Analysis of {γ-secretase [gamma-secretase] regulation by phosphatidylethanolamine using mammalian and Drosophila melanogaster in vitro and in vivo model systems [Elektronische Ressource] / presented by Iva Nesic

Sven Gensler

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 2 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-Biologin Iva Nesic
Born in: BanjalukaAnalysis of -secretase regulation by
phosphatidylethanolamine using mammalian and
Drosophila melanogaster in vitro and in vivo model
systems
Referees: Prof. Dr. Dr. h.c. Konrad Beyreuther
Prof. Dr. Werner BuselmaierIch widme diese Arbeit meinen Eltern.Acknowledgements
I would like to express my gratitude Prof. Dr. Carlos Dotti for giving me the
possibility to carry on this project in his group.
I want to thank my supervisor Prof. Dr. Dr. h.c. Konrad Beyreuther for supporting
me with his critical comments, his suggestions regarding my work and for giving
me the possibility to make my doctoral thesis at the University of Heidelberg.
I want to thank Sabine Paul, Prof. Dr. Jens Wiltfang and Dr. Laura Doglio for A!
peptide measurements.
I want to thank all the members of the Dotti and Beyreuther group: especially my
friends Cristian Galvan and Bianca Hellias for help and critics and for being
always next to me, than Dr. Stefan Kins, Dr. Anita Szodorai and Dr. Tomas
Gruebl for being present in emergency situations.
Thank you very much Giulia for your support and for teaching me how to work
with flies.
Ich möchte mich herzlich bei meiner Freundin, Melanie bedanken, die immer für
mich da war und diese Erfahrung mit mir geteilt hat.
Mein besonderer Dank gilt meinen Eltern und Sven, auf deren Unterstützung ich
mich immer verlassen konnte.Table of contents
Abbreviations
Summary
Zusammenfassung
1. Introduction 1
1.1. Alzheimer’s Disease (AD) 1
1.2. Amyloid Precursor Protein (APP) 3
1.2.1. Proteolytic processing of APP 4
1.2.2. Modulators of APP proteolytic processing 12
1.3. Goals of my PhD thesis 21
2. Materials and Methods 23
2.1. Cell biology 23
2.1.1. HEK 293-hAPP cells 23
2.1.1.1. Culturing HEK 293-hAPP cells 23
2.1.1.2. RNAi transfection of HEK 293-hAPP cells 23
2.1.2. Drosophila Schneider-2 (S2) cells 24
2.1.2.1. Culturing S2 cells 24
2.1.2.2. DNA transfection of S2 cells 24
2.1.2.3. RNAi transfection of S2 cells 25
2.1.3. Determination of cell viability 25
2.2. Biochemistry 26
2.2.1. Preparation of cell lysates for protein and lipid analysis 26
2.2.2. DRM separation using Triton X-100 27
2.2.3. Western blot analysis 27
2.2.4. Immunoprecipitation of A! peptides and their 29electrophoretic separation.
2.2.5. Enzyme-linked immunosorbent assay (ELISA) 31
2.2.6. Analysis of soluble APP∀ in the medium of HEK 293-hAPP 31
2.2.7. Lipid extraction and analysis by Thin Layer Chromatographie 32
(TLC)
2.3. Molecular Biology 33
2.3.1. Rapid Small Scale Isolation of Drosophila genomic DNA 33
(Walter, 1991)
2.3.2. RNA interference (RNAi) synthesis 33
2.3.2.1. RNAi synthesis for the treatment of S2 cells 33
2.3.2.1.1. Preparation of PCR template for RNAi synthesis 33
directed against Drosophila phosphoethanolamine
citidylyltransferase gene
2.3.2.1.2. Transcription reaction assembly 35
2.3.2.2. Synthesis of RNAi oligonucleotides directed 36
against human phosphoethanolamine citidylyltransferase,
(PECT), CDP- ethanolamine phosphotransferase (CEPT),
and ethanolamine kinase 1 and 2 (ETNK1 and ETNK2)
genes utilized for the transfection of HEK 293 cells
2.3.3. Propagation and isolation of the SC100 construct 37
2.4. Flies 37
2.4.1 Drosophila transgenic lines and mutants utilized in this work 37
2.4.2. Analysis of adult eye phenotype 40
2.4.3. Analysis of GFP expression 40
2.4.4. Membrane lipid analysis in flies 41
3. Results 42
3.1. Role of phosphatidyethanolamine (PE) in APP proteolytic 42
processing in HEK 293 cells3.1.1. Phosphatidylethanolamine distribution in the cell 42
membranes of HEK 293 cells
3.1.2. Localization of Amyloid Precursor Protein (APP) in the 45
membranes of HEK 293 hAPP cells
3.1.3. RNAi against PE synthesis enzymes in HEK 47
293 cells efficiently affect plasma membrane PE levels
3.1.4. RNAi against PE synthesis enzymes in HEK 293 49
cells alter APP cleavage efficacy
3.2. Role of phosphatidyethanolamine (PE) in APP proteolytic 54
processing in Drosophila melanogaster
3.2.1. Role of phosphatidyethanolamine (PE) in APP 55
proteolytic processing in Drosophila S2 cells
3.2.1.1. Phosphatidylethanolamine distribution in the 55
cell membranes of Drosophila S2 cells
3.2.1.2. Localization of hAPP (hC99) in the cell 58
membranes of Drosophila S2 cells
3.2.1.3. Altered membrane PE levels in Drosophila 60
S2 cells correlate with changes in hC99 processing
3.2.2. Role of phosphatidyethanolamine (PE) in APP 65
proteolytic processing Drosophila in vivo system
4. Discussion 79
4.1. In mammalian HEK 293 cells membrane PE modulates 79
proteolytic processing of APP by its cleaving enzymes ∀-, !- and
#-secretase
4.2. PE is implicated in the regulation of hAPP processing by 80
#-secretase in Drosophila in vivo system
4.3. PE depletion leads to downregulation of #-secretase in neuronal 83
and non neuronal cells
4.4. PE effect on #-activity differs in non-differentiated, embryonic 84
Drosophila cells and in adult flies. Does PE correlate with #-activity
depending on cell differentiation stage?4.5. Conclusion 85
5. References 90



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