The role of matrix metalloproteinase-3 and -12 in the

Bibliografische Informationen der Deutschen Bibliothek

Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen

Nationalbibliografie;

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2012

© 2012 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH,

Gießen

Printed in Germany

ISBN 978-3-86345-0

Verlag: DVG Service GmbH

Friedrichstraße 17

35392 Gießen

0641/24466

[email protected]

www.dvg.net

71-7

Department of Pathology

University of Veterinary Medicine Hannover

Center for Systems Neuroscience Hannover

The pathogenic role of matrix metalloproteinases in a virus-induced mouse model

of demyelinating diseases

THESIS Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD) awarded by the University of Veterinary Medicine Hannover

by

Florian Heinrich Hansmann Kiel

Hannover 2012

Supervisor: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ. Supervision group: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.

Prof. Dr. Peter Claus Prof. Dr. Rita Gerardy-Schahn

1st Evaluation: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.

Department of Pathology,

University of Veterinary Medicine Hannover, Germany

Prof. Dr. Peter Claus Institute of Neuroanatomy,

Medical School Hannover, Germany

Prof. Dr. Rita Gerardy-Schahn Institute for Cellular Chemistry,

Medical School Hannover, Germany

2nd Evaluation: PD Dr. Susanne Alldinger Deutsche Veterinärmedizinische Gesellschaft

Giessen

Date of final exam: 31.03.2012

Florian Hansmann received a scholarship from the National Academic Research

Foundation (Germany).

To my parents

Publications

Parts of the thesis have already been published or communicated:

HANSMANN, F., HERDER, V., KALKUHL, A., HAIST, V., ZHANG, N., SCHAUDIEN, D., DESCHL, U., BAUMGÄRTNER, W., ULRICH, R. (2012): Matrix metalloproteinase-12 deficiency ameliorates the clinical course and demyelination in Theiler’s murine encephalomyelitis Acta Neuropathol. Epub ahead of print, DOI: 10.1007/s00401-012-0942-3

HANSMANN, F., V. HERDER, H. ERNST, W. BAUMGÄRTNER (2011): Spinal epidermoid cyst in a SJL mouse: case report and literature review. J. Comp. Pathol. 145, 373-377

KUMNOK, J., R. ULRICH, K. WEWETZER, K. ROHN, F. HANSMANN, W. BAUMGÄRTNER, S. ALLDINGER (2008): Differential transcription of matrix-metalloproteinase genes in primary mouse astrocytes and microglia infected with Theiler's murine encephalomyelitis virus. J. Neurovirol. 14, 205-217 Oral presentation: HANSMANN, F., V. HERDER, H. ERNST, W. BAUMGÄRTNER (2012): Epidermoidzysten im Rückenmark von Mäusen – ein Zufallsbefund „55. Tagung der Fachgruppe Pathologie der DVG“, Fulda, Germany, March 10 - 11, 2012. Poster presentation: HANSMANN, F., ULRICH, R., HERDER, V., BAUMGÄRTNER, W. (2009): In vivo demonstration of matrix metalloproteinase-3, -9 and -12 mediated demyelination. "Abstracts of the 54th Annual Meeting of the German Society of Neuropathology and Neuroanatomy (DGNN): Neuropathology in the 21st Century", Düsseldorf, Germany, September 16 - 19, 2009. Acta Neuropathol. 118: 433-467

Table of contents

I

CHAPTER 1 GENERAL REMARK ......................................................................... 1

CHAPTER 2 GENERAL INTRODUCTION ............................................................. 3

2.1. MULTIPLE SCLEROSIS .......................................................................... 3 2.2. ANIMAL MODELS OF CENTRAL NERVOUS SYSTEM DEMYELINATION ........... 8

THEILER’S MURINE ENCEPHALOMYELITIS ........................................ 10 2.2.1.2.2.1.1. THEILER’S MURINE ENCEPHALOMYELITIS VIRUS .......................... 10 2.2.1.2. PATHOGENESIS OF EXPERIMENTALLY INDUCED THEILER’S

MURINE ENCEPHALOMYELITIS ................................................... 12 EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS ......................... 15 2.3.1. TOXIN-INDUCED DEMYELINATION .................................................... 16 2.3.2. GENETICALLY-MEDIATED DEMYELINATION ...................................... 17 2.3.3.

2.4. MATRIX METALLOPROTEINASES .......................................................... 17 CLASSIFICATION, FUNCTIONS AND ACTIVATION ................................ 17 2.4.1. TISSUE INHIBITORS OF MATRIX METALLOPROTEINASES .................... 20 2.4.2. MATRIX METALLOPROTEINASES IN DEMYELINATING CNS DISEASES .. 21 2.4.3.

CHAPTER 3 DIFFERENTIAL TRANSCRIPTION OF MATRIX-METALLOPROTEINASE GENES IN PRIMARY MOUSE ASTROCYTES AND MICROGLIA INFECTED WITH THEILER’S MURINE ENCEPHALOMYELITIS VIRUS ....................................... 23

CHAPTER 4 MATRIX METALLOPROTEINASE-12 DEFICIENCY AMELIORATES THE CLINICAL COURSE AND DEMYELINATION IN THEILER’S MURINE ENCEPHALOMYELITIS .................................................................. 25

CHAPTER 5 SPINAL EPIDERMOID CYST IN A SJL MOUSE: CASE REPORT AND LITERATURE REVIEW ........................................... 27

CHAPTER 6 DISCUSSION AND CONCLUSIONS ............................................... 29

6.1. MATRIX METALLOPROTEINASE TRANSCRIPTION IN VITRO ....................... 30 6.2. STEREOTAXIC INJECTION OF MATRIX METALLOPROTEINASES ................. 30 6.3. INFECTION OF MMP-3 AND -12 KNOCK-OUT MICE WITH TMEV .............. 32 6.4. EPIDERMOID CYSTS IN THE SPINAL CANAL OF MICE ............................... 34 6.5. CONCLUDING REMARK ....................................................................... 35

CHAPTER 7 SUMMARY ....................................................................................... 37

CHAPTER 8 ZUSAMMENFASSUNG ................................................................... 39

CHAPTER 9 REFERENCES ................................................................................. 41

CHAPTER 10 ACKNOWLEDGEMENTS ................................................................ 59

Abbreviation list

III

Abbreviation list

ADAMs = a disintegrin and metalloproteinases ADAMTs = a disintegrin and metalloproteinases thrombospondin BBB = blood brain barrier CCP = caudal cerebellar peduncle CNPase = 2',3'-cyclic nucleotide 3'-phosphodiesterase CDV = canine distemper virus CNS = central nervous system DTH = delayed type hypersensitivity reaction EAE = experimental autoimmune encephalomyelitis ECM = extracellular matrix ECs = epidermoid cysts Ig = immunoglobulin IL = interleukin JHM = John Howard Mueller MAG = myelin-associated glycoprotein MBP = myelin basic protein MOG = myelin oligodendrocyte glycoprotein MHC = major histocompatibility complex MMPs = matrix metalloproteinases MS = Multiple sclerosis MT-MMPs = membrane type matrix metalloproteinases TIMPs = tissue inhibitors of metalloproteinases TME = Theiler’s murine encephalomyelitis TMEV = Theiler’s murine encephalomyelitis virus TNF = tumor necrosis factor p.i. = post infection PLP = proteolipid protein PPMS = primary progressive multiple sclerosis PRMS = progressive relapsing multiple sclerosis RRMS = relapsing and remitting multiple sclerosis SPMS = secondary progressive multiple sclerosis

Chapter 1: General remark

1

Chapter 1 General remark

The main issue of this thesis was to elucidate the contribution of matrix

metalloproteinases (MMPs) to the process of demyelination in Theiler’s murine

encephalomyelitis (TME) as a model of demyelinating diseases such as multiple

sclerosis (MS) or canine distemper encephalitis. Special emphasis was given to the

question whether MMPs contribute to demyelination directly by destruction of myelin

and/or oligodendrocytes or indirectly by facilitating infiltration of inflammatory cells,

breakdown of the blood-brain-barrier (BBB) or alteration of the extracellular matrix.

To address these questions two animal experiments were performed: firstly, in vitro

activated, recombinant, murine MMP-3, MMP-9 and MMP-12 were stereotactically

injected into the brainstem of adult SJL-mice. Secondly, MMP-3 and MMP-12 knock-

out as well as wild-type mice were intracerebrally infected with the BeAn strain of

TME virus (TMEV) and the clinical course as well as histopathological alterations

were studied.

The results from these studies aimed to clarify whether MMP-3 and/or MMP-12 are

key mediators in the pathogenesis of demyelinating diseases and represent suitable

target molecules for future therapeutic approaches.

Chapter 2: General introduction

3

Chapter 2 General introduction

2.1. Multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system

(CNS) affecting more than 2 million people worldwide with a variable disease course

(KEMPPINEN et al. 2011, NOSEWORTHY 1999). The occurrence of MS is more

frequent in North America and Europe while Asians and Africans are less frequently

affected (COMPSTON et al. 2008, HAUSER et al. 2006, ROSATI 2001). MS was

firstly described in 1868 by Jean-Martin Charcot and named Encephalomyelitis

disseminata or Charcot’s disease (LASSMANN 2005). This disease is characterized

by the development of demyelinating lesions in the CNS (LUCCHINETTI et al. 2000).

A typical hallmark of MS is formation of sclerotic plaques, which represents the end

stage of a process including inflammation, demyelination, eventual remyelination

(shadow plaques), oligodendrocyte depletion, astrocytosis as well as neuronal and

axonal degeneration/loss (COMPSTON et al. 2008, FRANKLIN 2002, LASSMANN et

al. 2007). Demyelination, followed by neurodegeneration and axonal loss is

responsible for a wide range of symptoms including motor deficits, disturbances in

cognition and vision as well as urinary incontinence and sexual dysfunction

(PANITCH et al. 2011, ZWIBEL et al. 2011).

The etiology of MS is still unknown, but it is suspected to be an immune-mediated,

infectious, inflammatory or degenerative disease or a combination of these

processes (STEINMAN et al. 2006). Environmental risk factors including infectious

agents like Epstein-Barr virus (SERAFINI et al. 2007), human herpesvirus 6

(SOLDAN et al. 1997), measles virus and Chlamydophila pneumoniae (SRIRAM et

al. 1998), as well as vitamin D levels, diet, sunlight, smoking and a genetic

susceptibility are under discussion (COHEN 2009, COMPSTON et al. 2008,

KEMPPINEN et al. 2011, MIROWSKA-GUZEL et al. 2009). Genome-wide

association studies in MS revealed 16 loci with significant contribution to the disease

(KEMPPINEN et al. 2011). Genetic factors which have been associated with MS

include genes of the major histocompatibility complex (OKSENBERG et al. 2005),

however a genetic basis alone cannot be responsible for the disease as, for example

concordance in identical twins is less than 50% (STEINMAN et al. 2006). Recently


4

chronic cerebrospinal venous insufficiency (CCSVI) has been suggested to be

associated with MS (ZAMBONI et al. 2009, ZAMBONI et al. 2009). However, other

groups reported that CCSVI plays no role in MS risk and disease severity

(BARACCHINI et al. 2011, CENTONZE et al. 2011).

Diagnosis of MS includes clinical and laboratory investigation with a need to

demonstrate dissemination of lesions in space and time as well as excluding

differential diagnoses (POLMAN et al. 2011). MS typically starts at an age of 20 to 40

years and the disease is more frequent in females than in males. MS can be divided

into different categories based on the clinical course: relapsing-remitting (RRMS;

Figure 1A), primary progressive (PPMS, Figure 1B), secondary progressive (SPMS,

Figure 1C) and progressive-relapsing (PRMS, LUBLIN et al. 1996).

Figure 1: Clinical courses of MS (adapted from LUBLIN et al. 1996)


5

RRMS is thought to reflect occasional inflammatory bursts in the CNS, while the

accumulating disability in progressive MS is believed to be due to neurodegeneration

(KEMPPINEN et al. 2011). During RRMS recovery from each episode becomes

increasingly incomplete and persistent symptoms accumulate (FRANKLIN 2002).

Clinically, MS starts as RRMS with a highly variable relapse frequency per year in

about 85% of the patients (TRAPP et al. 2008). About 65% of patients enter the

secondary progressive phase while the illness is primary progressive in about 20% of

the patients (COMPSTON et al. 2008). Statistically, PPMS starts about 10 years later

than RRMS and female and male subjects are equally affected (TRAPP et al. 2008).

Ten years following disease onset about 50% of MS patients are not able to care of

oneself and are unfit for work (TRAPP et al. 2008). On average the life expectation of

MS patients is about seven to eight years shorter compared to the normal population

(TRAPP et al. 2008). Current therapies reduce the relapse frequency between 33 to

66% and moderately delay disease progression of RRMS and SPMS (ROPPER

2006, STEINMAN et al. 2006).

Histologically, MS is characterized by an infiltration of inflammatory cells followed by

demyelination, loss of oligodendrocytes, astrogliosis as well as axonal degeneration

and loss (HUNTER et al. 1995, LASSMANN et al. 2007, TSUNODA et al. 2002).

Different staging systems for MS lesions exist, e.g. “Bö/Trapp staging system” or the

“Lassmann/Brück staging system” (VAN DER VALK et al. 2000). The staging system

according to Lassmann/Brück discriminates between 5 different categories as shown

in Table 1.


6

Table 1: Lassmann/Brück staging system (adapted from VAN DER VALK et al. 2000)

Stage Characteristics

Early active lesions - Macrophages contain myelin proteins and lipids

- Macrophages express MRP14

Late active lesions

- Macrophages contain myelin debris which is

luxol fast blue, myelin basic protein and

proteolipid protein positive but myelin

oligodendrocyte glycoprotein negative

- Macrophages are 27E10 positive but myelin

related protein 14 negative

Inactive lesion

- Macrophages are PAS positive

- Macrophages are MRP14 negative and do not

contain myelin breakdown products

Early remyelinating lesions - Numerous lymphocytes and macrophages

- Clusters of thinly myelinated axons

Late remyelinating lesions

(shadow plaques)

- Few macrophages

- Astrogliosis

- Numerous thinly myelinated axons

In actively demyelinating MS lesions four different patterns of demyelination have

been described investigating the following criteria: myelin protein loss, plaque size

and distribution, pattern of oligodendrocyte destruction and immunopathological

evidence of complement activation (LASSMANN et al. 2001, LUCCHINETTI et al.

