University Of California, San Diego

NIH Research Projects · FY 2025 · 2021-06

Prion diseases are rare, invariably fatal neurodegenerative disorders with pathologic features in common with Alzheimer’s disease, including extracellular protein aggregates, synaptic loss, and neuritic dystrophy. In prion and Alzheimer’s disease models, depletion of neuronal cellular prion protein (PrPC) ameliorates synaptic impairment and clinical disease, strongly implicating neuronal PrPC expression in the altered signal transduction cascades that may underlie synaptotoxicity and endolysosomal dysfunction. We have engineered the first knock-in mouse model with a point mutation in Prnp that develops a striking and severe spongiform encephalopathy, neuritic dystrophy, and altered post-synaptic receptor phosphorylation, in the absence of prion aggregates. Cultured cortical neurons from these knock-in mice show an increased sensitivity to glutamate and dendritic varicosities, suggestive of excitotoxicity. Thus, this PrP knock-in model provides a unique opportunity to elucidate key PrPC interactions and altered signal transduction pathways at the synapse and to determine the molecular mechanisms that link PrPC to synaptic loss and endolysosomal dysregulation. Our long-term goal is to understand how PrPC triggers aberrant neuronal signaling that may drive impaired proteostasis and synaptotoxicity in prion disease. Using cultured primary neurons and mice, we will first determine how the mutant PrPC interactions impact pre- and post-synaptic neuronal protein levels and glutamate receptor function. We will then identify how mutant PrPC dysregulates endolysosomal and proteostatic activity. Finally, we use highly sensitive and quantitative proteomics to define the PrP interactome and phosphoproteome network alterations in the brain by tandem mass tag mass spectrometry analysis. For all aims, we will directly test how the findings from the mutant PrPC-expressing brain compare to prion-infected mouse and human brain. These studies are the first to target the neuronal endolysosomal and synaptic pathways in a knock-in mouse model expressing mutant PrPC, and outcomes are expected to provide key insights into the role of PrPC in synapse maintenance and the signaling pathways inciting synaptic loss, thus revealing new therapeutic targets for prion disease.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Malaria is currently the most impactful mosquito-borne disease worldwide, sickening 228 million people and killing over 405,000 in 2018, 2/3 of which are young children — the most vulnerable demographic. Several mosquito species of the Anopheles genus can act as vectors of the parasite causing malaria, and in recent years their increasing resistance to pesticides is hampering current control methods and blunting our response to eventual disease outbreaks. Globalization is further allowing both vectors and pathogens to move freely and in certain situations to permanently establish themselves in new locations. CRISPR-based gene drive technologies for mosquito population engineering are being developed as they represent a new promising addition to our arsenal for fighting this disease. These technologies are up-and-coming, yet few issues have come up during their development. Briefly, a gene drive system based on CRISPR is composed of a Cas9 and a gRNA gene inserted in the mosquito genome at the location where the gRNA targets it. The arrangement of this genetic cassette endowed it with self-replicating properties that allow it to propagate to the same location on a wild-type chromosome. This property can be harnessed to spread within a population a beneficial trait that would help reducing disease transmission (population modification), or a deleterious trait to help reduce the mosquito population (suppression). While this process is extremely accurate, it can result in the failure of self-propagating, and the generation of small mutations at the targeted locus preventing further conversion by the gene drive. These “resistance alleles” generated during the drive process have been identified as a major hindrance to field applications of these tools. In addition, due to the deposition of active Cas9 and gRNA in the developing embryo, the mosquito biology allows an extensive production of such resistance alleles when a gene drive is inherited from a female. The long-term goal of this project is to develop powerful gene drive tools that can be used for the fast and reliable engineering of wild Anopheles populations. In order for these tools to be ready to have an impact on the malaria morbidity worldwide, the two issues described above need to be overcome. To tackle these two problems, in the three Aims of the proposed research, we will develop and optimize three parallel technologies in the fruit fly Drosophila melanogaster and subsequently apply them to the major malaria vector Anopheles stephensi.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Our research program focuses on the mechanism of eukaryotic protein synthesis and translational control. The two projects we are currently studying are: (1) the mechanism of translational control by the fragile X mental retardation protein (FMRP), and (2) the mechanism of translation initiation on influenza A virus (IAV) mRNAs. Fragile X syndrome is a disease that afflicts about 100,000 Americans and about 3 million people worldwide, resulting in intellectual disability, childhood seizures, and autistic behavior in the patients. The disease is caused by the transcriptional silencing of the fragile X mental retardation 1 gene (FMR1). FMR1 gene codes for an RNA-binding protein, FMRP, which is highly expressed in the brain and is essential for the normal development of the brain. Mammals have two autosomal paralogs of FMRP designated as fragile X related 1 and 2 (FXR1 and FXR2) proteins. FMRP, FXR1 and FXR2 have been implicated in regulating the translation of several mRNAs. However, the precise mechanism by which these proteins regulate the expression of these mRNAs is unknown. The goal of the first project is to understand the molecular mechanism underlying the regulation of protein synthesis by FMRP, FXR1 and FXR2. We will use a robust in vitro translation system, biochemical techniques and quantitative biophysical methods to significantly advance our understanding of the molecular mechanism used by FMRP, FXR1 and FXR2 to regulate protein synthesis. Results of these studies will provide useful insights in identifying potential drug targets to treat fragile X syndrome. The goal of the second project is to investigate the mechanism of translation initiation by IAV mRNAs. IAV is responsible for several thousand deaths annually and is a severe threat to global public health. We have new data that indicate that IAV mRNAs may use a non-canonical mechanism of translation initiation. Our studies show that poly A binding protein 1 (PABP1) binds to the highly conserved sequences present in the 5’-UTR of IAV mRNAs. Additionally, we show that the translation of the IAV mRNA is more resistant to the inactivation of eukaryotic initiation factor 4E (eIF4E) compared to a control mRNA. We hypothesize that the recruitment of PABP1 to the viral 5’-UTRs tethers eIF4G and promote the assembly of the translation initiation complex in an eIF4E-independent manner. This may favor the translation of IAV mRNAs under cellular stress conditions in the cell, which is known to reduce the activity of eIF4E. We will determine whether the binding of PABP1 to the 5’- UTR of IAV mRNAs is essential for translation initiation and the viral cycle using in vitro techniques and cellular IAV infection studies. Our research will lead to fundamental new knowledge about the process of translation initiation on IAV mRNAs, which could help in the development of new antiviral drugs.

