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Developmentally-regulated localization and possible functions of HRP-3 in the murine nervous tissue [Elektronische Ressource] / vorgelegt von Heba Mahmoud Ahmed Eltahir

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162 pages
Developmentally-regulated localization and possible functions of HRP-3 in the murine nervous tissue Dissertation zur Erlangung des Doktorgrades (Dr. rer. Nat.) der Mathematische Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms Universität Bonn vorgelegt von Heba Mahmoud Ahmed Eltahir Aus Kena, Egypt Bonn, Oktober 2009 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. med. Volkmar Gieselmann 2. Gutachter: Prof. Dr. Klaus Mohr Tag der Promotion: 10. März 2010 Erscheinungsjahr: 2010 For My small family, & for the soul of my parents. Acknowledgement First, I would thank God for helping me to finish this work in the best I can way then the people who helped me in every step of this work. I would like to thank Prof. Dr. med. Volkmar Gieselmann for giving me the generous chance to do my doctoral thesis in his lab. His interesting ideas and constructive discussion have been of major importance for this work. Prof. Dr. Klaus Mohr for taking the responsibility of being my second referee I would also like to deeply thank my co-supervisor Dr. Sebastian Franken for his excellent supervision of my work. He was always able to invent new ideas that gave my work much better dimensions.
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Developmentally-regulated localization and
possible functions of HRP-3 in the murine
nervous tissue
Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. Nat.)
der
Mathematische Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms Universität Bonn
vorgelegt von
Heba Mahmoud Ahmed Eltahir
Aus
Kena, Egypt
Bonn, Oktober 2009 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn





















1. Gutachter: Prof. Dr. med. Volkmar Gieselmann
2. Gutachter: Prof. Dr. Klaus Mohr


Tag der Promotion: 10. März 2010
Erscheinungsjahr: 2010



For
My small family,
& for the soul of my
parents. Acknowledgement
First, I would thank God for helping me to finish this work in the best I can way
then the people who helped me in every step of this work.
I would like to thank
Prof. Dr. med. Volkmar Gieselmann for giving me the generous chance to do my
doctoral thesis in his lab. His interesting ideas and constructive discussion have
been of major importance for this work.
Prof. Dr. Klaus Mohr for taking the responsibility of being my second referee
I would also like to deeply thank my co-supervisor Dr. Sebastian Franken for his
excellent supervision of my work. He was always able to invent new ideas that
gave my work much better dimensions. His constructive criticism, great support,
help and cooperation were of great importance for my work and always gave me a
push forward. He was always there answering my questions and had made a
great effort correcting and revising until this work had come out in its final shape.
The group of Dr. Franken; Rainer Gallitzendörffer, Katharina Klein, Stephanie
Bremer, Robert and specially Angela sedlmeier for the precise reading of the last
manuscript of this work.
The members of the anatomy department for their great help and instructions in
preparing the Vibratome sections. And also I would like to thank them for the
facilities they provided in using the confocal laser microscope.
The group of Prof. Dr. Magin for the microscope facilities they provided.
Dr. Kappler, Dr. Eckhardt and Dr. Matzner for their help and cooperation.

All co-workers for their cooperation and for providing a friendly work atmosphere.
Mrs. Simonis for her great help in general DNA techniques, Mrs. Buttkau and Mr.
Rösel for their great help in technical work.
Mr. Pflüger for his great and brilliant help in the use of computer programs.
My husband, Mekky, for his incredible help and endless support. He sacrificed
many important things to provide me with the suitable atmosphere to do my work.
Without his help, love and patience, I would never be able to do this work.
My children, Ahmed and Radwa who pushed me forward with their love and
smiles.
My brother and sister and my big family in Egypt, they provided me with support
and encouragement and they have done too much for me especially my mother.
