Role of JAK-STAT signaling in the pathogenesis of astrogliosis in chronic demyelinating Theilers̉ murine encephalomyelitis

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ISBN 978-3-86345-183-7

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

E-Mail: info@dvg.de · Internet: www.dvg.de

Y anyong Sun Hannover

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Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie;

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

1. Auflage 2013

Printed in Germany

ISBN 978-3-86345-183-7

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466 info@dvg.de www.dvg.de

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University of Veterinary Medicine Hannover

Department of Pathology

Center for Systems Neuroscience Hannover

Role of JAK-STAT signaling in the pathogenesis of astrogliosis in chronic demyelinating Theiler’s murine

encephalomyelitis

THESIS

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by Yanyong Sun Born in Jilin province, China

Hannover, Germany 2013

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Supervisor: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Supervision Group: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Prof. Dr. Peter Claus Prof. Dr. Manuela Gernert

1^st Evaluation: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Department of Pathology,

University of Veterinary Medicine Hannover, Germany Prof. Dr. Peter Claus

Department of Neuroanatomy, Medical School Hannover, Germany Prof. Dr. Manuela Gernert,

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

2^nd Evaluation: Prof. Dr. Christiane Herden

Department of Veterinary Pathology, Justus Liebig University Giessen, Germany

Date of final exam: 25.10.2013

Yanyong Sun has received financial support from the China Scholarship Council (CSC) under the File No.2009617013.

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To my family

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“Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop questioning.”

Albert Einstein (1879-1955)

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Parts of the thesis have been published:

Publications:

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. 21(6), 1161-1175.

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1. Aims of study

Multiple sclerosis (MS) is an etiologically unresolved, demyelinating central nervous system (CNS) disease, affecting more than 2.5 million patients worldwide (TRAPP and NAVE, 2008). MS is characterized by an infiltration of inflammatory cells with varying amounts of demyelination, remyelination, astrogliosis and axonal injury (CHOI et al., 2009; KUMMERFELD et al., 2012; TRAPP and NAVE, 2008).

Pathomorphological changes in active demyelinating MS lesions suggest different pathomechanisms and a heterogenous etiology and/or pathogenesis of the disease (LUDWIN, 1994). Active demyelinating MS plaques can be divided in lesions suggestive of a primary immune-mediated pathogenesis and those possibly due to primary oligodendropathy. Accordingly, 30% of MS cases display an extensive destruction of oligodendrocytes, whereas in the remaining 70%, oligodendrocyte numbers are only reduced in areas of active myelin destruction and reappear in remyelinated areas (LUCCHINETTI et al., 2000). A subset of approximately 20% of all MS patients exhibit extensive remyelination, whereas especially those patients with a progressive clinical course display only limited or absent remyelination (CAO et al., 2010; LUO et al., 2010; PATRIKIOS et al., 2006; POMBO et al., 1999).

Approaches to study demyelination and remyelination include transplantation of oligodendrocyte precursor cells (OPCs) into the demyelinated CNS as well as the analysis of naturally occurring and experimentally induced disease (GRIFFITHS, 1996;

KLUGMANN et al., 1997; LI et al., 1998; NAVE, 1994). To fully exploit the potential of cell transplantation and to analyze the relevance of specific molecules, it would be important to use mutant animals for cell isolation or as a host receiving transplants.

Though the majority of available transgenic animals represent mice, most OPC transplantation studies and investigations using experimental demyelination of the CNS were carried out in rats (FRANKLIN et al., 1996; WOODRUFF and FRANKLIN, 1999). Historically, this is most likely due to the fact that isolation and culturing of OPCs became first feasible in the rat model, and that isolation and purification as well as marker expression of murine OPCs is different and more elaborated in mice than in

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rats (CHEN et al., 2007). Thus, little is known about chemically induced demyelination in the CNS of mice and the behavior of murine OPCs following transplantation.

Many MS lesions are known to contain OPCs. However, they frequently lack differentiation into myelinating oligodendrocytes (CAO et al., 2010; KISSELEVA et al., 2002; SOFRONIEW and VINTERS, 2010; XIA et al., 2002; YOSHIMATSU et al., 2006). A similar situation of a progressive demyelination with insufficient remyelination despite the presence of an at least transiently increased number of intralesional OPCs is reported in the Theiler´s murine encephalomyelitis (TME) virus (TMEV)-induced model of demyelination (WOLSWIJK, 1998). In contrast to TME, a fast and complete remyelination can be observed in most other models of MS, including experimental autoimmune encephalomyelitis (EAE) (DARNELL et al., 1994), cuprizone toxicity (MASON et al., 2000), lysolecithin-induced demyelination in the caudal cerebellar peduncle (ZAMANIAN et al., 2012), and murine hepatitis virus (MHV) A-59 strain infection of mice (RODRIGUEZ et al., 2006). The principal reasons for a failure of remyelination can either be a depletion of OPCs, inability of OPCs to proliferate and/or differentiate within the lesion due to a non-permissive environment; axonal loss or the inability of chronically demyelinated axons to be remyelinated (FITCH and SILVER, 2008; FRANKLIN, 2002; WOLSWIJK, 1998).

Cumulative experimental evidence suggests that a block of oligodendroglial differentiation seems to represent the key factor in remyelination failure in MS (FRANKLIN, 2002; KISSELEVA et al., 2002). Based on previous experiments that suggested a shift from oligodendroglial towards astrocytic differentiation of OPCs in TME (PRINGPROA et al., 2010; WOLSWIJK, 1998), we modified the hypothesis of inhibited OPC differentiation pointing towards a more active dysregulation process as the underlying mechanism. Therefore the aims of the present study were: 1.) transplantation of BO-1 cells into the demyelinated CCP was carried out to demonstrate whether this environment may trigger an even more pronounced oligodendrocytic differentiation. 2.) to identify the principal molecular pathways regulating the differentiation of OPCs in TME employing gene expression microarrays, 3.) to verify the cellular source and activation status of the most promising candidate

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pathway in situ, and 4.) to demonstrate the suggested molecular switch-like mechanism of action in vitro.

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2. Introduction

2.1 Theiler's murine encephalomyelitis (TME)

2.1.1 Animal models for multiple sclerosis

Multiple sclerosis (MS) is the most prevalent demyelinating disease in humans, with documented more than 2 million MS patients worldwide. Its geographic distribution depends on ethnic and racial differences (ROSATI, 2001), the ratio of affected women to men is 2.6:1 (HIRST et al., 2009; SCHWENDIMANN and ALEKSEEVA, 2007).

Psychiatric, cognitive and motor disorders in MS commonly cause a wide range of symptoms including depression (PATTEN et al., 2003), euphoria (PINKSTON et al., 2007), fatigue, impaired balance, tremor and ataxia (FREEMAN, 2001), pain (STENAGER et al., 1995) sexual dysfunction (KESSLER et al., 2009), bladder impairments (BETTS et al., 1993) and visual impairments (MCDONALD and BARNES, 1992). According to its clinical course, MS is divided into four categories: relapsing remitting (RR), primary progressive (PP), secondary progressive (SP), and progressive relapsing (PR; LUBLIN and REINGOLD, 1996). The pathological features of MS are demyelination, inflammation, gliosis, and axonal injury in the CNS (HAGEMEIER et al., 2012; TRAPP and NAVE, 2008). The etiology of MS is unknown, suggested causes include autoimmunity (BURNS et al., 1999; HAFLER and WEINER, 1987), virus infections (OWENS et al., 2011; TSELIS, 2011), environmental causes (SLOKA et al., 2011), and/or genetic factors (LYNCH et al., 1991; SEBOUN et al., 1989). Various animal models provide powerful tools to study the pathogenesis of demyelinating disease. Accordingly, experimental autoimmune (allergic) encephalomyelitis (EAE) is frequently used to investigate autoimmune mechanisms in MS (GOLD et al., 2006; TSUNODA and FUJINAMI, 1996). Furthermore, multiple viruses including canine distemper (BEINEKE et al., 2009), Theiler’s murine encephalomyelitis virus (TMEV; LIPTON, 1975; ULRICH et al., 2006; ULRICH et al., 2010; ULRICH et al., 2008), murine coronavirus (JHM strain)-infection (HERNDON et al., 1975), visna of sheep (SIGURDSSON et al., 1957), and Semliki forest

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virus-infection (FAZAKERLEY et al., 1983) are used to study pathogen-induced demyelination. Additionally, there are multiple toxic (RODRIGUEZ, 2007;

WOODRUFF and FRANKLIN, 1999) and genetic (PROBERT et al., 1995) models of demyelinating diseases.