NIH Research Projects · FY 2025 · 2021-06

Abstract: The overarching goal of this proposal is to understand the molecular pathology of inherited retinal degeneration (IRD) by (a) generating maps of human retinal cell type-specific regulatory elements, (b) utilizing these maps to identify non-coding IRD causative mutations within retinal regulatory elements, and (c) gaining insight into the molecular underpinnings of pathological non-coding IRD mutations using cellular and animal models. IRDs are the most common cause of irreversible blindness in young individuals affecting 1 in 3000 individuals. Mutations in coding and splice site sequences in known IRD associated genes contribute to about 60%-65% of cases while the remaining 40%-35% of cases are currently unresolved. Mutations in non-coding or regulatory sequences are suggested to be responsible for a large proportion of these unresolved cases. Although the ENCODE and Roadmap Epigenomics projects have generated detailed maps of regulatory elements for the majority of body tissues, retina is left out. Lack of these maps is a major limitation in identifying IRD causative mutations involving regulatory sequences in retinal cells. We have analyzed the whole genome sequence (WGS) of 125 pedigrees with IRD; of these, 49 remain unresolved with no candidate causative nucleotide changes or structural variants (SVs) in coding or splice site sequences. This leads us to hypothesize the involvement of non-coding variants in pathology. We also have access to more than 391 additional IRD pedigrees that remained unresolved after WGS analysis. In this application we propose to test the hypothesis that non-coding sequence changes are involved in IRD pathology for the majority of these unresolved pedigrees. We will conduct the following studies: Aim 1, establish human retinal cell type specific maps of regulatory elements using innovative single cell genomics methodologies we developed, Aim 2, rank prioritize candidate causative variants using the retinal cell type-specific regulatory element maps and WGS of unresolved pedigrees, Aim 3, validate the impact of high ranking non-coding candidate disease causing variants in the context of the genome architecture of retinal cell types by developing patient iPSC-derived retinal cell models and mouse models. These studies will result in the establishment of retinal cell type-specific high-resolution multi-omic maps and will potentially identify, for the first time, non-coding variants involved in the pathology of IRD. The outcomes of these studies will (1) significantly enhance our understanding of the architecture of retinal cell type-specific regulatory networks, (2) reveal the molecular pathology underlying IRD, (3) establish a highly valuable, publicly-available data set of cis-regulatory elements relevant to retinal degenerative diseases as a resource for retinal disease research, (4) improve mutation detection in patients, and (5) facilitate discovery and development of novel therapies for IRD. We have assembled a multidisciplinary team of outstanding investigators with expertise in epigenetics (Ren), genome sciences (Frazer) and IRD genetics and disease modeling (Ayyagari) who are well positioned to complete this ambitious project.

NIH Research Projects · FY 2025 · 2021-05

Project Summary OSA is defined by repetitive collapse of the upper airway, a process which leads to transient hypoxemia and arousals from sleep, and is associated with various cardiovascular, metabolic, and neurocognitive consequences. OSA is the most common respiratory disorder, affecting roughly 10% of middle aged men and women in the USA and up to 1 billion globally. Although continuous positive airway pressure (PAP) is an efficacious therapy, it is not always well tolerated and adherence is less than ideal. OSA is increasingly recognized as a multifactorial disorder that can occur in different people for different reasons, not only due to anatomical predisposition (collapsibility of the upper airway), but also related to low arousal threshold (wake up too easily), dysfunction in upper airway dilator muscles and instability in ventilatory control. Through careful measurement of these underlying factors and the symptoms experienced in OSA, this proposal seeks to understand how different mechanisms underlying OSA – endotypes – lead to different symptoms or consequences – phenotypes. These different phenotypes range from having no appreciable symptoms, to falling asleep at the wheel, to experiencing cardiometabolic consequences. Additionally, addressing the underlying cause might be useful to personalize therapy, to predict adherence to PAP, and to understand which symptoms will or will not improve with long term PAP therapy. A major challenge in clinical practice is understanding whether particular symptoms are due to OSA, and whether these symptoms will improve with treatment. Moreover, we are currently struggling to find alternative therapies for OSA which will likely depend on underlying mechanism. Similarly, we do not currently know which patients to put into clinical trials since it seems unlikely that, e.g., all OSA patients will have cardiovascular benefits to PAP therapy since not all are at risk of heart disease. Our goals are 1) To understand the contribution of the underlying cause, or endotype, of OSA to the symptoms experienced by people with OSA, 2) To elucidate how the endotype predicts response to non CPAP therapies, such as oxygen or sedative hypnotics, and 3) To define how underlying endotype (mechanism) mediates changes in phenotype (clinical manifestations of disease) with treatment of OSA. Ultimately we hope that our efforts will advance the OSA field and help to alleviate suffering or reduce/prevent disease burden.

