Case Western Reserve University

NIH Research Projects · FY 2025 · 2022-09

Abstract Kidney cancer is expected to affect 76,080 new patients with 13,780 deaths in the U.S. in the year 2021. Renal cell carcinoma (RCC) is the most common type of kidney cancer which imposes significant economic burden on healthcare system. A recent study based on SEER Medicare database reported that the total healthcare cost per RCC patient was $23,489 with a weighted total economic burden of $2.1 billion. RCC often presents as an incidentally detected, incompletely characterized renal mass. Many of these patients with incidental renal mass either undergo direct surgery or biopsy without further imaging evaluation as accurate histologic diagnosis with current imaging techniques is not always possible. However, upfront surgery or biopsy is not ideal as nearly 25% incidental renal masses are either benign (angiomyolipoma, oncocytoma) or low-grade (chromophobe RCC, low-grade clear cell RCC) and overtreatment of such masses adds to unnecessary morbidity and health care cost. Prior studies have shown low-grade RCC can be managed conservatively with active surveillance in select patients (elderly patients and patients who are poor surgical candidates), but at present there is a no non-invasive way to separate low-grade RCC from aggressive RCC (high-grade clear cell RCC, papillary RCC). Accordingly, there is an emergent need to develop novel non-invasive quantitative biomarkers for accurate characterization of renal masses so that more patients eligible for active surveillance could be identified. Recent studies have shown that MR tissue relaxometry mapping including T1, T2 and T2* mapping and fat fraction quantification can provide improved characterization of kidney diseases and correlate with tumor grade and biologic aggressiveness in RCC. However, the current kidney relaxometry mapping techniques still suffer from long breath-holds, limited spatial resolutions/coverage, and ability to mostly capture one tissue property at a time. Further, the quantitative measures are often susceptible to motion artifacts with poor repeatability and reproducibility. In this study, we propose to utilize the novel MR Fingerprinting (MRF) technique together with machine learning methods to mitigate aforementioned limitations in kidney imaging. In particular, we will develop a new 3D free-breathing kidney MRF method for simultaneous T1, T2, T2* and fat fraction quantification (Aim 1). We will combine this kidney MRF acquisition with novel deep learning approaches to accelerate data acquisition and improve tissue mapping efficiency (Aim 2). Finally, we will apply the MRF technique in patients with RCC to explore its diagnostic strength in characterizing kidney cancer (Aim 3). Upon successful development, the multi-parametric quantitative measures acquired with MRF could make MRI a more powerful tool for the diagnosis and predicting of tumor grade in RCC, with the ultimate goal to eliminate unnecessary biopsy/surgery in eligible patients with benign/low-grade RCCs and provide guidance towards the most appropriate treatment strategy.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Our long-term goal to establish sustainable, patient-centered interventions at the patient level to engage patients in safety and prevention by offsetting barriers such as physical and sensory limitations that prohibit them from actively helping themselves. Unfortunately, older adults are at highest risk for infections and yet while hand hygiene is the single most important way to prevent the spread of infection, mechanisms for older adults to minimize their own risk for infections is often overlooked. Many contamination elements exist in hospital settings and for older adults they will encounter high-touch surfaces and medical devices that harbor pathogens that lead to infections. However, without assistance, hand-hygiene practice rates are poor among hospitalized older adults’ due to frailty, limited dexterity and mobility, cognitive limitations, and risk of falling, which prohibits their independent use of visible hand- hygiene products (e.g., wall dispensers, towelettes, in-room sinks). New solutions are necessary. Therefore, in this proposal, we have updated our investigator-developed, technology-enhanced patient hand hygiene system. Clean Hands Accessible and Manageable for Patients (CHAMPs), our investigator-developed and pilot tested bed rail-affixed hand-sanitizing dispenser, which features verbal, auditory and visual reminders to remind patients to clean their hands. The safe and easily accessible motion-sensing system with usage tracking requires very little physical effort, as users need only to be able to freely move their upper extremities and reach over to the bed rail to clean their hands when prompted (e.g. before meal times). Our pilot results among both a small group of older adults and in a high-tech simulated environment demonstrated both efficacy and feasibility of the intervention. In this 4-year project, we propose a large heterogeneous randomized controlled trial (RCT) comparing two groups of hospitalized adults ≥ 65 years in two public hospitals, one group receives CHAMPs (n=125) and the other is the usual-care (UC) group (n=125). Our research team consists of early and late stage investigators who have a successful record of working together and are ready to address the following aims: (1) to determine the effect of CHAMPs as a method to improve hand- hygiene behavior and reduce patients’ hand contamination, (2) assess the implementation of our intervention, (3) examine factors that influence outcomes associated with our intervention and (4) costs and cost-effectiveness of CHAMPs. The primary outcome is hand contamination as measured by presence, type, and quantity of colony- forming units located on participants’ hands. Our preliminary results offer promise that the CHAMPs technology-enhanced intervention may be an effective approach to engage patients in infection prevention as a solution to reduce colonization and infection rates among older adults. Our proposal aligns with all four goals of NIH/NIA’s 2020-2025 strategic plan, which is to improve the health, well-being, and independence of adults as they age and to prevent or reduce the burden of age-related diseases, disorders, and disabilities.

NIH Research Projects · FY 2025 · 2022-09

Most stroke survivors walk slowly and are at an increased risk of falls. As a result, many adopt a sedentary lifestyle with limited functional independence that negatively impacts health and can be socially isolating. Physical therapy including advanced rehabilitation techniques have improved function, but there is no intervention available that enables stroke survivors with moderate and severe impairment to walk at speeds necessary for independent community ambulation. The long-term goal of this work is to restore stroke survivors’ ability to walk safely in the community at speeds necessary for independence. Our approach utilizes an implanted neuroprosthesis, that is a device inside the body that applies small electrical pulses to activate the nerves that cause the muscles serving multiple joints to contract in a coordinated manner for functional movement of the entire limb. The system measures volitional muscle activity and body motion and then coordinates stimulation at the different joints in the leg to produce the necessary movement for safe walking at functionally relevant speeds. The benefit of such an approach is that it is always available and does not require setup for individuals with impaired hand control. The implanted hardware also improves reliability and bypasses sensory fibers that can cause discomfort. Our team has shown in a case study that targeting muscles throughout the paretic limb can substantially improve walking speed and endurance. This study will expand this work through achieving the following Aims: 1) determining the clinical impact of an implanted multi- joint neuroprosthesis on post-stroke gait, and 2) developing and assessing an advanced neuroprosthesis cooperative control strategy. This study will implement an available neuroprosthesis that incorporates an external control unit and some external sensors in preparation for implementation of a fully implanted system that has been developed at our Center. Six participants will be implanted with devices that include 12-channels of stimulation and 2-channels for recording muscle activity. External sensors will measure limb motion. After the device is implanted, stimulation patterns will be generated and participants will undergo training to use the device. A simple triggering pattern will be created for home use and then we will implement our advanced controller in the laboratory via machine-learning techniques. Once a controller and stimulation pattern have been defined, we will determine how much faster, more safely, and easier walking is with the neuroprosthesis compared to without and confirm whether these effects are maintained over time. We will also determine if the advanced controller substantially improves walking ability over the simple triggering methods that have been previously implemented. Successful completion will confirm approaches for a post-stroke neuroprosthesis for walking and generate preliminary effect sizes for subsequent clinical trials to evaluate home and community use of a fully implanted system. This study may lead to a new clinical tool that can empower independent walking after stroke, improve quality of life, and enhance overall health of stroke survivors.

