University Of Arizona

NIH Research Projects · FY 2026 · 2020-06

PROJECT SUMMARY/ABSTRACT Centrosomes are organelles used to build microtubule-based protein machines, including mitotic spindles and cilia. At the centrosome core lies a pair of ‘mother-daughter’ centrioles, barrel-shaped structures that act as the duplicating elements of the organelle. Normally, the centriole pair duplicates only once each cell cycle and, during mitotic entry, centrioles recruit a shell of pericentriolar material (PCM) from which microtubules grow. Not only are they one of the largest protein complexes in eukaryotic cells but one of the most ancient of organelles, and have fascinated cell biologists since their discovery in the late 19th century. During the past 25 years, advances in imaging, proteomics and functional genomic screens have led to an explosion of discoveries in the centrosome field. At present, we have a complete inventory of the proteins comprising centrosomes. In our model system, Drosophila, centrosomes assemble from a surprisingly small number of components (approximately 20). Despite these advances, many important questions remain unanswered. For example, although Polo-like kinase 4 (Plk4) is recognized as the conserved master-regulator of centriole duplication, it is not known how Plk4’s catalytic activity is regulated specifically on centrioles. How are mother centrioles restrained to spawn only a single daughter once per cell cycle? What are the phosphorylation targets of Plk4 and how do the modified components then assemble a centriole? How is centriole length controlled? Understanding these processes at the molecular level is important because alterations in centrosome structure or number cause a number of serious pathologies, including birth defects, ciliopathies and cancer. Plk4 has been the centerpiece of our research program because it is both necessary and sufficient to induce centrosome overduplication (amplification) when overexpressed, a scenario observed in cancer cells. We have published a series of studies that have defined Plk4 structure, regulation and identified several of its substrates. Notably, Plk4 utilizes multiple mechanisms of control to restrain its activity and prevent rampant centrosome overduplication, using an elaborate combination of autophosphorylation, ubiquitination and autoinhibition. We continue to pursue two overarching goals in this renewal of R35 R35GM136265: 1) identifying the molecular mechanisms that suppress centrosome amplification, and 2) characterizing the inherent mechanisms that govern centrosome function and duplication. Building on our progress during the past four years, we propose to continue our studies that will define the mechanisms underlying four sequential steps in the centriole assembly process. Specifically, we will determine (i) how a single site of daughter centriole assembly is selected on mother centrioles, (ii) the composition of the pre-procentrioles, how it forms and its role in centriole duplication, (iii) how Plk4 induces nascent daughter centriole (procentriole) assembly, and (iv) how centriole length is controlled to promote elongation and maintain a terminal length.

NIH Research Projects · FY 2024 · 2020-06

Project Summary The central nervous system regulates gonadotropin secretion through neurosecretion of gonadotropin- releasing hormone (GnRH). GnRH is synthesized in preoptic and periventricular neurons, transported via intertwined processes that distribute as axonal-vascular terminals in the median eminence and released into the hypothalamic-hypophysial portal vasculature. GnRH binds receptors on gonadotropes to stimulate secretion and synthesis of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Neurosecretion of GnRH is pulsatile and is obligatory for sustaining normal gonadotropin secretion and synthesis. Kisspeptin (KISS1) neurons are a major GnRH afferent network that activates GnRH neurons during puberty, maintains GnRH release during adulthood, and transduces negative and positive feedback actions of gonadal steroids. In our studies we will test the hypothesis that KISS1 neurons, specifically those in the arcuate nucleus (ARC) that co-express the peptides neurokinin B (NKB) and prodynorphin (PDyn) (KNDy neurons), function as a GnRH pulse generator (Aim 1), with NKB mediating synchronized activation of KNDy cells (Aim 2) and dynorphin terminating each pulse discharge (Aim 3). These studies are significant because they will provide information on the cellular circuitries that govern GnRH pulse generation, and thus will shed new light on the pathophysiologic mechanisms underlying neuroendocrine-based fertility disorders, such as absent, delayed, or precocious puberty, functional hypothalamic amenorrhea, or polycystic ovary syndrome, and in the longer term, point to new potential targets for intervention in these reproductive impairments. The proposed studies will use powerful genetic tools to dissect and characterize the functional components of the kisspeptin-GnRH pulse generating system. To define the key elements of the pulse generator, we will utilize innovative genetically modified mouse models, each enabling an analysis of the consequences of kisspeptin neuron-specific gene deletion. We will first study the role of the KNDy neuronal population using a Kiss1fl/fl mouse recently created in our laboratory. This mouse will be bred to a prodynorphin Cre (Pdyn-IRES- Cre21,2) mouse, hereafter Pdyn-Cre, to eliminate Kiss1 in KNDy neurons. We have also developed a novel mouse bearing a doxycycline-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. Two other lines, a NKBfl/fl mouse (developed in our laboratory) and an Oprk1fl/fl mouse (Jackson labs) will be crossed to Kiss1-Cre or iKiss-Cre mice to generate mice that will allow us to analyze the effects of kisspeptin neuron-specific deletion of NKB and Oprk1 on GnRH- triggered LH pulses. We will also use the iKiss-Cre mouse to delete and study the roles of steroid receptors in kisspeptin neurons in the adult. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the axis.

NIH Research Projects · FY 2025 · 2020-04

ABSTRACT The long-term goal of this project is to improve clinical pain and symptoms for older adults with knee osteoarthritis (OA) using home-based nonpharmacological approaches. Knee OA is one of the most common pain conditions among people over 45 years old, and the management of OA pain is challenging because existing pharmacological approaches often produce significant adverse events, and the treatment benefits may decrease over time. Also, knee OA pain is characterized by increased pain-related brain activation, possibly explaining the limited success of existing peripherally based treatments that target the pain locally in the area of the knee. Therefore, innovative nonpharmacological interventions targeting pain-related brain function are needed. Two nonpharmacological pain treatments, transcranial direct current stimulation (tDCS) and mindfulness-based meditation (MBM), have been shown to improve pain-related brain function in older adults with knee OA. The rationale for the proposed research is that because tDCS promotes neuroplasticity, it may potentiate the effect of MBM, which also stimulates adaptive changes in the brain. However, no investigations to date have examined whether remotely supervised tDCS paired with MBM at home can enhance pain-related brain function and reduce OA-related clinical pain and symptoms. Home-based interventions are critical because older adults with knee OA have limited mobility, and recent technological advances have created the potential for home interventions with real-time monitoring through a secure videoconferencing platform. The central hypothesis is that remotely supervised tDCS paired with MBM at home will decrease clinical pain and OA-related clinical symptoms, improve physiopsychological pain processing, and increase participant satisfaction with treatment. This hypothesis will be tested by pursuing the following specific aims: determine the effects of active tDCS paired with active MBM on clinical pain and OA-related clinical symptoms (specific aim 1); determine the effects of active tDCS paired with active MBM on physiopsychological pain processing (specific aim 2); and determine the effects of active tDCS paired with active MBM on participant satisfaction with treatment (specific aim 3). The proposed study will directly investigate the effects of remotely supervised tDCS paired with MBM at home in 200 older adults with symptomatic knee OA using a double-blind, randomized, sham-controlled, phase II parallel group (1:1:1:1 for four groups defined by 2x2 factorial design) design. The proposed research is significant because it is expected to provide valuable insight into an exciting new modality of nonpharmacological pain self-management that is extremely easy, safe, and noninvasive with minimal side effects.

