understanding of epitranscriptomic regulation in cancer, the specific aims of this proposal are: (1) To define the functional requirement for SON and nuclear RNA methylation on cell fate in leukemia. In this aim we will use a new conditional knockout mouse model to dissect SON's stage-specific role, clarifying its impact on METTL3-related blood phenotypes. We will determine SON's importance in leukemia and unravel its downstream targets, mRNA regulatory mechanisms, and links to the RNA methylation. (2) To identify pathways and mechanisms that make cancer cells sensitive to METTL3 inhibition. Our preliminary data show that METTL3 inhibitors cause marked reorganization of nuclear architecture, and their effects can be overcome by forced expression of YTHDC1. We have also identified additional suppressors of METTL3 inhibitors by performing a base editor screen to generate >600 oncogenic mutations and probing how the mutations affect sensitivity to METTL3 inhibitors. We will broadly identify how YTHDC1 and these newly discovered oncogenic pathways influence the m6A pathway and METTL3 inhibitors. (3) To determine the function of m66A in MYC mRNA. Our studies revealed the presence of a previously unidentified epitranscriptomic mark, N6,N6-dimethyladenosine (m6,6A) as a highly prevalent evolutionarily conserved modification in MYC mRNA. We will determine the prevalence and functional significance of this new modification, and determine how it impacts the oncogenic function of MYC. Overall, this project develops new technologies and tests innovative mechanisms to uncover novel mechanisms of epitranscriptomic regulation of cancer.

NIH Research Projects · FY 2025 · 2013-09

PROJECT SUMMARY/ ABSTRACT Cigarette smoking is the greatest known single risk factor for the development of lung disease, being a dominant risk for the development of both emphysema and idiopathic pulmonary fibrosis. We have assembled a team of investigators who have worked synergistically to better understand the mechanism(s) by which cigarette smoke can induce either lung fibrosis or emphysema. Our team members are leaders in the field of COPD and IPF who are most committed to better understand the mechanism(s) by which cigarette smoke can induce either fibrotic or emphysematous phenotype in the lung. During the previous years of funding support by P01 HL114501 grant entitled “Distinct and Overlapping Pathways of Fibrosis and Emphysema in Cigarette Smokers”, we have integrated the expertise of investigators from the COPD and IPF communities, spanning basic, translational and clinical researchers, to come together to tackle this important challenge. This synergistic integration among the projects and cores have been impactful and will continue to be greater than the sum of each of its component parts. We employ both Ureductionist and mechanisticU approaches by utilizing cell culture and animal models (Project 1 and Project 2), and UdiscoveryU approaches using high throughput profiling (genomics, epigenetics) methods in human lung tissues and cells (Project 3) to discover new pathway(s) mediating the fibrotic and emphysema phenotypes in cigarette smoker. Hence, a PPG mechanism has been not only critical but absolutely necessary to best address the main objective of this fundamental question at hand: How can we better understand the mechanism(s) by which cigarette smoke mediates fibrotic or emphysematous phenotype in the lung? We will attempt to reach our goals by approaches described in the following Uprojects and cores: Projects: 1) Mitochondrial and Metabolic Dysfunction in COPD and IPF 2) Differential roles of Chi3l1 and its Receptors in Pulmonary Fibrosis and COPD 3) Integrating Omics, Networks, and Functional Studies in COPD and IPF Cores: A) Administrative Core B) Respiratory Computational Discovery Core C) Clinical Biorepository Core D) Molecular Characterization Core

