Grants

NIA - National Institute on Aging

PROJECT SUMMARY Effective therapeutics for several fatal neurodegenerative disorders, including frontotemporal dementia (FTD), an Alzheimer's Disease Related Dementia (ADRD) continue to remain elusive. Emerging evidence suggests that TAF15, an important RNA-binding protein (RBP) with a prion-like domain (PrLD), assembles into pathological fibrils in degenerating neurons of ~10% of all FTD cases (i.e., FTD-FET). These findings suggest that aberrant TAF15 phase transitions into pathological fibrils in the neuronal cytoplasm are problematic and difficult to resolve. Agents that prevent and reverse the aberrant phase transitions of TAF15 and restore functional TAF15 to the nucleus in the degenerating neurons of FTD-FET patients are likely to confer therapeutic effects. Indeed, such agents would simultaneously eliminate any toxic gain-of-function of aberrant TAF15 conformers in the cytoplasm, eliminate any prion-like TAF15 conformers that may spread pathology between neurons, and mitigate any toxic loss-of-function caused by depletion of TAF15 from the nucleus. However, TAF15 has been largely overlooked as a therapeutic target. Previously, we have established that short, specific RNAs (~25-34 nucleotides [nts]) provide a novel mechanism to antagonize neurotoxic phase transitions of two related RBPs with PrLDs that are also connected to neurodegenerative disease: TDP-43 and FUS. These short RNAs can engage TDP-43 or FUS, prevent aberrant TDP-43 or FUS phase separation, reverse the formation of existing TDP-43 or FUS aggregates, restore nuclear localization of TDP-43 or FUS, and protect human neurons against TDP-43 or FUS toxicity. Importantly, one short RNA penetrates into neurons, reverses TDP-43 proteinopathy, and mitigates neurodegeneration in mice. These short RNAs are similar in size to FDA-approved antisense oligonucleotides that can be delivered successfully to the CNS of patients to treat neurodegenerative disorders. Here, we propose to extend this approach to TAF15, which has emerged as a more important contributor to FTD-FET than previously appreciated. We hypothesize that short, specific, drug-like RNA oligonucleotides (25nts) can antagonize aberrant TAF15 fibrillization in a neuroprotective manner. Thus, we will pursue two aims: (1) Define RNA oligonucleotides that prevent and reverse aberrant TAF15 phase separation at the pure protein level; (2) Define RNA oligonucleotides that mitigate TAF15 toxicity in neuronal models of FTD-FET. Our studies hold the potential to yield the first therapeutic oligonucleotides that reverse TAF15 aggregation and mitigate toxicity in human neurons in culture. We envision a therapeutic strategy whereby specific short RNA oligonucleotides reverse TAF15 aggregation in FTD-FET and restore functional TAF15 to the nucleus.

NIGMS - National Institute of General Medical Sciences

The evolution of multicellularity was a pivotal transition that laid the foundation for cell biology, development, physiology, and immunity in animals. Building on our previous work in cell-cell adhesion and self/nonself recognition as unifying elements of multicellular development, in the next five years we will examine the evolutionary foundations of antibacterial immunity. Our work centers on sponges, invertebrate animals with unique relevance for identifying both anciently conserved and novel immune paradigms for regulating interactions with bacteria. Like all animals, sponges must defend against opportunistic pathogens, but like protists they feed by direct phagocytosis of bacteria. Also, living in aquatic habitats they are constantly bathed in environmental bacteria from which they cultivate complex communities of beneficial symbionts within their tissues. With genomic resources, a cell atlas, and transgenic tools in-hand, we are uniquely positioned to probe sponge immune pathways in unprecedented depth. Our preliminary findings have already revealed a novel family of glycan-binding pattern recognition receptors (PRRs) that have never before been studied. Characterizing their biochemical and functional properties will provide critical new insights into bacterial recognition and response. Furthermore, the glycan-binding region of these PRRs is composed of three-sided beta-helices that hold tremendous promise as novel diagnostic and therapeutic tools. We have also established that the versatile intracellular pathogen Legionella pneumophila can be used to infect sponges and thereby model an authentic immune response. As L. pneumophila infection has been extensively studied in human macrophages and amoebae, there exists a rich framework for comparing infection dynamics across nearly 1.8 billion years of evolutionary history, spanning the divide between unicellularity and multicellularity. Our research seeks to define foundational principles of immunity, bridging evolutionary biology and biomedical science to address critical challenges in health and disease.

NCI - National Cancer Institute

PROJECT SUMMARY The goal of this K08 Career Development Award is to train Dr. S. John Liu, a second year Assistant Professor in Residence at UCSF, in the skills he will need to become an independently funded physician-scientist capable of basic and translational investigation in neuro-oncology. Dr. Liu proposes to investigate the role of DNA-PKcs, a kinase well described in DNA repair but relatively underappreciated in non-canonical functions such as tumorigenesis and tumor-immune signaling. The proposal is an extension of his previous work on developing and implementing single cell functional genomics tools to identify and validate genetic regulators of radiotherapy resistance, which is a major problem in the treatment of patients with glioblastoma (GBM). Dr. Liu has shown through unbiased genome-wide CRISPR screens, in vivo perturb-seq experiments, intracranial xenografts, and spatial transcriptomics of patient tumors that inhibition of DNA-PKcs combined with radiotherapy results in decreased pro-growth gene expression and remodeling of the tumor immune microenvironment in GBM. In Aim 1, Dr. Liu will investigate the tumor cell-intrinsic mechanisms of how DNA-PKcs drives resistance to radiotherapy and cell survival in GBM, focusing on transcriptional regulation through chromatin binding and interactions between DNA repair and interferon signaling. In Aim 2, Dr. Liu will investigate how DNA-PKcs regulates the tumor immune microenvironment in syngeneic animal models of GBM. He will then determine the role of the cGAS/STING pathway in the anti-tumor effects of DNA-PKcs inhibition, followed by therapeutically targeting this pathway through STING agonism. In Aim 3, Dr. Liu will validate tumor immune microenvironment reprogramming using patient tumor biospecimens from a clinical trial of DNA-PKcs inhibition and radiotherapy for GBM. This proposal uses vertebrate animals because understanding glioblastoma, a complex disease that involves tumor, stromal, and immune cells, requires experimentation in mice in which the tumor immune microenvironment cannot yet be entirely recapitulated in vitro. Dr. Liu’s career development and research plans include a combination of formal coursework tailored to fill his specific knowledge gaps, national workshops, international conferences, and intensive hands-on research experience that will take place at UCSF, a leading NCCN Comprehensive Cancer Center with an excellent track record in basic and translational neuro-oncology research. Dr. Liu’s training plan specifically focuses on areas that are underdeveloped in his research skillset, specifically (1) molecular biology and radiobiology research methods, (2) tumor immunology knowledge and research methods, (3) translational research skills in neuro-oncology, and (4) leadership and laboratory management skills to achieve and sustain independence. This project will be mentored by David Raleigh, Associate Professor of Radiation Oncology, Neurological Surgery, and Pathology at UCSF who is an experienced mentor well suited to train Dr. Liu in these key areas. Dr. Liu has a highly distinguished but available advisor committee possessing synergistic expertise spanning basic, translational, and biostatistical sciences to guide his research and career trajectory. Successful completion of this proposal will train Dr. Liu for independence and also provide mechanistic understanding of RT resistance in GBM, while elucidating rationally informed combination therapy strategies that may improve outcomes for patients with GBM.

