Characterisation of cytosolic prion protein mediated putative cytotoxicity in neuronal cell lines [Elektronische Ressource] / von Jana Mehlhase

Characterisation of cytosolic prion protein-
mediated putative cytotoxicity in neuronal cell
lines



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



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




von
Jana Mehlhase
aus Mittweida



Langen (Hessen) 2006























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




Dekan: Prof. Dr. Rüdiger Wittig
Gutachter: Prof. Dr. Anna Starzinski-Powitz
Prof. Dr. Johannes Löwer



Datum der Disputation: …………………………………….
Table of contents

Table of contents

1 Summary......................................................................................................... I
1.1 German Summary..................................................................................... I
1.2 English Summary.................................................................................... IV
2 Introduction ................................................................................................... 1
2.1 Prion diseases and infectivity................................................................... 1
2.2 Cellular prion protein................................................................................ 2
C2.2.1 Characteristics and structure of PrP ..................................................... 2
C2.2.2 Biosynthesis and endosomal trafficking of PrP ..................................... 3
C2.2.3 Putative functions of PrP ...................................................................... 4
2.3 Neurotoxicity of pathological prion protein ............................................... 6
Sc2.3.1 PrP and neurotoxicity .......................................................................... 6
2.3.2 ER-stress and PrP misfolding ................................................................ 7
C2.4 PrP proteolysis and the proteasomal system ......................................... 8
2.5 Cytosolic PrP ........................................................................................... 9
2.5.1 Cytotoxicity of cytosolic PrP ................................................................. 11
2.6 Ecdysone-inducible expression system ................................................. 12
2.7 Objectives .............................................................................................. 15
3 Materials and Methods................................................................................ 16
3.1 Materials and chemicals......................................................................... 16
3.1.1 Materials and equipments .................................................................... 16
3.1.2 Chemicals ............................................................................................ 16
3.1.3 Enzymes .............................................................................................. 18
3.1.4 Cells and media.................................................................................... 18
3.1.5 Kits ....................................................................................................... 19
3.1.6 Antibodies 19
3.2 Molecular biology ................................................................................... 20
3.2.1 Plasmids............................................................................................... 20
3.2.2 Construction of vectors encoding transgene PrPs ............................... 21
3.2.3 Polymerase chain reaction ................................................................... 22
3.2.4 Oligonucleotides 23
Table of contents
3.2.5 Agarose gel electrophoresis................................................................. 23
3.2.6 Restriction and ligation......................................................................... 24
3.2.7 DNA purification ................................................................................... 24
3.2.8 Photometric determination of DNA concentration................................. 25
3.2.9 Working with bacteria........................................................................... 25
3.3 Cell biology and biochemistry ................................................................ 27
3.3.1 Cultivation of eukaryotic cells ............................................................... 27
3.3.2 Determination of the cell number.......................................................... 28
3.3.3 Flow cytometry analysis ....................................................................... 28
3.3.4 Viability assays..................................................................................... 29
3.3.5 Caspase-3 activity assay...................................................................... 29
3.3.6 Proteinase K digestion ......................................................................... 30
3.3.7 SDS-PAGE........................................................................................... 30
3.3.8 Immunoblot analysis............................................................................. 31
3.3.9 Proteasome activity assay.................................................................... 31
3.3.10 Immunofluorescence.......................................................................... 33
3.3.11 Immunoprecipitation........................................................................... 34
3.3.12 Cellular fractionation 34
3.4 Statistics and fitting ................................................................................ 34
4 Results ......................................................................................................... 35
4.1 Generation and expression of Cy-PrP and PM-PrP ............................... 35
4.1.1 Generation of Cy-PrP and PM-PrP....................................................... 35
4.1.2 Ecdysone-inducible expression system................................................ 37
4.2 Analysis of cytotoxicity in Cy-PrP expressing cells ................................ 39
4.2.1 Cell viability in Cy-PrP expressing N2a cells ........................................ 39
4.2.2 Cell viability in Cy-PrP expressing 293T cells ...................................... 42
4.2.3 Caspase-3 activity in Cy-PrP expressing N2a cells.............................. 43
4.3 Proteolysis of Cy-PrP and PM-PrP in N2a cells ..................................... 44
4.3.1 Kinetics of Cy-PrP and PM-PrP proteolysis.......................................... 44
4.3.2 Role of proteasome in Cy-PrP and PM-PrP proteolysis ....................... 47
4.3.3 Viability in Cy-PrP expressing N2a cells after proteasome inhibition ... 51
4.4 Cellular localisation of Cy-PrP and PM-PrP in N2a cells........................ 53
4.4.1 Intracellular localisation of Cy-PrP........................................................ 53
4.4.2 Cy-PrP co-localisation with Hsc70 in EEA1 positive vesicles............... 57
Table of contents
4.4.3 Binding of Cy-PrP by Hsc70................................................................. 60
4.5 Stable Cy-PrP expressing neuronal cell lines ........................................ 62
4.5.1 Cy-PrP and PM-PrP expressing N2a cells ........................................... 62
4.5.2 Phenotype of N2a-Cy-PrP cell lines ..................................................... 64
4.5.3 Localisation of Cy-PrP in N2a-Cy-PrP cell lines ................................... 65
0/04.5.4 Cy-PrP and PM-PrP expressing PrP neuronal precursors................ 67
5 Discussion................................................................................................... 69
5.1 Cy-PrP toxicity and proteasome in cell culture....................................... 69
5.1.1 Cy-PrP is per se not toxic to neuronal cells.......................................... 69
5.1.2 Stability and proteolysis of Cy-PrP ....................................................... 71
5.1.3 Retro-translocated Cy-PrP ................................................................... 73
5.1.4 Cy-PrP/membrane interaction as toxic event ....................................... 75
5.2 Cy-PrP localisation in early endosomal vesicles .................................... 75
5.3 Hsc70/Hsp70 - prevention against Cy-PrP toxicity................................. 78
5.4 Cy-PrP and N2a cell morphology........................................................... 82
5.5 Putative consequences for Cy-PrP expression in vivo........................... 83
6 References................................................................................................... 84
7 Abbreviations ............................................................................................ 102
Summary
1 Summary
1.1 German Summary
Prionenerkrankungen sind neurodegenerative Erkrankungen, neuropathologisch
charakterisiert durch spongiforme Vakuolenbildungen im Hirngewebe, den Verlust
neuronaler Zellen sowie die verstärkte Proliferation von Mikroglia und Astroglia.
Di