NIH Research Projects · FY 2026 · 2021-05

Project Summary/Abstract: We propose to conduct a first-in-human clinical trial of BDNF gene therapy in Alzheimer’s Disease (AD) and Mild Cognitive Impairment (MCI), aiming to reduce neuronal loss and to activate neuronal function. BDNF (Brain-Derived Neurotrophic Factor) is actively produced and utilized in cortical circuits throughout life to sustain neuronal function and circuits. In animal models of AD, BDNF builds new synapses, prevents neuronal death and activates neurons; thus, BDNF offers the potential to slow or actually reverse cognitive decline in established AD and MCI. Proof-of-concept studies have been performed in mice, rats and rhesus monkeys. Because BDNF is a relatively large and polar protein that does not cross the blood brain barrier, we will use intraparenchymal gene therapy to deliver BDNF directly into the entorhinal cortex. BDNF will be neuronally trafficked into the hippocampus. BDNF will be delivered using adeno- associated serotype 2 vectors (AAV2), which have now been utilized in hundreds of patients in CNS gene therapy trials. We will utilize start-of-the-art methods for gene delivery, employing real-time MR guidance and convection-enhanced delivery (CED) in collaboration with the world leaders in this technology at Ohio State University (OSU). A total of 12 patients (6 AD and 6 MCI) will be recruited from two clinical sites: UCSD and Case Western. All patients will undergo gene delivery at OSU. The primary outcome measure will be safety, together with secondary cognitive outcome measures that reflect memory-specific and global cognitive measures. Serum, CSF and imaging biomarkers will be collected. If AAV2- BDNF gene delivery is safe and well-tolerated, and exhibit possible cognitive benefits, we will advance to Phase 2 trials. An IND for this program is under review by the FDA, and the trial will begin upon FDA clearance. Two dose groups will be studied: 3x1011 vg/ml and 1x1012 vg/ml. Relevance: Effective disease-modifying therapies for AD and MCI have not been identified. BDNF gene delivery offers the potential to slow or reverse cognitive decline in established AD by building new synapses, stimulating neuronal function and reducing neuronal death. Our approach also offers the potential for combination therapy with amyloid- and tau-modifying therapies.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Generalized approaches for laryngopharyngeal reflux (LPR), a prevalent and heterogeneous syndrome in which laryngeal symptoms are attributed to gastroesophageal reflux, have led to poor health outcomes, inappropriate resource utilization, and tremendous healthcare costs. As a physician scientist with advanced training in esophageal physiology and health outcomes research, my long-term career goal is to discover phenotype guided care paradigms for esophageal conditions, such as LPR, focused on distinct disease mechanism. My preliminary findings from single-arm clinical trials, observational studies, and initial translational research in LPR have generated the central hypothesis that novel diagnostic biomarkers can identify clinical-physiologic phenotypes of LPR and guide a mechanism focused treatment strategy. Thus, the research goals of this proposal are to (1) measure the efficacy of the novel therapeutic upper esophageal sphincter assist device in a rigorous biomarker targeted randomized sham-controlled trial of 78 subjects with salivary pepsin positive LPR, (2) identify phenotypes of LPR using latent class analysis of comprehensive physiologic and clinical data, and (3) compare oral microbiome between subjects with and without pepsin positive LPR. The research aims are aligned with my training goals to (1) develop expertise in advanced clinical trial methods, (2) acquire skills in phenotype design and latent class analysis, and (3) obtain experience with translational microbiome research. My outstanding and diverse mentorship team is overseen by primary mentor Dr. Samir Gupta, an expert clinical trialist that has contributed seminal discoveries in colorectal cancer screening. Co-mentors include Dr. John Pandolfino, a leading international authority and clinical researcher in esophageal physiology, and Dr. Bernd Schnabl, a physician scientist that has pioneered work in host effects on the intestinal microbiome. The exceptionally supportive and conducive institutional environment at University of California San Diego (UCSD) is a key strength of this application. UCSD’s NIH supported Clinical Translational Research Institute will provide cutting-edge clinical trial infrastructure. UCSD’s Center for Esophageal Diseases and Center for Voice & Swallowing Disorders will ensure successful subject recruitment and access to state-of-the-art diagnostic techniques. UCSD’s P30 supported Digestive Diseases Research Center directed by Dr. Bernd Schnabl, will provide leading resources in microbiomics and bioinformatics. Immediate anticipated impacts of this proposal are to inform the clinical management of LPR and shed light on a novel mechanistic pathway of inflammation in LPR, as well as enhance my career by providing unique skills in clinical trial methods and microbiome research. Ultimately, this career development proposal will launch my career as an impactful leader in multi-disciplinary clinical-translational research in the field of aero- digestive esophageal diseases.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Genome-wide association studies (GWAS) in model organisms such as mice and rats have identified hundreds of genetic loci that are associated with addictive behaviors, but determining the causal genes remains challenging. Single nucleotide polymorphisms (SNPs) may not adequately tag other classes of variants such as structural and repetitive variants that have higher mutation rates. Thus, both panels of inbred strains and outbred populations will fail to identify a subset of loci and causal alleles. We are proposing to address this serious limitation by using cutting-edge methods to discover and genotype structural variants (SVs) and tandem repeats (TRs). Because they are technically challenging to analyze, SVs and TRs have not yet been adequately surveyed in rodents. However, there is already extensive evidence that SVs and TRs are prevalent in mice and rats, and that they have important functional consequences. Our proposal brings together complementary expertise in human genetics and bioinformatic analysis of SVs (Sebat) and repetitive variation (Gymrek) with established leadership in elucidating the genetic basis of behavioral phenotypes in model organisms (Palmer). This study will generate the first large-scale resource for analyzing the effects of complex variation on mouse and rat phenotypes. We use this resource to examine gene expression and behavioral traits in inbred mice (BXD recombinant inbred strains, the Hybrid Mouse Diversity Panel (HMDP), the Diversity Outbred (DO) mice, the Hybrid Rat Diversity Panel (HRDP) and the Heterogeneous Stock (HS) outbred rats. In Specific Aim 1 we will characterize SVs in inbred and outbred mice and rats by single-molecule sequencing. In Specific Aim 2 we will genotype TR in inbred and outbred mice and rats. Finally, in Specific Aim 3 we will perform GWAS using SV and TR for gene expression and behavioral traits. We will determine the phenotypic consequences of the SV and TR genotypes on gene expression and behavioral traits. We will impute SVs and TRs into outbred mice and rats and then perform GWAS using the wealth of preexisting gene expression and behavioral data that are available for the mouse and rat populations studied in this project. Completion of this project will characterize the SV and TR landscape in mice and rats, elucidate their role in gene expression and complex behavioral traits relevant to addiction, and create a community resource that will enhance numerous ongoing mouse and rat genetic studies.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract It has recently become clear that carcinogenesis and cancer progression involves dysregulation of immune cells within the tumor microenvironment. The particular interest of this proposal are how tumor cells develop tactics to manipulate immune cells to facilitate cancer progression, and how chronic inflammation induced immunosuppression accelerates carcinogenesis. Dr. Wettersten recently published that tumor cell expression of a cell adhesion receptor, integrin αvβ3, is associated with the enrichment of tumor-associated macrophages (TAMs), which are known to promote immune suppression and prevent the influx of cytotoxic T cells. Her new preliminary studies reveal that αvβ3 expression on lung cancer cells is sufficient to recruit TAMs and that inflammatory mediators can induce αvβ3 expression both on normal lung epithelium as well as lung cancer cells. Together, these findings suggest inflammation can induce tumor cell expression of αvβ3, which in turn leads to TAM accumulation, immune suppression, and tumor progression. Furthermore, inflammation induced αvβ3 expression on lung epithelium may contribute to the well-known relationship between inflammation and cancer. The overall goals of this study are to understand how αvβ3+ cancer cells promote a pro- tumor/immunosuppressive microenvironment during cancer progression and how αvβ3 expression on normal lung epithelium induces inflammation-mediated carcinogenesis. Aim 1 will determine if TAM enrichment factors from αvβ3+ cancer cells promote an immunosuppressive microenvironment. The goal of Aim 2 is to target integrin αvβ3+ cancer cells to shift the tumor immune profile from pro-tumor to anti-tumor. Aim 3 will assess if inflammation-induced expression of integrin αvβ3 on lung epithelial cells accelerates carcinogenesis. To achieve these aims, the expression of αvβ3 will be genetically modified in immunocompetent mouse models of lung cancer and inflammation to reveal how αvβ3+ cells exploit the immune system to enable cancer development and progression. Understanding this process will lead to the development of novel therapeutic strategies to strengthen the anti-tumor immune response. Dr. Wettersten’s career goal is to become an independent investigator in an academic setting. She will leverage her dual training as a veterinarian and cancer biologist to generate new therapeutic approaches targeting the communication between the tumor microenvironment and cancer cells. While her project is strongly supported by her recent findings with Dr. Cheresh, exploring the impact of αvβ3+ cancer cells in the tumor microenvironment is distinct from Dr. Cheresh’s existing work, and the novel mouse models established in this study will provide a foundation for her to further investigate the cancer cell-microenvironment interaction. Dr. Cheresh and her other collaborators at UCSD will give her access to world-class facilities, equipment, and animal models, providing her with an ideal environment to accomplish the proposed work and launch her career as an independent investigator.

