University Of California-Irvine

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Transposable elements (TEs) are genomic parasites that can negatively impact host viability and fertility. They have been identified as the causes of inherited human disorders and cancers. Despite their detrimental effects, TEs are prevalent across eukaryotic genomes and exhibit dramatic variation in abundance and genomic positions within and between species. For instance, the proportion of vertebrate genomes occupied by TEs ranges from only 6% in pufferfish to 65% in salamander. Over 45% of the human genome harbors TEs, and any two people differ by at least a thousand TE insertions. However, it remains unclear what evolutionary forces drive TE variation and how that influences functions and, thereby, host health. Most studies of the harmful effects of TEs have centered on TE-mediated physical disruption of DNA and changes in DNA sequences. While such genetic disturbances have important consequences, this paradigm overlooks the detrimental epigenetic effects mediated by TEs, including biochemical modifications of chromatin and reorganization of three-dimensional (3D) genome structures. My recent pioneering studies revealed, on a genome-wide scale, that epigenetically silenced TEs can perturb the function of neighboring genes through cis spreading of silencing marks (cis epigenetic effects of TEs) and alter 3D genome organization (3D epigenetic effects of TEs). These exciting observations offer a possibility to answer long-unresolved questions about why there are between-species differences in TE content and how these differences affect genome function and evolution—the overarching goals of my research program. My laboratory uses Drosophila as a primary model and integrates evolutionary genomics and cell biology to decipher the functional and evolutionary significance of TE variation. One major goal of my research program is to determine how TE variation influences genome evolution through my newly discovered 3D epigenetic effects of TEs. My research group will use integrative genomic analysis at multiple levels (DNA, RNA, epigenetics, and 3D genome structures) to investigate our hypothesis that the 3D epigenetic effects mediated by TEs can produce varying 3D genome organization. We further predict that this TE-mediated variation in 3D genome structures can shape genome evolution by affecting fundamental genetic processes. In addition, my laboratory seeks to identify the molecular and evolutionary mechanisms contributing to between-species differences in TE content. We will use Drosophila genetics and transgenics to identify host genetic factors that modulate the epigenetic effects of TEs in cis and in 3D nuclear space. Furthermore, we will combine comparative evolutionary genomics and experimental evolution to investigate our hypothesis that between-species variation in these host genetic factors contributes to varying epigenetic effects of TEs and ultimately drives the evolution of divergent TE content across Drosophila species. Our discoveries will provide a novel basis for understanding eukaryotic genome evolution and open new perspectives for TEs' roles in human health and disease.

NIH Research Projects · FY 2025 · 2021-08

Any human being has on average 5 million single-nucleotide variants and 13 million nucleotides of insertions, deletions, and other regions present in variable copy numbers compared to the human reference genome. Together these variants must account for all of the genetic contributions to every phenotype of that person, whether it is their height or their familial predisposition to complex diseases. While we can quickly measure the presence of these variants in a genome, we lack the framework to understand which of the variants impact genomic function or how they interact with each other in living, breathing organisms. A core mission of the IGVF Consortium is to identify variants that impact the expression of genes using single-cell techniques and computational modeling. Applying a single-cell genomics approach to selected diverse mouse strains can make a powerful contribution to the mission and to resources of the Consortium. Our Center for Mouse Genomic Variation at Single Cell Resolution will first use 38 mouse Collaborative Cross recombinant inbred lines that possess similar levels of sequence diversity to humans to identify variants that influence gene expression levels and chromatin accessibility at the single-nucleus level in 8 distinct tissues. We will sequence simultaneously a subset of single-nuclei with both short-read sequencing and long-read sequencing to identify variants that impact the expression of different transcript isoforms in different cells across the different mouse strains. We will also measure the relationship of variants in these CC Lines in the response of macrophages in these tissues in response to LPS stimulation. The resulting resource catalogs of cell-type expression QTL, chromatin accessibility QTL, splicing QTL, and response QTL maps will be useful for IGVF modeling groups; for characterizing important variants; and for use by the wider community studying the function of these tissues as well as for designing better pre-clinical models of human diseases.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Hypertension (HTN) is the most significant modifiable risk factor for cardiovascular disease and involves multiple pathways including those in the neuroendocrine and immune systems. Antihypertensive drugs manage HTN but do not address the central sympathetic and inflammatory pathways and approximately 50% of hypertensive (HTNive) patients do not have their blood pressure (BP) controlled. The World Health Organization has suggested acupuncture for HTN; however, prior clinical trials utilizing acupuncture have shown modest or null outcomes and not achieved clinically meaningful reductions in BP. These conflicting outcomes result from the lack of a mechanistic-based approach to using electroacupuncture (EA), a form of neurostimulation. The reasons for inadequate treatment and BP control are complex, but one reason for this therapeutic misalignment may be an incomplete understanding of the mechanisms underlying the development and progression of HTN including autonomic dysfunction and resulting low-grade inflammation. Using neuroanatomical mapping, phenotyping, and recording of neurophysiological responses that correspond to neuromodulatory mechanisms underlying effects of EA, we successfully selected specific acupoints that in combination (or combined (c)EA) show complementary mechanisms of sympathoinhibition and parasympathoexcitation and not only reduce BP to clinically meaningful levels but also improve underlying autonomic dysfunction and low-grade inflammation. Our proposed study addresses one of NCCIH’s top priorities, “Determine and analyze the neural pathways by which acupuncture exerts its therapeutic effects.” Our strong preliminary data, from Dahl Salt Sensitive (DSS) HTNive rats, show that our targeted acupoints, which simultaneously activate afferents in the median, tibial, and deep peroneal nerves then modulate CNS regulation by activation of neurons in the nucleus of tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV). This modulation resulted in an increase in descending peripheral parasympathetic splenic activity. Treatment with cEA also reduced presympathetic neuronal and splanchnic sympathetic nerve activities. In this study, we will investigate if cEA treatment leads to BP reduction by improving autonomic dysfunction and decreasing inflammation through the neural mechanistic pathways we have preliminarily discovered. Our main aim is to assess mechanisms of the BP lowering effect of cEA in HTNive animals and then validate the improvement in autonomic indices by translating it in mild-moderative HTNive patients. We plan to achieve this by: 1) direct assessment of the effect of cEA on neurons in the hypothalamic and brainstem regions controlling sympathetic and parasympathetic balance including NTS, DMV, paraventricular nucleus, and rostral ventrolateral medulla, as well as sympathetic and parasympathetic efferent activities; 2) investigating if cEA reduces inflammation through acetylcholinergic receptors (AChR) activity in DSS HTNive rats; and 3) using a parallel 2x2 factorial design in a human randomized control study, primarily assess effects of cEA compared to sham-EA (as well as secondarily compare sympathoinhibitory-EA, anti-inflammatory-EA, and cEA) on physiological alterations in autonomic function and secondarily on chronic inflammation. If successful, this study would address the mechanistic basis for the effects of EA as a therapeutic option for HTNive patients who are not at optimal BP goals with conventional therapy alone.

