University Of California, San Diego

NIH Research Projects · FY 2025 · 2020-10

PROJECT SUMMARY/ABSTRACT Chronic tic disorders (referred to here as Tourette syndrome: TS) are complex and often serious neurodevelopmental disorders characterized by motor and/or vocal tics. Tics are brief, repetitive, unwanted movements or noises, which can severely impinge upon quality of life. While TS was once thought to be relatively rare, recent epidemiological studies find that 1-6% of all children meet criteria for a chronic tic disorder, making it a significant public health problem. Typically in TS, tics begin around age 5-7 years old, peak in severity around age 10-14 years, and improve throughout adolescence into adulthood. However, not all patients show this improvement during adolescence, as ~30% continue to experience significant impairment into adulthood. Thus, the years during and immediately following peak symptom severity represent a critical time for TS, during which individuals may show considerable improvement or not. Surprisingly little research has targeted this critical developmental stage of TS. Moreover, longitudinal investigations of predictors of TS outcome have focused primarily on single variables (e.g., caudate nucleus volume or tic severity). Yet there is considerable evidence that the neurobiology of TS is quite complex, involving interactions within and between multiple brain networks. For example, our preliminary findings demonstrate stronger brain functional connectivity among cognitive control networks and motor networks, as well as altered white and gray matter volumes in prefrontal and subcortical regions in TS. Using this complex information may be more informative for understanding tic severity changes and predicting clinical outcome. We propose a longitudinal study in which we will capture the developmental stage of TS with the greatest likelihood of change in tic severity (beginning at age 10-12 years), and will follow these children to track the development of brain and cognitive features, and how they relate to symptom change, over time. To capture the complex neurobiology of TS, we will collect whole-brain resting state functional connectivity, structural MRI, cognitive, and clinical data from a group of children with TS. We will compare these children to tic-free controls (from the NIH’s ABCD Study Washington University site subject pool), as comparison to typical development will be essential for interpreting longitudinal changes in TS. We will target diagnostic differences and developmental changes in specific functional brain networks, regional brain volumes, and cognitive abilities. We will also use multivariate machine learning methods to unify this rich dataset to classify and make predictions about individual children. This approach analyzes complex patterns of multidimensional data rather than single variables, providing the potential for clinical utility and to contribute converging evidence about mechanism. Identifying mechanisms underlying symptom change will provide insight into why many children with TS improve while some do not, potentially yielding new targets for treatment and predictive indicators of persistent tics. Markers of symptom improvement could be targeted to treat children who do not improve. Being able to make predictions about individual children could identify those children who need those interventions most. We have expertise with every step of the proposed study, but the application to longitudinal data over the first half of the second decade of life is novel.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY During suppressive antiretroviral therapy (ART), chronic inflammation persists among people with HIV (PWH) and appears to be driven by low levels of HIV replication, monocyte/macrophage activation, and other mechanisms. This persistent inflammation has been linked to adverse neuropsychiatric (e.g., depression, psychological stress, emotion dysfunction), neurocognitive, and medical outcomes. Cannabis, which is used more commonly by PWH than people without HIV, appears to have anti-inflammatory effects and therefore may have therapeutic applications for inflammation and end-organ complications. Cannabinoids exert their effects in part via the endocannabinoid (EC) system (ECS), which includes cannabinoid receptors that are expressed on the surface of cells of the immune and central nervous systems. Cannabinoids have demonstrated clinical benefits for conditions like pain and nausea, but less is known about its effects on inflammation in HIV. Moreover, the range of cannabis exposure (e.g., dose and frequency of use) that may be effective, ineffective, or unsafe for different neuropsychiatric and medical conditions is unknown. In response to RFA-DA-20-022, this application proposes to address key scientific gaps by examining the effects of chronic cannabis use and the role of the ECS in persistent inflammation in PWH who are taking suppressive ART. We propose to assess 120 participants (60 PWH on suppressive ART and 60 HIV- persons) across a spectrum of cannabis use from persons who have never used cannabis to daily users. We will comprehensively evaluate participants with multimodal in vivo and ex vivo assessments of the biological pathways underlying the effects of chronic cannabis use on persistent inflammation in PWH and its corresponding impact on neuropsychiatric and neurocognitive complications. In vivo assessments will include standardized medical, neuropsychiatric, and neurocognitive assessments, including collection of cerebrospinal fluid and blood. Current cannabis users within this sample (n=80) will also be assessed with novel brain imaging of microglial activation with [(18)F]FEPPA translocator protein (TSPO) positron emission tomography. In the collected specimens, we will measure soluble (e.g., pro- and anti- inflammatory cytokines) and cellular (e.g., HLA-DR, CD38) biomarkers of inflammation and immune activation as well as components of the ECS (e.g., anandamide, CB2 receptors). Blood specimens will also serve as the source for primary cell cultures for ex vivo mechanistic experiments that will assess the effects of cannabinoids, HIV, and ART on biological pathways such as the TREM2 and the NLRP3 inflammasome. Data from all assessments and experiments will be integrated and analyzed centrally to identify cross-cutting signals and reduce noise from our multimodal assessments. Knowledge gained from this study will contribute to OAR priorities (e.g., comorbidities, end organ injury) and provide valuable insight into strategies for treatment of persistent inflammation in PWH and its health-related consequences.

