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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.

2031-03-31

health research

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Defining the cellular basis of neurological dysfunction in models of ALG8 Congenital Disorder of Glycosylation

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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.

Up to $37K

2028-12-03

health research

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Defining the contribution of HIV diversity to infant immunity

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NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

ABSTRACT Developing an effective HIV vaccine has been remarkably challenging in part because attempts to elicit robust antibody responses to HIV immunogens have largely failed. Thus, intense effort has focused on understanding how humans elicit such responses to HIV infection, which occurs in only a subset of individuals. These responses have been primarily studied in adults living with HIV where broadly neutralizing antibodies (bnAbs) often have high levels of somatic hypermutation (SHM) that takes years to develop. Studies in infants suggest they may hold important keys to understanding how to elicit a broadly neutralizing antibody response. Leveraging samples from the Nairobi Breastfeeding Trial (NBT) conducted before the availability of antiretroviral therapy, we showed that infants develop broad neutralizing antibody responses much more commonly and more rapidly than adults. A similar finding was observed in a second cohort from South Africa, which showed that while only 19% of adults develop a broad response, 75% of children infected as infants develop such responses. We further showed that antibodies isolated from infants with a broad neutralizing antibody response have more limited SHM and are part of a polyclonal response. Here we propose to explore the basis for these unique responses in infants, focusing specifically on whether viral diversity plays a role. We hypothesize that the higher levels of viral replication in infants leads to an accelerated arms race between the virus and the B cell responses, driving the breadth and potency of the antibody response. We will test this hypothesis by deeply sequencing the viral population over time in a cohort of infants and a cohort of adults from the same region, collected over a similar time period. We will examine the relationship between viral diversity metrics and the breadth and potency of the neutralizing antibody response. The proposed comparative analyses of infants and adults are possible because of our long-standing collaborative studies that resulted in archived longitudinal samples collected from both infants and adults prior to the availability of antiretroviral treatment. The proposed study will be the first to examine the role of viral evolution in the unique infant antibody response to HIV and the first to directly compare this process between infants and adults.

Up to $537K

2028-05-31

health research

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Defining the contributions of hypothalamic microglia and perineuronal nets to obesity pathogenesis

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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.

Up to $889K

2031-01-31

health research

Free to search & build · $99 one-time to unlock the application pack · No subscription

Defining the critical functions of the stem-loop II motif in the lifecycle of astrovirus VA1

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NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Astroviruses are RNA viruses that commonly cause disease in humans, including gastroenteritis and fatal cases of encephalitis. Despite their broad human impact, astroviruses are understudied and many of the critical steps of the viral lifecycle are poorly characterized. There is limited understanding of host-pathogen interactions that facilitate viral replication, including the role of RNA motifs in the viral genome. Our lab was the first to cultivate astrovirus VA1 (VA1), the most common cause of astrovirus encephalitis to date. In a region overlapping with ORF2 and the 3' untranslated region, VA1 is predicted to encode a stem-loop II motif (s2m). Similar motifs with the same secondary structure have been identified in astroviruses and viruses of other viral families, with its importance in the viral lifecycle being virus-dependent. Using SHAPE-MaP on the full-length VA1 genome, we confirmed the formation of the s2m and its secondary structure. Mutagenesis of the s2m in a novel reverse genetics system for VA1 revealed the s2m to be essential. Deletion of the s2m or mutations that disrupt guanine- cytosine base pairs (GC-bp) that are critical for the secondary structure of the s2m result in virus that cannot be propagated. The mutant genomes can be rescued when complementary mutations are introduced into the s2m that restore GC-bp in the secondary structure. Mutagenesis of a position not involved in GC-bp was important but not essential for the function of the s2m. Capsid expression could not be detected from transfected genomes containing s2m mutations. Translation of capsid was also reduced by mutations of the s2m using a reporter system, and we have identified putative proteins involved in translation that may also bind to the s2m. We are now uniquely positioned to study the mechanism of action for the s2m in promoting the VA1 lifecycle using our novel tools that we have developed. Our central hypothesis is that the s2m facilitates viral translation through RNA-protein interactions, mediated by the s2m sequence, structure, and location in the genome. To test this hypothesis, we will take a combination of genetic and biochemical approaches to mechanistically understand why the s2m is essential. In other viral stem-loop structures, the loop region often serves as an important interaction site. We will define the role of the VA1 pentaloop for the function of the s2m by mutagenesis. Next, we will determine whether the function of the s2m is dependent on location in the genome. We will also assess whether the s2m must be encoded on the expressed RNA strand or if it can function independently. Using an RNA-pulldown, we have identified putative proteins that bind to the s2m that also mediate translation. We will confirm s2m-protein interactions and determine the effects of loss of function of these candidate proteins on the viral lifecycle. The findings of this project will provide important insights into the function of the VA1 s2m, address gaps in our knowledge of the VA1 lifecycle, and contribute to our larger understanding in RNA motifs in viral biology. These results will set the foundation for further dissection of the molecular biology of VA1, ultimately accelerating development of antivirals and vaccine-based approaches.

Up to $389K

2031-06-30

health research

Free to search & build · $99 one-time to unlock the application pack · No subscription

Defining the determinants of long term CAR-T persistence

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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.

Up to $703K

2031-03-31

health research

Free to search & build · $99 one-time to unlock the application pack · No subscription