NIH

This proposal aims to advance our understanding of clonal hemopoiesis of indeterminate potential (CHIP) in the development of atherosclerotic cardiovascular disease (ASCVD). CHIP is a recently identified acquired risk factor for ASCVD. With aging, hematopoietic stem cells accumulate mutations that can lead to a proliferative advantage resulting in CHIP. Tet Methylcytosine Dioxygenase 2 (TET2) is a commonly mutated gene in CHIP and confers a 50% increased risk for incident coronary disease. How TET2 leads to ASCVD is in humans is not well understood and there is currently no ability to assess whether a specific TET2 mutation is high-risk. The central objective of this proposal is to (1) identify TET2 mutations that are high-risk for developing ASCVD to derive a comprehensive and clinically actionable risk score calculator and (2) identify the aberrant cell states and signaling pathways among TET2 mutated immune cells in the coronary vasculature. To identify high-risk TET2 mutations, the candidate will leverage a population-scale human genetics approach in >1 million people via the Million Veteran Program (MVP). To identify aberrant cell states and signaling pathways, the candidate will deploy their novel single cell lineage tracing protocol in coronary vascular tissue followed by validation experiments via population-based human genetic association studies. The candidate's career goals are to become an independently funded physician scientist focused on developing new ways of treating ASCVD. In addition to the proposed science, the training activities outlined in the candidate's career development plan are focused on the crucial skills and experiences necessary to enable an independent research program. Combined with the direct mentorship of Ors. Brent Ferrell and Adrianna Hung, Tennessee Valley Health System Nashville VAMC represents an ideal environment for the proposed work and leverage some of the world-class strengths of the Veterans Affairs resources. The Ferrell and Hung labs have deep experience in the methods used in this proposal and are prepared to support the candidate throughout the entirety of the grant period. Overall, this VA CDA-2 proposal represents a set of innovative and timely scientific aims combined with a tractable career development plan that will meaningfully contribute to human health research and catalyze the candidate's long-term career goal of developing into an independent investigator.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Neural circuit development and function depends on precise interactions between neurons and glia. Astrocytes, the primary peri-synaptic glia, mediate synapse formation, stability, and function. Neuron-astrocyte crosstalk is facilitated by complex protein-protein interactions, and loss of these interactions contributes to circuit instability in many neurological disorders. Thus, understanding the mechanisms that regulate neuron-astrocyte communication is of broad clinical importance. Glycosylation is a posttranslational modification that regulates protein stability and binding through addition of sugar groups to specific amino acids. Mutation of genes in glycosylation pathways cause congenital disorders of glycosylation (CDGs), a group of monogenic disorders associated with neurological dysfunction, including epilepsy, autism, and cerebellar degeneration. The mechanisms underlying neurological dysfunction in CDGs remain unknown. Here, I focus on ALG8, an enzyme in the N-glycosylation pathway. To explore the molecular underpinnings of ALG8-CDG, I first needed to develop models that reflect the patient population. To this end, I generated a predicted null zebrafish line (alg8stl973) and human embryonic stem cell (hESC) lines with a missense mutation (p.Thr47Pro) found in ALG8-CDG patients. My preliminary data revealed a decrease in astrocyte numbers in the brains of alg8 mutant zebrafish with no change in total cells, and reduced proliferation of ALG8 mutant hESC-derived astrocytes. Moreover, in alg8stl973 fish, astrocyte morphological complexity is reduced. As astrocyte-synapse association is necessary for neuronal signaling, I hypothesize that defective glycosylation disrupts specification and maturation of astroglia, which in turn drives circuit imbalance and CDG-associated behavioral deficits. To address this hypothesis, I will leverage preexisting transgenic tools in zebrafish to label astrocytes and test whether changes in proliferation and/or cell death result in reduced astrocytes in alg8stl973 fish (Aim 1). Furthermore, I will use biochemistry and in vivo imaging to characterize how loss of alg8 impacts the glycosylation status of one key regulator of astrocyte morphogenesis: NrCam (Aim 2). Finally, as ALG8 is expressed in all neural cell types, I will use cell-type specific rescue in fish and co-culture of hESC-derived neural cells to determine which cell type(s) drive changes in astrocyte morphology and synaptogenesis in ALG8-CDG (Aim 3). My long-term goal is to define common molecular changes in brain development across distinct CDGs. Critically, various CDG subtypes result in common neurological symptoms, but the cellular and molecular underpinnings of these phenotypes are largely unknown. Similar to my preliminary findings in ALG8-CDG models, recent work indicates that astrogenesis is altered in a mouse model of MGAT5-CDG, a CDG with defective N-glycosylation. Thus, I anticipate that my findings will be broadly applicable to the CDG community and will enhance our fundamental understanding of how glycosylation shapes brain development.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Our long-term goal is to understand the contribution of stress-induced neural signals to glucose regulation and the development of metabolic diseases with the aim of developing better therapies. To achieve this goal, we need a more detailed understanding of the neural circuits modulated by internal and external signals that, in turn, regulate metabolic function. In this proposal, wewill create a detailedand systematic analysis of the activity of specific amygdala neural populations in response to stress, determine their physiological roles, identify genetic markers for stress-responsive neural populations with defined projections and assess the contribution of these populations to metabolic regulation in acute and repeated stress. Our pilot data strongly support the activation of specific populations of amygdala neurons in response to stressors and chemogenetic activation of these neurons and circuits rapidly and robustly increases blood glucose. Our proposal will create new insights into the neural populations and circuits that regulate metabolism bring an unprecedented level of precision and specificity by combining cell-type specific neural tracing, spatial transcriptomics and targeted neuromodulation in which we have extensive experience. Without this detailed knowledge, our understanding the contribution of defined amygdala circuits to metabolic responses to stress will remain incomplete. The overall objective of this proposal is to understand the structure, genetic composition and function of defined amygdala circuits. Our central hypothesis is that defined medial amygdala neural populations and their hypothalamic projections are critical to the metabolic responses to stress. The rationale that underlies the proposed research is that internal and external stressors result in profound metabolic responses. However, we do not know the activity or genetic identity of the activated neural populations, their downstream circuits, or physiological roles. These represent major gaps in our understanding. To test our central hypothesis and attain the overall objective, we will a) determine the activity and physiological roles of defined medial amygdala neurons in the glucose response to acute and repeated stress and b) identify the neural activity, function and gene markers for specific downstream circuits that regulate blood glucose in responses to acute and repeated stress. To do so, we will use in vivo calcium imaging to assess neural activity, cell type and circuit- specific neuromodulation to define physiological roles of defined amygdala circuits in the regulation of glucose with stress, and spatial transcriptomics to identify gene markers for neural populations with defined projections contributing to glucose regulation in stress. The proposed studies will provide a comprehensive understanding of the unique biology of medial amygdala circuits regulating metabolic function in response to acute and repeated stress to form a crucial foundation for future studies to identifying potential therapies for stress-associated metabolic diseases such as type 2 diabetes. in response to homeostatic challenges. We will