Finally, I wanted to thank my mother for all what she had done for me, for being
always behind me and for all her words, advices and love even when she is not
there any more to read these words. Table of content
Table of content
Table of content ........................................................................... I
List of figures ............................................................................... V
List of tables ................................................................................. VIII
Abbreviations ............................................................................... IX
1. Introduction .............................................................................. 1
1.1 Development of the murine nervous system ................................ 1
1.2 Extracellular matrix ....................................................................... 5
1.3 Growth factors, cytokines and neurotrophic factors ........... 7
1.3.1 Nomenclature ................................................................................. 7
1.3.2 Roles of growth factors .................................................................. 9
1.3.2.1 General roles ...................................................................... 9
1.3.2.2 Neuron-related roles ........................................................... 9
1.3.3 Hepatoma-derived growth factor and its related proteins .............. 11
1.3.3.1 Family-member overview .................................................... 11
1.3.3.2 Hepatoma-derived growth factor related protein -3 ............ 13
1.4 Aim of work ................................................................................... 13
2. Materials .................................................................................... 15
2.1 Instruments ................................................................................... 15
2.2 Equipments .............. 16
2.3 Reagents ...................................................................................... 16
2.3.1 Chemicals ...................................................................................... 16
2.3.2 Standard solutions and buffers ...................................................... 19
2.3.3 Media ............................................................................................. 20
2.3.4 Antibodies ...................................................................................... 20
2.3.5 Restriction enzymes ....................................................................... 21
2.3.6 Bacteria, cell lines, and animals ..................................................... 21
2.3.7 Primers ........................................................................................... 22
3. Methods .................................................................................... 23
3.1 Cell culture protocols .................................................................... 23
3.1.1 Primary mouse cortical neurons culture ......................................... 23
3.1.2 Protein coating of tissue culture vessels ........................................ 23
3.1.3 Determination of neurite length ...................................................... 24
3.1.4 Neuron rescue by the addition of purified proteins ......................... 24
® 3.1.5 Quantitative determination of cell survival using Alamar blue ...... 25
3.1.5.1 Principle .............................................................................. 25
3.1.5.2 Protocol ............................................................................... 25
3.1.6 Antibody treatment of neurons ....................................................... 26
3.1.7 Gene silencing ............................................................................... 27
3.1.7.1 Definition and types ............................................................ 27
3.1.7.2 RNAi approach ................................................................... 27
ITable of content
3.1.7.3 Transfection of neurons with siRNA oligos using
®Lipofectamin 2000 ............................................................. 29
3.1.7.4 Transfection of neurons with vector-based siRNA using
®
MATra ............................................................................... 29
3.1.7.4.1 Principle ................................................................ 29
3.1.7.4.2 Protocol ................................................................. 30
3.1.8 Primary mouse sensory neuron culture ......................................... 30
3.1.9 B35 neuroblastoma cells ................................................................ 31
3.1.9.1 B35 cells in culture .............................................................. 31
3.1.9.2 Transient transfection of B35 cells with HRP-3 constructs . 32
3.1.10 Daoy medulloblastoma cells ........................................................ 32
3.1.10.1 Transient transfection of Daoy cells using calcium
phosphate ........................................................................ 32
3.1.10.2 Stable transfection and selection of Daoy cells ................ 33
3.1.10.3 Proliferation assay of Daoy cells using MTS .................... 34
3.1.10.3.1 Principle .............................................................. 34
3.1.10.3.2 Protocol ............................................................... 34
3.1.11 Freezing of cells ........................................................................... 34
3.1.12 Thawing of cells ........................................................................... 35
3.2 Immunolocalization protocols ............................................................ 35
3.2.1 Immunolocalization of interest proteins in mouse tissues .............. 35
