• Cellular and Molecular Players in Neuromuscular Junction (NMJ) Formation and Function

      Barik, Arnab; Institute of Molecular Medicine and Genetics (2014-04)
      There are three distinct segments in this dissertation. First, I attempted to address the role of Schwann cells in mammalian neuromuscular junction (NMJ) development and function. Schwann cells at the NMJs do not form myelin sheaths and are known as terminal Schwann cells. Terminal Schwann cells are thought to be analogous to astrocytes in the central nervous system. Schwann cells (as described in details in the next section) provide trophic support to motor axons and modulate synaptic activity by sensing neurotransmitter release at the nerve terminal. However, the role of Schwann cells in synapse formation and maintenance remains unknown. Second, during NMJ formation, anterograde signals from nerve to muscle, and retrograde signals from muscle to nerve are critical for the establishment of a functional synapse. Research over the last three decades has contributed to our understanding of the role of the anterograde signaling at NMJ. However, identification of muscle-derived retrograde signals involved in motoneuron terminal differentiation remains scarce. Recent work from our laboratory suggests that genes that are transcriptionally regulated by p-catenin in muscles might play a crucial role in pre-synaptic differentiation at the NMJ.2 Third, Agrin-LRP4-MuSK signaling is critical for NMJ formation. At the NMJ, LRP4-mediated activation of MuSK by neural Agrin is required for post-synaptic differentiation. Mice that lack any one of the three genes fail to form NMJs and die at birth. Due to perinatal lethality of these null mice, less is known about how Agrin-LRP4-MuSK might regulate NMJ maintenance. Moreover, mutations in Agrin, LRP4, and MuSK have been reported in patients diagnosed with congenital myasthenic syndrome (CMS), and autoantibodies against MuSK and LRP4 have been detected in patients with myasthenia gravis (MG). However, the role of Agrin-LRP4-MuSK in the etiology of these neuromuscular disorders is not clear.
    • Ceramide-mediated Regulation of Cell Polarity in Primitive Ectoderm Cells: A novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; Institute of Molecular Medicine and Genetics (2009-01)
      Ceramide is considered a key sphingolipid, regulating a variety of critical cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide, raised in rabbit (rabbit IgG). The novel antibody specifically recognizes ceramide in lipid overlay assays and detects ceramide containing different fatty acid chain lengths (i.e. C2-, C16-, C18-, C20- and C24 ceramide). The new antibody was compared with the commercially available anti-ceramide mouse IgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in intracellular compartments that are not well identified with the commercially available antibody. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine both the localization of ceramide, and its interaction with proteins by immunocytochemistry. To investigate the role of ceramide in early embryonic development, we used embryoid bodies (EBs) differentiated from mouse embryonic stem cells as a model. The primitive ectoderm cell layer of EBs represents the primitive ectoderm of the early embryo. In mammals, the primitive ectoderm is an epithelium of polarized cells that undergoes gastrulation and differentiates into all embryonic tissues. We find that in primitive ectoderm cells, ceramide was elevated and asymmetrically distributed to the apico-lateral cell membrane, where it was co-distributed with Cdc42 and F-actin. Pharmacological or siRNAmediated inhibition of ceramide biosynthesis impaired primitive ectoderm formation and concomitantly increased apoptosis in EBs. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog, indicating that the observed defect was due to loss of ceramide. Ceramide depletion also prevented membrane translocation of atypical PKC (aPKC), interaction of aPKC with Cdc42, and phosphorylation of GSK-3|3. Recombinant aPKC, when bound to ceramide-containing lipid vesicles, formed a complex with the polarity protein Par6 and Cdc42. Taken together, our data suggest a novel mechanism by which a ceramide-induced, apico-lateral polarity complex with aPKC regulates primitive ectoderm cell polarity and morphogenesis.
    • Characterization o f the DNA Ligase IV and XRCC4 complex in the DNA double-strand break repair

      Lee, Kyung-Jong; Institute of Molecular Medicine and Genetics (2002-11)
      DNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage. The nonhomologous end-joining (NHEJ) pathway is the principal mechanism for repairing DSBs in mammalian cells. It is also required for V(D)J recombination. There are at least four essential proteins in this pathway. These include Ku protein, DNA PKcs, and the DNA Ligase IV/XRCC4 (DNL IV/XRCC4) complex. This dissertation reports the determination of the quaternary structure of the DNL IV/XRCC4 complex, the mapping of a major human autoimmune epitope in XRCC4, the identification of DNAPKcs phosphorylation sites in XRCC4, and an investigation of the biochemical significance of XRCC4 phosphorylation. Biochemical characterization shows that DNA Ligase IV and XRCC4 form a stable mixed heterotetramer. This is the active form of the enzyme and is essential for in vitro DNA end joining in the presence of additional factors derived from cell extracts. Data shown here also demonstrate that the DNL IV/XRCC4 complex is a human autoantigen. The major autoimmune epitope maps to amino acids 251-266. This epitope coincides with several sites where XRCC4 is potentially modified in response to radiation or inflammation, including a DNA-PKcs phosphorylation site at serine 260. Results raise the possibility that radiation-induced post-translational modifications contribute to development of an autoimmune response in susceptible individuals. Previous work has shown that DNA-PKcs kinase activity is required for NHEJ, but the critical physiological target of this enzyme is not yet known. Current work shows that DNA-PKcs phosphorylates serine 318 of XRCC4, in addition to the serine 260 site described above. The presence of serine 260 increases phosphorylation at serine 318, suggesting that phosphorylation can occur sequentially. Mutation o f serine 260 reduced DNA end-joining activity and sensitivity to the PI3 kinase inhibitor (LY294002). These data provide preliminary evidence that phosphorylation of XRCC4 by DNA-PKcs contributes to regulation of DNA repair.
    • Characterization of Cervical and Head and Neck Squamous Cell Carcinomas by Proteomic Analysis