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary An emerging model of obesity pathogenesis posits a central role for the inflammatory activation of hypothalamic microglia localized to the arcuate nucleus (ARC, a key brain area for the control of food intake and body weight) in the pathogenesis of diet-induced obesity (DIO), a finding observed in mammalian species ranging from rodents to humans. While microglia activation in the ARC is a known determinant of weight gain in high fat diet (HFD)-fed mice, it paradoxically improves glucose tolerance in DIO, but the mechanisms underlying both of these metabolic effects remain unclear. Herein, we report the novel finding that in mice, DIO induces loss of specialized extracellular matrix (ECM) structures known as perineuronal nets (PNNs) in the same brain area where reactive gliosis occurs. PNNs can powerfully influence the activity of neurons that they enmesh, and in the ARC, a large proportion of AgRP and a subset of POMC neurons are among those enmeshed by PNNs. These neurons are central regulators of energy homeostasis, and their altered function in DIO is strongly implicated in obesity pathogenesis and glucose regulation. Importantly, ablating or silencing microglia reduces Npy and AgRP levels and increases POMC neuron excitability, suggesting a link between microglial activation and ARC neuronal function that promotes weight gain. Furthermore, DIO is associated with loss of hypothalamic PNNs in proportion to the degree of microglial activation, with PNN stability enhanced by interventions that ablate or limit the inflammatory capacity of microglia. Finally, removal of hypothalamic PNNs experimentally causes gliosis, hyperphagia and rapid weight gain with preserved glucose tolerance in rodents, providing strong evidence that microglia and PNNs are linked in a critical mechanism that underlies obesity pathogenesis. Here, we investigate the inter-related hypotheses that obesity-associated microglial activation induces loss of PNN enmeshment of ARC neurons, thereby altering AgRP and Pomc neuron function in ways that promote excess fat accumulation but maintain glucose tolerance. Proposed studies will first quantify the role played by microglia to regulate ARC PNN turnover. We will then 1) determine whether the effect of DIO to induce loss of ARC PNNs depends on microglial activation, 2) identify the roles of both microglial activation and PNN loss in obesity-associated dysfunction of AgRP and POMC neurons, and 3) determine the bidirectional contributions linking ARC PNN loss and microglial activation to DIO susceptibility.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT ABSTRACT (SUMMARY) Astrocytes, the most abundant cell type in the brain, contribute to neuroinflammation seen in neurodegenerative diseases like Parkinson’s disease (PD). Mitochondrial dysfunction is also a hallmark of PD, and occurs in both neurons and astrocytes. However, the consequences of mitochondrial dysfunction in astrocytes are not well understood. As accumulating data link mitochondrial damage to onset of inflammation, it is essential to our understanding of PD to elucidate astrocytic responses to mitochondrial damage. Here, I will (1) define a pathway astrocytes use to clear damaged mitochondria (PINK1/Parkin mitophagy), and (2) identify inflammatory signaling cascades resulting from mitochondrial damage in primary astrocytes. In PINK1/Parkin mitophagy, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase Parkin coordinate assembly and phosphorylation of ubiquitin chains on damaged mitochondria. Autophagy receptors, which facilitate autophagosome biogenesis, are recruited to this ubiquitin. Our lab demonstrated that the NF-κB essential modulator (NEMO) protein is also recruited to Parkin- assembled ubiquitin chains. This is a novel mechanism for initiation of NF-κB signaling, a major innate immunity pathway. Using oxidative phosphorylation (OXPHOS) inhibitors as mitochondrial damaging agents, I found that NEMO recruitment to damaged mitochondria occurs in cortical murine astrocytes ex vivo. Prolonged treatment results in heightened transcription of NF-κB-associated cytokines TNF-α and Il6, and TNF-α upregulation is ameliorated by NF-κB inhibition. Studies in HeLa cells suggest the autophagy receptor p62 is recruited to mitochondria alongside NEMO. I observe both p62 upregulation and recruitment to damaged mitochondria in astrocytes, suggesting p62 participates in astrocytic mitophagy. About 50% of p62 localizes to phospho-ubiquitin, indicating phosphorylation of ubiquitin may drive p62/NEMO recruitment. However, it is unclear if p62 is necessary for mitophagy in astrocytes, how it influences NEMO recruitment, and whether NF-κB is the major pathway activated. Based on my initial data, I hypothesize p62 is required for mitophagy and promotes NEMO recruitment to ubiquitin chains (Aim 1), and that NF-κB is the major inflammatory pathway activated (Aim 2a,b), resulting in secretion of neurotoxic signals that promote neurodegeneration (Aim 2c). In Aim 1, I will test whether knocking down p62 hinders mitophagy in astrocytes. I will also perform in vitro reconstitution assays with purified protein to determine if the efficiency of NEMO recruitment to damaged mitochondria is increased by p62 and phosphorylation of ubiquitin. In Aim 2, I will analyze bulk RNA-sequencing data from wild-type (WT) and PINK1 knockout astrocytes treated with OXPHOS inhibitors or a vehicle control. I will look at differential gene expression related to inflammatory signaling and NF-κB, and validate sequencing results via an NF-κB inhibitor and qPCR. Finally, I will compare the neurotoxicity of WT and PINK1 knockout astrocytes with and without OXPHOS inhibitor treatment. This project will define how astrocytes clear damaged mitochondria and the inflammatory signaling activated by this mitochondrial damage, which is critical to understand the role of astrocytes in PD pathology.

NCI - National Cancer Institute

While some patients with previously untreatable blood cancers have achieved long-term remissions following CAR T cell therapy, others relapse after months to years. While engraftment and expansion are well characterized clinical surrogates of a durable anti-tumor effect, the attributes and origin of CAR T cells with prolonged function remain unknown. Prior research on CAR T cell-intrinsic properties in the treatment of hematologic malignancies has primarily focused on the manufactured product, but did not capture or define the T cell clones that are directly responsible for functional persistence and durable remissions. We have now found that long-lived CAR T cells up to 5 years post-infusion have a distinct CD4- CD8a- “double-negative” (DN) TCRαβ+ effector memory T cell phenotype. Our multimodal single-cell analyses have revealed that (i) the clones that eradicate leukemia in the initial phase differ from those that perform long term immunosurveillance, and that (ii) long lived DN CAR T cells show specific transcriptional signatures, with remarkable similarity to persistent CAR T cells in small cohort of children with B-ALL, to rare TCRαβ+ DN T cells from healthy donors, and to melanoma patients responding to dual checkpoint blockade. The CD4 and CD8 glycoproteins are co- receptors in TCR recognition of class II or class I major histocompatibility (MHC) – peptide complexes, respectively, serving to stabilize the TCR:peptide/MHC. Since DN T cells accumulate in chronic inflammatory diseases, these observations raise the intriguing possibility that in some settings, chronic antigen exposure can lead to a unique and previously under-appreciated T cell differentiation trajectory that is distinct from that found in “classic” CD8 T cell exhaustion. This dual PI proposal unites experts in immune cell therapies and evolutionary biology to test the overarching hypothesis that the fittest CAR T cells adopt a DN phenotype to withstand the demands of long-term immunosurveillance. In Aim 1 we will define the contribution of DN T cells to the long-lived CAR T cell population in patients with hematologic malignancies using flow cytometry and single cell multiomic analyses of CAR T cells persisting >12 months after infusion in patients with CLL, NHL, and MM. By leveraging our extensive cryorepository and well-annotated clinical trial outcome data we will be able to correlate CAR T cell phenotype to their functional persistence. In Aim 2 we will identify the cellular origin and evolutionary paths of long-lived CAR T cells. To distinguish the possibilities that persistent CAR T cells originated as true DN T cells vs. conventional T cells that downregulate the CD4 or CD8 co-receptors, we will use complementary approaches to trace DN persistent clonotypes back to their infusion origin. In Aim 3 we will determine whether and how DN T cells resist exhaustion during chronic stimulation, using a novel high-fidelity T cell exhaustion model along with gain- and loss-of function interventions to illuminate this biology. Together, these studies will illuminate the lineage relationships of various endogenous and engineered T cells and guide the development of more effective CAR T cell therapies.