NIH Research Projects · FY 2025 · 2022-09

Tyrosine kinase targeted (TKI) therapies have revolutionized leukemia treatment, but TKIs are not able to kill leukemia stem cells (LSCs), which are responsible for propagating and disease recurrence, and believed to be the source of treatment failure. Our long-term goals are to further address how LSC persistence is regulated, and to develop new LSC-eliminating treatment strategies to improve cure rates and survival. The short-term goals of this research are to determine whether a N6-methyladenosine (m6A)--long non-coding RNA (lncRNA) axis regulates LSC stemness and persistence during TKI selection process, and to explore the therapeutic potential of targeting the m6A-lncRNA axis for eradicating TKI resistant LSCs and also decipher the underlying molecular mechanisms. The m6A methylation is the most common epitranscriptomic modification on RNAs (i.e., lncRNAs), and crucially regulates lncRNA-initiated gene expression. lncRNA abnormalities frequently associate with cancer disease progression and drug resistance. The preliminary evidence linking an m6A-lncRNA axis to resistant LSCs is from our proof of principle studies demonstrating that i) a dynamic and reversible m6A methylome determined by fat mass and obesity-associated protein (FTO) helps leukemia cells avoid TKI killing leading to TKI resistance; ii) there are many lncRNAs (annotated) that are differentially expressed in resistant versus sensitive cells. About 50% of these lncRNAs bear m6A motifs and have the changed m6A amounts in resistant cells, collectively, suggesting a unique lncRNA signature that is specific to TKI resistance and is regulated by m6A methylation; iii) upregulation of these m6A-associated lncRNAs in patients who do not respond to TKIs is predicative of worse outcomes, and knockdown of them impairs resistant cell growth and renders resistant cells sensitive to TKIs; iv) compared to sensitive ones, TKI-resistant cells highly express LSC markers (CD117, CD44, CD25, CD133) whose upregulation is associated with m6A reduction. Our hypothesis is that the FTO-m6A-lncRNA cascade may be a critical pathway to control LSC persistence to TKIs and a new druggable target to eradicate persistent LSCs improving TKI cure rates. We will test our hypothesis through three aims: 1) Determine how the FTO-m6A axis regulates lncRNA aberrations in TKI resistance; 2) Determine whether and how a dynamic m6A methylome regulates LSC persistence to TKIs; 3) Determine whether and how pharmacological targeting of the FTO-m6A-lncRNA cascade kills persistent LSCs using preclinical leukemia models. The proposed studies are conceptually innovative, because it targets a new pathway (the FTO-m6A- lncRNA cascade) in understanding LSC persistence to TKIs and in developing new regimens to eliminate TKI resistant LSCs. The proposed research is significant, because the findings will a) identify new pathways (i.e., FTO-m6A-lncRNA cascade) that regulate LSC persistence, deepening the molecular understanding of LSCs and lncRNA functions; b) develop new approaches (targeting the FTO-m6A-lncRNA cascade) to eliminate persistent LSCs improving the management of patients with refractory leukemia.

NIH Research Projects · FY 2024 · 2022-09

Project Summary This project will organize and host a two-day meeting for the NIH Stimulating Peripheral Activity to Relieve Conditions (SPARC) Phase 2 program from March 12-13, 2025. The meeting will bring together approximately 100 scientists who are working on active programs funded by SPARC to 1) present recent findings and 2) forge new collaborations between each other to develop future translational goals for their projects. The meeting agenda will begin on day 1 with research presentations from SPARC Phase 2 project teams to showcase their latest activities and discoveries. This will be followed by a series of workshops that enable different teams to form collaborations. Day 1 will also include a research poster session that is open to all attendees and end with research demonstrations. Day 2 will open with presentations from NIH officials about programs that are aligned with the future work of current SPARC investigators. Trainee travel award winners will also present their work. Day 2 will also feature rotating roundtable discussions on key issues suggested by SPARC investigators and report-outs to SPARC program officers.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT Natural killer (NK) cells are cytotoxic lymphocytes with important immune functions in killing virally infected cells and cancer cells. NK cells have been explored for cancer immunotherapy and have advantages over T cell- based therapies. However, their cytotoxicity and tumor immunosurveillance functions are often dysfunctional in cancer patients, in large part due to elevated levels of TGF-β, a potent immunosuppressive cytokine. Cyclin- dependent kinase 5 (Cdk5) is a Cdk family proline-directed serine/threonine kinase. Unlike other Cdk members, its kinase activity is primarily dependent on binding of the coactivator p35 or p39, and it has unclear cell cycle roles. Cdk5 was thought to primarily function in neuronal cells, but recent research has discovered new roles for Cdk5 and p35 in other cell types, including cancer and immune cells. For the first time, we have discovered that Cdk5 and p35 protein are both expressed in NK cells and appear to play an important role in regulating NK cell cytotoxicity. Additionally, TGF-β appears to induce p35 expression in NK cells in a dose-dependent manner. Based on our preliminary data, we hypothesize that Cdk5/p35 kinase activity negatively regulates NK cell cytotoxicity and is a key mediator of TGF-β-induced NK cell dysfunction, and we also hypothesize that Cdk5/p35 inhibition can be utilized to enhance NK cell immunotherapy. First, we will explore how the Cdk5/p35 and TGF- β signaling pathways overlap in NK cells. Using genetic tools to knock down p35, as well as the selective Cdk5 inhibitor roscovitine, we will determine whether Cdk5-inhibited NK cells can mitigate the various phenotypic changes caused by TGF-β treatment. We will measure any changes in the expression of NK cell activating/inhibitory receptors, lytic granule cytotoxic enzymes, and cytokine release. We will also determine how TGF-β induces p35 expression in NK cells, then investigate the molecular mechanism of how Cdk5/p35 activity regulates NK cytotoxicity. Whole transcriptome sequencing of p35 knockdown NK cells will be used to reveal differentially expressed pathways downstream of Cdk5 kinase signaling. We also wish to explore the therapeutic potential of Cdk5/p35 inhibition in enhancing NK cell immunotherapy. Using in vitro cytotoxicity assays against cancer cell lines, we will determine whether p35 knockdown will enable NK cells to resist TGF-β-induced suppression of cytotoxicity. Similarly, using established patient-derived xenograft mouse models, we will test whether p35 knockdown NK cells are able to enhance NK cell adoptive therapy against patient-derived B cell acute lymphoblastic leukemia (B-ALL), which is known to cause NK dysfunction through elevated TGF-β secretion. Discoveries from this project would advance our basic understanding of the signaling pathways that regulate NK cytotoxicity and mediate NK dysfunction, potentially leading to improved NK cell-based cancer immunotherapies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Neuronal ionotropic glutamate receptors (iGluRs) play key roles in mediating excitatory synaptic transmission in the brain and in a wide range of brain diseases, including Alzheimer’s and Huntington’s disease, schizophrenia, epilepsy, autism spectrum, major depression, and mood disorders. Glutamatergic signaling is pivotal for synaptic plasticity, learning, and memory formation. Kainate-type ionotropic glutamate receptors (KARs) are distributed throughout the brain and regulate the release of neurotransmitters and mediate excitatory synaptic transmission. KARs form homo- or hetero-tetramers composed of five homologous subunits of GluK1–5. Each subunit exhibits unique structural, functional, and pharmacological properties and subcellular localization. Moreover, misguided localization and dysfunction of KARs result in neuropathologies, therefore, KARs are a promising drug target. However, KARs are the least well understood group of iGluRs, and their molecular mechanisms remain elusive. The activities of neuronal KARs are regulated by pH, posttranslational modifications, lipid/cholesterol, and small molecules. Additionally, KAR function and localization are further modified by their auxiliary and accessory proteins. Thus, such brain lipids, modifications, and protein co-factors increase the diversity of KAR functional properties. The neuropilin and tolloid-like (NETO) auxiliary proteins, NETO1 and NETO2, are auxiliary proteins of KARs that are distantly homologous compared with auxiliary proteins associated with other iGluRs. How do such ligands and protein co-factors determine the gating of KARs and regulate synaptic signaling? How are the physiological brain lipid environment and posttranslational modifications contributing to receptor activities? To answer these questions, this proposed research will employ structural and electrophysiological approaches to develop our mechanistic understanding of the regulation of native postsynaptic GluK2/GluK5 KARs isolated from rat brains. The program will move forward in two major directions: In one project, I will determine high-resolution cryo-electron microscopy (cryo-EM) structures of native GluK2/GluK5 KARs in an activated state, but also in complex with ligands to capture multiple functional states. This will elucidate the conformational alternations of GluK2/GluK5 KARs by their ligands, which have not been well-observed in previously determined structures. Comparing our structures with other iGluRs will uncover how physiologically relevant heteromeric KARs are structurally and functionally distinct from other iGluR subfamilies. In a second concurrent project, we will elucidate the regulatory mechanisms of native KARs by NETO1 and NETO2 auxiliary proteins. Overall, our studies will provide fundamental insights into how neuronal KAR complexes are controlled by their ligands and auxiliary proteins, and how they mediate synaptic signaling, and thus neural activities. This information will facilitate the development of new therapeutic strategies for treating brain diseases.

