Vanderbilt University

NIH Research Projects · FY 2025 · 2022-07

Project Summary: The Vanderbilt Training Program in Genetic Variation and Human Phenotypes supports predoctoral students in human genetics. We request funding for 8 predoctoral students, funded for two years. The mission of this program is to prepare these students for a successful career in human genetics in an academic, industrial, or public service capacity. We prepare our students beginning with skills-focused classroom instruction and transitioning to experienced- based learning through research and active collaboration with more experienced mentors. Students are regularly exposed to leaders in human genetics research through their collaborations, research seminars, and attendance at national and international meetings. This training program has enjoyed broad success, with our students achieving excellent productivity (mean 5.1, range 2-11, publications during graduate school by recently funded students). Student excitement for human genetics training has rapidly increased, demonstrated by a 3-fold increase in the number of students entering the Vanderbilt Human Genetics (HGEN) PhD program over the past 5 years. Much of that increased excitement has grown from the unprecedented data available now to students. Previously, it could take years of career advancement to assemble even a single large-scale cohort of a single phenotype. However, with the advent of large biobanks with clinical data, cohorts can now be assembled relatively quickly by graduate students with the proper training, which our program provides. The Vanderbilt HGEN program has been at the forefront of training graduate students in this new type of research. With Vanderbilt’s own large- scale biobank (BioVU), and now the Data and Research Center for the NIH “All of Us” program located in the Vanderbilt University Medical Center, our graduate students have been getting both hands-on and formal didactic training in this new and groundbreaking type of research. With the establishment in 2015 of the Vanderbilt Genetics Institute, Vanderbilt has committed substantial resources for recruitment of faculty in genetics and genomics and additional investment in genotyping of biobank subjects. Our graduate students are well-rounded biologists, with most matriculating through the Interdisciplinary Graduate Program (IGP) at Vanderbilt and the rest from the Quantitative & Chemical Biology Program (QCB). They conduct research in an amazing variety of topics and publish as graduate students in the top scientific journals. Their education is clearly empowering them, as the students themselves are major designers of their own collaborative research within Vanderbilt, and they rightly feel that they are not just benefitting from the excitement in the field of human genetics today but are also driving it.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY An estimated 50-70 million Americans suffer from a sleep disorder, which is commonly comorbid with many psychiatric illnesses, such as alcohol use disorder (AUD). Individuals with AUD frequently report insomnia, and sleep difficulty significantly increases the likelihood of relapse. Despite the overwhelming need to control sleep disturbances to aid in the prevention of relapse, the underlying physiology of wakefulness, particularly in the context of acute and chronic alcohol use, is not well understood. The brain’s noradrenergic system mediates many behavioral states, such as arousal, alertness, and stress. Acute and chronic alcohol exposure differentially alter the activity of noradrenergic neurons as well as the release, synthesis, and turnover of norepinephrine (NE) in the brain. Recently, our lab alongside collaborators delineated a novel arousal circuit from the noradrenergic locus coeruleus (LC) to dopaminergic neurons in the ventral periaqueductal gray (vPAGDA), where stimulation of this circuit promotes wakefulness. Acute ethanol exposure also increases excitatory drive and the activity of wake-promoting vPAGDA neurons; however, the mechanism is unknown. Specific activation of alpha1-adrenergic receptors (α1ARs) increases excitatory drive onto vPAGDA neurons and subsequently causes arousal. Interestingly, α1AR expression is particularly enriched on vPAG astrocytes, and activation of astrocytic Gq signaling is sufficient to promote wakefulness. While astrocytes are essential in mediating synaptic transmission in other brain regions through purinergic signaling, the role of neuron- astrocyte interactions in the vPAG during ethanol exposure requires further investigation. This F30 aims to test the central hypothesis that purinergic transmission from neighboring astrocytes mediates the noradrenergic modulation of vPAGDA neurons, and that ethanol potentiates this signaling. The goal is to elucidate the mechanistic details mediating the increase in excitatory drive onto the wake-promoting vPAGDA neurons by activation of α1ARs and the pathologic changes to the noradrenergic system following acute and chronic alcohol exposure. In Specific Aim 1 I will utilize pharmacological and viral genetic approaches combined with ex vivo fluorescence imaging and whole cell patch-clamp electrophysiology to examine the neuron-astrocyte interactions underlying noradrenergic modulation of vPAGDA neurons. In Specific Aim 2 I will use similar approaches in addition to a mouse model of chronic intermittent ethanol exposure to determine the changes in synaptic transmission onto vPAGDA neurons following acute and chronic ethanol exposure. The results of these studies will allow our lab, our collaborators, and others to further investigate the role of neuron- astrocyte physiology related to sleep dysfunction in alcohol use disorder. This will further the work towards identifying possible therapeutic targets for the development of novel therapies to alleviate disordered sleeping and aid in relapse prevention.

