University Of Arizona

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Late onset Alzheimer’s disease (LOAD) is a neurodegenerative disease with a multifactorial etiology and intersecting genetic and environmental risks, making it a complex systems challenge. Brain functions are highly energy-dependent, with most of which generated by mitochondria via oxidative phosphorylation. While the association between LOAD and an early decline in brain glucose metabolism and changes in mitochondrial function is well-established, therapeutics that universally enhance brain mitochondrial function have yet resulted in favorable outcomes. As the greatest genetic risk factor for LOAD, the e4 variant of APOE (APOE4) was also found to affect brain bioenergetics and lipid metabolism via incompletely understood mechanisms. Considering the metabolically diverse cellular composition of the brain and APOE as an inter-cellular lipid carrier, we propose that cell type-specific intra-cellular bioenergetic shifts and inter-cellular metabolic uncoupling of fatty acid (FA) metabolism underlie APOE4-driven AD relevant metabolic phenotypes in the brain. Specifically, we hypothesize that APOE4-induced disruption to astrocytic clearance of neuronal FAs and subsequent degradation in astrocytic mitochondria could elicit lipid dysregulation, neuronal dysfunction, neuroinflammation and cognitive decline. Program of research proposed herein will determine the mechanisms, at the cellular level, by which APOE polymorphism alters brain bioenergetics and lipid homeostasis, and eventually LOAD risk. To test our hypotheses, we will implement three levels of investigations to understand the complex mechanisms underlying APOE regulation of metabolic coupling between neuron and astrocytes. Aim 1 will determine and differentiate the effect of APOE isoforms on cellular metabolic shift in neurons and astrocytes using single-cell transcriptomics and in vitro functional assessment. Using a neuron-astrocyte co-culture system, Aim 2 is designed to investigate the impact and mechanism by which different isoforms and origins (neuronal- vs. astrocytic) of APOE affect neuron-astrocyte metabolic coupling, focusing on fatty acid metabolism. Aim 3 will test determine how perturbations to neuron-astrocyte metabolic coupling mediate APOE4-induced LOAD at-risk phenotypes during aging in vivo. Projected outcomes from this research will elucidate how cell types with distinctive bioenergetic phenotypes jointly maintain the brain metabolic homeostasis, and how APOE4 increases risk of LOAD by disrupting the metabolic system of the brain. Translationally, this research will shed light on selective cell vulnerability in AD development and has the potential to identify APOE genotype-specific and cell type- specific therapeutic targets to sustain or restore a bioenergetic equilibrium and lipid homeostasis in the brain that are resilient against synaptic- and cognitive declines in LOAD.

NIH Research Projects · FY 2025 · 2021-05

Human cytomegalovirus (HCMV) establishes a life-long persistent infection by evading the immune system, in part, by direct cell-to-cell viral spread. In solid organ or stem cell transplant recipients, HCMV spread leads to end-organ diseases that can cause death. During pregnancy, HCMV spread causes congenital infection and is a leading cause of congenital disabilities. No HCMV treatment offers a cure, and there is no vaccine. Thus, there is a need for new treatments to limit infection based on novel discoveries in HCMV biology. Viral proteins required for HCMV cell-to-cell spread are known, but the host processes involved in HCMV cell-to-cell spread have received less attention. Clinical strains of HCMV spread most efficiently through cell-to-cell means, but the molecular mechanisms—including host metabolic ones—essential to HCMV cell-to-cell spread are largely unknown. Understanding host mechanisms regulating cell-to-cell spread may lead to new understandings of how to reduce HCMV infection. Our research has uncovered a novel role of metabolite signaling in promoting HCMV spread. This project's overall goal is to mechanistically understand virus-host interactions regulating metabolite signaling essential to HCMV cell-to-cell spread. We found a metabolite in tryptophan metabolism—kynurenine (KYN)—enhances HCMV spread. In addition to its metabolic role, KYN is a signaling messenger. KYN signals through aryl hydrocarbon receptor (AhR). We show that activation of AhR supports HCMV replication. Moreover, we found that hypoxia-inducible factor 1α (HIF1α), through its metabolic regulatory function, limits the production of KYN and suppresses HCMV infection. We hypothesize that metabolite-mediated signaling from infected cells to uninfected cells promotes HCMV cell-to-cell spread, which is attenuated by a HIF1α-dependent cellular response. The proposed research will determine molecular mechanisms involved in the enhancement of HCMV infection by KYN-metabolite signaling (aim 1) and define virus-host interactions regulating HIF1α attenuation of HCMV cell-to-cell spread (aim 2). The experimental approach will integrate virus assays, CRISPR/Cas9 engineering, and untargeted metabolomics to understand HCMV biology. Our findings will provide a mechanistic understanding of metabolite signaling and AhR activity in promoting HCMV cell-to-cell spread and the HIF1α-dependent host-response that targets metabolite signaling to reduce infection. Our studies will advance our knowledge in an understudied area of HCMV research that will provide significant steps-forward in developing novel strategies to treat HCMV infection and limit HCMV-related disease.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Type 2 diabetes (T2D), a major risk factor for poor bone quality and fractures, is associated with the premature accumulation of senescent cells and advanced glycation endproducts (AGEs; activators of the receptor for AGE [RAGE] pathway) in multiple tissues, including bone. Intuitively, senescent cells and RAGE could act independently or interact via cross-talk to contribute substantially to skeletal fragility in T2D, yet this concept has not been rigorously tested. This proposal is founded on innovative concepts, technology, and approaches to test our central hypothesis that targeting cellular senescence or RAGE can improve T2D-related skeletal fragility. To test our hypothesis, we will use novel transgenic mice and innovative technology, including mass cytometry as well as advanced histological and molecular tools. The interplay among bone, energy metabolism, and T2D has been a topic of research for years, yet few in vivo studies have rigorously interrogated the contributions of senescent cells or RAGE signaling to skeletal dysfunction in T2D. From a translational perspective, better understanding of the cross-talk between senescence and RAGE in bone will yield impactful advances and may reveal novel strategies to ameliorate accelerated skeletal aging in T2D. To this end, in Aim 1 we will identify, locate, and characterize bone-resident senescent cell populations in mice with T2D and define their T2D-specific senescence-associated secretory phenotype (SASP). In Aim 2, using mice harboring transgenes that enable the selective elimination of p16Ink4a+ or p21Cip1+ senescent cells, we will test the hypothesis that senescent cell clearance in mice with established T2D will normalize bone remodeling and quality. Thus, we will distinguish the causal roles of p16Ink4a and p21Cip1 in mediating skeletal dysfunction in T2D using our global p16- and p21-ATTAC mouse strains by comparing the effects of systemic clearance of p16Ink4a+ vs p21Cip1+ senescent cells. In addition, we will assess the relative impact of clearing senescent osteocytes, using our novel Cre-LoxP lines – p16-LOX-ATTAC and p21-LOX-ATTAC. Global and osteocyte- specific clearance of senescent cells will be compared with pharmacological elimination using “senolytics”. Finally, in Aim 3, using our novel Cre-loxP mouse that inhibits RAGE signal transduction via cell-specific cytosolic-domain deficient dominant-negative RAGE (DN-RAGE) expression, we will define the effects of inhibiting RAGE signaling in the osteoblast/osteocyte and myeloid/osteoclast lineages on skeletal fragility in mice with T2D. Collectively, these studies will rigorously test whether cellular senescence and RAGE signaling underlie T2D-related skeletal fragility. We will address these questions by leveraging our unique resources and expertise. We will build upon compelling preliminary data and innovative approaches, including novel analytical, transgenic, and pharmacological tools that we anticipate will significantly advance our understanding of the fundamental biology of skeletal dysfunction in T2D, leading to new mechanistic insights, and evidence- based therapeutic approaches to facilitate the translation of preclinical discoveries to clinical applications.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY. Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a severe congenital disorder associated with neonatal pulmonary hypertension (PH). Despite all available therapies and respiratory support, the vast majority of ACDMPV patients die from respiratory failure in the first month after birth. There is an urgent need for innovative therapeutic approaches for ACDMPV newborns. ACDMPV is associated with heterozygous loss-of-function mutations in the Forkhead Box F1 (FOXF1) gene encoding a transcription factor critical for lung angiogenesis. Based on human genetic data, there are 3 types of ACDMPV: type 1, with loss of coding FOXF1 sequences; type 2, with point mutations in FOXF1 exons; type 3, with loss of non-coding (regulatory) sequences. During previously funded periods, my laboratory generated the mouse models of type 1 (Foxf1wt/-) and type 2 ACDMPV (Foxf1wt/S52F) that recapitulate main features of the human disease and can be used for preclinical testing of potential ACDMPV therapeutics. However, the animal model of type 3 ACDMPV has not been generated. We recently used single-cell RNA and ATAC sequencing of mouse and human ACDMPV lungs to identify 4 evolutionarily conserved FOXF1 enhancers (FELs) that are often lost in type 3 ACDMPV. In Aim 1, we will focus on the FEL1 enhancer. In our preliminary data, the loss of the FEL1 enhancer is found in 9 out of 10 patients with type 3 ACDMPV. CRISPR/Cas9-mediated deletion of FEL1 in mouse embryonic stem cells (ESCs) disrupts differentiation of lung ECs in vivo, decreasing the numbers of capillary, arterial and venous ECs, and causing aberrant accumulation of non-mature “transitional” ECs characterized by decreased EC markers and upregulation of fibroblast markers. In Aim 1, we will test the hypothesis that the FEL1 enhancer is required for differentiation of lung ECs and if its loss causes ACDMPV. We will generate a novel mouse model of type 3 ACDMPV and identify molecular mechanisms through which FEL1 regulates EC differentiation. In the previously funded period, we used a high throughput screen to identify the TanFe small molecule compound, which prevents protein-protein interaction of FOXF1 protein with HECTD1 E3 ubiquitin ligase, and therefore, stabilizes the FOXF1 protein. In preliminary data for Aim 2, we chemically modified TanFe to produce a non-toxic and stable derivative, TanFe[F], which can be encapsulated into the P22- F1 PBAE/PEI/PEG nanoparticles and specifically delivered to lung capillary ECs in vivo. We also discovered that nanoparticle delivery of a known HECTD1 inhibitor, I3A, a plant-derived small molecule compound which is currently in clinical trials for cancer patients, increases lung FOXF1 protein levels and prevents neonatal PH caused by prolonged neonatal hyperoxia. In Aim 2, we will test the hypothesis that nanoparticle delivery of FOXF1-stabilizing compounds into lung capillary ECs will increase neonatal angiogenesis, improve respiratory function and protect from PH in mouse ACDMPV models. Altogether, we will develop a novel mouse model of type 3 ACDMPV, and test promising ACDMPV therapies based on stabilization of the FOXF1 protein in ECs.

