University Of Wisconsin-Madison

NIH Research Projects · FY 2026 · 2024-05

Fragile X Syndrome (FXS), caused by deficiency of RNA binding protein Fragile X Messenger Ribonucleoprotein 1 protein (FMRP), encoded by X-linked FMR1 gene, is the most common heritable cause of intellectual disability and a top contributor to autism spectrum disorders (ASD). The mechanisms underlying FXS are not fully clear. Our knowledge of FMRP functions in the mammalian brain are mostly obtained from rodent studies. However, there are significant differences between primate and rodent brains, particularly in the prefrontal cortex (PFC) where FMRP is highly expressed. PFC is critical for myriad higher-order brain functions, such as working memory, planning, decision-making, language, and creative intelligence, which are severely affected in FXS. Primate PFC is significantly larger proportionally and much more complex compared to other species and exhibit gene signatures unique to primates. To date, clinical trials based on rodent models have not achieved primary endpoints highlighting a critical need for complementary primate models to better understand FXS. FMRP binds many mRNAs in the brain. An established role of FMRP is that it promotes neuronal maturation and synaptogenesis during postnatal development,. On the other hand, FMRP is also robustly expressed in human and mouse cortex during prenatal development and its deficiency affects mouse cortical development. In contrast to its well-established role in postnatal brain development and function, the role of FMRP in prenatal brain development is not well studied, especially in primates. In addition, Developmental stage-specific roles of FMRP have not been fully investigated and primate-specific targets of FMRP have not been systematically identified. The goal of this project is to investigate functions of FMRP in primate prenatal brain development and unveil developmental stage-specific roles of FMRP in primate brains. We will test the hypothesis that FMRP regulates genes critical for functional maturation of PFC neurons, and its deficiency leads to altered gene expression and impaired cortical development. We determine the impact of FMRP-deficiency on gene expression changes that impair neuronal maturation during primate prenatal development using Patch-seq. We will identify FMRP-mRNAs and FMRP-protein interactomes in the PFC that regulate primate brain development using CLIP-seq and Co-IP-mass spectrometry, respectively. The proposed work will fill a major gap of our knowledge in understanding function of FMRP in primate brain development. Such understanding is critical for better therapeutic development for FXS as well as other neurodevelopmental disorders including ASD.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Over the past decade, dual use of combustible cigarettes and electronic cigarettes (e-cigarettes) has risen substantially. The evidence is mixed as to whether this use pattern helps dual users quit using combustible cigarettes. However, there is a clear need to help dual users quit smoking, given that use of combustible cigarettes is the leading preventable cause of death and disease in the US, causing one-third of all cancer deaths and exacting an enormous financial and personal toll. To date, no published studies have identified effective pharmacotherapy or counseling interventions to help dual users quit smoking. The goal of the proposed research is to identify the most effective pharmacotherapy and counseling approaches to help dual users quit smoking. Specifically, using the Multiphase Optimization Strategy (MOST), dual users of combustible cigarettes and e-cigarettes (N=500) who are motivated to quit smoking and willing to quit their e- cigarette use, if asked, will be enrolled in a randomized efficient 2x2x2 factorial clinical trial. Participants will be randomized to one of two levels of each factor: Pharmacotherapy (Varenicline vs. Nicotine Patch), Counseling Approach (a Dual Focused Cessation approach in which participants are encouraged to quit their e-cigarette use as part of their quit attempt vs. a Smoking Focused Cessation approach in which participants are encouraged to quit smoking and to use their e-cigarettes strategically to replace their cigarettes in service of harm reduction), and Counseling Intensity (4 vs. 1 session). All participants will complete baseline measures of demographics, tobacco use history, combustible and e-cigarette dependence, and potential mechanisms. Participants will use smartphones to complete ecological momentary assessments (EMA) for 2 weeks pre- target quit date (TQD) and 2 weeks post-TQD; participants will record smoking, vaping, and medication use in real-time and complete EMAs of potential mechanisms (e.g., craving reduction, self-efficacy) every evening. Follow-up assessments of combustible and e-cigarette use, as well as potential mechanisms, will occur 4, 8, 12, 18, 26, 39, and 52 weeks post-TQD. Participants will also complete biochemical assays of combustible tobacco use (carbon monoxide [CO]) and nicotine exposure (urine cotinine) at baseline, one-week pre-TQD, and 3 months post-TQD. Participants who report 7-day point-prevalence abstinence from smoking at Weeks 26 or 52 will be invited to the laboratory to provide biochemical verification of abstinence (CO<6 ppm) and provide a urine sample to assay nicotine exposure (cotinine). These data will allow us to identify the best medication approach as well as the best counseling approach and intensity to help dual users quit smoking.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Hospitalized children who experience cardiopulmonary deterioration are at increased risk for mortality and long- term morbidity. Because timely intervention increases survival in children, it is critically important to identify cardiopulmonary deterioration events as early as possible. However, the current paradigm for detecting these events in advance has several gaps. First, existing risk prediction methods can lead to fragmented care, as each unit employs different tools for predicting specific outcomes. For example, risk prediction within the emergency department (ED) is targeted toward triage; ward-based tools predict the risk of being transferred to the intensive care unit (ICU), while the ICU focuses on determining the likelihood of death or cardiac arrests. Transitioning from multiple, siloed risk assessment tools to a single, hospital-wide cardiopulmonary deterioration prediction model could significantly improve outcomes for children. A second critical gap is that current prediction model outputs are not accompanied by helpful explanations, a need unmet by standard machine learning (ML) explainers due to inherent limitations. Developing new algorithms that provide real-time interpretations of model outputs may increase situational awareness, decrease diagnostic delay, and enable better treatment selection. Third, to ensure high usage and effective decision-making, any new model should be accompanied by a user interface explicitly designed using human factors engineering principles. The long-term goal is to improve outcomes among children experiencing cardiopulmonary deterioration by enabling better quality of care. The overall objective of this project is to develop a new clinical decision support (CDS) tool that is accurate, interpretable, and actionable for early detection of cardiopulmonary deterioration events in children. In Aim 1, we will use electronic health record (EHR) data from pediatric admissions to four academic hospitals and ML to derive and externally validate a new hospital-wide cardiopulmonary deterioration prediction model and compare performances to our preliminary model. In Aim 2, we will develop novel algorithms that provide physiological explanations and clinical context for model predictions for a given patient. Finally, in Aim 3, we will create a new CDS tool that embeds the best-performing prediction tool and explainer algorithm outputs within a graphical user interface purposefully designed to facilitate increased user interaction. The proposed research is innovative because it incorporates deep learning-based pediatric risk prediction, real-time explainable algorithms with highlighted clinical context, and human factors engineering for developing the CDS tool. In addition, the proposed work is significant because it will result in a new, accurate, interpretable, efficient, and user-friendly CDS tool for risk assessment throughout a pediatric hospital. Ultimately, this powerful tool will enable early recognition of pediatric cardiopulmonary deterioration events, facilitating timely diagnosis and intervention to improve outcomes among hospitalized children.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Frontotemporal dementia (FTD) is the most frequent dementia in patients under 60 years old. Mutations in MAPT (the gene encoding for the Tau protein), including the autosomal dominant R406W missense point mutation, have been linked to FTD. Current evidence supports the concept that FTD neurodegeneration starts decades before symptom onset, yet genetic FTD studies in young adults or minors are seldomly done, due to the challenges of disclosing genetic condition and the burden of long-term testing. Defining when the pathological processes begin will be critical to administer neuroprotective therapies before substantial neuronal loss and, therefore, to prevent FTD onset. At the Wisconsin National Primate Research Center (WNPRC) we currently have 6 rhesus macaque carriers of an exact replica of the human MAPT R406W mutation. They are 3 adults (19, 6 & 7 yrs.), a young adult (4 yrs.), a juvenile (2 yrs.) and an infant (0.6 yrs.); 3 more offspring are due October/November 2023. These nonhuman primate (NHP) carriers provide a unique opportunity to longitudinally and systematically assess biomarkers of FTD-tau. The overarching goal of this proposal is to identify the earliest biomarkers of FTD and their timeline of onset across the lifespan of MAPT R406W rhesus, aiming to inform clinical translation. Based on our preliminary findings, we hypothesize that prodromal FTD-tau begins during the transition period of sexual maturation from juvenile to young adult. To test this hypothesis, we propose three Specific Aims: 1) To characterize across the lifespan the onset of FTD-like cognitive, mood and motor behaviors of rhesus MAPT R406W mutation carriers; 2) To identify early imaging predictors of brain neurodegeneration in rhesus MAPT R406W mutation carriers. 3) To assay CSF and blood fluid biomarkers of FTD-tau linked to neurodegeneration in rhesus MAPT R406W mutation carriers and assess their temporal relationship with imaging and behavioral biomarkers. We will access databases at WNPRC for NHP normative rhesus data. To facilitate harmonization between old and new datasets, additional MRI and biofluids assays will be collected from animals of the WNPRC colony. We will maximize the translational value of the NHP resource, by comparing the results to human datasets available through the Tau consortium. This rhesus study is timely due to the availability of the MAPT R406W monkeys generated by the R61/R33 NS115103 award, and fulfills a gap in clinical research. The NHP results will be enriched in the future by postmortem analyses, as more rhesus carriers become available. The findings generated by this rhesus project will inform on the impact of the tau mutation in early neurodevelopmental stages and assist in the design of future neuroprotective preclinical and clinical studies.

