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Browsing by Author "Nass, Richard M."
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Item Developing In Vitro and In Vivo Models for Lyme Neuroborreliosis (LNB)(2023-08) Alanazi, Fuad Fahad; Yang, X. Frank; Bauer, Margaret E.; Yu, Andy Q.; Relich, Ryan F.; Nass, Richard M.Lyme neuroborreliosis (LNB) is a neurologic disorder caused by infection with Borrelia burgdorferi, resulting in inflammation in the central and peripheral nervous systems. LNB remains poorly understood due to the lack of a suitable experimental model. The non-human primate model for LNB presents significant impediments, such as high costs, specialized training, ethical considerations, and low infection frequency. Finding alternative models is imperative to advance LNB research. This study aims to develop alternative in vitro and in vivo models for LNB. First, we developed an in vitro transwell assay to identify the factors required for the blood-brain barrier (BBB) transmigration of B. burgdorferi. Second, we established a middle-aged mouse model for studying neuroinflammation associated with LNB. Last, we further characterized a Caenorhabditis elegans (C. elegans) model to study B. burgdorferi-associated neuron damage. With these models, we discovered that the Rrp2-RpoN-RpoS pathway in B. burgdorferi is essential for B. burgdorferi to cross the BBB and that the outer surface protein C (OspC) controlled by this pathway plays a vital role in crossing the BBB. We found that B. burgdorferi is detectable in the brains of middle-aged mice but not in younger mice and triggers host immune response, resulting in elevated levels of cytokines such as TNF-alpha, IFN-γ, and IL-9 and reduction in microglia in the infected mice. Lastly, we demonstrated that C. elegans can feed on B. burgdorferi, which may result in neurodegeneration. This provides a powerful tool for screening pathogen and host factors involved in neuroborreliosis. Overall, the in vitro and in vivo models developed in this study will significantly advance LNB research, which may lead to the development of new treatments and improved patient outcomes.Item Identification and characterization of molecular modulators of methylmercury-induced toxicity and dopamine neuron degeneration in Caenorhabditis elegans(2014) VanDuyn, Natalia M.; Nass, Richard M.; Atchison, William D.; Brustovetsky, Nickolay; Cummins, Theodore R.; Sullivan, William J., Jr.; Wek, Ronald C.Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue.Item Identification of TgElp3 as an essential, tail-anchored mitochondrial lysine acetyltransferase in the protozoan pathogen toxoplasma gondii(2014-07-11) Stilger, Krista L.; Nass, Richard M.; Bauer, Margaret E.; Oxford, G. S.; Queener, Sherry F.; Sullivan Jr., William J.Toxoplasma gondii, a single-celled eukaryotic pathogen, has infected one-third of the world’s population and is the causative agent of toxoplasmosis. The disease primarily affects immunocompromised individuals such as AIDS, cancer, and transplant patients. The parasites can infect any nucleated cell in warm-blooded vertebrates, but because they preferentially target CNS, heart, and ocular tissue, manifestations of infection often include encephalitis, myocarditis, and a host of neurological and ocular disorders. Toxoplasma can also be transmitted congenitally by a mother who becomes infected for the first time during pregnancy, which may result in spontaneous abortion or birth defects in the child. Unfortunately, the therapy currently available for treating toxoplasmosis exhibits serious side effects and can cause severe allergic reactions. Therefore, there is a desperate need to identify novel drug targets for developing more effective, less toxic treatments. The regulation of proteins via lysine acetylation, a reversible post-translational modification, has previously been validated as a promising avenue for drug development. Lysine acetyltransferases (KATs) are responsible for the acetylation of hundreds of proteins throughout prokaryotic and eukaryotic cells. In Toxoplasma, we identified a KAT that exhibits homology to Elongator protein 3 (TgElp3), the catalytic component of a transcriptional elongation complex. TgElp3 contains the highly conserved radical S-adenosylmethionine and KAT domains but also possesses a unique C-terminal transmembrane domain (TMD). Interestingly, we found that the TMD anchors TgElp3 in the outer mitochondrial membrane (OMM) such that the catalytic domains are oriented towards the cytosol. Our results uncovered the first