Now showing items 1-20 of 61

    • SELF-ASSOCIATION IS A COMMON PROPERTY IN BOVINE, MOUSE AND XENOPUSARRESTIN

      Calvert, Peter; Qiu, Shuang (2014)
      The light-driven translocation of arrestin from rod inner segment to outer segment was indicated to involve free diffusion with binding affinityto light-activated phosphorhodopsin, however, it is still debatable how arrestin is excluded from the dark-adapted outer segment. Previousstudies demonstrated that bovine visual arrestin had the property of self-association. The self-association propertyof both wild-type and mutated purified visual arrestin of several species (bovine, mouse and Xenopus laevis) was studiedby performing analytical ultracentrifugation experimentswhich providedthe oligomer formation information and association constants.Theself-association parameters of purified bovine and mousevisual arrestinwere investigated and compared with other studies. Results showed that arrestin of both species could self-associate, forming dimersin a concentration-dependent manner, but tetramerswere not detected at the highest concentrations examined.Xenopus arrestin was shown to self-associateas well, existing in a monomer-dimer equilibrium with the dimer dissociation constantKD,dim=80.8μM, which suggestedthat self-association was also a feature of Xenopus visual arrestin. Interestingly, homologous mutations (F78A/Y84A/F193A)of Xenopusarrestin, which were supposed to beconstitutive monomers, failed to completely disrupt the oligomerizationof Xenopusarrestinwith the dimer dissociation constantKD,dim=200.7μM , indicating that these regions were not conserved in amphibian visual arrestin, includingXenopuslaevis. The percentage of the dimer was higher than that of monomer at physiological concentrations in all species of arrestins tested.
    • POSTNATAL ALCOHOL EXPOSURE INFLUENCES ADOLESCENT OLFACTORY RESPONSES TO THE DRUG

      Youngentob, Steven; Harrison, Danielle (2016)
      Human studies illustrate that alcohol exposure while breastfeeding produces a memory of the alcohol scent and modifies behavioral responses to the odor of the drug. The memory and modified behavioral response to alcohol odor suggest that the addictive attributes of alcohol may contribute to patterns of use that increases the risk for alcohol abuse later in life. There is a growing body of evidence that demonstrates prenatal alcohol exposure produces a memory and modified behavioral response to alcohol odor that persists into adolescence, and contributes to alcohol abuse. Given that both postnatal and prenatal alcohol exposure has lasting effects on infants, this study investigated whether rats exposed to alcohol while breastfeeding have a prolonged memory and modified behavioral response to alcohol odor. Long-Evans Hooded rat pups were exposed to alcohol during breastfeeding via the dams' liquid diet. Control animals received ad lib access to an isocaloric, iso-nutritive liquid diet after delivery of their litter up to weaning. To control for effects of malnutrition pair-fed animals were given a control liquid diet equivalent in quantity to the amount their matched animal provided with an alcohol diet consumed the day before. When litters reached adolescence, the behavioral and neurophysiological responses to alcohol odor in a male and female animal from each litter was examined. Relative to controls, animals exposed to alcohol postnatally displayed an altered breathing pattern response to alcohol odor specifically, and an altered breathing pattern and neurophysiological response to novel odorants. The findings of this study builds on the growing body of research that shows the consequences of postnatal alcohol exposure.
    • MECHANISM OF ANT1-INDUCED HUMAN DISEASES AND STRESS SIGNALING

      Liu, Yaxin (2016)
      Adenine nucleotidetranslocase(Ant) is a mitochondrial inner membraneprotein, the primaryfunction of which is to mediate the ADP/ATP exchange acrossthe inner membrane. Missense mutations in Ant1, the skeletal muscle-and heart-specificisoform, induce human disordersincluding autosomal dominant ProgressiveExternalOpthalmoplegia(adPEO), cardiomyopathyand myopathy. Several models were proposed to interpret the pathogenesis of mutant Ant1-induced diseases, but no consensus has been reached.Our lab has previouslyfound that mutant Aac2, the homologof Ant1 in yeast, causes cell death due to the mitochondrialbiogenesis defect. In the present study, we provided biochemical evidencesupporting the idea that themutant Aac2 proteins are misfolded, which derailsthe proteostasis on the inner membrane.We found that the assemblyand stability of multipleprotein complexeson the inner membrane are affected, including those involved in mitochondrialrespirationand protein transport. In human cells, the mutant Ant1 proteinshave reduced steady-state levels and increased degradation, consistent with misfolding and increased susceptibility to protein quality control machineries. In the second part of thework, we sought to identify the cellularsignalingpathways that respond to Ant1-induced proteostatic stress on the mitochondrialinner membrane. We generatedANT1alleles that have enhanced toxicity and are able to induce proteostatic stress on the inner membrane in human. Although these mutant Ant1 proteins only mildly affectmitochondrialrespiration, they trigger robust transcriptional responses in the nucleus to remodel cellular signaling. The upregulationof the cytosolic chaperone/ubiquitin-proteasome systems, and the downregulation of mTOR signaling may serveas adaptiveresponses to mitigate mitochondrial PrecursorOver-AccumulationStress (mPOS), a novel pathway of cell death recently discovered in yeast. We also found that humancells respond to mitochondrialinner membrane stress by increasingEgr1 signaling and the alternative splicing of many genes important for cell survival/deathcontrol. Insummary, the data suggested that protein misfoldingcauses Ant1-induced pathogenesisin human diseases. Our study provided support for mitochondria-induced proteostatic stress in the cytosol (or mPOS) in human cells. We also identified several novel mitochondria-to-nucleus signaling pathways, which may help in developingtherapeutic interventionsfor the treatmentof mitochondria-induced pathologies.
    • ALTERATIONS IN CELLULAR GLUTAMATE TRANSPORT DO NOT CONTRIBUTE TONEURONAL CELL DEATHIN A MIXED CORTICAL CELL CULTURE MODEL OF HYPOGLYCEMIA