2.1.2 Theiler’s murine encephalomyelitis virus (TMEV)

TMEV belongs to the Cardiovirus genus and Theilovirus species. They are members of the Picornaviridae family (LIANG et al., 2008). Various positive-stranded RNA viruses in this family, such as poliovirus, foot-and-mouth disease virus, rhinovirus, coxsackie virus, and hepatitis A virus induce diseases in humans and animals (TRACY et al., 2006). The CNS is rarely involved in natural TMEV infection (WELSH et al., 1990). However, experimentally infected mice of susceptible strains develop a demyelinating leukomylitis (LIPTON, 1975; ULRICH et al., 2008). There are two main subgroups of TMEV strains. One subgroup including GD VII and FA viruses induces an acute, mostly fatal polioencephalitis within one week. The second group including the DA, BeAn8386, WW, Yale and TO strains causes a biphasic disease course with mild polioencephalitis and chronic demyelinating leukoencephalitis (DAL CANTO and LIPTON, 1975, 1980; LIANG et al., 2008; LIPTON, 1975; LIPTON and DAL CANTO, 1976; RODRIGUEZ et al., 1987a; RODRIGUEZ et al., 1987b; THEILER, 1937).

These strains infect neurons and glial cells of the gray matter resulting in often subclinial acute polioencephalitis (CAMPBELL et al., 2001; JOHNSON et al., 2004;

NJENGA et al., 1997; SIEVE et al., 2004, 2006). After 2-3 weeks, a chronic progressive demyelinating lymphohistoplasmacytic leukomyelitis develops that represents a well-characterized model for multiple sclerosis (CLATCH et al., 1986;

DAL CANTO and LIPTON, 1982; KUMMERFELD et al., 2012; ULRICH et al., 2008).

2.1.3 Clinical Features of chronic demyelinating TME

Motor dysfunctions are the most obvious clinical feature in TMEV-infected susceptible mice. TMEV-infected mice show progressive ataxia, spastic paresis, postural disorder and an enhanced flexor reflex with maintenance of nociception (ULRICH et al., 2006).

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TMEV (BeAn strain) infected SJL mice develop clear clinical signs at 30 to 50 days after infection (dpi), such as wadding gait and hind limb paralysis (IWAHASHI et al., 1999). To some extent, incontinence of urine occasionally can be seen later on in the diseased animals (DAL CANTO and LIPTON, 1975). The functional deficiencies can be quantified by lack of horizontal and vertical activity (spontaneous box), loss of motor coordination (rotarod assay) beginning 6 weeks post infection (wpi) and stride length impairment (footprint analysis) appears around 12 wpi in TMEV-infected susceptible SJL/J mice (MCGAVERN et al., 1999b; ULRICH et al., 2010; ULRICH et al., 2008).

2.1.4 Pathohistological features of chronic demyelinating TME

In chronic demyelinating TME, pathohistological examination reveals severe inflammation characterized by lymphohistioplasmacytic inflammation of the meninges and perivascular spaces within the white matter of the spinal cord (leukomyelitis). The perivascular inflammatory cells consist of CD3⁺ T-lymphocytes, CD45R/B220⁺ B-lymphocytes, CD107b⁺ macrophages, and IgG⁺ plasma cells. CD107b⁺ macrophages and CD3⁺ T-lymphocytes are present in the parenchymal infiltrates, and numerous CD107b⁺ macrophages showed gitter-cell morphology in lesioned areas (ULRICH et al., 2010). The onset of demyelination coincides with the onset of leukomyelitis with first ultrastructural pathologic alterations of myelin sheaths beginning at 14 dpi (ULRICH et al., 2008). Multiple demyelinated foci are mainly found in the ventrolateral funiculi of cervical and thoracic spinal cord segments by light microscopy (ULRICH et al., 2008). The number and extend of demyelinated areas progressively increases until the late stage (ULRICH et al., 2010). Parenchymal macrophages/microglia phagocytosing myelin debris appear at 6 wpi, and dense aggregates of intralesional macrophages/microglia and abundant gitter cells are present within the demyelinated areas at 28 wpi (DAL CANTO and LIPTON, 1975;

ULRICH et al., 2008). Demyelination has been quantified in TME employing toluidin blue stained semi-thin sections, showing 5-10% of demyelinating within the white matter of the spinal cord at 180 dpi (MCGAVERN et al., 1999a). Moreover, at 180 dpi

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both Schwann cell-type and oligodendrocyte-type remyelination is present in 8% of the spinal cord of TMEV-infected SJL/J mice (MCGAVERN et al., 1999a).

Immunohistochemical quantification shows a decline in the number of 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNPase)-positive cells at 56, 98 and 196 dpi (ULRICH et al., 2008), and a 20-30% decrease in myelin basic protein (MBP) expression at 196 dpi in TMEV-infected mice as compared to mock-infected mice (ULRICH et al., 2010).

2.1.5 Pathogenesis of chronic demyelinating TME

Viral persistence and immunopathology represent key features of the pathogenesis of TME. Virus persistence is an obligatory prerequisite for the development of demyelinating lesions (RODRIGUEZ et al., 1997). Persisting viral replication in the mouse spinal cord is reported in multiple studies (TROTTIER et al., 2001; ULRICH et al., 2006). The host genetics play a major role for viral persistence (ALTINTAS et al., 1993; BRAHIC et al., 2005; LIPTON and MELVOLD, 1984; RODRIGUEZ et al., 1986).

All inbred mouse strains develop early polioencephalomyelitis after experimental intracerebral inoculation with TMEV. However, viral clearance can be observed at approximately 2 wpi only in resistant strains, whereas life-long viral persistence develops in susceptible mouse strains. Among several susceptibility genes, the H2 class I gene locus on chromosome 17 has a major effect on susceptibility to TMEV (BRAHIC et al., 2005; MELVOLD et al., 1987; RODRIGUEZ et al., 1986). Moreover, further gene loci numbered Tmevd1, 2, 3, 4, 5, 6, 7, 8, 9, Tmevp2 and 3 also play a role in susceptibility to demyelinating TME (BRAHIC et al., 2005).