      Merkley, Mark A.; Institute of Molecular Medicine and Genetics (2009-06)
      (First Paragraph) The oral cavity, oropharynx, larynx, esophagus, and ano-genital orifices are lined with stratified squamous nonkeratinized epithelium, which forms the barrier between the underlying tissue and the external environment. The proliferative nature of this epithelium, together with its potential exposure to environmental insults such as tobacco carcinogens, alcohol, or oncogenic viruses, makes it susceptible to carcinogenesis. Indeed, carcinomas of stratified squamous nonkeratinized epithelium are among the most common and deadly cancers worldwide. In particular head and neck squamous cell carcinoma and cervical squamous cell carcinoma together account for about 1 million new cases annually, worldwide. These cancers arise from similar tissues and share common risk factors, although they differ in that effective population-based screening exists only for cervical cancer.
    • The Clinical Phenotype of An Extended Pedigree with Late-onset Alzheimer's Disease

      Fennell, Eleanor M.; Institute of Molecular Medicine and Genetics (2009-03)
      Alzheimer's disease (AD) is a devastating neurodegenerative disease with a multifaceted etiology. This retrospective exploratory study used the family history method to develop a phenotype based on the personal history of cognitive decline, changes in behavioral characteristics, and the medical, social and environmental history of the five AD affected family members in one large extended family. Group family interviews provided a description of the phenotype and identified medical and environmental risk modifiers. Average age-of-onset for AD in 5 of 12 siblings, (all homozygous APOE4/4) was 69.2 years (range 66-72 years). Fisher's exact test identified neuropsychiatric behaviors as common phenotypic manifestations; delusions (p = 0.0455), hallucinations (p = 0.0101), irritability (p = 0.0455), personality change (p = 0.0013), pacing (p = 0.0013), aggressiveness (p = 0.0455), and poor judgment (p = 0.0013). Environmental variables that emerged as significant were a less than an eighth grade education (p — 0.0152), presence of stroke/TIA (p = 0.0455), presence of osteoarthritis (p = 0.0455), and vitamin B12 deficiency (p = 0.0013). Risk of stroke/TIA may be related to the increased risk from APOE4 while vitamin B12 deficiency may be associated with advanced age. There was no significant protective benefit from the management of hypertension (p = 0.5758), use of statins to control cholesterol (p = 0.9545), use of Vitamin C (p = 1.000), and/or Vitamin E (p = 0.9899) among family members. Potential modifiable health practices among the AD unaffected siblings demonstrated that 85% continued to engage in a sedentary lifestyle (p = 0.6818), 57% were overweight (p = 0.6894), 57% (p = 0.6894) consumed an unhealthy diet, and 56% smoked (p = 0.1591). Single nucleotide polymorphism (SNP) microarray Clinical Phenotype of Alzheimer's Disease analysis indicated that several SNPs in the gene, transient receptor potential cation channel, subfamily C, member 4 associated protein, (TRPC4AP), were significant. All five of the affected siblings and three unaffected siblings exhibited one haplotype; the unaffected siblings with the same haplotype as affected siblings were younger in age and did not have any cognitive problems at the time of the study (Poduslo, Huang, Huang, Smith, 2008).
    • Design, Synthesis, and Initial Evaluation of D-Glyceraldehyde Crosslinked Gelatin-Hydroxyapatite as a Potential Bone Graft Substitute Material