      Hewett, Sandra; Thorn, Trista (2013)
      Severe hypoglycemia is associated with neurological deficits thatwhen left untreated can lead to frank neuronal cell death. Despite longstanding evidence in both in vitro andin vivomodels that hypoglycemic neuronalcell death is mediated by glutamateexcitotoxicity, the cellularand molecular mechanisms involved remain incompletelydefined. Toward this end, werecently reported that glutamate efflux from astrocytes via the anionic cystine/glutamate antiporter, system xc-, contributed to glucose-deprivation (GD) induced neuronal cell death in vitro. However,the precise mechanism by which system xc-activity links to glutamate-mediated injury has yet to be determined. Thus, the overall purpose of this thesis was toinvestigate whetherchanges insystem xc-expression in our astrocyte and mixed cortical cell cultures and/or alterations in glutamate handling in a mixed cortical culture modelfollowing glucose deprivationoccur(s). Toward the former, no change in the expression of mRNA (GD up to 4 h) or protein(GD up to 8 h) ofxCT, the functional light chain of system xc-, in either astrocyte or mixed cortical cell cultureswas demonstrated via quantitative RT-PCR or western blot analysis, respectively. Further, aglycemic neuronal injury, induced by 6 or 8 h of glucose deprivation, was not prevented by the addition of either actinomycin D (10 μg/mL) or cycloheximide (1 μg/mL), demonstrating no requirement for transcription or translation, respectively. Toward the latter, alterations in classical glutamate re-uptake transporter function also did not appear to be altered. Media containing added glutamate taken from control astrocytes or astrocytes deprived of glucose (6 h) was equally toxic to pure neuronal cultures, demonstrating no alterations in glutamate removal between control and glucose-deprived cells. However, neurons in mixed cortical cell cultures deprived of glucose showed increased neuronal cell death over those maintained in glucose-containing medium when exposed directly to equimolar concentrations of either glutamate or NMDA.Similarly, this increased neuronal death in glucose deprived mixed cortical cultures was shown across several different time points using constant concentrations of either glutamate or NMDA. Lastly, we show that neurons in our mixed cortical cultures are fully protected from excitotoxic cell death when system xc-and NMDA receptor inhibitors are added up to two hours following the initiation of glucose deprivation. Overall, our data reveal that neither enhanced system xc-expressionnor impaired glutamate uptake could account for the neuronal cell death induced by glucose deprivation, but that energy deprived neurons appear simply more susceptible to excitotoxic insults. Therefore, physiological levels of glutamate releasedfrom astrocyte system xc-maybe sufficient to mediate neuronal cell death under aglycemic conditions.
    • GENOME-WIDESTUDIES OF AUTISM IDENTIFY ALTERED IMMUNOLOGIC AND NEURODEVELOPMENTAL PATHWAYS AND HIGHLIGHT PUTATIVE DIAGNOSTIC BIOMARKER SIGNALS

      Glatt, Stephen; Tylee, Daniel (2017)
      Autism is a complex neurodevelopmental syndrome that can be challenging to identify in young children. Family-based and genetic studies indicate that autism has a strong genetic component, though immunologic processes may also contribute to altered neurodevelopment. Over the past two decades, genome-wide investigations have provided critical insights into the genetic causes and molecular correlates of autism at the levels of both the individual and the population. Studies of DNA have identified highly penetrant genetic factors that appear to explain a sizable minority (20-40%) of autism-affected individuals. However, patho-developmental mechanisms are less-clearly understood for the remaining majority of individuals for whom no highly penetrant factors are identifiable(i.e., idiopathic autism). The present body of work contains three studies that used genome-wide assessment methods to predict the diagnosis of autism and to characterize its molecular correlates using samples that predominantly reflect idiopathic etiology. In Chapter 1, we demonstrate that large numbers of low-penetrance genetic factors(i.e., commonly varying single nucleotide polymorphisms; SNPs) can be harnessed with machine-learning methods to predict autism risk. Throughout this document, we provide a review and critical evaluation of DNA-and RNA-based autism biomarkers. In Chapters 2 and 3, we use microarray and RNA sequencing to identify consistent patterns of autism-related gene expression in peripheral blood samples. These patterns shed light on altered immunologic signaling and also implicate signaling pathways that are known to be involved in neurodevelopment and to influence autism-related clinical and neurobiological phenotypes. Importantly, our findings indicate that the molecular correlates of idiopathic autism may converge with mechanisms understood from high-penetrance genetic causes. Furthermore, our findings support the idea that immunologic mechanisms could contribute to perturbations of neurodevelopmental pathways. We integrate our findings in the context of existing literature, highlight current gaps in knowledge, and propose future experiments to address these questions.
    • Analysis of cdGAP in Extracellular Matrix Rigidity Sensing and Cell Migration

