Rockefeller University

NIH Research Projects · FY 2026 · 2022-08

Project Summary Cancer immunotherapy leverages a patient’s immune system to recognize and destroy tumor cells. However, a major challenge to current therapeutics is the immunosuppressive nature of the tumor microenvironment that limits the potency of antitumor immune responses. A new approach for overcoming immunosuppression is the delivery of natural signals that stimulate innate immunity and transform tumors into an immunoactivated state to promote the infiltration and cross-priming of cytotoxic T-cells. In humans, the cGAS-STING innate immune pathway controls a potent antitumor response by detecting tumor-derived cytosolic DNA and inducing proinflammatory signaling. Mechanistically, DNA sensing activates the enzyme cGAS (cyclic GMP-AMP synthase) to synthesize the nucleotide second messenger 2′3′-cGAMP which then induces immune signaling through the cyclic dinucleotide receptor STING (STimulator of INterferon Genes). Thus, controlling cGAS-STING activation is an exciting new strategy for stimulating antitumor immunity. The clinical promise of natural and synthetic STING agonists underscores the importance of discovering and defining the function of novel nucleotide second messenger signals that can expand the immunotherapy toolset. For the F99 phase of this proposal, I will describe my discovery that cGAS is part of a large family of diverse immune sensors named “cGAS-like receptors” (cGLRs). The remarkable diversity of uncharacterized cGLRs in humans and animals supports that many new nucleotide signals in innate immunity remain to be discovered. My research identified cGLR1 as a dsRNA sensor in Drosophila that controls an antiviral immune response through the novel nucleotide signal 3′2′-cGAMP, demonstrating that the cGLR enzyme family can sense ligands beyond dsDNA and signal through distinct nucleotide second messengers. My current research investigates how a new Drosophila cGLR responds to a unique molecular pattern and signals through an uncharacterized nucleotide messenger. Ultimately, my research builds a mechanistic framework to define new cGLR signaling pathways in animals and humans and understand the role of diverse nucleotide second messengers in immunity. For the K00 phase of this proposal, I will leverage our expanded knowledge of nucleotide second messenger signaling to discover new molecules that stimulate antitumor immunity. Using a bacterial screening platform to deliver diverse cGAS-like enzymes to tumors I will identify chemically unique nucleotide signals that activate human innate immunity. As part of this new discovery pipeline, I will use in vivo tumor models and a mechanistic dissection of signaling in human cells to define new immune pathways and develop novel immunotherapy strategies. My research will harness the chemical diversity naturally generated by cGAS-like enzymes to expand our immunological toolset for treating cancers. In sum, my proposal will define new animal immune signaling pathways and discover novel antitumor molecules as tools for cancer immunotherapy.

NIH Research Projects · FY 2025 · 2022-08

Project Summary / Abstract A number of recent observations suggest that complex brain functions in the mammalian brain emerge from highly parallel computation in which information about sensory inputs, internal states, and behavioral parameters are mapped onto highly distributed brain-wide neuronal populations. This calls for neurotechnologies that allow for large-scale recording of neuro-activity across tissue depths and brain regions at physiological timescales and cellular resolution in awake and behaving animals. While recent advancements in optical tool development based on the combination of two-photon scanning fluorescence microscopy (2p M) and genetically-encoded calcium indicators (GECIs) as reporters of neuro-activity have been aimed at addressing these needs by developing faster, larger-scale, and volumetric calcium (Ca2+) imaging technologies, a fundamental unsolved challenge in this context is navigating the inherent tradeoffs between speed, resolution, and the size of the recording volume in a principled and scalable manner. Our lab has recently established criteria for such optimal recording schemes which has led to the realization of a new high-speed volumetric Ca2+ imaging approach termed Light Beads Microscopy (LBM). Through LBM, we have demonstrated fluorescence lifetime limited volumetric recording of neuro-activity at a single-cell resolution of up to 1 million neurons within both cortical hemispheres of awake, behaving mice. In this project, we will pursue a multipronged strategy towards the optimization, biological applications, and effective dissemination of our LBM technology while extending its performance. This will result in a more robust, less complex, and more user-friendly version of our LBM technology. To enable its broad and effective dissemination, in the second part of the project, we will utilize feedback from our α-testers to design, build, and disseminate β-prototypes of our system that will be distributed to several end-user laboratories who will be testing and applying our LBM technology in the context of their biological questions. This β-prototype will also form the basis for commercial dissemination of our technology as well as a parallel effort for its open-source dissemination.