NIH Research Projects · FY 2025 · 2021-08

Project Summary (30 lines) Preterm birth (<37 weeks of gestation) increases the risk infant death, hospitalization, developmental disorders, and low educational attainment. Although non-Hispanic (NH) Black mothers show an increased risk (vs. NH whites) of delivering preterm, NH Black infants historically show―at each gestational age before term―improved health and survival relative to NH white infants. The main explanation for this counterintuitive finding assumes greater selection against frail NH Black fetuses. According to the selection argument, excess fetal loss among frail NH Black gestations results in a hardier cohort of survivors to birth but who are delivered preterm. Prior work describing this racial survival advantage has three important limitations. First, it continues to infer the survival advantage from data now nearly two decades old. Second, it includes no test of the fetal selection argument. Third, it fails to utilize a structural racism framework to understand the potential causes of, and changes over time and place in, racial differences in fetal loss and infant survival. Rapid changes in neonatal technology suggest that decades-old estimates of the survival advantage may have, since 2000, diminished―or even transformed into a disparity. We will use the universe of live births, infant deaths, and fetal deaths among NH Blacks and NH whites in the US (~65 million records, 1995 to 2018) to rigorously examine race-specific trends in preterm birth and infant mortality rates. We will link these records longitudinally by conception cohort to achieve several research objectives. First, we will determine whether NH Blacks (vs. NH whites) born preterm show a survival advantage—or a disparity—in infant mortality in the US. Second, we will investigate how the NH Black / NH white difference in preterm birth rates and infant mortality rates has changed over time, in response to fluctuations in fetal death rates and exogenous changes in neonatal technology (e.g., use of antenatal steroids). Third, we will use a structural racism theoretical framework to examine the extent to which dynamic race-based spatial indicators of inequality (e.g., segregation, incarceration rates) affect patterns across place and time in NH Black (vs. NH white) fetal loss, selection in utero, and infant mortality among preterm births. Our work is significant because we focus on the entire spectrum of perinatal outcomes, including the often neglected but quite large racial disparity in fetal death. Results are expected to advance the knowledge base on NICHD's high-priority research area to better understand racial/ethnic differences in infant health. Our approach will also inform our understanding of the extent to which structural racism may have maintained―or exacerbated―perinatal health disparities. Lastly, our place-based analysis will identify regions with potentially large disparities in fetal loss and perinatal survival that may benefit from targeted healthcare and non-healthcare resources.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Nearly 100,000 patients with kidney failure in the US await kidney transplantation. Living donation reduces the wait and offers superior survival compared with deceased donation. Yet only 6,000 living kidney donations are achieved every year with a substantial decline in donation among biologically related donors in across race/ethnicity. While prior programs were associated with a 6-fold increase in donor referral, efforts to convert donor candidates to donor nephrectomy have been largely unsuccessful. Access to a transplant center is a key barrier to engagement of donor candidates. Alas, the pandemic has amplified this existing barrier. Living kidney donor evaluation is a complex multiphase process that includes activities over approximately one year. We have previously reported that two-thirds of referred persons are deemed medically ineligible donors after reviewing medical history and/or laboratory screening results. Of the one-third who are deemed eligible donor candidates, only 54% complete donor evaluation and counseling (of whom 58% ultimately donate), whereas 35% cannot proceed to complete this initial outpatient clinic visit due to non-medical (personal, social, and arrangement) reasons. The initial outpatient clinic visit is thus a bottleneck as it involves the first obligatory trip to an in-person visit where access to a transplant center becomes a barrier for willing candidates facing geographic, financial, or logistics challenges to come for their initial in-person evaluations. Efforts to promote engagement of kidney donor candidates are needed. Telemedicine via synchronous video visits can facilitate coordination of care in the donor evaluation process. Donor evaluation may become more accessible, efficient, and convenient. The rapid adoption of telemedicine during the pandemic has created opportunities and challenges. In our national survey of US transplant centers, we found that 81% reported telemedicine challenges related to structure and processes of care. In primary care studies, telemedicine video visits have achieved high levels of patient satisfaction and similar outcomes compared to in-person visits. A knowledge gap exists regarding how donor candidates perceive telemedicine in care coordination, and how to best tailor telemedicine care coordination in donor evaluation. Our scientifically goal is to integrate a telemedicine care coordination intervention into the donor evaluation process to enhance engagement of donor candidates and support completion of their evaluation. This award will allow Dr. Al Ammary the protected time to complete his proposed research. Findings of this work will fill a knowledge gap about the new paradigm using telemedicine for donor evaluation and offer preliminary data for an R01-funded randomized clinical trial to test the effectiveness of telemedicine care coordination in increasing living donation and support his transition to independence.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY During homeostasis and proper acute immune responses, interleukin-18 (IL-18) acts as a facilitator of higher cerebral functions and as an alarmin molecule to signal incoming danger to cells. Our studies led us to discover endogenous counter-regulatory components in the IL-18 pathway that are impaired in Alzheimer's disease (AD), which results in excessive and prolonged signaling. More importantly, we found compelling evidence that this process plays a major role in the dysregulation of tau and accumulation of its pathological species. Investigating the underlying mechanisms by which the IL-18 pathway is affected by and drives tau pathology in AD could, therefore, yield novel means to mitigate this insidious disease. Here we propose three aims to investigate this important issue. In aim 1, we hypothesize that β-amyloid (Aβ) disrupts the endocytic protein Tollip (toll interacting protein), which is the leading regulator driving the homologous desensitization of the IL-18 receptor (IL-18R). We will genetically up- or downregulate Tollip in neuronal cell cultures and in an AD mouse model. The outcome of this aim may show that the buildup of pathological forms of tau induced by chronic IL-18 signaling in neurons is, at least in part, caused by the upregulation of IL-18R due to the impairment of its trafficking and degradation mediated by Tollip. For the second aim, we will test the hypothesis that the deficiency in the production of IL- 18's endogenous decoy receptor, IL-18 binding protein (IL-18BP), also plays a role in the chronic activation of this pathway in AD. We will use an adeno associated virus to upregulate IL-18BP levels in AD mice and determine its therapeutic efficacy on AD-like pathology. Finally, our last aim utilizes biochemical and proteomic methods to map the neuronal intracellular networks affected in response to IL-18. We will apply pharmacological and genetic tools to link potential candidates altered by IL-18, such as protein kinases and phosphatases, to the hyperphosphorylation of tau and subsequent synaptic loss and cognitive decline. Establishing these intracellular cascades could allow us to identify novel strategies to inhibit the pathological effects of IL-18, while preserving its relevant physiological functions. Overall, our studies are significant for two reasons: first, they will provide greater insights into the IL-18 signaling cascade and second, they will uncover the critical steps specifically involved in the IL-18 pathway in AD, and may result in the identification of new therapeutic candidates to potentially translate our discoveries into the clinic.