TMEV preferentially infects neurons of the hippocampus, cortex, and the ventral horns of the spinal cord during the first 2 wpi (BRAHIC et al., 1981). Later, the virus disappears from the brain to persist mainly in macrophage/microglial cells within the spinal cord, accompanied by the development of an anti-viral delayed-type hypersensitivity response (DTH; BRAHIC et al., 2005; BRAHIC et al., 1981). However, viral RNA was also detected in other cells by using in situ hybridization. Accordingly, 23.2% Ia-positive cells (microglia/macrophages), 26.1% glial fibrillary acidic protein

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(GFAP)-positive cells (astrocytes), and 28.4% MBP-positive cells (oligodendrocytes) were detected in frozen spinal cord sections from TMEV (DA strain) infected C. B-17 mice (NJENGA et al., 1997). Viral antigen was also found in BeAn-strain infected susceptible SJL/J mice and resistant C57BL/6 mice by immunohistochemistry. The phenotype of TMEV infected cells during the early phase of TME in susceptible mice represent 75% astrocytes, 10% oligodendrocytes and 15% macrophages/microglia (KUMMERFELD et al., 2012). Among glial cells, the main viral reservoir is found in macrophages originaling from infiltrating monocytes not from microglial cells (BRAHIC et al., 2005; LIPTON et al., 2005; LIPTON et al., 1995). TMEV disseminates in the CNS primarily due to axonal transport (BRAHIC et al., 1981; TSUNODA and FUJINAMI, 2002). The virus is transported via the axons of infected neurons from the brain to the spinal cord where it spreads to oligodendrocytes. Subsequently, macrophages become infected by phagocytosis of infected oligodendrocyte (BRAHIC et al., 2005). Moveover, viral spread through cerebrospinal fluid is suggested as an alternative route (GOINGS et al., 2008; KUMMERFELD et al., 2012; TROTTIER et al., 2001).

Axonal injury represents another major pathological feature of TME. Investigations indicate that axonal injury precedes or occurs simultaneously with the earliest ultrastructural changes of the myelin sheaths as early as 1-2 weeks after TMEV infection (KREUTZER et al., 2012; TSUNODA and FUJINAMI, 2002, 2010;

TSUNODA et al., 2007). Axonal injury (wallerian-like degeneration) has been shown to lead to the recruitment of inflammatory cells into the locations of axonal degeneration, and causes exacerbating lesion development in the CNS (TSUNODA et al., 2007). Consistent with this, the authors suggested that axonal degeneration heralds demyelination in TMEV-induced demyelinating disease, and lesions are triggered from the axons (inside) to the myelin (outside) “Inside-Out model”

(TSUNODA and FUJINAMI, 2002). In contrast, evidence supports that axonal loss during late phase of TME, results form primary demyelination followed by secondary progressive axonal degeneration and atrophy (MCGAVERN et al., 2000; MCGAVERN et al., 1999a; SATHORNSUMETEE et al., 2000). This leads to a theory that lesions

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develop from the outside (myelin) to the inside (axons), the “Outside-In model” (SATO et al., 2011b; TSUNODA and FUJINAMI, 2002) is suggested to be of major importance in progressive MS (CHANG et al., 2000a; RAINE and CROSS, 1989).

2.1.6 Immunopathology of chronic demyelinating TME 2.1.6.1 CD4⁺cells

In early TME, CD4⁺cells activate CD8⁺cells and also assist B cells in the production of anti-viral antibodies, which are important mediators of viral clearance (CLATCH et al., 1987; WELSH et al., 2009). A DTH reaction against virus and myelin epitopes occurs in the chronic demyelinating phase of TME mainly mediated by Th1 cells (BORROW et al., 1993; CHANG et al., 2000b; MILLER et al., 1990). The virus-specific CD4⁺ Th1 mediated DTH response spreads to encephalitogenic myelin epitopes in the later course of the disease in a process called epitope spreading (MILLER et al., 1997). Interestingly, all susceptible strains exhibit a highly TMEV-specific DTH response, that is not observed in resistant strains (CLATCH et al., 1987). Th1 cells secrete a numerous cytokines such as interleukin (IL)-2, interferon (INF)-γ, and tumor necrosis factor (TNF)-alpha (COFFMAN et al., 1986). Neutraliztion of IFN-γ by anti-IFN-γ monoclonal antibodies significantly accelerates the disease in the acute phase (PULLEN et al., 1994), whereas neutralization of IL-12 with anti-IL-12 monoclonal antibody during the chronic phase of TME leads to suppression disease severity (INOUE et al., 1998). Treatment with the Th2-type cytokines IL-4 during the early chronic phase of TME reduces the anti-TMEV antibody response and subsequentially ameliorates inflammation and demyelination (YAMADA et al., 1990).

2.1.6.2 CD8⁺T cells

CD8⁺T cells have been demonstrated to play an important role in TMEV induced demyelinating disease. CD8⁺ cytotoxic T cells (CTLs) are obligatory for clearance the virus (OLESZAK et al., 2004). CD8⁺T cell deficient mice failed to clear virus from the CNS and developed a severe demyelinating disease (BORROW et al., 1992). On the other hand, an ameliorated motor disorder was found in the disease process after inhibition of CD8⁺ virus peptide specific T cells (JOHNSON et al., 2001). Activated

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CTLs not only kill virus infected cells, but they can also kill non-infected oligodendrocytes, in which the CTLs recognize myelin antigens associated with MHC class I molecules contributing to demyelination (TSUNODA et al., 2005). Interestingly, diminished demyelination by application of anti-CD8 antibody is found in TMEV-infected wild type mice, but no influence on the extent of demyelination is observed in CD8⁺deficient SJL/J mice (MURRAY et al., 1998).

2.1.6.3 Th17 cells

Th17 cells are demonstrated to play a role in the pathogenesis of TME (HOU et al., 2009). A large number of Th17 cells producing IL-17 are detectable in TMEV-infected susceptible SJL/J mice and lipopolysaccharide (LPS)-treated C57BL/6 mice, compared to TMEV-infected resistant naïve C57BL/6 mice. Moreover, the application of anti-IL-17 neutralizing antibody inhibited viral persistence in the CNS, and promotes a CTL-response against virus in the TMEV-infected susceptible mice (SATO et al., 2011b).

2.1.6.4 Regulatory T cells (Treg)

Treg secrete transforming growth factor (TGF)-β and IL-10 and regulate proliferation and differentiation of other Th cells (MATSUI et al., 2010). Treg signaling can result in viral persistence in the CNS causing a chronic demyelinating disease (RICHARDS et al., 2011). On the other hand, Treg cells may ameliorate demyelinating disease by controlling antiviral as well as anti-myelin immune responses (SATO et al., 2011a).

2.1.6.5 Natural killer T (NKT) cells

NKT cells are important in regulating the immune response and clearing virus in the CNS. TMEV resistant C57BL/6 mice have a 50% higher NKT cells activity when compared to susceptible SJL/J mice (PAYA et al., 1989). NKT-deficient mice infected with the highly neurovirulent GDVII-strain exhibit increased number of viral antigen-positive cells, demyelination, disease severity and mortality as compared to wild type mice (TSUNODA et al., 2008; TSUNODA et al., 2009).

2.1.6.6 Macrophages/microglia

TMEV persists in the macrophages/microglia and initiates the innate immune response by increasing cytokine expression, such as tumor necrosis factor-alpha

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(TNF-α) and IL-6 (OLSON et al., 2001). SJL/J mice treated with dichloromethylene diphosphate for the depletion of blood-borne macrophages showed loss of viral persistence along with a lack of demyelination (ROSSI et al., 1997). It is suggested that activated macrophages are involved in the demyelination process, since these cells are massively increased in the lesions (POPE et al., 1998) and macrophages/microglia can release proteolytic factors that lead to MBP degradation in TMEV-infected mice (LIUZZI et al., 1995). Macrophages/microglia mediate bystander damage to myelin resulting in phagocytosis and processing of myelin debris as has been found during acute TMEV infection of SJL/J mice (KATZ-LEVY et al., 2000).

2.1.6.7 Antibodies

Anti-TMEV antibodies contribute to clearance of the virus from the CNS (FUJINAMI et al., 1989), but passive transfer of anti-TMEV antibodies also causes augmentation of demyelination in mice with EAE due to a cross-reaction of the anti-TMEV antibody with galactocerebroside, a major myelin lipid component (YAMADA et al., 1990).

