University Of Pennsylvania

NIH Research Projects · FY 2024 · 2021-09

Project Summary Gene regulation is fundamental for every aspect of human function and errors in this process are key drivers for many diseases, including cancer. Proper gene expression requires a large number of proteins to assemble and function in a highly coordinated manner, but how this is achieved is still not understood. Recent studies revealed that some transcriptional regulators form dynamic high local concentration assemblies termed transcriptional condensates or hubs. These condensates are driven by weak multivalent interactions, which have biophysical properties and regulatory mechanisms distinct from the long-studied ‘lock and key’-type of high-affinity interactions. As such, the discovery of transcriptional condensates offers a new molecular principle to explain how transcription is organized. However, it remains unclear how exactly transcriptional condensates from, how they are regulated and dysregulated, and how they impact gene control during development and in diseases. This is largely due to the lack of experimental strategies to precisely control condensate formation and its properties for functional and mechanistic interrogations. This project uniquely addresses these major gaps by leveraging naturally occurring disease mutations. We discovered a series of oncogenic mutations in a chromatin regulator that promote aberrant formation of transcriptional condensates. These ‘acquired’ condensates strongly correlated with oncogenic gene activation and tumorigenesis, providing one of the first examples linking aberrant condensate formation to human disease. Using these mutations as the stepping-stone, this project aims to develop a more in-depth and broad understanding of transcriptional condensates and establish their dysregulation as a new pathognenic mechanism. Specifically, we will ask three major questions: (1) How do transcriptional condensates form at the molecule level? (2) What are the regulatory functions enabled by transcriptional condensates? (3) Can transcriptional condensates be therapeutically targeted? We will integrate cutting-edge approaches in structural biology, single-molecule imaging, gene regulation, epigenetics, cancer biology, and high-throughput drug discovery to address these questions. Successful completion of this project would offer definitive evidence and mechanistic insights needed to establish a new model of gene control. It also has the potential to transform how we think about and target gene dysregulation in diseases. The ideal experimental system we discovered and extensive expertise of my lab put us in a unique position to embark on this highly ambitious but urgently needed research program.

NIH Research Projects · FY 2025 · 2021-09

Abstract Glioblastoma therapy. delivered to Despite promising clinical outcomes, significant (GBM) is the deadliest of all brain cancers with a dismal prognosis despite aggressive multi-modal T umor treating fields (TTFields) are a recently approved loco-regional and noninvasive therapy by placing transducer arrays on patient's shaved scalp close to the tumor. TTFields have been found improve survival outcomes in GBM patients without causing any adverse effects on the quality of life (QoL). inter-individual variability in treatment response to TTFieldsis observed. This isbecause only solid/contrast enhancing regions of tumors are targeted for TTFields delivery in the current clinical practice. This is highly inadequate as GBMs are extremely infiltrative tumors that invade extensively into adjacent normal brain regions beyond enhancing margins where inevitable recurrence occurs. In cellular by by deliver tumor positioning dose with choline/N-acetylaspartate computational patients randomized TTFields experimental array response end will acceptable paradigm contrast to conventional neuroimaging, proton MR spectroscopy derived choline (an indicator of tumor proliferation) can detect occult microscopic tumor spread more accurately. We have demonstrated that using advanced computational modeling, it i s possible to deliver three-fold increased TTFields dose to t umors readjusting the layout of transducer arrays. In this proposed academic-industrial partnership, we aim to enhanced TTFields dose to the entire viable tumor bed by precise mapping of this i nfiltrative (precision diagnostics) and subsequent delivery of enhanced TTFields dose by optimized of transducer arrays (personalized therapeutics) . We hypothesize that enhanced TTFields to tumor beds will achieve more effective cancer cell killing resulting in delayed tumor recurrence increased overall survival (OS) of these patients. Whole brain spectroscopic imaging (WBSI) derived maps will be employed to dentify the target volume. Then, sophisticated modeling will be used to design personalized placement of transducer arrays. A total of 155 GBM after being treated with standard-of-care therapy and willing to receive TTFields will be recruited and into two treatment arms prior to i nitiation of TTFields. Patients in control arm (n=77) will receive based on target volume defined by contrast enhancement only (conventional array layout) and in arm (n=78) will receive TTFields based on target volume defined by choline abnormality (alternate configuration). Dosimetry profile parameters will be computed from tumor beds to assess dose-clinical relationships. Time to progression (TTP) and OS will be considered as primary and secondary study points, respectively. Using WBSI, diffusion and perfusion MR imaging, a combined multiparametric approach be utilized to compare treatment response from patients enrolled in two study arms. Lastly, we will establish QoL profile in patients receiving enhanced TTFields dose. If successful, our study will cause a shift by developing a personalized treatment plan with improved clinical outcomes of GBM patients. i

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Single cell gene expression atlases are now routinely generated for human tissues and entire model organism embryos and have shed light on the diversity of cell types and regulation of gene expression. While these wild-type single cell atlases can predict candidate regulatory genes across development for focused studies, further work is needed to determine the regulatory mechanisms and functional importance of the observed expression patterns at scale. A key problem is how to identify homologous cells between datasets in which their expression may be altered, for example data from the same tissue across evolution, or from animals that have experienced a genetic or pharmacological perturbation. This project will use the widely used model organism Caenorhabditis elegans to develop and test methods to compare cells across such conditions. Our focus is on two biological problems. In Aim 1, we will compare expression in single cells between C. elegans and four other related nematode embryos. These nematode species have nearly identical embryonic lineages to C. elegans despite substantial sequence divergence (>1 substitution per neutral site), making them an ideal test case for alignment of single cell datasets across evolution. We will generate large single cell RNA-sequencing datasets for embryos of each species (C. remanei, C. brenneri, C. briggsae and C. nigoni). We will compare both automated homology transfer and de novo lineage inference methods to identify cell types in each species. We will use quantitative imaging approaches (smFISH and live imaging of GFP knock ins) to validate the results of the single cell experiments. The resulting data will allow us to classify genes and cell types by the conservation of their gene expression, providing insight into the evolution of cell types. In Aim 2, we will test the role of conserved regulators in the specification and diversification of the mesodermal “MS” lineage (which produces pharynx, body wall muscle, and some specialized mesodermal cell types). We will measure gene expression by scRNA-seq after conditional loss of these mesodermal regulators using an auxin degron approach. As in Aim 1, we will test and validate automated alignment methods for these datasets to identify cells. The resulting data will allow us to distinguish homeotic fate transformations from the formation of novel cell states, to distinguish likely direct or context specific targets from indirect targets of each regulator, and to generate a genome-wide mesodermal regulatory network of a developing animal embryo.

