The Role of Reactive Oxygen Species for the Demyelinating Process in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis and Theiler’s Murine Encephalomyelitis Virus Induced Demyelination

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Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie;

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

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ISBN 978-3-86345-509-5 1. Auflage 2019

Verlag:

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University of Veterinary Medicine Hannover Department of Pathology

Center for Systems Neuroscience

The Role of Reactive Oxygen Species for the Demyelinating Process in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis and

Theiler’s Murine Encephalomyelitis Virus Induced Demyelination

THESIS

Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD)

Awarded by the University of Veterinary Medicine Hannover

by

Friederike Attig Berlin

Hannover 2019

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Supervisor: Prof. Dr. W. Baumgärtner, PhD/Ohio State Univ.

Supervision Group: Prof. Dr. W. Baumgärtner, PhD/Ohio State Univ.

Prof. Dr. M. Gernert

Prof. Dr. K. Haastert-Talini

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

Department of Pathology

University of Veterinary Medicine, Hannover, Germany

Prof. Dr. M. Gernert

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

Prof. Dr. K. Haastert-Talini Department of Neuroanatomy

Hannover Medical School, Hannover, Germany

2^nd Evaluation: Prof. Dr. Dr. h.c. Marc Vandevelde University Bern, Vetsuisse Faculty

Department of clinical research & Veterinary public health

Bremgartenstrasse 109a 3012 Bern, Switzerland

Date of final exam: 25.10.2019

This study was in part supported by the Niedersachsen research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower Saxony.

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To my parents

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So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das möglichste getan

hat.

Johann Wolfgang von Goethe (1749 - 1832), italienische Reise (1787)

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v Table of Content

List of Publications and Presentations vii

List of Abbreviations viii

List of Figures xi

Summary xiii

Zusammenfassung xv

1. General Aim of the Study 1

2. General Introduction 2

2.1. Reactive Oxygen Species (ROS) 2

2.1.1. Reactive Oxygen Species in Health 3

2.1.2. Reactive Oxygen Species in Diseases of the Central Nervous System 5

2.2. Antioxidants 8

2.2.1. Endogenous Antioxidants 8

2.2.2. Exogenous Antioxidants 10

2.3. Canine Distemper Virus (CDV) 12

2.4. Theiler’s Murine Encephalomyelitis Virus (TMEV) 14 3. Manuscript: Reactive oxygen species are key mediators of demyelination in canine distemper leukoencephalitis but not in Theiler’s murine encephalomyelitis 16

4. General Discussion 90

4.1. Amount and Localization of ROS Products in CDV-Induced Leukoencephalitis

and TMEV-Induced Demyelination 93

4.2. Amount and Localization of Antioxidants and Important ROS Detoxifying Pathways in CDV-Induced Leukoencephalitis and TMEV-Induced

Demyelination 95

4.3. Transcriptional Activity of Important ROS-Generation Pathways in CDV-

Induced Leukoencephalitis and TMEV-Induced Demyelination 96

4.4. Conclusion and Perspective 98

5. References 100

6. Appendix 111

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Table of Content

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6.1. Materials and Methods 111

6.1.1. Analyzed Animals 111

6.1.2. Lesion Profile of Dogs 111

6.1.3. Lesion Profile of Mice 112

6.1.4. Light-Microscopy Histology 113

6.1.5. Double-Immunofluorescence 116

6.1.6. Evaluation of Immunofluorescence 118

6.1.7. Statistics 118

6.1.8. Microarray Analysis 119

6.1.9. Additional Results from Mice with Respect to Gray Matter Only and Gray

and White Matter Together 120

6.1.10. Solutions and Buffers 125

7. Acknowledgement 130

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vii List of Publications and Presentations

Parts of the thesis have been published in a peer-reviewed journal:

Friederike Attig, Ingo Spitzbarth, Arno Kalkuhl, Ulrich Deschl, Christina Puff, Wolfgang Baumgärtner, Reiner Ulrich (2019): Reactive oxygen species are key mediators of demyelination in canine distemper leukoencephalitis but not in Theiler’s murine encephalomyelitis. International Journal of Molecular Sciences, 20(13), 3217, doi:10.3390/ijms20133217.

Parts of the thesis have been presented at a national scientific congress:

As oral presentation:

Friederike Attig, Ingo Spitzbarth, Arno Kalkuhl, Ulrich Deschl, Christina Puff, Wolfgang Baumgärtner, Reiner Ulrich (2017): Die Rolle reaktiver Sauerstoffspezies in der Pathogenese der Staupevirus-induzierten Demyelinisierung. In: 60. Jahrestagung der DVG-Fachgruppe Pathologie, 04.-05.03.2017 in Fulda, Germany. Abstract published in: Tierärztliche Praxis Kleintiere/Heimtiere Abstracts 2017, 45(03), A19, doi: 10.1055/s-0038-1625020.

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List of Abbreviation

viii List of Abbreviations

ATP Adenosine triphosphate

BeAn BeAn8386

Ca²⁺ Calcium

CDV Canine distemper virus

CDV-DL Canine distemper virus-Induced demyelinating leukoencephalitis

CNPase 2',3'-cyclic-nucleotide 3'-phosphodiesterase

CNS Central nervous system

CAPE Caffeic acid phenethyl ester

CSF Cerebrospinal fluid

CV Cresyl violet

DA Daniels

DAB 3,3-diaminobenzidine-tetrahydrochloride

dpi Days post infection

EAE Experimental autoimmune encephalomyelitis EDTA Ethylenediaminetetraacetic acid

EGCG Epigal-locatechin-3-gallate

E06 Clone of oxidized phospholipids

Fe²⁺ Iron II

Fe³⁺ Iron III

FFPE Formalin-fixed, paraffin-embedded GFAP Glial fibrillary acidic protein

h Hours

H&E Hematoxylin and eosin

H2O2 Hydrogen peroxide

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8OHdG 8-hydroxyguanosine

8OHG 8-hydroxyguanosine

•OH Hydroxyl radical

Iba-1 Ionized calcium-binding adapter molecule 1

IgG Immunoglobulin G

IL-1 Interleukin 1

K⁺ Potassium

KEGG Kyoto Encyclopedia of Genes and Genomes

LFB Luxol-fast-blue

LFB-CV Luxol-fast-blue cresyl violet

LPS Lipopolysaccharide

MAG Myelin-associated glycoprotein

MBP Myelin basic protein

MDA Malondialdehyde

MHCII Major histocompatibility complex class II MHC-JHM Mouse hepatitis virus strain JHM

MPO Myeloperoxidase

MS Multiple sclerosis

Na⁺ Sodium

NaCl Sodium chloride

NADPH oxidase Nicotinamide adenine dinucleotide phosphate-oxidase

NOX Nicotinamide adenine dinucleotide phosphate-oxidase family of enzymes

NOS Nitric oxide synthase

PBS Phosphate-buffered saline

PKC Protein kinase C

PUFAs Poly-unsaturated fatty acids

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p.i. Post infection

RRMS Relapsing-remitting MS

ROS Reactive oxygen species

RNS Reactive nitrogen species

•O2- Superoxide

O2- Superoxide

SOD Superoxide dismutase

SOD2 Mitochondrial superoxide dismutase

SJL Swiss Jim Lambert

TME Theiler’s murine encephalomyelitis TMEV Theiler’s murine encephalomyelitis virus