NIH Research Projects · FY 2025 · 2021-05

Project Summary The plant hormone auxin regulates virtually all aspects of plant growth and development. We and others have demonstrated that auxin promotes degradation of transcriptional repressors called Aux/IAA proteins, via a family E3 ligases called SCFTIR1/AFB. Auxin is perceived by a co-receptor consisting of a TIR1/AFB protein, the F- box subunit of the E3, and the Aux/IAA protein. The formation of this complex promotes degradation of the Aux/IAA protein and transcription by ARF transcription factors. Although this basic pathway is well established, it is not clear how auxin regulates such a wide range of developmental and physiological processes. Currently in the second year of our RO1 grant, we are investigating this broad question using two complementary genetic systems; the flowering plant Arabidopsis thaliana, and the basal land plant Physcomitrella patens. Our focus has been on the function of the TIR1/AFB proteins, regulation of Aux/IAA level, and the architecture of the auxin signaling network. We recently demonstrated that 5 of the 6 member TIR1/AFB family in Arabidopsis act in an overlapping fashion. The 6th member, AFB1, functions in a novel transcription-independent auxin response pathway. Further, our recent ChIPseq experiments indicate that TIR1/AFB proteins (excluding AFB1) are recruited by auxin to chromatin adjacent to auxin-regulated genes. We speculate that this mechanism permits rapid de-repression of auxin responsive genes. We also made significant advances in our understanding of Aux/IAA proteins and their regulation. We recently discovered that Aux/IAA are substrates of E3 ligases called CRL3-BPM, in addition to SCFTIR1/AFB. The BPMs are orthologs of the human SPOP proteins. In addition, we showed that the Aux/IAA genes are regulatory nodes that integrate environmental signals with the auxin gene regulatory network. For example, we have shown that certain Aux/IAA proteins confer drought tolerance by regulating the levels of secondary products called glucosinolates. These compounds promote stomatal closure and drought tolerance. In the next 5 years we will continue to investigate the molecular basis of auxin signaling and to characterize the auxin-based regulatory networks that control plant growth and development. We are very interested in the novel rapid auxin response pathway and its role in growth. We will also investigate the regulation of Aux/IAA levels by both SCFTIR1/AFB and CRL3-BPM. One of our long-term goals is understand the specificity of the AFB, Aux/IAA, and ARF proteins in collaboration with Joe Ecker's lab at the Salk Institute. This effort will include the investigation of AFB recruitment to chromatin. These studies address a number of key issues in cellular regulation and will have important implications for human health.

NIH Research Projects · FY 2025 · 2021-05

Many enzymes catalyzing ubiquitin-like (Ubl) modifications have been identified as targets for the therapeutic development of life-threatening human diseases that lack a cure. However, Ubl modifications are poorly addressed by FDA approved drugs, highlighting the immense potential to exploit this type of post-translational modification to address unmet medical needs. Conjugation of Ubls to target proteins begins by activation of the C-terminus of a Ubl, a step catalyzed by enzymes generally known as activating enzyme or E1. Several E1 enzymes, including the SUMO-activating enzyme (SAE), have been validated as therapeutic targets by animal models and by early phase clinical studies. Our recent discovery of a conserved cryptic site on the SAE provides a paradigm-shifting allosteric approach to inhibit Ubl activating enzymes. In the next funding period, we will further elucidate the mechanism of the structure-activity relationship. Additionally, we will validate that the allosteric inhibition approach we discovered reduces cancer drug resistance for targeting Ubl activating enzymes. Furthermore, we will define the molecular mechanism of how SUMOylation regulates type I IFN expression and validate that SAE inhibition is an effective approach to induce anti-tumor immunity for immune cold colorectal cancers. The proposed studies build on our scientific progress in the previous funding cycle and our more than 20-year experience in studying SUMOylation. The proposed studies are expected to result in long- lasting impacts that will spur innovation in targeting Ubl for therapeutic development and result in new immune therapeutic strategies for immune cold tumors that do not respond to current immune therapies and are high unmet needs, such as colorectal cancers.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract The first change in the brains of patients with Alzheimer’s and the best biomarker of the disease is synaptic loss. Several studies have shown that PSD-95 (a major scaffolding protein at the synapse) is significantly depleted in brain tissue from patients with Alzheimer’s as well as in neurons exposed to Aβ. Our data indicate that elevated synaptic PSD-95 blocks Aβ-induced synaptic depression. The amount of synaptic PSD-95 is controlled by a process called ‘palmitoylation’ which mediate the insertion of PSD-95 in post-synaptic membranes. Using a commercially available inhibitor of PSD-95 depalmitoylating enzyme, Palmostatin B, we showed that this drug could rescue Aβ-mediated effects on dendritic spines in vitro. In female APP/PS1 mice, we found that in the hippocampus, palmitoylated PSD-95 was significantly lower than in WT littermates while total PSD-95 levels were barely affected. Importantly, Palmostatin B injections in the intraperitoneal cavity rescued this effect in a dose dependent manner, indicating that this drug can access brain synapses in vivo. The key focus of the parent grant is thus to test the potential of a novel drug target, PSD-95 depalmitoylating enzyme, as a new therapeutic avenue against Alzheimer’s disease (AD). To do so, we proposed several different experiments, some of which were completed in the first 3.5 years of the grant. However, some key experiments still need to be completed and due to unforeseen circumstances, require this Administrative Supplement. In some of the research described in Aims 1 and 2, fluorescence lifetime imaging (FLIM) experiments were proposed to understand how Aβ reduces synaptic PSD-95 (Aim 1.2), to test if increasing other synaptic scaffolding proteins can also protect synapses from Aβ (Aim 1.3) and determine if Palmostatin B can rescue Aβ-induced changes in PSD-95 interactions (Aim 2.3). These experiments require a functional two-photon laser and our laser unexpectedly broke in September 2024. This Administrative Supplement would allow us to replace our laser, complete these key experiments and continue to use and develop FLIM to understand molecular mechanisms in synaptic physiology as our lab is one of the world’s leaders using FLIM in neuroscience research. Moreover, in Aim 3, we proposed behavioral experiments to assess if Palmostatin B can rescue memory deficits in vivo. Characterizing the effect of a drug on mouse behavior is the most convincing way of demonstrating therapeutic potential. These experiments were planned with Dr. Rissman, a leader in AD model mouse research. Dr. Rissman unexpectedly left UCSD in June 2024, making it impossible for us to use his behavioral equipment. To complete these essential experiments, we need to purchase our own behavioral equipment, which would be possible with this Administrative Supplement. Importantly, all the proposed research in this Administrative Supplement request is to complete experiments originally planned in the main grant, and thus within its scope. In conclusion, this innovative project will improve our understanding of how synapses are affected during AD and test a pharmacological approach to make synapses stronger from within, which would be beneficial for both treating and preventing AD.

