Investigation of murine and canine glia cell differentiation in vitro and in vivo

ISBN 978-3-86345-153-0

Florian Heinrich Hansmann

Investigation of murine and canine glia cell

differentiation in vitro and in vivo

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1. Auflage 2013

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ISBN 978-3-86345-153-0

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University of Veterinary Medicine Hannover

Investigation of murine and canine glia cell differentiation in vitro and in vivo

Thesis

Submitted in partial fulfillment of the requirements for the degree -Doctor of Veterinary Medicine-

Doctor medicinae veterinariae (Dr. med. vet.)

by

Florian Heinrich Hansmann Kiel

Academic supervision: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ.

Department of Pathology,

University of Veterinary Medicine Hannover, Germany

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

Department of Pathology,

University of Veterinary Medicine Hannover, Germany

2. Referee: Prof. Dr. Marion Bankstahl

Department of Pharmacology, Toxicology and Pharmacy University of Veterinary Medicine Hannover, Germany

Day of oral examination: 17^th May 2013

This study was partly funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG).

To my family

Happiness is the only thing that doubles when you share it.

Albert Schweizer

Parts of the thesis have already been published or communicated:

F. HANSMANN*, 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. 21, 1161-1175

*both authors contributed equally to this publication.

I. GERHAUSER*, F. HANSMANN*, C. PUFF, J. KUMNOK, D. SCHAUDIEN, K. WEWETZER and W. BAUMGÄRTNER (2012):

Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro.

J. Neuroimmunol. 252, 49-55

*both authors contributed equally to this publication.

M. OMAR, P. BOCK, R. KREUTZER, S. ZIEGE, I. IMBSCHWEILER, F. HANSMANN, C. T. PECK, W. BAUMGÄRTNER and K. WEWETZER (2011):

Defining the morphological phenotype: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a novel marker for in situ detection of canine but not rat olfactory ensheathing cells.

Cell Tissue Res. 344, 391-405

Oral presentation:

F. HANSMANN, K. PRINGPROA, R. ULRICH, Y. SUN, V. HERDER, M. KREUTZER, W. BAUMGÄRTNER and K. WEWETZER (2011)

Transplantation einer oligodendroglialen Vorläuferzellinie in den entmarkten

C

1 G

... 1

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2.1. CELLULAR COMPOSITION OF THE CENTRAL NERVOUS SYSTEM ... 4

2.2. OLIGODENDROCYTES ... 6

2.3. SCHWANN CELLS AND ALDYNOGLIA ... 8

2.4. OLFACTORY ENSHEATHING CELLS... 13

2.5. MICROGLIA AND MACROPHAGES ... 15

C

3 D

: 2',3'-

3'-

(CNP

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... 19

C

4 H

-

... 21

C

5 T

’

... 23

C

6 D

C

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C

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List of abbreviations

A2B5 Cell surface ganglioside epitope A2B5 AP2 Activator protein 2

ATP Adenosine triphosphate

BDNF Brain-derived neurotrophic factor BMPs Bone morphogenic proteins

CC-1 Anti-Adenomatosis polyposis coli, clone CC-1 CCP Caudal cerebellar peduncle

CCR2 Chemokine receptor 2

CNPase 2’,3’-cyclic nucleotide 3’-phosphodiesterase CNS Central nervous system

CSCs Central Schwann cells CX3CR1 Chemokine receptor 1

EGFP Enhanced green fluorescence protein ETS Transcription factor family E-twenty six FGF-2 Fibroblast growth factor 2

FGFR3 Fibroblast growth factor receptor 3 Fizz 1 Resistin-like alpha

GalC Galactosylceramidase

GDNF Glial cell line-derived neurotrophic factor GFAP Glial fibrillary acidic protein

HGF Hepatocyte growth factor HNK-1 Human natural killer-1 Hpi Hours post infection IFN-γ Interferon-γ

IGFs Insulin-like growth factors IL10^high High expression of interleukin 10 IL12^low Low expression of interleukin 12 INOS Inducible nitric oxide synthase LPS Lipopolysaccharide

MAG Myelin associated glycoprotein MBP Myelin basic protein

MHCII Major histocompatibility complex class II

List of abbreviations

IV MS Multiple sclerosis

MSCs Mesenchymal stem cells NCAM Neural cell adhesion molecule

NF-κB Nuclear factor that binds the kappa-light-chain-enhancer in B cells NG-2 Nerve/glial antigen 2

NO Nitric oxide NRG1 Neuregulin 1 NSCs Neural stem cells NT3 Neurotrophin 3

O4 Seminolipid sulfatide antigen OECs Olfactory ensheathing cells

OCT6 Octamerbinding transcription factor 6 Olig2 Oligodendrocyte transcription factor 2 Omgp Outer membrane glycoprotein OPC Oligodendrocyte precursor cells P0 Protein zero

p75^NTR Neurotrophin receptor p75 PAX3 Paired box gene 3

PDGF Platelet-derived growth factor

PDGFR-α Platelet-derived growth factor receptor alpha PNS Peripheral nervous system

PSA-NCAM polysialylated-neural cell adhesion molecule PSCs Peripheral Schwann cells

SCI Spinal cord injury SCP Schwann cell precursor SCs Schwann cells

SOX2 Sex determining region Y-box 2 SOX10 Sex determining region Y-box 10 SPHK1 Sphingosine kinase 1

TGFβ Tumor growth factor β

TME Theiler’s murine encephalomyelitis TMEV Theiler’s murine encephalomyelitis virus TNF Tumor necrosis factor

YM1 Chitinase 3-like protein 3 ZNS Zentrales Nervensystem

List of tables and figures

Table 1: Marker expression of myelinating and non-myelinating Schwann cells as well as central Schwann cells ... 10 Table 2: Marker expression of olfactory ensheathing cells in vitro and in vivo... 13 Table 3: Classification, marker expression and function of M1/M2 cells ... 16

Figure 1: Summary of the most frequent cell types in the central nervous system .. 4 Figure 2: Oligodendrocyte differentiation ... 7 Figure 3: Development of central Schwann cells ... 9 Figure 4: Development and differentiation of peripheral Schwann cells ... 12

Chapter 1 General remarks

The main topic of this thesis was the characterization of murine and canine glia cell differentiation in vitro and in vivo. This is an important step for a better understanding of the pathophysiology of demyelinating diseases like multiple sclerosis (MS), Theiler’s murine encephalomyelitis (TME), canine distemper encephalitis and spinal cord injury (SCI). Transplantation of cells represents a promising treatment strategy for demyelinating diseases of the central nervous system (CNS). Therefore, a comprehensive in vitro characterization of cells represents the first and very important step before investigating the behavior and therapeutic potential of cells in vivo. In vitro assays are necessary for predicting and/or testing their proliferation, migration and differentiation potential. However, the best proof of principle is testing the potential and behavior of cells in vivo – in a demyelinated/injured environment as well as in the normal CNS. In addition, a sound knowledge regarding the environment at the lesion site is necessary for successful establishment of such treatment strategies in CNS diseases. Therefore the aims of this thesis were: i) a comparative in vitro and in vivo analysis of canine olfactory ensheathing cells using immunohistochemistry, quantitative PCR and western blot, ii) the establishment and characterization of an ethidium bromide-induced (toxic) demyelination model in the murine caudal cerebellar peduncle and iii) an in vivo characterization of the murine oligodendrocyte precursor cell line BO-1 after transplantation in the demyelinated and non-demyelinated murine caudal cerebellar peduncle.