2000). Investigations of MS autopsy cases show that patterns of demyelination are

homogeneous within active lesions from the same patient but heterogeneous

between patients leading to the hypothesis that MS possibly has a heterogeneous

etiology (Table 2, LUCCHINETTI et al. 2000). According to Lucchinetti et al. (2000),

pattern I is characterized by a T-lymphocyte- and macrophage-mediated

demyelination accompanied by myelin debris. In this pattern a delayed type

hypersensitivity (DTH) is suspected to be the main pathomechanism. Pattern II is

characterized by intralesional deposition of immunoglobulins, mainly IgG. A


7

hypersensitivity type II reaction is considered to contribute to lesion development in

this pattern. The striking feature of pattern III is a preferential loss of myelin

associated glycoprotein (MAG) while other myelin proteins like myelin basic protein

(MBP) and proteolipid-protein (PLP) are still present, suggesting a peripheral

oligodendropathy. In addition, a pronounced loss of oligodendrocytes at the border of

actively demyelinating plaques and an inactive plaque center almost devoid of

oligodendrocytes is characteristic for this pattern. Ischemia or virus-infection are the

suspected causes of this pattern. Pattern IV resembles demyelination following

oligodendrocyte death but in contrast to pattern III without preferential loss of the

peripheral MAG. Toxic processes are thought to be responsible for this pattern.

Table 2: Pattern of actively demyelinating MS lesions (according to LUCCHINETTI et al. 2000)

Pattern Morphological characteristics and suggested pathomechanism

I - T-lymphocytes, macrophages - Hypersensitivity type IV reaction

II - T-lymphocytes, macrophages - Immunoglobulin and complement deposition - Hypersensitivity type II reaction

III

- T-lymphocytes, macrophages, activated microglia cells - General loss and periaxonal alteration of myelin associated

glycoprotein-immunoreactivity - Pronounced loss of oligodendrocytes at the active plaque border - Inactive center of the plaque is almost completely devoid of

oligodendrocytes - Remyelinated shadow plaques are absent - Peripheral oligodendropathy (degeneration of myelin prior to

destruction of oligodendrocytes)

IV

- T-lymphocytes, macrophages - Deposition of immunoglobulins and complement is absent - Demyelination associated with oligodendrocyte death in a small

rim of periplaque white matter - Central oligodendropathy (myelin degenerates as consequence

of oligodendrocyte destruction)


8

2.2. Animal models of central nervous system demyelination

One important obstacle in MS research is, that only limited tissue samples of early

and immunologically active MS lesions are available. Accordingly, modifications of

experimental circumstances are much more limited in human studies compared to

studies in animal models (DENIC et al. 2011). Animal models are necessary to get

an insight into specific aspects of demyelinating diseases and to establish and/or test

novel therapeutic approaches (Table 3). One single animal model is not able to cover

and adequately incorporate all clinical, pathological and predisposing features of MS

(DENIC et al. 2011). The most commonly used animal models of CNS demyelination

and inflammation can be divided into virus-induced, autoimmune-mediated, toxin-

induced and genetic models. Important virus-induced animal models include infection

of mice with Theiler’s murine encephalomyelitis (DAL CANTO et al. 1996,

DRESCHER et al. 2008, OLESZAK et al. 2004), the John Howard Mueller (JHM)

strain of mouse hepatitis virus (BAILEY et al. 1949, CHEEVER et al. 1949, TIROTTA

et al. 2010), Visna/Maedi virus infection in sheep (PALSSON 1976, PETURSSON et

al. 1978), and canine distemper encephalitis (BEINEKE et al. 2009, SEEHUSEN et

al. 2009, VANDEVELDE et al. 2005). Experimental autoimmune encephalomyelitis

(EAE) represents a suitable model for studying immune-mediated de- and

remyelination (CROXFORD et al. 2011, DENIC et al. 2011, MIX et al. 2010). The

most commonly used agents inducing toxin-mediated demyelination include

cuprizone (BLAKEMORE 1972, BLAKEMORE 1973, HERDER et al. 2011), ethidium

bromide (HANSMANN et al. 2012, WOODRUFF et al. 1999), lysolecithin

(RODRIGUEZ 2007) and a mixture of anti-galactocerebroside antibody and guinea

pig complement (WOODRUFF et al. 1999). Animals showing mutations in myelin

proteins like PLP or MBP (DUPOUEY et al. 1979, GRIFFITHS 1996, GRIFFITHS et

al. 1990, PRIVAT et al. 1979), genetic defects leading to a reduced number of

oligodendrocytes (SKOFF 1976) or a disturbed cholesterol biosynthesis leading to a

severely reduced rate of myelin synthesis (SAHER et al. 2005) can be used to study

the effects of specific genes/gene products on the integrity of the myelin sheath,

axonal preservation and neuronal loss.


9

Table 3: Animal models of central nervous system demyelination

Category Agent Reference

Virus-induced

demyelinatinon

Mouse hepatitis virus, John Howard Mueller strain Coronaviridae, Betacoronavirus

BAILEY et al. 1949 CHEEVER et al. 1949 TIROTTA et al. 2010

Theiler´s murine encephalomyelitis virus Picornaviridae, Cardiovirus

DAL CANTO et al. 1996 DRESCHER et al. 2008 OLESZAK et al. 2004

Visna/Maedi virus Retroviridae, Lentivirus

PETURSSON et al. 1978 PALSSON 1976

Canine distemper virus Paramyxoviridae, Morbillivirus

BEINEKE et al. 2009 SEEHUSEN et al. 2009 VANDEVELDE et al. 2005

Semliki forest virus Togaviridae, Alphavirus

FAZAKERLEY et al. 2006 SUCKLING et al. 1978

Autoimmune-

mediated

demyelination

Crude brain preparation in Freund´s adjuvant Fragments of: - Myelin basic protein (MBP) - Myelin oligodendrocyte glycoprotein (MOG) - Myelin associated glycoprotein (MAG) - Proteolipid-protein (PLP)

CROXFORD et al. 2011 DENIC et al. 2011 MIX et al. 2008 MIX et al. 2010

Toxin-induced

demyelination

Cuprizone BLAKEMORE 1972 BLAKEMORE 1973 HERDER et al. 2011

Ethidium bromide HANSMANN et al. 2012 WOODRUFF et al. 1999

Lysolecithine RODRIGUEZ 2007

Anti-galactocerebroside antibody + complement WOODRUFF et al. 1999

Genetically-

mediated

demyelination

Rumpshaker mouse (PLP mutant) GRIFFITHS et al. 1990 GRIFFITHS 1996

Shiverer mouse (lacks MBP) PRIVAT et al. 1979 DUPOUEY et al. 1979

Jimpy mouse (reduced oligodendrocyte number) SKOFF 1976

Alterations in myelin-biosynthesis SAHER et al. 2005

Transgenic TNF overexpressing mice (Tg6074) TSEVELEKI et al. 2010


10

Theiler’s murine encephalomyelitis 2.2.1.

TME is an important, virus-induced model of MS for the following reasons: firstly the

host immune response plays a major role in the pathogenesis of myelin destruction,

secondly virus infection can be studied in its natural host and thirdly the chronic

progressive course of the clinical signs and morphological changes share many

similarities with the chronic progressive form of MS, whereas most other models

display a relapsing and remitting course (DAL CANTO et al. 1996, DAL CANTO et al.

1982, LIPTON 1975, STOHLMAN et al. 2001, ULRICH et al. 2008).

2.2.1.1. Theiler’s murine encephalomyelitis virus

TME virus (TMEV) was first isolated from the CNS of naturally infected young mice

with flaccid paralysis (THEILER 1934). TMEV is a naturally occurring mouse

pathogen belonging to the family of Picornaviridae genus Cardiovirus (DAL CANTO

et al. 1982, LIPTON et al. 2001). Virions have an icosahedral symmetry, are non-

enveloped, measure 25 to 29 nm in diameter and contain a single-stranded RNA of

positive polarity (RACANIELLO 2007). The normal route of transmission is oral/fecal

followed by a gastrointestinal infection that is only rarely complicated by a CNS

disease (THEILER 1937). However, the neurological disease can be experimentally

induced in a high percentage of mice employing intracerebral inoculation (BRAHIC et

al. 2005). TMEV strains can be divided into two subgroups such as high-

neurovirulent (GDVII, FA) and low-neurovirulent (Theiler’s original [TO]), including

BeAn, DA, Yale, WW] substrains (DANIELS et al. 1952, MICHIELS et al. 1995,

MONTEYNE et al. 1997, PEVEAR et al. 1988, ROZHON et al. 1983, THEILER 1937,

THEILER et al. 1940). High-neurovirulent TMEV strains are known to cause a fatal

polioencephalitis while the low-neurovirulent DA and BeAn strains induce a biphasic

disease characterized by a mild initial polioencephalitis followed by a chronic

progressive demyelinating leukoencephalomyelitis in susceptible mouse strains

(DANIELS et al. 1952, LIPTON 1975, LIPTON 1980, TSUNODA et al. 1996,

ZOECKLEIN et al. 2003). Genes which have been found to determine susceptibility

or resistance of different mouse strains to persistent TMEV infection (Table 4) include


11

major histocompatibility complex (MHC) and non-MHC genes (DAL CANTO et al.

1996, RODRIGUEZ et al. 1986).

Table 4: Susceptibility of different mouse strains to persistent TMEV infection (according to DAL CANTO et al. 1996)

Susceptibility to persistent TMEV infection Mouse strain

Highly susceptible SJL/J, DBA/1, DBA/2, SWR, PL/J, NZW

Intermediate susceptible C3H, CBA, AKR, C57BR

Mostly resistant BALB/c, C57BL/6, C57BL/10, C57/L, 129/J

In addition, gene loci which have been mapped to be associated with susceptibility to

TMEV induced demyelination include H-2D locus (on chromosome 17; RODRIGUEZ

et al. 1986), Tmevd-1 locus (on chromosome 6; KAPPEL et al. 1991, MELVOLD et

al. 1990) and Tmevd-2 locus (on chromosome 3; MELVOLD et al. 1990). Some gene

loci (e.g. H-2D and Tmevd-1) are involved in T-cell regulation which further

substantiates the hypothesis that demyelination in TME is immune-mediated (DAL

CANTO et al. 1996). Intracerebral infection of mice with the BeAn strain of TMEV

mice induces an acute polioencephalitis, characterized by prominent infection of

mainly neurons and some glial cells. This early and acute phase of the disease has a

duration of approximately two weeks, after which the virus is either cleared in

genetically resistant mice or persists in the CNS in genetically susceptible mice

(LIPTON 1975). In genetically susceptible mice first clinical evidence of white matter

involvement can be seen about four weeks post infection (p.i.) when mice start to

show a wobbling gait (DAL CANTO et al. 1996). Clinical signs like weakness of the

posterior limbs followed by continuously progressive ataxia leading to spastic

paralysis and in final stages urine incontinence can be observed (LIPTON 1975,

MCGAVERN et al. 2000, MCGAVERN et al. 1999). Cells of viral persistence include

glial cells of the spinal cord white matter, mainly microglia/ macrophages and to a

lesser extent oligodendrocytes as well as astrocytes (AUBERT et al. 1987, BRAHIC

et al. 1981, LIPTON et al. 1995, RODRIGUEZ et al. 1986). In addition, the

cytoplasmic channels of myelin are a site of viral expression during virus persistence

(RODRIGUEZ et al. 1983). Since TMEV infects oligodendrocytes in vivo and in vitro,


12

direct lytic infection of oligodendrocytes can result in demyelination without

inflammation (SATO et al. 2011). Furthermore, repeated restraint stress has been

shown to facilitate virus spread from the CNS to systemic organs like spleen, lymph

nodes, thymus, lung and heart compromising the ability of viral clearance within

those organs (MI et al. 2006). In vitro studies substantiate that glial progenitor cells,

oligodendrocytes, astrocytes, neurons as well as microglia/macrophages are

susceptible to TMEV infection (KUMNOK et al. 2008, O'SHEA et al. 1997,

PRINGPROA et al. 2010, WROBLEWSKA et al. 1979).

2.2.1.2. Pathogenesis of experimentally induced Theiler’s murine encephalomyelitis

The outcome of TMEV infection depends on mouse strain (Table 4), sex, virus strain

and age of the mice at time of infection (Figure 2; BRAHIC et al. 2005, MONTEYNE

et al. 1997). Following intracerebral TMEV infection of mice, initial virus replication

takes place in gray matter neurons causing a polioencephalitis followed by an axonal

virus spread during the first weeks post infection (DAL CANTO et al. 1982,

MARTINAT et al. 1999, TSUNODA et al. 2003). Depending on the mouse strain

(Table 4) and on the virus strain such as high-/low-neurovirulent the virus is

eliminated or the acute phase is followed by a progressive demyelinating disease in

the chronic phase (LIPTON 1975, ZOECKLEIN et al. 2003). Both, susceptible and

resistant strains can be infected by TMEV, but only those strains that can mount anti-

TMEV DTH-responses will develop demyelination and inflammation of the spinal cord

white matter (CLATCH et al. 1986, CLATCH et al. 1987, MILLER et al. 1987,

MILLER et al. 1990).


13

Figure 2: Outcome of TMEV infection (according to DAL CANTO et al. 1996).

Axons play an important role in virus distribution because they are necessary for

virus trafficking from brain to spinal cord. In addition, TMEV is able to traffic from the

axon into the surrounding myelin (ROUSSARIE et al. 2007). However, a

hematogenous and/or liquorogenic virus spread within the CNS cannot completely be

ruled out. Axonal degeneration prevents virus spread via axonal transport within the

CNS in hosts infected with low-neurovirulent strains (e.g. DA or BeAn) while it is

detrimental to hosts infected with high-neurovirulent TMEV strains (e.g. GDVII;

TSUNODA et al. 2008). For low-neurovirulent TMEV strains it has been shown that

axonal degeneration precedes demyelination, meaning that lesions develop from the

axon to the myelin sheath - inside-out model (TSUNODA et al. 2002). Since axonal


14

degeneration has been shown to be a self-destructive physiological process during

development, axonal degeneration in TMEV infected mice may be a self-destructive

defense mechanism that protects from the transport of toxic substances and viruses

in the CNS (DAL CANTO et al. 1975, SATO et al. 2011). In addition, TMEV causes

apoptosis of neurons and oligodendrocytes in the CNS in vivo (TSUNODA et al.

2008). In this context apoptosis of virus-infected cells and adjacent uninfected cells

can be interpreted as a mechanism protecting the CNS against virus spread

(TSUNODA et al. 2008).

In TMEV-infection cellular immune responses seem to play a protective and

pathogenic role. Most studies using the BeAn strain of TMEV suggest that CD4+

lymphocytes are directly involved in the disease because mice depleted of CD4+ T-

cells prior to TMEV infection die within 3-5 weeks (BORROW et al. 1993).