Intrathecal up-regulation of antibody production has been observed in TMEV-infected mice (PACHNER et al., 2007; ULRICH et al., 2010). In contrast, a down-regulation of humoral immune response related genes was found in the lymph node at later time points of TME suggesting a compartmentalization of the immune response within the CNS in chronic demyelinating TME (NAVARRETE-TALLONI et al., 2010).

2.2 Macroglia

2.2.1 Gliogenesis

In the CNS of mammals, glial cells are the most abundant cells (DOETSCH, 2003).

According to morphological and functional aspects, they are divided into astrocytes, oligodendrocytes, and microglia, which account for 17.3%, 75.6%, and 6.5% of all human cerebral cortical glial cells, repectively (PELVIG et al., 2008). Astrocytes and oligodendrocytes are the two main types of macroglia as compared to microglia. In general, gliogenesis follows neurogenesis (ORENTAS and MILLER, 1998; RAFF and

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LILLIEN, 1988), radial glia are the first identified glial population, followed by oligodendrocyte precursors (OPCs), oligodendrocytes and astrocytes (BENTIVOGLIO and MAZZARELLO, 1999; LEE et al., 2000). Oligodendrocytes, the myelin-producing cells in the CNS, originate from the precursor cells known as OPCs.

These precursor cells are derived from neuroepithelial precursors of the embryonic brain and spinal cord (MENN et al., 2006; PLUCHINO and MARTINO, 2008; SHER et al., 2008). The cells of the oligodendrocyte lineage are subdivided into various developmental stages based on the expression of stage-specific markers. OPCs express markers like ganglioside Q (A2B5; MIRON et al., 2011), nerve/glial antigen 2 (NG2; ZHU et al., 2008), and platelet-derived growth factor alpha receptor (PDGFαR;;

HALL et al., 1996; LEVINE et al., 2001). Expression of CNPase (BRAUN et al., 1988), galactocerebroside (GalC; NICOLAY et al., 2007) and antigen O4 (O4; NICOLAY et al., 2007) indicates pre-mature oligodendrocytes. Mature oligodendrocytes are characterized by the expression of myelin-specific proteins, including MBP, myelin-associated glycoprotein (MAG), myelin-oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP; GIELEN et al., 2006; STANGEL and HARTUNG, 2002).

Astrocytes are derived from neuroglial progenitors within the dorsal regions of the neural tube (FOK-SEANG and MILLER, 1994; PRINGLE et al., 1998), and neural stem cells (NSCs) within the rodent subventricular zone (BENNER et al., 2013). The astrocytes exhibiting phenotypic heterogeneity in later stage of development reside throughout CNS (CHABOUB and DENEEN, 2012; ROWITCH and KRIEGSTEIN, 2010). Microglia have important immunological roles in the CNS including scavenging, phagocytosis, antigen presentation and secretion of cytokines (DICKSON et al., 1991).

The origin of CNS microglia is still controversial. Microglial cells are derived from bone marrow hematopoietic stem cells during embryogenesis. Some of these stem cells differentiate into monocytes, hematogenously settle to the brain and further differentiate into microglia (RITTER et al., 2006). Furthermore, engraftment of circulating microglia progenitors to the brain has been shown in the diseased but not healthy CNS (AJAMI et al., 2007; HEPPNER et al., 2005; MILDNER et al., 2007).

However, evidence supports the view that microglia locally derived from their

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progenitors respond to various CNS damages, and do not require turnover from circulating precursors (CHAN et al., 2007; REZAIE and MALE, 2002).

2.2.2 Oligodendrocyte precursor cells (OPCs)

OPCs are present throughout the developing and mature mammalian CNS (KOMITOVA et al., 2009; NISHIYAMA et al., 2009). Moreover, in the rodent CNS, perinatal OPCs first express NG2 antigen during the last 3 to 4 days of embryonic life (LEVINE et al., 1993). These cells rapidly increase in number over the first 10 days of postnatal life and migrate to the developing white and grey matter before they give rise to myelin-forming oligodendrocytes (NISHIYAMA et al., 2009; POLITO and REYNOLDS, 2005). NG2 cells are ubiquitous in grey and white matter and constitute 2-9% of total cells in the adult rodent brain (DAWSON et al., 2003).

Adult OPCs persist in significant numbers throughout life (DE BIASE et al., 2010;

KARADOTTIR et al., 2008; NISHIYAMA et al., 1999; WOLSWIJK and NOBLE, 1992;

WOODRUFF et al., 2004; ZISKIN et al., 2007). OPCs were termed oligodendrocyte type-2 astrocyte (O-2A) cells since they can differentiate into oligodendroglia in serum-free medium or type-2 astrocyte in serum-containing medium (LOUIS et al., 1992; RAFF et al., 1983). Moreover, it has been shown that OPCs generate oligodendrocytes throughout the brain and spinal cord as well as protoplasmic astrocytes in the grey matter of the ventral forebrain and spinal cord (NISHIYAMA et al., 2009). OPCs mainly populate in the forebrain subventricular zone (SVZ), rostral migratory stream (RMS) and olfactory bulbs (OB) in the mouse brain (KOMITOVA et al., 2009). It is widely accepted that OPCs serve as the primary source of remyelinating cells in demyelinating lesions (NISHIYAMA et al., 2009), and respond to demyelination by rapid proliferation and they repopulate lesioned areas (REYNOLDS et al., 2002; WATANABE et al., 2002). OPCs are also involved in cellular homeostasis, including uptake of neuronally released glutamate (BERGLES et al., 2000), and an active glutamate transport (DOMERCQ et al., 1999; REYNOLDS and HERSCHKOWITZ, 1987). Processes of OPCs are in contact with nodes of Ranvier (BUTT et al., 1999),

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2.2.3 Oligodendrocytes

Oligodendrocytes produce the myelin sheaths that insulate the axons within CNS (BAUMANN and PHAM-DINH, 2001). Destruction of oligodendrocytes or myelin leads to disastrous consequences for CNS functions (MILLER and MI, 2007). The importance of myelin for the axonal function is clearly illustrated by demyelinating disease, in which the myelin sheath is damaged, leading to a conduction delay or block and thereby inducing a broad spectrum of clinical symptoms (COMPSTON and COLES, 2008). The CNS myelin contains about 40% water, the dry mass of myelin consists of 70-85% lipids and 15-30% myelin-related proteins, including MBP, MAG, MOG, myelin-associated oligodendrocyte specific protein (MOSP), carbonic anhydrase II (CA-II), CNPase, and PLP (CHRAST et al., 2011; MORELL P, 1999). In the white matter tracts of the CNS, mature myelin-producing oligodendrocytes are process bearing cells and each terminal oligodendrocyte process ensheaths axons with one internodal myelin segment of variable thickness and length (BUTT and RANSOM, 1989, 1993; RANSOM et al., 1991). Each oligodendrocyte is involved in the myelination of up to 50 axonal segments (BAUMANN and PHAM-DINH, 2001;

MILLER, 2002). During postnatal myelination, the GalC-positive premyelinating oligodendrocytes start myelination by the extension of profuse filipodia which engage axons and begin to express CNPase, MBP, and PLP (BUTT et al., 1997a; BUTT et al., 1997b; GARD and PFEIFFER, 1989; GRIFFITHS et al., 1998; HARDY and FRIEDRICH, 1996; TRAPP et al., 1987). In the maturational phase, MBP and PLP are redistributed within oligodendrocytes and there is increased expression of MOG and MAG (TRAPP, 1990). Oligodendrocytes and axons continue to be interdependent (TSUNODA and FUJINAMI, 2002; TSUNODA et al., 2007). For instance, after loss of oligodendrocytes and/or their myelin sheaths, axolemmal ion channels become dispersed and axonal conduction is impaired (WAXMAN, 2006), and loss of axonal function induces disruption of the transcription of myelin-related gene products in oligodendrocytes (YOOL et al., 2000).