NCI - National Cancer Institute

N6-methyladenosine (m6A) is the most common internal mRNA modification and has been suggested to impact the formation and fate of mRNA. METTL3, the enzyme that deposits m6A, is essential for development and has emerged as a promising therapeutic target in various cancers, including acute myeloid leukemia (AML). Despite the dynamic processes m6A is implicated in, our understanding has predominantly been informed by long-term knockdown experiments whereby the cell has had ample time to compensate. However, to understand the direct influence and mechanisms underlying m6A’s role in RNA regulation, this proposal sets out to combine acute perturbations with precise transcriptomic measurements. Surprisingly, despite numerous studies demonstrating that long-term knockdown of METTL3 impacts transcription, short term METTL3 inhibition causes no significant changes in transcription initiation or elongation across gene bodies. Instead, gene upregulation observed by RNA-seq upon METTL3 inhibition reflects increased RNA stability. Although the regulation of RNA stability by cytoplasmic m6A readers is one of the most well-established functions of m6A, these data suggest an alternative mechanism. At the upregulated genes, altered 3’ end processing and a shift towards more distal 3’ Untranslated Regions (UTRs) are observed. Sequences within 3’ UTRs are known to impact mRNA stability, location, and translation, raising the intriguing possibility that regulation of 3’ UTR choice, and the altered RNA fate conferred by shifts in 3’ end sequences is central to m6A function. In this proposal, the hypothesis that m6A deposition impacts AML cell survival by directly regulating 3’ end processing will be tested. First, fast-acting inhibitors of METTL3 in combination with direct measurements of RNA synthesis and transcript 3’ ends will be used to define the consequences of m6A loss in AML cells (Aim 1). The model that an altered 3’ UTR sequence landscape is linked to the upregulation of AML driver genes will be tested. Next, the underlying mechanism by which m6A deposition impacts 3’ end processing will be explored through acute degradation and proteomic techniques (Aim 2). Finally, a cellular reporter assay will be employed to directly assess how 3’ UTR selection influences transcript abundance, stability, and translation of genes that promote AML proliferation (Aim 3). These results have the potential to clarify current models that have centered cytoplasmic m6A readers in regulating mRNA output. In addition, these results should inform therapeutic contexts wherein METTL3 inhibitors would be most beneficial. The proposed work will be conducted in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, which provides a rich interdisciplinary training environment and robust infrastructure to facilitate the successful completion of the aims. In addition, the proposed plan will deepen the lead investigator’s technical and conceptual training in cancer biology and transcriptomics. These experiences will support the principal investigator on her path towards leading a team of epigenomics researchers developing cancer therapeutics.