NIH Research Projects · FY 2025 · 2019-09

Children with Down syndrome (DS) are known to be at very high risk for obstructive sleep apnea (OSA), with a prevalence of up to 66%. OSA has been associated with neurocognitive impairment and impaired health- related quality of life (HR-QOL) in children with DS. Current treatments for OSA in children with DS include adenotonsillectomy and positive airway pressure (PAP) therapy. Unfortunately, treatment effectiveness is limited by a high risk of residual OSA after adenotonsillectomy and poor adherence to PAP therapy. OSA is prevalent among children with DS and is associated with neurocognitive impairment and impaired HR-QOL. Targeted therapies are needed to mitigate these negative effects of OSA in children with DS. Airway hypotonia during sleep has been identified as a cause of OSA in children with DS. Consistent with this, OSA treatment aimed at improving airway tone via hypoglossal nerve stimulation appears to be effective in adolescents with DS. However, use of hypoglossal nerve stimulation may be limited in children given that multiple revision surgeries would likely be necessary in younger children to adjust for growth over time. The combination of atomoxetine and oxybutynin (ato-oxy) was shown to improve airway tone during sleep and treat OSA in adults without DS. Given that both drugs are routinely used and well-tolerated in children, we hypothesize that ato- oxy will be an efficacious treatment for OSA in children with DS and will lead to improvement in neurocognition and HR-QOL. This supplement will fund the addition of an additional clinical trial site as well as provide travel funds for participants. Specific aims of this project during the R33 phase include: Aim 3: To evaluate the long-term efficacy of ato-oxy treatment for OSA in children with DS. Aim 4: To evaluate the long-term efficacy of ato-oxy treatment on improving HR-QOL in children with DS and OSA. Aim 5: To evaluate the efficacy of ato-oxy treatment on improving neurocognition in children with DS and OSA. If successful, this project would identify a novel treatment for OSA in children with DS, as well as medication-based route to improve cognition in children with DS.

NIH Research Projects · FY 2025 · 2019-09

Asthma and COPD are the most commonly diagnosed chronic lung diseases in the United States. Studies have shown that asthma is the most important risk factor for COPD that develops through a course of low lung function from school age that tracks into adulthood. However, there is a fundamental gap in understanding the basic underlying mechanisms of this progression. In the first cycle of funding, we addressed this gap by focusing on the role of CC16 in mediating the immune response that yields protection against lung function decline. We published several key reports providing mechanistic and clinical evidence supporting the notion that persistent early life infections in the context of CC16 deficits may be a previously overlooked link in understanding progression of asthma into severe asthma with fixed airflow limitation. Additionally, we identified 2 novel ligands for CC16’s protective activity in different compartments, a4b1(VLA-4) in the circulation and a2b1 (VLA-2) in the respiratory tract. By engaging with VLA-4 in circulation on activated leukocytes, CC16 limits cellular migration into the airways and reduces airway hyperresponsiveness. By engaging with VLA-2 on epithelial cells, CC16 promotes antimicrobial and antiviral secretion thereby aiding in host defense against pathogens. In this renewal application, we will continue these translational studies and test the overall hypothesis that CC16 protects against Rhinovirus (RV) infection by promoting epithelial-driven antiviral host responses and that early life infections in the context of low CC16 leads to epigenetic changes resulting in enhanced airway remodeling. RV is the most common trigger of asthma exacerbations, especially in severe asthmatics and elicits acute exacerbations. Since CC16 is known to be lower in asthma patients, we will test the hypothesis that epithelial-driven antiviral host responses are decreased in asthma patient nasal epithelial cell samples via CC16- dependent mechanisms and that delivery of rCC16 and CC16-derived peptidomimetics offers a therapeutic benefit by enhancing antiviral host responses, thereby reducing RV infection, and limiting airway remodeling. This hypothesis will be tested by pursuing three specific aims: 1) Determine mechanisms by which CC16 promotes epithelial-driven host defense against respiratory pathogens, 2) Examine the impact of CC16 deficiency during early life viral infection on lung development and function in adult life and 3) Determine if circulating CC16 levels and asthma status impact nasal epithelial cell production of antimicrobials and antivirals and if exogenous CC16 can augment production in a VLA-2 dependent manner.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY The University of Arizona Cancer Prevention Clinical Trials Network (UA CP-CTNet) is at the forefront of advancing cancer prevention through early-phase clinical trials, with a focus on five major organ systems: skin, gastrointestinal, lung and upper aerodigestive, breast and gynecologic, and prostate and other urologic cancers. With strong scientific and academic leadership from Lead Academic Organization (LAO) at the University of Arizona has partnered with George Washington University and 20+ highly collaborative nationally recognized Affiliate Organizations (AOs), UA CP-CTNet rigorously evaluates preventive agents designed to prevent or intercept cancer at its earliest stages. High-impact studies led by the network have contributed to HPV vaccine guidelines, evaluated the preventive potential of low-dose apalutamide in prostate cancer, and are targeting at risk patients such as organ transplant recipient, populations including smokers, and community groups including firefighters. Additionally, UA CP-CTNet is exploring innovative strategies, such as topical immune modulators and microbiome-modifying agents, to prevent skin and cervical cancers. The four core objectives of UA CP- CTNet are: (1) to execute scientifically rigorous early-phase trials that target key molecular pathways involved in cancer prevention across these five organ systems, ensuring trials are aligned with the most current scientific advancements; (2) to characterize the clinical activity and biological effects of preventive agents, focusing on molecular targets, immune responses, and key carcinogenesis markers to assess their potential efficacy and safety; (3) to enhance trial efficiency and inclusivity by leveraging modern statistical and trial design methods and by employing a multipronged process involving stakeholder and investigators to consider eligibility and participation barriers and recruitment strategies toward optimizing access and inclusion; and (4) to formalize the UA CP-CTNet training and professional development pipeline to foster the growth of future leaders in cancer chemoprevention. Through these objectives, UA CP-CTNet aims to generate critical early phase clinical and biomarker guided efficacy and safety evidence on promising prevention agents that will drive forward cancer prevention efforts, influence public health policies, and ensure that future cancer prevention research continues to benefit diverse populations and at-risk groups.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY/ABSTRACT Therapeutics to prevent, delay and treat Alzheimer’s disease (AD) remain an unmet need. Proposed herein is a regenerative medicine, systems biology approach that targets the regenerative system of the brain while simultaneously activating systems to reduce burden of AD pathology. Allopregnanolone (Allo) is a pleiotropic neurosteroid that in preclinical discovery models of AD and aging promotes neurogenesis, restores cognitive function and reduces burden of AD pathology. Mechanisms by which Allo promotes neural stem cell regeneration and restoration of cognitive function are extensively characterized with a large margin of safety. Importantly, Allo promotes regeneration of human neural stem cells in vitro. Allo is a low molecular weight neurosteroid endogenous to the brain that is blood brain barrier penetrant with abundant existing safety data in animals and humans. Completed National Institute on Aging (NIA) Phase 1 clinical trial of Allo in persons diagnosed with MCI due to AD or mild AD, indicates that the regenerative treatment regimen of once per week via intravenous infusion is well tolerated with no indications of Allo-related adverse events. MRI brain imaging for regenerative surrogate markers and cognitive testing were well tolerated and feasible in this early AD cohort. Safety and tolerability findings in women and men are consistent with outcomes of IND-enabling chronic toxicology in two species indicating no adverse outcomes following 24 weeks of once per week Allo exposure at doses exceeding those to be tested in humans by 10-fold. Based on a foundation of discovery and mechanistic preclinical research, IND-enabling studies and Phase 1 clinical development in women and men, we propose a delayed start Phase 2 clinical trial of Allo administered in a regenerative treatment regimen for 18 months, which includes a placebo-controlled period of 12 months followed by a delayed-start (open label) period of 6 months. To advance clinical development, three specific aims are proposed. Aim 1 is designed to conduct a Phase 2, randomized, placebo-controlled, delayed start group, proof of concept clinical trial of Allo in APOEe4 positive participants diagnosed with mild AD. The primary outcome measure will be rate of change in ADAS-cog14 score after 12 months. Secondary analyses will assess change from baseline to 12 months on activities of daily living assessed by ADCS-iADL, MRI volumetric outcomes, and on cognitive function as determined by CANTAB AD battery, MMSE, and CDR-SB. Aim 1 exploratory analyses will assess cognitive clinical and functional outcomes during the delayed-start period (12-18 months). Aim 2 is exploratory and designed to develop surrogate MRI-based biomarkers of hippocampal regeneration and connectivity. Aim 3 is exploratory and is designed to establish a blood-based predictive biomarker of regenerative responders and non-responders. To be explored are Allo-induced regeneration of iPSC-derived neural stem cells and mitochondrial respiration. Secondary objective is to determine the cellular population with greatest predictive accuracy using participant derived iPSCs / neural stem cells, peripheral blood mononuclear cells and CD34+ cells.

