Organization of the Nerve Terminal by Synaptic Cleft Components
(Hiroshi Nishimune, Ph.D., P.I.)
Preimplantation Embryonic Secreted/Released Proteins: Embryo Quality Predictors
(Lane Christenson, Ph.D., PI)
Genetic Models of Congenital Vascular Malformations
(Jay Vivian, Ph.D., P.I.)
Regulation of Gene Expression in the TH2 Cytokine Locus
(Patrick E. Fields, Ph.D., P.I.)
Germ Cell Development in the atrichosis Mutant Mouse
(T. Rajendra Kumar, Ph.D., P.I.)
Transcriptional Mechanisms of Endothelial Function and Differentiation
(Soumen Paul, Ph.D., P.I.)
Functional analysis of histone demethylase activity in hypoxic cancer cells
(Adam Krieg, Ph.D., P.I.)
Targeting and regulation of O-GlcNAc transferase at M phase
(Chad Slawson, Ph.D., P.I.)
Impact of early experience on vulvovaginal sensitivity in adult mouse
(Julie Christianson, Ph.D., P.I.)
Molecular mechanism of THM1-mediated renal cystogenesis
(Pamela Tran, Ph.D., P.I.)
Role of cytoskeletal protein SPECC1L in facial morphogenesis and facial clefting
(Irfan Saadi, Ph.D., P.I.)
Prachee Avasthi, Ph.D., Assistant Professor, Department of Anatomy and Cell Biology “Cell Biology of ERK-Cilium Crosstalk”
Cilia and flagella are nearly ubiquitous organelles that protrude from the cell surface and perform functions related to signal transduction and motility. They consist of a microtubule core surrounded by a specialized extension of the plasma membrane. Ciliary microtubules are extensions of the basal body, which is derived from the mother centriole. The same centriole is a component of the spindle during cell division. Cilium formation is therefore tightly coupled to the cell cycle. While ciliogenesis and cell proliferation appear inextricably linked by their shared and mutually exclusive requirement for the centriole, the molecular mechanisms that mediate the balance between the two processes are unknown. Dr. Avasthi’s preliminary data using the flagella of the unicellular green alga Chlamydomonas reinhardtii as a model system show that regulation of extracellular signal regulated kinase (ERK) signaling can tune ciliary length and actin phosphorylation. Based on this and her previous finding demonstrating a requirement of actin dynamics in flagellar assembly, she proposes a model by which the ERK pathway modulates ciliary length via actin modification and redistribution. To test this model, her lab will 1) determine if ERK signaling is necessary and sufficient for flagellar disassembly, 2) determine the role of actin phos-phorylation in actin-mediated flagellar dynamics, and 3) test the effects of flagellum associated ERK signaling on actin distribution in Chlamydomonas. Functions of cilia include light sensing in photoreceptors, fluid flow detection in renal cilia, and mucus movement in tracheal cells. Defects in these functions result retinal degeneration, polycystic kidney disease, and primary ciliary dyskinesia respectively. Collectively, the diseases and pleiotropic disorders involving cilium dys-function are termed ciliopathies. Additionally, up to one-third of human cancers can be traced to mutations in the ERK pathway. Identifying mechanisms of crosstalk between ERK signaling and ciliogenesis will yield novel points of therapeutic intervention for ciliopathies as well as cancer.
Pinelopi Kapitsinou, M.D., Assistant Professor, Department of Internal Medicine “Oxygen sensing and endothelial metabolism in ischemic kidney injury.”