NIH Research Projects · FY 2025 · 2021-05

Abstract The effect of aging on the human brain shows wide individual variation ranging from early onset Alzheimer's disease (AD) to maintenance of cognitive clarity into the 10th decade. The challenge is to understand why aging can have such disparate outcomes, and why it contributes so profoundly to the risk of neurodegenerative disease. We have examined aging and AD from the perspective of molecular pathways that underlie memory consolidation and determined that a gene termed NPTX2 provides an important clue to human cognitive failure. NPTX2 is expressed by pyramidal neurons and secreted at their excitatory synapses on parvalbumin interneurons (PV) to control inhibitory circuit function. NPTX2 and markers of PV function are prominently down-regulated in the brain of humans with AD, and CSF levels of NPTX2 correlate with both disease state and cognitive performance. NPTX2 is not down-regulated in the brains of individuals who maintain cognitive clarity despite amyloid accumulation (asymptomatic AD). These and other findings support the hypothesis that NPTX2 is associated with brain resilience critical for cognition and fails in the shift from healthy to unhealthy aging. Aim 1 will identify signaling pathways associated with preserved or deteriorated NPTX2 expression across the spectrum from older individuals with exceptional cognition to those with AD. Studies use an approach of targeted proteomics combined with bulk and single nuclei RNAseq, and will specifically examine the hypothesis that NPTX2 loss-of-function is associated with changes in interneuron cell properties. Aim 2 extends the goals of Aim 1 to establish the cellular mechanism of NPTX2 down-regulation using isogenic human iPS neurons encoding familial mutations of APP and PS1. iPS neurons with fAD mutations show profound and specific reductions of NPTX2 expression and provide an extraordinary opportunity to isolate and validate critical disease pathways. Analyses will include TMT differential mass spectroscopy and RNAseq. Candidate pathways will be manipulated and tested using CRISPR and pharmacological approaches. Aim 3 will provide the first test of the hypothesis that NPTX2 loss of function (LOF) in the adult brain is causal for circuit dysfunction and cognitive decline in the context of AD pathogenesis. Experiments use a newly established rat genetic model for conditional deletion of NPTX2 in a transgenic APP/PS1 AD model (Tg344- AD). Analyses will include high density electrophysiological recordings in hippocampal subregions CA1 and CA3 together with behavior tests and histopathological assessments of AD markers. Single nuclei RNAseq performed in CA3 will define the signature of NPTX2 LOF in the context of amyloid pathology. These data will be cross-referenced with findings from Aims 1 and 2 as part of an integrated interspecies analysis of the cause and consequences of NPTX2 LOF. Combined studies will deepen our understanding mechanisms that can confer cognitive health or bias the brain towards disease.

