University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY We propose a research program in evolutionary genetics and genomics that emphasizes two distinct themes. The first theme focuses on the island rule – the widespread phenomenon of populations evolving unusual body sizes after colonizing islands. Advances in our laboratory have established mice from Gough Island, the largest wild house mice in the world, as a tractable system for understanding genetic mechanisms responsible for the evolution of extreme body size. Through comprehensive characterization of novel congenic strains, we will identify genes and mutations involved in this instance of the island rule. To elucidate causes and consequences of gigantism, we will extend this unique system to genetically dissect another trait associated with evolution on islands: exploratory behavior. This research direction will reveal genetic principles of complex trait evolution in novel environments. The second theme centers on recombination, a process that diversifies offspring genomes and ensures proper chromosomal segregation during meiosis in many species. Using single-cell methods that enable us to quantify recombination in individuals, we have discovered that house mice evolved substantial differences in genomic crossover number over short timescales, with females and males showing discordant trajectories. Motivated by this advance, we will reconstruct the evolutionary dynamics of the recombination landscape in house mice across genomic scales (from hotspots to whole genomes) and temporal scales (from thousands to millions of years). To identify cellular processes involved in the evolution of recombination, we will integrate high- resolution, sex-specific positioning of crossovers with cellular profiling of key meiotic phenotypes. This research direction will unveil mechanisms that drive the evolution of a primary determinant of genetic variation. These distinct themes of research showcase a program that exploits the power of genetics and genomics in house mice to address fundamental evolutionary questions with breadth and depth. Beyond their evolutionary significance, the traits of interest are connected to common human diseases. Defects in recombination are the leading genetic cause of birth defects, body size is related to the metabolic syndrome, and exploratory activity is associated with neurological disorders. Our research offers potential to illuminate these conditions by deciphering natural variation in the premier genetic model organism for human disease.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Millions of people worldwide suffer from neurological diseases such as Alzheimer’s disease, stroke, and brain cancer. Advances in protein/gene profiling techniques and high throughput drug screening technologies have spawned many new drug candidates. However, the blood-brain barrier (BBB) has impeded the development and clinical realization of this new generation of neurotherapeutics by restricting the brain uptake of most small molecule therapeutics, and prohibiting brain uptake of protein- and gene-based medicines. A promising noninvasive brain delivery strategy takes advantage of endogenous BBB transport mechanisms as a means to shuttle drug cargo from the blood to the brain. Such receptor-mediated transport systems can be targeted using the exquisite specificity of antibodies that are in turn linked to a drug payload that can include small molecules, proteins, or DNA therapeutics. After binding to the receptor on the blood side, the antibody- drug conjugate acts as an artificial substrate for the transporter and is transcytosed from the blood, across the BBB, and into the brain. However, current approaches have yielded limited brain uptake because the targeted transporters are ubiquitously expressed and the antibody targeting reagents have low BBB permeability. Therefore, this proposal is focused on the identification and validation of a new panel of antibodies and cognate transporters that can mediate BBB crossing of therapeutics. The antibodies were identified by mining a large phage display antibody library against a BBB model comprising human induced pluripotent stem cell- derived brain microvascular endothelial-like cells (BMECs). The barrier characteristics of these BMECs are well- suited to screen large antibody libraries for those antibodies capable of crossing the BBB in vitro. Moreover, at the transcriptome level, the transporter profiles in these cells correlate quite well to freshly isolated human BMECs. Finally, the human sourcing ensures that identified antibodies recognize human BBB transporters. Using this innovative platform, we have identified a panel of high value antibodies that preliminary data indicate are capable of binding both the human and murine BBB and delivering pharmacologically relevant amounts of drug cargo to the murine brain. We propose to further validate these brain targeting antibodies by identifying their cognate transporters. Next, we will determine their pharmacokinetic properties, biodistribution and brain regiospecificity. Finally, the antibodies will be tested for their ability to mediate brain uptake of conjugated drug cargo to normal brain and to diseased brain in the form of a murine stroke model. Those antibodies that exhibit significant and selective brain uptake would represent new, noninvasive drug delivery vectors with potential application in many neurological disease settings.