TMEV-DL Theiler’s murine encephalomyelitis virus-induced demyelinating leukoencephalitis

TNF Tumor necrosis factor

TO Theiler’s original

XO Xanthine oxidase

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xi List of Figures

Figure 2.1.: Sources of Reactive Oxygen Species (ROS)

Figure 2.2.: Mitochondrial Dysfunction and the Consequences for Axons

Figure 4.1.: Specific Markers for Damaged Lipid and DNA Induced by Reactive Oxygen Species (ROS)

Figure 6.1.: Relative Changes in the Proportions of Cells Immunopositive for ROS Products and Colocalization with Astrocytes, Oligodendrocytes and Macrophages/Microglia in the Spinal Cord Gray Matter of TMEV-DL

Figure 6.2.: Relative Changes in the Proportions of Cells Immunopositive for ROS Products and Colocalization with Astrocytes, Oligodendrocytes and Macrophages/Microglia in the Spinal Cord White and Gray Matter of TMEV-DL

Figure 6.3.: Antioxidant Enzymes and Colocalization with Astrocytes, Oligodendrocytes and Macrophages/Microglia in the Spinal Cord Gray Matter of TMEV-Infected Mice

Figure 6.4.: Antioxidant Enzymes and Colocalization with Astrocytes, Oligodendrocytes and Macrophages/Microglia in the Spinal Cord White and Gray Matter of TMEV-Infected Mice

.

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xiii Summary

The Role of Reactive Oxygen Species for the Demyelinating Process in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis and Theiler’s Murine Encephalomyelitis Virus induced Demyelination

Friederike Attig

Reactive oxygen species (ROS) are highly reactive molecules. ROS arise as by-product during metabolic activity or they are generated actively during phagocytosis. In general, ROS take part in physiological as well as pathological processes. Reactions with ROS can lead to degradation of lipids or nucleic acids. These can be detected as characteristic metabolites like for example malondialdehyde (MDA), oxidized lipids, and 8-hydroxydeoxyguanosine (8OHdG). Canine distemper virus (CDV) can cause CDV-induced demyelinating leukoencephalitis (CDV-DL) in dogs. The experimental infection of Swiss Jim Lambert (SJL) mice with Theiler´s murine encephalomyelitis virus (TMEV) induces an early polioencephalitis and late demyelinating leukomyelitis (TMEV-DL). Both diseases are an animal model for multiple sclerosis (MS). MS is a demyelinating disease of the central nervous system (CNS) in humans. The etiology of this disease is still under debate. Recent data suggest that ROS are involved in the pathogenesis of demyelination in MS.

The aim of the current study was to increase the knowledge upon pathogenesis of the demyelinating process of both, CDV-DL and TMEV-DL, and if ROS are key effector molecules in demyelination.

In the current study cerebellar specimen derived from diagnostic necropsy cases of acute, subacute and chronic CDV-DL were compared with control dogs. Furthermore, spinal cord tissue from experimentally TMEV-infected SJL mice was compared with mock-infected SJL mice killed 14, 42, 196 and 245 days post infection (dpi). Immunofluorescence was used to evaluate the localization and quantity of ROS-induced metabolites and antioxidant enzymes in formalin-fixed, paraffin-embedded (FFPE) tissue from CDV-DL and TMEV-DL, respectively. The employing antibodies reacted with oxidized phospholipids (clone E06), MDA and 8OHdG for ROS-induced metabolites and with mitochondrial superoxide dismutase (SOD2) and catalase for antioxidant enzymes. Double labelling experiments were performed using antibodies reacting with macrophages/microglia (ionized calcium-binding adapter molecule 1, Iba-1), oligodendrocytes (2',3'-cyclic-nucleotide 3'-phosphodiesterase, CNPase)

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and astrocytes (glial fibrillary acidic protein, GFAP). In parallel, data of the transcription of manually selected genes involved in the generation or detoxification of ROS was analyzed for CDV-DL and TMEV-DL, respectively.

Immunofluorescence revealed increased amounts of oxidized phospholipids (clone E06), MDA, and 8OHdG, mainly within macrophages/microglia and oligodendrocytes while the amount of antioxidant enzymes were decreased in CDV-DL. In contrast to CDV-DL, TMEV-infected mice did not show markedly increased up-regulation of ROS metabolites. The microarray analysis showed a trend towards down-regulation in ROS detoxification and up- regulation in ROS toxification in CDV-DL as well as TMEV-DL.

The present studies demonstrate similar results regarding microarray analysis, which in both diseases show an up-regulation of genes essential for ROS generation and a down- regulation of gene-expression for ROS detoxification. In contrast to the microarray analysis, immunofluorescence revealed a significant increase of ROS-induced metabolites exclusively in CDV-DL. Summarized results of the immunofluorescent analysis suggest a difference in the pathogenesis of demyelination between CDV-DL and TMEV-DL.

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xv Zusammenfassung

Die Rolle reaktiver Sauerstoffspezies in der Pathogenese der kaninen Staupevirus- induzierten Demyelinisierung und der Theiler´schen murinen Enzephalomyelitisvirus- induzierten Demyelinisierung

Friederike Attig

Reaktive Sauerstoffspezies (ROS) sind hoch reaktive Moleküle. Sie entstehen als Nebenprodukt während metabolischer Stoffwechselprozesse oder werden aktiv während der Phagozytose produziert. ROS spielen sowohl in physiologischen als auch in pathologischen Prozessen eine Rolle. Die Reaktionen mit ROS können zu degradierten Lipiden oder Nukleinsäuren führen. Diese können mit Hilfe von charakteristischen Metaboliten wie beispielsweise Malondialdehyd (MDA), oxidierten Lipiden und 8-Hydroxydeoxyguanosin (8OHdG) nachgewiesen werden. Das kanine Staupevirus (CDV) verursacht die Staupevirus- induzierte demyelinisierende Leukoenzephalitis (CDV-DL) bei Hunden. Eine experimentelle Infektion von Swiss Jim Lambert (SJL) - Mäusen mit dem Theiler´schen murinen Enzephalomyelitisvirus (TMEV) verursacht in der akuten Phase eine Polioenzephalitis und später eine demyelinisierende Leukomyelitis (TMEV-DL). Beide Erkrankungen stellen ein Tiermodel für die multiple Sklerose (MS) dar. MS ist eine demyelinisierende Erkrankung des zentralen Nervensystems (ZNS) des Menschen. Die Ätiologie der Erkrankung ist noch nicht vollständig geklärt und wird daher immer noch diskutiert. Verschiedene Studien suggerieren eine Beteiligung von ROS in der Pathogenes der Demyelinisierung bei der MS.

