Rutgers Biomedical And Health Sciences

NIH Research Projects · FY 2026 · 2021-09

PROJECT SUMMARY/ABSTRACT In order to understand neurological diseases, it is essential to identify the affected neuronal cell types, create model systems that accurately recapitulate normal function and disease phenotypes, and develop tools that allow cellular manipulations. Motor neurons in the spinal cord control body movement by communicating central motor commands with muscle targets. All spinal motor neurons are born and specified during embryonic development, but their molecular identities and electrophysiological properties evolve for weeks in mice and months in humans, until motor circuits and behavior become fully mature in post-natal life. In diseases like Amyotrophic Lateral Sclerosis (ALS), sarcopenia, or spinal cord injury, the degeneration of specific subsets of mature, adult motor neurons can lead to loss of muscle control, paralysis, and death. Although several studies have mapped the mechanisms of motor neuron diversification in the embryonic spinal cord, our understanding of motor neuron diversity in the adult spinal cord is in its infancy. This hinders the study of adult motor neuron diseases as the affected motor neuron subtypes are not thoroughly defined. Furthermore, in vitro models that faithfully recapitulate adult motor neuron identity do not exist, and adequate tools to access specific motor neuron subtypes in vivo are lacking. This research plan aims to map the trajectory of post-mitotic motor neurons from embryo to adulthood and use this data to both create viral tools that provide genetic access to specific subtypes of motor neurons in vivo, and develop methods for generating adult-like motor neurons in vitro. This will be done by first performing single cell transcriptome and chromatin profiling in mouse spinal motor neurons at various embryonic to adult ages. The temporal chromatin profiles will be used to develop AAV tools that provide genetic access to specific motor neuron subtypes at all ages, and to computationally identify candidate regulators of subtype- and adult-specific identity. The identified regulators will then be used to mature the age of mouse stem cell derived motor neurons in vitro. Finally, single cell transcriptomic and chromatin accessibility profiles of adult human motor neurons will be generated and a combination of mouse and human-specific regulators will be used to program the age of iPSC-derived motor neurons. This thorough approach will define the molecular features of motor neuron subtypes that contribute to their differential susceptibility in disease, and establish tools necessary for dissecting circuits, disease modeling, and the delivery of potential therapeutics. The training phase of the award will be conducted in the laboratory of Dr. Hynek Wichterle at Columbia University, and under the co- mentorship of Dr. David Gifford and Dr. Paola Arlotta. My career development plan describes a detailed timeline for acquiring all the technical and professional skills necessary for the successful transition into a career as an independent researcher. The completion of the proposed research plan will facilitate future research in my lab aimed at understanding the temporal dynamics of neuronal identity, circuits, and disease in motor neurons and other nervous system cells.

