Anatomy and Cell Biology
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Item Open Access Epigenetic Control of Ataxin-1 in Multiple Sclerosis(2022) Didonna, Alessandro; Ma, Qin; Oksenberg, Jorge R.Item Open Access The Germ Cell-Specific RNA Binding Protein RBM46 is Essential for Spermatogonial Differentiation in Mice(2022-09-21) Johnson, Taylor A.; Geyer, Christopher B.Item Open Access Modeling Mammalian Spermatogonial Differentiation and Meiotic Initiation in Vitro(2022) Kirsanov, Oleksandr; Johnson, Taylor; Malachowski, Taylor; Niedenberger, Bryan A.; Gilber, Emma A.; Geyer, Christopher B.; Fisher-Wellman, Kelsey; et alItem Open Access COVID-19 Infection Enhances Susceptibility to OxidativeStress–Induced Parkinsonism(2022-06-09) Eells, Jeffrey B.; Akula, Shaw M.; Sriramula, Srinivas; O’Rourke, Dorcas P.; Smeyne, Richard J.; et alItem Open Access Investigating Non-Tight Junction Functions of Claudin-7: Regulating Large Intestine Stem Cell Functions and Niche Maintenance, and Junctional Protein Nanoarchitecture(East Carolina University, 2022-12-12) Naser, Amna; Chen, Yan-Hua; Lu, Qun; Askew, Emily; Schwalbe, Ruth; Yang, Li; Anatomy and Cell BiologyEdit this abstract.Item Embargo Discovering mechanisms during spermatogonial differentiation that prepare male germ cells for meiosis(East Carolina University, 2022-12-05) Johnson, Taylor A; Geyer, Christopher B; Eells, Jeffrey B; Ellis, Jessica M; Schindler, Karen; Sperry, Ann O; Anatomy and Cell BiologySpermatogenesis, the male germ cell maturation process, allows men to become and remain fertile for decades. Male fertility relies on fate decisions of different types of spermatogonia – spermatogonial stem cells maintain the germline long-term, undifferentiated progenitor spermatogonia divide and await the differentiation signal (retinoic acid, RA), and differentiating spermatogonia proceed forward into meiosis as spermatocytes to ultimately become sperm. Spermatogonial differentiation and meiotic initiation are indispensable transitions in spermatogenesis that remain poorly understood. The three studies within this dissertation, using a combination of in vivo and in vitro approaches, detail spermatogonial requirements in these two transitions. The first study (chapter two) scrutinizes and redefines a presumed dogma – the requirement of RA for the spermatogonia-to-spermatocyte (mitotic-to-meiotic) transition. The second study (chapter three) deciphers the differential responsiveness of spermatogonia to RA in the adult testis. The third study (chapter four) introduces a novel RNA binding protein required for the commitment to and completion of spermatogonial differentiation. The collective findings from these three studies both enrich our understanding of spermatogonial biology and propose avenues for treatments that can enhance or discontinue spermatogonial differentiation.Item Restricted INVESTIGATING MECHANISMS REGULATING MAMMALIAN SPERMATOGONIAL DIFFERENTIATION AND MEIOTIC INITIATION(East Carolina University, 2022-06-27) Kirsanov, Oleksandr; Geyer, Christopher B; Litwa, Karen A; Eells, Jaffrey E; Neufer, Darrell P; Hu, Guang; Anatomy and Cell BiologySpermatogonia differentiation and meiotic initiation are complex processes essential for male fertility. Despite knowing for nearly 60 years that retinoic acid (RA) is instrumental for successful production of functional sperm, molecular mediators that regulate spermatogonial differentiation and prepare male germ cells for meiosis are poorly defined. The work presented here is organized into chapters and demonstrates my efforts to develop new tools to study germ cell development and to employ these tools to investigate the role and the developmental timeframes of RA activity during differentiation and meiotic initiation. In chapter 1, I examine published studies on development of germ cells starting at establishment of male germline in fetus to production of functional sperm in adult and highlight significant gaps in knowledge that are addressed with my work. In chapter 2, I describe negative effects of Everolimus, a pharmaceutical compound commonly used to treat cancer, on male fertility in mice. In chapter 3, I identify a new fluorescent reporter mouse model used to isolate large, highly enriched, precise population of germ cell at specific stages of their development and present a new culture system to study male germ cells in vitro. In chapter 4, I test the requirement for RA signaling during spermatogenesis.Item Open Access Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex(2021-09-28) Tate, Kinsley; Kirk, Brenna; Tseng, Alisia; Ulffers, Abigail; Litwa, KarenItem Open