NIH Research Projects · FY 2025 · 2021-04

Abstract: Multiple sclerosis, a chronic autoimmune inflammatory disease associated with demyelination of the central nervous system (CNS), remains a public health issue. Currently there is no known cure for multiple sclerosis. Although several disease-modifying treatments (DMTs) are available, relapsing of multiple sclerosis occurs frequently and DMTs often result in severe adverse effects such as liver failure and fetal outcomes. Novel therapies are needed to reduce the disease burden for multiple sclerosis patients. Recently, we published that Hectd3, an E3 ubiquitin ligase, is expressed predominantly in T cells of the immune system, which play a critical role in pathogenicity of experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis. Specifically, we found that Hectd3 controls pathogenic Th17 effector response in EAE by regulating ubiquitination of Malt1 and Stat3 in a non-degradative manner, resulting in stabilization of Malt1 and Stat3. In addition, Hectd3-mediated polyubiquitination of Stat3 promotes Stat3 activation. Moreover, Hectd3-deficient mice showed reduction in EAE disease scores, Th17 cell pathogenicity and effector Th17 cytokines. Furthermore, Hectd3 deficiency causes a cell-intrinsic defect in Th17 cell pathogenicity that is responsible for the attenuation of EAE in Hectd3−/− mice. Overall, our results demonstrate that Hectd3 is a critical modulator of Malt1 and Stat3 signaling in EAE. Based on these results, we hypothesize that compounds abolishing Hectd3- mediated ubiquitination of substrates can lower EAE severity. However, although Hectd3 plays significant roles in pathogenesis of multiple sclerosis, currently there is no chemical probe to further investigate the pathways and the implication in therapy of multiple sclerosis. Therefore, in this proposal, we aim to develop high throughput screening assays to identify and characterize chemical probes to investigate in depth the biochemistry of Hectd3- mediated Malt1 and Stat3 signaling pathways, and their therapeutic potential in pathogenic Th17 cells and EAE. This innovative work explores the novel function of Hectd3 in immune regulation, specifically in pathogenic Th17 cells, the identification of Malt1 and Stat3 as target substrates for Hectd3-mediated ubiquitination, and characterization of novel chemical probes for Hectd3, and their impact on EAE. The long-term sustained impact of this work is to identify compounds to modulate Hectd3 activity on its target substrates and its functions in EAE to open avenues for development of more specific and effective immune therapies to treat multiple sclerosis, a crucial need given current treatment challenges and limited therapeutic options. These combined approaches will lead to the development of unique Hectd3 inhibitors with novel inhibition mechanisms. This work will have a global reach by promoting fresh and effective strategies to treat multiple sclerosis. Hectd3 has also been implicated in promoting breast cancer drug resistance, cancer metastasis (unpublished results), and bacterial infections. Therefore, this project may also have significant impact on cancers and bacterial defense.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY To expand the kidney-related biomedical workforce and counter the increasing disparity between the growing prevalence of renal disease and the disproportionate level of trainees, researchers and practitioners in nephrology and kidney health, we developed the Arizona Technology Development and Clinical Education Program for Students in Kidney Health (ADVANCE Kidney Health). ADVANCE Kidney Health is an education- based, hands-on research, education and clinical experience that applies pillars of 1) science, medical and engineering education; 2) training in innovation, entrepreneurialism and scientific translation; 3) experiential learning, mentorship and clinical immersion; and 4) needs-based application and practical translation – all aimed at producing motivated, trained and committed biomedical trainees interested in renal health and science to advance the workforce and develop the new health-related therapies of the future. The program recruits undergraduate students from across 15 departments within the College of Engineering at the University of Arizona (UArizona) that include: Biomedical Engineering, Electrical and Computer Engineering, Mechanical Engineering and Chemical Engineering. ADVANCE Kidney Health is structured to provide trainees a medical school experience for early-stage undergraduate learners geared to instill an understanding of renal anatomy and kidney function. The core structure accesses a clinical experience to instill a motivation to pursue kidney- related patient care and/or translational research and progresses to an already established innovation bootcamp that culminates in an interdisciplinary capstone that has doubled in size over 10 years and accesses by more than 450 captive engineering students. The program leverages new infrastructure in medical and engineering education along with transdisciplinary programs aimed at innovation, technology development and entrepreneurialism with 15 physician navigators in kidney health and 23 engineering and scientific mentors spanning renal physiology, biomedical engineering, optical sciences and machine learning. The result is an interrelated program that bridges renal medicine, engineering and product development to develop new pipelines and on-ramps to impact career decisions and grow the future kidney-related workforce.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT It is well accepted that intrinsic action of the androgen receptor (AR) within the prostate epithelium drives prostate cancer proliferation and survival. Less appreciated is the fact that AR is also expressed in the stroma surrounding the epithelium. Stromal-expressed AR acts extrinsically to maintain the differentiated striated basal and luminal epithelium of the normal gland. During prostate cancer development, AR expression in the stroma is lost. How AR is lost from the stroma and how its loss promotes prostate cancer development is unknown. Our objectives are to define the mechanism that leads to stromal-specific AR loss and determine how AR loss in the stroma, in conjunction with epithelial oncogenesis, promotes prostate cancer development Based on our preliminary data, we hypothesize that tumor-derived TNFα/TGFβ1 transcriptionally suppresses AR expression in the stroma, causing loss of FGF10 and Wnt16 secretion, which are required to maintain the stratified epithelium through induction of luminal cells and maintenance of basal cells, respectively. To test this, we developed the first human Prostate-on-Chip model by culturing basal epithelial cells next to prostate stromal cells within a microfluidic device. Within this model, we can fully recapitulate the stromal AR-dependent induction of luminal epithelial cell differentiation. Furthermore, co-culturing normal stroma with tumor cells within this model leads to the induction of CAF phenotypes and reduced stromal AR expression, mimicking the tumor/host interactions seen in vivo. Models that recapitulate human glandular organization and its dysregulation during disease development are critical for our mechanistic understanding of how stroma and oncogenic epithelial interactions drive tumor development. We will test our hypothesis in three aims: 1) Determine the mechanism by which stromal AR maintains prostate epithelial cell differentiation. Our working hypothesis is that stromal AR signaling induces secretion of stromal FGF10 and Wnt16, which are required for induction of luminal epithelial cells and maintenance of basal epithelial cells, respectively. 2) Determine the mechanism by which AR expression is lost in the tumor stroma. Our working hypothesis is that tumor-secreted factors, TNFα and TGFβ1, acting through NF-κB signaling, suppress transcription of the stromal AR gene independent of CAF conversion. 3) Determine the functional consequence of tumor-induced stromal AR loss on prostate epithelial differentiation in a new de novo in situ human prostate cancer model. Our working hypothesis is that tumor-induced stromal loss of AR- dependent induction of Wnt16 and FGF10, via TNFα/TGFβ1, co-operates with epithelial oncogenes to accelerate tumor development and induce loss of basal epithelial cells. The proposed studies will be the first to demonstrate how TNFα/TGFβ-mediated suppression of stromal AR expression leads to the loss of Wnt16 and FGF10 to promote prostate cancer development. These studies will also provide the framework for further development of the first human Prostate-on-Chip model, which recapitulates human prostate biology, for basic and translation cancer research.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Appetite suppressing agents secreted from the gut, such as cholecystokinin (CCK), play a critical role in regulating feeding behavior. However, drugs based on CCK or its receptors failed to effectively treat obesity. These drugs lack a specific neural target because the central neural mechanism underlying how peripheral CCK regulates appetite is not fully understood. Our long-term goal is to understand the neural mechanisms that regulate appetite and body weight, and to develop corresponding therapies to treat obesity and eating disorders. Using novel genetic methods, we identified a specific population of central amygdala (CEA) neurons, marked by the expression of protein kinase C delta (PKC-δ), that are necessary for the effect of CCK on appetite suppression. We demonstrated that CEA PKC-δ neurons suppress feeding through inhibitory synaptic connections with CEA PKC-δ negative neurons. However, the identity of CEA PKC-δ negative neurons and their downstream targets for CCK-induced anorexia are unknown. The objective of this application is to determine the neural circuits in the central brain areas that are downstream of CEA involved in regulating CCK-elicited feeding suppression. The central hypothesis is that the intersectional brain regions, i.e. disynaptically disinhibited by CEA PKC-δ neurons and activated by CCK, mediate CCK-induced feeding suppression and appetite control. The rationale for the proposed research is that the identification of the central brain neural circuits for appetite control will advance our understanding of the neural mechanisms of CCK-induced anorexia and feeding control, and suggest novel strategies for developing effective therapies to treat obesity and eating disorders. Guided by strong preliminary data, the hypothesis will be tested by pursuing three Specific Aims: (1) Establish functional circuitry connections from CEA PKC-δ neurons to the downstream targets. (2) Determine the role of the neurons downstream of CEA PKC-δ negative neurons in CCK-induced feeding suppression. We will test the working hypothesis that feeding is regulated by the neurons that are downstream of CEA PKC-δ negative neurons and activated by CCK. (3) Determine the specific neural pathways through which CEA PKC-δ negative neurons regulate the effect of CCK on feeding suppression. We will test the working hypothesis that neural circuits project from CEA PKC-δ negative neurons to their downstream neurons to regulate CCK-elicited feeding suppression. The innovation of the proposed research includes the intersectional approach of using unique genetic marker labeling of neurons combined with a well-established appetite suppression agent to map the central brain neural circuits for feeding regulation, and will develop and apply new tools to dissect functional-specific neural circuits. Finally, the proposed research is significant because it will provide novel neural targets in the central brain regions and determine their role in the neural axis of appetite control. Such knowledge has the potential to inform the development of novel therapies that include specific neural targets to treat obesity and eating disorders.