NIH Research Projects · FY 2025 · 2021-09

The Severe Acute Respiratory Syndrome Coronavirus 2 (CoV-2) belongs to a family of pathogenic enveloped RNA viruses of the family Coronaviridae. The ongoing pandemic has caused a public health emergency worldwide, accompanied by dire health and economic consequences. There is evidence suggesting that CoV-2 may have relatively higher infection rates compared to previous epidemic strains of SARS and higher affinity to the receptor ACE2 than SARS-1. In addition, new CoV-2 variants have appeared recently with mutations that correlate with higher infection rates and the ability to escape specific immunity, causing major concerns. A major gap in knowledge remains as to how CoV-2 may have acquired the ability to spread more efficiently, and how new mutations may affect virus infectivity. The overarching goal of this proposal is to better understand the molecular mechanisms that regulate CoV-2 cell entry and replication, and how the appearance of new variants could lead to immune escape. We will focus on the role of the host Ubiquitin (Ub) system in promoting CoV-2 infection. This information could help predict appearance of more transmissible variants of coronaviruses, and to develop antiviral approaches by targeting specific steps of the ubiquitination process. Our data recently published in Nature, show that the envelope protein of flaviviruses is K63-linked polyubiquitinated, which enhances virus attachment to host cell receptors. Therefore, we asked whether a similar mechanism applies to SARS-CoV-2. Our preliminary data indicate that CoV-2 structural proteins are ubiquitinated on multiple lysine residue, some of which are not conserved in the original epidemic CoV strain. In addition, new variants of CoV-2 have appeared with mutations on these ubiquitination sites. Our data also suggest that ubiquitination of Spike (S) protein may play a role in stabilizing the CoV-2 S-ACE2 interaction, potentially leading to enhanced entry and pathogenesis. It is currently unknown whether any member of the Coronaviridae family, including SARS-CoV-2, utilize ubiquitination of viral structural proteins as a mechanism of virus attachment and entry. We have also identified E3-Ub ligases of the Tripartite Motif (TRIM) family of proteins, which ubiquitinates viral structural proteins. Our general hypothesis is that the variants of CoV-2 that have gained specific lysine residues provide new Ub acceptor sites on structural proteins, which can enhance virus replication and immune escape. By using in vitro biochemical approaches, novel recombinant mutant viruses, and in vivo models, we will assess how ubiquitination of structural CoV-2 proteins contribute to CoV-2 infectivity. In Aim 1 we will determine the mechanistic role of ubiquitination of the CoV-2 S protein in virus replication and antibody escape, and in Aim 2 we will determine the mechanistic role of ubiquitination of the CoV-2 Membrane protein in virus replication and IFN antagonism. The outcome of these studies may help explain how new more infectious viruses may appear by gaining ubiquitination sites and will provide the basis for the development of an antiviral approach that could be applied to a broad range of enveloped viruses.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The ovary and its functional unit, the follicles, are critical for the secretion of sex steroids and peptide hormones as well as the maturation and release of oocytes for ovulation and fertilization. Increasing evidence has revealed that a wide spectrum of environmental chemicals and pharmaceuticals cause ovarian toxicity (ovotoxicity) and heighten the risk of premature ovarian failure, hormonal imbalance, and infertility in both reproductive aged women and prepubertal girls. With over 85,000 chemicals used in consumer products as well as emerging contaminants such as harmful algal bloom (HAB) toxins, there are few means to screen for potential toxicity to the ovaries. We have previously developed an ovary-on-a-chip (OvaryChip) that maintains the 3D architecture of follicles and produces human menstrual cycle-like follicle development, hormone secretion, oocyte maturation, ovulation, and luteinization. Yet, one remaining challenge of this innovative model is the critical need to accelerate throughput. The central hypothesis of this research is that in vitro microfluidic follicular cultures can be used to identify novel mechanisms of ovarian toxicity for environmental toxicants. In Aim 1, we will integrate follicle vitrification, 3D in vitro follicle growth, and a new OvaryChip with high-content culture and imaging to develop a high-throughput ovotoxicity testing of growing follicles. In Aim 2, we will develop a new OvaryChip for ovotoxicity testing of primordial follicles by using triple transgenic mouse ovarian explants. In Aim 3, we will develop a liver-ovary-on-a-chip to incorporate liver metabolism into ovotoxicity testing. In these studies, we will assess follicle and oocyte reproductive outcomes at morphological, functional, and molecular levels for 1) chemicals with known ovotoxicities (validation studies) and 2) novel HAB toxins alone and in mixtures. These studies are critically significant because the OvaryChip models will (1) translate a bench assay for a single compound into a robust high-throughput ovotoxicity screening platform; (2) reveal novel insight into chemical- induced ovotoxicity (stage- and metabolite-dependent mechanisms); (3) minimize the cost and use of whole animals; and (4) prioritize chemicals of high ovotoxicity concern, including HAB toxins, for further risk assessment.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Placental abruption results in hemorrhage, ischemia, and fetal hypoxia, placing a tremendous health burden on both the mother and the newborn. Efforts to understand the etiology of this devastating obstetrical complication have been disap- pointing. This project will delineate environmentally-associated pathways to abruption and determine the impact of pol- lutant triggers that are implicated in acute versus chronic placental abruption. Given that one-fourth of all abruption cases have an acute etiology and 15% of abruptions may recur in future pregnancies, the role of environmental triggers is a critically important, yet unexplored, opportunity to understand the pathophysiology of this enigmatic obstetrical compli- cation. The project will capitalize on high resolution exposure and health outcome data as it aims to develop a birth linkage database that will include hospital discharges linked to both stillbirths and live births-infant deaths to resident women in California, Florida, Massachusetts, Michigan, and South Carolina (estimated 16 million births, including 155,000 abruption cases) between 2000–2016. For each pregnancy we will assign average daily ambient exposure to fine particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5), its constituents (elemental carbon and organic carbon, sulfate, nitrate, and ammonium), as well as gaseous pollutants (nitrogen dioxide and ozone), using spatiotemporally resolved models that predict exposure for each residential location. We will also assign every residence with average daily temperature, humid- ity, dew point, heat waves, and atmospheric air pressure. The project will focus on disentangling the relative contributions of ambient air pollution and weather-related conditions on acute abruption through a bi-directional, time-stratified case- crossover design, and those of abruptions with chronic underpinnings using a cohort design. We will apply distributed lag linear and non-linear models to identify critical windows of exposure, Bayesian Kernel Machine Regression to characterize associations based on multi-pollutant exposures, and causal interaction-mediation decomposition analyses through ischemic placental disease (preeclampsia, fetal growth disturbances). We will consider individual– and neighborhood–level con- founders derived from residential census tracts. All associations will be corrected for simultaneous exposure and outcome misclassification, as well as for exposure measurement error owing to maternal residential mobility through a regression calibration approach. The ubiquitous nature of air pollution and weather exposures and their potential impact on adverse perinatal outcomes, as well as the preliminary data supporting the associations, presents unprecedented opportunities to address implications of the adverse impact of air pollution and weather-related exposures on placental abruption and related obstetrical complications. This project aligns with 2 major critical areas of research–Co-exposures, and Data Sci- ence and Big Data–outlined in the 2018-2023 strategic goals of NIH-National Institute of Environmental Health Sciences.