NIH Research Projects · FY 2026 · 2024-05

Project Summary/Abstract: Colitis-associated colorectal cancer (CAC) is one of the most deadly types of colorectal cancer (CRC) that causes the most morbidity and mortality in patients with inflammatory bowel disease (IBD). The early detection of dysplastic or malignant lesions in patients with ulcerative colitis is challenging, which resulted in delayed diagnosis and limited therapeutic efficacy of CAC. Bile acids (BAs), as early dietary sensors, are critical mediators of gut physiology, partly by affecting their master receptor, Farnesoid X Receptor (FXR) 's activity. BAs overproduction is implicated in diarrhea associated with IBD and CAC, with which the function of FXR is severely compromised. The BAs-FXR axis is a convergent point of genetic and dietary risk factors of CRC. In addition, FXR is a crucial modulator of the pro-and anti-inflammatory responses in the intestinal epithelium. Intestinal macrophages constitute the largest pool of macrophages in the body and have emerged as crucial sentinels for gut microbiome and metabolite recognition. However, due to the exceptional plasticity of gut macrophages during cancer progression, there is a major lack of mechanistic understanding of how gut macrophages sense BAs and how FXR regulates gut macrophages’ function, and mediate its interaction with other cell types in CAC. Our preliminary studies revealed that epithelium damage and intestinal inflammation resulted in the loss of BAs homeostasis and compromised FXR signaling in CAC mice models. Our overarching hypothesis is the dysregulated BAs-FXR axis further induces pathological immune landscape changes, especially in macrophages, which crosstalked with Th17 cells, together promoting tumorigenesis. This proposal aims to evaluate the mechanisms of action (MOA) of FXR agonism including FDA-approved Obeticholic acid (OCA) on gut macrophages’ differentiation and function in vitro and in vivo, including expansion of animal testing to delineate breadth of this anti-cancer efficacy on both colitis and CAC, as an immunomodulator drug. Three specific aims will be pursued: Aim 1 will investigate the impact of inflammation- and tumor-induced, host- and microbial-derived BAs on macrophage and its crosstalk to T cell and intestinal stem cells. Aim 2 will investigate the gut macrophges’ dependence on FXR in modulating its function in CAC mice model. Aim3 of this proposal will determine the therapeutic utility of activation of FXR in gut macrophage for the treatment of CAC. Besides, we will also validate the combination therapies to maximize the efficacy of cancer treatments including rational combinations of anti-IL23 and FXR agonists. We believe investigating the role of the BAs-FXR axis in inflammation-induced and tumor-infiltrated Macrophages will facilitate our understanding of the etiology of colitis- induced colon cancer. The proposed study of FXR ligands will hasten the development of novel therapeutics for this debilitating and most lethal colorectal cancer.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY Wilms tumor (WT) is the second most common solid pediatric cancer that usually develops in young children before age 5. WT encompass three main cell types: epithelium, stroma, and blasterma. WT are typically treated with a combination of surgery, chemotherapy and radiotherapy. Although the survival rate for WT patients is generally favorable, prognosis for patients with blastema dominant histology remain poor. Patients with blastema dominant histology are more resistance to chemotherapy and have higher rate of cancer recurrence, therefore there is unmet clinical need to improve therapy. The major obstacle for developing novel therapies is the lack of cell lines and animal models representing high-risk blastemal WT. Because blastemal WT represents aberrant ESCs, we reprogrammed WiT49 WT cells of epithelial origin into induced pluripotent stem cells (iPSCs) and transplanted iPSCs into kidneys of mice. The tumors metastasized to the liver and lung. Thus, WiT49-iPSCs represent the first blastemal WT model suitable for mechanistic studies to understand the molecular changes distinguishing epithelial and blastemal types of WT and can be used to screen FDA-approved drugs to find better treatments for children with aggressive WT. Because WiT49-iPSCs acquire stem cell markers as well as metastatic traits as compared to WiT49, we hypothesize that WiT49- iPSCs serve as a blastemal WT model suitable for mechanistic studies and drug screening to develop novel therapies for metastatic blastemal WT. In this proposed study, we will (1) reprogram WiT49-iPSCs and perform the teratoma assay to determine stem cell potency. (2) identify the epigenetic and chromatin signatures distinguishing the blastemal and epithelial types of WT. (3) develop novel therapeutic strategies for the treatment of WT. We will screen WiT49 and WiT49-iPSC cells against FDA-approved drugs and epigenetic compounds to identify drugs effective against WiT49-iPSCs versus WiT-49 cells. Successful completion of this project is expected to have a great impact on understanding the biology of kidney cancer and establishing the invaluable experimental platform for the development of safer and more effective therapies for Wilms tumors.