      Florschutz, Anthony V; Institute of Molecular Medicine and Genetics (2012-07)
      Utilization of bone grafts for the treatment of skeletal pathology is a common practice in orthopaedic, craniomaxillofacial, dental, and plastic surgery. Autogenous bone graft is the established archetype but has disadvantages including donor site morbidity, limited supply, and prolonging operative time. In order to avoid these and other issues, bone graft substitute materials are becoming increasingly prevalent among surgeons for reconstructing skeletal defects and arthrodesis applications. Bone graft substitutes are biomaterials, biologies, and guided tissue/bone regenerative devices that can be used alone or in combinations as supplements or alternatives to autogenous bone graft. There is a growing interest and trend to specialize graft substitutes for specific indications and although there is good rationale for this indication-specific approach, the development and utility of a more universal bone graft substitute may provide a better answer for patients and surgeons. The aim of the present research focuses on the design, synthesis, and initial evaluation of D-glyceraldehyde crosslinked gelatin-hydroxyapatite composites for potential use as a bone graft substitutes. After initial establishment of rational material design, gelatinhydroxyapatite scaffolds were fabricated with different gelatin:hydroxyapatite ratios and crosslinking concentrations. The synthesized scaffolds were subsequently evaluated on the basis of their swelling behavior, porosity, density, percent composition, mechanical properties, and morphology and further assessed with respect to cell-biomaterial interaction and biomineralization in vitro. Although none of the materials achieved mechanical properties suitable for structural graft applications, a reproducible material design and synthesis was achieved with properties recognized to facilitate bone formation. Select scaffold formulations as well as a subset of scaffolds loaded with recombinant human bone morphogenetic protein-2 were implanted ectopically in a rodent animal model and histologically evaluated for biocompatibility, degradation, and bone formation in vivo. The gelatin-hydroxyapatite scaffolds retained dimensional structure over 28 days and did not elicit any undesirable systemic or local effects. Distinct areas of mineralization and osteoid/bone were noted in all the implanted scaffolds and quantitative differences were primarily dependent on the presence of hydroxyapatite.
    • Evidence Supporting Glial Derived TGF-B1 as a Modulator of Luteinizing Hormone-Releasing Hormone

      Buchanan, Clint D.; Institute of Molecular Medicine and Genetics (2000-03)
      The overall objective of this research is to elucidate mechanisms involved in glial cell regulation of reproductive function. Regulation of LHRH secretion is a complex process that involves a multiplicity of inputs of both excitatory and inhibitory nature, and recent evidence has demonstrated the significance of glial cell-neuron interactions in modifying the activity of LHRH producing neurons. Evidence exists indicating that glial derived growth factors may play a role in the functional control o f the LHRH neuronal network as conditioned medium from astrocytes has been shown to stimulate LHRH secretion from immortalized LHRH neurons (52-55,57,58). However, there is a controversy concerning the identity of the active factor from astrocytes that is responsible for the LHRH releasing activity of conditioned medium. Melcangi and colleagues have provided evidence that TGF-Pi may be responsible for astrocyte-conditioned medium induced LHRH release in the GTl-l cell line (55). However, many of these studies supporting a role for TGF-pi were performed using cortical astrocytes, and additionally, no attempt was made to measure TGF-Pi levels in astrocyte-conditioned medium and correlate it to conditioned medium ability to induce LHRH release. Furthermore, these studies did not discuss potential regulators of TGF-pi secretion and also failed to investigate whether TGF-p receptors, which are necessary for TGF-pi action, are expressed in the GT1 cell line or hypothalamic tissue of the female rat (55,57,58). A second group suggests that TGF-a rather than TGF-Pi may be the active astrocyte factor that regulates LHRH release (53). Although TGF-a mRNA expression and precursor peptide immunoreactivity have been reported in the female rat hypothalamus, these studies failed to demonstrate the ability of hypothalamic astrocyte cultures to produce Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 TGF-a and relied upon addition of exogenous TGF-a to astrocyte cell cultures (50ng/ml 16 hours) in formation of astrocyte TGF-a-conditioned medium (25,46,52,53,76,77).
    • Genomic and Functional Analysis of Vesicular Inhibitory Amino Acid Transporter During Mouse Embryogenesis

      Oh, Won-Jong; Institute of Molecular Medicine and Genetics (2006-01)
      The specification of particular neuronal phenotypes during embryonic development requires the appropriate activation and regulation of genes encoding the proteins required for neurotransmitter synthesis, vesicular packaging and re-uptake from the synaptic cleft. Each neurotransmitter is packaged into synaptic vesicles by its own distinct vesicular transporter. In addition, neurotransmitter packaging is well controlled by other co-factors (reviewed in Ahnert-Hilger et al., 2003). Components of GABAergic neurons GABAergic neurons are the principal inhibitory neurons in the mammalian central nervous system (CNS), where GABA is synthesized from glutamate by two glutamate decarboxylases (GAD), namely GAD65 (Gad2) and GAD67 (Gad1) (Erlander et al., 1991). GABA is then loaded into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT, also known as VGAT). Four GABA transporters (GAT 1-4) are responsible for the re-uptake of GABA from the synaptic cleft through the plasma membrane. Inhibitory GABAergic transmission is mediated by binding of GABA to its ionotropic receptors, GABAA and GABAC, which are ligand-gated chloride channels, and its metabotropic receptor, GABAB (Fig. 1).
    • Glutamic Acid Decarboxylase Expression And Function In The Developing And Neonatal Mouse