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Genetic discovery for schizophrenia and bipolar disorder lags behind that in other areas of medicine, where the identification of mutations responsible for familial forms of major disorders has yielded extraordinary biological insights. However, recent successes in gene identification from both rare and common variant analyses indicate what the field needs to do to catch up: expand the size, diversity and scope of genetic studies. Indeed, NIMH recognized this need, issuing PAR-20-027, “Genetic Architecture of Mental Disorders in Ancestrally Diverse Populations.” In response to this call, we will create the Populations Underrepresented in Mental illness Association Studies (PUMAS) Project, an international collaboration of investigators from the US, South America and Africa with the strongest track record of large-scale psychiatric genetic research in Latino and African populations, along with several of the field’s leaders in genetic data generation and analysis. PUMAS will be well powered to discover new genes for schizophrenia and bipolar; it will dramatically increase the diversity of genetic discovery efforts, an important step towards reducing health disparities; and it will expand the scope of psychiatric genomics by generating low-pass whole genome sequencing for 120,000 samples (which we will analyze together with 22,500 samples already sequenced by our team). Through these efforts we will also discover similarities and differences in genetic architecture of schizophrenia and bipolar across diverse ancestries and environments. The Aims of the PUMAS project are to: 1) Build the PUMAS sample bank of schizophrenia cases, bipolar cases and controls from Africa and from admixed populations in the Americas, achieving a total sample of 183,500 (88,600 cases and 94,900 matched population controls) by recruiting 17,000 new cases and 16,500 controls. 2) Generate low-pass whole genome sequencing (WGS) data and variant calls on 40,000 cases of schizophrenia, 40,000 cases of bipolar disorder and 40,000 matched controls from African, Native American and admixed ancestries b) perform extensive sample and variant quality control. 3) a) Systematically analyze the combined dataset to power discovery of the genetic basis of schizophrenia and bipolar across diverse ancestries and down the allele frequency spectrum; and b) through portals and browsers, share the data and results of the genetic studies with the world. The PUMAS 120,000 sample WGS dataset, together with data for 22,500 previously sequenced admixed (AA+EA) samples, provides sufficient statistical power for genetic discovery for SZ, BP, and combined across diverse ancestries. Our study will identify new genes and loci, increase the precision of fine-mapping of known loci, and form the foundational knowledge base for polygenic risk scores (PRS) of global value.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Schizophrenia, and other disorders of the psychosis spectrum (PS), most commonly emerge throughout development and are thought to be caused by disruptions to normative brain maturation occurring during this time. Critically, deviations from normative neurodevelopment are thought to precede the emergence of clinically significant PS symptoms by several years, highlighting the profound impact that their discovery would have for psychiatry research; if we can successfully identify the antecedent brain abnormalities, then we may be able to intervene early and reduce the risk of individuals developing schizophrenia. Uncovering these antecedent brain abnormalities requires predictive models built upon recent advances in network neuroscience and machine learning; moreover, such models must be coupled with large samples of longitudinal neuroimaging and clinical data to uncover truly prognostic biomarkers. Finally, sex differences are found in both the PS symptoms and neurodevelopment. Thus, studies that provide a precise understanding of how sex interacts with PS symptoms and abnormal neurodevelopment are needed. The purpose of the current study is to use advanced tools from network neuroscience and machine learning coupled with multi-modal neuroimaging to uncover biomarkers that can predict the emergence of PS symptoms throughout development. To achieve this goal, we will draw on multiple largescale cross-sectional and longitudinal neurodevelopmental datasets, including the Philadelphia Neurodevelopmental Cohort, the Healthy Brain Network, and the Adolescent Brain Cognitive Development study, to study brain structure and connectivity. We use Network Control Theory (NCT) to study connectivity. NCT treats the brain as a dynamical system allowing us to probe a region's capacity to control changes in brain states via white matter pathways. Compared to graph theory, NCT is a contemporary approach that posits an explicit model of how the brain's structure informs and constrains its function, enabling mechanistic insight into the dysconnectivity associated with the PS. We will quantify developmental abnormalities in NCT metrics using a nascent machine learning technique known as normative modeling. A normative model builds a growth chart of brain development that incorporates the expected variation in the relationship between age and the brain into its predictions. Then, deviations from these growth charts can be understood in terms of what is and what is not expected in a normative population. Here, we will build cross-sectional (Aim 1) and longitudinal (Aim 2) normative models of NCT metrics and use multivariate deviations to predict PS symptoms out-of-sample. Finally, Aim 3 will investigate the extent to which deviations from normative neurodevelopment mediate the relationship between sex and PS symptoms. The goal of this Pathway to Independence award is to build on my strong background in psychiatry, multimodal neuroimaging, network neuroscience, and machine learning by expanding my expertise to developmental psychopathology, NCT, and longitudinal neuroimaging data.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Cognitive effort is subjectively costly. It can cause people to discount valuable goals and avoid thoughtful planning and careful deliberation. Effort avoidance can thus be problematic, especially in disorders like depression and schizophrenia where excessive cost sensitivity undermines cognitive motivation. To address cost sensitivity, we first need to understand what the brain treats as costly. Traditional indices, like lateral frontal functional magnetic resonance imaging signals, and parietal alpha desynchronization in electroencephalography data, track cognitive load to a point, but often plateau or decline, even when subjective effort continues to rise. This project will examine a promising candidate mechanism underlying the experience of subjective cognitive effort: divergence from criticality. Criticality characterizes cortical dynamics at rest, and prior studies have shown that brains become increasingly sub-critical under the very conditions known to increase subjective effort: increasing working memory load, fatigue, sleep deprivation, novelty, and cognitive aging. In the Applicant’s K99 training phase, they found that brain dynamics diverge from a critical point, the harder that tasks become. Most importantly, however, people whos’ brains diverge the most from that critical point experience tasks as more subjectively effortful than people whos’ brains more closely approximate criticality. It is hypothesized that proximity to a critical point depends on the balance of cortical excitatory versus inhibitory signaling. Consistent with this hypothesis, the Applicant also found that artificially perturbing cortical excitability using transcranial magnetic stimulation also pushes brains farther away from a critical point and increases subjective effort on a subsequent cognitive task. The direction of the effect of transcranial magnetic stimulation was surprising, however, opening doors to new hypotheses about the effects of transcranial magnetic stimulation on brain function from the perspective of critical dynamics. This project will test the hypothesis that the cortical excitatory inhibitory balance regulates how closely to a critical state the brain operates, and thereby how sensitive people are to effort costs. In so doing, the project will also re-examine the effects of common transcranial magnetic stimulation protocols on cortical excitability with respect to critical, sub-critical, and super-critical dynamics. Thus, Aim 1 will combine electroencephalography and transcranial magnetic stimulation to test hypothesized mechanisms which may underlie cognitive effort, working memory, and cognitive motivation and also re-assess longstanding assumptions about the effects of transcranial magnetic stimulation on cortical plasticity.

