The 37kDa, 67kDa laminin receptor as a therapeutic target in prion diseases [Elektronische Ressource] : potency of antisense LRP RNA, siRNAs specific for LRP mRNA and a LRP decoy mutant / Karen Vana

Karen Vana

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation
zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München











The 37 kDa/67 kDa laminin receptor
as a therapeutic target in prion diseases:
potency of antisense LRP RNA,
siRNAs specific for LRP mRNA and a LRP decoy mutant




Karen Vana
aus
Altenburg



2006
Erklärung

Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar
1998 von PD Dr. Stefan Weiß betreut.











Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 17.02.2006


Karen Vana








Dissertation eingereicht am 17. Februar 2006

1. Gutachter: PD Dr. Stefan Weiß
2. Gutachter: Prof. Dr. Eckhard Wolf



Mündliche Promotionsprüfung am 06. April 2006





















Man kann niemanden überholen,
wenn man in seine Fußstapfen tritt.

Francois Truffaut (1932 - 1984)
frz. Regisseur, Schauspieler und Produzent IV CONTENTS
___________________________________________________________________________

ACKNOWLEDGEMENT VIII

SUMMARY IX

CHAPTER I Introduction
1 Prion diseases 2
1.1 History of prion diseases 2
1.2 Human prion diseases 5
1.3 Animal prion diseases 8
2 The prion protein (PrP) 10
c2.1 Role of PrP 12
c2.2 Trafficking of PrP 13
Sc2.3 Characteristics of PrP 16
c Sc2.4 Conversion of PrP to PrP 17
2.5 The prion-like protein Doppel (Dpl) 18
3 Prion-like elements in yeast, fungi and aplysia 19
4 Prion strains 20
5 Prion transmission barriers 21
6 In search of binding proteins and receptors for prions 21
6.1 Molecular chaperones 23
6.2 Protein X 24
6.3 The 37kDa/67kDa laminin receptor (LRP/LR) 24
7 Therapeutic approaches for the treatment of prion diseases 28
7.1 Chemical compounds 28
7.2 Immunotherapy 31
7.3 RNA interference (RNAi) and antisense RNA 33
7.4 Trans-dominant negative mutants 35

CHAPTER II BSE-die gebannte Gefahr? Epidemiologie, Diagnostik,
Therapie und Vakzinierung bei Prionenerkrankungen 37

ScCHAPTER III The 37 kDa/ 67 kDa laminin receptor is required for PrP
propagation in scrapie-infected neuronal cells 43


V CONTENTS
___________________________________________________________________________

CHAPTER IV Knock-down of the 37 kDa/ 67 kDa laminin receptor in
mouse brain by transgenic expression of specific antisense
LRP RNA 53

CHAPTER V A trans-dominant negative 37kDa/67kDa laminin receptor mutant impairs
ScPrP propagation in scrapie-infected neuronal cells 59

CHAPTER VI Generation of transgenic mice expressing a LRP decoy mutant as a
therapeutic approach in prion diseases 75

ScCHAPTER VII Inhibition of PrP formation by a lentivirus-based RNAi
delivery system in scrapie-infected neuroblastoma cells 81

CHAPTER VIII References 93

ABBREVIATIONS 122

CURRICULUM VITAE XI
VI
Die vorliegende Arbeit wurde in der Zeit von September 2002 bis Oktober 2005 im Labor von PD Dr.
Stefan Weiß am Genzentrum der Ludwig-Maximilians-Universität angefertigt.







Im Verlauf dieser Arbeit wurden folgende Manuskripte publiziert bzw. zur Veröffentlichung
eingereicht:

Vana K. and Weiss S. (2006) A trans-dominant negative 37kDa/67kDa laminin receptor mutant
Scimpairs PrP propagation in scrapie-infected neuronal cells. J Mol Biol, 358(1), 57-66

Vana K. and Weiss S. (2006) BSE-die gebannte Gefahr? Epidemiologie, Diagnostik, Therapie und
Vakzinierung bei Prionenerkrankungen. BIOforum, 29(1), 35-37

Vana K. and Weiss S. (2006) Generation of transgenic mice expressing a LRP decoy mutant as a
therapeutic in vivo approach in prion diseases, Transgenic Res., submitted

ScVana K., Nikles D., Messow D. and Weiss S. (2006) Inhibition of PrP formation by a lentivirus-
based RNAi delivery system in scrapie-infected neuroblastoma cells, J Virol., submitted

Leucht C., Vana K., Renner-Muller I., Dormont D., Lasmezas C.I., Wolf E., Weiss S. (2004) Knock-
down of the 37-kDa/67-kDa laminin receptor in mouse brain by transgenic expression of specific
antisense LRP RNA. Transgenic Res., 13(1): 81-5.

