ARC25 Agenda – Fri, Jan 31

Caraline New1, Sonia S. Ali1, Kylee Meece2, and Hilary R. Katz1

1Department of Biology, Western Kentucky University, Bowling Green, KY, 2College of Pharmacy, University of Kentucky, Lexington, KY

Introduction/Background. The larval sea lamprey, Petromyzon marinus, is a well-established model for successful spinal cord regeneration due to its unique ability to recover after a complete spinal transection. Immediately following a transection, the animal is paralyzed below the injury site, and over the course of 11 to 12 weeks, it exhibits substantial swimming recovery. Spinal injuries in non-regenerative species, such as mammals, produce many acute and chronic medical issues including progressive muscle atrophy. To our knowledge, it is unknown whether the lamprey exhibits muscle atrophy during their initial recovery period, and whether such muscle atrophy could reverse as animals regain their swimming ability. 

Hypothesis/Goal of Study. We sought to examine muscle physiology in the larval lamprey after a period of disuse (3 weeks post-injury), and after progressive swimming recovery (11 weeks post-injury).

Methods and Results. We developed a procedure to analyze lamprey muscle structure with transmission electron microscopy pre- and post-injury. Briefly, muscle tissue was collected from uninjured control, 3 weeks post-injury, and 11 weeks post-injury individuals. The tissue was then fixed, infiltrated in resin, thin-sectioned with an ultramicrotome, and imaged with a Transmission Electron Microscope (JEM-1400Plus). Preliminary observations of 11 weeks post-injury animals revealed shrunken sarcomeres with abnormal z-discs. In contrast, the muscle above the injury site maintains its integrity.

Discussion/Conclusions. These results suggest that lampreys experience muscle atrophy in response to traumatic nerve injury despite their regenerative capabilities.

Citation/Acknowledgements. KY INBRE Voucher

Jason A. Stewart1, Stephanie M. Ackerson2, Jaclyn S. Holbrooks1, Grayson H. Duvall1, and Meaghan Arnold2

1Department of Biology, Western Kentucky University, Bowling Green, KY, 2University of South Carolina, Department of Biological Sciences, Columbia, SC

Introduction/Background. Telomeres are repetitive DNA sequences bound by protective protein complexes at the ends of chromosomes. These structures protect chromosome ends from being recognized as DNA breaks and from degradation. Loss of telomere protection often leads to chromosome fusions and instability, which can promote cancer and aging. Previous work identified the human DNA binding protein CST (CTC1-STN1-TEN1) as a potential regulator of the telomeric DNA damage response (DDR). Deletion of the CTC1 or STN1 subunits of CST leads to the binding of DNA repair factors at telomeres, the activation of DNA damage signaling, and cell cycle arrest but surprisingly does not significantly increase chromosome fusions, suggesting a previously undiscovered mechanism of telomere protection.

Hypothesis/Goal of Study. The goal of this study is to determine how telomeres remain protected from DNA repair mechanisms while still promoting a DNA damage response sufficient to induce cell cycle arrest following CST deletion.

Methods and Results. Using conditional CTC1 and STN1 knockout cell lines, we determined that the DDR kinase ATR is necessary to prevent chromosome fusions and induce cell cycle arrest in CST-deleted cells. Furthermore, we identified that the DDR factor 53BP1, which can inhibit certain types of DNA repair, localizes to telomeres in an ATR-dependent manner. We are also conducting a phosphoproteomics screen through the Core Proteomics Laboratory at the University of Louisville to identify novel targets that are phosphorylated by ATR in CST-deleted cells. 

Discussion/Conclusions. Overall, this work defines a novel telomere protection mechanism mediated through the ATR kinase that prevents chromosome fusions and instability. 

Citation/Acknowledgements. This project was supported by a Start-up Award, Research Project Award, and a Core Utilization Voucher through KY INBRE grant P20GM103436-24 from the National Institutes of Health.

