University Of Colorado Denver

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Maintaining a stable brain vascular network is crucial for ensuring overall brain health throughout life. Perivascular cells, like pericytes and smooth muscle cells, are crucial to maintain the integrity of the brain vasculature. Loss of pericytes and smooth muscle cells are noted in Alzheimer’s Disease (AD) and affects vascular integrity, ultimately contributing to disease pathology. Perivascular fibroblasts (PVFs) are another cell population along the brain vasculature, however their role is largely unknown. PVFs express numerous extracellular matrix proteins that are uniquely found on arterioles and venules but not capillaries. My preliminary investigations indicate that PVFs maintain vessel structural stability, particularly along arterioles, in the healthy brain. Further, I find that arterioles are more tortuous in a mouse model of cerebral amyloid angiopathy (CAA), and this is associated with a significant reduction in PVFs. CAA is a small vessel disease characterized by the accumulation of amyloid- on vessels commonly observed in AD. Arterioles and their immediate off-shoots are important major regulators of blood flow into the brain. In doing so, they undergo extensive dilation and constriction events which is likely supported in part by extracellular matrix proteins expressed by PVFs. The goal of this proposal is to determine if PVFs regulate arteriole structure and dynamics in the healthy brain. Further, my goal is to understand if CAA contributes to PVF loss, altering arteriole structure and dynamics by affecting the expression of extracellular matrix proteins, ultimately exacerbating CAA. Understanding these important aspects of the brain vasculature could ultimately provide a potential for developing therapeutics aimed at limiting AD pathology and improve vascular function. The training I will receive under the guidance of Dr. Andy Shih, who is an expert in in vivo imaging and brain vascular physiology in health and disease, will enable me to achieve the goals of this proposal. My training is further supported by my advisory committee, consisting of Drs. Steven Greenberg, Richard Daneman, and Timothy Cherry who will enhance my training by providing guidance in CAA clinical pathology, PVF pathobiology and single-cell transcriptomics, respectively. Upon completion of these studies, I will have gained extensive knowledge of in vivo imaging, complex vascular physiology and single-cell transcriptomic approaches, in addition to PVF biology in heath and CAA pathology. These foundational studies and techniques are crucial components of my proposed independent phase described in this application and will propel my future goals of running an independent research group studying small vessel diseases in the brain. Further, with the support of Dr. Shih, my advisory committee, and the faculty at Seattle Children’s Research Institute in the Center of Developmental Biology and Regenerative Medicine, I will have expanded my experience in scientific communication, grantsmanship, networking, and mentorship. By continuing to strengthen these crucial skills during my training phase I will be well poised to guide a successful research group of my own.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT CANDIDATE: Matthew C. Babcock, Ph.D. is an Assistant Professor at the University of Colorado Anschutz Medical Campus (CU-AMC). Dr. Babcock aims to study the neural and renal contributions to hypertension with androgen deprivation therapy (ADT). He has developed preliminary data indicating that 1) men with lower testosterone concentrations have reduced baroreflex sensitivity compared to men with higher testosterone concentrations; and 2) baroreflex sensitivity in these individuals is correlated with circulating concentrations of the proinflammatory cytokine interleukin-6. In this proposal, he will translate these data by comprehensively assessing neural control of blood pressure in men who undergo ADT, and extend his observations to include renal control of blood pressure. The central hypothesis is that ADT increases inflammation and thereby reduces baroreflex sensitivity, increases sympathetic reactivity, and increases renovascular resistance. CAREER DEVELOPMENT PLAN: Dr. Babcock proposes to enhance his career development by: 1) acquiring new skills in the assessment of renal plasma flow, assessment of renal function biomarkers, and assessment of pro- inflammatory cytokines; 2) Advanced training renal diseases and physiology; 3) Working with a clinical population by recruiting men diagnosed with prostate cancer and studying them while they undergo ADT under the guidance of Dr. Elizabeth Kessler, a medical oncologist who specializes in prostate cancer; and 4) Refining his professional skills through formal course work, attendance and presentations at weekly Scientific Advancement and Grand Rounds, and at national scientific meetings. ENVIRONMENT: Dr. Babcock will train in an outstanding research environment supported by a multi-disciplinary team of mentors. The primary mentor, Dr. Moreau, is an NIA- funded professor at CU-AMC with a record of successful mentorship. She is an expert in the role of sex hormones and inflammation on cardiovascular function. She has extensive experience in utilizing the gonadal suppression study design proposed in the current study. Co-Mentor Dr. William Cornwell, III is also at CU-AMC and an expert in autonomic control of the circulation. Co-Mentor Dr. Jessica Kendrick and advisor Dr. Petter Bjornstad are also at CU-AMC and experts in studying renal function. Advisor Dr. Kessler is an expert in prostate cancer and ADT. Advisor Drs. Joyner and Farquhar are experts in autonomic function and the exercise pressor reflex. RESEARCH: ADT is a mainstay in the management of prostate cancer and the impressive survival rates among with men diagnosed with prostate cancer is largely attributed to the effectiveness of ADT. However, ADT increases the likelihood of developing hypertension and, accordingly, prostate cancer survivors who were treated with ADT are more likely to die early from cardiovascular diseases compared to men treated without the use of ADT. There is a critical need to elucidate the mechanisms underlying the increased cardiovascular disease risk in these patients to improve the lives of prostate cancer survivors.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Oligodendrocytes are glial cells in the central nervous system that form myelin, which are critical for the proper formation and function of neural circuits. During brain development, neural progenitor cells first give rise to excitatory neurons before gradually transitioning to the production of oligodendrocyte precursor cells (OPCs); a phenomenon known as the “neuron-glia switch”. Whereas oligodendrocyte differentiation from OPCs and myelin formation are well understood, we still do not know the earlier mechanisms that facilitate the neuron-glia switch and specify progenitors towards an OPC fate. This study aims to understand the developmental and molecular mechanisms that instruct neural progenitors to generate OPCs instead of neurons in the developing mouse neocortex. Our lab previously identified Sonic hedgehog (Shh) as a critical extracellular signal that initiates the neuron-glia switch during late embryonic development. However, while some progenitors generate OPCs in response to Shh, others continue to produce neurons. This suggests that co-existing neural progenitors differentially respond to Shh and require additional cell-intrinsic mechanisms to acquire an OPC fate. Our single cell RNA-sequencing analysis and my preliminary data indicate that both the Notch signaling pathway and the transcription factor Ascl1 are critical for promoting OPC specification from neural progenitors in response to Shh. Importantly, Ascl1 has recently been identified as a pioneer transcription factor capable of promoting chromatin accessibility to direct cell fates. Based on these data, I hypothesize that the Notch signaling pathway promotes OPC specification by regulating the response of neural progenitors to Shh in addition to establishing an epigenetic state primed for the OPC fate through Ascl1. I will test this hypothesis in two Specific Aims: 1) Define the functional role of Notch signaling in Shh-mediated OPC specification using in vivo genetic manipulations and ex vivo pharmacological approaches and 2) Test the hypothesis that Notch signaling cooperates with Ascl1 to promote an epigenetic state for specifying the oligodendrocyte lineage. Completion of these aims will significantly contribute to a better understanding of neural cell fate specification and oligodendrocyte development, which will allow for novel tools and methods to restore myelin following disease and injury.