NIH Research Projects · FY 2025 · 2021-09

Lymphedema is an incurable condition characterized by lymphatic obstruction, tissue swelling, immune dysfunction, and fibrosis after lymphatic injury. Lymphedema affects 5 million Americans and is associated with poor quality of life due to extremity disability, disfigurement, and risk for recurrent limb-threatening infection. In the US, lymphedema is most commonly a consequence of lymph node dissection for the treatment of solid tumors such as breast or pelvic cancer. Despite the fact that lymphedema is common and morbid, there are currently no effective drug treatments. Using preclinical rodent models of lymphedema, we have shown that 9-cis retinoic acid (RA) significantly accelerates lymphatic regeneration following injury, restores functional lymphatic drainage, and prevents development of lymphedema. Our overarching hypothesis is that RA-mediated lymphangiogenesis is a promising therapy for secondary lymphedema. The objective of this proposal, which is the first logical step towards implementing this treatment clinically, is to increase our understanding of the mechanisms by which RAs regulate lymphangiogenesis and develop a translational framework for the use of these compounds. The specific aims of this proposal include Aim 1: Determine how RA selectively induces lymphangiogenesis; Aim 2: Elucidate the roles of FGFR and VEGFR signaling in RA-mediated lymphangiogenesis; and Aim 3: Use early biomarkers of lymphatic insufficiency to develop a predictive model that can guide initiation of RA therapy. Based on the current lack of effective therapy, it is clear that there is a need to develop an etiology-focused treatment for post-surgical lymphedema. The proposed studies will address important mechanistic questions regarding RA mediated lymphangiogenesis and also develop an early biomarker based predictive model that will guide treatment windows for RA therapy. The proposed work will significantly improve our understanding of RA-mediated lymphangiogenesis as well as support clinical translation of a RA as a preventative treatment regimen for post-surgical lymphedema.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The current opioid epidemic is a pressing public health crisis. A key precipitating factor of reuse and relapse among people with opioid use disorders (OUD) is craving, or the intense, specific desire for the drug. While craving has been extensively studied, and is known to predict drug use, we still lack an explanatory and algorithmically-precise model that can directly link craving neurobiology to its observed consequences: the decision to pursue drugs over other valuable alternatives. Given that typical treatments for OUD do not adequately address craving and fail to prevent reuse in many patients, clarifying the precise, decision-relevant, mechanism of craving may critically inform more targeted ways to treat craving and improve clinical outcome. To address these important questions, we developed an experimental paradigm to study craving based on methods widely used in decision neuroscience to assess value-based decision-making. Decision neuroscience (or neuroeconomics) integrates concepts and methods from psychology, economics, and neuroscience to understand the neural architecture for decision-making, and has been increasingly applied in mechanistic studies of psychiatric disorders including addiction. Our paradigm constitutes a novel application of this framework by quantifying a subject’s in-the-moment (i.e., state-dependent) decision process during craving15. In pilot behavioral studies in healthy and opioid addicted subjects, we find that this paradigm captures 1) how value—the key determinant of the decision to pursue a particular option versus another—changes under craving, and 2) the selectivity of this effect to the object of craving. It also 3) provides an algorithmically-specific process (a mathematical description) of this change that can be used to tie behavior to its neural substrate. In the present study we aim to elucidate this neural substrate by identifying the specific neural computations through which craving modulates the value of drug and nondrug alternatives and thereby drug use decisions in human OUD. We propose to identify the neural substrate of opioid craving in N=89 OUD patients who will complete our paradigm during fMRI in a within-subjects cross-over design following a brief craving induction or a control manipulation16. Because decision circuits encode value in a reward-identity specific manner, our design will enable us to isolate the computations associated with drug-related value from those of nondrug value. Our study will for the first time determine whether and how experimentally-induced craving dynamically shifts such “identity-specific” neural encoding of drug-related value (Aim 1), and the parts of a putative ‘craving circuit’ involved in this shift (Aim 2). To test whether this mechanism is unique and reward-identity specific, we will also measure brain activity associated with experimentally-induced food craving and specific food-value in the same patients and N=89 healthy controls (Aim 3). If successful, this integrative approach will uncover precise targets for selectively mitigating craving-induced increases in drug-value that promote opioid reuse, laying the groundwork for precision interventions to treat craving in treatment unresponsive individuals.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Compelling evidence suggests that mitochondrial dysfunction is an early feature in susceptible neurons in the brains of patients with Alzheimer’s disease (AD) and plays a critical role in pathogenesis, yet the underlying molecular mechanisms remain incompletely understood. Phosphodiesterases (PDEs) are a superfamily of enzymes responsible for the hydrolysis of cAMP and cGMP, second messengers that regulate important cellular functions. Interestingly, recent studies demonstrated that cAMP/cGMP-PKA/PKG signaling is involved in the regulation of mitochondrial dynamics and expression/assembly of key enzymes in the electron transport chain (ETC) and mitochondrial respiration. Among the many PDEs, PDE2A is the most highly expressed PDE in the hippocampus and frontal/temporal cortex, brain regions vulnerable to AD. Our preliminary studies found increased PDE2A expression in the brains of AD patients and APP/PS1 mice (an AD model), accompanied by decreased cAMP and cGMP in both cytosol and mitochondria matrix, implicating the potential involvement of an aberrant PDE2A-cAMP/cGMP signaling in the pathogenesis of AD. Multiple studies, including ours, demonstrated cognitive enhancing effect of PDE2A inhibitors, although the underlying mechanism remains elusive. In this regard, our preliminary studies revealed that PDE2A overexpression impaired mitochondrial function accompanied by extensive mitochondrial fragmentation. Importantly, Aβ-induced mitochondrial fragmentation and respiratory deficits could be rescued by a PDE2A inhibitor, suggesting mitochondrial dynamics and function could be mechanism of action for PDE2A to influence cognition. Based on these studies, we hypothesized that aberrant PDE2A signaling caused mitochondrial dysfunction which adversely impacted neuronal/synaptic function and caused pathological/cognitive deficits in AD. Novel animal models with PDE2A conditional knockout in the forebrain will be crossed with different AD transgenic mouse models and carefully characterized. The role of PDE2A2, the PDE2A isoform uniquely localized to mitochondria, in brain function and behavior in AD mouse models will also be determined. Finally, based on the literature and our preliminary study, we propose to explore the mechanism underlying the effects of aberrant PDE2A expression on mitochondrial dysfunction with a focus on mitochondrial dynamics and the expression/assembly of mitochondrial ETC complexes. Our proposed studies will provide mechanistic insights into molecular mechanisms underlying mitochondrial dysfunction in AD and deepen our understanding of PDE2A in the regulation of cognition in the brain. The successful completion of this study will likely pave the way for future drug development of PDE2A inhibitors, specifically for the mitochondrial PDE2A2 isoform, as a promising treatment for AD.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Gastrointestinal malfunctions and immune activation remain significant health issues for people living with HIV/AIDS in the era of anti-retroviral therapy (ART). Immune activation fuels HIV reservoirs that are established early during infection and support virus rebound after ART interruption (ATI). Increasing evidence points toward sex differences in HIV pathogenesis, disease progression, and persistence. Identifying sex-specific immune parameters associated with viral persistence and rebound will be critical to our long term goal of developing personalized therapeutic strategies to reliably halt HIV-associated chronic diseases, and to target HIV reservoirs. In subjects without ART, women have higher levels of immune activation than men and have a higher risk of developing AIDS. Sex differences in immune activation in HIV-infected subjects on ART are less definitive, possibly due to confounding factors, including gender (involving social/behavior factors) differences in seeking treatment. Recent evidence identifying estrogen receptor 1 as a key regulator of HIV latency in CD4+ T cells suggests the involvement of estrogen signaling in sex-based differences in HIV persistence. However, the role of estrogen in HIV persistence in vivo remains unknown, as estrogen induces type I IFNs and other pro- inflammatory cytokines, resulting in immune activation. Research in this area has been lacking a reliable animal model. In that regard, we have developed a rhesus macaque (RM) model that mimics key features of human HIV infection providing promising avenues for pursuing sex-based difference studies. The model, which employs R5 SHIV C109 intrarectal challenge, displays a spectrum of disease outcomes similar to human AIDS. In this model, fast disease progression occurs in females only, and is accompanied by an uncontrolled robust mucosal immune response. All male RMs tested to date exhibit normal (slow/chronic) progression. We hypothesize that kinetics and magnitude of immune dysregulation that drive viral replication, persistence, and rebound exhibit sex-based differences and that estrogen may partially contribute to sex difference in immune responses and viral persistence. We will determine sex-specific immune cellular and molecular determinants relevant to HIV reservoirs and rebound longitudinally. The contribution of estrogen to sex differences in immune regulation and viral persistence will also be examined. The proposed work is significant because it promises to provide new insights into the cross-talk between intestinal immune activation and viral persistence that are differentially regulated in male and female RMs, which will be critical for developing personalized therapeutic strategies to reliably halt HIV-associated chronic disease, and to target HIV reservoirs.