NIH Research Projects · FY 2025 · 2012-03

SUMMARY Hypertension (HTN), affects over 60% of US population above 60. It increases the risk of Alzheimer's disease (AD) through compromised regulation of cerebral blood flow (CBF). Multiple studies failed to establish that lowering blood pressure (BP) entails consistent benefits for cognition and brain measures. This is probably due to a preexisting compromise of the vascular system, which does not compensate properly for relative perfusion pressure decrease caused by BP lowering. In a previous cycle, we showed that, in HTN, there is an optimal BP level that maximizes CBF. We also show 1) an optimal BP that decreases white matter lesion risk, 2) perfusion correlates with tau pathology, and 3) all these findings occur in older HTN subjects. Despite these new discoveries, there is a large variance in CBF and cognition due factors other than age and BP, suggesting the need for further fine-tuning of the model for optimal BP. In this competitive renewal of our NIH/NHLBI R01 HL111724 we propose to focus on variants of the circle of Willis (CoW). They adversely influence CBF and outcome in chronic and acute conditions. However, very little is known about how they affect perfusion, cognition and AD markers in HTN. Our data indicate that incomplete variants play a role in circumstances when there is already a pre-existing impairment of the vascular system (HTN). We offer that these variants in the setting of HTN necessitate higher perfusion pressure to maintain adequate blood flow, thus increasing the risk for hypoperfusion and AD-related pathology. Over a 2-year we will enroll 140 hypertensive, cognitively healthy subjects, 70-85 years old, with (n=70) and without (n=70) typical anatomy of the CoW. At baseline and 24-month follow-up, we will perform BP and cognition assessments, magnetic resonance imaging (including perfusion and vessel anatomy). 50% of the group will receive both tau (PI-2620) and amyloid (Neuraceq) positron emission tomography at baseline and follow-up. We will test whether: AIM1. H1. For the same BP, CBF is lower in subjects with an incomplete CoW than in subjects with a complete circle. H2. Longitudinally, in subjects with an incomplete CoW, for the same baseline BP, an equal reduction in BP entails greater reduction in CBF than in subjects with a complete circle. AIM2. H1. For the same BP, baseline amyloid and tau accumulation is higher in subjects with an incomplete CoW than in subjects with a complete circle. H2. AD biomarkers correlate with CBF. H3. Longitudinally, in subjects with an incomplete CoW, for the same baseline BP, an equal reduction in BP entails greater accumulation of amyloid and tau than in subjects who have a complete circle. Secondary AIM. H1. Variants of the posterior circulation (incomplete posterior circle, vertebral artery hypoplasia) are selectively related to lower hippocampal CBF, and to H2. higher hippocampal tau accumulation. We hope our research will contribute to fine-tuning of HTN management and help discover a novel AD risk.

NIH Research Projects · FY 2025 · 2011-07

PROJECT ABSTRACT The immune system at mucosal sites must be tightly regulated to mediate rapid immunity to invading pathogens, while limiting over-reactive responses that drive chronic inflammation. In particular, type 2 immune responses in the airway or gastrointestinal tract are essential to protect from helminth parasites, but if dysregulated, drive asthma and allergic inflammation. Despite this knowledge, we do not yet fully appreciate the complexity of cellular and molecular signals that control these responses, which will be critical for developing the next generation of preventative, therapeutic or curative treatments. The fundamental focus of this renewal application for the Mucosal Immunology Studies Team is to define novel pathways by which the type 2 immune response harnesses signals associated with the nervous system to regulate rapid mucosal immunity and inflammation. In this context, we will define: (i) the pathways that induce and regulate these neuronal signals, (ii) the functional significance of these pathways in type 2 mucosal immunity and inflammation, and (iii) whether it is possible to therapeutically target these signals to boost immunity to helminth infection or reduced chronic allergic inflammation. We will employ innovative approaches and develop new tools to address these fundamental gaps in knowledge, and where possible, translate our findings from mice into human samples. Results from these studies will significantly advance our understanding of the pathways that are essential to mediate rapid type 2 immunity and inflammation at mucosal sites and could provoke the next generation of preventative, therapeutic and curative treatment strategies.