3.2.2 Immunolocalization of interest proteins in neuron cultures ............ 36
3.3 DNA protocols .............................................................................. 37
3.3.1 Cloning of HRP-3 variants .............................................................. 37
3.3.2 Construction of HRP-3siRNA pSilencer vectors ........................... 38
3.3.3 Ligation of DNA .............................................................................. 39
3.3.4 Transformation ............................................................................... 40
3.3.4.1 Principle .............................................................................. 40
3.3.4.2 Protocol ............................................................................... 40
3.3.5 Colony screening ........................................................................... 41
3.3.6 Separation of DNA by agarose gel electrophoresis ....................... 41
3.3.6.1 Principle .............................................................................. 41
3.3.6.2 Protocol ............................................................................... 41
3.3.7 DNA sequencing ............................................................................ 42
®
3.3.8 Gel extraction of DNA (Qiagen )..................................................... 42
®3.3.9 Mini and midi plasmid DNA preparations (Qiagen )....................... 43
3.4 Protein chemistry protocols................................................................ 43
3.4.1 Purification of His-tagged proteins.................................................. 43
2+
3.4.1.1 Ni -affinity chromatography .............................................. 43
3.4.1.2 Choice of lysis buffer .......................................................... 43
3.4.1.3 Isolation of the recombinant proteins .................................. 43
3.4.2 Antibody purification protocol ......................................................... 44
3.4.3 Western blot analysis ..................................................................... 45
3.4.3.1 Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) ............................................... 45
3.4.3.2 Semi dry protein transfer (blotting) ..................................... 47
4. Results ...................................................................................... 48
Part I: Cellular localization of HRP-3 ...................................................... 48
I.1 Expression of HRP-3 under Physiological conditions ................. 48
IITable of content
I.1.1 HRP-3 expression in adult mouse nervous system ................ 48
I.1.1.1 HRP-3 expression in adult mouse brain ................... 48
I.1.1.2 HRP-3 expression in adult mouse spinal cord .......... 58
I.1.1.3 HRP-3 expression in adult mouse sciatic nerve ....... 60
I.1.2 HRP-3 expression in the embryonic mouse ........................... 62
I.1.2.1 General expression of HRP-3 in mouse embryo ...... 62
I.1.2.2 HRP-3 expression in embryonic mouse DRG ........... 65
I.1.2.3 HRP-3 expression in the developing mouse cerebral
cortex ......................................................................... 66
I.2 Expression of HRP-3 under pathological conditions ................... 68
I.2.1 HRP-3 expression in medulloblastoma tumour ...................... 68
I.2.2 HRP-3 expression in crushed sciatic nerve ............................ 70
I.3 HRP-3 expression in vitro ............................................................... 72
I.3.1 HRP-3 expression in mouse primary DRG (sensory) neurons 72
I.3.2 HRP-3 expression in mouse primary cortical neurons ............ 73
Part II: Possible roles of HRP-3 .............................................................. 79
II.1 Intracellular roles ............................................................................ 79
II.1.1 Knock down of endogenous HRP-3 protein level .................. 79
II.1.2 Increasing the endogenous HRP-3 protein level ................... 82
II.2 Extracellular roles ........................................................................... 89
II.2.1 Role of HRP-3 in neuronal cell survival ................................. 89
II.2.2 Role of HRP-3 in neuritogenesis ........................................... 93
II.2.2.1 Effect of soluble HRP-3 on neuritogenesis .............. 93
II.2.2.2 Effect of substrate bound-HRP-3 on neuritogenesis 94
5. Discussion ................................................................................ 99
5.1 HRP-3 expression in vivo and in vitro .............................................. 99
5.2 Intracellular roles of HRP-3 ............................................................... 105
5.3 Extrar roles of HRP-3 .............................................................. 107
5.4 HRP-3 expression in pathological conditions .................................. 110
5.5 Perspectives ................................................................................. 111
6. Summary ................................................................................... 112
7. Zusammenfassung .................................................................. 113
8. References ................................................................................ 114
9. Appendix ................................................................................... 136
9.1 Supplementary data ........................................................................... 136
9.1.1 HRP-3 expression in mouse E13.5 neural tube ............................. 136
9.1.2 Vector-based HRP-3siRNA reduced endogenous level of HRP-3
in primary cortical neurons .............................................................. 137
9.1.3 The effect of a reduced endogenous HRP-3 level on
neuritogenesis ......................................................................................... 138