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2.2.4 Astrocytes

Astrocytes assume multiple roles in the healthy CNS as they maintain an optimally suited milieu for neuronal function. Astrocytic functions include provision of a scaffold and trophic support, regulation of neurotransmitter secretion and ion homeostasis, detoxification and removal of debris from the cerebrospinal fluid (SIDORYK-WEGRZYNOWICZ et al., 2011). Moreover, astrocytes respond to different forms of CNS injury, such as infection, trauma, ischemia, and neurodegenerative disease (SOFRONIEW, 2009; SOFRONIEW and VINTERS, 2010). Astrocytes are omnipresent throughout the grey and white matter, and they exhibit a heterogeneous morphology including fibrillary, protoplasmic, and gemistocytic subtypes (MILLS, 2007;

SOFRONIEW and VINTERS, 2010). The expression of the intermediate filament GFAP is the prototypical immunohistochemical marker for the identification of astrocytes, especially in reactive astrogliosis and glial scar formation (HERRMANN et al., 2008; MIDDELDORP and HOL, 2011; PEKNY et al., 1995; PEKNY and PEKNA, 2004; SOFRONIEW and VINTERS, 2010). At the single-cell level, GFAP is not present uniformly throughout the astrocytic cytoplasm, and GFAP is not expressed in all portions of the astrocyte (SOFRONIEW and VINTERS, 2010). At the ultrastructural level GFAP immunoreactivity is usually localized to the filament-rich cell processes of fibrillary and pilocytic astrocytes, whereas in the gemistocytic astrocytes, strong positive signals are found in the filament bundles surrounding the nucleus (BOZOKY et al., 1993). Other molecular markers suggested for the immunohistochemical identification of astrocytes include glutamine synthetase, S100β (GONCALVES et al., 2008; NORENBERG, 1979), and aldehyde dehydrogenase 1 family member L1 (Aldh1L1; CAHOY et al., 2008).

2.2.5 BO-1 cell line

Permanent cell lines are versatile tools to study the neurobiology, and numerous experiments suggest the possibility to treat various neurological disease by cell transplantation (LENDAHL and MCKAY, 1990). Since primary cell cultures require regular cell isolations from fresh tissues and are difficult to maintain over multiple

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passages, cell lines provide a useful research tool for long term observations (BONGARZONE et al., 1996; VERITY et al., 1993). A major advantage for using cell lines is that they are readily available, and permit investigations of cell functions without influences of the immune system. A comprehensive list of currently described mouse, rat and human glial cell lines is presented by Pringproa et al (PRINGPROA, 2010). The permanent glial progenitor cell line, BO-1 was generated by spontaneous immortalization of OPCs isolated from neonatal SJL/J mice (PRINGPROA et al., 2008). BO-1 cells display a bi- to multipolar morphology with a varying degree of process length and arborization (PRINGPROA et al., 2008; PRINGPROA et al., 2010).

BO-1 cells proliferate in B104-conditioned Dulbecco's Modified Eagle's (DME) medium. The vast majority of BO-1 cells expresses early OPCs markers, such as A2B5 (97%) and NG2 (99%). A low amount of these cells can be induced to express immature oligodendrocyte markers, such as O4 (25%) and CNPase (35%), as well as the mature oligodendrocyte markers MBP (6%-20%) using 5 μM retinoic acid.

Astrocytic differentiation and GFAP expression (5%-10%) can be induced by 20%

fetal calf serum. This up-regulation of GFAP expression in serum-containing medium is accompanied by significant reduction of the in percentage of cells expressing A2B5, NG2, O4, and CNPase (PRINGPROA et al., 2008; PRINGPROA et al., 2010).

2.3 The JAK-STAT pathway

The janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways are essential pleiotropic cascades used to transduce a multitude of signals for numerous physiologic and pathologic processes in animals and humans (HARRISON, 2012; RAWLINGS et al., 2004). The first JAK-STAT signaling pathway was discovered in the 1990’s due to its association to type I interferon signaling (reviewed in DARNELL et al., 1994). In mammals, there are four non-receptor tyrosine kinases, JAK1, 2, 3 and the tyrosine kinase (Tyk) 2 that belong to the JAK family, and a class of seven structurally and functionally related transcription factors STAT1, 2, 3, 4, 5a, 5b, and 6 that belong to the STAT family (KISSELEVA et al., 2002). A variety of

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ligands including IFN-α/β, IFN-γ, and IL-6 (O'SHEA et al., 2002; SCHINDLER, 1999), ciliary neurotrophic factor (CNTF; BONNI et al., 1997), and growth homone (GH;

BOUTINAUD and JAMMES, 2004) can stimulate the JAK-STAT pathway. Involved receptors include interferon gamma receptor 1 (Ifnar1) and interleukin 6 signal transducer (gp130). The canonical JAK-STAT pathway starts with IFN-γ (extracellular ligand) binding to one of its receptors (type II IFN receptors) on the cell membrane, leading to the intracellular JAK1 and 2 phosphorylation, which phosphorylates downstream substrates including STAT1 monomers (Figure 1). In response to phosphorylation these STAT proteins homo- or hetero-dimerize, translocate into the nucleus, and bind to their specific DNA consensus sequence gamma interferon activation site (GAS), thereby inducing the expression of target genes like proviral integration site 1 (Pim-1; HORVATH, 2004).

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Figure 1. The canonical JAK-STAT signaling pathway (adapted from KEGG pathway, KANEHISA and

GOTO, 2000; KANEHISA et al., 2012).

Cb1 = cannabinoid 1; CBP = cAMP-response element-binding protein;

CIS = cytokine inducible SH2-containing protein; CNTF = cilliary neurotrophic factor;

CytokineR = cytokines receptor; EPO = erythropoietin; JAK = janus kinase; IFN = interferon;

IL = interleukin; +P = phosphorylation; -P = dephosphorylation; +U = ubiquitinylation; P48 = interferon regulatory factor 9;

PIAS = protein inhibitors of active STATs; Pim-1 = proviral integration site 1;

PTPs = protein tyrosine phosphatases; SHP = protein tyrosine phosphatase, no-receptor type;

SOCS = suppressors of cytokine signaling; STAT = signal transducer and activators of transcription;

UPD = uroporphyrinogen decarboxylase;

Activating processes are shown in line with arrows, inhibiting processes are represented in lines with perpendicular bar at the end, and indirect action are shown in dashed lines.

Activation of STATs can also occur independently of JAK activation, for example, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are capable of directly phosphorylating STAT proteins (AKIRA, 1999). Activation of STATs is characterized by phosphorylation upon activation of specific tyrosine or