NIH Research Projects · FY 2025 · 2021-08

Abstract The importance of elucidating the molecular mechanisms that underlie cell-to-cell communication is impossible to overstate. Intercellular communication formed the foundation for the evolution of metazoans; then as organisms grew larger, the transduction of signals over distance assumed crucial importance. Long-range signaling in which cells must communicate accurately over several cell diameters is vital both during development and for homeostasis. Deficiencies in the proper execution of the process often results in disease or death. The study of human cancers provides numerous examples of what can happen when a component of a signaling process goes bad. Numerous signaling pathways have been characterized over the past few decades, and a large number of the molecules involved in intercellular signal transduction, both extracellular ligands and their cognate cellular receptors, have been identified. However, gaps still exist at present in what is known of the means by which signals are propagated. The diffusion-based propagation model suggested more than 60 years ago has been the dominant paradigm. This has been particularly true in developmental biology regarding the impact of concentration gradients that are predicted to form as signaling molecules passively diffuse through tissues. Nevertheless, debate has continued on how well such models can explain the precision by which various signals are transmitted over distances of multiple cell diameters. Recently, several research groups including our own have shown that signaling molecules can also be delivered accurately over such distances by direct cell-cell contact that involves the extension of long, thin cellular protrusions (also referred to as signaling filopodia). We have identified a novel variety of such protrusions extending from zebrafish pigment cells that we have named ‘airinemes.’ Airinemes have been seen to be an indispensable component of stripe pattern formation in zebrafish epidermis based on their mediation of long-distance Delta-Notch signaling between pigment cell types. Intriguingly, we have also discovered that this airineme-mediated long-range intercellular signaling is dependent on participation of skin-resident macrophages. Collectively, our findings denote that mechanisms of signal propagation can involve much more than mere passive diffusion of molecules. Moreover, we subsequently found airinemes protruding from other cell types of the epidermis, e.g., keratinocytes, suggesting that airineme-mediated signaling may be a general mechanism in at least that tissue if not others. Although the airineme/macrophage-mediated signaling that we have described has been clearly validated as occurring in vivo, the molecular and cellular details of how this signaling is accomplished are still not sufficiently described. Those details are a crucial first step in linking anomalies in this mode of signaling with the occurrence of various pathologies in vertebrates. In this proposal we will address questions essential to establishing how airinemes achieve target specificity, whether there is a particular subset of skin-resident macrophages that promotes airineme-mediated signaling, and determining what functions airinemes provide in zebrafish keratinocytes. The answers to these questions will not only expand our view of fundamental cellular strategies employed in paracrine signaling but could also provide a basis for detecting where biomedicine might be able to intervene in the process when signaling goes awry in human pathophysiology.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Hepatocellular carcinoma (HCC) has been alarmingly increased, in part due to rapid increases in fatty liver disease. Owing to the lack of early diagnostic markers, HCC's 5-year survival is only 10%. Alcoholism is a well-known HCC risk factor, but the mechanism of how alcohol induces HCC is still unclear. Another HCC risk factor is consumption of dietary fructose, especially in liquid form (soft drinks), which has increased 100-fold over the past two centuries. Based on our preliminary data, we hypothesize that fructose alters hepatic alcohol metabolism via induction of acetyl-CoA synthetase 2 (ACSS2), which enhances the hepatic usage of alcohol carbons to generate carcinogenic metabolites and thereby creates a pro-tumorigenic environment. When alcohol reaches the liver, it is converted to acetate and mostly released into circulation. However, when ACSS2 is activated, flux is shifted from acetate release to acetate catabolism, resulting in excessive production of acetyl-CoA, a high-energy- charged, reactive metabolite. This alteration can initiate and support HCC in many ways, including carcinogenic metabolite production. Indeed, ACSS overexpression and increased acetate usage are commonly observed in cancers including HCC. We recently found that mice exposed to fructose showed strongly induced hepatic ACSS2 activity and acetate usage. Thus, fructose drinking will shift the metabolic fate of alcohol from release as acetate to usage of acetate within the liver. Fructose drinking also induces gut leakiness and alters microbiota metabolism. The resulting toxic microbiota metabolites cause chronic hepatic inflammation, which is a pro-tumorigenic microenvironment. We will test these hypotheses by systematically defining the impact of fructose-elicited ACSS2 induction and microbiota changes on liver alcohol metabolic flux. In Aim 1, we will determine whether and how fructose and alcohol synergistically increase HCC risk. Using in vivo stable isotope tracing of 13C- ethanol and hepatic-portal comparative metabolomics in mice, we will quantitatively define progressive changes of hepatic alcohol metabolism during HCC initiation and progression. This will build a comprehensive catalog of HCC-associated, alcohol-derived metabolites. We will then choose the top candidate metabolites and test whether these metabolites can trigger HCC. In Aim 2, we will test our hypothesis that fructose-induced hepatic ACSS2 activity and/or colonic microbiota changes enhance carcinogenic metabolite production in the liver, thereby triggering HCC. To this end, we will use our newly generated liver-specific ACSS2 knockdown mice and antibiotics treatment. These studies will provide molecular evidence for a clinically relevant question about how the two best-known dietary risk factors, alcohol and fructose, synergistically initiate and advance HCC. Our study's success will have direct impacts on public dietary guidelines and provide mechanistic insights into alcohol-induced HCC.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Once conceived, a woman’s cardiovascular system undergoes dramatic structural and functional changes to accommodate the increasing demands of the fast growing fetus, resulting in profound uterine artery dilation exemplified by dramatic rise in uterine blood flow (UBF). UBF is a rate-limiting factor for pregnancy health because an insufficient rise in UBF during pregnancy is causative for intrauterine growth restriction and preeclampsia (PE) characterized by systemic endothelial damage and vascular dysfunction. Since 1900’s, numerous studies have concluded that local endothelial nitric oxide (NO)-mediated vasodilation is the major mechanism controlling rise in UBF. However, blockade of local NO production only partially inhibits baseline pregnancy-associated rise in UBF, suggesting that other mediator(s) are involved. Endogenous hydrogen sulfide (H2S), mainly synthesized from L-cysteine by two key enzymes: cystathionine -synthase (CBS) and cystathionine -lyase (CSE), is an extremely potent proangiogenic vasodilator. We initially posited that local CBS/H2S production can fill the mechanism behind NO to mediate UA vasodilation during pregnancy. Indeed, we reported that pregnancy dramatically augments UA H2S biosynthesis by selectively upregulating EC and SM CBS but not CSE expression in animals (rats and ewes) and women in vivo and that H2S stimulates pregnancy-dependent dilation of pressurized UA ex vivo. In animal models of PE and women with PE, we found that pregnancy-augmented myometrial UA CBS/H2S is significantly downregulated. However, research on H2S in uterine hemodynamics is still in its infancy; many important key questions need to be answered before a physiological and a pathophysiological role of CBS/H2S signaling in normal pregnancy and PE can be determined. In this new RO1 we propose to test a novel hypothesis that enhanced UA EC and SM CBS/H2S production mediates pregnancy-associated UA dilation by interacting with vascular endothelial growth factor and EC eNOS-NO and downregulated UA CBS/H2S signaling contributes to the vascular dysfunction in PE. We will test this hypothesis by a multidisciplinary translational approach with biochemical, cellular, molecular, physiological, and pharmacological methods coupled with rat models in vivo, freshly isolated human and rat UA rings ex vivo, novel human UA EC (hUAEC) and smooth muscle cell (hUASMC) models in vitro, and myometrial UAs from normotensive vs. PE pregnant women. We have an outstanding team with a track record of long-term productive collaborative research in the field and unique tools needed to complete this exciting and important project. We believe that the novel studies outlined in this RO1 will provide new data to fill a knowledge gap on the physiological and pathophysiological role for H2S in in uterine hemodynamic regulation and this knowledge will provide a compelling rationale for clinical trials to explore the therapeutic potential of H2S in women in high risk of PE.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract Child maltreatment is widely recognized as a serious threat to children's well-being and health. In maltreatment cases, the fidelity and credibility of the child witness/victim's report is often critical to securing an outcome in the best interest of the child. Eliciting information from children about the time-course and sequence of alleged maltreatment is central in these cases. The field of child interviewing is actively debating how best to question children about sequence in these cases with little existing empirical research on which to draw. There is a pressing need to identify strategies for obtaining information from even young, cognitively vulnerable children about the time course and sequence of alleged events. The proposed research will determine (1) how children are questioned about event sequence, and how they respond, across age, in maltreatment investigations (2) how differences in children's age, comprehension, working memory (WM), attention, and episodic memory may impact their abilities to accurately recall event sequence, and (3) how questions and child responses about sequence impact the likelihood that jury-eligible adults' will understand and believe children's allegations of abuse. These aims will be achieved via four proposed projects. In Project 1, the research team will code a sample of 581 legal transcripts to assess the sequencing content included in the prompts used to question child witnesses about their alleged maltreatment experiences and confusion in children's responses to the questions. Of particular interest is identifying instances of potential ambiguity for young children. In Projects 2 and 3, the research team will conduct laboratory studies with 644 4- to 12-year-olds to test the roles of cognition and context in children's responses to sequencing questions like those identified in the maltreatment case transcripts. Memory and response biases are predicted to be most pronounced with decreasing age and WM, and when attention is divided. In Project 3, the research team will examine children's responses to sequencing questions with potentially ambiguous interpretations. Their interpretations of the questions' intent are expected to vary with age and WM in predictable ways. Finally, in Project 4 the research team will examine mock juror interpretations of sequencing questions and children's responses. Participants from across the U.S. (N = 300) will rate the credibility of adult questioners and child respondents selected from Projects 1-3 and the accuracy with which mock jurors understand various sequencing questions and responses from our laboratory studies will be determined. The proposed work is innovative in that it represents a multi-dimensional approach to examining the cognitive, developmental, and contextual appropriateness of varying sequencing questions asked of children in maltreatment investigations and determining the extent to which these questions may impact just decisions in maltreatment cases. This topic has been surprisingly understudied given the substantial implications for understanding the foundations of children's sequential knowledge and memory, and for improving health-relevant legal outcomes in cases of child maltreatment.