NIH Research Projects · FY 2025 · 2024-05

PROJECT ABSTRACT/SUMMARY People with Down syndrome (DS) have a 75-90% lifetime risk for developing Alzheimer’s disease (AD).2,3 However, there is marked variability in the timing of AD, with a 30+ year range in the age of onset of clinical dementia8, suggesting that factors beyond trisomy 21 may influence AD in DS. Outside of DS, mid-life obesity or being overweight is an identified risk factor for later life AD, such that efforts to reduce body mass index (BMI) and increase physical activity have been proposed to be promising AD prevention programs. Adults with DS are at risk for being overweight and obese40--42, yet virtually nothing is known about whether mid-life BMI is associated with the timing of AD pathology and symptomology in DS. To date, studies examining BMI and AD in DS have focused on BMI after a clinical AD dementia diagnosis,49,51 used primarily cross-sectional methods,49,50 and/or relied on small sample sizes.51 The proposed F31 will provide training on advanced statistical analytic methods to assess longitudinal data, grow my knowledge on the valid assessment of AD pathology and cognition in DS, and develop professional skills for engaging in multisite research consortiums. The project will leverage already existing data to address three specific aims: 1) evaluate the effect of BMI and weight change on AD biomarkers (PET Aβ and tau PET) across 36 months, 2) examine the effect of BMI and weight change on cognitive performance (episodic memory, executive functioning, motor planning and control, and dementia symptoms) and clinical dementia status (cognitively stable, mild cognitive impairment, and dementia) across 36 months, and 3) explore if inflammatory biomarkers (serum c-reactive protein, IL-6, IL-10, and tumor necrosis factor alpha) are related to BMI and mediate the link between BMI and AD pathology and cognitive decline. This work will help me launch an independent program of research on healthy aging in DS that can inform social policy and intervention for preventing or delaying AD in DS and other at-risk populations.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY The overall goal of this proposal is to improve histotripsy treatments of liver tumors by developing an accurate, clinically translatable workflow for targeting liver tumors transcostally. Globally, there are ~780,000 deaths from liver cancer each year and in the US, this is expected to rise by more than 150% in the next 30 years. Histotripsy is an emerging noninvasive, nonthermal and nonionizing focal tumor treatment that has recently undergone a successful Phase I human clinical trial for treatment of liver tumors. It uses focused, short duration ultrasound pulses to produce tissue cavitation at a focal point. Cavitation occurs from nanometer scale gas pockets present in tissues, which rapidly expand and collapse, causing a high stress and strain that mechanically disrupts adjacent tissues and cells. Histotripsy damage is a binary process where tissue is either destroyed or it isn’t, providing many benefits over current thermal ablation techniques. A critical limitation of histotripsy is the sole use of ultrasound to visualize and target the tumors. Targeting with diagnostic US limits treatments to a subcostal approach due acoustic blockage from the rib cage, leaving an estimated ~50% of liver tumors non-visualizable and therefore, untreatable. With the recent development of cone-beam CT-guided histotripsy, tumors can be visualized throughout the liver, including beneath ribs, but cannot be accurately targeted due to acoustic aberrations from intervening ribs potentially altering treatment size and location. In this proposal, we will develop an accurate, clinically translatable workflow for targeting liver tumors for transcostal histotripsy. This will be accomplished by first quantifying the effect intervening ribs have on targeting accuracy through ex-vivo and in- vivo experiments (porcine model) (Aim 1). Secondly, a robust machine learning model will be developed to predict targeting offsets, which will be trained and validated with ex-vivo, in-vivo, and in-silico datasets (Aim 2). Finally, the model will be incorporated and tested to deliver patient-specific treatment plans in rabbit VX2 liver tumors (Aim 3). This proposal includes radiographic image analysis of ex-vivo and in-vivo data, histopathologic assessment of in-vivo treatments (Aims 1 and 3) and development and validation of a robust machine learning algorithm to create a multi-dimensional analysis of a novel targeting method for histotripsy treatments. An accurate and robust targeting method for performing transcostal histotripsy of liver tumors is crucial to expanding the cohort of patients eligible for the therapy. It has the potential to almost double the number of patients with liver tumors eligible for the treatment and the technique is translatable to tumors in other organs (eg, kidney and pancreas) as well.

NIH Research Projects · FY 2026 · 2024-04

SUMMARY/ABSTRACT: Diabetes is a costly and complex chronic illness and a serious public health problem. The number of individuals with diabetes, particularly obesity-linked type 2 diabetes (T2D), is certain to increase over the next decades. Shockingly, the children of today have an estimated overall lifetime risk of developing diabetes of nearly 50%. Therefore, developing new methods to prevent T2D and properly treat T2D patients is exceptionally timely and of great significance. The progression to T2D is increasingly being linked with changes in cellular and molecular signaling pathways in the insulin-secreting pancreatic β-cell, preventing adequate insulin secretion to stimulate the body’s cells to take up glucose from the blood. Yet, few T2D drugs specifically target the β-cell, and those that do are not effective in all individuals and are controversially linked with β-cell failure long-term. Two molecules that are cornerstones of our research program are prostaglandin EP3 receptor (EP3), a G protein-coupled receptor (GPCR) for the arachidonic acid metabolite, prostaglandin E2 (PGE2), and its associated G protein alpha subunit, Gαz. Work from our group and others has definitively shown EP3 expression and signaling is increased in β-cells of T2D mice and human organ donors and blocking EP3 signaling can stimulate β-cell insulin secretion and proliferation. These exciting results suggest targeting EP3 and/or Gαz might increase the number of functional β-cells in T2D; yet, much more work is necessary to achieve this. Our long-term goal is to fully characterize the EP3 and Gαz activation and signaling pathways during the progression to T2D at the whole body, tissue, cellular, and molecular levels, providing us key information on how to target this β-cell pathway in T2D. The overall objective of this work, which is the next logical step in pursuit of our goal, is to define the molecular signaling pathways responsible for the impact of EP3 signaling, both Gɑz-dependent and -independent, on mouse and human β-cell compensation and β-cell failure. Our central hypothesis is EP3 and Gαz, when active, modulate intracellular signaling pathways critical for β-cell compensation in obesity but, when chronically active, contribute to β-cell dysfunction and loss in T2D. We will test our central hypothesis with a combination of innovative cellular imaging, metabolomics, and proteomics assays, correlating changes in EP3 and Gɑz signaling pathways with measurements of β-cell function in response to glucose and glucagon-like peptide 1: a well-accepted class of T2D drugs. We will accomplish this goal by pursuing the following three Specific Aims: 1. Determine effects of EP3 on discrete cellular pools of β-cell Ca2+ and cAMP and downstream β-cell function and mass; 2. Quantify Gɑz-dependent Rap1GAP translocation, Rap1GAP phosphorylation, and their effects on β-cell function and mass; and 3. Quantify changes in the human islet arachidonic acid metabolome, and the functional consequence of these changes, during the progression to T2D. This work, when completed, will provide a much more complete understanding of the role of the β-cell and its signaling molecules in the progression to and pathophysiology of T2D.