tail-anchored mitochondrial KAT reported for any species to date. We also discovered a shortened form of Elp3 present in mouse mitochondria, suggesting that Elp3 functions beyond transcriptional elongation across eukaryotes. Furthermore, we established that TgElp3 is essential for parasite viability and that its OMM localization is important for its function, highlighting its value as a potential target for future drug development.Item Investigations into the function of Elp3 in Toxoplasma gondii(2017-05-04) Padgett, Leah Rausch; Arrizabalaga, Gustavo; Jerde, Travis; Mosley, Amber; Nass, Richard M.; Sullivan, William J., Jr.The parasite Toxoplasma gondii causes life-threatening infection in immunocompromised individuals. Our lab has determined that Toxoplasma Elongator protein-3 (TgElp3) is required for parasite viability. While catalytic domains are conserved, TgElp3 is the only component of the six-subunit Elongator complex present in Toxoplasma; moreover, TgElp3 localizes to the outer mitochondria membrane (OMM). These unusual features suggest that TgElp3 may have unique roles in parasite biology that could be useful in drug targeting. The goals of this thesis were to determine the function of TgElp3 and how the protein traffics to the OMM. In other species, Elp3 mediates lysine acetylation of histones and alphatubulin, and its radical S-adenosyl methionine (rSAM) domain is important for the formation of tRNA modifications, which enhance translation efficiency and fidelity. Given its location, histones would not be an expected substrate, and we further determined that tubulin acetylation in Toxoplasma is mediated by a different enzyme, TgATAT. We found that overexpression of TgElp3 at the parasite’s mitochondrion results in a significant replication defect, but overexpression of TgElp3 lacking the transmembrane domain (TMD) or with a mutant rSAM domain is tolerated. We identified one such modification, 5-methoxycarbonylmethyl-2thiouridine (mcm5S2U) that is likely mediated by TgElp3. These findings signify the importance of TgElp3’s rSAM domain for protein function, and confirms TgElp3 activity at the OMM is essential for Toxoplasma viability as previously reported. To determine how TgElp3 traffics to the OMM, we performed a bioinformatics survey that discovered over 50 additional “tail-anchored” proteins present in Toxoplasma. Mutational analyses found that targeting of these TA proteins to specific parasite organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.Item Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylation(2014-04-02) Wang, Jiachen; Sullivan, William J. Jr.; Queener, Sherry F.; Arrizabalaga, Gustavo; Nass, Richard M.; Lu, TaoHistone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.Item Novel regulation of neuronal genes implicated in Alzheimer disease by microRNA(2013-12-11) Long, Justin M.; Zhou, Feng C.; Lahiri, Debomoy K.; Farlow, Martin R.; Nass, Richard M.; Du, YanshengAlzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.Item Resveratrol augments paclitaxel treatment in MDA-MB-231 and paclitaxel-resistant MDA-MB-231 breast cancer cells(2014) Sprouse, Alyssa A.; Herbert, Brittney-Shea; Flockhart, David A.; Nass, Richard M.; Pollok, Karen E.; Safa, Ahmad R.Resveratrol has been shown to inhibit cell growth and induce apoptosis, as well as augment chemotherapeutics and irradiation in multiple cancer types. However, it is unknown if resveratrol is beneficial for treating drug-resistant cancer cells. To study the effects of resveratrol in triple negative breast cancer cells that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the MDA-MB-231 breast cancer cell line. The resulting cell line, MDA-MB-231/PacR, exhibited a 12-fold increased resistance to paclitaxel but remained sensitive to resveratrol treatment. Resveratrol treatment reduced cell proliferation and colony formation and increased senescence and apoptosis in both the parental MDA-MB-231 and MDA-MB-231/PacR cell lines. Importantly, resveratrol treatment augments the effects of paclitaxel in both cell lines. The expression of the drug efflux transporter gene, MDR1, and the main metabolizing enzyme of paclitaxel gene, CYP2C8, was increased in the resistant cells. Moreover, pharmacological inhibition of the protein products of these genes, P-glycoprotein and CYP2C8, decreased paclitaxel resistance in the resistant but not in the parental cells, which suggests that the increase of these proteins are important contributors to the resistance of these cells. In conclusion, these studies imply that resveratrol, both alone