NIH Research Projects · FY 2026 · 2021-03

Alzheimer's disease and related dementias (ADRD) disproportionately impact populations exposed to adverse social exposome (i.e., socioeconomic contextual life-course) factors, yet the sociobiological mechanistic underpinnings of this risk are only partially known. Deep precision phenotypic evaluation of populations at greater ADRD risk fundamentally increases opportunity for discovery of novel mechanistic pathways and development of new and effective ADRD prevention strategies. Since most specific ADRD neurodegenerative diseases can only be diagnosed through examination of post-mortem tissue, and brain bank repositories with such tissue rarely offer linkage to deep social exposome phenotyping, progress has been limited. We have begun to address this critical gap through the Neighborhoods Study (R01AG070883), a unique collaboration first funded by the NIA in 2021. Incorporating 21 Alzheimer's Disease Research Center (ADRC) brain banks, the Neighborhoods Study is the largest study of its kind, linking life-course social exposome measures— including the Area Deprivation Index (ADI, metrics built 1910-present by our team)— to neuropathology (N=8,637), uniquely providing insights into the many ADRD neurodegenerative diseases that can only be diagnosed from brain tissue. This ADI— built, validated and updated annually by our team— incorporates poverty, education, housing and employment indicators and is a widely used measure of the social exposome. In the first funding period, we built unique infrastructure, grew the ADRD exposome research community, and derived residential histories to establish dosage and timing of ADI exposure across each life-course. The Neighborhoods Study has catalyzed the field through its innovative findings to date, including demonstrating that even after adjustment for individual risk factors, high ADI is strongly associated with cognitive function, neurodegeneration, AD biomarkers and post- mortem AD neuropathology; that high ADI exposure carries markedly increased odds of post-mortem limbic- predominant age-related TDP-43 encephalopathy (LATE) and Lewy body neuropathology; and that living in a high ADI neighborhood across the life-course increases risk of cerebrovascular neuropathology over eight-fold. Mechanistic underpinnings for these associations remain unknown but could be linked to toxic heavy metal and high-risk occupational exposures (e.g., agriculture, military, factory) more common in high ADI areas. Exposure to such factors is known to increase clinical dementia diagnosis risk, but the precise neuropathology and life- course windows of susceptibility remain unknown. Thus, a path to precision prevention and risk mitigation remains elusive. To better understand these mechanisms, we propose a renewal that further enriches the cohort and offers novel exposome measures including life-course assessment of lead-laced infrastructure exposure; life-course occupational history construction; and brain tissue measures of heavy metals. The proposed project leverages the majority of ADRCs to further broaden understanding of how adverse social exposome impacts ADRD risk, providing a potential pathway to new interventions and directly responsive to the NIA mission.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Chronic pancreatitis (CP), a painful, debilitating disorder of the exocrine pancreas, lacks treatments and strategies to prevent disease progression. Alcohol abuse and smoking are common causes of CP but the mechanisms underlying their toxic effects on the pancreas are unclear. Recently, adaptive mechanisms that prevent pancreatitis with stressors such as alcohol were identified in the pancreatic acinar cell. These adaptive mechanisms involve the endoplasmic reticulum (ER) Unfolded Protein Response (UPR) and the UPR transcription factor, X-box binding protein 1 (XBP1s) that upregulates ER chaperones, ER transporters, and quality control machinery to maintain ER function. Our previous work showed that alcohol administration induces oxidative stress but also upregulates XBP1s and a protective UPR that prevents pathology, while smoking inhibits alcohol induced XBP1s formation, and upregulates a pathologic UPR signal mediated by C/EBP homologous protein (CHOP) resulting in ER dysfunction and pancreatitis. This project investigates molecular determinants of pancreatitis associated with alcohol abuse, smoking and perturbed ER protein folding and trafficking. Reversible Nε-lysine acetylation regulates the efficiency of ER protein transit to Golgi and the secretory pathway. Acetylation of properly folded proteins enables ER-to-Golgi exit, while non- acetylated, misfolded proteins divert to protein degradation systems. Thus, disruption of ER protein acetylation perturbs ER function and protein trafficking. The acetyl CoA transporter, AT-1 mediates ER entry of acetyl CoA from cytoplasm to provide substrate for acetylation. In humans, the AT-1-S113R mutant reduces AT-1 transport capacity, and individuals with this mutant exhibit neurodegenerative disorders. We found that XBP1s regulates AT-1 levels in acinar cells. Moreover, AT-1S113r/+ or acinar-specific AT-1 deficient mice develop mild/ moderate CP and chronic ER stress with elevated XBP1s. Strikingly, AP induction in these mice decreases XBP1s levels and markedly exacerbates CP progression. Our results indicate AT-1 and XBP1s are interdependent and important for pancreas adaption in response to alcohol but when overwhelmed by environmental stressors these adaptive systems fail leading to pathology. Our overarching hypothesis is that chronic acinar cell stress and reduced XBP1s protective programs by drinking/smoking attenuate AT-1 expression, disrupting ER acetylation and ER function, and inducing severe CP. Using experimental models of alcoholic + smoking CP, we will pursue 3 aims. Aim 1 will test whether alcohol consumption/smoking converts mild/moderate CP into severe disease in acinar-specific AT-1 KO mice. Aim 2 will evaluate whether enhanced XBP1s expression or CHOP genetic deletion partially mitigates CP severity in AT-1 KO mice. Aim 3 will investigate ER acetylation pathway regulation of ER protein folding and trafficking as well as ER protein degradation. We expect this project to provide insights into the pathways driving CP development, and pinpoint molecular targets for strategies to halt CP progression.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Invasive aspergillosis (IA) caused by Aspergillus fumigatus is characterized by uncontrolled filamentous hyphal growth deep into host tissues and is a fatal disease of immunocompromised patients with mortality rates as high as 90%. This high mortality rate indicates the critical need for improved antifungal therapeutic strategies. We have uncovered a bidirectional lipid signaling system between the fungus and host that mediates invasive hyphal growth and phagocyte activation. Based on strong preliminary data, this communication system consists of structurally similar fungal and host ligands (e.g. oxylipins) that are recognized by specific fungal and host G protein coupled receptors (GPCRs). The fungal and host oxylipins work in opposition to regulate fungal growth and leukocyte functionality. We hypothesize that fungal and host oxylipins are cross-Kingdom molecular analogs that signal through specific GPCR cascades, inducing penetrating hyphal growth and manipulating host defense responses to drive IA progression. Our data not only provide new insight into how eukaryotic pathogens and their hosts communicate with one another directly during disease but also provide a new foundation for experimental approaches to decipher, manipulate, and control this communication system in favor of the host. Accordingly, we will (1) Identify the oxylipins and their transcriptional cascades that regulate invasive branching growth and (2) Characterize the receptors by which fungus and host recognize each other’s oxylipins and the consequences of this recognition. GPCR are particularly propitious targets for therapeutic design (40% of current pharmaceuticals target GPCR). Thus, upon completion of this work, we anticipate that we will have delineated a new fungal-host ligand-receptor communication language amenable to therapeutic intervention to inhibit filamentous invasive growth during human disease.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Human daylight vision is dominated by signaling in the fovea, a specialization unique to diurnal primates which is responsible for half of the retinal output and hence input to the higher visual centers. Our high-definition central vision is initiated in the cone photoreceptors which are packed in a dense and exquisite pixel array in the fovea. This unique arrangement together with the specialized retinal circuitry is key for the highest spatial and chromatic resolution attributed to our central vision. It is well known that the density and morphology of cone photoreceptors differ remarkably between foveal and peripheral primate retina, but our knowledge about the physiological and functional differences remain quite poor. Interestingly, our recent observations in primate retina have revealed that the time course of cone signals in the fovea is two-fold slower than in the peripheral retina consistent with the two-fold difference in the temporal sensitivity of our cone-mediated vision to high-frequency flicker. The broad goal of our project is to determine the full breadth of heterogeneities in cone signaling, “retinotopy of function”, across a range of visual inputs and functional properties. We will focus on three salient questions across three aims: 1) What are the differences in key functional properties of signals originating in the primate cone photoreceptors across the visual field? (2) Is cellular noise generated in cone phototransduction homogenous in cones across the visual field and what limits does it pose for cone function and perception? (3) Do foveal cones exhibit differences in function during natural vision compared to cones in rest of the primate retina? We will answer these questions using electrophysiological recordings of responses from cones in primate retina and models that describe cone function. The proposed work will provide a detailed insight into primate cone signaling especially in the fovea. Death of cone photoreceptors is the primary cause for vision loss in retinal diseases that attack the fovea such as macular degeneration. A therapy option that holds promise for such degenerative diseases is stem cell derived photoreceptor replacement therapy. Our study will provide the much-needed baseline information about foveal cone signaling to evaluate cone function in human stem cell derived retina for designing effective stem cell-based therapies as a way to ultimately cure degenerative retinal diseases such as macular degeneration and others.