      Turner, Chris; Wormer, Duncan (2014)
      CdGAP is a Rac1/Cdc42 specific GTPase activating protein that localizes to cell–matrix adhesions through an interaction with the adhesion scaffold α-parvin/actopaxin to regulate lamellipodia formation and cell spreading. In chapter 2 of this thesis, using a combination of siRNA-mediated silencing and over expression, I show that cdGAP negatively regulates directed and random migration by controlling adhesion maturation and dynamics through the regulation of both adhesion assembly and disassembly. Interestingly, cdGAP was also localized to adhesions formed in three-dimensional matrix environments and cdGAP depletion promoted cancer cell migration and invasion through 3D matrices. Cell migration in 3D CDMs more closely approximates the topography of in vitroconnective tissues, suggesting that cdGAP likely plays an important regulatory role in cell migration in vivo. Other aspects of the extracellular matrix also influence cell migration. Specifically, motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal adhesion-mediated rigidity sensing and durotaxis are poorly understood. In chapter three of this thesis, I utilizefibronectin-coated polydimethoxysiloxane gelsto manipulate substrate compliance, and show that cdGAP is necessary for U2OS osteosarcoma cells to coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. I also found that CdGAP was necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility.These findings highlight the importance of GAP proteins in the regulation of Rho family GTPases andprovide insight into how GAPs co-ordinate the cell migration machinery.
    • STUDIES ON PHOTORECEPTOR GENE EXPRESSION

      Knox, Barry; ZHUO, XINMING (2013)
      Rod photoreceptors are a group of specialized retinal neurons that convert light into a neuronal signal in low light condition. The phototransduction function of rodsrequires expression of a group of rod genes. The homeostasis of these genes is primarily regulated by a photoreceptor regulatory network, which contains two major retina-specific transcription factors, neural retina leucine zipper (Nrl) and cone-rod homeobox(Crx). Nrl and Crx synergistically activate the expression of several phototransduction genes, most notably rhodopsin. Base on the studies done in cell culture, the synergy is theconsequence of interaction of Nrl and Crx. However, the interaction of Nrl and Crx has not been studied in live rods. The goal of thesis is to develop methods that can be used for studying Nrl and Crx interaction in live rods.In order to study Nrl and Crx in live rods without altering cell fate and inducing cell degeneration, I developed a novel inducible system, G3U, which can regulate gene expression in live rods. In this chapter, we investigated the characters of G3U by using rhodopsin-mCherry as a reporter and monitoring the induction response in transgenic Xenopus. The results of live rod imaging suggest that the inducible system has negligible background expression before induction and significant fold increase of expression after induction. Moreover, the induction response of G3U is reproducible in rods. These findings suggest that G3U system is a good candidate for expressing Nrl and Crx temporally in rods. In the second part of my thesis, I developed a flow cytometry based FRET method, FC-FRET, to study Nrl and Crx interaction in live cells. This method allows non-invasively analysis of protein-protein interaction in large population of live cells in a very short time. Furthermore, researchers will be able to analyze the concentration effect of FRET conveniently. In this study, I investigated the orientation of Nrl-Nrl, Crx-Crx and Nrl-Crx interactions. Our studies revealed that the Nrl-Nrl homodimer has a head-to-head and tail-to-tai l conformation. Both the Crx-Crx and the Nrl-Crx dimers have a head-to-head conformation in interacting complex. I also performed structure-function studies on both Nrl and Crx and classified the role of their different domains in the interactions. In addition to the known Nrl basic leucine zipper domain (b-ZIP) and Crx homeobox domain (HD), we found that the Nrl extended homology domain (EHD) plays an important role in the Nrl-Crx interaction. Two methods presented in this thesis are my major achievement during my graduate studies. The G3U inducible can be used to generate transgenic animals carrying inducible Nrl and Crx in rods. These transgenic animals allow researchers to study the interaction of Nrl and Crx in live rods by FC-FRET assay.
    • ANALYSIS OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR-ERLIN1/2 COMPLEX-RNF170 AXIS MUTATIONS THAT RESULT IN NEURODEGENERATIVE DISEASE