NIH Research Projects · FY 2026 · 2021-04

PROJECT SUMMARY This project seeks to shed light on mechanisms underlying transdiagnostic risk for mental illness by integrating two traditionally disparate lines of research. One line of work indicates that repetitive negative thinking (RNT)—a transdiagnostic risk factor characterized by frequent, negative, self-focused thoughts— increases vulnerability for a range of mental health disorders, including depression and anxiety. A second line of work demonstrates that high-quality social relationships are associated with lower rates of mental and physical illness; conversely, relationship stress, hostility, and disconnection exacerbate loneliness, isolation, and mental and physical illness. This proposal will test a new model in which RNT and social connectedness work together as parts of an integrated whole. The central argument of this model is that RNT exerts its pernicious effects on mental health by impairing the ability to meaningfully connect with others through empathy—a critical component of social connection that involves sharing and understanding others’ emotions. Critical to this model is the hypothesis that RNT and empathy operate dyadically; that is, they affect both partners in a close relationship. To test this model, this project will implement a multilevel research design that integrates self-report, neuroimaging, and naturalistic observation to study RNT, social connection, and mental health in the context of established close relationships. Specifically, the project will employ a multi-method approach across 200 established romantic couples (young adults to those in middle age; N = 400) to assess the following aims: (1) Examine associations between RNT and partner-directed neural and behavioral empathy among romantic couples; (2) Determine the role of neural empathy in dyadic social-emotional and mental health outcomes; (3) Determine the role of RNT in dyadic mental health outcomes; and (4) Examine whether neural empathy mediates the dyadic association between RNT and longitudinal mental health outcomes. Advancing prior work, the proposed research will examine neural empathy in a novel and validated social feedback task using functional MRI in each member of the couple, to be modelled using dyadic statistics. Additionally, RNT and daily social behaviors will be assessed in everyday life using two mobile apps developed by the research team: Mind Window and the Electronically Activated Recorder (EAR). Finally, mental health will be assessed over 6 months to allow for prospective changes in the primary outcomes of interest. To tackle the study’s aims, this proposal brings together an interdisciplinary team of researchers with expertise spanning all facets of the proposed research: RNT, depression, neural empathy, social connectedness, dyadic modelling, and ambulatory assessment. Ultimately, this work holds promise for advancing scientific understanding of how individual and social risks for psychopathology operate together to shape emotional disorders. In turn, this research has the potential to help identify novel intervention targets to strengthen social connectedness in service of improving mental health.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Sleep disturbance is one of the primary complaints of gynecologic cancer survivors, with more than 80% of survivors reporting sleep difficulties. Sleep disturbance also contributes to worsened health-related quality of life (HRQOL) and increased symptom burden. Although non-pharmacological interventions for sleep disturbance have been explored among cancer populations, up to 50% of survivors do not adhere to treatment recommendations, possibly due to the behaviorally labor-intensive nature thereof. Therefore, there is a need for more effective and acceptable approaches to improve sleep outcomes in cancer. This application proposes a career development plan to support Dr. Rina Fox in establishing an independent research program focused on examining strategies to improve HRQOL and reduce symptom burden among cancer survivors, with particular consideration for the role of sleep and circadian rhythms in the overall disease experience. Her career development will be supported by a multidisciplinary group of researchers. Her mentors, Drs. Ong, Rini, and Zee, are recognized scholars in behavioral sleep medicine, cancer control and survivorship, and circadian rhythms and biology, respectively. They have lengthy histories of federal funding, multidisciplinary collaboration, and successful mentorship of trainees who go on to achieve independence. Dr. Fox’s training will be enhanced by expert consultants who will provide guidance in clinical gynecologic oncology (Dr. Tanner), biostatistics and optimization methodology (Dr. Siddique), qualitative research methods (Dr. Kaiser), circadian activity rhythms and execution of sleep-focused research among cancer survivors (Dr. Ancoli-Israel), and biobehavioral mechanisms of intervention efficacy and psychosocial intervention development specifically among gynecologic cancer survivors (Dr. Penedo). The aim of this application is to optimize a behavioral intervention to decrease sleep disturbance (primary outcome), and improve HRQOL and reduce symptom burden (secondary outcomes) among gynecologic cancer survivors. In Phase I, 15 participants will simultaneously receive three candidate intervention components known to reduce sleep disturbance (i.e., sleep restriction, stimulus control, and bright light), and will subsequently complete semi-structured individual interviews to provide feedback regarding barriers and facilitators to intervention adherence. In Phase II, 80 participants will be randomized to evaluate these intervention components within a 23 full factorial trial design, as guided by the Multiphase Optimization Strategy (MOST) framework, to identify the optimal combination of candidate intervention components, enhanced to promote adherence, that best affect the study’s primary and secondary outcomes. Sleep disturbance and circadian markers will also be evaluated as potential mechanisms underlying the candidate intervention components’ effects on study outcomes. Results of this innovative research will directly lead to an R01 application and an independent and programmatic line of research for Dr. Fox focused on HRQOL, symptom burden, and sleep and circadian disturbance among cancer survivors.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Exercise has well-documented benefits for systolic blood pressure (SBP) and cardiovascular health. Whereas current guidelines advocate ~150 min moderate intensity exercise/week, our preliminary data show ~5 min/day of inspiratory muscle strength training (IMST) for 6 weeks lowers casual (resting) SBP by ~12 mmHg. This simple approach to lowering BP could be applied to almost any population however we propose to study IMST in older adults with obstructive sleep apnea (OSA). OSA is an ideal population to target because OSA prevalence is growing and because snoring and apneas result in chronic intermittent hypoxemia that drives sympathetic nervous system (SNS) hyperactivity, endothelial dysfunction and hypertension. These substantive risks for cardiovascular disease are compounded by poor adherence to the mainstay treatment continuous positive airway pressure (<50%), obesity, fatigue and a robust intolerance for exercise. Our findings in healthy young adults (n=50) show IMST-related reductions in BP are mediated by decreases in systemic vascular resistance, suggesting changes in vascular tone and function. Consistent with this hypothesis, our results from a pilot clinical trial in adults with OSA (n=24) show IMST-related reductions in plasma norepinephrine levels (PNE) and muscle sympathetic nerve activity (MSNA), both markers of SNS activity. Our preliminary mechanistic assessments indicate IMST may lower circulating concentrations of other vasoconstrictor factors and increase nitric oxide (NO)-mediated endothelium-dependent dilation. And, findings in a novel endothelial cell culture model, point to increases in NO and declines in reactive oxygen species (ROS) and oxidative stress. However, it is unknown if: 1) IMST lowers casual and 24-h (ambulatory) SBP in older adults with OSA; 2) the reductions in SBP are long-lasting; 3) arterial stiffness, NO-mediated endothelial dilation and/or oxidative stress are improved; and 4) if adherence in this population is high long term. We propose a randomized, double-blind, placebo-controlled, clinical trial to establish the efficacy of IMST (75% maximum inspiratory pressure, [PImax]) 5 days/week for 24 weeks vs. placebo (15%PImax) (n=61/group) for lowering SBP in adults (>50 years) with above normal BP and OSA. We hypothesize that IMST will lower SBP via reductions in SNS activity and circulating vasoconstrictor factors, improvements in vascular function, and reductions in oxidative stress/inflammation and that reductions in SBP will be sustained after IMST. Aim 1: To determine casual and 24-h ambulatory BP; before/after, and 4- and 12-weeks post-IMST/placebo training. Safety, tolerability and adherence also will be assessed. Aim 2: To measure arterial stiffness, brachial artery flow-mediated dilation (FMDBA), plasma PNE, MSNA, vasoconstrictor factors and inflammation; before/after, and 4- and 12-weeks post-IMST/placebo training. Aim 3: To evaluate superoxide related suppression of FMDBA, and markers of oxidative stress and antioxidant defense in endothelial cells from subjects before/after, and 4- and 12-weeks post-IMST/placebo training.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The overarching goal of this project is to optimize, validate and implement a revolutionary and safe modality for noninvasive functional imaging of neural currents deep in the human brain through the skull at unprecedented spatial and temporal resolution. Transcranial Acoustoelectric Brain Imaging (tABI) is a disruptive technology that exploits pulses of ultrasound (US) to transiently interact with physiologic current, producing a radiofrequency (RF) signature detected by one or more sensors (e.g., surface electrodes). By rapidly sweeping the US beam and simultaneously detecting these RF modulations, 4D high resolution current density maps are generated. This approach overcomes limitations with electroencephalography (EEG), which suffers from poor spatial resolution and inaccuracies due to blurring of electrical signals as they pass through the brain and skull, and, unlike fMRI and PET that measure slow “intrinsic” signals, tABI directly maps fast time-varying current within a defined brain volume at the mm and ms scales. As a disruptive and scalable modality for noninvasive human brain imaging, tABI offers the following benefits: 1) High spatial resolution determined by the US focus (e.g., 0.3 – 3 mm); 2) Real-time, volumetric imaging of local field potentials and evoked activity; 3) 4D imaging of neural currents from deep brain structures without assuming the conductivity distribution; and 4) Co-registration of neural currents (tABI) with brain structure, motion (pulse echo US) and cerebral blood flow (Doppler). Our multidisciplinary team of engineers, physicists, neuroscientists, psychologists, and imagers will overcome the primary challenge of detecting weak interaction signals through skull at safe US intensities. To demonstrate tABI as a safe and reliable modality for electrical brain imaging at the mm and ms scales in healthy volunteers, we propose to 1) Optimize, calibrate, and validate tABI using established human head and in vivo swine models; 2) Develop and validate the first tABI platform for functional brain imaging in human subjects; 2a) Assess extraoperative tABI for mapping patients with intractable epilepsy referred for surgery; and 2b) Assess tABI for mapping somatotopic organization in healthy volunteers. If successful, this project will deliver a safe, revolutionary and mobile technology for noninvasive human brain imaging with the goal of transforming our understanding of brain function and help diagnose, stage, monitor and treat a wide variety of neurologic (e.g., epilepsy, Parkinson’s), psychiatric (e.g., depression) and behavioral (e.g., OCD) disorders.