NIH Research Projects · FY 2026 · 2019-08

ABSTRACT The goal of this work is to understand the fundamental biology of cellular response to different forms and combinations of stress. Cells are constantly subjected to intrinsic and extrinsic stresses—reactive oxygen species, protein misfolding, osmotic stress—that negatively impact cellular structure and function. In response, cells activate a range of molecular pathways to mitigate and repair damage—oxidative stress response, unfolded protein response, osmotic stress response. Several interventions that improve health, such as dietary restriction, both activate stress response pathways and promote multi-stress resistance. While individual stress response pathways are reasonably well defined, how stress responses differ when cells are challenged with multiple forms of stress simultaneously is less well understood and represents a critical knowledge gap. This gap has broad implications for medicine. Human diseases rarely involve a single form of stress—Alzheimer’s disease is characterized by neuroinflammation, increased oxidative stress, and accumulation of misfolded proteins, while cancer exhibits oxidative stress, DNA damage, and localized hypoxia. By understanding the network of molecular pathways that define cellular stress response, we aim to identify intervention points that can be targeted to activate distinct stress response profiles that improve health, combat disease, and enhance resilience. The long- term goal of this research program is to answer fundamental questions about the biology of stress response: (1) How is the molecular stress response network organized? (2) Which elements of this network are general (responsive to many types of stress) and which are specific (responsive to specific stressors)? (3) How does the cellular response to one type of stress alter an organism’s resistance to other types? (4) Are there key molecular nodes in the stress response network that can be targeted to improve health or treat specific diseases? Over the past five years we have examined the physiological and molecular response of the round worm Caenorhabditis elegans to a range of stress combinations. In parallel, we studied C. elegans and mice with elevated levels of the tryptophan metabolite 3-hydroxyanthanilic acid (3HAA) as models of multi-stress resistance. Evidence from these projects converged on heavy metal regulation and host-bacteria intercommunication as molecular processes responsive to many types and combinations of stress. We are now focused on answering several questions related to these molecular themes: (1) What role does heavy metal transport and storage play in the response to diverse stressors? (2) How does the response of intestinal bacteria to stress impact host health and stress resistance? (3) Can these processes be targeted to promote animal health and resilience? (4) How do changes in intestinal iron and zinc localization promote broad-spectrum stress resistance in animals with elevated 3HAA? Beyond these questions, we will continue our search for novel stress interactions in pursuit of our broader goal to comprehensively understand the cellular stress response network. Supporting each of these projects, we continue to build innovative, high-content tools for studying stress response in C. elegans.

NIH Research Projects · FY 2025 · 2019-05

PROJECT SUMMARY The most profound demographic change confronting our world is a dramatic increase in individuals over 65 years of age, projected to reach over 80 million people in the US and 1.5 billion worldwide by 2050. The increasing number of older adults has major socioeconomic ramifications. First, the increase in longevity dramatically increases numbers of older adults with multiple chronic diseases and with reduced quality of life. Second, the challenge of managing the health care of older adults consumes a disproportionally large segment of health care dollars. Therefore, biomedical innovations must target increasing resilience and healthspan in older adults. The COVID-19 pandemic starkly illuminated the heightened risk of viral infection in the elderly. Robust and expanded training in biomedical sciences pertinent to the biology of aging is critical to address the needs of the growing number of older adults. Our T32 renewal application is a multidisciplinary program designed to train pre-doctoral investigators (6 per year) to define mechanisms underlying the roles of Infection and Inflammation as Drivers of Aging (IIDA). The IIDA training program is comprised of basic scientists, clinicians, and public health mentors at the University of Arizona (UArizona), a top 20 public Research I University, recognized for its training history in the field of basic biomedical research and for its commitment to mentoring, diversity (designated Hispanic-Serving Institution) and cross-disciplinary training. To address the complex challenges of aging, we propose a graduate-level training program with 3 major synergistic themes: (i) inflammation and immunity in aging, (ii) persistent infection in aging, and (iii) inflammation and age-related pathology. The 27 NIH-funded faculty supporting this training program have expertise in infection, inflammation, immunology, and age-related pathologies. The training program is supported by a number of UArizona strengths in aging, basic sciences, clinical practice, and public health. The goal of the IIDA is to harness these strengths to train the next generation in the underserved area of infection and inflammation as drivers of the aging process. Extensive institutional support, state-of-the-art core facilities, and clinical and public health mentorship will enhance the training experience. IIDA trainees will be exposed to cutting-edge, innovative science through the participation in colloquia, seminars, data blitzes, and journal clubs, as well as through their engagement with the EAB, the training faculty, and distinguished scientists in the aging field. IIDA also provides unique opportunities to students to develop professional skills in advocacy and networking. IIDA also has a robust underrepresented population (URP) outreach plan and diversity-building component. IIDA program provides exceptional training for the next generation of diverse scientists to advance our understanding of the biological basis of aging processes and develop strategies to enhance resilience and healthspan.