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serine residues at the C-terminus (AKIRA, 1999). The functions of STAT proteins have been extensively studied, with each individual STAT exhibiting a variety of biological functions. JAK-STAT signaling plays a role in a variety of biological functions, such as embryogenesis (TAKEDA et al., 1997), cell survival, growth, apoptosis (CHIN et al., 1997; KISSELEVA et al., 2002), proliferation, differentiation (TAKEDA and AKIRA, 2000; VERA et al., 2011), hematopoiesis (IHLE et al., 1995), immune development (LIONGUE et al., 2012), mammary gland development, lactation (BOUTINAUD and JAMMES, 2004), adipogenesis (RICHARD and STEPHENS, 2011), sexually dimorphic growth (IGAZ et al., 2001; O'SHEA et al., 2002), inflammation, tumorigenesis (CALO et al., 2003), anti-viral immune response (GAO et al., 2012), ischemia/reperfusion injury (BOLLI et al., 2001), and reactive astrogliosis (HERRMANN et al., 2008). Generally, STAT1, 3, and 6 are thought to be massively involved in the cytokine signal transduction in inflammation and autoimmune diseases (PFITZNER et al., 2004). On the other hand, STAT2 and 5 seemed to be less involved in inflammation (LIU et al., 1998). Moreover, multiple enhancers and inhibitors of the JAK-STAT pathway were discovered in recent years (CHEN et al., 2004; O'SULLIVAN et al., 2007). Major endogenous negative regulators include the suppressors of cytokine signaling (SOCS), protein inhibitors of active stats (PIAS), and protein tyrosine phosphatases (PTPs; GREENHALGH and HILTON, 2001). Protein tyrosine phosphatase, non-receptor type 6 (SHP-1) is a protein tyrosine phosphatase with two Src Homology 2 (SH2) domains which acts as an inhibitor for STAT1 (DAVID et al., 1995; MASSA and WU, 1996) and 6 (HANSON et al., 2003; HUANG et al., 2005) signaling. The SOCS are a family of STAT suppressor proteins that mediate a negative feedback loop in the JAK-STAT pathway. Activated STATs stimulate transcription of SOCS genes and resulting SOCS proteins bind phosphorylated JAKs and their receptors to turn off the pathway (ALEXANDER, 2002).STAT1, 2 and 3 are involved in IFN-mediated immune responses through interferon receptors. In response to IFN-γ stimulation, STAT1 forms dimers with STAT3 that bind to the GAS promoter element (HORVATH, 2004), whereas in response to IFN-α/β stimulation, STAT1 forms dimers with STAT2 that bind to the interferon stimulated response

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element (ISRE) promoter (IGAZ et al., 2001). It is known that STAT3 gene knockout causes an embryonic lethal phenotype (TAKEDA et al., 1997). In addition, a constitutively activated STAT3 has been shown to potentiate tumorigenesis (BROMBERG et al., 1999). Abnormal constitutive activation of STAT3 and 5 is observed in intestinal T cells in Crohn’s disease (LOVATO et al., 2003). STAT4 is activated by IL-12, which plays a critical role in the development of the Th1 subet of Th cells, whereas, IL-4 stimulates STAT6 activation, and a shift towards a Th2-type immune response (KISSELEVA et al., 2002). Despite structural and functional similarities STAT5a and 5b knockout mice display a distinct phenotype. STAT5a knockout mice exhibit a defective prolactin dependent mammary gland development (LIU et al., 1997; TEGLUND et al., 1998), whereas, STAT5b knockout mice exhibit sexual dimorphism (TEGLUND et al., 1998; UDY et al., 1997; WAXMAN et al., 1995).

The STAT5a/b double knockout mice exhibit more severe defects than single knockout mice (KISSELEVA et al., 2002), including a lacking rapid influx of macrophages in inflammatory exudates (KIESLINGER et al., 2000). Erythrocytosis caused by SOCS3 null mice induces embryonic lethality at E12-E16 (MARINE et al., 1999). Moreover, SOCS3 transgenic mice die due to anemia through an inhibition of Epo-Stat5 signaling (MARINE et al., 1999). The PIAS proteins bind to activated STAT dimers, thereby blocking their ability to bind DNA. PIAS1 impedes activated STAT1 activity (LIAO et al., 2000), whereas PIAS3 negatively regulates STAT3 activity (CHUNG et al., 1997).

2.3.1 JAK-STAT signaling pathway in CNS

The JAK-STAT pathway is involved in neurogenesis (CAO et al., 2010), glial differentiation (HE et al., 2005) and CNS diseases (DE LA IGLESIA et al., 2009;

HERRMANN et al., 2008; MAIER et al., 2002). The various JAK and STAT molecules are widely expressed within different cell types of the brain and spinal cord (DE-FRAJA et al., 1998).

2.3.1.1 Neural stem cells (NSCs)

The mammalian cerebral cortex is derived from NSCs, which self-renew and give rise

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to neurons, astrocytes and oligodendrocytes during CNS development (TEMPLE, 2001). Cytokines including CNTF, leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) stimulate astrocytic differentiation in NPCs via the JAK-STAT pathway (FREEMAN, 2010; MILLER and GAUTHIER, 2007). CNTF receptor activates JAK1, STAT1 and 3 thereby promoting differentiation of cerebral cortical precursor cells into astrocytes and inhibiting the alternative differentiation along a neuronal lineage (BONNI et al., 1997; RAJAN et al., 1996). A similar differentiation can also be induced by prolactin via JAK2, STAT1 and 3 activation (MANGOURA et al., 2000;

YANAGISAWA et al., 1999). Conditional depletion of Stat3 in NSCs promotes neurogenesis while it inhibits astrogliogenesis (CAO et al., 2010). Similarly, suppression of STAT3 activity employing SOCS3 enhances neurogenesis and inhibits astrogliogenesis (CAO et al., 2006; GU et al., 2005). Plumbagin, a low molecular weight lipophilic phytochemical STAT3-activator promotes astrocytic differentiation of NSCs (LUO et al., 2010). Similarly, the endogenous STAT3 activator meteorin is known to promote astrocytic differentiation of cortical NSCs and retinal glial cells (LEE et al., 2010). Basically, STAT3 maintains NSCs in a proliferative stage, while combined with bone morphogenetic protein (BMP) and Notch it stimulates astrocytic differentiation (FUKUDA et al., 2007; TAYLOR et al., 2007). On the other hand, SOCS2 increases neuronal differentiation by inhibiting GH signaling (TURNLEY et al., 2002). JAK3 induces neuronal and oligodendroglial differentiation in NSCs (KIM et al., 2010). Platelet derived growth factor (PDGF) and EGF are potential ligands of the JAK-STAT pathway, which may induce NSCs proliferation during early embryonic development, while in stimulate NSCs differentiation into neurons or oligodendrocytes later (MONDAL et al., 2004). Deletion of Stat3 in murine embryonic stem cells (ESC) and NSCs, leads to impaired neurosphere formation and self-renewal (YOSHIMATSU et al., 2006). IL-15 is highly expressed in NSCs of the SVZ, promotes activation of STAT1, 3 and 5, and contributes to NSCs proliferation that can be blocked by JAK inhibits (BAUER, 2009; GOMEZ-NICOLA et al., 2011).

2.3.1.2 Oligodendroglial lineage cells

Oligodendrocyte transcription factor 2 (Olig2) depresses CNTF-STAT dependent

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astrogenesis. In addition, sonic hedgehog (Shh) signaling is needed for oligodendrocytic differentiation (DU et al., 2006). The presence and activation of JAK-STAT signaling is reported in OPCs (VALERIO et al., 2002). PDGF and CNTF stimulated OPCs respond with a rapid tyrosine phosphorylation of JAK1, 2, STAT1, and 3 (DELL'ALBANI et al., 1998). Methylprednisolone prevents the death of oligodendrocytes through the binding of the glucocorticoid receptor leading to STAT5 phosphorylation, which subsequently binds to the B-cell lymphoma 2-like 1 (Bcl-xl) promoter and activates the expression of Bcl-XL (XU et al., 2009). Klotho plays a role in OPC maturation and myelination via the STAT3 pathway (CHEN et al., 2013). A bulk of studies suggests that CNTF activates the JAK-STAT pathway in mature oligodendrocytes, which strongly promotes survival and maturation of oligodendrocytes (BARRES et al., 1996; MAYER et al., 1994; STANKOFF et al., 2002), and myelination (STANKOFF et al., 2002). Both fibroblast growth factor (FGF)-2 and PDGF-AA are signaling molecules (GORETZKI et al., 1999), and potential ligands of the JAK-STAT pathway (DELL'ALBANI et al., 1998; MCKINNON et al., 1993). Moreover, JAK1, STAT4 and 6 are expressed in oligodendrocytes of MS patients (CANNELLA and RAINE, 2004). Cytokine receptors of the CNTF and IFN receptor-family can initiate survival signals by an activation of the JAK-STAT pathway in oligodendrocyte lineage cells, thereby preventing cell death (CASACCIA-BONNEFIL, 2000). In contrast, an inhibition of the JAK-STAT pathway blocked the protective effects of CNTF in ammonia-exposed oligodendrocytes in vitro (CAGNON and BRAISSANT, 2009). IL-6 induced activation of STAT1 and 3 (EMERY et al., 2006; VALERIO et al., 2002) and glucocorticoid receptor induced activation of STAT5 (XU et al., 2009) promotes survival of oligodendrocytes, and these effects can be counteracted by SOCS3 (EMERY et al., 2006). SHP-1 blocks oligodendrocytic myelination through IL-6-STAT3 signaling (MASSA et al., 2000).