NIH Research Projects · FY 2024 · 2020-09

Project Summary. Human and nonhuman primates are highly visual animals that are predominantly active during the daylight hours. Yet our understanding of the neural mechanisms supporting spatial navigation is largely based on studies of nocturnal, burrowing rodents with poor vision. Indeed, studies of human and nonhuman primates have already demonstrated that spatial positions can be encoded in the hippocampus exclusively by visual inspection of a scene (i.e. spatial-view cells). At the same time, primate hippocampus also comprises populations of neurons that encode self-position in a scene during locomotion (i.e. place cells). Ultimately, primate representations of space must integrate these parallel threads of spatial information, but precisely how this occurs within the primate hippocampus is entirely unknown. Here we propose to address this fundamental question by leveraging several conceptual, technical and computational innovations to examine the neural basis of spatial representations in the hippocampus of marmoset monkeys. Aim 1 complements our previous work demonstrating canonical place cells in marmoset hippocampus during free-navigation to characterize whether neurons in this neural structure can also encode space through visual exploration of a scene. Aim 2 seeks to systematically characterize behavioral strategies in marmosets when searching for food in naturalistic 3-dimensional `forest' environments. Specifically, we will test how visual exploration and physical navigation of the landscape complement each other in marmoset spatial behavior. Experiments in Aim 3 build on these results to record the activity of hippocampal neurons of freely-moving marmosets in the same 3D naturalistic environments. By integrating head-mounted, wireless eye-tracking technology and video tracking of the animals position in space, we will explicate the role of different primate hippocampal subfields for exploration and navigation.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Despite a federal prohibition against marijuana, since 2012, recreational marijuana has been legalized in 11 states and Washington DC where over a quarter of US population live. Among these jurisdictions, ten states further opened or planned to open retail markets in near future to adults aged 21 years or older. The efforts of protecting public health in the new policy regime, however, have been complicated by the lack of knowledge regarding how individuals make purchase decisions and what regulatory measures would be effective to reduce problem marijuana use. Particularly, the rapid adoption of novel products and the coexistence of illegal markets pose unprecedented public health concerns and regulatory challenges. The overarching goal is to examine the relationships between recreational marijuana regulatory strategies and individual preferences for marijuana products. We will examine a wide range of policy measures that have potential to influence individual decisions, including those regulating product characteristics, restricting promotional features, modifying availability and context, and controlling price. We will innovatively test research hypotheses using two behavioral economics approaches with distinct yet complementary strengths, namely stated preferences approach and revealed preferences approach, and integrate them to provide calibrated estimates. Specifically, we aim: 1) To quantify the relationships of product, promotional, availability, and price attributes with individual hypothetical choices on marijuana products. 2) To quantify the relationships of product, promotional, availability, and price policies with individual real-world choices on marijuana products. 3) To correct hypothetical bias in stated preferences data with revealed preferences data. We will recruit representative adult samples of never users, former users, and current users of marijuana to complete a series of web-based surveys with rigorously developed discrete choice experiments on marijuana choices. To address concerns on hypothetical bias, we will complement these experiments with revealed preferences data on real-world choices through longitudinal cohort surveys in a subsample of the respondents. Particular attention will be given to the heterogeneities in policy impacts among never users, former users, and current users and among current users with medical, recreational, and dual purposes. The project will be the first rigorous and comprehensive investigation of the impacts of policy-relevant factors on marijuana use decisions at individual level. It will advance our understanding about the potential effectiveness of marijuana policies on choices between traditional and novel products in the emerging legal markets. It will also shed light on the unintended consequences of legal market regulations on demand in the coexisting illegal markets. Overall, the project has potential to advance the methodological framework for predicting marijuana demand and facilitate rational and informed design of marijuana regulatory strategies that can be directed to protect public health.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Maternal childbirth injury is the leading risk factor for pelvic floor muscle dysfunction and the resultant pelvic floor disorders, which include pelvic organ prolapse and urinary and fecal incontinence. Despite high prevalence, significant morbidities, and economic burden associated with pelvic floor disorders, preventative strategies are almost non-existent, and the available treatments are delayed and compensatory as they do not directly target the underlying pathophysiology. Thus, our long-term goal is the development of new, minimally invasive tissue- engineered therapies for the prevention and treatment of pelvic muscle dysfunction. Our pilot studies of pelvic floor muscle morphometric properties in parous women with pelvic organ prolapse and the rat model of simulated birth injury demonstrate substantial degeneration, specifically cell death, myofiber atrophy and fibrosis. The above alterations render muscles insensitive to rehabilitation and are associated with poor clinical outcomes. We developed a novel tissue-specific injectable extracellular matrix hydrogel, derived from decellularized porcine skeletal muscles, which promotes muscle regeneration. This proposal is centered around the overall hypothesis that the skeletal muscle matrix hydrogel, which contains tissue-specific cues, can be delivered alone at the time of birth injury to prevent pelvic floor muscle dysfunction or following a maladaptive post birth injury recovery to reverse pelvic floor muscle dysfunction. We will test this hypothesis in our translationally-relevant pregnant model by comparing untreated and treated pelvic floor muscle phenotypic, functional, and transcriptional signatures at multiple time points following birth injury and determining mechanisms by which the extracellular matrix hydrogel enhances regeneration. Collectively, this innovative study will provide comprehensive and functionally relevant assessments of the role of this low-cost acellular minimally invasive regenerative therapy in pelvic floor muscle recovery following birth injury and the fundamental knowledge of the biological processes involved in the regulation of pelvic floor muscle regeneration. The above has a high potential for the development of novel preventative and therapeutic strategies to counteract pelvic muscle dysfunction and the related pelvic floor disorders.

NIH Research Projects · FY 2024 · 2020-09

Development of patient-specific mathematical models for the transport of solute molecules in the cerebrospinal fluid (CSF) along the spinal canal PROJECT SUMMARY / ABSTRACT The cerebrospinal fluid (CSF) is predominantly secreted from the blood plasma and continuously bathes and circulates around the external surfaces of the brain and spinal cord. It maintains the electrolytic balance of the central nervous system (CNS), and serves as a medium for the supply of nutrients to neuronal and glial cells and the removal of waste products of cellular metabolism. It also transports hormones, neurotransmitters, and other neuropeptides throughout the CNS. The deregulation of the CSF circulation may compromise the transport of these solutes and the normal physiologic functions of the CNS contributing to the development of some cognitive and neurological diseases. CSF also provides a conduit for the delivery of potent analgesics and chemotherapy to the CNS, a drug delivery procedure often referred to as intrathecal or intraspinal drug delivery (ITDD). To date, there is no comprehensive methodology capable of predicting the patient-specific, long-term, motion of the CSF and the transport of solute molecules along the spinal canal. Thus, the main objective of this proposal is to develop a comprehensive modeling methodology capable of predicting the long-term transport of solute molecules along the spinal canal in each patient-specific anatomy and physiological conditions. The modeling approach combines the use of two time-scales asymptotic analysis of the Eulerian velocity field of the CSF in the spinal canal with in-vitro experimentation and detailed in-vivo validation with patient-specific radiological measurements. The proposed methodology is valid for a wide range of molecular diffusivities and accounts for convective effects of the CSF, including “shear-enhanced diffusion”, “steady-streaming”, and “Stokes drift”, to determine the long-time Lagrangian transport of the solute in the subarachnoid space (SAS) of the spinal canal. The expected outcomes of the proposed research are twofold: 1) it will provide a detailed understanding of the mechanisms regulating the transport of all important molecules key to the functioning of the CNS and 2) it will also provide the methodology necessary to optimize ITDD protocols.