NIH Research Projects · FY 2025 · 2023-08

Research Objective: The objective of our proposed research is to identify patients with rheumatoid arthritis associated interstitial lung disease (RA-ILD) that are at the highest risk for progressive disease and may potentially benefit from more targeted, and potentially less harmful, treatment. Unmet Health Need: Deaths from RA-ILD are not improving despite an overall decline in RA mortality. Novel, noninvasive methods are urgently needed to identify those with progressive RA-ILD so that interventions can delay the development of end-stage lung disease. Rationale: RA-ILD is a heterogeneous condition and the majority will experience disease progression resulting in lung transplantation and/or death. Despite the heterogeneity of RA-ILD, all RA-ILD is treated the same without taking in to account the known heterogeneity of this disease. This immunosuppressive-based treatment approach has led to unpredictable natural histories, inconsistent responses to treatment, and ultimately irreversible fibrosis leading to increased symptom burden, worse quality of life, and ultimately death. Hypothesis: Our overall hypothesis is that novel quantitative imaging and specific blood markers will be associated with a progressive phenotype in RA-ILD. Aims: We will evaluate the role of novel quantitative imaging (Specific Aim 1), peripheral blood telomere length (Specific Aim 2) and peripheral blood mononuclear cell (PBMC) gene expression (Specific Aim 3) in predicting progressive RA-ILD as defined by 12-month change in FVC% predicted. We will also explore the overlap and additive strength of each of these predictors in a composite profile (Specific Aim 4). Approach: To achieve the proposed aims, we will recruit 364 subjects with RA-ILD at the time of ILD diagnosis by an ILD pulmonologist and prior to treatment with lung-specific immunosuppression. Recruitment will occur at 4 expert ILD centers across the country with longitudinal collection of clinical, physiologic, and radiologic data with collection of serial biospecimens. The overall goal of this proposal is to identify the subset of RA-ILD patients that are at highest risk for disease progression following diagnosis by using novel imaging and specific blood markers. This risk stratification will help us determine whether or not immunosuppression should be started on an RA-ILD patient at the time of diagnosis. This knowledge will ultimately lead to less harm to patients with RA-ILD by decreasing exposure to immunosuppression in the subset that is most vulnerable. This proposal will lead to future precision-medicine based investigations for RA-ILD treatment and will lay the foundation to perform clinical trials that will determine the role of additional treatment pathways (e.g., observation, antifibrotic therapy, novel therapeutics and/or combination therapy) in RA-ILD, leading to reduction in harm and delaying the development of end- stage lung disease.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Inflammation is an evolutionarily conserved reaction with both beneficial and detrimental impacts on health. Excessive and prolonged inflammatory responses can promote damage to host tissues and contribute to numerous chronic human diseases while inadequate inflammation promotes susceptibility to infection. It is still not clear how the balance of these pro-and anti-inflammatory factors is determined and thus why inflammation persists and becomes chronic in some contexts, but not others. Due to the antimicrobial effects of inflammation, microbes have evolved strategies to interfere with the inflammatory response. Our published and preliminary studies have provided evidence that a secreted Listeria monocytogenes (Lm) virulence-promoting protein (p60) specifically targets the cDC1 subset of dendritic cells to promote the production of IL-10 by NK cells. IL-10 is a cytokine important for resolution of inflammation. Our proposed studies will dissect the mechanisms by which a specific domain present in p60 as well as proteins from numerous other bacteria acts to induce this response. Specifically, we investigate how newly identified receptors promote the response to p60, how p60 acts once in the cell, and unique features of p60 that support it ability to manipulate immune responses.

NIH Research Projects · FY 2026 · 2023-08

SUMMARY The enteric nervous system (ENS) is a complex network of neural crest-derived neurons and glia responsible for regulating key intestinal functions including motility, sensation, and secretion. Unfortunately, the ENS is frequently subject to injury leading to motor and other abnormalities. Often, this leads to debilitating disorders with few available treatment options. Excitingly, there is now mounting evidence of postnatal ENS injury-induced neurogenesis. Importantly, through work on adult animal models we have shown that Schwann cells (SC) can enter the gut alongside the extrinsic nerves and then differentiate into specific neuronal and glial subtypes (enteric neuro-gliogenesis). Thus, SC provide an unexpected source of cells to repopulate injured neurons and enteric glia. Furthermore, we have found that microbiome manipulation is a powerful method to induce Schwann cell-mediated enteric neuro-gliogenesis leading to functional recovery of the ENS and that this is mediated via the serotonin 5HT4 pathway. However, many aspects of postnatal ENS neuro-gliogenesis are not fully understood, including the functional impact of the neuro-gliogenesis from the SC, and the therapeutic potential for 5HT4 manipulation in human disease aiming for an enhanced SC-induced neuro-glial regeneration. Building on our published and preliminary results from mice and humans, our overarching hypothesis is that SC migrating into the gut from the gut’s extrinsic innervation are an important source for postnatal enteric neuro-gliogenesis, and that this ENS regenerative response is regulated by the microbiome via 5HT4. To test this novel hypothesis, we propose: Aim 1 will characterize postnatal SC-derived enteric neuro- gliogenesis after microbiome eradication/re-establishment using inducible, fluorescently labeled mice. We will also determine the functional effects of SC neuro-gliogenesis through extensive in vivo assays of motility and permeability and ex vivo characterization of cellular function using calcium imaging. Additionally, we will determine the functional effect of eliminating the SC entering the gut using a diphtheria toxin mouse model. In Aim 2, we will use two knockout mouse lines: (1) P0CreER/tdT::Tph1-/- and (2) P0CreER/tdT::Tph2-/- to determine the source of serotonin and the possible clinical applications of our findings by evaluating the SC response to a 5HT4 agonist, prucalopride. We will also identify specific metabolomic and transcriptomic profiles of the GI tract (mucosal and myenteric compartments). Finally in Aim 3, We will determine components of human microbiome-host crosstalk regulating SC-derived enteric neuro-gliogenesis in patients with slow colonic transit/dysmotility including the effect of 5HT4 agonists (i.e., prucalopride, tegaserod) on the ENS integrity/neuro- glial regeneration and function and determine metagenomic profiles in our patient cohort. Last, we will perform fecal transplants from these subjects into germ-free (GF) mice to evaluate ENS recovery. Results from this proposal will be key for the continued progress in targeted regenerative therapy for the treatment of congenital and acquired neuro-intestinal disease.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT My long-term career goal is to improve outcomes for pediatric patients with acute myeloid leukemia (AML), in part through development of improved modalities to detect residual disease and thus allow early identification and intervention for those patients at highest risk of relapse. My clinical experience as a pediatric oncologist specializing in treatment of myeloid malignancies informs my translational research focus in this area. This mentored career development award will facilitate my development into an independent translational physician- scientist by providing salary support and protected time to enhance my technical skills, knowledge base, and personal development in digital PCR (dPCR) assay development and validation, duplex NGS technology, NGS data analysis and bioinformatics, clinical trial design and development, networking, and collaboration. My mentoring team comprised of Dr. Craig Jordan (primary mentor), Dr. Dan Pollyea, Dr. Mike Verneris, and Dr. Chris Hourigan are all leaders in their respective fields and have a proven track record of fostering trainees and junior faculty to successful academic careers. The resource-rich environment on the Anschutz Medical Campus of the University of Colorado and the access to patient samples afforded me by the COG Myeloid Committee further contribute to a high probability of success for the proposed patient-oriented research. AML accounts for a disproportionate percentage of leukemia-associated morbidity and mortality in children, with relapse the leading cause of death in these patients. Measurable residual disease (MRD) has been shown in AML and other hematologic malignancies to be the single most valuable post-treatment predictor of relapse, but the existing clinical assays for MRD have significant limitations such that a high proportion of children who ultimately succumb to relapsed AML are actually MRD negative post-treatment. We hypothesize that application of molecular MRD assays to pediatric AML disease monitoring will be a sensitive predictor of disease burden and relapse. During the next 5 years I propose (1) to retrospectively evaluate the correlation between relapse and MRD positivity by mutation-based and chimerism-based dPCR assays in pediatric AML patients generally or post-transplant, respectively; (2) to retrospectively evaluate the correlation between relapse and MRD positivity by custom duplex NGS panels in pediatric AML patients; and (3) to prospectively validate the relapse predictive value of duplex NGS as a novel MRD modality in pediatric AML. Successful completion of this project will pave the way toward development of molecular tools such as dPCR and duplex NGS as improved MRD modalities that will enhance clinicians' ability to identify patients at highest risk of relapse and intervene to prevent its occurrence. This will lead to improved survival in pediatric patients suffering from myeloid leukemia.