Mayo Clinic Jacksonville

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY An estimated 3.1 million cases of myocarditis/cardiomyopathy were diagnosed in 2017. Patients with myocarditis are at risk of sudden death from acute heart failure and progression to dilated cardiomyopathy, often necessitating a heart transplant. The incidence and severity of myocarditis is higher in men than women. Additionally, there are no disease-specific therapies to reduce myocarditis. Enteroviruses including coxsackievirus group B3 (CVB3) are a common cause of myocarditis in the US. Viruses are frequently found in endomyocardial biopsies from patients with myocarditis. There is currently no clear understanding of why an enterovirus like CVB3 would target the heart or the mechanism for how a relatively mild viral infection like CVB3 leads to heart failure. Previously it was hypothesized that an overwhelming lytic CVB3 infection damages the heart. It was recently published that the predominant mode of viral egress for CVB3 from cardiomyocytes is in extracellular vesicle-like mitochondrial autophagosomes, and that CVB3 requires mitochondrial fission for viral replication. Interestingly, other viruses that cause myocarditis have been found to use mitochondria to promote viral replication including influenza A, HIV, poliovirus, hepatitis C virus and SARS-CoV-2. The high energy demands of cardiac muscle and abundant mitochondria in the heart provide for the first time an explanation for why such disparate types of viruses, with no obvious cardiac tropism, replicate in the heart. The overall goal of this proposal is to determine whether the CVB3 replicative cycle through mitochondria in cardiomyocytes induces sex differences in cardiac inflammation during myocarditis and to determine whether microRNA and/or protein from mitochondrial-derived extracellular vesicles influence viral replication and inflammation in a sex-specific manner.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT We seek to perform in-depth characterization studies to unravel the clinico-pathological variability associated with frontotemporal dementia (FTD) and related neurodegenerative diseases caused by an expanded repeat in the C9orf72-SMCR8 complex subunit (C9orf72). To this end, we will examine a range of neuroanatomical regions (e.g., frontal cortex, temporal cortex, motor cortex, spinal cord, and cerebellum) and span the full spectrum from frontotemporal lobar degeneration (FTLD) – a neuropathological diagnosis of FTD – to amyotrophic lateral sclerosis (ALS). For each region, we will assess features of C9orf72-linked diseases, such as levels of C9orf72 transcripts, RNA foci, and dipeptide repeat proteins (Aim 1). Moreover, we will use an innovative targeted long-read sequencing technology to measure the size, purity, and methylation status of the expanded repeat. Additionally, we will perform whole-tissue RNA sequencing to detect overall changes in a complex context, basically taking a snapshot (Aim 2). This data will be accompanied by single-nuclei transcriptomic data to acquire gene expression levels while deciphering the cellular diversity encountered in the central nervous system. Since splicing defects have been reported in C9orf72-related diseases, we will combine short- and long-read sequencing strategies. The inclusion of long-read sequencing data creates the opportunity to obtain the full-length transcriptome with highly accurate splice junctions, even for large transcripts. These combined approaches, therefore, will enable us to capture the entire landscape of transcriptomic changes. Top hits identified through our in-depth transcriptomic studies will be further investigated in our extensive collection of clinical and pathological specimens and tested as potential biomarkers. Taken together, our detailed examinations will uncover crucial region- and disease-specific similarities and/or differences, thus laying the foundation for improved understanding of the heterogeneity associated with the FTLD-ALS spectrum and the identification of disease modifiers, translatable biomarkers, and therapeutic targets.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY/ABSTRACT Development of cognitive problems at any age is devastating, but development of cognitive problems in working-age people with dependents represents a major public health concern. Young-onset Alzheimer's disease (YOAD) is defined as individuals who present before age 65 and lack mutations known to cause Alzheimer's disease (AD) pathology. Neuropathology and neuroimaging studies demonstrate greater tau accumulation in YOAD who often present with atypical, non-amnestic syndromes. Our preliminary data demonstrates that tau accumulation occurs through progressive maturity levels in tangle-bearing neurons, which disproportionately affects cortical more than limbic structures in YOAD. Moreover, we show younger age onset is associated with greater tangle accumulation and neuronal loss in nucleus basalis of Meynert (cholinergic hub) and locus coeruleus (noradrenergic hub) – two neuromodulatory hubs implicated in early stage of disease. As stereotypic amyloid-β plaque patterns are robustly observed regardless of age, and comorbid neuropathologies are less frequent in YOAD, this cohort is ideally suited for a targeted investigation of selective vulnerabilities to tangle pathology in AD. Regional vulnerabilities to advanced tangle maturity levels in corticolimbic structures and neuromodulatory hubs are hypothesized to underlie the syndromic heterogeneity observed in YOAD. The overall goal of this grant is to uncover signatures of regional and cellular vulnerabilities underlying syndromic heterogeneity in YOAD by investigating what modifies patterns of tangle accumulation and microglial activation. Our preliminary data from single-cell RNA sequencing underscores the importance of considering disease heterogeneity and the utility of quantitative neuropathology for validating gene expression changes. This proposal seeks to shift current research in AD by focusing on younger-aged individuals and demonstrating how regional variability can inform cellular biology even in the context of end-stage disease. To accomplish our goals and facilitate stratification by atypical and typical (amnestic) clinical syndromes, the MPI team has combined expertise and resources to amass one of the largest documented YOAD cohorts totaling 558 brains with available tissue for study. The goal of the grant is to test the following hypotheses: 1) Modifiers of the neuropathologic patterns of tau pathology in YOAD brains differ between cases stratified by atypical vs. typical (amnestic) clinical syndromes, 2) The most vulnerable neuronal populations to AD-tau share a similar molecular signature across corticolimbic regions reflective of syndromic heterogeneity in YOAD, and 3) Corticolimbic microglial activation patterns differ in the brains of YOAD cases stratified by atypical vs. typical (amnestic) clinical syndrome. Completion of this project will identify specific cell populations vulnerable to regional AD-tau pathology and identify modifiers of microglial activation patterns corresponding to aggressive tau accumulation in YOAD.