NIH Research Projects · FY 2024 · 2020-09

Women of advanced age (> 35 years of age) are at significantly higher risk for adverse outcomes during childbirth, compared to younger women. This problem is both critical and growing, as the number of births occurring in this age group have increased nine-fold over the last 40 years, escalating maternal morbidity and mortality in the US. Given that prolonged labor, Cesarean birth and postpartum hemorrhage are more common among older women, a decline in uterine function (contractility) with advancing age may be a source of labor- related dysfunction. Prior studies have shown that individuals’ biological ages often differ from chronological (i.e. actual) ages, raising the possibility that biological age could be a better predictor of age-related perinatal morbidity. A robust method of calculating biological age is the Epigenetic Clock, which determines an individual’s Epigenetic Age based on their specific DNA methylation patterns (common epigenetic modifications). Epigenetic Age has been shown to better predict morbidity or mortality over chronological age and epigenetic aging is also associated with social adversity and stress exposure. Given that social and economic stressors contribute to poor maternal outcomes (as evidenced by maternal health disparities), it is possible that epigenetic age is also a key mediator of birth related morbidity. Therefore, I will test the central hypothesis that epigenetic age will predict impaired uterine function more accurately than maternal chronological age (years) and that greater epigenetic age is associated with higher indices of psychosocial/ socioeconomic stressors during pregnancy. The career development goal of this application is to gain proficiency in genome wide epigenetic methods and epigenetic clock specifically in addition to expanding my training to include health disparities research methods. In this proposal, I seek to integrate these scientific fields and advance knowledge of the role of the environment on maternal health and morbidity related to childbirth and uterine function. In the first aim, using bio-banked tissues, I will apply Epigenetic Clock methods to extracted DNA from maternal uterine and blood samples to compare epigenetic age across tissue types and correlate with uterine mRNA for proteins responsible for uterine contractility and function during labor. In the second aim, I will use banked data and tissue from a large nationally representative sample of young (18-25 yo) and advanced age women (>35 yo). I will apply the Epigenetic Clock method to these DNA samples to 1) understand the relationship between phenotypes of socioeconomic and psychosocial stress (using mixture modeling) and epigenetic age in maternal blood and 2) examine the role of epigenetic age and uterine dysfunction during labor leading to greater maternal morbidity (prolonged labor leading to cesarean delivery, failed induction or postpartum hemorrhage). Finally, I will explore how maternal epigenetic age relates to infants’ gestational age and his/her own epigenetic age. Together, this study will advance our knowledge of how epigenetic aging can influence perinatal morbidity in the context of the maternal environment.