      Wojcikiewicz, Richard; WRIGHT, FORREST (2017)
      Inositol 1,4,5-trisphosphate receptors (IP3Rs) are endoplasmic reticulum (ER) proteins that assemble into tetrameric IP3- and Ca2+-gated Ca2+ channels. Activation of IP3Rs begins with stimulation of cell surface receptors that elevate cytosolic IP3 levels. IP3, with its co-agonist Ca2+, binds to IP3Rs and causes a conformational change that results in the opening of the channel aperture, allowing for Ca2+ ions to flow from stores within the ER lumen to the cytosol and thereby promoting a number of Ca2+-dependent cellular events, including secretion, neurotransmitter release and cell division. Intriguingly, it appears that the same conformational change that IP3Rs undergo during activation makes them a target for degradation by the ubiquitin-proteasome pathway. This processing allows the cell to fine-tune its internal Ca2+ responses to extracellular stimuli. In the Wojcikiewicz lab, it was discovered that processing of activated IP3Rs is mediated by the Erlin1/2 complex, a large (~2MDa) complex composed of the proteins Erlin1 and Erlin2. Constitutively-associated with the Erlin1/2 complex is the E3 ubiquitin ligase RNF170. Thus, we employed TALEN and CRISPR/Cas9-mediated gene editing technologies to abrogate expression of these three proteins to define their roles in this process. Remarkably, analysis of cells lacking RNF170 showed that it is required for all ubiquitination of activated IP3Rs. Investigation into the roles of Erlin1 and Erlin2 uncovered that Erlin2 is the “dominant partner” in the Erlin1/2 complex, mediating complex interaction with activated IP3Rs and bringing RNF170 into place to allow for ubiquitination to proceed. Mutations to RNF170 (R199C) and Erlin2 (T65I) have been identified as causative for progressive neurodegenerative diseases. Investigation of the R199C mutation on IP3R processing by RNF170 uncovered that while the mutation did not affect normal RNF170 function, it destabilized the RNF170 protein, resulting in a significantly reduced cellular complement of RNF170 and inhibition of IP3R degradation. Analysis of the Erlin2 T65I mutation showed that the effect of the mutation on Erlin1/2 complex function was two-fold. First, Erlin2 T65I interaction with activated IP3Rs was completely blocked, thereby inhibiting recruitment of RNF170 and subsequent ubiquitination and processing. Second, normal binding of the Erlin1/2 complex to phosphoinositol-3-phosphate (PI(3)P) – the significance and function of which remains undefined – was drastically inhibited. Examination of complex assembly and stability by SDS-PAGE and Native PAGE showed no destabilization of individual Erlin2 proteins nor of overall Erlin1/2 complex assembly. These data demonstrate that proper and tight control of IP3R levels in the cell are critical to overall cellular homeostasis, as disruptive mutations to requisite mediators of IP3R processing - the Erlin1/2 complex and RNF170 – result in the development of progressive neurodegenerative disease.
    • Investigating the Role of Paxillin in Mammary Gland Morphogenesis and Breast Cancer Progression

      Turner, Christopher; Xu, Weiyi (2020)
      Breast cancer is one of the most invasivecancers among women. Understanding the mechanisms contributing to breast cancer progression may identify potential ways to prevent and cure the disease. Meanwhile, mammary gland morphogenesis shares similar mechanisms to allow the ducts to invade and occupy the fat pad. Thus, it is equally important to investigate the normal mammary gland development as breast tumor progression. Paxillin, as a focal adhesion scaffold protein, has previously been implicated in multiple types of cancer cell migration and invasion through its role in cell-ECM signaling. Herein, I utilized a novel paxillin conditional knockout mouse model and paxillin knockout mouse crossed with PyMT breast tumor mouse model to show that paxillin is critical for both mammary gland morphogenesis and breast tumor progression. In Chapter 2, by evaluating the developing mammary gland morphology with immunohistochemistry and the three-dimensional cultured mammary organoids and acini, a critical role of paxillin was shown in facilitating apical-basal polarity formation in the luminal epithelial cells in part, through its control of HDAC6 activity and associated microtubule acetylation. Correct polarization and columnar shape of the epithelial cells potentially contributes to lumen formation and branching of the ducts. Investigation in Chapter 3 highlights a crucial role of paxillin in breast cancer invasion and distant organ metastasis, but did not affect the primary tumor growth rate. Further analysis revealed that paxillin is required for the endocytosis and recycling of E-cadherin, which is important for the maintenance of Adherens junction equilibrium during cancer cell collective migration. This thesis characterizes the roles for paxillin in mammary gland morphogenesis and breast cancer progression and reveals the importance of paxillin-dependent apical trafficking in normal epithelial cells, and E-cadherin trafficking in collective migrating tumor cells. Together, this work highlights a trafficking-dependent mechanism for paxillin during both physiologic and pathologic processes in the mammary gland.
    • Characterization of Hic-5 in Cancer Associated Fibroblasts: A Role in Extracellular Matrix Deposition and Remodeling