NIH Research Projects · FY 2026 · 2022-02

Postpartum-related breast cancers (PRBCs), herein defined as breast cancers (BCs) diagnosed within five years after a birth, augur a poor prognosis. Although early age at first birth and multiparity lower risk of late onset BCs, childbirth transiently increases risks of both estrogen receptor (ER)+ and ER- BCs for over two decades, with peak risks at 5 years postpartum (parous vs. nulliparous: HR=1.80, 95%CI=1.63-1.99). Peak risks are even higher among women with a family history of BC (parous vs. nulliparous: HR=3.53, 95%CI=2.91-4.29), and for women whose first birth is at later ages (pinteraction=0.03). In preclinical models, post-partum involution (PPI), a pro-inflammatory process which restores the breast to a non-lactating state after weaning, is linked to activation of cyclooxygenase-2 (COX-2), which increases production of prostaglandins and results in an immune suppressed, pro-carcinogenic “wound healing” microenvironment that drives BC progression; in these models, treatment with non-steroidal anti-inflammatory agents (NSAIDs) inhibits PRBC development. Among women, both postpartum normal breast tissues and BBD biopsies are characterized by significant inflammation. We and others have reported significantly reduced risk of BC among NSAID users with benign breast disease (BBD), suggesting a potential preventive benefit for women at risk of PRBC. We propose 3 aims to test the hypothesis that dysregulated PPI increases risk of BBD and PRBC, and that inhibition of deleterious inflammation following PPI can lower BC risk. In Aim 1, we will define and validate a PPI-specific immune signature score to distinguish normal breast tissues of young nulliparous from parous women (obtained within 5 years of a birth, matched by age) using 240 samples donated to the Komen Tissue Bank (KTB). We will also measure eicosanoids, including prostaglandin E2, in frozen tissues from a subset (n=100) of these women. We will define factors related to PPI immune signature score, hypothesizing that higher scores may indicate increased PRBC risk. In Aim 2, we will refine and independently test whether a PRBC immune signature score based on 8 previously defined markers with a combined AUC=0.76 predicts risk in previously untested BBD biopsies (75 cases and 75 matched controls). We will compare PRBC immune signature scores in 707 BCs from women aged 40 years or less by parity status, molecular subtype, and adjusted for potential confounders. In Aim 3, we will conduct a window of opportunity clinical trial to test if a 6-week course of aspirin 81 mg per day can reduce the deleterious inflammation associated with PRBC risk (i.e., PRBC immune score) and favorably alter other BC risk markers in breast tissues, blood and urine. This significant and innovative proposal seeks to define a unifying mechanism of pathogenesis for PRBC, which will provide the basis for developing a short-term, well-tolerated prevention strategy using immune-targeting and/or anti-inflammatory agents to prevent BC.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY/ABSTRACT The human trypsin isoforms, trypsin 1, trypsin 2, and mesotrypsin, are proteases that have been implicated in disease processes in cancer and pancreatitis, and may offer viable therapeutic targets. Trypsins belong to a large family of trypsin-like enzymes with similar active site topology, and hence existing inhibitors lack selectivity. There is a need for selective trypsin inhibitors and isoform-selective trypsin inhibitors as pharmacological tools to better define the functions of these individual enzymes in disease, and to evaluate trypsin inhibition as a therapeutic strategy in preclinical models of disease. In this project, we will take a multipronged approach to develop new strategies for potent and selective inhibition of each of the human trypsin isoforms. (1) Our preliminary data reveal a previously unsuspected auto-inhibited conformation of mesotrypsin with a ligand-targetable allosteric site that may be exploited for inhibitory effect. We will use high- throughput virtual screening and structure-based hit-to-lead optimization to develop potent and selective allosteric inhibitors of mesotrypsin. We will also use structural and molecular dynamics analyses to evaluate whether similar strategies may hold potential for trypsins 1 and 2. (2) Our published studies have shown that Kunitz domains can be engineered to create more selective protein-based inhibitors of trypsin-like proteases by using a yeast surface display (YSD) platform for directed evolution. To enable further optimization of such inhibitors, we seek to generate comprehensive maps of the binding specificity landscapes that can, for any possible combination of mutations within an inhibitor, predict the consequences on inhibitor affinity and specificity toward a set of target proteases. We will accomplish this task by integrating YSD combinatorial library screening with next-generation sequencing (NGS), machine-learning (ML) approaches, and experimental calibration to enable quantitative prediction of the impact of multiple potentially interacting mutations of an inhibitor. These data will enable us to identify the most potent and selective Kunitz domains that can be achieved for targeting each of the human trypsins. (3) Our preliminary data demonstrate an enhancement of trypsin affinity by bivalent inhibitors capable of binding simultaneously to two molecules of mesotrypsin. Here, we will dissect the mechanisms responsible for these affinity enhancements and design strategies to exploit this information toward development of more potent and selective polyvalent trypsin inhibitors. In addition to developing three complementary strategies, each of which has high potential to produce the desired selective inhibitors of human trypsins, our project will provide broader insights that can aid future development of inhibitors for many other important trypsin-like proteases. Finally, the novel methodology developed here for mapping protein-protein interaction (PPI) affinity and specificity landscapes will be of broad utility for characterizing the sequence and structural constraints governing affinity and selectivity of functional protein interactions in many other diverse systems.

NIH Research Projects · FY 2025 · 2021-09

CREST-2 (clinicaltrials.gov number: NCT02089217) is a pair of parallel, actively recruiting procedural trials to prevent stroke comparing: 1) carotid endarterectomy (CEA) and intensive medical management (IMM) to IMM alone, and 2) carotid artery stenting (CAS) and IMM to IMM alone. CREST-2 is designed to have ≈3-year average follow-up (maximum, 4 years). 1,735 patients have been randomized as of 10/29/2020. CREST-2 IMM involves centralized iterative titration to maximum tolerated effect of medications to treat hypertension and hyperlipidemia. We have already demonstrated that CREST-2 IMM results in significant improvements in rates of vascular risk factor control. We propose a highly cost-efficient, centralized process involving telephone and telehealth visits and review of medical records to monitor for stroke endpoints and home health visits to monitor control of vascular risk factors after patients graduate from CREST-2 to assess rigorously the transferability and real-world long-term effectiveness of intensive medical management with or without carotid revascularization. Our proposed long-term observational extension (LOE) is significant regardless of the CREST-2 results. If revascularization fails to show superiority (i.e., if IMM-only treatment is superior or not significantly different), proponents of revascularization will argue that the average follow-up was not sufficiently long to document the benefit of the procedure. The intersection of risk curves in the Asymptomatic Carotid Atherosclerosis Study, which supported benefit of CEA compared to medical management, was noted at ≈2 years following study entry. Contemporary IMM likely pushes the point of intersection outward. If revascularization is shown to be superior in CREST-2, then the durability of the benefit of revascularization beyond the 3-year average follow-up will be questioned due to possible restenosis. We propose obtaining written informed consent on all CREST-2 patients for continued follow-up for an additional 5 years, providing a 7.5-year average follow-up, with a subset of patients followed to >10 years. Cost-effectiveness will be achieved through innovative follow-up methodology. Our approach is also designed to minimize patient burden and maximize retention. The primary aim of this proposal is estimation of post- procedure treatment differences between revascularization and IMM vs. IMM alone. The currently funded CREST-2 trial will provide data on post-procedure treatment differences up to ≈3 years. Extending follow-up will enable us to assess whether post-procedure benefit is maintained during intermediate (4-6 years) and long-term follow-up (7-10 years); thereby, testing durability. The primary outcome for the CREST-2 LOE is the composite of stroke and death within 44 days after randomization and ischemic stroke ipsilateral to the randomized vessel thereafter. Our LOE approach is significant because it can be applied to a wide variety of stroke prevention trials where the therapeutic intervention is a device or procedure.

