University Of California-Irvine

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Biological tissues appear to “know” their intended final sizes and achieve them precisely and robustly. While, in principle, a simple negative signaling feedback should be sufficient to explain how a given stem cell lineage regulates its cellular outputs, in reality it cannot work because most tissues are physically large, with stem cells and their progeny spread out over centimeter-scale distances. How tissues overcome the microscopic decay limits of diffusible molecular signals to breach distances orders-of-magnitude in spatial scale remains elusive. This application is inspired by our serendipitous discovery that FGF and BMP mutant mice are able to grow super-long and highly imprecise hairs that can exceed the length of normal mouse hairs by 7-fold. Our lineage analyses suggest that hair stem cells continuously replenish short-lived transit-amplifying (TA) cells spatially located nearly 1 cm away from the stem cells. Interestingly, our single-cell RNA-sequencing analyses reveal previously unappreciated heterogeneity of the intermediate epithelial progenitor cells physically located between the stem and TA cells. Through an integrated mathematical and experimental approach, this application will focus on testing our new hypothesis that dynamic equilibrium between two or more intermediate cell states and their associated cell-cell communications enable feedback information propagation over large spatial scale from TA cells to stem cells to regulate the new progenitor cell production for hair size control. The first aim of the proposed research is to profile and quantify the heterogeneity of intermediate epithelial progenitors, and computationally and experimentally determine the functional link between specific intermediate progenitor states in the hair follicle and the hair length and its precision. The second aim is to define the cell-cell communication networks within the epithelial hair follicle lineage, and computationally and experimentally establish how multiple short-range signaling activities coordinate to form a long-range feedback mechanism that controls progenitor flux between distant stem and TA cell compartments for proper hair growth. The third aim is to determine the signaling impact of mesenchymal niche cells, which surround the hair follicle, on the epithelial lineage cells for hair size control. The study premise is based on novel and extensive preliminary experimental and computational data. The proposed studies are significant because they will establish new long-range signaling mechanisms and uncover novel roles of intermediate cell states in tissue size control. The proposed studies are innovative because they will establish new experimental models for studying tissue size regulation using super-long and extra-short hair mutant mice and will result in numerous new genetic mouse tools for epithelial stem cell research. They will also result in several novel mathematical and computational tools for analyzing single-cell RNA- sequencing data and new spatial models for complex cell lineages, such as in the hair follicle.

NIH Research Projects · FY 2025 · 2022-02

7. Project Summary/Abstract Therapy for ischemic brain injury is poor in part because of our limited understanding of mechanisms leading to neuronal loss. While roles of excessive glutamate release and neuronal Ca2+ accumulation have been much studied, recent evidence implicates critical contributions of another divalent cation, Zn2+. After ischemia or prolonged seizures, free Zn2+ accumulates in neurons and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria, powerfully disrupting their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. Using acute hippocampal slices subjected to oxygen glucose deprivation (OGD) to model ischemia, we recently made the first simultaneous measurements of cytosolic Zn2+ and Ca2+ changes, and found that Zn2+ accumulation is an early event in hippocampal pyramidal neurons that precedes and contributes to a subsequent sharp and terminal Ca2+ deregulation event, causatively linked to loss of membrane integrity. We have further found that the acute deleterious effects of Zn2+ seem to result specifically from its uptake into mitochondria via the mitochondrial Ca2+ uniporter (MCU). In ongoing slice studies, we find evidence for major differences between sources of the Zn2+ that accumulates in hippocampal CA1 and CA3 pyramidal neurons contributing to acute OGD induced damage, with considerable Zn2+ accumulation in mitochondria of CA1 but not of CA3 neurons at delayed time points after a sublethal episode of OGD. These differences in Zn2+ contributions may bear upon the differential vulnerabilities of CA1 vs CA3 neurons in disease conditions, with CA1 preferentially degenerating after transient global ischemia, and CA3 after recurrent limbic seizures. This proposal continues ongoing studies, generally organized around a Hypothesis: Mitochondrial Zn2+ accumulation is an early event after transient ischemia, which causes disruption of mitochondrial function and contributes to delayed cell death. Aim I applies imaging techniques to acute hippocampal slices to further clarify mitochondrial effects of Zn2+ in hippocampal neurons in the hours after transient oxygen glucose deprivation (as a model of ischemia), and to study events occurring after restoration of O2/glucose (“reperfusion”) that may be amenable to beneficial therapeutic interventions. Aim II seeks to make initial test of principle studies of our hypothesis in an in vivo rat global ischemia model. These studies will provide mechanistic insights that will aid the development of new and effective therapeutic interventions to be delivered after an episode of transient ischemia, that will disrupt the pathological cascade, enabling improved outcomes.