      Turner, Christopher; Goreczny, Gregory (2017)
      Hic-5 (TGFβ1i1) is a focal adhesion scaffold protein that has previously been implicated in many cancer-related processes. However, the contribution of Hic-5 during tumor progression has never been evaluated, in vivo. In Chapter 2 of this thesis, I crossed our Hic-5 knockout mouse with the MMTV-PyMT breast tumor mouse model to assess the role of Hic-5 in breast tumorigenesis. Tumors from the Hic-5 -/-;PyMT mouse exhibited an increased latency and reduced tumor growth. Immunohistochemical analysis of the Hic-5 -/-;PyMT tumors revealed that the tumor cells were less proliferative. However isolated tumor cells exhibit no difference in growth rate. Surprisingly, Hic-5 expression was restricted to the tumor stroma. Further analysis showed that Hic-5 regulates Cancer Associated Fibroblast (CAF) contractility and differentiation which resulted in a reduced ability to deposit and reorganize the extracellular matrix (ECM) in two-and three-dimensions. Furthermore, Hic-5 dependent ECM remodeling supported the ability of tumor cells to metastasize and colonize the lungs.The molecular mechanisms by which CAFs mediate ECM remodeling remains incompletely understood. In Chapter 3 of this thesis, I show that Hic-5 is required to generate fibrillar adhesions, which are specialized structures that are critical for the assembly of fibronectin fibers. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1, a scaffold protein that binds to β1 integrin and actin. Furthermore, this interaction was mediated by Src-dependent phosphorylation of Hic-5 in two and three-dimensional matrix environments to prevent β1 integrin internalization and subsequent degradation in the lysosome. This work highlights the importance of the focal adhesion protein, Hic-5 during breast tumorigenesis and provides insight into the molecular machinery driving CAF-mediated ECM remodeling.
    • Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration

      Blystone, Scott; Miller, Eric (2018)
      Metastasis is one of the leading causes of death in the world, affecting thousands every year. This is especially true of breast cancer, which can often result in the formation of secondary metastatic sites in the lung, liver, and bone marrow. There are many aspects to metastasis and an innumerable amount of molecular, biochemical, and cellular interactions contribute to its pathology. The ability of primary tumor cells to disseminate from the primary tumor, degrade the basement membrane, invade through the ECM, and eventually intravasate across the endothelial cell lining of the circulatory system or lymphatics requires a plethora of proteins, all working together in concert to achieve this. Nowhere in the cell is this more apparent than the actin cytoskeleton.Locomotion of cells requires several alterations in the actin cytoskeleton component of the cellular machinery. Generally speaking, cells must be able to polarize, form protrusions, adhere to the substratum, translocate, and then retract their tail, repeating this process as they continue to navigate to their destination. While there are many underlying aspects to this activity, spatiotemporal rearrangements of the actin cytoskeleton are key to the successful cellular motility. The mechanics behind dynamic actin cytoskeletal modifications are varied and complex, demonstrating the requirement for a variety of actin-associated, regulatory proteins.A crucial family of proteins involved in this process is the formin family of proteins. Formins are a relatively “new” group of actin modifiers which possess the unique ability to modify and generate linear actin filaments. While the members of this protein family all share some of the same actin modifying processes, many of these proteins also have functions exclusive to themselves. As a result, research into this field has blossomed and several novel features of different formins have been identified. Furthermore, alternative splice isoforms of several formins are often expressed in a variety of cell types, with specific functions attributed to each.The formin FMNL1 was originally identified in cells of a myeloid lineage and for many years was mostly thought to be involved in leukocyte adhesion and migration. Indeed, our lab has characterized many of the functions of this protein in both human and murine macrophages. However, as a result of the work in this dissertation, we have generated sufficient evidence suggesting that FMNL1 not only plays a role in breast cancer migration, but also exhibits functions unique to a specific alternative splice isoform of this protein.Our work on FMNL1 has pushed the field of study into this protein family in new directions. Herein, we have demonstrated that all three alternative splice isoforms of FMNL1 are expressed in a variety of cell types and the FMNL1ɣalternative splice isoform distinguishes itself from these isoforms via its ability to bundle linear actin filaments. Additionally, our data indicates that this is accomplished independently of the trademark FH2 domain, often thought to be the essential component of all formins. More specifically, we have identified a unique amino acid sequence in the C-terminal region of this isoform that most likely regulates this function. As a result, we have not only identified a potential therapeutic target for the treatment of metastasis via inhibition of cellular locomotion, but also pushed the field of formin research into a novel direction by providing insight which may foster new hypotheses and challenge classical theories regarding the relationship between formins and actin.
    • BRAIN SPECIFIC NEURAL EXTRACELLULAR MATRIX EXPRESSION AND MODIFICATIONS IN NEUROLOGICAL DISEASE AND DISORDERS