NIH Research Projects · FY 2024 · 2022-07

Project Summary All organisms must maintain a balance of nutrient metals to survive, including zinc (Zn). These metals are required as catalytic and structural cofactors for a variety of proteins, but in excess can lead to the generation of reactive oxygen species or inactivation of non-cognate enzymes through mismetallation. Therefore, tight control of metal levels through import, efflux, and storage is important for optimal growth and survival. Due to this requirement, bacterial metal homeostasis mechanisms are attractive targets for novel therapeutics. This proposal seeks to inform the development of metal-based therapies by identifying mechanisms used by the opportunistic pathogen Acinetobacter baumannii to prevent metal imbalance. Previous work in our laboratory has identified zigA which encodes a putative Zn metallochaperone with increased expression upon Zn starvation mediated by calprotectin, a metal sequestering protein of the innate immune system. Loss of ZigA results in a severe fitness defect upon calprotectin exposure, indicating the essentiality of ZigA under these conditions. Since ZigA is critical for A. baumannii to grow under Zn limitation, we hypothesized that proteins that receive metal from ZigA (clients) are equally important in mediating Zn stress. To identify these clients, I performed a genome- wide transposon mutagenesis screen in Zn limiting conditions comparing WT and ΔzigA libraries to identify genes whose fitness is influenced by Zn deficiency and that modulate the fitness of a ΔzigA mutant. I discovered several genes through this genetic interaction method and chose A1S_3027 for further characterization. A1S_3027 encodes a lytic transglycosylase that is predicted to tailor the bacterial cell wall. Strains lacking A1S_3027 are sensitized to Zn deficiency, and this can be reversed upon addition of ZnCl2. We hypothesize that to ensure the integrity of the cell envelope in conditions of Zn starvation, ZigA interacts with A1S_3027 to regulate its function. Characterizing A1S_3027 and its coordination with ZigA will be tested in two specific aims. In Specific Aim 1, I will study the biochemical properties and function of A. baumannii A1S_3027 using biochemical, genetic, and functional assays to probe how A1S_3027 helps to maintain Zn homeostasis. Experiments proposed in Specific Aim 2 will determine the functional role of A. baumannii A1S_3027 in maintaining appropriate nutrient metal balance by employing A1S_3027-deficient strains in a series of in vitro and in vivo experiments. Taken together, these aims will determine the impact of metal imbalance on A. baumannii pathogenesis and provide the first description of the contribution of a Zn metallochaperone and its clients to microbial virulence. Therapeutics that modulate bacterial metal levels will synergize with the immune system’s defenses, and A1S_3027 may be an attractive target for such metal-focused therapeutics.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Eosinophilic esophagitis (EoE) is a chronic inflammatory disease affecting the esophagus, and one of the most common causes of vomiting, feeding and swallowing difficulties, including esophageal food impaction in children. EoE is difficult to diagnose and distinguish from other common esophageal diseases in real-time using white light endoscopic imaging of the esophagus conducted via an esophagogastroduodenoscopy (EGD) procedure. Therefore, clinicians rely on multiple random esophageal biopsies which is obtained during this procedure for histologic confirmation (gold standard). However, since EoE is a patchy disease even the multiple biopsies may not yield accurate results. This can lead to a delay in diagnosis and ineffective treatment, and result in preventable complications such as esophageal remodeling requiring surgical interventions. As such there is an urgent need to develop alternative diagnostic tools which can quickly survey more regions of the esophagus, in situ, and provide tissue-specific inflammatory biomarker information for real-time identification of EoE. Raman spectroscopy (RS) is one such technology that can provide a solution as it relies on light interaction with molecules to provide a unique “fingerprint” of the biochemical and molecular composition of a specimen within seconds. Therefore, RS via an endoscope is uniquely suited for real-time biochemical assessment of active EoE. This proposal, presents the development and assessment of a depth specific dual-wavelength Raman endoscope to characterize both biochemical and water changes within the esophagus as it relates to active EoE. To accomplish these goals, Raman biomarkers will be characterized and identified spatially and correlated with known biomarkers of EoE in vivo as well as ex vivo. Computational modeling will be performed using a modified Monte Carlo simulation to optimize probe design based on tissue optical properties. Ex vivo non-linear imaging coupled with Raman maps will be performed on a subset of biopsies, obtained during the EGD procedures, to further study the spatial distribution of key biomarkers and help guide the endoscopic Raman probe design. This probe will be evaluated using optical tissue phantoms and in vivo patient measurements. Finally, a machine learning classification algorithm will be optimized and evaluated to provide a mechanism by which in vivo RS spectra can be classified into active EoE, inactive EoE, GERD (i.e., acid reflux disease) and other non-EoE control. Furthermore, we aim to track patient’s response to treatment and assess Raman spectral changes over time to determine the feasibility of using RS to provide a predictive treatment outcome model. The successful completion of this proposal will yield a novel diagnostic tool for real-time detection and monitoring of EoE in pediatric cases.

NIH Research Projects · FY 2026 · 2022-06

Project Summary Long-standing hypotheses have posited a critical role for dysregulated dopaminergic neuromodulation of prefrontal cortex in the development of alcohol use disorder (AUD). Despite intense interest in the mesocortical dopamine system, technical challenges have precluded direct, temporally resolved observation of dopamine release patterns in the prefrontal cortex until recently. Accordingly, questions regarding the basic function of mesocortical dopamine as well as its role in AUD have been notoriously difficult to address. Here we propose to use a recently developed genetically-encoded fluorescent dopamine sensor, which allows for direct assessment of dopamine dynamics in vivo with unprecedented specificity and temporal resolution, to dissect dopamine release patterns in the medial prefrontal cortex (mPFC) associated with alcohol self-administration behaviors. In male and female mice, we will first test the responsiveness of dopamine innervation of mPFC to a range of stimuli including alcohol to determine its contribution to basic behavioral processes. Next we will assess how these release patterns evolve during initiation of alcohol self-administration and repeated alcohol exposure to determine if deficits in this system are associated with the emergence of heightened alcohol drinking and seeking. Finally, we will mechanistically investigate mesocortical control of alcohol drinking behaviors by manipulating the activity of this system in vivo and determine alcohol’s direct actions on presynaptic dopamine terminals in the mPFC using ex vivo imaging. Completion of this proposal will provide unprecedented insight and understanding as to the contribution of dysregulated cortical neuromodulation in AUD-relevant behaviors.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract Fundamental gaps remain in our understanding of the cell biological mechanisms that drive mitochondrial decline and associated age-related diseases. Organelles like the mitochondria and endoplasmic reticulum (ER) are physically and functionally linked, in part via sites of membrane contact. These lines of communication between mitochondria and other organelles represent an understudied avenue by which to therapeutically target mitochondrial function. Our long-term goal is to understand the physiological roles of inter-organelle communication during aging and age-related disease. In pursuit of that goal, our objective in this application is to determine how the ER regulates mitochondrial health during aging through its role as a platform for calcium signaling. We have exploited the simple anatomy of C. elegans and experimental advantages in genetics and microscopy to lay a foundation in this model for the study of ER-mitochondrial interactions. Similar to mammals, the worm ER calcium efflux channel, inositol triphosphate receptor (InsP3R), exerts potent control over mitochondrial bioenergetics, and we have extended the roles of InsP3R to regulation of mitochondrial gene expression and dynamics in the worm as well. Furthermore, the InsP3R regulates lifespan in C. elegans through mechanisms that depend upon mitochondrial function. Here we will test the hypothesis that ER remodeling in aging animals acts to trigger mitochondrial dysfunction and organismal decline by promoting aberrant subcellular calcium signaling and dynamics. To test this hypothesis, we will first determine whether the InsP3R is a cell autonomous regulator of mitochondrial function and lifespan. Secondly, we will identify the molecular mechanisms linking InsP3R activity to the diverse changes observed in mitochondrial behavior. Finally, we will determine how organellar remodeling of the calcium flux machineries initiates age-onset mitochondrial dysfunction. By revealing the mechanisms by which ER signaling governs mitochondrial health at the organismal level, these results will open new therapeutic avenues in treating mitochondrial pathologies.