NIH Research Projects · FY 2025 · 2021-02

The age-related processes that contribute to Alzheimer's disease (AD) development, particularly in the prodromal period, are incompletely understood. Age-related reduction in gut microbiome alpha-diversity is apparent in the majority of older adults, and is suspected of contributing to brain changes, including the development of neurodegenerative disease. Our team published the first comprehensive report describing differences in the gut microbiome observed in AD dementia, including reduced diversity in gut microbiota and altered composition in people with AD dementia compared to age-matched controls. Furthermore, we found that differentially abundant genera were associated with cerebrospinal fluid biomarkers of AD, even among individuals who were cognitively unimpaired. Several studies in mouse models of AD indicate that gut microbiota play a role in the development of AD neuropathology, however to date, the mechanisms underlying these effects are virtually unknown. Recently it has also become clear that the innate immune response in AD plays a critical role in mediating the pathology associated with AD; however the interplay between systemic changes and the innate immune response in AD are not well understood, nor is it known how these factors impact the progression of AD pathology. Our overarching goal is to determine the extent to which alterations in the composition of gut microbiome exacerbate and/or accelerate the development of AD pathology. This proposal is based on the central hypothesis that age-associated gut dysbiosis and inflammation weaken gut barrier function, which in turn leads to the systemic dissemination of microbial components, driving an immune response and system wide changes that worsen AD pathology. To test this hypothesis we propose to study well-characterized participants enrolled in the Wisconsin Alzheimer's Disease Research Center as well as conventional and gnotobiotic APPPS1 mice, to address the following specific aims: 1. Determine the longitudinal relationship between gut microbiome (metagenome), gut inflammation and permeability, and the development of AD pathology in human participants, and 2. Determine the effects of modifying gut permeability on AD pathology in mice. We expect that alterations in gut microbiome composition and gut permeability exacerbate AD pathology in humans, and that impairment of intestinal barrier function and increased gut permeability alters brain homeostasis and exacerbates AD progression in mouse models of AD. Our research group has been working to determine the role of gut microbiome in the development of AD pathology for the past 5 years, and we are perfectly poised to address the proposed aims. We will leverage our expertise in clinical AD, neuroimmunology, and gut microbiology/gnotobiotic mouse models to successfully carry out the proposed project. Completion of the proposed experiments is expected to lead to the development of novel therapeutic strategies for AD and related dementias.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Nonsense mutations cause approximately 15% of genetically inherited retinopathies and inherited human diseases in general, accounting for 2.5 to 3 million patients in the U.S. For certain specific genes, nonsense mutation incidences can be as high as 40%. Because nonsense mutations cause premature termination (PTC) of protein translation, the disease phenotype is often severe. Currently, there are only a limited number of therapies for nonsense mutations being tested in human clinical trials, including gene therapy, small molecule read-through drugs, or genome editing. Associated challenges equal the promises of each of these therapeutic options. Looking forward, newer technologies may address these hurdles and provide more safe and efficacious treatments for patients. During protein translation, tRNA functions at the ribosomal site to incorporate a specific amino acid into the polypeptide sequence. We aim to develop the next generation of nucleic acid therapy based on anticodon encoding transfer RNA (ace-tRNA) that incorporates the correct wild type amino acid at the site of a disease-causing nonsense mutation. Because of the many anatomical advantages afforded by the eye, we seek to test the broad applicability of ace-tRNA therapeutics for nonsense mutations that cause retinopathies and related blindness due to defects in a variety of genes, including those encoding ion channel proteins. Specifically we will focus on nonsense mutation in ion channels expressed in photoreceptors (PR) which convert retinal light inputs and retinal pigment epithelium (RPE), which provide support for PR. These two cell types are primarily the site of blindness pathogenesis. In this project, we will: 1) Develop ace-tRNA therapeutics that target specific nonsense mutations across several PR and RPE ion channels. 2) Engineer both viral and non-viral ace-tRNA delivery systems for long-term editing. Using these we will determine the functional outcome of ace-tRNA treatment using cultured cells and human iPSC-derived RPE and iPSC-PR retinal organoids. 3) Test both our viral and non-viral ace-tRNA in vivo using mice harboring genetic defects that cause blindness in humans; and 4) Assess the safety and bioavailability of ace-tRNA therapeutics in our preclinical NHP model systems. There are no FDA-approved therapeutic drugs that target channelopathies because of the complexities associated with precise post-translational modifications, carefully regulated expression, and assembly. Our team’s combined expertise in ace-tRNA development, nanomaterial synthesis, human pluripotent stem cell biology, ion-channel physiology, and pathophysiological model systems is unique and ideally suited to advance ace-tRNA technology toward clinical trials for a wide range of genetic diseases that cause blindness.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract Opportunistic fungal pathogens are a leading cause of healthcare associated bloodstream infections. Candida yeasts, specifically, cause 80-90% of biofilm-associated invasive fungal infections and mortality rates can approach 50%. Furthermore, the incidence of Candida infections is rising with the increased use of catheter and other device-based interventions. To date, the majority of work related to fungal infections and potential treatments has focused on biofilms and their prevention. However, recent evidence suggests that the dispersion of yeast cells from the biofilm (into the bloodstream) and the persistence of these dispersed cells are perhaps more important virulence factors and represent significant but underutilized treatment targets. Further, existing assays and instrumentation are not amenable to measuring dispersion and phenotyping dispersed cells. Nor do they recapitulate in vivo conditions (e.g. flow, interaction with host cells). Thus, here we will develop a new type of under-oil open microfluidic system (UOMS) to quantify the dispersive capacity of biofilms and assess the phenotype of dispersed cells in Candida mutants and clinical isolates. The UOMS platform is built on the foundation of a newly observed phenomena called Exclusive Liquid Repellency (ELR). ELR provides a unique environment where liquid is completely repelled from a solid surface to eliminate biofouling. Additionally, ELR expands the capabilities of simple open microfluidic devices allowing us to overcome the limitations of current methods and provide a system capable of quantitatively studying fungal dispersion. We will first (Aim 1) develop an under-oil microchannel device to measure the dispersive capacity of Candida biofilms and virulence phenotypes. Second (Aim 2) we will automate the UOMS and develop a single- cell distribution assay to measure the phenotype of individual dispersed cells. And finally (Aim 3), we will use the UOMS platform to develop dispersion phenotype profiles for Candida mutant libraries and clinical isolates. We will measure the dispersive capacity and phenotype of dispersed cells from hundreds of Candida albicans clinical isolates and mutants available in existing libraries, providing clues to the genetic determinants of dispersion.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Growing advances in imaging and fluid-based assays of Alzheimer's disease (AD) biomarkers including amyloid, tau and neurodegeneration, confirm that AD processes begin decades before clinical impairment in cognitive function. Subtle changes to cognition are also likely to co-occur years before a clinical diagnosis of dementia due to AD. There is an urgent need to develop sensitive measures of subtle cognitive decline associated with AD biomarkers, particularly for monitoring response to early intervention treatments in clinical trials. The proposed investigation is highly innovative and designed to leverage existing data from three longitudinal cohort studies—Wisconsin Registry for Alzheimer's Prevention, Wisconsin Alzheimer's Disease Research Center, and BIOCARD–using a classic and widely used measure of cognition: the story recall task. We developed a novel scoring system that we hypothesize targets semantic and associative memory processes: measures that capture lexical categories and serial position. Our preliminary data shows that proper name recall and serial position scores from story recall are significantly associated with beta-amyloid status from positron emission tomography (PET), while the traditional total score was not related to amyloid status. In this proposal, our central hypothesis is that item-level analysis of existing story recall data from several longitudinal cohorts will yield one or more new measures of cognition that are uniquely associated with underlying preclinical AD pathology. The specific aims are: Aim1: Use data from multiple cohort studies to a) replicate preliminary findings that lexical-level and serial position markers from delayed story recall are associated with increased risk of amyloid positivity and b) extend analyses to investigate whether these variables are associated with PET tau, CSF Aβ and tau, or MRI neurodegeneration measures. Aim 2: Compare concurrent and predictive validity of measures to determine whether the novel measures are more strongly associated with biomarkers, cognitive decline, or progression to clinical levels of impairment than traditional total score measures. Aim 3: Enhance the lexical-level and serial position analysis with computational linguistic analysis of digitally recorded speech from story recall to determine whether semantic content, speech fluency, error-monitoring, and serial position recall explain unique variance in levels of amyloid and/or tau pathology. Impact: The proposed project leverages existing data and is expected to lead to the development of new outcome measures from a classic, commonly used test that has played a central role in detection of disease. We expect that our higher-level language and process-based measures will be sensitive to AD biomarkers in preclinical phases of cognitive decline. By utilizing existing resources from differing cohorts, we can validate our findings without adding participant burden, share these methods with other cohort studies, further develop a digital marker of speech and cognition, and contribute an improved understanding of the underlying mechanisms of memory and communication breakdowns in preclinical AD.