NIH Research Projects · FY 2025 · 2021-09

A fundamental question in biology is to understand how genetic variation affects genome function to influence phenotypes. The majority of genetic variants associated with human diseases are located within non-coding genomic regions and may affect genome functions and phenotypes through modulating the activity of cis-regulatory elements and cell-type specific gene regulatory networks (GRNs). However, our knowledge about the impact of genomic variants (alone or as combinations) on gene expression, GRN activity and ultimately cellular phenotypes are rather limited. Further, because transcription factors (TFs) and related cis-regulatory elements are known to have distinct functions based on cell-type and state, how genomic variants influence cell-type/state-specific activity of functional elements and phenotypes remains to be characterized in much greater details. This proposal aims to leverage a panel of well-characterized human induced pluripotent stem cell (hiPSC) lines as well as recent advances in single-cell time-resolved or multi-omics technologies, predictive modeling of regulatory networks by machine learning and high throughput perturbation methods to study the functional impact of genomic variations on regulatory network and cellular phenotypes. First, we will establish a robust experimental framework of deploying advanced time-resolved and multi-omic single-cell technologies for detecting functional genetic variants at single-cell level. Next, we will develop novel computational methods for integration of single-cell data across different modalities and for accurate reconstruction and predictive modeling of GRNs driving cellular identify, developmental dynamics (cardiac and neural lineage cell fate transition). Finally, we will apply high-throughput combinatorial genetic or epigenetic perturbation approaches to modulate activity of key genes or putative cis- regulatory elements at single-cell levels to improve our understanding of network level relationships among genomic variants and phenotypes.

NIH Research Projects · FY 2025 · 2021-09

Cognitive behavioral therapy involving exposure and ritual prevention (EX/RP) is a first-line treatment for obsessive compulsive disorder (OCD). Despite its efficacy, it remains unclear how EX/RP influences the neural mechanisms of the fear and anxiety brain networks to yield clinical improvement. Moreover, data indicate that EX/RP outcomes may be more variable in women. Studies in rodents and healthy humans show that estrogen (E) affects the brain regions involved in fear learning, extinction, and extinction retention (the fear extinction network); E also has been shown to enhance extinction memory retention. In addition, the structure and connectivity of these same brain regions predict OCD treatment outcomes, including EX/RP. These and other data lead to the hypothesis that this Collaborative R01 will begin to test: that delivering EX/RP to women with OCD during high E states improves extinction memory retention via enhanced engagement of the fear extinction brain network, resulting in better clinical outcomes. We will also explore whether EX/RP-induced extinction processes differ between women and men. Our specific aims are to examine: 1) the impact of menstrual-cycle phase and sex on extinction-induced neural responses pre and post EX/RP; 2) the impact of menstrual-cycle phase and sex on EX/RP outcome; and 3) the relationship between OCD symptom change and EX/RP-induced neuronal changes. Our long-term goal is to understand how sex as a biological variable affects specific neural processes and hence EX/RP treatment outcomes. To achieve our aims, 120 adults with OCD – 80 natural cycling women and 40 men--will be recruited across two sites: The University of Pennsylvania (UPenn) and the New York State Psychiatric Institute/Columbia University (NYSPI). Through a subcontract, New York University (NYU) will provide expertise in the fMRI imaging paradigm that will be used as an experimental tool to probe the fear extinction brain network. Study participants will complete fMRI scanning before and after receiving manualized EX/RP. The EX/RP protocol will consist of 8 (90 minute) daily sessions comprised of two introductory and six exposure sessions. Women will be randomly assigned to complete EX/RP during either: a) the first 10 days after the start of menstruation (early follicular phase), when E levels are low; or b) days 12-22 of the menstrual cycle (late follicular, early luteal phase), when E levels are elevated. OCD symptoms and E levels will be measured at multiple time points. This design will allow us to study the effects of hormonal variation during the menstrual cycle and sex on the fear extinction network and on EX/RP outcome. The results will elucidate treatment mechanisms and could lead to personalized treatment recommendations for women with OCD.

NIH Research Projects · FY 2024 · 2021-09

Abstract The nervous system requires tight control of transcription for processes such as learning and memory formation. The field of epigenetics seeks to understand how changes to gene transcription occur in response to environmental cues and external signals such as those that our brains experience during learning. This proposal lies at the intersection of neuroscience and epigenetics, with a particular focus on chromatin biology. Chromatin is the complex of DNA and the histone proteins that wrap up DNA into complex structures, recruit key transcriptional regulators, and in doing so, control gene expression. In recent years, it has become clear that disruptions to chromatin regulation lead to a range of neurological and mental health disorders such as post- traumatic stress disorder (PTSD). However, we have a limited understanding of how chromatin functions in the brain or how its disruption can lead to disease. We will apply the tools and techniques of the epigenetics field to the study of neuronal function. In doing so, we hope to elucidate the molecular mechanisms that allow our brains to perform incredibly complex tasks and how disruption of these mechanisms can lead to neuronal dysfunction. We propose overcome long-standing hurdles in the field using a combination of novel techniques to reveal how the epigenetic landscape encodes the transcriptional changes that underlie memory formation. Specifically, we seek to uncover the transcriptional signature of memory formation and memory maintenance within single neurons in an in vivo context. We then will examine the epigenetic underpinnings of this transcriptional signature and manipulate specific components of the chromatin environment to define their contribution to learning and memory maintenance. First, in order to elucidate the gene program associated with learning, we will use single-nucleus RNA-sequencing in combination with mouse models that label the specific neurons activated during learning. This will allow us to examine the transcriptional programs activated in neurons that form a memory engram compared to their neighboring cells at various times after learning. Next, we will employ a quantitative biochemical approach uniquely available to our group as part of the Epigenetics Institute to characterize the chromatin landscape changes the occur during memory formation, memory maintenance, and reversal learning. Finally, we will modify the chromatin landscape by manipulating specific histone proteins in combination with numerous sequencing approaches to elucidate how chromatin controls learning and the transcriptional program. Employing this novel combination of techniques will allow us to uncover the mechanisms through which the epigenome encodes information within neurons to modify behavior both in the context of normal learning and in the context of maladaptive responses that lead to disorders such as PTSD. If successful, these methods will 1) identify the transcriptional signature that encodes a memory in neurons, 2) map how this signature is encoded by specific epigenetic regulatory mechanisms, and 3) define how the chromatin landscape affects memory formation and contributes to mental health disorders.