NIH Research Projects · FY 2025 · 2022-07

Eukaryotic RNA polymerase II (Pol II) plays a pivotal role in transcription. Normal physiological processes depend upon precise transcriptional controls, whereas transcriptional dysregulation is the basis of numerous pathologies that include cancer. Pol II recruitment to specific promoters is regulated by multiple cofactors that include the multi-subunit Mediator, which directly binds both to enhancer/promoter-bound transcriptional activators and to Pol II to facilitate gene activation. Following initiation and promoter escape, Pol II remains subject to regulation by multiple elongation factors, acting either at Pol II pause-release or productive elongation steps. Pol II(G) is a recently described form of Pol II that contains the tightly associated, metazoan-specific Gdown1 polypeptide along with the normal 12 subunits. Our genetic- based studies of Pol II(G) have demonstrated that Gdown1 is essential for early embryonic development and for cell-specific transcription in quiescent hepatocytes, in which heavy localization to gene bodies of highly expressed liver-specific genes (e.g., albumin) is indicative of elongation functions and in which ablation leads to downregulation of both liver-specific and lipid metabolism genes, cell cycle re-entry and (in the absence of p53) a premalignant type of transformation. Studies in hepatocarcinoma and breast cancer cells have also indicated a key role for Gdown1 in cell growth and in expression of lipid metabolism genes, which are generally important for maintenance of cancer cell growth. Our biochemical studies have revealed that the Pol II-associated Gdown1 conditionally represses basal (activator- and Mediator-independent) transcription initiation by preventing association of TFIIB and TFIIF with Pol II, thereby establishing a potential checkpoint and eliciting a strong requirement for activator-bound Mediator to overcome repression. Our structural studies have defined Gdown1 interaction sites on Pol II and provided clues regarding Mediator interactions that might facilitate its reversal of the conditionally repressed initiation capacity of Pol II(G), although the underlying mechanism remains unclear. With the general objective of understanding the molecular mechanisms of action of Pol II(G) in conjunction with its roles in breast cancer and hepatocarcinoma cells, especially on Gdown1-regulated cell-specific and lipid metabolism genes, as a potential basis for new cancer therapeutics, our specific aims are: (i) to investigate the mechanisms underlying Mediator-dependent transcription initiation and post-initiation events by Pol II(G), including concomitant, newly described interactions with general transcription factors and elongation factor TFIIS, using powerful in vitro transcription and immobilized template assays and CX-MS and cryo-EM structural analyses of interacting complexes and (ii) to investigate Gdown1 functions in hepatocarcinoma cells in promoter-proximal pausing, pause release and transcriptional processivity using (a) a multiomics cell-based approach in conjunction with acute degradation of Gdown1 and (b) biochemical (in vitro reconstitution of these processes with purified factors and recombinant chromatin templates) and structural (CX-MS and cryo-EM) analyses of Pol II(G) elongation factor complexes.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY Combination antiretroviral therapy (ART) revolutionized the treatment and prevention of HIV-1 infection. However, ART does not eradicate established infection and worldwide HIV-1 incidence rates remain high and have been declining slowly. Thus, the search for novel preventive and therapeutic interventions remains a high priority. In recent years, broadly neutralizing antibodies (bNAbs) emerged as a long-acting alternative to daily ART and as a promising strategy to achieve long-term treatment-free HIV-1 control. bNAbs differ from ART in that they engage the host immune system by virtue of their Fc effector domains and therefore have the potential to mediate killing of infected cells and modulate or enhance HIV-specific immune responses. However, bNAbs are vulnerable to escape by HIV-1 variants. During HIV-1 infection, antibody responses co-evolve with a large population of rapidly mutating viruses, such that variants that are resistant to individual antibodies are frequently encountered. Consistent with this high level of diversity, several clinical studies have demonstrated that bNAb monotherapy leads to transient declines in viremia with rapid selection of bNAb-resistant viral strains. In contrast, a combination of two bNAbs targeting non-overlapping Env epitopes maintained viral suppression in participants harboring antibody sensitive viruses who had achieved viral suppression with ART and subsequently received repeated doses of bNAbs during ART interruption. These early studies demonstrate the potential therapeutic application of bNAbs but also highlight the need to better understand viral escape pathways leading to bNAb resistance. Although resistance to some bNAbs (i.e. anti-V3 loop) is predicated on known features of Env, the determinants of resistance are poorly defined for other bNAbs and for combinations of bNAbs. The overarching goals of this proposal are to understand the diversity of pathways leading to bNAb escape and use this information to guide the design of more effective optimized bNAb combinations that prevent emergence of resistant variants. This proposal has four interrelated aims directed at accomplishing these goals: (1) Determine the sequence elements that lead to viral resistance to bNAb administration in humans using newly developed next generation deep sequencing methods; (2) Systematically map all possible viable bNAb resistance mutations to identify mechanisms of escape across viral strains and subtypes by producing and testing complete libraries of Env mutants; (3) Determine the nature of clinically relevant bNAb-resistant HIV-1 variants that can be selected in cell culture in the presence or absence of autologous serum; (4) Develop computational models that define mechanisms of HIV-1 bNAb resistance by integrating the sequence information obtained from Aims 1-3.

NIH Research Projects · FY 2025 · 2022-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. This is a new application to support a pre-doctoral training program in Genetics and Cell Biology at The Rockefeller University, an institution with a rich history in these scientific areas. The mission of the program is to provide trainees with the instruction, experience, and career development support needed for successful careers in genetics and cell biology. Objectives of the program include recruiting outstanding graduate trainees to the training program and providing them with comprehensive didactic training, state-of-the-art research skills, and professional skills needed for careers in the biomedical science workforce. Incorporated into the training plan are recurring training in responsible conduct of research and mechanisms to increase retention and reduce time-to-degree. We propose to support 12 pre-doctoral trainees per year in their second and third year of training: 6 in year two and 6 in year three. The applicant pool is outstanding, including many students with accomplished undergraduate records, extensive research experience and a strong interest in genetics and cell biology. The 49 faculty trainers are accomplished scientists, including 3 Nobel laureates and 19 members of the US National Academy of Sciences, with a shared interest and experience in graduate education. The interdisciplinary nature of the program encourages trainees to perform collaborative work in various areas with different faculty. Trainees would be mentored by the Program Directors; a Program Advisory Committee of selected faculty for general curriculum and research advice; and a Faculty Advisory Committee, specifically designed for each trainee to provide detailed experimental guidance. An External Advisory Committee will evaluate the effectiveness of the program and provide advice on new initiatives. Our past trainees have been highly successful, with most continuing in biomedical research careers. Upon completion, we expect trainees to have a range of scientific knowledge, technical expertise, and professional development skills necessary to become leaders in their chosen field.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY Adult neurogenesis is emerging as an important player in maintaining brain homeostasis and normal functions. The dysfunctions of neurogenesis have been associated with aging and neurological disorders, including Alzheimer’s disease (AD). The ability to systematically map the molecular dynamics of neurogenesis at single- cell resolution could serve as a foundation for a systematic effort to better understand the molecular events that give rise to abnormal cell states in aging and diseases. While the rapid advances in single-cell genomics are creating unprecedented opportunities to explore molecular heterogeneity in mammalian brains, nearly all such methods are restricted to low throughput and fail to recover the heterogeneity and dynamics of the profoundly rare cell states in adult neurogenesis (e.g., less than 0.1% of the cell population in the brain). Herein, we propose to develop novel methodologies that enable a comprehensive view of temporal-spatial dynamics of neurogenesis during aging and Alzheimer's disease (AD) in both human and mouse brains. Specifically, we will first develop a novel high-throughput, low-cost single-cell genomics approach, sciNext1000, to profile the molecular heterogeneity of four million cells from post-mortem human hippocampal samples. This approach will be powerful because we can not only quantitatively characterize the frequency of human adult hippocampal neurogenesis at single-cell resolution, but also identify the transcriptome features associated with impaired neurogenesis in aging and AD at isoform resolution. In addition, we will develop another novel single-cell genomic technique, sci-Div- seq, to enhance the detection of newborn neurons, and identify the cellular differentiation trajectories and associated transcriptomic features of adult neurogenesis in young and aged mouse brains. The resulting dataset will advance our understanding of gene regulation in neurogenesis across different neural lineages and constitute a significant step towards a comprehensive characterization of the molecular mechanism underlying neurogenesis impairment in aging. In addition to the internal molecular programs, the neurogenesis process is controlled by aspects of environmental signals from the neural stem niche. We will apply a high-throughput spatial transcriptomic strategy to identify the cellular interactions and local microenvironment involved in adult neurogenesis in both human and mouse brains. These multi-pronged approaches will open a new paradigm for understanding the global molecular programs and environmental regulation of adult neurogenesis, thereby informing potential therapeutic targets to restore cell population homeostasis in aging and brain disorders.