NIH Research Projects · FY 2025 · 2022-06

Project Summary/Abstract Cocaine use disorder (CUD) has no FDA approved therapies putting stimulant use disorders at a unique treatment disadvantage, necessitating further research on stimulant use neural dysregulation for novel interventions. The nucleus accumbens (NAc) is at the core of valence-based stimulus processing and associative learning, and its dysregulation by cocaine is a primary component underlying the development of CUD in human and animal models. The NAc is not only an incredibly plastic area but receives numerous glutamatergic inputs from across the brain that integrate complex information in order to drive the activity of the NAc. Long-term cocaine exposure leads to plasticity in synaptic strength of glutamatergic inputs into the NAc and this plasticity has been directly linked to maladaptive behaviors associated with cocaine exposure. While a large body of work has highlighted the synapse-specific mechanisms that occur following cocaine exposure, this has been largely done in ex-vivo preparations and on individual inputs into the NAc; however, information encoding within these projection populations is a dynamic process that occurs on a fast timescale and understanding how their relationship encodes complex information requires their simultaneous recording in awake and behaving animals. I will be using a range of calcium imaging and viral-mediated expression approaches to 1. identify the glutamatergic inputs that modulate and drive neural activity within the NAc and 2. understand how these signals facilitate the encoding of stimuli to drive behavior at baseline and following cocaine use. First, using multisite fiber photometry in glutamatergic projections from the basolateral amygdala (BLA-NAc), Hippocampus (vHPC- NAc) and medial prefrontal cortex (mPFC-NAc) into the NAc, I will define how these circuits are simultaneously activated by unconditioned and conditioned stimuli to drive behavior (Aim 1). Next, using cocaine self- administration, I will define how these circuits are altered by repeated drug exposure, leading to neural and behavioral impairments in learning for non-drug stimuli (Aim 2). As a future physician-scientist, understanding the complex factors that contribute to addiction, especially as they relate to learning from drug and non-drug stimuli, are a critical component of effective CUD treatment and intervention. This proposal with provide the technical training needed to answer such questions in the laboratory, while also providing the theoretical training to provide optimal care for patients in my clinical practice.

NIH Research Projects · FY 2025 · 2022-05

Violence disproportionately affects African American youth. In addition to death and injury, violence exposure has significant psychological consequences, including traumatic stress symptoms and internalizing problems. Self-directed violence has shown startling and disproportionate growth among African American youth, with suicide rates nearly doubling from 2007 to 2018. Current prevention strategies have limited effectiveness, perhaps due to their failure to address root causes of violence. The primary aim of this proposed project is to examine the extent to which an intervention addressing problematic experiences in education reduces interpersonal violence and suicide among middle school-aged youth, with a focus on populations experiencing health disparities (African Americans; low-income communities). The proposed project will examine community-level changes using a multiple baseline experimental design that randomizes the start of the intervention in four communities, each comprising a police precinct and middle school. The intervention will consist of school-based intervention components including a culturally responsive, community-inclusive adaptation of a whole-school climate intervention (School-wide Positive Behavior Interventions and Supports) and culturally responsive practices training and coaching. Outcomes will be measured using archival data from the schools as well as survey data from youth and school personnel. Aim 1 is to evaluate the extent to which targeting disciplinary and pedagogical practices reduces interpersonal violence among youth, as measured by individual-level and school-level data. Aim 2 is to evaluate the extent to which targeting disciplinary and pedagogical practices reduces suicidality among youth, as measured by completed suicides and proximal precedents for suicide, including attempts and ideation. Aim 3 is to evaluate the extent to which targeting disciplinary and pedagogical practices reduces both overall rates and disproportionality of school-based exclusionary discipline practices and increases culturally relevant pedagogy. Aim 4 is to evaluate specific intervention components by determining their effects on hypothesized mechanisms of change at the individual, teacher, and school levels. This intervention has the potential to reduce morbidity and mortality among African American youth, promote overall quality of life, and reduce the societal costs associated with both interpersonal violence and suicidality. Furthermore, effective strategies to address shared causes of violence and suicidality have the potential to facilitate groundbreaking public health prevention of health disparities.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT The caregiving environment children experience is the most important modifiable feature for shaping brain development and influencing subsequent mental health. Building from knowledge of infant mental health and developmental neuroscience, this NIMH BRAINS award application is designed to advance our understanding of how children’s experiences with their caregivers during infancy, a developmental period characterized by heightened brain plasticity, influences brain and behavioral development. Specifically, we will use innovative methods to characterize multiple aspects of the early caregiving environment in relation to changes in brain structural and functional connectivity that are believed to contribute to the onset of mental disorders. The application of new tools, coupled with traditional metrics, will improve our measurement of children’s experiences using a child-centered approach (i.e., capturing children’s contact with multiple caregivers). To do this, we will recruit 150 women in pregnancy and, following birth, conduct assessments in children’s daily ecological context at ages 1, 6, 12, and 18 months. This project introduces a wearable device technology that can dynamically, unobtrusively, and continuously measure patterns of physical proximity between children and caregivers, regardless of physical location. In addition to children’s proximity to caregivers, we will obtain ecological assessments of language exposure and observation-based caregiver sensitivity and examine the convergence and divergence among these different ways of capturing children’s experiences (Specific Aim 1). While prior research suggests that greater environmental enrichment leads to lower symptoms of psychopathology, the lack of granularity in measurement (i.e., not capturing the full continuum of relative psychosocial neglect–enrichment) has precluded the ability to characterize the shape of these associations (e.g., linear, nonlinear). We will study children selected to range in experiences along the neglect–enrichment continuum and use repeated neuroimaging of infant brain structural and functional connectivity and repeated behavioral assessments to explore the possible profile of the associations between aspects of the caregiving environment and changes in brain and behavior (Specific Aim 2). Last, we will examine how changes in emotion regulation and emotion reasoning circuitry are associated with signs of emerging psychopathology at age 18 months in order to test whether, when, and how variations in early experience influence risk for psychopathology through changes in emotion-related circuitry (Specific Aim 3). Here, neuroimaging is particularly advantageous as it allows us to examine the maturation of emotion-related networks from birth and, importantly, prior to the onset of detectable mental health difficulties. Achievement of the aims of this proposal is expected to meet NIMH’s objectives to determine the biological and psychological mechanisms by which experience affects neural and behavioral development, with direct applications for prevention and early intervention.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY/ABSTRACT When learning in complex, realistic, or even real worlds, we have the benefit of using different strategies adaptively. For most primate brains, adaptive means adjusting as a function of where we are, who we are with, and what things of use are in view or in reach. Learning theories like Complementary Learning Systems (CLS) originally suggested that the hippocampus and neocortical structures contributed distinct computations to represent different kinds of memory. This theory relied heavily on assumptions about the finer structure of neurons in these areas, built largely from knowledge of these structures in rats and to some extent mice. Methodological limitations have prevented measuring in primates (human or monkey) the fine circuit computations predicted by these models. This has led to assumptions that the computations are similar to those in rodents, yet rodents have very different real-world behaviors from primates. We propose to check these assumptions and extend and/or revise the theory, by recording wirelessly in macaques who learn rules about objects in an immersive, real-world enclosure. We will use high- density, multi-site recordings in and around the hippocampus to test two major aspects of memory theory in need of resolution. First, we ask if there are differences in the two main hippocampal-CA1 inputs in supporting episodic and category learning. This question derives from an untested prediction of our expanded CLS model. We will record wirelessly as macaques make decisions about the assignment of object exemplars (‘FauXna’) on displays set up in their environment. The model predicts that CA3-CA1 inputs are particularly supportive of the arbitrary mappings required for episodic memory, whereas layer III entorhinal cortex ‘direct’ inputs are more involved in integrating information across trials, affording object category learning. Using high-definition linear arrays, we can resolve CA1 dendritic field currents as well as multi-site ensemble unit activity, allowing us to test our prediction for the first time. Second, we ask if the hippocampal and connected neocortical dynamics play a role in memory retrieval as a function of either memory age or of the episodic/semantic nature of the task. We will use closed-loop stimulation to interrogate the necessity of each region during recall, and the role of coordinated activity between hippocampus and neocortex for recall, across memory age and type. From these experiments we will (1) disambiguate several competing theories of the division of labor across nodes in the memory network, (2) create the first conceptual microcircuit model of these memory systems in the primate brain, and (3) contrast with our expanded computational model.