NIH Research Projects · FY 2025 · 2020-09

Project Summary/Abstract Over 2.2 million children aged 2–5 years have wheezing episodes that are severe enough to require Emergency Department (ED) visits each year in the United States, and 15% of these children require hospitalization. There is new evidence suggesting that harmful bacteria growing in the throat may play an important role in determining which preschoolers will wheeze and then go on to develop asthma. Bacteria and viruses are equally associated with the risk of acute episodes of wheezing in preschoolers, and antibiotics may be a potential treatment. Two large, well-designed clinical trials performed in outpatient clinics recently showed a significant reduction in severe symptoms when children were treated with the antibiotic Azithromycin (AZ) either before or after they started wheezing. Though these results are encouraging, we are not sure how this benefit occurs since AZ has both anti-bacterial and anti-inflammatory effects. In addition, we do not know if AZ could be effective in more severe cases, like those requiring ED visits. The relatively underprivileged preschoolers who present to the ED for care of their severe wheezing episodes are usually sicker and with greater risk factors for bacterial infections. They are, therefore, the population that may get the greatest benefit, if AZ is shown to be effective in this setting. We propose a trial in preschool age children coming to the ED with severe wheezing who will be treated with either AZ or placebo. We will also determine which bacteria are growing in the children’s pharynx. This will answer the question “Does Azithromycin make children with severe wheezing better sooner and, if so, is that benefit seen in all the children treated or only in those with potentially harmful bacteria in their throats?” There is concern that excessive use of antibiotics may cause bacterial resistance to their effects. We will thus determine if genetic factors or the populations of microbes present in the pharynx can identify children that are more likely to respond to AZ. This will allow us to target the use of AZ to these children in the future. By testing treatment of high risk children with severe wheezing in the ED with AZ and determining which bacteria are growing in their throats, our study may identify a new way to treat these severe, frightening, and difficult to treat illnesses.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Alzheimer's disease (AD) is the most common dementia in the elderly characterized by neurofibrillary tangles, senile plaques and a progressive loss of brain neurons. Compared with senile plaques, neurofibrillary tangles have a better correlation with the severity of cognitive impairment in AD. As intracellular lesions, neurofibrillary are largely composed of hyperphosphorylated microtubule-associated protein tau. Not surprisingly, considerable efforts have been devoted to tau-based AD drug development though the pathomechanism underlying tau toxicity remains largely unknown. Mitochondrial dysfunction and neuroinflammation are prominent early pathological features of AD and have been increasingly implicated as critical factors for AD pathogenesis. Despite both mitochondrial dysfunction and neuroinflammation have been repeatedly reported in animal models of tauopathies, there is limited study of their interplay. Interestingly, in our preliminary studies, we found that Mfn2, the mitochondrial outer membrane protein regulating mitochondrial morphology and association with endoplasmic reticulum, was significantly reduced in the widely used PS19 tau transgenic mice for AD and related tauopathies. Excitingly, the overexpression of Mfn2 in neurons is sufficient to remarkably suppress tau phosphorylation, mitochondrial dysfunction, neuroinflammation, neuronal loss and behavioral deficits in PS19 mice. In addition, lipopolysaccharide-induced neuroinflammation and even sudden death could also be greatly suppressed by overexpressing Mfn2 in neurons, together implying neuronal Mfn2 as a crucial mediator for both mitochondrial dysfunction and neuroinflammation. These exciting and promising preliminary studies suggest that a detailed investigation into the potential role of Mfn2 as a point of convergence for mitochondrial dysfunction and neuroinflammation in AD and related tauopathies is warranted. Using novel transgenic mouse models and a promising synthetic therapeutic peptide inhibiting Mfn2 degradation, this study will not only study whether and how Mfn2 regulates mitochondrial dysfunction and neuroinflammation, but also test the feasibility of targeting Mfn2 as a novel therapeutic approach against tau toxicity. Tau pathology is a prominent common histopathological feature of various major neurodegenerative diseases including but not limited to AD. Our proposed studies of Mfn2 and its convergent role in mitochondrial dysfunction and neuroinflammation will have very broad scientific and translational significance.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in neocortex and hippocampus. Currently, there is no effective treatment for AD. Less than 10% of AD cases are early onset with only a small fraction caused by autosomal dominantly inherited genetic changes in APP, presenilin 1 (PS1) or presenilin 2 (PS2), all of which are responsible for the overproduction of Aβ and the earlier formation of amyloid plaques. Though more than 90% of AD cases are referred to as sporadic AD without family history, they have the similar clinical and pathologic phenotypes as sporadic AD. Despite a large body of evidence suggests that Aβ deposition in the brain as the likely culprit playing a critical role in the pathogenesis of AD or related dementia, the molecular pathomechanisms of amyloid plaque formation remain largely elusive. Interestingly, in our recent study, we have identified a novel protein Aggregatin specifically accumulated within the centers of amyloid plaques. Aggregatin is predominantly expressed in the central nervous system and increased in brains of patients with AD or amyloid precursor protein (APP) transgenic mice for AD. Excitingly, Aggregatin physically interacts with Aβ with very high affinity, and remarkably facilitates Aβ aggregation even under near-physiologic nanomolar concentrations. Forced expression of Aggregatin resulted in increased amyloid deposition, whereas ablation of Aggregatin suppressed the formation of amyloid plaques in APP transgenic mice, further implying it as an important factor for Aβ aggregating to form amyloid plaques. These exciting and promising preliminary studies suggest that a detailed investigation into the potential role of Aggregatin in the formation of amyloid plaques in AD is warranted. Using a novel transgenic mouse model with conditional ablation of Aggregatin, this study will not only study whether and how Aggregatin regulates amyloid plaque formation and disease progression, but also test the feasibility of targeting Aggregatin as a novel therapeutic approach for AD. Amyloid plaque is a prominent common histopathological feature of in various major neurodegenerative diseases including but not limited to AD. Our proposed studies of Aggregatin and its connection with amyloid plaque will have very broad scientific and translational significance.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT From a single fertilized egg, the human genome must regulate an incredible succession of cellular divisions and fate decisions to give rise to the adult human body and its ~30 trillion cells [1]. The genome must also orchestrate highly diverse functions in these terminal cell types and in many instances allow for dynamic responses to a variety of stimuli - from white blood cells responding to stimulation [2] to hepatocytes responding to hormonal cues [3]. Furthermore, developmental processes are asynchronous and continue for many cell types into adulthood. Fundamental to our understanding of the causal links in all of these processes is the concept of time. While time course studies have a long history in genomics [4], single-cell genomic technologies are providing unprecedented views into the temporal dynamics of cellular differentiation and response at a genomic scale [5]. This will have widespread implications for our strategies of stem cell therapy, windows of intervention in disease progression, and our basic understanding of developmental biology. However, these inferences are to-date limited and rely on a concept called ‘pseudotime’ [5], which is difficult to validate and can be warped relative to real time. To truly understand how the genome coordinates development, differentiation, and disease we need new tools that allow us to better measure several key features of developmental trajectories: the ordering of regulatory cascades, the duration of the key genomic events in developmental processes, and the specific DNA sequences that can regulate temporal expression patterns. In order to address these concerns, we will develop a new suite of tools that leverage single-cell readouts to better understand the genomic regulation of time. In particular, we will focus on highly multiplexed assays to better understand the necessary and sufficient ordering of regulatory cascades in differentiation pathways, assays to convert pseudotime to real time, and genome scalable assays to identify and validate the exact regulatory sequences that define temporal patterns of gene expression.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY. We have recently developed a label-free biological and chemical sensing system known as a frequency locked optical whispering evanescent resonator (FLOWER) that integrates microtoroid optical resonators with frequency locking feedback control, which aids the suppression of noise. FLOWER is currently capable of highly sensitive and rapid (under 30 seconds) label-free detection down to the single macromolecule level, as demonstrated by the detection of single human interleukin-2 (IL-2) molecules. To date, FLOWER has achieved a signal-to-noise ratio of 5 using an anti-IL-2 antibody layer immobilized on a microtoroid to specifically capture IL-2. In addition to its high sensitivity, FLOWER has an advantage over other nanoscale sensing platforms such as cantilevers or nanowires in that it possesses a relatively large capture area, increasing the probability of analyte detection. We propose (1) using FLOWER to target a large number of current biomedical problems which would benefit from a rapid, sensitive, and accurate means to identify key microscopic, nanoscopic, or molecular markers specific to the problem; (2) miniaturizing and multiplexing FLOWER and making it part of a self- contained, compact portable device to quickly establish the prognosis of various conditions; and (3) further increasing FLOWER’s sensitivity and selectivity, making it capable of detecting even smaller molecules with societal interest such as insulin or more capable of detecting the small signal changes mentioned in (1). As such, FLOWER may be used to understand the process of olfaction in synthetic optical noses or disease progression in Alzheimer’s. FLOWER is a new method that has not been applied to these questions before and, with proof of concept, has great potential to advance several fields. If successful, this project would allow for a robust, extremely sensitive, and portable device that could be given to an EMT or a solider in order for them to rapidly detect diseases or viruses and bacteria in drinking water or food. These devices could empower citizen scientists to monitor their drinking supply or breathing air. Furthermore, these devices could be easily translatable to other labs, enabling robust assays for drug library screening, cell signaling studies, and clinical assays. Eventually, we envision these devices to be prevalent in drug stores throughout the country, creating a convenient, inexpensive, routine, accessible, and non-invasive means to impact the diagnosis and treatment of many diseases, including Alzheimer’s, cancer, pain from multiple sclerosis, diabetes, addiction, and depression.