Furthermore, treating mice with antibodies to CD4+ T-cells after viral infection but

before disease onset can prevent the development of a demyelinating disease

(MILLER et al. 1994). In addition, in the CNS of TMEV infected mice CD4+

lymphocytes in contrast to CD8+ lymphocytes have been shown to express IL-2

receptors (MILLER et al. 1994). IL-2 is known as an important indicator of

lymphocyte activation, supporting an important role of T-helper cells in TMEV-

induced demyelination (DAL CANTO et al. 1996). However, prevention of the

disease has also been shown after inoculation of antibodies to CD8+ T-cells

(RODRIGUEZ et al. 1988). In addition, in lymph nodes of mice immunized with

TMEV, production of IL-2, TNF-α and IFN-γ indicative of a Th1 response has been

observed. However, a production of IL-4, IL-6 or IL-10 indicative of a Th2 response

not detected (MILLER et al. 1994, PETERSON et al. 1993). Furthermore, anti-TMEV

antibodies produced by susceptible mice are mainly of the IgG2a or IgG2c subclass

in SJL/J mice (ULRICH et al. 2010) which is dependent on stimulation by Th1

cytokines (PETERSON et al. 1992). However, chronic progressive demyelination

during TME is dependent on virus persistence within the CNS (LIPTON et al. 2005).

Virus antigen can be localized by immunohistochemistry in macrophages in and

around white matter lesions as well as in other inflammatory cells, astrocytes and

oligodendrocytes (DAL CANTO et al. 1982). An important mechanism contributing to


15

demyelination during TME is a DTH-reaction which primarily targets virus epitopes

but later detects myelin epitopes, a process called epitope spreading (CLATCH et al.

1986, MILLER et al. 2001). Hereby a specific role of CD4+ T-cells is suspected (DAL

CANTO et al. 1996). CD8+ T-cells are important for virus clearance while virus-

specific and auto-reactive CD8+ T-cells have been suggested to contribute to

demyelination (TSUNODA et al. 2008). In addition, anti-TMEV antibodies can help to

eliminate the virus whereas some anti-viral antibodies cross-react with host myelin

molecules including galactocerebroside (TSUNODA et al. 2008).

Taken together, TME-induced demyelination resembles the clinical course observed

in PPMS or SPMS. Important underlying mechanisms have been shown to be a

MHC-II restricted, CD4+ T-cell mediated DTH-reaction (CLATCH et al. 1985, MILLER

et al. 2001) as well as an intrathecal antibody production (PACHNER et al. 2007,

PACHNER et al. 2007, YAMADA et al. 1990) resembling most features observed in

MS lesions (pattern I and/or II; LUCCHINETTI et al. 2000). In addition, depending on

the virus strain, a variable degree of demyelination due to virus-induced

oligodendrocyte loss can be observed (ZOECKLEIN et al. 2003). This indicates that

some features observed in TME may resemble pattern III or IV of MS lesions

according to Lucchinetti et al. (2000).

Experimental autoimmune encephalomyelitis 2.3.1.

EAE is a suitable model to investigate the autoimmune-mediated hypothesis of MS. It

is a cell-mediated disease which can be induced by injection of CNS-tissue, myelin or

myelin antigens like MBP, PLP or MOG dissolved in complete Freund’s adjuvant

(BEN-NUN et al. 1981, LASSMANN 2004, LININGTON et al. 1992, VAN DER VEEN

et al. 1986) or via transfer of primed T-lymphocytes (PATERSON 1960). EAE is

widely used to investigate pathogenic, diagnostic and therapeutic aspects of MS

(LINDSEY 2005). The immune response in EAE targeting myelin epitopes resembles

a MHC-II restricted DTH-reaction leading to an inflammation of the white matter

(ZAMVIL et al. 1985). Besides auto-reactive T-lymphocytes a variable amount of

humoral factors can also be involved in the process of demyelination, depending on


16

the experimental settings. Histopathology and clinical course of the disease show a

significant variation depending on the source of the antigenic material, the mode of

its application and the genetic background of the animals (MIX et al. 2008). Lesions

observed during EAE depend on the experimental setting (e.g. antigen used for

immunization) and are most similar to pattern I or II according to Lucchinetti et al.

(2000).

Toxin-induced demyelination 2.3.2.

Demyelination of the CNS can be induced by a systemic administration of a toxicant

(e.g. cuprizone; BLAKEMORE 1972, HERDER et al. 2011, LINDNER et al. 2008) or

by focal injection of toxic substances (e.g. ethidium bromide, lysolecithine, anti-

galactocerebroside antibodies combined with complement; HANSMANN et al. 2012,

RODRIGUEZ 2007, WOODRUFF et al. 1999). Toxin-induced demyelination

represents a suitable model for studying the process of de- and remyelination as well

as microglial responses and axonopathies, independent from the systemic immune

response (HANSMANN et al. 2012, LINDNER et al. 2009). The time course of de-

and remyelination depends on the target of the toxicant, especially if oligodendrocyte

precursor cells and myelin producing oligodendrocytes are affected. When both cell

populations are affected the process of remyelination is markedly delayed

(WOODRUFF et al. 1999). In detail, demyelination induced by anti-

galactocerebroside antibodies and complement application, targeting mainly

oligodendrocytes and myelin, is followed by a relatively fast oligodendrocyte-

mediated remyelination while ethidium bromide induced demyelination, targeting

mostly all cell types, is followed by a slow and sometimes incomplete process of

remyelination involving oligodendrocytes and Schwann cells (WOODRUFF et al.

1999). This model of focally induced demyelination can be used to study the effects

of therapeutic treatments like growth factor application or cell transplantation

employing stereotaxic injection of substances and/or cells into the same location

where initially demyelination has been induced. Toxin-induced models of

demyelination have the disadvantage that they are not helpful in elucidating the


17

primary cause of demyelination in MS, although they represent suitable approaches

to study the process of de- and remyelination. Depending on the specificity of the

toxicant targeting myelin and/or oligodendrocytes the observed lesions resemble

pattern III and/or IV according to Lucchinetti et al. (2000).

Genetically-mediated demyelination 2.3.3.

MS is believed to be a combination of environmental risk factors and genetic

susceptibility (COMPSTON et al. 2008). To date, 10 genome-wide association

studies in MS have been conducted which confirmed 16 loci of MS risk variants with

genome-wide significance (KEMPPINEN et al. 2011). In models of genetically-

induced demyelination like “rumpshaker mouse” (mutated PLP protein; GRIFFITHS

1996, GRIFFITHS et al. 1990) or “jimpy mouse” (lacks MBP; DUPOUEY et al. 1979,

PRIVAT et al. 1979) loss/lack of myelin takes place following a specific pattern due to

a lack or misfolding of a specific protein. These models can help to understand the

contribution of the respective protein and related biological processes to the integrity

of the myelin sheath/cytoplasmic membrane but they are not able to simulate such a

complex situation as it is present in MS.

2.4. Matrix metalloproteinases

Classification, functions and activation 2.4.1.

Metalloproteinases are a huge group of enzymes including the families matrix

metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a

disintegrin and metalloproteinases thrombospondin (ADAMTs). MMPs were first

discovered in 1962 during frog metamorphosis (GROSS et al. 1962). They are a

group of more than 20 zinc-dependent endopeptidases, which can be classified

based on their structural domain arrangement (Table 5, Figure 3). MMPs are

important enzymes involved in extracellular matrix (ECM) remodeling. They are able

to degrade nearly all proteins of the ECM (NAGASE et al. 1999) and are also

involved in execution of intracellular functions (CAUWE et al. 2010). In adult mice a


18

constitutive expression of Mmp2, -3, -7, -9, -11, -12, -13, -14, -15, -24 as well as

Timp1, -2, -3, -4 has been shown within the spinal cord under physiological

conditions while Mmp10 was not detectable (ULRICH et al. 2005). During

development or in pathological conditions (e.g. spinal cord injury or demyelination) an

immediate upregulation of MMPs can be observed (ULRICH et al. 2006, ULRICH et

al. 2005, WELLS et al. 2003). Due to their proteolytic capabilities, MMPs are tightly

regulated at the transcriptional, translational and post-translational level (YONG et al.

2001). Table 5: Classification of the mammalian family of matrix metalloproteinases based on their

domain arrangement (adapted from FANJUL-FERNANDEZ et al. 2010)

Group Subgroup Enzymes

Archetypical MMPs

Collagenases MMP-1, MMP-8, MMP-13

Stromelysins MMP-3, MMP-10

Other MMPs MMP-12, MMP-19, MMP-20, MMP-27

Gelatinases MMP-2, MMP-9

Matrilysins MMP-7, MMP-26

Furin activable

MMPs

Secreted MMP-11, MMP-21, MMP-28

Type I transmembrane MMPs MMP-14 (MT1-MMP) ,MMP-15 (MT2-MMP),

MMP-16 (MT3-MMP), MMP-24 (MT5-MMP)

Glycosyl phosphatidyl inositol

anchored MMPs MMP-17 (MT4-MMP), MMP-25 (MT6-MMP)

Type II transmembrane

MMPs MMP-23A, MMP-23B

Endogenous regulators of MMP-transcription include immediate early genes (IEGs),

interferons, cytokines (IL-1α, IL-1β, TNF-α), epidermal growth factor (EGF), nerve

growth factor (NGF), vascular endothelial growth factor (VEGF), platelet derived

growth factor and cell-cell interactions (GERHAUSER et al. 2005, NAGASE et al.

1999, ROSENBERG 2002, SEKINE-AIZAWA et al. 2001, STERNLICHT et al. 2001,


19

YONG et al. 2001). Important steps of post-translational MMP-regulation are

zymogen activation (NAGASE 1997, VAN WART et al. 1990) and inhibition (BREW

et al. 2000, BREW et al. 2010). In vivo latent MMPs are activated by proteases.

However, in vitro chemical agents, low pH and heat treatment can be used for MMP-

activation (VISSE et al. 2003). The mechanism which is responsible for activation of

latent MMPs is called “cysteine switch” (VAN WART et al. 1990). In many cases

proteolytic activation resembles a stepwise process which initially starts with a

proteolytic attack to the exposed region between the first and second helices of the

pro-peptide (VISSE et al. 2003).

Figure 3: Structural classification of the mammalian family of MMPs based on domain arrangement (adapted from FANJUL-FERNANDEZ et al. 2010, YONG et al. 2001).

As = amino acids FC = furin cleavage site GPI = glycosyl phosphatidyl insositol anchor MMP = matrix metalloproteinase Zn = zinc ion

C = catalytic domain FTII = fibronectin type II motif MT-MMP = membrane type-MMPs TD1 = transmembrane domain Type 1


20

Removing a part of the pro-peptide induces a destabilization of the remaining pro-

peptide, including the cysteine which is involved in the cysteine switch-zinc

interaction. This destabilization allows an intra-/inter-molecular processing by partially

activated MMPs (autocatalytic or intercatalytic) or other active proteases (NAGASE

et al. 1990, SUZUKI et al. 1990). Finally the MMP lacks its pro-peptide resulting in a

proteolytically active enzyme (VISSE et al. 2003). All MMPs have to be activated in

the extracellular milieu except MMP-11, -21, -28 and all membrane type (MT)-MMPs,

which are activated during secretion by enzymes like furin (SOMERVILLE et al.

2003). Furthermore, excretion of intracytoplasmic stored MMPs (e.g. MMP-9) can be

modulated (STERNLICHT et al. 2001). Molecules inhibiting MMP-activity include α2-

macroglobulin and tissue inhibitors of MMPs (TIMPs; BAKER et al. 2002, BREW et

al. 2010). The ADAMs family consists of enzymes that cleave a number of ECM

molecules. Most ADAMs are integral membrane proteins but by alternative splicing a

secreted form of some ADAMs can be generated (YONG et al. 2001). They can act

as “sheddases” by removing ectodomain molecules from the cell surface

(ROSENBERG 2002) which has been shown for TNF-α receptor, IL-6, L-selectin and

syndecans (YONG et al. 2001). In contrast to MMPs, some ADAMs (ADAM2, -7, -11,

-14, -18, -22 and -29) lack the intact zinc-binding site and the metalloproteinase

domain can also be retained in mature proteins of this family (e.g. ADAM1 and -2;

YONG et al. 2001). Therefore, ADAM1 and -2 are not considered true degradative

enzymes (YONG et al. 2001). ADAMTs are another metalloproteinase family

containing one or more thrombospondin type I motifs at the carboxyl terminus and

which have been shown to be involved in spinal cord injury and to degrade the ECM

molecule aggrecan (LEMONS et al. 2001). ADAMTs are distinguished from ADAMs

by the lack of epidermal-growth factor-like, transmembrane and cytoplasmic domains

(YONG et al. 2001).

Tissue inhibitors of matrix metalloproteinases 2.4.2.

Tissue inhibitors of MMPs (TIMPs) are small molecules with a molecular weight of

about 21 kDa and a variable glycosylation (BAKER et al. 2002). They consist of an


21

N-terminal (~125 amino-acids; GOMEZ et al. 1997) and C-terminal domain (~65

amino-acids; BREW et al. 2000). TIMPs are secreted inhibitors of MMPs but they are

also known to exert functions apart from MMP-inhibition (BREW et al. 2000, BREW

et al. 2010). Today the group of TIMPs comprises four proteins (TIMP-1 to TIMP-4;

BREW et al. 2010, VISSE et al. 2003, YONG et al. 1998) which vary in their affinity to

the respective MMPs. TIMPs are crucial in maintaining a balance between ECM

deposition and destruction under physiological conditions (BAKER et al. 2002,

BREW et al. 2000). Furthermore, TIMPs are essential in pro-MMP activation,

stimulation of cell growth and steroid-synthesis as well as induction of apoptosis

(BREW et al. 2010, STERNLICHT et al. 2001). TIMPs regulate MMP activity by

establishing a non-covalent, reversible complex-formation with the catalytic domain

of MMPs (BREW et al. 2000). Although different TIMPs are able to bind to most

MMPs there are some differences in their inhibitory properties (BREW et al. 2000).

MT-MMPs can be effectively inhibited by TIMP-2 and TIMP-3 while TIMP-3 (not

TIMP-1, -2 or -4) is a good inhibitor of TNF-α converting enzyme. In addition, some

TIMPs are essentially required for proMMP activation (e.g. TIMP-2 for MMP-2;

BREW et al. 2000).

Matrix metalloproteinases in demyelinating CNS diseases 2.4.3.

Zinc-proteases of the MMP system and serine-proteases of the plasminogen/plasmin

system act in concert in a number of physiological processes like neurogenesis,

myelinogenesis, angiogenesis, axonal growth, and wound healing but they are also

involved in pathological conditions including demyelination, inflammation and BBB

disruption (CUZNER et al. 1999, LO et al. 2002, ROSENBERG 2009, STOMRUD et

al. 2010). An increased expression of MMPs is reported in MS (LINDBERG et al.

