University Of Minnesota

NIH Research Projects · FY 2024 · 2021-06

Abstract Depression, non-suicidal self-injury (NSSI) and suicidal thoughts and behaviors (STB) commonly emerge in youth and each represent important risk factors for death by suicide. Early detection and intervention has the promise of altering trajectories, improving adult outcomes and preventing suicide. Brain networks implicated in depression (frontal-limbic threat, cortico-striatal approach/reward and default mode) undergo significant change during childhood and adolescence; the manner of how these changes unfold may be critical to understanding the onset and course of depression, NSSI, and STB. The Adolescent Brain Cognitive Development (ABCD) is a population-based study that is following over 11,000 children annually over 10 years, with clinical and neuroimaging data from the first three years already publicly available. Since ABCD data collection spans a critical developmental window notable for significant rises in depression, NSSI and STB, analyzing this data presents an ideal opportunity to characterize the links between neural network changes and unfolding risk for suicide in youth. Resting-state fMRI (rs-fMRI) can be used to characterize brain network structure and organization. While our laboratory and others have extensively applied standard functional connectivity methods to characterize strength within depression networks using cross-sectional designs, longitudinal designs are needed to understand now aberrant development in network strength may contribute to onset depression, NSSI and STB. Recently, novel approaches have emerged to estimate network flexibility from rsfMRI data. These include drawing from information theory to measure entropy of brain signals and from dynamic connectivity analyses to measure state-switching, or shifts between brain network configurations during rest. Our preliminary data point to inverse relationships between brain flexibility (entropy and state-switching frequency) and depression, NSSI and STB in adolescents, suggesting a potential neural mechanism for getting “stuck” in negative ways of thinking and feeling. We propose that individual differences in the trajectory of neural network strength and flexibility changes across childhood and adolescence may help explain the emergence of depression and suicide risk in adolescents. In our conceptual model, inherited and environmental factors shape network developmental trajectories, which in turn underlie the emergence of depression, NSSI and STB. This proposal seeks to delineate the neurodevelopmental trajectories of strength and flexibility in fronto-limbic threat, cortico-striatal approach/reward and default mode networks associated with the risk, onset and early course of depression, NSSI and STB in children and adolescents in the ABCD study using novel analytic strategies. New insights from this study will provide the foundation for designing personalized interventions to facilitate early detection of depression and suicide risk, and to guide interventions capable of restoring healthy brain development and averting serious negative outcomes including suicide.

NIH Research Projects · FY 2025 · 2021-06

Summary Pluripotent stem cells (ES and iPS cells) have the ability to self-renew and to differentiate into multiple lineages in vitro. This makes these cells a powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine and disease investigation. Our research group has pioneered methods to derive large quantities of skeletal myogenic progenitor cells from mouse and human pluripotent ES and iPS cells. Upon transplantation into dystrophic mice, these progenitors are not only able to generate new functional myofibers, but also to seed the satellite cell compartment, thus providing long-term regeneration. With prior NIH support, we defined the molecular signature of in vitro-generated PSC-derived myogenic progenitors by comparing their transcriptome profiles to those of primary skeletal myogenic progenitors isolated at different developmental stages. Our findings revealed that PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. Paradoxically however, they differ functionally from fetal myoblasts, as PSC-derived myogenic progenitors show much superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple re-injuries and contribute to long-term regeneration. These results led us to hypothesize that exposure to the adult host skeletal muscle environment may induce molecular changes in transplanted cells. We found this to be the case as transcriptome analysis of PSC-derived mononuclear cells (MNCs) re-isolated after engraftment revealed a shift in molecular signature from embryonic/fetal towards neonatal/adult stages. In this renewal application we propose studies to understand i) the interaction and molecular cues provided by the adult niche that favor the in vivo maturation of PSC-derived myogenic progenitors, ii) the role of post-transcriptional regulation in this process, and iii) the dynamics of engraftment and the quiescence status of specific donor-derived sub-fractions.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY In 2017, it was estimated that nearly 20% of the adult population in the U.S. live with a mental illness, yet much remains unknown about the pathophysiology of psychiatric disease and the specific behavioral symptomatology. Aberrations in decision making and information processing have been implicated in neuropsychiatric diseases like schizophrenia, obsessive-compulsive disorders, and depression. Importantly, dysregulations in nucleus accumbens core (NAc core) dopamine signaling onto medium spiny neurons (MSNs) constitute a key neural mechanism underlying these aberrant processes. Currently, NAc core research disproportionately focuses on the motivation and reward-learning aspects of decision making. There is, however, strong evidence that decision making involves more complex information-processing: making a decision requires evaluation of actual outcomes but also requires prospection and retrospection on imagined alternate outcomes. Therefore, it is important to investigate the role of dopamine release and MSN activity in information processing when agents prospectively imagine potential outcomes before making a decision through deliberation and when agents retrospectively imagine what could have been in the “path untraveled” (counterfactual reasoning) or imagine poor time investments (sunk cost processing) after making a decision through reevaluation. Our understanding of how NAc core dopamine encodes these imagined outcomes during prospective deliberation and retrospective reevaluation is lacking greatly because there have not been behavioral tools with the complexity to probe the mechanisms contributing to this type of information-processing in rodents. Innovative work in the Redish lab has led to the development of a rodent behavioral task, called Restaurant Row, whose intricacy allows for in-depth behavioral analysis of neuroeconomic behaviors. This proposal aims to elucidate how NAc core dopamine dynamics reflect deliberation, counterfactual reasoning and sunk cost evaluation by 1) simultaneous monitoring of in vivo dopamine release and MSN activity using fiber photometry 2) manipulating endogenous dopamine signaling at specific points in decision making. Restaurant Row has tremendous translational potential and has been adapted for non-human primates and humans. Thus, the work proposed here will contribute to a larger translational collaboration in the future to investigate these questions across species through collaboration with my clinical mentors in the Department of Psychiatry. My advisors Dr. David Redish and Dr. Patrick Rothwell are equipped to mentor me both technically and professionally and together we have curated a training plan that perfectly aligns with my career goals. By incorporating pioneering techniques into a translational project in such an interdisciplinary environment will provide the training I need to develop become a successful physician-scientist contributing to the field of computational psychiatry.

