Characterization of molecular alterations in normal appearing - edoc

196 Pages·2008·8.4 MB·English

Characterization of molecular alterations in
normal appearing white matter of Multiple
Sclerosis brain tissue and its animal model
experimental autoimmune encephalomyelitis

Inauguraldissertation

zur
Erlangung der Würde eines Doktors der Philosophie
vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät
der Universität Basel

von

Thomas Zeis

aus Fällanden, ZH

Basel, 2008
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät der Universität
Basel auf Antrag von:

Prof. Dr. Heinrich Reichert
Prof. Dr. Nicole Schaeren-Wiemers
Prof. Dr. Markus Rüegg

Basel, den 24. Juni 2008

Prof. Dr. H.-P. Hauri
Dekan
Content

Acknowledgements......................................................................................................6
List of publications........................................................................................................7
Abbreviations...............................................................................................................8
Abstract........................................................................................................................9
Introduction................................................................................................................10

Multiple Sclerosis...............................................................................................................10

History of Multiple Sclerosis.......................................................................................................10

The Epidemiology of Multiple Sclerosis.............................................................................13

The distribution of Multiple Sclerosis....................................................................................13

Environmental factors...........................................................................................................15

Genetic factors......................................................................................................................16

Clinical Features of Multiple Sclerosis.......................................................................................17

Symptoms of MS...................................................................................................................17

Disease course.....................................................................................................................17

Prognosis in MS....................................................................................................................19

MS variants...........................................................................................................................19

Pathology and Pathogenesis of Multiple Sclerosis....................................................................21

Immune system and the brain...............................................................................................21

The CNS immune privilege...................................................................................................24

The MS lesion.......................................................................................................................27

Heterogeneity of active demyelinating lesions......................................................................30

Cellular composition of MS lesions.......................................................................................32

The „normal“ white matter in MS...........................................................................................36

Axonal pathology..................................................................................................................37

Grey matter pathology..........................................................................................................38

Origin or cause of MS...........................................................................................................40

Animal models for MS................................................................................................................43

Experimental autoimmune encephalomyelitis.......................................................................43

Limitations and potential of EAE as a model for MS.............................................................45

Theiler’s virus-induced encephalomyelitis.............................................................................48

Cuprizone-induced demyelinating models............................................................................48

Impacts on therapy of MS.....................................................................................................49
Oligodendrocytes and MS..........................................................................................50
Aim of the work..........................................................................................................54
Results.......................................................................................................................55
Normal-appearing white matter in multiple sclerosis is in a subtle balance between

inflammation and neuroprotection.....................................................................................57

Abstract......................................................................................................................................58

Introduction................................................................................................................................59

Material and Methods................................................................................................................61

Results.......................................................................................................................................68

Discussion..................................................................................................................................79

Acknowledgements....................................................................................................................84
Molecular changes in white matter adjacent to demyelinating lesions in early Multiple

Sclerosis............................................................................................................................85

Abstract......................................................................................................................................86

Introduction................................................................................................................................86

Materials and Methods...............................................................................................................87

Results.......................................................................................................................................88

Discussion..................................................................................................................................95

Acknowledgements....................................................................................................................97

Molecular Changes in normal appearing tissue in an animal model of MS.......................99
Lame Ducks or Fierce Creatures?

- The Role of Oligodendrocytes in Multiple Sclerosis......................................................123

Abstract....................................................................................................................................124

Introduction..............................................................................................................................124

Oligodendrocytes - lame ducks?..............................................................................................125

Reactive or activated oligodendrocytes – pure defensive or even fierce creatures?...............130

Conclusions.............................................................................................................................136

RNA Profiling of MS Brain Tissues..................................................................................137

Abstract....................................................................................................................................138

Introduction..............................................................................................................................138

Microarrays in MS....................................................................................................................139

Summary..................................................................................................................................148

Acknowledgment......................................................................................................................148

A Chronic Relapsing Animal Model for Multiple Sclerosis...............................................149

Abstract....................................................................................................................................150

Introduction..............................................................................................................................150

Materials and Methods.............................................................................................................152

Results and Discussion............................................................................................................156

Conclusions.............................................................................................................................161

Acknowledgments....................................................................................................................162
Discussion................................................................................................................163

General discussion..........................................................................................................163
Final summary.................................................................................................................168

Outlook............................................................................................................................169
References...............................................................................................................170
Appendix A : Main Methods.....................................................................................186
Appendix B : Curriculum Vitae.................................................................................191
Acknowledgements

This work was carried out under the supervision of Prof. Nicole Schaeren-Wiemers
and supported by grants from the Swiss Multiple Sclerosis Society, the French MS
Society (ARSEP), the UK MS Society (grant number 619/01) and the National MS
Society of America (RG 3583A1/1).