NIH Research Projects · FY 2025 · 2021-07

Picornaviruses cause a wide range of significant human and animal diseases, including poliomyelitis, hepatitis, encephalitis, myocarditis, and the common cold. Since they are among the most genetically simple of all RNA viruses, members of the picornavirus family must usurp host cell functions and modify the cytoplasmic environment to facilitate viral translation, RNA replication, and virion biogenesis. Among the many ways that picornaviruses alter host cell functions is by re-localizing them from the nucleus to the cytoplasm where viral replication takes place. The nuclear versus cytoplasmic separation of the major processes that lead to the expression of protein-coding genes in eukaryotes requires a complex transport process that allows RNAs and proteins to move between these two cellular compartments. The Picornaviridae family is one of several virus families that disrupt the nucleo-cytoplasmic trafficking of cells to promote viral replication. Viral proliferation requires the activity of host RNA-binding proteins that normally function in cellular gene expression and are primarily localized to the nucleus. Picornaviruses alter nucleo-cytoplasmic trafficking to exploit these and other nuclear proteins that are subsequently delivered to the cytoplasm to facilitate efficient viral replication. Our recently-published analysis of the nuclear versus cytoplasmic proteome in human rhinovirus-infected cells has established the identities of a large cohort of nuclear proteins that re-localize to the cytoplasm during infection. In this application, state-of-the-art molecular and cell biology experiments are proposed to determine the functional significance of this protein redistribution on human rhinovirus replication, focusing on nuclear RNA binding proteins involved in mRNA splicing/processing and 3’ polyadenylation. A global analysis of protein distribution during rhinovirus infection of different human lung cell lines by quantitative mass spectrometry is also proposed to uncover novel virus-host interactions that may be respiratory cell-type specific. Results from these studies should reveal novel mechanistic insights into how a respiratory virus like human rhinovirus co-opts specific host nuclear functions and how these functions are altered or re-purposed for specific steps in viral replication. Detailed knowledge of such interactions, particularly at essential interfaces between host proteins and viral proteins and/or viral RNA sequences, promises to reveal new targets for antiviral therapies.