NIH Research Projects · FY 2026 · 2022-04

Project Summary Islet glucose-stimulated somatostatin (Sst) secretion is lost in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucagon and insulin secretion. It is generally accepted that Sst secretion from δ-cells occurs in response to elevated intracellular Ca2+, which primarily results from endoplasmic reticulum (ER) Ca2+ (Ca2+ER) release. However, the mechanisms that control δ-cell Ca2+ER handling and how they are altered in T2D are largely unknown. Data from our lab finds that the islet-enriched two-pore- domain K+ channel, TALK-1, is an ER localized channel in that provides a countercurrent for δ-cell Ca2+ER release and Ca2+ER leak. TALK-1-mediated augmentation of the electrochemical driving force for δ-cell Ca2+ER leak con- strains Ca2+ER storage, which limits glucose-stimulated Ca2+ER release and Sst secretion. Further data show that δ-cell Ca2+ER release and Sst secretion are amplified by glucose-induced allosteric activation of δ-cell Ca2+-sens- ing receptors (CaSRs). Finally, our preliminary data provide the first evidence that diabetic conditions diminish δ-cell Ca2+ER storage, which contributes to perturbations in glucose-stimulated Ca2+ handling and Sst secretion under diabetic conditions. Based on these exciting preliminary data, the overall objective of this proposal is to elucidate how δ-cell Ca2+ER is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that glucose-stimulated δ-cell Sst secretion is amplified by CaSR-mediated Ca2+ER release, which is controlled by TALK-1 channel constraint of Ca2+ER storage. The rationale that underlies this project is that understanding how CaSR and TALK-1 control δ-cell Ca2+ER handling and Sst secretion will expose novel therapeutic targets for restoring glucose-stimulated Sst secretion and islet hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine how δ-cell CaSR controls Ca2+ER handling, Sst secretion, and islet hormone secretion; and 2) Determine how TALK-1 channel control of Ca2+ER release modulates δ-cell function and dysfunction. Under the first aim, transgenic mice with δ-cell ablation of CaSR as well as human pseudoislets with ShRNA knockdown of δ-cell CaSR will be utilized to assess the roles of the Ca2+-sensing receptor during secretagogue modulation of δ-cell Ca2+ handling and Sst secretion. Aim1 will also determine how depletion of δ-cell Ca2+ER stores under diabetic conditions impacts CaSR signaling and Sst secretion. Under the second aim, the function TALK-1 channels on δ-cell Ca2+ER handling and function will be determined in mice with δ-cell specific ablation of TALK-1 and in human pseudoislets containing either δ-cells with knockdown of TALK-1 or expressing dominant negative TALK-1 channel subunits. Furthermore, Aim2 will determine how TALK-1 augmentation of δ-cell Ca2+ER depletion under the stressful conditions associated with diabetes contributes to δ-cell dysfunction. This project is significant because it is expected to illuminate mecha- nisms that alter δ-cell Ca2+ER handling and disrupt islet hormone secretion in T2D. Moreover, this project will identify pharmacological strategies for normalizing Sst secretion and reducing islet dysfunction in T2D.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY / ABSTRACT Our proposal investigates in the nonhuman primate (NHP) how muscarinic modulation enhances cognition, motivation and behavioral regulation and which neurochemical and cell-type specific mechanisms underlie these positive effects. We specifically will benchmark a positive allosteric modulator (PAM) for the centrally expressed muscarinic M1 receptor, developed at the Vanderbilt Center for Drug Discovery. M1-PAMs promise to overcome dose-limiting side effects and avoid agonist overstimulation that limit compliance, efficacy, and tolerability of existing compounds. M1 selective modulation can be antipsychotic, reduce negative symptoms (e.g. reduce lack of motivation) and ameliorate cognitive deficits in patients with schizophrenia. M1-PAMs may achieve this by gating intrinsic cholinergic signaling which is believed to regulate glutamatergic and dopaminergic release in the prefrontal cortex and striatum. We test these hypothesized working mechanisms by determining the neurochemical and electrophysiological consequences of M1 PAM action. First, we will determine the dose-response efficacy of M1 PAMs to enhance cognition, motivation, and behavioral regulation, comparing their effects to the agonist Xanomeline and the non-selective cholinergic drug Donepezil. We will assess primary cognitive functions (attention, working memory), primary motivational functions (effort control, resilience to loss), cognitive flexibility (set shifting, perseveration, reward learning), visuospatial problem solving, and the regulation of behavior video-captured when NHPs engage with the touchscreen assessment Kiosk in their home cages. The behavioral metrics evaluate five RDoC domains, tested in single sessions using a novel Multi-Task Test Battery for NHP. We will determine dose-response efficacy for each RDoC domain separately which clarifies how broad M1 PAMs enhance cognitive-motivational-behavioral functions and which domains suffer from dose-limiting side effects with a conventional agonist and a nonselective cholinergic drug. Second, we will determine the drug-dose dependent changes of extracellular concentrations of Acetylcholine, Dopamine, Serotonin, Glutamate, GABA, and of the systemically administered drug itself. We achieve this in NHPs in parallel in three brain areas that load differently on the five RDoC domains to determine the dose- response efficacy for each brain area separately. The dorsolateral prefrontal cortex is assessed to understand how M1 PAMs regulate glutamate and acetylcholine implicated to support cognitive RDoC constructs. The Striatum is assessed to understand how M1 PAMs regulate dopamine to support reward learning and cognitive flexibility. The anterior cingulate cortex is assessed to determine dose-efficacy for modulating serotonin and glutamate to mediate effort-control and motivation. Simultaneously, neural spiking activity is recorded to understand how M1 PAMs alter firing and synchronization of different interneuron types that we distinguish electrophysiologically. Together, the proposed studies elucidate the working mechanisms and strength of M1 PAMs relative to existing dose-limited drug regime and thereby inform treatment strategies for schizophrenia.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Extensive research has established clear, strong associations between human social relationships and health and illness. A lack of social connection, including isolation, loneliness, and conflict, is related to the onset and progression of cardiovascular disease, some forms of cancer, diabetes, and obesity among other acute and chronic health conditions. Neurodegenerative diseases have been relatively overlooked in this research despite having adverse effects on patients’ functioning that may disrupt a range of social relationships. Huntington’s disease (HD) is an exemplar neurodegenerative disease that it is a fully penetrant, autosomal dominant condition characterized by progressive cognitive, behavioral, emotional, and motor impairments that have the potential to negatively affect family functioning and community engagement. HD is likely to place a particular burden on the parent-child relationship given that the disease is most often diagnosed in middle adulthood, a period that includes the primary years for child rearing and parenting, and offspring of parents with HD have a 50% risk of inheriting the disease themselves. As children watch their parents’ disease progress, they observe their own potential future and may be tasked with significant caretaking demands. Notably, qualitative research highlights significant impairments to social relationships experienced by both HD parents and their offspring within and outside of the family. In response to PAR-21-145, the proposed study will address the gap in empirical research by documenting levels of the structure, function and quality of social connectedness in HD families and examine potential mechanistic targets for behavioral intervention. Our preliminary data emphasize the negative impact of HD on social connectedness, including the quality of communication, of parents with HD and their offspring. Further, our previous research and preliminary data suggest two potential mechanisms linking social relationships and psychological and physical health outcomes for parents with HD and their offspring: executive function (EF; e.g., working memory) and emotion regulation (ER) in response to stress (e.g., cognitive reappraisal, problem solving). We will examine the associations between social connectedness and quality of life and impairment in cognitive and emotional function in a sample of 200 patients with HD and their adolescent and young adult offspring (n = 200). A sample of parents without neurodegenerative disease (n = 200) and their adolescent and young adult offspring (n = 200) will serve as a comparison sample.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Preexisting cognitive deficits or exposure to stressors both increase the probability of alcohol use disorder (AUD). These relationships are bidirectional, and excessive alcohol consumption can also directly impact cognition and dysregulate stress systems. Individual differences in cognition and stress reactivity are thought to define phenotypes within the AUD spectrum which may differ in disease prognosis and responsivity across treatment strategies. As such, precisely defining the behavioral and neurobiological substrates mediating covariance across cognitive, stress, and drinking domains is critical for our understanding of AUD. However, until recently we have lacked technical approaches which would allow for determination of whether individual differences in these behaviors arise from the same neurons or from distinct populations within brain regions. To parse how these phenotypes manifest we must 1) quantify the complex individual differences that emerge at the intersection of stress, alcohol drinking, and cognitive function and 2) determine the precise neurons in the brain that control these interactions. To this end, we will first use deep phenotyping of both behavioral and neuronal features to computationally define individual differences across domains in mice. Previous studies have demonstrated that prefrontal cortex is a critical mediator of cognitive function, responses to stressors, and drinking, but the precise degree of shared circuitry between these behaviors is unclear. Thus, we will use a longitudinal design to define the neuronal plasticity signatures in prefrontal cortex that govern expression and interactions between these behaviors within the same subjects. Mouse models offer unique advantages for defining the precise cell-types within the prefrontal cortex that give rise to these behaviors, but it will also be essential to determine if these neurobehavioral relationships are conserved in higher-order species. Using a cross-species approach, conservation of relationships between plasticity in specific cortical cell-types and individual differences in cognitive, stress, and alcohol interactions observed in mice will then be directly tested in ex vivo brain slices from non-human primates. Successful completion of this proposal will provide novel insight into the circuit basis of alcohol and stress interactions and advance these hypotheses across species towards translational endpoints.