FU Berlin Digitale Dissertation
Johannes H. Bauer : Apoptotic Regulation by the Huntingtin Protein Apoptotische Regulation durch das Huntingtin Protein
|Abstract| |Table of Contents| |More Information|

Abstract Huntington's Disease is a neurodegenerative disease that leads to progressive cell death of a select set of neurons. It is caused by the expansion of a stretch of glutamines in the N-terminus of the Huntingtin (Htt) protein. This gain-of-function event results in increased apoptosis of striatal neurons. Wild-type (wt) Htt is an ubiquitously expressed protein whose function remains largely unknown. Mice with a targeted disruption of the Htt gene die early in utero. In this work, the question of the function of wtHtt and the apoptotic pathways engaged by the poly-Q expanded mutant (mu) Htt is addressed. Results show that wtHtt is an anti-apoptotic protein that protects striatal cell lines from various apoptotic stimuli. Htt acts downstream of mitochondrial Cytochrome c release and interacts with the catalytic domain of Caspase-9. This interaction leads to decreased Cytochrome c-dependent cleavage of pro-Caspase-9 and decreased catalytic activity, probably through inhibition of proper apoptosome formation. The results also show that muHtt does retain some of the function of the wt protein, most markedly interaction with Caspase-9. In addition, muHtt induces apoptosis in striatal cell lines by a mechanism that involves Caspase-3 activation. Most importantly, pro-apoptotic activity of muHtt is also observed in C. elegans.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title Title page and contents Chapter I - Introduction 1 A Matter of Death 1 1.1 Necrosis 1 1.1.1 Morphological Features of Necrosis 2 1.2 Apoptosis 2 1.2.1 Morphological Features of Apoptosis 3 1.3 Apoptosis and Necrosis: A Continuum? 4 2 The Molecular Components of Apoptosis 2.1 Death Receptors 5 2.2 Bcl-2 Family 6 2.3 Apaf-1 Family 7 2.4 Caspases 7 2.5 DNases 8 2.6 Cytochrome c 9 2.7 Inhibitors of Apoptosis 9 2.8 Other molecules 10 3 Apoptotic Signaling Pathways 3.1 Mitochondria-dependent Death 12 3.2 Death Receptor Pathway 15 3.3 The C. elegans death machinery 17 4 Huntingtons Disease 4.1 Disease Phenotype 19 4.2 Molecular Basis of HD 19 4.3 The Function of wtHtt 23 4.4 Other triplet expansion diseases 24 5 The goals of this study 26 6 Figures and Legends 28 7 References 44 Chapter II - The Function 1 Introduction 57 2 Preliminary Results 2.1 Effects of Htt on viability at the non-permissive temperature 59 2.2 WtHtt prevents the appearance of apoptotic markers 60 3 Results 3.1 The anti-apoptotic function of wtHtt 61 3.2 WtHtt acts downstream of Cytochrome c-release 63 3.3 WtHtt inhibits Cytochrome c-dependent Caspase-9 activity 65 3.4 WtHtt prevents Cytochrome c-dependent cleavage of pro-Caspase-9 66 4 Discussion 68 5 Materials and Methods 73 6 Figures and Legends 76 7 References 96 Chapter III - The Mechanism 1 Introduction 97/p> 2 Results 2.1 No upregulation of heat shock proteins by Htt 99 2.2 Htt is part of a large protein complex 99 2.3 Caspase-9 interacts with both wtHtt and muHtt 103 2.4 WtHtt does not interact with Apaf-1 104 2.5 WtHtt does not interact with Bcl-2 family members 104 2.6 WtHtt preferentially interacts with the zymogen form of Caspase-9 105 2.7 WtHtt interacts with long pro-domain caspases 105 2.8 Htt binds the catalytic domain of Caspase-9 106 2.9 Interaction between endogenous proteins 106 2.10 Htt is upregulated in some cancer cell lines 107 3 Discussion 108 4 Materials and Methods 114 5 Figures and Legends 116 6 References 137 Chapter IV - The Recombinant Trap 1 Introduction 138 2 Results 2.1 Production of recombinant wtHtt and recombinant Caspase-9 139 2.2 Binding experiments 140 2.3 Htt phosphorylation by Akt 140 2.4 The final straw 141 3 Discussion 143 4 Materials and Methods 145 5 Figures and Legends 147 6 References 151 Chapter V - WORMS! 1 Introduction 152 2 Results 2.1 Htt interacts with components of the C. elegans death machinery 157 2.2 Introduction of Htt constructs into wildtype C. elegans 157 2.3 Breeding of the Htt extrachromosomal arrays into different genetic backgrounds 159 2.4 Phenotypes 161 3 Discussion 164 4 Materials and Methods 268 5 Figures and Legends 271 6 References 187 Chapter VI - Conclusion 1 Discussion 189 2 References 198 Appendix Acknowledgements 200 Thanks 201 Special Thanks 202 Abbreviations 203 Curriculum Vitae of the Author 205

More Information:
Online available:	http://www.diss.fu-berlin.de/2001/248/indexe.html
Language of PhDThesis:	english
Keywords:	Huntington's Disease, huntingtin, neurodegenerative diseases, apoptosis, caspases, apoptosome
DNB-Sachgruppe:	30 Chemie
Date of disputation:	17-Dec-2001
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Peter A. Ward, MD
Second Referee:	Prof. Dr. Ferdinand Hucho
Contact (Author):	bauer@neuron.uchc.edu
Contact (Advisor):	pward@umich.edu
Date created:	03-Dec-2001
Date available:	19-Dec-2001

 

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