NIH Research Projects · FY 2025 · 2021-01

INTRODUCTION TO REVISED APPLICATION The A0 grant was submitted in May 2019, before Prof. Alexander Barnes moved to ETH-Zurich and before Prof. Chad Rienstra was recruited to UW-Madison. Now Rienstra is officially a Full Professor at UW-Madison, after having negotiated major investments in the solid-state NMR (SSNMR) program at NMRFAM (new and/or moving from Illinois) including three shielded 600 MHz magnets, one 750 MHz wide bore magnet, four spectrometers, several custom-designed magic-angle spinning (MAS) probes at 600-750 MHz, and upgrades to the 900 MHz spectrometer, which immediately have had an impact on data collection for DBP6 in late 2019 and for other experiments in progress during early 2020. Furthermore, the National Science Foundation Mid-Scale Research Infrastructure-2 proposal on "Network for Advanced NMR", which was submitted by UW-Madison PIs (Rienstra and Henzler-Wildman) in collaboration with Jeff Hoch at UConn Health and Art Edison at U. Georgia, is in late stages of negotiation and review with NSF. If funded, this grant would bring a 1.1 GHz dedicated SSNMR spectrometer to NMRFAM in ~2022-23. These developments have motivated several changes to this A1 application which more explicitly emphasizes the SSNMR program at NMRFAM: (1) Rienstra is now contact PI and Henzler-Wildman co-PI. (2) TR&D1 now includes sub-aims targeting development of micro-rotor packing and sample manipulation tools to leverage recent breakthroughs in ultra-fast MAS (>100 kHz) at <1 mm rotor diameters; this broadens the scope and impact of TR&D 1. Baselines and benchmarks for NMR under gradients are also more clearly described and proof-of-principle experiments are in place. (3) TR&D2 now addresses critical bottlenecks in SSNMR data collection, emphasizing: (a) automation for parameter optimization and spectrometer configuration; (b) new narrow bore magic-angle spinning probe designs at 600-900 MHz (that will be applicable at 1.1 GHz and higher in the future); and (c) real-time feedback interaction with data processing (TR&D3). (4) TR&D3 now leverages NMRFAM software products and continuing technology development for solid- state NMR, including assignment, structure determination, refinement and validation tools, and it is more clearly integrated with the rest of the proposal. (5) DBPs 5, 6 and 7 have been changed to include well-developed and impactful collaborations between Rienstra and Paul Kotzbauer (Wash. U. Medicine, Lewy bodies and synucleiopathies), Marty Burke (U. Illinois, antifungal drugs), and James Morrissey (U. Michigan, blood coagulation). Overall the proposal is now organized in (we think) a more logical/chronological manner, with TR&D1 emphasizing samples, TR&D2 the spectrometer and probes, and TR&D3 the software and analysis procedures. The proposal body further explains how these developments greatly augment the cost-benefit ratio for the project and integrate with the current user program and the long-term vision of NMRFAM. The revisions also address the overarching concerns of reviewers of the A0 application including "narrow scope/modest innovation in TR&D1"; "weak integration of TR&D3"; cost-benefit ratio; preliminary data; solution vs. solids emphasis of TR&D3; innovation; context (addressing competing ideas and precedents); and specificity of outcomes. Finally, we have clarified the premise and approach with respect to asymmetry in membrane proteins.

NIH Research Projects · FY 2026 · 2020-12

PROJECT SUMMARY/ABSTRACT Adult mammals poorly regenerate injured hearts. In contrast, adult zebrafish possess a remarkable capacity to regenerate damaged hearts. Combined with available genetic tools, this capacity makes zebrafish a powerful system for deciphering the innate mechanisms underlying heart regeneration. In the previous funding period, our laboratory uncovered that interleukin11a (il11a) signaling plays dual roles as a regenerative and fibrotic factor in adult zebrafish hearts. We demonstrated that these opposite outcomes are caused by cell-type-dependent effects. By integrating epigenome and transcriptome analyses with transgenic assays, we also identified the importance of enhancer and promoter pairing in transcriptional activation and cell types emerging in regenerating hearts and their molecular identity. However, critical gaps remain in our understanding regeneration-associated genetic and epigenetic mechanisms. First, we know little about transcriptional regulators of il11 signaling responsible for regenerative processes. Second, while numerous cardiac regeneration enhancers have been discovered, we still lack a comprehensive understanding of how DNA sequences and their composition encode regeneration-dependent expression. Third, there is a need to delineate the differences between injury- and regeneration-associated gene regulatory networks. The current application aims to address these central questions using cutting-edge genetic and epigenetic analysis tools. We hypothesize that a cardiac mitogen activates dedicated cardiac regeneration enhancers to initiate the repair process. In Aim 1, we will elucidate transcription factors (TFs) mediating il11a-triggered cardiomyocyte (CM) proliferation and related gene regulatory networks (GRNs). Epigenome and biochemical analyses with zebrafish genetic models will identify the foxm1/e2f8/mybl2b/znf367 complex and their related GRNs as key players for CM proliferation. In Aim 2, we will define a cluster of cardiac regeneration enhancers (regeneration-cistrome) that ensures precise transcriptional control of the regeneration response. Our computational analyses with zebrafish transgenic and regeneration assays will identify enhancer logics of cardiac regeneration enhancers and related cistrome in the zebrafish genome. In Aim 3, we will employ zebrafish genetic models and CUT&Tag approaches to identify regeneration-dedicated enhancers to discriminate injury and regeneration processes. Overall, this project will articulate TF networks and epigenetic factors for heart regeneration, illuminating innate mechanisms promoting heart repair and uncovering strategies for mammalian heart repair.