2001, LO et al. 2002, ROSENBERG 2001, ROSENBERG 2005, YONG et al. 2007),

TME (ULRICH et al. 2006), EAE (GONCALVES DASILVA et al. 2008, GONCALVES

DASILVA et al. 2009, TEESALU et al. 2001, TOFT-HANSEN et al. 2004), CDV

(GRÖTERS et al. 2005, MIAO et al. 2003), an experimentally-induced delayed type

IV hypersensitivity model of MS (ANTHONY et al. 1998), infection of mice with the


22

JHM strain of mouse hepatitis virus (ZHOU et al. 2005), cuprizone-induced

demyelination (SKULJEC et al. 2011), spinal cord injury (WELLS et al. 2003) and

traumatic brain injury (WANG et al. 2000). These proteases have different functions:

firstly, they are secreted by white blood cells to enable their entry into the CNS and

secondly, they are involved in myelin attack (KIESEIER et al. 1999). During the acute

phase MS patients show increased MMP levels in blood, liquor and CNS tissues

(ANTHONY et al. 1997, GIJBELS et al. 1992, LEE et al. 1999). MMPs may contribute

directly to the process of demyelination by cleaving essential proteins of the myelin

sheath (e.g. MBP; CHANDLER et al. 1995, CHANDLER et al. 1996). Structural

proteins which are important for development and maintenance of the myelin sheath

include MBP and PLP (ROUSSARIE et al. 2007). The cleavage of MBP by MMPs

has been shown in vitro (CHANDLER et al. 1995, SHIRYAEV et al. 2009) but

whether destruction of this protein is sufficient to induce a disintegration of the total

myelin sheath remains unknown so far. It remains also unclear how MMPs can

manage to cleave MBP in vivo because MBP binds to the cytoplasmic surface of the

myelin leaflet (ROUSSARIE et al. 2007) and MMPs are supposed to attack the

myelin sheath from the extracellular compartment. However, MBP essentially

influences the susceptibility of mice to TMEV because shiverer mice (DUPOUEY et

al. 1979, PRIVAT et al. 1979), carrying a large deletion of the MBP gene causing a

severe reduction of the amount of myelin, are completely resistant to chronic TMEV

infection (ROUSSARIE et al. 2007). Furthermore, MMPs may contribute to

demyelination indirectly by enhancing the migration of inflammatory cells into the

CNS and/or opening of the BBB causing an influx of plasma-proteins including

immunoglobulins (AGRAWAL et al. 2008, ROSENBERG 2009, YONG et al. 2001).

Besides demyelination MMPs have been shown to be involved in ECM remodeling in

MS (LINDBERG et al. 2001, MOHAN et al. 2010) and TME (HAIST et al. 2012).

Furthermore, MMPs and TIMPs have been implicated in regenerative processes

including axonal growth, oligodendrocyte maturation, remyelination and maintenance

of myelin (LARSEN et al. 2004, LEHMANN et al. 2009, OH et al. 1999, SKULJEC et

al. 2011, YONG 2005).

Chapter 3: MMPs in TMEV-infected astrocytes

23

Chapter 3 Differential transcription of matrix-metalloproteinase genes in primary mouse astrocytes and microglia infected with Theiler’s murine encephalomyelitis virus

KUMNOK, J., R. ULRICH, K. WEWETZER, K. ROHN, F. HANSMANN, W. BAUMGÄRTNER, S. ALLDINGER Abstract The BeAn strain of Theiler’s murine encephalomyelitis virus (TMEV) induces

demyelinating disease in susceptible mice comparable to human multiple sclerosis.

Recent in vivo studies showed that matrix metalloproteinases (MMPs) and their

inhibitors (tissue inhibitors of MMPs, TIMPs) are associated with demyelination in

Theiler’s murine encephalomyelitis. The present study was performed to evaluate the

in vitro MMP and TIMP expression in astrocytes and microglia following TMEV

infection. Brain cell cultures from SJL/J mice were infected with the BeAn strain of

TMEV and the expressions of 11 MMPs and 4 TIMPs were evaluated by reverse-

transcription quantitative polymerase chain reaction (RT-qPCR) at different time

points post infection (p.i.). In control astrocytes and microglia, a constitutive

expression of MMP-2, -3, -9, -10, -12, -13, -14, -15, -24 and TIMP-2 to -4 was

detected. In addition, TIMP-1 and MMP-11 was found in astrocytes only, and MMP-7

was absent in both cells cultures. RT-qPCR demonstrated high virus RNA copy

numbers in astrocytes and a low amount in microglia. In accordance, TMEV antigen

was detected in astrocytes, whereas it was below the limit of detection in microglia.

MMP-3, -9, -10, -12, and -13 as well as TIMP-1 were the enzymes most prominently

up-regulated in TMEV-infected astrocytes. In contrast, TMEV infection was

associated with a down-regulation of MMPs and TIMPs in microglia. Conclusively, in

addition to inflammatory infiltrates, TMEV-induced astrocytic MMPs might trigger a

proteolysis cascade leading to an opening of the blood-brain barrier and

demyelination in vivo.

Neurovirol. 2008 May: 14(3):205-217

www.informaworld.com

DOI: 10.1080/13550280802008305

Chapter 4: MMP-12 deficiency ameliorates TME

25

Chapter 4 Matrix metalloproteinase-12 deficiency ameliorates the clinical course and demyelination in Theiler’s murine encephalomyelitis

HANSMANN, F., HERDER, V., KALKUHL, A., HAIST, V., ZHANG, N., SCHAUDIEN, D., DESCHL, U., BAUMGÄRTNER, W., ULRICH, R.

Abstract Matrix metalloproteinases (MMPs) are a family of extracellular proteases involved in

the pathogenesis of demyelinating diseases like multiple sclerosis (MS). The aim of

the present study was to investigate whether MMPs induce direct myelin

degradation, leukocyte infiltration, disruption of the blood–brain barrier (BBB), and/or

extra-cellular matrix remodeling in the pathogenesis of Theiler’s murine

encephalomyelitis (TME), a virus-induced model of MS. During the demyelinating

phase of TME, the highest transcriptional upregulation was detected for Mmp12,

followed by Mmp3. Mmp12-/- mice showed reduced demyelination, macrophage

infiltration, and motor deficits compared with wild-type- and Mmp3 knock-out mice.

However, BBB remained unaltered, and the amount of extracellular matrix deposition

was similar in knock-out mice and wild-type mice. Furthermore, stereotaxic injection

of activated MMP-3, -9, and -12 into the caudal cerebellar peduncle of adult mice

induced a focally extensive primary demyelination prior to infiltration of inflammatory

cells, as well as a reduction in the number of oligodendrocytes and a leakage of BBB.

All these results demonstrate that MMP-12 plays an essential role in the

pathogenesis of TME, most likely due to its primary myelin- or oligodendrocyte-toxic

potential and its role in macrophage extravasation, whereas there was no sign of

BBB damage or alterations to extracellular matrix remodeling/deposition. Thus,

interrupting the MMP-12 cascade may be a relevant therapeutic approach for

preventing chronic progressive demyelination.

Acta Neuropathol. 2012: epub ahead of print

www.elsevier.com

DOI: 10.1007/s00401-012-0942-3

Chapter 5: Spinal epidermoid cyst in a SJL Mouse

27

Chapter 5 Spinal Epidermoid Cyst in a SJL Mouse: Case Report and Literature Review

HANSMANN, F., V. HERDER, H. ERNST, W. BAUMGÄRTNER Abstract This report is the first description of a spinal epidermoid cyst (EC) in a SJL mouse

and gives an overview on the occurrence of ECs in animals including dogs, horses,

mice and rats. The EC was not detected grossly and the mouse did not display

clinical signs or an altered rotarod performance. Microscopically, there was an oval

cyst lined by stratified squamous epithelium that was attached to the dorsolateral

meninges and caused moderate compression of the adjacent lumbar spinal cord.

ECs in mice and rats are mainly located in the caudal part of the spinal cord with a

variable, strain-dependent occurrence. ECs in mice and rats are not associated with

clinical signs and can be interpreted as incidental findings.

J. Comp. Pathol. 2011 November: 145(4):373-377

www.sciencedirect.com

DOI: 10.1016/j.jcpa.2011.03.002

Chapter 6: Discussion and Conclusions

29

Chapter 6 Discussion and Conclusions

This study is based on previous results from our group describing upregulation of

mainly MMP-12 and -3 as well as TIMP-1 in association with a demyelinating

meningoleukomyelitis in TME (ULRICH et al. 2006). These findings resulted in the

hypothesis, that MMP-3 and MMP-12 are key-molecules in the pathogenesis of

demyelinating diseases (Figure 4). The aims of this study were 1.) to determine the

influence of TMEV-infection on astroglial and microglial MMP-synthesis in vitro, 2.) to

elucidate whether injected MMPs contribute to demyelination by a direct destruction

of myelin and/or oligodendrocytes or indirectly by facilitating infiltration of

inflammatory cells or breakdown of the BBB, and 3.) to investigate the effect of

Mmp3 and Mmp12 deficiency using knockout mice upon the clinical and

morphological outcome during TME. Furthermore, the incidental finding of a spinal

epidermoid cysts (ECs) without associated clinical signs in one control animal has

been described. Figure 4: Possible mechanisms how MMPs can contribute to demyelination in the CNS

(adapted from YONG et al. 2001).


30

6.1. Matrix metalloproteinase transcription in vitro

MMPs are associated with demyelination in TME (ULRICH et al. 2006). The in vitro

experiment aimed to investigate the impact of TMEV-infection upon MMP

transcription in astrocytes and microglia cells. Both cell types represent besides

invading monocytes/macrophages an important source of MMP production in the

CNS (NUTTALL et al. 2007). In vitro, a constitutive expression of Mmp2, -3, -9,

-10, -12, -13, -14, -15, -24 and Timp2 to -4 in normal astrocytes and microglia cells

(derived from brain cultures of SJL/J mice) was detected while Timp1 and Mmp11

were found in astrocytes only, and Mmp7 was absent in both cell populations. TMEV-

infection of astrocytes induced a prominent upregulation of Mmp3, -9, -10, -12, and

-13 as well as Timp1 transcripts in vitro. The observed upregulation of Mmp3 and -12

as well as Timp1 is in accordance with described in vivo observations during TME

(ULRICH et al. 2006), while Mmp9 transcripts are not differentially expressed in vivo.

However, in contrast to observations in TME an upregulation of MMP-9 transcripts is

reported in EAE and MS (ANTHONY et al. 1997, AVOLIO et al. 2003, COSSINS et

al. 1997). This in vitro investigation revealed that TMEV-infection induces an

increased MMP transcription in astrocytes, while TMEV-infection of microglia cells

was associated with a down-regulation of MMP- and TIMP-transcription. However,

Mmp12 expression was about 100-fold higher in microglia compared with astrocytes.

In addition, demyelination in TME is associated with a progressive intralesional

astrogliosis as well as an increased number of microglia/macrophages (HAIST et al.

2012). Conclusively, MMPs produced by TMEV-infected astrocytes and inflammatory

cells might contribute to the process of demyelination and BBB disruption.

6.2. Stereotaxic injection of matrix metalloproteinases

This experiment aimed to elucidate the mechanisms by which MMPs especially

MMP-3 and -12 contribute to the process of demyelination. MMPs are known to

cleave MBP, an essential constituent of the myelin sheaths in vitro (CHANDLER et

al. 1995, SHIRYAEV et al. 2009). This raises the question whether these enzymes

will be able to destroy myelin sheaths in vivo. MMP-9 was injected in addition to


31

MMP-3 and -12 because this MMP is among others associated with demyelination in

MS (AVOLIO et al. 2003, COSSINS et al. 1997). In addition, a loss of LFB-staining

intensity is reported following stereotaxic injection of MMP-9 into the rat brain

(ANTHONY et al. 1998). Stereotaxic injection of 440 ng recombinant, in vitro

activated murine MMP-3, -9 and -12 into the caudal cerebellar peduncle (CCP) of

adult SJL/J mice induced a severe demyelination before infiltration of inflammatory

cells occurred (Figure 5). In addition to demyelination most prominent in MMP-3-

injected animals a severe loss of oligodendrocytes was noticed. To confirm these

histological findings transmission electron microscopy was performed.

Ultrastructurally, myelin sheath edema, fragmentation of myelin membranes and an

extracellular edema were found. These observations demonstrate that MMP-3, -9

and -12 are able to induce demyelination in vivo. However, the mechanism whether

MMPs induce demyelination by disintegration of the cell membrane possibly due to

cleavage of MBP or its intrinsic oligodendrocyte-toxic-potential remains unknown. In

addition, a leakage of the BBB, detected by extravasation of Evans blue and IgG,

was observed following stereotaxic injection of MMP-3, -9 and -12. Opening of the

BBB leading to extravasation of plasma proteins may be a second, important

mechanism involved in the process of demyelination, complement activation and

inflammatory cell extravasation. Figure 5: Consequences of MMP-3, -9 and -12 following stereotaxic injection into the caudal

cerebellar peduncle of adult SJL/J mice


32

6.3. Infection of MMP-3 and -12 knock-out mice with TMEV

Microarray analysis of transcriptional changes during TME revealed a marked and

progressive upregulation of Mmp12 (Figure 6). In addition, Mmp2, -3, -13, -14, -16 -

19 and Timp1, -2 were upregulated while Mmp-9, -17, -24, -28 and Timp4 were

downregulated at various time points (Figure 6). Furthermore, a variable expression

pattern was observed for Mmp23. Figure 6: Transcriptional changes associated with the ECM in the spinal cord of

TMEV-infected mice (adapted from HANSMANN et al. 2012)

Thermometer-like icons display the fold-changes of significantly expressed genes in TMEV-infected mice compared with mock-infected mice employing pairwise Mann-Whitney U-tests (p≤0.05) at four time points: (1) = 14 dpi, (2) = 42 dpi, (3) = 98 dpi and (4) = 198 dpi, respectively. Thermometer-like icons: red = upregulation, blue = downregulation; green arrows = positive functional interaction; red arrows = negative functional interaction; grey arrows = technical link; hollow green arrays = pathway start; B = binding; C = cleavage


33

These observations are supported by previous RT-qPCR investigations of TMEV-

and mock-infected mice (ULRICH et al. 2006). Stereotaxic injection of activated

MMP-3 and -12 demonstrated the potential of MMPs to induce demyelination as well

as a disruption of the BBB in vivo. Considering that MMP-3 and -12 are key-

molecules in the process of demyelination the next question was whether disruption

of Mmp3 or Mmp12 gene expression may abolish/reduce demyelination during TME.

To answer this question Mmp3-/- and Mmp12-/- mice (both on a SJL/J genetic

background) as well as SJL/J wild-type mice were infected with TMEV. Clinical

investigation employing a scoring system and rotarod-test revealed a significant

difference between TMEV and mock-infected animals starting at 70 dpi (wild-type

mice) and 91 dpi (Mmp3-/- mice) while significant differences between TMEV- and

mock-infected Mmp12-/- mice were lacking. This leads to the conclusison that Mmp12

deficiency ameliorates clinical signs observed during TME.

