University Of Michigan At Ann Arbor

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT With near-ubiquitous adoption of screen media among U.S. families, the average age of screen media exposure is becoming increasingly younger. Most infants are exposed to screen media by 4 months of age and 75-96% of infants are using screen media daily. The scale of screen media adoption underscores a pressing public health need to characterize infant screen media exposures, predictors, and effects. Despite decades of research highlighting the importance of the infants’ role in shaping caregiving behaviors, the contribution of infant characteristics to screen media exposures is greatly under-explored. Indeed, the National Academies of Science has highlighted that more attention needs to be paid to individual differences to screen media effects, given that not all children experience adverse impacts from screen media. Prior work has identified that infants likely have individual differences in their affective and behavioral response to screen media. However, only infant temperament has been explored as a predictor of screen media outcomes. Drawing from a parallel literature of obesity, detailed observational assessments of infant regulatory behaviors in eating contexts are stronger predictors of obesity than temperament alone. More rigorous and nuanced assessment of infants’ attentional, affective, and regulatory responses to screen media is urgently needed to move the science forward. This assessment would create a corpus of observable infant symptoms that predict individual differences to screen media. Therefore, this proposal seeks to test a novel paradigm characterizing infants’ behaviors during screen viewing, to be termed infant affinity toward screens. Specifically, this project will measure an infant’s attraction to screens, changes in visual attention, body movement, affect, and calming when viewing screen-based videos, in addition to negative affect during the transition away from screen media. The study will leverage a diverse existing cohort of infants in a longitudinal study (K23 HD105988) to test these predictors of infant affinity toward screens. This project will also test prospective associations with maladaptive screen media behaviors to identify at-risk infants. Among 100 mother-infant dyads, the aims are to: 1) characterize infant affinity toward screens in a behavioral task at 9 months of age; 2) identify infant temperamental and maternal predictors of infant affinity toward screens; 3) test prospective associations between infant affinity toward screens at 9 months of age and maladaptive screen behaviors at 12 months of age. This proposal will identify clinically relevant infant behaviors that shape screen media outcomes, salient aspects of an infant’s early environment that contribute to infant affinity toward screens, and potential targets for intervention to prevent maladaptive outcomes. In future work, this paradigm could be applied to scale in pediatric clinics, home visitation programs, and parent-child interventions. In summary, careful behavioral phenotyping of infant affinity toward screens has high translational impact to identify at-risk infants and precisely inform future interventions that shape healthy trajectories of screen media use.

NIH Research Projects · FY 2025 · 2025-08

Undesired immune responses are central to many chronic diseases, with these immune responses either responsible for disease and its progression (e.g., cancer) or limiting therapies (e.g., transplant rejection). However, a significant challenge with these chronic diseases is that they develop over time and often go undetected until the disease has reached a substantial burden such as tissue or organ failure, after which therapeutic efficacy is limited. We propose a living sentinel based on an injectable material for i) monitoring of the immune system to identify disease at the earliest stages and ii) monitoring of the response to immunotherapy. Type 1 diabetes mellitus (T1D) will be used as a model system, as patients, typically children and young adults, present acutely ill due to symptoms of hyperglycemia (polydipsia, polyuria, nocturia, and weight loss) or diabetic ketoacidosis, which occurs because of destruction of the insulin-producing -cells of the pancreatic islets. The system is based on employing an injectable scaffold that functions as an “immunological niche" (IN) to which immune cells home and contains many of the cell types found in the diseased tissue during progression, including neutrophils, inflammatory monocytes, macrophages, B, and T cells. INs have been used to monitor these cell populations during disease progression in cancer, and herein we are proposing to develop them for T1D. We propose that the IN can be used for longitudinal surveillance of systemic immune responses and can monitor for the initiation of diabetes and the response to immunotherapy. Teplizumab has emerged as an immunotherapy with the potential to delay T1D onset if applied at the appropriate time, and emerging immunotherapies such as those by the industry partner Cour Pharmaceutical can transform diabetes treatment. The proposed studies are captured in the following aims. Aim 1 will develop the design parameters for an injectable IN to non-invasively monitor alterations in immune responses in the onset of T1D. The injectable IN allows for easy delivery, as well as analysis of cells with a fine needle aspirate (FNA). We will analyze the gene expression dynamics at the IN, with comparison to the native pancreas, and will develop a gene expression signature from the IN to predict disease onset and progression. The IN will also be analyzed histologically comparison to the pancreas. Aim 2 will employ injectable INs modified with disease antigens to monitor response to immunotherapy and for surveillance of recurrence. INs are modified with disease antigens will enable the analysis of disease specific T cells. These studies will employ teplizumab and nanoparticles, that latter being developed for clinical trials in autoimmune disease. The assembled research team has expertise in biomaterials, T1D, and immunology that will contribute to the success of the project. This system offers the opportunity to treat autoimmune disease prior to tissue destruction and potentially avoid the lifelong mangament of disease that is often associated with severe macrovascular and microvascular complications (e.g., coronary artery disease, stroke, retinopathy, neuropathy, nephropathy).