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ ABSTRACT RNA localization is critical for a diverse set of biological processes. The localization of an RNA depends on cis-elements, features inherent to the transcript, and trans-factors, effectors independent of the target transcript, which are often RNA binding proteins. Cis-elements that regulate RNA localization, often called "zip-codes," are often found in the 3′ untranslated regions (UTRs) of transcripts. However, for the more than 99% of the known localized RNAs, the cis-elements that regulate their localization are unknown. Recent work from the Taliaferro lab identified several 260 nucleotide RNA sequences within the 3’ UTRs of some neurite-localized RNAs that were necessary and sufficient for neurite RNA transport. These were identified using a massively parallel reporter assay that screened approximately 10,000 RNA sequences drawn from endogenous 3’ UTRs for their ability to traffick a reporter transcript to neurites. Interestingly, 100 nt subsequences of these 260 nt active elements were not capable of directing RNA transport. This indicates that (1) the minimal regulatory elements are quite large (likely longer than 100 nt) and (2) the true character of the localization regulatory elements remains unknown. In this work, it is proposed to comprehensively characterize the previously identified RNA localization regulatory elements and zero in on their important features. This will be done by generating a pool of 10,000 RNA sequences based on the previously identified 260 nt zipcodes. Each sequence in this pool will contain defined deletions of varying sizes that span the length of the zipcode. By integrating these mutants into the 3’ UTR of a reporter transcript assaying which of them retain the ability to direct localization of the reporter to neurites, a quantitative readout of the functional importance of each nucleotide within the 260 nt zipcode will be obtained. From this, a clear picture of the important features that make up active localization elements will arise, facilitating their identification in other localized RNAs. The large size of these zipcodes suggests that their secondary may be important for their activity. To test this, their secondary structure will be determined using chemical probing techniques. To test the functionality of the structure, thousands of mutants that disrupt RNA structure as well as compensatory mutants that restore it will be generated. As above, the ability of each of these mutants to drive a reporter transcript to neurites will be tested. In this way, RNA structure and function will be directly related. Answering these questions will help in understanding the underlying mechanisms of RNA localization as very few examples of RNA localization have known mechanistic underpinnings. Identifying the mechanism of RNA localization under physiological conditions is important to being able to understand potential dysfunction in disease states.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Up to 20% - or nearly 7,000,000 – adults with diabetes (T2DM) concurrently experience disordered eating behaviors (DEBs, e.g., significant overeating with loss of control), resulting in higher body mass index and lower adherence to diet and exercise recommendations. DEBs are often disregarded by medical providers and patients, and therefore undertreated in clinical settings. Research is limited on whether treatment for DEBs impacts diabetes self-management and glycemic functioning. To address this, we developed the Balanced and Empowered EaTing (BEET) in Diabetes, or the BEET Diabetes Program, a novel cognitive-behavioral therapy- based program to designed to specifically treat DEBs in T2DM and improve diabetes self-management. By designing with implementation science methods and engaging integrated behavioral health providers (BHPs) in real clinical settings, we aim to 1) address an important treatment need for people with T2DM and concurrent DEBs, and 2) improve the integration of BHPs in collaborative diabetes care in clinical settings. Candidate and Mentors: I am an Assistant Professor in the Department of Psychiatry at the University of Colorado. As a clinical health psychologist with training in implementation science methods, my program of research aims to implement and evaluate evidence-based programs for mental health conditions that could worsen diabetes and other endocrinopathies. I have built a strong mentorship team to guide my training including primary mentor, Dr. C. Neill Epperson (neuroendocrinology of risk and resilience), and co-mentor, Dr. Bethany Kwan (dissemination & implementation science). Research and Training: I propose short term scientific, educational, and training goals that will build upon my prior research training through three research aims: 1) engage BHPs to refine the BEET Diabetes Program for implementation in clinical settings, 2) evaluate the feasibility of the BEET Diabetes Program, and 3) determine the reach and estimate the effectiveness of the BEET Diabetes Program on patient-reported and clinical outcomes. The complementary training goals to establish scientific independence through training in: 1) advanced mixed-methods analytical skills by learning configurational comparative methods for implementation science, 2) clinical trial design for behavioral research and longitudinal data analysis, and 3) methods for biological data collection, analysis, and interpretation in T2DM. Summary: DEBs in T2DM are a serious yet under-recognized clinical problem. There is a significant dearth of research examining associated risk factors and treatments of DEBs in T2DM. My ultimate career goal is to transform our approach to the prevention of endocrine- and metabolic-conditions by developing and implementing evidence-based mental/behavioral programs in clinical settings. Completion of this career development award will accelerate my path towards becoming a national expert in the biobehavioral management of endocrinopathies.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY Polymorphisms within major histocompatibility complex class II (MHC II) genes confer significant risk for developing type 1 diabetes (T1D) in both murine models and humans. MHC class II molecules function to present processed antigens to CD4 T cells, and recent studies have identified a number of different post-translational modifications (PTM) of self-antigens in T1D including peptide fusion, deamidation, citrullination, and disulfide bond formation (S-S). Autoimmune T cell responses to neoantigens formed in peripheral tissues may explain how and why T cell responses are not subject to usual thymic education and tolerance mechanisms. Disulfide bond formation is an important PTM, with implications for structure, function, and stability of numerous proteins. We have shown that an epitope from islet amyloid polypeptide (IAPP), which is co-secreted with insulin by pancreatic beta cells, forms a disulfide bond that activates diabetogenic CD4 T cells when presented by the non- obese diabetic mouse (NOD) MHC class II molecule, IAg7. Our overarching goal is to study the pathogenicity of CD4 T cells responding to disulfide modified self-peptides in mouse and human T1D. The first 20 amino acids of IAPP, termed, is the target antigen for a highly diabetogenic CD4 T cell clone, BDC-5.2.9. KS20-reactive CD4 T cells can be detected in the pancreatic islets of prediabetic and diabetic NOD mice. We showed that the KS20 N-terminal disulfide loop contributes to a large portion of TCR contact, necessary for T cell activation. Increasing evidence indicates that pancreatic beta-cells undergo oxidative and endoplasmic reticulum stress during T1D development that can lead to the generation of reactive oxygen species (ROS), which are capable of inducing post-translational modifications including disulfide bond formation. Thus a potential mechanism exists to form disulfide modified antigens within pancreatic beta-cells that are capable of activating diabetogenic CD4 T cells. Taken together, this leads us to hypothesize that disulfide bonds are a common post-translational modification formed during T1D development that activate islet-antigen specific CD4 T cells. Understanding T cell responses to disulfide modified antigens will enhance our understanding of T1D disease development and identify therapeutic targets to prevent tissue-specific disulfide bond formation and potentially prevent T1D. We propose to determine epitopes for disulfide-reactive CD4 T cells and their necessity for NOD diabetes development and identify T cells responding to disulfide modified self-antigens in patients at-risk and with newly diagnosed type 1 diabetes.