NIH Research Projects · FY 2024 · 2021-09

Project Summary The response to systemic infection and tissue injury requires the rapid adaptation of hematopoietic stem cells (HSCs) in the bone marrow, which proliferate and divert their differentiation towards the myeloid lineage. Significant interest has emerged in understanding the signals that trigger this emergency hematopoietic program. However, the mechanisms that terminate this response of the HSCs and restore tissue homeostasis remain unknown. The clinical success of proteasome inhibitors, bortezomib, and E3 ubiquitin ligase glues for the treatment of hematologic diseases has made the Ubiquitin pathway a bona fide target for cancer therapeutics. Thus, defining how novel E3 ligases function in the bone marrow and investigating their specific roles in normal and emergency hematopoiesis can lead to novel therapeutic interventions. We have demonstrated that the E3 ubiquitin ligase Spop restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeds in an unresolved manner and the sustained response in the HSCs results in a lethal phenotype reminiscent of hyper-inflammatory syndrome. Our proteomic/biochemical studies demonstrated that Spop restricts inflammation by targeting the signal transducer Myd88 for proteasome-dependent degradation. Myd88 accumulation in conjunction with an inflammatory stimulus leads to Myddosome formation, the hyper-phosphorylation of the Irak4 kinase and activation of a number of transcription factor pathways (NF-kB, Jun, Pu.1, Cebpb). This proposal defines: (a) the transcriptional and chromatin landscape changes imposed during initiation and termination of emergency hematopoiesis in the bone marrow HSC and progenitor cells, (b) the role of the myddosome assembly, signaling and termination in emergency hematopoiesis and gene regulation and (c) the structural details of myddosome assembly and termination. The findings of this grant proposal will uncover HSC-intrinsic mechanisms essential for reestablishing homeostasis following emergency hematopoiesis.

NIH Research Projects · FY 2025 · 2021-08

Abstract Kidney transplant is the treatment of choice for patients with end-stage renal disease (ESRD), as it extends survival, improves quality of life and is highly cost-effective. However, for about a third of patients on the waitlist, pre-existing anti-HLA antibodies (i.e. allo-sensitization) presents a major barrier to successful transplant. HLA antibody responses are maintained by memory B cells (Bmem) and plasma cells (PC). Unfortunately, desensitization approaches have been largely ineffective due to incomplete depletion of allo-specific B cells and PCs. WE HYPOTHESIZE that stringent depletion of donor-specific B cells and PCs is required for a clinically significant reduction of allo-antibodies necessary to achieve successful kidney transplantation. We have shown that engineered T cell immunotherapies employing synthetic chimeric antigen receptors (CARs) can induce durable remission of B cell lineage and plasma cell malignancies. Two CAR-T cell therapies that target CD19 (CART-19) and B cell maturation antigen (CART-BCMA) result in depletion of malignant cells but also physiologic B cells and PCs. Importantly, we have shown that CART-BCMA and CART-19 can be safely administered together. Based on this experience, our GOAL is to leverage this innovative platform to target Bmem and PCs and promote reduction of preformed anti-HLA antibodies, thus providing a window of opportunity for transplantation. Specifically, we propose a single-arm proof-of-concept CLINICAL TRIAL that combines CART-19 with CART-BCMA as a novel desensitization measure in kidney transplant candidates with a cPRA ≥99.9%. MECHANISTIC studies will evaluate the cellular, humoral and molecular immune correlates of CART-19 + CART-BCMA immunotherapy in highly sensitized kidney transplant candidates. These are focused on the CAR T cells, T- and B-cell immunity (both allo-specific and protective) and, in the event of successful transplantation, the allograft biology. An INFECTIOUS DISEASE STUDY proposal will evaluate vaccine response and immune function in our renal transplant candidates, including our study cohort. The multi-center team (Penn, NYU, MGH) brings together investigators with extensive experience in CART therapy, desensitization, and outstanding depth of laboratory expertise to carry out robust mechanistic studies.

NIH Research Projects · FY 2025 · 2021-08

The long-term objectives of this application are to establish the epidemiological association between methamphetamine use and pulmonary arterial hypertension. Methamphetamine and other stimulant abuse have been increasing across the United States and the world with several known complications. Pulmonary arterial hypertension has been suspected to occur as a sequela, however this has never been studied in a rigorous multicenter epidemiologic study. Preliminary findings have suggested that carboxylesterase 1 (CES1) may be an important genetic modifier in the setting of methamphetamine use leading to pulmonary arterial hypertension. The Specific Aims are 1) to determine whether methamphetamine use is associated with PAH in a case-control study and whether this association is modified by genetic variants in (or activity of) CES1; 2) to determine the risk factors for clinical worsening in patients with methamphetamine-associated pulmonary arterial hypertension in comparison with idiopathic pulmonary arterial hypertension in a prospective cohort study; and 3) to understand if reductions in CES1 activity result in human pulmonary endothelial cell damage in methamphetamine-associated pulmonary arterial hypertension. Patient-oriented research studies will be conducted in a multicenter study group to address these epidemiologic questions.