NIH Research Projects · FY 2025 · 2021-07

Chemotherapy-related cognitive impairment (CRCI, chemobrain), chemotherapy-induced peripheral neuropathy (CIPN) and gait changes are debilitating side-effects of cancer treatment with platinum agents (e.g., cisplatin), taxanes, and vinca alkaloids. Cisplatin is widely used as a chemotherapeutic agent to treat ovarian malignancies. Over 70% of women report experiencing CRCI, CIPN and/or falls during treatment or after completion, impairing their quality of life. These neurotoxic impairments can also compromise treatment with cisplatin, influencing disease progression. Currently, there are no FDA-approved clinical interventions for the treatment of CRCI and CIPN. Mechanistically, cisplatin-induced neuronal toxicity derives from nuclear and mitochondrial DNA damage, and oxidative stress, which induce the activation of the mitogen-activated protein kinases (MAPK), p38MAPK and c-Jun N-terminal kinase (JNK), leading to neuronal apoptosis. Our preliminary data show that in vitro pharmacological inhibition with small molecule inhibitors, i.e., neflamapimod for p38MAPK and SP600125 for JNK, prevents cisplatin-induced reduction in dendritic spine branching and density. Based on these data, we hypothesize that inhibition of the p38MAPK/JNK pathways will prevent cisplatin-induced neuronal apoptosis and damage, leading to attenuation of cognitive impairments, gait changes, and neuropathic pain associated with CRCI and CIPN. In this project, we propose to determine if: (1) cisplatin-induced p38 MAPK/JNK signaling underlies structural and functional neuronal damage, using in vitro pharmacological inhibition and siRNA silencing; (2) neflamapimod and SP600125 prevent cisplatin-induced neuropathy and gait alterations in the ID8 syngeneic epithelial ovarian cancer in C57BL/6 mice and the transgenic breast cancer model C3TAg in FVBN mice; and (3) cisplatin-induced neurotoxicity is attenuated by p38MAPK/JNK inhibition without compromising its anti-cancer activity. Our Approach includes in vitro analysis of 2 separate neuronal cell lines, behavioral analysis using sensory testing for CIPN, testing of cognitive impairment, and novel MouseWalker for gait changes in female mice using the two mouse cancer models. The proposed studies will demonstrate the role of the p38MAPK and JNK in cisplatin induced CRCI/CIPN, and translational potential for novel strategies to treat CRCI and CIPN. Due to health disparities, women suffer more disproportionately from cancer and pain-related treatment than men. Therefore, testing our hypothesis in female mice is expected to significantly advance the understanding and treatment of cisplatin-induced neurotoxic side effects and improve the quality of life for women with cancer. Nevertheless, we expect that these findings may also apply to cisplatin- induced neurotoxicity in males and to other cancers than ovarian and breast cancers.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Our R01 application entitled, “Improving pediatric brain tumor treatments using FLASH radiotherapy” is focused on translating a novel irradiation modality into clinical practice. Here we will test whether radiation delivered in ultra-high dose rate (one-tenth of second), which far exceed the dose rate used in current clinical practice (minutes), can significantly reduce normal tissue toxicities associated with the radiotherapeutic management of childhood medulloblastoma (MB). The overarching goal is to alleviate the long term neurocognitive and cerebrovascular complications that compromise the quality of life of MB survivors while maintaining tumor control. To achieve these goals, we will undertake studies using clinically relevant FLASH and conventional radiation regimens. Two distinct human MB tumor models will be orthotopically implanted in the brain of nude mice to investigate simultaneously tumor response and neurocognitive function after irradiation. In addition, for long-term follow up, tumor-free mice will also be subjected to cranial irradiation using FLASH or conventional dose rate irradiation. Short-term (1-month) and longer term (4-6 months) studies conducted on tumor bearing and tumor free mice respectively, will critically evaluate tumor control, neurocognitive, cerebrovascular and molecular outcomes in these cohorts. Preclinical studies investigating the response of MB tumors, behavioral performance on multiple learning and memory tasks, vascular structure and integrity and the sparing of the neurogenic niche will unambiguously elucidate many of the mechanisms underlying the neuroprotective effects of FLASH radiotherapy. Data derived from these collaborative studies between UCI and the CHUV will facilitate the clinical translation of FLASH-RT to pediatric oncology, where despite the favorable prognosis of children diagnosed with MB, survivors still suffer a lifetime of complications caused by their prior radiotherapy, a scenario we hope to ameliorate with our innovative FLASH technology.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Tumor heterogeneity is the main cause of resistance to current chemotherapy drugs as well as metastasis development, leading to patients' death. Within the same tumor from the same patient, tumor cells might be subtly or even dramatically different, making it harder to treat clinically. Understanding mechanisms driving cancer diversity is a critical step toward developing new strategies to attenuate tumor evolution and adaptation. Genomic instability is a prominent source of genetic diversity within tumors, generating a cell population subject to potential selection from a micro-environmental or therapeutic context. In recent years, next- generation sequencing technologies have begun to identify genomic signatures of DNA damage and errors in DNA repair processes, revealing new mechanisms causing an accumulation of mutations in cancer genomes. From the 30 mutational signatures identified across many cancer types to date, one is particularly dominant: the APOBEC signature. APOBEC3A (A3A) and APOBEC3B (A3B), two members of the APOBEC3 family, target TpC motifs on single-stranded DNA and are the major sources of the APOBEC mutational signature detected in patients' tumor samples. Our preliminary observation identified a discrepancy between A3A and A3B expression and mutation accumulation in cancer cells. On one hand, A3A is rarely found expressed, yet many of the tumors have a strong A3A-mutational signature. On the other hand, A3B is expressed in most cancer cells, but only a fraction has an A3B-mutational signature. Both A3A and A3B significantly increase mutations in tumors, but these observations have led us to propose that A3A and A3B expression is not a reliable way to assess the APOBEC status of cancer cells, as previously thought. We propose that A3A is tightly regulated at the transcription level and transiently expressed to generate mutations. Our study will explain why A3A is rarely found expressed in cancer but many cancers have a strong A3A mutational signature. In contrast, we propose that A3B is regulated at the protein level to protect the genome against A3B activity. Our goal is to uncover the molecular mechanisms that govern A3A and A3B regulation in cancer cells. Our overall hypothesis is that cells exploit two separate mechanisms to regulate A3A and A3B and to protect their genome against their activity. In addition, we propose that specific signals in cancer lead to the deregulation of these protective mechanisms, causing a surge of mutations. Our Specific Aims are to 1) define signaling pathways in cancer cells that regulate A3A expression and 2) identify protein complexes controlling A3B activity in cancer cells. Our long-term goal is to develop therapeutic strategies to suppress mutations in the genome caused by A3A and A3B, leading to tumor heterogeneity, metastasis, and drug resistance.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT The incretin-mediated potentiation of glucose-stimulated insulin secretion (GSIS) accounts for 50% of postprandial insulin secretion and is essential for physiologic glycemic regulation. Upon nutrient stimulus, the two principal incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted, respectively, from intestinal endocrine K- and L-cells and reach β-cells via the circulation, where they bind their receptors, GIP receptor (GIPR) and GLP-1 receptor (GLP-1R) to amplify GSIS. While circulating incretin hormone levels are not impacted by T2DM, incretin effects on β-cell GSIS are significantly diminished in T2DM patients. However, the precise mechanisms underlying diabetes-associated blunting of incretin signaling in β-cells remain unclear. In T2DM, pharmacologic means to increase circulating GLP-1 levels improves GSIS and glycemic control. In contrast, the GIP-mediated incretin effect is notably absent in T2DM; and pharmacologic means to increase circulating GIP concentrations is not met with any GSIS improvement. The molecular mechanisms underlying diminished response to GLP-1 and absent response to GIP in T2DM remain poorly understood. T2DM is in part linked to a chronic inflammatory state with increased levels of local and circulating inflammatory cytokines that negatively impact insulin action in peripheral tissues and negatively impact the endocrine pancreas. We have therefore examined the role of pro-inflammatory cytokines that are elevated in T2DM on incretin signaling in islet -cells. Conversely, we have also interrogated the anti-inflammatory cholinergic signaling system through nicotinic acetylcholine receptors (nAchR) in modulating incretin signaling. Based on our preliminary studies, we hypothesize that TNFα - signaling through its receptor TNFR1 on β- cells - activates the Ser/Thr G-protein receptor kinase 2 (GKR2) in a non-canonical manner to suppress Gαs- activation and cAMP synthesis by ligand activated GIPR and by GLP-1R. Thus, our findings establish a mechanistic link between inflammatory cytokines in T2DM with diminished incretin signaling in β-cells. Our preliminary studies also indicate that in a murine model of T2DM, stimulating the α7 nicotinic acetylcholine receptor (α7-nAchR) reactivates GIPR signaling in β-cells to potentiate GSIS and improve glycemia. Our mechanistic studies indicate that α7-nAchR signaling phosphorylates (TNFα-activated) GRK2 at serine 670 to counteract its inhibitory effects on GIPR and GLP-1R-mediated cAMP synthesis. We now seek to expand our novel and exciting findings specifically a) to understand the role of TNFR1- GRK2-mediated signaling on in vivo β-cell function, on GIP and GLP-1R action as well as how TNFR1 signaling modulates α-cell gene expression in the context of obesity and T2DM; b) to specifically understand the role of GRK2 in mediating resistance to β-cell incretin action in T2DM; and c) to understand the beneficial role of anti- inflammatory α7-nAchR signaling in β-cells in the context of T2DM. We will use complementary in vivo and in vitro approaches to elucidate these important signaling pathways in β-cells using newly generated unique mouse models as well as in vitro in human islets and β-cells. The outcomes of our studies will yield important insights into how inflammatory cytokines of T2DM impact incretin signaling and β-cell function. Our proposed studies will also provide new insights into the effects of nicotinic acetylcholine signaling in β-cells. Finally, our studies identify potentially new receptor targets with the potential to ameliorate β-cell dysfunction in T2DM. .