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Current migraine management lacks objective tools for predicting imminent migraine attacks, which could enable preemptive treatment and improved patient outcomes. This project aims to establish predictive measures of migraine onset through continuous monitoring of autonomic activity using wrist-worn devices in individuals with episodic migraine. Approximately 30-40% of migraine sufferers experience prodromal symptoms, yet the mechanisms and temporal dynamics of these symptoms are poorly understood. Advances in wearable sensor technology provide an opportunity to monitor physiological parameters correlated with these symptoms continuously. We will exploit these advancements to test specific hypotheses through the following aims: Aim 1: Identify predictive markers of migraine onset by analyzing continuous physiological data (e.g., skin conductance, blood volume pulse, motion, skin temperature) from 60 patients using wrist-worn sensors. Aim 2: Differentiate objective versus subjective evaluative measures to enhance prediction accuracy of migraine onset. We hypothesize that autonomic changes will be detectable hours before an attack and that combined objective and subjective data will yield robust predictive markers. The methodology includes noninvasive, continuous data collection and transmission, complemented by self-reported data on prodromal symptoms and migraine events via validated e-diaries. Utilizing machine learning, we will develop algorithms to forecast migraine onset, enhancing the timing and efficacy of prophylactic treatments. This research leverages existing technology validated for clinical use and promises to elucidate the pathophysiology of migraine prodromes, offering transformative potential for migraine management.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Epilepsy is characterized by recurrent seizures, but its associated comorbidities – including attention deficits, mood disorders, cognitive impairment, and sleep disorders – are prevalent and often highly detrimental to quality of life. Current treatments do not specifically prevent or reverse these epilepsy-associated comorbidities, and progress in developing targeted therapies has been hampered by an incomplete mechanistic understanding of how seizures remodel relevant networks. Cholinergic neurons, located in discrete nuclei within the brainstem and basal forebrain, play critical roles in regulating attention, mood, cognition, and sleep through widespread projections across the central nervous system. Case reports and preliminary data suggest altered cholinergic function in epilepsy, but a comprehensive understanding of these changes remains lacking. This proposal aims to mechanistically link acute seizures with chronic alterations in cholinergic networks, thereby identifying targets for next-generation therapeutics. Specifically, our primary objective is to delineate seizure-associated pathology in the cholinergic pedunculopontine nucleus (PPN) utilizing a mouse model (Scn1a+/-) of Dravet Syndrome, a severe developmental and epileptic encephalopathy. Our central hypothesis is that overactivation of cholinergic neurons by seizures chronically alters ascending cholinergic network function due to a downregulation of acetylcholine expression. In Aim 1, we will establish the causal role of neuronal activity in cholinergic neuron loss. We will test whether seizure-induced hyperactivation is necessary and sufficient to reduce cholinergic neuron numbers in the PPN by inhibiting these neurons during seizures in Scn1a+/- mice and activating them exogenously in wild-type mice. In Aim 2, we will identify seizure-induced functional changes in cholinergic PPN circuitry by quantifying downstream cholinergic neurotransmission and sleep architecture integrity. We will test these measures of PPN circuit functionality both across genotypes (Scn1a+/- versus wild-type littermate control mice) and within animals (before versus after repeated seizure induction). In Aim 3, we will test whether the observed decrease in cholinergic neuron number in Scn1a+/- mice results from an activity-dependent phenotypic switch from cholinergic to GABAergic expression. The results from these aims are expected to demonstrate that seizures induce an activity-dependent reduction in cholinergic neurons, impair cholinergic network activity, and alter neurotransmitter expression patterns in Scn1a+/- mice. These findings will identify vulnerable neuronal populations and circuits as targets for therapeutic intervention, potentially leading to novel strategies to protect against debilitating comorbidities in patients with epilepsy.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Glioblastomas (GBMs) are deadly incurable brain cancers that are treated with resection followed by radiation, which is often combined with chemotherapy. GBMs rewire their metabolism to fuel their growth, invasion, survival and treatment resistance. Therapeutic strategies to block altered GBM metabolism are being translated into the clinic, however we do not know which metabolic strategy is optimal for the treatment of GBM patients. Further, we lack methods to directly measure metabolic activity in human brain cancers, which makes it difficult to determine which patients are likely to respond to which metabolic treatment strategy. In this research proposal, we will develop new methods to directly measure metabolic pathway activity in mouse models and patients with GBM. We will then perform a therapeutic clinical trial to evaluate the safety, on-target activity, and potential efficacy of a new metabolic strategy for the treatment of GBM.

NIH Research Projects · FY 2026 · 2025-08

Hearing loss is a leading source of disability with 15% of U.S. adults (37.5 million people) suffering from hearing loss which predicts increased depression, social isolation, loneliness, increased risk of falls, cognitive decline, and dementia. Hearing aids are an effective treatment associated with improved quality of life, less loneliness, better relationship quality, and reductions in cognitive decline. Yet only 25% of adults with hearing loss have ever used hearing aids and these patterns are worse among older adults. Up to 65% of those 71 and older have hearing loss and less than a third report using hearing aids. The lack of insurance coverage and the high cost contribute to lack of hearing aid ownership, but hearing aid ownership is low even among individuals with hearing aid coverage. Many individuals who own hearing aids use them sporadically or not at all. There is an urgent need to understand the predictors of hearing aid use in daily life among individuals who own hearing aids. The scientific premise of this study is that social ties are key to both use and benefits of hearing aids. This project is guided by our newly developed Hearing Aid Use in a Social Context Framework, which suggests that individuals will wear their hearing aids more frequently when they have larger social networks and their social partners use positive rather than punitive social control efforts. Further, we predict that verbal communication when wearing hearing aids will be more cognitively complex, involve greater comprehension, and greater positive emotion all of which will lead to better social ties over time. The purpose of the proposed NIH stage 0 (i.e., basic science observational research) longitudinal study is to understand links between social networks, daily interpersonal processes (i.e., daily social interactions), and daily hearing aid use among older adults experiencing hearing loss who own hearing aids. We aim to enroll 300 older adults (65+) with bilateral moderate, moderate-severe, or severe age-related hearing loss (from 1000 to 4000 Hz in at least some of their audiogram) who obtained prescribed hearing aids in the last 3 to 6 months. The design includes a baseline interview regarding social networks, a 7-day ecological momentary assessment (EMA) study with mobile phone surveys every three hours regarding hearing aid use and social interactions, and a mobile app that records daily verbal communication (coded for complexity, comprehension, and emotion), along with12 subsequent monthly surveys regarding hearing aid use and relationships This study will address three aims: 1. Identify implications of social networks and daily social interactions for the frequency of hearing aid use in daily life. 2. Examine effects of hearing aid use in daily life for conversation. 3. Assess the consequences of hearing aid use reported in daily life and daily conversation experiences for social ties over time. Understanding how social ties predict hearing aid use in daily life and the long-term implications of daily hearing aid use for social ties will lead to actionable recommendations for interventions aimed at improving hearing aid use among older adults.