NIH Research Projects · FY 2024 · 2020-09

Project Summary The United States is facing an unprecedented opioid epidemic caused by the misuse and abuse of both prescription pain relievers and illegal opioids. This issue has devastating consequences for public health, including a significant increase in overdoses related fatalities, in neonatal withdrawal syndrome and spread of infectious diseases such as HIV and hepatitis. Numerous studies indicate that opioid use disorder (OUD) has a strong genetic component. However, the genes networks implicated in opioid addiction remain poorly understood. The primary goal of this proposal is to study the transcriptional regulatory mechanisms that underlie the development of distinct stages of oxycodone abuse disorder. We will leverage the power of quantitative epigenomic methods that will provide a comprehensive map of regulatory elements, transcription factors and downstream target genes that are dysregulated in specific stages along the OUD trajectories. Our major innovation is the use of capped small (cs)RNA-seq, a method that we developed to quantify newly initiated transcripts with high sensitivity and high spatial resolution directly from total RNA. This approach enables the unbiased annotation of Transcriptional Start Sites (TSS) of both activated genes and transcribed regulatory elements at single nucleotide resolution. Compared with other epigenomic profiling, csRNA-seq is highly sensitive to changes in transcription, and it can capture the dynamic regulation of both stable genes and unstable transcripts, such as enhancer RNA. To study regulatory changes in distinct stages of OUD, we will use a rat model of oxycodone self-administration under extended access conditions. This model recapitulates several aspects of the human addiction-like behaviors, including tolerance, dependence, and motivation. Thus, it enhances the translational relevance of our results. This proposal will use two inbred strains that exhibit large differences in their motivation to seek oxycodone during abstinence while having similar pharmacokinetic for oxycodone and similar exposure to oxycodone. Using transcriptional initiation profiling, in combination with other sensitive profiling techniques, we will investigate the transcriptional regulatory networks underlying different stages of the OUD, including initial exposure, escalation of use, acute and sustained abstinence. Together, our proposed studies will have a broad impact in the field by defining regulatory networks that underlie phenotypes associated with vulnerability to distinct stages along the OUD trajectory in rats, and it may lead to novel therapeutic targets to treat OUD.

NIH Research Projects · FY 2024 · 2020-09

Abstract The World Health Organization (WHO) recommends that all people with HIV (PWH) initiate ART and countries in Sub-Saharan Africa (SSA) have started to roll-out a universal test and treat (UTT) strategy. Recent trials have shown that expanded treatment does not always result in reduced HIV incidence at the population level. Questions remain on the role of underlying factors that may reduce the benefits of UTT and drive the HIV epidemic. According to a recent UNAIDS report, alcohol use is an overlooked factor in the HIV epidemic that requires greater attention. Alcohol use increases HIV risk through sexual behaviors and reducing uptake and adherence to ART. Thus, alcohol use may contribute to population level failure to achieve HIV elimination targets. My proposed research will estimate the long-term, population impact of harmful alcohol use on HIV transmission and mortality in the UTT era in Uganda. Uganda has one of the highest levels of alcohol use per capita in SSA and has a generalized HIV epidemic, with a prevalence of 5.7%. The UTT strategy took effect in Uganda in 2017. However, in 2019, 22% of PWH in Uganda were not on ART and 36% were not virally suppressed. Uganda has an opportunity address both burdens by reducing harmful alcohol use. My research will assess the value for money of investing in alcohol use interventions to reduce HIV transmission in the era of UTT in Uganda. This project will improve understanding of the relationship between harmful alcohol use and the HIV epidemic through the following aims: 1) Estimate the prevalence of harmful alcohol use and relationships with receipt of ART and viral load suppression in the era of UTT in Uganda 2) Quantify the contribution of harmful alcohol use to HIV transmissions and mortality in Uganda during the UTT era, using infectious disease modeling. 3) Determine the cost-effectiveness of alcohol reduction interventions to prevent HIV transmission, in Uganda. The candidate, Dr. Adriane Wynn, is well qualified to conduct this research because of her strong background in quantitative methods and track record of publications and successful grant writing. Over the 5-year award period, Dr. Wynn will achieve the following career development objectives: 1) Develop expertise in the epidemiology and intersection of alcohol use and HIV and alcohol reduction interventions in the era of UTT in Sub-Saharan Africa. 2) Gain skills in modeling the population impact of alcohol use on HIV transmission and disease progression. 3) Acquire expertise in health economic evaluation of alcohol reduction interventions incorporating HIV transmission prevention benefits. 4) Obtain expertise in the ethical conduct of research pertaining to alcohol-using populations and those at risk for or living with HIV. 5) Expand professional development skills in preparation for a successful academic career by further developing skills in grantsmanship, publication, and scientific collaborations. These aims are in line with the Office of AIDS Research high priority topics: “Reducing Incidence of HIV/AIDS” and the NIAAA’s Strategic Plan.

NIH Research Projects · FY 2024 · 2020-09

The transcriptional regulatory sequences communicate with each other dynamically in the 3D nuclear space to direct cell type specific gene expression. Currently, a major barrier to understanding the transcriptional regulatory programs is the lack of tools, models and maps to explore the chromatin architecture in diverse cell types and physiological contexts. We will address this pressing need by deploying transformative technologies to study the chromatin architecture in mammalian cells at an unprecedented resolution and scale. Specifically, we will generate navigable, cell-type-specific reference maps of chromatin architecture in the mouse, macaque and human brains by integrating high resolution and high throughput imaging and orthogonal single-cell-based genomic methods. We will also dissect the role of chromatin architecture in gene regulation through a set of controlled perturbation experiments in the mouse ES cells (ESC) and ESC-derived neural progenitor cells (NPC). We will develop structural models of chromatin organization with advanced polymer physics and statistical learning methods, and validate their predictive power in embryonic stem cells and in ex vivo brain slices. Finally, we will make the reference maps, analytical tools, visualization methods and structural models available to the broader community. The proposed research project will dramatically transform our ability to analyze the 4D Nucleome of complex tissues, and produce the much-needed maps, tools and models for understanding the gene regulatory programs encoded in the linear genome sequences.

NIH Research Projects · FY 2024 · 2020-09

The future of biomedicine will rely on the ability to integrate genotype and phenotype data contextually to identify biomarkers, decipher mechanisms, reconstruct networks, and develop quantitative models by biomedical and clinical researchers in a seamless manner. What would be extremely valuable is a webaccessible one-stop shop providing these capabilities. Creating such seamless infrastructure is the vision of our initiative. We propose the development of the Biomedical Data Commons Workbench (BDCW), which will help overcome the fundamental barriers to biomedical data integration. BDCW will address two fundamental barriers faced by biomedical researchers – the first deals with the question, are there data available that can answer a biomedical question or questions (also referred to from the research point of view as a Use Case) and second, how can such data be integratively analyzed without having to go through the tedious process of either developing or using a variety of tools (which in turn requires working knowledge of computational methods). Several Common Fund data sources attempt to help the end-user vis-à-vis the data they provide, but not the ability to seamlessly interoperate with another Common Fund data source. Even if one were to identify the appropriate data sources to address a given research question, the challenge of integration of these diverse data is non-trivial. Data integration refers to the task of combining information about the same entities managed in different information systems to present a unified data view across different systems. It also refers to integrative analyses of multiple types of data, including data collected at different biological scales, to discover new knowledge as to how biological systems function (“mechanisms”).