Chapter 2 General introduction

Demyelination and axonal degeneration or loss in the central nervous system (CNS) are common features observed in diseases like multiple sclerosis (MS;

LUCCHINETTI et al. 2000, COMPSTON et al. 2008), Theiler’s murine encephalomyelitis (TME; DAL CANTO et al. 1996, HANSMANN et al. 2012a, HERDER et al. 2012), canine distemper encephalitis (BEINEKE et al. 2009) and spinal cord injury (WELLS et al. 2003, SPITZBARTH et al. 2011, BOCK et al. 2013).

Cell transplantation is considered to be a promising treatment strategy for the therapy of CNS diseases (BLAKEMORE et al. 2000, KOCSIS et al. 2002, GRANGER et al.

2012). The CNS is mainly composed of glial cells which can be divided into macroglia and microglia. Transplantation of exogenous cells (e.g. mesenchymal stem cells (MSCs), neural stem cells (NSCs), olfactory ensheathing cells (OECs), oligodendrocyte precursor cells (OPCs) or Schwann cells (SCs) has been successfully applied in several demyelinating diseases (FRANKLIN et al. 1996a, SHIELDS et al. 2000, KOCSIS et al. 2004, ZAPPIA et al. 2005, GRANGER et al.

2012). The goal of transplantation should be the morphological and even more important the functional restoration of damaged CNS tissue. This can be achieved by transplantation of cells which should integrate into the recipient tissue and replace the destroyed tissue and/or form a microenvironment that supports endogenous regeneration. For transplantation of cells with the aim to replace damaged tissue, myelin producing cells like oligodendrocytes, SCs and OECs as well as their precursor cells have to be considered primarily (FRANKLIN et al. 1997, WOODHOO et al. 2007, MARTINO et al. 2010, RADTKE et al. 2010). Furthermore, MSCs as well as neural stem cells represent promising candidates for therapeutic approaches (JUNG et al. 2009, LEE et al. 2011, GUPTA et al. 2012). Application of the latter may support the establishment of a beneficial, anti-inflammatory microenvironment at the lesion site blocking further lesion progression and supporting endogenous repair mechanisms. However, the route of application, used cell type and applied cell amount has to be determined, depending on the type of disease to be treated.

General introduction Chapter 2

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2.1. Cellular composition of the central nervous system

The nervous system consists of a central and a peripheral part with complex interactions between both compartments. The CNS of vertebrates can be divided into the brain and spinal cord and is constituted mainly of three cell types:

i) neurons, ii) macroglia and iii) microglia (Figure 1). Historically the macroglia comprise two cell types: astrocytes and oligodendrocytes. Recently, an additional group of neuronal growth and regeneration promoting macroglial cells termed aldynoglia, consisting of central Schwann cells, radial glia, tanycytes, OECs, pituicytes, Bergmann glia and Müller glia of the retina, has been described and distinguished (Figure 1; GUDINO-CABRERA et al. 2000, NIETO-SAMPEDRO 2002, LIU et al. 2004, BLAKEMORE 2005, ZAWADZKA et al. 2010, IMBSCHWEILER et al.

2012, WOHLSEIN et al. 2013). A characteristic of this cell type is the expression of p75^NTR (low affinity neurotrophin receptor; GUDINO-CABRERA et al. 1999 and 2000, ROJAS-MAYORQUIN et al. 2010).

Figure 1: Summary of the most frequent cell types in the central nervous system.

The central nervous system consists of three main groups of cells: neurons, macroglia and microglia. The group of macroglia can be further divided into oligodendrocytes, astrocytes and aldynoglia. Aldynoglia consist of central Schwann cells, Bergmann glia, Müller Glia, olfactory ensheathing cells, pituicytes, radial glia and tanycytes.

The size of neurons ranges from large α-motor neurons to small interneurons (SUMMERS et al. 1995b). Four different morphologies of neurons exist: unipolar, bipolar, pseudo-unipolar and multipolar (SUMMERS et al. 1995b). Furthermore, neurons can be classified according to their localization within the CNS (upper/lower motor neuron, interneuron), produced neurotransmitters, and their function rather being excitatory or inhibitory (SUMMERS et al. 1995b).

Astrocytes, belonging to the macroglia cells, constitute 20 to 50 percent of the volume of most brain areas (LIU et al. 2004). According to morphologic criteria astrocytes can show a fibrous, protoplasmatic or gemistocytic phenotype (SUMMERS et al. 1995b, WOHLSEIN et al. 2013). Furthermore, astrocytes are called Alzheimer type II cells when they show a swollen cytoplasm, enlarged and swollen nuclei, a margination of the chromatin and prominent nucleoli (GRANT MAXIE et al. 2007). Astrocytes are necessary for the integrity of the blood brain and blood spinal cord barrier, nutrition of neurons, maintenance of the extracellular ion homeostasis and guidance of migrating cells during development (SUMMERS et al.

1995b, GOLDMAN 2004, BARTANUSZ et al. 2011). Astrocytes can be identified using immunophenotyping by antibodies targeting glial fibrillary acidic protein (GFAP;

BIGNAMI et al. 1972). In addition, astrocytes especially their progenitor cells, show an expression of vimentin and the calcium-binding protein S100 protein (BIGNAMI et al. 1972, SEEHUSEN et al. 2007).

Another important component of the CNS beside neurons and glial cells represents the meninges and the vasculature (WOHLSEIN et al. 2013). Brain and spinal cord are surrounded by meninges called dura mater, arachnoidea and pia mater (SUMMERS et al. 1995b). They take part in the protection of the CNS by forming a compartment for cerebrospinal fluid circulation, ensheathment of cranial and spinal nerves and by giving support to blood vessels (SUMMERS et al. 1995b). The CSF circulates within the ventricular system and the central canal, both lined by ependymal cells (SUMMERS et al. 1995b).

General introduction Chapter 2

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2.2. Oligodendrocytes

Oligodendrocytes are the resident, myelin-producing cells of the CNS (AGGARWAL et al. 2011). They form an insulating myelin layer around axons which is important for rapid propagation of action potentials within the vertebrate CNS (MIRON et al. 2011).

Oligodendrocytes are suspected to be lifelong regenerated by oligodendrocyte progenitor cells (OPCs; BAUMANN et al. 2001b, MENN et al. 2006, ZAWADZKA et al. 2010). In early embryonic life, OPCs originate from the neuroepithelium of the neural tube while during late embryonic and post-natal life OPC generation takes place in the hindbrain/telencephalon and dorsal spinal cord (NOLL et al. 1993, PRINGLE et al. 1998, FU et al. 2002, CAI et al. 2005, VALLSTEDT et al. 2005, MIRON et al. 2011). OPCs have the property to proliferate and to migrate. Once arrived at their destination, OPCs stop proliferation and start differentiation (NOBLE et al. 1990, FOK-SEANG et al. 1994). This process is accompanied by the expression of a subset of myelin-associated proteins like myelin-associated protein (MAG), myelin-oligodendrocyte protein (MOG), myelin basic protein (MBP) and CNPase (Figure 2; BAUMANN et al. 2001a, MILLER 2002, MATSUSHITA et al.

2005, ULRICH et al. 2008, MIRON et al. 2011). OPCs as well as mature oligodendrocytes are within the gray and white matter of the CNS (LEVINE et al.

2001, DAWSON et al. 2003). They can be isolated from mixed brain cultures of neonatal animals including mice (CHEN et al. 2007). Culturing of OPCs in serum-free media lead to their differentiation into oligodendrocytes while the addition of fetal calf serum induced a differentiation into GFAP-positive astrocytes (RAFF et al. 1983, 1984). However, OPCs show a wide variation with respect to antigen-expression and growth-factor responsiveness depending on the age of the donor (GENSERT et al.