NIH Research Projects · FY 2026 · 2022-02

The current pandemic has highlighted fundamental gaps in our knowledge about the replication strategies of coronaviruses, and how these are affected by the host at both the organismal and cellular level. There is a pressing need to understand how SARS-CoV-2 infection and host-cell responses trigger such a diverse set of pathologies, and the roles played by viral variation, host genetics and underlying preconditions. As studies of SARS-CoV-2 frequently utilize population-based assays that look hours to days post infection, information on cellular and spatial variability are lost. Furthermore, host responses are communicative spatial processes subject to signaling gradients that vary between cells. Thus, averages over populations obscure heterogeneity and spatial separations, and miss the earliest viral and host behaviors due to lack of sensitivity. To fill this gap, we developed experimental and computational approaches to quantify individual virion entrance, establishment of the first replicative events, and production of viral RNAs and host responses in single cells, all while maintaining sample spatial integrity. This project's long-term objective is to apply this novel approach to gain insights into SARS-CoV-2 biology distinct from those gleaned using traditional strategies. This knowledge will provide new insight into the spectrum of COVID-19 disease outcomes and help guide future therapeutic strategies. To this end, single-molecule in situ analyses, including single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) will be applied to the study of SARS-CoV-2. Aim 1 will quantify SARS-CoV-2 entry, replication and spread, and host transcriptional responses in cells of varying tissue origin. These data will be used to develop a stochastic computational model to address the determinants of early viral replication and the resulting cellular response. Aim 2 will examine the effect of host mutations or pre-existing conditions that affect the type I interferon (IFN) response and have been associated with severe COVID-19, as well as emerging viral Variants of Concern. Aim 3 will use mouse models, single cell epigenomics, primary cell culture systems and superinfection model systems in vivo to study long-term effects of respiratory infections, including SARS-CoV-2, influenza A virus and helminth pathogens, and particular the phenomenon, with a particular focus on antigen-independent innate immune memory. Together, our multidisciplinary approach utilizing techniques and information from systems-level virology, spatial transcriptomics, host genetics, computational biology, and innate immunity provides a powerful means of probing questions central to understanding clinical outcome, long-term effects and informing life-saving interventions.

NIH Research Projects · FY 2026 · 2022-01

Encephalitic flavivirus infections affect thousands of people globally every year causing acute encephalomyelitis and placing significant burden on healthcare systems. Currently, no virus-specific treatments are available for these life-threatening conditions. The central nervous system (CNS) encompasses dozens of cell types with diverse properties and functions. Limited by a lack of adequate tools that combine throughput, depth and resolution, the interactions between viruses and this complex environment remain largely a mystery. To complicate matters, the CNS is also extensively connected to the periphery by both physical neural projections and peripheral immune signaling. Our preliminary studies demonstrate that tropism of the important encephalitic virus West Nile (WNV) within the CNS after direct intracranial inoculation of mice differs from that seen after spread to the CNS following peripheral infection. We hypothesize that CNS tropism is largely determined by resident neural and glial innate immune profiles, which can be readily modified by immune signals generated during peripheral infection. To address this hypothesis, we will utilize WNV to study immune interactions between cell types in an in vivo mouse model and in vitro using human induced pluripotent stem cell (hPSC) models. Tissue clearing techniques and whole mount imaging will be used to visualize viral antigens across the entire brain and spinal cord using light sheet microscopy, creating a complete time-resolved 3-dimensional map of infection. Responses of single cells will be examined using a combination of cutting-edge nuclear RNA sequencing and microscopy-based spatial transcriptomics. Co-cultures of hPSC-derived neurons and glia will be interrogated using high-throughput microscopy and sequencing to identify resistance factors and responses in human cell types. Hits will be mechanistically studied using blocking antibodies and CRISPR-mediated knockouts. Lastly, by using systemic and cell-type specific knock out animals or cytokine neutralizing antibodies, we will investigate the role of type I interferon in modulating CNS tropism and disease. This project will provide new data on flaviviral encephalitis at unparalleled resolution to help bridge current information gaps and improve fundamental knowledge by defining cellular tropism and CNS inflammatory responses at the single cell level and evaluating how changes in peripheral signaling influence infection of the brain. Identified peripheral factors restricting CNS infection are possible targets for immunomodulatory therapy, thus promoting research that may improve treatment for other forms of viral encephalitis. Finally, the resulting experimental pipeline will be broadly applicable to the study of CNS stress and inflammation, with relevance to other diseases like Lyme neuroborreliosis or chronic debilitating conditions like traumatic brain injury.

NIH Research Projects · FY 2025 · 2021-09

Project Summary: Metastatic disease is a complex, dynamic and emergent process that requires collective and coordinated interactions between many cell types, metabolites and the host. There is substantial clinico- pathologic and experimental evidence for critical roles of neural innervation, lymphatic interactions, metabolites and endothelial cells in regulating metastatic progression by altering cancer and immune cell functions. As such, these cellular interactions likely shape metastatic progression, responses to therapy and metastatic dissemination. However, we have a limited understanding of how these components coordinately regulate metastatic progression. This application describes a series of highly innovative multidisciplinary molecular, cell- biological, metabolic, massively-parallel single-cell sequencing and organismal methods applied towards defining the dynamic and emergent mechanisms by which neural cells, lymphatics, immune cells and metabolites interact to coordinately regulate metastatic progression—contributing to a systems-level understanding of metastasis. We aim to (i) define the role of neural innervation on metastatic progression by characterizing neuro- tumor and neuro-immune interactions and identifying neural signals and their pro-metastatic mechanisms of action, (ii) determine how endothelial cells regulate innervation of metastatic tumors, (iii) define the role of regionalized lymphatic interactions in driving metastatic progression and anti-metastatic immunity, (iv) assess the role of neuro-immune and neuro-epithelial interactions on early metastatic dissemination and colonization, (v) identify metabolite and protein signals that drive metastatic colonization, (vi) discover tumoral transcription factors and RNA-binding proteins that act downstream of neural and metabolic signals to drive emergent pro- metastatic gene expression programs, and (vii) determine the impact of standard chemotherapy on these diverse cellular interactions and metabolic determinants of metastatic progression. Our proposed MetNet Center will enhance our understanding of how interactions and crosstalk between cancer cells with nervous system cells, lymphatics, vasculature and immune cells enables emergence of metastatic disease. We will also assess how therapy impacts specific cell-cell and metabolic interactions of metastatic cells and provide insights into the impact of specific cellular interactions in the primary microenvironment on metastatic dissemination, including early dissemination. These findings will generate an integrated, systems-level understanding of metastasis, enabling development of a new generation of anti-cancer therapies that prevent critical emergent coordinated pro-metastatic interactions.