NIH Research Projects · FY 2026 · 2024-04

Well-characterized longitudinal epidemiological cohorts are crucial resources for identifying novel risk factors, examining exposure-outcome associations, and determining the role of biomarkers in disease development and progression. The Survey of the Health of Wisconsin (SHOW) is a unique environmental epidemiological cohort where rigorous survey methodology was used to ensure state-wide participation from Wisconsin, resulting in a representative sample. Based on our successful experience in participant recruitment, we propose to initiate SHOW-Forward to further expand the cohort, enrich already collected data and enhance its access to the research community to facilitate novel environmental epidemiology and translational research aligned with the NIEHS mission. To accomplish these goals, we propose three specific aims. First, we will expand the cohort (renamed as REACH) through continued and enhanced outreach and engagement across Wisconsin. Novel approaches for participant-centric biospecimen collection will be implemented to reduce the burden for participants from remote areas. Second, we will develop a Real-World Data Collaborative infrastructure as a foundation for secure integrative environmental health research and translation. Third, we will establish the administrative infrastructure to develop, refine, and implement ethical and compliant participant recruitment, acquisition, linkage, usage, analysis and sharing of PHI- containing multi-dimensional data and annotated biospecimens in SHOW-Forward. Ultimately, this collaborative infrastructure will facilitate broad access to SHOW data and biospecimens, enable early detection and treatment of emerging health issues, improve understanding of the role of environmental and lifestyle factors, harmful chemical exposures, and genetics on human health, and unlock new pathways for evidence-based prevention of environmentally caused diseases.

NIH Research Projects · FY 2025 · 2024-04

Well-characterized longitudinal epidemiological cohorts are crucial resources for identifying novel risk factors, examining exposure-outcome associations, and determining the role of biomarkers in disease development and progression. The Survey of the Health of Wisconsin (SHOW) is a unique environmental epidemiological cohort where rigorous survey methodology was used to ensure state-wide participation from Wisconsin, resulting in a representative sample. Based on our successful experience in participant recruitment, we propose to initiate SHOW-Forward to further expand the cohort, enrich already collected data and enhance its access to the research community to facilitate novel environmental epidemiology and translational research aligned with the NIEHS mission. To accomplish these goals, we propose three specific aims. First, we will expand the cohort (renamed as REACH) through continued and enhanced outreach and engagement across Wisconsin. Novel approaches for participant-centric biospecimen collection will be implemented to reduce the burden for participants from remote areas. Second, we will develop a Real-World Data Collaborative infrastructure as a foundation for secure integrative environmental health research and translation. Third, we will establish the administrative infrastructure to develop, refine, and implement ethical and compliant participant recruitment, acquisition, linkage, usage, analysis and sharing of PHI- containing multi-dimensional data and annotated biospecimens in SHOW-Forward. Ultimately, this collaborative infrastructure will facilitate broad access to SHOW data and biospecimens, enable early detection and treatment of emerging health issues, improve understanding of the role of environmental and lifestyle factors, harmful chemical exposures, and genetics on human health, and unlock new pathways for evidence-based prevention of environmentally caused diseases.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Estrogen receptor positive (ER+) breast cancer (BC) accounts for the majority of all diagnosed BC for women in the United States. While traditional antihormone therapies for ER+ BC are initially effective, approximately 40% of ER+ BC will develop therapy resistant recurrence and metastasis, which contributes to most BC related deaths. The presence of the G-protein coupled estrogen receptor (GPER) is suggested to aid in disease progression and metastasis of BC and GPER is theorized to be a driver of therapy resistance due to the agonistic activity of tamoxifen, a traditional antihormone therapy for ER+ BC. GPER is expressed on cancer- associated fibroblasts, the main remodelers of the extracellular matrix (ECM) which plays an important role in disease progression. Previous work by Dr. Suzanne Ponik and collaborators has demonstrated dynamic interactions between hormone signaling and ECM stiffness to drive tumor cell invasion and metastasis. However, the mechanism responsible for these dynamic interactions is poorly understood. Therefore, the hypothesis for the proposed research is that GPER activated CAFs are priming the TME to drive tamoxifen resistant disease progression in ER+ BC. The research strategy proposes to establish the specific role GPER activated CAFs play on ECM protein deposition and organization in vitro prior to introducing GPER knockdown CAFs into an orthotropic ER+, tamoxifen resistant tumor model. The in vitro and in vivo work will be complemented with a novel tumor microarray (TMA) from BC patients where the association between GPER expression, ECM signatures, administered therapy and patient outcome can be validated. The specific aims of this proposal are: 1) identify how GPER signaling in CAFs regulates biophysical/biochemical cues and how GPER-mediated matrix deposition influences tumor cell behavior in vitro, 2) test the hypothesis that ECM stiffness and antihormone therapy synergize through GPER signaling in CAFs to drive tumor progression in vivo, and 3) define how GPER expression and ECM signatures relate to human disease/patient outcomes. The research training for this proposal will be conducted under the co-sponsorship of Dr. Suzanne Ponik (Sponsor) and Dr. Mark Burkard (Co-Sponsor) at the University of Wisconsin-Madison. Both Dr. Ponik and Dr. Burkard will provide mentorship on the research conducted by Shelby Fertal as well as provide guidance for her career development with the intention that Shelby will remain in academia. The success of this proposal will offer novel insights into the mechanisms of antihormone therapy driven disease progression in ER+ BC and provide a solid foundation for Shelby’s career as an independent research professor.

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract Breast surgeons have been highly successful at deimplementing some low-value surgical care but less successful at deimplementing care that is preference-sensitive. Sentinel lymph node biopsy for women ≥70 years of age (SLN over 70) and contralateral prophylactic mastectomy (CPM) have been deemed low-value by national surgical societies. However, use has remained unchanged or even increased over time. Patients are frequently motivated to pursue these operations because of perceived prognostic value or to minimize future cancer risk. However, while SLN over 70 and CPM have no impact on survival, they may have a substantial negative impact on psychosocial, physical, and financial outcomes. Surgeons endorse these operations should not be routinely performed, but also acknowledge that the perceived benefits of the procedures can outweigh the potential risks for some women. A critical gap towards supporting deimplementation of low-value preference-sensitive care is our understanding of how decisions are made within the patient-surgeon interaction. By directly studying patterns of conversation associated with deimplementation, we can understand whether patients’ preferences are driving the treatment plan and what tools may help surgeons support patients in these conversations. In an R01 funded study (NCT03766009), we audio-recorded 594 surgical consultations from a racially and socioeconomically diverse sample of breast cancer patients cared for at academic and community centers across the United States. These consults have been transcribed and the content categorized. The objective is to use this novel dataset to understand the relationship between what surgeons and patients say during the consult, and how deimplementation decisions for SLN over 70 and CPM are made. We have three aims: Aim 1, test the association between performance of shared decision making and deimplementation of preference-sensitive low-value care; Aim 2, identify patterns of surgeon-patient interactions that are associated with deimplementation of preference-sensitive low-value care; Aim 3, develop a conversation guide to support surgeons in discussing preference-sensitive low-value care. This R21 study will generate key understanding about how decisions to deimplement preference-sensitive low-value care are made within the patient-surgeon interaction. By combining our insights with the existing literature on surgeon and patient attitudes and beliefs, we will develop a multi-faceted strategy to support deimplementation of preference-sensitive low-value care which can then be tested in a future R01 study.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that is poised to reach epidemic proportions given our rapidly aging population. As such, the identification of risk and resilience factors that might curb the progression of AD and delay symptom onset is a national public health imperative. Multiple emerging lines of research demonstrate that sleep disturbance, and particularly obstructive sleep apnea (OSA, a very common disorder in older adults), and poor cardiorespiratory fitness (CRF) are associated with increased risk for AD pathology and cognitive decline. Furthermore, new evidence suggests that OSA and CRF may interact in their relation to AD; however, thus far, this potential interaction is poorly understood because of the dearth of relevant data and because AD pathology is present long before detectable signs of dementia are observed. The overall objective of this project is to fill a critical gap in AD research by deploying advanced methods to estimate the age of onset of AD biomarkers in the Wisconsin Sleep Cohort (WSC), relate this emergent pathophysiology to midlife OSA and fitness, and determine how midlife OSA and fitness influence trajectories of cognitive impairment in later life. The WSC has followed adult participants since the late 1980s and is the only longitudinal cohort with objective sleep, fitness, and neurocognitive data spanning decades to explain how OSA and CRF interrelate to predict AD onset. Accordingly, by leveraging the unique sleep and health data available in the WSC Study, results from this proposal will elucidate midlife risk transducers of AD pathology and dementia. In this investigation, we will prospectively collect blood plasma, MRI, and PET biomarkers of AD and neurocognitive data in a sample of 300 WSC participants who are now older aged, to address two Specific Aims with testable hypotheses supported by preliminary data: Aim 1 will identify the effects of more severe OSA and poorer midlife CRF on multiple AD biomarkers, determine whether midlife OSA and CRF delay the age of amyloid onset, a key indicator of AD pathology, and examine if trajectories of OSA and CRF interact to predict AD pathology in later life; Aim 2 will determine whether similar patterns of OSA and fitness are associated with clinical endpoints of cognitive decline and dementia. For all Aims, it is expected that higher CRF will serve as a protective factor, moderating relations between OSA and AD pathology and cognitive decline. Detailed sleep and health history data available in the WSC allows for key covariates to be evaluated in analyses interrogating our Aims. Addressing the Aims of this application will fill a significant and critical need in AD research by capitalizing on an existing cohort with extensive midlife phenotyping of two salient lifestyle factors associated with lower AD risk. In so doing, this project will ultimately lead to improved preventative and therapeutic strategies that target sleep and fitness as modifiable risk factors for AD.