NIH Research Projects · FY 2025 · 2022-01

Project Summary/Abstract Stargardt disease (STGD1) is the most common form of inherited juvenile macular degeneration. STGD1 is caused by autosomal recessive mutations in the ABCA4 gene, which encodes a membrane transporter that removes all-trans-retinals (atRALs) from photoreceptors as part of the retinoid cycle. Free atRALs or their bisretinoid condensation products promote photo-oxidative damage to the macula as seen in STGD1. The same atRAL-mediated damage can also be seen in age-related macular degeneration (AMD), which is expected to affect at least 18 million Americans by 2050. The production of atRAL starts at the level of opsin proteins, which reside within photoreceptor outer segment disc membranes. Light is captured by opsin- chromophore complexes, or visual pigments, causing their native bound chromophore, 11-cis-retinal (11cRAL), to be converted to atRAL and forming activated opsins. These activated opsins initiate phototransduction and are eventually spontaneously hydrolyzed to apo-opsin and atRAL. Exposure to intense light causes photoreceptor overstimulation and dangerously high levels of atRAL, potentially leading to photoreceptor damage and loss. Recently, a chromophore analogue, retinyl formate (RF), was found to irreversibly bind apo- opsin and form retinyl-opsins that can no longer form visual pigments with 11cRAL. These retinyl-opsins also absorb light outside the visible light spectrum and do not subsequently release atRAL upon light absorption. Thus, RF can potentially reduce the proportion of visual pigments in the retina and thereby reduce the atRAL burden during periods of intense light exposure. Therefore, I hypothesize that RF can serve as a molecular shade at the opsin level, providing long-lasting protection to photoreceptors from light-induced damage. In this proposal, I will characterize the site of the retinyl modification on opsin by RF, distinguishing whether RF binding is competitive or allosteric with 11cRAL. I will determine if and how retinyl-opsins also could initiate the phototransduction cascade. To investigate its applicability to a pre-clinical model, I will study whether RF treatment of an STGD1 mouse model provides neuroprotection to photoreceptors against intense light exposure via formation of retinyl-opsins and reduction of retinal atRAL and determine the relative proportion of retinyl-opsins and remaining natural visual pigments. This work thus serves as a proof-of-concept approach to determining whether disabling a proportion of opsins with an irreversible inhibitor of visual pigment formation could prevent light-induced damage to photoreceptors, and point to the development of future therapeutics and interventions for STGD1 and AMD.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease with a high mortality rate. In most cases, PAH-induced chronic pressure overload leads to right ventricle (RV) failure. Right heart catheterization (RHC) is currently the gold standard test for PAH diagnosis and follow-up. Furthermore, repeated RHC should be considered in patients who experience unexplained clinical deterioration during their clinical course or for periodic follow-up over time. Yet many centers across the world do not routinely perform RHC during follow-up, for a variety of potential reasons, including its invasive nature, cost, or availability. We have recently developed 3D echocardiographic particle image velocimetry (Echo-PIV), which allows high- temporal volumetric assessment of RV hemodynamics. In combination with standard 3D speckle tracking, 3D Echo-PIV enables a comprehensive assessment of hemodynamics and energy state (ventricular kinetic energy/work and viscous energy dissipation) in the RV. The RV energy state reflects the structural and functional changes in the RV that may occur due to PAH. Thus, it can be used as a load-independent measure to assess RV function and monitor disease progression/regression in PAH patients. The project's global hypothesis is that in PAH patients' follow-up, changes in the RV energy state: (1) reflect changes in RHC-derived pulmonary arterial pressures, and (2) are associated with changes in patients' clinical status such that it can be used to monitor disease progression and regression. Given that in more advanced disease, RV dysfunction is present, we will gather a prospective, ethnically diverse cohort of 210 subjects at Cedars-Sinai Medical Center: 70 PAH patients with normal RV function, 70 PAH patients with RV dysfunction, and 70 healthy control subjects. The following Specific Aims will be accomplished: SPECIFIC AIM 1: Investigate the accuracy of 3D Echo-PIV in characterizing 4D flow hemodynamics in the RV using 4D Flow CMR as a reference standard and assess test/retest reproducibility of 3D Echo-PIV in PAH. SPECIFIC AIM 2: Determine the relationship between the noninvasively derived RV energy state and RHC- derived parameters in following up PAH patients and test whether changes in the RV energy state reflect changes in the RHC-derived pulmonary arterial pressures in PAH patients with and without RV dysfunction. SPECIFIC AIM 3: Investigate the feasibility of noninvasive imaging-derived RV energy state for monitoring disease progression/regression in PAH patients. The success of the proposed studies will establish 3D Echo-PIV as a tool for assessment of PAH patients and will: (1) determine the relationship between noninvasive RV imaging and invasive RHC-based hemodynamics, (2) establish whether changes in noninvasive RV energy state reflect changes in the RHC-derived parameters in PAH patients, and (3) facilitate monitoring of disease progression/regression by noninvasive RV energy state in lieu of invasive RHC (current clinical gold standard).