NIGMS - National Institute of General Medical Sciences

Project Summary / Abstract Protein synthesis, also known a translation, is a highly regulated process with deep significance to public health. Dysregulation of translation is a major driver of diseases such as cancer, and mutations that prematurely halt translation cause 11% of all heritable human diseases. Despite its importance, we do not yet understand the molecular events used to evaluate messenger RNAs (mRNAs) during translation, which serves as a crucial branchpoint in cells. When translation concludes on a normal mRNA, the molecular machinery that drives translation in cells (ribosomes) is typically released from the mRNA to facilitate further rounds of protein synthesis. Yet, for reasons that remain unclear, translation of an aberrant mRNA instead recruits specialized machinery to the ribosome to halt translation and destroy the faulty mRNA. Although we understand that the concluding stages of translation play an outsized role in determining mRNA fate, these branchpoints have proven extremely difficult to study due to the intricate and transient nature of the underlying interactions. As a postdoctoral fellow, I used an in vitro-reconstituted yeast translation system composed solely of purified components to recapitulate translation termination, the process through which newly synthesized proteins are typically released from ribosomes. By attaching fluorescent dyes to the key cofactors that drive translation termination, I was able to watch this process unfold in real time using single-molecule fluorescence spectroscopy to obtain detailed, time-resolved movies of this core cellular process. During the ESI MIRA phase, my research group will apply in vitro systems, traditional biochemical techniques, single-molecule fluorescence spectroscopy, and structural approaches to determine how translation concludes on normal and aberrant mRNAs. In Area 1, we will uncover how translation termination is regulated by local mRNA sequence and proximity of the poly(A) tail to help ribosomes distinguish normal from aberrant mRNAs. We will also determine how recycling, the process through which ribosomes are removed from normal mRNAs after translation, is choreographed. In Area 2, we will characterize the interplay of two interrelated processes (the No-Go Decay and Ribosome Quality Trigger pathways) that are both activated by the collision of ribosomes during translation of aberrant mRNAs. Put together, our investigations will uncover the key molecular interactions that dictate the fate of mRNAs during translation. A detailed understanding of these fundamental gene expression processes could also uncover new ways to manipulate them for therapeutic benefit, paving the way to new treatments for cystic fibrosis, muscular dystrophy, Alzheimer’s, and cancer.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Despite countless significant advances in the field of cardiovascular disease, it remains the number one cause of death worldwide. Several systemic risk factors which increase disease risk and include hypertension, high cholesterol, obesity, diabetes, smoking, and chronic inflammation. An additional local risk factor is disturbed blood flow (d-flow), which activates the endothelium to permit of atherosclerosis, and is accelerated when other risk factors are present. Despite our best efforts to treat these conditions to improve outcomes, cardiovascular disease remains the number one killer, which suggests that there remain residual unaddressed risk factors. We have identified through previous studies using the partial carotid ligation (PCL) model of d-flow causing atherogenesis that CCAAT/Enhancer Binding Protein β (C/EBPβ), a protein previously identified to play critical roles in development, cell differentiation, and inflammation, was upregulated at the mRNA level in d-flow exposed inflamed endothelial cells in vivo. We found in orthogonal studies that C/EBPβ was upregulated at the protein level in regions of d-flow, and that the compared to regions of stable flow, that the protein had localized to the nucleus, suggested that it was playing a role in downstream signaling and transcription. Three isoforms comprise C/EBPβ, which is translated from a single intron. The first isoform is LAP*, the full length protein, with activating and repression domains, the second isoform is LAP, which is nearly identical to LAP* but lacks 23 amino acids (and one of the activating domains) at the N terminal region. The short isoform is LIP, which lacks all activating domains and is thought to have a repressive role on the function of LAP* and LAP. We found that flag-tagged LIP isoform co-immunoprecipitated with the Protein Subunit Beta type-9 immunoproteasome component (PSMB9, alternatively named LMP2) in vitro, which notably, has also been implicated in atherosclerosis. Finally, we found that in contrast to stable flow, d-flow induced PSMB9 activity in vitro. Given these findings, we will set out to define the role of C/EBPβ and PSMB9 in atherosclerosis. We will determine whether C/EBPβ and PSMB9 are required for endothelial cell inflammation caused by d-flow in vitro using CRISPR-i and small molecule inhibition. We will employ a mouse model of endothelial-specific C/EBPβ or PSMB9 deletion combined with PCL and hypercholesterolemia to determine if these proteins are required by endothelial cells for the propagation of disturbed blood flow leading to endothelial inflammation and atherosclerosis in vivo. Finally we will determine whether those two proteins are over-expressed in endothelial cells from atherosclerotic regions compared to healthy control regions in human coronary arteries. We hypothesize that C/EBPβ is a key protein required for atherosclerosis, and that it exerts its pro-atherogenic effects by binding and activating the downstream PSMB9 immunoproteasome. We hope that our findings will further elucidate unaddressed risk factors which contribute to cardiovascular disease, and will lead to improved treatments for the number one cause of death.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Cryptococcus neoformans, the etiological agent of cryptococcal meningitis (CM), is a globally distributed environmental yeast that mainly causes infections in immunocompromised individuals. Particularly in low- resource countries, the mortality rate of CM can reach 81% and accounts for 19% of HIV/AIDS-related deaths each year. Despite this high burden, CM treatment options are suboptimal, largely due to an incomplete understanding of the fungal biology and host-pathogen interactions. In immunocompromised individuals, once inhaled, C. neoformans escapes from the lungs and disseminates with special predilection for the central nervous system (CNS). Once in the brain, C. neoformans interacts with microglia, the tissue-resident macrophages of the CNS. Previous studies indirectly showed that microglia are ineffective at controlling this fungal infection. It has also been shown that many phagocytosed yeasts can survive, replicate, and avoid killing in the microglial phagosome, and this can be influenced by prior yeast opsonization. The mechanisms underlying this fungal survival and proliferation within the CNS, however, remain unclear. My preliminary studies have expanded on these previous findings, showing that immortalized and primary human microglia have decreased phagocytic and killing activity that is specific to C. neoformans. This has led to my central hypothesis that C. neoformans survives in the CNS by using both novel, capsule-independent, antiphagocytic mechanisms and phagosome maturation disruption within microglia, and that these mechanisms vary based on yeast opsonization. To test this, I will use an immortalized human microglia cell line to study the interactions between these immune cells and C. neoformans. This model is advantageous as it is more translational that rodent-derived cell lines, and it is easier to culture and obtain than primary human microglia. Experiments in Aim #1 will define the process of phagosome maturation in microglia after engulfment of wild-type C. neoformans. I will look at phagosome development, acidification, damage, and subsequent function. Experiments in Aim #2 will identify antiphagocytic cryptococcal proteins and determine their impact on microglia killing efficiency and phagosome maturation. Experiments in Aim #3 will define if yeast opsonization leads to differential receptor engagement and how this impacts the outcome of the infection. Specifically, I will study how opsonization impacts microglial phagosome maturation and killing efficiency, as well as study the receptors necessary for cryptococcal recognition and engulfment. Overall, my project takes advantage of the expertise in cryptococcal-phagocyte interactions and glia biology and function of my sponsor and co-sponsor laboratories, respectively, and will yield a better understanding of the interactions between C. neoformans and microglia, ultimately contributing to the knowledge of fungal biology and CM pathogenesis, making it more likely that effective therapeutics to treat CM will be developed.

NCI - National Cancer Institute

Ovarian cancer is the most lethal gynecological cancer in the United States with poor survival due to extensive peritoneal metastasis. We discovered a previously uncharacterized early response signature to matrix detachment of high-grade serous adenocarcinoma cells, the most common ovarian cancer histologic subtype. This signature is associated with poor patient outcomes and distinct pro-metastatic signaling nodes. We find that the atypical GTPase RHOV, the most significantly induced gene in this signature, is essential for peritoneal metastasis in vivo, promoting anoikis resistance, invasion and clearance of mesothelial cells, an essential step in peritoneal metastasis. Our data reveals RHOV functions as a novel TGF-β/SMAD signaling integrator in OC, a previously uncharacterized function of this understudied GTPase. This finding represents a critical mechanistic link between early detachment responses and established pro-metastatic signaling pathways in ovarian cancer. Our overall hypothesis is that the rapid induction of the detachment response signature is a crucial step for initiating downstream signaling events necessary for ovarian cancer metastasis. Leveraging the PIs' complementary expertise in ovarian cancer research, we will use innovative approaches including in vivo CRISPR screens, auxin-inducible degradation systems, transcriptomics and imaging strategies in cell lines, xenograft tumors, and patient-derived models. In Aim1 we will identify essential genes within the clinically significant detachment response signature and delineate their convergent signaling pathways using unbiased functional genomics. In Aim 2 we will determine how RHOV and the detachment signature reprograms endocytic trafficking to change cellular signaling as exemplified by the TGF-β pathway and evaluate RHOV expression and the detachment signature as a biomarker for TGF-β inhibitor sensitivity. Defining the essential genes and downstream signaling of the detachment response including the novel RHOV- TGF-β/SMAD axis will identify key adaptations of metastatic ovarian cancer that can be targeted therapeutically. This work shifts the paradigm from studying late-stage adaptations to understanding the earliest events in the metastatic cascade, potentially leading to novel biomarker-guided precision medicine approaches for ovarian cancer patients.