NIH Research Projects · FY 2024 · 2020-09

Abstract Macrophages in Greek means “big eaters" are powerful cellular components of innate immunity. They play a pivotal role in immune defense by ‘eating’ pathogens, dead or cancerous cells. They also contribute to tissue homeostasis, development and repair. When doing their job, macrophages react to their surroundings and trigger acute inflammation to resolve the problems. They do so by assuming one of the two states that have been widely recognized, i.e., immunoreactive (proinflammatory) and immunotolerant (a.k.a, M1 and M2, respectively). While finite degrees of reactivity and tolerance are desirable in physiology, excess of either state is undesirable and invariably associated with disease pathogenesis (i.e., the Goldilocks conundrum). For example, hyperreactivity is recognized as the root cause of tissue injury in a wide array of diseases (colitis, sepsis, NASH) and hypertolerance is a common determinant that drives most, if not all chronic diseases that are incurable, e.g., cancers. Consensus on the definition of these physiologic and pathologic macrophage states has not been reached, perhaps because of 4 major challenges: heterogeneity, biological robustness, the temporal evolution of the network, and artifacts (tremendous plasticity of macrophages as they drift rapidly when isolated from tissues). We have used a novel computational methodology, Boolean Implication Network [Sahoo 2008], to analyze pooled human macrophage gene expression datasets. This method, which identifies asymmetric gene expression patterns, blurs noise (heterogeneity/artifacts) but reveals a temporal model of events that is invariably seen across all datasets. The analysis revealed hitherto unknown continuum transition states between reactive to tolerant states along five paths; machine-learning identified one of them as the major path which subsequently stood the rigorous test/validation on multiple publicly available transcriptomic datasets, across species (mouse and human), macrophage subtypes and disease states. Most importantly, unlike other commonly used gene cluster signatures, the Boolean path can prognosticate outcomes across diverse diseases. Preliminary validation studies on a genetic model confirm that the path could be exploited for modulating macrophage polarization by altering LPS/TLR4 responses. We will now interrogate the impact of these discoveries using an iterative approach, i.e., model-driven experimentation and experiment-driven model refinement, through three aims: Unravel the importance of novel molecular drivers in the newly identified gene signatures of macrophage polarization using semi-HTP chemical/genetic screens on murine and human monocyte-derived macrophages (Aim 1), in murine disease models of hyperreactivity and hypertolerance (Aim 2) and in “Humanoids”, i.e., human organoid-based microbe/immune cells co-culture models (“gut-in-a-dish”; Aim 3). Although our focus is gastrointestinal infection and inflammation, the findings will define macrophage transition states in multiple organs/disease contexts and therefore, impact many fields. We expect to identify high-value therapeutic targets that can restrict and/or reset macrophage responses to infections and inflammation within the “Goldilocks zone.”

NIH Research Projects · FY 2024 · 2020-09

Abstract The study of cryopyrin associated periodic syndromes (CAPS) has shaped our view of innate immunity, and led to the clinical translation of therapies for CAPS and other NLRP3-dependent inflammatory diseases. Our preliminary in vivo genetic data now suggest that the assumed central role of monocytes and macrophages in CAPS may be overstated. Rather, the neutrophil lineage alone can drive lethal autoinflammation in CAPS neonatal mice, and this is indistinguishable from systemic NLRP3 activation. The molecular regulation of NLRP3 inflammasome activation is distinct in neutrophils compared to macrophages, so this research will study biochemical regulation of NLRP3 activation in mouse and human cells of the neutrophil and monocyte lineages. We will investigate cells from patients with CAPS, and from mouse models with activating NLRP3 mutations expressed specifically in neutrophils (Nlrp3PMN mice). Neutrophil precursors are elevated in successfully-treated CAPS patients and in Nlrp3PMN mice, suggesting developmental abnormalities of neutrophils in Nlrp3PMN mice, or a reduced threshold for pyroptosis induction in the neutrophil lineage - hypotheses that will be formally tested in this study. Single cell Western and RNA-Seq data from purified neutrophil precursor populations have revealed constitutive IL-1b expression in neutrophil progenitors and immature neutrophils in healthy mice, suggesting that these precursors, but not mature neutrophils, are the dominant source of IL-1b in organs of CAPS patients. We now hypothesize that this developmentally-regulated expression of pro-IL-1b enables neutrophil precursors with NLRP3 activating mutations to release processed IL-1b independent of signal 1 or signal 2. We also hypothesize that the absence of mature neutrophils from all organs and tissues of neutrophil- specific CAPS mice is a consequence of pyroptosis. This project seeks to define: (a) the differences in organ pathology and morbidity of mice with monocyte- or neutrophil-specific NLRP3 activation; (b) the cell-intrinsic effects of NLRP3 activation on neutrophil differentiation and lifespan; and (c) the sensitivity of cells of the neutrophil lineage in CAPS patients and neonatal CAPS mice to canonical and non-canonical activators of the inflammasome and pyroptosis; Our specific aims are therefore to: (1) compare organ involvement and morbidity of mice with monocyte-specific and neutrophil-specific activation of NLRP3; (2) understand why mature neutrophils are absent in neonates of neutrophil-specific CAPS mice; and (3) investigate inflammasome activation and pyroptosis induction in neutrophil lineage cells from patients diagnosed with CAPS. Identification of the key cell types causing disease in CAPS will help us to understand the development of other NLRP3-driven inflammatory diseases, and may also improve disease management and highlight novel biomarkers for autoinflammation.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Overall Despite the development of increasingly effective therapies to reduce elevated levels of atherogenic lipoproteins, cardiovascular disease (CVD) complications are projected to rise worldwide due in part to the increasing incidence of obesity and insulin resistance. An emergent question is the extent to which non-alcoholic fatty liver disease (NAFLD), which is a spectrum ranging from fatty liver to non-alcoholic steatohepatitis (NASH) to cirrhosis, contributes to CVD risk. Among patients with NAFLD, the leading cause of death is CVD, estimated to account for 31% of total mortality. The development of NAFLD and cardiovascular disease is influenced by combinations of genetic and environmental factors, some of which are disease-specific and others that affect both disease processes. The overall hypotheses of our PPG are that liver fat and fibrosis predict CVD risk and that interventions targeting Liver X receptors (LXRs) in macrophages, the farnesyl X receptor (FXR) in the gut, and oxidation specific epitopes (OSEs) in the liver and artery wall will reveal common mechanisms that contribute to the clinical association between NASH and CVD. Importantly, each of these interventions make use of representative small molecules or antibodies that have the potential to be advanced for clinical trials. Identifying mechanisms by which known and unknown risk factors promote both NASH and CVD would be of great significance, especially if targeting one or more of these mechanisms would produce beneficial effects on both diseases. To achieve this goal, we propose a PPG consisting of four highly inter-related projects and three cores. Project 1, led by Dr. Christopher Glass, will test the hypothesis that selective activation of LXRs in macrophages and Kupffer cells with desmosterol mimetics will result in reductions of atherosclerosis and NASH without causing steatosis or hypertriglyceridemia. Project 2, led by Dr. Ronald Evans, will investigate the hypothesis that selective activation of FXR in the gut or liver will result in reductions in atherosclerosis and NASH. Project 3, led by Dr. Joseph Witztum, will test the hypothesis that antibody-mediated reductions in OSEs will coordinately reduce both atherosclerosis and NASH. Project 4, led by Dr. Rohit Loomba, will investigate the relationships of liver fat content and fibrosis with cardiovascular risk in human subjects and enable translational extension of mechanistic findings made in Projects 1, 2 and 3. A Phenotyping Core will enable Projects 1, 2 and 3 to quantitatively evaluate extent of atherosclerosis and NASH in mouse models, and enable all projects to obtain targeted lipidomic profiles and cytokine levels from relevant samples. A Genomics and Bioinformatics Core will support the application of massively parallel sequencing-based assays, such as RNA Seq, by Projects 1, 2 and 3 and provide a shared resource for bioinformatics and statistical analysis. An Administrative Core will support the overall administrative and scientific needs of the PPG.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Alcohol-associated liver disease (ALD) is a major cause of cirrhosis and liver failure, and the 12th leading cause of death in adult patients in the United States. ALD progresses from fatty liver, to alcoholic steatohepatitis, fibrosis/cirrhosis and hepatocellular carcinoma (HCC). Pharmacological therapies for ALD are urgently needed as FDA-approved medications are currently available. In published work, we have demonstrated that the proinflammatory cytokine interleukin-17A (IL-17A) is a critical mediator of alcohol-related liver damage in both humans and mice. IL-17A is mainly produced by CD4+ Th17 cells. IL-17A production is regulated by IL-23, a cytokine that promotes the maintenance, survival, and proliferation of Th17 cells. Our preclinical studies clearly demonstrate that blocking IL-17 signaling with an anti-IL-23 antibody-based treatment significantly improves alcohol-related liver fibrosis, cirrhosis, and cancer. Here, we evaluate whether the IL-23 blocking antibody guselkumab effectively reduces serum levels of IL-23 and IL-17A as well as the number of circulating Th17 cells48,49 in samples from treated patients from our Phase I clinical trial. This Phase 1 trial will enroll adult participants who have a history of moderate to severe alcohol use disorder (AUD) along with documented clinical evidence of chronic liver disease due to alcohol but no evidence of cirrhosis or severe hepatic dysfunction or alcoholic hepatitis. It will follow a standard 3+3 Phase I dose escalation trial design with a maximum of 24 subjects. We will assess the drug’s safety, pharmacokinetics, and pharmacodynamics in a population that meets criteria for AUD and early signs of end-organ-damage to the liver, as made evident and quantified by advanced non-invasive MRI based biomarkers of liver fat and fibrosis. We will assess biomarkers for both guselkumab target engagement as well as biomarkers for early treatment response (ALT, ELF, Pro-C3). Aim 1: Assess safety and tolerability of guselkumab (anti-IL-23 monoclonal antibody) in a Phase 1 dose escalation study in patients with alcohol use disorder and alcohol-associated liver disease. Aim 2: Assess the pharmacokinetics of guselkumab in patients with alcohol-associated liver disease. Aim 3: Assess pharmacodynamics of guselkumab target engagement and biomarkers of early treatment response.