2001, CRANG et al. 2004, MASON et al. 2004). OPCs in rats and mice are characterized by the expression of nerve/glial antigen 2 (NG-2) and platelet-derived growth factor receptor α (PDGFR-α; MIRON et al. 2011). During CNS injury or demyelination, OPCs are known to up-regulate NG-2 and to start proliferation (LEVINE 1994 and 1999, WATANABE et al. 2002) which could be the initial step in CNS regeneration.

Figure 2: Oligodendrocyte differentiation (modified according to ULRICH et al. 2008, MIRON et al. 2011).

Development of oligodendrocytes from multipotent neural stem cells to mature oligodendrocytes. The following differentiation stages between the multi-potent neuroglial stem cell and a mature oligodendrocyte can be distinguished:

oligodendrocyte precursor cells, pre-oligodendrocytes and immature oligodendrocytes. Typical markers for the respective developmental stages are listed in the bottom row. GalC=Galactosylceramidase, MAG=myelin associated glycoprotein, MBP=myelin basic protein, MOG=myelin oligodendrocyte glycoprotein, NG-2=nerve/glial antigen 2, PDGFR-α=platelet derived growth factor receptor alpha, PSA-NCAM=polysialylated-neural cell adhesion molecule.

However, in demyelinating CNS diseases like MS or TME a remyelination failure occurs (FRANKLIN 2002b, KUHLMANN et al. 2008, ULRICH et al. 2008). In particular OPC differentiation fails because of a dysregulation of a complex cell-cell and cell-growth factor interaction (FRANKLIN 2002b and 2008, ULRICH et al. 2008).

This may be related to: i) an inadequate number of OPCs at the lesion site or ii) a dysregulation/blockage of OPC differentiation into myelinating oligodendrocytes (FRANKLIN 2002b). An inadequate number of OPCs at the lesion site may be due to

General introduction Chapter 2

8

targeting of OPCs (ARCHELOS et al. 1998, NIEHAUS et al. 2000, FRANKLIN 2002b). In this process the local environment at the lesion site plays an important role, not only for the differentiation of oligodendrocytes and maintenance of axons but also for the determination of the phenotype of microglia/macrophages (DAVID et al.

2011).

2.3. Schwann cells and Aldynoglia

Schwann cells (SCs) originate from the neural crest (SASAKI et al. 2011), develop from Schwann cell precursor cells (WOODHOO et al. 2007) and play an important role in peripheral nerve regeneration (KING-ROBSON 2011). SCs can be divided into distinct groups: myelinating and non-myelinating SCs (JESSEN et al. 2005). For the sake of clarity these SCs will be called peripheral Schwann cells (pSCs) throughout this thesis. Recently, a new group of SCs called central Schwann cells (cSCs), belonging to the group of aldynoglia has been described (GUDINO-CABRERA et al.

1999 and 2000, IMBSCHWEILER et al. 2012). These cells are also called Schwann cell-like glia, aldynoglial Schwann cells or CNS Schwann cells (ORLANDO et al.

2008, ZAWADZKA et al. 2010, IMBSCHWEILER et al. 2012). cSCs share characteristics with oligodendrocytes and astrocytes and resemble pSCs (GUDINO- CABRERA et al. 2000, NIETO-SAMPEDRO 2002, IMBSCHWEILER et al. 2012).

Most of the remyelinating cSCs have been shown to originate from Olig2/PDGFR-α expressing glial cells, presumably OPCs (Figure 3; BLAKEMORE 2005, ZAWADZKA et al. 2010). cSC development from Olig2/PDGFR-α expressing oligodendrocyte progenitor cells is favored by bone morphogenic proteins and an astrocyte-poor environment while in an astrocyte-rich environment development of oligodendrocytes from the same progenitor cells is preferred (Figure 4; ZAWADZKA et al. 2010). One characteristic of aldynoglia is the expression of p75^NTR (ORLANDO et al. 2008, IMBSCHWEILER et al. 2012). Aldynoglia can be found in several areas of the adult brain (NIETO-SAMPEDRO 2003). Under pathological conditions like axonal damage or demyelination a differentiation of tissue-resident precursor cells into p75^NTR expressing cSCs is described (BLAKEMORE 2005, IMBSCHWEILER et al. 2012).

Figure 3: Development of central Schwann cells (according to ZAWADZKA et al. 2010).

Remyelination of the CNS is carried out mainly by oligodendrocytes and to a lesser extent by Schwann cells (SCs). Most of the central SCs (cSCs) develop from oligodendrocyte progenitor cells which also contribute to the development of oligodendrocytes. cSC differentiation is encouraged by bone morphogenic proteins (BMPs) and an astrocyte-poor environment. Peripheral SCs can migrate into the CNS along the spinal nerves/routes and participate in CNS remyelination but this population has shown to be not the source of the central SC. In contrast to oligodendrocytes and SCs, reactive astrocytes originate from different, fibroblast growth factor 3 positive, progenitor cells.

CC-1=clone detects Adenomatous Polyposis Coli antigen in astrocytes and oligodendrocytes, FGFR3=fibroblast growth factor 3, NG-2=nerve glial antigen 2, PDGFRα=platelet derived growth factor alpha, Olig2=oligodendrocyte transcription factor 2, P0=protein zero, OCT6=octamer-binding transcription factor 6, SCIP/OCT6=POU-domain transcription factors SCIP/OCT6.

Peripheral SCs represent the major myelin producing cell type of the peripheral nervous system. In case of CNS lesions, SCs are known to participate in the process of remyelination. One specific feature of all developmental stages of SCs is the

NTR

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et al. 2005, BOCK et al. 2007). In addition, all types of SCs including their progenitor cells showed expression of O4, representing a typical oligodendroglial surface marker (IMBSCHWEILER 2009). An overview about the antigen expression of myelinating and non-myelinating pSCs as well as cSCs is given in table 1.

Table 1: Marker expression of myelinating and non-myelinating Schwann cells as well as central Schwann cells.

* also termed aldynoglial Schwann cells, Schwann cell-like brain glia or CNS Schwann cells.

** lower numbers of central Schwann cells expressed GFAP compared to peripheral Schwann cells (Imbschweiler et al. 2009).

GFAP=glial fibrillary acidic protein; HNK-1=human natural killer-1; MBP=myelin basic protein;

n.d.=not determined; O4=seminolipid sulfatide antigen; P0=protein zero; p75^NTR=neurotrophin receptor p75.

The process of myelination depends on various interactions between axons and the respective myelinating glial cells (JESSEN et al. 2005). pSCs myelinate axons with a diameter larger than 1µm while pSCs in contact with smaller axons form non- myelinating associations (JESSEN et al. 2005). SCs ensheathing non-myelinated axons are also called Remak cells by some authors (SUMMERS et al. 1995a, THOMAS et al. 1997). pSCs show a pronounced plasticity with most of the developmental steps being reversible, e.g. axonal alterations induce a phenotype switch of mature myelinating or non-myelinating SCs to a phenotype similar to Marker

Peripheral non-myelinating

Schwann cells

Peripheral myelinating Schwann cells

Central

Schwann cells* References In vitro In vivo In vitro In vivo In vitro In vivo

O4 + + + + + n.d. BOCK et al. (2007)

IMBSCHWEILER (2009) IMBSCHWEILER et al. (2012) JESSEN et al. (2005) NIETO-SAMPEDRO (2002) ORLANDO et al. (2008) SASAKI et al. (2011) TECHANGAMSUWAN (2009) WEWETZER et al. (2006)

p75^NTR + + + - + +

S100 + + + + + -

GFAP + + + - (+)** -

P0 - - - + n.d. n.d.