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Cerebral Autosomal Dominate Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a common small vessel disease (SVD) that is characterized by microvascular dysfunction leading to ischemic stroke and vascular dementia. CADASIL is caused by mutations in the NOTCH3 gene expressed by smooth muscle cells (SMCs), that are associated with larger vasculature such as arteries/arterioles, and pericytes that wrap around capillaries. These mutations induce extracellular receptor aggregation and complex formation with other proteins including matrix metalloproteinase inhibitor, TIMP3. TIMP3 therefore accumulates around the microcirculation causing impaired cerebral blood flow (CBF) regulation. Downstream of TIMP3 accumulation, the pathomechanism includes epidermal growth factor receptor (EGFR) inhibition, resulting in decreased neurovascular coupling (NVC) and ATP production. While this pathomechanism has been investigated in other vascular cell types (endothelial cells and SMCs), pericytes remain understudied. Pericytes are an abundant, highly heterogenous population, that includes mesh pericytes which display fast on/off contractile responses and typically reside on 1-4th order capillaries. Recent studies have revealed a KATP channel, Kir6.1, is uniquely highly expressed in pericytes compared to other cells in the microvascular domain. This channel has proven to be highly relevant in CBF regulation where activation of the channel from low ATP, results in pericyte hyperpolarization, ensuing vasodilation to enhance regional cerebral perfusion to meet energy demands. Due to the CADASIL’s early presentation of neurovascular dysfunction, this proposal focuses on mesh pericytes and their impairment. My preliminary data show that, in CADASIL, mesh pericyte Kir6.1 channels are hyperactive in basal conditions, indicating dysregulation of channel activity. However further characterization of the channel is needed to elucidate potential treatment targets. Specifically, how TIMP3 and EGFR signaling influence Kir6.1 channel activity. This proposal’s first Aim is to characterize Kir6.1 channel properties using a bimodal approach looking at both function and expression in cerebral mesh pericytes. The goal of the second Aim is to expose the mechanism underlying Kir6.1 channel dysfunction including TIMP3 exogenous application and genetic knockdown. This Aim will also employ rescue techniques via EGFR ligand, HB-EGF, to restore Kir6.1 channel function, which due to its prominent expression in cerebral pericytes may be key to restoring CBF dysregulation in CADASIL. Completion of this proposal will fill a significant gap in the literature involving cerebral mesh pericytes and the devastating cerebrovascular disease, CADASIL which currently has no cure and limited treatment options.

NIH Research Projects · FY 2025 · 2023-07

Effective clinical research into rare and ultra-rare neurologic and neurogenetic disorders requires a nationwide, collaborative network of neuroscience clinical research centers. The objective of the proposed University of Rocky Mountain NeuroNEXT Clinical Research Consortium (UNCOMON CRC) is to integrate expert from adult and child neurology, neurosurgery, neuroimaging, neuropsychology, and neurogenetics both the University of Colorado Hospital and Children's Hospital of Colorado on the University of Colorado Medical Campus into a large collaborative clinical research enterprise that exceeds start-up, retention, and operational goals of the NeuroNEXT network. The UNCOMON CRC is composed of recognized leaders in clinical research who have successfully participated in federally funded and multi-center clinical trials as well as local investigator-initiated therapeutic and biorepository The UNCOMON CRC will employ established clinical research and educational resources in conjunction a robust clinical research infrastructure to efficiently contribute to the NeuroNEXT network and advance the and research careers of the next generation of junior faculty nd fellows. We plan to achieve our goal the execution of the following specific aims: Aim 1: To establish a leadership network consisting of study adult and pediatric research directors, career enhancement director, DEI recruitment specialist, clinical coordinator, NeuroNEXT fellow, and operations manager; Aim 2: To establish an UNCOMON clinical consortium of collaborative co-investigators from adult and child neurology, neurosurgery, neuropsychology, and neurogenetics that encompass all subspecialty fields of clinical Aim 3: To establish a referral network to optimize recruitment and enhance outreach to minority and rural populations; and Aim 4: To develop and execute clinical research training and enhancement programs f or young i investigators using the collective expertise of the UNCOMON CRC. UNCOMON CRC will work efficiently to accelerate site qualification and IRB approval, employ multiple to optimize subject recruitment and retention, and carefully monitor research operations to ensure data a quality.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Immune checkpoint inhibitors (ICIs) are biologic drugs that have revolutionized cancer treatment by targeting specific inhibitory receptors, or their ligands, on T lymphocytes and thereby restoring immune system surveillance. Despite significant improvements in therapeutic responses, ICIs cause ‘immune-related adverse events’ (irAEs). ICI-induced immune-mediated damage to the kidneys exhibits two phenotypes including glomerulonephritis and acute kidney injury with interstitial nephritis. These kidney toxicities were not anticipated in preclinical testing but now occur in patients receiving ICIs, at a mean of 3 months of therapy. Within 5 years of receiving ICI therapy, new onset chronic kidney disease and declines in glomerular filtration rate have been observed in 20% of cancer patients. Several issues mask our understanding of ICI nephrotoxicity: 1) the ability to predict which patients will exhibit the toxicity, 2) how to sensitively detect subclinical injury prior to significant elevations in serum creatinine, and 3) poorly elucidated relationships between drug disposition, the immune system, kidney biology, and antitumor responses to inform nephrotoxicity mechanisms. There is an urgent need to develop preclinical models and assessments that can inform irAEs as ICIs are becoming the primary therapeutics for some cancers. This proposal will advance a novel mouse cancer model with a humanized immune system to identify mechanisms of kidney immunotoxicities associated with ICIs. Pharmacological interventions will evaluate the contributions of 1) tumor type, 2) drug exposure kinetics, 3) on-target versus off- target responses, and 4) human CD8+ and CD4+ lymphocyte signaling, in the mouse model of ICI nephrotoxicity. The animal model will bridge preclinical testing and clinical practice, in that the proposal will also evaluate cancer patients prescribed ICI biologics for kidney toxicities. For patients, mechanistic evaluations will be performed using quantitative systems pharmacology (QSP) and pharmacokinetic approaches. The central hypothesis of this proposal is that a novel humanized animal model recapitulates the renal pathology observed clinically with ICIs, and in combination with human biospecimens from cancer patients prescribed ICIs and novel QSP modeling can inform relationships between drug disposition, the immune system and kidney biology, antitumor responses, and nephrotoxicity to understand mechanisms of ICI renal irAEs. The proposal consists of two independent Specific Aims to systematically evaluate kidney irAEs in an animal model and clinical patients receiving ICIs. We have assembled a multidisciplinary team with expertise in clinical oncology, nephrology, immunology, pharmacokinetic and pharmacodynamic modeling, proteomics, and toxicology across two NCI-designated cancer centers to complete the proposed studies. The proposed research has high translational impact due to the current unmet need to predict, detect, and monitor kidney injury caused by ICIs and other immunomodulatory drugs with the goal of preventing long-term chronic kidney disease.