Das Ziel der vorliegenden Studie ist es, den Kenntnisstand über die Pathogenese des demyelinisierenden Prozesses in beiden Erkrankungen, CDV-DL und TMEV-DL, zu erweitern und herauszufinden, ob ROS dabei eine Schlüsselfunktion haben.

In der vorliegenden Studie wurde Kleinhirngewebe von natürlich mit dem kaninen Staupevirus infizierten Hunden verwendet. Im Kleinhirngewebe dieser Hunde wurden akute, subakute und chronische Läsionen unterschieden. Diese wurden mit nicht-infizierten Kontrollhunden verglichen. Die Proben stammen aus dem diagnostischen Sektionsgut. Im zweiten Teil der Studie wurde Rückenmark von experimentell mit TMEV-infizierten SJL Mäusen verwendet, das mit Mock-infizierten Mäusen verglichen wurde. Die Mäuse wurden jeweils 14, 42, 196 und 245 Tage nach der Infektion getötet. Mittels Immunfluoreszenz wurde

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Zusammenfassung

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die Quantität und die Lokalisation von ROS-induzierten Metaboliten und antioxidative Enzyme in Formalin-fixiertem und in Paraffin-eingebettetem (FFPE) Material bei CDV-DL und TMEV- DL evaluiert. Die verwendeten Antikörper reagieren mit oxidierten Phospholipiden (Klon E06), MDA und 8OHdG für ROS-induzierte Metaboliten und mit mitochondrialer Superoxiddismutase (SOD2) und Katalase für antioxidative Enzyme. Doppelfärbungen wurden verwendet um eine Kolokalisation mit Makrophagen/Mikroglia (ionized calcium-binding adapter molecule 1, Iba-1), Oligodendrozyten (2',3'-cyclic-nucleotide 3'-phosphodiesterase, CNPase) und Astrozyten (saures Gliafaserprotein, glial fibrillary acidic protein, GFAP) festzustellen. Parallel zur Immunfluoreszenz wurde die Transkription von manuell selektierten Genen, die in der Produktion oder Entgiftung von ROS involviert sind, für die Staupevirus- und Theilervirusinfektion untersucht.

Die Immunfluoreszenz zeigte ein vermehrtes Aufkommen von oxidierten Phospholipiden (Klon E06), MDA und 8OHdG bei Staupevirus-infizierten Hunden. Dieses war vor allem in Makrophagen/Mikroglia und Oligodendrozyten zu beobachten, während antioxidative Enzyme vermindert waren. Im Gegensatz dazu zeigten TMEV-infizierte Mäuse keine so deutlichen Ergebnisse. Die Mikroarray Studie zeigte, dass Gene, die für die ROS Produktion verantwortlich sind, heraufreguliert sind, während Gene, die für die ROS Entgiftung verantwortlich sind, herunterreguliert sind. Dieses Phänomen war in beiden Erkrankungen zu beobachten.

Die vorliegende Studie zeigt, dass bezüglich der Mikroarray-Analyse beide Erkrankungen Ähnlichkeiten aufweisen. Im Gegensatz dazu zeigte sich in der Immunfluoreszenz eine signifikante Zunahme von ROS-induzierten Metaboliten nur bei den Staupevirus-infizierten Hunden. Die Ergebnisse sprechen daher für einen Unterschied in der Pathogenese der Demyelinisierung bei CDV-DL und TMEV-DL.

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1 1. General Aim of the Study

Reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) are chemically highly reactive molecules and are involved in both physiological and pathological processes (Thannickal et al. 2000; Mittal et al. 2014). ROS and RNS have been highlighted to play a role in the pathogenesis of multiple sclerosis (MS) (Lassmann 1998; van Horssen et al.

2006; van Horssen et al. 2008; Haider et al. 2011; van Horssen et al. 2011; Fischer et al. 2012;

Schuh et al. 2014; Witte et al. 2014; Lassmann et al. 2015). MS is a chronic inflammatory and demyelinating disease in young adults. In young adults, it is also the most common non- traumatic disease of the central nervous system (CNS) (Prins et al. 2015). Various animal models have been established in order to mimic certain aspects of MS and in this context mice represent a commonly used species for experimental research upon MS. For instance, Theiler’s murine encephalomyelitis (TME) virus (TMEV)-induced demyelinating leukomyelitis (TMEV-DL) and experimental autoimmune encephalomyelitis (EAE) are two commonly employed mouse models for demyelinating disease including MS (Ercolini et al. 2006).

Furthermore, canine distemper virus (CDV)-induced demyelinating leukoencephalitis (CDV- DL), the most common nervous manifestation of canine distemper in dogs, represents one of the relatively rare non-experimental models that have been used as a model for demyelinating disease in humans (Amude et al. 2010).

The hypothesis of this study is that ROS are key effector molecules in the demyelinating process of both CDV-DL and TMEV-DL. So far, knowledge of the role of ROS in TMEV-DL is missing. For CDV-DL, a study points towards an involvement of ROS in the demyelination process due to antiviral antibodies (Burge et al. 1989). Antiviral antibodies bind to the Fc receptor on macrophages and initialize the ROS production in macrophages (Burge et al. 1989).

The aim of the present thesis is to comparatively evaluate the localization and the degree of ROS-induced metabolites in CDV-DL and TMEV-DL in situ. Furthermore, the cellular localization and amount of ROS-detoxifying enzymes will be evaluated in both diseases. The results of both diseases are not only compared to each other but also to published studies of MS and other animal models for this disease. Lastly, previously published and publically available microarray studies on TMEV-DL and CDV-DL (Ulrich et al. 2010; Ulrich et al. 2014a) will be analyzed for the transcriptional activity of ROS generation and ROS-detoxifying pathways in CDV-DL and TMEV-infection and conclusively discussed together with data on the respective protein expression in both animal models.

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General Introduction

2 2. General Introduction

2.1. Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS) are highly reactive molecules that arise from molecular oxygen (Turrens 2003). ROS have physiological signaling functions, but in case they overwhelm the antioxidant capacity, they can cause substantial tissue damage (Thannickal et al. 2000). Examples of ROS include superoxide (•O2-), the hydroxyl radical (•OH) and hydrogen peroxide (H2O2) (Datta et al. 2000). Superoxide (•O2-) is generated by reduction of oxygen (O2). This can occur by an addition of one electron to O2. Hydrogen peroxide (H2O2) represents an uncharged molecule. It is quite stable unless metal ions like iron or copper are present. During the so-called Fenton’s reaction, iron II (Fe²⁺) is oxidized by hydrogen peroxide to iron III (Fe³⁺), a hydroxyl radical and a hydroxide ion.

Fe²⁺ + H2O2 → Fe³⁺ + HO• + OH^- Fe³⁺ + H2O2 → Fe²⁺ + HOO• + H⁺

The hydroxyl radical is a highly reactive molecule that can react with every molecule at its side.

For instance, hydroxyl radical is generated as a product of radiolysis (Halliwell 1991; Halliwell 1992).