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT Age-related macular degeneration (AMD) is a disease that leads to the loss of visual acuity, resulting in a substantial medical and social burden. Many unique pathways lead to the pathogenesis of AMD. The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer which sits directly adjacent to the photoreceptor outer segments (POS) and is responsible for the maintenance of photoreceptor health. Among its functions include the generation of 11-cis-retinal chromophore for phototransduction, nutrient delivery, and diurnal phagocytosis of spent POS. Degeneration and dysfunction in the RPE is linked to the subsequent decline in photoreceptors seen in diseases such as AMD and Leber’s congenital amaurosis. Therefore, understanding RPE dysfunction is critical to understanding overall retinal health, but regulation of these key RPE roles is still incompletely understood. Recent research has pointed to the important role of microRNA (miR) regulation of gene expression, and miRs are critical for ophthalmic development and homeostasis. Of these miRs, miR-204 and miR-211 are among the most highly expressed miRs in the RPE and have been shown to both maintain its epithelial properties and modulate endosomal/lysosomal processing. These two miRs share the same seed sequence and are hypothesized to regulate many of the same genes, and are themselves regulated by light and the circadian clock. Previous studies have implicated the expression of these miRs in the control of RPE phagocytosis, but this has not been fully tested in a RPE specific manner. Thus, this proposal will examine the role(s) of miR-204 and miR-211 in an RPE specific manner and test the hypothesis that these two miRs regulate RPE phagocytosis. To pursue these objectives, we have generated miR-204fl/fl and miR-211fl/fl double knock-in mice. Induction of recombination in double knock-in mice will be achieved by (1) crossing them with RPE65-ERT2-cre mice and inducing recombination in the resultant triple knock-in mice by intraperitoneal injection of tamoxifen; and (2) subretinal delivery of AAV1-CMV-cre-GFP. The miR double knockout mice will be aged and assessed for phenotypic, electrophysiological, and histological changes. RPE and retina from these mice will also be collected and analyzed through bulk and single cell RNA sequencing to examine changes in gene expression resulting from miR-204/miR-211 deletion. The direct target genes of these two miRs will be assessed through the application of Halo-enhanced Ago2 pulldown (HEAP). We will also perform the in vitro culture of double knock-in mouse RPE and challenge them with POS to assess their phagocytic capacity. Lastly, we will correlate the results for mouse RPE to analogous results for human RPE through the use of RPE cultures derived from induced pluripotent stem cells. Overall, these studies will seek to define the role(s) of miR-204 and miR-211 in the RPE, identify their regulated genes, and suggest further genes and pathways to target in the development of AMD therapeutics and diagnostics.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ ABSTRACT Colorectal cancer (CRC) is the third most diagnosed cancer in the United States. Though CRC cases in adults (55 and older) have decreased, the incidence of CRC in young adults, ages 15-40, is on an alarming rise. It is estimated that by the year 2030, a staggering 11-12% increase in early-onset (EO) cancers will be observed. Adult cases of CRC typically harbor driver mutations in Apc, a tumor suppressor that regulates Wnt signaling, in addition to second hits in Kras, Braf, p53 and Smad4. Apc mutations are also found in early-onset CRC (EO- CRC), but a decrease in the typical second hit driver pathways has been reported. Therefore, there is an urgent need to better define the root cause of EO-CRC. Moreover, clinical evidence suggests that diet is likely a root underlying cause of the increased incidence in sporadic cases of EO-CRC. Interestingly, dietary challenge and timing of food intake directly impinge on the circadian clock, which is our internal pacemaker that governs sleep/wake cycles, feeding, hormonal and other cyclic rhythms. This suggests that disruption of the circadian clock could be a major risk factor for EO cancers. In further support of this idea, clinical data indicates that clock genes are broadly downregulated in human colorectal tumors, suggesting that suppression of the clock could be important for transformation in the intestinal epithelium. To directly address the potential links between the clock and CRC, we have developed a novel genetic mouse model to define how disruption of the circadian clock drives CRC pathogenesis. Our preliminary data demonstrates that disruption of the clock in the intestinal epithelium drives a statistically significant increase in polyp formation. Using our mouse model system, organoid cultures reveal that clock disruption accelerates transformation in the intestinal epithelium. Based on these findings, we hypothesize that clock disruption impinges on intestinal transformation and rewires cellular metabolism to sustain the heightened demand of hyperproliferative cells. Aim 1 will define how the clock machinery regulates genome instability and transformation in the intestine. Aim 2 will determine the role of the circadian clock in governing metabolism of intestinal epithelial cells in both mouse and human organoid systems, established from EO-CRC patient samples. Aim 3 will delineate how dietary paradigms that disrupt the circadian clock accelerate intestinal transformation. The broader impact of our findings will outline new prevention strategies for eradicating EO-CRC and other cancers that potentially relate to disruption of the circadian clock. Additionally, our long-term goal is to achieve targeted pharmacological approaches to regulate the circadian clock and therefore minimize behavioral and lifestyle factors that potentially impinge on tumorigenesis.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Prostate cancer (PCa) incidence and mortality rates are the highest in African American (AA) men compared to any other racial/ethnic population. These differences persist even after accounting for socioeconomic factors, suggesting genetics and unknown biological factors may contribute to PCa health disparities. However, common genetic alterations, such as TMPRRSS2-ERG gene fusions and PTEN loss, were found to occur much less frequently in AA PCa than in European American (EA) PCa. Instead, prominent differences in tumor immunobiology between AA vs. EA men were reported in several studies, including a clinical trial with a cancer vaccine, Sipuleucel-T, which AA men had a median nine-month of overall survival advantage over EA men. To mechanistically dissect the immunological and/biological factors that determine tumor cell sensitivity and resistance to immunotherapy of different races, we have developed primary cultures of AA and EA PCa patient-derived tumor organoids, normal organoids, carcinoma associated fibroblasts (CAFs) and benign- associated fibroblasts (BAFs) from many patients and cryopreserved their peripheral blood lymphocytes (PBLs) from the same patient over past years. Our preliminary data show that AA CAFs secrete increased levels of active TGF- in the culture medium than EA CAFs. In addition, we are the first to show that Glycoprotein A repetitions predominant (GARP), the docking receptor for the release of active TGF-β, is over expressed in the adjacent stroma of AA PCa compared to adjacent stroma of EA PCa and AA PCa tissues. Interestingly, the adjacent stroma of AA PCa has increased infiltration of cytotoxic CD8+T cells compared to the adjacent stroma of EA PCa and to the distant stroma of AA PCa, suggesting that immune response is higher in AA stroma but may not be effective due to the increased TGF-β1 and GARP. Our preliminary data of co-culture studies with CAFs and T cells lends further support to the scenario as we observed increased TGF-β1 and reduced IFN-  in these co-cultures. These results suggest that although AA PCa patients may be more responsive to immunotherapies, GARP/TGFβ signaling represents a vulnerable point in AA PCa and may be used as a target for developing more effective immunotherapies. Therefore, we hypothesize that the interaction between tumor and stroma in AA and EA PCa differentially affect the tumor reactivity of T cells and that GARP/TGF-β signaling contribute to the differences in the T cell tumor reactivity among patients. To test the hypothesis, first we will determine whether T cells from AA and EA PCa patients display differences in tumor reactivity in co-cultures with autologous tumor organoids and/or CAFs. Second, our preliminary data have shown that Dabigatran etexilate, an anticoagulant drug for preventing stroke in people with atrial fibrillation, effectively blocks GARP/TGF-1 signaling and that its combination with anti-CTLA4 results in a durable regression of Myc-CaP xenograft tumors. We therefore will determine whether Dabigatran can enhance the anti-PD1/anti-CTLA4’s anti-PCa efficacy in the HiMyc and TRAMP transgenic PCa mouse models. Third, to further enhance the clinical relevance of our study, we will utilize available formalin fixed paraffin embedded tissue blocks from 141 AA and 141 EA matched PCa cases by age and Gleason score and determine the relationship between GARP/TGF-β signaling and various infiltrating immune cells in tumor and stroma of AA and EA PCa. Impact: This proposal capitalizes on the development of unique organoids and cell resources, which allow dissecting factors and mechanisms (i.e. GARP/GARP/TGF-β) signaling leading to immune differences between AA and EA PCa. In addition, a novel strategy for enhancing immune checkpoint therapies in PCa in general and AA PCa in particular may be developed by repurposing Dabigatran etexilate through targeting GARP/TGF-β signaling that is highly activated in the tumor microenvironment of AA PCa, as suggested by our preliminary data. Dabigatran etexilate has shown similar safety profiles or side effects as Aspirin in humans.