MBP - - - + n.d. -

HNK-1 - - - + n.d. n.d.

immature SCs (JESSEN et al. 2005). Therefore, an increased expression of myelin- genes like P0 or MBP is correlated with a down-regulation of GFAP, NCAM and p75^NTR expression (MORGAN et al. 1991). pSC development and differentiation is a complex process which is mediated by numerous transcription factors, growth hormones and cytokines (Figure 4). In general, the sex determining region Y-box 10 (SOX10) is essential for the development and differentiation of all glial cells from the neural crest while bone morphogenic proteins (BMPs) block their development/differentiation (SHAH et al. 1994, BRITSCH et al. 2001, JESSEN et al.

2005). Neuregulin 1 (NRG1) has been shown to support Schwann cell precursor (SCP) survival and NRG1 as well as FGF-2 and Notch induce the differentiation of SCP to immature SCs (DONG et al. 1999, BRENNAN et al. 2000, GARRATT et al.

2000, MORRISON et al. 2000, WOLPOWITZ et al. 2000, LEIMEROTH et al. 2002, JESSEN et al. 2005). In contrast, transcription factor activator protein 2 (AP2) and endothelins delay SCP differentiation (BRENNAN et al. 2000, JESSEN et al. 2005).

The survival of immature SCs is supported by autocrine factors like NRG1, Ets and laminin, whereas TGFβ and p75^NTR induce SC death (GRINSPAN et al. 1996, TRACHTENBERG et al. 1996, MEIER et al. 1999, SYROID et al. 2000, PARKINSON et al. 2001, JESSEN et al. 2005, YU et al. 2005). Furthermore, the induction of myelination is promoted by NRG1, insulin-like growth factors (IGFs), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), laminin, octamerbinding transcription factor 6 (OCT6) and progesterone (BUNGE 1993, KOENIG et al. 1995, STEWART et al. 1995, CHAN et al. 2001, HÖKE et al.

2003, JAEGLE et al. 2003, MICHAILOV et al. 2004, JESSEN et al. 2005). On the other hand, myelination is inhibited by c-Jun, paired box gene 3 (PAX3), SOX2, Notch, Neurotrophin 3 (NT3) and ATP (KIOUSSI et al. 1995, FIELDS et al. 2000, CHAN et al. 2001, PARKINSON et al. 2004, JESSEN et al. 2005, LE et al. 2005).

Selecting SCPs for transplantation bears important advantages: Firstly, SCPs migrate through the normal CNS although they were not directly injected into the lesion and secondly, SCPs form an extensive amount of myelin (BACHELIN et al.

2005, WOODHOO et al. 2007). Disadvantages for selecting mature SCs for

General introduction Chapter 2

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SCs do not significantly migrate from the implantation site, iii) SCs fail to integrate with recipient oligodendrocytes and astrocytes and iv) the presence of an astrocyte- rich environment significantly reduces myelin-formation of SCs (WOODHOO et al.

2007).

Figure 4: Development and differentiation of peripheral Schwann cells (modified according to JESSEN et al. 2005).

The development and differentiation of peripheral Schwann cells (pSCs) from neural crest cells to myelinating and non-myelinating pSCs is regulated at several steps by numerous growth- and transcription factors. Green color indicates an induction of development/differentiation while red color stands for an inhibition/blockage. AP2α=transcription factor AP-2-alpha; ATP=adenosine triphosphate; BDNF=brain derived neurotrophic factor; BMP=bone morphogenic protein; Ets=transcription factor E-twenty six; FGF-2=fibroblast growth factor 2;

GDNF=glial cell line-derived neurotrophic factor; IGF=insulin-like growth factor;

NRG1=neuregulin 1; NT3=Neurotrophin 3; SOX-2=sex determining region Y-box 1; SOX-10=sex determining region Y-box 10; p75^NTR=p75 neurotrophin receptor; TGFβ=tumor growth factor β.

2.4. Olfactory ensheathing cells

Olfactory ensheathing cells (OECs) have been firstly described more than 100 years ago by Blanes and Golgi (GOLGI 1875, BLANES 1898, HIGGINSON et al. 2011).

They occur in the CNS (nervus and bulbus olfactorius) as well as in the peripheral nervous system (olfactory mucosa; RAISMAN 1985, DOMBROWSKI et al. 2006, SU et al. 2010). The high similarity between OECs and non-myelinating SCs suggests, that OECs may be a myelinating phenotype of SCs (FRANKLIN 2003). OECs are also named intermediate glial cells because they share many similarities with astrocytes and SCs (Table 2; BARBER et al. 1982, DOUCETTE 1984, WEWETZER et al. 1997 and 2005).

Table 2: Marker expression of olfactory ensheathing cells in vitro and in vivo.

Marker Rodent OECs Canine OECs References In vitro In vivo In vitro In vivo

p75^NTR + +¹ + -¹ BARTSCH (2003)

BOCK et al. (2007) HIGGINSON et al. (2011) IMBSCHWEILER (2009) IMBSCHWEILER et al. (2012) PELLITTERI et al. (2010) SASAKI et al. (2011) TECHANGAMSUWAN (2009) WEWETZER et al. (2002) WEWETZER et al. (2005)

HNK-1 n.d. n.d. - -

S100 + (+) + +

GFAP + + + -

L1 + n.d. + n.d.

PSA-NCAM + (-) n.d. n.d.

P0 - - n.d. n.d.

1p75 is expressed by a subpopulation of neonatal OECs, not by adult OECs (TECHANGAMSUWAN 2009, OMAR et al. 2011). GFAP=glial fibrillary acidic protein; HNK- 1=human natural killer 1; MBP=myelin basic protein; n.d.=not determined; P0=protein zero;

PSA-NCAM=polysialylated-neural cell adhesion molecule; p75^NTR=p75 neurotrophin receptor.

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In contrast to the in vitro situation, adult OECs do not express p75^NTR under physiological conditions in vivo (VICKLAND et al. 1991, RAMON-CUETO et al. 1998, BOCK et al. 2007). In addition, it is important that some of the aforementioned markers are only expressed transiently or by OEC subpopulations (OMAR et al.

2011). Proliferation of OECs and SCs can be effectively induced by addition of neuregulin, forskolin and fibroblast growth factor-2 in vitro (FGF-2; RAMON-CUETO et al. 1998, WEWETZER et al. 2001 and 2002, YAN et al. 2001, OMAR et al. 2013).

Cultured primary rodent OECs in absence of mitogens reach their senescence after approximately 5 weeks in culture, however a spontaneous immortalization was not observed in canine OECs compared to rodent OECs (RUBIO et al. 2008, TECHANGAMSUWAN et al. 2008). The decision whether an in general myelinating cell starts myelin-production depends primarily on the thickness of the respective axon (FRANKLIN 2003). SCs are known to myelinate individual axons with a diameter larger than 1µm while in the presence of smaller axons SCs form a non- myelinating association with multiple axons (FRANKLIN 2003, JESSEN et al. 2005).

OECs in their natural environment (olfactory mucosa and olfactory bulb) are associated with axons displaying a diameter of less than 0.5µm which is below the known threshold for myelination found in the CNS and PNS (DOUCETTE 1990, FRANKLIN 2003, JESSEN et al. 2005). In their natural in vivo environment OECs in contrast to SCs do not form myelin (FRANKLIN 2003). However, myelin-formation of OECs has been shown in vitro and in vivo under experimental conditions (FRANKLIN 2003, RADTKE et al. 2010, SASAKI et al. 2011). Several reports documented beneficial effects of OECs on axonal growth in vitro and in vivo (LEAVER et al. 2006, RUNYAN et al. 2009, JIAO et al. 2011, WINDUS et al. 2011, GARCIA-ESCUDERO et al. 2012, GRANGER et al. 2012, ZIEGE et al. 2013). Transplantation of OECs in the CNS caused a reduced degree of astrogliosis compared to SCs and a supportive effect on axonal regeneration is described (RADTKE et al. 2009). Therefore OECs have to be considered as promising candidates for therapeutic interventions in demyelinating/degenerative diseases of the CNS (GRANGER et al. 2012).