NIH Research Projects · FY 2024 · 2021-08

ABSTRACT We present herein a grant application focused on the late-stage development of preclinical candidate JSF-3285. Per our recently published research, we have disclosed the genesis of this program that led to the hit compound DG167 and identification of the essential β-keto acyl synthase KasA as its target. Building on this effort, our preliminary data detail the optimization to arrive at JSF-3285 which is efficacious in the acute and chronic models of M. tuberculosis infection in mice at doses as low as 5 mg/kg once-daily oral. This proposal seeks to build on this data by conducting the requisite drug combination and relapse studies to achieve clinical status and begin IND-enabling studies. In addition, we propose a second generation program based on preliminary data consisting of a structurally distinct amide series with promising in vitro efficacy, mouse PK, and X-ray structural data. The grant's second aim will evolve this series, leveraging our extensive X-ray structural data, SAR, and machine learning models, to produce at minimum novel early lead compounds if not compounds equal to or surpassing JSF-3285. The sum total of the two aims, featuring JSF-3285 and second generation candidate/s, will lend a high probability to a KasA inhibitor becoming clinically relevant in the next 5 years.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY There is a fundamental gap in our understanding of the complex processes that govern the interactions between Mtb and macrophages. The overall objective of this application is to investigate how the novel biophysical phenomenon of phase separation impacts biological processes, specifically in the context of Mtb infection. A detailed knowledge of the molecules that recognize and respond to pathogens is required to reveal how cells fight infection; therefore, there is a critical need to understand how phase separation may influence or control innate immune responses. Mycobacterium tuberculosis (Mtb) is an incredibly successful and deadly human pathogen that infects one-quarter of the world's population. While interaction of Mtb bacilli and macrophages activates numerous innate immune pathways, we have a limited understanding of how these complex networks of host sensing molecules are regulated to work cooperatively. Furthermore, only a small subset of the many secreted effectors used by M. tuberculosis have well-characterized functions. Recent studies have illuminated the biological and cellular importance of liquid-liquid phase separation, a process by which proteins condense into discrete droplets to alter their localization and function in a cell. Several proteins involved in the host response to M. tuberculosis infection, like cGAS, TBK1, p62, and LC3, have been found to phase separate in vitro, but how in vivo phase separation impacts host responses to infection is unknown. Preliminary studies have found that these and other innate immune proteins form circular puncta in M. tuberculosis-infected cells that resemble phase separated droplets. The central hypothesis of this proposal is that upon infection, pathogen- sensing and post-translational modifications induce phase separation of host proteins and that Mtb modulates these condensation events with its own phase-separating PE/PPE proteins. Here, a combination of novel optogenetics tools, live cell fluorescent imaging, and host and bacterial genetics will be employed to probe the biological consequences of phase separation of host proteins (Aim 1) and Mtb proteins (Aim 2). In addition, directed and unbiased genetics approaches will be used to probe how post-translational modifications, and especially ubiquitination in particular, contributes to phase separation during Mtb infection (Aim 3). This approach is innovative in that it uses novel tools to specifically and precisely modulate phase separation in order to link this biophysical process with meaningful cellular phenotypes. The proposed research is significant because it will greatly expand our understanding of how macrophages destroy Mtb and advance efforts to combat Mtb infection via enhancing host responses.

NIH Research Projects · FY 2025 · 2021-08

PROJECT ABSTRACT Anemia is the most common complication of inflammatory bowel disease (IBD). IBD-associated anemia is often refractory to treatment, and the role of dysregulated iron homeostasis in IBD pathology is unknown. Accordingly, a better understanding of intestinal iron homeostasis may facilitate new therapeutic strategies for IBD and IBD-associated anemia. Anemia and inflammation are directly linked by the hormone hepcidin, which critically inhibits iron release from intracellular stores via the iron transporter ferroportin. In new data, I've identified that hepcidin produced by dendritic cells (DCs) is critical for tissue healing after intestinal inflammation, and that hepcidin is highly expressed by DCs in inflamed tissues of pediatric Crohn's disease (CD) patients. Moreover, hepcidin promoted healing by limiting iron availability to tissue-associated bacteria via iron sequestration in intestinal myeloid cells. The focus of this research proposal is to investigate the hypothesis that intestinal iron homeostasis is regulated by hepcidin in chronic intestinal inflammation and in pediatric Crohn's patients to promote tissue healing. In Aim 1, I will interrogate the role of hepcidin and ferroportin in chronic intestinal inflammation, iron homeostasis, and myeloid cell biology. In Aim 2, I will define the regulation of hepcidin and ferroportin expression in humans, and I will probe correlations between this axis, iron homeostasis, and TNF blockers in pediatric Crohn's disease. I will employ innovative technical approaches to characterize anatomical iron levels in mice and IBD patient samples, and I will develop new genetic tools to study the role of hepcidin and ferroportin in myeloid cells. Collectively, results from these studies will define the regulation and functional significance of intestinal iron homeostasis in IBD, with the potential to define novel therapeutic strategies for pediatric CD patients.

NIH Research Projects · FY 2025 · 2021-08

Abstract Alzheimer’s Disease (AD) is the most common cause of ageing-dependent dementia in the world and is associated with cerebral amyloid plaques, mostly composed of Aβ peptides, and intraneuronal neurofibrillary tangles, mostly composed by hyperphosphorylated tau. The impacts of AD on patients, families, caregivers and society are shattering. Regrettably, AD-modifying drugs are unavailable underscoring the scant understanding of AD pathogenesis. ~5% of AD cases have early onset (<65 yo) and are due to Familial autosomal dominant mutations in APP, PSEN1 and PSEN2 (FAD); ~95% of cases are sporadic with late onset (>65 yo, SAD). Yet, commonly used animal organisms model FAD. This may be an issue if FAD and SAD present significant pathogenic differences. If so, therapeutics effective in FAD animals may have limited efficacy in SAD patients. Thus, models that reproduce the pathogenesis of SAD are needed to identify therapeutic targets and test SAD-modifying therapeutics. Variants of the microglia gene TREM2 increase the risk of SAD by 3 fold. To gain insights into the pathogenic mechanisms of SAD, we generated rats carrying the p.R47H pathogenic variant in the rat Trem2 gene (Trem2R47H). Rat and human APP differ by 3 amino acids in the Aβ region. These differences may be crucial in this model organism because: 1) human Aβ possesses higher propensity to form toxic species as compared to rodent Aβ; 2) the pathogenic role of the p.R47H TREM2 variant may be linked to deficits in microglia-mediated human Aβ clearance. To overcome this issue, we humanized the rat Aβ sequence (Apph allele); thus, our rat models produce human Aβ from the endogenous rat App gene. We choose a knock in (KI) approach rather than the more common transgenic overexpression approach because KI models make no preconceived assumption about pathogenic mechanisms, except the unbiased genetic one. In contrast, transgenic models, which produce high levels of Ab and can readily deposit amyloid plaques, are based on the hypothesis that plaques and/or other forms of toxic Ab have a central pathogenic role. We propose to dissect pathogenic mechanisms triggered by the p.R47H pathogenic variant using these KI rat models. We will also study the impact of Trem2R47H on the pathological processes triggered by the Apps and Psen1LF FAD mutations. We will analyze microglia function, cell-to-cell transcriptomic changes in the brain, APP processing, brain pathology, neuro-inflammation and neurodegeneration, synaptic transmission/plasticity, learning & memory. Dissecting pathogenic pathways set off by the Trem2R47H variant may pave the way to therapeutic approaches that can prevent/delay sporadic AD.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Breast cancer survivors are a growing population and their symptom burden is significant. Despite growing evidence on specific symptoms and risk management strategies, efforts to translate knowledge into practice have produced suboptimal results. Primary care is an ideal receptor site for breast cancer survivors, with studies demonstrating the effectiveness of primary care based survivorship care. Nevertheless, over the past decade, the emphasis on survivorship care plans and survivorship models has not had an evident impact on primary care breast cancer survivorship care processes. Over the past two decades, primary care practices have redesigned structures and care processes to deliver care to different complex populations; however, breast cancer survivors are not among them. Many currently proposed and tested strategies are considered oncology-centric and do not fit well within the real world contexts of primary care practices. Results from our recent research studies indicate that breast cancer survivorship is not considered clinically actionable even among primary care practices with advanced staffing models. Therefore, capacity building and stakeholder- informed strategies are needed to enhance the translational potential of survivorship evidence into primary care practices. This project uses a “designing for dissemination” perspective, blending the implementation Exploration, Planning, Implementation and Sustainment (EPIS) framework and the primary care Practice Change Model (PCM) as a theoretical basis for understanding multilevel factors impacting the implementation of cancer survivorship guidelines in primary care. These perspectives help frame and identify mismatches among existing policy, practice, provider, and patient motivators to translate the evidence into clinically actionable primary care practice change strategies. Using a blended implementation/primary care practice change conceptual framework, this project aims to: (1) engage diverse primary care stakeholders in identifying actionable, practice-based activities for provision of long-term breast cancer survivorship care in primary care using depth interviews; (2) prioritize, synthesize, and identify actionable activities for providing care to long- term cancer survivors in primary care by engaging key stakeholders using modified online Delphi methods and concept mapping; and (3) implement and evaluate actionable breast cancer survivorship symptom and risk management activities using a hybrid type 1 effectiveness-implementation cluster study design with waitlist control in 26 primary care practices. Study results are poised to have profound impact on implementation strategies used throughout the U.S. to provide long-term care for patients with a history of breast cancer.

