The role of K_1tnA_1tnT_1tnP channels in model systems of dopaminergic neuron loss in the ventral mesencephalon [Elektronische Ressource] / presented by Christian Scholz

ruprecht-karls-universitat_heidelberg - Christian Scholz

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129 pages

English

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Publié par	ruprecht-karls-universitat_heidelberg
Publié le	01 janvier 2008
Nombre de lectures	18
Langue	English
Poids de l'ouvrage	17 Mo

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Dissertation
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
presented by
Diplombiologe Christian Scholz
Born in Langen, Germany
Oral Examination: 31.01.2008The role of K channelsATP
in model systems of dopaminergic neuron loss
in the ventral mesencephalon
Referees: Prof. Dr. Konrad Beyreuther
Prof. Dr. Hilmar Bading
ITo my family and friends
To my love, who is both
(...)
There lies the port; the vessel puffs her sail;
There gloom the dark, broad seas. My
mariners,
Souls that have toil'd, and wrought, and thought with me,
That ever with a frolic welcome took
The thunder and the sunshine, and opposed
Free hearts, free foreheads,
you and I are old;
Old age hath yet his honor and his toil.
Death closes all; but something ere the end,
Some work of noble note, may yet be done,
Not unbecoming men that strove with Gods.
The lights begin to twinkle from the rocks;
The long day wanes; the slow moon climbs; the
Deep
Moans round with many voices.
(...)
Tho' much is taken, much abides; and tho'
We are not now that strength which in old days
Moved earth and heaven, that which we are, we are,
One equal temper of heroic hearts,
Made weak by time and fate, but strong in will
To strive, to seek, to find, and not to yield.
- Alfred Lord Tennyson, “Ulysses”
IITable of Contents
Table of Contents
1 INTRODUCTION 10
1.1 Overview 10
1.1.1 The engrailed phenotype 11
1.1.2 Possible roles of Foxa1 and Foxa2 11
1.1.3 K channels in systems of cellular stress 12ATP
1.2 The Development of Midbrain Dopaminergic Neurons 13
1.2.1 Early Development 13
1.2.2 Regionalisation of the Brain 14
1.2.2.1 Otx2 and Gbx2 14
1.2.2.2 Pax2, Pax5, Pax6 15
1.2.3 Defining the Ventral Midbrain 16
1.2.3.1 Fgf8 and Shh 17
1.2.3.2 Wnt1, Wnt3a and Wnt5a 18
1.2.3.3 Engrailed 1 and engrailed 2 (early function) 18
1.2.3.4 Tgfβ 19
1.2.4 Differentiation of the mesDA precursors 20
1.2.4.1 Lmx1a and Lmx1b 20
1.2.4.2 Engrailed 1 and engrailed 2 (late function) 21
1.2.4.3 Pitx3 23
1.2.4.4 Nurr-1 23
1.3 The mature mesDA system 26
1.4 Parkinson’s Disease (PD) 27
1.4.1 Genetic and environmental risk factors of PD 28
1.4.1.1 The recessive mutations (Parkin, DJ-1 and Pink1) 29
1.4.1.2 The dominant mutations (α-synuclein, LRRK2, UCH-L1) 31
1.4.2 Environmental risc factors 32
1.5 Major Toxic Insult systems of PD research 33
1.5.1 MPTP 33
1.5.2 Rotenone 34
1.5.3 6OHDA 35
1.6 Oxidative Stress and K channels 36ATP
1.6.1 K channels 36ATP
1.6.1.1 K channel function 36ATP
1.6.1.2 K channel structure 37ATP
1.6.1.3 Involvement of K channel function in mesDA neuron activity 37ATP
1.6.1.4 Genetic control of K channel expression in the midbrain 38ATP
1.6.1.5 Foxa1 expression is controlled by engrailed in the midbrain 38
1.6.1.6 Foxa2 38
1.7 Goals of this work 40
2 MATERIALS AND METHODS 42
2.1 Generation of mutant mouse strains and genotyping 42
2.1.1 Genomic DNA extraction 42
2.1.2 Primers and PCR settings for genotyping 43
2.2 Primary cell culture 44
2.2.1 Coating of coverslips 44
IIITable of Contents
2.2.2 Culture 44
2.2.3 Neurotoxins used on primary cell culture 45
2.2.4 Medium used for primary cell culture 45
2.3 Implantation of osmotic minipumps 45
2.4 Preparation of postnatal mouse brain sections 47
2.5 Immunohistochemistry 47
2.6 Cell counting procedures 48
2.7 Data management and statistics of primary cell cultures 48
2.8 Image processing 48
2.9 Additional methods applied in chapter 4: Miscellaneous Results 49
2.9.1 Cloning of a HNF3a/Foxa1 flox plasmid 49
2.9.1.1 Database search for Foxa1 cloning 49
2.9.1.2 Programs used for in silico cloning 49
2.9.1.3 PCR conditions and restriction digests for BAC cloning 49
2.9.1.4 Cell culture of ES cells 50
2.9.1.5 Genomic Southern Blots 51
2.9.2 Pet-1 DIG in situ hybridization on whole mount 52
2.9.3 En1/Pbx1a Tetracycline inducible expression vectors 52
2.9.4 ChIP assay 53
3 RESULTS 55
3.1 Pharmacological treatment of primary mouse ventral midbrain cultures 56
3.1.1 Culture system and concentrations for drugs and toxins 56
3.1.2 Tolbutamide alleviates rotenone-induced cell death 57
3.1.3 TEA has a positive effect on both rotenone and MPTP treated cells 58
3.1.4 Pinacidil enhances cell death in all toxin assays tested 60
