Caenorhabditis elegans as an experimental model organism to study Parkinson's disease related genes [Elektronische Ressource] : functional analysis of Parkin and α-synuclein / Wolfdieter Springer

Wolfdieter Springer

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 23
• Poids de l'ouvrage: 6 Mo

Extrait

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






Caenorhabditis elegans
as an Experimental Model Organism to Study
Parkinson’s Disease-Related Genes
-
Functional Analysis of Parkin and α-Synuclein














Wolfdieter Springer


aus


Los Alamos / New Mexico / USA




2005 Erklärung

Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Prof. Dr. Ralf Baumeister betreut.




Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.



München, 24.02.05


Wolfdieter Springer








Dissertation eingereicht am 24.02.05
1. Gutachter: Prof. Dr. Ralf Baumeister
2. Gutachter: Prof. Dr. Rudolf Grosschedl
Mündliche Prüfung am 07.07.05


Meinen Eltern und Großeltern




























Thus spake Zarathustra:
“Ye have made your way from the worm to man, and much within you is still worm…"
(Friedrich Nietzsche, Zarathustra’s Prologue, 1.3) Content I
1 Summary............................................................................................................ 1
2 Introduction ....................................................................................................... 3
2.1 Clinical Characteristics and Pathology of Parkinson’s Disease .......................... 3
2.2 Pathogenesis of Parkinson’s Disease ................................................................ 5
2.2.1 Mitochondrial Dysfunction and Oxidative Stress......................................... 5
2.2.2 Proteasomal Dysfunction............................................................................ 6
2.2.3 Dysfunction of the Endoplasmatic Reticulum ............................................. 8
2.3 Aetiology of Parkinson’s Disease ..................................................................... 10
2.3.1 α-synuclein............................................................................................... 11
2.3.2 Parkin ....................................................................................................... 12
2.3.3 Other PD-Associated Genes .................................................................... 15
2.4 The Model Organism Caenorhabditis elegans.................................................. 16
2.5 Aim of the Work................................................................................................ 19
3 Results ............................................................................................................. 21
3.1 C. elegans pdr-1 is the Homolog of Human parkin........................................... 21
3.1.1 Analysis of PDR-1/Parkin Proteins ........................................................... 21
3.1.2 pdr-1 Gene Structure................................................................................ 23
3.1.3 Comparative Genomics of the pdr-1 Operon............................................ 24
3.1.4 The Downstream Gene K08E3.8.............................................................. 26
3.2 Expression Analysis of the pdr-1 Gene ............................................................ 28
3.2.1 Alternative Splicing of pdr-1...................................................................... 28
3.2.2 pdr-1 Transcription is Developmentally Regulated ................................... 29
3.2.3 pdr-1 in vivo Expression Pattern............................................................... 30
3.3 Biochemical Analysis of PDR-1 Protein 33
3.3.1 Yeast-Two-Hybrid Protein Interaction Studies.......................................... 33
3.3.2 GST-Pull Down Experiments.................................................................... 39
3.3.3 Expression and Purification of Recombinant PDR-1 ................................ 40
3.3.4 PDR-1 Mediates E3 Ubiquitin Ligase Activity ........................................... 40
3.3.5 Antibody Generation and Purification ....................................................... 41
3.4 Analyses of pdr-1 Deletion Mutants.................................................................. 42
3.4.1 Identification of Different pdr-1 Deletion Mutants...................................... 42
3.4.2 Transcriptional Analysis of pdr-1................................... 43
3.4.3 Biochemical Analysis of Mutant PDR-1 Gene Product ............................. 45
3.4.4 Phenotypicis of pdr-1(lg101) ...................................................... 47
3.5 Analyses of pdr-1 Mutants under ER Stress Conditions 48
3.5.1 The pdr-1(lg103) Mutant is Sensitized to ER Stress................................. 48
3.5.2 Rescue of the Tunicamycin Hypersensitivity ............................................ 50
3.5.3 pdr-1 is Involved in the UPR..................................................................... 52
3.5.4 is Regulated by the UPR................................................................. 54 Content II
3.6 Ectopic Expression of α-synuclein in C. elegans.............................................. 56
3.6.1 Mutant α-Synuclein Expression Leads to
Developmental Arrest and Lethality of pdr-1(lg103) ................................. 57
3.6.2 Cytotoxicity Is Dependent on Levels of both Mutant Proteins................... 60
3.6.3 Blockage of the UPR is Not Sufficient
for α-Synuclein A53T Mediated Cytotoxicity............................................. 62
3.6.4 pdr-1(lg103) and α-synuclein A53T Mediated Toxicity
is Independent of Oxidative and Heat Stress Pathways........................... 65
4 Discussion ....................................................................................................... 67
4.1 C. elegans PDR-1 Is the Functional Equivalent of Human Parkin .................... 67
4.2 PDR-1 Is Part of the UPR Pathway .................................................................. 70
4.3 PDR-1 Is Involved in the Cytosolic Stress Response ....................................... 74
4.4 PDR-1/Parkin Loss-Of-Function Vs. Gain-Of-Misfunction ................................ 76
4.5 The Biological Role of PDR-1/Parkin................................................................ 80
4.6 Outlook ............................................................................................................. 82
5 Experimental Procedures ............................................................................... 87
5.1 Microbiology Techniques.................................................................................. 87
5.2 DNA Techniques .............................................................................................. 87
5.2.1 DNA Preparation and Purification............................................................. 87
5.2.2 Plasmid Isolation from S. cerevisiae......................................................... 88
5.2.3 Plasmid Excision from Phages ................................................................. 88
5.2.4 Preparation of Genomic DNA from C. elegans......................................... 88
5.3 RNA Techniques 89
5.3.1 In vitro Transcription................................................................................. 89
5.3.2 Preparation of RNA from C. elegans ........................................................ 89
5.3.3 RT-PCR.................................................................................................... 89
5.3.4 Northern Blot Analyses............................................................................. 90
5.4 Protein Techniques........................................................................................... 90
5.4.1 Yeast-Two-Hybrid Screen ........................................................................ 90
5.4.2 Expression and Purification of Proteins from E. coli ................................. 91
5.4.3 Preparation of Yeast Protein Extracts....................................................... 92
5.4.4 Expression and Purification of Proteins from SF9 Cells ........................... 93
5.4.5 Protein Extraction from C. elegans........................................................... 93
5.4.6 In vitro Translation.................................................................................... 93
5.4.7 GST-Pull Down......................................................................................... 94
5.4.8 In vitro Ubiquitylation ................................................................................ 94
5.4.9 Production of Antiserum ........................................................................... 94
5.4.10 Affinity Purification of Antibodies .............................................................. 95

Content III
5.5 C. elegans Methods.......................................................................................... 95
5.5.1 Breeding of C. elega