Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Pancreatic beta cell insulin production is the critical lynchpin that determines diabetes resistance or susceptibility. ER stress is one cause of beta cell dysfunction and failure, not only in T2D but also in T1D and some forms of monogenic diabetes. Many published reports show that the ATF6 pathway, one of three principal ER stress response pathways, plays important roles in cellular adaptation to stress. In particular, ATF6 is known to drive beneficial effects including increased ER capacity, cell survival in the face of stress, and more recently evidence from our group and several others implicate ATF6 in beta cell compensatory proliferation in response to insulin demand. For these reasons, activation of ATF6 has been proposed as a potential beta cell therapeutic approach that might improve beta cell mass and insulin production capacity. We have developed two novel, exciting tools that allow us to activate ATF6 in beta cells with temporal precision, either ex vivo or in vivo in live mice. Initial experiments, however, show that when we indiscriminately activate ATF6 for an extended period of time we observe a mix of beneficial and harmful effects, in some ways reminiscent of glucotoxic beta cell failure. Specifically, we do observe evidence of increased beta cell proliferation and survival, but activating ATF6 in vivo continuously for a 14-day period leads to frank glucose intolerance due to beta cell dysfunction. Remarkably, if we allow ATF6 to turn off, beta cell function gradually returns to normal. Molecular and morphological preliminary data suggest that in vivo chronic continuous ATF6 activation mimics, in many ways, chronic beta cell stress in T2D, with similarities to mouse and human observations. As such, this model represents a tremendous opportunity to study the proximate causes of beta cell failure after chronic activation of one ER stress response pathway (ATF6), as well as a unique and exciting chance to understand the in vivo recovery process if that stress pathway activation is able to shut off. In this project we will explore the causes of beta cell failure after ATF6 activation, with in-depth molecular, morphological and tissue homeostasis experiments. We will determine the cellular and molecular bases for beta cell recovery when ATF6 is allowed to turn off. Finally, we turn our attention to the molecular mechanisms driving benefits and harms of ATF6 activation and seek to identify conditions in which beneficial responses can be separated from harmful responses, to see whether it may be possible in the future to harness ATF6 for safe therapeutic potential in diabetes treatment or prevention. If successful, this project will lead to important new insight into beta cell stress-induced diabetes, the in vivo recovery process after ATF6-induced beta cell dysfunction, the molecular mechanisms by which ATF6 drives benefits and harms, and whether it may be possible to separate benefit from harm for future therapeutic benefit.

NIH Research Projects · FY 2025 · 2022-12

Project Summary/Abstract Alzheimer’s disease (AD) and other tau-related neurodegenerative diseases are characterized by neurofibrillary tangles which are composed of aggregates of the microtubule associated protein tau. The accumulation of these tangles contributes to neuronal death and cognitive decline. While millions nationally are plagued by Alzheimer’s, therapeutic efforts focusing on one of the main hallmarks of the disease, amyloid-beta (Aβ) plaques, have remained unsuccessful. The lack of correlation between treatment and robust cognitive improvement during these clinical trials highlight the urgent need to elucidate the preclinical mechanistic changes driving disease manifestation. The physiological roles of tau include the stabilization of microtubules, and bundling of F-actin filaments. While tau-microtubule interactions are well-studied, tau interactions with F- actin are more poorly understood, but are reported to drive the development of pathological species such as Hirano bodies, actin inclusions found in AD brains and other tauopathies. Tau can form other functional or pathological interactions, including recently reported binding to post-synaptic density protein 95 (PSD-95), which was shown to interfere with functional hyperemia and promote the neurotoxicity induced by amyloid- beta. Tau is also modified by a rich array of post-translational modifications (PTMs), which have been shown to alter normal and pathological tau interactions. This proposal will test the central hypothesis that tau PTMs within the critical PHF6 and PHF6* hexapeptide motifs modulate tau interactions with binding partners such as PSD-95 and F-actin and thereby contribute to the role of these partners, as well of changes in tau structure and function, in specific processes associated with disease manifestation. Using biophysical methods, including nuclear magnetic resonance (NMR) spectroscopy, I will characterize the interactions of tau with F-actin and PSD-95 and assess the effects of PTMs located within the PHF6 and PHF6* motifs on these interactions. Our structural observations, complemented by functional studies performed both by myself and our collaborators, will contribute to a deeper understanding of the mechanistic changes in tau behavior that drive the manifestation of AD and other tauopathies, and facilitate the development of novel therapeutic targets for the treatment of tau-based neurodegeneration.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY The overarching goal of this proposal is to identify novel addictions in mesenchymal colorectal cancer (CRC) that will create new vulnerabilities to be targeted therapeutically. This type of aggressive CRC shows a microsatellite stable phenotype, characterized by a strong reactive desmoplastic stroma and immunosuppressed microenvironment. Mesenchymal CRC is enriched in a transcriptomic CMS4 signature, affects about 30-40% of all CRCs with the poorest prognosis, and lacks effective therapies. Our recently published and preliminary data demonstrated that the loss of the two atypical protein kinase Cs (aPKCs; PKCλ/iota and PKCzeta, encoded by PRKCI and PRKCZ genes) drives the mesenchymal phenotype in mouse intestinal tumors. Furthermore, interrogation of human specimens and transcriptional datasets of CRC patients established the aPKCs as new suppressors of mesenchymal CRC. Our data also show that PKCλ/iota is the one whose inactivation unleashes transformation through the mesenchymal route, and new preliminary results, which constitute the foundation of this proposal, demonstrated that aPKC inactivation in several models results in the upregulation of a cholesterol biosynthetic signature and promotes cholesterol synthesis in vivo as evidenced by metabolic tracing experiments in mice. Analysis of human CRC data and samples demonstrate a positive correlation between the activation of the cholesterol biosynthetic pathway and mesenchymal CRC, associated with a negative correlation with aPKC levels. Also, PKCλ/iota directly phosphorylates SCAP, an obligate regulator of SREBP2 processing, which is the master regulator of cholesterol biosynthesis. Therefore, the central hypothesis of this application is that PKCλ/iota by phosphorylating SCAP restrains the cholesterol biosynthetic pathway to repress tumorigenesis in mesenchymal CRC. Building on our strong preliminary data, we will (Aim 1) determine the role of increased cholesterol metabolism in mesenchymal CRC initiation and progression. To that end, we will determine the impact of feeding a high-cholesterol diet (Aim 1.1) or blocking cholesterol metabolism in vivo (Aim 1.2) in tumor initiation and progression of mesenchymal CRC tumors driven by PKCλ/iota deficiency; and determine the human relevance of PKCλ/iota-regulated cholesterol metabolism in mesenchymal CRC (Aim 1.3). We will also (Aim 2) determine the molecular mechanisms whereby PKCλ/iota regulates cholesterol biosynthesis in mesenchymal CRC tumorigenesis. Therefore, we will (Aim 2.1) determine the impact of PKCλ/iota-mediated SCAP phosphorylation on regulating the SCAP/SREBP2 complex at a mechanistic level; and (Aim 2.2) determine the functional contribution of PKCλ/iota-mediated SCAP phosphorylation to CRC in vivo. The results from this proposal will allow the identification of new targets as therapeutic points for intervention in mesenchymal CRC.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY The lymphatic vasculature plays a critical role in fluid homeostasis, removing cellular waste and immune responses. Recent research has highlighted its integral contribution during the regenerative response after cardiac injury. The central nervous system was until recently, believed to be immune privileged and lacking a lymphatic system. Recent studies have revealed the lymphatic system extends into the mammalian and zebrafish brain, however the role it plays in neurogenesis and the response to injury is unknown. The need to better understand how to alleviate the detrimental responses to Traumatic brain injury (TBI) and stroke and promote regeneration is imperative in order to improve outcomes. This proposal aims to uncover the role of the meningeal lymphatic system in adult neurogenesis and regeneration and the mechanisms through which it contributes to generating new neurons during homeostasis and injury response. Previously, we have characterized the development of the zebrafish cardiac lymphatic system and identified the signaling pathways that regulate its specification and formation. We then demonstrated that the cardiac lymphatic vasculature expands post injury in the adult zebrafish heart. This work demonstrated that lymphatic vessels respond to injury and aid the regenerative response by trafficking immune cells. By blocking lymphangiogenesis we demonstrated that the lymphatic vasculature was required to promote regeneration and prevent scarring of heart tissue. The discovery of the meningeal lymphatics led us to hypothesize that meningeal lymphatics support adult neurogenesis and regenerative repair after injury by providing neurotropic factors, controlling cerebral spinal fluid (CSF) composition and the immune response. To test this hypothesis, we will pursue two aims in the zebrafish as a model of adult neurogenesis and brain regeneration. Aim 1 will characterize the lymphatic response to injury using time lapse imaging and determine how disruption of the lymphatic system impacts adult neurogenesis. Using mass spectrometry, we will analyze the composition of the CSF to identify lymphangiocrine factors that support neurogenesis. In Aim 2 we will manipulate the development of the meningeal lymphatic vasculature and use transcriptomic analyses to determine the impact on immune cell populations and response. Pursuit of these hypotheses will open new avenues for investigation of lymphatic support of neurogenesis in mammalian systems in health and after injury. In order to further our comprehension of adult neurogenesis, the regenerative response of the nervous system and potential therapies forward it is critical to understand the how the lymphatic system modulates the immune and environmental aspects of neurogenesis and regeneration.