NIH Research Projects · FY 2024 · 2021-09

SUMMARY/ABSTRACT Hypothalamic amenorrhea (HA) occurs during reproductive years and results in ovulatory dysfunction, anovulation and infertility which can be prolonged from months to years. There are multiple HA phenotypes including varying combinations of psychosocial stress, anxiety, high levels of physical activity and/or weight loss. Large population studies, including the original Nurses’ Health Study have related menstrual cycle irregularity/amenorrhea with cardiovascular disease (CVD) events. However, these analyses have not differentiated the HA phenotype from polycystic ovary syndrome (PCOS) and other lower prevalence menstrual disorders. Emerging data from our group indicates that one-third of women with HA (mean age 27 yrs) have preclinical CVD measured noninvasively as vascular dysfunction, and circulating cytokine patterns indicative of vascular inflammation compared to age-matched eumenorrheic controls not on hormone therapy. Our proposed research application responds to the funding opportunity announcement PA-20-281, “Fertility Status as a Marker for Overall Health” and will study HA, a marker of fertility status, related to cardiovascular health. In Aim 1, we will use innovative remote patient monitoring and patient reported outcomes to investigate HA specific phenotype(s) related to preclinical CVD (Aim 1a) and vascular inflammation (Aim 1b). We will then expand our analysis in Aim 2 to the Nurses’ Health Study II, a large prospective cohort study that can now phenotype women with HA in the premenopausal years and determine associations with subsequent 30 year incident CVD risk factors and clinical CVD events. Understanding the HA phenotype(s) related to CVD is a crucial next step to identify women at-risk in order to take preventive action and improve overall CVD health. Early identification of young at-risk women presents a unique opportunity to intervene earlier in life when CVD preventive approaches are most beneficial. Further, preclinical CVD is a multi-organ disease that left untreated can manifest in neurocognitive disorders, peripheral CVD, and chronic kidney disease that contribute to health disparities in women. The outcome of the proposed research will identify young women at risk of CVD using HA phenotyping to inform the design of next step intervention trials and, ultimately, to translate our findings to clinical care to address the CVD epidemic in younger women using existing and emerging cardiovascular preventive strategies.

NIH Research Projects · FY 2025 · 2021-07

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative diseases with no effective treatment. We have shown that transcripts of the expanded repeat undergo an unconventional mode of translation, resulting in the production of five dipeptide repeat proteins (DPRs). Our group and others demonstrated that DPRs form neuronal inclusions throughout the central nervous system of patients with C9orf72-associated ALS and FTD (“c9ALS/FTD”). The identification of this neuropathological hallmark shed new light on possible disease mechanisms, and our systematic assessment of DPRs in human tissues, along with their evaluation in mice and other preclinical models, indicate that the arginine-rich DPRs, poly(GR) and poly(PR), are especially toxic. Our data further suggest that this toxicity stems, at least in part, from the co-condensation and/or co-aggregation of these DPR proteins with proteins that regulate essential cellular functions, such as stress granule biology and nucleocytoplasmic transport. We have also shown that poly(GR) aggregation induces TDP-43 pathology, another hallmark feature of c9ALS/FTD and of the majority of sporadic ALS and FTD cases. Nevertheless, since poly(GR) and poly(PR) interact with more than 200 endogenous proteins, they are likely to adversely influence a host of cellular functions – a line of investigation that merits attention. Also of importance is determining the underlying factors that regulate poly(GR) and poly(PR) condensation and aggregation, and their interactions with other proteins. It is thus notable that our preliminary data show that poly(ADP-ribose) (PAR) promotes poly(GR) and poly(PR) condensation, and enhances their co-condensation or co-aggregation with disease-related proteins in vitro. Moreover, we observed that reducing PAR suppresses poly(GR)- or poly(PR)-mediated neurodegeneration in Drosophila. In light of these exciting findings, we hypothesize that PAR mediates the aggregation and toxicity of arginine-rich DPRs and the proteins to which they bind. Thus, the goal of the proposed project is to determine how PAR does so. To this end, we will identify proteins that interact with poly(GR) or poly(PR) in a PAR-dependent fashion, and investigate the contribution of PAR to arginine-rich DPR aggregation and toxicity using multiple preclinical models and brain tissues from C9orf72 expansion carriers. Overall, by availing the diverse expertise of our team members, we endeavor to elucidate the role of PAR and arginine-rich DPR proteins in c9ALS/FTD pathogenesis, and thus uncover novel therapeutic targets to expedite the development of effective treatments for c9ALS/FTD.

NIH Research Projects · FY 2025 · 2021-07

Project Abstract Lung cancer is the number one cause of cancer death in the United States. Non-small cell lung cancer (NSCLC), which accounts for the majority (80%) of lung cancer diagnoses, is divided into two major sub-types, lung adenocarcinoma (LADC) and lung squamous cell carcinoma (LUSC). New therapeutic strategies targeting major oncogenic drivers of LADC have led to improved response rates and patient survival. However, due to a lack of well-characterized, validated, and therapeutically-actionable oncogenic drivers, similar therapeutic advances for LUSC have not been forthcoming. Genomic analysis of primary human LUSC reveals that the most prevalent recurrent genetic alterations in LUSC are concurrent loss of TP53 and copy number gains (CNGs) at chromosome 3q26 (>85-90%). These genetic alterations are observed in both precancerous lesions and early-stage LUSC, indicating they are early promotive events in LUSC tumorigenesis. Our previous studies have demonstrated that the 3q26 genes, PRKCI, SOX2 and ECT2 are coordinately co-amplified and functionally collaborate to maintain the transformed phenotype of LUSC cells. PKCι, the protein product for PRKCI, directly phosphorylates and regulates the oncogenic function of SOX2 and ECT2 in LUSC cells. Furthermore, our preliminary data demonstrate that: 1) overexpression of SOX2, PRKCI and ECT2, in the context of Trp53 loss, is sufficient to transform mouse lung basal stem cells (LBSCs), a major cell of origin for LUSC, and drive formation of tumors with malignant LUSC characteristics; 2) PKCι-SOX2 signaling activates a master transcriptional program specifying lineage-restricted lung squamous transformation; 3) PKCι-ECT2 signaling functions to increase the proliferative potential of LUSC cells through control of MEK-ERK signaling and enhanced ribosome biogenesis; and 4) both human LUSC cell lines with 3q26 CNGs and SOX2, PRKCI and ECT2 transformed LBSCs are sensitive to the growth inhibitory effects of Auranofin (ANF), a potent PKCι inhibitor, and to small molecule inhibitors of oncogenic PKCι-SOX2 and PKCι-ECT2 driven effector pathways including Hedgehog, WNT, NOTCH, MEK-ERK and rRNA synthesis. Based on these data we hypothesize that: 1) PRKCI, SOX2 and ECT2 represent cooperative multigenic drivers of LUSC tumorigenesis; and 2) combined treatment with ANF and inhibitors of PKCι-SOX2 and PKCι-ECT2 effector pathways will synergistically inhibit transformed growth of LUSC tumors. These hypotheses will be tested through completion of two interrelated specific aims designed to: 1) evaluate the efficacy of novel drug combinations that target oncogenic PKCι effector pathways; and 2) characterize a novel genetically- tractable mouse model for PRKCI-driven LUSC. Successful completion of these studies will facilitate the design of novel therapeutic strategies to improve outcomes for patients with LUSC. Furthermore, our novel clinically-relevant genetically-engineered LUSC mouse model will enhance our understanding of LUSC biology, characterize LUSC initiation and progression from preneoplastic lesions to malignant LUSC, identify markers for early LUSC diagnosis and develop and test novel targeted therapies for improved treatment of LUSC.