NIH Research Projects · FY 2024 · 2020-09

Project Summary / Abstract Age-related hearing impairment (ARHI) is the most common cause of hearing loss, is heritable, and is one of the most prevalent conditions affecting the elderly globally. Twin and family studies reveal 25-75% heritability for ARHI (Momi et al., 2015). Estimates suggest approximately two‐thirds of people over the age of 70 in the United States experience ARHI, and that by 2020, over half of all people in the United States with hearing loss will be over 70 years of age (Bainbridge and Wallhagen, 2014). ARHI has been shown to be independently associated with cognitive decline, dementia, depression, and loneliness and results in an estimated annual economic burden of over $3 billion (Deal et al., 2017; Deal et al., 2018; Lin and Albert, 2014). Our overarching hypothesis, supported by preliminary data in both mice and humans, is that ARHI is a complex trait with many likely genes associated (Fransen et al., 2015; Friedman et al., 2009; Kalra et al., 2019; Wells et al., 2019). Greater than 100 genes have been identified for monogenic deafness; however, a substantial fraction of patients with ARHI have no identifiable mutation in any known deafness gene suggesting that there remain additional genes to discover (Bowl and Dawson, 2018). Mice continue to be the predominant organism for hearing research. Similarities in the auditory structure and physiology between mice and humans, the close evolutionary relationship of genomes (most genes in mice have a human homologue), relatively low housing costs, genetic standardization and the available genetic toolkit make the mouse a crucial model system for the study of the functional genomics of the auditory system (Bowl and Dawson, 2015). We are proposing to identify candidate genes by performing the first complete genome-wide association study (GWAS) of ARHI in CFW mice and examining gene expression in the inner ear. Although several labs including ours have used human subjects for GWAS, to date there exist no comprehensively characterized cohorts with sufficient power and therefore there exist limited replication studies of candidate genes. In Aim 1, we will measure auditory brainstem response and distortion product otoacoustic emission thresholds in 2,000 one-year-old CFW mice equally divided among males and females. We will genotype each mouse at more than 1,000,000 single nucleotide polymorphism markers and in Aim 2 perform GWAS to identify quantitative trait loci (QTL). In Aim 3, we will use RNAseq to assess differences in gene expression in the inner ears of 100 randomly selected mice from the CFW cohort and define expressed QTLs (eQTLs).The genetic variation within CFW mice presents a unique opportunity to elucidate the molecular mechanisms that underlie ARHI providing novel targets for drug development and providing a means for identifying patients at risk.

NIH Research Projects · FY 2025 · 2020-09

Global fertility has declined, with sperm count and quality dropping by 50-60% since the 1970s due to lifestyle, environmental, and metabolic factors. Women face reduced ovarian reserves from delayed maternal age and rising rates of reproductive disorders like polycystic ovarian syndrome and endometriosis. Pesticide exposure is linked to fertility issues in both sexes, with insecticides affecting sperm count, motility, and hormone levels in men, and ovarian reserve and menstrual cycles in women. Most existing studies have been cross-sectional, focused on specialized populations (e.g., fertility patients), had small sample sizes, included participants with limited pesticide exposure potential (urban settings), or lacked biomarker measurements for exposure assessment. Longitudinal population-based studies using biomarkers of exposure are needed. In the Secondary Exposures to Pesticides among Children, Adolescents, and Adults (ESPINA) study, an 18th-year follow-up is proposed for 2026, when participants will be 21-27 years old (N≈500), corresponding to their peak reproductive years. This study, based in Pedro Moncayo County, Ecuador, a region known for one of the largest concentrations of flower crops in the Americas, exporting roses to the USA and globally. Our objective is to assess the long-term impact of pesticide exposure on fertility and endocrine health from early adolescence to young adulthood. We propose to measure reproductive biomarkers in stored biospecimens collected during follow-up years (FUY) 8 (adolescence), 14 (adolescence- young adulthood), and 18 (young-adulthood) and leverage existing insecticide (neonicotinoid, pyrethroid organophosphate) and herbicide (2,4-D, glyphosate) urinary biomarker data at 7 exam periods (FUY 8, 14, 15a, 15b, 16a, 16b and 18 [new]). We propose the following specific aims: Aim 1: Pesticide Exposure and Female Fertility. We hypothesize that insecticide and herbicide biomarkers are associated with: 1a) decreased ovarian reserve. Primary: lower anti-Müllerian hormone concentration (AMH); Secondary: higher early follicular phase follicle-stimulating hormone (FSH) and estrone-3-glucuronide. 1b) endocrine alterations: higher testosterone (T), and thyroid stimulating hormone (TSH); lower free T4 (fT4). 1c) delayed ovulation, confirmed by daily measurements of reproductive hormones during 1 menstrual cycle; secondary: greater oligomenorrhea (cycles>35 days) and dysmenorrhea (pain). Aim 2: Pesticide Exposure and Male Fertility. We hypothesize that these pesticides are associated with: 2a) lower motile sperm concentration and progressive motility; 2b) higher LH, FSH, 17β-estradiol (E2), T and TSH, and lower fT4 assessed in FUY 8, 14, 18; 2c) higher prevalence of erectile dysfunction symptoms. This research builds on long-standing community-engaged efforts in a major agricultural region and is one of the largest studies on pesticide exposure and fertility. The findings will offer critical insights into how chronic pesticide exposure impacts human fertility, with implications for global public health and pesticide regulation.