These results may be explained by a significantly reduced degree of demyelination at

98 days post infection (dpi) in Mmp12-/- mice compared to wild-type and Mmp3-/-

mice. In accordance with a reduced demyelination a reduced leukomyelitis was

detected at 98 dpi while the degree of meningitis and poliomyelitis was similar

between the TMEV-infected groups. Immunohistochemistry revealed that

inflammatory cells in the white matter were mainly composed of

microglia/macrophages and lymphocytes. However, the density of

microglia/macrophages was significantly reduced in Mmp12-/- mice compared to wild-

type mice from 1 to 98 dpi. The observation that MMP-12 deficiency leads to a

reduced degree of demyelination in the spinal cord in association with a reduced

density of microglia/macrophages indicates that MMP-12 has a detrimental effect

upon myelin preservation. Although the number of microglia/macrophages in

Mmp12-/- mice was significantly reduced during the demyelinating phase of TME, the

activation/infiltration of these cells was not completely abolished. This is an important

finding because numerous studies show that several aspects of inflammation

following CNS injury are beneficial (GIULIAN et al. 1990, GONCALVES DASILVA et

al. 2009, MABON et al. 2000, POPOVICH et al. 1999, SKULJEC et al. 2011) and

macrophages also facilitate recovery (BATCHELOR et al. 1999, GUTH et al. 1994,


34

RAPALINO et al. 1998). These data indicated that an excessive infiltration of

macrophages may contribute to a detrimental outcome. Furthermore, it has been

shown that MMP-12 has a deleterious effect following spinal cord injury and

intracerebral hemorrhage (WELLS et al. 2003). Macrophages and gitter cells

contribute to demyelination and axonal damage in MS and TME by producing

mediators of tissue damage such as MMPs, reactive oxygen species and pro-

inflammatory cytokines (BATCHELOR et al. 1999). Macrophages represent an

important source of MMPs with a broader range and often higher amounts of

proteolytic enzymes compared to lymphocytes (BAR-OR 2008, BAR-OR et al. 2003,

ULRICH et al. 2006).

6.4. Epidermoid cysts in the spinal canal of mice

The incidental finding of a spinal epidermoid cyst (EC) in one control animal leads to

the first description of an EC in this respective mouse strain (HANSMANN et al.

2011). The cyst was attached to the meninges and compressed a part of the lumbar

spinal cord from dorsolateral. In general the caudal part of the spinal cord represents

a location where most of the described spinal ECs in other strains and species were

located (JUNG et al. 1981, KULWICH 1994, STROOP 1984). ECs in mice have a

small diameter and are not detected grossly. The occurrence of ECs in inbred mouse

strains is variable, with C58/J, AKR/J, B6C3F1 and C57L/J strains lacking reported

lesions, whereas albino swiss mice, Balb/c, quaking mice, C57BR/cdJ, Crl:CD® and

C57Bl/6J strains have incidences of up to 6.3% (JUNG et al. 1981, KULWICH 1994,

NOBEL et al. 1987, STROOP 1984). The incidences of ECs are variable among

different mouse strains indicating a genetic predisposition for their development.

Most studies dealing with animal models of CNS diseases include clinical

investigations like scoring systems behavioral tests or motor coordination tasks (e.g.

rotarod analysis). Clinical investigation is an important feature because most

histopathological results as well as effects of applied substances were correlated with

clinical outcome. The lack of clinical signs despite the spinal EC in the present case

is consistent with previously described cases in mice. Additionally, performed rotarod


35

tests confirmed the lack of motor coordination deficits. The lack of clinical signs in

mice and rats is in contrast to findings in dogs and man where clinical signs including

paraparesis, sensory loss and back and/or leg pain are reported (FERRARA et al.

2003, PLATT et al. 1999, STEINBERG et al. 2007). Dermoid cysts are an important

differential diagnosis to ECs. These cyst formations are rare in mice but in contrast to

ECs dermoid cysts can induce clinical signs (NGUYEN 1988). Dermoid cysts can be

distinguished from EC by histopathology because dermoid cyst lack adnexal

structures like hair follicles, sebaceous or sweat glands. Conclusively, the occurrence

of spinal ECs in mice seems to be strain dependent and ECs are mainly located in

the caudal part of the spinal cord. Furthermore, ECs in mice are neither detected

grossly nor associated with clinical signs and therefore the detection of an EC within

a mouse study should be interpreted as an incidental finding which will not influence

the results of clinical investigations.

6.5. Concluding remark

In this thesis in vitro analysis of MMPs secreted by astrocytes and microglia,

microarray analysis of transcriptional changes in the spinal cord of TMEV-and mock-

infected mice, stereotaxic injection of activated MMP-3, -9 and -12 into the caudal

cerebellar peduncle of mice and TMEV-infection of Mmp3- and Mmp12-knockout

mice has been performed. In vitro Mmp3, -9, -10, -12 and -13 transcripts were

upregulated in TMEV-infected astrocytes. Furthermore, in vivo Mmp3 and Mmp12

transcripts were upregulated in the demyelinating phase of TME. During TME

Mmp12 knockout mice showed reduced demyelination, macrophage infiltration and

motor deficits compared with wild-type mice. In addition, stereotaxic injection of

MMP-12 into the CCP revealed demyelination and a reduced number of

oligodendrocytes prior to the infiltration of leukocytes indicating a direct myelin and/or

oligodendrocyte-toxic mode of action.

Conclusively, these data indicate that MMP-12 plays an essential role in the

pathogenesis of demyelinating diseases and an inhibition of MMP-12 may be a

suitable approach for preventing chronic progressive demyelination.

Chapter 7: Summary

37

Chapter 7 Summary

The pathogenic role of matrix metalloproteinases in a virus-induced mouse model of demyelinating diseases Florian Heinrich Hansmann

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases which

are involved in the pathogenesis of demyelinating diseases like canine distemper,

Theiler’s murine encephalomyelitis (TME) or Multiple sclerosis. In this work the

following experiments have been carried out:

1.) Microarray analysis of differentially expressed genes in the spinal cord of

TMEV-infected compared with mock-infected SJL/J mice.

2.) In vitro investigation of MMP-transcription in normal and TMEV-infected

astrocytes and microglia from SJL/J mice.

3.) Stereotaxic injection of 440 ng in vitro activated, recombinant murine MMP-3,

-9 and -12 into the caudal cerebellar peduncle (CCP) of adult SJL/JOlaHsd

mice (3-4 female mice per group; necropsy at 12, 24, 72 and 168 hours post

MMP-injection) to investigate the impact of MMP-3, -9 and -12 on

demyelination, blood brain barrier leakage, and infiltration of inflammatory

cells.

4.) Intracerebral TMEV infection of Mmp3 and Mmp12 knock-out mice (in-house

backcrossed over 10 generations on a SJL/JOlaHsd genetic background; 6-7

female mice per group, necropsy at 0, 1, 28 and 98 days post infection).

Controls included mock-infected (medium only) knock-out animals as well as

TMEV- and mock-infected SJL/JOlaHsd (wild type) mice.

5.) First description of a spinal epidermoid cyst in a SJL/JOlaHsd mouse without

associated clinical signs.

Microarray analysis of transcriptional changes during TME in vivo revealed a marked

and progressive upregulation of Mmp12. In addition, Mmp2, -3, -13, -14, -16 -19 and

Timp1, -2 were upregulated, while Mmp9, -17, -24, -28 and Timp4 were

Chapter 7: Summary

38

downregulated at various time points. Furthermore, investigations of astrocytes and

microglia cells in vitro revealed a constitutive expression of Mmp2, -3, -9, -10, -12,

-13, -14, -15, -24 and Timp2 to -4. TMEV-infection of astrocytes in vitro induced a

prominent upregulation of Mmp3, -9, -10, -12, and -13 as well as Timp1 transcripts.

To clarify whether these MMPs contribute essentially to demyelination in vivo,

stereotaxic injection of in vitro activated, recombinant murine MMP-3, -9 and -12 into

the CCP of adult SJL/J was performed. This induced demyelination and loss of

oligodendrocytes prior to the infiltration of inflammatory cells indicating a myelin-

and/or oligodendrocyte-toxic mode of action of all three MMPs. Furthermore,

following MMP-injection a BBB-leakage was observed, indicating a role of MMPs in

the extravasation of inflammatory cells. To elucidate whether Mmp3 and/or Mmp12

are essential key molecules in the process of demyelination Mmp3 and Mmp12

knockout mice were intracerebrally infected with the BeAn strain of TMEV. Mmp12

knockout mice showed reduced demyelination, macrophage infiltration and motor

deficits during TME compared with wild-type mice, whereas Mmp3 knockout mice did

not.

In conclusion, MMP-12 plays an essential role in the pathogenesis of demyelinating

diseases and the interruption of its cascade may be a suitable therapeutic approach

to prevent chronic progressive demyelination or to ameliorate the disease process.

Chapter 8: Zusammenfassung

39

Chapter 8 Zusammenfassung

Die pathogenetische Bedeutung von Matrix Metalloproteinasen bei einem Virus-induzierten Mausmodell für demyelinisierende Erkrankungen Florian Heinrich Hansmann

Matrix Metalloproteinasen (MMPs) stellen eine Familie von Zink-abhängigen

Proteasen dar, die an der Pathogenese von demyelinisierenden Erkrankungen wie

Staupevirus-Enzephalitis, muriner Theilervirus-Enzephalomyelitis oder Multipler

Sklerose beteiligt sind. In dieser Arbeit wurden die folgenden Experimente

durchgeführt:

1.) Microarray-Analyse von unterschiedlich exprimierten Genen im Rückenmark

von Theilervirus-infizierten im Vergleich zu Mock-infizierten SJL/J Mäusen.

2.) In vitro-Untersuchung der MMP-Transkription in infizierten und nicht-infizierten

Astrozyten und Mikrogliazellen von SJL/J Mäusen

3.) Stereotaktische Injektion von 440 ng in vitro aktivierter, rekombinanter,

muriner MMP-3, -9 und -12 in den kaudalen Kleinhirnstiel von adulten

SJL/JOlaHsd Mäusen (3-4 weibliche Mäuse pro Gruppe;

Untersuchungszeitpunkte: 12, 24, 72 und 168 Stunden nach stereotaktischer

Injektion) zur Untersuchung der Auswirkungen von MMP-3, -9 und -12 auf

Entmarkung, Blut-Hirnschranken-Schaden sowie Infiltration von

Entzündungszellen.

4.) Intrazerebrale Theilervirus-Infektion von Mmp3 und Mmp12 knockout Mäusen

(über 10 Generation auf SJL/JOlaHsd genetischen Hintergrund rückgekreuzt;

6-7 weibliche Mäuse pro Gruppe; Untersuchungszeitpunkte: 0, 1, 28 und 98

Tage nach Infektion). Als Kontrolltiere wurden Medium-injizierte knockout

Mäuse sowie Medium-injizierte Wildtyp-Mäuse (SJL/JOlaHsd) verwendet.

5.) Erstbeschreibung einer spinalen Epidermoidzyste bei einer SJL/JOlaHsd

Maus ohne klinische Symptome.

Die Mikroarray-Analyse der transkriptionellen Veränderungen während der

Theilervirus-Enzephalomyelitis in vivo zeigte eine deutliche und progressive

Chapter 8: Zusammenfassung

40

Aufregulierung von Mmp12. Darüber hinaus waren an verschiedenen Zeitpunkten

Mmp2, -3, -13, -14, -16, -19, Timp1 und -2 hochreguliert, während Mmp9, -17, -24,

-28 und Timp4 herunterreguliert waren. Die in vitro-Untersuchung von Astrozyten und

Mikrogliazellen ergab, dass diese Zellen eine konstitutive Expression von Mmp2, -3,

-9, -10, -12, -13, -14, -15, -24 und Timp2-4 zeigen. In Astrozyten fand sich nach der

Theilervirus-Infektion eine starke Aufregulierung von Mmp3-, -9-, -10-, -12-, -13- und

Timp1-Transkripten. Um zu klären, ob diese MMPs im Wesentlichen zur Entmarkung

in vivo beitragen, wurden stereotaktisch in vitro aktivierte, rekombinante, murine

MMP-3, -9 und -12 in den kaudalen Kleinhirnstiel von adulten SJL/JOlaHsd Mäusen

injiziert. Die Applikation induzierte eine Entmarkung und einen Verlust von

Oligodendrozyten bevor eine Infiltration von Entzündungszellen festzustellen war.

Dies spricht für eine Myelin- und/oder Oligodendrozyten-toxische Wirkung aller drei

untersuchten MMPs. Darüber hinaus wurde mittels Quantifizierung von Evansblue

und Immunglobulin G-Extravasation ein Blut-Hirnschranken-Schaden festgestellt, der

auf eine Beteiligung der MMPs bei der Auswanderung von Entzündungszellen

hindeutet. Um herauszufinden, ob MMP-3 und/oder MMP-12 wichtige

Schlüsselmoleküle in der Pathogenese der Entmarkung darstellen, wurden Mmp3

und Mmp12 knockout Mäuse mit dem BeAn Stamm des Theilervirus infiziert. Mmp12

knockout Mäuse zeigten eine verminderte Entmarkung und Makrophageninfiltration

sowie geringere motorische Defizite im Verlauf der Theilervirus-Enzephalomyelitis im

Vergleich zu Wildtyp-Mäusen. Mmp3 knockout-Mäuse wiesen keine Unterschiede im

Vergleich zu Wildtyp-Mäusen auf.

Zusammenfassend lässt sich festhalten, dass MMP-12 eine wesentliche Rolle bei

der Pathogenese von demyelinisierenden Erkrankungen spielt. Die Hemmung von

MMP-12 könnte einen geeigneten, therapeutischen Ansatz darstellen um eine

chronisch-progressive Demyelinisierung zu verhindern oder den Krankheitsverlauf

günstig zu beeinflussen.