NIH Research Projects · FY 2025 · 2021-05

Project Summary The broad goal of the proposed work is to conduct a randomized controlled trial of a specialized computer- delivered cognitive-behavioral therapy (CBT) to supplement standard alcohol use disorder (AUD) treatment in patients with a co-occurring anxiety disorder (“comorbidity”). Comorbidity is both common in AUD treatment patients (up to 50%) and confers a substantial increase in the risk of a return to drinking in the months following treatment. Because research shows that simply adding a standard psychiatric treatment does not substantially improve the AUD outcomes of comorbid individuals, we developed a CBT-based intervention aimed at disrupting the positive-feedback loop (“vicious cycle”; VC) of mutually aggravating negative affect and drinking behavior/urges (the “VC-CBT”). In an RCT, AUD treatment patients who received the therapist- delivered VC-CBT demonstrated significantly improved alcohol use outcomes as compared to those who received a standard anxiety treatment. Unfortunately, most community-based AUD treatment programs do not have clinical staff with the specialized training and technical expertise needed to deliver the VC-CBT. To help bridge this “research-to-practice” gap, we went on to develop a fully autonomous and interactive computer- delivered version of the VC-CBT and have demonstrated its functionality in AUD patients. Now, we propose to test the clinical efficacy of the computer-delivered VC-CBT, as well as the mechanisms and processes by which it is hypothesized to work. Aim I is a randomized controlled trial comparing the computer-delivered VC- CBT to an intensity-matched computer-delivered active control intervention that focuses on healthy lifestyles. 256 individuals in residential AUD treatment who have a comorbid anxiety disorder will receive either the VC- CBT or the active control intervention to obtain 200 cases that complete a 1-, 4- and 8-month follow-up. We predict the VC-CBT group will demonstrate superior alcohol-related outcomes at follow-up relative to the control group. Aim II evaluates the extent to which the computer-delivered VC-CBT selectively imparts the skills and knowledge targeted and whether they convey (mediate) the interventions therapeutic effect. This entails a formal series of “causal steps” analyses of the associations of: treatmentskills/knowledge; skills/knowledge outcomes; and, treatmentoutcomes with vs. without statistically controlling the effect of skills/knowledge. Aim III will test the theoretically-derived prediction that the computer-delivered VC-CBT moderates (i.e., weakens) the association between levels of real-time negative affect and drinking behavior/ urge. This will be accomplished by analyzing a series of twice-daily ecological momentary assessments (EMAs) that participants record in their natural environment for the 7 days prior to each of the three follow-up assessments. The impact of this work would be to provide a scalable and inexpensive means of improving the otherwise poor AUD treatment outcomes of comorbid AUD treatment patients. The work will also provide new scientific knowledge about the mechanisms and processes of change in comorbidity treatment.

NIH Research Projects · FY 2025 · 2021-05

PROJECT ABSTRACT Rural Americans face disproportionate cardiovascular disease burden, yet they must travel farther to reach hospitals that can provide effective, guideline-concordant treatment for acute myocardial infarction (AMI). Transfer networks play a crucial role in care for rural ST-Elevation Myocardial Infarction (STEMI), the most severe form of AMI, moving patients from rural hospitals to hospitals that can perform primary Percutaneous Coronary Intervention (PCI), the preferred treatment for STEMI. Of the 925 hospitals have closed or been acquired in rural areas since 2007, 75% may have been part of a STEMI transfer network. Although closures and acquisitions of hospitals may disrupt STEMI transfer network, the impact on STEMI patients has not been explored. Existing research on hospital closures finds conflicting results on AMI mortality. Failure to account for networks disadvantages previous research in 3 ways. 1) Time to PCI depends on the geographic location and the transfer relationships of hospitals within STEMI transfer networks. 2) Disruptions can cause spillover effects to patients not directly affected by closures and acquisitions but in the same transfer network. 3) The characteristics of network affect time to PCI after disruption. Although not previously explored, disruptions in rural STEMI networks could worsen rural-urban disparities in AMI mortality rates and reverse reductions in rural cardiovascular mortality over the past four decades. This project will draw upon novel data resources collected by the research team including validated data on hospital closure status and timing and validated data on hospital-level PCI capabilities. Leveraging the expertise of the research team in innovative network analysis techniques, invasive cardiology, emergency medicine, rural health, Medicare claims data, and biostatistics we will estimate STEMI transfer networks using Medicare claims data from 2007-2021 and link networks to hospital closure and acquisition data collected and validated by the research team. We will leverage connections to stakeholders in STEMI systems of care and rural health as well as expertise in survey design and implementation to develop a survey of EMS agencies to collect information on the emergency medical services involvement in STEMI networks. We propose the following aims: 1) estimate the association of STEMI network disruption with through0network time to PCI; 2) estimate the association of STEMI network disruption with STEMI treatment and mortality; 3) identify network-level factors associated with changes in patient mortality after STEMI network disruption; and 4) quantify the role of STEMI transfer network disruption in rural-urban STEMI disparities. We will disseminate information on STEMI networks and their characteristics via a web-based app. Our findings will identify a previously unknown access barrier for rural patients and can inform policymakers and clinicians about how to improve the health of rural STEMI patients.

NIH Research Projects · FY 2026 · 2021-05

Scientific advances from pain research in basic models and chronic pain populations are rarely translated and applied to improve our clinical understanding of pain and disability among individuals with significant intellectual, motor, and communicative impairments associated with neurodevelopmental disorders. Despite the well document burden of chronic pain in cerebral palsy (CP), the most common cause of pediatric-onset lifelong motor and developmental disability, there is relatively little known about sensory function in relation to chronic pain. The heterogeneity in CP etiology, pathophysiology, and clinical appearance suggests new approaches may be warranted to identify measureable phenotypic patient characteristics predictive of individual variation in chronic pain outcomes. Current pain assessment approaches used in CP are limited in their ability to subgroup CP patients in relation to sensory function that may be relevant for understanding pathophysiological pain mechanisms. Most of the sensory testing research conducted with CP, while important and not in question, has relied on sensory stimuli designed to assess impaired discriminative tactile abilities such as two-point discrimination, texture and shape perception reflecting large sensory fiber afferent function. There has been little work incorporating sensory testing approaches that simultaneously evaluate loss and gain of sensory function reflecting both large and small fiber afferent integrity. The specific purpose of this application is to address the limited scientific understanding of sensory function in CP to ultimately reduce the burden of chronic pain. As a first step to close the gap between the well documented pain burden in CP and the relatively unexplored pain mechanisms in CP we are proposing to investigate an objective standardized (protocol-based) measurement approach by extending the application of a modified quantitative sensory testing (QST) protocol to characterize and compare sensory function in children with CP with and without chronic pain. Our reasoning is as follows. First, comparing sensory function between chronic pain and no chronic pain groups has the potential to reveal differences in underlying tactile/nociceptive sensory function with high relevance for improving our understanding of chronic pain in CP and other severe IDD populations. Second, applying QST to a large sample of children and adolescents with CP will provide the basis for investigating sensory function in relation to individual and clinical characteristics, and health outcomes to identify novel tailoring variables that could guide pain treatment target selection (no such guidelines exist right now). Third, using a protocol-based approach affords an important standardized context in which to investigate nociceptive and inflammatory relevant biomarkers. Finally, the logic of QST provides the basis for exploratory but highly novel tests of sensory subtype constructs (gain, loss of function) informed by the chronic/neuropathic pain research literature as a first step toward creating sensory-function based grouping of chronic pain in CP.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT The proposed research aims to reduce obesity-related health disparities by promoting healthy lifestyle behav- iors among African Americans (AAs), given the high disease burdens associated with low physical activity, in- sufficient sleep, and obesity. To this end, the candidate will seek to understand how social contextual factors impact sleep by using qualitative interviews and an existing quantitative data sets (K99). Knowledge gained from the K99 phase will inform the adaptation and testing of an existing sleep intervention (MDACC IRB Proto- col #2018-0568) (R00). During the K99 phase, the candidate will receive focused and intensive training involv- ing workshops, courses, lectures, and directed readings from renowned experts in the fields of health dispari- ties, sleep medicine, and obesity. Two studies will include: (Aim 1) conducting qualitative in-depth interviews to elucidate social contextual influences on sleep, while soliciting feedback to adapt a sleep intervention among obese, sedentary, and short-sleeping AA adults; and (Aim 2) empirically testing the associations between so- cial contextual factors, sleep, physical activity, and body weight using data from an ongoing studies of AAs (Project FAITH; American Cancer Society, RSG-17-158-01; PI: Dr. McNeill, mentor). Lastly, during the R00 phase will compile the knowledge gained in the K99 phase to adapt a sleep intervention and test its feasibility, satisfaction, and preliminary efficacy in improving sleep, increasing physical activity, and decreasing sedentary behavior among overweight/obese, sedentary, and sleep-deprived AA adults (Aim 3). The candidate is uniquely positioned to complete the proposed research based on his past clinical experience in treating insom- nia and his research on sleep and obesity among AAs. The sleep intervention is a promising and innovative approach to optimizing psychological and physical wellness to reduce obesity and cancer-related health dis- parities – a research priority at the National Institute on Minority Health and Health Disparities. Furthermore, the proposed training plan will be critical in expediting the candidate’s transition to an independent investigator with focused expertise in developing culturally informed interventions to reduce cancer health disparities among AAs.

