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Expression and function of GDNF family ligands and their receptors by human immune cells [Elektronische Ressource] / vorgelegt von Vivian Vargas-Leal

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Expression and function of GDNF family ligands and their receptors by human immune cells Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Vivian Vargas-Leal Venezuela München, Dezember 2003 Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe habe. Ich habe weder anderweitig versucht, eine Dissertation oder Teile einer Dissertation einzureichen beziehungsweise einer Prüfungskommission vorzulegen, noch eine Doktorprüfung durchzuführen. München, Dezember 2003 Dissertation eingereicht: 18. Dezember 2003 Tag der mündlichen Prüfung: 2. April 2004 Erstgutachter: Prof. Dr. Elisabeth Weiß Zweitgutachter: Prof. Dr. Georg Dechant Acknowledgments This work has been performed under the support of the “Fundación Gran Mariscal de Ayacucho” from the government of Venezuela, which provided me with a fellowship for four years. I am extremely grateful to the Foundation for making my dreams possible. Thanks are due to the max-Planck Institute of Neurobiology, Martinsried, where I performed all of my investigation. I am indebted to Prof. Dr.

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Published 01 January 2003
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Expression and function of GDNF family
ligands and their receptors by human
immune cells









Dissertation
zur Erlangung des
Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München



vorgelegt von
Vivian Vargas-Leal
Venezuela




München, Dezember 2003





























Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig
und ohne unerlaubte Hilfe habe.
Ich habe weder anderweitig versucht, eine Dissertation oder Teile
einer Dissertation einzureichen beziehungsweise einer
Prüfungskommission vorzulegen, noch eine Doktorprüfung
durchzuführen.


München, Dezember 2003











Dissertation eingereicht: 18. Dezember 2003

Tag der mündlichen Prüfung: 2. April 2004



Erstgutachter: Prof. Dr. Elisabeth Weiß
Zweitgutachter: Prof. Dr. Georg Dechant











































Acknowledgments

This work has been performed under the support of the “Fundación Gran Mariscal de
Ayacucho” from the government of Venezuela, which provided me with a fellowship for
four years. I am extremely grateful to the Foundation for making my dreams possible.

Thanks are due to the max-Planck Institute of Neurobiology, Martinsried, where I
performed all of my investigation. I am indebted to Prof. Dr. Hartmut Wekerle, head of the
department of Neuroimmunology , and prof. Dr. Reinhard Hohlfeld, head of the Institute
of Clinical Neuroimmunology – LMU.

I would like to thank my family, specially my mother, for their unconditional support and
help in each moment of my life, whenever and wherever they had been. Without them I
could have not finished these studies.

I whish to thank:
- Prof. Dr. Edgar Meinl for his experimental advice and supervision, stimulating
discussions and for directing my thesis over many years.
- Prof. Dr. Leonardo Mateu (from Instituto Venezolano de Investigaciones
Científicas), who even from a great distance has always helped me in any
difficulty, giving his wise advice and great understanding.
- Prof. Dr. Georg Dechant for his discussion and suggestions for my work.
- Prof. Frau Dr. Elisabeth Weiß for supporting this thesis at the Ludwig-Maximilian
Universität of Munich.
- Many thanks for the help received from my colleagues from the department of
Neuroimmunology, in particular: Martina Sölch, Dr. Tobias Derfuß, Dr. Roxana
Bruno and Dr. Alexander Flügel.
- Also many thanks to my closest friends: Dr. Marta Labeur, Dr. Seiko Kataoka, Ms.
Ute Sukop, Dr. Eva Vonasek.
- Finally very special thanks to Dr. Enrico Marchetti for having supported me in all
my difficulties.

And of course, many thanks to God and his Angels: they have been always around me.