NIH Research Projects · FY 2025 · 2021-12

Project Summary This proposal centers on the role of the paraventricular nucleus of the thalamus (PVT) in mediating the impact of early life adversity (ELA) on reward-seeking behaviors later in life. I will identify and characterize PVT neuronal populations that are engaged by ELA, test the hypothesis that these neurons execute the enduring effects of ELA on reward-seeking behaviors, and investigate a potential mechanism behind this process. Early life adversity (ELA) consisting of factors such as poverty, trauma, or chaotic environment impacts the lives of over 30% of children in the United States. ELA is associated with poor cognitive and emotional health and increased risk for affective disorders, including depression, PTSD, and addiction, which involve aberrant reward- circuit function. Studies in animals have demonstrated causal relations between ELA and impaired reward- seeking behaviors and enable the establishment of underlying neurobiological mechanisms. It is crucial to understand these associations, because ELA and its consequences, unlike genetic contributors to vulnerability to psychopathologies, may be amenable to prevention or mitigation. The paraventricular nucleus of the thalamus (PVT) is an important component of the reward circuit that encodes remote emotionally-salient experiences to influence future reward-related behaviors. However, it remains unknown if the PVT encodes experiences as remote as ELA and whether PVT neurons activated during ELA contribute to deficits in reward-seeking behaviors later in life. Building on my robust preliminary data and a potent validated naturalistic model of ELA in mice, which provokes deficits in reward-seeking behaviors later in life, my central hypothesis states that PVT neuronal populations are activated during ELA and contribute to ELA- provoked deficits in reward-seeking behaviors later in life. To test this hypothesis I will (a) capitalize on a transgenic mouse for activity-dependent genetic labeling [Targeted Recombination in Active Populations (TRAP2) mice] to determine the the location of neuronal populations within the PVT that are activated by ELA and establish their molecular identity; (b) use chemogenetics to probe the role of PVT neurons activated during ELA in governing reward behaviors later in life; (c) establish the differential PVT activation during reward-seeking behaviors in adult ELA and control mice, with a potential role in the observed behavioral impairments. Together, the proposed experiments will provide critical information about the developmental functions of the PVT and its contributions to the impact of ELA on reward-seeking behaviors, significantly advancing our understanding of the neurobiological mechanisms underlying reward-related psychopathologies.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Considerable data now suggests that inflammatory processes play a critical role in the pathogenesis of diabetic retinopathy. Leukocytes in particular appear to play a major role in the diabetes-induced degeneration of retinal capillaries (which sets up the conditions for eventual development of retinal ischemia, release of vaso- proliferative factors like VEGF, and ultimately, retinal neovascularization). How leukocytes mediate this capillary cell damage in diabetes is not known. Neutrophils contain large quantities of proteases, which they use to kill foreign invaders in the body. Neutrophils are known to release neutrophil elastase (NE) as a part of their response to injury, but failure to regulate their levels can result in tissue injury. We present evidence that neutrophil elastase plays and important role in the endothelial damage and cytotoxicity in diabetes, and postulate that the diabetes-induced induction of retinal inflammation and the vascular damage that is characteristic of early diabetic retinopathy are secondary to neutrophils via transport of NE in extracellular vesicles to endothelial cells, where the NE cleaves Gasdermin D (GSDMD) to cause cytotoxic pores in the endothelial cell membranes. We propose 3 specific aims: Aim 1. To investigate the effect of pharmacologic inhibition of NE on early stages of diabetic retinopathy. We will use structurally NE inhibitors, and will administer the therapies systemically as well as via eyedrops. Aim 2. To investigate mechanism(s) by which NE increases death of retinal endothelial cells in diabetes. Aim 3. Investigate the role of GSDMD in the pathogenesis of diabetic retinopathy. These studies will be conducted initially using GSDMD-/- mice. These studies will be conducted in vivo using pharmacological means to inhibit NE in diabetes and using mice genetically deficient in GSDMD. This proposal is novel because it focuses on (i) the role of a neutrophil protease in the pathogenesis of the retinopathy, and (ii) toxicity to retinal endothelial cells as a result of transfer of the protease from neutrophils to the endothelial cells via extracellular vesicles. This area is new and has not been previously been studied with respect to diabetic retinopathy. The insights learned from these studies can lead to development of novel and effective therapies that inhibit the development of diabetic retinopathy by targeting a protease secreted by neutrophils or the subsequent cleavage of GSDMD.

NIH Research Projects · FY 2025 · 2021-09

Subgroups within the House and Ballroom Community (HBC) account for at least half of all new HIV infections in the United States (U.S.). At the same time there is a significant disparity in knowledge of, access to and uptake of pre-exposure prophylaxis (PrEP) among these subgroups. Individuals in the HBC face stigma and individual, group, societal and structural challenges that put them at high risk of HIV, creating barriers to HIV prevention and care. This study will include a longitudinal qualitative phase, a longitudinal social epidemiologic phase, and an intensive longitudinal ecological momentary assessment phase. We propose to use an innovative and culturally relevant sampling strategy of web-based respondent driven sampling (webRDS) to recruit an online cohort of 900 people in the HBC. A subgroup of enrolled participants will participate in an ecological momentary assessment (EMA) phase to measure the immediate context of stigma and HIV prevention behaviors and HIV risk. These participants will receive short random ecological momentary assessments daily for 30 days via a mobile health application. Not only will this study examine stigma in context of the daily lives of individuals in the HBC, the EMA phase will also inform future development of a context aware, ecological momentary intervention to reduce HIV risk and optimize the HIV prevention and care continua.

NIH Research Projects · FY 2024 · 2021-09

This project centers on the development of statistical network models for understanding the formation of protein aggregates associated with disease states as well as critical biological processes. Systems of this type include amyloid fibrils and toxic oligomers, amorphous protein aggregates, and the large, dynamic complexes formed by small heat shock proteins. Our work combines modeling techniques from the mathematical social sciences with theoretical and experimental methods from biophysical chemistry, enabling us to approach biological problems in novel ways. Our technical innovations are focused on Hamiltonian-driven network models, extending methods originally developed for social networks to capture interactions among individual proteins in solution over time scales of hours to days. The project team comprises an established collaboration between a mathematical social scientist and statistician with expertise in computational statistics and network analysis, and an experimental biophysical chemist with relevant expertise in protein structure and function. Essential components of this research include both the creation of modeling techniques that can be used effectively with existing experimental data, and the collection of new data to validate our modeling work. This work will result in a collection of novel methods for the study of protein aggregation that are both statistically principled and empirically grounded, as well as biologically relevant empirical data.