NIH Research Projects · FY 2025 · 2022-09

The HEAL Data2Action (D2A) Program is a coordinated effort to promote the synthesis and real-world application of existing data to guide and monitor improvements in service delivery to prevent or treat opioid use disorder (OUD) and pain. Collectively, these projects will address gaps in the delivery of evidence-based practices in each of the four pillars of the HHS Overdose Prevention Strategy: primary prevention, harm reduction, treatment of opioid use disorder, and recovery support. The HEAL D2A Modeling and Economic Resource Center (MERC) will support the HEAL D2A Innovation Projects by providing expertise in simulation modeling and economic evaluation methods and will consult with the HEAL D2A Innovation Grant recipients on applying these methods and insights in their local implementation activities. The MERC will also conduct original research applying state-of-the-art economic and simulation modeling expertise to understand and address the overdose crisis. Since 2015, we have worked collaboratively to lead a highly successful multi- institutional NIDA-funded national center of excellence, the Center for Health Economics of Treatment Interventions for Substance Use Disorder, HCV, and HIV (CHERISH). We are thus uniquely qualified to lead the HEAL D2A MERC with the following aims: 1) to support the HEAL D2A Innovation Projects by providing methodological consultation on the application of rigorous modeling techniques, cost analyses, and behavioral economic strategies to support the projects' selection and implementation of evidence-based practices to address service delivery needs; 2) to develop tools that can be readily used by the Innovation Projects to make informed decisions about the relative costs of different implementation interventions to different stakeholders, to measure the overall costs of these efforts, and to inform the design of appropriate payment systems that support sustainability; 3) to use meta-modeling techniques to develop a user-friendly interface so that simulation model results generated for one system or community can be adapted for other systems/communities or adapted when local circumstances change; 4) to conduct novel research using advanced modeling and health economics approaches that build on existing data to provide insights into the complex dynamics of addressing the overdose crisis through strategic implementation of evidence-based practices. The HEAL D2A MERC will support the application of health economics and modeling research to real-world decision making about services to prevent or treat opioid use disorder and pain, will further scientific innovation in the field, and will establish a paradigm for integrating health economics and modeling into substance use treatment and prevention policies at the system level. The HEAL D2A MERC will be collaborative with the work of the HEAL D2A Research Adoption Support Center (RASC), including the consultations and catalog of measures and tools of the Implementation Support Cores, and will leverage and contribute to the resources of the HEAL D2A Data Infrastructure Support Center (DISC).