NIH Research Projects · FY 2024 · 2021-09

Project Summary In each cell cycle, DNA replication machinery encounters replication fork barriers including DNA lesions, secondary structure-forming repetitive sequences, and transcriptional machinery. Oncogenic transformation also perturbs normal replication and results in replication fork dysfunction commonly referred to as replication stress. Response to replication stress is an essential aspect of the DNA damage response in cells, and the consequences of inappropriate response results in genome instability and cancer. We have recently identified a novel regulatory pathway that is required for the protection of stalled replication forks and recovery from replication stress. We showed that the mammalian replisome contains a previously unidentified and completely unstudied protein, RTF2 (Replication Termination Factor 2), which must be removed for proper response to replication stress. We showed that RTF2 is removed from stalled forks in a process that is dependent on the proteasomal shuttle proteins DDI1 and DDI2, which interact with RTF2 and the proteasome. Persistence of RTF2 at stalled forks resulted in replication fork restart defects, hyperactivation of the DNA damage signaling, accumulation of single stranded DNA, sensitivity to replication drugs including hydroxyurea and aphidicolin, and chromosome instability. Our results establish that removal of RTF2 is necessary for cells to manage replication stress and maintain genome integrity. The first goal of the proposed studies is to fully understand how RTF2 functions during DNA replication. To this end, we will fully characterize replication without RTF2, using a conditional knockout mouse and cell model, and identify the mechanism of how RTF2 regulates DNA replication during unperturbed conditions. The second goal is to determine how RTF2 is itself regulated under replication stress and why it needs to be removed from the replisome. RTF2 ubiquitination is necessary for interaction with DDI1/2, thus we will identify the regulatory network of this ubiquitination and subsequent removal of RTF2 from the replication fork. The final goal in this project, is to leverage the idea that the removal of proteins during DNA damage response is as equally important as recruitment of DNA repair proteins to sites of DNA damage. Most published studies have concentrated on proteins traveling or recruited to sites of DNA damage. However, our work on DDIs and RTF2 suggests a large component of the DNA damage response network is missing, i.e. proteins that must be removed from the sites of damage to allow for proper DNA damage response and repair. In order to identify other proteins removed during replication stress, we will use an approach similar to the one we used for our DDI studies and detect proteins inappropriately enriched at stressed replication forks using a recently-developed technique, Isolation of Proteins On Nascent DNA (iPOND). We envision that our studies will identify yet unknown regulatory networks essential during the DNA damage response that prevents development of cancer-causing genome instability.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY The functions of mammalian organs are maintained by the behaviors and dynamics of individual cells. The ability to systematically map each cell type's temporal dynamics is central to the understanding of many aspects of biological changes that mammals undergo in development. However, conventional methods are restricted by inadequate throughput and the limited range of cellular contents that can be measured. While single-cell genomic techniques have been developed to characterize cell state heterogeneity with high resolution, nearly all such methods capture only a static snapshot at a single time point, with both temporal and spatial information lost during cell isolation. Herein, the proposed projects aim to develop novel methodologies that enable a comprehensive view of single-cell spatiotemporal dynamics across the lifespan of an entire mammalian organism. Specifically, I will expand on the high-throughput single-cell RNA-seq platform (sci-RNA- seq), to develop a novel method for concurrently profiling transcriptome, epigenome, and cellular temporal dynamics (e.g., proliferation, apoptosis) in each of millions of cells. The technique will be employed to investigate how aging regulates the status of a whole mammalian body by systematically monitoring single cell state dynamics across a broad range of tissues in young and aged mice. This approach will be powerful because we can not only visualize in-vivo proliferation and apoptosis behaviors of each cell type but also dissect its connection with internal transcriptome/epigenome states. In addition to the internal molecular programs, cell state dynamics are controlled by aspects of tissue architecture such as cell-cell interactions and extracellular matrix abundance. To profile single cell microenvironment with high throughput and accuracy, we will develop a novel technique called "microtissue-seq", for co-profiling single-cell molecular contents, cellular spatial interactions, and extracellular matrix (ECM) proteins across tens of thousands of spatial locations in a single experiment. We will employ this technique to interrogate how cellular microenvironment regulates organismal-scale cell state dynamics in different age groups of mice. Overall, the proposed projects will establish a technical framework for comprehensive profiling single-cell spatiotemporal dynamics at an unprecedented scale of a whole mammalian organism. By profiling cell-state specific dynamic behaviors across the lifespan of mice, these technologies and experiments would uniquely enable accurate modeling of the exquisite program underlying mammalian system maintenance and breakdown with age at single cell resolution. These multi-pronged approaches also open a new paradigm for understanding the global molecular programs regulating cell states and dynamics during aging, thereby informing potential pathways to delay the aging process as well as the rational design of effective therapies to restore tissue homeostasis for patients with aging-related diseases.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Social insects show robust and complex behaviors, and have served as important study systems in ethology for decades. However, because they are not genetically tractable, researchers have not been able to study these behaviors at the level of brain circuitry with cutting-edge neurogenetic tools. The proposed work will pioneer such tools in the clonal raider ant Ooceraea biroi, a species that uniquely combines experimental amenability with the fascinating behavior of social insects. It will then address an important biological question: how does variation in neural responsiveness give rise to consistent differences in how individuals respond to social and environmental stimuli? O. biroi is particularly suitable to study this question for a number of reasons. First, the ants reproduce asexually and clonally, implying that behavioral differences arise from phenotypic plasticity, rather than genetic differences. Second, unlike in conventional model systems like Drosophila or mice, differences in behavioral propensities are adaptive because they give rise to stable division of labor in a colony context. Accordingly, these differences are robust and predictable, and they have received a lot of theoretical and empirical attention at the behavioral level. Given that ants communicate almost exclusively via pheromones, we will focus on the antennal lobe, the primary processing area of chemosensory information in the insect brain, analogous to the mammalian olfactory bulb. In Aim 1, we will generate transgenic lines expressing the genetically encoded calcium indicator GCaMP in the antennal lobe to enable live imaging of neural activity with two-photon microscopy. We will also generate lines expressing the photoactivatable fluorescent protein CaMPARI2, enabling stable labeling of neurons active in freely behaving animals. In Aim 2, we will use these tools to create a functionally annotated map of chemosensory representation in the ant antennal lobe. We will also use single-cell RNA-sequencing of labelled neurons to identify odorant receptors responding to pheromones. We will then use the promoters of these receptors to generate additional, narrowly targeted transgenic lines. In Aim 3, we will study how differences in neural representation and sensitivity correlate with plastic differences in behavioral responses to identical social stimuli. Based on these data, we will build a predictive theoretical model of division of labor in insect societies. On a fundamental level, our results on the modulation of sensory perception will also inform our understanding of human disorders involving abnormal sensory sensitivity, such as autism and schizophrenia. Finally, we will make the tools and protocols developed under this proposal available to the scientific community, greatly advancing the field of social insect neuroscience and opening up a vast new experimental space. The robust and expansive behavioral repertoire of social insects combined with the simplicity of a compact invertebrate nervous system allows O. biroi to fill an important niche in neuroscience.