2.3.1.3 Astrocytes

Multiple JAK-STAT pathway activating ligands including CNTF, EGF, LIF, and IL-6 are known to trigger astrogliosis (AARONSON and HORVATH, 2002; MOLNE et al., 2000). STAT3 immunoreactivity is strongly present in astrocytes in MS (CANNELLA

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and RAINE, 2004). Genetic ablation or inactivation of STAT3 signaling massively attenuates astrocyte proliferation, migration and glial scar formation (HERRMANN et al., 2008; OKADA et al., 2006; XIA et al., 2002). In the human embryonic carcinoma NTera-2 cell line, STAT3 signaling facilitates astrocytic differentiation (CHENG et al., 2011). Depletion of LIF, a potent activator of Stat3 (BUGGA et al., 1998) or its receptor component gp130 (NAKASHIMA et al., 1999) causes impaired astrocytic differentiation in vivo. BMP, a potential activator of the JAK-STAT pathway is known to accelerate the differentiation and development of astrocytes (ADACHI et al., 2005;

NAKASHIMA et al., 2001; YANAGISAWA et al., 2001). JAK2-STAT1 signaling is involved in reactive astrogliosis in scrapie (NA et al., 2007). JAK1-STAT3 signaling is involved in the astroglial response to focal ischemia (JUSTICIA et al., 2000). Reactive oxygen species (ROS) activate STAT6 signaling in astrocytes as an oxidative stress sensor (PARK et al., 2012). SOCS2 expression is enhanced in astrocytes upon ischemia (CHOI et al., 2009). Genetic deletion of Socs3 leads to increased migration of astrocytes into the contused spinal cord (OKADA et al., 2006).

2.3.1.4 Microglia

Jak1 mRNA is expressed in cultured microglia (SPLEISS et al., 1998), and activated STAT3 is detectable in reactive microglia following focal cerebral ischemia (JUSTICIA et al., 2000; PLANAS et al., 1996). Curcumin induces SHP-2, which suppresses the JAK-STAT pathway thereby attenuating inflammatory changes of brain microglial cells in vitro (KIM et al., 2003). JAK1, STAT1 and 4 are expressed in microglia in MS (CANNELLA and RAINE, 2004). IFN-γ induced JAK1/2-STAT1 signaling is observed in classically-activated macrophages (M1 macrophages; CAO and HE, 2013;

FLAVELL et al., 2010; LAWRENCE and NATOLI, 2011). The latter is involved in the acute pro-inflammatory response by inducing pro-inflammatory cytokines such as TNF-α, IL-12, 23, 6 and chemotactic factors (CAO and HE, 2013; LAWRENCE and NATOLI, 2011). In contrast, IL-4 or IL-10 usually trigger JAK1-STAT3 signaling in alternatively-activated macrophages (M2 macrophages) and thereby they induce the secretion of cytokines such as IL-10 and TGF-β that regulating and suppressing inflammatory responses (BUTOVSKY et al., 2006a; BUTOVSKY et al., 2006b).

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Moreover, in vitro M1 microglia inhibit oligodendrocytic differentiation of NSCs (BUTOVSKY et al., 2006a; BUTOVSKY et al., 2006b). Pretreatment with IL-4 a potential trigger of the JAK-STAT pathway (also as an inducer of M2 microglia), promotes oliogodendrogenesis and differentiation of OPCs through inhibition of the production of toxic M1 molecules including nitric oxide (NO) and TNF-α (PAINTLIA et al., 2006).

2.3.1.5 Neurons

CNTF activates the JAK-STAT pathway in neurons in vitro (BUZAS et al., 1999).

Leptin regulates neuroproliferation both in vivo and in vitro and has neuroprotective effects through activation of JAK-STAT and thymoma viral proto-oncogene 1 (Akt) signaling (GARZA et al., 2008; TANG, 2008). SOCS2, 3 and 6 regulate neuronal differentiation and neurite outgrowth after induction of insulin-like growth factor-1 (IGF-1) and GH (GUPTA et al., 2011; TURNLEY et al., 2002). IL-9 signaling protects neonatal neurons form apoptosis by activation of the JAK-STAT pathway (FONTAINE et al., 2008). Jak2 and 3 mRNA are up-regulated in damaged hypoglossal motoneurons (YAO et al., 1997). STAT3 is massively expressed and activated in regenerating neurons after axonal injury (SCHWAIGER et al., 2000), and JAK1-STAT3 signaling is known to be involved in axonal pathfinding, branching, and muscle targeting in zebrafish motor neurons (CONWAY, 2006). Deletion of SOCS3, enables a robust and sustained axon regeneration (SMITH et al., 2009; SUN et al., 2011).

2.3.2 The JAK-STAT pathway in pathological conditions of the CNS 2.3.2.1 Inflammation

Tyrosine-protein kinase Lyn (Lyn), a member of the rous sarcoma oncogene (Src) family of protein tyrosine kinases is mainly expressed in hematopoietic cells (YAMANASHI et al., 1989), and neural tissues (UMEMORI et al., 1992). Lyn is associated with various cell surface receptor proteins including the B cell antigen receptor (BCR; YAMAMOTO et al., 1993) and CD40 (REN et al., 1994), triggers rapid signaling cascades such as the JAK-STAT pathway leading to cell proliferation and

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differentiation (WANG et al., 2007). STAT1 signaling is observed in the early phase of B cells stimulation (SU et al., 1999). It is suggested that STAT5 plays a role in the self-renewal of B cells (SCHEEREN et al., 2005). BCR and Lyn-mediated activation of STAT3 is JAK-independent in B cells (WANG et al., 2007). IL-12 family (IL-12, IL-23 and IL-27) cytokines secreted by dendritic cells (DCs) play an immune-regulating role by the induction of Th1, Th2, Th17 or Treg cells (EGWUAGU C, 2013). The JAK-STAT signaling pathways are involved in the signaling of multiple of these pro- and anti-inflammatory cytokines. For instance, IL-12 activates JAK2-STAT4 signaling which promotes T cell differentiation into Th1 cells that secrete pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6 (EGWUAGU C, 2013; ZHU et al., 2010).

Mice lacking STAT1 are susceptible to develop EAE, which suggests that STAT1 induction of Th1 and IFN-γ are essential for the inflammatory disease in the CNS (BETTELLI et al., 2004; NISHIBORI et al., 2004). Moreover, pro-inflammatory cytokines including TNF-α, IL-6, IL-17 and IL-22 are produced by Th17 cells, which is induced by IL-23-JAK2-STAT3 signaling (EGWUAGU C, 2013). Treg cells secrete anti-inflammatory cytokines such as IL-10, TGF-β1, and are activated by the IL-27-JAK1-STAT3 pathway (EGWUAGU C, 2013; REGIS et al., 2008). To some extent, expansion of Th1 and Th17 cells is involved in the pathogenesis of MS and EAE (AMADI-OBI et al., 2007; LAURENCE and O'SHEA, 2007). SOCS1 inhibits expansion of Th17 cells as observed in EAE (AHMED et al., 2010; FLOWERS et al., 2004). In brain ischemia IL-6 regulates survival-associated gene expression, via the JAK2-STAT1/STAT3 pathway (PLANAS et al., 2006).