3.1.5 Overall influence of drug treatment in toxin models 61
HT3.2 Targeted deletion of Sur1 rescues En mesDA phenotype 64
3.3 Preliminary results on engrailed mice and K channel action 68ATP
3.4 Pharmacological treatment of wild-type mice with osmotic mini-pumps 70
4 MISCELLANEOUS RESULTS 72
4.1 Engrailed genes are required for the development of dorsal raphe nucleus/locus coruleus
(Simon HH, Scholz C et al., 2005) 72
HT4.1.1 Serotonergic and noradrenergic cell loss in En mice 72
4.1.2 Engrailed is required at an early stage of DRN and LC development 73
4.1.3 Deficits of the DRN stem from a loss of precursor cells 74
4.2 In silico design and generation of Foxa1 knock-in targeting construct (technique
described in (Scholz et al., 2006)) 76
4.2.1 One step cloning of DNA framents of defined size 76
4.2.2 Designing and cloning the Foxa1 targeting vector 80
4.3 ChIP assay to verify binding of Pbx protein to AADC promoter 83
4.4 Lmx1b heterozygous phenotype of aged animals 85
IVTable of Contents
5 DISCUSSION 87
5.1 K channel function in cellular stress 88ATP
5.1.1 pmK activation in hypoxia and ischemia 88ATP
5.1.2 m K channels in hypoxia and ischemia 88ATP
5.1.3 K blocking and neurodegeneration 91ATP
5.1.4 Hypothesis of K function in mesDA oxidative damage 91ATP
5.1.4.1 Effect of pmK channels 92ATP
5.1.4.2 Effect of mK 92ATP
5.1.5 Differential effect of drugs and toxins 93
5.1.6 TEA: different mode of apoptosis? 93
5.1.7 Towards specifity: cellular differences between VTA and SNpc 94
5.1.8 Involvement of the engrailed genes in oxidative stress 95
5.2 Requirement of engrailed for DRN and LC 96
5.3 One step cloning – a fast way to obtain DNA fragments of defined size 97
5.4 Future perspectives 98
6 ABBREVIATIONS 100
7 INDEX 103
8 FIGURE INDEX 105
9 LITERATURE INDEX 106
VSummary
Summary
The dopaminergic neurons of the ventral midbrain (mesDA neurons) form several
distinct sub-populations which are involved in emotional control, reward behavior and
motor control. In patients with the neurodegenerative disorder Parkinsons Disease
(PD), one of these groups, the dopaminergic neurons of the substantia nigra, pars
compacta (SNpc) gradually die. One focus of research has been to find an explanation
why the SNpc neurons are more vulnerable to cellular stress than other neuronal
populations. In engrailed mutant mice, the absence of the homebox transcription
factors engrailed 1 and engrailed 2 (En1 and 2) causes a cell-autonomous and gene-
dose dependent loss of mesDA neurons. Recently, my lab identified several genes
differentially expressed between wild-type and engrailed mutant mice. Analysis of
mouse mutants of one of the genes, the forkhead containing trancription factor 1,
Foxa1 (formerly HNF3α) showed no phenotype in regard to the mesDA neurons. As
it is well known that members of the Fox/HNF family of genes can compensate for
each other, the focus of research shifted to one downstream target of Fox genes, the
expression of K channels in mesDA neurons. K channels consist of Sur1 or 2ATPATP
and Kir6.1 or 6.2 subunits and link the metabolic state of a cell to its membrane
potential. The hypothesis was, that misregulation or impairment of these channels
may lead to an increased electrical activity of the mesDA cells, putting them under
heightened physiological stress which then may in turn cause cell death. Analysis of
both Sur1 and Kir6.2 mouse mutants showed no change in TH+ cell number, cell
density or density of axonal projections, thus a loss of functional K channels hasATP
no effect on the survival of mesDA neurons. After Liss and colleagues found a
decreased cell loss when Kir6.2 mutant mesDA cells suffer a toxin insult, I revised
my hypothesis and postulated that the normal open-closed state of K channelsATP
influences cellular survival when the cell is under oxidative stress. In this work, I
show that blocking K channels in vitro by pharmaceutical means has a positiveATP
effect on cell survival when mesDA are under oxidative stress. Conversely, forced
activation of K channels under these conditions leads to an increased rate of cellATP
death. Furthermore, abolishment of K channel expression in En1-/+:En2-/- miceATP
leads to a complete rescue of the mesDA cell of the SNpc. This highlights the
importance of K channel function and may give a new direction in theATP
development of drugs targeting Parkinsons disease.
VISummary
Zusammenfassung
Die dopaminergen Neurone des ventralen Mittelhirn (mesDA) sind an der Kontrolle
von Emotionen, Belohnungserwartung und Motorfunktionen beteiligt. In Patienten
mit der neurodegenerativen Krankheit Morbus Parkinson sterben die dopaminergen
Neurone der Substantia Nigra, pars compacta, ab. Es ist unklar, warum die SNpcgegen