Key regulators of cellular adaptation to hypoxia are Hypoxia-Inducible-Factors (HIF)-1 and -2, basic-helixloop-helix transcription factors, whose stability is regulated by the PHO family of oxygen-dependent prolyl hydroxylases . Up-regulation of HIF in different models of ischemic injury such as limb ischemia, ischemic myocardium and renal ischemia-reperfusion injury (IRI) decreases post-ischemic inflammation and organ dysfunction. However, with regard to the cytoprotection, the cell specific responses regulated by HIF remain poorly understood. Endothelial cells are important players in the pathogenesis of IRI and express both HIF isoforms. Inactivation of HIF-1 and HIF-2 in endothelial cells augmented kidney injury following renal IRI in mice lacking HIF in endothelial cells, which was associated with increased expression of endothelial adhesion molecules and pronounced inflammatory response. Furthermore, disruption of endothelial cell derived HIF-2 abolished the cytoprotection afforded by pharmacologic inhibition of HIF prolyl-hydroxylation. These findings suggest that endothelial HIF-2 signaling plays a critical role in the context of ischemic injury and hypoxic preconditioning but the underlying mechanism remains unclear. The goal of the proposed research is to investigate the role of endothelial Phd2/HIF-2 signaling pathway in endothelial cell metabolism and its effects on post-ischemic kidney injury and repair. In the first aim, the effect of HIF on endothelial cell metabolism will be explored by performing comprehensive metabolic profiling studies. In the second aim, the role of the endothelial PHD2/HIF-2 axis on post-ischemic kidney injury will be examined by using transgenic mice in which endothelial HIF-2 is activated by PHD2 loss. In the third aim, the contribution of endothelial PHD2/HIF-2 signaling in post-ischemic kidney repair will be elucidated by utilizing a transgenic model of inducible recombination in the endothelium . Furthermore, the lab will employ endothelial cell function assays (migration, tube formation) to investigate the effect of PHD2/HIF-2 signaling on angiogenic responses and metabolic analyses to dissect the contribution of metabolic reprogramming in these functional effects.
Mary Markiewicz, Ph.D., Assistant Professor, Department of Microbiology, Molecular Genetics, and Immunology “The Role of NKG2D in Autoimmune Diabetes.”
There is conflicting data as to the importance of signaling through the NKG2D receptor on immune cells during the development of autoimmune diabetes. Genetic linkage studies demonstrate an association with polymorphism in the gene encoding a human ligand for NKG2D and autoimmune, or type 1, diabetes. Therefore, it is important to clearly understand the role NKG2D-NKG2D ligand interaction may have in the development of this disease. Preliminary data utilizing the best animal model of type 1 diabetes, the nonobese diabetic (NOD) mouse, generated the hypothesis that NKG2D signaling affects autoimmune diabetes development by two opposing mechanisms. First, engagement of NKG2D on immune cells by NKG2D ligands expressed in the pancreatic islets may enhance the immune response within the pancreas and increase diabetes development. Second, NKG2D signaling may alter the composition of the commensal microbiota, which is a critical factor in diabetes development, in a way that protects against disease. This proposal describes experiments designed to directly test whether one or both of these mechanisms are involved in diabetes development in the NOD mouse. Specific Aims 1-3 will address the first hypothesis using a variety of novel mouse models. In Specific Aim 1, NKG2D ligand expression in pancreatic islets during diabetes development in NOD mice will be thoroughly characterized. In Specific Aim 2, a novel transgenic mouse strain will be used to determine whether constitutive expression of a murine NKG2D ligand in pancreatic islets alters diabetes development in NOD mice. Conversely, in Specific Aim 3 the lab will determine whether expression of the NKG2D receptor is required for diabetes development in NOD mice. In Specific Aim 4, she will test the second hypothesis that NKG2D expression alters diabetes development via effects on the intestinal microbiota composition. At the conclusion of these studies, results should show whether engagement of NKG2D on immune cells by NKG2D ligands expressed inside or outside the pancreatic islets is critical for autoimmune diabetes in the NOD mouse, the best model of type 1 diabetes.
Rajasingh Johnson, Ph.D., Assistant Professor, Department of Internal Medicine “Molecular Regulation and Differentiation of iPSCs into Cardiomyocytes.”