NIH Research Projects · FY 2025 · 2021-07

Germline mutations in BRCA2 predispose carriers to breast, ovarian, pancreatic, and other cancers. The gene encodes a very large protein that plays critical roles in genome integrity control by promoting homologous recombination (HR)-mediated repair of DNA double strand breaks (DSBs), DNA damage-induced cell cycle checkpoint, and stability of stalled DNA replication forks, etc. Besides, BRCA2 may play a direct role in DNA replication, as its mutant cells have long been known to have the so-called radio-resistant DNA synthesis (RDS) phenotype, which reflects a defect in the intra-S phase checkpoint, a mechanism that slows down DNA replication after DNA damage presumably to allow time for DNA repair and prevent replication of damage DNA. However, the potential role of BRCA2 in DNA replication initiation or elongation has not been defined. In our preliminary studies, we found that BRCA2 interacts with an essential DNA replication factor, MCM10, and that this interaction restrains replication fork progression, promotes fork stalling and sustains origin firing after DNA damage. We hypothesize that BRCA2 regulates DNA replication kinetics after DNA damage through its interaction with MCM10 and that a dysregulation of replication kinetics after DNA damage, or intra-S phase checkpoint defect, leads to increased mutation rate, cancer development and therapy resistance. We propose 3 specific aims to test the hypotheses. In Aim 1, we will define the mechanisms of BRCA2 function in DNA replication kinetics after DNA damage. In Aim 2, we will determine the role of the BRCA2-MCM10 interaction in DNA replication fidelity and cancer cell response to DNA damaging therapies. In Aim 3, we will determine the role of BRCA2-MCM10 interaction in spontaneous and radiation-induced tumor development. Our findings and proposed studies will elucidate key mechanisms of BRCA2 function in DNA replication, provide new insights into the regulation of replication kinetics after DNA damage, and define the impact of RDS and replication kinetics dysregulation on cancer development and tumor relapse after radiation and chemotherapy.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT As a central controller of cancer growth and metabolism, mTORC1 pathway is commonly mutated and activated in human cancer, leading to uncontrolled growth. Cancer cells are ‘addicted’ to elevated mTORC1 signaling, rendering mTORC1 a desirable cancer drug target. The highly specific mTORC1 inhibitors rapamycin analogs (rapalogs, e.g. temsirolimus) are US FDA-approved oncology drugs. However, their clinical response has been moderate, which is in a large part due to incomplete understanding of mTORC1 signaling mechanisms underpinning rapamycin action. In this application, we will test the central hypothesis that nuclear mTORC1 signaling promotes aerobic glycolysis, or Warburg Effect, through a long non-coding RNA (lncRNA) NEAT1- dependent mechanism, which is important for mTORC1-driven tumorigenesis and rapamycin response. A successful completion of this project will provide a deeper understanding of this oncogenic pathway and therapeutic response to its blockage.

NIH Research Projects · FY 2025 · 2021-07

Project Summary This is a new program for “Training in Translating Neuroscience to Therapies” at Rutgers University. Its overall objective is to provide scientists in training performing laboratory-based experiments the skills and competencies needed to advance their discoveries to treatments for neurological disorders. The aims of the program are to: 1) provide a continuum of comprehensive training that advances translational neuroscience research towards therapeutic developments; 2) expand the pipeline of translational neuroscience researchers within a diverse cohort of trainees; and 3) leverage Rutgers’ strengths in neuroscience drug discovery and development to provide broad experiences to trainees in this program. The rationale for establishing this program is the increasing burden of neurological diseases globally, many of which have no good treatments much less cures, and the accelerated advances in scientific discoveries in the genetics and molecular pathogenesis of nervous system disorders. Thus, this is an opportune time to harness knowledge gained in our understanding of disease processes, fill in the gaps in this knowledge base, and translate basic discoveries to deliverables for patients suffering from these disorders. The design of the training program combines traditional coursework, seminars, conferences, a biostatistics curriculum, and an annual retreat, along with experiential learning through a series of apprenticeships with experts in fields that link scientific innovation with industry to advance laboratory discoveries to clinical trials. Trainees will learn from neurologists and trialists, and actively engage in clinical trials. The training period for each participant is two years. A total of two positions are requested in year one, and four positions in subsequent years. The trainees will include senior graduate students, MD/PhD students and postdoctoral fellows at a ratio of 1:1:2. Trainees will work in the laboratories of highly qualified neuroscientists and translational investigators employing multidisciplinary approaches to learn the principles of rigorous and reproducible research. They will also have mentors with expertise in medicinal chemistry, drug discovery and development, clinical research and trials, start-ups, and career opportunities. Faculty and trainees will benefit from a formal mentoring program in place at Rutgers Biomedical and Health Sciences. Program oversight includes internal and external advisory boards, and an Executive Committee consisting of senior leadership at Rutgers University with extensive experience in translational and clinical research, pharmaceutical development, commercialization of innovations, and graduate and postgraduate education. The quality and effectiveness of the training program will be assessed by an independent assessor. The intended outcome of this program is to train the next generation of leaders in translational neuroscience who have the knowhow and passion to take basic discoveries to clinical trials and treatments that benefit millions with neurological diseases.