NIH Research Projects · FY 2025 · 2022-09

Alzheimer disease (AD) is a devastating neurodegenerative disorder that affects millions of individuals in the U.S. It has so far resisted attempts to develop effective therapies despite numerous (failed) clinical trials based on known targets, most identified over 20 years ago. While genomic research (e.g. the Alzheimer’s Disease Sequencing Project; ADSP) has identified numerous additional risk loci, these results derive primarily from case- control datasets. In contrast, cohorts designed to identify variants that may protect from AD, and those using complementary study designs, are few. We used our extensive experience with the Amish communities in Indiana and Ohio to establish a cohort of older individuals at high risk of developing AD but who are cognitively unimpaired (CU). The Amish provide a unique opportunity to identify protective variants for AD because of their reduced background genetic variation and environmental risk factors. Their small founding population and endogamy provides enrichment for rare variants. Founder populations also enable testing for non-additive allelic effects and can magnify sub-significant association signals identified in case-control studies. The stability and engagement of our Amish participants enables longitudinal assessments of cognition and biomarkers. Our primary goals are to identify AD protective loci and characterize pre-clinical biomarkers of progression to cognitive impairment. Building on our existing large cohort, our replicated protective locus and several suggestive protective loci, and our existing biobank of DNA and plasma and databank of GWAS and WGS, we propose to: 1) Perform longitudinal assessment of cognition in our family-based Amish cohort; 2) Identify protective factors for AD and predictors of progression to cognitive impairment by analyzing genomic and longitudinal cognitive, biomarker, and SDOH data; and 3) Examine the functional implications of current and novel genes and variants by prioritizing loci using in silico annotation for functional consequences followed by in vitro functional characterization. Our results will identify potential druggable targets and accelerate the development of better treatments for AD.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY: Inflammation of the central nervous system (CNS) develops in both systemic infection as well as sterile lung injury. This neuro-inflammation mirrors systemic inflammation and affects the neural control of respiration. We have published that peripheral inflammation evokes neuro-inflammation, decreases synaptic efficacy of vagal sensory feedback and increases the predictability of ventilatory pattern variability (VPV). Thus, we have an overall perspective of the effects of systemic inflammation on respiratory control at molecular and functional levels, but the effects at the level of the respiratory network activity remain obscure. Our hypothesis is that brainstem neuro-inflammation alters synaptic strengths in the respiratory network which suppresses reflex modulation of the respiratory pattern to decrease ventilatory pattern variability. During systemic inflammation, respiratory rate and predictability of the ventilatory pattern increase. These maladaptive changes persist in the absence of peripheral sensory inputs in in situ brainstem preparations derived from rats with either sterile lung injury or systemic infection, indicating alterations in respiratory network activity. Further, respiratory rate increases in healthy rats following IL-1β microinjections in the nTS. Thus, our data indicate that neuro-inflammation of the brainstem respiratory network drives changes in VPV in these disease processes. We test our hypothesis in the following Specific Aims: 1) to determine anatomic and functional maps of the ponto-medullary respiratory circuits in lipopolysaccharide (LPS) treated rats; 2) to determine if the etiology of the inflammatory process affects neuro-inflammation, maladaptation of respiratory network activity and VPV; 3) to assess the causal relationships between neuro-inflammation, respiratory network activity and VPV and whether anti-neuro-inflammatory therapies will attenuate the increases in respiratory frequency and predictability of VPV and restore healthy respiratory network activity. We will apply recent advances in multi-electrode array technology and analysis to map respiratory local field potentials throughout the ponto-medullary respiratory network to investigate where and how peripheral inflammation maladapts central- and sensory-modulated respiratory pattern formation. We will also generate immuno-histochemical maps of the distribution of key early pro-inflammatory cytokines in the brainstem. Electro- physiologic and anatomic atlases will be co-registered to the Waxholm MRI atlas of the rat brain to enable analysis of their relationships. In total, we will use this innovative approach to quantitatively investigate the impact of systemic and neuro-inflammation on the function of an intact neural network. This project is a collaboration between Case Western Reserve University (CWRU, Cleveland, OH) and the Florey Institute (Melbourne, Australia). Frank Jacono (PI, Chief of Pulmonary Critical Care and Sleep Medicine), Yee-Hsee Hsieh & Thomas E. Dick (co-Investigators) at CWRU; and Rishi R. Dhingra & Mathias Dutschmann (co-Investigators) at Florey. This team has the needed expertise to perform the proposed project.