NIH Research Projects · FY 2025 · 2023-07

Intermuscular adipose tissue (IMAT) is marbled within skeletal muscle and appears to play a key role in the obesity-induced risk of type 2 diabetes. What is not known is how IMAT promotes decreased muscle insulin sensitivity. There is a critical need to understand how IMAT contributes to the risk of obesity-induced diabetes, and how to intervene to minimize IMAT-induced muscle metabolic dysfunction. The overall objective for this project is to determine the impact of weight loss on IMAT secretion of fibronectin and inflammatory cytokines and eicosanoids. Our central hypothesis is that IMAT secretion of fibronectin promotes muscle insulin resistance, and IMAT secretion of inflammatory cytokines and eicosanoids causes muscle inflammation, both of which are diminished by weight loss. The rationale that underlies the proposed research is that clarifying the extent to which weight loss alters the IMAT secretome will inform development of interventions to modify IMAT and improve muscle insulin sensitivity in obese individuals. We propose two specific aims: Specific Aim 1. Evaluate the impact of weight loss on IMAT secretion of fibronectin and the role of fibronectin in the IMAT secretome to decrease insulin sensitivity in vitro. Preliminary data inform our working hypothesis that IMAT secretes fibronectin that decreases muscle insulin sensitivity and is attenuated after weight loss in humans. In vitro experiments will measure the extent to which IMAT fibronectin secretion explains IMAT-induced muscle insulin resistance. We will study individuals with obesity before and after a 12-week weight loss intervention. IMAT will be sampled using an ultrasound guided IMAT biopsy technique, insulin sensitivity measured using insulin clamps, and IMAT content measured using MRI. Specific Aim 2 – Determine the influence of weight loss on IMAT secretion of pro-inflammatory cytokines and eicosanoids and potency to cause inflammation and decrease insulin sensitivity in vitro. We hypothesize, again based on preliminary data that the IMAT secretome is less inflammatory after weight loss in obese individuals, with decreased potency to promote muscle inflammation and insulin resistance. Muscle inflammatory response and insulin sensitivity with IMAT secretome exposure will be compared before and after weight loss in vitro. The proposed research is innovative because it represents a new and substantive departure from the status quo by testing specific IMAT secreted paracrine signals rather than clinical associations with IMAT content. These contributions will be significant by identifying the first IMAT paracrine signals that impact muscle and revealing the plasticity of IMAT through weight loss, providing proof of concept that IMAT is a therapeutic target to combat muscle metabolic dysfunction.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Clinical decision-making in subspecialties, like pediatric neuro-oncology, is becoming increasingly data- driven and complex. Artificial intelligence (AI) is a powerful tool that can help distill this expanding dataset to present the clinician with the right information at the right time. AI has had little success in clinical applications so far, but new methods (like OpenAI's DALL-E or GPT3 models) are now in the public eye, clearly demonstrating the power of the technology generally. Effective translation of that technology into the clinical setting requires a comprehensive understanding of the specific clinical setting, such as personnel/roles, data/technology used, clinical goals and workflows. Human-Centered Design (HCD) is a solution framework that emphasizes the needs of the people who perform a specific task and is well suited to facilitate the design of clinical AI. However, HCD is challenging to execute in this space because it is difficult and expensive to assemble a team of experts that spans clinical medicine, artificial intelligence, visualization, and social sciences. Academia and industry have established multidisciplinary HCD/AI teams but are seeking solutions for filling interdisciplinary leadership roles in these teams. I previously published a deep learning model for classifying pediatric suprasellar tumors from preoperative MRI. My model performed as well as human experts on the same dataset (86%), which was also congruent with previous studies on human expert performance on the task. In addition, pediatric suprasellar tumors are almost always diagnosed via surgical pathology, with roughly 8% of patients being radiographically diagnosed. Preliminary data (Aim 2) suggests that we can improve the deep learning model performance up to 95% by incorporating a Bayesian methodology to estimate model uncertainty. Additional preliminary data (Aim 3) indicates that embedding my model into Google's What-If Tool (WIT) can help clinicians radiographically diagnose these tumors with less perceived difficulty and greater perceived confidence. Therefore, this proposal's central hypothesis is that explainable AI solutions can improve human experts' pediatric suprasellar tumor radiographic diagnosis beyond the current performance levels. Moreover, using an HCD approach, Google's What-If Tool (WIT) can be adapted into the clinician's workflow in a manner that will result in adoption of this assistive technology. I will investigate my aims, specifically designed to provide functional knowledge in these topics to enable me to effectively lead a multidisciplinary team of subject-matter experts in developing robust clinical AI tools. I am guided by an expert team of mentors representing pediatric neuro-oncology, neurosurgery, AI, visualization, and HCD. Completion of this proposal will significantly contribute to my career goal: to be a leader in the application of HCD to develop clinical AI technology that supports pediatric neuro-oncology patient care.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Three-dimensional (3D) microscopy offers many promises for biological investigations and medical applications. However, it is currently limited to well-resourced laboratories in settings with established infrastructure. Many 3D microscopy techniques (e.g., confocal) rely on focusing light at an array of small locations within the sample, which requires expensive and specialized equipment. Optical Projection Tomography (OPT) is a 3D imaging technique that utilizes traditional microscopy equipment; instead of focusing the light at specific locations, OPT images a sample from many angles to reconstruct a 3D volume. It is a very effective method for 3D imaging of small translucent objects (e.g., mouse fetuses, parasites, and large bacteria). While it is possible for OPT to be a lower-cost method of 3D microscopy, existing systems remain expensive and large. We propose to take advantage of the ubiquitous and high-quality computing and imaging hardware available in smartphones to make an OPT device that is inexpensive and extremely portable. We will create a smartphone extension that robustly images a rotating sample and uses the phone’s computational hardware to reconstruct the 3D volume. Two imaging modalities will be pursued: visible-band attenuation microscopy (e.g., brightfield) and luminescent microscopy (e.g., bioluminescent). The components of the smartphone extension are either 3D printed, laser cut acrylic, or readily commercially available. Thus, the cost of manufacturing the device is extremely small and the total weight of the device is very low; the total cost of all components will be less than $50. The components of the extension are easily assembled on site, permitting the device to be transported with in a small package. Due to its low cost and size, the OPT microscope can also serve as a useful tool for educational purposes (e.g., as part of an undergraduate laboratory course involving optics) and for generating real data for tomographic algorithm development. To validate the device, we will build two phantoms with three-dimensional features that allow us to evaluate the Modulation Transfer Function: one for attenuation microscopy and another for luminescent microscopy. Aim 1: Build a visible-band tomographic microscope extension to a smartphone. Aim 1A: Implement 3D cone-beam reconstruction from visible-band sinogram data for samples approximately 10 mm in size with approximately 10 µm resolution. Aim 1B: Improve the image resolution with a multi-lens optical system. Aim 2: Implement bioluminescent tomographic microscopy by appropriately modifying the tomographic reconstruction algorithm. Aim 3: Build two shelf-stable 3D phantoms with features of sizes varying from 1 µm to 200 µm.