and in combination with paclitaxel, may be useful in the treatment of paclitaxel-sensitive and paclitaxel-resistant triple negative breast cancers.Item The role of SMF 1, SMF-2, SMF-3 in metal-induced whole animal vulnerability and dopamine neuron degeneration in Caenorhabditis elegans(2012-12-04) LeVora, Jennifer K.; Nass, Richard M.; Nicol, Grant D.; Hingtgen, Cynthia M., 1966-The etiology of many neurodegenerative diseases is unknown, but a number of studies indicate that a combination of both genetic and environmental factors contribute to the progression of disease. Exposure to environmental metals, such as Mn2+, Fe2+, Cu2+, and Al3+, has been shown to increase cell death that is characteristic of neurodegenerative disorders such as AD, PD, Wilson’s disease and Menkes disease. These metals are important in numerous biological processes in the brain and their homeostasis is regulated through multiple mechanisms of transport, storage, and secretion. The vertebrate divalent metal transporter-1 (DMT-1) has been implicated in transport and homeostasis of these divalent cations. In these studies I utilize Caenorhabditis elegans (C. elegans) to show that long term exposure to Mn2+ decreases animal viability in a dose-dependent manner, and I demonstrate that C. elegans homologues to DMT-1, SMF-1, SMF-2, and SMF-3, play specific roles in divalent metal ion-induced DA neurodegeneration. I show that SMF-1 contributes to Fe2+-induced DA neuron degeneration, SMF-3 contributes to Al3+-induced DA neuron degeneration, and both SMF-2 and DAT-1 contribute to Cu2+-induced DA neuron cell death. These studies utilize C. elegans as a powerful model to characterize molecules and pathways involved in metal toxicity and metal-induced DA neuron degeneration.Item THE ROLE OF THE NMDA RECEPTOR AND REVERSE SODIUM CALCIUM EXCHANGER IN CALCIUM DYSREGULATION IN GLUTAMATE-EXPOSED NEURONS(2012-10-29) Brittain, Matthew K.; Brustovetsky, Nickolay; Cummins, Theodore R.; Nass, Richard M.; Nicol, Grant D.; Vasko, Michael R.Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.Item Targeting Soluble Epoxide Hydrolase to Treat Choroidal Neovascularization(2022-05) Park, Bomina; Corson, Timothy W.; Bhatwadekar, Ashay D.; Jerde, Travis J.; Lu, Tao; Nass, Richard M.Neovascular or “wet” age-related macular degeneration (nvAMD) is a leading cause of blindness among older adults, affecting millions of people worldwide. Choroidal neovascularization (CNV) is a major pathological feature of nvAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible loss of vision. Currently, the effort to treat nvAMD is hampered by resistance and refractory responses to the current standard of anti-angiogenic care, anti-vascular endothelial growth factor biologics. Thus, there is a critical need to develop novel therapeutic strategies. Previously, we discovered an anti-angiogenic small molecule SH-11037, and identified soluble epoxide hydrolase (sEH) as a target of SH-11037 through a forward chemical genetics approach. sEH, encoded by the EPHX2 gene, is a lipid-metabolizing enzyme that hydrolyzes epoxy fatty acids into corresponding diols. I hypothesized that sEH is a key mediator of CNV. Given that the kinetic mechanism of sEH inhibition by SH-11037 and the cellular role of sEH in CNV are poorly understood, the objectives of my thesis project were to elucidate drug-target interactions through enzyme kinetics, investigate sEH mediated mechanisms that regulate CNV, and preclinically validate sEH as a therapeutic target. I discovered that SH-11037 is a mixed inhibitor of sEH with a binding affinity for both the enzyme and enzyme-substrate complex. I examined retinal spatial expression of sEH at both the protein and mRNA levels through immunohistochemistry and RNAscope in situ hybridization and investigated the efficacy of adeno-associated virus (AAV) serotype 8 vector expressing shRNA against Ephx2, in the mouse laser-induced (L-) CNV model with features of nvAMD. My study revealed sEH protein and mRNA overexpression in the retinal pigment epithelium (RPE), vasculature and photoreceptors under the disease state. The delivery of AAV8-Ephx2 shRNA, which has tropism towards RPE and photoreceptor cells, significantly reduced CNV. In addition, gene expression analysis showed normalized Vegfc and CNV-related inflammatory markers upon sEH knockdown. Thus, my study demonstrated sEH overexpression in disease-relevant cell types, highlighted a functional role of sEH in AMD pathophysiology, and provided a novel context to target these cell types for developing pharmacotherapies.