Jenna L. Kesselring, Abigail E. Santos, and Gary T. ZeRuth

Department of Biology, Murray State University, Murray, KY

Abstract embargoed

Eric J. Rellinger1, Michelle G. Pitts1, Beibei Zhu1, Michael D. Buoncristiani1, Lindsay T. Bryant1, Oscar Lopez-Nunez2, Juan Gurria3, Chi Wang4, Matthew S. Gentry5, Emiel Rossing6, Nathan R. Shelman7, Derek B. Allison7, B. Mark Evers1, Christian Büll7, Thomas J. Boltje8, Johan F. A. Pijnenborg8, and Ramon C. Sun5

1Department of Surgery, University of Kentucky, Lexington, KY, 2Department of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 3Department of Pediatric Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 4Department of Bioinformatics, University of Kentucky, Lexington, KY, 5Department of Biochemistry & Molecular Biology, University of Florida, Gainesville, FL, 6GlycoTherapeutics, Nijmegen, the Netherlands, 7Department of Pathology, University of Kentucky, Lexington, KY, 8Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands

Introduction/Background. Aberrant glycosylation is a hallmark of cancer but few have evaluated the N-linked glycome of neuroblastomas. MYCN-amplification is a frequent feature of high-risk neuroblastomas that metabolically transforms cancer cells. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to quantify N-linked glycans within human neuroblastoma tissues. MALDI-MSI revealed that core fucosylated glycans are enriched within MYCN-amplified neuroblastomas. GDP-mannose 4,6-dehydratase (GMDS) is the first committed enzyme of de novo fucose synthesis. 

Hypothesis/Goal of Study. Our central hypothesis is that GMDS is a critical mediator of MYCN-amplified neuroblastoma progression and immune evasion.

Methods and Results. MALDI-MSI was performed on five MYCN-amplified and four MYCN non-amplified neuroblastomas. Neuroblast-rich regions were defined by H&E overlay and demonstrated increased abundance of seven N-linked glycans. Six glycans featuring core fucosylation were enriched. Kaplan-Meier analysis revealed that high GMDS expression is associated with poor overall survival and MYCN-amplification in neuroblastomas. Chromatin immunoprecipitation and luciferase promoter assay demonstrated that N-MYC directly activates the GMDS promoter. Stable GMDS knockdown decreased subcutaneous growth in MYCN-amplified subcutaneous models (p<0.01). GMDS chemical blockade by intratumoral injection of Fucotrim I in immune competent 9464D tumors impedes tumor growth (p<0.01).  Flow cytometry-based immune profiling of Fucotrim I-treated tumors revealed enrichment of MHC-II+ macrophages (p<0.05).

Discussion/Conclusions. Core fucosylation is increased in situ within human MYCN-amplified neuroblastomas. High GMDS expression is associated with poor survival and MYCN-amplification. Genetic knockdown of GMDS blocked subcutaneous tumor formation. Fucotrim I decreased tumor growth and increased recruitment of macrophages featuring high MHC-II expression demonstrating that GMDS is a novel regulator of myeloid trafficking within the MYCN-amplified neuroblastoma tumor microenvironment.

Citation/Acknowledgements. This research was generously supported by the University of Kentucky Center for Cancer and Metabolism, funded through the NIH/NIGMS COBRE (P20 GM121327) and the Dick Vitale Pediatric Cancer Research Fund (V2023-026).

Sara Palacio1, Nicole Rummel2, James Campbell1, Allan Butterfield2, Subbarao Bondada3, Heidi Weiss4, John Villano5, Ines Batinic-Haberle6, Daret St. Clair1, and Luksana Chaiswing1

1 Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY, 2 Department of Chemistry, University of Kentucky, Lexington, KY, 3Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY, 4Department of Internal Medicine, University of Kentucky, Lexington, KY, 5Department of Neuro-Oncology, University of Kentucky, Lexington, KY, 6Department of Radiation Oncology, Duke University, Durham, NC 

Introduction/Background. Extracellular vesicles (EVs) are nanoparticles released by most cells. Their cargo depends on the biochemical characteristics of their cell of origin. We previously demonstrated that EVs can be an early indicator of neuronal injury after radiation therapy (RT) in mice. Glioblastoma (GBM) remains an incurable cancer, with surgery, chemotherapy and RT as the standard treatment. We found that GBM patients exhibit higher numbers of EVs, particularly following RT.