NIH Research Projects · FY 2025 · 2019-04

Summary This proposal focuses on titin, the largest protein known, in heart function and disease. Titin forms a novel and multifunctional myofilament in the striated-muscle sarcomere with important roles that include regulating the diastolic stiffness of the heart. Recent breakthrough studies revealed that titin is of high clinical importance in both heart failure with preserved ejection fraction (HFpEF), and heart failure with reduced ejection fraction (HFrEF). Although significant progress has been made in understanding the basic biology of titin, major gaps in our understanding still remain, including a mechanistic understanding of how titin causes/contributes to heart disease. An important focus of this proposal will be on titin’s role in diastolic dysfunction, motivated by recent studies on patients with HFpEF that revealed deranged phosphorylation of titin’s molecular spring elements and diastolic stiffening. The full spectrum of posttranslational modifications that occur in HFpEF will be studied and high-resolution time-resolved spectroscopic techniques will focus on uncovering the structural changes in titin’s spring elements triggered by posttranslational modification. Drug screens will focus on identifying compounds that mimic or block these structural changes and functional studies will test whether newly discovered and candidate drugs ameliorate titin-based diastolic stiffening in HFpEF. Post-transcriptional mechanisms will be investigated as well, taking advantage of our recent work that has shown that splicing of titin can be manipulated to upregulate complaint titin isoforms and restore diastolic function. The functional efficacy of identified compounds will be tested on engineered heart tissues as well as on animal models. The second major focus of this proposal will be on titin in HFrEF. Several recent sequencing studies in large groups of patients revealed that mutations in the titin gene (TTN) are causative in ~20% of studied dilated cardiomyopathy (DCM) patients. Many of the mutations are truncation mutations (TTNtv) and they have a preferential location in the A-band segment of titin. The A-band segment is the least well-studied part of titin and an important goal of our research is to critically examine the biology of titin in this region of the sarcomere where disease-causing mutations are prominent. These studies include a focus on the role of titin in interacting with cMyBP-C (cardiac myosin-binding protein C, a clinically important thick filament protein). Animal models will be investigated in which TTNtv have been introduced in different regions of titin’s A-band segment. The effects of the mutations will be studied under baseline conditions, when stressed, and when occurring in combination with mutations in other genes. Importantly, we will also test whether excision of the mutated titin exons ameliorates titin-based DCM. In summary, capitalizing on my >20-year track record of innovative titin research, and utilizing our team of experienced scientists and talented trainees, this proposal sets ambitious goals that are expected to further accelerate understanding of the biology of titin, its role in heart disease and titin’s potential to function as a therapeutic target.

NIH Research Projects · FY 2025 · 2019-01

PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) is a disease affecting more than 1 in 500 individuals and is an unmet medical need with limited FDA-approved treatments. ~40% of HCM cases are associated with mutations in the gene encoding cardiac myosin-binding protein C (MyBP-C). MyBP-C is a thick filament-associated protein that is critical for normal myocardial performance and centrally positioned in the sarcomere to regulate interactions between myosin and actin responsible for force development. We previously demonstrated that increased phosphorylation of MyBP-C, enhances actin-myosin interactions to accelerate contraction kinetics in myocardium, whereas the decreased MyBP-C phosphorylation, reduces actin-myosin proximity and decelerate contraction. However, it is remains unknown how MyBP-C functions under varied states, including myofilament activation, phosphorylation, and HCM mutations. The structural dynamics of MyBP-C and its interactions with actin and/or myosin to modulate force development in myocardium are key to understanding this mechanism of action. We have developed innovative biophysical tools that, for the first time, enable determination of these mechanisms by evaluation of: (1) MyBP-C structural dynamics, (2) how it interacts with actin and myosin in relaxed and activated muscle, (3) how these interactions are affected by phosphorylation and (4) known pathologic mutations. We will test the central hypothesis that MyBP-C function is determined by dynamic structural changes of its domains that determine interactions of MyBP-C with thin (actin) and thick (myosin) filaments and that the equilibrium of these interactions is affected by phosphorylation and HCM mutations. The proposed aims further develop our innovative biophysical tools to measure structural dynamics underlying MyBP- C regulation of contraction in normal and diseased states. These tools include site-directed fluorescence spectroscopy, computational simulations, thin and thick filament function, and mechanical measurements. We will examine how the activation state of the myocardium (Aim 1), phosphorylation of MyBP-C (Aim 2), and HCM mutations (Aim 3) affect MyBP-C’s structural dynamics and interactions to modulate cardiac contractility. The proposed studies resolve interactions in real myocardial space and capture structural dynamics in real time using high-resolution approaches during the contractile cycle. This involves monitoring distances between points on proteins and the order (or disorder) of those distances under physiological conditions, in interacting proteins and functioning myocardium. Fluorescence lifetime data components, thin and thick filament activation, mechanics, and simulations will be used to define models of MyBP-C regulation. The proposed aims offer unprecedented mechanistic resolution of MyBP-C for its functions in health and HCM disease. This mechanistic understanding is critical to lay the foundation for determining the qualitative and quantitative impact of MyBP-C-targeted drugs that are currently being identified by our group for the development of new therapies to treat HCM and other cardiac disorders.

NIH Research Projects · FY 2026 · 2018-09

PROJECT SUMMARY Approximately 25% of diabetic patients experience diabetic foot ulcers (DFUs). This is a significant clinical problem since there are no effective biomarkers for predicting outcomes, no drug candidates that have recently been FDA-approved and no therapies that are widely effective in treatment. Additionally, the prevalence of diabetes and non-healing DFUs are highest among minorities, such as in Native American and Hispanic populations, and associated with social deprivation and high mortality. The University of Arizona (UArizona) is the leading public research university in the American Southwest and an ideal transdisciplinary research community for studying DFU healing. The partnership between UArizona and Banner Health provided service to 5,680 patients with open wounds last year. Ranging from trauma to podiatry, Banner Health saw 3,770 individuals with diabetic foot ulcers (DFUs) in the past 3 years. Dr. Geoffrey Gurtner has an established history in studying both late-stage biomarker validation through clinical trials and also mechanistic and early-stage biomarkers identification through the collection of many high-quality biosamples and longitudinal data (Aim 1). This is supported by both the large patient population at UArizona as well as Dr. Gurtner’s history in identifying rate cell subpopulations in DFU samples collected from human patients. Next, we aim to create a unified Standard of Care through the execution of high-quality clinical trials for DFUs, which historically have been difficult to recruit, and also by collecting high-quality data to address the heterogeneity of DFUs with complex pathologies, co-morbidities, and social factors. At UArizona, Dr. Gurtner and Dr. Zhou conduct high-quality clinical trials and have access to a wide distribution of patients and a range of techniques to make sure that “No DFU Patient Goes Unstudied” (Aim 2). Next, our diverse patient pool will allow us to specifically understand how social and environmental contextual factors (“social determinants of health”; SDH) may affect diabetic healing and biomarkers, specifically be recruiting over a diverse and expansive patient pool (Aim 3). Finally, we have access to not just the entire Banner Health Network (BHN), but also to the Indian Health Network and outreach programs for Latino communities. Through this expansive network that incorporates people of all demographics and socio-economic statuses, we will establish effective collaborations between the CRU and new clinical sites to generate cooperative problem-solving and high-level training, with shared resources and values (Aim 4).