Chapter 9: References

41

Chapter 9 References

AGRAWAL, S. M., L. LAU and V. W. YONG (2008): MMPs in the central nervous system: where the good guys go bad. Semin. Cell Dev. Biol. 19, 42-51

ANTHONY, D. C., B. FERGUSON, M. K. MATYZAK, K. M. MILLER, M. M. ESIRI and V. H. PERRY (1997): Differential matrix metalloproteinase expression in cases of multiple sclerosis and stroke. Neuropathol. Appl. Neurobiol. 23, 406-415

ANTHONY, D. C., K. M. MILLER, S. FEARN, M. J. TOWNSEND, G. OPDENAKKER, G. M. A. WELLS, J. M. CLEMENTS, S. CHANDLER, A. J. H. GEARING and V. H. PERRY (1998): Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS. J. Neuroimmunol. 87, 62-72

AUBERT, C., M. CHAMORRO and M. BRAHIC (1987): Identification of Theiler's virus infected cells in the central nervous system of the mouse during demyelinating disease. Microb. Pathog. 3, 319-326

AVOLIO, C., M. RUGGIERI, F. GIULIANI, G. M. LIUZZI, R. LEANTE, P. RICCIO, P. LIVREA and M. TROJANO (2003): Serum MMP-2 and MMP-9 are elevated in different multiple sclerosis subtypes. J. Neuroimmunol. 136, 46-53

BAILEY, O. T., A. M. PAPPENHEIMER, F. S. CHEEVER and J. B. DANIELS (1949): A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin: II. Pathology. J. Exp. Med. 90, 195-212

BAKER, A. H., D. R. EDWARDS and G. MURPHY (2002): Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J. Cell Sci. 115, 3719-3727

BAR-OR, A. (2008): The immunology of multiple sclerosis. Semin. Neurol. 28, 29-45

BAR-OR, A., R. K. NUTTALL, M. DUDDY, A. ALTER, H. J. KIM, I. IFERGAN, C. J. PENNINGTON, P. BOURGOIN, D. R. EDWARDS and V. W. YONG (2003): Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis. Brain 126, 2738-2749

BARACCHINI, C., P. PERINI, M. CALABRESE, F. CAUSIN, F. RINALDI and P. GALLO (2011): No evidence of chronic cerebrospinal venous insufficiency at multiple sclerosis onset. Ann. Neurol. 69, 90-99

BATCHELOR, P. E., G. T. LIBERATORE, J. Y. WONG, M. J. PORRITT, F. FRERICHS, G. A. DONNAN and D. W. HOWELLS (1999): Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. J. Neurosci. 19, 1708-1716


42

BEINEKE, A., C. PUFF, F. SEEHUSEN and W. BAUMGÄRTNER (2009): Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet. Immunol. Immunopathol. 127, 1-18

BEN-NUN, A., H. WEKERLE and I. R. COHEN (1981): Vaccination against autoimmune encephalomyelitis with lymphocyte-T line cells reactive against myelin basic-protein. Nature 292, 60-61

BLAKEMORE, W. F. (1972): Observations on oligodendrocyte degeneration, the resolution of status spongiosus and remyelination in cuprizone intoxication in mice. J. Neurocytol. 1, 413-426

BLAKEMORE, W. F. (1973): Remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cuprizone. J. Neurol. Sci. 20, 73-83

BORROW, P., C. J. WELSH and A. A. NASH (1993): Study of the mechanisms by which CD4+ T cells contribute to protection in Theiler's murine encephalomyelitis. Immunology 80, 502-506

BRAHIC, M., J. F. BUREAU and T. MICHIELS (2005): The genetics of the persistent infection and demyelinating disease caused by Theiler's virus. Annu. Rev. Microbiol. 59, 279-298

BRAHIC, M., W. G. STROOP and J. R. BARINGER (1981): Theiler's virus persists in glial cells during demyelinating disease. Cell 26, 123-128

BREW, K., D. DINAKARPANDIAN and H. NAGASE (2000): Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim. Biophys. Acta 1477, 267-283

BREW, K. and H. NAGASE (2010): The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim. Biophys. Acta 1803, 55-71

CAUWE, B. and G. OPDENAKKER (2010): Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases. Crit. Rev. Biochem. Mol. Biol. 45, 351-423

CENTONZE, D., R. FLORIS, M. STEFANINI, S. ROSSI, S. FABIANO, M. CASTELLI, S. MARZIALI, A. SPINELLI, C. MOTTA, F. G. GARACI, G. BERNARDI and G. SIMONETTI (2011): Proposed chronic cerebrospinal venous insufficiency criteria do not predict multiple sclerosis risk or severity. Ann. Neurol. 70, 52-59

CHANDLER, S., R. COATES, A. GEARING, J. LURY, G. WELLS and E. BONE (1995): Matrix metalloproteinases degrade myelin basic protein. Neurosci. Lett. 201, 223-226


43

CHANDLER, S., J. COSSINS, J. LURY and G. WELLS (1996): Macrophage metalloelastase degrades matrix and myelin proteins and processes a tumour necrosis factor-alpha fusion protein. Biochem. Biophys. Res. Commun. 228, 421-429

CHEEVER, F. S., J. B. DANIELS and ET AL. (1949): A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. J. Exp. Med. 90, 181-210

CLATCH, R. J., H. L. LIPTON and S. D. MILLER (1986): Characterization of Theiler's murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEV-induced demyelinating disease - correlation with clinical signs. J. Immunol. 136, 920-927

CLATCH, R. J., H. L. LIPTON and S. D. MILLER (1987): Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. II. Survey of host immune responses and central nervous system virus titers in inbred mouse strains. Microb. Pathog. 3, 327-337

CLATCH, R. J., R. W. MELVOLD, S. D. MILLER and H. L. LIPTON (1985): Theiler murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is influenced by the H-2d Region - correlation with TMEV-specific delayed-type hypersensitivity. J. Immunol. 135, 1408-1414

COHEN, J. A. (2009): The future of multiple sclerosis treatment. J. Neurol. Sci. 277, S55-S61

COMPSTON, A. and A. COLES (2008): Multiple sclerosis. Lancet 372, 1502-1517

COSSINS, J. A., J. M. CLEMENTS, J. FORD, K. M. MILLER, R. PIGOTT, W. VOS, P. VANDERVALK and C. J. A. DEGROOT (1997): Enhanced expression of MMP-7 and MMP-9 in demyelinating multiple sclerosis lesions. Acta Neuropathol. 94, 590-598

CROXFORD, A. L., F. C. KURSCHUS and A. WAISMAN (2011): Mouse models for multiple sclerosis: historical facts and future implications. Biochim. Biophys. Acta 1812, 177-183

CUZNER, M. L. and G. OPDENAKKER (1999): Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system. J. Neuroimmunol. 94, 1-14

DAL CANTO, M. C., B. S. KIM, S. D. MILLER and R. W. MELVOLD (1996): Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination: a model for human multiple sclerosis. Methods 10, 453-461

DAL CANTO, M. C. and H. L. LIPTON (1975): Primary demyelination in Theilers virus-infection - ultrastructural study. Lab. Invest. 33, 626-637


44

DAL CANTO, M. C. and H. L. LIPTON (1982): Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler's virus infection. Am. J. Pathol. 106, 20-29

DAL CANTO, M. C. and S. G. RABINOWITZ (1982): Experimental-models of virus-induced demyelination of the central nervous-system. Ann. Neurol. 11, 109-127

DANIELS, J. B., A. M. PAPPENHEIMER and S. RICHARDSON (1952): Observations on encephalomyelitis of mice (DA strain). J. Exp. Med. 96, 517-530

DENIC, A., A. J. JOHNSON, A. J. BIEBER, A. E. WARRINGTON, M. RODRIGUEZ and I. PIRKO (2011): The relevance of animal models in multiple sclerosis research. Pathophysiology 18, 21-29

DRESCHER, K. M. and D. SOSNOWSKA (2008): Being a mouse in a man's world: what TMEV has taught us about human disease. Front. Biosci. 13, 3775-3785

DUPOUEY, P., C. JACQUE, J. M. BOURRE, F. CESSELIN, A. PRIVAT and N. BAUMANN (1979): Immunochemical studies of myelin basic protein in shiverer mouse devoid of major dense line of myelin. Neurosci. Lett. 12, 113-118

FANJUL-FERNANDEZ, M., A. R. FOLGUERAS, S. CABRERA and C. LOPEZ-OTIN (2010): Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. Biochim. Biophys. Acta 1803, 3-19

FERRARA, P., S. COSTA, D. RIGANTE, A. MULE, C. D'ALEO, S. PULITANO, P. BELLI, P. MENCHINELLI and M. CALDARELLI (2003): Intramedullary epidermoid cyst presenting with abnormal urological manifestations. Spinal Cord 41, 645-648

FRANKLIN, R. J. M. (2002): Why does remyelination fail in multiple sclerosis? Nat. Rev. Neurosci. 3, 705-714

GERHAUSER, I., S. ALLDINGER, R. ULRICH and W. BAUMGÄRTNER (2005): Spatio-temporal expression of immediate early genes in the central nervous system of SJL/J mice. Int. J. Dev. Neurosci. 23, 637-649

GIJBELS, K., S. MASURE, H. CARTON and G. OPDENAKKER (1992): Gelatinase in the cerebrospinal-fluid of patients with multiple-sclerosis and other inflammatory neurological disorders. J. Neuroimmunol. 41, 29-34

GIULIAN, D. and C. ROBERTSON (1990): Inhibition of mononuclear phagocytes reduces ischemic injury in the spinal cord. Ann. Neurol. 27, 33-42

GOMEZ, D. E., D. F. ALONSO, H. YOSHIJI and U. P. THORGEIRSSON (1997): Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur. J. Cell Biol. 74, 111-122


45

GONCALVES DASILVA, A. and V. W. YONG (2008): Expression and regulation of matrix metalloproteinase-12 in experimental autoimmune encephalomyelitis and by bone marrow derived macrophages in vitro. J. Neuroimmunol. 199, 24-34

GONCALVES DASILVA, A. and V. W. YONG (2009): Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation. Am. J. Pathol. 174, 898-909

GRIFFITHS, I. R. (1996): Myelin mutants: model systems for the study of normal and abnormal myelination. Bioessays 18, 789-797

GRIFFITHS, I. R., I. SCOTT, M. C. MCCULLOCH, J. A. BARRIE, K. MCPHILEMY and B. M. CATTANACH (1990): Rumpshaker mouse: a new X-linked mutation affecting myelination: evidence for a defect in PLP expression. J. Neurocytol. 19, 273-283

GROSS, J. and C. M. LAPIERE (1962): Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. USA 48, 1014-1022

GRÖTERS, S., S. ALLDINGER and W. BAUMGÄRTNER (2005): Up-regulation of mRNA for matrix metalloproteinases-9 and-14 in advanced lesions of demyelinating canine distemper leukoencephalitis. Acta Neuropathol. 110, 369-382

GUTH, L., Z. ZHANG, N. A. DIPROSPERO, K. JOUBIN and M. T. FITCH (1994): Spinal cord injury in the rat: treatment with bacterial lipopolysaccharide and indomethacin enhances cellular repair and locomotor function. Exp. Neurol. 126, 76-87

HAIST, V., R. ULRICH, A. KALKUHL, U. DESCHL and W. BAUMGÄRTNER (2012): Distinct spatio-temporal extracellular matrix accumulation within demyelinated spinal cord lesions in Theiler's murine encephalomyelitis. Brain Pathol. 22, 188-204

HANSMANN, F., V. HERDER, H. ERNST and W. BAUMGÄRTNER (2011): Spinal epidermoid cyst in a SJL mouse: case report and literature review. J. Comp. Pathol. 145, 373-377

HANSMANN, F., V. HERDER, A. KALKUHL, V. HAIST, N. ZHANG, D. SCHAUDIEN, U. DESCHL, W. BAUMGÄRTNER and R. ULRICH (2012): Matrix metalloproteinase-12 deficiency ameliorates the clinical course and demyelination in Theiler's murine encephalomyelitis. Acta Neuropathol. DOI: 10.1007/s00401-012-0942-3,

HANSMANN, F., K. PRINGPROA, R. ULRICH, Y. SUN, V. HERDER, M. KREUTZER, W. BAUMGÄRTNER and K. WEWETZER (2012): Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and non-demyelinated central nervous system. Cell Transplant. doi:10.3727/096368911X096627444


46

HAUSER, S. L. and J. R. OKSENBERG (2006): The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron 52, 61-76

HERDER, V., F. HANSMANN, M. STANGEL, T. SKRIPULETZ, W. BAUMGÄRTNER and A. BEINEKE (2011): Lack of cuprizone-induced demyelination in the murine spinal cord despite oligodendroglial alterations substantiates the concept of site-specific susceptibilities of the central nervous system. Neuropathol. Appl. Neurobiol. 37, 676-684

HUNTER, S. F. and M. RODRIGUEZ (1995): Multiple sclerosis: a unique immunopathological syndrome of the central nervous system. Springer Semin. Immunopathol. 17, 89-105

JUNG, H. J., C. S. RAINE and K. SUZUKI (1981): Spinal epidermoid cyst in the mouse. An ultrastructural study. Acta Neuropathol. 53, 81-85

KAPPEL, C. A., M. C. DAL CANTO, R. W. MELVOLD and B. S. KIM (1991): Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease. J. Immunol. 147, 4322-4326

KEMPPINEN, A., S. SAWCER and A. COMPSTON (2011): Genome-wide association studies in multiple sclerosis: lessons and future prospects. Brief. Funct. Genomics 10, 61-70

KIESEIER, B. C., T. SEIFERT, G. GIOVANNONI and H. P. HARTUNG (1999): Matrix metalloproteinases in inflammatory demyelination: targets for treatment. Neurology 53, 20-25

KULWICH, B. A. (1994): Epidermoid cysts in the central nervous system of rats and mice: an incidental finding in toxicity/oncogenicity studies. Vet. Pathol. 31, 475-478

KUMNOK, J., R. ULRICH, K. WEWETZER, K. ROHN, F. HANSMANN, W. BAUMGÄRTNER and S. ALLDINGER (2008): Differential transcription of matrix-metalloproteinase genes in primary mouse astrocytes and microglia infected with Theiler's murine encephalomyelitis virus. J. Neurovirol. 14, 205-217

LARSEN, P. H. and V. W. YONG (2004): The expression of matrix metalloproteinase-12 by oligodendrocytes regulates their maturation and morphological differentiation. J. Neurosci. 24, 7597-7603

LASSMANN, H. (2004): Experimental autoimmune encephalomyelitis. In: Myelin biology and disorders, R. A. Lazzarini, Ed, Vol. 2, Elsevier Academic Press, London pp. 1039-1063

LASSMANN, H. (2005): Multiple sclerosis pathology: evolution of pathogenetic concepts. Brain Pathol. 15, 217-222


47

LASSMANN, H., W. BRÜCK and C. LUCCHINETTI (2001): Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy. Trends Mol. Med. 7, 115-121

LASSMANN, H., W. BRÜCK and C. F. LUCCHINETTI (2007): The immunopathology of multiple sclerosis: an overview. Brain Pathol. 17, 210-218

LEE, M. A., J. PALACE, G. STABLER, J. FORD, A. GEARING and K. MILLER (1999): Serum gelatinase B, TIMP-1 and TIMP-2 levels in multiple sclerosis - a longitudinal clinical and MRI study. Brain 122, 191-197

LEHMANN, H. C., A. KOHNE, F. BERNAL, P. JANGOUK, G. MEYER ZU HORSTE, T. DEHMEL, H. P. HARTUNG, S. C. PREVITALI and B. C. KIESEIER (2009): Matrix metalloproteinase-2 is involved in myelination of dorsal root ganglia neurons. Glia 57, 479-489