NIH Research Projects · FY 2025 · 2022-09

In the US, we have had record high overdose deaths and high risk for future injection-related HIV and hepatitis c (HCV) outbreaks. Innovative approaches are urgently needed to expand access to syringes and naloxone, a drug used for opioid overdose reversal, for the prevention of HIV/HCV outbreaks and opioid overdose fatalities. Expansion and scale-up of mail-based safer drug use services may address access gaps by providing a more convenient and confidential way to obtain services. To inform scale-up these services, we aim to 1) describe policy barriers to national expansion of mail-based syringe services and fentanyl strip distribution, 2) conduct a national, longitudinal cohort study to examine predictors of uptake and long-term engagement in mail-based safer drug use services, and 3) assess the add-on safer drug use and health services preferences of mail delivery clients. For Aim 1, we will conduct a systematic legal review of relevant laws in each of the 50 states and the District of Columbia to determine whether and how state law may impact the legality of distributing syringes and fentanyl test strips by mail. We will develop a taxonomy of policies that may prevent legal expansion of mail-delivered syringes. To understand the perception of legal risks associated with mailing syringes among potential adopters, we will survey and interview 20 stakeholders (i.e., health departments and SSPs) from states representing different legal environments for mail-delivery. For Aim 2, we will conduct a social media-recruited survey examining uptake and acceptability of mail-delivered safer drug use services at 12 months. This will allow us to assess predictors of uptake and mail-delivered safer drug use service engagement over the long-term in order to fully understand who uses mail-delivered services and how these services are being used over time. For Aim 3 we will design and implement discrete choice experiment (DCE) surveys to determine mail-based service client preferences for receiving add-on safer drug use and health services. These results will be disseminated to policy stakeholders, potential adopters, and NEXT Distro affiliates to inform expansion and scale-up of mail-based safer drug use services, improve engagement with current NEXT users, and expand the type of services offered through these programs.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The over-arching aims of this research are to: (1) develop a novel primary care screening tool to identify elder neglect in patients with Alzheimer’s Disease and Related Dementias (AD/ADRD) and a point-of-care technology-driven intervention for caregivers [R61], and (2) conduct a NIH Model Stage III 3-arm randomized clinical trial to determine the impact of both screening itself and screening combined with the intervention compared to usual care [R33]. We are focusing exclusively on developing screening for neglect rather than all types of mistreatment given that neglect is very common in AD/ADRD patients, neglect is associated with the highest mortality and morbidity of all mistreatment types, and specific targets exist for interventions. Combining all types of mistreatment, the approach taken to date in screening tool design, has important disadvantages. It makes screening time-consuming and challenging. Additionally, different mistreatment types are very different phenomena, occur in different groups of older adults under different circumstances, have different impacts on health, and likely require different intervention strategies. Further, our research team has developed and studied a successful, evidence-based technology intervention for AD/ADRD caregivers. We believe that an adaptation of this intervention for caregiver neglect, which focuses on education and skill building and access to resources, may be highly impactful in reducing and preventing neglect. The Specific Aims of the R61 phase are: (Aim 1a) develop a novel, easy-to-use, brief screening tool to identify elder neglect in primary care and describe the tool’s test characteristics (KQ3), (Aim 1b) develop an innovative intervention for elder neglect by modifying a highly successful evidence-based technology-driven caregiving intervention, and (Aim 2) pilot-test the screening tool and intervention in primary care (NIH Model Stage 1b) to assess feasibility and acceptability. In the R33 phase, we will conduct a 3-arm randomized controlled trial (NIH Model Stage III) to compare: (1) screening + intervention, (2) screening only, and (3) usual care without screening. The Specific Aims of the R33 phase are: (Aim 3) evaluate the impact of screening for elder neglect on reduction in neglect exposure (KQ1) and other patient and caregiver-important outcomes by comparing to usual care and (Aim 4) evaluate the impact of screening tool paired with a technology-based caregiver intervention on reduction in exposure to neglect (KQ5) and other patient and caregiver-important outcomes by comparison to usual care and screening without intervention. Neglect screening in primary care with a novel screening tool will have a positive impact on outcomes including decrease in presence of neglect at 6 months and other patient and caregiver-important outcomes (e.g., neglect severity, neglectful ideation; caregiver burden) and that screening paired with a technology-based caregiving intervention will have a greater positive impact than screening alone. Our long- term goal is to develop a screening tool for neglect in AD/ADRD patients that may be integrated into primary care and paired with a scalable intervention with sufficient evidence to justify USPSTF endorsement.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Synaptic transmission and its plasticity are based on a core group of synaptic receptors that rapidly sense neurotransmitters such as glutamate and GABA. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are typically thought to signal on slower time scales, but have also been shown to regulate the induction and expression of synaptic plasticity. Despite both neurotransmitter and neurotrophin signaling underlying most forms of synaptic plasticity and serving as major mediators of the pathophysiology and treatment of neurogenerative and neuropsychiatric disorders, little is known about how these different receptor classes work in concert through direct and indirect forms of crosstalk. Motivated by the paucity of direct tests of neurotrophin/neurotransmitter crosstalk, our preliminary studies have indicated that the neurotrophin receptor, TrkB, and metabotropic glutamate receptor 5 (mGluR5) are able to mutually regulate each other in both native and heterologous systems. In contrast to previous studies which have focused on the ability of GPCRs to activate RTK signaling, we have identified one of the first examples of an RTK (TrkB) engaging a GPCR (mGluR5) as a signaling effector. In this proposal, we will test the hypothesis that mGluR5 is a critical mediator of TrkB effects by amplifying and altering the spatiotemporal dynamics of downstream signaling to canonical effectors to drive a unique form of crosstalk-dependent synaptic plasticity. Collectively, this new form of TrkB-mGluR5 signaling may contribute to the diverse higher order nervous system functions related to neural plasticity that have been attributed to the BDNF-TrkB neurotrophin system. Aim 1 will define BDNF-TrkB receptor mediated signaling crosstalk with mGluR5. Aim 2 will decipher the signaling mechanisms that regulate TrkB/mGluR5 crosstalk. Aim 3 will determine the impact of TrkB-mGluR5 synergy on BDNF-LTP and mGluR5-LTD.

NIH Research Projects · FY 2025 · 2022-09

The overall goal of research in the Geri lab is to map protein interactomes using discovery technologies that provide orders of magnitude improvements in spatiotemporal resolution over the current state-of-the-art. The motivation for this work is that advancing the resolution of protein interactome discovery technology beyond key milestones, such as single cell and single protein thresholds, will have a field-wide impact analogous to similar advances in transcriptomics and microscopy. The first three years of work in the lab will be focused on creating new technologies by combining photocatalytic proximity labeling, in which light-powered catalysts attached to an affinity handle drive the crosslinking of synthetic affinity probes with nearby proteins, with patterned light and interaction-gated activation to simultaneously enforce multiple dimensions of specificity. The fourth and fifth years of work will focus on applying the mature technologies. The overall strategy is divided along two thrusts, in which labeling specificity is obtained through extrinsic optical control or intrinsic chemical control, and has been designed to be programmatically robust by minimizing project interdependency. Intrinsically selective systems will exploit the high spatial resolution of photocatalytic labeling (5 nm) and use “split” systems that operate when defined protein targets are in proximity. Initial work will use natively expressed orphan peptides as proximity labeling loci to discover their currently unknown receptors. The effort will cover thousands of peptides by using label free ion mobility mass spectrometry for proteomics, maximally leveraged by using optimized labeling probe designs. Split systems will combine multiple photocatalysts targeted to different proteins of interest to make colocalization a dimension of specificity, and will be initially applied to map proteins present at membrane junctions. Extrinsically controlled systems will enable subcellular resolution labeling in human tissue sections and ms-resolution temporal control for the study of transient protein interactions. Both approaches are enabled by combining optical tools for spatiotemporal control of light itself with the total and instantaneously responsive (<1µs) dependence between photocatalytic efficiency and the local supply of light, and each allow for a three order of magnitude increase in resolution vs current tools. Spatially selective labeling will focus on identifying protein interactions unique to cell subpopulations in human tissues, with initial studies focusing on discovering location-conditional interactions driving lymphocyte function in human tonsil and thymus. Later studies will focus on discovering interactome differences between translationally relevant tissue samples. Temporally resolved labeling will combine optogenetic tools and photocatalytic proximity labeling to synchronize and interrogate transient protein interactions. The power of this approach will be fully exploited by studying exocytosis in neurons with millisecond resolution, one of the fastest dynamic biological processes known. Successful development and deployment of these systems for protein interaction discovery will enable the study of large interactome spaces for the first time, and is expected to have a broad impact on the molecular biology community.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY WNT pathway hyperactivation is a dominant oncogenic driver in colorectal cancer (CRC), and directly linked to disease progression and drug resistance in many other cancer types, including lung (LUAD), breast and prostate cancer. Indeed, there is strong evidence from pre-clinical model systems that targeting hyperactive WNT signaling can provide significant therapeutic benefit in multiple cancer models. We and others have shown that blocking the function of redundant Tankyrase enzymes (TNKS and TNKS2) can suppress hyperactive WNT signaling, impede cancer cell proliferation and drive differentiation, particularly in CRC. However, WNT signaling is also critical for the homeostatic maintenance of multiple organ systems. Consequently, drugs that effectively block WNT, like TNKS inhibitors, have shown a range of on-target toxicities in essential tissues such as the intestine and bone. Defining a strategy for tumor-restricted WNT pathway suppression is a major goal in precision oncology. Advanced cancers often contain significant disruptions to their genomes, including gains and losses of large chromosome segments. These large-scale alterations encompassing many genes are presumed to support cancer cell growth. However, they also lead to loss of ‘passenger’ genes that do not drive cancer progression, but may unintentionally ‘rewire’ the signaling networks. Such cancer-specific collateral damage may provide opportunities for therapeutic intervention. We will test the hypothesis that a large chromosomal deletion on chromosome 8p, which induce loss of TNKS, create a tumor-specific dependency on TNKS2 that can be exploited to block WNT only in cancer cells. Because normal cells express both redundant family members, they should remain unaffected by selective targeting. In Aim 1, using a panel of CRC, LUAD and breast cancer cell lines and patient-derived organoids, in combination with CRISPR-based genome editing, we will determine how heterozygous and homozygous chromosome deletions impact the response to TNKS2 inhibition. Further, we will define downstream protein targets that are most sensitive to TNKS disruption and TNKS2 suppression. In Aim 2 we will exploit a unique transgenic shRNA system we developed, to define the safety of systemic and selective TNKS2 inhibition in vivo and determine the efficacy of selective TNKS2 inhibition in immunocompetent animal models of aggressive CRC and LUAD. Aim 3 will determine the efficacy of novel TNKS2-selective small molecules in cancer cell lines and organoid models and evaluate potential mechanisms of therapy resistance to TNKS2 inhibition. Identifying a safe and effective approach to block hyperactive WNT signaling in multiple tumor types could have a profound impact on the clinical management of advanced cancers. Thus, we believe our work will contribute substantially to the overall goal of developing safe and effective targeted therapies for WNT-driven cancers.