NIH Research Projects · FY 2026 · 2021-05

Summary/Abstract The overall vision of our proposed research is to understand the structural, molecular, and cellular mechanisms by which germline mutations in protein kinase C gamma (PKCg) drive the neuro- degenerative disease Spinocerebellar Ataxia type 14 (SCA14). PKCg is a Ca2+/diacylglycerol-regulated kinase expressed only in neurons and primarily in Purkinje cells of the cerebellum. Purkinje cell degeneration is a hallmark of the almost 50 subtypes of SCA. We have assembled a team with extensive and complementary expertise in PKC mechanisms and structural biology to understand how PKCg mutations alter the structure and function of PKCg to contribute to the disease phenotype. In the previous funding period, we established that SCA14 mutations in PKCg break autoinhibitory contacts to produce an aberrant, more open, conformation resulting in enhanced basal activity. We now propose to determine whether disease pathology is caused by the leaky basal activity of this open conformation, driving aberrant phosphorylation, or by the aberrant conformation, driving an aberrant protein interactome. This will inform on how to target aberrant PKCg therapeutically. We aim to combine structural, biochemical, live-cell imaging, proteomics approaches and animal models to understand the molecular details of how disease-associated mutations impact function.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT The objective of this K23 Mentored Patient-Oriented Research Career Development Award is to assist the candidate in acquiring expertise and methodological skills to become an independent implementation science investigator focused on integrated substance use disorder and HIV care practices. The objective of the candidate’s research is two-fold: to reduce the detrimental impact of methamphetamine use while simultaneously supporting engagement in HIV care. People with HIV who use methamphetamine are susceptible to experiencing gaps at each stage of the HIV care continuum. Contingency management is an effective behavioral therapy for methamphetamine use disorder that is based on operant conditioning principles. Contingency management decreases the reinforcing effects of methamphetamine use by providing immediate, positive reinforcement following abstinence from substance use. Incentive-based interventions based on operant conditioning principles also demonstrate positive effects for improving antiretroviral therapy (ART) adherence. It remains unclear whether a contingency management program that integrates incentives for ART adherence (CM+ART, i.e., dually targeting methamphetamine use and ART adherence) is more acceptable and appropriate to people with HIV than a contingency management program targeting methamphetamine use only. The specific aims of the research plan for the five-year K23 award period are: 1) to identify resources required to implement contingency management programs in settings serving people with HIV, 2) to adapt contingency management to integrate incentives for adherence to ART (CM+ART), and 3) to evaluate whether CM+ART is acceptable and appropriate to people with HIV who use methamphetamine compared to a contingency management program targeting only reductions in methamphetamine use. Dr. Montoya’s mentored training plan, including formal didactics and other activities, aligns with the research aims and career development plans and has four key areas: 1) to deepen knowledge of evidence-based practices implemented at local and national levels to support patient engagement in HIV and substance use disorder care, 2) to apply an implementation science approach to guide adaptation of contingency management to integrate incentives for ART adherence, 3) to acquire advanced methodological skills to design and evaluate mixed-method studies and clinical trials, and 4) to engage in professional development activities geared toward development of a competitive NIDA R01 application and an independent research career. This proposal strongly aligns with NIDA Strategic Plan Objectives 3.1 (“Develop and test novel treatments based on the science of addiction”) and 3.4 (“Develop and test strategies for effectively and sustainably implementing evidence-based treatments”); the NIH Office of AIDS Research priority to address HIV- associated comorbidities such as substance use disorders; and the “Ending the HIV Epidemic: A Plan for America” initiative. The comprehensive training activities and research plan will effectively position the candidate for an independent research career focused on the integration of HIV and substance use disorder care.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Understanding how co-morbidities in persons with HIV (PWH) such as substance use affect risk-taking, decision- making, and other cognitive behaviors is important given implications for everyday functioning and transmission risk. The high prevalence of cannabis use in PWH, medicinally and recreationally, may indicate disease severity, impart therapeutic benefits, or adverse consequences. In fact, cannabis is recommended to those with HIV to alleviate nausea, improve appetite, relieve pain, and lift mood. To-date, the consequences of cannabis use in PWH remain unclear as do potential interactions with HIV treatments. In healthy participants, heavy cannabis use is associated with cognitive deficits e.g., risky decision-making, response disinhibition and inattention, but pro-cognitive effects in PWH may exist at mild use levels due to its anti-inflammatory and anti-excitotoxic properties. Furthermore, little has been done to determine the effects of cannabis use on the endocannabinoid (EC) system in general or in PWH. This area of study is especially germane to cognition since the virus affects brain regions rich in ECs. CNS relevance is of particular importance given that the EC system exerts regulatory effects over the dopaminergic system, critical for these cognitive processes. This application will utilize a cross- species approach to delineate the effects EC system activation has on HIV-relevant cognitive and motivational domains. Animal studies enable mechanistic insights on chronic and withdrawal effects in this system. Both behavioral and mechanistic overlap will occur between the human and animal studies. Specific Aim 1 will determine the effects of the two primary cannabis constituents (Δ9-tetrahydrocannabinol [THC], cannabidiol [CBD]) vs. placebo on risky decision-making, response inhibition, reward learning, temporal perception, and motivation, plus EC and homovanillic acid (HVA; a surrogate for dopamine activity) levels in HIV+ and HIV- subjects. Participants with infrequent cannabis use will undergo baseline cognitive testing and biomarker assays with antiretrovirals (ART) use quantified. They will be randomized to a 5-day course of either THC, CBD, or placebo and return for follow-up testing and re-assaying of ECs and HVA levels. Specific Aim 2 will conduct parallel experiments in a rodent model of HIV on acute, chronic, and withdrawal effects of 2 doses of THC vs. 2 doses of CBD, plus combined THC/CBD/ART (dolutegravir) on the same cognitive and motivational tests, plus EC and HVA levels to provide directionality and potential interaction of drug effects. Each experiment will train and test HIV-1 transgenic and wildtype littermate rats on cross-species versions of tasks used in Aim 1. Rats will be tested at baseline, immediately after acute, then chronic treatment, then during withdrawal. The brains of rats will be harvested and assessed for EC and dopamine receptor levels to determine potential mechanisms of the beneficial/negative effects of cannabinoid treatments on symptoms related to HIV. Disentangling the cognitive and biological effects of THC and CBD and their relation with ART is a much-needed advance in the HIV field and will inform development of therapeutics and policy advice for co-morbid substance use.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY As of January 2021, the World Health Organization (WHO) reports that 89 million cases of COVID-19 (SARS- CoV-2) have been confirmed and have resulted in more than 1.9 million deaths globally. Currently, the United States (U.S.) is the country with the largest number of infections and deaths due to COVID-19, with a total of 22 million infections and 373,167 deaths. Furthermore, early findings that have examined COVID-19 demographics show that racial and ethnic minorities in the U.S. are bearing a disproportionate number of COVID-19 cases and deaths irrespective of geographic region. While there's no evidence that people of color (POC) have genetic or biological factors that make them more likely to be affected by COVID-19, they are more likely to have underlying health conditions, live in multi-generational homes, live in densely populated areas, have limited access to healthcare, and have jobs that are considered essential and involve interaction with the public. All of these factors contribute to higher rates of infection and adverse outcomes due to COVID-19. Although COVID-19 preventive behaviors such as hand washing, mask wearing, and social distancing have been shown to be effective in curbing the spread of the virus, acceptance and uptake of COVID-19 vaccines will be instrumental to ending the pandemic. However, public confidence in vaccination is fragile, especially among racial and ethnic minorities. To this end, we have formed an intervention working group comprised of representatives from community and academic organizations to address challenges in COVID-19 vaccination uptake among Latinx and African American (AA) communities in Southern California by using a community-based participatory research (CBPR) approach. Project 2VIDA! (SARS-CoV-2 Vaccine Intervention Delivery for Adults in Southern California), is a multilevel intervention to address individual, social, and contextual factors related to access to, and acceptance of, the COVID-19 vaccine among Latinx and AA adults (>18 years old) across six highly affected communities in Southeast San Diego. 2VIDA! seeks to implement and assess a COVID-19 vaccination protocol to increase interest and uptake of COVID-19 vaccine, provide COVID-19 vaccines in the community, and establish a model for the rapid vaccination of Latino and AA adults that could be generalizable to other highly affected communities. 2VIDA! builds on our previous CBPR efforts and centers on conducting COVID-19 community outreach and health promotion, Individual awareness and education, and linkages to medical and supportive services and offering the COVID-19 vaccine to Latinx and AA adults (>18 years old) in community health centers (CHC) and mini-vaccination stations in communities highly impacted by the pandemic in San Diego County.