2.5. Microglia and macrophages

Microglia and macrophages are an essential part of the innate immune system, which also influence the adaptive immune system by e.g. lymphocyte activation or recruitment (SICA et al. 2008). Under physiological conditions microglia and macrophages are responsible for the detection of pathogens, phagocytosis of dying or dead cells as well as for the maintenance of tissue homeostasis (DAVID et al.

2011). Microglia represent the resident phagocytic cells of the CNS. They can be distinguished from activated microglia and monocytes by flow cytometric analysis showing low CD45 expression (DAVID et al. 2011). During inflammation, blood-born macrophages (monocytes) are able to leave the circulation and migrate into the CNS.

In mice inflammatory monocytes are characterized by high expression of chemokine receptor 2 (CCR2), Ly6 (GR1) and a low expression of chemokine receptor 1 (CX3CR1) while resting monocytes showed a contrary expression of these markers (GEISSMANN et al. 2003, MOSSER et al. 2008). Under inflammatory conditions blood-born macrophages and microglia showed a high CD45 expression and cannot be distinguished, neither by morphology nor by antigenic markers (DAVID et al.

2011). Therefore, under inflammatory conditions microglia and blood-born macrophages should be called microglia/macrophages or phagocytes (SHECHTER et al. 2013). Microglia/macrophages have been related to secondary tissue damage in CNS diseases, but they are also necessary for removing cellular debris and therefore exhibit protective/regenerative effects (GIULIAN et al. 1990, POPOVICH et al. 1999, YONG et al. 2009). The beneficial or detrimental behavior of these cells may be caused by different subsets of microglia/macrophages. Recently macrophages were classified into proinflammatory, classically activated cells called M1 and anti-inflammatory, alternatively activated cells called M2 (Table 3; GORDON 2003, KIGERL et al. 2009). M2 macrophages can further be subdivided into M2a, M2b and M2c. Similarly, a polarization of macrophages has been shown in non-CNS diseases like atherosclerosis or bacterial and parasitic infections (DAVID et al. 2011).

General introduction Chapter 2

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Table 3: Classification, marker expression and function of M1/M2 cells (modified according to KIGERL et al. 2009, DAVID et al. 2011).

Phenotype Marker Function

CD16 CD32 CD86 iNOS MHCII

- Phagocytosis

- Killing of intracellular pathogens

- Acidification of phagosome and release of reactive oxygen intermediates and NO

M2a

Arginase I CD163 CD204 CD206 Fizz 1

YM1

- Immunity against parasites - Growth stimulation - Tissue repair - Collagen formation

- Recruitment of Th2 cells, basophils and eosinophils

M2b

CD86 CD163 IL10^high

IL12^low MHCII SPHK1

- Pro- and anti-inflammatory function - B cell class switch and antibody production - Recruitment of regulatory T cell

M2c Arginase I CD163 CD204 CD206

- Debris scavaging - Pro-healing function - Iron sequestration

Fizz 1=resistin-like alpha; IL10^high=high expression of interleukin 10; IL12^low=low expression of interleukin 12; iNOS=inducible nitric oxide synthase; MHCII=major histocompatibility complex class II; NO=nitric oxide; SPHK1=sphingosine kinase 1; YM1=chitinase 3-like protein 3.

M2 microglia/macrophagesM1 microglia/macrophages

Differentiation of M1 macrophages is promoted by the influence of lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ; KIGERL et al. 2009). M1 cells produce proinflammatory cytokines such as tumor necrosis factor (TNF), IL-1, IL-6, IL-12 (DAVID et al. 2011) which are necessary for host defense and tumor cell destruction.

However, an over-expression of these cytokines may also induce a severe collateral damage to unaffected, normal tissue (DING et al. 1988). On the other hand, an activation of macrophages under the influence of cytokines such as IL-4 or IL-13 induces the M2 phenotype (DAVID et al. 2011). This group is composed of a heterogeneous subset of macrophages which are described as anti-inflammatory cells expressing IL-10, TGF-β, CD206, arginase I and they down-regulate proinflammatory cytokines (SICA et al. 2006 and 2008, DAVID et al. 2011). It was shown, that M2 macrophages isolated from uninjured spinal cords maintain their phenotype when transplanted into intact spinal cords while transplantation into lacerated spinal cords induced a 20-40% reduction of the M2 phenotype (KIGERL et al. 2009). This observation strongly indicates, that the local environment (transplantation site) determines the phenotype/differentiation of (transplanted) microglia/macrophages. Therefore, a predominance of M1 cells at sites of CNS injury may be expected (KIGERL et al. 2009), whereas the main phenotype of microglia/macrophages in the normal CNS displays M2 polarity (PONOMAREV et al.

2007). Interestingly, a fully differentiated microglia/macrophage population may reversibly change its phenotype and function in response to the local microenvironment (STOUT et al. 2004, MOSSER et al. 2008) or may be influenced by the exogenous application of microglia/macrophage differentiation-inducing molecules like LPS, IFN-γ, IL-4, IL-13 (KIGERL et al. 2009, DAVID et al. 2011).

Chapter 3 Defining the morphological phenotype: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a novel marker for in situ detection of canine but not rat olfactory ensheathing cells

M. Omar, P. Bock, R. Kreutzer, S. Ziege, I. Imbschweiler, F. Hansmann, C.T. Peck, W. Baumgärtner, K. Wewetzer

Abstract

Olfactory ensheathing cells (OECs) are the non-myelinating glial cells of the olfactory nerves and bulb. The fragmentary characterization of OECs in situ during normal development may be due to their small size requiring intricate ultrastructural analysis and to the fact that available markers for in situ detection are either expressed only by OEC subpopulations or lost during development. In the present study, we searched for markers with stable expression in OECs and investigated the spatiotemporal distribution of CNPase, an early oligodendrocyte/Schwann cell marker, in comparison with the prototype marker p75(NTR). Anti-CNPase antibodies labeled canine but not rat OECs in situ, while SCs and oligodendrocytes were positive in both species. CNPase immunoreactivity in the dog was confined to all OECs throughout the postnatal development and associated with the entire cell body, including its finest processes, while p75(NTR) was mainly detected in perineural cells and only in some neonatal OECs. Adult olfactory bulb slices displayed CNPase expression after 4 and 10 days, while p75(NTR) was detectable only after 10 days in vitro. Finally, treatment of purified adult canine OECs with fibroblast growth factor-2 significantly reduced CNPase expression at the protein and mRNA level. Taken together, we conclude that CNPase but not p75(NTR) is a stable marker suitable for in situ visualization of OECs that will facilitate their light-microscopic characterization and challenge our general view of OEC marker expression in situ. The fact that canine but not rat OECs expressed CNPase supports the idea that glia from large animals differs substantially from rodents.

Cell Tissue Res. 2011: 344(3):391-495

Chapter 4 Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and non-demyelinated central nervous system

F. Hansmann, K. Pringproa, R. Ulrich, Y. Sun, V. Herder, M. Kreutzer, W. Baumgärtner, K. Wewetzer Abstract

Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically-generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFRα, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.