NIH Research Projects · FY 2025 · 2020-09

Genome-wide association studies (GWAS) have identified thousands of genetic loci linked to complex traits, but determining the causal variants, target genes, and biological mechanisms responsible for each signal remains challenging. While traditionally GWAS has focused on single nucleotide polymorphisms (SNPs), the advent of biobank-scale next-generation sequencing datasets as well as advanced functional genomics techniques now enable systematic interrogation of the role of more complex variants in polygenic traits. We focus on the role of genetic variation at repetitive regions of the genome. Specifically, we consider two repeat types: short tandem repeats (STRs), consisting of repeated motifs of 1-6bp, and variable number tandem repeats (VNTRs), with motifs of 7+bp, which we collectively refer to as tandem repeats (TRs). TRs exhibit rapid mutation rates that render them one of the largest sources of genetic variation in humans. Increasing evidence suggests that TRs act as an important source of causal variants for complex traits and may drive some of the strongest GWAS signals identified to date. Yet, due to bioinformatic and experimental challenges in studying repeats, the genome-wide role of TRs on complex human traits is only beginning to be uncovered. We hypothesize that TR variants are key drivers of complex traits. We recently published the first genome-wide integration of TRs into the GWAS framework. This identified 93 STRs predicted to causally impact blood and serum biomarker traits and estimated STRs explain 5-10% of GWAS signals for these traits. We have experimentally interrogated the effects of thousands of promoter TRs by optimizing a massively parallel reporter assays (MPRA) to enable studying low-complexity sequences. Using our MPRA we were able to show widespread and cell-type specific TR effects on expression. While these findings offer intriguing evidence that thousands of TRs contribute to human phenotypes, they have been limited by the range of TRs that could be accurately imputed into available GWAS datasets and the biological mechanisms by which TRs affect complex traits remains unknown in most cases. Here, we leverage (i) newly available whole genome sequencing (WGS) for hundreds of thousands of individuals from UK Biobank (UKB) and All of Us (AoU) which will enable direct TR genotyping rather than imputation, (ii) a suite of computational tools we have developed for population-scale TR analysis and association testing, and (iii) our recently developed MPRA and genome editing frameworks for experimental interrogation of TR effects to systematically evaluate the contribution of TRs to complex traits in humans. Using these, we will develop scalable methods to perform TR-based GWAS in large biobanks to generate a comprehensive catalog of TRs associated with complex traits (Aim 1), use MPRA to investigate the effects of tens of thousands of TRs on gene regulation (Aim 2) and perform deep characterization of candidate medically relevant TRs in induced pluripotent stem cells and their differentiated derivatives in cell lines originating from ancestrally diverse donors (Aim 3).

NIH Research Projects · FY 2024 · 2020-09

I. Abstract The proposed UC San Diego MADURA (Mentorship for Advancing Diversity in Undergraduate Research on Aging) Program responds to the NIA ADAR R25 Training Program Announcement, to offer under-represented Hispanic/Latino undergraduates tailored, longitudinal mentorship, training and work experiences with researchers and clinicians focused on aging and Alzheimer's disease. It will provide effective, sustained academic and social support, skills training and supervision, to foster academic success and retention over the near term, and subsequent increased rates of application for aging-related graduate training or employment, thereby improving inclusion in the field. The MADURA project is designed to achieve these goals through a multi-component program comprised of: career-relevant Individual, Paid Aging and Alzheimer's Disease Research-related Internship Placements with researcher/clinician mentors (8 hours/week); Paid Weekly Group Mentorship/Training Meetings, facilitated by a team of doctoral level trainers and research faculty (2 hours per week); integrated, tailored Professional Development Experiences (some with additional funding support); Guided Outreach Experiences for a partner high school that serves potential first generation college attendees; and formal curriculum and process development activities and rigorous evaluation, enabling continuous quality improvements and future dissemination. UC San Diego is an emerging Hispanic Serving Institution with a deep field of diversity-promoting academic support and Hispanic/Latino student groups, centers and services which welcome collaboration with the MADURA Program. The MADURA Program is innovative in depth, comprehensiveness and integration of its evidence-based supportive elements: student pay, broad array of experiential placements, full integration of weekly Group Mentorship and tailored Training (provided by skilled aging research facilitators from similar cultural backgrounds), co-occurring peer mentorship and support, and finally, its fidelity to rigorous evaluation and dissemination of results and materials. MADURA is positioned for success, given the convergence of experienced program leadership, strong program development and evaluation teams, pay for students who must earn income in order to stay in school, exemplary willing advisors and complimentary training activity partners, and existing linkages with Hispanic/Latino student networks. The carefully conceptualized MADURA Program brings together the leadership, advisors, training and placement experiences to successfully promote diversity in Aging/Alzheimer's disease MSTEM careers for participating Hispanic/Latino undergraduates, within the nurturing context of a University energized around improving diversity and inclusion. 1