2.3.2.2 Neoplasia

STAT3 signaling exhibits a dual role in glial tumorigenesis. STAT3 is considered a tumor suppressor due to its functional relationship to the major tumor suppressor phosphatase and Tensin homolog (PTEN). STAT3 is stimulated by phosphorylation of LIFR-β and supresses IL8 expression, thereby inhibiting glioma cell proliferation and invasiveness (DE LA IGLESIA et al., 2008a). In contrast, Akt is constitutively activated in PTEN-deficient tumor cells, thus inhibiting FOXO3 function, and leading to LIFR-β down-regulation, followed by lacking STAT3 activation and subsequent IL-8

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up-regulation which drives glioma cell proliferation and invasiveness (DE LA IGLESIA et al., 2008a; DE LA IGLESIA et al., 2008b; DE LA IGLESIA et al., 2009). Furthermore, mutation of epidermal growth factor receptor class III variant (EGFRvIII) can lead to glioma formation (ZHANG et al., 2007). STAT3 and EGFRvIII regulate the transcription of oncogenic genes targets by the formation of a physical complex which can bind to oncogenic genes in nucleus (DE LA IGLESIA et al., 2009). Aberrant activation of STAT3 signaling is critically involved in glioblastoma pathogenesis, such as constitutive STAT3 activation in glioblastoma cells (IWAMARU et al., 2007;

RAHAMAN et al., 2002). Possible ligands of STAT3 signaling in glioblastomas include IL-6 and oncostatin M (VAN WAGONER and BENVENISTE, 1999). Inhibition of STAT3 signaling by JAK inhibitors AG490, WP1066 (IWAMARU et al., 2007;

RAHAMAN et al., 2002), and the STAT3 inhibitor PIAS3 (BRANTLEY and BENVENISTE, 2008) trigger apoptosis and inhibits survival of astroglioma cells in vitro (KONNIKOVA et al., 2003). Moreover, the IL-8-chemokine (C-X-C motif) receptor 2 (CXCR2)-STAT-3 signaling pathway is a regulatory mechanism promoting proliferation of astroglial tumor cells and regulating angiogenesis (KORKOLOPOULOU et al., 2012; PIPERI et al., 2011). Increased JAK1 expression was seen in low-grade gliomas (CATTANEO et al., 1998). STAT6 is also overexpressed in gliomas and promotes invasive growth (MERK et al., 2011).

2.3.2.3 Demyelinating diseases

A reduced severity of demyelination can be induced in EAE by blocking IL-12 signaling in T cells via the JAK-STAT pathway (MUTHIAN et al., 2006; NATARAJAN and BRIGHT, 2002). In MS, increased interferon-beta (IFN-β) induces STAT1 activation, which is considered a possible pathway that enhances the immune response (FENG et al., 2002). STAT1 proteins are markedly up-regulated in neurons and microglia in demyelinating EAE (JEE et al., 2001). The increased transcription of Stat1 RNA is widely distributed in neurons, astrocytes and microglia (MAIER et al., 2002). In MS patients activated STAT1 (FENG et al., 2002; FRISULLO et al., 2006;

GOBIN et al., 2001) mediates interferon signaling and is controlled by SHP-1 (FRISULLO et al., 2006; GOBIN et al., 2001; MASSA and WU, 1996; YETTER et al.,

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1995). Lower levels of SHP-1 in macrophages of MS patients may result in activation of STAT1 and downstream target genes (CHRISTOPHI et al., 2009). Moreover, multiple studies suggest that the activation of the STAT1 signaling pathway by IFN-γ signaling is involved in the pro-inflammatory changes in MS (DAVID et al., 1995;

FENG et al., 2002; MASSA et al., 2000). Up-regulation of activated STAT1, 3 and T-bet was found in peripheral blood CD4⁺ T-cells and monocytes in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and relapsing-remitting MS patients (MADIA et al., 2009). STAT3 enhances T cell differentiation and activity, which is a potential pathogenic factor in MS (LILL et al., 2012). Up-regualated STAT3 immunoreactivity in astrocytes is suggested to play an immune suppressive role in EAE (JEE et al., 2001). In vitro, increased expression of STAT3 signaling in SOCS3-deficient macrophages leads to shift these cells towards a M1 phenotype.

Adoptive transplantation of M2 macrophages into SOCS3-deficient mice with EAE ameliorates the disease process by an inhibition of STAT3 activation (QIN et al., 2012).

Massive expression of STAT4 and IL-12 in microglia is thought to accelerate the autoimmune inflammation in acute EAE (MATSUMOTO et al., 1992). TMEV-infection of Stat4 knockout mice results in delayed onset of severe demyelination after 180 days. In contrast, TMEV-infected Stat6 knockout mice show little demyelination at this time point (RODRIGUEZ et al., 2006). STAT6 activation and expression of STAT6-responsive elements in macrophages may play an important role in the pathophysiology of MS (CHRISTOPHI et al., 2009). Plumbagin inhibits EAE by affecting the differentiation of pathogenic Th1 and Th17 cells differentiation through a down-regulation of JAK-STAT signaling (JIA et al., 2011)

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3. Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and nondemyelinated central nervous system.

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

Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.

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

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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): 1161-75 doi: 10.3727/096368911X627444. Epub 2012 Mar 8

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4. Signal transducer and activator of transcription 3 represents a molecular switch inducing astroglial instead of oligodendroglial differentiation of oligodendroglial progenitor cells in Theiler’s murine encephalomyelitis

Yanyong Sun*^†, Arno Kalkuhl^‡, Ulrich Deschl^‡, Wenhui Sun*^†, Karl Rohn^§, Wolfgang Baumgärtner*^†, Reiner Ulrich*

*Department of Pathology, University of Veterinary Medicine Hannover, Germany,

†Centre for Systems Neuroscience Hannover, Germany,^‡Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma, Biberach (Riß), Germany, and

§Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Germany

Manuscript in preparation

Running title: STAT3 in TME

Keywords: Janus kinase; Microarray; Multiple sclerosis; Oligodendrocyte progenitor cell; Signal transducer and activator of transcription;; Theiler’s murine

encephalomyelitis

Correspondence: Dr. med. vet. Reiner Ulrich. Ph.D., Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany. Tel: +49511-953-8670; Fax: +49511-953-8675; E-mail:

Reiner.Ulrich@tiho-hannover.de

Role of JAK-STAT signaling in the pathogenesis of astrogliosis in chronic demyelinating Theilers̉ murine encephalomyelitis

ISBN 978-3-86345-183-7

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

E-Mail: info@dvg.de · Internet: www.dvg.de

Y anyong Sun Hannover

University of Veterinary Medicine Hannover

Role of JAK-STAT signaling in the pathogenesis of astrogliosis in chronic demyelinating Theiler’s murine

encephalomyelitis

THESIS

DOCTOR OF PHILOSOPHY (PhD)

To my family

“Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop questioning.”

Albert Einstein (1879-1955)

Contents

1. Aims of study

2. Introduction

3. Highly malignant behavior of a murine oligodendrocyte precursor cell line following transplantation into the demyelinated and nondemyelinated central nervous system.

4. Signal transducer and activator of transcription 3 represents a molecular switch inducing astroglial instead of oligodendroglial differentiation of oligodendroglial progenitor cells in Theiler’s murine encephalomyelitis