NIH Research Projects · FY 2026 · 2025-08

Project Abstract An estimated 6.7 million US adults live with Alzheimer’s disease (AD) and related dementias (ADRD); a figure that is expected to more than double by 2060 unless new prevention strategies or treatments emerge. Although later-life neuropsychiatric symptoms have been associated with a greater risk of dementia, they are all too often viewed and treated as independent of ADRD. This traditional approach overlooks the possibility that neuropsychiatric symptoms emerge due to AD/ADRD pathology build-up that drives structural changes in brain regions or networks that differ from those affected by the more common “memory first” phenotype. Thus, the proposed project leverages data from the National Alzheimer’s Coordinating Center (NACC) to evaluate a recently developed mild behavioral impairment (MBI) diagnostic framework that standardizes the assessment of neuropsychiatric symptoms in older adults during the pre-dementia phase(s). While MBI is typically linked with regional atrophy of the medial temporal lobes, this is likely an overly simplistic view. Thus, our central hypothesis is that MBI symptoms in early-stage clinical phenotypes (i.e., cognitively unimpaired and MCI) are associated with structural changes within brain networks implicated in the processing and integration of emotional experience--i.e., the salience and default-mode networks. To test this, we will define the network-level structural alterations linked to MBI in early clinical phenotypes (Aim 1) and quantify the predictive value of MBI-related structural network changes for dementia conversion (Aim 2). This is the first study to conduct a network-level analysis of the structural alterations associated with MBI in a nationally representative cohort and link longitudinal trajectories of network-level structural changes in those with MBI with the risk of conversion to dementia. Clarifying the neural correlates of MBI and their predictive value for dementia conversion may help with early detection of “at-risk” individuals and understanding of disease progression. These findings will pave the way for future studies integrating AD biomarkers and inform the development of targeted interventions for specific brain regions implicated in MBI. The current project aligns well with the NIA SCAN initiative to harmonize MRI data across AD Research Centers and will strengthen my plan to pursue a K award focusing on individualized treatments for MBI.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Morphologically and functionally appropriate limb development is critical for quality of life. There is compelling evidence that growth factors, including Bone Morphogenetic Proteins (BMPs), regulate limb development. However, the mechanism of how growth factor signaling is involved in early patterning of mesenchymal condensation and subsequent cell fate specification in mesenchymal stem/progenitor cell population remains unclear. We recently found a formation of massive ectopic cartilage in the craniofacial region of a mutant mouse line engineered to augment BMP-Smad signaling in a neural crest-specific manner. During preliminary characterization, we made three interesting findings: 1. augmented BMP signaling in cranial neural crest cells (NCCs) results in an increase of Xist expression, a non-coding RNA critical for X chromosome inactivation, (XCI) leading to ectopic XCI in cranial NCC-specific manner, 2. the ectopic XCI is functionally essential for ectopic cartilage formation, and 3. transient upregulation of Xist expression and ectopic XCI are found in wildtype limb development. Based on these experimental data, we hypothesize that transient ectopic XCI is an unappreciated unique mechanism essential for limb development during early mesenchymal condensation and subsequent skeletogenesis. We will fully utilize mutant mouse lines generated in our laboratories and ones from our collaborators, and unbiased bioinformatics approaches to test the hypothesis. In Aim 1, we will uncover the landscape of how differentially suppressed genes by ectopic XCI in a spatial-temporal manner. In Aim 2, we will identify the functions of ectopic XCI during limb development. Our preliminary data using limb bud ex vivo culture suggest that ectopic XCI is involved in early mesenchymal patterning. Our unique experimental tools and approaches will allow us to determine the role of BMP signaling in mesenchymal patterning and subsequent cell fate specification in limb development. Completing the proposed study will generate a novel knowledge informative to understand unique mechanisms of limb patterning and thus provide new insight for better treatment options for congenital limb abnormalities and directions toward limb regeneration.

NIH Research Projects · FY 2025 · 2025-08

Vascular cognitive impairment and dementia (VCID) after stroke increases risk for death and is highly disabling. New-onset VCID after stroke is common, affecting as many as one-third of stroke survivors. There are currently no treatments recommended to reduce VCID risk. Interventions to reduce risk of incident VCID after stroke are critically needed. One simple, inexpensive, accessible intervention that holds promise for VCID prevention among stroke patients is cognitively stimulating activities (such as reading, doing crossword puzzles, and word games). The performance of cognitively stimulating activities has been associated with lower risk of dementia in stroke-free adults. Whether the performance of cognitively stimulating activities after stroke reduces risk for VCID after stroke has not been elucidated. Other opportunities for practical interventions to prevent VCID may be available in the treatment of sleep disorders. Obstructive sleep apnea (OSA) is prevalent after stroke (~70% of stroke survivors) and disrupted sleep (more time spent in wakefulness during periods of attempted sleep) is more common after stroke than in stroke-free adults. Both OSA and disrupted sleep have been associated with cognitive decline in stroke-free adults, but their contribution to post- stroke VCID remains unclear. If these sleep disorders are similarly associated with VCID among stroke patients, other novel targets for VCID may become available. Currently, performance of cognitively stimulating activities and screening and treatment for sleep disorders are not standard of care. In this project, we will: 1) pool and harmonize longitudinal cohorts with repeated measures of cognition over time to quantify the association between the performance of cognitively stimulating activities after stroke and post-stroke cognitive decline and incident VCID. We will also leverage an established cohort of stroke participants with baseline post-stroke polysomnography measures to: 2) evaluate the association between post-stroke OSA, assessed by the apnea-hypopnea index and a threshold of hypoxemia, and post-stroke cognitive decline and incident VCID over 2 years post-stroke, and 3) evaluate the association between measures of disrupted sleep, assessed by wake after sleep onset and sleep efficiency, and post-stroke cognitive decline and incident VCID over 2 years post-stroke. This project is innovative because it will leverage and pool longitudinal cohorts with repeated measurements of cognition and cognitively stimulating activities. This project is significant because it is a step towards identifying a possible simple intervention (cognitively stimulating activities) and novel intervention targets (OSA and disrupted sleep) for post-stroke VCID.

NIH Research Projects · FY 2026 · 2025-08

Project Summary Biomedical research relies on complex, evolving, multidisciplinary collaborations driven by researchers who spend entire careers working at the moving frontiers of knowledge. The resulting innovations contribute dramatically to human health, wealth and well-being. But increasing evidence suggests that marquee successes are becoming more difficult and costly to achieve, a phenomenon commonly attributed to substantial increases in the scale, scope, velocity and competitiveness of science over several decades. This project (1) develops flexible computational, data and network science tools (2) integrated by a social scientific framework to (3) analyze discovery and innovation dynamics and (4) improve individual and collective capacities to identify and realize solutions for even wildly challenging problems. This use-inspired approach is applicable across science, technology, engineering and mathematics (STEM) fields. Findings will thus contribute to knowledge in network science, science of science, sociology and economics of science and innovation and related fields while simultaneously offering new possibilities for application to the long, complex, uncertain processes that characterize biomedical research and development (R&D). By employing advanced techniques from network science, information theory, and machine learning, we aim to identify and predict emerging opportunities for groundbreaking research—termed 'adjacent possibles'—and accelerate transformative breakthroughs in biomedical fields and in science policies designed to improve human health through research, innovation, and translation. We examine the evolving social and conceptual landscape of research and its implications for innovative possibility, the knowledge and collaborative dynamics of teams, their capacity to achieve novel research aims and the sources and trajectories that yield long-term high impact disruptive findings. Results will increase understanding of how biomedical R&D can more effectively harness diverse knowledge to achieve disruptive scientific outcomes in critical areas such as mental health, addiction, and aging-related diseases. Project outcomes will inform strategies to foster transformative biomedical research, identify and enhance trajectories for high-impact translation, and thus contribute to human health. The resulting models and tools will increase capabilities for identifying emerging research areas and assessing risk in research to help inform policy, funder and investigator decision-making. This project directly addresses the National Library of Medicine's call for innovative approaches to advance biomedical informatics and data science, potentially revolutionizing our understanding of scientific innovation dynamics and informing evidence-based science policy decisions.