      Maddox, Dennis M; Institute of Molecular Medicine and Genetics (2001-06)
      (First Paragraph) In the mouse, there are two distinct genes that encode isoforms of the enzyme glutamic acid decarboxylase (Gad). The G adl gene encodes the larger isoform that has a molecular mass of 67 kilodaltons and is termed "Gad67" (Erlander et al., 1991; Bu et al., 1992). The Gad2 gene encodes the other isoform that has a molecular mass of 65 kilodaltons and is termed "Gad65" (Erlander et al., 1991; Bu et al., 1992). These two isoforms are often co-expressed in GABAergic neurons (Feldblum et al., 1993; Esclapez et al., 1994; Katarova et al., 2000). The isoforms differ in subcellular localization, with Gad67 being found mainly in the soma and Gad65 being found mainly in axon terminals (Kaufman et al., 1991). Additionally, the two isoforms of Gad differ in their affinity for the required cofactor pyridoxal-5’-phosphate (PLP) (Kaufman et al., 1991). By catalyzing the decarboxylation of L-glutamic acid, the Gad enzymes are the rate-limiting step in the biosynthesis of y-aminobutyric acid (GABA) (Barker et al., 1998).
    • Identification of RAB11-Family Interacting Proteins (RAB11-F1Ps): Integral Components in Plasma Membrane Recycling

      Hales, Chadwick M; Institute of Molecular Medicine and Genetics (2003-05)
      Given the involvement of Rabl la in each of these cellular processes and given the potential impact of Rabl la on human health and disease, we sought to further establish a role for Rabl la in plasma membrane recycling. Since other Rab proteins have numerous characterized interacting proteins and because the repetoire for Rabl la is currently limited to three identified interacting proteins, we hypothesized that other Rabl la binding partners exist as putative downstream effectors for the small GTPase. We therefore proposed the following three aims: Aim 1: Identify R ab lla interacting proteins. Aim 2: Determine the effect of interacting proteins on membrane recycling. Aim 3: Establish an organizational model of a putative Rabl la complex. The progression of studies herein provides insight into the dynamic and complex process of plasma membrane recycling. Yeast two hybrid screening of a parietal cell cDNA library utilizing dominant active Rabl laS20V as the bait identified Rabl 1-Family Interacting Protein 1 (Rabll-FIPl), a novel R ab lla interacting protein. EST database searches with the R abll-FIPl sequence identified three homologous proteins with high carboxyl-terminal identity. Chapter 1 introduces the new family of Rabl la interacting proteins and provides the initial characterization. Interestingly, these studies indicated an interaction between Rabll-Family Interacting Protein 2 (Rabll-FIP2) and myosin Vb tail. Chapter 2 further describes the Rabl l-FIP2/myosin Vb tail binding and provides functional data placing Rabll-FIP2 as an integral component of the plasma membrane recycling system. Finally, recent studies have indicated a recycling system dependence on different kinase activities. Through kinase inhibitor studies and immunofluorescence imaging, evidence presented in Chapter 3 suggests that R ab lla along with multiple Rabll-FIP proteins function as a complex beginning at the process of endocytosis with movement dependent on multiple phosphorylation events. The ultimate goal throughout these studies is to provide a clearer picture of Rabl la function in plasma membrane recycling so that one day a positive impact on human health can be achieved.
    • Influence of DNA Ends on Structure and Function of the DNA-dependent Protein Kinase

      Jovanovic, Marko; Institute of Molecular Medicine and Genetics (2006-12)
      Non-homologous end joining is a major DNA double-strand break repair pathway in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer, is hypothesized to be a key regulator of the pathway. Available data suggest DNA-PKcs may exert this regulatory function by controlling access to the DNA termini and by phosphorylation of itself and other proteins. I further characterized DNA-PK-DNA interaction by studying binding of DNA-PKcs and Ku to oligonucleotides with chemically defined end structures under conditions that preclude synapsis between opposing DNA ends. Binding of DNA-PKcs to DNA varied with the end structure in a manner that suggests that partial melting of DNA ends is necessary for the formation of a stable, enzymatically active complex. Unexpectedly, these studies also revealed that ATP, as well as its nonhydrolyzable analog AMP-PNP, have an allosteric effect on the interaction of DNA-PKcs with DNA.
    • Mechanisms of Neuroprotection by Estrogen and Selective Estrogen Receptor Modulators

      Dhandapani, Krishnan M.; Institute of Molecular Medicine and Genetics (2003-03)
      Specific Aim #1: To determine whether 17b-E2 and SERMs directly influence neuronal survival. Specific Aim #2: To determine whether astrocyte-derived TGF-b protects neurons from cell death. Specific Aim #3: To determine whether 17b-E2 or SERMs regulate the release of TGF-b from astrocytes. Specific Aim #4:To elucidate the mechanism of TGF-b mediated neuroprotection in GT1-7 Neurons. Specific Aim #5: To identify genes potentially mediating the neuroprotective effects of 17b-E2 and/or tamoxifen through the use of high density gene chip arrays.
    • Mechanosensory Hair Cell Precursors in the Zebrafish Lateral Line