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Despite recent advances in cancer metabolism, whether and how nutritional interventions affect tumor development, metastasis and therapeutic response are still poorly understood. Thus, the goal of this study is to elucidate the effect of glutamine supplementation on tumor development and therapeutic responses, and eventually provide molecular evidence that nutritional interventions on cancer patients can inhibit tumor growth and sensitize tumors to treatments. Using metabolomic analysis, we and others have found that, compared to other amino acids, many solid tumor cells are situated in a glutamine poor environment in vivo. Interestingly, we found that glutamine deficiency in melanoma tumors resulted in cancer cell de-differentiation and resistance to treatment due to increased histone methylation levels. This finding further prompted us to test if increases in glutamine levels through dietary supplementation can be detrimental to tumor cells that have been well adapted to a low glutamine environment. Our preliminary data demonstrated that supplementation of glutamine in the diet is sufficient to increase tumoral α-ketoglutarate levels and leads to decreased histone methylation in melanoma patient-derived xenograft (PDX) tumors. Importantly, we found that high glutamine diet significantly hinders tumor growth and decreases expression of melanoma-associated oncogenes compared to control diet. In support with this, accumulating evidence from in vivo experiments demonstrate that glutamine is not an essential nutritional source to support TCA cycle and tumor growth. Thus, we hypothesize that dietary glutamine supplementation inhibits melanoma tumor growth and sensitizes tumor cells to current treatments via epigenetic reprogramming. In this proposal, we will 1) determine the effect of dietary glutamine supplementation on melanoma tumor growth, metabolism and oncogene expression in vivo; 2) determine the molecular mechanisms by which glutamine supplementation inhibits tumor growth; 3) investigate the effect of glutamine supplementation in response to BRAF/MEK inhibitors and immunotherapy for melanoma treatment. Despite many proven clinical benefits of glutamine supplementation to cancer patients, recent in vitro studies showing that tumor cells are avid glutamine consumers led to cautionary usage of dietary glutamine on cancer patients. Completion of the proposed studies will provide insight into glutamine driven epigenetic regulation and its effect on tumor growth. The results of these studies will reveal a novel therapeutic direction for using dietary glutamine supplementation to prevent tumor growth and enhance therapeutic responses without detrimental side effects.

NIH Research Projects · FY 2025 · 2021-06

Save date: 15 Jan 2020, 22:40 ABSTRACT Membrane Protein Folding and Assembly Many human diseases, such as cystic fibrosis, result from misfolding of membrane proteins (MPs) during their synthesis and targeting. It is therefore important to understand the principles and mechanism of MP folding and assembly. A largely unexplored part of the problem is to understand folding in the context of the cellular milieu. Toward that goal, we are studying the targeting, secretion, and insertion of membrane proteins along the so-called SecA post- translational pathway of living Escherichia coli. We have shown that the SecA motor ATPase, a significant drug target, can insert single-span membrane proteins (S-SMPs) across the E. coli inner membrane. This simplified in vivo model system eliminates the many unanswered questions about the folding of multi-span MPs along the signal recognition particle (SRP) pathway, because we gain direct access to the translocon-bilayer partitioning process. We have engineered two different chimeric protein families for probing systematically S- SMP stability using TM segments of the form GGPG-H-GPGG (used in an earlier study to determine a biological hydrophobicity scale using a cell-free eukaryotic system). To determine stabilities, we have developed methods for cleaving TM segments in vivo via native intramembrane proteases. We have discovered that many S-SMPs are stable across the membrane only because their periplasmic & cytoplasmic domains cannot cross the membrane. We have also discovered that translocon-to-membrane transfer energetics are not equal to membrane-to-cytoplasm transfer energetics and that stability depends upon growth temperature. An important aspect of our work is the use of Molecular Dynamics simulations in concert with experiments to understand the dynamics of the SecYEG translocon. Little is known about SecA function at the atomic level despite hundreds of papers on the subject. Calling upon our lab’s expertise in lipid-protein interactions, we have laid the foundation for electron cryomicroscopic (cryo-EM) studies of the structure of SecA bound to lipid nanodiscs. Our ambition is to obtain a complete structural view of the SecA-guided secretion process. SWhite_Abstract_MIRA_2020.docx, 15 January 2020

