Engrailed genes are cell autonomously required for the survival of the mesencephalic dopaminergic neurons [Elektronische Ressource] / presented by Lavinia Alberi

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• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2004
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 48 Mo

Extrait

DISSERTATION
submitted to the
Combined Faculties for Natural Sciences and for Mathemathics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree
of Doctor of Natural Sciences
Presented by
Lavinia Alberi
born in Trieste 24.07.1974
2004ENGRAILED GENES ARE CELL AUTONOMOUSLY
REQUIRED FOR THE SURVIVAL OF THE MESENCEPHALIC
DOPAMINERGIC NEURONS
Referees: Prof. Dr. Beyreuther
Prof. Dr. Witzemann"gnoqi s’ autón"
ÂokrathsTABLE OF CONTENTS
ACKNOWLEDGMENTS........................................................................................I
ABBREVIATIONS ................................................................................................ II
SUMMARY .............................................................................................................V
INTRODUCTION................................................................................................... 1
1. General introduction.................................................................................................................................2
2. The dopaminergic system.........................................................................................................................3
2-1. Location of the midbrain DA neurons in the rodent brain ................................................................3
2-2. The dopaminergic afferent projections to the striatum .....................................................................4
2-3. The afferent projection to the mDA neurons.....................................................................................5
2-4. Function of the midbrain DA system.................................................................................................6
3. Parkinson’s disease ...................................................................................................................................7
3-1. Epidemiology and Clinical traits........................................................................................................7
3-2. Causes and pathogenesis of PD..........................................................................................................8
3-3. Mechanisms of cell death in the nervous system ..............................................................................9
3-4. Apoptosis and Parkinson Disease ....................................................................................................10
3-5. Key molecules in neuronal apoptosis...............................................................................................11
3-6. Molecular pathways for neuronal cell death in PD .........................................................................14
3-7. Models for Parkinson disease MPTP, 6-OHDA, and Rotenone....................................................17
3-8. Targeting apoptosis in Parkinson disease ........................................................................................19
3-9. Clinical treatment of PD...................................................................................................................20
4. Development of the midbrain DA neurons..........................................................................................21
4-1. Midbrain patterning and specification .............................................................................................21
4-2. Induction of the midbrain dopaminergic neurons............................................................................22
4-3. The maturation phase........................................................................................................................25
4-3-I. Extracellular factors.......................................................................................................................25
4-3-II. Intracellular mediator ...................................................................................................................25
Nurr-1 .......................................................................................................................................................26
Lmx-1b ......................................................................................................................................................27
Ptx-3..........................................................................................................................................................28
Engrailed-1 and 2.....................................................................................................................................28
4-4. The dopaminergic phenotype ...........................................................................................................29 ...........................................................................................................30
MATERIALS AND METHODS .......................................................................... 32
1. Generation and maintenance of mice...................................................................................................33
2. Genomic DNA extraction ......................................................................................................................33
3. Derivation of En-1-/- En2-/- stem cells .................................................................................................34
3-1. Preparation of feeder layer cells from mouse embryonic fibrobloblasts........................................34
3-2. Mytomicin C treatment of fibroblast feeder cells............................................................................34
3-3. De novo isolation of embryonic stem cells from E3.5 blastocysts.................................................354. Immunohistochemistry...........................................................................................................................36
4-1. Processing of the embryonic specimen............................................................................................36
4-2. Processing of the whole mounts.......................................................................................................36
4-3. Immunohistochemistry .......................................................................................................................36
5. In situ Hybridization...............................................................................................................................37
5-1. Synthesis of mouse En-1 riboprobe .................................................................................................37
5-2. In situ hybridization with mouse En-1 riboprobe............................................................................38
6. BrdU labeling...........................................................................................................................................39
7. Coverslip coating.....................................................................................................................................39
8. Threedimensional collagen gels from rat tails.....................................................................................40
9. Primary cell culture ................................................................................................................................40
10. Neurointoxication.................................................................................................................................41
11. RNA interference in vitro.....................................................................................................................41
11-1. siRNA design..................................................................................................................................41
11-2. siRNA transfection .........................................................................................................................42
12. Caspase inhibition.................................................................................................................................42
13. Real time PCR on E12.5 and E14.5 ventral midbrain tissue...........................................................43
13-1. RNA isolation .................................................................................................................................43
13-2. Reverse transcription ......................................................................................................................43
13-3. Real time PCR ................................................................................................................................44
14. Promoter analysis..................................................................................................................................45
15. Retroviral construction and infection ................................................................................................45
16. Image processing ...................................................................................................................................46
RESULTS .............................................................................................................. 47
1. Cell autonomous requirement of En1/2 in the midbrain DA neurons.............................................48
1-1. The midbrain DA neurons are induced in the En-1/2-/-, but fail to survive...................................48
1-2. Developmental expression of Engrailed in the mDA neurons .....................................