NIH Research Projects · FY 2025 · 2022-09

The goal of this project is to discover fundamental epigenomic regulatory mechanisms that commit cells to defined fates during early stages of embryogenesis. Early in development, commitment of the epiblast to germ layers is followed by activation of key regulatory genes that drive lineage fate. These genes control normal development and underlie the genetic basis for a broad range of human structural birth defects. We have studied members of the TET family of hydroxylation enzymes, which regulate the demethylation of DNA, or block active methylation of DNA, to control gene expression. We discovered requirements for TET enzymes during early development in the zebrafish model, and for progenitor specification from human embryonic stem cells (hESCs). Major gaps in understanding include: i) whether common or distinct mechanisms control demethylation for progenitors from different germ layers, ii) whether different TET family members distinguish developmental programs, and iii) how TETs are targeted to regulatory regions such as bivalent promoters. Suspecting that additional proteins beyond TETs are needed to target DNA demethylation, we carried out a genome-wide CRISPR screen and discovered QSER1, a previously uncharacterized chromatin-binding protein. We showed that QSER1 is a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys, broadly across different cell fates. We found biochemical and genetic interactions between QSER1 and TETs, suggesting that they cooperate to safeguard transcriptional and developmental programs from methylation. QSER1 variant alleles were recently linked to coronary artery disease, while haploinsufficiency of a QSER1 paralog, PRR12, is associated with multi-organ developmental birth defect syndromes. We propose to fully explore the genetic relationships and downstream networks of TET/QSER1 (TQ) family members, including how they function to control methylation and impact chromatin structure in the context of two complementary model systems, zebrafish and hESCs. The zebrafish model allows full genetic analysis of potentially compensatory or cooperating family members (including tet1, tet2, tet3, qser1, and prr12), in an animal model with highly conserved developmental programs. The hESC model provides outstanding biochemical and “omics” capacity, and validation in developing human progenitor and differentiated cells. The multi-PI project represents a continued collaboration among investigators with complementary and overlapping expertise, with a strong record of productivity. Specific Aims are proposed to determine the relative contribution of these genes for directing early progenitor fate, discover the regulatory networks in which they function, and to test interacting factors as candidates for linking TQ methylation control to chromatin modification. Because regulation of methylation is a fundamental step of progenitor fate determination, our results will be broadly relevant to organogenesis and structural birth defects.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Despite the recognition of the contribution of the immune system to cancer response to ionizing radiation, successful translation to the clinic is lagging. We are proposing to adapt the ROBIN mechanisms of research to enable a deep dive into the field of radiation (RT) and immunity. Representatives from seven international academic centers already engaged in RT and immunity research have converged to participate in a small prospective clinical trial (accrual: 25 patients in US and 25 patients in Europe), to synergize and accelerate discovery. The setting of preoperative short course radiotherapy (SCRT) in rectal cancer, has emerged as ideal for the scientific questions posed. SCRT is a standard treatment, preceded and followed by colonoscopies to respectively assess tumor baseline extent and response (at the end of RT): during each colonoscopy consenting patients can donate a tumor biopsy, as well as stool and blood (PBMC) samples. The same set of specimens can be harvested, six weeks later at surgery, where research sampling of lymph nodes will also be possible, within and outside the RT field. These sequential sets of tissues will enable us to conduct cutting edge multiple “omics” approaches to study irradiated normal and cancer tissue and the microbiome in the RT field. The PBMC analysis will allow correlation at a single cell level between RT-induced oxidative stress, changes in immunophenotype and PBMC biology. Similarly, the ability to analyze lymph nodes harvested inside and outside the radiation field will allow to pinpoint at the single cell level the RT effects on each immune subpopulation. The longitudinal analysis on cancer biopsy, collected before and after RT and at surgery, will give a snapshot on the RT-induced “omics” changes. An orthogonal radiomic study will analyze MR images obtained before SCRT and before surgery (also standard imaging procedures in rectal cancer) together with images obtained at CT simulation. Compliance with international regulations for data sharing, standardization of procedures and data acquisition and harmonization of uploaded data will be essential to this effort. Advanced bioinformatics tools will be applied through a dedicated Data Sharing and Integrative Analysis Core, capable to deconvolute and interpret complex biological and imaging data, sorted by utilizing NCI FireCloud workspaces. By converging experienced clinical investigators, bio-scientists and bio-informaticians to address fundamental radiation biology questions, this ROBIN will rapidly enable unprecedented discovery that will be shared with the ROBIN network and the scientific community at large. Finally, since inception, ROBIN has revealed an optimal environment for cross-training and cross-fertilization of the scientists and clinicians involved in the grant preparation and has created a robust foundation for the proposed Cross Training Core, a novel structure to form future leaders in radiation oncology and biology, a task each of the three P.I.s consider crucial to the future of our discipline.