NIH Research Projects · FY 2025 · 2021-07

Abstract Tumor suppressor p53 is the most frequently mutated gene in human cancer, including colorectal cancer (CRC). Many tumor-associated mutant p53 (mutp53) proteins not only lose the tumor suppressive function of wild-type p53, but also gain new oncogenic activities to promote tumorigenesis, which is defined as the “gain-of-function” (GOF). Mutp53 proteins often become stable and accumulate to very high levels in cancer, which is critical for mutp53 GOF in tumorigenesis. Destabilizing mutp53 protein is being actively tested as a novel and promising strategy for cancer therapy. However, the mechanism of mutp53 accumulation in cancer is poorly understood, which hinders the development of effective strategies for cancer therapy. MDM2 is the most critical negative regulator of p53. MDM2 isoform B (MDM2B), a spliced isoform of MDM2, is frequently overexpressed in human cancer, and plays an important role in tumorigenesis. Currently, the mechanisms underlying MDM2B-mediated mutp53 accumulation and MDM2B overexpression in cancer are poorly understood. Identifying their underlying mechanisms has the direct potential to develop effective strategies to treat cancers carrying mutp53. The goal of this study is to determine the mechanism of mutp53 accumulation in CRC to provide novel therapeutic targets/strategies for CRC carrying mutp53. Based on our preliminary studies, we hypothesize that MDM2B overexpression is a critical mechanism underlying mutp53 protein accumulation and GOF in CRC, and furthermore, MDM2B and its signaling pathway can be targeted for therapy in CRC carrying mutp53. We will test our hypothesis by following specific aims: 1) determine whether MDM2B overexpression is a critical mechanism underlying mutp53 accumulation and GOF in CRC using different mouse colorectal tumor models, and identify mechanisms underlying MDM2B-mediated mutp53 accumulation; and 2) identify mechanisms underlying MDM2B overexpression in CRC, and test whether targeting MDM2B and its signaling pathway can inhibit mutp53 accumulation and GOF in CRC. We anticipate that this proposed study will provide new paradigms regarding mutp53 accumulation, tumor-promoting function of MDM2B, and MDM2B overexpression in CRC. If accomplished successfully, results from this study will have potential to develop MDM2B and its signaling pathway as novel therapeutic targets for CRC carrying mutp53.

NIH Research Projects · FY 2025 · 2021-07

Despite the widespread use of an attenuated vaccine and several antibiotics, tuberculosis (TB) continues to be a global public health problem. Over 1.2 million people died from TB in 2019. This dire situation is compounded by increasing prevalence of antibiotic resistant strains of Mycobacterium tuberculosis (Mtb), the main etiological agent of human TB. Central to Mtb success is its ability to evade, modulate, and even manipulate host immune defense response. Consequently, bacterial factors involved in undermining the immune system are potentially good targets for TB intervention. Like many other bacteria, Mtb actively produces extracellular vesicles (EVs) in vitro and in vivo. These are membrane enclosed spherical structures that allow the bacteria to concentrate and secrete a variety of molecules, and communicate with other cells in their environment. The release of EVs by Mtb infecting macrophages enables the delivery of pathogenicity factors and immunomodulatory molecules into the host cell, and the extracellular milieu. Strong evidence from in vitro studies indicates that EVs may allow Mtb to remotely influence bystander immune cells. However, the limited understanding of the molecular mechanisms involved in vesicle biogenesis, and the lack of mutants deficient in vesicle production have impeded progress in elucidating the relevance of vesicle secretion to Mtb virulence. Our preliminary work identified the dynamin-like proteins (DLP) of Mtb as essential factors for efficient EVs release and characterized a DLP mutant deficient in vesicle biogenesis. We are now well positioned to dissect DLP's function in vesicle formation and assess the role of EV production during infection, using a mouse model of TB; those are the main goals of this proposal. We anticipate the findings will advance the TB field by highlighting ways to target vesicle release, or disrupt the effects of vesicles in host-resistance to TB.

NIH Research Projects · FY 2025 · 2021-07

Project Summary The Latino Ancestry-Genomic Psychiatry Cohort (LA-GPC) is an expansion of the GPC into the Latino population. LA and other minority populations have been poorly represented in large-scale genomic studies, and yet these populations (i) suffer the largest disparities in health care and outcomes, and (ii) have the potential to broaden our knowledge of human genetics. In particular, LA genomes are admixed and are characterized by different haplotype blocks than European ancestry (EA) populations. As a consequence, genetic polymorphisms that are in perfect linkage disequilibrium (LD) in Europeans may be broken-up by recombination events in LA genomes, allowing the contributions of different genomic intervals to be assessed independently. Progress to date: The GPC has enrolled and assessed over 5,000 new LA participants and we have recently published a pilot GWAS including both LA and African Ancestry (AA) participants. GWAS meta- analysis of PGC-SCZ2 with pilot results for 4,324 (cases and controls) LA-GPC participants yielded 8 new schizophrenia-associated risk loci, and for 9 loci there was a concomitant reduction in the number of SNPs in the associated interval. Crucially, the resultant cross-ancestry summary statistics, informed by multiple ancestrally diverse cohorts, yielded individual-level polygenic risk scores that explained more variance than analogous scores based on either ancestry alone In Phase 1 of this renewal: we will study genome-wide common variation in our existing LA-GPC and VA- CS#572 combined cohort of 16,124 LA participants. In Phase 2, we will perform an expanded analysis by adding 8,000 new participants ascertained in the LA-GPC, 35,000 additional participants from the VA, 8,000 participants from the NeuroMex study (PI. Koenen), and 10,000 participants from the Colombian study (PI. Freimer). This meta-analysis will include over 77,000 LA participants, a sample of non-Caucasians with the potential for significant novel discovery.