NIH Research Projects · FY 2025 · 2018-08

PROJECT ABSTRACT Rates of maternal mortality have been increasing annually despite growing public attention and efforts to promote maternal health. Moreover, rates of pregnancy-associated mortality including homicide, suicide, and drug overdose are alarmingly high and rising as well, and disparities characterize all of these outcomes. Concurrently, state reproductive health policies have grown increasingly restrictive in recent years, and an unprecedented number of states have enacted total bans on pregnancy termination or narrow gestational limits on care since 2022. Extant research has failed to interrogate the interconnectedness of these two issues, and the role of state policies as determinants of maternal and infant health outcomes and disparities remains unknown. Our overall objective in this application is to fill a critical gap in the scientific evidence by evaluating the impact of the current policy environment on trends and disparities in maternal mortality; pregnancy-associated homicide, suicide, and drug overdose; infant mortality; and preterm birth. We have established and will continue to expand a national longitudinal geodatabase for monitoring trends in state policies, macrosocial contexts, maternal and infant mortality and other maternal population health outcomes US that includes and extends back 16 years from the most recently available data (2023). Our specific aims are to (1) identify the impact of state reproductive health policy changes on state trends in maternal health including pregnancy-associated mortality (homicide, suicide, drug overdose) and maternal mortality; (2) identify the impact of state policy changes on state trends in infant health including all-cause and cause-specific infant mortality and preterm birth; and (3) identify the impact of state policy changes on disparities in maternal and infant health outcomes. The research approach applies rigorous and innovative econometric methods that exploit a natural experiment framework to establish empirical evidence of the causal impact of state policies on trends in the most severe and salient indicators of maternal and infant population health.

NIH Research Projects · FY 2022 · 2018-08

Primary auditory afferent neurons conduct sound-evoked action potentials (APs) through surprisingly small diameter axons at remarkable speed and with millisecond precision. The response properties of the auditory neuron (AN) are phased-locked for low-frequency (<5 kHz) sounds, suggesting that conduction failure is a rarity. However, the neural mechanisms that enable swift and phase-locked conduction are poorly understood. We hypothesize that ANs utilize non-uniform nodal, internodal, and patchy nodal ionic channel distribution to maintain fast conduction velocity (CV). These features optimize action potential (AP) CV and prevent AP conduction failure despite the structural limitations of the AN. We propose to test the underlying hypotheses using various knockin and knockout mouse models and pharmacological strategies. We utilize innovations such as optogenetics, high-resolution microscopy, and multiple electrophysiological approaches. We aim to determine 1) AN axonal ion channels' expression, colocalization, and interactions. We will employ multidisciplinary approaches to assess the expression distribution of specific ionic channels in AN axons. 2) The functional and physical interactions between specific ionic channels in AN neurites. The proximity of certain channels shapes the APs of ANs for high-speed conduction. We will use proximity ligation assay (PLA), live-cell imaging, and spatial and temporal resolution recordings to quantify the protein-protein interactions. 3) Axonal ionic channels' ex vivo and in vivo functional roles of ion channels that shape AN AP CV will be examined. We will use patch- clamp analyses of the kinetics, voltage dependence, and conductance in AN neurons to determine the underlying mechanisms for the differences in response properties and CV using computational studies. Thus, we will address the complexity of anatomic projections and signal processing and subsequent alterations of the structural and ionic conductances that would alter AP CV. This information is a necessary step toward developing treatments for hearing loss.

NIH Research Projects · FY 2026 · 2018-07

PROJECT SUMMARY Metal dysbiosis is detrimental to any living system as approximately 40% of proteins use metals as a cofactor or structural component. Therefore, when pathogenic bacteria invade a host, there is a battle for metal micronutrients such as iron, calcium, manganese, and zinc that benefit each organism. While human hosts acquire metals through their diet, bacteria must acquire metals from within the host. However, for bacteria that exist at the host/pathogen interface, some host-utilized metals can be toxic to bacteria. For example, compared to iron, calcium, and manganese concentrations needed for survival, zinc and especially copper are toxic to bacteria even at lower concentrations. As such, bacteria have evolved import and export systems to maintain homeostasis. Complicating metal acquisition is mismetallation, when the unintended metal binds to the protein to diminish function (e.g., low enzymatic turnover or decreased substrate binding), specifically because the stability of complex formation with divalent metal is as follows: Cu >Zn > Fe > Mn > Ca. The human host sequesters beneficial metals (iron, calcium, and manganese) to restrict infection while also bombarding the bacteria with zinc and copper. How bacteria respond to copper + zinc stress and the different concentrations of these metals they encounter in the host are largely unknown. While metal toxicity has been the subject of other studies, most of these have focused on single concentrations of one metal, often in complex media. These media are more lavish than the host environment and may mask portions of the metal response. To address fundamental gaps in knowledge regarding how bacteria respond to metal dysbiosis, we used a multi-omics approach (transcriptomics, metabolomics, and suggest a proteomic arm) to investigate the pathways affected during bacterial disruption via copper and zinc at varying concentrations in a host-adjacent, minimal, and defined media in Streptococcus pneumoniae as the bacterial system. Using these data and leveraging our technical expertise in metallobiology and microbiology, we will determine how metal influx, efflux, and internal metal concentrations respective to copper and zinc affect the transcriptome and metabolome. These data will be used to determine not only which systems are affected but also when these systems are being turned on, with respect to concentration dependence. We will determine how some of these systems are regulated based on the mismetallation of proteins such as DNA transcription factors. We will determine how metal dysbiosis affects other metal transport. Lastly, we will determine if an affected system is poisoned by copper and/or zinc or used to overcome that stress based on examining the metabolite level respective to the direction of the system’s transcriptomic profile.

