Studies of the cell biological function of the amyloid precursor protein (APP) family in Drosophila melanogaster and mammals [Elektronische Ressource] / presented by Peter Šoba

Peter Baily

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2004
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and
for Mathematics
of the Ruperto-Carola University of Heidelberg,
Germany
for the degree of
Doctor of Natural Sciences
















presented by
Diplom-chemist Peter Šoba
born in Munich


Oral examination: …….……………………..









Studies of the cell biological function of the Amyloid
Precursor Protein (APP) family in Drosophila
melanogaster and mammals























Referees: Prof. Dr. Dr. h. c. Konrad Beyreuther
Prof. Dr. Renato Paro














Mojim staršem
Meinen Eltern
For my parents

Acknowledgements


In the first place, I would like to thank Konrad Beyreuther for giving me the
opportunity to work in his group. I am very grateful for his continuous support and
advice, and especially the freedom he offered me for designing and conducting the
project.
Very special thanks go to Sylvia Kreger, and also Anke Diehlmann, for a great deal of
technical support, and Friedrich Reinhard for friendship and introducing me to the
molecular biology field.
Many thanks go to Stefan Kins for sharing thoughts, for plentiful discussions, help,
and advice. I will definitely miss discussions with the “old guys” in the 128 office.
I would like to thank all the people from the Beyreuther lab, especially the whole
bunch working in the 128 lab, namely Simone Eggert, Tweety Kuan, Stefan Kins,
Tomas Grübl, Anita Szodorai, Peter Prior, Markus Uhrig, Annette Trutzel, and Anke
Diehlmann for the very relaxed and great lab environment, making one forget the
plenty of unsuccessful experiments.
I would like to thank Renato Paro for allowing me to work in his lab and for being my
referee, and Gunter Merdes for good collaboration and many advices. Further,
Alexander Löwer and Michaela Bili ć from the Paro lab for a lot of help, discussion,
material, and the nice atmosphere while working together. Many thanks also to the
whole Paro lab, especially Stefan Schönfelder, for creating a relaxed and exceptional
atmosphere on the first floor of the ZMBH.
Many thanks go to Klemens Wild and Irmgard Sinning for excellent collaboration on
the crystallization project.
I would also like to thank Kiminobu Sugaya for introducing me to the neural stem cell
field and his great hospitality during my visit in Chicago. Especially, thanks to Mike
Kim for a tremendous amount of help and hospitality, and for friendship.
I am most thankful to my parents for supporting everything I have done during my
studies, not only financially, but also morally.
Last, but definitely not least, I feel very lucky to share my time with Simone Eggert,
and I would like to thank her for her continuous support and belief.

Table of Contents
Table of Contents

I. Abbreviations………………………..………………………..i
II. Summary……………………………………..………………iii
III. Zusammenfassung………………………..……………..…iv
1 Introduction .........................................................................1
1.1 Alzheimer’s disease................................................................................... 1
1.2 Structure, expression, and processing of the Amyloid Precursor
Protein family.............................................................................................. 2
1.3 Functions of APP family proteins............................................................. 6
1.4 Drosophila melanogaster as a model system for APP function and AD9
2 Aim of this study...............................................................12
3 Results ...............................................................................14
3.1 Functional analysis of APP in Drosophila melanogaster ..................... 14
3.1.1 Generation and analysis of APP C-terminal deletion mutants............. 14
3.1.2 Generation and analysis of secretion defective APP constructs ......... 18
3.1.3 In vivo analysis of APP-M596I/F615P transgenic flies ........................ 21
3.2 Functional analysis of APLP2 in Drosophila melanogaster ................. 23
3.2.1 APLP2 induced wing notching............................................................. 23
3.2.2 Analysis of dorsal-ventral boundary formation in larval imaginal wing
discs expressing APLP2 ..................................................................... 25
3.2.3 Influence of mosaic expression of APLP2 on DVB formation and Notch
signaling.............................................................................................. 27
3.2.4 APLP2 induces wing disc compartment shrinkage.............................. 29
3.2.5 Enhancement of APLP2 induced wing notching.................................. 31
3.2.6 Rescue of APLP2 induced wing notching ........................................... 32
3.2.7 Genetic interaction of APLP2 and E11-Dally....................................... 34
3.2.8 Influence of E11-Dally on APLP2 processing in vivo........................... 36
3.3 Interaction partners of the APP intracellular domain............................ 37
3.3.1 APP binding to Drosophila PTB adaptor proteins................................ 37
ID3.3.2 In vitro pulldown of Drosophila Numb and Disabled with GST-APP .39
3.3.3 In vitro pulldown assays of human Numb and Disabled-2 with APP ... 41
3.3.4 In vivo binding of Disabled-2 and APP ................................................ 42
3.3.5 In vivo binding of human Numb and APP family proteins.................... 44
3.4 Trans-interaction properties of APP family proteins in cell adhesion. 45
3.4.1 Processing dependent cell clustering induced by expression of APP
family proteins..................................................................................... 45
3.4.2 Homophilic intercellular interaction of APP, APLP1, and APLP2 ........ 47
3.4.3 Heterophilic intercellular interaction of APP, APLP1, and APLP2 ....... 49
3.4.4 APLP1 and APLP2 mediate cellular uptake of secreted fragments..... 51
3.4.5 APLP1 ∆NPTY plasma membrane localization and intracellular
accumulation of sAPLP1 ..................................................................... 53
3.4.6 APP interacts with APLP1 in vivo and APLP1 protein levels are
upregulated in APP -/- mouse brain .................................................... 54
3.4.7 APP, APLP1, and APLP2 are enriched in synaptic plasma membranes
and APP interacts with APLP1 synaptic compartments ...................... 56
4 Discussion.........................................................................59
Table of Contents
4.1 APP interference with cell adhesion in vivo depends on membrane
anchoring and processing ...................................................................... 59
4.2 APLP2 interferes with Wingless signaling via interaction with the
Drosophila Glypican Dally....................................................................... 61
4.3 Numb and Disabled interact with the C-terminal NPTY motif of APP
family proteins.......................................................................................... 64
4.4 Trans-interaction of APP, APLP1, and APLP2....................................... 66
4.4.1 Homo- and hetero-trans-dimerization of APP family proteins promotes
cell adhesion ....................................................................................... 66
4.4.2 Trans-cellular interaction of APP family proteins is modulated by
proteolysis and correlating with in vivo phenotype strength ................ 68
4.4.3 APLP1 and APLP2 have a receptor-like function................................ 69
4.4.4 APP and APLP1 interact in synaptic compartments............................ 71
4.5 Conclusions and outlook ........................................................................ 73
5 Material and Methods .......................................................74
5.1 Chemicals ................................................................................................. 74
5.2 Materials.................................................................................................... 75
5.3 Cell lines ................................................................................................... 76
5.4 Antibodies 77
5.5 Oligonucleotides ...................................................................................... 78
5.6 Plasmids 79
5.7 Cloning of constructs .............................................................................. 80
5.7.1 Cloning of APP nt-myc into pUAST..................................................... 80
5.7.2 Cloning of APLP1 ct-myc into pUAST ................................................. 80
5.7.3 Cloning of APLP2 into pUAST............................................................. 80
5.7.4 Cloning of APP-Y682A, -N684A, and –Y687A .................................... 80
5.7.5 Cloning of APP-BASS