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Identification of Neurotrypsin-Dependent Changes in Synapses and Hippocampus Proteome

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Identification of Neurotrypsin-Dependent Changes in Synapses and Hippocampus Proteome Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2007 Identification of neurotrypsin-dependent changes in synapses and hippocampus proteome Walther, Pascal Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163682 Dissertation Published Version Originally published at: Walther, Pascal. Identification of neurotrypsin-dependent changes in synapses and hippocampus pro- teome. 2007, University of Zurich, Faculty of Science. Identification of Neurotrypsin-dependent Changes in Synapses and the Hippocampus Proteome DISSERTATION zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftliche Fakultät der UNIVERSITÄT ZÜRICH von Pascal Walther von Wohlen (BE) Promotionskomitee Prof. Dr. Peter Sonderegger (Vorsitz und Leitung) Prof. Dr. Ruedi Aebersold Dr. Detlev Boison Zürich 2007 Die vorliegende Arbeit wurde von der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich auf Antrag von Prof. Dr. Peter Soneregger und Prof. Dr. Heinz Gehring als Dissertation angenommen. Abbreviations 2DE 2D gel electrophoresis aa amino acid ACN acetonitrile AD Alzheimer’s disease AMPA α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid α-CCA α-cyano-4-hydroxycinnamic acid AK adenosine kinase ASAPRatio automated statistical analysis of protein abundance ratio CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CID collision-induced dissociation CNS central nervous system DAB diaminobenzidine tetrahydrochloride DTT DL-Dithiothreitol EAAT2 excitatory amino acid transporter 2 ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EtOH ethanol ER endoplasmatic reticulum Erk extracellular regulated kinase ESI-MS electrospray ionization mass spectrometry FT ICR Fourier transform ion cyclotron resonance GO gene ontology GFAP glial fibrillary acidic protein GluR2 glutamate receptor 2 Grp glucose-regulated protein GS glutamine synthetase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hNT human neurotrypsin HRP horseradish peroxidase ICAT isotope-coded affinity tag IEF isoelectric focusing IgG immunoglobulin IPG immobilized pH gradient kDa kilo Dalton KO knock-out L-LTP late long-term potentiation LTD long term depression µLC microcapillary liquid chromatography MALDI-TOF matrix-assisted laser disorption/ionization time-of-flight MAPK mitogen-activated protein kinase MPT minimal probability threshold mNT mouse neurotrypsin MetOH methanol m/z mass-to-charge ratio NMJ neuromuscular junction NMDAR N-methyl-D-aspartic acid receptor PAR protease-activated receptor PBS phosphate buffered saline PAK1 p21-activated kinase 1 PCR polymerase chain reaction PKC protein kinase C PSD post-synaptic density PVDF polyvinylidene fluoride ppm parts per million RF radio frequency RP reversed phase SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SCX strong cation exchange SIC single ion chromatogram TAMRA carboxytetramethylrhodamine TBS Tris buffered saline TCEP tris(2-carboxyethyl)phosphine hydrochloride TFA triflouracetic acid tPA tissue-type plasminogen activator uPA urokinase-type plasminogen activator VDAC voltage-dependent anion channel Table of contents AbbreviAtions 1 summAry 7 ZusAmmenfAssung 8 1. introduction 9 1.1 Proteolytic enzymes 9 1.1.1 Serine peptidases 9 1.1.2 S1 family peptidases and their catalytic mechanism 10 1.2 Peptidases are capable to modulate brain function 11 1.2.1 Plasminogen activators play a crucial role in the CNS 12 1.2.1.1 Tissue-type plasminogen activator 12 1.2.1.2 Urokinase-type plasminogen activator 16 1.2.2 tPA modulates neurotransmission and synaptic plasticity 16 1.2.2.1 tPA interacts with NMDA receptors 16 1.2.2.2 tPA induces late-LTP through the association with LRP-1 17 1.2.2.3 tPA facilitates late-LTP via the dopaminergic system 18 1.2.2.4 tPA is involved in drug addiction 19 1.2.3 Plasminogen in the brain – a versatile peptidase activated by tPA and uPA 19 1.2.4 Thrombin is involved in brain development 20 1.2.4.1 Proteolytic activation of thrombin receptors 21 1.2.4.2 Thrombin elicits proliferative and pathophysiological changes in the brain 22 1.2.4.3 Activated PAR-1 signaling in the CNS 23 1.2.5 Neuropsin – a neural activity-dependent serine peptidase 24 1.2.6 Calpains act in the pre- and postsynaptic boutons 25 1.3 Neurotrypsin – a brain specific extracellular serine peptidase 26 1.3.1 Neurotrypsin is a trypsin-like serine peptidase 26 1.3.2 The role of neurotrypsin in the CNS – some evidence for a function in synaptic plasticity 28 1.3.3 Cleavage of agrin by co-expression with neurotrypsin in cell culture and in vivo in the hippocampus 30 1.4 Agrin – an extracellular matrix heparan sulfate proteoglycan 31 1.4.1 