NIH Research Projects · FY 2025 · 2022-09

PROJECT ABSTRACT Host-microbe interactions profoundly impact cancer. This is exemplified by well-documented infections that promote cancer, and the ability to prevent these cancers through vaccination or pathogen avoidance. However, humans are densely colonized with trillions of normally beneficial microbes, termed the microbiota, which also have the ability to promote cancers through the induction of inflammation or genomic instability. Further, recent seminal studies demonstrated that intestinal microbiota are also required for anti-tumor immunity in the context of therapeutic interventions, such as checkpoint blockade. Despite these advances, the specific pathways by which microbiota shape pro- versus anti-tumor immunity remain poorly defined, and the potential relevance of these findings to specific types of cancer are unknown. The fundamental focus of this proposal is to mechanistically define a novel pathway that controls host-microbiota interactions to protect from tumor progression and promote the efficacy of immunotherapies in colorectal cancer (CRC). In recently published data (Goc et al., Cell, 2021), we have determined that group 3 innate lymphoid cells (ILC3s) are fundamentally altered in CRC and contribute to tumor progression and immunotherapy responsiveness by coordinating host-microbiota interactions. These data provoke a fundamental hypothesis that intestinal ILC3s are protective in cancer, but become inherently disrupted in CRC, subsequently driving dysfunctional adaptive immunity and alterations to the microbiota that support tumor progression and immunotherapy resistance. We will mechanistically test this hypothesis by asking the following specific questions: (1) What drives dysfunction of ILC3s in CRC?; (2) What are the microbial and host pathways by which ILC3s protect from tumor progression?; And (3) What are the microbial and host pathways by which ILC3s protect from immunotherapy resistance? Finally, we will directly test a number of interventional strategies that target the microbiota to limit tumor progression and break resistance to cancer checkpoint inhibitors. Results from these experiments will pave the way for a greater understanding of host-microbiota interactions in cancer, and could provoke novel preventative, therapeutic or curative strategies in cancer by modulating host-microbiota interactions.

NIH Research Projects · FY 2025 · 2022-09

Riverside Research, the University of Bristol (Bristol, United Kingdom), and the University Paul Sabatier (Toulouse, France) propose to develop the next generation of quantitative acoustic microscopy (QAM) systems. Specifically, data- science and coded-excitation approaches will be applied for the first time to QAM technology to yield better image quality, decreased scanning time, greater ease of use, as well as to pave the way for a new generation of novel, low-cost, user-friendly QAM instruments. QAM permits formation of fine-resolution (i.e., <7 µm at 250 MHz) maps of acoustic and mechanical properties of tissue sections that are <12 µm in thickness. These data can have great value in numerous preclinical investigations. Such property maps are not currently obtainable by any other microscopic-imaging modality, and the new generation of QAM technology made possible by success in this proposed project could become widespread in research laboratories and microscopy suites in commercial as well as academic research environments. Such new-generation QAM instruments could be used by technicians with limited knowledge of QAM and, in many ways, their use would be no more complicated than use of a conventional bright-field microscope. These novel approaches to QAM will be demonstrated using already available resolution targets, phantoms, and biological tissues (ocular-tissue samples from a guinea-pig model of myopia and cancerous human lymph nodes). During the course of this project, optimal methods will be incorporated in a prototype QAM (pQAM) instrument capable of producing ultra-fine spatial resolution (< 2 µm) images much faster (<1 min) and for a much lower cost (<$40k) than current state-of-the-art QAM systems. In addition, pQAM use will be ``turn-key'' (i.e., requiring no technical knowledge and less than 1 hour of training.)

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Chronic cancer-related pain is highly prevalent and international guidelines have long supported the use of opioid therapy for moderate to severe pain related to active disease. The strength of such consensus is strongest for patients experiencing advanced disease and limited life expectancy. For patients experiencing long-term remission, or, stable or indolent disease without the need for ongoing anti-cancer treatment (“long- term survivorship”), there is emerging consensus that opioid therapies should be addressed in a similar manner as for patients with chronic non-cancer pain. There is mounting ambiguity regarding best practices for patients receiving active anti-cancer treatment intended for cure. In the wake of the opioid epidemic, state policies have proliferated in an effort to reduce unsafe opioid prescribing. Prominent recent policies include state mandates for prescriber participation in the Prescription Drug Monitoring Programs (PDMPs) and state legislative limits on duration and/or dosage of opioid prescriptions for acute pain. These policies vary in their intended applicability to subpopulations of cancer patients, and, coupled with the ambiguity regarding clinical best practices, may have inadvertently impacted opioid use and related outcomes among the different subpopulations of people with cancer. We propose a study to evaluate intended and unintended consequences of the two types of state policies for opioid prescriptions and pain- and opioid-related adverse health events among cancer patients with advanced disease, long-term survivors, and patients receiving active cancer treatment. To help elucidate mechanisms underlying changes in response to policies, we will also explore the trajectories of opioids dispensed and clinical encounters within each subpopulation, using an innovative pattern mining approach. We will use the SEER-Medicare linked database and a large national commercial insurance database to achieve study aims. Findings will inform consensus-building, guideline and intervention development, and policy and practice changes by providers, health care organizations, and policymakers in optimizing opioid prescribing and pain management for cancer patients.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Despite its putative link to many mental illnesses, Nonsense-Mediated mRNA Decay (NMD) represents a relatively unexplored mechanism for regulating mRNA stability in brain function. NMD functions in a tissue-, cell type- and cell-state specific manner and modulates stability of selective mRNAs to fine-tune transcript abundance. There is dearth of knowledge regarding the identity of such NMD target RNAs, particularly in cells in their normal in vivo context. A particularly large gap in the field is the cell-specific function and targets of NMD in vivo. Our recent work has established that neuronal NMD regulates GLUR1 signaling and is required for proper synaptic plasticity, cognition, and local protein synthesis in dendrites, providing fundamental insight into the neuron-specific function of NMD within the brain. To date, no study has reported a specific function for NMD nor identified NMD substrates within glial cells in the brain. Astroglial control of synaptic activity translates into regulation of cognition making astrocytes a novel therapeutic target to treat cognitive dysfunctions. However, the mechanisms through which astrocytes regulate neuronal function are not well understood. Currently, it is not known whether mRNA degradation in astrocytes contribute to the regulation of synaptic plasticity and behavior. The goal of this application is to determine the contribution of astrocytic NMD to synaptic plasticity and cognitive performance. Several predicted ‘canonical’ and ‘atypical’ NMD targets are expressed in astrocytes. Our gene ontology analysis of these predicted NMD targets identified molecular function enrichment for Ca2+ signaling. Consistent with this, we have found that disruption of NMD in astrocytes resulted in elevated Ca2+ activity in vitro. Dynamic Ca2+ transients in astrocytes have been suggested to control proper basal synaptic transmission and modulate hippocampal LTP. We have also found that conditional ablation of NMD in astrocytes impaired memory in the adult mice. Based on the published literature and our preliminary studies, we hypothesize that NMD regulates Ca2+ activity in astrocytes, and astrocytic NMD is required for proper cognitive function and behavior in the adult brain. To test this hypothesis, we propose to determine 1) whether NMD is required for different aspects of learning and memory 2) the effects of astrocytic NMD ablation on neurons (e.g., assessing neuronal network connectivity and synaptic plasticity) and 3) functional deficits of NMD-deficient astrocytes (i.e., by assessing Ca2+ activity in vivo) and in vivo NMD targets in astrocytes. We will use a combination of techniques including an inducible-genetic mouse model, behavioral assays, electrophysiology, live-animal Ca2+ imaging by two-photon microscopy, stereotaxic viral labeling, Multielectrode Array Assay, in vivo RNAseq/bioinformatics, and in vivo HITS-CLIP. The successful completion of this research will provide a coherent view of how cell-specific mRNA degradation underlies the highly regulated synaptic and cognitive function in the mammalian brain and might be valuable for providing new insights into the astrocytic mechanisms of synaptic dysfunction and neurocognitive diseases.