      Matthews, Rick; Dwyer, Chrissa (2013)
      The central nervous system (CNS) is extraordinarily complex in both structure and function. The neural extracellular matrix (ECM) is one of the key classes ofmolecules that regulates thedevelopment of the CNS and maintains its structure and function in the adult.Thereby understanding the function of the neural ECMis key to understanding the CNS. The neural ECM is composed of several nervous-system specificproteins, which are hypothesized to uniquely contribute to the defining physiological functions of the CNS. However,work in this area has been hindered by the highly complex molecular properties of the neural ECM, which stem from alterations in expressionand modifications (resulting from glycosylation and proteolytic cleavage) of its constituents. Further defining mechanisms that alter the expression and modifications of neural ECM constituents are critical to fully understanding its complex array of functions. Often in neuropathologies, the neural ECM undergoes dynamic changes providing a valuable tool to further understand its function andthe opportunity to explore its contribution to disease pathology and utility as a therapeutic target. The work presented herein investigates the role of altered expression of the nervous-system specific ECM constituent, Brain Enriched Hyaluronan Binding (BEHAB)/ brevican(B/b), in glioma,and altered glycosylation of the nervous system enriched ECM constituent, RPTPζ/phosphacan, in O-mannosylrelated congenital muscular dystrophies (CMDs). Our work suggests that increased expression of B/b in the glioma tumor microenvironment (TEM) contributes to the pathological progression of these tumors, and reducing its expression is a valuable therapeutic strategy. Additionally, our work evaluates the transcriptional regulatory mechanisms leading to increases inB/b expression in glioma and highlights the potential value of these mechanisms as therapeutic targets. Our work also identifies the absence of O-mannosyl linked carbohydrates on RPTPζ/phosphacan in the brains of CMD models and suggests that altered glycosylation of RPTPζ/phosphacan may have a role in the neuropathologies underlying these disorders. Overall this work provides valuable insight intothe molecular complexities of the neural ECM stemming from changes in the expression and glycosylation of its constituents and furthers our understanding of its function in the normal CNS and in neuropathologies.
    • PHOSPHORYLATION AND UBIQUITINATION REGULATE PROTEIN PHOSPHATASE 5 ACTIVITY AND ITS PROSURVIVAL ROLE IN KIDNEY CANCER

      Mollapour, Mehdi; Dushukyan, Natela (2018)
      Protein Phosphatase 5 (PP5) is a serine/threonine phosphatase known to regulate many essential cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is a knownco-chaperone of the molecular chaperone heat shock protein 90 (Hsp90), and its regulation of Hsp90aidswiththe proper activation of Hsp90 clients and withsteroid hormone signaling.Hsp90 is also one of the strongest activators of PP5, as it releases the auto-inhibition of PP5 by interacting with the N-terminal tetratricopeptide repeat (TPR) domain of PP5. Our lab has recently shown that PP5 is phosphorylated at T362, and that this phosphorylation acts as an “on switch” resultingin the hyperactivation of PP5. Misregulation of this key phosphatase has been shown to aid in the tumor progression of ER-dependent and independent breast cancer. Elevated PP5 levels have also been linked to colorectalcancer, hepatocellular carcinoma (HCC), lymphoma, and prostate cancer. The work presented here reveals the pro-survival role that PP5 plays in kidney cancer. Clear cell renal cell carcinomas (ccRCC) are most often driven by mutations in the von Hippel-Lindau tumor suppressor (VHL). The data in this thesis shows that VHL binds and multi mono-ubiquitinates PP5 at two lysine residues K185 and K199. This post-translational modification negatively regulates PP5 likean “off switch” and ultimately leads to its degradation bythe proteasome. Mutations in the VHLgene that result in inactive mutants or a lack of VHL protein expression lead to ccRCC tumors. The data in this thesis shows that these VHL-nulltumors become dependent on elevated levels of PP5, and that both PP5 knockdown and inhibition lead to cancer cell death. The data further shows that the decrease in PP5 activity in VHL-null cells results in the induction of the extrinsic apoptotic pathway with a dramatic increase in the cleavage of PARP and caspases 3, 7, and 8.
    • POST-TRANSLATIONAL REGULATION OFCO-CHAPERONES AFFECTS HSP90 DRUG SENSITIVTY IN CANCER

      Mollapour, Mehdi; Dunn, Diana (2017)
      Heat Shock Protein-90 (Hsp90) is a molecular chaperone critical to thestability and activity of over 200 proteins known as “clients” including many oncogenes. Hsp90 chaperone function is linked to its ability to hydrolyze ATP and Hsp90 drugs inhibit its activity leading to the degradation of clients, thus making Hsp90 an attractive target for cancer therapy. The Hsp90 chaperone cycle is fine-tuned by another group of proteins called co-chaperones. They modifythe cycle, allowing Hsp90 to chaperone different pools of clients. Post-translational modifications (PTM) of Hsp90 and its co-chaperones can also regulate the chaperone cycle, and affect Hsp90 drug sensitivity. Here it is shownthat c-Abl kinase phosphorylates Y223in the co-chaperoneAha1, promotingits interaction with and stimulation of Hsp90 ATPase activity. Pharmacologic inhibition of c-Abl prevents the Aha1-Hsp90 interaction thereby, hypersensitizing cancer cells to Hsp90 inhibitors.Another co-chaperone of Hsp90, protein phosphatase-5 (PP5), mediates thede-phosphorylation of the co-chaperone Cdc37which is an essentialprocessfor the activation of kinase clients. The crystal structure of phospho-Cdc37 bound to the catalytic domain of PP5revealed elements of substrate specificity within the phosphatase cleft. Hyperactivityandhypoactivity of PP5 increasedHsp90 binding to its inhibitor, providing insight into increasingthe efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.PP5 is autoinhibited by intramolecular interactions that can be activated by anumber of cellular factors, includingHsp90. Casein kinase-1δ (CK1δ)-mediated phosphorylation of T362-PP5, was identified as an integral step for PP5 activation, independent of binding to Hsp90. Additionally, the tumor suppressor von Hippel-Lindau (VHL), the substrate recognition component of the VCB-E3-ubiquitin ligase, was found to interact with and multi-monoubiquitinate K185/K199-PP5 for proteasomal degradationin an oxygen-independent manner. Furthermore, VHL-deficient clear cell renal cell carcinoma (ccRCC) cell lines or patient tumors exhibit elevated PP5 levels. Down-regulation of PP5 caused apoptosis inccRCC cells, suggesting a prosurvival role for PP5 in ccRCC.Thisevidence suggests that inhibition of the enzymes that target and catalyze the PTM of Hsp90 and co-chaperones can act synergistically with Hsp90 inhibitors, providingnovel therapeutic strategiesto enhance the efficacy of Hsp90 inhibitors in cancer cells.
    • MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ACTIVITY IN SYSTEMIC LUPUS ERYTHEMATOSUS