9.1.4 Role of nuclear localization signals 1 & 2 in the neuritogenetic
effect of HRP-3 ............................................................................... 141
9.1.5 Neuron rescue by soluble HRP-3 .................................................. 142
9.1.6 Determination of protein amount bound to glass surface ............... 143
9.1.7 Effect of substrate-bound HRP-3 on neuritogenesis ...................... 144
IIITable of content
9.2 Vector maps ................................................................................. 146
9.2.1 pCDNA3 ......................................................................................... 146
9.2.2 eGFPC3 ......................................................................................... 146
9.2.3 pBlueskript ..................................................................................... 147
9.2.4 pSilencer ........................................................................................ 147
IVList of figures
List of figures
Number Title Page
Figure 1 Formation of the neural tube ..................................................... 1
Figure 2 A simplified view of corticogenesis of the developing cerebral
wall in mammals ......................................................................... 3
Figure 3 A schematic diagram presenting the steps of gene silencing
achieved by introducing siRNA to the cell .................................. 28
Figure 4 A photograph showing an overview of the exposed neural tube
of E14.5 mouse embryo where the attached DRG could be
seen on both sides ..................................................................... 31
Figure 5 Schematic diagram of a lobe of the cerebellum ......................... 49
Figure 6 Expression of HDGF and HRP-3 in granular cells of the mouse
cerebellum .................................................................................. 50
Figure 7 Expression of HDGF and HRP-3 in Purkinje cells of mouse
cerebellum .................................................................................. 51
Figure 8 Expression of HDGF and HRP-3 in astrocytes ......................... 52
Figure 9 Expression of HDGF and HRP-3 in oligodendrocytes ................ 52
Figure 10 Simultaneous expression of HDGF and HRP-3 in adult mouse
brain cerebellum ......................................................................... 53
Figure 11 A Schematic diagram showing the laminar structure of the
hippocampus .............................................................................. 54
Figure 12 Comparison of HDGF and HRP-3 expression in mouse
hippocampus ...…...................................................................... 55
Figure 13 HDGF and HRP-3 expression in adult mouse brain cortex ........ 56
Figure 14 Extranuclear expression of HRP-3 in different areas of adult
mouse brain ................................................................................ 57
Figure 15 Schematic diagram of the adult spinal cord in mammals ........... 58
Figure 16 Expression of HRP-3 in adult mouse spinal cord ....................... 60
Figure 17 Expression of HRP-3 and HDGF in adult mouse sciatic nerve .. 61
Figure 18 Expression of HRP-3 in transverse section of E11.5 mouse
embryo ....................................................................................... 63
VList of figures
Figure 19 Expression of HRP-3 in E15.5 embryo ....................................... 64
Figure 20 HRP-3 expression in embryonic mouse dorsal root ganglia ...... 65
Figure 21 A comparison of HRP-3 expression pattern between
developing and adult mouse brain cortex ................................... 67
Figure 22 Expression of HDGF and HRP-3 in a medulloblastoma tissue .. 68
Figure 23 Increased proliferation of Daoy medulloblastoma cells over-
expressing HDGF .............…................…................................... 69
Figure 24 A schematic diagram for a transversely cut nerve with a part of
an adjacent ganglion .................................................................. 70
Figure 25 Changes in HRP-3 expression pattern after sciatic nerve injury 71
Figure 26 HRP-3 expression in mouse primary sensory neurons at
different time points .................................................................... 73
Figure 27 HRP-3 expression in both axonal and dendretic compartments
of mouse primary cortical neurons ............................................. 74
Figure 28 HRP-3 expression dynamically changed over time in cultured
primary cortical neurons ............................................................. 76
Figure 29 Comparison between HDGF and HRP-3 expression patterns in
cultured mouse cortical neurons after 1 and 5 days ................... 77
Figure 30 HRP-3siRNAs reduced HRP-3 level in HEK293T cells .............. 80
Figure 31 Reduction of the intracellular HRP-3 level resulted in neurons
bearing shorter and fewer neurites ............................................. 81
Figure 32 B35 neuroblastoma cells differentiated into a neuron
phenotype on increasing cAMP level ......................................... 82
Figure 33 Endogenous HRP-3 expression in B35 cells .............................. 83
Figure 34 Expression pattern of wild type HRP-3 transfected into B35
showed a differentiation dependent distribution pattern ............. 84
Figure 35 Schematic diagram of NLS mutant HRP-3 constructs ............... 85
Figure 36 Subcellular localization of wild type or NLS-mutant HRP-3
constructs transfected into B35 neuroblastoma cells ………….. 86
Figure 37 NLS1 is responsible for the neurite outgrowth promoting effect
of HRP-3 in B35 cells ................................................................. 88
VI

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