NIH Research Projects · FY 2025 · 2022-09

Alzheimer’s disease and related dementias (ADRDs) are a group of age-related diseases affecting cognitive function for which no treatments are available. Neuroinflammation and neurovascular dysfunction have emerged as crucial drivers of disease progression in ADRDs. In particular, cerebral endothelial cells and microglia, brain resident innate-immune cells, have been implicated in the accumulation of hyperphosphorylated tau, a microtubule associated protein and a key feature of ADRD’s pathology. Dysregulation of the peripheral immune system could also play a role and may influence endothelial and microglial function contributing to cognitive impairment. However, how the peripheral immune system communicates with the cerebral microvasculature and microglia to alter cognitive function remains to be fully established. IL17-producing T-helper lymphocytes (Th17 cells), a subset of T-helper lymphocytes, and their signature cytokine, IL17, have been implicated in the mechanisms of cognitive impairment. Previous data indicate that circulating Th17-derived IL17 acts on cerebral endothelial cells to induce a deficit in endothelial nitric oxide (NO) promoting tau accumulation and cognitive impairment. Due to their abundance in the gut, Th17 cells are particularly sensitive to gut bacteria and alterations in the gut flora are associated with dysregulation of Th17 cells. Segmented filamentous bacteria (SFB) are commensal bacteria which potently induce gut Th17 cells in mice and alter the course of models of Th17 cell-associated diseases. Therefore, gut Th17 dysregulation induced by SFB could be used as a model to gain a better mechanistic understanding of how gut Th17 cells alter brain health. On these bases, we propose to test the central hypothesis that dysregulation of gut Th17 cells, induced by colonization of the small intestine with SFB, promotes an IL17-mediated inflammatory response in cerebral endothelial cells which, in turn, activates microglia, leading to tau accumulation and cognitive impairment. To this end, the present grant application will examine endothelial function, tau pathology and cognitive function in a mouse model of SFB colonization to test the following hypotheses in male, female and aging mice: (1) Gut SFB colonization promotes gut Th17 differentiation, increases circulating IL17 and induces cerebrovascular and cognitive impairment; (2) Circulating IL17 mediates cerebrovascular dysfunction and cognitive impairment through activation of brain endothelial IL17 receptors; (3) IL17-induced endothelial pro-inflammatory mediators activate microglia leading to tau accumulation and cognitive impairment. The proposed studies fill an obvious gap in the understanding of the effects of gut microbiota on cognition and of the role of circulating IL17, endothelial cells and microglia in mediating these effects. Furthermore, these findings may unveil a novel link between microbiota-induced dysregulation of intestinal Th17 cells, brain tau accumulation and cognitive impairment, with potential public health implications.

NIH Research Projects · FY 2025 · 2022-09

Whereas lipidomics assays in peripheral blood are relatively well-established, the application of spatially- resolved lipidomics in the brain is novel. Bulk lipidomics studies have revealed potential differences in lipid metabolism across aging and disease, but the regional and cell type-specificity of these changes remains unresolved. In particular, given that the brain comprises numerous cell types of multiple lineages with tightly regulated spatial organization and inter-connections, it is likely the lipid profiles, metabolism, and dysregulation are all dramatically non-uniform across the brain. Thus, we propose to provide a map of this non-uniformity in brain tissue would move the field forward substantially in terms of having a universal reference, much as the Allen mouse brain atlas revolutionized researchers' ability to query spatial patterns of gene expression in the brain. We believe that a lipidomics atlas will be transformative in a similar way, especially given the increased focus on lipid dysregulation in neurodegenerative disease. We will test the hypothesis that deficits in the acyl chain remodeling pathway may underlie the changes in metabolic profile such as fatty acid metabolism and functional effects mediated by genes ABCA7, PICALM and BIN1 which have recently been identified associated with Late Onset AD genetic risk. Our continuing studies on phosphoinositide metabolism and the gene network including Synaptojanin1 (Synj1) are highly relevant to PICALM, a phosphoinoistide binding protein, and BIN1, also known as amphiphysin2, which interacts with Synj1 and is likely to mediate phosphoinositide signaling. ABCA7 interestingly, has been shown to transport lysophosphatidylcholine (LPC) a major biochemical intermediate of the Land's cycle and acyl chain remodeling. Recently, ABCA7 haplodefeciency has been shown to disrupt microglia function. It is clear that functional studies to understand phospholipid regional brain distribution, cell specificity and roles in cell-specific functions are critical for gaining understanding of these genes as well as the pathogenesis and disease progression of AD. Ultimately, we will identify biomarkers based on lipids which are dysregulated in brain and show correlated (positive or negative) dysregulation in plasma, which is tractable in the clinic. We hypothesize that re-programming of lipid metabolism is likely to be based on early changes in the Lands Cycle, acyl chain remodeling. This early and stereotypically altered metabolic shift in the lipid profile could ultimately be used for biomarker or therapeutic target discovery in AD. Successful completion of these studies will lead to system-wide, biological insight into the contribution of lipid metabolism to Alzheimer's Disease and validation of a lipid discovery platform which can be applied to future studies for development of biomarkers as well as therapeutic targets.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract More than 100 million people in the United States currently show signs of clinical depression, approximately three times more than before the onset of the COVID-19 crisis. Currently, the main treatment options for such depressed individuals include pharmacological and psychological interventions, the acute and long-term effectiveness of which are significantly limited: up to one-third of patients develop treatment-resistant depression. Noninvasive neurostimulation therapies such as repetitive Transcranial Magnetic Stimulation (rTMS)–where a magnetic coil placed over the cortex is used to focally stimulate the brain–have recently emerged as promising low-risk interventions for treatment-resistant depression. However, the mechanisms and appropriate parameters for this treatment remain poorly understood. In the best cases, rTMS can have dramatic effects, changing the course of a patient's life in hours. In many cases, however, it has little to no measurable effect. This raises the obvious question: why do current rTMS protocols work well for some individuals but not for others? Could we adapt protocols to work well for everyone, potentially providing reliable personalized treatment or even a lasting cure? A growing literature suggests this is possible if we learn to tailor treatment to individual differences in human neurophysiology. Here we propose an innovative and unique approach towards precision psychiatric neurostimulation: personalized modeling of treatment using “Generative Digital Twins”. To affordably build and scale Generative Digital Twins, we propose combining high density electroencephalography (HD-EEG)–which is non-invasive, inexpensive, and easily deployable–to measure longitudinal changes in brain connectivity during an accelerated rTMS treatment protocol for depression. Then, using controllable generative neural networks that allow detailed predictive simulations of individual response trajectories given rTMS treatment, we can begin to predict outcomes prior to treatment, understand individual responses, and personalize treatment parameters.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Islet transplantation offers a potential cure for Type 1 diabetes (T1D). Wide adoption of a cell therapy requires abundant islet supplies and effective immune protection without long-term systemic immune suppression. My laboratory and others showed that it was feasible to derive insulin-secreting cells from renewable and abundant gastrointestinal (GI) stem cells. However, it has not been possible to mass-produce islet-like organoids from human GI tissues for detailed assessment of their translational potential. In preliminary studies, we established methods to culture human gastric stem cells (hGSCs) from biopsy samples and expanded them to billions. The hGSCs were engineered for transient activation of NGN3 and stable expression of PDX1 and MAFA (collectively referred to as NPM factors), leading to formation of thousands of GINS (Gastric Insulin Secreting) organoids. GINS organoids acquired glucose-stimulated-insulin-secretion (GSIS) within 10 days, and upon transplantation, rapidly reversed diabetes in mice and maintained normoglycemia for over 3 months, with no tumor formation. Human GINS organoids thus have favorable attributes as a potential cell product for T1D treatment with a scalable derivation method. GINS organoids contain 25-30% of cells that closely resemble pancreatic Beta-cells. In this project, we will evaluate the hypothesis that clonal hGSC lines yielding a higher percentage of Beta-like cells can be readily identified from donor tissues. We will develop a standard derivation protocol using genetic knockin of NPM and clonal selection with the aim to consistently produce highly functional GINS grafts from donors. GINS cells lack key autoantigens and may be naturally less immunogenic than islet Beta-cells. We will confer further autoimmune protection by constitutive expression of two potent immune regulators PD-L1 an CD47. Normal and immune-evasive organoids will be evaluated in vitro and in vivo with a panel of antigen-specific cytotoxic CD8 T cells, the main effector of Beta-cell demise in T1D. Together, these studies will create a technology for reliable production of autologous GINS grafts with strong autoimmune protection, suitable for long-term glycemic control without immune isolation or suppression.