Access Metabolic Reprogramming in Chondrocytes to Promote Mitochondrial Respiration Reduces Downstream Features of Osteoarthritis(2021) Knudson, Cheryl B.; Knudson, Warren; Ohashi, Yoshifumi; Takahashi, Nobunori; Terabe, Kenya; Tsuchiya, Saho; Kojima, Toshihisa; Imagama, ShiroItem Restricted INVESTIGATING MECHANISMS REGULATING MAMMALIAN SPERMATOGONIAL DIFFERENTIATION AND MEIOTIC INITIATION(East Carolina University, 2022-06-27) Kirsanov, Oleksandr; Geyer, Christopher B; Anatomy and Cell BiologySpermatogonia differentiation and meiotic initiation are complex processes essential for male fertility. Despite knowing for nearly 60 years that retinoic acid (RA) is instrumental for successful production of functional sperm, molecular mediators that regulate spermatogonial differentiation and prepare male germ cells for meiosis are poorly defined. The work presented here is organized into chapters and demonstrates my efforts to develop new tools to study germ cell development and to employ these tools to investigate the role and the developmental timeframes of RA activity during differentiation and meiotic initiation. In chapter 1, I examine published studies on development of germ cells starting at establishment of male germline in fetus to production of functional sperm in adult and highlight significant gaps in knowledge that are addressed with my work. In chapter 2, I describe negative effects of Everolimus, a pharmaceutical compound commonly used to treat cancer, on male fertility in mice. In chapter 3, I identify a new fluorescent reporter mouse model used to isolate large, highly enriched, precise population of germ cell at specific stages of their development and present a new culture system to study male germ cells in vitro. In chapter 4, I test the requirement for RA signaling during spermatogenesis.Item Open Access Mammalian SWI/SNF Chromatin Remodeler is Essential for Reductional Meiosis in Males(2021-11-12) Kirsanov, Oleksandr; Geyer, Christopher B.; Magnuson, Terry; Menon, Debashish U.Item Open Access Rare Genetic Disorders: Novel Treatment Strategies and Insights Into Human Biology(2021-08-06) Koch, Peter J.; Koster, Maranke I.Item Open Access Synaptic Hyaluronan Synthesis and CD44-Mediated Signaling Coordinate Neural Circuit Development(2021-09-28) Wilson, Emily S.; Litwa, KarenItem Open Access Quantitative Immunohistochemistry to Measure Regional Expression of Nurr1 in the Brain and the Effect of the Nurr1 Heterozygous Genotype(2021) Kummari, Evangel; Guo-Ross, Shirley X.; Partington, Heath S.; Nutter, Jennifer Makenzie; Eells, Jeffrey B.Item Open Access Synaptic Hyaluronan Synthesis and CD44-Mediated Signaling Coordinate Neural Circuit Development(MDPI, 2021-09-28) Wilson, Emily S.; Litwa, KarenThe hyaluronan-based extracellular matrix is expressed throughout nervous system development and is well-known for the formation of perineuronal nets around inhibitory interneurons. Since perineuronal nets form postnatally, the role of hyaluronan in the initial formation of neural circuits remains unclear. Neural circuits emerge from the coordinated electrochemical signaling of excitatory and inhibitory synapses. Hyaluronan localizes to the synaptic cleft of developing excitatory synapses in both human cortical spheroids and the neonatal mouse brain and is diminished in the adult mouse brain. Given this developmental-specific synaptic localization, we sought to determine the mechanisms that regulate hyaluronan synthesis and signaling during synapse formation. We demonstrate that hyaluronan synthase-2, HAS2, is sufficient to increase hyaluronan levels in developing neural circuits of human cortical spheroids. This increased hyaluronan production reduces excitatory synaptogenesis, promotes inhibitory synaptogenesis, and suppresses action potential formation. The hyaluronan receptor, CD44, promotes hyaluronan retention and suppresses excitatory synaptogenesis through regulation of RhoGTPase signaling. Our results reveal mechanisms of hyaluronan synthesis, retention, and signaling in developing neural circuits, shedding light on how disease-associated hyaluronan alterations can contribute to synaptic defects.Item Restricted THE HYALURONAN EXTRACELLULAR MATRIX CRITICALLY REGULATES SYNAPSE FORMATION IN DEVELOPING NEURAL NETWORKS(East Carolina University, 2021-04-21) Wilson, Emily S; Litwa, Karen; Anatomy and Cell BiologyThe majority of neurodevelopmental disorders present with an imbalance in synaptic signal transmission. The delicate balance of excitatory to inhibitory synaptic transmission is regulated by multiple extracellular and intracellular factors. Specifically, our work