NIH Research Projects · FY 2025 · 2021-08

Project Summary COVID-19, caused by the coronavirus SARS-CoV-2, continues to devastate the world. In less than a year, there have been more than 20 million cases with over 700,000 deaths. The viral RNA-dependent RNA polymerase (RdRp) is the central enzyme responsible for transcription and replication of the viral RNA genome. This enzyme is also a target for the current antiviral, remdesivir, used to ameliorate the severity and duration of this disease. The virus also encodes several nucleic acid processing enzymes, in addition to the RdRp, including a helicase, an endonuclease, an exonuclease, and methyltransferases. However, it is unknown how these enzymes coordinate to transcribe and replicate the viral genome. This proposal builds upon preliminary data of the structure of the helicase, nsp13, in complex with the RdRp and a primed substrate RNA (nsp13-replication/transcription complex or nsp13-RTC). The aims here include completing the structural analysis of this complex by utilizing additional data collected. The result of this aim will provide higher resolution (better than 2.7 Å in some parts the RdRp), providing a rich basis for the development of antiviral inhibitors. Also, having this structure in hand allows for the collaboration with expert developers of antimicrobials, also part of the aims, including the investigation of the structural details of the pre-incorporation state of remdesivir and antivirals produced by human microbiome. The models resulting from the structure of nsp13-RTC serve as foundations to test how the helicase and exonuclease function together with the RdRp. Specifically, real-time fluorescence assays, single-molecule fluorescence resonance energy transfer (FRET), and multi-color fluorescence microscopy will be used to probe the role of the helicase and the exonuclease in unwinding substrate RNA, backtracking, and proofreading. Another aim applies the pipeline used to characterize the nsp13-RTC assembly, which yielded a high- resolution structure of the complex, to other RTC assemblies. Specifically, native electrophoretic mobility assays will be used as a starting point to probe larger assemblies of the RTC. Native mass-spectrometry will then be used to determine the composition and stoichiometry of the complexes. Finally, cryo-EM will be applied to solve the structures of these macromolecular machines. The resulting structures will provide a starting point to elucidate the coordinated functions of these enzymes, provide insight into their mechanisms, and establish novel targets for therapeutics. In summary, this proposal aims to understand at the molecular and structural level how the SARS-CoV-2 nucleic acid processing enzymes coordinate to replicate and transcribe the viral genome, and to provide structure-guided targets for drug discovery, with the ultimate goal of providing relief for the COVID-19 pandemic.

NIH Research Projects · FY 2025 · 2021-08

Our long-term goal is to understand the molecular basis of a novel morphologically-conserved non-apoptotic developmental cell-death program we uncovered, and to determine its roles in mammalian development and disease. Programmed cell death is a major cell fate. Apoptosis, an extensively studied cell death process, requires caspase proteases and is accompanied by chromatin compaction and cytoplasmic shrinkage. Surprisingly, mice lacking apoptotic effectors survive to adulthood. These observations suggest that non- apoptotic cell death may play key roles in animal development. Although genes promoting necrotic cell death have been described, these are not required for development. Thus, whether alternative developmental cell death pathways exist, and if so, what molecular mechanisms govern their execution, is a major outstanding question. Our studies of the C. elegans linker cell provide direct evidence that caspase-independent non- apoptotic cell death pathways operate during animal development. Linker cell death occurs in the absence of C. elegans caspases, and other apoptosis genes are also not required, nor are genes implicated in autophagy or necrosis. The morphology of a dying linker cell is characterized by lack of chromatin condensation, a crenellated nucleus, and swelling of cytoplasmic organelles. Remarkably, cell death with similar features (linker cell-type death, LCD) also occurs in vertebrates, and is characteristic of neuronal degeneration in polyglutamine diseases. We recently described a pathway governing C. elegans LCD. This is the first such framework for a non-apoptotic developmental cell-death program. LCD is controlled by Wnt signals that function in parallel with a developmental-timing and a MAPKK pathway to control non-canonical activity of HSF-1, a conserved heat-shock transcription factor. let-70/Ube2D2, encoding a conserved E2 ubiquitin- conjugating enzyme, is a key target of HSF-1. The E3 components CUL-3/cullin, RBX-1, BTBD-2, and EBAX-1 function with LET-70/UBE2D2 for LCD. Our recent evidence suggests that histone methylation may be a target of this pathway, likely resulting in genome-wide chromatin opening, allowing nuclease access and DNA degradation. LCD pathway components promote vertebrate cell-degenerative processes. pqn-41, a glutamine- rich protein, is reminiscent of polyQ proteins causing neurodegeneration. and tir-1/Sarm and BTBD-2 promote distal axon degeneration following axotomy, supporting conserved cell dismantling roles. We recently showed that treatment of mammalian cells with the kinase inhibitor staurosporin (STS) causes LCD like death. Here we will build on these studies to uncover LCD pathway targets, and study relevance to mammals. We will: (1) Investigate the role of SAMS-4, a BTBD-2 target, and NUC-1, a DNaseII enzyme, in LCD, and test an hypothesized pathway for these in chromatin modification and DNA degradation. (2) Identify EBAX-1 target genes and assess roles in LCD control. (3) Characterize STS-induced death in mammalian cells, define conservation with C. elegans LCD, and identify relevant genes.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT The common marmoset provides a very relevant primate model for understanding the organization of the human nervous system and the diseases that affect it. Like humans, marmosets also demonstrate cooperative social behavior and have advanced cognitive processes, making them of great interest in the field for modeling developmental and psychiatric diseases and their therapies. They are also ideal for multigenerational genetic experiments as they give birth twice a year and mature faster than most primates. However, while the CRISPR/Cas9 system has been used to knockout genes and create knock-ins of single amino acids in a heritable manner in marmosets, it has been a challenge in the field to create germline transmissible models of gene reporters and trinucleotide repeat genes analogous to their murine counterparts. The very low efficiency of homologous recombination (HR) in primates has precluded knocking-in coding sequences by simply injecting Cas9 protein and a guide RNA into embryos during in vitro fertilization (IVF) as is done for creating knockouts. This limitation has prevented modelling of more genetically complex neurological diseases such as Huntington’s disease (HD) and for creating conditional reporters in marmosets, both of which are mainstays in the mouse neurogenetics field. In addition to low HR frequency, other barriers to creating germline transmission of knock- ins include the absence of a well annotated marmoset genome until recently, lack of protocols for derivation of ground state marmoset pluripotent stem cells (cjPSCs), the low percentage of marmoset pregnancies after embryo reimplantation, and a general deficiency of developmental biology expertise in the marmoset field. We propose to harness our labs’ expertise in developmental biology, IVF technologies, and transgenic stem cell biology to overcome this barrier to widespread use of marmosets. We aim to create transgenic knock-in cjPSCs, convert them into ground-state pluripotent stem cells and then inject them into IVF morula to create a chimeric founder marmoset that carries the modified genome. We then aim to screen the transgenic gametes from the founder marmosets to create the F1 progeny and use them to correlate the molecular-behavioral phenotype of HD. As proof-of-principle, we will focus on three knock-in reporter lines to broadly target excitatory, inhibitory, and peripheral neuronal populations. Together, if successful, our aims will result in creation of the first primate model with neuron-specific reporters, establish the marmoset as a valid model of HD, enable access to single- cell transcriptomic changes at the early stages of HD in a primate disease model, and finally correlate these molecular changes with the behavioral phenotype. These aims will provide fundamental insights into the biology of HD and the role of huntingtin protein in different classes of neurons. The outcome of this project will also influence a better understanding of poly-glutamine neurodegenerative diseases that affect humans. In addition, the transgenic marmosets that we generate will be broadly available to the research community and enable new studies into neural circuits, development, behavior, and a wide range of optogenetic applications.