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Ubiquitin is a 76 amino acid peptide that can be covalently conjugated to substrates to alter protein fate in diverse ways, regulating protein degradation, trafficking, subcellular localization and protein-protein interactions. Given its versatility, ubiquitin regulates many fundamental cellular processes, and its dysregulation is associated with many human diseases ranging from neurodegeneration to cancer. Ubiquitin networks include conjugating and deconjugating enzymes as well as effector pathways comprised of ubiquitin binding proteins that direct the fate of ubiquitin-modified substrates. All of these elements work together to “write”, “read”, and “edit” the ubiquitin code – which ultimately consists of ubiquitin polymers of different lengths and topologies that determine which effector pathways are engaged. Here, we describe two main research directions that will result in a deeper understanding of the ubiquitin code and how it regulates diverse cellular functions, including stress signaling and membrane trafficking. The first research direction will address how phosphorylation of ubiquitin at the Ser57 position regulates stress responses in yeast and human cells. The proposed studies will build on our recent discovery of a small group of Ser57 ubiquitin kinases conserved from yeast to humans and will include genetic, biochemical, and proteomic approaches. Specifically, we will determine how these kinases and Ser57 phosphorylation of ubiquitin contribute to the cellular stress response, and we will address how ubiquitin phosphorylation alters its interaction profile and engagement with effector pathways. This research will contribute transformative new insights into the biology of ubiquitin and proteostasis. The second research direction will address how human glucose transporters are regulated by ubiquitin modification and endocytic trafficking. Glucose transporters of the GLUT family are key regulators of cellular glucose homeostasis, and yet regulation of their trafficking and quality control remain poorly characterized. Here, we describe lines of investigation based on our recent findings that GLUT1 endocytic trafficking is regulated by specific ubiquitin modifications. These studies have important implications for cellular glucose homeostasis and human diseases including GLUT1 Deficiency Syndrome and many types of cancer. Together, these research directions will result in a deeper understanding of the ubiquitin code, membrane trafficking, and stress responses in eukaryotic cells.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Eukaryotic cells must maintain a specific protein and lipid composition of the plasma membrane and all of the internal membrane-bound organelles in order to function normally. Even though membrane biogenesis is crucial for life, mechanisms for establishing the composition and organization of membranes remain poorly understood. We study how membrane asymmetry is established, a fundamental feature of the eukaryotic cell plasma membrane defined by the enrichment of phosphatidylserine and phosphatidylethanolamine within the cytosolic leaflet, while sphingolipids and phosphatidylcholine are typically enriched in the extracellular leaflet of the bilayer. Regulated exposure of PS and PE on the extracellular leaflet contributes to cell signaling, cytokinesis, blood clotting, cell-cell fusion, apoptotic cell corpse removal and host-viral interactions. Membrane asymmetry is driven by type IV P-type ATPases (P4-ATPases), which are a large family of flippases that pump lipids from the extracellular leaflet to the cytosolic leaflet of the membrane bilayer. The P4-ATPase subfamily is highly conserved among eukaryotes and these transporters have been implicated in pathological conditions such as obesity-linked type 2 diabetes, cardiovascular disease, liver disease, hearing loss, immune deficiency, and severe neurological disease. In addition, P4-ATPases are critical components of the vesicle-mediated protein trafficking machinery within the Golgi complex and endosomal system. Through their role in protein trafficking, P4-ATPases help control the precise protein composition of the plasma membrane, Golgi complex endosomes and lysosomes. The proposed studies will determine how the P4-ATPases recognize and transport their lipid substrates to establish membrane asymmetry using structural, biochemical and molecular genetic approaches. These structure/function studies will include how P4-ATPase activity is regulated by post- translational modification and protein-protein interactions. We will also probe the cellular requirements for transport of specific substrate lipids, like glucosylceramide and phosphatidylserine, on cell morphogenesis, fungal pathogenesis, nutrient signaling, and protein trafficking. For the latter studies, we will probe how P4- ATPases help drive vesicle-mediated protein transport with a focus on carriers formed by COPI and retromer. Atypical roles for ubiquitination in the P4-ATPase- and COPI-dependent transport pathways will also be defined. In total, these studies should lead to a much better understanding of how P4-ATPases exert their essential function, and will be invaluable to our ability to understand and ultimately treat pathologies associated with P4-ATPase deficiency.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY The AMPA type ionotropic glutamate receptors (AMPARs) are ligand gated ion channels activated by the neu- rotransmitter glutamate. They mediate the majority of excitatory neurotransmission in the brain and the signals transduced by these complexes are critical for synaptic plasticity, learning and memory. AMPAR auxiliary sub- units regulate trafficking and gating modulation of AMPARs. In this proposal we will investigate the mechanism of AMPAR regulation by their auxiliary subunits. The core AMPAR auxiliary subunits are TARPs, GSG1L, and cornichons (CNIHs). The TARPs are extensively studied and therapeutic compounds to alleviate seizure are already available to target hippocampus enriched TARP gamma-8. GSG1L is a negative modulator of AM- PARs, while TARPs and CNIHs serve as positive modulators. In humans, various residues located at the inter- action interface between AMPAR and auxiliary subunits are intolerant to missense mutations, indicating their critical roles in brain function. We hypothesize that different auxiliary subunits can co-assemble with the chan- nel and produce a rich variety of gating modulations, which are fundamental in regulating synaptic transmission and plasticity. To establish the structural and mechanistic basis, we will study complex AMPAR assemblies that have high physiological relevance. In Aim 1 we hypothesize that fine structural differences among AMPAR assemblies are fundamental for producing characteristic gating modulation and propose to reveal the architec- tures of heterotetrameric AMPARs containing up to two types of auxiliary subunits at different functional states in detergent using cryo-EM. By comparing the structures, new mechanistic models that could explain how aux- iliary subunits control the time course and magnitude of gating are likely to emerge, which will be validated us- ing electrophysiology. Next, currently available cryo-EM structures revealed the presence of lipids surrounding the complex. We hypothesize that these lipids play important function in AMPAR gating modulation, which will be tested in Aim2. Finally, we suggest that AMPAR/auxiliary subunit complex prepared in near physiological conditions void of detergent must be studied to build more precise mechanistic models of its allosteric gating modulation. In Aim 3, we propose to solve high resolution cryo-EM structures of AMPAR/auxiliary subunit complex embedded in a lipid bilayer mimetic environment to resolve the known discrepancies between struc- tures obtained in detergent and electrophysiology data. The role of auxiliary subunits in tuning ion channel gat- ing kinetics is predicted to have significant impact on circuit dynamics. In summary, the outcomes of this study are expected to advance our mechanistic understanding of AMPAR function and assist developing new thera- peutic compounds that can alleviate dysregulation of AMPARs seen in neurological and psychiatric disorders, such as Alzheimer’s disease, stroke, autism, Rasmussen’s and limbic encephalitis, and seizure.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Aspergillus fumigatus is a major human fungal pathogen that infects – often killing – hundreds of thousands each year. A few closely related species are also pathogenic but cause fewer infections. In contrast, most other closely related species are not pathogenic. Pathogens have originated repeatedly from non-pathogens, suggesting that the ability to cause disease or pathogenicity has evolved multiple times independently in this lineage. The observed spectrum of pathogenicity cannot be explained by differences in species' ecologies or by ascertainment bias, indicating that the repeated evolution of Aspergillus pathogenicity has, at least partially, a genetic basis. Several key traits – and their underlying genes and pathways – are known to be associated with A. fumigatus pathogenicity, including virulence, growth at the human body temperature, and the production of secondary metabolites. In contrast, we know surprisingly little about the repeated evolution of pathogenicity in Aspergillus and the variation in the traits and genetic elements that contributed to its origins. Whether pathogenic species share traits and genetic elements that are absent in non-pathogens (“conserved pathogenicity” model) or each pathogen contains a unique suite of traits and genetic elements that distinguish it from its non-pathogenic relatives (“species-specific pathogenicity” model) remains unknown; a third model, essentially a mix of the other two, is also possible, under which some traits and genetic elements that contribute to pathogenicity are conserved and some are species-specific. Elucidating which model explains the repeated evolution of Aspergillus pathogenicity is key for developing strategies to combat infections, identifying genetic elements that contribute to virulence, and predicting new pathogen emergence. This project will test these models and advance our understanding of the evolution of Aspergillus pathogenicity by investigating: the variation of species (and strains within species) that span the pathogenicity spectrum with respect to virulence, growth at the human body temperature, and secondary metabolite production (Aim 1); the genomic and transcriptomic variation associated with the observed differences in pathogenicity (Aim 2), and; how genetic elements that vary between pathogens and non-pathogens have contributed to the evolution of fungal pathogenicity (Aim 3). The project is innovative because: it will address fundamental, largely unanswered, questions in medical mycology, such as how fungal pathogenicity evolved and why A. fumigatus infects hundreds of thousands yearly but its very close relatives do not; b) it will generate invaluable resources, such as comprehensive, in-depth examinations of variation for key disease-relevant traits and genetic elements in a lineage of closely related fungi that vary extensively in their pathogenicity, and; it will lead to the generation of novel, genetically tractable model organisms for studying how major fungal pathogens originate from historically innocuous organisms.