NIH Research Projects · FY 2025 · 2021-07

Abnormally phosphorylated microtubule-associated protein tau (p-tau) is one of the diagnostic hallmarks of AD pathologies, which strongly correlates with synaptic loss and cognitive decline in AD. Tau pathology first appears in the transentorhinal cortex and entorhinal cortex layer II (EC II), then spreads to the Cornu Ammonis 1 (CA1) field of the hippocampal region at the prodromal stage of AD (Braak stage I-II). However, no animal model has ever succeeded in showing tau propagation from EC II specific to CA1 as typically seen in the early Braak staging. A recent study has discovered that Wolfram syndrome-1 (Wfs1) positive cells in EC II project to CA1 via the stratum lacunosum moleculare along the temporammonic (TA) pathway. We hypothesize that misfolded tau propagates from Wfs1+ cells in EC II to CA1 via the TA pathway, and that the TA pathway is a novel therapeutic target to suppress tau propagation in the prodromal AD stage. Our exciting preliminary data showed that the stereotaxic injection of adeno-associated virus expressing Cre-inducible P301L tau into EC II of Wfs1-Cre mice induced: 1) robust human tau transfer from EC II to CA1 pyramidal neurons, 2) direct tau transfer between axonal terminals of Wfs1+ EC II neurons and dendrites of CA1 pyramidal neurons, 3) suppression of excitability of CA1 pyramidal neurons, and 4) impaired associative working memory. Thus, this new mouse model may recapitulate tau pathology progression from Braak I to II, and develop neurophysiological dysfunction and hippocampal learning impairment. The object of this current application is to fully characterize the pathology of this EC II-CA1 tau propagation mouse model and delineate the connectivity and mode of tau transmission from EC II to CA1. Our overarching goal is to invent therapeutics for prodromal AD using this mouse model. In Aim 1, we will i) characterize the post-translational modification of tau and cell types in EC II-CA1 mice using multiple p-tau antibodies and neuronal specific markers. ii) Validate the translatability of the findings in human AD brain tissues using early Braak stage and age/sex matched control specimens.iii) Investigate the gene expression profiles in EC, CA1, and prefrontal cortical regions of male and female mice to understand the molecular basis of sexual dimorphism in the behavioral outcome. In Aim 2, we will i) employ immuno-electron microscopy and super-resolution confocal microscopic imaging to capture tau transfer between EC II axonal terminals and radial dendrites of CA1 pyramidal neurons;ii) explore monosynaptic tracing between CA1 pyramidal cells and EC II neurons using Cre-dependent complementation of a modified rabies virus and WGA-GFP reporter system, and iii) determine the effect of activating/inhibiting neuronal firing on tau propagation, the role of neuronal extracellular vesicle release, and LDL Receptor Related Protein 1 as the predicted receptor for free tau secreted from the synaptic terminals. The proposed research project will develop a new understanding of tau propagation mechanism seen in the early Braak stages and provide a new insight for the sexual dimorphism in cognitive phenotype.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY (APOE U19: OVERALL) The overarching goal of this U19 project is to comprehensively understand the biology and pathobiology of apolipoprotein E (apoE) in aging and Alzheimer’s disease (AD) to inform therapeutic strategies. The ε4 allele of the APOE gene (APOE4) is the strongest genetic risk factor for AD impacting 50-70% of all AD patients, while the ε2 allele is protective compared to the common ε3 allele. APOE4 is also a strong risk factor for age-related cognitive decline and vascular cognitive impairment. To integrate existing knowledge and address critical gaps, we propose a unified ApoE Cascade Hypothesis that the structural differences and related biochemical properties among the three apoE isoforms initiate their differential effects on a cascade of events at the cellular and systems levels ultimately impacting aging-related pathogenic conditions including AD. Towards this, we have assembled a multi-disciplinary team to synergize expertise and resources across multiple institutions. By integrating five interactive Projects and seven robust Cores, we will create a nexus for apoE-related aging research, sharing the knowledge, expertise and resources with the broader scientific community. Project 1 will work closely with Core B to address the structural and biochemical properties of the three apoE isoforms to generate insights for functional outcomes. Projects 2, 3 and 4 will interactively study how apoE isoforms expressed in astrocytes, microglia, or vascular mural cells impact lipid metabolism, glial and vascular functions, AD-related pathologies, and cellular and molecular pathways using conditional mouse models and systems- based approaches. These studies will generate cell type-specific apoE/lipoprotein particles that will be collected through in vivo microdialysis for structural and biochemical studies. Project 5 will carry out genomic and genetic analyses to identify modifiers of APOE-related age at onset of AD. Studies in Projects 2-5 will be interactively supplemented by neuropathological studies using postmortem brains from healthy aging studies or with AD pathologies (Core C), biomarker studies using both human and mouse biospecimens (Core D), and functional studies using human iPSC-derived cellular and organoid models (Core E). This U19 proposal is supported by a comprehensive Multi-Omics Core (Core F) for centralized proteomics, lipidomics, and metabolomics studies on various animal and iPSC models, as well as human postmortem brains and fluid biospecimens. The Bioinformatics, Biostatistics, and Data Management Core (Core G) will provide critical supports for analyzing large datasets including those from single-cell RNA-seq and biostatistics supports to ensure scientific rigor. Core G will also work closely with the Administrative Core (Core A) to maintain an ApoE Web Portal designated as EPAAD where knowledge, resources, and data will be shared with the scientific community. Core A will also organize annual ApoE Symposium to promote collaboration and engage the ApoE Community. As such, this U19 will drive a team-based effort to generate essential knowledge to guide disease- modifying therapies for AD and other aging-related conditions.