Hypothesis/Goal of Study. Given that EVs can function as biomarkers and GBM patients experience severe cognitive impairments, we seek to elucidate the role of GBM-derived EVs in cognitive decline. 

Methods and Results. We characterized EVs collected from GBM patients. Size and concentration were assessed with ZetaView Nanoparticle Tracking Analysis (NTA) while morphology was assessed with Transmission electron microscopy (TEM). Then, we developed a murine orthotopic GBM model. EVs collected from tumor mice were significantly higher than their healthy counterparts. To further understand the function of EVs in cognition, we focused on EVs released by GBM after RT. These EVs contain high levels of 4HNE-adducted proteins as confirmed by western blotting and TEM with immunogold labeling. Then, we injected GBM-derived EVs intracranially in immunocompetent mice, which exhibited cognitive decline, DNA damage in cerebral tissue, reduced neuron markers and increased pro-inflammatory cytokines. In vitro studies suggest that GBM-derived EVs can activate microglia and cause neurotoxicity via H2O2 release.

Discussion/Conclusions. GBM-derived EVs induce microglia-mediated neuronal damage and cognitive impairment.

Citation/Acknowledgements. Work supported by LC start-up funding (MCC/COM/DCTB), University of Kentucky COBRE-CNS Metabolism NIGMS (P20 GM148326), 2023 Collaborative Bench to Bedside Pilot Grant MCC Radiation Medicine, and NIH Award KY INBRE P20GM103436-24.

Abdullah Masud1, Ethan A. Fernandez1, Qunfeng Huang1, Hu Huang2, James C. Zimring3, and Qingjun Wang1

1Department of Ophthalmology and Vision Sciences, University of Kentucky, Lexington, KY, 2Department of Ophthalmology, University of Missouri, Columbia, MO, 3Department of Pathology, University of Virginia, Charlottesville, VA

Introduction/Background. Glucose-6-phosphate dehydrogenase (G6PD) is a crucial enzyme in the pentose phosphate pathway (PPP), producing NADPH, essential for neutralizing oxidative stress, and pentoses, which contribute to nucleotide synthesis1. G6PD deficiency, the most common human enzymopathy, affects 400 million people globally and is linked to hemolysis due to insufficient NADPH in red blood cells (RBCs)2. Beyond RBCs, emerging evidence suggests G6PD deficiency may impact platelet function, particularly in cardiovascular disease patients over 603. However, its role in platelet activity remains underexplored.

Hypothesis/Goal of Study. This study aims to elucidate the effect of G6PD deficiency on platelet function using a G6PD Mediterranean mutation (Med) conditional knock-in mouse model and G6PD knock-out (KO) mice.

Methods and Results. We treated mouse platelets with a G6PD inhibitor and evaluated Ca²⁺ influx. Additionally, we analyzed platelet functionality in G6PD knockout (KO) mice by measuring platelet count, morphology, Ca²⁺ influx, and clot contraction following thrombin stimulation. Tail bleeding times were also assessed. Similar parameters, including clot contraction and tail bleeding time, were evaluated in G6PD Med-mutant mice.

G6PD inhibition reduced Ca²⁺ influx in a dose-dependent manner, while G6PD KO mice displayed significantly reduced Ca²⁺ influx, indicating impaired platelet activity. Clot contraction was markedly decreased in KO mice, and tail bleeding times were slightly prolonged, suggesting reduced platelet activation. In contrast, G6PD Med-mutant mice exhibited enhanced platelet activity, characterized by faster clot contraction and reduced tail bleeding times.

