Genome-wide analysis of C99-overexpressing human neuroblastoma cells [Elektronische Ressource] : effects of C99 cleavage products on gene expression, signal transduction and phosphorylation status / presented by Markus Uhrig

Markus Uhrig

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Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Zentrum für Molekulare Biologie
der Ruprecht-Karls-Universität Heidelberg


Genome-wide analysis
of C99-overexpressing human neuroblastoma cells:
Effects of C99 cleavage products on gene
expression, signal transduction and
phosphorylation status



Dissertation


submitted to the

Combined Faculties for the Natural Sciences
and for Mathematics of the University of Heidelberg,
Germany


for the degree of


Doctor of Natural Sciences


presented by
Markus Uhrig





Dissertation


Genome-wide analysis
of C99-overexpressing human neuroblastoma cells:
Effects of C99 cleavage products on gene
expression, signal transduction and
phosphorylation status

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 (Dr. rer. nat.)


presented by

Diplom-Humanbiologe Markus Uhrig
born in: Stuttgart, Germany
oral examination: ............................



This thesis was researched and written at the Center for Molecular Biology of the
University of Heidelberg (ZMBH) and supervised by
Prof. Dr. Tobias Hartmann
















Referees: Prof. Dr. rer. nat. Dr. h. c. Konrad Beyreuther
Center for Molecular Biology of the
University of Heidelberg (ZMBH)
Heidelberg, Germany

Prof. Dr. rer. nat. Tobias Hartmann
Instiue for Neurobiology and Neurodegeneration
Department of Neurology
Saarland University,
Homburg/Saar, Germany








Es ist nicht genug zu wissen,
man muss auch anwenden;
Es ist nicht genug zu wollen,
man muss auch tun.

(Johann Wolfgang von Goethe, 1749-1832)































To my parents























Table of Contents


TABLE OF CONTENTS

1 Abstract _______________________________________________________ 1

2 Zusammenfassung ______________________________________________ 3

3 Introduction____________________________________________________ 5
3.1 Overview ________________________________________________________ 5
3.2 Proteolytic processing of the amyloid precursor protein (APP)
and activation of transcription_______________________________________ 9
3.3 The amyloid cascade hypothesis ___________________________________ 12
3.4 A β, tau and the connection in between ______________________________ 16
3.5 Genes associated with Alzheimer's disease __________________________ 21
3.6 Selection of normalization algorithms _______________________________ 25
3.7 MIAME standards ________________________________________________ 29
3.8 Flow diagram of sample preparation and hybridization _________________ 30
3.9 Significance level, cut-off for fold change and normalization ____________ 31
3.10 Software________________________________________________________ 32

4 The aim of this Ph.D. thesis______________________________________ 33

5 Results_______________________________________________________ 35
5.1 Preface _________________________________________________________ 35
5.2 Flow chart of experimental steps ___________________________________ 36
5.3 Cloning and expression of APP-fragments (C99 wildtype and -mutants) ___ 37
5.4 Transcriptomics _________________________________________________ 39
5.4.1 Preface _____________________________________________________________ 39
5.4.2 Quality control________________________________________________________ 39
5.4.3 C99 as an internal control. The APP gene (overexpressed C99 respectively) was
detected among the top up-regulated genes ________________________________ 39
5.4.4 Genes identified using different algorithms and software _______________________ 41


i Table of Contents

5.5 Transcriptomics compared to proteomics ____________________________ 43
5.5.1 Preface _____________________________________________________________ 43
5.5.2 CRABP1 (cellular retinoic acid binding protein 1) and further genes
associated with retinoic acid (RA)_________________________________________ 43
5.5.2.1 CRABP1 was differentially expressed both on the transcript level
and on the protein level ____________________________________________ 43
5.5.2.2 CRABP1 was up-regulated in mutant C99I45F (A β /A β ↑) only, 42 40
whereas mutant C99V50F (A β /A β ↓) showed no differential expression 42 40
of CRABP1______________________________________________________ 46
5.5.2.3 CRABP1 was not differentially expressed in consequence of
C99-overexpression in contrast to neurofilaments________________________ 47
5.5.2.4 Increased A β /A β ratio up-regulated CRABP1 and 42 40
made cells less sensitive to all-trans retinoic acid 48
5.5.2.5 CRABP1 knockdown rescued the differentiation potential of
A β overexpressing human neuroblastoma cells after RA treatment _________ 50 42
5.5.2.6 Three further genes, associated with RA-metabolism were differentially
expressed in consequence of a changed A β /A β ratio and 42 40
may have influenced the effects mediated by RA ________________________ 50
5.5.2.7 Further genes, located on chromosomal regions adjacent to
CRABP1 (15q24), were differentially expressed _________________________ 51
5.5.3 Further overlapping transcripts/proteins ____________________________________ 51
5.5.3.1 Preface_________________________________________________________ 51
5.5.3.2 PREP (prolyl endopeptidase)________________________________________ 52
5.5.3.3 T-cell receptor, α and δ locus 52
5.5.3.4 VGF (VGF nerve growth factor inducible) ______________________________ 52
5.5.3.5 NADH dehydrogenase (ubiquinone) __________________________________ 53
5.5.3.6 DNCLI2 (dynein cytoplasmic light intermediate polypeptide 2) ______________ 53
5.6 Inversely regulated genes _________________________________________ 53
5.6.1 Preface _____________________________________________________________ 53
5.6.2 Genes up-regulated in mutant C99I45F versus C99WT (A β /A β ↑) and at the 42 40
same time down-regulated in mutant C99V50F versus C99WT (A β /A β ↓) _______ 54 42 40
5.6.3 Genes down-regulated in mutant C99I45F versus C99WT (A β /A β ↑) and at the 42 40
same time up-regulated in mutant C99V50F versus C99WT (A β /A β ↓) __________ 57 42 40
5.7 Increased A β /A β ratio down-regulated the imprinted region IGF2-H19 42 40
on chromosome 11p15.5 __________________________________________ 58
5.8 Phosphorylation status of proteins and total protein amounts in
consequence of an altered A β /A β ratio detected by 42 40
high throughput immunoblotting ___________________________________ 59
5.8.1 Immunoblots _________________________________________________________ 59
5.8.2 Quantification and graphical presentation of previously shown immunoblots________ 69
5.9 Models 77
5.9.1 Preface _____________________________________________________________ 77
5.9.2 Model: Downregulation of IGF2/IGF1R/PKC and PI3K/AKT signaling
by an increased A β /A β ratio __________________________________________ 78 42 40

ii Table of Contents

5.9.3 Model: Increased A β /A β ratio is expected to reduce the ATP level 42 40
and to induce phosphofructokinase upregulation _____________________________ 85
5.9.4 Model: Transcriptional control of blood coagulation and fibrinolysis
is influenced by an altered A β /A β ratio __________________________________ 86 42 40
5.10 Candidates for tau or GSK3 phosphorylation _________________________ 89
5.10.1 Preface _____________________________________________________________ 89
5.10.2 Kinases 90
5.10.3 Phosphatases________________________________________________________ 91
5.11 Genes co-regulated with APLP1 and APLP2 __________________________ 91
5.12 Identification of altered signal transduction pathways
and molecular functions___________________________________________ 92
5.12.1 Preface _____________________________________________________________ 92
5.12.2 Over and under-representation of transcripts in signa