NIH Research Projects · FY 2025 · 2021-05

Sepsis remains a major cause of death worldwide (11 million sepsis-related deaths were reported in 2017), and that costs associated with treating septic patients place a large burden on the healthcare industry. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Early stages of sepsis are marked by hyperinflammation driven by proinflammatory cytokines (i.e., IL-1β, IL-6, IFNγ, and TNF). Patients who survive the acute phase of sepsis display long-term impairments in immune function. This state of chronic immunoparalysis renders sepsis survivors increasingly susceptible to secondary infections. Consequently, there is a desperate need to better understand the cellular and molecular basis of acute sepsis pathophysiology and subsequent immune reprogramming that defines the prolonged immune suppression. CD4 T cells, essential for coordinating the cellular and humoral immune response to a range of pathogens under normal circumstances, are severely depleted during the acute stage of sepsis. The overall number of CD4 T cells gradually recover over time, but their functional capacity remains blunted for many months. For the past 10 years, we have focused our research to pursue the long-term goal of understanding how sepsis impacts the CD4 T cell compartment because of the key role played by CD4 T cells in the overall fitness of the immune system. We will continue our investigation of the cellular and molecular reprogramming of CD4 T cells during sepsis in three interconnected areas of future research: 1) Define the mechanism(s) by which regulatory CD4 T (Treg) cells expand during sepsis; 2) Perform an integrated discovery approach using genomics, proteomics, and metabolomics to elucidate the molecular basis of sepsis pathophysiology and CD4 T cell immunoparalysis; and 3) Determine how intestinal microbiota dysfunction during sepsis affects the magnitude of the cytokine storm and promotes CD4 T cell immunoparalysis and increased incidence of late-onset mortality. We will interrogate samples obtained from multiple cohorts of sepsis patients, as well as from preclinical mouse models of sepsis at the level of Ag-specific CD4 T cell populations. Our preclinical studies will be further strengthened by using a novel mouse model that mimics a critical aspect of human biology – exposure to multiple ongoing and resolved infections trains the immune system for robust responses to new pathogens – and will serve as an important and novel ‘transitional translational’ preclinical bridge between humans and SPF laboratory mice to mechanistically study CD4 T cell dysfunction and reprogramming during sepsis. Addressing these key gaps in knowledge regarding the effect of sepsis on CD4 T cell biology will likely reveal new points of intervention that can be exploited in the future to restore CD4 T cell-mediated immunity, and overall immune fitness, following sepsis.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Methamphetamine (meth) is an addictive psychostimulant. Our pilot studies show that chronic meth resulted in degeneration of substantia nigra pars compacta (SNc) dopamine (DA) and locus coeruleus (LC) norepinephrine (NE) but not ventral tegmental area (VTA) DA or dorsal raphe (DR) serotonin (5-HT) neurons in mice. DA, NE, and 5-HT are monoamine neurotransmitters synthesized and packaged into vesicles in axonal compartments. Meth increases cytosolic monoamines and MAO-dependent mitochondrial stress in SNc axons but not the soma. Pilot studies show that MAO inhibition prevented meth-induced SNc degeneration. These data suggest that degeneration is driven by MAO-dependent axonal mitochondrial stress. However, pilot data show meth also increased LC, VTA, and DR axonal mitochondrial stress yet chronic meth induced SNc and LC but not VTA or DR degeneration. We found L-type Ca2+ channel (LCC)-dependent mitochondrial stress in SNc but not VTA axons. We propose a `two-hit' hypothesis wherein both MAO- and LCC-dependent axonal mitochondrial stress are required for degeneration. The degeneration `trigger' is meth-induced MAO-dependent axonal mitochondrial stress and vulnerability is a consequence of axonal LCC-dependent mitochondrial stress. Since MAO- and LCC- dependent stress converge in axons, we propose that axonal loss precedes somatic loss in a `dying-back' pattern of degeneration. Pilot data indicate that SNc axonal mitochondrial stress was increased during abstinence. We hypothesize this stress is MAO-dependent and contributes to degeneration during abstinence. Lastly, meth- induced degeneration is expected to impair novel object recognition and fear conditioning which are dependent on DA and NE, respectively. Hypotheses will be tested using two-photon laser scanning microscopy in brain slices, genetically encoded biosensors, pharmacological and genetic manipulation of MAO/LCCs, and immunohistochemistry. Aim 1: Determine mechanisms of mitochondrial stress in monoaminergic axons, consequences of chronic meth on mitochondria, and whether mitochondrial stress continues during abstinence. We hypothesize that SNc, VTA, LC, and DR axons all have meth-induced MAO-dependent mitochondrial stress whereas SNc and LC but not VTA or DR axons will have LCC-induced mitochondrial stress. We also hypothesize that axonal stress will remain elevated throughout abstinence from chronic meth and will be MAO-dependent. Aim 2: Determine the roles of MAO and LCCs in chronic meth-induced degeneration and behavioral consequences. We hypothesize that pharmacologically inhibiting or genetically deleting MAO or LCCs will prevent chronic meth-induced degeneration of vulnerable (SNc and LC) neurons and deleting the Ca2+ buffering protein calbindin from resistant (VTA and DR) neurons will render them vulnerable to degeneration. We further hypothesize that degeneration will be progressive throughout abstinence and MAO- dependent. Lastly, we hypothesize that SNc and LC degeneration will result in cognitive impairments.