In contrast to ROS, the term “free radicals” encompasses any highly reactive molecule with one or more unpaired electrons. Many ROS are simultaneously free radicals, but by definition, not all ROS are free radicals (Lewen et al. 2000; Thannickal et al. 2000). Despite this fact, the term “ROS” and “free radical” are sometimes used interchangeably (Nordberg et al. 2001). The oxidation promoted by free radicals or specific ROS can lead to the generation of oxidized proteins, lipids or nucleic acids (Lewen et al. 2000).

ROS can arise from enzymes such as, nicotinamide adenine dinucleotide phosphate- oxidase family of enzymes (NOX), xanthine oxidase (XO), cytochrome P450 or myeloperoxidase (MPO) (Thannickal et al. 2000; Zhang et al. 2002; Bedard et al. 2007; Mittal et al. 2014). However, other possibilities where ROS can arise from are various metabolic processes including cellular respiration (Thannickal et al. 2000; Bedard et al. 2007; Brieger et al. 2012; Mittal et al. 2014) (Figure 2.1.).

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Figure 2.1.: Sources of Reactive Oxygen Species (ROS). ROS arise enzymatically or non- enzymatically in different cell types. ROS occur also during cellular metabolism. (Thannickal et al. 2000; Brieger et al. 2012; Mittal et al. 2014).

2.1.1. Reactive Oxygen Species in Health

Although the brain is only representing 2% of the body weight in humans, it needs 20%

of the body’s oxygen. This is in part attributed to the fact that the brain cannot work under anaerobic conditions. Therefore, the brain is prone to any metabolic changes regarding the oxygen and glucose support. Due to reduction of O2, ROS can arise.

O + 2e^- O^2-

ROS can arise enzymatically and non-enzymatically. Moreover, they can also be produced unintentionally as “by-products” (Kirkinezos et al. 2001). This can happen in 2– 5% of the total oxygen consumption in mitochondria (Chan 2001; Kirkinezos et al. 2001).

However, under physiological circumstances, substantial amounts of mitochondrial superoxide dismutase (SOD2), which converts O2- to H2O2, keep the concentration of superoxide within mitochondria on a relatively low level. H2O2 can diffuse through the membrane into the cytoplasm (Thannickal et al. 2000). In general, superoxide anions are produced by the reduction of oxygen. Within mitochondria, superoxide anions can be produced at the redox-active prosthetic groups within proteins (Halliwell 1992; Kirkinezos et al. 2001).

Another possibility for superoxide production are electron carriers, which are bound to proteins (Murphy 2009). Superoxide anions can also be produced by complex I-III of the electron chain

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transport mainly on the inner membrane of the mitochondrial membrane. Furthermore, glycerolphosphate can arise dehydrogenase on the outer side of the inner membrane, mono- amino-oxidase on the inner side of the outer membrane and dihydroorotate dehydrogenase on the outer surface of the inner mitochondrial membrane (reviewed by Turrens 2003).

It is suggested that the mitochondrial complex I (NADH- ubiquinone oxidoreductase) may only produce high amounts of ROS in pathological circumstances (Zorov et al. 2014).

Furthermore, ROS take part in cell signaling pathways. For instance, they can regulate ion channels, protein phosphorylation or transcription factors (Brieger et al. 2012). In the vascular system, oxidative signaling can stimulate monocytes to adhere to endothelial cells (Go et al.

2010). Experiments within the hippocampus demonstrate that O2- and H2O2 take part in the communication between neurons and glia cells like oligodendroglia (Atkins et al. 1999). These ROS are suggested that they are involved in the activation of protein kinase C (PKC) to regulate myelin basic protein (MBP) phosphorylation (Atkins et al. 1999). Based on the fact that ROS are involved in several mechanisms like signaling or regulation of the cell cycle and supporting immune functions it is also discussed to describe them as a “oxidative regulation” (Alfadda et al. 2012).

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2.1.2. Reactive Oxygen Species in Diseases of the Central Nervous System

As described above, ROS are generated during various physiological processes.

However, they simultaneously play a pivotal role in pathology. Generation of ROS may lead to damage of lipid, proteins and nucleic acids (Inoue et al. 1995; Brieger et al. 2012; Ayala et al.

2014; Mittal et al. 2014). In general, ROS are needed in cell signaling as well as in the defense of pathogens. However, if the control mechanisms of ROS or the antioxidative system fail to work appropriately, tissue injury will arise (Mittal et al. 2014). The exuberant generation of ROS in central nervous system (CNS) pathology has manifold consequences. For instance, hypoxia inhibits sodium (Na⁺)-potassium (K⁺) -ATPase activity in neurons because of a lack of adenosine triphosphate (ATP). Furthermore, ROS can lead to Na⁺-K⁺-ATPase degradation. The Na⁺-calcium (Ca²⁺) exchange channel reverses its activity and Ca²⁺ will enter the axons in higher amounts (Figure 2.2.). These disturbed activities may subsequently lead to a neurofilament fragmentation, loss of axonal transport and neuronal degeneration (Mao et al.

2010; Lassmann et al. 2012). The Na⁺-K⁺-ATPase activity can be a result of a defect in the complex IV of the respiratory chain within mitochondria. A defect complex IV is also the reason for a higher sensitivity to axonal injuries that are mediated by glutamate (Mahad et al. 2009) (Figure 2.2.).

In macrophages, the NADPH oxidase complex can for instance produce ROS. In fact, the NADPH oxidase complex has been shown to play a major role in the production of ROS within macrophages that phagocytose myelin. By the inhibition of the NADPH oxidase phagocytosis of myelin was decreased (van der Goes et al. 1998). During demyelinating diseases, ROS can have either direct effects on myelin or oligodendrocytes. Myelin is very prone to ROS damage because of its high lipid content. Myelin is composed of at least 70%

lipids of its dry weight (Finean et al. 1958; Aggarwal et al. 2011; Chrast et al. 2011).

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Figure 2.2.: Mitochondrial Dysfunction and the Consequences for Axons. a) The adenosine triphosphate (ATP)-dependent Na⁺-pump removes Na⁺ from the axon after normal action potential (AP); b) Mitochondrial dysfunction leads to a lack of ATP and Na⁺ accumulates within the axon. The additional Na⁺/Ca²⁺ exchanger works in opposite direction and Ca²⁺ enters the axon via glutamate receptors and voltage dependent calcium channels. The proteinase calpain is activated by Ca²⁺ and resolves cytoskeletal proteins and intra-axonal proteins. (Modified from Lassmann et al. 2012).

The myelin lipids itself do not differ from those within the cell membrane. However, the composition of the lipids is specific for myelin. It contains cholesterol, phospholipids and glycolipids in a ratio from 4:3:2 to 4:4:2 (Baumann et al. 2001). Furthermore, myelin contains myelin-specific proteins including myelin basic protein (MBP), proteolipid protein and myelin- associated glycoprotein (MAG) (Solly et al. 1996). In the CNS, oligodendrocytes and its progenitor cells produce myelin (Chrast et al. 2011; Carvalho 2013). ROS can react with both components of myelin, protein and lipid (Smith et al. 1999). In addition, it is discussed that the concentration of iron is relatively high within oligodendrocytes. If oligodendrocytes are destroyed, iron will be released into the extracellular space where macrophages/microglia will

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take it up. These processes can lead to Fenton’s reaction (Connor et al. 1996; Lassmann et al.