NIH Research Projects · FY 2024 · 2021-09

CSUF/UCI-CFCCC Cancer Health Equity Research Partnership (CHERP) Overall Component: Abstract California State University, Fullerton (CSUF) is an institution serving underserved populations and underrepresented students (ISUPS), and the University of California, Irvine Chao Family Comprehensive Cancer Center (UCI-CFCCC) is an NCI-designated comprehensive cancer center. Together, we plan to establish a collaborative partnership to develop pilot research projects between faculty members at CSUF and UCI-CFCCC that will generate preliminary data for R01 or other competitively funded grant applications, and educate undergraduates (at CSUF) and master's students (at CSUF and UCI) in cancer health disparities research. The Partnership plans to fund five pilot projects, and educate a total of 38 students during the length of the proposed grant period. The overall specific aims of this new cancer-specific partnership are to: plan, implement and evaluate a highly integrated and interactive cancer health disparities partnership between CSUF and UCI- CFCCC; conduct pilot cancer health disparities research projects involving at least one investigator at each institution that advance knowledge regarding cancer health disparities, leading to the submission of competitive grant applications by NIH/NCI and other federal/non-federal agencies; and provide cancer research education to underrepresented undergraduate and graduate students to increase their understanding of cancer health disparities, leading to a larger pool of highly talented future scientists from diverse backgrounds. To accomplish this we will undertake a set of activities organized under four components. The Administrative Core will be responsible for the overall administrative and fiscal aspects of all P20 activities, and will include the Executive Committee and Internal Advisory Committee. Activities will be implemented in three stages: initial planning, implementation, and evaluation. The evaluation effort will be spearheaded by an external evaluator with expertise in multi-component center reviews. The two pilot research projects each focus on a cancer health disparity: the first is a basic science study that will conduct lipidomic profiling, and DNA and RNA sequencing of tumor/NAT paired samples to optimize sample preparation and detect genetic/genomic and transcriptomic differences that are related to African American triple negative breast cancer risk. The second is an epidemiological study that will examine Asian sub-ethnic group differences in ovarian cancer mortality and treatment patterns. The long term goals of this collaborative partnership are to diversity the cancer research workforce and ultimately increase our scientific impact to address the cancer health disparity needs in Orange County, California.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract The ability of cells to efficiently communicate across organismal-level distances and difficult-to-surmount biological barriers is one of the most important fundamental phenomena in biology. A vast body of literature has revealed that such cell-to-cell communication is mediated via membrane-enclosed particles called extracellular vesicles, which are released by cells into their surroundings and contain lipids, ligands, nucleic acids, and proteins. Such extracellular vesicles represent one of the only natural non-viral structures by means of which “source” cells can reprogram the genetics and fate of “target” cells, and as such, they are implicated not only in the regulation of nearly every cellular process but also in the development of nearly every tissue within the human body. Moreover, these vesicles play essential roles in various disease states and pathological conditions, including cancer progression, neurodegeneration, musculoskeletal disorders, cardiovascular degradation, metabolic syndromes, and wound healing. Given their ubiquity and crucial biological roles, extracellular vesicles hold great promise for clinical diagnostics and therapeutics, but to date, such applications have been hindered by key challenges associated with 1) fundamentally understanding extracellular vesicle formation in source cells, 2) loading extracellular vesicles with biomolecular cargo in high yield, 3) controllably regulating extracellular vesicle production with external stimuli, 4) delivering extracellular vesicles to target cells over tissue-relevant length scales, 5) maintaining the long-term biological activity of extracellular vesicle-internalized cargo, and 6) implementing extracellular vesicle-based treatment strategies in living animals. Herein, by drawing inspiration from proteins and structures found in cephalopod skin cells and leveraging the technical foundation established for cephalopod-inspired bioelectronic devices, we propose to solve all of the scientific and technological challenges currently impeding clinical applications of extracellular vesicles. The envisioned research plan involves 1) validating electrical techniques for controlling the release of extracellular vesicles from genetically engineered cells interfaced with different bioelectronic devices and platforms, 2) developing strategies for remotely and wirelessly controlling the release of biomolecular cargo-loaded extracellular vesicles from engineered cells interfaced into implantable bioelectronic systems, and 3) demonstrating that remotely controlled release of clinically valuable cargo-loaded extracellular vesicles by implanted bioelectronic system-integrated source cells can guide target cell fate and tissue development in living animals. Altogether, the successful completion of the proposed work will enable harnessing of extracellular vesicle-mediated cell-to-cell communication pathway for the regulation of physiological processes and will thus furnish transformative opportunities for developing unprecedented next generation diagnostic and therapeutic technologies.

NIH Research Projects · FY 2024 · 2021-09

NIH Research Projects · FY 2025 · 2021-09

Multiple myeloma (MM) is the second most common hematological malignancy and is largely incurable. Black Americans experience an unexplained 2-fold excess risk compared to White Americans, while Asian Americans experience a lower risk. MM is preceded by a precursor state characterized by an accumulation of benign monoclonal plasma cells that secrete a monoclonal protein (monoclonal gammopathy of undetermined significance, MGUS), which occurs with the same racial/ethnic disparity as seen in MM. Thus, explaining the causes of the disparity in MGUS will shed light on the causes of the disparity in MM. We (MPIs Cozen, Desai and Bertrand) are submitting this proposal in response to PAR 19-279, MMDPQ1: “What risk factors, singularly or in cooperation, explain the variation in MGUS incidence among different races?” Our central hypothesis is that racial and ethnic differences in plasma cell growth/angiogenic factors, microbial translocation, chronic antigenic stimulation due to previous infection and lifestyle factors can explain the observed MGUS incidence disparity. By screening participants in 4 multiethnic NCI epidemiology cohorts (Black Women's Health Study, Women's Health Initiative, Multiethnic Cohort, Southern Community Cohort Study), we will identify 844 Blacks, 844 Non-Latino Whites, 146 Latino and 146 Asians with laboratory validated MGUS and an equal number of age-, sex- race/ethnicity- matched controls without MGUS by the same laboratory screening. We will measure levels of 18 biomarkers reflecting plasma cell growth, angiogenesis, inflammation and microbial translocation (IL6, IL17, XCL13, IL6-R, BAFF, APRIL, gp130, HGF, CCL-8, Angioprotein-2, LBP, sCD14, adiponectin, BCMA, IP-10, IL10, MIP1-a, CXCL12) (Aim 1). We will also examine exposures associated with B-cell activation including lifetime cumulative infection from chronic immune stimulating agents by measuring antibodies simultaneously in a multiplex system to Hepatitis B and C viruses, H. pylori, T. gondii, T. pallidum, C. trachomatis, HPV, and all 8 Herpes family viruses (Aim 2), and lifestyle factors obesity, physical activity, diabetes and use of anti-inflammatory medications metformin, statins and aspirin, known to affect B-cell response (Aim 3). To determine whether a given putative risk factor can (at least partly) explain the racial disparities in MGUS we will (1) determine whether the factor is consistently related to MGUS within all four ethnic groups and if so then; (2) estimate the strength of the relationship in a combined analysis and; (3) determine whether the prevalence of an MGUS-associated factor differs by race in a direction consistent with the known racial differences in MGUS risk. We will use logistic regression techniques that relate either continuous or binary factors (body mass index, diabetes, individual infections) to the log odds of MGUS. With our multidisciplinary team of co-investigators and collaboration of 4 racially/ethnically diverse cancer epidemiology cohorts, we are uniquely positioned to be able to identify causes of the MGUS disparity, about which there is little known. This study will provide critical information on the knowledge gap that exists in the causes of racial/ethnic disparity for MGUS.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Mechanisms of inter-organ signaling have been established as hallmarks of nearly every pathophysiologic condition, where many exist as related and complex diseases. While significant work has been focused on understanding how individual cell types contribute and respond to specific perturbations related to common, complex disease, an equally-important but relatively less-explored question involves how relationships between organs are altered in the context of an integrated living organism. Current technical advances, such as proteomic analysis of plasma or conditioned media, have allowed for a more unbiased visualization and discovery of additional inter-tissue signaling molecules. However, one important feature which is lacking from these approaches is the ability to gain insight as to the function, mechanisms of action and target tissue(s) of relevant molecules. To begin to address these constraints, we initially developed a correlation-based bioinformatics framework which uses multi-tissue gene expression and/or proteomic data, as well as publicly available resources to statistically rank and functionally annotate endocrine proteins involved in tissue cross-talk. Using this approach, we identified many known and experimentally validated several novel inter-tissue circuits. This was this first study to directly link an endocrine-focused bioinformatics pipeline from population data directly to experimentally-validated mechanisms of inter-tissue communication. While these validations provide strong support for exploiting natural variation to discover new modes of communication, these serve as simple proof-of-principle studies and, thus has promising potential for expansion. As a result, we have developed a series of in silico tools to guide discovery of endocrine interactions. Specifically, pathway-targeted enrichments, Bayesian network interrogation and scalable machine learning. The goal of this proposal is to closely bridge these computational tools with experimental methods to systematically dissect mechanisms by which tissues communicate and how these interactions are perturbed in metabolic disease settings. Given that we survey genetic variation to guide prediction of new modes of endocrine communication, these findings are likely to be robust across diverse backgrounds. We will implement high- throughput screening of specific tissue communication circuits which operate under disease-specific conditions of metabolism (ex. Obesity and Type 2 Diabetes), define which are conserved from mice to humans and mechanistically dissect pathophysiologic impacts of endocrine communication through in vivo experimentation. The success of these aims relies heavily on bridging computational and experimental approaches, justified by the training and focus of the PI. Collectively, these objectives will begin with unbiased computational approaches, validate using high-throughput in vitro assays and evaluate therapeutic potential of new endocrine interactions using mouse models of disease.