NIH Research Projects · FY 2024 · 2021-04

PROJECT SUMMARY/ABSTRACT Black women in the US are twice as likely to experience a preterm birth (PTB), a low birth weight (LBW) infant, or the death of a child before age one compared to white women. This profoundly disturbing racialized pattern of adverse reproductive outcomes has endured for as long as data have been available. Decades of medical and public health research have documented the magnitude of racial inequity in maternal and child health, and it persists although the total infant mortality rate has declined and despite efforts aimed at improving access to prenatal care and early childhood initiatives. The intractability of this problem suggests structural racism—the ways in which societies foster discrimination by reinforcing inequitable systems that in turn reinforce discriminatory beliefs, values, and distribution of resources—as a root cause of these racial health inequities. The overall objective of the proposed project is to understand the degree to which acts of police violence—a pervasive form of structural racism—negatively impact reproductive health outcomes among black women and exacerbate inequities in adverse birth outcomes at the population level. This project will test the hypothesis that incidences of racialized police violence have both a direct and an indirect link to negative birth outcomes among black women compared to white women. Understanding the mechanisms through which racialized police violence is a source of psychosocial stress for black women will lead to novel intervention targets that may reduce racial inequities in birth outcomes. The project has three specific aims. Aim 1: Quantify the risk of PTB and LBW related to spatial and social proximity to the killing of a black man by police and the ensuing civil unrest. This population-based cohort study leverages vital records from the Minnesota Department of Health to assess if black women experienced worse birth outcomes following the killing of Philando Castile by police and the subsequent civil unrest. It will also assess if spatial proximity to the site of the incident and civil unrest increased risk for worse birth outcomes among all women. Aim 2: Quantify the impact of police violence on PTB and LBW risk among black women. Using a quasi-experimental causal framework and leveraging 13 years of birth records available from every county in the US, this aim assesses if incidents of police violence across the US are associated with greater risk for PTB and LBW among black women. Aim 3: Illuminate the lived experience of how racialized police violence impacts black women during pregnancy. An online survey administered to black women (n=200) who were pregnant and living in two communities with incidents of high- profile police violence (Twin Cities, MN, and Baton Rouge, LA) will assess psychosocial stress related to police violence, followed by 50 in-depth interviews in each city with women to characterize the impact of police violence on birth outcomes and to identify patterns and mechanisms. The long-term goal of this project is to eliminate inequities in birth outcomes for black women. This timely and urgent project not only aligns with NICHD’s goal of preventing pregnancy- related complications and reducing maternal and infant death, but it will further underscore the negative impact of racialized police violence on society, bolstering arguments for large-scale change in police-community interactions.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Protein prenylation is characterized by the addition of farnesyl (C15) or geranylgeranyl (C20) isoprenoids to cysteine residues located near the C-termini of different proteins. Although originally considered to be a rare modification, it is now clear that protein prenylation is widespread in eucaryotes and is of critical importance for a variety of proteins involved in oncogenesis, secretion, nuclear structure, and signal transduction. It has been estimated that as many as 2% of all proteins in mammalian cells are isoprenylated. This prevalence, coupled with the central role that many of these modified proteins play in cellular signaling, underscores the significance of this post-translational modification. Prenylation inhibitors were initially developed as therapeutic agents for cancer treatment. With the development of precision medicine approaches that target specific Ras- driven cancers, several clinical trials are ongoing. These inhibitors are also being investigated for the treatment of a wide variety of other disease including malaria, viral infections, Parkinson's disease and progeria. Two closely linked critical questions in the field of prenylation research concern what proteins are prenylated and how do they change in disease? What are clearly necessary are global methods that can compare the prenylomes in normal and disease states and allow those prenylated proteins whose levels change to be identified. If this were possible, it would reveal new targets for therapeutic intervention in these debilitating diseases. To address this, new isoprenoid probes for improved metabolic labeling will be synthesized and methods to decrease sample complexity and improve probe delivery will be developed. These will be utilized in quantitative proteomic experiments in cell culture and mouse models for disease. Another critical set of questions is what controls prenylation efficiency and is how is prenylation regulated? To address these questions, what are needed are strategies that allow the process of protein prenylation to be assayed in real time in live cells. This would allow prenylation reactions to be studied in a holistic manner in the presence of all relevant cellular components. It could open also up additional avenues for therapeutic intervention since it could reveal new regulatory interactions that could be targeted. To accomplish this, cell penetrating peptides with caged cysteine residues that mask their site of prenylation will be prepared and their subsequent prenylation monitored via microscopy after uncaging in a temporally controlled manner. Complementary experiments with full-length proteins, prepared by sortase ligation, containing the same light activated trigger will be used to explore potential interactions with chaperone proteins that may not be observed with the simpler peptide-based models.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Virus-specific CD8 T cells exert antiviral activity against HIV-1/SIV in vitro and in vivo. Yet, despite these responses in HIV-1-infected humans and SIV- infected macaques, they are unable to fully suppress virus replication. This is likely due to the majority viral replication occurring in CD4+ T cells within B-cell follicles in secondary lymphoid tissues; where virus-specific CD8 T cells are relatively few in number. In fact, we found that in vivo effector virus-specific CD8 T cell to target SIV RNA+ cell ratios (E:T) were over 40-fold lower inside compared to outside of B cell follicles in lymphoid tissues. These findings indicate that B cell follicles are an immune privileged site in which low levels of virus-specific CD8 T cells permit ongoing viral replication. Furthermore, we found that few virus-specific CD8 T cells express the follicular homing molecule CXCR5, likely explaining their low levels in B cell follicles. These data suggest that the inability of virus specific CD8 T cells to fully suppress virus replication may be due to a deficiency of these T cells in B-cell follicles. These findings have led us to our central hypothesis that targeting HIV-specific immunotherapy to B cell follicles will lead to durable remission of HIV infection. In support of this hypothesis we have shown that increased levels of virus-specific CD8 T cells in B cell follicles is associated with lower viral loads. Although many immunotherapies utilize patient T cells to generate CAR-T therapies, there are special consideration in the treatment of HIV. One major short comings of CAR-T approaches is the fact that T cells need to be autologous due to the risk of graft versus host disease (GvHD), requiring complicated/expensive manufacturing processes of patient cells. This is also challenging in the HIV setting as the patient T cells are already compromised and processing cells which may contain active virus is risky. Alternatively, Natural Killer (NK) cells are highly suited for allogeneic use as they do not cause GvHD and thus hold significant clinical potential as an off-the-shelf cellular product. Thuts, we propose to evaluate NK immunotherapy that targets virus-specific CAR NK cells (expressing CD4-MBL-CAR and CXCR5) to B cell follicles. Moreover, we will use CRISPR/Cas9 to knockout negative regulators of NK cell function, such as PD1, which we have previously shown to enhance NK cell function. Our long-term goal is to develop an intervention that will lead to durable remission of HIV infection using CAR NK cells. To test our hypotheses, we propose the following aims. 1) Develop reagents and methods to generate human and rhesus macaque CAR/CXCR5/PD1KO NK cells. 2) Determine the ability of CAR/CXCR5/PD1KO NK cells to migrate into B cell follicles of SIV-infected rhesus macaques and to induce and maintain viral suppression. Our proposed studies targeting CAR NK cells to follicles will have a broad impact on the field by providing insights into cell trafficking, persistence, and pre-conditioning regimens for NK immunotherapy. Moreover, our methods for engineering rhesus macaque NK cells will enable studies assessing the therapeutic use of NK cells in preclinical NHP models. Moreover, these studies could result in an effective strategy to induce long-term sustained remission of HIV.