NIH Research Projects · FY 2025 · 2025-08

Abstract The protein-protein interaction involving menin and MLL1 (Mixed Lineage Leukemia 1, also known as KMT2A) plays a critical role in acute leukemias with upregulated HOX genes, including leukemias with translocations of the MLL1 gene, mutations in nucleophosmin (NPM1) gene or NUP98 rearrangements. Patients with these leukemia sub-types have very poor prognosis (~35% five-year survival), emphasizing the need for new therapies. Our group has developed the first-in-class menin-MLL1 inhibitors, which directly bind to menin at the MLL1 binding site and strongly inhibit this protein-protein interaction, abrogating leukemia progression in advanced pre-clinical models of high HOX Acute Myeloid Leukemia (AML). Our pioneering efforts in this field led to clinical translation of menin inhibitors that are currently under clinical evaluation in AML patients. However, clinical studies with the menin inhibitor Revumenib revealed somatic mutations M327I/V, T349M, G331D/R (which we called the MTG mutations) in the MEN1 gene (encoding menin) in ~39% of AML patients, leading to clinical resistance. These acquired mutations occur in the inhibitor binding site, resulting in reduced drug binding to menin and leukemia relapse. Since all menin inhibitors in clinical evaluation bind to the same binding site where the mutations occur, they are not effective against the menin patient mutants. Thus, a new generation of menin inhibitors is required to overcome resistance and provide an effective treatment for AML patients. In this project, we propose to develop the next generation of menin inhibitors with high potency (sub-nanomolar) against both menin WT and menin patient mutants to overcome resistance observed for the current menin inhibitors. To accomplish this goal, we developed a new class of very potent menin inhibitors with similar activity against menin WT and the most frequent menin mutants found in patients. These compounds demonstrate strong activity in leukemia cells and mouse xenograft leukemia models harboring the MTG patient mutations in menin. Here, we will apply highly interdisciplinary approach involving medicinal chemistry, structure-based design, pharmacokinetic (PK) studies and biological studies to develop highly optimized compounds effectively blocking menin WT and all (or at least majority) of menin patient mutants. Then, we will assess the efficacy of these compounds in xenografts and PDX models of high HOX leukemia with menin WT and mutants. We expect this study will result in the next generation of menin inhibitors that will overcome resistance resulting from menin patient mutations preventing leukemia relapse.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Zero effective options exist for treating individuals who are chronically infected with the protozoan parasite Toxoplasma gondii (Tg) as a source of reactivated toxoplasmosis. Reactivated Tg infection in the retina is a leading cause of infectious posterior uveitis, impairing the vision of approximately 3,600 Americans annually. Some individuals experience multiple episodes of reactivated ocular toxoplasmosis, with each bout further eroding their vision. Eliminating Tg tissue cysts could preclude reactivated ocular toxoplasmosis and preserve the vision of at-risk individuals. We have identified a Tg lysosomal protease (TgCPL) that is critical for chronic Tg infection, developed several promising TgCPL-targeted lead compounds based on multiple scaffolds, and established a mouse intravitreal ocular treatment model. Our long-term goal is to provide a safe and effective treatment that eliminates chronic Tg infection from the retina after 2-week administration of a TgCPL inhibitor. As a critical step toward this goal, the proposed studies will: (1) develop a computational structure-to-function model of TgCPL to guide rationale discovery of novel ligands; (2) optimize the potency, pharmacokinetics, and selectivity of our lead TgCPL inhibitors, including derived from novel ligands identified in aim 1; and (3) eradicate chronic Tg infection in a mouse intravitreal ocular treatment model. Completing the proposed studies will result in one or more advanced lead compounds suitable for further progression through the development pipeline, thereby progressing toward a key unmet need for curbing chronic Tg infection. 1