NIH Research Projects · FY 2026 · 2024-04

PROJECT ABSTRACT Voice disorders are among the most common communication disorders and estimated to affect 3-9% of Americans annually. These disorders are often associated with laryngeal inflammation secondary to epithelial injuries following phonotrauma, surgical resection, radiation, and various infectious processes. Serious injury can lead to disordered mucosal remodeling, scar formation, and a profound dysphonia. Bacterial dysbiosis, a disturbance in the microbial community structure, is implicated in a variety of inflammatory disease etiologies in mucosal systems, however, understanding of laryngeal microbiota and their protective mechanisms against mucosal inflammation remains limited. To date, laryngeal microbiome studies have been limited to characterization of microbial communities in disease states of the larynx. Research in regard to bacterial contributions to the maintenance and regeneration of laryngeal mucosa is still lacking. The overall goal of this work is to identify bacterial species associated with each stage of epithelial wound healing in the larynx and define the role of laryngeal microbiota in modulation of epithelial regeneration through a longitudinal study using a unique combination of gnotobiotic laryngeal model and naphthalene induced epithelial injury model. Aim 1 will investigate the role of resident microbiota in the acute wound healing of laryngeal epithelium by measuring the expression of inflammatory markers and barrier protection associated genes in the larynges of naphthalene injected germ-free mice and gnotobiotic mice colonized with laryngeal microbiota. Aim 2 will identify wound-associated bacterial species by delineating the longitudinal dynamics of bacterial compositions at multiple timepoints through wound healing process. Gnotobiotic mice colonized with candidate bacterial species will be assessed with and without antibiotics treatment to confirm their beneficial or pathogenic influence that promotes or inhibits laryngeal wound healing. Our overarching hypothesis is that laryngeal microbiota is a significant contributor to the healing of injured epithelium in the larynx. By clarifying the role of resident microbiota in laryngeal wound repair process, completion of our specific aims will have a direct significant impact on our current understanding of laryngology, bacteriology, and laryngeal immunology. Results of this novel application will lead to new facets of understanding for host-microbiome interactions and will ultimately be used to develop innovative prevention and treatment strategies for laryngeal diseases with mucosal injury.