NIH Research Projects · FY 2024 · 2021-09

Project Summary / Abstract Alzheimer's disease (AD) is the most common cause of human dementia that progressively worsens with age. Sporadic late-onset AD accounts for more than 90 percent of Alzheimer’s cases without clear documented familial history of the disease. However, the vast majority of existing transgenic and knock-in models incorporate disease-causing familial mutations in one or more genes associated with dementias, representing a major limitation. The RFA-AG-21-003 [New/Unconventional Animal Models of Alzheimer’s Disease] highlights the need to develop and characterize naturally occurring “non-murine models of AD that may represent improved translational potential by better replicating pathological features of the human disease”. We respond to the RFA to apply single cell epigenomic and transcriptomic technologies developed by our team to create cell-type- specific epigenome and transcriptome maps in frontal cortex and hippocampus that are associated with AD-like pathogenesis in two naturally occurring AD animal models: Octodon degus and Canis familiaris. These animals show age-dependent neuropathology and cognitive impairment similar to those observed in human AD, thus they are natural AD models. As both degus and mice are rodents, the studies of long-lived degus will be particularly valuable for a within-mammalian order comparison of which AD gene regulatory pathways are common to spontaneous AD-like features in degus versus different transgenic mouse models. While we generate the resources in alignment with the RFA goals, the proposed research will allow us to develop a comparative analysis to determine conserved epigenetic alterations in the unconventional animal models and bridge our existing databases of mouse models and humans. Maladaptive changes in accessible chromatin accessibility, chromatin organization and gene expression in disease relevant cell types will reveal species- specific and cross-species conserved mechanisms of AD pathogenesis, as well as new targets for AD prevention and treatment. This will provide new insights into the mechanisms of AD pathogenesis in humans. In addition to genome data sharing at the designated NIH depository, resources will be shared and curated at our UCI Center for Neural Circuit Mapping.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT: Glial immune signaling in radiation-induced brain injury. Cranial radiation therapy (CRT) for the treatment of CNS cancers often leads to unintended and debilitating cognitive impairments. CRT also remains the standard of care to counter brain metastases for other invasive cancers. However, the molecular and cellular mechanisms underlying CRT-induced cognitive decline are multifaceted and have not been completely resolved. Our past findings show that whole-brain, acute CRT induces progressive neurodegenerative changes, including oxidative stress, reduced neurogenesis, and increased neuroinflammation. Microglia and astrocytes form complex glial networks in the CNS by pruning and maintaining thousands of synapses that are actively involved in cognition. Yet, we have shown that CRT-induced cognitive disruption coincides with astrocytic hypertrophy, elevated expression of astrogliosis genes, and persistent microglial activation in rodent models. Therefore, we hypothesize that detrimental glial signaling significantly contributes to cognitive deficits. The complement system is a potent mediator of the glial activation, but it also has a range of non- immune functions in the CNS, including synaptic pruning and clearance of apoptotic cells and cellular debris which is detrimental if dysregulated. Particularly, global elevation in the expression of complement C1q and C3 in the CNS has been reported in neurodegenerative conditions. Our findings indicate that acute, whole-brain CRT-mediated chronic microglial activation and reactive astrocytes, elevated co-expression of complement proteins (C1q, C3) and specific receptors (C5aR1, TLR4) coincided with cognitive impairments. Reactive gliosis has been shown to upregulate complement cascade proteins that are destructive to synapses and associated with neurodegeneration. We hypothesize that brain cancer therapy-induced aberrant activation in the glial complement cascade leads to cognitive deficits. Our hypothesis is supported by two key preliminary data sets targeting complement signaling at the upstream (C1q) and the downstream (C5a) activation branch points. First, exposure of conditional microglia-selective C1q (knockdown) mice to CRT did not exhibit impaired cognition and showed a lack of neuroinflammation as compared to irradiated WT mice. Second, treatment with an orally active, BBB permeable, C5a receptor (C5aR1) antagonist ameliorated acute CRT-induced cognitive deficits and alleviated microglial activation in the irradiated brain. Our hypothesis will be addressed using a clinically relevant, fractionated, focal cranial irradiation paradigm ± temozolomide, transgenic and glioma- bearing syngeneic mouse models, and pharmacologic approaches designed to test mechanisms and therapeutic interventions to restore cognitive function in the impaired animals.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The Virtual Reality Unmet Clinical Needs course offered at the junior level will engage undergraduate engineering students using online and immersive active learning techniques to develop intuition, teamwork skills, and unmet clinical needs evaluation prior to their senior capstone course, the BioENGINE program. This course will be offered through online websites and virtual reality applications to allow large institutions and those without access to medical centers to be able to perform clinical immersion and unmet clinical needs finding for all undergraduate biomedical engineering students. Through immersive technologies such as virtual reality, simulations, and online lectures using manikins and staged real-life simulations, we will provide a large class size of students the opportunity to understand how medical devices are used in the real world, and identify novel potential commercializable solutions that they will develop during their senior capstone course. To accomplish this, students will work in multidisciplinary teams to develop a proposed innovative medical solution identified through the immersive clinical environments exploration and filmed clinician interviews. By having all students in the curriculum the ability to perform unmet clinical needs identification and evaluation through virtual immersion, we will provide an inclusive online active learning program to all students in our program as well as those at other institutions. Through this strategy, all biomedical engineering students will gain more practice, depth, and experience in listening, learning, and designing a solution to an unmet clinical need, we hope to gain a more effective method of teaching biomedical engineers how to solve problems in a real world situation, and how to communicate in a team more effectively.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Using single-particle cryogenic electron microscopy (cryoEM) we can study proteins and protein complexes to atomic resolution and elucidate dynamics and distinct functional states in native or near-native environments. But this is virtually impossible to do with small proteins. My lab is interested in understanding protein function by observing their structures at high-resolution in native states. We leverage expertise in cryoEM and computational protein design to study proteins, both soluble and membrane, and their complexes that play major roles in human diseases. Membrane proteins are cellular gatekeepers and one of the most important class of membrane proteins are the G protein-coupled receptors (GPCRs), which act as signal conduits through ligand-induced binding on outside cells to the recruitment of binding complexes inside cells to relay signals. As they are involved in most cellular processes they are of considerable interest in drug development. While cryoEM has gained momentum in structural biology, it has a fundamental limitation that proteins smaller than 40 kDa cannot be studied effectively because the signal in the images is too low. This means that most proteins in the human genome cannot be studied by cryoEM. Using a designed approach, I was recently able to solve the high-resolution structure of a 17kDa protein by cryoEM, almost 3 times smaller than current cryoEM size limits. I succeeded because we used computational design to attach the 17 kDa protein to a scaffold which helped imaging by increasing the mass of the particle and the higher symmetry afforded better reconstruction. This proof-of-principal experiment demonstrates the powerful combination of using computational design for cryoEM. While exciting, this scaffold approach is still at its infancy and further design and development are needed to realize its full potential. New scaffolds capable of displaying important membrane proteins like GPCRs will be developed, tested and optimized. These new nanomaterials will be specifically tailored to address cryoEM needs. With this approach we will investigate membrane protein structure, describe functional dynamics in near native environments and facilitate rapid structure-guided drug design to help against devastating diseases.