NIH Research Projects · FY 2025 · 2020-12

Scientific abstract Epilepsy is a chronic and debilitating disease, leading to refractory seizures in up to 40% of patients. A better understanding of the neural mechanisms that cause recurrent seizures could lead to improved diagnostic markers and new neuroprotective therapies. My recent research suggests that abnormal slow-wave activity (SWA) patterns during sleep may constitute a promising diagnostic marker to locate the seizure onset zone (SOZ). In a high-density electroencephalogram (hdEEG) study of fifteen focal epilepsy patients, I found increases in sleep SWA that were maximal in the SOZ and were correlated with seizure and interictal spike frequency. Building on a wealth of studies validating sleep SWA as a marker of synaptic strength, my results suggest that seizures and spikes induce synaptic potentiation in the human brain. To further validate sleep SWA as a diagnostic marker for the SOZ, I aim to make use of the higher spatio-temporal resolution of direct intracranial EEG (iEEG) recordings. In Aim 1, I will analyze continuous iEEG recordings in patients with focal epilepsy to quantify sleep SWA in the SOZ, in the seizure propagation network (SPN, areas secondarily recruited in the ictal rhythm), and in the periphery (areas not involved in the ictal rhythm). I hypothesize that 1) in the SOZ, SWA will increase maximally; 2) in the periphery, sleep SWA will have lower values; and 3) the SPN will show intermediate patterns. In Aim 2, I will also analyze single-unit (SU) recordings to identify the neuronal contributors to sleep SWA during sleep and their alterations across seizure territories. I will use existing long-term microelectrode recordings from epileptic patients to quantify SU firing rates and multi-unit activity (MUA) synchrony during sleep (two markers of synaptic strength) in the SOZ, the SPN, and the periphery. To shed light on the relationship between increased sleep SWA and ictal firing rates, I will use the SU recordings to separate the SPN into areas of high vs. low ictal firing rates (the ictal core vs. ictal penumbra, respectively). I hypothesize that 1) in the SOZ, SU firing rates and MUA synchrony will increase maximally; 2) in the periphery, SU firing rates and MUA synchrony will show lower values; and 3) the SPN will show intermediate patterns, but with more normal values overall in the ictal penumbra compared to the ictal core. If this project is successful, it will provide mechanistic evidence for a link between chronic hyperexcitability in the epileptic network and synaptic potentiation due to seizures, which can be sensitively detected using intracranial sleep EEG. It will also allow researchers and clinicians to develop new diagnostic tools to localize the SOZ, paving the way for new therapeutic interventions targeting sleep to decrease seizure frequency in patients with epilepsy.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY Parkinson disease (PD) is devastating to communication, which is also impacted by concurrent cognitive and affective impairments. The hallmark pathology, loss of dopamine, has guided therapy for decades; however, dopamine-centered treatments do not improve vocal communication, cognition, or affect. In fact, prior to classic dopamine loss, there is significant degeneration in the locus coeruleus, a norepinephrine-rich brainstem region that is vital to communicative and cognitive behaviors. Here, we propose to study three different therapeutic approaches that modulate norepinephrine (exercise, drugs, socialization) based on the rationale that modulating noradrenergic brain systems will improve PD-related communication deficits, as well as cognition and affect. We hypothesize that the benefit of these therapies will be improved communication, cognition, and affect as well as neuroprotection (i.e. sparing of neurons, increased neurotrophins). However, as with any treatment, there may be unwanted side effects such as anxiety, increased motor errors, or enhanced neuroprogression (loss of neurons) due to neurotoxicity of drugs. For superior experimental control and to study underlying neural mechanisms with increased scientific rigor, we will use a well-established translational rat model. The Pink1-/- rat is based on a genetic form of early and progressive PD (PARK6) that is nearly identical to idiopathic PD. We have shown communication, motor, cognitive, and affective deficits and neural abnormalities that are analogous to humans, including early loss of norepinephrine in brain regions important to communication. Aim 1 will analyze the benefits and side effects of intervention on communication, cognition, and affect. Pink1-/- rats will be treated with either: (1) cardiovascular exercise, (2) targeted vocal exercise, (3) methylphenidate, (4) propranolol, (5) social enrichment, or (6) control conditions at 10 months of age, which is equivalent to human age at time of diagnosed and treatment initiation. Vocalization, attention, accuracy, memory, anhedonia, and anxiety will be assayed, and effect sizes of each treatment will be calculated to determine the impact of treatment on all outcomes. Aim 2 will quantify changes to the brain with these interventions. Rats from Aim 1 will undergo in vivo microPET scanning to determine how interventions modulate norepinephrine. Ex vivo, brain tissues will be analyzed for neurotransmitter content, cell numbers, and changes to neurotrophins/receptors in regions associated with vocalization, cognition, and affect using high pressure liquid chromatography and immunohistochemistry. We hypothesize that interventions will result in either neuroprotection (e.g., increases in neurotrophic factors) or neurodegeneration (e.g., cell death/loss of neurotransmitter). This work is innovative because it is the first controlled study to robustly assess behavioral responses to noradrenergically-based interventions and concurrently measures in vivo modulation of norepinephrine and other important brain mechanisms as a result of intervention. Findings will be readily translated to directed human clinical trials to combat this devastating human health problem.