NIAID - National Institute of Allergy and Infectious Diseases

Inflammatory Bowel Diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are life-long diseases characterized by cycles of active flares and remission. Disease flares result in significant inflammation in the colon, as well as pathology in other organ systems, including the brain. Central nervous system (CNS) dysfunction is a well-established comorbidity in patients with IBD. We have recently demonstrated CNS inflammation, and specifically inflammasome activation, is induced in the CNS of mice in an acute colitis model using a novel mouse model in which inflammasome activation induces tissue bioluminescence. In this grant, we seek to understand the mechanisms by which CNS inflammation is induced in this and similar models. We seek to extend exciting preliminary data which reveal that colitis results in the presence of a factor or factors in serum that drive CNS inflammation to identify this factor and the mechanism by which is induces inflammasome activation in the CNS. We also propose to use a newer version of our mouse model, in which expression of our luciferase based caspase-1 biosensor is restricted to specific cell lineages in the CNS, allowing us to understand the impact of colitis and CNS inflammation on specific cell populations.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The leading cause of hospital-acquired, infectious diarrhea in the United States is a gastrointestinal pathogen known as Clostridioides difficile. C. difficile can be found in the guts of many healthy individuals but typically only becomes problematic after disruption of the gut microbiota and is thus often associated with the administration of broad-spectrum antibiotics. The pathology that accompanies infection varies from one person to the next, ranging from relatively mild symptoms like diarrhea to severe, and potentially life-threatening conditions, known as pseudomembranous colitis and toxic megacolon. All these symptoms are the product of three toxins secreted by the bacterium during infection known as Toxin A, Toxin B, and the C. difficile Transferase (CDT). While Toxins A and B have historically been considered the drivers of the disease that accompanies infection, recent studies have suggested a potential synergy among all three toxins in the most severe cases. These observations have underscored the need to develop a better understanding of how CDT contributes to disease. The goal of this proposal is to expand our understanding of the intoxication mechanism governing CDT activity by addressing two key questions: 1) how is toxin assembly regulated and triggered? and 2) what is the mechanism underlying CDT delivery into host cells? We will use a combination of cryogenic electron microscopy (Cryo-EM), biochemical/biophysical assays, and cellular biology to describe these two points in molecular detail thereby generating a framework that can be used to deduce the precise role of CDT during infection. We also anticipate our analysis will uncover new structures and activities that will be exploited in future studies aimed at developing CDT inhibitors. As a third goal, we aim to leverage the toolkit we have assembled to probe the activity of two extrachromosomal CDT variants. Our plan is to use our full array of biochemical/biophysical assays, cellular biology, and Cryo-EM to generate a detailed description of the activity of these variants to better understand their potential to cause disease. Our work will thereby inform future studies that seek to understand how variation among toxins leads to different clinical outcomes. Together, this proposal will develop a thorough understanding of the function of CDT, an often overlooked, yet problematic, virulence factor associated with a notorious pathogen.