First, I would like to thank Nicole for the opportunity to perform this thesis under her
supervision and for her support during this time. Furthermore, I thank Prof. Heinrich
Reichert who enabled my thesis at the University of Basel. Also, I thank Prof. Markus
Rüegg for co-referring my work.

A very HUGE thank you goes to my better half Kris, who supported and encouraged
me during my whole thesis and who was always there when I needed her……

For all the support during the years, all the good and somehow carefree times I was
able to experience, a BIG thank you goes to my parents, Josef & Johanna Zeis.

For huge support, good times in the lab, interesting & helpful discussions and
especially for many laughters, a heartfelt thank you to all the people in the lab,
namely:
Jochen, Frances, Beat, Andres, Anna, Owen, Nicole, Richard, Svenja, Eva, Bettina,
Daniela and Marie-Francoise.

Many thanks go to Dr. Anna Stalder for the indefatigable reading of my
manuscripts….

Last but not least, a special thank goes to Dr. Jochen Kinter for all the tea and coffee
time discussions ;)
List of publications

This thesis is based on the following articles submitted
to, or published in scientific journals:

Manuscripts

Zeis, T., U. Graumann, R. Reynolds, and N. Schaeren-Wiemers. 2008. Normal-
appearing white matter in multiple sclerosis is in a subtle balance between
inflammation and neuroprotection. Brain. 131:288-303. Imp. Factor: 7.61
Zeis, T., and N. Schaeren-Wiemers. 2008. Lame Ducks or Fierce Creatures? - The
Role of Oligodendrocytes in Multiple Sclerosis. J Mol Neurosci. Imp. Factor: 2.96
Zeis, T., A. Probst, A.J. Steck, C. Stadelmann, W. Brück, and N. Schaeren-Wiemers.
Molecular changes in white matter adjacent to demyelinating lesions in early
Multiple Sclerosis. Manuscript submitted
Zeis, T., J. Kinter, E. Herrero-Herranz, and N. Schaeren-Wiemers. Characterization
of autoprotective mechanisms in an animal model of Multiple Sclerosis.
Manuscript in preparation
Kinter, J., Zeis, T., and N. Schaeren-Wiemers. RNA profiling in MS brain tissue.
Int.MS.J. In press

Book Chapters

Kinter, J., Zeis, T., Schaeren-Wiemers, N. 2007. A chronic relapsing model of
Multiple sclerosis: experimental autoimmune encephalomyelitis in DA rats.
BioValley Monogr. Basel, Karger Verlag.
Abbreviations