NIH Research Projects · FY 2025 · 2021-08

Pulmonary arterial hypertension (PAH) is characterized by limitation of physical activity even with current effect treatments. Most observational studies, clinical trials, and outpatient clinical assessments of patients with PAH focus on exercise capacity (the maximal effort a person can achieve under controlled circumstances), measured by cardiopulmonary exercise or six minute walk testing. However, these artificial tests do not capture the intensity, frequency, duration, context, and pattern of physical activity throughout the day or week in the patient’s life. Such an activity “signature” or phenotype may more accurately reflect an individual’s function and perceived health-related quality of life (HRQOL) by providing insight into multisystem function, side effects, and treatment benefits and burdens. A physical activity intervention which is personalized for a PAH patient could lead to improvements in psychosocial function, symptoms, HRQOL, fitness, and even survival. Unfortunately, there have been very few published studies of physical activity in PAH patients, none of which have assessed multidimensionality in a large multicenter cohort. We have performed preliminary studies using traditional accelerometry which have shown that patients with PAH cluster into low, medium, and high activity phenotypes which show differences in six minute walk distance and HRQOL. Functional principal components analysis has identified “signatures” of physical activity patterns throughout the day in PAH. Novel biosensors which continuously capture multiple streams of data in real time (including accelerometry) would provide an innovative approach to remote clinical monitoring and may increase the efficiency and pertinence of clinical trials in PAH. The Pulmonary Hypertension Association Registry (PHAR) has been prospectively collecting data from adult and pediatric PAH patients from centers throughout the United States since 2015. The PHAR has enrolled 1400 patients with 2000 patient-years of follow-up at 52 centers, representing one of the largest multicenter registries of patients with PAH. We propose to measure accelerometry for one week periods biannually for > 1400 patients over four years (~7000 assessments) in the PHAR with high efficiency and low patient burden. We aim to determine the predictors of physical activity phenotype and whether physical activity patterns are associated with health-related quality of life, emergency department visits, hospitalizations, and time to lung transplantation or death in PAH. We will estimate the “minimally important difference” in physical activity which could be used as an end point in clinical trials in PAH. We will incorporate a novel wearable biosensor which could be used to advance these models of physical activity in PAH.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Acne is one of the most common diseases worldwide, affecting 85% of adolescents and often persisting into adulthood. Acne is responsible for a greater global burden of disease than psoriasis, cellulitis, and melanoma. Although mild acne can usually be managed with topical medications, moderate to severe acne often requires treatment with systemic medications such as oral antibiotics, spironolactone, and isotretinoin. While these medications are a standard part of care, there are very few randomized clinical trials showing efficacy and none that show comparative effectiveness between these commonly used treatment options. The Institute of Medicine has identified this area as one of the top priorities for comparative effectiveness research. In addition, little is known about the effects of these different treatments on the microbiome. Previous work by our group has shown oral antibiotics are the most common systemic medication used in the treatment of acne and patients frequently use them for more than one year. In fact, dermatologists prescribe more antibiotics per capita than any other specialty. However, prolonged antibiotic use may be associated with a variety of adverse outcomes including bacterial antibiotic resistance, pharyngitis, collagen vascular illnesses, inflammatory bowel disease, and breast and colon cancer. As a result, there is growing international interest and attention specifically from the Centers for Disease Control regarding improving antibiotic stewardship in the outpatient setting and multiple clinical guidelines for acne have recommended reducing the use of antibiotics through the use of non-antimicrobial therapies and by limiting the duration of therapy. For women with moderate to severe acne, spironolactone may represent an effective, well-tolerated therapeutic alternative to oral antibiotics. Originally developed as a potassium-sparing diuretic, for many years it has also been used off-label for acne due to its potential impact on hormonal pathogenesis of this disease. However, despite expert opinion supporting the use of spironolactone in the treatment of acne, its use remains relatively uncommon and clinical evidence demonstrating the effectiveness of spironolactone is limited to small, often retrospective studies. Trials comparing the effectiveness of spironolactone to that of other medications such as oral antibiotics are lacking. In Specific Aim 1, we will conduct a double-blind randomized controlled non-inferiority comparative effectiveness study of spironolactone versus doxycycline for women with acne. Since oral tetracycline-class antibiotics like doxycycline are currently the most common systemic medication prescribed for acne, the results of this trial will have substantial implications for the treatment of acne. In Specific Aim 2, we will evaluate the impact of spironolactone versus doxycycline treatment on the microbiome, which will provide valuable insights regarding the relative effects of antibiotic (doxycycline) versus non-antibiotic (spironolactone) acne treatments on the microbiome. This trial will significantly influence healthcare practice with respect to the treatment of moderate to severe acne in women and inform policy regarding more appropriate use of antibiotics throughout medicine.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The ability of recently activated T cells to express the cell surface molecule CD40L allows them to communicate with other immune and non-immune populations. This molecule is of particular importance in the gut to help control the parasitic infection caused by Cryptosporidium. Here we leverage a novel, natural mouse model of Cryptosporidium to dissect the impact of the CD40-CD40L interaction in T cell-mediated resistance to infection in the gut. In this model, WT mice (like humans) develop sterile immunity mediated by T cell production of IFN-γ, but mice that lack CD40L mice (like humans) do not resolve infection. In addition, treatment of chronically infected CD40L-deficient mice with soluble (s)CD40L results in rapid parasite clearance. We will test if protective effect of CD40L may be explained by either I. its ability to promote T cell responses essential for resistance and/or II. because CD40L directly activates EC to limit parasite growth. We are uniquely equipped to utilize parasite transgenesis, combined with sophisticated genetic approaches to define the key cellular interactions that allows CD40L to determine the outcome of an enteric infection.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT The actin cytoskeleton is a major cellular component with key functions in virtually every aspect of cell physiology including cell motility, shape and mechanics, cell and tissue morphogenesis, cell-cell and cell-matrix interactions, and dynamics of membrane organelle. Aberrations in actin cytoskeleton structure, functions and/or dynamics contribute significantly to human pathologies, especially to cancer and neurodegenerative, immune and cardiovascular diseases. The actin cytoskeleton plays indispensable roles in cells due to its ability to generate large pushing, pulling and resistance forces in many different combinations. To perform these diverse functions, actin filaments are organized into diverse structural arrays by multiple accessory proteins. Despite extensive research, the exact organization of these actin-based molecular machineries is frequently unknown. The main barrier toward this goal is the difficulty of resolving actin cytoskeleton architecture at a single-filament level. Without knowledge of the structure, functional understanding of the machinery is incomplete. My lab uses a distinctive approach to overcome this problem. We take advantage of platinum replica electron microscopy (PREM), which is uniquely able to combine high resolution imaging of the cytoskeleton with full coverage of the whole cell and to efficiently correlate the cytoskeleton structure with live cell dynamics. With help of PREM, my lab has made multiple fundamental contributions toward understanding of cytoskeleton functions in a range of generic and specialized cell types. In this application, we propose in the course of the next five years to address the following questions representing each of the four major categories of actin cytoskeleton functions: (1) Protrusion – How microtubules regulate protrusive activity of the actin cytoskeleton for directional migration; (2) Contraction – How an interplay between nonmuscle myosin II paralogs regulates polarized subcellular distribution of contractile forces; (3) Cell mechanics – How differences in the molecular architecture of the actin cortex are linked to different mechanical properties of normal and cancer cells; (4) Membrane dynamics – How branched actin networks promote invagination of clathrin-coated membrane domains. Our expertise in PREM in addition to a broad range of other cell biological, imaging, functional, biochemical, and molecular biological methods puts us in unique position to significantly advance our understanding of actin cytoskeleton functions. In turn, this knowledge may provide important new insights into how to combat human diseases associated with actin cytoskeleton malfunctions.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The striatum is an evolutionarily conserved structure involved in cognitive and limbic regulation of motor control. Striatal circuits are implicated in the initiation and execution of ethologically relevant motor output, ranging from exploratory actions to highly stereotyped motor patterns. Dysfunction of these circuits leads to motor control abnormalities that frequently manifest as excessive repetitive behaviors. Self-directed grooming, a highly stereotyped repetitive motor pattern, is observed in virtually all animals, serving vital functions in hygiene maintenance, thermoregulation, de-arousal, stress reduction, and social communication. Abnormally repetitive grooming is a central behavioral phenotype observed in numerous models for neurological and neuropsychiatric diseases. A better understanding of the neural control of grooming may thus yield fundamental insights into how the brain controls repetitive motor output in both normal and diseased conditions. Our preliminary work suggests that an understudied population of interneurons within the olfactory tubercle (OT; the most ventral part of the striatum), predominantly in the Islands of Calleja (IC), is involved in mediating this behavior. The striatum has a fairly uniform cellular composition, with ~95% of the neurons being spiny projection neurons (SPNs), classified as D1- or D2-type according to the dopamine receptors they express. One exception to this uniformity is the existence of evolutionarily conserved IC, clusters of densely- packed, GABAergic granule cells, which express the D3 dopamine receptor. By means of optogenetic manipulations, we have shown that activation of OT D3 neurons initiates robust grooming behavior via arrest of other alternative ongoing behaviors. In contrast, inactivation of these neurons halts ongoing grooming. These findings lead to the central hypothesis that OT D3 neurons play critical roles in controlling grooming behavior. Through an array of modern neuroscience approaches (optogenetics, ex vivo and in vivo electrophysiology, fiber photometry, neural circuit tracing, and behavior), we will pursue three specific aims to determine (1) in vivo activity patterns of OT D3 neurons and SPNs in grooming and other behaviors, (2) contributions of OT D3 neurons to grooming in relation to other brain regions, and (3) the effects of dopamine release into the OT on grooming behavior. Overall, this project will provide insights into the neural circuitry of the IC/OT D3 neurons and its role in neurobiological control of a highly important motor pattern.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics and composition are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including skeletal muscle. Along with increased fibrosis, the regenerative function of skeletal muscle decreases with aging. Muscle stem cells (MuSCs) are responsible for maintaining and repairing muscle throughout life and are known to be acutely mechanosensitive, losing their stem cell potential when cultured on stiff substrates. Thus, the stiffened, fibrotic microenvironment may contribute to the diminished regenerative capacity of aged MuSCs. The goal of this project is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials and to employ this model to identify molecular mechanisms of MuSC mechanosensing that are implicated in MuSC dysfunction in aging. The mentored phase of this proposal will provide advanced technical training in aging biology, transgenic mouse models, cellular traction force measurement, and machine learning approaches for bioinformatics. This training will enable an independent research program leveraging dynamic biomaterials to deconvolve the complex interactions of mechanical forces, matrix biochemistry, and cell-cell signaling that dictate the progression of aging and disease. Additional structured training in scientific writing, grantsmanship, and research management will facilitate the transition to independence, supported by a committee of faculty from the Stanford Schools of Medicine and Engineering. Aim 1 will optimize a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to elucidate mechanisms of mechanosensing in MuSCs, using FRET-based force sensors and transgenic mouse models. Aim 2 will model muscle aging in vitro, using dynamically stiffening gels modified with ECM components characteristic of aging. Single cell RNA sequencing and machine learning bioinformatics approaches will identify unique mechanically regulated drivers of cell fate that reduce MuSC regenerative potential in aging. Aim 3 will develop novel materials for 3D cell culture with dynamic tuning of viscoelastic properties to establish the first human model of muscle “aging in a dish.” This project stands to identify new therapeutic targets to improve muscle function with aging and to develop engineered platforms to study numerous heritable diseases and aging in diverse tissues.