NIH Research Projects · FY 2025 · 2021-04

The G protein-coupled chemokine receptor, CXCR4, and the atypical chemokine receptor, ACKR3, play critical roles in cell migration during immune responses and organ development, through coordinated responses to a shared ligand, CXCL12. Both receptors contribute to numerous inflammatory and autoimmune diseases and are under active investigation as therapeutic targets. Nevertheless, there is currently only one FDA-approved CXCR4 antagonist (AMD3100/Plerixafor), and its use is limited to mobilizing hematopoietic stem cells for bone marrow transplants, because many of its properties are suboptimal. Therapeutic targeting of ACKR3 is at a less mature stage than CXCR4; in fact, most known compounds are agonists, and it is unclear how to antagonize this receptor. Improved compounds targeting both CXCR4 and ACKR3 are therefore needed. As an "atypical" receptor, ACKR3 is widely assumed to function only through β-arrestin (and not G proteins), and is best known for its ability to “scavenge” CXCL12 from the extracellular environment. By doing so, ACKR3 prevents downregulation of CXCR4 and maintains its responsiveness to CXCL12 gradients. When co-expressed in the same cell, ACKR3 can also alter CXCR4 signaling and trafficking via heterodimerization, sequestration of β-arrestin, and other as-yet-undeciphered mechanisms. Given that ACKR3 binds CXCL12 in an architecture similar to CXCR4, undergoes similar conformational changes upon activation, and shares all of the conserved G protein-coupling determinants, its presumed Gi incompetency is striking. Even more striking is the exceptional robustness of ACKR3 activation to ligand and receptor modifications, whereas CXCR4 activation is abrogated by the subtlest of such changes. Because of this activation-prone nature, most non-chemokine (and even small molecule) ligands activate ACKR3 association with β-arrestin, with unknown downstream consequences. Despite the role of the two receptors in disease, the structural and molecular mechanisms underlying their individual functions and their cellular crosstalk remain elusive. In this MPI proposal, the Handel and Kufareva labs combine their experimental and computational expertise, respectively, with their in-depth knowledge of chemokine receptors, to explain the distinct activation mechanisms of CXCR4 (Aim 1) and ACKR3 (Aim 2) from the standpoint of structure and dynamics, to understand how to inhibit these receptors (Aims 1 and 2), and to understand how ACKR3 regulates the function of CXCR4 (Aim 3). To achieve these aims, specific mechanistic hypotheses are probed with a combination of structural (cryo-EM and crystallography), computational (modeling and MD) and cell-based functional experiments, and complemented by unbiased discovery proteomics. These studies will deliver unprecedented insight into the function of CXCR4 and ACKR3, which will have a direct impact on the development of small molecule therapeutics and provide the rationale for blocking one or both receptors. By revealing general principles, the proposed studies will also advance the understanding and targeting of other therapeutically important chemokine receptors, which are considered challenging targets.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Alternative splicing of pre-mRNAs is extensively employed by the nervous system to expand the transcriptomic manifold. Regulated in specific cellular contexts by multiple RNA-binding proteins (RBPs), this process is a major contributor to cellular identity that acts orthogonally to transcriptional regulation and has been implicated in many neurodevelopmental disorders. Understanding and integrating the impact of alternative RNA splicing on the establishment and organizational hierarchy of interneuron subtype specification is therefore of high value, though there has been relatively little progress in this direction. To address this gap, we have designed a high throughput system to determine the role of alternative splicing regulatory networks in cortical interneuron subtype specification. We hypothesize that alternative splicing is an informative component of interneuron cell fate decisions. Using scRNA-seq datasets with in-depth and full transcript read coverage across diverse neuronal cell types, we will identify key RBPs by highly correlated expression with cell-type specific splicing patterns together with integrative analysis of RBP target networks. We will then test the role of these RBPs by CRISPR knockout in mESCs differentiated into interneuron subtypes using a dual interneuron lineage fluorescent reporter line that we have developed. Training in splicing analysis in the expert environment of the Zhang lab is an ideal complement to the trainee’s previous extensive training in interneuron biology. The proposed studies and training plan will provide a fertile basis for a productive independent research program synthesizing traditional neurodevelopment with state of the art bioinformatics. Alternative splicing and cellular diversity are known to be closely intertwined and important in mature neurons; a better understanding of the inception of these relationships will be greatly impactful to our understanding of the nervous system in health and neurodevelopmental disease.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Now in its 13th year, the Psychiatric Genomics Consortium is perhaps the most innovative and productive experiment in the history of psychiatry. The PGC unified the field and attracted a cadre of outstanding scientists (802 investigators from 157 institutions in 41 countries). PGC work has led to identification of ~500 genetic loci in the 11 psychiatric disorders we study. Our work has led to 320 papers, many in high-profile journals (Nature 3, Cell 5, Science 2, Nat Genet 27, Nat Neurosci 9, Mol Psych 37, Biol Psych 25). As summary statistics are freely available, psychiatric disorders often feature prominently in papers by non-PGC investigators. To advance discovery and impact, we propose to continue the work of the PGC across 11 disorder groups. Considerable new data are coming in the next five years. We thus can rapidly and efficiently increase our knowledge of the fundamental basis of major psychiatric disorders. Aim 1: we will continue to advance genetic discovery for severe psychiatric disorders in all working groups, systematically interface with large biobank studies to ensure maximal comparability, and aggressively promote new studies of individuals with psychiatric disorders from diverse ancestries to increase discovery and improve fine-mapping. Aim 2: most studies analyze common variation (Aim 1), rare CNV (Aim 2), and rare exome/genome resequencing results (via collaboration) in isolation: we will apply an integrative framework to rigorously evaluate the contributions of all measured types of genetic variation on risk for psychiatric disorders. Aim 3: we will move beyond classical case-control definitions to a more biologically-based and nuanced understanding by enabling large trans-diagnostic studies, convene trans-disciplinary teams to use genetics to address unresolved questions about the nature of psychiatric disorders, and to promote large studies of the severest cases seen in psychiatric practice (leveraging the global reach of PGC investigators). Aim 4: we will work to maximize the impact of our work via translational efforts: close collaborations with neuroscience consortia to understand the biological implications of our findings; work to identify modifiable causal risk factors; and work to robustly predict clinical outcomes and identify patient subsets. Aim 5: we will increase impact of our work by extending and formalizing outreach to different communities (including pharma and biotech), via digital media (Twitter, Facebook, Wikipedia), and by developing, distributing, and updating resources/educational material for patients, families, and medical professionals. We will convene a Scientific Advisory Board to ensure we respond positively to those invested in our results Successful completion of this body of work will greatly advance knowledge of the genetic basis of psychiatric disorders with potentially major nosological and treatment implications. These goals are consistent with a core mission of the NIMH, and the central idea of the PGC: to convert the family history risk factor into biologically, clinically, and therapeutically meaningful insights.