NIH Research Projects · FY 2025 · 2025-08

Abstract Arrays of large, nearly identical, palindromic DNA sequences in mammals are rare in the genome. What benefit large palindrome arrays have to the genome, or an organism remains unclear. A few studies of large palindrome arrays on the mouse X and Y chromosomes reveal they can result in selfish chromosome transmission and be necessary for male fertility. Due to the large megabase size, high levels of nucleotide identity between palindrome arms, and multicopy nature, large palindrome arrays have been challenging to resolve and study experimentally. Moreover, our understanding of large palindrome arrays on autosomes is largely nonexistent. Here, we propose to overcome these barriers by using recent haplotype-resolved genome assemblies to identify, genome-wide, and comprehensively characterize seven of the 14 large palindrome arrays in mice. We hypothesize large palindrome arrays evolve to favor their own inheritance and are necessary for fertility. We will test our hypothesis via two aims. Aim 1 will determine whether two autosomal large palindrome arrays contribute to selfish chromosome transmission when heterozygous and are necessary for fertility when homozygous. Aim 2 will determine whether the five large palindrome arrays on the X chromosome cooperate or compete to influence selfish chromosome transmission or fertility. The simultaneous characterization of large palindrome arrays on autosomes and the sex chromosomes can provide a unifying view of palindrome arrays in selfish chromosome transmission and necessity in fertility. Our studies will enable new studies of these largely overlooked genomic regions and their associated genes in all species, including humans. Our research efforts will provide the foundation for mechanistic insights into their associated genes’ roles in influencing fertility, chromosome evolution, and speciation.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Spinocerebellar ataxia type 3 (SCA3) is a debilitating neurodegenerative disorder characterized by impaired balance and coordination, and it currently has no effective treatment. SCA3 is the most prevalent dominantly inherited ataxia and is caused by a CAG repeat expansion in the ATXN3 gene. Although ATXN3 is expressed throughout the body, its’ aggregates cause dysfunction in specific cell types, including neurons and glia, in vulnerable central nervous system regions. Previous research suggests that neurons play a crucial role in driving SCA3, as overexpression of mutant ATXN3 specifically in neurons is sufficient to cause ataxic symptoms in a mouse model. However, it is still unknown whether neuronal mutant ATXN3 expression is necessary for SCA3, which could be key for therapeutic development. To investigate this, our lab created a novel conditional “off” knock-in Atxn3cQ300/Q6 mouse model that allows for the selective silencing of mutant ATXN3 in specific cell types. Validation of this model in the absence of Cre expression, shows they exhibit the well-established SCA3 pathophysiological features of disease such as progressive motor impairment, nuclear ATXN3 accumulation, and significant pathological and transcriptional dysregulation in neurons, oligodendrocytes, astrocytes, and microglia. By breeding this model with mice expressing neuron-specific Cre, we will determine whether neuronal expression of mutant ATXN3 is necessary for the motor deficits and pathological signatures in neurons and glia. These studies are expected to provide critical insights into the cellular mechanisms of SCA3, which could have a direct impact on therapeutic development. Additionally, this project will provide me with extensive experience and training in experimental design, bioinformatic data analysis, and scientific communication, which will prepare me for a career as an independent scientific investigator focusing on neuron-glia interactions in neurodegeneration.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY: Rabbits offer great potential as translationally relevant models of human disease for many biomedical research applications thanks to their evolutionarily closeness to humans, favorable reproduction features, relatively long lifespan which allows for clinically relevant studies, a body size that is similar to that of human infants allowing for more relevant surgical models, and an economically feasible housing cost in a research setting. Rabbit models have been critical to diverse scientific breakthroughs, such as the development of in vitro fertilization techniques, the HPV vaccine, and a multitude of antibodies for various scientific applications. Over the last 30 years, rabbits have been surpassed by mice in part due to ease of generating genetically engineered mice from embryonic stem cells (ESCs), which are not available in rabbits. CRISPR/Cas9 has made it possible to generate gene edited (GE) rabbit models analogous to those in mice for use in research. Other GE large animal models including pigs and non-human primates have also been generated, but the extended generation times, cost of housing, and high level of technical care required greatly limit how widely they can be utilized. GE rabbits overcome many of these barriers and allow for the development and expansion colonies in limited space and in a short time frame, making them a large animal that is practical for wider adoption. More than 50 GE rabbit models have been published thus far highlighting their usefulness, yet the cost of generating these models can be significantly higher than mice and rats. We have developed a genome editing pipeline coupled with an improved rabbit reference genome for efficient generation of GE rabbits. We have generated more than 40 different translationally relevant GE rabbit lines, including whole body knockouts, humanization of disease associated alleles, and targeted knock-ins of proteins of interest. Our lab has expansive expertise in characterizing these GE rabbits to study human disease and have demonstrated that for multiple diseases rabbits are superior to rodents due to increased translational relevance. However, rabbit models have increased costs and require advanced technical skills to generate which has limited the overall number of GE rabbits generated and distribution of models. A centralized resource to produce and distribution of GE rabbits would help overcome this limitation and increase the implementation of rabbits in biomedical research. The overall mission of this grant is to leverage our expertise to create the National Center of Rabbit Models for Translational Research (NCRMTR) and expand access to GE rabbit models across the United States for biomedical research. We propose to generate rabbit models with direct translational relevance to human diseases including cardiovascular, ocular, neurologic, immunology and infectious diseases. This proposed centralized NIH resource center would improve the generation of translationally relevant GE rabbits. We also aim to increase the dissemination and maintenance of these rabbit models to the wider research community.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Spinocerebellar ataxia type 3 (SCA3) is the most common dominantly inherited ataxia and currently has no effective treatment. This progressive, multisystem disorder is typified by the degeneration of critical parts of the central nervous system including the cerebellum, brainstem, basal ganglia, and spinal cord. Moreover, neuropathological studies show widespread involvement of the peripheral nervous system (PNS). Despite the focus of past research on the central nervous system, peripheral neuropathy symptoms are prominently observed in over half of SCA3 patients. This research aims to deeply understand SCA3 disease, particularly exploring its impact on the PNS and its connection to peripheral neuropathy. Our preliminary data in two SCA3 mouse models confirm that both exhibit peripheral neuropathy that mirrors human features. Postmortem tissue analysis and preliminary SCA3 mouse studies point towards the dysregulation of selective subtypes of dorsal root ganglion sensory neurons and demyelination as potential instigators of peripheral neuropathy onset in SCA3 patients. This research employs SCA3 mouse models to analyze peripheral neuropathy onset and progression through functional, histological, and transcriptional studies. Ultimately, our research aims to illuminate a cell-type specific mechanistic understanding of SCA3 peripheral neuropathy, paving the way for potential therapeutic interventions in this crucial area that might have implications for other multisystem neurodegenerative disorders.

NIH Research Projects · FY 2025 · 2025-08

Characterizing Cancer Associated Fibroblast Evolution, Programming, and Fibroblast-Epithelial Cell Crosstalk in Pancreatic Ductal Adenocarcinoma Pancreatic adenocarcinoma (PDAC) remains one of the deadliest cancer diagnoses in the United States with an abysmal overall 5-year survival rate of less than 15%. Unlike many other solid tumors such as breast or colorectal cancers, treatment of pancreatic cancer is not dictated by molecular subtypes of disease. A major barrier to developing precision-based treatments for pancreatic cancer has been the pancreatic tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) comprise >80% of the TME and can either be tumor-promoting or tumor-suppressive in a highly context- dependent manner. There is a critical unmet need to characterize CAF development and polarization in PDAC to help develop effective treatments. In this proposal, we will test the central hypothesis that CAFs co-evolve with epithelial cells during tumor progression, and that the molecular subtype of PDAC can dictate stromal CAF composition. This hypothesis will be investigated through the following specific aims: (1) Characterize the evolution of fibroblasts from normal pancreatic tissue to adenocarcinoma. (2) Dissect the effects of fibroblast polarization by PDAC and assess subtype specific programming. Together, this work will generate a temporal map of CAF evolution and can help clarify subtype-specific TME features that can pave the way for new therapies.

