Characterization of the protein phosphatase 1E (PPM1E) [Elektronische Ressource] : localisation and truncation in brain tissue and effects on neuronal morphology in primary neuronal culture / submitted by Anne Lene Jessen

Sujets

• Biologie

Informations

• Publié par: georg-august-universitat_gottingen
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Characterization of the
protein phosphatase 1E (PPM1E)

Localization and truncation in brain tissue and effects on
neuronal morphology in primary neuronal culture


Dissertation

in partial fulfillment of the requirements
for the degree “Doctor rerum naturalium”
at the Georg August University Göttingen,
Faculty of Biology


submitted by

Anne Lene Jessen

born in

Kiel, Germany




Göttingen 2010




































First Referee: Prof. Dr. Nils Brose
Second Referee: Prof.em. Dr. Rüdiger Hardeland
Advisor: Dr. Heinz von der Kammer

Date of submission of the PhD thesis: September 21, 2010
Date of thesis defence: November 2, 2010
2


Declaration

This dissertation has been written independently and with no other sources and aids
than quoted.
I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no materials previously published or written by
another person nor material which to a substantial extent has been accepted for the
award of any other degree of the university or other institute of higher education,
except where due acknowledgement has been made in the text.



Anne Lene Jessen
Göttingen, September 21, 2010

3
TABLE OF CONTENTS

TABLE OF CONTENTS

ABSTRACT.................................................................................................................. 7
ABBREVIATIONS ........................................................................................................ 9
LIST OF FIGURES..................................................................................................... 13
LIST OF TABLES....................................................................................................... 16
1 INTRODUCTION.................................................................................................17
1.1 Alzheimer’s disease.....................................................................................18
1.1.1 Cognitive phenotype, diagnostic and treatment.....................................18
1.1.2 Ante-mortem diagnostic........................................................................18
1.1.3 Treatment of Alzheimer’s disease – state of the art............................... 19
1.1.4 Treatment of Alzheimer’s disease - outlook........................................... 20
1.1.5 Histological phenotype..........................................................................20
1.1.6 Post-mortem diagnostic.........................................................................21
1.1.7 Etiology of Alzheimer’s disease.............................................................22
1.2 Neuronal and dendritic spine morphology.................................................... 24
1.2.1 Neurons in the hippocampus.................................................................24
1.2.2 Dendritic spine morphology...................25
1.2.3 Dendritic spine motility..........................................................................26
1.2.4 Synapses on dendritic spines................................................................27
1.2.5 Compartimentalization and organelles in spines...................................27
1.2.6 The cytoskeleton...................................................................................28
1.2.7 The actin cytoskeleton in dendritic spines and the neuronal nucleus..... 29
1.2.8 Synapse and dendritic spine morphogenesis........................................30
1.2.9 Synapse maturation.....................................31
1.3 A screen for differentially regulated genes in early stages of Alzheimer’s
disease................................................................................................................... 31
1.4 The protein phosphatase 1E - PPM1E......................................................... 34
1.4.1 A general introduction of phosphatases................................................34
1.4.2 The structure of the PPM1E gene and the PPM1E protein.................... 36
1.4.3 Cellular localization and tissue specificity of PPM1E............................. 37
1.4.4 Upstream effectors and regulators of the PPM1E gene......................... 38
1.4.5 Proposed PPM1E effector kinases and binding partners....................... 38
1.4.6 Phenotypes induced by PPM1E............................................................45
1.5 Model systems and intentions of this study.................................................. 45
1.5.1 Animal and cellular models for Alzheimer’s disease.............................. 46
1.5.2 Intentions of this study..........................................................................47 TABLE OF CONTENTS


2 MATERIAL AND METHODS...............................................................................48
2.1 Material........................................................................................................48
2.1.1 Chemicals and consumables................................................................48
2.1.2 Biological material.................................................................................48
2.1.3 Animals..................48
2.1.4 Oligonucleotides...................................................................................49
2.1.5 Vectors..................................................................................................54
2.1.6 Plasmids, as generated in this study..................................................... 55
2.1.7 Antibodies and markers........................................................56
2.2 Methods.......................................................................................................58
2.2.1 General genetic, molecular biological and biochemical methods........... 58
2.2.2 His-hPPM1E protein expression, purification and in vitro activity
determination ...................................................................................................... 62
2.2.3 PPM1E Antibody production and characterization.................................64
2.2.4 Effects of PPM1E expression in Drosophila melanogaster.................... 66
2.2.5 Influence of PPM1E in cell lines and primary neuronal culture.............. 68
2.2.6 Expression and localization of PPM1E in human, rat and mouse brain. 76
3 RESULTS...........................................................................................................78
3.1 Characterization of tools for this study.......................................................... 78
3.1.1 Polymorphisms in the PPM1E sequence...............................................78
3.1.2 Specificity of polyclonal anti-PPM1E antibodies....................................79
3.1.3 Endogenous and ectopic expression of PPM1E in different cell lines.... 83
3.1.4 Characterization of human brain samples.............................................84
3.2 Characteristic truncation and localization of the PPM1E phosphatase ......... 88
3.2.1 PPM1E truncation and expression levels..............................................88
3.2.2 Cellular and sub-cellular localization of endogenous PPM1E................ 94
3.3 Effects of PPM1E in vitro and in primary neuronal culture...........................106
3.3.1 Eukaryotically expressed PPM1E exhibits phosphatase activity in vitro106
3.3.2 PPM1F levels do not compensate for PPM1E overexpression.............107
3.3.3 Dephosphorylation of downstream kinases in primary neuronal culture107
3.3.4 Increased PPM1E levels reduce mushroom spine density and dendritic
arborization ........................................................................................................118
3.3.5 Down-regulation of PPM1E with RNA interference affects spines and
dendritic arborization..........................................................................................125
3.3.6 Coexpression of hARHGEF6 had no effect on dendritic spines............129
4 DISCUSSION.....................................................................................................131
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TABLE OF CONTENTS


4.1 Characterization of PPM1E.........................................................................131
4.1.1 Assessment of differential PPM1E expression is based on well-
characterized human brain samples...................................................................131
4.1.2 The truncation of PPM1E is conserved in all investigated organisms...132
4.1.3 The levels of PPM1E are comparable in R. norvegicus and H. sapiens133
4.1.4 The PPM1E expression rises during development...............................134
4.1.5 PPM1E changes its localization during development...........................136
4.1.6 PPM1E is enriched in the post-synaptic compartment and at sites of high
metabolic activity................................................................................................137