NIH Research Projects · FY 2025 · 2021-04

The human placenta is a semi-allogeneic tissue whose growth and development requires tolerance by the maternal immune system. Placental cells come in close contact with maternal blood and uterine tissues yet are able to evade immune recognition during the course of a normal pregnancy. The maternal immune system faces a challenge during pregnancy: to maintain tolerance toward foreign fetal alloantigens while simultaneously staging a response to potential pathogens at the maternal-fetal interface. The mechanisms through which placental cells evade maternal immune recognition are poorly understood, particularly in the context of human pregnancy. The uterine lining, called decidua, is a particularly understudied and important microenvironment, because it is the interface where placental cells called extravillous trophoblast (EVT) come in close contact with maternal immune cells, of which decidual natural killer (dNK) cells are the most abundant. EVT are highly invasive cells which are required for proper remodeling of the maternal uterine lining, including vascular remodeling which leads to establishment of maternal blood flow to the placenta. Interactions between placental EVT and decidual leukocytes are known to facilitate maternal vascular remodeling by EVT and limit the extent of EVT invasion into the uterine wall. Indeed, problems in preterm birth could result from inappropriate responses by dNK cells. Unfortunately, interactions between dNK and trophoblasts are difficult to study in an ongoing pregnancy, due to lack of access to the decidual compartment, where these important interactions occur. While animal models have offered some insights into these processes, they do not accurately model human placentation and pregnancy. This proposal aims to evaluate the decidual cell population in both term and preterm birth in a systematic, detailed manner, then to combine this knowledge with the latest technologies in regenerative medicine in order to develop in vitro models for the study of dNK-EVT interactions. Over the past few years, our collaborative team of investigators has established optimized methods for differentiation of pluripotent stem cells into both NK cells and EVT. We now propose to generate matched maternal and placental induced pluripotent stem cells (iPSC), and differentiate these cells into dNK cells and EVT, respectively, in order to model interactions between these two cell types. Successful completion of this proposal will establish a reproducible and manipulatable model system for studying interactions between the placenta and maternal immune system and has the potential to lead to identification of mechanisms through which abnormalities in these interactions increase the risk of idiopathic spontaneous preterm birth.

NIH Research Projects · FY 2025 · 2021-04

Obesity-induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of Type 2 diabetes mellitus and is thus the driving force behind the emerging diabetes epidemic. Current anti-diabetic therapeutics are available, but are inadequate to control the disease in most patients and there is a large unmet medical need for better methods of treating diabetes to prevent morbidity and mortality. Our recent work has led to the discovery that obesity induces a dynamic change in secretion of hepatic extracellular vesicle (EV) miRNAs that exert profound impacts on insulin producing cells and peripheral insulin sensitivity. Depletion of hepatic extracellular miRNAs in the hepatocyte-specific Rab27KO mice resulted in impaired glucose tolerance and insulin sensitivity at the early onset of obesity. We further demonstrated that 4wks high-fat diet feeding (4wks-HFD)-induced hepatic EV miRNAs can reduce the insulin resistance of obese recipient mice. In addition, the 4wks-HFD EV treatment significantly enhanced the insulin secretion and proliferation of beta cells in vitro and in vivo. By contrast, prolonged obesity induced secretion of pathogenic hepatocyte EV miRNAs that blunted insulin sensitivity of lean recipient WT mice. Consistently, the mice without hepatic extracellular miRNAs by knockout of hepatic Rab27 showed a reduction in insulin resistance after 16 weeks HFD feeding. miRNA-free EVs derived from YBX1KO hepatocytes had minimal effects on the metabolic phenotypes of recipient mice, suggesting miRNAs as key cargoes within these EVs. Using a novel thiouracil tagging method, we identified that miR-3075-5p, a highly enriched miRNAs in 4wks-HFD EVs, can be efficiently incorporated into target cells and improves cellular insulin responses through repressing Fa2h expression. In addition, the miR-434-3p-Map2k6 regulatory axis plays a critical role in promoting proinflammatory activation of macrophages, which can subsequently exacerbate tissue inflammation and insulin resistance. These results lead to the conclusion that hepatic EV miRNAs are important endocrine molecules regulating functions of insulin-producing and -targeting cells in obesity. This proposal sees to build on this newly identified hepatic EV miRNAs regulatory system to reveal the underlying cellular and molecular mechanisms of obesity-induced insulin resistance. We will further determine the mechanisms by which hepatic EV miRNAs modulate functions of beta cell and insulin sensitizing cells in response to obesity. With the proposed experiments, we will develop miR-3075-5p as an insulin sensitizer molecule and explore the pathogenic effect of miR-434-3p in obesity. This therapeutic strategy could be used for the treatment of obese patients with insulin resistance pre-diabetic state. This would lead to improved glycemic control adding a new component in our therapeutic armamentarium for the treatment of this widespread metabolic disease. Finally, using the thiouracil tagging method, we will identify the hepatic extracellular miRNAs in circulation as biomarkers predicting the insulin resistance state in obesity.

NIH Research Projects · FY 2025 · 2021-04

Targeting Systems Vulnerabilities in the Gαq/GNAQ Oncogenic Signaling Circuitry: New Precision Therapies for Uveal Melanoma G protein-coupled receptors (GPCRs) represent the largest family of cell surface proteins involved in signal transmission. GPCRs play key physiological roles and their dysfunction contributes to some of the most prevalent human diseases, making them the target of >25% of all therapeutic drugs. Strikingly, our recent analysis of human cancer genomes revealed an unanticipated high frequency of mutations in G proteins and GPCRs in most tumor types. Indeed, nearly 30% of human cancers harbor mutations in GPCRs or G proteins. While their tumorigenic potential is under investigation, activating mutations in GNAQ and GNA11 (herein referred as GNAQ oncogenes, which encode GTPase deficient and constitutively active Gαq proteins), were identified in ~93% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM), respectively, where they act as oncogenic drivers. UM is the most common primary cancer of the eye in adults, affecting more than 2,500 patients each year in the US alone, nearly 50% of which will die from liver metastasis. To date, there are no effective therapeutic options to treat metastatic UM disease (mUM). We recently demonstrated that YAP activation is central to UM growth and uncovered a novel direct link between Gαq-FAK driven tyrosine phosphorylation networks and YAP activation. Our central hypothesis is that this signaling specificity may represent a systems vulnerability that can be exploited for the development of new precision therapies for mUM. Our overall hypothesis is that our proposed studies targeting FAK, which acts downstream from Gαq, and its compensatory (resistance) or synthetic lethal (sensitizing) mechanisms will provide an oncogene-specific therapeutic approach for advanced and mUM, resulting in increased antitumor activity with lower toxicities and fewer side effects. Ultimately, our premise is that FAK is an integral part of the GNAQ oncogenic pathway and that in turn, FAK blockade with clinically relevant FAK inhibitors (FAKi) may represent a precision therapeutic approach for the treatment of mUM, alone or as part or as part of novel signal transduction-based precision co-targeting strategies. This will be investigated in 3 aims: Aim 1: To exploit GNAQ-synthetic lethal and gene interaction networks to expose systems vulnerabilities resulting in UM cell death as a precision therapeutic approach to treat mUM. Aim 2. To establish the therapeutic potential of co-targeting the Gαq-FAK regulated pathway in vivo. Aim 3. Characterization of FAKi/MEKi tolerant persister populations and mechanisms of acquired resistance