NIH Research Projects · FY 2025 · 2021-07

White and brown adipocytes not only play a central role in energy storage and combustion, but are also dynamic secretory cells that produce signaling molecules that link levels of energy stores to other vital physiological systems. Disruption of the signaling properties of adipocytes, as occurs in obesity, contributes to insulin resistance, type 2 diabetes, and other metabolic disorders. Fat cells have been estimated to secrete more than 1,000 polypeptides and microproteins and even large number of small molecule metabolites. The great majority of the adipocyte secretome has not been defined or characterized and addressing this gap in knowledge is the main goal of this collaborative, interdisciplinary team project. A major obstacle has been the lack of suitable technologies to quantitatively identify circulating proteins and metabolites, determine their cellular origin, and elucidate their function. Building on compelling preliminary data and key innovations from members of this team, we will generate the first encyclopedia of the white and brown adipocyte secretome in mouse models and humans. Specifically, we will (1) Generate an encyclopedia of the secretome of murine adipocytes, (2) Characterize the adipocyte secretome in response to physiological stress and in pathological states, (3) Characterize the adipose secretome in humans, and (4) Characterize the function of the adipocyte secretome. These studies, which span from basic biology to human subject investigation will only be possible by optimizing tools within diverse disciplines and at their intersection. This project has the potential to address questions central to the mission of the NIDDK such as the molecular basis for the links between obesity and type 2 diabetes and understanding whether the anti-diabetic benefits of brown fat are conveyed by secreted mediators. Our studies have the potential to identify new secreted mediators with roles in obesity, type 2 diabetes and metabolic diseases, catalyze the development of new technologies, provide a crucial new resource for researchers and clinicians, and lead to new biomarkers and therapies.

NIH Research Projects · FY 2025 · 2021-07

Project Summary There is immense interindividual clinical variability in humans infected with SARS-CoV-2, ranging from silent infection to lethal COVID-19. The first breakthrough to crack this enigma came from the field of inborn errors of immunity (IEI). In an international cohort of 659 patients, we reported 23 patients with IEIs at eight influenza susceptibility loci that govern TLR3- and IRF7-dependent type I interferon (IFN) immunity (3.5%), including four unrelated patients with autosomal recessive IRF7 or IFNAR1 deficiency. We also reported an additional 101 patients with neutralizing autoantibodies (auto-Abs) against type I IFN (10.2% of 987), who were auto-immune phenocopies of the patients with IEI. Interestingly, 94% of the patients with auto-Ab against type I IFN were men, and one of the six sick women had X-linked dominant incontinentia pigmenti (IP), suggesting X-linked inheritance in at least some of the patients. Collectively, these patients account for about 13.5% of life-threatening COVID- 19 cases studied. We now hypothesize that other IEI that result in abnormal (i) production or amplification of type I IFN, (ii) activity of soluble type I IFNs (via neutralizing auto-Abs), or (iii) response to type I IFN (in terms of interferon stimulated gene (ISG) activity), can underlie life-threatening COVID-19 in other patients. To tackle these three specific aims, we benefit from an international recruitment from the COVID Human Genetic Effort (https://www.covidhge.com). Our preliminary data are very strong. First, we have found 215 patients with predicted loss-of-function (pLOF) variants at 157 loci associated with production or amplification of type I IFN, including one patient homozygous for a pLOF variants in NLRC3, two patients heterozygous for pLOF variants in DDX58/RIG-I, and six patients heterozygous for pLOF variants in subtypes of type I or III IFNs. Second, among patients with auto-Ab against type I IFN, we identified a patient hemizygous for a pLOF in X-linked SASH3. In addition, we found that 25% of patients with IP, which is associated with severely skewed X-inactivation, have auto-Ab against type I IFN, further suggesting an X-linked basis of auto-Ab to type I IFN production. Third, we found 24 patients with pLOF variants in 18 ISGs. We have shown that the international path-breaking program we established in only 6 months is highly efficient, as it resulted in a paradigm-shifting discovery. Our new program will benefit from this momentum. Our future discoveries of new inborn errors of type I IFN immunity underlying life-threatening COVID-19 pneumonia will pave the way for new diagnostic and therapeutic strategies to better manage patients infected with SARS-CoV-2 at risk of severe disease. Selected patients may benefit from subcutaneous or nebulized IFN-a or IFN-b (defect in type I IFN production or amplification), plasmapheresis and/or B cell depletion (neutralizing auto-Abs against type I IFNs), or other therapies, including mAbs against SARS-CoV-2 (defects of ISGs).

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Although the past decade was marked by the tremendous success of immunotherapeutics in oncology, few have provided significant benefit to patients with pancreatic cancer. The long-term goal of this project is to develop therapeutically useful immunotherapies for the clinical treatment of pancreatic cancer. The overall objectives in this application are to (i) characterize the role for MARCO in the TME, (ii) to evaluate the therapeutic efficacy of antibodies blocking this receptor, and (iii) improve antigen presentation within the pancreatic TME with a combination of Fc-enhanced antibodies targeting MARCO and CD40. Our central hypothesis is that pancreatic cancer repolarizes tumor associated macrophages to an M2 phenotype an this can be reversed through engagement of MARCO with antibodies, therefore promoting anti-tumor responses. The rationale for this project is that a determination of the immunosuppressive role of TAMs expressing MARCO in pancreatic cancer will likely offer a strong scientific framework whereby new immunotherapies can be developed. The central hypothesis will be tested by pursuing three specific aims: 1) Develop anti-human TAM antibodies for cancer immunotherapy and set up novel assays for large-scale evaluation of anti-TAM antibodies, 2) Define the role for MARCO in the polarization of TAMs in pancreatic cancer, and 3) Augment TAM repolarization in the pancreatic cancer TME using the Fc-enhanced CD40 agonist antibody 2141-V11, alone or in combination with anti- hMARCO antibodies.. Under the first aim, novel antibodies will be generated against human MARCO and optimized for Fc-receptor binding. In the second Aim, mice genetically engineered to express human MARCO will be studied for their role in the TME of pancreatic cancer. Following characterization immune cells expressing MARCO that can be targeted by antibody therapy, a panel of antibody variants will be tested to determine the Fc-requirements for optimal in vivo activity. MARCO expression and characterization of the immune microenvironment will also be evaluated in human pancreatic cancer specimens. Finally, in the third aim, we will use knowledge gained in Aims 1 and 2 to test novel combinations targeting the myeloid axis in pancreatic tumors. Here, the anti-tumor activity of the Fc-enhanced anti-CD40 antibody will be tested alone or in combination with anti-MARCO antibodies in pancreatic cancer models. The research proposed in this application is innovative, in the investigator’s opinion, because it focuses on defining novel pathways on myeloid cells contributing to the immune suppressive TME of pancreatic cancer, while identifying lead therapeutic candidates through Fc- engineering. The proposed research is significant because it is expected to provide strong scientific justification for the development of macrophage targeting immunotherapies. Ultimately, such knowledge has the potential of offering new opportunities for the translation of innovative therapies to the treatment of pancreatic cancer.

