The pathological role of synphilin-1 and the therapeutic potential of Hsp70 in models of Parkinson's disease using viral vectors [Elektronische Ressource] / von Antje Krenz, geb. Elstner

Akrenz

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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therapeutic potential of Hsp70 in models of
Parkinson’s disease using viral vectors



der Fakultät für Biologie
der EBERHARD KARLS UNIVERSITÄT TÜBINGEN

zur Erlangung des Grades eines Doktors
der Naturwissenschaften

von

Antje Krenz, geb. Elstner
aus Heilbad Heilgenstadt
vorgelegte
D i s s e r t a t i o n
2010















Tag der mündlichen Prüfung: 04.12.2009
Dekan: Prof. Dr. H.A. Mallot

1. Berichterstatter: Prof. Dr. J.B. Schulz
2. Berichterstatter: Prof. Dr. O. Rieß
3. Berichterstatterin: Dr. C. Klein






Dedicated to my Husband









This study was conducted from January 2004 to December 2005 at the Hertie-
Institute for Clinical Brain Research inTübingen and from January 2006 to
December 2007 at the Department of Neurodegeneration and Restorative
Research in Göttingen under supervision of Prof. Dr. J.B. Schulz and Dr. B.H.
Falkenburger.

























“A question that sometimes drives me hazy: am I or are the others crazy?”
Albert Einstein
CONTENTS
CONTENTS
CONTENTS ................................................................................................................ 6
ABBREVIATIONS .................................................................................................... 10
1. INTRODUCTION ............................................................................................... 12
1.1. Parkinson’s disease ............................................................................................................................... 12
1.1.1. History ............................................................................................................................................... 12
1.1.2. Clinical Picture ................................................................................................................................... 12
1.1.3. Prevalence and incidence ................................................................................................................... 13
1.1.4. Diagnosis of PD ................................................................................................................................. 13
1.1.5. Pathological changes in PD ................................................................................................................ 14
1.2. Pathogenesis of PD ................................................................................................................................ 15
1.2.1. Oxidative stress and mitochondrial dysfunction ................................................................................ 16
1.2.2. Protein aggregation and impairment of the ubiquitin-proteasome system ......................................... 18
1.2.3. Cell death mechanisms....................................................................................................................... 19
1.3. PD genetics ............................................................................................................................................ 20
1.3.1. α-synuclein ......................................................................................................................................... 21
1.3.2. Synphilin-1 ......................................................................................................................................... 22
1.4. Therapeutic strategies .......................................................................................................................... 25
1.4.1. Pharm acotherapy ................................................................................................................................ 25
1.4.2. Surgical therapies ............................................................................................................................... 25
1.5. Experimental therapies................ 26
1.5.1. Neuroprotective therapies .................................................................................................................. 26
1.5.2. Molecular chaperones ........................................................................................................................ 27
1.5.2.1. Heat shock proteins ....................................................................................................................... 29
1.5.3. Viral vector-mediated gene transfer ................................................................................................... 36
1.5.3.1. Adenoviral vectors (AdV) ............................................................................................................. 37
1.5.3.2. Adeno-associated viral vectors (AAV) ......................................................................................... 39
1.5.3.3. Lentivirus (LV) ............................................................................................................................. 40
1.6. PD models .............................................................................................................................................. 42
1.6.1. Cellular models of PD ........................................................................................................................ 42
1.6.2. Animal models of PD .............. 43
1.6.2.1. Toxin-induced models ................................................................................................................... 43
1.6.2.2. Genetically modified models ........................................................................................................ 45
1.7. Objectives .............................................................................................................................................. 47
2. METHODS ........................................................................................................ 48
2.1. Molecular biology ................................................................................................................................. 48
2.1.1. Propagation and preparation of plasmid DNA ................................................................................... 48
2.1.1.1. Bacteria culture conditions ......... 48
2.1.1.2. Heat shock transformation ............................................................................................................ 48
2.1.1.3. Plasmid mini preparation .............................................................................................................. 49
2.1.1.4. id Midi, Maxi and Mega preparations ................................................................................. 49
2.1.2. Isolation of genomic DNA from mouse tail biopsies ......................................................................... 50
2.1.3. DNA precipitation .............................................................................................................................. 50
2.1.4. PCR .................................................................................................................................................... 51
2.1.5. DNA restriction, electrophoresis, gel extraction ................................................................................ 52
6 CONTENTS
2.1.6. Cycle sequencing of PCR-amplified DNA ........................................................................................ 52
2.1.7. Quantitative real-time PCR (qPCR) ................................................................................................... 53
2.1.8. Plasmid construction .......................................................................................................................... 55
2.1.8.1. Cloning into pLV-plasmid ............................................................................................................ 55
2.1.8.2. nto pAAV-plasmid ......................................................................................................... 56
2.2. Cell culture ............................................................................................................................................ 57
2.2.1. Culture conditions, transient transfection ........................................................................................... 57
2.2.2. Viral Infection .................................................................................................................................... 58
2.3. Cell death assays ................................................................................................................................... 58
2.3.1. LDH release assay ................. 58
2.3.2. Trypan blue exclusion assay .............................................................................................................. 58
2.3.3. Caspase-3 activity (DEVD-cleavage assay) ....................................................................................... 59
2.4. Proteinbiochemie .....................................................