NIH Research Projects · FY 2025 · 2021-05

Neurofibrillary tangles, composed of intracellular aggregates of hyperphosphorylated microtubule-associated protein tau (tau), are by far the most correlated pathology for clinical symptoms of Alzheimer disease (AD). Emerging evidence suggests that extracellular vesicles (EVs, such as exosomes and microvesicles), transfer pathological tau between cells as vehicles, propagating tau pathology. It is urgently important to find the molecular basis of brain-derived EVs, which may critically regulate EV uptake by neurons and aggregation of tau protein in EVs and/or recipient neurons. We have recently established the method for isolating EVs from human brain samples and successfully performed their proteomic profiling. We found that selective molecules from the EV proteomics datasets were able to differentiate human AD-EV from healthy control (CTRL)-EV with 88% accuracy by machine learning analysis, confirming pathogenic character of AD-EV molecules. Furthermore, our exciting preliminary data have shown that AD-EV have significantly higher tau seeding activity compared to CTRL-EV by FRET sensor tau seeding assay with subsets of EV molecules showing significant association with tau seeding activity. This proposed project will fortify these preliminary results and find the converging or specific mechanisms among tauopathies for mediating tau aggregation and its seeding via EV uptake through proteomics and biological examination of brain-derived EV samples. To meet this challenge, we assembled a multi-disciplinary team of investigators who have a strong record of accomplishments in biologic (Ikezu), proteomic (Emili) and bioengineering and bioinformatic analysis (Issador). In Aim1, we will examine EV samples from 240 new brain specimens (40 AD, 40 CTE, 40 LBD, 40 PSP, 40 CBD and 40 CTRL) for precision mass-spectrometry-based proteomics and tau-interactomes, and analyze those datasets by the machine learning approach. Aim 2 will examine the efficiency of tau propagation using tau fibrils, oligomers and EVs isolated from the same donors of the 5 different tauopathies and control cases in vitro and in vivo using FRET-based tau seeding assay and EV uptake by primary cultured mouse cortical neurons in vitro. EV-associated tau will be further characterized by the biochemical and microscopy-based analysis for their conformational and posttranslational changes. We will evaluate the difference in tau propagation after the intracranial injection of the tau seeds from different tauopathy brains using our recently established mouse models. Aim3 will identify candidate molecules most likely involved in EV uptake and tau seeding activity by bioinformatic analysis of the proteome dataset (Aim 1) and biological datasets (Aim 2). We will then test the functional roles of the identified molecules on EV uptake, tau seeding activities and neuronal firing activities in vitro. The candidate molecules will be specifically targeted by gene silencing or antagonists for their therapeutic potential to halt tau propagation in vitro and in vivo. Successful identification of responsible molecules for tau propagation will serve as a foundation for understanding EV-mediated disease progression.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT We seek to discover novel biomarkers and drug targets for frontotemporal dementia (FTD) and related disorders. FTD is a fatal neurodegenerative disorder that demonstrates substantial clinical, genetic, and pathological overlap with amyotrophic lateral sclerosis (ALS). Although both diseases can display TAR DNA- binding protein 43 (TDP-43) pathology, much remains unknown about the underlying mechanisms. It has been suggested that RNA processing pathways play a vital role, which is exemplified by the description of mutations in genes encoding RNA-binding proteins and the abundance of splicing defects in TDP-43 proteinopathies. Given the fact that long-read sequencing techniques have a higher accuracy in splice junctions, a better recovery of large transcripts, and detect more alternative splicing events than traditional sequencing methods that rely on short reads, we will produce full-length long-read transcriptomic data. We will examine a well- characterized pathological cohort of patients belonging to the FTD-ALS spectrum for whom frontal cortex and cerebellar tissue are available. Additionally, we will create single-nuclei long-read sequencing data, enabling us to determine in which cell type specific transcript variants are detected. This innovative approach will allow us to capture transcriptomic diversity, aiding the identification of novel, disease-specific, and/or disease-relevant transcript variants (Aim 1). We will compare the RNA signature observed in the brain to that seen in a large collection of clinical blood specimens. Moreover, we will assess differences between presymptomatic and symptomatic individuals and evaluate changes over time. These studies give us the ability to reveal interesting biomarker candidates, which will be validated in our extensive biospecimen collection (Aim 2). To elucidate the mechanisms underpinning these diseases, we will also perform in-depth mechanistic studies using various cell culture models, in vivo systems, and post-mortem tissues from patients along the FTD-ALS spectrum (Aim 3). Our original strategy, thorough characterization, and precious sample collection, will accelerate the discovery of pathological mechanisms, druggable targets and translatable biomarkers, which are highly valuable in preparation of future clinical trials for FTD and related disorders.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT MAYO CLINIC JACKSONVILLE Impairments of cerebral blood supply and blood-brain barrier (BBB) integrity cause neuronal damage, synaptic dysfunction, and white matter injuries, which eventually lead to the pathogenic condition referred to as vascular cognitive impairment and dementia (VCID). Importantly, aging is a strong risk factor for the disease pathogenesis. In general, aging is predicted to be caused by accumulation of senescent cells, in which the increase of p16INK4a is one of the key mechanisms triggering cellular senescent phenotypes. Thus, the major goal of our project is to define molecular mechanisms in which cerebrovascular senescence to the pathogenic pathways of VCID using mouse models. Apolipoprotein E (apoE) isoforms are also critically involved in the cognitive decline seen in the elderly. While APOE4 is the strongest genetic risk factor for late-onset Alzheimer’s disease, APOE4 increases the risk for mild cognitive impairment and VCID. Furthermore, APOE4 also causes neurovascular dysfunction, including BBB breakdown and the reduction of small vessels. Therefore, we hypothesize that p16INK4a expression in endothelial cells triggers vascular senescence which disturbs the homeostasis of the cerebrovascular system during aging, resulting in VCID and that APOE4 exacerbates VCID phenotypes. To reach our goals, we propose three specific aims. In Aim 1, we will determine the impact of adeno-associated virus (AAV)-mediated p16INK4a expression in cerebrovascular endothelial cells on VCID-related phenotypes in apoE3-tareget replacement (TR) and apoE4-TR mice. We have found that cerebrovascular endothelial cell senescence induced by transient p16INK4a expression through AAV leads cerebrovascular dysregulation in young wild-type mice. In Aim 2, we will examine how systemic endothelial cell-specific expression of p16INK4a in conditional mouse models affects VCID-related phenotypes depending on APOE4, accompanied with cerebrovascular single cell RNA-sequencing. In Aim 3, we will examine the effect of senolytics on APOE4- and/or endothelial p16INK4a-mediated VCID-related phenotypes to investigate the contribution of senescence in the AAV-based mouse and the conditional mouse models. Collectively, these studies should provide novel insights into the cellular and molecular mechanisms that underlie VCID pathogenesis.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT The MMPs have long been recognized as potential targets for cancer therapy, but drugs developed to target these enzymes have been unsuccessful. A primary reason has been inadequate selectivity, since most MMP inhibitors cannot discriminate among MMPs that drive cancer progression and other MMPs that prevent cancer progression. We have recently developed a new approach, expertise, and methodology for engineering much more highly selective MMP inhibitors based on a human protein, tissue inhibitor of metalloproteinases-2 (TIMP2). In our recently published work, we have created an engineered variant of the TIMP2 N-terminal domain (N-TIMP2) with greatly improved selectivity toward MMP-9, an enzyme critically involved in triple- negative breast cancer (TNBC) progression and metastasis. In preliminary studies, we find that this prototype inhibitor shows enhanced activity for blocking TNBC cellular invasion. We propose to further engineer N- TIMP2 for increased selectivity toward MMP-9 and also for enhanced affinity toward α3β1 integrin, a second natural target of TIMP2 through which TIMP2 mediates inhibition of tumor growth. We will define the structural basis for selective MMP binding of engineered N-TIMP2 variants to enable yet greater molecular improvements, and we will evaluate the therapeutic potential of these engineered proteins in multiple complementary preclinical models of TNBC. In Aim 1, we will use a combination of structural insights, computational design and yeast surface display (YSD) technology to engineer N-TIMP2, further optimizing selectivity toward MMP-9 and enhancing beneficial integrin binding activity. In Aim 2, we will elucidate structures of the engineered proteins with target and anti-target MMPs using X-ray crystallography, to uncover the structural basis for engineered selectivity and to facilitate yet greater refinements of our engineering platform and our selective MMP-9 inhibitors. In Aim 3, we will use complementary mouse orthotopic, transgenic, and patient-derived xenograft (PDX) models of TNBC to evaluate the utility of engineered N-TIMP2 variants as a therapeutic strategy in TNBC, and identify candidate biomarkers of response with potential for directing this therapeutic approach to patients who will most benefit from it. Our proposal is both conceptually and technically innovative in the combination of approaches toward generating novel protein therapeutics. The proposed research is highly significant because it has substantial potential to develop an entirely new approach for targeted treatment of TNBC by selectively inhibiting MMP-9, a well-validated target with key roles in tumor growth, invasion, metastasis, and angiogenesis.