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Mutations in tafazzin (Taz, also known as G4.5) cause Barth syndrome (BTHS, MIM 302060), a life-threatening disorder disrupting metabolism of the mitochondrial-specific phospholipid cardiolipin (CL). Cardiomyopathy is the major clinical feature in BTHS, highlighting the importance of Taz and the CL metabolism pathway in cardiomyocytes (CMs). Taz encodes a mitochondrial phospholipid-lysophospholipid transacylase, which is essential for CL remodeling to achieve the characteristic fatty acid composition of mature CL. Mutations in Taz found in BTHS patients result in low total CL concentrations, abnormal CL fatty acyl composition, and elevated monolyso-CL (MLCL) to CL ratios. However, little is known as to the detailed molecular mechanisms by which Taz deficiency and consequent CL abnormalities lead to the progression of cardiomyopathy. Thus far, there is no curative therapy for BTHS. Although it has been established that Taz deficiency causes BTHS, lack of a Taz knockout mouse model has hindered studies of molecular pathology and developments of therapeutic approaches for BTHS. To elucidate the molecular pathogenic mechanism of BTHS cardiomyopathy, and to identify potential targets for therapeutic intervention, we have generated Taz CM-specific knockout (cKO) mice and observed dilated cardiomyopathy (DCM) phenotypes, as well as mitochondrial malformations and dysfunction in Taz cKO mice. Our data strongly suggest a critical role of Taz and CL in cardiac and mitochondrial function. Our Taz cKO mouse provides us with a unique model to investigate the molecular basis for and potential therapeutic approaches to BTHS. Studies in cultured cells suggest that linoleic acid (LA) supplementation increases mature CL levels in Taz-deficient cells by increasing incorporation of linoleoyl groups into de novo synthesized CL and also ameliorating the increase in MLCL. Inhibition of the mitochondrial phospholipase A2 (PLA2) by bromoenol lactone (BEL) also ameliorates increased MLCL in Taz-deficient cells by blocking generation of MLCL from nascent CL. However, these potential therapeutic approaches have not been studied in an in vivo mammalian model of BTHS. Moreover, no study has explored if a combination of LA supplementation and BEL treatment can act synergistically to ameliorate BTHS. Accordingly, our hypothesis is that Taz-mediated CL remodeling is essential to maintain mitochondrial homeostasis and CM function, and that linoleic acid (LA) and/or bromoenol lactone (BEL) treatment will provide beneficial effects to ameliorate BTHS cardiomyopathy. Our specific aims are: (1) To investigate the role and molecular mechanisms by which Taz- mediated CL remodeling is required in maintaining CM mitochondrial homeostasis and normal cardiac function by histological, physiological, biochemical, and molecular analyses of Taz cKO mice; and (2) To assess therapeutic effects of linoleic acid (LA) and mitochondrial PLA2 inhibitor bromoenol lactone (BEL), as single agents or in combination, on BTHS cardiomyopathy by utilizing Taz cKO mice.

NIH Research Projects · FY 2025 · 2021-04

Project summary: The overall goal of our research is to uncover the molecular and cellular mechanisms by which mTOR signaling is spatially regulated and to elucidate the contribution of subcellular mTORC1 signaling to tumorigenesis and cancer therapy resistance. The signaling pathway regulated by phosphatidylinositol 3- kinase (PI3K) and mechanistic target of rapamycin (mTOR) regulates a number of processes that are critical to cell physiology, and therefore is often dysregulated in diseases, including cancer. In particular, persistent activation of the PI3K/mTOR signaling circuitry is the most frequent dysregulated signaling mechanism in oral squamous cell carcinoma (OSCC), a disease that results in ~300,000 deaths each year worldwide, with 5-year survival estimates of approximately 60%, despite aggressive multimodality therapies. Spatial compartmentalization of PI3K/mTOR is not only critical for enhancing the signaling specificity, but also required for proper functioning of the pathway. However, the mechanisms underlying spatial regulation of PI3K/mTOR signaling remain poorly understood and it is not clear which subcellular pools of the signaling molecules contribute to tumorigenesis and therapy resistance. We have assembled a strong interdisciplinary team with complementary expertise, including Dr. Jin Zhang, an expert in chemical biology and kinase signaling, Dr. J. Silvio Gutkind, a renowned cancer biologist whose lab has focused on the study of oncogenic signaling pathways driving OSCC initiation and progression. In our previous studies, we have created novel tools for studying the spatial regulation of mTOR signaling, including a fluorescent biosensor for tracking mTOR Complex 1 (mTORC1) activity in living cells and an approach for achieving subcellular inhibition of kinase signaling. Using these tools, we discovered novel mechanisms underlying regulation of nuclear mTORC1. In the context of OSCC, we have shown that mTOR inhibition exerts potent antitumor activity in a large series of genetically-defined and chemically-induced OSCC models and favorable clinical responses in a recently completed clinical phase II trial (NCT01195922). The current proposal will develop new molecular tools to interrogate the spatiotemporal regulation of mTORC1 signaling in living cells, elucidate the regulatory mechanisms of nuclear mTORC1 signaling, and determine the functional roles of subcellular mTORC1 signaling in tumorigenesis and Cetuximab resistance in OSCC. Unravelling the function and regulation of subcellular mTORC1 signaling should offer a path toward selective targeting of pathway components and yield therapies with reduced toxicity and resistance.

NIH Research Projects · FY 2025 · 2021-04

Cytoplasmic aggregation of TDP-43 has been reported in almost every age-dependent neurodegenerative disease, including in >40% of frontal temporal dementia (FTD), in the hippocampal neurons of Alzheimer's disease (AD) patients, in >90% of ALS, and in ~100% of a recently recognized AD-like dementia in the oldest of the elderly, an AD-like syndrome identified in 2019 and named Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). We have demonstrated that TDP-43 phase separation and aggregation can drive neuronal death independent of RNA binding, stress granule formation, and TDP-43 association with stress granules. We have subsequently identified that acetylation of TDP-43 (which abolishes its RNA binding) drives its separation into liquid spherical annular bodies. These nuclear annuli have liquid annular shells enriched in TDP-43 and liquid centers highly enriched in HSP70 family molecular chaperones. Use of inhibitors of known deacetylases or the proteasome (to mimic the known age-dependent declines in deacetylase and proteasome activities) provokes cytoplasmic TDP-43 aggregation. We propose to determine the biological and pathological role(s) of acetylated TDP-43 and how HSP70 chaperone activity regulates nuclear TDP-43 function and its aggregation in the cytoplasm. We will determine the regulation and biological consequences of acetylated TDP-43 in neurons by identifying the key regulatory enzymes (acetyltranferases and deacetylases) of acetylated TDP-43 and alter TDP-43’s function in RNA splicing and its subcellular localization/aggregation. To understand how HSP70 family molecular chaperones regulates phase transition of TDP-43, we will use Hsc70 (encoded by the HSPA8 gene and the most abundant HSP70 in neurons) and determine how Hsc70 interacts with TDP-43. We will also determine if enhancing the activity of HSP70 (such as HSPA8, which is highly expressed in neurons) ameliorates TDP-43 pathology. We will also develop a potential therapeutic approach for TDP-43 proteinopathies in which rapid proteasome-mediated degradation of aggregated TDP-43 is achieved through an engineered E3 ubiquitin ligase linked to a synthetically evolved nanobody (a single chain antibody derived from an antibody heavy chain) recognizing either acetylated or phosphorylated TDP-43. Outcomes of these efforts will provide key insights for understanding basic aspects of TDP-43 biology and pathobiology in common dementia, and for developing a new concept of therapy that specifically targets TDP- 43 pathology that could potentially benefit aged patients with TDP-43-related dementia.