NIH Research Projects · FY 2025 · 2025-08

Meaningful impact across the continuum of cancer control requires an array of evidence-based interventions that address individuals’ changing needs, circumstances, and strengths. This can be achieved via Adaptive Interventions—evidence-based protocols that guide how information about an individual can be used in practice to decide whether and how to deliver intervention-related services. Recent years have seen explosive growth in research to develop adaptive interventions in cancer control, including cancer risk reduction, treatment, survivorship, and palliative care. This growth was powered by the rapid development of randomized trial designs used to answer important scientific questions about how to best construct adaptive interventions. These novel designs, which were developed by our team, include the sequential multiple assignment randomized trial (SMART) and the micro-randomized trial (MRT). Among new and established cancer control scientists alike, there is overwhelming and growing demand for new methodological skills for constructing adaptive interventions. Existing training programs are not meeting this demand. A recent NCI workshop1 to increase rapid cycle intervention research in cancer care delivery, as well as a systematic analysis of NCI-funded research grants on the topic,2 called for greater adoption of experimental designs for optimizing adaptive interventions such as SMARTs and MRTs, and identified a need for more methodological training in this area. A review of the 2024 scientific program for the American Society of Preventive Oncology Annual Meeting3 identified 17 out of 20 scientific program sessions that included at least one presentation related to personalized behavioral interventions. Yet no didactic training sessions were offered on adaptive interventions at this well-attended meeting. This project will build nationwide investigator capacity in methods for constructing adaptive interventions in cancer control by developing the widest-reaching and most comprehensive training program of its kind. Specifically, we will: (Aim 1) develop an online short course that introduces different types of adaptive interventions for cancer control and research methods for developing them; (Aim 2) Provide opportunities for scientists who have completed the short course to gain the deeper conceptual and practical expertise necessary to obtain NIH funding and apply the methods successfully in cancer control; and (Aim 3) catalyze independent research programs in adaptive interventions for cancer control. Over five years, this program will reach 150 learners in-person and far more online via the free online course and web-based materials. We will increase the number of scientists proficient in and funded for research to develop adaptive interventions in cancer control. The training program will support an optimized cancer control scientific workforce, that will test interventions to prevent and detect cancer early, deliver optimal care, and enhance engagement.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Dental pulp necrosis, whether caused by tooth decay or injury, can progress to apical periodontitis (AP) if left untreated. AP is a common condition characterized by inflammation of the tissues surrounding the tooth roots and bone destruction, affecting about 52% of the global population. In the United States, this condition leads to at least 15 million root canal procedures annually for treatment. Suppose the tooth is a mature permanent tooth with a closed apex. In that case, treatment typically involves orthograde root canal therapy by mechanical instrumentation, and the use of outdated and toxic chemical agents, such as sodium hypochlorite. In contrast, when pulpal necrosis occurs in immature permanent teeth in children, the death of odontoblasts disrupts normal root development. Treatment options in such cases include apexification or regenerative endodontic procedures (REP). However, these approaches often involve harmful substances for root canal cleaning and antibiotic combinations in pastes that are highly detrimental to mesenchymal stem cells, ultimately compromising the tooth's regenerative potential. Thus, there is a pressing need to develop clinical therapies that can predictably resolve AP in permanent teeth, regardless of their developmental stage. To address this issue, I will split the effort into three specific aims. First, I will identify a non-toxic synthetic high- density lipoprotein (sHDL)-antibiotic-loaded formulation with enhanced antimicrobial and immunomodulatory properties (Aim 1). Then, I will identify the mechanisms and pathways these nanoparticles activate when treating necrotic mature permanent teeth with AP in rats (Aim 2), and I will determine their ability to promote pulp and dentin regeneration and root formation in rats’ partial or fully necrotic immature permanent teeth (Aim 3). A highly experienced advisory team has been assembled to guide the candidate. Dr. Marco Bottino is the primary mentor, offering valuable support throughout the training, scientific plan, academic job search, and career transition. The advisory team also includes Dr. Anna Schwendeman, an expert in the manufacturing of potent and safe sHDL nanomedicines; Dr. Joshua D. Welch, a leading expert in computational methods related to high-throughput sequencing technologies; and Dr. Hajime Sasaki, a world-renowned scientist specializing in inflammation related to apical and marginal periodontitis, who serves as an advisor/consultant. The candidate, a dentist-scientist, is currently a postdoctoral research fellow at the University of Michigan School of Dentistry. This proposal outlines a comprehensive mentorship and training plan to enhance the candidate’s expertise in lipid nanoparticle manufacturing, high-throughput transcriptomics, and bioinformatics.

