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CHARACTERIZATION of a NOVEL ISOFORM of NOS1AP: Nos1apc

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CHARACTERIZATION of a NOVEL ISOFORM of NOS1AP: Nos1apc CHARACTERIZATION OF A NOVEL ISOFORM OF NOS1AP: NOS1APc by Michael O’Brien Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia March 2011 © Copyright by Michael O’Brien, 2011 DALHOUSIE UNIVERSITY DEPARTMENT OF PHARMACOLOGY The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled “CHARACTERIZATION OF A NOVEL ISOFORM OF NOS1AP: NOS1APc” by Michael O’Brien in partial fulfillment of the requirements for the degree of Master of Science. Dated: March 29, 2011 Supervisor: _________________________________ Readers: _________________________________ _________________________________ _________________________________ ii DALHOUSIE UNIVERSITY DATE: March 29, 2011 AUTHOR: Michael O’Brien TITLE: CHARACTERIZATION OF A NOVEL ISOFORM OF NOS1AP: NOS1APc DEPARTMENT OR SCHOOL: Department of Pharmacology DEGREE: MSc CONVOCATION: May YEAR: 2011 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights, and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author iii TABLE OF CONTENTS LIST OF FIGURES…………………………………………………………………….. viii ABSTRACT……………………………………………………………………………... ix LIST OF ABREVIATIONS AND SYMBOLS USED……………………………….... x ACKNOWLEDGEMENTS……………………………………………………………… xiii CHAPTER 1 INTRODUCTION………………………………………………………. 1 1.1 NOS1AP…………………………………………………………………… 1 1.1.1 NOS1AP Domain Architecture………………………………….. 1 1.1.2 NOS1AP as an nNOS-targeting Protein……………………….. 3 1.1.3 NOS1AP and nNOS Function through the Formation of Two Separate Ternary Complexes……………... 4 1.1.4 NOS1AP Associates with a Scribble, Git1, β-Pix, PAK Complex to Regulate Dendritic Spine Morphology……... 7 1.1.5 NOS1AP Isoforms and Disease Linkage………………………. 9 1.2 NOS1AP within the CNS…………………………………………………. 11 1.2.1 Components of a synapse………………………………………. 11 1.2.2 Actin Dynamics …………………………………………………... 12 1.2.3 Dendritic Nucleation Model……………………………………… 13 1.2.4 Rho GTPases in Actin Dynamics ……………………………… 15 iv 1.3 The Cerebellum…………………………………………………………… 17 1.3.1 Anatomy and Circuitry…………………………………………….. 18 1.3.2 NO Activity in the Cerebellum: 20 Implications in Schizophrenia……………………………………. 1.3.3 The Cerebellum in Schizophrenia: 21 Human Studies …………………………………………………... 1.3.4 Schizophrenia: Synaptic Implications with the Cerebellum ……………………………………………………….. 22 1.4 Glial Cells ………………………………………………………………... 23 1.4.1 Astrocyte Characterization and Identification………………….. 24 1.4.2 Astrocytes and NO Signaling……………………………………. 25 1.5 The Cell Cycle……………………………………………………………. 27 1.5.1 Phases of the Cell Cycle………………………………………….. 28 1.5.2 Cyclins and Cyclin-dependant Protein Kinases………………… 29 1.5.3 Scribble negatively regulates Cell Cycle Progression…………. 30 1.6 Summary and Rationale………………………………………………… 32 1.7 Hypothesis………………………………………………………………... 33 1.8 Objectives………………………………………………………………… 33 CHAPTER 2 METHODS………………………………………………………… 34 2.1 Cell Culture……………………………………………………………….. 34 2.2 Northern Blotting for NOS1APc RNA………………………………….. 34 2.2.1 Isolation of Tissue…………………………………………………. 34 2.2.2 Isolation of Cell Lines……………………………………………… 34 2.2.3 Isolation of RNA from Animal Tissues and Cell Lines…………. 35 2.2.4 Nothern Blotting……………………………………………………. 36 v 2.2.5 Generation of a NOS1APc-specific cDNA probe………………. 37 2.2.6 Labeling of cDNA Probes and Hybridization……………………. 38 2.3 In Situ Hybridization……………………………………………………… 39 2.3.1 Tissue Preparation………………………………………………… 39 2.3.2 Generation of NOS1APc-specific RNA probes…………………. 40 2.3.3 Hybridization and Detection………………………………………. 41 2.4 Cortical Astroglia Cultures………………………………………………. 42 2.5 BrdU labeling……………………………………………………………... 44 2.6 Antibodies………………………………………………………………… 45 2.6.1 Generation of a NOS1APc-specific GST-fusion Antibody…….. 45 2.6.2 Generation of a NOS1APc-specific peptide Antibody…………. 47 2.7 NOS1APc Immunohistochemistry……………………………………… 48 2.8 NOS1APc Immunoprecipitation and Western Blotting……………….. 49 CHAPTER 3 RESULTS………………………………………………………… 51 3.1 Identification of NOS1APc……………………………………………… 51 3.2 Northern Blotting confirms the existence of a novel 52 NOS1APc isoform……………………………………………………….. 3.3 In situ hybridization: NOS1APc is enriched in the adult cerebellum………………………. 55 3.4 NOS1APc protein is expressed in the adult rat brain………………... 57 3.5 Characterization of various NOS1APc antibodies …………………... 60 3.6 NOS1APc is enriched in the cerebellum ……………………………... 