2012).

ROS contribute to demyelination by the induction of lipid peroxidation. This process represents a reaction of ROS or free radicals with the carbon-carbon double bond of lipids.

Polyunsaturated fatty acids (PUFAs) are prone to lipid peroxidation (Ayala et al. 2014). Lipid peroxidation can arise non-enzymatically or enzymatically and consists of three steps (Marnett 1999; Bochkov et al. 2010; Ayala et al. 2014). The first step is the initiation. This reaction produces a fatty acid radical. Because of this unstable molecule the second step, propagation, occurs and produces peroxyl fatty acid radicals by adding molecular oxygen. These molecules are similarly not very stable and initiate other lipids to start the lipid peroxidation or they react with themselves and a reaction circle starts. In order to end this reaction, the third and last step of the reaction is needed, which is called the termination step. For termination of the reaction, an antioxidant is needed to catch the free radical. Alternatively, the fatty acid is expended or the radical molecules react to a non-radical molecule (Marnett 1999; Bochkov et al. 2010; Ayala et al. 2014)

Both oxidized DNA and oxidized lipids have been described in MS lesions. Oxidized phospholipids were present in oligodendrocytes as well as in some astrocytes and spheroids.

Oxidized phospholipids were similarly found within neurons and macrophages (Haider et al.

2011). Some genes encoded mitochondrial DNA as well as the respiratory chain especially for the complex I and IV are downregulated in MS-lesions, as demonstrated by microarray studies (Fischer et al. 2012). The aforementioned two studies indicate that a mitochondrial defect might be involved in the pathogenesis of MS, which in turn leads to an increased presence of ROS.

However, interestingly, the reproduction of these findings in MS is very limited in animal rodent models (Schuh et al. 2014). Mice with chronic inflammatory demyelination, induced by an infection with the mouse hepatitis virus strain JHM (MHV-JHM), show increased amounts of oxidized lipids. This result parallels the findings in MS. In contrast to MS, rats suffering from acute EAE show only sparse amount of oxidized phospholipids within few degenerated neurons (Schuh et al. 2014). In chronic relapsing EAE in rats, nearly none oxidized phospholipids are detected (Schuh et al. 2014).

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8 2.2. Antioxidants

Antioxidants can be either endogenous or exogenous compounds. These compounds are able to reduce the production of ROS and free radicals or they neutralize them and may interrupt the reaction chain, respectively. Antioxidants can be localized within specific organelles or operate freely (Delanty et al. 2000). Furthermore, antioxidants can be classified according to the fact, whether they are produced in situ or need to be externally supplied. Supply can be through food or special supplements (Pham-Huy et al. 2008). The fact that antioxidants can be externally supplied makes them an interesting candidate for therapeutic treatments.

2.2.1. Endogenous Antioxidants

The body has some potent endogenous antioxidative systems. One is the superoxide dismutase (SOD), which was discovered by McCord and Fridovich (McCord et al. 1969). This enzyme catalyzes the reaction from superoxide anions to hydrogen peroxide and oxygen (McCord et al. 1969):

O2- + O2- + 2H^-^ O2 + H2O2

There are different types of superoxide dismutase. First, a manganoenzyme that is, similar to SOD located within mitochondria. The second form is a cuprozinc enzyme which is located in the cytosol (Weisiger et al. 1973a, b). Catalase is a tetrametic enzyme which converts hydrogen peroxide to oxygen and water (Kirkman et al. 1984).

2H2O2 2H2O + O2

Catalase is localized within peroxisomes of several cells. Moreover it was shown to be localized within neurons, oligodendrocytes and astrocytes in rat brains (Moreno et al. 1995).

Gene thereapy with SOD and catalase for optical neuritis provoked by EAE in DBA/1J mice showed a decreased retinal ganglion cell loss in comparison to the control eye (Qi et al.

2007).

One study showed that MS patients have a higher SOD activity within erythrocytes (Polidoro et al. 1984). In contrast, catalase is decreased within granulocytes of MS patiens (Jensen et al. 1980). In addition to SOD and catalase, glutathion peroxidase represents an important antioxidant enzyme (Pigeolet et al. 1990). Glutathion peroxidase converts hydrogen peroxide to oxygen and water and might fulfill an equal function as catalase in human

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9

erythrocytes (Gaetani et al. 1989). Glutathion itself cannot cross the blood-brain-barrier, but precursors of it can (Schulz et al. 2000). NAD(P)H:quinone oxidoreductase 1 is a flavoprotein and is thought to catalyze detoxification of ROS in all tissuses. Within the CNS higher oxidative stress and seizures are observed in the absence of this protein (Stringer et al. 2004). Moreover, NAD(P)H:quinone oxidoreductase 1 is upregulated within chronic active MS lesions (van Horssen et al. 2006). Urinic acid can be a water soluble antioxidant (Maples et al. 1988; Simic et al. 1989; Hooper et al. 1998). It can bind free iron and build stable complexes with iron (Davies et al. 1986). Furthermore, it is known to be a potent scavenger of peroxynitrite (Rentzos et al. 2006). α-Lipoate is a low molecular weight molecule, which can be taken up by diet and is able to cross the blood-brain barrier. Within cells it is reduced to dihydrolipoate and can be exported into the extracellular space. α-Lipoate and its reduced form act as antioxidant molecules that can exert their functions both within the cell as well as extracellularly (Packer et al. 1997). Moreover, neuroprotective efficiency was shown. An animal model of MS, EAE, reveals less demyelination within the CNS if the animals were treated with a-lipoic acid (Morini et al. 2004). Bilirubin can reduce superoxide formation. It has been shown that bilirubin has a higher antioxidant effect than α-tocopherol. Furthermore, it has a positive effect on the clinical outcome of EAE (Liu et al. 2003).

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10 2.2.2. Exogenous Antioxidants

Antioxidants can be absorbed from external sources. This can happen through food or special supplements (Pham-Huy et al. 2008). There are several studies on exogenous antioxidants that were tested to be beneficial in several diseases.

Sud'Ina et al. (1993) showed that caffeic acid phenethyl ester (CAPE) has antioxidative properties. CAPE is a component of honeybee propolis, which acts like an antioxidant.

Furthermore, anti-inflammatory activities are discussed. In general CAPE inhibits the oxygenation of linoleic acid and arachidonic acid that is catalyzed by 5-lipoxygenase and similar reactions that are catalyzed by 15-lipoxygenase (Sud'Ina et al. 1993). One study also showed that rats suffering from EAE and treated with CAPE showed only moderate clinical symptoms in comparison to non-treated animals. Besides there was less inflammatory infiltration. This may indicate an antioxidant effect of CAPE in this disease (Ilhan et al. 2004).

In SJL mice with EAE, epigal-locatechin-3-gallate (EGCG) has beneficial effects.