NIH Research Projects · FY 2024 · 2022-09

There is a great need for treatments for diseases caused by nonsense mutations. In the context of Cystic Fibrosis (CF), caused by mutations in the CFTR gene, patients with two nonsense alleles lack CFTR-specific treatments. CF provides a useful context in which to study nonsense mutation biology as CF is a well characterized genetic disease, has numerous model systems, and has dozens of nonsense mutations within CFTR. Nonsense-mediated decay (NMD) is the cellular process that degrades transcripts containing premature termination codons caused by nonsense mutations. NMD acts as a treatment barrier in nonsense mutation conditions by preventing protein from being translated as few nonsense transcripts remain in the cell. NMD, however, does not affect all nonsense transcripts equally. NMD efficiency has been suggested to vary by tissue, cell type, and even by patient. Furthermore, nonsense mutations in specific locations within the transcript escape NMD. This proposal aims to study the heterogeneity of NMD in CF nonsense mutations and the pharmacological correction of CF nonsense mutations. Novel mouse models containing CF nonsense mutations predicted to escape NMD will be developed. Using these NMD resistant models, NMD efficiency will be measured and compared to existing CF models containing NMD susceptible nonsense mutations. Using existing and new CF nonsense mouse models, pharmacological correction of nonsense mutations will be studied. NMD inhibitors will be investigated, as well as readthrough agents (capable of triggering the readthrough of premature stop codons), and CFTR modulators (which promote CFTR protein folding and function) will be tested independently and in combination in mouse and human intestinal organoids and ultimately in vivo in nonsense mutation mouse models. The best pharmacological combination may differ for each genotype or by NMD phenotype. Studying the role of NMD in CF heterogeneity and CF nonsense mutation correction may elucidate better therapeutic strategies and provide evidence for pursuing a precision medicine approach in CF. The training plan outlined in this proposal will strongly support a successful transition to a career in translational research for the trainee. The training plan includes activities relating to science communication, clinical and translational research, preclinical in vivo animal techniques, and bioinformatics.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Subclinical (asymptomatic) malaria with Plasmodium falciparum (Pf) is common in children who live in moderate- to-high transmission areas of sub-Saharan Africa. Although subclinical malaria may be submicroscopic, affected school-age children (ages 8-15 years) often have positive blood smears that include millions of parasites per mL of blood. While subclinical malaria is typically attributed to acquired adaptive immunity that tightly controls the Pf biomass, patent parasitemia (blood smear+) exceeds any reasonable estimate of the pyrogenic threshold. How is it possible to remain without fever and overt malaria symptoms with patent parasitemia? In contrast to symptomatic malaria, temporally persistent (chronic) subclinical malaria seems to be maintained by a complex balance of pro-inflammatory and anti-inflammatory cytokines and immune cells. The scientific premise of this proposal is that epigenetic modifications of innate immune cells, including blood monocytes (Mo), modulate inflammatory pathways underlying subclinical malaria. Furthermore, we hypothesize that an immune homeostasis network involving anti-inflammatory Pf-induced type 1 T regulatory cells (Tr1) and IL-10 as well as enhanced IL-1RA production suppress innate immune inflammatory pathways. Subclinical malaria is of high epidemiologic significance as parasitemia persists for months in schoolchildren who serve as the major reservoir of gametocytes required to sustain Pf transmission to local anopheline vectors. Indeed, it is estimated that ~ 60% of new mosquito infections can be attributed to this demographic. To test our hypotheses, we will enroll Kenyan schoolchildren (ages 8-15) in a longitudinal cohort study to compare and analyze differences in immune parameters between those with A) chronic subclinical malaria (Pf+ smear at baseline and who remain afebrile despite repeatedly smear+ x 16 weeks) relative to B) children who develop febrile clinical malaria up to 2 weeks after an afebrile Pf+ smear. The specificity of immune parameters for chronic Pf exposure in these cohorts will be interrogated by comparison to age and sex matched children residing in a nearby highlands area where malaria endemicity is ~ zero. PBMC and isolated Mo from children will be analyzed by RNA-seq to determine activated gene expression pathways. We will define the differences in immune cell subsets, the transcription factors that are activated and their effector cytokine expression profiles using mass cytometry (CyTOF). In addition, we will use chromatin immunoprecipitation (ChIP) DNA sequencing to determine if the epigenomes of children with chronic subclinical malaria are modified in order to silence proinflammatory genes or conversely, to activate anti-inflammatory ones. Finally, we will identify and compare by Assay for Transposase-Accessible Chromatin with high-throughput Sequencing (ATAC-seq) open chromatin sites in key gene expression pathways in Mo from the comparator groups and align these regions with RNA-seq data from the same child. The successful completion of this project should give us new and important insights as to the mechanism of subclinical malaria and how this disease state can be modified to facilitate malaria eradication.

NIH Research Projects · FY 2025 · 2022-08

Protein tyrosine phosphatase receptor T (PTPRT) is frequently mutated in human cancers, including colorectal cancer. PTPRT has two tyrosine phosphatase domains. While the membrane-proximal domain is an active protein tyrosine phosphatase, it has been thought that the C-terminal domain is a pseudo-phosphatase lacking enzymatic activity. Our preliminary data demonstrated that the pseudo-phosphatase domain of PTPRT is an active enzyme, termed denitrase, that removes nitro-groups (NO₂ at the 3-carbon position of the phenol ring of tyrosine) from the Y333 residue in ERK and Y404 residue paxillin. We demonstrated that nitro-Y333 (nY333) Erk increases its kinase activity, whereas nitro-Y404 (nY404) paxillin is likely to transduce cell signal through a “reader” (nY binding protein). Further, we generated denitrase-inactivating mutant knockin mice and showed that the mutant mice are susceptible to carcinogen-induced colon tumor development. Recently, several recent bioinformatics studies demonstrated that PTPRT mutations, including those in the denitrase domain, are enriched in metastatic colorectal cancers, suggesting that inactivation of PTPRT denitrase promotes tumor metastasis. Thus, we hypothesize that the PTPRT-regulated ERK and paxillin nitration signaling pathways play a critical role in colorectal progression and metastasis. Three aims are proposed to test this central hypothesis by determining the role of: (1) PTPRT denitrase-regulated paxillin nitration signaling in colorectal cancer progression and invasion; and (2) PTPRT denitrase-regulated Erk nitration signaling in colorectal cancer progression and metastasis. Protein tyrosine nitration is currently believed to be a byproduct of reactive oxygen/nitrogen species and not regulated by enzymes. Success in our proposed studies will establish protein tyrosine nitration as a critical player in colorectal tumor progression and metastasis.