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically heterogeneous sample of the US population. To accomplish this, the Healthy Brain and Child Development (HBCD) Consortium has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,200 mothers and infants enrolled at 27 sites across the United States (US). The HBCD Study will carry out a common research protocol under direction of the HBCD Consortium Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD Data Coordinating Center (HDCC). The overarching goal of the HBCD Study is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a varied cohort that is representative of the US population; 2) pregnant woman with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD Study will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state-of-the-art methods. The HBCD Study will inform public policy to improve the health and development of children across the nation. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT The mesolimbic dopamine system is at the core of reinforcement learning, and its dysregulation by stimulants is a major factor in the development of cocaine use disorder (CUD). While dopamine is often linked to valence- based learning, emerging data - as well as preliminary data in this proposal - has indicated that dopamine release in the NAc core is evoked by both rewarding and aversive stimuli, thus challenging the idea of bi- directional valence coding. It is likely that dopamine release in the NAc core signifies how salient – or important – a stimulus is independ`ent of its positive or negative emotional value (valence). Salience is a key driver of the speed at which information is learned in an environment; thus, deficits in a system that encodes saliency would slow many forms of learning - both drug and non-drug associated. If NAc core dopamine signals saliency, rather than a valence-based signal, aberrations in this system could explain several CUD-associated learning deficits. For example, deficits in salience attribution would slow learning of new contingencies while leaving previously learned reward-seeking behaviors, such as drug seeking, intact. Indeed, individuals with CUD exhibit deficits in fundamental behavioral functions following repeated drug exposure that extend to non-drug associated stimuli. These deficits in fundamental behavioral functions negatively affect the lives of individuals suffering from CUD, and the severity of these symptoms is strongly associated with disease progression and treatment outcomes. Thus, to determine the contribution of NAc core dopamine deficits to CUD symptomology, it is critical to first understand the role of NAc core dopamine in basic stimulus processing and learning. Next it will be important to understand how repeated drug exposure dysregulates these basic processes to cause these deficits. In both rodents and humans, long-term cocaine exposure leads to reduced responsiveness of NAc dopamine at baseline and to environmental stimuli. Our overarching framework is that cocaine use increases behavior directed towards drug-associated stimuli, in part, by weakening of the salience of non-drug associated events. We will combine optical tools for recording and manipulating dopamine release in the NAc core of mice during behavioral tasks that dissociate valence from behavioral action and saliency to understand how dopamine drives learning. Next, we will conduct a series of experiments to understand how valence-based and valence-free learning recruits dopamine release to influence behavior. Finally, we will outline how cocaine self-administration dysregulates dopamine responses to non-drug stimuli to drive punishment resistance and deficits in new reward learning. Together these studies will define how cocaine self-administration alters stimulus processing to drive behaviors characteristic of CUD.