Cell Transplant. 2012: 21(6):1161-1175

Chapter 5 Theiler’s murine encephalomyelitis virus induced phenotype switch of microglia in vitro

I. Gerhauser, F. Hansmann, C. Puff, J. Kumnok, D. Schaudien, K. Wewetzer, W. Baumgärtner Abstract

The present in vitro study aimed to define the involvement of astrocytes and microglia in the initial inflammatory response of Theiler's murine encephalomyelitis (TME), a virus-induced mouse model of multiple sclerosis, and whether intralesional microglia exert pro- (M1) or anti-inflammatory (M2) effects following TME virus (TMEV) infection. Therefore astrocytes and microglia were purified from neonatal murine brains and inoculated either with TMEV or mock-solution. Gene expression of IL-1, IL-2, IL-10, IL-12, TNF, TNF receptors (TNFR1, TNFR2), TGFβ1, IFNγ and transcription factors NF-κB (p50, p65) and AP-1 (c-jun, c-fos) were quantified using RT-qPCR at 6, 48, and 240 h post infection (hpi). In addition, IL-1, IL-10, IL-12, TNF and TGFβ1 mRNA transcripts were investigated at 168 hpi in TMEV- and mock- infected SJL/J mice. Overall in vitro astrocytes showed a significant higher amount of viral RNA compared to microglia. In addition, TMEV-infected astrocytes showed higher numbers of IL-1, IL-12 and TNF transcripts at 48 hpi. In microglia high IL-10 and low IL-12 mRNA levels were detected at 48 hpi, while the opposite was the case at 240 hpi. In addition, TNF mRNA was increased in microglia at 240 hpi. In addition, the observed up-regulation of IL-1, IL-12 and IL-10 in the early phase of TME in vivo substantiates the relevance of these cytokines during the disease induction.

Summarized data indicate that TMEV infection of microglia induces a switch from the anti-inflammatory (M2) during the early phase to the pro-inflammatory (M1) phenotype in the later phase of the infection. The simultaneous expression of TNF and its receptors by both cell types might generate autocrine feedback loops possibly associated with pro-inflammatory actions of astrocytes via TNFR1.

J. Neuroimmunol. 2012: 252(1-2):49-55 www.elsevier.com/locate/jneuroim DOI: 10.1016/j.jneuroim.2012.07.018

Chapter 6 Discussion and Conclusions

Inflammation, demyelination, neuronal degeneration, axonal loss and an insufficient or exhausted regenerative capacity represent the hallmarks of demyelinating diseases like MS, canine distemper encephalitis, Theiler’s murine encephalomyelitis or spinal cord injury (COMPSTON et al. 2008, BEINEKE et al. 2009, HANSMANN et al. 2012a, BOCK et al. 2013, ZHANG et al. 2013). For none of these diseases a sufficient therapy leading to a “restitutio ad integrum” is available. Possible therapeutic options are exogenous replacement of damaged cells/tissue or the creation of a beneficial environment at the lesion site favoring endogenous regeneration (KEIRSTEAD et al. 1999, JUNG et al. 2009, LU et al. 2010, DARLINGTON et al. 2011, GRANGER et al. 2012). In this context, the transplantation of non-myelinating cells in order to stimulate remyelination by either modulation of the immune system and/or enhancement of endogenous repair processes represents a new, very promising therapeutic approach (KANG et al.

2012b, WANG et al. 2012). Recently, several studies investigated the therapeutic efficiency of mesenchymal and neural stem cells (UCCELLI et al. 2011, AL JUMAH et al. 2012, KANG et al. 2012a, RYU et al. 2012, SHER et al. 2012, UCHIDA et al.

2012). MSCs have been shown to exert neuroprotective effects by induction of neurotrophic factors and modulation of the immune responses (KANG et al. 2012b, WANG et al. 2012). In addition, MSCs are suggested to be able to replace damaged cells by a process called trans-differentiation (EDAMURA et al. 2012).

If the goal is to repair myelin by transplantation of exogenous cells the following issues should be addressed: Firstly, used cell type and their number and secondly route and time point of application. The selection of appropriate criteria largely depends on the pathogenesis of the respective disease and on the planned time point for the intervention. Therefore, a promising opportunity is the selection of cells that have been shown to produce myelin in vitro and/or in vivo. Most attention has been given to OPCs, oligodendrocytes, olfactory ensheathing cells as well as SCPs and SCs (FRANKLIN et al. 1996b, FRANKLIN 2002a, HALFPENNY et al. 2002,

Discussion and Conclusions Chapter 6

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2005, RAISMAN et al. 2007, GRANGER et al. 2012). Each cell type has specific advantages and disadvantages. However, currently no cell type has evolved as universal method of choice and further studies are needed.

Characterization of canine olfactory ensheathing cells as possible candidates for transplantation

Olfactory ensheathing cells, which are non-myelinating glial cells of the olfactory nerve and bulb, represent a suitable cell type for a regenerative approach (FRANKLIN et al. 1996b, SU et al. 2010). A major obstacle for the identification and purification of OECs is their different antigen expression depending on the developmental stage, localization (in vivo, in culture) and culture conditions (WEWETZER et al. 2011). In the present study canine glial cells have been investigated, because the dog represents a suitable translational animal model and canine OECs share many similarities with human OECs (JEFFERY et al. 2005, JEFFERY et al. 2006, WEWETZER et al. 2011, GRANGER et al. 2012). At present, no stable marker for the in vivo identification of OECs is known. Rat OECs were detected using anti-p75^NTR antibodies, however a major disadvantage of this marker is its down-regulation during post-natal development (GONG et al. 1994, FRANCESCHINI et al. 1996). The present study showed that p75^NTR is mainly expressed by perineural cells and only few neonatal OECs in the dog. This leads to the conclusion that p75^NTR is not suitable for the in vivo identification of canine OECs.

CNPase represents a well-established marker for the detection of oligodendrocytes and SCs of many species including rats and dogs (MIRON et al. 2011). In the present study, anti-CNPase antibodies labeled the entire cell body of all canine OECs including their processes during the postnatal development. It seems unexpectedly, that non-myelinating glial cells such as OECs or non-myelinating SCs should express CNPase. However, this enzyme has been shown to exhibit a variety of functions in oligodendrocytes and SCs. An explanation for this observation could be that these cells are differentiating and currently show a premyelinating status. In this context it can be speculated that OECs are closely related to SCs and that both cell types may participate in myelin production in demyelinating diseases.

Furthermore, in this study purified adult canine OECs were treated with FGF-2, which has been previously shown to act as potent OEC and SC mitogen in vitro (TECHANGAMSUWAN et al. 2008, 2009). Under this condition CNPase expression was significantly reduced at the protein and mRNA level. Since CNPase expression is known to be upregulated by OPCs during differentiation and not during proliferation it is presumed that CNPase expression in OECs and SCs is related to cellular differentiation. In addition, dissociation and organotypic slice cultures of canine olfactory bulb were investigated for CNPase and p75^NTR expression. CNPase antigen was detected at 4 and 10 days while p75^NTR was only detectable after 10 days in vitro cultivation. All canine OEC populations lacked or lost their p75^NTR expression during development in vivo. In contrast, long term cultivation of canine OECs leads to an upregulation of this marker in vitro. Therefore, p75^NTR may serve as a useful in vitro marker for the detection of OECs.

Conclusively, CNPase but not p75^NTR can be used as suitable marker for in vivo visualization of canine OECs. It is important to point out that species specific differences regarding the marker expression of cells exist and have to be taken into consideration. The present study demonstrated that canine but not rat OECs express CNPase in vivo. The differences between canine and rodent glia further favors the use of dogs as translational large animal model. Beside this, further studies have to determine whether CNPase can be used as marker for the discrimination between rat OECs and SCs in vivo.