NIH Research Projects · FY 2025 · 2021-08

Pancreatic adenocarcinoma (PDAC) is a particularly lethal form of cancer that kills over 40,000 Americans every year. PDAC is most often diagnosed when disease is advanced, with metastases that lead to death. Patient outcomes are further negatively-impacted by a typical poor response to currently-available treatments. It is thus critical to develop a stronger understanding of the processes which lead to PDAC development and metastasis, as well as to determine novel, more-efficacious targets for therapies. Low-level, constitutive endoplasmic reticulum (ER)-to-mitochondria transfer of calcium is required for optimal bioenergetics and cancer-cell survival. We hypothesize that this pathway contributes to pancreatic cancer development, metastasis, and tumor maintenance, and may therefore present a viable anticancer target. The ER-localized IP3R calcium-release ion channel and the mitochondrial calcium-uniporter ion channel, MCU, mediate calcium transfer between the two organelles at membrane-contact sites. However, it has been impossible to target this pathway in vivo because of the lack of selective, cell-permeable pharmacological agents against these ion channels. We therefore propose to examine the role of ER-to-mitochondria calcium transfer in PDAC development, metastasis, and tumor maintenance through the use of novel animal and cell-culture models. We will genetically delete MCU during early development in a murine genetic-model of PDAC, the KPCY mouse, to observe the role of this protein in tumor development. In addition, we will use tumor cells as well as genetically- modified cells using Cre/lox and CRISPR/Cas9 systems, as well as patient-derived cell lines and the established human PDAC cell line, Panc-1. We will assay proliferation, cellular bioenergetics, oxygen consumption rates, and mitochondrial calcium homeostasis, using biochemical, cell biological and biophysical approaches, including electrophysiology, live-cell imaging and fluorimetry, to define the role of ER-to- mitochondria calcium transfer in these processes. To determine the role of MCU in metastasis, we will quantify metastasis in the KPCY model using the sensitive YFP-reporter gene, and we will use an in vivo tail-vein metastasis model with genetically-modified Panc-1 cells expressing luciferase in NOD/SCID mice, as well as in vitro transwell-invasion and gel-degradation assays, and biochemical and morphological assessment of metastasis-associated markers of epithelial-to-mesenchymal transition. To observe the role of ER-to- mitochondria calcium transfer in tumor maintenance and thus its therapeutic potential for more advanced disease, we will use an inducible CRISPR/Cas9 cell-culture model of murine PDAC in vitro and an in vivo inducible orthotopic model to observe the effects of acute MCU ablation in already-growing tumors and cells as a method to simulate profound pharmacological inhibition. These studies will elucidate the role of ER-to- mitochondria calcium transfer in PDAC development, metastasis, and maintenance, and they may inform future development of novel therapeutic targets in PDAC, potentially saving lives.