      Floyd, Tiffany L.; Institute of Molecular Medicine and Genetics (2009-07)
      The vertebrate inner ear mediates the senses of hearing and balance. Contained within both the auditory and vestibular compartments of the inner ear are specialized mechanosensory hair cells that function as receptors and transducers of environmental stimuli. In all vertebrates, these sensory hair cells are particularly susceptible to ototoxic insults resulting in cell death and, in mammals, the irreversible loss of hair cells underlies deafness and balance disorders. In contrast to mammals, several non-mammalian vertebrates (including zebrafish) possess the innate capacity to produce new hair cells throughout life as well as regenerate hair cells that have been lethally damaged. A long-term strategy of the hearing research field is to determine the molecular mechanisms of hair cell regeneration using regenerating model systems such as zebrafish, then to apply this information to mammalian models where sensory hair cell regeneration is limited or nonexistent. During embryogenesis, sensory hair cell fates are specified through a mechanism of Notch-Delta-mediated lateral inhibition. The gamma secretase complex is an upstream regulator of Notch signaling, responsible for proteolytic\ cleavage and activation of the Notch receptor. Recent evidence suggests that Notch signaling may also play a role during the process of hair cell regeneration in zebrafish (Ma et al., 2008). I used a chemical inhibitor of the gamma secretase complex to examine the role of Notch signaling in the regulation of hair cell number maintenance in larval zebrafish. Results presented in this thesis provide novel insight into the mechanisms regulating the maintenance of resident hair cell precursors within the sensory epithelium. Moreover, this new information is directly relevant to research efforts in mammalian models by providing the molecular framework for therapeutic strategies designed to replace or regenerate lethally damaged hair cells in the mammalian cochlea by reactivating resident precursors to differentiate into hair cells.
    • Myr 8: The First Member of a Novel Myosin Class Is Expressed During Brain Development

      Patel, Krishna G.; Institute of Molecular Medicine and Genetics (2000-05)
      Neuronal cell migration and cellular differentiation, major phases in the assembly process o f the mammalian neocortex, involve considerable organelle and cellular motility. While the cytoskeletal organization of migrating neurons is well documented, and the involvement of the cytoskeleton in modulating intracellular membrane transport events during neuronal cell differentiation is well appreciated, identification of selective cytoskeletal components underlying these processes is only beginning to emerge. Observations over the past two decades reveal that myosin motors are involved intimately in multiple actin-dependent membrane movements, including vesicular trafficking, organelle localization and organization, endocytosis, exocytosis, phagocytosis, lamellopodial extension, and the more classically defined functions such as cytokinesis, contractility, and cell motility or migration. Accordingly, our studies have been directed toward the identification and characterization of unconventional myosins that may participate in neuronal cell migration and/or differentiation events within the developing mammalian brain. Our analyses identified two myosin isoforms that contribute to a novel unconventional myosin class. We have cloned, sequenced, and designated these myosin isoforms as myr 8a and 8b (8th unconventional myosin from rat). Structurally, the head domain o f myr 8 contains a large N-terminal extension composed of multiple ankyrin repeats similar to myosin phosphatase. The motor domain is followed by a single putative light chain binding domain. The tail domain of myr 8a is comparatively shortwith a net positive charge, whereas the elongated tail domain of myr 8b bears an overall neutral charge and reveals several streches o f poly-proline residues. Phylogenetic analysis indicates that myr 8 is sufficiently divergent from known myosins as to comprise a new class of myosins. Northern analyses demonstrate that the m yr 8 myosins are expressed predominantly in the nervous system, and are detected principally at developmental timeperiods. Indirect-im munofluorescent studies reveal a pattern of im m unoreactivity within forming neuronal and astroglial cell processes located throughout the developing brain. Taken together these data suggest that this novel myosin may play a crucial role in membrane biogenetic events during neuronal and astroglial cell differentiation. Given the increasing identification of neurological dysfunctions that arise as a consequence of defective myosins, as well as from other cytoskeletal components, it is essential to unravel the selective roles in which this novel unconventional myosin may participate during neocortical development.
    • Neurotransmitter Gases as Modulators of GnRH and the Preovulatory LH Surge