APOE - Apolipoprotein E
CD - Cluster of Differentiation
CNP - Cyclic nucleotide phosphodiesterase
CNS - Central Nervous System
DA - Dark Agouti
EAE - Experimental Autoimmune Encephalomyelitis
EBV - Epstein-Barr Virus
HHV-6 - Human Herpes Virus 6
HLA - Human Leukocyte Antigen
Ig - Immunoglobulin
IL - Interleukine
LT - Lymphotoxin
MAG - Myelin-Associated Glycoprotein
MHC - Major Histocompatibility Complex
MOG - Myelin Oligodendrocyte Glycoprotein
MRI - Magnetic Resonance Imaging
MS - Multiple Sclerosis
MSRV - Multiple Sclerosis-associated Retrovirus
NAGM - Normal Appearing Grey Matter
NAWM - Normal Appearing White Matter
nNOS - neuronal Nitric Oxide Synthase
OG - Oligodendrocyte
RT-PCR - Reverse-Transcription - Polymerase Chain Reaction
STAT - Signal Transducer and Activator of Transcription
Tc1 - cytotoxic T cell type 1
Th1 - T helper cell type 1
Th2 - T helper cell type 2
Th17 - T helper cell type 17
TNF - Tumor Necrosis Factor
Abstract
Multiple sclerosis is a chronic, inflammatory and demyelinating disease of the CNS.
Although diffuse inflammatory damage as well as progressive axonal injury has been
shown in the chronic phase of the disease, little is known about the molecular
mechanisms underlying these pathological processes. In order to identify such
mechanisms, the gene expression profile in MS normal appearing white matter
(NAWM), was studied. Furthermore, the presence of such changes in a MS animal
model was analyzed.
A differential gene expression analysis on NAWM revealed the upregulation of genes
involved in anti-inflammatory mechanisms, such as STAT6, and genes involved in
pro-inflammatory mechanisms, such as STAT4. By immunohistochemistry, a
predominant expression of the components of the STAT6 signalling pathway in
oligodendrocytes was demonstrated. These findings suggest an endogenous
inflammatory activation throughout the whole MS NAWM, in which oligodendrocytes
actively participate. Whether such changes represent also earliest pathological
processes in MS or are due to a long, chronic disease course is unknown. Therefore,
differential gene expression of a biopsy with NAWM taken from a 17 year-old woman
during her first clinical incident was analyzed. This revealed a strong upregulation of
neuronal nitric oxide synthase (nNOS) as well as STAT6, and genes involved in
neuroprotection against oxidative stress. These findings suggest that intrinsic
inflammatory- as well as neuroprotective mechanism activation are early events in
MS NAWM, which sustain over time. To study these mechanisms in more detail, a
gene expression study in an animal model for MS was performed. For this, normal
appearing white and grey matter of DA rats with recombinant MOG-induced EAE was
analyzed. However, an induction of immune-modulating or neuroprotective genes
was not evident in EAE NAWM. Therefore, we conclude that MOG-induced EAE in
DA rat may not be a suitable model to investigate the immune-modulating or
neuroprotective mechanisms observed in MS NAWM. In contrast, a comparable
downregulation of glutamate receptors and genes encoding mitochondrial proteins as
in MS NAGM was detected in EAE NAGM.
In summary, gene expression changes characteristic of endogenous inflammatory as
well as neuroprotective mechanisms were identified in the MS NAWM, whereas
these mechanisms were not present in an animal model of this disease, leading to
the conclusion that in MS intrinsic mechanisms may take place, independent of
acute, autoimmune-mediated inflammation.
General Introduction
Introduction

Multiple Sclerosis

Multiple sclerosis is a chronic inflammatory, demyelinating disease of the CNS. It is
one of the most common diseases of the CNS in young adulthood. The hallmark of
this disease is the inflammatory plaque. Despite extensive research, the clinical
cause of MS is still unknown.
A major goal of this thesis was to unravel certain molecular aspects of this disease
with the focus on the normal appearing white matter (NAWM), which is one of the
most promising tissue to study earliest pathogenic mechanisms possibly leading to or
protecting from the formation of lesions.

History of Multiple Sclerosis
There exist several historical reports of people probably suffering from MS. One of
the first documented cases probably suffering from MS is thought to be the case of
Saint Lidwina of Schiedam (1380-1433). The Dutch nun developed from the age of
sixteen until her death at age 53 intermittent pain, weakness of the legs and vision
loss, which are typical symptoms for MS. There exists also an Icelandic saga about a
possible early case of multiple sclerosis in which a woman lost sight of both eyes and
her speech which on the following days recovered again.
However, the first illustrations and descriptions of clinical details of multiple sclerosis
were made by Robert Hooper (1773-1835), Robert Carswell (1793-1857) and Jean
Curveilhier (1791-1873) during the 1830’s (Figure 1). But it was Jean-Martin Charcot
(1825-1893) who putted previous work and his own clinical and pathological
observations together and developed the disease concept of MS (Charcot, 1868).
Later, several subforms of MS were introduced, such as Devic’s type of neuromyelitis
optica, Marburg’s acute MS and Balo’s concentric sclerosis (Balo, 1928; Devic, 1894;
Marburg, 1906).
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