NIH Research Projects · FY 2024 · 2020-09

PROJECT DESCRIPTION The ability of glioblastomas to proliferate in an uncontrollable manner and disperse widely within normal brain define the malignant phenotype and make this disease uniformly lethal. Effectively treating glioblastoma therefore requires finding targets that drive these two components of the malignant phenotype. We have identified one such target—the myosin II family of molecular motors. We show that myosin II family members can be targeted with a non-toxic small molecule inhibitor that is CNS permeant, and that this drug suppresses tumor progression and significantly prolongs survival in murine models of glioblastoma. However, we also find that targeting myosin II family members leads to a compensatory upregulation of a variety of proliferation- stimulating signaling pathways, and that inhibiting these pathways is synthetically lethal when myosin II function is blocked. In this proposal, we will develop strategies to enhance the efficacy of a myosin II targeting strategy in treating glioblastoma that build on our novel findings. Results from these translational studies will be vital to our ongoing efforts to develop effective treatment approaches that block both glioblastoma invasion and proliferation.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Diffusely infiltrative glioma is the most common primary brain tumor in adults. Most glioma patients experience at least one seizure during the course of their disease, and over 30% suffer from repeated seizures, known as tumor-associated epilepsy (TAE). Current front-line treatment for TAE is levetiracetam (LEV) (a.k.a. Keppra®), but this fails to control seizures in over 50% of patients. Such patients then require more powerful second-line antiepileptic drugs that often have greater side effects. TAE is more common in World Health Organization (WHO) grade II-III gliomas than in grade IV glioblastomas, but the reason for this is not clear. The vast majority of grade II-III gliomas contain mutations in isocitrate dehydrogenases 1 and 2 (collectively “IDHmut”), which lead to the production and release of large amounts of D-2-hydroxyglutarate (D2HG). D2HG bears a great deal of structural similarity to glutamate, an excitatory neurotransmitter that binds to N-methyl-D-aspartate receptor (NMDAR) on neurons. Our data show that D2HG increases in vitro neuronal membrane depolarization and neuronal network activity, and that this can be completely blocked by an NMDA receptor (NMDAR) antagonist. We also found that IDHmut glioma increases seizure activity in engrafted mice compared to IDHwt glioma, and that this is greatly reduced by treatment with IDHmut enzyme inhibitor. Finally, we found that IDHmut gliomas are much more likely to cause seizures compared to IDHwt gliomas. This is the first direct evidence of a mechanistic link between IDHmut and seizures; therefore, our hypothesis is that D2HG contributes to an increased incidence of seizures in patients with IDHmut gliomas, and that new targeted therapeutic strategies can decrease seizures in these patients. In Aim 1, we will explore the mechanisms by which D2HG triggers neuronal depolarization and increased neuronal network activity. Our two main hypotheses are: (i) D2HG directly stimulates NMDA receptors; (ii) D2HG inhibits glutamate reuptake transporters that normally prevent the pathologic accumulation of glutamate in the synaptic cleft. We will use patch clamping and multi-electrode arrays to study the effects of D2HG on the electrical activity of cultured mouse cortical neurons, as well as on mouse brain slices. In Aim 2, we will explore the effects of IDHmut glioma on the surrounding nonneoplastic tissue in vivo, focusing on changes that are characteristic of epilepsy, including neuronal loss, NMDAR downregulation, oxidative stress, inflammation, hippocampal damage, and altered mouse behavior. Results will be validated in patient-derived IDHwt and IDHmut gliomas. In Aim 3, we will compare the anti-seizure effects of two next- generation IDHmut inhibitors, AG-120 and AG-881, as well as memantine, an NMDAR antagonist that is already used to treat Alzheimer’s Disease. Each of these drugs will be tested as monotherapy and in combination with LEV. Successful completion of these Aims will establish the D2HG product of IDHmut as an epileptogenic agent, will shed more light on how IDHmut alters the nonneoplastic neural tissues surrounding glioma, and will foster clinical trials to determine the efficacy of IDHmut inhibitors, and memantine, against seizures in these patients.