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Population aging is leading to a public health crisis in the growing number of people affected by Alzheimer’s disease (AD) and Related Dementias (ADRD). It has been more than 15 years since the last FDA approval for a new treatment for ADRD. To continue the dramatic progress over the last decade in understanding ADRD pathophysiology we will require significant preclinical and clinical translational research to bring candidate therapies to clinical use and grow a therapeutic armamentarium sufficient to curb the public health impact of ADRD. Essential to this success will be a new generation of ADRD scientists, especially scientists with the unique training and skills necessary to design and perform translational research. This training is rarely provided through the traditional course of medical, clinical psychology, basic science, or biostatistical education. As a result, there is a dearth of well-trained ADRD translational investigators. Moreover, there is inadequate diversity among the current group of active ADRD translational investigators, limiting the unique perspectives and team science synergies that could propel the field toward critical solutions. We propose here a new training program in translational ADRD research titled “Training in Translational ADRD Neuroscience (TITAN)” at the University of California, Irvine (UCI). This new training program will be broadly inclusive of promising graduate students and junior translational scientists, such as medical doctors, clinical psychologists, epidemiologists, biostatisticians, bioethicists, and neuroscientists. We have assembled an outstanding and similarly diverse team of preceptors who will ensure the success of this new training program. The program will develop novel training opportunities in ADRD translational research while also leveraging the considerable scientific and training resources at UCI, including the NIA P30-funded Alzheimer’s Disease Research Center (ADRC), which since April 2020 includes a Research Education Component with complementary objectives. Similarly, the UCI Institute for Clinical and Translational Science (ICTS) is UCI’s NCATS-funded CTSA program that offers synergistic training options to be leveraged, such as the K-club, responsible conduct of research workshops, and KL2 funding mechanisms. We will recruit diverse trainees from multidisciplinary specialties as well as individuals from nationally underrepresented racial and ethnic groups, individuals with disabilities, individuals from disadvantaged backgrounds, and women.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Neural stem/progenitor cells (NSPCs) lie in close proximity to blood vessels in brain stem cell niches and after transplant to treat neurological conditions. This adjacency allows for considerable interaction between NSPCs and the endothelial cells (ECs) that form blood vessels, creating an interdependent and complex relationship between these cell types. Human NSPC (hNSPC) and human EC (hEC) interactions have not been well studied, creating knowledge gaps that hinder our understanding of human brain function and repair after injury. We used a tissue engineering approach with a a 3D scaffold mimicking brain properties to study hNSPC-hEC interactions. We found hEC contact with hNSPCs induced the formation of GFAP+/ SOX2+ cells, which could be type B adult neural stem cells. Type B cells are slowly dividing NSPCs that prevent depletion of the NSPC pool and may be activated after injury to help replace lost brain cells. We found hNSPCs stimulate hEC vessel formation (vasculogenesis) and this effect is mediated by hNSPC secreted components rather than hNSPC contact with hECs. These data lead to the hypothesis that hEC contact promotes a type B adult neural stem cell phenotype while hNSPC secreted components stimulate human vessel formation in 3D niches. We will test this hypothesis with the following Aims: Aim 1 - determine how contact with hECs affects hNSPCs; Aim 2 - determine how hNSPC secreted components impact human vessels; Aim 3 - test whether hNSPCs and hECs promote type B adult neural stem cells and vessels in vivo. By investigating the crucial interactions between hNSPCs and hECs in 3D tissue engineered niches, we will better understand how their relationship impacts human brain function. This knowledge could be used in the future to optimize co-transplants of hNSPCs and hECs in scaffolds to recreate critical niche interactions leading to formation of type B cells and vessels that stimulate brain repair.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Genetic sex determination and sex chromosomes have evolved in both animals and plants. In mammals, males are XY and females are XX. Degeneration of the Y chromosome and the difference in the number of X- chromosome would lead to dose imbalance of X-linked gene products between male and female cells. To resolve this problem, various dosage compensation mechanisms have evolved in heterogametic species. In mammals, one of the two X chromosomes is transcriptionally silenced in females during X-inactivation. While X-inactivation has been extensively studied as a paradigm for epigenetic regulation of sex chromosomes, it is largely controlled by long noncoding RNAs (lncRNAs) that remain elusive for their role in the evolution of dosage compensation mechanisms. In general, human lncRNAs impact early development and affect human disease. However, whether lncRNAs represent “byproducts” of transcription or essential biomolecules in gene regulation, e.g. X- inactivation, has been under continuous debate. Experimental models for functional lncRNA and evolution of RNA-mediated mechanisms are scarce. Our research focuses on a cluster of lncRNA genes functional for X-inactivation. This cluster of lncRNA genes have evolved concomitantly on the X chromosome, from ancestral protein-coding genes through pseudogenization and gain of RNA functions, coincidental with the evolution of X-inactivation along the divergence of eutherian and marsupial mammals. Our recent data have demonstrated that, within this gene cluster in the mouse genome, the lncRNA Jpx activates lncRNA Xist and functions as a molecular switch for mouse X-inactivation. We have also reported, by comparing mouse and human homologs, functional conservation of Jpx in X-inactivation despite overall sequence and RNA structural divergence. But questions regarding 1) what sequence variations are determinant of lncRNA function for X-inactivation, 2) are there regulatory features of lncRNA essential for X-inactivation, 3) what drives the function of Xic locus in X- chromosome silencing, are unresolved. These are questions directly related to the evolution of dosage compensation in mammals. In this project, we will (1) determine the regional sequence motifs of Jpx lncRNA that are essential for its function in X-inactivation, (2) determine whether both trans and cis activities of Jpx lncRNA are necessary for its function in X-inactivation, and (3) understand the function of Xic in chromosome silencing and the requirement of its cis and trans activities by integrating the Xic sequence into an autosome. The proposed research is anticipated to uncover insights into the molecular features of lncRNA for function in X-inactivation, which will contribute to our understanding about not only lncRNA evolution, but also dosage compensation evolution in mammals.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Rare forms of familial Alzheimer disease (fAD) are known to be caused by life-long, genetically determined perturbations in the production of the amyloid ß-protein (Aß), but the cause(s) of the vast majority of so-called sporadic AD (sAD) cases remains remarkably poorly defined. This proposal will use state-of-the-art mouse models of sAD together with several highly innovative approaches to address key temporal and spatial aspects of sAD pathogenesis for the first time, with critical implications for the development of effective therapies. Whereas fAD is attributable to chronic perturbations in the production of Aß, we hypothesize that sAD is triggered by impairments in the clearance of Aß—specifically by transient impairments in Aß clearance. This hypothesis is consistent with evidence showing that several established risk factors for sAD, such as brain trauma, stress, or poor sleep, lead to short-lived or episodic increases in cerebral Aß levels due to reduced Aß clearance. To model this novel mechanistic hypothesis, we employ innovative methods to inhibit Aß clearance transiently and reversibly by blocking either blood-brain barrier transport of Aß or its proteolytic degradation. Because we aim to define the triggers for sAD, we require an animal model that does not develop AD-type pathology on its own, as most AD mouse models do. To this end, we will use an innovative new sAD mouse model, the APPNL-F/hAß mice, which expresses wild-type human Aß only, under the control of the endogenous murine App promoter, with the minimal genetic mutations needed to model sAD. As is true for normal humans, this sAD mouse model develops diffuse deposits of human Aß in an age-dependent manner, and forms very minimal dense-core plaques only at very advanced ages. Accordingly, these mice are ideal for investigating the pathophysiological mechanisms responsible for triggering the conversion of “normal” Aß deposition to the pathological type in sAD. We hypothesize further that the Aß-dependent pathological mechanisms most relevant to sAD occur much earlier than the ages studied in clinical trials, with clinical symptoms emerging only much later, specifically in the context of aging. Accordingly, we will define the temporal window most relevant to the emergence of AD by increasing Aß levels in APPNL-F/hAß mice transiently at various ages, then evaluating the consequences for the development of AD-type pathology longitudinally, up to and including old age. Finally, we will test the novel hypothesize that spatially distinct pools of Aß (e.g., extra- vs. intracellular) impact the pathogenesis of AD in qualitatively different ways. Specifically, we postulate that intracellular Aß is more relevant than extracellular Aß to the neurodegeneration and memory loss that characterize AD. To test this, we will selectively increase extra- vs. intracellular pools of Aß by reversibly downregulating Aß-degrading proteases that, as our preliminary results show, selectively regulate these distinct pools of Aß. Collectively, these experiments will allow us to investigate, cleanly and for the first time, many critical temporal and spatial aspects of AD pathogenesis, yielding novel insights that will inform improved approaches to the treatment of sAD.