NIH Research Projects · FY 2025 · 2022-08

Project Summary / Abstract Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), is a disease that kills 1.4 million people every year. There is no reliable vaccine to prevent TB, yet many latently-infected individuals are protected from progressing to active TB despite heavy Mtb exposure living in an endemic setting. CD4+ T cells are critical for host protection against TB as they interact directly with Mtb-infected cells, secrete cytokines and cytolytic molecules, and recruit or augment other immune cells. However, the candidate and others find that not all T cells specific for Mtb antigens are able to recognize Mtb-infected macrophages, the niche cell for Mtb. A critical unmet need for vaccine development is to define the antigen specificities and functions of T cells that can recognize infected macrophages and prevent progression to active TB. In Aim 1, the candidate uses autologous ex vivo co-culture and T cell antigen receptor (TCR) sequencing to determine the proportion, antigen specificities and functions of memory CD4+ T cells that recognize Mtb-infected macrophages. In Aim 2, the candidate expands on this system to compare the repertoires of memory CD4 T cells that respond to infected macrophages among two groups of individuals who live in a setting endemic for TB yet differ in their susceptibility to active disease. Using single-cell transcriptomics, the candidate compares the functions and TCR repertoires of Mtb- specific memory CD4+ T cells isolated from exposed individuals who do not develop active TB (“stable” latent Mtb infection) vs. individuals who will later progress to active TB (“pre-TB” progressors). Results from this project will define key features of protective memory CD4+ T cells that are linked to the prevention of active TB, providing benchmarks for vaccine development and improvement of TB risk stratification. This 5-year K08 program provides mentoring, training in human immunology, translational research and single-cell transcriptomics for Dr. Stephen Carpenter, a T cell immunologist and infectious disease physician at Case Western Reserve University (CWRU). The institutional environment at CWRU combines an established TB research unit, BSL-3 flow cytometry, cell sorting, single-cell RNA sequencing, and expertise in bioinformatics together with a premier graduate program for scientific interaction and courses. The longstanding Uganda- CWRU Research Collaboration for TB enables clinical and translational work in a TB endemic setting. The candidate is establishing a lab with the long-term goals of understanding the defining features of protective memory T cell responses to Mtb. He has recruited mentors with substantial expertise in translational TB research, T cell biology and single-cell transcriptomics, including Drs. Henry Boom and Mark Cameron at CWRU, and Dr. Sam Behar (UMass Medical School). Completion of this K08 project will transition the candidate into an independent investigator, positioning his research program to use powerful single-cell immune profiling approaches to track and study antigen-specific human memory T cells.

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract In the past decade, traumatic brain injury (TBI) has been rising in incidence, and is linked to a 2-4 fold increase in developing Alzheimer's disease and related dementias later in life1–5. TBI presents as a progressive neurodegenerative injury characterized by cognitive deficits and neuropsychiatric impairment4,6. Currently, there are no available treatments to prevent, slow, or reverse the chronic progression of neurodegeneration and accelerated AD after TBI. In many neurodegenerative conditions, including AD, Parkinson's disease, Huntington's disease, aberrant mitochondrial fission has been identified as a critical component of pathogenesis7. It has also been implicated in the acute stages of concussive TBI8–10. The goals of this project are to characterize the changes in regulation of mitochondrial fission that occur in acute and chronic TBI, and to determine whether pharmacologically limiting aberrantly high mitochondrial fission after TBI provides a neuroprotective strategy for TBI and TBI-induced accelerated AD. To address my goal, I propose the following aims, using an established mouse model of TBI: 1. Determine how TBI affects expression, modification, and activity of the key mediator of mitochondrial fission, dynamin-related protein 1 (Drp1). I will measure expression of Drp1 across a comprehensive list of brain regions at early and late timepoints after TBI. I will also measure post-translational modifications, oligomerization activity, and expression of regulatory proteins of Drp1. 2. Determine how pharmacologic inhibition of mitochondrial fission after acute TBI mitigates pathology and symptoms at acute and chronic timepoints after TBI. I will administer injured mice with P110, a small peptide inhibitor of the key mitochondrial fission protein Drp1 which has already been determined to be neuroprotective in TBI, and then investigate pathological changes across an array of histological measures, ultrastructural changes via transmission electron microscopy, and bioenergetic changes via Seahorse analysis. 3. Determine the efficacy of P110 treatment in mitigating TBI-induced acceleration of Alzheimer's disease. I will administer P110 to 5xFAD mice following mild TBI, and use behavioral testing to determine P110's efficacy in reducing accelerated neurocognitive deficits. I will also use histological methods to monitor changes in plaque deposition as a function of TBI and P110 treatment. Through completing this project, I will acquire new lab techniques, including behavioral testing, immunohistochemistry, biochemical assays, and both confocal and electron microscopy. I will also foster collaboration with researchers in related fields of neurodegeneration, and develop clinical insight into the field of neurodegeneration. This proposal outlines a rigorous training plan by which I will establish the skills needed for a successful career as a physician-scientist in the fields of neurodegeneration and neuropsychiatry.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract To obtain grants and successfully conduct biomedical research with human subjects, researchers must recruit and retain sufficient numbers of participants. Literature on ethical concerns with coercion and undue influence abounds, but there is little consideration of whether it is ethical to employ behavioral economics techniques called “nudges” that can encourage participation, such as screening surveys that predispose participants to consent, structured choice architecture in consent forms, certain forms of community engagement to generate group support for participation, and certain positive personal behaviors by recruiters. In addition, little prior empirical research has examined what recruitment nudges are actually being used in recruitment into human subjects research and their effects. This project addresses to what extent recruitment nudges are being used in recruitment into clinical trials, whether they impact participation, the views of human subjects about their use, and whether their use is ethical in research studies with varying ratios of risk to human subjects. To address these issues, this interdisciplinary investigator team of translational scientists, bioethicists, and legal scholars at Case Western Reserve University and the University of Utah will first identify use of different types of recruitment nudges in clinical trials with varying ratios of risk (minimal risk vs. more than minimal risk) and benefit (direct benefit vs. no direct benefit) to human subjects. Aim 1 will characterize use of recruitment nudges in clinical trials with varying risk/benefit ratios by conducting interviews and a survey with clinical trials recruiter. Aim 2 will examine the effects of a set of nudges on recruitment into a minimal risk interview study for healthy adult volunteers and views of participants on the use of nudges for themselves and others. Aim 3 will utilize the empirical data generated in Aims 1 and 2 to identify the normative, legal and ethical considerations for different recruitment techniques used in clinical trials and propose policy and practice recommendations.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Streptococci are the most abundant microbes in the human oral cavity, with most people harboring multiple species, and often even multiple strains of the same species (1-6). Further, while most species of oral streptococci are not pathogenic, studies with selected strains have shown that streptococci can influence the physical location and the virulence factor expression of oral pathogens such as Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (2, 3, 7-11). These interactions influence the progression and severity of disease, as well as its resistance to treatment (7, 12-16). However, these interactions also rely are governed by the traits that are known to vary across the genus, such as cell surface structures and secreted organic acids (17, 18). Further, transcriptional heterogeneity resulting in the existence of subpopulations, can further alter the behavior of streptococci (19-23). Thus, while the abundant streptococcal genus is thought of as an important mediator of pathogen behavior, it is not well understood how streptococcal-pathogen interactions vary across the genomic and transcriptomic diversity of the genus. In this study, the overarching hypothesis is that genomic, phenotypic, and transcriptional heterogeneity of commensal streptococci alters interactions with pathogens and impacts infections. This proposal aims to expand our understanding of streptococcal-pathogen interactions by investigating diversity at two levels: genomic and transcriptional. First, it proposes to systematically characterize the interactions formed by taxonomically diverse streptococci with the pathogens Pg and Aa in a phylogenomic framework (Aim 1). This work will identify the scale at which interactions vary across the streptococcal genus and potentially identify new functions that mediate interactions with these pathogens. Second, this proposal will characterize the transcriptional heterogeneity in commensal streptococci that result in subpopulations and ask how subpopulations influence, and are influenced by, interactions with oral pathogens (Aim 2). This aim will significantly advance our understanding of the role of transcriptional heterogeneity in oral commensal streptococci, how it varies taxonomically, and how it is altered by environmental changes. Further, both Aims will consider the interplay of streptococcal diversity and interactions on spatial patterning at the micron-level. Overall, the proposed research will broaden our understanding of the role of streptococci during oral disease.