Index


1 INDEX
1 INDEX.....................................................................................................................................................1
2 SUMMARY.............................................................................................................................................5
3 INTRODUCTION..................................................................................................................................9
3.1. GDNF FAMILY LIGANDS ...........................................................................................................9
3.1.1. General description of GFLs .....................................................................................................9
3.1.2. GDNF-Family Ligand receptor complex .................................................................................10
3.2. INTERACTIONS BETWEEN THE CNS, PNS AND THE IMMUNE SYSTEM ............................................12
3.3. GDNF ...........................................................................................................................................14
3.3.1. Description and characteristics ...............................................................................................14
3.3.2. The GDNF gene .......................................................................................................................15
3.3.3. GDNF knock-out mice .............................................................................................................15
3.3.4. Signaling ..................................................................................................................................15
3.3.5. Expression and physiological functions...................................................................................16
3.3.6. Regulation of GDNF expression ..............................................................................................19
3.3.7. GDNF during pathological conditions ....................................................................................19
3.4. NEURTURIN.........24
3.4.1. Description and characteristics ...............................................................................................24
3.4.2. NTN knock-out mice.................................................................................................................24
3.4.3. Signaling............24
3.4.4. Expression and physiological functions25
3.4.5. Regulation of expression of NTN .............................................................................................26
3.4.6. NTN under pathological conditions .........................................................................................27
3.5. PERSEPHIN...........27
3.5.1. Description and characteristics ...............................................................................................27
3.5.2. PSP knock-out mice....28
3.5.3. Signaling ..................................................................................................................................28
3.5.4. Expression and physiological functions...................................................................................28
3.5.5. PSP under pathological conditions..........................................................................................28
3.6. RET TYROSINE KINASE RECEPTOR ................................................................................................29
3.6.1. Description and characteristics29
3.6.2. The RET gene...........................................................................................................................31
3.6.3. c-RET re-arrangements............................................................................................................38
3.6.4. RET knock-out mice .................................................................................................................39
3.6.5. Expression and distribution .....................................................................................................39
3.6.6. Signaling............40
3.6.7. Physiological functions of RET42
3.6.8. RET under pathological conditions..........................................................................................45
3.7. GDNF FAMILY RECEPTORS-α ................................................................................................46
3.7.1. General characteristics............................................................................................................46
3.8. GFRα-1.........................................................................................................................................47
3.8.1. Description and characteristics ...............................................................................................47
3.8.2. GFRα-1 knock-out mice...........................................................................................................47
3.8.3. Signaling ..................................................................................................................................48
3.8.4. Expression and physiological functions...................................................................................48
3.8.5. GFRα-1 under pathological conditions49
3.9. GFRα-2............50
3.9.1. Description and characteristics50
3.9.2. The GFRα-2 gene ....................................................................................................................50
3.9.3. GFRα-2 knock-out mice52
3.9.4. Signaling............52
3.9.5. Expression and physiological functions...................................................................................52
3.9.6. GFRα-2 under pathological conditions53
1Index