NIH Research Projects · FY 2026 · 2021-04

Our long-term goal is to develop new chemical approaches to dissect the molecular mechanisms associated with epigenetic complexes and transcription. Defining mechanisms for the dynamic regulation of gene expression remains a significant challenge for the field of epigenetics. Our research program in chemical epigenetics is addressing these challenges by developing selective chemical probes and proximity-inducing molecules, defining new biomolecular interactions, and developing enabling structural biology approaches to quantify these dynamic multivalent interactions. These approaches are helping to create a more detailed picture of diverse epigenetic complexes for developing epigenetic therapies. PHD and bromodomain-containing proteins are a subset of epigenetic reader proteins and are an emerging protein-drug class for epigenome therapy. During the last funding period, my research program has developed new chemical probes and degraders (PROTACs) to study bromodomain-containing proteins, BPTF and BRD4. One specific innovation pioneered in our lab for ligand discovery uses protein-based 19F NMR. Due to the hyper-responsiveness of fluorine to ligand binding and simplified spectra, this structural biology tool is ideally suited to rapidly screen small molecules, characterize transient interactions with native transcription factor- protein or DNA, and to quantify the dynamics and allosteric effects induced by small molecules. Over the next five years, we will further develop this approach for enabling small molecule discovery, with new proteins of therapeutic interest, including E3 ligases and quantify the affinity and dynamics of large multidomain bromodomain-containing proteins associated with DNA and synthetic nucleosomes. Our new chemical probes for the BPTF PHD and bromodomains have potential for high impact as chemical tools for deciphering the role of the chromatin-binding domains in diverse disease contexts and a starting ligands for the synthesis of new proximity-inducing molecules. More specific BRD4 chemical probes and PROTACs with improved selectivity profiles will help uncover new biology in cancer and inflammation obscured by less specific tool compounds. We will use these tools to dissect the functional roles of these bromodomains on transcription, including gene regulation, chromatin engagement, and novel-protein-protein interactions in our own lab, while continuing to make our chemical tools available to the biomedical research community. Finally, next generation chemical probes will be further developed including a new direction developing tissue specific degraders. This proposal has broad biomedical significance for early lead discovery and studies of epigenetic reader domains important in human health and disease. Given that epigenetic proteins and transcription factors represent major classes of new drug targets, we envision our innovations in chemical epigenetics and ligand discovery could significantly increase the repertoire of targets and thereby open up new avenues for drug discovery while uncovering new molecular mechanisms in biology.

NIH Research Projects · FY 2025 · 2021-04

PROJECT ABSTRACT The number of women incarcerated in the United States has increased more than 600% over the past three decades. At last count, more than 225,000 women – a majority of whom were of reproductive age – were behind bars. As the number of incarcerated women has risen, so too has the need for prisons to address women’s health needs, including pregnancy, childbirth, and postpartum care. Providing incarcerated pregnant women with enhanced pregnancy and postpartum supports may improve both maternal and neonatal outcomes. One promising approach is to use doulas to provide this comprehensive support. As non-medical companions, doulas provide an array of supports and have been shown to reduce labor length and obstetric complications, improve maternal satisfaction, and promote breastfeeding initiation among low-income women. Several state prisons are now partnering with local organizations to offer enhanced pregnancy and postpartum support; yet, key questions that are critical to successful program expansion remain. The overall objective of this project is to conduct a multi-state study of enhanced pregnancy and postpartum support programs for women incarcerated in six geographically-diverse prisons, with the overall goal to provide valuable, practical, and actionable information to prisons about how to implement pregnancy and postpartum support programs to promote maternal and neonatal health. We will accomplish this goal by pursuing the three specific aims: 1) identify facilitators and barriers to implementation of pregnancy, childbirth, and postpartum support programs for women in prison, 2) evaluate the pregnancy and birth outcomes of program participants, and 3) evaluate the postpartum outcomes of program participants. Results from this study will fundamentally advance our understanding of pregnancy and postpartum support programs and the maternal and neonatal outcomes among program participants. Identifying key modifiable factors across prisons that facilitate program implementation and sustainability and documenting the outcomes of participants across sites will directly inform ongoing program expansion. Ultimately, this work will yield critical information to address the complex health needs of pregnant women in prison, reduce health disparities, and promote health equity among justice- involved women and their infants.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Neuroblastoma is the most common solid tumor in infants. About 25% of patients have high-risk neuroblastoma, a devastating disease with poor prognosis and few treatment options. The primary driver of high-risk neuroblastoma is the oncogene MYCN, a MYC-family transcription factor that has no druggable pockets and has long eluded drug development efforts. Recently, the protein kinase Aurora A (AurA) was shown to bind to the N-Myc protein in neuroblastoma cells and interfere with its ubiquitination by the SCF ubiquitin ligase complex, preventing N-Myc from being degraded by the proteosome. Blocking complex formation between AurA and N-Myc results in rapid N-Myc degradation and cell death in neuroblastoma cell lines. The same AurA/N-Myc complex has now been shown to drive neuroendocrine prostate cancer (NEPC), and AurA also forms a similar complex with the closely- related c-Myc protein in liver cancer. These recent discoveries point to a new paradigm for targeting Myc- family transcription factors in cancer using inhibitors that trigger structural changes in AurA that block Myc protein binding and promote Myc degradation. Our lab has recently shown that most existing AurA inhibitors, including the current clinical candidate alisertib, do not have a strong enough allosteric effect on AurA to be effective at weakening N-Myc binding. In agreement with this, alisertib has inconsistent effects on N-Myc levels in cell lines, and has not performed well in ongoing clinical trials in neuroblastoma and NEPC. The weakness in our current understanding of how AurA binds to c-Myc and N-Myc and how these interactions are affected by inhibitors represents a major impediment to this therapeutic strategy for targeting Myc-driven cancers. The goal of this project is to provide the missing molecular picture of the interactions between AurA and Myc transcription factors and how they can be modulated by inhibitor binding. We plan to use new experimental tools and approaches to define how the binding of c-Myc and N-Myc alters the conformation (shape) and dynamics (protein motion) of AurA, and to delineate the specific structural changes an inhibitor must trigger to efficiently destabilize these complexes. We will a) define the structure of the AurA/Myc complexes at atomic resolution using x-ray crystallography, magnetic resonance spectroscopies and molecular modeling, b) determine how these interactions alter AurA conformation and dynamics by tracking key structural elements of the kinase in solution, c) correlate the effects of a large panel of kinase inhibitors on AurA conformation with their ability to alter the binding affinities of N-Myc and c-Myc, and d) test the efficiency of the strongest AurA allosteric modulators in a series of N-Myc- and c-Myc-dependent cancer cell lines including neuroblastoma, NEPC and liver cancer cells. The insights will pave the way for the repurposing of existing kinase inhibitors and the development of new inhibitors as a new treatment modality for Myc-driven cancers.

NIH Research Projects · FY 2025 · 2021-04

This proposal presents a five-year research career development program focused on the study of antigen specific CD4 T cells in tuberculosis (TB), with the long-term goals to reveal new information about immune protection in TB and inform the development of an effective new vaccine for this disease. The candidate is currently an Assistant Professor of Medicine at the University of Minnesota (UMN) in the Division of Infectious Diseases. The outlined proposal builds on the candidate’s previous experience by adding new domains of expertise in advanced T cell immunology, international collaborative patient-oriented research and human immunology, and single cell transcriptomics. The training objectives are represented by the mentorship team of Marc Jenkins and David Boulware, and other key collaborators at UMN. The proposed experimental and didactic work will position the candidate with a unique set of cross-disciplinary skills that will enable his transition to independence as a physician scientist focused on the immunology of TB. A better understanding of the factors that govern CD4 T cell function in TB could help guide the development of vaccine candidates more likely to elicit protective immunity against TB disease. It is becoming clearer that IFN-g secretion is only one protective function of CD4 T cells in TB. The other key effector mechanisms of CD4 T cells in TB are still being defined, and the determinants of these immune functions remain to be fully characterized. The overall scientific objectives of this particular project are to characterize CD4 T cell functions at the site of infection and to define how antigen availability and T cell receptor (TCR)- epitope affinity govern these functions. The premise of this proposal is that effector activity of CD4 T cells at the site of TB infection is determined by characteristics of the Mtb antigen targeted. The central hypotheses are 1) that CD4 T cells with lower affinity TCRs, targeting Mtb antigens with moderate abundance, have more diverse and superior functions, and 2) that antigen specific CD4 T cells at the site of infection perform key effector functions outside the Th1 paradigm. To test these hypotheses, the following aims are proposed: Aim 1: Determine how antigen-intrinsic factors govern CD4 T cell function in TB, and Aim 2: Characterize CD4 T cell function and diversity at the site of human TB infection. To accomplish these aims, innovative approaches are proposed including: modulation of Mtb gene expression in vivo, identification of lower affinity TCRs, and unbiased identification of CD4 T cells that have recently received TCR stimulation in vivo. By pairing these approaches in the mouse model with studies of human T cells from the cerebrospinal fluid of individuals with HIV-associated TB meningitis, using single cell transcriptomic profiling, these studies will characterize how Mtb antigen-intrinsic factors elicit CD4 T cell populations of varying functional capacity and diversity. This K08 project will guide new TB vaccine development and promote the candidate’s transition to independence.