NIH Research Projects · FY 2026 · 2022-08

Abstract Arginine methylation is one of the most common posttranslational modifications (PTMs), which is comparable to phosphorylation and ubiquitination. Protein arginine methyltransferases (PRMTs) correspond to “writers” that generate three types of methylated arginine residues: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). PRMT1 is the main type I enzyme for catalyzing ADMA, while PRMT5 is the predominant type II enzyme for generating SDMA. PRMT1/5 methylates many downstream substrates to regulate a variety of fundamental cellular processes, such as transcription, DNA repair and cell signaling transduction. Deregulation of PRMT1/5 is frequently observed in various cancers and is correlated with poor prognosis and survival of cancer patients. Like many other PTMs, arginine methylation is a reversible process. Demethylases function as “erasers” to remove methyl groups from targeted proteins. In addition, effector proteins called “reader” bind to methylarginine and mediate signals transduction in cells. Although tremendous efforts have been made in the past three decades, there are still many outstanding questions/gaps in the field of arginine methylation. How is PRMT activity regulated by upstream signals/regulators? Are there specific arginine demethylases? What are the readers for numerous of arginine methylated proteins? In this proposal, we will explore three projects to address these questions. Project 1 will elucidate the molecular mechanism by which amino acids regulate PRMT1 subcellular localization, activation, and function, revealing a novel upstream stimulus/regulator of PRMT1. Project 2 will dissect roles of the ubiquitination pathway in regulation of PRMT5, defining a novel interplay between arginine methylation and ubiquitination. Project 3 will identify novel demethylases and readers of arginine methylation, filling the key gap in the field of arginine methylation. We will use a range of complementary methods including biochemistry, mass spectrometric (MS) analysis, molecular and cellular biology, and mouse models in our studies. Our short-term goal is to advance our understanding of arginine methylation biology by completing proposed studies, and log-term goal is to identify novel targets/strategies/inhibitors to target arginine methylation signaling pathway for cancer therapy. To achieve these goals, I will be committed to this program at 51% “research effort”.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Despite advances in the treatment of inflammatory bowel disease (IBD), many patients fail to respond to therapy. Thus, there is a need to find new therapeutic approaches against IBD. The CD40-CD154 pathway is a known target against IBD and other inflammatory disorders. Clinical trials indicated that CD40 blockade with anti-CD154 antibodies reduced inflammation. However, the anti-CD154 antibodies caused thrombosis (unrelated to inhibition of CD40). Moreover, other approaches to cause global inhibition of CD40 are predicted to increase the risk of opportunistic infections. Identification of a strategy to inhibit CD40-induced inflammation that does not induce thrombosis or opportunistic infections can be a major advance in the treatment of IBD. We uncovered that blocking the interaction between CD40 and an intracellular adaptor protein inhibits pro- inflammatory responses induced by CD40 while leaving protection against an opportunistic pathogen intact. We identified a small molecule that binds the adaptor protein, blocks CD40 signaling, reduces pro- inflammatory responses in vitro and diminishes intestinal inflammation in mouse models of IBD. The compound did not impair resistance against an opportunistic pathogen. The compound has suboptimal solubility and microsomal stability. While some analogs designed to date showed some improvement in solubility or microsomal stability, further optimization is necessary. The objective of this application is to develop an optimized inhibitor that will be tested in mouse and human IBD systems. The central hypothesis is that a potent analog with improved solubility and microsomal stability will optimally block CD40 signaling, and markedly suppress inflammation in mouse models of IBD as well as CD40-driven inflammatory responses in intestinal cells from patients with IBD. To test this hypothesis, we will design and generate analogs of the compound, test their properties, perform signaling studies in reporter cells and intestinal cells and test the lead inhibitor in animal models of IBD. In the first specific aim we will use structure activity relationships with the aid of a docking model to design and generate analogs of the compound in order to improve solubility and microsomal stability. We will examine their ability to inhibit CD40 signaling and their affinity for the adaptor protein. In the second aim, we will test the most potent analogs to determine if they inhibit CD40 signaling in vivo. In the third aim, we will determine if the lead analog reduces intestinal inflammation in mouse models of IBD and inhibits CD40-induced expression of inflammatory molecules in intestinal cells from IBD patients. The proposed work may lead to a new strategy to treat IBD based on a novel approach to inhibit CD40 signaling.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Asprosin is a recently discovered, fasting-induced hormone that stimulates hepatic glucose production and appetite. Plasma asprosin crosses the blood-brain-barrier and directly activates orexigenic AgRP neurons, resulting in downstream anorexigenic POMC neuron inhibition, in a GABA-dependent manner. This asprosin- mediated chain of events leads to appetite stimulation and a drive to accumulate adiposity and body weight. Genetic deficiency of asprosin in humans results in Neonatal Progeroid Syndrome (NPS), characterized by low appetite and extreme leanness, a result mimicked by mice carrying similar mutations. Obese humans and mice display pathologically elevated circulating asprosin levels, and depletion of plasma asprosin using monoclonal antibodies reduces appetite and body weight in such mice, in addition to improving their glycemic profile. Thus, asprosin, in addition to performing a glucogenic function, is an orexigenic hormone, and anti-asprosin antibodies show therapeutic potential as anti-obesity agents. A central question the above observations have led to is – what is the identity of the asprosin cell-surface receptor? Recently, through unbiased mass spectrometry on the mouse brain we identified a candidate asprosin receptor (AR) as a putative brain receptor for asprosin. AR is robustly expressed in AgRP neurons but not expressed in the liver. Conversely, the recently discovered liver receptor for asprosin (OLF734), while being highly expressed in the liver, is not expressed in AgRP neurons and Olfr734-/- mice do not display leanness or reduced appetite. Thus, Olfr734 cannot account for asprosin’s stimulatory effect on AgRP neurons and appetite. Besides confirming AR as a binding partner for asprosin, we present preliminary studies demonstrating the necessity of AR for asprosin mediated AgRP neuron activation, appetite stimulation and body weight maintenance. This proposal seeks to build on these preliminary results to determine the contribution of AR as an asprosin receptor in the brain. We seek to do so within the following 3 aims: 1) Determine the necessity of AR for asprosin’s orexigenic effects 2) Validate the AR soluble ligand binding domain for efficacy against metabolic syndrome 3) Determine the molecular mechanisms by which asprosin neuralization reduces appetite and body weight and interplay with leptin signaling. At the completion of these aims we expect to definitively validate AR as the brain receptor for asprosin through an exploration of necessity, druggability, function and in vivo mechanism.