NIH Research Projects · FY 2026 · 2021-05

Abstract I have been studying neurodegenerative disease for more than 30 years. While a graduate student, I identified the cause of X-linked spinal and bulbar muscular atrophy (SBMA) as the expansion of a CAG repeat in the androgen receptor (AR) gene. As the first disorder shown to be caused by a CAG – polyglutamine (polyQ) repeat tract, this discovery led to the emergence of a new field. My research program began with emphasis on 2 polyQ disorders: SBMA and spinocerebellar ataxia type 7 (SCA7). My early work established transcription dysregulation as a key factor in polyQ disease pathogenesis. I initiated research on Huntington’s disease (HD), and linked mitochondrial dysfunction and metabolic deficits in HD to transcription dysregulation of PGC-1a, a transcription co-activator. In 2016, I documented an interaction between PPARd and huntingtin (htt) protein in striatal-like neurons and in the cerebral cortex of HD mice, and I demonstrated that PPARd repression contributes to HD neurotoxicity. These findings led me to repurpose a selective and potent PPARd agonist, KD3010, as capable of rescuing htt neurotoxicity in HD transgenic mice and in medium spiny neurons from HD patient stem cells. Concomitant with my HD research, I uncovered a central role for skeletal muscle in SBMA by demonstrating that excision of mutant AR transgene from skeletal muscle in BAC conditional transgenic mice prevented the development of neuromuscular SBMA phenotypes, establishing the importance of skeletal muscle – motor neuron (MN) communication at the neuromuscular junction (NMJ) for SBMA lower MN disease. I have thus continuously maintained NINDS R01 funding to support my SBMA research since 2000, and have held NINDS R01 funding to support my HD research since 2010. During this time frame, my research has increasingly focused on identification of targets and pathways for development of therapies. As a R35 recipient, I will continue my research on the cellular and molecular basis of polyQ neurodegeneration, embracing opportunities to extend our findings to more common neurodegenerative diseases, including AD, PD, and ALS. One major focus will be to define the basis of SBMA muscle-driven MN disease through skeletal muscle and NMJ transcriptome analysis of SBMA model mice and stem cell modeling to recapitulate non-cell autonomous SBMA MN degeneration. I also intend to pursue studies of HD and PPARd by determining the normal function of PPARd in CNS and defining how PPARd activation achieves neuroprotection. I will follow up on exciting findings linking PPARd neuroprotection to regulation of neuronal activity-dependent gene expression, and will test if blunting of rapid primary response gene expression can ameliorate HD phenotypes. As PPARd is highly expressed in microglia and represses neuroinflammation, I will study PPARd function in microglia and test if PPARd dysregulation plays a role in neurodegenerative disease. R35 funding would provide me with the flexibility to pursue novel, ambitious studies of polyQ disease, expand my research program to encompass emerging areas of pathogenesis, and maintain a lasting commitment to translational research and therapy development for neurodegeneration.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Antibiotic resistant bacteria pose a global threat to human health and wellbeing. New strategies for combating resistance are urgently needed because current drug development pipelines are not keeping up with the dwindling supply of effective antibiotics. I propose to investigate and manipulate the ecology of antibiotic resistance acquisition and host-to-host transmission. Specifically, I will determine how the physical structure of bacterial communities within the intestine—which is a major reservoir of antibiotic resistant bacteria—affects the evolution of resistance traits and transmission of resistant cells. A motivation for this research direction is that antibiotic resistance fundamentally dependends on the spatial and temporal organization of host–microbe systems. For example, the sharing of resistance genes between bacteria through lateral gene transfer often requires cells to be in close proximity to one another. In addition, the transmission of resistant bacteria between hosts requires that they are physically displaced and expelled into the environment. Thus, altering the spatiotemporal organization of gut bacterial communities could be used to prevent and contain resistant bacteria before they become agents of infection. However, dissecting the spatially and temporally complex mechanisms governing antibiotic resistance is a significant challenge using current approaches. My solution to overcome this limitation is to combine synthetic biology, genetically engineered bacterial communities, and live imaging to track and control bacterial behavior inside the intestines of living animals. I will employ larval zebrafish as a vertebrate host model because they enable studies of host–microbe systems across scales of complexity, space, and time that are difficult to perform in mice or humans. Using this experimental approach, I previously discovered that intestinal flow, bacterial swimming motility, and sublethal antibiotics represent host, bacterial, and environmental factors, respectively, that can modulate the spatiotemporal organization and physiological landscape of gut bacteria. I will harness these factors and my experimental approach to address the following three hypotheses. First, I will test the hypothesis that the spatiotemporal organization of gut bacteria controls the acquisition and persistence of resistance traits within the intestine. Second, I will test the hypothesis that the spatiotemporal organization of gut bacteria regulates the transmission of antibiotic resistant cells between hosts. And third, I will test the hypothesis that bacteria coordinate both the acquisition of resistance traits and host-to-host transmission through specific genetic pathways. My proposed research has the potential to inspire ecology-based strategies for curtailing antibiotic resistance through the therapeutic manipulation of the intestinal microbiome’s physical structure. Such ecology-informed and antibiotic-free strategies would preserve the potency of current antibiotics for when they are needed most and avoid the unintended side effects of antibiotics on beneficial resident bacteria.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract: Understanding the aggregation of the β-amyloid peptide (Aβ) to form toxic oligomers is fundamental to understanding the molecular basis of Alzheimer’s disease (AD). Despite decades of research, the structures of Aβ oligomers remain a mystery, constituting a significant gap in understanding AD. This proposal seeks to address this knowledge gap through the structural, biophysical, and biological profiling of a diverse group of Aβ oligomer models and correlation of these models with biogenic Aβ oligomers. My laboratory has developed an approach for create structurally defined Aβ oligomer models composed of peptide fragments from Aβ constrained into a β-hairpin. The X-ray crystallographic structures of these Aβ β- hairpin peptides reveal the structures of oligomers that the peptides can form and key intermolecular contacts that the peptides make in the oligomeric state. These contacts reveal sites that can be crosslinked to create covalently stabilized Aβ oligomer models that mimic the crystallographic oligomers. Studying the crosslinked oligomers then allows detailed correlation between oligomer structure and biophysical and biological properties. We will characterize how our Aβ oligomer models interact with and affect neurons, microglia, and astrocytes, to provide detailed insights into how our Aβ oligomer models impact different brain cell types and thus help shed light on the relationship between Aβ oligomer structure and cellular events that occur in AD. We will use fluorescence microscopy and fluorescence-assisted cell sorting (FACS) to visualize and quantify the interactions and uptake of our Aβ oligomer models with neurons, microglia, and astrocytes. We will evaluate downstream biochemical and cellular effects elicited by Aβ oligomer models in neurons, microglia, and astrocytes and evaluate how treatment affects apoptosis, necrosis, calcium homeostasis, endoplasmic reticulum stress, oxidative stress, neurite length, tau phosphorylation and aggregation, and proinflammatory responses in microglia and astrocytes. To elucidate the relationship between the structures of our Aβ oligomer models and biogenic Aβ oligomers, we will generate polyclonal antibodies against each Aβ oligomer model and then examine the immunoreactivity of these antibodies with brain protein extract and brain slices from 5XFAD mice. We will discover new Aβ oligomer models, by creating new Aβ β-hairpin peptides that contain more of the Aβ peptide sequence and alternate β-strand alignments. We will create new crosslinked Aβ oligomer models by identifying key contacts in existing and newly discovered Aβ oligomer models and then engineering in disulfide bonds to stabilize the oligomers. To characterize the structures and oligomerization properties of the new Aβ oligomer models that we generate, we will use X-ray crystallography and a variety of other biophysical experiments, including CD spectroscopy, SDS-PAGE, size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), NMR spectroscopy, and Förster resonance energy transfer (FRET) studies. The biological and immunological properties of these new Aβ oligomer models will be studied as described above.

NIH Research Projects · FY 2025 · 2021-05

Recent AD GWAS studies have uncovered a number of risk single nucleotide polymorphisms (SNPs) within genes predominantly expressed or upregulated in microglia in association with pathology suggesting that altered microglia function plays a critical role in disease etiology. Each of these polymorphisms can either confer increased or decreased risk and it will be important to understand how inheritance of each of these polymorphisms impact microglia function to modify disease risk. One identified AD GWAS risk SNP rs3865444 is near the CD33 gene, confers protection from AD, and is found in linkage disequilibrium with rs12459419 SNP, which leads to skipping of exon 2 that encodes the sialic acid-binding domain. CD33 is a member of the Sialic acid-binding immunoglobulin-like lectin (Siglecs) family of receptors in which sialic acid binding transmit signals from the extracellular microglial environment leading to downstream microglia signaling and a subsequent microglia cellular response/change. Because CD33 lacks a true murine ortholog, the study of CD33 requires human cells. The ability to generate microglia-like cells (iMGLs) from iPSC and development of microglia functional assays in my lab will enable investigation of the role of exon 2 skipping on microglia function. Patient and CD33 isogenic induced pluripotent stem cells (iPSCs) will be used to derive iMGLs to interrogate microglia function in the context of differing CD33 genotype. Using liposomal nanoparticles that bring together ligands of two receptors, we will examine the role of CD33 as a co-receptor in modulating Dap12 and FcRg signaling. In addition, we will investigate the functional significance of an interferon microglia signature uncovered by whole transcriptome analysis of patient-derived microglia-like cells. Lastly, a novel AD mouse model facilitating xenotransplantation will be employed to examine the CD33-D2 interferon signature in vivo and whether it improves behavior, reduces AD pathology, and therefore can be targeted as a novel AD therapeutic.

NIH Research Projects · FY 2025 · 2021-05

Retinal pigment epithelium (RPE) and photoreceptors depend on each other for normal function and survival. Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) lead to irreversible loss of vision in millions worldwide, due to RPE dysfunction and photoreceptor degeneration. Our long-term goal is to reverse vision loss by a combined transplant of neural retina sheets containing photoreceptor progenitors, plus a polarized functional RPE monolayer to support the photoreceptors. Human embryonic stem cells (hESCs) differentiated into RPE and retina organoids (RO) - which would substitute for neural retina sheets - can provide an unlimited supply of clinically applicable retinal donor tissue. Retinal sheet transplants - dissected from hESC-ROs – develop photoreceptors, improve visual acuity, responses to light, and integrate with the host retina. However, RO’s are generally devoid of a polarized RPE layer. Therefore, cografting of hESC–derived neural retina and a healthy RPE monolayer is needed for treatment of advanced disease conditions; but have not been performed on a large scale. To advance cell replacement therapy, the mechanism of action of the transplant should be well understood. Although synaptic connectivity of retinal transplants has been ascribed a role for visual improvement, details regarding the circuitry between transplant and host retina remain unknown. To obtain better insight, retinal degenerate (RD) rats that express a specific label to identify defined host retinal neuron populations need to be used. Thus, the goal of this project is to improve the efficiency of transplants and analyze the functional recovery in relationship to neural connectivity. The proposed studies are aimed (1): To determine the efficacy of cografting “complete transplants”. We hypothesize that “complete” transplants consisting of hESC-neural retina together with RPE will improve visual recovery - compared to transplanting hESC-neural retina or RPE alone. Royal College of Surgeons (RCS) rats, a model of a hereditary (chronic) RPE defect, at an advanced stage of RD will receive either “cografts”, or transplants of ROs or RPE alone, and analyzed long-term (6-8 months) after grafting. The successful outcome of this Aim should allow us to quantitatively evaluate the improved outcome of cografting. (2): To determine mechanisms of functional recovery by detailing transplant-host connectivity. We hypothesize that visual improvement will correlate with integration and synaptic connectivity of neural retinal sheet transplants in the host retina. This will be tested by labels for both the donor and specific retinal neurons of the host retina. In collaboration with Envigo, we will produce immunodeficient rhodopsin mutant (Rho S334ter-3) RD recipients expressing Td-Tomato either universally (cre/lox inducible) or in CaMKII retinal neurons (using CRISPR-Cas9 technology), which will allow us to examine in detail the connectivity of RO transplants with specific retinal cell types in the host retina, and to correlate visual functional improvement with transplant-host connectivity. This project directly addresses the NEI Audacious Goals Initiative to regenerate the eye and visual system.