NIH Research Projects · FY 2025 · 2021-03

Project Summary Pyrazinamide (PZA) is a critical component of first-line tuberculosis (TB) therapy because it has dramatically reduced relapse rates and treatment duration. PZA is weakly active in vitro, but potently sterilizing in vivo, due to its outstanding activity against slow and non-replicating populations of Mycobacterium tuberculosis that are phenotypically tolerant to most other TB drugs. Understanding the basis for the in vivo sterilizing activity of PZA represents one of the most important unmet needs in TB drug discovery. While the mode of action of PZA remains under investigation, our knowledge of factors that govern susceptibility and resistance has advanced considerably in recent years. PZA is a pro-drug that must be converted to the active form of pyrazinoic acid (POA) by the M. tuberculosis amidase PncA, and loss-of-function mutations in pncA account for at least 70% of clinical resistance. Other genetic variations associated with resistance have been described, but most have not been evaluated in isogenic strains to determine their role in resistance, and have not been assessed for association with PZA resistance in animal models of infection. Importantly, PZA lacks antitubercular activity in athymic nude mice, indicating a key role for T cell-mediated immunity in efficacy. Consistent with these observations, our lab and others have shown that PZA is inactive against M. tuberculosis in resting macrophages, but contributes to bacterial killing in macrophages activated by interferon-gamma (IFN-ɣ). Host- dependent stressors that are associated with IFN-ɣ stimulation, such as exposure to low pH, nutrient limitation and reactive oxygen species, have been implicated as contributors to PZA action. Our recent studies have revealed a central role for specific M. tuberculosis stress responses in PZA conditional susceptibility. Through these studies, we have identified a network of functions that modulates PZA susceptibility when expression of the corresponding genes is altered. We have also identified specific host factors that contribute to PZA action in macrophages and in infected mice. Based on these findings, we have identified means to bolster drug action under conditions where PZA typically lacks activity. Through this proposal, we aim to characterize novel molecular mechanisms for PZA resistance, determine host factors that modulate PZA susceptibility, and assess opportunities in host- and microbe-targeted PZA potentiation. These studies will advance our understanding of mechanisms that govern PZA susceptibility and resistance of M. tuberculosis. As a substantial proportion of TB disease results from impaired T cell responses, it is of fundamental importance to understand how these responses relate to PZA efficacy and how we can use this information to optimize PZA action in the context of impaired immunity.

NIH Research Projects · FY 2025 · 2021-03

Project Summary Natural killer (NK) cells are potent, effector lymphocytes with the ability to kill malignant and virally infected cells by releasing lytic granules without the need for antigen specificity. Acute myeloid leukemia (AML) and multiple myeloma (MM) are highly susceptible to NK cell-mediated killing. Strategies using donor NK cells to treat these malignancies have yielded clinical responses in more than a third of patients, even allowing some refractory AML patients to eventually get a curative bone marrow transplant. Despite these successes, NK cell therapy has been limited by the short life span of infused cells and the occurrence of functional exhaustion that occurs when NK cells are exposed to the tumor microenvironment. A subset of NK cells, termed ‘adaptive’ develops in response to cytomegalovirus (CMV) infection. Adaptive NK cells live longer than conventional NK cells, have a robust capacity to secrete cytokines, and are resistant to suppression in the tumor microenvironment. We demonstrated that adaptive NK cell expansion after transplant is associated with a 26% reduction in AML relapse and a 53% reduction in MM relapse. Our lab has developed a reliable method to expand adaptive NK cells from peripheral blood of CMV seropositive donors; however, our preliminary data also shows that chronic stimulation of adaptive NK cells through the activating receptor NKG2C, in combination with inflammatory cytokines, induces high expression of checkpoint inhibitory receptors. We seek to characterize the in vivo behavior of adaptive NK cells given as therapy for AML or MM. In Aim 1, we will determine whether adaptive NK cells survive longer than conventional NK cells and traffic to the bone marrow after allogeneic infusion. Patient samples will be collected from an ongoing phase I/II clinical trial using allogeneic, adaptive NK cells to treat relapsed AML. In addition, we will test adaptive NK cell persistence and longevity compared to conventional NK cell therapy in a murine model of MM. In Aim 2, we will identify mechanisms of NK cell exhaustion and test whether checkpoint receptor blockade restores NK cell function. These studies will be led by Dr. Aimee Merino, at the University of Minnesota Masonic Cancer Center, under the mentorship of Dr. Jeffrey Miller. Dr. Merino is currently a postdoctoral fellow, but will become an instructor upon completion of her fellowship training. Dr. Miller is a leader in NK cellular therapy with a track record of translating discoveries in NK cell biology into novel clinical applications. The University of Minnesota Masonic Cancer Center offers an exceptional environment for cultivating a career in translational cancer research. To achieve the long-term goal of becoming an independent investigator, Dr. Merino has recruited an advisory committee of leading scientists and developed a training plan aimed at broadening her knowledge base, developing her technical expertise, and cultivating her leadership skills.