Leucht C., Simoneau S., Rey C., Vana K., Rieger R., Lasmézas C.I. and Weiss S. (2003) The 37 kDa/
Sc67 kDa laminin receptor is required for PrP propagation in scrapie-infected neuronal cells. EMBO
reports, 4, 290-295. VII

Teile dieser wissenschaftlichen Arbeit wurden auf Kongressen als Posterbeiträge präsentiert und sind
im Folgenden aufgelistet:

Töpper D., Vana K., Nikles D. and Weiss S.
Delivery of siRNAs directed against LRP mRNA by recombinant lentiviruses into mice as a
therapeutic strategy for the treatment of TSEs, Treffen des Bayerischen Forschungsverbundes
ForPrion, 04.-05. Juli 2005, Hohenkammer

Vana K. and Weiss S.
Transdominant negative LRP/LR mutants as alternative therapeutic tool in TSEs, Treffen des
Bayerischen Forschungsverbundes ForPrion, 04.-05. Juli 2005, Hohenkammer

Vana K., Töpper D. and Weiss S.
Small interfering (si) RNAs directed against LRP mRNA as anti-prion tools and their delivery by
lentiviral vectors, Treffen des Bayerischen Forschungsverbundes ForPrion, 15.-16. Juli 2004,
Würzburg

Vana K., Gauczynski S., Leucht C. and Weiss S.
Laminin receptor decoy mutants and small interfering (si) RNAs directed against the 37kDa/67kDa
laminin receptor as powerful tools in therapy of prion diseases, International Prion-Conference, 08.-
10. Oktober 2003, München

Vana K., Gauczynski S., Leucht C. and Weiss S.
Secretion of a transmembrane delition mutant of the 37kDa/67kDa laminin receptor prevents PrP
binding and internalization in mammalian cells, Jahrestagung der Gesellschaft für Virologie, 26.-29.
März 2003, Berlin

Vana K., Gauczynski S., Leucht C. and Weiss S.
A transmembrane deletion mutant of the 37kDa/67kDa laminin receptor abolishes PrP binding and
internalization in mammalian cells, International Prion-Therapeutics-Conference, 01.-03. Dezember
2002, Paris





VIII ACKNOWLEDGEMENT
___________________________________________________________________________

THANK YOU


DANKE



I would like to thank PD Dr. Stefan Weiß for giving me the opportunity to intensively study the world
of prions and for the creation of the first certificate. In addition, I am thankful for having the chance to
visit a lot of national and international congresses and learn to know the prion scientific community.

I thank Prof. Dr. Eckhard Wolf for the preparation of the second certificate.

I want to thank all present members of the Weiß group, namely Daphne Nikles, Clémence Rey,
Chantal Zuber, Katharina Krüger and Tina Hallas, for the cushy working atmosphere, several Ladies
Nights and All-u-can-eat-Sushi events. Special thanks go to Daphne for the initiation of the “YFSCC
(Young Female Scientific Champagne Circle)”, which later turned into a non-champagne circle due to
the pregnancies of three members.

I’d like to thank Sabine Gauczynski for correcting my PhD manuscript and providing me helpful hints
to survive the PhD exam.

A big thankyou to Diana Messow (nee Töpper) for perseveringly establishing the lentivirus system
within the scope of her diploma thesis.

I thank Sigrun Jacklin (Sigrünchen) for the funny skating evenings during wintertime including the
compulsory Children’s punch.

Many thanks to Sigi Kastenmüller who excellently cared about all administrative issues and always
had an sympathetic ear.

A big thank to my husband Thomas for his love and patience and his spontaneity and continuous
motivation. Thank you for sportive and relaxing weekends during stressful times and for your
uncomplicated attitude to life.

Mein ganz besonderer Dank gilt meinen Eltern, die mir erst ein Studium und die Promotion ermöglicht
und mich in jeder Hinsicht beständig unterstützt haben. Ich danke Ihnen, dass Sie Vertrauen hatten,
dass ich meinen Weg finden würde und dass sie immer für mich da sind.
IX SUMMARY
___________________________________________________________________________

Prion diseases are a group of rare, fatal neurodegenerative diseases, also known as transmissible
spongif