      Perl, Andras; Doherty, Edward (2014)
      Systemic lupus erythematosus (SLE) is an autoimmune disorder, characterized by T cell and B cell dysfunction. SLE mitochondria have been shown to be dysfunctional with increased mass, mitochondrial potential, decreased ATP, elevated reactive oxygen species (ROS) and reactive nitrogen species (RNS) concentrations, and altered Ca2+ stores. Drug treatments that target the mitochondria have shown efficacy in treating SLE. Here we have investigated electron transport chain (ETC) activity in SLE, to better understand the causes of mitochondrial dysfunction in SLE. We have found that mitochondrial complexes I and IV of the ETC have elevated respiration in SLE compared to healthy controls after both overnight resting and anti-CD3/CD28 stimulation. We have also shown that SLE complex I is resistant to NO inhibition of respiration. SLE peripheral blood lymphocytes (PBL) have increased S-nitrosylation (SNO) while immunoprecipitated complex I had decreased SNO of proteins compared to healthy controls. The drug Nacetylcysteine (NAC) was able to inhibit complex I activity in SLE, and was found to reduce the amount of complex I protein NDUFS3 after 15 minutes as measured by western blotting. These results have led us to the conclusion that SLE mitochondrial complex I is in an active form which is resistant to SNO and is driving the production of ROS and RNS that are associated with SLE. The drug NAC is able to inhibit complex I respiration which may have therapeutic efficacy by reducing the ROS and RNS stress in SLE.
    • Preclinical Development of Anti-Cancer Drugs from Natural Products.

      Huang, Ying; Sun, Qing (2014)
      Cancer has been and will continue to be the common concern in the United States and worldwide. As a conventional treatment to fight cancer, new anti-cancer drugs with more efficiency and less toxicity are extremely required. In this study, we have identified two novel compounds with anti-cancer properties from two traditional Chinese medicinal plants. One is Lappaol F that was extracted from the seeds of the plant Actium Lapp L., which has been used in China for centuries as anti-viral and anti-bacterial medicine. Another is M-9 that was extracted from the stem of Marsdenia tenacissima,a plant that has been applied to treat inflammation and cancer in China. Our results showed that Lappaol F inhibited cancer cell growth by regulating a series of cell cycle related proteins and inducing cell cycle arrest at G1 and/or G2 phase. p21 played a critical role in Lappaol F-induced cyclin B1 and cyclin-dependent kinase 1 (CDK1) suppression as well as G2arrest. Lappaol F also induced cell death in a number of cancer cells through caspases activation. Lappaol F-mediated cell growth inhibition was p53-independent. Notably, results from animal studies showed that Lappaol F effectively inhibited tumor growth in vivo, while being well tolerated by the mice. Thus, Lappaol F has a strong potential to be developed as a novel anti-cancer chemotherapeutic. Our studies showed that M-9 successfully sensitized several tumor cells but not non-tumorigenic cells to paclitaxel (Taxol) treatment. Additionally, M-9 reversed chemotherapeutic resistance in a number of multidrug resistant cells. Further results suggested that M-9 functioned, at least to a certain extent, via inhibiting drug efflux by competitively binding to P-glycoprotein (P-gp), a protein that accounts for multidrug resistance. Importantly, results from the in vivostudies demonstrated that M-9 strongly enhanced Taxol-induced growth suppression against xenografts derived from HeLa cells. Moreover, mice tolerated the treatment of Taxol and M-9 well. Therefore, M-9 is a novel chemosensitizer candidate to overcome P-gp-mediated multidrug resistance. Taken together, our studies provide a solid basis for further development of these two compounds as anti-cancer remedies.
    • RELN AS A CANDIDATE GENE FOR AUTISM SPECTRUM DISORDER (ASD)

      Howell, Brian; Lammert, Dawn (2017)
      Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.
    • A matter of life and death: human cytomegalovirus induction of monocyte survival and differentiation into macrophages through manipulation of the PI3K/Akt pathway