EGCG is included in green tea. The mice that were treated with EGCG showed a better recovery. Furthermore, neurological deficits in the late phase of the disease were significantly lower (Aktas et al. 2004). In patients suffering from relapsing-remitting MS (RRMS) omega-3 fatty acids have demonstrated to have beneficial effects in a 6 months study. Omega-3 fatty acids seem to decrease matrix metalloproteinase-9 (MMP-9) secreted by immune cells, thus acting as an immune modulating agent (Shinto et al. 2009). Vitamin E (synonym: α-tocopherol) can compete for peroxyl radicals. The reaction with peroxyl radicals is faster than with polyunsaturated fatty acids. Therefore, vitamin E can act as an antioxidant molecule to protect fatty acids (Burton et al. 1990). Balb/c mice, which were infected with lipopolysaccharide (LPS) intraperitoneally, showed decreased lipid peroxidation after treatment with α-tocopherol (Godbout et al. 2004). Vitamin C is a water-soluble antioxidant. Its antioxidative properties are attributed to the direct reaction with radicals or by indirect mechanisms in terms of renewing vitamin E (Bendich et al. 1986). Within the CNS, it is reported that vitamin C can have neuroprotective effects in rats suffering from seizures (Xavier et al. 2007). Due to the observation that MS occurs more often in northern countries, which might be related to a lower time of sun exposure, vitamin D is considered to play a role in MS (Smolders et al. 2008;

Melcon et al. 2014). Indeed, female mice suffering from EAE showed significantly improved clinical signs, if they were fed with vitamin D3 (Spach et al. 2005).

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11

In summary, several mechanisms and pathways result in ROS or free radical production.

However, under normal circumstances multiple enzymes and proteins that have an antioxidant effect and are capable of protecting cells and tissue from ROS damage effectively. In MS as well as in animal models for MS (e.g. EAE) antioxidant therapies have shown positive effects, thus arguing for a pathogenic relevance of ROS in demyelinating disease (Jensen et al. 1980;

Polidoro et al. 1984; Spitsin et al. 2002; Liu et al. 2003; Morini et al. 2004; Yadav et al. 2005;

Rentzos et al. 2006; van Horssen et al. 2006; Qi et al. 2007).

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12 2.3.Canine Distemper Virus (CDV)

Canine distemper virus (CDV) is a morbillivirus and belongs to the family of Paramyxoviridae (Baumgärtner et al. 1989). It is related to measles virus as well as rinderpest virus and peste-des-petites-ruminants virus (Baumgärtner et al. 1989; Burge et al. 1989;

Beineke et al. 2009; Lempp et al. 2014). Several members of the order carnivores (terrestrial and aquatic carnivores) are susceptible to canine distemper virus. CDV can cause gastrointestinal and respiratory signs as well as neurological disease (Carvalho et al. 2012). The virus is usually transmitted by aerosol infection of the upper respiratory tract. CDV replicates first in lymphoid tissues, which leads to immunosuppression. Subsequent to primary replication in lymphoid tissue, it replicates in various epithelial cells, causing respiratory, intestinal and dermatological signs (Vandevelde et al. 2005; Beineke et al. 2009). The virus may enter the brain on the hematogenous route and spread along the cerebrospinal fluid (CSF) (Vandevelde et al. 1985b; Beineke et al. 2009). Another way for viral spread is the cell-to-cell spread using astrocyte processes (Wyss-Fluehmann et al. 2010). Viral antigen can be detected in astrocytes, microglia cells, neurons, ependymal, leptomeningeal and choroid plexus cells (Wünschmann et al. 1999; Vandevelde et al. 2005; Beineke et al. 2009; Lempp et al. 2014). Clinically the neurological signs can be very variable. They include a tendency to fall, head tilt, nystagmus, head bobbing, conscious proprioceptive deficits, ataxia with hypermetria, seizures (partial or generalized), paraplegia, quadriplegia, tremor, coma, circling or blindness (Dunkin et al. 1926;

Tipold et al. 1992; Zurbriggen et al. 1997).

The virus is a single-stranded, enveloped, negative-sense RNA virus. It has six structural proteins and two nonstructural proteins. The structural proteins are the nucleocapsid- (N), the matrix- (M), the hemagglutinin- (H), the fusion- (F), the large- (L) and the phosphor-(P) protein.

The C and V protein represent the nonstructural proteins (Beineke et al. 2009; Carvalho et al.

2012). CDV leukoencephalitis (CDV-DL)-lesions occur predominantly in the cerebellum, and can be subdivided in acute, subacute non-inflammatory, subacute inflammatory and chronic lesion (Baumgärtner et al. 2005; Ulrich et al. 2014b). Acute CDV-DL show vacuolization of the white matter and mild gliosis (Vandevelde et al. 1995; Seehusen et al. 2010; Ulrich et al.

2014b). Subacute lesions are characterized by demyelination. Subacute non-inflammatory lesion show only demyelination. In contrast to subacute non-inflammatory lesions subacute inflammatory lesions show a mild perivascular infiltration of up to three layers of inflammatory cells. Chronic lesions are characterized by the ongoing demyelination as well as a severe perivascular inflammatory infiltrate of more than four layers of inflammatory cells. The

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13

inflammatory infiltrate is mainly composed of lymphocytes and macrophages (Vandevelde et al. 1995; Seehusen et al. 2010; Ulrich et al. 2014b).

The demyelination that occurs in subacute to chronic lesions might be a secondary or

“bystander” phenomenon. This can arise because of an interaction between macrophages/microglia and antiviral antibodies or because of autoreactive T cells (Beineke et al. 2009). Vandevelde and Zurbriggen reviewed that oligodendrocytes are only sparsely infected within CDV-DL. In addition, demyelination is found before oligodendrocytes get lost.

Thus, oligodendrocyte destruction may occur due to another pathogenic mechanism (Zurbriggen et al. 1997; Vandevelde et al. 2005; Lempp et al. 2014; Ulrich et al. 2014b).

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14

2.4.Theiler’s Murine Encephalomyelitis Virus (TMEV)

Theiler’s murine encephalomyelitis virus (TMEV or Theiler’s virus) is a positive, single-stranded RNA virus and belongs to the genus of Cardiovirus (Ozden et al. 1986; Pevear et al. 1987) of the family Picornaviridae (Oleszak et al. 2004). Theiler first reported infection of mice in 1937. He described an intracerebral inoculation of mice with TMEV and a clinical outcome of a flaccid paralysis of the hind limbs. Young mice are more susceptible than adult ones. Theiler (1937) initially described that after intracerebral infection, mice show first clinical signs of weakness of the limbs after seven to more than 30 days. Natural infection was initially described in mice around 6 to 7 weeks of age. The natural manifestation of the virus within the CNS occurs only sporadically and represents a rare finding (Theiler 1937). Naturally, TMEV is found in the gastro-intestinal tract (Olitsky 1939, 1940; Theiler et al. 1940; Theiler 1941).

TMEV strains are subdivided into two subgroups. The GDVII and the FA strain belong to the first subgroup, which is very neurovirulent (Ozden et al. 1986; Pevear et al. 1987). Infection with these strains in mice leads to acute encephalitis and only few animals survive the infection.