demonstrates that the major extracellular component of the brain, hyaluronan, critically regulates synaptic formation and the emerging balance between inhibitory and excitatory neurotransmission. Furthermore, our results suggest that these effects are in part mediated by hyaluronan's interaction with its receptor, CD44, leading to actin cytoskeleton rearrangements that alter synapse formation. For the first time, we demonstrate that human brain models secrete an endogenous hyaluronan matrix through expression of hyaluronan synthase. Hyaluronan is present at the synaptic cleft of nascent developing synapses. Through both genetic and pharmacological regulation of hyaluronan levels, we demonstrate that hyaluronan antagonizes excitatory synapse formation, preventing the emergence the hyperexcitability in developing neural networks. Furthermore, excitatory synapses contain the HA-receptor, CD44. In other tissue systems, the interaction between HA and CD44 activates RhoA signaling leading to actomyosin contractility. In our research, we demonstrate that similar to HA, RhoA signaling through its effector kinase, ROCK, also antagonizes excitatory synapse formation in developing neural networks. Together, our data supports a model in which synaptic HA suppresses excitatory synaptogenesis through interaction with CD44 and activation of RhoA/ROCK, resulting in the destabilization of synaptic contacts. We propose that HA-mediated regulation of synapse formation, critically regulates neural network development, and prevents the emergence of hyperexcitability in neural networks, which is characteristic of neurodevelopmental disorders.Item Open Access PPP1R2 COORDINATES KINASE AND PHOSPHATASE ACTIVITY TO REGULATE THE CENTROSOME AND MIDBODY(East Carolina University, 2021-04-21) Bresch, Alan-Michael; Sperry, Ann O; Anatomy and Cell BiologyCentrosomes are the primary microtubule organizing centers of the cell and prepare the cell for division by establishing the bipolar spindle during mitosis. A balance of kinase and phosphatase activity regulates centrosome number and mitotic spindle function. PPP1R2 is a negative regulator of Protein Phosphatase 1, PP1, and an activator of Aurora A Kinase, AURKA. Both PP1 and AURKA play critical roles in centrosome regulation, however PPP1R2's role at the centrosome has not been examined. Given that PPP1R2 interacts with PP1 and AURKA, critical regulators of the centrosome, I hypothesized that PPP1R2 is a key regulator of the centrosome cycle through its interaction with AURKA and PP1. I tested this hypothesis through an overexpression model using PPP1R2, PPP1R2 truncation mutant, PPP1R2 phosphomutant, AURKA, and PP1 plasmid constructs. PPP1R2, AURKA, and PP1 overexpression resulted in both supernumerary centrosomes and [gamma]-tubulin mislocalization. In addition, PPP1R2 truncation mutant overexpression resulted in similar effects at the centrosome. Only PPP1R2 phosphomimetic mutant overexpression resulted in increased centrosome number. PPP1R2 and PPP1R2 mutant overexpression also resulted in disruption of midbody architecture as well as polyploidy. PPP1R2 truncation mutant overexpression significantly decreased PP1 midbody localization while PPP1R2 as well as PPP1R2 phosphomimetic, PPP1R2E, mutant overexpression increased PP1 midbody localization.PPP1R2's regulation of PP1 midbody recruitment further supported PPP1R2's role in midbody regulation. Overall, I found that PPP1R2 does coordinate PP1 and AURKA activities to regulate the centrosome protein recruitment as well as centrosome number through midbody architecture maintenance. In addition, PPP1R2 was shown to regulate microtubule nucleation from the centrosome and this regulation was dependent on PPP1R2's phosphorylation state. Overall, this dissertation demonstrates that PPP1R2 is a critical regulator of centrosome as well as midbody structure and function through AURKA and PP1 activity coordination.Item Restricted FUNCTIONS OF CLAUDIN-7 IN THE SELF-RENEWAL OF INTESTINAL EPITHELIUM(East Carolina University, 8/5/2020) Xing, Tiaosi; Chen, Yan-HuaTight junctions (TJ) are located at the most apical region of the junctional complex and contain many integral membrane proteins. Claudin 7 (Cldn7) is a TJ membrane protein located at the apical TJ and basolateral side of intestinal epithelial cells. Deletion of Cldn7 by gene-targeting leads to the inflammatory bowel disease (IBD)-like phenotype in mice, which includes weight loss, diarrhea, mucosa ulceration, and severe intestinal epithelial damage. We hypothesize that Cldn7 plays a critical role in regulating intestinal crypt stem cell functions. In this study, we