The role of ephrinB signaling during synaptic plasticity [Elektronische Ressource] / Clara Luise Essmann

Clara Luise Essmann

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Für Hans & Karin



The role of ephrinB signaling during synaptic
plasticity



Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München

Angefertigt am Max-Planck-Institut für Neurobiologie, Martinsried
in der Arbeitsgruppe „Signal Transduction“




Clara Luise Essmann
aus Freiburg i. Br.
München * 16. Dezember 2008
1. Gutachter: Prof. Dr. Rüdiger Klein
2. Gutachter: Prof. Harry MacWilliams
Tag der mündlichen Prüfung: 16. Juli 2009



















The work presented in this thesis was performed in the laboratory of Dr. Amparo
Acker-Palmer, Junior Group – Signal transduction, at the Max-Planck-Institute of
Neurobiology, Martinsried, Germany.

Erklärung

Ich versichere hiermit, dass ich die vorgelegte Dissertation „The role of ephrinB signaling
during synaptic plasticity“ selbständig und ohne unerlaubte Hilfe angefertigt habe. Ich habe
mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Hilfen und Quellen
bedient.
Hiermit erkläre ich, dass ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg
unterzogen habe. Die Dissertation wurde in ihrer jetzigen oder ähnlichen Form bei keiner
anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient.

München, den
(Unterschrift)


Table of Contents
1 Publications .................................................................................. 9
2 Abbreviations .............................................................................. 11
3 Summary ..................................................................................... 15
4 Introduction ............................................................................... 17
4.1 Synaptogenesis .......................................................................................... 17
4.2 Postsynaptic density .................................................................................. 20
4.3 Dendritic spines ........................................................................................ 22
4.4 From filopodia to spines ............................................................................ 23
4.5 Synaptic plasticity ..................................................................................... 26
4.5.1 LTP and LTD ............................................................................................... 27
4.6 AMPA receptors ........................................................................................ 30
4.6.1 AMPA-receptor trafficking ............................................................................... 32
4.7 PDZ-proteins ............................................................................................. 35
4.7.1 Glutamate receptor interacting protein (GRIP) ...................................................... 37
4.8 The Eph receptors and ephrin ligands ...................................................... 39
4.8.1 Classification and structure ............................................................................... 40
4.8.2 Eph/ephrin interaction .................................................................................... 46
4.8.3 Eph/ephrin signaling outside the nervous system .................................................... 50
4.8.4 Eph/ephrin signaling in the nervous system ........................................................... 50
5 Results ......................................................................................... 55
5.1 Grb4 and GIT1 transduce ephrinB reverse signals modulating spine
morphogenesis and synapse formation ................................................................. 55
5.1.1 EphrinB-reverse signaling promotes spine morphogenesis ......................................... 55
5.1.2 Interference with ephrinB-reverse signaling impairs spine formation ............................ 57
5.1.3 Spine morphogenesis downstream of ephrinB mediated via Grb4 and GIT1 .................... 62
5.2 Serine phosphorylation of ephrinB2 regulates trafficking of synaptic
AMPA receptors .................................................................................................... 68
5.2.1 EphrinB2 reverse signaling regulates AMPA-receptor trafficking ................................. 68
5.2.2 Lack of ephrinB2 leads to enhanced AMPA-receptor internalization and reduced synaptic
transmission ............................................................................................................. 73
5.2.3 GRIP molecules link ephrinB ligands to AMPA receptors .......................................... 78
5.2.4 GRIP molecules are required for ephrinB ligand-mediated AMPA-receptor stabilization .... 80
5.2.5 GRIP binding to ephrinB ligands is regulated by activation through EphB receptors .......... 85
5.2.6 Serine phosphorylation in ephrinB ligands regulates PDZ-interactions ........................... 86
5.2.7 Serine phosphorylation of ephrinB ligands regulates AMPA-receptor internalization ......... 90 Publications 1
6 Discussion ................................................................................... 95
6.1 Grb4 and GIT1 transduce ephrinB reverse signals modulating spine
morphogenesis and synapse formation ................................................................. 95
6.1.1 EphrinB ligands induce spine morphogenesis ......................................................... 95
6.1.2 Grb4 and GIT1 transduce ephrinB reverse signals ................................................... 98
6.2 Serine phosphorylation of ephrinB2 regulates trafficking of synaptic
AMPA receptors .................................................................................................. 100
6.2.1 GRIP as the bridging molecule .......................................................................... 103
6.2.2 PICK1 as a ephrin-AMPA receptor linker ............................................................ 105
6.2.3 GRIP binding to ephrinB regulated via serine phosphorylation ................................... 106
6.2.4 Serine phosphorylation of ephrinB ligands ............................................................ 107
6.2.5 How does ephrinB2 exert its function during synaptic plasticity? ................................ 108
6.2.6 Concluding remarks ...................................................................................... 109
7 Material and methods ................................................................ 111
7.1 Material ................................................................................................... 111
7.1.1 Chemicals, Reagents, Commercial Kits & Enzymes ................................................ 111
7.1.2 Antibodies .................................................................................................. 113
7.1.3 Consumable Material ..................................................................................... 115
7.1.4 Equipment .................................................................................................. 116
7.1.5 Oligonucleotides .......................................................................................... 119
7.1.6 Plasmids ..................................................................................................... 121
7.1.7 Cell lines and bacteria .................................................................................... 123
7.1.8 Primary cells and tissue ................................................................................... 123
7.1.9 Media and standard solutions ............................................................................ 124
7.1.10 Solutions and buffers for Western Blot analysis.................................................. 131
7.2 Methods ................................................................................................... 135
7.2.1 Molecular Biology ......................................................................................... 135
7.2.2 Cell culture ................................................................................................. 140
7.2.3 Biochemistry ............................................................................................... 143
7.2.4 Postsynaptic density fractionation ...................................................................... 149
7.2.5 Tandem affinity purification (TAP) and mass spectrometry ....................................... 149
7.2.6 Electrophysiology-patch-clamp recordings ........................................................... 149
7.2.7 Data analysis ................................................................................................ 150
8 Acknowledgements ................................................................... 153
9 Curriculum Vitae ....................................................................... 155
10 Bibliography .............................................................................. 157