NIH Research Projects · FY 2025 · 2021-08

Penn ADRC Overall Project Summary The mission of the University of Pennsylvania’s Alzheimer’s Disease Research Center (Penn ADRC) is to increase research and education on Alzheimer’s disease (AD) and its links to related dementias (ADRD) with the goal of identifying the causes of and cures for AD/ADRD. To do so, the Penn ADRC will address one of the fundamental barriers to effective treatment or prevention, which is the significant phenotypic, pathological, and sociodemographic heterogeneity of AD. We will embrace and seek to characterize and understand this heterogeneity to ultimately achieve a precision medicine approach leading to targeted interventions that will facilitate realization of the National Alzheimer’s Project Act’s (NAPA) ambitious goal of effective prevention or treatment by 2025. Indeed, the Penn ADRC is constructed to directly contribute to a number of the milestones of NAPA necessary to achieve this goal. Emerging from the 30-year history of the Penn Alzheimer’s Disease Core Center (ADCC), the Penn ADRC benefits from a rich scientific milieu in which there is significant integration and collaboration across Penn’s neurodegenerative disease centers. This construction is critical to the understanding of AD heterogeneity which is driven, in part, by overlapping pathologies and mechanisms, such that cross-degenerative disease studies are of increasing importance in capturing the full spectrum of disease. This environment has led to a history of transformative research that has influenced understanding of disease definition and mechanisms, diagnostic approaches and biomarker development, statistical and bioinformatics methodology, and ethical, social and legal perspectives of those suffering from this condition and their care partners. It has also created an intellectual, cultural, and physical setting dedicated to training the next generation of investigators and clinicians, as well as partnering and educating the community. To achieve our mission, the Penn ADRC will bring together eight cores (Administrative; Biomarker; Clinical; Data Management and Statistical; Genomics; Neuroimaging; Neuropathology; Outreach, Recruitment, and Engagement) and the Research Education Component (REC). These highly integrated cores will support development of a phenotypically, pathologically, and ethno-racially diverse cohort which will be deeply characterized through cognitive assessments, measures of social determinants of health, genetics, biofluid and neuroimaging biomarkers, and autopsy. All these data will be stored within the Integrated Neurodegenerative Disease Database (INDD), which is linked to the other neurodegenerative centers at Penn and will contribute to our understanding of the upstream factors and processes that lead to AD heterogeneity and its downstream manifestations. Further, the ADRC supports robust sharing of these data and participation in larger NIA and national programs. The REC leverages these cores and their research programs for training new investigators. Together, the Penn ADRC will advance our ultimate mission to reduce the tremendous burden of AD.

NIH Research Projects · FY 2025 · 2021-08

Project Summary In the nematode C. elegans as in other animals, sickness is associated with reduced movement, reduced feeding, and increased sleep. Sickness behavior in worms is induced by environmental stressors including heat shock, ultraviolet light, and infection. This behavioral program during sickness is regulated by worm central neurons that are activated by the cytokine epidermal growth factor (EGF). EGF causes reduced activity in worms, flies, fish, and mice, but the mechanisms of EGF activation itself during sickness are not clear. We will use C. elegans to study the mechanism of EGF regulation during sickness. In Aim 1, we will determine where (from which cells) and when EGF is released to promote sickness behavior. To identify other regulators of EGF activation, in Aim 2, we will perform genome-wide discovery screens for genes required for sickness behavior. In Aim 3, we will test the hypothesis that sickness behavior supports survival during sickness. A long- term goal of these studies is to identify candidate signaling molecules for developing diagnostics and treatments of sleepiness during sickness and to understand the importance of sleep in promoting recovery from illness.

NIH Research Projects · FY 2025 · 2021-08

Modified Project Summary/Abstract Section Patients’ health expectations lie at the foundation of their preference-sensitive choices, such as whether to pursue comfort-oriented therapies as a primary or concurrent strategy. To achieve high-quality shared decision making, patients with serious illnesses must form accurate expectations for their future health. However, many patients have inaccurate expectations, limiting their ability to make decisions concordant with their values. Chronic obstructive pulmonary disease (COPD) is an incurable lung disease of older adults and a leading cause of death worldwide. Patients with COPD and their caregivers frequently experience a high burden of symptoms, mood disorders, and difficulty coping, yet underuse advance care planning and palliative treatments. Existing evidence reveals that patients with COPD are at risk for expectation inaccuracies and our preliminary work reveals that overly optimistic expectations are associated with worse quality of life over time. Our overarching objective is to promote the well-being of patients living with COPD through the delivery of care concordant with individual patients’ values. This work proposes a novel application of behavioral theories of decision making and innovative methodologies to the common problem of goal-discordant care. This is a prospective cohort study among 420 patients with severe COPD plus their family caregivers recruited from Wake Forest Baptist Health, Geisinger Health System, and the University of Pennsylvania Health System. These three health systems have robust research infrastructures and serve communities where COPD is disproportionately burdensome. The specific aims of this study are to: 1) identify patient and caregiver characteristics associated with inaccurate health expectations, 2) quantify associations between patients’ and caregivers’ expectation accuracy and well-being, and 3) identify mechanisms through which clinician communication influences expectation accuracy. The study team has the requisite content expertise, methodological expertise, and prior success conducting investigations of this type. Completion of this project will provide mechanistic insights into ways to promote optimal shared decision making in serious illness using state-of-the art methods to test a novel conceptual model. This work will result in key targets for intervention development to improve expectation management and decision making among patients with serious illness.