NIH Research Projects · FY 2026 · 2025-08

Project Summary / Abstract Bone age (BA) is a measure of maturation of a child’s skeleton, and, as such, a key clinical indicator of growth used by pediatricians and pediatric endocrinologists. As a person’s body ages, from birth through childhood, puberty, and adulthood, the size and shape of the bones of the skeleton change. Growth plates, initially wide open, fuse progressively in childhood. The BA is meant to be the “average” age at which the skeleton reaches a certain degree of maturation. In combination with other measures, it can be used to predict future adult height and detect possible growth disorders or abnormal pubertal maturation. The estimation of BA with a radiological image of the left hand and wrist describes the degree of maturation of a child’s skeleton. The most commonly available standards for the BA estimation, such as the Greulich and Pyle (G&P) Atlas (1959) and the Tanner-Whitehouse method, involve visual inspection of X-ray images of the person’s left hand and wrist, followed by its comparison with the set of reference images. This manual inspection is not only time- consuming, but subjective, and the estimation among radiologists may vary depending upon experience / expertise. Moreover, the data collected in these approaches is outdated: the current population of the United States has been much reshaped in the last 60 years, due to a larger number of children of international ancestry and a nutritional environment, particularly during the COVID pandemic, that has resulted in an obesity pandemic that affects the growth and rate of physical maturation of children. Thus, there is a need to renovate these bone age standards such that the reference is representative of the current population and develop an AI-assisted system for the accurate prediction of bone age. Moreover, the research team for the proposed project will develop an adjustment factor to incorporate the BMI Z-score for more clinical relevance of the AI- assisted outcome. The proposed technical approach is three-pronged. Specific Aim 1 is to establish a database for BA assessment in children that addresses racial and ethnic disparities and reduces spacing between available standards. Specific Aim 2 is to develop and validate an AI-assisted classification system for BA readings from the X-ray images. Finally, Specific Aim 3 is to enhance BA determination by an adjustment factor reflecting the impact of BMI Z-score on skeletal maturation. In support of all three aims, a web designer will build and deploy a web application capable of incorporating the AI algorithm for the adjusted Bone Age determination with qualitative data provided by users.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT The cerebral cortex – the brain’s center for conscious perceptions, thoughts, and actions – is supported by a myriad of cells distinct identities. These identities are established during cortical development in a temporally organized manner: neural progenitor cells (NPCs) first give rise to deep layer neurons, then to upper layer neurons, and finally transition into gliogenesis to produce astrocytes. To generate the correct type of cell at the correct time, NPCs must keep track of where they are in the sequence. However, the mechanisms that directs when NPCs transition from one production mode to another during sequential neurogenesis remain incompletely understood. Some groups have proposed the Polycomb Repressive Complex 2 (PRC2) as a potential mechanism. PRC2 is thought to be involved in the mitotic inheritance of the histone post-translational modification (PTM) H3K27me3, which is associated with transcriptional repression. In fact, when essential PRC2 subunit Eed was deleted at the onset of neurogenesis, NPCs bypassed generation of late-born upper-layer neurons and prematurely transitioned to astrocyte generation. To directly test if PRC2 is the mechanism that transitions NPCs through the neurogenesis sequence, we first conditionally deleted Eed at the onset of neurogenesis, then re-expressed it by in utero electroporation (IUE) at E15.5 – 5 days after conditional deletion of Eed – when upper layer neurons are typically generated. Remarkably, re-expression of Eed in NPCs enabled them to correctly generate upper-layer neurons appropriate for E15.5, effectively rescuing cKO phenotypes. This surprising finding supported the possibility that temporal information successfully progressed in NPCs over the 5 days during which H3K27me3 deposition was absent. These exciting preliminary data form the basis of my hypothesis that PRC2 is required for interpreting the correct stage of neurogenesis, but NPC temporal progression is mediated by non-PRC2 mechanisms. Here, I aim to investigate this hypothesis by: 1) mechanistically separating the roles of PRC2 in transcriptional repression from its functions in histone PTM inheritance across NPC divisions; and 2) identifying non-PRC2 transcriptional mechanisms and histone PTMs that can progress the neurogenic sequence. Together, the proposed work will delineate the roles of PRC2 in neurogenesis, deliver developmentally-staged NPC-specific transcriptomic and functional genomic data, and identify candidate mechanisms of sequential neurogenesis for future follow-up studies. Importantly, these studies will provide excellent conceptual and technical training towards my long-term goal of pursuing academic research in molecular and developmental neuroscience.

NIH Research Projects · FY 2025 · 2025-08

Abstract This is the first study to evaluate the effects of personalized brain stimulation as a potential treatment for those with posterior cortical atrophy (PCA), a condition that is most often caused by Alzheimer’s disease (AD). Individuals with PCA generally have intact vision but, as pathology spreads through the brain, have increasing difficulty “making sense” of what they see. Examples of this visuospatial impairment include difficulty locating objects in crowded environments (e.g., locating items in a full refrigerator) or traveling from one location to another (i.e., spatial navigation). Both recent findings in the literature and our preliminary data suggest that the Dorsal Attention Network (DAN) is affected early in the course of PCA, which means that treatments targeting this network may be able to improve functioning in everyday life. We test this possibility by providing personalized brain stimulation for those with PCA. Specifically, we will use each participant’s functional magnetic resonance imaging (fMRI) brain scans to identify the DAN and measure how well it is functioning relative to other brain networks. We will then develop a personalized high-definition transcranial direct current stimulation (HD-tDCS) montage that delivers electrical current to key regions of the DAN, ensuring they are aligned with the participant’s fluorodeoxyglucose (FDG) positron emission tomography (PET) scan. Importantly, our novel methods allow us to equate the the amount of electricity delivered to the DAN for each participant, thereby markedly reducing the variability seen with traditional one-size-fits all brain stimulation approaches. Using a double blind randomized controlled trial format (i.e., active vs. sham), we will evaluate HD-tDCS induced changes in the DAN using fMRI and a combination of tasks that reflect real-world performance. For example, we will use using eye-tracking to evaluate participants’ ability to identify objects in crowded environments as well as an innovative immersive virtual reality (iVR) spatial navigation paradigm. Moreover, we will use functional near infrared spectroscopy (fNIRS) to evaluate changes in brain activity during this spatial navigation task. Importantly, we will evaluate these changes after relatively brief (i.e., 8 session Randomized Controlled Trial) and a subsequent open-label 6-month period of HD-tDCS. The extended duration is possible thanks to our first-of-its-kind personalized 3D- printed headgear and validated training program that teaches informants to administer HD-tDCS remotely as we supervise through videoconference. The open-label format allows us to refine methods and determine optimal treatment parameters necessary for a subsequent large-scale trial. Finally, we will perform exploratory analyses to identify participant-level factors associated with treatment response in order to refine a subsequent late-stage clinical trial. Our approach builds on decades of efforts to enhance early detection of AD pathology in vivo by translating this knowledge into personalized treatment protocols that target dysfunctional networks. Our plans to explore response heterogeneity will further promote precision brain stimulation for those with PCA and, ultimately, other neurodegenerative conditions in subsequent Phase II/III trials.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT The goal of this proposal is to explore the mechanism of Notch inhibition resistance in glioblastoma (GBM) in order to develop novel therapy for this deadly disease. Cancer Stem- like cells (CSCs) are thought to be critical for the engraftment and long-term growth of GBM. They are at least partially spared by traditional chemo- and radiation-therapies, and finding new treatments to target CSCs may be critical for improving patient survival. We recently demonstrated that Notch pathway blockade by gamma-secretase inhibitor (GSI) depleted CSCs, inhibited neurosphere growth in vitro and propagation in vivo, and prolonged the survival of mice bearing the intracranial xenografts. A subsequent Phase I clinical study showed that 24% malignant glioma patients responded well to GSI treatment and have stabilized disease for more than four months. Another Phase I trial showed that GSI indeed cross the blood brain barrier (BBB) and reduce Notch activity in treated GBM CSCs. However, the molecular mechanism of GSI treatment resistant in GBMs are largely unknown. One main possible mechanism of GSI resistance is different genetic background of GBM, such as status of PTEN, may result in different responses to GSI. Our preliminary data showed that GBM with loss of PTEN resistant to GSI treatment. Interestingly, we found that Akt inhibitor can reduce these GSI-resistance GBM neurospheres propagation in vitro and in vivo. We hypothesize that GBM with wild type of PTEN will be sensitive to GSI treatment and that GBM with loss of PTEN will be sensitive to pAkt inhibition. In the present proposal, we will identify GBM patient subsets that will be benefit from Notch pathway inhibition or Akt inhibition in term of targeting CSCs based on individual genetic background. To test our hypothesis, we will pursue the following specific aims: Aim1: Define the mechanism of targeting GBM CSCs by Notch pathway blockade based on tumor's genetic background of PTEN. Aim2: Examine a combination of Notch inhibitor and pAkt inhibitor plus chemo- and radiation-therapy in a pre-clinical GBM intracranial xenograft mouse model. Success in this proposal will enhance our understanding of glioblastoma biology and have significant impact on GBM therapy.