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The world around us has a statistical structure that we can use to improve our choices. Learning the underlying structure by identifying key features, such as the rate of change, is useful for adapting and optimizing our decision-making strategies. However, learning these features requires accumulating evidence across multiple timescales: a short timescale that considers explicit evidence for the current decision, and a long timescale that supports latent environmental feature inference. In the brain, evidence accumulation across timescales necessary for flexible decision-making should therefore engage contextual memory in regions such as hippocampus (HC). This proposal aims to identify cognitive strategies and neural mechanisms humans use to accumulate evidence across timescales for adaptive decision-making. Using an interdisciplinary approach that utilizes computational modeling to develop and validate human behavioral and human electrophysiological experiments, I will 1) identify the variety of decision strategies humans use to support multi-timescale inference, 2) model plausible neural mechanisms of human cognitive strategies, and 3) define HC’s role in implementing multi-timescale inference. This work is in line with the BRAIN initiative’s mission to link behavior and function and priority research areas 5 (Theory and Data Analysis tools) and 6 (Human Neuroscience). With my outstanding mentor team, who have combined expertise in theory and experimental work, the mentored phase of this grant will provide me with 1) additional research skills in both static inference models and neural-circuit modeling and 2) career development through personalized mentorship, writing, and scientific communication training. The University of Colorado Boulder offers an ideal environment for this work, with numerous resources between the departments of Applied Math, Psychology and Institute for Cognitive Science. Additionally, with the availability of many programs and seminars online, resources at co-mentor institutions University of Pennsylvania and University of Houston are also accessible. The independent phase research will combine this additional training with my previous experience in human electrophysiology and signal processing to study the role of HC in flexible decision-making, analyzing human neural recordings from epilepsy patients while they perform a multi-timescale decision-making task recorded by my collaborators at University of Utah. My long term goals are to launch my own lab that applies a multimodal approach of theory, human behavior, and human neural electrophysiology to identifying the cognitive and neural strategies associated with flexible decision-making and the impacts that pathological disruptions have on these processes.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Multiple Sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system that afflicts nearly 1 million people in the United States alone. While MS is classically regarded as a white matter disease, gray matter lesion load may exceed that of the white matter in patients with MS. The pathological features of lesions differ between gray and white matter lesions. Interestingly, in lesions that span white and gray matter regions, display pathological hallmarks of white matter-only lesions, suggesting that cellular environments distinctly regulate oligodendrocyte loss and regeneration. Furthermore, attempts at remyelination are more frequent in cortical versus white matter lesions regardless of patient age or disease duration. These findings suggest that remyelination of gray matter regions may be specifically limited by decreased functional integration of new oligodendrocytes compared to white matter regions. Understanding the regional differences in oligodendrocyte loss and regeneration represents a clear unmet need in the MS research community. A major limitation to understanding these regional differences is the inability to monitor the dynamics of oligodendrocytes in white matter in the living brain. To overcome this obstacle, we will use new optical methodologies to determine regional variability in oligodendrocyte cell behavior. We propose to use the superior penetration depth of three- photon excitation fluorescence and longitudinal in vivo imaging to determine the effects of circuit-specific neuronal activity and demyelinating injury on oligodendrocyte lineage cells in both superficial and deep areas of the adult brain. The objectives of this proposal are: 1) evaluate how behaviorally-relevant neuronal activity regulates gray and white matter oligodendrogenesis, 2) to elucidate whether behavioral interventions can equally promote oligodendrocyte regeneration of the gray and white matter. The overall hypothesis of this proposal is region-specific rates of oligodendrocyte precursor differentiation and integration govern the proportion of myelination in healthy, adaptive, and regenerative contexts. This proposal represents a novel synthesis of cutting-edge approaches in optical physics and oligodendrocyte biology, and breaks new ground in understanding the mechanisms underlying the regulation of gray and white matter oligodendrocyte plasticity and regeneration.

NIH Research Projects · FY 2025 · 2023-07

Research Summary Epidemiological data have indicated that the use of products with cannabidiol (CBD) and other cannabinoids have increased dramatically among adults over the age of 65. Recent survey data collected in Colorado indicate that older adults who use cannabinoids believe that it helps alleviate pain, helps improve sleep quality, and decreases negative affect (i.e., depression, anxiety). Older adults may also be taking cannabinoids like CBD because they believe it might have a positive impact on the progression of dementia and cognitive decline, as popularized by a recent Discover magazine article. Given the aforementioned socioeconomic trends, the preclinical data suggesting that CBD may be neuroprotective, and our preliminary data suggesting that CBD impacts key biomarkers of inflammation and oxidative stress, it is clearly time to assess the impact of these products on the cognitive health of older adults who are at high risk for AD. The significance of this question is underscored by both the rapidly aging population in the U.S. (>60 million adults over the age of 65 by 2025), the prevalence of MCI (~15–20%) and Alzheimer’s (about 10% or 6-7 million), as well as the enormous mortality, morbidity, and socioeconomic costs of AD. The proposed research will address this public health research need with a gold standard, 24 week, randomized, double-blind, placebo-controlled clinical trial (RCT) comparing full spectrum hemp-derived CBD, to CBD alone, and to placebo. The proposed study will determine whether CBD impacts the progression of biomarkers related to neurodegeneration and Alzheimer’s disease and determine whether CBD impacts measures of anxiety, depression, sleep, and pain in a population at high risk for AD. The research will also determine whether any effects of CBD on outcomes are mediated by the effect of CBD on biomarkers of inflammation and oxidative stress and/or changes in endocannabinoids. Given the number of older adults at risk for AD who are using CBD products and given that these individuals believe that these products are helpful, the proposed well-controlled trial will have a significant impact by informing the public about the effects of CBD, regardless of the outcome of the analyses (positive or negative).