NIH Research Projects · FY 2024 · 2021-07

Project Summary We propose a 5-year renewal of our African Ancestry-Genomic Psychiatry Cohort (AA-GPC) R01- MH104964. AA and other minority populations have been poorly represented in large-scale genomic studies, and yet these populations (i) have the largest disparities in health care and outcomes, and (ii) have the potential to broaden our knowledge of human genetics. In particular, AA genomes have nearly a million more variants per individual and are characterized by shorter haplotype blocks than European ancestry (EA) populations. As a consequence, genetic polymorphisms that are in perfect linkage disequilibrium (LD) in Europeans are broken-up by recombination events in AA genomes, allowing the contributions of smaller genomic intervals comprised of fewer variants to be assessed independently. Progress to date: During the initial funding period we will have enrolled and assessed ~10,000 new AA participants and have already completed 8,000 AA whole genome sequences (WGS). We recently demonstrated that among the 128 associated SNPs identified in the PGC-2 schizophrenia analysis, 41 increased in significance when combined with data from 10,000 AA-GPC participants, and for 12 of these regions, there was a reduction in the number of SNPs in the associated interval. In addition, this trans-ancestry meta-analysis of PGC-2 schizophrenia and AA-GPC results yielded 10 newly genome-wide significant loci. Similarly, the trans-ancestry meta-analysis results yielded the best polygenic risk score “training” dataset, explaining more variance in individuals of European and African ancestry, than scores based on either ancestry alone. In Phase 1 of this renewal: we will study genome-wide common variation in our existing AA-GPC and VA-CS#572 combined cohort of 36,322 AA participants. In Phase 2, we will perform an expanded analysis by adding 5,000 new participants ascertained in this project, 27,500 additional controls from the VA, and 34,000 cases and controls from the NeuroGap-Psychosis study (NeuroPsychiatric Genetics in African Populations; PI. Koenen). This meta-analysis will include over 100,000 AA participants, a sample of non-Caucasians with the potential for significant novel discovery.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Pathological hypertrophy can progress to failing heart. During the transition, fatty acid utilization is decreased, while utilization of other substrates, such as ketone body, is increased. Multiple lines of evidence indicate that increased myocardial ketone body utilization is an adaptive response against cardiac pathology. Furthermore, although ketoacidosis is life-threatening, short-term administration of exogenous ketone body enhances myocardial oxygen consumption with increases in both ketone body oxidation and overall ATP production in the heart. This intervention improves cardiac function and remodeling in humans and mice with heart failure (HF). Although ketone body serves as not only a fuel source but a modulator of lysine acetylation, the effect of ketone body-mediated acetylation against hypertrophy and HF remains poorly understood. Elucidating the molecular mechanisms of ketone body action beyond fueling, which mediates anti-hypertrophic and pro- energetic effects without provoking detrimental effects, is the most important issue in establishing ketone body as a therapeutic option for HF. We recently found that lysine acetyltransferase 6A (KAT6A) is acetylated in the heart by a low-carbohydrate (LC) diet-mediated increase in ketone body, which is negatively associated with hypertrophy and HF after pressure overload. Thus, we here ask whether acetylation of KAT6A is critically involved in ketone body action against cardiac pathology. Our study provided evidence that acetylation of KAT6A inhibits phenylephrine-induced hypertrophy and improves energy homeostasis in cardiomyocytes in vitro. However, it remains unknown how KAT6A acetylation regulates cardiac morphology and function. Based on these exciting observations we propose a novel role of KAT6A acetylation in pathological hypertrophy and its transition to HF. Together with the surprising findings from our studies using proteomics and genomics analyses, we hypothesize that ketone body promotes acetylation of KAT6A, which stimulates the AMPK signaling in the heart to suppress protein synthesis and maintain energy homeostasis, thereby inhibiting pathological hypertrophy and a transition to HF. To address this hypothesis, we will conduct the following experiments. In Aim 1, we will determine the significance of KAT6A acetylation in pressure overload-induced hypertrophy and HF in vivo by using newly generated KAT6A acetylation-resistant knock-in mice and an AAV- KAT6A acetylation-mimicking mutant. In Aim 2, we will demonstrate the critical involvement of AMPK in KAT6A action by pharmacologically and genetically inhibiting AMPK. We will further elucidate the mechanism by which AMPK is activated by KAT6A by using molecular signaling and biological assays. The long-term goal of this project is to identify the therapeutic targets to specifically modulate the ketone body-KAT6A-AMPK pathway relevant to the strategies for the primary and secondary prevention of cardiac hypertrophy and HF.

NIH Research Projects · FY 2025 · 2021-07

Modified Project Summary/Abstract Section Rare loss-of-function (LoF) mutations in SETD1A are strongly associated with schizophrenia (SZ), a debilitating mental disorder affecting 1% of the population, and other severe neurodevelopmental disorders. SETD1A encodes a component of the histone methyltransferase complex producing mono-, di, and trimethylated histone H3 at Lysine 4 (H3K4). H3K4 trimethylation (H3K4me3) and H3K4me1 are epigenomic marks of active gene transcriptional promoters and enhancers, respectively. Interestingly, histone methylation has also been suggested as one of the most enriched gene pathways in common variant-based genome-wide associations studies (GWAS) of major psychiatric disorders. Furthermore, a recent mouse model with heterozygous knockout of SETD1A exhibited working memory deficits and showed transcriptional changes that overlap with those implicated in neurodevelopmental disorders, however, seemingly independent from a H3K4me3 mechanism. Therefore, it remains largely unclear whether and how SETD1A causes SZ-relevant molecular and cellular changes in a human brain. Our central hypothesis is that human induced pluripotent stem cell (hiPSC)-derived neuronal cells and cortical organoids recapitulate key SZ-relevant epigenetic, molecular and cellular properties of SETD1A LoF in the human brain. Using CRISPR/Cas9 gene editing, we have generated isogenic hiPSC lines carrying heterozygous LoF mutations (in exon 4 and exon 16, on different genetic backgrounds) of SETD1A. Preliminary results showed that mutant lines were defective in cortical organoid development with premature neuronal differentiation at early developmental stages. Furthermore, morphological, electrophysiological and transcriptomic analyses of hiPSC neurons carrying SETD1A LoF mutation showed defective synaptic neurotransmission. Interestingly, genes showing differential expression in both 3D cortical organoids and 2D cultures from mutant lines are enriched for common GWAS risk variants of SZ and other neuropsychiatric disorders/traits, suggesting possible convergent pathways shared by SETD1A LoF and common GWAS risk variants of major psychiatric disorders. Leveraging our respective expertise in hiPSC models and neurogenesis, synaptic physiology and functional genomics within our team, we propose to characterize the molecular and cellular mechanisms underlying the deficits associated with SZ-associated LoF mutations in SETD1A in human neural systems. We will identify the cell-type-specific and developmental stage-specific cellular and molecular phenotypes associated with SETD1A LoF in cortical organoids, and then investigate the synaptic phenotype(s) of SETD1A LoF mutations in human neurons and associated transcriptome changes. The proposed study will enable us to perform a well-controlled assessment of the impact of SETD1A LoF mutations on the molecular and cellular mechanisms underlying deficits in early neurodevelopment and synaptic properties.