NIH Research Projects · FY 2025 · 2021-03

Project Summary The University of Minnesota (UMN) and Photonic Pharma (PP) a Minnesota-based drug discovery start-up, have partnered to optimize, field-test, and deploy at industrial scale, an innovative new approach to developing allosteric kinase inhibitors (AKI). These molecules have high potential as novel cancer therapeutics that circumvent clinical resistance to conventional orthosteric kinase inhibitors (OKI). We have developed high- throughput screening (HTS) technology based on nanosecond fluorescence lifetime (FLT) detection of Förster resonance energy transfer (FRET), that tracks ligand-driven kinase allostery with angstrom precision by monitoring structural changes of the activation loop, the key regulatory element in all kinases. This is the first HTS-amenable technology that accurately resolves allosteric effects of kinase inhibitors, relying on high-quality nanosecond FLT readouts unavailable from conventional fluorescence plate readers (PR). PP have developed a proprietary HTS platform that uses FRET biosensors and a state-of-the-art FLT-PR to detect structural readouts in <2 min for 384-well and <5 min for 1536-well plates. By partnering with PP, we will transform our kinase FRET sensor technology into a broadly applicable drug-discovery platform for identifying AKIs. We propose drug-discovery programs on two different targets to demonstrate broad utility and accelerate large- scale adoption of our technology for drug development. In AIM 1, we identify Aurora A inhibitors that downregulate the undruggable c-Myc oncoprotein by inhibiting the scaffolding interaction of Aurora A with c- Myc. These molecules would represent a novel treatment strategy for the large number of cancer patients with c-Myc-driven tumors. In AIM 2, we identify allosteric inhibitors of the c-MET receptor tyrosine kinase as a novel therapeutic strategy for patients with c-MET-driven lung cancer. These patients invariably develop resistance to current MET inhibitors through acquired mutations in the ATP site, and allosteric inhibitors that bind outside the ATP-site would circumvent this mode of resistance, filling an unmet clinical need. This UMN-PP partnership translates decades of biophysics research by two world-leading experts – Levinson and Thomas – toward drug- discovery by resolving ligand-driven allostery in kinases. This is enabled by the FLT-PR instrumentation and know-how required to implement nanosecond FLT detection in assays that resolve allosteric inhibitors in true HTS mode. This overcomes key drawbacks of conventional kinase inhibitor screens, which detect kinase inhibition or binding without regard to allosteric mechanism. The platform is broadly applicable, as almost all kinases undergo the large-scale allosteric structural changes our technology detects. Success of this project will catalyze adoption of this technology targeting a wide range of biomedically important kinases, as highlighted by Photonic Pharma’s successful partnership with Bristol-Myers Squibb on drug discovery for other high-priority therapeutic targets.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy including immune checkpoint blockade. Meanwhile, immunotherapy targeting the PD-1:PD-L1 pathway is inducing stunning clinical outcomes in other advanced malignancies. Despite decades of cancer immunology research, the underlying mechanisms governing immune surveillance in pancreas cancer are largely unknown. This is due, in part, to a lack of animal models that permit the detection of tumor-specific T lymphocytes during disparate clinical outcomes commonly observed in patients. We have filled this knowledge gap by creating novel animal models that permit the interrogation or the rare tumor-antigen specific T cells over time. Based on our recent discoveries, we are now uniquely poised to identify how to safely promote antigen-specific T cell-mediated destruction of pancreas cancer, and our proposal will use these results to develop a novel preclinical combination immune-based therapy to inform a clinical trial. Our compelling preliminary data support the hypothesis that immune-mediated pancreas cancer eradication requires a combination of a 1) high affinity tumor specific T cell, 2) modifying suppressive intratumoral myeloid cells, and 3) overcoming chronic inflammatory signaling mediated by TNFα. We screened a series of immunotherapies and identified that agonistic αCD40 or PD-L1 blockade has only transient antitumor activity whereas the combination leads to tumor eradication in 63% of animals. PD-L1 blockade failed to reinvigorate intratumoral T cell functions and instead changed the peripheral tumor-specific T cell repertoire and expands a unique peripheral T cell subpopulation enriched for pro-survival genes and Ikzf2. In contrast, agonistic αCD40 promotes potent, yet short-lived, cytolytic intratumoral T cells which correlates with a decrease in intratumoral myeloid cell IL-27 production. Finally, abrogating Tnfr1 expression by non-tumor/host cells overcomes tumor escape resulting in 100% of tumor eradication in αCD40+αPD-L1-treated animals. In Aim 1, we will identify the functional significance of the altered TCR repertoire and the Ikzf2+ T cell subpopulation induced following PD-L1 blockade. In Aim 2, we will test if agonistic αCD40 promotes potent cytolytic effector T cells by abrogating intratumoral myeloid cell production of IL-27 and/or CD8 T cell production of IL-10. In Aim 3, we will identify how IFNγ and TNFα signaling on non-tumor/host cells lead to disparate outcomes following combination immunotherapy of pancreas cancer and test a novel combinatorial immunotherapy that includes TNFα blockade in combination with a CD40 agonist and a PD-1 inhibitor. The studies are designed to identify a mechanistic basis for T cell dysfunction in pancreas cancer and to create a feasible clinical strategy to overcome it with the goal to create a safe and effective immune-based treatment for pancreas cancer patients.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract This project, titled the Indigenous Cultural-understandings of Alzheimer’s – Research and Engagement (ICARE) focuses on the urgent need to address the increasing burden of Alzheimer’s disease and related dementias (ADRD) in American Indian (AI) and First Nations (FN) populations. Our central hypothesis is that culture and community-specific context shape ADRD illness experiences in Indigenous populations significantly enough to create distinct impacts of ADRD requiring culturally tailored approaches to diagnosis, care and education. Our goal is to create a foundational ethnographic database of AI/FN lived experience of ADRD that can be examined to inform the creation of culturally appropriate and safe approaches to improve dementia diagnostics, care and outreach. Our findings will inform our longer-term goal to create culturally safe clinical guidelines and dementia diagnosis and care tools for North American Indigenous populations. AI/FN ADRD rates are approximately three times higher, with a 10-year earlier onset, compared to majority populations. Higher rates of co-morbidities and limited access to social, economic, and health resources increase Indigenous health disparities. Culture and community context influence Indigenous peoples’ experience with dementia and culturally grounded approaches/resources increase awareness and improve outcomes. Currently, there is little information to guide culturally appropriate efforts to address ADRD. Using community-based participatory research (CBPR), ICARE engages AI/FN communities in Minnesota, Wisconsin and Ontario. We will undertake a CBPR qualitative ethnographic examination of the AI/FN lived experience across the illness trajectory including: cultural understandings of ADRD; experiences with diagnosis and care; and AI/FN community strengths and challenges. Specific methods include participant observation and semi-structured in-depth interviews with people with dementia (PWD), caregivers, and healthy seniors. Our qualitative analytic approach incorporates both biomedical and Indigenous understandings of ADRD. Our research has three specific aims. First, we will document and examine the lived experiences of ADRD across the disease trajectory in 3 diverse AI/FN regions (Red Lake Nation and Grand Portage, MN; Oneida Nation, WI; Manitoulin Island, ON) and identify cultural, health systems, and community factors influencing ADRD diagnostic and care pathways for PWD. Second, we will use this ethnographic data to delineate AI/FN specific explanatory models of ADRD and understandings of ADRD Quality of Life, diagnosis and staging to identify appropriate approaches to diagnose and assess ADRD in AI/FN populations. Third, we will conduct collaborative knowledge translation of ethnographic knowledge into culturally appropriate health promotion/education tools (fact sheets, videos or training modules) to respond to community needs. This study represents an important step in identifying effective, culturally-grounded approaches to address dementia- related inequities in AI/FN populations.

NIH Research Projects · FY 2025 · 2021-03

Project Summary With medical progress and growing efforts to empower individuals in making life-impacting choices, older individuals are thus more engaged in their behavioral and socioeconomic choices than ever before. These decisions are multifaceted and nuanced, but unfortunately, older adults often make poor socioeconomic decisions. Progress in medicine has not only led to increased life expectancy but it has also contributed to a rise in Alzheimer's disease (AD), the most common form of dementia, due to rapid increase in population aging and the current absence of AD-modifying therapies. Exploiting recent advances in judgement and decision-making neuroscience, we now propose a three-pronged effort designed to uncover whether and how Aβ-dependent mechanisms induce changes in circuits underlying various forms of decision-making and we have formed an investigative multi-PI team uniquely qualified to pursue these questions. Leveraging exciting new results from our joint group, we will test the central hypothesis that decision-making in AD mice is altered in a multifaceted economic- and sex-specific manner. In the light of novel findings reported in the preliminary results, we will i) test the hypothesis that aging impairs decision-making differentially in male and female mice, ii) test the prediction that decision-making is impaired in mouse models of AD and worsens with aging and iii) test the hypothesis that genetic, pharmaceutical and optogenetic intervention will improve decision-making in AD mouse models, thereby providing a preclinical proof-of-principle that decline in decision-making can be ameliorated.