NIH Research Projects · FY 2026 · 2018-07

Synaptic disruption is a prelude to and often a primary cause of neurological disease, but we have few strategies to correct dysmorphic synapses, even if they occur early in the degenerative process. Growth control pathways, i.e. those that promote protein and lipid synthesis while reducing catabolism, regulate synaptic form that in turn ensures efficient function and plasticity. The 7-pass endosomal membrane protein TMEM184B regulates synaptic structure and function across species; accordingly, its loss causes exuberant synaptic sprouting, swollen nerve terminals, and altered excitability. In humans, disruption of conserved amino acids in TMEM184B is linked to nervous system disruptions including microcephaly, intellectual disability, corpus callosum hypoplasia, and epilepsy. While TMEM184B genetic disruptions are rare, the disorders produced by TMEM184B disruption are common, suggesting an intersection with key neurological pathways. Our long-term goal is to define the mechanisms underlying TMEM184B variant-associated nervous system disorders in order to provide mechanistic guidance for their treatment. Our overall objectives in this proposal are to determine how TMEM184B directs key signaling pathways supporting neuronal structure and function and to illuminate how patient variants of TMEM184B alter synaptic transmission and resultant behavior. TMEM184B has sequence similarity to bile acid and sterol transporters, but this proposed molecular function remains untested. Preliminary data and published studies suggest an intersection between TMEM184B and mTOR, but how TMEM184B influences mTOR pathway activity is completely unknown. We hypothesize that TMEM184B is an endosomal transporter whose function impacts mTORC1 signaling to promote synaptic structure and function. In Aim1, we will evaluate the specific contributions of TMEM184B to the mTORC1 pathway using primary cortical neuron cultures from wild type and TMEM184B mutant mice. We will evaluate the function of upstream activators and downstream effectors of mTORC1 using functional readouts as well as lipidomic and phospho-proteomic tools. In Aim 2, we will model human disease-linked TMEM184B variants in Drosophila by introducing patient mutations into conserved amino acids. With these flies we will evaluate synaptic form, function (electrophysiological recording), and behavior. In Aim 3, we will establish the molecular function of the TMEM184B protein using a combination of in silico, thermodynamic, and proteomic assays to reveal candidate transport substrates and other metabolites most affected by TMEM184B disruption. Overall, our multifaced approach will illuminate the mechanism through which TMEM184B acts to ensure synaptic morphology and function while enabling a better classification of TMEM184B-associated disorders with others of similar etiology, facilitating improved diagnosis and treatment.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY. Advances in neonatal critical care have greatly improved the survival of preterm infants but the long-term complications of prematurity, including Bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. After premature birth, transition of the lung to a non-aqueous environment appears sufficient to disrupt subsequent alveolar growth and the attendant vascular structures required for effective gas exchange. This is further exacerbated when the preterm lung is exposed to supplemental oxygen and positive pressure ventilation. Irreversible loss of alveolar capillaries and vascular remodeling after oxygen exposure cause pulmonary hypertension (PH) seen in patients with severe BPD (BPD-PH). There is an urgent need for innovative therapeutic approaches to stimulate neonatal lung angiogenesis and preserve respiratory function in BPD-PH infants. My laboratory recently created PEI600-MA5/PEG-OA/Cho nanoparticles that can deliver non-integrating expression plasmids with pro-angiogenic genes into pulmonary microvascular endothelial cells with the purpose of stimulating neonatal lung angiogenesis. We also identified a specialized subpopulation of pulmonary endothelial progenitor cells (EPCs), FOXF1+ EPCs, that are a subset of recently discovered general capillary cells (gCAPs). Transplantation of FOXF1+ gCAPs increased neonatal lung angiogenesis and alveolarization in mice with congenital deficiency of alveolar capillaries. We propose to test the hypothesis that increasing neonatal lung angiogenesis via the nanoparticle FOXF1 gene therapy or the FOXF1+ gCAP cell transplantation will prevent PH and improve lung function in mouse and rat models of BPD- PH. In Aim 1, we will determine whether the nanoparticle FOXF1 gene therapy has a long-term beneficial effect in BPH-PH by preventing PH and right ventricular (RV) hypertrophy, and accelerating lung regeneration after neonatal hyperoxic lung injury. We will also identify novel downstream targets of FOXF1 in regenerating endothelial cells and test whether FOXF1 recruits STAT3 to the chromatin to activate endothelial enhancers. Our studies will determine if the FOXF1-STAT3 protein-protein interactions are required for lung regeneration in BPH-PH models. In Aim 2, we will determine whether transplantation of donor FOXF1+ gCAPs has a long- term beneficial effect by preventing PH and RV hypertrophy in mouse model of BPH-PH. We will also test requirements of the DLL4/NOTCH signaling pathway for the ability of donor FOXF1+ gCAPs to stimulate proliferation and tube formation in recipient endothelial cells during lung regeneration after hyperoxic injury. Finally, we will produce mouse FOXF1+ gCAPs from embryonic stem cells (ESCs) in vitro (via directed differentiation of ESCs into FOXF1+ gCAPs) and in vivo (via interspecies mouse-rat chimeras). Mouse ESC- derived FOXF1+ gCAPs will be used for cell therapy to prevent or delay PH and RV hypertrophy in mouse BPD-PH model. Altogether, the proposed preclinical studies will directly test whether endothelial delivery of the FOXF1 vector or cell therapy with FOXF1+ gCAPs have therapeutic potential in BPD-PH.

NIH Research Projects · FY 2026 · 2017-12

Abstract: The usage of metabolic pathways is tailored to meet the specific functions and demands of a given cell type. Of particular interest is how metabolism supports the survival and antibody secretion of plasma cells, the primary cell type that is responsible for humoral immunity. The lifespan of these cells dictates the duration of antibody-mediated immunity after infections or vaccines—a particularly relevant topic in the midst of this pandemic. During the previous funding period, our work suggested a surprisingly minimal role for transcriptional pathways in controlling plasma cell lifespan. Instead, metabolic pathways functionally distinguish plasma cells of differing lifespans. Using newly created genetic tools, we will rapidly define and dissect essential plasma cell metabolic pathways in vivo. We will use newly generated plasma cell Cre-drivers, lentiviral bone marrow chimeras, and CRISPR/Cas9 approaches to functionally define mitochondrial dynamics, essential metabolic pathways, and vesicular maturation pathways that promote plasma cell lifespan and antibody secretion. These approaches will be coupled with sensitive imaging mass spectrometry and stable isotope-tracing experiments to provide mechanistic insight. Specifically, our experiments will define the importance of mitochondrial fission and fusion in plasma cell energy metabolism, antibody production, and survival. Physiological experiments using viral infections and immunizations will define key factors that promote plasma cell metabolic re-programming as these cells become progressively longer lived. Second, based upon results of a completed genome-wide CRISPR/Cas9 screen, we will pursue the importance of V-type ATPases in amino acid uptake, plasma cell lifespan, and antibody secretion in vivo.

NIH Research Projects · FY 2026 · 2017-09

PROJECT SUMMARY/ABSTRACT Up to 50% of cases of chronic obstructive pulmonary disease (COPD) develop the disease through a lung function trajectory characterized by an impaired lung function growth in childhood, without necessarily experiencing a faster decline in adult life. This “low flier” trajectory is associated with early low-grade systemic inflammation and can lead to the development of COPD through an intermediate stage of “pre-COPD” in which lung function deficits, respiratory symptoms, and small airway abnormalities are present and identify at-risk individuals years before the disease is fully manifest. However, at present the factors that confer resilience to the low flier trajectory and may, in turn, modify its sequelae on pre-COPD remain unknown. Here, we propose that Club cell secretory protein (CC16), a pneumoprotein with critical anti-inflammatory properties, plays a key role in protecting from the risk of early airflow limitation and other pre-COPD phenotypes linked to impaired lung function growth in childhood. Although it is mainly produced by club cells and other non- ciliated airway epithelial cells, CC16 can be readily measured in circulation and work completed during the previous funding period has demonstrated that this molecule is likely to exert direct protective effects against airway obstruction partly through anti-inflammatory properties by reducing adhesion of activated leukocytes to endothelial cells and, in turn, preventing their lung infiltration. In this renewal application, we intend to extend these tantalizing findings by establishing the role of circulating CC16 in childhood as a resilience factor against early airflow limitation and pre-COPD phenotypes in young adult life. In epidemiological and experimental studies, we will also evaluate whether these CC16 effects counteract those induced by early systemic inflammation (assessed by an inflammatory score of 5 proteins that we have recently linked to low lung function growth). To accomplish these goals, we will expand our CC16 Epi Consortium to include eight international birth cohorts and establish one of the largest respiratory epidemiological consortia to date. Aim 1 - To determine whether deficits in circulating CC16 in childhood predict the presence of airflow limitation and other pre-COPD phenotypes in young adult life in a large international consortium of birth cohorts. Aim 2 - To determine whether low circulating CC16 and systemic inflammation in childhood have combined effects on risk for pre-COPD phenotypes by young adult life. Aim 3 - To determine whether leukocytes from low fliers with early systemic inflammation have increased propensity to adhere to the endothelium ex vivo, and whether their adhesion to endothelial cells can be reduced with recombinant CC16 (rCC16). The proposed work will shed new light on the role of CC16 in resilience against pre-COPD and may pave the way to novel CC16-centered strategies to prevent and/or mitigate the long-term sequelae of early COPD.