NIH Research Projects · FY 2025 · 2025-08

Molecular profiling of gliomas led to their genetic classification and a better understanding of their pathophysiology. Adult mutant IDH1 gliomas are characterized by a mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) at active site residue R132. The mutation imparts a gain-of-function catalytic activity which leads to the conversion of α-ketoglutarate (αKG) to (R)-2-hydroxyglutarate (2HG). 2HG is a competitive inhibitor of histone 3 (H3) lysine-specific demethylases (KDMs) and of DNA demethylation by ten-eleven translocation methylcytosine dioxygenases (TETs). KDM and TET inhibition lead to an overall increase in methylation with concurrent epigenetic changes that influence numerous downstream pathways. IDH1 mutation (mIDH1) is an early event in gliomagenesis and imparts favorable prognosis compared to wild-type IDH1 (wt- IDH1). Due to the epigenetic changes mediated by mIDH1 and manifold affected pathways, the survival advantage of mIDH1 patients may arise via several mechanisms, identification of which may, (i) reveal therapeutic interventions for mIDH1, and (ii) by comparison, suggest approaches for wt-IDH1 therapeutics. We developed an immune competent murine mIDH1 glioma model by incorporating genetic lesions into the genomic DNA of neural progenitor cells using the Sleeping Beauty (SB) transposase system. Intracranial tumors develop de novo and exhibit hallmarks of human mIDH1 astrocytoma, including 2HG production, hypermethylation, lower cellular differentiation within the tumor, and longer median survival when compared to wt-IDH1. Total RNAseq followed by gene-set enrichment analysis (GSEA) revealed that tumors harboring mIDH1 exhibited upregulated DNA damage response, upregulated immune response mechanisms and downregulated differentiation in mIDH1. Also, differences arose in cytokines key to immune cell regulation. These mechanisms and signaling pathways were confirmed by analyzing scRNA-seq data from both genetically engineered mouse models (GEMMs) and human samples. Gliomas are known for their immunosuppressive tumor microenvironment (TME), an obstacle to successful immunotherapy. We found substantial recruitment of immature myeloid cells (IMCs) into the TME of both mIDH1 and wt-IDH1. However, our data indicates that mIDH1 IMCs do not exhibit immunosuppressive properties as the wt-IDH1 counterparts do. Thus, we hypothesize that this could account for a functional immune system capable of mounting effective anti-mIDH1 glioma immunity. To uncover neuroimmune mechanisms at play in mIDH1 gliomas, we propose three Specific Aims (SA). SA 1 will uncover the epigenetic and transcriptomic remodeling affecting the myeloid compartment in mIDH1 gliomas. SA2 will uncover the mechanistic and functional impact of combining immunotherapy with mIDH1 inhibition in mIDH1 mouse glioma models. SA 3 will uncover the ontogeny, spatial phenotyping, and cellular and neighborhood interactions within the TME in mIDH1 gliomas, and their reprogramming elicited by treatment with immune- mediated gene therapy and mIDH1 inhibition.

NIH Research Projects · FY 2025 · 2025-08

The Ras-Mitogen-activated protein kinase (MAPK) signaling pathway is a critical regulator of blood vascular development and function. Dysregulated Ras-MAPK signaling in blood vascular endothelial cells (EC) leads to different types of vascular malformations, including arteriovenous malformations in which arteries connect di- rectly with veins. Ras is a small GTP-binding protein that cycles between inactive GDP-bound and active GTP- bound states in response to growth factor stimulation. Ras cycling is mediated by Ras guanine nucleotide exchange factors that exchange Ras-bound GDP for GTP, and Ras GTPase-activating proteins (RasGAPs) that promote Ras hydrolysis of GTP to GDP. RASA1 is one member of a family of RasGAPs that is essential for normal Ras cycling in the vasculature, and loss of function of RASA1 in EC results in AVM. In addition to its catalytically active GAP domain, RASA1 contains several modular binding domains, including a protein kinase C2 homology domain. Missense mutations in the C2 domain have been identified in families with a type of brain AVM known as Vein of Galen AVM (VGAM), which is the most common pediatric cerebrovascular mal- formation that carries the risk of hemorrhage, stroke, epilepsy, and other complications. The role of the C2 domain in RASA1 function has hitherto been unclear. However, our preliminary data, which comprises in vitro enzymatic assays, crystal structure determination, structure-based conservation assessment, and mutational analyses, indicate that the C2 domain directly contacts Ras and promotes GAP domain-mediated Ras cycling. We generated a VGAM-associated RASA1 C2 domain mutation in mice and found that homozygous mutant mice show similar embryonic vascular defects to those observed in RASA1 null mice. Together, our preliminary findings lead us to our central hypothesis that the RASA1 C2 domain is required for normal regulation of Ras, which is necessary to prevent vascular malformations such as VGAM. In this new MPI grant, we will address this hypothesis in three specific aims that combine highly complementary and synergistic expertise in the King and Boggon laboratories. In the first aim, we will employ enzymatic analyses to understand the mechanism by which the C2 domain promotes RASA1 catalytic activity against different Ras isoforms. In the second aim, we will further interrogate RASA1 mutant mouse models to provide additional support for our proposed mechanism in vivo, including its role in protection from the development of AVM. In the third aim, we will conduct RASA1- Ras mutant complementation studies in vitro and in vivo and will perform structural analyses of a RASA1-Ras complex to provide a detailed understanding of how RASA1 C2 domain interaction with Ras promotes normal vascular development. Our proposed studies are highly innovative and significant in that they will explore a heretofore unappreciated mechanism involved in Ras cycling that is relevant to an understating of the patho- genesis and potential treatment of vascular malformations such as VGAM.