NIH Research Projects · FY 2026 · 2021-02

PROJECT SUMMARY Bacteria must sense and respond to rapidly changing and often stressful environments and their ability to enact rapid changes in gene expression that alter cell structure and function is key to their survival. Post-transcriptional mechanisms of regulation of gene expression are pervasive in bacteria and represent an important stress response strategy. Small RNAs (sRNAs) that base pair with mRNAs and regulate transcription elongation, translation, and mRNA stability are common mediators of bacterial post-transcriptional regulation and sRNA activity often requires RNA chaperone proteins. Many bacterial genomes encode hundreds of sRNAs, yet their roles in bacterial physiology and virulence remain poorly understood in all but a few model organisms. For nearly 20 years, my group has been engaged in research to identify sRNAs and the factors they work with, including small proteins and RNA chaperones, and characterize their molecular mechanisms of action and their roles in an array of physiological processes. Our overall approach is to combine genomics with classical genetics and biochemistry to study sRNAs and small proteins and their molecular and cellular functions in Escherichia coli Salmonella, and Bacteroides. Our work has defined the target regulons of multiple sRNAs, revealing new molecular mechanisms of sRNA-mediated regulation of transcription elongation, translation, and mRNA stability. We have uncovered mechanisms of hierarchical regulation of multi-target sRNA regulons. We have also found stress response and metabolic phenotypes for sRNA mutants, linking the molecular mechanisms of regulation to physiological outcomes. In the next funding period, we will build on a strong and productive foundation of previous work to continue investigating novel mechanisms of sRNA-dependent regulation in E. coli and Salmonella. We will extend our work to investigate RNA-based mechanisms of regulation by novel RNA chaperones in Bacteroides. We will also characterize mechanisms of prophage-encoded sRNA and small protein-mediated phage defense and more broadly explore the roles of prophages in protection of their hosts. A strong team of collaborators with a diverse skillset will allow us to interrogate RNA-RNA, RNA-protein, and protein-protein interactions on a global scale and at the level of individual molecules. Our work focusing on RNA will continue to connect molecular biology to cell physiology and reveal fundamental new insight into the biology of microbes.

NIH Research Projects · FY 2025 · 2021-02

Project Summary A hallmark of alcohol use disorder is continued seeking and consumption of alcohol despite negative consequences. Neuroadaptations in corticostriatal projections to nucleus accumbens are critical for the development of compulsive-like alcohol use behaviors, including in an aversion-resistant drinking model. Yet it remains unclear how potentiated activity in nucleus accumbens neurons alters sensitivity to aversive outcomes during consumption and seeking of alcohol. Pre-existing individual differences in aversion-related circuits have been shown to predict future compulsive-like alcohol consumption. Yet, we are not aware of any demonstrations of alcohol-induced neuroadaptations in aversion-related circuits that track the development of aversion-resistant drinking, and that account for the emergence of this compulsive phenotype after alcohol exposure. Our long-term goal is to identify dynamics changes in the activity of aversion-related neural circuits that drive the emergence of compulsive alcohol use. Here, we will examine glutamatergic basal forebrain neurons that project to the lateral habenula. These neurons are found in an anterior-posterior continuum from the ventral pallidum to the lateral hypothalamus and are targeted by nucleus accumbens inhibitory projection neurons. We hypothesize that the emergence of aversion-resistant drinking requires selective inhibition of these neurons during alcohol consumption. A crucial first step in our investigation of this circuit is to determine whether the emergence of aversion- resistant drinking is correlated with cell-type specific alterations in drinking-related neural activity in the glutamatergic basal forebrain. We will assess this question, and whether these neural correlates are downstream of nucleus accumbens mechanisms in Aim 1. Our second goal is to examine lateral habenula glutamate activity dynamics during aversion-resistant drinking. Therefore, our Aim 2 experiments will utilize a fluorescent glutamate sensor combined with fiber photometry to identify temporally-specific neural correlates of aversion-resistant drinking. Finally, in Aim 3 we will use chemogenetic approaches to assess the functional contributions of basal forebrain projections to lateral habenula in aversion-resistant drinking and determine which inputs to lateral habenula from regions of the glutamatergic basal forebrain most effectively modulate compulsive alcohol consumption. Together, these experiments will yield novel insights into the neural circuits mediating compulsive alcohol use, as well as aversion-related constraint of reward-seeking more broadly.

NIH Research Projects · FY 2025 · 2021-01

Abstract Total pancreatectomy with islet autotransplant (TPIAT) is performed to treat the severe, intractable pain of chronic pancreatitis for patients who have failed medical or endoscopic therapies. The TP relieves the source of pain, while the IAT reduces risk or severity of post-operative diabetes; after 1 year, up to 40% of patients are off insulin and nearly 90% have islet function (C-peptide positive). However, little is known about the long-term function of the islet graft. Rigorous studies are needed to determine what proportion of patients maintain islet function long term and whether islet function improves glycemic control and reduces diabetes complications in this population. This carries high potential for impact in clinical care: currently some patients are denied coverage for IAT due to lack of rigorous studies establishing the benefit of IAT. Furthermore, we know little about how changes in gut anatomy and associated hormones (GLP-1) and alpha cell dysregulation (glucagon) of intraportally transplanted islets impact long-term glycemic regulation. Hypoglycemia has been increasingly reported after TPIAT, with exaggerated incretin response and/or defective glucagon counterregulation suggested as possible mechanisms. We propose to study islet function, glycemic control, diabetes complications, and mechanisms impacting glycemic control (incretin hormone axis, counterregulatory hormones) in patients who are 5-20 years out from TPIAT. The study’s overall aim is to determine the long-term benefit of IAT. To assess islet graft function, we will use C-peptide levels from mixed meal tolerance testing (MMTT) as the marker of endogenous islet function. We will enroll at least 200 participants in this cross-sectional study, who are 5-20 years after TPIAT for chronic or recurrent acute pancreatitis. The study's first aim is to determine the proportion of patients who maintain islet graft function 5-20 years after TPIAT and to determine whether C-peptide levels from a MMTT are associated with concurrent glycemic control measures. The second aim is to determine whether islet graft function is inversely associated with diabetes-specific complications (severe hypoglycemia, diabetic ketoacidosis, and micro- or macrovascular disease). The third aim is to determine other mechanisms that impact long-term glycemic control in TPIAT, including incretin function, alpha cell function, and markers of beta cell stress. As an exploratory aim we will recruit a subgroup of patients who are 5-20 years out from TP alone to undergo the same testing protocol for comparison with TPIAT recipients without and with graft function. This study's significance lies in its potential to directly impact clinical care and access to IAT when TP is needed. We hypothesize that islet graft function improves glycemic control and reduces diabetes complications even in recipients who are not insulin-independent, but that dysfunctional incretin and counter-regulatory responses will impact hypoglycemia risk.