In vitro and in vivo characterization of histone deacetylase inhibitors as potential therapeutics for autosomal recessive proximal spinal muscular atrophy (SMA) [Elektronische Ressource] / vorgelegt von Markus Rießland

In vitro and in vivo characterization of
histone deacetylase inhibitors as potential
therapeutics for autosomal recessive proximal
spinal muscular atrophy (SMA)











Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln

vorgelegt von
Markus Rießland
aus Köln

Köln
2009
The Doctoral Thesis "In vitro and in vivo characterization of
histone deacetylase inhibitors as potential therapeutics for autosomal recessive proximal spinal
muscular atrophy (SMA)" was performed at the Institute of Human Genetics, Institute of
Genetics and Centre for Molecular Medicine Cologne (CMMC) of the University of Cologne from
July 2005 to 2009.

























Berichterstatter/in Prof. Dr. rer. nat. Brunhilde Wirth
Prof. Dr. rer. nat. Manolis Pasparakis

Tag der letzten mündlichen Prüfung: 20.11.2009




























Für meine Eltern

ACKNOWLEDGEMENTS
First, I wish to thank my supervisor Professor Dr. Brunhilde Wirth, for giving me the opportunity
to work on various very interesting projects, for helpful discussions and encouragement, and for
generous support to attend scientific congresses and further education. I appreciate that I could
perform my thesis in Brunhilde Wirth´s laboratory.

I thank my examiners, Prof. Dr. Manolis Pasparakis and Prof. Dr. Ansgar Büschges.

A very big “Thank You!” to all past and present members of the Institute for Human Genetics and
especially of the “SMA group” for ever being nice colleagues. I would like to thank Anja Förster
for her excellent technical support and for always being motivated and interested. I thank Lutz
Garbes for very helpful discussions about virtually everything and for carefully reading this
manuscript. I thank Irmgard Hölker for staying by my left side since a quite long time and for her
helpfulness. I thank Bastian Ackermann and Sandra Kröber, for always being nice and helpful.
With you it was never boring in the lab! Thank You! A big “Thank You!” to Ylva Mende and
Miriam Jakubik, for their nice and patient way to introduce me into “mouse-work”. Moreover, I
thank Lars Brichta for all that he taught me at the beginning of my scientific carreer.

I am very grateful to Dr. Eric Hahnen for various helpful discussions, his help with the “Erlangen-
project” and supplying me with new HDACis. I thank Jan Hauke and Sebastian Seufert for being
always helpful in the lab.
I thank Karin Boß for carefully reading the manuscript.

I thank Darius Zlotos for synthesizing SAHA. I thank Prof. Sendtner for supplying us with SMA-
like mice. A big thank to J.J. Buggy and Gloucester pharmaceuticals for supplying us with PCI-
34051 and FK228. I thank Christian Tränkle for performing the in vitro HDAC activity test.

I thank Christoph Patsch for motivating discussions, being a perfect fellow student and being a
good friend.

I wish to thank Sandra Stelter for her great support in everything and for just being there.

I thank parents and my grandmother. Without their everlasting support, it would never have been
possible to study biology or to perform this work. Thank you! I thank Sonja Antweiler for being
the best big sister one can imagine. Table of contents

Table of contents

List of abbreviations ...........................................................................................................V
1 Introduction..................................................................................................... 1
1.1 The clinical picture and diagnosis of proximal spinal muscular atrophy.......1
1.1.1 Classification of proximal SMA............................................................................... 6
1.1.1.1 SMA type I (Werdnig-Hoffmann disease, MIM #253300).......................................... 6
1.1.1.2 SMA type II (intermediate form, MIM #253550)........................................................ 6
1.1.1.3 SMA type III (Kugelberg-Welander, MIM #253400) .................................................. 7
1.1.1.4 SMA type IV (adult SMA, MIM #271150).................................................................. 7
1.2 The molecular basis of proximal spinal muscular atrophy (SMA) ...................8
1.2.1 The SMN gene.......................................................................................................... 9
1.2.2 The SMN transcript ................................................................................................ 10
1.2.3 The SMN protein .................................................................................................... 13
1.2.3.1 Housekeeping functions of SMN............................................................................ 13
1.2.3.2 Neuron specific functions of SMN.......................................................................... 15
1.2.3.3 Muscle specific functions of SMN........................................................................... 16
1.3 SMA animal models.................................................................................................17
1.3.1 SMA mouse models............................................................................................... 17
1.3.1.1 Classic knock-out of murine Smn........................................................................... 17
1.3.1.2 SMN2 transgenic mice modeling an SMA phenotype ............................................. 18
1.3.1.3 Conditional Smn knock-out mice............................................................................ 19
1.3.2 Further SMA animal models .................................................................................. 19
1.4 SMA therapy..............................................................................................................20
1.5 Epigenetic chromatin modifications ....................................................................22
1.5.1 Class I histone deacetylases................................................................................. 24
1.5.2 Class IIA histone deacetylases.............................................................................. 25
1.5.3 Class IIB histone deacetylases.............................................................................. 25
1.5.4 Class IV histone deacetylase................................................................................. 25
1.5.5 Histone acetyltransferases (HATs)........................................................................ 25
1.5.6 Histone deacetylase inhibitors.............................................................................. 26
2 Aims............................................................................................................... 30
3 Materials and Methods................................................................................. 31
3.1 Fibroblasts derived from SMA patients...............................................................31
3.2 Mouse inbred strains ..............................................................................................31
3.3 Cell lines derived from SMA patients...................................................................32
3.4 Equipment and Chemicals.....................................................................................32

I Table of contents

3.4.1 Chemicals...............................................................................................................34
3.4.2 Kits..........................................................................................................................34
3.4.3 Reagents, enzymes and additional supplies for cell culture procedures............35
3.4.3.1 Reagents...............................................................................................................35
3.4.3.2 Enzymes................................................................................................................36
3.4.3.3 Additional materials for cell culture procedures.......................................................36
3.4.3.4 Additional materials for laboratory mouse in vivo procedures..................................37
3.4.3.5 Purchased human cDNA clones.............................................................................37
3.5 Antibodies.........................................