NIH Research Projects · FY 2024 · 2020-08

OVERALL PROGRAM SUMMARY One of the most significant challenges for understanding genetic control of blood pressure (BP) is that the vast majority of BP-associated single nucleotide polymorphisms (SNPs) in humans are located in noncoding regions of DNA. Many of these noncoding SNPs are located in haplotype regions thousands of base pairs away from any protein-coding gene and their effects on BP cannot be explained by any currently known coding or other functional sequence variant, making it nearly impossible to link these noncoding SNPs to genes or physiological pathways that regulate BP based on genomic sequence. Understanding the effect of intergenic noncoding SNPs on gene expression and the underlying mechanisms is a major challenge not just for BP and hypertension research, but for research on nearly all complex traits and common diseases. The goal of this PPG proposal is to begin to address this major challenge and test the overall hypothesis that noncoding SNPs associated with human BP but located far from any protein-coding gene regulate gene expression in specific BP relevant cell types through epigenetic mechanisms and these mechanisms can influence BP. We have developed three projects that each address one aspect of this overall hypothesis. Project 1 will use precision genome editing to identify the effect of specific BP- associated noncoding SNPs on gene expression in BP-relevant human cell types. Project 2 will test the hypothesis that BP-associated noncoding SNPs influence the expression of BP-relevant genes through epigenetic mechanisms including chromatin looping, enhancer function and noncoding RNA in human cells and tissues. Project 3 will take this line of research to animal models in vivo to test directly the novel hypothesis that chromatin conformation plays a role in BP regulation. The three projects will interact with, and inform, each other extensively and, together, will achieve the overall goal of the program. All three projects will rely on Core A for administrative support and Core B for sequencing coordination and data analysis. We have published at least 16 papers in the last few years that provide direct support for key aspects of the conceptual validity and technical feasibility of this PPG. In addition, we have obtained a large amount of preliminary data to further support the feasibility of the wide range of sophisticated and new technologies that we will use and the validity of proposed novel hypotheses. This PPG represents a fundamentally new direction for hypertension research. It will establish several novel approaches and technologies, generate unique and extensive datasets, and provide new biological insights, all of which will help to advance genetic and epigenetic research in hypertension and other disease areas.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY: The distal arthrogryposes (DA) are a heterogeneous group of disorders characterized by congenital nonprogressive joint contractures associated with muscle weakness. Depending on the gene involved and the specific mutation, inheritance is typically autosomal dominant with variable expression and incomplete penetrance. Current clinical classification identifies eleven different discrete syndromes with several associated with mutations in sarcomere genes including slow skeletal myosin binding protein-C (MYBPC1). Recently, a homozygous recessive mutation in MYBPC1 was linked to a severe form of DA, lethal congenital contracture syndrome type 4 (LCCS4). Despite the increasing association of DA syndromes with specific genetic mutations, molecular mechanisms that underlie skeletal muscle weakness that presumably lead to disabling contractures are poorly understood. As these mechanisms are unknown and, specifically, little is known about how sMyBP-C regulates muscle function in vivo, current therapies are largely ineffective and relegated to symptomatic physical therapy. The overall long-term goal of our research program has been to define the contribution of the myosin binding protein-C (MyBP-C) proteins in health and disease. These sarcomeric-specific proteins are known to regulate striated muscle contractility via modulating actomyosin function. Three MyBP-C paralogs exist, namely slow skeletal MyBP-C (sMyBP-C), fast skeletal (fMyBP-C), and cardiac MyBP-C, and encoded by separate genes. The specific goal of this proposal is to define the physiologic mechanisms underlining how mutations in sMyBP- C lead to muscle dysfunction and contractures. In our preliminary studies, we determined that mouse pups that are homozygous global sMyBP-C null (Mybpc1-/-), similar to the human LCCS4 phenotype, all died within the first day of birth and exhibited tremors secondary to muscle atrophy. We demonstrated that muscle creatine kinase Cre- and human a-skeletal actin-Cre/Tamoxifen-mediated sMyBP-C ablation (Mybpc1fl/fl) resulted in significant muscle weakness in postnatal and adult stages, respectively. Finally, we showed in transgenic mice overexpressing Mybpc1Tg under the control of the human a-skeletal actin promoter that sMyBP-C replaces fMyBP-C impairing fast muscle type function. Based on these data, we hypothesize that sMyBP-C acts as a key regulator of striated muscle formation and function in both slow and fast muscle types. The planned experiments will systematically define whether (i) sMyBP-C is essential for normal formation of muscle in prenatal and perinatal stages, (ii) sMyBP-C is required for skeletal muscle function in postnatal and adult stages, and (iii) sMyBP-C and fMyBP-C transcomplement each other. We anticipate that addressing these key questions will drive mechanistic understanding of how sMyBP-C regulates skeletal muscle physiology across developmental stages. Consequently, this proposal will identify therapeutic targets to improve muscle function in those afflicted with DA diseases.