NIH Research Projects · FY 2024 · 2021-08

Quantitative Radiology holds great promise to transform our ability to diagnose, monitor, stage, prognosticate, and detect diseases as well as to plan and guide patient therapeutic interventions. However, the process of locating and delineating anatomic organs and pathologic regions in medical images, known as image segmentation, at a high level of automation has remained a major hurdle to these advances. Most developments on image segmentation have focused on a specific organ or a small group of objects in a specific body region. A new method or a major adaptation of an existing method is engineered when any of these parameters changed. Such an approach is not sustainable and becomes a stumbling block when dealing with whole-body systemic diseases where body-wide image analytics is required. A critical advance is needed in this field to overcome two main challenges: (1) Although prior information about normal anatomy is deemed vital for image segmentation and analysis, its creation and utilization body-wide on a massive scale have not been attempted and are sorely lacking. (2) Techniques to employ such information and methods for body-wide disease quantification at high levels of automation do not exist. The overarching goal is to overcome these challenges by developing a body-wide and generalizable anatomy-guided deep learning image segmentation methodology and demonstrate its application in the study of patients with diffuse large B cell lymphoma (DLBCL) for which PET-based staging and response assessment are of paramount importance. The project has three specific aims. Aim1: To develop a family of body-wide anatomy models representing the entire human adult age spectrum. Existing FDG PET/CT scans of 600 patients from two institutions (Penn and New York Proton Center) covering 10 age groups will be utilized to build anatomy models involving 50 organs and 50 lymph node zones in the extended body torso including neck, thorax, abdomen, and pelvis. A family of 40 anatomy models representing the 4 body regions and 10 age groups will be created from roughly 60,000 3D object samples. Aim2: To develop, implement, and validate a methodology for localizing objects and to quantify disease without explicitly delineating organs and lesions. Gender- and age-specific anatomy models will be utilized for automatically locating the above 100 objects in any given patient PET/CT image and to quantify disease in each body region, organ, and lymph node zone. The methods will be tested on 400 PET/CT images of DLBCL patients. Aim3: To develop and validate an automated method of DLBCL disease staging and prognosis. The disease quantity information will be utilized to develop automated staging and outcome prediction algorithms which will be tested on the above 400 cases in comparison to current clinical methods. Two key outcomes of this project will be: an unprecedented well-curated database of body-wide images, segmented objects, and family of models; and a validated methodology for automatic body-wide disease quantification and disease staging in DLBCL.

NIH Research Projects · FY 2024 · 2021-08

Project Summary Ischemic heart disease is the most common cause of death in the western world, largely due to myocardial infarction (MI), the irreversible damage of myocardial tissue induced by the blockage in coronary arteries. After MI, formation of new blood vessels, i.e., neovascularization, is crucial for ischemic tissue reperfusion and repair. However, the newly formed vasculatures in infarcted tissue are characterized by functional and structural abnormalities, which compromise vessel delivery function and cardiac repair after MI. Likewise, aberrant non-productive neovascularization represents a promising therapeutic target for MI treatment. Here, by utilizing endothelial lineage tracing and single-cell RNAseq technology, our preliminary studies with a murine MI model reveal robust endothelial cell (EC) plasticity mediated through endothelial mesenchymal transformation (Endo-MT, i.e., partial endothelial mesenchymal transition) during cardiac repair after MI. We show that ECs acquire mesenchymal phenotypes including high proliferation and motility after MI, leading to vascular abnormalities and non-productive neovascularization. We identify a PDGF/NF- kB/HIF-1a/Snail-mediated axis that controls Endo-MT. Notably, EC-specific deletion of PDGF receptor-b promotes post-MI tissue repair and cardiac function recovery in mice. Finally, pharmacological inhibition of PDGF improves cardiac function recovery after MI. In addition, Snail is expressed in human MI-associated ECs. Based on these findings, we hypothesize that endothelial plasticity drives non-productive neovascularization and impedes cardiac repair after MI. To test this hypothesis, we will pursue the following aims: 1) To define the molecular mechanisms for endothelial plasticity after MI; 2) To determine the in vivo role of endothelial plasticity for aberrant neovascularization and cardiac repair after MI; and 3) To test experiment therapy that targets PDGFR-mediated endothelial plasticity for MI treatment. Thus, targeting EC plasticity may offer a promising therapeutic opportunity to recondition vascular microenvironment and improve cardiac repair and function recovery after MI. Successful completion of this project will provide new insights into the mechanism for aberrant neovascularization and may lead to development of new therapeutic revenue for treating ischemic heart disease.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY In nearly all studies of comparative effectiveness, the investigators seek to estimate how an in- tervention changes outcomes on average. That is, are outcomes for treated subjects better on average than for untreated subjects? While the average treatment effect (ATE) is a useful sum- mary of the treatment effect, the treatment effect may vary from patient to patient. The ATE is a low-dimensional summary of a treatment effect, since it summarizes the overall effect of the treatment using a single quantitative measure and ignores possible effect heterogeneity. Many in- vestigators seek to go beyond low-dimensional summaries by estimating heterogenous treatment effects (HTEs). The most common approach to the estimation of HTEs relies on simple statistical methods. Specifically, regression models are widely used but may be biased due to the linear functional form especially where HTEs that are nonlinear or based on complex combinations of patient subgroups. Currently, there is considerable interest in developing more flexible methods for the estimation of the HTEs. In this project, we will use the the doubly robust machine learning (DRML) framework to develop improved methods for a variety of HTEs. The DRML framework is a combination of semiparametric theory, machine learning (ML) methods, and doubly robust estimators. The key advantage of the DRML framework is that it allows one to reduce bias using ML estimation methods, while retaining the efficiency of parametric models.