NIDCR - National Institute of Dental and Craniofacial Research

ABSTRACT Epstein-Barr virus (EBV) is a latent, recurring herpesvirus that replicates in the oral cavity and is transmitted by saliva. While most infections are relatively benign, EBV can cause malignancies in the immune suppressed, such as transplant patients and HIV-infected individuals, and is also a potential trigger for certain autoimmune diseases. In addition, rare germline genetic variants lead to increased susceptibility to EBV- associated diseases. EBV is a highly successful virus with nearly 95% of adults worldwide being latently infected. This efficiency is due to the unique and evolutionarily conserved ability of EBV to mimic B cell signaling pathways and evade the innate immune response. The balance of these activities determines whether EBV is successful in establishing latency and in some genetic backgrounds, the consequences range from autoimmunity to cancer. It is therefore our ultimate goal to define the molecular mechanisms for EBV latency establishment and persistence in the oral cavity. In this proposal, we aim to characterize how EBV evades innate immune signaling and usurps intrinsic B-cell signaling to promote latency establishment and pathogenesis. It is our central hypothesis that EBV establishes B-cell latent infection through antagonizing innate immune signaling and mimicry of constitutive B-cell signaling pathway activation. We have formulated our central hypothesis based on preliminary data including a CRISPR screen following up on our recent early infection scRNA/ATACseq experiments in primary human B cell infection leading to mechanistic studies demonstrating a positive role for the ubiquitin like molecule, ISG15, and restrictive roles for BCR signaling regulators in EBV-driven B cell immortalization. We further found that ISG15 acts as a regulator of type I interferon signaling such that ISGylation enzymes were suppressors of B cell outgrowth and STAT1/2 or IFNAR1/2 loss rescued ISG15 loss. Excitingly, we also identified patients with poor control of EBV harboring deleterious variants of these EBV regulators suggesting that our CRISPR screen identified clinically relevant molecules as arbiters of EBV infection in vivo. Therefore, the rationale for this proposed research is that understanding how EBV regulates innate immune and BCR signaling to establish latency and transform B cells provides insight into therapeutic modalities to eliminate EBV-infected cells from the oral cavity. We plan to test our central hypothesis and complete the objectives in this proposal through the following three specific aims: i) to determine the molecular mechanism by which EBV regulates type I interferon signaling to promote latency establishment and tumorigenesis, ii) to determine the unique role of B-cell receptor signaling regulators in EBV transformation and tumorigenesis, and iii) to determine the mechanistic roles of rare germline variants in regulatory genes from patients with EBV-associated diseases impacting ISGylation, signaling, and B cell fate decisions.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY / ABSTRACT The airway epithelial surface is constantly exposed to the environment and serves as a barrier to many types of injury - toxins, infections, allergens, and other insults. The airway epithelial surface must regenerate after denuding injury. Both these aspects – barrier function and regeneration – can become dysregulated in diseases such as asthma, COPD, and bronchiectasis. We have discovered a new airway epithelial cell type, called the hillock, which survives various airway injuries and regenerates the epithelial surface to restore mucociliary transport. The overall hypothesis of this proposal is that the hillock plays a critical role in barrier function and regeneration in the airway and may become dysregulated in disease. Through single cell RNA sequencing and histological characterization, we find that the hillock highly expresses keratin 13 and desmoglein 3. These proteins are important in barrier function in of the skin, and we hypothesize that they similarly play a major role in the airway hillock. In Specific Aim 1 of this proposal, we will define the mechanism of hillock injury resistance. Using a hillock specific driver mouse we have developed, we will test this hypothesis by disrupting keratin 13 and desmosomes and assess for injury resistance by live/dead staining, proliferation, and electron microscopy. Hillocks also play an essential role in airway regeneration. We find when stimulated with retinoic acid, hillocks regenerate ciliated cells. In Specific Aim 2 of this proposal, we define the mechanism of ciliated cell differentiation from hillocks and restoration of mucociliary transport. It has been shown that different retinoic acid receptors may affect ciliogensis and oncogenesis. We hypothesize that retinoic acid receptor alpha and best leads differentiation of hillocks into ciliated cells which could then restore mucociliary transport. We will test this hypothesis in both mouse and human hillock explants. After performing luminal injury, we will regenerate the epithelia in the presence of retinoic acid receptor selective agonists and characterize ciliogenesis by immunocytochemistry, electron microscopy, cilia length measurements and functional assessment by micro-optical coherence tomography. This work is potentially paradigm shifting as it implicates a specific cell type, hillock cells, in disease pathology of multiple airway diseases and interrogates this cell type to uncover novel therapeutic targets. Dr. Shah will carry out this work under the mentorship of Dr. Jay Rajagopal, a leader in stem cell biology with an outstanding record of guiding young investigators to independence. Through this proposal, Dr. Shah will obtain training in (1) inducible genetic models, (2) developmental and stem cell biology, (3) explant models of disease, and (4) functional real-time imaging, which is only possible in the environment of the Rajagopal lab and collaborators at MGH. An advisory team of complementary and diverse scientists have been assembled to provide breadth and depth to the training plan. Hands-on training will be supplemented with coursework in stem cell biology and advanced imaging, necessary for Dr. Shah to become an independent R01 funded investigator.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Obesity is one of the most pressing public health challenges of the 21st century. Weight gain occurs in the setting of nutrient excess, which stimulates adipose tissue expansion via both healthy hyperplastic and pathologic hypertrophic processes. The metabolic consequences of obesity, including diabetes and fatty liver disease, stem from a relative deficiency in hyperplastic expansion of adipocyte progenitor cells. GLP1 medications have drastically changed the therapeutic landscape, enabling millions of patients to achieve significant weight loss, however much remains to be learned about the pathophysiology of obese and post- weight loss adipose. Using single-cell sequencing we identified a population of maladaptive FAPs arising in obese adipose as well as an anatomically-distinct population of connective-tissue-resident macrophages marked by Lyve1. Preliminary data indicate that the Lyve1+ macrophages serve as niche-defining cells that maintain FAP homeostasis in lean adipose, however their number are diminished in obesity. Genetic depletion of Lyve1+ macrophages leads to pathologic lineage allocation of maladaptive FAPs, that express a transcriptional profile analogous to obesity-induced FAPs, including the matrix-modifying genes such as Timp1. We discovered corresponding ECM stiffening and decreased compositional diversity in obesity using highly precise rheological quantification and ECM proteomics of mouse and human adipose. We developed a tunable ex vivo 3D collagen hydrogel model and found that FAP adipogenic differentiation capacity is tightly regulated by ECM biomechanics. Most notably, many of the cellular changes and ECM properties induced by obesity persist following weight loss. The Aims of this grant are to: (1) Determine the role of Lyve1+ macrophage in directing the lineage allocation of maladaptive FAPs in obesity and weight loss. (2) Characterize the activity of FAP-expressed Timp1 in maladaptive ECM remodeling and its consequences for post-weight loss adipose physiology. To attain these objectives, we will utilize novel mouse models, advanced transcriptomic techniques, metabolic and biomechanical phenotyping to investigate of the mechanisms regulating the development of adipose ECM-encoded “metabolic memory” of previous obesity that persists despite subsequent weight loss. This work will address long-standing questions in the field regarding adipose-intrinsic cellular and molecular mechanisms promoting rebound weight gain.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Breathing disorders affect most individuals with obesity—and while respiratory muscle dysfunction occurs with elevated BMI, its underlying pathophysiology and potential therapeutic targeting in obesity-associated pulmonary compromise are largely uninvestigated. Mice subjected to diet-induced obesity (DIO), develop diaphragm weakness. In these animals, intra-diaphragmatic adiposity, and extracellular matrix (ECM) content quantitatively correlate with reductions in muscle contractile force. Resident mesenchymal cells called fibro-adipogenic progenitors (FAPs) are the source of all adipocytes and many ECM-depositing cells in the obese diaphragm. With DIO, diaphragm FAPs assume a proliferative, fibrogenic phenotype, and differentiate toward a Thy1- expressing sub-population previously linked to muscle degeneration. Thrombospondin-1 (THBS-1) is a matricellular protein produced by cell types including macrophages, adipocytes and FAPs. Physiologically induced by injury and ischemia, THBS1 levels chronically increase in obesity. A FAP mitogen and activator of TGF signaling, THBS-1 has been linked to fibrotic and degenerative changes in genetic myopathies. Global Thbs1 knockout subjected to DIO gain equivalent weight to WT controls, but do not exhibit FAP pool expansion or shift toward the Thy-1 expressing sub-type. Moreover, Thbs1-null diaphragms are protected from obesity-induced increases in adiposity and ECM deposition. Strikingly, Thbs1 knockout mice maintain normal diaphragm contractile force and motion with DIO. We hypothesize mesenchymal THBS1 promotes TGF-mediated, FAP-driven stromal expansion that can be targeted to mitigate diaphragm weakness and respiratory dysfunction in obesity. We will test the hypothesis through cell and tissue-based systems, in genetically engineered mouse models, and in human subjects: (1) First, we will employ primary cell culture to interrogate TGF and other putative THBS1 effectors as drivers of FAP proliferation, fibrogenesis and adipogenesis. We will then leverage a FAP-myobundle organoid system to determine whether the negative impact of THBS1 on muscle contractile function depends on FAPs. (2) Next, we will apply mouse models of inducible cell type-specific Thbs1 ablation (macrophage, adipocyte, FAP) to establish the cellular source of pathogenic THBS1 in obesity. We will also treat DIO mice with agents targeting THBS1/TGF-driven fibrosis to test whether this maneuver is sufficient to forestall or reverse pathological diaphragm remodeling and respiratory dysfunction. (3) Finally, we will analyze a translational weight loss cohort to define the relationship among circulating THBS1 levels, diaphragm motion (ultrasound measurement of diaphragm excursion amplitude), and diaphragm fibro-adipogenic remodeling (ultrasound measurement of muscle echo intensity). Together, the proposed experiments will define targetable cellular and molecular mechanisms underlying obesity-induced respiratory dysfunction.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Leishmaniasis, caused by Leishmania spp., is a neglected tropical disease with a significant global health burden. It is estimated to affect over 6.2 million people globally, with over 1 million incident cases anually. While cutaneous leishmaniasis is considered endemic to tropical and subtropical regions of South America, Central America, and Mexico, Leishmania has also been quietly spreading northward and establishing itself in the United States. Autochthonous transmission of Leishmania occurs in several areas, including Texas, where diagnosed cases of cutaneous leishmaniasis are predominately acquired locally. Understanding the impact of leishmaniasis in the US is compounded by the challenge of early detection and diagnosis, as symptoms resemble those of other common skin conditions, so cases are often misdiagnosed or missed completely. Thus, passive surveillance is ineffective for detecting the true burden of disease, and even the mandatory reporting in Texas frequently misses cases. We critically need multi-level epidemiologic studies that include human, vector, and animal infection dynamics to fully understand the specific risk of Leishmania transmission. However, such studies do not currently exist. Therefore, we propose conducting a One Health investigation into the transmission of Leishmania in two major metropolitan areas in Texas: Houston and Dallas. We will first analyze hospital-based data of locally acquired leishmaniasis from major medical systems in Houston and Dallas, which will inform our sandfly surveillance investigations to elucidate this parasite’s burden and distribution in these areas. Additionally, we will test shelter dogs to identify enzootic transmission. Our preliminary data suggests that the prevalence of Leishmania in Texas vectors and animals is significantly higher than previously published. Our central hypothesis is that Leishmania is endemic in sandfly vectors throughout Texas, with a disproportionate burden in north- central Texas, and is causing disease in major metropolitan areas. Our aims are as follows: (1) Determine the prevalence of Leishmania in sandfly vectors through untargeted and targeted collections informed by locations of autochthonous human cases in Harris and Dallas County, Texas, and (2) Determine the prevalence of Leishmania-positive domestic dogs in Harris and Dallas County, Texas. The proposed research represents a foundational study to evaluate disease transmission and provide critical data for public health decision making. Our long-term goals are to define the geospatial epidemiology of leishmaniasis in the southern United States, and to design targeted public health interventions to prevent transmission. This study brings together a collaborative team to begin to tackle a growing public health problem with no active surveillance testing funded by other means. Our findings will critically impact our understanding of leishmaniasis as this disease continues to invade the US and will allow us to identify high-risk populations for targeted prevention and intervention efforts.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Acari (mite and tick)-borne illnesses severely impact human health, and it is essential to understand the mechanisms maintaining the pathogens within their vectors for the development of countermeasures. Transovarial transmission (ToT) is used by mites and ticks to keep vector populations persistently colonized, but virtually nothing is known about the mechanisms of vertical transmission. While most work focuses on ixodid ticks, mites and argasids are understudied vectors most likely because of their complex biology. We developed the argasid–relapsing fever (RF) spirochete model (Ornithodoros turicata–Borrelia turicatae), which is one of the most comprehensive systems to study reproduction and ToT. We reported O. turicata vertically transmits B. turicatae by autogenous reproduction (laying of eggs without a blood meal), a unique aspect of argasid reproduction. This means that endemic foci of RF spirochete-infected ticks can be established quickly without needing a blood meal host. We have also identified an isolate of B. turicatae that is infectious by tick bite but fails to be vertically transmitted to offspring ticks. This phenotype enables us to study the mechanisms of ToT in this complex non-model system through comparative genomics. Also, our work in O. turicata genomics identified vitellogenins and the vitellogenin receptor, proteins that can be targeted by plant and veterinary microbial pathogens for ToT. Building on prior work and our developed entomological, bacterial, genomics, and genetics resources, we can now identify the molecular players involved in ToT. This application implements a functional genomics approach to test the hypothesis that ToT of RF Borrelia occurs throughout the reproductive cycles of female ticks and is driven by B. turicatae binding to vitellogenins and/or the vitellogenin receptor. The first aim will define the reproductive cycles of O. turicata and assess the dynamics of ToT for B. turicatae. This will be accomplished through the utilization of developed in vitro and in vivo tick feeding systems, tick colonies, and a diverse collection of B. turicatae isolates. The second aim will identify the molecular constituents of ToT. We identified gene loci and plasmids associated with a vertical transmission phenotype. We will build on these findings through comparative genomics and transcriptomics and identify B. turicatae surface proteins that bind to vitellogenins and/or the vitellogenin receptor. Using our developed genetics, we will transform the non- vertically transmitted B. turicatae isolate with plasmid and/or gene candidates to restore a ToT phenotype. While rickettsial, viral, parasitic, and RF Borrelia undergo ToT in mites and ticks, the molecular mechanisms are unknown. The completion of this project will result in the first identification of the molecular constituents involved with ToT of an Acari-borne pathogen. These findings will likely be broadly applicable and move the field closer to finding interventions that disrupt the life cycle of pathogen and vector.