      Lamar, Charisee; Institute of Molecular Medicine and Genetics (1998-04)
      Until recently nitric oxide (NO) and carbon monoxide (CO) were viewed only as toxic substances. However, there has been a substantial amount of evidence in the past decade that has redefined these gaseous molecules as physiological messengers. Along these lines, NO and CO are now recognized as modulators of immunological defense, vasodilation, endocrine signaling, and neurotransmission (1-8). As neurotransmitters, NO and CO are unique when compared to classical neurotransmitters. For instance, unlike all other classical neurotransmitters NO and CO are, 1) lipophilic gases with short half lives, 2) not stored in synaptic vesicles, 3) their effects are not mediated through classic receptor proteins - rather their effects result from NO and CO binding to the heme moiety of heme-proteins such as guanylate cyclase and cyclooxygenase, and 4) their effects are terminated by diffusion from target tissues ( 1,2,5,7,9). Production of NO and CO relies on the activity of the enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), respectively. NOS uses the substrate Larginine to generate NO (2, 10), while HO uses the substrate heme to generate CO (9,11-16). NOS exists as three isoforms, macrophage NOS (mNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (17-21). HO also exists as three isoforms, heme oxygenase-1 (HO-1), heme oxygenase-2 (HO-2), and heme oxygenase-3 (HO-3) HO-1 is inducible, while HO-2 and HO-3 are constitutive enzymes; however, HO-3 is currently viewed as a poor heme catalyst (12,13,22). There is a growing body of evidence that suggests that NO and CO regulate hypothalamic function. For example, recent studies have shown that the hypothalamus produces significant quantities of NO, primarily through the actions of nNOS (23,24). Likewise, the hypothalamus possesses one of the largest production rates of CO in the brain (25,26). That NO and CO can regulate neurohormone release from the hypothalamus is evidenced by findings demonstrating that NO and CO regulate corticotropin-releasing hormone (CRH) (27-33), vasopressin (29-31,34,35), and oxytocin secretion (29,36). With respect to reproductive function, numerous studies (23,37-45) have provided evidence for a significant role of NO in the control of the hypothalamic releasing factor, gonadotropin-releasing hormone (GnRH). For instance, it has been demonstrated (37,46,47) that NO neurons are located in close proximity to GnRH generating cells bodies in the hypothalamus and thus strategically located to exert regulatory effects over GnRH neurons. That NO can exert regulatory effects on GnRH neurons was demonstrated in studies where exogenously applied NO markedly stimulated GnRH release from male hypothalamic fragments (1,48,49) and immortalized GnRH (GT1-7) cells in vitro (1,48). A physiological role for NO in the steroid-induced luteinizing hormone (LH) surge has been suggested based on studies in which the LH surge was attenuated by the administration of NOS inhibitors (43,44) or NOS antisense oligonucleotides (38) No studies, however, have reported nNOS mRNA, protein, or NOS activity levels in the hypothalamus during the LH surge so as to verify that an increased NO tone actually occurs during this critical time. To address this deficit, Aim 1 of this study was designed to examine NO tone on proestrus in the cycling female rat-the day the natural preovulatory LH surge occurs. Since heme oxygenase, the enzyme that produces CO from heme molecule metabolism, is located in the hypothalamus (2,11,12,14,15), and CO production in the hypothalamus is one of the highest in the brain (25,26), it is conceivable that CO could play a role in regulating GnRH secretion. This possibility has not been investigated and thus studies on this issue appear warranted. Therefore, Aims 2-4 of this study were designed to assess the possible role of CO in the control of GnRH and LH secretion.
    • Novel Role of Heat Shock Protein (HSP) 90 in Regulating ATR-CHK1 DNA Damage Response Pathway in Cancer Cells

      Ha, Kyungsoo; Institute of Molecular Medicine and Genetics (2011-10)
      DNA damage caused by environmental mutagens or reactive metabolic byproducts induces DNA damage response (DDR), which regulates cell cycle transit, DNA repair and apoptosis. DDR involves the phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM) and ATM and RAD3-related (ATR) proteins. ATR regulates the firing of the replication forks during S phase, and the repair of damaged replication forks to prevent premature onset of mitosis. ATR phosphorylates and activates CHK1 which phosphorylates and inactivates CDC25, thereby inhibiting CDK1 activation and cell cycle progression. In the present studies, we determined that treatment with an hsp90 inhibitor AUY922, without affecting the mRNA levels, dose-dependently depletes the protein levels of p-ATR (Ser 428), ATR and CHK1 in human breast and cervical cancer cells. Additionally, treatment with the pan-histone deacetylase inhibitor panobinostat (PS), which is known to induce hyperacetylation and inhibition of hsp90 function, also depleted ATR and CHK1 levels in cancer cells. Co-treatment with the proteasome inhibitor bortezomib (BZ) partially reversed AUY922- or PS-mediated depletion of ATR and CHK1 expression, indicating proteasome-mediated degradation of ATR and CHK1. Treatment with either AUY922 or PS markedly inhibited the binding of ATR with hsp90, induced polyubiquitylation of ATR, and decreased the half-life of both ATR and CHK1 proteins. Treatment with AUY922 also abrogated ionizing radiation (IR)-induced cell cycle arrest and increased the amount of DNA damage in the cancer cells following IR. Treatment with AUY922 also inhibited the recruitment of p-ATR, ATR and 53BP1 to the site of DNA damage. In addition, HDAC3 binds to and deacetylates hsp90 in the nucleus. Depletion of HDAC3 by either short hairpin RNA or genetic knockout induced hyperacetylation of nuclear hsp90, resulting in the inhibition of chaperone association of ATR with hsp90 and depletion of ATR. These findings demonstrate that 1) ATR is chaperoned by hsp90, 2) Inhibition of chaperone function of hsp90 results in proteasomal degradation of ATR and inhibition of DDR, 3) pan-HDAC inhibitors abrogate ATRCHK1 cell cycle checkpoint pathway by modulating chaperone activity of hsp90 and 4) HD AC3 plays a critical role in the regulation of DNA damage response by stabilizing the chaperone activity of nuclear hsp90.
    • ParaDIME: Genome-wide differential DNA methylation analyses using next generation sequencing