NIH Research Projects · FY 2024 · 2020-04

PROJECT SUMMARY Glioblastoma (GBM) is the most common cancer arising in the adult brain, and is lethal in nearly all cases. A key contributing factor to poor outcomes for GBM patients is a subpopulation of cells, known as cancer stem cells (CSCs), that are highly resistant to routinely used genotoxic/cytotoxic therapies, and ultimately manifest as recurrent tumor. Inhibiting tumor recurrence from CSCs that survive therapy would therefore improve GBM patient outcomes. Our preliminary and recently published work show that CSC survival is dependent on Tissue Factor (TF), a conserved transmembrane and secreted protein involved in blood clotting. TF activates protease- activated receptor 2 (PAR2), a G-protein-coupled receptor on GBM cells, which promotes CSC maintenance and expansion, as indicated by analysis of marker expression, self-renewal capacity, and in vivo growth of CSCs. TF suppression greatly reduces CSC subpopulations, in some cases even leading to complete tumor eradication in vivo. Protective and proliferative effects of TF-PAR2 signaling on CSCs appears to be through activation of multiple classes of oncogenic receptor tyrosine kinases (RTKs) like EGFR, although the mechanism by which TF-PAR2-RTKs stimulate CSCs is unclear. Our preliminary data also show that TF positively correlates with expression of Junctional Adhesion Molecule-A (JAM-A), a protein that promotes cell-cell adhesion by stabilizing integrin β1. CSCs depend on such cell-cell adhesion, and our data show that JAM-A is necessary for CSC behavior in GBM. Because TF can also signal through integrin β1, our overarching hypothesis is that TF upregulates JAM-A expression, which stabilizes integrin β1 and enhances the ability of TF to act on integrin β1 and promote self-renewal in GBM. This has therapeutic relevance, because the pro-CSC effects of TF are independent of its role in hemostasis, and blocking JAM-A could potentially reduce the pro-tumor effects of TF, without causing bleeding that would result from targeting TF directly. In Specific Aim 1, we will test the hypothesis that TF drives JAM-A expression through PAR2-RTK signaling. We will identify components of the TF-PAR2 complex that are essential for TF-PAR2 pro-tumor activities, and will test the ability of TF to trigger JAM-A expression while inhibiting each major downstream pathway of RTK signaling. In Specific Aim 2, we will determine whether JAM-A requires serpin B3, a serine-protease inhibitor that we have shown binds to JAM-A, for its pro-CSC activities. In Specific Aim 3, we will use molecular knockouts to determine whether JAM-A is an effective therapeutic target against aggressive, high TF-expressing GBM. Completion of this project will advance our understanding of how CSC subpopulations are maintained in GBM, could well be applicable to a wide range of cancers, and would demonstrate a compelling new therapeutic target in treating numerous malignancies, including GBM.

NIH Research Projects · FY 2026 · 2019-07

PROJECT ABSTRACT Hereditary pancreatitis (HP) is an autosomal-dominant disease that causes recurrent acute pancreatitis (AP) and eventually progresses to chronic pancreatitis (CP). Unfortunately, HP patients also have a 44% cumulative risk of pancreatic cancer by age 70. No targeted intervention is currently available. Lack of animal models poses a big challenge to study the disease pathogenesis. Since the discovery of the gain-of-function mutant PRSS1R122H as a major cause of human HP nearly 26 years ago, many attempts to develop HP animal models have not been so successful. Recently, we were able to generate a novel mouse HP model by expressing both human PRSS1R122H and PRSS2. Heterozygous PRSS1R122H-PRSS2 HP mice are more sensitive to various stimuli in developing more severe AP than wild-type mice, while homozygous HP mice develop AP spontaneously at ~20 days old after birth which gradually progress to CP. During the studies of our new HP models, we observed that, in addition to the gain-of-function mutations, PRSS1 expression levels may also control the initiation of HP. Furthermore, a lower dose of cerulein (7.5 ug/kg), which activates less trypsin than high doses (100 ug/kg), paradoxically induced hemorrhagic AP, a more severe and often lethal form of AP. In contrast, high doses of cerulein only cause edematous AP. With these new findings, we hypothesize that the expression levels of trypsinogens and the location of active enzymes determine the spontaneous initiation and severity of HP. In Specific Aim 1, we will elucidate the mechanisms that upregulate the expression of pancreatic trypsinogens during the spontaneous initiation of HP. Various diets, hormones, and neurotransmitters, and intracellular signaling pathways in the regulations of PRSS1 expression and the initiation of HP will be studied using both pharmacological and genetic approaches. In Specific Aim 2, we will determine the role of active trypsin location in the development of hemorrhagic AP. We will characterize the histopathologic features including damage of pancreatic ducts and vasculature in the hemorrhagic models, determine the dose- dependent effects of cerulein and bombesin on trypsin activation and localization, define the molecular mechanisms through which trypsin damages the pancreatic duct and vasculature, and evaluate the therapeutic effects of inhibiting extracellular trypsin on hemorrhagic AP. Impact: This study will provide novel insights into the spontaneous initiation of HP and the pathogenesis of hemorrhagic AP. Some of the pathway inhibitors tested in this study are FDA-approved drugs for other disease conditions. Therefore, they can be readily translated to these diseases. Our study will also pave the ways for developing other novel effective preventive and therapeutic interventions.

NIH Research Projects · FY 2025 · 2017-12

Project Summary The long-term objective of this application is to understand the molecular pathways of myocardial infraction (MI)- mediated cardiac fibrosis and eventually heart failure. In our last grant cycle, we elucidated thoroughly the role of Neuropilin-1 (NRP1), a multi-ligand receptor/adaptor and mediator of different signaling pathways including TGFβ, in MI-induced cardiac pathogenesis, in particular fibrosis, and inflammation. Our preliminary data suggest that conditional knockdown of NRP1 in the adult mice, either in cardiomyocytes (CM) or in endothelial cells (EC) resulted varied phenotypes following cardiac injury (MI). In addition, our preliminary data from the single-nucleus RNA sequencing (snRNA seq) using the cardiac tissues in a cell-specific manner and revealed marked changes in gene expression patterns of key genes previously known to be associated with inflammation, cardiac hypertrophy, fibrosis. In addition, we revealed microRNA (miR30s) showed differentially expressed in ECNRP1-/- vs. CMNRP1-/- cells that correlated with HF genes. Hence, the scientific premise of this proposal is to reveal the mechanistic role of divergent NRP1 spatiotemporal signaling in MI pathogenesis, and eventually to develop new therapeutic targets and strategies to overcome progressive cardiac dysfunctions and overall heart failure. In this regard, we will examine the spatiotemporal influence of NRP1 pathways for cardiac fibrosis following MI injury as well as define the tissue-specific role of microRNAs regulated by NRP1 pathways on MI-induced fibrosis. Finally, we will develop new therapeutic approaches to overcome cardio fibrosis following MI injury combining novel NRP1 inhibitor (NRP1i) using novel targeted liposomal formulations. The current application is a multi- disciplinary approach to reveal the spatiotemporal role of NRP1 in cardiac fibrosis/remodeling and identify new targets to develop novel therapies to overcome one of the most common myocardial pathologies, which is an unmet clinical need.