NIH Research Projects · FY 2024 · 2020-09

In this proposed project, we plan to fill the knowledge gap of the relationships between microscopic self-assembled structures, collagen-molecule interactions and macroscopic fiber morphologies of type-I collagen, the primary component of most human tissues and a commonly used biomaterial for tissue engineering. By investigating collagen-water and collagen-protein interactions in in vitro systems that mimic basic aspects of physiologically relevant three- dimensional fibrillar tissue architectures, we aim to fill knowledge gaps in fundamental collagen research. We will achieve this goal by developing a hyperspectral imaging technique – vibrational sum frequency generation (VSFG) microscopy – at high repetition rates (400 kHz) and apply it to collagen. The long-term vision is to develop new biophysics methods to reveal molecular-level structures and interactions for pericellular space research and other complex biological environments, and eventually applying it to study various pericellular environment related diseases. In order to correlate spectral features to microscopic and macroscopic structures of type I collagen, we plan to apply machine-learning techniques to analyze our data and extract spectral signatures of collagen’s micro/macrostructures. We will two major scientific focuses: (A) understanding molecular signatures of microscopic self-assembly fibrils structures and its relationship to the macroscopic morphology (plan 1 and 2); and (B) investigating molecular level collagen-molecule interactions (plan 3 and 4). Specific plans include: 1. Obtaining hyperspectral VSFG images of collagen tissues to study their morphology in a label free and non-invasive manner 2. Establishing molecular spectral signatures of self-assembled collagen fibril structures 3. Understanding collagen-water interaction in first solvation layer of collagen fibers. 4. Imaging spatial locations of chemicals and peptides that interact with collagens. If successful, the significance is that a label free, vibrational mode specific imaging technique specific for pericellular space will be available, which can reveal molecular level insights of collagen structures and its interactions with surrounding molecules, pertinent to fibrosis and cell— pericellular space interaction related diseases. This proposed project contributes to the scope of NIGMS by developing new technology to reveal fundamental molecular-level principle, mechanism and signatures related to morphology of collagen I at both micro- and macroscopic scales, and collagen-molecule interactions, laying foundations for biophysical/biochemical principles for future biomedical applications related to collagens.

NIH Research Projects · FY 2024 · 2020-09

Abstract There is little doubt that we are in the midst of a worldwide epidemic of obesity and diabetes. Recent data suggest an inflammatory link between obesity and insulin resistance. This proposal will focus on adaptive changes in energy expenditure during caloric overload and restriction, and explore a novel hypothesis that may directly lead to new therapeutic approaches. We hypothesize that that activation and induction of the protein kinase TBK1 in obesity and fasting reprograms metabolism to promote anabolic and repress catabolic pathways, tipping the scales towards energy storage and away from expenditure. These conclusions are derived from changes observed in mouse knockout mice, and in patients treated with the TBK1 inhibitor amlexanox. Specifically, activation or induction of TBK1 represses AMPK activity, inhibiting lipid oxidation and mitochondrial biogenesis while increasing lipogenesis. We will explore this hypothesis with three aims: 1) We will evaluate the temporal and spatial aspects of TBK1 activation and induction during obesity, and characterize the underlying mechanism; 2) We will explore the activation and induction of TBK1 during fasting, and elucidate the molecular mechanisms; 3) We will assess the role of the TBK1/AMPK axis in controlling metabolism during obesity and fasting via development of compound knockout mouse models, and evaluate the temporal and spatial aspects of this regulation. These findings may reveal how energy expenditure is repressed during caloric excess and restriction, and provide new therapeutic approaches to these devastating diseases.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Glaucoma is the world's leading cause of irreversible blindness and will affect >110 million people by 2040. Early detection and treatment are critical, as symptoms typically do not present until the disease is advanced. A data-driven precision medicine approach is needed to better identify individuals who are at greatest risk of developing the disease and who are at greatest risk of progressing quickly to vision loss. While there has been considerable progress in eye imaging and testing to improve glaucoma monitoring, precision management of glaucoma is incomplete without accounting for patients' co-existing systemic conditions, concurrent systemic medications and treatments, and adherence with prescribed glaucoma treatment. Understanding how systemic conditions, and specifically vascular conditions such as hypertension, impact glaucoma presents growing public health importance given the increasing co-morbidities facing aging populations. Preliminary studies have demonstrated the predictive value of systemic data, even without ophthalmic endpoints. Similarly, measuring medication adherence is important for guiding patient counseling and engagement and avoiding downstream interventions such as surgeries, which carry high cost and morbidity. These factors are important for providing a more comprehensive perspective of glaucoma management and for improving patient outcomes, yet they are relatively understudied. I propose applying multi-modal advancements in health information technology (IT) to address these gaps and achieve the following specific aims: (1) Develop machine learning-based predictive models classifying patients at risk for glaucoma progression using systemic electronic health record (EHR) data from a diverse nationwide patient cohort; (2) evaluate how integrating blood pressure (BP) data from novel smartwatch-based home BP monitors enhance predictive models for risk stratification in glaucoma, and (3) measure glaucoma medication adherence using innovative flexible electronic sensors to validate their use for future interventions aimed at improving adherence and clinical outcomes in glaucoma. These studies would leverage state- of-the-art methods in big-data predictive modeling as well as cutting-edge advancements in sensor technologies. This multi-faceted approach will build a foundation for a health IT framework geared toward improving risk stratification and generating novel therapeutic targets for glaucoma patients.

NIH Research Projects · FY 2024 · 2020-09

This proposal outlines plans for the creation of K-CORE, a new core for SPARC that will provide curation of SPARC datasets, knowledge management and services for the SPARC Knowledge Graph. The latter is a semantic store that combines information on SPARC datasets annotated according to the SPARC Minimal Information Standard (MIS) with functional and anatomical topologically based connectivity knowledge for the autonomic nervous system (ANS). Included in the ANS are the sympathetic, parasympathetic and enteric nervous systems, with reference to sensory and motor pathways as necessary. Technology development in SPARC is provided by the SPARC Data and Resource Center (DRC), currently comprising DAT-CORE, which provisions the data platform, MAP-CORE, which develops 2 and 3D mapping tools and a map-based GUI, and SIM-CORE which fields the simulation platform. To date, the curation and knowledge management functions have been conducted under the purview of MAP-CORE, led by Dr. Peter Hunter at the Auckland Bioinformatics Institute with a subcontract to UCSD. However, these services are sufficiently broad and cross cutting that they should be managed through a separate core. The K-CORE team has been providing curation and knowledge management services for SPARC since 2018 and thus are deeply familiar with the project.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Two out of every three adults has overweight or obesity, which is associated with significant medical and psychological consequences. To date, the most successful weight loss treatment is behavioral weight loss (BWL), which includes nutrition and physical activity education, as well as behavior therapy techniques. Although some adults lose weight in these programs, one third to one half do not respond with even bleaker rates of weight loss maintenance over time. These low success rates suggest that there are underlying mechanisms, such as appetitive traits, that may interfere with response to BWL and underscore the critical need to develop targeted models for the treatment of obesity. Our data suggest that high food responsiveness (FR) is a risk factor for failure in BWL. We have developed a new model for the treatment of obesity based on the Behavioral Susceptibility Theory, called Regulation of Cues (ROC), that focuses on decreasing FR and improving satiety responsiveness (SR). In this study, we propose to recruit adults with overweight and obesity who also exhibit high levels of FR to test the efficacy of an enhanced ROC treatment program (ROC+) for this specific behavioral phenotype. We propose a 3 arm randomized controlled trial that will compare ROC+, BWL and an active comparator (AC). We will recruit and randomize 300 adults with overweight and obesity and will assess them at baseline, during treatment, post-treatment, and at 6- and 12-month follow-up. Primary and secondary aims are as follows. Primary Aim 1: Compare ROC+ to AC on body mass index (BMI) over the course of treatment and follow-up. Primary Aim 2: Compare ROC+ to BWL on body mass index (BMI) over the course of treatment and follow-up. Secondary Aim 1: Compare BWL to AC on body mass index (BMI) over the course of treatment and follow-up. Secondary Aim 2: Compare ROC+, BWL and AC on sensitivity to satiety, sensitivity to food cues, inhibition, restriction, caloric intake, and overeating over the course of the treatment and follow-up. Exploratory aim 1: Evaluate effects of mediators (FR, SR, restriction, overeating) and moderators (demographics, baseline BMI) of treatment effects on weight loss over time. This program of research is an important next step in the development of treatments for specific phenotypes of adults with overweight/obesity, and could change the paradigm of obesity treatment for these individuals. This study will contribute to the study of appetitive phenotypes of obesity, will provide a targeted treatment for individuals with high FR, and could inform clinical decision making for adults with obesity.