report for the first time that Cldn7, a TJ membrane protein, controls intestinal stem cell survival, self-renewal, and differentiation, an unexpected and novel role for a TJ proteins. Gene-deletion of Cldn7 in intestines displayed the significant alteration of expression profiles with striking downregulation of intestinal crypt stem cell markers, such as olfm4, dislocated proliferative cells, and disrupted epithelial cell differentiation. In addition, Cldn7- deficient crypts from small and large intestines were unable to survive and form organoids, highlighting the functional impairment of crypt stem cells in the absence of Cldn7. Remarkably, the Cldn7 expressing organoids with buddings underwent the rapid cell degeneration within days after turning off Cldn7 expression in the culture. We identified that activation of Wnt/[beta]-catenin signaling rescued the organoid defects caused by Cldn7 deletion in small intestine. Using a genome-wide gene expression approach and subsequent verifications by cell and molecular analyses, we have also identified notch signaling, Wnt signaling and hippo signaling as the key pathways involved in Cldn7 deletion-mediated defects in the large intestine. Disrupted cell differentiation of intestinal epithelium could be due to the suppression of notch signaling. Olfm4 was significantly decreased in Cldn7 KO small intestines, whereas it was increased in the large intestines, suggesting the different mechanism that Cldn7 regulates the self-renewal on mouse large and small intestines. Overall, our current study highlights a novel function of Cldn7 in regulating intestinal epithelial stem cell functions and epithelial self-renewal. This study could open a door to study roles of TJ proteins in stem cell regulations in other tissues and organs.Item Open Access The therapeutic effect of Rho kinase inhibitor Y-27632 on protection from chemotherapy-induced peripheral neuropathy(2018-04-17) Elliott, ZacharyCisplatin often causes loss of touch sensitivity in the hands and feet of cancer patients as well as tingling, numbness, and a shooting or burning pain; these clinical symptoms are referred to as chemotherapy-induced peripheral neuropathy (CIPN). CIPN frequently results in a reduction or cessation of chemotherapy, and there is currently no effective intervention or prevention for CIPN. Therefore, it is important to understand the mechanism of CIPN pathogenesis and determine associated signaling pathways to identify potential therapeutic targets. Previous in vitro studies generated by other labs indicated that aberrant activity of RhoA signaling pathway was involved in the onset of CIPN. In order to fully capture the clinical situation, in this study, we created a CIPN mouse model in 4 month old C57/BL6 mice by intraperitoneal injections of 6 mg/g cisplatin and 30 mg/g Y-27632 weekly for 4 weeks for each respective group. To investigate the potential therapeutic effect of RhoA signaling pathway inhibition in CIPN, Y-27632, which selectively inhibit Rho kinase/p160ROCK (a downstream effector of the RhoA signaling pathway), was applied to selective group of mice. The peripheral nerve function of mouse hind paw was evaluated by Von Frey monofilaments and acetone evaporation assay. Our data indicated that Y-27632 treatment could potentially protect mice from cisplatin-induced peripheral nerve damage. Additionally, immunohistochemical analysis of intraepidermal nerve fibers (IENFs) in the footpad tissue demonstrated that concurrent treatment of cisplatin with Y-27632 could alleviate the loss of touch sensory-associated IENFs caused by cisplatin. Therefore, Y-27632 can protect peripheral nerve function in a CIPN mouse model.Item Open Access Do Pesticides cause Obesity and Diabetes?(2019-08-05) Elliott, ZacharyWe have an epidemic of obesity that presents with serious health and economic consequences. Obesity is the most common disease in the U.S. that has developed rapidly in only a matter of decades. Glyphosate (RoundupTM) is a popular pesticide with private and commercial use that has dramatically increased in recent decades. The geographic distribution and rise of obesity and glyphosate over time are similar (Fig 1). Recent evidence suggests that chronic glyphosate/AMPA (Fig. 2) exposure causes hepatorenal toxicity and oxidative stress. Our previous LABS study has 4700+ blood samples from across the country (Fig.1) with multiple time points before and after bariatric surgery (6, 12, 24, 36, 48, 60, 72 months). These samples give us the power to determine if there is a regional relationship between obesity and pesticide use once a method of validation is established.