NIH Research Projects · FY 2025 · 2016-09

We have previously reported that microglia and extracellular vesicles (EVs) play pivotal roles in tau propagation. Our findings have been reproduced in numerous studies showing involvement of microglia and EVs in the development of tau pathology in animal models and human biospecimens. During the current funded period (1RF1 AG054199), we identified: 1) Enrichment of glial and disease-associated proteins in Alzheimer’s disease brain-derived EVs (AD EVs) compared to control EVs, 2) Highly transmissible nature of AD EVs demonstrated by increased neuronal uptake and tau aggregation potency, 3) Robust tau propagation throughout the entire hippocampus by inoculating AD EVs containing just 300 pg of tau in aged C57BL/6 mouse brains. We have yet to elucidate the underlying mechanism of AD EVs gaining pathogenic functions. We hypothesize that Ab-induced inflammatory conditions in AD leads to microglial activation, which alters molecular compositions of microglial EVs, leading to their increased transmission to neurons. APOE4 enhances this process by augmented Ab deposition and MGnD induction. The hypothesis has been developed based on the previous studies and our preliminary data showing that (1) Clec7a+ neurodegenerative microglia (MGnD) surrounding Ab plaques play a key role in accelerating tau propagation in APPNL-G-F/NL-G-F knock-in (APP KI) mice; (2) MGnD upregulates EV markers; (3) AD EVs and APP mouse brain-derived EVs show elevated MGnD and cell adhesion molecules compared to controls; (4) AD EVs show increased tau seeding activity and neuronal uptake compared to controls, which is exacerbated in APOE4 over non-APOE4 AD cohorts; and (5) APOE4 knock-in APP/PS1 mice accelerates Ab deposition, microglial activation, and expression of MGnD markers compared to APOE3 knock-in APP/PS1 mice. The rationale of the proposed research is that identification of EV protein and lipid composition uniquely expressed in AD and APOE4 background will serve as the basis of molecular understanding of EV-mediated disease progression. Our study will fill the missing link between MGnD induction and tau propagation by microglial tau-seeding EV production. In Aim 1, we will delineate the chemical and molecular structure of aggregation-prone tau in AD EVs and tau- interacting molecules. We will also characterize the protein and lipid compositions of EVs from APOE4/4 and APOE3/3 EVs to characterize the difference in cellular origin, involvement of inflammatory and toxic molecules, and tau seeding activity in vitro and in vivo. In Aim 2, we will knock down Apoe, involved in microglial activation and Smpd3, involved in EV synthesis, to determine the role of MGnD induction and glial EV production on tau propagation. In Aim 3, we will differentiate isogenic human iPSCs in APOE3/3 and APOE4/4 background to microglia-like cells (iMGL), and characterize pathogenicity of iMGL EVs under MGnD condition. We will perform proteomic and lipidomic profiling of iMGL EVs to validate our findings from human AD EVs in Aim 1. The proposed work is expected to obtain the fundamental molecular bases for EV biology in tauopathy.

NIH Research Projects · FY 2025 · 2016-08

Project Summary This is an application for K24 award renewal for Dr. Lea T. Grinberg, a neuropathologist at the University of California, San Francisco (UCSF). Dr. Grinberg is an Associate Professor in Residence and co-lead the UCSF/Memory and Aging Center's Neuropathology Core. She is an established researcher in the patient- oriented clinical research of dementia. A distinctive hallmark of her research is her direct involvement in creating, managing, and analyzing well-characterized postmortem collections of brains belonging to people at-risk or already with dementia. Dr. Grinberg proposes to use K24 dedicated time to mentor USCF as well as international investigators in patient-oriented dementia research. Her mentees will gain hands-on research experience, expertise in age-related human neuropathology, training in data analysis, manuscript preparation, and grant writing, as well as career, mentoring. Mentee training will leverage the infrastructure and resources of the UCSF/Memory and Aging Center Autopsy program, which is part of ongoing longitudinal cohort studies, research portfolio, and her collaborations with multidisciplinary researchers in the areas of dementia domestically and worldwide. Dr. Grinberg intends to conduct K24-supported Alzheimer's disease research studies that will serve as training vehicles for mentees and expand her research. These studies, using clinical, genetic, and neuropathological data, will be conducted using data from ongoing UCSF/Memory and Aging Center's NIH- funded cohort studies of persons with Alzheimer's disease (AD). She will examine the factors underlying selective neuronal vulnerability in AD. In summary, this K24 will enhance Dr. Ginberg's active research program with extensive infrastructure at UCSF to support her goal to remain a leader in neurodegenerative diseases, especially in the field of neuropathology, and to develop a program of excellence for training medical students, trainees, and junior faculty in POR related to age-related neuropathology that is also intended to close the gaps caused by interruption of neuropathology training for neurologists and neurodegenerative disease training for neuropathologists.

NIH Research Projects · FY 2025 · 2014-03

PROJECT SUMMARY The Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis Trial (CREST-2, U01 NS080168) consists of two parallel randomized trials in patients with asymptomatic high- grade carotid artery stenosis. One trial compares Intensive Medical Management (IMM) plus carotid endarterectomy to IMM alone, and the other compares IMM plus carotid artery stenting to IMM alone. The trial is registered with clinicaltrials.gov (NCT02089217). Monitoring plays a crucial role in ensuring the protection of subjects and the validity of the science. Additionally, monitoring is required for the CREST-2 trial by the FDA and NINDS. As of July 31, 2024, CREST- 2 has enrolled a total of 2,486 patients: 1,241 in the endarterectomy trial and 1,245 in the stenting trial. Patients have been enrolled across 183 sites (171 US and 12 international). To be cost-effective in monitoring these clinical sites, the CREST-2 Clinical Coordinating Center has taken on the responsibility of hiring and managing the monitor rather than outsourcing the responsibility to a Clinical Research Organization. During the course of the trial, monitoring has adapted for various reasons most notably the COVID-19 pandemic. Most monitoring visits were conducted on-site prior to the COVID-19 pandemic, remote monitoring and quality control checks were implemented during the COVID-19 pandemic, and a combination of remote and on-site monitoring have been used since March 2023. The approval of a previous administrative supplement request provided funding to partially fund and support travel for our current Monitor. Through that approval, we were able to resume site-level source document verification in the CREST-2 trial. From March 2023 to July 2024, a total of 41 monitoring visits have been conducted, including 25 onsite and 16 remote visits. Furthermore, we have collaborated with our Statistical and Data Coordinating Center to develop forensic and risk-based monitoring approaches, developed and deployed a survey on remote vs. in person monitoring preferences to all CREST-2 sites, refined our site closeout monitoring process, developed a risk-based monitoring prioritization system, built reports and dashboards in Smartsheet to track monitoring efforts, and increased the frequency of data quality meetings to enhance data quality and completeness as we near the end of the trial. With approximately two years remaining of grant activities, we request additional funding through an Administrative Supplement to continue monitoring efforts. The goals of this supplement are to continue onsite and remote monitoring, close out CREST-2 sites once final follow-up visits occur, and help ensure a complete and verifiable dataset.