NIH Research Projects · FY 2025 · 2021-09

Project Summary The pathways that regulate processes necessary for wound healing, such as proliferation and migration, are often co-opted by tumors, leading to their description as wounds that do not heal. Therefore, identifying novel genes involved in wound healing and understanding how they are dysregulated in cancer may provide new targets for therapeutic efforts. We recently discovered thousands of small open reading frames that encode proteins <100 amino acids, dubbed microproteins. Among these was a 10 kDa microprotein encoded on the lncRNA Terminal Differentiation-Induced non-coding RNA (TINCR), which is a critical inducer of terminal differentiation in the epidermis and regulator of cancer cell proliferation and migration. TINCR microprotein (TINCR-MP) is highly conserved across mammals, strongly suggesting that it is functional. Compelling preliminary data demonstrate that in human skin models TINCR-MP downregulates signaling pathways involved in wound healing, and that it interacts with histone modifying enzymes that have functions in differentiation and cancer. Furthermore, while TINCR-MP is expressed during epidermal differentiation, it is unnecessary for differentiation, making its function separate from the differentiation promoting activity of TINCR RNA. The central hypothesis is that TINCR-MP acts as a brake for proliferation and migration during wound healing through alterations to the epigenetic landscape, and that this function is hijacked in cancer cells to promote tumor progression. The goals of this proposal are thus: 1) to determine the role of TINCR-MP in cutaneous wound repair processes, and establish whether TINCR-MP effects on wound healing are driven by epigenetic modifications, 2) to determine the extent to which TINCR-MP regulates proliferation and migration in prostate and breast cancer cell lines, and establish its effects on epigenetic and subsequent gene expression changes, and 3) to identify additional microproteins regulated during differentiation that affect cancer proliferation. Successful completion of these aims will reveal an important microprotein that functions in wound healing and tumor progression, as well as new microproteins to investigate. The candidate, Dr. Thomas Martinez, plans to develop an independent research program focused on characterizing microproteins that function in both differentiation and cancer. The opportunities offered by this Career Development Award will allow Dr. Martinez to deepen his knowledge of developmental biology, epigenetics, and cancer biology. He will also gain experience utilizing 3D ex vivo skin models, in vivo mouse models, and techniques for analyzing the epigenetic landscape. Dr. Martinez will conduct these studies under the mentorship of Dr. Alan Saghatelian, expert on peptide biology, as well as co-mentors Dr. Diana Hargreaves and Dr. George Sen, experts on chromatin remodeling complexes in disease and epidermal homeostasis, respectively. Dr. Martinez’s Advisory Committee will also help foster his scientific and academic career goals. The Salk Institute provides an ideal environment with expertly run core facilities and ample seminars and workshops to prepare trainees for independent academic careers.