LEMONS, M. L., J. D. SANDY, D. K. ANDERSON and D. R. HOWLAND (2001): Intact aggrecan and fragments generated by both aggrecanse and metalloproteinase-like activities are present in the developing and adult rat spinal cord and their relative abundance is altered by injury. J. Neurosci. 21, 4772-4781

LINDBERG, R. L. P., C. J. A. DE GROOT, L. MONTAGNE, P. FREITAG, P. VAN DER VALK, L. KAPPOS and D. LEPPERT (2001): The expression profile of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lesions and normal appearing white matter of multiple sclerosis. Brain 124, 1743-1753

LINDNER, M., J. FOKUHL, F. LINSMEIER, C. TREBST and M. STANGEL (2009): Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination. Neurosci. Lett. 453, 120-125

LINDNER, M., S. HEINE, K. HAASTERT, N. GARDE, J. FOKUHL, F. LINSMEIER, C. GROTHE, W. BAUMGÄRTNER and M. STANGEL (2008): Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelination. Neuropathol. Appl. Neurobiol. 34, 105-114

LINDSEY, J. W. (2005): EAE: history, clinical signs, and disease course. In: Experimental models of multiple sclerosis, E. Lavi and C. S. Constantinescu, Eds, Springer US, New York, pp. 1-10

LININGTON, C., B. ENGELHARDT, G. KAPOCS and H. LASSMAN (1992): Induction of persistently demyelinated lesions in the rat following the repeated adoptive transfer of encephalitogenic T-cells and demyelinating antibody. J. Neuroimmunol. 40, 219-224

LIPTON, H. L. (1975): Theilers virus-infection in mice - unusual biphasic disease process leading to demyelination. Infect. Immun. 11, 1147-1155


48

LIPTON, H. L. (1980): Persistent Theiler's murine encephalomyelitis virus infection in mice depends on plaque size. J. Gen. Virol. 46, 169-177

LIPTON, H. L., B. S. KIM, H. YAHIKOZAWA and C. F. NADLER (2001): Serological evidence that Mus musculus is the natural host of Theiler's murine encephalomyelitis virus. Virus Res. 76, 79-86

LIPTON, H. L., A. S. KUMAR and M. TROTTIER (2005): Theiler's virus persistence in the central nervous system of mice is associated with continuous viral replication and a difference in outcome of infection of infiltrating macrophages versus oligodendrocytes. Virus Res. 111, 214-223

LIPTON, H. L., G. TWADDLE and M. L. JELACHICH (1995): The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease. J. Virol. 69, 2525-2533

LO, E. H., X. Y. WANG and M. L. CUZNER (2002): Extracellular proteolysis in brain injury and inflammation: Role for plasminogen activators and matrix metalloproteinases. J. Neurosci. Res. 69, 1-9

LUBLIN, F. D. and S. C. REINGOLD (1996): Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis. Neurology 46, 907-911

LUCCHINETTI, C., W. BRÜCK, J. PARISI, B. SCHEITHAUER, M. RODRIGUEZ and H. LASSMANN (2000): Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann. Neurol. 47, 707-717

MABON, P. J., L. C. WEAVER and G. A. DEKABAN (2000): Inhibition of monocyte/macrophage migration to a spinal cord injury site by an antibody to the integrin alphaD: a potential new anti-inflammatory treatment. Exp. Neurol. 166, 52-64

MARTINAT, C., N. JAROUSSE, M. C. PREVOST and M. BRAHIC (1999): The GDVII strain of Theiler's virus spreads via axonal transport. J. Virol. 73, 6093-6098

MCGAVERN, D. B., P. D. MURRAY, C. RIVERA-QUINONES, J. D. SCHMELZER, P. A. LOW and M. RODRIGUEZ (2000): Axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis. Brain 123, 519-531

MCGAVERN, D. B., L. ZOECKLEIN, K. M. DRESCHER and M. RODRIGUEZ (1999): Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination. Exp. Neurol. 158, 171-181


49

MELVOLD, R. W., D. M. JOKINEN, S. D. MILLER, M. C. DAL CANTO and H. L. LIPTON (1990): Identification of a locus on mouse chromosome 3 involved in differential susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease. J. Virol. 64, 686-690

MI, W., C. R. YOUNG, R. W. STORTS, A. J. STEELMAN, M. W. MEAGHER and C. J. WELSH (2006): Restraint stress facilitates systemic dissemination of Theiler's virus and alters its pathogenecity. Microb. Pathog. 41, 133-143

MIAO, Q., W. BAUMGÄRTNER, K. FAILING and S. ALLDINGER (2003): Phase-dependent expression of matrix metalloproteinases and their inhibitors in demyelinating canine distemper encephalitis. Acta Neuropathol. 106, 486-494

MICHIELS, T., N. JAROUSSE and M. BRAHIC (1995): Analysis of the leader and capsid coding regions of persistent and neurovirulent strains of Theiler's virus. Virology 214, 550-558

MILLER, S. D., R. J. CLATCH, D. C. PEVEAR, J. L. TROTTER and H. L. LIPTON (1987): Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins. J. Immunol. 138, 3776-3784

MILLER, S. D., S. J. GERETY, M. K. KENNEDY, J. D. PETERSON, J. L. TROTTER, V. K. TUOHY, C. WALTENBAUGH, M. C. DAL CANTO and H. L. LIPTON (1990): Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. III. Failure of neuroantigen-specific immune tolerance to affect the clinical course of demyelination. J. Neuroimmunol. 26, 9-23

MILLER, S. D., W. J. KARPUS, J. G. POPE, M. C. DAL CANTO and R. W. MELVOLD (1994): Theiler´s virus-induced demyelinating disease. In: Autoimmune disease models: a guidebook, I. R. Cohen and A. Miller, Eds, Academic Press, San Diego/New York, pp. 23-38

MILLER, S. D., J. K. OLSON and J. L. CROXFORD (2001): Multiple pathways to induction of virus-induced autoimmune demyelination: Lessons from Theiler's virus infection. J. Autoimmun. 16, 219-227

MIROWSKA-GUZEL, D., G. GROMADZKA, A. CZLONKOWSKI and A. CZLONKOWSKA (2009): Association of MMP1, MMP3, MMP9, and MMP12 polymorphisms with risk and clinical course of multiple sclerosis in a Polish population. J. Neuroimmunol. 214, 113-117

MIX, E., H. MEYER-RIENECKER, H. P. HARTUNG and U. K. ZETTL (2010): Animal models of multiple sclerosis-potentials and limitations. Prog. Neurobiol. 92, 386-404

MIX, E., H. MEYER-RIENECKER and U. K. ZETTL (2008): Animal models of multiple sclerosis for the development and validation of novel therapies - potential and limitations. J. Neurol. 255 Suppl 6, 7-14


50

MOHAN, H., M. KRUMBHOLZ, R. SHARMA, S. EISELE, A. JUNKER, M. SIXT, J. NEWCOMBE, H. WEKERLE, R. HOHLFELD, H. LASSMANN and E. MEINL (2010): Extracellular matrix in multiple sclerosis lesions: fibrillar collagens, biglycan and decorin are upregulated and associated with infiltrating immune cells. Brain Pathol. 20, 966-975

MONTEYNE, P., J. F. BUREAU and M. BRAHIC (1997): The infection of mouse by Theiler's virus: from genetics to immunology. Immunol. Rev. 159, 163-176

NAGASE, H. (1997): Activation mechanisms of matrix metalloproteinases. Biol. Chem. 378, 151-160

NAGASE, H., J. J. ENGHILD, K. SUZUKI and G. SALVESEN (1990): Stepwise activation mechanisms of the precursor of matrix metalloproteinase-3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry (Mosc). 29, 5783-5789

NAGASE, H. and J. F. WOESSNER (1999): Matrix metalloproteinases. J. Biol. Chem. 274, 21491-21494

NGUYEN, H. T. (1988): Intraspinal Dermoid Cyst in a C57BL6 Mouse. Lab. Anim. Sci. 38, 739-740

NOBEL, T. A., A. NYSKA, M. PIRAK, M. SKOLNIK and A. MESHORER (1987): Epidermoid cysts in the central nervous system of mice. J. Comp. Pathol. 97, 357-359

NOSEWORTHY, J. H. (1999): Progress in determining the causes and treatment of multiple sclerosis. Nature 399, A40-47

NUTTALL, R. K., C. SILVA, W. HADER, A. BAR-OR, K. D. PATEL, D. R. EDWARDS and V. W. YONG (2007): Metalloproteinases are enriched in microglia compared with leukocytes and they regulate cytokine levels in activated microglia. Glia 55, 516-526

O'SHEA, H., J. CRANG, P. TONKS, A. A. NASH and J. K. FAZAKERLEY (1997): The PI capsid region of Theiler's virus controls replication in mouse glial cell cultures. Arch. Virol. 142, 1521-1535

OH, L. Y., P. H. LARSEN, C. A. KREKOSKI, D. R. EDWARDS, F. DONOVAN, Z. WERB and V. W. YONG (1999): Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J. Neurosci. 19, 8464-8475

OKSENBERG, J. R. and S. L. HAUSER (2005): Genetics of multiple sclerosis. Neurol. Clin. 23, 61-75


51

OLESZAK, E. L., J. R. CHANG, H. FRIEDMAN, C. D. KATSETOS and C. D. PLATSOUCAS (2004): Theiler's virus infection: a model for multiple sclerosis. Clin. Microbiol. Rev. 17, 174-207

PACHNER, A. R., J. BRADY and K. NARAYAN (2007): Antibody-secreting cells in the central nervous system in an animal model of MS: phenotype, association with disability, and in vitro production of antibody. J. Neuroimmunol. 190, 112-120

PACHNER, A. R., L. LI and K. NARAYAN (2007): Intrathecal antibody production in an animal model of multiple sclerosis. J. Neuroimmunol. 185, 57-63

PALSSON, P. A. (1976): Maedi and Visna in sheep. In: Slow virus diseases of animals and man, R. H. Kimberlin, Ed, North-Holland Pub. Co., Amsterdam/New York, pp. 17-43

PANITCH, H. and A. APPLEBEE (2011): Treatment of walking impairment in multiple sclerosis: an unmet need for a disease-specific disability. Expert Opin Pharmacother 12, 1511-1521

PATERSON, P. Y. (1960): Transfer of Allergic Encephalomyelitis in Rats by Means of Lymph Node Cells. J. Exp. Med. 111, 119-143

PETERSON, J. D., W. J. KARPUS, R. J. CLATCH and S. D. MILLER (1993): Split tolerance of Th1 and Th2 cells in tolerance to Theiler's murine encephalomyelitis virus. Eur. J. Immunol. 23, 46-55

PETERSON, J. D., C. WALTENBAUGH and S. D. MILLER (1992): IgG subclass responses to Theiler's murine encephalomyelitis virus infection and immunization suggest a dominant role for Th1 cells in susceptible mouse strains. Immunology 75, 652-658

PETURSSON, G., N. NATHANSON, P. A. PALSSON, J. MARTIN and G. GEORGSSON (1978): Immunopathogenesis of Visna. A slow virus disease of the central nervous system. Acta Neurol. Scand. Suppl. 67, 205-219

PEVEAR, D. C., J. BORKOWSKI, M. CALENOFF, C. K. OH, B. OSTROWSKI and H. L. LIPTON (1988): Insights into Theilers virus neurovirulence based on a genomic comparison of the neurovirulent GDVII and less virulent BeAn strains. Virology 165, 1-12

PLATT, S. R., J. GRAHAM, C. L. CHRISMAN, A. ADJIRI-AWERE and R. M. CLEMMONS (1999): Canine intracranial epidermoid cyst. Vet. Radiol. Ultrasound 40, 454-458

POLMAN, C. H., S. C. REINGOLD, B. BANWELL, M. CLANET, J. A. COHEN, M. FILIPPI, K. FUJIHARA, E. HAVRDOVA, M. HUTCHINSON, L. KAPPOS, F. D. LUBLIN, X. MONTALBAN, P. O'CONNOR, M. SANDBERG-WOLLHEIM, A. J. THOMPSON, E. WAUBANT, B. WEINSHENKER and J. S. WOLINSKY (2011): Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann. Neurol. 69, 292-302


52

POPOVICH, P. G., Z. GUAN, P. WEI, I. HUITINGA, N. VAN ROOIJEN and B. T. STOKES (1999): Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury. Exp. Neurol. 158, 351-365

PRINGPROA, K., K. ROHN, M. KUMMERFELD, K. WEWETZER and W. BAUMGÄRTNER (2010): Theiler's murine encephalomyelitis virus preferentially infects immature stages of the murine oligodendrocyte precursor cell line BO-1 and blocks oligodendrocytic differentiation in vitro. Brain Res. 1327, 24-37

PRIVAT, A., C. JACQUE, J. M. BOURRE, P. DUPOUEY and N. BAUMANN (1979): Absence of the major dense line in myelin of the mutant mouse "shiverer". Neurosci. Lett. 12, 107-112

RACANIELLO, V. R. (2007): Picornaviridae: the viruses and their replication. In: Fields Virology, D. M. Knipe and P. M. Howley, Eds, Lippincott Williams & Wilkins, Philadelphia, pp. 795-839

RAPALINO, O., O. LAZAROV-SPIEGLER, E. AGRANOV, G. J. VELAN, E. YOLES, M. FRAIDAKIS, A. SOLOMON, R. GEPSTEIN, A. KATZ, M. BELKIN, M. HADANI and M. SCHWARTZ (1998): Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat. Med. 4, 814-821

RODRIGUEZ, M. (2007): Effectors of demyelination and remyelination in the CNS: Implications for multiple sclerosis. Brain Pathol. 17, 219-229

RODRIGUEZ, M., J. LEIBOWITZ and C. S. DAVID (1986): Susceptibility to Theilers virus-induced demyelination - mapping of the gene within the H-2d region. J. Exp. Med. 163, 620-631

RODRIGUEZ, M., J. L. LEIBOWITZ and P. W. LAMPERT (1983): Persistent infection of oligodendrocytes in Theiler's virus-induced encephalomyelitis. Ann. Neurol. 13, 426-433

RODRIGUEZ, M. and S. SRIRAM (1988): Successful therapy of Theiler's virus-induced demyelination (DA strain) with monoclonal anti-Lyt-2 antibody. J. Immunol. 140, 2950-2955

ROPPER, A. H. (2006): Selective treatment of multiple sclerosis. N. Engl. J. Med. 354, 965-967

ROSATI, G. (2001): The prevalence of multiple sclerosis in the world: an update. Neurol. Sci. 22, 117-139

ROSENBERG, G. A. (2001): Matrix metalloproteinases in multiple sclerosis: is it time for a treatment trial? Ann. Neurol. 50, 431-433

ROSENBERG, G. A. (2002): Matrix metalloproteinases in neuroinflammation. Glia 39, 279-291