NIH Research Projects · FY 2025 · 2021-04

Project Summary / Abstract This grant focuses on how very recent experiences––over the past few seconds to minutes––allow brains to update expectations about the world and then use these expectations to guide behavior. The ability to flexibly adjust one's course of action in this manner is a hallmark of adaptive human behavior. At the neural level, relevant cellular-activity correlates have been described in non-human primates and other vertebrate model systems. For example, ramping neural activity has been observed in the few hundred milliseconds, or seconds, leading up to behavioral decisions and the rate of rise of these ramps tracks the gradual accumulation of information relevant for the decision being made. Ramping activity is thus a correlate of an increasing expectation that an important decision needs to be made and the moment at which the ramp reaches a threshold level typically signifies when a final decision is taken. Another salient correlate of internal expectations are reward-prediction error signals: bursts of dopamine-neuron activity when an animal receives an unexpected reward or a reward is surprisingly omitted. Reward-prediction error signals seem well poised to adjust animal and human behavior based on learned expectations. A clearer picture of how quantitative internal signals––like ramping and reward-prediction error activity––contribute to behavioral flexibility would be an important step forward for cognitive neuroscience. Here, we propose to develop two new behavioral tasks in tethered Drosophila, where we can perform simultaneous neurophysiology. Our first aim is to use one of these tasks to test the hypothesis that ramping neural signals are fundamental in forming behavioral decisions over tens-of-seconds to minutes timescales in ethologically relevant contexts, rather than just on much shorter timescales and in laboratory defined tasks (as has been shown to date). Such a discovery would argue that expectations built over minutes in real-world conditions are ultimately fed into slowly ramping neuronal signals so as to guide natural decision-making. Our second aim is to discover reward-prediction error signals in fruit flies actively performing a trial-by-trial conditioning task and to define a circuit mechanism through which such signals allow brains to form quantitatively precise expectations––updated on a trial-by-trial basis––on the likelihood of rewards arriving or not arriving in the near future. Such discoveries in a genetically tractable model will inform our thinking on how our brains generate expectations that allow for flexible, adaptive behaviors, ultimately informing new therapeutic approaches to neurological conditions in which flexibility is impaired, such as obsessive-compulsive disorder.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY The proposed research addresses a critically important question in autism spectrum disorder (ASD) research: how defects in cerebellar circuits contribute to ASD. In particular, it examines the role of the predominantly cerebellar gene ASTN2 in cerebellar circuit function and ASD-related behaviors. Copy number variations (CNVs) in ASTN2 have been identified as a significant risk factor for ASD (Lionel et al, 2014), suggesting that ASTN2 mutations such as those found in patients with ASTN2 CNVs, lead to altered cerebellar synaptic function. In addition, we recently reported a family with a paternally inherited intragenic ASTN2 duplication, which caused a heterozygous loss of function of ASTN2. The family manifested a range of neurodevelopmental disorders, including ASD, learning difficulties and speech and language delay (Behesti et al, 2018). Our cellular and molecular studies on mouse cerebellum show that ASTN2 binds to and regulates the trafficking of multiple synaptic proteins, including Neuroligins, which have been genetically linked to ASDs, and modulates cerebellar Purkinje cell (PC) synaptic activity (Behesti et al, 2018). To provide a genetic model to study cerebellar circuit function, we generated both a global loss of function Astn2 line and a floxed Astn2 line for conditional knockout experiments. New, preliminary evidence indicates that PCs in mice lacking Astn2 have a decrease in evoked excitation relative to inhibition in PCs and reduced PC dendritic spine density, suggesting specific cerebellar circuit defects. In addition, preliminary evidence shows mild motor deficits and defects in USVs and an open field assay, ASD-related behaviors. As other preliminary findings do not indicate major defects in cerebellar development, we hypothesize that the behavioral defects we observed relate to defects in the cerebellar circuitry with underlying changes in receptor trafficking. In the proposed research, we will 1) test how loss of Astn2 alters intrinsic excitability in PCs and the synaptic efficacy of their presynaptic inputs from GCs and molecular layer interneurons, 2) use proteomics to identify changes in the levels of synaptic proteins and live imaging to assess whether such changes relate to changes in the rate of endocytosis, 3) compare changes in PC dendritic branching as well as the regional distribution of PC spines in wild type and mutant animals to provide insight on whether there are changes in the organization of PC inputs during the establishment of the cerebellar circuitry, and 4) analyze changes in social behavior and ultrasonic vocalization in Astn2 wild type, heterozygous and mutant animals. Taken together, the proposed research will provide a new mouse model that allows us to link an ASD-related gene that is predominantly expressed in the cerebellum with specific cerebellar circuit function and molecular pathways.

NIH Research Projects · FY 2024 · 2020-09

The role and mechanism of action of Vpr (Viral Protein R), an accessory protein encoded by HIV- 1, has been enigmatic for decades. Vpr causes cell cycle arrest at G2/M, triggers a DNA damage response, and enhances viral gene expression. It exerts these activities by targeting host protein(s) for degradation, hijacking cullin4-based E3 ubiquitin ligase complex (CRL4) to induce their depletion. We recently identified a host protein CCDC137, also known as cPERP-B, as a key target protein depleted by Vpr in a CRL4 complex dependent manner. Specifically, CCDC137 depletion by RNA interference recapitulates the aforementioned effects of Vpr on host and virus. In this project we seek to study the molecular details of how CCDC137 represses HIV-1 gene expression as well as how it controls cell cycle progression and the DNA damage response. In Aim 1, we will determine whether CCDC137 depletion is a conserved feature of Vpr proteins from diverse HIV and SIV strains, map the CCDC137 determinants required for Vpr-induced depletion, and assess the effect of Vpr from diverse viruses on viral gene expression. In addition, we will define host proteins required for CCDC137 depletion by Vpr. Aim 2 is centered on the mechanisms of CCDC137-mediated repression of HIV-1 gene expression. We will delineate cis- acting sequences required for CCDC137-mediated repression and evaluate the effect of integration and integration site selection on the Vpr/CCDC137-regulated HIV-1 gene expression. We will also combine screening methods (proteomics, yeast 2-hybrid, and CRISPR functional screens) to identify CCDC137 interacting cofactor(s) to illuminate the mechanism of how CCDC137 inhibits HIV-1 gene expression. In Aim 3 we will investigate how CCDC137 prevents DNA damage response and controls cell cycle progression. In particular, we will determine whether CCDC137 protects chromosomal DNA and delineate host factor(s) cooperating with CCDC137 to modulate the DNA damage response.