NIH Research Projects · FY 2025 · 2021-05

Project Summary This proposal seeks to engineer an allogeneic zygapophyseal (facet joint) articular resurfacing (AZAR) system by following a rational experimental approach in three specific aims: the AZAR system will consist of a cartilage component (chondro-portion) robustly integrated with a bone-like scaffold component (osteo-portion); the replacement will be secured in situ in the recipient bone bed of the facet joint. In Aim 1, three levels of human and minipig facet joints from both sexes will be fully characterized to define design criteria for the AZAR system. The AZAR system will be designed and fabricated through three phases in Aim 2. First, cytochalasin-D and hyaluronidase will be applied to minipig passaged chondrocytes to engineer the chondro-portion of the implant with compressive properties mimetic to native tissue values. In the same phase, methods for using lysyl oxidase like protein-2 (LOXL2) and tensile stimulation (CoTenS) will be identified to improve the tensile properties of the chondro-portion to native tissue levels. In Aim 2, Phase II, the pore size of the osteo-portion (i.e., the bone portion) directly beneath the engineered cartilage will be fine-tuned for optimum cartilage integration. In Aim 3, minimally invasive surgical methods will be developed for implanting a total facet replacement in the minipig, analogous to the ones currently employed in human patients. Finally, the AZAR system will be implanted into both male and female minipigs to examine its efficacy prior to moving to bipedal models in future studies. Successful completion of this proposal will allow, for the first time, resurfacing of an entire articular surface in a diarthrodial joint using a tissue engineered construct. It will also shed light on as-of-yet unexplored structure- function relationships in the poorly studied facet joint. Last, but not least, it will contribute toward new therapies for facet-related ailments.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY The human gene DNMT3A encodes one of the three enzymes that carry out DNA methylation in humans. Clonal expansion of blood cells with acquired mutations in DNMT3A is common in older adults, occurring in 5-10 % of healthy individuals aged 60 or above. Carriers of DNMT3A mutations have an approximately tenfold increased risk of developing hematologic cancers and are twice as likely to develop coronary heart disease. Given the rapidly aging population in the United States and worldwide, understanding the mechanistic basis of the association between acquired DNMT3A mutations in blood cells and increased susceptibility to cancer and cardiovascular disease is tremendously important for public health. Recent studies found evidence of increased inflammation mediated by myeloid cells when the ortholog of DNMT3A was perturbed in animal models. However, the molecular mechanisms underlying this phenomenon and whether DNMT3A mutations affect the inflammatory response of human myeloid cells remain poorly understood. To address this gap in knowledge, we established an experimental system based on myeloid cells differentiated from human pluripotent stem cells. Using this system, we found that human macrophages with DNMT3A mutations displayed altered inflammatory response compared to wild-type macrophages, characterized by augmented expression of IL-6, a potent proinflammatory cytokine. The IL6 promoter was one of the most significantly hypomethylated loci in DNMT3A-mutated macrophages, suggesting a direct mechanistic link between DNA methylation and inflammatory response in our model. In this application, we propose to characterize the molecular signature of the inflammatory response associated with DNMT3A mutations using genetically defined human macrophages and neutrophils and to dissect the epigenetic mechanisms underlying DNMT3A-mediated gene expression regulation. In addition, we will examine the impact of harboring clonally expanded blood cells with DNMT3A mutations on the inflammatory response of primary myeloid cells using a novel single-cell transcriptomic technique. We are in an ideal position to pursue this project given the availability of human pluripotent stem cell-based human myeloid cell models that we have developed and validated, our access to a large biobank representing extremely diverse populations, and the assembly of a strong scientific team consisting of investigators with complementary expertise. Findings from the proposed study will provide critical new insights into the consequence of acquiring DNMT3A mutations on inflammation, and help us develop novel strategies to prevent and treat pathologic conditions related to DNMT3A mutations.

NIH Research Projects · FY 2025 · 2021-04

We propose a circuit-level principal underlying how brains acquire 'episodic' memories and reprocess them into compact, efficient 'schemas': The attributes or 'contents' of experience are represented primarily in the deeper layers of neocortex (NC), whereas the superficial layers are dedicated to encoding the contexts in which the attributes occur. Synaptic associations between superficial context codes and deep attribute codes permit contexts to evoke appropriate attribute output hence enabling memory recall and predictive behavior. The hippocampus (HC) is essential for acquisition of memories and for their reprocessing into efficient, schematic representations of the world. NC exhibits dense local but sparse long-range connectivity, which severely limits its ability to make rapid, long-range associations. HC likely solves this dilemma by merging the totality of the brain's current internal state (i.e., sensory input and internal variables such as hunger, fear, current goals) into a unique, 'index' code that is projected to NC, and associated with its current, distributed, attribute representation. Retrieval of an index code evokes the corresponding attributes. Such HC-orchestrated retrieval may enable the gradual rewiring of NC circuitry in a manner that captures the overall statistics of experience, much the same way as deep, artificial, neural networks learn incrementally by small connection weight adjustments directed by the overall statistics of the input. Our hypothesis on the laminar division of labor in this process is based on the facts that HC output is directed primarily to upper layers of NC, which implements a 'spatial' coding scheme that is lost after HC lesions; and that the deeper layers of NC frequently exhibit more robust responsiveness to and discrimination of sensory inputs than the superficial ones. We propose to record cellular level, neural ensemble activity simultaneously from deep and superficial layers in primary and association cortex, using high-density, electrophysiological recording. First, we attempt to establish the 'attribute vs index' principal by showing that deep cells shift their firing locations with shifts in the relevant sensory attributes, whereas superficial cells do not. Next we test the hypothesis that, as NC accumulates large amounts of diverse experience, attribute representations in deeper layers becomes sparser and more categorically organized, whereas superficial layer coding is relatively unchanged. To accomplish this, we employ a recent chemogenetic advance that enables us to acquire large amounts of resting-state cellular data, in which we expect the predicted changes will be most easily observed. We also explore the statistics of excitatory-inhibitory cell functional connectivity that may underlie such coding statistics changes. The expected advances in understanding cortical memory and schema encoding circuits will ultimately improve clinical assessment of, and intervention in memory and cognitive disorders.