NIH Research Projects · FY 2024 · 2020-07

Post-traumatic headache (PTH) commonly occurs following mild traumatic brain injury (mTBI), also known as concussion. PTH is a secondary headache that often presents with a migraine-like phenotype and is subdivided as acute or persistent (PPTH) depending on whether it resolves within 3 months after injury. The pathophysiology of PTH and PPTH is not understood and no evidence-based treatments exist for these conditions. Critically, PPTH might differ from PTH, not only in the duration but also in underlying mechanisms and responsiveness to treatment. The reasons for emergence of PPTH in some patients remain unclear but may be related to risk factors including pre-existing migraine and the experience of a previous mTBI. We have developed an approach to investigate the mechanisms of PTH and PPTH as well as potential strategies for treatment. Using a weight drop method in male and female mice that recapitulates biomechanical properties and clinical features of mTBI, we have shown that a single mTBI is sufficient to induce clinically relevant PTH symptoms including an acute period of allodynia, elevated CGRP blood levels and lowered thresholds for induction of cortical spreading depression (CSD). Additionally, we have explored the concept that the transition from acute to chronic pain states may rely on a “pain memory” that can be studied using the “two-hit” model of hyperalgesic priming where a prior insult confers vulnerability to a subsequent provocative stimulus. Thus, following resolution of acute allodynia, mTBI mice transition into a long-lasting persistent phase (PPTH) where, remarkably, allodynia can be reinstated by physiologically relevant and common migraine triggers, including stress. CGRP is established in migraine pathogenesis and our data also suggest an important role in promoting PTH. Treatment with either a CGRP antibody or with onabotulinum toxin A (botox) prevents mTBI-related allodynia (PTH) as well as subsequent provoked allodynia representative of PPTH. However, blockade of CGRP after mTBI sensitization is established is ineffective in blocking provoked allodynia, while botox still maintains efficacy. We have hypothesized that mTBI results in CGRP release from meningeal afferents promoting PTH and central sensitization that underlies the development of PPTH, but that PPTH may be maintained in a CGRP-independent fashion. Additionally, we hypothesize that existing sensitization prior to a mTBI event will promote vulnerability to the development of CGRP-independent PPTH. We explore these hypotheses with two related but, independent, aims using behavioral, neurochemical, immunohistochemical and electrophysiolgical analyses. Aim 1 will determine whether, and when currently available therapies can block mTBI-related outcomes relevant to PTH and if these treatments can prevent the expression of PPTH. Aim 2 will determine if prior sensitization promotes more severe, long-lasting and CGRP-resistant PPTH. Our studies will fill in significant knowledge gaps about the role of CGRP in promoting PTH and the importance of pre-existing sensitization in establishing CGRP- independent PPTH. Such information will influence treatment as well as guide the discovery of new therapies.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT The ability for cells to detect and respond to metabolic cues is critical to maintaining homeostasis, and perturbations in the sensing mechanisms that respond to oscillations in metabolic flux are the root cause of many diseases, including sepsis, autoimmunity, cancer, and diabetes. There is mounting evidence that protein post- translational modifications (PTMs) are the critical sensors for these metabolic fluctuations and are often dysregulated in disease. Currently, we have a fundamental gap in our understanding of the composition, abundance, and enzymatic control of PTMs and how they are altered in disease. My laboratory focuses on the identification and characterization of PTMs and how they are regulated in both health and disease. To accomplish this goal, we have developed sensitive methods to identify and quantify global changes in PTMs across a broad spectrum of biological samples. Using this approach, we have identified a novel lysine PTM that is derived from a glycolytic by-product. These PTMs are elevated when glyoxalase 2 (GLO2) is inhibited, resulting in reduced glycolytic output and disrupted one-carbon metabolism. Our primary goal is to establish the therapeutic efficacy of a GLO2 inhibition strategy for the treatment of metabolic disorders. My research program is dedicated to understanding four fundamental questions: 1) How does GLO2 control one-carbon metabolism and cellular redox? GLO2 knockout cells have reduced glutathione and increased oxidative stress. We will quantify the role of GLO2 in the regulation of de novo glutathione synthesis. In addition, the role of GLO2 in the regulation of antioxidant responses will be evaluated in a cellular model for oxidative stress and inflammatory signaling. 2) How are LactoylLys modifications regulated? We will employ quantitative proteomics using CRISPR-Cas9 knockout cell lines of candidate proteins to identify enzymatic regulators of LactoylLys modifications in cells. 3) Is GLO2 a viable target for the treatment of glycolysis- dependent disease states? A xenograft model will be employed using GLO2 knockout cell lines to quantify proliferation and metabolic regulation in vivo. This will determine the therapeutic feasibility of targeting GLO2 for the treatment of disease. 4) Are LactoylLys modifications functional histone marks? We have identified histones as targets for modification by LactoylLys modifications in unstimulated cells. The presence of these PTMs basally suggests a putative role in transcriptional regulation. We will use proteomics to identify site-specific modifications and putative ‘reader’ domains for LactoylLys modifications in cells. Our primary goal is to establish the role of GLO2 and LactoylLys modifications in cell metabolism and chromatin biology. This project will address a fundamental gap in our basic understanding of how cell metabolism is regulated. Understanding how these PTMs regulate homeostasis is a critical first step to understanding their role in disease. Due to the far-reaching implications of this project and the broad applications for the treatment of highly glycolytic disease states, this research program is an ideal fit for the ESI MIRA Award.

NIH Research Projects · FY 2025 · 2020-07

BEAMS ABSTRACT: Overall Prevalence of childhood asthma has significantly increased in the last decades, and one potential explanation for this upsurge is decreased exposure to protective environmental microbes due to improvements in sanitation and use/overuse of antimicrobial products during pregnancy and early life. In support of this contention, several studies in isolated rural communities have reported lower prevalence of asthma among children of animal farmers more heavily exposed to environmental microbes than among their non-farmer peers. If these observations are applicable to more mainstream US populations is unknown. We recently observed that Mexican American children living in Tucson, Arizona have a prevalence of childhood asthma that is fourfold higher than in Nogales, Mexico. This dramatic difference in rates of asthma suggests that, even in this limited geographic region and among an ancestrally similar population, differential exposures may exist which account for relative protection against asthma in Mexico. Tucson is only 70 miles north of Nogales, Mexico, which is a 200k inhabitant city located just south of the US-Mexico border. Nogales, Mexico lacks public sanitation facilities and poverty rates are very high. In preliminary data, we observed marked differences in microbial communities present in dust, drinking water, and in stools of one-month-old children between the two cities. Based on these findings, we propose the Binational Early Asthma and Microbiome Study (BEAMS). The BEAMS overall goals are: to identify divergent early-life microbial and immune developmental trajectories associated with asthma protection in Nogales, Mexico compared with Tucson, Arizona, and the environmental factors that promote them; to isolate the specific microbes, genes and their products that confer such protection; and to ascertain the mechanisms by which these microbial communities or their products promote asthma protection. To accomplish these goals, BEAMS will have three Projects and four Cores. We will enroll 250 pregnant mother/offspring dyads of Mexican ancestry in each city. We will thoroughly assess environmental microbial exposure, maternal gut microbiota and microbial gene expression, maternal immune markers and meconium microbiome. We will relate these characteristics to child’s fecal microbiome and metabolome in early life, to asthma-related immune markers in the child’s blood, as assessed by mass cytometry analysis and single cell epigenetic and gene expression studies, and to asthma-related clinical phenotypes by age 2 years. We will also assess in mouse models the specific molecular mechanism that explain the protective effects against the development of childhood asthma of specific microbial strains and metabolites present in Nogales, Mexico. We expect BEAMS to offer a better understanding of the early origins of asthma and new asthma prevention strategies applicable to Mexican- Americans, and potentially to all Americans.

NIH Research Projects · FY 2025 · 2020-07

PROJECT SUMMARY The Biochemistry, Molecular and Cellular Biology (BMCB) graduate program at the University of Arizona is an interdepartmental and interdisciplinary graduate program that seeks to equip students with a broad understanding of modern approaches in Biochemistry, Molecular and Cellular Biology, and Systems/ Quantitative Biology, and the way these approaches can be combined to tackle important unsolved problems in Biology and Medicine. To achieve this goal, we focus on ensuring our students develop eight interrelated skills: (1) A broad knowledge of Biochemistry, Molecular Biology and System/Quantitative Biology; (2) A broad interdisciplinary knowledge of experimental/computational approaches; (3) An ability to communicate (and thus think) clearly; (4) An ability to read, evaluate and integrate the scientific literature (5) A deep knowledge in the field of thesis research; (6) An ability to plan and execute experiments; (7) An ability to generate new insights and ideas; (8) An ability to recognize important research problems and questions. Students start developing their foundational skills (1-5) in first year classes; using group work to study key literature, build up their understanding of experimental methods and approaches, and develop good communication skills. At the same time, students do three eight-week rotations to learn the intricacies of experimentation and identify a suitable faculty mentor. Then, as students start their second year, they enroll in a Scientific Communication course, where they are guided though the process of developing their thesis project, writing it up and submitting it as a fellowship, and presenting it in oral form. Student also take a Quantitative Methods course at the start of their second year, where they learn to program in R, carry out a range of statistical tests, and use Al to analyze images, movies, and large Omics datasets (helping with the rigor and reproducibility of their future research). Students then move on to their Oral and Written exams and begin focusing on their thesis project. At the same time, however, they continue to participate in a number of activities designed to help them refine their skills, remain connected to the BMCB community, and identify and secure a career that works for them. The most important of these are: (1) a Journal Club and associated Idea Development Workshops that provide students multiple opportunities to develop and refine new ideas and questions each semester; and (2) "Student Only" retreats, career development workshops, and a BMCB internship program, that expose students to a wide variety of professions and help them make the contacts they need to move forward in their career. To help support these activities, we are requesting funds to cover the costs of eight BMCB students per year, approximately 1/3 of our TGE students. Students will be appointed to the training grant for one, or subject to renewal, two years; preferably in their second and third year. Our expectation is that ~90% of students that enter the BMCB program will graduate and go on to have a career in Biological or Biomedical research--regardless of their gender or race- a goal we have met for the last 15 years.