Role of PrP_1hnC in neuronal differentiation and propagation of its infectious isoform PrP_1hnS_1hnc by microvesicles [Elektronische Ressource] / von Maria Grazia Barenco Montrasio

Pei-Lokal

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

CRole of PrP in neuronal differentiation and
Scpropagation of its infectious isoform PrP
by microvesicles



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe-Universität Frankfurt am Main


von
Maria Grazia Barenco Montrasio
aus Bellinzona (TI-Schweiz)


Langen (Hessen) 2008



















vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität
als Dissertation angenommen.



Dekan: Prof. Dr. Volker Müller
Gutachter: Prof. Dr. Jürgen Bereiter-Hahn
Prof. Dr. Johannes Löwer



Datum der Disputation: 8. September 2008
TABLE OF CONTENTS

1 SUMMARY .........................................................................................................I
ZUSAMMENFASSUNG ..................................................................................... V
2 INTRODUCTION ............................. 1
2.1 Transmissible Spongiform Encephalopathies (TSEs) ........................................... 1
2.2 PrP-gene structure and expression ........................................ 4
2.3 Normal cellular function of PrP ............................................ 7
2.4 Neurotoxic effects of PrP 32-134 ........................................ 8
2.5 Peripheral prion pathogenesis ............................................... 9
2.6 Prion transmission ............................................................................................... 10
2.7 Outline of the work described in this thesis ........................ 11
C2.7.1 Role of PrP in neuronal differentiation ................................. 11
2.7.2 Plasma membrane released microvesicles as carriers of infectious prions ........... 12
3 MATERIALS AND METHODS ..................................................................... 14
3.1 Cell culture .......................................... 14
3.1.1 Reagents .................. 14
3.1.2 Media and buffers .................................................................................................... 14
3.1.3 Cell lines .................. 15
0/03.1.3.1 Generation and maintenance of the PrP ML cell line............... 15
3.1.3.2 Maintenance of N2a cells ............................................................................................. 16
3.1.3.3 Maintenance of Neuro-2a PK1 cells ............................................................................ 16
3.1.3.4 Maintenance of N2a58/22L cells .................. 16
3.1.4 Thawing procedure ................................................................. 16
3.1.5 Splitting cells ........................................... 17
3.1.6 Freezing procedure . 17
0/03.1.7 Subcloning (limited dilution) of PrP cells (heterogeneous population) .............. 17
3.1.8 Proliferation Assay .................................................................................................. 18
0/03.1.9 Stable transfection of PrP ML cell line 18
3.1.9.1 Isolation of single cell-derived clones .......... 18
3.1.10 Isolation of microvesicles ...................................................................................... 19
3.1.11 Infection of cells with microvesicles and brain homogenates ............................... 19
3.2 Molecular and biochemical analysis ................................................................... 20
3.2.1 Reagents .................................................. 20
3.2.2 Media and buffers .... 20
3.2.2.1 General buffers ............................................................................................................. 20
3.2.2.2 Media for bacteria and supplements ............ 21
3.2.2.3 Bacterial strain ............. 21
3.2.3 Vectors ..................................................................................................................... 21
3.2.4 Molecular size markers ........................... 21
3.2.5 Antibodies ................ 22
3.2.5.1 Primary antibodies ....................................................................................................... 22
3.2.5.2 Secondary antibodies ... 22
3.2.6 PCR genotyping ....................................... 23
3.2.7 Cell viability assay .................................................................. 23
3.2.8 Immunoprecipitation ............................... 23
3.2.9 High-performance-thin-layer-chromatography and gangliosides immunostaining 23
3.2.10 Western blot analysis ............................................................................................ 24
3.2.11 Cell blot assay ....................................... 24
3.2.12 PCR using the pPrPHG plasmid and the pPrPHG.F vector as templates ........... 25
3.2.13 Agarose gel electrophoresis .................................................................................. 25
3.2.14 Extraction of DNA fragments from agarose gels 26
3.2.15 Ligation of PCR products with pIRESpuro2 vector .............. 26
3.2.16 Chemical transformation of bacteria .................................................................... 26
3.2.17 Plasmid preparation .............................................................. 26
3.2.18 Restriction enzyme analyses .................................................. 27
3.2.19 DNA sequencing .................................... 27
3.3 Immunological analysis ...................................................... 27
3.3.1 Reagents .................................................................................. 27
3.3.2 Media and buffers .................................... 28
3.3.2.1 General buffers ............................................................................. 28
3.3.3 Antibodies ................................................ 29
3.3.3.1 Primary antibodies ....................................... 29
3.3.3.2 Secondary antibodies ................................................................... 29
3.3.4 Immunofluorescence analysis of specific cell markers ........................................... 29
C3.3.5 Immunofluorescence PrP .................................... 30
3.3.6 Fluorescent activated cell sorting (FACS) .............................. 30
3.3.7 Immunoelectron microscopy ................................................................................... 31
3.4 In vivo infectivity bioassay ................. 31
4 RESULTS ........................................................................................................ 33
4.1 The role of the cellular prion protein in the process of neuronal differentiation 33
4.1.1 Generation of a novel PrP-knockout cell line by SV40 Large T Antigen- mediated
immortalization ..................................................................................................... 33
0/04.1.2 Proliferative potential of the PrP ML cell line .................. 36
0/04.1.3 Temperature - and growth factor-dependent phenotypic changes of the PrP ML
cells ........................................................................................................................ 36
0/04.1.4 Reconstitution of the PrP ML cell line with the full-length prion protein or
PrPΔ32-134 ........................................................................................................... 44
4.1.4.1 Construction of the pPrP-IRESpuro or the pPrPΔ32-134-IRESpuro vector .......... 44
0/04.1.4.2 Generation of full-length prion protein- or PrPΔ32-134-reconstituted PrP ML
cells ......................................................................................................................... 45
0/0 4.1.5 Neuronal differentiation process in PrP- and PrPΔ32-134-reconstituted PrP
ML cells ................. 47
4.1.6 Activation of p59Fyn kinase in PrP-mediated neuronal differentiation ............... 53
4.1.7 Neural cell adhesion molecule (NCAM) and PrP-mediated neuronal
differentiation ........................................................................................................ 56
4.2 The role of plasma membrane-released microvesicles in the paracrine diffusion
C of PrP and propagation of prion infectivity ...................................................... 58
C4.2.1 Neuronal cells release membrane-derived microvesicles bearing PrP and other
lipid rafts components ........................................................... 58
C4.2.2 Paracrine transfer of PrP by microvesicles ........................................................ 62
4.2.3 Prion infected neuronal cells release membrane-derived microvesicles bearing
Sc the pathogenic isoform PrP ................................................ 65
Sc4.2.4 In vitro prion infectivity transmission by PrP -bearing microvesicles 66
Sc4.2.5 In vivo prion infectivity transmission by PrP -bearing microvesicles ................. 67
4.2.6 Establishment of a novel cell-based in vitro prion replication model................... 69
4.2.6.1 Susceptibility of the PrPA36 cell line to MV-mediated prion infection .................. 69
4.2.6.2 Susceptibility of the PrPA36 cell line to prion infection: a coculture approach .... 71
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