Alternative splicing of agrin 31 1.4.2 Agrin is an important organizer of the neuromuscular junction 32 1.4.3 The function of agrin in the brain 33 1.5 mass spectrometry and protein analysis 35 1.5.1 Quadrupole ion trap vs. Fourier Transform Ion Cyclotron Mass Spectrometry 36 1.5.2 Interpretation of MS/MS spectra 38 1.5.3 MS analysis of partially purified proteins 40 1.5.4 Strategies for quantitative peptide analysis 41 1.5.5 Isotope-coded affinity tag (ICAT) 43 2. Aim of this work 45 3. Results 46 3.1 Neurotrypsin in the mouse brain 46 3.1.1 Neurotrypsin is expressed in synaptosomes and the hippocampus 46 3.1.1.1 Isolation of synaptosomes 46 3.1.1.2 Subcellular fractionation of the hippocampus 47 3.1.1.3 Detection of neurotrypsin in wild-type and overexpressing mouse lines 47 3.2 Neurotrypsin cleaves agrin in cell culture and in the CNs 49 3.2.1 Agrin processing by neurotrypsin in synaptosomes and in the hippocampus 49 3.3 Proteomic profiling of synaptosomes and the hippocampus comparing wild-type and transgenic mice 51 3.3.1 Sample validation for the proteomic profiling 52 3.3.2 Isotope-coded affinity tag (ICAT) labeling and multidimensional peptide separation 52 3.3.3 Quantitative proteome analysis of synaptosomes from P20 and P40 mice using the ICAT method 53 3.3.3.1 Data filtering with Peptide- and ProteinProphet (I) 55 3.3.4 Quantitative proteomic profiling of the hippocampus from P10 mice using ICAT 56 3.3.4.1 Data filtering with Peptide- and ProteinProphet (II) 58 3.3.4.2 Visual inspection and comparison of data sets obtained from repeated MS runs 59 3.3.4.3 Computational analysis of peptide identifications 61 3.3.5 Categorization of total protein identifications from synaptosomes and the hippocampus 62 3.4 Differentially regulated proteins in synaptosomes and the hippocampus 64 3.4.1 Neurotrypsin-dependent changes of protein abundance in synaptosomes and the hippocampus proteome 64 3.4.2 Quantitative proteomic profiling using 2D gel electrophoresis 72 3.4.3 Regulatory Changes of molecular functions 74 3.4.3.1 Cytoskeleton reorganization, cell proliferation/differentiation 74 3.4.3.2 Cell adhesion 75 3.4.3.3 Synaptic transmission and synaptic plasticity 76 3.4.3.4 Exocytosis 78 3.4.3.5 Endocytosis 79 3.4.3.6 Neuroendocrine, synapse remodeling, and neurotransmitter clearance 80 3.4.3.7 Metabolic processes 81 3.4.3.8 Neuron migration and differentiation of the nervous system 83 3.4.3.9 Apoptosis 83 3.4.3.10 Varia 83 3.4.4 GO annotation of differentially expressed proteins 84 3.5 Verification of candidate proteins 86 3.5.1 Verification of three candidates found in synaptosomes 86 3.6 Verification of regulated proteins found in the hippocampus 91 3.6.1 Copine VI (N-copine) is a Ca2+ sensor in postsynaptic events 91 3.6.2 Glutamine synthetase is responsible for the conversion of L-glutamate 93 3.6.3 Neuron specific alpha3 Na+/K+-ATPase is essential for the excitability of nerve cells 95 3.6.4 Differential protein expression of various other proteins 98 4. discussion 99 4.1 Neurotrypsin – Agrin interaction in synaptosomes and in the hippocampus 99 4.2 Quantitative proteomic profiling of a transgenic mouse line 100 4.3 Multifaceted neurotrypsin-dependent changes in protein expression 102 4.4 Pros and Cons of two proteomic approaches to investigate brain proteins of a transgenic mice 106 5. MAteRiAl AND MethoDs 108 5.1 subcellular fractionation 108 5.1.1 Isolation of synaptosomes 108 5.1.2 Hippocampus fractionation 108 5.2 isotope-coded Affinity tag (iCAt) 108 5.3 Mass spectrometry (Ms) 109 5.3.1 Reversed-Phase microcapillary Liquid Chromatography Electrospray Ionization Mass Spectrometry (RP-µLC ESI-MS) 109 5.3.2 Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) 110 5.3.3 Protein identification and quantification 110 5.3.3.1 Mascot search engine 110 5.3.3.2 SEQUEST search algorithm 110 5.3.3.3 Peptide and Protein Probability Assignment 111 5.3.3.4 Automated Statistical Analysis on Protein Ratio (ASAPRatio) 111 5.3.3.5 XPRESS software 111 5.4 2D Gel electrophoresis 112 5.4.1 First Dimension: Isoelectric Focusing (IEF) 112 5.4.2 Second Dimension: SDS-PAGE 112 5.4.3 2D Gel Analysis software 112 5.4.4 Spot Excision, Digestion and Spotting on MALDI target plate 113 5.5 Antibodies 113 5.6 Gel electrophoresis and immunoblotting 114 5.6.1 SDS-PAGE 114 5.6.2 NuPAGE 4 – 12% Bis-Tris and 10 – 20% Tricine Gel 114 5.6.3 Immunoblotting 114 5.7 Cleavage of agrin by co-expression with neurotrypsin in hela cells 115 5.8 immunohistochemistry 115 5.9 Generation of transgenic mouse lines overexpressing neurotrypsin 116 5.10 Polymerase Chain Reaction (PCR) 118 5.10.1 PCR genotyping 118 5.10.2 Real-time PCR 118 6. RefeReNCes 120 Acknowledgments 134 Summary Summary Neurotrypsin is a multi-domain serine peptidase expressed in the developing and adult nervous system.
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