      Pabla, Sarabjot; Institute of Molecular Medicine and Genetics (12/27/2016)
      Epigenetic modifications are key players in the regulation of a plethora of cellular and physiological processes. DNA methylation is one of the most widely studied epigenetic modification. Genomic abnormalities in DNA methylation have been implicated in various complex diseases including cancer and autoimmunity. With advent of next generation sequencing, investigating DNA methylation patterns at genome-wide scale has become increasingly feasible. However, the pace of developing appropriate statistical methods to analyze large scale DNA methylation data has been slower. This can be attributed to both statistical and computational challenges faced by current methods. In order to overcome these statistical and computational shortcomings, we developed ParaDIME, a web application for differential DNA methylation analysis. ParaDIME tests CpG dinucleotide sites or pre-defined regions of CpG sites for differential DNA methylation using Rao-Scott chi squared test. ParaDIME not only uses a nonparametric test that accounts for differential sequencing coverage but also uses permutation testing to compute exact p values. In order to overcome computation challenges of large amount of permutations, we use parallel computing to share the workload and decrease execution time significantly. To test ParaDIME in-silico, we initially simulated bisulfitesequencing data and tested it against two most widely used methods: MethylSig and MethylKit. It performed equal or better at accurately detecting differentially methylated regions than both the methods. Especially, at important, low differences of percent methylation, ParaDIME performed better than existing tools. In order to test ParaDIME’s ability to detect biologically relevant differentially methylation regions (DMRs), it was then tested on publically available methylation data from chronic lymphocytic leukemia patients. Our method was able to detect previously known and experimentally verified DMR in CLL, especially DMRs located in Nfatc1 and FOXA2 genes. Additionally, it was able to detect other DMRs in genes present in caner related pathways. Due to ParaDIME’s ability to detect biologically relevant DMRs, we employed it in an integrative analysis study to identify epigenetically regulated genes in Sjogren’s syndrome mouse model, B6.NOD aec1/aec2. We performed reduced representation bisulfite sequencing and RNA sequencing on salivary glands of four and eighteen weeks old B6.NOD aec1/aec2 compared to age and gender matched C57BL/6 mice. After removing age and mouse model effect, we discovered 89 differentially expressed as well as differentially methylated genes. Spearman rank order correlation analysis found a significant correlation between DNA methylation and gene expression. Autoimmunity related genes Klf9 and Nfkbid showed significant negative correlation whereas, other genes like Fgf12 and Coll11a2 genes showed significant positive correlation. Subnetwork enrichment using MATISSE showed three jointly active connected subnetworks that were highly enriched in Immune system related pathways, especially, T cell and B cell activation along with cytokine signaling and endocrine system development. Evidence presented in this report presents a novel and a robust differential DNA methylation analysis method with high accuracy to detect disease-relevant DMRs. ParaDIME is a user-friendly and scalable web application with appropriate test statistic to analyze large-scale DNA methylation studies.
    • Regulation of Embryonic Stem Cell Pluripotency and Differentiation by Notch Signaling

      Noggle, Scott A; Institute of Molecular Medicine and Genetics (2004-05)
      (First Paragraph) Mammalian development prior to implantation is primarily involved with establishing the support tissues needed for interaction with the mother’s uterine tissue and blood stream as the uterine tissue prepares to receive the embryo. Development of the embryo proceeds slowly while initiating differentiation of the first cell lineages, trophectoderm and the primitive endoderm, that are necessary for interactions with the mother’s uterine tissue. In mice, development from fertilization to the initiation of implantation of the blastocyst into the uterine wall takes about four and a half days (Fig. 1) whereas this period in human development requires about 7 days. This is in contrast to the much-accelerated development, for example, of amphibian species that produce embryos that develop in isolation of maternal support after fertilization.
    • Role and Significance of Bradykinin in Reproduction