NIH Research Projects · FY 2026 · 2024-04

Abstract Targeted protein degradation by chimeric molecules has emerged as a novel therapeutic modality. The bifunctional chimeric molecules usually have one end binding to the protein of interest (POI) and the other end directing the ternary complex towards the degradation in proteasome or lysosome. The PROteolysis TArgeting Chimera (PROTAC) received the most attention to date. PROTACs contain an E3 ubiquitin ligase ligand to direct the POI for ubiquitination and route it to the proteasome for degradation. Because of this, PROTACs are only capable of depleting intracellular proteins. About 40% of the proteome are extracellular secreted and membrane proteins and many of them are related to cancers. To broaden the scope of targets to include extracellular proteins, the LYsosome TArgeting Chimeras (LYTAC) were recently reported by us and others. LYTACs are created by conjugating a ligand of a lysosome targeting receptor (LTR) on the cell surface with a ligand that can bind to the extracellular POI. The first two LTRs employed for LYTACs are lectins or carbohydrate binding proteins: cation-independent mannose 6-phosphate receptor (CIM6PR) and asialoglycoprotein receptor (ASGPR). The receptor-ligand interaction triggers the internalization of the extracellular proteins through receptor-mediated endocytosis, further inducing the degradation of the targets in the lysosome. However, CIM6PR is ubiquitously expressed in most types of cells, while ASGPR is mainly expressed on liver. It would be ideal to recruit a LTR that is overexpressed on cancer cells to degrade extracellular POIs associated with cancers to achieve high efficiency and selectivity. After examining a series of cell surface receptors that are overexpressed on cancers, we found that chimeric molecules that recruit folate receptor (FR) could degrade extracellular POIs on cancer cells highly effectively. In this application, we will demonstrate for the first time that the conjugation of FR ligand, folate (FA), to binders of POI on the membrane of cancer cells can create degraders that are capable of selectively degrading the endogenous membrane POI on cancers. We propose to exploit the potential of this strategy to develop novel FA-antibody conjugates that can degrade check point inhibitors, such as programmed death ligand 1 (PD-L1), for the treatment of various cancers with a focus on head neck cancers (HNC). In Aim 1, we will synthesize various FA-antibody conjugates and test their degradation activity in cells. In Aim 2, we will evaluate the in-vivo degradation activity and anti-tumor efficacy of the PD-L1 degrader in syngeneic and humanized mouse models with a focus on HNC. In Aim 3, we propose to study the mechanism of the FR-mediated degradation of PD-L1 and the underlying principle for improved anti-tumor efficacy. These studies will facilitate the establishment of a general platform for the development of efficient and tissue-selective degraders for cancer associated extracellular POIs, which will lead to effective therapeutics for the treatment of many types of cancers.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY: This R21 project will develop new classes of synthetic liquid-infused porous surfaces (or `SLIPS') that address challenges related to bacterial fouling and virulence in clinical and healthcare settings. Our objectives will be accomplished by the pursuit of two focused and synergistic Aims: (1) to explore designs of novel drug-patterned `proto-SLIPS' that can release potent and highly water-soluble antibiotics and thereby reduce biofouling and bacterial load, and (2) to explore new designs of `proto-SLIPS' that release synthetic anti-virulence agents that can block bacterial communication and attenuate virulent behaviors. Colonization and fouling of surfaces by bacteria pose persistent and costly threats to human health. These problems are urgent, and the potential societal and economic impacts of robust methods to prevent bacterial fouling and virulence are nearly impossible to overstate. Many strategies have been used to design materials that resist bacterial fouling, but all of them ultimately fail when deployed in real-world scenarios. Fundamentally new approaches to the design of antifouling or `anti-virulence' surfaces that move beyond conventional design strategies are desperately needed and would have substantial impacts on human health and well-being. One promising approach to prevent bacterial fouling is to exploit the inherent anti-biofouling properties of polymer-based SLIPS coatings. These liquid-infused coatings have enormous potential in healthcare settings, but are generally passive materials—i.e., they can strongly repel bacteria with which they come into contact, but cannot reduce microbial load or attenuate the virulent behaviors of organisms in surrounding environments. This proposal seeks to advance innovative new designs of `drug-eluting' SLIPS that can address this challenge and enhance inherent anti-biofouling properties by releasing either antimicrobial or anti-virulence agents. The proposed work is based on our recent and unexpected discovery that the infusion of hydrophobic oils into porous polymer coatings patterned with small spots of a potent and highly water-soluble antibiotic leads to drug-patterned `proto-SLIPS' that can (i) release antibiotic into aqueous environments and, subsequently, (ii) transform or self-heal into SLIPS coatings that are uniformly slippery and strongly antifouling to pathogenic bacteria. This approach is unprecedented, conceptually simple, and has potential to lead to innovative polymer coatings that can both strongly prevent surface biofouling (via inherent slippery character) and reduce bacterial load or alter bacterial behavior in ways that could advance the application of SLIPS in healthcare contexts (e.g., to prevent fouling or infection on or around interventional devices). Our cross-disciplinary research plan seeks to explore these new ideas and test hypotheses to create a foundation for the design of new synthetic polymer coatings with superior anti-biofouling properties. The proposed studies embody novel questions and associated levels of risk and reward appropriate for an R21-level study and unite a team of established and actively collaborating investigators to demonstrate and explore the feasibility of this new approach.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Cognitive abilities have a profound impact on life outcomes. As such, there has been much scientific interest in identifying genes that are critical for regulating cognitive phenotypes. The miR-132 microRNA is among the few studied microRNAs that impacts learning and memory in rodents, is dysregulated in multiple human disorders typified by cognitive impairment, and regulates synaptic structure and function. Human genome wide association studies (GWAS) have identified numerous genes of potential relevance to general cognitive function, yet a challenge is identifying which genes, among the many, are likely to have direct effects on learning and memory. Genes that are targets of miR-132, and that have also been identified in GWAS of human cognitive function, could have a high likelihood of directly impacting learning and memory. The overlay of empirically identified high probability miR-132 gene targets with a list of genes that exhibit genome-wide significant association with human cognitive abilities lead to the identification of two genes, one of which is ARHGEF11. ARHGEF11 encodes PDZ- RhoGEF, a guanine nucleotide exchange factor (GEF) for the RhoA small GTPase. Within the brain, PDZ- RhoGEF is most highly detected in the cerebral cortex, including the prefrontal cortex (PFC). PDZ-RhoGEF is enriched in the dendrites and dendritic spines of pyramidal neurons, with minimal detectable expression in glia. Despite being among the most potent known activators of RhoA, no studies have investigated the functions of PDZ-RhoGEF in the brain. One overarching theory guiding this proposal is that PDZ-RhoGEF is a critical regulator of dendrite and dendritic spine stability, and that it also regulates synaptic function, neuronal engagement and cognition. A second overarching theory guiding this proposal is that PDZ-RhoGEF is a previously unrecognized component of multiple signaling pathways of known importance for controlling synaptic and cognitive phenotypes. Aim 1 will use viral-mediated gene transfer to determine if increased levels of PDZ- RhoGEF in the prefrontal cortex affects synaptic structure, synaptic function, neuronal engagement, and cognition in a manner that requires RhoA activation. Aim 2 will determine if the effects of altered miR-132 expression on dendritic spine stability and cognition are due, at least in part, to miR-132's ability to control levels of PDZ-RhoGEF. As part of the experimental design of Aim 2, we will also simultaneously determine the effects of PDZ-RhoGEF knockdown in the mouse prefrontal cortex on baseline dendritic spine and cognitive phenotypes. Aim 3 will use enzyme activity assessments, super resolution synaptic imaging, and viral gene transfer to characterize the physical and functional interaction between PDZ-RhoGEF and the scaffolding-like protein DISC1. As part of Aim 3, we will determine if the effects of DISC1 loss on synapse destabilization and cognitive impairment are due to excessive PDZ-RhoGEF activity. If our hypotheses are correct, these studies could identify a previously unrecognized role for PDZ-RhoGEF in controlling synaptic and cognitive phenotypes via multiple signaling pathways, and illuminate RhoA signaling as a target for future therapeutics.

NIH Research Projects · FY 2026 · 2024-04

Proposal Summary/Abstract During mouse embryonic development, approximately 40 cells in the post-implantation epiblast are designated as Primordial Germ Cells (PGCs) and destined to become sperm or egg. PGCs undergo global epigenetic remodeling, which is not seen in the surrounding somatic cells. The repressive histone modification H3K9me2 is depleted during PGC specification. In parallel, DNA is demethylated in two waves: a global erasure followed by a loci specific depletion. The only locations to escape this first wave of global erasure are a few germ cell specifying genes, imprinted loci, and transposable elements. How these loci are protected from the first phase of DNA demethylation is still unknown. Previous data from my thesis lab has shown that in somatic cell reprogramming to induced pluripotent stem cells, there is coordinated removal of the same repressive epigenetic marks of H3K9me2 and DNA methylation. I propose that H3K9me2 demethylases KDM3A and KDM3B have important roles in PGC development which I will investigate using an in vitro PGC-like cell differentiation model and Next-generation sequencing experiments. This research will elucidate the importance of removal of repressive modifications to prevent transgenerational epigenetic inheritance which could be disruptive to embryonic development, germ cell specification, and fertility.