NIH Research Projects · FY 2025 · 2022-09

Coronary heart disease (CHD) and heart failure (HF) disparities persist in the US despite decades of research and national public health campaigns. The role of structural social determinants of health (SDH) in health disparities has been studied in only 1% of published work. Few studies of SDH have taken a life course perspective related to risk of CHD and HF. The ability to recover after the stress of an acute health event, or resilience, is an important patient-centered outcome, but the influence of SDH on resilience is unknown. Few past studies have examined both structural and intermediary SDH in the World Health Organization's (WHO) Commission on SDH (WHOCSD) conceptual framework in a longitudinal national sample with rigorously adjudicated CHD and HF endpoints, which is one of the aims of this application. We propose a series of studies to fill these gaps, while also providing rigorously adjudicated CHD and HF events and causes of death to a host of investigators interested in using these data (to date, over 500 have used these data). We build on a track record of mentoring to propose a formal year-long career development program for early-stage investigators (ESI). The specific aims of this application are to: (Aim 1) conduct studies examining underlying mechanisms of health disparities, guided by the WHOCSD conceptual framework, in three thematic areas: a) the incidence and recurrence of CHD; b) the incidence and recurrence of HF with preserved ejection fraction and HF with reduced ejection fraction; c) reserve and resilience after an incident or recurrent CHD or HF event. (Aim 2) To continue to adjudicate CHD events, HF hospitalizations, and causes of death to support a wide range of studies by investigators beyond our group, and to link the cohort with Medicare data to support investigators conducting health services utilization studies. (Aim 3) To support the development of researchers in CVD epidemiology through a new mentored research program for a cohort of early stage investigators, including analytic and statistical support and an annual 2-day Summer Research Institute of presentations, training, mock study section, and networking. Early stage investigators will graduate with specific competencies in CVD epidemiology research. The proposed grant will inform policy, advocacy, and the design of interventions by generating new evidence on which SDH and disparities in health services lead to population-level disparities in incident and recurrent CHD and HF, and on resilience in recovery after a CHD or HF event. Our multidisciplinary team includes epidemiologists, sociologists, health services researchers, biostatisticians, clinicians, and a participant Advisory Board, assuring rigor and relevance of the proposed research. Continued funding for the REGARDS-MI infrastructure will support a host of additional studies led by an expanding group of investigators. Funding will also expand the cadre of researchers dedicated to discovering strategies to achieve optimal health for all US adults, a goal supported by NHLBI, the National Academy of Sciences, the CDC, the American Heart Association, and all major professional societies in the US.

NIH Research Projects · FY 2025 · 2022-09

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with multiple cancers, including Burkitt lymphoma, Hodgkin lymphoma, primary central nervous system lymphoma and post-transplant lymphomas. EBV-associated B-cell lymphomas occur at significantly higher frequency in the setting of HIV co-infection, even with the use of antiretroviral therapy. AIDS-related lymphomas (ARLs) are most commonly diffuse large B cell lymphomas (DLBCLs), followed by Burkitt lymphoma and classical Hodgkin lymphoma (cHL), all frequently associated with EBV infection. Each of these cancers is linked to a viral latency program that is used as EBV- infected B-cells navigate the B-cell compartment to colonize memory cells, the reservoir for lifelong infection. Yet, much remains to be learned about epigenetic mechanism that control viral oncoprotein expression, and how this can ultimately be exploited in novel therapeutic approaches. We therefore recently performed CRISPR and chemical genetic analysis to identify host factors that tightly regulate the expression of EBV oncoproteins in B-cells. These analyses highlighted host DNA and histone methyltransferases with key roles in regulation of EBV latency and lytic gene expression. Characterization of top screen hits revealed multiple layers of EBV oncoprotein control, yet, how DNA and histone methyltransferases target specific EBV genomic promoter sites remains largely unknown. In parallel, we integrated these studies with tumor genome mutation analysis to identify host genes that are mutated at high frequency in EBV-infected and EBV-uninfected B-cell lymphomas. These analyses recently identified that linker H1 histone genes, which are highly recurrent in B cell lymphomas, are genetic driver mutations in lymphomagenesis. They also demonstrated that histone H1 mutation drives malignant transformation via three-dimensional genome reorganization, resulting in epigenetic reprogramming and de-repression of developmentally silenced genes. Notably, our proteomic analysis identified that EBV strongly downmodulates expression of multiple linker histone 1 isoforms, though it remains unknown how this H1 subversion alters the viral and host genome landscape in EBV-associated lymphomas. We hypothesize that germinal center microenvironment cues orchestrate B-cell epigenetic programs that together with EBV oncoprotein effects on linker histone expression dictate tumor latency programs. While immunological selective pressures also contribute to latency, we propose that T-follicular helper signals and cell intrinsic factors control EBV latency patterns. Our specific aims are therefore to: 1) Identify how key germinal center cytokines affect epigenetic writers to alter the EBV epigenome and dictate latency program selection. 2) Identify dynamic histone H1 roles in EBV epigenome and latency program regulation. 3) Define the relationship between B-cell differentiation state, epigenetic profile, and EBV latency pattern in HIV+ DLBCL. Collectively, these studies address long-standing question in the EBV tumor virology field and lay the foundation for novel therapeutic approaches to EBV-driven human malignancies.