NIH Research Projects · FY 2026 · 2020-09

Project Summary The intestine hosts a complex microbiota that is essential for various physiological processes and immune stimulation. Secretory Immunoglobulin A (IgA), produced by plasma cells (PCs) originating predominantly from gut-associated germinal centers (gaGCs) in Peyer’s patches (PPs) and mesenteric lymph nodes (mLNs), is central to intestinal adaptive immunity. Despite the critical role of IgA in maintaining intestinal homeostasis, microbiota composition, and preventing diseases such as inflammatory bowel disease and colorectal cancer, significant gaps remain in our understanding of how gaGC B cells select microbial targets among thousands of potential antigens and why only IgA+ B cells selectively colonize the intestinal lamina propria (LP). In our current funding cycle, we have addressed the fundamental aspects of gaGC function and IgA biology. We demonstrated that robust clonal selection occurs within gaGCs, even under steady state conditions. Our data revealed distinct “winner” clones exhibiting clear affinity maturation towards commensal microbiota antigens. Importantly, we discovered two distinct types of B cell clones: “specialist” clones targeting single bacterial species and “generalist” clones recognizing multiple species simultaneously. Additionally, we identified DMBT1 as a critical epithelial receptor for IgA, which is crucial for restraining epithelial proliferation and colorectal cancer progression. In our comprehensive preliminary analyses, our “clonality-aware fate-mapping” approach demonstrated that LP IgA+ PCs originate exclusively from PP gaGCs, whereas mLN gaGCs predominantly produce locally retained IgG+ plasmablasts, highlighting a fundamental difference in gaGC output. This renewal leverages our complementary expertise—the Victora lab in B cell receptor dynamics and clonal selection, and the Mucida lab in mucosal immunology and gnotobiotic models—to dissect the mechanistic basis of IgA+ B cell colonization in the LP and comprehensively map gaGC B cell specificity to defined microbial consortia. Aim 1 will determine whether unique signaling properties of the IgA B cell receptor (BCR) or imprinting signals during class-switch recombination dictate selective colonization of IgA+ PCs to the LP, employing novel genetically- engineered mouse models as well as novel proximity-based methods for labeling cell–cell interactions. Aim 2 will define gaGC B cell specificities using advanced single-cell sequencing, recombinant antibody technologies, bacterial binding assays, and phage-display techniques to systematically characterize specialist and generalist responses to defined microbiota consortia (Oligo-MM12 and hCom2). By integrating our multidisciplinary strengths and state-of-the-art experimental approaches, we aim to elucidate the fundamental mechanisms governing gaGC B cell selection, antigen specificity, and plasma cell localization in the intestine, significantly advancing our understanding of mucosal immunity with direct implications for human health and disease prevention.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple pathologies, such as proteinaceous brain inclusions and neuroinflammation. The vascular system is also recognized as a factor in AD, yet there are few models to study the mechanism. We and others have found that the Aβ peptide, a known driver of AD, can activate the plasma contact system, which can lead to blood clot formation and inflammation via generation of bradykinin upon cleavage of high molecular weight kininogen (HK). There are three main lines of evidence that the contact system is involved in AD pathology: 1) Aβ activates factor XII (F12), which initiates the contact system; 2) AD patient plasma has increased contact system activation compared to that of age-matched, non-demented individuals; and 3) Knockdown of the contact system using an anti-F12 antisense oligonucleotide ameliorates AD pathology in a mouse model. HK circulates in blood as a complex with other coagulation factors, and it serves as a non-enzymatic co-factor for the activation of these proteins. Compared to other components of the contact system, depletion of HK offers more robust protection from blood clotting and inflammation due to its central role in both pathways. We have generated antibodies that are specific for cleaved HK that could help identify AD patients with contact system involvement. We also have developed antibodies that block HK cleavage, which might be beneficial to patients as they might ameliorate some of the pathologies of AD. It is important to note that people who lack a contact system are not prone to bleeding, and therefore, blocking this system in AD patients would not risk intracerebral hemorrhage. Despite decades of research, there are no effective treatments that slow or prevent AD. Progress in treating AD requires a multidisciplinary approach. We hypothesize that blocking the contact system could reduce vascular and inflammatory pathologies in AD patients. We propose to further develop our anti-HK antibodies for AD patient diagnostic and therapeutic use.

NIH Research Projects · FY 2024 · 2020-08

Project Summary/Abstract How accurate cell fate specification occurs in the context of dynamic tissue-scale rearrangements is one of the most exciting questions in developmental biology. In this proposal we aim at deciphering the interplay between fate acquisition, patterning and morphogenesis of the ectodermal germ layer in the context of human neurulation. We have recently developed a robust protocol which allows for the generation of extremely reproducible human neuruloids: self-organized human Embryonic Stem Cell (hESC) assemblies that recapitulate the organization of the ectodermal compartment at neurulation stages by organizing neural, neural crest, placodes and epidermis populations within the same colony on adhesive micropatterns. This self-organization is extremely reproducible and can be quantified with sub-cellular resolution and in real time over hundreds of colonies. Armed with this novel technology, we propose three specific aims. The first is to unravel the mechanism of cell-cell signaling driving self-organization. The second is to integrate signaling with fate acquisition and morphogenesis through live reporter imaging and time dependent single cell RNAseq. Finally, the third aim focuses on the full characterization of the origin and sub-populations of ectodermal derivatives and their in vivo validation by performing side by side comparisons with stage-matched marmoset fetal samples and grafting experiments in chick embryos. The generation of large numbers of homogenous human neuruloids, where self-organization of ectodermal fate can be followed dynamically for a period of one week, with sub-cellular resolution, not only solves the inherent heterogeneity observed in cerebral organoids, but provides us a unique opportunity to study these events in models of human embryos. This will have a high impact in both basic research as well as clinical application, a prospect already on the horizon. !

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Monoclonal antibodies have played a pivotal role in the diagnosis and treatment of cancer for nearly two decades and continue to grow at an exponential pace. Initially developed for their exceptional ability to target tumor antigens and elicit antibody-dependent cellular cytotoxicity (ADCC), they have more recently been used to modulate a patient’s immune system for anti-cancer immunotherapy. While the generation and development of antibodies targeting various cell surface proteins has rapidly progressed, appropriate model systems for pre- clinical testing of such therapeutics has lagged. This is because human antibodies i) don’t fully engage murine or non-human primate Fc receptors (FcγRs), ii) are foreign proteins that are rapidly rejected in allogeneic hosts and iii) are often inappropriately tested in immunodeficient xenograft models lacking adaptive immune cells or homologous FcγR. Thus, our studies have focused on the generation and testing of clinically relevant models to better understand the in vivo activity of diagnostic and therapeutic antibodies. The current proposal aims to now generate and fully characterize novel murine models that allow better preclinical testing of human antibodies by engineering our previously developed humanized FcγR mouse strains to express human FcRn and IgG1. Expression of human FcRn will allow more accurate pharmacokinetic analysis of human antibodies and assessment of methods aimed at generating antibodies with extended in vivo half-life. By replacing the mouse heavy chain with the constant regions of human IgG1, this model will also allow chronic administration of human IgG-based therapeutics without developing anti-drug antibody responses. By addressing two major hurdles in the field of antibody therapeutics, these models will allow more rapid and efficient pre-clinical toxicology testing and potentially uncover novel mechanisms of Fc-engineered antibodies. Additionally, given the growing interest in immunotherapy, having an immunocompetent model provides an additional advantage over current xenograft models. Finally, as recent data suggest an important role for Fc-FcγR in radiolabeled antibody diagnostics, these models will provide a clinically relevant model to help improve the development and testing of innovative antibody-based molecules for the in vivo detection and localization of neoplasms.