NIH Research Projects · FY 2024 · 2021-09

The liver is near-unique among the body organs in its ability to extract glucose and store it as glycogen under postprandial conditions, as well as to maintain a substantial rate of glucose production under fasting or hypoglycemic conditions. Our previous work has clearly shown that a morning period of hyperinsulinemia enhances hepatic glucose uptake and glycogen storage during an afternoon hyperinsulinemic hyperglycemic clamp mimicking a second meal. The goal of our current proposal is to identify factors influencing meal responses that can be utilized in improving the care of those with insulin resistance and type 2 diabetes. Specifically, in Aim 1 we will determine which aspect of morning hyperinsulinemia (direct effects on the liver or indirect effects brought about by insulin’s impact on the brain and/or fat tissues) is the key element involved in enhancement of afternoon hepatic glucose uptake. Furthermore, in Aim 2 we will determine the importance of the route of insulin delivery (intraportal vs peripheral circulation) in the morning on the afternoon response. This question is highly relevant to the treatment of those with insulin-dependent diabetes since most of the currently available insulin therapy involves delivery by a peripheral route. In addition, we will determine what factor or factors in the afternoon response are most clearly impacted by morning hyperinsulinemia (Aim 3). We will utilize the conscious, chronically catheterized dog model which is near-unique in that it not only makes possible the delivery of glucose and insulin into the portal circulation, the usual route of entry for ingested nutrients and secreted insulin, but also permits sampling from the hepatic and portal veins to allow the assessment of the liver’s response to the treatments. Importantly, the canine model has been shown to display many physiologic responses very similar to those in the human. The innovative aspects of the proposed experiments include the fact that the model allows us to clearly separate factors that may impact the response to the initial and subsequent meals of the day and examine the effect of each factor individually. The proposed studies are highly significant because the knowledge gained through these experiments has great potential to contribute to the development of new therapeutic approaches, more accurately tailored to bring about normal nutrient disposition, for those with impaired glucose tolerance (prediabetes) and diabetes.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Mononegavirales is a taxonomic order of viruses, so classified for their negative sense single stranded RNA genome and their pleomorphic membrane-enveloped virions. Mononegavirus life cycles involve a number of different events, including entry of virions into host cells, viral mRNA transcription, genome replication and virus assembly, and viral budding from host cells. These events are carried out through molecular interactions between virus and host cell machinery; elucidating these interactions is key to understanding viral life cycles and identifying potential therapeutic targets. Studies of viral machinery are typically limited to isolated particles or assemblies; this removes them from their native environments and strips away important molecular interactions. To preserve biological context, viral machinery must be studied in situ, i.e. under near-native conditions, such as within intact virions or cells. These environments are a complex, disordered mixture of molecules, making it particularly difficult to obtain molecular resolution information. Here, we propose to study three mononegaviruses: measles, rabies, and Ebola viruses. Each serve as prototypical viruses for their taxonomic families, and each are pathogens important to global health. To carry out our proposed research, we will use and develop in situ structural biology methods. Our primary method will be cryo-electron tomography (cryo-ET), a type of cryo-electron microscopy (cryo-EM) that allows for visualization of three-dimensional volumes. This overcomes the typical cryo-EM requirement of thin monolayers of purified particles, allowing for the acquisition of molecular-resolution information in near-native environments. We will develop data collection and computational methods for cryo-ET to enable rapid, automated data collection, high-resolution structure determination, and accurate molecular identification. We will also use and develop methods complementary to cryo-ET including focused ion-beam milling and correlative light and electron microscopy approaches. Our research will provide novel biological insights three important viruses, but more broadly, it will demonstrate a transformative approach for studying viruses. Rather than trying to tease apart function and interactions through indirect biochemical means, our research will provide an infrastructure to directly observe the virus and host cell machinery with complete biological contexts under near-native conditions.