      Chan, Gary; Cojohari, Olesea (2017)
      Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus infecting up to 80% of the US population and reaching 100% seroprevalence in many parts of the world. In mostindividuals HCMV infection is usually asymptomatic. In contrast, in immunodeficient or immunonaive people, such as transplant recipients and the developing fetus, the virus is a major cause of morbidity and mortality. During a primary infection, HCMVcan spread very effectively in the body infecting many organ types and monocytes are believed to be the principal cell type responsible for HCMV dissemination throughout the body. Monocytes, however, are naturally programmed to undergo apoptosis after 48h in the circulation and are not permissive for viral replication. Our lab has shown that in order to combat these biological hurdles, HCMV promotes survival of these short-lived cells past their 48h “viability gate”. Besides inducing survival, the virus also mediates the differentiation of monocytes into macrophages skewed towards an M1 pro-inflammatory phenotype with select M2 anti-inflammatory features, which are long-lived cells, permissive for viral replication. However, the mechanisms used by HCMV to concomitantly induce survival and macrophage differentiation -two linked but separate processes, are not fully understood. The studies in this thesis reveal that upon binding and entry, HCMV initiates a survival program in monocytes by inducing a rapid and sustained activation of the PI3K/Akt pathway, which isdifferent from that induced by myeloid growth factors. Moreover, after inducing cellular survival across the 48-h viability gate, the virus also employsthe PI3K/Akt pathway to regulate caspase 3 activation which mediatesthe atypical M1/M2 polarization. Our work suggests that virus not only makes use of the PI3K/Akt pathway, but manipulates it at multiple levels toallow for viral-specific downstream functional changes.Deciphering how the virus uniquely maneuvers signaling pathways in monocytes to drive their survival and differentiation might allow us to develop new treatments targeting HCMV-infected monocytes and preventing viral spread and disease.
    • The Role of TET Methylcytosine Dioxygenase 2 in Myeloid Malignancies

      Mohi, Golam; Nath, Dipmoy (2017)
      TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate stage in the DNA demethylation processthat controls the transcription of genes. TET2 is highly expressed in the hematopoietic system and is suggestedto regulate the maintenance and differentiation of hematopoietic stem/progenitors. Mutations in TET2 has been found in different hematological malignancies including Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndrome (MDS), etc. However, the mechanisms by which TET2 controls hematopoiesis and contributes to myeloidmalignancies remainunknown. The direct targets of TET2 have not been elucidated yet. In order to identify the direct targets of TET2, we have performed chromatin immunoprecipitation using TET2 specific antibody followed by genome-wide sequencing. We have found enrichment in binding of TET2 in the promoters of SHP1, SOCS3 and PLZF among other targets. Interestingly, the expression of these genes is also significantly downregulated in the hematopoietic progenitors of TET2 deficient mice. Furthermore, we have found that CMML patients with TET2 mutations also have decreased expression of these genes. Knockdown of TET2 resulted in downregulation of these genes in leukemic Molm14 and murine Ba/F3 cells. Conversely, overexpression of TET2 in monocytic U937 cells increased the expression of these genes. Using methylation specific PCR, we also have observed increased methylation in the promoters of SHP1, SOCS3 and PLZF in TET2 deficient Molm14 and Ba/F3 cells. Also, using methyl/hydroxymethyl-DNA immunoprecipitation, we observed an increase in the 5mC level and decrease in the 5hmC level in the promoter region of these genes suggesting that TET2 directly regulates the expression of these genes by regulating the methyl and hydroxymethyl level of the promoter of these genes. Although loss of function of TET2 has been associated with multiple hematopoietic malignancies, TET2 is most commonly mutated in CMML with almost 50% patients bearing TET2 mutations. Interestingly, TET2 mutations are frequently associated with CBL mutations in CMML. In order to assess the concurrent effects of TET2 and CBL deficiencies, we generated TET2 CBL double knockout mice. We observed that simultaneous deletion of TET2 and CBL resulted in increased leukocytes and neutrophil and enhanced splenomegaly compared to control mice. The double knockout mice showed increase in the granulocyte macrophage progenitors and a significant expansion of the stem progenitor cell population. The overall survival of these mice also reduced substantially. It suggests that concurrent deletion of TET2 and CBL increased the severity of the CMML like disease in mice and thus TET2 and CBL deletion may cooperate in the pathogenesis of CMML. Although most studies suggest a tumor suppressor function of TET2, we also have found a tumor promoter function, especially in MLL rearranged leukemia. We have found that knockdown of TET2 resulted in decreased proliferation in MLL-AF9 positive Molm14 leukemia cells and murine Ba/F3 MLL-AF9 expressing cells. Conversely overexpression of TET2 significantly increased the proliferation level of Molm14 cells. In orderto understand the in vivo role of TET2 in MLL-AF9 mediated leukemia, we performed a retroviral BMT experiment. Whereas the expression of MLL-AF9 in wild type BM resulted in marked increase in WBC and NE and splenomegaly, the deletion of TET2 reduced the white blood cell and neutrophil count and also caused reduction in the spleen size. MLL-AF9 overexpression resulted in the increase in hematopoietic stem/progenitor cells and granulocyte macrophage progenitors and granulocyte/monocyte precursors which was significantly reduced in TET2 KO MLL-AF9 mice. There was a significant reduction of the hematopoietic colony formation ability mediated by MLL-AF9 in TET2 KO mice. The overall survival of the knockout mice was markedly improved compared to the control MLL-AF9 mice. Together these results suggest a tumor promoter role of TET2. Taken together, all the results indicate a dual role of TET2 in myeloid leukemia.