Molecular mechanisms underlying presynaptic plasticity...

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    Molecular mechanisms underlying presynaptic plasticity: characterization of the RIM1α and

    SV2A interactome

    Dissertation

    zur

    Erlangung des Doktorgrades (Dr. rer. nat.)

    der

    Mathematisch-Naturwissenschaftlichen Fakultät

    der

    Rheinischen Friedrich-Wilhelms-Universität Bonn

    vorgelegt von

    Ana-Maria Oprişoreanu

    aus

    Târgovişte, Rumänien

    Bonn 2014

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    Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn

    1. Gutachter Prof. Dr. Susanne Schoch

    2. Gutachter Prof. Dr. Albert Haas

    Tag der Promotion: 13.01.2015

    Erscheinungsjahr: 2015

    Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn unter http://hss.ulb.uni-bonn.de/diss_online electronisch publiziert.

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    Erklärung

    Diese Dissertation wurde im Sinne von § 4 der Promotionsordnung vom 17.06.2011 am

    Institut für Neuropathologie und Klinik für Epileptologie der Universität Bonn unter der

    Leitung von Frau Prof. Dr. Susanne Schoch angefertigt.

    Hiermit versichere ich, dass ich die vorliegende Arbeit selbständig angefertigt habe und keine

    weiteren als die angegebenen Hilfsmittel und Quelle verwendet habe, die gemäß § 6 der

    Promotionsordnung kenntlich gemacht sind.

    Bonn, den

    Ana-Maria Oprişoreanu

       

  • Table of contents  

    IV   

    1.Introduction .......................................................................................................................... 1

    1.1 The synapse ....................................................................................................................... 1 1.2 Cytometrix at the active zone (CAZ) ................................................................................ 1

    1.2.1 Active Zone Ultrastructure ....................................................................................... 1 1.2.2 Active Zone composition ......................................................................................... 3

    1.3 The synaptic vesicle cycle ................................................................................................. 4 1.4 Synaptic plasticity .............................................................................................................. 5

    1.4.1 Presynaptic dormancy .............................................................................................. 6 1.4.2 Molecular mechanisms involved in presynaptic LTP .............................................. 7

    1.5 Two major players in synaptic plasticity ........................................................................... 7 1.5.1 RIMs ......................................................................................................................... 8

    1.5.1.1 RIM gene structure ...................................................................................... 8 1.5.1.2 RIM protein structure and binding partners................................................. 9 1.5.1.3 RIM function ............................................................................................. 11

    1.5.1.3.1 RIM in invertebrates (C.elegans and D.melanogaster) ................ 11 1.5.1.3.2 RIM in vertebrates (M.musculus) .................................................. 12

    1.5.1.3.2.1 RIM1α knock-out mice ..................................................... 12 1.5.1.3.2.2 RIM1αβ double knock-out mice ....................................... 13 1.5.1.3.2.3 RIM2α knock-out mice ..................................................... 13 1.5.1.3.2.4 RIM1α/RIM2α double knock-out mice ............................ 13 1.5.1.3.2.5 RIM conditional knockout mice ....................................... 14

    1.5.2 Synaptic vesicle protein 2A (SV2A) ...................................................................... 15 1.5.2.1 SV2A function ........................................................................................... 15 1.5.2.2 SV2A knock-out mice ............................................................................... 16

    1.6 Aim of the study ............................................................................................................... 17 2. Materials .............................................................................................................................. 18

    2.1 Equipment ....................................................................................................................... 18 2.2 Chemicals ......................................................................................................................... 19 2.3 Cell culture media ............................................................................................................ 20 2.4 Kits .................................................................................................................................. 20 2.5 Enzymes .......................................................................................................................... 20 2.6 Inhibitors ......................................................................................................................... 20 2.7 Diverse materials ............................................................................................................. 20 2.8 Cloning primers ................................................................................................................ 21 2.9 Sequencing primers .......................................................................................................... 22 2.10 Site-directed mutagenesis ............................................................................................... 22 2.11 Oligonucleotides used for HA-tag cloning ................................................................... 22 2.12 Oligonucleotides used for shRNA cloning ................................................................... 22 2.13 Generated constructs ...................................................................................................... 23 2.14 Plasmids obtained from other sources and used in this thesis ....................................... 23 2.15 Primary and secondary antibodies ................................................................................. 24

    3. Methods ............................................................................................................................... 25 3.1 Molecular Biology ........................................................................................................... 25

  • Table of contents  

    V   

    3.1.1 RNA extraction and cDNA synthesis .................................................................... 25 3.1.2 Polymerase chain reaction (PCR) .......................................................................... 25 3.1.3 Site directed mutagenesis ....................................................................................... 25 3.1.4 Sequencing ............................................................................................................. 26 3.1.5 Cloning technique .................................................................................................. 26

    3.1.5.1 Oligonucleotides cloning .......................................................................... 26 3.2 Cell Culture ..................................................................................................................... 26

    3.2.1 HEK (AAV) 293T cell culture ............................................................................... 26 3.2.2 HEK (AAV) 293T transfection methods .............................................................. 27

    3.2.2.1 Ca2+ -phosphate method ............................................................................. 27 3.2.2.2 Lipofectamine method ............................................................................... 27

    3.2.3 Neuronal primary cell culture ................................................................................ 27 3.2.3.1 Generation of primary cell culture ............................................................. 27 3.2.3.2 Transfection of neurons ............................................................................. 28 3.2.3.3 Infection of neurons ................................................................................... 28

    3.3 Virus Production .............................................................................................................. 28 3.3.1 rAAV serotype 1/2 and 8 production (Ca2+-phosphate method) ........................... 28 3.3.2 rAAV serotype 8 purification ............................................................................... 29 3.3.3 P0-P3 animal injection ........................................................................................... 29

    3.4 Biochemistry ................................................................................................................... 30 3.4.1 Preparation of crude synaptosomes ....................................................................... 30 3.4.2 Protein-protein interaction assays .......................................................................... 30

    3.4.2.1 Protein induction and purification from BL21 bacteria ..............