Noonan syndrrome (NS) is an inheritable relatively common autosomal dominant congenital disease and seen in both male and females with the incidence of 0.1%. The incidence of cardiac abnormalities is higher and approximately 80% of patients with NS. Successful generation of induced pluripotent stem cells (iPSCs), differentiation towards cardiomyocytes from NS patient’s fibroblast and high-throughput screening of cells provide hope for identifying the biological mechanisms of diseases. These advances have been instrumental in helping understand rare disorders, and reassure parents and patients by identifying a biologic basis for these diseases. The objective of this proposal is to use the patient materials to create the cellular model for human rare disease NS. This model will allow Dr. Johnson to more clearly determine if an identified variant causes the clinical phenotype and providing information about the cellular and molecular pathogenesis. Studies have shown that iPSC-derived CMCs represent a potentially renewable source of cells for therapy and also for disease modeling and drug discovery. He has already generated a non-viral, efficient, safe and reliable iPSCs and subsequently differentiated into functional CMCs from normal cells, and the manuscript is under review (Unpublished manuscript). His main goals are to generate clinically safe iPSCs and iPSC-derived CMCs from NS-patient cells, and identify the genetic basis of the NS and understand the cellular and biochemical pathologies. He will use the genomic editing tool of Clustered regularly interspaced short palindromic repeat (CRISPR) technology to repair the defect in NS-iPSCs and introduce mutant p.Arg522Gly into normal iPSCs. This will allow his lab to understand and study the importance of genetic variant during NS disease prognosis. He hypothesizes that the CMC-derived from NS corrected iPSCs behave like normal iCMCs and the introduction of NS mutation in normal iPSC-derived iCMCs behave like natural mutated CMCs. He plans to test this hypothesis by pursuing the following two aims: Specific Aim 1. Generation of safe and efficient iPSCs from NS-patients fibroblasts. SpecificAim 2. Gene Correction, differentiation of iPSCs derived from NS-iPSCs into NS-iCMCs and analyzing its functional properties.
Alan Yu, M.D., Professor of Internal Medicine “Protective Role of tight junctions in glomerular podocytes.”
The broad, long-term objective of this proposal is to understand the mechanisms of glomerular podocyte injury and develop novel therapies for glomerular diseases. The glomerular barrier is formed by slit diaphragms, which are unique intercellular junctions with similarities to adherens junctions. Impairment of the glomerular barrier leads to proteinuria and nephrotic syndrome. Interestingly, tight junction proteins are also expressed in the podocyte but do not localize at intercellular sites. During podocyte injury, the slit diaphragm is lost, foot processes become effaced and closely opposed to each other, and tight junction proteins condense to form bona fide tight junctions. Why tight junctions appear during podocyte injury and what role they play is completely unknown. Dr. Yu hypothesizes that podocyte tight junctions function to attach neighboring podocytes and anchor them to the epithelial monolayer, thereby protecting individual podocytes from detachment. To test this hypothesis, he proposes to generate a podocyte-specific conditional knockout of claudin-5 in mice. Claudin-5 is the principal claudin expressed in podocytes and is likely to be a critical component of podocyte tight junctions. He therefore predicts that these mice will either develop nephrotic syndrome spontaneously or exhibit increased susceptibility to podocyte injury. In Aim 1, his lab will generate podocyte-specific and tamoxifen-inducible claudin-5 knockout mice. He will use CRISPR/Cas9 technology to generate claudin-5 floxed mice and cross them to a podocin promoter-driven tamoxifen-inducible Cre transgenic mouse. In Aim 2, he will analyze the mice for evidence of nephrosis, and test their susceptibility to adriamycin-induced injury in vivo, and to acute adriamycin- and TNF -induced injury in an in vitro glomerular permeability assay. This pilot grant will make use of two COBRE-supported cores (Transgenic Core and EM Lab) and generate a mouse model and provide the preliminary data needed to support an R01 proposal aimed at understanding the underlying mechanisms by which podocyte tight junctions protect against glomerular injury.
Funded by NIH grant 9P20GM104936 from the National Institute of General Medical Sciences.