NIH Research Projects · FY 2024 · 2021-08

Summary The growth differentiation factor 15 (GDF15), formerly known as macrophage inhibitory cytokine-1 (MIC-1), is a cytokine that shows expression and serum rise in response to many conditions and diseases, including pregnancy, obesity, diabetes, and cancer. GDF15 signaling has gained significant attention in recent years with multiple papers in 2017 identifying the GDNF family receptor α-like ( GFRAL ) receptor as binding GDF15 selectivelyand with high affinity. However, the reported restrictive expression of the GFRAL receptor to the area postrema (AP) and nucleus tractus solitarius (NTS) of the brainstem, areas highly critical to both energy balance and emesis/nausea/malaise suggests that GDF15-GFRAL signaling could be an important factor not only in long-term body weight regulation, but also in short-term processing of emesis and illness. Behavior. Thus, understanding what role GDF15-GFRAL signaling plays in illness behavior and anorexia is paramount to determining the mechanism of GDF15 action. Compelling evidence links GDF15 signaling with chemotherapy- induced nausea and anorexia, which remain important clinical problems despite relatively well-controlled chemotherapy-induced emesis, by showing that: 1) GDF15 signaling causes nausea and emesis; 2) an AP/NTS site of action is responsible for mediating the feeding effects of GDF15 signaling through binding of the GFRAL- RET receptor complex, and 3) obesity, cancer, and chemotherapy increase circulating GDF15 in rodents and humans. We hypothesize that a functional dynamic change in the expression of central GDF15 levels in the NTS and AP will occur following energy balance dysregulation and/or administration of emetic stimuli, and that we can mitigate/treat such through the unique molecular and behavioral assays and patented peptide-based technology employed here (i.e. our peptide-based inhibitor bind nausea novel GFRAL-RET antagonist “GRASP”). The GRASP antagonist is a small, sequence with our in vivo and conformational binding models supporting it to be an allosteric to the GFRAL-RET complex. We have also shown that GRASP can penetrate into the brainstem and to GFRAL-expressing neurons in the AP/NTS, and consequently attenuate GDF15- and cisplatin-induced behaviors in rats.To further explore the GDF15-GFRAL system, we propose complimentary studies by a multi-PI team of established investigators with extensive collaborative experience to investigate the following aims: Aim I will characterize brainstem circuitry and unbiased single cell transcriptomics for endogenous GDF15 production and GFRAL/RET-expressing neuronal phenotypes. Aim II will characterize GDF15-induced emesis, nausea behavior, and anorexia as well as characterize the GRASP lead compound against these behaviors with a multi-species approach. Aim III will characterize the critical mechanistic and stability parameters of GRASP through rational design of analogs based on functional, computational and structural data to build upon our successful technology to-date that seeks to block the GFRAL receptor to treat sickness measures that include unwanted anorexia, nausea and emesis.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Ovarian cancer remains the most lethal gynecologic malignancy and the fifth most frequent cause of cancer- related mortality in women in the United States. Advanced-stage high-grade serous ovarian carcinoma (HGSOC) is the most commonly diagnosed subtype and constitutes the majority of ovarian cancer deaths. Despite a high response rate to initial platinum-taxane chemotherapy, patients with HGSOC frequently relapse and become increasingly resistant to platinum analogues. PARP inhibitors have been recently approved to treat HGSOC; however, the greatest clinical benefit from PARPi monotherapy has been mainly observed in tumors with homologous recombination (HR) deficiency. Therefore, there is an urgent unmet medical need to develop alternative therapeutic strategies for patients with HGSOC. Given that the transcriptional program is remarkably dysregulated in cancer, resulting in cancer dependency on specific components of the transcriptional program (termed “transcriptional addiction”), targeting transcriptional CDKs is emerging as a new strategy for cancer treatment. Among the transcriptional CDKs, CDK7 shows the most significant copy number loss (dominantly hemizygous) across multiple cancer types with the highest deletion score in HGSOC. Importantly, HGSOC cells with hemizygous loss of CDK7 are highly sensitive to CDK7i treatment. Additionally, CDK7 loss is correlated with increased sensitivities to DNA damaging drugs such as PARPi and platinum. Inhibition of CDK7 preferentially represses the expression of genes in the DNA damage repair pathways and impairs the activity of HR. Therefore, we hypothesize that hemizygous loss of CDK7 is a targetable vulnerability in HGSOC, and that CDK7i alone or in combination with DNA damaging agents is a novel strategy to treat patients with HGSOC. Low-dose CDK7i treatment can preferentially repress a group of HGSOC-associated genes that are driven by super enhancers, serving as an effective and tolerable treatment for a select population of patients with HGSOC. The central goal of this application is to study the mechanistic basis and translational potential of CDK7-targeted therapy in HGSOC. We have assembled a team of collaborators with added expertise and resources to test the above hypothesis through three specific aims. Specific Aim 1. Evaluate whether hemizygous loss of CDK7 is a targetable vulnerability in HGSOC. Specific Aim 2. Characterize the molecular mechanisms of low-dose CDK7i treatment in DNA damage response. Specific Aim 3. Evaluate CDK7i in combination with FDA-approved therapies in preclinical models of HGSOC. Our proposed studies may provide strong rationale for clinical development of CDK7 inhibitors for ovarian cancer treatment.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY/ABSTRACT The number of Americans with Alzheimer's disease and related dementias (ADRD) is projected to reach 13.8 million by 2050. Given the current lack of disease-modifying treatments for ADRD coupled with evidence that ADRD develops over decades, there is growing interest in developing prevention strategies. Middle age (i.e., 45-64 years) is increasingly recognized as a key life course period to target modifiable risk factors for cognitive decline and ADRD. Prior studies have identified individual-level risk factors in middle age, including chronic conditions such as hypertension and obesity and health-related behaviors such as excessive alcohol use. The 2020 Lancet Commission estimated that addressing these mid-life risk factors could result in up to 15% of dementias being prevented or delayed. However, recent data suggests the need to look beyond individual- level factors to examine the potential role of environmental exposures to improve health outcomes in mid-life and reduce overall dementia burden. Since 1990, the prevalence of chronic conditions that are risk factors for ADRD has increased in middle-aged adults, particularly in individuals with lower socioeconomic status (SES). The reasons for these trends are not yet fully understood, but a leading hypothesis is that lower SES in middle age is leading to premature onset of aging-related conditions through exposures in the social and physical environment (e.g., levels of social support, crime, food insecurity, pollution). A growing number of studies have linked such environmental factors to risk of cognitive decline and ADRD in older adults, and these factors could also be impacting cognitive trajectories and risk of ADRD in middle age. However, key knowledge gaps remain. First, it is unknown if cognitive outcomes are worsening in middle-aged adults, nor if environmental exposures increase the risk of cognitive decline in middle age. Additionally, whereas cognitive decline and ADRD in older adults result from the interplay of environmental and genetic risk factors, it is unclear how genetic risk contributes to cognitive decline in middle age. A longitudinal study of middle-aged adults that includes measures of environmental exposures and genetic data is needed to address these questions. The objective of the proposed project is to examine if cognitive outcomes are worsening in middle age and how environmental and genetic risk factors contribute to cognitive decline in this age group. The specific aims are to: (1) examine the epidemiology of cognitive impairment in middle-aged adults in the U.S., including identifying cognitive trajectories and incidence of “cognitive impairment no dementia” (CIND) over time; (2) determine if environmental and genetic factors predict cognitive trajectories and incident CIND; and (3) replicate our analyses in a second dataset. We will complete these aims using longitudinal nationally representative data from the NIA-funded Health and Retirement Study. Findings will help elucidate the burden and mechanisms of cognitive decline in middle age and inform policy planning to mitigate the projected burden of dementia.