3.10. MYOBLASTS ..............................................................................................................................55
4 OBJECTIVES.......................................................................................................................................57
5 MATERIALS AND METHODS.........................................................................................................58
5.1. BUFFERS AND REAGENTS........................................................................................................58
5.2. CELL PREPARATIONS..............................................................................................................60
5.2.1. Cell line cultures ......................................................................................................................60
5.2.2. Isolation of peripheral blood mononuclear cells from fresh blood..........................................61
5.2.3. Isoland activation of sub-types of immune cells from PBMCs........................................62
5.2.4. Purity of selected sub-sets........................................................................................................66
5.3. PROLIFERATION ASSAYS .......................................................................................................67
5.3.1. Protocol....................................................................................................................................67
5.3.2. Human myoblast proliferation .................................................................................................68
5.4. ENZIME LINKED IMMUNOSORBENT ASSAY (ELISA) .......................................................68
5.4.1. ELISA – GDNF ........................................................................................................................68
5.4.2. ELISA - TNF-α.........................................................................................................................69
5.4.3. ELISA - IL-10...........................................................................................................................70
5.5. WESTERN BLOTTING...............................................................................................................70
5.5.1. Cell lysates............71
5.5.2. Polyacrylamid-Gel electrophoresis (PAGE)............................................................................71
5.5.3. Antibodies used in Western blot...............................................................................................72
5.6. IMMUNOFLUORESCENCE.......................................................................................................73
5.6.1. Staining for adherent cells .......................................................................................................73
5.6.2. cells in suspension.74
5.7. FACS ANALYSIS........................................................................................................................74
5.7.1. Extracellular Immunostaining .................................................................................................75
5.7.2. Intracellular Immunostaining ..................................................................................................76
5.8. RT-PCR ........................................................................................................................................77
5.8.1. RNA extraction.........................................................................................................................77
5.8.2. First-strand cDNA synthesis78
5.8.3. RT-PCR reaction......................................................................................................................79
5.9. PRIMER SEQUENCES80
5.9.1. β-Actin......................................................................................................................................80
5.9.2. GDNF.......................................................................................................................................80
5.9.3. NEURTURIN............................................................................................................................80
5.9.4. PERSEPHIN......81
5.9.5. RET ..........................................................................................................................................81
5.9.6. GFRα-1....................................................................................................................................82
5.9.7. GFRα-2.......82
5.10. STATISTICAL ANALYSIS.........................................................................................................83
6 RESULTS..............................................................................................................................................84
6.1. PURITY OF CELLS .....................................................................................................................84
6.2. PROLIFERATION ASSAYS .......................................................................................................85
6.3. GDNF EXPRESSION...................................................................................................................86
6.3.1. RT-PCR: GDNF.......................................................................................................................86
6.3.2. ELISA-GDNF......87
6.3.3. Western blot-GDNF .................................................................................................................90
6.4. NEURTURIN EXPRESSION ......................................................................................................92
6.4.1. RT-PCR: NTN ..........................................................................................................................92
6.4.2. Western blotting - NTN ............................................................................................................94
6.4.3. Immunofluorescence - NTN......................................................................................................95
6.5. PERSEPHIN EXPRESSION101
6.5.1. RT-PCR: PSP.....101
6.6. RET EXPRESSION102
6.6.1. RT-PCR: RET.....102
2 Index


6.6.2. Western blot - RET.................................................................................................................118
6.7. GFRα-1 EXPRESSION..............................................................................................................119
6.7.1. RT-PCR - GFRα-1 .................................................................................................................120
6.7.2. FACS-GFRα-1 .......................................................................................................................121
6.7.3. Western blot- GFRα-1 ...........................................................................................................126
6.8. GFRα-2 EXPRESSION128
6.8.1. RT-PCR: GFRα-2 ..................................................................................................................128
6.8.2. FACS - GFRα-2 .....................................................................................................................130
6.8.3. Indirect immunofluorescence – GFRα-2................................................................................131
6.9. GDNF AND RECEPTORS IN HUMAN MYOBLASTS.............................................................134
6.9.1. GDNF.....................................................................................................................................134
6.9.2. GFRα-1..................................................................................................................................134
6.9.3. RET ........................................................................................................................................135
6.10. FUNCTIONAL EXPERIMENTS...............................................................................................137
6.10.1. Proliferation Assays ..........................................................................................................137
6.10.2. ELISA-TNFα137
6.10.3. ELISA-IL-10 ......................................................................................................................142
6.10.4. FACS for IL-4 and IFN-γ ..................................................................................................142
6.10.5. FACS: Expression of surface molecules............................................................................143
7 DISCUSSION .....................................................................................................................................144
7.1. EXPRESSION OF GDNF...........................................................................................................146
7.2. OF NEURTURIN...............................................................................................147
7.3. EXPRESSION OF PERSEPHIN ................................................................................................147
7.4. OF RET RECEPTOR.........................................................................................148
7.5. EXPRESSION OF GFRα-1 RECEPTOR...................................................................................155
7.6. OF GFRα-2 RE156
7.7. FUNCTIONAL EXPERIMENTS158
7.8. MYOBLASTS ............................................................................................................................158
8 CONCLUSIONS.................................................................................................................................160
9 REFERENCES...................................................................................................................................162
10 APPENDICES...............181
10.1. ABBREVIATIONS ....................................................................................................................181
10.2. CURRICULUM VITAE.............................................................................................................183

















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