53

ROSENBERG, G. A. (2005): Matrix metalloproteinases biomarkers in multiple sclerosis. Lancet 365, 1291-1293

ROSENBERG, G. A. (2009): Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. Lancet. Neurol. 8, 205-216

ROUSSARIE, J. P., C. RUFFIE and M. BRAHIC (2007): The role of myelin in Theiler's virus persistence in the central nervous system. PLoS pathogens 3, e23

ROUSSARIE, J. P., C. RUFFIE, J. M. EDGAR, I. GRIFFITHS and M. BRAHIC (2007): Axon myelin transfer of a non-enveloped virus. PloS one 2, e1331

ROZHON, E. J., J. D. KRATOCHVIL and H. L. LIPTON (1983): Analysis of genetic variation in Theiler's virus during persistent infection in the mouse central nervous system. Virology 128, 16-32

SAHER, G., B. BRÜGGER, C. LAPPE-SIEFKE, W. MÖBIUS, R. TOZAWA, M. C. WEHR, F. WIELAND, S. ISHIBASHI and K. A. NAVE (2005): High cholesterol level is essential for myelin membrane growth. Nat. Neurosci. 8, 468-475

SATO, F., H. TANAKA, F. HASANOVIC and I. TSUNODA (2011): Theiler's virus infection: pathophysiology of demyelination and neurodegeneration. Pathophysiology 18, 31-41

SEEHUSEN, F. and W. BAUMGÄRTNER (2009): Axonal pathology and loss precede demyelination and accompany chronic lesions in a spontaneously occurring animal model of multiple sclerosis. Brain Pathol. 20, 551-559

SEKINE-AIZAWA, Y., E. HAMA, K. WATANABE, S. TSUBUKI, M. KANAI-AZUMA, Y. KANAI, H. ARAI, H. AIZAWA, N. IWATA and T. C. SAIDO (2001): Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum. Eur. J. Neurosci. 13, 935-948

SERAFINI, B., B. ROSICARELLI, D. FRANCIOTTA, R. MAGLIOZZI, R. REYNOLDS, P. CINQUE, L. ANDREONI, P. TRIVEDI, M. SALVETTI, A. FAGGIONI and F. ALOISI (2007): Dysregulated Epstein-Barr virus infection in the multiple sclerosis brain. J. Exp. Med. 204, 2899-2912

SHIRYAEV, S. A., A. Y. SAVINOV, P. CIEPLAK, B. I. RATNIKOV, K. MOTAMEDCHABOKI, J. W. SMITH and A. Y. STRONGIN (2009): Matrix metalloproteinase proteolysis of the myelin basic protein isoforms is a source of immunogenic peptides in autoimmune multiple sclerosis. PLoS ONE 4, e4952

SKOFF, R. P. (1976): Myelin deficit in the jimpy mouse may be due to cellular abnormalities in astroglia. Nature 264, 560-562


54

SKULJEC, J., V. GUDI, R. ULRICH, K. FRICHERT, O. YILDIZ, R. PUL, E. V. VOSS, K. WISSEL, W. BAUMGÄRTNER and M. STANGEL (2011): Matrix metalloproteinases and their tissue inhibitors in cuprizone-induced demyelination and remyelination of brain white and gray matter. J. Neuropathol. Exp. Neurol. 70, 758-769

SOLDAN, S. S., R. BERTI, N. SALEM, P. SECCHIERO, L. FLAMAND, P. A. CALABRESI, M. B. BRENNAN, H. W. MALONI, H. F. MCFARLAND, H. C. LIN, M. PATNAIK and S. JACOBSON (1997): Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA. Nat. Med. 3, 1394-1397

SOMERVILLE, R. P., S. A. OBLANDER and S. S. APTE (2003): Matrix metalloproteinases: old dogs with new tricks. Genome Biol 4, 216

SRIRAM, S., W. MITCHELL and C. STRATTON (1998): Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS. Neurology 50, 571-572

STEINBERG, T., K. MATIASEK, A. BRUHSCHWEIN and A. FISCHER (2007): Imaging diagnosis - intracranial epidermoid cyst in a Doberman Pinscher. Vet. Radiol. Ultrasound 48, 250-253

STEINMAN, L. and S. S. ZAMVIL (2006): How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis. Ann. Neurol. 60, 12-21

STERNLICHT, M. D. and Z. WERB (2001): How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463-516

STOHLMAN, S. A. and D. R. HINTON (2001): Viral induced demyelination. Brain Pathol. 11, 92-106

STOMRUD, E., M. BJÖRKQVIST, S. JANCIAUSKIENE, L. MINTHON and O. HANSSON (2010): Alterations of matrix metalloproteinases in the healthy elderly with increased risk of prodromal Alzheimer's disease. Alzheimers Res Ther 2, 20

STROOP, W. G. (1984): Spinal leptomeningeal epidermoid cysts in inbred mouse strains. Acta Neuropathol. 63, 301-305

SUZUKI, K., J. J. ENGHILD, T. MORODOMI, G. SALVESEN and H. NAGASE (1990): Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry (Mosc). 29, 10261-10270

TEESALU, T., A. E. HINKKANEN and A. VAHERI (2001): Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice. Am. J. Pathol. 159, 2227-2237


55

THEILER, M. (1934): Spontaneous encephalomyelitis of mice - a new virus disease. Science 80, 122

THEILER, M. (1937): Spontaneous encephalomyelitis of mice, a new virus disease. J. Exp. Med. 65, 705-719

THEILER, M. and S. GARD (1940): Encephalomyelitis of mice, III. Epidemiology. J. Exp. Med. 72, 79-90

TIROTTA, E., K. S. CARBAJAL, C. S. SCHAUMBURG, L. WHITMAN and T. E. LANE (2010): Cell replacement therapies to promote remyelination in a viral model of demyelination. J. Neuroimmunol. 224, 101-107

TOFT-HANSEN, H., R. K. NUTTALL, D. R. EDWARDS and T. OWENS (2004): Key metalloproteinases are expressed by specific cell types in experimental autoimmune encephalomyelitis. J. Immunol. 173, 5209-5218

TRAPP, B. D. and K. A. NAVE (2008): Multiple sclerosis: an immune or neurodegenerative disorder? Annu. Rev. Neurosci. 31, 247-269

TSEVELEKI, V., R. RUBIO, S. S. VAMVAKAS, J. WHITE, E. TAOUFIK, E. PETIT, J. QUACKENBUSH and L. PROBERT (2010): Comparative gene expression analysis in mouse models for multiple sclerosis, Alzheimer's disease and stroke for identifying commonly regulated and disease-specific gene changes. Genomics 96, 82-91

TSUNODA, I. and R. S. FUJINAMI (2002): Inside-out versus outside-in models for virus induced demyelination: axonal damage triggering demyelination. Springer Semin. Immunopathol. 24, 105-125

TSUNODA, I., Y. IWASAKI, H. TERUNUMA, K. SAKO and Y. OHARA (1996): A comparative study of acute and chronic diseases induced by two subgroups of Theiler's murine encephalomyelitis virus. Acta Neuropathol. 91, 595-602

TSUNODA, I., L. Q. KUANG, J. E. LIBBEY and R. S. FUJINAMI (2003): Axonal injury heralds virus-induced demyelination. Am. J. Pathol. 162, 1259-1269

TSUNODA, I., T. TANAKA, F. HASANOVIC and R. S. FUJINAMI (2008): Axonal degeneration as a self-destructive defense mechanism against neurotropic virus infection. J. Neuropathol. Exp. Neurol. 67, 491-492

ULRICH, R., W. BAUMGÄRTNER, I. GERHAUSER, F. SEELIGER, V. HAIST, U. DESCHL and S. ALLDINGER (2006): MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis. J. Neuropathol. Exp. Neurol. 65, 783-793


56

ULRICH, R., I. GERHAUSER, F. SEELIGER, W. BAUMGÄRTNER and S. ALLDINGER (2005): Matrix metalloproteinases and their inhibitors in the developing mouse brain and spinal cord: A reverse transcription quantitative polymerase chain reaction study. Dev. Neurosci. 27, 408-418

ULRICH, R., A. KALKUHL, U. DESCHL and W. BAUMGÄRTNER (2010): Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis. J. Cell. Mol. Med. 14, 434-448

ULRICH, R., F. SEELIGER, M. KREUTZER, P. G. GERMANN and W. BAUMGÄRTNER (2008): Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)-positive putative oligodendroglial progenitor cells. Neuropathol. Appl. Neurobiol. 34, 603-620

VAN DER VALK, P. and C. J. DE GROOT (2000): Staging of multiple sclerosis (MS) lesions: pathology of the time frame of MS. Neuropathol. Appl. Neurobiol. 26, 2-10

VAN DER VEEN, R. C., R. A. SOBEL and M. B. LEES (1986): Chronic experimental allergic encephalomyelitis and antibody-responses in rabbits immunized with bovine proteolipid apoprotein. J. Neuroimmunol. 11, 321-333

VAN WART, H. E. and H. BIRKEDAL-HANSEN (1990): The cysteine switch - a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. USA 87, 5578-5582

VANDEVELDE, M. and A. ZURBRIGGEN (2005): Demyelination in canine distemper virus infection: a review. Acta Neuropathol. 109, 56-68

VISSE, R. and H. NAGASE (2003): Matrix metalloproteinases and tissue inhibitors of metalloproteinases - structure, function, and biochemistry. Circ. Res. 92, 827-839

WANG, X., J. JUNG, M. ASAHI, W. CHWANG, L. RUSSO, M. A. MOSKOWITZ, C. E. DIXON, M. E. FINI and E. H. LO (2000): Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J. Neurosci. 20, 7037-7042

WELLS, J. E. A., T. K. RICE, R. K. NUTTALL, D. R. EDWARDS, H. ZEKKI, S. RIVEST and V. W. YONG (2003): An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice. J. Neurosci. 23, 10107-10115

WOODRUFF, R. H. and R. J. M. FRANKLIN (1999): Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study. Glia 25, 216-228


57

WROBLEWSKA, Z., S. U. KIM, W. D. SHEFFIELD and D. H. GILDEN (1979): Growth of the WW strain of Theiler virus in mouse central nervous system organotypic culture. Acta Neuropathol. 47, 13-19

YAMADA, M., A. ZURBRIGGEN and R. S. FUJINAMI (1990): Monoclonal antibody to Theiler's murine encephalomyelitis virus defines a determinant on myelin and oligodendrocytes, and augments demyelination in experimental allergic encephalomyelitis. J. Exp. Med. 171, 1893-1907

YONG, V. W. (2005): Metalloproteinases: mediators of pathology and regeneration in the CNS. Nat. Rev. Neurosci. 6, 931-944

YONG, V. W., C. A. KREKOSKI, P. A. FORSYTH, R. BELL and D. R. EDWARDS (1998): Matrix metalloproteinases and diseases of the CNS. Trends Neurosci. 21, 75-80

YONG, V. W., C. POWER, P. FORSYTH and D. R. EDWARDS (2001): Metalloproteinases in biology and pathology of the nervous system. Nat. Rev. Neurosci. 2, 502-511

YONG, V. W., R. K. ZABAD, S. AGRAWAL, A. GONCALVES DASILVA and L. M. METZ (2007): Elevation of matrix metalloproteinases (MMPs) in multiple sclerosis and impact of immunomodulators. J. Neurol. Sci. 259, 79-84

ZAMBONI, P., R. GALEOTTI, E. MENEGATTI, A. M. MALAGONI, G. TACCONI, S. DALL'ARA, I. BARTOLOMEI and F. SALVI (2009): Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 80, 392-399

ZAMBONI, P., E. MENEGATTI, B. WEINSTOCK-GUTTMAN, C. SCHIRDA, J. L. COX, A. M. MALAGONI, D. HOJANACKI, C. KENNEDY, E. CARL, M. G. DWYER, N. BERGSLAND, R. GALEOTTI, S. HUSSEIN, I. BARTOLOMEI, F. SALVI and R. ZIVADINOV (2009): The severity of chronic cerebrospinal venous insufficiency in patients with multiple sclerosis is related to altered cerebrospinal fluid dynamics. Funct. Neurol. 24, 133-138

ZAMVIL, S. S., P. A. NELSON, D. J. MITCHELL, R. L. KNOBLER, R. B. FRITZ and L. STEINMAN (1985): Encephalitogenic T-cell clones specific for myelin basic-protein - an unusual bias in antigen recognition. J. Exp. Med. 162, 2107-2124

ZHOU, J., N. W. MARTEN, C. C. BERGMANN, W. B. MACKLIN, D. R. HINTON and S. A. STOHLMAN (2005): Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis. J. Virol. 79, 4764-4773

ZOECKLEIN, L. J., K. D. PAVELKO, J. GAMEZ, L. PAPKE, D. B. MCGAVERN, D. R. URE, M. K. NJENGA, A. J. JOHNSON, S. NAKANE and M. RODRIGUEZ (2003): Direct comparison of demyelinating disease induced by the Daniel's strain and BeAn strain of Theiler's murine encephalomyelitis virus. Brain Pathol. 13, 291-308


58

ZWIBEL, H. L. and J. SMRTKA (2011): Improving quality of life in multiple sclerosis: an unmet need. Am. J. Manag. Care 17 Suppl 5 Improving, S139-145

Chapter 10: Acknowledgements

59

Chapter 10 Acknowledgements

I like to thank…. … my main supervisor Prof. Dr. Wolfgang Baumgärtner for providing the interesting

topic of my project, his academic guidance, fruitful discussions and that he gave me the opportunity to propose and realize own ideas.

… my co-supervisors Prof. Dr. Gerardy-Schahn and Prof. Dr. Claus for their critical

appraisal of my work, suggestions, support as well as interesting and helpful discussions.

… Dr. Reiner Ulrich, PhD. for his collaboration, scientific advice and helpful

discussions that contributed to the realization of this work. … Prof. Dr. Hagen Gasse for his dedication and support. … the National Academic Research Foundation (Germany) for providing a

scholarship and giving financial support. … Kerstin R. for a nice time at the sunny side of the second floor, for sharing the lab

and office with me and tender, love and care. … Siggi for his support and a lot of funny hours in the necropsy hall. … Bettina, Petra, Claudia, Caro, Kerstin S. and Danuta for their active support and

help with all minor and major problems in the lab. … all my other colleges at the Department of Pathology for support, help, good ideas

and interesting conversations especially Robert, Annika, Verena, Christina, Mihaela, Ingo G., Ingo S., Frauke, Dirk and Björn. Special thanks to Suliman for sharing the office with me and Max & Johannes for a lot of coffee breaks with interesting discussions in the first floor.

Chapter 10: Acknowledgements

60

… Jan and Marc for being their friend and many shared experiences during a great time in Giessen and Hannover.

… Vanessa ;-* … my parents, who always support me in every possible way and who are always

there for me.

Documents

The role of matrix metalloproteinase-3 and -12 in the