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The University of Colorado MD/PhD Program (CU-MSTP) was established in 1983 and has successfully trained 263 dual-degree students to expand the physician-scientist workforce. CU-MSTP has been continuously funded by NIGMS since 1992, with our program recently growing from ~ 75 students to a current total of 86 students. Our Program Mission is to train a diverse cadre of dual-degree students to become outstanding physician- scientists and future leaders in biomedical research. To accomplish this mission, we select students from an increasing national applicant pool (from 386 in 2017 to 567 in 2021), seeking out those candidates whose record of research, academic, and leadership achievements are exemplary. CU-MSTP recruits 10-11 diverse applicants per year from across the nation, being one of two programs serving the nine Mountain West states. CU-MSTP uses a holistic review process to identify applicants with experiences, attributes, and metrics that project likely success in our program and as physician scientists. Our current roster of 86 students (48% female; 16% URM, 8% disabled, 15% 1st generation college, and 25% disadvantaged) come from across the nation, from a variety of educational backgrounds, and with significant prior research experience and publications. Several recent institutional changes enhance the training environment for CU-MSTP students. Our MSTP students benefit from an innovative new medical school “Trek” curriculum implemented in 2021 that incorporates novel early longitudinal integrated clinical training. The resulting new MSTP “Switchback” curriculum connects rigorous research training with cutting-edge medical training in a highly integrated, flexible, and individualized manner, maintaining the strengths of the prior CU-MSTP training plan and keeping the ~8-year timeline for completion of both degrees. For thesis research, our students choose from a wide variety of fields with MSTP faculty in 15 graduate training programs across three campuses (Anschutz Medical Campus, National Jewish Health, and the University of Colorado-Boulder). Participating CU-MSTP faculty have a combined annual grant income of ~$60M. To enhance the success of our students, we provide career guidance throughout the training period, and we work diligently to place our graduates in elite residencies and fellowships. Our students have excelled in metrics that predict career success as physician scientists, including publishing impactful peer-reviewed manuscripts, obtaining early research funding, and matching in competitive, research-focused residencies. CU School of Medicine provides strong support to CU-MSTP, with a total of $16.97M total support in the 2017-2021 five-year cycle. The growing Anschutz Medical Campus provides state-of-the-art education, research and clinical facilities, and enjoys significant momentum with recent major campus initiatives including the new Center for Health AI, RNA Biosciences Initiative, and Department of Biomedical Informatics. In sum, the continuous improvement of the Program, enlarging applicant pool, quality of recruited students and training faculty, student outcomes, institutional support, research funding and environment justifies our request for 21 training slots/year.

NIH Research Projects · FY 2025 · 2023-07

Single-stranded circular RNAs (circRNA) are enriched in cancers, yet the regulation and biological function of most circRNA remains unclear. Our long-term goal is to understand the role of both human papillomavirus (HPV)-derived and endogenous circRNAs in both infectious and neoplastic diseases. The specific goal of this proposal is to generate a more comprehensive understanding of the function and regulation of circRNA that are present in head and neck squamous cell carcinoma (HNSCC). Our central hypothesis is that many circRNA are coding RNA that differ in regulation and function from their corresponding full-length RNAs in ways that promote the development of HNSCC. In preliminary studies, we have developed innovative circRNA-Seq and Polysome RNA-Seq protocols and found that many circRNA with coding potential are enriched in HNSCC tumors compared to adjacent non-tumor mucosa. In addition, our discovery of HPV16 circular E7 RNA (circE7), which is translated to the E7 oncoprotein, offers a novel tool to understand the mechanistic and physiological regulation of circRNA formation and function. With these innovative tools and extensive preliminary evidence supporting feasibility, we propose to expand our understanding of circRNAs in HNSCC in three related aims. First, a comprehensive transcriptomic profile from up to 50 HPV+ and 50 HPV- HNSCC tumors and adjacent tissue will be generated, including RNA-Seq approaches that enrich for circRNAs. Moreover, circRNA identified in this transcriptomic profiling and circE7 will be assessed for their potential as prognostic biomarkers in a validation cohort of archived HNSCC. Second, using the HPV16 circE7 locus, we will determine the cis and trans elements that regulate circRNA formation in HNSCC and determine how they are physiologically regulated. Third, we will combine HNSCC circRNA-Seq datasets with recently completed polysome RNA-Seq to identify endogenous circRNA that are likely to be translated. These prioritized candidates will be validated and characterized through rigorously controlled overexpression and knockdown experiments including the use of circRNA-derived specific peptide antibodies. The investigative team includes experts in circRNA metabolism, HPV biology, pathology, statistical genetics, head and neck cancer, and epidemiology, thus ensuring we have the skills and resources to execute the proposal. Completion of the proposal will provide both a global and detailed understanding of circRNA and their regulation in HNSCC.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT The overall goal of this Program is to understand the role of MUC5B in establishing a vulnerable lung and the transition of a vulnerable lung to a lung characterized by persistent injury of bronchoalveolar epithelia and activation of lung fibroblasts. While our findings have identified a novel molecule (MUC5B) and target (bronchoalveolar epithelia) for IPF, only ≈5% of individuals with this genetic variant develop usual interstitial pneumonia (UIP) on HRCT scan, suggesting the need for another insult (a ‘second hit’) to initiate and intensify the fibroproliferative process. Based on our preliminary findings, we postulate that while overexpression of MUC5B places individuals at risk of developing IPF by causing persistent homeostatic ER stress of bronchiolar epithelia, fibroblast recruitment and pro-fibrotic programming requires a second hit to the bronchiolar epithelia resulting in detrimental ER stress and recruitment and activation of fibroblasts. Our Program includes 3 Scientific Projects and 4 Cores, and our unifying scientific themes include: 1) IPF is initiated by enhanced expression of MUC5B (first hit) that establish a vulnerable lung characterized by persistent homeostatic ER stress (without substantial UPR or apoptosis); 2) secondary injury to the bronchoalveolar epithelia results in transition of a vulnerable lung to a lung characterized by detrimental ER stress (involving substantial UPR and apoptosis) and the development of microscopic bronchiolar-centric fibroproliferation; and 3) understanding etiologic and initial biological responses in distal airway epithelia and AEC2 cells, and the interaction of bronchoalveolar epithelia with lung fibroblasts will create opportunities for disease prevention and early intervention. The overarching hypothesis of our Program is that the development of IPF requires two hits, MUC5B overexpression in bronchiolar epithelia that induces a homeostatic, priming response and subsequent injury of the bronchiolar epithelia that results in detrimental ER stress, aberrant epithelia, and fibroblast activation. Project 1 will definitively address the drivers of MUC5B overexpression, Project 2 will identify the determinants of epithelial injury and detrimental ER stress, and Project 3 will investigate the molecular interface between MUC5B-induced epithelial injury and fibroblast activation. At the completion of this highly integrated Program, we will have: 1) established the basic molecular mechanisms that regulate MUC5B-induced injury/repair process in fibroproliferation; 2) defined mechanisms that will create a roadmap for primary and secondary intervention in IPF; and 3) provided a rationale and targets for early intervention in a disease that remains a significant public health problem and may increase post-Covid.