Usually the animals die 1 to 2 weeks post infection (p.i.) (Oleszak et al. 2004). Furthermore, GDVII and FA strains do not persist in the animals that survive (Pevear et al. 1987; Oleszak et al. 2004). The second subgroup (Theiler’s Original; TO) includes DA (Daniels) BeAn8386 (BeAn) and WW strain (Oleszak et al. 2004; Tsunoda et al. 2010). The DA and the BeAn strain induce a biphasic disease. In the acute disease phase, the gray matter is predominantly affected.

Both TMEV-strains lead to a chronic demyelinating disease in susceptible mouse strains (Lipton 1975; Lipton et al. 1984; Oleszak et al. 2004; Kummerfeld et al. 2012). However, clinical signs develop differently. First clinical signs are present in mice infected with the BeAn-strain 30-40 days p.i. (dpi) while in mice infected with the DA-strain become clinically apparent at 140-180 dpi (Oleszak et al. 2004). Histologically DA and BeAn appear similar, as they both show demyelination of the white matter within the spinal cord and perivascular as well as parenchymal inflammatory infiltrates (Lipton 1975; Oleszak et al. 2004). The outcome of infection with TMEV critically depends on the mouse strain. B6 mice can clear the virus (Oleszak et al. 2004; Gerhauser et al. 2007; Prajeeth et al. 2014). They develop the early stage of the disease but are able to clear the virus and do not develop a chronic demyelinating disease.

In contrast, SJL mice are sensitive to TMEV-infection and develop a chronic demyelinating disease after the acute phase (Oleszak et al. 2004). Besides C3H/He and CBA mouse strains are also susceptible to TMEV-infection. However, SJL mice show the most severe lesions (Lipton et al. 1979).

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After intracerebral inoculation, the first inflammatory infiltrate is present at 14 dpi within the meninges and the white matter of spinal cord. This inflammatory infiltrate is mainly composed of lymphocytes as well as macrophages. At 42 dpi, demyelination within the ventrolateral funiculi of the spinal cord is present. The amount of inflammatory cells as well as the demyelination increases in the course of infection (Ulrich et al. 2008; Ulrich et al. 2010;

Sun et al. 2015). The demyelination process itself might be different in TMEV-DL mice in contrast to CDV-infected dogs. CD4+ T helper cells and macrophages initiate myelin damage in mice infected with TMEV. Therefore, demyelination might be a result of a delayed hypersensitivity reaction (Dal Canto et al. 2000). However, it could be shown that there is an increase oligodendroglia apoptosis in the early phase of TMEV-DL. This may indicate a primary demyelination due to oligodendroglia damage (Akassoglou et al. 1998; Gerhauser et al. 2018). The myelin damage due to damage of oligodendrocytes represents the outside-in model of demyelination (Tsunoda et al. 2002). Furthermore, TMEV-DL leads also to axonal damage. Axonal damage can be a result of an impairment of neuronal protein metabolism, failure of axonal transport or due to a lack of ATP (Kreutzer et al. 2012; Lassmann et al. 2012).

A primary axonal damage would represent an inside-out model of demyelination (Tsunoda et al. 2002).

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Manuscript

16

3. Manuscript: Reactive oxygen species are key mediators of demyelination in canine distemper leukoencephalitis but not in Theiler’s murine encephalomyelitis

Friederike Attig^1,2,*, Ingo Spitzbarth^1,2,*, Arno Kalkuhl³, Ulrich Deschl³, Christina Puff¹, Wolfgang Baumgärtner^1,2, Reiner Ulrich^1,2,4

1Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany

2Center for Systems Neuroscience, Hannover, Germany

3Department of Non-clinical Drug Safety, Boehringer Ingelheim Pharma GmbH & Co.KG, Biberach, Germany

4Institute for Veterinary Pathology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany

The first two authors contributed equally to this article and should be considered as co-first authors.*

Correspondence: Prof. Dr. Wolfgang Baumgärtner, PhD., Dipl. ECVP, DACVP (hon.) Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17

D-30559 Hannover, Email: wolfgang.baumgaertner@tiho-hannover.de, Phone: 0049 511 953 8620, Fay: 0049 511 953 8675

International Journal of Molecular Sciences, 20(13), 3217, doi:10.3390/ijms20133217

Permission of reprint:

International Journal of Molecular Sciences is an open access journal. The author is allowed to re-use published material without obtaining permission.

Author Contributions: W.B. and R.U. conceived and designed the experiments; F.A. performed the experiments; F.A. and I.S. analyzed the data; W.B., R.U., U.D., A.K. and C.P. contributed reagents, materials, analysis tools; F.A. and I.S. wrote the paper. W.B., R.U., U.D., A.K., and C.P. finalized the draft.

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46 Supplementary Table S1

Case No Breed Gender Age (month) Lesions

1 West

Highland White Terrier

Not determined Not determined 8 acute

2 Podenco female juvenile 11 acute

3 Mixed breed female 46 5 acute

4 Not

determined Not

determined Not determined 6 acute

5 Mixed breed female Not determined 2 acute, 8 subacute non-inflammatory

6 Mixed breed female 8 2 acute, 2 subacute

non- inflammatory

7 Miniature

Pinscher male 84 2 acute, 7 subacute

non-inflammatory

8 Siberian husky male 12 11 acute, 1 subacute

non-inflammatory

9 Not

determined Not

determined Not determined 1 subacute inflammatory

10 Mixed breed female 12 6 subacute

inflammatory 11 Mixed breed male Not determined 1 subacute

inflammatory

12 German

shepherd female 9 1 subacute

inflammatory

13 Pitbull female 6 2 acute, 4 subacute

non-inflammatory, 3 subacute

inflammatory, 3 chronic

14 Mixed breed female Not determined 2 subacute inflammatory, 1 chronic

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47 Supplementary Table S1 Continued

Case No Breed Gender Age (month) Lesions

17 Beagle male 20 Control

18 Beagle female 17 Control

21 Rhodesian

Ridgeback male 26 Control

22 Labrador

Retrievers male 110 Control

23 Bernese

Mountain Dog Female

neutered 96 Control

24 Mixed breed male 51 Control

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Manuscript

48 Supplementary Table S2

Primary antibody Species Dilution Antigen

retrieval Company

E06 Mouse

(monoclonal antibody, mAb)

1: 25 Citrate

buffer Avanti Polar

Lipids, Inc.