NIH Research Projects · FY 2025 · 2022-07

Abstract Atrial fibrillation is the most common form of cardiac arrhythmia, with prevalence estimated to be 5.2 million in 2010 and predicted to increase to 12.1 million in 2030. Atrial fibrillation is a major risk factor for blood clots and stroke, independently increasing stroke risk 4- to 5-fold throughout all ages. Therefore, the vast majority of patients with atrial fibrillation require some form of stroke prevention therapy. Current first line stroke prevention therapy for atrial fibrillation patients is life-long use of oral anti-coagulation medications, which are associated with increase in bleeding risk by approximately 2- to 2.5-fold, including intracranial hemorrhage, that may lead to hospitalization, transfusion, surgery, and death. The purpose of the present study is to improve stroke prevention treatment for non-valvular atrial fibrillation by transforming the Left Atrial Appendage Occlusion (LAAO) procedure into a first line therapy for a larger segment of patient populations, especially for younger patients with 20+ year of life expectancy who are likely to experience bleeding problems in their lifetimes. LAAO is a minimally invasive procedure where an implant delivered using an intravascular catheter is used to permanently seal off the left atrial appendage mechanically to reduce the risk of blood clots. The barriers preventing LAAO from becoming a first line therapy are primarily safety and cost. The investigators aim to overcome these barriers and transform LAAO into a first line therapy by developing a real-time Magnetic Resonance Imaging (MRI)-guided robotic intravascular catheter system for performing LAAO procedures by synergistically combining novel medical imaging, robotic catheter control, and advanced visualization technologies to improve the safety, cost, and workflow of LAAO procedures. The proposed technology expands on novel approaches initiated by the investigators in earlier work in the areas of real-time MRI image acquisition and reconstruction, robotic catheters actuated using the magnetic field of the MRI scanner, advanced visualization, and volumetric planning of LAAO. The project is organized into three Specific Aims, each focusing on one key aspect of the procedure workflow, namely, procedure planning, transseptal puncture, and LAAO implant delivery. These Specific Aims build on crosscutting technical research on MRI, robotics, and human-machine interface technologies, where the investigators will develop novel technologies for rapid and flexible 2D/3D cardiac MRI imaging, robotically controlled MRI-compatible dexterous cardiac catheters, and human-machine interfaces with advanced visualization. The end result of this proposal will be the complete prototype of an MRI-guided robotic catheter system for performing LAAO procedures combining real-time intraoperative MRI, robotic catheter control, and advanced visualization technologies to facilitate safer, more efficient, and cost-effective LAAO procedures. The developed system and the underlying technologies will be validated by experts in interventional cardiology in vertebrate animal and non-clinical human studies.

NIH Research Projects · FY 2025 · 2022-07

Project Abstract Pancreatic β-cells is essential for the regulation of blood glucose. One major hope for diabetes therapy is to generate a large number of functional, transplantable beta-cells from patient-derived pluripotent cells. In the past decade, a few in vitro protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into functional β-like cells, which also serve as fantastic tools for the study of human pancreatic development to reveal the etiology of relevant diseases. However, the major limitations to use β-cell differentiation system in research and therapeutics is that the protocol is still not robust. (i) The differentiation generates heterogenous cell populations; (ii) Differentiation efficiency is variable between different hPSC lines, and also between batches. (iii) The resulting β-like cells are still not quite equivalent to primary β-cells from human islets at molecular and physiological levels. To address this problem, we propose to use the latest single cell and low-input genomic technology to generate a reliable map of lineage determination in this system. Importantly, we will for the first time map the individual variation between the differentiation of 24 hPSC lines. To ensure robust comparison, we have devised a pooling- demultiplexing single cell genomic approach that allows simultaneous mapping of many hPSC lines in one scRNA-seq or scATAC-seq experiment. This strategy minimizes the batch variation and significantly reduces the experimental cost. In Aim 1, we will use this approach and scRNA-seq to map the dynamics and variation of single cell transcriptome while differentiating 24 hPSC lines towards pancreatic β-cells. In Aim 2, we will map the dynamics and variation of open chromatin using scATAC-seq, and we will also use a low-input Hi-C technology to reveal the dynamic 3D genome during β-cell differentiation. In aim 3, we will perform high-throughput CRISPR screen and locus-specific genome editing to discover and validate key differentiation regulators at both gene and enhancer levels. This project is built upon a rich set of published and preliminary data, which already led to improved differentiation protocol and better understanding of disease genetics. Completion of this project will deliver a comprehensive data resource of transcriptome, epigenome, and 3D genome during the β-cell differentiation, which will shed light on the disease etiology, and reveal novel therapeutic opportunities.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/ Abstract Distinct epigenetic mechanisms, including histone modifications, DNA methylation, chromatin remodeling and histone variant exchange, collaboratively and dynamically shape local epigenetic landscape and control gene activities. Alteration of epigenetic pathways and mutations in epigenetic regulators contribute to the pathogenesis of various human diseases. Several studies suggest that these epigenetic pathways are orchestrated to regulate activity-gene expression. Current tools to simultaneously manipulate multiple epigenetic pathways in temporal and locus specific are lacking. Such tools are necessary to precisely dissect the functional interplays and crosstalk between these distinct epigenetic mechanisms for gene regulation. To address this limitation, we propose to establish unique chemical inducible methods that integrate dCas9/gRNA-guided targeting with the orthogonal chemically induced proximity (CIP) technology to simultaneously control different epigenetic regulations including all major epigenetic pathways. We will focus on the following three aims: (i) Develop inducible dual editing platforms for diverse histone modifications; (ii) Develop chemical inducible platforms for chromatin remodeling and nucleosome editing; (iii) Develop chemical inducible editing platforms for DNA methylation and validate the orthogonal manipulation of multiple mechanisms across distinct epigenetic pathways. After finishing this work, we expect to establish a unique toolset that can be readily used by researchers in the field. This platform can be easily adapted and expanded to manipulate new epigenetic factors not tested in this study. This new epigenome editing/remodeling platform will facilitate the advancement of epigenetic research and potentially offer new directions in developing new epigenetic-based therapies.