NIH Research Projects · FY 2025 · 2020-09

The long-term goal of the proposal is to develop new therapeutic strategies using mechanistic insights drawn from understanding astrocyte-motor neuron interaction in amyotrophic lateral sclerosis (ALS). In particular, the primary objective of this proposal is to better delineate the mechanisms responsible for the protection conferred by enhancing nicotinamide adenine dinucleotide (NAD+) availability in ALS models. ALS or Lou Gehrig's disease accounts for about 1 in 500 to 1 in 1,000 adult deaths in the United States and is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Motor neuron death leads to muscle weakness and paralysis causing death in one to five years from the time of symptoms onset. Most ALS cases are sporadic (SALS) and exposure to yet unidentified environmental toxicants might be responsible for SALS. About 5-10% of the cases are inherited (familial ALS, FALS) but FALS and SALS are phenotypically indistinguishable, and a significant share of our understanding come from the study of rodent models over-expressing ALS-linked mutant human superoxide dismutase 1 (hSOD1). Several lines of evidence underscored the contribution of non-neuronal cells in the neurodegenerative process and astrocytes appear to have a decisive role in the progression of the disease. Accordingly, primary astrocytes isolated from mutant hSOD1 over-expressing mice induce motor neuron death in co-culture, and it has been demonstrated that astrocytes differentiated from spinal cord autopsy-derived neuronal progenitor cells from FALS and SALS patients are also toxic for motor neurons in co-culture. We have shown that over-expression of the NAD+-synthesizing enzyme, NAMPT, or increasing the activity of two sirtuins (SIRT3 and/or SIRT6) is protective in a co-culture model of ALS. Sirtuins are a family of enzymes capable of catalyzing NAD+-dependent deacylation and mono(ADPribosyl)ation reactions, and play a key role in transcription, DNA repair, metabolism, and oxidative stress resistance. Our previously published data and ongoing experiments demonstrate that modulating NAD+ metabolism and signaling is protective in a co-culture model of ALS, while the expression of enzymes involved in NAD+ synthesis and NAD+-dependent signaling is altered in ALS patients. Moreover, enhancing NAD+ levels by dietary supplementation with a metabolic precursor (nicotinamide riboside) exerts neuroprotective effects in an ALS mouse model. Thus, we seek to better define the role of NAD+-dependent signaling during motor neuron degeneration. The results obtained during the previous funding period rationally support the development of cell-type specific approaches to better target NAD+ metabolism and signaling in ALS. Since we have shown that therapeutic targets identified in our astrocyte-motor neuron co-culture system have a beneficial effect when translated into animal models of ALS, the proposal is likely to provide a mechanistic insight and in vivo proof of the value of modulating NAD+ metabolism and signaling as a therapeutic approach in ALS.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY The rate of substance use-related hospital visits in the US continues to increase, and now outpaces visits for heart disease and respiratory failure. The prevalence of substance misuse (nonmedical use of opioids and/or benzodiazepines, illicit drugs, and/or alcohol) in hospitalized patients is estimated to be 15%-25% and far exceeds the prevalence in the general population. With over 35 million hospitalized patients per year, tens of millions of patients are not screened for substance misuse during their stay. Despite the recommendation for self-report questionnaires (single-question universal screens, Alcohol Use Disorders Identification Test [AUDIT], Drug Abuse Screening Tool [DAST]), screening rates remains low in hospitals. Current screening methods are resource-intensive, so a comprehensive and automated approach to substance misuse screening that will augment current clinical workflow would therefore be of great utility. In the advent of Meaningful Use in the electronic health record (EHR), efficiency for substance misuse detection may be improved by leveraging data collected during usual care. Documentation of substance use is common and occurs in 97% of provider admission notes, but their free text format renders them difficult to mine and analyze. Natural Language Processing (NLP) and machine learning are subfields of artificial intelligence (AI) that provide a solution to analyze text data in the EHR to identify substance misuse. Modern NLP has fused with machine learning, another sub-field of AI focused on learning from data. In particular, the most powerful NLP methods rely on supervised learning, a type of machine learning that takes advantage of current reference standards to make predictions about unseen cases In our earlier version of an NLP and machine learning tool, our opioid and alcohol misuse classifiers successfully used data from clinical notes collected in the first 24 hours of hospital admission to reach a sensitivity and specificity above 75% for detecting alcohol or opioid misuse. We will improve the performance of our baseline, individual NLP single-substance classifiers for alcohol and opioid misuse by implementing multi-label and multi-task machine learning methods. These methods will take advantage of information shared across different types of substance misuse and better capture the state of a patient within a single model. The resulting classifier will be capable of jointly inferring all types of substance misuse (alcohol misuse, opioid misuse, and non-opioid illicit misuse) including polysubstance use, and cater to each individual patient’s substance use treatment needs. We aim to train and test our substance misuse classifiers at Rush in a retrospective dataset of over 35,000 hospitalizations that have been manually screened with the universal screen, AUDIT, and DAST. The top performing classifier will then be tested prospectively to: (1) externally validate its screening performance in a hospital without established screening; and (2) test its effectiveness against usual care at a hospital with questionnaire-based substance misuse screening. We hypothesize that a single-model NLP substance misuse classifier will provide a standardized, interoperable, and accurate approach for universal screening in hospitalized patients and guiding interventions.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Individuals with Alzheimer’s disease and related dementias (ADRD) demonstrate heterogeneous cognitive, functional, and behavioral responses to disease progression, yet underlying sources of these variations remain poorly understood. Fluctuations in communication abilities in particular have important implications for care and attitudes toward individuals with ADRD. Of particular interest is the occurrence of episodes of lucidity (EL) in advanced disease stages, which are characterized by the manifestation of spontaneous, meaningful, and relevant communication abilities that were previously believed to be irretrievable. Reports suggest EL are transient and most likely to occur near end of life, yet empiric evidence documenting these events is extremely limited. Efforts to better understand, and ultimately define EL, are hindered by underdeveloped methodological approaches for capturing and characterizing these events. While underutilized in ADRD research, audiovisual observation is well-suited to addressing these gaps, as these data provide an objective data source and enable a robust assessment of verbal and nonverbal communication, the primary means through which EL are observed. Our long-term objective is to clarify conceptual, operational and epidemiologic understandings of EL in ADRD. Our short-term objective is to establish the necessary foundational data and infrastructure to accelerate systematic investigation of EL. To advance these goals, we will develop feasible and acceptable procedures to enable capture of longitudinal audiovisual data of targeted populations and timeframes to maximize opportunities for detecting EL; these rich data sources will then be analyzed via computational linguistic and sequential analysis (timed-event) methods, to assemble a robust, fully characterized understanding of the linguistic, non- linguistic, and non-verbal communication features of EL along with the specific temporal qualities of these events. In the R21 Phase, we will demonstrate the feasibility of collecting and sharing longitudinal audiovisual data among PLWD near end of life through a mixed-methods feasibility study to determine key ethical and practical considerations (R21 Aim 1); and evaluate usability, reliability and capture rate for coding of verbal/nonverbal indicators of EL from collected audiovisual data using computational linguistic and timed-event methods (R21 Aim 2). The R33 Phase will harness procedures optimized in the R21 phase to expand data collection and develop more robust estimates of EL frequency. Specifically, we will expand longitudinal audiovisual observations of persons with ADRD near the end of life to obtain initial estimates of EL frequency, describe event attributes, and inform the development of definitions for EL (R33 Aim1); and establish a repository of longitudinal audiovisual observational data and a secure data sharing platform (R33 Aim 2). Impact: Findings from this research will set the stage for extension and validation of urgently needed objective measurement for EL in ADRD and provide a critically needed foundation for future systematic investigation of lucidity in ADRD. The objectives of this proposal are directly responsive to national ADRD research priorities and the NIA mission.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Despite many years of research in humans and model organisms, there remains no clear consensus about which diet is most compatible with human health. However, the premise of this statement is that a single diet is ideal for everyone. Yet, among humans, there is a wide range in metabolic response to various diets, including body weight, glucose tolerance, and plasma lipids. Genetically diverse mice show this same metabolic variability, suggesting that genetics plays a key role in driving diet-responsiveness. In addition, the microbiome in both humans and mice contributes to diet responsiveness through its metabolism of dietary nutrients and production of potent metabolites. The premise of this project is that genetic interactions with diet and the gut microbiome affect metabolic health. Using an outbred mouse model system that has as much genetic diversity as the entire human population, the Diversity Outbred population, we will genetically map the gene loci that interact with diet and the microbiome to affect cardiometabolic phenotypes. We will test two diets, a low-fat/high carbohydrate diet and a high-fat/low carbohydrate diet. The mice will be phenotyped for glucose tolerance, insulin resistance, weight gain, and circulating levels of lipids and metabolites. Using 15 stable isotope tracers, we will conduct metabolic flux measurements using mass spectrometry-based isotopomer analysis, enabling us to interrogate the major pathways of carbohydrate, lipid, and protein metabolism in multiple tissues. This will be the first time metabolic flux has been subjected to a genetic screen. We will also map gut microbial composition, and gene regulation in key metabolic tissues: liver, adipose, muscle and intestine. These studies will deliver comprehensive genetic maps of these phenotypes. Through the identification of phenotypes that co-map, we will perform mediation analysis to construct causal networks that link gene loci, metabolites, microbiome taxa and physiological phenotypes. We will prioritize loci that are syntenic to human loci with significant metabolic associations in GWAS. These results will provide metabolic markers that can help predict an individual’s metabolic response to specific diets, the first step towards matching diets to individuals.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY Critical knowledge gaps in our understanding of benign genitourinary (GU) diseases exist, such as their etiology and development, pathophysiology, and mechanisms of progression. The phenotypic expressions of many GU diseases remain undefined, which stymies both basic and clinical research. Clinical challenges include lack of accurate and objective diagnostic tests for many of these conditions, dearth of effective multidisciplinary and interdisciplinary approaches to management, impact of current treatments and therapies on comorbid disease processes and quality of life, and lack of reimbursement for strategies to prevent onset and progression. Moreover, the true public health burden of GU diseases overall is unknown, and the impact of disparities in health outcomes is under-appreciated and under-studied. The proposed Interactions Core for the Community of NIDDK-funded benign GU Centers and Programs known as CAIRIBU (Collaborating for the Advancement of Interdisciplinary Research in Benign Urology) will foster and support integrated basic and translational science that answers critical questions in benign GU disease and builds the breadth, depth, and visibility of the urology research community. Building on our prior successes, we will provide leadership, coordination, infrastructure, and logistic support for the CAIRIBU research community by identifying and facilitating opportunities for cross-CAIRIBU collaboration, promoting research findings and resources to the broader GU research community, and raising the visibility and support for GU research. We will: 1) provide centralized organization and coordination to leaders, investigators, and trainees in the CAIRIBU Community to enhance and promote highly meritorious convergent research; 2) develop and nurture research collaborations by identifying and fostering opportunities for knowledge exchange, resource-sharing, transdisciplinary interaction, and networking across CAIRIBU and the entire GU research community; 3) increase the research capacity of GU investigators, particularly of trainees and early-stage investigators; and 4) amplify GUD research by building a multi-faceted communications, networking, and outreach strategy across the broader urology research community, among patients, and within the public. Our Interactions Core (IC) is led by an established benign GU disease investigator who has functioned in the capacity of CAIRIBU IC Director since July 2018, during a 2- year pilot phase, and then since 2020 after obtaining a competitive award. Due to its multiple resources and strengths in urology research, the University of Wisconsin is an ideal environment for supporting the IC. The Department of Urology is ideal for the home of the IC due to its strong commitment to GU research as evidenced by its ranking within the top five for research funding to urology departments. We will use innovative and engaging approaches to leverage investigators’ common passion for addressing critical gaps in knowledge in benign GU disease and building the numbers and diversity of the urology research workforce.