NIH Research Projects · FY 2024 · 2021-09

Cellular metabolism is the crux of all organismal biology. Therefore, uncovering fundamental knowledge regarding how metabolism is controlled will have far-reaching implications. Metabolic systems are traditionally depicted as linear or circular pathways in textbooks. In reality, these processes are intricately governed by complex, higher-order networks of macromolecules including proteins and lipids. A metabolon is a dynamic cluster of proteins, cofactors, and small molecules that interact to control a metabolic process. Importantly, metabolons are found across multiple biological systems from plants to humans, indicating their fundamental importance in biology. Heme is an essential and conserved biomolecule that is produced by the community of proteins forming the heme metabolon. Heme not only transports oxygen in red blood cells, but it also serves as a catalytic cofactor for proteins governing multiple cellular signaling processes across all kingdoms of life. Thus, determining how proteins assemble and disassemble to control heme metabolon formation will provide insight into production of this critical molecule and also form the basis for studying other key metabolons. Specifically, we will 1) isolate and solve the structure of the heme metabolon, 2) determine dynamics of metabolon formation, and 3) investigate how defects in specific assembly steps alter metabolic output. We will accomplish this by integrating high-resolution cryo-EM with time-resolved proteomics and metabolomics experiments to reveal metabolon dynamics. The combination of these approaches will unite multiple hierarchies of cellular signaling, transforming the static textbook snapshot of metabolism into a 3D movie of a living, breathing metabolic machine. Addressing the fundamental and unknown question of how metabolic networks are controlled via coordinated protein organization will have major impacts in broad areas of research, including cancer progression, diabetes, and the immune response.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Approximately 43 million adults in the U.S. struggle to comprehend basic texts. This is a major public health crisis given the strong association between reading comprehension (RC) ability and broad health and economic outcomes, including more than 300 billion annual economic burden related to low literacy in the US. Despite the prevalence of low RC, meta-analyses of behavioral interventions and national statistics in the US reveal no significant RC gains over the past 30 years. This is due in part to limitations of behavioral approaches to characterize the complex processes involved in RC. Brain research has identified more granular mechanisms of RC/RC ability. This includes my own research that has identified brain pathways that promote behavioral resilience in RC disorders, i.e. brain pathways that mitigate severe symptoms in RC disorders. However, brain research has thus far been unable to apply brain network science for a direct, clinical benefit. The goal of the current proposal is to address the need for brain-based RC interventions by integrating recent breakthroughs in two separate fields: brain network science of RC resilience and non-invasive brain network stimulation. I have established a line of research that uses functional MRI and EEG to characterize brain signatures of RC ability and resilience. I have found that cross-network communication between the reading- language brain network (RLN) and a brain network responsible for goal-oriented thought (the cognitive control network; CCN) is more predictive of RC outcomes than within-network communication (e.g. RLN alone). These results mirror findings in a range of disorders that connect the CCN to resilience and provide a compelling target for brain intervention. In a separate field, brain stimulation has also seen a recent breakthrough: using EEG-guided, individually-tuned stimulation of full networks results in recovered memory capacity in older adults that outlasts stimulation, but this has not been applied to other domains. The proposed project will take advantage of recent advances in RC brain network science and non-invasive brain stimulation to develop a safe, brain-based RC intervention protocol. I hypothesize that promotion of cross-network connectivity will result in increased RC ability particularly in low RC groups. In this project, I will determine: the causal effects of cross- vs within-network stimulation on readers' RC ability and brain metrics (Aim 1); how stimulation outcomes interact with individual differences in baseline RC ability/brain metrics (Aim 2); and the efficacy of stimulation beyond behavioral training effects (Aim 3). N = 225 adults with good and poor RC ability will be tested across 3 visits: a pre-intervention visit for baseline behavior and fMRI/EEG metrics; a stimulation visits (randomized/subject for cross- vs. within-network targets) with behavior and fMRI/EEG metrics, and a post-intervention visit to measure prolonged behavior and fMRI/EEG effects. Before stimulation, subjects will receive a short RC intervention. This approach will allow us to establish the efficacy of cutting-edge stimulation approaches in adult literacy, with potential applications in a range of other disorders.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY Inositol 1,4,5-triphosphate receptors (IP3Rs) integrate diverse signals generated by hormones, growth factors, neurotransmitters, and changes in metabolic state to modulate downstream signaling in all cell types. IP3Rs are ligand-gated ion channels that are further regulated by allosteric and covalent mechanisms, mediating Ca2+ release from the endoplasmic reticulum (ER). The resulting increases of cytoplasmic and mitochondrial Ca2+ concentrations regulate many physiological processes (e.g., learning, memory, membrane trafficking, synaptic transmission, secretion, motility, membrane excitability, gene expression, cell division, and apoptosis). Furthermore, pathological dysregulation of IP3Rs and calcium signaling is implicated in cancer, neurodegenerative, autoimmune, and metabolic diseases, making IP3Rs promising targets for treatment of these diseases. Despite recent advances in structural studies, fundamental questions regarding the mechanisms of ligand interactions and channel gating remain mostly unanswered, in part because of the large size and complexity of IP3Rs and the limited availability of specific pharmacological tools. In this proposal, we will (Aim 1) combine cryo-electron microscopy (cryo-EM) and X-ray crystallography in conjunction with functional IP3R assays based on fluorescence-based calcium imaging to elucidate the general themes of IP3R gating cycle and molecular basis for receptor inhibition by small molecules. Our recently published data revealed that the IP3 binding site is occupied by a loop that we have termed the self-binding peptide (SBP), which is located distantly in the primary sequence. We hypothesize that the SBP is a novel regulatory site in IP3Rs that can modulate the apparent affinity for IP3, and thereby Ca2+ channel activity, and that the divergence of SBP sequences between IP3R subtypes contributes to their distinct regulatory properties. We will perform (Aim 2) functional and structural studies on IP3R subtypes and SBP mutants to test this hypothesis and identify the structural determinants of this interaction. Completion of these aims will yield unparalleled mechanistic insight into IP3R gating and regulation, potentially leading to the development of novel and specific pharmacological modulators of IP3Rs. In addition to being used as a long-sought research tools to study IP3Rs, these compounds will serve as a starting point for development of novel therapeutic approaches to treat diseases associated with aberrant IP3R activity.