      Shi, Beien; Institute of Molecular Medicine and Genetics (1998-04)
      The ultimate goal o f reproduction is to maintain the life and well-being o f the species In mammals, it includes the nurturance and maturation o f the individual male and femaie germ cells, their successful union, the subsequent growth and development of the newly created individual within the body o f mother, and the birth o f a new' offspring In order to achieve these goals, the female reproductive system undergoes a series of closely interrelated events involving follicle development, ovulation, fertilization, implantation, pregnancv. and parturition Ovulation, the discharge of the ovum ( female germ cell) from a mature follicle tor fertilization, is a prerequisite and central process for a new lite ot the species, and it occurs in a cyclic fashion It has been noted for a long time that the ovulatory cycle requires a highly coordinated and complex interplay ot events among the components ot the hypothalamus-pituitarv-ovarv axis (1) The central endocrinological component in the ovulatorv cycle is the preovulatory gonadotropin (luteinizing hormone, LH, and follicle stimulating hormone, FSH) surge (1-3). The LH surge induces ovulation o f a mature ovum for potential fertilization (1-3) The activity o f the pituitary gonadotrophs which secrete LH and FSH is controlled by its principal regulator, gonadotropin-releasing hormone (GnRH) (4.5) GnRH is a decapeptide secreted in a pulsatile manner by a specific group o f neurosecretory neurons in the hypothalamus (6,7). Once released into the pituitary portal capillaries in the median eminence, it is delivered to the anterior pituitary' via the hypophyseal penal veins GnRH binds to its receptor on the plasma membrane o f the gonadotrophs and stimulates the secretion o f LH and FSH ( The gonadotropins travel to the ovary via the bloodstream where they stimulate estradiol and progesterone production and initiate the process o f ovulation (1) The o \arian steroids, estradiol and progesterone, regulate GnRH and LH secretion at the hvpothalamus and pituitary level There is a large release o f GnRH shortly before ovulation on proestnjs day in the rat in response to the elevated levels o f estradiol and progesterone 0-M !> This GnRH surge results in the gonadotropin surge and subsequent ovulation Therefore, it is now d ear that estradiol and progesterone are actually the ke\ regulators o f the preovu!atorv gonadotropin surge (12-16) There is a voluminous literature that indicates that ovarian estradiol is a positive stimulus for the gonadotropin surge ( whereas progesterone is thought to synchronize, potentiate, and limit the uonadotropin surge to a single day (20-24) Although the roles o f ovarian steroids are clear, the site and mechanisms underlving their actions remain poorly understood. Since GnRH neurons do not possess steroid hormone receptors (25-27). steroid regulation o f GnRH secretion appears to be through an indirect neuronal circuitry involving several neurotransmitters and/or neuropeptides, which in turn regulate GnRH secretion. Within the last decade, rapid progress has been made concerning the mechanisms by which GnRH neurons are regulated Information available so far has already shown a clearer picture o f the control o f G nR H secretion However, the real challenge still lies in our ability to understand how G nRH secretion is regulated and how information is integrated into coherent patterns o f regulation Recently, bradykinin has been demonstrated to be present in the central nervous system (CNS) (2S-30). and work presented in this dissertation demonstrates that bradykinin is present in key hypothalamic sites responsible for the control of GnRH secretion This finding raises the possibility that bradykinin may be an important transmitter in the control o f GnRH secretion. This possibility was the foundation for manv o f the aims proposed and work conducted in this study A second focus o f this dissertational work was to examine the role o f bradykinin in the reproductive processes o f implantation and parturition. Implantation is the process wherebv the embryo becomes fixed in the uterus in physical contact with the maternai oruanism Implantation includes adhesion o f the blastocyst to the uterine epithelium, increased permeabilitv at implantation sites, the decidual cell reaction (deciduaiization). and loss o f epithelial cells surrounding the implanting blastocyst (31-33) file process 01 implantation, especiallv decidual transformation is very similar to an inflammatory response (34). in that there is hyperemia and tissue remodeling. Steroid hormones are major regulators o f implantation, and various inflamm atory mediators, such as cytokines and growth factors, are also involved in this process (35,36). During pregnancy, the fetus continues to grow until term, at which point the uterus acts to expel the mature fetus. This event is called parturition. Uterine smooth muscle contractility is reduced during pregnancy and dramatically increases during parturition (37) Since bradykinin is a well know n inflammatory mediator during inflammation and tissue injury, and a potent stimulator o f uterine smooth muscle contraction, the physiological roles o f endogenous bradykinin in the processes o f implantation and parturition were examined in addition to its potential role in the control o f GnRH secretion.
    • Role of the NR2 Subunit and its Molecular Motifs on Memory and Cognition and the Physiological Outcomes after Experimental Intracerebral Hemorrhage

      Jacobs, Stephanie A.; Institute of Molecular Medicine and Genetics (2013-10)
      The N-methyl-D-aspartate receptor is the main coincidence detector in the brain. It is known to be necessary for many forms of learning and memory. Interestingly, the NR2 subunit composition of the NMDA receptor is modulated endogenously according to the location in the brain and the age of the animal, with the NR1 subunit being ubiquitously expressed. In the forebrain regions, including the cortex, hippocampus, striatum, and amygdala, the NR2A and NR2B subunits are the primary subunits expressed. While it is known that a high NR2B:NR2A ratio enhances memory and cognition, it is not directly known, the effects of a low NR2B:NR2A ratio. In this project, the effects of modulating the NR2A:NR2B ratio on multiple forms of learning and memory are explored by the use of a NR2A transgenic mouse. Our transgenic mice overexpress the NR2A subunit in the forebrain regions, driving the NR2A:NR2B ratio toward the expression of the NR2A. As the NR2B subunit is known to favor learning and memory; we also further explore the role of the N-terminal and membrane domains and the Cterminal domain in the observed enhancements. Our data indicate that a high NR2A:NR2B ratio constrains multiple forms of long-term memory in our transgenic animals. Additionally, we have observed additional forms of enhanced learning and memory in the NR2B transgenic mice that were not tested previously. We were able to show that the NR2B C-terminal tail, and thus the intracellular signaling cascades, is responsible for the enhancements seen in the NR2B animals. Using the NR2A and NR2B transgenic mice, we also investigated the long-held hypothesis that a low NR2B:NR2A ratio would be beneficial to hemorrhagic stroke recovery. Until now this hypothesis has only been investigated by the use of pharmaceuticals, whereby the NR2B subunit is antagonized. We found that while several physiological factors, including neurological deficit and survival rate were unchanged, lesion size and percent edema were significantly less in the NR2A transgenic mice than the NR2B transgenic mice. This demonstrates that a low NR2B:NR2A ratio may be beneficial for some aspects of hemorrhagic stroke recovery.