NIH Research Projects · FY 2024 · 2022-09

Despite advances in prenatal and maternal care, the incidence of pregnancy complications and preterm births remains high in the United States. Maternal immune tolerance toward the fetus is critical to promote fetal growth, but the regulatory mechanisms underlying fetal immune cell development remain largely undefined. Interferons are key regulators for placentation, but excessive interferon responses contribute to fetal resorption. In addition, maternal neutropenia is frequently associated with pregnancy complications such as preeclampsia, which has been demonstrated to affect the fetus and newborn adversely. Our understanding of the functions of neutrophils at the maternal-fetal interface, beyond their conventional role in infection, is still very limited. The gut microbiome undergoes drastic changes during pregnancy. However, the role of maternal gut microbiome immune tolerance and fetal immune regulation remains poorly understood. We have developed novel techniques to isolate and characterize placental immune cells in pregnant mice. Built upon our novel discovery of the maternal MDSC- IFNγ axis at the maternal-fetal interface that is orchestrated by the gut microbiome, the current proposal aims to define the role of maternal gut microbiome-driven MDSC-IFNγ axis in fetal and neonatal immune development. Leveraging the methodologies established in our lab to isolate and profile the transcriptome of immune cells, we will use an unbiased and comprehensive approach to fully appreciate functional changes in the fetal immune landscape due to perturbations of the gut microbiome. The transcriptomic studies will be coupled with ex vivo functional studies of tissue immune cells isolated from the placentas and fetal/neonatal intestines, as well as investigation of specific gut bacteria and metabolites that drive the immune cell changes at the maternal-fetal interface. First, we will employ an unbiased approach to profile the transcriptome of the fetal immune cells in the fetal liver and intestine to determine the role of the maternal MDSC-IFNγ axis in the regulation of immune cell development in these fetal tissues. Secondly, we will investigate how lacking maternal MDSCs during gestation impacts the postnatal immune development in the intestine, and investigate immune imprinting of T cells by in utero excessive IFNγ signaling and assess how it impacts the susceptibility to gut inflammation in the offspring. Upon completion, these studies will have defined the role of maternal gut microbiome, via regulation of the MDSC-IFNγ axis, shapes the fetal immune landscape and neonatal immune development in the intestine. Our studies will identify specific fetal immune subsets that are susceptible to perturbed MDSC-IFNγ axis that could be potential therapeutic/interventive targets. The findings from our studies will provide the framework for future studies of targeted manipulation of the gut microbiome to modulate these immune pathways to promote fetal immune cell development.

NIH Research Projects · FY 2024 · 2022-09

Abstract Clonal outgrowths are observed across a wide range of normal human tissues. They also appear during the course of cancer evolution, leading to clonal heterogeneity that fuels the development of treatment-resistant disease. Clones harbor somatic mutations in known cancer driver genes and show evidence of positive selection. Nevertheless, how these driver mutations alter the cellular states of cells to allow clones to outcompete wildtype counterparts remains poorly understood. To date, efforts to chart clonal outgrowths in normal or malignant human tissues have been largely limited to genotyping. This is due to the fact that these clones often affect a minority of cells in a sample without distinguishing cell-surface markers. To address this challenge, we developed an array of multi-omic single-cell technologies that are capable of capturing multiple layers of information (e.g., genotypes, transcriptomes, methylomes, protein expression) from the same single cells. Moreover, we addressed the specific challenge of genotyping in scRNA-seq in single cells at high throughput by developing Genotyping of Targeted loci (GoT). Importantly, GoT turns the admixture of mutant and wildtype hematopoiesis from a limitation to an advantage, enabling the direct comparison of mutant (“winner”) and wildtype (“loser”) cells within the same individual. Given the increasing adoption of our GoT platform, we now aim to extend the multi-omics single-cell toolkit to study how somatic mutations lead to clonal growth advantage. We will integrate GoT with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) to yield GoT-CITE, which will add the critical layer of cell surface marker phenotyping to single-cell whole transcriptomes. As mutations in splicing factors are specifically associated with greater risk of malignant transformation, we will develop and implement GoT- Splice, where long-read sequencing will be used to define splicing variation as a function of cell identity. Given the high frequency of epigenetic mutations in cancer, we will also develop and apply targeted single-cell genotyping in the context of chromatin accessibility (GoT-ChA). Finally, as clone growth will also be determined by its interaction with the microenvironment, to define clonal driver genotypes in its spatial context, we will adapt spatial transcriptomics (ST) to add the critical feature of genotyping (GoT-ST). Our overarching goal is to invoke multi-omic comparisons at the single-cell level between wildtype and mutant cells to comprehensively identify the underpinnings of fitness advantage in clonal outgrowth. The proposed comprehensive GoT toolkit will enable the linking, at high throughout, single-cell genotypes with transcriptional, protein, epigenetic and spatial phenotypes. We anticipate that these advances will transform the study of clonal mosaicism as a harbinger of cancer, as well as resistance to cancer therapies.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Dysregulation of transactivating response region DNA-binding protein-43 (TDP-43) has been linked to many neurodegenerative diseases, including frontotemporal dementia, amyotrophic lateral sclerosis, and Alzheimer’s disease (AD). TDP-43 has a variety of functions linked to its RNA-binding motif, including regulation of transcription, splicing, and RNA transport. Along with these effects, TDP-43 alters expression of interferon (IFN)- related and other immune genes essential for antiviral responses. The relationship between viral pathogens and TDP-43 is bidirectional, as exposure to poly(I:C), which simulates viral pathogens, can promote subcellular mislocalization of TDP-43. Viral pathogens, like TDP-43 dysregulation, are linked to AD and other dementias; AD has been associated with increased presence of viral pathogens, like herpes simplex virus 1 (HSV-1), and altered IFN-related signaling and neuroimmune cascades. Our laboratory found that, like neuronal TDP-43, astrocytic TDP-43 can be mislocalized to the cytoplasm in AD. Dysregulation of astrocytic TDP-43 in mouse models caused neural deficits and cell-autonomous changes in antiviral and IFN-inducible factors. Further, dysregulated TDP-43 increased astrocytic susceptibility to HSV-1. Astrocytic susceptibility to HSV-1 associated with overexpression of human TDP-43 was reduced by blocking the ability of human TDP-43 to bind RNA. Previous studies also show that the RNA-binding domain on TDP-43 is necessary for its other disease-linked effects. Based on this evidence, I will test the hypothesis that dementia-related TDP-43 dysfunction affects antiviral pathways and increases neural susceptibility to HSV-1 by altering TDP-43 binding to host RNA, resulting in aberrant host antiviral and immune gene expression and impaired innate antiviral signaling. I propose to use a variety of cellular and molecular techniques to examine in vitro (Aim 1.1) and in vivo (Aim 1.2) susceptibility to HSV-1 following cell-specific expression of TDP-43 variants that either maintain nuclear localization, mislocalize to the cytoplasm, cannot bind to RNA, or both. I will also determine cell-specific molecular mechanisms that promote differences in antiviral pathways via single-cell RNA sequencing (Aim 2.1), and conduct targeted analysis of alternative splicing (ScISOr-Seq), transposable element expression (TEtranscripts), and protein levels (Western blotting). Finally, I will examine the physiological functions of differential genes of interest identified in Aim 2.1 using genetic and pharmacological approaches. Uncovering the mechanistic links that connect TDP-43 dysregulation to antiviral pathways and viral susceptibility may define new pathobiological mechanisms and therapeutic targets to prevent neurodegenerative disease onset and progression.