Establishment of an ethidium bromide-induced demyelination model in the murine caudal cerebellar peduncle and in vivo characterization of BO-1 cells The analysis of mechanisms involved in CNS demyelination and remyelination are essential for the establishment of regenerative therapeutic approaches. In this study the species mouse was selected for the following reasons: several natural and genetically induced mouse mutants exist (GRIFFITHS et al. 1990, GRIFFITHS 1996, KLUGMANN et al. 1997, DIMOU et al. 1999, EDGAR et al. 2010) which can be evaluated with this model and secondly the previously established murine

Discussion and Conclusions Chapter 6

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investigated in vivo. The caudal cerebellar peduncle (CCP) was chosen as transplantation site because this localization has several advantages: i) it displays a well circumscribed area, ii) most of the axons show a similar diameter and orientation and iii) myelin sheaths have a similar thickness (WOODRUFF et al. 1999). In the present study ethidium bromide concentrations between 0.1% and 0.01% have been applied. It is important to select a toxicant concentration which causes a marked demyelination with limited axonal damage. Ethidium bromide-induced lesions showed a concentration-dependent increase in size. Finally, a concentration of 0.025% ethidium bromide in a volume of 2µl was selected because at this concentration a marked and significant demyelination was shown by myelin basic protein immunohistochemistry and a mild to moderate axonal damage as detected by phosphorylated- and non-phosphorylated neurofilament immunohistochemistry was observed (HANSMANN et al. 2012b). The occurrence of axonal alterations following ethidium bromide application was interpreted as a mouse specific feature because similar studies investigating the caudal cerebellar peduncle of rats lacked significant axonal alterations (WOODRUFF et al. 1999, HANSMANN et al. 2012b).

The next step was the in vivo investigation of BO-1 cells. To ensure the detection of the transplanted BO-1 cells, they were transfected with enhanced green fluorescent protein (eGFP). It was hypothesized that the local environment of the demyelinated CCP would stimulate differentiation of BO-1 cells to remyelinating oligodendrocytes.

Therefore BO-1 cells were stereotactically transplanted into the demyelinated CCP 4 days following ethidium bromide-induced demyelination. Transplanted animals showed a reduced general behavior, weight loss and were euthanized for ethical reasons 17 days post stereotaxic injection of BO-1 cells. Macroscopically and histologically an invasive and liquorogenic metastasizing, murine giant cell glioblastoma was detected in all cell transplanted animals. Furthermore, BO-1 cells were investigated for their marker expression in vivo. BO-1 cells showed a strong expression of eGFP, NG-2, PDFGRα and p53 while only few cells expressed GFAP and MBP. Moreover, the expression of CNPase was markedly reduced compared to the in vitro situation. The reduced expression of CNPase can be interpreted as a reduced degree of differentiation of BO-1 cells in vivo. It can be speculated that the

reason for this may be an insufficient environmental signaling and/or a BO-1 cell intrinsic factor. To confirm that the tumor formation was not the consequence of the ethidium bromide application, BO-1 cells were also transplanted into the normal, non- demyelinated CCP. This revealed that all BO-1 transplanted animals showed tumor formation at the transplantation site.

Conclusively, instead of an expected remyelination by BO-1 cells a murine giant cell glioblastoma occurred in the transplanted animals. Thus BO-1 cells are not suitable for a regenerative therapy. However, they may serve as a versatile experimental model to study tumorigenesis of glial tumors.

Determination/modulation of the phenotype of microglia in TME

The local environment plays an essential role for the blockage of remyelination in demyelinating CNS diseases. In this context the immune system and maybe other glial cells like astrocytes have a major impact on the behavior of microglia and macrophages. Therefore, the present in vitro study aimed to identify the role of astrocytes and microglia in the early inflammatory response to TME. TME represents a well-established, virus-induced animal model of MS. The major goal was to illuminate whether microglia exert pro- (M1) or anti-inflammatory (M2) properties following TMEV infection. In addition, the cytokine profile of astrocytes was measured for the detection of a possible determining role of astrocytes upon the phenotype of microglia. Therefore, astrocytes and microglia were isolated from neonatal SJL/J mice, inoculated with TMEV or mock-solution and gene expression including pro- and anti-inflammatory cytokines, TNF receptors and transcription factors NF-κB (p50, p65) and AP-1 (c-jun, c-fos) was quantified at 6, 48 and 240 hours post inoculation.

Glia cells were isolated from SJL/J mice because this inbred mouse strain is highly susceptible to TMEV infection (DAL CANTO et al. 1996, MECHA et al. 2013). Firstly, microglia and astrocytes were both infected by TMEV with the latter showing a general higher amount of viral RNA. In the early phase post infection (48hpi) astrocytes showed an upregulation of IL-1, IL-12 and TNF transcripts resembling a Th-1 dominated immune response. At the same time high IL-10 and low IL-12 mRNA

Discussion and Conclusions Chapter 6

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M2-phenotype of microglia at this time point. However, at 240 hours post infection (hpi) microglia showed high IL-12 and TNF mRNA levels but low IL-10 levels indicating a proinflammatory M1 phenotype of microglia. Taken together, astrocytes showed an early, Th-1-like dominated cytokine expression profile following TMEV infection while microglia at the same time maintained their normal, anti-inflammatory M2 phenotype. It can be speculated, that astrocytes being highly susceptible to TMEV-infection, favor a very early phenotype switch of microglia in vivo which may contribute to the development of the chronic demyelinating myelitis in vivo. However, monocultures of microglia showed, that the phenotype switch also occurs without the presence of other cells (glial or immune cells). This finding indicated that the phenotype switch may also be interpreted as a direct, virus-induced phenomenon.

Conclusively, TMEV infection of microglia induced a switch from the anti- inflammatory (M2) during the early phase to the pro-inflammatory (M1) phenotype in the later phase of the infection. The simultaneous expression of TNF and its receptors by both cell types might generate autocrine feedback loops possibly associated with pro-inflammatory actions of astrocytes via TNFR1.

Chapter 7 Summary

Investigation of murine and canine glia cell differentiation in vitro and in vivo

Florian Heinrich Hansmann

Cell transplantation represents a promising treatment strategy for demyelinating and degenerative diseases of the central nervous system (CNS) like multiple sclerosis and spinal cord injury. A comprehensive in vitro characterization of cells represents the first and very important step before investigating the behavior and therapeutic potential of transplanted cells in vivo. Therefore, the aims of this thesis were threefold:

1.) Characterization of canine olfactory ensheathing cells as promising candidates for transplantation in vitro and in vivo.

2.) Establishment of an ethidium bromide (toxic) induced mouse model to study de- and remyelination and to test the behavior of the oligodendrocyte precursor cell line BO-1 in vivo.

3.) Studying astrocytes and microglia following infection with Theiler’s murine encephalomyelitis virus (TMEV) in vitro for the characterization of the phenotype of microglia and their possible impact upon the inhibition or progression of inflammatory/demyelinating diseases of the CNS.

Firstly, canine olfactory ensheathing cells (OECs) were characterized in vitro and in vivo employing the cell markers p75^NTR and CNPase using quantitative real-time PCR, western blot and immunohistochemistry. OECs were selected because these cells represent suitable candidates for a regenerative therapeutic approach. At present no stable marker for the in vivo identification of OECs exists. The present study revealed that CNPase represents a suitable marker for the in vivo detection of canine OECs. In contrast to CNPase, p75^NTR was mainly expressed by perineural cells and only in some neonatal OECs in dogs in vivo. However, canine OECs showed an upregulation of this marker during long term culturing and therefore this

Summary Chapter 7

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Furthermore, an important species specific difference was detected. Rat OECs in contrast to canine OECs lacked CNPase expression. Thus this marker may be used to discriminate between SCs and OECs in rats.

Secondly, an ethidium bromide-induced (toxic) demyelination model in the murine caudal cerebellar peduncle was established. Thismodel represents an important tool for the investigation of de- and remyelination and can be further used to evaluate the impact of transplanted cells and/or substances upon the progression of demyelinating diseases. The caudal cerebellar peduncle was chosen because of the following reasons: i) it is a well circumscribed anatomical structure, ii) axons in this localization have a similar diameter and orientation and iii) myelin sheaths show a similar size. Ethidium bromide concentrations between 0.1 and 0.01% in a volume of 2µl were applied stereotactically. A concentration dependent lesion size following ethidium bromide application was observed. For the establishment of a suitable demyelination model it is important to select a concentration which is high enough to induce significant demyelination but the toxicant should spare most of the axons.