NIH Research Projects · FY 2024 · 2020-09

Project Summary This application is being submitted in response to NIH's INCLUDE (OT-OD-20-025) Notice of Special Interest. The purposed R01 provides the first longitudinal investigation of the time-ordered effect of four lifestyle factors - physical activity, sleep, cognitive stimulation, and social engagement - on early Alzheimer's disease (AD) neuropathology and the transition to clinical AD in adults with Down syndrome (DS). These lifestyle factors will be assessed at a total of three time points, each spaced 16 months apart, in 140 adults with DS enrolled in the NIH-funded Alzheimer's Biomarker Consortium in DS (ABC-DS; https://www.nia.nih.gov/ research/abc-ds). Adults with DS are at genetic risk for AD due to the triplication of chromosome 21, which contains the gene for the amyloid precursor protein and thus results in an overproduction of amyloid-bet (Aβ). Despite this genetic risk, there is variability in the age of onset of clinical AD in the DS population. Lifestyle factors may contribute to this variability, as has been found in non-DS populations including adults with early-onset familial forms of AD. Indeed, as a group, adults with DS have been found to engage in a relatively high rate of sedentary behavior, experience a high rate of sleep problems, and to have lifestyles marked by low levels of cognitive stimulation and social engagement. In the proposed study, we will collect information on physical health, sleep, cognitive stimulation, and social engagement across a 7-day/night period at three time points (spaced 16 months apart). Self/information report and objective measures (actigraph and WatchPAT) are used to assess these lifestyle factors. This data collection will correspond in time with ABC-DS data collection of AD biomarkers, cognitive functioning, and dementia symptoms and status. Time-ordered associations between lifestyle factors and AD biomarkers (PET Aβ, PET tau, PET FDG, structural and functional MRI, and CSF Aβ and tau, and blood) and cognitive functioning and dementia will be examined. The specific aims of the study are to: 1) Examine the association between lifestyle factors –physical activity, sleep, cognitive stimulation, and social engagement – and AD biomarkers longitudinally (T1 to T3; each spaced 16-months apart); 2) Determine the association between lifestyle factors and cognitive functioning and dementia symptoms and status longitudinally (T1 to T3); 3) Evaluate the moderating role of lifestyle factors on the relation between early AD neuropathology (indexed by biomarkers) and cognitive functioning and dementia symptoms and status longitudinally (T1 to T3). These lifestyle factors may be important modifiable resiliency mechanisms for delaying clinical AD in adults with DS despite their genetic risk.

NIH Research Projects · FY 2024 · 2020-09

Project summary The overarching goal of this study is to use complementary Drosophila and inducible pluripotent stem cells (iPSC)-derived neuronal models to understand how mutations in the Ubiquilin 2 (UBQLN2) gene cause amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). UBQLN2 and closely related UBQLN1 belong to a family of eukaryotic ubiquitin (Ub)-binding proteins that function, in part, as chaperones for proteins that are destined for proteasomal degradation. Missense mutations within a unique, functionally orphan proline-repeat region (PRR) of UBQLN2 cause X-linked, forms of ALS/FTD, with some patients exhibiting a pure dementia onset. In addition, ubiquilin histopathology, comprised of dense aggregates of UBQLN2 and UBQLN1 are observed in most instances of ALS/FTD regardless of UBQLN2 mutation status. To address pathomechanisms of UBQLN2-associated FTD we exploited the upstream activating sequence (UAS)/GAL4 system to generate isogenic Drosophila strains expressing wild-type (WT) and ALS mutant forms of UBQLN2 in different tissues and cell types. We found that UBQLN2ALS mutants elicited dose-dependent phenotypes— including eye degeneration, motor defects, and lifespan shortening—that were more severe than phenotypes caused by equivalent expression of UBQLN2WT. UBQLN2ALSmutants, but not UBQLN2WT, formed intraneuronal aggregates characteristic of ubiquilin inclusions found in ALS/FTD patients. Unbiased genetic screens identified more than 30 genetic intervals that either reduced or enhanced UBQLN2ALS mutant toxicity. Gene mapping studies suggest that endolysosomal pathways are centrally involved in UBQLN2ALS-mediated neurodegeneration and point toward axon guidance genes and neuronal dependence receptors as potentially novel disease modifiers. In this R01 grant proposal we will continue genetic studies of UBQLN2-mediated neurodegeneration in Drosophila, focusing on endolysosomal trafficking and axon guidance as lead pathways for discovery efforts. Modifier pathways identified in Drosophila will, in turn, inform studies of iPSC-derived motor neurons (iMNs) expressing endogenous UBQLN2ALS alleles. The specific objectives of Aim 1 are to: (a) investigate the Rab5 gene and endolysosomal defects in UBQLN2ALS flies; (b) investigate the contributions of the Unc-5 axon guidance pathway to UBQLN2-mediated neurodegeneration; and (c) map and validate of UBQLN2ALS modifier genes in Drosophila. The specific objectives of Aim 2 are to: (a) establish functional defects in UBQLN2ALS iMNs; (b) carry out proteomic analysis of UBQLN2ALS iMNs; and (c) evaluate genes emerging from Drosophila screens in Aim 1 as genetic modifiers of UBQLN2ALS toxicity in iMNs. The combined genetic, cellular, and biochemical studies using Drosophila and mammalian iMN models will provide important new insights into UBQLN2-associated neurodegeneration in ALS/FTD. In addition, pathways identified in our study are likely to overlap with and inform toxicity pathways instigated by other aggregation-prone, ALS/FTD- associated genes.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Chronic pain is pervasive and devastating. It diminishes quality of life for 20% of adults. An integral feature of persistent pain is increased excitability in nociceptive sensory neurons. The molecular mechanisms that orchestrate plasticity in nociceptors are unclear. The integrated stress response (ISR) is a master regulator of neuronal function. Activation of the ISR triggers global suppression of translation and initiates pain through direct effects on sensory neurons. Four upstream kinases trigger the ISR through phosphorylation of the regulatory subunit of eukaryotic initiation factor 2. We recently discovered that one of these kinases, general control nonderepressible 2 (GCN2) is uniquely critical. Virtually nothing is known about the function of GCN2 in pain signaling. We will focus on resolving this problem in the following two aims: Aim 1 – Define the role of GCN2 in pain. Aim 2 – Examine the landscape of translation in the DRG during neuropathic pain. This work is broadly significant for the following reasons. The targets of translational regulation in neuropathic pain are unclear. We apply a transformative genomics tool that reveals molecular underpinnings of translational regulation with astonishing molecular clarity. Parallel experiments will identify relevant factors using a novel CRISPR/Cas9 in vivo perturbation strategy. The master regulatory factors that govern translational regulation in neuropathic pain are unclear. Our preliminary data at the molecular, cellular, and organismal levels provide strong evidence that GCN2 is critical.