(Alabama, United States of America) Malondialdehyde

(MDA, ab6463) Rabbit (polyclonal antibody, pAb)

1:100 Citrate

buffer Abcam (Cambridge, United Kingdom) Hydroxyguanosin

e (8OHdG/8OHG, ab10802),

Goat (pAb) 1:100 Proteinase K Abcam (Cambridge, United Kingdom) Superoxide

dismutase 2 (SOD, ab13533),

Rabbit (pAb) 1:200 Citrate

buffer Abcam (Cambridge, United Kingdom) Catalase

(ab50434), Goat (pAb) 1:50 Citrate

buffer Abcam (Cambridge, United Kingdom) 2',3'-Cyclic-

nucleotide 3'- phosphodiesteras e (CNPase)

buffer Abcam (Cambridge, United Kingdom) CNPase Mouse (mAb) 1:500 Citrate

buffer Millipore Merck KGaA, Darmstadt, Germany

Ionized calcium- binding adapter molecule 1 (Iba-1)

buffer Wako Pure

Chemical Industries, Richmond, USA Iba-1 Mouse (mAb) 1:100 Citrate

buffer Abcam (Cambridge, United Kingdom) Glial fibrillary

acidic protein (GFAP)

buffer DakoCytomation, (Hamburg, Germany)

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49 Supplementary Table S2 Continued

Supplementary Table S3

Primary antibody Species Dilution Antigen

retrieval Company GFAP Mouse (mAb) 1:400 Citrate

buffer (GFAP, clone G-A- 5, Sigma-Aldrich, Taufkirchen, Germany) CDV D110 Mouse (mAb) 1:2000 Citrate

buffer Zurbriggen

Secondary antibody Dilution Company Goat-anti Mouse IgG

Cy3 1:200 Jackson ImmunoResearch,

DIANOVA, Hamburg, Ger- many

Goat-anti Rabbit Alexa

Fluor 488 1:200 Invitrogen, Thermo Fisher

Scientific Waltham, Massachusetts, USA

Donkey-anti Goat IgG

Cy3 1:200 Jackson ImmunoResearch,

DIANOVA Hamburg, Ger- many

Donkey-anti Rabbit

Alexa Fluor 488 1:200 Jackson ImmunoResearch,

DIANOVA, Hamburg, Ger- many

Goat anti-mouse-

biotinylated, GaM-b 1:200 Vector Laboratories,

Peterborough, United Kingdom, BA-9200

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Supplementary Table S4 (CDV)

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Supplementary Table S4 Continued (CDV)

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Supplementary Table S4 Continued (TMEV)

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General Discussion

90 4. General Discussion

Previous studies revealed ROS-induced metabolites such as oxidized lipids or oxidized DNA and RNA within active MS lesions. These were detected by immunohistochemistry and were present within oligodendrocytes, macrophages, astrocytes, myelin, and neurons (Haider et al. 2011; Schuh et al. 2014). A microarray study showed that genes, which are related to ROS production, are up-regulated (Fischer et al. 2012). Furthermore, increased amounts of ROS products are also described for Alzheimer´s disease (Markesbery 1997; Christen 2000;

Butterfield et al. 2002), and spinal cord as well as traumatic brain injuries (Lewen et al. 2000;

Bains et al. 2012).

Observation of studies regarding ROS production within MS lesions lead to the hypothesis that ROS may also take part in the pathogenesis of CDV-DL and TMEV-DL. CDV- DL as well as TMEV-DL are both well established animal models for MS (Amude et al. 2010;

Procaccini et al. 2015). While CDV-DL mimics MS lesions by acute demyelination followed by inflammatory infiltrates and progressive demyelination (Vandevelde et al. 1982; Amude et al. 2010; Lempp et al. 2014), the immune response in TMEV-infected mice is a key feature, which is highly similar to MS (Lipton 1975; Dal Canto et al. 1982; Dal Canto et al. 1996; Mecha et al. 2013). The present thesis shows that ROS products are present in both models, thus unraveling another pathogenetic factor that has similarities to MS.

In general, the CNS has a high sensitivity to ROS damage. The CNS consists of high lipid component in form of myelin. Lipids are preferred molecules to react with ROS.

Furthermore, the CNS is dependent on aerobic metabolic processes that make the CNS also susceptible to hypoxia (Smith et al. 1999). Due to the oxidative metabolism of the CNS, high amounts of superoxide arise intracellularly. Therefore, antioxidants are needed to detoxify these metabolites (McCord et al. 1971; Kirkman et al. 1984; Smith et al. 1999; Auten et al. 2009;

Brieger et al. 2012).

The detection of ROS damage often needs the usage of antibodies detecting specific ROS products. These markers can be oxidized lipids, oxidized RNA and DNA (Palinski et al.

1990; Palinski et al. 1996; Horkko et al. 1999; Haider et al. 2011; Hametner et al. 2013; Pisoschi et al. 2015). To detect oxidized lipids, malondialdehyde (MDA) and oxidized phospholipid (clone E06) are commonly used markers (Figure 4.1.) (Palinski et al. 1990; Palinski et al. 1996;

Horkko et al. 1999; Marnett 1999; Haider et al. 2011; Schuh et al. 2014). MDA occurs naturally as a product of lipid peroxidation or as a by-product of prostaglandin synthesis (Figure 4.1.)

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(Marnett 1999; Marnett et al. 1999). It reacts with nucleic acid bases, phospholipid and protein.

Furthermore, mutagenic characteristics are described (Draper et al. 1986). To detect other oxidized lipids that are not MDA-related the marker for oxidized phospholipids (clone E06) can be used (Palinski et al. 1990; Palinski et al. 1996). Finally, the markers 8- hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) are commonly used to detect ROS damage of DNA (Figure 4.1.) or RNA (Wu et al. 2004; Pisoschi et al. 2015). It is a change of the 8^th position of guanosine that alters the binding (Wagner et al. 2000). Normally, guanosine binds with cytosine, but hydroxyguanosine binds incorrectly with adenosine. This changes the base sequence irreversibly and can lead to mutations. 8OHdG is also used as a tumor-marker. The incorrect binding lead to the suggestion of a mutagenic effect (Wagner et al. 2000). However, it is known that abundance of 8OHdG also increases during aging. The reason for this may be reduced repair mechanisms (Wagner et al. 2000).

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General Discussion

92

Figure 4.1.: Specific Markers for Damaged Lipids and DNA Induced by Reactive Oxygen Species (ROS). Inflammatory cells as well as mitochondria can produce ROS. ROS lead to generation of oxidized phospholipids that can be detected by the antibody E06 that represents a clone of oxidized phospholipid (Palinski et al. 1990; Palinski et al. 1996; Horkko et al. 1999).

Malondialdehyde (MDA) can occur naturally as a product of lipid peroxidation or as a by- product of prostaglandin synthesis (Marnett 1999; Marnett et al. 1999). 8- hydroxydeoxyguanosine (8OHdG) is produced by hydroxylation in the 8^th position of guanosine (Wagner et al. 2000).

The mitochondria and especially complex I and III (NADH dehydrogenase and ubisemiquinone) of the electron transport chain are one of the main sources of superoxide (Thannickal et al. 2000; Dröge 2002; Han et al. 2003; Mittal et al. 2014).

Another source of ROS is represented by macrophages/microglia. The release of ROS can be either related to direct virus effects or due to the release of pro-oxidant cytokines like interleukin-1 (IL-1) and tumor necrosis factor (TNF) (Griot et al. 1989; Schwarz 1996;

Peterhans 1997). The production of ROS within macrophages/microglia arises either through NADPH oxidase (Bedard et al. 2007; Mittal et al. 2014) or myeloperoxidase (Mao et al. 2010).

NADPH oxidase displays one enzyme (NOX2) of the group of NOX enzymes. This group of