NIH Research Projects · FY 2026 · 2022-07

SUMMARY Stroke is a leading cause of death and long-term disability. Brain edema is the most common life- threatening complication following an acute stroke event. It leads to elevated intracranial pressure that will affect the spared areas surrounding the stroked tissue. The preservation of such areas is critical in reducing brain injury and promoting long-term recovery. Understanding the pathophysiology of brain edema is key to identifying therapeutic targets and reducing the final disability burden related to acute stroke. The glymphatic system is a recently discovered waste clearance pathway in the brain. It removes interstitial metabolic waste products, as well as excessive interstitial fluid (ISF), by facilitating the exchange of ISF and cerebrospinal fluid (CSF). With drastically increased metabolic byproducts and the development of cerebral edema, the glymphatic system may play a critical role in post-stroke recovery. Further, aquaporin-4 (AQP4), a water channel protein that has been recognized to be involved in cerebral edema, also drives the glymphatic system. However, due to the limited means of evaluating the glymphatic function in vivo, our current understanding of the role of AQP4 and the glymphatic system in post-stroke recovery is still quite limited. The goal of this project is to develop novel MRI methods for quantitative assessment of the glymphatic function in vivo and to apply these methods to investigate the role of AQP4 and the glymphatic system in post-stroke edema formation and reabsorption. Specifically, we will develop and validate a 3D magnetic resonance fingerprinting (MRF) method for dynamic and simultaneous tracking of a gadolinium (Gd)-based, large molecular weight (MW) paravascular tracer (GadoSpin, MW=200 kDa, primarily a T1 contrast agent) and oxygen-17 (17O) enriched water (H217O, MW=19 Da, a T2 contrast agent) in mouse brain (Aim 1). This approach will enable the simultaneous evaluation of CSF flow in the paravascular space and CSF-ISF exchange between the paravascular space and brain parenchyma in a single MRF scan. Kinetic analysis methods will be developed for quantitative assessment of the glymphatic function from MRF measurements, including the CSF flow in the paravascular conduits and the CSF-ISF exchange rate and water transport in the glymphatic pathway (Aim 2). These methods will be applied to evaluate the effects of AQP4 knockout and inhibition on edema formation and reabsorption in two mouse models of ischemic (cytotoxic edema) and hemorrhagic (vasogenic edema) stroke (Aim 3). Successful completion of the project will give rise to a novel MRI method for in vivo quantification of glymphatic function. Application of this method to the investigation of post-stroke pathophysiology will lead to new insights into the role of AQP4 and the glymphatic system in post-stroke edema.

NIH Research Projects · FY 2025 · 2022-07

Abstract / Project Summary The brain extracellular matrix (ECM) is a complex three-dimensional milieu that has a profound influence on synaptic plasticity and myelination during development. The pathways and cell types driving ECM generation during brain development are poorly understood. This proposal investigates a recently identified cellular pathway that controls ECM composition from the oligodendrocyte progenitor cells (OPCs). This discovery was made through studies of the transcription factor THAP1, which is mutated in the neurodevelopmental movement disorder DYT-THAP1 (DYT6) dystonia. Previous work identified a critical role for THAP1 in developmental myelination. Loss of THAP1 in the oligodendrocyte (OL) lineage impairs the progression of OPC into mature myelinating OLs. Recent work has established that THAP1 mediates OL differentiation in large part by controlling the composition of secreted chondroitin sulfate (CS) GAGs, a class of long unbranched polysaccharides and core-constituents of the ECM, which are inhibitory to OL differentiation. The hypothesis that OPCs regulate ECM composition is new, and the transcription factor THAP1, together with its partner YY1, provides an inroad to delineating the cellular mechanisms that regulate ECM composition during CNS development. The proposed studies will establish a molecular understanding of the regulation of ECM and myelination by THAP1. Aim 1 will use ChIP-seq and transcriptomic studies to test the hypothesis that THAP1 and its co-regulatory transcription factor YY1 regulate GAG metabolism through a shared pathway in differentiating OPCs. Both transcription factors have established roles in myelination and cause human dystonia when mutated. Aim 2 will test the hypothesis that OPCs are important contributors to developmentally regulated CS-GAG composition of the brain ECM and define the relative contribution of other CNS cell types, and the role of THAP1 in this process. Aim 3 test the hypothesis that THAP1- dependent OL maturation is required for normal motor learning and define THAP1-dependent changes in ECM pathway genes induced by motor learning. These studies will advance our understanding of the mechanisms by which glia and ECM contribute to CNS motor function and will shed light on how disruption of these processes contribute to pathogenesis of dystonia and other neurodevelopmental disorders.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY / ABSTRACT Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders among the aged population. Studies showed the critical impacts of apolipoprotein E (APOE) ε4 isoform on susceptibility and pathogenesis of late-onset AD (LOAD). APOE proteins modulate amyloid beta (Aβ) in extracellular plaque formation in an isoform dependent manner. Mitochondrial deficits were also shown associated with risk APOE ε4 and Aβ in AD. The interplay of APOE ε4 and Aβ likely modulates the inter-organelle crosstalk, which justifies the investigation into such crosstalk in AD pathogenesis. The pilot study found increased contacts formed between mitochondria and endolysosomal system in AD neurons compared to non-AD controls, both in human brain and the mouse model. Furthermore, many more contacts were observed among human AD APOE ε4 carriers than in AD APOE ε3 carriers. In vitro studies showed APOE AD risk isoform affected the mito- endolysosomal contacts and mitochondrial Aβ levels and identified STARD3 as an important mediator. These exciting data unveiled a novel abnormality in mito-endolysosomal contacts, which was associated with APOE ε4 regulated Aβ toxicity in AD. Therefore, we hypothesized that APOE ε4 caused abnormally increased mito-endolysosome interaction and mi- tochondrial translocation/accumulation of Aβ through enhanced interaction with STARD3 which led to mitochondrial dysfunction and neurodegeneration in sporadic LOAD. To test this hypothesis, the detailed examination of the mito- endolysosomal contact by in vivo and in vitro AD models is required. The long-term goal of this study is the pursuit of abnormal inter-organelle crosstalk that likely underlies mitochondrial dysfunction and neuronal degeneration in LOAD. In this regard, the efforts will strive to decipher these three specific aims: 1) whether and how the abnormal crosstalk be- tween mitochondria and endolysosomal system causes mitochondrial deficits, 2) what is the role of the interplay of APOE ε4 and its endosomal cargo Aβ in such inter-organelle crosstalk, 3) whether the underlying mechanism supports a thera- peutic potential for AD. The proposal will focus on the pathogenesis in general sporadic AD population associated with common AD risk APOE isoform. Importantly, the proposed novel mechanism will also pave the road for discovery of a yet uncharacterized therapeutic target for common AD population.