NIH Research Projects · FY 2026 · 2017-09

OVERALL- Abstract Infectious disease, cancer, and autoimmune disorders affect hundreds of millions of older adults. They reduce length and quality of life across the globe and inflict a massive economic burden on society, as vividly exemplified by the SARS-CoV-2 pandemic, that has claimed 93.1% of its victims amongst those 50 years and older, and 74.4% in those 64 and older. Yet, despite decades of research, restoring protective immunity in older adults has remained elusive. One critical factor contributing to age-related immune decline is a loss of naïve T (Tn) cell numbers and function, and their rejuvenation is highly desirable in order to enhance protective immunity and overall healthspan in older adults. The renewal of this T cell Rejuvenation Program Project is centered on two key questions: (1) why do Tn cell numbers and function deteriorate with age; and (2) what can be done about it? The premise of the program is that Tn cell aging is multifactorial and that it can only be resolved by targeting multiple defects. Thymic involution and the resulting decline in T cell production is the earliest event leading to immunosenescence. This reduction is compounded by a decline in bone marrow function, as well as by defects in Tn cell maintenance and function in the periphery. These deficiencies combine to erode the ability of the older immune system to detect and eliminate infectious agents and neoplastic cells, and to properly guard against autoimmunity. In the first program period, we strongly confirmed our initial hypotheses that lymphoid organ stromal elements deteriorate earlier than previously thought, and in a manner to decisively erode immunity, with aging. We will build on the synergy and success of the initial program period, where discoveries in one project are critically informing science in others, and continue to identify mechanistic reasons behind reduced central and peripheral lymphoid organ function with aging. We will then develop combined strategies to ameliorate these defects to improve immune defense in the elderly. Our hypothesis is that mechanistic dissection of defects in both thymic production AND peripheral Tn cell maintenance is required to devise and test effective interventions for T cell rejuvenation in the elderly. Three integrated projects led by experts in the field, supported by four cutting-edge cores, will test this hypothesis and achieve the following Program Goals: 1. Define mechanistic changes in primary and secondary lymphoid organ aging; 2. Determine the endogenous regenerative capacity of thymic and secondary lymphoid organ stroma over the lifespan; 3. Relate the progression of murine thymus, lymph node (LN) and T cell aging phenotypes to humans; and 4. Devise and test rejuvenation strategies to improve thymopoiesis, T cell survival and peripheral T cell maintenance and function, so as to enhance protective immunity. Over this support period, the above goals will provide a wealth of basic knowledge that will be translated to preclinical models and be poised for translation to older adults.

NIH Research Projects · FY 2025 · 2017-07

Primary auditory afferent neurons conduct sound-evoked action potentials (APs) through surprisingly small diameter axons at remarkable speed and with millisecond precision. The response properties of the auditory neuron (AN) are phased-locked for low-frequency (<5 kHz) sounds, suggesting that conduction failure is a rarity. However, the neural mechanisms that enable swift and phase-locked conduction are poorly understood. We hypothesize that ANs utilize non-uniform nodal, internodal, and patchy nodal ionic channel distribution to maintain fast conduction velocity (CV). These features optimize action potential (AP) CV and prevent AP conduction failure despite the structural limitations of the AN. We propose to test the underlying hypotheses using various knockin and knockout mouse models and pharmacological strategies. We utilize innovations such as optogenetics, high-resolution microscopy, and multiple electrophysiological approaches. We aim to determine 1) AN axonal ion channels' expression, colocalization, and interactions. We will employ multidisciplinary approaches to assess the expression distribution of specific ionic channels in AN axons. 2) The functional and physical interactions between specific ionic channels in AN neurites. The proximity of certain channels shapes the APs of ANs for high-speed conduction. We will use proximity ligation assay (PLA), live-cell imaging, and spatial and temporal resolution recordings to quantify the protein-protein interactions. 3) Axonal ionic channels' ex vivo and in vivo functional roles of ion channels that shape AN AP CV will be examined. We will use patch- clamp analyses of the kinetics, voltage dependence, and conductance in AN neurons to determine the underlying mechanisms for the differences in response properties and CV using computational studies. Thus, we will address the complexity of anatomic projections and signal processing and subsequent alterations of the structural and ionic conductances that would alter AP CV. This information is a necessary step toward developing treatments for hearing loss.

NIH Research Projects · FY 2026 · 2017-03

Our earlier mechanistic analyses of estrogen action in brain led to the discovery that estrogen is a master regulator of the bioenergetic system in brain that promotes glucose transport, glucose metabolism, mitochondrial respiration and ATP generation. Collectively, the data provided compelling evidence for estrogen as a systems biology metabolic regulator in brain and illuminated compensatory mechanisms consistent with an aging female brain that is starving. For estrogen to function as master regulator of the bioenergetic system in the female brain, estrogen must be integrating nuclear and mitochondrial genomic responses. Further from a systems level perspective, it would be necessary for estrogen to also regulate cytoplasmic signaling mechanisms for real time feedback on the functional outcomes of nuclear and mitochondrial gene transcription. The fundamental issues to be investigated are the mechanisms whereby estrogen integrates bioenergetic responses across two genomic compartments while simultaneously monitoring energetic demand and performance in real time. The proposed program of research is designed to test two hypotheses. First, estrogenic control of the bioenergetic system in the female brain requires: 1) both nuclear and mitochondrial genomes; 2) integration of gene expression across both genomic compartments and 3) activation of rapid signaling cascades to provide real time feedback on bioenergetic performance. Second, we hypothesize that loss of estrogen in the aging female brain leads to a systematic dis-integration of estrogenic control of nuclear and mitochondrial genomes followed by decline in bioenergetic sensing mechanisms. Estrogenic control of the bioenergetic system of the brain and the dismantling thereof has basic, translational and clinical significance. From a discovery perspective the proposed program of research is unique in exploring the mechanisms underlying estrogenic integration of nuclear and mitochondrial gene expression and the real time feedback mechanisms that control the bioenergetic system of the brain. Further, the process by which this control system is dismantled in the aging female brain is uncharted territory of high significance for understanding bioenergetic aging in brain. Translationally, determining the mechanisms underlying the systematic dismantling of estrogenic integration of bioenergetic compartments in brain has the potential to detect therapeutic targets to sustain bioenergetic function in the aging female brain. Clinically, the aging transition of menopause, unique to the female, is a process that dismantles both reproductive ability and potentially bioenergetic capacity in brain. This is particularly relevant to age-related neurological conditions associated with deficits in glucose hypometabolism such as Alzheimer's, depression and multiple sclerosis which have greater prevalence in postmenopausal women. Research proposed herein aligns with NIA Strategic Research Goals A and C and the “need to better distinguish patterns of brain aging” https://www.nia.nih.gov/about/living-long-well-21st- century-strategic-directions-research-aging and to objectives of Office of Research on Women's Health.