NIH Research Projects · FY 2025 · 2024-03

Project Summary Chronic inflammation is highly prevalent and has deleterious effects on brain health, including links to brain structural abnormalities1, neural metabolic changes2, psychiatric disorders2, cognitive dysfunction3, and aging- related diseases4,5. Given the increasing prevalence of chronic inflammatory diseases6–9, a better understanding of interventions that may reduce the impacts of inflammation on brain health is imperative10. A growing body of literature demonstrates positive impacts of mindfulness-based interventions (MBIs) on mental and physical health11–15, brain structure and function14,16–19, and chronic inflammation20. Asthma is a chronic inflammatory disease that is uniquely well-suited to examine the protective effects of MBIs across multiple dimensions of health because it is sensitive to changes in stress21–23, highly prevalent and comorbid with depression24,25, and has been associated with compromises in brain structure26–28 and function29,30. The first aim of this proposal is to 1) assess changes in brain health indices in individuals with asthma from pre- to post- 8-week Mindfulness-Based Stress Reduction (MBSR) intervention and at 3-month follow-up (n=46). I will utilize a novel, whole-brain voxel- wise assessment of DWI, modeled using diffusion tensor (DTI) and neurite orientation dispersion and density imaging (NODDI), which provides meaningful, clinically relevant tissue parameters of white and grey matter microstructure and will enable detection of changes at a high spatial resolution. Additionally, I will examine changes in biomarkers of neuroinflammation and neurodegeneration in concert with DTI and NODDI metrics, a novel approach which will strengthen our confidence in the underlying meaning of changes in DWI metrics. The second aim of this proposal is to 2) assess the association of these brain health indices with indices of whole-person health and wellbeing, from pre- to post- 8-week MBSR intervention and at 3-month follow-up (n=46). While previous work has reported structural and functional brain changes related to MBIs, a deeper understanding of how these changes are related to other aspects of whole-person health is needed. High stress and poor sleep are drivers of systemic inflammation31 and brain microstructural deterioration32–35, and their improvement may link MBIs to salubrious outcomes. Sleep quality, life stress, and self-reported wellbeing will serve as indices of a psychological dimension of whole-person health, and asthma severity and airway inflammation will serve as indices of a disease- specific dimension of whole-person health, contributing to our understanding of how changes in brain health after MBI relate to experiential outcomes. The impact of a behavioral intervention on the interrelations of brain- and whole-person health indices in asthma has not previously been investigated and represents a potential for substantial public health impact given the growing evidence of the effects of asthma on the brain and the growing prevalence of asthma. Finally, this training fellowship will facilitate valuable training opportunities in DWI analysis, statistical methods, psychoneuroimmunology and contemplative neuroscience education, experimental design and data collection, writing, public-speaking, mentorship, and ethics.

NIH Research Projects · FY 2026 · 2024-03

Project Summary / Abstract: Significance: Spinal Cord Injury (SCI) is a devastating trauma that leaves approximately 10,000 to 20,000 people paralyzed every year in the U.S., costing the health care system $40.5 billion annually. Although it has been shown that the inflammatory response after SCI is beneficial in removing debris and releasing neurotrophic factors, there is an overreaction of the inflammatory response causing further neural destruction and inflammatory macrophages remain for a prolonged time period. Using anti-inflammatory cytokines to attenuate inflammation after SCI has shown some encouraging results. However, there are several limitations that need to be overcome to use anti-inflammatory cytokines as a treatment for SCI including, a short half-life, inability to cross the blood spinal cord barrier, rapid clearancefrom the injury site, and higher risk of infection when using large systemic doses. Therefore, it would be beneficial to have a local sustained delivery of anti-inflammatory cytokines, coinciding with critical stages of the ensuing inflammatory response. Innovation: Emerging cytokine delivery approaches are often limited by sub-optimal release characteristics and poor biological activity of the cytokine when delivered in vivo. We hypothesize that: mineral coated microparticles (MCMs) releasing anti-inflammatory cytokines can be injected at clinically relevant treatment times after SCI, which will reduce inflammation coinciding with critical stages of the ensuing secondary damage resulting in smaller lesions and a higher level of function retained after SCI. We have preliminary data showing that IL-4, IL-10, and IL-13 retain their bioactivity when bound to MCMs and are capable of reducing inflammation after SCI. Our preliminary results also show a clear synergistic effect at reducing inflammation when using the combination of IL-4, IL-10, and IL-13. Aims: The proposed plan is to 1) optimize IL-4, IL-10, and IL-13 dosage in a rat contusionmodel; 2) test the influence MCMs delivering IL-4, IL-10, and IL-13 have on immune cells during important stages of inflammation and glial scar development; and 3) explore the influence a sustained release of IL-4, IL-10, and IL-13 has on reducing inflammation and improving the amount of function retained below the level of injury after SCI. Impact: Successful completion of the proposed research will produce three direct outcomes. First, it will establish a method to attenuate inflammation after SCI using locally delivered biologically active anti- inflammatory cytokines with optimized dosage. Second, it will explore fundamental synergies between cytokines that influence specific stages of inflammation. Third, it will develop a controllable drug delivery system for biologically active molecules after SCI. In view of the importance of soluble cytokines involved in the inflammatory response after SCI, as well as growth factors for signaling and guidance cues for axonal growth, the proposed MCMs are expected to be a uniquely enabling technology in neural tissue engineering.

NIH Research Projects · FY 2026 · 2024-03

Study Title: Sleep and emotion processing in adolescent Post-traumatic stress disorder Principal Investigators: Stephanie Jones, PhD and Ryan Herringa, MD, PhD Project Summary Although nearly all affective disorders are associated with sleep pathology, PTSD is singularly conceptualized as one in which functionally impairing symptoms, including trauma re-experiencing and hyperarousal, are present across the 24- hour period—during both wake and sleep. As such, sleep pathology has been reconceptualized from a highly comorbid symptom with little mechanistic value to a function that is fundamentally involved in the development, maintenance, and severity of the disorder. Remarkably, sleep research in youth with PTSD has relied almost exclusively on subjective assessments of sleep, which afford no information about the sleeping brain. This gap is particularly relevant given the overlap between the functional impairments, both neural and behavioral, associated with PTSD and those associated with poor quality sleep. In this proposal, we will explore sleep’s relationship to emotion processing and daily affect in 165 adolescents between 15-18 years spilt into 3 groups: PTSD, TEC (trauma-exposed comparison) and TD (typically developing). We will use advanced sleep assessment methodologies including: (1) high-density EEG (256 channels) in the laboratory to explore the regional distribution of sleep before and after an emotional learning task and; (2) a sleep-wearable EEG recording headband, SmartSleep, to record sleep in the ecologically relevant home-environment, longitudinally, (14 days) and explore its relationship to daily measures of affect and symptom severity. Finally, to determine whether the deepest sleep (e.g. slow-wave activity (SWA); EEG frequency 1-4 hertz) of non-rapid eye-movement sleep (NREM)) can be reliably increased over time in youth, during one 5-day period of sleep recording, we will increase SWA using SmartSleep’s acoustic enhancement algorithms. These algorithms represent the first validated, non-pharmacological method available for enhancing slow-waves. Our long-term goal is to understand the role sleep plays in the maintenance and progression of adolescent PTSD. We expect our results will have a significant impact on our understanding of sleep pathophysiology in adolescent PTSD and to lay groundwork for larger clinical trials to test novel sleep neuromodulatory interventions.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by an epigenetic silencing of the X- linked FMR1 gene, which leads to loss of its protein product fragile X messenger ribonucleoprotein (FMRP). Currently, FXS is the largest single-gene contributor to autism affecting about 1 in 4,000 males and 1 in 7,000 females in the U.S. Despite FXS being a monogenic disorder, FMRP controls many aspects of neurodevelopment leading FXS patients to present a wide range of symptoms that include intellectual disability, language impairment, anxiety, hyperactivity, and aggression. Many studies investigating the molecular mechanisms affected in FXS have been performed in Fmr1 knockout (KO) rodent models and drosophila. However, many of the treatments that proved to be successful in animal models failed in human clinical trials, leaving a large unmet need for treatment targeting FXS. Human induced pluripotent stem cells (iPSCs) have been a useful model in the study of neurodevelopmental disorders and studies have shown that FXS iPSC derived neurons have defects in neuronal maturation and differentiation. Recently, 3-dimensional cortical organoids (3DOs) derived from iPSCs have become an increasingly promising tool for the study of human neurodevelopment because they resemble human brain formation and can be cultured for more than one year. However, a study examining the effect of FMRP deficiency throughout development has not been done. Therefore, I propose to study cortical development, electrical activity, and genetic signatures using 3DOs derived from FMR1 deficient iPSC lines at different developmental stages. My preliminary data already shows increased cell proliferation in FXS patient-derived 3DOs, which corresponds to previous findings in FXS neural progenitor cells (NPCs), and hyperexcitability, which is consistent with our previous studies in FXS 2D neurons. I have also found upregulation of a putative FMRP target SPTBN1 (Spectrin Beta, Non-Erythrocytic 1) across three developmental stages. I hypothesize that FMRP deficiency leads to dysregulation of SPTBN1, a gene important for neuronal maturation, which results in disrupted development of FXS cortical organoids. To test this hypothesis, in the first aim I propose to identify developmental deficits in FXS 3DOs by performing immunohistochemistry of developmental markers in early, middle, and late developmental time-points along with the analysis of electrical activity using calcium imaging. In the second aim, I propose to determine whether and how FMRP regulates SPTBN1 and investigate how dysregulation of SPTBN1 may contribute to developmental deficits that we have found in FXS 3DOs. I also plan to confirm these previous results and identify novel differentially expressed genes through single nucelus RNA sequencing (snRNAseq) at different developmental time points. Overall, this project will further the understanding of the developmental and molecular mechanisms disrupted in FXS patients throughout development and lead to better-targeted treatments.