NIH Research Projects · FY 2025 · 2010-09

ABSTRACT Potassium (K+) channels are major determinants of cell excitability and play crucial roles in physiological processes. Large conductance and Ca2+-activated K+ (BK) channels, have the ability to couple intracellular Ca2+ to membrane potential variations, play major physiological roles in vascular smooth muscle tone maintenance, regulation of circadian rhythms, hearing, neurotransmitter release. BK channels can associate with tissue-specific accessory subunits, endowing the channels with different functional properties. b2 and b3 subunits induce N-type (or ball-and-chain) inactivation of the otherwise non-inactivating BK channels. BK channel dysfunction has been associated with many pathophysiological conditions, so understanding how they gate can have major therapeutic consequences. In the previous grant cycle, we determined the structural correlates of gating and ball-and-chain inactivation in K+ channels, using a prokaryotic homolog of BK channels, MthK, from Methanotropicum thermoautotrophicum. We also found that lipid bilayer thickness strongly affects MthK inactivation. Unlike BK, MthK channels inactivate using an intrinsic N-terminal “ball” domain, similar to Shaker K+ channels. The overall objective of this grant is to determine the structural correlates of ball-and-chain inactivation in BK channels and understand how lipids modulate inactivation in both MthK and BK. Since membrane lipid composition cannot be controlled in cells, we will use a bottom-up approach of purified channels in reconstituted systems to rigorously control lipid composition. We will use structural, functional, and computational analysis (the last with a collaborator, see letter) on both MthK and BK channels to reach these goals. Our first aim is to determine the mechanism by which lipids modulate ball-and- chain inactivation in MthK. Our hypothesis, predicted by MD simulations, is that thicker bilayers bind the amphipathic ball domain better than thinner bilayers, which will be tested with mutagenesis of the ball domain, constructs with different chain lengths, and membrane manipulations. This aim was proposed in the previous grant cycle, we made considerable progress but the mechanism was more complex than anticipated. Hence, this aim still needs investigation. During the last cycle, we also treated another unexpected finding from the structure of closed MthK, still in the scope of the grant but not explicitly proposed, which was just published. Our second aim is to determine the structural correlates of ball-and-chain inactivation in BK channels as well as how bilayer-thickness and other bilayer properties modulate these channels. We will use b2 and a chimeric b4-b2 subunit for both structural studies and electrophysiology in HEK cells and liposomes. We will investigate lipid-dependence of BK channel gating with and without b subunits with stopped-flow assays and single- channel recordings of purified BK channels reconstituted in liposomes. Channel structures in complex with lipids will be solved using cryo-EM. The accomplishment of these aims will provide a comprehensive picture of ball-and-chain inactivation and lipid modulation in calcium-gated potassium channels.

NIH Research Projects · FY 2025 · 2009-07

Aggregates of lipoproteins that are tightly crosslinked to the extracellular matrix are the major type of lipoprotein in atherosclerotic lesions. The majority of the cholesterol in these aggregates is unesterified, but it has been unclear how the cholesteryl esters in the core of retained and aggregated extracellular LDL are hydrolyzed – especially because a lysosomal hydrolase has been reported to be involved. Our recent studies demonstrate a novel mechanism for the hydrolysis of cholesteryl esters in the core of retained and aggregated LDL in which macrophages (M) create tightly sealed compartments that surround portions of the aggregated LDL. They then acidify these compartments and secrete lysosomal enzymes into them, creating a lysosomal synapse. It has been shown that the extracellular hydrolysis of cholesteryl esters by lysosomal acid lipase, in a process called digestive exophagy, leads to production of unesterified cholesterol outside the cell, and transport of this cholesterol into the cell leads to foam cell formation. The high concentrations of cholesterol in the aggregated LDL were observed in a preliminary study to lead to the formation of extracellular cholesterol crystals, which can cause inflammatory responses in M . The overarching hypothesis of this proposal is that this mechanism for degrading lipoproteins has significant differences as compared to conventional phagocytic or endocytic mechanisms and that these differences have important consequences in the pathophysiology of atherosclerosis. Furthermore, understanding this process may lead to improved therapeutic interventions. Work in the first aim will characterize the molecular mechanisms of digestive exophagy. This will include a study of the Rab and SNARE proteins that are required for lysosomal exocytosis. Signaling by Tlr4, Myd88, PI3-kinase, Akt, Syk, Vav, Cdc42, and other molecules has been shown to be important for digestive exophagy, and the roles of additional signaling molecules will be explored. High concentrations of cholesterol are generated in aggregated LDL, and in Aim 2 formation of cholesterol crystals and resulting inflammatory activation of M will be examined. Work in the third aim will use optical imaging and 3-D electron microscopy (FIB-SEM) to examine the 3D structures of lysosomal synapses in a mouse atherosclerosis model. Formation of lysosomal synapses, association of cholesterol crystals with retained and aggregated LDL, and inflammatory activation will be studied in various mouse models of atherosclerosis by optical microscopy. Better understanding of the cellular and molecular events occurring in atherosclerotic lesions can lead to better risk assessments and potentially new therapies.