62 3.7 NOS1APc is located in the nucleus of a subpopulation of 63 proliferating astrocytes………………………………………………….. 66 vi 3.8 NOS1APc localizes to the nucleus of cycling cells…………………... CHAPTER 4 DISCUSSION…………………………………………………… 70 4.1 Summary of major findings…………………………………………….. 70 4.2 NOS1APc is a legitimate NOS1AP isoform distributed throughout multiple tissues ……………………… 70 4.3 NOS1APc is a NOS1AP isoform ……………………………………… 71 4.4 NOS1APc is widely distributed in the CNS…………………………… 71 4.4.1 NOS1APc mRNA………………………………………………….. 71 4.4.2 NOS1APc protein…………………………………………………. 72 4.5 Identification of novel NOS1APc isoforms?………………………….. 73 4.6 NOS1AP isoforms in the nucleus……………………………………… 75 4.7 NOS1AP isoforms in the cell cycle……………………………………. 76 4.8 NOS1AP isoforms: nuclear nitric oxide regulators?.......................... 76 4.9 NOS1AP isoforms in disease………………………………………….. 77 4.9.1 Schizophrenia……………………………………………………… 77 4.9.2 Long Q/T syndrome and diabetes……………………………….. 78 CHAPTER 5 CONCLUSION…………………………………………………. 80 REFERENCES…………………………………………………………………………. 81 vii LIST OF FIGURES Figure 3.1 Complete peptide sequence of NOS1APc………………………… 53 Figure 3.2 Identification of NOS1APc transcript through Northern blot…….. 54 Figure 3.3 Localization of NOS1APc mRNA in the 56 hippocampus and Cortex of adult rat …………………………….. Figure 3.4 Localization of NOS1APc mRNA in adult Rattus cerebellum…… 58 Figure 3.5 Localization of NOS1APc mRNA in adult Rattus brainstem…….. 59 Figure 3.6 Characterization of various NOS1APc antibodies………………... 61 Figure 3.7 NOS1APc is localized in the molecular layer of the cerebellum…. 64 Figure 3.8 NOS1APc is localized in the nucleus of glial cells………………. 65 Figure 3.9 NOS1APc is confirmed in the nucleus of glial cells..…..………… 67 Figure 3.10 NOS1APc is confirmed in the nucleus of astroglia………………. 68 Figure 3.11 NOS1APc is expressed in the nucleus of a subpopulation of 69 proliferating astroglial cells ………………………………………. viii ABSTRACT The current study characterizes a novel isoform of the Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP), herein NOS1APc. NOS1APc was identified in a proteomic screen for Scribble interacting proteins. Northern blot analysis revealed a 7kb transcript that was present in a number of different tissues and cell lines. Highest levels were detected in the cerebellum. In situ hybridization studies revealed NOS1APc mRNA throughout the cortex and hippocampus. In addition, cerebellar Purkinje cells and different brainstem nuclei also contained NOS1APc mRNA. Antibodies directed against NOS1APc revealed a 100 kDa protein, while immunohistochemical staining revealed high levels of this protein within the molecular layer of the cerebellum. Finally, immunocytochemical studies using isolated primary astrocytes revealed a subset of BrdU positive nuclei that co-expressed NOS1APc, suggesting that this protein may function in some capacity in cell-cycle progression. ix LIST OF ABBREVIATIONS AND SYMBOLS USED μm micrometer [α- 32P]dCTP [alpha-32Phosphorus]Deoxycytidine 5'-triphosphate β-Pix β-p21-activated kinase interacting exchange factor ADP Adenosine diphosphate AMPAR α-amino-3-hydroxyl-5-methyl-4-isoxazote-propionic acid receptor AP Alkaline Phosphatase APC Adenomatous polyposis coli ARF ADP-ribosolation factor Arp2/3 Actin-related protein 2 and 3 Asp Asparagine ATP Adenosine triphosphate BCIP bromo-4-chloro-3-indolyl-phosphate BrdU 5-bromo-2-deoxyuridine CAPON Carboxyl-terminal PDZ ligand of nNOS CAZ Cytoskeletal matrix at the active zone Cdc42 Cell division cycle 42 Cdk Cyclin-dependant protein kinase cDNA complimentary DNA CFA Completet Freunds Adjuvant cGMP cyclic Guanosine Monophosphate CKI Cyclin-dependant kinase inhibitor CNS Central nervous system CP Capping Protein C-terminal Carboxyl-terminal DAB Diaminobenzene DEPC diethylpyrocarbonate Dexras1 Dexamathasone induced Ras DIG Digoxigenin Dlg Discs-Large DMEM Dulbecco’s Modified Eagles Medium DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid eNOS endothelial Nitric Oxide Synthase Erc Extracellular signal-related kinase F-actin Filamentous actin FBS Fetal Bovine Serum FMOC 9H-fluoren-9-ylmethoxycarbonyl fMRI functional magnetic resonance imaging G-actin monomeric actin GAP GTPase activating protein GC Guanylyl Cyclase x GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GFAP Glial fibrillary acidic protein GFP Green Fluorescent Protein Git1 G-protein coupled receptor kinase interacting protein1 GST Glutatione-S-Transferase
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