Discussion/Conclusions. Our findings demonstrate that G6PD deficiency impairs platelet function by reducing Ca²⁺ influx and clot contraction, which may contribute to prolonged bleeding times. Conversely, G6PD Med mutations appear to enhance platelet activity, potentially increasing cardiovascular risks through faster clot formation and reduced bleeding times. These results highlight the critical role of G6PD in regulating platelet function and its implications for hemostasis and thrombotic disorders.

Citation/Acknowledgements. 

1. 1. D’Alessandro, Angelo, et al. Haematologica 106.5 (2021): 1290.

   2. D’Alessandro, Angelo, et al. JCI insight 6.14 (2021).

   3. Ding, Yangyang , et al. Nature Communications 14.1 (2023): 4829.

Funding support: R01HL160910 and pilot grants from P30GM127211 and P30DK020579 (to Q.J.W.)

Barbara Whitt1, Kirsten Scoggin1, Mats Troedsson1, Yatta Boakari2, and Hossam El-Sheikh Ali1

1Gluck Equine Research Center, University of Kentucky, Lexington, KY, 2College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 

Abstract Embargoed

Ethan B. Tiller1, John M. Allen2, and Elizabeth M. Duncan2 

1Transylvania University, Lexington, KY, 2Department of Biology, University of Kentucky, Lexington, KY 

Introduction/Background. The canonical wnt/β-catenin pathway is highly conserved across species, but disruptive changes to the pathway results in species-specific phenotypes. As found in numerous papers, RNA interference (RNAi) of adenomatous polyposis coli (APC) and notum in Schmidetea mediterranea (S. med) results in tail formation in both the anterior and poster ends of the worm (Gittin and Peterson, 2022; Petersen and Reddien, 2011). However, there are no papers involving a double knockdown of both APC and notum in S. med. Additionally, none of these knockdowns have been performed in Girardia guanajuatiensis (G. gua). I am in the process of performing a double-knockdown of APC and notum in S. med and will later perform knockdowns of APC, notum, and APC/notum in G. gua.

Hypothesis/Goal of Study. Given our knowledge of the wnt/β-catenin pathway, we hypothesize that the double-knockdown of APC and notum in S. med will yield a double tail-phenotype as well. Since notum is upstream of APC, the double knockdown will result in the pathway being constitutively active regardless of APC expression. 

Methods and Results. PCR reactions can be loaded onto an agarose gel with a standard to few the size of the amplicon. After isolating the amplicon from the gel, the next step being used is cloning. Cloning involves annealing the amplicon, or insert, into a vector. Once the vector and amplicon are annealed, the plasmid can now be grown on bacteria. Once the plasmid copies are isolated from the bacteria, the plasmids can be opened to form double-stranded RNA (dsRNA). Once dsRNA is formed using PCR and RNA nucleotides, the dsRNA can be mixed with food or injected directly into the organism. RNAi takes advantage of endogenous virus-protection mechanisms. When dsRNA binds to the RISC complex, a single RNA fragment is left behind. This serves as a template. Complementary mRNA binds to the template RNA and is cleaved. This prevents the mRNA from being translated into protein. This is an RNAi knockdown. 

Discussion/Conclusions. All of the necessary S. med amplicons have been cloned and will be ready for RNAi shortly. I am in the process of making dsRNA for all amplicons. S. med APC dsRNA has been made. I am working on unc-22 (control), notum, and wnt-1. S. med RNAi will start during August 2024. G. gua APC and wnt-1 have been amplified via PCR and are ready for cloning. G. gua notum has not been amplified successfully. Once I finish the S.med RNAi experiments, I will shift my focus to the G. gua RNAi experiments. Once I finish, I will compare the results of the two species. 

Citation/Acknowledgements. P20GM103436