Therefore, an ethidium bromide concentration of 0.025% was selected at which a marked demyelination and mild to moderate axonal damage was observed.

Interestingly, the occurrence of axonal alterations was in contrast to previous published studies in rats and indicates a species specific difference with respect to the mode of action. Next, enhanced green fluorescence protein (EGFP) labeled BO-1 cells were stereotactically transplanted into the CCP 4 days post ethidium bromide application. 17 days post transplantation all BO-1 cell transplanted animals showed reduced general behavior and weight loss and were euthanized due to ethical reasons. In all BO-1 cell transplanted animals an infiltrative murine giant cell glioblastoma was detected at the transplantation site. Immunohistochemistry revealed a strong EGFP expression in the neoplastic cells confirming their BO-1 identity. Furthermore, the majority of BO-1 cells expressed PDGFR-α, NG-2 and p53 while only few cells showed glial fibrillary acidic protein or myelin basic protein expression. In addition CNPase expression was reduced compared to in vitro findings. To confirm, that the observed tumor formation was not the consequence of the ethidium bromide application, BO-1 cells were transplanted into the normal, non-

demyelinated CCP. This approach again resulted in the development of a murine giant cell glioblastoma. The observed marker expression of BO-1 cells following transplantation revealed, that the demyelinated environment was insufficient to support the differentiation of BO-1 cells into myelinating oligodendrocytes.

Nevertheless BO-1 cells are unsuitable for a regenerative therapy; however they may be used as a versatile model for the investigation of malignant gliomas.

Thirdly, murine microglia and astrocytes were investigated regarding their cytokine expression profile following TMEV infection in vitro. This experiment aimed to determine the phenotype of microglia (M1=pro-inflammatory, M2=anti-inflammatory) following TMEV infection. Microglia are supposed to have a major impact upon the prevailing milieu of the CNS and are therefore important for the progression or inhibition of inflammatory/demyelinating diseases. In the present study astrocytes showed an early upregulation of IL-1, IL-12 and tumor necrosis factor (TNF) mRNA transcripts following TMEV infection indicating a Th-1-like immune response. In contrast, microglia at the same time maintained high IL-10 and low IL-12 mRNA levels suggesting a normal, anti-inflammatory M2 phenotype. At 240 hours post infection microglia showed high IL-12 and low IL-10 mRNA levels indicating a phenotype switch to M1. It remains speculative, whether astrocytes being highly susceptible to TMEV infection favor a phenotype switch of microglia which may contribute to the development of a chronic progressive, demyelinating myelitis. In addition, the simultaneous expression of TNF and its receptors by astrocytes and microglia might generate autocrine feedback loops possibly associated with pro- inflammatory actions of astrocytes via TNF receptor 1.

Chapter 8 Zusammenfassung

Untersuchungen zur in vitro und in vivo Differenzierung muriner und kaniner Gliazellen

Florian Heinrich Hansmann

Zelltransplantation stellt eine erfolgversprechende Behandlungsstrategie für demyelinisierende und degenerative Erkrankungen des zentralen Nervensystems (ZNS) wie Multiple Sklerose oder Rückenmarksverletzungen dar. Eine umfassende in vitro Charakterisierung der zu transplantierenden Zellen stellt einen ersten und wichtigen Schritt vor der Untersuchung des Verhaltens und des therapeutischen Potentials der Zellen in vivo dar. Daher verfolgte diese Arbeit im Wesentlichen drei Ziele:

1.) In vitro und in vivo Charakterisierung von kaninen, olfaktorischen Hüllzellen, die einen vielversprechenden Kandidaten für die Zelltransplantation darstellen.

2.) Die Etablierung eines Ethidiumbromid (toxischen) induzierten Mausmodells, um die Vorgänge während der De- und Remyelinisierung zu untersuchen und um das Verhalten der oligodendroglialen Vorläuferzelllinie BO-1 in vivo zu testen.

3.) “Theiler’s murine encephalomyelitis virus“ (TMEV) infizierte Astrozyten und Mikroglia der Maus im Verlauf der Infektion in vitro zu untersuchen, um den Phänotyp der Mikroglia und dessen möglichen Einfluss auf die Hemmung oder Progression von entzündlichen/demyelinisierenden Erkankungen des ZNS zu charakterisieren.

In der ersten Studie wurde eine Charakterisierung von kaninen, olfaktorischen Hüllzellen (OECs) in vitro und in vivo unter Verwendung der Zellmarker p75^NTR und CNPase mittels quantitativer PCR, Western Blot und Immunhistochemie durchgeführt. Als Zellen wurden OECs verwendet, da diese geeignete Kandidaten für

Zusammenfassung Chapter 8

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Marker für die in vivo Detektion von kaninen OECs beschrieben. Die vorliegende Studie zeigte, dass CNPase einen geeigneten Marker für die in vivo Identifikation von kaninen OECs darstellt. Im Gegensatz zu CNPase wurde der Neurotrophin-Rezeptor p75 bei Hunden vor allem in perineuralen Zellen und nur von einigen neonatalen OECs in vivo exprimiert. Allerdings wiesen kanine OECs eine Hochregulation dieses Markers bei längerer Kultivierung in vitro auf. p75^NTR könnte somit als Marker zur Detektion von kaninen OECs in vitro verwendet werden. Im Gegensatz zu den kaninen OECs zeigten Ratten OECs keine CNPase-Expression. Hierbei handelte es sich um einen wesentlichen, Spezies-spezifischen Unterschied. CNPase könnte somit als Marker für die Unterscheidung von OECs und Schwann Zellen (SCs) bei der Ratte verwendet werden.

In der zweiten Studie wurde ein Ethidiumbromid induziertes (toxisches) Entmarkungsmodell im kaudalen Kleinhirnstiel (CCP) der Maus etabliert. Dieses Modell stellt ein wesentliches Werkzeug für die Untersuchung der Pathogenese von De- und Remyelinisierung dar und kann unter anderem verwendet werden, um den Einfluss von Zellen und/oder Substanzen im Verlauf von Entmarkungserkrankungen zu untersuchen. Der CCP wurde aus folgenden Gründen gewählt: 1) Es handelt sich um eine anatomisch gut umschriebene Region, 2) die Axone in dieser Lokalisation weisen einen ähnlichen Durchmesser sowie eine ähnliche Orientierung auf und 3) die Myelinscheiden zeigen eine ähnliche Dicke. In dem Versuch wurden Ethidiumbromid-Konzentrationen zwischen 0,1 und 0,01% in einem Volumen von 2µl stereotaktisch appliziert. Die Mäuse wiesen im CCP eine Konzentrations-abhängige Läsionsgröße nach Ethidiumbromid-Applikation auf. Für die Etablierung eines geeigneten Entmarkungsmodells ist es wichtig, eine Ethidiumbromid-Konzentration zu wählen, die hoch genug ist um eine signifikante Entmarkung hervorzurufen aber keinen ausgeprägten Axonschaden induziert. Für die weiteren Experimente wurde eine Ethidiumbromid Konzentration von 0,025% gewählt, da bei dieser Konzentration eine ausgeprägte Entmarkung sowie ein gering- bis mittelgradiger Axonschaden beobachtet wurde. Das Auftreten von Axonschäden bei der Maus steht im Widerspruch zu publizierten Daten bei der Ratte und stellt einen wichtigen, Spezies- spezifischen Unterschied dar. Des Weiteren wurde die murine Oligodendrozyten-