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The olfactory epithelium is one of three major regions within the mammalian nervous system where new neurons are added throughout life. In humans, a failure to maintain olfactory sensory neurogenesis is associated with olfactory dysfunction, which afflicts an estimated 12.4 percent of adults in the U.S. and can adversely affect health and quality of life. A key barrier to treating olfactory dysfunction is our incomplete understanding of how persistent olfactory sensory neurogenesis is regulated and maintained. A related deficiency lies in our understanding of why neurogenesis persists within the olfactory epithelium. Life-long olfactory sensory neurogenesis is presumed to function solely to replace damaged olfactory sensory neurons. However, work from our laboratory has demonstrated that the birthrates of neurons that express a fraction of odorant receptors are accelerated upon stimulation by specific odors, leading to the central hypothesis of this proposal: that persistent neurogenesis within the olfactory epithelium serves, in part, an adaptive function. Our results are not readily explained by the current model of olfactory sensory neurogenesis, which predicts that the relative birthrates of neurons expressing each of the hundreds of different receptor genes encoded in the genome are determined stochastically by a process in which each post-mitotic neural precursor randomly ‘chooses’ a single odorant receptor gene for expression. Accordingly, the relative birthrates of distinct olfactory sensory neuron ‘subtypes’ are expected to be impervious to olfactory experience. The overall objective of this proposal is to determine how odor stimulation selectively accelerates the birthrates of specific olfactory sensory neuron subtypes. Our working model is that a fraction of subtypes have a special capacity, upon stimulation by odors with potential salience, to amplify themselves by selectively promoting the proliferation of mitotic neural progenitors that are of the same lineage and predisposed toward the same subtype fate. This model will be tested through three specific aims. Aim 1 will test the hypothesis that olfactory stimuli that selectively promote the neurogenesis of specific neuron subtypes are discrete, salient odors that selectively stimulate those subtypes. This will be tested by identifying, via a selective single-cell sequencing-based approach, the scope of neuron subtypes whose birthrates are accelerated by sex-specific odors. Aim 2 will test the hypothesis that some mitotic neural progenitors are predisposed toward specific odorant receptor fates that can be selectively amplified via cell proliferation. This will be tested by mapping the subtype fates of individual progenitors using genetic barcoding and in situ sequencing strategies. Aim 3 will test the hypothesis that mature olfac- tory sensory neurons of specific subtypes have a special capacity to promote the proliferation of progenitors within the same lineage via odor stimulation-dependent signaling. This will be tested through functional analyses of genes that have been found to be selectively expressed by neuron subtypes that undergo stimulation-dependent neurogenesis. The proposed experiments are expected to elucidate key aspects of persistent olfactory sensory neurogenesis, including how it is regulated, why it occurs, and how it may be manipulated to enhance human health.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Group B Streptococcus (GBS), is an opportunistic pathogen that asymptomatically colonizes the urogenital and female reproductive tract of approximately 25-30% of individuals. However, GBS can cause serious infections in immunocompromised individuals including those with diabetes. Diabetic wound infections are a major public health burden, with approximately 25% of diabetic individuals developing a wound in their lifetime, 25% of these wounds not healing and 28% requiring surgical amputation. Poor infection outcomes are correlated with the presence of numerous bacterial pathogens, and GBS, along with Staphylococcus aureus, is one of the most common bacteria found in these wounds. Despite its prevalence, no prior work has been done on GBS pathogenesis in the diabetic wound environment. Recently, we developed a Type 2 diabetic murine model of GBS diabetic wound infection in leprdb mice, and demonstrated that GBS forms a robust wound and persists in this environment. Further observations found that GBS colonies recovered from diabetic wound tissue were hyper-pigmented/hemolytic, suggesting selection of more virulent GBS mutants during diabetic infection. These phenotypes mimic those of a covR mutant, as CovR is a major repressor of GBS virulence factors such as the GBS hemolysin/pigment, nuclease (NucA), and surface adhesin plasminogen binding protein PbsP. Dual RNA-sequencing of GBS and the murine wound revealed that these same CovR regulated genes were highly upregulated in the diabetic wound. In addition, GBS infection triggered the recruitment of neutrophils, neutrophil activation and NET formation at the site of infection. Finally, we have shown in our murine model that the presence of S. aureus promotes GBS persistence in the diabetic wound. With these preliminary data, we have formulated hypotheses which address multiple mechanisms by which GBS may survive and persist in the diabetic wound environment. These hypotheses will be addressed in the following specific aims: Aim 1: Determine how CovR regulation contributes to diabetic wound infection, Aim 2: Characterize the contribution of PbsP to GBS diabetic wound formation, persistence, and dissemination, Aim 3: Examine the contribution of nuclease activity in promoting GBS immune evasion and wound persistence. These studies will increase our understanding of the pathogenesis of GBS diabetic wound infection and will provide a platform for additional studies.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Type 1 diabetes (T1D) is characterized by infiltration of autoreactive T cells in pancreatic islets, leading to autoimmune destruction of insulin-producing beta cells and diabetes. Initiation of autoimmunity and substantial beta cell loss may begin years prior to symptomatic onset. Therefore, there is a crucial need to develop diagnostics and therapeutic interventions directed towards this often-lengthy presymptomatic phase of T1D. Limited success has been demonstrated in clinical trials for therapeutics directed towards presymptomatic T1D. While anti-CD3 has shown promise, anti-CD3 only prevented diabetes onset in a subset of the study cohort and is not directed specifically against T cells reactive to beta cell antigens, such as insulin. An approach that has gained substantial traction in preclinical studies is the usage of peptide therapeutics to provide tolerance towards antigens targeted by autoreactive T cells. Administration of insulin peptide therapeutics in mice has been shown to prevent diabetes onset by expanding insulin-reactive regulatory T cells, which are anti-inflammatory and are essential for proper immune tolerance and regulation. Despite their therapeutic potential, insulin peptides have shown mixed results amongst different groups and have only been effectively administered via surgical implantation of an infusion pump. Therefore, optimizing therapeutic efficacy through targeted delivery and incorporation with diagnostics is warranted. This could be accomplished with ultrasound contrast agents (UCAs), which are small gas-filled bubbles that can be visualized using contrast enhanced ultrasound (CEUS) and are safe, easy to formulate, and clinically approved. A novel, submicron, ‘nanobubble’ ultrasound contrast agent has been developed and prior work has demonstrated enhanced accumulation of nanobubbles in islets of mice with presymptomatic T1D as a result of inflammation-associated microvascular permeability. My overall goal is to develop and apply submicron UCAs to both target therapeutic agents specifically to the disease site and track the effect of therapeutics on T1D progression. I hypothesize that submicron UCAs can be applied to both predict therapeutic induced disease prevention and as vehicles for targeted peptide delivery. I will examine this via two specific aims: I aim 1, I will predict therapeutic-induced disease prevention using submicron UCAs, using CEUS to detect changes in islet accumulation of submicron UCAs following therapeutic intervention. In aim 2, I will apply UCAs as therapeutic peptide delivery vehicles. Preliminary data indicates that peptide can be incorporated into nanobubbles and nanobubbles can target peptide to islets. I will characterize effect of nanobubble ablation on peptide cellular uptake characterize dynamics of peptide-nanobubble islet extravasation, and assess immunological and disease-modifying effects of peptide-nanobubble treatment. Developing an agent that allows for accumulation of therapeutic peptides in islets, enhanced therapeutic efficacy, and disease- reversal-predicting diagnostics can serve as a major advancement in T1D prevention.