NIH Research Projects · FY 2025 · 2016-07

Abstract Poor language skills are associated with numerous negative outcomes ranging from higher rates of tantrums and difficulty developing friendships to school failure, contact with the justice system, and increased victimization. Although language deficits may be noticed as early as toddlerhood, effective treatment may not begin this early and there is relatively little time to close the language gap before these children are faced with the increased language demands of formal education and the cumulative effects of academic struggle. For the 7-13% of children with impaired language skills, language treatments that are faster and more effective are urgently needed. This competing renewal addresses this need with a series of studies that translate basic research in statistical learning to treatment contexts. The Statistical Learning Framework posits learners extract word meaning and grammatical structure from the language input they receive, and the statistical structure of the input accounts for rapid, implicit language learning. Six proposed studies translate statistical learning principles to a treatment context. Theoretically-motivated treatment factors are tested in two groups of children with poor language skills. “Late Talkers” are children (ages 2-3 years) who are identified by the very limited number of vocabulary words that they understand and use. Preschool children with Developmental Language Disorder (ages 4-5 years) show marked deficits in the use of grammatical morphemes. Parallel studies targeting vocabulary treatment (for Late Talkers) and morphosyntax treatment (for children with DLD) will test whether leveraging prior learning can improve treatment methods by making learning faster and more effective. We will also directly address the issue of non-responders (i.e., children who make limited improvement despite treatment that is effective for others), an unaddressed problem inherent to all treatment research. We leverage our previous findings to predict which children are highly likely to be non-responders and propose alternative treatment methods that might assist this subset of children. These studies represent the necessary work for principled language treatment that is supported by evidence, and can provide insights into the nature of learning in a range of children with poor language skills.

NIH Research Projects · FY 2025 · 2016-06

Clearance of cytoplasmic RNA, protein and mRNA-protein (mRNP) granules maintains homeostasis and prevents the accumulation of toxic species. Stress granules (SGs) and P-bodies (PBs) are mRNP granules enriched in mRNAs, RNA binding proteins and signaling proteins, that often aid cell survival during stress. This may reflect regulation of the transcriptome and signaling pathways. Aberrant SG clearance is implicated in many cancers, viral infections, and Amyotrophic Lateral Sclerosis (ALS), where SGs may promote cytoplasmic mis- localization and aggregation of TAR DNA-binding protein 43 (TDP-43); this is toxic to neurons. SGs are likely cleared by various disassembly and degradative means, with roles for chaperones, the proteasome, and a selective autophagic pathway termed granulophagy. In contrast, PB clearance has barely been studied. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal trafficking pathway (distinct from autophagy), which, when induced, suppresses TDP-43 toxicity. Understanding of the mechanisms and consequences for SG, PB and TDP-43 clearance remains at an early stage. It is also known that large amounts of RNA decay occur in vacuoles and lysosomes, though the RNA molecules targeted, trafficking mechanisms used and impacts of such decay on gene expression are unknown. Key gaps in understanding include determining how different clearance pathways function, co-operate and affect the degradation or disassembly of mRNP granules, cytoplasmic RNA and TDP-43. The impact of such clearance pathways on cell function and disease also requires elucidation. The aims of this grant are: 1.) define the usage, importance and co-operativity of reported SG and PB clearance mechanisms under disease-relevant stress, and identify the mechanism of granulophagy; 2.) determine the extent, specificity and trafficking mechanism(s) underlying vacuolar/lysosomal RNA decay; 3.) mechanistically assess TDP-43 endolysosomal degradation and evaluate consequences to neuronal and TDP-43-related RNA phenotypes. Using genetic, biochemical and cell biology assays, a granulophagy model based on a prior unbiased yeast screen will be tested. These efforts will be aided by a novel SG purification method, which will identify SG-localized granulophagy effectors. RNA-sequencing and vacuole isolation will be combined to quantify the vacuolar RNA degradome, while genetics and single-molecule imaging will identify RNA vacuolar decay trafficking mechanism(s). Finally, supported by an unbiased yeast screen identifying regulators of TDP-43 abundance, a model of TDP-43 degradation involving endosomal membrane invagination will be tested. Yeast, human, and neuronal cell models will be used. This proposal is innovative in that it will generate basic understanding of how novel vacuolar/lysosomal trafficking mechanisms affect RNA and protein homeostasis. The value of this work is that the knowledge obtained will offer paradigms for clearance of similar cellular substrates and globally reveal targets of an unappreciated RNA decay pathway. Finally, understanding clearance of SGs and cytoplasmic TDP-43 may identify therapeutic targets in ALS and cancer.

NIH Research Projects · FY 2025 · 2016-06

In this AADCRC program renewal, we will focus on three critical and understudied innate immune factors and how they impact viral infections in asthma: the anionic phospholipids of surfactant (palmitoyl- oleoyl-phosphatidylglycerol, POPG, and phosphatidylinositol, PI), Toll interacting protein (Tollip), and surfactant protein-A (SP-A). Because these mediators have complementary functions to modulate inflammation and immunity in asthma and infection, we propose three interrelated, synergistic, self-standing projects to investigate how these mediators orchestrate novel innate immune responses associated with viral infections in asthma. We will study three viruses with a spectrum of effects in airway disease, and determine how innate responses protect against them. Specifically, we will focus on rhinovirus C (RV-C), a known exacerbator of asthma that can cause severe disease; influenza A, a virus whose effect in asthma remains ambiguous and SARS-CoV-2, a virus that can cause severe lung disease, but for which asthma may not be a risk factor, and may in fact confer protection. We show innovative preliminary data indicating that 1) POPG, PI and SP-A attenuate RV-C infection; 2) Tollip exhibits protective effects as it is required for IL-13 to generate soluble ST2 that in turn attenuates the effects of IL-33 during influenza A infection; and 3) SP-A and type 2 cytokines confer protection in the effector and initiation phases of SARS-CoV-2 infection in asthma by inhibiting the expression and function of ACE2, the SARS-CoV-2 receptor, through effects upon transcription, receptor binding and downstream pro-inflammatory signaling. Thus, all these innate immune components appear to protect against viral infections in asthma. Our exciting preliminary data underpin our program’s overall hypothesis that POPG/PI, Tollip and SP-A function as unique immune modulators that attenuate the impact of specific viral infections (RV-C, Influenza A and SARS-CoV-2) in type-2 asthma. Therefore, supplementation of functional POPG/PI, SP-A and the IL-33 decoy receptor sST2 may be novel strategies against asthma exacerbations due to viral infections. Project 1 will critically test the activity of POPG/PI and SP- A supplementation as a novel molecular tool for disrupting infections due to RV-C, a virus known to exacerbate asthma. Project 2 will determine how Tollip protects against viral exacerbations caused by influenza A in asthma through inhibition of IL-33 signaling. Project 3 will determine how type-2 cytokines and SP-A synergize to protect against SARS-CoV-2 infection through inhibition of ACE2-mediated infection and IL-6 signaling pathways. We also include an Administrative Core and a Clinical Core, both which serve all projects equally. We build upon productive collaborations of over 20 years on innate molecular mechanisms underlying the interaction between type 2 inflammation and viral exacerbations of asthma. The strong synergy among our three projects will accelerate progress toward novel therapies by demonstrating that the innate immune components under study protect against viral infection in asthma.