NCI - National Cancer Institute

PROJECT SUMMARY Epstein-Barr virus (EBV) is a human DNA tumor virus that is present in nearly all cases of the endemic form of Burkitt lymphoma (eBL). In addition to the presence of an oncogenic gammaherpesvirus (GHV), eBL is characterized by translocations between the immunoglobulin locus and c-Myc proto-oncogene. Interestingly, this cancer occurs primarily in areas of the world where infections with the human malaria parasite Plasmodium falciparum (P. falciparum) are common. The epidemiology of these diseases supports the hypothesis that coinfections with EBV and P. falciparum cooperate to promote eBL development, but direct evidence to support this hypothesis is lacking. Since neither EBV nor P. falciparum readily infect mice, we coinfected mice with two well-established model pathogens, murine gammaherpesvirus 68 (MHV68) and P. yoelii, in an effort to better understand mechanisms by which GHVs and Plasmodium species might synergize to promote oncogenic mutations. MHV68 is a murine gammaherpesvirus that is genetically related to EBV and exhibits similar pathogenic phenotypes, especially coopting germinal center (GC) B cell reactions to establish long-term chronic infections in B lymphocytes. Like P. falciparum, P. yoelii stimulates polyclonal B cell activation and achieves high percentages of parasitemia prior to immune-mediated clearance. Using a newly developed PCR-based assay, we evaluated B cells following coinfections and detected the presence of Igh/Myc translocations that were only present in mice infected with both MHV68 and P. yoelii. This indicates that the defining eBL chromosomal abnormality can be generated through coinfecting mice with a GHV and Plasmodium parasite. Moreover, we found that ~90% of cells infected with MHV68 have expressed the GC mutagenic enzyme activation-induced cytidine deaminase (AID). These findings support our major hypothesis that MHV68 and P. yoelii interactions in GC B cells promote oncogenic Igh/Myc mutations. Experiments proposed in this application seek to understand how coinfection with P. yoelii impacts MHV68 latency, reactivation and transcription to promote translocations. We will identify viral gene products required and define the capacity of P. yoelii to stimulate B cell activation that leads to genomic instability. We will determine how GC B cell enzyme AID and coinfection-induced transcription synergize to facilitate non-immunoglobulin locus mutations, and we will define the oncogenic potential of coinfections over time. Results of this work will provide a more complete understanding of how altered infection dynamics due to GHV and Plasmodium species coinfections promote mutations such as those that define eBL.

NIGMS - National Institute of General Medical Sciences

Abstract Phosphatidylserine (PS), which constitutes only 2-10% of plasma membrane lipids, is a key phospholipid involved in intercellular communication/signaling. PS is actively sequestered to the inner leaflet of the plasma membrane and cells spend significant energy to maintain this asymmetry. PS externalization to the outer leaflet under specific contexts, such as after induction of apoptosis, allows phagocytes to recognize and engulf the dying cells via efferocytosis. Further, the engagement of PS on apoptotic cells by PS receptors on phagocytes (such as macrophages) induces anti-inflammatory signaling, which is a hallmark of efferocytosis during homeostasis. Despite our recognition of PS as a key apoptotic marker, nearly all studies consider PS as an isolated entity, and the PS-proximal proteins influencing PS recognition and signaling remain largely unexplored. Our exciting preliminary studies reveal that PS exposure on apoptotic cells occurs in patches and occurs adjacent to specific proteins, which can, in turn, influence efferocytosis. Our overarching hypothesis tested in this proposal is that proteins in the PS neighborhood on apoptotic cells critically influence/modulate the efferocytosis of dying cells, and the downstream signaling/responses elicited within phagocytes. In Aim 1, based on preliminary data identifying specific PS-proximal proteins, we test how they differ between apoptotic versus 'live’ cells engineered to expose PS (without caspase activation), the proximity of PS to specific scramblases that mediate PS exposure, and how different cell death modalities may alter PS- proximal proteins. In Aim 2, we test specific PS-proximal tetraspanin proteins in regulating efferocytosis ex vivo and in vivo, via models of tissue inflammation. The novel approaches taken here to define the PS neighborhood on apoptotic cells and its influence on efferocytosis address a long-standing key gap in our knowledge, with relevance to auto-inflammatory diseases.