NIH Research Projects · FY 2024 · 2020-09

Title: Preventing cognitive impairment in mouse genetic models of Down syndrome by early postnatal suppression of Kir3.2 channel signaling Abstract Neural mechanisms responsible for cognitive impairment in DS are still unclear, and there is no treatment to prevent this most common genetic disability. Our long-term goal is to develop treatments ameliorating cognitive impairment in DS. Recently we discovered that synaptic plasticity and cognition can be restored in DS models by genetic removal of an extra copy of Kcnj6, the gene encoding Kir3.2 (Girk2) subunits of potassium channels. These channels, known for their critical role in neuronal hyperpolarization, are highly expressed during early postnatal development suggesting their involvement in the formation of neural circuits. Our central hypothesis is that, in DS models, increased signaling through Kir3.2 channels reduces the spontaneous activity of neonatal neurons, thus affecting synaptogenesis and the formation of neural circuits, which ultimately leads to cognitive impairment. Accordingly, suppression of Kir3.2 channel signaling during the early postnatal period can normalize the neural circuits, thereby reducing neuronal, synaptic, and cognitive abnormalities in DS. Two Specific Aims will used to test this hypothesis. Aim1. To examine the effects of the Kcnj6 gene dose on the development and maturation of neural circuits in mouse genetic models of DS. Formation and maturation of GABAergic and glutamatergic synaptic connections will be examined in the hippocampus of DS mice containing either 2 or 3 copies of Kcnj6 (Ts65Dn/Kcnj6++- vs. Ts65Dn/Kcnj6+++ mice). Electrophysiological, biochemical, and histochemical techniques will be used to assess the effects of Kcnj6 gene dose on: (i) Neuronal properties affecting the activity of neonatal neurons; (ii) The efficiency of synaptic depolarizing GABA action before the ‘excitatory-to-inhibitory GABA switch’ (P5 - P12); (iii) The efficiency of GABAergic inhibition after the ‘excitatory-to-inhibitory GABA switch’ (P14 – P22); (iv) Development of synchronized neonatal neuronal activity indicative of the formation of neural circuits; (v) Developmental changes in synaptic plasticity. These studies will show the role of the Kcnj6 gene in the formation of neural circuits and give a basis for monitoring post-treatment changes in the Aim 2. Aim 2. To examine the effects of early postnatal treatments reducing signaling through the Kir3.2 subunit-containing potassium channels on the development and maturation of neural circuits in DS models. Kir3.2 channel signaling will be reduced in a time- and region- specific manner via: (i) Neonatal intraventricular AAV delivery of Kcnj6 siRNA; (ii) Intraperitoneal injections of selective GABAB receptor antagonists. Consequences of these treatments will be examined in neonatal and adult mice. Following properties will be assessed: changes in Kir3.2 channel signaling, properties of neonatal neural circuits, post-treatment changes in GABAergic synaptic transmission and synaptic plasticity. This data will provide information on the role of Kcnj6 gene in synaptic, structural, and functional abnormalities in DS, and provide a ‘proof of principle’ that time-limited postnatal suppression of Kir3.2 channel signaling can be used to improve brain functions and cognition in DS.

NIH Research Projects · FY 2025 · 2020-09

Komor – Project Summary/Abstract Advances in sequencing technologies have made the detection of genetic variants in patients increasingly routine, and identification of clinically actionable genes and pathogenic mutations has revolutionized the field of precision medicine. However, less than 0.5% of the 759 million genetic variants (96% of which are single nucleotide variants, or SNVs) currently in the Genome Aggregation Database have a defined clinical interpretation, highlighting the need for new strategies to functionally characterize their impact in higher throughput. New methods capable of interpreting the full range of human genetic diversity in high throughput would have the potential to advance the field of precision medicine in a more equitable manner. A major goal of our research program is to combat this “variant interpretation problem” through the development of new precision genome editing tools and strategies and their application in generating cellular models of human genetic variants. We focus here on base editors (BEs), which utilize single-stranded DNA (ssDNA)-specific editing enzymes tethered to a catalytically impaired Cas9 (Cas9n) protein to install SNVs with high efficiency and precision. Current BEs can facilitate the introduction of C∙G to T∙A base pairs (called cytosine BEs, or CBEs) or A∙T to G∙C base pairs (called adenine BEs, or ABEs) using cytidine or adenosine deaminase enzymes, respectively. There are a variety of opportunities upon which to improve, optimize, and expand the technology, which we seek to address with this MIRA proposal. Specifically, we aim to expand the genome editing toolbox through the study and characterization of nucleic acid editing enzymes (Direction 1), development of novel directed evolution platforms (Direction 2), and the improvement and engineering of current editing tools (Direction 3). Direction 1 research aims to use new machine learning methods in combination with next-generation sequencing-based profiling assays to characterize the RNA editing profiles of a diverse set of nucleic acid editing enzymes. These data will be of high interest to the nucleic acid editing community and provide us with ideal starting points for our directed evolution efforts. Direction 2 research seeks to develop mammalian cell-based directed evolution platforms for genome editors, as we have found that inherent differences between bacteria (the host for most directed evolution platforms) and mammalian cells have caused setbacks in our development of new genome editing tools. This modular system will then be applied to evolve new BE tools and improve upon existing ones. Finally, Direction 3 research aims to improve upon existing BEs by eliminating bystander editing (when multiple Cs or As are edited concurrently) and the propensity of CBEs to install C∙G to non-T∙A edits. While we currently have the tools to begin work in all three areas, our research Directions are designed such that progress in any one Direction can be integrated into the other Directions to exponentially advance the research. The successful completion of the proposed work will produce new genome editors that will be of immediate use to basic science researchers, and can act as starting points for the development of therapeutics.