NIH Research Projects · FY 2024 · 2020-09

Abstract In the past 40 years, the prevalence of obesity has increased at an alarming rate. Even modest weight loss of at least 5% improves clinical parameters and quality of life. Efficacious behavioral weight loss programs teach participants behavioral strategies to create and maintain a caloric deficit. The two strongest predictors of long- term weight loss in such programs are initial weight loss and dietary self-monitoring. Over time, these phenomena decline, limiting program effectiveness. Financial incentives to increase initial weight loss and self- monitoring are appealing because they can be delivered to large populations with relative ease and at low cost. Employers and payers have begun to provide financial incentives for health behaviors and outcomes despite an inadequate evidence base to inform the optimal design of such interventions. The proposed study will evaluate which incentive approach has the greatest impact and durability—incentivizing interim weight loss, dietary self-monitoring, or both. Studies testing the effects of incentivizing these phenomena have showed some promise for increasing short-term weight loss. Few studies have evaluated incentive effects on long-term weight loss; examined the mediating role of intrinsic or extrinsic motivation; or calculated program costs or cost-effectiveness of financial incentive interventions. In the proposed two-site, randomized, single-blinded, longitudinal 2x2 factorial study, known as “Log2Lose,” we address these limitations by evaluating the individual and joint effects of incentivizing, in near real-time, weekly weight loss and dietary self-monitoring on 6-month weight loss and subsequent weight loss maintenance. People with obesity from the communities of Madison, WI and Durham, NC will receive an 18-month intervention comprising: 1. An incentivized weight loss program for 6 months (Phase I), 2. An incentivized weight maintenance program for 6 months (Phase II), and 3. A non- incentivized weight maintenance program for 6 months (Phase III). Participants will be randomized to receive adjunctive incentives for weekly weight loss, dietary self-monitoring, both, or neither. We will measure the proportion of participants achieving clinically significant weight loss of ≥5% at 6 (primary endpoint), 12, and 18 months. We will assess whether extrinsic and intrinsic motivation mediate intervention effect; calculate intervention costs; and calculate cost-effectiveness ratios across the four study arms. Our financial incentives intervention was designed to be scalable by using available technology and automating the process of analyzing data to provide incentives in near real-time. The intervention could be paired with various weight loss interventions offered by clinicians or payers, or integrated into a variety of patient and consumer-facing technologies. Completion of this study will contribute to our long-term goal of identifying and implementing efficacious, cost-effective, scalable approaches to reduce the prevalence of obesity and associated health outcomes.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Not applicable to the application.

NIH Research Projects · FY 2024 · 2020-09

Project Summary: Rates of FDA approval for oncology drugs in clinical trials are low and often clinical trial failures are driven by pre-screening of therapies in models that cannot adequately replicate patient physiology. The tumor microenvironment (TME) is highly complex consisting of multiple cell types including stromal cells, immune cells and vasculature. The interplay between tumor cells and neighboring cells in the TME results in environmental changes that can support tumor growth, vascularization and metastasis and, thus, plays an important role in prognosis and treatment efficacy (e.g. by modulating resistance). It is important for clinical prescreening models to include the TME to assess how treatment efficacy can be impacted by this multicellular crosstalk. For men with advanced castrate resistant prostate cancer (CRPC) that have progressed to metastasis, the disease is invariably lethal as current therapies are not curative. 90% of these patients have developed bone metastases but the bone microenvironment has been historically difficult to model in animal models or traditional co-culture. Therefore, in vitro models of the bone marrow TME are urgently needed to improve pre-screening of novel therapeutics, improve clinical trial design, outcomes and expedite much needed treatments to the clinic. Here we propose to create a tissue chip model of the bone marrow microenvironment for testing metastatic CRPC therapeutics. Patient-derived prostate tumor spheroids model the solid tumor embedded in a collagen hydrogel surrounded by multiple resident bone marrow stromal cells derived from bone marrow aspirates, immune cells and iPSC endothelial cell vasculature. Cell-surface targeted therapies, such as IMMU-132 have great potential for treatment of metastatic cancers. IMMU-132 is an antibody drug conjugate, with an antibody against Trop 2, a receptor expressed on tumor cells, coupled to the drug SN-38. SN-38 is a topoisomerase inhibitor that induced apoptosis in rapidly proliferating cells. We have access to samples and data from a Phase II trial of IMMU-132 in metastatic CRPC which will allow us to validate our bone marrow tissue chip model. In the UG3 phase, we will optimize our bone marrow tissue chip model and demonstrate that normal and disease chip environments replicate the in vivo physiology. We will also validate the chip for measuring responses to cell surface targeted therapies. In the UH3, we will use clinical trial data to build tissue chips that represent patients who respond and do not respond to IMMU-132 and validate these models. These chips will be used to determine mechanisms of TME-induced treatment resistance and identify signatures of response for use in stratifying patients for more efficient clinical trials. The chips can also be used to screen multiple different cell-surface targeted therapies helping direct therapy choice in future trials. The bone marrow tissue chips can be easily adapted for any cancer type that has bone metastases and can measure a range of cell surface targeted therapies. These chips have the potential to be a powerful tool for improving clinical trial success rates in therapies for metastatic cancer.