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Aging is a non-modifiable risk factor of stroke. Notably, females are more likely to experience a stroke at an older age and tend to have worse recovery outcomes than males post stroke, especially in motor recovery. In addition to baseline weakness, increased neuromuscular fatigability (an acute, exercise-induced reduction in maximal voluntary force or power generation) of the paretic musculature following a stroke limits task endurance of activities such as walking. Recently, our laboratory showed that fatigability may be exacerbated in females, but not males, with stroke as compared to neurologically intact controls during a sustained isometric contraction. However, little is known regarding stroke-related sex differences in fatigability during dynamic tasks despite their greater relevance to everyday activities like walking. In people without stroke, older females show a relatively blunted vascular response during dynamic exercise and are more fatigable than older males, but it is unknown if this persists post stroke. The F99 phase research aims to quantify sex differences in fatigability during a dynamic task post stroke and examine the role of sex differences in peripheral muscle perfusion as a mechanism. We propose that impaired muscle perfusion contributes to the greater fatigability during dynamic knee extension contractions in females versus males with chronic stroke. If muscle perfusion is a mechanism, then manipulating blood flow during exercise would modulate sex differences in fatigability among people with chronic stroke. Aim 1 will test the hypothesis that females will have greater fatigability than males post stroke and that fatigability will be negatively correlated with the metrics of blood flow to the exercising musculature. Aim 2 will test the hypothesis that sex differences in fatigability post stroke will be lessened during the same exercise with blood flow occlusion via an inflatable cuff to the exercising limb. Aim 3 will test the hypothesis that sex differences in fatigability post stroke will also be minimized during the same exercise following a single session of ischemic conditioning (IC) procedure. IC is a well-established non-invasive procedure which has been used to enhance blood flow through facilitating vasodilation. Results from the proposed F99 research will contribute to targeted rehabilitative interventions to optimize motor recovery post stroke, especially for females. For the K00 phase, I will shift focus from stroke-related peripheral perfusion impairments to aging-related changes in cortical neurovascular function to better understand the cortical manifestations of neurological disorders on motor performance. With guidance from my K00 mentor, I aim to establish robust quantitative imaging biomarkers of neuromuscular fatigue to study aging-related neurological diseases and the response of individual patients to personalized therapeutic interventions using non-invasive techniques such as functional magnetic resonance imaging and transcranial Doppler ultrasonography. Completion of the proposed F99 and K00 will be a springboard for my future success as an independent extramurally funded aging researcher.

NIH Research Projects · FY 2026 · 2024-03

SUMMARY The microtubule poison paclitaxel (PTX) is standard-of-care for breast cancer treatment for all breast cancer subtypes in both early stage and metastatic disease. However, ~50% of breast cancer patients do not benefit from PTX. Though PTX causes mitotic arrest at high concentrations in cell culture, our data from two biomarker studies demonstrate that PTX levels in patient tumors are too low to cause mitotic arrest. Instead PTX causes abnormal multipolar mitotic spindles. Division on multipolar spindles causes unequal chromosome segregation (termed chromosomal instability, or CIN). While low rates of CIN are common in tumors, increasing the rate of CIN above a maximally tolerated threshold is lethal. Since PTX increases CIN, these data support a model in which PTX is effective when it increases CIN over the maximally tolerated threshold. Our long term goal is to use this novel mechanistic insight to convert PTX from generic chemotherapy into precision medicine by developing a) a biomarker to predict which tumors will respond to PTX, and b) method(s) to sensitize the ~50% of resistant cancers. Based on our preliminary data, we hypothesize that there are two major determinants of PTX sensitivity. First, tumors that have endogenous CIN close to the maximally tolerated CIN threshold are more sensitive to PTX. Second, some tumors can focus PTX-induced multipolar spindles into near-normal bipolar spindles by mid mitosis and dramatically reduce PTX-induced cytotoxicity—even when PTX affects spindles in early mitosis. Since PTX treatment increases the incidence of multipolar spindles in early mitosis, this implicates mid mitosis focusing of PTX-induced multipolar spindles as a second mechanism of PTX resistance. Aim 1 will test the hypothesis that pre-treatment CIN correlates with PTX response in primary breast cancer, as determined in an ongoing biomarker study of neoadjuvant single-agent PTX and a retrospective analysis of I-SPY2 samples. Additionally, Aim 1 will test whether experimentally inducing CIN enhances PTX response. Aim 2 will enable state-of-the-art quantitative measure of CIN by a) employing single-cell DNA sequencing (scDNAseq) to quantify all chromosomes; and b) accounting for dropout of highly aneuploid cells from the tumor population through a technique known as Approximate Bayesian Computation. Aim 3 will experimentally test how pole focusing controls PTX response. Additionally it will test the hypothesis that alisertib, an inhibitor of AurkA, can prevent pole focusing. Preventing pole focusing is expected to sensitize tumors that are resistant to PTX by forcing cells to undergo multipolar divisions, markedly elevating CIN. Finally, a CRISPR screen will be used to identify genes that confer this mechanism of primary resistance. A gene signature for pole focusing could improve a CIN-based predictive biomarker of PTX response. Together, this translational work based on the conceptual advance provided by our previous translational studies will provide basic knowledge of the biological mechanisms regulating cell death in response to a cornerstone of anti-cancer treatment. This will provide a sound basis for the future development of a predictive biomarker of PTX and strategies for sensitizing PTX-resistant cancers.